Bacteria in Agrobiology: Crop Ecosystems

Bacteria in Agrobiology: Crop Ecosystems . Dinesh K. Maheshwari Editor

Bacteria in Agrobiology: Crop Ecosystems Editor Prof.(Dr.) Dinesh K. Maheshwari Gurukul Kangri University Deptt. of Botany and Microbiology 249404 Haridwar (Uttarakhand) India [email protected]

ISBN 978-3-642-18356-0 e-ISBN 978-3-642-18357-7 DOI 10.1007/978-3-642-18357-7 Springer Heidelberg Dordrecht London New York

Library of Congress Control Number: 2011926231

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Cover illustration: Optical micrograph showing cross sections of intercellular colonization rice calli and regenerated plantlets by A. caulinodans: CS view of root uninoculated control; magniﬁed cross section view of leaf colonized by A. caulinodans in regenerated rice plant; possible sites of infection and colonization of rice root (from left to right); see also Fig. 3.1 in “Endophytic Bacteria – Perspectives and Applications in Agricultural Crop Production”, Senthilkumar M, R. Anandham, M. Madhaiyan, V. Venkateswaran, Tong Min Sa, in “Bacteria in Agrobiology: Crop Ecosystems, Dinesh K. Maheshwari (Ed.)”

Background: Positive immunofluorescence micrograph showing reaction between cells of the rhizobial biofertilizer strain E11 and specific anti-E11 antiserum prepared for autecological biogeography studies; see also Fig. 10.6 in “Beneficial Endophytic Rhizobia as Biofertilizer Inoculants for Rice and the Spatial Ecology of this Bacteria-Plant Association”, Youssef Garas Yanni, Frank B. Dazzo, Mohamed I. Zidan. in “Bacteria in Agrobiology: Crop Ecosystems, Dinesh K. Maheshwari (Ed.)”

Cover design: deblik, Berlin

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com) Preface

Bacteria are among the most adaptable organisms. Their evolutional passage across the long timescale, extremely short generation time, and aptitude to adapt to diverse and often hostile environments, combined with the remarkable power of natural selection have made these microorganisms the most resilient of life forms on this planet. As such, bacteria and fungi abound in the soil are the essential contributors in maintaining the ecological balance. One of the most remarkable developments of the twentieth century vis-a`-vis microorganisms is the discovery of the plant growth promoting bacteria (PGPB) that offers a vast array of beneficial attributes to plants, and thereby facilitating enhancement of crop productivity in a sustainable manner. More than 97% of our food requirements are realized from terrestrial ecosystems through agricultural productivity. Diversified populations of bacterial species occur in agricultural fields and contribute to crop productivity directly or indirectly. Plants provide a substantial ecological niche for microorganisms and below ground (roots) portions of plants and soil are constantly associated with a larger number of microorganisms reaping several benefits from such associations. This volume is accordingly con- ceived to provide consolidated information on the subject. The book entitled Bacteria in Agrobiology: Crop Ecosystems has chapters that cover studies on various aspects of bacteria–plant interactions. Better understand- ings of the challenges in development of PGPB as efficient commercial bioinoculant have met in enhancing crop production. A large number of bacterial genera interplay with rhizosphere communities in different crops ecosystems, in particular, the oil-yielding crops, cereals, fruits and vegetables, forest trees, etc. Keeping in fitness with such important crops, the developmental challenges faced in the management of growth and soil and seed borne diseases associated with food crops such as rice, sesame, peanut, along with horticultural, sericultural plant ecosystems as well as in forestry are aptly covered in this volume. Detection of PGPR and biocontrol of postharvest pathogens as suitable alternatives to agrochemicals for sustainable crop production and protection, and restoration of degraded soils has also been duly addressed. I believe that this book will be useful not

v vi Preface only for researchers, teachers, and students, but also for those who are interested in the subjects of applied microbiology, plant protection, ecology, environmental science, and agronomy. I would like to express my gratitude to all the authors for their scholarly contributions. I recognize with credit the continuous support that I received from my research students Mr. Abhinav Aeron, Mr. Rajat Khillon, Mr. Pankaj Kumar, and Dr. Sandeep Kumar in the preparation of this volume. I am also thankful to Council of Scientiﬁc and Industrial Research (CSIR), New Delhi; and Director, Uttarakhand Council of Science and Technology (UCOST), Dehradun, India for their support in implementation of my research projects on PGPB that served as a prolog to arrange base for compilation of this book. I extend my earnest appreciation to Dr. Jutta Lindenborn of Springer for her valuable support to facilitate completion of the task.

Haridwar, Uttarakhand, India Dinesh K. Maheshwari Contents

1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology ...... 1 Abhinav Aeron, Sandeep Kumar, Piyush Pandey, and D.K. Maheshwari

2 Bacillus as PGPR in Crop Ecosystem ...... 37 Ankit Kumar, Anil Prakash, and B.N. Johri

3 Endophytic Bacteria: Perspectives and Applications in Agricultural Crop Production ...... 61 M. Senthilkumar, R. Anandham, M. Madhaiyan V. Venkateswaran, and Tongmin Sa

4 PGPR Interplay with Rhizosphere Communities and Effect on Plant Growth and Health ...... 97 Gabriele Berg and Christin Zachow

5 Impact of Spatial Heterogeneity within Spermosphere and Rhizosphere Environments on Performance of Bacterial Biological Control Agents ...... 111 Daniel P. Roberts and Donald Y. Kobayashi

6 Biocontrol Mechanisms Employed by PGPR and Strategies of Microbial Antagonists in Disease Control on the Postharvest Environment of Fruits ...... 131 Anjani M. Karunaratne

7 Plant Growth-Promoting Bacteria Associated with Sugarcane ..... 165 Samina Mehnaz

vii viii Contents

8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops ...... 189 Ahmet Esitken

9 Commercial Potential of Microbial Inoculants for Sheath Blight Management and Yield Enhancement of Rice ...... 237 K. Vijay Krishna Kumar, M.S. Reddy, J.W. Kloepper, K.S. Lawrence X.G. Zhou, D.E. Groth, S. Zhang, R. Sudhakara Rao, Qi Wang M.R.B. Raju, S. Krishnam Raju, W.G. Dilantha Fernando, H. Sudini B. Du, and M.E. Miller

10 Beneﬁcial Endophytic Rhizobia as Biofertilizer Inoculants for Rice and the Spatial Ecology of This Bacteria–Plant Association ...... 265 Y.G. Yanni, F.B. Dazzo, and M.I. Zidan

11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation ...... 295 Clara Pliego, Faina Kamilova, and Ben Lugtenberg

12 PGPR in Coniferous Trees ...... 345 Elke Jurandy Bran Nogueira Cardoso, Rafael Leandro de Figueiredo Vasconcellos, Carlos Marcelo Ribeiro, and Marina Yumi Horta Miyauchi

13 Perspectives of PGPR in Agri-Ecosystems ...... 361 Meenu Saraf, Shalini Rajkumar, and Tithi Saha

14 Ecofriendly Management of Charcoal Rot and Fusarium Wilt Diseases in Sesame (Sesamum indicum L.) ...... 387 Sandeep Kumar, Abhinav Aeron, Piyush Pandey, and Dinesh Kumar Maheshwari

15 Crop Health Improvement with Groundnut Associated Bacteria .. 407 Swarnalee Dutta, Manjeet Kaur, and Appa Rao Podile

Index ...... 431 Contributors

Abhinav Aeron Department of Botany and Microbiology, Faculty of Life Sciences, Gurukul Kangri University, Haridwar 249404, Uttarakhand, India, abhinavaeron@ gmail.com

Rangasamy Anandham Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625104, Tamil Nadu, India, [email protected]

Gabriele Berg Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria, [email protected]

Elke Jurandy Bran Nogueira Cardoso Soil Microbiology Laboratory, Depart- ment of Soil Science, Luiz de Queiroz College of Agriculture, Sa˜o Paulo State University, Piracicaba Sa˜o Paulo, Brazil, [email protected]

Frank B. Dazzo Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA, [email protected]

B. Du Department of Microbiology, Shandong Agricultural University, Taian Shandong Province, China

Swarnalee Dutta Department of Plant Sciences, School of Life Sciences, Univer- sity of Hyderabad, Hyderabad 500046, India

Ahmet Esitken Department of Horticulture, Faculty of Agriculture, Ataturk Uni- versity, 25240 Erzurum, Turkey, [email protected]

W.G. Dilantha Fernando Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada, [email protected]

ix x Contributors

D.E. Groth LSU AgCenter, Rice Research Station, Baton Rouge, LA, USA

Bhavdish N. Johri Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, Madhya Pradesh, India, bhavdishnjohri@ rediffmail.com

Faina Kamilova Koppert Biological Systems, Veilingweg 14, PO Box 155, 2650 AD Berkel en Rodenrijs, The Netherlands, [email protected]

Anjani M. Karunaratne Department of Botany, Faculty of Science, University of Peradeniya, Peradeniya, Sri Lanka, [email protected]

Manjeet Kaur Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

Joseph W. Kloepper Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA

Donald Y. Kobayashi Sustainable Agricultural Systems Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, USDA-ARS, Beltsville, MD 20701, USA; Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA

Sandeep Kumar Department of Botany and Microbiology, Faculty of Life Sciences, Gurukul Kangri University, Haridwar 249404, Uttarakhand, India, [email protected]

K. Vijay Krishna Kumar Department of Entomology and Plant Pathology, Au- burn University, Auburn, AL, USA; Acharya N G Ranga Agricultural University, Hyderabad, India

Ankit Kumar Department of Biotechnology and Bioinformatics Centre, Barkatul- lah University, Bhopal 462026, Madhya Pradesh, India, [email protected]

K.S. Lawrence Department of Entomology and Plant Pathology, Auburn Univer- sity, Auburn, AL, USA

Ben J.J. Lugtenberg Sylvius Laboratory, Institute of Biology, Leiden University, Sylviusweg 72, PO Box 9505, 2300 RA Leiden, The Netherlands, Ben.Lugtenberg @gmail.com

Munuswamy Madhaiyan Department of Agricultural Chemistry, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea Contributors xi

Samina Mehnaz Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan; Institute of Pharma- ceutical Biology, Bonn University, Bonn 53115, Germany, [email protected]. pk, [email protected]

M.E. Miller Department of Biological Sciences, Auburn University, Auburn, AL, USA

Tongmin Sa Department of Agricultural Chemistry, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea, [email protected]

Marina Yumi Horta Miyauchi Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Sa˜o Paulo State University, Piracicaba, Sa˜o Paulo, Brazil

Senthilkumar Murugesan Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India, senthiltnj@ rediffmail.com

Piyush Pandey Department of Biotechnology, S. B. S. P. G. Institute of Biomedi- cal Sciences and Research, Balawala, Dehradun 248161, Uttarakhand, India

Clara Pliego Instituto de Hortofruticultura Subtropical y Mediterrnea “La Mayora”, Universidad de Mlaga – Consejo Superior de Investigaciones Cientı´ﬁcas (IHSM- UMA-CSIC), A´ rea de Gene´tica, Universidad de Mlaga, Campus de Teatinos s/n, 29071 Mlaga, Spain; Division of Biology, Department of Life Science, Imperial College London, Imperial College Road, SW7 2AZ London, UK, [email protected], [email protected]

Appa Rao Podile Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India, [email protected], apparaopodile@ yahoo.com

Anil Prakash Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, Madhya Pradesh, India, [email protected]

Shalini Rajkumar Institute of Science, Nirma University, S. G. Highway, Ahmedabad 382481, Gujarat, India

M.R.B. Raju Andhra Pradesh Rice Research Institute, Maruteru, India

S. Krishnam Raju Andhra Pradesh Rice Research Institute, Maruteru, India xii Contributors

R. Sudhakara Rao Acharya N G Ranga Agricultural University, Hyderabad, India

M. Sudhakara Reddy Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA, [email protected]

Carlos Marcelo Ribeiro Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Sa˜o Paulo State University, Piracicaba, Sa˜o Paulo, Brazil

Daniel P. Roberts Sustainable Agricultural Systems Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, USDA-ARS, Beltsville MD 20701, USA; Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA, [email protected]

Tithi Saha Institute of Science, Nirma University, S. G. Highway, Ahmedabad 382481, Gujarat, India

Meenu Saraf Department of Microbiology, Gujarat University, Ahmedabad 380009, Gujarat, India, [email protected]

H. Sudini International Crops Research Institute for the Semi-Arid Tropics (ICRI- SAT), Hyderabad, India

Rafael Leandro de Figueiredo Vasconcellos Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Sa˜o Paulo State University, Piracicaba, Sa˜o Paulo, Brazil

V. Venkateswaran Ministry of Food Processing Industries, New Delhi 110049, India

Qi Wang China Agricultural University, Beijing, China

Youssef Garas Yanni Department of Microbiology, Sakha Agricultural Research Station, Kafr El-Sheikh 33717, Egypt, [email protected]

Christin Zachow Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria, [email protected]

Shouan Zhang Tropical REC, University of Florida, Homestead FL, USA, szhang0007@uﬂ.edu

Mohamed I. Zidan Department of Plant Nutrition, Sakha Agricultural Research Station, Kafr El-Sheikh 33717, Egypt . Chapter 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology

Abhinav Aeron, Sandeep Kumar, Piyush Pandey, and D.K. Maheshwari

1.1 Introduction

Declining crop productivity due to unsuitable agricultural practices over the years and a galloping rate of population growth have both put up a severe strain on the food supply situation in the world. To meet the food requirements of the growing population, a second green revolution has become imperative due to “loss of dynamism” in agriculture as pointed out in global economic survey during the year 2007–2008. This has two obvious objectives, ﬁrstly to rejuvenate the agricultural sector and secondly to improve the income of those dependent on it. Pertaining to massive population pressure, increase in food grain production is an uphill task in today’s world. The need of the day is sustainable agriculture without harming the delicate balance of soil ecology as well as unlocking the mystery of biota inﬂuencing plant growth by using plant growth promoting rhizobacteria (PGPR). PGPR are nowadays applied in a wide array of agro and allied industries in the form of inoculants in a range of agro-economically important plants including leguminous and nonleguminous crops, trees and plants of forest, horticulture, sericulture, medicinal, fodder, oilseed, and cash crops for enhancing their growth and productivity. Green revolution was achieved as it resulted in increased yield due to extensive use of chemical based components. The indiscriminate use of these components imparted pesticide resistance in pests and made presence in plant produce. Presence of residual pesticides cause disruption and degradation of agro-ecosystem resulting in decreased soil fertility. Excessive application of fertilizer for obtaining higher production was not only undesirable from the economic point of view, but also

A. Aeron, S. Kumar, and D.K. Maheshwari (*) Department of Botany and Microbiology, Gurukul Kangri University, Haridwar 249 404, Uttarakhand, India e-mail: [email protected] P. Pandey Department of Biotechnology, S. B. S. P. G. Institute of Biomedical Sciences and Research, Balawala, Dehradun 248 161, Uttarakhand, India

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 1 DOI 10.1007/978-3-642-18357-7_1, # Springer-Verlag Berlin Heidelberg 2011 2 A. Aeron et al. exerted an adverse effect on the environment and crop quality (Kenny 1982). It led to nutrient imbalance, whereas inefficient and overuse of chemical fertilizers resulted in considerable economic loss to the farmers (Ayala and Rao 2002). It is widely believed that agrochemicals including chemical fertilizers reduce the population of beneficial microorganisms thus having an all-embracing effect (Smiley 1981). Reduction in the population of desirable beneficial microbes alters the topology of top soil and reduces the productivity of fertile soils. Thus, an important factor in this respect is to maintain the enhancement of soil fertility through appropriate sustainable technology, which should be achieved to replenish the nutrients so as to build up the nutrient status of soils (Hera 1996). The challenges of meeting the food requirements of the burgeoning population and plateauing productivity of agricultural lands can only be met by a second green revolution or ever green revolution. Some of the strategies that can be channeled to second green revolution include micro-irrigation system, organic farming, precision farming, green agriculture, eco-agriculture, white agriculture, straw revolution, and use of PGPR and their combinations. The aim of this chapter is to elaborate the need of PGPR applications in agriculture-based industries for economic development in an eco-friendly manner.

1.2 Soil and Rhizosphere in Sustainable Agriculture

Agricultural industries are mainly soil based because they extract nutrients from the soil. Effective and efficient approaches to slowing the removal and returning nutrients to the soil is required in order to maintain and increase crop productivity apart from efforts to sustain agriculture for the long term. The overall strategy for increasing crop yields and sustaining them at high level required natural or artificial inputs. The soil is managed by both biological and nonbiological factors known to have a major impact on plant growth, soil fertility, and agricultural sustainability. The physical, biological, and chemical characteristics of soil, such as organic matter content, pH, texture, depth, and water-retention capacity, are factors that influence soil fertility. A soil’s potential for producing crops is largely determined by the environment that soil provides for root growth, such as nutrients and the surrounding microflora that may be beneficial or deleterious. Roots need air, water, nutrients, and adequate space to develop. Soil quality is defined by capacity to store water, acidity, depth, and density that determine how well roots developed. Changes in soil quality affect the health and productivity of the plants and can lead to lower yields and/or higher costs of production. Organic matter content is important for the proper management of soil fertility and helps growth by improving water-holding capacity and drought resistance. Moreover, it permits better aeration, enhances the absorption and release of nutrients, and makes the soil less susceptible to leaching and erosion. The higher plant root system significantly contributes to the establishment of the microbial population in the rhizosphere. The rhizosphere has attracted much interest 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 3 as it is a habitat of several biologically important processes and their interactions. Acidification of the rhizosphere as a result of exudation of organic acids from root plays a pivotal role in determining the surrounding population (Dakora and Philips 2002). The rhizosphere is populated by diverse microorganisms including bacteria, fungi, actinomycetes, protozoa, algae, etc.; therefore, modifying plant root systems is considered as a means of crop improvement targeted toward low-resource environments, particularly low nutrient and drought-prone agriculture. Microbial processes and properties in the rhizosphere are crucial to support functional agriculture.

1.3 Beneﬁcial Bacteria

The microbe–plant interaction in the rhizosphere is dynamic and complicated. Some microbes contribute to plant health by mobilizing nutrients, while some are detrimental to plant health as they compete with the plant for nutrients or cause disease and some stimulate plant growth by producing hormones or by suppressing pathogens. The bacteria useful to plants are characterized into two general types: bacteria forming a symbiotic relationship with the plant and the free-living ones found in the soil but are often found near, on, or even within the plant tissues (Kloepper et al. 1988a; Frommel et al. 1991). Different authors have found different origins with the classification and definition of beneficial rhizobacteria. Beneficial free-living soil bacteria that enhance plant growth are usually referred to as “plant growth promoting rhizobacteria” (Kloepper et al. 1989) or yield increasing bacteria (YIB) (Tang 1994). PGPR originally defined (Kloepper and Schorth 1978) as root-colonizing bacteria (rhizobacteria) cause either growth promotion or biological control of plant diseases. Bashan and Holguin (1998) proposed that the PGPR can be categorized as biocontrol-plant growth promoting bacteria (PGPB) and phytostimulating PGPB. Root-associated bacteria have a great influence on organic matter decomposition which in turn is reflected in soil nutrient availability for plant growth (Glick et al. 1994). The phosphorus- and potassium-solubilizing bacteria (PSB) may enhance plant nutrient availability by dissolving insoluble phosphorus and releasing potassium from silicate minerals (Goldstein and Liu 1987). PGPB often help increase root surface area to increase nutrient uptake and in turn enhance plant production (Mantelin and Touraine 2004). The premier example of PGPR agents occur in many genera including Actinoplanes, Agrobacterium, Alcaligens, Amorphospor- angium, Arthrobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Cellulomonas, Enterobacter, Erwinia, Flavobacterium, Gluconacetobacter, Microbacterium, Micromonospora, Pseudomonas, Rhizobia, Serratia, Strepto- myces, Xanthomonas, etc., as stated by several workers (Kloepper et al. 1989; Tang 1994; Okon and Labandera-Gonzalez 1994;Glicketal. 1999; Mayak et al. 2001;Lucyetal. 2004; Tahmatsidou et al. 2006; Aslantas et al. 2007; Lee et al. 2008; Pedraza et al. 2010). 4 A. Aeron et al.

One of the dominant genera among PGPR is Pseudomonas spp. reported in the biological control of different phytopathogenic fungal species such as Rhizoctonia, Fusarium, Sclerotonia, Pythium, Erwinia, Macrophomina, etc. (Defago et al. 1990; Gupta et al. 2001a; Garbeva et al. 2004; Validov et al. 2005). Interestingly, certain rhizobia have also been noticed for the biological control of M. phaseolina (Arora et al. 2001; Deshwal et al. 2003), F. oxysporum, F. solani, R. solani, Pythium spp., etc. (Chao 1990). Rhizobia had been reported to produce several secondary metabolites for biocontrol activity, similar to fluorescent pseudomonads in their mode of action. Pseudomonads confer active role in biocontrol and yield promotion of plants, and therefore are the most widely used genera among PGPR (Gupta et al. 2001a; Kumar et al. 2005a, b, c). Among free living bacteria, various species of Azotobacter have been reported for the biological control of different phytopathogens such as Alternaria, Helminthosporium, Fusarium, etc., under in vitro conditions (Laxmikumari et al. 1975; Joshi et al. 2006a). Free nitrogen-fixing bacteria were probably the first rhizobacteria used to promote plant growth. Other bacterial genera capable of nitrogen fixation that may be responsible for growth promotion effect are Azoarcus sp., Burkholderia sp., Gluconacetobacter diazotrophicus, Herbaspirillum sp., Azotobacter sp., and Paenibacillus polymyxa. These genera have been isolated from a number of plant species such as rice, sugarcane, corn, sorghum, other cereals, pineapple, and coffee bean (Vessey 2003). Azoarcus has recently gained attention due to its great genetic and metabolic diversity. Because of their competitive advantages in a carbon- rich, nitrogen-poor environment, diazotrophs become selectively enriched in the rhizosphere (Reinhold-Hurek and Hurek 2000). Azotobacter spp. is also being applied as bioinoculant due to its several direct PGP activities including asymbiotic nitrogen fixation, phosphate solubilization, growth hormones production, and vitamins production (Shende et al. 1977). The first reports on Azotobacter appeared in 1902 and it was widely used in Eastern Europe during the middle decades of the last century (Gonza´lez and Lluch 1992). As previously suggested, the effect of Azotobacter and Azospirillum is attributed not only to the amounts of fixed nitrogen but also to the production of plant growth regulators such as indole acetic acid (IAA), gibberellic acid, cytokinins, and vitamins (Rodelas et al. 1999). Azotobacter is also known to produce antifungal compound that inhibits the production of conidia of Botrytis cinerea (Doneche and Marcantoni 1992). Similarly, Azospirillum is also known to secrete phytohormones, induce root cell differentiation, and increase water uptake. Azospirillum associates with polysaccha- ride degrading bacteria (PDB) in rhizosphere, establishing a metabolic association (Bashan and Holguin 1997). The sugar-degrading bacteria produce degradation and fermentation products that are used by Azospirillum as a carbon source that in turn provides PDB with nitrogen. In fact, here the symbiosis is extended to multiple prokaryotic interactions. Other example includes the association between Azospir- illum and Bacillus that degrades pectin, Azospirillum and Cellulomonas degrade cellulose, and Azospirillum and Enterobacter cloacae that ferment glucose (Kaiser 1995; Khammas and Kaiser 1992; Halsall 1993). Production and release of plant growth regulators by bacteria causes an alteration in the endogenous levels of the plant growth regulator. Other growth regulators such as cytokinins are less common 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 5 among PGPR, while gibberellin production in high concentrations has only been described from the genus Bacillus. Different genera of bacteria, such as Proteus mirabilus, P. vulgaris, Klebsiella pneumoniae, B. cereus, Escherichia coli, etc., produce auxins, cytokinins, gibberellins, and abscisic acid (Griffith and Ewart 1995). Symbiotic bacteria generally termed as rhizobia for a broad group of nodule- inhabiting symbionts have been used as inoculants for well over a century. These organisms were used to enhance nodulation and N-fixation among legumes. Their roles were limited earlier, but have been extended to an extent as major solubilizers of inorganic phosphate, making it available for the plants. Further their biocontrol credentials were proved when the genera Bradyrhizobium and Rhizobium reported to produce antibiotics effectively controlling fungal pathogens (Chakraborty and Purkayastha 1984; Briel et al. 1996). Sinorhizobium meliloti showed antagonism toward F. oxysporum and M. phaseolina regardless of their symbiotic effectiveness in presence of pathogen and increased overall growth of groundnut (Arora et al. 2001). Deshwal et al. (2003) isolated bradyrhizobia from the root nodules that antagonized M. phaseolina in vitro which increased under iron-limited conditions. One of the mechanisms of biocontrol by rhizobia and bradyrhizobia was established due to the production of siderophores resulting in increased growth of Arachis hypogaea. The adaptability of introduced strains to achieve equilibrium within an aboriginal niche is limited, but identification, screening, and application of local strain have been advocated (Bashan 1998; Aeron et al. 2010). Various strains of species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus are known as potential elicitors of induced systemic resistance (ISR) and exhibit significant reduction in the incidence or severity of various diseases on diverse hosts (Kloepper et al. 2004). Certain volatile compounds, especially 3-hydroxy-2-butanone (acetoin) and 2, 3- butanediol, released by the B. subtilis and B. amyloliquefaciens in rhizosphere play a crucial role in the elicitation of ISR (Ryu et al. 2003). More recently, Choudhary and Johri (2008) have reviewed the significance of ISR by Bacillus spp. in relation to the biological control of pathogenic organisms. Bacillus species are believed to enhance the plant growth through synthesis of plant growth regulators such as auxins (indole-3-acetic acid) and gibberellic acid (Wipat and Harwood 1999). However, more recently representatives of B. subtilis/B. amyloliquefaciens group have been shown to produce substances with IAA-like activity; reasonable amount of IAA was produced by B. amyloliquefaciens FZB42 when fed with tryptophan (Idris et al. 2004). Based on studies of wheat rhizosphere colonization by Bacillus species, it seems that rhizosphere competent genotypes occur in this bacterium (Milus and Rothrock 1993; Mavingui et al. 1992; Maplestone and Campbell 1989; Juhnke et al. 1987). Enhancement of plant growth by root-colonizing species of Bacillus and Paenibacillus is well documented and PGPR members of the genus Bacillus can provide a solution to the formulation problem encountered during the development of biocontrol agents to be used as commercial products, due in part to their ability to form heat- and desiccation-resistant spores (Kloepper et al. 2004; Emmert and Handelsman 1999). 6 A. Aeron et al.

The organic forms of phosphorous are estimated to comprise between 30 and 50% of total soil phosphorous. This reservoir can be mineralized by microorganisms, making it available to the plant as soluble phosphates. Different PGPR genera are capable of solubilizing phosphate and include Pseudomonas, Bacillus, Rhizo- bium, Burkholderia, Achromobacter, Agrobacterium, Micrococcus, Aerobacter, Flavobacterium, Chryseobacterium, and Erwinia. The solubilization of phosphate occurs due to the involvement of organic acids and/or by releasing phosphatases responsible for releasing phosphate groups bound to organic matter. Most of these bacteria are able to solubilize the Ca–P complex, and there are others that operate on the Fe–P, Mn–P, and Al–P complexes. Results with PGPR capable of solubilizing phosphate are sometimes erratic, probably due to soil composition. While the inorganic forms occur in minerals as insoluble calcium, iron, or aluminum phosphates, organic phosphates are derived from the decaying plants, animals, and microorganisms. Organic matter is an important reservoir of immobilized phosphate that accounts for 20–80% of soil phosphorus (Goldstein 1986) and only a small portion (0.1%) is available to plants. Phosphatases are known to play a key role in transforming organic forms of phosphorous into plant available inorganic forms. Conversion of the insoluble forms of phosphorous to a form accessible by plants such as orthophosphates is an important criterion. Plant may poorly/not possess an innate ability to acquire phosphorus directly from soil phytate which is a major phosphorous source. The production of enzyme phytase leads to an increase in the availability of phosphorus to plants and in turn the plant uptake (Gyaneshwar et al. 2002). It is known to be secreted by many microorganisms and is involved in the stepwise degradation of phytate to lower phosphate esters. Although plants are known to produce phytase, they display poor activity in roots and other plant organs (Greiner and Alminger 2001). As zinc is a limiting factor in crop production, study on zinc solubilization by bacteria has an immense application in zinc nutrition to plants. Zinc-solubilizing potential of few bacterial genera has been studied along with mobilization of potassium (Sarvanan et al. 2003; Sperberg 1958). Potassium (K) is an essential soil nutrient that performs a multitude of important biological functions to maintain plant growth and quality. Although silicon (Si) is still not recognized as an essential element for plant growth, the beneficial effects of this element on the growth, development, yield, and disease resistance have been observed in a wide variety of plant species. However, plants cannot directly use mineralic K and Si unless they are released by weathering or dissolved in soil water. Studies have documented the release of K and Si during the degradation of silicate minerals by bacteria (Barker et al. 1998; Welch and Vandevivere 1994; Sheng and He 2006). Iron in the Earth’s crust is present in a highly insoluble form of ferric hydroxide (Fe3+), and thus unavailable to microorganisms and plants. Some bacteria have developed iron uptake systems (Neilands and Nakamura 1991). These systems involved a siderophore – an iron binding legend – and an uptake protein needed to transport iron into the cell. Siderophores are low molecular weight (~400–1,000 Da) iron-chelating compounds that bind Fe3+ and transport it back to the cell and make it available for the microbial cells (Briat 1992). The secreted siderophore molecules 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 7 have a very high affinity (kd ¼ 1020 to 1050) for iron and bind most of the Fe3+ that is available in the rhizosphere and prevent the pathogens present in immediate vicinity from proliferation because of lack of iron (O’Sullivan and O’Gara 1992). Antagonists can prevent the proliferation of fungal phytopathogens by producing siderophores that bind most of the Fe3+ in the rhizosphere. The resulting lack of the iron prevents any fungal pathogen from proliferating in this immediate vicinity. Kloepper et al. (1980) have supported this mechanism and stated that the production of siderophores that chelate and thereby scavenge the ferric iron in the rhizosphere may result in growth inhibition of other microorganisms whose affinity for iron is lower. It has been suggested that the ability to produce specific siderophores and/or to utilize a broad spectrum of siderophores may contribute to the root-colonization ability of biocontrol strains. In addition, siderophores also mediated the iron uptake by plant roots in iron- limiting conditions (Wang et al. 1993). Root colonization is an important first step in interaction of PGPR group of bacteria with host plant (Kloepper and Beauchamp 1992). Effective colonization of plant roots by PGPR plays an important role in growth promotion irrespective of the mechanism of action, i.e., production of metabolites, antibiotics against pathogens, or ISR or even nutrient uptake (Bolwerk et al. 2003). It is now common knowledge that bacteria in natural environments persist by forming biofilms (Davey and O’Toole 2000). The use of microorganism for biological control is a nonhazardous strategy to reduce crop damage caused by plant pathogens. The antagonistic microorganisms are ideal biocontrol agents, as the rhizosphere provides the front- line defense for roots against infection by the pathogens (Lumsden et al. 1987). Biocontrol research has gained considerable attention and appears promising as a viable alternative to chemical control strategies (Rebafka et al. 1993). The protection of root from the attack of the pathogen was due to the production of diverse metabolites like siderophore (Arora et al. 2001) and antifungal metabolites such as rhizobitoxine (Chakraborty and Purkayastha 1984). One of the most effective mechanisms, which antagonists employ to prevent proliferation of phytopathogens, is the synthesis of antibiotics. A large number of antibiotics have been reported from different fluorescent pseudomonads including agrocin-84, agrocin-434, 2, 4- diacetyl phloroglucinol, herbicolin, oomycin, phenazine, pyoluteorin, pyrrolnitrin, pyrroles, etc., and they have a role to play in inhibition of pathogens (Colyer and Mount 1984; Gutterson et al. 1986; James and Gutterson 1986). Many Bacillus strains are considered as natural factories of cyclic lipopeptides, including iturins, fengycins, and surfactins, and their involvement in control of plant microbial diseases has been proved (Li et al. 2007; Romero et al. 2007; Yoshida et al. 2001; Asaka and Shoda 1996). Recently, the hydrolytic enzymes have received considerable attention because they play a role in controlling diseases due to plant pathogens. Microorganisms capable of lysing other organisms are widespread in natural ecosystems. The enzymatic digestion or deformation of cell wall components of phytopathogenic fungi by the enzymes chitinase, b-1, 3-glucanase produced by antagonistic bacteria and the hyperparasitism, lysis of phytopathogen propagules present in soil is one of the few logical methods of biological control of soil-borne plant pathogens 8 A. Aeron et al.

(Vaidya et al. 2001). Hyperparasitism occurs when a fungus exists in intimate association with another fungus which derives some nutrients while conferring no beneﬁt in return. Hyperparasitism and lysis of propagules in soil is a logically satisfying method of biological control of soil-borne plant pathogens by microbial antagonists. The production of cell wall degrading or lytic enzymes, such as chitinase, chitosanase, b-1, 3-glucanase, b-1, 4-glucanase, b-1, 6-glucanase, proteases, and lipase (Fridlender et al. 1993;LimandKim1995; Dunne et al. 1997;Vaidyaetal. 2001; Vivekanathan et al. 2004), degrades fungal cell walls, resulting in lysis of wall material, leading to cell death. Induction of the systematic resistance against many pathogens has also been reported inducing long-lasting and broad-spectrum systemic resistance against disease-causing agents (Zehnder et al. 2001). Plants do not have an immune system but have evolved a variety of potent defense mechanisms, including the synthesis of low-molecular-weight compounds such as proteins and peptides that have antifungal activity.

1.4 Crop Ecosystem

PGPR can influence plant growth directly but may differ from species to species and even at strain level. Symbiotic plant colonizers and certain free-living bacteria contribute to plant growth by nitrogen fixation. The symbiotic bacteria form a host- specific symbiosis with legumes. Molecular signal molecules (lipo-oligosaccha- ride) secreted by these bacteria play a critical role in this process (Lange 2000; Spaink 2000; Perrot et al. 2000). Species of Bacillus are common inhabitants among the resident microflora of inner tissues of various species of plants, including cotton, grape, peas, spruce, and sweet corn, where they play an important role in plant protection and growth promotion (Berg et al. 2005; Shishido et al. 1999; Bell et al. 1995; McInroy and Kloepper 1995; Huang et al. 1993; Hallaksela et al. 1991; Misaghi and Donndelinger 1990). Little work has been done to date concerning the beneficial relationship of Rhizobium and nonleguminous plants, although Wiehe and Hoflich (1995) demonstrated that rhizobia can multiply and survive under field conditions as well as in the rhizosphere of nonhost legumes. The attachment of bacteria with maize, wheat, rice, oat, sunflower, mustard, and asparagus has been reported along with improved growth of certain nonlegumes when inoculated with rhizobia (Planziski et al. 1985; Terouchi and Syono 1990; Yanni et al. 1995; Biswas et al. 2000a, b; Peng et al. 2002). Recently, Chandra et al. (2007) reported that a successful rhizospheric competent Mesorhizobium loti MP6 induced root hair curling, inhibited Sclerotinia sclerotiorum, and enhanced growth of mustard. Specific rhizobacteria have the ability to improve plant growth and/or root health (Kloepper et al. 1980; Suslow and Schrolh 1982; Schippers et al. 1987; Sikora 1988; Weller 1988). A key factor of all PGPR is that they colonize seed and root, or behave as endophytes. Such traits are desirable for considering them suitable for biocontrol activity (Lugtenberg and Bloemberg 2004; Compant et al. 2005). 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 9

Further, the phenomenon of chemotaxis, flagellar mobility, lipopolysaccharides (LPS) structure, the outer membrane protein OprF and to a lesser extent, presence of pili, all are important for competitive root colonization (Lugtenberg and Bloemberg 2004). However, in field soil, environmental conditions and competition or displacement by the myriad of microorganisms present in the rhizosphere limit colonization. Certainly, use of mutants and promoter probe techniques are the beginning to identify genes in bacteria that are important to root colonization and these are often related to nutrient uptake (Roberts et al. 1997; Rediers et al. 2005). Variation for interaction with PGPR is often dependent on environmental conditions. For example, phosphorus deficiency provokes a differential response to Rhizobium inoculation among common bean cultivars (Vadez et al. 1999; Christiansen and Graham 2002). Such phenotypic variation among cultivars may be, in part, the result of genetic variation and suggests genetic improvement of the host as an approach for development of superior plant growth promoting (PGP) strategies in conjunction with rhizosphere microbial inoculants.

1.5 PGPR in Agrobiology

PGPR are most commonly used in agriculture, and their application in various crops resulted in an average approximate increase of 20–40% in yield across multiple crops all over the world when various reports were combined over last decade. In general, PGPR-carried plant growth benefit owing to increase in seed germination rates, root growth, leaf area, chlorophyll, proteins, and hydraulic activity, fluid movement within the plant, tolerance to drought, low temperature, delayed leaf senescence, disease resistance, and finally enhanced grain size and crop yield of crop, as elaborated for some of the crops in this chapter.

1.5.1 Cereals

In recent years, crop roots association with bacteria and their proliferation in the rhizosphere has been found to be beneficial in most of the cereals (Terouchi and Syono 1990; Biswas et al. 2000a, b; Peng et al. 2002). However, the selection of effective strains is of prime importance for the growth promotion of cereals (Westcott and Kluepfel 1993; Siddiqui and Ehteshamul-Haque 2000). In rice, endophytic strains of Rhizobium leguminosarum br. trifoli E11 and E12 increased grain yield of rice in field inoculation experiment (Yanni et al. 1997). Biswas et al. (2000a, b) reported that rhizobial inoculation increased rice grain yield at different N rates. The benefit of early seedling development could carry over to a significant increase in grain yield at maturity. Earlier, Datta et al. (1982) reported that a P-solubilizing and IAA-producing strain of B. firmus increased the grain yield 10 A. Aeron et al. and P-uptake of rice in a P-deficient soil amended with rock phosphate. Similarly, increased yield was obtained in wheat, sorghum, and barley due to application of A. brasilense (Okon and Labandera-Gonzalez 1994; Dobbelaere et al. 2001;Saubidet et al. 2002)andwithBeijerinikia mobilis and Clostridium sp. in wheat and barley, respectively (Polyanskaya et al. 2000). It was interesting to note that Pseudomonas spp. increased yield of winter wheat by 27% (de Frietas and Germida 1990). Application of P. cepacia, P. fluorescens, and P. putida in winter wheat exhibited antagonism against Rhizotonia solani and Leptosphaera maculans and enhanced yield indirectly in soil reported to be relatively infertile (De Frietas and Germida 1990; 1992). Inoculation of P. cepacia R55, R85 and P. putida R104 increased root and shoot dry weight of winter wheat in R. solani infested soil (de Freitas and Germida 1991). On the other hand, P. chlororaphis 2E3 and 06 when applied on spring wheat showed increased emergence of seedlings. Both strains could also inhibit a dreaded pathogen F. culmorum (Kropp et al. 1996). In another study on winter wheat, application of P. fluorescens enhanced length of seedling and significant increase in plant height and grain yield in Pythium infested soil (Weller and Cook 1986). Iswandi et al. (1987) observed an increased yield in wheat along with maize and barley by Pseudomonas spp. 7NSK2. Rice, wheat, corn, millet, sweet potato, cotton, etc., also showed average yield increase by 10–22.5% after application of YIB (Mei et al. 1990). Chabot et al. (1993) demonstrated R. trifolii inoculation on increased yield in maize by reducing dose of phosphorous fertilizers. Seed treatment with rhizobacteria or their formulations increased the growth of maize (Jacoud et al. 1999), wheat (Khalid et al. 2004), rice (Yanni et al. 1997), and several other crops (Vidhyasekaran and Muthamilan 1995; Rabindran and Vidhyasekaran 1996; Vidhyasekaran et al. 1997a, b; Podile and Dube 1988; Kloepper et al. 1991). Recently, Ashrafuzzaman et al. (2009) isolated bacterial strains with successful root colonizer and increased plant height, root length, and dry matter production rice seedlings. Some novel efforts were made to elucidate the molecular responses of rice to P. fluorescens treatment through protein profiling (Kandasamy et al. 2009). However, the mechanism underlying such promotional activity is not yet fully understood clearly. Application of several genera, such as B. licheniformis RC02, Rhodobacter capsulatus RC04, P. polymyxa RC05, P. putida RC06, Bacillus OSU-142, B. megaterium RC01, and Bacillus M-13, showed increased root and shoot weight along with nutrient uptake in barley (Cakmacki et al. 1999). Similarly, Bacillus observed increase in yield of rice (Sudha et al. 1999), barley (Sahin et al. 2004), wheat (de Freitas 2000), canola (de Freitas et al. 1997), and maize (Pal 1998; Pal et al. 2001). Lalande et al. (1989) observed increased yield in maize by using Serratia liquifaciens, Pseudomonas spp., and Bacillus sp., but B. megaterium induced yield in rice and barley (Cakmacki et al. 1999; Khan et al. 2003). Gholami et al. (2009) reported maize seeds inoculated with bacterial strains significantly increased plant height, seed weight, number of seed per ear and leaf area along with significant increase in ear and shoot dry weight of maize. Recently, more efforts have focused on beneficial rhizobacteria in cereals that are endophytic in nature especially in the regions where legume crop season is 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 11 followed by cereals (Ashrafuzzaman et al. 2009). Nodule-inhabiting bacteria are now known to colonize the cereals such as rice, wheat, and sorghum. Several field inoculation trials have been conducted to assess the agronomic potential of rhizobial group in nonlegumes (Chandra et al. 2007). More efforts are required to focus on Rhizobium–cereal associations under field conditions, with the long-term goal of identifying, developing, and implementing superior PGPR inoculants for the growth promotion of rice and wheat productivity in real-world cropping systems while reducing their dependence on nitrogen fertilizer inputs.

1.5.2 Oilseeds

The importance of PGPR applications in oil seed crops production was demonstrated by several workers. The growth promotion and health of canola, sesame, and peanut were supported by using different genera of PGPR (Kumar et al. 2005a; Chandra et al. 2007; Bhatia et al. 2008; Kumar et al. 2009). Pseudomonas putida, P. fluorescens, Arthrobactro citreus, Azospirillum spp., and Serratia liquefaciens demonstrated growth promotion of canola (Brassica campestris and B. napus)in field conditions (Kloepper et al. 1988a, b; 1989). Kloepper et al. (1989) observed an increase in the yield of mustard with the application of Azospirillum spp. Selected bacterial strains showed increased seedling emergence, vigor, and yield. Non-nitrogen fixing mutants provide greater root elongation effects and greater phosphate uptake in canola (Lifshitz et al. 1987), while P. putida inoculation increased yield of canola. Van Peer and Schippers (1998) found that inoculation of Pseudomonas spp. increased root and shoot weight in canola under hydroponic growth chamber. Belimov et al. (2001) observed that inoculation of B. napus seeds with Alcaligenes sp., B. pumilus, Pseudomonas sp., and Variovorax paradoxus showed vigorous growth. Bertrand et al. (2001)observed significant increase in root dry weight due to aggressive effect of Phyllobacterium sp. apart from Variovorax sp. and Agrobacterium sp. It was demonstrated that Methylobacterium fujisawaense promoted root elongation in canola (Madhaiyan et al. 2006). Earlier, Ghosh et al. (2003) observed that B. circulans DUC1, B. wrmus DUC2, and B. globisporus DUC3 enhanced root and shoot elongation in B. campestris. Differential response of sesame under influence of indigenous and nonindigenous rhizosphere competent fluorescent pseudomonads were observed recently by Aeron et al. (2010). The results of root colonization stated the difference of using indigenous and a nonindigenous strain and the successful colonization by fluorescent pseudomonads in sesame rhizosphere promoted growth which proved efficacy of indigenous microflora over nonindigenous microflora (Table 1.1). Integrated use of organic and inorganic biofertilizers has been reported to sustain productivity of sesame by improving soil physical conditions and also reduce the costly inorganic fertilizer needs (Duhoon et al. 2001; 12

Table 1.1 Plant growth promoting attributes of fluorescent pseudomonads and their effect on plant growth parameters of sesame Treatments PGP attributes Plant growth parameters Isolate Rhizospheric IAA ACC P (mg of S U/ml/h HCN (OD Antagonism (%) G (%) RDW SDW RL SL G/P m origin ( g/ml) deaminase P/ml) at 625 nm) 12 (gm) (gm) (cm) (cm) P. aeruginosa Potato + (31) – + (67) + (29) – + (55) – 78.8** 16.2** 43.2** 26.2* 170.3** 42.6** rif+ tet+ GRC1 P. aeruginosa Groundnut + (36) – + (55) + (17) + (0.09) + (61) – 76.3** 13.5** 40.7** 24.7* 168.9** 38.8** PS2str+ P. aeruginosa Sunflower + (30) – + (71) + (12) + (0.05) – – 77.9** 14.1** 42.5** 25.6* 173.8** 39.2** PS (II) neo+ P. aeruginosa Tomato + (42) – + (75) + (22) – + (72) + (68) 80.3** 14.7** 44.3** 26.2* 180.7** 41.3** LES4tet+ P. aeruginosa Velvet Bean + (40) – + (68) + (15) – + (78) – 79.1** 12.4** 39.8** 22.8* 165.3** 40.8** PRS4gen+ P. aeruginosa Sesame + (41) + + (76) + (19) – + (70) + (63.3) 83.5** 17.3** 45.6** 27.1* 185.9** 47.3** PSI5azi+ kan+ Control na na na na na na na 61 10.1** 18.6** 21.6 141.3 26.3 PGP attributes are mean of three independent experiments; Field data is a mean of two year trials; Values are mean of 15 randomly selected plants G Germination, RDW Root dry weight, SDW Seedling dry weight, RL Root Length, SL Shoot Length, G/P seed yield per plant; + attribute positive; attribute negative; IAA Indole acetic acid; P Phosphate solubilization; Sid Siderophore; ACC 1-aminocyclopropane-3-carboxylic acid; 1 Macrophomina phaseolina; 2 Fusarium oxysporum; (%) pathogen inhibition percentage (control – treatment/control 100) [Adapted from Aeron et al. (2010)] *Significant at P > 0.01 level of ANOVA **Significant at 0.01 level of LSD as compared to control .Arne al. et Aeron A. 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 13

Kumar et al. 2009). Siddiqui et al. (2001) reported inoculation of P. fluorescens along with chemical fertilizers is an effective way to reduce the infestation of Meloidogyne spp. in sesame. Groundnut (Arachis hypogaea L.) is a major oilseed and food crop of the semiarid tropics. The late leaf spot disease of groundnut caused by the fungus Cercosporidium personatum almost co-exists with the crop and contributes to significant loss in yield throughout the world. Leaf spots can cause up to 53% loss in pod yield and 27% loss in seed yield (Patel and Vaishnav 1987). Smith (1992) reported pod loss of 10–50% by late leaf spot disease. Control of this disease mainly depends on fungicides, although considerable effort has been invested in developing biocontrol methods (Meena et al. 2002). On the other hand, Jadhav et al. (1994) reported a Rhizobium isolate that increased plant growth and chlorophyll content in groundnut. Earlier, Howell (1987) explained in part the rhizobia- enhanced mineral uptake in groundnut tissues. Pal et al. (2000) reported increased pod yield following seed treatment with Pseudomonas sp. Gupta et al. (2002) found reduced disease incident, better vegetative growth parameters, and ultimately enhanced grain yield in peanut by the addition of P. aeruginosa GRC2 in M. phaseolina-infested field soil. Recently, Bhatia et al. (2008) reported increased seed germination, growth promotion, and suppression of charcoal rot due to M. phaseolina with fluorescent pseudomonads. Earlier, Arora et al. (2001) observed enhanced seed germination, seedling biomass, and nodule weight with reduced disease incidence in groundnut. Simi- larly, Meena et al. (2006) applied P. fluorescens for plant growth and in biocontrol of late leaf spot caused by C. personatum in groundnut. Seed treated with P. fluorescens strain Pf1 recorded the highest seed germination percentage and the maximum plant height with significantly controlled late leaf spot disease of groundnut resulting in increased pod yield. In another study, B. subtilis strain AF1, isolated from soils suppressive to pigeon pea (C. cajan) wilt caused by F. udum, was presumed to induce resistance against Aspergillus niger on peanut. Strong experimental evidence that AF1 elicited ISR came from the findings of Sailaja et al. (1997) who reported a noteworthy reduction in the incidence of crown rot of peanut caused by A. niger corresponding to the increase in lipoxygenase activity, a phenomenon associated with ISR.

1.5.3 Fruits, Vegetables, and Cash Crops

Several workers have reported successful management of plant disease and increased yield in various horticultural crops such as strawberry (Tahmatsidou et al. 2006; Pedraza et al. 2010), chillies (Bharathi et al. 2004), mango (Vivekanathan et al. 2004),tobacco(Panetal.1991), pea (Chang et al. 1992), potato (Geels et al. 1986), red pepper (Lee et al. 2008), banana (Gunasinghe and Karunaratne 2009), and apple (Karlidag et al. 2007;Aslantasetal.2007) with the application of PGPR. Increased seedling growth was observed in sugar beet with the application of 14 A. Aeron et al.

Pseudomonas spp. (Williams and Asher 1996). Reddy et al. (2001) reported foliar application of PGPR bioformulation which promoted plant growth besides effectively controlling tomato bacterial spot, cucumber angular leaf spot, tobacco blue mold, and wild fire. Sarvanankumar et al. (2007)usedPseudomonas and Bacillus bioformulation against blister blight disease of tea caused by Exobasidium vexans while Chakraborthy et al. (2009) studied talc-based bioformulation of Ochrobacterium anthropi TRS-2 for plant growth promotion and management of brown root rot disease of tea. Recently, Karakurt and Aslantas (2010) investigated the effects of Agrobacterium rubi A-18, B. subtilis OSU-142, B. gladioli OSU-7, and P. putida BA-8 on growth and leaf nutrient content of apple cultivars and found interesting variations that support the application of PGPR. The role of PGPR in vegetative crops production has got less attention in comparison to that of other crops. Raupach and Kloepper (2000) reported that seed treatment of cucumber with B. amyloliquefaciens IN937a, B. subtilis GB03, and a mixture of the two strains resulted in significant increases in plant growth and reductions in disease severity. Han and Lee (2005) reported PSB B. megaterium and potassium solubilizing bacteria (KSB) B. mucilaginosus inoculated in nutrient- limited soil planted with eggplant. Inoculation of these bacteria in conjunction with amendment of its respective rock P or K materials increased the availability of P and K in soil and enhanced N, P, and K uptake, and growth of eggplant. The early seedling emergence and significant increase in yield of potato was observed with the application of PGPR. Increase in yield of potato was reported by several workers after the application of different species of Pseudomonas (Howie and Echandi 1993; Geels et al. 1986; Kloepper et al. 1989). Kloepper et al. (1980) demonstrated larger root system and significant increase in yield in different soil types. Frommel et al. (1993) found the application of Pseudomonas strain Ps JN increased whole plant dry weight. Suppression of Erwinia caratovora causing soft rot in potato was seen after the inoculation with P. putida W4P3 (Xu and Gross 1986). Raupach and Kloepper (2000) demonstrated the effect of B. amyloliquefaciens In937a and B. subtilis GB03 individually as well as in combination for plant growth promotion and reduction of disease severity on seeds of cucumber treated with these antagonists. A nonfluorescent Pseudomonas of onion rhizosphere showed significant increases in root dry weight, stem length, and lignin and enhanced stem hair formation (Frommel et al. 1991). A disease complex by Meloidogyne incognita and F. oxysporum was suppressed by the application of fluorescent pseudomonads in tomato (Santhi and Sivakumar 1995; Kumar et al. 2005a) (Table 1.2). Ekin et al. (2009) applied Bacillus sp. OSU-142 as compared to three different levels of N fertilization. The beneficial effect of Bacillus sp. OSU- 142 on tuber yield of potato was reported in two successive years over fertilizers. Several strains of P. fluorescens, P. cepacia, and P. aeruginosa have been used for the biological control of several plant diseases in a wide range of horticultural crops (Weller 1988; Chandel et al. 2010). Yusran et al. (2009) reported biological control of F. oxysporum f. sp. radicis-lycopersici that causes crown and root rot in tomato. The inoculation also increased the N yield and fixed N in association with banana roots subsequently increased the yield, improved the physical attributes of fruit 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 15

Table 1.2 Effect of Pseudomonas EP10 on root disease complex and growth of tomato after 60 days Treatment Plant length Plant fresh Shoot dry Root Knot Infection (%) of (cm) weight (g) weight (g) Index F. oxysporum F. oxysporum 26.5 18.0 4.5 6.0 100 Pseudomonas 55.0** 51.0** 15.0** 3.0 – EP10 Pseudomonas 58.5** 55.0** 17.5** 2.5 4 EP10 + F. oxysporum Control 32.8 27.5 6.5 7.0 – **P < 0.01, Values average of ten replicates during three trials (Modiﬁed and adapted from Kumar et al. 2005b) quality, and initiated early ﬂowering. More recently, PGPR proved effective as a bioenhancer and biofertilizer for banana cultivation (Mia et al. 2005). B. subtilis S499 is involved in suppression of gray mold disease caused by Botrytis cinerea on wounded apple fruits (Ongena and Jacques 2007; Jacques et al. 1999). Recently, Romero et al. (2007) showed the involvement of iturin and fengycin antibiotics from four B. subtilis strains UMAF6614, UMAF6616, UMAF6639, and UMAF8561 in suppression of powdery mildew of cucurbits caused by Podosphaera fusca. Arrebola et al. (2010) reported the production of iturin from B. amyloliquefaciens PPCB004 which inhibited seven different postharvest pathogens of citrus, avocado, and mango fruits. Recently, Choudhary and Johri (2008) implicated the mechanisms and role of Bacillus species as inducers of systemic resistance in relation to plant–microbe interactions and explicated the pathways involved in their regulation.

1.5.4 Legumes

A unique relationship was observed between two bacterial isolates Burkholderia sp. MSSP and Sinorhizobium meliloti PP3, where commensalisms between them resulted in increased IAA production in mixed-species culture and significant increase in seedling length and weight of pigeon pea (Cajanus cajan) (Pandey and Maheshwari 2007a). When wheat-bran-based bioformulation comprising consortium of PGPR was applied in field trials, significant improvement in growth and yield of pigeon pea was obtained (Pandey and Maheshwari 2007b). Kumar et al. (2010) obtained wilt disease management and enhancement of growth and yield of Cajanus cajan (L) var. Manak by bacterial combinations using root nodulating Sinorhizobium fredii KCC5 and P. fluorescens LPK2 isolated from nodules of host plant and disease suppressive soil of tomato rhizosphere, respectively (Table 1.3). Mishra et al. (2009) studied application of several potential PGPR strains on Cicer arietinum. All isolates showed significant increase in shoot length, root length, and dry matter of seedlings. Even the application of P. cepacia caused an early soybean 16 A. Aeron et al.

Table 1.3 Effect of S. fredii KCC5, P. fluorescens LPK2, bacterial consortium (KCC5 þ LPK2) on post harvest parameters of C. cajan var. Manak, after 120 days of sowing Treatments *Pods Grain yield A soluble Stover yield Harvest plant1 (Kg ha1) protein (mg g1) (Kg ha1) index (%) KCC5 115.4** 962.1** 211.1* 4,230* 18.53 LPK2 116.3** 955.2** 197.2* 4,200* 18.52 Consortium 118.6** 988.3** 212.3* 4,280* 18.76 (KCC5 þ LPK2) Control 51.2 710.0 179.1 3,150 18.03 Average value of ten plants from each treatment; a soluble protein content of seed (g1) *Significant at 5% (ANOVA) **Significant at 1% as compared to control (ANOVA) [Modified and adapted from Kumar et al. (2010)] growth and enhanced seed germination (Cattelan et al. 1999). Ma et al. (2003) reported R. leguminosarum bv. viciae 128C53K enhanced nodulation in pea (Pisum sativum L.). Moreover, Penã-Cabriales and Alexander (1983) found that strains of rhizobia and bradyrhizobia grew readily in the presence of germinating seeds and developing root systems of soybean (Glycine max (L.) Merr.), kidney bean (Phaseolus vulgaris L.), red clover (Trifolium pretense L.), and cowpea (Vigna unguiculata L.), but Pseudomonas sp. and Bradyrhizobium sp. increased growth and promoted nodulation in mung bean (Shaharoona et al. 2006). Wiehe and Hoflich (1995) demonstrated that R. leguminosarum bv. trifolii can multiply and survive under field conditions in the rhizosphere of nonhost legumes (Lupinus albus L. and Pisum sativum L.) and nonlegumes such as corn, rape, canola, and wheat; some strains of B. subtilis that have been integrated into pest management strategies, such as biocontrol strain GB03 of B. subtilis, could inhibit the fusarial wilt caused by Fusarium species more effectively on semiresistant cultivar of chick pea than on susceptible variety (Jacobson et al. 2004;Hervasetal.1998). There are several reports which reveal that efficacy of rhizobia could be enhanced by co-inoculation with PGPB. Co-inoculation with symbiotic and rhizosphere bacteria may improve nodulation by a number of mechanisms. Different mechanisms for such activity by Gram-positive and Gram-negative bacteria include siderophore chelating insoluble cations, LPS, flavonoids, phytoalexins, antibiotics, and colonization of root surfaces by outcompeting pathogenic organisms, and thus increase nodulation and growth (Garcia Lucas et al. 2004; Parmar and Dadarwal 2000). A common attribute, although, is efficient colonization of roots by PGPR strain to reduce the ethylene concentration inside the plant. That is so if it is able to utilize ACC as a sole nitrogen source, thereby increasing the root surface in contact with soil. Therefore, it is highly expected that presence of PGPR containing ACC deaminase on the roots of legume could suppress accelerated endogenous synthesis of ethylene during the rhizobial infection and thus may facilitate nodulation. So, co-inoculation of legumes with competitive rhizobia and PGPR-containing ACC deaminase could be an effective and novel approach to achieve successful and dense nodulation in legumes. It is highly expected that inoculation with rhizobacteria containing ACC-deaminase hydrolyzed endogenous ACC into ammonia and alpha-ketobutyrate instead of ethylene. Consequently, root and shoot growth of the 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 17 legume plant as well as nodulation can be promoted (Garcia Lucas et al. 2004;Remans et al. 2007). Earlier, the use of specific PGPR mutant strains has indicated that bacterial indole-3-acetic-acid production and 1-aminocyclopropane-1-carboxylate deaminase activity play an important role in the host nodulation response. Tittabutr et al. (2008) conducted such a study to evaluate effect of ACC deaminase on nodulation and growth of Leucaena leucocephala. Further, Remans et al. (2007)examined the potential of ACC deaminase producing PGPR to enhance nodulation of common bean (Phaseolus vulgaris). Shaharoona et al. (2006) observed that co-inoculation with Pseudomonas and Bradyrhizobium species significantly improved root length, total biomass, and nodulation in mung bean. Belimov et al. (2009) evaluated the effect of root-associated bacterium containing ACC deaminase on pea (Pisum sativum)plants grown in dry soil. Huang and Erickson (2007) tested the effectiveness of R. leguminosarum for improving growth and yield of pea and lentil. They found improved seedling growth, nodule biomass, and shoot and root biomass in peas as we observe in velvet bean. Similarly, the effect of different methods of rhizobial inoculation on yield, root nodulation, and seed protein contents of two lentil varieties and improvement in nodulation was observed in peanut by inoculation with Rhizobium species (Ahmad et al. 2008; Dey et al. 2004). Rhizobia and other microorganisms employ various mechanisms to acquire essential nutrients such as iron, which includes production of iron-chelating molecules known as siderophores. Despite their efficient nitrogen-fixing potential, most of the times they fail to increase plant yield under field trials in agricultural soils. This has been attributed to their ineffi- ciency to successfully colonize the rhizosphere. Iron availability is one of the limiting factors for poor rhizospheric colonization. The successful performance of rhizobial inoculant strain depends upon their capability to outcompete the indigenous soil bacteria, survive, propagate, and enter into effective symbiosis with host plant. Many studies have indicated that efficient utilization of siderophores by rhizobia is a positive fitness factor with respect to its soil survival (Carson et al. 2000). Further, Joshi et al. (2009) observed increase in nodule occupancy and higher rhizospheric colonization by pigeon pea-nodulating rhizobia expressing engineered siderophore cross-utilizing abilities. Since survival under iron limitations in soils is an important quality which every biofertilizer strain must possess, the iron sufficiency of any organism therefore largely depends on its ability to utilize siderophores present in large and small concentrations in its vicinity that may be of plant, microbial, or soil origin (Carson et al. 2000; Joshi et al. 2009; Joshi et al. 2008; Joshi et al. 2006b; Khan et al. 2006). Thus, iron availability is one of the major factors determining rhizospheric colonization. This fact is further evidenced by work of Mahmoud and Abd-Alla (2001). They showed that co-inoculation of siderophore-producing PGPR significantly enhanced nodulation and nitrogen fixation of mung bean compared to plants infected with rhizobial strain alone. Thus, siderophore plays an important role in the competition between microorganisms and may act as growth promoters as the rhizosphere is heavily populated with siderophore-producing microorganisms. There are more reports that specific siderophore producing microorganisms stimulated the nodulation, nitrogen fixation, 18 A. Aeron et al. and plant growth of leguminous plants (Grimes and Mount 1987; Omar and Abd- Alla 1994; Shenker et al. 1999). A nodulated legume has an increased need for iron compared to non-nodulated plant since this metal is a constituent of key proteins involved in nitrogen fixation such as nitrogenase and leghemoglobin. Although scientists have reported both direct and indirect ways of growth stimulation by PGPR, there is no clear separation between these two mechanisms. A bacterium influencing plant growth by regulating synthesis of plant hormones can also play a role in controlling plant pathogens and diseases and vice versa. The presence of PGPR in the root vicinity may also improve ability of rhizobia to compete with indigenous populations for nodulation. Parmar and Dadarwal (2000) reported that increase in root growth provides more number of active sites and access to nodulation for rhizobia in chickpea. Co-inoculation of Bradyrhizobium with P. striata has also been observed to enhance biological nitrogen fixation in soybean (Dubey 1996). Rosas et al. (2006) studied the promising action of two phosphate solubilizing Pseudomonas strains on the symbiosis of rhizobial strains (S. meliloti and B. japonicum) with alfalfa and soybean. Further, differential effects on chick pea plant growth were also observed under co-inoculation with a PSB (Pseudomonas) strain and rhizobia alone (Valverde et al. 2006). There is a great advantage of using PSB in co-inoculation with rhizobia. This is because increased P mobilization in soil alleviates P deficiency. Deficit P severely limits plant growth and productivity particularly with legumes, where both plants and their symbiotic bacteria are affected. This may have a deleterious effect on nodule formation, development, and function (Robson et al. 1981). Similarly, dual inoculation of rhizobia with PGPR promoted nodulation, plant growth, and N2 fixation in Vigna radiate (Gulati et al. 2001; Gupta et al. 2003).

1.5.5 Forestry

Worldwide efforts to increase green cover and reforestation of abandoned, barren, and wasteland can benefit from a wider application of PGPR in both angiosperm and gymnosperm. There are currently very few reports on forestry-PGPR research. As a consequence, there is currently no field data for deciduous trees and still comparatively little field data for gymnosperms (Chanway 1997). Hindrance in successful application of PGPR in forestry includes aspects such as low pH conditions of forest soil, perennial nature of trees, soil type, forest environment, and survival of PGPR with trees in colder regions. In contrast to agricultural crops, the inoculation of PGPR on tree species and with special reference to their effect on seedling emergence, reduction in seedling transplant injury during the transfer from nursery to field, biomass increase due to inoculation apart from raising disease free plantlets in nursery, and increased strength of plantlets to withstand storm of antagonists in the form of pathogens have scope for investigation. 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 19

Earlier, Pokojska-Burdziej (1982) and Beall and Tipping (1989) demonstrated increase in height and biomass in black spruce, jack pine, and white spruce by using Arthrobacter citreus, P. fluorescens, and P. putida under greenhouse conditions, while Chanway and Holl (1994) used A. oxydans and P. aureofaciens in Douglas Fir and recorded improved height and biomass. On the other hand, Arthrobacter sp. increased the shoot length of pine. Leyval and Berthelin (1989) found that a strain of Agrobacterium radiobacter increased biomass of beech and pine. Accord- ing to one of the reports from authors group, Pinus roxburghii was found to show luxuriant growth due to application of Bacillus subtilis BN1. Seed treatment resulted in significant increase in seed germination, early seedling emergence, increase in biomass besides reduction in charcoal root rot in chir-pine seedlings (Singh et al. 2008; Singh et al. 2010) (Table 1.4), and reduction in the total, pre- and postemergency mortality of the P. radiata seedlings in nursery trial (Valiente et al. 2008). Inoculation of A. brasilense Cd increased root growth while inoculation of A. chroococcum increased biomass in oak (Akhromeiko and Shestakova 1958; Zaady et al. 1993; Zaady and Perevoltsky 1995), A. brasilense in river oak (Rodriguez-Barrueco et al. 1991), A. chroococcum in ash (Akhromeiko and Shestakova 1958), Quercus serrata (Pandey et al. 1986), and Eucalyptus (Mohammad and Prasad 1988). Enebak et al. (1998) studied inoculation of B. polymyxa and P. fluorescens in loblolly pine and slash pine under green house conditions. A significant increase in seedling emergence was observed along with total biomass. The postemergence damping-off was reduced in loblolly pine. B. polymyxa and Staphylococcus hominis significantly increased growth of hybrid spruce (O’Neill et al. 1992). Chanway et al. (2000) observed that B. polymyxa and P. fluorescens overwinter on the roots of field-planted trees such as spruce. Shishido and Chanway (2000) observed a significant increase in plant biomass of hybrid spruce when inoculated with Pseudomonas strain at all sites apart from reduction in seedling injury after transplant. B. licheniformis CECT5105 and B. pumilis CECT5106 led to increase in the plant growth and nitrogen content in silver spruce (Porbanza et al. 2002). Mafia et al. (2009) reported B. subtilis and Pseudomonas sp. in controlling mini-cutting rot of eucalyptus caused by Cylindrocladium candelabrum and R. solani. In fact, trees with mycorrhizal associates, with associative, or symbiotic N2-fixers, or with

Table 1.4 Effect of P. aeruginosa strain PN1 on the growth of chir-pine (90DAS/Pot assay) Treatments Germination Shoot length Root length Fresh weight (g) (%) (cm) (cm) Shoot Root P. aeruginosa PN1 84* 7.2ns 10.1ns 0.92ns 0.266ns P. aeruginosa 72* 6.5ns 9.1ns 0.727ns 0.236ns PN1 þ M. phaseolina M. phaseolina 54ns 5.9ns 8.1ns 0.52ns 0.182ns Control 66 6.2 8.8 0.625 0.212 Values are the mean of triplicates; ns non-significant *Significant at 5% LSD Modified and adapted from Singh et al. 2010 20 A. Aeron et al. rock-weathering capacities have significant impacts on biogeochemical processes, affecting recovery of degraded ecosystems and forest sustainability.

1.5.6 Mulberry (Sericulture)

For sericulture industry, mulberry is food plant of silkworm (Bombyx mori) grown in 1, 70,000 ha in India under different agro-climatic conditions. The sustainable leaf production, silkworm rearing, and cocoon production are dependent on soil fertility of mulberry gardens maintained through periodical application of either organic manures or chemical fertilizers in required quantity. Former is an approach wherein crop can be raised without imparting any adverse effect on soil and other beneficial microbial ecology. Therefore, a shift toward nonchemical strategies has to be evolved. The biofertilizers enriched with bacteria and fungi have proven to be great importance in improving the yield and quality of mulberry (Morus alba L.) More than three decades ago, Vasantharajan and Bhat (1967) studied the interactions of beneficial microorganisms and mulberry and reported an increase in shoot length and root length of seedlings and saplings due to the application of different genera of PGPR such as Pseudomonas spp., Acetobacter, Flavobacterium, Achromatobacterium, Micrococus, Bacillus, Arthrobacterium, etc. Later, Kasivis- wanathan et al. (1977) observed inoculation of Azotobacter to soil proved beneficial to increase the growth and yield of mulberry. Vijayan et al. (2007) evaluated the effect of biofertilizers Azotobacter and Azospirillum on establishment of mulberry and revealed that inoculants improved the mulberry growth and development over control in saline conditions. Even the P-solubilizing Bacillus megaterium, Bacillus sp., Aspergillus awamuri, and A. niger enhanced the growth and yield parameters of mulberry (Nagendra Kumar and Sukumar 2001). Gangwar and Thangavelu (1992) isolated the nitrogen-fixing bacteria Azotobacter and Beijerinckia from phyllosphere and rhizosphere of mulberry. The first pair of leaf was inoculated with both the bacterial isolates and showed the airborne nature of the nitrogen fixers. In rhizosphere, the population of both the genera increased corresponding to increase in the age of the plant. Yadav and Nagendra Kumar (1989) observed reduction of nitrogen fertilizer to half or one-third dose along with Azospirillum, which improved the mulberry plant growth and leaf yield at par with full dose of nitrogen fertilizer. Umakant and Bagyaraj (1998) reported improvement of plant growth in mulberry nursery with dual inoculation of Azotobacter chroococcum and Glomus fasciculatum. During several studies, Das et al. (1990, 1994) reported that biological nitrogen fixation mediated through Azotobacter is considered to be the potential system for mulberry cultivation for economizing up to half dose of N fertilizer in different mulberry cultivars without any reduction in leaf yield and quality. Rangarajan and Santhanakrishnan (1995) demonstrated the combined effect of P. fluorescens and Azospirillum more superior than that of single inoculation or uninoculated controls. This enhanced the quality of mulberry leaf and consequently improved 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 21 the silkworm growth and silk production. Chandrashekar et al. (1996) studied the effect of co-inoculation in mulberry with Acaulospora sevis, B. megaterrium var. phosphaticum, and A. brasillense using two sources of phosphorous and attained improvement of leaf growth, yield, and quality. Gupta et al. (2008) reported the sustainability of mulberry leaf production by reducing the application of chemical fertilizers and biofertilizers, such as Azotobacter, Azospirillum, PSB, and mycorrhizae. Baqual and Das (2006) demonstrated that dual inoculation of mulberry with Azotobacter and VAM could curtail use of fertilizers by 50% besides improving the leaf yield, cocoon production, and quality. Azotobacter inoculation to the roots caused better increase in root and shoot weight in comparison to that of leaf inoculation. The proliferation of Azotobacter in nutrient solutions was highly stimulated in the presence of mulberry plants, but the similar stimulation in the natural condition was not observed. Sudhakar et al. (2000a, b) studied the role of biotic and abiotic (seasonal variation) factors in contributing toward population buildup of diazotrophs and other microorganisms both on phylloplane and rhizosphere of mulberry. It was concluded that rainy season and the shoot age of 30–40 days after pruning appear to be ideal for the increase of diazotrophs both in phylloplane and rhizosphere by foliar application of Azotobacter and Beijerinckia. Similarly, PGPR proved to be an effective tool for increase in biomass production in som (Machilus bomycina), which in turn has an impact on the growth of silkworms to produce more silk fiber of good quality. Unni et al. (2008) isolated and exploited PGPR from rhizosphere of som plants. Muga silkworm larvae fed on some leaves of the plant treated with PGPR showed growth-promoting activities in plants. The shell weight of the cocoons formed from the larvae fed with treated som leaves was significantly higher than that of the control. Such cocoons used for fiber estimation showed considerable increase in fiber content which were not only longer but had higher nonbreakable filament length (Unni et al. 2008). B. subtilis strain Lu144 was isolated as an endophyte from the surface sterilized leaves of mulberry (Ji et al. 2008). Strain Lu144 exhibited strong in vitro antagonistic activity against Ralstonia solanacearum which causes bacterial wilt on mulberry plants and displayed reducing the disease incidence.

1.6 Limitations Associated with PGPR

In fact, the inconsistent response of field-grown crops to PGPR has limited commercial development. In natural ecosystems, the behavior of introduced bacterial inoculants (e.g., PGPR) and the subsequent expression of PGP represent a complex set of multiple interactions between introduced bacteria, associated crops, and indigenous soil microflora. These interactions are, in turn, influenced by multiple environmental variables such as soil type, nutrition, moisture, and temperature (Kloepper et al. 1989; Glick 1995). Thus, the ability of a bacterial inoculant to promote plant growth can only be fully evaluated when they are tested in 22 A. Aeron et al. association with all of the components of the rhizosphere (Schroth and Weinhold 1986). Inconsistent responses to beneficial bacteria are frequently reported (Brown 1974; Broadbent et al. 1977; Schroth and Hancock 1982; Howie and Echandi 1983; Schroth and Weinhold 1986; Schippers et al. 1987; Kloepper et al. 1988a, b;de Frietas and Germida 1990, 1991, 1992a, b). Moreover, in a compilation of reports of crop yield responses to bacterial inocula in field studies after 1974, Kloepper et al. (1989) noted that in 13 of 26 studies bacterial inocula previously identified as PGPR caused significant yield reductions as compared to the control. It has been suggested that inconsistencies associated with microbial inoculants for field applications are not surprising because physical and chemical factors, such as soil texture, pH, nutrient status, moisture, temperature, organic matter content, and biological interactions in the rhizosphere, may affect the establishment, survival, and activity of certain organisms whereas other organisms may remain unaffected (Schroth and Weinhold 1986; Kloepper et al. 1989). Thus, apparent discrepancies in experimental results likely reflect differences in experimental conditions including soil and associated indigenous soil microorganisms. Therefore, workers have devised new strategies to overcome these inconsistencies. Recent studies showed a promising trend in the field of inoculation technology, which is the use of mixed inoculants or application of consortia (combinations of microorganisms) that interact synergistically are currently being devised. Micro- bial interaction studies performed without plants indicate that some bacterial genera allow each other to interact synergistically providing nutrients, removing inhibitory products, and stimulating each other through physical or biochemical activities that may enhance some beneficial aspects of their physiology such as nitrogen fixation (Pandey and Maheshwari 2007b; Arora et al. 2008). Plant studies have shown that these beneficial effects of Azospirillum on plants can be enhanced by co-inoculation with other microorganisms (Alagawadi and Gaur 1992; Belimov et al. 1995). Co-inoculation frequently increased growth and yield compared to single inoculation, which provided the plants with more balanced nutrition and improved absorption of nitrogen, phosphorus, and mineral nutrients (Kumar et al. 2009). Application of PGPR could not only produce significant benefits that require minimal or reduced levels of fertilizers but also consequently produce a synergistic effect on root growth and development (Kumar et al. 2009).

1.7 Conclusion and Future Prospects

The application of PGPR, in recent times, is gaining attention mainly due to the environment friendly nature of bioinoculants and increased acceptability of natural “organic” plant products globally. In practice, alien strains suitable to different crops grown in various climatic conditions and their use are limited due to the unavailability of suitable climate-based microbial inoculants. In view of the knowledge of ecological specificity associated with naturally occurring microorganisms, consistent efforts from research laboratories are required for selecting and 1 Emerging Role of Plant Growth Promoting Rhizobacteria in Agrobiology 23 developing microbial performance always in field suited in the specific set of climatic conditions. Prior to registration and commercialization of PGPR products, a number of hurdles must be overcome. These include scale up and production of the organism under commercial fermentation conditions while maintaining quality, stability, and efficacy of the product. Formulation development must consider factors such as shelf life, compatibility with current application practices, cost, and ease of application. Health and safety testing are also required to address such issues as nontarget effects on other organisms including toxigenicity, allergenicity and pathogenicity, persistence in the environment, and potential for horizontal gene transfer. Capitalization costs and potential markets must be considered in the decision to commercialize the product for their application in agro and allied industries.

Acknowledgement Thanks are due to UCOST (Dehradun), UGC, and CSIR (New Delhi) for providing ﬁnancial support in the form of research project to DKM.

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Ankit Kumar, Anil Prakash, and B.N. Johri

2.1 Introduction

Plant growth promoting rhizobacteria (PGPR) are beneficial bacteria which have the ability to colonize the roots and either promote plant growth through direct action or via biological control of plant diseases (Kloepper and Schroth 1978). They are associated with many plant species and are commonly present in varied environments. Strains with PGPR activity, belonging to genera Azoarcus, Azospirillum, Azotobacter, Arthrobacter, Bacillus, Clostridium, Enterobacter, Gluconacetobacter, Pseudomonas, and Serratia, have been reported (Hurek and Reinhold-Hurek 2003). Among these, species of Pseudomonas and Bacillus are the most extensively studied. These bacteria competitively colonize the roots of plant and can act as biofertilizers and/or antagonists (biopesticides) or simultaneously both. Diversified populations of aerobic endospore forming bacteria (AEFB), viz., species of Bacillus, occur in agricultural fields and contribute to crop productivity directly or indirectly. Physiological traits, such as multilayered cell wall, stress resistant endospore formation, and secretion of peptide antibiotics, peptide signal molecules, and extracellular enzymes, are ubiquitous to these bacilli and contribute to their survival under adverse environmental conditions for extended periods of time. Multiple species of Bacillus and Paenibacillus are known to promote plant growth. The principal mechanisms of growth promotion include production of growth stimulating phytohormones, solubilization and mobilization of phosphate, siderophore production, antibiosis, i.e., production of antibiotics, inhibition of plant ethylene synthesis, and induction of plant systemic resistance to pathogens (Richardson et al. 2009; Idris et al. 2007; Gutierrez-Manero et al. 2001;

A. Kumar (*), A. Prakash, and B.N. Johri Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal 462026, Madhya Pradesh, India e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 37 DOI 10.1007/978-3-642-18357-7_2, # Springer-Verlag Berlin Heidelberg 2011 38 A. Kumar et al.

Whipps 2001). It is very likely that plant growth promotion by rhizosphere bacilli may be a result of combined action of two or more of these mechanisms (Fig. 2.1). Pathogenic microorganisms affecting plant health are a major threat to food production, and traditional methods, viz., crop rotation, breeding for resistant plant cultivars, and application of chemical pesticides, seem to be insufﬁcient to control root diseases of important crop plants (Johri et al. 2003). Further, it appears inevitable that fewer pesticides will be used in future and that greater reliance will be laid on biotechnological applications including use of microorganisms as antagonists. Therefore, interest in biological control has been increased in the past few years partly due to change in the public concern over the use of chemicals and the need to ﬁnd alternatives of chemicals used for disease control. Both Bacillus and Paenibacillus species express antagonistic activities by suppressing the pathogens and numerous reports covering this aspect both under in vitro and in vivo conditions are available (Arrebola et al. 2010; Chen et al. 2009; Joshi and McSpadden Gardener 2006). Enhancement of plant growth by root-colonizing species of Bacillus and Pae- nibacillus is well documented and PGPR members of the genus Bacillus can provide a solution to the formulation problem encountered during the development of BCAs to be used as commercial products, due in part to their ability to form heat- and desiccation-resistant spores (Kloepper et al. 2004; Emmert and Handelsman 1999). In the past few years, research has been directed more toward the induced systemic resistance (ISR), a process by which PGPR stimulate the defense

Fig. 2.1 Schematic illustration of important mechanisms known for plant growth promotion by PGPR. Different mechanisms can be broadly studied under (1) Biofertilization, and (2) Biocontrol of pathogens. Biofertilization encompasses: (a) N2 Fixation, (b) Siderophore production, (c) Pinorganic solubilization by rhizobacteria. Biocontrol involves: (a) Antibiosis, (b) Secretion of lytic enzymes, and (c) Induction of Systemic Resistance (ISR) of host plant by PGPR 2 Bacillus as PGPR in Crop Ecosystem 39 mechanisms of host plants without causing apparent harm to the host. More recently, Choudhary and Johri (2008) have reviewed ISR by Bacillus spp. in relation to crop plants and emphasized on the mechanisms and possible applications of ISR in the biological control of pathogenic microbes. Various strains of species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus are known as potential elicitors of ISR and exhibit significant reduction in the incidence or severity of various diseases on diverse hosts (Choudhary and Johri 2008; Kloepper et al. 2004). It is believed that plants have the ability to acquire enhanced level of resistance to pathogens after getting exposed to biotic stimuli provided by many PGPRs and this is known as rhizobacteria- mediated ISR (Choudhary et al. 2007). The aim of this chapter is to perpetuate the ecological perspectives and role of Bacillus species studied in the past few years, pertaining to its plant growth promotory activities with emphasis on the biocontrol mechanisms and possible implications in crop ecosystem. Published and some previously unpublished work have been summarized in this chapter, showing that strains of Bacillus and Paeni- bacillus species, including B. subtilis, B. cereus, B. amyloliquefaciens, B. pumilus, B. pasteurii, B. mycoides, B. sphaericus, P. polymyxa, P. azotofixans, and some other newly discovered species (B. endophyticus), influence the growth, development, and yield of crops under controlled and varied natural conditions either directly or indirectly following various mechanisms.

2.2 Ecology of Bacillus and Paenibacillus Species

Most species of Bacillus and Paenibacillus are distributed globally and the widespread occurrence of subspecies of B. subtilis and B. cereus with their ability to suppress the plant pathogens has been widely recognized.

2.2.1 Distribution, Diversity, and Population Dynamics

Plant growth promoting strains of Bacillus and Paenibacillus have been widely studied for enhancement of plant growth (Choudhary and Johri 2008; Kloepper et al. 2004). Cultivation-dependent approaches have revealed the occurrence of multiple isolates of phylogenetically and phenotypically similar species of B. subtilis and B. cereus ranging from log 3 to log 6 counts (CFU) per gram fresh weight (Vargas-Ayala et al. 2000). While culture-independent studies of soil confirmed the uncultured diversity of both Bacillus and Paenibacillus rRNA lineages, there are contradictions about the relative abundance of culturable and unculturable representatives of these genera in different soils (McSpadden Gardener 2004; Smalla et al. 2001). Though multiple species of Bacillus and Paenibacillus are frequently found in the soil and rhizosphere, only limited 40 A. Kumar et al. information is available about the most commonly isolated species of this genus. In some cases, B. megaterium has been found as the most abundant species, but it is improbable that a single species will dominate numerically in most soils (Liu and Sinclair 1992). Species of B. polymyxa group, recently renamed Paenibacillus, are autotrophs, commonly associated with rotting plant materials, composts, and the rhizosphere. Some of them are able to fix nitrogen and thus contribute significantly to the acquisition of nitrogen by crops such as Canadian wheat (Priest 1993). Members of B. brevis group, renamed Brevibacillus, are found in both soil and water habitats. The species B. sphaericus is most noted as an insect pathogen and is found in the sediments of pools, lakes, and drainage ditches where insect larvae thrive. Limited attempts have been made to study the diversity of bacterial populations in and around the rhizosphere, probably due to lack of appropriate techniques required to isolate sufficient number of strains belonging to the same species. Due in part to the unavailability of suitable methods to explore the community dynamics, our understanding of the variation in microbial community dynamics in response to soil type, plant type, or stage of plant development is limited as yet (McSpadden Gardener 2004; Duineveld et al. 1998). In fact, bacterial communities residing in the rhizosphere respond, in particular, with respect to density, composition, and activity, to the plethora and diversity of organic root exudates, resulting in plant species-specific microflora which may eventually vary with the stage of plant growth (Wieland et al. 2001 and references therein). To come to an improved understanding of factors affecting the ability of bacteria to colonize the rhizosphere, the plant has to be taken into account. Rhizospheric competence is a necessary prerequisite for PGPR. It comprises of effective root colonization combined with the ability to survive and proliferate along the growing plant roots in the presence of indigenous microbiota over a period of time. Given the importance of rhizospheric competence as a prerequisite, understanding the plant–microbe communication as affected by genetic and environmental factors in the context of their ecological niche can contribute significantly toward understanding the mechanisms of action (Bais et al. 2004; Whipps 2001). Bacillus species are believed to be less rhizosphere competent than Pseudomonas species. As a consequence, most research even today is aimed at the development of BCAs based on Pseudomonas species (Weller 1988). However, studies on the genetic diversity of Bacillus from soil as well as from the wheat rhizosphere implied that rhizosphere competence is a characteristic of the strain (genotype) not exclusive to the genus or species. Based on studies of wheat rhizosphere colonization by Bacillus species, it seems that rhizosphere competent genotypes occur in this bacterium (Milus and Rothrock 1993; Maplestone and Campbell 1989). Experiments with different wheat varieties conducted by Juhnke et al. (1987) and Milus and Rothrock (1993) have revealed that seeds bacterized with selected strains of Bacillus could successfully establish in the rhizosphere. But, whether the colonization attained by introduced strains was on the entire root or only the top few centimeters of root below the seed could not be confirmed. However, in another 2 Bacillus as PGPR in Crop Ecosystem 41 study, high populations of B. mycoides and B. pumilus in the rhizosphere of wheat at a depth of 20–30 cm below the site of bacterial inoculation (200 ml/seed) at the time of planting have been reported; the bacterial population was believed to be carried downward either in conjunction with water infiltration or along with elongating tips of growing roots (Maplestone and Campbell 1989).

2.2.2 Spatiotemporal Aspects

Variations are known to exist in the genetic microdiversity within the species of Bacillus and Paenibacillus (McSpadden Gardener 2004). Wieland et al. (2001) studied the spatiotemporal variation among the microbial communities from soil, rhizosphere, and rhizoplane with respect to crop species (clover, bean, and alfalfa), soil type, and crop development following a comparative study of 16S rRNA sequences employing temperature gradient gel electrophoresis (TGGE). According to their study, the type of plant species had profound effects on microbial community dynamics, with the effect of soil type typically exceeding that of plant type. Plant development had only minor habitat-dependent effect and insignificant variations were observed in time-dependent shifts among the microbial communities compared to the soil type or plant type in all the habitats under study. Systematic community shifts could not be recognized in samples from bulk soil; however, some variations in the TGGE patterns could be correlated to time of development in the rhizosphere and rhizoplane. Nearly, similar findings were reported by Mahaffee and Kloepper (1997) who used fatty acid methyl ester analysis (FAME) to determine the community shifts in the rhizosphere of cucumber. However, only an altered window of observations generated by the use of specific primers could possibly reveal a stronger time-dependent stimulation of certain bacterial groups. McSpadden Gardener (2004) studied the population structure of these two groups by terminal restriction fragment length polymorphism (TRFLP) using group specific primers Ba1F and Ba2R and characterized the plant growth promoting population of PGPR; only minor differences were observed in the number and relative abundance of Bacillus-like ribotypes from different sites all the way through Ohio (USA). Despite environmental constraints and interactions with other microorganisms, some bacteria are able to colonize the phylloplane with higher frequency than others. Arias et al. (1999) evaluated the diversity and distribution of Bacillus spp. from soybean phylloplane wherein a decline was observed in the population of Bacillus spp. from 80% of total bacterial isolates in early stages to 0% at the time of harvesting. In addition, the diversity of Bacillus spp. decreased from nine species at 45 days to just one species at 133 days, shortly before harvesting. B. pumilus was reported as the most prominent species from soybean phylloplane at all sampling times till the end of cropping season, followed by B. subtilis as second most abundant species from 15 to 108 days after sowing. Several other Bacillus spp., such as B. subtilis, B. brevis, B. firmus, and B. circulans, were found as regular or as dominant microflora at an early stage of plant growth, but were no longer detected 42 A. Kumar et al. after 85 days from the phylloplane of trifoliate leaves. The cause of apparent reduction in Bacillus spp. populations at the end of soybean cropping season, however, remained unclear. The genus Paenibacillus encompasses several species described as nitrogen- fixing bacilli, including P. polymyxa, P. azotofixans, and P. macerans (Ash et al. 1993). In contrast to other species of this genus, strains belonging to P. azotofixans are efficient nitrogen fixers and are prevalent in the rhizosphere of maize, sorghum, sugarcane, wheat, banana, and forage grasses (Rosado et al. 1998a; Seldin 1992). Rosado et al. (1998b) showed that bacterial diversity of P. azotofixans was high in bulk soil compared to the rhizosphere. Seldin et al. (1998) determined the diversity of P. azotofixans strains isolated from the rhizoplane, rhizosphere, and nonroot associated soil of maize grown in two different field soils of Brazil (Cerrado and Varzea). On the basis of phenotypic traits, 60 strains from Varzea soil and 46 strains from Cerrado were identified as P. azotofixans and they could be categorized into six groups for each soil. Fifteen different hybridization patterns were obtained in 60 P. azotofixans strains from Varzea while only two patterns were obtained from 46 strains of Cerrado when specific plasmids for nifH genes were used as probes. Data from the phenotypic and hybridization studies were used to construct a dendrogram; all strains could be distributed into 29 groups. Strains isolated from Varzea soil were more heterogeneous than those obtained from Cerrado soil. This heterogeneity is believed to be a result of difference in soil type but it remained unclear whether the difference in soil type could account for differences demonstrated by the heterogeneity between Varzea and Cerrado soil populations. These observations were in agreement with the findings of Berge et al (1991) who also reported variations in the population structure of B. circulans from the rhizosphere of maize with the soil type.

2.2.3 Rhizospheric Effect and Host Speciﬁcity

It is not certain if plants actively select beneficial soil microbial communities in their rhizosphere through rhizodeposition, though earlier studies showed that plants select for taxonomic functional groups in the rhizosphere (Grayston et al. 2001; 1998). Although some field studies with mixed plant communities did not find such selections in the rhizosphere, there are reports that suggest a strong correlation between plant and soil microbial communities (Duineveld et al. 2001; Smalla et al. 2001). The root exudation is believed to be plant specific and this specificity may reflect the evolution or specific physiological adaptation to conditions of a particular soil habitat (Crowley and Rengel 1999). The type of root exudates is crucial for the ecosystem distribution and niche specificity of certain plants. Composition of root exudates was shown to vary with plant species and stage of plant growth (Jaeger et al. 1999). Concomitantly, the plant is supposed to influence the population structure of indigenous rhizobacteria as well as the population dynamics of introduced BCAs. Under certain conditions, many compounds present in the root 2 Bacillus as PGPR in Crop Ecosystem 43 exudates (sugar, amino acids, or organic acids) stimulate a positive chemotactic response in bacteria (Somers et al. 2004). Being a major driving force for microbial root colonization, plant root exudation could be engineered precisely to stimulate specific microbial colonization on the roots. Oger et al. (1997) demonstrated that genetically engineered plants producing opines have an altered rhizosphere community. In fact due to high diversity of chemical influences in the rhizosphere of different plants, roots drive specific selections of microbes out of indefinite pool of soil microbial diversity. Nevertheless, the cultivation practices being followed have also been recognized as an important determinant of rhizospheric microbiota (Mittal and Johri 2007). Agriculture management strategies can induce clear shifts in the structures of plant- associated microbial communities (Garbeva et al. 2004). For example, plant genotype can exert strong effects on the bacterial communities associated with the plants (Gu and Mazzola 2003; Adams and Kloepper 2002). Growth stage of plant is another important factor that provides shape to the rhizobacterial community structure and as reported in case of potato rhizosphere it could be the strongest one affecting the bacterial communities (van Overbeek and van Elsas 2008). Besides, land use, soil history, cultivation practices, and plant growth stage are some of the other factors which govern the structure of plant-associated microbial communities (van Overbeek and van Elsas 2008 and references therein, Mittal and Johri 2007). Among the existing practices, use of biofertilizer is of utmost importance in crop ecosystem pertaining to agriculture production. A study was carried out to evaluate the effect of cultivation practices (traditional and modern), on the community structure of culturable bacteria antagonistic toward soilborne pathogenic fungus Sclerotinia sclerotiorum, associated with the soybean (Glycine max L.) rhizoplane and rhizosphere/endorhizosphere and bulk soil (Kumar et al. 2009). The cultivation parameters for both kinds of practices were otherwise similar except that the traditional system of cultivation involved use of farmyard manure (FYM) as fertilizer input while modern cultivation system was based on application of commercially available inorganic chemical fertilizers. The community structure of bacterial antagonists isolated following traditional system of cultivation was structurally more diverse than modern system. Further, traditional system of cultivation was found to support higher population density of the antagonists. The bacterial diversity was found to increase with the stages of plant growth gradually from seedling up to maturation stage and then eventually followed a decline with only transient changes. Little variation was observed in bulk soil for community structure, implying that the bulk soil was highly stable while the gradual shifts observed in bacterial diversity may be a consequence of change in composition of root exudates excreted from the plant roots which are known to change the chemistry and biology of root microenvironments (Hartmann et al. 2009). The nature of organic amendments used in traditional system of cultivation may account for the occurrence of high bacterial diversity of antagonists in the traditional system. As a matter of fact, these organic substrates can act as ideal source of nutrients for the antagonists in soils and offer an opportunity to introduce 44 A. Kumar et al. and establish specific BCAs into soils, which in turn leads to sustainable disease control based on activities of microbial communities. Smalla et al. (2001) demonstrated for the first time that roots of each model plant species are colonized by its own bacterial communities using cultivation-independent methods on three phylogenetically different and economically important crops – strawberry (Fragaria ananassa Duch.), potato (Solanum tuberosum L.), and oilseed rape (Brassica napus L.). It was possible to differentiate the plant species on the basis of the rhizosphere communities using DGGE in a randomized field trial (Smalla et al. 2001). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere. All rhizobacteria showed some bands in common, and also specific bands intriguingly, e.g., Nocardia populations were identified as strawberry-specific bands.

2.2.4 Endophytic Colonization and Plant Growth Promotion

Bacteria residing in the rhizosphere of plants may gain access into the root interior and establish endophytic populations. Several bacteria can transcend the endodermis barrier, reach the vascular system by crossing through the root cortex, and subsequently thrive as endophytes in plant tissues, viz., stem, leaves, tubers, etc. (Compant et al. 2005). The endophytic colonization of host plant by bacteria reflects on their ability to selectively adapt themselves to these specific ecological niches resulting in an intimate association without any apparent harm to the plant (Compant et al. 2005 and references therein). Bacterial endophytic communities are presumed to be a product of colonization process initiated in the root zone but they may originate from other sources, viz., phyllosphere, anthosphere, or spermosphere (Sturz et al. 2000). Species of Bacillus are common inhabitants among the resident microflora of inner tissues of various species of plants, including cotton, grape, peas, spruce, and sweet corn, where they play an important role in plant protection and growth promotion (Berg et al. 2005; Shishido et al. 1999; Bell et al. 1995). Almost all the endophytic, aerobic, spore forming bacteria described so far belong to the species generally recognized as free-living soil organisms, such as B. cereus, B. insolitus, B. megaterium, B. pumilus, B. subtilis, and P. polymyxa, though in some cases the bacteria have not been identified beyond the genus level (Shishido et al. 1999; Benhamou et al. 1996; Sturz et al. 1997; Bell et al. 1995). Reva et al. (2002) studied the diversity of endophytic AEFB in the inner tissues of healthy cotton plants (Gossypium sp. Dushanbe, Tajikistan). A total of 76 strains were characterized phenotypically and majority of them were identified as B. amyloliquefaciens, B. licheniformis, B. megaterium, B. pumilus, and B. subtilis; four strains could not be assigned to any known species. Among the isolates, B. subtilis was most abundant (43 strains) followed by B. licheniformis (15 strains), B. megaterium (eight strains), and B. pumilus (six strains). Phenotypically all the four unusual strains appeared similar and showed some resemblance to B. insolitus, another well-known colonizer of plants but differed from the latter in some 2 Bacillus as PGPR in Crop Ecosystem 45 physiological properties (Sturz et al. 1997; Bell et al. 1995). Molecular typing of these four strains revealed similar RAPD patterns that were different from those of the reference strains of common plant-associated species such as B. licheniformis, B. megaterium, B. pumilus, and B. subtilis. Based on similarity level of RAPD profiling, the four strains were grouped into single distinct taxon but two different amplification profiles were obtained when the hypervariable spacer regions between 16S and 23S rRNA genes were targeted, suggesting that these four bacteria encompass two lineages within the same taxon. Complete 16S rRNA sequencing of the two representatives unravelled the distinction between them; one of these was characterized as a new species, B. endophyticus.

2.3 Phtyostimulation and Biofertilization Effects

The physiology of plant and signaling are affected by bacterial hormones in different ways depending upon the physiological role played by hormone or recalcitrance of the plant tissues to change in hormonal level and the concentration of the hormone being produced. Biofertilizing PGPR, in particular, refers to the rhizobacteria that are able to promote plant growth by increasing nutrient uptake by plants.

2.3.1 Phytostimulation

Enhancement of plant growth by root colonizing species of Bacillus and Paeniba- cillus is well known (Idris et al. 2007; Kloepper et al. 2004). It is also very likely that growth promoting effects of various PGPRs are due to bacterial production of plant growth regulators such as indole-3-acetic acid (IAA), gibberellins, and cytokinins (Bottini et al. 2004; Bloemberg and Lugtenberg 2001). A large proportion (80%) of bacteria colonizing the rhizosphere have been reported positive for IAA production, but reports depicting IAA production by Gram-positive soil-living bacteria are only few (Loper and Schroth 1986). However, Idris et al. (2004) showed production of substances with auxin (IAA)-like bioactivity from strains of B. subtilis/B. amyloliquefaciens including strain FZB42. Further, gibberellin production was confirmed from B. pumilus and B. licheniformis (Gutierrez-Manero et al. 2001). Tryptophan has been identified as main precursor molecule for biosynthesis of IAA in bacteria. IAA controls a diverse array of functions in plant growth and development and acts as a key component in shaping plant root architecture such as root vascular tissue differentiation, regulation of lateral root initiation, polar root hair positioning, and root gravitropism (Aloni et al. 2006). Idris et al. (2007) first demonstrated the production of reasonable quantities of IAA from Gram-positive bacterium B. amyloliquefaciens FZB42 and IAA production was enhanced when the bacterium was fed with tryptophan. Production of IAA was dramatically reduced in the mutants deficient in trp gene responsible for biosynthesis 46 A. Kumar et al. of IAA, suggesting that main route of IAA biosynthesis in this bacterium was dependent on tryptophan. Spaepen et al. (2007) reviewed different pathways involved in the biosynthesis of IAA based on the chemical nature of intermediate molecules produced using tryptophan as precursor. The plant beneficial Gram-negative bacteria synthesize IAA following different pathways that involves indole-3-pyruvic acid (IPA), indole-3-acetamide (IAM), or indole-3-acetonitrile (IAN) as important inter- mediates (Patten and Glick 1996; Kobayashi et al. 1995). However, in Gram-positive bacteria the main route for biosynthesis of IAA involves IPA (Vandeputte et al. 2005). Plant hormones affect the spatial and temporal expression of various phenotypes such as cell elongation, division, and differentiation. Besides they are believed to play an important role in plant’s response to biotic and abiotic stresses. Many bacteria are capable of producing more than one type of plant hormone; however, some of them can produce and degrade the same hormone, produce one, and degrade the precursor of another, thus affecting the physiology of plant in several ways (Boiero et al. 2007; Leveau and Lindow 2005). Further, bacterial production of IAA may be beneficial or detrimental to the plant health. For example, IAA production by P. putida GR12-2 has been found to improve the root proliferation of Azospirillum brasilense resulting in increased root surface area which helps in augmentation of nutrient and water uptake from soil (Patten and Glick 2002). On the other hand, in some reports IAA production has been found necessary for pathogenesis (Yang et al. 2007; Vandeputte et al. 2005). There is a growing body of literature showing that IAA can act as a signal molecule, indicating that use of hormones as signaling molecules is not confined only to the plants but also takes part in communication between bacteria and other microorganisms (Spaepen et al. 2007).

2.3.2 Biofertilization

PGPR stimulate the plant growth directly through increase in nutrition acquisition, such as phosphate solubilization, or more generally by rendering the inaccessible nutrients available to the plants (Persello-Cartieaux et al. 2003). After nitrogen, perhaps the essential mineral element that most frequently limits the growth of À plants is P, which is taken up from soil solution as phosphate (Pi, H2PO4 ). Although soils generally contain a large amount of total P but only a small proportion is available for uptake by the plants. On an average, most of mineral nutrients in soil are present in millimolar amounts but P is present in micromolar or even lesser quantities (Khan et al. 2006). However, plants are well adapted to uptake of P from low concentration soil solution (Jungk 2001). Therefore, it is presumed that the supply and availability of P to the root surface is influenced by the root and microbial processes. Phosphate-solubilizing microorganisms (PSM) include a wide range of symbiotic and nonsymbiotic organisms, such as Pseudomonas, Bacillus, and Rhizobium 2 Bacillus as PGPR in Crop Ecosystem 47 species; actinomycetes; and various fungi-like Aspergillus and Penicillium species (Richardson et al. 2009 and references therein). Phosphate-solubilizing bacteria have already been applied in the agronomic practices as potential bioinoculants to increase the productivity. For example, in Soviet Union, a biofertilizer product under the trade name “phosphobacterin” was prepared and commercialized for agricultural applications. Phosphobacterin contained Bacillus megaterium var. phosphaticum and later on it was also introduced to other countries, like Eastern Europe and India (Khan et al. 2006). Similarly, in India, a consortium, termed as Indian Agricultural Research Institute (IARI) microphos culture, has been developed containing two very efficient phosphate-solubilizing bacteria (Pseudomonas striata and Bacillus polymyxa) and three phosphate-solubilizing fungi (Aspergillus awamori, A. niger, and Penicillium digitatum) (Gaur 1990). Application of phosphate solubilizers alone or in combination with nitrogen fixers has been found beneficial for cotton and wheat fields (Zaidi and Khan 2005; Kundu and Gaur 1980). A study had been carried out under green house conditions to explore the effects of combined inoculation of Rhizobium and phosphate- solubilizing P. striata or B. polymyxa with or without added fertilizers on chickpea yield and nutritional contents (Algawadi and Gaur 1988). Whereas, inoculation with Rhizobium alone was found to increase nodulation, addition of phosphate solubilizers increased the phosphorus content of the soil. Combined inoculation increased the nodulation and available phosphorus of the soil coupled with improved grain yield and phosphorus and nitrogen uptake by the plants. Natarajan and Subramainan (1995) suggested that following a combined inoculation of Rhizobium (strain Tt 9) with B. megaterium var. phosphaticum could meet with about 50% of the phosphatic fertilizer requirement of the groundnut. This consortium was found very effective for groundnut, resulting in increased nodulation, increased root and shoot length, as well as increased pod yield. Tomar et al. (1993) reported that inoculation with the phosphate-solubilizing bacterium B. firmus resulted in significant increase in seed yield in field trials on lentil (Lens esculentus) and black gram (Vigna mungo). Similarly, Bethlenfalvay (1994) demonstrated the impact of a consortium comprising Glomus mosseae, Bacillus sp., and Rhizobium sp. on plant growth and soil aggregation upon Pisum sativum cultivation and observed a dramatic increase in plant growth and soil aggregation. While in case of P. sativum, inoculation of Rhizobium, B. polymyxa, and Glomus faciculatum 3À resulted in enhanced dry matter production and PO4 uptake, no significant response of soybean to dual inoculation was observed (Kloepper et al. 1980).

2.4 Biological Control: Gram-Positive Perspectives

Biological control, using microorganisms to suppress plant disease, offers a powerful alternative to the use of synthetic chemicals. The rich diversity of the microbial world provides a seemingly endless resource for this purpose. While a diverse array 48 A. Kumar et al. of microorganisms contribute toward the biological control of plant pathogens, most research has utilized species of Bacillus, Trichoderma, and Pseudomonas (McSpadden Gardener and Driks 2004). There are eight species of microorganisms registered by U.S. Environmental Protection Agency for commercial use against soilborne plant pathogens in the United States (Cook et al. 1996). These include two fungi (Gliocladium virens G-21 and Trichoderma harzianum KRL-AG2), three Gram-negative bacteria (Agrobacterium radiobacter K84, Pseudomonas ﬂuores- cens EG1053, and Burkholderia cepacia type Wisconsin), and three Gram-positive bacteria (Bacillus subtilis GB03, B. subtilis MBI 600, and Streptomyces griseovir- idis K61). Other than A. radiobacter K84, all others are used to control damping-off diseases and improve stand establishment and seedling vigour. There is a growing body of literature which describes different mechanisms for biocontrol ability of Bacillus, viz., siderophore production, secretion of hydrolytic enzymes, antibiosis, ISR, etc. However, discussion on all these aspects of biocontrol is beyond the scope of this chapter, hence antibiosis, quorum quenching (QQ), and ISR, the mechanisms of major importance being emphasized in current scenario involved in biocontrol, will be discussed in detail. Moreover, numerous reports on in vitro antimicrobial activity of Bacillus species are available, but here we emphasize on the selective studies that combine the successful in situ demonstration of antagonism in addition to in vitro studies, i.e., success stories of Bacillus species used as BCAs in the ﬁeld.

2.4.1 Success Stories of Bacillus Species as Biocontrol Agents

Extensive research including the field testing of different Bacillus strains has led to the development of a number of products widely used as commercial BCAs (McSpadden Gardener and Fravel 2002). There is a list of biopesticides (available online: http://www.oardc.ohio-state.edu/apsbcc) registered for pests and disease control in the United States, approved by the U.S. Environmental Protection Agency (EPA), wherein the commercial formulations of different Bacillus strains used as BCAs are specified. The products are available as different formulations, viz., liquid or suspension in a liquid, wettable powder, or dry cakes depending upon the compatibility of the biocontrol strain with the carrier molecule. Products like Companion, Kodiak, Serenade, Subtilex, and Taegro are based on exploitation of different strains of B. subtilis as BCAs. Although Companion and Kodiak manufactured by Growth Products Ltd, NY, and Gustafson Inc., TX, of the United Staes, respectively, use the same strain B. subtilis GB03, the formulations used differ; the former is used as liquid while the latter as dry flakes. While Kodiak is labeled for the control of root pathogens of cotton and legumes (soybean) such as Rhizoctonia solani, Fusarium spp., Aspergillus spp., and Alternaria spp., Companion is known to control the diseases caused by species of Rhizoctonia, Phytophthora, Pythium, and Fusarium. The principal component of Subtilex (Becker Underwood, Ames, IA) is B. subtilis MBI600 and is marketed for control of root- and 2 Bacillus as PGPR in Crop Ecosystem 49 seed-borne infections of ornamental and vegetable crops, such as root rot of soybean and Botrytis species, infection of vines, strawberry and cucumber, and brown rust of cereals. Likewise, Serenade (AgraQuest, Davis, CA, USA) containing B. subtiis strain QST713 has been proposed to mitigate the downy mildew, Cercospora leaf spot, and early blight and late blight diseases associated with various crop plants. However, until today the genetic basis of biocontrol ability of B. subtilis strains is not clearly understood and much has been emphasized on the antibiotic production (Joshi and McSpadden Gardener 2006).

2.4.1.1 Antibiosis

Bais et al. (2004) demonstrated the protective action of surfactin produced by B. subtilis against infection caused by Pseudomonas syringae in Arabidopsis thaliana and suggested that surfactin was necessary not only for root colonization but also provided protection against the pathogen. The disease suppression was correlated with inhibitory concentrations of surfactin produced by the organism on roots. Moyne et al (2001) identified B. subtilis strain AU195 capable of producing antifungal peptides showing similarity with bacillomycin (group iturin A). The strain AU195 exhibited strong antagonistic activity against Aspergillus flavus and a broad range of other plant pathogenic fungi. In another study, B. amyloliquefaciens strain A1Z isolated from soybean rhizosphere was found to produce iturin-like compounds, which successfully inhibited three taxonomically diverse fungal pathogens, Sclerotinia sclerotiorum, Macrophomina phaseolina, and Fusarium oxysporum, the causal agents of sclerotinia stem rot, charcoal rot, and fusarial wilt of soybean plants, under controlled conditions. Chromatographic analysis and mass spectrometric studies showed that the principal antifungal components show similarity with iturin-like compounds (Kumar et al. unpublished). However, the efficacy of antifungal compounds has not been evaluated in the field as yet. Romero et al. (2007) showed the involvement of iturin and fengycin antibiotics from four B. subtilis strains UMAF6614, UMAF6616, UMAF6639, and UMAF8561 in the suppression of powdery mildew of cucurbits caused by Podo- sphaera fusca. The culture supernatant could successively inhibit the powdery mildew at levels previously reported for vegetative cells (Romero et al. 2004). The chemical analysis of culture filtrate together with the recovery of inhibitory components (surfactin, fengycin, and iturin A or bacillomycin) from the melon leaves treated with two strains (UMAF6614 and UMAF6639) strongly supported the evidence of in situ production of these antimicrobials.

2.4.1.2 Quorum Quenching and Biological Control

Bacteria sense their population density and coordinate the expression of target genes, including the virulence factors in Gram-negative bacteria, by N-acylhomoserine lactones (AHLs) dependent mechanism known as quorum sensing (QS). While 50 A. Kumar et al.

AHLs and other substituted g-butyrolactones are synthesized by Gram-negative bacteria, certain oligopeptides and substituted g-butyrolactones are the primary signal molecules found in Gram-positive bacteria (Faure et al. 2009). The most widely studied signal molecules involved in quorum sensing are the AHLs (Whitehead et al. 2001). In Gram-positive bacteria, QS signaling molecules are generally peptides, except for the universal pheromone LuxS found in both Gram-positive and Gram- negative bacteria (Schauder et al. 2001). QS is believed to play a crucial role in bacterial physiology including regulation of rhizospheric competence factors such as antibiotic production, horizontal gene transfer, and control of those functions that are directly or indirectly related to plant–microbe interactions (Whitehead et al. 2001). However, several soil bacteria are able to interfere with the QS by enzymatic degradation of AHLs, a process known as QQ. AHL inactivation has been reported in a-proteobacteria (e.g., Agrobacterium, Bosea, and Ochrobactrum), b-proteobacteria (e.g., Variovorax, Ralstonia, Comamomonas, and Delftia), and g-proteobacteria (e.g., Pseudomonas and Acinetobacter)(Faureetal.2009). In case of Gram-positive bacteria, AHL degradation occurs in both low G + C% strains, i.e., Firmicutes, such as Bacillus, and in high G + C% strains or actinobacteria, such as Rhodococcus and Arthrobacter. Acylhomoserine lactonase activity (AiiA) that hydrolyzes the lactone ring of AHLs was first observed in a Bacillus isolate from soil (Dong et al. 2001, 2000). Until now, two types of enzymes that inactivate AHLs have been identified in several species/genera of bacteria: the AHL lactonases that cause lactonolysis (opening of the gamma-butyrolactone ring) resulting in acyl-homoserine with reduced biological activity and AHL acylases that break the amide linkage of AHLs to produce homoserine lactone and fatty acids with no biological activity (Uroz et al. 2008; Zhang and Dong 2004). QQ covers various phenomena that lead to perturbation of expression of QS- regulated functions. Dong et al. (2007) evaluated the mechanisms and functions of QQ in vivo and threw light on the possible applications of this phenomenon in control of plant diseases and promotion of plant health. It has been suggested by many researchers to take advantage of QQ to develop novel biocontrol strategies for plant pathogens (Dong et al. 2007). For example, Park et al. (2008) identified a potential AHL-degrading enzyme, AiiA, from B. thuringiensis which could effectively attenuate the virulence of Gram-negative bacterium Erwinia carotovora in the root system of pepper plant by QQ. Recent studies on B. thuringiensis show that many subspecies of this organism produce AiiA homolog enzymes to degrade AHLs (Dong et al. 2004, 2000). In another case, genetically modified plants which expressed AHL lactonase, AiiA of Bacillus, were found to be more resistant to Pectobacterium carotovorum infection than their parental, wild-type plants (Dong et al. 2001). Moreover, studies carried out by Molina et al. (2003) clearly demonstrated the role of AHL-lactonase enzyme in biocontrol of phytopathogens. A significant reduction was observed in the severity of soft rot of potato caused by P. carotovorum and crown gall of tomato caused by A. tumefaciens when applied with soil bacterium Bacillus sp. A24 or P. fluorescens P3 modified with lactonase gene AiiA, suggesting that disease inhibition was a result of QQ. 2 Bacillus as PGPR in Crop Ecosystem 51

Thus, QQ, in a way, can be used under antivirulence/antidisease strategies to develop novel medical/animal therapies or novel biological control strategies for phytopathogens (Dong et al. 2007). These studies elegantly suggest that QQ can be used as a potential weapon for biological control of pathogenic microorganisms targeting the QS pathway, however, little is known toward ecological aspects of QQ enzymes under in situ conditions. All QQ strategies have so far been developed under in vitro or under the green house conditions and their efﬁcacy under ﬁeld conditions remains to be evaluated. Assessment of interconnections in the signal molecules is a future challenge that needs the help of advanced analytical tools and techniques including transcriptomics, proteomics, and metabolomics to account for the intra- and inter-species communications in the rhizosphere and their ecological impact on the rhizospheric microbiota.

2.4.1.3 Induced Systemic Resistance: Ecological Signiﬁcance and Applicability

Induced resistance may be defined as a physiological “state of enhanced defensive capacity” elicited in response to specific environmental stimuli and consequently the plant’s innate defenses are potentiated against subsequent biotic challenges (van Loon 2000). In addition, there is another defined form of induced resistance, popularly known as systemic acquired resistance (SAR) which is different from ISR in context to the nature of elicitor and regulatory pathways involved. While ISR relies on pathways regulated by jasmonic acid (JA) and ethylene (ET), SAR involves accumulation of salicylic acid (SA) and pathogenesis related (PR) proteins – chitinase and cellulase. PGPRs are among the various groups of plant-associated microorganisms that can elicit the plant defense systems resulting in reduction of disease severity or incidence of diseases caused by pathogens which are spatially different from the inducing agent (van Loon and Glick 2004). Recently, Choudhary and Johri (2008) explicated the mechanisms and role of Bacillus species as inducers of systemic resistance in relation to plant–microbe interactions and demarketed the pathways involved in their regulation. Available reports suggest that specific strains of the species B. amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts including greenhouse studies or field trials on tomato, bell pepper, muskmelon, watermelon, sugarbeet, tobacco, Arabidopsis species, cucumber, loblolly pine, and tropical crops (Kloepper et al. 2004).

2.4.1.4 Greenhouse Studies on Induction of Plant Resistance Systems

A greenhouse test was performed for ISR study of B. mycoides strain Bac J isolated from sugarbeet leaves infected with Cercospora beticola, the causal agent of Cercospora leaf spot on sugarbeet. The strain was sprayed (1.0 log 8 CFU/ml) 52 A. Kumar et al. onto one leaf of test plant and bagged. After 3 days of treatment with Bac J, plants were challenge inoculated with the spore suspension of pathogen. There was a significant reduction in disease severity in plants treated with Bac J on a highly susceptible and a moderately susceptible variety of sugarbeet (Bargabus et al. 2002). In another study performed by Krause et al. (2003), bacterial strains isolated from compost were screened for their capacity to elicit systemic protection against Xanthomonas campestris py. armoraciae. A total of eleven isolates were found to elicit significant reduction in the disease severity in two of the three repeated experiments: four of the top performing strains were characterized as members of Bacillus species. A comparative study of the results obtained by microtiter-based bioassays to assess elicitation of ISR and pot experiments was conducted in greenhouse against blue mold of tobacco caused by Peronospora tabacina Adam (Zhang et al. 2002). The disease incidence was significantly reduced in terms of mean percentage of leaf area under infection from P. tabacina Adam when strains of B. pasteurii C-9 and B. pumilus SE34 and T4 were applied as soil drenches on three tobacco cultivars. Also, the sporulation of the pathogen was significantly decreased when compared with the treated strains and nonbacterized control. To explore the relationship between elicitation of plant growth promotion and ISR, the three strains were further evaluated and applied separately as seed treatment. Tobacco growth was significantly increased by strains SE34 and C-9 but not by T4. It was found to be induced by C-9, not by SE34 and T4. However, application of bacteria by seed treatment following soil drenches resulted in elicitation of ISR by all three strains, in addition to the enhancement of plant growth. In another study, B. subtilis strain AF1, isolated from soils suppressive to pigeon pea (Cajanus cajan) wilt caused by Fusarium udum, was presumed to induce resistance against Aspergillus niger on peanut (Arachis hypogea) (Podile and Dube 1988). Further, it was found that strain AF1 stimulated production of phenylalanine ammonia lyase (PAL) and peroxidase activity, indicating that AF1 elicited ISR (Podile et al. 1995). Strong experimental evidence that AF1 elicited ISR came from the findings of Sailaja et al. (1997) who reported a notable reduction in the incidence of crown rot of peanut caused by A. niger corresponding to increase in lipoxygenase activity, a phenomenon associated with ISR.

2.4.1.5 Field Experiments for Protection Against Systemic Disease

It is not surprising that many biological control agents showing promising results under the controlled environmental conditions of greenhouse fail to exhibit same results in the field under natural environments where competition is more severe. Therefore, shifting from greenhouse to field trials is an important step to evaluate the efficacy of PGPR eliciting ISR and Bacillus. Species were found effective in reduction of disease incidence or plant growth promotion have been examined under field conditions. 2 Bacillus as PGPR in Crop Ecosystem 53

In a field trial conducted on sugarbeet for six consecutive growing seasons, the disease severity due to Cercospora leaf spot was reduced significantly when sprayed with B. mycoides strain Bac J (log 7.0 CFU/ml). About 38–91% reduction in disease severity was found in comparison to the nontreated control. However, in 2 of the 6 years, reduction in disease severity achieved by treatment with Bac J was not significantly different from that attained by using triphenyltin hydroxide, the most commonly used fungicide for Cercospora leaf spot. It has been suggested previously that ISR was presumed to be the mechanism of disease control in greenhouse test that provided spatial separation of pathogen and PGPR but spatial separation was not maintained in the field experiments (Bargabus et al. 2002). In addition to the bacterial and fungal diseases, reduction in the incidence or severity of viral diseases has also been studied in the field employing selected strains of ISR-eliciting Bacillus species. Zehnder et al. (2000) assessed three strains, B. subtilis IN937b, B. pumilus SE34, and B. amyloliquefaciens IN937a for ISR activity against CMV on tomato plants under field conditions for two consecutive cropping seasons. The PGPR strains were applied as seed treatments at the time of transplanting to the pots prior to their transplantation in the field, while CMV inoculation was done on plants 1 week before transplantation to the field. Treatment with all three Bacillus strains resulted in significant reduction of disease compared to the nonbacterized control. Resistance-inducing rhizobacteria offer an attractive alternative, providing a natural, safe, effective, persistent, and durable type of protection. But protection based on biological agents is not always trustworthy and is seldom as effective as chemical treatments. However, different treatments may be combined and combinations of BCAs that suppress diseases by complementary mechanisms may further reduce the incidence or severity of disease. Rhizobacteria-mediated ISR thus may be a valuable addition to the alternatives available for environmentally friendly plant disease control.

2.5 Conclusions

Considerable efforts toward understanding the ecology and management of PGPR have been directed, yet their development as inoculants remains a considerable challenge. The rhizospheric community is highly complex, comprises of a myriad of organisms interacting in various ways, acting upon each other and reacting to the external environment. Several isolates of Bacillus spp. have been developed as BCAs of plant pests and pathogens. However, to be used as successful BCAs a greater understanding of their ecology is desired. In this context, greater knowledge of the diversity, distribution, and physiology of Gram-positive species will be helpful for identification of new strains compatible with the cropping systems. Paramount to success of PGPR is a need to better understand the ecology of rhizobacteria either indigenous or introduced within the rhizosphere. Exploration and identification of traits involved in the ability of certain bacteria to establish 54 A. Kumar et al. themselves into the rhizosphere at levels sufficient to exert effects on plant growth, effectively compete with the indigenous microflora, cooperatively interact with other beneficial members of rhizospheric biota, and understand the mechanisms (signaling, growth promotory actions, disease suppression etc.) that occur between plants and bacteria are also required. Clearly, the taxonomic and physiological diversity of Bacillus spp. appears capable of reducing the disease incidence or severity but also indicates that much remains to be done on the mechanisms by which these bacteria promote plant growth. The molecular mechanisms involved in the root colonization are under study nowadays and advancement in the molecular and genomic tools offers new possibilities for improving the selection, characterization, and management of biological control. Development of proteomics and functional genomics will be helpful to determine and follow expression of crucial genes of BCAs during mass production, formulation, and application. Transformation of BCAs by inserting genes that improve the tolerance of antagonists to abiotic stresses, such as increased tolerance or resistance to cold, heat, drought, high salinity, heavy metal rich soils, or acidic soils, etc., could be another exciting and challenging task and may provide with better opportunities to implement the concept of biocontrol in the field under the dynamic natural environments.

Acknowledgements This work was supported in part by grant received in the form of Silver Jubilee Fellowship to BNJ from Madhya Pradesh Council of Science and Technology, Bhopal. The authors are thankful to Dr. Shipra Singh, DST Young Scientist for critical reading of the manuscript and Mr. Sandeep Saini, Research Fellow, Department of Biotechnology and Bioinformatics Centre, Barkatullah University, Bhopal for help in preparation of the manuscript.

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M. Senthilkumar, R. Anandham, M. Madhaiyan, V. Venkateswaran, and Tongmin Sa

3.1 Introduction

Both aerial and subterranean plant organs are constantly exposed to intimate contacts with a plethora of microorganisms, including members of phyla as diverse as viruses, bacteria, oomycetes, fungi, and eukaryotic protozoans. The outcome of interactions between plants and microbes can be neutral, detrimental, or even beneﬁcial for the photoautotrophic organisms. Disadvantageous encounters typically manifest themselves as diseases, which in extreme cases can result in full collapse of plant tissues (Volker and Panstruga 2005). By contrast, benign contacts usually give rise to symbiotic relationships that typically support the plant’s nitrogen metabolism and mineral uptake. Surprisingly, although many microbes have a principal phytopathogenic potential, the majority of interactions between plants and microbes remain macroscopically symptomless. Beneﬁcial plant–microbe interactions that promote plant health and development have been the subject of considerable study. Endophytes, microorganisms that reside in the tissues of living plants, are potential sources of novel natural products for exploitation in medicine, agriculture, and industry but these are relatively unstudied. It is noteworthy that, of the nearly 300,000 plant species that exist on earth, each individual plant is host to one or more endophytes. Only a few of these plants have been completely

M. Senthilkumar Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India R. Anandham Department of Agricultural Microbiology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Madurai 625 104, Tamil Nadu, India M. Madhaiyan and T. Sa (*) Department of Agricultural Chemistry, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea e-mail: [email protected] V. Venkateswaran Ministry of Food Processing Industries, Government of India, New Delhi 110 049, India

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 61 DOI 10.1007/978-3-642-18357-7_3, # Springer-Verlag Berlin Heidelberg 2011 62 M. Senthilkumar et al. studied relative to their endophytic biology. Consequently, the opportunity to ﬁnd new and interesting endophytic microorganisms among myriads of plants in different settings and ecosystems is great.

3.2 “Endophytes” What It Means?

For more than 50 years, bacteria have been observed to exist inside plants without causing apparent disease symptoms (Tervet and Hollis 1948). Various reports indicate that such bacteria exist in a variety of tissue types within numerous plant species, suggesting a ubiquitous existence in most plants. Taken literally, the word endophyte means “in the plant” (endon Greek, within; phyton, plant). Since the discovery of endophytes in Darnel, Germany, in 1904 (Tan and Zou 2001), various investigators have defined endophytes in different ways, which is usually dependent on the perspective from which the endophytes were being isolated and subsequently examined. Bacon and White (2000) gave an inclusive and widely accepted definition of endophytes as “microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effects.” While the symptomless nature of endophyte occupation in plant tissue has prompted focus on symbiotic or mutualistic relationships between endophytes and their hosts, the observed biodiversity of endophytes suggests that they can also be aggressive saprophytes or opportunistic pathogens. The usage of the term is as broad as its literal definition and spectrum of potential plant hosts and inhabitants includes bacteria (Kobayashi and Palumbo 2000), fungi (Stone et al. 2000), algae (Peters 1991), and insects (Feller 1995). Any organ of the host can be colonized. The endophytic partners and their relationships to each other vary. There are pathogenic endophytic algae (Bouarab et al. 1999), parasitic endophytic plants (Marler et al. 1999), mutualistic endophytic bacteria (Chanway 1996), ectomycorrhizal helper bacteria (Founoune et al. 2002), as well as endophytic bacteria in pathogenic and commensalistic symbioses (Sturz and Nowak 2000). Both fungi and bacteria are the most common microbes existing as endophytes (Strobel and Daisy 2003). It seems that other microbial forms, e.g., mycoplasmas and archaebacteria, exist in plants as endophytes, but no evidence for them has yet been reported. Kloepper et al. (1992) called bacteria found within tissues internal to the epidermis as endophytes. However, quiescent endophytic bacteria can become pathogenic under certain conditions and within different host genotypes (Misaghi and Donndelinger 1990). James and Olivares (1997) adjusted the definition and stated that all bacteria that colonize the interior of plants, including active and latent pathogens, can be considered to be endophytes. Considering all bacteria that colonize the interior of plants, one should also take into account those bacteria that reside within living plant tissues without doing substantive harm or gaining benefit other than securing residency (Kado and Kado 1992), as well as those bacteria that establish endosymbiosis with the plant, whereby the plant receives an ecological benefit from the presence of the symbiont (Quispel 1992). 3 Endophytic Bacteria: Perspectives and Applications 63

The definition of endophytic bacteria should, in accordance with the definition of endomycorrhizal fungi, also include bacteria that reside in the cortex of the root. These subdefinitions may provide an operational overview of what is considered to be an “endophyte,” and consequently this might be regarded as the most general definition for which the term “endophyte” stands.

3.3 Sources of Endophytic Bacteria

Two of the most frequently raised questions in connection with endophytic bacteria are what is the origin of endophytes and how do they enter plant tissues in nature? The sources of endophytes are various. These can appear to originate from seeds (McInory and Kloepper 1995), vegetative planting material (Sturz 1995), rhizosphere soil (Patriquin et al. 1983), and the phylloplane (Beattie and Lindow 1995). The importance of seeds as a source of endophytic bacteria is still controversial. The role of the spermosphere as a source of bacterial endophytes is also evident by observations that bacterial endophytes introduced as seed treatments colonized the internal tissues of root radical newly emerged from the seed coat (Musan et al. 1995). In detail, endophytic colonization appeared to begin with the migration of bacteria through the germination slit and into the starchy endosperm, from which these bacteria colonized the radicle and coleoptile and finally spread systematically through the plant. Besides seeds and spermosphere, several observations favor the rhizosphere soil as the primary source for endophytic colonization. Axenic potatoes planted into field soil mainly harbored genera of commonly found soil saprophytes (De Boer and Copeman 1974). Comparing the internal and external bacterial communities of cucumber, cotton, and potato, almost all endophytic bacteria were found also in the rhizosphere, thus supporting the hypothesis that there is a continuum of root-associated microorganisms from the rhizosphere to rhizoplane to epidermis and cortex (Kloepper et al. 1992). Statistical analysis of the bacterial diversity from rhizosphere (zone outside roots) and endorhiza (root interior) indicated that the initial composition of the endorhiza was dependent on the rhizosphere community. However, the endorhiza community quickly differentiated from that of the rhizosphere with fewer genera present, suggesting that the endorhiza is a distinct habitat from the rhizosphere. Rosenblueth and Martıńez-Romero (2006) published a list of endophytic bacteria reported in various plant species (Table 3.1) but the list continues.

3.4 Modes of Entry of Endophytes

With the exception of seed transmitted bacteria, which are already present in the plant, potential endophytes must ﬁrst colonize the root surface prior to entering the plant. Potential internal colonists ﬁnd their host by chemotaxis, electrotaxis, or 64 M. Senthilkumar et al.

Table 3.1 Reported putative endophytes in cultivated plants (Rosenblueth and Martıńez-Romero 2006) Endophytes Plant species References a-Proteobacteria Azorhizobium Rice Engelhard et al. (2000) caulinodans Azospirillum brasilense Banana Weber et al. (1999) Azospirillum Banana, pineapple Weber et al. (1999) amazonense Bradyrhizobium Rice Chantreuil et al. (2000) japonicum Gluconacetobacter Sugarcane, coffee Cavalcante and Dobereiner€ (1988); diazotrophicus Jimeńez-Salgado et al. (1997) Methylobacterium Citrus plants Araujo et al. (2002) mesophilicuma Methylobacterium Scots pine, citrus plants Araujo et al. (2002); Pirttil€a et al. (2004) extorquens Rhizobium Rice Yanni et al. (1997) leguminosarum Rhizobium Carrot, rice Surette et al. (2003) (Agrobacterium) radiobacter Sinorhizobium meliloti Sweet potato Reiter et al. (2003) Sphingomonas Rice Engelhard et al. (2000) paucimobilisa b-Proteobacteria Azoarcus sp. Kallar grass, rice Engelhard et al. (2000); Reinhold-Hurek et al. (1993) Burkholderia pickettiia Maize McInory and Kloepper (1995) Burkholderia cepaciab Yellow lupine, citrus plant Araujo et al. (2001); Barac et al. (2004) Burkholderia sp. Banana, pineapple, rice Weber et al. (1999); Engelhard et al. (2000) Chromobacterium Rice Phillips et al. (2000) violaceuma Herbaspirillum Sugarcane, rice, maize, Olivares et al. (1996); Weber et al. (1999) seropedicae sorghum, banana Herbaspirillum Sugarcane Olivares et al. (1996) rubrisubalbicans g-Proteobacteria Citrobacter sp. Banana Martıńez et al. (2003) Enterobacter spp. Maize McInory and Kloepper (1995) Enterobacter sakazakiia Soybean Kuklinsky-Sobral et al. (2004) Enterobacter cloacaea Citrus plants, maize Araujo et al. (2002); Hinton and Bacon (1995) Enterobacter Soybean Kuklinsky-Sobral et al. (2004) agglomeransa Enterobacter asburiae Sweet potato Asis and Adachi (2003) Erwinia sp. Soybean Kuklinsky-Sobral et al. (2004) Escherichia colib Lettuce Ingham et al. (2005) Klebsiella sp. Wheat, sweet potato, rice Engelhard et al. (2000); Iniguez et al. (2004); Reiter et al. (2003) Klebsiella pneumoniaeb Soybean Kuklinsky-Sobral et al. (2004) (continued) 3 Endophytic Bacteria: Perspectives and Applications 65

Table 3.1 (continued) Endophytes Plant species References Klebsiella variicolab Banana, rice, maize, Rosenblueth et al. (2004) sugarcane Klebsiella terrigenaa Carrot Surette et al. (2003) Klebsiella oxytocab Soybean Kuklinsky-Sobral et al. (2004) Pantoea sp. Rice, soybean Kuklinsky-Sobral et al. (2004); Verma et al. (2004) Pantoea agglomerans Citrus plants, sweet potato Araujo et al. (2001, 2002); Asis and Adachi (2003) Pseudomonas Marigold (Tagetes spp.), Sturz and Kimpinski (2004); Surette chlororaphis carrot et al. (2003) Pseudomonas putidaa Carrot Surette et al. (2003) Pseudomonas Carrot Surette et al. (2003) fluorescens Pseudomonas Soybean Kuklinsky-Sobral et al. (2004) citronellolis Pseudomonas synxantha Scots pine Pirttil€a et al. (2004) Salmonella entericab Alfalfa, carrot, radish, Cooley et al. (2003); Guo et al. (2002); tomato Islam et al. (2004) Serratia sp. Rice Sandhiya et al. (2005) Serratia marcescensa Rice Gyaneshwar et al. (2001) Stenotrophomonasa Dune grasses (Ammophila Dalton et al. (2004) arenaria and Elymus mollis) Firmicutes Bacillus spp. Citrus plants Araujo et al. (2001, 2002) Bacillus megaterium Maize, carrot, citrus plants Araujo et al. (2001)McInory and Kloepper (1995); Surette et al. (2003) Clostridium Grass Miscanthus sinensis Miyamoto et al. (2004) Paenibacillus odorifer Sweet potato Reiter et al. (2003) Staphylococcus Carrot Surette et al. (2003) saprophyticusb Bacteroidetes Sphingobacterium sp.a Rice Phillips et al. (2000) Actinobacteria Arthrobacter Maize Chelius and Triplett (2000) globiformis Curtobacterium Citrus plants Araujo et al. (2002) flaccumfaciens Kocuria varians Marigold Sturz and Kimpinski (2004) Microbacterium Marigold Sturz and Kimpinski (2004) esteraromaticum Microbacterium Maize Zinniel et al. (2002) testaceum Mycobacterium sp.b Wheat, Scots pine Conn and Franco (2004); Pirttil€a et al. (2004) Nocardia sp.b Citrus plants Araujo et al. (2002) Streptomyces Wheat Coombs and Franco (2003) aOpportunistic human pathogenic bacteria bCommon human pathogenic bacteria 66 M. Senthilkumar et al. accidental encounter. Motility of beneficial associative rhizosphere bacteria has been described for several bacteria such as Alcaligenes faecalis, Azospirillum brasilense, and Pseudomonas fluorescens (Bashan 1986; You et al. 1995). In general, entry into a plant tissue can be via the stomata, lenticels, wounds (including broken trichomes), areas of emergence of lateral roots and germinating radicles. However, the main entry for endophytic bacteria appears to be through wounds that naturally occur as a result of plant growth or through root hairs and at epidermal conjunctions. Several authors have reported extensive colonization of the secondary root emergence zone (site of root branches) by bacterial endophytes. Because of breaks in the endodermis at these points, bacteria colonizing the cortex can be observed to extend to and across the epidermis into the vascular tissue. Plant wounds, in general, induced either by biotic or abiotic factors, are ubiquitous in any agroecosystem and are probably a major factor for bacterial entrance. Besides providing entry avenues, wounds also create favorable conditions for the approach- ing bacteria by allowing leakage of plant exudates, which serve as a food source for the bacteria (Hallmann et al. 1997). Wounds and lateral roots are not, however, absolutely required for entrance of endophytic bacteria. Several workers have proposed that the enzymatic degradation (cellulolytic and pectinolytic enzymes) of plant cell walls by these bacteria was only observed when they colonized the root epidermis but never after colonizing intercellular spaces of root cortex. These results suggested that endophyte induced production of cellulose and pectinase was only for penetration into the host plant. Although these observations demonstrate the possibility of active penetration mechanisms for some endophytic bacteria, very little is known about the origin and regulation of these enzymes. Nevertheless, active penetration is still controversial and the fact that soil bacteria show a higher frequency of hydrolytic enzymes than xylem- inhabiting bacteria suggests that it is unlikely for systemic endophytic bacteria to gain plant entry primarily via production of hydrolytic enzymes (Bell et al. 1995). Benhamou et al. (1996) concluded that hydrolytic enzymes might only be produced by endophytes during the early invasion phase and not after residing in plant tissues. In addition, one must differentiate between bacterial enzyme production in vitro and in planta. However, constitutive release of plant cell degrading enzymes by endophytic bacteria is undesirable as this would confer plant pathogenicity (Collmer et al. 1982). Therefore, endophytic bacteria must have some regulatory mechanisms to specifically regulate their enzyme production in terms of quantity and time of expression, a fact well known for Xanthomonas campestris where virulent and avirulent strains differ in their cellulose activity (Knosel and Garber 1967). Although the root zone offers the most obvious site of entry for many endophytes, entry may also occur at sites on aerial portions of plants. Sharrock et al. (1991) suggested that, in some cases, endophytic populations within fruit may arise by entry through flowers. Endophyte penetration is also believed to occur through natural openings on the leaves (e.g., stomata) or through stem lenticels (Kluepfel 1993). A completely different way of penetration is described by Ashbolt and Inkerman (1990) for sugarcane, via the mealybug, and by Kluepfel (1993), via a range of different insects. 3 Endophytic Bacteria: Perspectives and Applications 67

3.5 Endophytic Movement Inside the Plant

Once inside the plant tissue, endophytes either remain localized in a specific plant tissue like the root cortex or colonize the plant tissues systematically by transport through the conducting elements or apoplast (Hurek et al. 1994). The prevailing thought that systematic colonization of the vascular system from cortical tissues is limited appears to be due to the belief that the endodermis represents a physical barrier to the apoplastic movement of bacterial endophytes into the vascular tissue (Kloepper et al. 1992). This theory is based upon the research demonstrating that the root endodermis functions physiologically to regulate chemical flow into the vascular region and that casparian strip, when intact, limits apoplastic movement of solutes from the cortex to the stele. Therefore, it was believed that endophytes present in the root cortex could not traverse the endodermis to enter the vascular system. However, as discussed previously, the endodermis is not an unbroken barrier, as epidermal disruption occurs at the sites of secondary root formation, providing an apoplastic route to the root stele, which can be visualized by fluorescent dyes (Peterson et al. 1981). Bacterial endophytes may also follow this same path to colonize the vascular tissue. The endodermal barrier can also be surpassed bacterial entrance via the undifferentiated cells of the root tissues (Mahaffee and Kloepper 1994). Once inside the plant tissue, endophytic bacteria remain localized in a specific plant tissue, such as the root cortex, or colonize the plant systematically by transport or active migration through the conducting elements or the apoplast (Hurek et al. 1994; James et al. 1994; Hallmann et al. 1997; Patriquin and Dobereiner€ 1978). The different mechanisms of distribution might be due to interactions with other bacteria or to the different requirements of each microorganism that allows them to inhabit different niches, represented by tissue and, more specifically, by the intercellular spaces inside each tissue.

3.6 Endophytic Colonization in Plant Tissues

The high population density of endophytes in carrot crowns indicates the preferential colonization of these tissues by bacterial endophytes. Surette et al. (2003) speculated that the greater abundance of colony-forming units (CFU) in the crown tissues may perhaps be due to the higher sugar content of crowns which can vary from 6 to 8% compared to the slender slicer carrot which varies from 3 to 4%. Fisher et al. (1992) found similar accumulations in the base of corn stems. Preferential colonization of crown tissues may also be a function of proximity to the soil surface. In this instance, bacterial colonization may be inﬂuenced by environmental factors such as oxygen concentration (Sitnikov et al. 1995). Higher oxygen concentrations for the multiplication and survival of the more aerobic bacterial species would be found in host tissues that were located nearer the soil surface. 68 M. Senthilkumar et al.

The fact that colonization is especially abundant in root tissue may reflect the fact that the root is the primary site where endophytes gain entry into plants. As mentioned earlier, except for bacteria transmitted through seeds other potential endophytes must first colonize the root surface prior to entering the plant. This might explain the close relationship between endophytic and rhizosphere colonizing bacteria, that is, many facultative endophytic bacteria can also survive as rhizosphere bacteria. Potential internal colonists find their host by chemotaxis, electrotaxis, or accidental encounter. The fact that bacteria seem to be capable of colonizing the internal tissues of plants could confer an ecological advantage over bacteria that can only colonize plants epiphytically. The internal tissues of plants are thought to provide a more uniform and protective environment for microorganisms than plant surfaces, where exposure to extreme environmental conditions, such as temperature, osmotic potentials, and ultraviolet radiation, is a major factor limiting long-term bacterial survival. The plant interior could also be a hideout from grazing by soil protozoa. However, there are probably other limiting factors that must be overcome when establishing populations in the internal tissues of plants. Endophytic colonization of the shoot and root seem to differ. For most of the endophytes that have been investigated to date, colonization of the shoot is either intracellular and then confined to individual cells or intercellular but localized. Colonization of roots by endophytes, on the other hand, is usually extensive but may also be inter- or intracellular. A criterion for some endophytes to colonize the plant is these must find their way through cracks formed at the emergence of lateral roots or at the zone of elongation and differentiation of the root. The evidence for the penetration and root colonization by the rhizosphere bacteria came from the recovery of rhizosphere bacterial populations from the endodermis and root cortex of plants (Quadt-Hallman et al. 1997). The means of infection by the endophyte into the host plants in most cases is not clearly understood, where no specialized structures such as root nodules are formed as in legume–rhizobia symbioses. According to the model proposed by Darbyshire and Greaves (1973) and supported by Old and Nicolson (1978), the root cortex becomes an integral part of the soil–root microbial environment, resulting in a continuous apoplastic pathway from the root epidermis to the shoot which favors the movement of microorganisms into the xylem (Peterson et al. 1981). Thus, a continuum of root-associated microorganisms, which are able to inhabit the rhizosphere, the root cortex, and other plant organs, exists (Kloepper et al. 1992). The major reason for better root colonization in contrast to the above-ground organs of the plants may be due to the fact that roots are in intimate contact with an environment harboring many different mainly degradatively active microorganisms that can potentially provide the plants with water and essential minerals. Hence, a mutualistic interaction has been developed between microorganisms and the roots, because the roots as a natural carbon sink of the plants can supply dual and multiorganism symbioses with nutrients. In return the host can be supplied with minerals and water by the microorganisms (Schulz and Christine 2005). Another important fact is that the organisms occupying the endosphere are not accidentally there but most probably have been selected for this niche by the plant, because of the beneficial effects they offer their host and their 3 Endophytic Bacteria: Perspectives and Applications 69 abilities to resist the effects of plant defense products. The energy lost by the plant in the production of endophyte biomass is in all likelihood adequately compensated for by the improvements in plant health derived from the presence of mutualistic microorganisms. It has also been proposed that cell wall-degrading enzymes, such as endogluconase, polygalacturonase, pectate lyase, cellulose, and pectinolytic enzymes, produced by endophytes are involved in the infection process (Hallmann et al. 1997; Kovtunovych et al. 1999; Compant et al. 2005b). Some endophytic plant growth promoting rhizobacteria (PGPR) may utilize other organisms as vectors to gain access to apoplastic spaces in their host. For example, both the pink sugarcane mealybug (Saccharicoccus sacchari) (Franke et al. 2000) and arbuscular mycorrhizae (Isopi et al. 1995) have been implicated in the infection of host plants by the endophytic diazotroph, Gluconacetobacter diazotrophicus. Endophyte colonization has also been visualized with the use of the b-glucuroni- dase (GUS) reporter system. A GUS marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedlings. GUS staining was most intense on coleop- tiles, lateral roots, and also at some of the junctions of the main and lateral roots (James et al. 2002). This study by James et al. (2002) showed that endophytes entered the roots through cracks at the point of lateral root emergence. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. The xylem vessels in leaves and stems were also colonized. Currently, one can only speculate on the reasons for these different colonization patterns, because many factors may be involved, e.g., anatomical differences, source–sink relationships, and differences in permeability or nutrients supplied by the micropartner or by the host (Schulz and Christine 2005).

3.7 Ecology of Endophytic Bacteria

Abundant microorganisms live both within and on crop plants. They possess functional diversity – the diversity of metabolism and interspecific relationships. The crop plant and its microorganisms constitute a holistic system. The complex relationships between plant and microorganisms and among the microorganisms themselves constitute the key to plant health and growth. Moreover, selective utilization of the functional diversity of microbial species provides us with several opportunities to improve agricultural production. Roots, stems, leaves, flowers, and fruits of plants all provide suitable habitats for microbial populations (Campbell 1989; Atlas and Barthar 1993). Plants can be considered to be complex micro- ecosystems where different habitats are exploited by a wide variety of bacteria (McInroy and Kloepper 1994). These habitats are not only represented by plant external surfaces, where epiphytic bacteria predominate, but also by internal tissues, where many microorganisms penetrate and survive. Inside the plant micro- ecosystem, different microbial species, both bacterial and fungal (Fisher et al. 1992) 70 M. Senthilkumar et al. are able to interact and establish an equilibrium. Some of these microorganisms can be considered to be dominant species (Van Peer et al. 1990) and may be represented by those that are most frequently, and in large numbers, isolated from the host plant. In addition to the dominants, there is a large variety of species that cannot be isolated easily because of their low numerical consistency. These are considered to be rare species. Endophytic bacteria have been isolated from both monocotyledonous and dicotyledonous plants, ranging from woody tree species, such as oak (Brooks et al. 1994) and pear (Whitesides and Spotts 1991), to herbaceous crop plants, such as sugar beets (Jacobs et al. 1985) and maize (Fisher et al. 1992; Gutierrez-Zamora and Martinez-Romero 2001). Diversity associated with bacterial endophytes exists, not only in the plant species colonized but also in the colonizing bacterial taxa. Plants can be colonized simultaneously by a large variety of endophytic bacteria. The variation in bacteria that has been reported as endophytes spans a significant range of Gram-positive and Gram-negative bacteria and include genera such as Acidovorax, Acinetobacter Actinomyces, Aeromonas, Afipia, Agrobacterium, Agromonas, Alcaligenes, Alcanivorax, Allorhizobium, Alteromonas, Aminobacter, Aquaspirillum, Arthrobacter, Aureobacterium, Azoarcus, Azomonas, Azorhizobium, Azotobacter, Azospirillum, Bacillus, Beijerinckia, Blastobacter, Blastomonas, Brachymonas, Bradyrhizobium, Brenneria, Brevundimonas, Burkholderia, Chelatobacter, Chromobacterium, Chryseomonas, Comamonas, Corynebacterium, Delftia, Derxia, Devosia, Enterobacter, Flavimonas, Flavobacterium, Flexibacter, Frankia, Halomonas, Herbaspirillum, Matsuebacter, Mesorhizobium, Moraxella, Nevskia, Nocardia, Ochrobactrum, Pantoea, Pectobacterium, Phenylbacterium, Phyllobacterium, Photobacterium, Porphyrobacter, Pseudoalteromonas, Pseudo- monas, Psychrobacter, Ralstonia, Renibacterium, Rhizobacter, Rhizobium, Rhizomonas, Rhodanobacter, Rhodococcus, Shewanella, Sinorhizobium, Sphingo- bacterium, Sphingomonas, Spirillum, Stenotrophomonas, Streptomyces, Thauera, Variovorax, Vibrio, Xanthomonas, Xylella, Zoogloea, Zymobacter, Zymomonas,or members of the group of the pink-pigmented facultatively methylotrophic bacteria, such as Methylobacterium. Even though it remains difficult to compare earlier and more recent studies that identify bacteria, certain trends are apparent with predominant bacterial types isolated as endophytes (Kobayashi and Palumbo 2000).

3.8 Endophytes and Their Role in Plants

From literatures available, endophytic bacteria were shown to have beneficial effects on plant growth and health, and the main modes of action described are diazotrophy, production of plant growth hormones and antifungal compounds, and induced systemic resistance (Benhamou et al. 1996; Hallmann et al. 1997). Inter- actions of endophytic bacteria with their host plants are not only beneficial for the host, but provide enough nutrients for the endophytes to extensively colonize the 3 Endophytic Bacteria: Perspectives and Applications 71 host’s roots and potentially for growth in the rhizosphere, which in turn could improve the host’s mineral and nutrient supply as in the case of mycorrhizal fungi. Mutualistic endophytic associations have been reported more frequently in associations with roots than with the aerial plant organs, may be due to the fact that colonization of the aboveground organs is frequently localized, whereas that of the roots is more often extensive and sometimes systemic (Stone et al. 2000). Most of the endophytes that have been investigated to date revealed colonization of the shoot is either intracellular and then confined to individual cells or intercellular but localized. On the other hand, colonization of roots by endophytes is usually extensive, but may also be inter- or intracellular. Specialized structures that are presumed to improve the exchange of metabolites have been observed in both shoots and roots. An endophyte cannot improve the nutrient status of the photosynthetic organs directly. Thus, in general, a mutualistic systemic interaction with the roots of a putative host is more probable than with the aboveground organs. And, recently a molecular basis for mutualistic interactions of roots with microorganisms was found (Imaizumi-Anraku et al. 2005). The aboveground organs – leaf community is colonized by endophytes in the internal tissues and epiphytes on the surface. These colonizers are of bacterial or fungal community and may be of three types: (1) pathogens of another host that are nonpathogenic in their endophytic relationship, (2) nonpathogenic microbes, and (3) pathogens that have been rendered nonpathogenic but still capable of colonization by selection methods or genetic alteration (Imaizumi-Anraku et al. 2005). Endophytic bacteria in a single plant host are not restricted to a single species but comprise several genera and species. So far, no studies have indicated endophytic communities’ interaction inside the plants, and it has been speculated that beneficial effects are the combined effect of their activities. The population density of endophytes is highly variable, depending mainly on the bacterial species and host genotypes and also in the host growth stage, inoculum density, and environmental conditions. Generally, bacterial populations are larger in roots and decrease in the stems and leaves (Lamb et al. 1996). Natural endophyte concentrations can vary À1 between 2.0 and 6.0 log10 CFU g for alfalfa, sweet corn, sugar beet, squash, cotton, and potato, as described by Kobayashi and Palumbo (2000). Similar results were obtained for endophytic bacteria inoculated by root or seed drenching, with À1 the population levels reaching between 3.0 and 5.0 log10 CFU g of plant tissue for tomato and potato (Kobayashi and Palumbo 2000). Population densities of bacterial endophytes have been shown to be greatest in plant roots (McInory and Kloepper 1995) with densities ranging from 104 to 106 CFU gÀ1 fresh weights in cotton and sweet corn roots. In potato, the average bacterial densities over two seasons were 5.6 Â 107 in the rhizosphere, 2.2 Â 106 in the endorhiza, and 5.2 Â 105 phyllo- À sphere and were lowest in the endosphere 3.9 Â 104 CFU g 1 fresh weight basis (Berg et al. 2005). The total number of endophytes present at a particular time is being controlled by the plant and environment (Hallmann et al. 1997). Recent papers indicate that the phyllosphere is much more diverse than previously thought. Using molecular techniques, studies by Yang et al. (2001) evaluating cultivated citrus, and Lambais et al. (2006) evaluating nine tree species in an ancient 72 M. Senthilkumar et al. subtropical forest, determined a high level of diversity with many unculturable and often unidentifiable species. To learn more about the functions of endophytes inside plants and the conditions to which they are exposed there, it will be crucial to develop appropriate methods to localize bacterial gene expression in plants. To study endophytic gene expression in situ in uninoculated natural systems, immunolocalization of specific enzymes (such as nitrogenase) or in situ hybridization studies (Hurek et al. 1994) have recently been introduced. The interrelationship and the ecological role of these organisms and how these functions are related to the metabolic capabilities of microbe and plant are just now beginning to be studied.

3.9 Beneﬁcial Effects on Plant

There have been vast studies describing potential advantage of plant-associated bacteria as agents inducing plant growth and maintaining soil and plant health. Because of various factors such as small size, diversity, and culturable nature, the endophytes are unnoticed in the plants. This makes plant physiologists to consider the plants as a single organism. As a result, the latter’s crucial roles were sometimes overlooked or unnoticed. Endophytic bacteria ubiquitously inhabit most plant species, and have been isolated from a variety of plants. Among the plant-associated microorganisms, endophytic bacteria are regarded as a largely unexplored potential resource for the discovery of isolates with novel antibiotic substances and PGP traits (Lodewyckx et al. 2002; Rosenblueth and Martı´nez-Romero 2006).

3.10 Endophytic Diazotrophs

Much progress has been made in the area of biological nitrogen fixation (BNF) with nonleguminous plants over the last 10 years. Several new species of nitrogen-fixing bacteria have been identified but special attention has been given to endophytic diazotrophs. The concept of diazotrophic endophytes was introduced to the area of BNF by Doobereiner€ (1992) although the term endophyte was coined more than 150 years ago by Leveille (1846) to define a special class of fungi living inside plant tissues. The term was later extended to bacteria by Chanway (1996) who also observed that some bacteria colonize the interior of plant tissues without causing disease symptoms. Although there are several definitions of bacterial endophytes, the role of the endophytic diazotrophs in association with graminaceous plants is still not yet well understood. Splitting the term diazotrophic endophytes into facultative and obligate was suggested to distinguish, respectively, strains that are able to colonize both the surface and root interior and to survive well in soil from those that do not survive well in soil but colonize the root interior and aerial parts. 3 Endophytic Bacteria: Perspectives and Applications 73

In nature, legumes benefit directly from biologically fixed nitrogen, provided that they are in symbiotic association with root-nodulating bacteria, such as Rhizobia. However, nonleguminous plants, most of which belong to the Gramineae, do not have this symbiosis (Hurek and Reinhold-Hurek 2003). Studies of plant bacteria that might make reliable contributions to the growth of nonlegumes such as cereals revealed that there are groups of bacteria which intimately associate with nonleguminous crops called diazotrophic endophytes. In general, the term “endophyte” includes all the microorganisms that are capable of colonizing the inner tissues of plants. The term endophyte was first introduced to the area of nitrogen fixation research associated with graminaceous plants by Doobereiner€ (1992). Among nonleguminous plants, several diazotrophic endophytes have been isolated and characterized as nitrogen fixing endophytes including Acetobacter (Sevilla et al. 2001), Azoarcus spp. (Reinhold-Hurek and Hurek 1998), Serratia spp. (Gyaneshwar et al. 2001), Burkholderia spp. (Baldani et al. 2000), and Herbas- pirillum spp. (Elbeltagy et al. 2001; Gyaneshwar et al. 2002). The interaction of endophytic diazotrophic bacteria with plants has been extensively studied through the inoculation of sugarcane and rice plants grown under sterile conditions followed by microscopic analysis. These bacteria enter the plant tissues primarily through the root zone. They colonize the spaces at the junctions of the lateral roots and the intercellular spaces of the root epidermis (Roncato-Maccari et al. 2003) and penetrate deeply to enter the internal tissues of the roots and basal stem (James et al. 2000; Zakria et al. 2007) and colonize the aerial parts by entering in the xylem tissues of the roots and stem (Hurek et al. 1994; James et al. 2000). The nitrogen-fixing bacteria are reported to provide biologically fixed nitrogen to their hosts, but the amounts of nitrogen that they supply are highly variable, for example, in rice it ranged from 0 to 36% (Malarvizhi and Ladha 1999; Shrestha and Ladha 1996) and in sugarcane from 4 to 70% of the host plant’s nitrogen requirement (Yoneyama et al. 1997). The variation in the amount of fixed nitrogen is believed to depend on variety, plant stage, endophyte strain, inoculation method, and environmental conditions. Several diazotrophic endophytes have been isolated from rice and they can provide fixed N (Gyaneshwar et al. 2001, 2002; Verma et al. 2001). Many endophytes appear to have a broad host range. For example, H. seropedicae has been found in a variety of crops, including maize, sorghum, sugarcane, and other Gramineae plants (Baldani et al. 1986; Olivares et al. 1996). This indicates that an endophyte isolated from one host family member can colonize other nonhost members of the same family, which is suggestive of host nonspecificity in Gramineae plants. Endophytes also show host nonspecificity among families. Burkholderia sp. isolated from the onion can colonize grapes (Compant et al. 2005b), potatoes, and vegetables (Nowak et al. 1995). However, interaction studies involving rice and endophytes isolated from other host families have not been done in an efficient way, and the issue needs to be addressed. The nitrogen supplied by indigenous endophytic N2-fixing bacteria is inadequate for crop growth (Nishiguchi et al. 2005). Asis et al. (2000) isolated several putative strains of G. diazotrophicus and Herbaspirillum from sugarcane, and reported that only 40% of these strains had acetylene reduction activity. Thus, there is potential 74 M. Senthilkumar et al. to introduce nonindigenous endophytes that can contribute to plant growth. There- fore, studies of the interactions among the host, endophyte, and environmental conditions are very important in defining the nitrogen-fixing associations between hosts and endophytes. Researchers are interested in finding bacterial strains with enhanced PGP capabilities. As new beneficial bacterial strains are identified, the delivery of these strains to specific plant tissues becomes an issue. To facilitate the use of endophytic bacteria in practical agronomic production, reliable and practical methods of inoculum delivery must be developed. The ability of an endophyte to fix atmospheric nitrogen within a host has been proved using different approaches: acetylene reduction assay, 15N isotope dilution 15 15 experiments, N2 reduction assays, or N natural abundance assays. These experiments have conclusively shown that an increase in the host–plant N content as high as 30–45 mg of N per plant (6-week-old seedlings) in rice and to 170 kg of N per hectare per year in sugarcane was a result of BNF (Iniguez et al. 2004). Due to their ability to colonize the root surface and interior of many cereals and forage grasses, the first report showing the presence of Azospirillum in cells of the cortex, in intercellular spaces between the cortex and endodermis, and in the xylem cells of maize roots by applying the tetrazolium reduction staining technique (Patriquin and Dobereiner€ 1978). Many inoculation experiments with Azospirillum spp. have shown a positive effect of the bacteria on crops (Boddey and Dobereiner€ 1995) but still a debate remains about the exact mode of action by which endophytic diazotrophs contribute to the nitrogen accumulated in the plants. Effects of PGP substances (Zimmer et al. 1988), nitrogen fixation per se, or the ability of the bacterial nitrate reductase to help in the incorporation of the nitrogen assimilated from soil by the plant have been demonstrated (Ferreira et al. 1987). Despite the different mechanisms exerted by Azospirillum in association with graminaceous plants, increases in the range of 5–30% in yield have been observed in several inoculation experiments (Okon and Labandera-Gonzalez 1994). Systematic studies by various workers in Brazil over the years led to the observation that some sugarcane varieties grown for decades or even a century do not show any decline in the soil N reserve or yield despite the supply deficit of N (Boddey et al. 1995). In 15 the wild rice variety Oryza officinalis, acetylene reduction and N2 gas incorporation were deployed to determine the in planta nitrogen fixation after inoculation with endophytic Herbaspirillum sp. strain B501 was 381% as compared to 0.4% of the uninoculated plant, which proved the role of nitrogen fixation by Herbaspi- rillum sp. strain B5 (Elbeltagy et al. 2001). Nitrogen-fixing Burkholderia vietnamiensis (Gillis et al. 1995) has been isolated from the rhizosphere of rice in Vietnam. It was the first species of Burkholderia reported to fix nitrogen. More recent reports suggest that many species of this genus actually contain diazotroph strains. A phytohormone-producing diazotroph Enterobacter of sugarcane inoculated to roots of micropropagated sugarcane assimilated 29% of nitrogen by atmospheric fixation (Mirza et al. 2001). In all the above cases, the bacteria that colonized and invaded the plant upon inoculation contributed the fixed nitrogen (Boddey et al. 1995; Oliveira et al. 2002). The crucial for the understanding of functions of diazotrophic endophytes is the long-standing question, whether the 3 Endophytic Bacteria: Perspectives and Applications 75 host plants profit from nitrogen fixation. This has been successfully demonstrated for Azoarcus sp. BH72 and its host Kallar grass (Leptochloa fusca L. Kunth). The comparison of wild type and a nif mutant showed a significant gain of plant nitrogen after inoculation with the wild type; total N balance and natural 15N abundance corroborated that fixed nitrogen was contributed, moreover nifH-mRNA of strain BH72 was found to be predominant in plant roots (Hurek et al. 2002). Thus, grass endophytes are able to supply fixed nitrogen to the plant, as has also been demonstrated for G. diazotrophicus in sugar cane (Sevilla et al. 2001). This makes the Azoarcus sp. – grass system a highly interesting model system for a novel type of plant–microbe interaction. If several diazotrophs are found on one plant, it becomes difficult to judge their relative significance. In the case of Kallar grass, different nitrogen-fixing bacteria associated with distinct root zones (Reinhold et al. 1986). This result suggests that different diazotrophs might be adapted to colonize different root zones of their host plants and to contribute there to the association. It may be considered that the dominant diazotrophs, i.e., those occurring in highest numbers, to be the most important ones for an association, because they may be able to make a significant contribution to the nitrogen supply of the plant.

3.11 Endophyte’s Physiological Role

Plants infected with endophytes are often healthier than endophyte-free ones (Waller et al. 2005). This effect may be partly due to the endophytes’ production of phytohormones (such as indole-3-acetic acid (IAA) (Lee et al. 2004), cytokinins, and other PGP substances) and/or partly owing to the fact that endophytes can enhance the hosts’ absorption of nutritional elements such as nitrogen (Reis et al. 2000) and phosphorus (Guo et al. 2000) and that they regulate nutritional qualities such as carbon–nitrogen ratio. Moreover, a number of other beneficial effects on plant growth have been attributed to endophytes and include siderophore production (Costa and Loper 1994), supply of essential vitamins to plants (Pirttil€a et al. 2004), osmotic adjustment, stomatal regulation, modification of root morphology, enhanced uptake of minerals, and alteration of nitrogen accumulation and metabolism (Compant et al. 2005a, b). Protective effects on endophyte-infected host plants greatly enhance their resistance to unfavorable challenges. The evidence suggests that plants infected with endophytes often have a distinct advantage against biotic and abiotic stress over their endophyte-free counterparts. Beneficial features have been offered in infected plants, including drought acclimatization, improved resistance to insect pests and herbivores, increased competitiveness, enhanced tolerance to stressful factors such as heavy metal presence, low pH, high salinity, microbial infections, biocontrol of phytopathogens in the root zone (through production of antifungal or antibacterial agents, siderophore production, nutrient competition and induction of systematic acquired host resistance, or immunity), or enhanced availability of minerals (Sturz et al. 2000). Endophyte infected plants also gain protection from herbivores and pathogens due to the bioactive secondary metabolites 76 M. Senthilkumar et al. secreted by the endophytes in plant tissue. An increasing number of antimicrobial metabolites biosynthesized by endophytic microorganisms, such as alkaloidal mycotoxins and antibiotics, have been detected and isolated (Strobel 2003; Strobel and Daisy 2003). But only a few studies have been published describing the molecular basis of the interactions between endophytic bacteria and plants. Adapt- ing strategies that have been used to study bacterial gene expression in the rhizosphere and phyllosphere such as in vivo expression technology (IVET) and recombination IVET (Leveau and Lindow 2001; Zhang et al. 2006) may provide an insight into genes that are required by bacteria to enter, compete, colonize the plant, suppress pathogens, and generally survive within the plant.

3.12 Biotization

Plant propagation technology via tissue culture has been developed over the last 30–40 years as a spinoff of in vitro studies on differentiation. Typically, aseptic explants are grown under low light intensity in small containers, on artificial culture media containing sucrose, mineral salts, vitamins, and growth regulators, in concentrations exceeding levels recorded under natural environments. During the last decade, some researchers have searched for natural inhabitants of plants, epiphytes, and endophytes to enhance adaptation of tissue culture propagules to environmental stresses (Herman 1996). In nature, microorganisms inhabit the interior and exterior of plant organs (McInroy and Kloepper 1994). Some of these microorganisms, plant-beneficial bacteria, and vesicular-arbuscular mycorrhizae in particular can improve plant performance under stress environments and consequently enhance yield. The tissue culture approach is one way of method to introduce the selected endophytes into the host plant (Nowak 1998). Biotization, in the current context, may be defined as the metabolic response of in vitro grown plant material to microbial inoculants which promote developmental and physiological changes that enhance biotic and abiotic stress resistance in subsequent plant progeny. Such systems allow for mutual adaptation between the host plant and the introduced bacteria. Induction of stress resistance in plant propagules produced in vitro prior to transplanting is a primary target of several research groups attempting utilization of microbial inoculants in micropropagation (Nowak et al. 1995). Such responses under in vitro conditions are referred to as “biotization” (Herman 1996). Biotization of potato plantlets enhanced the transplant stress tolerance and eliminate an expensive greenhouse hardening step (Herman 1987). Greenhouse experiments also demonstrated that plants derived from dual cultures of potato and pseudomonad bacterium had larger root system, set stolons, tuberized earlier, and gave better tuber yield than nonbacterized control. Both in vitro and ex vitro benefits of biotization depended on plant species, cultivar, and growth conditions (Nowak et al. 1997; Pillay and Nowak 1997). In vitro cocultivation of soybean cotyledon explants with two strains of Pseudomonas maltophilia stimulated development of 3 Endophytic Bacteria: Perspectives and Applications 77 nodular callus with high regeneration potential. Improvement of somatic embryogenesis by selected bacterial inoculants in genotypes recalcitrant to regeneration has further been reinforced by Visser-Tenyenhuis et al. (1994). The in vitro growth responses depend on the degree of endophytic colonization, and a certain threshold of the bacteria concentration is required to trigger beneficial responses (Pillay and Nowak 1997). Sharma and Nowak (1998a) postulated similar induction patterns for the resistance to pathogens. A study by Senthilkumar et al. (2008) aimed at developing biotized rice investigated the effects of Azorhizobium strain isolated from the stem nodule of Aeschynomene aspera on the rice calli colonization, dispersion, colonization in roots, leaves, and leaf sheath, growth physiology, and yield under greenhouse condition. In this study, a natural endophytic association between A. caulinodans and tissue culture rice plants was observed with migration of rhizobia to roots, stem, and leaves with nitrogen fixation, plant growth promotion, and increased yield (Fig. 3.1). There appears to be some variation in the colonization pattern of rice calli and plantlets by the A. caulinodans strains studied. Several factors like host specificity, geographical distribution, plant age and tissue type may explain these differences. These strains also confirmed the Koch’s postulate when they were able to recolonize the original plant host. Our findings have shown that the qualitative parameters of rice calli such as protein and total nitrogen content can be modulated by microbial co-bionts when imbibed with calli. Although the mechanism for the growth promoting capacity has not been established, evidence gained so far suggests that the growth and development stimulatory properties of the isolated strains were mediated through microbial-derived compounds. Qualitative evidence based on callus induction, tissue subculture, and plantlet regeneration experiments suggests that the strains-induced growth and cell proliferation are mediated by microbial-derived cytokinin-like or auxin-like substances. Endophytic bacterial associations with rice are generally nonspecific (Reddy et al. 2002), and the size of the bacterial population density in rice tissues is too low to support adequate N2 fixation. Hence, it is important to develop strategies that enable enhanced diazotrophic bacterial colonization for significant endophytic BNF in rice. N2 fixation by inoculated diazotrophs per se seems to have minimal but significant effect on the nitrogen content of the biotized plantlets, as it was clear from the data of percent N content of inoculated samples (Muthukumarasamy et al. 2007; Senthilkumar et al. 2008). Bacterized plantlets did not only grow faster than unbacterized plantlets (Chanway 1997; Bensalim et al. 1998) but are also sturdier and have a better developed root system (Nowak 1998) and a significantly greater capacity to withstand adverse biotic stresses (i.e., drought) and low level disease pressures (Sharma and Nowak 1998b). Frommel et al. (1991) reported that the endophyte bacteria can be translocated to successive generations of potato plants during multiplication through stem explants (Fig. 3.1). If the culture conditions are refined, artificial associations between callus and individual microorganisms or their groups can be created. Such microorganisms not only would act as inducers of the stress resistance responses but also could occupy microsites on host plants, making them unavailable to pathogens. An example of successful artificial association between plants and a nitrogen-fixing bacterium 78 M. Senthilkumar et al.

Fig. 3.1 Optical micrograph showing cross section of intercellular colonization rice calli and regenerated plantlets by A. caulinodans.(a) Embryogenic calli (b) Cross section of rice calli colonized by A. caulinodans 15 days after inoculation (c) CS view of colonized rice leaf (d) Magnified cross section view of leaf colonized by A. caulinodans in regenerated rice plant (e) CS view of root uninoculated control (f) Possible sites of infection and colonization of rice root (g) Shoot and (h) Root development (i) Regenerated plantlets 3 Endophytic Bacteria: Perspectives and Applications 79 has been described by Varga et al. (1994). The authors created a symbiotic culture system, callus-bacterium, between Daucus carota L. and Azotobacter zettuovii (CRS-H6) which could grow for 4 years on a nitrogen-free medium with lactose as a carbon source. The bacterium, located in intracellular spaces, could also be transmitted to and fix nitrogen in newly regenerated plantlets (Varga et al. 1994; Preininger et al. 1997). The authors were also able to establish similar associations in tomato, potato, wheat, sugarcane, and poplar, using 11 strains of 8 Azotobacter species and in strawberries with Azomonas insignis (Preininger et al. 1997). The fact that we can culture probably only a few percent or even less of naturally occurring microorganisms is one of the challenges of the utilization of microbial inoculants in plant production. Development of new culture methods will allow establishment of stable association between plants and beneficial organisms in vitro and ex vitro and understanding of mechanisms of signal recognition and transduction in plant–microbial associations under different environments are probably the most critical elements of this challenge.

3.13 Biological Control

The intimate association of bacterial endophytes with plants offers a unique opportunity for their potential application in plant protection and biological control. Although biocontrol activity of microorgansims involving synthesis of allelochemicals has been studied extensively with free-living rhizobacteria, similar mechanisms apply to endophytic bacteria (Lodewyckx et al. 2002), as they can also synthesize metabolites with antagonistic activity toward plant pathogens. Certain endophytic bacterial isolates may play a significant role in plant protection against soilborne pathogens and in the overall productivity of an agricultural ecosystem (Hallmann et al. 1997; Sturz et al. 2000). Establishing a stable microbial endoplant communities may induce disease resistance through de novo synthesis of structural compounds and inhibition of fungal penetration (Benhamou and Nicole 1999), the induction and expression of general molecular-based plant immunity (Benhamou and Nicole 1999), or a simple exclusion of other organisms (phytopathogens and colonists) by niche competition (Sturz et al. 2000). The widely recognized mechanisms of biocontrol mediated by both organisms are competition for an ecological niche or a substrate, production of inhibitory allelochemicals, and induction of systemic resistance (ISR) in host plants to a broad spectrum of pathogens and/or abiotic stresses. But the effectiveness of endophytes as biological control agents is dependent on many factors, which include the host specificity, the population dynamics and pattern of host colonization, the ability to move within host tissues, and the ability to induce systemic resistance (Backman et al. 1997). For example, Chen et al. (1995) found five endophytic species, Aureobacterium saperdae, Bacil- lus pumilus, Phyllobacterium rubiacearum, Pseudomonas putida, and Burkholderia solanacearum, which could significantly reduce vascular wilt in cotton caused by Fusarium oxysporum f. sp. vasinfectum. Pseudomonas sp. strain PsJN, an onion 80 M. Senthilkumar et al. endophyte, inhibited Botrytis cinerea Pers. and promoted vine growth in colonized grapevines, demonstrating that divergent hosts could be colonized (Barka et al. 2002). Colonization of multiple hosts has been observed with other species of endophytes and plants. For example, P. putida 89B-27 and Serratia marcescens 90-166 reduced Cucumber Mosaic Virus in tomatoes and cucumbers (Raupach et al. 1996) as well as anthracnose and Fusarium wilt in cucumber (Liu et al. 1995). Mahaffee and Kloepper (1994) have shown that biological control by endophytic bacteria is possible and can involve induced resistance to soilborne pathogens. Inoculation with some bacterial endophytes has also been demonstrated to reduce disease incidence and symptoms of F. oxysporum in cotton (Chen et al. 1995). Castillo et al. (2002) demonstrated that munumbicins antibiotics produced by the endophytic bacterium Streptomyces sp. strain NRRL 30562 isolated from Kennedia nigriscans can inhibit in vitro growth of phytopathogenic fungi, P. ultimum and F. oxysporum. Subsequently, it has been reported that certain endophytic bacteria isolated from field-grown potato plants can reduce the in vitro growth of Strepto- myces scabies and X. campestris through production of siderophore and antibiotic compounds (Sessitsch et al. 2004). Interestingly, the ability to inhibit pathogen growth by endophytic bacteria, isolated from potato tubers decreases as the bacteria colonize the host plant interior, suggesting that bacterial adaptation to this habitat occurs within their host and may be tissue-type and tissue-site specific (Sturz et al. 1999). Aino et al. (1997) have also reported that the endophytic P. fluorescens strain FPT 9601 can synthesize 2, 4-diacetylphloroglucinol (DAPG) and deposit DAPG crystals around and in the roots of tomato, thus demonstrating that endophyte can produce antibiotics in planta. Most reports of plant growth promoting bacteria (PGPB)-mediated ISR involve free-living rhizobacterial strains, but endophytic bacteria have also been observed to have ISR activity. For example, ISR was triggered by P. fluorescens EP1 against red rot caused by Colletotrichum falcatum on sugarcane (Viswanathan and Samiyappan 1999), Verticllium dahliae on tomato (Sharma and Nowak 1998b), P. fluorescens against F. oxysporum f. sp. radicis- lycopersici on tomato (M’Piga et al. 1997), B. pumilus SE34 against F. oxysporum f. sp. pisi on pea roots (Benhamou et al. 1996), and F. oxysporum f. sp. vasinfectum on cotton roots (Conn et al. 1997). In all the reports that exist, the exact mode of action of disease suppression by particular endophytes has not been studied. Additional research is needed in order to better understand the parameters of this biocontrol system. The ISR in plants following treatment with endophytic fungi or bacteria and the mechanism of action opens a new line of research on the biochemical and genetic nature of signaling and gene induction.

3.14 Rice Endophytes

Diazotrophs, in rice cultivation, can be broadly grouped into two existing BNF systems, with the possibility of an additional third system. The existing systems include (1) indigenous (autochthonous) BNF systems comprising of heterotrophic 3 Endophytic Bacteria: Perspectives and Applications 81 and phototrophic bacteria as well as cyanobacteria native to soil–plant–floodwaters and (2) exogenous (allochthonous) BNF systems include Azolla that harbor symbiotic N2-fixing cyanobacteria, and aquatic legumes like Sesbania and Aeschynomene species that form symbioses with heterotrophic and phototrophic rhizobia. These exogenous BNF systems are not ubiquitous and hence need to be applied/inoculated to rice fields. The third system, endogenous (in planta) BNF, would come about € from transforming rice to an autonomous N2-fixing plant. Dobereiner et al. (1993) speculated that endophytic diazotrophs in certain rice genotypes may, in fact, be responsible for the substantial contributions of BNF to rice. Studies have shown that rice indeed harbors a wide range of endophytic diazotrophs (Barraquio et al. 1997; Gyaneshwar et al. 2001; James et al. 2000). Some or all of them may be responsible for supplying plants with fixed N. Inoculation experiments with the endophytes S. marcescens and Herbaspirillum sp. in nonsterilized soil under greenhouse conditions have shown that they can be readily introduced into the rice plant by applying bacterial cultures on seeds or roots prior to planting. Endophytic associations with rice are nonspecific and the size of the bacterial populations in rice tissues is low. To overcome some of these problems, de Bruijn and his associates examined the possibility of creating “biased rhizospheres” to selectively encourage the growth of introduced microorganisms (Rossbach et al. 1994). Rice has been intensively studied in the last decade because of their potential to associate with endophytic diazotrophs and contribute to nitrogen nutrition of the plant through BNF. The discovery of endophytic diazotrophs such as H. seropedicae (Baldani et al. 1986) and Herbaspirillum rubrisubalbicans (Baldani et al. 1986) and the recent isolation of a new N2-fixing bacterium within the genus Burkholderia, provisionally named Burkholderia brasiliensis (Baldani et al. 1997b) based on morphological and physiological characteristics (Baldani et al. 1997a) and 23S rDNA oligonucleotide probes (Kirchhof et al. 1997), could explain the BNF observed in certain cereal genotypes. Azoarcus originally isolated from Kallar grass (Leptochloa fusa Kunth) growing in saline-sodic soils of Pakistan, a strain of Gram-negative nitrogen-fixing bacterium Azoarcus sp BH72 has been described by Reinhold-Hurek et al. (1993) that also colonizes rice in laboratory experiments (Hurek et al. 1994). These diazotrophs colonize roots, stems, and leaves of cereals endophytically (Olivares et al. 1996; Baldani et al. 1997a) and therefore probably suffer much less competition from other microorganisms for C substrates than rhizosphere bacteria, and possibly excrete part of their fixed N directly into the plant (Stoltzfus et al. 1997). The finding of a beneficial PGP association of Rhizo- bium leguminosarum bv. trifolii with rice roots (Yanni et al. 2001) is particularly relevant to assessments of whether rhizobia can fix N2 endosymbiotically in cereals. It was concluded that the benefits of this association leading to greater production of vegetative and reproductive biomass more likely involved rhizobial modulation of the root architecture for more efficient acquisition of certain soil nutrients rather than biological N2 fixation. By inhabiting the interior of the plants, these bacteria are thought to (1) avoid competition with bacteria of the rhizosphere and (2) derive nutrients directly from host plants (Baldani et al. 1997a; Boddey et al. 1995; James and Olivares 1997). In return, the plant interior (which is low in O2 and relatively 82 M. Senthilkumar et al. high in carbon) provides an environment conducive to N2 fixation allowing the bacteria to efficiently transfer fixed N products to the host (James and Olivares 1997). Dobereiner€ et al. (1993) speculated that endophytic diazotrophs in certain rice genotypes may, in fact, be responsible for the substantial contributions of BNF to rice as reported earlier by App et al. (1984). Studies have shown that rice indeed harbors a wide range of endophytic diazotrophs (Barraquio et al. 1997; Gyaneshwar et al. 2001; James et al. 2002; Stoltzfus et al. 1997). Mano and Morisaki (2008) reported in their recent review the occurrence of diverse endophytic bacteria throughout the rice plant irrespective of cultivar (Tables 3.2 and 3.3).

3.15 Sugarcane Endophyte

A variety of diazotrophic bacteria have been isolated from rhizosphere (Beije- rinckia) and roots (Azospirillum, Bacillus, Klebsiella, Enterobacter, Erwinia)of sugarcane plant. Diazotrophs such as G. diazotrophicus and Herbaspirillum spp. grow endophytically in the stems and leaves of sugarcane. In addition, Azospi- rillum, Burkholdaria, and perhaps other diazotrophs also inhabit sugarcane. Even though it is still not clear which of these endophytes is the main contributor to the observed BNF in sugarcane, evidence, nevertheless, suggests that the total endophytic BNF, according to N-balance studies, can be as high as 150 kg N haÀ1 yearÀ1 in sugarcane (Boddey et al. 1995). In 1988, Cavalcante and Dobereiner€ (1998) reported an acid-tolerant N-fixing bacterium, Acetobacter diazotrophicus, associated with sugarcane which contributed abundant N to sugarcane crops, with a capability to excrete almost half of the fixed N in a form that is potentially available to plants. A. diazotrophicus has also been isolated from other plants, viz., Cameroon grass (Pennisetum purpureum), sweet potato (Ipomoea batatas), coffee (Coffea arabica), tea, banana, ragi, rice, and pineapple (Table 3.4) and even from insects that infest sugarcane. The close association between a plant and an endophyte may provide suitable conditions for nutrient transfer between the bacteria and their host, than the association between predominantly rhizosphere bacteria and plants. It was reported that up to 80% of the plant nitrogen in certain sugarcane varieties has been derived as a result of BNF (Boddey et al. 1995). Application of G. diazotrophicus to sugarcane has been proved beneficial where the plant height, nitrogenase activity, and yield of the inoculated plants were higher than the control. Plant inoculation studies revealed the abundant population of G. diazotrophicus in the artificially inoculated sugarcane plantlets, reflecting through enhanced ARA, supporting the suggestion of James et al. (1994) that direct inoculation of G. diazotrophicus is possible. Inocula- tion of G. diazotrophicus was reported to enhance leaf N, biomass, and yield. Field trials conducted in sugarcane system revealed the usefulness of G. diazotrophicus with other diazotrophs, which have contributed to the yield equal to that of control (275 kg N ha–1). Mixed inoculation of VAM spores and G. dizaotrophicus also proved beneficial in improving the yield of different sugarcane varieties. The yield 3 Endophytic Bacteria: Perspectives and Applications 83

Table 3.2 The endophytic bacteria isolated from various parts of the rice plant (Mano and Morisaki 2008) Rice part Rice species Bacterial taxa Reference Seed Oryza alta Pantoea ananatis Elbeltagy et al. (2000) Seed Oryza Herbaspirillum seropedicae, Elbeltagy et al. (2000) meridionalis Methylobacterium sp. Seed Oryza sativa Klebsiella oxytoca Elbeltagy et al. (2000) Seed Oryza sativa Acidovorax sp., Bacillus pumilus, Mano et al. (2006) B. subtilis, Curtobactrium sp., Methylobacterium aquaticum, Micrococcus luteus, Panibacillus amylolyticus, Pantoea ananatis, Sphingomonas melons, S. yabuuchiae, Xanthomonas translucens, B. cereus, Azosprillum amazonense, Flavobacterium gleum Seed Oryza sativa Sphingomous echinoides, Okunishi et al. (2005) S. parapaucimobilis Seed Oryza sativa Ochrobactrum anthropi, Pantoea Verma et al. (2001) agglomerans, Pseudomonas boreopolis, P. fulva Leaf Oryza sativa Aurantimonas altamirensis, Bacillus Mano et al. (2007) gibsonii, B. pumilis, Curtobacterium sp., Diaphorobacter nitroreducens, Methylobacterium aquaticum, Methylobacterium sp., Pantoea ananatis, Sphingomonas echinoides, S. melonis, S. yabuuchiae, Stenotrophomonas maltophili, Streptomyces sp. Leaf Oryza sativa Methylobacterium sp. Elbeltagy et al. (2000) sheath Stem Oryza alta Methylobacterium sp. Elbeltagy et al. (2000) Stem Oryza barthii Methylobacterium sp. Elbeltagy et al. (2000) Stem Oryza Methylobacterium sp. Elbeltagy et al. (2000) brachyantha Stem Oryza Methylobacterium sp. Elbeltagy et al. (2000) glandiglumis Stem Oryza latifolia Methylobacterium sp. Elbeltagy et al. (2000) Stem Oryza longiglumis Herbaspirillum seropedicae Elbeltagy et al. (2000) Stem Oryza Rhodopseudomonas palustris Elbeltagy et al. (2000) meridionalis Stem Oryza minuta Methylobacterium sp., Sphingomonas Elbeltagy et al. (2001) adheasiva Stem Oryza ofﬁcinalis Azosprillum brasilense, Enterobacter Elbeltagy et al. (2000) cancerogenus, Herbasprillum seropedicae Stem Oryza ridleyi Cytoohagales str. MBIC4147, Elbeltagy et al. (2001) Methylobacterium sp. Stem Oryza ruﬁpogon Azosprillum lipoferum, Ideonella Elbeltagy et al. (2001) dechloratans (continued) 84 M. Senthilkumar et al.

Table 3.2 (continued) Rice part Rice species Bacterial taxa Reference Stem Oryza ruﬁpogon Agrobacterium vitis, Azorhizobium Elbeltagy et al. (2000) caulinodans, Azosprillum sp., Bacillus megaterium, B. subtilis, Pseudomonas cepacia Stem Oryza sativa Herbaspirilium seropedicae Stoltzfus et al. (1997) Stem Oryza sativa Serratia marcescens Olivares et al. (1996) Stem Oryza sativa Klebsiella sp. Gyaneshwar et al. (2001) Leaf Oryza sativa Azoarcus sp. Engelhard et al. (2000) Stem, Oryza sativa Gallionella sp. Engelhard et al. (2000) Root Stem, Oryza sativa Azocarcus sp. Engelhard et al. (2000) Root Root Oryza granulate Sphingomonas paucimobilis Engelhard et al. (2000) Root Oryza minuta Azoarcus sp., Azoarcus indigens, Engelhard et al. (2000) Azorhizobium caulinodans, Azosprillum brasilense, A. lipoferum, Burkholderia sp., Hespaspirillum sp. Root Oryza nivara Klebsiella pneumoniae Engelhard et al. (2000) Root Oryza ofﬁcinalis Ochrobactrum sp., Mano et al. (2007) Sphingomonas paucimobilis Root Oryza sativa Azosprillum irakense, Bacillus Singh et al. (2006) luciferensis, B. megaterium, Bradyrhizobium elkanii, B. japonicum, Brevibacillus agri, Burkholderia kururiensis, Caulobactor crescentus, Chryseobacterium taichungense, Enterobactor cloacae, E. ludwigii, Hyhomicrobium sulfonivorans, Methylocapsa acidiphila, Micrococcus luteus, Mycobacterium petroleophilum, Paenibacillus alvei, Rhizobium loti, Roseateles depolymerans Root Oryza sativa Burkholderia cepacia, Yanni et al. (1997) Rhizobium leguminosarum, R. leguminosarum 3 Endophytic Bacteria: Perspectives and Applications 85

Table 3.3 Bacterial diversity in rice plants as revealed by culture independent method (Mano and Morisaki 2008) Rice part Rice species Bacterial group Bacterial taxa Surface and Oryza sativa g-Proteobacteria Erwinia amylovora inside of Pseudomonas ﬂuorescens milled rice Firmicutes Xanthomonas sacchari Actinobacteria Staphlococcus sp. a-Proteobacteria Nocardia globerula Brevundimonas diminuta, Caulobacter sp. Kaistiana koreensis, Methylobacterium sp., Novosphingobium tardaugens, Sinorhizobium terangae Inside root Oryza sativa b-Proteobacteria Achromobacter xylosoxidans Acidovorax facilis Burkholderia fungorum Burkholderia sp. Comamonas testerone Curvibacter gracilis Delftia acidovorans D. tsuruhatensis Duganella violaceinigra Gallionella ferruginea, Herbaspirillum frisingense Hydrogenophaga taeniospiralis Methyloversatilis universalis Sterolibacterium denitriﬁcans Variovirax sp. Acinetobacter baumannii Alkanindiges illinoisensi g-Proteobacteria Enterobacter sp. Methylophaga marina Pantoea sp. Plesiomonas shigelloides Pseudomonas stutzeri Stenotrophomonas maltophilia Stenotrophomonas sp. Bdellovibrio bacteriovorus Geobacter sp. Sulfurospirillum multivorans d-Proteobacteria Flavobacterium frigoris e-Proteobacteria F. psychrophilum Sphingobacterium sp. Bacteroidetes Acidaminobacter hydrogenoformans Clostridium sp. Firmicutes Lachnospiraceae bacterium Deinococcus- Planomicrobium okeanokoites Thermus, P. mcmeekinii Acidobacteria Deinococcus indicus Holophaga foetida 86 M. Senthilkumar et al.

Table 3.4 Sources of Source Part G. diazotrophicus Sugarcane Root, root hair, stem, leaf (Muthukumarasamy Cameroon grass Root, stem et al. 2002) Sweet potato Root, stem tuber Coffee Root, rhizosphere, stem Ragi Root, rhizosphere, stem Tea Root Pineapple Fruit Mango Fruit Banana Rhizosphere Others: mealy bugs, VAM spores Internal environment

was also not reduced even under 50–100% reduction from the recommended dose of chemical N compared to the control, attributing the role of inoculated G. diazotrophicus in N contribution (Muthukumarasamy et al. 1999). It has been reported that inoculation of micropropagated sugarcane seedlings would make the plants not only grow faster, but also ensure efficient N-fixing plants in fields (Reis et al. 2000). The 15N incorporation experiments, using sterile sugarcane plants, have also demonstrated the potential for N fixation in G. diazotrophicus– sugarcane interaction (Sevilla et al. 1998). Use of mutant strains (carrying nif D::kan interposan mutation that prevents N fixation entirely) in plant experiments proved the participation of G. diazotrophicus in N fixation. It is an established fact that the growth hormones, viz., auxins, cytokinins, and gibberellins, play a role in enhancing the growth of grasses associated with diazotrophs. Apart from N fixation, G. diazotrophicus is also reported to benefit sugarcane through production of PGP factors (Fuentes-Ramirez et al. 2001).

3.16 Conclusion

Currently, endophytes are viewed as an outstanding source for undescribed microbes because there are so many of them occupying literally millions of unique biological niches (higher plants) growing in so many unusual environments. Exploitation of endophyte–plant interactions can result in the promotion of plant health and can play a signiﬁcant role in low-input sustainable agriculture applications for both food and nonfood crops. An understanding of the mechanisms enabling these endophytic bacteria to interact with plants will be essential to fully achieve the biotechnological potential of efﬁcient plant–bacterial partnerships for a range of applications.

Acknowledgment This study was supported by Rural Development Administration (RDA), Republic of Korea and Tamil Nadu Agricultural University, Coimbatore, India. 3 Endophytic Bacteria: Perspectives and Applications 87

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Gabriele Berg and Christin Zachow

4.1 Introduction

The interface between soil and plant roots – the rhizosphere – is, due to root exudates and the resulting high nutrient content, a unique microenvironment in terrestrial ecosystems (Sørensen 1997; Raaijmakers et al. 2009). Although ubiquitous and cosmopolitan rhizosphere-associated bacterial genera are known, e.g. Pseudomonas, Bacillus and Methylobacterium, specific populations have been detected for each plant species (Berg and Smalla 2009). The function of rhizosphere-associated bacteria is only partly understood. Firstly, bacteria play a role for plant growth. They can supply macro- and micro-nutrients. The most prominent example is bacterial nitrogen-fixation. The symbiosis between rhizobia and its legume host plants is an important example for plant growth-promoting rhizobacteria (PGPR). Bacteria of this group metabolise root exudates (carbohydrates) and in turn provide nitrogen to the plant for amino acid synthesis. The ability to fix nitrogen also occurs in free-living bacteria like Azospirillum, Burkholderia and Stenotrophomonas (Dobbelare et al. 2003). Another nutrient is sulphate, which can be provided to the plant via oxidation by bacteria (Banerjee and Yesmin 2002). Bacteria may contribute to plant nutrition by liberating phosphorous from organic compounds such as phy- tates and thus indirectly promote plant growth (Unno et al. 2005). Mineral supply is also involved in plant growth promotion and includes synthesis of siderophores and siderophore uptake systems. Poorly soluble inorganic nutrients can be made available through the solubilisation of bacterial siderophores and the secretion of organic acids. Recently, de Werra et al. (2009) showed that the ability of Pseudomonas fluorescens CHA0 to acidify its environment and to solubilise mineral phosphate is strongly dependent on its ability to produce gluconic acid. In the processes of plant growth, phytohormones, e.g. indole-3-acetic acid (IAA), ethylene, cytokinins, and gibberellins, play an important role. Furthermore, plant-associated bacteria can

G. Berg (*) and C. Zachow Graz University of Technology, Environmental Biotechnology, Petersgasse 12, A-8010 Graz, Austria e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 97 DOI 10.1007/978-3-642-18357-7_4, # Springer-Verlag Berlin Heidelberg 2011 98 G. Berg and C. Zachow influence the hormonal balance of the plant (Costacurta and Vanderleyden 1995). An interesting phenomenon is the enhancement of stress tolerance by lowering the ethylene level (Glick et al. 1998). Another important function is the involvement of rhizosphere-associated bacteria in pathogen defence. Whereas resistance against leaf pathogens is often encoded in the plant genome, it is difficult to find resistance genes against soil-borne pathogens. Cook et al. (1995) suggest that antagonistic rhizobacteria fulfil this function. Interestingly, besides direct antagonism, plant- associated bacteria can induce a systemic response in the plant, resulting in the activation of plant defence mechanisms (Pieterse et al. 2003). However, several studies suggest that there are many more plant–microbe interactions and resulting functions. A fascinating example is endophytic methylobacteria, which use C1 compounds from the plant for their energy production (Zabetakis 1997). The (intermediate) catabolic product hydroxypropanol is given back to the plant and works as precursor of the flavour compounds mesifuran and 2,5-dimethyl-4- hydroxy-2H-furan (DMHF). The latter posses additional antifungal activity and can be responsible for pathogen defence. Methylobacterium treatment resulted in both a statistically significantly higher content of flavour compounds and a better taste of strawberries (Verginer et al. 2010). Interestingly, an earlier report provided evidence that hormone-producing methylobacteria are essential for germination and development of protonema of the moss Funaria hygrometrica (Hornschuh et al. 2002). Another function of rhizobacteria can be the degradation of root exudates with allelopathic or even autotoxic functions (Bais et al. 2006). To study plant-associated bacteria and their structure and functions is important not only for understanding their ecological role and the interaction with plants and plant pathogens, but also for any biotechnological application. In biotechnology, rhizosphere-associated bacteria can be applied directly for biological control of plant pathogens as biological control agents (BCAs), for growth promotion as PGPR or as biofertilisers and rhizoremediators (Whipps 2001; Lugtenberg et al. 2002). During the last years, there are an increasing number of products based on microbial inoculants on the market (rev. in Berg 2009). However, for many promising candidates the translation into practical approaches failed due to tech- nical problems but also due to (1) their human pathogenic potential or (2) due to negative interactions with the environment (Fig. 4.1). Risk assessment studies at an early stage of the product development can avoid these problems.

4.2 Interplay with Eukaryotic Hosts: The Human Pathogenic Potential of PGPR

PGPR interact intensively not only with their host plant but also with other eukaryotic hosts living in the rhizosphere. The interaction will be analysed not only to understand the human pathogenic potential of PGPR but also to develop assays, which allowed assessing the latter. 4 PGPR Interplay with Rhizosphere Communities 99

Disease Soil-borne Plant pathogens Induced resistance

PGPR Root exudation INTERPLAY Biocontrol Plant growth promotion Competition Hormomal stimulation Change of composition

Plant Growth Promoting Humans Rhizobacteria PGPR

Impact? Impact? i) Human health Microbial communities ii) Impact on environment

Fig. 4.1 Interplay of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere: key players, modes of action and impact on (i) human health and (ii) on the environment

4.3 The Rhizosphere as Reservoir for Potential Human Pathogenic Bacteria

During the last few years, it has been shown that plants, especially in the rhizosphere, can harbour not only beneficial bacteria, but also those that potentially can cause diseases in humans (Berg et al. 2005; Opelt et al. 2007). These pathogens are called opportunistic or facultative human pathogens and they cause diseases only in patients with a strong predisposition to illness, particularly in those who are severely debilitated, immuno-compromised or suf- fering from cystic fibrosis or HIV infections (Parke and Gurian-Sherman 2001; Steinkamp et al. 2005). This group of bacteria cause the majority of bacterial infections associated with significant case/fatality ratios in susceptible patients in Europe and Northern America. A special group are those bacteria responsible for hospital-acquired diseases which are called nosocomial infections. For example, in intensive care units in Europe, 45% of the patients were infected by opportunistic pathogens (Vincent et al. 1995). In the last two decades, the impact of opportunistic infections on human health has increased dramatically. Many plant-associated genera, including Burkholderia, Enterobacter, Herbas- pirillum, Ochrobactrum, Pseudomonas, Ralstonia, Serratia, Staphylococcus and Stenotrophomonas contain root-associated bacteria that enter bivalent interactions with plant and human hosts (Fig. 4.1). Several members of these genera show plant growth-promoting as well as excellent antagonistic properties against plant 100 G. Berg and C. Zachow pathogens and therefore were utilised as PGPR and for the development of biological control products (Whipps 2001). However, many strains also successfully colonise human organs and tissues and thus cause diseases. The mechanisms behind these interactions are similar; they include recognition, adherence, colonisation as well as survival (Rahme et al. 1995; Cao et al. 2001). Furthermore, the rhizosphere is a tool for naturally occurring antibiotics produced by bacteria and their resistance genes (Martinez 2009). The problems with biofungicides based on strains of the genus Burkholderia underlines the importance of thorough risk assessment studies prior to registration (Govan et al. 2000; Parke and Gurian- Sherman 2001). Another example is given by Stenotrophomonas maltophilia,a PGPR and emerging pathogen in humans (Ryan et al. 2009). Interestingly, for Stenotrophomonas strains a study indicate that clinical environments select bacterial populations with high mutation frequencies from rhizosphere populations (Turrientes et al. 2010). In a study published by Alonso et al. (1999) it was shown that clinical and environmental isolates of P. aeruginosa, which is the major cause for morbidity and mortality in cystic fibrosis patients, share several phenotypic traits with respect to both virulence and environmental properties. Several studies support the view that the environmental Pseudomonas strains are indistinguishable from those from clinical sources in terms of genotypic, taxonomic or metabolic properties (Kiewitz and Tummler€ 2000; Wolfgang et al. 2003). But there is also an impact of the clinical environment: a study showed that P. putida as well as P. stutzeri strains acquired the antibiotic resistance genes under selective pressure of antibiotic exposure in the hospital environment (Carvalho-Assef et al. 2010). Rhizosphere-associated bacteria with a high capacity for biocontrol and plant growth promotion can be potentially dangerous for human health. Therefore, it is important to understand the mode of action and specific properties of the PGPR. It is well known that antagonistic properties and underlying mechanisms are highly strain-specific (Berg et al. 2002, 2006) but identification of bacteria is based mainly on 16 S rDNA sequencing. Thus, from sequencing information it is difficult to draw conclusions about potential pathogenicity: neutral bacterial strains can be dangerous due to pathogenicity islands or pathogenic bacteria can be harmless because of the absence of any pathogenicity factor. Therefore, models to assess the pathogenicity of individual BCAs are important as risk assessment studies.

4.4 Caenorhabditis elegans: A Model to Assess Potential Human Pathogenic Bacteria

To assess the pathogenic potential is particularly difficult in many opportunistic human pathogens as well as BCAs because of the lack of adequate animal models. Until now, this procedure for BCAs is based on rules originally developed for 4 PGPR Interplay with Rhizosphere Communities 101 synthetic pesticides (EU Council Directive 91/414/EEC, see also http://www. rebeca-net.de). Methods adopted from standardised tests for chemical-based agents, including elaborate animal tests are not only time-consuming and expensive but also their results are difficult to interpret. Pathogenicity and the mode of action of facultative pathogens such as Burkholderia and Stenotrophomonas could not be analysed in traditional animal models. Therefore, alternative models using the slime mould Dictyostelium discoideum (Alonso et al. 2004), duckweed (Lemna minor L.; Radic´ et al. 2010), the zebrafish Danio rerio (van der Sar et al. 2003) or the nematode C. elegans (Tan et al. 1999) were developed. The model organism C. elegans has valuable advantages, enabling it to be used in many bacteria–pathogen interaction analyses to evaluate the pathogenic potential of these bacteria (Aballay and Ausubel 2002; Cardona et al. 2005). C. elegans is a free-living terrestrial nematode that feeds on bacteria in its environment (Beale et al. 2006). Extensive information about C. elegans research is available in well-resourced internet databases (http:// www.wormbook.org, http://www.wormbase.org). In an extensive study, we applied a broad range of BCAs, pathogens and plant- associated bacteria to a rapid and inexpensive bioassay, using C. elegans to estimate the risk of bacteria to harm human health (Zachow et al. 2009). The nematode killing assay described as slow killing assay by Kothe€ et al. (2003) was used. Movement and reproduction behaviour of C. elegans with BCAs were compared with those fed with the human pathogen P. aeruginosa QC14-3-8 (positive control) and Escherichia coli OP50 (negative control). In Fig. 4.2, the kinetics of killing C. elegans under slow killing conditions is shown (a) for different Pseudomonas strains: P. fluorescens L13-6-12, P. trivialis RE*1-1-14 and Pseudomonas sp. ¼ # Proradix [product produced by Sourcon-Padena GmbH & Co.KG] and by (b) different enterics: Pantoaea agglomerans L24-6-12, Rhanella aquatilis G3SM41, Serratia liquefaciens N1SM25, S. grimesii N1SM34, S. marcescens and S. ply- # muthica HRO C48 ¼ RhizoStar [product produced by e ~ nema Gesellschaft fur€ Biotechnologie und biologischen Pflanzenschutz mbH]. All strains were isolated from the rhizosphere but some of them are selected according to their identification and grouping into risk group 2 (P. aeruginosa, P. agglomerans, R. aquatilis, S. grimesii, S. liquefaciens and S. marcescens). Indeed, these bacteria from risk group 2 showed a significantly higher rate of killing. In contrast, results obtained from other pseudomonads and S. plymuthica gave hints to a low risk. Altogether, results indicate that C. elegans provides a reliable model system to assess the human pathogenic potential of BCAs prior implementation of extensive studies using animal test systems. The C. elegans assay can be integrated into initial screens for BCAs and is useful to exclude pathogens in a very early stage of the product development. There are some restrictions for the C. elegans assay. The model of pathogenicity is limited by the amount of bacteria infecting the worm, which was shown for P. aeruginosa, P. fluorescens, S. marcescens, Burkholderia cepacia, B. pseudomallei, B. thailandensis, Salmonella spp. and Bacillus megaterium (Tan and Ausubel 2000). Therefore, in this study we used an overnight bacterial culture with approximately 107 cells/ml, which provides an appropriate thin cell layer to evaluate the 102 G. Berg and C. Zachow

a 100 Pseudomonas aeruginosa QC14-3-8 Escherichia coli OP50 90 Pseudomonas fluorescens L13-6-12 Pseudomonas trivialis RE*1-1-14 80 Pseudomonas sp. 70

30 Dead worms(%) 20

0 0 h 24 h 48 h 72 h Time (hour) b 100 Pseudomonas aeruginosa QC14-3-8 Escherichia coli OP50 90 Pantoea agglomerans L24-6-12 Rahnella aquatilis G3SM41 80 Serratia liquefaciens N1SM25 Serratia grimseii N1SM34 70 Serratia marcescens Serratia plymuthica C48 60

30 Dead worms(%) 20

0 0 h 24 h 48 h 72 h Time (hour)

Fig. 4.2 Kinetics of killing of Caenorhabditis elegans by different PGPR under slow killing conditions. Worms grown on NGMII and feeding on Pseudomonas aeruginosa QC14-3-8 (positive control, black circle), Escherichia coli OP50 (negative control, black square). The PGPR were represented by (a) different Pseudomonas strains: Pseudomonas fluorescens L13-6-12 (white square), Pseudomonas trivialis RE*1-1-14 (white circle) and Pseudomonas sp. ¼ Proradix# (white triangle), and by (b) different enterics: Pantoaea agglomerans L24-6-12 (black cross), Rhanella aquatilis G3SM41 (white circle), Serratia liquefaciens N1SM25 (white square), Serratia grimesii N1SM34 (grey square), Serratia marcescens (white triangle), and Serratia plymuthica HRO C48 ¼ RhizoStar# (grey triangle). Data points represent means standard errors of at least five independent experiments 4 PGPR Interplay with Rhizosphere Communities 103 behaviour of the transparent worm on the Petri dishes. In a pilot study, this concentration of cells was found to permit detection of differences in survival among worm strains after 24 h (Schulenburg and Ewbank 2004). Furthermore, the developmental stage of the applied worms influenced the slow killing. Adult worms were more sensitive and died faster than fourth-stage larvae. Therefore, in this study we used second-stage larvae in the assay, exactly 48 h after egg preparation. Another restriction is associated with B. thuringiensis, a well-known BCA. Although B. thuringiensis is used world-wide against insect pests without reports that it has caused harm to humans, the bioactive toxin does act against C. elegans (Devidas and Rehberger 1992).

4.5 Interplay with Rhizosphere Communities: The Impact on the Environment

Although originating from plant-associated microenvironments themselves, beneficial bacteria, if applied to plant roots in adequate numbers, may perturb indigenous microbial populations and their associated important ecological functions (Fig. 4.1). Therefore, unwanted, unspecific actions of the introduced beneficial microbes against nontarget organisms have to be assessed. To this end, sufficient knowledge about the microbial ecology of the target habitats is necessary for reasonable risk assessment studies concerning the release of beneficial microorganisms. As only a small proportion of the microorganisms can be analysed by common cultivation techniques, several DNA-based, cultivation-independent methods, have been developed to overcome the limitations of cultivation (Smalla 2004). The use of such molecular methods is urgently needed in order to include the highest possible number of total microorganisms in risk assessment studies to determine non-target effects of introduced beneficial bacteria (Winding et al. 2004). Several studies using cultivation-independent methods exist. They focus mainly on the effects of genetically modified microorganisms (GMOs) such as Pseudomonas (Viebahn et al. 2003; Glandorf et al. 2001) and Sinorhizobium (Schwieger and Tebbe 2000) on non-target microorganisms. Examples of studies, which analysed the fate and ecosystem effects of introduced PGPR and antagonistic bacteria, are given in Table 4.1. Generally it can be concluded from these studies that the impact of bacterial inoculants is either negligible or small compared with effects of general agricultural practises, and more or less all effects are transient. However, for strains with a strong production of antifungal antibiotics or genetically modified strains with additional genes to synthesise antibiotics, effects were observed (Viebahn et al. 2003; Walsh et al. 2003; Blouin-Bankhead et al. 2004). Interestingly, also pathogenic bacteria are able to persist in plant-associated microenvironments, especially in the rhizosphere (Table 4.1). As their non-pathogenic counterparts they caused only transient effects on microbial communities. Examples for risk assessment studies for PGPR and facultative human pathogenic bacteria

Table 4.1 Zachow C. and Berg G. 104 Strains Plant/pathosystem Results Reference PGPR Pseudomonas putida QC14-3-8 Potato No differences between the inoculated Lottmann et al. (2000) Serratia grimesii L16-3-3 and non-inoculated communities Pseudomonas fluorescens CHA0 Cucumber Differences in the composition and/or Girlanda et al. (2001) and GMO relative abundance of species in the fungal community, no effect on species diversity indices, and species abundance Impact of treatments was smaller than the effect of growing cucumber repeatedly in the same soil Pseudomonas putida WCS358r Wheat Transient change in the composition of Glandorf et al. (2001) and GMOs the rhizosphere microflora Viebahn et al. (2003) No influence on soil microbial activities Pseudomonas fluorescens F113Rif Clover No influence on the structure of the Walsh et al. (2003) Rhizobium community Small influence on the proportion of Phl-sensitive isolates Microbispora sp. strain EN2 Wheat Treatment with mixture disrupted the Conn and Franco (2004) Streptomyces sp. strain EN27 natural actinobacterial endophyte Nocardioides albus EN46 population, reducing diversity and Single isolates; mixture colonisation levels Treatment with single isolates – population was not adversely affected Pseudomonas fluorescens strains Wheat Inoculation with Z30-97 resulted in Blouin-Bankhead et al. (2004) 2-79, Q8rl-96, and a several shifts in rhizosphere bacterial recombinant strain, Z30-97 community structure Serratia plymuthica HRO-C48 Strawberry and potato – Only negligible, short-term effects due Scherwinski et al. (2006) Streptomyces sp. HRO-71 Verticillium dahliae to the bacterial treatments Serratia plymuthica 3Re4-18 Lettuce – Rhizoctonia Only negligible, short-term effects due Scherwinski et al. (2007, 2008) Pseudomonas trivialis 3Re2-7 solani to the bacterial treatments Pseudomonas fluorescens L13-6-12 GRItrlywt hzshr omnte 105 Communities Rhizosphere with Interplay PGPR 4 Pseudomonas fluorescens SBW25 Wheat Only minor impacts were found on native J€aderlund (2008) microflora due to bacterial (GMM or wild-type) inoculation Collimonas spp. Soil The introduction of collimonads altered Hoppener-Ogawa€ et al. (2009) the composition of all fungal communities studied but had no effects on fungal biomass increase, cellulose degrading activity or plant performance Pseudomonas jessenii RU47 Lettuce – Rhizoctonia RU47 established as the dominant Adesina et al. (2009) solani Pseudomonas population in the rhizosphere but reduced the impact of R. solani on fungal communities Pseudomonas fluorescens strains Maize A persistence study of the three strains Von Felten et al. (2010) F113, CHA0 and Pf153 indicated that the strains persisted differently over a period of 5 weeks Facultative human pathogenic bacteria Burkholderia cepacia, E. coli, Hydroponically While B. cepacia and P. aeruginosa Morales et al. (1996) Pseudomonas aeruginosa, grown wheat strains showed considerable growth Streptococcus pyogenes in the rhizosphere, E. coli and Staphylococcus aureus survived without substantial growth and Streptococcus pyogenes cells died Escherichia coli O157:H7 Lettuce The time for pathogens to reach detection Mark Ibekwe et al. (2009) limits (real-time PCR) on the leaf surface by plate counts was 7 days after planting in comparison with 21 days in the rhizosphere Escherichia coli O157:H7 Spinach Escherichia coli O157:H7 persisted in soil Patel et al. (2010) for at least 28 days, not on the plant 106 G. Berg and C. Zachow

4.6 Conclusions

Rhizosphere-associated bacteria are as PGPR or BCAs an interesting bio-resource for biotechnological applications. On the other hand, problems with opportunistic infections, which are originally from rhizosphere, will become even more severe due to the increasing numbers of at-risk individuals in the human population. Therefore, it is important to understand the ecological behaviour of rhizosphere- associated bacteria. Further, it is essential to exclude potential pathogenic bacteria at an early stage of product development. Criteria are growth at 37C, grouping in risk groups (http://www.dsmz.de or Dir. 2000/54 EC) or any toxic effect in the C. elegans assay. Otherwise, more research and toxicological data are necessary for risk assessment. In all studies assessing the risk for the environment, mainly transient short-term effects were reported. Due to the fact that a broad spectrum of microorganisms was already investigated, it is not necessary to perform such studies with each individual strain.

Acknowledgements The work has been ﬁnanced in part by the Austrian Science Fund (FWF) and by KWS SAAT AG.

References

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Daniel P. Roberts and Donald Y. Kobayashi

5.1 Introduction

There has been considerable effort directed at finding alternatives to chemical pesticides for suppression of soilborne pathogens due to environmental and human health risks associated with their use (Larkin et al. 1998; Raupach and Kloepper 1998). Interest in developing these alternative disease control measures has increased as there is no guarantee that registered chemicals may remain available for commercial growers as exemplified by the phase-out in use of the broad-spectrum chemical fumigant methyl bromide (Martin 2003). There is added concern since nonregistered chemical compounds currently in development will not be made available for commercial use (Duniway 2002; Noling 2002). Biological control, applied alone or as a component of an integrated pest management strategy, is one tactic being investigated as a means to reduce reliance on chemical pesticides for control of soilborne plant pathogens. The use of biological controls instead of more traditional chemical methods has great appeal (Pielach et al. 2008) due to the view that these biologicals are more environmentally benign and of less risk to human health than synthetic chemical compounds. Numerous small-scale experiments have demonstrated the potential of bacterial biological control agents for control of soilborne plant pathogens (Compant et al. 2005; Kloepper et al. 1989; Larkin et al. 1998; Lugtenberg et al. 1991; Weller 1988). Unfortunately, there is a great deal of inconsistency in performance by biocontrol agents when used in larger scale field trials that more accurately mimic production agriculture (Compant et al. 2005; Handelsman and Stabb 1996). While many factors are attributed to inconsistent biocontrol performance, one major factor receiving increased attention is the effect of spatial heterogeneity occurring among the

D.P. Roberts (*) USDA-ARS, Sustainable Agricultural Systems Laboratory, Henry A. Wallace Beltsville Agricultural Research Center, Beltsville, MD 20705, USA e-mail: [email protected] D.Y. Kobayashi Department of Plant Biology and Pathology, Rutgers University, New Brunswick, NJ 08901, USA

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 111 DOI 10.1007/978-3-642-18357-7_5, # Springer-Verlag Berlin Heidelberg 2011 112 D.P. Roberts and D.Y. Kobayashi physical, chemical, and biological components of the spermosphere and rhizosphere environments (Pielach et al. 2008). The spermosphere and rhizosphere are the regions of soil directly under the influence of seeds and roots, respectively, and are the critical interfaces between plants and microbes where beneficial and detrimental interactions that lead to disease and disease suppression occur (Rudrappa et al. 2008). It will be a challenge to develop effective strategies to decrease inconsistencies in performance of bacterial biocontrol agents in field settings without a better understanding of how these individual physical, chemical, and biological components of the spermosphere and rhizosphere influence these bacterial agents. Without adequate or consistent performance, the use of bacterial and other biocontrol agents in commercial agriculture will most likely remain limited (Pielach et al. 2008). As a result of extensive work on a range of experimental systems, significant advances have been made with regard to our understanding of mechanisms and traits required for suppression of soilborne plant pathogens by biocontrol agents (Haas and De´fago 2005; Pielach et al. 2008). Standard mechanisms of biocontrol include exclusion of the pathogen through competition for limited resources, production of antibiotics or other inhibitory molecules, predation and parasitism, and induction of plant host defense pathways (Pielach et al. 2008). It is widely held that in most applications bacterial biocontrol agents must also colonize the developing rhizosphere to effectively express these mechanisms (Pielach et al. 2008; Haas and De´fago 2005; Compant et al. 2005; Gamalero et al. 2004; Lugtenberg et al. 2001; Weller 1988) and that biocontrol agents are capable of colonizing the rhizosphere only if they have the appropriate traits to defend against harmful chemicals, utilize available nutrients, and contend with the indigenous microflora (Hartman et al. 2009). However, our knowledge of biological control at the whole systems level remains limited. Despite shortfalls in our knowledge, there is solid evidence demonstrating the impact of various rhizosphere environmental factors on expression of genes contributing to biocontrol activity (Haas and De´fago 2005; Kiely et al. 2006). In this chapter, we discuss the heterogeneity of the physical, chemical, and biological components within the spermosphere and rhizosphere as they impact (1) the expression of traits and mechanisms associated with biocontrol, and (2) the performance of bacterial biocontrol agents.

5.2 The Spermosphere and Rhizosphere Inﬂuence Behavior of Bacterial Biological Control Agents

In Vitro Expression Technology (IVET)- and microarray-based experiments provide snapshots at the genetic and genomic levels of the impact that various soil and plant influences have on bacterial biocontrol agents during colonization of seeds and roots (Matilla et al. 2007; Rainey 1999; Silby and Levy 2004). These studies suggest a highly dynamic environment that necessitates the adaptation of bacteria 5 Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere 113 to detrimental environmental stresses and a varied nutritional environment (Matilla et al. 2007; Rainey 1999). IVET experiments with Pseudomonas fluorescens in soil show differential upregulation of genes functioning in nutrient transport and utilization, detoxification, and regulation (Silby and Levy 2004). Pseudomonas genes differentially expressed during colonization of the rhizospheres of sugar beet and corn include those functioning in nutrient utilization and stress responses and reflect the effects of the rhizosphere environment on general metabolism and the need for protection against detrimental factors encountered within this environment (Matilla et al. 2007; Rainey 1999). Expression of genes functioning in type III secretion in the plant rhizosphere suggests an intimate association of the biocontrol bacterium with the plant (Jackson et al. 2005; Mark et al. 2005; Rainey 1999).

5.3 Soil Factors Inﬂuence Biological Control Agents in the Spermosphere and Rhizosphere

Soils consist of aggregates that vary spatially in size, composition (clay, silt, sand, organic matter), and chemistry, as well as in the voids between and within these aggregates. The voids can be occupied by gases, differing aqueous solutions, and a variety of soil microbial communities (Buyer et al. 1999; Foster 1988; Garbeva et al. 2004; Hattori and Hattori 1976; Wieland et al. 2001) thereby creating vastly diverse microenvironments that can profoundly affect the distribution, persistence, and physiology of biocontrol agents. As an example, in the absence of percolating water, which in itself can greatly influence downward movement of bacteria on roots, soil matric potential becomes a major factor controlling the downward movement of bacteria (Liddell and Parke 1989; Parke et al. 1986; Scott et al. 1995). Voids of smaller sizes represent microenvironments that retain water longer during dry periods, decreasing exposure of inhabiting microbes to desiccation stress (Foster 1988). Void size can also influence predation of bacteria by protozoans (Postma and van Veen 1990; van Veen et al. 1997). Abiotic soil factors, such as pH, temperature, high osmotic conditions, or matric tension, are also known to impose stresses on microorganisms that influence their survival (van Elsas and van Overbeek 1993; van Veen et al. 1997). Chemical compositions of soil appear to influence survival of microorganisms in the soil. Bashan et al. (1995) demonstrated that concentrations of nitrogen, potassium, and phosphate in soil are correlated with survival of Azospirillum brasilense. In addition to influencing survival, abiotic soil factors have demonstrated their impact on the expression of biocontrol genes in bacterial agents (Haas and De´fago 2005). For example, a number of soil factors influence secondary metabolite production in P. fluorescens. Excess iron concentrations repress biosynthesis of the iron siderophores pyoverdine and pyochelin, while low oxygen tensions and low iron availability are required for production of volatiles like hydrogen cyanide in certain pseudomonads (Haas and De´fago 2005). Some other factors such as oxygen concentration, pH, and temperature were shown to impact expression of the 114 D.P. Roberts and D.Y. Kobayashi antibiotic phenazine-1-carboximide in P. chlororaphis (van Rij et al. 2004). These in vitro studies on gene expression were corroborated by studies where certain physical and chemical factors in soil were shown to influence disease suppression by bacterial biocontrol agents (Duffy and De´fago 1997; Duffy et al. 1997; Haas and De´fago 2005; Ownley et al. 2003). It is clearly evident from the examples described earlier that variation in numerous soil factors, including aggregation, voids, chemistry, and resident microflora, can impose heterogenous conditions in a relatively small volume of the field such as a single plant rhizosphere. The end result is a spatially diverse collection of unique and localized microenvironments (Smiles 1988; van Elsas and van Overbeek 1993), each with the potential to influence persistence, distribution, gene expression, and ultimately the performance of bacterial biocontrol agents in different ways.

5.3.1 Plants Inﬂuence the Complexity and Heterogeneity of the Spermosphere and Rhizosphere

As a consequence of the growth and development of the root system, an extremely diverse range of organic and inorganic compounds can be taken up or released by seeds and roots into the soil. The uptake and release of these compounds has a profound effect on the physical and chemical properties of the soil and on the indigenous microflora (Badri and Vivanco 2009; Rougier 1981), and add to the complexity and spatial heterogeneity of microenvironments encountered by bacterial biological control agents. For example, protons and electrons are secreted within carbon compounds as undissociated acids or compounds with reducing capabilities. Acidification of the surrounding soil can occur with the release of protons and organic acids from the seed and root, and uptake of nutrient ions by the plant (Hartman et al. 2009). While pH levels represent one example of how plants can significantly alter soil properties, there are other examples of how plants influence soil changes. Oxygen consumption, due to respiration by the root and associated microflora, can result in steep redox gradients in the rhizosphere (Hartman et al. 2009). Organic material released into the soil by the plant in the form of rhizodeposits significantly influences the nutritional environment encountered by bacterial biocontrol agents. Estimates regarding the percentage of photosynthate released vary with the methods employed in the analysis (Farrar et al. 2003) and have been reported to be as high as 30–40% in young seedlings (Lugtenberg et al. 1999; Meharg 1994; Whipps 1990). Root products and rhizodeposits entering the soil probably consist of almost every type of plant compound as sloughed cells from the root cap and root hairs decay or the rhizodermis degrades in older portions of roots. Root products with specific biological activity can also be actively secreted (De-la Penã et al. 2008; Hartman et al. 2009; Wen et al. 2007). Reduced carbon compounds can be divided into low- and high-molecular-weight compounds with the low-molecular-weight compounds consisting of a diverse mixture of 5 Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere 115 carbohydrates, amino acids, organic acids, phenolics, and secondary metabolites. High-molecular-weight components such as proteins and mucilage have been reported (Badri and Vivanco 2009; Wen et al. 2007). The quantity and quality of root exudates are determined by plant species, age of the plant, soil nutritional environment, and biotic agents and abiotic soil factors that cause plant stress (Badri and Vivanco 2009). For example, plants under iron stress release phytosiderophores or protons and chelators (phenolics, carboxylates) to acquire iron while phosphorous deficiency in many plants enhances the production and release of phenolic and carboxylate compounds (Hartman et al. 2009). Organic compounds released by seeds and roots can impact bacterial biological control agents in many ways, serving as important nutrients, attractants, and deterrents (Badri and Vivanco 2009; Pielach et al. 2008). Arguably, one of the most significant influences plants impose on bacterial biocontrol agents residing in the spermosphere and rhizosphere, however, is on nutritional content, which is discussed in detail in the following section.

5.3.1.1 Plant Inputs Serve as Nutrients and Inﬂuence the Behavior of Biological Control Bacteria

Soluble reduced carbon compounds within rhizodeposits are thought to have the greatest stimulatory impact on growth and metabolic activity of microbes in the spermosphere and rhizosphere (Kraffczyk et al. 1984; Lynch and Whipps 1990). Sugars, amino acids, and organic acids are typically the dominant soluble reduced carbon compounds in rhizodeposits (Farrar et al. 2003; Lynch and Whipps 1990) and their availability affects growth and metabolism of biocontrol agents in the soil. The importance of reduced carbon inputs from plants into soil is perhaps most easily seen in studies determining the significance of these nutrients on bacterial colonization. Roberts et al. (1999a, 2000) studied the impact of nutrients released from cucumber and pea seeds on spermosphere populations of Enterobacter cloacae. These two seed types differ dramatically in the quantity, and therefore availability, of reduced carbon nutrients within their exudates to colonizing bacterial biocontrol agents. Pea seeds release two to three orders of magnitude more carbohydrate and amino acid during the first 24 h after imbibition than cucumber seeds. Consequently, pea seeds support substantially greater growth and population sizes of E. cloacae than cucumber seeds (Roberts et al. 1999a, 2000). Further evidence that differences in populations of E. cloacae carried by pea compared with cucumber seeds are directly related to the quantity of reduced carbon nutrients in exudate comes from studies using E. cloacae mutant strains that are impaired in catabolic capabilities. Mutants disrupted in key enzymatic steps of catabolic pathways are no longer capable of using certain compounds in exudate as nutrient sources. The net result is one that mimics a reduction in exudate nutrients available for use during growth in the spermosphere or rhizosphere (Liu et al. 2007; Roberts et al. 1999a, 2000, 2007). E. cloacae A-11 contains a mutation in pfkA,which encodes the key glycolytic enzyme phosphofructokinase. With the exception of fructose, this mutant cannot catabolize hexose sugars found in seed exudate resulting 116 D.P. Roberts and D.Y. Kobayashi in a reduction in sugar compounds in the spermosphere available to the mutant compared with the wild-type strain 501R3. Indeed, seed colonization experiments demonstrated that strain A-11 is significantly reduced in population levels in the cucumber spermosphere relative to the wild-type strain (Fig. 5.1b). When exogenous fructose is supplied to cucumber spermosphere to compensate for the nutritional deficit, colonization ability by strain A-11 can be restored to levels comparable to the wild type (Roberts et al. 1999a, 2000). In addition to the glycolytic mutant A-11, colonization effects have been observed with E. cloacae catabolic mutant strains containing mutations in sdhA and aceF, which encode subunits for succinate dehydrogenase and the pyruvate dehydrogenase complex, respectively (Liu et al. 2007; Roberts et al. 2007). The sdhA mutant strain M2 is severely impaired in in vitro growth on almost all amino acids and organic acids detected in seed exudates but relatively unaffected in growth on the carbohydrates. The aceF mutant strain M43 is more severely affected, with only limited in vitro growth in almost all components of seed exudate. The resulting reduction in nutrient utilization capabilities of these strains is reflected in their colonizing populations in cucumber spermosphere. The sdhA mutant strain M2 is significantly reduced in colonization relative to wild-type strain 501R3, while the aceF mutant strain M43 is completely deficient in colonization (Roberts et al. 1996, 2007). Along with cell population levels, metabolic activity (as measured by metabolic energy status) of colonizing bacteria has been found to correlate with plant exudate content. Using a bioluminescence-based reporter system, it was shown that E. cloacae displayed significantly higher metabolic energy status per cell on pea seed than on cucumber seed (Roberts et al. 2009), corresponding directly with differences in quantities of exudate between the two seed types. In vitro experiments using oxygen consumption to measure metabolic activity by E. cloacae corroborated the role of pea seed exudate in supporting higher metabolic activity in pea spermosphere. Additionally, the pfkA mutant strain A-11 had lower average metabolic energy status per cell than the wild-type strain in both cucumber and pea spermosphere due to its inability to use the full range of sugars available to the wild type (Fig. 5.1a, c). These results indicate that greater exudation by pea seed not only supports higher populations of cells, but that the average bacterial cell colonizing a pea seed is more active metabolically than that colonizing a cucumber seed due to this greater exudation. These observations suggest profoundly different influential effects of microenvironments that vary in nutrient levels on expression of traits by biocontrol bacteria. When analyzed further, the example described previously illustrates the complexity of the influence that plant exudation imposes on the behavior of bacterial biocontrol agents. In addition to quantity of exudate, individual components of the exudate have been shown to differentially influence colonization and metabolic activity by biocontrol agents. Roberts et al. (2000, 2009) demonstrated that qualitative differences in seed exudate not only influenced growth rate during seed colonization by E. cloacae, but also the average metabolic energy status per cell. In this particular study, hexose sugars had the greatest stimulatory effect on the 5 Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere 117

a 7 A11(pGL3.1) 501R3(pGL3.1) A11(pGL3.1)+Fructose 6 501R3(pGL3.1)+Fructose C C C B B 5 C B

B B RLU/ seed A 10 4 A A A

Log A A A A

2 b (2 hours) (18 hours) (45 hours) (72 hours) 8

7 B C C C C B 6 B B B B A 5 A A A CFU/ seed A A A 10

Log 4

2 (2 hours) (18 hours) (45 hours) (72 hours) c 1.0

0.9 B C B A B C B B 0.8 A CFU A B A A 10 A A A A 0.7 RLU/Log 10 0.6 LOg

0.5

0.4 (2 hours)(18 hours)(45 hours)(72 hours)

Fig. 5.1 Colonization and metabolic energy status of Enterobacter cloacae strains 501R3 and A-11 in cucumber spermosphere in the presence and absence of exogenous fructose. Both strains 118 D.P. Roberts and D.Y. Kobayashi enteric bacterium, E. cloacae. However, the metabolic influence by both the type and concentration of exudate components may be completely different in taxonomically unrelated bacteria, such as fluorescent pseudomonads, which favor organic acids over simple sugars as preferred carbon sources for growth. While greater exudation or nutrient availability may prolong metabolic activity, extend colonization persistence, and enhance expression of certain traits; its influence on overall biological control performance is directly dependent on the pathosystem, the biocontrol agent, and the mechanism by which disease suppression occurs (Pielach et al. 2008). An excellent example of the confounding influence of exudation on performance by bacterial biocontrol agents comes from a series of studies investigating the mechanism by which E. cloacae suppresses damping-off caused by Pythium ultimum.vanDijkandNelson(2000) demonstrated that degradation of certain fatty acids by E. cloacae was responsible for suppression of P. ultimum damping-off. These plant-derived fatty acids serve as signals for spore germination by P. ultimum and therefore are important for infection and disease. A subsequent study by Kageyama and Nelson (2003) demonstrated that variation in nutrient availability between spermospheres of different plant species had a profound effect on biocontrol of P. ultimum damping-off. Biological control activity by E. cloacae was much less effective in nutrient-rich spermospheres, such as pea, compared with nutrient poor spermospheres, such as cucumber. Winstam and Nelson (2008) demonstrated that concentrations of sugars in the nutrient-rich spermospheres were sufficient for catabolic repression of fatty acid degrading enzymes in E. cloacae, resulting in loss of biocontrol activity. This variable disease suppression behavior by E. cloacae underscores the impact of differential plant inputs and/or availability of certain nutrients in plant-associated environments on biocontrol activity. Hence, differential plant inputs of certain nutrients may have an effect on the pathogen as well and thus indirectly impact performance of the biocontrol agent. It has been posed that the metabolically active portion of colonizing populations of plant-beneficial bacteria is most important for success in their biotechnological applications (Heijnen et al. 1995; Ramos et al. 2000; Unge et al. 1999) as metabolic energy is necessary for expression of required traits (Crowley et al. 1996; Sørenson et al. 2001). Clearly, metabolic activity is essential for biological control to occur successfully; however, the above studies with E. cloacae represent a caveat regarding how one addresses the importance of bacterial metabolic activity to biocontrol performance. The relevance of metabolic activity is system dependent, further emphasizing the need to better understand the mechanisms by which biocontrol bacteria suppress disease.

Fig. 5.1 (continued) contained the bioluminescence reporter plasmid pGL3.1 (Roberts et al. 2009). (a) Relative luminescence units (RLU) from total populations of E. cloacae strains. (b) Colony-forming units (CFU) of E. cloacae strains. (c) Average metabolic energy status per cell of E. cloacae strains. Experiments were performed as described in Roberts et al. (2009) with the exception that strains were not starved prior to application to cucumber seed 5 Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere 119

5.3.1.2 The Distribution and Bioavailability of Nutrient Compounds to Bacterial Biological Control Agents Are Nonuniform in the Spermosphere and Rhizosphere

The previous section emphasizes the impact of the variability in types and concentrations of plant-derived nutrients from different plant host species on the populations and physiology of bacterial biocontrol agents. Nutrient availability also varies spatially within the spermosphere and rhizosphere of an individual plant leading to impacts on the biocontrol agent situated in different locations in the spermosphere or along the root. The spermosphere and rhizosphere can be thought of as gradient systems where diffusible compounds released from seeds and roots influence microbes in regions of the soil that extend for millimeter distances radially from the plant (Helal and Sauerbeck 1983, 1986;Toaletal. 2000). As distance from the plant increases, the concentration of plant-released compounds decreases. There are many factors that contribute to the formation of this gradient, including binding of these compounds by soil particles and their degradation or uptake by the associated microflora (Farrar et al. 2003;Hartman et al. 2009). In contrast, concentrations of soil-derived nutrients such as nitrogen or phosphorus tend to increase with increasing distance from the root due to their uptake by roots. Exudation of organic compounds into the soil varies along the root longitudinally with the maturation phase of the root system (Badri and Vivanco 2009;Walkeretal. 2003). Most roots can be divided into four classes ranging from the tip to the base of the root: (1) the root tip, consisting of the root cap and meristematic region; (2) the elongation zone; (3) the maturation zone; and (4) the mature zone (Gilroy and Jones 2000). Experiments involving 14C-labeling and the use of reagents such as ninhydrin to detect organic compounds released from roots have identified exudation sites along the root system (McDougall and Rovira 1970; Van Egeraat 1975). In general, the zone immediately behind the root tip is considered a major site of exudation (Badri and Vivanco 2009). This is not surprising as the xylem and phloem are not yet mature necessitating the flow of nutrients through the apoplast to the root tip. These nutrients are readily released into the adjoining soil due to diffusion, as this region of the root is not yet suberized (Cardon and Gage 2006). Regions of older roots, such as points of secondary root emergence, release exudate as well (Van Egeraat 1975). Additionally, the root cap, border cells, and root hair cells have been reported to be involved in the release of compounds into the rhizosphere (Czarnota et al. 2003; Farrar et al. 2003; Nguyen 2003; Pineros et al. 2002; Wen et al. 2007). Therefore, the distribution of reduced carbon compounds and other nutrients in the rhizosphere vary both radially away from the root as well as longitudinally along the root. Distribution of nutrients in the spermosphere and rhizosphere at a given point in space will also vary over time due to the growth and maturation of the root and to the development of attendant soil microbial communities. This is evidenced by wave-like patterns of indigenous bacterial populations developing while root tips move through soil; pulsing the localized soil environment with nutrients (Semenov et al. 1999; van Bruggen et al. 2008). 120 D.P. Roberts and D.Y. Kobayashi

Molecular tools, based on whole-cell bioreporter systems, are being constructed to better determine the availability of spermosphere and rhizosphere nutrients to microbes on micrometer scales, which is representative of the scale that individual microbial cells sense their environment (Leveau and Lindow 2001). Although current biosensors have been constructed to detect only a small portion of compounds in the spermosphere and rhizosphere (Farrar et al. 2003), work with these biosensors is providing insight into the spatial distribution of nutrients that are available for uptake by bacterial biocontrol agents. Working with an ice-nucleating-reporter-based system, Jaeger et al. (1999) demonstrated that availability of sucrose was maximal near the root tip and decreased dramatically with increasing distance along the root away from the root tip. On the other hand, bioavailability of the amino acid tryptophan was minimal at the root tip and increased along the root until 12–16 cm back from the tip. Bringhurst et al. (2001) detected bioavailable galactosides around root hairs of legumes and in regions where lateral roots emerged from main roots, but not at root tips. Joyner and Lindow (2000) demonstrated substantial heterogeneity in iron bioavailability to Pseudomonas syringae in the rhizosphere. The emerging picture is that the spermosphere and rhizosphere are spatially and temporally heterogeneous on micro and macro scales with regard to the concentration of individual nutrients and their availability to biocontrol agents. The impact of this spatially heterogeneous distribution of nutrients on bacterial biocontrol agents is readily seen when analyzing the distribution of these bacteria in the rhizosphere and their metabolic activity. Several studies, including those using ﬂuorescent pseudomonads and E. cloacae on roots of different crop species, indicate that these bacteria have a wide, but nonuniform distribution in the rhizosphere (Bahme and Schroth 1987;Bulletal.1991; Dandurand et al. 1997; Lohrke et al. 2002; Loper et al. 1984; Roberts et al. 1999a, b, 2003; Weller 1988). Metabolically active cells of bacterial biocontrol agents are nonuniformly distributed in the rhizosphere and form a subset of the colonizing population. Microbes are known to have greater activity in association with plants than in bulk soil (Kroer et al. 1998; Rattray et al. 1995). However, only a small portion of the bacterial population maintains high levels of metabolic activity in the rhizosphere; the majority of microbial cells having metabolic characteristics similar to those of starved cells (Marschner and Crowley 1996; Normander et al. 1999; Ramos et al. 2000). It is thought that variations in nutrient availability as well as differences in the compositions of plant-derived compounds result in this spatial heterogeneity of metabolic activity (Brennerova and Crowley 1994; Crowley et al. 1996; Heijnen et al. 1995; Kragelund et al. 1997; Ramos et al. 2000; Rattray et al. 1995; Sørenson et al. 2001). Speciﬁc studies have indicated that regions along roots where root exudation is greatest, such as behind root tips and near lateral roots, support the highest metabolic activity by bacterial cells (Boldt et al. 2004; Brennerova and Crowley 1994). At the single-cell level, only small portions of the colonizing population within these regions are metabolically active (Ramos et al. 2000). 5 Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere 121

5.3.1.3 Molecules Released by Plants into the Spermosphere and Rhizosphere Can Have Detrimental Effects on Residing Microorganisms, Including Biological Control Bacteria

Roots produce a number of organic compounds with antibacterial and/or antifungal properties that can be released into the rhizosphere, and some of these compounds have broad-spectrum activity (Bais et al. 2004b). These compounds most likely function in protecting the plant from infection by pathogens but also may be detrimental to bacterial biocontrol agents. In particular, border cells sloughed from the root tip appear to have a fundamental role in protecting the plant from pathogen attack (Wen et al. 2007). Border cells release a number of compounds that attract, repel, and influence gene expression in soilborne microbes (Farrar et al. 2003). The border cell secretome of pea was shown to contain a complex mixture of proteins that potentially function in protection of the vulnerable root cap from pathogen infection (Wen et al. 2007). The root cap is surprisingly devoid of infection by pathogens (Olivain and Alabouvette 1999; Turlier et al. 1994; Wen et al. 2007) or colonization by beneficial microbes (Assmus et al. 1997; Gamalero et al. 2005) despite being a major site of organic carbon input into the soil. In hydroponic culture, 98% of organic material released into the medium is thought to be derived from the root apex (Farrar et al. 2003). Rudrappa et al. (2007) present findings that demonstrate the impact of root- produced detrimental compounds on biofilm formation and disease suppression by the biocontrol agent Bacillus subtilis FB17. Strain FB17 did not form biofilms on the surface of roots of the NahG transgenic line of Arabadopsis. Inhibition of biofilm formation was shown to be a response by strain FB17 to the presence of catechol on the surface of roots; catechol leading to the production of reactive oxygen species. The presence of this compound led to the downregulation of the yqx -sipW-tasA and epsA-O operons which are important for biofilm formation by this bacterium. Biofilm formation was previously demonstrated to be of importance in disease suppression by B. subtilis (Bais et al. 2004a). Evidence suggests that plants can selectively detect a pathogen and respond through the release of antimicrobial compounds (Bais et al. 2004b). Earlier, Bais et al. (2002) demonstrated that release of rosmarinic acid by roots was elicited by cell walls from the fungal plant pathogen Phytophthora cinnamoni. This compound is inhibitory to a number of fungal and soilborne microorganisms, indicating that the ability of biocontrol agents to escape such inhibitory activity may be of primary importance for successful plant associations to occur.

5.3.1.4 Indigenous Microbial Communities Are Distributed Nonuniformly in the Spermosphere and Rhizosphere

Plant inputs into soil also affect the local soil environment by stimulating the development of indigenous microbial communities in the rhizosphere. Plant exudates have been shown to select bacteria from the indigenous bacterial community 122 D.P. Roberts and D.Y. Kobayashi in bulk soil, resulting in microbial communities within the rhizosphere that are different from those observed in bulk soil. Such communities not only can vary between plant species, but also between locations along the root. This effect is probably linked to root exudate composition which, as discussed above, varies not only between plant species and regions of the root, but also as the result of environmental influences (Garbeva et al. 2004; Haichar et al. 2008; Wieland et al. 2001; Vandenkoornhuyse et al. 2007; Yang and Crowley 2000). The combined effect of plant and soil factors on the indigenous microflora is a spatially diverse collection of microbial communities with different capacities for nutrient competition and production of inhibitory metabolites. Green fluorescent protein (gfp)- tagged cells of the biocontrol bacterium P. fluorescens introduced into the soil as a treatment on barley seeds were often physically associated with indigenous bacteria on barley roots (Normander et al. 1999). This would allow for negative interactions such as competition for nutrients and other resources, antibiosis, and disruption of regulatory networks that control expression of genes involved in disease suppression mechanisms (Haas and De´fago 2005). Thus, bacterial biocontrol agents must also contend with indigenous microflora in order to express intended suppressive effects on pathogens.

5.3.1.5 Biological Control Agents Must Colonize the Rhizosphere for Effective Disease Suppression to Occur

As discussed in the preceding sections, biocontrol agents can only colonize the rhizosphere if they have the appropriate traits to defend against harmful chemicals and other adverse environmental conditions, utilize available nutrients, and contend with the indigenous microﬂora (Hartman et al. 2009). If a bacterial biocontrol agent does not effectively colonize and become widely distributed throughout the rhizosphere, it is thought that effective disease suppression will not be achieved in most cases. This is because many pathogens are believed to infect plants near root tips, in the zone of root elongation, or through root hairs, which represent infection courts that can be relatively distant from the point of introduction of the biocontrol agent into the soil. Close proximity of biocontrol agents to pathogens is likely to be important in cases where they suppress pathogens through the production of secondary metabolites, such as antibiotics or siderophores, so that these inhibitory compounds are present in sufﬁcient concentration to be effective (Gamalero et al. 2004). Occupation of the same microenvironment by the bacterial biocontrol agent and pathogen is also likely to be important in cases where they suppress pathogens through competition for resources such as nutrients or through predation/parasitism. Even with biocontrol agents that suppress disease through induction of plant defense responses, it is likely that redistri- bution of populations from the dead seed coat to other plant tissues capable of a physiological response is necessary. Close contact of the bacterial elicitor to the plant cell membrane is thought to be needed for induction of a plant response (Leeman et al. 1995). 5 Impact of Spatial Heterogeneity Within Spermosphere and Rhizosphere 123

Strong evidence of a role for extensive colonization of the rhizosphere by biocontrol agents came from Lugtenberg’s group in The Netherlands working with suppression of Fusarium oxysporum f.sp. radicis-lycopersici on tomato with P. chlororaphis PCL1391 (Chin-A-Woeng et al. 2000). This pathogen approaches the root via root hairs, colonizes the root surface, and then infects at random sites throughout the root system (Lagopodi et al. 2002). For this work, three classes of colonization mutants of strain PCL1391 were constructed that were deficient in traits or loci previously demonstrated to be important for root colonization (Lugtenberg et al. 1999). All mutants produced wild-type levels of phenazine- 1-carboximide, an antibiotic crucial for control of F. oxysporum f.sp. radicis- lycopersici (Chin-A-Woeng et al. 1998). Production of other inhibitory compounds potentially involved in disease suppression (hydrogen cyanide, chitinase, protease) was shown to be unaffected by these mutations. All colonization mutants were reduced in disease suppression despite being unaffected in production of these metabolites (Chin-A-Woeng et al. 2000). In subsequent work, it was shown that autofluorescent protein-tagged PCL1391 and F. oxysporum f.sp. radicis-lycopersici strains compete for the same sites on tomato roots and that PCL1391 readily colonized the hyphae of this pathogen (Bolwerk et al. 2003). Colonization density of the pathogen was reduced when in close proximity with PCL1391. Only with damping-off pathogens has the importance of extensive spatial distribution of bacterial biocontrol agents throughout the rhizosphere been demonstrated to be of lesser importance (Roberts et al. 1997). With damping-off diseases the infection courts are the seed coat, endosperm, embryo, the emerging radicle, hypocotyl, and cotyledons (Paulitz 1992). These tissues have a limited spatial distribution in soil at the end of a brief period of high susceptibility to disease. Consequently, large populations of biocontrol bacteria can be readily delivered directly to the infection court as seed treatments (Paulitz 1992). We demonstrated that it is possible to suppress pre-emergence and post-emergence damping-off of cucumber caused by P. ultimum with E. coli S17R1 in the absence of extensive or persistent root colonization (Roberts et al. 1997). This bacterium was root-colonization deficient on cucumber plants, being limited to the seed coat and upper 1 cm of cucumber root at 7 days and nondetectable after 42 days. E. coli S17R1 and a root-colonization- proficient strain of E. cloacae provided similar and effective levels of biological control of damping-off of cucumber seeds and seedlings (Roberts et al. 1997).

5.4 Conclusion

In this chapter, it is highlighted that variations in the numerous soil- and plant- derived factors within the spermosphere and rhizosphere impose heterogeneous conditions in relatively small volumes of the field. On field scales, the end result is a collection of plant rhizospheres containing myriad unique and localized microenvironments, each microenvironment with the potential to influence persistence, distribution, expression of biocontrol traits, and ultimately the performance of 124 D.P. Roberts and D.Y. Kobayashi bacterial biocontrol agents in different ways. Inconsistent performance in disease suppression on field scales occurs when the influences of these microenvironments (1) preclude sufficient colonization of certain rhizospheres by the biocontrol agent necessary for effective disease suppression and/or (2) result in subpopulations of biocontrol bacteria residing in certain rhizospheres that are not in the correct physiological state for disease suppression. Overcoming inconsistent performance by bacterial and other biocontrol agents at the field-scale level is necessary if the use of these biological controls is to become widespread in production agriculture. Approaches to overcoming inconsistent performance by biocontrol agents resulting from the spatial heterogeneity in physical, chemical, and biological components of the spermosphere and rhizosphere include their integration into multitactic disease management strategies, where different biocontrol agents are combined with each other or with other biologically based disease management technologies such as cover crops. These approaches have been documented in the literature for some time (Lemanceau and Alabouvette 1991; Lemanceau et al. 1993; Pierson and Weller 1994; Raupach and Kloepper 1998). A combination of biocontrol agents is more likely to have a greater variety of traits responsible for suppression of the pathogen. Further, it is likely to have these traits expressed over a wider range of microenvironmental conditions due to the different ecological adaptations of the producing strains. Likewise, combining biocontrol agents with cover crops with disease suppressive capabilities will increase the likelihood of disease suppressive activity over a wider range of microenvironmental conditions. The current challenge is to develop a greater understanding of spermosphere and rhizosphere factors, and their impacts on biocontrol agents, so that strategic combinations of biocontrol agents or other biologically based technologies can be made; the combinations being made to overcome the known susceptibilities of each individual component to given microenvironmental conditions.

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Anjani M. Karunaratne

6.1 Introduction

The immediate microenvironment surrounding a plant root in the plant–soil interface, having much microbial activity (due to bacteria, yeast and fungi), is referred to as the rhizosphere. When referring to plant growth promotion, bacteria are given a special place although the rhizosphere fungi (as ectomycorrhizae and endomycor- rhizae) also play a significant role in nutrient acquiring function of roots. The term “rhizobacteria” decribes the ability of certain bacteria to colonize the rhizosphere aggressively (Rao 1993). Certain strains of rhizosphere bacteria are known as plant growth promoting rhizobacteria (PGPR) because their application can stimulate growth and improve plant stand under stressful conditions (Van Loon et al. 1998). Well over a decade ago, demonstrating the attention PGPR have attracted, Bashan and Holguin (1998) have reported that close to 4,000 publications have appeared in the decade prior to their publication in the mid-1998. There is no reason to believe that the attention has declined over the years thereafter, as different aspects of PGPR have been reviewed in the recent past as well (Lucy et al. 2004; Van Loon 2007; Lugtenberg and Kamilova 2009). The term PGPR dates back to the 1970s, and this term has been used in the early days to describe the biocontrol ability of these bacteria along with their aggressive colonization and plant growth stimulation (Kloepper and Schroth 1978). Davison (1988) has mentioned that the beneficial effects of PGPR fall into two categories according to whether the bacteria benefit the plant directly or indirectly by antagonizing a phytopathogen (for which the term “biocontrol” is used) or removing a growth inhibitor. Hence a decade ago, two new terms for general scientific usage as biocontrol-PGPB (PGPB standing for “plant growth promoting bacteria”) and PGPB have been suggested (Bashan and Holguin 1998). These two authors argued that since many beneficial bacteria are not rhizosphere bacteria, replacing the term

A.M. Karunaratne Department of Botany, University of Peradeniya, Peradeniya, Sri Lanka e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 131 DOI 10.1007/978-3-642-18357-7_6, # Springer-Verlag Berlin Heidelberg 2011 132 A.M. Karunaratne

“rhizobacteria” with “bacteria” to coin the term PGPB is more appropriate. How- ever, not all publications thereafter have adhered to this terminology. Bashan and Holguin (1998) correctly pointed out that the term PGPR does not cover nonrhizosphere interactions like biocontrol in the phyllosphere. It is also striking that the term PGPB has been used in connection to phyllosphere, anthosphere or spermosphere (Hallman et al. 1997; Compant et al. 2005). Bashan and Holguin (1998) further pointed out that at the early stages its broad reference (meaning PGPR in biocontrol) was an advantage, but today (referring to 1998) the term was too general and nonspecific. Interestingly, chronological detection of Burkholderia phytofirmans initially on root surfaces, next in root internal tissues, then in the internode and on xylem vessels and substomatal chambers of leaf tissue has been described (Compant et al. 2006) giving weightage to the idea proposed by Bashan and Holguin. Some PGPR being established as endophytic population in stem, leaves, tubers, and other organs, after transcending the endodermis barrier, crossing from the root cortex to the vascular system has also been noted (Compant et al. 2005; Kloepper et al. 1999), further substantiating the wisdom of the statement by Bashan and Holguin, indicating the absence of clear demarcations for their location on plants. Postharvest scientists also have noted that the antagonists encountered in the fructoplane may have come from other closely related or unrelated sources, and phylloplane has been a good source of antagonists (Janisie- wicz and Korsten 2002). This phenomenon has been expressed for the occurrence of pathogens too. For instance, the presence of pathogen conidia on dead floral parts as debris on the ground has been recorded for the banana fruit pathogen Colleto- trichum musae (Adikaram 2005) and avocado fruit pathogen C. gloesporioides (Karunaratne et al. 1999) in twigs, leaves, flowers, and debris under the tree. Further Pinto et al. (2000) have documented that the postharvest pathogens of banana C. musae and Fusarium moniliforme live endophytically in plant tissues asymp- tomatically and could have pathogenic activity (in causing banana crown rot by a pathogen complex or anthracnose by C. musae) at a later stage, causing visible symptoms. Initial emphasis on biocontrol has been on soilborne plant pathogens (Cook 1990). The very first mention of postharvest disease control by a biological agent is the control of Botrytis rot of strawberry with a fungal antagonist Trichoderma (Tronsmo and Dennis 1977) which was more than 40 years ago. However, much focus on the biocontrol of postharvest pathogens of fruits has been observed in the last decade of the twentieth century and the first decade of the twenty-first century. For instance, in his text book Campbell (1989) has mentioned that there is little research on the biological control of diseases of flowers and fruit and even less practical applications. As late as 2002, Janisiewicz and Korsten have compared the long-standing interest in biological control of soilborne pathogens, with research into biological control of postharvest diseases and noted that the latter is in its infancy. A recent review by Droby et al. (2009) states that while in the early 1980s one could find 1–2 publications per year on postharvest biocontrol, now a literature search on the topic will bring up at least a hundred related publications per year. Of the postharvest environment, fruits have attracted more attention than vegetables. 6 Biocontrol Mechanisms Employed by PGPR 133

In fact, Wilson and Wisniewski (1989) have reported that since 1983, an explosion of research has occurred in the area of postharvest biocontrol of mainly fruit diseases. Therefore, in retrospective it appears that the accelerated interest in biocontrol of postharvest pathogens is more recent. Unlike research on PGPR, research on biocontrol for postharvest pathogens appeared to have gained interest, as a pressing need to search for alternatives to replace agrochemicals. An impetus to look for biological control strategies to control postharvest diseases of fruits and vegetables was a report of the US National Academy of Sciences that indicated the dangerous presence of oncogenic compounds in pesticides (NAS 1987). In fact, the use of agrochemicals has been the main means of preventing postharvest pathogen attack (Eckert and Ogawa 1985) until the need to look for alternatives was felt and even thereafter due to the lack of suitable alternatives to takeover their role completely. Wilson and Wisniewski (1989) have correctly pointed out that postharvest diseases have not received the attention that the magnitude of the problem warrants. By late 1980s and early 1990s, a number of book chapters and reviews on biological control of postharvest disease (Wilson and Pusey 1985; Chalutz et al. 1988; Janisiewicz 1988; Jeffries and Jegger 1990; Janisiewicz 1991; Wilson and Wisniewski 1989; Wisniewski and Wilson 1992; Barkai-Golan 2001) indicate the attention on this agrochemical-free alternative to combat disease on harvested produce. Since then, biological control still remains as a potential area requiring further investigation for successful ﬁeld application. It is reported that about 90% of the 2,000 major diseases of the 31 major crops in the USA are caused by soilborne plant pathogens (Wilson 1968; Lewis and Papaviza 1991). This gives the cue that answers may lie in the rhizosphere. The long-standing interest on PGPR appears to present a wealth of knowledge to applications on biocontrol strategies. In fact Lucy et al. (2004) have expressed similar sentiments by stating that PGPR present an alternative to the use of chemicals for plant growth enhancement in many different applications. By and large, it appears that the development of research on postharvest applications of biocontrol has taken a technological route with applied aspects taking a lead role. On the other hand, research on PGPR, having a longer history seems to have taken a fundamental approach and may have much to offer to this relatively younger area of research on biocontrol. Therefore, it may be timely to look at the development of these two areas of research in perspective for a mutual beneﬁt for both disciplines. Some selected milestones for the present discussion in the two disciplines are summarized in Table 6.1. It appears that other than the rhizosphere bacteria, early emphasis on plant tops to look for antagonists has been on the phylloplane. Up to 1984 weightage has been on basic research with regard to rhizosphere bacteria, particularly PGPR. In the latter part of 1980s, there has been interest among postharvest scientists to experiment with biocontrol strategies to control pathogens as a result of a report indicating health issues related to use of agrochemicals (NAS 1987). While other biocontrol applications have been concentrating on molecular biological aspects, postharvest work has been on the modes of antagonist selection and application. In the decade of 1990s, there has been an obvious emphasis on 134 A.M. Karunaratne

Table 6.1 Some selected, historically important key observations and findings during different periods in the fields of rhizosphere biocontrol and postharvest biocontrol Upto 1984 1985–1990 1991–2000 2001–2010 Auxin production by Fungicides are a Induced systemic Use of root exudate as phylloplane fungi primary means of resistance of energy to degrade (Buckley and Pugh controlling Pseudomonas pollutants (Kuiper 1971) postharvest mentioned (Van et al. 2001) diseases (Eckert Peer et al. 1991) and Ogawa 1985) Fungi showing Antagonist selection Commercialized PGPR mixtures to resistance to from the Pseudomonas combat multiple fungicides (Lewis phylloplane syringae used in pathogens 1977) (Andrews 1985) the fruit industry (Jetiyanon and (Janisiewicz and Kloepper 2002) Marchi 1992) PGPR induced Phylloplane Bacillus Antagonists show Integrated increased plant spp. control greater success management with growth is related anthracnose in when applied after biocontrol for in part to antibiosis mango and harvest PH disease control in root zones avocado (Korsten (Wisniewski and suggested (Spadaro displacing some and Kotze 1992) Wilson 1992) and Gullino 2004) microorganisms (Kloepper and Schroth 1981) Plant growth was Phylloplane Erwinia Phylloplane PH disease control by enhanced by herbicola as a Pseudomonas inducing resistance siderophores of biocontrol agent fluorescens from by applying PGPR (Kloepper (Kempf and Wolf mango leaves jasmonate (Yao et al. 1980) 1989) controlled latent and Tian 2005) fungal infections (Koomen and Jeffries, 1993) PGPR produce Soilborne plant Use of UV light to Method to enrich for extracellular pathogens produce induce resistance in biocontrol strains siderophores antimicrobial PH disease control of rhizobacteria (Neilands 1981) compounds (Wilson et al. (Kamilova et al. (Bruehl 1987) 1994) 2005) Fluorescent Pseudomonas spp. is Antagonist selection Root exudates Pseudomonas best mentioned from the composition known exclusively as a phylloplane for PH reviewed (Uren microorganisms PGPR (Schippers avocado fruit 2007) with PGPR activity 1988) anthracnose (Kloepper et al. (Stirling et al. 1980) 1995) Soil pH is crucial in Strain improvement by Bacillus thuringiensis Concern on invasive disease suppression genetic engineering looked at as a microbes (Van der (Scher and Baker suggested (Davison biopesticide Putten et al. 2007) 1980) 1988) (Cannon 1996) Genetic manipulation Rhizobacteria elicited Suggestion for the of pseudomonads induced systemic need to occupy to show antibiosis resistance activates specific niches on (Thomashow and signal transduction the rhizosphere by Weller 1988) pathways involving antagonists for (continued) 6 Biocontrol Mechanisms Employed by PGPR 135

Table 6.1 (continued) Upto 1984 1985–1990 1991–2000 2001–2010 jasmonic acid and successful ethylene (Van biocontrol (Pliego Loon et al. 1997) et al. 2007) Microorganisms in the Influence of a mineral Importance of rhizosphere are ion on biocontrol molecular tools in ideal for use as (Duffy and Defago rhizosphere biocontrol agents 1997) microbiology (Weller 1988) (Sorensen et al. 2009) Several unsuccessful Use of antagonist Genetically modified attempts of mixtures in PH organisms as transferring research biocontrol agents effective biocontrol (Janisiewicz 1998) may be a way from the lab to field to achieve in PHa research commercial use of (Janisiewicz 1988) biocontrol agents for PH use (Droby et al. 2009) Need to explore Labeling rhizobacteria chemical and with fluorescent physical elicitors tags (Bloemberg among biocontrol et al. 2000) agents that could induce resistance in harvested commodities (Wilson and Wisniewski 1989) Suggestion that nonindigenous antagonists might be successful in the aerial plant surfaces (Pusey 1990) Competition for niches and nutrients as a mechanism of biocontrol rhizobacteria (Lemanceau and Alabouvette 1990) aPH postharvest

Pseudomonas spp. as biocontrol agents, and Bacillus thuringiensis also has been in the limelight. While postharvest scientists have concentrated on applications, rhizosphere scientists have continued on basic research and molecular biological aspects. A slight change in the emphasis by both groups is seen in the decade of 2001–2010. While work on rhizosphere biocontrol has shown an interest on 136 A.M. Karunaratne applications, as late as 2009, postharvest scientists have critically evaluated research done and have suggested ways to go forward (Droby et al. 2009). It appears that both groups of scientists will move towards molecular biological applications in the future.

6.2 Biocontrol Strategies of PGPR of the Rhizosphere and of Antagonists in the Postharvest Environment

The term biocontrol appears to have different nuances of meaning based on where it is applied. While there is diversity among the antagonists in the postharvest environment, the modes of action of the antagonists too appear to be diverse.

6.2.1 The Philosophy of Biocontrol

Biological control has been defined by Campbell (1989) giving a broad definition including crop rotation, direct addition of microbes antagonistic to pathogens or favorable to the plant, use of chemicals to change the microflora, and plant breeding as it is known that changes in the plant genome may affect disease resistance and also the surface microflora. In a broader sense, the positive and negative factors governing the sizes of bacterial populations in a natural habitat have been identified as (1) the effect of nutrients and growth factors and the ability to invade and (2) predation and desertion, respectively (Watanabe and Baker 2000). When the above definition is applied to biocontrol of pathogens in the postharvest environment, manipulation of the microenvironment to favor the needs of the antagonists and hindering the progress of pathogens by a variety of means have been practiced over the years, for the antagonists to win the “battle” against the pathogens. Elsewhere, in unifying the terminology of biocontrol, four strategies of biological control as classical, inoculation, inundation, and conservation have been mentioned (Eilenberg et al. 2001). Of these the intentional introduction of an exotic, usually co-evolved, biological control agent for permanent establishment and long-term pest control, which is referred to as classical biological control, has been the main strategy in the past. In fact looking back, the term biological control has been initially used to describe suppression of insect pests by natural enemies (Smith 1919). In a critical look at the practices adopted over the past years, Droby et al. (2009) have noted that plant pathologists have mainly adopted the entomo- logists’ definition of biocontrol which involves the control of one organism with another, and they further pointed out that a plant disease is not an organism, but a process influenced at different levels: the pathogen, microenvironment, and host. Therefore, the need for a broader approach to combat plant pathogens has been highlighted. When reviewing the methods employed, selection of one antagonist to control one pathogen referred to as the “silver-bullet” approach 6 Biocontrol Mechanisms Employed by PGPR 137

(Spurr and Knudsen 1985) has been the trend in a majority of publications on biocontrol of postharvest pathogens. Here the emphasis has been only on the pathogen and antagonist (in line with controlling insect pests by natural enemies in the past). Spurr et al. (1990) have mentioned that a “non-silver-bullet” approach is important to make progress toward achieving biological control. Among the criticisms of the “silver-bullet” approach are that it omitted testing of antagonism in the natural environment (in planta) and it did not determine the antagonists impact on disease reduction (inoculum dose: disease index) (Edwards et al. 1994). The “silver-bullet” method could be considered a simplistic method without considering the interactions that occur simultaneously with the antagonist effect on the pathogen. Alterna- tively, making the system favorable to the biocontrol agents over the pathogen by determining the population sizes has been suggested by Edwards et al. (1994)as a more appropriate strategy. This calls for the need for a broader understanding of the microbial ecology of the environment in question to target biocontrol strategies based on the knowledge of how a mixed population multiplies and survives. Further substantiating this idea, the importance of studies of soil microorganisms at the community level in addition to research on population dynamics and metabolic activity of individual strains has been highlighted by researchers working on rhizosphere bacteria (Gilbert et al. 1993, 1994). In their review on biological control of postharvest fruit diseases, Spadaro and Gullino (2004) have stated that biological control fits in well with the concept of sustainable agriculture because it mostly exploits natural cycles with reduced environmental impact. This idea also is biased towards ecological conservation and appears to fit in with the definition of conservation biological control, which is modification of the environment or existing practices to protect and enhance specific natural enemies or other organisms to reduce the effect of pests (Eilenberg et al. 2001). Confining to the biological control methods adopted in postharvest technology, a broad definition of the term biological control encompasses constitutive or induced resistance, natural plant products, and antagonistic microorganisms to control pathogens (Baker 1987; Wilson and Wisniewski 1989). The current review will focus mainly on antagonistic microorganisms with a brief account of host resistance.

6.2.2 Antagonist Candidates in the Postharvest Environment

Although the emphasis is on antagonist bacteria, this section explores briefly other microbial antagonists with the intension of identifying the key favorable characteristics of potential antagonist. In the postharvest environment among potential antagonists, yeast has com- manded a special place (Chalutz et al. 1988) as they could colonize the surface for long periods under dry conditions by rapidly using available nutrients and thereby they may restrict not only colonization sites for pathogens, but also flow of germination cues to fungal propagules (Janisiewicz 1988). Thus, yeasts have been the target of interest for postharvest biocontrol in the late 1980s and early 1990s and 138 A.M. Karunaratne they also form the major component of flora of fruit surfaces (Janisiewicz 1991). Yeasts do not seem to have got a special mention in the rhizosphere probably because they are not the dominant flora in the rhizosphere. There is another more important reason for yeasts to have a special place in postharvest applications. The treated product (fruit) is often consumed raw, and the antagonists used to control pathogens should be absolutely safe. Although there are exceptions of pathogenic yeasts, their general inability to produce toxic metabolites (antibiotics) also has been considered as a reason for yeasts to be preferred as biocontrol agents. An overview of commercial products of yeasts is given elsewhere (Droby et al. 2009; Sharma et al. 2009). The biocontrol potential of bacteria and fungi has been investigated with much promise. Fungi, especially certain groups in the Fungi Imperfecti, are promising candidates as antagonists, and isolates of mainly Trichoderma, Gliocladium, Peni- cillum, and Aspergillus have been observed to be the primary colonizers of steamed, pasteurized, or fumigated soils (Cook and Baker 1983; Lewis and Papavizas 1991) which indicate their competitive edge. Among the fungal antagonists, the very first biocontrol agent tried out on a postharvest environment (Tronsmo and Dennis 1977), the Genus Trichoderma has got a special place. Trichoderma has been a well-known mycoparasite and has been commercially available from 1987 (Campbell 1989). Many investigations using Trichoderma spp. have been documented elsewhere (See references listed in Droby et al. 2009 and Sharma et al. 2009). Other than Trichoderma spp., mycotoxin nonproducing strains of fungi have been the popular topics of interest with regard to fungal antagonists. Use of nontoxigenic/nonpathogenic strains of the same species to control toxigenic/ pathogenic strains has been reported to control aflatoxin producing Aspergillus flavus (Ichielevich-Auster et al. 1985; Herr 1988; Cotty 1989). The philosophy behind this is that there is competition between nonpathogenic/nontoxic strains to occupy the same ecological niche as the toxigens/pathogens where the more aggressive antagonist could dominate. In the postharvest environment, the ability of bacteria to grow very rapidly in wounds but not on undamaged fruit surfaces, and therefore bacteria being more effective as biocontrol agents in wounds, has been highlighted (Smilanick 1994). Looking at the antagonist bacterial genera common to the postharvest environment and the rhizosphere, both Bacillus spp. (Edwards et al. 1994)andPseudo- monas spp. have been mentioned, among other genera. More recently, Compant et al. (2005) have remarked that in spite of the focus on free-living rhizobacterial strains especially on Pseudomonas and Bacillus much remains to be learned from nonsymbiotic endophytic bacteria that have unique associations and apparently a more pronounced growth enhancing effect on host plants. Of the above the most commonly mentioned are the pseudomonads. Many bacilli have been used as postharvest biocontrol agents with successful results and Bacillus spp. in the rhizosphere have been demonstrated to promote induced systemic resistance (ISR) and plant growth (Kloepper et al. 2004). Currently, the use of B. thuringiensis seems to have surpassed all other biocontrol agents as it has almost become a household name. 6 Biocontrol Mechanisms Employed by PGPR 139 6.2.3 Diverse Mechanisms of Action

The common strategies mentioned for PGPR and biocontrol of postharvest pathogens in several publications include competition for nutrients and/or space, antibiotic protection and synthesis of cell wall lysing enzymes. Additionally, siderophore production has been noted in bacteria (Rao 1993). Fungal antagonists have demonstrated the ability to attach to the pathogenic fungal hyphae (parasitism) (Campbell 1989), although information on this on subsequent publications appears to be scarce. The above overlap of strategies in the two disciplines is not surprising considering the species mentioned as PGPR and as biocontrol agents of postharvest pathogens. As there are specific conditions favorable for pathogens, the necessity of specific conditions that are favorable for antagonists should be acknowledged. Eckert and Ogawa (1985) have noted that the number of infectious propagules at a potential infection is usually a major determinant of disease severity following either field inoculation or postharvest wound inoculation. The existence of an inoculum threshold implies that a reduction in the amount of inoculum on the harvested crop will result in less postharvest disease. Similarly, the threshold for effective biocontrol by antagonists should be expected. However, information on the mechanisms of action employed by a majority of antagonists is still incom- plete probably because of the difficulties encountered in planning long-term longitudinal studies, under controlled conditions incorporating the complex interactions between host, pathogen, antagonist, other associated microorganisms, as well as the environmental effects. The biocontrol strategies to be used on a pathogen would vary depending on the stage of pathogenicity, i.e., quiescent infection, wound infection, primary infection, secondary infection, etc. Even preharvest applications of biocontrol agents for postharvest pathogen control have been suggested (Ippolito and Nigro 2000). Preharvest applications may have an added advantage as it would be possible to transfer some of the findings on research on PGPR of the rhizosphere directly to the postharvest environment. The idea of manipulation of rhizosphere bacteria to combat plant pathogens is not a new concept in spite of the fact that the results have been variable (Chet and Baker 1980;Mazzola2007). Additionally, the preharvest application of biocontrol agents seems an appropriate strategy also where postharvest handling is unacceptable because the produce may appear less appealing. Looking at the problem from an environmental approach, growth prevention of pathogens can be achieved by making the microclimate undesirable. The strategies employed could be limiting the essential requirements such as space, nutrients, and physical factors such as pH, water activity, and temperature. In fact, soil pH has been shown to be an important criterion for disease suppression (Scher and Baker 1980). Interestingly, it has been noted that the suppressiveness of some soils could be lost by a change of pH (Haas and Defago 2005). It should be acknowledged that even the presence of a single undesirable condition/compound would hinder growth, even if all other essential requirements are available. 140 A.M. Karunaratne

From the organisms’ point of view, Wilson and Wisniewski (1989) have mentioned two basic approaches of using antagonistic microorganisms to control postharvest diseases: promoting and managing natural antagonists that already exist on fruit and vegetable surfaces or artificially introducing antagonists against postharvest pathogens. Plants already harboring pathogens remain healthy most of the time, as the pathogens are suppressed by natural means. Referring to previous work on natural antagonists in the phyllosphere and rhizosphere of plants that could suppress disease development, Wilson and Wisniewski (1989) have queried the presence of natural epiphytic antagonist populations on the surfaces of fruits and vegetables that might be managed to control postharvest diseases. An interesting finding in this regard was when concentrated washings from surfaces of citrus fruit were plated on an agar media, only bacteria and yeast appeared initially, but when these washings were diluted further, rot fungi appeared on the plates indicating that the yeasts and bacteria may be suppressing the growth of the latter (Chalutz 1990). To draw an analogy, the existence of disease suppressive soils has been reported (Schroth and Hancock 1982) and pasteurizing such soils is known to lose their suppressiveness, and when disease suppressive soil is transferred to another location, conducive soil has shown to establish disease suppression (Campbell 1994; Haas and Defago 2005). The above observations show the “behind the screen” role played by the antagonists in natural systems. Extending a similar idea to pesticide applications, Ippolito and Nigro (2000) have commented that pesticide applications can have a harmful effect on nontarget, nonpathogenic phylloplane microbial populations as well. The role of these populations in natural disease suppression is largely unknown and unacknowledged. Additionally, while nonspecific agrochemicals may target both pathogens and antagonists, resistance to them too could be pathogen oriented or antagonist oriented. Combining of antagonists with different modes of action has also been suggested and is thought to have at least three main advantages: broaden the spectrum of activity, enhance efficacy, and allow combination of various mechanisms of biocontrol strategies (Janisiewicz 1998; Ippolito and Nigro 2000). Treatment of plants with a combination of rhizobacteria antagonistic to various soilborne plant pathogens could have a marked effect in reducing root disease if the rhizobacteria are not mutually inhibitory (Kloepper and Schroth 1981). Even more recently, the idea of development of antagonist mixtures consisting of complementary and noncom- petitive antagonists to combat postharvest pathogens has been put forward as a promising approach (Spadaro and Gullino 2004). In our experience in combating crown rot of banana caused by a pathogen complex, two different antagonists investigated had different modes of action (Gunasinghe and Karunaratne 2009). However, there appears to be a word of caution from the scientists working on PGPR. Lugtenberg and Kamilova (2009) in their review on PGPR pointed out that although its logical to inoculate seeds with two strains that use different mechanisms of biocontrol, in their experience such mixtures (so called cocktails) never resulted in better disease control. Their explanation for this unexpected result was that the cell numbers of each of the two bacteria on the root are reduced below the threshold level required to cause control. Ippolito and Nigro (2000) have also 6 Biocontrol Mechanisms Employed by PGPR 141 mooted the idea that if populations of bacteria decline that could lead to densities insufficient to control disease. Poor rhizoplane colonization has been considered as a factor that can limit biocontrol efficacy and is reported to be required for some biocontrol mechanisms (Lugtenberg and Kamilova 2009 and references listed therein). However earlier findings have shown enhanced plant root elongation as a consequence of initial binding of bacteria to seed rather than the roots, under controlled experimental conditions (Hong et al. 1991). It has also been noted by Van Loon (2007) that in 1938, Van Luijk has reported that grass seeds germinated to a higher percentage in nonsterile than in sterilized soil. It turned out that nonpathogenic Pythium spp. took over and counteracted the actions of pathogenic Pythium spp. and other deleterious soil microorganism. Inadequate biocontrol in field experiments has often been correlated to poor root colonization (Bloemberg and Lugtenberg 2001). Chin-A- Woeng et al. (2000) using colonization mutant strains showed that root colonization played a crucial role in biocontrol, disputing earlier claims that colonization is not important for biocontrol (Gilbert et al. 1993; Roberts et al. 1994). Motility, later refined to chemotaxis toward root exudates, appeared to be an important colonization characteristic (Lugtenberg and Kamilova 2009). In retrospect it appears that the motility of the organisms in question would have contributed to the above dispute. Additionally, the phenomenon discussed earlier in this section of the “behind the screen” role of antagonists also would have had a confounding effect. Unfortunately, on many postharvest biocontrol applications, the role of colonization has not been monitored and chemotaxis has not been considered as a trait of importance for potential biocontrol agents. Selection of enhanced root tip colonizers after plant growth in a gnotobiotic system has been tried out successfully using grass root tips (Kuiper et al. 2001; Kamilova et al. 2005). The other characteristics required to accomplish successful colonization by antagonists were noted as the high level of competitive capability and niche overlap (Ippolito and Nigro 2000). While concepts in biocontrol such as antagonism, antibiosis, resistance, and suppressiveness had already been established by about 1935 (Lewis and Papavizas 1991), competition for nutrients and niches as a mechanism of biocontrol bacteria in the rhizosphere has been a comparatively new finding (Lemanceau and Alabouvette 1990). More recent work has focused on biofilms proposed earlier by Casterton (1990) where colonies of microbes attached to the host exert their effects on the pathogens and host. It is now known that most bacteria grow as complex multicellular-like communities attached to surfaces and immersed in polysaccharides, known as biofilms of slime (Marques et al. 2005). Attachment to the fruit surface is probably an important trait for the antagonists to possess for successful preharvest applications because persistent attachment would contribute to a better colonization and avoiding their dislodging due to wind, rain, or water level fluctuations (Dickinson 1986; Ippolito and Nigro 2000). Furthermore, root colonizing Psedomonas bacteria have been shown to alter plant gene expression in roots and leaves to different extents, indicative of recognition of one or more bacterial determinants by specific plant receptors (Van Loon 2007). The above phenomena demonstrate the 142 A.M. Karunaratne complexity of the events leading to biocontrol involving the plant host, microbes on the host plant and the soil in which the host plant grows. More than 2 decades ago, expressing similar sentiments as Lemanceau and Alabouvette (1990), Wilson and Wisniewski (1989) have expressed the view that other than antibiosis little information exists on other modes of action of biocontrol. Even as late as 2009, successful biocontrol by certain bacteria has been reported but the explanation of the control mechanism was inadequate (Sharma et al. 2009; Droby et al. 2009). We demonstrated that a strain of Erwinia herbicola (synonym to Pantoea agglomerans) suppressed C. musae on banana (Gunasinghe et al. 2004). Antibiotic production could not have been the mode of suppression as there was no conclusive evidence for the presence of antibiotics. It was noted that previous researchers too have mentioned the existence of nonantibiotic producing strains of P. agglomerans that could also suppress disease (Wilson et al. 1992) but their mode of action remained elusive. The plant roots are known to offer a niche for the proliferation of soil bacteria that thrive on root exudates and lysates (Van Loon 2007). Fungi too are known to utilize root exudates (Campbell 1989). The fruit surface has been described as a moist, nutrient-rich environment in which resistance to disease decreases as maturation progresses (Janisiewicz 1988). The gradual abundance of nutrients on the fruit surface (among other factors related to ripening) may help the fungal pathogens to progress. Therefore, the presence of competitors for these nutrients (and space) would help in disease control. In postharvest biocontrol, competition for nutrients and space by antagonists has been demonstrated by using radiolabeled glucose, where the deprivation of pathogenic conidial germination due to consumption of glucose by antagonists has been demonstrated (Spadaro and Gullino 2004; Filonow 1998). Another dimension to this aspect is offered by researchers on rhizosphere colonizers. It is reported that in addition to the root surface bacteria utilizing the nutrients that are released from the host for their growth, it also secretes metabolites into the rhizosphere and several of these metabolites could act as signaling compounds that are perceived by neighboring cells within the same microcolony or by cells of other bacteria or by root cells of the host (Van Loon 2007). The presence of a complex network of genetic signaling is apparent from the above findings. Perhaps related to genetic signaling, it is also interesting that different sources of information show the involvement of metal ions in pathogenesis. For instance, Duffy and Defago (1997) have reported that zinc improved biocontrol of Fusarium crown and root rot of tomato. More recently, Conrath et al. (2006) noted that almost 2+ + immediately after pathogen recognition, ion fluxes (Ca ,NO3 ,Cl ,K) and changes in the electrical potential differences across the plasma membrane can be detected in plant cells, which they attributed to the enzyme dependence of metal ions. There may be similar applications with benefits to the plant in question. In the postharvest environment, the effects on mineral ions (except the siderophores) have not attracted the attention of researchers. However, addition of nutrients prefer- ably metabolized by the antagonist and not by the pathogen has been suggested (Janisiewicz 1998; Spadaro and Gullino 2004). Even the mobile signal which moves from the rhizosphere bacteria to the specific aerial organ for systemic 6 Biocontrol Mechanisms Employed by PGPR 143 resistance on the plant has remained elusive (Van Loon 2007). However, this is a cue for postharvest scientists to exploit the possibility of improving postharvest shelf-life of fruits by a preharvest mode of treatment perhaps initiating from the rhizosphere through a mobile signal.

6.3 The Role of Host Plant in Biocontrol of Pathogens

While earlier literature has concentrated on compounds formed as strategies of defense by host plants, newer information has ventured into signal transduction and genetics.

6.3.1 Strategies of Defense by Host Plants

In the postharvest environment, an intriguing question has been why ripe fruits become more susceptible to disease unlike unripe fruits. The presence of preformed antimicrobial compounds (currently referred to as phytoanticipins) in unripe mature fruits which breakdown when the fruit ripens has been demonstrated (Sivanathan and Adikaram 1989; Cojocaru et al. 1986; Adikaram et al. 1993; Terry and Joyce 2004). Phytoalexins can be elicited to protect the plant by the use of nonpathogenic microorganisms as well as by other means. One of the earliest recorded studies in this regard has been on peanuts. Being a susceptible commodity to aflatoxins, peanut is reported to produce phytoalexins in response to invasion by storage fungi (Vidhyasekeran et al. 1972). Later, Wotton and Strange (1985) determined how phytoalexin synthesis by peanuts affected invasion by A. flavus. They found that conditions which promoted invasion of peanuts by A. flavus also inhibited phytoalexin production. In addition, Fujita and Yoshizawa (1989) have recorded that mycotoxins such as T2 toxin, deoxynivalenol, and ochratoxin A, which, have the ability to induce phytoalexins in sweet potato discs. In a time course experiment, these authors have shown the metabolization of mycotoxins into other unknown compounds. However, the safety of having phytoalexins in edible commodities has also been questioned (Frank 1987). ISR (Jetiyanon and Kloepper 2002; Kloepper et al. 2004; Manonmani et al. 2007) and systemic acquired resistance (SAR) (Van Loon 2007) have been mentioned as biocontrol mechanisms of PGPR. Specific defense mechanisms may be expressed as reinforcement of plant cell walls, production of antimicrobial phytoalexins, or synthesis of pathogenesis-related (PR) proteins (Van Loon 2007). Induced or acquired resistance of plants has been long documented by Chester (1933)as reported by Terry and Joyce (2004). It is interesting to note how resistance of plants has been reported by scientists working on rhizosphere microorganisms and on postharvest produce; it is perhaps indicative of the limited sharing of information within the two disciplines. While SAR and ISR are referred to as biologically 144 A.M. Karunaratne induced defense responses, by Pieterse and Van Loon (2004) (who have published on rhizosphere bacteria), they are referred to as not only of biological origin, but also as chemical or physical origin, by Terry and Joyce (2004). In fact SAR has been defined by Conrath et al. (2002) as the systemic resistance response activated upon infection of plants with necrotizing pathogens. Van der Ent et al. (2008) noted that phenotypically ISR resembles SAR that develops upon primary infection with a necrotizing pathogen, unlike SAR, ISR is not marked by transcriptional activation of PR genes encoding PR proteins. Additionally, while Terry and Joyce (2004) refer to local acquired resistance (LAR), Pieterse and Van Loon (2004) refer to wound induced defense typically elicited upon tissue damage, i.e., feeding insects, probably meaning LAR. Later, Van Loon (2007) has discussed the role of nonpathogenic rhizobacteria in antagonizing pathogens through many mechanisms including activating inducible defense mechanisms. Various induced resistance phenomena are reported to be associated with an enhanced capacity for the rapid and effective activation of cellular defense responses, induced only after contact with a (challenging) pathogen, and the responses include the hypersensitive reactions, cell-wall strengthening, oxidative burst, and expression of defense-related genes (Conrath et al. 2002). By analogy with a phenotypically similar phenomenon in mammalian monocytes and macrophages, the augmented capacity to mobilize cellular defense responses has been called the “primed” or “sensitized” state of the plant (Conrath et al. 2002). In their review titled “Priming: getting ready for battle”, Conrath et al. (2006) noted that plants can be primed for more efficient activation of cellular defense responses. These authors have defined “primed state” as the physiological condition in which plants are able to better or more rapidly mount defense responses, or both, to biotic or abiotic stress. Interestingly Terry and Joyce (2004) have used the word hormesis (after Luckey 1990) to describe the involvement of stimulation of a beneficial plant response by low or sub-lethal doses of an elicitor/agent, such as a chemical inducer or a physical stress. Conrath et al. (2006) hypothesized that the primed state could be based on accumulation or post- translational modification of one or more signaling proteins that, after being expressed or modified, still remained inactive, they pointed out that molecular mechanisms underlying priming are not understood. Terry and Joyce (2004) mentioned that considerably more applied and basic research is required to fully understand the role that induction of LAR, SAR, and/or ISR could play in achieving practical suppression of postharvest diseases, and they have acknowledged the need for enhanced fundamental and applied knowledge.

6.3.2 Signal Transduction and Genetic Involvement

Many root colonization genes and traits from Pseudomonas biocontrol species have been identiﬁed (Bloemberg and Lugtenberg 2001). Genetic manipulation of pseudomonads dates back to 1988 (Thomashow and Weller 1988). Simons et al. (1996) developed a monoaxenic system to demonstrate rhizosphere colonization. 6 Biocontrol Mechanisms Employed by PGPR 145

P. fluorescens can be labeled with fluorescent tags for in situ detection (Bloemberg et al. 2000). Using the above technology, Lugtenberg and Kamilova (2009)have reported sequence of events leading to biofilm formation by P. fluorescens WCS365 and induction of systemic signaling. Aiding in signaling, the plant hormones, such as salicylic acid, jasmonic acid, and ethylene that accumulate in response to pathogen infection or herbivore damage, are noted as major players in the regulation of signaling networks involved in induced defense responses against pathogens and insects (Pieterse and Van Loon 2004). Interestingly, both salicylic acid and jasmonic acid (additionally methyl jasmonate and acibenzolar, a functional analogue of jasmonic acid) as well as other plant hormones such as cytokinins and gibberellic acid have been tried out as chemical elicitors of natural disease resistance to postharvest pathogens in horticultural produce as reported by Terry and Joyce (2004). Ethylene could induce fruit ripening which is detrimental as it shortens shelf-life, and therefore, with respect to ethylene in postharvest technology, only methods of reducing levels attract attention unless it is specifically used to induce ripening of fruits. Fuqua et al. (1994) coined the term quorum sensing (QS) to describe communications occurring between bacterial communities occupying a single niche. Among the QS bacteria of PGPR, Pseudomonas spp., Erwinia spp., and Burkhol- deria cepacia have also been used successfully as postharvest antagonists. Interest- ingly, B. cepacia isolated from banana fruit skin has been used as biocontrol agents on controlling banana fruit pathogens (De Costa and Erabadupitiya 2004). It appears that understanding the molecular basis of biocontrol mechanism has much to offer to fill in the missing gaps. For instance, a better understanding of QS would perhaps be the key to harness benefits of biocontrol strategies displayed by different biocontrol agents. True to this point, Defago and Moenne-Loccoz (2006) have mentioned that discovery of alternative mechanisms by QS interference based on signal degradation shows that not all biocontrol mechanisms are documented yet. These authors further pointed out the need to understand the social life and biogeography of biocontrol pseudomonads along with other indigenous plant- beneficial bacteria to determine how they could confer disease suppressiveness to soils and expressed the need to understand the microevolution of biocontrol pseudomonads. QS has been shown to influence biofilm development for several species (Parsek and Greenberg 2005). QS of Gram-negative PGPR has been reported to produce N-acyl-homoserine lactones (Sharma et al. 2003). Thus, QS within and between bacterial populations is a major regulatory mechanism in bacteria to adjust their metabolism to crowded conditions or other changes in the biotic and abiotic environment (Whitehead et al. 2001; van Loon 2007). Parsek and Greenberg (2005) have also noted that there is growing appreciation within the biofilm field that individual cells of a variety of bacterial species are capable of actively leaving a biofilm and in crowded conditions QS would be an ideal way to mediate exodus from a biofilm. The influence on genes of the host plant by rhizobacteria has been reported (Verhagen et al. 2004). Conversely, plants could alter root exudation and secrete compounds that interfere with QS (van Loon 2007). It could be assumed that this two-way sensing need not be restricted to only the rhizosphere. 146 A.M. Karunaratne

6.4 Challenges in the Postharvest Environment

The postharvest environment is unique as the harvested organ is independent of the parent plant and should be able to survive independently in the absence of any nutritional input. While continuing life’s processes is essential to maintain fresh- ness, the slower and steady this process is, the longer it can stay fresh. Janisiewicz (1988) has reported that fruits have a moist, nutrient-rich environment in which resistance to disease decreases as maturation progresses. Very broadly fruits would have a lower pH compared to vegetables, resulting in their pathogens to be of mainly fungal origin, and vegetables in general would have a pH near neutral being susceptible to both bacteria and fungi. Earlier research on biocontrol in plant tops (unlike control of root pathogens) has been less attractive because of more severe and fluctuating conditions which adversely affect many biocontrol agents resulting in inconsistent control (Janisiewicz 1991). However, unlike the rest of the plant, in harvested commodities disease should be easier to control than preharvest disease because conditions encountered in storage are less variable (Spurr et al. 1990) and could be maneu- vered to suite the needs. For instance, postharvest diseases of fruits and vegetables can be suppressed by low temperature storage, low oxygen atmosphere and treatment with growth regulators that delay tissue senescence, but these beneficial practices may not adequately protect the crop from microbial attack especially during prolonged storage (Eckert and Ogawa 1985). Turning to the pathogen, some pathogens not only spoil the commodity but also make it dangerous for consumption by the presence of toxins, i.e., toxins from fungi (mycotoxins) and bacteria which can cause bacterial intoxications and infections. The deleterious effects of toxigenic organisms can be overcome by preventing its growth, if not by preventing toxin production or by metabolizing the preformed toxins into harmless compounds. Of these three options, the first is obviously the ideal although it is not possible all the time. Additionally, many postharvest diseases are known to be caused by a complex of pathogens. These pathogens have distinct roles to play and therefore their control has to be carefully planned by understanding their modes of action (Gunasinghe and Karunaratne 2009). Eckert (1990) has noted three types of infections that may lead to postharvest disease, as latent (quiescent) infections (i.e. anthracnose and stem-end rots) initiated on immature fruit in the field, infection of unripe fruit at an advanced stage of development in the field, where progressive development of the disease is suppressed for a while (i.e., Botrytis rot), and infection through wounds. Based on the above information, in planning disease control strategies, the above types of infections are taken into account. To compete successfully with pathogens at the wound site, the microbial antagonist should be better adapted to various environmental and nutritional conditions than the pathogen (Barkai-Golan 2001; Sharma et al. 2009). Thus, the focus on initial attempts on biological control of postharvest diseases has been on the control of wound pathogens on a selected number of fruits (Wilson and Wisniewski 1989; Eckert 1990). 6 Biocontrol Mechanisms Employed by PGPR 147

The inability to control previously established infections, quiescent infections and incipient infections occurring through wounds by potential biocontrol agents has been of concern. Quiescent infections have been the most difficult to control, of all pathogen infections even with the use of agrochemicals. Eckert and Ogawa (1985) have noted that the development of benzimidazole fungicides (i.e., dicloran, imazalil, prochloraz, etaconazole and guazatine) in the late 1960s was a breakthrough as they could penetrate into fruit tissues to prevent development of quiescent infections and other subsurface inocula without injuring host cells. If biocontrol agents are to replace benzimidazole fungicides, they should demonstrate the same capability. For this, knowledge of the epidemiology of the target disease is crucial for choosing the right time for the application of biocontrol agents (Ippolito and Nigro 2000). It has been stated that preharvest, harvest (transition) and postharvest activities impact epiphytic microflora and in postharvest disease control, proper management of this microflora determines success (Spurr et al. 1990). Therefore, getting a biocontrol agent established before the pathogen arrives (i.e., preharvest application of biocontrol agents for controlling postharvest pathogens) has been suggested as more effective (Smilanick 1994; Ippolito and Nigro 2000). Surprisingly, in spite of the skepticism, several investigations have shown that postharvest applications of biocontrol agents can accomplish at least partial control of quiescent infections (Chuang and Yang 1993; Janisiewicz and Korsten 2002;De Costa and Erabadupitiya 2004; Gunasinghe et al. 2004; Sharma et al. 2009). Overall, Janisiewicz (1991) has mentioned that most fruit and vegetable storage diseases are caused by fewer than 30 pathogen species, giving the idea that controlling them by biological means would not be a difficult target to achieve. Lack of consistent control of pathogens by antagonists has been reported in the past and the reasons for this have been identified as lack of survival of antagonists in the environment, effects of environmental and edaphic (soil related) factors on the live organisms and interactions with other microorganisms (Campbell 1994). However, being able to maneuver storage conditions in the postharvest environment gives the opportunity to switch the host pathogen antagonist equilibrium towards the antagonist (Spadaro and Gullino 2004), provided the strategies of pathogens and antagonists are better understood.

6.5 Ecology of the Microbial Environment

At a very early stage, Janisiewicz (1988) has contributed immensely to applications of biocontrol technology to the postharvest environment. It was correctly pointed out that the outcome of biocontrol by antagonists will be subject to biotic and abiotic factors and stresses. Recently, in the control of postharvest diseases of tropical fresh produce, Korsten (2006) has noted that natural antagonistic populations could be exploited to attain natural disease control with a broader understanding of population dynamics and ecological interactions. With this regard, in spite of the complexity of planning even short-term experiments, it appears that not having 148 A.M. Karunaratne a holistic understanding of the microbial population dynamics could be a major limitation in harnessing maximum benefits obtainable from the approach of biological control. Looking back at the biocontrol methods employed on harvested produce, the “silver-bullet” approach cannot be considered farsighted as the aspect of microbial ecology involving associations between microfloras has been neglected. Turning to research on the rhizosphere, synergistic effects of arbuscular mycorrhizal fungi, PGPR and yeast on root colonization have been documented (Linderman and Paulitz 1990; Singh et al. 1991; Bhowmik and Singh 2004). In spite of the comparatively better awareness among researchers working on the rhizosphere microflora, Van Loon (2007) noted that the ecological diversity and its consequences for metabolic activity of the rhizosphere bacteria deserve further investigation. With regard to the postharvest environment too it appears that this aspect has not received due consideration. The approach of biocontrol by introduction of antagonists has been looked at by certain earlier workers with much skepticism, as nonconductive habitats will not sustain introduced microflora (Garrett 1956; Lewis and Papavizas 1991). However, studies of the multiple interactions among saprophytes, pathogens, nutrients and fungicides have been used for the development of a simulation model to guide the introduction of biocontrol agents (Spurr et al. 1990; Knudsen et al. 1988). There- fore, the trend thereafter was that environments must be altered to accept biocontrol agents or that formulations must be developed which allow the antagonist to survive, proliferate, become active (produce toxins or lytic enzymes), and establish themselves in an alien environment (Lewis and Papavizas 1991). This idea appears to be in par with conservation biological control (Eilenberg et al. 2001) and seems to fit in with traditional cultural practices used for pest control (Kean et al. 2003). In postharvest applications of fruits and vegetables, the ecology of the microflora on exudates of the intact fruit has not received much attention while root exudates have been reviewed extensively (Uren 2007). Although addition of nutrients pref- erably metabolized by the antagonists and not likely to be metabolized by the pathogen has been suggested (Janisiewicz 1998; Spadaro and Gullino 2004), determining the composition of fruit exudates with this regard has not received much attention. The fruit surface is considered as a very good food base for epiphytic microorganisms as it is rich in nutrients coming from plant leakage, outside deposits of pollen, organic debris and honeydew (Janisiewicz 1988). Later works reported on fruit exudate have been to determine exudates from plum and nectarine fruit to relate to Botrytis cinerea causing rot development (Fourie and Holz 1998) and to determine exudates from cucumber slices to design a method to measure chilling injury (Cabrera and Saltveit 1992). The need for this aspect to be investigated is further intensified by the fact that exudates found on the rhizosphere are composed of organic acids such as malic acid (Rudrappa et al. 2008) which are found in abundance in the pulp of many fruits. The rhizosphere microbes are known to benefit by metabolizing nutrients that plant roots secrete (Lugtenberg and Kamilova 2009). It is also noted that plants could alter root exudation and secrete compounds that interfere with QS regulation in the bacteria 6 Biocontrol Mechanisms Employed by PGPR 149

(Van Loon 2007). The ability of plants to react to root colonization by rhizobacteria by increasing the release of exudates and the ability to change quantity and composition of root exudates with the developmental stage of the plant have been noted (Van Loon 2007). The author is not aware of any comprehensive study on fruit or vegetable exudates, which may explain the microbial ecology on their surfaces. Such information would be valuable, having acknowledged the need of beneficial bacteria to colonize the root surface to exert their effect (Lugtenberg and Kamilova 2009). In one of the few such studies done on fruits, it is mentioned that sugars in fruit exudates of plum and nectarine may contribute to susceptibility of fruits to B. cinerea infection and noted that their concentrations increased as the fruit ripened (Fourie and Holz 1998). An interesting account involving P. fluorescens in a postharvest environment is reported by Campbell (1989), which again emphasizes the need to have a holistic approach. It is on a failed attempt by Swinburne (1986) to control C. musae, which causes latent infections on the banana skin. It is said that its germination is stimulated by leachates from the fruit, especially anthranilic acid, which may act as a siderophore, sequestering iron. Germination and appressorium formation are also reported to be stimulated by the siderophores produced by P. fluorescens and other bacteria on the fruit surface. The exact reverse of the mode of action proposed for some biocontrol agents whose siderophores reduce infection by starving the pathogen of iron is reported on the banana skin as free iron, and various chelates in which iron is available to the plant, inhibit germination, possibly by stimulating phytoalexin production. Here, instead of the pathogen being starved of iron, the removal of iron by other microorganisms stimulates pathogen germination. This shows the significance of ecology of the surface microorganisms in question, as well as acknowledging the different modes of interactions of pathogens and antagonists. Wisniewski and Wilson (1992) have remarked on the difficulties involved with biological control agents tested in the laboratory to the field largely because of problems of ineffectiveness when exposed to the “uncontrolled” environment of the latter. Campbell (1994) too has cautioned against selection of organisms by known modes of action, as selection based on tests in vivo has not shown such inhibition when tested in vitro. Managing naturally occurring population of microorganism of the surfaces of fruits and vegetables to enhance resistance of harvested commodities to disease has been postulated by Wilson and Wisniewski (1989). Cook and Baker (1983) suggested that antagonists should be sought from where disease does not occur in spite of considerable inoculum pressure. Since the fruit surface supports the growth of a variety of interacting microorganisms, it has also been postulated that a form of biological control occurs in fruits in nature and that some of these may be potential biocontrol agents for fruit pathogens (Janisiewicz 1991). In retrospect, the inability to transfer technology from the laboratory to the field is likely due to the “silver-bullet” approach adopted by postharvest scientists. In fact, Halverson et al. (1993) working on rhizosphere microorganisms have shown the need to perform multiple year field experiments for successful results. In spite of the fact that the postharvest environment could be considered to be less variable and well defined, compared to the field conditions of a plant which depend on climatic 150 A.M. Karunaratne and weather conditions, the host characteristics may be influenced by field conditions, thus having an indirect effect on the influence directed at the microbes on the surface. Considering the interactions between the rhizobacteria and the host plants, it is possible that the fruit physiology and hence the organoleptic qualities of the fruit too is influenced by the composition of the surface microflora. It is reported that microbial populations in the natural environment are much more diverse than microorganisms so far isolated in the laboratory (Watanabe and Baker 2000). The complexity of microbial interactions that occur and the importance of investigating the microbial ecology on the surface in question are apparent for successful biocontrol. To conclude this section, it appears that two decades ago some of the questions raised by Wilson and Wisniewski (1989) appear to be very pertinent currently. They are as follows: “What are the effects of antagonists on wound healing and host resistance? How important and widespread in nature are the direct effects of antagonists on pathogens? How do incidental microorganisms or mixtures of antagonists affect the pathogen/antagonist interaction? And how does the nutrient/chemical composition at the wound site affect antagonists, other microflora and the infection process and the fruit wound response?” The authors have correctly stated that a greater understanding of the microecology of fruit and vegetable surfaces would shed light to this area of research. It is timely therefore to survey the strategies of PGPR in controlling postharvest pathogens, to draw parallels between the control strategies, so that we could obtain a better understanding of the mechanisms involved in postharvest biocontrol strategies. One major attribute seen when talking of PGPR and biocontrol agents is that the emphasis on the latter is on its direct effect on the pathogen. PGPR, on the other hand, may influence indirect growth promotion of crops. In a recent review (Droby et al. 2009), the need for a more thorough understanding of the microbial ecology of fruit surface has been emphasized.

6.6 Nature and Role of Bacterial Antagonists

Wilson and Wisniewski (1994) have mentioned that an ideal antagonist should be genetically stable, should be efficient at low concentrations and against a wide range of pathogens on various fruit products, should have simple nutritional requirements and survive in adverse environmental conditions. Also, when used on edible plant matter, being 100% safe in terms of not being toxic or allergenic and not producing toxic metabolites are important considerations. The necessity to manage the epiphytic microflora to control postharvest diseases has been recognized (Spurr et al. 1990) and this management has to be at different phases of progression of disease. Therefore, it appears that an ideal antagonist should have multiple mechanisms of action such as niche and nutrient competition, enzymes to attack pathogen cells, induction of resistance, antimicrobial production and additionally help the host tissue to maintain metabolic processes. Additionally at a time 6 Biocontrol Mechanisms Employed by PGPR 151 when agrochemicals are still in use for postharvest applications, in spite of fungicide resistance of pathogens has been of concern (Campbell 1989) the resistance of biocontrol agents to fungicides may be useful in integrated disease control programs (Ippolito and Nigro 2000). It is apparent from the remarks made by different authors that the attention on Gram-positive bacteria on the rhizosphere has been studied much later (Kloepper and Schroth 1981; Gilbert et al. 1993; Halverson et al. 1993). At an early stage, the emphasis was on Gram-negative rhizobacteria (which are the more dominant population in the rhizosphere), mainly the pseudomonads. The importance of Gram-positive bacteria, mainly of Bacillus spp. is discussed much later, with the note that the rich diversity of the microbial world provides a seemingly endless resource for biocontrol (Emmert and Handelsman 1999). However, the extensive use of B. thuringiensis (a Gram-positive bacterium) that has been patented for use on control of pests of several agricultural crops, trees, and ornamental plants (Falcon 1971) is unparalleled to any other bacterial biocontrol agent. Gram-positive bacteria are known to utilize signaling molecules such as short peptide pheromones to inhibit pathogens (Sharma et al. 2003). Parsek and Greenberg (2005) mentioned two most thoroughly described QS systems of which are the acyl-homoserine lactone systems of many Gram-negative species and peptide-based signaling systems of many Gram-positive species. Also mentioned are AI-2 systems common to several Gram-positive and Gram-negative species. Among the PGPR, P. fluorescence has received much attention (Schippers 1988; Van Loon et al. 1997; Lugtenberg and Kamilova 2009), and this species has been mentioned exclusively in determining survival in soil and rhizosphere (Mazzola et al. 1992, 2001; Mazzola 2007). Interestingly, the word “pseudomonads” (referring to Pseudomonas fluorescence-putida group) has even been used synonymously with PGPR (Kloepper et al. 1980). The awareness of suppression of plant diseases by fluorescent Pseudomonas spp. has a long history dating back from 1976 and has made a steady progress over the years (Cook and Rovira 1976; Weller and Cook 1983; Baker et al. 1986; Parke et al. 1991). Antibiotics produced by Pseudomonads are known to be effective against not only bacteria but also fungi (Howell and Stipanovic 1979). Additionally, Davison (1988) has reported that many rhizosphere bacteria (such as P. fluorescens and P. putida) have been isolated and screened for inhibition of phytopathogenic fungal or bacterial growth. Much information on biocontrol strategies of microbial antagonists has been unraveled by using pseudomonads. On the postharvest environment, the studies on antagonistic activity of Pseudo- monas spp. by their production of antibiotics are reviewed by Spadaro and Gullino (2004). P. cepacia has been analyzed on postharvest rot of apples (Janisiewicz and Roitman 1988) and on pome fruit (Janisiewicz et al. 1991). It is reported that poorly soluble inorganic nutrients that are rate limiting for growth could be made available through the solubilizing action of bacterial siderophores or the secretion of organic acids (Vessey 2003; Van Loon 2007). The role of siderophores which efficiently complex environmental iron, making it less available to certain native microflora has been documented (Kloepper et al. 1980). 152 A.M. Karunaratne

Postharvest fruit pathogens are described mainly as necrotrophs and depend on exogenous nutrients for germination and initiation of the pathogenic process (Janisiewicz 1991). On the fruit surface, application of fungicides is prophylactic and therefore application of biocontrol agents to a fruit surface should serve the same purpose. No mention has been made of the ability of pathogen antagonists to have a direct effect on growth of the host plant in postharvest applications. This is in spite of several reports that have appeared on the effect of phytohormones produced by antagonists in the rhizosphere. For instance, reducing ethylene production during the postharvest shelf life of a fruit is a major requirement for increased shelf-life. Ethylene is considered a menace as it hastens fruit ripening and senescence of the host tissue, and promotes growth of pathogens. 1-aminocyclopropane-1-carboxylate (ACC), the immediate precursor of ethylene can be metabolized to prevent formation of ethylene. Breakdown of ethylene by bacterial ACC deaminase has been reported (Glick et al. 1998;Lucyetal.2004). The bacterial enzyme ACC deaminase is known to be the only nonplant enzyme that metabolizes ACC (Grichko et al. 2000). It is noted that transforming the ACC deaminase gene, which directly stimulates plant growth by cleaving the immediate precursor of plant ethylene into P. fluoresens CHAO, not only increases plant growth but can also increase biocontrol properties of PGPB (Glick et al. 1998;Wangetal.2000; Compant et al. 2005). The use of antagonists capable of metabolizing ACC in the postharvest environment would be a very valuable tool in postharvest applications. Apart from lowering ethylene levels, production of plant hormones like auxins, cytokinins and gibberellins (Glick et al. 1998; Lucy et al. 2004) known as juvenile phytohormones may be helpful in the postharvest environment. Incidence of enhanced pathogen control when microbial antagonists are applied in the presence of certain additives, including phytohormones has been demonstrated by several investigations on postharvest applications (Sharma et al. 2009). Other stimulants of plant growth include certain volatiles and the cofactor pyrrolquinoline quinine in several enzymes involved in antifungal activity and induction of systemic resistance (Lugtenberg and Kamilova 2009). Among the other attributes listed for PGPR are increases in chlorophyll content, magnesium content, nitrogen content, protein content, hydraulic activity, tolerance to drought and delayed leaf senescence (Lucy et al. 2004). Such attributes would also have a direct positive effect in the postharvest environment of specific fruits and vegetables. With regard to mycotoxin detoxification, one of the earliest records known is the work of Ciegler et al. (1966) who screened about 1,000 microorganisms which included yeasts, fungi, actinomycetes, bacteria, and algae. These authors reported that Flavobacterium aurantiacum was capable of removing aflatoxins from test substrates. Later various scientists have determined the capability of this bacterium to detoxify aflatoxin contaminated food substrates with positive results (Lillehojetal.1971; Hao and Brackett 1988). Much later work concentrated on other nontoxigenic microorganisms capable of detoxifying preformed mycotoxin (Cotty 1989). It has been correctly reported by Gilbert et al. (1993)thatan improved understanding of the influence of the introduced organism on microbial 6 Biocontrol Mechanisms Employed by PGPR 153 communities is required for biocontrol or growth promotion of plants to be accomplished through the introduction of microorganisms.

6.7 Application and Commercialization of Microbial Antagonists

While postharvest applications could be accomplished by drenching or dipping in an antagonist suspension, pressure infiltration of fruit with the suspension or mixing the antagonists with waxes on sorting lines is also suggested (Janisiewicz 1991). Vacuum infiltration and high pressure processing are also more complex methods which have given positive results in the laboratory, but they need to be further developed to suit field applications. Preparations of biocontrol formulations such as dusts, wettable powders, granules, pellets, prills, pastes, tablets, emulsifiable liquids, fluid-drill gels have been discussed (Vidhyasekaran 2004; Lewis and Papavizas 1991). Several microbial antagonists have been patented and evaluated for commercial use of which ASPIRE, YieldPlus and BIOSAVE-110 are used worldwide for controlling postharvest diseases of fruits and vegetables and the continuous increase in the use of BIOSAVE without failure since 1996 indicates that current biological control practices can be cost effective in large packing houses (Sharma et al. 2009). Some commercial products using free-living PGPR are listed elsewhere (Lucy et al. 2004). By 2005 more than 33 products of PGPR have been registered for commercial use in greenhouse and field in North America (Nakkeeran et al. 2006). There have been suggestions to formulate mixtures containing biocontrol agent(s) with chemical fungicide(s) to broaden the spectrum of activity (Eckert 1990). Such investigations have provided substantial evidence of success (Chand-Goyal and Spotts 1997; Sharma et al. 2009). Also suspending the conidia of antagonists in nutrients, such as malt and yeast extractions, and use of various inorganic salts as the suspension medium have given desired results (Ippolito and Nigro 2000).

6.8 Current Challenges in the Field of Biocontrol

At an early stage of investigation although there has been concerns regarding public reaction in the application of “living fungicides” to food (Wisniewski and Wilson 1992), the number of commercial products that are available now bears testimony to the fact that it has been accepted by the public. However, there are several issues to be addressed currently, if we are to make progress. It appears that many researchers are ready to move out for field trials, but the necessary wherewithal for such an attempt is not there. Except for a few countries who have commercialized products, scientists in other countries have restricted their know-how only to 154 A.M. Karunaratne the laboratory and publications restricted to the scientific community. Droby et al. (2009) have noted that large-scale production of formulated biocontrol agents requires costly trials and no serious attempts have been made to address the large-scale production and formulation technology of biocontrol agents. It appears that the problem is deeper and wider than that. Inability to move forward after laboratory investigations can be cited as a major drawback for scientists who have found positive results in the laboratories of many countries. The absence of a central body giving guidelines on specific tests to be conducted for safety, in terms of health, and the absence of a ready repository of biocontrol agents and their information, may be cited as snags in the process. Droby et al. (2009) have noted that even with the large number of researchers all over the world working on biocontrol applications, currently the use of chemical agents remains the major method of choice for managing postharvest rots and the few postharvest biocontrol products commercially available have limited use, mostly in niche markets. It appears a battery of standard protocols to be adopted to facilitate the link research findings and field applications. Issues on concerns of safety and environmental impact should be addressed following a parallel protocol for commercializing novel agrochemicals. Linked to this is the concern posed by Van der Putten et al. (2007) on moving plants, animals, or microbes around the globe as invasive microbes (plants and animals) could be a major threat to the composition and functioning of ecosystems and can have a major impact on the abundance of individual species, affecting the diversity of native communities. Additionally, agrochemical companies do not seem to have felt the pressing need to replace their current products with biocontrol agents, in spite of the effort of the researchers. The lack of knowledge of the current issues to be addressed among law makers to facilitate such a process also could be cited as another dimension to the failure. With the concept of a global village, suitable means of sharing such information of isolated scientists, on the pretests required and registration protocol for new biocontrol agents, at a central body (perhaps in par with the Codex Alimentarius Commission of WHO/FAO), would benefit the entire world to address global food insecurity at large.

6.9 Concluding Remarks

In this chapter, of the several benefits for PGPR, biological control of plant pathogens, one of the main benefits, was reviewed by comparing biological control strategies employed in postharvest technology of fruits. Biological control of postharvest pathogens has become a popular research topic over the years, with a high demand for immediate applications, and several biocontrol formulations are now allowed in the postharvest environment. In spite of this, many of the positive findings in research on the postharvest environment cannot be explained. Besides, many of the postulations have given opposite results. Revisiting ideas mooted by two postharvest scientists Wilson and Wisniewski (1989), who have been 6 Biocontrol Mechanisms Employed by PGPR 155 publishing on the subject for the last 20 years, it is anticipated that our knowledge will increase rapidly by drawing and building upon the foundation of information developed in other areas of biocontrol and phylloplane research. According to Wilson and Wisniewski (1989) understanding the mode of action of antagonists is important for two reasons, as it will allow the development of more reliable procedures for effective application of known antagonists and it should provide a rationale for selecting more effective antagonists. It is interesting to note that the current strategies to combat pathogens by using microbial antagonists have revolved round the broader definition for biocontrol given by Campbell (1989) with the last option gaining ground with the refining of molecular biological tools. Work on PGPR has a longer and a more comprehensive development, and has ventured into molecular biological applications as seen in several reviews (Bloemberg and Lugtenberg 2001; Conrath et al. 2002; Duffy et al. 2003; Pieterse and Van Loon 2004; Haas and Defago 2005; Compant et al. 2005; Van Loon 2007; Sorensen et al. 2009). It appears that some of the fundamental research ideas put forward by scientists working on PGPR could be applied by scientists working on postharvest applications of biocontrol strategies. On the other hand, work on postharvest biocontrol, which has concentrated more on applications having many unexplained positive results, may have answers from the work on PGPR. Additionally, such applications also may give research ideas for scientists working on PGPR. Marques et al. (2005) have indicated that novel pesticides and disinfectants, and/ or decontamination procedures should be designed which could attack biofilms as current pesticides and protocols are based on killing of single-celled organisms. Applying this idea into antagonists of postharvest pathogens, it appears that a novel way of thinking is needed. It is known that conducive soil can establish disease suppression by transferring an inoculum of 0.1–10% of suppressive soil (Haas and Defago 2005). This shows the potential of the elusive ideal antagonist to be encountered by future scientists. One area that has not been addressed by researchers on postharvest application of biocontrol is the capability of the fruit surface microflora to influence gene expression of the host. Whether it happens and how it happens remains elusive. This may be an interesting area to explore, considering the numerous gene related events that could occur in relation to fruit maturation and ripening. Van Loon (2007) has cited several examples to indicate that root-colonizing Pseudomonas spp. may activate signaling pathways. The reader is referred to comprehensive reviews which discusses current applications and future opportunities for improving pseudomonad-based biological control (McSpadden Gardener 2007) and on the genetics of disease suppression by fluorescent pseudomonads (Haas and Defago 2005). The use of genetic engineering for enhancement of biological control efficacy has been suggested (Spadaro and Gullino 2004). If future scientists could consider genetically engineering an ideal antagonist with multiple mechanisms of actions with characteristics of being environmentally safe and being nontoxic, and also perhaps having some prebiotic characteristics, that would be a breakthrough. Meanwhile it is obvious that there is much to give and take from the two disciplines. 156 A.M. Karunaratne

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Wilson CL, Wisniewski M (1994) Biological control of postharvest diseases: theory and practice. CRC Press, Boca Raton, FL, 182 pp Wilson CL, Wisniewski ME, Biles CL, McLaughlin R, Chalutz E, Droby S (1991) Biological control of postharvest diseases of fruits and vegetables: alternatives to synthetic fungicides. Crop Prot 10:172–177 Wisniewski ME, Wilson CL (1992) Biological control of postharvest diseases of fruits and vegetables: recent advances. Hort Sci 27:94–98 Wotton HR, Strange RN (1985) Circumstantial evidence for phytoalexin involvement in the resistance of peanuts to Aspergillus ﬂavus. J Gen Microbiol 131:487–494 Yao HJ, Tian SP (2005) Effects of a biocontrol agent and methyl jasmonate on postharvest diseases of peach fruit and the possible mechanisms involved. J Appl Microbiol 98(4):941–950 . Chapter 7 Plant Growth-Promoting Bacteria Associated with Sugarcane

Samina Mehnaz

7.1 Introduction

Sugarcane is an important industrial and cash crop in many countries of the world. It is grown in over 110 countries, in tropical and sub-tropical regions, in a range of climates from hot dry environment near sea level to cool and moist environment at higher elevations. Besides sugar production, sugarcane produces numerous valuable by-products like ethanol, bagasse, press mud, molasses, and essential items for industries like chemicals, plastics, paints, synthetics, ﬁber, insecticides, and deter- gents (http://www.pakissan.com). This crop is perhaps the most economically competitive source of ethanol and can effectively contribute to a cleaner environment. Ways of improving its productivity are subject to investigation in several countries. Worldwide climate change due to the intense use of greenhouse gas- producing energy sources has resulted in the development of sustainable energy. Consequently, sustaining and enhancing the growth and yield of sugarcane have become a major focus of research. Sugarcane and other grasses such as rice, wheat, maize, and sorghum, currently have much of their nitrogen (N) needs supplied by costly mineral fertilizers. It has been a general practice to apply 250 kg N haÀ1 yearÀ1, or more in most of the sugarcane cultivating countries. In 2008, an estimated 1,743 million metric tons of sugarcane were produced worldwide, with about 50% of production occurring in Brazil and India. In India, sugarcane is grown over 4.2 million ha, producing about 250 million tons of canes annually and the nitrogen requirement of Indian sugarcane ranges from about 250 to 350 kg haÀ1. Brazil is the largest sugarcane producer in the world, with the crop occupying more than ﬁve million hectares with a yield of 495 M tons in 2007/2008 (UNICA 2009), 16 million m3 of alcohol in 2006 (Mendes

S. Mehnaz Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan and Institute of Pharmaceutical Biology, Bonn University, Bonn 53115, Germany e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 165 DOI 10.1007/978-3-642-18357-7_7, # Springer-Verlag Berlin Heidelberg 2011 166 S. Mehnaz et al. 2007; Oliveira et al. 2006) and annual application of nitrogen fertilizer for sugarcane is around 50 kg N haÀ1, with a cost near US$ 500 tÀ1 (http://www.udop. com.br). Researchers in Brazil are intensively working on further reducing the use of N-fertilizer application by one half (<125,000 t N yearÀ1) due to the biological nitrogen ﬁxation (BNF), so the producers could save estimated US$ 62.5 m yearÀ1 (Oliveira et al. 2006). This approach can signiﬁcantly reduce the cost of bio-energy in the whole world.

7.2 Plant Growth-Promoting Rhizobacteria

The biological reaction that counterbalances the loss of nitrogen from soils or agroecosystems is the BNF, which is the enzymatic reduction of the atmospheric dinitrogen (N2) to ammonia, catalyzed by nitrogenase and this process is unique to Bacteria and Archaea. Bacteria which fix nitrogen can also be beneficial for the plants by using other mechanisms such as phytohormone production, phosphate solubilization, etc. They are grouped under the name plant growth-promoting rhizobacteria (PGPR) also known as plant growth-promoting bacteria (PGPB) defined as “free-living soil, rhizosphere, rhizoplane, endophytic, and phyllosphere bacteria that under certain conditions are beneficial for plants” (Bashan and de Bashan 2005). They are capable of promoting plant growth through different mechanisms, including BNF, phytohormone production, phosphate solubilization, siderophore production, and biological control. PGPR belong to diverse genera including Azospirillum, Azotobacter, Herbaspirillum, Bacillus, Burkholderia, Pseudomonas, Rhizobium, and Gluconacetobacter, among others. In this article, only those PGPRs will be discussed which have been isolated from sugarcane. A complete list of genera, species, source of isolation, country of origin and references are provided in Table 7.1.

7.2.1 Azospirillum

Azospirillum belong to the facultative endophytic group of bacteria which colonizes the surface and interior of the roots. Bacteria are micro-aerophilic, gram-negative rods and often associated with roots of cereals and grasses (Grifoni et al. 1995). They are very well known for nitrogen fixation and higher production of indole acetic acid. In the group of bacteria responsible for associative nitrogen fixation, Azospirillum is as important as Rhizobium in the bacterial group known for symbiotic nitrogen fixation. It is a very well-studied PGPR. Several reports have been published about its beneficial effect due to nitrogen fixation and growth hormone production. Due to its nonpathogenic behavior, it is the safest choice to use as a biofertilizer for any crop. Three species of this genus namely, Azospirillum amazonense, A. brasilense, and A. lipoferum have been isolated from sugarcane. In 1976, Dobereiner and Day 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 167

Table 7.1 List of the bacterial genera and species isolated from the sugarcane Bacteria Source Country References Acinetobacter Apoplast Cuba Vela´zquez et al. (2008) baumanii Agrobacterium Stem China Xing et al. (2006) tumefaciens Azospirillum sp. Rhizosphere, Egypt, South Africa, Gangwar and Kaur (2009), roots Brazil, India Hegazi et al. (1979), Purchase (1980), Ruschel (1981) A. brasilense Rhizosphere, Spain, Pakistan, Graciolli et al. (1983), Mehnaz root, stem, Brazil et al. (2010), Reis et al. leaves (2000), Tejera et al. (2005) A. lipoferum Root, stem, Brazil Dobereiner and Day (1976), leaves Reinhardt et al. (2008), Reis et al. (2000), Tejera et al. (2005) A. amazonense Roots, stem Brazil Cavalcante and Dobereiner (1988), Reis et al. (2000) Azotobacter Roots Spain Tejera et al. (2005) chroococum A. vinelandii Rhizosphere, Egypt, Brazil Graciolli et al. (1983), Hegazi roots et al. (1979), Rennie (1980) Bacillus spp. Rhizosphere, South Africa, India, Antwerpen et al. (2002), roots, Stem Pakistan Gangwar and Kaur (2009), Hassan et al. (2010) B. cereus Apoplast Cuba Vela´zquez et al. (2008) B. pumilus Apoplast Cuba Vela´zquez et al. (2008) B. subtilis Apoplast, Cuba, Pakistan Hassan et al. (2010), Vela´zquez rhizosphere et al. (2008) Beijerinckia sp. Root Brazil Vendruscolo (1995) B. ﬂuminensis Rhizosphere Brazil Dobereiner (1961), Dobereiner and Alvahydo (1959) B. indica Rhizosphere, Brazil Dobereiner et al. (1972) roots Brevibacillus sp. Stem, leaves Brazil Magnani et al. (2010) Burkholderia spp. Stem, leaves South Africa, Brazil Antwerpen et al. (2002), Perin et al. (2006b) B. ambifaria Rhizosphere, South Africa Omarjee et al. (2008) roots B. cepacia Rhizosphere, Brazil, South Africa Luzivotto et al. (2010), Mendes roots, stem et al. (2007), Omarjee et al. (2008) B. cenocepacia Roots, stem Brazil Mendes et al. (2007) B. fungorum/ Rhizosphere, South Africa Omarjee et al. (2008) graminis roots B. gladioli Rhizosphere, South Africa Omarjee et al. (2004, 2008) roots, stem B. plantarii/glumae Stem Papua New Guinea Omarjee et al. (2004) B. sacchari Rhizosphere Brazil Bramer et al. (2001) B. silvatlantica Rhizosphere, Brazil Omarjee et al. (2008), Perin roots, leaves et al. (2006a) B. tropica Rhizosphere, South Africa, Omarjee et al. (2008), Perin roots Mexico et al. (2006b), Reis et al. (2004) (continued) 168 S. Mehnaz

Table 7.1 (continued) Bacteria Source Country References B. unamae Stem Papua New Guinea, Caballero-Mellado et al. Brazil, Mexico (2004), Omarjee et al. (2004), Perin et al. (2006b) B. vietnamiensis Stem India Govindrajan et al. (2007) Caulobacter Roots Pakistan Mehnaz et al. (2010) crescentus Citrobacter sp. Rhizosphere Brazil Magnani et al. (2010) Comamonas Apoplast Cuba Vela´zquez et al. (2008) testosteroni Curtobacterium sp. Stem Brazil Magnani et al. (2010) Delftia acidovorans Stem, leaves Pakistan Mehnaz et al. (2010) Derxia gummosa Rhizosphere Brazil Graciolli et al. (1983), Rennie (1980) Enterobacter sp. Rhizosphere, Brazil, Australia Li and Macrae (1992), Magnani roots et al. (2010) E. aerogenes Stem Pakistan Mehnaz et al. (2010) E. cloacae Rhizosphere, Pakistan, Brazil Graciolli et al. (1983), Mehnaz roots, stem et al. (2010), Mirza et al. (2001), Rennie (1980), Rennie et al. (1982) E. oryzae Stem Pakistan Mehnaz et al. (2010) Erwinia cypripedii Apoplast Cuba Vela´zquez et al. (2008) E. herbicola Stem Brazil Graciolli et al. (1983), Rennie et al. (1982) Gluconacetobacter Roots, stem, Brazil, Australia, Asis et al. (2000), Bellone et al. diazotrophicus leaves, India, Egypt, (1997), Cavalcante and apoplast, Cuba, Mexico, Dobereiner (1988), Dong bud, Philippines, et al. (1994), Fuentes- sugarcane Argentina Ramirez et al. (1993), Gillis juice et al. (1989), Li and Macrae (1991), Muthukumarasamy et al. (1994), Prabudoss and Stella (2009), Reis et al. (1994), Vela´zquez et al. (2008), Youssef et al. (2004) G. saccharii Leaf sheath Australia Franke et al. (1999) Herbaspirillum Stem, leaves Brazil, Philippines Asis et al. (2000), Baldani et al. seorpedaceae (1992), Olivares et al. (1996) H. rubrisubulbicans Leaves Brazil, Philippines Asis et al. (2000), Olivares et al. (1996), Pimentel et al. (1991) Klebsiella spp. Stem Brazil, South Africa Antwerpen et al. (2002), Magnani et al. (2010) K. oxytoca Rhizosphere, Pakistan Mehnaz et al. (2010), Mirza roots, stem et al. (2001) K. pneumoniae Roots, stem India, Brazil, Govindrajan et al. (2007), Australia Graciolli et al. (1983), Li and Macrae (1992), Rennie et al. (1982) K. variicola Stem Mexico Rosenblueth et al. (2004) (continued) 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 169

Table 7.1 (continued) Bacteria Source Country References Kocuria kristinae Apoplast Cuba Vela´zquez et al. (2008) Lactococcus lactis Leaves Colombia Cock and de Stauvenel (2006) subsp. lactis Microbacterium Apoplast Cuba Vela´zquez et al. (2008) oleivorans M. testaceum Stem Brazil Mendes et al. (2007) Micrococcus luteus Apoplast Cuba Vela´zquez et al. (2008) Ochrobactrum Rhizosphere Pakistan Hassan et al. (2010) intermedium Paenibacillus Roots Brazil, Hawaii Cavalcante and Dobereiner azotoﬁxans (1988), Seldin and Penido (1986) P. polymyxa Roots, stem Brazil Graciolli et al. (1983), Rennie (1980), Rennie et al. (1982) Pannonibacter Root Pakistan Mehnaz et al. (2010) phragmitetus Pantoea sp. Stem, leaves Cuba, Brazil Loiret et al. (2004), Magnani et al. (2010) P. ananatis Stem Brazil Mendes et al. (2007) P. herbicola Roots, stem, Brazil Graciolli et al. (1986) leaves P. stewartii Stem Brazil Mendes et al. (2007) Pseudomonas spp. Rhizosphere, Brazil, South Africa, Antwerpen et al. (2002), roots, stem, Australia, India Gangwar and Kaur (2009), leaves Li and Macrae (1991), Magnani et al. (2010) P. aeruginosa Stem India Viswanathan et al. (2003) P. aurantiaca Stem Pakistan Mehnaz et al. (2009b) P. ﬂuorescence Roots, stem India, Pakistan, Mehnaz et al. (2009a), Mendes Brazil et al. (2007), Viswanathana and Samiyappan (2002) P. putida Rhizosphere, India, Pakistan Mehnaz et al. (2009a), roots, stem Viswanathana and Samiyappan (2002) P. reactans Stem Pakistan Mehnaz et al. (2010) Rahnella aquatilis Roots Pakistan Mehnaz et al. (2010) Rhizobium sp. Roots Pakistan Mehnaz et al. (2010) R. rhizogenes Apoplast Cuba Vela´zquez et al. (2008) Saccharibacillus Apoplast Spain Rivas et al. (2008) sacchari Serratia spp. Stem South Africa Antwerpen et al. (2002) Staphylococcus sp. Stem, leaves Brazil Magnani et al. (2010) S. epidermidis Apoplast Cuba Vela´zquez et al. (2008) S. saprophyticus Apoplast Cuba Vela´zquez et al. (2008) Stenotrophomonas Rhizosphere Pakistan Hassan et al. (2010), Mehnaz maltophilia et al. (2010) S. pavanii Stem Brazil Ramos et al. (2010) Xanthomonas spp. Stem South Africa, Antwerpen et al. (2002), Pakistan Mehnaz et al. (2010) X. campestris Apoplast Cuba Vela´zquez et al. (2008) X. oryzae Apoplast Cuba Vela´zquez et al. (2008) Zymomonas sp. Stem South Africa Antwerpen et al. (2002) 170 S. Mehnaz reported the isolation of Spirillum lipoferum (now known as A. lipoferum)from sugarcane roots. Dobereiner also isolated A. amazonense from sugarcane roots but data were not published. The information was communicated without any detail in 1988, when isolation of Gluconacetobacter diazotrophicus was reported (Cavalcante and Dobereiner 1988). A. brasilense was also isolated from roots of Brazilian cultivars by Graciolli et al. (1983). Reis et al. (2000) isolated A. brasilense, A. lipoferum, and A. amazonense from four genotypes of Brazilian sugarcane and found them in all parts of the plant except that A. amazonense was not found in leaves. Other than Brazil, isolation of Azospirillum spp. have been reported from Egypt, India, Pakistan, South Africa, and Spain (Gangwar and Kaur 2009;Hegazietal.1979;Mehnazetal.2010;Purchase1980;Tejeraetal.2005).

7.2.2 Azotobacter

Azotobacter is a gram-negative, polymorphic, obligate aerobic bacterium, although it can grow under low pO2. It is very well known for its nitrogen-fixing ability and can fix at least 10 mg N gÀ1 of carbohydrate (Becking 1992). Azotobacter is a poor competitor for nutrients in soil and mostly isolated from roots of the grasses. Two species, namely Azotobacter chroococum and A. vinelandii, have been isolated from sugarcane. Hegazi et al. (1979) and Rennie (1980) isolated the A. vinelandii from sugarcane rhizosphere. Graciolli et al. (1983) reported its isolation from roots. A. chroococcum is recently reported from roots of sugarcane cultivars growing in south of Spain (Tejera et al. 2005). The author could not find any other report about the isolation of this organism from sugarcane.

7.2.3 Beijerinckia

The occurrence of nitrogen-fixing bacteria in this genus was mentioned for the first time in Brazil. Dobereiner and Alvahydo (1959) and Dobereiner (1961) reported the first observations of selective stimulation of nitrogen-fixing bacteria in sugarcane in Brazil. Additional studies on the occurrence of this genus in soil of several Brazilian States (Rio de Janeiro, Saõ Paulo, Pernambuco and Parana´) led to the description of a new species named B. fluminensis (Dobereiner and Ruschel 1958). Analysis of 158 samples collected in different regions of Brazil showed that this species occurred predominantly in soils where sugarcane was cultivated (Dobereiner 1959a), as 95% of sugarcane soil samples contained Beijerinckia.In the sugarcane rhizosphere and on the root surface there were 20–50 times more Beijerinckia and two to five times less other microorganisms than in control soil (Dobereiner 1961). Additional studies showed that roots, leaves, and stems had positive influence on Beijerinckia population. A direct influence of the plant on the development of bacteria was suggested (Dobereiner€ 1959b). 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 171

In 1970s, the introduction of acetylene reduction methodology stimulated further studies involving Beijerinckia and sugarcane. The nitrogenase activity in sugarcane roots was much higher than rhizosphere and nonrhizospheric soil (between the plant rows) and Beijerinckia indica was the most abundant bacterial species in roots and soil samples (Dobereiner et al. 1972). Quantitation of BNF in sugarcane based on the extrapolation of the nitrogenase activity data indicated a contribution of 50 kg N haÀ1 yearÀ1 (Dobereiner et al. 1973). Vendruscolo (1995) also isolated Beijerinckia sp. from the roots of sugarcane. Before the discovery of G. diazotrophicus, Beijerinckia was considered as the most important genus, responsible for nitrogen ﬁxation in sugarcane growing in Brazil. Isolation of this bacterium, from sugarcane, is not reported from any other country although it has been isolated from other crops.

7.2.4 Burkholderia

The genus refers to a group of ubiquitous Gram-negative, motile, obligate aerobic rod-shaped bacteria including animal, human, and plant pathogens as well as some environmentally-important species. Some of these organisms are useful for promoting plant growth and bio-remediation. However, the problem about the threat to human health remains open. Until very recently, the genus Burkholderia included 30 properly described species, but the number of novel Burkholderia species has continuously increased (Perin et al. 2006b). Five of them, Burkholderia vietnamiensis, B. kururiensis, B. unamae, B. tropica, and B. xenovorans can fix atmospheric nitrogen, three species namely B. tuberum, B. phymatum, and B. caribensis help the formation of nitrogen-fixing tubers of bean in tropical regions whereas B. vietnamiensis, B. ambifaria, and B. phytofirmans are known to synthesize vitamins and phytohormones that help crop growth and development (Stoyanova et al. 2007). More than ten species of this genus are reported to be associated with sugarcane plants. These were found in all parts of the plant and rhizosphere as well. Most of the reports are from South Africa and Brazil. The association of B. tropica, B. unamae, and B. cepacia has been reported more frequently as compared to the rest of the species. B. vietnamiensis is reported only from Indian sugarcane cultivars (Govindrajan et al. 2007). Complete list of the species name and their references are given in Table 7.1. Although the species isolated from sugarcane are nonpathogenic to this host at least five of them namely, B. cepacia, B. gladioli, B. graminis, B. glumae, and B. plantarii are reported pathogens for other crops (Stoyanova et al. 2007). Of all Burkholderia species, B. cepacia is of greatest importance. It is an extremely versatile and flexible microorganism, which can be considered like friend or foe of humans. Although B. cepacia is known as a plant pathogen, today it is accepted as one of the most important agents for plant protection and plant growth promotion but its use as a biofertilizer/biocontrol agent in fields at “commercial level” still seems to be very difficult. 172 S. Mehnaz 7.2.5 Enterobacter, Klebsiella, and Pantoea

These three genera belong to the Enterobacteriaceae, a large family of bacteria. Members of Enterobacteriaceae are rod-shaped, gram-negative, facultative anae- robes, motile or non-motile and most of them reduce nitrate to nitrite. Isolation of members of Enterobacteriaceae has been reported from several crops including rice, wheat, sorghum, sugarcane, grasses, and dicotyledonous plants (Li and Macrae 1992). Most of these isolates are capable of nitrogen fixation. Since 1980, there are several reports about isolation of nitrogen-fixing members of Enterobacteriaceae from sugarcane. Rennie et al. (1982) described the members of Enterobacteriacea as the dominating nitrogen-fixing bacteria isolated from sugarcane roots as most of them belonged to the genera Enterobacter and Klebsi- ella. The most commonly isolated species of these two genera are Enterobacter cloacae and Klebsiella pneumoniae. E. cloaceae has been reported from Brazil and Pakistan (Graciolli et al. 1983; Mehnaz et al. 2010; Mirza et al. 2001; Rennie 1980; Rennie et al. 1982). Li and Macrae (1992) also reported the isolation of Entero- bacter but species were not identified. E. oryzae and E. aerogenes have been reported from Pakistan (Mehnaz et al. 2010). Isolation of K. pneumoniae has been reported from Australia, Brazil, and India (Govindrajan et al. 2007; Graciolli et al. 1983; Li and Macrae 1992). K. oxytoca has been reported from Pakistan, and bacteria were isolated from root, stem and rhizosphere (Mehnaz et al. 2010; Mirza et al. 2001). Magnani et al. (2010) and Antwerpen et al. (2002) also reported the association of Klebsiella spp. with sugarcane. Rosenblueth et al. (2004) isolated a new nitrogen-fixing species of Klebsiella from different crops including sugarcane and named it as K. variicola. Pantoea, another nitrogen-fixing member of Enterobacteriacea, has been isolated from all parts of the sugarcane plants. Three species of this genus, Pantoea ananatis, P. herbicola, and P. stewartii have been isolated from roots, stem, and leaves of Brazilian sugarcane plants (Graciolli et al. 1986; Mendes et al. 2007). The unidenti- fied strains of Pantoea sp. have been reported by Loiret et al. (2004) and Magnani et al. (2010) from Cuba and Brazil, respectively. Other members of Enterobacter- iacea, isolated from sugarcane are Citrobacter sp., Erwinia herbicola, E. cypripedii, and Serratia sp. (Antwerpen et al. 2002; Graciolli et al. 1983; Magnani et al. 2010; Rennie et al. 1982; Vela´zquez et al. 2008). These were isolated from roots and stems of Brazilian and South African sugarcane plants.

7.2.6 Gluconacetobacter

This genus belongs to the family Acetobacteraceae. Members of this family are known to produce acetic acid, which are usually acid-tolerant and grow well below pH 5.0. They are gram-negative, aerobic, and rod-shaped bacteria. Gluconaceto- bacter is a nitrogen-fixing and acetic acid-producing bacterium. The first nitrogen- fixing Gluconacetobacter was isolated and described in Brazil by Cavalcante and 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 173

Dobereiner (1988). Initially a new genus and species was suggested for this organism and it was named as Saccharobacter nitrocaptans. When paper was in press, based on the results of DNA/RNA Tm and DNA/DNA binding values, it was named as new species of Acetobacter, i.e., Acetobacter nitrocaptans (Cavalcante and Dobereiner 1988). Later on, it was changed to A. diazotrophicus (Gillis et al. 1989) and then renamed as G. diazotrophicus (Yamada et al. 1997). It has been isolated from all parts of sugarcane, including apoplast, in trash of sugarcane and also from a mealy bug associated with sugarcane plants (Pedraza 2008). In addition to Brazil, it has been reported from Mexico, India, Cuba, Egypt, Argentina, Philippines, and Australia (Table 7.1). Isolation of this organism is not easy as it is slow growing and affected by several factors including the presence of high nitrogen fertilizer which decreases its population. This organism has lack of nitrate reductase and only partial inhibition of nitrogenase activity by ammonium ion, enables it to fix nitrogen in the presence of soil nitrogen. The minimum use of nitrogen fertilizer for sugarcane crop in Brazil is believed to be due to natural occurrence of this organism in their soils. In addition to nitrogen fixation, G. diazotrophicus is also a phytohormone producer. There is so much research done on this organism in Brazil and other countries and its effect on sugarcane growth in labs and field has been studied extensively. The whole genome of this organism is sequenced (http://www.biomedcentral.com/1471-2164/10/450). Several scientific papers and reviews have been written about the isolation and significance of this organism (Boddey et al. 2003; James and Olivares 1997; Pedraza 2008). A recent review by Pedraza (2008) provides detailed information about all the work done up till now on this organism. Recently, another species of Gluconacetobacter,i.e.,G. sacchari has been isolated from leaf sheath of Australian sugarcane crop (Franke et al. 1999). Unfortunately, this bacterium does not fix nitrogen. There are no reports about isolation of any other species of Gluconacetobacter from sugarcane.

7.2.7 Herbaspirillum

The genus comprises several diazotrophic species, some of which exhibit the potential of endophytic and systemic colonization of a number of plants. Two of them, namely Herbaspirillum seropedicae and H. rubrisubulbicans are repeatedly isolated and reported from sugarcane. H. seropedicae could be detected on root surface and as endophyte in intercellular spaces, as well as within intact root cells (Olivares et al. 1997). H. rubrisubalbicans was described as a diazotrophic endophyte with slight pathogenicity in some sugarcane varieties (Baldani et al. 1996;Olivaresetal.1997). The bacteria are gram-negative, curved rods with polar ﬂagella and grow best on dicarboxylic acids, gluconate, glucose, and mannitol, ﬁx N2 at a pH range of 5.3–8, and very high sucrose concentrations (up to 10%), even though they cannot metabolize this substrate (James and Olivares 1997). 174 S. Mehnaz

H. seropedicae was originally thought to be a new Azospirillum species because of similar growth characteristics in the semi-solid, N-free, malate NFb medium devised for the isolation of Azospirillum spp. (Tarrand et al. 1978). However, further analyses showed that it was a completely new genus, i.e., Herbaspirillum (Baldani et al. 1986). The similarity of Herbaspirillum and Azospirillum made further isolation of Herbaspirillum difficult, and therefore, Baldani et al. (1992) devised a new semi-solid malate medium (JNFb medium) to more easily distinguish Herbaspirillum from Azospirillum spp. Gillis et al. (1990) reported that H. seropedicae was very closely related by phenotypical and genotypical characteristics to a mild pathogen of sugarcane and sorghum called “Pseudomonas” rubrisubalbicans, which also fixes N2. After further analyses, “Pseudomonas” rubrisubalbicans was renamed as Herbaspirillum 15 rubrisubalbicans. It was proven to be able to incorporate N from labeled N2 gas (Baldani et al. 1992) and is only the second confirmed diazotrophic plant pathogen, the first being Agrobacterium tumefaciens (Kanvinde and Sastry 1990). Most of the reports about these two organisms are from Brazil. James and Olivares (1997) published a very comprehensive review, describing the details of these two organisms and G. diazotrophicus, with special emphasis on their association with sugarcane.

7.2.8 Pseudomonas

The genus belongs to the family Pseudomonadaceae and itself contains large number of species which are distributed into subgroups. Pseudomonas is known for different beneficial and pathogenic characteristics. It is a very well known PGPR due to its ability to produce phytohormones, siderophores, antibiotics, phosphate solubilization, and production of antifungal compounds. Some species also fix nitrogen in addition to above-mentioned characteristics. Pseudomonas fluorescens and Pseudomonas putida are very well known and well-studied species of this genus. These species have been isolated very frequently from different crops and also used in several studies as inoculum to promote plant growth. Pseudomonas aurantiaca and Pseudomonas chlororaphis are known to be used as biocontrol agents, due to the production of antifungal phenazine compounds. Pseudomonas spp. have been isolated from stem, root, leaves, and rhizosphere of sugarcane growing in Australia, Brazil, India, Pakistan, and South Africa (Table 7.1). P. fluorescence and P. putida have been frequently isolated and reported from India (Gangwar and Kaur 2009; Kumar et al. 2002; Viswanathana and Samiyappan 2002). Viswanathan et al. (2003) isolated P. aeruginosa,in addition to P. fluorescence and P. putida from sugarcane stalk. In 2009, these species have also been reported from Pakistani sugarcane cultivars (Mehnaz et al. 2009b). P. aurantiaca and P. reactants have been recently isolated from stem of sugarcane plants (Mehnaz et al. 2009a, 2010). Magnani et al. (2010) reported the Pseudomonas spp. as dominant bacterial community in leaves of Brazilian 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 175 sugarcane cultivars. Although there are several reports for the isolation of Pseudo- monas spp. from sugarcane, reports for identified Pseudomonas species are not many.

7.2.9 Other Bacteria

Although the genera described above are those which are very frequently reported and represent dominating bacterial community associated with sugarcane, however there are several other bacteria which have been isolated from sugarcane. These include the diazotrophic and nondiazotrophic organisms. Bacillus spp., Bacillus subtilis, B. cereus, and B. pumilus have been isolated from rhizosphere, root, stem, and apoplast of sugarcane (Antwerpen et al. 2002; Gangwar and Kaur 2009; Hassan et al. 2010; Vela´zquez et al. 2008). Brevibacillus sp. was isolated from stem and leaves of Brazilian cultivars (Magnani et al. 2010). Caulobacter crescentus and Delftia acidovorans from rhizosphere, Curtobacterium sp. from stem and leaves and Derxia gummosa from root and rhizosphere have been isolated (Graciolli et al. 1983; Mehnaz et al. 2010; Rennie 1980). Cock and de Stauvenel (2006) isolated a lactic acid-producing bacteria, Lactococcus lactis subsp. lactis, from leaves of sugarcane. Recently, this bacterium has started getting attention due to its pro- biotic nature. Paenibacillus azotofixans and P. polymyxa have been isolated from root and stem (Graciolli et al. 1983; Seldin and Penido 1986). Rivas et al. (2008) isolated a bacterium from apoplastic fluid of sugarcane and identified it as Sacchar- ibacillus sacchari, a new genus and species of Paenibacillaceae. The closely related genus to the Saccharibacillus is Paenibacillus. Two nitrogen-fixing bacteria Rah- nella aquatilis, Rhizobium sp., and R. rhizogenes have been isolated from roots and apoplast (Mehnaz et al. 2010; Vela´zquez et al. 2008). Recently, nitrogen-fixing species of Stenotrophomonas have been isolated from stem and apoplast (Mehnaz et al. 2010; Ramos et al. 2010). Xing et al. (2006) isolated Agrobacterium tumefaciens from sugarcane stalk. Strains of Acinetobacter, Comamonas, Mycobacterium, Micrococcus, Staphylococcus, Xanthomonas, Zymomonas, Ochrobactrum, Kocuria, and Pannonibacter have also been reported from sugarcane (Antwerpen et al. 2002; Hassan et al. 2010; Magnani et al. 2010; Mehnaz et al. 2010; Mendes et al. 2007).

7.3 Role of PGPR in Sugarcane Growth

Beneﬁcial effects savored by the host plant in a PGPR–plant interaction have been speculated to be the result of BNF by the colonizing bacteria, plant growth- promoting substances produced by the rhizobacteria, antifungal, and antibacterial compounds or biocontrol agent. In some cases, a cumulative participation of all of the above mechanisms was observed. 176 S. Mehnaz 7.3.1 Biological Nitrogen Fixation and Phytohormones

Systematic study by various workers in Brazil over the years led to the observation that some sugarcane varieties grown for decades or even a century do not show any decline in the soil nitrogen reserve or yield despite the supply deficit of nitrogen (Boddey et al. 1995). In some varieties of sugarcane, grown in well-irrigated and fertilized tank (with proper supply of K and P) without nitrogen, yield increase was in the range of 170–230 t haÀ1 in the first year. In sugarcane varieties CB45-3, SP70-1143 and Krakatau, the trend of yield increase continued for three subsequent years. Researchers were convinced that in these varieties, 60–80% of the nitrogen accumulated was a result of BNF (Boddey et al. 1995). Studies have shown that BNF by the endophytic bacteria has contributed significantly to the nitrogen nutrition of some sugarcane cultivars in Brazil (Boddey et al. 1991) and Australia (Li and MacRae 1991). Nitrogen balance and 15N-aided dilution studies have confirmed nitrogen nutrition benefits by sugarcane; however, there have been cultivar differences in amounts of fixed nitrogen ranging from 4 to over 70% Ndfa (nitrogen derived from atmosphere) of the total nitrogen from the atmosphere (Lima et al. 1987; Urquiaga et al. 1992; Yoneyama et al. 1997; Asis et al. 2002). In the last decade, numerous studies were undertaken to optimize conditions and reap maximum benefit from various bacteria–non-legume interactions. However, most of the experiments to test the performance of bacteria were conducted under controlled conditions. For sugarcane, these studies include the tissue culture, pot and field experiments but only few PGPR have been used in these experiments. Most of these experiments are conducted with inoculums of G. diazotrophicus, H. ruribulbicans, and H. seropedicae. G. diazotrophicus increased 26% plant dry weight of micropropagated sugarcane plants in green house (Munõz-Rojas and Caballero-Mellado 2003), plant biomass almost 19–50% in a pot trial (Suman et al. 2005, 2007) and 13–16% yield increase in field trials (Govindarajan et al. 2006). Oliveira et al. (2006) used H. rubrisubalbicans and H. seropedicae in a green house experiment and observed 35% increase in dry matter whereas Govindarajan et al. (2006) used H. seropedicae in a field experiment and reported 5–12% increase in yield. B. vietnamiensis increased 19.5% yield and Klebsiellea sp. GR9 enhanced the plant biomass 13–19.5% in field trials (Govindrajan et al. 2006). Burkholderia MG43 inoculation in sugarcane resulted in an effect greater than increasing the fertilizer from half to the full recommended rate, saving the cost of ~140 kg haÀ1 N fertilizer (Govindarajan et al. 2006). Enterobacter, inoculated to roots of micropropagated sugarcane, assimilated 29% of nitrogen by atmospheric fixation (Mirza et al. 2001). PGPR can be used discretely or as a mixture for inoculating plants in pots or fields. A mixture of bacterial isolates used as an inoculum gave a synergistic result in terms of plant growth and development (Govindarajan et al. 2008). A mixture of G. diazotrophicus LMG7603, A. amazonense, and Burkholderia sp. when applied to sugarcane gave a comparatively lower yield than individual inoculation of B. vietnamiensis MG43 and G. diazotrophicus LMG7603 (Govindarajan et al. 2006; 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 177

Oliveira et al. 2002). Oliveira et al. (2002) inoculated micropropagated sugarcane with five different strains of nitrogen-fixing bacteria (G. diazotrophicus, H. seropedicae, H. rubrisubalbicans, A. amazonense and Burkholderia sp.) originally isolated from sugarcane. These strains were used together in various combinations. Plantlets were transferred to pots containing N15 for assessment of nitrogen fixation by the N15 isotope dilution technique. The bacterial inoculation documented a maximum rise of 39% in total biomass over the uninoculated control and assimilated 30% nitrogen by BNF (Oliveira et al. 2002). These studies emphasize the importance of strain selection in a mixed inoculum for obtaining higher performance in the plant. Some of the factors that may affect the performance of PGPR are nitrogen content of the soil, soil type, host plant age, and variety. Soil provided with high amount of nitrogen fertilizer (ammonia) reduced the colonization of sugarcane by both G. diazotrophicus and H. seropedicae (Fuentes-Ramı´rez et al. 1999; Reis et al. 2000; Muthukumarasamy et al. 1999, 2002). Munõz-Rojas and Caballero-Mellado (2003) observed a drastic decrease in the G. diazotrophicus population with the age of the plant and the genotype. In some sugarcane varieties, apparently, the persistence of the endophyte was for a longer period and in higher numbers. Lima et al. (1987) compared four varieties and found that IAC 52-150 yielded only a small positive N balance that was only one-eighth of that shown by CB 47-89. Environmental factors like the soil hydric stress and seasonal changes also contribute to the observed variation in diazotrophic bacteria number (Reis et al. 2000). Oliveira et al. (2006) observed the influence of the soil type, inoculation mixture, and nitrogen fertilization level in the yield response and BNF contribution of two sugarcane varieties. Inoculation promoted increases as well as decreases in the productivity of the sugarcane, with regard to the interaction of the soil classes, sugarcane varieties, and nitrogen rates. The inoculants showed better growth- promoting effects in the soils with lower and medium fertility, and without nitrogen fertilizer. More field trials are therefore required to optimize these parameters including time and way of application of the PGPR and environmental factors. Moutia et al. (2010) described the influence of genotype and drought stress on plant growth promotion by Azospirillum sp. Two agronomically contrasting sugarcane cultivars R570 and M1176/77 adapted to different agro-climatic zones were inoculated with Azospirillum sp. with and without stress. After 103 days of planting, cultivar M1176/77 responded positively with 15% improved growth in shoot height and 75% more root dry mass when subjected to drought stress whereas R570 responded negatively particularly in the absence of drought stress. Some workers observed that the overall growth promotion and nitrogen assimilation in a plant inoculated with PGPR is not solely due to BNF. Sevilla et al. (2001) suggested the participation of other growth-promoting factors in addition to nitro- À gen fixation as both wild and nifH mutants of G. diazotrophicus promoted growth of sugarcane in the presence of nitrogen. Similarly, P. fluorescence, P. putida, indole acetic acid-producing strains, increased the plant biomass of micro-propagated sugarcane, from 2- to 5-folds as compared to un-inoculated plants in in vitro, experiments (Mehnaz et al. 2009b; Fig. 7.1). Moutia et al. (2010) reported the 75% 178 S. Mehnaz

Control P. putida (OK-St) P. putida (QR2) P. fluorescense (PB-St1)

Fig. 7.1 Effect of sugarcane isolates, Pseudomonas ﬂuorescence (PB-St1) and Pseudomonas putida (OK-St and QR2) on root growth of sugarcane plantlets under gnotobiotic conditions increase in root dry weight of sugarcane plants due to auxin production by Azospir- illum sp. rather than nitrogen ﬁxation. Most PGPRs are producers of phytohormones: indoleacetic acid, gibberellins, and cytokinins (Biswas et al. 2000a, b; Verma et al. 2001; Yanni et al. 2001), iron-sequestering siderophores (Verma et al. 2001; Yanni et al. 2001), phosphate-solubilizing enzymes, (Verma et al. 2001) and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Khalid et al. 2005). Growth hormones produced by the bacteria enhance the development of lateral roots and improve the plant’s nutrient uptake from the rhizosphere.

7.3.2 Biocontrol Agent

PGPR induce resistance in plants against fungal, bacterial and viral diseases, insects and nematodes. Induced resistance (IR) is defined as an enhancement of the plant’s defensive capacity against a broad spectrum of pathogens and pests that is acquired after appropriate stimulation. PGPR bring about IR through fortifying the physical and mechanical strength of the cell wall as well as changing the physiological and biochemical reaction of the host leading to the synthesis of defense chemicals against the challenge pathogen. PGPR provide different mechanisms for suppressing plant pathogens. They include competition for nutrients and space, antibiosis by producing antibiotics, viz., pyrrolnitrin, pyocyanin, 2,4-diacetyl phloroglucinol and production of siderophores, viz., pseudobactin which limits the availability of iron necessary for the growth of pathogens. Other important mechanisms include production of lytic enzymes such as chitinases and b-1,3-glucanases which degrade chitin and glucan, respectively, present in the cell wall of fungi, present in the cell wall of fungi, HCN production and degradation of toxin produced by pathogen. Environmental and health concerns about the extended use of pesticides in agriculture necessitate the finding of alternative control approaches for eliminating or controlling pathogens from crops. Several authors have reported on the use of bacteria or fungi as a biocontrol agent. Strains that inhibit the growth of pathogens 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 179 and also have nitrogen-fixing properties could be inoculated into sugarcane varieties and thereby enhance the growth of the crop in the field. The well-known diseases of sugarcane are smut (Ustilago scitaminea), stem rot (Fusarium spp.), red rot (Colletotrichum falcatum), and Nematodes (Melaidogyn sp.). Red rot disease caused by the fungus C. falcatum is one of the major production constraints. It is responsible for the deterioration of sugarcane cultivars and continues to be problem in other countries such as USA, Australia, Taiwan, Thailand, India, and Bangladesh. Plant protection chemicals are currently not recommended for this disease. One approach adopted by the farmer is the use of disease-free seed canes for planting. Such measures are impractical due to the difficulty in diagnosing the dormant fungal infection in seed canes. One key to overcome this situation is the use of biocontrol agent to contain this disease. Researchers are already working on this aspect and several published reports are available. Antwerpen et al. (2002) checked the antifungal activity of Burkholderia isolates from the sugarcane rhizosphere, against U. scitaminea (sugarcane smut) and Fusarium spp. (stalk rot). Forty-seven strains inhibited the growth of Ustilago while 72 strains inhibited the growth of Fusarium in vitro. Twenty-one of these bacterial strains inhibited the growth of both Fusarium and Ustilago (Fig. 7.2a and b). Kumar et al. (2002)isolatedP. fluorescens strains from sugarcane and reported antifungal activity against Fusarium oxysporum and Rhizoctonia bataticola. Hassan et al. (2010) reported the antifungal activity of sugarcane isolates, Ochrobactrum intermedium, P. putida, B. subtilis, Bacillus sp., and Stenotrophomonas maltophilia against local strains of Colletotrichum falcatum. Malathi et al. (2002)conducteda study on the possible detoxification of phytotoxin produced by the sugarcane red-rot pathogen C. falcatum Went by antagonistic fungal and bacterial strains. Eleven P. fluorescens strains and two Trichoderma harzianum strains, isolated from sugarcane rhizosphere, were grown on a medium containing the partially purified toxin from the C. falcatum pathotype Cf 671. Results of this study confirm the efficacy of some strains of biocontrol agents in detoxifying the pathogen toxin. Viswanathan et al. (2003) isolated and checked the antifungal activity of P. putida, P. fluorescens, and P. aeruginosa isolates against C. falcatum and observed that seven isolates of these three species were strong inhibitors. P. aurantiaca, isolated from sugarcane stalk, also

Fig. 7.2 Antifungal activity of sugarcane isolates against fungal pathogens of sugarcane. Fusar- ium sp. (a), Ustilago scitaminea (sporidia) (b) and C. falcatum (c) 180 S. Mehnaz showed antifungal activity against four local isolates of C. falcatum (Mehnaz et al. 2009a; Fig. 7.2c). Ramamoorthy et al. (2001) published a review about the ability of PGPR to induce systemic resistance in plants against diseases and pests. The authors also mentioned that use of an endophytic PGPR strain for inducing systemic resistance is more beneficial for vegetatively-propagated crops like banana, sugarcane, and tapioca. There are several bacterial determinants involved in the induction of systemic resistance by PGPR, the most important being lipopolysaccharides present in the outer membrane of bacterial cells, siderophore, and salicylic acid production (Van Loon et al. 1998). PGPR bring about ISR through fortifying the physical and mechanical strength of the cell wall as well as changing the physiological and biochemical reaction of the host leading to the synthesis of defense chemicals against the challenge pathogen (Viswanathan and Samiyappan 1999a, b). Up till now, most of the reports about induced systemic resistance in sugarcane are from India. Viswanathan and his group worked on PGPR-mediated induced systemic resistance in sugarcane against C. falcatum and published several papers. Viswanathan (1999) and Viswanathan and Samiyappan (1999a) revealed the utility of endophytic P. fluorescens strain EP1 isolated from stalk tissues of sugarcane in inducing systemic resistance against red rot (C. falcatum). In sugarcane, due to PGPR-mediated ISR against C. falcatum, enhanced levels of chitinase and peroxidase were noticed and specific induction of two new chitinase isoforms were found when inoculated with C. falcatum (Viswanathan and Samiyappan 1999a, b). Viswanathana and Samiyappan (2002) also reported that application of PGPR, as sett-treatment, induced systemic resistance against C. falcatum in addition to enhanced sett germination, tillering, and growth of the cane both under controlled conditions as well as field conditions. The Pseudomonas-mediated ISR was significantly higher in the disease susceptible cultivars than in the moderately resistant and moderately susceptible cultivars. Less pathogen-induced invertase enzyme activity was recorded in cane tissues from bacteria-treated stalks, and higher juice characters viz. sucrose percent and sugar yield as compared to the untreated stalk tissues, after pathogen inoculation. These studies clearly show that PGPR-mediated ISR and plant growth promotion can operate under field conditions. Arencibia et al. (2006) described a new role for G. diazotrophicus. According to their report G. diazotrophicus induce systemic resistance against Xanthomonas albilineans-cause leaf scald disease of sugarcane. G. diazotrophicus passes and/or produce elicitor molecules which activate the sugarcane defense response resulting in plant resistance to X. albilineans, controlling the pathogen transmission to emerging agamic shoots. The disease was not observed in the presence of G. diazotrophicus. Their results point toward a form of induced systemic resistance which protects the plant against X. albilineans attack. Defense mechanisms induced against insect pests in plants are different from that against pathogens. PGPR do not kill insects, but application of PGPR brings about some physiological changes in the host plant that prevents the insects from feeding. In nematode control, PGPR induce resistance by altering root exudates or inducing the host to produce repellents that affect nematode attraction or recognition 7 Plant Growth-Promoting Bacteria Associated with Sugarcane 181 of the host (Oostendorp and Sikora 1990) and altering the syncytial development or sex ratio in the root tissue (Wyss 1989). Guyon et al. (2003) isolated B. cepacia complex, B. graminis, B. gladioli, B. caribensis, B. fungorum and B. tropicalis from sugarcane and observed that all isolates show anti-nematode activity against Mel- aidogynae strains of nematodes. Unfortunately, other than B. tropicalis, all strains are also human pathogen. Strain of B. tropicalis that was able to paralyze nematodes also fix atmospheric nitrogen and was isolated from the rhizosphere of sugarcane. This isolate can serve as an alternative of chemical control for nematodes. An interesting strategy to use PGPR as a biocontrol was suggested by Omarjee et al. (2008). The authors studied the relationship between Burkholderia populations and plant parasitic nematodes in sugarcane and observed that more pathogenic nematode, Xiphinema elongatum was associated with B. graminis, B. silvatlantica, B. gladioli, and B. fungorum whereas the less pathogenic species, Helicotylenchus dihystera and Pratylenchus zeae were associated with B. tropica. On the basis of their results, the authors suggested that the B. tropica might be used to reduce nematode damage in sugarcane by promoting certain nematode species to create a less pathogenic nematode community.

7.4 Conclusion

Large number of PGPR have been isolated from sugarcane. Most of them are nitrogen-fixing bacteria and several species can infect the internal tissues of sugarcane. The endophytic nature of these PGPR makes them suitable for the use in vegetatively propagated crops such as sugarcane because of their capability to colonize and persist in the intercellular space of epidermal cells, also reducing the need for further application if the same vegetative parts are used as propagation material. The beneficial effects of PGPR include direct plant growth promotion through BNF and phytohormone production, biological control, and inducing systemic resistance in host plants. Complex interactions between plant genotype, specific environment for N2 fixation and highly efficient diazotrophs are necessary to stimulate BNF in sugarcane but these are not clearly defined yet. It is also observed that instead of using single strain, it would be more effective to apply a mixture of strains to get good growth and broad spectrum activity against multiple pathogens and pests. Considering all the studies carried out for sugarcane, might be researchers are close to developing a biofertilizer for this crop but there is still need to carry out more studies, as most of the scientific literature available on sugarcane is dominated by Brazilian researchers and they are more focused on G. diazotrophicus and H. seropedicae. Brazilian soils are rich in these organisms and they are doing well there but several countries do not have the same climate as Brazil nor same bacterial community. Therefore, scientific community should focus on other PGPR like Azospirillum, Pseudomonas, Klebsiella, etc., and their performance should be evaluated in the field experiments. These organisms have greater potential to be used as biofertilizer due to their abilities of nitrogen fixation, 182 S. Mehnaz phytohormone production, and acting as biocontrol agent. These organisms are easy to isolate, well known for their ubiquitous distribution and association with grasses as compared to G. diazotrophicus.

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Ahmet Esitken

8.1 Introduction

The rhizosphere, soil surrounding roots and influenced chemically, physically, and biologically by plant roots, is a highly favorable habitat for the proliferation of microorganisms and exerts a potential impact on plant health and soil fertility. The rhizoplane is the plant root surfaces strongly adhering soil particles (Kennedy 2005; Antoun and Prevost 2006; Podile and Kishore 2006). In the rhizosphere, very important and intensive interactions are taking place among the plant, soil, microorganisms, and soil microfauna. These interactions can positively or negatively affect plant growth and crop yields (Antoun and Prevost 2006). Deleterious bacteria are assumed to negatively affect plant growth and development through the production of metabolites such as phytotoxins and plant growth inhibitory compounds and/ or competition for nutrients and/or inhibition of the beneficial effects of mycorrhiza (Nehl et al. 1996; Sturz and Christie 2003; Antoun and Prevost 2006). An important group of these microorganisms that exert beneficial effects on plant growth upon root colonization was first defined by Kloepper and Schroth (1978) and termed as plant growth promoting rhizobacteria (PGPR). However, these rhizobacteria have not only beneficial influence in the rhizosphere but also in the phyllosphere of the plants. Thus, PGPR are free-living microorganisms having beneficial effects on plants by colonizing in the rhizosphere or phyllosphere of plants (Bashan and de-Bashan 2005). PGPR may improve plant growth and yield by direct and indirect mechanisms. Direct mechanisms may act on the plant itself and effect growth by means of plant growth regulators (PGRs) such as auxins, cytokinins, and gibberellins or lowering of the ethylene in plant, solubilization of inorganic phosphate and mineralization of organic phosphate and/or their nutrients, asymbiotic fixation of atmospheric nitrogen, and stimulation of disease-resistance mechanisms (induced systemic resistance) (Zahir et al. 2004; Bashan and de-Bashan 2005; Antoun and Prevost 2006; Podile and Kishore 2006). In the indirect mechanism, PGPR act like

A. Esitken Department of Horticulture, Ataturk University, 25240 Erzurum, Turkey e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 189 DOI 10.1007/978-3-642-18357-7_8, # Springer-Verlag Berlin Heidelberg 2011 190 A. Esitken biocontrol agents reducing disease or stimulate other beneficial symbioses or protect the plant by degrading xenobiotics in contaminated soils (Jacobsen 1997). Addi- tionally, they improve plant’s tolerance to stresses, such as drought, high salinity, metal toxicity, and pesticide load (Bashan and de-Bashan 2005). PGPR especially influencing fruit crops have remained considerably limited while the use of rhizobacteria as inocula has dramatically increased during the last couple of decades. Most of the studies with PGPR are related to field crops. Nevertheless, in recent years, the use of PGPR in horticultural crops to promote plant growth, development, and yield has increased in various parts of the world. Several authors have reported that root inoculation and/or spraying with PGPR can result in increased germination, seedling emergence, and modified growth and yield of various horticultural crops. In addition, PGPR have been used for different purposes in horticultural crops, for instance, improving grafting union in grape (Kose et al. 2005), fruit setting (Esitken et al. 2006), fruit thinning (Esitken et al. 2009), and runner plant production in strawberry (Aslantas et al. 2009; Pirlak and Kose 2010). Furthermore, some studies have been conducted on the effects of PGPR on growth and productivity in different horticultural crops, such as tomatoes (Mayak et al. 2004; Woitke et al. 2004), lettuce (Han and Lee 2004; Barassi et al. 2006), radish (Yildirim et al. 2008a, b), and strawberry (Karlidag et al. 2011) under salinity and calcareous soil (Ipek et al. 2011) conditions.

8.2 Modes of Action

PGPR are beneficial for plant growth and they can affect plant growth and yield in a number of ways in many crops. PGPR have been divided into two groups: biocontrol PGPR that indirectly benefit the plant growth and PGPR that directly affect plant growth, seed emergence, or crop yields (Glick et al. 1999). The growth stimulation in plants by PGPR can be a direct effect on production of secondary metabolites such as phytohormones, riboflavin, and vitamins (Dakora 2003). Some PGPR can produce different phytohormones such as auxins, cytokinins, and gibberellins or inhibit ethylene production. These stimulate growth of plant organs via cell division and expansion (Taiz and Zeiger 2002) or by improving nutrient availability (Glick 1995; Chabot et al. 1996a; Yanni et al. 1997). Enhanced plant nutrition by PGPR is mainly through increased phosphorous uptake by solubilization of inorganic phosphate or mineralization of organic phosphate. They also release organic acids, which help to make available forms of nutrients (Biswas et al. 2000a) and often lead to increased plant growth through uptake of water and mineral nutrients. In contrast, through antibiosis, parasitism, competition for nutrients, especially Fe, and space within the vicinity of plant roots, PGPR indirectly benefit the plant growth by suppression of deleterious microorganisms or root pathogens (Glick 1995; Wei et al. 1996; Vidhayasekaran and Muthamilan 1999). However, Kloepper (1993) reported that there is often no clear separation between direct growth promotion and biological disease control promoting indirect growth. 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 191

Details of these mechanisms and their individual contribution to plant growth promotion are discussed below. In general, free-living rhizobacteria usually do not rely on a single mechanism of plant growth promotion (Glick et al. 1999) and may involve two or more of the below-listed individual mechanisms. Understand- ing the environmental factors that regulate the biosynthesis of growth promoting and antimicrobial compounds by PGPR is an essential step toward improving the level and reliability of their growth promoting activity.

8.2.1 Direct Plant Growth Promotion

Direct growth promotion occurs when a rhizobacterium produces a metabolite(s) such as phytohormones or improves nutrient availability that directly promotes plant growth.

8.2.1.1 Biological Nitrogen Fixation

Despite the fact that 78% of the earth’s atmosphere is composed of nitrogen, it is often a limiting factor in plant growth. Some PGPR can fix N2 in phyllosphere. Biological N2 fixation (BNF) by soil microorganisms is considered one of the major mechanisms by which plants benefit from the association of micropartners (Zahir et al. 2004). BNF is the process by which dinitrogen is reduced to ammonia by a specialized group of prokaryotic organisms called diazotrophs (Dalton and Kramer 2006). The nitrogen fixation reaction is catalyzed by the nitrogenase enzyme. One of the benefits that diazotrophic microorganisms provide to plants is fixed nitrogen in exchange for fixed carbon released as root exudates (Glick 1995). Microbial nitrogen fixation rates are highest in carbon-rich environments because of high energy requirement and many diazotrophs are frequently concentrated in the root zone of plants. Depending on the nitrogenase activity, many rhizobacteria were obtained as N2 fixing bacteria including Alcaligenes, Azoarcus, Azospirillum, Azotobacter, Bacillus, Beijerinckia, Klebsiella, Paenibacillus, Pantoe, Pseudomo- nas, Rhodobacter, and Stenotrophomonas (Hurek et al. 1994; Baldani et al. 1997; Riggs et al. 2001; Cakmakci et al. 2008). Cakmakci et al. (2008) investigated isolation and identification of diazotrophic bacteria from the rhizosphere of wild red raspberries (Rubus ideaus L.) grown in northeastern region of Turkey and found 68 bacterial strains which showed nitrogenase activity and were able to grow in N-free basal medium. Similar to other crops such as rice, maize, and wheat, free- living bacteria strains can promote the growth of horticultural plants such as apricot, raspberry, and apple by contributing to N economy through their ability to fix N2. However, most of N2-fixing PGPR can also have other promoting mechanisms such as phytohormone production. Therefore, it is difficult to determine whether the impacts resulted from BNF by diazotrophic PGPR or not from BNF PGPR strains may be a contributing factor of horticultural plant growth 192 A. Esitken promotion in addition to other mechanisms. More detailed information on the BNF contribution in promoting plant growth is reviewed by Ladha and Reddy (2000), Yanni et al. (2001), Kennedy and Islam (2001), and Dalton and Kramer (2006).

8.2.1.2 Phytohormones Production or Inhibition of Ethylene

Plant hormones are a group of naturally occurring organic substances that influence physiological processes at low concentrations. They are often effective at internal concentrations lower than 1 mM. These phytohormones have beneficial effects on plant growth and development (Davies 2004). Some PGPR are capable of synthesizing plant hormones and inhibiting ethylene synthesis in pure culture and soil (Glick et al. 1994; Costacurta and Vanderleyden 1995; Frankenberger and Arshad 1995; Patten and Glick 1996; Arshad and Frankenberger 1998). It is estimated that as much as 80% of all soil bacteria have capacity to secrete indole-3-acetic acid (IAA). Microorganisms can synthesize IAA from tryptophan (Normanly et al. 2004). Production of phytohormones by PGPR has been suggested as one of the most plausible mechanisms of action affecting plant growth. Numerous studies have shown an improvement in plant growth and development in response to seed or root inoculation with various microbial inoculants capable of producing phytohormones. In addition, PGPR appear to be capable of IAA production in the phyllosphere (Esitken et al. 2002, 2009). The type and amount of phytohormone production by microorganisms is variable. Cakmakci et al. (2008) reported that 56 bacterial isolates that IAA produces were collected from the rhizosphere of wild raspberry plants. Under gnotobiotic conditions, Noel et al. (1996) showed the direct involvement of IAA and cytokinin production by PGPR in the growth of canola and lettuce. Gutierrez-Manero et al. (2001) isolated Bacillus pumilus and B. lichenoformis from the rhizosphere of Alnus glutinosa L. which had strong growth promoting activity on alder. Bioassay data indicated that the dwarf phenotype of alder seedlings induced by paclobutrazol (an inhibitor of gibberellin [GA] biosynthesis) was effectively reversed by applications of extracts from a medium incubated with both bacteria and also by exogenous gibberellic acid (GA3). Full-scan GC-MS analysis of the extracts of this medium showed the presence of GA1,GA3,GA4, and GA20 in addition to the isomers 3-epi-GA1 and iso-GA3. Different amounts of PGRs produced by PGPR were also confirmed by Garcia de Salamone et al. (2001). They reported that five PGPR strains produced the cytokinin and dihydrozeatin riboside (DHZR) in pure culture. P. fluorescens G20-18 produced higher amounts of three cytokinins: isopentyl adenosine (IPA), trans-zeatin ribose (ZR), and DHZR. Isopentyl alcohol was the major metabolite produced, but the proportion of ZR and DHZR accumulated by the mutants (CNT1 and CNT2) increased with time. It is now commonly accepted that microbial production of phytohormones is one of the major mechanisms in modifying the growth and yield of plants. Various workers have suggested the involvement of an enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase produced by P. putida GR12-2 in modifying the root growth of different plants (Glick et al. 1994; Hall et al. 1996; Glick et al. 1998; Li et al. 2000; Belimov et al. 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 193

2001, 2002). They found that this bacterium hydrolyzes ACC, the immediate precursor of ethylene (C2H4) in higher plants. ACC deaminase might act to stimulate plant growth by sequestering and then hydrolyzing ACC from germinating seeds, thereby lowering the endogenous levels of ACC which subsequently results in plant growth promotion. PGPR inﬂuenced the growth and yield of inoculated plants including horticultural crops by producing IAA in the rhizosphere upon the release of tryptophan in the root exudates, although other mechanisms of action might have also contributed. Likewise, Asghar et al. (2002) reported a signiﬁcant correlation between L-TRP-derived auxin production by PGPR in vitro and grain yield, number of pods, and number of branches per plant of Brassica juncea.

8.2.1.3 Solubilization of P and Enhanced Nutrient Uptake

An adequate supply of mineral nutrients is necessary for optimum plant growth. However, when adequate amounts of essential nutrients are present in soil, plants may still show deﬁciencies due to the nonavailability of these mineral nutrients. Phosphorus (P) is an essential macronutrient. Most soils contain substantial total P reserves but, in general, less than 10% of this total P pool is accessible to plants (Kucey et al. 1989). When P fertilizer is added, only a small portion tends to be utilized by plants while the remainder reacts with the constituents of soil to form less-soluble compounds (Sundra et al. 2002). Microorganisms play an important role in enhancing P availability to plant roots. Solubilization of mineral nutrients such as phosphorus by PGPR makes them more readily available for plant uptake, and this should be considered as a mechanism for enhanced plant growth (Glick 1995). Dominant phosphate forms are generally Fe and Al complexes in acidic soil and Ca phosphate in calcareous soil. Microorganisms are mainly agents in natural P cycle. A large number of heterotrophic and autotrophic microorganisms including bacteria, fungi, and cyanobacteria have been studied for their ability to solubilize hydroxyapatite, tricalcium phosphate, and rock phosphate due to the production of organic acids such as citric, glutamic, succinic, lactic, oxalic, malic, fumaric, and tartaric acid (Bhattacharya et al. 1986; Goldstein 1986, 1995; Leyval and Berthelin 1989; Salih et al. 1989; Hadler et al. 1990). It is known that microorganisms could be solubilized to inorganic phosphate materials (Jones and Darrah 1994; Nautiyal et al. 2000) and organic acid like metabolites play role in the solubilizing (Kucey et al. 1989; Gadd 1999; Kumar and Narula 1999; Vassileva et al. 2000; Whitelaw 2000). Phosphate-solubilizing bacteria (PSB) are ubiquitous (Gyaneshwar et al. 2002), and Bacillus, Enterobacter, Erwinia, and Pseudomonas spp. are among the most potent strains. Pietr et al. (1990) tested 748 bacterial strains isolated from the rhizoplane of different ﬁeld crops and found that 25.6% strains were capable of dissolving calcium phosphate. They suggested that production of organic acids was the major mechanism of action by which insoluble phosphorus compounds were converted to more soluble forms. 194 A. Esitken

Apart from mineral phosphate, organic matter is the second main source of P. In many soils, organic P occurs in 30–50% of total P and is generally present in inositol phosphate, phosphomonoester or phospholipid, nucleic acid, and phospho- triester forms. For uptake of organic P by plant, it must be hydrolyzed to inorganic P where phosphatase enzyme plays a key role in phosphate cycling. It has been shown that phosphatase (El-Sawah et al. 1993; Bishop et al. 1994; Kremer 1994; Sarapatka and Kraskova 1997) and microbial phosphatase (Kirchner et al. 1993; Kucharski et al. 1996) present in soils and acid phosphatase have main function in this process. In situations where P is limiting crop growth and soil P buffering capacity is large, addition of phosphate-solubilizing microorganisms might increase P availability and fertilizer P recovery, offsetting high costs for P fertilizer. Sundra et al. (2002) stated that the increase in plant-available P is due to a microbial-induced increase in P concentration of the soil solution. Several reports have suggested that PGPR can stimulate plant growth by increasing solubilization (via releasing siderophores or organic acids) and facilitate the uptake of mineral nutrients by the plant (Kloepper et al. 1987; Glick 1995; Chabot et al. 1996a, b; Biswas et al. 2000b). Toro et al. (1997) evaluated the interactive effect of PSB (Enterobacter sp. and B. subtilis) and arbuscular mycorrhizal (AM) fungi (Glomus intraradices) on onion (Allium cepa L.) with a soil of low P content. Co-inoculation of G. intraradices and B. subtilis significantly increased the vegetative biomass and N and P accumulation in plant tissues. This study revealed that the mycorrhizosphere interaction between bacteria and fungi can affect on P cycling, thus promoting a sustainable nutrient supply to plants. Several other researchers have reported a similar role of other bacteria in P and other mineral nutrient solubilization enhancing nutrient uptake and subsequently plant growth (Belimov et al. 1995; Noel et al. 1996; Glick et al. 1998; Biswas et al. 2000a). PGPR may also alter the solubility of mineral nutrients by releasing organic and sugar acids and creating acidity via CO2 (respiration). The rhizosphere is a favorable habitat for acid-producing bacteria (Rouatt and Katznelson 1961; Louw and Webley 1959). Many other scientists have also reported that PGPR can create an acidic environment to promote mineral nutrient solubilization (Webley and Duff 1962; Moghimi et al. 1978; Alexander 1977). Iron is an essential micronutrient of plants as it serves as a cofactor of many enzymes with redox activity. A large portion of iron in soils is in the highly insoluble form of ferric hydroxide, thus iron acts as a limiting factor for plant growth even in iron rich soils. The availability of iron in soil solutions is 1018 M, a concentration which even cannot sustain the microbial growth. Several soil microorganisms produce siderophores and low-molecular-weight iron-chelating compounds that bind Fe3+ with very high affinity and help in iron uptake. It is possible for the rhizosphere microorganisms to use siderophores provided they contain the appropriate uptake protein (Raaijmakers et al. 1995). It was reported in many studies that PGPR may increase the mobility and availability of micronutrients by the formation of high affinity siderophores. Siderophores are low-molecular-weight compounds that complex with Fe+2 and render it available to microorganisms (Leong 1986). Some fluorescent pseudomonads produce a yellow-green pigment, a siderophore 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 195 which Kloepper et al. (1980) designated as “pseudobactin.” Pseudomonas spp. are the potent siderophore producers among Gram-positive PGPR and they produce pseudobactin, pyochelin, pyoverdine, quinolobactin, and salicylic acid, and the structure of the outer membrane receptor proteins complementary to some of these siderophores has been determined (David et al. 2005). The role of pseudobactin in promoting the growth of potato was demonstrated when 10 mg of pseudobactin increased plant growth to the same extent as when the fluorescent pseudomonad was applied to potato seed pieces. The widespread production of siderophores by microbes at low iron levels is reviewed by Neilands (1986). Organisms as diverse as Bacillus, Rhizobium, Pseudomonas, Agrobacterium, Escherichia coli, and many fungi produce a wide range of iron-chelating compounds. Numerous plants are capable of using bacterial Fe siderophore complexes as a means of obtaining Fe from soil (Wang et al. 1993). This view is supported by the findings of Hughes et al. (1992) who reported enhanced Fe uptake in oat due to siderophore production. More details on microbial production of siderophores and their role in enhancing Fe uptake have been reported by Loper and Schroth (1986), Mori et al. (1991), and Biswas et al. (2000a).

8.2.2 Indirect Plant Growth Promotion

Diverse PGPR antagonize the root pathogens through one or more of the different mechanisms identified, for example, by production of volatile or nonvolatile antibiotics, siderophores, enzymes, and other secondary metabolites such as HCN. Production of these compounds is highly influenced by the qualitative and quantitative nutrient availability and is also subjected to quorum sensing (Haas and Keel 2003). The antibiotics commonly produced by different antagonistic bacteria include ammonia, butyrolactones, 2,4-diacetyl phloroglucinol (DAPG), kanosamine, oligomycin A, oomycin A, phenazine-1-carboxylic acid, pyoluteorin, pyrrolnitrin, viscosinamide, xanthobaccin, and zwittermycin A (Whipps 2001). Many of these antibiotics possess a broad-spectrum activity, and DAPG was the most potent and most extensively studied (Raaijmakers et al. 2002). Siderophores produced by PGPR inhibit the root pathogens by creating iron- limiting conditions in the rhizosphere. Siderophore-mediated growth promoting activity of PGPR is associated with the suppression of root pathogens by competitive exclusion and the activity is reversed by the addition of Fe EDTA. A siderophore overproducing mutant of P. putida is more effective than the wild type in suppression of Fusarium wilt in tomato (Vandenbergh and Gonzalez 1984), while a siderophore- deficient mutant of P. aeruginosa lost its biocontrol ability (Buysens et al. 1994). However, the suppression of pathogens by majority of the siderophore-producing PGPR is due to a combination of various other traits (Ongena et al. 1999). Certain PGPR strains produce volatile antibiotics, of which HCN that inhibit the cytochrome oxidase of many organisms is most important. The producer strains 196 A. Esitken possess an alternate cyanide-resistant cytochrome oxidase and are relatively insen- sitive to HCN. Baker and Schippers (1987) and Schippers et al. (1987) established that 4% of the total aerobic bacteria and 40% of the pseudomonads from the rhizosphere of potato grown in short rotation were able to produce HCN in vitro. They demonstrated that the cytochrome respiratory pathway of potato roots was particularly sensitive to cyanide. PGPR compete with deleterious microorganisms and pathogens for limited available nutrients in root exudates and suitable colonization niches, and finally outnumber them. Populations of PGPR established on the plant roots could act as a sink for the available nutrients and limit the nutrient availability for pathogen stimulation and its subsequent root colonization. This mechanism is most often used by fluorescent pseudomonads due to their nutritional versatility and high growth rates in the rhizosphere (Walsh et al. 2001). Apart from root colonization, the PGPR should be able to compete for nutrients with native microbial populations in the rhizosphere for successful elimination of the pathogens. Parasitism of pathogenic fungi by PGPR is facilitated through the production of hydrolytic enzymes that degrade the fungal cell walls. Chitinases, among the hydrolytic enzymes, are of prime importance, as chitin, a linear polymer of b-(1, 4)-N-acetylglucosamine, is a major cell wall constituent in majority of the phytopathogenic fungi. Another important group of hydrolytic enzymes, glucanases degrade the b-1, 3-glucans of the fungal cell walls. Pathogenic fungi produce extracellular hydrolytic enzymes, which degrade the polymers present in plant cell walls and facilitate the fungal infection by disintegrating the cell wall. These hydrolytic enzymes include pectolytic enzymes (exo- and endo- polygalacturonases, pectin lyases), cellulases, and cutinase. A reduction in the activity of these enzymes correlates with a reduction in virulence (Beraha et al. 1983). A few strains of rhizobacteria activate plant defense responses against a broad spectrum of plant pathogens, termed as induced systemic resistance (ISR). Rhizobacteria-mediated ISR has been demonstrated in many plant-pathogen systems wherein the bacterium and the challenging pathogen remained spatially separated, and these observations indicate that ISR is genetically determined (Pieterse et al. 2001). PGPR trigger host defense responses through two different signaling pathways, and are broadly classified as salicylic acid (SA) dependent and independent. The later pathways involved jasmonic acid (JA) and ethylene to trigger defense responses (Pieterse et al. 2001). Signaling molecules like SA, JA, and ethylene accumulate in activated plants and coordinate the defense responses.

8.3 Application of PGPR in Horticulture

PGPR could be used for various purposes such as propagation, biocontrol, and promotion of growth and development in horticultural crops. They were used to improve growth, development, and biocontrol especially in vegetable 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 197 crops until 2000s, but hereafter, studies with fruit crops have been rigorously started.

8.3.1 Effects on Vegetative Propagation of Fruit Crops and Grapes

The majority of fruit species are heterozygous and many are also self-sterile. For this reason, almost all commercially produced fruit cultivars do not develop true-to-type if propagated from seeds. Generative (seedling) methods of propagation are, therefore, of no value in propagating fruit species. New plants identical to a parent plant are usually produced by techniques of vegetative propagation such as cuttings and grafting (Webster and Wertheim 2005). Thus, it is believed that the vegetative propagation of fruit species or rootstocks with superior horticultural characteristics and adaptation ability for given locations and conditions is useful and necessary for overcoming drawbacks, and providing genetically uniform and cost-effective planting material (Perry 1987). Vegetative propagation of fruit crops and grapevine could be helped by PGPR inoculation. Just as, various researchers have reported beneﬁcial effects of PGPR inoculation on many different fruit crops and grapevine (Table 8.1). With cutting techniques, young plant parts are separated from the mother plant and then induced to form roots (Webster and Wertheim 2005). As is commonly known, the rooting of cuttings of temperate zone fruit species is very difﬁcult. So far, there have been many attempts to stimulate the rooting of cuttings by various treatments including treatment with PGRs, carbohydrates, and various other chemical substances (Doud and Carlson 1972; Couvillon 1988). It is well known that auxins have an important role in the rooting of hardwood cuttings on many temperate species (Sims and Lawes 1981). Recent studies have showed that some bacteria in the genera of Agrobacterium, Bacillus, Streptomyces, Pseudomonas, and Alcaligenes might induce root formation in stem cuttings (Bassil et al. 1991; Jacob et al. 1991; Jacob and Hamdam 1992; Hatta et al. 1996; Rinallo et al. 1999). It has been reported that these bacteria produce IAA (Goto 1990), and several studies have also shown that the rooting of bacteria-inoculated cuttings can be further accelerated by exogenous indole-3-butyric acid (IBA) application (Bassil et al. 1991; Falasca et al. 2000). Esitken et al. (2003) evaluated the effects of Agrobacterium rubi A1, A16, and A18 and/or in combination with IBA on rooting of wild sour cherry softwood and semihardwood cuttings. Four different levels of IBA were applied at 0, 250, 500, and 750 ppm. They obtained that no rooting was observed on the cuttings of wild sour cherry with control treatment (no IBA or bacterial treatment) in both types of cuttings, whereas different rooting percentages were observed on the cuttings treated with IBA and bacteria. The highest rooting percentages were 65% for softwood and 70% for semihardwood cuttings when they were treated with 250 ppm IBAþA16 treatments. Moreover, they suggested that the combination of 198

Table 8.1 Response of fruit crops and grapevine to PGPR for vegetative propagation Crop Material PGPR and other applications Result Reference Sour cherry Softwood and Agrobacterium rubi A16 þ 250 ppm IBA 70% rooting Esitken et al. (2003) semihardwood cutting Kiwifruit Hardwood cutting Agrobacterium rubi A18 þ 4,000 ppm IBA 78% rooting Ercis¸li et al. (2003) Grapevine rootstock Hardwood cutting Bacillus BA-16, Bacillus OSU-142 80–100% rooting Kose et al. (2003) Rosa spp. Hardwood cutting Agrobacterium rubi A16 þ 4,000 ppm IBA 65–91% rooting Ercis¸li et al. (2004) Agrobacterium rubi A18þ2,000 ppm IBA Tea Single node cutting Bacillus simplex RC19, Paenibacillus polymyxa 40–76.5% Rooting Erturk et al. (2008) RC05, Comamonas acidovorans RC41, Bacillus megaterium RC01 Apple rootstock Hardwood cutting Agrobacterium rubi A18+sorbitol 30% Rooting Karakurt et al. (2009) Bacillus OSU-142 þ sorbitol þ 2,000 ppm IBA, A18þsorbitolþ2,000 ppm IBA, A18þsorbitolþ4,000 ppm IBA Pistachio Seedling Agrobacterium rubi A1 7.8 lateral root Orhan et al. (2007) Almond Seedling Agrobacterium rubi A18 8.89–9.6 lateral root Orhan et al. (2006a) Grapevine Grafting Pseudomonas BA-8, Bacillus BA-16, Bacillus 80–93% grafting success Kose et al. (2005) OSU-142 Strawberry Runner Bacillus spp., Variovorax sp., Paenibacillus sp., 124–449% increase over Aslantas et al. (2009) Pseudomonas sp. non-inoculated control Strawberry Runner Bacillus OSU-142, Bacillus M3, Pseudomonas 32.8% increase over Pirlak and Kose, (2010) BA-8 non-inoculated control .Esitken A. 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 199

IBA + bacteria was highly effective in increasing rooting capacity when compared to the control, or bacteria and IBA treatments alone. Similarly, Ercis¸li et al. (2003) assessed the influence of A. rubi A1, A16, and A18 and/or combination with IBA on rooting of kiwifruit cuttings. The results showed that treatments of hardwood cuttings of kiwifruit cv. Hayward with bacteria, IBA, and IBA plus bacteria were found to promote rooting and the highest rooting percentage was obtained from cuttings treated with 4,000 ppm IBA plus A18. In another study with Rosa spp., Ercis¸li et al. (2004) investigated the effect of A. rubi on rooting of cuttings of Rosa canina ERS 14 and R. dumalis ERS 15 and showed that treatment of A16 combined with 4,000 ppm IBA in ERS 14 and A18 þ 2,000 ppm IBA resulted in a significant stimulation of rooting of cuttings. Kose et al. (2003) conducted an experiment to study the effect of PGPR on rooting of various grapevine rootstocks. Basal end of 41B and Rupestris du Lot rootstock’s cuttings were inoculated with Bacillus BA16 and OSU-142 alone or in combination. According to their results, bacterial applications alone had no significant effects on rooting in both rootstocks whereas they reported an increase in rooting rate and rooting degree in 41B but decrease in Rupestris du Lot cuttings when cuttings were treated with BA16 + OSU-142 combination compared with the control. In a different study, Erturk et al. (2008) tested seven bacteria, namely Bacillus RC23, Paenibacillus polymyxa RC05, B. subtilis OSU142, Bacillus RC03, Comamonas acidovorans RC41, B. megaterium RC01, and B. simplex RC19, for their potential to form adventive roots of tea (Camelia cinensis) cutting. The cuttings were prepared from three commercial tea clones, namely Pazar-20, Dere- pazari-7, and Tuglali-10. They reported that all bacteria showed IAA producing capacity and higher rooting percentages than control treatment. B. simplex RC19 and P. polymyxa RC05 were more effective on rooting for Pazar-20 clone (76.06–76.50%), P. polymyxa RC05 and C. acidovorans RC41 for Derepazari-7 clone (57.20–63.00%), and P. polymyxa RC05 and B. megaterium RC01 treatments for Tuglali-10 clone (40.10–44.09%). Similarly, Karakurt et al. (2009) studied on the effects of PGPR on rooting of hardwood cutting of MM106 apple rootstock. They evaluated the effects of IBA (1,000, 2,000, and 4,000 ppm), A. rubi A18, and B. subtilis OSU-142 bacterial strains, which have IAA producing capacity and carbohydrate sources (glucose, sucrose, sorbitol, and mannitol) alone or in combination on rooting of cuttings and found that bacterial application alone or combined with IBA and/or sorbitol significantly increased rooting of cuttings of MM106 apple rootstock compared to the control. Some nut species, such as pistachio and almond, have a typical tap root system, but do not have enough lateral roots. In these species, seedling survival is an important problem after transplanting in the field (Ak et al. 1992; Kaska et al. 1992; Nikpeyma and Kaska 1995). In nursery tree production, a well-developed and branching root system is a prerequisite for a high percentage of bud development (Mulas et al. 1989). Growers therefore attempt to eliminate the tap root in pistachio and other seedling species using radicle-tip cutting (RC) treatment (Kaska et al. 1992; Gungor et al. 1995; Nikpeyma and Kaska 1995; McCreary 1996). RC methods to encourage lateral rooting are no longer time consuming and require 200 A. Esitken

Fig. 8.1 Effect of Agrobacterium rubi A1 on lateral root formation in Pistachio

extensive labor. PGPR that produce IAA can be used for lateral root induction in these species. Similarly, Orhan et al. (2007) conducted a research to determine the effects of each of three strains of nonpathogenic A. rubi (A1, A16, and A18), a nonpathogenic strain of B. subtilis (OSU-142), and RC alone, or a combination of bacteria and RC on the number of lateral roots, plant height, stem diameter, root length, and fresh and dry root weights of Pistacia vera seedlings. Treatments with RC and bacterial inoculations alone or in combination increased average lateral root numbers from 2.1 in untreated controls up to 7.8 in A1 treatment (Fig. 8.1). Similar results were obtained from almond by Orhan et al. (2006a). Grapevine cultivars are propagated easily with cuttings, but grafting on rootstocks that are resistant to phylloxera inheres in the infected soils. Some grapevine rootstocks such as Vitis berlandieri have great potential to be used as grape rootstock owing to its resistance to calcareous soils and phylloxera, but their use is very limited due to the difficulties in propagation and grafting. For a successful propagation, increasing callus formation, grafting union, and grafting success are very important, and PGPR can have effects on success rate. The effectiveness of grafting zone inoculation with Pseudomonas BA-8 (cytokinin producer) and Bacillus BA16 and OSU-142 (IAA producers) to improve successing rate, callusing rate, callusing degree, and full callusing rate on four different rootstock-scion combinations including 41B-Beyaz Cavus, 41B-Italia, 5BB-Beyaz Cavus, and 5BB- Italia were studied by Kose et al. (2005). Inoculation with BA-8 in 41B-Beyaz Cavus (83.3%) and 5BB-Beyaz Cavus (80.0%), OSU-142 in 41B-Italia (93.3%), 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 201 and BA16 and OSU-142 in 5BB-Italia (86.7%) significantly increased success rate of grafting compared to the noninoculated control. Strawberries readily proliferate asexually via runners (Hancock 1999) but desire high production efficiency, and runner plant quality has an important impact on yield and growth in the field. In a study by Aslantas et al. (2009), application of some bacterial strains (including Bacillus spp., Variovorax sp., Paenibacillus sp., Pseudomonas sp.) isolated from wild raspberry and tomato, to strawberry plant shows enhancement of sibling number, stolon number and length, and daughter plant number and quality. Bacterial treatments caused the increases of daughter plant between 124 and 449% depending on bacterial strains and years. Similar results of increased runner production in strawberry have also been reported with Bacillus spp. (OSU-142 and M3) and Pseudomonas sp. (BA-8). Two years of replicated field trials were conducted on strawberry in high altitude of Turkey. Root application in Selva increased the number of runner plant per stolon compared to the control. But in Sweet Charlie the most important increment was observed by foliar þ root application in comparison with the control. In addition, the highest usable runner plant was obtained from foliar + root application in Selva (Pirlak and Kose 2010).

8.3.2 Effects on Growth and Development

PGPR can improve growth and development by direct or indirect effect mechanisms. In general, PGPR treatments were given to roots, but recently, sprayed to aerial part of plant.

8.3.2.1 Vegetable Crops

In plant species propagated with seed like vegetables, high germination ratio and rate and synchronization have important effects on seedling growth, development and productivity. Moreover, it is important that seeds and seedlings protect themselves against pathogenic microorganisms before, during, and after germination. Priming with PGPR increases germination and improves seedling establishment. It initiates the physiological process of germination, but prevents the emergence of plumule and radicle. Initiation of physiological process helps in the establishment and proliferation of PGPR on the spermosphere (Taylor and Harman 1990). Bio- priming of seeds with bacterial antagonists increases the population load of antagonist to a tune of tenfold on the seeds thus protected rhizosphere from the ingress of plant pathogens (Callan et al. 1990). Their versatile metabolism, fast growth, active movement, and ability to readily colonize the root surface make these rhizobacteria suitable for seed bacterization. Further, seed treatments provide targeted application of PGPR, allowing earlier protection than with foliar sprays. Microbial inoculants can be applied during three possible phases: (1) at the seed processing plant as 202 A. Esitken a seed coating, months before the actual sowing, (2) “on site,” as a seed application just before sowing, or by inoculant delivery directly onto the seeds in the furrow, and (3) after seedlings emerge (Bashan 1986). Several studies indicate that PGPR may act as natural elicitors for improving the growth and yield of vegetable crops. Ribaudo et al. (2006) inoculated tomato seeds with the PGPR A. brasilense FT326 and evaluated changes in parameters associated with plant growth 15 days after inoculation. According to their results, an increase in shoot and root fresh weight, main root hair length, and root surface indicated that inoculation with A. Brasilense FT 326 led to plant growth improvement. The levels of IAA and ethylene were higher in inoculated plants. Moreover, they found that exogenously supplied ethylene mimicked the effect of inoculation, and the addition of an inhibitor of its synthesis or of its physiological activity completely blocked A. brasilense growth promotion. Therefore, they proposed that the process of growth promotion triggered by A. brasilense inoculation involves a signaling pathway that has ethylene as a central, positive regulator. It is well known that ethylene leads to triple response in seedlings of dicotyl plants (Taiz and Zeiger 2002) and thus improved seedling quality. Kloepper et al. (2007) suggested that photoperiod regulates elicitation of growth promotion and ISR by PGPR and they conducted a greenhouse experiment to study the effect of a commercially available formulation ® of PGPR strains B. subtilis GB03 and B. amyloliquefaciens IN937a (BioYield )on growth of tomato and pepper under short-day (8 h of light) (SD) and long-day (12 h of light) (LD) conditions. The results indicated that under LD conditions, BioYield consistently elicited significant increases in root and shoot mass as well as in several parameters of root architecture in both species. However, under SD conditions, such increases were not elicited. They suggested that differential root colonization of plants grown under LD and SD conditions and changes in leachate quality partially account for these results. Nevertheless, BioYield elicited ISR in tomato and pepper under both LD and SD conditions, indicating that although growth promotion was not elicited under SD conditions, induced resistance was. Similar results were obtained from rocket (Dursun et al. 2008a), spinach (Dursun et al. 2008b), and lettuce (Ekinci et al. 2008). Many researchers have reported pronounced effects of PGPR inoculation on the growth and yield of tomato (Gagne et al. 1993; Kokalis Burelle et al. 2002; Turan et al. 2004; Mena-Violante and Olalde-Portugal 2007; Gul et al. 2008; Kidoglu et al. 2008, 2009). Gagne et al. (1993) conducted a greenhouse experiment to study the effect of PGPR on tomato yield. The bacteria were inoculated into a commercial peat-based substrate. The bacterial strains increased fruit yield up to 9.6% in the spring crop, but these results were statistically nonsignificant. However, in the fall experiment where plants were grown under suboptimum environmental conditions, P. fluorescens strain 63–28 significantly increased fruit yield by 18.2%. The average size of the harvested fruit was also increased by 11.1%. Significant increases in tomato and pepper transplants were also reported by Kokalis Burelle et al. (2002) in response to a formulation of PGPR. Transplant vigor and survival in the field was improved by PGPR treatments in both tomato and pepper with an 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 203 increase of 395% in dry root weight of tomato and 565% in dry root weight of pepper compared to the noninoculated control. Mena-Violante and Olalde-Portugal (2007) demonstrate that inoculation of tomato (Lycopersicon esculentum Mill.) roots with B. subtilis BEB-lSbs (BS13) significantly increased yield per plant and marketable yield, as well as fruit weight and length when compared to the control treatment. Texture of red fruits was also enhanced by the BS13 treatment compared to that of the control treatment. Jagadeesh et al. (2006) tested the influence of deleterious bacteria and PGPR on germination and growth of tomato in vitro. Deleterious bacteria were found to significantly inhibit seed germination as evidenced by the reduced length of radicle of tomato, but PGPR (Pseudomonas sp. RDV 108) significantly suppressed the deleterious bacteria and seed germination, root and shoot length increased by RDV 108 inoculation. Pot experiments were conducted by Adesemoye et al. (2008) with B. subtilis and P. aeruginosa in tomato (Solanum lycopersicum L.), okra (Abelmoschus esculentus), and African spinach (Amaranthus sp.). Inoculation of these vegetables with B. subtilis and P. aeruginosa produces higher dry biomass, which increased 31% for tomato, 36 and 29% for okra, and 83 and 40% for African spinach, respectively, over the nontreated control. Kidoglu et al. (2008) studied to evaluate the effects of different strains of PGPR on cucumber, tomato, and pepper. P. putida 18/1K, Enterobacter cloacae 21/1K, Serratia marcescens 62, P. fluorescens 70, Bacillus sp. 66/3, and P. putida 180 were compared with two commercial products (B. amyloliquefaciens FZB24 and FZB42). All PGPR strains had the ability to produce IAA and to solubilize phosphate except Bacillus 66/3. The results suggested that P. putida 18/1K, S. marcescens 62, P. fluorescens 70, and Bacillus sp. 66/3 significantly increased seedlings’ fresh and dry weight in cucumber, pepper, and tomato compared to the control. In another study, same PGPR and commercial products were tested on hydroponically grown tomato in a PE covered greenhouse during fall and spring (Kidoglu et al. 2009). They found that Bacillus sp. 66/3 was effective for increasing tomato yield. Increase in marketable yield with Bacillus 66/3 inoculation was 37 and 18% in fall and spring compared with the control, respectively. In a pot experiment under greenhouse condition, Cakmakci et al. (2007) investigated the effect of different N2 fixing, phytohormone producing, and P-solubilizing bacterial species on spinach (Spinacia oleracea L.) growth and enzyme activities. Seeds of spinach were inoculated with nine PGPR (Bacillus cereus RC18, B. licheniformis RC08, B. megaterium RC07, B. subtilis RC11, Bacillus OSU-142, Bacillus M-13, P. putida RC06, P. polymyxa RC05, and RC14) and determined that all bacterial strains applications significantly improved growth of spinach. Inocula- tion with PGPR increased shoot fresh weight by 2.2–53.4%, leaf area by 5.3–49.3%, and plant height by 1.9–36.8% compared with the noninoculated control. In addition, depending on bacterial strains, they showed that the bacterial inoculation significantly increased enzyme activities in spinach and a close relationship between plant growth and enzyme activities such as glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), glutathione reductase (GR), and glutathione S-transferase (GST). 204 A. Esitken

8.3.2.2 Fruit Crops

Apart from vegetables that are cultivated annually, fruit species and grapes are perennial crops and have huge root system. Therefore in mature plants, root inoculation of PGPR in orchard is considerably difficult. Thus, root inoculation with PGPR in these plants should be applied before planting to orchard or PGPR should be sprayed to aerial parts of plants to reach promoting effects. For this reason, PGPR studies with fruit crops and grapes are considerably limited, but in recent years, studies for determining effects of PGPR on fruit crops have been initiated. In an earlier study, Caesar and Burr (1987) isolated PGPR strains from roots of apple and rosaceous host and investigated the effects of a total of 226 strains on apple seedling by seed inoculation before sowing and rootstocks M7 and M26 by dipping of roots in greenhouse and field. The obtained treatment of PGPR strains resulted in significant growth up to 65% increasing in seedling and up to 179% in rootstocks; PGPR inoculation led up to 102% more active lateral root nodes in rootstocks. Orhan et al. (2006b) conducted a field experiment to evaluate the potential of two isolates of Bacillus (isolated from maize rhizosphere soil) on raspberry under organic growing conditions. Root inoculation of M3 and M3 + OSU-142 significantly increased yield (33.9 and 74.9%), cane length (13.6 and 15.0%), number of cluster per cane (25.4 and 28.7%), and number of berries per cane (25.1 and 36.0%) compared with the control, respectively. In addition, N, P, and Ca contents of raspberry leaves with OSU-142+M3 treatment and Fe and Mn contents of the leaves of raspberry with M3 and OSU-142+M3 applications significantly improved under organic growing conditions. Bacterial applications also significantly effected on soil total N, available P, K, Ca, Mg, Fe, Mn, Zn contents, and pH. Available P contents in soil were determined to be increased from 1.55 kg P2O5/da at the beginning of the study to 2.83 kg P2O5/da by OSU-142, to 5.36 kg P2O5/da by M3, and to 4.71 kg P2O5/da by OSU-142þM3 treatments. Nevertheless, OSU-142 inoculation alone had negative effects on growth and yield due to high amount auxin production. Similarly, Karlidag et al. (2007) reported that root inoculation of Bacillus M3 and/or Bacillus OSU-142 and/or Microbacterium FS01 combinations before planting stimulated plant growth and resulted in significant yield increases in apple cv. Granny Smith. Root inoculation of PGPR strains significantly increased cumulative yield (26.0–88.0%), fruit weight (13.9–25.5%), shoot length (16.4–29.6%), and shoot diameter (15.9–18.4%) compared with the control. Although biological N2 fixation by rhizobacteria is generally in rhizoplane, PGPR also fixed N2 in the phyllosphere of plants. Furthermore, the foliar application of biofertilizers has many advantages: (1) the nitrogen is being fixed close to its place of assimilation, (2) the nitrogen-fixing bacteria (NFB) on the phylloplane can act as antagonists to many plant pathogens if the right strains are used, (3) there is enough food material for NFBs on the phylloplane in the form of leaf leachates and degrading cuticle which is better suited to symbiotic NFB, and (4) the NFBs face less competition by other microflora on the phylloplane than in soil (Sudhakar et al. 2000). Spraying with N2-fixing PGPR such as Azotobacter, Azospirillum, 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 205 and Beijerinckia to mulberry significantly increased leaf yield and leaf quality when compared to the noninoculated control. The addition of Azotobacter resulted in significantly greater yield than that given by the recommended dose of N and the Beijerinckia treatment resulted in a leaf yield equal to that from the recommended dose of N. The results also suggested that foliar application of biofertilizers especially Azotobacter could safely be used with half the normal dose of chemical nitrogen fertilizer to improve mulberry leaf production (Sudhakar et al. 2000). Esitken et al. (2002, 2003) studied the impact of Bacillus OSU-142 on the growth and yield of apricot cv. Hacihaliloglu in field trials. The Bacillus OSU-142 inoculants were sprayed at full bloom and 30 and 60 days after full bloom. They demonstrated significant increases in yield, shoot length, and nutrient element contents of leaves only on trees treated at the full bloom stage. The average increase in yield was between 30 and 90% as compared to the untreated control. Similarly, higher shoot length and N, P, K, Ca, and Mg contents of leaves were found OSU-142 spraying at full bloom stage. Similarly, Esitken et al. (2006) conducted a field trial to assess the potential of PGPR to growth and yield of sweet cherry. Alone or in combination of two PGPR strains belonging to the genera Bacillus (OSU-142) and Pseudomonas (BA-8) were used to inoculate sweet cherry trees growing in the field. Spraying with PGPR increased yield per trunk cross-sectional area (10.9–21.7%), fruit weight (1.24–5.37%), and shoot length (11.3–29.6%) as compared to the control. In addition, N, P, K, Fe, Mn, and Zn contents of sweet cherry leaves with PGPR treatments significantly increased. Spraying with Bacillus OSU-142 and Pseudomonas BA-8 alone or in combination significantly increased yield per trunk cross-section area (TCSA; 13.3–118.5%), fruit weight (4.2–7.5%), shoot length (20.8–30.1%), and shoot diameter (9.0–19.8%) in “Starkrimson” and yield per TCSA (14.9%) and fruit weight (6.5–8.7%) in “Granny Smith” as compared to the control. The results also suggested that bacterial spraying significantly affected all nutrient elements’ contents (N, P, K, Mg, Ca, Fe, Mn, and Zn) investigated in both cultivars, except Mg in “Starkrimson” when compared with the control (Pirlak et al. 2007). In addition, PGPR can inoculate to root of fruit crops or combine with spraying and root inoculation. Pirlak and Kose (2009) studied the impact of Bacillus OSU- 142 (foliar) and M3 (root inoculation) on the growth and yield of strawberry in field trials. They found that foliar + root application of PGPR strains significantly increased yield per plant as compared with the control. Root application of PGPR strains also significantly increased total soluble solids, total sugar, and reduced sugar, but decreased titratable acidity. Similarly, Esitken et al. (2010) investigated the effects of combination of root inoculation and floral and foliar spraying of PGPR on strawberry under organic growing conditions. Inoculation experiments showed that root inoculation of Bacillus M3 and floral and foliar spraying of Bacillus OSU-142 and Pseudomonas BA-8 bacteria stimulated plant growth resulting in significant yield increases. M3þBA-8, BA-8þOSU-142, M3, M3þOSU- 142, and BA-8 applications increased cumulative yield by 33.2, 18.4, 18.2, 15.3, and 10.5%, respectively. Similar results were realized from PGPR, AVM, and fungi inoculation in strawberry (Kokalis-Burelle 2003; Malusa et al. 2006). 206 A. Esitken

Many fruit tree species bear plenty of flowers, even after poor pollination conditions, and produce too many fruits that the tree is unable to support. Thus, production of excess fruits can cause negative effects such as biennial bearing and low fruit quality. Therefore, excess blossoms, flowers, and/or fruits should be removed, defined as “thinning,” from the trees to increase fruit quality and to prevent biennial bearing. Flowers or fruits can be thinned manually or chemically (Dennis 2000; Wertheim 2000). Plant hormones such as auxin, cytokinin, and ethylene can be used in chemical thinning (Wertheim 2000). In addition, PGPR that produce IAA may be an alternative for fruit thinning. Similarly, Esitken et al. (2009) tested fruit thinning effects of three PGPR strains (Bacillus OSU-142, Microbacterium R23, and Bacillus T7), which produce IAA and NAA (10 and 20 ppm) in apple cvs. Golden Delicious and Braeburn. The results showed that NAA and bacterial treatments decreased fruit setting and number of fruit per tree at various ratios in both cultivars. Among the bacterial treatments R23 (24.1% in 2006 and 39.1% in 2007) in Golden Delicious and R23 (11.6% in 2006) and T7 (17.3% in 2007) in Braeburn gave the lowest fruit setting ratio while fruit setting ratio in Golden Delicious and Braeburn were 36.7 and 46.2% (2006) and 12.8 and 28.2% (2007) in the control, respectively. In addition to yield promotion, PGPR inoculation also enhanced nutrition of plant. Gunes et al. (2009) tested a phosphate-solubilizing bacterium and fungus to evaluate their effects on strawberry plant grown under greenhouse conditions and in pots. They reported that P-solubilizing bacterium and fungus were able to improve P nutrition of strawberry and thus could stimulate plant growth under conditions of low P levels. In a pot trial of the same study, root dipping of a bacterial suspension containing 109 CFU/ml before transplanting increased fruit yield of strawberry up to 90% and P uptake of the shoot was increased by 67.8% with Bacillus FS3 compared with the noninoculated control. They suggested that at the maximum yield with P addition but without microorganism addition, P-fertilizer saving of Bacillus FS-3 was 149 kg P/ha, reflecting greatly enhanced fertilizer-P recovery and P-uptake efficiency. Similarly, Attia et al. (2009) evaluated the effect of phosphate-solubilizing bacteria (B. megaterium NRC 131 and P. fluorescens RM3M) on banana in Egypt. Four different levels of N fertilizer were applied at 25, 50, 75, and 100% P2O5 of recommended dose. They reported that PSB enhanced plant growth (pseudostem length and circumference, number and area of green leaves) and increased yield (bunch weight and length, number of hands/bunch, and number of finger/bunch) under 25% P2O5 treatment conditions (percentage of recommended dose). Thus, they suggested that PSB inoculation with mineral phosphorus raised the efficiency of P fertilizer and required P rate to plants by about 25%. Similar results were reported by Baset Mia et al. (2009) and Adesemoye et al. (2009).

8.3.3 Enhance to Tolerance Under Abiotic Stress

Environmental stresses are among the most limiting factors to plant growth and productivity. PGPR can prevent the deleterious effects of one or more stressors 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 207 from the environment. In addition, the identification, selection, and application of suitable beneficial microorganisms can increase the options to deal with growing problems (Kilian et al. 2000) and can also be environmentally sound (Woitke et al. 2004). Although several studies have been conducted on effects of PGPR on growth and productivity in other plants, fewer reports have been published on PGPR as elicitors of tolerance to abiotic stresses, such as drought, salt and nutrient deficiency, or excess in horticultural crops (Table 8.2). Yildirim et al. (2006) studied the impact of commercial bioinoculants (AgBlend, SoilBuilder, Yield Shield, PlantShield, Inoculaid, and Equity) on the growth of squash in greenhouse under salinity stress (0, 50, and 100 mM of NaCl). They found that biological treatments significantly increased fresh weight compared to nontreated plants that were challenged with salt stress. Furthermore, the most effective biologicals increased the K+/Na+ ratio, which was positively correlated with plant growth, and suggested that alteration of mineral uptake may be one mechanism for the alleviation of salt stress. Yildirim et al. (2008a) assessed the effects of inoculation with Staphylococcus kloosii EY37 and Kocuria erythromyxa EY43 on some agrophysical properties of radish. The results showed that EY37 and EY43 treatments under saline conditions might ameliorate the deleterious effects of salt stress on nutrition and on the growth parameters of radish plants. Similar results were also obtained from radish (Yildirim et al. 2008b; Kaymak et al. 2009), lettuce (Han and Lee 2004), and tomato (Woitke et al. 2004) with various PGPR. Beneficial effects of inoculation with PGPR on the growth and yield of strawberry under saline conditions have also been demonstrated. Karlidag et al. (2011) isolated 44 rhizobacteria strains from the rhizosphere of various crops naturally grown in high salty soils in Upper Coruh Valley, Erzurum, Turkey. Five bacteria were selected for their ability to grow on a saline culture medium (10% NaCl). B. subtilis EY2, B. atrophaeus EY6, B. spharicus GC subgroup B EY30, S. kloosii EY37, and K. erythromyxa EY43 were selected as inoculants. Plant growth promoting effects of these rhizobacteria strains were tested on strawberry cv. Fern under saline conditions. Results of pot trials showed 54.4, 51.7, and 94.9% greater fruit yield in response to inoculation with EY30, EY37, and EY43, respectively, than the noninoculated control under saline conditions. In addition, the LRWC increased from 72.0% in 35 mM NaCl treatment without PGPR to 83.5–88.4% by PGPR. Root inoculation of bacteria also significantly decreased membrane permeability. More- over, nitrogen content of leaves was significantly increased by bacterial treatments whereas Na and Cl contents of leaves and Cl content of roots were significantly decreased by root inoculation with all bacterial treatments in comparison to 35 mM NaCl treatment with no inoculation. The use of PGPR and symbiotic microorganisms, especially arbuscular- mycorrhizal (AM) fungi, may prove useful in developing strategies to facilitate plant growth in saline soils. As a matter of fact, Kohler et al. (2009) investigated the influence of inoculation with a plant growth-promoting rhizobacterium (P. mendocina Palleroni) alone or in combination with an arbuscular mycorrhizal (AM) fungus (G. intraradices or Glomus mosseae) on antioxidant enzyme activities (catalase and total peroxidase), phosphatase activity, solute accumulation, growth, Table 8.2 Response of some horticultural crops to PGPR under abiotic stress 208 Crop Stress condition PGPR Specific comments Reference Squash Salinity AgBlend, SoilBuilder, Yield Shield, Inoculation with biologicals Yildirim et al. (2006) PlantShield, Inoculaid, and Equity significantly promoted fresh weight Radish Salinity Staphylococcus klosii EY37, Kocuria Seed inoculation increased shoot fresh Yildirim et al. (2008a) erythromyxa EY43 weight, root fresh weight, shoot dry weight, root dry weight, LRWC, emergence percent and chlorophyll content; decreased electrolyte leakage Radish Salinity Bacillus subtilis EY2, Bacillus Seed treatment increased fresh and dry Yildirim et al. (2008b) atrophaeus EY6, Bacillus spharicus weight, uptake of minerals, GC subgroup B EY30 chlorophyll contents, LRWC; decreased Na and electrolyte leakage Lettuce Salinity Pseudomonas mendocina Root inoculation increased shoot Kohler et al. (2009) biomass, water content, and antioxidative enzymes Lettuce Salinity Serratia proteamaculans ATCC35475, Root inoculation increased photosynthesis, Han and Lee (2004) Rhizobium leguminosarum bv. stomatal conductance, fresh weight, viciae 128C56G leaf area, total chlorophyll, N, Ca, and decreased ascorbate peroxidase (APX) and glutathione reductase (GR) Radish Salinity Agrobacterium rubi A16, Burkholderia Seed inoculation improved seed Kaymak et al. (2009) gladii BA 7, Pseudomonas putida germination BA 8, Bacillus subtilis BA 142, Bacillus megaterium M3 Tomato Salinity Bacillus subtilis FZB24 Root inoculation improved vegetative Woitke et al. (2004) growth Strawberry Salinity Bacillus subtilis EY2, Bacillus Root inoculation increased fruit yield, Karlidag et al. (2011) atrophaeus EY6, Bacillus LRWC, N, and decreased membrane Esitken A. spharicus GC subgroup B EY30, permeability, Na and Cl of leaves Staphylococcus klosii EY37, and Cl content of roots Kocuria erythromyxa EY43 s fPatGot rmtn hzbcei nHriutrlCos209 Crops Horticultural in Rhizobacteria Promoting Growth Plant of Use 8 Lettuce Drought Pseudomonas mendocina Root treatment increased growth, foliar del Mar Alguacil et al. (2009) K, and LRWC Tomato, Drought Achromobacter piechaudii ARV8 Root inoculation increased fresh and dry Mayak et al. (2004) pepper weights, and reduced production of ethylene Pea Drought Pseudomonas putida, P. fluorescens Seed coating increased grain yield, Arshad et al. (2008) delayed ripening of pods, and decreased endogenous ethylene production Strawberry High Ca Alcaligenes 637Ca, Agrobacterium Root inoculation increased fruit yield, Ipek et al. (2011) A18, Staphylococcus MFDCa1 N, P, K, Fe, Mn, and Cu and MFDCa2, Bacillus M3 and Pantoea FF1 Tomato Low Ca Pseudomonas corrugata Root inoculation promoted the fresh Lee et al. (2010) weight, height and dry matter, and reduced blossom-end rot Apple Replant disease Bacillus subtilis BACT-1, EBW-4 Root inoculation increased trunk Utkhede and Li (1989) and B10, Enterobacter diameter aerogenes B8 Apple Replant disease Pseudomonas spp. Root inoculation stimulated growth Biro et al. (1998) Apple Replant disease Bacillus OSU-142 and M3, Root inoculation increased yield, shoot Aslantas et al. (2007) Bukholderia OSU-7 and number, length, and diameter Pseudomonas BA-8 Strawberry Freezing INA- P. syringae, P. fluorescens Spraying inhibited growth of INA+ Lindemann and Suslow (1987) biovar I P. syringae and protected to freeze Pear Freezing Pseudomonas fluorescens A506 Spraying reduced INA+ bacteria Lindow et al. (1996) population and freeze injury about 40% Grapevine Low Burkholderia phytofirmans PsJN Inoculation increased growth, starch, Ait Barka et al. (2006) temperature proline and phenolics, and enhanced CO2 fixation and O2 evolution 210 A. Esitken and mineral nutrient uptake in leaves of Lactuca sativa L. cv. Tafalla affected by three different levels of salt stress. Shoot biomass increased up to 47% in response to P. mendocina inoculation compared with the control plants at both salinity levels, whereas the mycorrhizal inoculation treatments only were effective in increasing shoot biomass at the medium salinity level. At the highest salinity level, the water content was greater in leaves of plants treated with P. mendocina or G. mosseae. Combined with P. mendocina and G. mosseae presented higher concentrations of foliar K and lower concentrations of foliar Na under high salt conditions. The PGPR strain also induced a higher increase in these antioxidant enzymes in response to severe salinity. Water scarcity in the root zone imposes serious reduction in crop yield and is one of the greatest limitations. Under stress conditions, including drought, the plant hormone ethylene endogenously regulates plant homeostasis and results in reduced root and shoot growth (Apelbaum and Yang 1981; McKeon et al. 1982; Hoffman et al. 1983; Graves and Gladon 1985; Mayak et al. 2004). 1-aminocyclopropane- 1-carboxylic acid (ACC) is the immediate precursor of ethylene in higher plants (Yang and Hoffman 1984). However, degradation of the ethylene precursor ACC by bacterial ACC deaminase releases plant stress and rescues normal plant growth. It has been discovered that certain microorganisms contain an enzyme ACC deaminase that hydrolyzes ACC into ammonia and a-ketobutyrate (Glick et al. 1994, 1998; Mayak et al. 1999; Shaharoona et al. 2006) instead of its conversion into ethylene. The uptake and cleavage of ACC by ACC deaminase containing rhizobacteria decrease the amount of ACC, as well as ethylene, in the spermosphere and rhizoplane, thereby acting as a sink for ACC. In a study with tomato and pepper, Mayak et al. (2004) conducted pot trials to assess the effect of PGPR under salt stress. The isolated bacterial strain, Achromobacter piechaudii ARV8 that has ACC deaminase activity, was used to inoculate tomato and pepper. Inoculation had a significant positive effect on fresh and dry weights of both tomato and pepper seedlings exposed to transient water stress. In addition, the bacterium reduced the production of ethylene by tomato seedlings, following water stress. During water deprivation, the bacterium did not influence the reduction in relative water content; however, it significantly improved the recovery of plants when watering was resumed. Inoculation of tomato plants with the bacterium resulted in continued plant growth both during the water stress and after watering was resumed. Arshad et al. (2008) investigated the potential of PGPR with 1-aminocyclopropane- 1-carboxylate (ACC) deaminase to ameliorate the effects of drought stress on growth, yield, and ripening of pea (Pisum sativum L.). Inoculation had a significant positive effect and resulted in better grain yield (up to 62%) than the respective uninoculated control. Ripening of pods was also delayed in plants inoculated with PGPR. The effectiveness of soil inoculation with AM fungus (G. intraradices)or the PGPR (P. mendocina) for improving lettuce growth, plant physiological parameters, and gene expression of one PIP aquaporin in roots under deficit water and elevated CO2 conditions was studied in pot experiments by del Mar Alguacil et al. (2009). The inoculation with PGPR produced the greatest growth in lettuce plants under all assayed treatments as well as the highest foliar potassium concentration 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 211 and leaf relative water content under elevated (CO2) and drought. However, under such conditions, the PIP2 gene expression remained almost unchanged by PGPR inoculation while G. intraradices showed enhanced expression of the PIP2 gene. Lime-induced iron chlorosis is a term often used for chlorosis associated with disturbed Fe metabolism on high Ca-containing soil. This is a widespread and damaging nutritional disorder in several important horticultural crops. In general, horticultural crops are also considerably sensitive to lime-induced chlorosis. In wet calcareous soils, the dynamics of bicarbonate formation is much higher and depends on high CO2 pressure in the soil and hydrolysis of CaCO3, which requires the presence of water. Economically, production of several horticultural crops needs regular irrigation. Thus, lime-induced Fe chlorosis can be a great problem in particular irrigated area. Although PGPR can appear to decrease in sensitivity when they are inoculated to root, little or no study exists on this subject. Recently, Ipek et al. (2011) had studied the impact of PGPR inoculants on the growth, yield, and nutrition of strawberry in greenhouse under high calcareous soil conditions. They selected some PGPR which ruptured to bicarbonate in in vitro including Alcaligenes 637Ca, Agrobacterium A18, Staphylococcus MFDCa1 and MFDCa2, and solubilized phosphate including Bacillus M3 and Pantoea FF1. These bacteria inoculated to strawberry root before planting. Plant growth responses were variable and dependent on the inoculant strain. However, all bacterial strains significantly increased fruit yield and mineral nutrition especially N, P, K, Fe, Mn, and Cu. Bacterial inoculation promoted fruit yield, leaf area, N, P, K, Fe, Mn, and Cu concentrations of leaves up to 48, 16, 32, 52, 25, 68, 58, and 223%, respectively, over the noninoculated control. Contrary to high Ca, calcium deficiency often caused blossom-end rot (BER) in tomato plants. BER can also occur under suboptimal growth conditions such as drought (Nishio and Morita 1991) and high salinity (Ehret and Ho 1986), even when the calcium concentration is sufficient for fruit development under normal conditions. Lee et al. (2010) investigated the effect of Pseudomonas sp. LSW25R on tomato growth and BER, because this bacterium stimulate uptake of Ca as well as growth promotion. They found that root inoculation of LSW25R significantly promoted the fresh weight, height, and dry matter of tomato plants and also reduced blossom-end rot of tomato fruits (up to 61%) as compared to the control. Replant disease is one of the components of the fruit tree problems and contributes to the poor growth of fruit trees planted on old same fruit tree orchard sites. This disease suppresses initial growth of young trees and reduces yield of pome and stone fruits in all the major fruit growing areas of the world. Both biotic and abiotic causal agents have been associated with this disease. Utkhede and Li (1989) conducted field trials to test four selected rhizobacterial isolates (B. subtilis BACT-1, EBW-4, B10, and Enterobacter aerogenes B8) for growth promotion of apple in sites where there is a replant problem. The response was measured by increase in trunk diameter of Spur McIntosh on M26 apple trees. They found that bacterial applications with other treatments such as monoammonium phosphate and formalin significantly increased tree growth and suggested that B. subtilis EBW-4 and E. aerogenes B8 have potential for field control of apple replant disease in 212 A. Esitken orchards of British Colombia. Similar results were also obtained from apple by Biro et al. (1998) who determined more than 100% plant growth promotion effects. Recently, Aslantas et al. (2007) assessed the growth promoting activity of Bacillus OSU-142 and M3, Bukholderia OSU-7, and Pseudomonas BA-8 in apple cv. Granny Smith on M9 and MM106 in newly uprooted old apple orchard. These bacteria were capable of producing IAA and cytokinin, but three of them (OSU-7, BA-8, and M-3) were also able to dissolve phosphate. They reported that root inoculation of apple trees with OSU-142, OSU-7, BA-8, and M-3 increased average shoot length by 59.2, 18.3, 7.0, and 14.3% relative to the control and fruit yield by 116.4, 88.2, 137.5, and 73.7%, respectively. A low temperature is a major factor limiting the productivity and geographical distribution of many species, including important agricultural crops. Cold-hardy plants increase their freezing tolerance upon exposure to low, nonfreezing temperatures by a phenomenon known as cold acclimation (Thomashow 1999). Nevertheless, many of horticultural crops such as vegetable crops or some parts of fruit crops such as flowers and fruitlets are more sensitive to low temperatures, namely, cold sensitive. Epiphytic bacterial species with ice-nucleating activity (Ice-bacteria), such as P. syringae and Erwinia herbicola, contribute to the frost injuries of many cold-sensitive plants by reducing the plants’ ability to supercool, a process that prevents the formation of membrane-damaging ice crystals (Hirano and Upper 2000; Lindow and Leveau 2002). Therefore, biological control of these bacteria will lead to a corresponding decrease in frost injury. Several studies reported that a reduction in frost injury at various horticultural crops, such as almond, citrus, tomato, pear, and avocado, was realized by chemical and biological control of P. syringae and E. herbicola (Lindow 1983, 1986, 1995; Lindow et al. 1983; Lindow and Connell 1984; Lindemann and Suslow 1987). For example, ice nucleation-deficient (INA ) mutants of Pseudomonas strains were tested for their efficiency as biological control agents of frost injury on blossoms of greenhouse- grown strawberry plants (Lindemann and Suslow 1987). The INA- deletion mutants of P. syringae and P. fluorescens biovar I inhibited growth of their ice nucleation- þ active (INA ) parental strains but only the INA P. syringae strain protected blossoms against freezing in strawberry. In addition, antifreeze proteins (AFPs) are a structurally diverse group of proteins that have the ability to modify ice crystal structure (Raymond and DeVries 1977) and inhibit recrystallization of ice, which is the growth of ice at high subzero temperatures (Knight et al. 1988). Polar fish, insects, fungi, plants, and bacteria can produce AFPs. AFPs which produced PGPR may lead to reduction of frost injury in the inoculated plant. Just as, Ait Barka et al. (2006) assessed the effects of in vitro inoculation with B. phytofirmans PsJN on grapevine (Vitis vinifera L. cv. Chardonnay) growth at a low temperature. The results indicated that in vitro inoculation of grapevine explant with bacteria increased grapevine growth and physiological activity at a low temperature. They also found that there was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26C) and low (4C) temperatures and their sensitivities to chilling. The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 213 control. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics and they suggested that these increases correlated with the enhancement of cold tolerance of the grapevine plantlets.

8.3.4 Biocontrol

Soil-borne and also foliar and floral pathogens are well known for their devastating effects on plant health and yield. For successful disease management, it is important to find the most effective and economical ways to protect the plant from various pests or diseases. Protection of plants to harmful organisms by synthetic chemicals is costly and has deleterious effects on environment. Therefore, it requires methods with little or no harmful effects on environment and economy. PGPR can have important effects on pathogenic microorganisms, insects, and nematodes. Bio- control agents are easy to deliver, improve plant growth, activate resistance mechanism in the host, and increase biomass production and yield. These antagonists act through antibiosis, secretion of volatile toxic metabolites, mycolytic enzymes, parasitism, and competition for space and nutrients (Whipps 2001; Bent 2006). Some PGPR also elicit physical or chemical changes related to plant defense, a process referred to as “induced systemic resistance” (ISR). ISR elicited by PGPR has suppressed plant diseases and pests caused by a range of pathogens in both the greenhouse and field (Van Loon et al 1998; Ramamoorthy et al. 2001; Van Loon and Bakker 2006). There are a number of studies related to biological control of pest in horticultural crops, especially vegetable. Significant reductions in damping-off diseases caused by Pythium spp., R. solani, and Sclerotium rolfsii and rot root or wilt due to Aphonomyces euteiches, Fusarium oxysporum, Ralstonia solanacearum, and Thielaviopsis basicola on several vegetable crops have been reported under field, greenhouse conditions, and closed system (Ahl et al. 1986; Becker et al. 1990; Benhamou et al. 1996, 1997; Di Pietro et al. 1992; Elad and Chet 1987; Hadar et al. 1983; Perke 1990; Leeman et al. 1995a, b; Liu et al. 1995; Ordentlich et al. 1987, 1988; Perke et al. 1991; Rankin and Paulitz 1994; Reddy et al. 1993; Zhou and Paulitz 1993, 1995; M’Piga et al 1997; Ongena et al. 2000; Jetiyanon and Kloepper 2002; Alsanius et al. 2004; Guo et al. 2004; Akkopru and Demir 2005; Nakkeeran et al 2006; Omar et al. 2006; Szczech and Dysko 2008). For example, Ongena et al. (2000) investigated the interaction between P. putida BTP1 and its sid- mutant M3 and cucumber in terms of phytoalexins productions. Inoculation experiments showed that these cucumber- associating bacteria could protect against Phytium aphanidermatum by accumulation of antifungal phenolics in the treated root and these phytoalexins were produced systemically. Several antifungal molecules accumulated in both treated and nontreated root parts of plants protected against P. aphanidermatum with BTP1 or M3. Furthermore, increased amounts of fungitoxic molecules also revealed in leaves of PGPR-treated plants. Likewise, Alsanius et al. (2004) evaluated the effects of 214 A. Esitken inoculation of tomato seed with P. fluorescens 5.014 and 5-2/4 on Pythium ultimum (Pu) under closed system conditions (nutrient film technique). The results indicated that the accumulated yield and fruit size were positively affected by seed inoculation with 5.014 and 5-2/4 compared to the Pu inoculated control and similar to the noninoculated control. In addition, inoculation with 5.014 and 5-2/4 significantly decreased the titer of re-isolated Pu and had amelioration in disease index. Induced resistance is defined as an enhancement of the plant’s defensive capacity against a broad spectrum of pathogens and pests that is acquired after appropriate stimulation. The resulting elevated resistance due to an inducing agent upon infection by a pathogen is called induced systemic resistance (ISR) or systemic acquired resistance (SAR) (Hammerschmidt and Kuc 1995). The induction of systemic resistance by rhizobacteria is referred as ISR and typically do not cause any necrotic symptoms on the host plants (Van Loon et al. 1998). ISR is the activation of latent resistant mechanisms that are expressed upon subsequent or challenge inoculation with a pathogen (Van Loon 1997). Raupach and Kloepper (2000) studied the impact of PGPR inoculants on cucumber plant growth and on naturally occurring cucumber diseases with and without methyl bromide fumigation. Seven PGPR treatments including single and mixtures of B. pumilus INR7, Curtobacter- ium flaccumfaciens ME1, and B. subtilis GB03 were inoculated to seed. In the absence of methyl bromide, all seven PGPR treatments significantly promoted plant growth, compared to the nontreated control. In addition, all PGPR treatments significantly reduced severity of foliar disease and mixtures of PGPR strains showed a higher level of disease protection, compared to the nontreated control, with and without methyl bromide. Jetiyanon and Kloepper (2002) tested to PGPR mixtures on the specific diseases and hosts included bacterial wilt of tomato (L. esculentum) caused by R. solanacearum, anthracnose of long cayenne pepper (Capsicum annuum var. acuminatum) caused by Colletotrichum gloeosporioides, damping off of green kuang futsoi (Brassica chinensis var. parachinensis) caused by R. solani, and cucumber mosaic virus (CMV) on cucumber (Cucumis sativus). Results indicated that four mixtures of PGPR and one individual strain treatment significantly reduced the severity of all four diseases compared to the nonbacterized control: 11 mixtures reduced CMV of cucumber, 16 mixtures reduced bacterial wilt of tomato, 18 mixtures reduced anthracnose of long cayenne pepper, and 7 mixtures reduced damping off of green kuang futsoi. Most mixtures of PGPR provided greater disease suppression than individual PGPR strains. Some strains of PGPR can elicit systemic disease protection and this is an important issue for practical application in agriculture and horticulture, and combine PGPR having different modes of action with organic amendments can occur positive results. Such an integrated system could be used for transplanted vegetables to produce more vigorous transplants that would be tolerant of nematodes and other diseases for at least a few weeks after transplanting to the field. In fact, Kloepper et al. (2004) tested the specific combination of B. subtilis GB03, B. amyloliquefaciens IN937a, and B. subtilis IN937b together with chitosan. Strain GB03 produces antibiotics while IN937a and IN937b elicit ISR. The results demonstrated that the combination of two bacilli strains (GB03 and IN937a) with chitosan resulted in significant 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 215 growth promotion in tomato (L. esculentum), bell pepper (C. annuum), and cucumber (C. sativus) and then the preparation has been commercialized under the name “BioYield.” Many other recent studies have confirmed positive effects of rhizobacterial inoculation on various diseases via ISR in different horticultural crops (Yan et al. 2002, 2003; Radjacommare et al. 2004; Romeiro et al. 2005; Ji et al. 2006; Jeun et al. 2007). Phenolic compounds are the natural constituents in all plants investigated until now. Besides several other classes of compounds, antibiotic phenols have been implicated in plant defense mechanisms (Nicholson and Hammerschmidt 1992; Kuc 1995). A study in India with two PGPR (P. fluorescens Pf4 and P. aeruginosa) and infection by Erysiphe pisi in pea (P. sativum) showed that synthesis of phenolic compounds was enhanced in all the treated plants. Especially gallic, ferulic, and cinnamic acids significantly induced with bacteria inoculation compared to the control. Maximum accumulation of phenolic compounds was observed in plants raised from PGPR-treated seeds and infection with E. pisi. It suggested that under pathogenic stress, P. aeruginosa performed better because it induced a relatively higher amount of phenolics (Singh et al. 2002). Many plant enzymes are involved in defense reactions against plant pathogens. These include oxidative enzymes such as peroxidase (PO) and polyphenol oxidase (PPO), which catalyze the formation of lignin and other oxidative phenols that contribute to the formation of defense barriers for reinforcing the cell structure (Avdiushko et al. 1993). Other enzymes such as tyrosine ammonia-lyase (TAL) and phenylalanine ammonia-lyase (PAL) (Bashan et al. 1985; Beaudoin-Eagan and Thorpe 1985) are involved in phytoalexin or phenolic compound biosynthesis. When plant roots were treated with PGPR, activity of these defense enzymes can be stimulated in root tissues. Similarly, Chen et al. (2000) studied the impact of P. corrugata 13 or P. aureofaciens 63-28 inoculation on PAL, PO, and PPO activity in cucumber plants. Inoculation with P. corrugata 13 or P. aureofaciens 63 28 of cucumber roots stimulated PAL activity in root tissues in 2 days and PO and PPO activities were increased in roots 2–5 days after bacterization with P. corrugata strain 13. Likewise, Bharathi et al. (2004) evaluated the efficacy of 13 PGP antagonistic rhizobacterial strains against chilli (Capcicum annum) fruit rot and dieback incited by Colletotrichum capsici under greenhouse and field conditions. They developed talc-based formulations of P. fluorescens (Pf1) and B. subtilis either single or mixed along with or without chitin and neem amendments and tested under greenhouse and field conditions. The PGPR mixed bioformulation Pf1 þ B. subtilis þ neem þ chitin was found to be the best for reducing the fruit rot incidence besides increasing the plant growth and yield parameters under both greenhouse and field conditions. In addition, plants treated with mixed formulation accumulated much more chitinase, b-1, 3 glucanase, PO, PPO, PAL, and phenol than uninoculated control. Similarly, Sendhilvel et al. (2007) conducted greenhouse trials of grapevine to assess the effect of PGPR on powdery mildew caused by Uncinula necator (Schw.) Burn. They found that foliar application of talc- based P. fluorescens Pf1 at 2% had a significant positive effect on reduction of disease incidence (76.7%) compared to the noninoculated control. Moreover, Pf1 216 A. Esitken application induced defense gene products such as PO, PPO, and chitinase in host plant. Similar results were obtained from different PGPR strains and horticultural crops such as pepper (Kavitha et al. 2005), tea (Saravanakumar et al. 2007; Chakraborty et al. 2009), and tomato (Girish and Umesha 2005; Latha et al. 2009). There are number of reports about biological control of bacterial disease in vegetable crops (Cooksey and Moore 1982; Anuratha and Gnanamanickam 1990; Phae et al. 1992; Cuppels et al. 1999; Donmez et al. 2000; Sahin et al. 2000; Wilson et al. 2002; Ji and Wilson 2003; Byrne et al. 2005; Ji et al. 2006). For instance, integration of foliar bacterial biological control agents and PGPR was investigated to determine whether biological control of bacterial speck of tomato, caused by P. syringae pv. tomato, and bacterial spot of tomato, caused by Xanthomonas campestris pv. vesicatoria and X. vesicatoria under field conditions (Ji et al. 2006). The results indicated that the foliar biological control agent P. syringae strain Cit7 was the most effective of the three foliar biological control agents, providing significant suppression of bacterial speck and bacterial spot. When applied as a seed treatment and soil drench, PGPR strain P. fluorescens 89B-61 significantly reduced foliar severity of bacterial speck. PGPR strains 89B-61 and B. pumilus SE34 both provided significant suppression of bacterial spot. Combined use of foliar biological control agent Cit7 and 89B-61 provided significant control of bacterial speck and spot. As is fungi and bacteria, PGPR induce systemic resistance against viruses as well as to enhance plant growth in horticultural crops. Recently, Kavino et al. (2009) investigated the effects of P. fluorescens (Pf1 and CHA0) on controlling Banana bunchy top virus (BBTV) in banana. The results suggested that banana plants treated at planting and at 3, 5, and 7 months after planting had significantly reduced bunchy top incidence (up to 54%) under field conditions compared with the control. In a different study, PGP rhizobacterial and endophytic bacterial strains were used to induce systemic resistance against BBTV in tissue-cultured banana plantlet (Harish et al. 2009). They assessed PGPR (P. fluorescens Pf1 and CHA0) and endophytic bacteria (B. subtilis EPB5 and EPB22) alone and in combination for their effects on controlling BBTV. Plants applied with mixtures of PGPR and endophytic bacteria showed minimum infection of 20% (100% in the control). In addition, the expression of defense-related chemicals and enzymes, such as phenol, PAL, PO and PPO, and pathogenesis-related proteins such as chitinase and b-1,3 glucanase, was more pronounced in the endophytic bacteria-treated banana plant than in the control. Similarly, Murphy et al (2000), Zehnder et al. (2000), Kandan et al. (2005), and Kirankumar et al. (2008) showed that PGPR inoculation had significant effects on controlling of various virus diseases in tomato. Nematodes are a threat to many horticultural crops, especially to young plants after transplanting to a field. Root-knot nematodes (Meloidogyne spp.) limit several horticultural crop productions in many areas of the world. Due to environmental concerns and increased regulations on use of chemical fumigants, more management strategies for control of root-knot (Meloidogyne spp.) nematodes are currently being investigated (Nico et al. 2004). Biological control using microbial antagonists is one potential alternative to chemical nematicides. Among the biological control 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 217 agents that being assessed are egg-parasitic fungi, nematode-trapping fungi, bacteria, and polyphagous predatory nematodes (Gray 1988; Kerry 1988; Kerry and Hidalgo-Diaz 2004; Kiewnick and Sikora 2005). These antagonists can limit nematode abundance. Burkett-Cadena et al. (2008) tested commercially available rhizobacterial inoculants (Equity®, BioYield®, and AgBlend®) and FZB42 strain in the product RhizoVital® for its ability to suppression of root-knot nematodes (M. incognita) in tomato. They demonstrated that the inoculants Equity (multiple strains), BioYield (two strains), and FZB42 induced significant reductions in nematode eggs per gram root, juvenile nematodes per ml of soil, and galls per plant on tomato. AgBlend, containing microbial metabolites, reduced number of galls. Treatment with each of the inoculants also increased root weight. Biocontrol agents which affect nematodes can use multiple mechanisms and therefore the control achieved by biocontrol agents with several distinct mechanisms of control may be additive or synergistic. Similarly, Siddiqui and Akhtar (2009) assessed antagonistic fungi (Aspergillus niger CA and Penicillium chrysogenum CA1), PGPR (Burkholderia cepacia 4684 and B. subtilis 7612), and AM fungi (G. intraradices KA and Gigaspora margarita AA) alone and in combination for their effects on the growth of tomato and on the reproduction of M. incognita in glasshouse experiments. They found that application of Bu. cepacia 4684, B. subtilis 7612, A. niger CA, Gl. intraradices KA, Gi. margarita AA, and P. chrysogenum CA1 caused 36.1, 32.4, 31.7, 30.9, 29.9, and 28.8% increases in shoot dry mass of nematode-inoculated plants, respectively. Use of Bu. cepacia 4684 with A. niger CA caused a highest (65.7%) increase in shoot dry mass in nematode-inoculated plants followed by B. subtilis 7612 plus A. niger CA (60.9%). Bu. cepacia 4684 greatly reduced (39%) galling and nematode multiplication, and the reduction was even greater (73%) when applied with A. niger CA. They suggested that Bu. cepacia 4684 with A. niger CA may be useful in the biocontrol of M. incognita on tomato. Similar results for tomato and capsicum have also been reported by Siddiqui et al. (2000), Rao (2007), and Parveen et al. (2008). Several rhizobacteria capable of controlling aphids, thrips, and whiteflies through ISR have been reported (Bharathi et al. 2001). Jagadeesh et al. (2007) tested certain isolates of PGPR (Pseudomonas B15, B21, B25, and B26) to evaluate their biocontrol ability against aphid (Aphis goosypii) and leafhopper (Amrasca biguttula) of okra grown under field conditions. The populations of aphids and leafhoppers were reduced by about 79 and 81%, respectively, when sprayed with B25, and the yield was also improved by over 53% compared with the noninoculated control. In a different study with pepper, a commercial soil amendment ® (BioYield ) containing a mixture of two species of Bacillus PGPR (B. subtilis and B. amyloliquefaciens) was evaluated for impact on germination and initial growth of bell pepper plants, efficacy against the green peach aphid (Myzus persicae Sulzer), and yield enhancement under greenhouse and field conditions during 2003–2005 (Herman et al. 2008). They found that pepper germination rate and dry weight of seedlings grown with or without Bacillus spp. in the greenhouse did not differ significantly. In the field, the PGPR did not significantly reduce aphid populations compared to control plants, whereas chemical control was highly 218 A. Esitken effective, but an increase in yield was observed in the 2003 season compared with control plants. Aphid pressure was high in 2003, and plants grown in the presence of Bacillus spp. exhibited substantial tolerance to aphids. Although there were significantly higher populations of the green peach aphid on both control and PGPR-treated plants compared with chemical control, fruit yield in the Bacillus spp. treatment was significantly greater than yield in the control treatment and similar to yield in insecticide-treated plots. Several studies reported that PGPR including P. fluorescens, Pantoea agglomerans (E. herbicola), and B. subtilis had potential effects on biological control of fire blight (Erwinia amylovora) in pear and apple (Vanneste et al. 1992; Wilson and Lindow 1993; Lindow et al. 1996; Momol et al. 1999; Pusey 1997, 1999, 2002; Mercier and Lindow 2001; Thomson and Gouk 2003; Ozaktan and Bora 2004; Broggini et al. 2005; Pujol et al. 2006; Schmoock et al. 2008). For instance, Mercier and Lindow (2001) assessed the effects of inoculation with antagonistic (P. fluorescens) and nonantagonistic (E. herbicola and Acinetobacter) PGPRs on control of fire blight on pear under field conditions. The results showed that four antagonistic strains (strain 22, 73, 198, and 257) reduced the incidence of flower infection by 43–73%, in addition, only one nonantagonistic strain (strain 59) resulted in a similar reduction in disease incidence. Recently, Ozaktan and Bora (2004) conducted a field experiment to evaluate the potential of talc-based formulation of Pantoea agglomorans Eh-24 on controlling fire blight disease of pear. Bacterial formulation was sprayed on naturally infected pear trees at 30 and 100% bloom stage. They found that bacterial application reduced the percentage of blighted blossoms on pear orchards by up to 76% and more effective than chemical control. Grey mold caused by Botrytis cinerea is one of the most destructive diseases of many fruit crops such as strawberry, raspberry, grapevine, and apple worldwide. Gray mold is also a major cause of postharvest losses of fruits during storage, transportation, or shipment. In biological control of B. cinerea, beneficial fungus and yeast were used widely but not PGPR until recently. In earlier study, Janisiewicz and Roitman (1988) conducted postharvest trial of apple and pear fruit to assess the effect of Pseudomonas cepacia on controlling B. cinerea and Penicillium expansum (blue mold). The results indicated that P. cepacia strongly inhibited development of gray mold and blue mold lesions on apple and pear. Application of apple and pear with P. cepacia decreased average lesion diameter by 93 and 25% relative to the control, respectively. In addition, Janisiewicz (1987) found similar results in postharvest control of blue mold in apple. Recently, Donmez et al. (2011) tested various PGPR isolates including Bacillus lentimorbus, B. megaterium, B. pumilis, B. subtilis, Enterobacter intermedius, Kurthia sibirica, P. polymyxa, and P. agglomerans to evaluate their effects on suppressing gray mold (B. cinerea)on strawberry fruit. They indicated that responses were variable and dependent on the inoculants strain; however, Entrobacter MFD-45 treatment significantly decreased disease incidence (conidia germination on fruit) (20.8%) compared with the control (79.2%). Similar observations related to gray mold were reported for different fruit crops, vegetable crops, and grapevine by Swadling and Jeffries (1996), 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 219

Dik et al. (1999); Moline et al. (1999), Helbig (2001), Ait Barka et al. (2002), Guetsky et al. (2002a, b), and Essghaier et al. (2009). However, biological control studies by PGPR in other fruit crops and related pathogens were considerably limited. In a study with mango, the talc-based formulation of PGPR (P. fluorescens and B. subtilis) and yeast antagonistic strains (Saccharomyces cerevisiae) with or without chitin amendment was evaluated against anthracnose (Vivekananthan et al. 2004). The results suggested that a preharvest application of P. fluorescens (FP7) with chitin formulation at monthly spray intervals through aerial spray significantly reduced the pre and postharvest anthracnose incidence. The strain FP7 containing chitin treated mango tree revealed the maximum panicle initiation and yield attributes. In storage trials, P. fluorescens (FP7) þ chitin preharvest treatment reduced the anthracnose incidence of 67% over untreated control and its efficacy was superior to standard fungicide carbendazim treatment (39%). Esitken et al. (2002) conducted an experiment to study the effect of Bacillus OSU-142 on control of shot-hole disease of apricot trees. Bacte- rial suspension was sprayed to apricot trees at full bloom and 30 and 60 days after full bloom stage. The results indicated that reduction in disease incidence and severity was between 15 and 71% depending on years. In a different study with shot-hole disease in apricot, Karlidag et al. (2006) reported that Bacillus OSU-142, Burkholderia OSU-7, and OSU-142 + OSU-7 spraying at full bloom stage significantly decreased disease incidence and severity compared with the noninoculated control. Likewise, Altindag et al. (2006) tested various isolates of antagonistic bacteria to evaluate their effects on brown rot (Monilia laxa) in apricot and compared with chemical control under field conditions. Apricot trees were sprayed with each of the three biological control agents (Burkholdria gladii OSU 7, B. subtilis OSU 142, and P. putida BA 8) at full bloom stage and also applied commercial disease management. They reported that disease incidence for untreated control was 9.94, which was significantly higher than disease incidence for commercial application (2.57%) or bacterial treatments (2.82–5.00%) in the first year. In the second year, the lowest disease incidence was observed in OSU 7 treatment (6.80%) while disease incidence rates for chemical control and untreated control were 9.45 and 28.46%, respectively. Similar to apricot, PGPR (Burkholdria OSU 7 and Bacillus OSU 142 and ERZ) significantly suppressed (up to 93%) brown rot (Monilia linhartina) development on quince (Cydonia oblanga) fruit (Sahin et al. 2002). Tahmatsidou et al. (2006) evaluated the effect of a commercial PGPR inoculant containing a B. subtilis FZB24 on control of Verticillum wilt caused by Verticullum dahliae of strawberry in the field. The results showed that marketable fruit yield (94.7 g/plant) was significantly reduced by approximately 60% in V. dahliae inoculated control plants whereas the yield of plants from runners inoculated with the PGPR inoculants (300.7 g/plant) did not differ significantly from that of the noninoculated controls (285.2 g/plant). Recently, Gupta et al. (2008) have evaluated the potential of various individual strains of PGPR and synthetic analogs of naturally occurring plant activators to elicit ISR against either brown leaf spot (Cercospora moricola) or leaf rust (Cerotelium fici) disease in mulberry. Three PGPR strains, Azotobacter chroococcum Azc-3, B. megaterium 220 A. Esitken

Bm-1, and P. ﬂuorescens Psf-4 and plant activators acetyl-salicylic acid (ASA), sodium salicylate (NaS), and 4-amino-n-butyric acid (ABA) were applied, as alone and in combination, to mulberry plants by way of soil application and foliar spray. The results suggested that disease suppression was signiﬁcantly high with integrated application of PGPR strains and plant activators when compared to their individual applications. Integration of Azc-3 þ ASA (82.60%) provided greater suppression to multiple infections of brown leaf spot and leaf rust diseases during the entire growth period of mulberry plants.

8.4 Conclusions and Future Prospects

PGPR are defined as free-living soil, rhizosphere, and rhizoplane bacteria; however, they can be present in the phyllosphere, which under some conditions is beneficial to plants. Most of the activities of PGPR have been studied in the rhizosphere, and to a lesser extent on the leaf surface. PGPR can affect plant growth directly or indirectly. Indirect promotion of plant growth occurs when introduced PGPR lessens or prevents deleterious effects of one or more phytopathogenic organisms in the rhizosphere and/or phyllosphere. Direct mechanisms may act on the plant itself and effect growth by means of PGRs such as auxin, cytokinins, gibberellins, and ethylene, solubilization of inorganic phosphate and mineralization of organic phosphate and/or their nutrients, and asymbiotic fixation of atmospheric nitrogen. In addition to rhizosphere, PGPR can introduce the promoting effects in phyllosphere when spraying to plant. Therefore, PGPR have more area in use upwards of present time in particular fruit crops like fruit tree. PGPR present an alternative to the use of chemicals such as PGRs and pesticides for plant growth enhancement in many different applications, and also may increase input efficiency like fertilizer especially under sustainable and organic growing conditions. Horticultural crops are one of the areas where PGRs are widely used for various purposes. Together with growth promoting influence, PGPR may serve as PGRs in horticultural crop production. For example, auxins are largely used for rooting of cutting, fruit setting, and thinning; similarly PGPR which produce IAA can use same areas. Thus, differently in other agricultural crops, PGPR can be used to stimulate rooting of cutting, grafting union, fruit setting, and thinning, as well as growth and yield promotion in horticultural crops. In addition, under stress conditions such as drought, salinity, lime-induced chlorosis, and low temperature, PGPR can help in increasing tolerance in inoculated plants. Consequently, PGPR play an important role in horticultural crop production especially sustainable and organic growing conditions. Accordingly, further investigation is needed to improve the performance and also use in phyllosphere as bacterial inoculants. Greater attention should be focused on spraying to aerial parts of plants and phytohormone production properties of PGPR in horticultural crops for improved results, because PGRs are used widely in horticultural crops. Therefore, PGPR can be substituted instead of PGRs in sustainable and organic growing. For instance, 8 Use of Plant Growth Promoting Rhizobacteria in Horticultural Crops 221 partenocarpic fruit formation by PGPR which produce IAA should be a matter of further research in greenhouse vegetable production. Stimulating of partenocarpic fruit formation in fruit crops especially grapevine by PGPR that produce GAs could also be investigated. In addition, the effects of PGPR which produce IAA on improve to reduction of excessive crop loading and of preharvest fruit drop in fruit crops such as apple could be exemined. Moreover, PGPR which have ACC- deaminase activity could be investigated to provide less fruit drop in fruitlet stage (i.e., walnut) or in preharvest stage (i.e., apple). The latter could also be essayed in postharvest period to extend the storage or shelf life of fruits and vegetables. Trees purchased from nurseries should be adequately branched and treatments with PGRs (cytokinin and GAs) can improve branching substantially. PGPR that produce cytokinin and GAs could also be used to provide branching of trees in the nurseries. In future studies, performance of the PGPR under stress conditions such as drought, salinity, high and low Ca, and low temperature in particular fruit crops and vegetable crops in greenhouse should be investigated. In addition, the effects of PGPR which especially fixed N2 and solubilized phosphate on efficiency of fertilization and mineral nutrition should be studied in the future. PSB could also be tested in soilless growing systems like nutrient film technique (NFT) for improved P uptake.

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K. Vijay Krishna Kumar, M.S. Reddy, J.W. Kloepper, K.S. Lawrence, X.G. Zhou, D.E. Groth, S. Zhang, R. Sudhakara Rao, Qi Wang, M.R.B. Raju, S. Krishnam Raju, W.G. Dilantha Fernando, H. Sudini, B. Du, and M.E. Miller

K.V.K. Kumar Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA and Acharya N G Ranga Agricultural University, Hyderabad, India M.S. Reddy (*), J.W. Kloepper, and K.S. Lawrence Department of Entomology and Plant Pathology, Auburn University, Auburn, AL, USA e-mail: [email protected] X.G. Zhou Agri-Life Research and Extension Center, Texas A and M University, College Station, TX, USA D.E. Groth LSU AgCenter, Rice Research Station, Rayne, LA, USA S. Zhang Tropical REC, University of Florida, Homestead, FL, USA R.S. Rao Acharya N G Ranga Agricultural University, Hyderabad, Andhra Pradesh, India Q. Wang China Agricultural University, Beijing, China M.R.B. Raju and S.K. Raju Andhra Pradesh Rice Research Institute, Maruteru, Andhra Pradesh, India W.G.D. Fernando Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada H. Sudini International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, Andhra Pradesh, India B. Du Department of Microbiology, Shandong Agricultural University, Taian, Shandong Province, China M.E. Miller Department of Biological Sciences, Auburn University, Auburn, AL, USA

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 237 DOI 10.1007/978-3-642-18357-7_9, # Springer-Verlag Berlin Heidelberg 2011 238 K.V.K. Kumar et al.

9.1 Introduction

Rice (Oryza sativa L.) is an important staple food crop for a larger part of the world’s population and is produced around the globe. Global rice production was approximately 680 million tons in 2009. More than 90% of rice is produced in Asia, with China and India being the lead producers. The other major rice-producing countries are Indonesia, Bangladesh, Vietnam, Thailand, Myanmar, Philippines, Brazil, and Japan (Table 9.1). Rice production in the USA, which started 300 years ago, now has an annual production of 9.2 million tons. Major rice-producing states of the USA are Arkansas, California, Louisiana, Mississippi, Missouri, and Texas. The forecasted increase in global population in the coming years is demanding a need for increase in productivity of rice, although there is only a limited scope for expansion of crop-growing area especially in densely populated countries such as Asia (Meunchang et al. 2006). Use of chemical fertilizers for enhancing rice production is a common practice. However, indiscriminate use of chemical fertilizers to increase grain yields in rice has several concerns such as leaching of fertilizers into ground water, change of microbial balance in soil–root-ecosystem, increased susceptibility of the crop to pests and diseases, and acidiﬁcation or alkalization of soils. Rice production is affected by many biotic and abiotic stresses including fungal pathogens that attack the crop from seeding to harvest and cause severe yield losses. Seed-borne pathogens often reduce the germination and inﬂict qualitative and quantitative yield losses (Haque et al. 2007). Among important fungal diseases, blast (Magnaporthe oryzae, formerly M. grisea or Pyricularia oryzae), sheath blight (Rhizoctonia solani AG 1-1A), brown spot (Bipolaris oryzae), sheath rot (Acrocylindrium oryzae), stem rot (Sclerotium oryzae), and bakane (Gibberella fujikuroi) cause severe yield losses in rice. Major bacterial diseases include bacterial leaf blight (Xanthomonas campestris pv. oryzae) and bacterial leaf streak (X. campestris pv. oryzicola) (Bangura and John 1991). Important viral diseases include tungro, grassy stunt, ragged stunt, yellow dwarf, orange leaf, and hoja

Table 9.1 Production details of major rice-producing countries in the worlda Rank Country Rice production (million tons) 1 China 187.40 2 India 144.57 3 Indonesia 57.15 4 Bangladesh 43.06 5 Viet Nam 35.94 6 Thailand 32.10 7 Myanmar 31.45 8 Philippines 16.24 9 Brazil 11.06 10 Japan 10.89 aFAOSTAT 2007 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 239 blanca. Other important ones include diseases caused by nematodes such as white tip (Aphelenchoides besseyi) and ufra (Ditylenchus angustus) (Datta 1981). Sheath blight (ShB) is an economically significant disease of rice in all growing areas of the world. Yield losses of up to 50% are reported when susceptible varieties are grown (Prasad and Eizenga 2008). Soil bacteria in rice ecosystems typically exert a significant fungistatic effect on mycelia and sclerotia of the ShB pathogen (Luo et al. 2005). Effective management of ShB with PGPR application has been reported (Mew and Rosales 1986; Vasantha Devi et al. 1989; Kanjanamaneesathian et al. 1998); however, the field results were not consistent due to varying reasons. This review focuses on recent developments in the management of rice ShB with PGPR. The topics covered in the chapter include PGPR application in rice, greenhouse, and field efficacy of PGPR and the scope of applying them in conjunction with chemical fungicides under integrated disease management system (IDM) of ShB. The overall goal of this chapter is to introduce the multistep process that leads to the development of a new microbial inoculant product and its use and to outline the beneficial strategies specifically for ShB disease management of rice. In addition, it attempts to define the major efforts under way to help stimulate the process. Because product development is integrally related to several tasks including intellectual property issues and to regulatory and liability concerns, these topics are also included. Data on product development for rice ShB management are not systematically available. We have, therefore, used information based on our own research efforts and, when possible, made comparisons.

9.2 Symptomatology

Initial ShB symptoms appear on lower rice leaf sheaths when the crop is in late tillering or early internode elongation phase. These lesions appear as green–grey water soaked at 0.5–3 cm below the collar region as circular, oblong, or ellipsoid and about 1 cm long. As the disease progresses, the lesions expand with bleached appearance and a brown border. Under favorable conditions (95% relative humidity and temperature of 28–32C), the disease spreads by runner hyphae to upper parts of plants including leaf blades (Fig. 9.1a). The pathogen also infects the panicle (Fig. 9.1b) and causes chafﬁness of lower grains (Lee and Rush 1983).

9.3 Disease Cycle

The pathogen survives from one crop season to another as sclerotia and mycelia in plant debris and also through weed hosts in tropical environments (Kobayashi et al. 1997). In temperate regions, the primary source of inoculum is sclerotia produced in previous rice crops (Kozaka 1961). The sclerotia ﬂoat in water during ﬁeld preparation and attack newly planted crop. The pathogen produces lesions on 240 K.V.K. Kumar et al.

Fig. 9.1 Sheath blight symptoms on rice leaf blades and panicle (a) leaf blades. (b) Formation of sclerotia on panicle leaf sheaths and leaf blades. The disease is more aggressive when the crop advances to the reproductive phase, and the pathogen also infects the rice panicles. New sclerotia are produced as the lesions mature and these sclerotia drop into the soil during harvesting, perpetuate, and infect a newly planted crop in the next season (Suparyono et al. 2003).

9.4 Use of Microbial Inoculants

Currently, ShB is managed through cultural and chemical control methods. Mostly, disease management is through use of systemic and non-systemic fungicides. Most widely used fungicides include azoxystrobin, hexaconazole, propiconazole, tebu- conazole, carbendazim, trifloxystrobin, validamycin, and jinggangmycin. Use of chemicals in ShB management is creating concerns over environmental pollution, escalated costs, and pathogen resistance to chemicals. Biological control is a viable alternative in ShB disease management. However, the use of biocontrol agents in managing rice diseases is still at its infancy due to varying reasons. A successful bioagent, when applied to rice ecosystem, should be able to survive, establish, proliferate, and control target pathogens. Fungal and bacterial biocontrol agents have been used for control of rice diseases. The popularly used fungal bioagents against ShB include Trichoderma spp. and Gliocladium spp. These bioagents were applied either as seed treatment, root dip, or foliar spray (Nagaraju et al. 2002). The other effective fungal bioagent is Helminthosporium gramineum that produces a toxin called “ophiobolin.” The toxin is effective in reducing ShB incidence under field conditions (Duan et al. 2007). The prevailing anaerobic conditions in rice are unfavorable for the fungal bioagents to survive, establish, and proliferate in the soil. Rice ecosystems are rich in bacteria (Yin and Mew unpublished data; Mew et al. 2004). They also have greater adaptability to rice ecosystems compared to fungal antagonists. Of them, plant growth-promoting rhizobacteria (PGPR) have been 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 241 used in controlling rice diseases. Besides, these PGPR also contribute to enhanced growth of the seedlings, induction of systemic resistance against diseases and thereby increases yields (Pathak et al. 2004). Bacterial strains of the genera such as Aeromonas, Azoarcus, Azospirillum, Azotobacter, Arthobacter, Bacillus, Clos- tridium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, and Serratia were identified as PGPR (Tripathi et al. 2005; Raj et al. 2004; Dey et al. 2004; Jaizme-vega et al. 2004; Joo et al. 2004; Bonaterra et al. 2003; Cezon et al. 2003; Esitken et al. 2003; Garica et al. 2003; Munir et al. 2003; Kokalis-Burelle et al. 2002; Khalid et al. 2003; Murphy et al. 2003; Preeti et al. 2002; Gupta et al. 1995; Bertand et al. 2001; Hamaoui et al. 2001; NandaKumar et al. 2001b; Pan et al. 1999; Arndt et al. 1998; De Freitas et al. 1997; Shishido et al. 1996; Babalola et al. 2003; Mirza et al. 2001; Podile and Kishore 2006). In addition to enhancement in plant growth, PGPR were also contributed to increase N uptake, phytohormone synthesis, phosphate solubilization, and acquisition of ferric iron through production of siderophores (Lalande et al. 1989; Glick 1995; Bowen and Rovira 1999). Use of PGPR in rice to control major diseases and to enhance yields was earlier reported (Lucas et al. 2009). A variety of beneficial bacteria were found to colonize the rhizosphere and aerial parts of rice. Nitrogen-fixing activity and indoleacetic acid (IAA) production was detected in roots and submerged shoots of field-grown rice due to these beneficial bacteria (Mehnaz et al. 2001). Rhizosphere bacterial isolates of rice have an excellent potential of producing biofertilizers. Inoculation of PGPR in rice increased total dry weight of plants, total N and P uptake through N fixation, P solubilization capacity, and IAA production (Meunchang et al. 2006). Use of biofertilizers in cereals was found to significantly increase plant growth and yields (Boddey et al. 1986; Fages 1994; Kapulnik et al. 1981; Kennedy and Tchan 1992; Pereira et al. 1988). Frequent rhizosphere colonizers of cereal crops and grasses include N-fixing bacteria such as Azospirillum, Acetobacter, Azoarcus, Herbaspirillum spp. (Baldani et al. 1986; Bally et al. 1983; Bilal et al. 1990; Dobereiner and Day 1976; Gillis et al. 1989; Reinhold-Hurek et al. 1993), Aero- monas, and Enterobacter spp. (Mehnaz et al. 2001).

9.4.1 Mode of Delivery

Field efﬁcacy of a PGPR strain partly depends on the method of delivery. PGPR and their formulations are generally delivered as seed treatment, soil amendment, or root dip in bacterial suspensions prior to transplanting. Other important methods also include foliar spray or through drip irrigation in different crops (Podile and Kishore 2006). Success of the PGPR strain is dependent on understanding the use of speciﬁc delivery system and its advantages over other methods. In rice, PGPR is delivered through seed, as soil amendment, seedling dip, and foliar spray, and through combinations of these methods. Against rice ShB, the popular delivery systems are through seed, soil, and foliar applications (Nakkeeran et al. 2005). 242 K.V.K. Kumar et al.

9.4.1.1 Seed Treatment

An ideal bacterial antagonist when treated to seed should colonize the rhizosphere during seed germination (Weller 1983), and several application methods can be used to accomplish this. Treating seeds with different PGPR was found to be highly effective in managing rice ShB disease. Seed coating of P. fluorescens (B41) was found to be comparatively more effective than soil drenching and foliar sprays against ShB under greenhouse conditions (Kazempour 2004). Seed bacterization of Pseudomonas strain GRP3 followed by root dipping resulted in ShB reduction in rice up to 46% (Pathak et al. 2004). Seed treatment with PGPR mixtures also resulted in effective ShB management. Soaking rice seeds in P. fluorescens mixture of strains PF1 and PF2 at 108 cfu g1 for 24 h were effective in reducing ShB incidence under field conditions (Nandakumar et al. 2001a). Seed bacterization with fluorescent Pseudomonads such as P. fluorescens and P. putida V14i was highly effective in reducing ShB severities by 68 and 52%, respectively, in seed bed and field experiments (Malarvizhi 1987) due to protection of the plants from infection. Subsequent planting in the same field after planting the first crop, in which the seeds were treated with bacteria, also showed reduced ShB severity (Mew and Rosales 1986). Seed treatment with peat-based formulation of P. fluorescens (PfALR2) at the rate of 20 g kg 1 resulted in ShB disease control effectively under greenhouse and field conditions (Rabindran and Vidhyasekaran 1996). Induced systemic resistance, plant growth promotion, and sheath blight control was observed by treating rice seeds with three isolates of Pseudomonas aeruginosa. The biocontrol agents were also found effective in reducing blast and brown spot diseases in rice due to increased accumulation of salicylic acid and pathogenesis- related peroxidases (Saikia et al. 2006).

9.4.1.2 Seedling Dip

The ShB pathogen is soil-borne, attacks the rice seedlings, and establishes host– pathogen relationship by root entry (Nakkeeran et al. 2005). Seedling root dip treatment of rice prior to transplanting for a period of 2 h in talc-based formulations of PGPR mixtures at 20 g/L reduced ShB incidence effectively (Nandakumar et al. 2001a). Earlier, seedling root dip in a talc-based formulation of P. fluorescens before transplantation into main field suppressed ShB disease and improved grain yields (Rabindran and Vidhyasekaran 1996). A novel application method of B. megaterium multiplied in empty fruit bunches (EFB) as carrier was reported. The rice seedlings when treated with bacterial inoculum multiplied in EFB carrier had significantly enhanced plant height, number of roots, and dry matter of root and shoot. The method offered a scope of developing new delivery system and granula- tion of bioinoculants for effective control of diseases as well as for enhancing grain yields (Al-Taweil et al. 2009). 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 243

9.4.1.3 Soil Application

Soil application of PGPR has also been reported to be an effective method of controlling soil-borne diseases of rice (Rabindran and Vidhyasekaran 1996). For effective suppression of ShB, the population thresholds of the antagonist in soil should be higher than 1 106 cfu g1 during early stages of infection by R. solani (Li et al. 2003). Effective management of ShB is feasible only when the applied bioagents survive, establish, proliferate, and control pathogen populations in soils. A strain of B. licheniformis (CHM1) isolated from rice fields was found to be highly effective in protecting rice seedlings from ShB disease as well as in plant-growth promotion when applied as soil drenching around root zone (Wang et al. 2009). Soil application of peat formulation of P. fluorescens (PfALR2) effectively controlled ShB disease under greenhouse and field conditions (Rabindran and Vidhyasekaran 1996). Broadcasting of talc-based formulation mixtures of Pf1 and Pf7 at 30 days of transplanting of rice seedlings reduced ShB and increased grain yields significantly under field conditions (Nandakumar et al. 2001a). The population levels of PGPR in rice fields are an important factor for effective control of ShB disease. Mixing the potting soil with bacterial suspensions of different P. aeruginosa mutants coupled with a soil drench at a concentration of 5 107 cfu g 1 elicited ISR in rice seedlings to blast and ShB diseases under greenhouse conditions (Vleesschauwer and Hofte 2005).

9.4.1.4 Foliar Application

Survival rates and application efficiencies of PGPR as foliar sprays against plant diseases is generally affected by variations in microclimate. Nutrient concentrations of amino acids, organic acids, and sugars that exude through lenticels, stomata, and hydathodes vary in the phyllosphere (Nakkeeran et al. 2005). The efficacy of PGPR against ShB under greenhouse and field conditions is dependent on time of application. Spraying of P. fluorescens at 7 days before pathogen inoculation resulted in effective ShB reduction (59–64%) over simultaneous application at 7 days after inoculation. Further, grain yields and 1,000 grain weight were also enhanced with the prophylactic sprays (Rajbir Singh and Sinha 2005). Commercial formulations of P. fluorescens (Ecomonas and Florezen P) when sprayed three times at 10-day interval after disease initiation under field conditions resulted in ShB control by 14–38% besides significant increase in grain yields (Vijay Krishna Kumar et al. 2009). Foliar sprays with floating pellet formulation of B. megaterium were effective in rice ShB suppression under greenhouse conditions (Wiwattanapatapee et al. 2007). Spraying of antifungal metabolites of Streptomyces spp. (SPM5C-2) at the rate of 500 mgml1 significantly decreased ShB and blast disease development by 82 and 76%, respectively, under greenhouse conditions (Prabavathy et al. 2006). Broadcasting of floating pellets formulation and spraying of water-soluble formulation of B. megaterium resulted in effective control of rice ShB disease under greenhouse and field conditions (Kanjanamaneesathian et al. 2007). 244 K.V.K. Kumar et al.

9.4.1.5 Multiple Delivery Systems

Protection of spermosphere, rhizosphere, and phylloplane from infection courts of plant pathogens through multiple delivery systems of PGPR offers a comprehensive means of plant disease management (Nakkeeran et al. 2005). Talc-based formulations of two P. fluorescens strains (PF1 and PF7) when applied through seed, root, soil, and foliar sprays significantly reduced ShB and pest (leaf-folder) incidence in rice under greenhouse and field conditions. The bacterial mixture performed better than individual strains with a reduction of 62% of ShB and 47–56% of leaffolder incidence (Radja Commare et al. 2002). Combined applications of P. fluorescens strains (PF1, FP7, and PB2) as bacterial suspensions or as talc-based formulations through seed, root, foliar, and soil application significantly reduced the ShB incidence (45%) under greenhouse and field conditions over their individual applications. Further, a significant increase in yield was obtained with application of mixtures over their individual applications. Fluorescent Pseudomonas application (PF1 and FP7) either as a suspension or talc-based formulation through seed, root, soil, and foliar means effectively reduced rice ShB incidence and promoted plant growth and grain yields (Nandakumar et al. 2001a). Similar results on ShB disease suppression and enhanced yields were reported with peat-based formulations of P. fluorescens (PfALR2) as seed treatment, root treatment, soil application, and foliar spraying. Further, the efficacy of combined application methods was comparable with fungicide treatments (Rabindran and Vidhyasekaran 1996).

9.4.2 Formulations

A formulated PGPR should ideally possess high rhizosphere competence, plant growth promotion, ease for large-scale multiplication, wide range of plant disease control, consistent in disease control, and compatible with environment and other rhizobacteria (Nakkeeran et al. 2005). Besides, the bacterial inoculants should be able to tolerate desiccation, heat, oxidizing agents, and UV radiations (Jeyarajan and Nakkeeran 2000). The formulated product should meet the important criteria such as satisfactory shelf life, non-phytotoxic nature, water solubility, ability to withstand environmental ﬂuctuations and compatibility with other agrochemicals. Besides, it should be cost-effective with ready availability of carriers at a cheaper rate and should not impart mammalian toxicity (Nakkeeran et al. 2005; Jeyarajan and Nakkeeran 2000). The carrier materials used in PGPR formulations are broadly categorized into organic and inorganic ones. The commonly used organic carriers are peat, turf, talc, lignite, kaolinite, pyrophyllite, zeolite, montmorillinite, alginate, press mud, sawdust, and vermiculite (Nakkeeran et al. 2005). In general, PGPR survive longer in carriers with smaller particle sizes than in those with larger particle sizes. Carriers with smaller size will have more surface area that enables increased resistance to desiccation of PGPR through increased coverage of bacterial cells (Dandurand et al. 1994). 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 245

Commonly available PGPR formulations are talc formulations, peat formulations, press mud formulations, vermiculite formulations (Nakkeeran et al. 2005), water-soluble granular formulations, liquid formulations, ﬂoating pellet formulations, and formulations with EFB as carriers. Details of different PGPR formulations that exhibited effective control of rice ShB disease under greenhouse or ﬁeld conditions are given in Table 9.2.

9.4.3 Shelf life

Effective disease control by PGPR is possible only when the formulated product delivers a sufficient number of viable cells. So, determining the shelf life and viability of a commercial bio-product is a crucial step. The shelf life of PGPR in the formulated product is dependent on the type of carrier material used. Talc is an excellent carrier material for PGPR with low moisture equilibrium, relative hydro- phobicity, reduced moisture absorption, and chemical inertness (Nakkeeran et al. 2005). The population levels of PGPR (fluorescent Pseudomonads) did not decline in talc powder with 20% xanthan gum after storage for 2 months at 4C (Kloepper and Scroth 1981). Vermicompost is comparatively a better carrier material than lignite for bioinoculants with high nitrogen, phosphorus, potassium, copper, manganese, and iron besides possessing an ideal pH. The shelf life of vermicompost-based formulations is greater than that of lignite-based ones. The population levels of B. megaterium and P. fluorescens were very high (7.6 108 and 1 108 cfu g1 of dry weight, respectively) at the end of 360 days when vermicompost was used as carrier (Gandhi and Saravanakumar 2009). The shelf life of peat-based formulations depends on the availability of good quality peat. Heat sterilization of peat results in release of toxic substances that are detrimental to bacteria thus affecting their population levels in the formulation (Bashan 1998). The population levels of P. fluorescens (2.8 106 cfu g 1) in peat- based formulation was maintained up to 8 months (Vidhyasekaran and Muthamilan 1995), whereas the shelf life of P. chlororaphis and B. subtilis were more than 6 months (Kavitha et al. 2003; and Nakkeeran et al. 2004). The use of press mud and vermiculite-based PGPR formulations are also in practice. The viability of Azos- pirillum spp. in press mud formulation is higher than in lignite (Muthukumarasamy et al. 1997) whereas in vermiculite, its viability is retained up to 10 months (Saleh et al. 2001).

9.4.4 Root Colonization

Of different soil microbial populations, bacteria residing in the rhizosphere are the most beneﬁcial. Bacterial communities in the rhizosphere vary in different root 4 ...Kmre al. et Kumar K.V.K. 246

Table 9.2 Currently used PGPR formulations against rice sheath blight disease PGPR strain Type of formulation Method of application References Bacillus megaterium EFB (empty fruit bunches) powder Seedling dip Al-Taweil et al. (2009) Bacillus licheniformis CHM1 Bacterial cell suspension Soil application Wang et al. (2009) Bacillus megaterium (No 16) Floating pellets Broadcasting Kanjanamaneesathian et al. (2007) Bacillus megaterium (No 16) Water soluble granules Foliar spray Kanjanamaneesathian et al. (2007) Pseudomonas fluorescens (Pf1 and FP7) Bacterial suspension/talc-based Seed + root + soil + foliar Nandakumar et al. (2001a) Pseudomonas fluorescens and Bacterial cell suspension Seed + root + soil Vleesschauwer and Hofte (2005) Pseudomonas. Aeruginosa Pseudomonas fluorescens (PfALR2) Peat based Seed + root + soil + foliar Rabindran and Vidhyasekaran (1996) Pseudomonas fluorescens (Pf1 and FP7) Talc-based Seed+root+soil+foliar Radja Commare et al. (2002) Beneficial bacteria (NF1, NF3, NF52, Bacterial cell suspension Foliar spray Lai Van E et al. (2001) NF49, CT6-37, and W23) Fluorescent and nonfluorescent Bacterial cell suspension Seed treatment Mew and Rosales (1986); Vasantha Pseudomonads Devi et al. (1989) Bacillus megaterium Floating pellets Broadcast + spray Wiwattanapatapee et al. (2007) Pseudomonas fluorescens Talc-based Foliar spray Vijay Krishna Kumar et al. (2009) Pseudomonas aeruginosa Bacterial cell suspension Foliar spray Saikia et al. (2006) Antagonistic bacteria Bacterial cell suspension Seed + foliar Chen et al. (1996) 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 247 zones and their composition can be altered by changes in root exudate composition (Yang and Crowley 2000). Root exudates of rice plants were found to exert a positive influence on the motility of these bacteria toward plant roots (Bacilio- Jiminez 2003). Earlier studies indicated that the rhizosphere isolates of rice were able to induce IAA production and have phosphate solubilization capacity. Further, these PGPR isolates were found to promote seed germination, root length, plant height, and dry matter production of shoot and roots in rice (Ashrafuzzaman et al. 2009). Application of bio-inoculants was found to enhance rice growth through production of total sugars, reducing sugars, amino nitrogen content, PGP substances in the root exudates, and biological nitrogen fixation. The microbial consortium viz., Azospirillum lipoferum-Az204, B. megaterium var. phosphaticum, and P. fluorescens Pf1 when applied to rice improved the colonization potential, sustainability within the inoculants, and enhanced plant growth when compared to their application individually (Raja et al. 2006). Mirza et al. (2006) reported a nitrogen-fixing, phytohormone-producing Pseu- domonas isolate (strain K1) that had a capacity of fixing nitrogen in inoculated rice plants and its efficacy was comparable to non-Pseudomonas nitrogen-fixing PGPR. Use of PGPR also alleviates zinc deficiency in rice plants. Zinc deficiency is a serious problem in rice production (Anon 1993). Inoculation of rice fields with PGPR had a significant positive impact on root length (54% increase), root weight (74%), root volume (62%), root area (75%), shoot weight (23%), panicle emergence index (96%), and Zn mobilization efficiency, thereby reducing the cost incurred in the application of chemical Zn fertilizers (Muhammad et al. 2007). Application of diazotrophs such as Rhizobium leguminosarum bv. trifolii (E11), Rhizobium spp. (IRBG74), and Bradyrhizobium sp. IRBG271 in lowland rice fields enhanced N, P, and K uptake by 10–28% due to rhizobial inoculation. In addition, the uptake of Fe was enhanced by 15–64%. Further, the growth promotion in rice was due to changes in growth physiology or root morphology rather than biological nitrogen fixation (BNF) (Biswas et al. 2000).

9.5 Sheath Blight Management

In rice, PGPR offer a promising means of controlling plant diseases besides contributing to the plant resistance, growth, and grain yields (Mew and Rosales 1992). Of different PGPR, ﬂuorescent Pseudomonads and Bacillus spp. group of bacteria are widely used against ShB. Their application promotes plant growth by direct and indirect mechanisms. Direct growth promotion is due to production of phytohormones, solubilization of phosphates, increased uptake of iron through production of siderophores, and volatile metabolites. Indirect way of plant growth promotion is due to mechanisms of antibiosis, competition for space and nutrients, parasitism or lysis of pathogen hyphae, inhibition of pathogen-produced enzymes or toxins, and through induced systemic resistance (ISR). The ISR in rice against ShB is either due to enhanced chitinase or peroxidase activity (Nandakumar et al. 248 K.V.K. Kumar et al.

2001b). However, no correlation was observed between chitinase production and ShB suppression (Thara and Gnanamanickam 1994). Strains of P. fluorescens were found to produce siderophores, volatile metabolites, extracellular secretions, and antibiotics that were inhibitory to ShB pathogen. Further, the strains reduced germination and caused lysis of sclerotia (Kazempour 2004). Rhizosphere isolates of P. fluorescens produced b-1,3-glucanase, salicylic acid, and HCN, and a significant relationship was observed between antagonism of the bacterium and the production of these substances (Nagarajkumar et al. 2004). Bacillus spp. are endospore-producing gram-positive bacteria, and some strains have been used in biocontrol of rice diseases. Strains of B. subtilis and B. megaterium exhibit inhibition of Rhizoctonia solani (Luo et al. 2005). The fermented product of Bacillus (Drt-11) is highly inhibitory to the sclerotial germination, hyphal growth, and colony diameter besides enhancing rice seedling growth (Chen and Hui 2006). Strains of Bacillus produce a thermo and proteinase – stable antagonistic substance (P1) that is effective against rice ShB and blast pathogens (He et al. 2002). The B. subtilis (AUBS1) strain produces phenylalanine ammonia- lyase (PAL), peroxidase (PO), and certain pathogenesis-related (PR) proteins in rice leaves when applied against ShB disease. Accumulation of thaumatin-like proteins, glucanases, and chitinases are the other important substances in plants against ShB by these bioagents (Jayaraj et al. 2004). The other promising bacteria against rice ShB include Streptomyces spp. and Serratia marcescens. The antifungal metabolites of Streptomyces spp. (PM5, SPM5C-1, and SPM5C-2) were highly effective against mycelial growth of rice ShB and blast pathogens under in vitro conditions. Greenhouse studies revealed that spraying of the strain SPM5C-2 at 500 mgml 1 significantly reduced ShB and blast diseases by 82 and 76%, respectively (Prabavathy et al. 2006). Culture filtrates of S. marcescens exhibited enhanced reduction of sclerotial viability of ShB pathogen, when applied with reduced doses of fungicides such as flutolanil, pencycuron, and validamycin (Someya et al. 2005). In order to identify a potential biocontrol agent, researchers have been spending their time on several microbes in areas of isolation, identification, and purification which is routine. This is a laborious process demanding efforts of time and man- hours. Here, we have provided our own selection of a potential microbial inoculant against rice ShB.

9.5.1 Screening of Different PGPR Against ShB Pathogen and Seedling Growth Promotion Under Laboratory Conditions

Seventy PGPR strains that belong to Bacillus, Paenibacillus, Brevibacillus, and Arthrobacter were selected from the bacterial culture collection of the Phytopathol- ogy Laboratory of Auburn University. These PGPR strains were found to be highly effective in inducing growth-promoting effects in various crops. These strains were screened against rice ShB pathogen, ShB lesion spread and in promoting rice 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 249

Table 9.3 Benefits and use rates of Integral in different crops Crop Method of Rates of application Target pathogens application Peanut In-furrow 0.1–1.2 fl oz/acre Rhizoctonia, Fusarium, Aspergillus Cotton, vegetables, soybean In-furrow 0.1–1.2 fl oz/acre Rhizoctonia, Fusarium corn Non-bearing plants (Cherry) Soil mix 1.3–13 fl oz/acre Fusarium, Rhizoctonia in greenhouses Cotton Seed 0.6–2.4 fl oz/100 lb seed Fusarium Rhizoctonia Soybeans Seed 0.13 fl oz/100 lb seed Fusarium Rhizoctonia Green beans, snap beans, Seed 0.6–2.4 fl oz/100 lb seed Fusarium Rhizoctonia lima beans, kidney beans, navy beans, pinto beans, wax beans, pole beans, garden beans, peas, field beans Alfalfa, forage, turf Seed 0.2–12 fl oz/100 lb seed Fusarium Rhizoctonia Wheat, barley Seed 0.1–0.6 fl oz/100 lb seed Fusarium Rhizoctonia Field corn, sweet corn Seed 0.6–2.4 fl oz/100 lb seed Fusarium Canola Seed 1.6–3.8 fl oz/100 lb seed Fusarium Rhizoctonia seedling growth under laboratory conditions. The mycelial growth inhibition of R. solani was as high as 83% with B. subtilis MBI 600, compared to the control. Only four strains completely inhibited the germination of sclerotia. The ShB lesion spread was determined by highest relative lesion height method (HRLH) and for effective strain (B. subtilis MBI 600) was found to be only 2.9 as against control (100). Highest seedling vigor of 13,600 was recorded in comparison to that of control (4,867) on 10-day-old seedlings. The PGPR strain, B. subtilis MBI 600 was found to be highly effective in all the screening assays and was selected for further studies. To further test the efficacy of B. subtilis MBI 600, the strain was produced in a commercial proprietary liquid formulation by Becker Underwood, Ames, Iowa, USA. The formulated strain MBI 600 has a proprietary trade name as Integral®. The product is stored at room temperatures prior to use. The minimum concentration of Integral in liquid formulation is 2.2 1010 cfu ml1. The details of different application methods of Integral are shown in Table 9.3.

9.5.2 Efﬁcacy of Integral

To assess the biocontrol suppression of ShB by using antagonistic bacteria and their combination with fungicide under ﬁeld conditions for a long time, antagonistic bacteria and fungicide used to control ShB must be evaluated for durability effect. Improved plant growth and health by PGPR is either due to direct mechanisms such as improvement in plant uptake through solubilization of mineral phosphates 250 K.V.K. Kumar et al. and other nutrients (De Freitas et al. 1997; Gaur 1990), nitrogen ﬁxation (Boddey and Dobereiner 1995), and phytohormone production such as indole 3-acetic acid, gibberellic acid, cytokinins, and ethylene (Arshad and Frankenberger 1993; Glick 1995). Indirect growth promotion is through biological control of plant pathogens by producing siderophores (Scher and Baker 1982), antibiotics (Shanahan et al. 1992), hydrogen cyanide (Flaishman et al. 1996), lytic enzymes, and competition for nutrients and space.

9.5.2.1 In-Vitro Inhibition of ShB Pathogen

The B. subtilis MBI 600 strain of Integral was further characterized for determining its mode of action against ShB pathogen. The PGPR strain was isolated from the formulation on TSA and confirmation of its purity was carried out using 16s rDNA sequence homology and by measuring the 16s rDNA sequence with 1,409 base pairs of the isolate. The BLAST analysis of the sequencing results confirmed 100% similarity with B. subtilis. The MBI 600 strain was highly effective against the ShB pathogen, R. solani and repeatedly shown significant results in inhibiting mycelial growth (Fig. 9.2) and germination of sclerotia (Fig. 9.3) under in-vitro conditions. A strong zone of inhibition (3 mm) between mycelial growth of pathogen and bacterium was observed. Inhibition of sclerotial germination was about 98% at a concentration of 2.2 109 cfu ml 1 whereas at a concentration of 2.2 108 cfu ml 1, the inhibition was 37%. Integral was not effective in inhibiting sclerotial germination at concentrations of 2.2 106 and 2.2 107 cfu ml1. Highest inhibition of sclerotial growth was obtained at a concentration of 2.2 109 cfu ml 1 (79%), followed by at 2.2 108 cfu ml 1 (72%). Integral was also effective at lower concentrations with sclerotial growth inhibitions ranging from 29 to 60% (Table 9.4). Efficacy of Integral was evaluated in reducing ShB lesions on rice leaves under in vitro conditions by detached leaf piece assay. Integral concentrations of

Fig. 9.2 Inhibition of mycelial growth of Rhizoctonia solani challenged with Integral 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 251

Fig. 9.3 Inhibition of sclerotial germination of Rhizoctonia solani by Integral

Table 9.4 Efﬁcacy of Integral on sclerotial germination and sheath blight lesion symptoms of rice Concentration1 % Inhibition of % Inhibition of sclerotial ShB lesion sclerotial germination2 growth compared to control3 spread4 2.2 106 CFU/ml 0c 28.5d 92.6b 2.2 107 CFU/ml 0c 59.5c 71.6c 2.2 108 CFU/ml 36.7b 71.8b 22.7d 2.2 109 CFU/ml 97.7a 78.8a 4.7e Control 0c – 99.2a Means followed by a common letter in the columns are not signiﬁcantly different at p 0.05 1Integral applied at these concentrations to test on sclerotial germination, growth of mycelia, and suppression of ShB lesions 2Sclerotial germination was recorded at 3 days after incubation 3Sclerotial growth was recorded at 5 days after incubation and 4ShB lesion spread was recorded by Highest Relative Lesion Height method at 7 days after inoculation

2.2 106 through 2.2 109 cfu ml1 were sprayed onto rice leaf pieces (8 cm) separately. Later the leaves were inoculated with 1-week-old sclerotia of R. solani at the centre and leaves were incubated in Petri dishes containing moistened ﬁlter papers. ShB lesion length around sclerotium was recorded after 5 days and disease severity was assessed by highest relative lesion height (HRLH) method. As shown in Fig. 9.4, Integral at 2.2 109 cfu ml1 signiﬁcantly reduced ShB lesion spread on detached rice leaves (4.7) (Table 9.4). At other concentrations, the lesion spread ranged from 23 to 93 as against untreated control (100). Integral was tested positive for production of siderophores. However, production of IAA, HCN, cellulase, chitinase, and phosphate solubilizing capacity when tested were negative. Siderophores are low-molecular-weight iron-chelating agents produced by PGPR that can create iron nutrient competition in soils to plant pathogens. Since the element iron is present in low quantities in soils, siderophore production is a strategy by the PGPR to compete with soil-borne plant pathogens. 252 K.V.K. Kumar et al.

Fig. 9.4 Suppression of sheath blight lesions in a detached leaf assay with Integral

9.5.2.2 Rice Plant Growth Promotion

Seed treatment with Integral was highly effective in promoting rice seedling development both under laboratory and greenhouse conditions. Under in vitro conditions, significantly higher root and shoot lengths were observed with Integral over untreated control. Increase in root and shoot lengths was noticed with an increase in Integral concentration from 2.2 106 cfu ml1 to 2.2 109 cfu ml1. As shown in Fig. 9.5, highest root and shoot lengths (47.5 and 39.1 mm, respectively) were recorded at a concentration of 2.2 109 cfu ml1 as against control (14.3 and 7.6 mm of root and shoot lengths respectively). Under greenhouse conditions, seed treatment with Integral significantly improved rice seed germination, seedling emergence, and plant growth. The percent germination of seeds sown in 15 cm pots filled with field soil was highest at a concentration of 2.2 109 cfu ml1 (88.9%) as against untreated control (61.1%) at 7 days after sowing (DAS). Integral application significantly improved root and shoot lengths at 15 DAS. Highest root length and shoot length were recorded at a concentration of 2.2 109 cfu ml1 (166 and 335 mm respectively) as against untreated control (73 and 222 mm respectively) (Fig. 9.6).

9.5.2.3 Chemical Compatibility

Currently, ShB disease management strategy is through use of systemic fungicides and also with certain non-systemic fungicides (Pal et al. 2005). Pathogen resistance to these systemic fungicides is of concern, thus demanding integration of PGPR in IDM. Since, host plant resistance to ShB range only from very susceptible to moderately susceptible levels in rice (Groth and Bond 2007), use of chemical fungicides has become a necessary component for an effective ShB management. For effective functioning of PGPR under the ambit of IDM, their compatibility with commonly used fungicides and insecticides in rice is also mandatory (Mew et al. 2004). 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 253

Fig. 9.5 Effect of Integral on rice seed development

Fig. 9.6 Efﬁcacy of Integral on rice seedling growth

Combined applications of PGPR with chemical fungicides are an important IDM package against ShB. Of different PGPR, Pseudomonads and Bacillus spp. were found to be very effective as a supplement in IDM. Greenhouse and ﬁeld studies against rice ShB with different PGPR isolated from farmyard manure, rice seed, phyllosphere, and rhizosphere proved that three bacteria, P. ﬂuorescens (PF-9), Bacillus sp. (B-44), and a chitinolytic bacterium (Chb-1) are compatible with carbendazim at 500 and 1,000 ppm concentrations. Of these, PF-9 was most effective in reducing ShB severity either alone or in combination with one spray of 0.1% carbendazim, followed by combination of PF-9 and B-44 (Laha and 254 K.V.K. Kumar et al.

Venkataraman 2001). The B. subtilis (Bs-916) when applied along with jinggangmycin was found to colonize the root system effectively. Further, the population density of BS-916 was maintained in its presence without any further decline (Chen et al. 2003). In order to use Integral in ShB management, it has to be compatible with existing agronomic practices and commonly used chemical fungicides in rice production systems. In our ongoing research, we have attempted to study our classical product Integral according to the assays described by Shanmugam and Narayanasamy (2009) under in vitro conditions. Brieﬂy, in this assay, a loop full of MBI 600 strain onto Nutrient Agar (NA) plates amended with various concentrations (100–1,000 ppm) of fungicides such as propiconazole, validamycin, benomyl, carbendazim, tricyclazole, mancozeb, azoxystrobin, and hexaconazole. Plates were later incubated at room temperature for 48 h and growth of bacterium was monitored. Further compatibility studies with azoxystrobin and carbendazim were carried out according to Omar et al. (2006) wherein 100 mL of bacterial inoculum was added to 250 ml yeast peptone glucose (YPG) liquid medium amended with fungicides at concentrations at 200 and 400 ppm and incubated on a shaker, growth of bacterium was enumerated on NA after serial dilution. Integral exhibited good tolerance to hexaconazole, propiconazole, and validamycin, moderately to tricyclazole and slightly to benomyl and mancozeb at 1,000 ppm. It was highly compatible with carbendazim and azoxystrobin up to 400 ppm whereas complete inhibition was obtained with these fungicides at 800 ppm (Table 9.5). Compatibility to azoxystrobin and carbendazim showed up to 400 ppm, and good growth of Integral was in carbendazim- and azoxystrobin- amended medium (Figs. 9.7 and 9.8).

9.5.2.4 Efﬁcacy of PGPR Against ShB Under Greenhouse and Field Conditions

The time of application of PGPR has signiﬁcant inﬂuence in the management of ShB disease. Ren et al. (2006) reported that the optimum time of application

Table 9.5 Compatibility of Integral with commonly used fungicides Fungicides Fungicide concentrations (ppm)a 100 200 400 600 800 1,000 Propiconazole +++ +++ +++ +++ +++ +++ Validamycin +++ +++ +++ +++ +++ +++ Benomyl +++ +++ +++ +++ ++ + Carbendazim +++ +++ +++ + Tricyclazole +++ +++ +++ +++ +++ ++ Mancozeb +++ +++ +++ ++ + + Azoxystrobin +++ +++ +++ + Hexaconazole +++ +++ +++ +++ +++ +++ aGrowth of Integral in NA amended with fungicides: +++ ¼ Good; ++ ¼ Moderate; + ¼ Slight; ¼ No growth 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 255

Fig. 9.7 Compatibility of Integral with Carbendazim. Values are means of ﬁve replications. Means followed by a common letter are not signiﬁcantly different at p 0.05

Fig. 9.8 Compatibility of Integral with Azoxystrobin. Values are means of five replications. Means followed by a common letter are not significantly different at p 0.05 of PGPR against ShB under field conditions was during the first day of inoculation of R. solani. Reduction in ShB severity under field conditions by PGPR is also dependent on the bacterial concentration. It is interesting to note that the PGPR when applied as consortia and in conjunction with other fungal antagonists offered synergistic effect over their individual applications in ShB disease reduction. Talc- based formulations of two P. fluorescens strains (PF1 and PF7), when applied as seed, soil, and root dip treatments and foliar sprays, significantly reduced ShB and leaf-folder incidence under greenhouse and field conditions. The PGPR mixture proved to be more effective over their individual applications (Radja Commare et al. 2002). Combined use of P. fluorescens and Trichoderma viride was effective 256 K.V.K. Kumar et al. in rice ShB reduction and enhanced seedling growth (Mathivanan et al. 2006). Bacillus spp. exhibited synergistic effect when used in conjunction with T. viride (Das et al. 1998) and Gliocladium virens (Sarmah 1999) against ShB. The fermented product of Bacillus strain Drt-11 when applied in combination with biofungi- cide, Jinggangmeisu WP (20%) proved to be more effective against ShB over their individual applications (Chen and Hui 2006). Our own studies with Integral under greenhouse and field studies effectively reduced ShB incidence in rice. In a typical greenhouse assay, Integral was evaluated at concentrations of 2.2 106 to 2.2 109 cfu ml 1 as seed treatment (ST), seedling root dip (SD), and foliar sprays (FS). Seed treatment with Integral at concentrations of 2.2 108 and 2.2 109 cfu ml1 significantly improved percent germination over untreated control. Further, the root and shoot lengths were significantly improved (12.2 and 40.7 cm respectively) at 2.2 109 cfu ml 1 as against untreated control (7.9 and 33.8 cm respectively) at 25 DAS. Significant reduction in ShB severity (9.2 vs. 24.1 in untreated control) (Fig. 9.9), increase in plant height (73.2 vs. 62.7 cm in untreated control) and number of tillers/plant (11.9 vs. 8.0 in untreated control) was obtained when Integral was applied at 2.2 109 cfu ml1 as seed treatment (ST) þ seedling root dip (SD) þ foliar sprays (FS). Our field studies at Andhra Pradesh Rice Research Institute (APRRI), India during 2009 indicated significant improvement in root and shoot lengths or rice seedlings in nursery with Integral seed treatment at 2.2 108 and 2.2 109 cfu ml 1 over untreated control. On a transplanted crop, Integral application as ST þ SD þ FS at 2.2 109 cfu ml1 significantly reduced sheath blight severity (19.2 vs. 69.7 in control) and percent diseased tillers (25.1 vs. 99.4 in control)

Fig. 9.9 Suppression of sheath blight severity with Integral under greenhouse conditions 9 Commercial Potential of Microbial Inoculants for Sheath Blight Management 257

Fig. 9.10 Sheath blight severity under ﬁeld conditions

(Fig. 9.10). Plant height (98.1 vs. 78.5 cm in control), tillers/plant (12.8 vs. 10.0 in control), and grain yields were maximum with Integral application at 2.2 109 cfu ml1 as ST þ SD þ FS. Integral was also effective in reducing ShB severity and promoting growth and grain yields at a concentration of 2.2 108 cfu ml1.

9.6 Conclusions

As shown by the examples discussed in this chapter, PGPR have good potential in the management of rice ShB. It is generally believed that the field efficacy of a particular PGPR strain is dependent on its root colonization capacity. According to this reasoning, rhizosphere competence of a PGPR strain is a desirable trait for its effective root colonization and subsequent disease control. Earlier reports indicated that diversity of PGPR in rice rhizosphere is changing according to soil salinity. With increase in soil salinity, the population levels of Pseudomonas spp. decreased. In non-saline sites of rice rhizosphere, fluorescent Pseudomonads are the dominant species whereas in saline sites, these were replaced by salt tolerant species such as P. alcaligens and P. pseudoalcaligens. Further, organic farming was found to significantly enhance the diversity of PGPR populations in saline soils (Rangarajan et al. 2002). It should be noted that although rhizosphere competence is considered important for effective PGPR biocontrol agents, there could be exceptions. For example, if a particular PGPR-based product is applied as a foliar spray, rhizosphere colonization would not be strictly required. For example, with the product Intergral, which was highly effective in biocontrol of ShB in field trials in India, one application method was a foliar spray. To date, there are no published studies examining possible rhizosphere competence or root colonization on rice by the PGPR strain contained in Integral (B. subtilis strain MBI600). 258 K.V.K. Kumar et al.

Several biotic and abiotic factors also have significant impact on the field consistency of a formulated PGPR strain in rice ShB management. Since gram- positive bacilli produce endospores that can withstand desiccation and have a long shelf life, they are considered to be ideal candidates for commercial use against ShB. Fungicidal compatibility of selected PGPR strain is another important factor that determines the efficacy of PGPR under IDM. Consistent efforts are therefore needed to select PGPR strain with all the desirable traits that contribute to effective rice ShB management. Although several advantages have been reported with the use of microbial inoculants in rice, variability in effectiveness of field performance remains a constraint. To overcome this, comprehensive basic research is essential in the areas of selection of microbial agents that focus on identifying strains that occupy the same ecological niche with that of pathogens such as roots, the phylloplane, and vascular systems. Application of novel techniques such as PCR, RFLP, and RAPD for rapid identification of bacterial strains with desirable traits like biocontrol and growth-promoting mechanisms are therefore necessary. Integrating these basic research concepts with studies on greenhouse and field studies are therefore essential before devising IDM approaches for ShB management in rice. The PGPR that are identified in these respects should be maintained as important genetic resource, which will be useful for future studies that form an alternate to the presently available chemical control of ShB.

Acknowledgments The primary author is thankful to the authorities of Acharya N G Ranga Agricultural University, India for granting sabbatical to pursue his Ph.D. program at Auburn University, USA. The authors are thankful to the Associate Director of Research, A. P. Rice Research Institute, India for extending help in carrying out greenhouse and ﬁeld studies. The support of Rice Research Station, LSU AgCenter, USA in providing the seed material and pathogen for our investigations, and of Becker Underwood, USA in providing the Integral formulation is highly appreciated.

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Y.G. Yanni, F.B. Dazzo, and M.I. Zidan

10.1 Introduction

In view of the severe lack of capacity in rice production, which is stifling the development of Africa’s rice sector, participants at the 2010 Africa Rice Congress held in Bamako, Mali [http://beta.irri.org/news/images/stories/ricetoday/9-3/Africa_ story.pdf] called for a “Marshall Plan” to overcome this weakness, suggested strategies to significantly increase rice production in Africa, develop competitive and equitable rice value chains, reduce imports, and enhance regional trade. They enthusiastically supported the newly proposed Global Rice Science Partnership, an initiative of the Africa Rice Center (AfricaRice), the International Rice Research Institute (IRRI), and the International Center for Tropical Agriculture (CIAT) to harmonize national and international rice research agendas worldwide for increased impact in Africa. The Congress delegates highlighted that rice has become a strategic commodity that can potentially fuel economic growth and reduce hunger and poverty across the continent. Rice consumption in Africa is growing at 6–7% per year. To meet this demand, Africa imports almost ten million tons of rice grain each year, which is equivalent to one-third of the rice traded in the world market at a cost of US$4 billion in foreign exchange. Africa has sufficient land and water resources and favorable growth environments to

Y.G. Yanni (*) Department of Microbiology, Sakha Agricultural Research Station, Kafr El-Sheikh 33717, Egypt e-mail: [email protected] F.B. Dazzo Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA e-mail: [email protected] M.I. Zidan Department of Plant Nutrition, Sakha Agricultural Research Station, Kafr El-Sheikh 33717, Egypt

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 265 DOI 10.1007/978-3-642-18357-7_10, # Springer-Verlag Berlin Heidelberg 2011 266 Y.G. Yanni et al. close the gap between rice consumption and production, and local rice production can be competitive vis-a`-vis imported rice. The capacity of national programs has to be strengthened with support from regional and international organizations. There is a pressing need to increase technological innovations supported by an appropriate policy environment including rice genetic diversity and improvement, ecological intensification and diversification of rice-based systems, developing competitive rice value chains, new alliances and tools for rural learning and innovations and policy implications, integrated management of pests, diseases, and weeds in rice-based systems, and rice physiology and modeling. However, the continent seeks a “Marshall Plan” for capacity building by working together to unlock the region’s potential to increase rice production. This needs expert groups working on international agricultural research to pool resources, build capacity, and align national and international research agendas, thus enabling greater efficiency and efficacy in rice research. A close inspection of the previous situation and suggestions revealed that soil and plant microbiologists are close to implementing a targeted strategy for transitional research and technology work plan. In 1994, we congregated “on one table” a group of international researchers containing microbiologists and molecular biologists who were able to assess and introduce to the scientific community a newly described, natural endophytic association that exists between rhizobia, the micro-partner of the legume–rhizobia symbiotic N-fixation interrelationship, and rice, the most important cereal crop worldwide. Later, Egypt national agronomists and plant nutrition specialists joined the group and the outcome was a “contentious” internationally published paper describing this newly discovered interrelationship (Yanni et al. 1997). Later, independent research groups from all over the world (Jha et al. 2009; Mano and Morisaki 2008; Peng et al. 2008; Prayitno et al. 1999; Sun et al. 2008; Singh et al. 2009) reached the same conclusion: a beneficial endophytic association naturally exists between various rhizobial strains and different cereal plants, and this association is pursuing a strain-variety basis, strongly promising that some of those strains can be used as high-performing biofertilizer inoculants for cereal crops! No doubt then that the relationship exists, and the field is then open for other investigators to assess this endophytic association between many other rhizobial strains belonging to different species and various varieties of different cereal crops growing under various worldwide agro-ecosystems. However, this discovery and its worldwide validation are far from the end of the story, and much more assessment of this interrelationship is needed for development of biofertilizer preparations that are ready for daily use. Extensive field experimentation using diverse bacterial strains, different rice varieties, various soil types, numerous geographical locations and climatic variations, multiple growing seasons, different farmer’s experi- ences, etc., must be included in a program in order to take the next step towards implementing the technological approach of biofertilization in order to benefit the agriculture landscape worldwide. 10 Beneficial Endophytic Rhizobia as Biofertilizer 267

10.2 Can Biofertilization Help Recover a Deteriorating Global Environment?

Adoption of biofertilization can manage nutrient dynamics and pathogenesis to more environmental biosafety, while improving crop production and lowering inputs of expensive and hazardous agrochemicals. More than one century after the first commercial use of pure cultures of rhizobial inoculant in 1896, biofertilization is relatively well established in some developed countries while few developing countries have experienced the technology or even know about it. In general, biofertilization was undertaken just as a commodity to reduce chemical-N input while other benefits were not considered extensively. This led to a situation in which the less-educated farmers carelessly considered utilizing biological nitrogen-fixing (BNF) inocula in their fields especially when chemical N-fertilizers are available in the market. In Egypt, applications of BNF systems started 45 years ago by legume seed inoculation with rhizobia and rice seedling inoculation with cyanobacteria, and more recently, inoculation of different nonlegume crops with azospirilla. Their utilization was mostly based on high efficiencies in BNF and plant growth-promoting (PGP+) activities. Recent emerging issues include stimulation of cereal performances by certain strains of root inhabiting Rhizobium sp. (Yanni et al. 2001, 2010). Constraints facing the biofertilization technology are very similar to those in most of the developing nations. They involve unsatisfactory information about potential of indigenous N-fixing candidates and adverse effects of excessive amounts of fertilizers and pesticides. Also, most of the agronomic studies for evaluating the biofertilization process reside in a “black box” type of research in which the basis of positive results is deduced from indirect evidence. The technology also suffers from unsatisfactory extension programs, inadequate training and lack of advanced lab facilities and experimental data that describe the magnitude of adverse effects of chemical fertilizers on the man, animal, plant, environment, and national economies. Major constraints also involve improper soil and water management, disagreements between microbiologists and agronomists about the value of biofertilization with consequential less adoption of their use. High priority topics to enhance the technology may involve: (1) development of rapid assays that can predict performance of biofertilizer formulations in the field; (2) evaluation of treatments that promote cell survival on inoculated seed; (3) determination of effects of amendments, chemical fertilizers and pesticides; (4) development of new extension strategies and initiation of innovative, informative methods that are statistically sound in design; (5) adoption of molecular biological methods to identify desirable characteristics, manipulate and introduce more efficient strains for specific inoculation purposes; (6) exploration of new plant–microbe associations that capitalize on their diversity, level of PGP and ecology (survival, colonization, endophytic state, biogeography, dispersal, etc.); and (7) strengthen the electronic connection of information between researchers and extension experts working on active, effective and modern research and extension programs. Involvement of farmers in such a bio-informatic system must be, starting from now, a near future strategy! 268 Y.G. Yanni et al.

10.3 Rice and Biofertilization

Cereals are the world’s major source of food for human nutrition. Among these, rice (Oryza sativa L.) is most prominent and represents the staple diet for more than two- fifths (2.8 billion) of the world’s population. High grain production of rice has only been possible with high inputs of chemical nitrogen fertilizers. This unfortunately creates many environmental, economical, and health risk problems, making it a practice that must be greatly minimized considering environmental biosafety and agriculture sustainability. The current status of cereal biofertilization is unsatisfactory because of the relatively low diversity of candidate microorganisms under consideration (Yanni and Abd El-Fattah 1999; Yanni and Mohamed 1985). Although some agricultural biofertilizers (Azospirillum and cyanobacteria) are already used in Egypt and elsewhere in crop production, none till now have satisfactorily achieved the ideal goal of replacing most of the demand for chemical fertilizers to obtain figures near the yield potential. Therefore, discovery and development of more effective biofertilizers that can reduce the dependence on chemical fertilizer inputs to maximize crop yields is a high priority area to help achieve the goals of global sustainable agriculture. The production of rice up to a level of global food security is currently not possible without major nutrient inputs, especially N. Indirectly, rice is able to utilize a basal level of fixed-N through N2-fixing activities of diverse diazotrophs in its agroecosystem. However, serious economic and environmental problems associated with the use of inorganic fertilizer application to enhance production of major cereal crops including rice could be mitigated if cereal crops could establish a more direct and intimate association with beneficial bacteria that promote their growth and increase their grain yield with less dependence on chemical N-fertilizer input. Our development of new, reliable agricultural biofertilizers addresses the important real-world problem of limited biologically available nitrogen in cereal crop production. This strategy exhibits exploitation of natural beneficial associations, is environmentally sound, and has high intrinsic probability for success. This chapter describes our novel finding that certain strains of clover-nodulating rhizobia (Rhizobium leguminosarum bv. trifolii) participate in a beneficial, natural endophytic association with rice in fields in the Nile Delta where the crop has been rotated with berseem clover since antiquity, and that some of these native adapted rhizobia have the potential to promote rice growth. We intend to develop, improve and evaluate the ecology and performance of biofertilizer inoculants containing superior Rhizobium strains that can enhance rice production under real-world agricultural field conditions.

10.4 Justiﬁcation of the Transitional Research and Technology Program

Our studies at both the lab and field levels established the potential for effective using of selected strains of rhizobia that are natural endophytes of rice, as biofertilizers capable of significantly enhancing its production in the agroecosystems of 10 Beneficial Endophytic Rhizobia as Biofertilizer 269 the Nile delta (Yanni et al. 1997, 2001) while reducing the requirement for additional N-fertilizer inputs. By sharing of our strains, we established a major multinational network of investigators to create a broad-based understanding of the physiology of PGP and the plant–microbe ecology of rhizobia–rice association. We envision that the most important impact will be the development and implementation of new microbial biofertilizers that can reduce the major real- world constraint of limited biologically available nitrogen to increase rice productivity. These contributions advanced scientific knowledge on beneficial plant–microbe associations, inevitably assisting low-income farmers who produce rice on marginally fertile soils deficient in N and many other nutrients as well as benefit manufacturers of agricultural biofertilizer inoculants in the private sector. Assuming that our results accurately reflect the potential benefits of this new agricultural biotechnology, we predicted the following outcomes to agricultural infrastructure in both Egypt, US and worldwide in the future: (1) increase the cereal yield by about 15–25% over the yield which can be reached using the full recommended amount of chemical fertilizer-N for the crop, with a reduction of some 33–67% of this chemical fertilizer input to obtain similar yield that can be obtained without inoculation; (2) decrease environmental pollution and health risks originating from excessive use of chemical N-fertilizers; (3) relieve the stress on manufacturing, distribution and marketing of chemical fertilizers during certain periods of the year when the availability of chemical fertilizers is mostly controlled by black-market rather than the legitimate normal market; (4) decrease the energy consumed to manufacture N-fertilizers and direct it to other necessary socioeconomic and industrial uses; (5) increase the opportunities for export of chemical fertilizers via reducing its local consumption; (6) promote a better understanding of the value of sustainable agriculture to farmers by utilizing biofertilizers in their agricultural practices as an environment friendly safe tool alternative to chemical fertilization; and (7) promote better cooperation between research institutions in Egypt and the USA on one side and both private and governmental sectors represented by farmers and agricultural biotechnology industries on the other side. To transform optimistic dreams to the stage of reality, beneficial plant–microbe associations are being examined, with a focus on assessment of their potential to promote plant growth and enhanced yield, and identification of superior candidate strains that can be developed into new improved biofertilizers that come closer to achieving the targeted results. Our guiding hypothesis has been that natural endophytic associations of rhizobia and cereal roots would most likely occur in regions of the world where the cereal is successfully rotated with a legume crop that could enhance the soil populations of the corresponding rhizobial symbiont(s). Such natural Rhizobium–cereal associations would be maintained if they were mutually beneficial. If this were correct, the cereal roots growing at these sites should contain, among other types of microorganisms, a reservoir of endophytic rhizobia that are already adapted to be highly competitive for colonization of this habitat, protected from stiff competition with other rhizosphere microorganisms under field conditions. Here, the endophytic rhizobia are strategically located where a more 270 Y.G. Yanni et al. rapid and intimate metabolic exchange is possible within the associative interrelationship between a microbial agent and host plants. One of the ideal places to address this question has been in the Nile Delta of Egypt, where for more than seven centuries, most of the rice has been in productive rotation with the legume Egyptian berseem clover (Trifolium alexandrinum L.). In this region, Japonica and recently Indica cultivars of rice are cultivated by transplantation in submerged lowlands. There, 60–70% of the 500,000 ha of land area used for rice production is in rice-clover rotation. The high yield, protein content, and N2-fixing capacity of berseem clover enhance its use as a forage and “green manure” plant. An interesting enigma for this ancient scenario of a successfully farming practice is that the rotation with berseem clover replaces 25–33% of the recommended amount of fertilizer-N application for optimal rice production when cultivated after a cereal like wheat or barley (Zidan and Yanni 1984). This benefit cannot be explained solely by the increased availability of biologically fixed N through mineralization of the N-rich clover crop residues, despite that scenario being mentioned in many research publications (Abd El-Wahab et al. 1993; Ebaid and Ghanem 2000; George et al. 1992; Reid and Goss 1981; Tisdall and Oades 1982) as the basis of improved rice cultivation and enhanced paddy yield when rice followed berseem clover in the same field area. This led to an important question about whether there is a natural endophytic Rhizobium–rice association evolved that contributes to this added benefit of clover–rice rotation. Our studies indicated a natural endophytic association does exists between the rice root and the clover root- nodule occupant, R. leguminosarum bv. trifolii, without induction of root nodules or obvious symptoms of disease on that cereal crop (Yanni et al. 1997, 2001). Furthermore, this association can significantly improve rice plant growth, resulting in an increase in rice grain productivity and agronomic fertilizer N-use efficiency (kg grain yield/kg fertilizer-N), making it possible to increase rice grain yields with less dependence on nitrogen fertilizer inputs. Our guiding hypothesis has now been independently validated worldwide by many research teams (Chaintreuil et al. 2000; Gutierrez-Zamora and Martinez- Romero 2001; Hilali et al. 2001; Jha et al. 2009; Lupway et al. 2004; Mano and Morisaki 2008; Matiru and Dakora 2004; Mishra et al. 2008; Peng et al. 2008; Singh et al. 2006, 2009; Tan et al. 2001) who independently verified the ability of rhizobia to occupy a third agriculturally important ecological niche as endophytic colonizers of cereal roots and can promote their growth in agroecosystems that host legume–cereal rotations. This closes rhizobia’s triangle of natural history (Fig. 10.1). Since that fundamental discovery, many tests of the generality of our original finding have indicated that this type of association is more widespread than just in the Nile delta. Following our initial publication (Yanni et al. 1997), other associations of endophytic PGP rhizobia have been found for wheat, barley, rice, wild rice, maize, sorghum, and millet that are rotated with legumes in Senegal, Canada, Mexico, Morocco, Kenya, India, Pakistan, China and elsewhere, respectively (Chaintreuil et al. 2000; Dazzo et al. 2000; Matiru and Dakora 2004; Gutierrez-Zamora and Martinez-Romero 2001; Perrine et al. 2001; Hilali et al. 2001; Tan et al. 2001; Lupway et al. 2004; Mishra et al. 2008; 10 Beneficial Endophytic Rhizobia as Biofertilizer 271

Fig. 10.1 The triangular natural history of Rhizobium that includes its ecological niches with legume and cereal crops in rotation

Matiru and Dakora 2004; Singh et al. 2006; Jing, personal communication). Thus, there is no longer any scientiﬁc basis upon which the existence and potential beneﬁt of the Rhizobium–cereal association can be a matter of doubt.

10.5 The Transitional Route Between Research and Technology

We successfully adopted a direct ecological approach to isolate a diverse collection of rice-adapted rhizobial strains that exhibit a high potential to perform as agricultural biofertilizer inoculants that can improve rice production with less additional consumption of chemical N-fertilizer. In situations exemplified by our field experiments at the Kafr El-Sheikh Governorate, inoculation of rice with rhizobia followed rather than replaced crop rotation with the berseem clover legume crop (Yanni et al. 1997, 2001). Rice could then gain benefits from the two biological associations of rhizobia, as the N2-fixing root nodule occupant of clover and as an active PGP in rice roots. This strategy is intended to augment the anticipatory colonization potential of superior inoculant strains in competition with other rhizosphere microorganisms for endophytic niches in this cereal, above the natural level of inoculum potential carried over from decay of remains of the previous legume crop. The strategy is appealing from the environmental impact standpoint since it makes greater use of the natural resource of beneficial plant–microbe interactions. The ability of this rhizobia–cereal association to increase the agronomic fertilizer N-use efficiency, i.e., higher productivity of paddy yield using the existing native and applied N sources, is fully consistent with ecologically sound sustainable agriculture practices and therefore, highly desirable. However, since a transitional research program in farmer’s fields is also a major requirement to justify further proceeding toward production of biofertilizer preparations containing strains of rice root- adapted rhizobia that can improve rice growth and crop performance, we established a national large-scale program in the Egypt Nile delta in which 24 further field experiments were conducted in nine counties in Kafr El-Sheikh governorate at 272 Y.G. Yanni et al.

Fig. 10.2 Experimental locations used in ﬁeld inoculation trials in the Kafr El-Sheikh governorate, north Nile delta of Egypt

the middle and northern part of the Nile delta (Fig. 10.2). We there tested the performances of seven genotypically distinct inoculant strains of R. leguminosarum bv. trifolii used individually or in consortia for inoculation of five rice varieties along five rice growing seasons (May through October) during 2000, 2002, 2003, 2004, and 2005. As the results continued to be promising year after year, we conducted ascending numbers of experiments (1, 4, 5, 6, and 8 during the five seasons, respectively). These field experiments were performed at two scales: 20 m2 replicated experimental plots at the Sakha Agricultural Research Station and larger plots of up to 210 m2 in farmer fields.

10.6 Methodology for Field Inoculation Experiments

Important information on the methodology used in this research and technology program is summarized in the following contextual part.

10.6.1 Rice Varieties

Seeds of the blast (Pyricularia grisea)-resistant Indica and Japonica rice varieties: Yasmein (Indica var.) and Giza 177, Giza 178, Sakha 101, and Sakha 104 (Japonica vars.) were used (Table 10.1). The first, second and fourth varieties are recommended for fertile nonsaline soils while the third and the fifth varieties are recommended for fertile saline soils. Their grain-specific weights are 26, 23, 19, 26, and 25 g/1,000 grain, and their milling percent are 65.0, 73.3, 70.9, 71.6, and 71.6, respectively. Durations (seed–seed) are 150, 125, 135, 140, and 130 days, respectively. Their National yield averages in Egypt are 8.4, 8.8, 10.7, 11.9, and 11.9 ton/ha, respectively. These varieties are more N-responsive (up to 144 kg N/ha) than the 0BnfiilEdpyi hzbaa ifriie 273 Biofertilizer as Rhizobia Endophytic Beneficial 10

Table 10.1 Location, replicated plot size, pertinent agronomic and inoculant information for each ﬁeld inoculation experiment Site Near city Year Plot size (m2) Previous crop Rice variety Inoculant strain(s) of rice-adapted R. leguminosarum bv. trifolii 1 K. El-Sheikh 2000 20 Berseem cover Jasmine E11 + E12 2 Baltem 2002 20 Berseem clover Giza 178 E11 + E12 3 Beila 20 Berseem clover Giza 178 E11 + E12 4 Metobas 20 Wheat Giza 178 E11 + E12 5 Sidi Salem 20 Wheat Giza 178 E11 + E12 6 Metobas 2003 20 Berseem clover Sakha 104 E24 7 Qalien 20 Wheat Sakha 101 E24 8 Desouk 20 Wheat Giza 177 E39 9 Fowa 20 Berseem clover Giza 178 E39 10 Sidi Salem 20 Wheat Giza 178 E39 11 Qalien 2004 20 Fababean Sakha 101 E18, E26, E36 12 Fowa 20 Berseem clover Giza 178 E18, E26, E36 13 Metobas 20 Berseem clover Giza 178 E18, E26, E36 14 Sidi Salem 20 Wheat Giza 178 E18, E26, E36 15 Desouk 20 Berseem clover Sakha 104 E18, E26, E36 16 El-Read 20 Wheat Sakha 104 E18, E26, E36 17 Sidi Salem 2005 52.5 Wheat Giza 178 E11 + E12 + E18 + E24 + E26 18 Fowa 52.5 Berseem clover Giza 178 E11 + E12 + E18 + E24 + E26 19 Desouk 52.5 Berseem clover Sakha 101 E11 + E12 + E18 + E24 + E26 20 Metobas 52.5 Berseem clover Giza 178 E11 + E12 + E18 + E24 + E26 21 Desouk 52.5 Berseem clover Sakha 104 E11 + E12 + E18 + E24 + E26 22 Sakha 52.5 Berseem clover Sakha 101 E11 + E12 + E18 + E24 + E26 23 Qalien 52.5 Berseem clover Sakha 101 E11 + E12 + E18 + E24 + E26 24 Qalien 52.5 Wheat Giza 178 E11 + E12 + E18 + E24 + E26 274 Y.G. Yanni et al. older varieties Giza 171 and 172 (96 kg N/ha), which were discontinued because of their lower grain yields, high water consumption requirements and high susceptibility to blast.

10.6.2 Bacterial Test Strains and Preparation of Inoculants

Seven different rice endophytic strains of R. leguminosarum bv. trifolii representing a strain diversity obtained in pure culture collections from two cycles of isolation from within surface-sterilized field-grown rice roots in the Nile delta (Yanni et al. 1997, 2001) were used as single-strain or consortia inoculants (Table 10.1). Dis- crimination according to their genotypic relatedness was based on BOX-PCR and plasmid profiling analyses, and comparison to the 16 S rDNA sequence of the type strain. Gnotobiotic culture tests indicated that they are Nod+ Fix+ on berseem clover and fulfillment of Koch’s postulates proved their endophytic colonization ability and PGP activities on several rice varieties (Yanni et al. 1997, 2001). Inoculants were prepared with peat carrier neutralized from its original acidic pH of 5.0–5.5 using powdered CaCO3 (Yanni and Dazzo 2010).

10.6.3 Field Inoculation Trials

The first field inoculation experiment was conducted at the experimental farm of the Sakha Agricultural Research Station (Kafr El-Sheikh) during the rice-growing season of year 2000 (April/May to September/October). The subsequent 23 field experiments were conducted during the four consecutive annual rice cultivation seasons of 2002–2005, at various paddy rice farms in nine counties of the Kafr El- Sheikh Governorate, located in the north-west-central regions of the Nile delta (Fig. 10.2). The rice cultivation area in this governorate represents approximately 18–25% of the total area used annually for rice cultivation in Egypt. Table 10.1 lists the identification number assigned to each field inoculation experiment, year of the rice growing season, the nearest central city, the previous crop grown in the same field, the rice variety and inoculant strain(s) tested, and the replicated plot area of the subplot. To assess the nitrogen fertilization economy in the presence and absence of bacterial inoculation, three N fertilization doses were placed as the main-plot treatments, representing 1/3, 2/3 and the full amount of fertilizer-N recommended by the Agriculture Research Center, Egypt (http://www.arc.sci.eg/) for use when no biofertilization practice was implemented. These 24 large-scale field experiments plus three previous small-scale field experiments performed at the Sakha Agricultural Research Station (Yanni et al. 1997, 2001) represent a total of 27 field inoculation trials for nine varieties of rice using seven genotypically distinct, indigenous rhizobial endophytic strains (Yanni et al. 2010). 10 Beneficial Endophytic Rhizobia as Biofertilizer 275

Soil textures ranged between clay, clay-loam, silty-clay, silty-clay loam and silty-loam. Soil pH (Black et al. 1965; Jackson 1967) ranged between 7.8 and 8.1, CaCO3 content between 1.4 and 5.4%, and organic matter content between 1.9 and 2.3%. The top soil (0–15 cm) salinity ranged between <2,500 and 8,609 ppm. Further field management information is provided in Yanni and Dazzo (2010). The experimental/statistical design was the split-plot with the three N-fertilization rates as the main-plot treatments and inoculation (or not) as the subplot treatments. Four replicates were used for each subplot treatment. The replicates, main and subplot treatments were randomly distributed. All plants in the subplot area were harvested and weighed to obtain the yield data (no subsampling). The mean differences were compared to their corresponding least-significant differences at the confidence level of 95%. Samples for yield comparison were taken from identical, noninoculated field areas cultivated with the same crop varieties during the same period in the adjacent field areas managed according to the farmer’s own conventional farming practices followed for decades to centuries, without supervision from the program research and technology teams. Importance of this estimation is clearly highlighted later in the text.

10.7 A “Live and in Full Color” Recognition of the Biofertilization Contribution to Rice Performance

In 19 of the 24 conducted field experiments, inoculation significantly increased grain yield compared to the corresponding noninoculated controls that received the same experimental research practices except inoculation (confidence level of 95%). These significant increases over the corresponding noninoculated controls ranged from 0.268 to 1.499 ton/ha (mean std. dev. of 0.737 0.281 ton/ha). The average inoculation-dependent increases in grain production in the three other trials where differences were not statistically significant ranged from 0.108 to 0.582 ton/ha. These comparisons are based on the means of the inoculated vs. the noninoculated subplots for all three ascending rates of fertilizer-N, as appeared by analysis of variances. The performance of Rhizobium biofertilizers plus different N-fertilizer doses on rice paddy yield (tons/ha) is shown in Fig. 10.3a–d. These include the mean performance at three doses of N-fertilizer application (Fig. 10.3a) and the mean of differential effects of inoculation plus N-fertilizer application at low (Fig. 10.3b, 48 kg N/ha), medium (Fig. 10.3c, 96 kg N/ha), and fully recommended (Fig. 10.3d, 144 kg N/ha) fertilizer doses. Unlike the well-known suppression of symbiotic nitrogen fixation resulting from application of N-fertilizers, the degree of benefit to rice by inoculation with rhizobia was enhanced rather than suppressed with supplementation with N-fertilizer, consistent with previous results (Yanni et al. 1997, 2001). Interestingly, inoculation significantly increased grain yield with application of the intermediate doses of 48 or 96 kg fertilizer-N/ha in 9 out of the 24 field trials. Inoculation could replace the need for higher amounts of 276 Y.G. Yanni et al.

Fig. 10.3 (a–d) Performance of Rhizobium biofertilizers plus different N-fertilizer doses on rice paddy yield (tons/ha). (a) Mean performance at three doses of N-fertilizer application. (b–d) Mean of differential effects of inoculation plus N-fertilizer application at b, 48 kg N/ha, at c, 96 kg N/ha, and at d, 144 kg N/ha chemical N-fertilizer to achieve the same or higher grain yields than with application of the full recommended amount of 144 kg N/ha. Especially interesting were the results of field trials #2, 6, and 9 where inoculation caused statistically significant increases in grain production even when no statistically significant increases were obtained in response to the maximum recommended dose of 144 kg N/ha without inoculation. Comparison of Fig. 10.4a, b shows the rice variety specificity in growth promotion (Jasmine is +, Giza 178 is ) using the same bacterial inoculant strains. Comparison of Fig. 10.4b, c shows the rhizobial strain specificity (E11 and E12 are , E39 is +) in growth promotion of the Giza 178 rice variety. Comparison of Fig. 10.4c, d shows the site-specific growth promotion of Giza 178 rice variety by the inoculant consortium of E11 and E12 resulting in increased rice grain productivity even when nitrogen is not the major limiting factor. Generally speaking, results of this inoculation program indicated that certain consortia of rhizobial strains (experiments #1 through 5, and #11 through 24, Table 10.1) performed better as rice biofertilizer inoculants than inoculants formulated with only a single rhizobial strain (experiments #6 through 10). The mean of the 10 Beneficial Endophytic Rhizobia as Biofertilizer 277

Fig. 10.4 a–d: Grain yield responses of rice cultivars to N-fertilizer application with and without inoculation using different endophytic strains of Rhizobium leguminosarum bv. trifolii statistically significant increases in rice grain yield due to biofertilization with multistrain consortia as inocula was 11.2% higher than with inoculants containing only a single strain (0.706 vs. 0.635 ton/ha). The overall best-performing biofertilizer that enhanced rice grain production in these studies was a consortium of strains E11 + E12, which increased paddy yield by a mean of 22.2% in the five inoculation experiments that received this consortium as the inoculant. This is consistent with our previous studies (Yanni et al. 1997, 2001) indicating that the beneficial endophytic association between the rhizobial strains and rice varieties followed a strain/variety specificity in which a rhizobial strain performed better when used for inoculation of certain rice varieties and response to inoculation of a given rice variety differed when inoculated with different rhizobial strains. The increased performance of multi-strain preparations is mostly due to an increased opportunity of successful association between the rice cultivar and one or more of the strains in the consortia preparation than when inoculated with only a single rhizobial strain. Since this is the first field experimentation of our strains in consortia inoculants, support for this explanation from independent research groups is needed. Another benefit of rhizobial inoculation found was its ability to increase the potential for certain rice varieties to exceed the Egyptian 2008 national production average of 10.04 tons paddy rice yield/ha (world’s highest) recorded by the USDA using the full recommended amount of N fertilizer (144 kg N/ha) without 278 Y.G. Yanni et al. inoculation (http://beta.irri.org/solutions/index.php?option¼com_content&task¼ view&id¼250). This increased production capacity that exceeded the previous world’s record in national mean of rice grain yield occurred in 7 of the 24 inoculation experiments (#8, 10, 15, 19, 22–24) when it could not be achieved by chemical N-fertilizer application alone. The number of experiments in which inoculation with rhizobia promoted grain yield to levels that exceeded that national production figure were 8 with 48 kg fertilizer-N/ha, increased to 15 with 96 kg fertilizer-N/ha, and finally in 21 experiments with the full recommended application rate of 144 kg N/ha (Yanni et al. 2010).

10.7.1 Contribution of Biofertilization in Salt-Affected Fields

Saline soils are very common in the northern coastal region of the Egypt Nile delta where the program has been performed. Submerged rice is predominantly grown not only for its importance as an economically staple cereal crop, but also because soil reclamation in those areas can be facilitated by percolation of water through the soil proﬁle that washes the high salt content downwards to groundwater and then to drainage canals. The effect of soil salinity on rice production in the 24 experimental locations is indicated in Table 10.2. Mean of grain yield obtained with inoculation exceeded what could be obtained in the corresponding noninoculated controls under all the soil salinity levels. The percentage increases in grain yield due to inoculation followed a special tendency in which – with few exceptions – it increased proportionally with salinity intensiﬁcation! The higher soil salinity in the county that includes the northern city of Baltem (experiment #2) most likely contributed to the lower grain yield of the variety Giza 178 obtained in the noninoculated treatments when compared to the higher grain yield of the same

Table 10.2 Mean of rice grain yield under different levels of soil salinity, N-fertilizer rates and inoculation with Rhizobium biofertilizer Soil salinity Locations N-fertilizer Mean of paddy yields (ton/ha) Mean of (ppm) (kg N/ha) Non-inoculated Inoculated increase (%) 0–2,500 3, 4, 5, 7, 8, 9, 10, 11, 48 9.204 9.680 5.2 12, 13, 14, 15, 17, 96 10.002 10.671 6.7 18, 19, 20, 21, 22, 144 10.662 11.122 4.3 23, 24 2,501–5,000 1, 6 48 9.763 10.627 8.5 96 9.885 10.815 9.4 144 10.519 11.241 6.9 5,001–7,500 16 48 9.154 9.857 7.6 96 9.826 10.309 4.9 144 10.056 10.439 3.8 7,501 2 48 6.959 8.134 16.9 96 6.424 8.623 34.2 144 6.881 8.305 20.7 10 Beneﬁcial Endophytic Rhizobia as Biofertilizer 279 rice variety obtained in noninoculated treatments at other locations with less soil salinity. Interestingly, the mean increase in yields resulting from inoculation was highest at that same location, suggesting that inoculation of rice with certain endophytic rhizobial strains may help to alleviate the adverse effects of salinity stress on rice grain production. Similar trends have been previously found for the root nodule symbiosis between Bradyrhizobium spp. (Cajanus) with pigeon pea and Rhizobium loti with chickpea (Rao and Sharma 1995; Subbarao et al. 1990). This raises the interesting possibility that rhizobial inoculation may help farmers obtain higher rice yields during and just after performing reclamation processes to saline soils (as long as the beneﬁt-to-cost ratio is acceptable).

10.7.2 Comparison Between Outcomes of Researcher’s and Farmer’s Rice Farming Managements

Result of comparisons between yields of the researcher’s best experimental treatment and those in the farmer’s adjacent fields using their conventional experience acquired through history is presented in Table 10.3. The results show increases in paddy yields in all the locations using the researcher’s integrated rice production system. The increases ranged between 2.7 and 47%, with 15 of the 23 experiments showing increases of paddy yield that exceeded 15% of the farmer’s yields, 8 scored increases of 20%, 6 scored increases of 25%, 6 scored increases of 30%, and 2 scored increases of 40% in paddy grain production. The experiment #1 was conducted in the experimentation farm and so no comparison with farmer’s yield could be scored. However, those increases cannot be attributed exclusively to biofertilization since other factors are also involved such as use of optimal field stand densities, balanced NPK fertilization treatments, hand-weeding, integrated pest management, and others that are normally followed by the researchers. In addition, the efficiency, frequency and fluency of direct and indirect exchange of information from the researcher to the cooperator field supervision specialists and finally to the farmer are important factors that contribute to the high increases in crop yield that sometimes extended beyond expectations. It is important to emphasize here that in most cases, inoculation with our cereal-adapted rhizobial endophytes increased grain yield even when the crop was fertilized with the full recommended dose of combined nitrogen that was recommended when no inoculation treatment was applied. This clearly indicates that inoculation with our biofertilizer strains transforms the plant to make better use of the available N and other plant nutrient resources in the root zone that are not accessible by the plant without inoculation (Fig. 10.4a–d). The outcome of this experimentation program supports our previous studies showing benefits of inoculation in expanding the plant root architecture (biovolume, biomass and cumulative root length, and surface area), increased absorption of nutrients by the plant, enhanced production of plant growth regulators, and efficient use of insoluble inorganic phosphates in the rhizosphere area and organic phosphates inside the root (Yanni et al. 2001) as the probable 280 Y.G. Yanni et al.

Table 10.3 A comparison between rice grain yields in the best experimental treatments and those obtained simultaneously in adjacent fields using the farmer’s conventional practices at 23 locations in the Nile delta Expt. Applied Best experimental grain yield Farmer’s % increase compared to no. N (Kg/ha) (ton/ha) grain yielda the farmer’s grain yield Non-inoculated Inoculated(tons/ha) Non-inoculated Inoculated 1 144 8.725 9.551b –– – 2 96 6.424 8.623b 8.330 – 3.5 3 96 10.609 11.309 9.520 11.4 18.8 4 96 11.483 12.400b 9.520 20.6 30.3 5 144 11.118 10.250 9.068 22.6 13.0 6 144 12.312 13.078b 12.729 – 2.7 7 48 12.406 12.805 10.880 14.0 17.7 8 144 9.008 10.008b 9.520 – 5.1 9 144 12.400 12.828b 12.240 1.3 4.8 10 144 9.520 10.472 9.520 0.0 10.0 11 144 10.870 11.282b 9.520 14.2 18.5 12 144 11.287 12.372b 9.044 24.8 36.8 13 144 11.245 12.372b 9.520 18.1 30.0 14 144 10.807 11.287b 8.330 29.7 35.5 15 144 9.738 10.369b 9.196 5.9 12.8 16 144 10.056 10.494b 7.140 40.8 47.0 17 144 11.110 11.986b 8.400 32.3 42.7 18 144 11.262 12.305b 10.320 9.1 19.2 19 144 9.934 10.343b 8.400 18.3 23.1 20 144 10.576 11.481b 9.600 10.2 19.6 21 144 10.338 10.586b 9.120 13.4 16.1 22 144 9.657 10.124b 9.600 0.6 5.5 23 144 9.729 10.076b 8.760 11.1 15.0 24 144 9.648 10.291b 8.44 14.3 21.9 aYield obtained without researcher supervision bStatistically different increase (95% confidence level) due to inoculation over the non-inoculated counterpart assessed by least significant differences between means mechanisms by which this growth promotion was obtained. However, the increases in grain yields reported in those comparisons most likely represent the minimum increases that can be obtained when the researcher’s recommendations have been followed.

10.7.3 Rice Straw, a Debit or Credit to National Economy and Environmental Soundness

Inoculation increased straw production in 19 of the 24 field experiments, with 14 being statistically significant at the 95% confidence level. The range of statistically significant increases resulting from inoculation was 0.417–1.037 with a mean of 0.740 ton/ha. The increases in straw yield resulting from inoculation at all levels of 10 Beneficial Endophytic Rhizobia as Biofertilizer 281

N-fertilizer application are consistent with earlier studies showing that rice shoot height, leaf area, and photosynthetic capacity can be significantly increased by inoculation with certain endophytic strains of rhizobia (Biswas et al. 2000a, b; Chi et al. 2005; Yanni et al. 1997, 2001). Straw biomass production was more responsive to chemical N-fertilization than to rhizobial biofertilization, and in contrast, biofertilization boosted rice grain production more so than it increased straw biomass production. This result can be considered as the preferred, positive benefit of inoculation (more grain yield) and inevitably leads one to hypothesize that the rhizobial interaction with rice intensifies the plant’s reproductive physiology in ways that increase grain production. Under certain conditions, application of more mineral N than the recommended dose may lead to a disproportional increase of above-ground vegetative growth and cause a detrimental phenomenon known as “lodging.” Lodging with a decline of rice grain yield may also occur when the plant is provided with an excess of N fertilizer in addition to fixed N supplemented by N-fixing inoculants like cyanobacteria or azospirilla (Yanni 1992a, b, 1996; Yanni and Abd El-Rahman 1993; Yanni and Abd El-Fattah 1999). In our first field trial (Yanni et al. 1997), detrimental lodging occurred when one of the main-plot treatments was inoculated with rhizobia and supplemented (for comparison reasons) with mineral N-fertilizer above the recommended dose for the rice variety tested. However, lodging did not occur in any of these 24 recent field experiments because no more than the recommended amount of N-fertilizer was applied and most of the varieties used were of the short stature type (heights at harvest 102–105 cm) that does not typically experience lodging damage. Rice straw is utilized in Egypt as a valuable fodder for domestic farm animals, in manufacture of byproducts including building bricks used in small villages, processed synthetic wood and as a packaging filler material. For animal feed, portions of the national produced straw are consumed directly or processed to a softer nutritive material in a closed system containing gaseous ammonia, a technology started in 1974. All the above treatments collectively cannot consume the annual produced quantity of rice straw byproduct. Farmers currently burn the unused straw to clear and prepare their fields for the next crop. This produces abundant smoke pollution causing eye and respiratory irritations and also road accidents. Proper technologies are needed to make better use of the increased rice straw!

10.7.4 Plant Growth Tendencies as Affected by Biofertilization

Although the harvest index (grain yield/above-ground harvested grain plus straw biomass, expressed as a %) recorded for all three levels of N-fertilizer applications was higher with inoculation in 19 of the 24 field trials, the differences were statistically significant in only five experiments. Table 10.4 shows increases in harvest index resulted from inoculation and/or application of the ascending doses of fertilizer-N. Increasing the N-fertilization dose significantly and typically decreased 282 Y.G. Yanni et al.

Table 10.4 Influence of Rhizobium inoculation on harvest index at six locations in the Nile delta Expt. N Non- Inoculated Mean Expt. N Non- Inoculated Mean no. (kg/ha) inoculated no. (kg/ha) inoculated control control 1 48 37.2 40.3 38.8a 11 48 46.4 46.0 46.2a 96 34.5 40.2 37.4a 96 47.2 46.4 46.8a 144 33.5 37.8 35.7a 144 43.0 42.7 42.9b Mean 35.1a 39.4b Mean 45.5a 45.0a 2 48 31.1 33.3 32.2a 12 48 46.1 47.0 46.6a 96 30.7 32.4 31.6a 96 43.9 44.3 44.1b 144 30.9 33.9 32.4a 144 44.9 45.5 45.2c Mean 30.9a 33.2b Mean 45.0a 45.6a 6 48 55.0 54.2 54.6a 13 48 47.8 46.0 46.9a 96 54.5 53.1 53.8a 96 44.6 45.4 45.0b 144 52.4 51.8 52.1b 144 43.8 45.6 44.7b Mean 54.0a 53.0a Mean 45.4a 45.7a 10 48 41.3 43.6 42.5a 14 48 44.2 44.1 44.2a 96 39.7 44.7 42.2a 96 42.2 41.6 41.9b 144 38.2 46.3 42.3a 144 40.3 40.0 40.2c Mean 39.7a 44.9b Mean 42.2a 41.9a Details of the Rhizobium leguminosarum bv. trifolii strains, rice cultivars, cultivation season and location are provided in Table 10.1. Harvest index: % of grain yield/grain + straw yields. Means superscripted by different letters in the same column (for N-fertilization rates) or highlighted in bold (for inoculation) for each experiment are statistically different at the 95% confidence level (assessed by least significant differences between corresponding means)

the harvest index, indicating that unlike for inoculation, N-fertilization increased straw production more so than increased grain production, consistent with results reported above on straw yield. In the remaining 16 experiments, no signiﬁcant changes in harvest index were observed due to inoculation or increasing the N-application dose. Lower harvest indices indicate that the recommended N-dose used in those experiments was within the acceptable range that balanced vegetative and reproductive growth (Ye et al. 2007). Inoculation and N-fertilization worked together synergistically in providing the plant’s demand for N and other nutrient requirements without decreasing the harvest index values in most cases. This result is consistent with our previous work (Yanni et al. 1997, 2001) showing that rhizobial inoculation and N-fertilization contributed to rice vegetative growth and grain yield in parallel, with the previously mentioned single exception where a N-fertilization dose above the recommended resulted in detrimental lodging with decreased grain yield (Yanni et al. 1997).

10.7.5 Inoculation and N Economy in the Rice Agroecosystem

As anticipated, the agronomic N-use efficiency (kg grain yield/kg fertilizer N) decreased sharply with increased applications of fertilizer-N for both the inoculated 10 Beneficial Endophytic Rhizobia as Biofertilizer 283 and noninoculated treatments in all the 24 field experiments, with the significant mean differences between inoculated and noninoculated treatments (over the three N-fertilizer doses) occurring in 23 of the trials (Table 10.5). Its values were significantly higher for inoculated treatments in 18 (75%) of the 24 field experiments,

Table 10.5 Influence of Rhizobium inoculation on rice agronomic N-use efficiency (kg grain yield/kg fertilizer-N input) at 24 locations in the Nile delta Expt. N Non- Inoculated Mean Expt. N Non- Inoculated Mean no. (Kg/ha) inoculated no. (Kg/ha) inoculated control control 1 48 152 180 166a 13 48 197 210 204a 96 81 92 87b 96 114 121 118b 144 61 65 63c 144 78 84 81c 2 48 145 170 158a 14 48 200 216 208a 96 67 90 79b 96 107 112 110b 144 48 58 53c 144 75 79 77c 3 48 203 213 208a 15 48 175 189 182a 96 111 118 115b 96 96 101 99b 144 73 76 75c 144 68 72 70c 4 48 210 229 220a 16 48 191 205 198a 96 120 129 125b 96 103 108 106b 144 86 86 86c 144 70 73 72c 5 48 195 205 200a 17 48 190 205 198a 96 108 105 107b 96 104 116 110b 144 77 71 74c 144 77 83 80c 6 48 255 263 259a 18 48 194 205 200a 96 125 133 129b 96 106 118 112b 144 86 91 89c 144 78 86 82c 7 48 259 267 263a 19 48 176 181 179a 96 127 131 129b 96 95 102 99b 144 78 85 82b 144 69 72 71c 8 48 150 173 162a 20 48 185 193 189a 96 86 97 92b 96 101 111 106b 144 62 70 32c 144 74 80 77c 9 48 223 241 232a 21 48 173 194 184a 96 124 129 127b 96 98 108 103b 144 86 89 88c 144 72 74 73c 10 48 193 181 187a 22 48 164 181 173a 96 99 98 99b 96 90 103 97b 144 66 73 70c 144 67 70 69c 11 48 205 210 208a 23 48 171 179 175a 96 110 116 113b 96 92 101 97b 144 76 77 77c 144 68 70 69c 12 48 196 211 204a 24 48 165 170 168a 96 109 117 113b 96 88 98 93b 144 78 85 82c 144 67 72 70c Details of the Rhizobium leguminosarum bv. trifolii strains, rice cultivars, cultivation season and location are provided in Table 10.1. Means superscripted by different letters in the same column (for N-fertilization rates) or highlighted in bold (for inoculation) for each experiment are statistically different at the 95% confidence level (assessed by least significant differences between corresponding means) 284 Y.G. Yanni et al. and by extrapolation, the grain yield of rice inoculated with rhizobia plus intermediate doses of fertilizer-N was close or equal to that obtained with the full fertilizer-N dose without inoculation. The agronomic fertilizer N-use efficiency is an important metric that helps to evaluate the mineral fertilization strategy for field crops and reflects their agronomical, economical and environmental efficiencies in using that resource input for grain production. It is not intended to represent the absolute values of N-use efficiency since computation of that physiological parameter requires 15N analyses to measure how much of the whole plant’s nitrogen (including roots, shoots and grain) was derived from the applied chemical fertilizer. Its values in most experiments indicated that rhizobial inoculation can benefit grain production by counter-balancing the tradeoff in reduced agronomic use efficiency of N fertilizer with increasing fertilizer-N doses, and provided the desired result that rhizobial inoculation can reduce the need for fertilizer-N application to achieve higher grain yield.

10.8 Comparisons of Rhizobia with Other Biofertilizer Candidates for Rice

Previous field inoculation studies on rice have used N2-fixing cyanobacteria, Azospirillum and Azotobacter as biofertilizer inoculants (Arora 1969; Gupta et al. 1989; Jack and Roger 1977; Omar et al. 1993; Rajarmamohan et al. 1978; Roger and Kulasooriya 1980; Shahaby et al. 1993; Subrahmanyan et al. 1965; Venkataraman 1966; Yanni and Abdallah 1990; Yanni 1991; Yanni and Abd El-Rahman 1993; Yanni and Hegazy 1990; Yanni and Osman 1990; Yanni et al. 1996). Like with rhizobia, fertilizer-N supplements are required to obtain maximum grain yields when rice is inoculated with cyanobacteria, Azospirillum and Azotobacter (Yanni and Abd El-Fattah 1999 and references therein). However, the performance of rhizobial inoculants differs from those other plant growth-promotive rhizobacteria in two major ways. First, the appropriate rhizobia is expected to benefit both the legume and the cereal host as in the rice–berseem clover crop rotation under which this research program was performed, whereas the other inoculants are only used for the cereal crop. In Egypt, 67% of rice is cultivated in rotation with berseem clover, and so that rotation can help to perpetuate the populations of clover rhizobia involved in both plant–microbe interactions. Second, the benefits of the rhizobial inoculants to rice occur at each stage of its development, beginning with seed germination and extending through grain maturity (Biswas et al. 2000a, b; Dazzo and Yanni 2006; Dazzo et al. 1999; Yanni et al. 1997, 2001), whereas the benefits to rice provided by the other biofertilizer inoculants are expressed only during its maximum “N-limitation stress period” when an external source of N is required at the tillering stage which extends between 15 and 40 days post transplanting (Yanni and Abd El-Fattah 1999 and references herein). 10 Beneficial Endophytic Rhizobia as Biofertilizer 285

10.9 Biosafety, a Pertinent Issue in the Biofertilization Technology

It is very important to test the biosafety of the inoculum strain(s) to assure that they do not have any adverse effect(s) to any of the crop plant species scheduled for rotation in the field. In earlier gnotobiotic culture studies (Yanni et al. 1997, 2001), we found that some natural rice endophytic strains of clover rhizobia were active root nodulators and efficient N-fixers on berseem clover but were inhibitory to rice growth, whereas other natural rice endophytic strains of rhizobia were efficient PGPR+ on rice but lethal pathogens on berseem clover. Since all these beneficial, neutral and harmful types of endophytic rhizobia–plant interactions exist in nature, it is important to screen for and identify possible detrimental combinations before- hand to avoid catastrophic inoculation outcomes under field conditions. All of the seven strains of rhizobia used in this experimental program were pretested under þ þ gnotobiotic conditions and found to be Nod and Fix on berseem clover and PGPRþ on rice (Yanni et al. 2001).

10.10 Conclusions and Recommendations Derived from Field Inoculation Studies

This translational research can serve as a successful example of how deployment of well-selected biofertilizers represents an integral component of sustainable nutrient management for rice production and can help farmers grow better, more successful rice crops in an environmentally safe way when soil fertility (especially available N) is a limiting factor. Thus, in addition to its well-known use as a micro-partner in the N2-fixing root-nodule symbiosis with legumes, rhizobia deserve serious consideration as a microbial biofertilizer that can significantly enhance rice production. The benefit provided by the rhizobial biofertilizer is enhanced rather than suppressed with N-fertilizer supplementation, consistent with a mechanism that involved bacterial induction of an increased ability of the plant to sequester and utilize N from the available soil N pool rather than from de novo biological nitrogen fixation. However, cases where inoculation increased grain production despite no statistically significant increase obtained in response to the maximum recommended dose of fertilizer-N indicate that the benefits of rhizobial inoculation on rice grain production extended beyond its alleviation of N-limitation (Yanni et al. 2010). These translational studies indicate that use of these environmentally friendly biofertilizer inoculants can now be recommended with sufficient supporting data to improve agriculture economy and sustainable agro-ecosystem maintenance of rice grain production where the benefits of such biotechnology are most urgently needed. However, because rice production often remains “N-responsive” even when combined with rhizobial inoculation, further efforts must be exerted to reach the ultimate goal of fully eliminating the need for fertilizer-N inputs to obtain maximum rice 286 Y.G. Yanni et al. yields. Perhaps the solution to achieve that goal is a critically formulated biofertilizer containing a mixed inoculant consortia that includes our best-performing rhizobial strains plus highly selected free-living N-fixers like cyanobacteria, “associative” diazotrophs like azospirilla, and other plant-growth promoting rhizobacteria like Pseudomonas. We are currently pursuing this hypothesis.

10.11 Spatial Ecology of Rice–Rhizobia Interactions

The strain-variety specificity exhibited by endophytic rhizobial strains on rice suggests that components of compatibility may influence the ability of these bacteria–plant associations to express beneficial growth promotion. This has prompted us to consider the spatial ecology of candidate inoculant strains within various agroecosystems of the Egyptian Nile delta to fully exploit the benefits of biofertilization for sustainable agriculture. A thorough understanding of their spatial ecology would reflect their colonization potential and behavior, should assist our biofertilization strategy program in predicting how necessary and effective will inoculation be to enhance cereal growth and performance in a specific location area, and should be useful in the interpretations of the field inoculation responses. For instance, a marginal yield gain by inoculation is expected to occur in field test sites where the same strain is already indigenous and abundant, as evidenced by the work of Bashan (1998) and Aeron et al. (2010). We, therefore, are performing a thorough investigation of the autecological biogeography of a top candidate of R. leguminosarum bv. trifolii biofertilizer (strain E11). This is being done at macro and micro spatial scales, the first throughout the main rice-cultivation regions of Egypt at the kilometer scale, and the second on patterns of its colonization on rice roots at the micrometer scale.

10.11.1 Distribution of a Candidate Rhizobial Inoculant Strain in Rice-Cultivation Fields in Egypt

The distribution of the inoculant strain is being mapped in numerous rice–clover field sites scattered throughout the Nile Delta, plus other sites in Al-Fayoum, Egypt located southwest of Cairo where cereals are cultivated in a fertile area surrounded by desert (Fig. 10.5). For these studies, we have prepared a polyclonal antibody that exhibits strain specificity and high immunoreactivity with surface antigens of the test strain (Fig. 10.6). This antibody is being used to detect the strain within many nodules collected from roots of noninoculated (naturally nodulated) berseem clover sampled at the various sampled field sites. As the genotype diversity of rhizobia may differ when isolated directly from soil and from legume nodules, we are using the legume trap approach since the nodules 10 Beneficial Endophytic Rhizobia as Biofertilizer 287

Fig. 10.5 Approximate location of 42 sites (white circular symbols) sampled for spatial distribution analysis of a candidate R. leguminosarum bv. trifolii inoculant strain in a satellite image of the northeastern region (including the Nile delta) of Egypt and in Al-Fayoum, southeast of Cairo. The equivalent rectangular area is indicated within the boxed insert of Egypt

Fig. 10.6 Positive immunoﬂuorescence reaction with cells of the biofertilizer strain used in our autecological biogeography studies

contain large rhizobial populations that can easily be sampled directly in the field, preserved antigenically, transported intercontinentally, and analyzed without a large background community of highly diverse microbes. The added advantage of this approach is that only isolates relevant to agriculture will be selected, even when present in low numbers if they are competent nodulators. For these macro-scale field ecology studies, we introduced an “autecological biogeography index” that is admirably suited for use as the Z-variate in geostatistical analysis of its spatial abundance (Dazzo et al. 2003). This metric ranges on a scale between 0 and 4, and takes into account the number of nodules sampled, the proportion of sampled nodules at each location that have rhizobial occupants that immunoreact with the antiserum, and the relative intensity of immunofluorescent brightness of the bacterial cells indicative of their degree of antigenic relatedness to the targeted rhizobial strain against which the antibody was prepared (Dazzo et al. 2003). 288 Y.G. Yanni et al.

Results obtained so far indicate that the spatial abundance of the test strain is clustered rather than uniformly distributed throughout this region, with an autecological biogeography index ranging from 0.22 to 1.41 and a mean std. dev. of 0.81 0.31. These Z-variate data will be used in geostatistical analyses to map the abundance of the test strain interpolated over the entire spatial domain, even at sites not sampled. We plan to evaluate how correlated is its spatial abundance with other environmental variables (e.g., soil salinity) predicted to inﬂuence the distribution of the test strain in the area under examination.

10.11.2 Spatial Analysis of Rhizobial Colonization on Rice Roots

The second spatial analysis is designed to operate at single cell resolution and is directly relevant to bacteria–bacteria and bacteria–plant interactions where spatial patterns of rhizobial colonization of rice roots are defined in situ. For this study, we are developing a suite of computer-assisted microscopy and digital image analysis software to analyze the spatial patterns of root colonization by inoculant strains. The software is named CMEIAS [for Center for Microbial Ecology Image Analysis System;(http://cme.msu.edu/cmeias)]. Recently, we released a component of CMEIAS designed to alleviate the laborious task of editing images so the foreground objects are separated from background, especially in complex color micrographs of microorganisms in environmental samples (Gross et al. 2010). That CMEIAS application is useful for segmenting images of immunofluorescence microscopy when the bacteria of interest are surrounded by background fluorescence of similar color. We using powerful point-pattern and geostatistical techniques to analyze and quantitatively model the spatial distributions of the attached bacteria on plant roots and the in situ spatial scale of microbe–microbe interactions that influence their colonization behavior over the entire spatial domain. For example, the scanning electron micrograph of Fig. 10.7a is an image quadrat that illustrates the colonization pattern of the targeted rhizobial strain on the rice root surface and its portal of “crack entry” to breach the epidermal barrier and gain access into the root interior as an endophyte. Figure 10.7b, c is spatial point-pattern data extracted by a CMEIAS digital analysis of the electron micrograph image showing that the bacterial colonization pattern has a clustered rather than random or uniform distribution on the root surface. Local clustering of bacteria is the most common distribution of their colonization on roots. Such interactions are best examined using quantitative geostatistical methods that test georeferenced data for spatial autocorrelation. Patterns displaying spatial autocorrelation indicate that operations involve a spatial process rather than occur independently. From the microbial ecology perspective, spatial colonization patterns that are autocorrelated imply a colonization behavior involving cell-to-cell interactions that affect their distribution over the spatial domain. To take advantage of this powerful statistical approach, we developed a CMEIAS cluster index to serve as a Z variate for the geostatistical analysis of root colonization (Dazzo et al. 2003). That spatial parameter represents the locally clustered density (increased proximity) of 10 Beneficial Endophytic Rhizobia as Biofertilizer 289 a b

c d 59.1

Cluster Index m) 40.2 1.00 u 0.94 0.89 0.83 0.77 0.72 0.66 0.60 0.55 21.2 0.49 0.43 Y coordinate ( 0.38 0.32 0.26 0.21 0.15 2.3

2.4 27.6 52.8 78.0 X coordinate (um)

Fig. 10.7 (a–d) Image quadrat of rhizobial colonization of the rice root and CMEIAS image analysis of its spatially clustered distribution. (a) Scanning electron micrograph showing bacterial cells colonized on the root surface and their “crack entry” through open gaps between epidermal cells into the root interior. Bar scale is 10 mm. (b) Ghat empirical distribution of nearest neighbor attributes, and (c) separation distances between each bacterium and its first and second nearest bacterial neighbors indicating clustered patterns. (d) Geostatistical kriging map of the autocorrelated strength of bacterial cell clustering over the same spatial domain. Note the high intensity of bacterial clustering behavior at their portal of entry into the root bacteria over the spatial domain of the substratum surface upon which they have colonized, all analyzed at single-cell resolution. CMEIAS computes this cluster index as the inverse of the separation distance between each bacterial cell and its nearest cell neighbor, and its intensity is designed to represent the degree of cell-to- cell interactions that influence their colonization by growth in situ (Dazzo et al. 2003). Validation of this concept is supported by recent geostatistical studies that used fluorescent reporter strains and CMEIAS image analysis to produce direct evidence indicating that positioning of cells within gradients of signal molecules (hence, cell proximity) strongly influences bacterial cell-to-cell communication during colonization of plant roots (Gantner et al. 2006; Gross et al. 2010). Geosta- tistical analysis of cell-positioning data tests whether bacterial colonization has spatial dependence, and if so, it can then (1) quantitatively define the spatial scale of separation distance at which bacterial interactions influence their spatial distribution, (2) predict the most probable pattern of colonization behavior, and (3) produce a statistically 290 Y.G. Yanni et al. defendable interpolation (kriging) map of the continuous spatial distributions of each organism’s influence on colonization by its nearest bacterial neighbors. The geostatistical analysis of this image quadrat (Fig. 10.7a) provided unambigu- ous evidence of spatial dependence in bacterial distribution colonized on the root surface. The semivariogram fitted to the geostatistical cluster index data (figure not shown) indicated a spherical isotropic model with strong autocorrelation (r2 of 0.949), indicating that individual bacterial cells influence each other’s spatial distribution of colonization over a range of separation distances up to 10.6 mm. The spherical model often is the best for spatial distribution with patches of larger/smaller values of approximately similar size. The average diameter of the clusters commonly corresponds to the spatial correlation range. The corresponding two-dimensional kriging interpolation map (Fig. 10.7d, pseudocolored with higher resolution in the online version) depicts the strength of autocorrelated, spatially dependent clustering by individual bacterial cells colonized on the root surface. This interpolation map clearly shows the increased strength of bacterial clustering (with predicted cell-to-cell interactions affecting their colonization) in the region where they are in the process of breaching the host barrier during crack entry between the root epidermal cells. In another related study, we collaborated with colleagues at the National Acad- emy of Plant Sciences in Beijing, China to analyze the colonization potential of rhizobia on the whole rice plant. Various wild type species of rhizobia were genetically tagged with the green fluorescent protein (Gfp reporter), then inoculated on rice roots and examined for colonization of rice plants in gnotobiotic culture and in potted soil. Laser scanning confocal microscopy indicated that the rhizobia colonize the root surface (especially at lateral root emergence, consistent with earlier studies of Reddy et al. 1997), then invade the root interior, and ascend in migration up into the stem and lower leaves (Chi et al. 2005). CMEIAS analysis of the confocal image stacks indicated that endophytic populations of the rhizobial inoculants reached local densities as high as 1010 bacterial cells per cubic centimeter of plant tissue. Those density values are extremely high and obtainable by direct methods of CMEIAS computer-assisted microscopy that exclude uninfected plant tissues to achieve a very high signal-to-noise ratio. Further biochemical analysis of the inoculated plants indicated elevated levels of several phytohormone growth regulators, providing important information on the physiological response of rice to endophytic rhizobia. This study was significant since it indicated that the rhizobia– rice association is significantly more invasive than originally thought, initially an epiphytic colonization of the root surface followed by endophytic colonization of both below-ground and above-ground plant tissues.

10.12 Concluding Statements

It is clear from the above summary of laboratory and field work conducted on a collaborative multinational scale that the natural association between Rhizobium and rice is intimate, highly evolved, widespread worldwide, exhibits compatibility 10 Beneficial Endophytic Rhizobia as Biofertilizer 291 and specificity traits, and involves invasive interactions that impact on plant growth physiology. It serves as an interesting experimental model of a beneficial plant–microbe interaction that can be exploited to increase grain yield with less N-fertilizer inputs. With proper laboratory and field testing, it can be optimized to carry biosafe and proven performance characteristics that are fully compatible with sustainable agriculture and production economy.

Acknowledgments This work was supported by projects BIO2-001-017, BIO5-001-015, and BIO10-001-011 of the US-Egypt Science and Technology Joint Fund. We thank the numerous rice farmers and ﬁeld experimentation and technology transfer specialists for their cooperation in this study, and to colleagues who contributed to the development of CMEIAS image analysis software.

References

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Clara Pliego, Faina Kamilova, and Ben Lugtenberg

11.1 Introduction

The rhizosphere was deﬁned by Hiltner (1904) as “the soil compartment inﬂuenced by the roots of growing plants.” Scientists agree that this area is not more than a few millimeters thick. When bacteria are present on the root plane or near the plant root we call them rhizosphere bacteria or rhizobacteria. When present on the leaf or inside the plant tissues we speak about phyllosphere bacteria and endophytes, respectively. When they colonize the rhizosphere they can be present on the root surface (the “rhizoplane”) or close to the rhizosphere. The bacteria Rhizobium and Bradyrhizobium can be present in special organs, so-called root nodules (Spaink et al. 1998). In comparison to bulk soil, the rhizosphere is rich in nutrients because of rhizodeposition. Rhizodeposits consist of the total carbon transferred from the plant root to the soil. They consist of small molecules as well as of macromolecules including enzymes, lysates from dead cells, and mucilage. The pH of this exudate is around 5.5 (Kamilova et al. 2006b). The result of this richness in nutrients is that the

C. Pliego Instituto de Hortofruticultura Subtropical y Mediterra´nea “La Mayora”, Universidad de Ma´laga - Consejo Superior de Investigaciones Cientı´ﬁcas (IHSM-UMA-CSIC), A´ rea de Gene´tica, Universidad de Ma´laga, Campus de Teatinos s/n, 29071 Ma´laga, Spain and Present address: Imperial College London. Div. of Biology, Department of Life Science, Imperial College Road, London SW7 2AZ, UK e-mail: [email protected], [email protected] F. Kamilova Koppert Biological Systems, Veilingweg 14, PO Box 155, 2650 AD Berkel en Rodenrijs, the Netherlands e-mail: [email protected] B. Lugtenberg (*) Leiden University, Institute of Biology, Sylvius Laboratory, Sylviusweg 72, PO Box 9505, 2300 RA Leiden, the Netherlands e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 295 DOI 10.1007/978-3-642-18357-7_11, # Springer-Verlag Berlin Heidelberg 2011 296 C. Pliego et al. bacterial concentration in the rhizosphere is 100- to 1,000-fold higher than that in bulk soil. Bacteria that are introduced in the rhizosphere should be able to cope with an environment which contains other microbes (bacteria, fungi, and viruses) and predators such as nematodes and protozoa. Also, the amount of available water changes, especially in case of irrigation, rain, and drought. When studying the behavior of a microbe in the rhizosphere, one should realize that exudate collected from a gnotobiotic rhizosphere allows bacteria to grow to only a 100-fold lower cell concentration than a laboratory medium, meaning that microbes in the rhizosphere are often in a state of nutrient starvation. In horticulture, plants are not only grown in soil but also in various other substrates such as cocopeat, stonewool, perlite, and vermiculite.

11.2 Organisms in the Rhizosphere

The rhizosphere represents a highly dynamic site for interactions between roots, pathogenic and beneficial soil microbes, invertebrates, and other competitors of root systems. Interactions between organisms on the root may be classified as positive, neutral, or negative associations. Positive interactions, which can contribute to plant growth promotion and biological control of plant diseases, include symbiotic associations with epiphytes, mycorrhizal fungi, and root colonization by biocontrol agents (BCAs). Negative interactions include competition or parasitism among plants, pathogenesis by bacteria or fungi, and by invertebrate herbivores. Plant–microbe interactions in the rhizosphere are responsible for a number of intrinsic processes such as carbon sequestration, ecosystem functioning, and nutrient cycling. The composition and quantity of microbes in the soil influence the ability of a plant to obtain nitrogen and other nutrients. On the other hand, plants can influence these net ecosystems changes through release of secondary metabolites that attract or inhibit the growth of specific microorganisms (Lugtenberg and Bloemberg 2004; Vivanco et al. 2004). In addition, the rhizosphere is subject to dramatic changes on a short temporal scale, which can be caused by fluctuations in salt concentration, pH, osmotic potential, water potential, and soil particle structure. Over longer temporal scales, the rhizosphere can change due to root growth and interactions with other soil biota. Various bacterial species have adapted to the ever-changing conditions of the rhizosphere and are capable of starting root colonization by multiplication and eventually by forming microcolonies on different parts of the roots, from tip to elongation zone. Such microcolonies eventually grow on roots to form mature biofilms (Bloemberg and Lugtenberg 2004; Rudrappa et al. 2008). Nonpathogenic plant growth-promoting rhizobacteria (PGPR) associated with plant root surfaces are known to contribute toward increases in plant quality and yield by mechanisms such as improved mineral uptake, phytohormone production (Biswas et al. 2000; Ryu et al. 2003; Ryu et al. 2004; Fujishige et al. 2006), competitive suppression of 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 297 pathogens by production of antibiotics (Chin-A-Woeng et al. 1998; Keel et al. 1992; Haas and De´fago 2005), and induction of secondary metabolite-mediated systemic resistance (Ryu et al. 2004; Van Loon 2007). Besides, bacterial biofilms established on plant roots can protect the colonization sites and act as a sink for the nutrients in the rhizosphere, thereby reducing the availability of nutritional elements in root exudates required for pathogen stimulation or subsequent root colonization (Weller and Thomashow 1994; Kamilova et al. 2005). In addition to the interaction with the plant, beneficial microorganisms interact and compete with the endogenous microflora, consisting of other bacteria, fungi, and/or mycorrhizal fungi. It is the dynamic nature of the rhizosphere that makes it an interesting setting for the interactions that lead to plant growth promotion and biocontrol of the disease. Significant biological control most generally arises from manipulating mutualisms between microbes and their plant hosts or from manipulating antagonisms between beneficial microbes and pathogens. To do this successfully, we need to know the players and understand their interactions with each other and with the growth substrate. Moreover, the effect of abiotic factors should be taken into account. The analysis of these interactions requires a strong interdis- ciplinary approach and an extensive set of tools.

11.3 Nutrients in the Rhizosphere

Compounds released by the plant root vary in relative and absolute amounts. The composition is influenced by the plant species, plant cultivar and age, and environmental properties such as the presence of microbes and some of their products, the growth substrate, and the levels of physical, biological, and chemical stresses. It should be realized that an exudate composition usually reflects the net result of secretion, conversion by enzymes, and uptake by microbes and the plant root. An excellent monograph on the rhizosphere was published recently (Pinton et al. 2007). The bulk of root products are carbon compounds derived from products of photosynthesis. An excellent overview of exudate compounds is written by Uren (2007). Organic compounds released by the root include organic acids, sugars and polysaccharides, amino acids, fatty acids, sterols, vitamins, enzymes, and nucleotides. Moreover, compounds as diverse as auxins, ethanol, flavonoids, and the osmoprotec- tant glycinebetaine are secreted. In many cases, compounds are only detected because one screens for their presence. Exudate compounds can also be detected accidentally. Kuiper et al. (2001a) studied a competitive colonization mutant and the identification of the knocked out gene suggested that putrescine could be an exudate compound. Subsequent tests confirmed this notion. The rhizosphere is also an important place for microbe–plant and microbe–microbe signaling (Perry et al. 2007). Rhizobacteria (Kamilova et al. 2006b), fungi (Meharg and Killham 1995), and rhizobacterial products such as phenazine, 2,4 diacetylphloroglucinol and zearale- none (Phillips et al. 2004), as well as the growth substrate (Kamilova et al. 2006a; Lipton et al. 1987), can influence the exudate composition. 298 C. Pliego et al.

Extensive studies have been carried out on the exudate composition of gnoto- biotically grown tomato, cucumber, and sweet pepper. For all three crops, the organic acid fraction was larger than the carbohydrate fraction which, in turn, was larger than the amino acid fraction (Kamilova et al. 2006a). The observation that a mutant of Pseudomonas unable to utilize organic acids is a poor colonizer (De Weert et al. 2007) but a mutant in sugar utilization can colonize the tomato root normally (Lugtenberg et al. 2001) is consistent with the notion that organic acids are a major carbon source. The presence of a disease-causing amount of the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici in the gnotobiotic tomato system resulted in a decrease of the amount of citric acid and an increase in the amount of succinic acid in such a way that the total amount of organic acid was not influenced. In contrast, when the Pseudomonas fluorescens biocontrol strain WCS365 was added to the gnotobiotic tomato system in amounts sufficient to cause biocontrol, a strong increase in the total amount of organic acid, especially of citric acid, but a dramatic decrease in the amount of another organic acid, succinic acid, was found. When both microbes were present under biocontrol conditions, the amounts of succinic acid decreased but the amount of citric acid was similar to that of the untreated control. Under all three conditions, the amounts of sugars were reduced by around 50% (Kamilova et al. 2006b). In contrast to the three dicots cucumber, sweet pepper, and tomato, the monocot grass produces equal amounts of organic acids and sugars in its root exudate (Kuiper et al. 2002). Whether this difference reflects a fundamental difference in exudate composition between monocots and dicots remains to be established. Although root exudates play a crucial role in plant growth and health, studies on composition, the influence of environmental factors on composition, and function of root exudates have hardly been performed in the past 4 decades. Such studies should be considered as a challenge for future research.

11.4 Root Colonization

Root colonization is crucial for the application of beneﬁcial bacteria. Recently, technologies for following root colonization have been developed and traits and genes involved in colonization have been identiﬁed.

11.4.1 Recently Developed Technologies for Studying Interactions in the Rhizosphere

Until recently, most research on biological control has focused on the interactions between two trophic levels: the BCA and the pathogen. However, multitrophic interactions among organisms belonging to three or more trophic levels are increasingly gaining the attention of microbial ecologists and plant pathologists, driven by 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 299 the need for improving biological control during the management of crop diseases. As we have mentioned previously, successful biocontrol operates at the population level, not at the level of an individual organism. Consequently, a detailed understanding of species interactions across at least three levels, the plant, the pathogen, and the BCA, seems to be necessary (Pliego et al. 2008). During the last years, the development of technologies such as the bioreporter technology has dramatically influenced our knowledge of biocontrol in the rhizosphere. Bioreporters can be classified into three categories (Lugtenberg and Leveau 2007). One class of bioreporters are those that allow direct visualization of individual organisms in vivo. The most common reporter used for this purpose is the green fluorescent protein (GFP), which does not depend on cofactors or additional substrates for activity. In combination with a constitutive promoter, the gfp gene is expressed independently of environmental factors and accumulation of its product renders the cells green fluorescent and detectable by fluorescence microscopy or confocal laser-scanning microscopy (CLSM). This has been exploited to study the direct visualization of the interactions established among various organisms, i.e., BCAs and pathogenic fungi. The second class allows monitoring the general well-being or activity of BCAs in the rhizosphere. Some groups have used the lux genes, which only results in bioluminescence when the cells are metabolically active, to show that the metabolic activity of Pseudomonas inoculants is higher in rhizosphere soil than in bulk soil and that metabolic activity decreases over time after introduction of the inoculated BCA (Porteous et al. 2000). The third group of bioreporters is aimed at providing information on specific activities of BCAs in the rhizosphere. The expression of the reporter gene is driven by promoter sequences that are selected based on their biological function. Besides gfp and lux, other reporter genes can be used for this, including lacZ, xylE, gusA, and inaZ (Loper and Lindow 1997). Finally, Transposon-Based-Strategies (TSBs) and the development of functional genomic techniques such as In Vivo Expression Technology (IVET), Signature Tagged Mutagenesis (STM), and DNA microarrays allow the identification of microbial genes potentially involved in biocontrol. Application of these techniques is largely increasing our knowledge of the molecular mechanisms involved in the interactions established among different trophic levels.

11.4.2 Autoﬂuorescent Proteins (AFPs)

Microscopic visualization of AFP-tagged BCAs in their natural environment during their interaction with plants and/or with target pathogens provides crucial information about their functioning and about the possible successful application of commercial inoculants. The GFP is the most widely studied AFP but the number of other proteins and variants with different ﬂuorescence characteristics has increased. This also applies for the development of improved variants of existing AFPs. Important GFP derivatives are Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP) (Yang et al. 1998; Tsien 1998; Matus 1999; Ellenberg et al. 1999). 300 C. Pliego et al.

A wide range of autofluorescent proteins with useful information is available on the Web site of the Clontech company (http://www.clontech.com). Taking into account that genes encoding AFPs have to be transformed into the organism of interest, the use of these markers is dependent on whether appropriate vectors and transformation protocols are available for a specific species or strain. In bacteria, AFP genes are usually delivered using plasmids or transposons (Bloemberg 2007). The advantage of a plasmid is that it can be present in multiple copies, which can improve the level of AFP molecules and does not disrupt hosts genes by chromosomal integration (Bloemberg 2007). In recent years, AFP technology in combination with CLSM has become an important tool for the analysis of processes such as microbe–plant interactions, biosensor design, biofilm formation, and horizontal gene transfer (Larrainzar et al. 2005). Besides, the ability to use AFPs with different fluorescent spectra allows the simultaneous visualization of different species and populations at the same time. The use of red fluorescent protein (RFP), isolated from Discosoma striata,in combination with eGFP, is very suitable as the excitation and emission spectra of these proteins are well separated (Matz et al. 1999). Because of the functional limitations of DsRed such as slow maturation and low photostability, new and improved variants have recently been produced, of which mCherry is one of the best (Shaner et al. 2004). For example, Lagendijk et al. (2010) were able to distinguish simultaneously in the tomato rhizosphere the presence of mixed microcolonies originated from two different populations of P. putida PCL1445 cells tagged with EGFP and mCherry. Most of the studies using AFP as markers for BCAs have been directed to their localization in the rhizosphere. These studies have shown that individual bacteria grow out to microcolonies and form biofilms, usually at junctions between epidermal root cells and at sites where side roots emerge. These are supposed to be microhabitats with enhanced exudation, humidity, and mucigel (Lugtenberg and Bloemberg 2004). Studies using mixed populations of two Pseudomonas species, P. chlororaphis PCL1391 and P. fluorescens WCS365, revealed that mixed colonies were formed and are present mostly on the upper part of the root and that P. fluorescens WCS365 was found preferentially on root hairs (Lagopodi et al. 2002). These studies revealed that different bacteria can have a preference for colonizing different parts of the roots. The most significant studies related with the use of AFPs to study the interaction of Pseudomonas spp. with plant roots are given in Table 11.1. Epifluorescence studies also showed the close interaction established between rhizosphere fungi and bacteria. Using this technique, bacterial cells have been shown to attach and colonize the hyphae of beneficial and phytopathogenic fungi. Colonization of fungal hyphae by antagonistic bacteria has also been postulated to enhance biocontrol in combination with the bacterial production of antifungal metabolites such as antibiotics, chitinases, or proteases (Hogan and Kolter 2002; Bolwerk et al. 2003; Lagopodi et al. 2002). For example, the biocontrol strains Collimonas fungivorans, P. fluorescens WCS365, and P. chlororaphis PCL1391 have not only been shown to be excellent root colonizers but appeared 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 301

Table 11.1 Microscopic visualization of AFP-tagged Pseudomonas strains in the plant rhizo- spherea Pseudomonas strain System Analyzed trait Promoter:: Reference gfp-version

P. fluorescens WCS365 Tomato Localization (Plac: gfp) Bloemberg et al. (1997) P. chlororaphis Lotus Localization (PpsbA::gfp) Tombolini et al. (1997) P. fluorescens Barley Localization and (PA1/03/04: Normander et al. DR54-BN14 activity gfpmut3b) (1999) P. fluorescens WC5365 Tomato Localization (Plac::rfp;Plac:: Bloemberg et al. egfp;Plac::ecfp; (2000) Plac::eyfp) P. fluorescens SBW25 Wheat Metabolic activity (PpsbA-gfp luxAB) Unge and Jansson and localization (2001) P. putida Barley Activity (PrrnBP1::gfp Ramos et al. (2000); Localization [AGA]) Boldt et al. (2004) (PA1/03/04:gfpmut3 P. fluorescens F113 Alfalfa Localization (Plac:gfpS65T) Villacieros et al. (2003) P. fluorescens WCS365 Tomato Localization (Plac::rfp) Bolwerk et al. (2003) and P. chlororaphis PCL1391 P. brassicacearum Arabidopsis Distribution (PA1/03/04: Achouak et al. (2004) thaliana RBSIIgfpmut3) (PA1/03/04:RBSII- dsred) € P. putida Tomato Localization (PA1/03/04:gfpmut3) Gotz et al. 2006 P. fluorescens 32 Wheat Localization (Plac:gfp) Van Bruggen et al. (2007) P. fluorescens CHA0 Set of plant Gene expression (PphlA-gfp3) De Werra et al. (2008) species (PprnA-gfp3) P. pseudoalcaligenes Avocado Distribution (PA1/03/04:gfp) Pliego et al. (2008) and P. alcaligenes P. putida PCL1445 Tomato Localization (Ptac:gfp;Ptac: Lagendijk et al. (2010) mcherry) a PpsbA, Plac,Ptac,PA1/03/04 (a Plac-derivative) are constitutive promoters; PrrnBP1 is a ribosomal promoter; PphlA contains the promoter region of the genes encoding the antifungal metabolite 2,4-diacetylphloroglucinol; PprnA contains the promoter region of the genes encoding the antifungal metabolite pyrrolnitrin; gfp, egfp, gfpmut3b, gfpmut3, and gfpS65T are genes encoding stable versions of the GFP protein; gfp[AGA] is a gene encoding an unstable version of the GFP protein; dsred and rfp are genes encoding derivatives of the GFP protein emitting red fluorescence; yfp is a gene encoding a yellow fluorescent protein; luxAB genes encode luciferase from Vibrio fischeri also to be able to colonize the hyphae of the phytopathogenic fungus F. oxysporum f.sp. radicis lycopersici (Forl) (Lagopodi et al. 2002; Bolwerk et al. 2003; Kamilova et al. 2007). Furthermore, abundant colonization of hyphae of the fungus Rosellina necatrix, the causal agent of avocado white root rot, was observed for the biocontrol strain P. pseudoalcaligenes AVO110 (Pliego et al. 2008). Moreover, the latter strain also colonized profusely pyriform swellings, characteristic structures of R. necatrix hyphae that seem to be involved in chlamydospore formation (Pe´rez- Jimeńez et al. 2003). Since fungi are frequently part of the endogenous microflora and can be the direct target for the PGPR effect as in case of biological control, it is also of great 302 C. Pliego et al. relevance to tag fungi and study their interaction with the PGPR (Bloemberg 2007). Genetic transformation of filamentous fungi is usually more difficult and less developed than transformation of bacterial cells. However, GFP expression vectors have already been developed for all major classes of filamentous fungi: basidio- mycetes, ascomycetes, and oomycetes. The vector to be used depends on the fungus to be transformed. Transformation of filamentous fungi has traditionally been performed using protoplasts, e.g., F. oxysporum f.sp. radicis lycopersici has been tagged with different autofluorescent proteins by co-transformation of two plasmids, one of which contained a hygromycin resistance gene and the second an afp gene (Lagopodi et al. 2002; Bolwerk et al. 2003). Until now, several root-infecting fungal pathogens have been tagged with GFP to study developmental processes occurring during root infection, for example, Magnaporte grisea (Sesma and Osbourn 2004), Forl (Lagopodi et al. 2002), Fusarium verticillioides (Oren et al. 2003), and Rosellinia necatrix (Pliego et al. 2009). These studies have made significant contributions to the understanding of how BCAs function during their beneficial interactions with plants. For example, Lagopodi et al. (2002) observed that the biocontrol bacteria P. fluorescens WCS365 and P. chlororaphis PCL1391, applied to seedlings, and the pathogenic fungus Forl, applied in the soil, occupy the same sites on the tomato root mostly in the junctions between cells. Furthermore, the observation that the biocontrol strain occupies these sites faster that the pathogenic fungus presumably underlies a crucial aspect of the bacterium’s biocontrol ability (Bolwerk et al. 2003). More recently, CLSM studies revealed that the biocontrol strain P. pseudoalcaligenes AVO110 is able to control the avocado white root rot disease caused by R. necatrix by colonizing profusely the same sites as used by the pathogen to penetrate the root such as root wounds and intercellular spaces (Pliego et al. 2008).

11.4.3 Colonization Genes and Traits

One of the major challenges for researchers is to understand the role of bacteria in their natural environment. One way to do this is to identify genes that are specifically expressed in natural surroundings. As mentioned earlier, efficient protection of plant roots by BCAs firstly requires sufficient and competitive colonization of the rhizosphere. In this regard, most studies have been directed to the identification of bacterial genes involved in rhizosphere colonization. Two different approaches were used to identify traits involved in root colonization and rhizosphere competence for the model strain P. fluorescens WCS365, one of the best colonizers among biocontrol strains (Lugtenberg et al. 2001). These studies were facilitated by the development of a gnotobiotic system developed by Simons et al. (1996) which offers a sterile environment for comparing the wild type with various mutants. The first approach deals with bacterial genes predicted to have an effect on colonization. These genes can be disrupted by directed mutagenesis using homolo- gous recombination. Prior to studying their root colonizing abilities, growth rate 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 303 and other differences between mutant and wild type should be established. Using this approach, the importance of, for example, motility and chemotaxis (Lugtenberg and Bloemberg 2004) for root colonization was demonstrated. In addition, using cheA mutants of various P. fluorescens strains, De Weert et al. (2002) showed that the presence of flagella is not sufficient for competitive colonization but that chemotaxis in addition is essential. Motility is required for the chemotactic response of microbes toward exudates compounds as a first step in the process of root colonization (De Weert et al. 2007). Major chemoattractants in tomato root exudates for P. fluorescens WCS365 are dicarboxylic acids and amino acids. Based on concentrations estimated to be present in the tomato rhizosphere (Simons et al. 1997; Kravchenko et al. 2003), malic acid and citric acid were concluded to be the major chemoattractants. As a second approach, random transposon mutagenesis can be performed using transposons such as Tn5lacZ (Lam et al. 1990)orTn5luxAB (Wolk et al. 1991). Selected mutants can be studied individually for impaired root colonization (Simons et al. 1996). Applying of the transposon approach resulted in the discovery of relevant traits for rhizosphere colonization. An overview of these traits is presented in Table 11.2. Reviews of most of these traits can be found in Lugtenberg and Dekkers (1999), Lugtenberg et al. (2001), De Weert et al. (2007), and Lugtenberg and Kamilova (2009). Functional genomic techniques allow the simultaneous in vivo examination of the expression of many genes. These techniques can be divided into three broad

Table 11.2 Functions of Pseudomonas genes required for root colonization Function Gene References Motility Involved in biosynthesis of flagella De Weger et al. (1987) Pilus retraction pilT Camacho Carvajal (2001) Chemotaxis cheA De Weert et al. (2002) Cell wall structure rhs De Weert et al. (2007) Synthesis of O-antigen Not identified Dekkers et al. (1998) of LPS Outer membrane integrity Homolog to hrtB Dekkers et al. (1998) Permeability of outer colR/colS required for expression De Weert et al. (2006) membrane of wapQ Increased pyrimidine pyrR Camacho Carvajal biosynthesis (2001) Utilization of exudate Homolog of mqo encoding malate Lugtenberg et al. (2001) compounds dehidrogenase De Weert and Homolog of cis-aconitate hydratase Bloemberg (2007) Downregulation of Binding site for regulator protein Kuiper et al. (2001a) putrescine uptake Phase variation xerC/sss Dekkers et al. (1998) Proton motive force nuo operon Camacho Carvajal et al. (2002) Secretion pathways secB, homolog of hrcC, hrcD encoding De Weert et al. (2007) components of the TTSS Adaptation to certain Homolog of mutY De Weert et al. (2004a) environments 304 C. Pliego et al. classes (1) IVET, (2) STM, and (3) transcriptomics, i.e., global gene expression analysis using DNA microarrays. IVET technology is a promoter-trap strategy designed to identify genes whose expression is induced in a specific environment, typically that encountered in a host. This technique was, for example, used to identify genes of P. fluorescens specifically expressed within the sugar beet rhizosphere (Rainey 1999; Gal et al. 2003) and to analyze gene expression in P. putida during colonization of the maize rhizosphere (Ramos-Gonza´lez et al. 2005). Rhizosphere-induced fusions identified genes with probable functions in nutrient scavenging, chemotaxis and motility, intracellular metabolism, regulation, secretion, cell envelope structure, nucleic acid metabolism and stress. One of the most important findings was the discovery of the hrC gene, encoding a component of the Type Three Secretion System (TTSS) of P. fluorescens (Rainey 1999), suggesting that the TTSS has functional significance in both pathogens and PGPR. Although TTSS in P. fluorescens was demonstrated to be functional, a hrcC mutation did not affect root colonization capacity (Preston et al. 2001). On the other hand, De Weert et al. (2007) compared the competitive root-colonizing abilities of both hrcD and hrcD genes of the TTSS of P. fluorescens SBW25. It appeared that both mutants were impaired in competitive tomato root tip colonization but were not impaired when analyzed alone on the root. They hypothesized that the mutants are using the TTSS for either involvement in attachment to seed and/or root or for feeding on the root surface cells by means of the TTSS. Because no difference was found in attachment to seeds or roots, in competition with the wild type, they suggested that the TTSS is not only used to inject proteins into plant cells but also to suck nutrients from the plant cell (De Weert et al. 2007). These results support the finding that biocontrol activity of P. putida against Pythium ultimum on cucumber was lower that that of the wild type when the hrcC gene was mutated (Rezzonico et al. 2004). Interestingly, application of IVET technology also led to the identification of the P. fluorescens wssE gene, being part of the wss operon involved in the synthesis of acetylated cellulose polymers (Rainey and Preston, 2000; Gal et al. 2003). The wssE gene encodes a cellulose synthase subunit. Acetylated cellulose polymers are known to play a role in colony development and bacterial cell–cell contact, which are known to be involved in the initial steps of biofilm development (Spiers et al. 2003). Mutational analysis of the wss operon has shown that it contributes to ecological fitness in the rhizosphere. Besides, an increased expression of oxidative stress genes (glutathione peroxidases and a paraquat inducible protein) was observed in P. fluorescens for protection against oxidative stress encountered in the sugar beet rhizosphere (Gal et al. 2003). In the functional genomic technique STM, the use of DNA signature tags facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Besides, STM allows the identification of fitness or competition mutants, usually missed by standard mutagenesis approaches, which do not cause a total phenotype knock- out. This technique was recently applied in Burkholderia vietnamiensis strain G4 to identify genetic determinants involved in colonization of the pea rhizosphere and in 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 305 phenol degradation. Seventy mutants involved in rhizosphere colonization were mapped to genes with the following putative functions: amino acid biosynthesis (36%), general metabolism (26%), transport and stress (2.8%), regulatory genes (5.7%), and hypothetical proteins (13%) (O’Sullivan et al. 2007). The majority of the mutants were associated with amino acid biosynthesis and metabolism. Amino acids are known to be present in root exudates but at limiting concentrations and not readily accessible (Lugtenberg et al. 2001). Several amino acid auxotrophs were also found for P. fluorescens WCS365 among competitive colonization mutants. Even when tested alone, these mutants were unable to colonize the root tip (Lugtenberg and Bloemberg 2004). One of the most interesting discoveries mediated by the rhizosphere-STM screen was the identification of three mutants, inactivated within a single virulence- associated autotransporter adhesion gene (O’Sullivan et al. 2007). This mutation consistently produced a hypercolonization phenotype. They suggests that this massive surface-exposed protein (229–368 Da) masks part of the surface adhesins that are actually responsible for plant root cell binding, hence these protein adhesins would be more freely available for binding to plant cells in these three mutants. Alternatively, this adhesion could also play a role in the plant host response, in a way analogous to the way the known immunogenicity YadA homologs act in animal hosts (Cotter et al. 2005). Therefore, it may be possible that the plant is more tolerant to the adhesin mutants and allows them to colonize in greater numbers (O’Sullivan et al. 2007). Using a transposon mutant library of the efficient colonizer P. fluorescens WCS365 in combination with the enrichment procedure developed by Kuiper et al. (2001a), De Weert and co-workers (2004a) observed that competitive root tip colonization can be enhanced. The best colonizing mutant was shown to be mutated in a mutY homolog. This gene is involved in the repair of A:G mismatches caused by spontaneous oxidation of guanine. Since these mutants are defective in repairing their mismatches, it is hypothesized that such cells harbor an increased number of mutations and that some of these mutants, with a beneficial combination of mutations, can adapt faster to the environment of the root system. In order to investigate how Pseudomonas populations readjust their genetic program upon establishment of a mutualistic interaction with plants, Matilla et al. (2007) performed a genome-wide analysis of gene expression using microarrays of the root-colonizing bacterium P. putida KT2440. Genes involved in amino acid uptake and metabolism of aromatic compounds were found to be preferentially expressed in the rhizosphere (22%). It was also found that bacterial efflux pumps and enzymes related with glutathione metabolism were induced in the rhizosphere, indicating again that adaptation to adverse conditions and to oxidative stress is crucial for bacterial life in this environment. A gene encoding a protein containing GGDEF/EAL domains was identified among the induced genes. These domains are involved in c-di-GMP metabolism. Numerous studies have shown that c-di-GMP regulates biofilm formation and motility. Moreover, c-di-GMP also activates the production of extracellular polysaccharides (EPS), specifically those that serve as an extracellular matrix for formation and support of biofilm architecture 306 C. Pliego et al.

(Tamayo et al. 2007). Results shown by Matilla et al. (2007) suggest a role for the turnover of c-di-GMP in root colonization. Besides, genes encoding ﬂagellar proteins and chemotaxis proteins were also upregulated in the interaction of P. putida KT2440 with maize roots, conﬁrming a role of chemotaxis in root colonization.

11.5 Bacteria Which Can Promote Plant Growth Directly

Some beneﬁcial bacteria can promote plant growth in the absence of a pathogen. They will be discussed here.

11.5.1 Introduction

Some rhizobacteria are plant beneﬁcial and can promote plant growth directly, e.g., by providing the plant with nutrients or growth hormones, whereas others can promote plant growth indirectly, e.g., by decreasing the growth of pathogens. Direct plant growth promotion will be discussed in this section whereas indirect plant growth promotion will be treated in the next section. Direct plant growth promoters can be divided into four classes: biofertilizers, rhizoremediators, phytostimulators or plant growth regulators, and stress controllers.

11.5.2 Biofertilizers

Biofertilizers are best known for their ability to provide the plant root with nutrients such as nitrogen, phosphorous, and iron. Biological nitrogen fixation is an extremely energy intensive process. The reduction of one molecule of N2 catalyzed by the nitrogenase enzyme complex requires between 16 and 42 molecules of ATP. It is therefore not surprising that N2-fixing bacteria only reduce N2 when they are starved for nitrogen (De Bruijn et al. 1990). Rhizobium and Bradyrhizobium bacteria can induce special organs, so-called root nodules on the roots of leguminous plants in which they, in the form of modified bacteria called bacteroids, fix atmospheric nitrogen and convert it into ammonia which can be utilized by the plant as a nitrogen source. In return, the plant provides the bacterium with a carbon source, probably malate. Both rhizobia are economically important. Bradyrhizobium is important in agriculture as it can nodulate soybean. Rhizobium can nodulate crops such as pea, peanut, and alfalfa. Egamberdieva et al. (2010) recently showed that co-inoculation of fodder galega with Rhizobium and biocontrol pseudomonads improves shoot and root dry matter of the plant. One of these strains, the cellulose-producing P. trivialis strain 3Re27, 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 307 significantly increased nodule numbers and nitrogen content of the co-inoculated plant (Egamberdieva et al. 2010). The authors coined the term “rhizobium helper bacteria” for this biocontrol strain. Klebsiella pneumonia and Azospirillum are free-living nitrogen-fixing rhizosphere bacteria. In the past, the plant growth-promoting properties of Azospirillum were thought to be due to its N2-fixing property, but recent developments show that this property is mainly due to its ability to produce the root architecture influencing hormone auxin. Therefore, Azospirillum will be discussed under “Phytostimulators.” In some soils, phosphorous is the limiting factor for plant growth. Some bacteria are able to solubilize bound phosphorous from organic or inorganic molecules, thereby making it available for the plant (Lipton et al. 1987; Vassilev et al. 2006). Production of organic acids such as gluconic acid is a major factor in the release of phosphorous from mineral phosphate (Rodrı´guez et al. 2006). All organisms need iron ions for their metabolism. Iron in the form of ferric hydroxide is an abundant element on the earth crust but it is insoluble and therefore not suitable for uptake by living organisms. The concentration of Fe3+, the form of iron ions available for living organisms, is only 1018 M. Therefore, bacteria growing þ þ under low Fe3 concentrations produce a variety of siderophores, which are Fe3 chelators able to bind this ion with high affinity. Subsequently, the Fe3þ-siderophore complex is bound to specific iron starvation induced bacterial cell surface receptors and the Fe3þ ion is transported into the cell’s interior, where it, in the form of Fe2þ,is involved in metabolic processes (Neilands 1981;Leong1986). Competition for ferric iron ions is a well-documented example of competition of biocontrol bacteria with pathogenic fungi for nutrients (Leong 1986; Lugtenberg and Bloemberg 2004). In the rhizosphere, there is often a limitation of solubilized Fe3þ which, in many microorganisms, is solved by the production of siderophores, þ i.e., Fe3 -binding ligands, which have a high affinity to sequester iron from the microenvironment. The relevance of siderophore production as a mechanism of biological control of Erwinia carotovora by P. fluorescens strains was first described by Kloepper et al. (1980). As with antibiotics, mutants not producing siderophores such as pyoverdine showed a reduced capacity to suppress different plant pathogens (Keel et al. 1989; Loper and Buyer 1991; Duijff et al. 1994). This phenomenon is not only a form of plant fertilization but also of biocontrol (see Sect. 6.2.3).

11.5.3 Rhizoremediators

Pollution of soil and water is an enormous problem worldwide. In the USA alone, the costs of restoration of all contaminated sites are estimated at 1.7 trillion USDs. The major approaches to solve these problems are incineration and removal of land. However, these approaches are not safe. Incineration results in air pollution and leaches from landfills can reach ground water and drinking water wells. Microbes at a site that is being polluted adapt to this situation and start degrading the pollutants. However, the degradation rate is very low. Scientists have isolated the microbes 308 C. Pliego et al. responsible for this process. For most contaminants it is possible to find microbes which can degrade them. The application of such microbes has been tested for decades. However, strains which were successful in the laboratory failed in soil. Due to lack of nutrients they go in a starvation mode soon after application and stop degrading the pollutants. Apparently, they are unable to multiply on the pollutants as their only carbon source. In order to degrade the pollutant molecules they have to co-metabolize it together with a regular C source (Cases and De Lorenzo 2005). With the notion, that it is possible to find bacteria able to solve almost any problem, Kuiper et al. (2001b) selected bacteria which combine two properties, namely (1) to utilize root exudate – the best carbon source available in soil and (2) are also able to degrade pollutants. Grass was used as the exudate source because the roots of some grasses can grow as deep as 5 m into the soil. The polyaromatic hydrocarbon compound naphthalene was chosen as the model pollutant. Starting with the crude rhizosphere of the roots, presumably containing a mixture of hundreds of thousands different rhizobacteria, the mixture was grown first on naphthalene as the only carbon source. The naphthalene users were subsequently coated on grass seeds, which were allowed to grow until the roots were 10 cm in length. Subsequently, the bacteria residing on the last centimeter of the root tip, i.e., the best root colonizers, were collected. This cycle was repeated twice (See- Fig. 11.1 for the principle of the enrichment procedure). In this way, strain P. putida PCL 1444 was isolated (Kuiper et al. 2001b). This strain combines the following properties (1) stable degradation of naphthalene; (2) efficient utilization of grass root exudate; (3) protection of coated grass seeds from poisoning by naphthalene, resulting in healthy plants. Mutants unable to degrade naphthalene are inactive in this respect; (4) roots are able to move bacteria through layers through which bacteria normally cannot penetrate (Kuiper et al. 2001b; Kuiper et al. 2002; Kuiper et al. 2004). In addition to rhizosphere bacteria, endophytes are often used in rhizoremediation. Reports on the ability of several endophytic bacteria to degrade some pollutants (i.e., explosives, herbicides, or hydrocarbons) have been published (Van Aken et al. 2004; Germaine et al. 2006; Phillips et al. 2008; Segura et al., 2009). Also, endophytic bacteria resistant to high concentrations of heavy metals, benzene, toluene, ethylbenzene and xylenes (BTEX), trichloroethylene (TCE), or polyaromatic hydrocarbons (PAHs) have been identified (Moore et al. 2006; Doty 2008).

11.5.4 Phytostimulators/Plant Growth Promoters

The plant can produce phytohormones, i.e., compounds which can regulate plant growth and development. There are ﬁve classes of plant hormones, namely auxins, cytokinins, gibberellins (GAs), abscisic acid (ABA), and ethylene (ET). See Fig. 11.2 for structures. They are usually effective at concentrations lower than 1 mM (Garcı´a de Salome et al. 2006). In addition to plants, many rhizosphere 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 309

Fig. 11.1 Enrichment of bacteria which compete efﬁciently for nutrients and niches. The principle of the enrichment procedure was published by Kuiper et al. (2001b). Starting from a seed on which a crude mixture of rhizosphere bacteria has been applied, enhanced competitors are selected by repeatedly selecting those bacteria which reach the root tip ﬁrst after application to a sterile seed. Strains isolated from the root tip after three cycles are subsequently tested on antibiotic activities toward pathogens, which results in (1) strains which are not antagonistic and easy to register as a product (Kamilova et al. 2005) or (2) in strains which have at least two mechanisms of biocontrol, namely CNN and antibiosis (Pliego et al. 2007; Pliego et al. 2008)

bacteria can produce plant growth regulators. The ﬁrst three classes of plant hormones will be discussed under “phytostimulators/plant growth promoters” whereas the last two classes will be discussed under “stress controllers.” For details on hormones produced by plants and rhizosphere bacteria, the reader is referred to excellent reviews by Spaepen et al. (2009) and Garcı´a de Salome et al. (2006). 310 C. Pliego et al.

COOH C NH2 OH

N N N

CH3 Phenazine-1-carboxylate Phenazine-1-carboxamide Pyocyanin

O O OH NH

H C CH 3 3 HO OH

Cl O CL HO OH NO2 HN Cl Cl 2,4-Diacetylphloroglucinol Pyoluteorin Pyrrolnitrin CH3

CH2

O NH2 HO OH O H O H N O H H O N N N OH H H NH HN OH O O O CH O 2 HN O CH2 N Viscosinamide N H O H CH3 O OH 2-hexyl-5- propyl resorcinol

O N H

H O HCN AHL structure Hydrogen cyanide

Fig. 11.2 (Continued) 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 311

O OH

OH CO H CH2 HO

O COOH N H CH3

Gibberellins (GA3) Auxins (IAA)

H3C NH

CH CH3 H3C 3 N OH N

N N

H O CH3 O OH

Zeatin Abscisic acid

OH O OH

OH OH

H2C CH2 OH OH

Ethylene D-gluconic acid

Fig. 11.2 Structures of important metabolites which play a role in the interaction between plant- beneﬁcial bacteria and plants

11.5.4.1 Auxins

Auxins are involved in several processes such as establishment of polarity in embryogenesis, cell elongation, vascular differentiation, floral and fruit development, lateral root formation, and determination of root and shoot architecture. Indole-3-acetic acid (IAA) (Fig. 11.2) is the most abundant member of the auxin family. It has been estimated that as much as 80% of the rhizosphere bacteria can synthesize IAA (Khalid et al. 2004; Patten and Glick 1996). Rhizosphere bacteria use several different pathways for IAA biosynthesis. Most of them use tryptophan 312 C. Pliego et al. secreted in the rhizosphere as a precursor (Costacurta and Vanderleyden 1995; Spaepen et al. 2009). Indeed, Kamilova et al. (2006a) observed that P. fluorescens biocontrol strain WCS365, which can produce IAA in the presence of tryptophan, is able to stimulate root growth of radish, a plant which secretes high amounts of tryptophane, but not of tomato, sweet pepper, or cucumber, plants which secrete at least tenfold less tryptophan in the growth medium. Azospirillum brasilense is an N2 fixer, which promotes plant growth by increasing its root surface through shortening the root length and enhancing root hair formation. It has been thought for a long time that its plant growth-promoting ability was based on N2 fixation. However, the present notion is that auxin production is the major factor responsible for its root changes and therefore for its plant growth-promoting properties. This notion is based on the following observations (1) Dobbelaere et al. (1999) showed that the effect of the wild-type strain on the root can be mimicked by the addition of pure auxin. (2) A mutant strain strongly reduced in IAA production did not induce the root changes. (3) A strain constitutive for IAA production showed the same effect on the root changes as the wild-type strain but already at lower bacterial cell concentrations (Spaepen et al. 2008). Interestingly, when the amount of root exudate becomes limiting for growth, A. brasilense cells increase their IAA production, thereby triggering lateral root and root hair formation, which results in more exudation and therefore in further bacterial growth. In this way, a regulatory loop is created which connects plant root proliferation with bacterial growth stimulation (Spaepen et al. 2009).

11.5.4.2 Cytokinins

Cytokinins consist of a group of molecules, of which zeatin is the major representative (Fig. 11.2). These compounds have the capacity to induce division of plant cells in the presence of auxin. Plants use ADP and ATP as substrates for cytokinin biosynthesis while bacteria use AMP. In all cases, an isopentenyl side chain is incorporated at the N6 position of the adenine ring. The balance between the amounts of auxin and cytokinin determines whether root or shoot differentiate from callus tissue. High auxin promotes root differentiation whereas high cytokinin promotes shoot morphogenesis. Equimolar concentrations induce cell proliferation. Cytokinins are synthesized in root tips and developing seeds. They are transported to the shoot where they regulate important processes such as chloroplast development, leaf expansion, and delay of senescence. Exogenous application of cytokinins to the plant can cause profuse lateral branching. Many rhizosphere bacteria can produce cytokinins, e.g. Agrobacterium, Arthro- bacter, Bacillus, Burkholderia, Erwinia, Pantoea agglomerans, Pseudomonas, Rhodospirillum rubrum, Serratia, and Xanthomonas (Garcıá de Salome et al. 2001). The spectrum of cytokinins produced by rhizobacteria is similar to that produced by the plant (Barea et al. 1976;Garcıá de Salome et al. 2001; Frankenberger and Arshad 1995), of which isopentenyladenine, trans-zeatin, cis-zeatin, and their 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 313 ribosides are the most commonly found. Concerning the mechanism of action, one speculates that cytokinin produced by rhizosphere bacteria becomes part of the plant cytokinin pool, and thus influences plant growth and development. Evidence for a role of cytokinin of rhizosphere bacteria in plant growth promotion has been published. Garcıá de Salome et al. (2001) have produced mutants of P. fluorescens strain G20–18 which produce reduced amounts of cytokinin and normal amounts of auxin. In contrast to the wild-type strain, the mutants are unable to promote growth of wheat and radish plants (Garcıá de Salome 2000). Garcıáde Salome and Nelson (2000) showed that cytokinin production is linked to callus growth of tobacco and suggested that this test can be used as a screening method for cytokinin-producing bacteria. For the pathogen Agrobacterium tumefaciens, the ability to produce auxins and cytokinins is a virulence factor. The strain produces crown galls. The genes for production of auxins and cytokinins are transferred to the plant and incorporated in its DNA (Spaink et al. 1998). Another bacterium from this genus, A. rhizogenes, modifies cytokinin metabolism causing the appearance of masses of roots instead of callus from infection site (Hamill 1993).

11.5.4.3 Gibberellins (GAs)

These hormones consist of a group of terpenoids with 20 carbon atoms, although active GAs only have 19 carbon atoms (Fig. 11.2). This group of compounds consists of over 120 different molecules. GAs are mainly involved in cell division and cell elongation within the subapical meristem, thereby playing a key role in internode elongation. Other processes affected by these hormones are seed germination, pollen tube growth, and ﬂowering in rosette plants. Like auxins and cytokinins, GAs mainly act in combination with other hormones. Hardly anything is known about gibberellin synthesis in rhizosphere bacteria. Bacteria that produce gibberellins, such as A. brasilense, A. lipoferum, Bacillus species, Bradyrhizobium japonicum, and Rhizobium phaseoli, secrete them in the rhizosphere (Frankenberger and Arshad 1995; Gutie´rrez Manero et al. 2001; Rademacher 1994). The mechanism of plant growth stimulation by gibberellins is still obscure. Fulchieri et al. (1993) speculate that gibberellins increase root hair density in root zones involved in nutrient and water uptake.

11.5.5 Stress Controllers

Plants can be subject to stress. Bacteria which can decrease plant stress are treated here. 314 C. Pliego et al.

11.5.5.1 Abscisic Acid (ABA)

ABA is a 15-carbon compound (Fig. 11.2) and, like ethylene, it is involved in plant responses to biotic and abiotic stresses. It inhibits seed germination and ﬂowering. It is involved in protection against drought, salt stress, and toxic metals. It also induces stomatal closure. ABA can be produced by several bacteria such as A. brasilense (Cohen et al. 2008) and B. japonicum (Boiero et al. 2007). The effect of inoculation with ABA- producing bacteria on plant growth is experimentally poorly underpinned. Since ABA inhibits the synthesis of cytokinins (Miernyk 1979) it was speculated that ABA increases plant growth by interfering with the cytokinin pool (Spaepen et al. 2009). It could also alleviate plant stress by increasing the root/shoot ratio (Boiero et al. 2007).

11.5.5.2 Ethylene (ET)

Ethylene, the gaseous hormone (Fig. 11.2), is best known for its ability to induce fruit ripening and ﬂower senescence. It generally inhibits stem elongation in most dicots favoring lateral cell expansion and leading to swelling of hypocotyls. However, it promotes growth in submerged aquatic species. ET also breaks seed and bud dormancy. ET is synthesized under biotic stress conditions following infection by pathogens, as well as by abiotic stress conditions such as drought. It is therefore also known as the stress hormone. In the plant, ethylene is produced from S-adenosylmethionine (SAM) which is enzymatically converted to 1-aminocyclopropane-1-carboxylate (ACC) and 50-deoxy-50methylthioadenosine (MTA) by ACC synthase. The enzyme ACC deaminase is present in many rhizosphere bacteria. Such bacteria can take up ACC secreted by the plant root and convert it into a-ketobutyrate and ammonia. This results in the decrease of ACC levels, and therefore also of ethylene levels in the plant and in decreased plant stress. Inoculation of plants with ACC deaminase producing bacteria can protect plants against stress caused by ﬂooding, salination, drought, heavy metals, toxic organic compounds, and pathogens (Glick 2005; Glick et al. 2007a; Glick et al. 2007b; Belimov et al. 2005). In addition to a direct role of ethylene on plant growth, this hormone can also act as a virulence factor and a signaling molecule in plant protection against pathogen attack. Ethylene production was reported to act as a virulence factor for bacterial pathogens, e.g., P. syringae (Weingart and Volksch 1997; Weingart et al. 2001). Furthermore, ethylene acts as a signaling compound in induced systemic resistance (ISR) caused by some rhizobacteria (Van Loon 2007).

11.6 Bacteria Which Can Control Plant Diseases

Many plant diseases are caused by fungi. Some beneﬁcial bacteria can reduce such diseases. 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 315 11.6.1 Introduction to Biocontrol Microbes

The story of microbes which can protect plants against pathogens begins with the discovery by Schroth and Hancock (1982) of so-called suppressive soils. In these soils, pathogens can be present without causing diseases to the normally susceptible plants. These soils contain microbes which protect the plant from the pathogen. When the disease-suppressing microbe is absent we speak about “conducive soils” in which pathogens can cause diseases. A small amount of suppressive soil can, when mixed with conducive soil, make the latter suppressive. Many of the disease-suppressive microbes that have been isolated belong to the genera Pseudomonas and Bacillus. Fluorescent pseudomonads can easily be detected on Fe3þ poor medium because, under Fe3þ limiting conditions, e.g., in Kings medium B (King et al. 1954), they secrete a siderophore, i.e., a molecule which sequesters Fe3þ. In the case of ﬂuorescent pseudomonads, (one of) the siderophore(s) is pyoverdin (Fig. 11.2).

11.6.2 Mechanisms of Biocontrol

Researchers have focused on characterizing the mechanisms involved in biocontrol operating under different experimental situations. In all cases, pathogens are antagonized by the presence and activities of other organisms they encounter (Haas and De´fago 2005; Pal and Gardener 2006; Alabouvette et al. 2006; De Weert and Bloemberg 2007). The most important pathogens are fungi, but there are also some pathogenic bacteria and nematodes. The modes of action of beneﬁcial microorganisms can be based on either a direct or an indirect antagonism. However, both mechanisms are not mutually exclusive as they have been frequently described as co-occurring within the activity of the same BCA (Castoria and Wright 2009). Direct antagonism results from physical contact and/or from a high degree of selectivity of the mechanism(s) expressed by the BCA(s), in relation to the pathogen, i.e., parasitism and predation, production of antibiotics, and signal interference. In contrast, indirect antagonism results from activities that do not involve sensing or targeting of a pathogen by the BCA(s), i.e., competition for nutrients and niches, production of siderophores, and ISR.

11.6.2.1 Antibiosis

Antibiotics produced by microorganisms have been shown to be particularly effective in suppressing plant pathogens and diseases. Most biocontrol strains of Pseu- domonas spp. with a proven effect in plant bioassays produce one or several antibiotic compounds, e.g., P. fluorescens strains CHAO (Laville et al. 1998; Haas et al. 2000) and Pf-5 (Thompson et al. 1999) produce complex cocktails of 316 C. Pliego et al. these secondary metabolites. In vitro, these antibiotics inhibit the growth of the fungal pathogen, and for this reason, strains acting through antibiosis are usually identified by screening them for antagonistic activity on plates on which the target pathogen is also inoculated (Lugtenberg and Bloemberg 2004). The contribution of antibiotics to biological control of root disease is documented in five experimental steps (1) Purification and chemical identification of the antibiotic compound. (2) Detection and quantification in the rhizosphere of the secondary metabolite. (3) Identification and characterization of the structural and principal regulatory genes controlling the expression of the antibiotic compound. (4) Poor biocontrol strains can acquire biocontrol activity by the introduction of antibiotic biosynthetic genes not present in the original strain. (5) Detection of the expression of the antibiotic biosynthetic genes through the use of detectable reporter genes fused to structural genes for antibiotic biosynthesis (Haas and Keel 2003).Well-characterized antibiotics (Fig. 11.2) with biocontrol properties include phenazines (Phz), phloroglucinols (Phl), pyoluteorin, pyrrolnitrin, hydrogen cyanide (HCN), cyclic lipopeptides (Perneel et al. 2008; Keel et al. 1992; Thomashow and Weller 1988; Haas and Keel 2003; Raaijmakers et al. 2006), and the most recently discovered 2-hydroxymethyl-chroman-4-one (Kang et al. 2004), D-gluconic acid (Kaur et al. 2006), and 2-hexyl-5-propyl resorcinol (HPR) (Cazorla et al. 2006). Phenazines are analogs to flavin coenzymes, inhibiting electron transport, and are known to have various pharmacological effects on animal cells (Ran et al. 2003). 2-4-Diacetylphloroglucinol, the best known Phl compound, causes membrane damage to Pythium spp. and is particularly inhibitory to zoospores of this oomycete (De Souza et al. 2003). Gleeson et al. (2010) provided evidence that Phl acts through impairing the function of mitochondriae. Dikin et al. (2007) reported that pyrrolnitrin causes the loss of mitochondrial activity in the fungal cytoplasm, inhibiting succinate oxidase and NADH-cytochrome reductase. Pyrrolnitrin also interferes with cellular processes such as oxidative stress, blockage of electron transport as well as inhibition of DNA and RNA synthesis (Dikin et al. 2007). The cyanide ion derived from HCN is a potent inhibitor of many metalloen- zymes, especially copper-containing cytochrome c oxidases (Blumer and Haas 2000). Finally, cyclic lipopeptides have surfactant properties and are able to insert themselves into membranes and perturb their function, resulting in broad antibacterial and antifungal activities (Haas and De´fago 2005; Perneel et al. 2008). Recently, Mazzola et al. (2009) have shown that in the wheat rhizosphere the cyclic lipopeptides viscosin and massetolide not only protect the plant against fungi but also against protozoan predation. In fact, the protozoa Naegleria americana de- represses the syntheses of these antibiotics. To our knowledge, no mode of action has been described for pyoluteorin, HPR, and D-gluconic acid. The syntheses of antifungal metabolites (AFMs) are extremely sensitive to environmental conditions in the rhizosphere, e.g., soil mineral content, oxygen tension, osmotic conditions, carbon sources, as well as fungal, bacteria, and plant metabolites can all influence the expression of secondary metabolites (Haas and 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 317

Keel 2003; Lugtenberg and Bloemberg 2004; Duffy and De´fago 1999; Van Rij et al. 2005).

11.6.2.2 Predation and Parasitism

Soilborne bacteria and fungi are able to produce extracellular enzymes such as chitinases, ß-1-3 glucanases, lipases, cellulases, and proteases. These lytic enzymes can hydrolyze a wide variety of polymeric compounds, including chitin, proteins, cellulose, hemicelluloses, interfering with pathogen growth and/or activities. Pro- duction and secretion of these enzymes by different microbes can result in biocontrol abilities (Markowich and Kononova 2003). Ordentlich et al. (1998) showed that chitinase of Serratia marcescens is involved in the biocontrol of Sclerotium rolfssi. Beta-1,3-glucanase contributes signiﬁcantly to the biocontrol activities of Lysobac- ter enzymogenes strain C3 against Bipolaris leaf spot caused by Phytium spp. (Palumbo et al. 2005). Chitinases of the mycoparasitic Trichoderma species have also been shown to play an important role in biocontrol and antagonistic activity against phytopathogens (Harman et al. 2004), including R. necatrix (Hoopen and Krauss 2006). Sometimes, these enzymes act synergistically with antibiotics playing an important role in the antagonistic effect on phytopathogenic fungi (Schirmbock€ et al. 1994; Fogliano et al. 2002). Predation and parasitism are not always related to biocontrol, as it is the case for the fungus eater C. fungivorans,of which the major mechanism of action for controlling tomato foot and root rot (TFRR) probably is competition for space and nutrients and niches (Kamilova et al. 2007).

11.6.2.3 Competition for Nutrients and Niches

To successfully colonize the rhizosphere, a microbe must effectively compete for the available nutrients. Competition for nutrients and niches (CNN) between pathogens and beneficials has been shown to be important for limiting disease incidence and severity (Kamilova et al. 2005). Enrichment for enhanced competitive tomato root tip colonizers was used to select for bacteria, not producing antibiotics, which control TFRR by CNN (Fig. 11.1) (Kamilova et al. 2005). The enhanced competitive root tip colonizers grow efficiently on root exudates. How- ever, Kamilova et al. (2005) observed that these two characteristics were not sufficient for biocontrol, as one of the best competitive root-tip-colonizing strains did not control TFRR. A similar enrichment procedure was used by Pliego et al. (2007) for the isolation of competitive avocado root tip colonizers strains displaying biocontrol ability against R. necatrix. Two Pseudomonas sp. strains, P. pseudoalcaligenes AVO110 and P. alcaligenes AVO73, were selected for their efficient colonization abilities. However, only AVO110 demonstrated significant protection against avocado white root rot. Further analysis revealed that both strains colonize different sites on the 318 C. Pliego et al. root: biocontrol strain AVO110 was observed to colonize the root at preferential penetration sites for R. necatrix infection (intercellular crevices between neighboring plant root epidermial cells and root wounds) while P. alcaligenes AVO73 was predominantly found forming dispersed microcolonies over the root surface and in the proximity of lateral roots, areas not colonized by this pathogen (Pliego et al. 2008). These results strongly suggest that biocontrol bacteria acting through CNN must efficiently colonize the same mini-niche as the pathogen. Competition for ferric iron ions is a well-documented example of competition of biocontrol bacteria with pathogenic fungi for nutrients (Leong 1986, Lugtenberg and Bloemberg 2004). This topic has been treated under 5.2.

11.6.2.4 Induced Systemic Resistance

Some PGPR have been identified as potential ISR elicitors, for their ability to induce resistance in plants toward pathogenic fungi, bacteria, and viruses (Van Loon et al. 1998; Van Loon 2007). The inducing rhizobacteria triggered a reaction in the plant roots that gave rise to a signal that spread systemically throughout the plant and enhanced the defensive capacity of distant tissues to subsequent infection by the pathogen (Van Loon 2000). ISR is distinct from systemic acquired resistance (SAR) in several key physiological and biochemical phenotypes that are best defined in A. thaliana (Van Wees et al. 1997). Studies with A. thaliana mutants indicated that the jasmonate/ethylene-inducible defense pathway is important for ISR. Many bacterial determinants induce ISR, i.e., flagella, siderophores (pycholin and pyocyanin), LPS, salicylic acid (Van Loon 2007), cyclic lipopeptides (Ongena et al. 2007), N-acyl homoserine lactone (AHL) molecules (Shuhegger et al. 2006), the bacterial volatile 2,3-butanediol produced by Bacillus spp. (Ryu et al. 2003), and antibiotics such as Phl (Lavicoli et al. 2003). In several ISR-competent strains of fluorescent pseudomonads, it has been difficult to identify specific ISR elicitors, possibly because a combination of siderophores, O-antigen, and flagella might account for the ISR effect (Bakker et al. 2003). It has also been shown that several Pseudomonas spp. are able to induce ISR in a wide range of plants toward different pathogens (Van Loon 2007). Generalization of the signal transduction pathways that are involved in ISR are further complicated by the fact that an ISR response to a given PGPR depends on the plant species and cultivar. For example, in Arabidopsis thaliana, the PGPR strain P. fluorescens WCS417r elicited ISR on all ecotypes examined, except ecotypes Wassilewskija and RLD (Van Wees et al. 1997).

11.6.2.5 Signal Interference

In Gram-negative bacteria, one type of communication system functions via small, diffusible N-acyl homoserine lactone (AHL) signal molecules (Fig. 11.2). Such a regulatory system allows bacteria to sense the density of cells of their own kind and to express target genes in relation to their cell density. This cell–cell 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 319 communication mechanism regulates a variety of physiological processes, including swarming, swimming and twitching motilities, production of pathogenicity/virulence factors, and rhizosphere colonization (Gray and Garey 2001; Miller and Bassler 2001). Several groups of AHL-degradation enzymes have recently been identified in a range of organisms, including bacteria and eukaryotes. Expression of these enzymes was identified to interfere with the quorum-sensing system of pathogenic bacteria. E. carotovora produces and responds to AHL quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains abolished the accumulation of the AHL signal by the expression of a AHL-lactonase, which is a potent AHL-degrading enzyme. In plants, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptoms development of potato soft rot caused by the pathogen (Dong et al. 2004). Discovery of these enzymes has not only provided a promising means to control bacterial infections, but also represents new challenges to investigate their roles in host organisms and their potential impacts on ecosystems (Dong et al. 2004).

11.6.3 Role of Root Colonization in Biocontrol Mechanisms

Selection of bacterial biocontrol strains by their ability to inhibit fungal growth in vitro is not always correlated with efficient control of plant diseases. Different steps are known to be involved in root colonization (1) attachment to the plant surface, in which flagella and different types of pili can be involved (De Weert et al. 2004b) and (2) surface motility which confers potential benefits to bacteria in the rhizosphere, including increased efficiency in nutrient acquisition, avoidance of toxic substances, ability to translocation to preferred hosts, and ability to access optimal colonization niches (Andersen et al. 2003). Motility has been reported to depend on the soil type, the plant, and the bacterial strains used (Weller and Thomashow 1994). Pliego et al. (2008) reported that strains differing in surface motility can show differences in the architecture of the biofilms developed by these strains in the rhizosphere which, at the same time, can reflect differences in their root colonization patterns. As we explained earlier, in case of BCAs acting through CNN, colonization strategies of the biocontrol strain must be correlated with penetration sites used for the phytopathogenic fungi to infect the root. In the case of AFM production, efficient root colonization is essential for the delivery of the AFM along the root system at the right time and place (Lugtenberg et al. 2001; Haas and Keel 2003). Insertional mutagenesis studies demonstrated that colonization can be a limiting step in some biocontrol strains. P. chlororaphis PCL1391 controls TFRR through the production of the antifungal metabolite phenazine-1-carboxamide (PCN) (Fig. 11.2). Nonmotile mutants of this strain impaired in root colonization, but not in production of extracellular metabolites, did not show biocontrol activity (Chin-A-Woeng et al. 2000). 320 C. Pliego et al.

In the case of microorganisms inducing the ISR response on the root, a decrease in root colonization does not clearly result in loss of biological control, i.e., P. ﬂuorescens WCS365, which acts through ISR (Kamilova et al. 2005), colonization of seedlings for a brief period is sufﬁcient to induce ISR in the whole plant (Dekkers et al. 2000). As is mentioned earlier, certain antifungal metabolites can induce ISR. This can explain the biocontrol activity of poorly colonizing PGPRs which produce antibiotics (Gilbert et al. 1994).

11.6.4 Mycelium Colonization by Bacteria

As mentioned previously, biocontrol bacteria in the rhizosphere compete for nutrients and niches with endogenous microorganisms. Therefore, to gain a better understanding of how biological control functions in the rhizosphere, the interaction with other microbes, such as root pathogenic fungi, must be taken into account. It has become clear that biocontrol bacteria can interact with phytopathogenic fungi in a variety of ways, all converging on the same objective, i.e., to derive nutrition from these fungi (Leveau and Preston 2008). This interaction can be split into several stages including (1) detection of the fungal host, (2) attachment to the fungal cells, and (3) growth of bacteria on biotic fungal surfaces (Hogan et al. 2009). Microscopic visualization of bacterial–fungal interactions showed that at least some antagonistic bacteria exhibit chemotaxis toward fungal exudates allowing bacteria to congregate around populations of fungi in soils. For example, fusaric acid (Fig. 11.2), a secondary metabolite secreted by Fusarium hyphae, acts as a chemoattractant for cells of biocontrol strain P. fluorescens WCS365, which can actively move toward and colonize the surface of fungal hyphae (De Weert et al. 2004a). Bacteria showing chemotaxis to specific fungal compounds may use a variety of mechanisms to reach and attach to the fungus, including pili, exopolysaccharides, and biosurfactans. The biosurfactants reduce surface tension, enhance swarming motility, and facilitate contact between bacteria and fungal cells (Nielsen and Sorensen 1999; Braun et al. 2001). Once bacteria reach the fungus, they may scavenge nutrients from the fungal cell wall, consume products secreted by the fungus, or induce lysis of the fungal cells, thereby liberating the intracellular contents for consumption by the local bacterial population. Bacterial communities associated with fungi seem to be under selection to develop fungi-specific traits that confer a competitive advantage during colonization of fungal surfaces. Such traits could be the ability to detect fungus specific compounds, the utilization of nutrients that are specific to, or particularly abundant in, fungal exudates, or the ability to tolerate or suppress the production of antibacterial metabolites by fungal cells. Several studies support the idea that hyphal colonization by bacteria can play an important role in biocontrol activity (Bolwerk et al. 2003, Pliego et al. 2008). It has been shown that the ability of P. fluorescens WCS365 to inhibit pathogenic activity, survival, and germination of the pathogen significantly contributes to the reduction 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 321 of TFRR after introduction of this bacterium into the plant nutrient solution (Kamilova et al. 2008). Co-inoculation of the biocontrol strain P. pseudoalcaligenes AVO110 with R. necatrix (the causal agent of avocado white root rot) showed that AVO110 utilizes hyphal exudates more efficiently for proliferation than the nonbiocontrol strain P. alcaligenes AVO73 (Pliego et al. 2008). Although it is not always clear whether bacterial consumption of fungal exudates contributes to reduction of the disease (Kamilova et al. 2007), bacteria can inhibit the growth of harmful fungi by feeding on them, thus supporting their own growth, which in turn leads to a better biocontrol activity (Leveau and Preston 2008). While several studies have been focused on identifying bacterial genes involved in root colonization, little attention has been paid to genes involved in interactions with fungi. Bacterial genes involved in the interaction established between biocontrol strain P. putida 06909 and the phytophatogenic oomycete Phytophthora have been identified using IVET. Several P. putida promoters were induced during the growth of this biocontrol bacterium on the surface of the fungus, corresponding to genes involved in carbon catabolism, amino acid/nucleotide metabolism, and membrane transport processes (Lee and Cooksey 2000; Ahn et al. 2007) (Table 11.3). Separate studies showed that genes involved in trehalose utilization may be important for bacterial growth when associated with fungi. P. fluorescens sp. have been shown to induce genes involved in trehalose metabolisms when exposed to fungal culture supernatant, and the presence of this sugar enhances inhibition of Pythium debaryanum in a radial growth assay (Gaballa et al. 1997; Rincoń et al. 2005). Recently, Pliego (2008) used STM, a powerful genomic mutagenesis technique which has been widely applied to bacterial pathogens (Hensel et al. 1995; Shea et al. 2000; Autret and Charbit 2005) to identify genes involved in growth and survival of a biocontrol Pseudomonas strain in fungal exudates. These genes were predicted to be involved in central metabolism, regulation of the second messenger cyclic (c)-di-GMP, and bacterial protection against fungal defenses.

Table 11.3 Pseudomonas proteins upregulated during colonization of Phythophthora myceliaa Energy metabolism Nucleotide biosynthesis Membrane proteins/ transporters Nitrogen regulatory protein PII-2 Carbamoyl-phosphate synthase C4-dicarboxylate transport protein Flavin-dependent oxidoreductase Phosphoribosylaminoimidazol Arginine-/ornithine-binding (AIR) synthetase protein Malic enzyme Carbamoylphosphate Leucine-,isoleucine-, synthetase large subunit valine-binding protein Succinate-semialdehyde ABC transporter dehydrogenase Probable glyceraldehyde- Outer membrane porins 3-phosphate dehydrogenase Ribulose-phosphate-3-epimerase Transaldolase aAdapted from Lee and Cooksey (2000) and Ahn et al. (2007) 322 C. Pliego et al.

In summary, further studies must be performed to identify bacterial genes involved in fungal colonization or acquisition of nutrients from fungi. This will gain a broader view on genes and mechanisms involved in biological control and in future design of new and improved BCAs. One of the most exciting aspects is that genes similar to those involved in colonization of fungal hyphae were also identiﬁed in other IVET or STM assays involving phylogenetically diverse pathogenic and nonpathogenic microorganisms residing in diverse complex environments, in particular plant or animal host. These results suggest that similar or common mechanisms might be involved in processes as different as controlling and causing diseases. The identiﬁcation of bacterial genes necessary for biocontrol of fungal pathogens will expand our ability to design new biocontrol strategies and, above all, feed our growing appreciation of the enormous diversity and adaptability of bacteria.

11.6.5 Resistance of the Pathogen Toward Biocontrol Agents

Although there are hardly any reports about pathogenic fungi that have become resistant against BCAs, it is good to consider this possibility. It is clear that the mechanism of action used by the BCA determines the possible resistance strategies. Since little is known about the interaction between the pathogenic fungus and the plant when ISR is used as the biocontrol mechanism, it is difficult to predict possible defense strategies for this case. When CNN is used, it is hard to envisage resistance strategies of the pathogen. In contrast, for antibiosis as a mechanism of biocontrol several defense strategies can be envisaged. To do this, it is best to look at the much better studied resistance mechanisms that bacteria are using against antibiotics. Here, known resistance strategies can be based on (1) detoxification of the antibiotic, e.g., by degradation or modification, (2) modification of the target of the antibiotic, (3) making penetration to the target molecule more difficult, e.g., by making entry pores narrower or by shielding the surface with an additional less penetrable layer, and (4) actively pumping the antibiotic out of the cell. Duffy et al. (2003) published an excellent review about defense mechanisms used by pathogens against microbial antagonism. Detoxification of the antifungal compound 2,4-diacetylphloroglucinol produced by Pseudomonas species has been reported. Schouten et al. (2004) reported the screening of 76 plant-pathogenic and 41 saprophytic F. oxysporum strains for sensitivity to 2,4-diacetylphloriglucinol. They found that 18 and 25%, respectively, of these strains were tolerant to the antibiotic. Of course, an antibiotic-producing strain should protect itself against the antibiotic it produces. Indeed, Bacillus subtilis strain UW85 which produces the antibiotic zwittermycin A can inactivate the antibiotic by acetylation (Milner et al. 1996). Another mechanism that can be used by a pathogenic fungus is efflux of the antibiotic. Upon exposure to phenazines, Botrytis cinerea induces an efflux pump 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 323 for the antibiotic. Mutants lacking pump activity are more sensitive toward the antibiotic (Schoonbeek et al. 2002). A very interesting mechanism of defense is the ability of a pathogen to repress the biosynthesis of the antibiotic produced by the BCA. Duffy and De´fago (1997) found that fusaric acid (Fig. 11.2), a small molecule secreted by many Forl strains, represses the synthesis of Phl in the biocontrol bacterium P. fluorescens CHAO by repression of the phlA promoter, a process for which an intact phlF gene is needed. Van Rij et al. (2004) observed that fusaric acid also suppresses the synthesis of another antibiotic, namely, PCN produced by P. chlororaphis strain PCL1391. This inhibition correlates with reduction of the level of the AHL autoinducer N-hexanoyl- L-homoserine lactone. The inhibition in P. chlororaphis strain PCL1391 takes place at or before the level of AHL production. It is striking to see that in two different Pseudomonas biocontrol strains inhibition of antibiotic production by fusaric acid has evolved, although at different molecular levels. Apparently, these mechanisms have evolved independently because of the need to protect against antibiotic- producing bacteria. It should be noted that P. fluorescens strain CHAO does not produce AHL and therefore the mechanism to suppress Phl production at the level at or before AHL production could not be used in this strain. In practice, there are very few examples of resistance of pathogens against BCAs (Duffy et al. 2003). This may have several reasons, for example, that a BCA uses a mechanism to which no resistance exists or that it uses several different mechanisms to which no cross-resistance exists. The relevance of resistance in biocontrol was studied by Mazzola et al. (1995). They screened a collection of 66 isolates of the pathogen Gaeumannomyces graminis var. tritici against the antibiotics Phl and PCA, which are produced by many biocontrol strains which act through antibiosis. A large variation in sensitivity was found. Most importantly, it appeared that the Phl- or PCA-producing Pseudomonas strains could not control take-all caused by the Phl- or PCA-resistant G. graminis var. tritici isolates, respectively.

11.7 From Laboratory to Industrial Application

It is important to realize that a beneﬁcial bacterium, which is active under laboratory conditions, not necessarily is a good basis for a commercial product. Here, we discuss criteria that industrial products should fulﬁll.

11.7.1 Introduction

The application of beneﬁcial microbes for plant growth stimulation and biocontrol makes horticulture and agriculture sustainable and will, in contrast to many chemicals, not be a burden for the ecology and for the next generations of human beings. This notion, supported by public, politicians, and some supermarkets, is a major 324 C. Pliego et al. driving force for the agricultural and horticultural industry to use, where possible, environmentally friendly products for plant growth stimulation and crop protection. In this section we will discuss how, starting from promising beneﬁcial microbes with known mechanisms of action, the industry will make the decision to carry out further research and which steps are required to develop a marketable product. Because this chapter focuses mainly on biocontrol microbes, we discuss only biocontrol products in this section. Furthermore, rules about registration as a product differ among countries. We will mainly focus on guidelines of the European Union (EU).

11.7.2 Evaluation of Opportunities

How does the biocontrol industry come to the decision which strain should be used to make it a proﬁtable product? What are the prerequisites for such a decision? What does the grower need? How does the development of a plant protection product occur? How can such a product be protected, registered, and commercialized? The biocontrol industry keeps constant contact with growers, the current users of their products. Collecting data on existing and emerging diseases of crops and search for solutions against these diseases is the starting point for decision making. The size of the market is another important factor. Therefore, the market potential for a new microbial product will be evaluated in terms of market size in relation to (1) the importance of the crop(s) and (2) the severity and geographical spread of the disease. What are the present biological and/or chemical products for disease prevention? Who are the competitors? How strong are they? Would the new product have a chance to successfully penetrate the market?

11.7.3 Industry Takes over

In case the above-mentioned evaluation is positive, a good strain has to be found. If the biocontrol industry has no useful strains available, it can approach academic parties to evaluate whether they have promising strains that are effective against the target disease/pathogen combination. Academic research, as described in the previous paragraphs, is usually directed toward the discovery of the potential plant- beneﬁcial strains by using approaches such as screening for antagonistic properties, studying root colonization, and identifying traits and metabolites involved in biocontrol and plant growth promotion. Most of this research is done in vitro or in planta under laboratory, growth chamber, or experimental greenhouse conditions. Academic scientists usually focus on one pathogen/crop model system as such experiments can easily be made suitable for publication. Industry should ensure that the selected strain is safe for environmental release and should come 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 325 to an agreement with the owner on intellectual property rights and the degree of exclusivity and/or licensing.

11.7.4 Industrial Research

In contrast to the usually limited plant experiments done by academic researchers, industry would like to develop a product applicable on a broader range of crops and/ or pathogens. This implies further research as well as the development of a suitable formulation for the strain. After positive evaluation of marketability of the potential biocontrol product, the following considerations are taken into account: strain safety and efﬁcacy, potential for upscale production and product development, and registration costs.

11.7.5 Strain Safety

The beneficial strains have usually already been taxonomically identified in an early state of the research. According to guidelines (Anonymous 1998) the strain can then be classified in risk groups which predict their degree of safety. Before a BCA can be registered as a product, cost-intensive, time-consuming pathogenicity tests have to be carried out. There is a good reason to do this since several strains isolated as potential BCAs are potential human pathogens (Berg et al. 2005; Egamberdieva et al. 2008). Thus, strain identification is crucial for safe applications in agriculture and horticulture. According to European Commission Directive 2001/36/EC amending Council Directive 1991/414/EEC, concerning the introduction of plant protection products on the market, the best available technology should be used to identify and characterize the microorganism at the strain level. The use of molecular biological techniques, mostly at the DNA level, allows the identification of potential pathogens which subsequently will be excluded from further research. It should be noted that a promising new tool for future evaluation of the pathogenic potential of putative biocontrol strains could be the use of an assay with the nematode Caenorhabditis elegans (Zachow et al. 2009). Work will only be continued with strains from the risk group 1 (Anonymous 1998). In this stage also evaluation of the mode of action of the beneficial strains is very important, because it will give a clue to whether this would cause additional studies and, consequently extra costs, for registration of the biocontrol product. For example, for strains with mechanisms of actions based on production of new antibiotics and/or other toxic compounds, the regulatory authority will ask to provide very detailed investigations on metabolite production, toxicological and residue studies. 326 C. Pliego et al. 11.7.6 Large-Scale Production and Formulation

Often the high cost of production for most of BCAs is a limiting factor for commercialization of a biocontrol product (Fravel 1998). High costs of the growth substrate, low productivity of the microbe, or limited economies of scale could be the reasons to stop development (Fravel et al., 2005). One of the goals of an industrial production unit is to minimize the fermentation costs and to produce the highest quantities of the microbe without negatively inﬂuencing the important traits involved in biocontrol such as (1) proliferation of spores in case of spore- forming bacteria and fungi, (2) ability to colonize rhizosphere or phyllosphere, or (3) competition with phytopathogenic organisms. Multiple tests on cell viability and plant root colonization should be performed in order to optimize a manufacturing process and to produce a good quality product. Beneﬁcial microbes are formulated in order to preserve the BCA, to deliver the microbes to their targets and, after their delivery, improve their activity (Burges 1998). Formulation usually consists of the addition of a combination of various additives, such as carriers, preservatives, nutrients, etc. Furthermore, the choice of dry or liquid formulation to be used depends on the biological and physical properties of the microorganism, and on its abilities to survive the formulation process and to maintain the desired properties for a certain period of time (Burges 1998). Evidently, for producing powder and granular formulations, spore-forming strains such as Bacillus have an advantage over non-spore-forming strains such as Pseudomonas which have to be formulated as vegetative cells. Spores are more robust and resistant to elevated temperature and high concentrations of chemicals that are part of the spray-drying process. Moreover, the shelf life of biological products based on bacterial spores can be up to 1–3 years. A disadvantage of the use of spores is that, after application, they need time to return to the metabolic active stage of a vegetative cell. Vegetative cells of nonspore formers are more sensitive than spores toward dry formulation processes. This makes dry formulation of vegetative cells more expensive than formulation of spores. For example, it has been shown for Pseudomonas by Validov et al. (2007) that freeze-drying provides a more stable formulation product with a longer shelf life than the less expensive spray drying. A liquid formulation can be an option for vegetative cells. However, it can be a challenge to keep these bacterial cells in a low metabolic state in the presence of water for a substantial amount of time without losing their viability. For a seed company, seed coating with biocontrol strains is an interesting option. Coating means here the introduction of BCAs during seed priming and/or seed pelleting. In fact, this is a process for upgrading the value of seeds. An advantage for the user is that the seeds do not have to be treated with the bioproduct immediately prior to sowing by means of spraying or by soaking the seeds at site. The choice of the formulation type is dependent not only on the biological properties of the strains but also, in many cases, on the desired application methods and existing irrigation systems. For example, for a drench application, slurry, spray 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 327 or soaking treatment of seeds, liquid or wettable powder products are more suitable. A granular formulation would be appropriate for mixing with horticulture potting mix or for distribution in-furrow (Fravel 1998).

11.7.7 Registration in the EU: Legal Basis and Requirements

Many biocontrol products have been registered. See Table 11.4 for examples. Active microorganisms used in the plant protection products in the EU are regulated according to the EU Council Directive 1991/414/EEC. This Directive provides deﬁnitions of the plant protection products and requirements for their authorization

Table 11.4 Examples of active microorganisms registered as biopesticides in the EU and/or the USA (as at March 1, 2010) Active microorganism Formulation Pathogen Crop type Bacillus subtilis FZB24b Liquid, Fusarium, Rhizoctonia, Potato, vegetables, powder, Pythium ornamentals, turf granule Bacillus subtilis Powder Botrytis, Sclerotinia, Vegetables, QST 713b Sphaerotheca macularis soft fruits Bacillus subtilis GB03b Powder Aspergillus, Pythium, Cotton, vegetables, Rhizoctonia, Fusarium soybean Pseudomonas Liquid Fusarium, Septoria, Cereals chlororaphis MA 342b Tilletia Streptomyces K61a,b Powder Fusarium Vegetables Gliocladium catenulatum Powder Rhizoctonia, Pythium, Vegetables, strain J1446a,b Phytophthora, Fusarium, herbs, Didymella, Botrytis, ornamentals, Verticillium, Alternaria, trees, Cladosporium, shrubs, Helminthosporium, turf Penicillium, Plicaria Trichoderma harzianum Powder, Fusarium, Pythium, Vegetables, strain T-22a,b granule Rhizoctonia ornamentals, turf Trichoderma asperellum Powder Fusarium, Pythium, Vegetables, turf strain ICC012a,b Rhizoctonia Trichoderma atroviride Powder, Fusarium, Pythium, Vegetables, turf strain IMI206040a granule Rhizoctonia Trichoderma gamsii strain Powder Fusarium, Pythium, Vegetables ICC080a,b Rhizoctonia, Verticillium Trichoderma polysporum Powder Botrytis, Chondrostereum Soft fruits, strain IMI206039a ornamentals Pythium oligandruma,b Liquid Alternaria, Botrytis, Canola, ornamentals, Fusarium, Sclerotinia vegetables Verticillium albo-atrum Liquid Ophiostoma nova-ulmi Elm strain WCS850a,b aEU Annex I listed strains (http://ec.europa.eu/sanco_pesticides/public) bUS-EPA registration (http://www.epa.gov/oppbppd1/biopesticides/ingredients) 328 C. Pliego et al. within the EU. Directive 1991/414/EEC was amended by Commission Directive 2001/36/EC regarding to the data requirements for the Annex I inclusion of microorganisms as active substances and national authorization of products (Annex II and III, respectively, of the directive). Council Directive 2005/25/EC lays down the Uniform Principles for evaluation and authorization of plant protection products containing microorganisms. Briefly, a registration dossier prepared in order to register a biopesticide in the European Union must contain all requested information on the active microorganism and the product, such as the biological properties of the microbe, the physical and chemical properties of the product, its safety for humans and for the environment, and its efficacy. In contrast to Europe, registration in the USA (http://www. epa.gov) does not require the efficacy data. In Table 11.4, we provide examples of microorganisms that have been registered as active substances in commercial biopesticides registered in the EU and/or the USA. Many physical and chemical studies of the product, the analysis of relevant metabolites and potential toxins in the preparations, the analysis of residues on treated plants and food, as well as toxicological and ecotoxicological studies should be performed under Good Laboratory Practice (GLP) conditions. In most of the cases, industry outsources these tasks to specialized GLP-certified laboratories and insti- tutes. Toxicological studies include evaluation of acute toxicity studies performed on certain laboratory animals such as rats, rabbits, and guinea pigs. Genotoxicity testing includes both in vivo (carcinogenic) and in vitro (mutagenic) studies. Characteristics such as impact of the product on the environment, particularly its effects on birds, aquatic organisms, bees, and other nontarget arthropods, earthworms, and soil microorganisms, are analyzed by direct observation of the tested organisms under laboratory conditions. Information from the relevant literature on the ecology of biocontrol microorganisms as well as closely related strains can help to evaluate the impact of this strain on soil microflora by extrapolation. Providing this information therefore can somewhat reduce laboratory studies on this subject. For example, there is a number of useful publications about the analysis of microbial communities of various soils after introduction of different bacterial strains with antagonism against Rhizoc- tonia solani (Adesina et al. 2009; Garbeva et al. 2004;Gotz€ et al. 2006; Scherwinski et al. 2008), E. carotovora, and Verticillium dahliae (Lottmann et al. 2000). However, this reduction of actual laboratory studies would be rather an exception, since the vast majority of studies must be performed with the strain to be registered. The efficacy part of the dossier is based on data obtained in the trials, performed for registration purposes, as well as those carried out in the research laboratory. The latter ones are considered only as preliminary data and treated by the experts accordingly. In the EU, efficacy trials are required to be carried out by efficacy testing organizations officially recognized by the national authorities. Efficacy tests and analyses include field, glasshouse, or laboratory trials and tests to determine the effectiveness and crop safety of plant protection products. Official Recognition is also known as Good Experimental Practice (GEP). This depends on the national regulations. Efficacy tests can be required to be performed during a number of seasons and at different locations. 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 329

In the framework of the European project on regulation of biological control agents (REBECA, 2008), an analysis on registration issues of the most important products (Annex I – listed) was performed. Data indicates that the mean registration time for EU Annex I inclusion is 75 months. Country registrations vary widely and range from a few months up to over 100 months, averaging around 24–36 months. Overall registration cost for EU Annex I inclusion is estimated to be about 1,890,000 Euro; out of that 970,000 Euro are external costs (fees, etc.). The breakdown of total costs to different kinds of tests averaged as efﬁcacy tests (21%), toxicological tests (43%), ecotoxicological studies (23%), and other required studies (13%) (REBECA 2008). In case of use of several microbial strains in one product (so-called microbial cocktails), registration costs will increase because an individual dossier for each active microorganism must be submitted. Registration strategy may include several options to minimize costs. For example, several companies that manufacture products with the same active microorganism(s) can unify into a task force team for inclusion of these organisms into Annex I list and share costs of this procedure. In this case member companies also share their studies and submit a joint dossier (Annex I) for the active microorganism. Individual access to certain protected data (mostly on acute toxicity and analysis of metabolites) belonging to other companies can be an option to save time for registration of some microorganisms, although ﬁnancial considerations must be taken into account.

11.7.8 Intellectual Property Rights and Patenting

Usually intellectual property rights on biocontrol strains belong to universities and small companies in which the basic research was performed. If a biocontrol company decides to commercialize a strain, both parties should come to an agreement on how the strain owner should be reimbursed. Protection is a rather sensitive subject for the biocontrol industry. In Europe, patenting of biocontrol strains per se is not possible due to the fact that these strains are naturally occurring living organisms. This is why the subject of invention usually is a combination of the strain with the bioproduct formulation(s) containing the viable cells or of the strain with applications against certain pathogenic organisms on certain crops. Another possibility for patenting is a combination of the strain and its speciﬁc metabolites involved in biocontrol activity and being produced during the fermentation and incorporated into the bioproduct formulation. Patenting can take place in various stages of the product development but should certainly take place prior to registration.

11.7.9 Marketing

Commercialization of the biocontrol products is not a simple task. First of all, demonstration trials at sites should convince farmers that the proposed bioproduct 330 C. Pliego et al. can provide a sound protection which is preferentially better than, or at least comparable with, other biological or chemical products (if these exist). Commer- cialization should also include a strong education element with emphasis on the preventive character of the biopesticide. Also, the new product should be easy to use. It would be ideal if the bioproduct can be integrated in grower’s routine schemes for the use of pesticides. Last but not least, the price of the new biocontrol product must be competitive and affordable for farmers without compromising the desired sales margins.

11.7.10 Integrated Pest Management (IPM)

In 2005, biopesticides had a share of approximately US$ 300–600 million of the US $ 35 billion agrochemical market (Cherry, 2005). Over the next 10 years, the market for biopesticides is expected to grow to US$10 billion (GIA, 2008). This is mainly due to the great public awareness of the risks of chemical fungicides and the clear trend toward sustainable agriculture, both in the EU and the USA. A good example of an effect of the public opinion is an activity of many supermarket chains and retailers toward to pesticide poor or pesticide free food. Within GLOBALG.A.P., a private sector body, many food retailers and supermarket chains, farmers and crop protection specialists collaborate in this respect. This organization sets standards for the certiﬁcation of agricultural products worldwide. The GLOBALG.A.P standard is primarily designed to reassure consumers about how food is produced on the farm by minimizing detrimental environmental impacts of farming operations, reducing the use of chemical inputs, and ensuring a responsible approach to worker health and safety as well as animal welfare (www.globalgap.com). Increasingly more attention is paid to IPM. This includes, among other means, a balanced use of chemicals and biologicals in order to provide healthy food without loosing or, preferentially, with even increasing productivity. Commercial biocontrol products must address not only efﬁcacy and, broadly speaking, safety requirements, but also be compatible with existing agricultural practices, including the use of fertilizers and chemical plant protection products. The ultimate intention of the biocontrol industry is to minimize and eventually eliminate the use of chemicals by replacing them with environmentally friendly biocontrol products.

11.8 Lessons from the Past to Create a Shining Future

In the past 3 decades, insight in fundamental aspects of microbe–plant interactions has enormously increased, especially at the molecular level. Understanding of these interactions at the molecular level is important for (1) improving the efficacy of the applied microbes, (2) enhancing the registration process, and (3) marketing microbes as reliable products to the users. In the recent past, mechanisms of biocontrol 11 Plant Growth-Promoting Bacteria: Fundamentals and Exploitation 331 through antibiosis have been elucidated, and the biocontrol mechanism CNN has been proven to exist. The most obscure biocontrol mechanism known so far, ISR, is presently being studied intensively and major breakthroughs may be expected in the next decade. Plant growth promotion through hormones needs further attention. In the past decade, endophytic microbes received a lot of interest, also for applications as beneficial microbes. The possible advantage of the use of endophytes is two-fold. (1) The plant’s interior may harbor a novel type of microbes. (2) Within the plants, beneficial bacteria are much better protected against competition by rhizosphere bacteria than when they are present in the rhizosphere. It should, however, be noted that a bacterium, which has been isolated as an endophyte, does not automatically resume its endophytic lifestyle when applied to the plant’s exterior. In addition, once inside the plant, it may have to compete with endogenous endophytes. A bottleneck in the understanding of microbe–plant interactions is our limited knowledge about the composition and function of root exudates. This requires a thorough study in which experts in the fields of microbiology, plant physiology, engineering, and soil science should work together. They should use the most modern technology combined with knowledge of the facts from the literature. In addition, a role of volatile organic compounds in biocontrol is becoming clear. The characterization of these compounds and the elucidation of their functions should become a new area for future research. Many studied and commercialized biocontrol strains have been isolated only on the basis of antibiotic activity whereas important traits such as root colonization and compatibility with formulation and industrial practice were not taken into account. Considering the complex and expensive process required to commercialize a plant- beneficial microbial product, it is crucial that, before large research efforts are undertaken, one evaluates all steps of the process, from isolation strategy and laboratory experiments to efficacy, registration, and marketing. Weak and expensive steps should be identified, evaluated and, if necessary, tested first. In this way a lot of time can be saved if one wants to commercialize a strain. A major driver for the application of microbes in the developed world is the fact that the public opinion is opposed against the use of agrichemicals. Several supermarket chains have adopted a policy of zero or very low tolerance against chemical pesticides. Also politicians support this policy, at least in theory. In practice the process to register biological products in the EU is difficult. Here, the products should be registered separately in every member country and this process lasts from several months to several years, depending on the country. Beneficial microbes often have a clearer effect on plants grown under suboptimal conditions than on plants growing under optimal conditions. Suboptimal conditions include limitation for nitrogen, phosphorous, or iron; elevated salinity; and water disbalance. Such conditions are often accompanied by plant diseases. An example is cucumber grown in salinated and naturally infested soil in Uzbekistan. Here, as many as 17% of the cucumber plants were diseased by the indigenous pathogen Fusarium solani. Several European biocontrol strains were very effective in reducing the percentage of diseased plants. 332 C. Pliego et al.

The area of salinated soil will increase, for example, at places with intensive irrigation. Also the predicted climate change will lead to an increase of the area of salinated agricultural soil, especially in the poorer areas in the world. Here, the yields are often low and chemicals are therefore too expensive. It can be expected that the need for the application of the relatively cheap beneficial microbes will largely increase in these areas. Therefore, it is comforting to know that many beneficial microbe isolates from plants growing on nonsalinated soil are salt tolerant and that they can be successfully applied in salinated soil and also in a warm climate, despite the fact that they have been isolated from a cold climate and from other plants than cucumber (Egamberdieva et al., submitted). This strong beneficial effect of bacterization can be explained by the low level of endogenous microbes in this salinated soil (Egamberdieva et al. 2008), which therefore has a low buffering capacity against pathogens. Nowadays many African, Asian, and South American countries with limiting financial resources for chemical fertilizers and pesticides clearly demonstrate their determination to use more microbial products as alternatives. Development of “cottage” type small production units helps local farmers to improve plant’s health and productivity (Harman et al. 2010). This low tech approach, as well as the increase in traditional sales of high-tech microbial products, indicates that the use of beneficial microbes in agriculture will play a more prominent role in the near future. The development of “cottage” type small production units is a clear example of how modern knowledge of general biology and of production and use of beneficial microbes, mainly collected in universities and biocontrol industries in developed countries, can contribute directly to sustainable agricultural practice and environmental protection in the economically less developed part of the world.

Acknowledgments Clara Pliego thanks MEC, grant numbers AGL-2005-06347-C03-01, AGL2008-0543-C02-01 and Junta de Andalucia, Grupo PAI CVI264. Ben Lugtenberg thanks Leiden University, The European Commission, INTAS, the NWO departments of ALW, CW, STW as well as the Netherlands (NWO) – Russian Center of Excellence for support. All of us want to express our sincere gratitude to Prof. Fernando Pliego Alfaro for critical reading and helpful comments on the manuscript.

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Elke Jurandy Bran Nogueira Cardoso, Rafael Leandro de Figueiredo Vasconcellos, Carlos Marcelo Ribeiro, and Marina Yumi Horta Miyauchi

12.1 Introduction

Conifers belong to the class Gymnospermae and include the important and widespread families Araucariaceae and Pinaceae. The Genera Pinus and Araucaria are especially important both economically and ecologically, occur in many regions of the world, and are often associated with several kinds of bacteria known as PGPR during the seedling stage. PGPR help protect plants against microbial pathogens, improve nutrient availability, synthesize mycorrhizae, and promote plant growth, thereby reducing nursery time. The first reports of bacteria stimulating growth in conifers date from the 1960s and 1970s (Table 12.1). An especially important early contribution was Timonin’s (1964) findings that disinfecting seeds of Pinus bank- siana and P. glauca caused a significant reduction in seedling emergence. The reason for this phenomenon was not understood at the time, and only later several authors discovered that the absence of certain microorganisms on the seed surface could have a negative effect on seed germination and adverse plant growth and development. Bowen and Theodorou (1979) subsequently investigated the interaction of ectomycorrhizal fungi (EMF) and soil bacteria, and reported that the bacteria had a stimulating effect on the fungi. Recently, such bacteria came to be known as mycorrhiza helper bacteria (MHB) (Frey-Klett et al. 2007). By the end of the 1970s many papers had reported positive interactions between plants and bacteria and it had become common practice to refer to these bacteria by the name PGPR (Vessey 2003). Since then, there has been a steady increase in studies on interactions between conifers and PGPR (Fig. 12.1). One of the reasons that interest in these associations has a difficulty that came across in demonstrating their economical benefits. Conifers are widely used in forestry, because they produce wood valued for construction and furniture, for producing charcoal, and most importantly, as a source of cellulose for industries.

E.J.B.N. Cardoso (*), R.L. de F. Vasconcellos, C.M. Ribeiro, and M.Y. Horta Miyauchi Soil Microbiology Laboratory, Department of Soil Science, Luiz de Queiroz College of Agriculture, Sa˜o Paulo State University, Piracicaba, Sa˜o Paulo, Brazil e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 345 DOI 10.1007/978-3-642-18357-7_12, # Springer-Verlag Berlin Heidelberg 2011 4 ... ads tal. et Cardoso E.J.B.N 346

Table 12.1 The most studied PGPR in conifers, their mode of action, the host plants, and references Action PGPR Effects on plants Conifer species References Hormones Bacillus sp. Pseudomonas Root length; shoot dry weight; Pinus pinaster; Barriuso et al. (2005); Bent et al. fluorescens M20; root weight; seed P. pinea; (2001); Dubeikovsky et al. P. fluorescens BSP53a; germination P. roxburghii (1993); Singh et al. (2008, P. polymyxa L6; 2010) Chryseobacterium balustinum; Arthrobacter oxydans Siderophores Bacillus sp; Pseudomonas Root length; shoot dry weight; P. pinaster; Barriuso et al. (2005); Singh fluorescens; Staphylococcus sp; root weight; seed P. pinea; et al. (2008, 2010) Arthrobacter oxydans germination P. roxburghii Phosphate Curtobacterium sp.; Burkholderia Shoot height and dry P. pinaster; Barriuso et al. (2005); Rincoń solubilization sp.; Staphylococcus sp.; mass P. pinea; et al. (2008); Singh et al. Pseudomonas fluorescens P. halepensis; (2008, 2010) P. roxburghii MHB B. cereus; Root length; Shoot length; No. P. sylvestris; P. contorta; Bending et al. (2002); Frey- B. sphaericus; leaves initiated; Shoot dry P. taeda; P. elliottii; Klett et al. (1999); Schrey P. fluorescens; Streptomyces sp. weight; Root dry weight Pseudotsuga menziesii; et al. (2005) Picea abies Induced systemic Streptomyces sp. – Picea abies Lehr et al. (2008) resistance P. fluorescens; Chryseobacterium Root length; shoot dry weight; P. pinea; Garcia et al. (2004) balustinum; Enterobacter root weight; neck root intermedius; diameter; stem length; Phosphorobacillus latus Incorporation of thymidine and leucine Antagonism B. subtilis; – P. roxburghii Singh et al. (2008, 2010) P. aeruginosa ACC degradation Staphylococcus sp. – P. pinaster; P. pinea Barriuso et al. (2005) 12 PGPR in Coniferous Trees 347

Fig. 12.1 Publications about PGPR on conifer species from 1960 to 2010

Generally conifers are not very tedious plants, and most of them grow well on poor soils. Several species, however, are currently at risk of extinction due to widespread deforestation and land use change related to the spread of development and the production of paper pulp (Farjon and Page 1999). Another reason to study conifer–PGPR interactions is thus related to ecological considerations. The increasing cultivation of conifers naturally results in increased levels of plant disease and the increased application of industrial fertilizers and pesticides, generating expenses that must then be offset by higher yields and lumber prices. Several mechanisms to promote growth have been identiﬁed in different PGPR groups or isolates (Table 12.1). In this chapter we present an overview of the importance of coniferous trees and of their possible interactions with PGPR.

12.2 Economic, Environmental, and Cultural Aspects of Conifers

Conifers belong to the phylum Pinophyta, class Pinopsida, order Pinales, with eight or nine families, comprising 70 genera and 630 species (Earle 2009). They are ubiquitous on the planet, with the exceptions of deserts and certain small sea- islands (Fig. 12.2). Conifers have diversiﬁed to tolerate a broad range of climatic conditions and soil types, and some of them can thrive even in very severe 348 E.J.B.N Cardoso et al.

Fig. 12.2 The worldwide distribution of conifers, with gray areas indicating the occurrence of conifer forests [adapted from Farjon and Page (1999)] environments. For example, some species are adapted to survive extreme droughts or soils containing high concentrations of heavy metals. Conifers are sometimes considered fossil plants, having inhabited the planet for at least 320 million years and having survived the rise of the now-dominant phanerogamic plants. The oldest and the tallest trees on Earth are conifers: a 9,550-year-old Picea abies in Switzerland and a 115.56-m tall Sequoia sempervi- rens in the USA, respectively. Many coniferous trees are currently at risk of extinction because of large-scale forest exploitation for a variety of purposes, especially during the second half of the last century. While the most important conifer genus in the world is Pinus,in Australia and South America the genus Araucaria predominates. In Brazil, vast stands of forests dominated by the species Araucaria angustifolia have been destroyed to harvest its valuable wood and edible seeds, and today only 3% of the original Araucaria forest remains. Such intensive deforestation modiﬁes the chemical, physical, and biological attributes of soil, reducing its fertility and its potential for plantations of any kind (Nogueira et al. 2006; Miyauchi 2008; Bini 2009). Of the 630 known species of conifers, 200 are currently listed as endangered and 20 of those as critically endangered (Farjon and Page 1999). While some pine species are now strictly protected, their remnant populations may be too small to possess enough genetic variability to guarantee their survival (Farjon et al. 1993). Coniferous trees have also played a prominent role in many different cultures and are well represented in mythology and folklore. In some religions they are considered sacred and used as amulets or in rituals and spiritual ceremonies to protect against bad spirits. Some conifers have been adopted as national symbols and are featured on countries’ ﬂags, while in the arts they are often associated with certain artists like van Gogh or types of art like millenarian Japanese bonsai culture (FAO 1995). 12 PGPR in Coniferous Trees 349

Conifers produce a broad range of timber suited for various purposes with some species growing fast and producing soft wood and others producing very hard woods. Therefore, the plantation of coniferous trees is utmost important. Pinus accounts for the second largest plantation area in the tropics, estimated at almost ﬁve million ha in 1990 (Earle 2009) and surpassed only by Eucalyptus. The most widely cultivated species in the USA are P. contorta and P. nigra, while P. radiata dominates in New Zealand and P. elliottii in Brazil. These species are rustic and grow well on poor soils. Their wood is light colored and contains long ﬁbers, which makes them ideal for producing strong paper. Some kinds of pines also produce commercially important resins. Brazil, the fourth-largest timber producer in the world, had 1.94 million ha of land planted in Pinus in the year 1999. Besides wood, conifers can also produce essential oils, resins, bio-fuels, edible seeds, and medical products, in addition to their use for ornamental purposes. Conifers play an important ecological role such as maintaining soil cover and interacting with many macro- and microorganisms, and they are sometimes an important food source for animals. For instance, in Brazil’s Araucaria forest, seeds of the dominant tree A. angustifolia are the primary food source for the Azure Jay (Cyanocorax caeruleus), as well as for many rodents and other animals.

12.3 Mechanisms of PGPR in Conifers

There are several possible mechanisms through which bacteria can affect plant growth and development, some due to actions exerted directly on the host plant, as, for example, liberation of auxins in the rhizosphere, called direct mechanisms, and others by means of their inﬂuence on other organisms, as can be exempliﬁed by antagonism against pathogens of the host plant, affecting it indirectly through the biocontrol of disease agents (Fig. 12.3).

12.3.1 Direct Mechanisms

Plant growth-promoting rhizobacteria (PGPR) can beneﬁt plant development through a variety of direct mechanisms, typically by producing certain metabolites that act on the plant or by increasing the availability of plant nutrients in the rhizosphere. These metabolites are mostly substances that regulate plant growth. While their activity is well documented in agricultural crops, similar research with conifers remains incipient (Teixeira et al. 2007). Interactions between PGPR and conifers have been studied in the genera Araucaria, Picea (spruce), Pinus, Pseudotsuga (Douglas ﬁr), and Tsuga (hemlock) by number of workers (Bent et al. 2001; Brunetta et al. 2007; Vasconcellos and Cardoso 2009; Singh et al. 2010). The best-studied PGPR belong to Arthrobacter, 350 E.J.B.N Cardoso et al.

Fig. 12.3 Direct and indirect bacterial plant growth promotion mechanisms studied in conifer trees

Curtobacterium, Bacillus, Burkholderia, Chryseobacterium, Enterobacter, Paenibacillus, Phosphorobacillus, Pseudomonas, Staphylococcus, Serratia,and Streptomyces (Enebak et al. 1998; Garcia et al. 2004;Barriusoetal.2005). An extensive screening of PGPR in conifers was carried out by Barriuso et al. (2005) in the rhizosphere of Pinus pinea and Pinus pinaster, when these were colonized by ectomycorrhizal fungus (EMF) Lactarius deliciosus. Of the 720 initially isolated bacterial strains, 389 were tested for their ability to degrade 1-aminocyclopropane-1-carboxylic acid (ACC) and to produce auxins and siderophores, or to dissolute phosphates. Thirty-eight percent of the isolates scored positive for at least one of the screened plant growth-promoting (PGP) characteristics. While the majority of the isolates capable of mobilizing nutrients were associated with the mycorrhizosphere of P. pinaster, the bacteria that produced growth regulators were mostly isolated from P. pinea. One hundred and forty-seven isolates were submitted to PCR-RAPD (random amplified polymorphic DNA), rendering ten groups with 85% similarity, a result that suggests low diversity in 12 PGPR in Coniferous Trees 351 this system. Finally, one isolate of each group was characterized by sequencing the gene 16SrRNA, which revealed similarities of isolates with Arthrobacter, Bacillus, Burkholderia, Curtobacterium, and Staphylococcus. Other advantageous characteristics for a PGPR are the ability to produce indole acetic acid (IAA) or to dissolve phosphate. These abilities were found in bacteria of the genus Pseudomonas isolated from the rhizosphere of Pinus roxburghii. The isolate Pseudomonas aeruginosa PN1 was a good growth promoter for this host plant and demonstrated a strong chemotaxis towards its root exudates (Singh et al. 2010). Earlier, growth promotion of P. pinea by PGPR was reported by Garcia et al. (2004). The PGPR isolates tested in that experiment were originally isolated from Lupinus hispanicus, L. albus, and Lactuca sativa, and were classified as Chryseo- bacterium balustinum, Enterobacter intermedius, Pseudomonas fluorescens, and Phosphorobacillus latus ATCC 33085. All were able to produce auxins and dissolve calcium phosphate, and established well in the rhizosphere of the host plant, sometimes modifying the chemical composition of the plant. However, they affected different growth parameters, suggesting their involvement in other nonspecific strategies. PGPR with the ability to produce plant growth-regulating hormones, like auxins and cytokines, were tested on P. contorta (lodgepole pine) (Bent et al. 2001). Paenibacillus polymyxa Pw2 and L6 (originally isolated from the P. contorta rhizosphere) produced the greatest biomass and lateral root growth, along with the highest IAA synthesis, whereas the application of P. fluorescens M20 resulted in high levels of dihydrozeatin riboside cytokine (DHZR) in the roots. It is well known that exogenous application of auxins stimulates the production of lateral roots in plants, while cytokines inhibit this growth (Charlton 1996). Nevertheless, the proportions of the two hormones produced by these PGPR could not explain the different responses obtained by following their application to the host plant (Bent et al. 2001). Experiments to date have not been able to determine to what degree isolates may cause different effects in their original hosts compared to other hosts. Some PGPR isolates produce different effects when applied to different species of the same conifer genus. For example, several PGPR isolates originating in the rhizospheres of P. taeda and P. caribea var. hondurensis were added to commercial plant growth substrates to evaluate their effect on P. taeda, P. caribea var. hondurensis, P. elliottii,andP. oocarpa. All the isolates performed best on P. taeda. Some incompatibilities may have arisen from the dynamics of the inoculant and cell concentrations, or from competition with native rhizosphere inhabitants (Vonderwell and Enebak 2000). On the other hand, studies have demonstrated the specificity of PGPR in conifers, attributing this effect to differences in root exudates and plant genotypes (Bent et al. 2001, 2002). The growth-promoting potential of 12 PGPR isolates was shown by Enebak et al. (1998), who reported that all of them promoted seedling emergence in Pinus. This is a very desirable trait, since seedlings are highly susceptible to plant pathogens during their early development and the shortening of this phase that could 352 E.J.B.N Cardoso et al. help decrease seedling mortality. Following emergence, however, different PGPR isolates caused contradictory responses, some inhibiting root growth and others promoting it on the same host plant. These results suggest that stimulating the emergence of seedlings and stimulating the growth of established plants are differential and sometimes host-specific effects. However, Vasconcellos and Cardoso (2009) documented solely beneficial effects when inoculating P. taeda with PGPR isolated from the rhizosphere of A. angustifolia. The most effective of the five actinobacterial isolates tested was Streptomyces A43, which promoted root and shoot growth, suggesting a lack of specificity for this isolate. The endophytic behavior of PGPR can be very advantageous because it protects bacteria from competition, predation, and other negative effects of the native microbiota. This internal colonization of the host plant has been shown for P. polymyxa Pw2R and P. fluorescens Sm3-RN on a hybrid host plant (Picea glauca P. engelmannii)asdemonstratedbyShishidoetal.(1999). However, endospore-producing rhizobacteria such as Bacillus and Paenibacillus may be isolated erroneously as endophytes because most methods to disinfect plant tissues are not effective for these genera (Bent and Chanway 2002). These studies illustrate the effectiveness of PGPR and their potential for plant nurseries or reforestation programs. Additional studies are urgently needed to identify the mechanisms of PGPR for every host plant, since one isolate may show multiple lines of action.

12.3.2 Indirect Mechanisms

The only two conifers that have been used in in vivo experiments on bio-control of plant diseases using PGPR were P. roxburghii and Picea abies, and the bacterial genera involved were Bacillus, Pseudomonas, and Streptomyces. In the first phase of the experiments, several bacterial strains were isolated from the rhizosphere of P. roxburghii and their antagonism was tested in vitro, using the dual-culture method, against some pathogenic fungi. In these tests, the best-performing isolates were Bacillus subtilis BN1 and P. aeruginosa PN1. The Bacillus isolate BN1 was classified as PGPR due to its potential to inhibit the mycelial growth of several pathogenic fungi of economic importance in plant nurseries, such as Macropho- mina phaseolina, Fusarium oxysporum, and Rhizoctonia solani. The only control of such diseases until then consisted of soil fumigation, which also eliminates beneficial soil microorganisms. The isolate B. subtilis BN1 showed effective control of M. phaseolina in vitro and in vivo. P. aeruginosa also demonstrated antagonism against this pathogen, and it was shown to be capable of colonizing the roots of the host plant in the absence as well as in the presence of M. phaseolina (Singh et al. 2008, 2010). Many actinobacteria have also been classified as PGPR. Thus, the isolate Strepto- myces AcH 505 has been called a MHB because it promoted the colonization of host plants by mycorrhizal fungi and also had antagonistic action against most isolates of 12 PGPR in Coniferous Trees 353

Heterobasidion abietinum on Picea abies. However, this PGPR was not considered efficient because it did not control the pathogens significantly to which it was exposed (Lehr et al. 2007). Another Streptomyces isolate, A43 (AB024441), obtained from the rhizosphere of A. angustifolia, was highly antagonistic against the plant pathogen F. oxysporum (Fig. 12.4) and Armillaria sp., which cause disease in A. angustifolia and Pinus spp. The isolate inhibited the mycelial growth of both pathogens in dual culture and showed the development of rhizomorphs in liquid culture. A cell free culture filtrate of this actinobacterial strain maintained the similar antagonistic action (Vasconcellos 2008) (Figs. 12.4 and 12.5). Some bacterial isolates produce, in their secondary metabolites, toxic substances such as hydrocyanic acid (HCN) and enzymes b-1,3 glucanase, chitinase, besides iron chelating siderophores. Of 20 strains of Bacillus and Pseudomonas isolated from the roots of chir-pine, only three inhibited the growth of pathogenic fungi

Fig. 12.4 Interaction of a Bacillus and an actinobacterial strain isolated from A. angustifolia Xa pathogenic Fusarium oxysporum in culture medium

Fig. 12.5 Colony of an actinobacterium covered by spores growing on soil in the neighborhood of a P. taeda seedling 354 E.J.B.N Cardoso et al. through the production of HCN, and all belonged to the genus Pseudomonas. The ability to produce b-1,3 glucanase was detected in both genera. This enzyme, as well as chitinase, is involved in the lysis of the hyphal wall of pathogenic fungi. As mentioned previously, the isolates PN1 and BN1 were the most effective in antagonism tests against conifer pathogens, and also produced the greatest number of different substances that inhibit the development of fungi. This may have been the main cause for their effectiveness as control agents of the pathogenic fungi (Singh et al. 2008, 2010). Streptomyces aurantiacogriseus and S. setonensis isolated from A. angustifolia were good producers of chitinases and effective in inhibiting the growth of Armillaria mellea in vitro (Vasconcellos and Cardoso 2009). The synthesis of siderophores is another interesting mechanism of biological control. Siderophores have no direct effect on the pathogens, but they scavenge all iron present in the rhizosphere, causing pathogens in the neighborhood to starve. The isolates BN1 and PN1 synthesized great amounts of siderophores, a characteristic that they have in common with the isolates of Bacillus and Staphylococcus obtained from the mycorrhizosphere of P. pinea and the mycosphere of P. pinaster (Barriuso et al. 2005), respectively. We have also found in our laboratory siderophore producing actinobacteria from the rhizosphere of A. angustifolia, showed ability to control F. oxysporum, Cylindrocladium candelabrum, C. pteridis, and Armillaria sp. (unpublished data). The lack of information about bio-control in vivo raises many doubts about the effectiveness of these control agents in the field. In the meantime, the information is limited to their potential as bio-control agents in vitro and to a few mechanisms responsible for this action. There is thus a great need for continued experimentation, especially with seedlings but also with adult plants, to test their effects in vivo. Other reports about the interaction of PGPR and conifers could not be found. Most studies are limited to laboratory tests, and therefore, almost impossible to foresee the real effectiveness of these PGPR in the field, where they are exposed to fierce competition from native microorganisms and unfavorable conditions, which may alter their bio-control potential. Another very interesting mechanism by which some PGPR achieve bio-control of some conifer diseases has been recently reported, and consists of the induction of local and systemic resistance in the host plant. In P. abies the bacterial isolate GB 4-2 (Streptomyces drozdowiczii) increased the survival of seedlings in the presence of plant pathogens. When confronted with the pathogen Heterobasidium abietinum in dual culture or in spore germination tests, however, the bacterial isolate stimulated the elongation of the germination tube and hyphal growth (Lehr et al. 2007, 2008). The seedlings inoculated with this pathogen maintained their resistance to the disease, thus demonstrating that it was not a simple interaction between two microorganisms, but involved more complex situation. Other strategies were examined until it was finally discovered that the isolate induced local and systemic resistance in the host plant by thickening and lignifying the cell walls in roots, which restricted fungal growth in the vascular system, and also by increasing the size of the xylem and the production of peroxidases. Peroxidase is an enzyme responsible for the exclusion of certain pathogens, such as Botrytis 12 PGPR in Coniferous Trees 355 cinerea, from the host plant. This bacterial isolate is thus able to control fungi that survive or escape the toxic compounds produced by PGPR. The effectiveness of this isolate has been confirmed repeatedly, including in experiments with adverse conditions, such as nonsterilized soil, and with other host plants. These findings make this actinobacterial isolate a very strong candidate for future use as biocontrol agent. With the isolate AcH 505, however, Lehr et al. (2007)reporteda result contrary to the one described above. As previously mentioned, this isolate acted as a growth inhibitor for most isolates of the pathogen Heterobasidion and also as a MHB. Nevertheless, when tested in vivo on Pinus abies,thisactinobac- terium suppressed the defense mechanisms of the plant by reducing the expres- sionofthegenePaSpi2,agenelinkedtodefense against pathogens, as well as the production of the enzyme peroxidase. For this reason, this isolate does not seem to be promising for future use as PGPR in conifers. These two reports on systemic, induced, or suppressed plant resistance open new prospects for research on the mechanisms underlying the different kinds of interactions between PGPR and host plants. Much more data from greenhouse and nursery studies are needed to complement these findings before specific isolates can safely be recommended for protecting plants. Among the various indirect mechanisms responsible for the action of PGPR, it is also important to consider bacteria that help mycorrhizal fungi establish on plant roots, therefore facilitating another phenomenon which promotes the growth of host plants. A. angustifolia has been shown to be very dependent on AMF (Souza and Cardoso 2002), although other conifers are normally associated with EMF, which results in better plant nutrition and development. Actinobacteria of the genus Streptomyces have been reported to promote mycelial growth of Amanita muscaria and Suillus bovinus, two EMF. The isolates AcH 505 (Strepto- myces sp.) and AcH 1003 (Streptomyces annulatus), besides promoting fungal growth in vitro, also increased root colonization of P. abies by A. muscaria and of P. sylvestris by S. bovinus in vivo. The underlying mechanism for this increase in growth and root colonization of EMF may be the modification of its gene expression. Some DNA sequences with modified gene expressions were cloned, and alterations in certain genes with known or unknown functions were observed. Some of the known genes were related to cellular growth, basic metabolism, and transduction of signaling molecules. The increase of the AmAacs gene expression induced by the bacterial isolate AcH 505 is responsible for increased production of ergosterol, which is fundamental for fungal growth. One of the most activated genes was AmCyp40, up to six times, which demonstrates its key role in hyphal growth. Few genes involved in the orientation of cellular growth were also altered, suggesting that there may be a direct interaction between the fungus and the PGPR. Confirmation of this hypothesis, however, awaits more detailed studies (Schrey et al. 2005). The effectiveness of co-inoculation of EMF and MHB has been studied with the goal of decreasing the amount of EMF inoculant required for optimal colonization of conifer seedlings (Frey-Klett et al. 1999). P. fluorescens (a MHB), when co- inoculated in small amounts, showed a positive response and allowed for a 356 E.J.B.N Cardoso et al. significant reduction of the EMF inoculant. This MHB, at the low dose of 102 UFC g1 in co-inoculation with the EMF, resulted in a root colonization rate of 70% in Pseudotsuga menziesii, while the same concentration of the EMF inoculant alone in the same host plant achieved only about 30% colonization rate. When EMF was applied at double concentration but without the MHB, the highest root colonization rate recorded was 64%. One of the drawbacks of this technique has been the low survival rate of most of these bacteria after their inoculation into soil. However, in these experiments, even 1 year after the inoculation of small doses of these MHB, the plants which had been inoculated with EMF and MHB together showed much better growth kinetics than the ones which had only received EMF. These findings indicate that it should be possible to produce vigorous and well- developed seedlings within a short time at a reasonably low cost (Frey-Klett et al. 1999). The mycorrhizosphere of P. sylvestris with Suillus luteus mycorrhiza was dominated by the genera Bacillus and Burkholderia, which normally do not favor root colonization by EMF. The latter even inhibited mycorrhiza formation by 97%. In spite of this, many of these isolates play a role as PGPR in conifers, but not because of their positive effect on root colonization or on the growth of the EMF (Bending et al. 2002). With regard to AMF, the symbionts of A. angustifolia, it was found that Streptomyces isolated from the Araucaria rhizosphere promoted the germination of Gigaspora rosea spores in vitro, when both organisms were incubated in the same Petri dish (Vasconcellos 2008). It has also been observed that geosmin, a highly volatile by-product of most actinobacteria, has a stimulating effect on AMF (Carpenter-Boggs et al. 1995). Based on available literature, our research group is the only one now examining this line of research. The influence of bacterial growth metabolites on the preferential direction (chemotaxis) of the germ tube of AMF spores has also been observed, but these findings still require confirmation.

12.4 Conclusions and Perspectives for the Future

The practical use and routine employment of PGPR remains a dream, and it may be many years before our understanding is sufﬁcient to warrant its successful application in different systems. Especially with regard to the conifers, we need to improve basic knowledge while reﬁning and addressing a large number of still-vague questions. Another problem to be overcome is competition with the chemical plant protection compounds that currently dominate the market. Several modes of activity of PGPR have already been thoroughly studied, and a few commercial inoculants are available in some countries, but only for restricted uses in agriculture. Their application for environmental problems, especially in forestry, is still very limited. It is also true that most forestry companies have traditionally relied on agrochemicals and are not easily convinced to try biological products. 12 PGPR in Coniferous Trees 357

Seedlings are currently produced via cloning, diseases and pests controlled with pesticides, and fertilization, when needed, carried out with commercially produced synthetic compounds. Modifying these habits will require a paradigm shift. For many years, traditional methods have generated large profits for forestry. It is still not known whether the PGPR, after inoculation into plants, will maintain their viability and effectiveness in the soil for long time periods. Another challenge is to produce inoculants that are well adapted or even specific to each conifer species or cultivar. Inoculants must be resistant to a broad range of environmental conditions and soil properties such as fertility, temperature, and moisture, so that they can survive with conifers around the whole world. Finally, it will not be easy to incorporate these new products into existing management practices. There is great interest in today’s world in reducing environmental impacts by means of sustainable interactions with nature and a higher quality of life. Used incorrectly, nitrogenous or phosphate fertilizers may leach into the subsoil, lead to acidification or denitrification of the soil, and cause water, soil, and air pollution. Combined with other anthropogenic impacts, such pollution can lead to the eutrophication of watersheds, resulting in the growth of algae which may liberate toxins into the water. The generalized use of herbicides, insecticides, and fungicides affects the soil microbiota and especially those organisms that live in close contact with plants, such as the mycorrhizal fungi and rhizobacteria that promote nutrient cycling and nutrient availability and protect plants via several mechanisms. In this context, switching to PGPR inoculants would appear to be beneficial and studies on their economic viability are urgently needed. Governmental institutions also have a very important role to play in facilitating research and development of PGPR inoculants for conifers. Another barrier to be overcome in the development of inoculants concerns the inoculation media. Normally bacteria are applied in lyophilized condition and dispensed in substrates as peat, coconut fiber, rice grains, or alginate, which keep living bacterial cells alive and active before their use in agriculture. Innovative ideas about inoculation media and new strategies, such as mixing PGPR with other elements like chemical fertilizers, are needed. It is also a priority to investigate the effects of continuous applications of such inoculants on the health, interactions, and function of native soil organisms. As shown in Fig. 12.1, only a small number of papers on this subject have been published over the last decades, especially on PGPR in conifers. Although there are still many barriers to be surmounted, the biotechnological future of PGPR seems to be promising and may 1 day prove of vital importance for both economic and environmental reasons.

Acknowledgments The authors are grateful to FAPESP for a research grant (BIOTA- Project No. 2001/050146-6) and acknowledge the support of the technicians Fernando Baldesin and Denise Mescolotti. EJBNC also expresses her recognition for a CNPq grant. 358 E.J.B.N Cardoso et al.

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Lehr NA, Schrey SD, Hampp R, Tarkka MT (2008) Root inoculation with a forest soil streptomy- cete leads to locally and systemically increased resistance against phytopathogens in Norway spruce. New Phytol 177:965–976 Miyauchi MYH (2008) Propriedades microbiolo´gicas e bioquı´micas do ciclo do carbono em solo sob diferentes coberturas vegetais. http://www.bibliotecadigital.uel.br/document/?code¼ vtls000128853. Cited 15 Jun 2010 Nogueira MA, Albino UB, Brandaõ-Junior O, Braun G, Cruz MF, Dias BA, Duarte RTD, Gioppo NMR, Menna P, Orlandi JM, Raiman MP, Rampazo LGL, Santos MA, Silva MEZ, Vieira FP, Torezan JMD, Hungria M, Andrade G (2006) Promising indicators for assessment of agroecosystems alteration among natural, reforested and agricultural land use in southern Brazil. Agric Ecosyst Environ 115:237–247 Rincoń A, Valladares F, Gimeno TE, Pueyo JJ (2008) Water stress responses of two Mediterranean tree species influenced by native soil microorganisms and inoculation with a plant growth promoting rhizobacterium. Tree Physiol 28:1693–1701 Schrey SD, Schellhammer M, Ecke M, Hampp R, Tarkka MT (2005) Mycorrhiza helper bacterium Streptomyces AcH 505 induces differential gene expression in the ectomycorrhizal fungus Amanita muscaria. New Phytol 168:205–216 Shishido M, Breuil C, Chanway CP (1999) Endophytic colonization of spruce by plant growth- promoting rhizobacteria. FEMS Microbiol Ecol 29:191–196 Singh N, Pandey P, Dubey RC, Maheshwari DK (2008) Biological control of root rot fungus Macrophomina phaseolina and growth enhancement of Pinus roxburghii (Sarg.) by rhizosphere competent Bacillus subtilis BN1. World J Microbiol Biotechnol 24:1669–1679 Singh N, Kumar S, Bajpai VK, Dubei RC, Maheshwari DK, Kang SC (2010) Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine. Crop Prot 29:1142–1147 Souza MMSR, Cardoso EJBN (2002) Dependeˆncia micorrı´zica de Araucaria angustifolia (Bert.) O. Ktze sob doses de fo´sforo. R Bras Ci Solo 26:905–912 Teixeira DA, Alfenas AC, Mafia RG, Ferreira EM, de Siqueira L, Maffia LA, Mounteer AH (2007) Rhizobacterial promotion of eucalypt rooting and growth. Braz J Microbiol 38:118–123 Timonin MI (1964) Interaction of seed-coat microflora and soil microorganisms and its effects on pre- and post-emergence of some conifer seedlings. Can J Microbiol 10:17–22 Vasconcellos RLF (2008) Actinobacte´rias da rizosfera de Araucaria angustifolia com potencial biotecnolo´gico. http://www.teses.usp.br/teses/disponiveis/11/11138/tde-18112008-150538. Cited 15 Jun 2010 Vasconcellos RLF, Cardoso EJBN (2009) Rhizospheric streptomycetes as potential biocontrol agents of Fusarium and Armillaria pine rot and as PGPR for Pinus taeda. BioControl 54:807–816 Vessey JK (2003) Plant growth promoting rhizobacteria as biofertilizers. Plant Soil 255:571–586 Vonderwell JD, Enebak SA (2000) Differential effects of rhizobacterial strain and dose on the ectomycorrhizal colonization of loblolly pine seedlings. Forest Sci 46:437–441 . Chapter 13 Perspectives of PGPR in Agri-Ecosystems

Meenu Saraf, Shalini Rajkumar, and Tithi Saha

13.1 Introduction

One of the ﬁnest success stories of post independent era is the green revolutions in 1960s, which transformed the country from “begging bowl” to “breadbasket.” This has been possible because of the use of chemical fertilizers and hybrid crops. However, in the long run, the use of chemical fertilizers had led to many serious problems which have forced the scientists to explore the other alternatives. One approach in this direction has been the use of biofertilizers, better known as plant growth-promoting rhizobacteria (PGPR). PGPR are those bacteria which are able to colonize plant root systems and promote plant growth (Kloepper and Schroth 1978). PGPR can affect the plant growth directly by: (1) production or changing the concentration of phytohormones such as IAA (Mordukhova et al. 1991), gibberellic acid (Mahmoud et al. 1984), cytokinins (Tien et al. 1979), and ethylene (Glick et al. 1995); (2) solubilization of mineral phosphates and other nutrients (De Freitas et al. 1997); (3) asymbiotic N2 ﬁxation (Kennedy et al. 1997), or indirectly by showing antagonism against phytopathogenic microorganisms, e.g., Fusarium spp. by production of siderophores (Scher and Baker 1982), b-1,3-glucanase (Fridlender et al. 1993), cyanide (Flaishman et al. 1996), chitinases (Renwick et al. 1991), and antibiotics (Shanahan et al. 1992). The rhizobacteria may be present (1) in the soil surrounding roots, utilizing the metabolites leaked from roots as the growth nutrients; (2) on the root surface or rhizoplane; (3) in the root tissue, inhabiting spaces between cortical cells; and (4) inside the cells in specialized root structures or nodules. Thus, based on their root proximity and intimacy of association, PGPR are categorized into two different classes: (1) extracellular PGPR (ePGPR), present in rhizosphere; and (2) intracellular

M. Saraf (*) Department of Microbiology, Gujarat University, Ahmedabad 380009, Gujarat, India e-mail: [email protected] S. Rajkumar and T. Saha Institute of Science, Nirma University, S. G. Highway, Ahmedabad 382481, Gujarat, India

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 361 DOI 10.1007/978-3-642-18357-7_13, # Springer-Verlag Berlin Heidelberg 2011 362 M. Saraf et al.

PGPR (iPGPR), exist inside the cells in specialized nodular structures (Gray and Smith 2005). The early reports of iPGPR dating back to 1880s, talk about the isolation of rhizobia from the root nodules and demonstrated their ability to convert the atmospheric nitrogen into plant usable forms. Since that time, intensive researches have taken place in this field which has laid the foundation upon which the agricultural use of PGPR is based. To know the PGPRs better, the investigations are going on at genomic, proteomic, cellular, whole plant, and environmental level. However, the field performance of PGPR has not been very satisfactory and only a few studies have shown consistent performance of these PGPR in the soil. This may be due to various abiotic factors like composition and properties of the soil, availability of soluble organic products and molecular oxygen, etc.; and biotic factors like interactions between PGPR and resident microorganisms, protozoan predation, and bacterial parasitism. The most important factor determining the survival of PGPR is the competition for the limiting resources between the PGPR and natural fauna of the soil. However, PGPR inoculation has been found to be effective in a nutrient deficient or stressed soil, where the development of the resident microorganism is poor (Strigul and Kravchenko 2005).

13.2 Rhizospheric Biodiversity

Biodiversity of soil microbes has been regarded as human and vegetation life resource, especially the one connected with biological and environmental resources because of their vital role in various biogeochemical cycles running through the flora, fauna, and the life of microbes itself. Microbe community composes one of the important components of the soil. A large variety of microbial species reside in the soil. This biodiversity and their activity depend upon the physical and chemical properties of the soil along with the climate and vegetation of that area. Soil microbes are one of the biota communities, which are very interesting to be studied in order to find out their existence and uses. It is well known that the soil microbes show symbiosis and commensalism with their host, however not every soil microbe is compatible with its host and habitat. Each type of soil microbe fills as a unique niche and plays a different role in nutrient cycling and soil structure. The microorganisms colonizing in the rhizosphere can be grouped into bacteria, fungi, actinomycetes, algae, and protozoa. Out of these, the microbes that are useful as biofertilizer as the examples cited are mostly Biofertilizers and not Biocontrol agents are Klebsiella, Nitrosomonas, Thiobacillus, Lactobacillus, Azotobacter, Azospirillum, Rhizobium, Bacillus, Pseudomonas, Enterobacter, Arthrobacter, Alcaligenes, Serratia, Streptomyces, and Frankia (Sri Widawati et al. 2004). Now-a-days, the rhizobacterial community structure can be characterized by the advanced phenotypic and genotypic approaches. Phenotypic methods that rely on the ability to culture microorganisms include standard plating methods on selective 13 Perspectives of PGPR in Agri-Ecosystems 363 media, community level physiological profiles (CLPP) using the BIOLOG system (Garland 1996), phospholipid fatty acid (PLFA) (Tunlid and White 1992), and fatty acid methyl ester (FAME) profiling (Germida et al. 1998). Culture-independent molecular techniques are based on direct extraction of DNA from soil and 16 S- rRNA gene sequence analysis, bacterial artificial chromosome, or expression cloning systems (Rondon et al. 1999). An understanding of the mechanisms is imperative to utilize its potential in agri ecosystems plant productivity. The mechanism of action of PGPR falls into two categories – direct and indirect. Direct mechanisms are those that occur inside the plants and directly affect the plants metabolism like nitrogen fixation, phosphate solubilization, production of siderophores, induction of systemic resistance (ISR), etc. whereas, indirect mechanisms include improved nutrient availability to plant, free nitrogen fixation, inhibition of microbes that have a negative growth on plants. Some of the important mechanisms have been discussed in detail.

13.3 Mechanisms of Plant Growth Promotion

A thorough understanding of the PGPR action mechanisms is fundamental to manipulating the rhizosphere in order to maximize the process within the system that strongly inﬂuence the plant productivity in different agri-ecosystems. These includes both direct and indirect mechanisms to support plant growth.

13.3.1 Nitrogen Fixation

The supply of nitrogen to arable plants is about 42 million tons of fertilizer which is being used annually on a global scale for the production of cereals such as wheat, rice, and maize alone as per an estimate. The global demand of fertilizers is estimated to increase by 3.2% reaching to about 175.8 Mt (Heffer and Prud’homme 2008). Of this, only 50% is lost from the soil–plant system through processes such as leaching, volatilization, and denitriﬁcation leading to adverse environmental effects such as (1) methemoglobinemia in infants due to NO3 and NO2 in water and food; (2) cancer due to secondary amines; (3) respiratory illness due to aerosols of NO2,NO3, and HNO3; (4) eutrophication of surface water; (5) material and ecosystem damage due to HNO3 in rainwater; (6) plant toxicity due to high levels of NO2 and NH4 in soils; (7) excessive plant growth due to more available N; and (8) depletion of stratospheric ozone due to NO and N2O(Doranetal.1996). This has led to resurgence in the use of biological preparations containing nitrogen-ﬁxing organisms as fertilizers for assimilation of nitrogen in the nonlegumes, sustaining environmental health and soil productivity. 364 M. Saraf et al.

Rhizobium is the most predominant genus of bacteria which colonize the roots of legumes forming nodules within which they change to a nonreproductive form called bacteroids and start fixing nitrogen. Specificity in this respect is exhibited by both plants and bacteria. Initially it was thought that rhizobia only fixed nitrogen in symbiosis but several investigators reported nitrogenase activity in species of rhizobia grown in pure culture, particularly the slow-growing “cowpea” strains. Since then, symbiotic associations between nonlegumes and nitrogen-fixing bacteria have been reported. These host plants include tropical grasses, rice, sugarcane, and maize. Notable examples of nonsymbiotic diazotrophs include Azotobacter paspalum which colonizes under a mucilaginous sheath on the roots of Bahia grass (Paspalum notatum) (Dobereiner et al. 1972) and Spirillum lipoferum which colonizes the roots of Digitaria decumbens and also some kinds of maize (Von Bulow and Dobereiner 1975). Blue-green algae are also agriculturally important for fixation of N (as they are distributed widely on soil surface in all kinds of environments and are tolerant of wide ranges of temperature and oxygen tension). Symbi- otic associations between blue-green algae and higher organisms are very effective nitrogen-fixing systems. In all biological nitrogen fixation systems studied, dinitrogen reduction is catalyzed by a highly conserved enzyme complex called nitrogenase, which consists of two component proteins: the iron (Fe-) protein (MW 64,000) and the molybdenum iron (MoFe-) protein (MW 220,000). Together, the nitrogenase proteins catalyze the ATP-dependent reductions of dinitrogen to ammonia and of protons to hydrogen as well as the reduction of alternative substrates such as acetylene or cyanide. From genetic studies, it is clear that nitrogen fixation needs a whole set of auxiliary proteins and regulatory genes to function inside a cell (Burgess and Lowe 1996). Regulation of nitrogen fixation at the transcriptional level is affected by factors such as environmental levels of oxygen and ammonium. Nitrogenase components are oxygen labile and the metabolically expensive nitrogenase system is repressed when the cellular level of fixed nitrogen is sufficiently high. Free-living diazotrophs are more sensitive to the cellular ammonium levels (Merrick 1992). Post-translational modes of regulation operate on the nitrogenase to prevent unproductive fixation of nitrogen. In Rhodospirillum rubrum, R. capsulatus (Purple, nonsulfur photosynthetic bacteria), Azospirillum brasilense, A. lipoferum (microaerophilic, associative bacteria), and Chromatium vinosum (a purple sulfur bacterium), the nitrogenase complex is rapidly, reversibly inactivated by ADP-ribosylation of Fe protein. Optimizing the procedure for screening of efficient diazotropic organisms is essential for obtaining high crop yields. In a study screening for competent diazotrophs, Azospirillum associated with the rhizosphere of maize was isolated using semi-solid N-free and solid selective media. The chromosomal DNA of the most promising isolates was extracted and nifH gene was amplified and sequenced (Fernando et al. 2006). Nitrogen fixation of the isolates is determined in nitrogen-free medium by acetylene reduction assay and ethylene produced is measured using a gas chromato- graph. Field trials of PGPR that act as nitrogen-fixing and phosphate-solubilizing bacteria have indicated increased yields in the rice crop (Sudha et al. 1999), sugar 13 Perspectives of PGPR in Agri-Ecosystems 365 beet (Cakmakci et al. 1999), wheat (de Freitas 2000), canola (de Freitas et al. 1997), maize (Pal 1998), and conifer species (Bent et al. 2002). Another PGPR known for nitrogen fixation properties in addition to P solubilization, production of antibiotics, cytokinin, hydrolytic enzymes, colonization of root hair and cortical cells is Bacillus polymyxa,nownamedPaenibacillus polymyxa (Timmusk et al. 1999). Field trials of Bacillus OSU-142, Bacillus M3, and Microcbacterium FS01 for determining their competency as PGPR from the year 2002 to 2006 have demonstrated highest N content from combined inoculations of Bacillus OSU-142 and Microcbacterium FS01 application (Karlidag et al. 2007). N2-fixing and P-solubilizing abilities of Bacillus OSU-142 and Bacillus M3 have been reported by many workers. Studies conducted to study the effects of coinoculation of PGPR with N2-fixing bacteria such as Bradyrhizobium has shown the positive effects of PGPR on nitrogen fixation by an increase in nodule number or mass (Polenko et al. 1987). However, the coinoculation trials conducted by Lucas Garcia et al. (2004) indicated that coinoculation of PGPR with Sinorhizobium fredii has no positive effect on nitrogen fixation or nodulation. Furthermore, it is demonstrated that mechanism of biological nitrogen fixation, nodulation, and growth effects on plants is different in gram-positive and gram-negative bacteria.

13.3.2 Phosphate Solubilization

Phosphorus, in spite of being a macro-nutrient essential for energy metabolism, sugar production, regulation of a number of enzymes, membrane physiology as well as structure of the genetic material (Saber et al. 2005), is present only in micromolar or even lesser concentrations in the soil. It has been suggested that 0.3–0.5% of P is required on a dry matter basis for optimal growth of a plant during its vegetative stages whereas the amount of P in soil seldom exceeds 10 mM. Phosphorus in the soil is of two types: organic, which constitutes 20–80% of the total soil content, and inorganic, which is complexed with cations and converted into insoluble P. (Fixation of phosphorus with calcium is a process observed in calcareous soils and that with iron and aluminum compounds constitutes acidic soils making the soil P unavailable for the plants.) Limited availability of phosphorus is a global problem. Acidic soils are found in all continents and constitute 30% of the soils worldwide. The deficiency of P is most prominent in acidic soils of the tropical and sub-tropical regions because fixation of P is double the amount added compared to neutral or calcareous soils in acidic soils. Also, in the semiarid tropics bearing sandy soils, deficiency of Pi severely affects crop productivity (Sanchez et al. 1997). The use of artificial P fertilizers to supplement its unavailability requires farmers to apply four times the quantity of fertilizer required otherwise, because plants are able to fix only 5–25% of the applied fertilizer. This global demand of chemical P fertilizers requires US$ 4 billion per annum (Goldstein et al. 1993) posing an 366 M. Saraf et al. economic burden on the society. This rate of employing artificial P fertilizers will deplete the nonrenewable P reserves in the coming 60–90 years (Runge-Metzger 1995). The situation is further worsened by the entry of excessive P onto the surface water posing threats of eutrophication and degradation of the environment making the discovery of alternatives for fixation of the unavailable P imperative. Soil microorganisms generally act as reserves of compounds of labile minerals including phosphorus and their ability to enrich the soil with available P has been reported since the year 1903. The ubiquitously found phosphate solubilizing microorganisms (PSM) increase the yields by enhancing fixation of N by the plants, protecting against pathogens, increasing the availability of trace elements such as iron, zinc, etc., and producing plant growth hormones in addition to making P available to them (Ponmurugan and Gopi 2006) and hence provide lucrative options for use as biofertilizer. The phosphate solubilization activity of microorganisms has been attributed to production of H+ ions and organic acids such as acetate, lactate, oxalate, tartarate, succinate, citrate, gluconate, ketogluconate, glycolate, etc. (Lal 2002). The mechanism of action of the organic acids includes acidification of rhizosphere soil, chelation of anions, and ligand exchange reactions (Omar 1998). A higher concentration of PSM is found in the rhizosphere compared to other sources (Vazquez et al. 2000). Ability of various bacteria and fungi to solubilize insoluble P compounds has been demonstrated. Among bacteria, the most powerful phosphate solubilizers include strains of Pseudomonas, Bacillus, and Rhizobium and the most important fungal genera include Aspergillus and Penicillium (Motsara et al. 1995). Kucey in 1983 has demonstrated that phosphate-solubilizing bacteria constitute 1–50% while fungi only represent 0.5–0.1% of the total respective population. However, fungi have been reported to be better phosphate solubilizers than bacteria by producing larger quantities of organic acids. In addition to this, bacteria have been demonstrated to lose their phosphate-solubilizing capacity on repeated subculturing unlike the phosphate-solubilizing fungi (Kucey 1983). In the laboratory, most of the PSMs are isolated depending on their ability to solubilize various compounds of P such as dicalcium phosphate (DCP), tricalcium phosphate (TCP), and hydroxyapatite (HAP), and a smaller number of PSMs have been shown to solubilize ferric and aluminum phosphates (Halder and Chakrabartty 1993) by production of acids. One of the very first screening media devised for checking the efficacy of PSMs isolated from nonrhizosphere and rhizosphere soils, the rhizoplane, and from other soil and marine environments, is the Pikovskaya’s medium, on which the PSMs form halos/clear zones surrounding their colonies if phosphate is solubilized. The organisms are cultured by serial dilution/enrichment techniques on a media containing insoluble mineral phosphates such as TCP or HAP. A modified screening assay using bromophenol blue was later devised on which the phosphate solubilizers too yielded yellow halos, leading to generation of clear and visible results (Gupta et al. 1994). The soluble phosphate concentration in the culture medium used above has been shown to be directly proportional to the titrable acidity and organic acid (principally 13 Perspectives of PGPR in Agri-Ecosystems 367 gluconic acid) concentration and inversely related to pH. The effects of C and N sources and their concentrations on the screening assays have been marked. Greater solubilization has been observed when ammonium salts are used as the sole source of N compared with use of nitrate salts (Whitelaw 2000); an exception to which was observed by Reyes et al. (1999). In case of C sources, maximum growth yield was observed with sucrose and galactose when Ca–P and Al–P were used as P sources, respectively, in case of the fungus Aspergillus niger (Nahas 1996). All the plate-screening assays are indirect assays, results of which are variable and reliability of which is questionable. Hence, Nautiyal (1999) devised a defined media called the National Botanical Research Institute’s Phosphate Growth Medium (NBRIP) which is about threefold more efficient compared to the Pikovskaya’s medium in broth assay. Recently, Modified Illmer and Schinner (MIS) media has been devised for isolation of PSB from a typical black wheat growing soil from the North-Western wheat belt of New South Wales (Illmer and Schinner 1992). It is a differential media containing insoluble phosphates and reduced amounts of sugars using root exudates as the inoculums. The results of the same were validated through pot trials (Harris et al. 2006). Most of the media discussed above have utilized unbuffered media for isolation of PSMs. However, the natural conditions prevailing in the soil are highly buffered leading to their failure when subjected to field trials because of secretion of 10- to 20-folds less organic acids. Therefore, a buffered media was formulated to mimic the alkaline vertisol conditions (Patel et al. 2007). For the commercialization of the PSM as biofertilizers, inoculants of efficient PSM, also called microphos, need to be developed. The efficiency of various materials as suitable carriers such as peat, pearlite, farm yard manure (FYM), soil, and cow dung cake powder has been evaluated (Kundu and Gaur 1981) for the same. Cultures for mass-supply are packaged in polybags and have a shelf-life of up to 3 months if stored at a temperature of 30 Æ 2C. In India, a microbial preparation termed Indian Agricultural Research Institute (IARI) microphos culture (Gaur 1990) has been developed that contains two efficient phosphate-solubilizing bacteria (Pseudomonas striata and B. polymyxa) and three phosphate-solubilizing fungi (Aspergillus awamori, A. niger, and Penicillium digitatum). Combined inoculation of Rhizobium, a phosphate-solubilizing B. megaterium sub sp. phospaticum strain-PB and a biocontrol fungus Trichoderma spp. produced effects such as increased germination, nutrient uptake, plant height, number of branches, nodulation, pea yield, and total biomass of chickpea compared to either individual inoculations or an uninoculated control (Rudresh et al. 2004). Moreover, it has been demonstrated that plants inoculated with arbuscular mycorrhizal fungi utilize more soluble phosphate from rock phosphate than noninoculated plants (Guissou et al. 2001) and hence they can be used in consortium with other PSM to develop better P biofertilizers. The use of molecular biology techniques for rapid and easy generation, detection, and characterization of PSM can be highly advantageous and promising (Igual et al. 2001). For improvisation of the solubilization of the organic P reserves in the soil, modification of the genes coding for enzymes such as nonspecific 368 M. Saraf et al. phosphatases, phytases and phosphonatases, and C–P lyases have been carried out. A large number of important genes have been cloned including acid phosphatase genes from gram-negative bacteria (Rossolini et al. 1998), thermally stable phytase genes (phy) from Bacillus sp. DS11 (Kim et al. 1998) and B. subtilis VTT E-68013 (Kerovuo et al. 1998), acid phosphatase/phytase genes from Escherichia coli (appA and appA2) (Golovan et al. 2000), neutral phytase genes from B. subtilis and B. licheniformis (Tye et al. 2002), phyA gene from the FZB45 strain of B. amyloliquefaciens. Table 13.1 enlists few other clone genes involved in solubilization of organic and inorganic phosphate.

13.3.3 Siderophore Production

Iron is present abundantly on earth but is mostly unavailable to the microorganisms. This is due to the aerobic atmosphere of this planet which has converted the surface iron into its oxyhydroxide form having very less solubility. It is found that maxi- À mum of 10 18 M of free ferric ion is present in solution at biological pH. However iron is one of the essential nutrients required by the microorganism for a variety of functions like reduction of oxygen for synthesis of ATP, reduction of ribotide precursors of DNA, for formation of heme, etc., which has forced the microorganisms to produce certain chemical compounds which can chelate ferric ions from the complexes and make them available for their use. Siderophores are chemical compounds of low molecular weight (<1,000 molecular weight) which act as ferric ion-specific chelating agents. They scavenge iron, present in the environment as complexes, and make available to the microorganisms (Neilands 1995). More than 500 different siderophores have been described so far. Siderophores are produced by all bacteria and fungi growing under low iron stress condition. Structurally, siderophores are classified into hydroxymate (e.g., aerobactin, ferrichrome) and catechol [e.g., enterobactin (Fig. 13.1), based on its iron ligation group]. Most of the siderophores have a peptide backbone with several nonprotein amino acid analogs like modified and D-amino acids (Drechsel and Jung 1998). However despite their structural differences, all form an octahedral complex with six binding coordinates for Fe3+.

13.3.3.1 Synthesis

Synthesis of siderophores is similar to that of the antibiotics, i.e., they are assembled by nonribosomal, cytoplasmic peptide synthetases (Wandersman and Delepelaire 2004). In Ustilago maydis, a pathogenic fungus causing corn smut disease in plant, the sid1 which is responsible for initiating siderophore biosynthesis, is present on the genomic DNA (Mei et al. 1993), whereas aerobactin, siderophore ﬁrst isolated from Aerobacter aerogenes (Gibson and Magrath 1969) is a product of 3Prpcie fPP nAr-csses369 Agri-Ecosystems in PGPR of Perspectives 13

Table 13.1 Cloning of genes involved in solubilization of P reserves Organism from Host organism Gene transferred Feature Reference which the gene is isolated Erwinia herbicola E. coli Mps Produces gluconic acid and solubilizes mineral P in Goldstein and Liu (1987) E. coli HB101; probably involved in PQQ synthesis Pseudomonas E. coli Gab Y Produces gluconic acid and solubilizes mineral P in Babu-khan et al. (1995) cepacia E. coli JM109; no homology with PQQ genes Enterobacter E. coli pKKY Solubilizes P in E. coli JM109; does not lower the Kim et al. (1997) agglomerans pH Rahnella aquatilis E. coli pK1M10 Solubilizes P and produces gluconic acid in E. coli Kim et al. (1998) DH5x; probably related to PQQ synthesis Serratia marcescens E. coli pKG3791 Produces gluconic acid and solubilizes phosphates Krishnaraj and Goldstein (2001) Morganella Burkholderia nAP phosphatase An increase in extracellular phosphatase activity Fraga et al. (2001) morganii cepacia IS-16 gene Morganella Azospirillum spp. phoC phosphatase Increased phosphatase activity Fraga et al. (2001) morganii gene Aspergillus niger Arabidopsis plants Phytase gene (phyA) Improved P nutrition and utilization of inositol Richardson et al. (2001) phosphates B. amyloliquefaciens B. amyloliquefaciens phyA High extracellular phosphatase activity Iddris et al. (2002) DS11 strain FZB45 370 M. Saraf et al.

Rhizosphere Plants microorganisms

Inorganic P Root morphology Altering pH

Altering pH Organic acid anions

Capacity/affinity of Organic P P transporters Phosphatases Phytase Organic acid anions

Phosphatases

Fig. 13.1 Plant–microbe mechanism to increase phosphorus availability in rhizosphere (Richard- son 2007) plasmid pColV-K30. Thus the gene encoding siderophore may be present on the genomic DNA or on the plasmid varying on the organism to organism. Iron present in excess amount is also harmful for the bacterial cell. Therefore, the gene encoding the biosynthetic enzyme are iron regulated, i.e., they are expressed only during iron stress conditions and once the intracellular iron concentration rises, the genes are repressed. In Salmonella typhimurium, the gene fur (ferric uptake regulation) controls the expression of siderophores (Ernst et al. 1978). The genes responsible for synthesis of siderophores are often clustered with genes involved in its uptake. For uptake of siderophores, receptors are present on the cell membrane – speciﬁc membrane anchored binding proteins in case of gram-positive cell and speciﬁc outer membrane receptors for gram-negative cells (Wandersman and Delepelaire 2004). But these receptors may also act as entry sites for large number of lethal agents such as bacteriophage, bacteriocin, and antibiotics.

13.3.3.2 Uptake

Siderophores have high afﬁnity for Fe (III) (calculated afﬁnity constant >1030 MÀ1), but at the same time, have weak complexing ability for Fe (II). Thus they take up Fe (III) from the environment and release them inside the cell by reduction by free intracellular electron donors or by extracellular reductases (Schroder et al. 2003). The release of Fe (II) inside the cell may follow two different mechanisms: 1. Reduction and release of Fe (III) from siderophores intracellular electron donors by intracellular and siderophore recycling 2. Cleavage of iron and release of siderophores. In this case, siderophores are used only once and cannot be recycled 13 Perspectives of PGPR in Agri-Ecosystems 371

Since iron is an essential growth element for all organisms and is present in a complexed form, there exists a furious competition for it among the microorganisms present in the soil habitat and on the plant surfaces. By the production of siderophores, PGPR competitively take up the iron from the soil and thus are able to survive and colonize on the rhizoplane. Some PGPR are even able to secrete siderophores which draws iron from heterologous siderophores produced by coha- biting microorganisms (Whipps 2001). The production of siderophores by PGPR indirectly increases the plant growth by depriving the pathogenic bacteria and fungi of iron and thus inhibiting their growth.

13.3.4 Phytohormone Production

One of the major forms of host plant–microbial interactions is synthesis of phytohormones by plant growth-promoting bacteria which is for plant growth, development, and productivity. Use of bioinoculants-producing phytohormones is gaining importance around the globe as a means of sustainable crop production (Narula et al. 2006). Phytohormones or plant growth regulators are organic substances that inﬂuence the physiological processes of plants at very low concentrations. Soil microorganisms produce a variety of phytohormones such as auxins, gibberellins, cytokinins, ethylene, and abscisic acid (ABA).

13.3.4.1 Auxin

Auxins inﬂuence many cellular functions, orientation of root and shoot growth in response to light and gravity, differentiation of vascular tissue, apical dominance, initiation of lateral and adventitious roots (Malamy and Benfey 1997), stimulation of cell division, and elongation of stems and roots (Kende and Zeevaart 1997). Some of the auxin-producing organisms are phytopathogens while others are involved in promoting growth. Vandeputte et al. (2005) demonstrated auxin production by the actinomycete Rhodococcus fascians which brings about a number of malformations in monocotyledonous and dicotyledonous host plants.

13.3.4.2 Gibberellin

Gibberellins (GA) are another group of phytohormones that are diterpinoid acids. GA is involved in all phases of plant growth and development from germination to senescence. However, the most prominent physiological effect of GA is in shoot elongation. Some other plant growth-related functions of GA include overcoming dormancy and dwarﬁsm in plants, inducing ﬂowering in some photoperi- odically sensitive and other low-temperature-dependent plants and contributing to fruit ripening. Several soil microbes are known to produce gibberellins and 372 M. Saraf et al. gibberellin-like substances (Steenhoudt and Vanderleyden 2000). The common bacterial genera are Arthrobacter, Azotobacter, Azospirillum, Pseudomonas, Rhi- zobium, Bacillus, Brevibacterium, and Flavobacterium, Actinomyces and Nocardia are the important actinomycetes and Fusarium, Gibberella, Alternaria, Aspergillus, Penicillium, and Rhizopus are known fungi.

13.3.4.3 Cytokinin

Cytokinins, N6-substituted aminopurines, regulate cell division and differentiation in certain plant tissues. They play an important role in nodule development and formation. Symbiotic N2-ﬁxing bacteria, Rhizobium, free-living N2-ﬁxing bacteria Azospirillum and Azotobacter, and mycorrhizal fungus, Rhizopogon roseulus, are known to produce cytokinins in the rhizosphere along with other growth-promoting substances. Other bacteria that produce cytokinins or cytokinin-like substances include Agrobacterium, Bacillus, Paenibacillus, and Pseudomonas.

13.3.4.4 Ethylene

Ethylene (C2H4) is considered to be a promoter of senescence and an inhibitor of growth and elongation. It can also promote ﬂowering, fruit ripening, and cell elongation in certain plants. Bacterial species of Aeromonas, Citrobacter, Arthro- bacter, Erwinia, Serratia, Klebsiella, Streptomyces, and fungal species of Acre- monium, Alternaria, Mucor, Fusarium, Pythium, Neurospora, and Candida are capable of producing ethylene (Subba Rao 1999). However, a sustained high level of ethylene in the plants inhibits root elongation which in turn causes improper germination of the seeds. Glick (1995) has shown that certain free-living bacteria in the rhizosphere release an enzyme called ACC deaminase. ACC (1-aminocyclopropane-1-carboxylate), which is the precursor for ethylene, is broken down by ACC deaminase and thus the concentration of ethylene goes down in a stressed plant and this restores the proper growth in the plant.

13.3.4.5 Abscisic Acid

ABA is generally involved in deceleration or cessation of plant growth. ABA production in two bacterial species A. brasilense and Rhizobium spp. and several phytopathogenic fungi such as Cercospora, Fusarium, Cladosporium, Monilia, Pestatoria, and Verticillium has been demonstrated. Barea et al. (1976) demonstrated that phosphate solubilizers and nonsolubilizers produced plant growth regulators. Phosphate solubilization bacteria would probably be more effective as inoculants if they produced several growth regulators. Plant growth regulators are effective at very low concentrations; even less than 1 mM. Barea et al. (1976) reported concentrations of plant growth regulators 13 Perspectives of PGPR in Agri-Ecosystems 373 produced by microorganisms. These ranged in mg/ml, from 0.01 to 0.09 of IAA, 0.008 to 0.2 of GA, and 0.01 to 0.1 of kinetin.

13.4 Metabolites

PGPR are effectively used in different ecological niches to control phytopathogens by production of secondary metabolites. These may include production of inhibitory allelochemicals, ISR in host plants, and other metabolites in response to biotic and abiotic stresses. It is well known that some soils are naturally suppressive to some soil-borne plant pathogens. This may be due to both the physicochemical properties and microbial composition of the soil. Two types of suppressiveness are known. General suppression is due to the activity of the total microbial population of the soil and is not transferable between soils. Specific suppression is due to the activity of specific individual or groups of microorganisms and is transferable. Bacterial protection of plants depends upon three properties: (1) ecological fitness to maintain an effective population size in situ; (2) rapid root colonization; and (3) stable production of secondary metabolites, e.g., siderophores, b-1,3-glucanase, chitinase, cyanide, antibiotics, etc., under variable growth conditions which antagonize to phytopathogens. Secondary metabolite production by PGPR as a basis for bioinoculants has become increasingly popular over the last two decades (Sessitsch et al. 2005). In the last few years, relatively few studies concentrate on the use of PGPR for controlling bacterial diseases that affect plants. One example is the use of nonpathogenic strains of Streptomyces to control scab of potato (Solanum tuberosum L.) caused by Streptomyces scabies. The volume of literature on the use of PGPR for control of fungal diseases and their interactions with fungal pathogens continues to increase. Although, a variable number of bacterial genera and species have been used for such studies, a large number of papers involve the use of Pseudomonas species. The features that make this genus so effective and the choice of so many workers are (1) fast growth, (2) ease in culture and genetic manipulation in the laboratory, (3) utilization of a range of organic compounds, and (4) adaptation to rhizosphere.

13.4.1 Antibiotic Production and Regulation

The antibiotics can be classified as polyketides, heterocyclic nitrogenous compounds, cyclic lipopeptides, lipopeptides, aminopolyols, aldehydes, alcohols, ketones, etc. Several factors are involved in modulation of production and efficacy of antibiotics such as type of carbon source, pH, temperature, and other environmental stimuli, growth phase, and cell density. P. fluorescens HV37a produces different antibiotics in the presence and absence of glucose (Gutterson and James 1986). It is important to note that many strains produce a variety of different secondary metabolites under different environmental conditions. This bestows 374 M. Saraf et al. upon the strain a degree of flexibility when confronted with changing environmental conditions. Furthermore, plant growth, development, and genotype may influence antibiotic production by the antagonistic. DAPG production is induced by exudates of older plants which results in selective pressure against other rhizosphere organisms. Regulation of antibiotic synthesis involves GacA/GacS (global antibiotics and cyanide production) or GrrA/GrrS, RpoD and RpoS, and N-acyl-homoserine lactone derivatives. The GacA/GacS, a two-component regulatory system, is found in many plant-associated Pseudomonads. The two-component regulatory system and N-acyl-homoserine lactone (AHL)-mediated regulatory system function dependent and independent of each other. At a threshold concentration of AHL, which is reached only when a certain density of bacterial cells is present, the AHL will sufficiently bind to and activate a transcriptional regulator which then stimulates gene expression. Microbial metabolites also play a role in the regulation of antibiotic synthesis. Salicylate, fusaric acid, and pyoluteorin have negative effect on DAPG production. Salicylate interacts with repressor PhlF and stabilizes its interaction with phlA promoter (Abbas et al. 2002). Pyoluteorin, an aromatic polyketide antibiotic, is produced by several Pseudomonas species that suppress plant diseases caused by phytopathogenic fungi (Sharma 2008).

13.4.2 Cyanide Production

Cyanide is another secondary metabolite produced from glycine by HCN synthase (Castric 1994). Production of HCN occurs among widely divergent organisms, in many species of fungi but only a few species of bacteria of the genera Chromo- bacterium and Pseudomonas. However, action of HCN is ambiguous as it is known to inhibit or enhance plant establishment or inhibit development of plant disease.

13.4.3 Antifungal Activity

Production of antifungal metabolites by PGPR is well documented. A large number of such compounds have been identified including 2,4-di-acetylphloroglucinol (DAPG), hydrogen cyanide, oomycin A, phenazine, pyoluteorin, pyrrolnitrin, ten- sin, tropolone, viscosinamide, oligomycin A, zwittermycin A, xanthobaccin, and several others that are as yet uncharacterized. Antagonism may also operate by parasitism, which involves the production of several hydrolytic enzymes that degrade cell walls of pathogenic fungi. Chitinases and b-1,3-glucanases are considered key hydrolytic enzymes in the lysis of cell walls of higher fungi. Bacteria of the genera, Aeromonas, Serratia and Enterobac- ter, and fungi from the genera Gliocladium and Trichoderma are known to produce chitinolytic enzymes. These enzymes cause the release of elicitors which in turn 13 Perspectives of PGPR in Agri-Ecosystems 375 elicit various resistance reactions in the plant. Chitinase and cellulase produced by Pseudomonas strains showed mycelial growth inhibition of different fungal pathogen on PDA medium (Sindhu and Dadarwal 2001). Production of extracellular b-1,3-glucanases, chitinases, and proteinase increases significantly when Pseudo- monas spp. are grown in media supplemented with either autoclaved mycelium or isolated purified host fungal cell walls (Viswanathan and Samiyappan 2000). Such induction of chitinases and their antifungal activity together with the fact that chitin, b-1,3-glucan, and protein are the main structural components of most fungal cell walls suggested that hydrolytic enzymes produced by some fluorescent pseudomonads play an important role in the destruction of plant pathogens. Chitinase and b-1,3-glucanase are the key enzymes associated with the decomposition of the fungal hyphal wall. Chitinase (E.C. 3.2.1.39) and b-1,3-glucanase (E.C. 3.2.1.39) have been involved in degradation of fungal cell walls since chitin and b-1,3-glucan are the major components of most fungal cell walls. It has also been demonstrated that extracellular chitinase and laminarinase synthesized by P. stutzeri digest and lyse mycelium of Fusarium solani (Kumar et al. 2010). Saraf et al. (2008) studied that Pseudomonas spp. (M1P3) showed maximum chitinase activity (40 mg/ml N- acetyl glucosamine production) and also induced maximum cellulase production. Arora et al. (2008)studiedfluorescentPseudomonas (PGC 1 and PGC 2) for their antifungal potential against R. solani and P. capsici. The results of this study indicated the role of chitinase and b-1,3-glucanase in the inhibition of R. solani, however antifungal metabolites of nonenzymatic nature were responsible for inhibition of P. capsici.

13.4.4 Elicitors

Elicitors are defined as substances of biotic origin that has the ability to trigger hypersensitive response in a plant. Elicitors may be protein and peptides, carbohydrates, b-glucans (especially heptaglucan), xylans, oligogalacturans (especially 10–15-mers), chitosan (>hexamers), fatty acids, and glycosyl lipids, while most frequently encountered elicitors are polysaccharides, small proteins, or lipids associated with the fungal or bacterial cell wall. Even the pectic fragments resulting from microbial damage to the plant’s own cell walls may also act as elicitors. The elicitors interact with the plasma membrane of undamaged cells and trigger activation of genes involved in the defense response. The elicitors play a major role in the antifungal activity incorporated in the plant by PGPR. When pathogenic fungi attack plant, the PGPR secrete enzymes like chitosanase and chitinase which are capable of hydrolyzing b-1,4-linkages between N-acetyl-D-glucosamine and D-glucosamine residues in a partially acetylated fungal cell wall polymer. This hydrolytic action is exploited by many plants as a component of larger post attack defense (Agrios 1997). The fungal cell wall fragments released after hydrolysis with chitinase and chitosanase act as elicitors of plant defense responses such as stomatal closure (Lee et al. 1999), lignifications (Vander 376 M. Saraf et al. et al. 1998) and PR gene transduction (Jabs et al. 1997). It has been reported by Vander et al. (1998) that the responses elicited by these molecules depend upon the length and acetylation of the fragments released. More specifically, shorter the fragments, more heightened will be the reaction. Long fragments or intact fungal cell wall will cause little or no reaction.

13.4.5 Biosurfactants

Microorganisms produce a wide variety of secondary metabolites that have a diverse spectrum of activity in environmental remediation and biocontrol. Rham- nolipids are examples of bioactive molecules which have found application against phytopathogenic fungi. A variety of biosurfactants like Alasan (Acinetobacter radioresistens), Arthofactin (Arthobacter sps), Rhamnolipid (Pseudomonas aeruginosa), Surfactin (B. subtilis), etc., have a broad range of applications including enhanced oil recovery, bioremediation of water insoluble pollutants, emulsiﬁcation, phase separation, viscosity reduction, etc. (Sharma 2008). Rhamnolipids are effective against three genera of zoosporic plant pathogens Pythium aphanidermatum, P. capsici, and Plasmopara lactunae. These mono- and di-rhamnolipids were produced by P. aeruginosa and caused cessation of motility and lysis of culture in less than 1 min (Sharma 2008).

13.5 Exopolysaccharide

The outer membrane proteins, lipopolysaccaharides (LPS) of several bacteria, have been demonstrated to stimulate plant growth. De Weger et al. (1987) characterized the chemical composition of LPS of three plant-associated Pseudomonads and found it to be different. LPS production may facilitate efficient colonization of the plant-associated bacteria. The location of individual rhizobacteria on the root can be monitored by using confocal laser scanning microscopy with the help of molecular markers such as green florescent protein or florescent antibodies (Bloemberg and Lugtenberg 2001). This approach in combination with an rRNA targeting probe, that monitors the metabolic activity of a rhizobacterial strain in the rhizosphere, has been used to find out that the bacteria located on the root tips are the most active (Sorensen et al. 2001).

13.6 Production of Insecticide

Jousset et al. (2008) have demonstrated that production of secondary metabolites in P. ﬂuorescens CHA0 is crucial for competition for resources and predation control. Keel et al. (2008) have identiﬁed a novel genomic locus encoding an insect toxin Fit 13 Perspectives of PGPR in Agri-Ecosystems 377

(P. ﬂuorescens insecticidal toxin) that is related to the insect toxin Mcf (Makes Caterpillars Floppy) of the entomopathogen Photorhabdus luminescens. This emphasizes anti-insect properties of plant-associated bacteria which remain to be explored. However, antibiosis as a biocontrol mechanism is challenged by low production of metabolites in natural environment, broad spectrum activity, and production of different metabolites under different conditions.

13.7 Plant Responses to PGPR

Plant roots offer a niche for the proliferation of soil bacteria that thrive on root exudates and lysates. Population of rhizospheric microflora is much higher in the rhizosphere than elsewhere in the soil. These bacteria utilize the nutrients and secrete the metabolites which help in growth promotion of plants. The best studied example of signal exchange is the rhizobium–legume symbiosis. Plant microbe interaction in the rhizosphere lead to increased growth (biofertilizer) and also are deleterious to the pathogenic microflora found in the root system (biocontrol) and leading to induced systemic resistance (van Loon 2007). P. aurantiaca SR1 produced compounds that could stimulate the growth of both wheat and alfa-alfa cultivars (Rovera et al. 2008). On the other hand, Tank and Saraf (2009) have reported enhancement of plant growth and decontamination of nickel spiked soil using P. stutzeri. While Glick et al. (2007) concluded that ACC-deaminase containing rhizobacteria can increase root growth by lowering endogenous ACC levels, Remans et al. (2007) have studied the effect of four PGPR on symbiotic interactions between Rhizobium and Phaseolus vulgaris under deficient versus sufficient phosphorus supply and reported that IAA production and ACC deaminase activity play an important role under low P conditions. The use of PGPR has been reported to ameliorate salinity stress (Tank and Saraf 2010). Whereas, the role of PGPR as biocontrol agents was reported by Singh et al. (2008) in controlling the root rot caused by Macrophomina phaseolina using B. subtilis.

13.8 Challenges and Future Prospects

A lot has been known regarding the beneficial aspects of the use of PGPR as biofertilizers, and its commercialization can reach much greater heights reaping large benefits to the ecology, environment, and economically. However, there is a lack of efficient screening methods for selection of PGPR and methods to validate their success in field trials. It is important to consider the host plant specificity or adaptation to a particular soil, climatic condition, or pathogen in selecting the isolation considered and screening assays. The traits of PGPR-like ACC deaminase activity, root colonization; antibiotic, and siderophore production can be used for 378 M. Saraf et al.

Table 13.2 List of few siderophores and the genes encoding them Name of Organism Type Gene encoding Reference siderophore Aerobactin E. coli, Hydroxamate pColV-K30, pSMN1 (plasmid) Loper et al. (1993) Enterobacter aerogenes Ferrichrome Aspergilllus, Hydroxamate fso 1 Welzel et al. (2005) Ustilago, Penicillium Yersiniabactin Yersinia pestis Catechol irp1, irp2, irp3, irp4, irp5 Pelludat et al. (1998) (13 kb chromosomal DNA) Alanguibactin Vibrio anguillarum Catechol Ang R (plasmid encoded) Chen et al. (1996) Mycobactin Mycobacterium Hydroxamate mbt E LaMarca et al. (2004) Enterobactin Salmonella Catechol ent F Gehring et al. (1997) typhimurium Bacillibactin Bacillus subtilis Catechol dhbABCD Raza et al. (2008) Vibriobactin Vibrio cholerae Catechol Vib F (chromosomal) Butterton et al. (2000) Pseudobactin Pseudomonas B 10 Hydroxamate psb A (monocistronic) Ambrosi et al. (2000) Erythrobactin Saccharopolyspora Hydroxamate nrps 3 and nrps 5 Oliveira et al. (2006) erythraea Pyochelin Pseudomonas Hydroxamate pchDCBA and pchEF Reimmann et al. aeroginosa (2001) Pyoverdin P. aeroginosa Mixed pvd (gene cluster of 103 kb Tsuda et al. (1995) of chromosomal DNA) Ornibactin Burkholderia Hydroxamate pvd A and ecf 1/Orb S Agnoli et al. (2006) cepacia Francobactin Frankia Hydroxmate Chincholkar (2000) Parabactin Paracoccus Catecholate Chincholkar (2000) the selection of PGPR strains. The failure of biofertilizers on the field can be attributed to competition for resources among the rhizobacteria, complex abiotic and biotic factors, and constantly varying environmental conditions (Table 13.2). Successful commercialization is hindered mainly due to lack of awareness regarding its use in the developing countries and stringent policies governing its use in the developed countries such as Canada and USA. Prior to registration and commercialization, the PGPR products must overcome few hurdles like scale up and production of the organism under commercial fermentation conditions while maintaining quality, stability, and efficacy of the product. Health and safety testing may be required to address the effects on other organisms including toxigenicity, allergenicity, and pathogenicity, persistence in the environment, and potential for horizontal gene transfer. In the process of commercialization, capitalization costs, and potential markets must be considered. Avenues that still need to be explored for the development of biocontrol strains include screening of microorganisms other than Pseudomonads for traits that characterize a PGPR. Moreover, efforts need to be directed toward screening of PGPR for traits, as yet unexplored, such as production of insecticidal toxins, heavy metal resistance, and phytoremediation. Genetic manipulation of the PGPR strains for the transfer of one or more traits for plant growth promotion and that of the host crops for root-associated traits to enhance establishment and proliferation of the beneficial microorganisms may lead to the formation of more effective and stable PGPR strains. The use of multistrain inocula of PGPR with known functions is a 13 Perspectives of PGPR in Agri-Ecosystems 379 topic of interest for today’s scientists working in the field of biofertilizer. Construc- tion of competent strains of PGPR using molecular biology tools is still in its infancy; focused efforts in this direction will lead to huge success stories.

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Sandeep Kumar, Abhinav Aeron, Piyush Pandey, and Dinesh Kumar Maheshwari

14.1 Present and Future Prospective of Sesame

By 2020, the edible oil requirement will be 28.8 million tons, equivalent to 60 million tons of oilseeds (El-Bramawy and Shaban 2007). Sesame (Sesamum indicum L) is recognized as one of the oldest oil seed crops in the world. Archeological records indicate that it has been used in India for more than 5,000 years (Bedigian 2004). It is an excellent source of vegetable oil and contains oil (43.4–58.8%), protein (20–28%), sugar (14–16%), and minerals 5–7%. Its seeds are rich source of food, nutrition, edible oil and have uses in health care and biomedicines; the oil is quite stable due to the presence of antioxidants such as sesamolin, sesamin and sesamol (Suja et al. 2004). Therefore, it has long shelf life and used to blend with less stable vegetable oils to improve their stability and longevity (Chung and Choe 2004). Due to the presence of low level of saturated fatty acids and antioxidants, sesame oil reduces the incidence of cancer (Hibasami et al. 2000; Miyahara et al. 2001), hypertension and lowers the cholesterol level in human beings (Lemcke- Norojarvi et al. 2001; Sankar et al. 2004). Adverse effects of different groups of chemicals have been observed on sesame by Gricher et al. (2001a, b). It has been reported that pesticides accumulate in sesame seeds, oil and oil cake (Bhatnagar and Gupta 1998). The presence of chemicals in sesame had been major impediment in the promotion of sesame export. Export consignments of sesame are sometimes unsuitable in the international market due to the presence of pesticide residue, resulting in loss of revenues (Duhoon et al. 2004). Thus the use of chemicals as fungicide and pesticide is found unsuitable because these chemicals subvert the soil ecology, disrupt environment, degrade soil fertility, pollute water resources and consequently pose harmful effects on human health (Ayala and Rao 2002).

S. Kumar, A. Aeron, P. Pandey, and D.K. Maheshwari (*) Department of Botany and Microbiology, Gurukul Kangri University, Hardwar, Uttarakhand, India e-mail: [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 387 DOI 10.1007/978-3-642-18357-7_14, # Springer-Verlag Berlin Heidelberg 2011 388 S. Kumar et al.

The international demand and export of sesame seeds and oil is continuously increasing with the growing health consciousness and increasing knowledge on the dietary and health benefits. Consequently, it has recently emerged as an important oilseed crop, presently earning over Rs. 1,000 crores of valuable foreign exchange from the export of 2.5 lakh tones of sesame seeds. India’s share in world’s trade has gone up to 60% during 2002–2003 (Duhoon et al. 2004). Sesame has been listed as a crop with high potential but remains neglected and underutilized as stated by the International Plant Genetic Resources Institute (IPGRI) (Were et al. 2006). Use of chemical fertilizers and plant protection measures in general has been observed to be the critical inputs holding the production of oilseeds (Ankineedu and Reddy 1983). The WHO has reported that globally at least three million people are poisoned by pesticides every years with a mortality of 20,000. A majority of pesticide death is reported to be occurring in developing countries which use only 25% of the global pesticides. Thus, organic farming is safe for providing clean environment (Sengar and Pant 1999). The use of organic manures and biofertilizers is essential to maintain soil fertility, reduce the cost of cultivation and pressure on the ecosystem. Declining soil organic matter and increasing fertilizer’s use are the serious threats to sustainability in agriculture (Duhoon et al. 2004). The major reasons of decline in production of sesame are the imbalances in the use of chemical fertilizers, ignorance towards biofertilizers, susceptibility to pests and diseases, low harvest index, seed shattering and stiff competition from exotic crops, mainly sunflower (Das 1997;Ashri1998), besides unfavorable natural conditions and abiotic stresses. The average productivity of sesame has been only 350 kg/ha which is mainly owing to its cultivation in marginal and sub-marginal lands having low organic matter and poor soil fertility without any nutrient management under rainfed conditions (Duhoon et al. 2001). However, adverse consequences have occurred due to excessive application of chemical fertilizers, pesticides, etc., on soil structure and health. Hence, adherences to the scientific principles of soil and plant health management in order to sustain the benefits of enhanced productivity over long period are utmost desirable. Thus, sustainable management of natural resources and progressive enhancement of soil quality, biodiversity and productivity are some of the steps that may lead towards productivity enhancement. This can achieve higher productivity in perpetuity without accompanying ecological harm, water quality, biodiversity, atmosphere and renewable energy sources (Kesavan and Swaminathan 2006).

14.2 Sesame Pathogens: Macrophomina phaseolina and Fusarium oxysporum f. sp. sesami

Quarantine processing of 2,240 germplasm samples of sesame introduced in India during last three decades resulted in the interception of 12 pathogenic fungi of high economic signiﬁcance. Among these Alternaria longissima, A. sesami, Cercospora sesami, C. cassiicola and Macrophomina phaseolina are seed borne in sesame, 14 Ecofriendly Management of Charcoal Rot 389 while Cephalosporium sp., Colletotrichum auctatum, C. coffeanum, Corticium rolfsii, Phomopsis sp. and Verticillium albo-atrum were not reported to have sesame as host and have very wide host range (Agarwal et al. 2006). Though there are a number of pathogens that infect sesame, viz. virus, mycoplasma, fungi, insects, etc., this section deals with only two pathogens.

14.2.1 Charcoal Rot

Wherever sesame is grown, it is liable to attack by various pathogenic fungi (Abd- El-Ghany et al. 1974). Among the fungal diseases, charcoal rot of sesame caused by M. phaseolina is the most devastating disease (Dinakaran and Manoharan 2001)of the crop in India with reports of about 50% diseases incidence resulting in heavy yield losses (Chattopadhyay and Kalpna 2002). The most common symptom of the disease is the sudden wilting of plants throughout the crop growth mainly after the ﬂowering phase. Due to severe infection, the stem becomes black and the roots rot with large number of black micro-sclerotia are formed on the affected portion of host tissue. The pathogen survives as sclerotia in the soil produced during parasitic phase and in crop residues during saprophytic phase. It has also been reported to be seed borne. Due to its soil-borne nature, practically no ﬁeld control is available so far.

14.2.2 Fusarium Wilt

Fusarium oxysporum f. sp. sesami is a serious pathogen of sesame which causes wilt disease and limits sesame production. It was reported for the ﬁrst time from USA in 1950 (Armstrong and Armstrong 1950). Sesame is infected by several pathogens from the seedling stage to the maturity of crops but the most drastic disease is sesame wilt caused by F. oxysporum f.sp. sesami, which drastically declines production of sesame. F. oxysporum is a soil-borne, root pathogen colonizing the xylem and blocking the food and water-conducting vessels resulting in the wilt (Bateman et al. 1996). Management of Fusarium wilt is mainly through soil fumigation and using resistant varieties. The broad-spectrum biocides used to fumigate soil before planting damage soil microbiota (Blancard 1993).

14.3 Resistant Varieties of Sesame: An Effective Strategy for Disease Control

Significant research has been done on breeding for high seed yielding varieties of sesame (Uzun and Cagirgan 2006). Sesame growing in varied agro-climatic conditions is infected by at least eight economically important fungal diseases (Kolte 1985) including F. oxysporum f. sp. sesami, M. phaseolina, and Alternaria sesami 390 S. Kumar et al. resulting in great yield losses. The yield losses have been estimated to vary from 25 up to 40% as reported by different workers (El Deeb et al. 1987; Avila and Pineda 1996; Dinakaran et al 1996; Ragab et al. 2002; El-Bramawy 2003; El-Bramawy 2006). On the other hand, great differences in resistance level of Fusarium wilt, charcoal rot and Alternaria leaf spot disease were observed in different breeding genotypes of sesame (Kavak and Boydak 2006; El-Bramawy and Wahid 2006; Ragab et al. 2002; El-Shakhess-Sammar 1998). Kavak and Boydak (2006) screened resistance in 26 breeding lines against Fusarium wilt for 5 years. Sanliurfa-63189 was the most resistant genotype with 6.6% infection rate. However, half of the breeding lines displayed this disease at the level of below 20% infection rate and was recognized in resistant category. Infec- tion rates of five lines within this group were lower than 10%. The most susceptible genotype was Sanliurfa-63283 local line with the average infection rate of 40.8% and the third scale value. They concluded that breeding lines in resistant category may include the resistance genes of sesame to Fusarium wilt. El-Shazly et al. (1999) evaluated 25 cultivars of sesame germplasm against Fusarium wilt. During the first season, the percentage of infection varied from 0 to 53.33% with an average of 22.13% among the evaluated genotypes. Out of 25 genotypes, only 3 genotypes were found highly resistant (HR), while 11 genotypes were ranked as resistant (R) and only five were ranked as moderately resistant (MR). The highest infection was noticed in three genotypes, with infection percentages more than 50% ranked as susceptible (S). However, some variation in the ranking of the genotypes tested was observed during second season. Fusarium is a common and permanent pathogen of sesame plants particularly in irrigated areas and contaminated fields are important sources of disease. Considerable variability in the reactions of sesame germplasm to Fusarium was reported and research has concentrated on evaluation of resistant variety with germplasm (Bakheit et al. 1988;Raghuwanshi et al. 1992;Xiaoetal.1992). Some of the wild species of sesame such as S. radiatum, S. occidentale and S. mulayanum possess resistance to the diseases viz. Alternaria leaf spot (Lee et al. 1991), Cercospora leaf spot (Mehetre et al. 1993) and insect pest like sesame shoot webber (Antigastra catalaunalis) (Thangavelu 1994).

14.4 Fertilizers Versus Biofertilizers for Sustainable Approaches

The bacteria useful to plants are characterized into two general types: bacteria forming a symbiotic relationship with the plant and another the free-living ones found in the soil but are often found near the root, on or even within the plant tissues (Kloepper et al. 1988; Frommel et al. 1991). Beneficial free-living soil bacteria that enhance plant growth are usually referred to as “plant growth promoting rhizobacteria” (Kloepper et al. 1989) or yield increasing bacteria (YIB) (Tang 1994). PGPR is originally defined (Kloepper and Schroth 1978) as root colonizing bacteria (rhizobacteria) that cause either growth promotion or biological control of plant 14 Ecofriendly Management of Charcoal Rot 391 diseases. Bashan and Holguin (1997) proposed the division of the plant growth promoting rhizobacteria into two categories (I) biocontrol-PGPB (plant growth promoting bacteria) and (II) PGPB, and both showed “rhizosphere competence”. Research on plant growth promoting bacteria (PGPB) has gradually increased since the first usage of the term by Kloepper and coworkers in the late 1970s (Kloepper and Schroth 1978; Suslow et al. 1979). Kloepper indicated those bacteria as plant growth promoting rhizobacteria (PGPR) which live in the vicinity of plant rhizosphere. Suslow (1982) further coined the term rhizobacteria for those bacteria that aggressively colonize root system in the rhizosphere region. The premier example of such bacterial genera include Actinoplanes, Agrobacterium, Alcaligens, Amorphosporangium, Arthrobacter, Azotobacter, Bacillus, Burkholderia, Cellulo- monas, Enterobacter, Erwinia, Flavobacterium, Gluconacetobacter, Micromonos- pora, Pseudomonas, Rhizobia, Serratia, Streptomyces and Xanthomonas as stated by a number of workers (Kloepper et al. 1989; Weller and Thomashow 1994; Glick et al. 1995, 1998, 1999; Lucy et al. 2004). Recently, a novel PGPR, Delftia tsuruhatensis HR4 has been reported both as diazotroph as well as biocontrol agent against various plant pathogens (Han et al. 2005). Biocontrol agents such as PGPR (Kloepper and Schroth 1981) produce antifungal compounds, siderophore, hydrogen cyanide (HCN), antibiotics, lytic enzymes (chitinase, b-1,3-glucanase and protease) and toxins, which directly inhibit the deleterious phytopathogens (Glick et al. 1995, 1999). PGPR has also been reported to facilitate the enhancement of crop plant by several direct mechanisms such as atmospheric nitrogen fixation, phytohormone secretion and insoluble phosphate solubilization (Glick 1995). A particular PGPR may inhibit phytopathogens and enhance plant growth indirectly using any one or more of these mechanisms. Pseudomonads rapidly and aggressively colonize the root system and suppress pathogenic microorganisms improving plant growth and grain yield (Schippers et al. 1987;Weller1988). Organic substances produced either endogenously or applied exogenously that regulate plant growth are called plant hormones. They regulate growth by affecting physiological and morphological processes at very low concentrations (Arshad and Frankenberger 1991). Involvement of plant hormones in growth and development of plants produced by bacteria such as indole acetic acid (IAA) (Park et al. 2005; Gupta et al. 1999; Kumar et al. 2005a), gibberrelic acid (Mahmoud et al. 1984), cytokinins (Garcia de Salamone et al. 2001) and ethylene (Glick et al. 1995; Ma et al. 2002) is well established. Phytohormones such as IAA and cytokinin producing PGPR have been found to cause growth promotion of non-leguminous plants (Hirsch et al. 1997; Patten and Glick 2002; Kumar et al. 2005b).

14.4.1 Cost-effective and Eco-friendly Strategies as Sustainable Approaches

Use of chemical fertilizers and plant protection measures in general has been observed to be critical in holding the production of oilseeds (Ankineedu and 392 S. Kumar et al.

Reddy 1983). The advanced agriculture practices have definitely increased crop productivity but their improper use is creating disorder and making the soil sick. Nearly, 2,000 ha land is becoming saline every year due to imbalanced use of chemical fertilizers and unmanaged cropping system. Application of biofertilizers not only helps to proliferate beneficial microbes in soil but also provides residual effect for subsequent crops and help in the recycling and decomposition of organic matter (Kumar et al. 2006). Intensive efforts have been made to improve the quality of crop by employing tools of physiological, biochemical and microbiological relevance. Under natural environmental conditions, successful plant development and high yield depend on the genetic constitution of crop species, the availability of nutrients, the presence of certain beneficial microorganisms and the absence of phytopathogens in the surrounding soil. But, due to intensive cropping and imbalanced diet, the soil health, in most part of country, is rapidly declining, which is an alarming situation for our food security. Biological control research has gained considerable attention and appears promising as a viable alternative to chemical control strategies (Rebafka et al. 1993; Trigalet et al. 1994). Bio-agents reduce the ability of the pathogen to cause infection and its disease-producing activities have induced resistance in hosts and has created an environment for unsuccessful disease development. Different microorganisms including both beneficial and deleterious ones heavily populate the rhizosphere region. Therefore, there is an opportunity to find rhizosphere- competent bacteria which may act as potential biocontrol agents. Proper and balanced fertilizer schedule is important amongst the various technologies for higher crop production (Lal et al. 1995). As chemical fertilizers are becoming costly day-by-day and further excessive use of these cause health hazards and soil health deterioration, there is an urgent need to adopt integrated nutrient management (INM) system for agriculture. Biofertilizers play an important role in INM because they not only contain a wide range of naturally chelated plant nutrients and trace elements, carbohydrates, amino acids and other growth promoting substances, but also act as soil conditioners by stimulating microbial activity in soil resulting in improved air water relationship in soil, improved fertility and making soils less prone to compaction and erosion (Kumar et al. 2006). Use of synthetic chemicals for control of parasites and pathogens has proved to be effective and convenient but their use and abuse are causing serious ecological, economical and social problems. The alternative or substitute of these in the form of bacterial fertilizers and bio-pesticides are the need of the hour. For sustainable soil fertility, blending of chemical fertilizers with chemical adaptive bacterial strains is one of the approaches that may derive synergistic benefits (Vargas et al. 2000; Joshi et al. 2006). For sustainability in agriculture, India does not need more fertilizers or total organic approaches but a blend of both is required (Ayala and Rao 2002). Krishnamurthi and Kumar (1987) highlighted the establishment of a judicious combination of organic and inorganic fertilizers recommended for increase in yield, improvement of crops, and physical, chemical and microbiological properties of the soils. Integrated nutrient management (INM) is an endeavor to blend ecology and economy in a cost benefit framework. It takes systemic and simultaneous 14 Ecofriendly Management of Charcoal Rot 393 account of the environmental aspects and the quality of the product. The rhizosphere harbors a large number of saprophytic bacteria with symbiotic, neutral and deleterious properties affecting crop production (Astrom 1990). Crop microbial system can, therefore, be energized in sustainable agriculture with considerable ecological stability and environmental quality, i.e., nutrients. Nutrient deficiency and enrichment over cease create imbalance in their activities. It is becoming apparent throughout the world that efficient and productive agriculture is practiced at great cost to the environment. There are increasing reports of loss of pesticides from the market due to resistance and revocation of registration. The biological management of plant productivity is still at an early stage of development, while the approach appears to have tremendous potential and many of the basic concepts necessary for the implementation of INM are in place, but apparent obstacles such as information of biomass, formulation of a product and site of application and registration difficulties exist. Being a new technology, most of the studies to date have been carried out under controlled laboratory, growth chamber or green house conditions (Wu et al. 2005;Cakmakcietal.2006). Therefore, there is an urgent need to investigate on integrated crop management of disease control and increase in plant productivity utilizing both biological and chemical methods during the field trials. Further, it seems to be essential not only for increasing the crop productivity but also for the maintenance and improvement of soil fertility for sustainable crop productivity. Multifunctional formulation needs to be developed for the enhancement of plant productivity by utilizing PGPR under the influence of chemical fertilizers. Biofertilizers cannot replace chemical fertilizers, but certainly they are capable of reducing their input (Singhal et al. 2003). Long-term fertilization showed that continuous application of chemical fertilizers to soil had a deleterious effect on soil productivity. However, integrated use of organic manures with optimum level of chemical nutrients such as N, P and K fertilizers not only improve the nutrient status and soil health but also stabilize the crop yield at higher level (Belay et al. 2002). In India, biofertilizers constitute the best renewable source of nutrient supply to plants and as supplements to chemical fertilizers and organic manures (Gahukar 2006). They enhance the availability of mineral nutrients to plants on application to seed or soil and offer an ecofriendly and economically viable and socially acceptable means of reducing external input of chemical fertilizers and the profitability of the agriculture (Luc 2000).

14.4.2 Biological Control of Charcoal Rot and Fusarium Wilt

Plant growth-promoting fluorescent pseudomonads applied through seed bacterization checked charcoal rot caused by M. phaseolina in Arachis hypogea. About 45–68% reduction in disease symptoms has been recorded in comparison to peanut crop raised in M. phaseolina-infested soil (Bhatia et al. 2003). This clearly indicates the role of fluorescent pseudomonads in the suppression of charcoal rot disease. Similarly, Gupta et al. (2002) observed reduction in disease incidence, better vegetative plant growth and enhanced grain yield in peanuts with the application 394 S. Kumar et al. of P. aeruginosa GRC2 in M. phaseolina-infested field soil. Fluorescent pseudomonas is reported for the biological control of fungi namely, Rhizoctonia, Fusarium, Sclerotonia, Pythium, Erwinia, Macrophomina, etc. (Defago et al. 1990; Gupta et al. 2001; Gua et al. 2004; Validov et al. 2005; Pandey et al. 2005). One of the potential strains of P. aeruginosa GRC1 proved efficient biocontrol agent, enhanced plant growth and showed long-term residual effect on subsequent crops (Deshwal et al. 2006). Although, not much information is available on rhizobia for the biological control (Arora et al. 2001; Deshwal et al. 2003), root nodulating bacteria secrete several secondary metabolites and toxins similar to that of fluorescent pseudomonads. Further, pseudomonads and rhizobia have also been reported for direct plant growth promoting activities leading to yield promotion (Deshwal et al. 2003). Attachment of rhizobia with non-leguminous crops such as maize, wheat, rice, oat, sunflower, mustard and asparagus has been observed (Biswas et al. 2000;Pengetal. 2002; Chandra 2004) to promote plant growth and enhance yield (Yadav et al. 2002).

14.4.2.1 Application of Phytostimulant and Biocontrol Agents

The bacteria useful to plants are categorized into two general types: symbiotic bacteria and free-living ones found in the soil but are often found near the root, on or even within the plant tissues (Kloepper et al. 1988). PGPRs have been introduced as microbial inoculants for biofertilization, phytostimulation, biopesticides and sometime in bioremediation (Bloemberg and Lugtenberg 2001). Large-scale application of PGPR to crops as inoculants seems to be attractive as it would substantially reduce the use of chemical fertilizers and pesticides (Singhal et al. 2003).

14.4.2.2 Integration of PGPR Amended with Chemical Fertilizers

In intensive cropping systems, supplementing soil nutrients using chemical fertilizers is considered inevitable for obtaining optimum yield of crops. But it has been observed that continuous use of chemical fertilizers may affect soil health and may lead to a negative impact on soil productivity (Poul and Savithri 2003). Thus biofertilizers can supplement the chemical fertilizers for meeting the nutrient needs and help in improving the yield and quality of crop plants (Stephan and Nybe 2003). Therefore, there is an urgent need to develop an integrated biological and chemical approach to enhance the yield of sesame. Green agriculture, a system of cultivation with the help of integrated nutrient supply, integrated pest management and integrated natural resource management system, does not exclude the use of minimum essential quantities of mineral fertilizers and chemical pesticides. Plant growth enhancement is virtually possible using microbial inoculants along with low concentrations of chemical fertilizers. This will lead to the development for each farm an evergreen revolution as quoted by Kesavan and Swaminathan (2006). 14 Ecofriendly Management of Charcoal Rot 395

14.4.2.3 Application of BioChemo-Formulations

Agrochemicals reduce the population of beneficial microorganisms due to the inhibitory effect on bacterial growth (Smiley 1981). The abnormal morphological changes in Rhizobium meliloti cells have been observed by Strzeleowa (1970)by growing R. meliloti on Thrton’s agar medium containing 2.5 mg ml1 urea. Excessive nitrogenous fertilization increased the generation time and disrupted protein synthesis in Acetobacter diazotrophicus (renamed as Gluconoacetobacter diazotrophicus) as visualized by Becking (1995). Further, Muthukumarasamy et al. (2002) examined higher population of G. diazotrophicus from low fertilized rhizosphere soil as compared to excessive N-fertilized soil. Such effects of different chemicals on microbes have been documented by a number of workers (Kantachote et al. 2001; Saraf and Sood 2002; Joshi et al. 2006). Similarly, the application of pesticides on sesame resulted in the accumulation of their residues in seeds, oil and oil cake (Bhatnagar and Gupta 1998). On the other hand, Gricher et al. (2001a) reported reduction in the height of sesame up to 66% and plant stand (biomass) 8–98% as compared to control, after the application of different herbicides. The seedling damage and reduction in the crop stand have been reported by several workers (Shukla 1984; Ibrahim et al. 1988; Bansode and Shelke 1991; Gricher et al. 2001b). Deshmukh et al. (2002) obtained the highest seed yield of sesame through the integrated use of organic and inorganic fertilizers. The beneficial effects of the combined application of urea, MRP-enriched farmyard manure (FYM) with phosphobacteria and rhizobia on nutrient uptake and postharvest soil nutrient availability have also been reported by numerous workers (Mishra et al. 1984; Ramamoorthy et al. 1994). Thaunanthan et al. (2001) observed the combined use of flyash, FYM and inorganic fertilizers (N, P, and K) on the enhancement of growth and yield of sesame. Earlier, Tiwari et al. (1995) found combined use of inorganic (NPK) and organic fertilizers increased crop production. The principal goal of agriculture is the production of quality, safe and affordable food for the ever-increasing worldwide population. Furthermore, agriculturists have the additional constraints of economic profitability and sustainability with the increasing problems associated with the use of synthetic chemicals in agriculture. There has been increasing interest in the use of native and non-native beneficial microorganisms to improve plant health and productivity, while ensuring safety for human consumption and protection of the environment. Scant information is available regarding the nature of biochemical changes occurring due to the application of INM, growth hormones and biofertilizers (Thiruppathi et al. 2001). Reduction in the dose of chemical fertilizers in combination with PGPR has recently been reported by Kumar et al. (2009) and Adesemoye et al. (2009). The interactive effect of biofertilizers and INM protocol with reduced dose of chemical fertilizers gives satisfactory results as compared with full dose of chemical fertilizers (Table 14.1). 9 .Kmre al. et Kumar S. 396

Table 14.1 Interactive effect of biofertilizers and integrated nutrient management protocol on yield and yield component of S. indicum L. after 120 DAS Treatments Average seeds yield (kg/ha) 1,000 seed weight (g) Oil Oil yield Protein Protein yield 2004 2005 Mean 2004 2005 Mean content (%) (kg/ha) content (%) (kg/ha) T-1 P. aeruginosa LES4 649 661 655 2.73 2.84 2.78 49.6 370 19.0 136 T-2 P. aeruginosa LES4 of half dose 987 1,011 999 3.01 3.04 3.02 50.4 496 20.0 208 fertilizers T-3 Half dose of fertilizers 719 763 741 2.59 2.65 2.62 47.2 372 18.6 141 T-4 Full dose of chemical fertilizers 986 998 992 2.92 3.03 2.96 49.6 495 20.0 203 T-5 Control 462 476 469 2.40 2.48 2.44 45.8 215 18.3 97 SEM 0.338 1.514 1.432 0.845 0.115 0.158 1.095 1.309 1.303 1.105 CD at 1% 1.459 6.539 6.183 0.365 0.500 0.685 4.729 5.654 5.626 4.773 CD at 5% 1.041 4.666 4.412 0.260 0.357 0.489 3.374 4.034 4.015 3.406 Adapted from Kumar et al. (2009) Data are mean of 2 years. Values of each year are mean of ten randomly selected plants from each treatment. Full dose of chemical fertilizers N40 þ 40 þ 40,P30, K30; half dose of chemical fertilizers N20 + 20 + 20 ,P15,K15 14 Ecofriendly Management of Charcoal Rot 397

14.4.2.4 Others

Plant and microorganism interaction in the rhizosphere influenced crop yield significantly (Antoun et al. 1998). Ongena et al. (2002) reported that Pseudomonas putida BTP1 induced resistance in bean to Botrytis cinerea after inoculation. Some species of pseudomonads such as P. putida and P. fluorescens increased root and shoot elongation in canola, lettuce and tomato (Glick et al. 1997), as well as crop yield in potato, radish, rice, sugar beet, tomato, lettuce, apple, citrus, bean, ornamental plants and wheat (Suslow 1982; Kloepper et al. 1988; Lemanceau et al. 1992). Dey et al. (2004) selected nine different isolates of PGPR from a pool of 233 rhizobacteria isolated from the peanut rhizosphere based on their siderophore, IAA, ACC-deaminase activity, etc. The bacterial strain showed the suppression of soilborne fungal diseases such as collar rot caused by Aspergillus niger, A. flavus and stem rot caused by S. rolfsii of peanut and germinating seed bioassay enhanced significantly over the untreated control. P. fluorescens produced the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) that plays a key role in the suppression of soil-borne pathogens (La Fuente et al. 2006). Kumar et al. (2001) tested PGPR strains alone and in combinations with each for the suppression of rice sheath-blight disease and plant growth promotion under greenhouse and field conditions. Burelle et al. (2002) evaluated tomato and pepper transplant amended with formulations of PGPR. Disease incidence was reduced in response to most of the formulations tested in comparison to individual PGPR strains. Recently, La Fuente et al. (2006) observed the colonization effect of the introduced strains and in wheat and pea rhizosphere. It is interesting to note that the introduction of their equidensity varied their effect according to the host plant species. In recent years, the use of PGPR for biocontrol and disease management has been demonstrated in pots (Kapur and Naik 2004; Kumar et al. 2005a, b), under greenhouse (Nandkumar et al. 2001) and field conditions (Gua et al. 2004; Cakmakci et al. 2006). The interactive effect of biofertilizers and INM have been studied on oil seed crops such as mustard (Glick et al. 1997; Gudadhe et al. 2005), sesame (Kumar et al. 2009), soybean (Singh and Abraham 2001), groundnut (Gupta et al. 2002; Meena and Gautam 2005), and sunflower (Rao and Soren 1991). The combined application of bio-fertilizers and reduced (low) dosages of chemical fertilizers increased growth and yield of wheat (Idris and Mohammad 2001). Saraf and Sood (2002) raised the pesticide adoptive mutant of rhizobial strains and observed the PGP activities. Reasonable results have been observed from fertilized field using fertilizer-tolerant strains of indigenous rhizobial population (Vargas et al 2000). Joshi et al. (2006) developed chemical fertilizer adaptive variants of P. aeruginosa and A. chrococcum on growth and yield of mustard (B. juncea). Recently, Adesemoye et al. (2009) reported enhanced yield of tomato (Solanum lycopersicon) and Kumar et al. (2009) of sesame (S. indicum) by reduced dose of chemical fertilizers supplemented with microbial inoculants. Biological control is considered as an alternative and a supplement for reducing the use of chemicals in agriculture (Postma et al. 2003; Webaum et al. 2004). 398 S. Kumar et al.

Kapur and Naik (2004) studied the effect of mixed culture liquid formulation of biofertilizers along with the pure culture of Azotobacter and phosphate solubilizing bacteria and significant improvement has been observed in the N, P and chlorophyll content of leaves of chickpea and available P in soil. The interactive effect of integrated nutrient management with biofertilizers examined in peanut and horse gram (Parasuraman and Mani 2003) and positive effect on all the growth and yield parameters were observed. Recently, Dey et al. (2004) reported that fluorescent Pseudomonads isolates increased the growth and yield of Peanut (Arachis hypogaea L.) upto 18–28%, whereas Amir et al. (2003) reported that inoculation of the rhizobacteria in combination with inorganic N enhanced stimulation of oil-palm seedlings. Similarly, Shehata and El-khawas (2003) examined the effect of two biofertilizers on sunflower (Helianthus annus L. cv. Vedock) and showed increase in seed yield, nutrient content of seeds, nitrogen and all nitrogenous compounds, mineral and seed oil contents; decrease in the saturated fatty acids such as palmitic and stearic; increase in the main unsaturated fatty acids (oleic, linoleic and linolinic); and induced the synthesis of two new proteins of low molecular weight (2.1 kDa for biogien and 14.9 kDa for microbien).

14.5 Conclusion

Sesame is an important oil seed crop, next to soybean and ground nut, but has low yield potential due to the infection by fungal and other pathogens from the seedling stage to harvest. Signiﬁcant differences in the resistance level of sesame germplasm against charcoal rot and Fusarium wilt are reported by various workers. Sesame improvement program alone cannot depict the possible improvement of character achievable through selection. Signiﬁcant reduction in disease incidences, increased growth and yield attributes have been observed along with reduced dose of fertilizers and co-inoculation of PGPRs. Further studies are needed to understand the nature of infection and ecological behavior of M. phaseolina and F. oxysporum f. sp. sesami to improve the yield and quality of high potential oil seed sesame crop.

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Swarnalee Dutta, Manjeet Kaur, and Appa Rao Podile

15.1 Groundnut: An Economically Important Legume

Groundnut (Arachis hypogaea L.) or peanut is a major oilseed crop widely grown in tropical and subtropical regions of the world. It is an important source of protein and cultivated around the globe in different agronomical systems with the worldwide production estimated at around 30 million tons. Asia accounts for 55.2% of the global area and 66.7% of the global production of the crop compared to 40.3% of the area and 25.6% of the production in Africa. According to a survey by the USDA, China is the largest producer of groundnut with a share of about 37.5% of overall world production. India is the second largest producer followed by USA and Nigeria. Groundnut is an economically important crop as every plant part is useful for different purposes. It can be consumed as food in either raw, roasted, or boiled form and is used for extraction of cooking oil; pressings, seeds, green material, and the straw of the plant are all used as animal feed; the shells or pods as feed for livestock along with other purposes such as manufacture of plastic, wallboard, abrasives, fuel, cellulose (used in rayon and paper) and mucilage (glue), thereby making it a highly efficient industrial raw material. However, groundnut is primarily utilized as seed as they are a rich source of edible oils containing mostly fat (40–50%), protein (20–50%) and carbohydrate (10–20%). Besides, several other important dietary components are present in groundnut oil such as calcium, magnesium, phosphorus, zinc, iron, potassium, niacin, folacin, vitamin E, riboflavin, and thiamine (Fabra et al. 2010). Glycerides of palmitin and olein are also found in groundnut oil. It is the fifth most important oilseed after soybean, palm, colza, and sunflower oils. China and India are the two major producers of groundnut and its oil. Paint, varnish, lubricating oil, leather dressings, furniture polish, insecticides, and nitroglycerin are also made from groundnut oil. Soap is made from saponified oil and many

S. Dutta, M. Kaur, and A.R. Podile (*) Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India e-mail: [email protected]; [email protected]

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 407 DOI 10.1007/978-3-642-18357-7_15, # Springer-Verlag Berlin Heidelberg 2011 408 S. Dutta et al. cosmetics contain groundnut oil and its derivatives. The protein portion of the oil is used in the manufacture of some textile ﬁbers. Groundnuts contain more protein than meat, about two and a half times more than eggs and far more than any other vegetable food except soybean and yeast. Therefore, groundnut and their products are used to help ﬁght malnutrition in developing countries by organizations such as World Health Organization, UNICEF, Project Peanut Butter and Doctors Without Border. High protein, high energy and high nutrient groundnut-based pastes were also developed to be used as a therapeutic food to aid in famine relief. In China, the nuts are considered demulcent, pectoral, and peptic; the oil aperient and emollient can be taken internally in milk for gonorrhea and externally for rheumatism (Duke and Ayensu 1985). In Zimbabwe, it is used in folk remedies for plantar warts.

15.2 Major Constraints of Groundnut Production

The total production of groundnut has been inconsistent during the past few years. According to the reports of FAO (December 2009), the yield was 15,535 hg/ha from an area of 24,590,075 ha in 2008 whereas it was 16,908 hg/ha in 2007 from a comparatively less area. A gradual increase in the area harvested since 2006 was not reciprocated in the yield/production. Groundnut production in Argentina is expected to drop to 6,50,000 t for the year 2010–2011 from 7,50,000 estimated for 2009–2010. With about seven million hectares under cultivation, India has the largest area of groundnut in the world (Fig. 15.1). However, its productivity has stagnated to less than 1 t ha1, which is far below the productivity levels achieved elsewhere and the actual potential (3–4 t ha 1) of the crop. Despite its long history of cultivation, its importance in oil economy of India and as an important source of livelihood for millions of small and marginal farmers, the productivity of the crop has remained very low. Conventional breeding contributed signiﬁcant progress

Fig. 15.1 Area under groundnut cultivation in different countries around the world with India leading the chart followed by China and Nigeria 15 Crop Health Improvement with Groundnut Associated Bacteria 409 towards improving productivity and quality of groundnut. However, there remains a large gap in potential yield and realized yield at farm level, particularly in rainfed agriculture. Essentially, increase in the productivity of all important food crops, including groundnut, is an absolute requirement to meet the demand of the increasing population. Increase in cultivable land is difficult because of the constraint of space and, therefore, maximum yield from already available crop area is a challenge for agriculturists today. Several biotic, abiotic, and socioeconomic constraints have been identified (Balaji et al. 2003;Basu2003; Gadgil et al. 1996, 2002; Reddy et al. 1992) for the decline in groundnut productivity. These constraints include unpredictable weather in terms of onset of rainy season, amount of rainfall and its distribution during groundnut- growing period; cultivation of the crop on marginal and sub-marginal lands under rainfed conditions subjected to frequent drought; poor agronomic practices and low levels of input; use of low yielding and late maturing cultivars; high infestation by insects, pests, and diseases; inadequate availability of high-quality seed of improved varieties; and low levels of adoption of recommended technologies by the farmers. Soil fertility, unpredictable terminal drought and high incidence of fungal diseases are some of the major concerns of groundnut cultivation, especially in Afro-Asian countries. In India, it is mostly grown in areas with soils deficient in essential plant nutrients and therefore, yield losses due to imbalanced nutrition are significant. Throughout the cultivation of groundnut, from planting to storage, different types of biotic and abiotic factors form major threats. The abiotic factors include physiological and environmental stresses such as temperature, soil pH, and drought, and the biotic threats include insects, fungi, bacteria, virus, nematodes, and weeds. Of the various biotic stresses, pathogens causing diseases pose a serious limitation towards the crop health. The majority of the small-scale farmers are reluctant to invest in chemical fertilizers because of the uncertain crop returns besides being expensive. Moreover, increasing concern over the use of chemicals for crop improvement causing environmental threats, have led agriculturists to find viable and cost-effective biological alternatives. Thus, the need of the hour is to enhance the efficiency of the meager amount of external inputs by employing the best combinations of beneficial microbes for sustainable production of groundnut. In this context, there is a greater scope for development and popularization of bioinoculants in these groundnut production systems.

15.3 Major Diseases Affecting Groundnut Crop Health

Groundnut is prone to attack by more than 55 pathogens including fungi, bacteria, viruses, mycoplasma, and nematodes, of which fungal diseases cause the majority of yield loss. Important fungal diseases of groundnut and their causal organisms are listed in Table 15.1 (Podile and Kishore 2002). Symptoms of some of the diseases, listed in Table 1, are shown in Fig. 15.2. Early leaf spot (Fig. 15.2a) causes light to dark brown lesions on the upper surface of the 410 S. Dutta et al.

Table 15.1 Major fungal diseases of groundnut and their causal organisms Disease Causal organism Foliar diseases Leaf blight Alternaria tenuis Act. A. tenuissima (Kunze ex Pers.) Wiltshire A. arachidis Kulk. Cercospora canescens Ellis and Martin Bipolaris spicifera (Bainier) Subramanian Phoma arachidis P. microspora Balasubramaniam and Narayanasamy sp nov. P. sorghina (Sacc.) Boerema, Dorenbosch and V. Kest Leaf spot A. arachidis Kulk. A. arachidis (Fr.) Keissler Alternaria sp. Choanephora cucurbitarium (Berk. and Rav.) Thaxt Choanephora sp. Early leaf spot Cercospora arachidicola Hori Late leaf spot Phaeoisariopsis personata (Berk. and Curt.) v. Arx Leptosphaerulina crassiasca (Sechet) Jackson and Bell Pestalotiopsis arachidis Satya P. adusta (Ell. and Ev.) Steyaert P. versicolor (Speg.) Steyaert Didymella arachidicola (Chock.) Taber, Pettit and Philley Cristulariella pyramidalis Waterman and Marshall Rust Puccinia arachidis Speg. Scab Sphaceloma arachidis Bitancourt and Jenk. Seed and seedling diseases Crown rot Aspergillus niger van Tieghem Collar rot A. pulverulentus (McAlpine) Thom. Lasiodoplodia theobromae (Pat.) Grif. and Maub. Damping-off and Foot Rhizoctonia solani Kuhn rot Fusarium solani f.sp. phaseoli (Burk.) Sny. and Hans. F. oxysporum Schl. Emend Sny. Y Hans. Fusarium spp. Pythium myriotylam Dreschsler P. debaryanum Hesse P. irregular Buisman P. ultimum Trow Macrophomina phaseolina (Tassi.) Goid. Sclerotium rolfsii Sacc. Pre-emergence seed Rhizopus arrhizus Fischer and seedling rots R. stolonifer (Her. Ex Fr.) Vuillemin R. oryzae Went and Gerlings F. solani f.sp. phaseoli (Burk.) Sny. and Hans. F. oxysporum Schl. Emend Sny. Y Hans. Fusarium spp. Pythium myriotylam Dreschsler P. debaryanum Hesse P. irregular Buisman P. ultimum Trow P. butleri Subramaniam Rhizoctonia solani Kuhn Macrophomina phaseolina (Tassi.) Goid. (continued) 15 Crop Health Improvement with Groundnut Associated Bacteria 411

Table 15.1 (continued) Disease Causal organism Sclerotium rolfsii Sacc. Aspergillus niger van Tieghem A. flavus Link ex Fries Botryodiplodia theobromae Pat. Cochliobolus bicolour Paul and Par. Penicillium citrinum Thom. P. funiculosum Thom. Yellow mold, blue A. flavus Link ex Fries mold, aflaroot A. parasiticus Speare A. pulverulentus (McAlpine) Thom. Stem, root and pod diseases Blackhull Thielaviopsis basicola (Berk. and Br.) Ferr. Blacknut, charcoal rot, Macrophomina phaseolina (Tassi.) Goid. ashy stem Blight, dry rot, dry wilt R. bataticola (Taub.) Butler Blue damage Concealed damage S. rolfsii Sacc. Diplodia gossypina Cooke Aspergillus niger van Tieghem Macrophomina phaseolina (Tassi.) Goid. Cylindrocladium Cylindrocladium crotalariae (Loos) Bell Sobers (anamorph) black rot Fusarium wilt F. oxysoprum Schl. Emend. Sny. and Hans. Pythium vascular wilt Pythium myriotylam Dreschsler Root rots, pod rots, R. solani Kuhn pod breakdown F. solani f.sp. phaseoli (Burk.) Sny. and Hans. F. oxysoprum Schl. Emend. Sny. and Hans. Fusarium spp. Pythium myriotylam Dreschsler P. debaryanum Hesse P. irregular Buisman P. ultimum Trow Macrophomina phaseolina (Tassi.) Goid. S. rolfsii Sacc. Sclerotinia blight Sclerotinia minor Jagger S. sclerotiorum (Lib.) de Bary Stem rot, white mold, Sclerotium rolfsii Sacc. southern blight, Sclerotium wilt Verticillium wilt, Verticillium albo-atrum Reinke and Bert. floury rot V. dahlia Kleb.

leaflets with a chlorotic halo surrounding the lesions. Infected leaves finally necrotize and defoliate. Late leaf spot (Fig. 15.2b) symptoms occur as circular, dark lesions on the lower leaflet surface but without the halo formation. The leaves defoliate after necrosis. During severe infections, oval elongate lesions are formed on the petioles and stems. Leaf rust of groundnut (Fig. 15.2c) causes orange colored pustules on the lower surface and, in severe cases, on the upper surface of leaflets. 412 S. Dutta et al.

Fig. 15.2 Symptoms of different diseases caused by fungal pathogens in groundnut plant. (a) Early leaf spot, (b) late leaf spot, (c) leaf rust, (d) stem rot, (e) collar rot, and (f) yellow mold

Among stem diseases, stem rot and collar rot are the most dreadful diseases causing yield loss in groundnut. In stem rot (Fig. 15.2d), whitish mycelium is visible around the infected stem in the initial stages followed by brown sclerotia in the later stages. The leaves become yellow and wilt. Infected stems rot and break at the site of infection. White mycelium on the stem, pegs, and pods during the time of harvest causes subsequent pod decay, thereby causing heavy loss in production. Collar rot pathogen, Aspergillus niger, can attack the plant at different growth stages. When the fungus attacks before germination, the seed becomes completely rotten and fails to germinate. Young infected plants become wilted and the collar region becomes dark brown and disrupted (Fig. 15.2e). Pre-emergence infection of yellow mold causing fungus leads to rotting of the seeds. A. flavus, causal organism colonizes the cotyledons leading to rapid decay of emerging radical and hypocotyls (Fig. 15.2f). The seedlings appear stunted with small, pointed chlorotic and pale yellow leaves (aflaroot). A. flavus invades the groundnut seeds and produces aflatoxins both at pre- and post-harvest stages. Aflatoxins are potent carcinogens and are hazardous to human and animal health.

15.4 Beneﬁcial Microbes for Improved Health

Several attempts were made to achieve biological control of groundnut diseases using biocontrol agents such as fungi and bacteria. Most of these studies were conﬁned to greenhouse environments and a few have reached the point of 15 Crop Health Improvement with Groundnut Associated Bacteria 413 commercial application. Although progress has been made in the experimental biological control of groundnut diseases, the mechanisms involved are yet to be fully understood. There is great potential for control of major groundnut diseases using growth-promoting bacterial strains, thereby cutting the cost of agrochemicals applied and consequently, the yield loss. Nitrogen-ﬁxing Rhizobium and its role as a potential biofertilizer in leguminous plants has been known since its discovery by Bejeirinck in the 1880s. Fungal strains such as Trichoderma have been successfully applied to various crops for growth promotion and disease control (Rojo et al. 2007). A diverse group of free-living soil bacteria that can stimulate the growth of plants by one or more of a number of different mechanisms are known as plant-growth promoting rhizobacteria (PGPR) (Podile and Kishore 2006; Glick 1995; Klopper et al. 1989).

15.5 Different Plant Parts as Sources of Beneﬁcial Bacteria Affecting Crop Improvement

Plant-associated complex bacterial communities are diverse in their genotypic and phenotypic characteristics, their phylogeny, and their ability to affect plant health. Many plant-associated microbes, irrespective of their strength in the community, can have important and decisive functions affecting the agricultural output. Bacte- ria can attain densities as high as 109 cells per gram of plant tissue of roots based on culturing, and 1010 cells per gram based on cultivation-independent methods. Although eukaryotic microﬂora including ﬁlamentous fungi, yeasts, algae, protozoa, and nematodes are also present (usually at low densities), we will restrict this chapter to the role of bacteria associated with groundnut with special focus on exploiting such bacteria for the control of plant pathogens and improvement of groundnut health. Groundnut offers a wide variety of habitats that support microbial growth ranging from moist sites rich in nutrients, as well as sites that are nutrient poor. The surfaces of seeds, roots, leaves, and fruits provide space for proliferation of diverse bacterial communities. To exploit groundnut-associated bacteria for control of fungal diseases of crops and also to promote the growth and yield, several different habitats have been approached in different parts of the world (Kishore et al. 2005a; Baig et al. 2002). Beattie (2006) described the different habitats of plants (Fig. 15.3) that harbor different microbial communities as below:

15.5.1 The Spermosphere

The spermosphere is the zone that is inﬂuenced by a seed and extends 1–10 mm from the seed surface. Nutrients released during seed imbibitions support the 414 S. Dutta et al.

Fig. 15.3 Diagrammatic representation of habitat diversity of bacteria in different parts of the plant growth of bacteria, with the maximum nutrient release from the embryo end and from seeds that are cracked or damaged. Bacteria that establish populations on seeds can subsequently colonize the emerging roots.

15.5.2 The Rhizosphere

The rhizosphere is the zone inﬂuenced by the root, often deﬁned as the soil adhering to the root. Root growth changes the physical and chemical properties of the nearby soil, including the mineral and organic content, water potential, pH, and salinity. This region contains root exudates, secretions, and compounds released by plant cell lysis. Microorganisms do not grow on the root tip, due to the rapid rate at which border cells are shed and also the population of microbes in the mature roots are less, as they produce less mucilage and fewer cell lysates. Therefore, the developing roots generally support fast growing microorganisms such as bacteria, whereas mature roots support slow growing microorganisms such as fungi and actinomyce- tous bacteria.

15.5.3 The Phyllosphere

The phyllosphere denotes the external regions of the above-ground parts of plants, including leaves, stems, and fruits. Leaves are the dominant tissue in the phyllosphere based on the surface area available for colonization. The waxy plant cuticle ensures that water loss occurs primarily through the stomata, thereby ensuring presence of rich leaf surface microﬂora. Unlike the rhizosphere, the phyllosphere is subject to large and rapid ﬂuctuations in temperature, solar radiation, and water availability, which increases with rain, dew, or fog and decreases with wind. 15 Crop Health Improvement with Groundnut Associated Bacteria 415

In contrast to the rhizosphere, which often supports at least 109 bacteria per gram of root, leaf surfaces typically support fewer than 107 bacteria per gram of leaf.

15.5.4 Endophytic Sites

Endophytic sites include any region internal to the plant epidermis and endophytic microorganisms are usually found within the intercellular, or apoplastic, spaces. Intercellular air spaces comprise a significant fraction of the tissue inside roots and leaves. Microorganisms that reach these intercellular regions must contend with plant defense responses, which are triggered when bacteria are in close proximity to the plant cells. Some symbiotic bacteria such as rhizobia have evolved sophisti- cated entry mechanisms that include directing the plant to form a channel, called an infection thread that promotes bacterial penetration into the plant tissue. The rhizobial strains form nodules in the legumes and provide fixed nitrogen to the plants. Free-living bacteria residing in the nodules also benefit the growing plants through interaction with the rhizobial strains.

15.6 Groundnut-Associated Beneﬁcial Bacteria

Groundnut-associated bacteria mostly involve microorganisms that have both direct and indirect promoting properties (Bhatia et al. 2008; Arora et al. 2001). This includes rhizobial strains with biocontrol activities (Deshwal et al. 2003; Arora et al. 2001) and other beneﬁcial bacteria having multiple activities such as phosphate solubilizing activity, IAA and HCN production, and biocontrol properties such as production of siderophore and antibiosis (Kishore et al. 2005a; Dey et al. 2004; Pal et al. 2000).

15.6.1 Rhizobial Strains

The most important and abundant of beneﬁcial bacteria associated with groundnut belong to the rhizobial group. Being a leguminous plant, groundnut interacts with slow-growing rhizobia belonging to the genus Bradyrhizobium to form nodules. The analysis through morphophysiological and molecular methods of this group of bacteria from groundnut grown in different geographical regions revealed high level of diversity and heterogeneity (Taurian et al. 2008;YangandZhou 2008; Saleena et al. 2001; Zhang et al. 1999;UrtzandElkan1996; van Rossum et al. 1995). Besides Bradyrhizobium, bacteria belonging to the genus Rhizobium were also associated with groundnut nodules (El-Akhal et al. 2008;Iban˜ez et al. 2008; Taurian et al. 2006). The detailed interaction of groundnut plants with 416 S. Dutta et al. rhizobial strains have been discussed elaborately in a most recent review by Fabra et al. (2010).

15.6.2 PGPR Strains

Groundnut-associated PGPR were isolated from different parts of the plant including rhizoplane, phylloplane, and geocaroposhere; beneﬁcial effects and the possible mechanisms involved in growth promotion or improved health have revealed involvement of several factors in the observed improvement in health.

15.6.2.1 Groundnut Crop as a Source of PGPR

Besides the rhizosphere, bacterial strains of growth promoting and disease control ability have been isolated from other plant parts of groundnut such as phylloplane, geocarposhere, root nodules, and seed endophytes (Fig. 15.4). Some PGPR strains are reported to inhabit the inner tissues and generate endophytic populations not only in the roots but also in leaves and stems (Compant et al. 2005). Bacteria that are inhabitants of plant external surfaces and internal tissues are commonly named epiphytic and endophytic, respectively (Andrews and Harris 2000; Kuklinsky-Sobral et al. 2004). Both can contribute to the health, growth, and development of plants. Bacterial strains isolated from groundnut rhizosphere have the ability to improve plant growth and control diseases in groundnut. Pseudomonas strains from rhizosphere of groundnut efficiently colonized growing roots of bacteria thereby exerting growth promoting and biocontrol effects on the plant (Kishore et al. 2005a; Gupta et al. 2002; Pal et al. 2000). Another study showed efficient colonization of the spermosphere by the B. licheniformis strain MML2501, isolation of groundnut rhizosphere, thereby significantly increasing seed germination and other growth parameters under in vitro conditions (Prashanth and Mathivanan 2010). However,

Fig. 15.4 Diagrammatic representation of population of beneﬁcial bacteria from different habitats of groundnut plants 15 Crop Health Improvement with Groundnut Associated Bacteria 417

Shoot height Root length 45 80 a Fresh Weight b 40 ab a Dry weight T 70 T Germination percentage 35 b 60

Fresh & Dry weight b Seed germination (%) c 30 d 50 25 a 40 b T 20 T bc c

30 (g) / 15 20 10 Shoot hieght & Root length (cm) b a 5 c c 10 a a b c 0 0 P.elgii P.elgii with Chitosan Chitosan alone Control

Fig. 15.5 Effect of P. elgii with or without chitosan on germination and growth of groundnut seeds in terms of shoot height, root length, fresh, and dry weight (Das et al. 2010). Different letters on each bar represent values that are statistically different. (P ¼ 0.05). The vertical bars indicate standard error a similar increase in the growth and yield of groundnut was observed with phylloplane isolates of P. aeruginosa GPS 55 and GPS 21 when applied as seed treatment (Kishore et al. 2005b). Both the strains were potent siderophore producers, while GPS 21 was also a broad-spectrum antifungal strain. Since phylloplane bacteria are exposed to higher ﬂuctuations of abiotic factors such as temperature and moisture and have limited nutrient availability, these bacterial strains have better ability to survive and multiply in the nutritionally rich rhizosphere soil. Recently chitinolytic strains isolated from unconventional sources such as chitin- rich soils showed improved growth in groundnut (Das et al. 2010). Although the strain of Paenibacillus elgii SMA-1-SDCH02 was not isolated from plant parts, it showed tremendous potential in groundnut growth promotion (Fig. 15.5) and antifungal activities similar to other reported PGPR. This provides ample evidence for utilizing bacterial strains from unconventional sources, especially chitin-rich soils (with an added quality of chitinolysis), to facilitate plant growth directly or indirectly.

15.6.2.2 Mechanisms Involved in Growth Promotion and Disease Control by PGPR

PGPR can affect plant growth either directly or indirectly (Fig. 15.6). The direct promotion of plant growth by PGPR mostly includes either providing the plant with 418 S. Dutta et al.

PGPR

Antibiosis

Induced Competition Resistance Growth Promotion Improved Plant strength Pathogen Plant

Disease Escape

Fig. 15.6 Direct and indirect mechanisms of plant growth promotion and disease control by PGPR in plants

a compound that is synthesized by the bacterium or facilitating the uptake of certain nutrients from the environment. The indirect promotion of plant growth occurs when PGPR lessens or prevents the deleterious effect of one or more phytopathogenic organisms. Although the mechanisms of PGPR-mediated enhancement of plant growth and yield are not yet fully understood, various studies provided possible explana- tions. The major outcomes established through these studies described that the improved plant growth by PGPR is due to its ability to produce phytohormones such as indole acetic acid (IAA) (Patten and Glick 2002; Mordukhova et al. 1991), gibberellic acid (Mahmoud et al. 1984), and cytokinins (Tien et al. 1979) and its ability to produce ACC-deaminase to reduce the level of ethylene in the roots of the developing plants thereby increasing the root length and growth (Penrose and Glick 2001;Lietal.2000; Glick et al. 1995;Jacobsonetal.1994). The asymbiotic nitrogen ﬁxation by PGPR also adds to its growth-promoting traits (Figueiredo et al. 2008; Kennedy et al. 1997). Solubilization of mineral phosphates and mobilization of other essential nutrients by PGPR also helps in growth improvement of plants (Taurian et al. 2010; De Freitas et al. 1997). Besides, antagonism against phytopathogenic microorganisms by producing siderophores, b-1,3- glucanase, chitinases, antibiotics, ﬂuorescent pigment, and cyanide (Pal et al. 2001; Catellan et al. 1999; Renwick et al. 1991; Voisard et al. 1989; Scher and Baker 1982) provides indirect methods of improving crop health by deleting the deleterious effects of harmful microbes. 15 Crop Health Improvement with Groundnut Associated Bacteria 419

PGPR strains, isolated from different plant parts of groundnut, are reported to have one and/or another of the above-mentioned characteristics (Taurian et al. 2010, Kishore et al. 2005a, b).

15.6.2.3 Direct Growth Promotion by Bacteria

It was observed that Bacillus subtilis strains increased the emergence vigor and yield in Florunner groundnuts (Jaks et al. 1985). Similar results of improved germination, emergence, increased nodulation by groundnut Rhizobium, and increased root growth along with consistent colonization of B. subtilis were reported by Turner and Backman (1991). The rhizosphere of groundnut has since then been elaborately studied with varied agro-climatic conditions and found to be rich in bacterial biodiversity. Baig et al. (2002) estimated Gram-negative bacteria to be about 65% of the total bacterial population as compared to only 35% of Gram- positive bacteria. Pseudomonas was most predominant followed by Bacillus, Enterobacter, Micrococcus, Proteus, and Klebsiella. Yield increase of groundnut by Pseudomonas isolates from rhizosphere positively correlated with the ability of these strains to increase available soil phosphorus (Dey et al. 2004). The number of nodules in treated plants was also found to be higher than untreated control and, therefore, it was hypothesized that the energy needed for this symbiotic process is facilitated by the availability of high soil phosphorus content. Kishore et al. (2005a) reported various bacterial isolates from the geocarposphere, rhizosphere, phylloplane, and seeds of groundnut to significantly enhance shoot and root length, dry biomass, and yield under greenhouse and field conditions. Most of the bacterial strains produced IAA and solubilized phosphate under in vitro conditions (Fig. 15.7). Compared to phosphate-solubilizing bacteria, the majority of the auxin producers showed better results in terms of plant growth. However, there was no correlation between auxin production in vitro and growth improvement under field condition. The strains also produced siderophore besides showing broad-spectrum

Fig. 15.7 In vitro auxin production and mineral 30 phosphate solubilization by groundnut-associated bacteria. (Black bars) 20 Percentage of isolates positive for individual characteristic among the total 10

393 bacterial isolates was isolates of Percentage compared with (White bars) percentage of isolates positive 0 among the 27 growth- Auxin production Mineral phosphate promoting isolates solubilization 420 S. Dutta et al. antifungal activity (Kishore et al. 2005b). Similarly, bacterial strains isolated from groundnut rhizosphere by Dey et al. (2004) and showing growth-promoting characteristics exhibited multiple PGPR traits such as IAA production, ammonification, phosphate solubilization, and siderophore production besides ACC-deaminase activity. Endophytic isolates, such as Pantoea, from root nodules increased plant biomass of groundnut which was comparable to the effects of rhizobacterial strains (Taurian et al. 2010). This was the first report that described groundnut root nodule bacteria with PGPR activity. Apart from that, they also described 37 epiphytic and 73 endophytic isolates showing phosphate solubilizing ability from around 433 rhizosphere, phyllosphere, and other plant tissue isolates of groundnut. All the PGPR activities such as production of siderophores, IAA, and antibiosis against fungal pathogens were identified in a high number of endophytic bacteria, especially in those residing in the root nodules. Nodule endophytic bacteria are poorly studied compared with the endophytes living in other plant tissues (Li et al. 2008). Earlier reports demonstrated that root nodules harbor opportunistic gammaproteobacteria associated to root nodules (Iban˜ez et al. 2009). Several fast growing bacteria such as species of Pseudomonas, Enterobacter, and Klebsiella were isolated from surface- sterilized root nodules. Methylotrophic bacteria with growth promoting and nitrogen fixing ability have been isolated from the rhizosphere and phyllosphere of groundnut (Madhaiyan et al. 2010, 2006). The Methylobacterium sp. PPFM-Ah was originally isolated from groundnut leaves and applied through seed imbibitions to stimulate germination and plant growth under greenhouse conditions (Madhaiyan et al. 2006). Groundnut associated PGPR strains appear to have more than one growth- promoting activity and the involvement of a specific mechanism in enhancing plant growth and yield cannot be ruled out.

15.6.2.4 Indirect Growth Promotion by Bacteria

Apart from direct growth-promoting characteristics, exclusion of deleterious microorganisms from the vicinity of plants and induction systemic resistance (ISR) have also been reported by PGPR strains isolated from groundnut. Potential strains of fluorescent Pseudomonas and Bacillus antagonistic to A. flavus were isolated from the geocarposphere of groundnut and used successfully for the control of pre-harvest groundnut seed infection by A. flavus (Anjaiah et al. 2006). The employment of B. subtilis counteracted the destructive effects of the seed-borne pathogen Sclerotium rolfsii on nodulation, leghemoglobin, and nitrogenase activity of groundnuts (Abd-Allah and El-Didamony 2007). Two strains of fluorescent pseudomonads showing in vitro antifungal activity against charcoal rot causing fungus Macrophomina phaseolina produce siderophores, IAA, and HCN besides solubilizing phosphate, enhancing seed germination, and grain yield (Bhatia et al. 2008). 15 Crop Health Improvement with Groundnut Associated Bacteria 421

Sailaja et al. (1998) were the first to report induction of lipoxygenase (LOX) activities in groundnut on treatment with a PGPR strain B. subtilis AF1. This induction of LOX during activation of growth and pathogen infection was discussed in the light of the reported involvement of LOX both in growth and development as well as in plant pathogen interaction, particularly ISR. The increase in LOX activity led to the production of 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-hydroperoxyoctadecatrienoic acid (13-HPOTrE) as major products with linoleic acid (LA) and linolenic acid (ALA), respectively. Both the hydroperoxides are inhibitory to the growth of A. niger as measured in micro titre plates. Metabolic products of polyunsaturated fatty acids have been variously implicated in control of microbial pathogens. Bacterial strains isolated from various parts of groundnut plant by Podile’s group (Podile and Kishore 2006; Kishore et al. 2006; Kishore et al. 2005b) showed broad- spectrum antifungal activity and suppressed collar rot of groundnut caused by A. niger. The strains were also tested against a number of phytopathogenic fungi including soil/seed and phyllosphere pathogens. Five of the Pseudomonas strains and their cell-free supernatants showed fungicidal activity against all the pathogens tested by inducing mycelial deformations and inhibiting the spore germination. The possible explanation for the biocontrol ability could be effective rhizosphere colonization, production of extracellular antibiotics, lytic enzymes or siderophores, and activation of host defense responses. Kishore et al. (2005c) showed the ability of these strains to inhibit the activities of cell wall degrading enzymes produced by S. rolfsii, thereby reducing the severity of stem rot disease of groundnut. The chitinolytic strains of Bacillus circulans GRS243 and Serratia marcescens GPS5 control late leaf spot (LLS) by activating the defense-related enzymes chitinase, b-1,3-glucanase, peroxidise (PO), and phenylalanine ammonia lyase (PAL) in the leaves previously supplemented with 1% colloidal chitin (Kishore et al. 2005d). A 55-kDa chitinase was purified from the cell-free culture filtrate of GPS 5 by affinity chromatography and gel filtration. Purified chitinase of GPS 5 inhibited the in vitro germination of P. personata conidia, lysed the conidia, and effectively controlled LLS disease in greenhouse tests (Manjula et al. 2004). Similar increases in defense- related enzymes were observed in groundnut plants whose leaves were sprayed with Bacillus sp. prior to the root inoculation with fungal pathogen S. rolfsii (Furlan et al. 2008) and in plants treated with methylotrophic bacteria Methylobacterium and challenge-inoculated with A. niger/S. rolfsii (Madhaiyan et al. 2006). However, increase in defense-related enzymes could not make the plants disease free in the former case but, in the latter, the percent disease index value was reduced. There- fore, control of the disease due to ISR is a probable explanation for disease resistance by Methylobacterium. The experiments of Zhang et al. (2001) tend to differ in that the ISR is not always true against all pathogens. Greenhouse and field evaluation for the ISR resistance against LLS (caused by Cercosporidium personatum) by PGPR and chemical elicitors such as salicylic acid, sodium salicylate, isonicotinic acid, benzo thiadia- zole-7-carbothioc acid S-methyl ester, and DL-b-amino-n-butyric acid (BABA) showed inconsistent results. This indicated that LLS resistance in groundnut is 422 S. Dutta et al. not systemically inducible in the same manner as it is in other crops by these PGPR and chemical inducers (Zhang et al. 2001).

15.6.2.5 Compatibility of Bacteria with Seed/Root exudates

Root colonization by introduced bacteria in sufficient population is essential for exertion of beneficial effects on the host plant. A variety of bacterial traits and specific genes contribute to the process of root colonization by PGPR, but only a few have been identified (Benizri et al. 2001; Lugtenberg et al. 2001). These include motility, chemotaxis to seed and root exudates, production of pili or fimbriae, production of specific cell surface components, ability to use specific components of root exudates, protein secretion, and quorum sensing (Lugtenberg et al. 2001). Host-specific effects of root exudates on rhizobacteria occur at a metabolic level (Das et al. 2010; Dutta and Podile 2010). In the initial stages of the multiplication and establishment of rhizobacteria in the rhizosphere, it is essential for the rhizosphere bacteria to have the capability of utilizing the seed exudates as the sole source of C and N (Weller 1988). This ability gives an added advantage to the inoculant strains and renders rhizosphere competence as compared to the ones which cannot utilize the seed exudates as the sole source of C and N and, therefore, are less competent. Dey et al. (2004) studied utilization of seed exudates by inoculant bacterial strains in groundnut and showed that the majority of the PGPR isolates, applied as seed treatment, could utilize the seed exudates. The importance of photosynthates in root exudates for proper colonization of bacteria in the roots was also shown. The strain which was least able to utilize seed leachate and thus, was supposed to be the least competent, was one of the best in enhancing the growth, yield, and nutrient uptake of groundnut under potted and field conditions. Therefore, it was concluded that the seed leachate may provide the sources of carbon and nitrogen in the initial few days but later on, the translocation of the quantum and qualitative and quantitative nature of photosynthates in the form of root exudates plays a crucial role in the establishment of the inoculant bacteria. This ability to utilize complex sets of carbon sources of the root exudates by the organisms ultimately determines the outcome of PGPR–plant interactions. Thus, the least ability of utilizing seed leachate under in vitro conditions does not always infer the performance under in situ conditions. Similar kind of results was reported in groundnut Kishore et al. (2005a). The auxin-producing PGPR did not show desired growth-promoting ability under field condition. This difference in the performance could be due to the greater dependency on the availability of L-tryptophan in the root exudates for production of bacterial auxins. Moreover, the optimal concentration of auxin required for plant growth promotion is extremely narrow (Xie et al. 1996) and doses of auxin above the threshold levels are deleterious for root growth. However, it was confirmed that bacterial isolates from phylloplane, which are not familiar with the rhizosphere environment, successfully colonized groundnut roots and enhanced growth besides reducing disease incidence. Similarly, bacteria from chitin-rich soil colonized groundnut roots and 15 Crop Health Improvement with Groundnut Associated Bacteria 423 performed well. These results indicate that these strains, not isolated from rhizosphere, adapted well to the new environment and might utilize the exudates released by the roots.

15.7 Interaction of Rhizobial and PGPR Strains for Growth Improvement

Li and Alexander (1988) suggested co-inoculation of Rhizobium with antibiotic producing microorganisms as a promising technique for stimulating nodulation and nitrogen fixation through legume–bacterial symbiosis. Co-inoculation of some PGPR strains increased the nodulation of legumes by nitrogen fixing rhizobia (Kloepper et al. 1991) and were designated as nodulation promoting rhizobacteria (NPR). Inoculation of legumes with root colonizing bacteria and Rhizobium has been demonstrated to affect symbiotic nitrogen fixation by enhancing root nodule number or mass and increased nitrogenase activity (Dashti et al. 1998; Saxena and Tilak 1994; Alagawadi and Gaur 1988; Polonenko et al. 1987; Grimes and Mount 1984). Growth promotion and increase in defense-related enzymes were also reported in pigeon pea on co-inoculation of PGPR and rhizobial strains (Dutta et al. 2008). Growth enhancement was observed in groundnut on combined application of rhizobial and PGPR strains. Increase in crop vigor index, total nitrogen content, and survivability of both Rhizobium and B. subtilis, a PGPR strain, have been related to compatibility and even an occasional synergism between them in groundnut (Abd-Allah and El-Didamony 2007). Combined application of a P. fluorescens strain FPD-15 along with Bradyrhizobium in groundnut significantly enhanced groundnut root and shoot dry weight, nodule number, nodule dry weight, and per cent nitrogen content of shoot (Vikram et al. 2007). Madhaiyan et al. (2006) observed significant increase in plant growth, nodulation, and yield attributes of groundnut on combined inoculation of Methylobacterium with Rhizobium as compared to individual treatment of either Rhizobium or Methylobacterium. More recently, several fast growing bacteria isolated from groundnut nodules were unable to induce nodule formation after storage (Iban˜ez et al. 2009). However, they enhanced plant growth and, on co-inoculation with Bradyrhizobium strain, colonized pre-formed nodules. The exact mechanisms involved in the interaction of PGPR and rhizobial strains resulting in growth promotion are yet to be fully explored. Lucas-Garcia et al. (2004) had proposed the possibility that metabolites other than phytohormones, such as siderophores, phytoalexins, and flavonoids produced by PGPR might have a role in enhanced nodule formation in co-inoculated legumes. Another mechanism of co-inoculation, which is proposed to benefit the rhizobia, is the reduction of ethylene level in plants. Decreased levels of nodulation have been observed after application of exogenous ethylene or ACC prior to or at the same time as the addition of rhizobia (Nukui et al. 2000); conversely, nodulation 424 S. Dutta et al. can be promoted when plants are treated with ethylene inhibiters or antagonists (Yuhashi et al. 2000). As already established, PGPR has the ability to reduce ethylene level in plants (Glick et al. 1998). ACC deaminase-producing PGPR strains when co-inoculated with Rhizobium decrease the ethylene levels in plants (Ma et al. 2002). This may be due to better nodulation by rhizobia in the host plant leading to increased growth. Another limitation affecting the growth of rhizobia in legumes is the competition with low nitrogen yielding indigenous rhizobia (Brutti et al. 1999). Presence of PGPR incompatible with these indigenous rhizobia ensures their deletion from the roots and thereby providing space for the efficient rhizobial strains. Moreover, removal of harmful bacteria from the rhizosphere also helps in comfortable establishment of rhizobia in root nodules. Mobilization of nutrients and vitamins essential for rhizobial growth by PGPR benefits the residing population so as to have a better effect on crop health.

15.8 Carrier-based Formulations for Growth Promotion by PGPR

Different carrier-based chitin/chitosan-supplemented formulations (Manjula and Podile 2005, 2001) were effective against several phytopathogens and also significantly increased plant growth. We have shown the extensive damage caused to major fungal pathogens of groundnut by chitinolytic biocontrol strains and the partially purified enzymes (Manjula and Podile 2005; Manjula et al. 2004; Podile and Prakash 1996), and exploited the chitinolytic potential of the biocontrol PGPR strains to improve both the shelf life and effectiveness of the formulations (Manjula and Podile 2005; 2001;Kishoreetal.2005a, b). Increased growth of groundnut on application of chitinolytic Paenibacillus sp. together with chitosan has also been observed (Das et al. 2010). Kokalis-Burelle et al. (1992)demonstrated that foliar sprays of chitin resulted in reduced leaf spot damage, presumably due to enhanced populations of indigenous antagonists. Chitin-supplemented application of B. circulans GRS 243 and S. marcescens GPS 5 resulted in improved biological control of leaf spot disease under green house and field condition (Kishore et al. 2005d). Similarly, Meena et al. (2002) showed seed treatment and foliar spray with the talc-based powder formulation of P. fluorescens strain Pf1 alone effectively reduced the severity of leaf spot and rust in groundnut under glasshouse and field trials. In both the cases, the antagonist was able to multiply in the rhizosphere/ phyllosphere. Combined application of the P. fluorescens formulation to seed and foliage showed an enhanced control of leaf spot and rust besides an improvement in the pod yield. Peat-based formulations of groundnut-associated bacteria showed improved growth on application as seed treatment under field conditions (Kishore et al. 2005a). Therefore, PGPR formulations effectively work on plants on supplementation of chitin, or related products such as chitosan. 15 Crop Health Improvement with Groundnut Associated Bacteria 425

15.9 Conclusions

Groundnut yield loss is attributed to a number of factors including harmful diseases. Application of biological means for crop improvement and disease control is the need of the hour as it is cost effective and environment friendly. For this, the exploration of beneficial bacteria such as PGPR from plant sources such as rhizosphere, phyllosphere, etc. is essential. However, inconsistent results of plant-associated bacteria under varied climatic conditions and problems regarding host specificity are some of the major hurdles for successful application of PGPR. Therefore, application of native PGPR strains to the host plant is an ideal method to ensure colonization of bacteria in sufficient population in the host roots thereby exerting growth promotion and disease control in groundnut. The information summarized in this chapter projected the rich source of beneficial bacteria associated with groundnut and through different experiments their potentiality has been confirmed as plant growth promoters and biocontrol agents. Further studies are required to specify the exact mechanisms involved in this interaction. Compared to PGPR studies in other plants, beneficial bacteria associated with groundnut have not been fully explored. The examples show their diversity and ability to perform under varied nutritional and physiological conditions. Further studies on the groundnut–PGPR interactions through genomics, proteomics, and metabolomics approach will bring out more useful information to exploit bacteria associated with groundnut for overall crop health improvement.

Acknowledgements The ﬁnancial support under Department of Science and Technology-Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions (DST-FIST-level II) to the Department of Plant Sciences, University Grants Commis- sion-Centre for Advanced Studies (UGC-CAS) to the School of Life Sciences are highly acknowledged. SD thanks the Department of Biotechnology for Post Doctoral Fellowship.

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A Azospirillum, 3, 4, 11, 20–22 Abscisic acid (ABS), 372–373 A. brasilense, 364 ACC degradation, 346 A. lipoferum, 364 ACC-deaminase, 397 Azotobacter chroococcum, 19, 20 Acetobacter,20 A. paspalum, 364 Achromatobacterium,20 Achromobacter,6 B Actinoplanes,3 Bacillibactin, 378 Aerobacter,6 Bacillus, 37–54, 241, 246–248, 253, 256, Aerobactin, 368, 378 350–355 Agrobacterium radiobacter,19 B. amyloliquefaciens, 5, 14, 15 Agrobacterium rubi,14 B. cereus,5 Alanguibactin, 378 B. gladioli,14 Alcaligenes,11 B. licheniformis, 10, 19 Alternaria,4 B. megaterium, 10, 14, 20 Amanita muscaria, 355 B. mycoides,5 Amorphosporangium,3 B. pasteurii,5 Antagonism, 346, 349, 352 B. polymyxa, 365 Antibiotics, 37, 49 B. pumilus,5,11 production and regulation, 373–374 B. sphaericus,5 Antifungal activity, 374–375 B. subtilis, 5, 13–16, 19, 21 Apoplast, 67 Banana, 132, 140, 142, 145, 149 Aquatic legumes, 81 Biocontrol, 38, 39, 48–50, 54, 171, 174, 175, Arachis hypogaea,5,13 178–182 Araucaria angustifolia, 348 agent, 296, 322–323 Araucariaceae, 345 industry, 324, 329, 330 Armillaria mellea, 354 Biofertilizers, 265–291, 306–307, 361, 362, Arthrobacter, 349, 351, 362, 372 366, 367, 377, 378, 388, 390–398 Arthrobacter citreus, 11, 19 Biogeography, 267, 286–288 Arthrobacterium,20 Biological control, 79–80, 111–124, 240, 250 Aspergillus awamuri,20 Biological nitrogen ﬁxation (BNF), 72, 74, Aspergillus niger, 13, 20 77, 80–82 Autecology, 286–288 Bioreporter, 299 Auxins, 349–351, 371 Biosurfactants, 376 Azoarcus sp., 4 Biotization, 76–79

D.K. Maheshwari (ed.), Bacteria in Agrobiology: Crop Ecosystems, 431 DOI 10.1007/978-3-642-18357-7, # Springer-Verlag Berlin Heidelberg 2011 432 Index

Blue-green algae, 364 Enterobacter, 346, 350, 351 BNF. See Biological nitrogen fixation Enterobacter cloacae, 115, 117 Botrytis cinerea, 4, 14, 355 Enterobacter cloacae,4 Bradyrhizobium, 365 Enterobactin, 378 Brassica campestris,11 Environmental risk, 106 Brazil, 348, 349 Erwinia, 3, 4, 6, 14, 61 14 Burkholderia, 3, 4, 6, 15, 350, 351, 356 Erythrobactin, 378 Escherichia coli,5 C Ethylene (C2H4), 372 C2H4. See Ethylene Eucalyptus, 349 Caenorhabditis elegens, 100–103 Evergreen revolution, 394 Cajanus cajan, 13, 15, 16 Exobasidium vexans,14 Cell wall-degrading enzymes, 69 Exopolysaccharide, 376 Cellulomonas,3,4 Extracellular spaces, Center for Microbial Ecology Image Analysis Exudate, 115, 116, 118, 119, 122, 295–298, System (CMEIAS), 288–290 303, 305, 308, 312, 317, 320, Cercosporidium personatum,13 321, 331 Charcoal rot, 387–398 Chemical fertilizers, 388, 391–397 F Chir-pine, 353 Fatty acid methyl ester (FAME), 363 Chitin/chitosan, 424 Ferrichrome, 368, 378 Chromatium vinosum, 364 Field inoculation, 272–275 Chryseobacterium, 346, 350, 351 Flavobacterium,3,6,20 Chryseobacterium,6 Formulations, 424 Cicer arietinum,15 Francobactin, 378 Clostridium,10 Fruit crops, 190, 197–201, 204–206, 212, Colletotrichum sp., 132 218–221 Colonization, 112, 113, 115–118, 121, 123, Fruit set, 190, 206, 220 124, 296–306 Fruit thinning, 190, 206 Community level physiological profiles Functional genomics, 299, 303, 304 (CLPP), 363 Fungicide, 387 Computer-assisted microscopy, 288, 290 Fusarium oxysporum, 4, 5, 12, 14, 15, 352–354 Conifer, 345–357 Fusarium oxysporum f. sp. Sesame, 388–389 Crop yield, 268, 279 Fusarium solani, 4, 10, 19 Curtobacterium, 346, 350, 351 Fusarium wilt, 387–398 Cyanide production, 374 Cylindrocladium candelabrum, 19, 354 G Cylindrocladium pteridis, 354 Gibberellin (GA), 371–372 Cytokinin, 372 Gigaspora rosea, 356 Glomus fasciculatum,20 D Gluconacetobacter,3,4 Diazotrophs, 72–75, 77, 80–82, 86 Gluconacetobacter diazotrophicus,4 Douglas fir, 349 Glycine max,16 Grapevine, 197–200, 209, 212, 213, 215, E 218, 221 Ectomycorrhizal fungi (EMF), 345, 350, Groundnut, 407–425 355, 356 GUS marker, 69 Elicitors, 375–376 Gymnospermae, 345 Endophyte colonization, 69 Endophyte-plant interactions, 86 H Endophytes, 61–76, 79–82, 86, 352 Helminthosporium,4 Endophytic bacteria, 61–86 Hemlock, 349 Endophytic diazotrophs, 72–75, 81, 82 Herbaspirillum sp., 4 Index 433

Heterobasidion, 353, 355 Nitrogen fixation, 363–365 Hormones, 346, 351 Nitrogenase, 364 Human pathogenicity, 98–106 Nitrosomonas, 362 Non-target effects, 103 I Nonsymbiotic diazotrophs, 364 Immunolocalization, 72 Indian Agricultural Research Institute (IARI), 367 O Indole acetic acid (IAA), 351 Ornibactin, 378 Induced systemic resistance, 38, 51, 346 INM. See Integrated nutrient management P Inorganic fertilizers, 392, 395 Paenibacillus, 350–352 Input efficiency, 220 Paenibacillus polymyxa, 365 Insecticide, production of, 376–377 Parabactin, 378 Integrated nutrient management (INM), 392, Pathogenic fungi, 388, 389 393, 395–398 PCR-RAPD (random amplified polymorphic Intercellular spaces, 66, 67, 69, 73, 74 DNA), 350 Performance, 111–124 K Pesticides, 387, 388, 392–395, 397 Klebsiella, 362 PGP, 397 Klebsiella pneumoniae,5 PGPB. See Plant growth promoting bacteria PGPR. See Plant growth promoting L rhizobacteria Lactarius deliciosus, 350 Phaseolus vulgaris, 5, 16, 17 Lactobacillus, 362 Phosphate solubilization, 346, 365–368 Lactuca sativa, 351 Phosphate solubilizing microorganism (PSM), Lateral root formation, 200 366, 367 Leptosphaera maculans,10 Phospholipid fatty acid (PLFA), 363 Leucaena leucocephala,17 Phosphorobacillus, 346, 350, 351 Lodgepole pine, 351 Phyllobacterium sp., 11 Lupinus albus, 16, 351 Phylloplane, 132–134, 140, 155 Lupinus hispanicus, 351 Phytohormone production, 371–373 Phytohormones, 75, 152, 171, 174, 176–178 M Phytostimulator, 306–313 Macrophomina phaseolina, 352, 388–389 Picea, 348, 349, 352–354 Meloidogyne incognita,14 Pinaceae, 345 Mesorhizobium loti,8 Pinales, 347 Metabolites, 373–376 Pinophyta, 347 Methylobacterium fujisawaense,11 Pinopsida, 347 Microbacterium,3 Pinus, 345, 348–351, 353–356 Micrococcus,6,20 Pinus roxburghii,19 Micromonospora,3 Pisum sativum, 16, 17 Motility, 66 Plant growth promoting bacteria (PGPB), 391 Mycobactin, 378 Plant growth promoting rhizobacteria (PGPR), Mycorrhiza helper bacteria (MHB), 345, 346, 37–54, 166–175, 239–258, 285, 390, 352, 355, 356 391, 393–395, 397, 398, 413, 416–425 N effects on plants, 361 N-use efficiency, 270, 271, 282–284 exopolysaccharide, 376 National Botanical Research Institute’s genes encoding siderophores, 378 Phosphate Growth Medium insecticide, production of, 376–377 (NBRIP), 367 mechanism Nile delta, 268–272, 274, 278, 280, 282, 283, nitrogen fixation, 363–365 286, 287 phosphate solubilization, 365–368 434 Index

phytohormone production, 371–373 Root endophyte, 288 siderophore production, 368–371 Root exudates, 350, 351, 414, 422–423 metabolites antibiotic production and regulation, S 373–374 Salinity stress, 279 antifungal activity, 374–375 Sclerotinia sclerotiorum,8 biosurfactants, 376 Secondary metabolites, 75 cyanide production, 374 Serratia, 99, 101, 103, 104, 350 elicitors, 375–376 Serratia liquefaciens,11 plant responses to, 377 Sesame (Sesamum indicum L.), 387–398 rhizospheric biodiversity, 362–363 Sesamum mulayanum, 390 Plant growth promotion, 171, 177, 180, 181 S. occidentale, 390 PLFA. See Phospholipid fatty acid S. radiatum, 390 Podosphaera fusca,15 Sheath blight, 237–258 Postharvest, 131–155 Siderophores, 346, 353, 354 Proteus mirabilus,5 genes encoding, 378 Pseudobactin, 378 production, 368–371 Pseudomonas, 97, 99–106, 346, 350–355 Sinorhizobium meliloti, 5, 15, 18 P. aeruginosa, 394, 397 Soil fertility, 387, 388, 392, 393 P. ﬂuorescens, 10, 11, 13–16, 19, 20 Soil fumigation, 389 P. polymyxa, 4, 10, 19 Soil health, 392–394 P. putida, 10, 11, 14, 19 Soil microbes, 362 P. vulgaris, 5, 16, 17 Spermosphere, 63, 111–124 Pseudotsuga, 346, 349, 356 Spirillum lipoferum, 364 PSM. See Phosphate solubilizing Spraying, 190, 204, 205, 209, 219, 220 microorganism Spruce, 349 Pyochelin, 378 Staphylococcus, 346, 350, 351, 354 Pyoverdin, 378 Streptomyces, 3, 346, 350, 352–356 Pythium,4,10 Sugarcane, 165–182 Suillus bovinus, 355 Q Sustainable agriculture, 393 Quercus serrata,19 Synthetic chemicals, 392, 395 Quorum quenching, 48–51 T R Thiobacillus, 362 Ralstonia solanacearum,21 Trifolium pretense,16 Rhizobacteria, 295–297, 306, 308, 312, Tsuga, 349 314, 318 Rhizobium, 267–271, 275–279, 282, 283, V 290, 362, 364 Variovorax paradoxus,11 R. leguminosarum br. trifolii,9 Variovorax sp., 11 R. leguminosarum bv. trifolii,16 Vegetable crops, 201–203, 212, 213, 216, Rhizoctonia solani, 238, 243, 248–251, 218, 221 255, 352 Vegetative propagation, 197–201 Rhizoremediator, 306–308 Vibriobactin, 378 Rhizosphere, 111–124 Vigna unguiculata,16 Rhizospheric biodiversity, 362–363 Rhizotonia solani,10 X Rhodobacter capsulatus,10 Xanthomonas,3 Rhodospirillum capsulatus, 364 Rhodospirillum rubrum, 364 Y Rice, 237–258, 265–291 Yersiniabactin, 378 Root colonization, 288 Yield increasing bacteria (YIB), 390