Diversity and biological control of Sclerotium rolfsii, causal agent of stem rot of groundnut Cuong N. Le Thesis committee Thesis supervisor Prof. dr. ir. F.P.M. Govers Personal Chair at the Laboratory of Phytopathology Wageningen University Thesis co-supervisor Dr. J.M. Raaijmakers Associate Professor, Laboratory of Phytopathology Wageningen University Other members Dr. P.A.H.M. Bakker, Utrecht University Prof. dr. T.W. Kuyper, Wageningen University Dr. M.H. Nicolaisen, University of Copenhagen, Denmark Dr. Ir. A.J. Termorshuizen, BLGG AgroXpertus, Wageningen This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences Diversity and biological control of Sclerotium rolfsii, causal agent of stem rot of groundnut Cuong N. Le Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 16 December 2011 at 1:30 p.m. in the Aula Cuong N. Le Diversity and biological control of Sclerotium rolfsii, causal agent of stem rot of groundnut PhD Thesis, Wageningen University, Wageningen, The Netherlands (2011) With summaries in English and Dutch ISBN 978-94-6173-107-4 Contents Chapter 1. Introduction and outline of the thesis 7 Chapter 2. Genetic and phenotypic diversity of Sclerotium rolfsii Sacc. in 29 groundnut fields in central Vietnam Chapter 3. Involvement of phenazine antibiotics and lipopeptide surfactants 49 in suppression of stem rot disease of groundnut by Pseudomonas species Chapter 4. Diversity and antifungal activity of groundnut-associated bacteria 71 Chapter 5. Biological control of groundnut diseases and plant growth 93 promotion by beneficial Pseudomonas and Bacillus species Chapter 6. Diversity of endophytic bacteria of groundnut nodules and their 113 effects on plant growth and groundnut diseases Chapter 7. General discussion 131 Summary 143 Samenvatting 145 Acknowledgements 147 Curriculum vitae 150 Education certificate of Graduate School EPS 151 Chapter 1. Introduction and outline of the thesis Chapter 1 Introduction and outline of the thesis Parts of this chapter are integrated in the review: "Stem rot of groundnut caused by the soil- borne pathogen Sclerotium rolfsii” (to be submitted) Chapter 1. Introduction and outline of the thesis 8 Chapter 1. Introduction and outline of the thesis Introduction and outline of the thesis Introduction The central theme in this thesis is biological control of stem rot disease of groundnut in Vietnam. Groundnut (Arachis hypogaea L.) is an annual leguminous plant that is cultivated in many countries around the world. In Vietnam, it is the most important oil seed crop with a total area of 249,200 ha and a production of 0.53 million ton in 2009 (FAO 2011). Groundnut cultivation is hampered by a wide range of pests and diseases. One of the most important soil- borne fungal diseases of groundnut is stem rot caused by Sclerotium rolfsii. Control of stem rot disease mostly relies on cultural practices and fungicide treatment. However, cultural practices are not always effective due to the wide host range of the pathogen, and fungicides are often too expensive for local groundnut farmers in Vietnam. Biological control has been proposed as a sustainable, affordable and supplementary measure to control S. rolfsii, but has not been explored and exploited in detail. The overall aim of the research described in this thesis was to study the feasibility of biological control of stem rot disease on groundnut. The first part of this introduction describes several features of groundnut, in particular its biology, distribution, its symbiosis with nitrogen- fixing bacteria, and the agronomic and economic importance of this crop. Subsequently, the major yield-limiting factors in groundnut cultivation will be presented, followed by a detailed description of stem rot disease on groundnut, the life cycle and characteristics of the pathogen S. rolfsii. Then the current state-of-the-art of biological control of stem rot is summarized. The last section of this chapter describes the outline of this thesis. Groundnut Groundnut (Arachis hypogaea L.), also known as peanut, earthnut, monkey nut, or goobers, is an annual leguminous plant believed to originate in South America in a region encompassing Bolivia, Paraguay, Peru and parts of Western Brazil and Northern Argentina (Hoammons 1994). Groundnut production is important in several countries which are highly populated and where groundnut plays an important role as a food crop (Florkowski 1994). Today, groundnut is widely distributed and is Figure 1. Groundnut growing regions in the world. Solid line: center of origin; dotted line: area of cultivation; cultivated in more than 80 countries in black: areas of intensive cultivation. This figure is tropical and sub-tropical regions of the reproduced from Weiss (2000). world (Fig. 1). Groundnut requires a 9 Chapter 1. Introduction and outline of the thesis warm, sunny climate with a well-distributed rainfall of at least 500 mm and temperatures ranging from 25 to 30ºC. It thrives best in well-drained sandy loam soils with a pH ranging from 5.5 to 7.0 (Weiss 2000). Groundnut belongs to the family of Leguminoceae and the genus Arachis. Arachis is derived from the Greek ‘a-rachis’, meaning without spine and refers to the absence of erect branches. The species name hypogaea is derived from ‘hupo-ge’ which in Greek means below earth. Cultivated groundnut is an annual, herbaceous plant growing to a height of 30-60 cm with an angular hairy stem with spreading branches (Fig. 2). Leaves occur alternately, one at each node; they are pinnate with two pairs of ovate leaflets (Fig. 2). Flowers are self-pollinating. After pollination, the perianth withers and at the base of the ovary, a meristematic region grows into a stalk-like peg which pushes the ovary into the soil (Fig. 2). Groundnut has a taproot system Leaflet that is often covered with root nodules resulting from a symbiosis with nitrogen-fixing bacteria (collectively called rhizobia). On groundnut, root Stem Flower nodules develop at sites where lateral roots emerge (Uheda et al. 2001). The rhizobia penetrate the Peg Young pod root tissue, induce cell division and settle inside Soil level root cells where they convert atmospheric nitrogen Mature pod Tap root (N2) into ammonia, which in turn is used by the plant (Broughton et al. 2000). Among the rhizobia Figure 2. Schematic drawing of the groundnut identified on groundnut, Bradyrhizobium species plant. This figure was reproduced from Rao are the most prominent ones (van Rossum et al. and Murty (1994). 1995; Urtz and Elkan 1996; Zhang et al. 1999; Saleena et al. 2001). Other bacterial genera associated with groundnut nodules include Rhizobium, Pseudomonas, Enterobacter, Klebsiella, Sphingomonas, Bacillus, and Paenibacillus (Zakhia et al. 2006). Most of these bacteria were unable to induce nodule formation on groundnut plants, but some did promote plant growth (Ibanez et al. 2009). All parts of the groundnut plant can be used for a variety of purposes. The seeds are rich in protein and vitamins (A, B and some B2 types) and can be eaten raw, roasted or sweetened. They contain 44-52% edible oil which is also used in making soap, cosmetics and lubricants. The residual oil cake contains considerable amounts of nitrogen, phosphorous and potassium and is used as a fertilizer. The cake is also an important protein source in cattle and poultry feeds and can also be used for manufacturing artificial fiber. The haulms of groundnut can be fed to livestock and the peanut shell is used for manufacturing coarse boards and cork substitutes. In terms of economic importance, groundnut ranks thirteenth on the list of world food crops and tops the list of oil seed crops both in terms of acreage and production. In 2009, it was grown on 23.5 million hectares with an estimated total production of 35.5 million tonnes (FAO 2011). Currently, China is the leading groundnut producer with a share of about 37.5% of 10 Chapter 1. Introduction and outline of the thesis the overall world production, followed by India, the United States of America (USA), Argentina and Vietnam (Fabra et al. 2010). India has the largest groundnut cultivation area, but has a low average yield of approximately 1.0 ton ha-1. The USA have the highest average yield of 3.8 ton ha-1 (FAO 2011). Yield limiting factors in groundnut cultivation Several abiotic and biotic factors affect the growth and development of groundnut leading to qualitative and quantitative yield losses. Temperature, relative humidity, and soil characteristics are the major abiotic factors that can directly or indirectly influence productivity. In addition to the direct impact on growth, these abiotic factors can also have an impact on the proliferation and damage caused by pests and diseases. The major insect pests that attack groundnut are termites, white grubs, thrips, aphids, leaf miners, and caterpillars. The pathogens parasitizing on groundnut include viruses, mycoplasmas, bacteria, oomycetes, fungi, nematodes, and parasitic plants (Middleton et al. 1994; Wightman and Ranga Rao 1994). The diseases that are most damaging and cause the largest economic losses are stem rot, damping-off, black collar rot, early and late leaf spot, rust and bacterial wilt (Middleton et al. 1994; Podile and Kishore 2002). Stem rot caused by the fungus S. rolfsii will be discussed in detail in the next section. The fungus Rhizoctonia solani causes seed decay, pre- and post-emergence damping-off of seedlings, hypocotyl and root necrosis, root rot of young seedlings, peg and pod rot, and foliar blight of mature plants (Porter et al. 1984). The fungus Aspergillus niger causes black collar rot, an important disease found in all major groundnut-growing areas of the world.