Soil Microbes: the Invisible Managers of Soil Fertility 1

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Soil Microbes: The Invisible Managers of Soil Fertility 1

Arumugam Sathya , Rajendran Vijayabharathi , and Subramaniam Gopalakrishnan

Abstract Soil health is represented by its continuous capacity to function as a vital living system. Since soil health is the major driving factor for sustainable agriculture, it has to be preserved. Microorganisms are an essential and integral part of living soil inﬂ uencing various biogeochemical cycles on major nutrients such as carbon, nitrogen, sulphur, phosphorous and other minerals and play superior role in maintaining soil health than other biological component of soil. They also have the capacity to suppress soil borne pathogens and indirectly help in agricultural productivity. Besides contribution of speciﬁ c microbes to soil health by participating on nutrient cycles, certain other microbes directly/indirectly promote plant growth through the production of phytohormones, enzymes and by suppressing phytopathogens and insects. The vast functional and genetic diversity of microbial groups including bacteria, fungi and actinomycetes supports in all the above ways for soil health. This book chapter gives an outline of such microbes and their contribution in promoting soil health and its role as soil health indicators.

Keywords Soil health • Microorganisms • N ﬁ xation • Nutrient cycling • Climate change

1.1 Introduction

Soil, a ﬁ nite and non-renewable resource, supports numerous terrestrial life forms through its A. Sathya • R. Vijayabharathi S. Gopalakrishnan (*) critical functions. Soil health is deﬁ ned as ‘the International Crops Research Institute for the continued capacity of soil to function as a vital Semi-Arid Tropics (ICRISAT) , living system, within ecosystem and land-use Patancheru 502 324 , Telangana , India boundaries, to sustain biological productivity, e-mail: [email protected]

© Springer India 2016 1 D.P. Singh et al. (eds.), Microbial Inoculants in Sustainable Agricultural Productivity, DOI 10.1007/978-81-322-2644-4_1 2 A. Sathya et al. promote the quality of air and water environ- (Hoorman and Islam 2010 ). Typical soil samples ments and maintain plant, animal and human have about thousands of individual taxa (also health’ (Doran and Safl ey 1997 ). In the context known as operational taxonomic units, OTUs) of of sustainable agriculture, soil health is meant for bacteria, archaea and fungi. It is understood from crop productivity and protection via the functions some estimates that there can be >106 individual such as N2 fi xation and phosphorus (P) solubili- species-level OTUs in a single soil sample (Fierer zation, homeostasis of biogeochemical cycles, et al. 2007 ). During the analysis on genome size maintenance of soil structure, detoxifi cation of of microbial community among soil samples by pollutants and suppression of plant pathogens. In re-association of community DNA, it is known the absence/ineffi ciency of these functions, the that, the microbial community genome size soil is regarded as an inanimate entity with min- equals the size of 6,000─10,000 Escherichia coli erals and chemicals. In addition, soil regenera- genomes in unperturbed organic soils, and tion through chemical and biological process/ 350─1,500 genomes in arable or heavy metal weathering of underlying rock requires geologi- polluted soils. Still, the rare and unrecovered cal time (Huber et al. 2001; Buscot and Varma microorganisms may not be included in the anal- 2005 ). So, maintenance of soil health is crucial ysis. In contrast, the genomic complexity recov- for sustainable productivity. ered by culturing methods was less than 40 The following 14 nutrients are vital for a genomes. This complexity in microbial commu- proper plant growth and development – macro- nity genome size denotes the diversity in terms of nutrients, which are further divided into (1) struc- genetic information present in the soil and also tural nutrients: C, H, O; (2) primary nutrients: N, the overall functional variability (Torsvik et al. P, K and (3) secondary nutrients: S, Ca, Mg; and 1998 ; Øvreås 2000 ). micronutrients: Fe, B, Cu, Cl, Mn, Mo, Zn, Ni. Among the microbial groups, fungus have Besides the structural nutrients (which are higher tolerance and surviving capacity against obtained from air and water), the remaining 11 fl uctuating soil disturbances, untilled or no-till nutrients are obtained through soil and absorb soils than bacteria and actinomycetes; though the only in some specifi c/available forms as follows: latter groups also have the tolerance (Hoorman + − N – NH 4 (ammonium) and NO 3 (nitrate); P – and Islam 2010 ; Meliani et al. 2012 ). Besides the − −2 + H2 PO4 and HPO4 (orthophosphate); K – K ; smaller voluminous nature, soil microbes are the −2 +2 +2 S – SO4 (sulphate); Ca – Ca ; Mg – Mg ; Fe – key drivers of biogeochemical cycles on major Fe+2 (ferrous) and Fe+3 (ferric); Zn – Zn+2 , Mn – nutrients such as C, N, S, P and other mineral +2 −2 +2 Mn ; Mo – MoO4 (molybdate); Cu – Cu , cycles (Bloem et al. 1997 ). They also suppress − − Cl – Cl ; B – H3 BO3 (boric acid) and H2 BO3 the soil pathogens via various antibiosis com- (borate). Though many of the soil fl ora and fauna pounds and helps in plant disease protection are responsible for bringing these nutrients, (Haas and Défago 2005 ). This book chapter deals microorganisms are the drivers behind various with role of microbes in improving soil fertility biogeochemical cycles and making the organic and also the available techniques for indicating and inorganic nutrients in their available form to soil microbial activity. the plants (Lucas and Davis 1961; Mengel and Kirkby 2001 ). Microbes are the largest population that exists 1.2 Carbon Cycle in soil with a high diversity index. However, the microbial groups vary in their number vs. bio- Carbon (C) in the atmosphere is transferred to mass. The number (number/g soil) and biomass soil by photosynthetic plants and photo/chemo (g/m 2) of various microbial groups are, bacteria: autotropic microorganisms for the synthesis of 10 8 ─109 vs. 40─500; actinomycetes: 10 7 ─108 vs. organic materials. Hence, the largest carbon pool 40─500; fungi: 105 ─106 vs. 100─1,500; algae: on the earth’s surface (2,157─2,293 Pg) is/ 10 4 ─ 105 vs. 1 ─50; protozoa: 103 ─104 vs. varies becomes soil. The reverse process, i.e., 1 Soil Microbes: The Invisible Managers of Soil Fertility 3

decomposition of organic material built in plants teria (Baldrian et al. 2010). Role of these lytic and microbes was carried out by organic C utiliz- enzymes in maintaining soil health is previously ing heterotropic microorganisms as a substrate reviewed by Das and Varma (2011 ) and hence a for their metabolism and energy source. The brief note on some essential enzymes is described remaining C is liberated as metabolites or CO2 to here. the atmosphere (Prentice et al. 2001 ). The decomposition product termed as soil organic carbon Amylase: Starch hydrolyzing enzyme breaks the (SOC) is the largest pool within the terrestrial C complex polysaccharides and releases low cycle with an annual turnover of about 60 Gt molecular weight simple sugars which acts as (Schlesinger 1997 ). During the SOC formation, an energy source for microbes (Rahmansyah the organic materials were either mineralized to and Sudiana 2010 ) and it is conﬁ rmed by the

CO2 or humiﬁ ed. Since the SOC affects plant positive correlation between as enzyme activ- growth by serving as energy source and by inﬂ u- ity and SOM (Kujur et al. 2012 ). encing nutrient availability through mineraliza- Cellulase: Cellulose in plant debris is degraded tion, it is one of the most important constituents by a group of enzymes called cellulases into of the soil. glucose, cellobiose and high molecular weight It is understood that microbes transfer the C oligosaccharides. Soil fungus is the major primarily for their survival. Under oxic condi- contributors of this enzyme activity. Report of tions, i.e., in surface of soil and oxic layers of Arinze and Yubedee (2000 ) supports this by wetland systems, aerobic methane-oxidizing bac- documenting negative correlation between teria play the role (Chistoserdova et al. 2005 ; increasing fungicide concentration in agricul- Gupta et al. 2013 ), whereas under waterlogged tural soils and cellulase activity. Previous anoxic soils, CO 2 is reduced by hydrogenotropic studies by Vincent and Sisler (1968 ) and Atlas archaea and methoanogenic bacteria (Lu and et al. (1978 ) also documented the same effects. Conrad 2005 ; Trumbore 2006 ). Typically micro- Chitinase: Chitin is a major component of fungal bial C accounts for a minimum of 100─1,000 μg cell wall, exoskeleton of insects and many g−1 in arable soils and a maximum of 500─10,000 arthropods. As already quoted, the higher fun- μg g−1 in forest soils with the intermittent values gal biomass present in soils will be degraded in other ecosystems such as grasslands and semi- by the chitinases after the cell death with the arid regions (Kandeler et al. 2005 ). Besides the release of simple organic molecules. Besides considerable variations, microbial biomass C contributing for nutrient cycling, it serves generally accounts for about 0.9─6 % of total majorly for the control of soil borne fungal organic C with an indirect relationship for phytopathogens such as Sclerotium rolfsii and increasing soil depth. Rhizoctonia solani . This indirectly helps in Formation of soil organic matter (SOM), a increasing plant growth and yield (El-Tarabily major fraction containing SOC is aided by the et al. 2000 ; Sindhu and Dadarwal 2001 ). decomposition process through various lytic Oxidase: In contrast to the hydrolytic enzymes, enzymes including, amylase, glucosidase, prote- oxidases were produced for a variety of func- ases, cellulase, chitinase and phenol oxidase. tions including ontogeny, defence and the These enzymes convert the complex macromol- acquisition of C and N by microorganisms ecules into low molecular weight compound for (Sinsabaugh 2010 ). Representative of these the ready assimilation of microbial components enzymes include fungal laccases and prokary- or for their transformation into CO2 for energy otic laccase-like enzymes (Baldrian 2006 ; (Burns and Dick 2002 ). Though the enzymes Hoegger et al. 2006 ). were released from plants/animals/microorgan- Dehydrogenase: It is related during microbial isms, the latter are major contributors (Tabatabai respiration, where it oxidizes soil organic mat- 1994 ). Among the microbial groups, fungi are ter by transferring protons and electrons from reported to have higher enzyme activity than bac- substrates to acceptors and the activity 4 A. Sathya et al.

depends on soil type and soil air–water condi- The fi rst step in N cycle, assimilation, i.e., N tions (Wolińska and Stępniewska 2012 ; fi xation (also known as biological nitrogen fi xa- Kumar et al. 2013 ). tion, BNF) is aided by a group of bacteria called diazotrophs including cyanobacteria, green sul- Sequential changes in climatic conditions and phur bacteria, Azotobacteraceae, rhizobia and related ecosystem factors in the current situation Frankia at various ecosystems in which the for- affect all of the nutrient cycles. Hence, the mer three occurs by/through non-symbiotic pro- research trend has been directed towards (1) cess and the latter two through symbiotic process. effect on climate change including seasonal vari- BNF occurs through a cascade of reactions ations, elevated CO2 and long-term climate involving complex enzymes systems and change disturbances (Durán et al. 2014 ; Haugwitz accounts for about 65 % of N currently used in et al. 2014 ); (2) effect of fertilizers (Strauss et al. agriculture (Thamdrup 2012 ; Peoples et al. 2014 ), soil amendments (Anderson et al. 2011 ) 1995 ). Major quantity of N fi xed under the con- on long-term (Tyree et al. 2006 ) and short-term trol of legume–rhizobia is harvested as grains. scales (Tyree et al. 2009 ) and (3) effect of SOM The left out N in the soil, roots and shoot residues (Schmidt et al. 2011) etc. It is understood that, supports the succeeding crops for N supply. though the importance of soil microorganisms Hence legume–rhizobial symbiosis substantially for global C cycling is well known; only few reduces the N requirement from external sources research attempts have been made to evaluate the (Bhattacharyya and Jha 2012 ). Crops like wheat, chemical and microbiological views of C cycling rice, sugarcane and woody species also have the (Kandeler et al. 2005 ). capacity to fi x atmospheric N using free living or associative diazotrophs. However, the contribution of legume–rhizobia symbiosis (13–360 kg N 1.3 Nitrogen Cycle ha−1 ) is far greater than the non-symbiotic systems (10–160 kg N ha−1 ) (Bohlool et al. 1992 ).

Nitrogen (N), an essential element for the synthe- Review of Herridge et al. (2008 ) on global N 2 sis of amino acids and nucleotides is required by fi xation estimated from FAO databases and other all forms of life in large quantities. It is also experimental reports also indicates the higher involved in several respiratory energy metabo- contribution of legume–rhizobia than other sys- lisms in which N compounds may serve as either tems in N fi xation (Table 1.1 ). However, N fi xa- oxidant or reductant. Atmosphere is the largest tion effi ciency of legumes depends on the host reservoir of N (78 %) in the form of triple bonded genotype, rhizobial effi ciency, soil conditions,

N2 gas, though it is not freely available to most and climatic factors (Belnap 2003 ). Difference in living organisms. It is accessible only by N2 fi x- N fi xation effi ciency of various legumes is shown ing bacteria and archaea which pave the way for in Table 1.2 . other organisms to use the fi xed N for its incorpo- BNF is an energy demanding process through ration into their biomass. This fi xed N constitutes which atmospheric N is converted to plant usable less than 0.1 % of the N2 pool and is able to limit organic N and plays an important role in the N the primary production in both terrestrial and cycle. This can be understood by the complexity marine ecosystems. Within the organisms, N of the enzyme nitrogenase, a major enzyme exist in most reduced forms and during the cell involved in the nitrogen fi xation, which has two lysis it is nitrifi ed to nitrate which in turn denitri- components – dinitrogenase reductase, the iron

fi ed to N 2 gas. So, a balanced N cycle requires the protein and dinitrogenase (metal cofactor). The dual action of assimilatory (N fi xation and incor- iron protein provides the electrons with a high poration into biomass) and dissimilatory (recy- reducing power to dinitrogenase which in turn cling of fi xed nitrogen to N2 ) transformations reduces N2 to NH3 . Based on the availability of (Vitousek and Howarth 1991 ; Canfi eld et al. metal cofactor, three types of N fi xing systems 2010 ). viz. Mo-nitrogenase, V-nitrogenase and 1 Soil Microbes: The Invisible Managers of Soil Fertility 5

Table 1.1 Comparison of symbiotic and non-symbiotic nodB- chitooligosaccharide deacetylase, N fi xation in agricultural systems nodC- N- acetylglucosaminyltransferase, nodD- Crop N transcriptional regulator of common nod genes, Agricultural Area fi xed (Tg/ nodPQ , nodX , nofEF , nodIJ- Nod factors trans- Agent system (Mha) year) port, NOE- synthesis of Nod factors substituents, Legume–rhizobia Crop (pulse 186 21 and oilseed) nol genes- several functions in synthesis of Nod legumes factors substituents and secretion); (2) nitrogen Pasture and 110 12–25 fi xation (including nifA, nifHDK- nitrogenase, fodder fi xLJ, nifBEN- biosynthesis of the Fe-Mo cofac- legumes tor, fi xK-transcriptional regulator, fi xABCX- Azolla- Rice 150 5 electron transport chain to nitrogenase, cyanobacteria fi xGHIS- copper uptake and metabolism, fdxN- Endophytic, Sugar cane 20 0.5 fi x NOPQ- associative & Crop lands 800 <4 ferredoxin and - cytochrome oxidase) free-living other than and (3) other essential elements (including hup- bacteria used for hydrogen uptake, exo- exopolyssacharide produc- legumes and tion, gln- glutamine synthase, nfe- nodulation rice effi ciency and competitiveness, dct- dicarboxylate Extensive, 1390 <14 tropical transport, ndv- β -1,2 glucan synthesis, pls- savannas lipopolysaccharide production) (Laranjo et al. primarily 2014 ). used for It is a well-known fact that rhizobia belong to grazing the families Rhizobiaceae (excluding the Frankia Source: Herridge et al. (2008 ) sp.), Bradirhizobiaceae and Phyllobacteriaceae. Rhizobia have a unique association with root nod- Table 1.2 Comparison data for N fi xation effi ciency of ules of leguminous plants and induce plant growth various legumes in many ways. They also have capacity to induce Crop N plant growth of non-leguminous plants (Mehboob Shoot N Crop N fi xed Legume (Tg)a (Tg)b %Ndfa (Tg)c et al. 2012). The number of species reported in Common 1.03 1.45 40 0.58 Rhizobiaceae family increased considerably from bean 8 in 1980 to 53 in 2006. This drastic increase was Cowpea 0.27 0.37 63 0.23 mainly due to dispersion of leguminous plants to Chickpea 0.48 0.96 63 0.60 new geographical locations. The other possible Pea 0.65 0.90 63 0.57 reasons could be: (1) only 57 % of 650 genera of Lentil 0.24 0.33 63 0.21 leguminous plants have been studied for nodula- Faba bean 0.27 0.38 75 0.29 tion and nitrogen fi xation, and (2) recent advance- Groundnut 2.16 3.03 68 2.06 ments in the taxonomic research with the aid of Soybean 16.11 24.17 68 16.44 specifi c molecular tools (Willems 2006). Besides Source: Herridge et al. ( 2008). %Ndfa – Percentage of its role in effi cient N fi xation, they have multiple plant N derived from N 2 fi xation plant growth promoting traits such as mineral aUsing %N shoots of 3.0 % for soybean, 2.3 % for groundnut, 2.2 % for fababean and 2.0 % for the remainder enhancing capacity, phytohormone production and b Multiplying shoot N by 2.0 (chickpea), 1.5 (soybean) and alleviating biotic and abiotic stress (Gopalakrishnan 1.4 (remainder) to account for below-ground N et al. 2014a). All these help in developing formula- c Crop N × %Ndfa tion of rhizobial inoculants to achieve substantial increases in legume nodulation, grain and biomass Fe-nitrogenase were documented. Complexity of yield, nitrogen fi xation and post-crop soil nitrate nitrogen fi xation can be further understood by levels for succeeding crops (GRDC 2013). It is participation of multiple gene clusters as follows: already reported that, inoculation of soybean with (1) nodulation (including nodA- acyltransferase, rhizobial inoculants showed substantial increases 6 A. Sathya et al. in nodulation, grain and biomass yield and N fi xa- rhizobial species in the context of common core tion (Thuita et al. 2012 ). symbiotic genes (Masson-Boivinemail et al. Besides the rhizobia, the associative and free- 2009 ) and invasive mechanisms behind the sym- living nitrogen fi xing bacteria were also formu- biotic process (Kiers et al. 2003 ) are going on. lated and commercialized as biofertilizers. The However, the understanding of ecological con- genus Azospirillum, an associative N fi xing bac- trols on N fi xation is sparse (Vitousek et al. 2002 ) teria comprises nearly 15 species: A. lipoferum, and it is essential for developing a commercial A. brasilense, A. amazonense, A. halopraeferans, microbial inoculants. Current research trend is A. irakense, A. largimobile, A. dobereinerae, A. looking over the effect of various environmental oryzae, A. melinis, A. canadense, A. zeae, A. factors that limit N fi xation, such as soil moisture rugosum, A. palatum, A. picis and A. thiophilum . defi ciency, osmotic stress, extremes of tempera- Reis et al. (2011 ) also reported for its multiple ture, soil salinity, soil acidity, alkalinity, nutrient plant growth promoting traits. The next impor- defi ciency, overdoses of fertilizers, pesticides and tant genus is Azotobacter , a free-living nitrogen contaminants (Vance 2001 ; Galloway et al. 2004 ; fi xer which comprises of seven species: A. Mohammadi et al. 2012 ; Peoples et al. 2012 ). chroococcum, A. vinelandii, A. beijerinckii, A. paspali, A. armeniacus, A. nigricans and A. salinestri (Jiménez et al. 2011 ). Besides the N 1.4 Sulphur Cycle fi xing capacity, this genus has the history of more than 35 years in promoting plant growth through The sulphur (S) present in soil (>95 % of total S) multiple phytohormone production, enzymes, is in the bound form with organic molecules, and enhanced membrane activity, proliferation of the it is not directly available to the plants, i.e., inor- root system, enhanced water and mineral uptake, ganic S which constitutes about only 5 %. This mobilization of minerals, mitigation of environ- minimal part of available S in agricultural soils mental stress factors, and direct and indirect bio- leads to S defi ciency symptoms in plants (Schnug control against numerous phytopathogens and Haneklaus 1998 ). Besides the contribution of (Bashan and de-Bashan 2010 ). plant and animal-derived organic S, in situ syn- The N fi xed in the form of ammonium during thesis is also observed, which is mainly mediated assimilation process, is further dissimilated by by microbial process via immobilization of inor- two-step microbial process, i.e., nitrifi cation (the ganic S to organic S, interconversion of various aerobic oxidation of ammonium to nitrite and organic S forms, mineralization of inorganic sul- then to nitrate) and denitrifi cation (the respiratory phur in order to support plant growth. Rhizospheric anaerobic reduction of nitrate via nitrite, nitric microbes are the major players in allowing plants oxide, and nitrous oxide to N2 , coupled with the to access soil organosulphur (Kertesz 1999 ). oxidation of organic matter, hydrogen, or reduced Besides the mineralization and immobilization, iron or sulphur species) (Simon 2002 ). oxidation and reduction reactions also infl uence S Nitrifi cation is further carried out by two sets of cycling. Oxidation of elemental S and inorganic S microbial groups: (1) ammonia oxidizers (nitrosi- compounds to sulphate is carried out by chemoau- fyers) which convert ammonia to nitrite by the totrophic (Thiobacillus sp., T. ferrooxidans and T. activity of ammonia monooxygenase, e.g. thiooxidans ) and photosynthetic (Green and pur- Nitrosomonas, Nitrosospira and Nitrosococcus ; ple bacteria, Chlorobium and Chromatium.) bac- and (2) nitrite oxidizers (the true nitrying bacte- teria. Besides this, heterotrophic bacteria such as ria) which convert nitrite to nitrate by the activity Bacillus, Pseudomonas , and Arthrobacter , fungi of nitrite oxidoreductase, e.g. Nitrobacter and such as Aspergillus and Penicillium and some Nitrococcus (Vaccari et al. 2006 ). actinomycetes are also reported to oxidize sul- Though the physiology of nitrogen fi xation phur compounds. The process of sulphate/sulph- process is reasonably well characterized, still uric acid formation has the following advantages: research studies on the phylogenetic diversity of (i) it is the anion of strong mineral acid (H2 SO 4 ) 1 Soil Microbes: The Invisible Managers of Soil Fertility 7 which can render alkali soils fi t for cultivation by acids, the signifi cant contributors for solubiliza- correcting soil pH; and (ii) solubilize inorganic tion of metal compounds though the excretion of salts containing plant nutrients and thereby other metabolites, siderophore also contribute to increase the level of soluble P, K, Ca, Mg, etc. for the solubilization process (Sayer et al. 1995 ). Low plant nutrition. Dissimilatory sulphate reduction molecular organic acid – 2-ketogluconic acid – also occurs in order to balance the contents, where with a P-solubilizing ability has been identifi ed in sulphate-reducing bacteria such as Desulfovibrio, R. leguminosarum (Halder et al. 1990 ) and R. Desulfatomaculum and Desulfomonas play the meliloti (Halder and Chakrabarty 1993 ). key roles through the enzyme activity of desulfu- Mineralization of organic P takes place by several rases/bisulphate reductase. Among them, enzymes of microbial origin, such as acid phos- Desulfovibrio desulfuricans can reduce sulphates phatases (Abd-Alla 1994), phosphohydrolases at rapid rate in waterlogged/fl ooded soils, while (Gügi et al. 1991 ), phytase (Glick 2012 ), phos- Desulfatomaculum – a thermophilic obligate phonoacetate hydrolase (McGrath et al. 1998 ), anaerobes – can reduce sulphates in dry land soils D-α-glycerophosphatase (Skrary and Cameron (Tang et al. 2009 ). Though many studies have 1998) and C-P lyase (Ohtake et al. 1996 ). Other been conducted to evaluate the role of microbes in mineral elements also turn into their available S cycle, now the research focus has been moved form by any of the above mechanism. in to deal with enzymes, organisms, pathways, comparative approaches, symbiosis, and environments factors related to the S nutrition (Klotz 1.6 Suppression of Soil Borne et al. 2011 ). and Other Phytopathogens

Soil health is not only based on abundance and 1.5 Phosphorous Cycle diversity of total soil microbes but also on high population of beneﬁ cial organisms. Incidence Phosphorous (P) is a key component of nucleic and severity of root diseases is an indirect assess- acids, energy molecule ATP and membrane com- ment of soil health (Abawi and Widmer 2000 ). ponent phospholipids. P accounts for about Certain rhizospheric microorganisms are known 0.2─0.8 % of the plant dry weight, but only 0.1 % to have antagonistic activities against soil borne of this P is available for plants from soil (Zhou and other phytopathogens. This may be achieved et al. 1992). The P content of agricultural soil by lytic enzymes cellulase, chitinase, protease solutions are typically in the range of 0.01─3.0 mg and β-1, 3-glucanase which either induces direct P L−1 representing a small portion of plant require- suppression of plant pathogens or indirectly by ments. The remaining must be obtained through enhancing the host plant resistance. Some oligo- intervention of biotic and abiotic processes where saccharides derived from fungal cell wall break- the phosphate solubilizing activity of the microbes down contribute to indirect mechanism (Pliego has a role to play (Sharma et al. 2013 ). Soil et al. 2011; Kilic-Ekici and Yuen 2003 ). Role of microbes help in P release to the plants that absorb the genus Pseudomonas in disease suppression is − only the soluble P like monobasic (H2 PO 4 ) and reviewed by Haas and Défago (2005 ) in the con- 2− dibasic (H2 PO 4 ) forms (Bhattacharyya and Jha text of antifungal antibiotic production, induction 2012). Many bacteria (Pseudomonas and of systemic resistance in the host plant or inter- Bacillus ) (Rodriguez and Fraga 1999 ), fungi ference on fungal pathogenicity factors. ( Aspergillus, Penicillium and Trichoderma ) Mycorrhizal associations are one among them (Mittal et al. 2008) and actinomycetes which are found in all ecological situations (Streptomyces and Nocardia) (Tallapragada and including normal cropping systems and in natural Seshachala 2012 ) are noticed for P solubilizing ecosystems. Among them arbuscular mycorrhi- capacity and enhancement of plant growth. This is zas (AM) are the most common (Harley and aided by the synthesis of protons and organic Smith 1983 ; Gianinazzi and Schüepp 1994 ), but 8 A. Sathya et al. the excellency depends on its pre-establishment 1.7 Indicators of Soil Health and extensive development on plant roots before the pathogen attack. Still, AM’s broad-spectrum It is understood from the literature that soil health inhibition was noticed against pathogens such as is the result of continuous conservation and deg- Aphanomyces, Chalara, Fusarium, radation processes in an ecosystem with the Gaeumannomyces, Phytophthora, Pythium, unique balance of chemical, physical and bio- Rhizoctonia, Sclerotium and Verticillium (Azcón- logical (including microbial) components. So, Aguilar and Barea 1996 ). Another soil fungus evaluation of soil health requires indicators of all Trichoderma, a well-known avirulent plant sym- these components. Since microbes quickly biont, characterized as biocontrol agent against respond to changes in the soil ecosystem and vice broad range of phytopathogens works via compe- versa, they are the excellent indicators of soil tition, mycoparasitism, induced resistance, anti- health. Changes in microbial populations or biotic and enzyme production. Beside this, it acts activity can precede detectable changes in soil as plant growth promoting agents (Howell 2003 ; physical and chemical properties, thereby pro- Harman et al. 2004 ). Others such as Bacillus, viding an early sign of either soil improvement or Paenibacillus and Streptomyces were also found an early warning of soil degradation (NERI to have inhibitory activity against soil borne and 2002). The techniques were improved on the other phytopathogens (Cao et al. 2011 ; Köberl basis of the continuous identiﬁ cation and docu- et al. 2013 ). A list of available commercial for- mentation of microbial processes. Some of the mulations of these microbes has been summa- analytical and molecular techniques available are rized by Junaid et al. (2013 ). summarized in Table 1.3 .

Table 1.3 Biological, physical and chemical indicators used for determining soil health Indicator Analytical techniques Molecular techniques Microbial biomass Direct microscopic counts Fluorescence microscopy Chloroform fumigation Computerized image analysis SIR Soil DNA estimation

CO 2 production FISH Microbial quotient Fungal estimation PLFA Microbial activity Bacterial DNA synthesis RNA measurements using Bacterial protein synthesis RT-PCR

CO 2 production FISH Carbon cycling Soil respiration SIP

Metabolic quotient (qCO2 ) FISH Decomposition of organic matter Soil enzyme activity Nitrogen cycling N-mineralization SIP Nitrifi cation FISH Denitrifi cation N-fi xation Biodiversity and microbial Direct counts – resilience Selective isolation plating Carbon and nitrogen utilization patterns Extracellular enzyme patterns PLFA (continued) 1 Soil Microbes: The Invisible Managers of Soil Fertility 9

Table 1.3 (continued) Indicator Analytical techniques Molecular techniques Genetic and functional – DGGE biodiversity TGGE T-RFLP mRNA diversity using RT-PCR BIOLOGTM assay Microbial resilience – Equitability (J) index Bioavailability of contaminants Plasmid-containing bacteria RNA measurements Antibiotic-resistant bacteria Geochemical indicators Physical and chemical properties Bulk density – Soil physical observations and estimations pH EC CEC Aggregate stability and soil slaking Water holding capacity Water inﬁ ltration rate Macro/micronutrient analysis SOM lipid analysis – PLFA(GC-MS) SOM humic substances analysis – Non-destructive techniques: 15 N-NMR, 13C NMR UV/Vis and IR spectroscopy Destructive techniques: Pyrolysis-GC-MS Chemolysis-GC-MS Source: Arias et al. (2005 )

1.8 Work at ICRISAT (Vessey 2003 ). Many reviews were periodically available on these PGP microbes (Loon 2007 ; Microbes play positive roles in plant growth pro- Bloemberg and Lugtenberg 2001 ; Saharan and motion in addition to its direct or indirect partici- Nehra 2011 ; Bhattacharyya and Jha 2012 ). So pation in the nutrient cycles. These are called this book chapter just gives a glimpse on those plant growth promoting (PGP) microbes which and the related studies conducted by our research reside in rhizosphere/rhizoplane and promotes group. plant growth: (1) by using their own metabolism ICRISAT has identifi ed over 1,500 microbes (solubilizing phosphates, producing hormones or including bacteria and actinomycetes, isolated fi xing nitrogen) or directly affecting the plant from various composts and rhizospheric soil, in metabolism (increasing the uptake of water and which at least, one out of six has documented minerals), enhancing root development, increas- either single or multiple agriculturally favourable ing the enzymatic activity of the plant or helping traits. Our research group has a collection of 59 other benefi cial microorganisms to enhance their PGP bacteria and actinomycetes isolated from action on the plants; and (2) by suppressing plant various herbal vermi-composts and organically pathogens (Pérez-Montano et al. 2014 ). cultivated fi elds with documented PGP traits in Representative genera are Bacillus, Pseudomonas, vitro and also at fi eld conditions (Gopalakrishnan Trichoderma, Rhizobium, Bradyrhizobium, et al. 2014b ). PGP bacteria such as Pseudomonas Sinorhizobium, Mesorhizobium and Streptomyces plecoglossicida, P. monteilii, Brevibacterium 10 A. Sathya et al. antiquum, B. altitudinis, Enterobacter ludwigii Table 1.4 Extracellular enzyme profi le identifi ed for and Acinetobacter tandoii isolated from rhizo- PGP bacteria and actinomycetes spheric soil of system of rice intensifi cation (SRI) Isolates Cellulase Chitinase Lipase Protease fi elds has documented in vitro PGP traits and also PGP bacteria under fi eld conditions on rice. Enhanced growth SRI-156 + + + + performance was observed via increased tiller SRI-158 + + + + numbers, panicle numbers, fi lled grain numbers SRI-178 + + + + and weight, stover yield, grain yield, total dry SRI-211 + + + + matter, root length, root volume and root dry SRI-229 + + + + weight (Gopalakrishnan et al. 2012 ). Similar type SRI-305 + + + + of enhanced growth performance on rice by acti- SRI-360 + + + + nomycetes such as Streptomyces sp., S. cavisca- SBI-23 + - - + bies, S. globisporus subsp. caucasicus, S. SBI-27 + - - + griseorubens is also recorded. In addition, up- PGP actinomycetes KAI-26 + + + + regulation of PGP genes such as indole acetic KAI-27 + + + + acid and siderophore producing genes were doc- KAI-32 + + + + umented (Gopalakrishnan et al. 2014c ). A PGP KAI-90 + + + + bacterium Pseudomonas geniculata IC-76 KAI-180 + + + + showed its capacity on chickpea under fi eld con- SAI-13 + + - + ditions by enhanced plant growth performance SAI-25 + + + + and also agronomic performance via increased SAI-29 + + - + nodule number, nodule weight, pod number, pod Source: Gopalakrishnan et al. (2014b ) weight, seed number and seed weight (Gopalakrishnan et al. 2014d ). Besides increasing plant growth, they signifi - B. antiquum, B. altitudinis, E. ludwigii, A. tandoii cantly enhanced rhizospheric total nitrogen and P. monteilii, and actinomycetes Streptomyces (8─82 %), available phosphorous (13─44 %) and sp., S. tsusimaensis, S. caviscabies, S. setonii and S. organic carbon (17─39 %). Production of lytic africanus were found to have inhibitory activity enzymes such as cellulase, chitinase, lipase and against soil borne pathogens such as Fusarium oxy- protease by these microbes (Table 1.4 ) is an addi- sporum f. sp. ciceri, and Macrophomina phaseo- tional evidence for the enhanced soil organic car- lina under greenhouse conditions. Antagonistic bon and nitrogen contents (Gopalakrishnan et al. activity of these PGP actinomycetes on Fusarium 2014b , 2014c ). Analysis of soil health microbial wilt-sick fi elds has also been demonstrated indicators recorded enhanced microbial biomass (Gopalakrishnan et al. 2011a , b). These strains are carbon (23─48 %), microbial biomass nitrogen already reported for lytic enzymes in the context of (7─321 %) and dehydrogenase activity biocontrol such as chitinase and β-1,3 glucanse (14─278 %) on experimental plots over the un- (Gopalakrishnan et al. 2014b ). inoculated control during our fi eld studies on crops such as rice (Gopalakrishnan et al. 2012 ; 2013 ; 2014c ), chickpea (Gopalakrishnan et al. 2014d ) 1.9 Future Outlook and sorghum (unpublished results). Figures 1.1 , 1.2 , and 1.3 illustrate the combined results of our Microbes have multiple functions and features in published reports on rhizospheric PGP microbes infl uencing soil health and also in promoting plant on increasing soil health during the fi eld trials. growth and controlling diseases. Hence mainte- Apart from the PGP traits, they also have the nance of benefi cial microbial load will help in capacity to act as biocontrol agents by suppressing replacing inorganic fertilizer, pesticides and artifi - soil pathogens, one of the keystone logic for healthy cial plant growth regulators which have numerous soil. Our PGP bacteria such as P. plecoglossicida, side effects to sustainable agriculture. Beside this, 1 Soil Microbes: The Invisible Managers of Soil Fertility 11

Control IC-76

Control KAI-180

Control KAI-27 KAI-26 PGP isolates

Control SRI-360 SRI-305 SRI-229 SRI-211 SRI-178 SRI-158 SRI-156 0 500 1000 1500 2000 2500 Total N (ppm)

Fig. 1.1 Effect of PGP bacteria and actinomycetes on (Note: Control indicates the treatment groups without any soil total N under ﬁ eld conditions of chickpea and rice PGP bacterial inoculation, Gopalakrishnan et al. 2012 , cultivation 2013 , 2014c , d )

1.8 140 1.6 120 1.4 100 1.2

1 80

0.8 60

0.6

Organic carbon (%) 40 0.4

20 Available Phosphorous (ppm) 0.2

0 0

IC-76 Control Control Control Control SRI-156SRI-158SRI-178SRI-211SRI-229SRI-305SRI-360 KAI-26KAI-27 KAI-180 PGP Isolates

Fig. 1.2 Effect of PGP bacteria and actinomycetes on right axis. Control indicates the treatment groups without soil organic carbon and available phosphorous under ﬁ eld any PGP bacterial inoculation (Gopalakrishnan et al. conditions of chickpea and rice cultivation 2012 , 2013 , 2014c , d ) Solid bars ( ) are the % organic carbon on the left axis and solid triangles ( ) are the available phosphorous (ppm) on 12 A. Sathya et al.

200 4000

180 3500 160 3000 140 2500 120

100 2000

80 MBC 1500 60 1000 40

MBN and Dehydrogenase activity 500 20

0 0

Control SRI-156SRI-158SRI-178SRI-211SRI-229SRI-305SRI-360Control KAI-26KAI-27 KAI-180Control PGP Isolates

Fig. 1.3 Effect of PGP bacteria and actinomycetes on soil on left axis and solid diamond ( ) is the microbial biomass health indicators during ﬁ eld trials of rice cultivation Solid carbon (μg g−1 soil) on right axis. Control indicates the bars ( ; ) are the microbial biomass nitrogen (μg g−1 soil) treatment groups without any PGP bacterial inoculation and dehydrogenase activity (μg TPF g−1 soil 24 h−) (Gopalakrishnan et al. 2012 , 2013 , 2014c ) understanding the responses of terrestrial ecosys- microbial community change: Implications for biogeochemical cycling of carbon, nitrogen and phospho- tems to global climatic changes and modern agri- rus. Pedobiologia 54(5–6):309–320 cultural practices remains a major challenge, Arias ME, González-Pérez JA, González-Vila FJ, Ball AS since soil has a mixed interaction with physical, (2005) Soil health – a new challenge for microbiolo- chemical and biological component along with gists and chemists. Int Microbiol 8:13–21 Arinze AE, Yubedee AG (2000) Effect of fungicides on the inﬂ uence of water, air/atmosphere, soil Fusarium grain rot and enzyme production in maize amendments etc. So research in each of this con- (Zea mays L.). Glob J Appl Sci 6:629–634 text individually and also in combination at vari- Atlas RM, Pramer D, Bartha R (1978) Assessment of pes- ous ecosystem levels is necessary. ticide effects on non-target soil microorganisms. Soil Biol Biochem 10:231–239 Azcón-Aguilar C, Barea JM (1996) Arbuscular mycorrhizas and biological control of soil-borne plant References pathogens – an overview of the mechanisms involved. Mycorrhiza 6:457–464 Baldrian P (2006) Fungal laccases e occurrence and prop- Abawi GS, Widmer TL (2000) Impact of soil health man- erties. FEMS Microbol Rev 30:215–242 agement practices on soil borne pathogens, nematodes Baldrian P, Merhautová V, Cajthaml T, Petránková M, and root diseases of vegetable crops. Appl Soil Ecol Šnajdr J (2010) Small-scale distribution of extracellu- 15(1):37–47 lar enzymes, fungal, and bacterial biomass in Quercus Abd-Alla MH (1994) Solubilization of rock phosphates petraea forest topsoil. Biol Fertil Soils 46:717–726 by Rhizobium and Bradyrhizobium. Folia Microbiol Bashan Y, de-Bashan LE, (2010) How the plant growth- 39:53–56 promoting bacterium Azospirillum promotes plant Anderson CR, Condron LM, Clough TJ, Fiers M, Stewart growth – a critical assessment. Adv Agron 108:77–136 A, Hill RA, Sherlock RR (2011) Biochar induced soil 1 Soil Microbes: The Invisible Managers of Soil Fertility 13

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