Microbes for Sustainable Agriculture

MICROBESFOR

SUSTAINABLE AGRICULTURE

K.V.B.R. TilakPh.D., FNASc., FNAAS, FAPASc., FBS, FAMI

Senior Scientist, National Acadmy of Sciences, IndiaDepartment of Botany, Osmania University, Hyderabad, A.P.

(Formerly Head, Division of Microbiology,Indian Agricultural Research Institute, New Delhi)

K.K. PalPh.D., Senior Scientist,

National Research Centre for GroundnutJunagadh, Gujarat

Rinku DeyPh.D., Senior Scientist,

National Research Centre for GroundnutJunagadh, Gujarat

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Preface

Intensive agriculture depends on the use of fossil fuel-based inputs like chemicalfertilizers, pesticides, herbicides, etc. While the application of these inputs contributedtowards increased production, there is a growing awareness and concern over theiradverse effect on soil productivity and environmental quality. The high cost of chemicalfertilizers and pesticides, the widening gap between supply and demand, the lowpurchasing power of small and marginal farmers and their adverse effect on environmenthas led the scientists to look for alternate strategies for enhancing productivity. Onesuch approach is the use of soil microorganisms, which can supply nutrients or stimulatethe plant growth through synthesis of growth promoting substances and controllingsoil-borne phyto-pathogens as microbial fertilizers/pesticides to relieve strain on thepart of chemical fertilizers and pesticides.

Legume-Rhizobium association has a special role to play in improving the pulseproduction and soil fertility through biological nitrogen fixation. Selection and use ofefficient strains of Rhizobium together with other agronomic practices would lead toincreased supply of quality protein to the population and maintain soil fertility at a highlevel. Apart from nodulated legumes, forest trees like Casuarina and Alnus are benefitedfrom N2-fixing symbiosis through an actinomycetous microsymbiont designated as Frankia.

Certain nitrogen-fixing bacteria like Azotobacter, Beijerinckia, Azospirillum, Acetobacterdiazotrophicus and photosynthetic nitrogen-fixing blue-green algae are also equallyimportant in promoting plant growth. In waterlogged paddy soils, blue-green algaeare important source of nitrogen. Besides contributing to the nitrogen economy of thesoils, these algae have other effects of ecological significance. Besides the free-livingblue-green algae, an additional biological system which is increasingly attractingattention as a biological nitrogen input in rice cultivation is the tiny water-fern, Azolla,which harbours a blue-green alga Anabaena. This water fern has been used in the FarEast for centuries, indeed long before its capacity to fix nitrogen was realized.

Some of the rhizosphere inhabiting microorganisms are known to synthesize plantgrowth-promoting substances, siderophores and antibiotics and as such are importantin enhancing plant growth, supporting soil and in combating plant diseases.

Phosphorus differs fundamentally from the carbon, nitrogen and sulphur cycles in thatno natural channel exists for the return of the annual net loss. Many of our tropical soilslock up large amounts of phosphorus that are made available after very long periods.Microorganisms play an important role in the mobilization and immobilization of phosphorus.

It is well known that the soil surrounding the plant roots is a zone of intense microbial

vi

activity. The plant roots are known to be intimately associated with specific soil fungi,mycorrhizae (fungus roots). Mycorrhizal associations are of importance to plant nutrition,especially in nutrient-deficient soils. They have also been accredited with other benefits to thehost plants, such as increasing disease resistance, combating stress against soil moisture andother adverse soil factors like high acidity, alkalinity, high temperature and toxic substancesand improving soil aggregation besides serving as bioremediation and biocontrol agents.

Microorganisms are also the most befitting candidates for use as biocontrol agentsand many genera belonging to bacteria, fungi, actinomycetes and viruses are used asbiocontrol agents to fight against important plant diseases.

Most of the tropical soils are poor at the organic matter content due to high rate ofdecomposition and repeated cultivation. Recycling of organic wastes in agriculture bringsin the much needed organic and mineral matter to the soils. The integration of organicmanure and microbial inputs with chemical sources is necessary to maintain the soilbiological health for sustainable crop production. Organic farming largely excludes theuse of synthetic fertilizers and pesticides, plant growth regulators, and livestock feedadditives in an eco-friendly and sustainable manner.

The production and disposal of large quantities of organic and biodegradable waste, withoutproper treatment, result in widespread environmental pollution. Biomethane is generated fromdecaying organic materials with the help of methanogenic bacteria in anaerobic digestors andprovides greater economic, environmental and energy-saving waste treatment.

Recent researches have brought us to an understanding of some of the difficultproblems of microbial sources through the use of powerful tools of microbial genetics,molecular techniques and biotechnological approaches.

In this book, there are sixteen chapters covering various aspects of microorganismsas natural resources for sustainable agriculture. We have provided selected referencesand suggested readings. The composition of media used for culturing the microorganismsand methods are also enlisted in this book. A glossary of terms used is given at the endof the book.

The book is written as per the syllabi of the various universities. This will form atextbook for both UG and PG level of traditional universities, agricultural universities,industrial microbiology, microbial biotechnology, microbial ecology, applied microbiologyand plant pathology. This also forms a very good source for general reading for allcategories including the candidates appearing for various competitive examinations. Thisbook has figures with updated information.

A word of appreciation is due to our teachers, friends and well wishers forcooperation and support to bring out this book. We are also thankful to the NationalResearch Centre for Groundnut (NRCG), Junagadh, Gujarat, and the Indian Council ofAgricultural Research (ICAR), New Delhi, for encouragement and the University GrantsCommission for offering Emeritus Scientist position to the senior author.

Lastly, the senior author (KVBRT) is grateful to the National Academy of Sciences,India, for awarding the Senior Scientist-Platinum Jubilee Fellowship which is a sourceof encouragement to bring out this publication.

K.V. B. R. TilakK. K. Pal

R. Dey

Preface

Contents

Preface v

Authors ix

1. Biological Nitrogen Fixation 11.1 Introduction 1

2. Legume (Rhizobium) Inoculants 62.1 Introduction 62.2 Isolation and Growth Habit of Rhizobium 72.3 Systematics of Rhizobium 92.4 Characteristics of Rhizobium 112.5 Mechanism of Rhizobial Infection in Legumes 122.6 Rhizobium Inoculation 152.7 Preservation of Bacterial Cultures 162.8 Revival of Preserved Cultures 182.9 Production of Rhizobium Inoculants 18

2.10 Quality Control of Rhizobial Inoculants 222.11 Method of Application of Rhizobium Inoculants 252.12 Factors Affecting Legume – Rhizobium Symbiosis 272.13 Response of Legumes to Rhizobium Inoculation 302.14 Benefits of Rhizobial Inoculation to Subsequent Crops 312.15 Genetic Manipulation of Rhizobia 322.16 Interaction of Rhizobium with Beneficial Soil Microorganisms 332.17 Effect of Pesticides on Legume-Rhizobium Symbiosis 35

3. Free-Living Nitrogen Fixers 383.1 Introduction 383.2 Azotobacter Inoculant 383.3 Diversity of A. Chroococcum in Different Soils 393.4 Isolation of Azotobacter 393.5 Characteristics of Azotobacter 393.6 Classification of the Genus Azotobacter 41

viii Contents

3.7 Factors Influencing Azotobacter Growth 413.8 Production and Methods of Application of Azotobacter Inoculant 413.9 Production of Growth Promoters 42

3.10 Azotobacter-Crop Responses 433.11 Associative Effects of Azotobacter with Rhizosphere Microorganisms 443.12 Azotobacter–Host Specificity 45

4. Azospirillum Inoculant 464.1 Introduction 464.2 Isolation 474.3 Characterization of Azospirillum Strains 474.4 Establishment of Azospirillum in Plants 484.5 Carriers for Azospirillum 494.6 Enumeration of Azospirillum in Carrier Materials 494.7 Inoculation Studies 494.8 Potential Contribution of Azospirillum 514.9 Interaction with Other Agro-Microbes 51

4.10 Survival of Azospirillum Under Adverse Soil Conditions 52

5. Endophytic Bacteria 545.1 Introduction 54

6. Plant Growth Promoting Rhizobacteria (PGPR) 576.1 Introduction 576.2 Mechanisms of Growth Promotion 586.3 PGPR on Plant Growth and Yield 64

7. Blue-Green (Cyanobacterial) Inoculants 677.1 Introduction 677.2 Production of Algae for Field Application 687.3 Current Approaches of BGA Multiplication 687.4 Response of Algal Inoculation on Rice Crop 70

8. Azolla Biofertilizer 728.1 Introduction 728.2 Large-Scale Production of Azolla 738.3 Application in Fields 73

9. Phosphate Solubilizing Microorganisms 769.1 Introduction 769.2 PSM as Biofertilizers 78

10. Actinorrhizal Associations 8010.1 Symbiosis of Frankia with Higher Plants 80

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10.2 The Microsymbiont Frankia 8010.3 The Macrosymbiont: Host Species and Symbiotic Specificity of Frankia 8210.4 Inoculation Groups 8210.5 Structure and Function of Actinorrhiza 8310.6 Ecological and Agricultural Aspects 84

11. Mycorrhizae 8511.1 Introduction 8511.2 Taxonomy and Ecology of AM Fungi 8711.3 Role of AM Fungi 89

12. Organic Matter Recycling and Composting 9212.1 Introduction 9212.2 Composting 9212.3 Vermicomposting 93

13. Organic Farming 9513.1 Introduction 9513.2 History 9613.3 Methods 9613.4 Standards 9713.5 Productivity 9713.6 Issues 9813.7 Principles of Organic Agriculture Systems 9913.8 Components of Organic Farming 10013.9 Pest Management in Organic Farming 102

14. Biological Control 10414.1 Introduction 10414.2 Biological Control of Insect Pests 10414.3 Biological Control of Soil-Borne Fungal Pathogens 11214.4 Future Prospects 129

15. Biomethanation 13015.1 Introduction 13015.2 Biomass Gasification and the Production of Biomethane 13015.3 Biomethane from Anaerobic Digesters 13215.4 Use of Different Inputs for Biogas Production 137

16. Molecular Techniques for Ecological Studies 13816.1 Introduction 13816.2 Molecular Detection Methods for Microorganisms in Soil 13816.3 Future Perspectives 150

Contents

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Appendix 1: Media 153

Appendix 2: Some Universal Methods for Detection and Quantification ofPlant Growth Promoting Attributes of Rhizobacteria 161

Selected References and Suggested Reading 167

Glossary 187

Index 197

Contents

Authors

PROF. K. V. B. R. TILAK

Prof. K.V.B.R. Tilak, Retired Head, Division of Microbiology, IndianAgricultural Research Institute, New Delhi (Presently Senior Scientist,the National Acadmy of Sciences, India, Department of Botany,Osmania University, Hyderabad, AP). Prof. Tilak has a verydistinguished background in the field of soil microbiology, biologicalnitrogen fixation, biofertilizers and plant-microbe interactions. He wasa coordinator of Indo-US Science & Technology Initiative (SeniorScientific Panel) programme during 1984-91 and visited variousuniversities in USA in a bilateral programme on Biological Nitrogen Fixation andBiofertilizers including Mycorrhizae. Dr. Tilak held various positions at the IndianAgricultural Research Institute, New Delhi as Professor of Microbiology; NationalCoordinator, Indian Council of Agricultural Research (ICAR) of Biological NitrogenFixation Programme; Project Director, National Facility for Blue-Green Algal Collectionsand Head, Division of Microbiology for over a period of 25 years besides serving anumber of organizations in the country on programmes dealing with Biofertilizers andSoil (Agricultural) Microbiology. Presently, he is working as Emeritus Scientist in theDepartment of Botany, Osmania University, Hyderabad, AP. For his contributionstowards research in Soil Microbiology and Biofertilizers, he was awarded the prestigiousProf. S. R. Vyas Memorial Award by the Association of Microbiologists of India. He wasthe recipient of the Best Teacher Award of the Indian Agricultural Research Instituteand Late Prof. Uma Kant Sinha Memorial Award by the Indian Botanical Society. He isa Fellow of National Academy of Sciences, India; Fellow of National Academy ofAgricultural Sciences, India; Fellow of Andhra Pradesh Academy of Sciences, India;Fellow of Indian Botanical Society and Fellow of Association of Microbiologists of India.He has guided 30 students for doctoral degrees in Microbiology besides teaching atIARI, New Delhi and Osmania University, Hyderabad. He has published over 200research papers in peered journals.

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DR. K.K. PAL

Dr. K. K. Pal, Senior Scientist, Microbiology, National Research Centrefor Groundnut (ICAR), Junagadh, Gujarat. Dr. Pal has a long experienceof working in the field of soil/agricultural microbiology. He is workingin the areas of biological nitrogen fixation, bio-inoculants, variousaspects of plant-microbe interactions, formulation and delivery systemsof inoculants, microbial diversity, biocontrol of phytopathogens, etc.He has identified several Rhizobium and PGPR cultures suitable forgroundnut cultivation. Besides working in the institute projects, hehas worked in projects funded by CSIR, ICAR, NAIP, NATP and DBT. At present, he isalso working in AMAAS (Application of Microorganisms in Agriculture and AlliedSectors), network project on studying microbial diversity, funded by ICAR. Under thisproject he is studying the microbial diversity in the Kutch eco-region of Gujarat. He hasinitiated work under an NAIP funded project on “Diversity analysis of Bacillus and otherpredominant genera in extreme environments and its utilization in agriculture“. He wasawarded the Merit Medal for outstanding research work during his Ph.D. in 1997 by theIndian Science Congress Association (ISCA). He was also awarded with IARI MeritGold Medal for outstanding academic performance during M.Sc. programme. Dr. Palworked on genes of Bacillus subtilis involved in the biological control of plant pathogenicfungi in the Department of Plant Pathology, Ohio State University, Wooster, Ohio, USAunder the DBT Overseas Associateship Award. He has guided the dissertation works offive PG students and two doctoral students. He has published many research papers,review articles, etc. in national and international journals. Dr. Pal is also life member ofAMI, ISOR, ISCA besides Guest faculty of Department of Life Science, Kannur University,Kerala and recognized guide for PG students in the discipline of AgriculturalMicrobiology, JAU, Junagadh.

DR. RINKU DEY

Dr. Rinku Dey, Senior Scientist, Microbiology, National ResearchCentre for Groundnut (ICAR), Junagadh, Gujarat. Dr. Dey has beenworking as agricultural/soil microbiologist at the National ResearchCentre for Groundnut, Junagadh since 1997. Her research interestsinclude plant-microbe interactions, plant growth-promotingrhizobacteria, development of microbial consortia and formulations,biological nitrogen fixation, utilization of agricultural residues,fermentation for enzyme production, etc. She has been associated withCSIR, DBT, ICAR funded projects. She was the principal investigator of a DST fundedproject under Young Scientist Scheme. She has developed Rhizobium and PGPR culturesfor use as biofertilizers in groundnut cultivation. She has guided dissertation works ofmany PG students. She has published many research articles, reviews, etc. in nationaland international journals.

Authors

1.1 INTRODUCTION

The microbial inoculants are the preparations containing primarily active strains of micro-organisms in sufficient numbers. They are used either to fix atmospheric nitrogen or tosolubilize plant nutrients like phosphates, or to stimulate plant growth throughproduction of growth-promoting substances and/or biocontrol agents to suppress plantpathogens. Preparations containing these can also be considered as fertilizers under abroad term biofertilizers or biopesticides. Of the different biofertilizers, Rhizobiuminoculant, specific for different leguminous crops, is the most important one. In additionto rhizobia, blue-green algae (Cyanobacteria) also fix atmospheric nitrogen if they areinoculated into the soil and established in paddy fields. Other biofertilizers producedare Azotobacter, Azospirillum, Gluconacetobacter diazotrophicus (Syn: Acetobacterdiazotrophicus) and phosphate solubilizers. The wider usage of these biofertilizers willhelp in utilizing the atmospheric nitrogen available in such large amounts (about 80,000tonnes over an hectare of land) and thus supplement the meagre quantities of organicand inorganic fertilizers available for crops.

Nitrogen is the most important and at the same time most limiting nutrient forincreasing crop productivity. Input efficiency of N fertilizer is low (Prasad et al., 1990)and in turn, contributes substantially to environmental pollution. The indiscriminateuse of nitrogenous fertilizers amounts to depletion of non-renewable fossil fuels used infertilizer production. Sustainable agriculture depends largely on renewable resourcesand on-farm nitrogen contributions are achieved largely through biological nitrogenfixation (BNF), which helps in maintaining and/or improving soil fertility by usingatmospheric nitrogen.

Annually biological nitrogen fixation (BNF) is estimated to be around 175 milliontonnes of which close to 79% is accounted for by terrestrial fixation (Burns and Hardy,1975). This illustrates the importance of BNF in the context of global N cycle. The BNFoffers an economically attractive and ecologically sound means of reducing external Ninputs and improving the quality and quantity of internal resources. Legumes havebeen an important component of agriculture since ancient times. It is widely believedthat legumes improve soil fertility because of their N2-fixing ability.

Biological Nitrogen Fixation

CHAPTER

1

2 Microbes for Sustainable Agriculture

Crop legumes alone occupy 11% land globally under cultivation of permanent crops(Table 1.1). However, legumes are also present worldwide in some of the 3541 millionhectares estimated to be temporary and permanent pastures and may provide viableinputs of N in natural plant communities, forest ecosystems, tree plantations andintensive cropping systems (Peoples et al., 1995). Inputs of N by free-living diazotrophsin soil and plant residues, and non-symbiotic N2-fixing associations with cereals and C4grasses have been extensively reviewed by several workers (Boddey et al., 1995; Okon etal., 1998; Tilak and Saxena, 2000; Lata et al., 2002).

Table 1.1. Global distribution of cultivated land for different crops

Cultivated crops* Per cent land area covered

CerealsWheat 16Rice 10Maize 9All other cereals 13Total 48LegumesPulses 5Oilseeds 6Total 11Other cropsOther oilseeds 6Beverages / Tobacco 7Roots and tubers 4Sugar crops 2Vegetables and fruits 2Fibres/Rubber/Oil palm 1Total 22Temporary pastures / Fodder crops 14Others 6

Source: Peoples et al. (1995); * Distribution of 1442 million ha estimated to be arable land and underpermanent crop in 1991-1992 (FAO, 1993)

Legumes have a special role to play in areas where the diet is poor in protein becausethey are an excellent source of high-quality protein. Rhizobia are widely distributed insoils of the tropics. Even in newly developed lands, not previously supporting economiclegumes, these bacteria are found. However, many indigenous bacteria are wholly orlargely incapable of bringing about appreciable fixation on roots of the appropriate host.Selection and use of efficient strains of Rhizobium together with other agronomic practiceswould lead to increased supply of quality protein to the population and maintain soilfertility of a high level (Tilak, 2007).

Nitrogen fixed by legume-Rhizobium symbiosis and its contribution to subsequentcrops is well documented. Nitrogen fixation capacity of legumes is harnessed byinoculating the crops with appropriate competitive and efficient Rhizobium strain (s).

Among the asymbiotic nitrogen-fixing bacteria, Azotobacter, Beijerinckia, Azospirillum,Gluconacetobacter diazotrophicus and photosynthetic nitrogen-fixing blue-green algae are

Biological Nitrogen Fixation 3

important. In waterlogged paddy soils, blue-green algae are important source of nitrogen(Venkataraman, 1977). Besides contributing to the nitrogen economy of the soils, thesealgae have other effects of ecological significance. They have been shown to prevent thegrowth of weeds and they add to the soil organic matter content aiding particleaggregation, and help increased availability of phosphorus. The presence of blue-greenalgae in the immediate vicinity of rice seeds can decrease sulphide and iron injury also.The extent to which blue-green algae contribute to the nitrogen requirements of ricecrop is determined by a number of factors, the most important of which are the standingcrop, rate of nitrogen fixation per unit area, turnover of the nitrogen fixed, the extent towhich any nitrogen released becomes available to the rice plant and the soil properties.Nitrogen-fixing blue-green algae have a potential of approximately 25-30 kg N/ha/crop. Evidences have been adduced for the availability of the fixed nitrogen to the ricecrop both under lowland and deep water situations.

The role of photosynthetic bacteria on tropical soils was shown to be highlysignificant, particularly in association with other asymbiotic nitrogen fixers likeAzotobacter. Some of these organisms are known to synthesize growth-promotingsubstances, siderophores and antibiotics and as such are important in enhancing plantgrowth and in combating plant diseases, though the amount of nitrogen fixed may notbe large. Besides the free living blue-green algae, an additional biological system whichis increasingly attracting attention as a biological nitrogen input in rice cultivation isthe tiny water-fern, Azolla, which harbours a blue-green alga Anabaena. This water fernhas been used in the Far East for centuries, indeed long before its capacity to fix nitrogenwas realized. Azolla is subject to the same limitations as free-living blue-green algae,but it has the advantage in being readily distinguishable by the peasant farmer, can fixnitrogen in the presence of combined nitrogen, and in general, more tolerant of low pHthan are free-living blue-green algae. Azolla can also be utilized as a green manure source(Singh, 1987; Kannaiyan and Subramani, 1987).

Field experiments conducted with Azotobacter showed that in the majority ofexperiments sizable increase in crop yields were obtained (Shende, 1987). Although thevalue of inoculation might be considered due to the result of an enhancement of nitrogenfixation with subsequent release of fixed nitrogen in forms available to plants, thebeneficial effects appear to be more due to production of growth-promoting substances,biocontrol agents and solubilization of insoluble phosphates (Saxena and Tilak, 1998).

Occurrence of nitrogen-fixing Azospirillum in association with a number of crop plantshas been well documented by Dobereiner and Day (1976). The use of this inoculant fordifferent crops has been extensively reviewed by different workers (Saxena and Tilak,1998). Azospirillum biofertilizer has been recommended to the farmers in India for cropslike sorghum and millets. By the use of Azospirillum as a seed inoculant, savings of 20-30 kg N/ha equivalents could be achieved in crops like barley, sorghum and millets(Subba Rao, 1986; Tilak and Subba Rao, 1987; Bashan et al., 2004).

Other free-living nitrogen-fixing bacteria, like Gluconacetobacter diazotrophicus,Azoarcus, and several other plant growth-promoting rhizobacteria like Pseudomonas,Bacillus, Enterobacter, Burkholderia, Bacillus, etc. are important. Reports indicate that G.diazotrophicus fixes considerable amount of nitrogen in sugar-rich crops like sugar cane,

Microbes for Sustainable Agriculture

Publisher : IK International ISBN : 9789380026886 Author : K.K. Pal & Rinku Dey

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