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Nov 21, 2015 - Co-PI : Hari P. Deka Baruah, Archana Yadav. CSIR-North East Institute of Science .... Mahadev Swamy. University of Agricultural Sciences, ...
Application of Microorganisms in Agriculture and Allied Sectors

(AMAAS)

Annual Report (2015-2016)

Application of Microorganisms in Agriculture and Allied Sectors (AMAAS)

Annual Report (2015-2016)

Contents

Theme : Microbial Diversity 1. Microbial DiversityAnalysis of Extreme Ecological Niches

PI : Co-PI :

Sushil K. Sharma1 Pawan K. Sharma1, Karthikeyan Nanjappan1, Hillol Chakdar1, Sakthivel Krishnan2 1 ICAR- National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 2 ICAR-Central IslandAgricultural Research Institute, Port Blair

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2. Exploration of the Endophytic Microbial Diversity in Horticultural Crops through Metagenomics and Cultivation

PI : Pious Thomas ICAR- Indian Institute of Horticultural Research, Bengaluru

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3. Mapping of Deep-sea and Hypersaline Microbiota and their Characterization using Meta-omics Approaches for Bioprospecting of Novel Gene

PI : Pradeep MA2 1 Co-PI : KK Vijayan , Krupesh Sharma 1 ICAR-Central Marine Fisheries Research Institute, Kochi 2 ICAR- Central Institute of BrackishAquaculture, Chennai

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4. Analysis of Bacterial Community and their Functional Diversity from Rice Rhizosphere Employing Genomics and MetagenomicsApproaches

PI : Ashok Kumar1 Co-PI : M. B.Tyagi2, R. P. Sinha2 1 School of Biotechnology, Banaras Hindu University, Varanasi 2 Department of Botany, Banaras Hindu University, Varanasi

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5. Microbial Diversity of Bio-Films in Dairy Niche

PI : Co-PI :

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6. Functional Characterization of Salt tolerant Bacteria using Multi-Omic Approaches and their Exploitation for Alleviation of Salt Stress in Crop Plants

PI : Co-PI :

Kamlesh Kumar Meena P.S. Minhas, J. Rane, D, K.K. Krishanani, V. Govindasamy ICAR-National Institute ofAbiotic Stress Management, Baramati

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7. Culturable and Unculturable Microbial Diversity of Insects and their Role in Insecticide Resistance and other Fitness Attributes

PI : Mahesh S. Yandigeri Co-PI : G. Sivakumar, R. Rangeshwaran, M. Mohan ICAR-National Bureau ofAgricultural Insect Resources, Bengaluru

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8. Molecular Characterization and Exploration of Rumen Microbial Diversity in Black Bengal Goat at North Eastern State of Tripura

PI : Asis Bhattacharya Co-PI : Bikas Chandra Debnath,Aparajita Das,Ankan De Department of Microbiology, College of Veterinary Sciences & Animal Husbandry, Tripura

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9. Microbial Metagenomic Analysis of Hot Springs Situated a top the Himalayan Ranges at Manikaran, Himachal Pradesh, India

PI : Rup Lal Co-PI : Mallikarjun N. Shakarad Department of Zoology, University of Delhi, Delhi

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10. Diversity of Arbuscular Mycorrhizal Fungi and Dark Septate Endophytes in Woody Species and Shifting Cultivation Crop of Manipur, North East India

PI : R. R. Pandey Department of Life Sciences, Manipur University, Imphal

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11. Exploring Myxobacteria from the North Western Himalayas for Novel Enzymes and Secondary Metabolites

PI : Meenu Katoch Co-PI : BhahwalAli Shah CSIR-Indian Institute of Integrative Medicine, Jammu

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12. Exploration and Mass Culture of Freshwater Cyanobacteria for Development of Nutraceutical Feed for Freshwater Fish Hatchlings

PI : O.N. Tiwari Co-PI : R.N. Mandal DBT- Institute of Bioresources and Sustainable Development (IBSD), Imphal

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13. Reference and De Novo Draft Genome Sequencing of Potential AIMs and their FunctionalAnnotation

PI : Alok K. Srivastava 2 1 1 Co-PI : Prem L. Kashyap , Hillol Chakdar , K. Pandiyan 1 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 2 ICAR-Indian Institute of Wheat and Barley Research, Regional Station, Flowerdale, Shimla

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14. Biomolecules and Industrially Important Enzymes from Extremophilic Bacteria

PI : Anil Kumar Saxena Co-PI : Rajeev Kaushik ICAR- IndianAgricultural Research Institute, New Delhi

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R.K. Malik Chand Ram, S.K. Tomar, Surajit Mandal, Diwas Pradhan ICAR-National Dairy Research Institute, Karnal

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15. Draft Genome Sequencing of P-solubilizing Pseudomonas striata PS27 and Functional Validation of MPS Genes

PI : Anil Kumar Saxena Co-PI : Rajeev Kaushik, Geeta Singh ICAR- IndianAgricultural Research Institute, New Delhi

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16. Genomics and Proteomics of N2-fixing Cyanobacteria: with Special Reference to Discovering Abiotic Stress Tolerant Novel Protein Genes 17. Exploiting the Diversity of Extreme Halophiles by Functional and Comparative Genomics for Isolating Novel Genes and Alleles for Affording Salinity Tolerance to Crop Plants

PI : LC Rai Department of Botany, Banaras Hindu University, Varanasi

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PI : K. K. Pal Co-PI : Rinku Dey ICAR-Directorate of Groundnut Research, Junagadh

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18. Isolation and Selection of Naturally Occurring Cyanobacterial Strain and their Potential use in Biodegradation of Agricultural Pesticides (Organophosphate)

PI : A.K. Mishra1 Co-PI : Alok K. Srivastava2 1 Centre of Advance Study in Botany, Banaras Hindu University, Varanasi 2 ICAR National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan

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19. Bioprospecting for Microbial Products that Effects Cold Alleviation and Growth

PI : Pankaj Kumar Mishra ICAR-Vivekananda Parvatiya KrishiAnusandhan Sansthan,Almora

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20. Pathogenomics- Genome Sequencing of Puccinia striiformis

PI : RashmiAggarwal Co-PI : Vaibhav K. Singh ICAR, IndianAgriculture Research Institute, New Delhi

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21. Archaea and Actinobacteria in Vertisols of Central India – Assessment of Diversity, Bigeochemical Processes and Bioinoculant Potential

PI : D.L.N. Rao Co-PI : SR Mohanty, K Bharati, TK Radha ICAR-Indian Institute of Soil Science, Bhopal

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22. Mining the Metagenome for Bacterial Diversity in Extreme Areas of North-East India

PI : Ratul Saikia Co-PI : Hari P. Deka Baruah,Archana Yadav CSIR-North East Institute of Science & Technology, Jorhat

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23. Microbial Diversity of Glacier Samples and Peat Profile of Himalayan Region

PI : S.M. Singh1 Co-PI : Alok K. Srivastava2 1 ESSO-National Centre forAntarctic and Ocean Research, Goa 2 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan

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Theme : Taxonomy, Identification and Diagnostics of AIMs 24. Development of Microarray based Gene Chip for Major Fungal Plant Pathogens under the Background of DNA Barcodes using Multilocus Gene Phylogeny

PI : Co-PI :

Hillol Chakdar1 Prem Lal Kashyap2, Alok K. Srivastava1, Sanjay Goswami1 1 ICAR- National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 2 ICAR-Indian Institute of Wheat and Barley Research, Regional Station, Flowerdale, Shimla

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25. Exploration of Plant Growth Promoting Rhizobacteria, Antagonistic and Plant Pathogenic Microbial Resources from High Altitude Agro-climate/Cropping System of Jammu & Kashmir State for SustainableAgriculture

PI : Vishal Gupta Co-PI : V.K. Razdan, Prachi Sharma, Reyazul Roul Mir Sher-e-Kashmir University of Agricultuiral Sciences and Technology of Jammu, Jammu

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26. Development of Diagnostic Kit for Detection of Karnal Bunt and Loose smut of Wheat

PI : M.S. Saharan Co-PI : Indu Sharma, Sudheer Kumar, Pradeep Sharma ICAR - Indian Institute of Wheat and Barley Research, Karnal

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27. Development of Microbial Formulations for Biological Control of Grape Diseases

PI : Indu S. Sawant Co-PI : S. D. Sawant ICAR-National Research Centre for Grapes, Pune

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Theme : Plant Microbe Interactions, Nutrient Management, Disease Control and Agrowaste Management 28. Investigating the Potential of Root-Colonizing Microbial Population for Stress Tolerance in Chickpea through Proteomics

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PI : B.K. Sarma1 2 Co-PI : H.B. Singh , D.P. Singh 1 Banaras Hindu University, Varanasi 2 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan

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Mahaveer P. Sharma 2 3 4 Minakshi Grover , Sushil K. Sharma , DK Agarwal , 1 G. Satpute 1 ICAR-Directorate of Soybean Research, Indore 2 ICAR-Central Research Institute for DrylandAgriculture, Hyderabad 3 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 4 ICAR-Indian Institute of Seed Science, Maunath Bhanjan PI : Anil Kumar Sharma Department of Biological Science, CBSH, GBPUA&T, Pantnagar

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31. Development of Practicable Technologies for Field Level Exploitation of Consortia of Microbial Agents for Amelioration of Biotic andAbiotic stresses in Crops

PI : R. D. Prasad Co-PI : P. Lakshmamma,Aziz. Quereshi ICAR-Indian Institute of Oilseeds Research, Hyderabad

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32. Development of PGPR Inoculant Bioformulations for Rhizosphere Management in Enhancing Biomass of Fodder Crops

PI : Srinivasan, R Co-PI : H. V. Singh ICAR-Indian Grassland and Fodder Research Institute, Jhansi

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33. Endophytic Microorganisms for Management of Drought in Rainfed Maize and Groundnut

PI : Minakshi Grover Co-PI : S.K.Yadav, S. Desai ICAR-Central Research Institute for DrylandAgriculture, Hyderabad

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34. Mass Production of Bacillus thuringiensis (Bt) and Beauveria bassiana, Formulation as Oil Based Suspension Concentrates Singly and in Combination and Field Evaluation

PI : P. S. Vimala Devi Co-PI : P. Duraimurgan ICAR- Indian Institute of Oilseeds Research, Hyderabad

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35. Development of Formulations of Beauveria bassiana, Metarrhizium anisopliae and Lecanicillium spp. and Paecilomyces fumosoroseus for Management of Certain Sucking Pests in Vegetable Crops

PI : B. Ramanujam Co-PI : A.N. Shylesha ICAR-National Bureau ofAgricultural Insect Resources, Bengaluru

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36. Role of Potential Microorganisms in Seed and Crop Health of Rice, Wheat and Mustards

PI : Co-PI :

Madan Kumar 1 2 S. Rajendra Prasad , 1Pawan K. Sharma , Umesh 1 1 Kamble , Ramesh K.V , Ragvendra D . 1 ICAR- Indian Institute of Seed Science , Maunath Bhanjan 2 ICAR- National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan

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37. Development of Effective Salt Tolerant Microorganisms to Mitigate Salt Stress for Crop Production in Salt affected Soils

PI : P.K. Joshi ICAR-Central Soil Salinity Research Institute, Karnal

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38. Production and Formulation Technology Refinement of Bacterial Bio-agents for Soil-Borne Plant Disease Management under Coastal and Plain Ecosystems

PI : R. Ramesh ICAR- Central CoastalAgricultural Research Institute, Old Goa

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39. Prospecting the Potential of Cyanobacteria based Formulations as Plant Growth Promoting and Biocontrol agents in Cereal-Legume Cropping System and Selected Vegetables

PI : Radha Prasanna Co-PI : Lata and B. Ramakrishnan ICAR-IndianAgriculture Research Institute, New Delhi

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40. Degradation and Effective Utilization of Agrowastes through Technologies Involving Mushrooms or Macrofungi

PI : Co-PI :

Sachin Gupta Moni Gupta, Arvind Kumar Ishar, S.A. Mallick, Ranbir Singh, S. K. Singh Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu

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41. Metagenomics and Cultural Approaches for Identification of Novel Microbial Genes/Alleles and Microbes for Bioconversion of Lignocellulosic Biomass at Extreme Physiological Conditions of Low Temperature

PI : Rajeev Kaushik Co-PI : Anil Kumar Saxena, Livleen Shukla ICAR-IndianAgricultural Research Institute, New Delhi

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42. Development of Bioformulations with PGPR, PGPF and AMF for Induction of Resistance in Cereals and Pulses against Fungal Pathogens and Improvement of their Health Status

PI : B.N. Chakraborty Co-PI : U. Chakraborty Department of Botany, University of North Bengal, Darjeeling

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43. Identification of Salt Tolerant Microbes and Development of Dynamic Substrate for Cultivation of Commercial Crops in Sodic Soils

PI : T. Damodaran Co-PI : S.K. Jha, V.K. Mishra, D.K. Sharma, Y.P. Singh ICAR-Central Soil Salinity Research Institute, Regional Research Station, Lucknow

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44. Development of Microbial Consortia as Biocontrol for Corm rot in Crocus sativus

PI : Jyoti Vakhlu 2 Co-PI : Alok K. Srivastva 1 University of Jammu, Jammu 2 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan

29. Identification of High Trehalose-Producing Soybean Rhizobia and their Integration with AM Fungi for Enhanced Drought Tolerance in Soybean

30. Mapping and Creation of Gene Bank of Arbuscular Mycorrhiza and their Exploration for Enhancing Productivity of Underutilized Crops

PI : Co-PI :

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45. Exploration of Microbial Diversity in Seed Spices through MetagenomicsApproaches for Crop Improvement

PI : Sharda Choudhary Co-PI : B. K. Mishra ICAR-National Research Centre on Seed Spices, Tabiji, Ajmer

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46. Formulation of Coleoptera Active cry Toxins of Bacillus thuringiensis

PI : R.Asokan ICAR-Indian Institute of Horticultural Research, Bengaluru

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47. Influence of Arbuscular Mycorrhizal Fungi and Rhizobia in Conferring Drought Tolerance to Soybean

PI : D. J. Bagyaraj1 Co-PI : B. Mohan Raju2 1 Centre for Natural Biological Resources and Community Development, Bengaluru 2 University ofAgricultural Sciences, Bengaluru

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48. Exploitation of PGPR and Biocontrol Agents for Integrated Management of Biotic Stresses of Chilli in Irrigated and Dry land Ecosystem

PI : Co-PI :

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49. Studies on Rhizospheric Microbial Diversity in Relation to Different Sugar Profile Varieties for Growth Promotion and Disease Management

M. K. Naik Amaresh Y.S. , Savithaa. S., Arun Kumar Hosamani, Mahadev Swamy University ofAgricultural Sciences, Raichur PI : Dinesh Singh ICAR-Indian Institute of Sugarcane Research, Lucknow

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Theme : Microbe Based Green Energy 50. Synthetic Biology and Metabolic Engineering Opportunities for Enhanced Production of Biofuels through Microbes

PI : AnjuArora Co-PI : Lata Nain, Surender Singh ICAR-IndianAgricultural Research Institute, New Delhi

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51. Thermophilic Fungi from Diverse Ecosystems and their Potential for Producing Industrially Important Enzymes

PI : B.S Chadha Co-PI : Amarjeet Kaur Guru Nanak Dev University,Amritsar

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52. Whole Genome and Transcriptome Sequencing of Selected Cyanobacterial Genomes for Identification of the Role their Genes in Metabolic Pathways related to Bio-energy Production

PI : Sucheta Tripathy CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata

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53. Developing Enzyme Catalyzed-Micobe Mediated Processes for Biofuel from Mango Waste (peel and kernel)

PI : Neelima Garg ICAR-Central Institute for Subtropical Horticulture, Rehmankhera, Lucknow

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PI : Alok K. Srivastava1 Co-PI : P. L. Kashyap2, Hillol Chakdar1, K Pandiyan1 1 ICAR-National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath Bhanjan 2 ICAR-Indian Institute of Wheat and Barley Research, Regional Station, Flowerdale, Shimla

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Theme : Microbial Genomic Resource Repository 54. Microbial Genomic Resource Repository

Publications

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PREFACE Microorganisms represent the richest gamut of molecular and chemical diversity in nature, as they comprise the most diverse forms of life. India is home to billions of microbes, many of which are found nowhere else in the world. Interest in the exploration of microbial diversity has been spurred by the fact that microbes are essential for life since they perform numerous functions essential for the biosphere that include nutrient cycling and environmental detoxification. The exploited microbial activities are augmentation, supplementation and recycling of plant nutrients, so vital to sustainable agriculture. Soil biodiversity influences a huge range of ecosystem processes that contribute to the sustainability of life on earth. Soil biodiversity plays a role in soil fertility, soil erosion, nutrient uptake by plants, formation of soil organic matter, nitrogen fixation, the biodegradation of dead plant and animal material, reducing hazardous waste, the production of organic acids that weather rocks, and control of plant and insect populations through natural biocontrol. Microbial diversity and its richness to the environment provide a huge reservoir of resources, which we can utilize for our benefit. The microbes have developed an extensive range of metabolic pathways, which have traditionally been exploited by man in processes such as fermentation, production of antibiotics, vitamins, etc. From ancient period of time the microbes have been utilized in preparing variety of beverages, the dairy and bakery products. With the use of modern biotechnnology, the potential

applications of diverse microorganisms are vast. Scientists are experimenting with genetically engineered bacteria that are capable of producing products such as biodegradable plastics, and fibres. Maize, rice, potato and cotton are among the crops that have been genetically engineered to produce insecticidal Proteins from a common soil bacterium, Bacillus thuringiensis (Bt). The use of bacterial insecticides has reduced the use of chemical insecticides, which is both a cost savings to the producer and less stressful on the environment. Other bacterial enzymes and constituents of the organisms are utilized to produce materials such as plastic. The ICAR network project on 'Application of Microorganisms in Agriculture and Allied Sectors' is in the second phase during the XII five year plan. It encompasses 55 projects distributed in six thematic areas: 1. Microbial diversity 2. Taxonomy, Identification and Diagnostics of AIMs 3. Plant microbe interactions, nutrient management, disease control and agrowaste management 4. Microbe based green energy 5. Microbial genomic resource repository and 6.Human resource development. The significant achievement on annual basis have been compiled. I would like to thank the former Secretary, DARE and Director General, ICAR, Dr. S. Ayyappan; and present Secretary, DARE and Director General, ICAR Dr. Trilochan Mohapatra; DDG (Crop Science), Dr. J. S. Sandhu and ADG (PP&B) Dr. P. K. Chakrabarty for their valuable guidance in giving meaningful shape to the network project.

Anil K. Saxena Director & National Coordinator AMAAS

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EXECUTIVE SUMMARY promoting microorganisms and biocontrol agents. There is need to work on isolation of drought tolerant rhizobia from different crops for effective nitrogen fixation under drought conditions. Another symbiotic microorganism that forms tripartite relationship with plant and rhizobia is arbuscular mycorhizal fungi (AMF) responsible for contributing phosphorus nutrition and drought tolerance to plants. The rapid diagnosis and detection of microorganisms and pathogens is an emerging area of study. The diagnostic tools are useful to identifying microorganisms which are difficult to distinguish either by morphology or other features. PCRbased markers have been developed for plant pathogenic fungi like Alternaria and Rhizoctonia solani. DNA barcodes of multi-locus gene phylogeny is also an emerging technology for high-throughput detection and discrimination of fungal plant pathogens. During 12th Plan period the network project on “Application of Microorganisms in Agriculture and Allied Sectors” (AMAAS) has focused on different aspects of molecular microbiology and development of microbial products and technology to harness the potential of microorganisms for benefit of Indian agriculture with the modified thematic areas under six components. In the second year (2015-16), following significant achievements have been made from 55 subprojects of AMAAS which are running at different ICAR, DBT institutes, SAUs, CAUs, CSIR institutes and others. THEME 1: Microbial Diversity & Identification l An antagonistic Bacillus sp. JCR-21 isolated from soil samples of Rambha village, Manipur was found positive for production of hydrolytic enzymes viz. cellulase, protease and amylase and showed antagonism against fungal phytopathogens Macrophomina phaseolina, Fusarium oxysporum and Rhizoctonia solani. l Six species of Bacillus isolated from Andaman and Nicobar Islands showed antagonism against Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, Colletotrichum capisici, Fusarium spp. and Verticillium spp and are being used for management of disease at field level. l Endophytic bacterial diversity in shoot-tip tissue of Banana cv. Grand Naine was investigated through culture dependent and 16S rRNA metagene technique. Cultivable bacteria included 37 isolates belonging to 16 genera and three phyla (Proteobacteria, Actinobacteria, Firmicutes). 16S rRNA metagenome profiling revealed enormous endophytic bacterial diversity spanning over 20 phyla with Proteobacteria as the predominant

Indian agriculture is highly dependent on agrochemicals for enhancing productivity to meet food and nutritional requirements of burgeoning population. This type of practice for last many decades has deteriorated soil quality, depleted soil organic carbon and micronutrients, and thereby made soil unproductive and unhealthy. It is now time to gradually shift to the organic agriculture utilizing natural resources for enhancing agricultural productivity while maintaining better environmental conditions. Among organic inputs, agriculturally important microorganisms (AIMs) occupy central position in crop productivity, soil health and management of biotic and abiotic stresses. So far, most of the information on diversity of AIMs is available on culturable microorganisms which designate a fraction of vast microbial resources and huge chunk of microorganisms are yet to be cultured from explored and unexplored areas of the globe including India. Such unexplored areas are rich in microorganisms with high metabolic diversity that may be utilized in agriculture. The extreme and unique niches including hot springs, cold deserts, saline area, extreme dry area, acidic area as well as mangroves are reservoir of metabolically diverse microorganisms that could be utilized in traditional and modern agriculture practices to improve productivity. Hence, extreme niches are continuously getting attention for microbial diversity analysis. Some bacteria namely Klebsiella, Corynebacterium, Rothia, Exiguobacterium, Staphylococcus arlettae, Lampropedia cohaerens, Fictibacillus halophilus with potential attributes from extreme and unique environments are being used in agriculture. Soil microbial communities present in cold areas have capability of degrading lignocellulosic residues at low temperature. Penicillium ubiquetum SF3 and Phoma sp. LF1 were recovered from cold deserts of Himalayan region and found to be potent lignocellulolytic fungi at 10°C. Cultural diversity of microbial community from such habitats can be used for composting process, biofuel production and other industrial purposes at lower temperature. Since only 1-5% microorganisms are culturable, metagenomic tools are successful in deciphering unculturable diversity of microorganisms and revealing genes with novel and unique traits that can be utilized in agriculture directly or indirectly. A large number of genes with such unique traits are being mined from extremophiles and could be used in the transgenic program of the country for mitigating abiotic stresses. Microbes play an important role in nutrient management and biological control of various diseases. There is a need to identify efficient strains of nitrogen fixing; phosphorus, potassium and zinc solubilising bacteria; plant growth

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not been done. In its absence, SSR markers were mined from the closely related species i.e. Ustilago hordei and U. maydis. Six SSRs from U. hordei and 10 SSRs from U. maydis showed polymorphism against the Tilletia indica (Karnal bunt) isolated from infected wheat grain samples. THEME 3: Plant Microbes Interactions, Nutrient Management, Disease Control and Agro waste Management l Gene expression studies revealed induction of heterotrimeric G-protein expression in Pseudomonas fluorescens OKC and Trichoderma asperellum T42 inoculated chickpea plants after inoculation of pathogen Fusarium oxysporum. G-proteins are involved in plant defense signaling process. l Bradyrhizobium japonicum and Bacillus sp. strains were isolated from drought tolerant cultivars of soybean. The trehalose sugar which is responsible for drought tolerance mechanism was detected in nodules of 21 drought-tolerant cultivars. l Different strains of AMF Acaulospora laevis , Funneliformis mosseae, Gigaspora margarita, Glomus mosseae, Glomus caledonicum showed alleviation of drought in wheat and chickpea crops. l Different Trichoderma isolates (T. harzianumTh4d, T. asperellumTN13, TV5, TA5 and TA7) with salinity tolerance up to 1.5M NaCl and drought tolerance up to 12 bar PEG were identified. T. asperellumTN13 was effective in imparting salinity tolerance in sunflower at 6 dS/m EC while T. asperellumTA5 was found imparting drought tolerance in castor when 9 days of stress imposed by withholding irrigation. l Eight maize seed endophytes were evaluated as seed inoculants in kharif maize (rainfed) which influenced plant growth, physiological and biochemical parameters under drought conditions. Lytic enzyme production (in vitro) profile from these endophytic bacteria was generated for amylases, lipases, esterases, proteases, cellulases, and pectinases. l Ball milling particles (559 nm) of Bt-127 (technical) were optimized for mortality of Spodoptera litura larvae. Suspension concentrate (SC) formulations of Bt127 and Bt + Nomuraea rileyi were found effective against S. litura on castor at RARS-Palem, TCRSYethapur and IIOR, Hyderabad (83-99%) as well as Helicoverpa armigera on sunflower at RARS-Nandyal, ORS-Latur and IIOR, Hyderabad (90-100%) by 5 days after spray. l Three promising isolates viz., Beauveria bassiana (Bb5a), Metarhizium anisopliae (Ma-4) and Lecanicillium lecanii (Vl-8) were identified as promising isolates against aphids of cabbage, brinjal, chilli and french bean in the field trial which reduced 50-60% of the aphid population. Two isolates of B. bassiana (Bb-5a and Bb45) and M. anisopliae (Ma-4 and Ma-35) were

phylum. Pseudomonas, Klebsiella, Salmonella, Enterobacter, Citrobacter, E. coli, Staphylococcus aureus and Listeria ivanovii were isolated from diverse dairy niches. l Different bacterial genera including Bacillus, Pantoea, Marinobacterium, Acinetobacter, Enterobacter, Pseudomonas, Sinorhizobium and Rhizobium were isolated from terrestrial halophytic weed. The response of these bacteria as seed-inoculant for germination and growth of sorghum and wheat was found consistent under a wide range of osmotic stress (EC 5-10 dSm-1). l Two novel bacterial strains isolated from hot water spring, Manikaran were identified as Lampropedia cohaerens CT6T (=DSM 100029 T=KCTC 42939 T =MCC 2711 T) and Fictibacillus halophilus AS8 T (= MCC 2765 T = DSM 100124 T = KCTC 33758 T) by employing the polyphasic approach. l Bacteria capable of producing thermotolerant enzymes (45 0 C) enzymes including amylase, protease, acrylamidase, nitrilase and xylanase were identified. l Complete genome sequencing of Pseudomonas striata PS27 with 4th generation sequencing technology revealed the presence of three chromosomes and one plasmid in the strain. Several genes involved in phosphate solubilisation and transport were annotated. Fourteen different primers were designed and genes responsible [pqqEDCB (pyrrolquinone gene cluster), gltA2 (citrate synthase), prpC (2-methyl citrate synthase 1), prpC2 (2-methyl citrate synthase 1), pitA1, pitA2 and pitA3 (phosphate transporter)] for P solubilisation were amplified. l Comparative whole genome analysis of five different Mesorhizobium sp. with Mesorhizobium ciceri Ca181and annotation of nif family genes revealed four hypothetical proteins which belong to nif family. l The genomes of Bacillus sp. MSP5.4, Bacillus sp. MSP13, Halomonas cesinilyticus WD26, Haloarcula salaria H5-DGR, Netrinema altunense1A4-DGR and Aspergillus sp. WD1 were sequenced and annotated. By comparative genomics of the complete genome sequences of extreme halophilic archaea, serine glyoxalate cycle was identified as a potential candidate pathway for osmotolerance, carbon assimilation and anabolism in extreme haloarchaea. l A cyanobacterial strain Fischerella sp. (NAIMCC-C00130), efficient in removal and degradation of organophosphorus pesticide methyl parathion (MP) was isolated from agricultural field. THEME 2: Taxonomy, Identification & Diagnosis ofAIMs l Mating type idiomorphs of Alternaria were identified through PCR technique. Specific primers (RS10F:RS10R & AG1F1: AG1R1) for identification of Rhizoctonia solani were designed and validated. l Till date whole genome sequencing of Tilletia indica has

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characterize different ligno-cellulolytic glycosyl hydrolases from Acrophialophora sp. A complex cellulosic medium for hyper-cellulase production by Penicillium sp. dal 5 was devised for high expression of cellulases and xylanase. l The production of tannase enzyme was found in Penicillium restrictum AN4 (KJ820679), P. crustosum AN3 (KJ820682), Aspergillus niger, A. oryzae and A. fumigatus. In other study, conditions were (temp- 40°C, pH- 5.5, nutrient addition 2% and inoculums size 1.19 *106 cells/ mL) optimized for production of ethanol from mango kernel starch by Saccharomyces cerevisiae using response surface methodology. THEME 5: Microbial Genomics Resource Repository l Xylanase gene from Bacillus subtilis was cloned and expressed in E. coli BL21 using pET28a cloning vector. Chitinase and β-tubulin genes from Trichoderma sp. were amplified and cloned for preservation and further expression studies. About 234 rRNA genes from Pseudomonas sp. and Bacillus sp.; 33 internal transcribed sequences (ITS) from Trichoderma sp. and Fusarium sp. were amplified and preserved in Microbial Genomic Resources Repository.

established as endophytes in cabbage leaf tissues by artificial inoculation for management of sucking pests. l Out of 300 bacterial strains from rice, wheat and mustard from different agro-climatic zones of India, 35 bacterial strains were found to have potential against Fusarium oxysporum f. sp. ciceri, Ustilaginoidea virens, Magnaporthe oryzae , Drechslera teramera and Sclerotium rolfsi pathogens. Talc based bio-formulation were prepared for testing of bacterial strains against M. oryzae and U. virens under pot and field condition in susceptible rice variety Pusa Sugandh (PS). l Granular formulations were tested against larvae of white root grubs as well as Areca nut white root grubs (Leucopholis lepidophora Blanchard and Leucopholis burmeisteri). Various sources for the mass culturing of Bt strains were attempted. Corn Stalk and Amaranthas stalk proved to be the best source for mass culturing of BtA (1.12 x 10-9) and Bt CRY1I (1.01 x 10-9), Bt MD (9.58 x 10-9) respectively. THEME 4: Microbe Based Green energy l Two strains of Kodamaea were isolated from the Lagenaria siceraria flowers by enrichment on pentose rich medium. Kodamaea strains grew well on biologically pretreated hydrolysates, utilized (>90%) sugars and produced ethanol (GC analysis). l A rapid approach was developed to purify and

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AMAAS : Themes l

Draft genome sequencing of P-solublizing Pseudomonas striata PS27 and functional validation of MSP Genes l Genomics and proteomics of N2-fixing cyanobacterium with special reference to discovering abiotic stress tolerant novel genes and protein Exploiting the diversity of extreme halophiles by l functional and comparative genomics for isolating novel genes and alleles for affording salinity tolerance to crop plants l Isolation and selection of naturally occurring and genetically modified cyanobacterial strains for their potential use in biodegradation of agricultural pesticides (organophosphorous) l Bioprospecting for microbial products that affects cold alleviation and growth l Pathogenomics-genome sequencing of Puccinia striiformis Sub Theme : Microbial Diversity with Special Focus on Microbes from Farm, Fisheries andAnimals l Microbial management in freshwater aquaculture Sub Theme : Microbial Diversity with Special Focus on Metagenomic based CommunityAnalysis l Archaea and actinobacteria in vertisols of central India – assessment of diversity, biogeochemical processes and bioinoculant potential l Mining the metagenome for bacterial diversity in extreme areas of north eastern India l Microbial diversity of glacier sample and peat profile of Himalayan Region

Theme : Microbial Diversity Sub Theme : Microbial Diversity with Special Focus on Extremeophilic and Unique Niches

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Microbial diversity analysis of extreme ecological niches l Exploration of the endophytic microbial diversity in horticultural crops through metagenomics and cultivation l Mapping of deep-sea and hypersaline microbiota and their characterization using meta-omics approaches for bioprospecting of novel genes l Analysis of bacterial community and their functional diversity from rice rhizosphere employing genomic and metagenomics approaches l Microbial diversity of bio-films in Dairy Niche l Functional characterization of salt tolerant bacteria using multi-omic approaches: Towards the development of likelihood model of microbial amelioration of salt stress in wheat crop l Culturable and unculturable microbial diversity of insects and their role in insecticide resistance and other fitness attributes l Molecular characterization and exploration of rumen microbial diversity in Black Bengal goats at north eastern state of Tripura l Microbial metagenomic analysis of hot spring situated atop the Himalayan ranges at Manikaran, Himachal Pradesh, India l Diversity of arbuscular mycorrhizal fungi and dark septate endophytes in woody species and shifting cultivation crop of Manipur, India Sub Theme : Microbial Diversity with Special Focus on Biomolecules, Industrially Important Enzymes l Exploring myxobacteria from the North-Western Himalayas for novel enzymes and secondary metabolites l Exploration and mass culture of fresh water cyanobacteria for development of nutraceutical feed for freshwater fish hatchlings Sub Theme : Microbial Diversity with Special Focus on Bio prospecting for Novel Genes andAlleles l Reference and de novo draft genome sequencing of potentialAIMS and their functional annotation l Biomolecules and industrially important enzymes from extremophilic bacteria

Theme : Taxonomy, Identification and Diagnostics of AIMs

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Development of microarray based Gene Chip for fungal plant pathogens under the background of DNA barcodes using multilocus gene phylogeny Exploration of plant growth promoting rhizobacteria, antagonistic and plant pathogenic microbial resources from high altitude agro-climate/ cropping system of Jammu & Kashmir state for sustainable agriculture. Development of diagnostic kit for detection of Karnal Bunt and Loose smut of Wheat

AMAAS - Annual Report 2015-16

Theme : Plant Microbe interactions, Nutrient management, disease control and Agro waste management

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Investigating the potential of root-colonizing microbial population for stress tolerance in chickpea through proteomics Identification of high trehalose-producing soybean rhizobia and their integration with AM fungi for enhanced drought tolerance in soybean Mapping and creation of gene bank of arbuscular mycorrhizal fungi and their exploration for enhancing productivity of underutilized crops Development of practicable technologies for field level exploitation of broad spectrum biocontrol agents for amelioration of biotic/abiotic stresses in crops Development of PGPR inoculant bioformulations for rhizosphere management in enhancing biomass of fodder crops Exploring endophytic and rhizospheric microorganisms for management of abiotic stress (drought) in rainfed maize and groundnut Mass production of Bacillus thuringiensis (Bt) and Beauveria bassiana formulation as oil based suspension concentrates singly and in combination and field evaluation Development of formulations of Beauveria bassiana, Metarhizium anisopliae , Lecanicillium spp. and Paecilomyces fumosoroseus for management of certain sucking pests in vegetable crops Role of potential microorganisms in seed and crop health of rice, wheat and mustard Development of effective salt tolerant microbes to mitigate salt stress for higher crop production in salt affected soils Production and formulation technology refinement of bacterial bioagents for soil-borne plant disease management under coastal and plain ecosystems Prospecting the potential of cyanobacteria based formulations as plant growth promoting and biocontrol agents in cereal-legume cropping system and selected vegetables Degradation and effective utilization of agrowastes through technologies involving mushrooms or macrofungi Metagenomics and cultural approaches for identification

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of novel microbial genes/alleles and microbes for bioconversion of lignocellulosic biomass at extreme physiological conditions under low temperature. Development of bioformulations with PGPR, PGPF and AMF for induction of resistance in cereals and pulses against fungal pathogens and improvement of their health status Identification of salt tolerant microbes and development of dynamic substrate for cultivation of commercial crops in sodic soils Development of microbial consortia as biocontrol for corm rot in Crocus sativus Exploration of microbial diversity in seed spices through metagenomics approaches for crop improvement. Formulation of coleoptera active cry toxins of Bacillus thuringiensis Influence of arbuscular mycorrhizal fungi and rhizobia in conferring drought tolerance to soybean Exploitation of PGPR and biocontrol agents for integrated management of biotic stresses of chilli in irrigated and dry land ecosystem

Theme : Microbe Based Green Energy

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Synthetic biology and metabolic engineering opportunities for enhanced production of biofuels through microbes. Thermophilic fungi from diverse ecosystems and their potential for producing industrially important enzymes Whole genome and transcriptome sequencing of selected cyanobacterial genomes for identification of the role their genes in metabolic pathways related to bio-energy production Developing enzyme catalyzed- microbe mediated processes for biofuels from mango waste (peel and kernel)

Theme : Microbial Genomic Resource Repository

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Microbial Genomic Resource Repository

Theme : Coordinating Unit and Human Resource Development

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Coordination and HRD

Theme : Microbial Diversity Microbial DiversityAnalysis of Extreme Ecological Niches PI : Sushil K. Sharma1 Co-PIs : Pawan K. Sharma1, Karthikeyan Nanjappan1, Hillol Chakdar1, Sakthivel Krishnan2 1 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 2 ICAR-Central Island Agricultural Research Institute, Port Blair Loktak Lake and Siroi Lily plant of Manipur state and Andaman and Nicobar Islands (Fig. 1).

Rationale Extremophiles are microorganisms that can grow and thrive in extreme environments like high or low temperature, high or low pH, high salinity, high metal concentrations, very low nutrient content, very low water activity, high radiation, high pressure, and low oxygen tension which were formerly considered too hostile to support life. These organisms have evolved several structural and chemical adaptations, which allow them to survive and grow in extreme environments. The enzymes of these microbes, which function in extreme environments (extremozymes) have several biotechnological applications. Antibiotics, compatible solutes and other compounds obtainable from these microbes have been found to have variety of uses. The extreme niches of India include hot springs (Manikaran, Chumathang, Barkeshwar, Balarampur and Vashisht) and cold deserts (Leh), salty area (Rann of Kutch), acidic area (Andaman and Nicobar Islands), dry area (Jaiselmer), as well as mangroves forest (Bhitarkanika and Sunderbans). In this project, the soil samples were collected from Jhum cultivation sites, Siroi Hill and Phumdis in Loktak Lake of Manipur state; Mangroves forest of Sundarban Delta (West Bengal); and Andaman and Nicobar Islands for isolation of microorganisms.

l Among the bacterial strains recovered from Manipur region, Bacillus sp. JCR-3, Bacillus aerophilus JCR-5,

(A)

(B)

(D)

Objectives

l To assess the culturable and unculturable microbial

Fig. 1: Sampling sites: (A) Phumdis (Loktak Lake), (B) Heritiera fomes (Sundari), (C) Sea shore rocks & (D) Sea sediments (Car Nicobar Island)

diversity from selected ecosystems with focus on NEH and Andaman and Nicobar Islands.

Bacillus sp. JCR-21, Bacillus safensis JCR-44 were found positive for cellulase and protease production and also showed antagonism against fungal phytopathogen Macrophomina phaseolina. (Fig. 2 & 3). A Streptomyces sp. JCT-87 was found positive for amylase, cellulase xylanase production and showed antagonism against Macrophomina phaseolina.

l Functional screening of genomic and metagenomics library and identification of clones expressing novel genes/ alleles for antimicrobial hydrolytic enzymes, and other metabolites for application in agriculture. SignificantAchievements

l A total of 411 bacterial morphotypes were recovered from

l 16S rRNA gene sequencing of bacteria recovered from

the soil samples collected during exploration to Mangroves forest of Sundarban Delta (West Bengal), Jhum sites,

Jhum cultivation sites of Manipur revealed

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AMAAS - Annual Report 2015-16

(A)

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(C)

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Fig. 2: Stereomicroscopic images of bacteria and actinomycetes strains (A) Bacillus pumilus JCR-3, (B) Bacillus aerophilus JCR-5, (C) Streptomyces sp.

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Fig. 5: Antagonism and functional traits of Curtobacterium sp. SL-2 (A) Antagonism against Macrophomina phaseolina (B) siderophore production (C) protease production (D) cellulase production

(C)

Fig. 3: Antagonism against Macrophomina phaseolina by (A) Bacillus sp. JCR-21, (B) Bacillus aerophilus JCR-5, (C) Streptomyces sp. JCT-87

l Staphylococcus, Pseudomonas, Bacillus, Curtobacterium, Halobacillus, Sphingobacterium, Terribacillus, Serratia, Acinetobacter, Arthrobacter and Oceanobacillus were recovered genera from samples of Andaman and Nicobar Islands.

predominance of Bacillus genus in Jhum sites.

l Bacillus subtilis LLP-2 isolated from Loktak Lake, Manipur was found positive for cellulase, amylase, protease, and pectinase enzyme (Fig. 4) whereas Serratia marcescens LLP-32 showed cellulase and amylase production; and phosphate solubilisation.

l Total eight bacterial strains Enterobacter cancerogenus JCR-4, Bacillus aerophilus JCR-5, Staphylococcus arlettae JCR-6, Staphylococcus warneri JCR-13, Pseudomonas koreensis JCR-18, Micrococcus yunnanensis JCR-24, Enterobacter ludwigii JCR-38, Bacillus safensis JCR-44 have been deposited to NAIMCC.

l Curtobacterium sp. SL-2 isolated from rhizosphere soil of plant Siroy Lily cultivated exclusively in Manipur state were showing multiple traits like antagonistic activity and production of siderophore, protease, and cellulase (Fig. 5). Bacillus aerophilus SL-18 showed amylase, pectinase, cellulase, and protease production while Bacillus safensis SL-40 showed both protease production and phosphate solubilization.

l One hundred seventy (170) isolates were isolated from rhizosphere of vegetable, spice and mangroves all over South Andaman, Little Andaman, Middle, North Andaman and Car Nicobar Islands at ICAR-CIARI, Port blair for assessment of their plant growth promoting (PGP) and antagonistic traits. Among 170 isolates, 21 Bacillus isolates were showed better in vitro plant growth promoting traits viz., IAA, siderophore production and P solubilization. Also the antagonistic potential studies of those selected 21 Bacillus spp. against five native important phytopathogens revealed that 30% isolates showed good in-vitro antagonism against Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae (66.6%), Colletotrichum capisici (57.1%), Fusarium spp. (38.0%) and Verticilium spp. (38.0%). Six promising antagonistic and PGP isolates were subjected to 16S rRNA gene sequence based identification and Biolog based phenotypic fingerprinting. All the six isolates were identified as Bacillus subtilis, Lysinibacillus sphaericus, and Bacillus amyloliquenciens. Further, the 21 bacterial isolates possessing in vitro antimicrobial properties were screened for their antimicrobial peptide gene (AMP gene) amplification using 13 peptide gene primers. The results

l Isolates GW-7, GW-23 and GW-24 recovered from Sundarban region showed both siderophore production and phosphate solubilization.

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Fig 4: Functional traits of Bacillus subtilis LLP-2, production of (A) Cellulase (B)Amylase (C) Protease, (D) Pectinase.

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AMAAS - Annual Report 2015-16 Diversity to Utility form 11th -20th January, 2016

revealed that two of the Bacillus strains (BM13 & C19) were able to amplify nine AMP biosynthetic genes and HR4b strain could amplify eight AMP genes. Other remaining Bacillus isolates were able to amplify between two to seven number ofAMP genes.

l National Workshop on Bioinformatics-based Genomic & Proteomic Data Analysis in Microbial Domain form 4-9th March, 2015

l Dr. Sushil K. Sharma, a member of National Advisory

Conclusion

Committee, has participated in Satellite Workshop on PGPR for Sustainable Crop Productivity by the Asian PGPR Society on 25th Feb., 2016 at NASC, New Delhi.

Diverse bacterial gene pool recovered from Manipur, Sunderbean Delta and Andaman and Nicobar Island has potential of plant-growth promotion and biocontrol activity. These strains could be utilized for crop production in different niches after proper evaluation.

l Ankita Verma, Sandeep Saini, Priyam Mehrotra, Pallavi, Megha Singh, Amrita Gupta, Abhishek Bharti, Mahaveer P. Sharma, Aketi Ramesh, Hillol Chakdar, Pawan K. Sharma, Rup Lal and Sushil K. Sharma (2016). Exploring plant growth promoting and hydrolytic activity of bacteria isolated from Jhoom cultivation system of Manipur. In: IPS 6th International Conference on Plant, Pathogens, and people from Feb., 23-27. Pp 580.

Conference and workshop attended l Workshop on Renovating Bacterial Taxonomy with Bioinformatics at Delhi University, North Campus 14-15 May 2015

l Bioinformatics: Tools and Techniques for Genome

l Sakithivel K, Manigundam K, Gautam R K, Nakkeeran S,

Analysis at IIT Delhi from 14-16 July 2015

Kumar A, Sushil K. Sharma and Dam S Roy (2016) Detection of antimicrobial peptide genes in antagonistic Bacillus species isolated from soil of Andaman Islands, India. In: IPS 6th International Conference on Plant, Pathogens, and People from Feb., 23-27. Pp 584.

l National Workshop on Relevance of Microscopy in Microbial Characterization at Amity University, Noida from 11th-14th Aug., 2015

l Advances in Plant Growth Promoting Rhizobacetria:

Exploration of the Endophytic Microbial Diversity in Horticultural Crops through Metagenomics and Cultivation PI : Pious Thomas ICAR-Indian Institute of Horticultural Research, Bengaluru

l To assess the endophytic diversity in tomato with reference

Rationale

to the susceptibility/resistance reaction to the wilt pathogen Ralstonia solanacearum.

The term endophytes denote microorganisms that colonize the plants internally without any apparent adverse effects on the host. Bacterial endophytes have by and large been studied based on cultivation-based approaches. It is now emerging that majority of endophytes are not amenable for cultivation. Thus, the application of cultivation-independent approaches to study them is gaining importance. Metagenomics form a new molecular tool to study the microorganisms that cannot be cultured. The method has been applied successfully to different environments revealing considerable biodiversity and functional novelties. The project aims at exploring the endophytic microbial diversity in horticultural crops through cultivation and metagenomics and other aspects relating to the exploitation of endophytes in agri/ horticulture.

l Basic investigations on intra-plant and intracellular colonization by bacterial endophytes and related aspects in applied microbiology. SignificantAchievements

l The endophytic bacterial diversity in shoot-tip tissue of banana cv. Grand Naine was investigated through cultivation and 16S rRNA gene ribotyping in comparison with 16S rRNA metagene profiling. Tissue homogenate plating showed 2102 to 2106 bacterial cfu g-1. Cultivable bacteria from 20 suckers included 37 isolates under 16 genera and three phyla. Proteobacteria formed the major phylum (41.7%) with Klebsiella, Cedecea, Erwinia and Paracoccus spp. followed by Actinobacteria (Rothia, Arthrobacter, Cellulomonas, Corynebacterium, Kocuria, Rhodococcus, Streptomyces spp.) and Firmicutes (Staphylococcus spp. and Bacillus spp.).

Objectives

l Exploring into host-endophyte association in horticultural crops through cultivation and metagenomics.

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AMAAS - Annual Report 2015-16

l Cultivation independent approach of 16S rRNA gene 799f

several candidate phyla besides the domain Euarcheota. The OTU distribution at class level indicated high diversity with Gamma- and Alphaproteobacteria followed by Actinobacteria and Clostridia (Fig. 2). Analysis of single shoot tip of banana cv. Grand Naine through 16S rRNA

and 1492R PCR amplicon cloning and ribotyping (100 clones) showed only three genera with Serratia marcescens constituting 76.7% of the clones and the rest constituted by Achromobacter and Staphylococcus spp.

l 16S rRNA metagene V3-V4 taxonomic profiling approach was adopted on the pooled DNA from 10 shoot-tips of banana. This was initially hampered with high interference from chloroplast and mitochondrial sequences with limited bacterial diversity (Fig. 1A). A modified bioinformatics approach with the filtering of the chloroplast and mitochondrial sequences revealed abundant endophytic bacterial diversity (Fig. 1B). Proteobacteria formed the predominant phylum succeeded by Firmicutes, Actinobacteria, Bacteroidetes, Planctomycetes, Cyanobacteria and minor shares of 14 phyla including

Fig. 2: Extent of endophytic bacterial diversity documented in the shoottip tissue of banana cv. Grand Naine at class level as per 16S rRNA gene V3-V4 region taxonomic profiling

gene V3-region profiling confirmed the prevalence of high endophytic bacterial diversity within individual shoottips at phylum and family (Fig. 3) and genus levels. Firmicutes formed the predominant phylum followed by Actinobacteria, Proteobacteria and Bacteriodetes.

Fig. 3: Extent of endophytic bacterial diversity in individual shoot-tip tissue of banana cv. Grand Naine at phylum level as per 16S rRNA gene V3 region taxonomic profiling

l Cultivation vs 16S rRNA metagene taxonomic profiling has thus brought out that the vast majority of endophytes are not amenable for cultivation on common bacteriological media.

l Effects due to rhizospheric soil application of an

Fig. 1: Distribution of OTUs as per 16S rRNA gene V3-V4 region profiling of DNA from the shoot-tip tissue of banana Grand Naine in QIIME analysis showing a high abundance of plant chloroplast and mitochondrial sequences (A) and the revelation of high endophytic bacterial diversity with the modified bioinformatics analysis (B)

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antagonistic bacterial endophyte on native bacterial community and its survival in soil were undertaken employing 'GNS.13.2a' strain of Pseudomonas aeruginosa from banana as a model system. Monitoring of uninoculated control versus P. aeruginosa inoculated soil from banana rhizosphere showed a significant reduction in native bacterial cfu soon after inoculation compared with control soil. Sampling on days 4-28 indicated a significant reduction in P. aeruginosa cfu in inoculated soil and a continuous dip thereafter registering >99% reduction within one week while the native bacterial population resurged with cfu restoration on par with control. P. aeruginosa showed static cfu or

AMAAS - Annual Report 2015-16 identification of shoot-tip associated endophytic bacteria from banana cv. Grand Naine and testing for antagonistic activity against Fusarium oxysporum f. sp. cubense. American Journal of Plant Sciences 6: 943-954

proliferation in axenic-soil. Lateral introduction of soil microbiome in P. aeruginosa established soil under axenic conditions, its co-incubation with soil microbiota in suspension and agar-plate challenge assays with environmental bacterial isolates indicated significant adverse / interactive effects between P. aeruginosa and native microbial community. The multiple pathogen antagonistic putative biocontrol agent employed in this study caused an unwarranted disturbance to the soil microbial community upon its soil inoculation besides proving to be a poor survivor in field agricultural soil.

l Thomas, P., Sadashiva, A.T., Upreti, R. and Mujawar, M. M. 2015. Direct delivery of inoculum to shoot tissue interferes with genotypic resistance to Ralstonia solanacearum in tomato seedlings. Journal of Phytopathology 163: 320-323; doi: 10.1111/jph.12281

l Thomas, P, Sekhar, A.C., Upreti, R., Mujawar, M.M., Pasha, S.S. 2015. Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples. Biotechnology Reports 8: 45-55

Conclusion The elucidation of enormous prokaryotic diversity in banana shoot tissues (46 classes, 269 genera and 656 species) reinforces the microscopic documentation of abundant bacteria in the intracellular niches. The vast majority of endophytes are not amenable for cultivation. The immense microbial ecosystem intra-plant implies a significant role of endophytes in plant holobiome and hologenome with possible involvement in host plant biology and clonal transmission to successive generations. In effect, the application of P. aeruginosa in rhizospheric soil, which is employed as model organism in this study, did not serve any net benefit in terms of sustained survival. Conversely, it caused a disturbance to the native soil bacterial community. The findings highlight the need for monitoring the bioinoculant(s) in field-soil and assessing the interactive effects with native microbial community before commercial recommendation.

Conference and workshop attended

l Dr. Pious Thomas attended National Seminar on Biological Products from Crop, Animal and Human Health: Problems and Prospects, Organized by the National Academy of Biological Sciences and the University of Mysore on 21-22 August 2015, at University of Mysore and made an oral presentation entitled: Disturbance of field microbial community by introduced putative biocontrol bacterium and its survival in soil: A case study with Pseudomonas aeruginosa

l Dr. Pious Thomas the Satellite Workshop on PGPRs for Sustainable Crop Productivity by the Asian PGPR Society on 25th Feb., 2016 at NASC, New Delhi, and presented an invited paper: Endophytic Bacteria: Their Roles in Plant Health and Biocontrol

Paper Published fromAMAAS work

l Thomas, P. and Upreti, R. 2015. Evaluation of tomato seedling root-associated bacterial endophytes towards organic seedling production. Organic Agriculture Doi: 10.1007/s13165-015-0111-9

l Dr. Pious Thomas attended the Second World Noni Congress-at Chennai, 19-21 March 2016 and made an oral presentation entitled: Endophytic microorganisms: Their significance in Noni and other medicinal plants

l Sekhar, A.C. and Thomas, P. 2015. Isolation and

Mapping of Deep-sea and Hypersaline Microbiota and their Characterization using Metaomics Approaches for Bioprospecting of Novel Gene PI : Pradeep MA1 Co-PIs : KK Vijayan2, Krupesh Sharma1 1 2

ICAR- Central Marine Fisheries Research Institute, Kochi ICAR- Central Institute of Brackish Aquaculture, Chennai find the functional coding genes of microbes within an ecosystem grown in extreme condition.This technique is a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new

Rationale Metagenomics based experiments can provide more definitive information on the relative importance of specific genes and organisms in an environment-the approach helps to

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AMAAS - Annual Report 2015-16 initiatives to conduct genomic studies that link phylogenetic and functional information. Mangroves are shrubs or small trees that grow in coastal saline or brackish water. Mangrove forests play a central role in transferring organic matter and energy from the land to marine ecosystems. Mangroves are complex and dynamic ecosystems varying in salinity, water level and nutrient availability; they also contain diverse and distinct microbial communities. Salt evaporation ponds, also called salterns or salt pans, are shallow artificial ponds designed to extract salts from sea water or other brines. The microbial populations of these synthetic systems have been the subjects of several studies, and many of the halotolerant microbes available through culture collections are from solar salterns. Logical explanation accordance to the work includes.

Fig. 1 : PCR amplification of amylase gene

insert and the positive colonies were grown overnight for plasmid isolation and finally sequencing (Fig. 2).

Metagenomics is a valuable molecular technique for identifying and characterizing not only diversity but also functionally relevant genes. The presence of novel gene families from the uncultured and highly diverse microbial populations represents a challenge for the understanding and exploitation of the biology of the ocean and hyperhaline environment. Cloning and expression of these selected genes from cultured and uncultured bacteria will lead to likely discovery of novel bioactive molecules as well as the complex microbial diversity.

Fig. 2: Colony PCR for obtaining positive clones

l The blast result of the plasmid sequenced showed 88% similarity with the amylase gene of Bacillus amyloliquefaciens.

Objectives

l cDNA libraries from sediments of Sanikatta Salt pans in

l To collect environmental samples and to extract total

Gokarna were constructed as per the protocol mentioned below.

community DNAfrom the samples.

l To generate culturable and metagenomics library from the

l Soil DNA extraction was done from Sanikatta soil samples

isolated DNAusing a suitable cloning vector.

(2 g) directly without enrichment using the protocol described in Power soil DNA Isolation kit (MO BIO).The metagenomic DNA library was constructed using the copy control HTP fosmid library construction kit (Epicenter Biotechnologies, USA) according to the manufacturer's instruction. The purified high-molecular-weight DNA was end-repaired using end repair enzyme mix (T4 DNA polymerase and polynucleotide kinase) and size selected in low-melting point agarose (1%) along with the size control DNA having a molecular weight of 42 kb. The gel consisting of size fractionated DNA (∼ 42 kb) was recovered and ligated with fosmid vector pCC2FOS. The ligated construct was packed in Lambda packaging extract and used to transfect the competent E. coli EPI300 cells and plated on LB plates having chloramphenicol.

l Functional screening of the individual clones for the presence of proteins/enzymes from the environmental DNAfragments.

l High throughput metagenomic sequencing and bioinformatics analysis. SignificantAchievements

l Genomic DNA was isolated using the phenol-chloroform isolation method. Primers for amylase gene were designed and subsequently the gene was amplified from the culture TU 44 (Bacillus sp from Tutucorin salt pans) which was mentioned in the earlier reports (Fig. 1).

l The amplicon size was around 1800bp, which was purified and ligated into vector and transformed into E. coli cells. Colonies after overnight incubation was screened for the

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AMAAS - Annual Report 2015-16 further characterization including recombinant expression. The metagenomic library constructed has now to be screened for different enzymes and salt tolerant genes.

Conclusion The full length of ORF of the amylase gene amplified from the Bacillus sp. from Tutucorin salt pans has to be obtainedfor

Analysis of Bacterial Community and their Functional Diversity from Rice Rhizosphere Employing Genomics and MetagenomicsApproaches PI : Ashok Kumar1 Co-PIs : M. B.Tyagi2, R. P. Sinha2 1 School of Biotechnology, Banaras Hindu University, Varanasi 2 Department of Botany, Banaras Hindu University, Varanasi isolates showed remarkable metabolic and molecular diversity and certain strains proved to be potential candidate for use as biofertilizer. However, the presence of non-culturable population of bacteria remained unexplored. To fill this gap, we intended to analyze the whole bacterial community present in the rhizosphere of rice-wheat cropping system with emphasis on search for novel genes with new function. The present works describe isolation of metagenomic DNA from soil samples and amplification of certain important plant growth promoting genes by PCR.

Rationale Discovering new microbes and characterizing their functions are major goals in the study of microbial diversity. Of all the agriculturally important microbes, plant growth promoting rhizobacteria (PGPR) occupy central position in crop productivity and agriculture management. PGPR are capable of stimulating plant growth, e.g. by improving plant nutrition through N2 fixation, P solubilization and siderophore production, by the production of plant growth regulators such as indole acetic acid (IAA), cytokinin, GA3 or by preventing the attack of pathogenic microorganisms. Induced systemic tolerance (IST) to abiotic stresses in a number of crop plants by PGPR has also been reported. IST can result through production of bacterial 1-aminocylopropane-1-carboxylate (ACC) deaminase, antioxidants, cytokinin or volatile organic compounds (VOCs). It is presumed that PGPR may have a number of other beneficial traits as yet not known.

Objectives

l Analysis of total bacterial communities from rice rhizosphere employing genomics and metagenomics (culture-independent techniques) approaches so as to obtain microbial map. Our study shall concentrate mainly on PGPR.

l Characterization and isolation of not-yet culturable

Till date, most information on PGPR diversity (mainly community analysis) has been obtained by using culturedependent approaches. Results obtained have greatly advanced our knowledge of rhizosphere microbiology, several new bacteria with unique traits have been isolated and characterized in detail. For example, bacteria with efficient plant growth promoting characters like Acetobacter, Azoarcus, Derxia, Herbaspirillum, Serratia, Zoogloea, Burkholderia etc. which were missing from the list are now important component of rhizosphere. However, due to the unknown conditions for growth requirements of many bacteria and the presence of cells which are in a viable but non-culturable state, the organisms in culture represent a minor fraction (less than 1% of the bacterial species present) of the microorganisms occurring in situ. Truly, the statement that within rhizosphere there is a “black box” seems valid and there is a need to open the box if we intend to exploit the microbial deposits present therein.

PGPRs.

l Search of novel PGPR activities, either by functional screening of library clones or based on DNA sequence information.

l Documentation of new genes/enzymes from the rhizosphere metagenome. SignificantAchievements

l As the major part of works involve the use of metagenomic DNA, the fidelity of soil DNA extracted by using the Power Soil DNA Isolation Kit (MO BIO Laboratories, Inc., USA) was confirmed by performing PCR. It is evident from the bands appearing in the gel that the template used in PCR is very effective in the amplification of desired fragment of DNA (Fig. 1). A band of 200 bp (with GC clamp) was amplified from the DNA extracted from 12 soil samples collected from different locations. DGGE performed with this amplified fragment showed diversity in bacteria of different soil samples.

During the last five years we have accumulated vast information on culturable plant growth promoting bacteria from rice rhizosphere of Eastern UP and Western Bihar. These

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AMAAS - Annual Report 2015-16

l Amplification of sidA gene which plays key role in siderophore biosynthesis was also performed from the metagenomic DNA of different soil samples. Notably, amplification of sidA was not present in all the samples suggesting that all the bacteria present therein are not capable to produce siderophore. This is expected as culturable bacteria also showed siderophore production in about 35% isolates (Fig. 4).

Fig. 1: Effectiveness of soil metagenomic DNA in PCR. 200 bp DNA fragment (with GC clamp) was routinely amplified from DNA extracted from soil samples of Chandauli (Left) and Jaunpur districts (Right)

l Screening of plant growth promoting (PGP) traits such as IAA production, P solubilization and siderophore production of all the 62 strains isolated by using culturedependent approach has been completed. However, with a view to confirm the presence of PGP characters in both culturable and non-culturable bacteria, amplification of certain genes of PGP characters namely nifH and sidA was done by PCR using DNA extracted from soil of different rice fields. It is evident from the results of (Fig. 2) that metagenomic DNA of all the 10 soil samples of Chandauli district rice fields show amplification of a 396 bp amplicon of nifH. Similarly, amplification of nifH was achieved in seven samples out of 12 soil samples DNA tested in the study (Fig. 3). This clearly suggests that the occurrence of N2-fixing bacteria varies in different soil which may be due to the differences in physico-chemical properties of the soil as there is not much differences in agro-climatic conditions of both the districts. Selected amplicons of nifH are processed for sequencing so as to get better insight of diversity and richness of bacteria.

Fig. 4: Amplification of sidA gene from soil DNA of Jaunpur (A), and Chandauli (B) districts rice fields

l With a view to isolate novel metabolites/enzymes, two bacteria namely, Klebsiella pneumoniae and Alcaligenes spp. have been isolated which are capable to produce the skin pigment melanin. HPLC analysis of the partially purified melanin showed two prominent peaks corresponding to the standard degradation product of melanin in the chromatogram (Fig. 5).

Fig. 2: Amplification of nifH from DNA extracted from 10 soil samples of rice fields of Chandauli district. PCR conditions were kept identical for all the samples and equal amount of template DNAwas used in all the reactions

Fig. 5: HPLC chromatogram of melanin isolated from Alcaligenes spp. peaks obtained at 7 and 11 min denote degradation product of melanin

l Additionally, one of the genes, tyr (396 bp) involved in the biosynthesis of melanin has been successfully amplified which confirms the presence of the pigment in the bacteria (Fig. 6). Attempts are being made for large scale production and possible applications of melanin.

Fig. 3: Amplification of nifH from DNA extracted from 12 soil samples of rice fields of Jaunpur district. Other conditions as in Fig. 1.

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AMAAS - Annual Report 2015-16 Conclusion Effectiveness of metagenomic DNA extracted by using commercial kit has been proved by performing PCR. Amplification of 200 bp (with GC clamp) of 16S rDNA followed by DGGE suggested molecular diversity among bacteria of different soil samples. Presence of PGP genes namely nif H and sidA has been attained and results showed significant differences in distribution of both the genes in DNA of soil collected from different places. To certain extent data support the findings of culturable bacteria. Bacteria capable of melanin production have been isolated and the validity of melanin production has been confirmed by HPLC and PCR. Presence of tyr (396 bp) gene which is involved in melanin production has been reported in the test bacteria. Next generation sequencing of 16S rDNA amplified from DNA of three soil samples is under process.

Fig. 6: Amplification of tyr gene from Alcaligenes spp. (1) and Klebsiella pneumoniae (2). Band of 396 bp represents tyr gene

Microbial Diversity of Bio-Films in Dairy Niche PI : R.K. Malik Co-PIs : Chand Ram, S.K. Tomar, Surajit Mandal, Diwas Pradhan ICAR-National Dairy Research Institute, Karnal disinfectants and other antimicrobials as wellas its transfer mechanism in planktonic state and bio-films.

Rationale Presence of biofilms in dairy processing line pose a threat for food borne pathogens and spoilage microflora. Bio-films increases resistance of microbes towards commonly used antimicrobial to an extent of 10-1000 X as compared to their free floating counterparts. Biofilms also interfere with processing efficiencyand consume high energy to get the desired results. Molecular observations have indicated up- and down-regulation of many genes in biofilm forming microbes responsible for their development. The presence of diverse microbial communities also adds additional complexity to attachment & biofilms development. The mechanisms of increased resistance include delayed penetration of antimicrobial, altered growth rate of microbes in biofilm and physiological existence “persisters”. Hence, it is worth to investigate microbial diversity in dairy biofilms w.r.t. biochemical and molecular characterization for effective control of biofilms to avoid huge economic losses.

l Assessment of biofilm formation patterns and resistant profiles of selected isolates.

l Preservation of these cultures and submission to NBAIM. SignificantAchievements

l Atotal of 279 presumptive colonies picked up during 201415 from selective media were analyzed for microscopic, biochemical and molecular profiles. On the basis of colony morphology on Baired Parker agar and Gram's reaction, 46 isolates identified as S. aureus were sub-cultured to obtain pure cultures. Gram-stained smears of pure cultures under microscope showed arrangement of grape like bunches of gram +ve cocci.

l Amongst 46 presumptive S. aureus isolates, only 29 could ferment mannitol as indicated by change in color of mannitol salt agar whereas 17 colonies did not utilized mannitol and thus were negative. These 46 isolates were also tested for blood heamolysis patterns on agar media enriched with 5% (v/v) sheep blood, 6 and 23 isolates showed alpha and beta-heamolysis and rest 17 cultures were found non-haemolytic. These strains were also subjected to coagulase test, 23 strains each were observed to be coagulase +ve and -ve, respectively however all

Objectives

l Isolation of biofilm forming microbes from diversified dairy niche.

l Biochemical and molecular characterization of biofilm forming microbes and their molecular biodiversity.

l Resistant profiles of isolates towards biocides,

9

AMAAS - Annual Report 2015-16 showed presence of catalase. Biochemical observations of API staph kit revealed that 23, 17 & 6 isolates belong to S. aureus, S. epidermidis and S. capitis.

sequences– invAF (5'-GTGAAATTATCGCCACGTTC G G G C A A - 3 ' ) & i n v A R ( 5 ' TCATCGCACCGTCAAAGGAACC-3').

l Twenty three biochemically identified coagulase +ve S.

l Ten presumptive colonies of green metallic sheen of E. coli

aureus strains were also confirmed by PCR technique. The amplicon of 894bp was successfully amplified using species specific primer sequence - forward SAS2F (5'AGCGAGTCTGAATAGGGCGTTT-3') & reverse SASR (5'-CCCATCACAGCTCAGCCTTAAC-3') and genomic DNAof coagulase +ve S. aureus cultures.

confirmed by API test were also subjected to molecular confirmation, however, only 3 isolates could be amplified using primer sequences- forward (5'-AAAGCCGTGGC ACAGGCAAGCGT-3') and reverse (5'-TCAATTTG TTATCGCTATCCAGTTGG-3') with product size-348bp.

l The MIC and MBEC of selected strains of Bacillus,

l Three and six isolates of Enterococcus were confirmed as

Pseudomonas, E. coli, Listeria, Salmonella and Staphylococcus as well as their consortia against biocides were determined. The preliminary values for MIC and MBEC of selected strains against iodophore ranged from 20-70 ppm and 250-550 ppm, respectively

E. faecalis and E. faecium biochemically byAPI test kit.

l One presumptive colony of Listeria from PALCAM agar was identified as Listeria ivanovii byAPI Listeria Kit.

l Forty five presumptive colonies of gram +ve rods from

l During second phase of isolation, 354 more representative

PEMBA showed presence of endospore on spore staining as well as utilized glucose and mannitol anaerobically and thus were designated as Bacillus.

colonies from different selective agar media (Pseudomonas isolation, Eosin methylene blue, Klebsiella selective agar, Xylose Lysine Deoxycholate, PEMBA, PALCAM, MRS and Baired Parker agar) were purified and observed for microscopic and biochemical attributes. Microscopic examination revealed predominance of gram –ve rods (152) , whereas only 90 and 67 out of total 354 isolates were gram +ve rods and cocci, respectively. Out of 67 gram +ve cocci, 38 and 29 isolates showed their arrangement in grape like bunches and chains, respectively. Further work on identification, MIC & MBEC and biofilm formation capacity is in progress.

l The biochemical observations of API 20E kit of gram -ve rods revealed that 4, 11 and 12 isolates belonged to Citrobacter freundii, Enterobacter and Klebsiella pneumoniae, respectively. Klebsiella pneumoniae isolates exhibited capsule and catalase +ve reaction.

l Sixteen isolates of gram -ve rods belonged to Pseudomonas aeruginosa, which were catalase and oxidase +ve. Genomic DNA of 15 Pseudomonas cultures was amplified using primer sequences-PA-GS-F (5'-GACGGGTGAG TAATGCCTA-3') & PA-GS-R (5'-CACTGGTGTTCC TTCCTATA-3') having product size of 618bp.

Conclusion Observations on biochemical and molecular identification of dairy biofilm forming bacteria confirmed presence of diverse group of microflora, which may act as possible source of contamination of dairy foods and thus may pose a serious public health concern.

l Ten presumptive colonies of black color on XLD Salmonella agar were confirmed by API test kit, when analyzed for molecular confirmation, only three isolates were able to amplify product of 285bp using primer

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AMAAS - Annual Report 2015-16

Functional Characteriazation of Salt Tolerant Bacteria using Multi-Omic Approaches and their Exploitation forAlleviation of Salt Stress in Crop Plant PI : Kamlesh Kumar Meena Co-PIs : K. K. Krishnani, J. Rane, G. C. Wakchoure, P. S. Minhas ICAR-National Instutite ofAbiotic Stress Management, Malegaon, Baramati

l Seventy nine different bacterial morphotypes isolated from

Rationale

different plant parts viz., leaf (surface), root (surface and inside), stem (inside), root nodules and rhizosphere havesalt tolerance within the optimum range of 5-7% of NaCl (Fig.1).

The wild plants and weeds thrive even under worst environmental circumstances. The role of associated microflora is indespensible in the sustainance of host plant in such habitat. These microorganisms are capable to survive extreme environments; therefore they can recycle and solubilize the nutrients; produce plant growth promoting substances and even participate in stress alleviation of the host. Such resilient microbes could be exploited for growth promotion as well as stress mitigation in agricultural practices. It is therefore important to acquire the knowledge regarding the metabolic potential of such microorganisms and the probability of field-level implementation in agriculture.

Objectives

l To identify the whole composition/ total microbial community structure of halophytes associated microbiome.

l To isolate and characterize dominant group of halophytesroot associated microbes from different saline habitats of the country.

Fig. 1: Total no. of isolates v/s salt tolerance

l The isolates were functionally characterized for their plant

l To analyze the chemical composition of root exudates/

growth promotion ability. Almost all of the root nodulating population was siderophoregenic (Fig. 2), indicating the presence of active iron-chelation system to cope up with iron deficient situation.

rhizodeposition of the halophytes rhizosphere.

l To identify and validate the specific secondary metabolites produced by halophytes associated microbes under salinity.

l To Develop Crop Specific Microbial Consortium formulations for alleviation of salinity stress in crop plants. SignificantAchievements

l A total of 544 halotolerant bacterial isolates were obtained from the soil and weed samples collected from salinity affected regions of western Maharashtra. They were enriched selectively using the respective media. The isolates were grouped on the basis of their functional traits, which includes 49 exopolysaccharide producers; 101 nitrogen fixers (malate and mannitol utilizers); 82 Psolubilizers.

l Psoralea corylifolia L., a luxuriantly growing weed in the

Fig. 2: Quantitative estimation of siderophores

high-salinity area was targeted for the selective isolation of plant growth promoting halotolerant bacteria with the result of obtaining 40 leaf epiphytes; 52 root endophytes; 30 stem endophytes; 70 root nodulating bacteria and 120 rhizosphere-inhabiting bacteria.

l In vitro nitrogen fixation was more evident in root epiphytes as well as root endophytes. The nitrogen fixation was more predominant in root associated population. Similar was the case with exopolysaccharide production,

11

AMAAS - Annual Report 2015-16 influence the seedling development under salinity stress, exploring the possibility of using microbial community of wild origin in agriculture for enhancement of crop productivity (Fig. 5).

where root endophytes dominated.

l The rhizosphere isolates showed highest salt tolerance and

No. of Isolates

IAA production (Fig. 3). Presence of such plant growth promoting traits in the weed associated isolates underlines their agricultural importance and hence, a in-depth investigation needs to be done to look for the possibility of use of these isolates in routine agricultural practice for enhancement of productivity under diverse stress-prone environmental situations.

Fig. 5: Effect of culture extract on seed germination, seedling length and Vigour index

l The candidate isolates were subjected for molecular identification using 16S rRNA gene sequencing, that revealed the presence of multiple genera including Bacillus, Pantoea, Marinobacterium, Acinetobacter, Enterobacter, Pseudomonas, Sinorhizobium and Rhizobium. The sequences were deposited to DDBJ with accession numbers LC027447- LC027459 and LC128410.

Fig. 3: Plant growth promoting traits of the isolates

l The candidate isolates were also tested for the ability to utilize variety of carbon sources including different monosaccharides, polysaccharides, sugar-alcohols and amino acids using Biolog; this showed the metabolic flexibility of the isolates. The plant growth promotional ability of the candidate isolates was evaluated using the impact of culture extract of the isolates on seed germination and seedling growth of wheat crop under increased salinity. Significant enhancement in vigour index was noted in case of seeds grown in presence of organic metabolites extracted from these isolates (Fig. 4).

l Detailed biochemical and phylogenetic analysis of the isolates highlighted the novelty of some of the isolates, which were therefore subjected for further analysis. FAME analysis of promising novel species was also carried out; while further identification upto species level is underway.

l The impact of microbial metabolites on seed germination, root and shoot development was studied under salinity stress; where during germination, the seeds of wheat and sorghum were exposed to the culture filtrates rich in microbial metabolites. This indicated the role of microbial metabolites in enhancement of seed germination and sustainability under saline environment. UHPLC analyses of metabolites elaborated by potential halotolerant PGP isolates in response to the gradient of salt stress were performed. This revealed presence of diverse stress specific unknown metabolites in the culture extracts of the isolates (Fig. 6). These stress-responsive metabolites could have been playing some role in host-stress alleviation in saline environment; such culture extracts are therefore need to be keenly studied to reveal their exact role with respect to host the organism is associated with.

Fig. 4: Influence of cell free culture extract on seed gemrmination in Wheat

l The shoot-root development pattern and vigour index in wheat crop clearly highlighted the ability of the isolates to

12

AMAAS - Annual Report 2015-16 Book Chapter

l K. K. Krishnani, V. Kathiravan, K. K. Meena, B. Sarkar, Satish Kumar, M.P. Brahmane, Neeraj Kumar, B. P. Mohanty and M. Kailasam (2016). Bioremediation of aquatic toxicants: application of multi-omic approaches. Advances in Fish Research, Vol. VII, Pages 1–28 Chapter 20.

Fig. 6: UHPLC analysis of bacterial derived metabolites under different NaCl concentrations

Conference and workshop attended

l National conference on Inventions, innovations and

Conclusion

Regulations in Crop Sciences (IIRCS 2015) held at CSIRIICT, Hyderabad during June 24-25, 2015.

The wild plants and weeds thriving in the high salinity areas have consistently remained ignored in the view of harvesting the potential microbial population. One such weed, Psoralea corylifolia L, studied under this project so far, yielded quite a significant count of the associated halotolerant plant growth promoting bacterial isolates, highlighting the need to explore such entities for harvesting the novel microbial diversity. Such components from saline ecosystems may also provide the opportunity to elucidate the mechanism of microbe-mediated alleviation of salinity stress in plants.The isolates also have ability to alleviate salinity stress in agricultural crops including wheat.The isolates have potential to elaborate important biomolecules that have diverse utility in both agriculture and industry.

l Workshop on Fruit cracking and Soil Health Management held at ICAR-NRCP, Solapur on 3rd October, 2015.

l Krishnani KK, Meena KK, Sarkar B, Kumar Neeraj, Minhas PS. Biotechnology tools to enhance biodegradation of persistent organic compounds. 3rd International IUPAC Conference on Agrochemicals protecting crops, health and natural environment; New chemistries for phytomedicine and crop protection chemicals. Abstract No. IL-29, pp. 43

l Krishnani KK, Sarkar B, Meena KK, Maurya U. Prehnite trapped with nanosilver for alleviation of multiple stressors in aquaculture. Nano-2015.Abstract No. 989, Pp. 245.

Culturable and Unculturable Microbial Diversity of Insects and their Role in Insecticide Resistance and other FitnessAttributes PI : Mahesh S. Yandigeri Co-PIs : G. Sivakumar, R. Rangeshwaran, M. Mohan ICAR-National Bureau ofAgricultural Insect Resources, Bengaluru with these insects and their role in fitness attributes. Taking in to account the role played by microflora/endosymbionts in host fitness, this project was conceived to characterize the diversity of culturable and unculturable microflora of insects and assess their role in insecticide resistance/fitness attributes.

Rationale Insects harbour diverse microorganisms. Among the insects the different lines show different variability of symbionts. It is reported that symbionts play various roles in insects like a role in host plant use, reproduction, fecundity, resistance to abiotic stresses and as vectors in transmission of plant viruses. Most of the gut microbes of insects are unculturable and they are better studied through their genomes referred to as metagenomics. In India, the work accomplished on microbial flora of insects is less than a decade old and till date, meagre research works have been carried out to document the insect microbial diversity and their role in insect fitness attributes. Hence, the current investigation has been undertaken to determine the diversity of microflora associated

Objectives

l Diversity analysis of culturable and unculturable microflora of major insects across the different geographical locations.

l To identify the role of culturable and unculturable microflora for insecticide resistance mechanism and other fitness attributes of the major insects.

l To develop a metagenomic library of the insects especially insecticide resistance genes and their expression.

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AMAAS - Annual Report 2015-16

l Dilutions of insecticides were embedded in minimal

SignificantAchievements

l A total of 3 survey tours were accomplished in the current

medium with pour plate technique with imidocloprid 375, 750, 1500, 3000, 6000ppm and λ-cyhalothrin 5000, 10000, 20000, 40000 and 80000 ppm concentrations as final concentrations. Among 123 culturable bacteria of insects, isolates SPL 1-6, WFB 5a-1, WFB 4a-2, BM 3-1 and BM 34 were potent in degradation of both insecticides as evident from growth of microflora on plates. Further quantitative assays and identification of these strains is yet to be accomplished.

year and different types of insects were collected across different orders from various geographical locations in Karnataka, and Tamil Nadu. Insects were identified taxonomically using morphological keys and based on the host plants.

l A total of 123 microflora endosymbionts were isolated on various culture media like nutrient agar medium, yeast extract peptone dextrose medium and potato dextrose agar from surface sterilized insects. Isolated microflora were purified by subculturing using quadrant plate streaking method. The isolated microflora belonged to bacteria and none of them were fungi or yeasts. Biochemical characterization of microflora was accomplished for enzyme assays like protease, amylase, cellulase and ethanol production. A total of 55 bacteria were positive for protease, 25 for amylase, 17 for cellulase and 24 for ethanol production.

l Development of aposymbionts was initiated by rearing S. litura for many generations on artificial diet amended with antibiotics ampicillin and rifamycin in bioassay trays (Fig. 1). One generation of rearing has been completed successfully. Further rearing is under process for standardization of generations and concentration of antibiotics, till symbionts are completely eliminated.

l A total of 22 culturable bacteria from Aphis craccivora (cow pea aphid), cotton aphid, bean aphid, termmite and Erionotathrax (banana skipper) were subjected to molecular identification using 16S rRNA gene sequencing and homology search. GenBank accession numbers were obtained for identified sequences.

l Culturable microbial diversity from polyphagus pest Spodotera litura was assessed through 16S rRNA gene sequencing and identification. The bacteria were identified as Staphylococcus sciuri, Staphylococcus saprophyticus, Enterobacter cloacae, Bacillus jeotgali and Lysinibacillus macrolides. The nucleotide sequences of these organisms were deposited at Gen Bank and accession numbers KU867635 and KT818800-KT818805 were obtained.

l Identification of unculturable microflora was accomplished using metagenomic approaches. For identification of bacteria from metagenome amplicons of 790 bp were generated using universal primers 27F 5'AGAGGTTTGATCMTGGCTCAG3' and 806R 5'GGACTACHVGG-GTWTCTAAT3'. Further amplicons were cloned using pGEM-T vector and transformed in to E. coli XL1 blue cells. A total of 10 clones were subjected to sequencing and identification. NCBI-BLAST homology search revealed the clones as Gilliamellaapicola (2 clones),Gilliamella sp ., Enterococcus termitis, Enterococcus rotai, Enterococcus moraviensis, Parvimonasmicra, Erysipelato clostridium ramosum and Dysgonomonas sp.

Fig. 1: Development of aposymbionts of Spodoptera litura on bioassay plates with artificial media and antibiotics

l The tobacco caterpillar, Spodoptera litura is a major pest in India and causes severe economic loss on many crop plants. It attacks a wide variety of economically important crops such as tobacco, cotton, groundnut, castor, chilli, potato, soybean, cauliflower, cabbage, tomato, beans, sunflower and onion. Pyrethroids are among the most commonly used synthetic insecticides worldwide, accounting for more than 30% of global use. Cypermethrin is a synthetic, pyrethroid insecticide that is extremely effective against a wide range of insect pests including Spodoptera litura. The newer molecule flubendiamideis also recommended for control of S. litura.

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AMAAS - Annual Report 2015-16

l Bioassay experiment was initiated for S. litura using

logarithmic dose and 80-1280 ppm logarithmic dose for cypermethrin (25% EC). After drying vertically under shade, the discs were transferred to plastic containers and 10 freshly molted 2nd instar larvae were released in each container. Four such replications were maintained for each concentration and the number of concentrations varied from five to seven. Observations on larval mortality were recorded after 72 h and subjected to Probit analysis to estimate LC50, slope and fiducial limit values (Table 1).

flubendiamide and cypermethrin. Leaf dip assay IRAC method No.8 was adopted to test the insecticide resistance status of laboratory reared population. Larvae of S. litura originally collected from cabbage plants were reared under laboratory conditions on artificial diet. The freshly molted 2nd instar larvae were used in bioassays. Cabbage leaf disc (5cm diameter) were prepared and dipped in different concentrations of insecticides. The concentrations of flubendiamide (39.355 SC) used ranged from 0.5-16ppm

Table 1. Susceptibility status of Spodoptera litura to flubendiamide (39.355 SC) and cypermethrin (25% EC) insecticides (72 h bioassay data) Insecticide Flubendiamide Cypermethrin

LC50 (ppm)

Slope ±SE

1.9 636.0

2.73±0.31 2.06±0.29

Fiducial limits Lower Upper 1.59 2.31 503.6 851.8

l From the bioassay experiment S. litura was found resistant

χ (DF) 2

1.0 4.8

associated microflora to play in insecticide resistance in insects. Bioassay for S. litura revealed that S. litura was found resistant to cypermethrin as evident from LC50 values.

to cypermethrin as evident from LC50 values. The recommended field dose for cypermethrin against S. litura is 0.05%. However, S. litura is susceptible to flubendiamide at the recommended field dose. The bioassays are still under progress and will be evaluated for more field populations.

Paper published fromAMAAS work

l G. Sivakumar, R. Rangeshwaran, M. S. Yandigeri, M. Mohan, T. Venkatesan and Abraham Verghese (2016) Diversity of culturable gut bacteria associated with the field populations of cotton leafhopper (Amrasc abiguttula biguttula) in India. Indian Journal of Agricultural Sciences 86 : 208–15.

Conclusion A total of 29 culturable and 10 unculturable bacteria were identified up to species level using traditional and molecular approaches. Among 123 screened culturable bactrial isolates, 74 were positive for protease, 25 for amylase, 52 for cellulase and 32 for ethanol production. Isolates SPL 1-6, WFB 5a-1, WFB 4a-2, BM 3-1 and BM 3-4 were found potent in degradation of both insecticides. Further, the insecticide degradation assay will be confirmed through LCMS assay and establish insecticide degradation/utilization ability of

Conference and workshop attended

l Presented paper 'Diversity of microflora of aphids and their role in fitness' in UGC Sponsored & co-sponsored KSTA National Conference 'New Approaches and Concepts in Microbial Biotechnology' on 29-30 September, 2015 organized by Department of Microbiology, Maharani's Science College for Women, Bengaluru, Karnataka.

Molecular Characterization and Exploration of Rumen Microbial Diversity in Black Bengal Goat at North Eastern State of Tripura PI : Asis Bhattacharya Co-PIs : Bikas Chandra Debnath,Aparajita Das,Ankan De Department of Microbiology, College of Veterinary Sciences &Animal Husbandry, R.K. Nagar, West Tripura diversity of the black Bengal goats in the State of Tripura using molecular biological techniques. The data generated on molecular ecology of rumen microbiota are likely to provide better insight into the interaction between different enzymes among the microbes and their application of agriculture and allied industries.

Rationale The proposed project aims to study the rumen microbial ecosystem of black Bengal goats which are not fully explored and the majority of rumen microbes are unculturable. Hence there is a need to generate information on the microbial

15

AMAAS - Annual Report 2015-16

Objectives

SignificantAchievements

Whole metagenome sequencing and analysis of black Bengal goat rumen samples using illumine nextseq 500 platform. The following work will be done -

l Thirty Five (35) rumen samples were collected from black Bengal goats of different age (ranges from 4 months to 5 years) from slaughter houses from the Gomati and Sipahijala district, Tripura.

l DNAextraction from all the samples. l Library preparation of samples by bar coding of each

l A total of fifteen (15) rumen samples were submitted to the Bioserve Biotechnologies ( India) Pvt. Ltd. #3-1-135/1A, CNR Complex, Mallapur Main Road, R.R Dist., Hydrabad-500076, Telengana, India for the purpose of whole metagenome sequencing using Illumina Next seq 500 platform and analysis.

sample.

l Raw data generation of minimum 3 GB/ sample with an average read length of 2X 150bp PE on nextseq 500 illumina platform.

l Reads generated will be filtered using Bioinformatics for microbial diversity and functional analysis.

Microbial Metagenomic Analysis of Hot Springs Situated Atop the Himalayan Ranges at Manikaran, Himachal Pradesh, India PI : Rup Lal Co-PIs : Mallikarjun N. Shakarad Department of Zoology, University of Delhi, Delhi diversity cultured from hot spring water and microbial mat.

Rationale

l Community genomic analysis of hot spring microbial mat

The geological survey of India has identified 350 hot springs in India. Manikaran is one of them which lie in the Parvati Valley, 45 km from Kullu. With the advent of culture independent approaches the study of these hot water springs acquire special significance towards the thermophilic microbial communities, microbial diversity, production of thermostable enzymes and physicochemical condition. Additionally, cultivation dependent studies that are valuable for isolating novel organisms exploring their properties for important products can also supplement the cultureindependent findings. Among all the bacterial diversity isolated so far, strains belonging to the genus Thermus have been isolated from different thermal environments around the world. Apart from their applications in producing thermal enzymes during the past few years, efforts have been directed toward sequencing complete genomes of these eubacteria to understand how biochemical processes and bacterial life are sustained under high temperature. In this project we propose a culture independent approach to resolve the taxonomical and functional dynamics of the microbial communities assembled across hottest hot-springs of the country located atop (1700m) the Himalayan ranges.

and water samples e.g. taxonomical and functional analysis.

l Reconstruction of the rare biosphere, i.e. lesser abundant but highly diverse genotype of the ecosystem SignificantAchievements

l Genotypic, phenotypic, and chemotaxonomic characterization of two novel bacterial strains following the polyphasic approach.

l Lampropedia cohaerens : A biofilm forming, Gramnegative, aerobic, catalase positive but oxidase negative strain, designated CT6T was isolated from the microbial mats (~45ºC) of a hot spring, located atop the Himalayan ranges at Manikaran, Himachal Pradesh, India. Strain CT6T formed white coloured, smooth colonies with irregular margins. Transmission electron microscopy revealed coccoid, non-flagellated cells with wavy boundaries. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain CT6T belongs to the genus Lampropedia with sequence similarity value of 95.4% to the sole member of this genus, Lampropedia hyalina ATCC 11041T. Strain CT6T was found to have phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. The major cellular fatty acids were C16:0, summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C14:0, C19:0 ω8c cyclo and summed feature 3 (C16:1 ω7c

Objectives

l Physicochemical analysis of hot spring's water and microbial mat.

l Identification and characterization of novel microbial

16

AMAAS - Annual Report 2015-16 showed DNA-DNA similarities of 52.7 % with F. T nanhaiensis DSM 23009 , 50.7 % with F. phosphorivorans Ca7T, 34.8 % with F. barbaricus V2-BIII-A2T, and 38.0 % with F. arsenicus Con a/3T. In spite of the high 16S rRNA gene sequence similarities, the DNA-DNA hybridization and gyrB gene sequencing results (≤ 87%) supported by physiological and biochemical tests demonstrated that strain AS8T is a representative strain of a novel species for which the name Fictibacillus halophilus sp. nov. is proposed. The type strain is AS8T (= MCC 2765T = DSM 100124T = KCTC 33758T).

and/or C16:1 ω6c). The major respiratory quinone was ubiquinone-8. The major polyamines were putrescine, spermidine and the betaproteobacterial specific 2hydroxyputrescine. The DNA G+C content was 63.5%. Based on the genotypic, phenotypic, physiological and biochemical data, strain CT6T was considered to be a novel species of the genus Lampropedia, for which the name Lampropedia cohaerens sp. nov. is proposed (=DSM 100029T =KCTC 42939T =MCC 2711T).

l Fictibacillus halophilus : A Gram positive, motile, endospore forming, and moderately halophilic bacterium, designated as strain AS8T, was isolated from a microbial mat deposited at thermal discharges of Manikaran hot spring (with surface water temperature ~95°C) located in Himachal Pradesh, India. 16S rRNA gene sequence based phylogenetic analysis revealed that strain AS8T belonged to genus Fictibacillus with the highest sequence similarity to F. nanhaiensis JSM 082006 T (99.9 %) and F. phosphorivorans Ca7T (99.9 %), followed by F. barbaricus V2-BIII-A2T (99.1 %) and F. arsenicus DSM15822T (97.4 %). The polar lipids fraction consisted of diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The cell wall peptidoglycan was of the type A1γ based on directly cross-linked mesodiaminopimelic acid (Dpm). The DNA G+C content of strain AS8T was found to be 46.9%. The quinone system of strain AS8T consisted of MK-7 predominantly and the polyamine pattern primarily contained spermidine and spermine. The major cellular fatty acids in strain AS8T were iso-C15:0, anteiso-C15:0, and iso-C16:0. The strain

Conclusion Manikaran hot springs represent an unexplored ecological niche with specialized bacterial community including novel bacterial strains. These hot springs can also be explored for novel bacterial strains with biotechnological applications after characterization such as Fictibacillus and Lampropedia ecotypes. Paper published fromAMAAS work

l Tripathi et al., 2015. Lampropedia cohaerens sp. nov., a biofilm forming bacterium isolated from the microbial mats of a hot water spring, located atop the Himalayan ranges at Manikaran, India. International Journal of Systematic and Evolutionery Microbiology doi: 10.1099/ijsem.0.000853 Conference and workshop attended

l Workshop was conducted at Department of Zoology, University of Delhi on 14th-15th May, 2015 on “Renovating bacterial taxonomy using Bioinformatics”

Diversity of Arbuscular Mycorrhizal Fungi and Dark Septate Endophytes in Woody Species and Shifting Cultivation Crop of Manipur, North East India PI : R. R. Pandey Department of Life Sciences, Manipur University, Imphal of AMF in different woody species and shifting cultivation crops of Manipur, NE India.

Rationale Arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) are the essential components of sustainable plant root-soil system and play a crucial role in ecosystem functioning. Besides the beneficial roles played by these fungi in forestry and agro-ecosystems very little work has been done on related aspects in the North East (NE) India, in general and Manipur, in particular which is a part of Indo-Burma hot spot region. Therefore, the present project work will provide information on the composition of AM and DSE fungi, their root colonization patterns and the growth promoting efficiency

Objectives

l Assessment of AMF species compositions in rhizosphere soils of dominant woody tree species and shifting cultivation crops; Evaluation of AMF and DSE colonization patterns in the root samples; Analysis of physicochemical properties of collected soil samples.

l Maintenance of AMF spores in trap cultures using suitable host plants; Extraction of AMF spores from the pot soil and

17

AMAAS - Annual Report 2015-16 periods from Senapati District (Location: 25° 16′43.3′′–25° 16′ 45.3′′ N Latitude; 94° 01′ 11.1′′–94° 01′ 18.7′′ E Longitude), at elevations between 1122° and 1154 m a.s.l., and 62 km north of Imphal, the capital city of Manipur, India. Trap pot cultures were maintained. A total of 18 AM fungal spore morphotypes belonging to different g e n e r a v i z . A c a u l o s p o r a , C l a ro i d e o g l o m u s , Funneliformis, Glomus, Rhizophagus, Sclerocystis and Scutellospora were isolated from natural field (Fig. 1) and trap culture soils and identified to species level. Glomus was the dominant genus that was represented by 8 different species.

their pure culture establishment.

l Evaluation of efficient AMF species for their ability to improve seedling growth of important tree species and crops in nursery condition. SignificantAchievements

l Rhizosphere soil and root samples of several shifting cultivated vegetable and staple food crops i.e. cowpea (Vigna unguiculata), Naga King chilli (Capsicum chinense), tomato (Lycopersicon esculantum) and 4 indigenous rice (Oryza sativa) cultivars viz. scented black rice- Chakhaoamubi, Chakhao white, Phourelamuba and Phourelangouba were collected during different growth

Fig. 1: AMF spores isolated from rhizosphere soil. (a) Acaulospora sp.; (b) A. spinosa; (c) Claroideoglomus Claroideum; (d) Funneliformis geosporum; (e) Glomus aggregatum; (f) G. ambisporum; (g) G. glomerulatum; (h) G. goaensis; (i) G. macrocarpum; (j) G. magnicaule; (k) G. multicaule; (l) G. versiforme; (m) Rhizophagus fasciculatus; (n) R. intraradices; (0) Sclerocystis rubiformis; (p) S. taiwanensis; (q) Scutellospora auxillary cell.; (r) Scutellospora sp. (Scale Bars: 2040μm).

l All the examined test plant roots had dual association of

(Fig. 3). However, the root colonization patterns of the fungal structures varied with different test plant species. Extent of percentage root length with total AM colonization (% RLTC) was highest in C. chinense (94.8%), whereas maximum percentage of root length with total DSE colonization (% DSTC) was recorded in Phourelangouba rice cultivar.

both AM and DSE fungi. Arum-, Paris- and Intermediatetypes of AM morphology were observed in different crop plant species. AM fungal structures within the roots were characterized by hyphae, hyphal coils, arbuscules, arbusculate coils and vesicles (Fig. 2), while in case of DSE presence of darkly pigmented, septate hyphae and microsclerotia or moniliform structures were observed

18

AMAAS - Annual Report 2015-16 solubilizing bacteria (PSB) were evaluated in pot condition (Fig. 4). A total of 9 treatments were prepared (C- Control, T1- R. fasciculatus, T2- F. geosporum, T3- R. fasciculatus + Rhizobia, T4- R. fasciculatus + PSB, T5- F. geosporum+ Rhizobia, T6- F. geosporum+ PSB, T7- R. fasciculatus + F. geosporum + Rhizobia, T8- R. fasciculatus + F. geosporum + PSB and T9- R. fasciculatus + F. geosporum + Rhizobia + PSB). The findings of different treatments revealed significant enhancement in growth parameters viz. shoot length, root length, shoot dry weight and root dry weight of cowpea (Vigna unguiculata) as compared to controls. Maximum growth enhancement of cowpea was recorded with T9 in which both AMF species, Rhizobia and PSB were inoculated (Fig. 5).

Fig. 2: AM fungal colonizing structures (Appresorium, Arbuscules, Vesicle, Arbusculate coils and Hyphal coils) in roots of different crop plants.

Fig. 3: DSE fungal colonizing structures (Septate Hyphae, Moniliform structure and microsclerotia) in crop plants roots.

Fig. 5: Growth response of Vigna unguiculata after 90 days of inoculation with AMF species, rhizobia and phosphate solubilizing bacteria in nursery bag condition.

l Effects of inoculation of 2 dominant AMF species viz.

l A total of 9 DSE fungi were isolated from different test

Rhizophagus fasciculatus and Funneliformis geosporum alone and in combination with rhizobia and phosphate

plant roots and were characterized by molecular methods which are as follows i.e. Alternaria brassicae, Cladosporium cladosporiodes, C. tenuissimum, Epicoccum nigrum, Fusarium oxysporum, Phialophora mustea, Penicillium ochrochloron, P. sclerotiorum and Talaromyces verruculosus. Further works related with their morphological features are going on. The pure cultures of some DSE fungi and mycelial characters are presented in Fig. 6.

Fig. 4: Cowpea growth in nursery bags during inoculation experiment.

Fig. 6: Pure cultures of some isolated DSE fungi and mycelial characters.

19

AMAAS - Annual Report 2015-16 presented by K. Surendira Kumar, SRF-AMAAS Project, in Technical Session ′′Taxonomy of fungi, bacteria and viruses′′ has been awarded Third prize for the Best Poster presentation in 6th International Conference on ′′Plant, Pathogens and People′′, held during Feb. 23-27, 2016, at NASC Complex, New Delhi, India].

Conclusion Our findings suggest that this hot spot region harbours a diverse AMF community relative to other tropical and subtropical habitats. Presence of both AM and DSE fungal associations in the examined plant species indicate the wide occurrence of mycorrhizae in this region. Further, experimental studies are in progress to observe the role of dominant AMF species and other beneficial microbes for their suitability in enhancing the plant growth performances.

l K. Surendira Kumar, R.R. Pandey (2016). Arbuscular mycorrhizal and dark septate endophyte fungal associations in three indigenous rice (Orizasativa L.) cultivars of Manipur. In: National Symposium on ′′Microbial Diversity and its Impact′′ organized by Indian Mycological Society, University of Calcutta, Kolkata during February 18-19, 2016. p. 83.

Paper published fromAMAAS work

l R.R. Pandey, I. Chongtham, T. Muthukumar (2016). Influence of season and edaphic factors on endorhizal fungal associations in subtropical plantation forest trees of Northeastern India. Flora 222: 1–12. (Impact Factor-1.47).

l K. Surendira Kumar, I. Chongtham, R.R. Pandey (2015). Arbuscular mycorrhizal fungal associations in Vignaunguiculata(L.) Walp. from jhum field of Senapati District, Manipur. In: International Conference on Biotechnological Advances in Environmental Health and Biodiversity Conservation, organized by DBT sponsored State Biotechnology Hub, Department of Biochemistry, Manipur University, Imphal, during May 21-23, 2015, p. 69.

Conference and workshop attended

l K. Surendira Kumar, I. Chongtham, R.R. Pandey (2016). Arbuscular mycorrhizal and dark septate endophyte fungal association in three vegetable and staple crops of Manipur, Northeastern India. In: 6th International Conference on ′′Plant, Pathogens and People′′ organized by Indian Phytopathological Society, ICAR, IARI, New Delhi during February 23-27, 2016. pp. 216-217. [This Research Paper

Exploring Myxobacteria from the North Western Himalayas for Novel Enzymes and Secondary Metabolites PI : Meenu Katoch Co-PIs : BhahwalAli Shah Indian Institute of Integrative Medicine, CSIR Canal Road, Jammu Soce56 revealed the potential to produce more than the known compounds previously described from both strains.

Rationale Myxobacteria are obligate aerobic, chemotropic Gramnegative proteobacteria that most commonly inhabit the soil. The recent discovery of novel myxobacterial species from marine as well as moderate halophytic environments proves the ability of myxobacteria to adapt towards differing environmental conditions. The vegetative cells of myxobacteria are typically rod shaped, 3-12 μm in length and 0.7-1.2 μm in width. Many myxobacterial species secrete lytic enzymes which allow them to prey on other bacteria and yeasts and to digest the released proteins, lipids and nucleic acids.

In order to harness the microbial biodiversity for various industrial applications we would like to work on Myxobacteria keeping in context their potential for providing us with novel enzymes and secondary metabolites. This group of gram negative bacteria is largely unexplored especially in India. Our goal is to isolate myxobacteria form the soil and water samples collected from all over the north western Himalayas. The isolated strains will be characterized and screened for the production of novel enzymes of industrial importance like cellulases, proteases, lipases and amylases.

Myxobacteria are a rich source for secondary metabolites with interesting biological activities. Although several compounds have been isolated from different myxobacterial strains, genome analysis of the two model strains Myxobacteria xanthus DK 1622 and Sorangium cellulosum

Objectives

l Isolation of myxobacteria for diverse habitats of north western Himalayas.

20

AMAAS - Annual Report 2015-16

l Screening of myxobacteria for various industrial enzymes

l These cultures were screened for different enzymes viz

like cellulases, proteases, lipases and amylases.

cellulase, amylase, protease, glutenase. Two Myxococcus sp. were found significantly positive for cellulase and amylase enzymes while Myxococcus sp. 11556 were found significantly positive for glutenase enzyme. Byssoborax cruenta is found significantly positive for protease. Optimization and purification of glutenase enzyme is in process.

l Screening of myxobacteria for bioactive metabolites like antimicrobial activities, antioxidant activity and cytotoxic activity.

l Purification of the enzymes and bioactive metabolites from the respective strains.

l Cloning and expression of genes encoding enzymes of

l These cultures were screened for antimicrobial activity

interest and bioactive metabolites.

against Bacillus subtilis (MTCC No. 121), Pseudomonas aeruginosa (MTCC No. 424), Salmonella typhimurium (MTCC No. 98), Escherichia coli (MTCC No. 118), Klebsiella pneumonia (MTCC No.109), Staphylococcus aureus (MTCC No. 737). Few of cultures showed significant antimicrobial activity (Table 1).

SignificantAchievements

l Myxobacterial cultures were isolated from different zones of North Western Himalayas (Kashmir, Jammu, Himachal Pradesh) (Fig. 1)

l These cultures were screened for antioxidant activity also but none of them showed positive result with DPPH assay.

l Byssoborax cruenta was grown in bulk (28 L) and extract was prepared. Purification of the secondary metabolites from the respective strain is in progress.

l Polyketide synthase gene was amplified from Byssoborax cruenta genomic DNAand its sequencing is going on.

Fig. 1:Coloured fruiting bodies appearing on dung pellets

Table 1 : Some myxobacterial isolates showing antianicrobial activity against bacteria and fungi. Culture code 2a

Salmonella P. aeruginosa Typhimurium 50

1d ST/p/71

MIC-50

E. coli

B. subtilis

S. aureus

K. Pneumonia

50

-

-

MIC-50

MIC-100

MBC-100/ MIC-25 MBC-100/ MIC-25 MBC-100/ MIC-25 -

MBC-100/ MIC-25 MBC-100/ MIC-50 MBC-100/ MIC-50 -

MIC-100

MIC-25

MBC-100/ MIC 12.5 MIC-25

MBC-100/ MIC-50 MIC-100

MBC-100/ MIC-25 MIC-50

ST/sri/13(2)

-

MBC-100/ MIC-50 MIC-100

ST/sri/13(2b)

-

MIC-100

MIC-100

MIC-50

ST/sri/2(1)

-

-

MIC-100

-

21

Candida albicans MFC-100/ MIC-50 MFC-100/ MIC-50 MIC-50 -

AMAAS - Annual Report 2015-16

l Arushi Sharma, Project Fellow attended the conference

Conclusion

“New Vistas in Plant and Microbial Sciences” March 1112, 2016held at Jammu University, Jammu and presented a poster entitled“Isolation and phylogenetic characterization of myxobacteria from North Western Himalayas and their antimicrobial potential”.

Myxobacteria can be explored for bioactive metabolites and industrial enzymes. Conference and workshop attended

l National Workshop on “Relevance of Microscopy in Microbial Characterization” held at Amity University, Noida.

Exploration and Mass Culture of Freshwater Cyanobacteria for Development of Nutraceutical feed for Freshwater Fish Hatchlings PI : O.N. Tiwari Co-PIs : R.N. Mandal DBT- Institute of Bioresources and sustainable development (IBSD), Imphal conditions and enhancement of synthesis of high value products and intended to focus on the biodiversity of cyanobacteria in various environments, recent application and new developments that are diversifying the directions for commercial exploitation.

Rationale Cyanobacteria are source of a high number of chemicals of industrial interest, such as carotenoids and fatty acids among others, as well as biomass for feed (including aquaculture) and food (mainly nutraceuticals). Various methods have been adopted such as selection of the most adequate strains for the production; identifying the conditions that lead to maximal productivities; obtaining super-producing mutants by either classical mutagenesis or genetic engineering in the selected strains; and studying the corresponding metabolic pathways and their regulation. In order to maintain a culture successfully and to optimize the culture growth conditions, various environmental and nutritional parameters need to be taken into account. The most commonly studied parameters are light quality and quantity, pH, salinity, temperature, and macronutrients, mainly nitrogen (N) and phosphorus (P). Worldwide attention is drawn towards cyanobacteria for their possible use in aquaculture, food, feed, fuel, fertilizer, colourant, production of various secondary metabolites including vitamins, toxins, enzymes, pharmaceuticals, pharmacological probes and pollution abatement. Basic research is needed to identify new cyanobacterial strains of high value products, strain improvement using molecular tools for rapid growth rate, ability to withstand varied environmental

Objectives

l Screening and characterization of cyanobacteria available in germplasm of IBSD, Imphal for value added nutritional products and fatty acid composition and lipid profiling

l Process optimization for large scale biomass cultivation in indoor and outdoor natural freshwater bodies

l Technology development for value added nutraceutical feed for freshwater fish hatchlings SignificantAchievements

l Based on the earlier investigation, four (04) different cyanobacterial strains (Nostoc sp. BTA1075, Nostoc sp. BTA989, Phormidium sp. BTA975 and Calothrix sp. BTA1089) with high contents of chlorophyll-a, total carbohydrates, total soluble proteins and total carotenoids were selected for optimization for biomass production under different nutrients concentration (N, P and K), light quality (white, yellow, blue, green, red) and pH (6.0-9.0) for indoor as well as outdoor cultivation (Fig. 1).

22

AMAAS - Annual Report 2015-16

Fig. 2: Optimization of the strains under different light qualities

Fig. 3: Carotenoids production in different light quality

Fig. 1: Growth of cyanobacteria for biomass cultivation production

l The most efficient pH was found to be 8.0 for the

l Maximum production of chlorophyll-a was observed in

production of biomass (Fig. 4).

Nostoc sp. BTA1075 and a very good amount of carotenoid was produced by Nostoc sp. BTA989 in twice the normal concentration of NPK. Calothrix sp. BTA1089 yield high protein content and total carbohydrates production was maximum in Phormidium sp. BTA975 in half the normal concentration of NPK. However, comparing indoor and outdoor cultivation, the later was found to be more efficient (Table 1). Table- 1: Cyanobacteria showing maximum production of chlorophyll-a, total carbohydrates, total carotenoids and total soluble proteins in different NaNO3 concentration

Name of Estimation of the strains fine chemicals Nostoc sp. Chlorophyll-a BTA1075 Phormidium Total sp. BTA975 carbohydrates Nostoc sp. Carotenoids BTA989 Calothrix sp. Total soluble BTA1089 proteins

Values obtained (μg/ml) 37.42

Fig. 4: Biomass production in different pH range

l Fatty acid composition using FAME as standard by GCFID methods was done. Based on the chromatogram, the predominant and comparable amounts of fatty acids namely; pentadecanoic acid methyl ester- 59.49%, myristoleic acid methyl ester- 12.75% and methyl heptadecenoic acid methyl acid- 3.18% were found in Calothrix sp. BTA1089. One hundred (100) unialgal cyanobacterial strains belonging to 14 genera have been deposited to ICAR-NAIMCC, NBAIM, Mau.

Nitrogen concentration 2N

157.30

1/2N

85.22

2N

Conclusion

202.66

1/2N

Primary importance of successful cultivation and increased production of algae is the optimization of the culture conditions. Since natural conditions sometimes hinder the growth, optimization becomes an essential requirement in research for the enhancement of growth and production of fine chemicals from potentially important cyanobacterial strains which are of commercial benefits.

l Optimization under varying light quality (yellow, blue, green, red) showed diverse results. All four (04) potent strains showed maximum chlorophyll-a production, total soluble proteins, carotenoids and total carbohydrates yield in white light followed by yellow light (Fig. 2 & 3).

23

AMAAS - Annual Report 2015-16 evaluation of the genera Nostoc and Anabaena (Nostocales) from Loktak Lake. International Journal of Advanced Research, 3(5): 1597-1607.

Paper published fromAMAAS work

l Tiwari ON, Indira DW, Silvia Ch, Thadoi DA, Gunapati O, Avijeet SO, Ojit SK, Indrama Th, Subhalaxmi SA, RomiKh, Minerva Sh, Miranda L, Prasanna R (2015) Modulation of phycobiliprotein production in Nostoc muscorum through culture manipulation. Journal of Applied Biology & Biotechnology, 3(4): 11-16.

l Silvia Ch, Indira WD, Gunapati O, Avijeet SO, Ojit SK, IndramaTh, Subhalaxmi SA, RomiKh, Thadoi DA, Miranda L, Tiwari ON, Kalita MC (2015) Pigment production and growth metabolites amongst Nostoc strains of unexplored areas of Manipur, India falling under IndoBurma biodiversity hotspots. International Journal of Advanced Research, 3(10): 1752-1761.

l Tiwari ON, Ojit SK, Avijeet SO, IndramaTh, Gunapati O, Subhalaxmi SA, Silvia Ch, RomiKh and Sharma GD (2015) Isolation, morphological and biopigments

Reference and De novo Draft Genome Sequencing of PotentialAIMs and their FunctionalAnnotation PI : Alok K. Srivastava1 Co-PIs : Prem Lal Kashyap2, Hillol Chakdar1, K. Pandiyan1 1 ICAR National Bureau ofAgriculturally Important Microorganisms, Kushmaur Maunath Bhanjan 2 ICAR-Indian Institute of Wheat and Barley Research, Regional Station, Flowerdale, Shimla Rationale

Objectives

Genome sequencing has become an important tool for studying microbial properties, shedding light on phenotypes specific to different strains and environments as well as on evolutionary patterns. Recently, many microbial cultures have been sequenced across the globe and many more are coming from next generation sequencing. These data were providing a good source for deeper study into various aspects of microorganisms for their beneficial characteristics in agriculture including nitrogen fixation, phosphate solubilisation etc. Using computational and genomic information from a single genome is insufficient to provide insights into the life style and extended view of the gene pool of a species. Multiple genomes could enrich our understanding of the relatedness and variations in organisms. Comparative genome analysis of close relatives have yielded exciting insight into the sources of microbial genome variability with respect to gene content, gene order and evolution of genes with unknown functions. Microorganisms protect themselves from extreme environment through various mechanisms. Different enzymes and protein helps these microbes to cope with harsh environmental conditions. Heat/cold shock proteins and antifreeze proteins play a crucial role in microbe's survival at hot/low temperature. Microbes living in cold environments produce antifreeze proteins/ polypeptides (AFPs) to protect themselves from freezing damage. Antifreeze proteins have two main activities: thermal hysteresis and ice recrystallization inhibition activity.

l To produce high quality draft genome assembly of selected microorganisms such as phosphate solubilising Pseudomonas fluorescens and extremophilic microbes like Exiguobacterium and Penicilliopsis from India

l To perform assembly, annotation and bioprospecting for novel genes and alleles

l To conduct transcriptome analysis for the expression profiling SignificantAchievements

l A consensus phylogenetic tree topology was obtained for six Mesorhizobium species, M. austalicum WSM 2073, M. oppotunistum WSM2075, and M.ciceri biovar biserrulae WSM1271, clustered M. loti MAFF303099 and M. huakuii 7653R in a monophyletic clade with M. ciceri ca181 by Average Nucleotide Index (ANI) based pair-wise comparison and tetra correlation.

l The tree has clustered M. austalicum WSM 2073, M. oppotunistum WSM2075 and M.ciceri biovar biserrulae WSM1271 in a separate clade, showed close relationship among them. Furthermore, it clustered M. loti MAFF303099 and M. huakuii 7653R in a monophyletic clade with M. ciceri ca181, forming a close relationship (Figure 1A-1C).

l 16S rRNA phylogentic tree was constructed for the above six Mesorhizobium species and it showed that M. ciceri biovar biserrulae WSM1271, M. austalicum WSM 2073,

24

AMAAS - Annual Report 2015-16 found that the genes, nifS, nifX, nifN, nifE, nifK, nifD, nifH and nifB were found to be highly conserved during evolution. Overall, four hypothetical proteins were annotated during the analysis of nif family proteins.

M. huakuii 7653R and M. oppotunistum WSM2075 fall in same clade having close relation with M. ciceri ca181. The phylogenetic tree obtained by 16S rRNA classified strains differently than obtained byANI and tetra (Fig. 1 B).

l This could be attributed with the fact that the 16S rRNA phylogeny does not consider horizontal gene transfer. Further the size of sequence is negligible when compared to ANI method as it covers the whole genome. Moreover, since the whole genome based phylogeny is much more reliable than 16S rRNA based phylogeny, the phylogenetic tree by ANI and tetra is considered to depict the true relationship among them.

(A)

Fig. 2: Genome wide blast comparison of Mesorhizobium ciceri ca181 with related Mesorhizobium species. Higher color intensity represents higher percentage identity i.e., darker shades show higher sequence identity. Abbreviations- mcic: M. ciceri ca181, mcibi: M. ciceri biovar biserrulae WSM1271, mlot: M. loti MAFF303099, mhua: M. huakuii 7653R, maus: M. austalicum WSM 2073, mopp: M. oppotunistum WSM2075

l For evaluation of plant growth promoting efficiency of

(B)

Psudomonas korrensis (T-3) and cold tolerant bacterial isolate Exiguobacterium spp. (T-4), field experiment was conducted with tomato crop. After one month of experiment results showed that Pseudomonas and Exiguobacterium spp. enhanced the plant growth of tomato over un- inoculated control (Fig. 3 and 4).

(C) (C) Fig. 1: (A-C):Phylogenetic relationships of Mesorhizobium spp. (A) Phylogenetic tree obtained from Tetra (B) 16S rDNA and (C) ANI method based phylogeny.

l Genome-wide alignment of six Mesorhizobium species revealed that M. ciceri ca181 and M. ciceri biovar bissurale WSM1271 were quite similar to each other (Fig. 2).

l Nineteen nif cluster identified by Ortho MCL was analyzed

(a) (b) Fig. 3(a-b): Plant growth promoting efficiency of the selected strains: comparison between inoculated plant with uninoculated control

by Scan Prosite to detect the functional motifs. Fifteen nif genes (nifA, nifB, nifD, nifE, nifH, nifK, nifN, nifQ, nifR3, nifS, nifU, nifV, nifW, nifX, nifZ) were analyzed. It was

25

AMAAS - Annual Report 2015-16

l Pseudomonas isolates were screened for heavy metal tolerance and their correlation with siderophore production was studied. Out of 38 isolates, 27 showed positive siderophore production. Chemical assay of produced siderophore were also done. Out of 27 isolates, 13 isolates showed production of hydroxamate siderophore (Fig. 8). Fig. 4: Effect of bacterial inoculums on growth of tomato (Lycopersicon esculentum)

l The results of field experiments supported the data obtained previously by pot experiments.

l Antifreeze proteins/polypeptides (AFPs) are produced to protect themselves from freezing damage. Antifreeze proteins minimize freezing damage by inhibiting formation of large ice crystals. Screening of cultures was done on the basis of freezing of culture (broth) after different time interval. Out of eleven isolates, 4 isolates showed antifreeze activity. Quantification of positive culture was performed by different percentage (20%, 30% and 40%) of ammonium sulphate (Table 1). Table 1:Antifreeze culture of different cultures Culture B2P3 B2P6 HF5 B2P1

Antifreeze activity after 8 hours +ve +ve +ve +ve

Fig. 8: Bar chart showing siderophore production on the basis of halo zone formation and picture below dipicts hydroximate type sideophere production.

Total protein (ug/ul) NA 98 62 NA

l 27 siderophore producing isolates were screened for Cadmium and Nickel tolerance (0.1mM, 0.5mM, 1mM, 2.5mM,5mM,7.5mM,10mM). Out of 27 isolates, 25 isolates showed resistance on 10mM CdCl2 concentration and 10 isolates showed resistance on 10mM NiCl2 concentration.

l With an aim to develop a SCAR marker for Pseudomonas, 34 Pseudomonas isolates were characterized by RAPDPCR assay. Out of 10 primers used, one primer produced a monomorphic band of ~1Kb size. This monomorphic band has been cloned and being sequenced to develop SCAR marker (Fig. 6 and 7).

l To evaluate the use of bacteria as bioinoculants for promoting growth of maize, three Exiguobacterium strains Exiguobacterium indicum (NAIMCC-B-01146), Exiguobacterium sp. (NAIMCC-B-01168) and Exiguobacterium profundum (PHM 11) were used in pot experiment. Seeds of hybrid maize were used as host plant to evaluate the performance of Exiguobacterium. Bacterial cultures (109CFU ml-1) of each strain were used as bioinoculants.

1000

l The 60 days results of in vitro pot study showed that the co-

Fig. 6: Agarose gel electrophoresis of DNA amplified by RAPD (OPB-1)

inoculation of maize with strain Exiguobacteium sp. B01168 and E. profundum PHM11 significantly increase the plant shoot length (34.8 & 30.6%), root length (30.9 & 38.8%), shoot fresh weight (96 &131.2%), root fresh weight (157.6 & 118.6%), shoot dry weight (114.3 & 95.5%) and root dry weight (122.5 & 106.5%) over uninoculated control respectively (Fig. 9). Fig. 7: Gel electrophoresis image showing the PCR amplification of clone

26

AMAAS - Annual Report 2015-16

Fig. 9: Effects of inoculation with Exiguobacterium on shoot length (A), Root length (B), Shoot fresh weight (C), Root fresh weight (D) Shoot dry weight (E), Root dry weight (F) of maize plants grown under controlled conditions, 30 days and 60 days after sowing. C: uninoculated control, T1 (Exiguobacterium indicum-NAIMCC-B-01146), T2 (Exiguobacterium sp.NAIMCC-B-01168), T3 (Exiguobacterium profundum-NAIMCC-B-01146: inoculated. Data represent mean ± standard error of mean (SE). whole genome. Screening of cultures was done for antifreeze proteins/polypeptides (AFPs) on the basis of freezing of culture (broth) after different time interval. Out of eleven isolates, 4 isolates showed antifreeze activity. The evaluation of Exiguobacterium strains as bioinoculants for promoting growth in maize were used as bioinoculants. The coinoculation of maize with strain Exiguobacteium sp. B-01168 and E. profundum PHM11 significantly increased the plant growth on different parameters.

Conclusion A consensus phylogenetic tree was obtained for six Mesorhizobium species by Average Nucleotide Index (ANI) based pair-wise comparison, tetra correlation and 16S rRNA. The phylogenetic tree obtained by 16S rRNA classified strains differently than obtained by ANI and tetra. This could be attributed with the fact that the 16S rRNA phylogeny does not consider horizontal gene transfer. Further the size of sequence is negligible when compared to ANI method as it covers the

27

AMAAS - Annual Report 2015-16

Biomolecules and Industrially Important Enzymes from Extremophilic Bacteria PI : A.K. Saxena Co-PIs : Rajeev Kaushik ICAR- IndianAgricultural Research Institute, New Delhi different nutrient combinations was carried out by using Taguchi statistical method. For protease production, 27 nutrient combinations (medium) were generated using different nutrient ingredients. Autoclaved medium was inoculated with 1% inoculum and incubated at 55 ºC at 150 rpm for 72 hours. After incubation, microbial cells were separated through centrifugation at 10,000 rpm for 10 minutes and supernatant was collected. Enzyme assay was carried out using 0.6% casein solution and the results are presented in Fig. 1. Maximum protease activity of 507.32IU/mL was obtained in medium no. 21 using a nutrient combination of skim milk powder, maltose, beef extract, proline, (NH4)2SO4, KCl,AlCl3, and Tween 20.

Rationale Extremophiles are microbes that can live and reproduce in harsh environments. They are found in hot springs, volcanic areas, the deep sea, in the Antarctic biotopes and in other particular geothermal sites. The ability of these microorganisms to survive in such extreme conditions was strictly related to special features, which mainly consisted of novel enzymes and biochemical pathways. Microorganisms inhabiting extremes of temperature are capable of accumulating polymers as reserve source of food. In like manner to extremoenzymes, biopolymers are also gaining much more interest in industrial sectors worldwide. Microbes growing in extreme habitats under oligotrophic conditions could have genes responsible for hydrolyzing complex polysaccharides. Genes/alleles of biopolymers and hydrolytic enzymes can be used for developing genetically engineered organism capable of producing target compound at industrial level.

Objectives

l Characterization of microbes from extreme habitats capable of producing biomolecules and biopolymers of industrial importance.

Fig. 1: Production of protease in different nutrient combinations.

l Optimization of process parameters for pilot scale

l The relative activity of protease during optimization was

production of biomolecules and biopolymer.

calculated. Combination of different nutrients designated as Medium which exhibited maximum protease production among various medium was used as 100% relative activity.

l Mining of novel genes/alleles for industrially important biomolecules and biopolymers through cultural and metagenomic approach.

l Significant variation in PHB accumulation was observed

SignificantAchievements

among isolates over different period of time ranging from 0.20 to 29.50% on cell dry weight (CDW) basis. Maximum PHB accumulation was observed in isolate B11 (16.40%) followed by B15 (15.70%) and KB7 (11.58%) after 48 h of incubation. However, 9 isolates (1S1, 1S4, B4, B5, B16, B18, B22, B24, B28) produced PHB after 72 h of incubation. Among all the isolates producing PHB at 72 h of incubation, isolate B11 (29.5% PHB) and KB7 (23.80% PHB) produced significantly higher PHB than other isolates. With increase in time from 72 to 96 h significant reduction in PHB production by the isolates. Although amount of PHB significantly declined in isolate B11 and KB7 (18.90% and 17.60%, respectively) after 96 h but still the amount was significantly higher than other isolates (Table 1).

l Optimization of different nutrient combinations for efficient production of protease was carried out by employing thermotolerant bacteria MT-4. Following parameters like different carbon sources (glucose, sucrose, maltose), nitrogen sources (Yeast extract, peptone, beef extract), amino acids (asparagine, proline, glutamine), inorganic nitrogen source (ammonium sulphate, sodium nitrite, potassium nitrate), salts (NaCl, MgCl2, CaCl2), trace metal ions (CuCl2, AlCl3, MnCl2), substrate (Caseine, gelatin, skim milk powder), surfactants (Tween20, Tween80, Triton X-100) and pH (7.0, 8.0, 9.0) were selected by using the concentration of 1%, 0.1%, 0.05%, 0.1%, 0.5%, 0.005%, 1% and 0.1% respectively. pH of the medium was adjusted by adding 1N NaOH or 1N HCl. Optimization of

28

AMAAS - Annual Report 2015-16 Table 1: Quantitative estimation of PHB production by selected bacterial isolates at different time interval Isolate No. 48 h

PHB (%) 72 h 96 h

0.00

B10

0.00

0.00

B11

1.80 (±0.03) 1.40 (±0.01) 8.10 (±1.36) 3.10 (±0.07) 14.90 (±2.31) 3.70 (±0.05) 0.00

B13

18.60 (±2.35) 3.60 (±0.39) 5.10 (±0.69) 17.60 (±1.27) 0.00

9.20 (±0.21) 15.00 (±1.36) 8.00 (±1.59) 11.00 (±1.32) 17.60 (±2.39) 2.20 (±0.36) 1.50 (±0.08) 10.60 (±1.06) 13.80 (±1.09) 4.80 (±0.06) 10.60 (±1.51) 0.00

0.00

0.00

0.00

0.00

1S4

0.00

2S3

B1

4.00 (±0.51) 8.00 (±1.20) 5.00 (±0.98) 2.00 (±0.0.35) 11.58 (±0.3.67) 0.00

19.70 (±2.33) 15.30 (±1.69) 15.70 (±2.18) 12.90 (±2.19) 19.37 (±3.38) 15.30 (±1.38) 23.80 (±3.67) 0.00

B2

0.00

0.00

B3 B4

4.00 (±0.38) 0.00

B5

0.00

B6

11.00 (±2.69) 6.80 (±1.68) 3.00 (±0.34)

KB1 KB3 KB7

B7 B9

48 h

0.00

1S1

4S4

Isolate No. 120 h

B14 B15 B16 B17

4.80 (±0.89) 16.40 (±2.69) 15.70 (±1.97) 0.20 (±0.06) 10.30 (±2.19) 0.00

B18

9.80 (±2.36) 0.00

B22

0.00

6.90 (±0.91) 0.00

B24

0.00

B27

0.00

0.00

B28

0.00

3.40 (±0.29) 0.00

B29

0.20 (±0.06) 9.20 (±3.98)

B30

PHB (%) 72 h 96 h 11.30 (±2.68) 29.50 (±3.51) 12.70 (±1.03) 2.90 (±0.05) 11.40 (±0.67) 3.40 (±0.21) 8.40 (±0.83) 6.10 (±0.69) 2.90 (±0.06) 11.60 (±1.16) 0.00 11.30 (±0.62) 2.60 (±0.09) 10.10 (±1.11)

120 h

0.00

0.00

18.90 (±2.36) 5.40 (±0.53) 3.79 (±0.16) 0.00

13.43 (±1.61) 0.00

0.90 (±0.06) 1.10 (±0.08) 1.90 (±0.03) 16.40 (±1.06) 6.90 (±0.36) 19.50 (±2.59) 4.80 (±0.45) 3.26 (±0.07) 0.00

0.00

0.50 (±0.07) 0.00

0.30 (±0.09) 0.00 3.0 (±0.94) 0.00 12.89 (±3.64) 0.40 (±0.03) 0.00 0.00

LSD(p=0.05): Between isolates: 4.86 and between time: 4.32 (*Figures in parenthesis are standard deviation) enhanced production of enzymes by bacteria. PHB production at different time interval reveals that 72 hour time period of incubation is most favourable among most of the isolates. Based on quantitative estimation of PHB, B11 isolates showed the maximum PHB production (29.50%).

Conclusion Thermal springs represent important ecological niche for isolation of bacteria capable of producing enzymes active at 55oC. Optimization of nutrient conditions is important for

29

AMAAS - Annual Report 2015-16

Draft Genome Sequencing of P-solubilizing Pseudomonas striata PS27 and Functional Validation of MPS Genes PI : A.K. Saxena Co-PIs : Rajeev Kaushik, Geeta Singh ICAR- IndianAgricultural Research Institute, New Delhi

l Further functional characterization discovered 4 genes for

Rationale

Hsp, 5 for CSP and 28 genes coding for chaperons. It also resulted in 1654 hypothetical proteins, 8 having the function of antiporter and 13 responsible for symporter. 370 genes involved in transcriptional regulation. PS27 also has 13 genes (pstS, pstC, pstA, pstsB, phoU, phoR, phoB and phoH) responsible for phosphate transportation.

Pseudomonas striata PS27 is an efficient P bioinoculant widely used by the farmers and there is an urgent need to understand the genetic mechanism responsible for the Psolubilization. Pseudomonas striata is an efficient bacterium which has been tested on a variety of crop plants throughout India and found to be beneficial. It is capable of solubilizing 24-58.4% TCP and 5.26% MRP. To understand mechanisms responsible, it is essential to study the genomic and proteomics in totality. The genome sequencing and characterization of proteins is required for studying the complete mechanism of P solubilization.

l Fourteen different primers were designed for genes responsible for pqq (pqqB, pqqC, pqqD, pqqE) and gdh (glucose dehydrogenase-1) (gaby) genes and phosphate uptake regulators viz; pqq EDCB (Pyrrolquinone gene cluster), gltA2 (Citrate synthase), prpC (2-Methyl citrate synthase 1), prpC2 (2-Methyl citrate synthase 1), pitA1, pitA2 and pitA3 (Phosphate transporter) from data available in the online database. Primers were used for amplification and got the positive results for desired genes (Fig. 1&2). Further analysis of gene is in progress (cloning and sequencing).

Objectives

l Draft genome sequencing of efficient and standard phosphate solubilizing Pseudomonas striata PS27.

l Functional validation of genes responsible for P solubilization in P. striata PS27. SignificantAchievements

l A second approach was used for genome sequencing with 4th generation sequencing technology (SMRT, PacBio) which has an advantage to generate long read sequencing data. About 10 billion (1088622054) nucleotide bases were sequenced and assembled in four large contigs having 7666116 (7.66 Mb) bases through PacBio assembly tool Canu v1.2 (Table 1).

l Three contigs belong to three different chromosomes and fourth one has the similarity with plasmid of approximately 175 kb (175361 bases).

Fig. 1:Amplification of A) pqqC gene and B) pqqD gene and C) gabY gene in Pseudomonas striata PS27 and other P solubilizing bacteria using primers designed

l Annotation of assembled 4 contigs gave better results in comparison to previous annotations. Total predicted genes (7094) includes CDS:6969, tRNA:82, rRNA:18, Misc RNA:25, tmRNA:1, Pseudogenes: 86 and 2302 unique genes (Table 1). Table 1 : Total predicted genes in the genome of P. striala PS 27. Scaffolds Total number of bases CDS tRNA Unique genes Pseudogenes Total number of gene

269 8325967 (8.3 Mb) 7407 71 2209 77 7478

04 contigs 7666116 base (7.66 Mb) 6969 82 2302 86 7094

Fig. 2: (A). Amplification of pqq EDCB gene cluster in P. striata PS27 and other isolates, (B). Amplification of prpC gene in P. striata PS27 and other isolates. M1= 100bp DNA marker ladder, M= 1.0kb DNAmarker Ladder

30

AMAAS - Annual Report 2015-16 predicted genes is in progress. Primers used for amplification are validated and positive results have been obtained. Purification of the amplified gene products was carried out for cloning in expression vector.

Conclusion: Improvement of genome assembly and scaffolding yielded less number of scaffolds and gaps and help to achieve the circular chromosomal genome. Functional assignment of

Genomics and Proteomics of N2-fixing Cyanobacteria with Special Reference to Discovering Abiotic Stress Tolerant Novel Genes and Protein PI : L.C. Rai Department of Botany, Banaras Hindu University, Varanasi Rationale

SignificantAchievements

Cyanobacteria form a prominent component of microbial population in paddy fields, and bear the onslought of various biotic and abiotic stresses where salinity and desiccation constitute the major stresses. The Anabaena species are thought to be very useful in agriculture because of their (i) N2 fixation potential, (ii) extracellular polysaccharide, (iii) photo synthetic and carbon assimilation system, and (iv) high adaptive potential against abiotic stresses; all these together enrich the fertility of nutrient-poor soils. The adverse effects of abiotic stresses have been studied on a variety of cyanobacterial species, however, there is still a lacuna regarding the molecular mechanism underlying stress tolerance Little is known about the impact of salinity and drought on the proteome of cyanobacterial. Proteomics is a powerful tool to uncover cellular responses and acclimation strategies to stresses. The present work aims to analyse the proteome of Anabaena under salt and desiccation stress which is envisioned to bring into focus the proteins involved in stress management of cyanobacterial. some stress tolerance protein genes may be used in developing stress tolerance crop plants.

l Selected physiological and biochemical parameters such as ATP, NADP, catalase, SOD, lipid peroxidation have been estimated for Anabaena sp. PCC7120 under desiccation (Table 1). Eighty five differentially expressed proteins have been identified as a desiccation responsive protein from diazotrophic cyanobacterium Anabaena sp PCC 7120 (Fig. 1). A significant increase in superoxide anion level, SOD, APX and CAT except H2O2 upon rehydration was noted. Increased MDAand proline content supposedly aids in maintaining structural integrity during prolonged state of dehydration. Up-accumulated antioxidative defense proteins OR, Mn catalase, All1124, ahpC in cytosolic and sodA, sodB and GST in cytosolic proteome helps in mitigating oxidative damages during rewetting. Severe impairment in photosynthesis because of rapid decrease in antenna proteins and thylakoid membrane complexes including PSI, PSII and cytochrome b6f complex protein was observed during dehydration to reduce energy consumption. Other vital functions like nitrogen fixation, energy metabolism, amino acid and nucleic acid biosynthesis were also seemed halted during prolonged dehydration (Fig. 2). Further severe down accumulation of Alr1819, Alr2903, Alr3514, Alr2751 and All3324 throughout dehydration serves as the marker for dehydration stress. The dynamic changes of these proteins under dehydration might provide invaluable information toward understanding the underlying sophisticated cellular and molecular processes in stress response and tolerance. The results, thus showed a remarkable tendency of Anabaena PCC7120 to cope extreme dryness, by ceasing all of its metabolic activities but remaining intact and revive after rewetting. Further alr3904 a glyoxalase like gene from salt stress have been identified and characterized

Objectives

l Determination of LC50 values of NaCl and maximum desiccation level for Anabaena sp. PCC 7120 as reflected through physiological and biochemical variables.

l Stress induced changes in proteome of Anabaena sp. PCC 7120 using SDS-PAGE and 2D-PAGE.

l MALDI TOF MS/MS analysis of selected up and downregulated proteins using PD Quest software.

l Cloning of stress responsive novel protein genes and assessment of their stress acclimation potential by heterologous expression in E. coli.

31

AMAAS - Annual Report 2015-16 from Anabaena sp PCC 7120 by using in silico and wet lab approaches such as cloning and heterologous expression in E. coli. Furthermore, one more salt stress responsive

hypothetical gene alr2321 and two novel desiccation stress responsive gene (alr0750 and all1122) has been identified for characterization (Fig. 3).

Table 1: Physiological variables of Anabaena sp. under desiccation treatment Time in hours

PS-I activity (μmol O consumed μg protein h ) 19.56±0.08 10.37±0.02* (-1.89) 9.84±0.9* (-1.99) 6.64±0.06* (-2.94) 2

”1

0 Hr 0.5 Hr 6 Hr 24 Hr

”1

PS-II activity (μmol O evolved μg protein h ) 20.85±0.11 10.37±0.14* (-2.01) 9.99±0.05* (-2.08) 8.70±0.02* (-2.39) 2

”1

”1

Respiration (μM O consumed mg protein min ) 3.64±0.03 3.0±0.16* (-1.21) 2.49±0.11* (-1.46) 2.4±0.26* (-1.52) 2

”1

”1

NADPH (mM NADPH mg protein”1) 0.47±0.3 0.69 ±0.23* (+1.47) 0.84±0.13* (+1.79) 0.71±0.33* (+1.03)

Fig. 1: Protein profile of Anabaena at different time point under dehydrated condition

Fig. 2: Distribution of identified proteins under functional categories

32

ATPcontent (ngATPmg protein ) 23.62±0.06 20.01±0.2* (-1.18) 18.99±0.15* (-1.24) 5.59±0.11* (-4.22) ”1

AMAAS - Annual Report 2015-16

Fig 3: Cloning and expression of salt responsive gene alr3904 in E. coli (argG, DHAD, glnA, PGDH) and nucleic acid ( DHO, PurH, NadA, inorganic pyrophosphates, Adk) biosynthesis, cellular intermediary metabolism (nifH) were exclusively identified. The results showed that down accumulation of proteins were more pronounced during prolonged dehydration while up accumulation in late rehydration. Furthermore, study of the subset of proteins provides novel insight highlighting their role in stress tolerance and management.

Conclusion Measurement of alteration in physiological and biochemicals parameters under salt and desiccation stress of Anabaena at LC50 value at different time points followed by 2D gel analysis make it possible to identified stress tolerant genes for salinity and drought and inserted into the target organisms for enduring stress tolerance in plants using genetic engineering. Proteome based identification of some novel genes followed by their cloning expression, structural and functional characterization may provides a new insight to our understanding of abiotic stress tolerance. Creating transgenic cyanobacterial using these genes can open further possibilities of their use as biofertilizer. Present work provides a comprehensive analysis of cytosolic proteins that is envisioned to identify novel proteins in Anabaena sp. strain PCC7120 which may be key players in transport, signaling and other processes during desiccation/ rehydration process. Results at physiological, biochemical and proteomic levels demonstrated that dehydration is a quiescent state with low metabolic activities while upon rehydration, sudden resumption of metabolic activities will be observed. In cytosolic fraction proteins e.g. energy metabolism (6PGD, Fbp, Pgi, pdhE1-B), cellular process (Tig, GroEL, ClpP, ClpB, DnaK) amino acid

Paper published fromAMAAS work

l S. Sen , C. Agrawal , Y. Mishra , S. Rai , A. Chatterjee , S. Yadav , S. Singh, L.C. Rai. 2015. Exploring the membrane proteome of the diazotropic cyanobacterium Anabaena PCC7120 through gel-based proteomics and in silico approaches. Journal of Proteomics 127 : 161–168 Book Chapter

l A. Chatterjee, A.K. Shrivastava, S. Sen, S. Rai, S. Yadav and L.C. Rai, 2016. UVR8 signaling, mechanism and integration with other pathways, Wiley, UK, ( in press)

l Chatterjee, S. Yadav, S. Rai, P. K. Singh, A. K. Shrivastav, S. Sen and L.C. Rai 2016. Biodiversity and Agriculture, Elsevier, USA(in press).

33

AMAAS - Annual Report 2015-16

Exploiting the Diversity of Extreme Halophiles by Functional and Comparative Genomics for Isolating Novel Genes andAlleles forAffording Salinity Tolerance to Crop Plants PI : K. K. Pal Co-PIs : Rinku Dey ICAR-Directorate of Groundnut Research, Junagadh Extreme halophilic Haloarcula species would be the candidate for exploration to identify the possible sources of suitable candidate genes that can impart tolerance to osmotic stress, considering the existence and evolution of this group of primitive organisms billions of years ago by which these can survive in extreme conditions only. Generation of such information would be indispensable to our ability to engineer crop plant with more effective stress tolerance phenotypes to meet the demand of 21st century agriculture.

Rationale The genomics and proteomics of the extreme halophilic archaea, bacilli and fungi are still in their infancy. As these organisms can tolerate differential level of NaCl (upto saturated NaCl), these will be the most ideal candidates for exploration. Understanding the biochemical and molecular bases of extreme osmotolerance in these organisms would lead to identification of possible mechanisms of osmotolerance. Besides role of transporters; novelty in osmolytes, if any; roles of inorganic anions; possible osmotolerant genes; etc., extreme enzymes of industrial importance, etc. are proposed to be studied in details for identifying candidate gene(s) of interest for developing salinity tolerant transgenic crop in future.

Objectives

l To understand the biochemical and molecular bases of osmoadaptation and osmoregulatory mechanisms of selected extreme halophilic bacilli, archaea and fungi on evolutionary perspective.

To decipher the physiological, biochemical and molecular basis of osmotolerance in relatively unexplored microorganism like Haloarcula species, bacilli and fungi, multiple strategies need to be employed. As salinity tolerance is a polygenic trait it is assumed that different genes are linked to different physiological functions leading to expression of different genes related to osmotolerance. Thus, it would be of great significance to precisely define roles of each component of the traits responsible for expression of salt tolerant phenotypes.

l To identify candidate gene(s) having relevance to salinity tolerance for future exploitation in development of crops tolerant to salinity. SignificantAchievements

l To understand the mechanisms of osmotolerance on evolutionary perspectives and isolation of relevant genes, genomes of extreme halophilic bacilli, archaea and fungus were sequenced at draft level and annotated (Fig. 1 & Table 1). This includes Bacillus sp. MSP5.4 (40 contigs), Bacillus sp. MSP13 (13), Halomonas caseinilyticus WD26 (63 contigs), Haloarcula salariaH5-DGR (195 contigs)), Netrinema altunense1A4-DGR (215 contigs) and extreme halophilic fungus, Aspergillus sp. WD1 (11 scaffolds).

Functional and comparative genomics, proteomics, transcriptomics and systematic inactivation of the targeted genes by developing and integrating gene specific inactivation vectors would be useful in understanding the phenomenon.

Table 1 - Special features of draft genome of Bacillus and archaebacteria Features Length (bp) RNA CDS Hypothetical proteins G+C (%) Stress responsive genes NaCl tolerance

MSP5.4 3878495 82 4533 1239 45.89 90 ~15%

MSP13 4344864 87 4065 1409 43.87 104 ~20%

WD26 3464059 62 4768 1340 58.64 192 8-30%

34

H5-DGR 4091570 51 4460 2117 65.3 40 8-35%

1A4-DGR 3718350 50 4507 2482 68.1 65 10-35%

AMAAS - Annual Report 2015-16 validation of the expression of key enzymes of the serine glyoxalate cycle are underway to validate the finding. This is different from reported involvement of methyl aspartyl pathway in imparting osmotolerance in dead sea archaea which is missing in archaea of salt pan of Rann of Kutch.

l Orthologs of methylglyoxalate reductase, methyl malonate Haloarculasalaria H5-DGR

semialdehyde dehydrogenase, methylmalonyl-CoA epimerase and methylmalonyl-CoA mutase of serineglyoxalate pathway of haloarchaeon 3A1-DGR have been found in other extreme halophilic bacteria of Rann of Kutch. Real time analysis is going on to ascertain the expression patterns in archaea and bacteria in different osmolarity and their variability and gene flow across archaea, bacteria and fungi of the Rann of Kutch.

Halomonascesinilyticus WD26

Conclusion Bacillus sp. MSP5.4

Five genomes of extreme halophilic bacilli and archaea and that one fungus were sequenced to understand the mechanisms of Osmotolerance in evolutionary perspective. Comparative genome analysed indicated the involvement of serine glyoxalate cycle in imparting osmotolerance, carbon assimilation and anabolism in archaea of the Rann of Kutch which is different from that reported in Dead Sea archaea.

Bacillus sp. MSP13

Fig. 1: Draft genome of halophilic archaea and bacilli

l To ascertain the probable role of serine, leucine and isoleucine in imparting osmotolerance in extreme haloarchaeon, three genes from Halofer axvolcanii H1DGR (glycerol kinase, 2-oxo-methyl valerate dehydrogenase, hypothetical protein) were cloned to expression vector.

Conference and workshop attended

l Pal, K. K., Dey, R., Patel, M. B., Thomas, M. and Sherathia, D. N. (2016). Insight into the genome sequence of haloarchaeon sp. 3A1-DGR for understanding the mechanisms of extreme salinity tolerance. In 56th Annual Conference of AMI & International Symposium on “Emerging Discoveries in Microbiology”, December 7-10, 2015 at Jahawarlal Nehru University, New Delhi, India. Abstract No.AMP 98.

l By comparative genome analysis utilizing complete genome sequence data of haloarchaeon 3A1-DGR with the existing genomes of archaea, serine glyoxalate cycle has been identified as likely candidate in imparting osmotolerance, carbon assimilation and anabolism in extreme haloarchaea of the Rann of Kutch. Real time

Isolation and Selection of Naturally Occurring Cyanobacterial Strain and their Potential use in Biodegradation of Agricultural Pesticides (Organophosphate) PI : A.K. Mishra1 Co-PIs : Alok K. Srivastava2 1 Centre ofAdvance Study in Botany, Banaras Hindu University, Varanasi 2 ICAR-National Bureau ofAgriculturally Important Microorganisms, Maunath Bhanjan birds, reptiles and mammals including humans. In India, 76% of the total used pesticides are actually insecticide and around 20% pesticides are used particularly in the rice fields. Only 0.1% of total insecticide applied in the fields targets the insects and 99.9 % part remains and seeps in the environment which affects the other non-target organisms. Although, the vigorous use of OPs improves the quantity of agricultural products but it potentially affects their quality. Pesticides enter the human diet

Rationale Most of the synthetic insecticides used in the household system and agricultural fields, are organophosphate compounds (OP). OP compounds constitute nearly 34% of total pesticide produced worldwide at industrial scale. Due to their regular use OP compounds contaminate food and different types of ecosystems throughout the world which finally impose threat to the non target organisms like insect,

35

AMAAS - Annual Report 2015-16 and cause a number of chronic degenerative diseases including human cancers. OP. poisoning is a worldwide health problem because around 3 million poisonings and 3,00,000 deaths occur per year due to OP pesticides. Microbial degradation decides the fate of OP pesticides in soil and water. Many microbes (bacteria and fungi) having the potential to mineralize OPs have been isolated and characterised from soil but majority of them, due to their heterotrophic nature, require additional and specific nutrients for growth. Thus, it is difficult to maintain such microbes in natural conditions. In addition to these heterotrophic organisms, cyanobacteria are also known to degrade OP compounds up to a certain extent. Since cyanobacteria are autotrophic organisms and widely distributed in all the possible realms of the earth including highly polluted ecosystems, they can be used as alternative of those heterotrophic organisms in the removal of pesticides. However, majority of cyanobacteria fix atmospheric nitrogen, solubilise phosphorus and produce plant growth promoting hormones. Therefore, their application in the agricultural fields will not only help remediate pesticide pollution but will also enhance the nutrient dynamics and crop production.

Fig. 1: Removal of MP by cyanobacterial biomass with respect to time

Biosorption is a metabolically independent, passive, complex-chemical process which includes absorption, adsorption, surface complexation, ion exchange and precipitation. To understand the kinetics of removal of MP from the medium by cyanobacterium, two commonly used biosorption kinetics models i.e. the pseudo first order (equation 1) and pseudo second order (equation 2) were used.

Objectives

log(q e - q t ) = logq e -

l Assessment of microbial dynamics of pesticidecontaminated sites using a metagenomic approach.

K1t 2.303

t 1 1 = + t 2 q t K 2q e qt

l Isolation of naturally occurring cyanobacterial species with enhanced level of biodegradation and/ or removal capabilities of pesticides.

.................1

.................2

Where qe is sorption capacity at apparent equilibrium, K1 is langergren rate constant, K2 is a rate constant of pseudosecond order adsorption and qt is the concentration of pesticide at time t. The values of K1 and qe were determined from the slope and intercept of plot between log (qe-qt) and time (t) (Fig. 2) using equation (1). There was a large difference in the value of qe (exp) (obtained from pseudo first

l Proteomic and transcriptomic sequencing to identify gene(s)/enzymes involved in degradation of pesticide.

l Application of screened cyanobacterial strains in field having high amount of pesticide for their potential degradation/ field application and evaluation of their pesticide biodegradibility. SignificantAchievements

l Among the cyanobacterial isolates, a branched filamentous and heterocystous strain identified as Fischerella sp. and was found to be most efficient in degrading organophosphorus pesticide methyl parathion (MP). The optimum growth of cyanobacterium in terms of chlorophyll was observed at 20 mg/L of MP. Therefore, at this concentration removal of MP from the medium by the cyanobacterium was measured using HPLC (Waters, USA). Removal of methyl parathion from the medium inoculated with cyanobacterium was initially rapid and approached equilibrium after 24 hours of incubation (Fig. 1). The Pattern of rapid removal of MP during initial 24 hours was quite similar to that of the biosorption process.

Fig. 2: Regression Plot between log (qe-qt) and t.

36

AMAAS - Annual Report 2015-16 compared and analyzed using PDQuest software (Bio-Rad Laboratories USA) (Fig. 4). In the 2D gels of the cyanobacterium, 439, 339, 262 and 281 spots were detected under +P, –P, +PMP and –PMP conditions respectively. A total of 34 differentially expressed spots were identified by MALDI-TOF MS/MS analysis which

order kinetics model) and qe (cal) (amount of pesticide removed at the apparent equilibrium) (table 1), whereas the values of qe (exp) obtained from slope and intercept of plot t/qe versus t (Fig. 3) using pseudo-second order adsorption kinetics equation (2) was almost equal to the qe (cal). The

Fig. 3: Regression Plot between log t/qt and t.

value of correlation coefficient (R2) obtained from pseudo second order kinetics was also higher than that obtained from pseudo first order kinetics model (Table 1). Thus it is apparent from this result that the pseudo second order kinetics model is more appropriate to describe the process of biosorption of MP by cyanobacterium in comparison to pseudo first order kinetics model.

Fig. 4: 2D gel images of cyanobacterial proteins in four condition +P (supplemented with phosphate as K2HPO4), –P (phosphate deficient), –PMP (supplemented with MP in absence phosphate source) and +PMP (supplemented with methyl parathion in presence of P)

Table 1: Parameters of biosorption kinetics. Kinetic Parameters

Pseudo First Order -1

Pseudo Second Order

qe (calcu) (mg g cyanobacterial wt)

27.14

27.14

qe (exp) (mg g-1 cyanobacterial wt) K1

21.08

27.77

0.310

___

K2

___

0.04

0.930

0.998

R

2

were of different functional categories such as proteins related to photosynthesis, carbon metabolism, translation, amino acid metabolism, cellular processes and hypothetical proteins. Under phosphate limiting condition, expression of β subunits of FoF1 ATP synthase was observed to be up regulated by almost two folds whereas proteins related to carbon metabolism and photosynthesis showed the down regulation (Table 2). Treatment of methyl parathion in presence of inorganic phosphate also showed the down regulation of proteins involved in photosynthesis and carbon metabolism along with the FoF1 ATP synthase subunits. Results obtained from 2D analysis showed that phosphate deficiency affects both photosynthesis and energy metabolism which is appeared to be the possible reason of reduction of cyanobacterial growth.

l To know the mechanisms by which the cyanobacterial isolate Fischerella sp. modulated its metabolic processes in response to 20mg/L of MP, protein expression pattern was evaluated through 2D PAGE analysis. Protein spots appeared on the 2D gels of control (+P), phosphate deficient (–P) and MP treated cyanobacterium were

37

AMAAS - Annual Report 2015-16 Table 2: List of identified spots by MALDI-TOF MS/MS analysis and fold change in expression Spot change No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Protein annotation

F0F1 ATP synthase subunit alpha glutamine synthetase fructose 1,6-bisphosphatase peptidase S8 peptidase S8 F0F1 ATP synthase subunit beta F0F1 ATP synthase subunit beta elongation factor Tu hypothetical protein N9414_14278 hypothetical protein hypothetical protein cysteine synthase lectin ESA-2 ribulosebisophosphatecarboxylase fructose-bisphosphatealdolase ferredoxin-NADP reductase ferredoxin-NADP+ oxidoreductase hypothetical protein hypothetical protein superoxide dismutase c-phycocyanin alpha subunit phycocyanin hypothetical protein hypothetical protein histidinol dehydrogenase, partial thioredoxin hypothetical protein hypothetical protein photosystem I subunit VII hypothetical protein 2OG-Fe(II)oxygenase family oxidoreductase196 glyceraldehyde-3-phosphate dehydrogenase 36 molecular chaperone DnaK DSBA oxidoreductase

Score (Da) 340 427 273 172 463 594 507 130 106 91 40 49 220 145 192 107 50 123 37 95 74 286 120 42 36 91 52 66 259 107 35750 37637 169 119

l Amendment of MP in phosphate deficient media (–P)

Fold

Protein mass -P

+PMP

-P MP

54527 0.5 53259 0.2 37498 0.8 62181 2.0 62181 1.1 51807 2.1 51807 0.6 44740 0.5 41026 0.35 41026 1.0 33138 0.8 34235 28055 1.4 53547 0.5 39253 0.5 47808 0.3 48584 0.2 24777 0.5 16853 0.5 22514 0.6 5864 0.3 17421 0.5 17437 14423 9237 1.1 11752 1.3 8188 1.6 7707 0.6 9408 0.8 17398 0.6 unique to MP treated condition 68148 2.04 29243 -

0.1 0.5 1.2 1.3 0.5 70.5 1.1 1.8 1.0 0.8 1.4 0.5 0.4 0.4 1.2 1.4 1.3 0.2 0.3 1.9 0.6 1.3 -

0.6 1.2 1.3 0.5 0.5 1.4 1.8 1.1 1.1 3.4 0.6 0.6 0.8 1.5 1.6 2.8 1.3 0.3 0.1 0.2 1.4 -

1.7 -

0.8 -

0.7 -

oxidative response system). So it can be assumed that the phosphate released after biodegradation of MP was consumed by the cyanobacterium which further boosted the metabolic processes and supported the growth of the cyanobacterium in absence of inorganic phosphate source (K2HPO4). Out of 34 selected protein spots, 10 spots were of hypothetical proteins. Hypothetical protein represented by spot number 10 was quite interesting because of the presence of two conserved domains (i) Lamin tail domain

supported the cyanobacterial metabolic processes by up regulating the key metabolic enzymes such as ferredoxinNADP+ oxidoreductase (helps in generation of NADPH during photosynthesis which is used as reducing agent during Calvin cycle), peptidase S8 (modify precursor proteins by proteiolytic action), translational elongation factor Tu, cysteine synthase (cysteine synthesis), superoxide dismutase and thioredoxin (part of anti

38

AMAAS - Annual Report 2015-16 deficiency affects the photosynthetic efficiency and carbon metabolism by down regulating the proteins related to respective metabolic processes, (iv) phosphate released during the degradation of MP supports the metabolic processes under phosphate deficient condition and leads to the up regulation of some key enzymes and (v) the hypothetical protein with Lamin tail domain and Ricin-type beta-trefoil domain may have some role in pesticide degradation.

and (ii) Ricin-type beta-trefoil domain. Lamin tail domain has an immunoglobulin (Ig) fold which is generally found in several bacterial proteins: especially in membrane associated hydrolases and calcineurin-like proteins belonging to the metallo-beta-lactamase and phosphoesterase superfamilies respectively. Whereas Ricin-type beta-trefoil domain is a carbohydrate-binding domain which is found in a variety of molecules serving as enzymes, detoxification agents and signal transducers. Hence, it can be predicted that this protein may have some role in the degradation of MP.

Conference and workshop attended

l National Conference on Cryptogam Research in India: Progress and Prospect. (28-29 September, 2015, Indian Lichenological society and CSIR- National Botanical Research Institute, Lucknow, (India)

Conclusion Based on the obtained results following conclusions are made : (i) the initial rapid removal of pesticide by cyanobacterium is due to biosorption, (ii) the pseudo second order kinetics is the most preferable technique to explain the MP biosorption of by cyanobacterium, (iii) phosphate

l International Conference on Human Implication of Biotechnology (12-14 February, 2016, Central University of South Bihar, Patna, (India)

Bioprospecting for Microbial Products that Effects ColdAlleviation and Growth PI

: Pankaj Kumar Mishra

ICAR-Vivekananda Parvatiya KrishiAnusandhan Sansthan,Almora

l To validate the cold alleviating effect of the gene by

Rationale

transformation of non producing strains.

The N.W. Himalayan region that extends over the states of Jammu & Kashmir, Himachal Pradesh and Uttarakhand (fall within 29o31' – 36o58' N latitude and 73o26' – 83o30'E longitude with total geographical area of 33.49 mha. The rainfall ranges between 350-3000 mm per annum with dry and cold climate, presents a virgin opportunity for exploration and characterization of psychrophilic and psychrotolerant microorganisms, due to the unique ecological niche of the region that includes glaciers, alpine and sub alpine regions. Therefore, this study aims to explore the culturable/ unculturable psychrophilic and psychrotolerant microorganisms of the N.W. Indian Himalayas for the presence of novel genes encoding for cold shock proteins (Csp's)/ cold acclimation proteins (Caps)/ antifreeze proteins (Afp's) with a long term idea of creating an indigenous pool of expression systems that can be used for transgenic creation, in microbes and plants.

l To develop the delivery system and expression system for enhanced production of cold alleviating biomolecules. SignificantAchievements

l A total of twenty one soil samples of crop field/ forest/ grasses etc., collected from high altitude locations (3002–4018 meter amsl) of H.P. (Rohtang Pass, Kalong and Lahaul Spiti) of N.W. Indian Himalayas.

l Physiochemical analysis of collected soil samples revealed pH (ranged from 5.8 to 7.8) & EC (ranged from 0.009 to 1.128 dS/m).

l Organic carbon content ranged from 0.065 to 4.3 %, N 69.6 to 1568.9 kgha-1, P - 2.88 to 228.16 kgha-1, K- 11.25 to 588.25 kgha-1 and micronutrient Zn ranged from 0.67 to 1.95ppm and Fe - 3.83 to 94.15 ppm.

l Soil samples having microbial biomass Cmic ranged from

Objectives

l To identify cold alleviating genes (culturable/ unculturable

9.0 to 210.0μg g-1, Nmic - 7.06 to 24.55μg g-1 and Pmic - 0.57 to 17.14μg g-1.

microorganism) from higher hills of the N. W. Indian Himalayas.

l Dehydrogenase enzyme activity in the range of 1.4 to 880.0 μg TPF.g-1dm.16h-1 and total phosphomonesterase enzymes activity ranged from 57.1 to 533.4 μg NP.g-1dm.h-1.

l To sequence the gene(s) involved in the production of cold alleviating biomolecules.

39

AMAAS - Annual Report 2015-16

l A total of 291 psychrotolerant/ psychrophilic bacteria have

enzyme activities. Microbial biomass and activity are the main biological indicators of soil quality and respond rapidly to changes resulting from climatic variation/ changes. Microbial biomass and soil enzyme activity are useful indices for assessing changes in soil ecosystems as well as more responsive than organic C to an ecosystem establishment and sunstainability. A total of 291 psychrotolerant and psychrophilc bacteria were isolated strictly at 4ºC.

been isolated from rhizosphere/ rhizoplane/ endorhizosphere, strictly at 4ºC (Fig. 1 & 2). Conclusion The samples collected from high altitude (3002 – 4018 meter amsl) locations of N. W. Indian Himalayas revealed variations in physicochemical and available macro & micro nutrients and soil (dehydrogenase & phosphomonoesterase)

Fig. 2: Diversity of Psychrotolerant/ Psychrophilic Bacteria isolated form high altitude of Indian Himalayas

Fig. 1: Isolation of Psychrotolerant/ Psychrophilic bacteria from rhizosphere, rhizoplane & endorhizosphere

Pathogenomics- Genome Sequencing of Puccinia striiformis PI : RashmiAggarwal Co-PIs : Vaibhav K. Singh ICAR-IndianAgricultural Research Institute, New Delhi

l A total of 293 differentially expressed genes leading

Rationale

towards incompatible interaction through HR-specific and basal defence due to pathotype 38S102 were identified, out of which 146 were up regulated and 147 were down regulated. Of these, major up-regulated genes are calcium ion binding and its transport, small GTPase mediated signal transduction and transmembrane transport etc.

Pathotype 38S102 was picked up in 1973 from Nilgiri Hills. Yr9 virulences have now emerged as wide spread in North West plains where major area is under cultivation with varieties having gene Yr9, and pathotype 38S102 is avirulent on Yr9.

Objectives

l A total of 722 differentially expressed genes leading

l To characterize the transcriptional changes involved during

towards compatible interaction through basal defence and biotrophic interaction due to pathotype 46S119 were identified, out of which 450 were up regulated and 272 were down regulated. Of these major up-regulated genes are hydrolase activity (hydrolyzing O-glycosyl compounds),ATP binding proteins etc.

an incompatible pathotype-specific resistance interaction and a compatible interaction with Pst pathotypes. SignificantAchievements

l More than 10 million 125 bp paired-end clean reads for each library (control, 38S102-inoculated, 46S119inoculated) through RNA-seq sequencing were obtained.

l A gene for 1,3-beta-D-glucan synthase complex was found

l Of these, 89.5% for mock inoculated, 97.4% for 38S102-

to be upregulated in response to incompatible interaction and down-regulated in compatible interaction

inoculated and 95.9% for 46S119-inoculated were mapped to the wheat genome using STAR aligner.

l Copper ion binding gene was also upregulated due to 38S102 pathotype infection.

40

AMAAS - Annual Report 2015-16 'Plant, Pathogens and People' (Challenges in Plant Pathology to benefit humankind) conducted by Indian Phytopathological Society held at Delhi from 23-27 Feb. 2016 pp 502.

Conclusion There is a need to enhance our understanding on determinants of virulence of pathotype 38S102 and its restriction to Southern Hills only and to understand which factors determine the avirulence on Yr9. This will help in understanding host pathogen interaction at molecular level which will ultimately lead to more comprehensive understanding of Pst pathogenesis system, an important step towards developing more effective surveillance and management strategies for one of the most devastating pathogens of wheat.

l Gupta, S., Sharma, S., Kulshreshtha, D., Bashyal, B. M., Singh, V. K. and Aggarwal, R. (2015). Development of PCR based marker for the detection of stripe rust of wheat caused by Puccinia striiformis tritici. Poster paper presented in national Symposium on Germplasm to genes: Harnessing Biotechnology for food security and Health, held at Delhi from 9-11,August, 2015 pp93.

l Kulshreshtha, D., Kadimi, P., Singh, I., Shrivastava, S.,

Conference and workshop attended

l Kulshreshtha, D., Sharma, S., Manjunatha, C., Nigam, D.,

Sharma, S., Singh, V. K. and Aggarwal, R. (2015). Exploring miRNA like small RNAs in Puccinia striiformis using in silico approach. Poster paper presented in 6th World Congress ( Journal of Biotechnology & Biomaterials) on Biotechnology held at New Delhi from 57 October 2015.

Singh, V.K., Bhardwaj, S.C., Kumar, N., Khurana, S.M.P., Aggarwal, R. (2016).Transcriptome analysis of compatible and incompatible interaction in Wheat-Puccinia striiformis. Poster paper presented in 6th International Conference on

Archaea and Actinobacteria in Vertisols of Central India – Assessment of Diversity, Biogeochemical Processes and Bioinoculant Potential PI : D LN Rao Co-PIs : S R Mohanty, K Bharati, T K Radha ICAR-Indian Institute of Soil Science, Bhopal (soybean, maize, rice, wheat, chickpea) for development of bioinoculants.

Rationale Much uncertainty lies in our understanding of the role of archaea in agricultural soil ecosystems. Actinobacteria are a ubiquitous group of microorganisms involved in decomposition of organic matter and suppression of soil borne plant pathogens. Exploration of actinobacteria in enhancing crop production is limited. It hypothesize that these microbial groups are sources of useful genes that can be deployed in agriculture. The focus of the work is to explore the culturable and unculturable diversity of archaea and actinobacteria, their role in biogeochemical cycle and soil functions with the ultimate goal of enhancing nutrient transformation and agricultural productivity through developing of suitable microbial technology.

l To make multiple-repositories of the elite strains of archaea andActinobacteria. SignificantAchievements

l Soil samples from different cropping systems and agronomic practices were collected during the peak vegetative phase (Table 1) in rabi season. Rhizospheric soil samples were further analyzed for the physical and chemical attributes. Table 1: Characterization of rhizospheric soil samples collected from different locations Sampling Site Geelakhedi Jabalpur Chinndwara Bhopal

Objectives

l Define metagenomic diversity of archaea and actinobacteria to assess the community structure in relation to soil attributes in Vertisols of central India.

l Assess the community dynamics of archaea during

Crop Wheat Chickpea Wheat Chickpea

pH 7.5 6.6 7.7 7.4

Organic C (%) 0.81 1.04 0.76 0.56

l Culturable diversity of microbial groups was enumerated

different biogeochemical processes in soil.

in triplicate samples using nutrient agar for aerobic bacteria, Czapek Dox medium for fungi and

l Isolation and characterization of archaea and actinobacteria in the rhizosphere of selected crops

41

AMAAS - Annual Report 2015-16 cycloheximide (0.01%). Total N 2 fixing bacterial population estimated by using the N free Jensen's medium. Results are presented in (Table 2).

Actinomycetes in humic acid vitamin agar medium. Arthrobacter were enumerated on Arthrobacter medium with addition of methyl red (150 μg/ml), NaCl (2%) and

Table 2: Microbial abundance (CFU ± SD) in the rhizospheric samples of different sampling sites.

Site

Bacteria

Fungi 6

3

5.7 ± 2.1 X 10

Actinomycetes

Arthrobacter

6

7.7 ± 2.0 X 10

28.0 ± 4.4 X 10

Jabalpur

43.7 ± 3.8 X 10

6

16.3 ± 4.9 X 10

21.2 ± 7.2 X10

6

22.0 ± 2.8 X 10

Chhindwara

20.3 ± 3.1 X 106

9.7 ± 1.5 X 103

17.8 ± 2.3 X106

12.3 ± 4.9 X104

6

3

6

Bhopal

17.1 ± 0.5 X10

4

Geelakhedi

3

27.3 ± 3.8 X 10

5.0 ± 3.5 X 10

l Arthrobacter sp as major representatives of the cultural

25.0 ± 3.7 X10

5

29.7 ± X 105

attributes are characterized. The isolates are screened for duplicates based on colony morphology on Arthrobacter specific medium, tryptone soy agar (TSA) and 0.4% TSA and growth pattern in nutrient broth. A total of 200 isolates were screened for growth promotion of maize seedlings and PGPR characteristics like indole acetic acid production, siderophore production and phosphate solubilization. The characteristics of 13 effective strains and one ineffective strain are listed in Table 3 below. These fourteen strains have been shortlisted for sequencing of 16S rRNAgene.

fraction of actinobacteria. To explore them for providing better ecosystem services as well as for use as inoculants in agriculture we isolated them from various soils using synthetic medium. Based on the cultural characteristics, diversity of Arthrobacter followed the trend of Chickpea (Jabalpur) ≥ Chickpea (Bhopal) > maize (Chhindwara) > soybean (Geelakhedi) ≥ soybean (Bhopal) > Wheat (Chhindwara) = Wheat (Geelakhedi) > Rice (Jabalpur) (50, 50, 30, 20, 20, 12, 12 and 5 morpho-types respectively). Their morphological, physiological and biochemical Table 3: PGPR attributes of 14 Arthrobacter strains

Arthro- Rhizosphere bacter Strains AR1 AR2 AR3 AR4 AR5 AR6 AR7 AR8 AR9 AR10 AR11 AR12 AR13 AR14

Chickpea Soybean Chickpea Soybean Soybean Soybean Wheat Wheat Maize Rice Chickpea Soybean Soybean Soybean

Place

Bhopal Geelakhedi, Rajgarh Jabalpur Geelakhedi, Rajgarh Bhopal Bhopal Chhindwara Geelakhedi, Rajgarh Chhindwara Jabalpur Bhopal Bhopal Geelakhedi, Rajgarh Bhopal

pH

7.5 7.5 5.3 7.5 7.4 7.4 7.1 8.0 7.7 6.6 7.5 7.4 7.5 7.4

PGPR Attributes Organic Carbon PO4 IAA Vigor Siderophore (%) (μg/ml) index solubiliza- production tion 0.59 0.81 0.94 0.81 0.59 0.59 0.86 0.85 0.76 1.21 0.59 0.59 0.81 0.59

++ + NA -

l Thirteen strains of Arthrobacter (12 effective and one

++ +++ + +++ + ++ NA -

10.10 11.80 0.42 10.60 1.70 7.81 4.67 3.73 5.86 2.03 0.60 NA 1.61 6.54

4700 4730 5230 5940 5490 5740 5540 5440 5481 4650 4710 1140 4740 5880

% Increase of vigor index 51.6 52.3 68.7 91.6 77.1 85.2 78.7 75.5 76.8 50.0 51.9 -63.2 52.9 89.7

kharif 2015, significant results were obtained towards grain and straw yields, and total uptake of N, P and K by paddy crop due to inoculation with Arthrobacter isolates of AR6, AR8 and AR10, followed by AR5 over FUI. On

ineffective) were field tested for inoculation effect on growth of soybean, maize and rice in kharif and chickpea and wheat in rabi seasons at JNKVV, Jabalpur. During

42

AMAAS - Annual Report 2015-16 -

activity of NO3 reducers, Fe3+ reducers, SO42- reducers, and CH4 producers in that many species of anaerobes that reduce other terminal electron acceptors are capable of Fe3+ reduction. It is reported that many NO3- reducers are Fe3+ reducers and even some SO42- reducers are methanogens which also can reduce Fe3+ . Concentration of NO3 in chickpea rhizospheric soil was 0.4 mM. Nitrate concentration declined to undetectable level after 4 days of incubation. Fe3+ reduction started after 5 days and peaked to 0.08mM after a week. Initial SO42- was 0.14mM g-1 soil. Reduction of SO42- occurred after 12 days and decreased to 0.25Mm after 20 days. CH4 production started after 40 days of flooding and steadily increased to 300 ug g-1 soil (Fig 1). Sulfadiazine inhibited reduction of terminal electron acceptors and the temporal variation followed similarly as was without sulfadiazine. Rice soil exhibited sequential reduction of terminal electron acceptors differently than chickpea. In rice, initial NO3 concentration was 0.28mM and denitrification was completed after 2 days. Fe3+ reduction started after 3 days and peaked to 0.07mM at 6 day. Initial SO4 concentration in rice soil was 0.15mM and sulfate reduction started after 12 days. Methanogenesis started after 35 days and peaked to 600mg g-1 soil after end of incubation. Sulfadiazine inhibited TEAPs (Fig 2). To compare the relative impact of sulfadiazine on TEAPs, potential rate of reduction was determined (Table 1). Potential NO3 reduction rate (PNR) estimated as mM NO31reduced g-1 soil was 4.10 in chickpea and 1.80 in rice soil. Addition of sulfadiazine inhibited PNR significantly at P < 0.05. Potential iron reduction rate (PIR) estimated as mM Fe3+ reduced g-1 soil was more in rice soil than chickpea. It was 0.63 without sulfadiazine while, the PIR and inhibited to 0.21 mM Fe3+. Potential sulfate reduction (mM SO42reduced g-1 soil) was high in chickpea than rice. It ranged from 0.306 in chickpea and 0.029 in rice soil treated with sulfadiazine. Cumulative CH4 production was high chickpea than rice. It ranged from 690 ug and 214 ug g-1 soil in the head space of vials after end of incubation. Copy number of bacterial 16S rRNA gene was estimated from soil samples before after each TEAPs (Table 2). Rhizospheric soil of chickpea had higher bacterial population than rice soil irrespective of treatments. Interestingly we observed that bacterial abundance decreased over TEAPs. Experiment was based on the fact that sulfadiazine inhibits growth of ammonia oxidizing bacteria. Thus the linkage of ammonia oxidizing archaea was linked with TEAPs by restricting the growth of AOB by sulfadiazine. Real time PCR of bacterial gene also

considering the average performance of 13 Arthrobacter isolates towards grain yield, it was 16% higher over fertilized uninoculated (FUI) control. Arthrobacter isolates AR10, AR3 and AR12 exhibited prominent result to increase yields of grain and stover of maize and total uptake of N, P and K by crop over FUI. Average increase in grain yield of maize due to Arthrobacter inoculation was 19% higher over FUI. Arthrobacter isolates AR 2 responded significantly to increase all parameters viz., nodulation, grain and straw yield and total uptake of nutrients N, P and K by soybean crop, followed by isolates AR4 and AR7 for some of the observations over FUI. On an average, the number of nodules, oven dry weight of nodules and grain yield were 6, 17 and 12%, higher over FUI respectively in soybean. During rabi, the average increase of grain yield was 21% and 24% higher in chickpea and wheat.

l Ammonia oxidation in soil is carried out by both nitrifying bacteria and archaea. In an earlier experiment we have shown that nitrification is contributed almost equally by both ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in tropical Vertisols. N2O produced during nitrification was equally contributed by both AOB and AOA. This was confirmed following selective inhibition of AOB with the antibiotic sulfadiazine. Soil embodies different microbial groups for different ecological processes. Most of the species inhabiting soil are interlinked with other species. For example nitrifying bacteria often are linked with denitrifiers and sulfate reducers in biofilms in soil ecosystems. Similarly ammonia oxidizing bacteria have been found to coexist even with methanogenic microbes in flooded soil ecosystem. However it is unclear if ammonia oxidizing archaea (AOA) also have similar coexistence and function in flooded soil ecosystem. We hypothesize that akin to AOB, the AOA populations also regulate redox metabolism. Experiments were conducted to entail the differential role of AOB and AOA on terminal electron accepting processes in flooded soil ecosystem.

l Under flooded condition soil undergoes microbially mediated anaerobic respiratory redox processes with alternative electron acceptors being sequentially reduced in the order of NO3-, Fe3+, SO42- and CO2. Terminal electron accepting process in both the chickpea and rice soils followed this classical sequential reduction pathway. We observed overlapping redox process in slurry samples during the reduction processes- Fe3+, SO42- reduction, and methanogenesis. It is presumed that microbial groups responsible for specific redox processes also tend to overlap leading to unclear temporal distinction in the

43

AMAAS - Annual Report 2015-16 the metabolic response was restricted to AOA. Our experiment indicated that the linkage between nitrification and TEAPs is generally established by AOB and AOA. If AOB nitrification is blocked then AOA would initiate the nitrification and modulate TEAPs in flooded soil ecosystem.

indicated that sulfadiazine decreased abundance of total bacterial number. We also observed that bacterial population decreased over reductive phases. It could be due to niche specificity of bacteria during the later stage of soil reduction. Soil without sulfadiazine represented metabolic response of both AOB and AOA, while with sulfadiazine

-

Fig. 1: Redox metabolism of rhizospheric soil collected from rice plant. Soil samples were incubated under flooded condition with CH3COO as electron donor to 13+ 2induce sequential reduction of NO3 , Fe , SO4 , and methanogenesis (CH4 production). Treatments represent soil with sulfadiazine (sphere) or without sulfadiazine (cube). X axis represents incubation period in days and Y axis represents concentration of analytes. Each data point represents arithmetic mean with standard deviation as error bar of three replicated observations.

-

Fig. 2: Redox metabolism of rhizospheric soil collected from Chickpea. Soil samples were incubated under flooded condition with CH3COO as electron donor to 13+ 2induce sequential reduction of NO3 , Fe , SO4 , and methanogenesis (CH4 production). Treatments represent soil with sulfadiazine (sphere) or without sulfadiazine (cube). X axis represents incubation period in days and Y axis represents concentration of analytes. Each data point represents arithmetic mean with standard deviation as error bar of three replicated observations.

44

AMAAS - Annual Report 2015-16

Mining the Metagenome for Bacterial diversity in ExtremeArea of North-East India PI : Ratul Saikia Co-PIs : Hari P, Deka Baruah, Archana Yadav CSIR–North East Institute of Science and Technology, Jorhat was recorded 0-10 PPM and 5-25 PPM respectively (Fig.1). Concentration of heavy metals were also estimated (mg/L) in soil and water of Gelipung and Borpung which was recorded as Na(15.5 –392), K(1.7 –28.2), Zn(0.66 – 4.8), Cd(>0.04), Mg(1.0 - 31.7) and Ca(1.0–15.7).

Rationale Microbial diversity is an unseen National and International resource that deservesgreater attention. The vast majority of microorganisms on earth have not been cultured. The small proportion (>1%) that have been successfully cultured are the source of most of the functional properties. Therefore sequencing DNA directly from the environment, a technique commonly referred to as Metagenomics, is an important tool for cataloguing microbial life. In recent study by 16S rRNA genes amplified directly from soil indicated that novel phyla of bacteria and archeae are present. In this project, a typical metagenomics sequencing experiment will analyse diversity of bacterial community in few ecologically important sites as north eastern India is the best known for its rich biodiversity and its untapped bio resource. Till date no report or very few reports were published on bacterial diversity through metagenomics approach from ecological niche like natural hot water spring (Borpung and Gelipung, Golaghat district, Assam), cold adapted region (e.g. Tawang etc.) of N.E India. Therefore, bacterial community profiling in this area through metagenomics is essential.

Fig. 1: Physico-chemical properties of soil in natural hot water spring (Gelipung and Borpung).

l An insight into the soil microbial community deviations of medicinally important Rhododendron arboreum plant pertaining to altitudinal zonation along cold adapted Eastern slope of Himalayan Tawang region, India have been studying. Sampling had been done from different sites (Fig.2). Short reads of 16S r DNA (V3 region)

Objectives

l Construction of 16S rDNAmetagenomic library l Bacterial community profiling SignificantAchievements

l Sampling (soil and water) from natural hot water spring Nambor Wild Life Sanctuary (Borpung and Gelipung), Golaghat, Assam was done in different seasons. DNA was extracted from the collected samples and V3 region of 16S rRNA was amplified and process for sequencing by Next Generation Sequencing techniques (Illumina MiSeq).

l Determination of Physico-chemical properties of water/ soil of natural hot water springs, i.e. Borpung and Gelipung (Micronutrient) have been done to observe any bacterial community differences in relation to environmental factors. Water temperature of Borpung was recorded 45 47oC in the month of May whereas it was found 43oC during 2nd week of March. pH was recorded 5 – 6; the concentration of hydrazine was found 0.2 PPM and 0.1 PPM in water of Borpung and Gelipung, respectively. However, 25-45 PPM recorded in calcium harness test and fluoride was recordedas 2 PPM. Nitrate ion and nitrite ion

Fig. 2: A sampling site at Tawang (AP).

generated from Illumina Miseq from four rhizosphere sites which did not reveal significant linkage of sampling site inclination with community difference. Soil samples

45

AMAAS - Annual Report 2015-16

l During 2015-16, we have submitted 20 numbers of ACC-

collected from Bum La area was harboured significantly rich bacterial community compared to other sites at lower altitudes. Top phyla identified were Actinobacteria, Acidobacteria and Proteobacteria amongst others (Fig. 3).

deaminase producing rhizobacterial strains along with their passport data to the NAIMCC, NBAIM, Mau, UP. Conclusion Metagenomics has changed the way microbiologists approach many problems, redefined the concept of a genome, and boosted the rate of gene discovery. Hot springs are a vast source of new and diverse thermophilic enzymes, many with potential uses in industry. Apart from bacterial community profiles, metagenomics considers the effect of physico chemical conditions in community diversity from hot spring; while temperature seems to be the major factor, geochemical compositions and geographical distances are significant in several cases. Exploration of thermal environments has increased the knowledge about the evolution not only of bacteria, but also of archaea and viruses.

Fig. 3: Taxonomic composition of bacterial communities (phyla) associated with four different Rhododendron arboreum rhizosphere sites in Tawang.

Conference and workshop attended

l Conference and workshop attended: A. Dasgupta, P.

This represented with a core microbiome with abundances of Rhodoplanes, Rhodococcus, Candidatus Solibacter, Candidatus Koribacteria, Streptomyces and significant proportion of unclassified genera in the neighbourhood. We observed that main community differences were relating to soil factors when environmental factors were scaled on to response variables. Principal component analysis (PCA) revealed differences in soil bacterial community profiles was primarily determined by temperature, Mg, Silt and Clay content explained by PC1 which accounted for 43.43% of total sample variance.

Dutta, M. Goswami, R. Debnath, J. Saikia, P. Buragohain, A. Gohain, M. Kakoti, M. Hazarika, E. Kashyap, A. Yadav, R. K. Sarma, R. Saikia* (2016) Microbial Diversity study in the Genomic Era. Proceeding of UGC sponsored National Seminar on Biodiversity Degradation and Its Impact with Special Reference to North East India. Madhabdev College, Narayanpur, Lakhimpur, Assam, 18th& 19Th Feb., 2016, Pp. 22-23.

Microbial Diversity of Glacier Samples and Peat Profile of Himalayan Region PI : S.M. Singh1 Co-PIs : Alok Srivastava2 1 ESSO-National Centre forAntarctic and Ocean Research, Goa 2 ICAR-National Bureau ofAgriculturally Important Microorganisms, Maunath Bhanjan Rationale

Objectives

Glaciers are river of ice and archives microbes of immense importance for the prospect of biotechnology. Though our ancestors inherited the wealth of knowledge about Devalok Himalaya for health, agriculture, Industry and peace. but microbial investigations from Himalaya is fragmentary and still a thrust area for research for human welfare. Himalayas is the origin of many glaciers and important rivers of Asia including motherly river Ganga. During 1st year of project in 2014 glacier cryoconites and a pit profile samples were collected from Hamtah glacier of Himalayan region

l Microbial diversity study by isolating psychrophilic microorganisms (bacteria, fungi, yeast) from Chhota Shigri glacier samples.

l Screening of psychrophiles for their cold active enzymes such as lipases, cellulases, amylases, proteases etc.

l Production and optimization of cold active enzymes and pigments

l Cloning of genes responsible for cold active enzymes l Mass production and purification of cold active enzymes

46

AMAAS - Annual Report 2015-16

l Maximum colonies obtained on 1/10 NAand 1/10 Luria

and pigments

l Biochemical and molecular characterization

Bertani (LB) medium from patsio cryconite water and sediment sample respectively, whereas least colonies in TSA and ZMA.This suggests that most of cultures are Oligotrophic in nature. At the same time most of the different colonies based on morphological characteristics found on Antarctic biological medium (ABM) and Nutrient agar medium while least on ZMA and LB medium.

SignificantAchievements

l Glacier sediment and water samples were collected from Chhota shigri, and Patsio from Himalayan region during year 2015 (Fig. 1).

l In case of Chhota Shigri cryconites soil sample-1 there is a significant difference among the bacterial count along varied temperatures.

l There is a significant difference between the bacterial load along the media used in case of both sediment and water samples of Chhota Shigri cryconites soil sample-2.

l Randomly, 43 bacteria, 7 yeast and 13 molds were screened for extracellular enzymatic activity.

l Out of 43 bacterial isolates,69.77% of isolates were positive for Amylase and 41.86%, 44.19%, 25.58% of isolates were positivefor protease, cellulase and lipase. The highest enzymatic activity was observed at 4 and 15°C for most of the isolates. Isolate RN129 showed highest amylase activity at 15°C (Fig 2) and cellulase activity at 1°C. Isolate RN157 showed all the four enzymatic activity.

Fig. 1: a) Chota Shigri, b) Dhaka Sutri, c) Patsio glaciers, d) Cryoconites

l The Chhota Shigri and Patsio glacier of Himalaya showed diversified microbial population including bacteria, yeasts and molds (Table 1).

l Among 7 yeast isolates, 14.28% isolates were positive for

Table 1:Total number of bacteria, yeast and Mold isolate from Chhota Shigri and Patsio glacier Sample name Bacteria Chhota Shigri(G2L4A4a) 185 Chhota Shigri(G2SN3a) 72 Patsio (GPSA) 105

Yeast 46 38 65

cellulase, 85.71% isolates was positive for cellulase and 57.14% isolates showed lipase activity.

Mold 42 34

l The first cryconites soil samples (G2L4A4aof Chhota Shigri glacier showed highest CFU/gm at 1°C and lowest at 15°C whereas water samples showed highest CFU/ml at 22°C and least at 4°C.

l The second soil samples (G2SN3a) of Chhota Shigri glacier showed highest CFU/gm at 1°C and lowest at 22°C,whereas water samples showed highest CFU/ml at 15°C and least at 4°C.

Fig. 2: Amylase quantification of Chhota Shigri isolates

l Out of 13 selected molds, 23.07% of isolates were positive

l The Patsio cryconites soil sample showed highest CFU/gm at 1°C and lowest at 22°C, whereas water samples showed highest CFU/ml at 4°C.

for protease and 76.92%, 15.38% of isolates were positive for cellulase and lipase.

l Total 417 isolates were isolated from two Chhota Shigri

l Among 43 bacterial isolates (Chhota Shigri-G2SN3a) four isolates were selected on the basis of zone of clearance on starch agar plates. Quantitative assay of amylase(DNSA method) revealed that the isolate RN129 showed maximum amylase activity (17.9814 U/ml)at 15°C.

cryconites samples,included 257 bacteria, 84 yeast and 76 molds and 105 bacterial isolates from Patsio sample.

l The Chhota Shigri cryconites glacier samples showed highest colony forming units in 1/10 Zobell Marine Agar (ZMA), 1/10 Nutrient agar(NA) and least in trypticase soya agar (TSA) and zobell marine agar.

l For identification of isolates DNA isolation, PCR and sequencing are under process.

47

Theme : Taxonomy, Identification and Diagnostics of AIMs Development of Microarray Based Gene Chip for Major Fungal Plant Pathogens under the Background of DNABarcodes using Multi locus Gene Phylogeny 1

PI : Hillol Chakdar 2 1 1 Co-PIs : Prem Lal Kashyap , Alok K. Srivastava , Sanjay Goswami 1 ICAR-National Bureau of Agriculturally Important Microorganisms, Kushmaur, Maunath Bhanjan 2 ICAR-Indian Institute of Wheat and Barley Research, Regional Station, Flowerdale, Shimla Rationale

SignificantAchievements

Accurate and precise identification of fungal plant pathogens currently relies upon a wide range of techniques and skills, from classical culturing and taxonomic skills to modern molecular based methods. The wide range of methods employed reflects the great diversity of fungi and the hosts they infect. Further, a well documented decline in taxonomic expertise, along with the need to develop rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and technique. Microarray based gene chip under the background of DNA barcodes using multi locus gene phylogeny is emerging as one such generic platform technology that is well suited to high-throughput detection and discrimination of fungal plant pathogens. Keeping this in view, the present project has been initiated to develop a DNA based gene chip for the identification of major fungal plant pathogens. The DNA chip will be very useful in identifying pathogens that are difficult to distinguish either by morphology or other features. The chip also offers high accuracy in identifying quarantine pathogens and reduces the risk of spread. In addition to diagnosis, it also contributes to the fundamental understanding of pathogen phylogeography and relationship with host and contributes to the development of diseases management tactics.

l Surveys had been conducted in different districts of eastern Uttar Pradesh. A total 99 isolates of different plant pathogenic fungi like Rhizoctonia , Sclerotinia , Helminthosporium and Alternaria had been isolated and characterized (Fig. 1). Thirty one (31) isolates (NAIMCCF-03206 to NAIMCC-F-03236) had been submitted in NAIMCC. A

B

Rhizoctonia infected sheaths of A. Rice; B. Maize

Rhizoctonia

Sclerotinia

Alternaria

Helminthosporium

Objectives

l To characterize fungal plant pathogens of agriculturally important crops using multigene analysis.

Hyphae and spore of Helminthosporium

l To develop DNA barcoding system for identification of

Hyphae and conidia of Alternaria

Fig. 1: Isolation of different pathogenic fungi and their microscopic analysis

fungal pathogens.

l Following in silico approaches, primers have been

l To develop microarray based gene chip for rapid

designed for specific identification of R. solani. Two primer pairs viz. RS10F:RS10R and AG1F1: AG1R1 were found highly specific as no amplification signal was observed from other closely related and unrelated fungal and bacterial taxa (Fig. 2).

identification of plant pathogens.

l To validate gene chips for taxonomy, diagnostics, and plant quarantine.

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AMAAS - Annual Report 2015-16 Conference and workshop attended

600 bp

l Pallavi Rai, Prem Lal Kashyap, Hillol Chakdar and Alok K. Srivastava 2016 “Identification and characterization of microsatellites from whole genome of hemibiotrophic phytopathogenic fungus Colletotrichum gloeosporioides to assess cross-genus transferability and application as a diagnostic marker”. National Conference on Emerging trends in Fungal Biology and Plant Protection and 42nd Annual meeting of the Mycological Society of India Feb 16-18, 2016 organized by Centre of Advanced Study in Botany, Institute of science, Banaras Hindu University, Varanasi.

250 bp

Fig. 2: PCR amplification using primer pair RS10 and AG1F1showing amplification product of 600bp and 250bp Lane 1-41 Rhizoctonia solani, 42Alternaria alternata, 43-Trichoderma viride, 44-Fusarium oxysporum 45Colletotrichum capsici, 46-Colletotrichum falcatum, 47-Colletotrichum gloeosporioides 48-Bacillus subtilis 49-Pseudomonas sp.

l Pallavi Rai, Prem Lal Kashyap, Hillol Chakdar and Alok

l Mating type idiomorphs of Alternaria spp. have been

K. Srivastava 2016 “Development of a specific PCR assay for the detection of Rhizoctonia solani AG1”.International conference on recent advances in biotechnology and nanobiotechnology” February, 10-12, 2016, organized by Amity Institute of Biotechnology, Gwalior, Madhya Pradesh.

identified using different primer sets (Fig. 3).

Amat1R/Ama1cF

l Pallavi Rai, Prem Lal Kashyap, Hillol Chakdar and Alok K. Srivastava 2016 “Molecular diversity and phylogenetic analysis of Colletotrichum falcatum causing red rot of sugarcane in Eastern plains of India.” 6th International conference Plant, Pathogen and People Challenges in Plant Pathology to Benefit Humankind Feb 23-27 New Delhi.

Amat1R/Ama1aF

Amat1R/Ama1dF Fig. 3: PCR amplification using primer Lane1: Amat1R & Amat1cF, Lane2: Amat1R & Amat1aF and Lane3; Amat1R & Amat1dF : 1-HPIT3;2- BAB-18; 3-OTA-38; 4-F-1295; 5-Bab-39;6-UPT-1;7-ABDF-1282;8HPIT-6:9-BAB-2: 10-F-0973;11-BAB-47;12-JIT-3:13-F-96;14-BAB3;15-OSA-23

l Pallavi Rai, Prem Lal Kashyap, Hillol Chakdar, Alok. K. Srivastava, Arun K. Sharma 2015 “Molecular marker for rapid detectation and identification of Colletotrichum capsici responsible for anthracnose and fruit rot of chilli” national conference on 3rd Utter Pradesh Agricultural Science Congress, June 14-16 Organized by SHIATS, Allahabad and UPCAR and UPAAS.

Conclusion Isolates of fungal plant pathogens affecting various crops, from different geographical sites were collected and identified using phenotypic and genotypic methods. Genus or species specific genetic markers for R. solani were developed and mating type idiomorphs of Alternaria spp. were deciphered.

Exploration of Plant Growth Promoting Rhizobacteria,Antagonistic and Plant Pathogenic Microbial Resources from High Altitude Agro-Climate / Cropping System of Jammu & Kashmir State for SustainableAgriculture PI : Vishal Gupta Co-PIs : V.K. Razdan, Prachi Sharma, Reyazul Roul Mir Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, Jammu productivity. Therefore, existence of agriculturally potential microbial strains, having PGPR and bio-control properties, can be explored from these regions.They may be interesting source for novel biocatalysts that are beneficial for combating the diseases and pest, thereby enhancing the food production.

Rationale The state of Jammu & Kashmir, having sub-tropical, intermediate, temperate and cold semi-arid zones, has potential for biodiversity and is abode to large unexplored microbial diversity which can be utilized in enhancing the agricultural

49

AMAAS - Annual Report 2015-16 from 33.46 to 275.50 percent.

Objectives

l Isolation, identification and characterization of

l Isolates of plant growth promoting rhizobacteria also

microorganisms from different altitudes/agro-climatic/ cropping systems.

showed positive for the production of HCN and siderophore production (Fig. 2 & 3).

l Screening potential isolates against major soil/ seed/ air borne plant pathogens, and insect pests of important crops.

l Evaluation of rhizo-bacteria and their primary and secondary metabolites for plant growth promotion and pathogen suppression.

l Identification of strains with broad host range or specific to a group of plant pathogens and insect pests.

l Developing formulations suitable for various deliver systems and their evaluation SignificantAchievements

l Of total plant growth promoting rhizobacteria (PGPR)

Fig. 2: Production of HCN

collected from different altitudes and agro-climatic/ cropping systems of Jammu & Kashmir State based on the phenotypic, morphological and biochemical tests, fifty and twenty five isolates were identified as Pseudomonas fluorescens and Bacillus spp. respectively.

l Bio- assay studies indicated that isolates of P. fluorescens and Bacillus spp. showed 20-70 percent inhibition in the growth of Rhizoctonia solani, 11-78 percent in Fusarium oxysporoum f. sp. gladioli and 15-80 percent in Colletotrichum capsici, pathogens responsible for causing sheath blight of paddy, corm rot of saffron and anthracnose of chilli, respectively.

Fig. 3: Siderophore production

l Out of 50 isolates of P. fluorescens collected, thirty were

l The germination of sclerotia of R.solani was significantly

tested for the production of indole acetic acid (IAA). The maximum amount of IAA (20.75µg/ml) was shown by I-55 isolate (saffron) whereas, the minimum (6.1µg/ml) was produced by I-2 isolate (paddy).

inhibited by all the tested isolates, which ranged between 49.05-90.87 percent (Fig. 4). Isolates I-88 and I-35 showed the maximum inhibition of 90.87 and 88.21 percent with growth of 8.00 and 10.33mm, respectively, whereas the mycelial growth was 87.67mm in untreated control. The lowest inhibition of 49.05 percent was recorded by isolate I-27.

l Fifty isolates of plant growth promoting rhizobacteria exhibited phosphate solubilisation activity on Pikovskaya's agar medium by inducing clear zones (Fig.1) which varied

l The inoculation of rice seed (Basmati-370) with fluorescent pseudomonad isolates induced significant enhancement in germination percent, shoot length, root length and vigour index in 10-day old rice seedlings. Highest germination (83.33%), shoot length (11.10cm), root length (10.0cm) and vigour index (1683.27) was recorded by I-88 isolate, followed by I-35 and I-34 isolates with germination of 80.56 and 77.78 percent, shoot length of 10.20 and 8.90 cm, root length of 8.5 and 8.0cm and vigour index of 1466.19 and 1454.19, respectively, whereas minimum germination (38.39%), shoot length (5.20cm), root length (5.30cm) and vigour index (595.05) was observed in I-27 isolate.

Fig. 1: Phosphate solubilisation activities by plant growth promoting rhizobcteria isolates.

50

AMAAS - Annual Report 2015-16 Conclusion The isolates exhibited in vitro biological control ability and plant growth promoting properties against the economically important plant diseases (corm rot of saffron, sheath blight of rice and fruit rot of chilli). The selected isolates shall be subjected to molecular identification and field experiments. Conference and workshop attended

l Attended 6th International Conference 'Plant Pathogens and People” organized by Indian Phytopathological Society (IPS), New Delhi, Feb., 23-27, 2016 at New Delhi.

Fig. 4: Effect of plant growth promoting rhizobcteria on germination of sclerotia of Rhizoctonia solani

Development of Diagnostic Kit for Detection of Karnal Bunt and Loose Smut of Wheat PI : M.S. Saharan Co-PIs : Sudheer Kumar, Pradeep Sharma ICAR - Indian Institute of Wheat and Barley Research, Karnal Rationale

SignificantAchievements

Among biotic stresses of wheat, Karnal bunt and loose smut are important seed borne diseases. Though they have got restricted distribution and are important in certain regions they are responsible for isolated and localized failure of the crops. Loose smut is generally distributed throughout the country but it appears to be more in the Northen India than Southern. The disease is internally seed borne and recognizable only at the ear head stage by the conspicuous smutted heads. Similarly, Karnal bunt is also seed borne disease affecting kernels of wheat grown in some arid or semi-arid areas of several countries with hot summers and mild/cool winters. In India, loose smut and Karnal bunt causes average losses of 2-4% along with quality deterioration in grain and flour. Karnal bunt is a major deciding factor in export of wheat. Thus the detection is very important as per quarantine is concerned for International trading of wheat. Keeping in view the importance of these pathogens in wheat a rapid and early detection systems are required; therefore, this investigation is undertaken to study genetic diversity and development of rapid detection tool for these pathogens.

l Wheat grain samples were collected from different wheat growing areas of India. Tilletia indica was isolated from the infected seeds. The bunt pathogens whole genome data are not yet available under such condition for molecular variability assessment SSR were mined from the closely related species Ustilago hordei. The SSR from Ustilago hordei are being used to analyse variability among Karnal bunt pathogen because SSR markers are highly transferable cross closely related species. The Ustilago hordei whole genome of 21.15 Mb was downloaded from NCBI and mined for SSR motifs using online programme WebSat. Total 8226 SSR motifs have been identified consisting Mono (22%), di (14%), tri (43%), tetra (3%), penta (6%) and hexa (12%) repeats (Table 1, Fig. 1). Primers for 36 randomly selected SSR motifs have been custom synthesized and are being used for variability in Tilletia indica. In order to test the SSRs derived from loose smut, cross transferability studies were conducted with Karnal Bunt isolates. For this study, 17 isolates of Karnal bunt representing different geographic areas/regions were used. Only 6 SSRs showed polymorphism among the isolates. Further work on SSRs validation is in progress. A set of differential was inoculated with different T. indica isolates and compatibility among monosporidial lines was studied by inoculating in different combination on Karnal bunt susceptible cultivar of wheat.

Objectives

l Detection of genetic variation among the pathogens of wheat (Karnal bunt and loose smut).

l Development of a rapid PCR-based diagnostic method to detect seed borne pathogens (Karnal bunt and loose smut) in wheat.

l Evaluation of markers for early detection of smut and bunt pathogens.

51

AMAAS - Annual Report 2015-16 Table. 1 Computational analysis of simple sequences repeats in U. hordei SSR motif SSR motif numbers Frequency (%) of individual SSR SSR motif with highest frequency SSR motif with highest repeat Individual SSR relative density Individual (SSR) relative abundance Total (SSR) relative density Total (SSR) relative abundance

Mono 1800 21.88 T(687) A(50)

Di 1103 13.41 CT(169) CT (86)

Tri. 3553 43.19 GAG(255) GTT (43)

1.19 1.11 2.51 0.08 0.05 0.17 8226/21150.702 kb = 0.39 kb 142035bp/21150.702kb = 6.71bp/kb

Tetra Penta Hexa 237 523 1010 2.88 6.36 12.28 CAGT(14) TTTCT(15) TTTTTC(22) ATTT(17) CTTTT(13) ACCAAC (19) & GATGCG (19) 0.23 0.46 1.18 0.01 0.02 0.05

screened for SSR motifs using SSRIT online software (Temnykh et al. 2001), which is accessible through internet. The frequency of occurrence, relative density and relative abundance of the repeats motifs were analyzed. A total 2327 SSR motifs were identified that includes 1149 di-repeats (49%), 861 tri-repeats (37%), 76 tetra-repeats (3%), 67 penta-repeats (3%) and 174 hexa-repeat motifs (8%). Details are presented in the Tables 2-5. Primers were designed using PRIMER3 online software from flanking region. SSR primers were randomly selected for PCR amplification. Only 65 SSRs for loose smut were screened to know the genetic variation amongst the isolates. DNA from 17 isolates of loose smut was isolated using CTAB method. Quality of the DNA was assessed in 0.8% agarose gel with EtBr. Out of 65 SSRs, 10 SSRs showed polymorphism in tested isolates.

Fig. 1. Distribution of frequency and percentage of SSRs in U. hordei

l The whole genome sequence data of Ustilago maydis (19.68 Mb) were obtained from National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) and

Table 2. Longest SSR of U. maydis

Table 3. SSR motifs in genes of U. maydis

Di Tri Tetra Penta Hexa Others ag (60) tta (34) gctt(22) ttgct (25) tagggt (62) aattgtg (68) ag (37) ttg(31) gaaa (21) aggca (12) cagcaa (21) attgtga (48) ag (35) tgt(28) ctca (18) ggaga (12) tgcttc (21) atcgtga (32)

Orga Sequence Di Tri Tetra Penta Hexa Others Total nism analyzed (Mb) U. 19.68 1149 861 76 67 174 376 2703 maydis Mb

Table 4. Relative abundance and density* of SSR in U. maydis

Table 5. Most frequent SSR motifs* in genome of U. maydis

Di Tri Tetra Penta Hexa 58.3 43.5 3.8 3.4 8.8 (116) (131) (15) (17) (53) *Relative density of SSR (in paranthesis)

Di ag (161) tc (158) ct (137)

Organism U. maydis

Total 117.8 (332)

Tri gct (75) cag(72) tgc(71)

Tetra catc(4) ctgg (4) agcc (4)

Penta

Hexa

caaaa (2) caaag (2) caagc (2)

cagcaa (10) tgctgt (6) tcaacag (4)

*Relative density of SSR (in paranthesis)

52

AMAAS - Annual Report 2015-16 Conclusion

Conference and workshop attended

Tilletia indica isolates and monosporidial lines have been isolated from Karnal bunt infected grains. Compatible monosprodial lines have been identified based on host pathogen interaction. Agrressiveness among T. indica isolates have also been studied on a set of wheat varieties. DNA isolated from T. indica and Ustilago segatum tritici isolates is being used for further research work on developing diagnostic for these pathogens.

l Sharma P., Saharan M.S., Kumar Sudheer, Kumar Satish, Shefali Pandey, Bharti K, Senthil Kumar, Sharma I, and Gupta, R.K. 2016. Mining and validation of SSR markers for wheat fungal pathogens. IN: 6th Int'l Conf. Plant, P a t h o g e n s a n d P e o p l e o rg a n i z e d b y I n d i a n Phytopathological Society at NASC Complex, New Delhi, Feb. 23-27, 2016: 508-509.

Development of Microbial Formulations for Biological Control of Grape Diseases PI : Indu S. Sawant Co-PIs : S. D. Sawant ICAR-National Research Centre for Grapes, Pune powdery mildew disease after veraison stage as compared to untreated vines. T. koningii provided slightly better control than B. licheniformis.

Rationale In Indian viticulture, frequent applications of fungicides are required to prevent crop losses due to the high incidences of fungal diseases under wet and warm weather conditions. This project aims to minimize fungicide use by identifying efficient biocontrol agents which can be used in IDM.

l ISR: Four strains each of Trichoderma and Bacillus were identified as promising for control of powdery mildew disease through induction of systemic resistance in grapevines in earlier studies. These strains were further evaluated in 2015-16 in two field trials on V. vinefera cv. Manjri Naveen. Three soil applications of Trichoderma strains at 5×106 spores/ml and Bacillus at 1×108 cfu/ml were made at 15 day intervals during November to December, in the drip region at 2L/vine. T. harzianum 5R alone or mixed application of T. harzianum 5R + T. viride (NAIMCC-F-01812); and Bacillus strain TP-232 alone and mixed application of Bacillus strains TP-232 + TL-171 restricted development of powdery mildew on bunches and leaves as compared to untreated vines. On bunches, mixed applications of Bacillus strains DR-92 + TS-45 was most effective.

Objectives

l To test the efficacy of promising microorganisms in large scale field trials for control of downy mildew, powdery mildew and anthracnose diseases of grapes.

l To activate the plant defence mechanism through induction of systemic resistance by promising microorganisms.

l To enhance the biocontrol efficacy of microorganisms using compatible fungicides.

l To control fungicide resistant pathogen population using highly antagonistic isolates.

l To develop liquid culture methods for production of most efficient bacterial and fungal biocontrol agents.

l The Trichoderma strain 5R, earlier found useful in

l To develop liquid/ solid formulations with optimum

managing postharvest decay and enhancing the shelf life of grapes, was characterized. Conidia were globose and with distinctly warty surface as seen using scanning electron microscope. The ITS sequence showed homology to both T. asperellum and T. asperelloides, but the tef1 gene showed higher homology to T. asperelloides sequences available in NCBI database. A fingerprint of strain 5R was generated using (GTG)5, (GACA)4 and phage M13 core sequence oligonucleotide primers.

efficacy and shelf life. SignificantAchievements

l Biocontrol: In earlier studies a fungal and a bacterial antagonist were identified as promising for biocontrol of powdery mildew disease. Large scale field trials were conducted during 2015-16 using simple aqueous formulations of these two strains, T. koningii (NAIMCC-F01938) containing 5x10 6 spores/ml and Bacillus licheniformis–TL-171 containing 1x108 cfu/mlon V. vinefera cv Thompson Seedless. Three foliar applications made at weekly intervals restricted the development of

l Bacillus amyloliquefaciens strain TS-45 was isolated from healthy grapevine, evaluated in vitro, and in planta and found suitable for biocontrol of grapevine anthracnose at low concentrations of 104 CFU/ml.

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AMAAS - Annual Report 2015-16

l A simple talc based formulation of Trichoderma strain 5R

distinct from each other (Fig. 1). At p value< 0.01 and fold change >2 as differential analysis parameter, 62 masses in T. harzianum / T. koningii and 34 and 52 in T. viride and Trichoderma strain 5R, respectively were found to be differentially expressed.

was prepared. The strain was grown by liquid state fermentation method. The population was maintained at 0.70 ± 0.08 × 108 spores/ml over the 20 week period at room temperature.

l The sensitivity of four biocontrol strains of Trichoderma to eleven fungicides and four insecticides commonly used in grapes was studied. Isolates were compatible with sulphur, myclobutanil, copper hydroxide, dinocap, mancozeb, fosetyl-Al, iprovalicarb+ propineb, cymoxanil+mancozeb and insecticides; but not to carbendazim, hexaconazole and tetraconazole.

l A method for detection of fungal metabolites was developed on UHPLC-high resolution Orbitrap mass spectrometer. Trichoderma strain 5R, T. harzianum (NAIMCC-F-01965), T. koningii (NAIMCC-F-01938), and T. viride (NAIMCC-F-01812) were taken for analysis. Target analysis was done by in house developed Trichoderma metabolites database and Tracefinder 3.0 was used for metabolites detection from raw data. Many known compounds specific to isolates were detected. Sieve 2.2 and Mass frontier softwares were used for differential, statistical and non targeted analysis. PCA grouped T. harzianum and T. koningii together and other isolates were

Fig. 1. Principle component analysis of four Trichoderma strains using Siene Software.

Conclusion Foliar and soil applications of selected strains of Trichoderma and Bacillus can be used in vineyards for control of powdery mildew.

54

Theme : Plant Microbe Interactions, Nutrient Management, Disease Control and Agrowaste Management Investigating the Potential of Root-Colonizing Microbial Population for Stress Tolerance in Chickpea through Proteomics PI : B.K. Sarma1 Co-PIs : H.B. Singh1, D.P. Singh2 1 Banaras Hindu University, Varanasi 2 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan

l Similarly, growth parameters were also increased

Rationale

following treatments with the bioagents. Similar to the flower count, biomass was also highest in the consortium treatment of OKC and T42 compared to the other treatments. However, biomass of the microbial treated plants was significantly high compared to the control. The trend was similar with both root and shoot biomass. The plants showed increased biomass accumulation even after 45 days of sowing (Fig. 2).

The rationale of the study was to assess how beneficial rhizosphere microbes in consortia manipulate chickpea metabolism to ward off the threat of soil borne pathogens.

Objectives

l To understand the plant mediated signalling by root colonizing rhizosphere microbes in biotic and abiotic stress tolerance in chickpea through proteome analyses. SignificantAchievements

l The rhizosphere microbes Pseudomonas fluorescens OKC

Radicle length (cm)

Fig: Effect of bio-agent treatment on seed germination of chichkpea. Where O=Psendomonas, T=Trichoderma, OT=Consortium of O and T, C=control Seed germination (%)

Root Shoot

Stem length in cm

DW in g/plant

and Trichoderma asperellum T42 when applied singly and in combinations in chickpea and challenged with the pathogen Fusarium oxysporum f. sp. ciceris various responses were observed. The microbial treatments enhanced germination of chickpea seeds and radical length significantly compared to the control. Among the microbial treatments, combined applications of the rhizosphere microbes Pseudomonas fluorescens OKC and Trichoderma asperellum T42 caused maximum germination and stimulated highest radical development in chickpea. The difference was visible even at 48 h of seed treatment (Fig. 1).

Fig. 2: Effect of bio-agent treatments on plant growth in field condition. Where O=Pseudomonas, T= Trichoderma, OT= Consortium of O and T, C= control.

Fig. 1: Relative germination of chickpea seeds and radical length after microbial treatments. Where O=Pseudomonas, T= Trichoderma, OT= Consortium of O and T, C= control

55

AMAAS - Annual Report 2015-16

l Interestingly, the microbial treated plants showed more the number of flowers per plant particularly in the combined treatment of OKC and T42 compared to the other treatments. It was followed by OKC and T42 and the flower counts were significantly high in all treatments compared to control after 95 days of sowing (Fig. 3).

(A)

Fig. 3: Effect of bio-agent treatment on flower count of chickpea. Where O=Pseudomonas, T= Trichoderma, OT= Consortium of O and T, C= control

l Trichomes of plant are involved in the defense. Presence of glandular trichomes on leaf supports plant to defend them against different pathogens. We have observed increase in number of glandular trichomes in the bio-agent treated plants compared to control. Highest glandular trichomes were found in the consortia treatment compared to single treatments and control plants as represented in the bar diagram after 15 days of sowing (Fig. 4a).

l Further, damage in the glandular trichomes were observed after 48h of pathogen (Fusarium oxysporum f. sp. ciceris) inoculation in the control plants, whereas in the microbial treated plants, no such damage was found. It was concluded that the microbial treatments not only increased glandular trichomes on chickpea leaves but also protected them from pathogen infection. From the results it can also be concluded that despite the pathogen attacking roots of the chickpea plants. It can also make the leaves susceptible to other foliar pathogens by damaging the glandular trichomes (Fig. 4b).

(B) Fig. 4: (a) Glandular trichome count on leaves. Where O= Pseudomonas, T= Trichoderma, OT= Consortium of O and T, C= control. (b) Structure of glandular trichomes on leaves. Where O=Pseudomonas, T= Trichoderma, OT= Consortium of O and T, C= control.

l In the field conditions highest seed germination and seedling establishment was observed in the consortia treatment compared to single treatments and control plants.

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AMAAS - Annual Report 2015-16

l Proteome analysis of chickpea following inoculation with

The plant stand and growth both were significantly high in the consortium treatment compared to the single microbial treatments and control (Fig. 5).

the rhizospheric microbe T42 showed differential expression of some vital proteins. When only T42 was treated without challenge of Fusarium oxysporum f. sp. ciceris expression of proteins such as defensin, PR-5, NADPH oxidase, glucanase, peroxidase, calmodulin, NBS-LRR, NAC transcription family, WRKY transcription family, PAL, Rubisco and Thaumatin expression were more than doubled. Similarly, when chickpea plants were challenged with the pathogen F. oxysporum f. sp. ciceris expression of the proteins such as Thaumatin, glucanase, peroxidase, NBS-LRR, NADPH quinone oxidoreductase, NAC family transcription factors, superoxide dismutase, MAP Kinase, chitinase, autophagy, lipoxygenase, multidrug resistance protein, and WRKY were expressed more than doubled. Similarly, when the T42 treated plants were challenged with the pathogen then it was observed that apart from some of the already mentioned genes there was increase in expression levels of NBS-LRR, glycolate oxidase, ascorbate peroxidase, peroxidase, and NADPH oxidase.

Fig. 5: Growth of chickpea plants in microplotfield conditions. Where O=Pseudomonas, T= Trichoderma, OT= Consortium of O and T, C= control.

Conclusion Pseudomonas fluorescens OKC and Trichoderma asperellum T42 combined treated increased plant growth parameters, biomass, total number of flowers and glandular trichomes. Chickpea heterotrimeric G-protein was involved in chickpea's response towards the invading pathogen Fusarium oxysporum f. sp. ciceris and Gβ and γ components were involved but not the Gα component. Proteome analysis showed that there was activation of the antioxidant enzymes, photosynthetic enzymes and resistance genes in pathogen challenged plants compared to control plants.

l Heterotrimeric G-proteins are involved in plant defense signalling process. We checked the transcript accumulation pattern of the three subunits (Gα, β and γ) of chickpea by amplifying the cDNA samples made from harvested RNA after 48h of pathogen inoculation through semiquantitative RT-PCR. The results showed activation of Gβ and Gγ whereas activation of Gα was basal level in all the treatments. In Gβ and Gγ expression was increased after pathogen inoculation. The results indicate the role of Gβ and Gγ mediated signal transduction in chickpea following infection with Fusarium oxysporum f. sp. ciceris (Fig. 6).

Conference and workshop attended

l Sarma BK. 2016. Harnessing benefits of microbial consortium for enhancing disease resistance in chickpea. 6th International conference on 'Plant, Pathogens and People', 23-27 February, 2016, NASC Complex, New Delhi. Fig. 6: Expression pattern of chickpea heterotrimeric G-protein in leaves. Chickpea plants were treated with OKC and T42 in singly and in consortium and challenged with Fusarium oxysporum f. sp. ciceris.

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AMAAS - Annual Report 2015-16

Identification of High Trehalose-Producing Soybean Rhizobia and their Integration with AM Fungi for Enhanced Drought Tolerance in Soybean PI : Mahaveer P Sharma1 Co-PIs : Minakshi Grover2, Sushil K. Sharma3, DK Agarwal4, G. Satpute1 1 ICAR-Indian Institute of Soyabean Research, Indore 2 ICAR-Central Research Institute for Dryland Agriculture, Hyderabad 3 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 4 ICAR-Indian Institute of Seed Science, Maunath Bhanjan lines/cultivars based on RWC.

Rationale

l To optimize methods for extraction and quantification of

Central India is the hub for soybean cultivation in India. However, average productivity of soybean in India is almost only one-third as compared to that of other countries. The reason for this is largely, drought stress among several other abiotic factors. Nitrogen fixation gets adversely affected in soybean during drought conditions. Hence, it is crucial to develop technologies which can improve nitrogen fixation and mineral nutrition in soybean during stress conditions. Being a legume, rhizobial symbiosis can provide the necessary nitrogen to the soybean plants for growth and AM fungi symbiosis can enhance plant growth through the ability of extrametrical fungal hyphae to take up water, supplying lowdiffusing nutrients such as P from soil. Further, it has been reported that trehalose and the trehalose biosynthetic pathway are important contributors and regulators of stress responses in plants. Accumulation of trehalose, a non-reducing disaccharide, protects the cell by stabilizing cell structures and enabling protein to maintain their native conformation under drought stress. Its occurrence in plants colonized by AMF and nitrogen fixing nodules has also been reported. Trehalose content in nodules under drought stress correlates positively with increasing plant drought tolerance. However, trehalose dynamics in the Mycorrhiza-Rhizobium-Legume tripartite symbiosis is largely unknown. Higher trehalose–producing rhizobia can be used to improve the shelf life of rhizobial formulations and their integration with AM fungi to increase the trehalose level eventually to enhance the tolerance of soybean plants under drought conditions.

trehalose in root nodules of drought tolerant lines.

l Isolate and characterize (biochemical and molecular) soybean rhizobia and AMF from the rhizosphere of soybean lines harbouring higher trehalose in their nodules.

l To assess in-vitro trehalose accumulation in rhizobial cultures andAMF-ROC system and

l To integrate soybean rhizobia with AM fungi and evaluate for enhance drought tolerance in soybean. SignificantAchievements

l Based on FAME's profiling, isolates (previously recovered from the root nodules of highly drought tolerant soybean lines) mainly belong to Bradyrhizobium sp. & Bacillus sp. These isolates were tested for physiological bearing traits viz. Nitrate & nitrite reductase assay, ACC deaminase assay, Indole Acetic acid assay, proline accumulation assay and acetylene reduction assay) under in-vitro conditions. The 16S rRNA gene amplicons have been obtained using the universal primer sets viz. rD1& fD1 and 27F&1492R.

l Trehalose has been detected in nodules of all the 21 drought-tolerant lines through a modified GC-MS method and as per the relative values of area covered under the trehalose peak is concerned, root nodules of the lines DSR4 (PK-472), DSR-8 (NRC-37), DSR-9 (Jackson), DSR-15 (Bragg) & DSR-16 (JS 97-52) have higher trehalose content as compared to others, that in DSR-15 being the highest (Table 1).

Objectives

l To select drought tolerance soybean germplasm

Table 1:Trehalose derivatives obtained in nodules of the selected drought-tolerant lines through metabolic profiling in the GC-MS column Sample No. DSR 1 DSR 4 DSR 8 DSR 9 DSR 15 DSR 16

RT 24.564 24.581 24.592 24.583 24.587 24.597

A(Area ) 53,53,076 1,51,33,551 1,47,71,499 81,29,210 1,56,32,558 1,47,00,867

Area (%) 0.58 2.28 1.99 1.24 2.75 2.37

58

H (Height) 1606588 3209165 3436131 2051912 3644836 3678917

H% (A/H) 0.533.33 1.294.72 1.264.3 0.833.96 1.654.29 1.594

AMAAS - Annual Report 2015-16

l Two genotypes (DSR 2; CAT 53828 and DSR 12-Hardee)

Trehalose has been detected in root nodules of all the 21 lines. Various other osmolytes viz., fatty acids, amino acids, organic acids and sugars are also present according to the metabolic profiling obtained via GC-MS system.

were found to have higher affinity to AM fungi (reported by CNRBDC Bengaluru) where G. leptotichum was identified as the best AM fungus partner for inoculating soybean genotype (Hardee).

Two drought tolerant genotypes supplied to Bengaluru centre (DSR 2; CAT 53828 and DSR 12-Hardee) were found to have higher affinity to AM fungi (reported by CNRBDC Bengaluru) where G. leptotichum was identified as the best AM fungus partner for inoculating soybean genotype (Hardee). B. liaoningense strain supplied to Bengaluru centre for testing its synergistic effect withAMF, G. leptotichum.

l The predominant indigenous AMF species ( G. intraradices) identified from soybean rhizosphere is being attempted for its mass multiplication under in-vitro using Ri-T DNA hairy root cultures. Some activity has been seen in the A. rhizogenes (MTCC-532; ATCC-15834)-infected Amaranthus ex-plants (internodes) (Fig 1). Further growth is awaited so as to confirm whether or not these are hairyroots. Better results were seen in the ex-plants injected with limited amount (30μl) of overnight grown Agrobacterium culture as compared to the ones that were dipped in the culture for 20 minutes.

Virulence capacity (hairy-root formation) has been detected in the MTCC-532 strain obtained from IMTECH, Chandigarh using Amaranthus as host plant. The 16S rRNA gene has been amplified in the isolates and the amplicons are to be outsourced for sequencing for the purpose of molecular identification of the strains. Book Chapters

l Priyanka Gupta, Rita Sharma, Manoj K. Sharma, Mahaveer P. Sharma, Gyanesh K. Satpute, Shivani Garg,Sneh L. Singla-Pareek, Ashwani Pareek (2016) Signaling cross talk between biotic and abiotic stress responses in soybean. In: Abiotic and biotic stresses in soybean production (Ed. Mohammad Miransari). Volume I Oxford: Academic Press; p. 27-52. http://dx.doi.org/ 10.1016/B978-0-12-801536-0.00002-5. Fig. 1: Emerging hairy-root like structures from the internodal ex-plants of Amaranthus

Conference and workshop attended

l Mahaveer P Sharma (2015) Participated in two days

Conclusion

ICAR-NBAIM & Delhi University joint workshop on Renovating Bacterial Taxonomy with Bioinformatics at Delhi University, Delhi from May 14-15, 2015.

Various species of nodule-inhabiting Bradyrhizobia have been obtained from the drought tolerant lines and could be potential strains based on physiological traits.

Mapping and Creation of Gene Bank of Arbuscular Mycorrhiza and their Exploration for Enhancing Productivity of Underutilized Crops PI : Anil K. Sharma Department of Biological Science, CBSH, GBPUA&T, Pantnagar Rationale

Objectives

Identification and characterization of AMF and PGPR for exploring their yield enhancement potential in underutilized crops. Since, these crops are challenged with extreme environment like drought and temperature, exploration of indigenous microbes might therefore help in better plant sustainability.

l Mapping of arbuscular mycorrhizal diversity in underutilized crops.

l Evaluating the change in microbial diversity and their significance on the functioning of AMF in underutilized crops.

l Mass multiplication ofAMF and microbes.

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AMAAS - Annual Report 2015-16

l Evaluation of selected microorganisms and AMF on the

l Net house experiment on wheat and chickpea to study the plant microbe interaction during irregular watering condition in a mixed cropping was set up. Treatments: Control, AMF, DPB 16 (ACC producing bacteria), DPB16+AMF. Crop: Wheat (DBW 88, irrigated); ChickPea (PG 114). Each treatment had three replicates and whole experiment has two sets. Set 1 and Set 2. Set 1 was regularly irrigated and 70% field capacity was maintained, Set 2 was irregularly watered. The study was conducted to check the sustainability of crops in favorable and adverse conditions in the presence of microbes inoculated (Fig. 3).

growth of selected crops in field. SignificantAchievements

l AMF spore identification from the established traps of different locations. The samples collected from different sites and different time intervals were brought back to lab and a mixture of vermiculite and perlite was used to propagate AMF from the soil (Fig. 1). Different plants like Maize, Moongbean, etc were used as host in pots for five crop cycles of 60 days each.

Fig. 1: Spores from Loction Tajus showing difference in morphology when stained in Meltzer's reagent.

l A comparative experiment was done on Vigna mungo (PU 31) Urd bean to study the effect on plant growth promotion when the bacterial inoculation method was different. In these experiments, 6 bacterial isolates were used as sole treatment and also in combination with AMF soil based culture. The two experiments are no. as Exp 1 and Exp 2. In Exp 1, 1 ml of liquid medium culture of bacterial isolates (cfu 107) was given and, In Exp 2, the similar cultures was used for seed priming using talc and CMC as carrier material (Fig. 2). Soil based AMF culture was used containing 77-80 spores/g.

Fig. 3: Set I & II, regularly watered and irregularly watered respectively.

l Field experiment set up with 6 treatments and three replicates on wheat at GBPUA&T research station at Almora (Fig. 4).

Fig. 4: Figure showing experimental set up in different stages from field preparation to the crop growth at Manjhera research station, GBPAU&T, Almora

l Similar experiment was set up with 6 treatments and three replicates on wheat and chickpea at SPC, GBPUA&T, Pantnagar (Fig. 5).

Fig. 2: Experiment to test the difference in inoculation methods. 1. Direct inoculation of bacteria withAMF. 2. Bioprimed seeds withAMF

60

AMAAS - Annual Report 2015-16 Dehydrogenase

Urease

300 250 200 150 100 50 0

c

m

p

p

p

p

m

m

m

m

Treatments

Fig. 6: Effect of Treatments on Enzyme activity in organic farming system

Conclusion AMF traps have showed varying diversity morphologically. Bio priming is a better method of seed inoculation to study plant growth promotion. In net house experiment on wheat and chickpea mixed cropping, ACC deaminase producing bacteria DPB16+AMF treated plants showed plant growth promotion and sustenance towards minimum soil moisture in respective sets I & II. Isolated bacteria and their combination with AMF showed better response in field experiments in both locations, i.e, Manjhera research station, Almora and SPC Organic farming block, Pantnagar, GBPUAT. Soil enzyme assay studies showed improved soil health at the organic farming block, SPC, Pantnagar.

Fig. 5: Figure showing crop of wheat and chickpea in SPC field

l The farming system trial was set up at the Organic Farming

Conference and workshop attended

Block of Seed Production Centre of G.B. Pant University of Agriculture & Technology, Pantnagar, U.S. Nagar in July 2002 and still going on till date. The experiment was set up in randomized block design with three replications. Each plot was of 16m2 having 10 treatments, which are: The crop rotation followed was of Okra (Hibiscus esculentus L.), Pea (Pisum sativum L.) and cowpea (Vigna unguiculata). AMF spore count and enzyme study was conducted from this experiment (Fig. 6).

l Suvigya Sharma and A.k Sharma, Poster presentation on “Evaluation of total microbial diversity and characterization of bacteria under organic and intensive maize based farming systems in Uttarakhand and their potential use with AMF in enhancing crop growth” in 6th International Conference on Technology and Management for sustainable development-2016 held at ITM University, Gwalior, M.P

Development of Practicable Technologies for Field Level Exploitation of Consortia of Microbial Agents forAmelioration of Biotic andAbiotic Stresses in Crops PI : R. D. Prasad Co-PIs : P. Lakshmamma, M. Aziz Qureshi ICAR-Indian Institute of Oilseeds Research, Hyderabad growth. Trichoderma and Pseudomonas commercial products available in India are having limited bioefficacy, tested locally but not in different agroclimatic regions of the country. This has resulted in inconsistent performance of these products. Though there are more than 200 producers of these agents in India, the formulated product available for farmers for seed treatment is about 2000 MT only. To treat every seed sown in this country we need to produce lakhs of tones of Trichoderma/

Rationale Intensive research work is being carried out at ICAR-IIOR, Hyderabad on identification of potential antagonists and their efficacy against few soilborne and foliar fungal pathogens. In India, very limited research has been done to select the antagonists which have endophytic root colonizing and broad spectrum action. Not much is known on abiotic stress tolerance abilities of these strains and their role in promotion of plant

61

AMAAS - Annual Report 2015-16 Pseudomonas based biofungicides. To have increased demand and acceptance by farmers, there is a need to identify microbial agents that are; Root endophytes, Tolerant to salinity, drought and high temperature, Able to enhance plant growth, Induce defense response against biotic/ abiotic stresses. The project aims to address these problems and identify Trichoderma or consortia of microbials with broad spectrum abilities, develop bioformulation and integrating with ICM technologies under farmers field conditions.

untreated control (Fig.3b). Vigour index was also higher in T. asperellum TN13 (1292) treated plants compared to untreated control (887)

l High Vigour index (9591) has been recorded in plots where seed treatment with T. asperellum TN13, T. harzianum Th4d, and T. asperellum A5 with was done

l High root DMW was noticed in Trichoderma treated plants over control (Fig.2).

Objectives

l To identify strains of microbial agents with broad host range, ameliorators of biotic and abiotic stresses, enhance plant growth.

l To isolate and characterize bioactive metabolites (BAM) from potential strains. Fig. 2: Evaluation of Trichoderma strains in soils with EC= 6dS/m at AICRP on Management of salt affected soils, ARS. UAS-R, Gangavati, Karnataka A: Plant growth; B: Root growth

l To develop formulations of potential bioagents/ consortia/ BAM and evaluation in different agro-climatic zones of India.

l Among different Trichoderma isolates tested, seed

l Generate data required for registration with CIB & RC and

treatment with T. asperellum TaA5, T. harzianum Th4d resulted in better seed germination, seedling growth compared to untreated checks in both irrigated and drought stressed pots.

commercialization of potential technologies. SignificantAchievements

l Trichoderma isolates tolerant to salinity and drought (T.

l Relative water content in castor plants at 9 days stress was

harzianum Th4d, T. asperellum TN13 , TV5, TA5 and A7) were identified which showed saline tolerance upto 1.5M NaCl concentration and drought tolerance upto -12 bar PEG (i.e. 32.62% PEG)

65% in T. asperellum TaA5 treated plants as compared to 45% in untreated check. Membrane stability index was improved with T. asperellum TaA5 treatment (70%) against untreated control (32%).

l Seed treatment with T. asperellum TN13 resulted in better

l T. harzianum ThN1 showed least collar rot incidence

seed germination and seedling growth at EC=4 compared to untreated control greenhouse (Fig.1)

(10%) followed by T. harzianum Th4d (20%) and T. harzianum N2 (40%) compared with pathogen check (70.0%) and T. harzianum ThN1 showed high reduction percent (55.7%) over pathogen (Aspergillus niger) in groundnut. (Fig. 3).

Fig. 1: Growth of sunflower at different salinity levels with Trichoderma seed treatment A: at EC=4 ds/m; B: at EC= 6 ds/m

Fig. 3: Effect of Trichoderma seed treatment against A.niger in groundnut in germination towels; ThN1- T.harzianum ;ThN2-T.harzianum ; Th4dT.harzianum and pathogen A.niger

l At soil salinity of EC=6ds/m T. asperellum TN13 seed

l Under greenhouse conditions seed treatments, T.

treatment resulted in 80% plant stand compared to 40% in

harzianum Th4d and T. harzianum ThN2 showed least

62

AMAAS - Annual Report 2015-16 asperellum TN13 , TV5, TA5 and A7 were found to be saline and drought tolerant as they showed saline tolerance upto 1.5M NaCl concentration and drought tolerance upto -12 bar PEG (i.e. 32.62% PEG). In natural saline soil (EC = 6 dS/m)T. asperellum TN13, T. harzianum Th4d, T. and T. asperellum A5 were found superior in green house as well as in field as these strains resulted in improvemeny ingermination, vigour index and total dry matter of sunflower in natural saline soil compared to untreated control. T. asperellum TA5,T. harzianum Th4d were found to impart drought tolerance in castor in green house conditions as there was increase in germination, improvement in relative water content and Membrane stability index compared to untreated control at 9D stress. T. harzianum Th4d, T. harzianum ThN1 and T. harzianum ThN1 were found to have broad spectrum biocontrol ability against P. nicotinae in castor and A. niger in groundnut. Bioactive metabolites isolated from potential Trichoderma strains T. harzianum Th4d and T. longibrachiatum TaDOR 673 were found efficient in inhibiting growth of Sclerotium rolfsii and Fusarium oxysporum f. sp. ricini in vitro. In the compatibility studies of seed coat polymers with biocontrol agent Trichoderma harzianum Th4d, the combination of chitosan with biocontrol agent gave the highest germination percentage and vigour index than the polymers and biocontrol agent used alone.

disease severity of Phytophthora leaf blight (15.0%) whereas pathogen check recorded 60% disease severity. The study clearly showed induction of defense in castor by Trichoderma strains against Phytophthora seedling blight. (Fig.4).

Fig. 4: Induced systemic resistance against P. nicotianae in castor, severity of leaf blight in treatments Viz., (a) Seed treatment with T. harzianum Th4d and leaf inoculated with P. nicotianae; (b) Seed treatment with T. harzianumThN1 and leaf inoculated with P. nicotianae; (C) Seed treatment with T. harzianum ThN2 and leaf inoculated with P. nicotianae; (d) Untreated control leaf inoculated with P. nicotianae.

l Bioactive metabolites were isolated from potential strains of Trichoderma ( T. harzianum Th4d and T. longibrachiatum TaDOR 673) and their efficacy was tested against Sclerotium rolfsii, Fusarium oxysporum f. sp. carthami, Macrophomina phaseolina and Aspergillus niger and they were found to effective in inhibiting growth of S. rolfsii and F. carthami under in vitro conditions.

Paper published fromAMAAS work

l Prasad RD, Navaneetha T and Venakateswar Rao L (2016) Plant growth promotion and Induced defence response in safflower (Carthamus tinctorius L.) by Trichoderma. Journal of Biological Control, 30:40-48

l In the compatibility studies of seed coat polymers with Trichoderma harzianum, treatment of seeds with combination of chitosan with biocontrol agent gave the highest germination percentage (95%) (Fig.9) and vigour index (3673) than the polymers and biocontrol agent used alone. Fusarium oxysporum f. sp. ricini incited seed and seedling root rot is significantly low in combination of chitosan with biocontrol agent (20%) compared to pathogen check (70%).

Conference and workshop attended

l IPS 6th International Conference “Plant, Pathogens and People” “Challenges in Plant Pathology to Benefit Humankind” February 23-27, 2016 at New Delhi

l ISPP National Symposium on “Climate Challenges: Status and Management of Plant Diseases” from 1 to 3rd December, 2015 at Hyderabad.

Conclusion

l UNEP-GEF-MoEFF & CC-ABS Project workshop on

Seventy five Trichoderma strains were isolated from different agroclimatic regions and were screened in vitro for salinity and drought tolerance. T. harzianum Th4d, T.

Strengthening the implementation of biodiversity act and rules with focus on its Access and Benefit sharing provisions” on March 4th 2016 at Vijayawada,AP.

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AMAAS - Annual Report 2015-16

Development of PGPR Inoculant Bioformulations for Rhizosphere Management in Enhancing Biomass of Fodder Crops PI : Srinivasan, R Co-PIs : H. V. Singh ICAR-Indian Grassland and Fodder Research Institute, Jhansi

l Standardizing parameters / conditions of bioformulations

Rationale

that retain large number of viable cells.

Livestock rearing is one of the major occupations of small and marginal farmers in India, which contributes to about 25% of agricultural GDP. At present fodder availability shows a net deficit of 62.7% green fodder and 23.4% dry forages. Fodder and food demand is ever-increasing due to continuous increase in populations, degradation of land and natural resources, and st yield stagnation. Major challenge of 21 century is requirement of environmentally sound & sustainable crop production technologies. Plant growth-promoting potential can be influenced by managing the indigenous microbial potential, by the introduction of organic or inorganic amendments, etc and by applying autochthonous microorganisms as plant growthpromoting or biocontrol agents. Microbial inoculants have several advantages viz ., supply macronutrients and micronutrients. Bacterial nitrogen (N2) fixation by rhizobia legume fodders, free-living bacteria - Azotobacter, Azospirillum, and Burkholderia - fix N2 in various fodder crops. Microorganisms are integral to soil P cycle, specific microorganisms enhances P availability in soil, can play key role in developing a more sustainable fodder production. Arbuscular mycorrhizal fungi (AMF) produces glomalin, an organic substance (glycoprotein) is important for soil aggregate stability. Exploitation of higher glomalin producers will be beneficial. Thus, microorganisms can be used as biofertilizers, plant strengtheners, phytostimulators and biocontrol agents for environmentally friendly strategies for enhancing fodder yield. We do not have a suitable PGPR bioinoculants specific for fodder crops, which can enhance the biomass production. With this background, this project has been formulated with following objectives.

l Manipulation of rhizosphere of selected fodder crop(s) with developed PGPR bioformulations and evaluation of its performance in vitro in different fodder crop(s).

l Testing for soil fertility enhancement and improving plant biomass yield of important fodder crops by the use of promising inoculants at different experimental sites.

l To generate data required for registration with FCO, 1985 and CIB & RC of potential technologies of PGPR based microbial inoculants suitable for fodder crops. SignificantAchievements

l A total of 15 Azotobacter isolates have been isolated from rhizosphere soil of Stylosanthes, Clitoria, Cenchrus grass, Sehima, Brachiaria grass, Dichanthium, Heteropogan, Desmanthus grasses.

l A total of 25 fluorescent Pseudomonas isolates have been isolated from roots of guinea grass, Sehima, rhizosphere soils of oats, peas, guinea grass, stylo, clitoria.

l 35 Rhizobium isolates have been isolated from root nodules of berseem, lucerne, groundnut, soybean, Sesbania.

l All Rhizobium isolates have been tested with their hosts for nodulation potential by host inoculation study and found nodulating successfully.

l Screening for biocontrol ability of the PGPR isolates has been done and about 7 PGPR bacterial isolates were found inhibiting various plant pathogens such as Alternaria, Rhizoctonia bataticola, Sclerotium, Fusarium sp. (Fig. 1).

l Four PGP fungi have been isolated from rhizosphere soils

Objectives

l Development of PGPR based microbial inoculants for

of Cenchrus ciliaris, Heteropogon, Dichanthium and their root infection studies are being carried out under in vitro condition (Fig. 2).

multi-nutrients supplement suitable for important fodder crops.

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AMAAS - Annual Report 2015-16

Fig. 1: Inhibition of growth of Alternaria sp. by PGPR

Fig. 2: Influence of PGP Fungi on berseem root growth

of fodder and grasses. A total 7 PGPR bacterial isolates were found with bio-control activity.

Conclusion

Fifteen Azotobacter, 25 fluorescent Pseudomonads, 35 Rhizobium & 4 fungi have been isolated from rhizosphere soil

Endophytic Microorganisms for Management of Drought in Rainfed Maize and Groundnut PI : Minakshi Grover Co-PIs : S. K. Yadav, S. Desai ICAR-Central Research Institute for DrylandAgriculture, Hyderabad

l To study the mechanisms behind microbially induced

Rationale

abiotic stress tolerance in host plants through omics (proteomics/genomics) approaches.

Maize and groundnut are important rainfed crops cultivated on large areas across India. These crops are exposed to drought very often during growth period, consequently resulting in significant yield losses. Rainfed agricultural production systems generally are low input systems due to risks associated with uncertainties of weather. Any attempt to manage abiotic stresses with low cost methods like use of abiotic stress alleviating microorganisms (ASAM) will contribute to sustainable production of rain-fed crops and minimize the risk to the farmers. Hence, this project aims to utilize the biodiversity of endophytic microorganisms available in rainfed agroecosystem for management of abiotic stresses in maize and groundnut plants through a systematic programme of isolation, screening, characterization, greenhouse and field evaluation

SignificantAchievements

l Forty isolates selected (out of 80) on the basis of primary screening were subjected to secondary screening with maize (Bioseed 9681) under drought stress conditions using sterile soil. Eleven isolates selected were further evaluated using non-sterile soil and for growth promotion of maize under drought stress (Fig. 1) Eight promising isolates selected based on plant growth and physiological (chlorophyll, relative water content, proline and total sugar) parameters.

Objectives

l To explore endophytic (seed/ stem) microflora of maize and groundnut for selection of multi-functional microorganisms having abiotic stress tolerance with multiple PGP traits.

Control

Treated

Fig. 1: Maize plants without and with MSEB treatment under drought

l Out of 101 isolates tested, 32 isolates subjected to

l To evaluate stress tolerant isolates on maize/groundnut

secondary screening using both sterile and non sterile soil under drought conditions (induced after 60 days of germination). Promising isolates (8) selected based on growth, physiological parameters to be evaluated further.

under controlled drought stress conditions by pot culture/phenomics platform. Field evaluation (rainfed conditions) of selected strains/combinations

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AMAAS - Annual Report 2015-16

l Selected eight maize seed endophyte evaluated as seed

produce 5 or 6 lytic enzymes. Number of isolates able to produce amylases, lipase, esterase, protease, cellulase and pectinase were 47, 17, 16, 25, 24, 45 respectively.

inoculants in kharif maize (rainfed). Intermittent drought spells at vegetative stage, caused wilting and rolling of leaves. Inoculated treatments showed lesser extent of wilting and rolling of leaves over control treatments probably due to high soil moisture content over control treatment in the root zone (Fig. 2). Inoculated treatments showed significant improvement in plant biomass parameters. All the inoculated treatments exhibited higher chlorophyll, sugar, and relative water content when compared with uninoculated control. Inoculation also improved yield parameters including cob length, diameter, no. of seeds per cob, 100 seeds weght weigh, total grain yield (upto 23% increase) and stover biomass. Control plot

Lipase

Esterase

Amylase

Pectinase

Cellulase

Protease

Treated Plot 21 Days after sowing

Fig. 3: Lytic enzymes production by endophytic bacteria

l Rifampicin was used as marker for studying endophytic nature of selected maize seed endophytic strains. Rifampicin resistant mutant for selected eight maize seed bacterial endophytic strains were generated by spontaneous mutagenesis. Rifampicin resistant strains were used as seed inoculants for maize just before sowing. After 15 days of germination, re-isolation of seed inoculated isolates from surface sterilized root, stem and leaves confirmed true endophytic nature of selected strains (MSEB-72, MSEB-78, MSEB-8) (Fig. 4).

48 Days after sowing

Fig. 2: Maize plants with and without MSEB treatments, response to moisture stress

l Production of lytic enzymes (amylase, esterase, lipase, protease, cellulase and pectinase) by maize seed and groundnut stem endophytic bacteria was studied by using suitable substrate and following standard protocols (Fig. 3). Among 80 MSEB, no of isolates positive for amylase, esterase, lipase, protease, cellulose and pectinase were 55, 41, 46, 55, 47, 53 respectively. Of 80, 10 isolates could produce all the six lytic enzymes. Majority of the isolates (51) could produce four or more lytic enzymes. among 101 GSEB, 24 isolates could produce none of the lytic enzyme, majority could produce one or two, whereas few could

Fig. 4: Tracking of Rif resistant MSEB strains in different plant parts under non-sterile conditions

66

AMAAS - Annual Report 2015-16 (2016). Actinomycetes as mitigators of climate change and abiotic stress. In: Gopalakrishnan S, Satya A, Vijayabharathi R (Ed), Plant growth-promoting actinomycetes: A new avenue for enhancing the productivity and soil fertility of grain Legumes. SpringerVerlag Berlin Heidelberg.Accepted

Conclusion Screening at multiple levels under drought stress could help in narrowing down the number of promising isolates for further evaluation. Inoculation influenced plant growth, physiological and biochemical parameters under drought conditions in the field. Yield benefits (upto 23%) due to bacterial inoculation recorded.

l Grover M, Venkateswarlu B, Desai S, Gopinath KA, Srinivasarao Ch (2016). Application of Microbiology in Dryland Agriculture. In: Muhammad Farooq and Kadambot HM Siddique (Ed), Dryland Agriculture. Springer-Verlag Berlin Heidelberg.Accepted

Lytic enzymes production (in vitro) profile of endophytic bacteriarevealed maize seed endophytes (80 isolates) more diverse and efficient than groundnut stem endophytes (101 isolates) in terms of no. of lytic enzymes produced by each isolate.

Conference and workshop attended

l Shrey Bodhankar, H. Sunaina, Minakshi Grover, Gopal Reddy, S. Kavita and Sk. Rasul (2015) Endophytic diversity for sustainable agriculture and human nutrition. Presented in SUSFANS seminar held at IICT Hyderabad 1st – 2nd December 2015 P.65 (SUSFANS-2015) (Oral Presentation)

Rifampicin resistance can be used as biochemical marker for tracking inoculated bacteria in different parts of plant. Book Chapters

l Grover M, Bodhankar S, Maheswari M, Srinivasarao Ch

Mass Production of Bacillus thuringiensis (Bt) and Beauveria bassiana, Formulation as Oil Based Suspension Concentrates Singly and on Combination and Field Evaluation PI : P. S. Vimala Devi Co-PIs : P. Duraimurgan ICAR- Indian Institute of Oilseeds Research, Hyderabad of microbials available commercially nor any research in the direction, more so for Bt with Beauveria bassiana. No information exists on relation of Bt particle size to efficacy. Hence this project has been undertaken with an aim to mass produce virulent isolates of Bt and B. bassiana and develop oil based SC formulations singly and in combination with a view to come out with superior formulations of these microbials that are comparable to chemical insecticides in efficacy.

Rationale Under the completedAMAAS project (2007-2014) entitled “Development of suitable formulations of potential bioagents for management of important diseases and pests of Sunflower, Safflower and Castor”, we developed a storable combination formulation of Bt + B. bassiana with an extended shelf-life of 2 years (patent application no. 1118/CHE/2013). The formulation has been field tested against Helicoverpa armigera (Hübner) at 2 AICRP centres of sunflower and several centres of AICRP (pigeon pea) and found to be highly effective with performance on par with Spinosad and Profenofos. One new Bt isolate - DOR isolate no.127 (NAIMCC accession no. B-01463) effective against Spodoptera litura (Fabricius) has also been identified, used in the combination formulation and found effective against S. litura in lab and in the field.

Objectives

l Mass production of virulent isolates of Bt and B. bassiana / Nomuraea rileyi

l Milling of Bt to fine particle size of 5-20 μm and formulation as a suspension concentrate

l Microencapsulation of B. bassiana conidia through spray drying with humic acid and formulation development

l Formulation of Bt singly and Bt + B. bassiana/ N. rileyi in

We also conducted studies on efficacy of Bt particles of different sizes (ranging micron to submicron) through larval bioassays against 6 days old H. armigera larvae. The studies revealed an inverse correlation of efficacy to particle size i.e., efficacy increases with decrease in particle size. Reduction in particle size could therefore lower the effective dose requirement.

combination as stable suspension concentrates

l Large scale field evaluation on oilseed crops (castor & sunflower) as components of IPM against major lepidopteran pests SignificantAchievements

l A novel isolate of Bacillus thuringiensis var. kurstaki DOR

As on date there are no storable combination formulations

Bt-127 with a broad host range was identified with

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AMAAS - Annual Report 2015-16 potencies of 34,833 and 50,200 IU/mg against H. armigera and Achaea janata (Linnaeus) respectively. The isolate was also highly effective against S. exigua (Hübner) and S. litura with potencies of 46,205 and 71,722 SU/mg respectively. Hence this isolate could be a candidate for development into a reference standard since the International Btk reference standard HD-1-S-1980 is exhausted and no longer available. This would promote identification of more virulent Btk isolates with high potency against several lepidopteran pests for effective insect pest management.

(3ml contains 1g of Bt). Similarly shelf life studies at 3, 6, 9 and 12 revealed that there is no decrease in the heat viable spore count.

l Field testing of the Bt-127 and Nomuraea rileyi SC formulation was undertaken during kharif 2015 against S. litura at RARS-Palem, TCRS-Yethapur and IIORHyderabad. Treatments included Bt-127 SC formulation @ 3ml/l, Combination SC formulation of Bt-127 + N. rileyi @ 3ml/l, Combination SC formulation of Bt-127 + Beauveria bassiana @ 3ml/l, Bt-127 @ 1g/l, N. rileyi @ 1 x 1010 spores/l, B. bassiana @ 1 x 1010 spores/l, Profenofos @ 1ml/l, Untreated control. Treatments were imposed on 40 days old crop against larval masses of 4-5 days old larvae (Fig. 2).

l Bt-127 technical powder (105μ) was milled with 3mm zirconia balls at 300 rpm in a planetary ball mill. Dynamic light scattering analysis of samples drawn at 30, 60, 90, 120, 150, 180, 210, 240 min revealed particle sizes lower than 5μ viz., 1171, 559, 252, 210, 157, 112, 36, 35 nm respectively. Particle size with 559 nm was identified as optimum particle size of Bt-127 against S. litura, H. armigera, A. janata (Fig. 1).

Unsprayed Control plot

Bt-127 SC formulation sprayed plot

Fig. 2: Field testing of Bt-127 SC formulation against S. litura on castor

l Field testing of the Bt-127 and N. rileyi SC formulation was undertaken during kharif 2015 against H. armigera at Nandyal, ORS-Lathur and IIOR-Hyderabad. Treatments included Bt-127 SC formulation @ 2ml/l, Combination SC formulation of Bt-127 + N. rileyi @ 2ml/l, Combination SC formulation of Bt-127 + B. bassiana @ 2ml/l, Bt-127 @ 1g/l, N. rileyi @ 1 x 1010 spores/l, B. bassiana @ 1 x 1010 spores/l, Profenofos @ 1ml/l, Untreated control. Treatments were imposed on 40 days old crop against larval masses of 4-5 days old larvae. Conclusion

Fig. 1: Powders of Bt-127 drawn after different milling durations

Particle size reduction to micron/submicron-scale through ball milling is a promising approach for increasing the efficacy of Bt. SC formulations of Bt-127 and Bt + N. rileyi were found effective against S. litura on castor RARS-Palem, TCRSYethapur and IIOR-Hyderabad (83-99%) at the test locations by 5 days after spray. All three SC formulations - Bt-127, Bt + N. rileyi and Bt + B. bassiana were effective against H. armigera on sunflower at Nandyal, ORS-Lathur and IIORHyderabad (90-100%) at the test locations by 5 days after spray

l Bt particles of size 559 nm with polydispersity index 0.338, CFU 3.3 x 1017/g and protein 178 μg/g resulted in higher mortality of 7 days old Spodoptera litura larvae 72 h post treatment @ 1.0 mg/ml i.e., 83.3% in comparison to 56.7% mortality caused by unmilled Bt powder. These particles can be used for development of formulations with good stability. 5 Kg of Bt-127 technical powder was produced through SSF out of which 1.5 kg was milled in planetary ball mill

l Bt particles of 559 nm and Bt 105μ were formulated as

Conference and workshop attended

suspension concentrates singly and in combination with N. rileyi and B. bassiana and evaluated against S. litura and H. armigera in lab and LC50 values were generated.

l Attended the follow-up meeting on 'Epidemic of whitefly in cotton in Punjab, Haryana and Rajasthan' and 'factors responsible for lower production of Soybean in Madhya Pradesh" on 27th October, 2015 in the NASC Conference Hall, Pusa, New Delhi under the Chairmanship of Secretary, DARE & DG, ICAR

l Shelf life studies were carried out with the samples that were stored at room temperature. At 0 month, Bt-127 SC formulations and Bt-127 + N. rileyi contained heat viable Bt spores of 3.3 x 1017/ 3ml and 1.7 x 1017/3ml respectively

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Development of Formulations of Beauveria bassiana, Metarhizium anisopliae and Lecanicillium spp. and Paecilomyces fumosoroseus for Management of Certain Sucking Pests in Vegetable Crops PI : B. Ramanujam Co-PIs : P. Duraimurgan ICAR- Indian Institute of Oilseeds Research, Hyderabad Rationale For sucking insects, entomopathogenic fungi are the most appropriate microbial bioagents as they infect the insects directly by contact and do not require ingestion for infection as in case of bacterial and viral pathogens. Several commercial formulations of B. bassiana, M. anisopliae, Lecanicillium spp. and P. fumosoroseus have been developed in other countries and used for management of sucking pests in the open field cultivation and also in the protected cultivation. There is a need to develop formulations of these fungi suitable for dry weather conditions with better field persistence and efficacy for the management of sucking pests.

Ma-4 isolate treated brinjal leaf

Higher population of brinjal aphids in untreated control brinjal leaf Fig. 1: Effect of Ma-4 isolate on brinjal aphids

l Cabbage Aphids (Brevicoryne brassicae): Among the nine isolates tested, B. bassiana, Bb-5a showed significantly highest pest reduction of 68.64 % over control (Fig. 2). With regard to yield, no significant difference was observed among the treatments, even though higher yield (58542 Kg/ha) was noticed in Bb-5a isolate compared to the yield of untreated control (44236 kg/ha)

Objectives

l Development of oil formulations of B. bassiana, M. anisopliae, Lecanicillium spp. and P. fumosoroseus with longer shelf life and consistent field persistence and efficacy.

l Testing of oil formulations of B. bassiana, M. anisopliae, Lecanicillium spp. and P. fumosoroseus for management of sucking pests like Aphis craccivora, Bemisia tabaci, Myzus persicae and Scirtothrips dorsalis in vegetable crops in open field conditions and protected cultivation.

Mycosis of Bb-5a on Brevicoryne brassicae in cabbage leaf

SignificantAchievements

Brevicoryne brassicae on untreated control cabbage leaf

Fig. 2: Effect of Bb-5a isolate on cabbage aphids

l Field trial with oil formulations of entomofungal pathogens

l Chilli Aphids (Aphis gossypii): Maximum percent

against sucking pests in vegetable crops was carried out with nine promising isolates viz., Beauveria bassiana (Bb5a, Bb-12, Bb-68), Metarhizium anisopliae (Ma-4, Ma-6, Ma-11) and Lecanicillium lecanii (Vl-8, Vl-34 & Vl-35) at ICAR-NBAIR, Attur Research Farm, Bengaluru during kharif season, 2015.

reduction of chilli aphids was noticed in Bb-5a (47.82 %) followed by Vl-8 (41.89 %) and Ma-4 (35.62 %) which was on par with each other. Higher yield was recorded in Bb-5a (14383 kg/ha), Ma-4 (14111 kg/ha) and Vl-8 (13691 kg/ha) compared to the yield of control plot (12037 kg/ha).

l Brinjal Aphids (Aphis gossipii): Significantly higher

l French bean Aphids (Aphis fabae): Among all the isolates

percent reduction of brinjal aphids was observed in Ma-4 isolate (58.51 %) (Fig. 1), followed by Bb-5a isolate (53.39 %) and Vl-8 isolate (48.59 %) and these were on par with each other. Significantly higher yield was noticed in Ma-4 (25173 kg/ha), Bb-5a (24901 kg/ha) and Vl-8 (24506 kg/ha) in comparison with the yield in control (17815 kg/ha).

tested, significantly higher percent reduction of French bean aphid was observed in Bb-5a (54.52 %) followed by Ma-4 (52.10 %) which was on par with each other. The maximum yield was observed in Bb-5a (18851 kg/ha) followed by Ma-4 (17955 kg/ha) which are on par with each other and the untreated control gave the lowest yield (15735 kg/ha).

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AMAAS - Annual Report 2015-16

l Cauliflower Aphids (Brevicoryne brassicae) Rabi-2015-

genomic DNA extracted from untreated leaf tissues failed to amplify any PCR product (Fig. 4). The positive results of colonization of four isolates of B. bassiana and M. anisopliae in leaf tissues observed in plating technique were confirmed by the PCR amplification.

16: Field trial with oil formulations of entomofungal pathogens against cauliflower aphid ( Brevicoryne brassicae) was carried out with nine isolates (B. bassiana Bb-5a, Bb-12, Bb-68, M. anisopliae Ma-4, Ma-6, Ma-20 and L. lecanii Vl-23, Vl-31 & Vl-36) at NBAIR, Attur Research Farm, Bengaluru during Rabi season, 2015. Among the nine isolates tested, B. bassiana, Bb-68 showed significantly highest pest reduction of 60.15 % and was on par with Bb-5a (56.04 %) and Ma-20 (52.37 %). No significant differences in yield were observed in among the treatments including control, even though higher yield was noticed in Bb-68 (61009 Kg/ha) compared to the yield in control (43496 kg/ha).

l Cabbage: A glasshouse experiment was conducted to establish the ability of isolates of B. bassiana (NBAIR-Bb5a and Bb-45) and M. anisopliae (NBAIR-Ma-4 and Ma35) as endophytes in cabbage through foliar application of conidial suspension. The colonization and persistence of the four isolates were studied in leaf tissues during 15, 30, 45, 60 and 75 days after treatment (DAT). Detection of B. bassiana and M. anisopliae in cabbage leaf tissues was carried out using plating technique and PCR method. Among the four isolates tested, Ma-4 isolate showed leaf colonization upto 60 days after treatment where as Bb-5a during 30-45 days after treatment, Bb-45 in 15 and 45DAT and Ma-35 during 15-30 DAT showed colonization (Fig. 3). No colonization of B. bassiana and M. anisopliae was observed in the untreated cabbage leaf tissues (Fig. 3). The genomic DNA extracted from treated leaf tissues showed amplification of B. bassiana and M. anisopliae using specific primers at 450bp respectively and the

Fig. 4: PCR amplification of genomic DNA extracted from treated and untreated cabbage leaf tissues: 1-Bb-5a, 2-Bb-45, 3-Ma-4, 4-Ma-35, 5Control, 6-100bp ladder.

l Cauliflower: Field experiment was conducted at NBAIR Attur farm during rabi season for the establishment of entomofungal pathogens as endophytes using oil formulations in cauliflower leaf tissues. The NBAIR six isolates (Bb-5a, Bb-12, Bb-68, Ma-4, Ma-6 and Ma-20) were tested for endophytic ability in cauliflower leaf tissues through foliar application. Detection of B. bassiana and M. anisopliae in cauliflower leaf tissues was carried out using plating technique and PCR method. Among the six isolates tested, Bb-5a and Bb-68 isolates showed colonization during 15-30 days after treatment (DAT), whereas, Ma-6 isolate showed colonization at 15DAT (Fig. 5). No colonization of B. bassiana and M. anisopliae was observed in the untreated cauliflower leaf tissues (Fig. 5).

Fig. 3: Beauveria bassiana and Metarhizium anisopliae fungal growth from treated cabbage leaf tissues and no fungal growth from untreated cabbage leaf tissues

Fig. 5: B. bassiana and M. anisopliae fungal growth from treated cauliflower leaf tissues and no B. bassiana and M. anisopliae fungal growth from untreated cauliflower leaf tissues

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AMAAS - Annual Report 2015-16 (Lepidoptera: Pyralidae) and the effect of solid substrates on conidial production and virulence. Journal of Pure and Applied Microbiology 9:2979-2986.

The genomic DNA extracted from treated leaf tissues showed amplification of B. bassiana and M. anisopliae using specific primers at 450bp respectively and the genomic DNA extracted from untreated leaf tissues failed to amplify any PCR product (Fig. 6). The positive results of colonization of B. bassiana and M. anisopliae in leaf tissues observed in plating technique were confirmed by the PCR amplification.

l B. Ramanujam, B. Poornesha, K.R. Yatish and S. Renuka. 2015. Evaluation of pathogenicity of different isolates of Metarhizium anisopliae (Metchnik off) Soroking on maize stem borer Chilo partellus (Swinhoe) using laboratory bioassay. Biopesticides International 11:89-95.

l Renuka. S and Bonam Ramanujam. 2016. Fungal endophytes from maize (Zea mays L.): Isolation, identification and screening against Maize stem borer, Chilopartellus. Accepted in Journal of Pure and Applied Microbiology.

l B. Ramanujam, B. Poornesha and K.R. Yatish. 2016. Screening of Beauveria bassiana and Metarhizium anisopliae isolates against Sesamia inferens (Walker). Accepted in Indian Journal of Entomology.

l Renuka. S, Ramanujam. B and Poornesha. B 2016. E n d o p h y t i c a b i l i t y o f d i ff e r e n t i s o l a t e s o f entomopathogenic fungi Beauveria bassiana (Balsamo) Vuilleminin stem and leaf tissues of maize (Zea mays L.) Accepted in Indian Journal of Microbiology.

Fig. 6. PCR amplification of genomic DNA extracted from treated and untreated cauliflower leaf tissues:1-100bp ladder, 2-control, 3-Bb-5a, 4-Bb12, 5-Bb-68, 6-Ma-4, 7-Ma-6, 8-Ma-20, 6-100bp ladder.

Conclusion

Conference and workshop attended

Based on the field trials, three promising isolates viz., Beauveria bassiana (Bb-5a), Metarhizium anisopliae (Ma-4) and Lecanicillium lecanii (Vl-8) were identified as promising isolates against brinjal aphid, cabbage aphid, chilli aphid, French bean aphid. For cauliflower aphid, Bb-68 isolate of B. bassiana was identified as promising candidate. Isolates of B. bassiana (Bb-5a and Bb-45) and M. anisopliae (Ma-4 and Ma35) were established as endophytes in cabbage leaf tissues by artificial inoculation. In cauliflower, Bb-5a, Bb-68 and Ma-6 isolates were established as endophytes in cauliflower leaf tissuesthrough foliar application.

l B. Ramanujam, B. Poornesha, T.G. Avinash, S. Renuka, A.N. Shylesha and R. Rangeshwaran. 2015. Endophytic establishment of Beauveria bassiana (Balsamo) Vuillemin in sorghum. Paper presented at 56th Annual Conference of Association of Microbiologists of India during December 7-10, 2015 at Jawaharlal Nehru University, New Delhi. AMP no.47 p.252.

l Renuka. S, Ramanujam. B and Poornesha. B. 2015. Endophytic colonization of Entomopathogenic fungus Beauveria bassiana (Balsamo) Vuilleminin Maize (Zea mays L.). Paper presented at 56th Annual Conference of Association of Microbiologists of India during December 7-10, 2015 at Jawaharlal Nehru University, New Delhi. AMP no.48 p.253.

Paper published fromAMAAS work

l Renuka. S, Bonam Ramanujam and Poornesha. B. 2015. Screening of Beauveria bassiana (Balsamo) Vuillemin isolates against maize stem borer, Chilo partellus

Role of Potential Microorganisms in Seed and Crop Health of Rice, Wheat and Mustard PI : Madan Kumar Co-PIs : S. Rajendra Prasad1, Pawan K. Sharma2, Umesh Kamble1, Ramesh K.V 1, Ragvendra D1 1 ICAR- Indian Institute of Seed Science, Maunath Bhanjan 2 ICAR- National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan Several major fungal and bacterial diseases affect the yield of these crops due to which 20-30% losses in production. The use of synthetic chemicals in agriculture during the last three

Rationale Wheat, rice and mustard are important crops of the India and occupy the premier place among cereals and oil seeds.

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AMAAS - Annual Report 2015-16 chosen for pot and field trial analysis of susceptible rice variety Pusa Sugandh (PS-5).

decades has raised a number of ecological problems. After scrutinizing available reviews and reports, it has been found that there is lack of comprehensive and systematic literature of northern India with special reference to growth promotion and disease management for rice, wheat and mustard. Therefore, keeping in view the harmful effect of disease controlling and growth promoting chemicals, there is urgent need to discover alternate growth promotion and control strategies with novel and exiting microorganisms like Trichoderma sp., Pseudomonas sp. and Bacillus sp.for successful healthy plant establishment and disease control.

l Talc based bioformulation were prepared for testing of these four bacterial cultures.

l There were five type of treatment i.e. Biopriming with bacteria, Biopriming with bacteria and pathogen, Biopriming with consortia, root dipping and soil application for all four selected strains along with positive and negative control were planned for pot trial (Fig.1) and field trial analysis (Fig.2).

Objectives

l Isolation of different microbes for growth promotion and disease management in wheat, rice and mustard from rhizosphere, phylloplane & endosphere and procurement of microbes from National Bureau of Agriculturally Important Microorganisms, Mau.

l Characterization of potential isolates for growth promotion and diseases management in wheat, rice and mustard.

l Green house assay for growth promotion and diseases management of few selected and superior microorganisms for wheat, rice and mustard.

l Field trial of few selected and superior micro-organisms for growth promotion and diseases management in wheat, rice and mustard at DSR, Mau.

Fig. 1: Poly bag trial of rice (PS-5) treated with LWR19 strain

l Development of cost effective and environmentally safe

l A nursery was planted with 22 treatment combinations

bio-pesticide formulations for sustainable agriculture.

along with control in first week of July. After one month seedlings were transplanted in to the field having three replicate for each treatment combinations (Fig.2).

SignificantAchievements

l A total of 300 bacterial strains were isolated from rhizosphere, endosphere and phylloplane ofrice, wheat and mustard from various places of different agro-climatic zones of India. All the strains were tested for the biocontrol assay against several pathogens causing diseases in rice, wheat and mustard.

l Dual culture test for antagonistic activity were performed for screening of potential strains within isolated bacterial strains.

l A total 35 bacterial strains showed potential against Fusarium oxysporum sp. ciceri, Ustilaginoidea virens, Magnaporthe oryzae, Drechslera teramera and Sclerotium rolfsi pathogens.

l Four potential bacterial strains among two (URR7 and

Fig. 2: Field trial of rice (PS-5) treated with URR7, LWR19, BRR10 and BRR15 strain

LWR19) against pathogen Magnaporth eoryzae and two (BRR10 and BRR15) against Ustilaginoidea virens were

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AMAAS - Annual Report 2015-16

l Agronomical data of rice pot experiment viz. Root length,

Ustilaginoidea virens as well as showed good result for plant growth promotion of rice crop. So these strains could be useful for plant growth promotion as biofertilizer and in disease management as bio-control agent. But the field experiment should be repeated before the recommendation at former level.

shoot length, germination percent; field experiment viz. No. of tillers, plant height, 50% flowering were recorded and biochemical analysis is under progress.

l 16S rRNA gene sequence based identification of 35 potential bacteria is in progress.

Conference and workshop attended

l National conference 'MEEDTA' at HNB Garhwal

Conclusion

University on 29-30 October 2015

Among 300 bacterial isolates four bacteria showed potential among two (URR7 and LWR19) against pathogen Magnaporthe oryzae and two (BRR10 and BRR15) against

l International Conference on Int. BIONANO-2016 at Amity University, Gwalior, MP on 10-12 Feb, 2016

Development of Effective Salt Tolerant Microorganisms to Mitigate Salt Stress for Crop Production in Salt affected Soils PI : P.K. Joshi ICAR-Central Soil Salinity Research Institute, Karnal Rationale

SignificantAchievements

High concentration of salts in soil has a detrimental effect on crops and microorganisms. Salt tolerant microbes help plants in overcoming the effect of salt stress by different mechanisms. They can alter the availability of nutrients so as to maintain Na, K ratio in plants. They are also involved in production of anti-oxidants to prevent injury to the plants because of salt stress. They can alleviate salt stress by production of plant growth promoting (PGP) substances like indole acetic acid (IAA), phosphorus solubilization, siderophore and ammonia production. This ability of microbes to alleviate salt stress in crops varies greatly among microorganisms in salt affected soils. Hence, there is need to develop effective salt tolerant microorganisms for higher crop productivity in salt affected soils.

l Fifty salt tolerant bacterial and fourteen actinomycetes isolates were screened for PGP traits like indole acetic acid production, ammonia excretion and phosphorus solubilization. Uptake of sodium from liquid medium was also studied by salt tolerant bacterial and actinomycetes isolates. Salt tolerant bacterial isolates 10STB3C(2) and 15STB2C showed maximum Na uptake (513 and 323 mg/g) from nutrient broth containing 700 ppm of Na. Maximum ammonia excretion was observed by salt tolerant bacterial isolates 15STB1 (0.082μg/ml) and STB110 (0.078 μg/ml) in comparison to control (0.036 μg/ml). Highest indole acetic acid (IAA) was produced by salt tolerant bacterial isolates STB38 (11.40 ppm) and STB105 (8.90 ppm). Maximum P solubilization was observed by salt tolerant bacterial isolates 5STB19 (17.41 ppm P2O5) and 10 STB 7B (16.93 ppm P2O5) in liquid medium containing tricalcium phosphate.

Objectives

l Isolation of salt tolerant microorganisms from salt affected soils.

l Highest sodium uptake was observed in salt tolerant

l Screening of salt tolerant microbes for PGP traits. l Evaluation of efficient salt tolerant microbes on crops in

actinomyces isolate STAc8 (73.17 mg/g) and STAc3 (64.70 mg/g). Maximum ammonia excretion was observed by salt tolerant actinomycetes isolates STAc1 (0.188 μg/ml) and STAc2 (0.136 μg/ml). Highest indole acetic acid (IAA) was produced by salt tolerant actinomycetes isolates STA8 (9.67) and STAc8 (5.77ppm). Maximum phosphorus solubilization was observed by salt tolerant actinomycetes isolates STAc18 (31.13 ppm) and STAc17

pots in salt affected soils.

l Characterization of effective salt tolerant microbes by biochemical and molecular methods.

l Field testing of efficient salt tolerant microbes on rice, wheat and mustard in salt affected soils.

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AMAAS - Annual Report 2015-16

l Consortia of salt tolerant bacteria (C1 and C2) were tested

(30.65 ppm). Indole acid production by salt tolerant actinomycetes isolates has been depicted in Fig. 1.

in saline soil at Nain in Panipat and reclaimed soil at CSSRI, Karnal on salt tolerant wheat (KRL-210) at different doses of recommended fertilizer. Both the consortia performed better in terms of straw and grain yield of wheat at different doses of RDF in saline soil.

l Identified seven salt tolerant bacterial isolates (Bacillus aerophilus (STB-1)-NAIMCC-B-01857, Bacillus aerophilus (10STB-7B)-NAIMCC-B-01858, Bacillus licheniformis (STB-80)-NAIMCC-B-01859, Bacillus licheniformis (10STB3C(1))-NAIMCC-B-1860, Bacillus sonorensis (15STB5C), Bacillus stratosphericus (15STB5C), Bacillus licheniformis (STB133) with highest PGP activities and deposited four of these potential isolates to NAIMCC, NBAIM, Mau and got their accession number.

Fig. 1: Indole acid production by salt tolerant actinomycetes isolates

l Maximum grain yield of salt tolerant basmati rice CSR30 was observed by salt tolerant bacterial isolate STB80 (5.27g/plant) and 5STB19 (5.04 g/plant) in saline soil (ECe 5.53) in pots. Similarly, highest plant biomass was recorded by bacterial isolate 15STB5C (18.6 g/plant) and STB80 (18.5 g/plant) in comparison to control (12.6 g/plant) (Fig. 2).

Conference and workshop attended

l Joshi, P.K. 2015. Microorganisms for bioremediation of wastewaters for heavy metals. Presented in National Conference on Emerging trends in Host-Microbe Interactions held at DAV University, Jalandhar from April 17,18, 2015

l Joshi, P.K., Sharma, Vandana and Manjari. Isolation of salt tolerant microorganisms from salt affected soil. Presented in 4th National seminar on Innovative Saline Agriculture in changing environment held at RVSKV, Gwalior from 1214, December, 2015

l Joshi, P.K., Sharma, Vandana and Manjari. Microbial technology for removal of heavy metals from wastewaters. Presented in 4th National seminar on Innovative Saline Agriculture in changing environment held at RVSKV, Gwalior from 12-14, December, 2015

l Joshi, P.K., Sharma, Vandana and Manjari. Optimum conditions for removal of heavy metals from wastewater by agrowastes along with microbial consortium. Presented in 79th Annual Convention of ISSS held at Prof Jayshankar Telangana State Agril. University, Hyderabad from 24-27, November, 2015

Fig. 2: Response of salt tolerant bacterial isolates on salt tolerant basmati rice (CSR-30) in saline soil.

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Production and Formulation Technology Refinement of Bacterial Bioagents for Soil Borne Plant Disease Management under Coastal and Plain Ecosystems PI : R. Ramesh ICAR- Central CoastalAgricultural Research Institute, Old Goa screening and field experiments. The strain was identified based on morphological, biochemical and 16S rRNA gene sequence (NCBI- KU682845). The culture is deposited in the national repository of NBAIM, Mau with the accession number NAIMCC-B-01889 and with MTCC, IMTECH with the accession number MTCC 12535. Application to register this strain with NBAIM is submitted.

Rationale Due to heavy rainfall in the shortest duration along with hot and humid conditions favour development of diseases in crops plants in the coastal region. Nature and type of soil also favours the severity of soil borne diseases in the coastal region. Bacterial wilt in solanaceous vegetables, root rot in chilli, Fusarium wilt in watermelon and chillies, quick wilt in black pepper, seedling rot in mango nurseries are some of the economically important soil borne diseases in the coastal regions. Majority of the fungicides are not effective in the management of the above diseases. Though many reports of biological control of the above diseases are reported, they are either limited to experimental fields or based on the products from the research laboratories. Hence, there is a need for the standardization of production and formulation system to produce quality formulations.

l Bacillus methylotrophicus strain STC-4 is a plant growth promoter and is tolerant to salt concentration of up to 1.5M. The strain was identified based on morphological, biochemical and 16S rRNA gene sequence (NCBIU682846). The culture is deposited in the national repository of NBAIM, Mau with the accession number NAIMCC-B-01890.

l Synthetic medium for the multiplication of RCh6-2b was standardized. Population in the synthetic medium was at par with the control medium and hence this medium could be used for mass multiplication.

Bacterial antagonists are given much importance in the commercial formulations as most of the available products are only from Trichoderma or to a limited extend to Pseudomonas fluorescens. Almost all the commercial formulations in India are based on talc powder and the survival of the biocontrol agent and the shelf life is not up to the standards (viability and population reduces drastically within 4 months, which makes the product not efficient at field level). Hence, it is necessary to improve the formulation and the efficiency.

l Carrier based and liquid formulation of RCh6-2b was standardized. Population in the talc formulation is over 8.0 Log CFU g-1 and in the liquid formulation is over 7.0 Log CFU g-1 after 16 months. Population of RCh6-2b in sodium alginate formulation was above 9.0 Log CFU mL-1 till 345 days.

l In planta evaluation of talc, liquid and sodium alginate

Objectives

formulations of RCh6-2b improved plant growth in brinjal, tomato and other crops. Soil application of talc formulation was found to be superior to other application methods.

l Selection of promising wide spectrum bacterial biocontrol agent(s) with plant growth promoting activity against wilts and root rot diseases.

Conclusion

l Refinement of production technology to enhance the shelf

A promising bioagent, Bacillus methylotrophicus Strain RCh6-2b was characterized and its mass production technology was standardized. Formulations (talc, liquid, and alginate) of this strain showed enhanced shelf life of at least one year with high population of bacterium. Further, these formulations improved plant growth in brinjal, tomato and other crops.

life of the various forms of dry and liquid formulation.

l To develop appropriate dry and liquid formulation technology of biocontrol agent for increased viability and shelf life.

l To standardize delivery methods of formulation for enhanced survival and multiplication.

l To validate biocontrol and plant growth promoting

Conference and workshop attended

l Ramesh, R., Siddhesh Menon, Neelima Dasari and Singh,

efficiency of the biocontrol formulations under coastal ecosystems.

N.P. 2016. Bacillus spp., a promising rhizosphere bacterium in plant health management in Coastal ecosystem. 6th International conference “Plant, pathogens and people: Challenges in Plant Pathology to Benefit Human Kind” 23-27, February, 2016, held in NASC Complex, New Delhi, India pp 103 (Abs).

SignificantAchievements

l A rhizobacterium (Bacillus methylotrophicus strain RCh62b) has been identified as one of the promising bioagent in suppression of various plant pathogens during the

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AMAAS - Annual Report 2015-16

Prospecting the Potential of Cyanobacteria based Formulations as Plant Growth Promoting and Biocontrol agents in Cereal-Legume Cropping System and Selected Vegetables PI : Radha Prasanna Co-PIs : Lata, B. Ramakrishnan ICAR-IndianAgriculture Research Institute, New Delhi dressings. Activities of defense and antioxidant enzymes from the leaves at 60 DAS (Days after sowing) showed significant differences, with respect to control, with Anabaena-Trichoderma biofilm and AnabaenaProvidencia consortium.

Rationale Cyanobacteria have tremendous potential as a source of several bio-active compounds and pigments. Despite the extensive knowledge and information available on ubiquity of cyanobacteria in diverse habitats and rice fields, research on the PGP potential and disease suppressive effects of cyanobacterial formulations in other crops has received less attention. There exists a need to look at these organisms for as eco-friendly options for providing multiple benefits – for better nutrition of C, N, & P, micronutrients and improved soil aggregation/soil health, and as sources of molecules/enzymes with biocidal activity against pests/diseases in cereal, leguminous and vegetable crops.

l All the three formulations significantly influenced the availability of nitrogen and phosphorus and nitrogen, as compared to control. Ferrous iron concentration in soil was highest in V6 (48400) treated with Anabaena-Trichoderma biofilm (Fig. 1). The highest increment in the nitrogen, phosphorus and organic carbon was also recorded in Anabaena-Trichoderma biofilm treatment (Table 1). Nitrogenase activity was highest in the treatments inoculated with BF1-4 (Anabaena-Nostoc consortium), while dehydrogenase activity was significantly higher in Anabaena-Trichoderma biofilm, compared to other treatments.

Objectives

l To unravel the mechanism of colonization and effect on the qualitative and quantitative traits of plant and its microbiome in selected crop(s).

l To develop and evaluate foliar formulations / seed/ soil dressings using promising cyanobacteria/cyanobacteriabacteria consortia/ formulations for their PGP and biocontrol properties in maize, selected legumes and vegetables. SignificantAchievements

l An experiment was undertaken to study the influence of three cyanobacteria based inoculants (Anabaena-Nostoc consortium, Anabaena - Trichoderma biofilm and Anabaena-Providencia consortium) in maize inbred lines during kharif 2015. The inoculants were applied as seed

Fig. 1: Iron concentration (mg Fe 2+ g-1 soil) in the rhizosphere of different maize cultivars (V1-V10) treated with three promising cyanobacterial inoculants.

Table 1: Mean performance of cyanobacterial inoculants on soil nutrient and microbiological parameters in maize crop. Treatments

Available Nitrogen (kg/ha soil)

Available Phosphorus (kg/ha soil)

Organic Carbon %

B1 Control - No inoculation B2 Anabaena-Nostoc consortium (BF1+2+3+4) B3 Anabaena sp.- Trichoderma sp. Biofilm B4 Anabaena sp.+ Providencia sp. (CR1+PR3) CD (5%) CV%

95.4 195.0

14.7 18.4

0.34 0.43

143.2 264.0

20.8 21.2

198.3

22.2

0.48

247.0

21.3

191.1

21.7

0.46

231.1

21.4

11.8 13.5

1.9 19.3

0.01 5.85

25.8 22.6

0.1 0.1

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ARA (n moles Dehydrogenase activity ethylene/g (µg/g/d) soil/h)

AMAAS - Annual Report 2015-16

l Highest yield, in terms of g/plant or 100 seed weight was

enzyme activities and biometrical evaluation of harvest parameters revealed that Anabaena-Providencia consortium, followed by Anabaena-Trichoderma biofilm were the top ranking inoculants for maize cultivars tested.

recorded with the Anabaena -Trichoderma biofilm (Table 2). The overall analyses of soil nutrient and microbiological parameters, measurements of selected plant

Table 2: Mean performance of cyanobacterial inoculants on plant growth, yield and quality related parameters in maize crop. Treatments

Days to Male flowering

Plant height (cm)

100 seed weight (g)

Yield (g/row)

51.1 46.9 44.9 45.5 0.5 2.1

136.6 146.2 147.3 150.3 4.4 5.9

26.8 27.5 27.9 27.7 0.6 4.3

236.08 242.66 257.36 246.32 10.93 8.66

B1 Control - No inoculation B2 Anabaena-Nostoc consortium (BF1+2+3+4) B3 Anabaena sp.- Trichoderma sp. Biofilm B4 Anabaena sp.+ Providencia sp. (CR1+PR3) CD (5%) CV%

l The real-time PCR quantification of 16S rDNA showed

were also found to be influenced by the application of the cyanobacterial formulations, except AnabaenaTrichoderma formulation (Fig 2). Interestingly, the application of BF1-4 (Anabaena-Nostoc consortium) significantly increased the abundance of nifH genes in the five selected maize cultivars except V10. These results corroborated the measurements of nitrogenase activity in the maize rhizospheres.

that the three formulations tested with five different maize cultivars (V1, V4, V7, V8 & V10) had significantly influenced the abundance of rhizosphere eubacterial populations. The maize cultivars of V1 and V4 had higher population densities of eubacteria due to the application of Anabaena-Providencia formulation while those of V8 and V10 had higher densities due to the application of Anabaena-Trichoderma biofilm.

l A set of three microbial inoculants - Anabaena laxa,

l The copy numbers of nifH genes in the maize rhizospheres

Mesorhizobium ciceri and biofilmed Anabaena torulosa M. ciceri were tested for their influence on two each of desi and kabuli varieties of chickpea in terms of plant growth, soil functioning and the diversity of rhizosphere microbial communities. Significant increases in plant growth parameters, including fresh weight, the number of nodules, the concentration of nodular leghaemoglobin and the number of pods per plant have been observed. The Anabaena laxa inoculant was superior in its performance. The kabuli cultivar BG1108 when inoculated with the biofilmed Anabaena-Mesorhizobium inoculant recorded the highest concentration of leghaemoglobin in the nodules (Table 3). The potential for nodulation and nitrogenase activity by the acetylene reduction assay (ARA) differed widely among the cultivars tested

Fig. 2: Abundance of nifH gene copies (copy number g-1 soil) in the rhizosphere of five different maize cultivars (V1, V4, V7, V8 & V10) treated with three promising cyanobacterial inoculants.

Table 3: Mean performance of microbial inoculants on plant root parameters in Chickpea crop. Treatments

B1 An-Rhizobium biofilm B2 Anabaena laxa (RPAN8) B3 Mesorhizobium ciceri B4 No-inoculation (only carrier coating) CD (5%) CV%

ARA (n moles Leghaemoglobin Total Nodule Root fresh wt. ethylene/g content (µg/g (number/plant) perplant (g) nodule/h) f.wt. of Nodule) 609.6 683.2 689.3 494.9 69.1 21.7

77

117.8 109.0 112.6 74.8 9.2 17.3

16.4 21.3 19.1 16.9 1.4 15.1

5.09 5.06 4.46 4.67 0.20 8.17

AMAAS - Annual Report 2015-16

l Significant increases in the 100-seed weight, which ranged

cyanobacterial communities showed distinct and characteristic changes due to the microbial inoculation (Fig. 4). The abundance of Gram negative bacteria (in terms of PLFAs) differed between the desi and kabuli varieties inoculated with M. ciceri alone. Cyanobacterial inoculants, either alone or as biofilmed with M. ciceri showed distinctive influences on cv. BG1053.

from 22.9 to 46.3 g and the crude proteins ranging from 13.6% to 29.2 % were recorded due to microbial inoculation (Fig. 3). Differential effects of inoculation on the concentrations of available nitrogen and phosphorus, and those of iron, zinc and copper suggested their increased cycling in the rhizosphere.

l Aset of three biofilmed inoculants were tested in Capsicum (variety Swarna), along with two controls (carrier alone and uninoculated) under greenhouse conditions. Plant and soil parameters were evaluated at 60 days after inoculation (DAI). The Anabaena-Azotobacter biofilm inoculant (AnAz) elicited significantly higher values of catalase and statistically at par values with Anabaena-Trichoderma biofilm inoculant (An-Tr), in terms of SOD and ascorbate peroxidase activity (Fig. 5 a-d and Table 4). A similar trend was recorded in terms of polysaccharide content and available N, P in soil.

Fig. 3: Influence of microbial inoculation on the concentration of total crude protein and the 100-seed weight of chickpea inoculated with different microbial inoculants.

l The DGGE profiles of archaeal, bacterial and

Fig. 4: PCR-DGGE profiling (a) and dendrogram (b) of the cyanobacterial community structure in the rhizosphere of two each of desi and kabuli varieties of chickpea as influenced by different microbial inoculants (Anabaena laxa, Mesorhizobium ciceri and biofilmed Anabaena torulosaM. ciceri).

Fig. 5: Influence of microbial formulations in capsicum crop on (a) Superoxide dismutase (SOD) activity in leaves; (b) Polysaccharide content of soil; (c)Available N in soil and (d)Available P in soil.

Table 4. Anti-oxidant plant enzyme activities elicited by the application of microbial formulations in capsicum. Treatments

Ascorbate Peroxidase (IU/mg protein)

T1(Anabaena-Azotobacter biofilm) T2 (Anabaena -Trichoderma biofilm) T3 (Trichoderma-Azotobacter biofilm) T4 (Carrier) T5 (Control) SEM CD (0.05)

0.094 0.148 0.089 0.081 0.058 0.016 0.043

78

Catalase (IU/mg protein) 0.290 0.173 0.156 0.146 0.087 0.016 0.043

AMAAS - Annual Report 2015-16

l Scanning electron microscopy revealed the distinct

bioavailability of nutrients and yield of okra. Heliyon 10.1016/j.heliyon.2016.e00066

presence of biofilm–like aggregates and short pieces of filaments/mycelia of cyanobacteria /Trichoderma, and cells of Azotobacter in the roots of plants from treatments receiving microbial formulations (Fig. 6a)

l Babu, S., Prasanna, R., Bidyarani, N., Nain, L., Shivay, Y.S. (2015). Synergistic action of PGP agents and Rhizobium spp. for improved plant growth, nutrient mobilization and yields in different leguminous crops. Biocatalysis and Agricultural Biotechnology 4: 456-464

l PCR DGGE profiles of bacterial communities present in the soil samples of capsicum crop (Fig. 6b), showed distinct changes as a result of inoculation, illustrating the influence of microbial formulations on the rhizobacterial communities.

l Prasanna, R., Hossain, F., Babu S., Bidyarani, N., Adak, A., Verma, S., Shivay, Y.S., Nain, L. (2015). Prospecting cyanobacterial formulations as plant growth promoting agents for maize hybrids. South African Journal of Plant and Soil 32 (4): 199-207 Book Chapter

l Radha Prasanna, Nain, L., Rana, A., Shivay, Y.S. (2016). Biofortification of crop plants using microorganisms: present status and challenges. In: Biofortification of food crops, Singh, U. et al. (Eds.). pp. 249-262, Springer Science + Business Media B.V., Springer. Conferences and Workshop attended

l Workshop on “Small Enterprises in Microbiology” organized on 15-16th February 2016, at the Department of Microbiology, Maharshi Dayanand Saraswati University, Ajmer, in academic collaboration with Algae Biofuel and Biomolecules Centre and Krishi Vigyan Kendra, Tabizi.

Fig. 6: a. Scanning electron micrographs of the root tissues of capsicum, showing spherical aggregates of cells; b. PCR-DGGE profiles of bacterial communities in the rhizosphere of capsicum as influenced by the application of microbial formulations. Lanes 1-5: 1- An-Az; 2- An-Tr; 3 – Tr-Az; 4- Carrier control and 5- Control

l Prasanna, R., Ramakrishnan, B., Kaur, S., Kanchan, A., Ranjan, K., Hossain, F. and Nain, L. (2015). Interactive effects of cyanobacterial inoculants with chickpea leads to improved nodulation and pod yields. . In: National Seminar on Recent Advances in Agricultural-, Biomedical and Environmental Biotechnology, held at Department of Biotechnology, Anand Engineering College, Agra (1-2 May, 2015).

Conclusion The use of formulations containing cyanobacterial/ cyanabacterium-bacterium consortia or biofilms illustrated their beneficial role in improving plant growth and nutrient availability in soil. Microscopy and molecular tools aided in understanding their colonisation and interaction with microbial communities in the rhizosphere.

l Ramakrishnan, B., Kanchan, A., Ranjan, K., Prasanna, R., Venkatachalam, S., Hossain, F. and Nain, L. (2015). Influence of cyanobacterial formulations on soil microbial communities and crop growth and yields in maize hybrids. In: 56th Annual Conference of AMI and International symposium on Emerging Discoveries in Microbiology, held at JNU, New Delhi (7-10 Dec, 2015).

Paper published fromAMAAS work

l Manjunath, M., Kanchan, A., Ranjan, K., Venkatachalam, S., Prasanna, R., Ramakrishnan, B., Hossain, F., Nain, L., Shivay, Y.S., Rai, A.B. and Singh, B. (2016). Beneficial cyanobacteria and eubacteria synergistically enhance the

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Degradation and Effective Utilization of Agrowastes through Technologies Involving Mushrooms or Macrofungi PI : Sachin Gupta Co-PIs : Moni Gupta,Arvind Kumar Ishar, S.A. Mallick, Ranbir Singh, S. K. Singh Sher-e-Kashmir University ofAgricultural Sciences and Technology, Jammu Rationale Edible fungi or mushrooms are natural recycler which converts lignocellulosic wastes into protein rich health food. Lignocellulosic wastes such as paddy straw, saw dust, wheat bran, maize bran, sugarcane bagasse, water hyacinth etc. are available in abundance and are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. Degradation of different lignocellulosic substrates is due to the enzymes produced by different mushrooms. There is a need to identify the responsible enzymes and to standardize the parameters for their mass production, so as to put them to alternative uses. Use of Spent Mushroom Substrate for different uses like farm yard manure, animal feed, plant disease management needs to be exploited so that the agrowastes can be put to diverse uses for maximum usage efficiency.

Sparassis spp.

Russula spp.

Fig. 1: Diversity and cultivation of mushroom on lignocellulosic wastes

l Antioxidant profiling viz., 2,2 Diphenyl dipicryl hydrazil radical (DPPH) scavenging activity, Maximum reducing power and Highest chelating ion power as well as Enzymatic profiling viz. activity of cellulase, xylanase, laccase was assayed in wild edible mushrooms and these mushrooms have been found to be rich source of antioxidants (Table 1 & 2).

Objectives

Table 1: Enzymatic profile of wild edible mushrooms Xylanse Cellulase Wild b 1,3 content content mushroom Glucanase (U/mgprotein) (U/mgprotein) (U/mgprotein)

l Screening of various locally available cheap substrates (agricultural and horticultural wastes) for their degradation by macrofungi and their utility in mushroom production in terms of yield and nutritional aspects.

Ramaria spp. Flammulina spp. Sparassis spp.

l Assessment of production of various lignocelluloytic and other enzymes during the degradation process of agrowastes by mushroom fungi.

2.34 1.49 1.98

4

8.1 × 10 1. 9 ×102 3.5×102

0.112 0.08 0-103

Table 2: Antioxidant profile of wild edible mushrooms

l Standardization of cultural and nutritional requirements

Wild mushroom

for enhanced enzyme production.

l Characterization of different enzymes produced during the process and their alternative uses.

l Exploitation of spent mushroom substrate for soil health

DPPH Radical Chelation Reducing scavenging Power Ic50 power Ec50 activity IC50 value(µg/ml) value (µg/ml) value(µg/ml)

Ramaria spp. 462.4± 0.04 302.2 ± 0.12 Flammulinaspp. 1754.2± 0.08 608.1 ± 0.02 Sparassis spp. 567.3± 0.04 514.5 ± 0.02

improvement, bioremediation of contaminated industrial sites, animal feed, plant disease management and other uses.

0.568±0.05 0.54±0.04 0.527±0.02

l Antimicrobial and anticancer potential of wild as well

SignificantAchievements:

commonly cultivated mushrooms was assayed against diverse plant pathogens. Significant inhibition of Fusarium oxysporum, Rhizoctonia solani, Macrophomina phaseolina was observed under in vitro conditions. Anticancer activity against cancer cell lines viz. Lung A549, Colon HCT 116 and Breast T47D was exhibited by the mushroom extracts.

l Surveys were conducted in Rajouri, Doda, Udhampur districts of Jammu to collect the edible wild mushroom flora. The collected samples have been identified as Flamullina velutipes, Russula spp., Helvella spp., Ramaria spp., Coprinus spp. etc. Mushroom samples and their cultures have been preserved. Efforts are being made to cultivate these mushrooms on different substrates (Fig. 1).

l Paddy straw based compost formulation for cultivation of

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AMAAS - Annual Report 2015-16 Pacific Journal of Food Safety and Security, 2: 3-15.

button mushroom under Jammu conditions has been standardized and the initial trials revealed biological efficiency of paddy straw based compost at par with wheat straw based compost. Use of paddy straw based compost could effectively reduce the cost of mushroom cultivation.

l Bharti, V. and Gupta, S. 2016. Casing awaran mein purkoon ka prayog. Chatrak (Published by DMR, Solan) (in press). Conference and workshop attended

l Trials conducted for the use of spent mushroom substrate

l Attended 4th Jammu and Kashmir Agricultural Science

directly as manure, vermicompost preparation and for plant disease management revealed its utility in increasing the crop yield and protection from plant diseases.

Congress at SKUAST-Jammu w.e.f. 28-30th Oct., 2015 and presented a paper on Antioxidant activity and Nutraceutical profiling of Calocybe indica and its value added products.

Conclusion Wild edible mushrooms collected from different parts of Jammu were assayed for nutritional, enzymatic, antioxidant, antimicrobial and anticancer properties. These mushrooms were found to be rich source of antioxidants and have good anticancer potential. In vitro analysis also revealed their possible utility as eco friendly plant disease management measure. Standardization of formula for paddy straw base compost could considerably reduce the cost of mushroom cultivation.

l Attended International Conference of Indian Ecological Society at SKUAST-Jammu w.e.f. 18-20th Feb., 2016 and presented a paper on Mushrooms: A potential tool for bioremediation.

l

Attended 6th International Conference of Indian Phytopathological Society at New Delhi w.e.f 23-27th feb., 2016 and presented a paper on “Evaluation of grain substrates for quality spawn production of mushrooms”.

l Attended National Seminar on New Vistas in Plant and

Paper published fromAMAAS work

Microbial Sciences held at University of Jammu w.e.f. 1112th March, 2016 and presented a paper on “Studies on mycelial growth and cultivation of Pleurotus spp.”

l Gupta, S., Summuna, B. and Gupta, M. 2016. Mushroom Cultivation: A means of nutritional security in India. Asia

Metagenomics and Cultural Approaches for Identification of Novel Microbial Genes/Alleles and Microbes for Bioconversion of Lignocellulosic Biomass at Extreme Physiological Conditions of Low Temperature PI : Rajeev Kaushik Co-PIs : Anil K. Saxena, Livleen Shukla ICAR- IndianAgricultural Research Institute, New Delhi

l Isolation, cloning and expression of β -1, 4–

Rationale

endoglucanase, endo-β-1, 4 xy lanases and laccase gene from selected cold tolerant isolate.

Soil microbial communities present in cold deserts of Himalayan region must have capability of degrading lignocellulosic residues at low temperature. Cultural diversity of microbial community from such habitats can be used in developing consortium that can carry out composting process at low temperature in short time. Genes/alleles coding for cellulase, hemicellulases and lignin degrading enzyme in these microbial communities (presumed to be evolved by overcoming temperature stress) can be cloned for their subsequent use in production of stress tolerant biocatalysts for research programmes engaged in production of value added products from agrowaste.

l Mining of soil metagenome for laccase, cellulase and xylanase genes.

l To develop microbial consortium for composting of lignocellulosic biomass at low temperature. SignificantAchievements

l Sampling was conducted in regions of high altitudes across the Himalayan range starting from high humidity Sikkim to low humidity places in Leh-Ladakh regions. From these regions naturally decomposed plant samples and forest soils were collected.

Objectives

l Psychrophilic and Psychrotolerant fungal and bacterial

l Identification of microbial species cultivated from cold

isolates capable of hydrolysing lignocellulose components viz. CMC, Xylan and lignin at low temperature were isolated using spread plate technique prior to which the

environments that could hydrolyse lignocellulosic biomass at low temperature.

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AMAAS - Annual Report 2015-16 degradation of paddy straw in a 60 d experiment (Fig. 2). Glucose 0.9 and xylose 0.22 mg ml-1were detected after 30 d in aqueous extract of fungal treated rice straw through HPLC (Fig. 3).

samples were enriched at 10°C in respective enrichment mediums.

l As some lignin degrading microorganisms were unable to grow in pure lignin augmented medium, for which nutrient agar, tryptone yeast extract agar and actinomycetes isolation agar were used for isolation of microorganisms and further characterized them enzymatically for lignin degradation.

l Two step screening procedures were adapted for determination of the enzymatic activities responsible for lignocellulose degradation. The primary screening strategy using plate assay successfully screened bacteria and fungi capable of utilizing cellulose supplemented in form of carboxy methylcellulose sodium salt and beech wood xylan as determined through halo zone formation. The secondary screening procedure estimated the enzyme production in quantitative terms, prior to which pH and temperature optimization was performed.

Fig. 2: Degradation of rice straw at 10°C in 60 d by LF1

l For quantitative estimation of cellulose degradation, werechosen both amorphous (CMC) and crystalline (Avicel) cellulose as substrates. Analysing the reducing sugars (using DNSA method) released from the substrates, it was found that few microorganisms could degrade only crystalline cellulose, which was a breakthrough in this study as such microorganism possess the capability of hydrolysing the recalcitrant crystalline cellulose.

l Quantitative estimation of xylanolytic enzymes revealed that some bacteria had only xylanolytic activity and no cellulolytic activity. Such pure xylanase positive cultures with no cellulase producing ability have high industrial importance.

Fig. 3: HPLC chromatogram of aqueous extract obtained from paddy straw degradation after 30 days of the process (X axis is in minutes) indicates the peaks for glucose and xylose

l Potent lignocellulolytic bacteria identified as gram positive Bacillus megaterium and Paenibacillus polymyxa respectively and lignocellulolytic fungi identified as Penicillium sp. (SF3), Phoma sp.(LF01) and Mucor plumbeus (LF09) respectively.

l FT-IR spectra of the lignocellulose substrate treated with LF1 is shown in (Fig. 4) and it demonstrates the comparison of untreated substrate with the treated one. The ratio of peak areas from 1510 cm-1 to 900 cm-1 was calculated to find the lignin/cellulose content of the lignocellulosic substrate. It was found that the control had

l Penicillium ubiquetum (SF3) and Phoma sp. LF01 were found to be potent lignocellulolytic fungi. Out of the two isolates, LF0 1 showed the maximum lingocellulolytic activity (Fig. 1). It was used as test inoculants for

Fig. 4: FTIR-spectroscopy analysis of degraded paddy straw after 60 days

Fig. 1: Lignin degradation by isolates LF01 and SF03 at 10°C and Rh 60%

82

AMAAS - Annual Report 2015-16 a value of 4.049 as compared to the LF1 treatment with 3.161. From the spectra, the carbohydrate fingerprint region identified from the peaks 1066.9, 1106.6 and 1157.6 cm-1was found in the control untreated lignocellulosic substrate while the same was missing in the treated substrate. Similarly the regions 1646.5 and 1558.6 cm-1 which depicts the amide band regions was also missing in the treated sample.

Conclusion Penicillium ubiquetum SF3 and Phoma sp. LF1 were found to be potent lignocellulolytic fungi at 10°C. Isolate LF 1 could degrade rice straw significantly in comparison to control at 10°C in 60 d. Conference and Workshop attended

l Kaushik R, Senapati A, Shukla L and Saxena A K (2015). Development of a microbial accelerator for composting post-harvest crop waste residues at cold climate. Proceedings of CRYOBIOTECH International Conference on Low Temperature Science and BiotechnologicalAdvances,April 27-30, pg. No. 106

l Comparison of the values obtained from FTIR spectroscopy and HPLC analysis showed a higher degrading and saccharification rate of LF1 as compared to other psychrophilic fungal isolates at similar period of 60 days.

Development of Bioformulations with PGPR, PGPF and AMF for Induction of Resistance in Cereals and Pulses against Fungal Pathogens and Improvement of their Health Status PI : B.N. Chakraborty Co-PIs : U. Chakraborty University of North Bengal, Darjeeling bioformulations, singly and in combinations on plant growth promotion of cereals and pulses.

Rationale Among the beneficial microorganisms, plant growth promoting rhizobacteria (PGPR), plant growth promoting fungi (PGPF) and the mycorrhizal fungi (AMF) are agriculturally important. Plant growth promoting rhizobacteria include those bacteria, which, on inoculation into the soil, colonize the roots of plants and enhance plant growth as well as suppress diseases. AM fungal association also increase productivity and protect plants from soil-borne diseases. One of the common means of application of bacterial inoculants to soil is in the form of bioformulations.The combined activity of PGPR, PGPF and AMF in the form of bioformulation is generating ample evidence to act as bioprotector as well as biofertilizer which plays a significant role in sustainable agriculture. Keeping these in view, the present study was undertaken to determine how does a combination of bioformulations with PGPR, PGPF and AMF, singly or in combination, influence the growth of cereals and pulses as well as induction of resistance in host plants against fungal pathogens.

l Evaluation of these bioformulations singly and in combinations on their biocontrol potential against fungal diseases of cereals and pulses.

l Determination of cell defense responses associated with localized and systemic resistance to phytopathogens following induction by microbial formulations. SignificantAchievements

l Among six PGPR isolates [Bacillus methylotrophicus (NAIMCC-B-01492; NCBI JQ765577), Burkholderia symbiont (NAIMCC-B-01489; NCBI JQ765578), Bacillus altitudinis (NAIMCC-B-01484; NCBI HQ849482), Bacillus megaterium (NCBI JX312687), Bacillus pumilus (NAIMCC-B-01483; NCBI JF836847), Paenibacillus polymyxa (NAIMCC-B-01491; NCBI KC703775)] tested on sorghum plants , Bacillus methylotrophicus markedly reduced spot blotch disease intensity. Higher accumulation of phenolic compounds in spotch blotch infected sorghum plant was evident through HPLC analysis.

Objectives

l Mass production and development of package of

l Combined application of three biocontrol agents

bioformulations of PGPR (Bacillus altitudinis, B. pumilus and B. megaterium ), PGPF ( Talaromyces flavus, Trichoderma harzianum, T. asperellum and T. erinacium) and AM fungi (Glomus fasciculatum and Glomus mosseae) for field demonstration.

( Talaromyces flavus - NAIMCC-F-01948; NCBI GU324073, Trichoderma harzianum- NAIMCC-F01961; NCBI GQ995194 & Trichoderma asperellumNAIMCC-F-01963; NCBI HQ265418) as soil amendments were effective in reducing root rot incidence of Cicer arietinum caused by Thanatephoruscu cumeris

l Field trial and assessment of the efficacy of these

83

AMAAS - Annual Report 2015-16 (NAIMCC-F-02903) when applied prior to artificial inoculation of the pathogen. T. harzianum was found to control disease more efficiently than T. flavus and T. asperellum when applied singly. Biocontrol efficacy (BE%) was found to be as high as 75% when all the three biocontrol isolates were applied jointly (Fig.1).

Fig. 3A: a-c=Activities of defense enzymes in pathogen inoculated, bioinoculant treated and pathogen inoculated leaves in comparison to healthy control in CWL6702 genotype: a= PAL; b= CHT and c= GLU. Fig. 3B: a-d= HPLC analysis of p-coumaroyl agmatine (phytoalexin) in CWL6702 susceptible genotype: a= Healthy control; b= B. sorokiniana inoculated; c= Bioinouclant treated; d= Bioinoculant+B. sorokiniana treated.

l Immunolocalization of chitinase in bioinoculant treated and pathogen inoculated leaf tissue was confirmed by transmission electron microscopy using PAb of chitinase and gold labelled conjugate. Bioinoculants treated and pathogen inoculated wheat leaves labelled with PAb of chitinase showed distribution of intense black particles of gold throughout the cell structure (Fig.4).

Fig. 1: Disease index of Cicer arietinum and biocontrol efficacy % of BCA isolates following inoculation with T. cucumeris

l Talc based formulation of Bacillus methylotrophicus (NAIMCC-B-01492), wheat bran based formulation of Trichoderma asperellum (NAIMCC-F-01963) and mass multiplied AMF were used as soil application for wheat, where combination of all three formulations were found to be most effective in disease reduction (Fig. 2:A&B)

Fig. 4: Transmission electron micrographs of Immuno-gold labeled wheat leaves (probed with PAb of chitinase) following induction of resistance using bioinoculants. Fig. 2A: Disease suppression and biocontrol efficacy of bioinoculants.

l Analysis of expression of defense genes (CHT, GLU, POX

Fig. 2B: In vitro antagonism of Bacillus methylotrophicus (NAIMCC-B-01492)(b), B. pumilus (NAIMCC-B-01483)-(c) & B. altitudinis (NAIMCC-B-01484)(d) against B. sorokiniana (a).

and PAL) in susceptible wheat cultivar (CWL-6702) following induced resistance was done by microarray and the gene expression levels when compared.Atotal number of 461 genes were differentially expressed in bioinoculants treated wheat genotype following challenge inoculation with Bipolaris sorokiniana in comparison to control in which 284 genes were up regulated and 177 genes were down regulated (Fig. 5).

l Time course accumulation of chitinase, β-1,3-glucanase and phenyl alanine ammonia lyase following 12, 24, 48, 72 and 96 h of challenge inoculation in wheat plant with Bipolaris sorokiniana increased markedly in bioinoculants treated wheat plants in comparison to healthy control (Fig 3A). Induction of phytoalexin (ρ-coumaroyl agmatine) in treated and inoculated plants were further confirmed by HPLC (Fig. 3B).

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AMAAS - Annual Report 2015-16 asperellum) were found to be most effective in reducing root rot incidence of Cicer arietinum caused by Thanatephorus cucumeris. Paper published fromAMAAS work

l Bhattacharjee P., Acharya A., Chakraborty U. and Chakraborty B. N. (2015). Evaluation of value added Vermicompost on field grown rice plants. Journal of Botanical Society of Bengal, 69 (2): 99-108.

l Dey, P. L. and Chakraborty B. N. (2015). Screening of phosphate solubilizing isolates of actinomycetes for in vivo assay for antagonistic activity against fungal pathogens. Journal of Scientific Research and Advances, 2(4): 137140.

l Khati S. and Chakraborty B. N. (2015). Morphological

Fig. 5: Venn diagram showing up and down regulated genes

characterization of rice cultivars their root colonization with Arbuscular Mycorrhizal fungi and screening for field resistance caused by brown spot disease. NBU Journal of Plant Sciences, 9(1):78-86.

l Most significant upregulated genes were those of pathogenesis related proteins 4 and 10, phenylalanine ammonia lyase, glucanendo-1,3beta–D-glucosidase, 5enolpyruvylshikimate 3-phosphate synthase, β 1,3glucanase and peroxidase.

Book Chapter

l Chakraborty, B.N., Chakraborty, U., Sunar. K. and Dey,

l Relative expression of defense genes (CHT, GLU, PAL and

P.L. (2015). Molecular phylogenetic analysis of Talaromyces flavus, a phosphate solubilizing fungus. In: Molecular and Biotechnological Approach to Resource Utilization: Microbes to Angiosperm (Eds. S. Ray and S. K. Sen) VisvaBharati and Levant Books, Kolkata. pp. 21-28.

POX) through real-time PCR with cDNA from leaf tissue increased 2.98, 2.38, 2.70 and 2.25 folds inbioinoculant treated and inoculated plants than control (Fig. 6).

l Chakraborty, B.N. (2016) Scoping the potential uses of beneficial microorganisms for biopesticide industry and entrepreneurship development in crop protection. In: Perspectives of Plant Pathology in Genomic Era (Eds. P. Chowdappa, Pratibha Sharma, Dinesh Singh and A. K. Misra), Today and Tomorrow Publishers, New Delhi. Conferences and Workshop attended

l National Symposium on 'Emerging trends in Plant Sciences' organized by Department of Botany, University of Rajasthan, during October 26-28, 2015.

Fig. 6: Relative expression of defense genes through Real time PCR analysis.

l National Workshop on “Exploitation of Plant and

Conclusion

Microbial Resources of North Bengal”; organized by Department of Botany, University of North Bengal on December 14-16, 2015.

Among the PGPR tested in cereals sorghum and wheat, (Bacillus methylotrophicus NAIMCC- B-01492) was most effective in reducing spot blotch disease. Induction of resistance towards

l National symposium on 'Microbial Diversity and its Impact'; jointly organized by the Department of Botany, Kolkata and Indian Mycological Society on February 1819, 2016.

Bipolaris sorokiniana in wheat plants by combined application of bioinoculants (B. methylotrophicus, T. asperellum and AMF) was confirmed by immuno gold localization of defense enzymes using TEM and microarray analysis along with Real Time PCR analysis of defense genes. In case of pulses, combined application of three biocontrol agents (Talaromyces flavus,Trichoderma harzianum and T.

l Sixth International Conference on 'Plant, Pathogens and People: Challenges in Plant Pathology to Benefit Humankind', organized by Indian Phytopathological Society, IARI, New Delhi, during February 23-27, 2016.

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AMAAS - Annual Report 2015-16

Identification of Salt Tolerant Microbes and Development of Dynamic Substrate for Cultivation of Commercial Crops in Sodic Soils PI : T. Damodaran Co-PIs : V.K. Mishra, D.K. Sharma, S.K. Jha, Y.P. Singh ICAR-Central Soil Salinity Research Institute, Regional Research Station, Lucknow combination with dynamic media and validate them with on-farm demonstrations covering normal as well as degraded soils.

Rationale One of the important categories of waste land in India is salt affected waste land, which occupies extensive area in the world and in India (6.73 mha) as well, presenting a serious impediment to crop production. Uttar Pradesh alone accounts for 1.37 mha of sodic soils. High alkalinity and high exchangeable sodium percentage (ESP>15) of the soil and poor soil structure render it inhospitable for normal crop production and there is minimal bio-productivity in such soil. Poor hydraulic conductivity and poor biological activities are common features of these soils. The utilization of salt-affected soils for agriculture has become necessary to meet the rise in food demand and one possible strategy to counteract the adverse effect of salinity is to exploit the avenues of bio-agents or bio-inoculants. Under salt stress, PGPR have shown positive effects in plants on parameters such as germination rate, tolerance to drought, weight of shoots and roots, yield and plant growth. Many microbes have been detected in saline/sodic soils. Though PGPR are more commonly known to induce resistance against pathogen infection, reports are now available on their ability to elicit 'induced systemic tolerance' against abiotic stresses. One of the possible measures to improve crop health in saline soil conditions is to use saline-tolerant bacterial inoculants which promote plant growth and control diseases. It has been well established that the AM fungi enhance the ability of plants to cope with the environmental stresses generally prevalent in the degraded ecosystems. Apart from the growth regulation, bioamelioration through production of organic acids on interaction with appropriate substrate, the microbes through their PGP secretions facilitate in conversion of unavailable phosphorous, potassium and ferrous to available forms.

Significant achievements

l A total of 47 isolates were screened for PGPR potential and salinity tolerance. Thirteen bacterial and one fungal isolate which exhibited higher PGPR characters and salt tolerance in 10% NaCl media were further characterized using 16S rDNA. Purified PCR products from the 16S rRNAgenes of pure culture isolates representing all major RFLP patterns were sequenced on an ABI3730 automated sequencer (Applied Biosystems, USA) with primers 27 f and 1492 r. All reference sequences were obtained from the National Center for Biotechnology Information (NCBI) and the 16S rDNAsimilarity sequences searches were performed using the BLASTN tool in the NCBI website.

l Endophytic bacterial isolates were analysed for production of four enzymes that is, protease, amylase, cellulase and lipase by plate methods under (0.1, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 M) NaCl stress. Fourteen (16) newly isolated endophytic bacterial isolates were screened for production of extracellular enzymes. The of 0.1 to 3.0 M of sodium chloride concentration indicated that actively hydrolysing enzymes with higher salt concentration could be detected in qualitative form by direct correlation of the diameter of the zone of substrate hydrolysis and colony growth. Nine isolates showed positive hydrolytic activity. These nine isolates were further selected for screening in sodic soils in tomato var. NS585.

l A total of nine strains were assessed for their efficacy of growth promotion in tomato var,.NS585 under sodic soils of the pH 9.23 and Ec 0.515. A loopful of bacterium was inoculated into the CSR patent protected culture media (Rai et al., 2011) and incubated in a rotary shaker at 150 rpm for 48 h at room temperature (28±2oC). After 48h of incubation, the broth containing 6 x 108 CFU ml-l was used for treating the tomato seeds @ 1 % for 2 h and for foliar application during flowering and fruiting stage of the crop. The culture grown in CSR patent media were inoculated in FYM @ 3 L / 100 kg of FYM / acre. Significant higher yield and plant height were observed in treatments involving the strains CSR-A-16, CSR-A-11, CSR-M-16 and CSR-A-18 (Table 1). Application of rhizobacteria and endophytic strains increased the root and shoot dry weight.

Objectives

l To isolate and phenotypically characterize native strains of bacteria and fungi by surveying the sodic / saline soil / problem soils.

l To screen and select suitable bio- growth enhancers under stressed environment (high pH).

l To standardize suitable cost effective culture media for mass multiplication and commercialization.

l To develop commercial formulation (s) with the objectives of growth enhancement, nutrient mobilization and biocontrol with a consortia of microbes and its

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AMAAS - Annual Report 2015-16 Table 1: Efficacy of the bacterial strains in tomato var.NS585 grown in sodic soils S. no.

1 2 3 4 5 6 7 8 9 10

Strains

Yield (Q/ha)

Plant Height (cm)

e

CSR-A-1 CSR-A-9 CSR-A-11 CSR-A-13 CSR-A-16 CSR-A-17 CSR-A-18 CSR-A-20 CSR-M-16 Control

Root Dry weight (g) Shoot dry weight (g)

c

44.2 c 93.4 b 154.5 e 42.1 b 163.5 d 67.4 b 155.5 d 52.5 a 186.5 f 21.1

b

32.23 c 36.32 a 65.55 c 38.12 a 66.15 c 39.56 a 65.89 b 57.22 a 69.56 d 19.11

l Consortia of compatible microbes among the four bacterial

0.28 b 0.21 d 0.69 b 0.35 d 0.66 bc 0.42 d 0.64 c 0.48 e 0.83 a 0.18

a

2.40 a 2.54 c 6.20 b 3.76 c 6.52 ab 3.20 c 6.40 b 4.10 c 7.20 a 1.98

of incubation, the broth containing 6 x 108 CFU ml-l was used for treating the tomato seeds @ 1 % for 2 h and for foliar application during flowering and fruiting stage of the crop. The culture grown in CSR patent media were inoculated in FYM @ 3 L / 100 kg of FYM / acre. Significant higher yield and plant height were observed in treatments involving the consortia C5, C4 and C2 (Table 2). The consortia comprising of C-5 (CSR-M-16, CSR-A11 and CSR-A-16) did not had any sodicity (soil pH 9.12) effect in the crop. gave the highest yield of 55.5 tonnes/ha with higher lycopene content (166.57mg/kg) while the control exhibited the highest mortaility index with an yield of 12.00 t/ha and lycopene content of (44.22 mg/kg).

strains were made and assessed for growth and yield parameters in tomato var. NS585 grown in sodic soils of pH 9.14 in field experiment conducted at the sodic soil experimental research farm of Central Soil Salinity Research Institute, Regional Research Station, Lucknow, India. The experiment was laid out in randomized block design (RBD) with plot size 5m x grown during two successive rabi seasons (November – Febrauary) of the year 2015-16.

l A loopful of bacterium was inoculated into the CSR patent protected culture media and incubated in a rotary shaker at 150 rpm for 48 h at room temperature (28±2oC). After 48h

Table 2: Efficacy of the bacterial strains in tomato var. NS585 grown in sodic soils Consortia C1 C2 C3 C4 C5 CONTROL

Plant height (cm)

Yield / Plant (kg)

b

71.67 82.33c 75.00b 94.33d 101.67e 59.33a

Lycopene mg/100g. Dry wt. (Shoot in “g”)

Yield (t/ha)

b

d

1.49 1.66c 1.32b 1.92d 2.24e 0.36a

32.34 31.63cd 26.44bc 47.74e 55.47f 12.79a

l The activities of the stress related enzymes, Superoxide

9.43b 12.62c 9.92b 12.87c 16.65d 4.43a

41.19b 53.74b 41.48b 90.23c 123.48d 15.36a

plants showed nearly 2-3 times higher activity than the control. Among the six treatments the treatments involving consortia 5 (C5) showed higher activities of the SOD and proline than the other treatments and control.

Dismutase (SOD), and proline assessed in various treatments showed highly significant differences between treatments (Fig.1 & 2). Invariably, the microbial inoculated

Fig. 2: SOD activity content in the roots of the tomato var. NS585

Fig. 1: Proline content in the roots of the tomato var.NS585

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AMAAS - Annual Report 2015-16

l The Na+/K+ ratio (Fig. 3) in the shoot of tomato was

system in a three tier bio-priming protocol for in-vitro biohardening of banana TC plantlets with salt tolerant microbes (CSR-M-16 – Bacillus licheniformis: CSR-A11 - Lysinibacillus fusiformis and CSR-A-16 – Lysinibacillus sphaericus) and VAM (rooting is induced without any external source of harmone only through auxins produced by endophytes) .

significantly lower in the untreated control (0.914) as compared to the treatments. Among five consortia evaluated, the lowest Na+/K+ ratio was recorded in the C5 (0.149) followed by C2(0.187). Higher potassium contents were observed in consortia C4 and C5 strain treated plants than other consortia and untreated control.

l Each treatment was taken with 30 numbers of bottles comprising of 4 plantlets in each bottles. For all the experiments, the data was analysed using Completely Randomized Design model of experiment using SAS 9.2 software and Duncans mean was used to assess the significance.

l The response of plants for bacterization with different consortia of microbes varied widely with the control which was treated with IBA, a rooting harmone in the growing media. There was significant difference between the treatments and the control for all assessed parameters suggesting the effectiveness of treatments irrespective of consortia. Among the bacterial treatments the treatment with consortia 5 (C5) exhibited higher root length and also number of roots which was also on par with the treatment with C4 for root length and plant height (Table 3)

Fig. 3: Na/K ratio in the shoots of the tomato var. NS585

l Five effective consortia developed at this centre under the project were used to assess the efficacy in growth promotion and induction of salt tolerance in the plant

Table 3: Growth parameters at 45 days after inoculation Microbial consortia C1 C2 C3 C4 C5 Control (IBA)

Plant ht. (cm) a

6.5 7.7ab 6.8a 8.2c 8.0c 6.2a

No.of leaves / plant a

a

4.0 4.3a 4.6a 4.0a 5.0a 4.0a

l The technology reduced the duration from 80 to 45 days for

Root length (cm) 5.5 7.4b 6.8b 8.8c 9.5c 4.2a

No. of roots 9.8b 11.6c 10.2b 12.5c 14.0d 8.0a

tomorrow, held at NBRI, Lucknow from 09-10th January 2016.

hardening and resulted in profuse rooting. There was a significant difference in the root length and number of roots among the bio-primed and the control where rooting was induced with the use of IBA. Bio-hardened plantlets produced 40 percent more root bio-mass than the control.

l Dassanayaka, M.P., Damodaran, T., Mishra, V.K., Sharma, D.K., Kannan, R. and Ashish Sahu (2016) Molecular identification of cynobacterial diversity in sodic soils of Uttar Pradesh, In: International Seminar on indigenous technologies for sustainable agriculture and better tomorrow, held at NBRI, Lucknow from 09-10th January 2016.

Paper published fromAMAAS work

l T. Damodaran, R.B. Rai, B.K. Pandey, D.K. Sharma, V.K. Mishra, S.K. Jha and Prabhat Kumar (2015). CSR-BIO a boon for horticultural crops in sodic soils. Indian Horticulture. 3:16-19

Patents filed

l Damodaran, T., Mishra, V.K, Jha, S.K., Pandey, B.K.,

Conference and workshop attended

Sharma, D.K., Rai, R.B, Praveen Kumar and Singh, D.B. In-vitro bio-priming technology of potential antagonistic and salt tolerant bacteria and mycorrhizae for inducing tolerance to biotic and abiotic stress. (Submitted for patent).

l Damodaran, T. (2016). Innovative approaches in organic production systems for economic and sustainable livelihood. In: International Seminar on indigenous technologies for sustainable agriculture and better

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AMAAS - Annual Report 2015-16

Development of Microbial Consortia as Biocontrol for Corm rot in Crocus sativus PI : Jyoti Vakhlu1 Co-PIs : Alok Srivastva2 1 University of Jammu, Jammu 2 ICAR-National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan consortia.

Rationale

l Development of suitable formulations as delivery systems

Saffron (Crocus sativus L.) is an important low volume, high value perennial crop. The intensive cultivation and monoculturing of saffron in saffron growing belts of Jammu and Kashmir together with the continual use of diseased material has resulted in the frequent occurrence of the corm rot diseases caused by the pathogens such as Rhizoctonia crocorum, Phomacrocophila, Fusarium moniliforme and non-sporulating basidiomycetous fungi, Macrophomin aphaseolina, Fusarium oxysporum, F. solani, F. pallidoroseum, F. equiseti, Mucorsp., Penicillium sp., and Sclerotium rolfsii. Trichoderma viride, a well known biocontrol agent, has been evaluated for its biocontrol activity against corm rot causing pathogens. It was observed that T. viride reduced the growth of F. oxysporum f. sp. gladioli, Fusarium solani, S. rolfsii and P. corymbiferum by 88%, 76%, 63% and 74%, respectively. In the present study, biocontrol agents will be isolated from rhizosphere, cormosphere and field soil of saffron and will be analyzed for suppression of pathogenic fungi.

for commercialization.

l Field evaluation of selected biocontrol agents for their potential in disease suppression SignificantAchievements

l Eight hundred (800) isolated Bacillus strains were screened for siderophore activity, phosphate solublization and anti-fungal activity against the already known pathogen Fusarium oxysporum (KF663598) (Fig. 1).

In recent years, biological methods have been successfully used as an effective pathogen control. The biological control is provided by these bacteria or fungi by producing antimicrobial and phytotoxins against the pathogens. Biocontrol against plant pathogens provide advantage over the chemical methods because there is little chance of resistance development, as in case of chemical, due to multiple mechanisms involved. The other most important attribute is that these biocontrol agents are highly specific, eco-friendly and do not affect non-target organisms.

Fig. 1: Bacillus aryabhattai (KT228251)showing anti-fungal activity against Fusarium oxysporum

l Out of eight hundred (800), only thirteen (13) Bacillus

Objectives

strains showed positive results (Halo diameter more than 4 cm in in-vitro).

l Isolation and screening of microflora isolated from field soil and rhizosphere of saffron for biocontrol activity against fungal pathogens.

l Selected Bacillus strains were analyzed microscopically

l Identification of selected efficient BCA showing multiple

l Sequences were submitted at the NCBI database with

biocontrol activity by biochemical and molecular phylogeny.

Accession No: KF702277, KF702278, KF702284, KF702283, KF702286, KF702291, KF702293, KF702296, KT228251, KT228252, KT228256, KT228263 and KT228264.

and biochemically.

l Pot assays and confirmation of biocontrol activity of the

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l Phylogenetic tree was constructed to get idea about the

Book Chapter

l Magotra, S., Trakroo, D., Ganjoo, S., & Vakhlu, J. (2016).

similarity of Bacillus aryabhattai with other Bacillus species (Fig. 2).

Bacillus-Mediated-Induced Systemic Resistance (ISR) Against Fusarium Corm Rot. In Microbial-mediated Induced Systemic Resistance in Plants (pp. 15-22). Springer Singapore. Conference and workshop attended

Fig. 2: Phylogenetic tree of selected Bacillus species

Conclusion

l

Attended Two days workshop on “Renovating bacterial taxonomy with Bioinformatics” organised by Department of Zoology, University of Delhi, Delhi.

l

Presented poster entitled Biocontrol of Corm Rot in Crocus sativus at National Seminar on “New Vistas in plant and Microbial Sciences” organized by Department of Botany, University of Jammu, Jammu (11-12 March, 2016)

Bacillus aryabhattai (KT228251), out of 13 species was found to be potential candidate as Biocontrol agent showing maximum siderophore and phosphate activity along with antifungal activity against Fusarium oxysporum (KF663598) (Fig. 3).

Fig. 3: Functional profile of selected Bacillus species with Bacillus aryabhattai showing maximum activity

Exploration of Microbial Diversity in Seed Spices through Metagenomics Approaches for Crop improvement PI : Sharda Choudhary Co-PIs : B. K. Mishra ICAR-National Research Centre on Seed Spices,Ajmer at very small scales. If the same genotypes are not repeated at other locations, the large-scale diversity is greatly multiplied. It remains to be seen to what extent this large genotypic diversity actually affects functional diversity, microbial ecology, or biotechnological significance. Here we present a framework of methods for opening the soil black box that

Rationale Soil probably harbours most of our planet's undiscovered biodiversity. Recent results from both, culturable and unculturable based approaches indicate that soil microbial diversity is even higher than previously imagined. One reason for the high diversity is that much of the diversity can be found

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AMAAS - Annual Report 2015-16 showed amylase activities; 47 isolates were showed carbohydrate degradation activities (Fig. 1).

provides different levels of resolution of both microbial community structure and activity. The understanding of the populations responsible for these processes has been relatively neglected, yet it is these populations that are the fundamental units responsible for the processes. Research at this level has, however, largely not been tractable, but with the rapid development of the tools of molecular biology, new efforts are now feasible. Objectives

l Microbial profiling of rhizosphere soil of cumin and coriander growing areas

l Molecular characterization of beneficial rhizobacteria l Screening of rhizosphere microbial isolates for their attributes for plant health management. Fig. 1: Bacterial and fungal cultures from the collected soils. Bacterial cultures isolated on nutrient agar medium and fungal cultures isolated by using potato dextrose agar.

l MetagenomicAnalysis. Significant achievements

l Soil samples were collected from rhizosphere of Cumin

Conclusion

and Coriander of Jodhpur, Barmer, Jaisalmer, Pali, Ajmer, Kota, Bundi, Jhalawar, Baran districts of Rajasthan State and Udhanpur, Radhapur, Gamdi, Satalpur, Fatehgarh, Raper, Balasar, Ajar, Bhuj, Songarh, Murbi, Porbandar, Junagarh, Somnath, Rajkot, Surendra nagar, Mehsana of Gujarat State. A total of 96 bacterial isolates (NRCSS 1NRCSS 96) were isolated from the semi arid and arid zone of Rajasthan and Gujarat from rhizospheric soils of cumin and coriander seed spices. Among them all isolated bacterial strains (NRCSS1- NRCSS 96) were showing positive for catalase, 25 bacterial isolates were phosphate solubilizers; 63 bacterial isolates were citrate utilizers whereas 30 bacterial isolates showed auxin production; 59

All isolated bacterial colonies/ strains were identified by gram staining, morphological and biochemical characterization (catalase test, phosphate solubilizing test, starch hydrolysis, indole production, nitrogen fixing ability, citrate utilization). Sixteen unique microbial cultures identified based on morphological and biochemical characterization and in process for sequencing. Conference and workshop attended

l Attended National conference on “ Microbes in extreme environments: Diversity and Translational Applications (MEEDTA- 2015) on Oct 30-31, 2015 organized by H. N. B. Garhwal University, Srinagar, Uttrakhand, India.

Formulation of ColeopteraActive Cry Toxins of Bacillus Thuringiensis PI : R. Asokan ICAR-Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Bengaluru biopesticides over the chemical pesticides, their rate of consumption has remained extremely low in India mainly due to efficaciousness, short shelf life, high cost of production system and longer action time.

Rationale Bacillus thuringiensis (Bt) has become the main microorganism used in biological control. The application of Bt to combat invertebrates of human interest gained momentum with the growing demand for food free of chemical pesticides and with the implementation of agriculture methods that were less damaging to the environment. The toxicity of Bt is due to its capacity for the production of a crystalline protein concomitantly with endospore; and this observation led to the development of biopesticides, based on Bt for the control of certain insect species among the order Lepidoptera, Diptera and Coleoptera. In spite of the several benefits of Bt as

“Bt” biopesticide is one of the most promising alternatives over conventional chemical pesticides. As far as farmers are concerned, the production cost of Bt toxin is the crucial factor. Generally, raw Bt toxin mixed with a suitable surfactant is being formulated for applications into the agricultural fields. This would further increase the total cost. The farmers expect the availability of low-cost Bt toxin without compromising its entomotoxicity values. The use of Bt based insecticides offer

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AMAAS - Annual Report 2015-16 various advantages over harmful chemical insecticides being target specific, biodegradable and economical. With this intuition present investigation was carried out to evaluate the (bio) efficacy of different formulations of Bt based insecticides against Coleopteran insect pest with the following objectives

Table 1: Corn stalk extract

Bt Isolate Control Control 3A* cry1I MD CM AU

Objectives

l Expression of cry1I & cry3A in Cry-B strain of Bt l Insect bioassay studies with various species of Coleoptera

OD 600nm 0.000A 0.000A 1.490A 0.586A 0.402A 0.966A 1.202A

Bacterial Cells/Ml 1.12 x 10-9 4.69 x 10-8 3.22 x 10-8 7.73 x 10-8 9.62 x 10-8

Table 2: Amaranths stem extract

l Formulation Type-Solid, liquid, encapsulated form

Bt Isolate Control Control 3A CRY1I* MD* CM AU

SignificantAchievements

l Native Bt strains were tested for starch analysis (Clear area around the growth of the culture after the addition of the iodine indicates the breakdown of starch by the organism due to its production of amylase, an extracellular enzyme) Fig. 1, in order to use suitable source for the mass culturing of Bt. Heat stable colony forming Bt strains were studied

OD 600nm 0.000A 0.000A 0.868A 1.259A 1.197A 0.940A 0.992A

Bacterial Cells/Ml 1.12 x 10-8 1.01 x 10-9 9.58 x 10-9 7.52 x 10-8 7.94 x 10-8

l Field evaluation of liquid formulation of Bt were carried out for one season. Further efficacy of the different liquid formulation was in progress.

l Evaluated of granular formulations of Bt against Leucopholis burmeisteri (Coleoptera: Scarabaeoidea) under field trials at Huncha and Sagara, Shivamogga district, Pethri, Bhramavara taluk, Udupi district resulted comparative control of the larvae in field (Fig 2).

Fig. 1: Production of Amylase

for the development of suitable formulations.

l Granular formulations were tested against larvae of white

l Various sources for the mass culturing of Bt strains were

root grubs as well as Arecanut white root grubs (Lecuphoris burmerstri). Early instars as well as late instar larvae were more susceptible to the granular formulations (Fig 3).

attempted. Corn stalk and amaranthas stalk proved to be the best source for mass culturing of native Bt strains (table 1&2

Fig. 2: Preliminary evaluation of bioefficacy of granular formulations of native B. thuringiensis under field trials

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AMAAS - Annual Report 2015-16 Conclusion:

l Successfully developed the liquid and granular formulations of Bt. The efficacy of the formulation were tested against different coleopteran insect pest. Bt formulations effective against coleopterans seem to offer great promise.

Fig. 3: Evaluation of granular formulations of Bt against Leucopholis burmeisteri (Coleoptera: Scarabaeoidea) under field trials cry1I granulated formulations

Influence of Arbuscular Mycorrhizal Fungi and Rhizobia in Conferring Drought Tolerance to Soybean PI : D. J. Bagyaraj1 Co-PIs : B. Mohan Raju2 1 Centre for Natural Biological Resources and Community Development, Bengaluru 2 University of Agricultural Sciences, Bengaluru with drought tolerance.

Rationale

l To validate the results of pot trials under field conditions,

The need for new alternatives for drought tolerant plants would be a practical solution to alleviate the problem of moisture stress. To achieve this aim, the biological interventions for drought tolerance need to be explored. Soybean is an important legume crop which exhibits symbiosis with root nodule bacteria as well as AM fungi. As symbiotic microbes are shown to improve the drought tolerance of crop plants, it is worthwhile examining what physiological trait/ drought adaptive traits are improved and how one can exploit the symbiotic microbes, AM fungi and rhizobia, for improved productivity of soybean under drought conditions.

to assess the stress tolerance besides growth rates leading to the identification of drought tolerant soybean line/s. SignificantAchievements

l Through the first year (2014-2015), 11 varities of soybean were screened for their ability to withstand water stress both under pot culture and field conditions. Two genotypes viz. DSR-2, DSR-12, exhibited tolerance to water stress and the genotypes susceptible to water stress, whereas genotypes MAUS 2 and MAUS 212were found to be susceptible for water stress.

Objectives

l This year (2015-2016) a glasshouse experiment was

l To screen soybean germplasm lines/cultivars for drought

conducted with all these 4 varieties of soybean (DSR 2, DSR 12, MAUS 2 and MAUS 212). All the 4 varieties of plants were inoculated with 10 different AM fungi. The experiment also had control with no AM fungal inoculation. Each treatment had 6 replications. Morphological recordings like plant height, stem girth, total leaf area, pod weight, seed weight, total shoot and root dry biomass were recorded. Based on the soybean yield and total plant dry weight G. fasciculatum proved to be the best symbiont for inoculating DSR 2 and G. leptotichum for inoculating DSR 12. In the drought susceptible varieties MAUS 2 and MAUS 212, best plant growth response was obtained with G. leptotichum. Studies on other parameters like mycorrhizal spore

adaptive traits such as roots and WUE and identification of trait donor genotypes and contrasts.

l To screen different AM fungi and select the best inoculant that imparts greater droughttolerance.

l To obtain the best rhizobia (based on higher trehalose accumulation) that can confer drought tolerance to soybean from Dr. Mahaveer Sharma, Network Partner from DSR, Indore and to integrate best AM fungi with best Rhizobium to further enhance drought tolerance of soybean- pot culture studies.

l To characterize plants treated with microbial symbionts for physiological and biochemical parameters associated

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AMAAS - Annual Report 2015-16 parameters like plant height, shoot girth diameter, number of leaves in each plant, leaf area index and number of branches were recorded. From 31st DAS the plants are being subjected to water stress at 75% and 50% field capacity for respective treatments in comparison with 100% field capacity. This has already been done for varieties MAUS 212 and DSR 12. The water stress was induced for 15 days (the plants are now 5 days after inducing water stress in these two varieties) (Fig. 1 & 2) and physiological parameters like leaf membrane damage and chlorophyll status were recorded after inducing stress for 15 days. After the induction of stress for 15 days, all the plants were watered to 100% field capacity till harvest. At harvest growth parameters like plant height, shoot girth diameter, number of leaves, number of branches, total leaf area, leaf area index, pod number, pod weight, seed yield, and physiological parameters like stomatal conductance, chlorophyll status, transpiration rate, etc will be determined. The variety MAUS 2 was sown little later and the water stress will be induced after 30 DAS (Fig. 3).

numbers in root zone soil and plant P concentrationare underway.

l Interaction between the best AM fungus G. leptotichum and Bradyrhizobium liaoningense (obtained from DSR, Indore) is currently being studied using the drought susceptible varieties MAUS 2 and MAUS 212 to improve drought tolerance and in the drought tolerant variety DSR 12 for further enhancement of drought tolerance. This pot culture experiment is being conducted under glass house conditions with the following treatments. (1) Uninoculated control, (2) Inoculation with B. liaoningense, (3) Inoculation with G. leptotichum and (4) Inoculation with B. liaoningense + G. leptotichum. These 4 treatments are being studied using all the 3 varieties mentioned above by inducing 2 levels of water stress i.e. 75% field capacity and 50% field capacity in comparison with 100% field capacity. Hence, a total of 12 treatments is set up and replicated 5 times.

l Initially for 30 days after sowing (DAS),all the plants were watered to 100% field capacity and plant growth

Fig. 1: Experiment currently underway with2 levels of water stress (75% & 50%) in comparison with 100% field capacityin drought susceptible variety MAUS 212 inoculated with G. leptotichum and B. liaoningense

Fig. 2: Experiment currently underway with 2 levels of water stress (75% & 50%) in comparison with 100% field capacity in drought susceptible variety DSR 12 inoculated with G. leptotichum and B. liaoningense

Fig. 3: Experiment currently underway with 100% field capacity in drought susceptible variety MAUS 2inoculated with G. leptotichum and B. liaoningense

drought tolerance. This pot culture experiment is being currently conducted under glass house conditions.

Conclusion The two selected drought tolerant (DSR 2 and DSR 12) and two highly drought susceptible varieties (MAUS 2 and MAUS 212), based on first year results, were screened for their symbiotic response with 10 different AM fungi. Based on the yield and total plant dry weight, it is concluded that G. fasciculatum and G. leptotichum are the best symbionts for inoculating DSR 2and DSR 12 drought tolerant varieties. Similarly G. leptotichum was the best symbiont for inoculating MAUS 12 and MAUS 212 drought susceptible varieties.

Conference and workshop attended

l International conference on “Low temperature science and biotechnological advances” organized by NBPGR/ NBAIM at New Delhi on April 27-30, 2015.

l Group meeting on “Developing network of microbial culture collection of India” at NBAIM, Mau on Aug 3, 2015.

l International workshop on “Innovation of environmental

Interaction between the AM fungus G. leptotichum and Bradyrhizobium liaoningense (obtained from DSR, Indore) is being studied using the drought susceptible varieties MAUS 2 and MAUS 212 to improve drought tolerance and in the drought tolerant variety DSR 12 for further enhancement of

stress management of spice crops” at Bogor, Indonesia on Aug 25-27, 2015.

l Asian Mycological Congress, 2015 at Goa University, Goa on Oct 7-10, 2015.

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AMAAS - Annual Report 2015-16

Exploitation of PGPR and Biocontrol Agents for Integrated Management of Biotic stresses of Chilli in Irrigated and Dry Land Ecosystem PI : M. K. Naik Co-PIs : Amaresh Y.S., SavithaA.S.,Arun K. Hosamani, Mahadev Swamy University ofAgricultural Sciences, Raichur and Trichoderma were obtained by serial dilution spread plate method.

Rationale Over the years many potential antagonistic isolates have been identified to demonstrate their efficacy under laboratory and glass house condition. However, not many of them have been tested under field condition. Many a times, even if tested under field condition they are on a limited scale not under multi location conditions and leading to inconsistency in their performance. Hence it has become all the more important to see their efficacy at different locations under field conditions. Although, bio agents are much talked about and are in high demand but at present situation, their level of production and usage is very much limited. Even if seed treatment with bio agent is made mandatory for all crops, we need to produce several lakh tonnes of PGPR and bio agents such as Trichoderma and Pseudomonas to meet their requirement. In addition, their use in IPM as an integral component requires the production of huge quantity of bio agents. Furthermore, consortium approach would fulfil the successful bio agent for various requirementthrough antagonism, induction of systemic resistance, plant growth promotion and several other wide arrays of mechanisms of control of plant pathogens in managing a broad spectrum of plant pathogens. Objectives

l Among 135 isolates, 75 isolates produced fluorescent pigmentation under UV illumination. These 75 isolates were further used for biochemical studies (Fig. 1). Among 90 Trichoderma isolates, 82 isolates were selected based on the growth rate and sporulation. These 82 isolates were further used for cultural and morphological studies.

Fig. 1: Fluorescent pseudomonads isolates

l The Pseudomonas isolates were positive for oxidase, catalase test and citrate utilization, H2S production and was -ve for indole production, MR test.

l Further, Morphological characters of Trichoderma were varied in colour from dark green, light green to yellowish green with flat to raised growth pattern.

l To collect and characterise fluorescent Pseudomonads and isolates of Trichoderma species for identification of potential antagonists for broad spectrum of plant pathogen suppression

l Among 75 isolates, seven isolates PF-103, PF-105, PF-

(IPM) in chilli with bio agents of major diseases in irrigated and dry land conditions.

104, PF-109, PF-120, PF-121 and PF-126 were positive for HCN production and 15 isolates were positive for H2S production, 35 isolates positive for siderophore production. Among them, 5 isolates produced higher siderophore, 8 isolates moderate and remaining were less in siderophore production. Among 5 isolates, PF-115 isolate was found to produce maximum siderophore units (71.25%), followed by isolate PF128 (49.24%), PF-121 (47.25%) and PF-124 (40.78%).

l To generate data required for registration with CIB & RE

l Bioefficacy potentiality of fluorescent pseudomonads

l To develop efficient formulations, determination of shelf life and compatibility studies of Pseudomonas fluorescens and Trichoderma species consortium with commonly used fungicides, insecticides and plant products

l To undertake Integrated Disease and Pest Management

isolates tested, PF-8 showed the maximum per cent inhibition of mycelium (60.65%) the second highest per cent inhibition by PF-28 (55.29%) and remaining were moderate in inhibition of pathogen (Fig.2).

for commercialisation. SignificantAchievements

l A total of 135 soil samples were collected from different regions of northern Karnataka. Isolates of Pseudomonas

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Fig. 4: Amplification for pltB gene encoding plyoluterion antibiotic

Fig. 2: Bio efficacy of fluorescent pseudomonads against Sclerotium rolfsii

l The production of 2,4-DAPG antibiotic was detected by

l The Trichoderma isolates were screened for antagonistic

ethyl acetate extract on HPTLC sheets and the 2,4-DAPG antibiotic was detected at retention factor (Rf) value 0.77 in PF-131and PF-135strains.

and recorded by Tri-9 (83.6 %) isolate followed by Tri-17 (80.6 %) (Fig. 3). The Tri-18 isolate recorded highest inhibition of Aspergillus flavus by 80.8% growth. Dual culture assay against R. solani revealed that per cent inhibition of growth of the pathogen ranged from 61.1 to 90.0%.

l The production of pyoluteorin antibiotic was detected in PF-104 and PF-119 strains as confirmed by TLC at Rf value 0.50.

l The DNA from Trichoderma isolates were amplified and the size of amplified DNA was 600-650bp length. The Trichoderma isolates were sequenced. Among all, only sixteen diversified isolates sequences were deposited in the NCBI database under the accession no KT153650KT153665 (Fig 5).

Fig. 3: Screening of Trichoderma isolates for antagonistic

l Different formulations and shelf life of Pseudomonas and Trichoderma were developed in different carrier materials and the mean population of PF-131 isolate was significantly high in talc at both room and refrigerator temperature over the entire period of storage (10x107 to 14x107cfu/g) followed by vermicompost (11x107 to 12x107cfu/g).

Fig. 5: The size of amplified DNA was 600-650bp length

l An experiment was conducted at UAS Raichur Research Farm for testing the various modules on chilli diseases under field conditions. The experiment was conducted with judicious use of pesticides, bioagents, botanicals and other IPM interventions and following modules were tested.

l The DNA was isolated and purified from Pseudomonas isolates by using HiPure Bacterial DNA Purification Kit following manufacturers protocol (HIMEDIA).

l The result showed that IPM module recorded the least preand post-emergence damping off, wilt/root rot incidence of 4.65, 7.56 and 7.96 per cent respectively as against control followed by farmers practice. Biointensive module recorded almost on par with IPM module with respect to damping off, wilt/root rot with an incidence of 4.92, 7.15 per cent, respectively. In control, damping off (18.2%) wilt/root rot (19.0%), anthracnose (12%) was very high as compare to the biointensive module. However, the

l The extracted DNA was amplified with pltB and pvdAprimers (PltBf and PltBr for PltB) and (PvdAf and PvdBr for pvdA). The seven fluorescent pseudomonads isolates were amplified and amplified and the size of amplified DNA showed 1500 bp length. PF-104 and PF119 showed positive amplification for pltB gene encoding plyoluterion antibiotic (Fig.4).

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AMAAS - Annual Report 2015-16 chemical control recorded the minimum PDI for powdery mildew (12.48%) and fruit rot (13.28%) followed by a consortium of bioagents.

growth. In different formulations, PF-131 isolate was significantly high in talc at both room and refrigerator temperature.

l The incidence of leaf spot (16.94), powdery mildew

Pseudomonas isolates PF-104 and PF-119 showed positive amplification for pltB gene and in TLC (PF-104)and HPLC 2,4-DAPG antibiotic pyoluteorin antibiotic was detected. Trichoderma isolates accession no: KT153650KT153665.

(27.01), root rot/wilt (5.50) was brought down significantly in the IPM plot as compared to the non IPM (leaf spot 27.30, powdery mildew 35.84, wilt 8.25). Similarly, the leaf curl index, the mites infestation, percent fruit damage, and thrips infestation got reduced in the IPM plots as compared to Non IPM plot. Overall average yield of 31.26 q/ha in IPM plot as against 24.6 q/ha in non-IPM plot fetching a net gain of Rs. 54662 per ha over non-IPM apart from preventing untold ecological damage.

In different modules, IPM module recorded the least preand post-emergence damping off, wilt/root rot incidence respectively as against control followed by farmers practice. Biointensive module recorded almost on par with IPM module with respect to damping off, wilt/root rot. However, the chemical control recorded the minimum PDI for powdery mildew and fruit rot followed by a consortium of bioagents. As in case of large scale demonstration, the incidence of leaf spot, powdery mildew, root rot/wilt was brought down significantly in the IPM plot as compared to the non IPM. Overall average yield in IPM plot recorded highest against non-IPM.

Conclusion A total of 135 soil samples were collected from different regions of northern Karnataka. A total of 135 Pseudomonas and 90 Trichoderma isolates were isolated and characterized based on morphological, biochemical and cultural characters. PF-103, PF-105, PF-104, PF-109, PF-120, PF-121 and PF-126 were positive for HCN production and 35 isolates positive for siderophore production.

Conference and Workshop attended

l Naik M.K, Chennappa G, Vinay J.U (2016) Potentials

Bioefficacy potentiality of fluorescent pseudomonads isolates tested, PF-8 showed the maximum per cent inhibition of Sclerotium rolfsi and Trichoderma Tri-18 isolate recorded highest inhibition of Aspergillus flavus by 80.8 per cent

antibiotics of fluorescent pseudomonads in sustainable crop disease management. Contemporary Plant Pathology, IPS 6th International Conference on Plant, Pathogens and People.

Studies on Rhizospheric Microbial Diversity in Relation to Different Sugar Profile Varieties for Growth Promotion and Disease Management PI : Dinesh Singh ICAR-Indian Institute of Sugarcane Research, Lucknow and crop protection through biological agent is the global consensus to reduce inputs of different chemicals which are perceived as being hazardous. Therefore, the biological diversity of sugarcane rhizosphere of different sugar profile varieties will be explored for the growth promotion through microorganisms and potential bio-control agents (BCAs) of fungal, bacterial, and yeast origin.

Rationale Sugarcane (Saccharum spp. hybrid) accorded a premier place among commercial crops because of the vast acreage devoted to its cultivation and part and parcel of daily life. It is regarded as integral component of world trade, which is destined to be consumed either as food or as energy or used to grow the crop. On the other hand it is victim and the vehicle of disease propagules also. Sugarcane crops are raised true to the type because of its vegetative propagation with a serious drawback of concomitant propagation of many serious diseases. Pathogens within the seeds, serves as source of inoculum for secondary infection and keeps on building for increased incidence of disease. Seed cane borne pathogens remain viable within stalks due to rind and nodal barriers even after intensive application of pesticides. Growth promotion

Objectives

l Isolation of sugarcane rhizospheric microbes from different sugar content level varieties for growth promotion, nutrient and disease management in sugarcane and screening of microbes obtained from NAIMCC, NBAIM, Mau for growth promotion, nutrient and disease management.

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l Characterization of potential isolates for growth

0.049×105 while Khakai has yielded minimum number of fungal population 0.005×105 at 50 DAT. Similarly at 100 DAT, maximum bacterial population was recorded in CoLk 94184 (14.11×106), whereas SES 594 yielded the minimum number of bacterial population (03.31×106). In case of fungi, maximum population was recorded in CoJ 64 (0.091×105) whereas minimum number of fungal population was recorded in Khakai (0.012×105). Similar

promotion, nutrient and diseases management in sugarcane.

l Green house/glass house assay for growth promotion and diseases management of few selected and superior microorganisms in sugarcane for 2-3 most popular varieties.

l Field trial of few selected and superior microorganisms for growth promotion, nutrient and diseases management in sugarcane at IISR, Lucknow and two more locations. SignificantAchievements

l A field experiment was conducted with nine varieties of sugarcane namely CoJ 64, CoLk 94184, CoS 8436, Co 1148, CoLk 8102, CoSe 92423, SES 594, Baragua and Khakai had been laid out. Out of these nine varieties, CoJ 64 and CoLk 94184 are the early maturing high sugar varieties; CoS 8436 and Co 1148 are the medium sugar varieties; CoLk 8102 and CoSe 92423 are the low sugar varieties whereas SES 594, Baragua and Khakai are the standard check (Fig. 1).

Fig. 2: Some bacterial and fungal isolates recovered from rhizosphere of sugarcane varieties having different sugar profile. CoJ 64

pattern was recorded after 150 and 250 DAT (Fig. 2).

CoLk 94184

l At the age of 50 days old crop, a high sugar early maturing

SES 594

variety CoJ 64 found associated with maximum number of fungal genera in its rhizospheric region which was 13 in numbers. The fungal genera were Fusarium, Alternaria, Rhizoctonia, Trichoderma, Penicillium, Cladosporium, Acremonium, Chaetomium, Rhinocladiella, Candida, Aspergillus, Rhizopus and Mucor (13). A variety SES 594 belonging to Saccharum spontaneous species and designated for very less sugar content was found association with minimum 05 genera of mycoflora in its rhizospheric zone. Trichoderma, Acremonium, Aspergillus, Rhizopus and Mucor was the mycoflora with SES 594. Rest of the varieties, resulted in between rhizospheric association of mycoflora. Similar pattern was recorded in the fungal population build-up at 100 and 150 DAT. While in 250 days old crop, only 14 genera were recovered. Epicoccum and Candida were disappeared from rhizospheric zone of CoJ 64. No changes were recorded in case of SES 594. Rest of the varieties resulted in between rhizospheric association of mycoflora.

Khakai

Fig. 1: Field cultivation of sugarcane varieties having different sugar profile.

l Rhizospheric microbial diversity was worked out from above mentioned different sugar profile varieties following the sampling schedule of 50 days after transplanting; 100 days after transplanting; 150 days after transplanting; 150 days after transplanting; 250 days after transplanting and 350 days after transplanting (DAT).

l A total of 16 mycoflora genera and 120 numbers bacterial isolates have been isolated using Potato Dextrose Agar, Rose Bengal Agar and Nutrient Agar media with serial dilution factor 10-3 to 10-6. Minimum number of bacterial population 01.42×106 g-1 of soil was recorded in the variety CoLk 94184, whereas, in case of fungal mycoflora, variety CoJ 64 yielded maximum number of fungal population

l Juice analysis of the different varieties has also been conducted but the final results are awaited. Curvularia, Pythium, Verticillium, Nigrospora, Paecilomyces,

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AMAAS - Annual Report 2015-16 association was found increasing with the age of variety up to 150 days and keep on stable up to 205 days. It start decline after attending the maximum sugar content as per their genetic potential. The association of mycoflora (fungi) in rhizospheric region keeps on building with the age of crops up to 250 days. Grand growth phase of the crop resulted highest numbers of rhizospheric microbial association with all the experimental varieties including three different checks.

Epicoccum, Acremonium, and Cladosporium were the genera found different from the germination and establishment phase (at 50 days) to starting of tillering Phase. These genera of mycoflora got build up in rhizospheric microbial association during the grand growth phase and keep on continuing up to ripening and maturation phase of the crop especially with the high sugar early maturing varieties as compare to low sugar varieties.

Conference and workshop attended

l Attended “Advances in plant growth promoting

Conclusion

rhizobacteria: Diversity to Utility” from January 11-20, 2016 at ICAR-NBAIM, Mau (Attended by Mr. Aashish Tiwari, SRF)

Overall, CoLk 94184, a high sugar (early maturing) variety was associated with maximum number of bacteria in their rhizospheric region followed by CoJ 64 which is also a high sugar variety. While in the case of fungal mycoflora association of rhizospheric region, CoJ 64 (high sugar early maturing) variety yielded the maximum number of mycoflora followed by Co 1148 a medium sugar (medium maturing) variety. The trained of rhizospheric region microbial

l Attended the 6th International conference on “Plant Pathogens and People”: Challenges in Plant Pathology to Benefit Humankind organized by Indian Phytopathological Society New Delhi. February 23-27: 2016

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Theme : Microbe Based Green Energy

Synthetic Biology and Metabolic Engineering Opportunities for Enhanced Production of Biofuels through Microbes PI : Anju Arora Co-PIs : Lata Nain, Surender Singh

ICAR-Indian Agricultural Research Institute, New Delhi Rationale

Objectives

Development of sustainable biomass based fuels and chemicals is promising technology for replacing petro-based economy. Biomass processing and production of biofuels presents a distinct set of challenges. Therefore, efforts are on to optimize pretreatment and saccharification process for lignocellulosic biomass into monomeric sugars which can be fermented into fuels and chemicals. Microorganisms and biotechnological approaches are hugely applied to obtain these goals. However, the ability to utilize biomass utilizing and biofuel producing capacity in native organisms is limited and subject to tight regulations. Moreover, some additional traits may be desired in these organisms for them to be efficiently exploited in biorefineries. An ideal biomass processing organism should possess all the desirable characteristics consolidated into it, such as efficient conversion of biomass into polysaccharides and subsequent utilization of most of the sugars produced and tolerance to inhibitors but it is extremely difficult to find such native organism as the conditions applied in industry do not occur naturally for such organisms to evolve. Advances in metabolic engineering and synthetic biology promise to provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels.

l To isolate yeast and bacterial strains from diverse extreme habitats with properties selected to biofuel production.

l To characterize the selected microbial strains and elucidate metabolic and biochemical properties.

l In silico analysis of metabolic networks and metabolic flux analysis.

l Engineering the selected strains for pentose utilization and enhanced biofuel production.

l Validation of the engineered strains for pentose utilization and biofuel production SignificantAchievements

l A native strain of S. cerevisiae LN was found to grow and weakly assimilate xylose but did not show xylose fermentation. It showed highest glucose fermentation efficiency of ~78% at 48 h when 10% glucose was provided in minimal medium. Its potential to carry out mixed substrate fermentation was studied by providing mixed substrates (5% glucose + 5% xylose), in minimal medium supplemented with different amounts of yeast extract and peptone. Highest mixed fermentation efficiency of ~50% was obtained at 96 h with 1% yeast extract and peptone (Table 1).

Table 1: Stimulation of sugar consumption and fermentation efficiencies of S. cerevisiae due to supplementation of yeast extract and peptone. Time (h)

Sample name

96 h

0.1% (YE+P)* 0.5% YE 1% (YE+P) 0.1% (YE+P) 0.5% YE 1% (YE+P) 0.1% (YE+P) 0.5% YE 1% (YE+P)

120 h

144 h

Sugar consumed (mg/ml) Fermentation efficiency (%) 51.81 57.58 61.12 58.74 58.27 62.42 84.83 54.09 62.32

5.33 22.58 48.14 16.11 21.40 42.35 9.54 29.26 38.63

*(YE+P), Yeast extract+Peptone

100

Ethanol yield (g/g) 0.03 0.12 0.25 0.08 0.11 0.22 0.05 0.15 0.20

AMAAS - Annual Report 2015-16

l Optimum level of supplementation, sugar concentration

xylose concentration, time of fermentation, yeast extract concentration, peptone concentration while ethanol produced, biomass and sugars consumed were chosen as responses. Results of optimization predicted the combination of inputs with the possible responses as shown in Table 2.

and time were determined using response surface methodology. The three responses considered were ethanol concentration, sugar consumption and biomass produced. For Response Surface Methodology experiments, CCD layout was obtained using Design Expert Software and chosen input variables were: Glucose concentration,

Table 2: Optimization of ethanol production from S. cerevisiae strain LN through response surface methodology Name

Level

Glu

Expected responses Observed responses Input variables Xyl Yeast Peptone Ethanol Biomass Sugar Ethanol Biomass Sugar Time Extract consumed consumed

5.36

3.30

84.55

0.36

0.25

2.99

l Since this strain exhibits xylose assimilation, xylose

1.83

81.67

2.36

1.80

74.33

strain possesses very low XR and XDH activities. XR: XDH is 3:1, so the redox balance is shifting the mechanism towards ethanol formation rather than xylitol formation.

reductase (XR) and xylitol dehydrogenase (XDH) activities were determined (Table 3). Specific activities were determined (Table 3) and it was observed that this

Table 3: Specific xylose reductase (XR) and xylitol dehydrogenase (XDH) activities of S. cerevisiae strain LN. Sp. activity (U/mg protein)

Enzyme extract S. cerevisiae (2% Glucose+ 2% Xylose) Xylose(2%) Glucose (2%)

XR

XDH

0.18 0.0097 0.15

0.047 0.0066 0.036

l Since pretreatment procedures lead to generation of a variety of inhibitors- phenolics, furfurals, organic acids etc., Kodamaea and S. cerevisiae strain LN were checked for tolerance to main species of inhibitors like furfural, HMF, acetic acid and formic acid. Their growth pattern in presence of HMF, furfural and acetic acid and formic acid were evaluated as compared to controls. Both Kodamaea (Fig. 1) and Saccharomyces (Fig. 2) showed sufficient growth for fermentation on lower concentrations of HMF (upto 1 mg/ml) and furfural (upto 0.5 mg/ml). However, for

Fig. 1: Effect of HMF and acetic acid on strain 5 (A,C) and strain 6 (B,D)

acetic acid, a sudden rise in growth was observed after 72 h for 5 mg/ml concentration.

l In case of S. cerevisiae strain LN, effect of formic acid was observed to be deteriorating its growth. However, when pH of the fermentation medium was adjusted to normal, i.e. 5, its effect was annulled and the strain showed good growth on formic acid, suggesting that growth was hampered due to lowering of pH.

Fig. 2: Effect of HMF, furfural, acetic acid and formic acid (without pH adjustment) on the growth of S. cerevisiae strain LN.

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l Growth and ethanol production by and Kodamaea strains

l Biomass hydrolysates prepared from steam and

and S. cerevisiae strain LN on pretreated paddy straw hydrolysates: Kodamaea and Saccharomyces strains were evaluated for growth on biomass hydrolysates obtained from biologically pretreated paddy straw and ethanol production. Total sugar content in the hydrolysates was ~1.3% (with 2% glucan loading and 57% saccharification efficiency). Growth on hydrolysates was comparable to the control, maximum sugar consumption and ethanol production occurred within 24 h (Fig. 3).

biologically pretreated paddy straw having 11 g L and 16 -1 g L of total sugar concentration, respectively, were evaluated for supporting growth and ethanol production using S. cerevisiae strain LN. Growth of yeast strain on hydrolysates was much slower as compared to controls comprising of defined medium with same concentration of glucose (Fig. 4).

-1

Fig. 4: Growth of S. cerevisiae on steam and biologically pretreated paddy straw hydrolysates.

l Maximum sugar consumption and ethanol production occurred within 48 h. 7.92 g L-1 (72%) total sugar was consumed from steam pretreated hydrolysates whereas about 6.27 g L-1 (40%) of sugar was consumed from biologically pretreated hydrolysates (Table 4). Ethanol production from steam and biologically pretreated hydrolysates was 1.13 g L-1 and 0.86 g L-1 giving about 28 and 27% fermentation efficiency, respectively at 48 h. The ethanol production in both steam and biologically pretreated hydrolysates was statistically at par but significantly lower than controls comprising of defined medium.

Fig. 3: Growth and % sugar consumption by Kodamaea strain 5 (A) and strain 6 (B) on biologically pretreated hydrolysates

Table 4: Sugar consumption and ethanol production from steam and biologically pretreated hydrolysates

Treatments

-1

24 h Steam Biological Controls Glucose (1.1%) Glucose + Xylose (0.8% + 0.4%) SE (±) CD@5%

Ethanol (g L-1)

Sugar consumed (g L ) 48 h

6.16 4.84 11.50 8.41

7.92 6.27 11.53 9.08 0.52 1.44

m

“*” indicates alcohol produced below detection limit by gas chromatograph.

102

24 h 1.66 * 4.76 3.16

48 h 1.13 0.86 5.14 3.28 0.47 1.30

AMAAS - Annual Report 2015-16

l For protoplast fusion, native strain of Saccharomyces cerevisiae and Pichia stipitis were considered. For efficient construction of fusants in intergeneric species, selection traits need to be characterized for both the strains. The main distinguishing traits like temperature tolerance, ethanol tolerance, and antibiotic resiatance were studied which may be used for selection and to be used for screening of fusants.

applied. Out of 10 different yeast strains, 8 strains were identified through ITS sequencing.

l Using 2% glucose, fermentation of these ten strains was carried out and it was observed that ~42% sugars were consumed by most of the strains and maximum ethanol produced was around 3% by strainAX1.

l 27 bacterial strains were isolated from rotten apple and flower samples. Out of these, 6 have shown to produce ethanol. These strains are further being characterized.

l Same profile was obtained for temperature tolerance for both the strains. Growth was observed by both strains till 35°C and thereafter growth was diminished at higher tempereture. Following profile was obtained for both the strains on different antibiotics with hygromycin B as the distinguishing trait amongst all. Both S. cerevisiae and P. stipitis tolerated ethanol concentrations upto 9% (Fig. 5). Ethanol tolerance of S. cerevisiae

Conclusion

From the above findings, Kodamaea strains, which possess natural potential to utilize and ferment xylose and glucose are good candidates for further improvement. In silico studies are underway to understand their metabolic network and identify targets for knockout to divert metabolism towards ethanol production. Paper published fromAMAAS work

l Shalley Sharma, Sonia Sharma, Surender Singh, Lata, Anju Arora (2016). Improving yeast strains for pentose hexose Co-fermentation: Successes and Hurdles. Ed., Sachin Kumar et al. Proceedings of the First International Conference on Recent Advances in Bioenergy Research. 10.1007/978-81-322-2773-1_3. Conference and workshop attended

l Shalley Sharma, Anju Arora, Pankhuri Sharma, Surender Singh, Lata, Debarati Paul (2015). Notable mixed substrate fermentation ability of native Kodamaea strains isolated from Lagenaria siceraria flowers, published in the proceedings of International conference on “New Horizons in Biotechnology” held at Hotel Residency Palace, Trivandrum, organized by Biotechnology Research Society of India (BRSI).

Ethanol tolerance of P.stipitis

l Participated in International Conference on “New Horizons in Biotechnology”, held at Hotel Residency palace, organized by Biotechnology Research Society of India (BRSI).

Fig. 5: Ethanol tolerance of S. cerevisiae strain LN and P. stipitis NCIM 3498.

l Pentose utilizing microbes were enriched from rotten apple

l Shalley Sharma working as SRF in this project participated

sample using xylose as sole C source in the medium and two different pH 5 (for yeasts ) and (7 for bacteria) were

103

in a workshop “Synthetic and Systems Biology Applications in Bioenergy” organized by ICGEB, New Delhi from 29th February to 9th March 2016.

AMAAS - Annual Report 2015-16

Thermophilic Fungi from Diverse Ecosystems and their Potential for Producing Industrially Important Enzymes PI : B.S. Chadha Co-PIs : Amarjeet Kaur Guru Nanak Dev University, Amritsar

l A complex cellulosic medium for hypercellulase

Rationale This project would focus on isolation, screening and developing enzyme producing thermophilic fungal strains that can produce very high levels of enzymes for their commercial applicability.

Objectives

production devised resulting in high expression of cellulases and xylanase (Fig. 2). 97.4 66.2 45.6

l Isolation and screening of diverse thermophilic fungi from high temperature ecological niches for production of thermostable enzymes (cellulases, hemicellulase).

31.0 21.0 M

l System biology studies (proteome based analysis) of the potentially important thermophilic fungi for identification and expression analysis of novel enzymes (glycosyl hydrolases).

l Overproduction of the cellulases/xylanases from selected strains using classical and molecular approaches.

MS) of Penicillium sp. Dal 5 secretome identified 108 different proteins constituting an array of CAZymes including glycosyl hydrolases (GH) belonging to 24 different families, polysaccharide lyases (PL), carbohydrate esterases (CE), lytic polysaccharide monooxygenases (LPMO) in addition to swollenin and a variety of carbohydrate binding modules (CBM) indicating and elaborate genetic potential of this strain for hydrolysis of lignocellulosics (Fig. 1). Composition of proteins in secretome 34% 6%

6%

48%

l A method for evaluating the efficacy of produced cellulases using an array of lignocellulosics substrates was developed.

6%

l Eight cellulolytic fungal cultures submitted in NAIMCC, after molecular characterization Conclusion The cellulase levels of the developed strain is useful in hydrolysis of different pre-treated substrates Paper published fromAMAAS work

l Rai, R., Kaur, B., Chadha, B.S (2016) A method for rapid purification and evaluation of catalytically distinct lignocellulolytic glycosyl hydrolases from thermotolerant fungus Acrophialophora sp. Renewable Energy (In press)

Protein profile based on molecular weight

GHs

70 KdA

Conference and workshop attended

l International Conference on New Horizons in

Others (a)

Biotechnology (NHBT-2015) held at NIIST, Trivandrum, India November 22-25, 2015.

(b) Protein profile based on pI

1 2 3 4 5 6 7

screening carried out to develop high levels of cellulase producing strain.

SignificantAchievements

l The mass spectroscopy analysis (LTQ-Velos-Orbitrap LC-

1 2 3 4 5 6 7

l Penicillum Dal5 Mutants (three cycles of mutagenesis and

l Optimization of the enzymes production by the developed strains at shake flask and their validation at fermenter level.

1 2 3 4 5 6 7

Fig. 2: (a) SDS-PAGE showing protein expression profile of Penicillium sp. Dal5 cultured on CWR medium under shaking flasks (b) and (c) Zymogram showing expression of β glucosidase and cellobiohydrolase I against Methyl umbelliferryl β glucoside (MUG) and Methyl umbelliferryl β cellobioside (MUC)as respective substrateLane 1: Day1; Lane 2: Day2; Lane 3: Day3; Lane 4: Day4; Lane 5: Day5; Lane 6: Day6; Lane 7: Day7. Lane M represents the low molecular range marker.

l International Conference on Advances in Bioprocess Technology” scheduled from 26-28th November 2015 at MACFAST (Mar Athanasios College for Advanced Studies Tiruvalla), Kerala.

Acidic (>6.5) Basic (>7.5) Neutral (6.5-7.5) (c)

Fig. 1: (a) Functional categorization of CAZymes proteins in the secertome of Penicillum sp. Dal 5 (b) and (c) categorization of proteins in secretome on the basis of molecular weight and pI, respectively.

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AMAAS - Annual Report 2015-16

Whole Genome and Transcriptome Sequencing of Selected Cyanobacterial Genomes for Identification of the Role their Genes in Metabolic Pathways Related to Bio-energy Production PI : Sucheta Tripathy CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata Rationale

Conclusion

Since cyanobacteria genomes are under explored in Indian context and many of the genomes are unknown yet, we delve into deciphering genomes of selected cyanobacteria species followed by discovery of novel genes for biometabolites.

These organisms have promising metabolic as well as bioremediation potential. In addition to this, some of these organisms have huge fatty acid producing potential. Reverse genetics is a huge advantage for exploring these organisms commercially.

Objectives

l Grow the organisms and sequence the entire genome to study the genetic blue prints of these organisms.

Paper published fromAMAAS work

l Das S, Singh D, Madduluri M, Chandrababunaidu MM,

l Use computational methods for predicting genes and

Gupta A, Adhikary SP, Tripathy S (2015) Draft Genome Sequence of Bioactive-Compound-Producing Cyanobacterium Tolypothrix campylonemoides strain VB511288. Genome Announcement.2:3. pii: e00226-15. doi: 10.1128/ genome A.00226-15. PubMed PMID: 25838485; Pub Med Central PMCID: PMC4384489.

constructing metabolic pathways.

l Identify gene belonging to novel pathways. l Study the transcriptomics and profile them under control and treated conditions.

l Chandrababunaidu MM, Singh D, Sen D, Bhan S, Das S,

SignificantAchievements

l Genomes of Tolypothrix boutellei and

Tolypothrix campylonemoides strain VB511288 were sequenced using 300 base pairs and 400 base pairs single end sequencing using Ion Torrent as a platform. Illumina HiSeq sequencing of the above mentioned genomes were sequenced. The biosynthesis of the siderophore group of nonribosomal peptide synthetic pathways was predicted using KAASKEGG. The enzymes necessary for salicylate biosynthesis, isochorismate synthase, isochorismate pyruvate lyase, DhbF, and EntF are present in this assembly version. Eight genes from β-lactams and 7 genes from vancomycin resistance pathways were predicted in this genome. This organism also poses unique evolutionary challenges, and taxonomists opine that Tolypothrix and Scytonema should be merged (http://www.algaebase.org). The genome sequences of S. tolypothrichoides VB-61278 will undoubtedly be a very rich resource for biologists, genomicists, and evolutionary biologists (Table 1).

Gupta A, Adhikary SP, Tripathy S (2015). Draft Genome Sequence of Tolypothrix boutellei Strain VB521301. Genome Announcement. 19:3(1). pii: e00001-15. doi: 10.1128/genomeA.00001-15. Pub Med PMID: 25700407; PubMed Central PMCID: PMC4335316. 8486. Book Chapter

l Tripathy S, Singh D, Mathumalar C, Das A, 2015. Dissecting transcriptomes of cyanobacteria for novel metabolite production. In. Genomics, Proteomics and Metabolomics in Nutraceuticals and Functional Foods. John Wiley & Sons, Ltd, 557-72. Conference and workshop attended

l Cloning of glycogen synthase from the filamentous cyanobacterium Tolypothrix bouteillei in E. coli and determination of glycogen content. Diya Sen, Sushma Bhan, Mayuri Mukherjee, Vineeta Vinni, Rahul Gajbhiye, P. Jaisankar and Sucheta Tripathy. Advances in Algal Biotechnology. workshop conducted by IIT Mumbai on 21st November 2015.

Table 1: Genome sizes of all the genomes sequenced so far. Name of organism Hassallia byssoidea Tolypothrix campylonemoides Tolypothrix bouteillei Scytonema millei Scytonema tolyphothrichoides

Code 170 288 268 283 278

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Estimated genome size (MB) 13 10.6 11.5 11.63 10

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Developing Enzyme Catalyzed- Microbe Mediated Processes for Biofuel from Mango Waste (Peel and Kernel) PI : Neelima Garg ICAR-Central Institute for Subtropical Horticulture, Lucknow respectively. The experimental data revealed that the highest value of reducing sugar (35.87%) from mango kernel at temperature (40°C), pH (6.5%), and cocktail of enzymes viz. α- amylase (1980U), amyloglucosidase (930U) and tannase (2.40 U).

Rationale Mango (Mangifera indica L.) is one of the most important tropical fruits. During the manufacture of mango products large quantities of waste are generated; they account for 35–55% of the fruit depending on the variety. Actual figures on the quantity of mango waste generated commercially are not readily available. However, a rough estimate of the waste available annually in India alone based on the quantities of mango processed (0.2 million tonne) is of the order of 90,000 tonnes. Mango peel along with pulper waste sometimes referred to as peel waste constitutes 20%–30%. The peel waste is a rich source of fiber. The seed content of different varieties of mangoes ranges from 9% to 23% of the fruit weight and the kernel content of the seed ranges from 45.7% to 72.8%. Kernel contains carbohydrate (69.22%–78%) mainly starch. These raw materials are in abundance and may be utilized for biofuel production.

l This work was aimed to investigate the fermentation conditions viz temperature, pH, inoculum size and nutrient addition (NPK) for optimal production of bio-ethanol from the mango kernel substrate under the solid state fermentation by using the Central Composite Rotatable Design (CCRD) and Response Surface Methodology.The mango kernel was saccharified into the reducing sugars through cocktail of commercial α-amylase. Consequently, the time period for maximal production of bio-ethanol was performed at 40⁰C and pH 5.5 by optimizing the incubation time from 1 to 12 days. Maximum bio-ethanol production of 9.14 % was obtained at 40°C temperature, 5.5 pH, 2% nutrient addition and by inoculating 8.0 X 105 cfu /mL for 8 days under anaerobic culture conditions. Ethanol concentration gradually increased with increasing the incubation period up to 10 days, and a maximal ethanol production of 9.50% was obtained. Beyond ten days of incubation the ethanol production became constant. Gas chromatographic analysis revealed that alcohol produced contained 98% ethanol.

The aim of the project is to develop protocol for production of biofuel from mango peel and kernel through enzyme catalyzed- micobe mediated processes and minimize environmental concerns with regard to mango waste disposal.

Objectives

l Enzyme based microbes mediated alcohol production from mango kernel.

l Optimization of enzyme-mediated saccharification conditions of acid pretreated mango kernel starch.

l Five amylolytic fungal isolates (from fruit composts and farm yard manure) were grown on specific growth medium i.e. mango kernel broth (5%). These were tested for amylase activity. Three fermentation protocols were followed.

l Optimization of fermentation conditions for the production of ethanol from Mango kernel starch by Saccharomyces cerevisiae.

l Screening of microbe /microbial consortia for hydrolytic enzyme activity.

l Molecular identification of potent yeast strains. SignificantAchievements

l The objective of this study was to maximize the reducing sugar yield through the pretreatment of mango kernel starch with acid/enzymatic hydrolysis using cocktails of enzymes viz. α-amylase, amyloglucosidase and crude tannase using response surface method Box- Behnken Design with 46 experimental run. The ranges of the factors were selected between the 30-50°C (temperature), 5.5-7.5 (pH), 990 U –2970U (α-Amylase), 510U -930U (amyloglucosidase) and 1.30 – 4.80U (tannase)

(a) Mango kernel 10% (w/v) inoculated with consortia (2% v/v) at 37 °C for 3 days and then inoculated with S. cerevisiae (2%) and incubated at 37 °C for 20 days. Amylase activity was measured 0.47UmL-1 after 3 days fermentation. Reducing sugar gradually increased from initial 0.00 to final 2.6 % after 3 days fermentation with fungal consortia and alcohol from 0.011 (1st day) to 0.97% after 20 days of fermentation. (b) Mango kernel suspension 5% was taken and added with consortia culture and incubated at 37 °C for 5 days. After 5 days incubation, 10% mango kernel suspension was added and boiled at 70°C for 15 min and after cooling inoculated with consortia culture and incubated at 37 °C for 3 days. Observed value of amylase activity, TSS and reducing

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AMAAS - Annual Report 2015-16 Table 1: Identification and clear zone diameter of fungal isolates Sample Code used Microscopic Diameter of for fungi identification clear zone BD-505 Ct-B 3.0cm Penicillium sp.

sugar. Fermented mango kernel suspension were inoculated with 2 % S. cerevisiae and incubated at 37 °C for 20 days and analysed alcohol production. Amylase activity was found 0.84 UmL-1 after 3 days fermentation. Reducing sugar produced from 0.00 (initial stage) to 6.7 % (final stage) after 3 days fermentation with consortia fungus culture. Alcohol production increased from 0.014% (1st day) to 3.62 % after 20 days of fermentation.

(c) Fermented mango kernel suspension inoculated with 2 % S. cerevisiae and incubated at 37 °C for 20 days and production of bio-ethanol was observed at after 5 days intervals up to 20 days. Amylase activity was 0.97 UmL-1 after 3 days fermentation. Reducing sugar was produced 8.6 % after 3 days fermentation with consortia fungus culture and 4.57 % alcohol after 20 days fermentation.

BD-502

Ct-L

Acremonium sp.

1.8cm

Vermicompost

Ct-M

Aspergillus sp.

2.0cm

Vermicompost

Ct-N

Penicillium sp.

3.5cm

Table 2: Cellulase enzyme activity of fungal isolates individually or in combination, using mango peel as a substrate Combination of culture Ct-B

l Four fungal isolates from biodynamic preparations based on high clear zone in CMC agar plates using congo red, were identified and screened for cellulolytic activity individually and in combination based on clear zone and enzyme activity (Table 1 and 2). Combination of Ct-B, CtM and Ct-N was found to have highest cellulolytic activity i.e. 2.88 IUmL-1 for carboxymethyl cellulose using mango peel as substrate by solid state fermentation.

Enzyme activity (IU/ml) 1.502

Ct-M

0.989

Ct-N

1.578

Ct-L

1.212

Ct-B + Ct+N

2.641

Ct-N + Ct-M

2.466

Ct-B + Ct-M

2.603

Ct-B + Ct-M + Ct-N

2.886

l One of the main limitation with Saccharomyces cerevisiae is its inability to grow at high sugar concentrations. Since after hydrolyzing kernel, sugar concentration goes as high as 35OB is achieved, it needs to be diluted to 22OB in order to get optimum yeast growth, which involves cost and time

exploited at industrial level for ethanol production.

l Six yeast strains isolated from the fruit juice, distillaries, sugar rich sources were subjected to molecular characterization.

l An obligate osmophilic yeast that requires high sugar concentrations for growth was isolated from mango slices stored in sugar syrup.

l Lengths of amplified fragments using the primers ITS1 and

l The colonies were smooth, round, convex and white to cream coloured, with a diameter of 0.2 – 0.5 mm at 3 – 7 days. It was identified as Zygosaccharomyces rouxii by molecular characterization. The sugar tolerance of yeast isolate was determined by incubating the organisms in a series of sugar media. Optimum growth for this strain was in 40oB sugar solution while it could grow at 60 oB as well. Optimum growth temperature was 30°C, while the optimum pH was 4.5. The isolate was found to be heat sensitive as could not survive at 60oC for 5 min and could tolerate 12% alcohol in the medium. It was found to be sensitive to 100 ppm sodium benzoate and SO2. However, it exhibited invertase activity 2.179 IU/ml and had the ability to produce alcohol. It could produce 8.7 % alcohol in 3060oB. The main alcohol produced was ethanol as revealed by GC analysis. Since this organism has alcohol producing property even at high sugar concentrations, it may be

ITS4 ranged from 550 bp to 880 bp (Fig. 1). Sequences were analysed through NCBI BLAST search and the samples Y1, Y2, Y3 Y4 and Y5 showed high similarity (88 – 90%) to Saccharomy cescerevisiae, Y6 Fission Yeast and Y7 (98%) to Zygosachharomyces rouxii.

M

Y1

Y2

Y3

Y4

1000 1000 1000 900 800 700 600 500 400 300 200 100

Fig. 1: PCR assay for yeast identification

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Y5 Y6

Y7

AMAAS - Annual Report 2015-16 Sonipat, Haryana, from 17-18th March 2016, page no. 109, PP-46.

Conclusion Highest value of reducing sugar (35.87%) from mango kernel at temperature (40°C), pH (6.5), and cocktail of enzymes viz. α- amylase (1980U), amyloglucosidase (930U) and tannase (2.40 U). Maximum bio-ethanol production of 9.14 % was obtained at 40°C temperature, 5.5 pH, 2% nutrient addition and by inoculating 8.0 X 105 cfu /mL for 8 days under anaerobic culture conditions. Conference and workshop attended

l Neelima Garg, Priti, Kaushlesh K. Yadav and Preeti Singh.

l International Symposium on Sustainable Horticulture, Department of Horticulture, Aromatic & Medicinal Plans, Mizoram University, Aizawl, India, from 14-16th March, 2016.

l Training-cum-Workshop on Intellectual Property Rights for Innovation in Agricultural Research organized by National Bureau of Fish Genetic Resources, Lucknow, on 29th March 2016.

l National Conference on “Innovation & Biopreneurship

Characterization of osmophilic yeast isolated from mango slices stored in sugar syrup. International Conference on Food Value Chain: Innovations and Challenges, NIFTEM,

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for Sustainable & Rural Development “ on 9th May,2015 organized by CSTUP and UPTU at S.R. Group of Institutions, Lucknow.

Theme : Microbial Genomic Resource Repository

Microbial Genomic Resource Repository PI : Alok K. Srivastava1 Co-PIs : Prem Lal Kashyap2, Hillol Chakdar1, K. Pandiyan1 1 ICAR National Bureau of Agriculturally Important Microorganisms, Maunath Bhanjan 2 ICAR-Indian Institute of Wheat and Barley Research, Regional Station, Flowerdale, Shimla

l Characterization, validation and molecular typification of

Rationale Microbial genetic materials (DNA/RNA, plasmids, vectors, clones, cDNA) are the research tools for microbiology and biotechnology sciences. The vast majority of microorganisms and their gene pool around the globe still remain hidden and need to be explored, identified, conserved and utilized for the benefits of mankind. Microbial genetic resources are established in many counties around the world having a variety of purpose. These range from small specialized collections that support small groups of researchers to the large international public service repositories that provide reference materials and services to the scientific community and bio-industries. The huge gap between the discovery of new microorganisms and their potential numbers in nature has stimulated an interest in microbial diversity and the harnessing of their genes, properties and products. The operations of microbial collections have changed over the last twenty years as a result of the advancement of bioinformatics and the facility to present electronic data over the internet. This makes even the smaller collection resources more accessible. ICAR has taken up an initiation to establish MGRR at NBAIM, Mau. MGRR is a facility that preserve and conserves the genetic material of the agriculturally important microorganism, maintained in selected hosts or cloned and maintained in plasmids, accompanying the data details. This new organizational structure indicates the high importance and visibility that NBAIM places on our role as custodians of microorganisms and its related genetic resources. The policies and procedure represent evaluation, maintenance, regeneration, distribution and maintains genetic material at MGRR. MGRR maintains genetic material like, DNA/RNA, plasmids, vectors, clones, cDNAetc.

the reference microbial cultures and generation of molecular dataset and generation of barcode as reference.

l Exploration for collection of environmental microbial samples from different agro climatic regions and direct DNAisolation through metagenomic approaches.

l Collection of DNA materials from microorganisms and other relevant organisms which result from the various molecular genetics and genomics research programs.

l Acquisition of gene constructs from various sources. l Production/multiplication and quality control for distribution SignificantAchievements Cloning and expression of xylanse gene from Bacillus subtilis CG13:

l A total of 32 Bacillus spp. previously isolated from saline soil of eastern Indo-gangetic plain (IGP) of Uttar Pradesh, India were screened for in-vitro xylanase production. Out of 32 strains, one strain (CG13) was found to be positive for xylanase production and selected for further study.The strain CG13 was confirmed as Bacillus subtilis by phenotyping and sequencing of 16S rRNA gene sequence analysis.

l A set of two primers Xyl_Fexp (5' G C G G AT C C AT G T T TA A G T T TA A A A A G 3 ' ) Xyl_Rexp1 (5' ATGAATTCCCACACTGTTACATT 3') with restriction site BamHI and EcoRI were designed and xylanase gene (~700 bp) was successfully amplified in Bacillus subtilis CG13.

l The amplified gene product was purified and cloned in E.

Objectives

l Nationwide survey and collection of information about Microbial Genetic Resources.

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coli DH5α using pJET 1.2 cloning vector.Transformed colonies of E. coli DH5α for desired insert of xylanase gene were screened through colony PCR and confirmed through sequencing analysis.

AMAAS - Annual Report 2015-16

l Xylanase gene was also cloned in E. coli BL21 using pJET

through colony PCR and confirmed through sequencing analysis (Fig.1).

vector and the transformed colonies of E. coli BL21 for desired insert of xylanase expression gene were screened

Fig. 1: Cloning and expression of xylanase gene in E. coli DH5α and E. coli BL21 host cell, respectively using pJET 1.2 cloning vector

l Xylanase gene expression in recombinant bacterium was

l In silico sequence analysis and stereo chemical evaluation

confirmed through quantitative estimation of the xylanase enzymes produced in LB medium (supplemented with kanamycin by the addition of isopropyl-β-Dthiogalactopyranoside [IPTG]) and SDS-PAGE which revealed that one distinct band with molecular weight of approx. 24kDa (Fig.2) was present in the enzymes produced by the bacterium.

of modeled B. subtilis xylanase protein 3D structure shows high quality of predicted structural model, without any residue in disallowed region (Fig.3).

Schematic representation showing the arrangement of the alpha helices, β sheet and loop region in the predicted 3D structure of the xylanase protein

Ramachandran plot showing the validation of xylanase protein using procheck. Residue distribution: 84.2%, allowed region (15.8%), generic (0.0%) and disallowed region (0.0%).

Fig 3: Evaluation of B. subtilis xylanase structural model with verify-3D, Ramachandran plot analysis of predicted structure model of B. subtilis.

Characterization of PGPRs from tomato and potato rhizospheric soil of Eastern Uttar Pradesh

l Rhizospheric soil samples of tomato and potato were collected from various districts of Eastern Uttar Pradesh (Gazipur, Varanasi, Mirzapur and Gorakhpur etc.) and bacterial strains were isolated.

Fig. 2: SDS-PAGE analysis for xylanase

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l On the basis of KOH test, a total of 61 gram positive bacterial

40 and 11 isolates were positive for IAA (2.5-166.0µg/ml), siderophore, ammonia and HCN production, respectively. Elevan and 23 isolates were found to solubilize mineral phosphate and zinc and 17, 27 and 14 isolates were positive for lipase, protease and amylase (0.67-28.7IU/ml) production, respectively (Fig. 4). Nine isolates showed antagonism against Fusarium oxysporium f. sp . lycopersici.

strains and 125 gram negative bacterial strains were isolated. The gram positive bacterial strains (61) were characterized for morphological, physiological, biochemical, plant growth promotion and antagonistic properties.

l All the 61 isolates were able to grow at pH range of 7-10. Five isolates are able to grow at 15% NaCl, one isolate at 20% NaCl and 8 isolates at 4°C. Among 61 isolates, 16, 5,

Spread

IAA

Pure culture

P-solubilization

Stereomicroscopy

Ammonia Production

Siderophore production

Gram Staining

Lipase

Starch hydrolysis

HCN Production

Fig. 4: Morphological and biochemical characterization of Gram positive strains

l Genomic DNA from gram positive bacterial isolates

l Pseudomonas genus specific primers, PA-Gs-F- 5'

were extracted and 16S ribosomal DNA was amplified and subjected to PCR-RFLP analysis with restriction enzyme HaeIII (Fig.5). Upon RFLP analysis 7 operational taxonomic units (OTU) were obtained.

GACGGGTGAGTAATGCCTA 3') PA-Gs-R- (5' CACTGGTGTTCCTTCCTAT 3') were used for selective amplification of Pseudomonas sp. Out of 125 isolates screened, 40 isolates were found to be Pseudomonas sp. Characterization of Trichoderma and Fusarium species from rhizospheric region of chickpea and pigeonpea:

l A total of 22 isolates of Trichoderma sp. and 10 isolates of Fusarium sp. were isolated from rhizospheric region of chickpea and pigeonpea from different locations of Uttar Pradesh.

l Morphological and biochemical characterization of all the isolates were performed including plant growth promoting properties (Fig. 6).

l The internal transcribed spacers (ITS) sequences were amplified using the primers ITS1 and ITS-4 (Fig. 7). Further chitinase and β-tubulin gene sequences from Trichoderma sp. were amplified (Fig. 8 & 9) and purified and preserved at -80 ⁰C.

Fig. 5: Restriction Fragment Length Polymorphism (RFLP) for HaeIII. Enzyme PCR products of 16S rDNA gene digested by Hae III enzyme are shown in lanes 1, 2, and 3; M is DNAmolecular size marker

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AMAAS - Annual Report 2015-16

Fig. 6: Bright field and Fluorescent microscopy images of Trichoderma sp. (spores and conidiospores) and Fusarium sp.

Fig. 7: ITS 1-4 primer amplification of Trichoderma sp. and Fusarium sp.

Fig. 9: β-tubulin gene amplification from Trichoderma sp.

Fig. 8: Chitinase gene amplification from Trichoderma sp.

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l Pathogenicity test was performed in two wilt susceptible

l Wheat grain inoculum of each isolate of F. oxysporum f. sp.

chickpea varieties, L550 and JG315. Fusarium oxysporum f. sp. ciceris inoculum was prepared on wheat grains.About 100 g water soaked grains were taken into 500 ml Erlenmeyer flask, sealed by cotton plug and were sterilized for 30 min and inoculated with 10 mycelia discs (5 mm) obtained from 4 day old cultures of F. oxysporum f. sp. ciceris.

ciceris was thoroughly mixed with the soil at the rate of 20 g/kg soil. Observations on number of plants wilted in each pot were recorded at 30, 45 and 60 days after sowing. The plants that showed drooping petioles, rachis and leaflets without any external rotting in the roots, but dark brown discoloration of internal xylem was considered as wilted (Fig. 10).

Fig. 10: Pathogencity test F. oxysporum f. sp. ciceris by root dip method

Conclusion Bacillus subtilis CG13 was found to be positive for xylanase production in which xylanase gene ~700 bp was successfully amplified by aA set of two primers Xyl_Fexp (5' GCGGATCCATGTTTAAGTTTAAAAAG3') Xyl_Rexp1 (5' ATGAATTCCCACACTGTTACATT 3') with restriction site BamHI and EcoRI. The amplified gene product was purified and cloned in E. coli DH5α using pJET 1.2 cloning vector. Transformed colonies of E. coli DH5α for desired insert of xylanase gene were screened through colony PCR and

confirmed through sequencing analysis. Xylanase gene expression in recombinant bacterium was confirmed through quantitative estimation of the xylanase enzymes produced in LB medium. 125 isolates from IGP were screened with Pseudomonas genus specific primers,PA-Gs-F- 5' G A C G G G T G A G TA AT G C C TA 3 ' ) PA - G s - R - ( 5 ' CACTGGTGTTCCTTCCTAT 3') out of which 40 isolates were found to be Pseudomonas sp. The chitinase and β-tubulin gene sequences from Trichoderma sp. were also amplified and purified and preserved at -80 ⁰C.

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Publications

Articles 1. Babu S, Prasanna R, Bidyarani N, Nain L and Shivay YS (2015) Synergistic action of PGP agents and Rhizobium spp. for improved plant growth, nutrient mobilization and yields in different leguminous crops. Biocatalysis and Agricultural Biotechnology, 4:456-64 2. Bhattacharjee P, Acharya A, Chakraborty U and Chakraborty BN (2015) Evaluation of value added vermicompost on field grown rice plants. Journal of Botanical Society of Bengal, 69: 99-108 3. Chandrababu naidu MM, Singh D, Sen D, Bhan S, Das S, Gupta A, Adhikary SP, Tripathy S (2015) Draft genome s e q u e n c e o f To l y p o t h r i x b o u t e l l e i s t r a i n VB521301.Genome Announcements, 3: 00001-15 4. Damodaran T, Rai RB, Pandey BK, Sharma DK, Mishra VK, Jha SK and Kumar P (2015) CSR-BIO a boon for horticultural crops in sodic soils. Indian Horticulture, 3:16-19 5. Dey PL and Chakraborty BN (2015) Screening of phosphate solubilizing isolates of actinomycetes for in vivo assay for antagonistic activity against fungal pathogens. Journal of Scientific Research and Advances, 2: 137-140 6. Gupta S, Summuna B and Gupta M (2016) Mushroom cultivation: A means of nutritional security in India. Asia Pacific Journal of Food Safety and Security, 2: 3-15 7. Khati S and Chakraborty BN (2015) Morphological characterization of rice cultivars their root colonization with arbuscular mycorrhizal fungi and screening for field resistance caused by brown spot disease. NBU Journal of Plant Sciences, 9:78-86 8. Krishnani KK, Kathiravan V, Meena KK, Sarkar B, Kumar S, Brahmane MP, Kumar N, Mohanty BP and Kailasam M (2016) Bioremediation of aquatic toxicants: application of multi-omic approaches. Advances in Fish Research, 3:1-28 9. Manjunath M, Kanchan A, Ranjan K, Venkatachalam S, Prasanna R, Ramakrishnan B, Hossain F, Nain L, Shivay YS, Rai AB and Singh B (2016) Beneficial cyanobacteria and eubacteria synergistically enhance bioavailability of soil nutrients and yield of okra. Heliyon, 2:e00066

10. Pandey RR, Chongtham I and Muthukumar T (2016) Influence of season and edaphic factors on endorhizal fungal associations in subtropical plantation forest trees of North-eastern, India. Flora-Morphology, Distribution, Functional Ecology of Plants, 222: 1–12 11. Prasanna R, Hossain F, Babu S, Bidyarani N, Adak A, Verma S, Shivay YS and Nain L (2015) Prospecting cyanobacterial formulations as plant-growth-promoting agents for maize hybrids. South African Journal of Plant and Soil, 32:199-207 12. Prasad RD, Navaneetha T and Venakateswar Rao L (2016) Plant growth promotion and Induced defence response in safflower (Carthamus tinctorius L.) by Trichoderma. Journal of Biological Control, 30:40-48 13. Rai R, Kaur B, and Chadha BS (2016) A method for rapid purification and evaluation of catalytically distinct lignocellulolyticglycosyl hydrolases from thermotolerant fungus Acrophialophora sp. Renewable Energy, doi: 10.1016/j renene.2016.02.011 14. Ramanujam B., Poornesha B. and Yatish K.R. (2016) Screening of Beauveria bassiana and Metarhizium anisopliae isolates against Sesamia inferens (Walker). Accepted in Indian Journal of Entomology 15. Ramanujam B, Poornesha B, Yatish KR and Renuka S (2015) Evaluation of pathogenicity of different isolates of Metarhizium anisopliae (Metchnikoff) Sorokin on maize stem borer Chilopartellus (Swinhoe) using laboratory bioassay. Biopesticides International, 11:89-95 16. Renuka S, Ramanujam B and Poornesha B (2016) Endophytic ability of different isolates of entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin in stem and leaf tissues of maize (Zea mays L.) Accepted in Indian Journal of Microbiology 17. Renuka S and Ramanujam B (2016) Fungal endophytes from maize (Zea mays L.): Isolation, identification and screening against Maize stem borer, Chilopartellus. Journal of Pure and Applied Microbiology, 10: 523-528 18. Renuka S, Ramanujam B and Poornesha B (2015) Screening of Beauveria bassiana (Balsamo) Vuillemin isolates against maize stem borer, Chilo partellus (Lepidoptera: Pyralidae) and the effect of solid substrates on conidial production and virulence. Journal of Pure

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and Applied Microbiology, 9: 2979-2986 19. Salunkhe VP, Sawant IS, Banerjee K, Wadkar PN and Sawant SD (2015) Enhanced dissipation of triazole and multi-class pesticide residues on grapes after foliar application of grapevine associated Bacillus species. Journal of Agricultural and Food Chemistry, 63: 1073610746 20. Sawant IS,Wadkar PN, Rajguru YR, Mhaske NH, Salunkhe VP, Sawant SD and Upadhyay A (2016) Biocontrol potential of two novel grapevine associated Bacillus strains for management of anthracnose disease caused by Colletotrichum gloeosporioides. Biocontrol Science and Technology, 26: 964-979 21. Sharma S, Sharma S, Singh S, Lata, Arora A (2016) Improving yeast strains for pentose hexose cofermentation: Successes and hurdles. Ed., Sachin kumar et al. Proceedings of the First International Conference on Recent Advances in Bioenergy Research. 10.1007/97881- 322-2773- 1_3 22. Sekhar AC and Thomas P (2015) Isolation and identification of shoot-tip associated endophytic bacteria from banana cv. grand naine and testing for antagonistic activity against Fusarium oxysporum f. sp. cubense. American Journal of Plant Sciences, 6:943-954 23. Silvia CH, Indira WD, Gunapati O, Avijeet SO, Ojit SK, Indrama TH, Subhalaxmi SA, Romi KH, Thadoi DA, Miranda L, Tiwari ON and Kalita MC (2015) Pigment production and growth metabolites amongst Nostoc strains of unexplored areas of Manipur, India falling under Indo-Burma biodiversity hotspots. International Journal of Advanced Research, 3: 1752-1761 24. Sivakumar G, Rangeshwaran R, Yandigeri MS, Mohan M, Venkatesan T and Verghese A (2016) Diversity of culturable gut bacteria associated with the field populations of cotton leaf hopper (Amrasca biguttula biguttula) in India. The Indian Journal of Agricultural Sciences, 86: 208–15 25. Thomas P, and Upreti R (2015) Evaluation of tomato seedling root-associated bacterial endophytes towards organic seedling production. Organic Agriculture, 1-10 26. Thomas P, Sadashiva AT, Upreti R and Mujawar MM (2015) Direct delivery of inoculum to shoot tissue interferes with genotypic resistance to Ralstonia solanacearum in tomato seedlings. Journal of Phytopathology, 163: 320-323 27. Thomas P, Sekhar AC, Upreti R, Mujawar MM, and Pasha SS(2015) Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured

method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples. Biotechnology Reports,8: 45-55 28. Tiwari ON, Indira DW, Silvia CH, Thadoi DA, Gunapati O, Avijeet SO, Ojit SK, Indrama Th, Subhalaxmi SA, Romi KH, Minerva SH, Miranda L and Prasanna R (2015) Modulation of phycobiliprotein production in Nostoc muscorum through culture manipulation. Journal of Applied Biology and Biotechnology, 3: 11-16 29. Tiwari ON, Ojit SK, Avijeet SO, Indrama TH, Gunapati O, Subhalaxmi SA, Silvia CH, Romi KH and Sharma GD (2015) Isolation, morphological and biopigments evaluation of the genera Nostoc and Anabaena (Nostocales) from Loktak Lake. International Journal of Advanced Research, 3: 1597-1607 30. Tripathi C, Mahato NK, Singh AK, Kamra K, Korpole S and Lal R (2015) Lampropedia cohaerens sp. nov., a biofilm forming bacterium isolated from the microbial mats of a hot water spring, located atop the Himalayan ranges at Manikaran, India. International Journal of Systematic and Evolutionary Microbiology, 10990.000853 Book Chapters 1. Chakraborty BN (2016) Scoping the potential uses of beneficial microorganisms for biopesticide industry and entrepreneurship development in crop protection. In: Perspectives of plant pathology in genomic era (eds. P. Chowdappa, Pratibha Sharma, Dinesh Singh and A.K. Misra). New Delhi, IsBal: 81-7019-526-4 2. Chakraborty BN, Chakraborty U, Sunar K and Dey PL (2015) Molecular phylogenetic analysis of Talaromyces flavus, a phosphate solubilizing fungus. In: Molecular and biotechnological approach to resource utilization:Microbes to angiosperm (eds. S. Ray and S.K.Sen).Visva Bharati and Levant Books, Kolkata, 2128 3. Grover M, Bodhankar S, Maheswari M and Srinivasarao CH (2016) Actinomycetes as mitigators of climate change and abiotic stress. In: Plant growth-promoting actinomycetes: A new avenue for enhancing the productivity and soil fertility of grain legumes (eds. Gopalakrishnan Subramaniam, Sathya Arumugam and Vijayabharathi Rajendra). Springer-Verlag Berlin, 203-212 4. Grover M, Venkateswarlu B, Desai S, Gopinath KA and Srinivasarao CH (2016) Application of Microbiology in Dryland Agriculture. In: Dryland agriculture (eds. Muhammad Farooq and Kadambot HM Siddique).

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5.

6.

7.

Springer-Verlag Berlin Heidelberg Gupta P, Sharma R, Sharma MK, Sharma MP, Satpute GK, Garg S, Sneh L, Pareek S and Pareek A (2016) Signaling cross talk between biotic and abiotic stress responses in soybean. In: Abiotic and biotic stresses in soybean production (eds. Mohammad Miransari). 27-52 Gurikar C, Naik MK and Sreenivasa MY (2016) Azotobacter: PGPR activities with special reference to effect of pesticides and biodegradation. In: Microbial inoculants in sustainable agricultural productivity. Springer India, 229-244 Magotra S, Trakroo D, Ganjoo S and Vakhlu J (2016) Bacillus-mediated-induced systemic resistance (ISR)

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