CHARACTERIZATION OF INDIGENOUS SOIL ISOLATES OF ASPERGILLUS NIGER AGGREGATE AND DEVELOPMENT OF THEIR COMMERCIAL FORMULATIONS FOR THE MANAGEMENT OF WILT DISEASE COMPLEX OF CHICKPEA AND TOMATO CAUSED BY FUSARIUM SPP. AND MELOIDOGYNE INCOGNITA THESIS SUBMITTED FOR THE DEGREE OF Doctor of Philosophy IN (Agriculture) PLANT PROTECTION (Plant Pathology & Nematology) BY MD. ARSHAD ANWER DEPARTMENT OF PLANT PROTECTION FACULTY OF AGRICULTURAL SCIENCES ALIGARH MUSLIM UNIVERSITY ALIGARH 202 002, INDIA 2010 ACKNOWLEDGEMENTS First and foremost, I offer my heartiest thanks to Almighty ALLAH, the most Merciful and the most Beneficent who showered upon me the power and means to complete this thesis. It is my privilege to acknowledge with deep sense of gratitude and humble submission to my research supervisor, Dr. Mujeebur Rahman Khan, Associate Professor, Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh, India for his able guidance, affectionate attitude and authenticate support in bringing up this work in present form. His indebted oracle, unobjectionable counseling, enduring inquisitiveness and affectionate demeanour during accomplishment of this task served as reinforcement to wrap up the present thesis. I wish to acknowledge with thanks Prof. P.Q. Rizvi, Chairman and Prof. Akhtar Haseeb, ex-Chairman, Department of Plant Protection, Faculty of Agricultural Sciences, Aligarh Muslim University, Aligarh for providing facilities to carry out the research and also to Mr. Mohd. Ali Khan, Chairman, PHET for providing space for my pot trial and some other lab facilities. I am highly grateful to Dr. M.S. Ansari, Dr. S. Ashraf for time to time support, advice and encouragement and also to other teachers of the department especially Dr. R.U. Khan, Dr. (Mrs.) M. Haseeb from Aligarh Muslim University, Aligarh. I owe a very special thanks and pious gratitude to Dr. Masood Ali (ex-Director, IIPR, Kanpur), Dr. R. Ahmad (ex-Principal Scientist), Dr. S.S. Ali (Emeritus Scientist, CSIR), Mr. Naimuddin (Senior Scientist), Dr. N.P. Singh, Dr. P.R. Choudhury and Dr. Subhojit Datta (Senior Scientist) of the Indian Institute of Pulses Research, Kanpur for providing necessary facilities and support required during molecular analysis work. I wish to extend my sincere thanks to Prof. Jawed Khan, Jamia Millia Islamia, New Delhi for supporting me to complete molecular analysis and Prof. Saleem Jawed (Jamia Hamdard, New Delhi) for help in the mycotoxin analysis. I esteemingly acknowledge the help by my endearing friends, batchmates and seniors especially Dr. F.A. Mohiddin, Mr. Haidar Ali and Dr. Tufail Ahamd, who were a constant source of enthusiasm and zeal for me and their presence tranquillized my work. I wish to i extend special thanks to Mrs. Reshu, Mr. Nazrussalam, Ms. Rumanna, Ms. Huma Naz, Dr. Wajid Hassan, Mrs. Uzma Khan, Mr. Nadeem Ahmad, Mr. Salman Ahmad and Mr. Fazil Hasan who had a yawning effect and helped me in one way or the other during my research. Very special thanks go out to Mr. Mohd. Mahmood Khan my research colleague and Mr. Ziaul Haq my junior for their extreme support. My thanks are due to the technical staff, in particular the help rendered by Mr. Amaan-ul-Rehman and Mr. Raghib Ali. I acknowledge Darakshan Apa for issuing me desired books and journals for quiet a longer time with her precious dua and encouragement. I also wish to thank Guddu Bhai, Amir Bhai, Akbar Bhai, Nahid Madam and Anjum Madam for their regular assistance and cooperation in laboratory work. My special thanks are due to Waheed bhai, Fazal Bhai, Zarar Bhai and Shaba Apa for their time to time support. I also wish to thank Bano Baji, Shanaz Baji, Anjum, Sabir, Ansar and Babu Bhai especially for the preparation of research fields. At last I am fumbling for words to express my feelings for my family. I salute my parents who always enthused my nerves and sinews with the dreams and the hopes of realizing them. I am extremely thankful to my father in-law who inspired and encouraged me for dedication in research and felt pride whatever little I achieved ever. I wish to give special thanks to my brothers for their moral support and cooperation during critical periods of the work. The love and affection for me expressed by my loving son, Huzaifa and daughter, Aamna have been invaluable assets. I am extremely thankful to my wife for patience, support and also for being serviceable towards me at every moment, whenever I looked up to her. Without her help it would not have been possible for me to finish my work and apologize for not been able to give time due to spending hours to prepare this thesis. Md. Arshad Anwer ii TABLE OF CONTENTS Introduction 1-6 Review of literature 7-73 Materials and Methods 74-102 Isolation and identification of the wilt fungus 74 Mass culture of the wilt fungi and pathogenicity test 74 Isolation, identification and mass culture of root-knot nematode, 75 Meloidogyne incognita Isolation, identification, characterization and pure culture of 76 Aspergillus niger aggregate isolates Aspergillus niger as biocontrol agent 77-82 Screening against wilt fungi 77-79 Nematicidal effect 79-80 Compatibility with pesticides 80-81 Biosorption of toxic heavy metals by Aspergillus niger 81-82 aggregate isolates Biochemical characterization of Aspergillus niger aggregate 82-85 Molecular characterization of efficient Aspergillus niger isolates 85-86 Mass culture of efficient isolates of Aspergillus niger 87 Pot culture experiment to test effectiveness of Aspergillus niger 87-93 isolates against the wilt, root-knot and wilt disease complex Field trial 93-94 Preparation of biopesticides 94-95 Shelf life of A. niger biopesticides 95 Experiment on evaluation of performance of biopesticides of selected 95-103 isolates of Aspergillus niger aggregate under field condition Observations recorded 97-101 1. Biochemical tests of plant material 97- i. Estimation of leaf pigments 97 ii. Estimation of total phenol 97-98 iii. Estimation of salicylic acid 98 iv. Estimation of lycopene in tomato 98-99 2. Soil population of fungi 99 3. Soil population of root-knot nematode 99-100 4. Root-nodulation 100 5. Wilt incidence and severity 100 6. Root-knot severity 100-101 7. Dry weight of plants 101 8. Weight of seeds or fruit/plant 101 9. Seed index of chickpea 101 10. Seed health test 101 i. Viability test by Tetrazolium chloride 101 ii. Germination test 101 iii. Detection of pathogen associated with seeds 101 iii Results 103-205 Experiment-I Collection, isolation and identification (morphological and biochemical 103-110 basis) of soil isolates of Aspergillus niger aggregate from different agricultural fields of Uttar Pradesh, India. Experiment-II RAPD profiling of effective isolates of A. niger aggregate and their 111-125 characterization for ochratoxin A production, phosphate solubilization, heavy metal bioadsorption and in vitro pathogen suppression (Fusarium oxysporum f. sp. ciceri, F. oxysporum f. sp. lycopersici and Meloidogyne incognita). Experiment-III Evaluation of Aspergillus niger isolates for antagonism against 126-162 Fusarium spp. and Meloidogyne incognita and for promotion of plant growth and yield of chickpea and tomato under pot condition. Experiment-IV Evaluation of selected isolates of Aspergillus niger for effectiveness 163-183 against Fusarium wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato under field condition. Experiment-V Preparation of biopesticides of selected isolates of Aspergillus niger 184-205 (based on performance in Expt.-IV), and their field trial for effectiveness against the wilt, root-knot and fungus-nematode wilt disease complex of chickpea and tomato. Discussion 206-220 References 221-274 Appendices 275-285 iv LIST OF TABLES Table Caption of Table Page No. No. 1 Amino acids composition (mg/100 g protein) of protein 08 from different sources. 2 Protein contents in pulses. 08 3 Area and Production of chickpea in different states of 10 India. 4 Nutritional value of chickpea and tomato per 100 gram of 12 edible portion. 5 Essential amino acid composition of seed protein fractions 13 of chickpea. 6 Nutritional composition of whole seed and Dhal 14 component of chickpea. 7 Improved varieties of chickpea recommended for general 17 cultivation in different states in India. 8 Area, production and productivity of tomato in major 19 countries of the world. 9 Area, Production and productivity of tomato in different 20 states of India. 10 Temperature requirements for different stages of tomato. 23 11 Varieties of tomato recommended for different states or all 25 over the country. 12 Estimated annual crop losses caused by pests and 27 diseases worldwide. 13 Disease of chickpea caused by various pathogens. 28 14 Disease of tomato caused by various pathogens. 29 15 Base material/carriers used for mass production of fungus 69 biocontrol agents. 16 Colony forming unit load of inocula of Fusarium spp., 89 Rhizobium and Aspergillus niger isolates at the time of application. 17 Some biochemical characteristic of Aspergillus niger 106 isolates. 18 Summary of polymorphism produced by OPA and 112 Fusarium specific synthetic primers. 19 Ochratoxin A production and phosphate solubilization of 116 efficient Aspergillus niger isolates. 20 In vitro compatibility of selected efficient Aspergillus niger 117 isolates with some common fungicides and nematicides. 21 Minimum inhibitory concentrations (MIC) of Ni+2, Cd+2, or 118 Cr+6 for some selected isolates of Aspergillus niger. 22 Biosorption of heavy metals by some selected isolates of 120 Aspergillus niger (mg/g of biomass ) in single metal system of Ni+2, Cd+2, or Cr+6. 23 Biosorption of heavy metals by efficient Aspergillus niger 121 v Table Caption of Table Page No. No. isolates (mg/g of biomass ) in multi metal system of Ni+2, Cd+2, or Cr+6. 24 Inhibition in the colonization by Fusarium oxysporum f. sp.