DEVELOPMENT AND EVALUATION OF DIAGNOSTIC TOOLS FOR Nucleopolyhedroviruses (NPVs) INFECTING MAJOR LEPIDOPTERAN PESTS OF LEGUME CROPS IN THE SEMI-ARID TROPICS DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY BY SRIDHAR KUMAR CHITNENI DEPARTMENT OF BIOCHEMISTRY ACHARYA NAGARJUNA UNIVERSITY Dr. M. R. APPAROW CAMPUS NUZVID - 521 201, ANDHRA PRADESH, INDIA SEPTEMBER-2008 DEVELOPMENT AND EVALUATION OF DIAGNOSTIC TOOLS FOR Nucleopolyhedroviruses (NPVs) INFECTING MAJOR LEPIDOPTERAN PESTS OF LEGUME CROPS IN THE SEMI-ARID TROPICS Thesis submitted to ACHARYA NAGARJUNA UNIVERSITY in partial fulfillment of the requirement for the award of the degree of DOCTOR OF PHILOSOPHY in BIOCHEMISTRY by SRIDHAR KUMAR CHITNENI M.Sc., M.Phil. under the supervision of Prof. C. Rambabu Co-ordinator Department of Biochemistry Acharya Nagarjuna University Dr. M. R. Apparow Campus Nuzvid - 521 201, A.P., India and Dr. G.V. Ranga Rao Special Project Scientist Integrated Pest Management (IPM) Entomology - Division International Crops Research Institute for the Semi-Arid Tropics (ICRISAT ) Patancheru - 502 324, A.P., India www.icrisat.org ICRISAT CGIAR DEPARTMENT OF BIOCHEMISTRY ACHARYA NAGARJUNA UNIVERSITY Dr. M. R. APPAROW CAMPUS NUZVID-521 201, ANDHRA PRADESH, INDIA OCTOBER, 2008 ACHARYA NAGARJUNA UNIVERSITY Dr. M. R. APPAROW CAMPUS NUZVID-521 201, KRISHNA DISTRICT, ANDHARA PRADESH, INDIA Off : +91- 08656 - 235848 Prof. C. Rambabu, M.Sc., Ph.D. Res : +91- 08656 - 232155 Fax : +91- 08656 - 235200 CO-ORDINATOR E-mail : [email protected] DEPARTMENT OF BIOCHEMISTRY Date……………………….. CERTIFICATE Mr. SRIDHAR KUMAR CHITNENI has satisfactorily prosecuted the course of research and that the thesis entitled, “Development and Evaluation of Diagnostic Tools for Nucleopolyhedroviruses (NPVs) Infecting Major Lepidopteran Pests of Legume Crops in the Semi-Arid Tropics” submitted to “ACHARYA NAGARJUNA UNIVERSITY” in partial fulfilment of the requirement for the award of the degree of “DOCTOR OF PHILOSOPHY” in “BIOCHEMISTRY” is the result of original research work done under my guidence and is of sufficiently high standard to warrant its presentation to the examination. I also certify that the thesis or part there of has not been previously submitted by him for a degree of any University. Date : (C. RAMBABU) RESEARCH DIRECTOR Dr. G. V. Ranga Rao Special Project Scientist Off : 040-30713598 Integrated Pest Management (IPM) Res : 040-30713675 Entomology Division E-mail : [email protected] ICRISAT CERTIFICATE This is to certify that the thesis entitled “Development and Evaluation of Diagnostic Tools for Nucleopolyhedroviruses (NPVs) Infecting Major Lepidopteran Pests of Legume Crops in the Semi-Arid Tropics” submitted to “ACHARYA NAGARJUNA UNIVERSITY ” in partial fulfilment of the requirement for the award of the degree of “DOCTOR OF PHILOSOPHY ” in “ BIOCHEMISTRY” is a record of the bonafide research work carried out at ICRISAT by Mr. SRIDHAR KUMAR CHITNENI under my guidance and supervision. No part of the thesis has been submitted by the student for any other degree or diploma. The published part has been fully acknowledged. All assistance and help received during the course of the investigation have been duly acknowledged by the author of the thesis. Date : (G. V. RANGA RAO) CO-RESEARCH DIRECTOR DECLARATION I, SRIDHAR KUMAR CHITNENI hereby declare that the thesis entitled “Development and Evaluation of Diagnostic Tools for Nucleopolyhedroviruses (NPVs) Infecting Major Lepidopteran Pests of Legume Crops in the Semi-Arid Tropics” submitted to “ACHARYA NAGARJUNA UNIVERSITY” in partial fulfilment of the requirement for the award of the degree of “DOCTOR OF PHILOSOPHY” in “BIOCHEMISTRY” is the result of the original research work done by me. It is further declared that the thesis or any part there of has not been published earlier in any manner. Date : (SRIDHAR KUMAR CHITNENI ) Dedicated to My Beloved Teachers Dr. G.V.Ranga Rao and Dr. P. Lava Kumar ACKNOWLEDGEMENTS It is with great pleasure and respect, I wish to express my gratitude and sincere thanks to my major advisor Prof. C. Rambabu , Coordinator, Department of Biochemistry, for his valuable suggestions, constant encouragement and moral support throughout my study. His confidence in my work provided me with the motivation and stimulus to complete this work. I thank him for giving me the opportunity to work at ICRISAT, Patancheru. I would like to express my profound gratitude and heartfelt thanks to my Co- advisor Dr. G. V. Ranga Rao, Special Project Scientist in Integrated Pest Management (IPM), Entomology Division, ICRISAT, Patancheru for providing all facilities and meticulous guidance during the course of investigation also in the preparation of this thesis. I admit that it has been great fortune and proud privilege for me to be associated with him during my Ph.D. work. I wish to express my hearty gratitude and sincere regards to Dr. P. Lava Kumar , Principal Scientist in Virology, West and Central African Programme, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria for his excellent guidance and spending his valuable time at every stage of my Ph.D. project during his tenure at ICRISAT. His timely help, constant supervision and encouragement enabled me to overcome several stumbling hurdles during the period of investigation. Once again I would like to express special thanks for his great help and giving valuable suggestions even at every stage of this thesis preparation. I feel it is a great opportunity for me to work under the guidance of such an eminent scientist. It gives me immense pleasure to express my sincere thanks to Dr. Farid Waliyar , Principal Scientist in Plant Pathology and Regional Director, ICRISAT – Niamey, Niger, West Africa for providing the laboratory facilities and encouragement to my research especially in pathology diagnostics. Also I wish to express my sincere thanks to Dr. Varsha Wesley, Special Project Scientist, Virology, Mycotoxicology and Pathology, ICRISAT for her moral support and suggestions during preparation of this thesis. I am extremely thankful to Dr. C. L. L. Gowda , Global Theme Leader; Crop Improvement and Dr. V. Balaji, Head, Knowledge Management and Sharing (KMS) for the opportunity to undertake my Ph. D. work at ICRISAT. It would be great pleasure to me to express my heartful thanks to Dr. M. Subba Rao, Assistant Professor, Department of Biochemistry, for his valuable suggestions and constant encouragement during my entire Ph.D. Also I sincerely extend my profound gratitude to all my teachers and members of the department of Biochemistry, Acharya Nagarjuna University, Dr. M. R. Apparow Campus, Nuzvid, for their cooperation and help during my studies. I wish to express my profound gratitude to Dr. Y. G. Prasad, Principal Scientist, Entomology, Central Research Institute for Dry Land Agriculture (CRIDA), Hyderabad, for his valuable suggestions and guidance to my Ph.D. Also, I express my heartful thanks to Dr. K. Sireesha , Scientist, Entomology, All India Networking Project on Beetle wine, Bapatla, for her valuable guidance and suggestions during her stay at ICRISAT. Also I would like to extend my sincere thanks to Dr. D. Srinivas Reddy , Visiting Scientist, Genetic Transformation Laboratory (GTL), ICRISAT for his excellent guidance and help especially in phylogenetic studies. Also I thankful to Dr. Nagamalleswari, Associate Professor, Anatomy and In-charge of Ruska Laboratory, Sri Venkateswara Veterinary University , Rajendra Nagar, for her timely help in electron microscopic studies. I sincerely extend my profound gratitude to Mr. A. Sudarshan Reddy , Senior Scientific Officer, Virology Lab, ICRISAT for his valuable suggestions and technical support during my research work. Also I would like to express my sincere thanks to Senior Scientific Officers Mr. V. Rameshwar Rao (Entomology), Mr. S. Veera Reddy (Micotoxicology) and Mr. R. Kanaka Reddy (Groundnut Pathology) for their valuable suggestions during my stay at ICRISAT. Also I wish to express my heartful thanks to Senior Scientific associate Mr. V. Anjaiah and Administrative Associates Mr. S. R. Venkateswarlu, Mrs. I. Radha, Mrs. S. Rohini and Mrs. A. Krishna Priya for their timely help and support during my study at ICRISAT . I affectionately acknowledge my friends Kishore, Subba Reddy, Siva Prasad, Peter Vijay, Siva Kumar, Keerthi Kiran, Gopal Reddy, Charles devenish, Subramanian, Sridhar Rao, Venkat, Vardhan, Anitha, Haritha, Archana, Surekha, Padmaja, Harini, Kalyani, Sowmini, Jana Kholova, Namitha, Paramasiva and Ratna Kumari for their help and encouragement during my research. Also I wish to thank to Research Technicians Prabhakar Reddy, Satish Chandra, Md. Moinuddin, Ramachandraiah, Md. Sofimyia, Bhaskar Raju , and Ravinder Rao and Regular Work Force Anasuya and Swaroopa for their timely help and Cooperation during my research work at ICRISAT. I acknowledge my loving Parents Smt. Chitneni. Suguna Kumari and Sri Chitneni. Venkata Koteswara Rao and my brother Sri Chitneni. Babu Srinivas and sister-in-law Smt. Chitneni. Nagalakshmi and other family members for their concern, support and everlasting affection and continuous encouragement without which I could not have achieved this. I would like to thank World Bank for rendering partial financial support to my research from the Special Project ( World Bank, DM-2005) on Biopesticides during my study at ICRISAT. - SRIDHAR KUMAR CHITNENI Name of the author : SRIDHAR KUMAR CHITNENI Title of the thesis : “Development and Evaluation of Diagnostic Tools for