Studies on Cymbidium rot and its management by biocontrol agents Thesis Submitted for the Degree of Doctor of Philosophy (Science) of the University ofNorth Bengal Surjit Sen, M. Sc. Department of Botany University ofNorth Bengal, India 2006 524.15 ;3 41'-f 5 UNIVERSITY OF NORTH BENGAL DEPARTMENT OF BOTANY ~ . ~~do M. Sc. , Ph. D. MOLECULAR PLANT PATHOLOGY AND FUNGAL BIOTECHNOLOGY LABORATORY Reader P. 0 . • N.B.U., SIUGURI -7340 13 WEST BENGAL, INDIA Phone: 91 353 2699106 (0) 91 353 2580257 (R) Fax: 91 353 2699001 E-mail: drishtanta @ yahoo.com TO WHOM IT MAY CONCERN YMJ /J to ce«9Y tAd eKr. cfaya ~ &Kcfa. ~ «10~ iUUUn mg YLpeW;Jion at IAe !.lJepadment if 9/otang, CZ/a«;eu«g if c&;un !Llengat and 6-r wpew;Jion if !l),. %u~ ~~a, !lJepadment if 9/otang, CZ/a«;eu«g if t!lakaHa f~ 9'0. !l>. ~ enUIIed ~k«Ne.f ew {j~:z. WJI tutd it! ~ .t5r ~ a.9f81'h : ff rD» ftUIIIMdiAg /d1 fAe5;J ft'l} /Ae 9'4 !l). degree ((§denceJ if tAe CZiaU~&M;tg if c&;un 9/engm. ff wcomnu?Ad tAat Ae ~ .fo!/'dkd aH ~ ~ to Me ~ if tAe CZiaU~&M«g if c&;un !/Jengat regtMdiAg tAe LUOfhJ emOodied in /UJ tAe!iJ. ~~v~~ (DR. ANIRUDDHA SAHA) Supervisor & Reader, Department of Botany, N.B.U. Phone • (091) (033) 2461-4891 (0) ff~~ 9434567752 (M) M. Sc. (Bot.}, M. Tech. (Biotech) , Ph . D. Fax (091)(033)24764419 R der in Botany E-mail . krishnendua@rediffmailcom TO WHOM IT MAY CONCERN ~ r;) tu ee/(1!/J, tit at f)J1;: {j?k:?"lt, {j]kn, 9Jr' {j}k.. 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Dr. Krishne du Acharya Co-Supervis r Jepartment of Botany:: University of Calcutta:: Taraknath Siksha Prangan :: 35, Ballygunge Circular Road:: Kolkata 700 019 :: India Dedicated ~(]. fn!t ~ ~ ~. ~~metfw_~ Acknowledgement I have much pleasure in expressing my deepest sense of gratitude to my supervisor Dr. Aniruddha Saha, Reader and Head, Department of Botany, University of North Bengal for his inspiration, encouragement and guidance. I sincerely acknowledge the inspiration and cooperation extended by my co-supervisor Dr. Kri~hncndu Acharya, Reader in Botany, University of Calcutta, for his constant encouragement and stimulating practical guidance which kept me inspired throughout the course of this work. I fee l very much fortunate to have had him as my guide without his untiring help it would not possible for me to complete this work. I extend my heartiest gratitude to Dr. (Mrs.) Rupa Acharya, Scientist, University of Calcutta for her assistance and advice at all the time. I am grateful to Dr. (Mrs.) Dipanwita Saha, Lecturer, Department of Biotechnology, University of North Bengal, for her kind co-operation. I am also indebted to Prof. K.K. Tamang , Dr. K.K. De, Dr. B.C. Sharma, Dr. D.R. Chettri, Dr. P.C. Lama, Mr. R.P. Bhattacharyya, Mr. N.C. Karmakar, Mr. D.N. Palit of Post Graduate Department of Botany, Darjeeling Govt. College, Prof. A.P. Das, Prof. P.K. Basu, Prof. B.N. Chakraborty, Dr. (Mrs.) U. Chakraborty, Prof. P.K. Sarkar, Dr. A. Sen, Mr. P. Mandol and Mr. S.C. Roy of Department of Botany, University of North Bengal, Prof. N.D. Paria (Head), Prof. N. Samajpati, Dr. A.K. Manna, Department of Botany, University of Calcutta for their valuable encouragement throughout the course of my work. Special thanks go to Dr. (Mrs.) A. Pande, ARI, Puna, for identifying some of my organisms, j, Dr. S. Ghosh, Department of Geology, University of Calcutta for SEM facilities, Ms. D. Rai, Prakash Nursery, Ging, Mr. Rai, Kurseong, for providing samples and also permitting me to do various field survey as well as providing valuable information. I am also thankful to Mr . Nishit Roy for his photographical works to compose the thesis. I also express wholeheartedly to my colleagues Ms. Manjula Rai, Mr. C.K. Maiti, Mr. A. Banerjee, Mr . K. Bhattacharyya, Mr. J. Sarkar, Ms. S. Ghosh, Ms. M. Bhattacharyya, Mr. G. Biswas, Dr. S. Dasgupta, Mr. P. Mandai, Md. M. Isha, Ms. S. Dutta for their help in various ways. Finally, I am very much grateful to my parent, my beloved elder brother, bouthan and sweet Rani for their untiring inspiration and mental supports, which were the foundations upon I could make this thesis work successful. Surjit Sen CONTENT§ Chapter I Introduction 1.1. Orchid: An Overview ............................................................................ 1 1.2. Global Occurrence of Orchid Diseases: A Review .............................................. 6 1.3. Biological Control: A Concept .................................................................. 14 1.4. Mechanisms of Biocontrol. ...................................................................... 17 1.5. Thesis Objective ................................................................................. 34 1. 6. References ....................................................................................... 3 5 Chapter II Cymbidium Rot: Isolation, characterization and iden'tijication of the causal oraanisms 2.1.Introduction .................................................................................... 55 2.2. Materials and Methods ......................................................................... 58 2.2.1. Field survey and collection of samples .................................................. 58 2.2.2. Symptomological study ................................................................. 58 2.2.3. Histopathological study ................................................................. 58 2. 2.4. Isolation of pathogens .................................................................... 60 2.2.5. Characterization and Identification ofpathogen ......................................... 60 2.2.6. Pathogenicity test ........................................................................ 61 2.3. Results and Discussion .......................................................................... 62 2.3.1. Histological studies............................................ ......................... 64 2. 3. 2. Isolation, characterization and identification of pathogens .............................. 65 2.3.3. Pathogenicity test ......................................................................... 67 2.4. Reference ........................................................................................ 70 Chapter III Isolation, characterization and screenin9 of potential antanonists aoainst causal ornanism 3 .1. Introduction ..................................................................................... 73 3.2. Selection of efficacious organism ............................................................... 75 3.3. Materials and Methods .......................................................................... 77 3. 3 .I. Isolation of fluorescent pseudomonads ................................................. 77 3. 3. 2. Characterization of fluorescent pseudomonads ........................................ 77 3. 3. 3. Preliminary screening of antagonists ................................................... 82 3.3.4. Interaction of potential (screened) fluorescent pseudomonads against Cymbidium rot pathogens by dual culture technique ............................... 83 3.4. Results and Discussion .......................................................................... 85 3.4.1. Morphological characterization .......................................................... 85 3.4.2. Biochemical and physiological characterization ......................................... 85 3.4. 3. Cultural characteristics ................................................................... 90 3.4.4. Preliminary screening of potential antagonists ................... , ....................... 92 3.4.5. In vitro antagonistic activity of fluorescent Pseudomonas BRL- 1 ....................... 95 3.4.6. In vitro antagonistic activity of fluorescent Pseudomonas Dj-5 ......................... 96 3.4. 7 . .!n Yi!!:.9 antagonistic activity of fluorescent Pseudomonas BB-9 ......................... 98 3. 4. 8. ]1J_ vitro antagonistic activity of fluorescent Pseudomonas Sg-5 .................. , , ..... _.·.99 3 .4.9 . .!n_ vitro antagonistic activity of fluorescent Pseudomonas Sg- 1 ......................... f01 3.4 .1 0. In Y!!!:Q antagonistic activity of fluorescent Pseudomonas Sili-15 ..................... 102 3.5. References........................................................................... ... .. .... 106 Chapter IV Mechanism ofaction offluorescent Pseudomonas BRL-1 4.1. Introduction .................................................................................. 111 4.2. Plant Disease Suppression Mechanisms ...................... , ............................... 111 4.2.1. Siderophore mediated suppression ..................................................... 111 4.2.2. Antibiotic mediated suppression ....................................................... 112 4.2.3. Parasitism ...... : ........................................................................ 114 4.2.4. Colonization ............................................................................ 114 4.2.5. Induced Resistance ....................................................................