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Biology, Epidemiology, and Biological and Chemical Control of Phytophthora Vignae

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Biology, Epidemiology, and Biological and Chemical Control of Phytophthora Vignae AN ABSTRACT OF THE THESIS OF W. Gerard Dilantha Fernando for the degree of Doctor of Philosophy in Botany and Plant Pathology presented on October 4, 1990. Title: Biology, Epidemiology, and Biological and Chemical Control of Phytophthora vignae. Redacted for Privacy Abstract approved:_ Dr. Robert G. Linderman Phytophthora vignae, causal agent of stem and root rot of cowpea (Vigna unguiculata), was reported for the first time in Sri Lanka. The pathogen was found in cowpea field soils from 3 of 5 geographic regions sampled. Only one site however, had plants exhibiting disease symptoms. Of the eight cowpea varieties grown in Sri Lanka, four were shown to be relatively resistant; all other legumes inoculated were completely resistant. Two morphologic and physiologic races of P. vignae were identified among the 24 isolates recovered, based on differential pathogenicity on cowpea varieties. Bacteria isolated from field soils, and other known bacterial biocontrol agents, inhibited P. vignae in culture, but only three Sri Lankan isolates considerably suppressed the disease in greenhouse tests. Volatile substances produced by most bacteria inhibited mycelial growth and sporangial production by P. vignae. The increased pH of the exposed medium suggested the involvement of ammonia. Volatile inhibitors were produced by these bacteria in soil, but only with added substrate; Strain DF-3101 also reduced oospore germination in soil. Cowpea plants inoculated with the VA mycorrhizal (VAM) fungus Glomus intraradices in P. vignae-infested soil were larger than non-mycorrhizal plants, but only at low levels of the pathogen. VAM colonization was reduced at high levels of the pathogen, and root infection by the pathogen was reduced by VAM. The fungicides metalaxyl, fosetyl-Al, Banrot, and Manzate-200DF reduced in vitro mycelial growth, but at different concentrations. Sporangia formation and germination, and oogonia formation by P. vignae, was reduced significantly by metalaxyl and fosetyl-Al. In greenhouse tests, metalaxyl, even at low concentrations, reduced disease; Fosetyl-Al was effective at high concentrations; Manzate-200DF was effective as a soil drench but not as a foliar spray; Banrot effectively reduced disease at 50 mg a.i./L. Exposure of a bacterial biocontrol agent to these fungicides in vitro did not affect its capacity to subsequently produce volatile inhibitors, but exposure to 10 ug/ml of metalaxyl and 50 ug/m1 of Manzate-200DF reduced its capacity to subsequently inhibit mycelial growth of P. vignae. Biology, Epidemiology, and Biological and Chemical Control of Phytophthora vignae by W. Gerard Dilantha Fernando A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Completed October 4, 1990 Commencement June 1991 APPROVED: Redacted for Privacy Professor of Botany and Plant Pathology in charge of major Redacted for Privacy Head of Department of Botany and Plant P hology Redacted for Privacy Dean of Graduate hool \.1 Date thesis is presented October 4, 1990 Typed by W. Gerard Dilantha Fernando Dedicated with Love to Pushpini and Iru ACKNOWLEDGEMENTS Financial supportfor this thesis was provided by a scholarship through USAID, from the Department of Agriculture, Sri Lanka and from Dr. Robert Linderman by allowing me to use his lab and greenhouse facilities. I thank the good Lord for having been with me in every decision and step I took in my life. His guidance, love and forgiveness has made my life worthwhile in every way. I feel it was God's choice that brought me to work with Bob Linderman, my mentor and major professor. Words cannot describe how grateful I am for his guidance throughout my project, and mostly for allowing me to think independently and always valuing my suggestions or comments. At times he wasmy teacher and leader and at times he was my friend, who would listen to all my problems. I extend my appreciation for all this and my admiration for his knowledge of Rhizosphere Biology. I am grateful to the members of my doctoral committee, Drs. Joyce Loper, Peter Bottomley, Russ Ingham, and Kermit Cromack for their help and advice throughout the dissertation. I have especially enjoyed talking to Joyce about my work, and learning a lot from these discussions. To Dr. Tim Paulitz for his helpful suggestions in biocontrol research, to Dave Ianson, Mac McDaniel, and Tom Thompson for computer help, to Dr. Jack Pinkerton for providing his expertise on the 'MAC', to Rohini Ekanayake and Rod Poppleton for their greenhouse help, and to Phil Hamm for sharing his knowledge on Phytophthora. To all those in the Horticultural Crops Research Unit who always obliged with a helping hand when I needed their help technically or otherwise. They never made me feel I was far from home! I am indebted to all of them. During our four year stay in Corvallis, we made a lot of friends who have helped to make our transition to American life much easier. We are indebted to many, in fact too many to list. I especially thank my dear parents Matilda and Gerald Fernando who have always been behind me in every decision I made and offered their daily prayers for my studies and happiness in life. Though I may not say it enough, I thank them for their love, support and understanding. To my 'second' parents Sriya and Piyasoma Attanagoda and 'bro' Prabat for their love and help in our times of need. Finally I give my deepest thanks from the bottom of my heart to my wife Pushpini, for her love, support, friendship, and also for providing my favorite `distraction' Iru. As small as he is, Iru contributed to my graduate life in his own way of love and care when I needed them most. Both Pushpini and Iru went through all of it for me, and I doubt whether I could have ever made it this far without them! CONTRIBUTION OF AUTHORS R.G.Linderman assisted with the experimental design, interpretation of results and critically reviewing the thesis, and appears as a co-author on these manuscripts. The manuscript titled "Phvtophthora root and stem rot of cowpea in Sri Lanka" (chapter 1) was an investigation conducted in Sri Lanka, which reported the presence of P. vignae in Sri Lanka for the first time. B. Sivakadadcham assisted with the identification of the fungus and setting up experiments. TABLE OF CONTENTS Page INTRODUCTION 1 CHAPTER 1 Phytophthora Root and Stem Rot of Cowpea 6 in Sri Lanka Summary 6 Introduction 7 Materials and Methods 8 Results 10 Discussion 12 References 15 CHAPTER2 -Distribution and Pathogenicity of 16 Phytophthora vignae in Soils of Sri Lanka Summary 16 Introduction 17 Materials and Methods 18 Results 27 Discussion 29 References 41 CHAPTER3 -Biological Control of Phytophthora vignae 45 by Soil Bacteria Summary 45 Introduction 47 Materials and Methods 50 Results 61 Discussion 65 References 99 CHAPTER 4 - Chemical Control of Phytophthora vicinae 104 Stem and Root Rot of Cowpeas Summary 104 Introduction 106 Materials and Methods 108 Results 116 Discussion 120 References 134 CHAPTER 5 Effects of the Mycorrhizal Fungus Glomus 138 intraradices on Phytophthora vignae Root and Stem Rot of Cowpea Summary 138 Introduction 139 Materials and Methods 142 Results 145 Discussion 147 References 152 SUMMARY AND CONCLUSIONS 156 BIBLIOGRAPHY 159 LIST OF FIGURES FIGURE PAGE CHAPTER 1 1-1 Phytophthora root and stem rot disease of 14 cowpea and the incitant P. vignae.(A) Stem lesion (arrow) on symptomatic cowpea (B) oospore (C) sporangium of the pathogen, produced after 10 days on cowpea agar. CHAPTER 3 3-la Inhibition of Phytophthora vignae isolates 72 P001 and P006 by Sri Lankan strain AGB-3 on PDA. 3-lb Inhibition of P. vignae by production of 73 antibiotics by Psudomonas fluorescens (PF-5) on PDA. 3-1c Production of siderophores and inhibition of 74 P. vignae by P. fluorescens (PF-5) on KB. 3 -id Inhibition of P. vignae on different media 75 by P. fluorescens (PF-5). 3-2a Inhibition of P. vignae through volatile 76 compounds by strain DF-1481 on TSA. 3-2b Inhibition of P. vignae through volatile 77 compounds by Salmonella sp. (JL-4085) on KB. 3-2c Inhibition of P. vignae through volatile 78 compounds by DF-7104 strain on NA. 3-3a Inhibition of P. vignae by Enterobacter 79 aerogenes (B-8) by production of volatile compounds on TSA and NA with removal of the bacterium, before inoculation with the pathogen. 3-3b Inhibition of P. vignae by PGPR Psudomonas sp. 80 (JL-4084) by production of volatile compounds on TSA and KB with removal of bacterium before inoculation with the pathogen. 3-4a Inhibition of P. vignae mycelial growth by 81 production of volatile compounds by Bacillus subtilis (AB-6) in soil amended with TSB. 3-4b Inhibition of P. vignae mycelial growth by 82 production of volatile compounds by P. fluorescens (PF-5) in soil amended with Tryptic Soy Broth. 3-4c Inhibition of P. vignae mycelial growth by 83 production of volatile compounds by E. aerogenes (B-8) in soil amended with cowpea seed extract. 3-4d Inhibition of P. vignae mycelial growth by 84 production of volatile compounds by Salmonella sp. (JL-4085) in soil amended with cowpea seed extract. 3-5 Rhizoplane colonization in cowpea by DF-1124. 85 Log CFU per gram of root on Blackeye and Mississippi purple varieties. 3-6 pH changes on PDA medium exposed to 86 volatile compounds produced by DF-3101 grown on TSA, NA, and KB media. LIST OF TABLES TABLE PAGE CHAPTER 2 2-1 Isolation of Phytophthora vignae from 37 soil collection sites within five districts of Sri Lanka. 2-2 Percent mortality of cowpea 'California 38 Black-eye' plants grown in test soils collected from sites within five districts of Sri Lanka, and the calculated Disease Potential Index.
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