SEPARATION AND PHYTOTOXICITY OF SOLANAPYRONE COMPOUNDS PRODUCED BY ASCOCHYTA RABIEI (PASS.) LABR. AND THEIR METABOLISM BY CHICKPEA (CICER ARIETINUM L.) BY KHALID HAMID A thesis submitted for the degree of Doctor of Philosophy of the University of London 1999 DEPARTMENT OF BIOLOGY UNIVERSITY COLLEGE LONDON GOWER STREET LONDON WC1E6BT 1 ProQuest Number: 10797703 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10797703 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT An isolate of Ascochyta rabiei secreted the phytotoxins, solanapyrones A, B and C when grown on Czapek Dox nutrients supplemented with five cations. The toxins were identified and quantified by high performance liquid chromatography with diode array detection and isolated from culture filtrates by partitioning into ethyl acetate and flash chromatography on silica gel. Cells isolated from leaflets of 12 chickpea cultivars differed by up to five fold in their sensitivity to solanapyrone A and this compound was 2.6-12.6 times more toxic than solanapyrone B, depending on cultivar. When chickpea shoots were placed in solanapyrone A, the compound could not be recovered from the plant and symptoms developed consisting of turgor loss of stems and flame-shaped, chlorotic zones in the leaflets. In similar experiments with solanapyrone B, only 9.4% of the compound taken up was recovered and stems remained turgid but their leaflets became twisted and chlorotic. Glutathione reacted with solanapyrone A, rapidly reducing the amount of free toxin and forming a Sol.A-glutathione conjugate as well as reducing its activity when incorporated in the cell assay. Measurement of reduced glutathione concentration and GST activity among cultivars showed that the differences of their means were highly significant and both were negatively correlated with their relative sensitivity to solanapyrone A. Treatment of shoots with solanapyrone A enhanced total, reduced and oxidized glutathione content as well as GST activity 1.26, 1.23, 1.50 and 1.94 fold, respectively. 2 Similarly, treatment of shoots with the safener, dichlormid, also raised total, oxidized and reduced glutathione levels and GST activity. Cells isolated from shoots treated with dichlormid at 150 pg/shoot and 300 pg/shoot were 2.45 times and 2.66 times less sensitive to solanapyrone A with LD 50 values of 71.5 pg/ml and 77.8 pg/ml, respectively as compared to 29.2 pg/ml for controls. In preliminary experiments designed to identify microbial genes capable of detoxifying the solanapyrones a basal mineral salts medium caused demethylation of solanapyrone A. Demethylated solanapyrone A was 16.4 fold less toxic than solanapyrone A in the cell assay, requiring 514.0 pg/ml to kill 50% of the cells compared with 31.3 pg/ml for Sol.A. Dedicated to my parents 4 ACKNOWLEDGMENTS I am very grateful to Dr. Richard N. Strange for his advice and guidance throughout the course of this project and for his constructive suggestions and criticism in the preparation of this thesis. I am also thankful to Dr. Sarwar Alam, Dr. Moncef Harrabi and Dr. Habib Halila for supplying the chickpea seeds used in these studies. I would also like to thank Dr. Ahmad Saleem Akhtar and my brothers for moral support and Dr. Subba Rao for technical help during these studies. I am thankful to Dr. Roger Wotton for his help in doing statistical analysis of the data. Also my thanks to my colleagues for their help and suggestions and lastly but not least to my wife for her support. The heartfelt blessings of my father had been a major significance in my achievements whose encouragement, personal interest and moral support helped in achieving this goal. I am thankful to Government of Pakistan for providing a scholarship for these studies. 5 TABLE OF CONTENTS ABSTRACT 2 ACKNOWLEDGMENT 5 INDEX TO TABLES, FIGURES AND APPENDICES 12 ABBREVIATIONS 17 CHAPTER 1. INTRODUCTION 1.1. THE CHICKPEA 18 1.1.1. The agricultural importance of chickpea 19 1.1.2. Nutritional value 20 1.1.3. Constraints to chickpea production 20 1.2. CHICKPEA DISEASES 22 1.2.1. Chickpea blight 24 1.2.2. Nomenclature of causal organism 24 1.2.2.1. Imperfect stage 25 1.2.2.2. Perfect stage 26 1.2.2.3. Races of A. rabiei 26 1.2.2.4. Symptoms 27 1.3. EPIDEMIOLOGY 27 1.3.1. Survival of the fungus in crop residues 29 1.3.2. Survival of the fungus in infected seed 30 1.3.3. Dispersal of the pathogen 31 1.3.4. Mode of infection 32 1.3.5. Host range 33 1.4. CONTROL 34 1.4.1. Cultural practices 34 6 1.4.2. Chemical control 34 1.4.3. Difficulties in the development of resistant varieties 36 1.4.3.1. Inoculation techniques 36 1.4.3.2. Disease rating scales 37 1.4.3.3. Sources of resistance 39 1.5. TOXINS 41 1.5.1. Host selective toxins (host specific toxins) 43 1.5.2. Non host selective toxins 45 1.5.3. Toxins produced by the genus Ascochyta 46 1.6. DETOXIFICATION OF TOXINS 49 1.6 .1. Detoxification by plants 56 1.7. AIMS OF THE PROJECT 58 CHAPTER 2. PRODUCTION, SEPARATION AND SOME CHEMICAL REACTIONS OF THE SOLANAPYRONE TOXINS 2.1. INTRODUCTION 59 2.2. MATERIALS AND METHODS 60 2.2.1. Toxin production 60 2.2.2. Partial purification of solanapyrone toxins by solid phase extraction (SPE) 60 2.2.3. Partial purification of solanapyrone toxins by liquid phase extraction (LPE) 61 2.2.4. Separation of the solanapyrone toxins 62 2.2.4.1. By chromatotron 62 2.2.4.2. By flash chromatography using a glass column 65 2.2.4.3. By flash chromatography using a commercial apparatus 66 2.2.5. Observation of fractions on a spectrophotometer 66 2.2.6. Analytic HPLC 67 2.2.7. Detection of solanapyrones on TLC plates 70 2.3. RESULTS 71 2.3.1. Production of the solanapyrone toxins 71 7 2.3.2. Separation and purification of the solanapyrone toxins 71 2.3.2.1. By the chromatotron 71 2.3.2.2. By flash chromatography using a glass column 71 2.3.2.3. By flash chromatography using a commercial apparatus 72 2.3.2.4. Confirmation of purity of solanapyrones A, B and C 72 2.3.3. The effect of delaying or omitting the addition of zinc ions to cultures of A. rabiei growing on CDLMC 93 2.3.3.1. Toxin production 93 23 3 .2 . Dry weight of mycelium 93 2.3.3.3. pH of culture filtrates 93 2.3.4. The detection of the solanapyrone toxins on TLC plates 98 2.4. CONCLUSIONS 106 CHAPTER 3. THE SENSITIVITY OF CHICKPEA CULTIVARS TO SOLANAPYRONES A AND B 3.1. INTRODUCTION 109 3.2. MATERIALS AND METHODS 111 3.2.1. Plants 111 3.2.2. Preparation of holding buffer and digestion solution 111 3.2.3. Isolation of cells 112 3.2.4. Bioassay 112 3.3. RESULTS 114 3.4. CONCLUSIONS 128 CHAPTER 4. CHEMICAL REACTIONS OF SOLANANPYRONE A 4.1. INTRODUCTION 132 4.2. MATERIALS AND METHODS 137 4.2.1. Testing the stability of Sol.A to autoclaving in basal medium 137 4.2.2. Chemical stability of Sol.A 137 4.2.2.1. Effect of different carbon sources in basal medium (BM) on Sol.A 137 8 4.2.2.1.1. Extraction of hydrophobic compounds from basal medium and supplemented basal medium 138 4.2.2.2. Growing of bacteria in basal mineral medium (BMM) containing Sol.A 138 4.2.2.3. Effect of individual constituents of basal mineral medium 139 4.2.2.4. Effect of NH4OH and NaOH on Sol.A 139 4.2.2.5. Effect of basal medium and mineral basal medium without Na2H P04 139 4.2.2.6 . Effect of glutathione on Sol.A 140 4.2.3. Isolation of reaction products of solanapyrone A 140 4.2.3.1. Isolation of SLC-4 140 4.2.3.2. Isolation of Sol.A-glutathione conjugate 141 4.2.3.3. Mass spectrometry of Sol.A-glutathione conjugate 142 4.2.4. Phytotoxicity 142 4.2.4.1. Testing the phytotoxicity of SLC-4 142 4.2.4.2. Effect of different concentrations of glutathione on the phytotoxicity of Sol.A to cells 142 4.2.4.3. Toxicity of Sol.A with or without glutathione 143 4.3. RESULTS 144 4.3.1. Effect of autoclaving Sol.A in basal medium 144 4.3.2. Effect of basal medium supplemented with different carbon sources on Sol.A 144 4.3.3. Degradation of Sol.A by basal mineral medium (BMM) 149 4.3.3.1. Purification of SLC-4 by TLC 149 4.3.3.2. Phytotoxicity of SLC-4 149 4.3.4. Effects of individual constituents of BMM on Sol.A 161 4.3.5. Effect of NaOH and NH4OH 161 4.3.6. Effect of basal medium and basal mineral medium without Na 2H P04 on Sol.A 161 4.3.7.