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"Drippy Gill" : an Ooze Disease of the Cultivated Mushroom Agaricus Bisporus Caused by Pseudomonas Agarici

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'DRIPPY GILL' - AN OOZE DISEASE OF THE CULTIVATED MUSHROOM AGARICUS BISPORUS CAUSED BY PSEUDOMONAS AGARICI A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology in the University of Canterbury by Warwick Murray Gill -;:: University of Canterbury {1994} Christchurch, New Zealand i .( ,., . CONTENTS page Abstract 1.0 General Introduction 2 1.1 General Introduction 3 1.2 Bacteria in Mushroom Culture 3 1.3 The Fluorescent Pseudomonads 5 2.0 'Drippy Gill' Symptomology and the Biology of the Causal Organism 7 2.1 Introduction 8 .2.1.1 'Drippy Gill' Symptomology 8 2.1.2 Recorded Outbreaks of 'Drippy Gill' Disease 9 2.1.3 Description and Taxonomic Status of Pseudomonas agarici Young the 'Drippy Gill' Causal Organism 10 2.1.4 PV29: A Bacterium Causing Symptoms Similar to P. agarici 11 2.1.5 Evidence Supporting the Intrahyphal Occurrence of P. agarici 11 2.1.6 The Dolipore-Parenthesome Septum: A Barrier to Intrahyphal Transmission? 12 2.2 Materials and Methods 14 2.2.1 'Drippy Gill' Symptomology 14 2.2.2 Isolation and Maintenance of the 'Drippy Gill' Pathogen 16 2.2.3 Comparison of the 'Drippy Gill' Pathogens 17 2.2.4 Proof of Pathogenicity 18 2.2.5 Toxin Assay 19 ii page 2.2.6 Microscopic Examination of 'Drippy Gill' Bacteria and Agaricus Mycelium 20 2.2.7 Growth Room Trials 21 2.3 Results 23 2.3.1 'Drippy Gill' Symptomology 23 2.3.2 Description and Comparison of P. agarici and PV29 27 2.3.3 Proof of Pathogenicity 31 2.3.4 Toxin Assay 32 2.3.5 Microscopic Examination of 'Drippy Gill' Bacteria and Agaricus Mycelium 32 2.3.6 Growth Room Trials 33 2.4 Discussion 70 3.0 Ultrastructural Studies of 'Drippy Gill' 74 3.1 Introduction 75 3.1.1 The Fungal Wall 75 3.1.2 Agaricus Wall Chemistry and Ultrastructure 76 3.1.3 Chitin and Chitinase 80 3.1.4 Glucan and Glucanase 81 3.1.5 Intrahyphal Bacteria 82 3.2 Materials and Methods 85 3.2.1 Petri Dish Assay 85 iii page 3.2.1.1 Preparation of Chitin 85 3.2.1.2 Preparation of Chitin Agar Plates 86 3.2.1.3 Standard Chitin Agar Plates 87 3.2.1.4 Modified Chitin Agar Plates 87 3.2.1.5 Cellophane Culture of A. bisporus on Chitin Agar Plates 88 3.2.2 Examination of 'Drippy Gill' Sporocarps 89 3.2.2.1 Examination of the outer envelope of 'Drippy Gill' Bacteria 89 3.2.3 Colloidal Gold-Wheat Germ Agglutinin Labelling of Fungal Wall Chitin 90 3.2.3.1 Glassware Preparation 95 3.2.3.2 Preparation of the Colloidal Gold Sol 95 3.2.3.3 Preparation of the Ovomucoid-Colloidal Gold Conjugate 96 3.2.3.4 Sample Preparation 96 3.2.3.5 Indirect Localization of N-Acetylglucosamine 97 3.2.3.6 Controls and Specificity Trials 98 3.2.3.7 Analysis of Gold Labelling 99 3.2.4 Detection of Glucan Degradation 100 3.2.4.1 Thiery's Test For Polysaccharides 101 3.2.4.2 Colorimetric Assays 102 iv page 3.2.5 Isolation and Diges~ipn of Agaricus Wall Fractions 106 3.2.6 The Enzymatic Degradation of Agaricus Mycelium 107 3.2.7 The Adhesion of P. agarici and PV29 to Agaricus Mycelium 107 3.3 Results 109 3.3.1 Petri Dish Assay 109 3.3.2 T.E.M. Ultrastructure 109 3.3.3 Colloidal Gold-Wheat Germ Agglutinin Labelling of Fungal Wall Chitin 112 3.3.4 Detection of Glucan Degradation 114 3.3.5 Isolation and Digestion of Agaricus Wall Fractions 118 3.3.6 The Enzymatic Degradation of Agaricus Mycelium 118 3.3.7 Adhesion Assay 120 3.4 Discussion 165 4.0 Concluding Discussion 170 4.1 Concluding Discussion 171 4.1.1 General Considerations 171 4.1.2 Development of 'Drippy Gill' Disease 173 References 175 Appendices 199 v page Appendix A: PMS164: 'Cavity Cap' causal organism 200 Appendix B: Identification of S. Iiquefaciens 211 Appendix C: Raw data and ANOVA table for testing the variance between two gold labelling trials Appendix D: Raw data and nested ANOVA table testing the significance the presence of bacteria has on gold particle density. 213 Appendix E: ANOVA table for reducing sugars liberated from B-(1,3)-glucan 214 Appendix F: ANOVA table for reducing sugars liberated from 0:-(1 ,3)-glucan 215 Appendix G: ANOVA table for reducing sugars liberated from B~(1 ,3)/(1 ,6)-glucan 216 Acknowledgements 217 vi LIST TABLES page 1.1 The Biotic Pests of Mushroom Crops 4 2.1 Morphological and Biochemical Comparison of P. agarici and PV29 28 2.2 Antibiotic Sensitivity of P. agarici and PV29 30 3.1 Wall Chemistry of Agaricus bisporus 78 3.2 Wall Chemistry of Armillaria mellea 79 3.3 Glucose Production from Various Glucans 117 3.4 Reducing Sugar Determination HBH Method 117 5.1 Differentiating Characteristics of PMS164 and ATCC25416 204 vii LIST FIGURES page 2.1 Location of tissues sampled from a 'drippy gill' affected sporocarp. 15 2.2a Hymenium of A. bisporus sporocarp affected by P. agaric!. 34 2.2b Infected Agaricus sporocarp demonstrating the two major symptoms of 'drippy gill'. 34 2.2c Symptom expression on the stipe of Agaricus. 34 2.2d Bacterial droplets on Agaricus hymenium. 34 2.3a 'Drippy gill' bacteria in Agaricus central cap tissue. 36 2.3b Agaricus central cap hyphae from a 'clean'sporocarp. 36 2.4a 'Drippy gill' bacteria in Agar/cus peripheral cap tissue. 38 2.4b Agaricus peripheral cap hyphae from a 'clean' sporocarp. 38 2.5a 'Drippy gill' bacteria in Agaricus hymenial tissue. 40 2.5b Agaricus hymenial tissue from a 'clean'sporocarp. 40 2.6a 'Drippy gill' bacteria in Agaricus outer stipe tissue. 42 2.6b Agar/cus outer stipe tissue from a 'clean'sporocarp. 42 2.7a 'Drippy gill' bacteria in Agaricus outer stipe tissue (L.S.). 44 2. 7b Agaricus outer stipe tissue from a 'clean' sporocarp (L.S.). 44 2.8a 'Drippy gill' bacteria in Agaricus inner stipe tissue (L.S.). 46 viii page 2.8b Agaricus inner stipe tissue from a 'clean' sporocarp (L.S.). 46 2.9a 'Drippy gill' bacteria in Agaricus central cap tissue. 48 2.9b Agaricus outer stipe tissue from a 'drippy gill' affected sporocarp. 48 2.10a Extrahyphal 'drippy gill' bacteria in the stipe of an Agaricus sporocarp. 50 2.1 Db 'Drippy gill' bacteria in the stipe of an Agaricus sporocarp. 50 2.1 Dc 'Drippy gill' bacteria in close proximity to an Agaricus stipe cell wall. 50 2.10d 'Drippy gill' bacterium against an Agaricus cell wall. 50 2.11 a Extrahyphal 'drippy gill' bacteria in the hymenium of an Agaricus sporocarp. 52 2.11 b Agaricus hyphal wall artefact. 52 2.11 c Extrahyphal 'drippy gill' bacteria in the cap of an Agaricus sporocarp. 52 2.11 d 'Drippy gill' bacteria in an Agaricus sporocarp cap. 52 2.12a Extrahyphal 'drippy gill' bacteria in the cap of an Agaricus sporocarp. 54 2.12b 'Drippy gill' bacteria passing through a broken hyphal wall. 54 2.13 A bacterium entering an Agaricus hyphal cell. 56 2.14a 'Drippy gill' isolate PMS601 (Type Strain) streaked on KB agar. 58 2.14b Isolate PV29 streaked on KB agar. 58 2.15a Isolate PMS601 inoculated into a tobacco leaf. 60 2.15b Isolate PMS603 inoculated into a tobacco leaf. 60 ix page 2.15c Isolate PMS752 inoculated Into a tobacco leaf. 60 2.15d Isolate PV29 Inoculated into a tobacco leaf. 60 2. 15e Tobacco hypersensitive reaction positive control. 60 2.16a Rifampicin resistant PMS601 from Agaricus hymenium. 62 2.16b Rifampicin resistant PMS603 from Agaricus hymenium. 62 2.16c Rifampicin resistant PMS752 from Agaricus hymenium. 62 2.16d Rifampicin resistant PV29 from Agaricus hymenium. 62 2.16e Rifampicin control plate. 62 2.17a PV29 toxin assay. 64 2.17b PMS601 toxin assay. 64 2.17c PMS603 toxin assay. 64 2.17d PMS752 toxin assay. 64 2.18a 'Drippy gill' bacteria on Agaricus vegetative mycelium. 66 2.18b I ntrahyphal 'drippy gill' bacteria in Agaricus mycelium. 66 2.18c 'Drippy gill' bacteria on Agaricus mycelium. 66 2.19 The dolipore parenthesome septum. 68 3.1 The colloidal gold-Ovomucoid-wheat germ agglutinin labelling system. 93 3.2 'Drippy gill' isolates on standard chitin agar plates. 121 3.3 'Drippy gill' isolates on chitin agar amended with mushroom extract. 121 3.4a 'Drippy gill' bacteria in Agaricus hymenial tissue. 123 x page 3.4b 'Drippy gill' bacteria in Agaricus hymenium. 123 3.4c Agaricus hyphal extracellular matrix. 123 3.4d 'Drippy gill' affected tissue in an Agaricus sporocarp. 123 3.5a Multiple breaches of a fungal cell wall in a 'drippy gill' affected Agaricus sporocarp. 125 3.5b Agaricus hyphal wall breaks with associated 'drippy gill' bacteria. 125 3.5c 'Drippy gill' bacteria and associated hyphal wall breach. 125 3.5d 'Drippy gill' bacterium entering fungal cell already occupied by bacteria. 125 3.6a Site of penetration of an Agaricus hyphal cell by a 'drippy gill' bacterium. 127 3.6b Hyphal wall breaks in a 'drippy gill' afflicted sporocarp. 127 3.6c Bacteria and associated large micro­ fibrils, presumed to be flagella, in the hyphal intercellular space of an Agaricus sporocarp. 127 3.6d 'Drippy gill' bacteria within the Agaricus hyphal intercellular space. 127 3.7a 'Drippy gill' bacterium adjacent to an Agaricus hyphal wall. 129 3.7b 'Drippy gill' bacterium in close association with an Agaricus hyphal wall.
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