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Biological Control of Late Blight of Potatoes: in Vivo and in Vitro Evaluation of Microbial Antagonists Against Tuber Blight

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Biological Control of Late Blight of Potatoes: in Vivo and in Vitro Evaluation of Microbial Antagonists Against Tuber Blight Biological Control of Late Blight of Potatoes: in vivo and in vitro evaluation of microbial antagonists against tuber blight Jane C Hollywood BSc Biology and Geography (University of Bristol) Biology Department Darwin Building Gower Street London Presented for the degree of Doctor of Philosophy, University of London UMI Number: U602517 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 complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U602517 Published by ProQuest LLC 2014. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT The cost of losses and control measures attributed to late blight of potatoes caused by Phytophthora infestans, are estimated to exceed $5 billion annually. Breeding for resistance is difficult owing to the tetraploid genotype of potato and current strains of the pathogen have developed resistance to chemical control. Consequently the search for biological control has assumed greater importance. In this investigation an in vivo bioassay was used to select soils antagonistic to late blight of potatoes, caused by Phytophthora infestans. Four out of eight samples demonstrated reproducible antagonism as determined by a reduction in the volume of tuber tissue rotted. A total of 292 bacterial and yeast samples and 20 fungal samples were recovered from suppressive soils using a variety of non- selective and selective media. When these organisms were tested individually against P. infestans in the assay, 15 isolates suppressed tuber rotting by >85% in at least three out of four assays. The antagonists were characterised as Pseudomonas spp. (3 strains), Enterobacter spp. (4 strains), Bacillus spp. (1 strain), Pantoea spp. (2 strains), Citrobacter spp. (1 strain), Buttiauxella spp. (1 strain), Trichosporon spp. (2 strains) and Geotrichum spp. (1 strain) by routine bacteriological tests, fatty acid profiling and partial sequencing of the gene encoding 16S or 18S (where appropriate) ribosomal RNA. Subsequently the possible mechanisms by which the potential biocontrol agents inhibited the disease were examined. Nine isolates showed some evidence of antibiotic production with a Pantoea spp. producing a compound that caused the hyphae of P. infestans to kink and permanently cease growth. Three isolates colonised hyphae of the pathogen and eleven produced siderophores in liquid culture. Hydrogen cyanide, proteolytic, cellulolytic and beta-1,3-glucanase activity was also evident in some species. Significant promotion of axenically grown tomato seedlings, as determined by increased stem and main root elongation, was achieved by ten of the isolates. Three population levels of the isolates were retested for disease inhibition at the end of the investigation. Isolates 3, 7 and 14 exhibited the highest levels of consistent inhibition at the lowest population levels and were therefore tested in 2 combination. This achieved disease suppression that, at an antagonist concentration of 25 cfu/nL, was more consistent than isolate 3 alone and was over 30% greater than either isolate 7 or 14. 3 ACKNOWLEDGEMENTS I am very grateful to The Perry Foundation, without whose funding this PhD would not have been possible. I would like to thank Dr Richard Strange for his invaluable advice and continuous support throughout my entire PhD. I would also like to thank Professor Jonathan Nugent for his guidance and the Plant Pathology Lab for their help. Also many thanks to Sam Whiting for her help with identifying eukaryotic microorganisms. Many thanks to Richard Shattock and Diana Earnshaw of Bangor University and Louise Cooke of The Queen’s University of Belfast for the kind provision of Phytophthora infestans isolates. I am also grateful to my dad, mum, Anna, Caroline, Rob and all my grandparents for their patience and support. 4 CONTENTS ABSTRACT 2-3 ACKNOWLEDGEMENTS 4 CONTENTS 5-8 LIST OF TABLES 9 LIST OF FIGURES 10-13 ABBREVIATIONS 14-15 CHAPTER I 16-30 Introduction 1.1 The pathogen, Phytophthora infestans 16 1.2 Late blight: origin and historical impact 16 1.3 Evolution of P. infestans 16-17 1.4 The pathogen life cycle 17-21 1.5 Molecular aspects of the infection 21 -22 process 1.6 Mating types 22 1.7 Chemical control of late blight 22-23 1.8 Organic control 23 1.9 Resistant potato cultivars 24-26 1.10 The P. infestans EST projects 26 1.11 Biocontrol: the advantages 27 1.12 Suppressive soils 27-28 1.13 Biocontrol using rhizobacteria 28 1.14 Why tuber not foliage? 29 1.15 Biocontrol of late blight 29 1.16 Aims 30 CHAPTER II 31-60 Detection of Antagonists, their Isolation and Confirmation of their Antagonism 2.1 Introduction 31-32 2.2 Materials and Methods 31-41 2.2.1 Phytophthora infestans isolates 33 2.2.1.1 Culture, inoculum preparation and storage 33 2.2.1.2 Inoculation and testing of pathogenicity 33-34 2.2.2 Bioassay for in vivo selection of suppressive soils 35-39 2.2.2.1 Soil material 35 2.2.2.2 Bioassay 36-39 2.2.2.3 Cause of suppressiveness 39 2.2.3 Isolation 39-41 2.2.4 Assay of isolated microorganisms 41 2.3 Results 42-52 2.3.1 Growth of P. infestans 42-43 2.3.2 Bioassay of soils 44-45 2.3.3 Bioassay of test soils with no P. infestans (control 4) 46 5 2.3.4 Source of suppressiveness 47 2.3.5 Isolation of microorganisms from soil samples 48-50 2.3.6 Bioassay of isolated microorganisms 51-52 2.4 Discussion 53-59 2.4.1 Phytophthora infestans isolates 53-54 2.4.2 Bioassay for in vivo selection of suppressive soils 54-59 CHAPTER III 60-88 Identification and Characterisation of Antagonists 3.1 Introduction 60-62 3.2 Materials and Methods 63-67 3.2.1 Phenotypic characterisation of isolates 63 3.2.2 Fatty acid methyl ester (FAME) analysis 63 3.2.3 Genotypic identification of isolates by partial sequencing and 63-67 analysis of the 16S or 18S rRNA gene 3.2.3.1 DNA isolation 63-64 3.2.3.2 PCR 64-66 3.2.3.3 Sequencing and analysis 67 3.3 Results 68-80 3.3.1 Phenotypic characterisation 68-72 3.3.2 FAME analysis 73-74 3.3.3 DNA extraction, PCR amplification, sequencing and analysis 74-80 3.4 Discussion 81-88 CHAPTER IV 89-151 Mode of Action 4.1 Introduction 89-91 4.2 Materials and Methods 92-101 4.2.1 Antibiosis 92-93 4.2.1.1 in vitro antagonism between P. infestans and potential 92 biocontrol agents 4.2.1.2 Antibiosis by culture filtrates of potential biocontrol agents 93 4.2.2 Interaction between the antagonists and P. infestans 93 4.2.3 Hyphal colonisation 93-94 4.2.4 Siderophore production 94-95 4.2.4.1 Siderophore production in liquid medium 94 4.2.4.2 Chrome azurol S agar 94 4.2.4.3 Modified chrome azurol S agar 94 4.2.4.4 Siderophore production in liquid medium 95 4.2.5 Indoleacetic acid production 95 4.2.6 13-1,3-glucanase activity 95-97 4.2.6.1 Alkaline copper method 96 4.2.6.2 Glucose oxidase 97 4.2.7 Protease activity 97-98 4.2.8 Effect of enzymes on P. infestans zoospores 98 4.2.9 Induced systemic resistance 99-101 4.2.9.1 Adapted method from Simons et ai. (1996) 99-100 4.2.9.2 Method of Simons etal., (1996) using Magenta vessels 100 4.2.9.3 Magenta vessels using layers of medium 100 4.2.9.4 Magenta vessels without layers of medium 100-101 4.3 Results 102-136 6 4.3.1 Antibiosis 102-116 4.3.1.1 In vitro antagonism between P. infestans and potential 102-110 biocontrol agents 4.3.1.2 Antibiosis by culture filtrates of potential biocontrol agents 111-116 4.3.2 Visible interactions between the antagonists and P. infestans 116-120 4.3.3 Hyphal colonisation 120 4.3.4 Siderophore production 121-130 4.3.4.1 Siderophore production in liquid medium 121 4.3.4.2 Chrome azurol S agar 121 4.3.4.3 Modified chrome azurol S agar 122 4.3.4.4 Siderophore production in liquid medium 123-130 4.3.5 Indoleacetic acid production 131 4.3.6 13-1,3-glucanase activity 131-132 4.3.6.1 Alkaline copper method 131 4.3.6.2 Glucose oxidase 132 4.3.7 Protease activity 133 4.3.8 Effect of enzymes on P. infestans zoospores 134 4.3.9 Induced systemic resistance 135-136 4.3.9.1 Adapted method from Simons et al. (1996) 135 4.3.9.2 Method of Simons et al., (1996) using Magenta vessels 135 4.3.9.3 Magenta vessels using layers of medium 135 4.3.9.4 Magenta vessels without layers of medium 136 4.4 Discussion 137-151 CHAPTER V 152-175 Multiple Biocontrol Agents, Plant Growth Promotion Potential and Multiple Target Pathogens 5.1 Introduction 152-154 5.2 Materials and Methods 155-157 5.2.1 Determination of a consortium of biocontrol agents 155 5.2.2 Plant growth promotion of tomato seedlings 155 5.2.2.1 Promotion of shoot and foliage development 155 5.2.2.2 Promotion of root development 155 5.2.3 Potato root development and colonisation by antagonist 155 5.2.4 Testing of antagonists against tomato late blight 156 5.2.4.1 Treated tomato seedlings against tomato late blight 156 5.2.4.2 Treated tomato seedlings maintained in soil 157 5.3 Results 158-168 5.3.1 Determination of a consortium of two or three biocontrol 158-165 agents 5.3.2 Plant growth promotion of tomato seedlings 166-167 5.3.2.1 Promotion of shoot and foliage development 166-167 5.3.2.2 Promotion of root development 167 5.3.3 Potato root development and colonisation by antagonists 168 5.4 Discussion 169-175 CHAPTER VI 176-184 Overall discussion
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