The role and diversity of Pythium and Phytophthora in UK gardens Geoffrey James Denton Division of Ecology and Evolution Department of Life Sciences Imperial College London Silwood Park Campus PhD degree of Imperial College London & Diploma of the Imperial College Submitted September 2013 Page 1 This page has been intentionally left blank. Page 2 Abstract Gardens are little studied particularly in relation to major plant pathogen genera such as Phytophthora, or the closely related Pythium. UK gardens harbour a wide diversity of plants of worldwide origin, compared to the relatively few native in the UK, and are frequently the endpoint of the worldwide trade in plants and sometimes, as fellow passengers their associated pathogens. Samples from a plant clinic were surveyed for the presence of Phytophthora by three methods. DNA extracted from symptomatic tissue followed by a semi-nested PCR (DEN) gave the highest detection rates with approx. 70% of tests positive. A commercial immunoassay test kit (PocketDiagnositic™) was the fastest; with results in less than 10 min. Apple baiting gave the lowest detection rates (9%), but provided cultures vital for further studies. An unexpected and novel result was the widespread detection of Pythium causing much the same symptoms as Phytophthora. The phylogenetic trees, created using the elision method, of the Phytophthora and Pythium rDNA sequences revealed 46 named or well defined species, 21 and 25 respectively. The phylogeny of both genera was in general accordance with previous publications. Frequently identified species included Ph. cryptogea, Ph. cinnamomi, Py. intermedium and Py. sylvaticum, all ubiquitous with wide host ranges. Occasional occurrences included Ph. ramorum, Ph. tropicalis, Ph. austrocedri and Ph. “niederhauseri”. Twenty putative new species were also detected, based on the Phytophthora and Pythium phylogenies, 11 and 9 species respectively. In pathogenicity tests Phytophthora and Pythium caused root rot, and Py. intermedium caused Hebe death within 3 days of soil inoculation. Not all plants infected with Phytophthora or Pythium died, indicating disease development may involve additional interactions. Pythium foliar blight of mature woody plants was identified. Koch’s postulates were satisfied, indicating new symptoms previously not associated with Pythium infections. Page 3 This page has been intentionally left blank. Page 4 Declarations Originality I declare that this work, submitted as part of a PhD thesis towards a doctoral degree at Imperial College undertaken during 2006 – 2013, is my own original research. I have to the best of my abilities, referenced and acknowledged all materials, raw data and assistance which have contributed towards this body of work. This account of my work has not previously been submitted to any other university or institution. Signed: Dated: Copyright The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. Page 5 This page has been intentionally left blank. Page 6 Table of contents CONTENTS PAGE Title 1 Abstract 3 Declarations (Originality and Copyright) 5 Table of contents 7 List of Figures 15 List of Tables 21 Abbreviations 27 Acknowledgements 29 Dedication 30 1. Introduction 31 1.1. The diversity of gardens 31 1.2. The classification of Pythium and Phytophthora 32 1.2.1. Morphological based classification 33 1.2.2. Molecular based classification 37 1.3. Habit and habitat 40 1.3.1. Vegetative stage 40 1.3.2. Sexual Reproduction and long-term survival 41 1.3.3. Asexual Reproduction and dispersal 43 1.4. The importance of studying Phytophthora and Pythium 45 1.5. Methods of isolation and detection 49 1.5.1. Isolation methods 49 1.5.1.1. Isolation by selective culture plating 49 1.5.1.2. Isolation by baiting 50 1.5.2. Molecular detection methods 52 1.5.2.1. Immunodetection 52 1.5.2.2. PCR detection methods 52 1.6. Aims and objectives 54 2. General Methods 57 Page 7 2.1. Sampling method 57 2.2. Preparation of reagents, buffers and media 58 2.3. Apple baiting technique 58 2.4. Hemp seed baiting method 59 2.5. Immunoassay technique (PocketDiagnostic™) 60 2.6. Preparation of mycelium cultures from baiting for DNA extraction 60 2.7. DNA extraction from mycelium and plant samples 60 2.8. DNA extraction from soil samples 61 2.9. Semi-nested PCR 61 2.10. Sequencing and alignments 63 2.11. Species identification 63 3. Comparison of baiting, immunoassay and nested PCR methods for detecting Phytophthora in ornamental plants 65 3.1. Introduction 65 3.2. Methods 67 3.3. Results 67 3.4. Discussion 74 4. Survey of Phytophthora species in UK gardens 79 4.1. Introduction 79 4.2. Methods 80 4.2.1. Sampling and baiting methods 80 4.2.2. Sequence analysis and phylogeny 81 4.2.3. Growth rates 82 4.2.4. Mating compatibility and oogonia measurements 82 4.3. Results 83 4.3.1. Host genera and families associated with Phytophthora 83 4.3.2. Sequencing data 85 4.3.3. Results from temperature-growth profiles 92 Page 8 4.3.4. Results of mating compatibility tests and oogonial measurements 94 4.4. Discussion 96 5. Phytophthora spp. and Pythium intermedium isolates recovered from Taxus baccata: virulence and genetic variation 115 5.1. Introduction 115 5.2. Methods 117 5.2.1. Phytophthora and Pythium isolates 117 5.2.2. Pathogenicity of Phytophthora spp. and Pythium intermedium to Hebe 117 5.2.2.1. Hebe plants 117 5.2.2.2. Inoculum production 117 5.2.2.3. Inoculation and set up of trial 118 5.2.2.4. Assessments 119 5.2.3. AFLP analysis of Phytophthora spp. and Pythium intermedium isolates to assess genetic variability between isolates of the same species 119 5.2.3.1. DNA extraction 119 5.2.3.2. AFLP protocol 119 5.2.4. Pathogenicity of Phytophthora spp. and Pythium intermedium to Taxus baccata 121 5.2.4.1. Taxus plants 121 5.2.4.2. Phytophthora spp. and Pythium intermedium isolates 122 5.2.4.3. Trial randomisation and plan 122 5.2.4.4. Inoculum production 122 5.2.4.5. Inoculation and set up of trial 123 5.2.4.6. Assessment: Plant heights and foliage browning 125 5.2.4.7. Assessment: Root rotting and colonization 125 5.2.4.8. Apple-baiting of roots 126 Page 9 5.2.4.9. Foliage, root and soil dry weights 127 5.2.4.10. Analysis of results 127 5.3. Results 127 5.3.1. Comparison of pathogenicity of Phytophthora spp. and Pythium intermedium isolates to Hebe 127 5.3.2. AFLP analysis of the Phytophthora spp. isolates 131 5.3.3. Comparison of pathogenicity of Phytophthora spp. and Pythium intermedium to Taxus baccata 134 5.3.3.1. Selection of Phytophthora spp. and Pythium intermedium isolates for pathogenicity on Taxus trial 134 5.3.3.2. Height assessment of the Taxus plants and visual browning of the foliage 135 5.3.3.3. Visual root assessment results 137 5.3.3.4. Microbial presence following clarification and staining of root samples 138 5.3.3.5. Re-isolation of Phytophthora spp. and Pythium spp. from all treatments using apple baiting 142 5.3.3.6. Dry weight of Taxus foliage 143 5.4. Discussion 143 6. Detection and isolation of Pythium species associated with dying plants in UK gardens 153 6.1. Introduction 153 6.2. Methods 155 6.2.1. Sampling and hemp baiting methods 155 6.2.2. Phylogenetic analysis of the sequences 155 6.2.3. Temperature-growth relationship 156 6.3. Results 156 6.3.1. Results of baiting with hemp seeds 156 Page 10 6.3.2. Host data from PCR, apple and hemp bait data 159 6.3.3. Phylogenetic analysis of the sequences recovered 160 6.3.4. Pythium species identified from data collected through this study 164 6.3.5. Analysis of the growth rates 170 6.4. Discussion 171 6.4.1. Hemp baiting as a detection method for Phytophthora and Pythium 171 6.4.2. Range of hosts associated with Pythium spp. 172 6.4.3. Discussion of the phylogenetic analysis based on the sequences recovered through DEN and HBN 173 6.4.4. Discussion of phylogenetic results 174 7. Investigation of the pathogenicity of Pythium intermedium 191 7.1. Introduction 191 7.2. Methods 192 7.2.1. Selection of Pythium and Phytophthora isolates used for pathogenicity assays 192 7.2.2. Host plants used to study pathogenicity of Pythium spp. 193 7.2.3. Trials to study the effect of Pythium and Phytophthora on lettuce and radish seed germination 194 7.2.3.1. Inoculum production and application 194 7.2.3.2. Assessment of the effect of Pythium and Phytophthora on lettuce and radish seed germination 195 7.2.4. Trials to study the effect of Pythium and Phytophthora on pea seedlings 195 7.2.4.1. Inoculum production and application 195 Page 11 7.2.4.2. Assess of trials to study the effect of Pythium and Phytophthora on germinated pea seedlings 196 7.2.5. Trials to study the effect of Pythium on Hebe 196 7.2.5.1. Inoculum production and application 196 7.2.5.2. Assessment of trials to study the effect of Pythium on Hebe plants 197 7.2.6.