Biological control of grapevine trunk diseases using bacterial endophytes from grapevines Jennifer Millera Niem BSc Agriculture (Plant Pathology) University of the Philippines Los Baños, Philippines MSc Plant Pathology Washington State University, USA A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy National Wine and Grape Industry Centre School of Agricultural and Wine Sciences Faculty of Science Charles Sturt University April 2020 2 Table of Contents Table of contents............................................................................................................... 3 List of figures .................................................................................................................... 8 List of tables ..................................................................................................................... 11 Certificate of Authorship ................................................................................................. 12 Acknowledgements..........................................................................................................13 Publications arising from the research .......................................................................... 16 Author contribution.......................................................................................................... 17 Abstract ............................................................................................................................ 19 List of abbreviations ....................................................................................................... 23 Chapter 1: Introduction and literature review ............................................................... 24 1.1. Importance of grapevine trunk diseases… .............................................................. 24 1.2. Symptoms and causal pathogens… ........................................................................ 25 1.3. Current management practices ............................................................................... 31 1.3.1. Chemical control .......................................................................................... 31 1.3.2. Cultural methods…………………………………………………………………..33 1.3.3. Biological control agents………………………………………………………….35 1.4. Grapevine endophytes ...........................................................................................38 1.5. Potential of Pseudomonas spp. as biocontrol agents ............................................. 39 1.5.1. Mechanisms for biocontrol activity exhibited by Pseudomonas spp .............. 40 1.6. Summary and project aims ...................................................................................... 44 1.7. Research objectives ................................................................................................ 45 3 Chapter 2: Journal paper ................................................................................................. 47 Niem, J., Billones-Baaijens, R., Savocchia, S., Stodart, B. (2020). Diversity profiling of grapevine microbial endosphere and antagonistic potential of endophytic Pseudomonas against grapevine trunk diseases. Front. Microbiol. 11:477. doi: 10.3389/fmicb.2020.00477. Chapter 3: Biocontrol potential of endophytic Pseudomonas against Neofusicoccum luteum, a fungal pathogen causing Botryosphaeria dieback, and its persistence in grapevine tissues ............................................................................................................ 67 3.1. Introduction .............................................................................................................. 67 3.2. Materials and Methods ............................................................................................ 69 3.2.1. Plant materials .............................................................................................. 69 3.2.2. Bacterial strains, culture conditions, inoculum preparation ............................ 69 3.2.3. Pathogenicity tests of Pseudomonas poae BCA17 strain .............................. 70 3.2.4. Suppression of Neofusicoccum luteum infection in planta by strains of Pseudomonas ........................................................................... 71 3.2.4.1 Fungal isolates .................................................................................... 71 3.2.4.2 Challenge inoculations using detached canes .................................... 72 3.2.4.3 Challenge inoculations using potted grapevines ................................. 73 3.2.4.3.1 Assessment of N. luteum infections by isolations ............................ 74 3.2.4.3.2 Assessment of N. luteum infections by quantitative PCR ............. 75 3.2.5 Pseudomonas colonisation and establishment in grapevine tissues ............... 76 3.2.5.1 Development of rifampicin-resistant strain ...................................... 76 3.2.5.2 Pre-planting application of RifMut strain ......................................... 77 3.2.5.3 Post-planting application of RifMut strain ........................................ 79 4 3.2.6 Data analysis… ..................................................................................................... 80 3.3. Results… ................................................................................................................ 81 3.3.1. Pathogenicity tests on grapevine tissue ............................................................ 81 3.3.2. Suppression of N. luteum infection in planta by Pseudomonas strains .............. 83 3.3.2.1. Challenge inoculation using detached canes ............................................... 83 3.3.2.2. Challenge inoculation using potted grapevine plants… ................................ 84 3.3.2.2.1. Assessment of N. luteum infection by pathogen isolation .................... 84 3.3.2.2.2. Assessment of N. luteum infections by qPCR ....................................... 85 3.3.3. Pseudomonas colonisation and establishment in grapevine tissues… .............. 86 3.3.3.1. Pre-planting application of Pseudomonas RifMut strain… ........................... 86 3.3.3.2. Post-planting application of Pseudomonas .................................................. 87 3.4. Discussion ........................................................................................................... 88 Chapter 4: Elucidating the mechanism of action of Pseudomonas in the control of grapevine trunk disease pathogens .............................................................................. 94 4.1. Introduction ............................................................................................................ 94 4.2. Materials and Methods .......................................................................................... 98 4.2.1 Bacterial strains............................................................................................. 98 4.2.2 Fungal isolates .............................................................................................. .98 4 2 3. Elucidation of antagonistic mechanisms of Pseudomonas poae BCA17 against GTD pathogens…......... ............... …..99 4.2.3.1. Siderophore production ........................................................................... ..99 4.2.3.2. Bioactivity of the Pseudomonas poae culture filtrate on mycelial growth and spore germination of GTD pathogens… .............. 99 5 4.2.3.2.1 Preparation of culture filtrates ........................................................... 99 4.2.3.2.2 Effect of culture filtrates on mycelial growth .................................... .100 4.2.3.2.3 Effect of culture filtrates on spore germination ................................... 101 4.2.3.3 Test for production of bioactive volatile compounds… ............................ 102 4.2.3.4 Test for production of the antibiotics 2,4 diacetylphloroglucinol and phenazine thru thin layer chromatography (TLC) ............................ 102 4.2.3.5 Identification of biocontrol genes… ..................................................... ..104 4.2.3.6 Detection of bioactive lipopeptides… .................................................. ..104 4.2.3.7 Statistical analysis… ............................................................................. 105 4.3 Results… ............................................................................................................... 105 4.3.1 Test for siderophore production ................................................................... ..105 4.3.2 Bioactivity of the Pseudomonas poae culture filtrate on mycelial growth and spore germination of GTD pathogens… ....................................................... 106 4.3.2.1 Effect of culture filtrate on mycelial growth ............................................ .106 4.3.2.2 Effect culture filtrate on spore germination ........................................... .107 4.3.3 Test for production of bioactive volatile compounds… ................................. 109 4.3.4 Test for production of the antibiotics 2,4 diacetylphloroglucinol (DAPG) and phenazine………………………………………………………………………... 109 4.3.5 Identification of biocontrol genes… ............................................................. .110 4.3.6 Detection of bioactive lipopeptides ............................................................. .114 4.4 Discussion ............................................................................................................ 117 Chapter 5: General discussion.....................................................................................