Biology and Chemical Ecology of Spongospora subterranea During Resting Spore Germination Towards a germinate/exterminate control approach for Spongospora diseases of potato by Mark Angelo O. Balendres BSc in Agriculture, Major in Plant Pathology Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy University of Tasmania, May 5, 2017 Statements and Declarations Statement of Originality This thesis contains no material which has been accepted for a degree or diploma by the University or any other institution, except by way of background information and duly acknowledged in the thesis. To the best of my knowledge and belief, no material previously published or written by another person, except where due acknowledgement is made in the text of the thesis, nor does the thesis contain any material that infringes copyright. Authority of Access The publishers of the papers (as indicated in next section) hold the copyright for that content, and access to the material should be sought from the respective journals. The remaining non-published content of the thesis is not to be made available for loan or copying for two years following the date this statement was signed. Following that time, the remaining non-published content of the thesis may be made available for loan and limited copying and communication in accordance with the Copyright Act 1968. Mark Angelo O. Balendres University of Tasmania May 5, 2017 i Statement of Co-Authorship and Contribution The following people and institutions contributed to the publication of work undertaken as part of this thesis: Mark Angelo Balendres Tasmanian Institute of Agriculture, UTAS, Australia Calum Rae Wilson Tasmanian Institute of Agriculture, UTAS, Australia Robert Steven Tegg Tasmanian Institute of Agriculture, UTAS, Australia David Scott Nichols Central Science Laboratory, UTAS, Australia Paper 1: Balendres MA, Tegg RS and Wilson CR. 2016. Key events in pathogenesis of Spongospora diseases in potato: a review. Australasian Plant Pathology. Volume 45, Issue No. 3, pages 229-240 (Chapter 2). Paper 2: Balendres MA, Tegg RS, and Wilson CR. 2016. Resting spore dormancy and infectivity characteristics of the potato powdery scab pathogen Spongospora subterranea. Journal of Phytopathology, Volume 165, Issue 5, pages 323-330 (Chapter 3). Paper 3: Balendres MA, Nichols DS, Tegg RS and Wilson CR. 2016. Metabolomes of potato root exudates: compounds that stimulate resting spore germination of the soil- borne pathogen Spongospora subterranea. Journal of Agricultural and Food Chemistry. Volume 64, pp 7466-7474 (Chapter 4). Paper 4: Balendres MA, Nichols DS, Tegg RS and Wilson CR. 2017. Potato root exudation and release of Spongospora subterranea resting spore germination-stimulants are affected by plant and environmental conditions. Journal of Phytopathology Volume 165, Issue 1, pages 64-72 (Chapter 5). Paper 5: Balendres MA, Tegg RS and Wilson CR. 2016. The potential of resting spore germination stimulating-compounds for the management of Spongospora diseases in potato. Manuscript in preparation for journal submission (Chapter 6). ii Conference Presentations Balendres MA, Nichols DS, Tegg RS, Wilson CR. 2016. Potato root exudates contain stimulants of resting spore germination of Spongospora subterranea. 9th Australasian Soilborne Diseases Symposium, Hammer Springs, North Canterbury, New Zealand. November 14-17, 2016. Balendres MA, Nichols DS, Tegg RS and Wilson CR. 2014. Potato root exudates stimulate zoospore release of Spongospora subterranea. The 8th Australasian Soilborne Diseases Symposium. Hobart, Tasmania, Australia. November 10-13, 2014. Balendres MA, Tegg RS and Wilson CR. 2014. Somaclonal cell selection as a tool for developing resistance against soil-borne potato pathogen, Spongospora subterranea. The 8th Australasian Soilborne Diseases Symposium. Hobart, Tasmania, Australia. November 10-13, 2014. Balendres MA, Tegg RS and Wilson CR. 2014. Somaclonal selection for enhanced resistance to Spongospora subterranea tuber and root infection in potato and studies on zoospore dormancy, release and chemotaxis. The 2nd International Powdery Scab Workshop. Pretoria, South Africa. July 28-August 1, 2014. Mark Balendres (the candidate) was the primary author and contributor of all papers, contributed to the design of the study, acquisition, analysis and interpretation of data, drafting, revising and final approval of all articles. David Nichols contributed to the design of the study, acquisition and interpretation of data, technical support, critical revision of the articles for important intellectual content and final approval of the article (Paper 3 and 4). Robert Tegg contributed to the conception, formalisation and design of the study, interpretation of data, critical revision of the articles for important intellectual content and final approval of all articles. Calum Wilson contributed to the conception, formalisation and design of the study, interpretation of data, provision of study materials, obtaining of funding, critical revision of the articles for important intellectual content and final approval of all articles. iii Declaration of Agreement We the undersigned agree with the above stated “proportion of work undertaken” for each of the above published peer-reviewed manuscripts contributing to this thesis: ______________________ ______________________ Mark Angelo Balendres Calum Rae Wilson Candidate Supervisor School of Land and Food School of Land and Food University of Tasmania University of Tasmania Date: May 5, 2017 Date: May 5, 2017 ______________________ ______________________ Robert Steven Tegg David Scott Nichols Co-Supervisor Co-Supervisor School of Land and Food Central Science Laboratory University of Tasmania University of Tasmania Date: May 5, 2017 Date: May 5, 2017 iv Abstract Resting spores are important for the spread and survival of Spongospora subterranea, the causative agent of potato powdery scab and root disease. However, resting spores must germinate and release zoospores (the infective agents) to cause infection. Understanding of the germination process and factors is, therefore, important. The knowledge can potentially have implications for resting spore-inoculum management and Spongospora disease control. The biology and ecology of S. subterranea during resting spore germination is not well understood. Knowledge of factors influencing resting spore germination have been limited. This thesis studied the properties of S. subterranea resting spores and investigated some chemical factors stimulating resting spore germination. The role of germination- chemical stimulants was also examined. This study used a combination of tomato-plant and zoospore in vitro bioassays, light microscopy, a targeted hydrophilic interaction ultra- high performance liquid chromatography–mass spectrometry metabolomic approach, and a greenhouse chemical-soil treatment study. Some of S. subterranea resting spores exhibited dormant spore characteristics, which required specific stimuli to germinate. Although a proportion retains constitutive dormancy characteristics, chemical germination-stimulants were found in potato root exudates and in Hoagland’s solution. The low-molecular weight organic amino acids – tyramine and L-glutamine – and sugars – cellobiose and L-rhamnose – compounds stimulated resting spore germination at 0.1 mg/ml solution. The release of these compounds in potato roots were influenced by the plant’s physiology and growth conditions. Hoagland’s solution contains Iron-EDTA, which stimulated resting spore germination. Germination stimulant Fe-EDTA, in Hoagland’s solution, in the presence a susceptible host plant, enhanced root infection. However, Fe-EDTA and Hoagland’s solution added into S. subterranea-infested soil, a month prior to planting, reduced pathogen inoculum DNA levels in the soil. Further field studies underpinning the use of germination stimulant compounds could lead to a novel, safe and sustainable chemical approach for the management of S. v subterranea inoculum in the soil and thus will augment other Spongospora disease control measures. This thesis advances our understanding of S. subterranea biology and chemical ecology during resting spore germination. Substantial development is now beginning to be made in the facet of resting spore germination biology and chemical ecology, which are important aspects of Spongospora disease epidemiology and disease control development. There remains much to be learned, but the knowledge presented here will encourage additional studies and research efforts. KEYWORDS: Powdery scab, Spongospora root infection, plasmodiophorid, chemical ecology, potato metabolomics, HILIC UHPLC-MS, resting spore germination, spore dormancy. vi Acknowledgments I am most grateful to the University of Tasmania (UTAS), for the financial support, through the Tasmanian Graduate Research Scholarship program, which made it possible for me to pursue this PhD degree. I wholeheartedly thank my supervisors at the School of Land and Food (SLAF), Calum Wilson and Robert Tegg, for their excellent supervision, constant encouragement, for providing an ideal research environment, helpful comments on the manuscript, without their guidance and critical reviews this thesis would have not been written. I am particularly indebted to Calum, firstly for accepting my application five years ago, and for the extended financial support when my scholarship has ended. I feel privileged to be one of his PhD students. I also thank my supervisor at