Peronospora Sparsa Biology and Drivers of Disease Epidemics in Boysenberry
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Peronospora sparsa biology and drivers of disease epidemics in boysenberry A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University by Anusara Mihirani Herath Mudiyanselage Lincoln University 2015 i Abstract of a thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy Peronospora sparsa biology and drivers of disease epidemics in boysenberry Anusara Mihirani Herath Mudiyanselage Downy mildew, caused by the biotrophic oomycetes, Peronospora sparsa, is a major disease of boysenberries, with crop loss from disease for conventional growers being up to 45% and for organic growers up to 100% in some years. Most boysenberry plant material (including tissue culture propagated plants) are systemically infected with the pathogen. Disease expression by sporulation of naturally infected leaves, stems/canes and calyxes at 15ºC under high humidity in the field was identified as the source of inoculum in the wider Nelson area in 2010 and 2011 with peak spore dispersal in mid-November. A strong relationship between rainfall pattern, humidity and temperature and P. sparsa spore dispersal was observed. Spore dispersal was triggered by the frequency (%) of rainy days, RH and warm temperatures (16-23ºC) in early spring where early wet periods with high moisture levels promoting sporulation and a subsequent dry period allowed spore release. In vitro, the optimum temperature for spore germination, infection, sporulation and lesion expansion were 20ºC (24 h darkness), 15 or 20ºC (12 h/12 h light/ dark), 15 or 20ºC (12 h/12 h light/ dark) under high humidity, respectively. The optimum spore numbers for infection was 200. Similarly in vivo evaluations showed that disease expression on young foliage, stems/canes, calyxes/sepals, petals and stamen by symptoms and sporulation was favoured at temperatures ranging from 5-15ºC, under high relative humidity (90-100%) in potted systemically infected boysenberry plants. An existing nested PCR for detection of P. sparsa was optimised and limits of detection determined in different plant tissues. The optimised Plant & Food Research (P&FR) protocol (using modified Aegerter buffer) extracted more P. sparsa DNA with a higher purity than the commercial PowerPlant® DNA isolation kit. The optimised nested PCR could detect as little as 0.4 pg of P. sparsa genomic spore DNA from a range of asymptomatic boysenberry tissues including primocane tips, leaves, leaf buds, canes/ stems, roots, flower buds, flowers and berries. This was approximately equivalent to 40 spores. The method was improved to a more rapid and robust one step nested PCR method with an equivalent sensitivity for detecting latent ii infection of P. sparsa. The results showed that the most robust tissues for reliable detection of latent infection were root or crown. In vitro and in vivo evaluations indicated that dryberry is caused by spore infection of flowers or berries by the spores produced on systemically infected canes/ stems, calyx, and petals under favourable environmental conditions. Accordingly two infection pathways resulting in dryberry were identified: 1) spores produced on petals and calyx may infect pollen followed by fertilisation of infected pollen then systemic infection of the drupelets of the developing berry initials, 2) spore infection of drupelets at the red partially ripe stage. Movement of the pathogen from the systemically infected cane to berries across the pedicel and calyx was not observed. Of the ten fungicides investigated in vitro, chlorothalonil, mandipropamid, fluazinam, azoxystrobin and dimethomorph were the most effective to inhibit P. sparsa spore germination and infection. In vivo evaluations on young disease-free boysenberry plants showed that dimethomorph, azoxystrobin and metalaxyl-M+mancozeb were the most effective at protecting leaves from infection. Three applications of phosphorous acid (PA) reduced both incidence and severity of dryberry whereas, acibenzolar-s-methyl reduced only incidence in systemically infected plants. A tissue culture protocol was developed for the production of P. sparsa pathogen free clean boysenberry propagation material by heat alone. As this method does not rely on treatment of infected plants with fungicides, there is no subsequent risk of fungicide resistant strains developing. Distribution of clean boysenberry planting material in new gardens or replanting areas in existing gardens in New Zealand is an important initial step to avoid disease epidemics in the field. Keywords: Peronospora, spores, fungicides, rain, temperature, humidity, dispersal, PCR, CTAB and dryberry. iii Acknowledgements I would like to express my sincere gratitude to my supervisor Dr Eirian Jones for her guidance, expertise, encouragement, patience and commitment. I am very much thankful to you for directing me to opportunities in New Zealand to a career in science. I express my deepest gratitude for my associate supervisor Dr Hayley Ridgway for guiding all molecular work throughout my study, precious advice on how to write a thesis logically and the great courage in finalising the thesis. I also extend my heartfelt thanks to my other associate supervisor, Associate Prof. Marlene Jaspers, who always helped with precious advice and for helping with sample collection. I would not have contemplated this road if not for this team of supervisors. I am extremely grateful to Dr Simon Hodge for statistical advice and special thanks goes to Dr Monika Walter (Plant and Food Research, Riwaka) for her active support, aspiring guidance and invaluably constructive criticism based on her knowledge and experience. I also would like to thank my industry mentors Mr Geoff Langford (Plant and Food Research, Lincoln) for his valuable guidance, Mr Julian Raine, and boysenberry growers throughout New Zealand for sending me samples. I thank all Plant Microbiology staff and colleagues at Lincoln University, especially Ms Candice Barclay for all the technical support and quickly responding to all my queries, facilitating smooth running of my research and Mr Brent Richards and Mrs Leona Meachen for taking good care of my potted boysenberry plants in the nursery. Thanks to Mr Ben Shunfenthal (Plant and Food Research, Riwaka) for tissue culture work and Mr Robert Lawry (Lincoln University) for fluorescent microscopy. My PhD research was funded by the Collaghon Innovation and New Zealand Boysenberry Council Ltd and I would like to thank both organisations, as well as Fruitfed who supplied PerkSupa fungicide. I am grateful to ICPP-New Zealand for the conference travel grant, NZPPS for the 2012 research scholarship award and the APPS for the best talk on applied research award, 2013. I gladly dedicate this thesis to my dear husband, Tharaka Devinda Jayalath who was always with me, giving loads of encouragement and guidance with patience and support. I also extend my sincere gratitude to my loving parents for their love and support in my education, and also my loving sisters and brothers, mother-in-law and father-in law for their encouragement. iv Table of Contents Abstract ………………………………………………………………………………………………... ii Acknowledgements ………………………………………………………………………...................... iv Table of Contents ………………………………………………………………………........................ v List of Tables ……………………………………………………………………………….................... x List of Figures ………………………………………………………………………………………….. xii Chapter 1 Introduction …………………………………………………………………....................... 1 1.1 The New Zealand boysenberry industry 1 1.2 Boysenberry production in New Zealand 2 1.2.1 Varieties 2 1.2.2 Site selection 3 1.2.3 Climate 3 1.2.4 Propagation 3 1.2.5 Irrigation 6 1.2.6 Pruning 6 1.2.7 Desuckering 6 1.2.8 Training 6 1.2.9 Harvesting 7 1.3 Health benefits of boysenberries 9 1.4 Downy mildew disease (dryberry disease) 9 1.5 Downy mildew symptoms on boysenberry 11 1.6 Causative agent – Peronospora sparsa 12 1.7 Epidemiology of downy mildew 13 1.8 Identification of Peronospora sparsa 15 1.8.1 Polymerase Chain Reaction (PCR) for detection of Peronospora sparsa 15 1.8.2 Development of species specific PCR for P. sparsa 16 1.9 Control of downy mildew 17 1.10 Aim and Objectives 20 Chapter 2 Factors affecting sporulation and infection……………………………………………… 21 2.1 Introduction 21 2.2 Materials and Methods 22 2.2.1 Inoculum potential in the field 22 2.2.1.1 Timing of spore release in the field 22 2.2.1.2 Disease assessment under field conditions 24 2.2.1.3 Potential sources of inoculum under field condition 24 2.2.2 Sporulation potential of naturally infected boysenberry plant tissues at different temperatures and relative humidities 24 2.2.2.1 Sporulation potential of leaves 25 2.2.2.2 Sporulation potential of canes 26 2.2.2.3 Sporulation potential of fruit 27 2.2.3 In vitro production of asexual spore inoculum 28 2.2.3.1 Surface sterilisation methods for non-symptomatic plants 28 2.2.3.2 Development of a method