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On the Nature, Symptoms and Genetic Diversity of Monilinia Isolates and Their Viruses in Western Australia

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On the Nature, Symptoms and Genetic Diversity of Monilinia Isolates and Their Viruses in Western Australia ON THE NATURE, SYMPTOMS AND GENETIC DIVERSITY OF MONILINIA ISOLATES AND THEIR VIRUSES IN WESTERN AUSTRALIA Thesis is presented by Thao Thi Tran For the degree of Doctor of Philosophy at School of Veterinary and Life Sciences Murdoch University, Perth, Western Australia, Australia 2019 DECLARATION I declare that this work is my own account of my research, and it contains as its main content work which has not previously been submitted for a degree at any tertiary education institution. To the best of my knowledge, it contains no material or work performed by others, published or unpublished without reference being made within the text. Date: Thao Thi Tran 1 ABSTRACT Monilinia species occur in stone fruit (Prunus species) production areas in many parts of the world, where they cause the serious disease brown rot. Incursion into Western Australia (W.A.) by Monilinia fructicola was declared in 1997, and along with Monilinia laxa has subsequent spread throughout the state has cost the stone fruit industry millions of dollars in lost production and the cost of fungicidal sprays. Until this study, no studies had been made in W.A. of Monilinia species identity, genetic diversity, distribution, virulence, fungicide tolerance or presence of mycoviruses. This project aimed to address these knowledge gaps. A collection of Monilinia isolates was made across the major stone fruit production regions in W.A., and the fungal species were identified. Both species were present, but they were not evenly distributed between and within production regions, indicating the main agent of spread is people. High levels of genetic diversity have been reported for WA population, and both pathogens are not recently introduced in this state. ISSR (Inter Simple Sequence Repeat) markers were used to identify intra-specific diversity in populations, and compare modern populations with the strains that first invaded W.A.. This study revealed that the original incursive strain of M. fructicola has probably become extinct, replaced by new genotypes. Conversely the incursive strain of M. laxa remains widely distributed within the state. New strains appear to have subsequently invaded W.A. and pose ongoing serious threats to the industry. Although commercial stone fruit growers routinely spray their crops with fungicides to control brown rot, small scale and ‘organic’ growers may not. A study was done to assay the relative tolerance to three fungicides of Monilinia isolates collected from sprayed and unsprayed trees. No resistant isolates have been found in WA. Sprayed isolates exhibited a significantly greater range of responses to fungicides than unsprayed isolates, indicating that there is strong positive selection for tolerance in the orchards that routinely apply fungicides. We found there were significant differences between M. fructicola and M. laxa isolates in average tolerance to propiconizole-based fungicides. Assays for virulence revealed the existence of highly virulent, moderately virulent and avirulent strains of both species, and these occurred in both sprayed and unsprayed orchards. 2 On nutrient agar plates, M. laxa isolates presented as four distinct morphologies, while M. fructicola colonies were very similar in appearance across all isolates tested. A survey of 28 isolates of both species revealed three mycoviruses co-infecting three M. laxa isolates and one M. fructicola isolate. The complete or partial sequences of one isolate each of all three viruses was obtained. One M. fructicola isolate was co-infected with all three viruses: Sclerotinia sclerotiorum hypovirus 2 (SsHV2, genus Hypovirus), Fusarium poae virus 1 (FPV1, genus Betapartitivirus), and Botrytis virus F (BVF, genus Mycoflexivirus). To test the influence of these viruses on fungal pathogenicity, several methods were applied to cure isolate M196 of one or more mycoviruses. Of these treatments, hyphal tip culture either alone or in combination with antibiotic treatment generated isogenic lines free of one or more mycoviruses. Morphology and virulence assays were carried out to determine how mycoviruses influence growth of the fungal host. Surprisingly, growth of fungal mycelia was promoted by the presence of three viruses when they were cultured on nutrient agar medium in vitro, but did not influence fungal virulence after inoculation to fruits of sweet cherry. 3 TABLE OF CONTENTS DECLARATION...................................................................................................................... 1 ABSTRACT .............................................................................................................................. 2 TABLE OF CONTENTS ........................................................................................................ 4 LIST OF ABBREVIATIONS ................................................................................................. 8 LIST OF PUBLICATIONS .................................................................................................. 10 First author: ........................................................................................................................... 10 Chapter 1. Literature review ................................................................................................ 14 1.1. Stone fruit production ....................................................................................................... 14 1.2. Economic losses of brown rot disease .............................................................................. 15 1.3. Monilinia species .............................................................................................................. 15 1.3.1. Taxonomy and host range .......................................................................................... 15 1.3.2. Monilinia life cycle .................................................................................................... 16 1.3.3. Identification of Monilinia species............................................................................. 17 1.3.4. Geographical distribution .......................................................................................... 18 1.4. Genetic diversity ............................................................................................................... 19 1.5. Management of brown rot disease .................................................................................... 20 1.5.1. Fungicide application and development of fungicide tolerance ................................ 20 1.5.2. Potential alternative control measures to fungicides ................................................ 21 1.6. Mycoviruses ...................................................................................................................... 22 1.6.1. Identification of mycoviruses ..................................................................................... 23 1.6.2. Influence of mycoviruses on host fungi ...................................................................... 23 1.6.3. Mycoviruses of Monilinia .......................................................................................... 24 1.7. Research gaps in Australia ................................................................................................ 25 1.8. The five aims of this research project ............................................................................... 25 1.9. Notifications ...................................................................................................................... 25 Chapter 2. Spatial distribution of Monilinia fructicola and M. laxa in stone fruit production areas in Western Australia ................................................................................ 27 Statement of contribution of the authors contributed to the work ........................................... 28 Abstract .................................................................................................................................... 29 Introduction .............................................................................................................................. 29 4 Material and Method ................................................................................................................ 31 Result and Discussion .............................................................................................................. 35 References ................................................................................................................................ 39 Addendum to Chapter 2 ........................................................................................................... 42 Chapter 3. Genotypic structure of Monilinia populations in Western Australia two decades after incursion .......................................................................................................... 43 Statement of contribution of the authors contributed to the work ........................................... 44 Abstract .................................................................................................................................... 45 Introduction .............................................................................................................................. 45 Materials and methods ............................................................................................................. 47 Fungus collection ................................................................................................................
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