Downy mildew on oilseed Brassicas – understanding the drivers of disease epidemics and potential of novel host resistances Akeel Emad Mohammed Bachelor of Agricultural Science, Master of Plant Protection This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia School of Agriculture and Environment Plant Pathology 2018 THESIS DECLARATION I, Akeel Emad Mohammed, certify that: This thesis has been substantially accomplished during enrolment in the degree. This thesis does not contain material which has been accepted for the award of any other degree or diploma in my name, in any university or other tertiary institution. No part of this work will, in the future, be used in a submission in my name, for any other degree or diploma in any university or other tertiary institution without the prior approval of The University of Western Australia and where applicable, any partner institution responsible for the joint-award of this degree. This thesis does not contain any material previously published or written by another person, except where due reference has been made in the text. The work(s) are not in any way a violation or infringement of any copyright, trademark, patent, or other rights whatsoever of any person. This thesis contains published work and/or work prepared for publication, some of which has been co-authored. Signature: Date: 10/04/2018 ii ABSTRACT Downy mildew disease caused by (Hyaloperonospora brassicae syn. H. parasitica) is a major disease limitation to oilseed Brassica production (particularly rapeseed, canola, mustard) worldwide and also causes significant damage in vegetable Brassica crops. Infection can be high as 100% on some Brassica crops especially in the early seedling stage. Up to date, no Brassica varieties have been specifically developed with effective resistance to this important disease. Screening for the host resistance to H. brassicae of 131 Brassicaceae varieties at the cotyledon stage, including 109 Australian canola varieties (Brassica napus and B. juncea) and 22 diverse Brassicaceae (including B. napus, B. carinata, B. juncea, B. nigra, B. rapa, Crambe abyssinica and Raphanus sativus) highlighted new excellent resistance to downy mildew. Using a mixture of 10 H. brassicae isolates collected from southern Australia areas severely affected by downy mildew disease in 2015, new high level resistances were identified across R. sativus, B. carinata, B. napus, B. juncea and C. abyssinica. Cluster analysis revealed six distinct clusters (highly resistant, resistant, moderately resistant, moderately susceptible, susceptible, and very susceptible) based on disease index (%DI) values, and this opens the way for breeders having to only select a single genotype from within each of the clusters determined as highly resistant or resistant in developing new resistant commercial varieties. This is the first study to demonstrate the existence of these very high levels of pathotype-independent resistance to H. brassicae, particularly in Australian canola varieties. The above studies were followed up with additional search for further new sources of resistance to H. brassicae, but this time across more diverse iii Brassicaceae including 78 B. napus, 38 B. carinata, 25 B. juncea and 13 miscellaneous Brassicaceae including (three of Raphanus sativus, two of each of Rapistrum rugosum, B. incana and one each of C. abyssinica, B. fruticulosa, Hirschfeldia incana, B. insularis, B. oleracea and Sinapis arvensis. Further new sources of effective resistance were identified to H. brassicae among these, particularly in R. sativus; B. carinata, B. juncea B. carinata, B. incana and C. abyssinica. To examine the role of environmental factors on the development of downy mildew epidemics, the effects of temperature (14/10⁰C and 22/17⁰C day/night) and plant age (15, 23, 31 and 40 day-old-plants) on the severity of downy mildew on B. juncea and B. napus varieties were determined. There were significant effects of temperature, plant age and their interaction, with more severe disease under warmer conditions and on very young seedlings. Findings explain the recent increase in severe disease epidemics in canola as seasonal temperatures increase and why most severe epidemics are on youngest plants. H. brassicae isolates collected 2006-2008, and more recently, were inoculated onto cotyledons of 28 diverse Brassicaceae genotypes to identify and select suitable Brassica spp. differentials to enable characterisation of H. brassicae pathotypes and to define phylogenetic relationships among isolates across Australia. Using octal classification, the six Brassicaceae host genotypes most suitable as host differentials to characterise pathotypes of H. brassicae were identified and then used to define eight distinct pathotypes occurring in Australia. Phylogenetic relationships, determined across 20 H. brassicae isolates collected in 2006-2008 and 88 isolates collected in 2015-2016, highlighted seven distinct clades. These are the first studies to define the phylogenetic relationships and pathotype structure among H. brassicae isolates in Australia and set a iv benchmark for understanding current and future genetic and phenotypic pathotype shifts within pathogen populations. In summary, these studies are the first to determine the expression of pathotype-independent host resistances across an extensive set of canola varieties and diverse Brassicaceae to H. brassicae isolates. Highly resistant varieties identified have resistance(s) that can, in some instances, be directly deployed into downy mildew prone regions and, in all instances, provide resistances for use in Brassicaceae breeding programs. Studies of the effect of temperature and plant age explain, for the first time, why the incidence and severity of downy mildew is most severe on seedlings and why both have increased over the past decade and a half when season temperatures have been increasing as a consequence of climate change. The new set of Brassicaceae host differentials developed provides the first practical means for monitoring changes in pathotype structure within H. brassicae populations. Further, these host differentials not only allow early warning of new pathotypes able to overcome current and future host resistances, but provide breeders with the relevant pathotype information needed to develop and deploy appropriate and effective host resistances to counter the inevitable changes in pathotypes of H. brassicae over coming decades. v TABLE OF CONTENTS Thesis declaration……………………………………………………………………...ii Abstract……………………………………………………………………….………..iii Table of contents….............................................................................................vi Acknowledgements ………………….…….…………………………………….….viii Authorship declaration: Co-authored publications…………..……….………..…..ix Chapter 1: Literature review …………………………………………………………1 1.1 Introduction……………………………………………………..……………….…2 1.2 Brassicas (particularly oilseed brassicas rapeseed, canola, mustard)...……3 1.3 Oilseed brassica downy mildew disease……………………………………….4 1.3. 1 Classification of Hyaloperonospora brassicae……………………….…4 1.3. 2 Life cycle of Hyaloperonospora brassicae…………………...………….5 1.3. 3 Symptoms of downy mildew on brassicas……………………...……….6 1.3. 4 Host range of Hyaloperonospora brassicae on cruciferous species....7 1.3. 5 Downy mildew on canola and mustard………………………….………8 1.3. 6 Severity and impact of downy mildews on canola and mustard………8 1.3. 7 Physiological specialisation within Hyaloperonospora brassicae populations………………………………………………………………………...9 1.3. 8 Impact of environmental factors on development of downy mildew…………………………………………………………………………….11 1.3. 9 The effect of plant age on downy mildew epidemics …………….…..12 1.3. 10 Management of downy mildew disease…………………………..….13 1.4 Sources of resistance in oilseed brassicas and other brassicas……………14 1.5 Concept and aims of work………………………………………………………16 Bibliography for literature review……………………………………….…………..19 vi Chapter 2: New resistances offer opportunity for effective management of the downy mildew (Hyaloperonospora parasitica) threat to canola………..…..……35 Chapter 3: Resistances to downy mildew (Hyaloperonospora brassicae) in diverse Brassicaceae offer new disease management opportunities for oilseed and vegetable crucifer industries………………………………………………..….45 Chapter 4: Temperature and plant age drive downy mildew disease epidemics on oilseed Brassica napus and B. juncea…………………………………………80 Chapter 5: Pathotypes and phylogenetic variation determine downy mildew epidemics in Brassica spp. in Australia……………………………………………90 Chapter 6: General Discussion…………………………………………....………105 Bibliography for general discussion………………………………...…….………113 Appendix- Presentations-Proceedings………………………………...…………118 vii ACKNOWLEDGEMENTS First of all, I would like to acknowledge my supervisors Professor Martin Barbetti and Dr Ming Pei You for their efforts, guidance and continuous support during my PhD journey. It is a wonderful opportunity to complete my study under the supervision of a great pathologist and respectful scientist, who treats his students respectfully to achieve their goals. A huge thank to my family, my mother, father, my lovely wife and my dear sons (Emad, Alaa and Karar) for their support and their patience in the difficult circumstances of my study. I am grateful to my colleagues, Hebba, Margaret, Dolar and Solomon for their assistance and encouragement. My friends in Iraq, who support me by their sincere wishes are deeply appreciated.