Studies of pathogenicity in Plasmodiophora brassicae and segregation of clubroot resistance genes from Brassica rapa subsp. rapifera by Junye Jiang A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science Department of Agricultural, Food and Nutritional Science University of Alberta © Junye Jiang, 2020 Abstract The planting of clubroot resistant (CR) canola (Brassica napus) is the most effective method to manage clubroot, a soilborne disease caused by Plasmodiophora brassicae. In recent years, many P. brassicae isolates capable of overcoming resistance have been detected, often in mixtures with avirulent isolates. To improve understanding of the effect of low concentrations of virulent isolates on host resistance, three CR canola cultivars (‘45H29’, ‘L135C’ and ‘L241C’) were inoculated with pairs of isolates representing virulent/avirulent pathotypes (2*/2, 3*/3 and 5*/5) of P. brassicae, collected after or before the introduction of CR canola, respectively. Clubroot severity was significantly higher in all nine experimental treatments (low virulent + high avirulent) than in the negative control NC1 (high avirulent), and higher in seven of nine experimental treatments than in the negative control NC2 (low virulent). Disease severity was positively correlated with P. brassicae biomass in planta, as determined by quantitative PCR analysis 28 - 35 days after inoculation (dai). These results suggest that low concentrations of virulent isolates compromised the clubroot resistance in canola, facilitating infection by avirulent isolates. In a second study, the expression of 205 P. brassicae genes encoding putative secreted proteins was compared following inoculation of the canola ‘45H29’ with pathotypes 5I (avirulent) and 5X (virulent) of the pathogen. Sixteen of these genes were differentially expressed at 14 dai, and additional monitoring at multiple timepoints (7, 14 and 21 dai) indicated that, collectively, 12 of the 16 genes were upregulated in pathotype 5X. The relative expression of the same 16 genes was also compared in the interaction between the canola ‘Westar’ and pathotype 5I (virulent on ‘Westar’), with 12 of the genes showing similar expression patterns as in the ‘45H29’/5X interaction. Given their common expression patterns in two compatible ii interactions, it is possible that these genes play a role in clubroot pathogenesis. In a third and final study, clubroot resistance was introgressed from the European Clubroot Differential (ECD) 02 into the B. rapa accessions CR 2599 and CR 1505 (‘Emma’), and into the B. napus accession CR 1054 (‘Westar’). The distorted segregation ratios suggest that the two resistance genes are on different chromosomes and that two genes interact in an epistatic manner to confer resistance. Genotyping was conducted with 144 PCR-based markers in two of the three F2 populations. Linkage and QTL analysis with the polymorphic markers identified two QTLs on chromosome A03 associated with resistance to P. brassicae pathotypes 5X and 5G in Popl#1, whereas only the second QTL on chromosome A03 was associated with resistance to these pathotypes in Popl#2. The two QTLs clustered in genomic regions on the A03 chromosome of B. rapa where the CRa/CRbKato gene(s) are mapped. In addition, the Crr1 gene on the A08 chromosome of B. rapa was detected in the two populations. Therefore, the phenotypic and molecular data confirm the existence two CR genes in ECD 02. iii Preface This thesis is an original work by Mr. Junye Jiang, who conducted the experiments and wrote the first drafts of all the chapters. Mr. Jiang’s supervisors, Dr. Stephen E.Strelkov and Dr. Sheau- Fang Hwang, reviewed and edited each chapter. The chapters were also reviewed by Dr. Rudolph Fredua-Agyeman, a member of Mr. Jiang’s supervisory committee. Dr. Fredua-Agyeman also assisted with the data analysis and provided feedback and constructive suggestions on the experimental design of some of the studies. Dr. Robert Conner (Agriculture and Agri-Food Canada) and Mr. George Turnbull (Alberta Agriculture and Forestry) also reviewed Chapter 3 and provided suggestions for improvements. The Brassica accessions used in the experiments described in this thesis were obtained from the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany. The field and single-spore isolates of the clubroot pathotgen, Plasmodiophora brassicae, were from the research collections of Drs. Strelkov and Hwang. Technical staff and students from the Plant Pathology Lab, University of Alberta, assisted with the inoculation experiments described in Chapters 2 and 4. Financial support for this research came through grants awarded by Agriculture and Agri-Food Canada and the Canola Council of Canada through the Growing Forward 2 and Canadian Agricultural Partnership (CAP) programs to Drs. Strelkov, Hwang, and Fredua-Agyeman. Additional funding for earlier parts of this work was provided by the Alberta Crop Industry Development Fund. In-kind support, including access to facilities and equipment, was provided by the University of Alberta and Alberta Agriculture and Forestry. A version of Chapter 2 has been published as: Jiang, J., Fredua-Agyeman, R., Strelkov, S.E., and Hwang, S.F. 2020. Suppression of canola (Brassica napus) resistance by virulent isolates of iv Plasmodiophora brassicae (clubroot) during primary infection. Plant Dis. 104: 430-437. https://doi.org/10.1094/PDIS-03-19-0659-RE v Acknowledgements I would like to express my deep and sincere gratitude to my supervisor Dr. Stephen E. Strelkov and co-supervisor Sheau-Fang Hwang, for providing me with the opportunity to do research at the University of Alberta and offering invaluable guidance throughout this program. They instructed me from research methodology to the presentation of my work. In my opinion, what I learned most from them is how to present the research results. Their attitudes to research as well as life have deeply inspired me during my Ph.D. Therefore, it was a great privilege and honor to work and study under their guidance. I would also like to acknowledge Dr. Rudolph Fredua-Agyeman for his kind and patient guidance and support throughout my Ph.D. program, and we have become very close friends during my Ph.D. I would also like to thank Dr. Robert Conner and Mr. George Turnbull for their review of Chapter 3. I appreciate the assistance received from staff at the Crop Diversification Centre – North, Alberta Agriculture and Forestry, and from the many summer students at the University of Alberta Plant Pathology Lab. I would like to thank Dr. Genyi Li (University of Manitoba) for serving as my External Examiner, and Dr. Nadir Erbilgin for serving as my Arm’s Length Examiner, in my defense and for providing suggestions for this thesis. I must express my thanks and love to my wife, Jie Liu, for her support and love to me, especially when I was down and she stood behind me to get through the difficulties. She is the love of my life. I also need to thank my lovely and active boys, Leif and Livan, they are precious gifts in my life, I love them more than they imagine. vi I would also like to thank my parents Dr. Chongzhi Jiang and Dr. Yumei Liu because they are always supportive and stand behind me. I know they love me as much as I love my boys and I love them too. I would also like to thank my parents in-law, Xinhua Wang and Junhua Shi, for their support as well as for taking care of my boys. Finally, I would like to acknowledge the financial support received from Agriculture and Agri-Food Canada and the Canola Council of Canada through the Growing Forward 2 and Canadian Agricultural Partnership (CAP) Programs, and for the earlier support from the Alberta Crop Industry Development Fund. I also thank Alberta Agriculture and Forestry, and the University of Alberta for their generous in-kind support. vii Table of Contents Chapter 1: Introdcution and Literature Review........................................................................ 1 1.1 Introduction to the Brassicas ............................................................................................. 1 1.2 Development of Canola ....................................................................................................... 1 1.3 Economic Importance of Canola ....................................................................................... 2 1.4 Canola Agronomy and Pests .............................................................................................. 3 1.4.1. Environmental factors affecting canola growth ....................................................... 3 1.4.2 Weeds and canola ......................................................................................................... 6 1.4.3 Insects and canola ......................................................................................................... 7 1.4.4 Canola diseases ............................................................................................................. 8 1.5 Clubroot of Canola .............................................................................................................. 8 1.5.1 History of clubroot disease .......................................................................................... 8 1.5.2 Clubroot symptoms ...................................................................................................... 9 1.5.3 Taxonomy and lifecycle of P. brassicae....................................................................