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Powdery Mildew, Podosphaera Xanthii and Potato - Botrytis Cinerea

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Powdery Mildew, Podosphaera Xanthii and Potato - Botrytis Cinerea Confidential Histological study of the interactions between Cucumber - powdery mildew, Podosphaera xanthii and Potato - Botrytis cinerea Anindita Chakraborty Wageningen UR i Histological study of the interactions between Cucumber - powdery mildew, Podosphaera xanthii and Potato - Botrytis cinerea Anindita Chakraborty Registration number: 811206156020 MSc Minor Thesis (PBR-80424) for the partial fulfilment of Master of Sciences in Plant Biotechnology Specialization of Molecular Plant Breeding and Pathology Supervisors Dr. Yuling Bai Daily Supervisor Dr. Michela Appiano Kaile Sun Examiner Dr. Anne-Marie Wolters Dr. Evert Jacobsen July, 2015 Laboratory of Plant Breeding, Wageningen University and Research Centre, Wageningen, The Netherlands. ii Acknowledgements I would like to take the opportunity to convey my heartfelt gratitude to my supervisor Dr. Yuling Bai for her constructive supervision, valuable suggestions and relentless support to initiate and accomplish this research work. Her patience, wisdom and critical guidance to the project approach allowed me the freedom to work in a spectacular way. I would also like to express my sincere appreciation to Dr. Michela Appiano and Kaile Sun, my daily supervisors, for their special instructions on operating instruments and other laboratory knowledge required to my lab-work, as well as continuous guidelines for writing a scientific report during this research, motivation and encouragement to reach my destination. I am very thankful to Dr. Evert Jacobsen and Dr. Ageeth van Tuinen for their valuable time to provide academic guidance, insightful comments and critical remarks in improving the manuscript. Importantly I should not forget to express my sincere thanks to Dr. Anne-Marie Wolters and Dr. Jan van Kan for their advice, cooperation and valuable suggestion to carry out these experiments. I am also indebted to many student colleagues and the technical staff of the laboratory of plant breeding, WUR for their consistent support throughout my project. Moreover, I could not but mention the name of Patrick Hendrickx, for his helps in microscopy techniques. In a nutshell, I got myself as a successful student of the project having support from the above mentioned respectful people; such accomplishment would not have been possible without their contribution. iii Thesis outline This minor thesis is comprised of two experiments with the aim to provide insights into plant microbes interaction through histological analysis. In the first experiment, histological study was carried out to elucidate the mechanism of interaction of plants of a F2 population of cucumber (F2-pm3) with cucumber powdery mildew, Podosphaera xanthii. This pathogen is an obligate biotrophic fungus. The staining for the histological analysis was performed using trypan blue solution. Development of pathogen structures (appressorium, haustorium, hyphae) and response of host tissue (Hypersensitive response, HR) were examined by light microscopy in susceptible and resistant plants. In the second experiment, the infection progress of Botrytis cinerea, a necrotrophic fungus, was examined on potato plants. Leaf samples were stained in a medium using X-gluc. Histological study was performed to compare the development of the infection of Botrytis cinerea in susceptible control and DND1 silenced potato transformants. iv Contents 1 Histological study of Podosphaera xanthii on cucumber .............................................................. 1 1.1 Introduction ................................................................................................................ 1 1.1.1 Cucumber ............................................................................................................... 1 1.1.2 Cucumber Powdery Mildew ........................................................................................ 1 1.1.3 Disease symptoms.................................................................................................... 2 1.1.4 Life cycle of Powdery mildew pathogen ........................................................................ 2 1.1.5 Host-pathogen interaction ......................................................................................... 4 1.1.6 Objective of the present research ............................................................................... 6 1.2 Materials and methods .................................................................................................. 7 1.2.1 Plant materials ......................................................................................................... 7 1.2.2 Sample collection ..................................................................................................... 7 1.2.3 Staining of sample .................................................................................................... 7 1.2.4 Histological analysis .................................................................................................. 7 1.3 Results ....................................................................................................................... 9 1.3.1 Conidia germination and development ......................................................................... 9 1.3.2 Primary haustorium formation .................................................................................. 10 1.3.3 Host reaction ......................................................................................................... 11 1.3.4 Secondary structures and host reaction ..................................................................... 12 1.4 Discussion ................................................................................................................ 13 1.4.1 Infection structure development ............................................................................... 13 1.4.2 Host responses ...................................................................................................... 14 1.5 Summary ................................................................................................................. 17 1.6 References ................................................................................................................ 18 1.7 Appendices ............................................................................................................... 20 2 Histological study of Botrytis cinerea on potato ....................................................................... 21 2.1 Introduction .............................................................................................................. 21 2.1.1 Botrytis cinerea ...................................................................................................... 21 2.1.2 Infection cycle of Botrytis cinerea ............................................................................. 21 2.1.3 Resistance mechanism of the host plants against B. cinerea ......................................... 22 2.1.4 Susceptibility (S) genes ........................................................................................... 24 2.1.5 DND1 ................................................................................................................... 25 2.1.6 Objective of present study ....................................................................................... 25 2.2 Materials and methods ................................................................................................ 26 2.2.1 Plant materials ....................................................................................................... 26 2.2.2 Pathogens description ............................................................................................. 26 2.2.3 Inoculation and sample collection ............................................................................. 26 2.2.4 Staining of samples ................................................................................................ 26 2.2.5 Histological analysis ................................................................................................ 27 v 2.3 Results ..................................................................................................................... 28 2.3.1 Observation at 0.5 hours post inoculation (hpi) ........................................................... 28 2.3.2 Observation at 3 hours post inoculation (hpi) ............................................................. 28 2.3.3 Observation at 6 hours post inoculation (hpi) ............................................................. 29 2.3.4 Observation at 10 hours post inoculation (hpi) ............................................................ 30 2.3.5 Observation at 24 hours post inoculation (hpi) ............................................................ 31 2.3.6 Observation at 48 hours post inoculation (hpi) ............................................................ 32 2.4 Discussion ................................................................................................................ 35 2.5 Summary ................................................................................................................. 38 2.6 References ................................................................................................................ 39 2.7 Appendices ............................................................................................................... 43 vi 1 Histological study of Podosphaera xanthii on cucumber 1.1 Introduction 1.1.1
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