Biology and host-pathogen interaction of Stagonosporopsis tanaceti, the cause of ray blight in pyrethrum Md Abdullahil Baki Bhuiyan Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy Faculty of Veterinary and Agricultural Sciences The University of Melbourne April 2017 i Declaration I declare that this thesis includes only my original work in the direction of the degree of Doctor of Philosophy. I also acknowledge all other materials use in the text. The words of this do not exceed 100,000 words. This thesis fulfils the stipulations set out for the degree of Doctor of Philosophy by the University of Melbourne. Md Abdullahil Baki Bhuiyan April 2017 ii Acknowledgements My grateful thanks to my major supervisor Professor Paul Taylor and co-supervisor Dr Marc Nicolas for their scholastic academic guidance and continuous help to accomplish this thesis. My special thanks to Paul for his friendly support, guidance and encouragement. My special thanks to Tim Groom, Manager- Agricultural Businesses, Botanical Resources Australia (BRA) Pty. Ltd. for his judicious suggestions, inspirations and invitation at BRA to discuss my findings with the industry people which made this research worthwhile. Many thanks to our lab managers Carolyn Selway, Michelle Rhee and Martin Ji; Stephen and Priya Chand (Faculty staff), Steven (Glasshouse) for their assistance and continuous support throughout the research period. I would like offer special gratitude to my lab colleagues especially Niloofar, Dina, Eden, Mee-Yung, Sophia, Azin, Jiang, Dilani, Ruvini and Aruni for their friendship and continuous support. I would like to acknowledge with special gratitude the Melbourne International Research Scholarship (MIRS) and Melbourne International Fee Remission Scholarship (MIFRS) awarded by the University of Melbourne and financial support from BRA. I am especially grateful to my wife Farhana Jenny and my son Ahnaf Rayan Bhuiyan for their sacrifice, mental support and continuous inspiration during my whole study period in overseas. Special thanks to my parents in law Nizam Uddin Patwary and Jahanara Begum, sister in law Fariha and brother in law Arnab for their continuous support to my family. I would like to acknowledge the support and inspiration from my brother Md. Ashiqur Rahman Bayzid and also thanks to his wife Shamsun Nahar Suchi for her wish. iii Finally, I would like to acknowledge my parents Md Abdul Awal Bhuiyan and Ayesha Begum for their wish, inspiration, sacrifice and pray during my study period in abroad. iv Abstract Ray blight (Stagonosporopsis tanaceti) is a major biotic constraint for pyrethrum production in Australia. The biology and host-pathogen interaction of S. tanaceti in seed, seedlings and mature pyrethrum plants were studied in glasshouse experiments. Molecular detection and quantification of infection within pyrethrum tissues were determined using TaqMan PCR. Direct penetration into the epidermal cells of leaves resulted in the development of brown to black necrotic lesions. Hyphae of S. tanaceti then colonised the cortical tissues intra- and intercellularly resulting in degradation of tissues and deposition of extra- cellular material in necrotic cells. Pycnidia formed within 12 days after infection in the epidermis and hypodermis of pyrethrum leaves. Quadruple staining technique was more suitable than single or dual staining for visualising fungal hyphae within the tissue because the combination of four stains enabled the hyphae to stain blue-green in contrast to the cell tissues. Stagonosporopsis tanaceti infected only the seed coat and not the embryo with infected seed being symptomless and not deformed or discoloured. After germination, S. tanaceti infected the embryonic tissues in the seed coat and depending on the level of infection of these tissues, resulted in pre-emergence and post-emergence damping off. Degradation of whole embryonic tissues by S. tanaceti resulted in pre-emergence death while disintegration of hypocotyl/crown tissues resulted in post-emergence death. Some of the infected embryos developed into seedlings with S. tanaceti being present in the hypocotyl/ crown tissues without these tissues showing visible symptoms thus indicating a latent stage in the life cycle of the pathogen. v Stagonosporopsis tanaceti infected the cauline and petiolate leaves, crown tissues, flower stems, flower buds and rays of the flower but not the roots of pyrethrum plants. The necrotic region of flower stems extended from the peduncle to 8-11 cm down the flower stems where the epidermis, hypodermis and cortical tissues were degraded. Pycnidia that developed in the necrotic tissue of flower stems released pycnidiospores, which dispersed through wind and water-splash and then presumably settled onto the flower buds and infected the flower rays before infecting the developing seed. Pycnidia that also formed on the infected foliage released pycnidiospores that were then deposited, by water splash dispersal to the region of the crown tissue where the petioles emerged. At this point the spores germinated and infected the parenchyma cells of the hypodermis and cortical tissues of the crown. Throughout the infection cycle, vascular tissues of all plant organs were not colonised with the endodermis acting as a barrier to hyphal infection. The results of the infection and colonisation of various plant tissues has enabled a complete disease cycle of ray blight in pyrethrum to be described. The effect of S. tanaceti on growth and development of pyrethrum was studied in two glasshouse trials where the first trial optimised the inoculation concentration to establish ray blight and the second trial determined the effect of S. tanaceti on five cultivars 5 3 6 inoculated at10 spores/ mL. At inoculation concentrations of 10 and 10 spores/ mL there was a significant reduction in biomass (above and below ground dry weights) of cultivar BR1, 6 months after inoculation (mai). At 106 spores/ mL there was a significant reduction in the above ground biomass at 1 and 2 mai but only a significant reduction in below ground biomass at 2 mai. At 103 spores/ mL there was no significant difference in biomass (above and below ground) to the non-inoculated control plants at 1 and 2 mai although dry weights were lower in the infected plants. Infected pyrethrum vi cultivars BR1, BR2, Pyper, Pyrate and RS5 had significantly reduced plant height, above ground biomass, shoot and flower numbers over two growing cycles. Although cultivar BR1 was affected by S. tanaceti, this cultivar had significantly higher biomass and flower production in two consecutive growing seasons than the other cultivars indicating that BR1 may have moderate resistance/tolerance to S. tanaceti. The performance of BR1 needs to be further assessed under field conditions with natural levels of inoculum. A TaqMan probe-based polymerase chain reaction (PCR) assay was developed to quantify the level of S. tanaceti inoculum in pyrethrum seed and seedlings. Primer pair St_qF3 and St_qR2 was designed based on the intergenic spacer (IGS) region of S. tanaceti, which produced a 125 bp amplicon specific to S. tanaceti. TaqMan PCR assay using these primers and probe St_qP was highly specific against the genomic DNA of S. tanaceti and did not amplify the genomes of 14 related Stagonosporopsis species. The sensitivity limit of this assay was measured using the cycle threshold (Ct) value, which ranged from 17.59 for 10 ng to 36.34 for 100 fg of genomic DNA of S. tanaceti. There was a significant negative correlation (r= -0.999, P≤ 0) between the Ct value and the percent of S. tanaceti infected seed. This TaqMan PCR assay detected S. tanaceti in hypocotyl/ crown tissue of symptomless seedlings demonstrating the efficacy of this assay to detect latent infection of seedlings. In summary, this study has provided better understanding of the biology and infection process of S. tanaceti in pyrethrum seed, seedlings and mature plants and identified moderate level of resistance/tolerance in cultivar BR1. In addition, the amount of infection within pyrethrum seed and seedlings was able to be quantified using TaqMan PCR which will enable the pyrethrum industry to develop a seed testing service. The vii outcomes from this project will enable the development and application of more efficient targeted control measures and provide a basis to develop varietal resistance breeding programs to S. tanaceti. viii Table of contents Declaration ....................................................................................................................... ii Acknowledgements ........................................................................................................ iii Abstract ............................................................................................................................ v Table of contents ............................................................................................................ ix List of tables.................................................................................................................. xiv List of figures ................................................................................................................. xv List of abbreviations .................................................................................................. xviii Preface ........................................................................................................................... xxi Chapter 1 ........................................................................................................................