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S43021927 Phd Thesis.Pdf Plant pathogenic Magnaporthales in Australia, with particular reference to Pyricularia oryzae on wild and cultivated rice Wanporn Khemmuk Master of Science (Plant Pathology) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 Queensland Alliance for Agriculture and Food Innovation 1 Abstract The Magnaporthales is an order of fungi that contains plant pathogens and saprobes. This order consists of three families, Pyriculariaceae, Magnaporthaceae and Ophioceraceae, which are phylogenetically, morphologically and ecologically distinct. To date, about 200 species have been described in Magnaporthales, of which approximately 50% are plant pathogens. Some species are important pathogens of grasses and cereals such as the rice blast fungus Pyricularia oryzae (syn. Magnaporthe oryzae) and the take-all pathogen of cereals Gaeumannomyces graminis. The study of classification and identification of Magnaporthales in Australia and pathogenicity of Pyricularia oryzae are reported in this thesis. The genus Pyricularia comprises species that cause blast diseases on various hosts, especially grasses (Poaceae) that include crops such as rice, wheat, barley and grasses. This study used morphology, phylogenetic concordance, ecology and pathogenicity, to study Pyricularia and allied genera in the Magnaporthales. The fungi associated with blast diseases on eleven monocot hosts in Australia were identified by morphological characters and DNA sequence analysis in Chapter 2. Three species of Pyricularia, namely, Pyricularia angulata, Pyricularia pennisetigena and Pyricularia oryzae, were confirmed as present in Australia. Another species, Pyricularia rabaulensis was found to belong to a recently established genus Barretomyces. A formal transfer of this fungus to Barretomyces has been proposed. In Chapter 3, the phylogenetic relationships of some other Australian Magnaporthe-like fungi were investigated based on morphology and DNA sequence analysis of multiple genes. The root pathogenic fungi of Cynodon dactylon and Agrostis stolonifera as well as holotype specimens of Magnaporthe garrettii and Magnaporthe griffinii were examined. Two novel genera, Budhanggurabania and Wongia, were described as well as a new species, Magnaporthiopsis agrostidis. Rice (Oryza sativa L., Poaceae) is the most important cereal crop globally and a primary source of starch for more than half the world’s population. The major constrain to rice production worldwide is rice blast disease, which is caused by the fungus Pyricularia oryzae Cavara (syn. Magnaporthe oryzae Hebert). Rice blast disease is a problem in over 85 countries in all continents where rice is cultivated in both paddy and upland situations. Unpredictable epidemics can cause significant economic losses. 2 Foliar disease surveys of wild and cultivated rice in northern Queensland were completed in 2014 and 2015. The results presented in Chapter 4 show that a total of 109 fungal isolates was recovered and classified into seven genera, viz. Bipolaris, Colletotrichum, Curvularia, Nigrospora, Phoma, Pestalotiopsis and Pyricularia. Isolates from three genera, Pyricularia, Bipolaris and Curvularia were identified to species level by multigenes analysis because of their potential to cause important diseases on rice. The rice blast pathogen, Pyricularia oryzae, brown spot pathogen, Bipolaris oryzae and a large diversity of Curvularia species were recovered. To remove uncertainty about the identity of the host plants from which the fungi were isolated, a DNA barcoding strategy was developed using regions of the chloroplast genome. In Chapter 5, pathogenicity and cross infectivity of Pyricularia species are investigated. Four Australian isolates of Pyricularia oryzae, two isolates from domesticated rice and two isolates from wild rice, were tested onto nine commercial rice cultivars namely, Doongara, langi, Kyeema, Illabong, Sherpa, Reiziq, Koshi, Opus and Quest. The results showed that infection occurred on all rice varieties with Quest the most susceptible and Sherpa the least susceptible. The host ranges of four Australian isolates of Pyricularia oryzae from rice and one isolate from barley, were tested on barley, oats, rice and wheat. None of the isolates infected wheat, and one isolate from rice infected oats. Isolates of Pyricularia oryzae from Setaria italica and Pennisetum clandestinum were shown to cause blast disease on their original hosts and barley but did not infect rice and wheat. The research described in this thesis presents the identification and classification of plant pathogenic fungi in order Magnaporthales in Australia. Three species of Pyricularia were identified in this study based on a phylogenetic species concept as well as two novel species of root pathogens in family Magnaporthaceae. Phylogenetic analysis placed two Australian root pathogen species, Magnaporthe garrettii and Magnaporthe griffinii, in the new genus, Wongia, Diaporthomycetidae. Conidial morphology is a primary character to distinguish genera. The Magnaporthales is a diverse order and more novel species certaintly await discovery. Future researches that might provide better understanding of Pyricularia oryzae and other closely related fungi in the Magnaporthales are discussed in Chapter 6. 3 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. 4 Publications during candidature Peer-reviewed papers Khemmuk, W., Roger G. Shivas, Robert J. Henry and Andrew D.W. Geering. 2016. Fungi associated with foliar disease of wild rice and cultivated rice (Oryza spp.) in northern Queensland. Australasian Plant Pathology 45, 297-308. doi:10.1007/s13313-016-0418-3 Khemmuk, W., Geering, A. D. W. and Shivas, R.G. 2016. Wongia gen. nov. (Papulosaceae, Sordariomycetes), a new generic name for two root-infecting fungi from Australia. IMA Fungus 7(2), 247–252. doi:10.5598/imafungus.2016.07.02.04 Peer-reviewed papers (ancillary papers) Wong, P.T.W., Khemmuk, W., Geering, A. D. W. and Shivas, R.G. (2015). Budhanggurabania cynodonticola Fungal Planet No. 359. Persoonia 34, 240‒241. Wong, P.T.W., Khemmuk, W., Geering, A. D. W. and Shivas, R.G. (2015). Magnaporthiopsis agrostidis. Fungal Planet No.398. Persoonia 35, 322‒323. Conference abstracts Khemmuk, W., Geering, A. D. W. and Shivas, R.G. (2014) The diversity of Pyricularia in Australia. The 10th International Mycological Congress, 3rd–8th August 2014, Bangkok, Thailand. Khemmuk, W., Geering, A. D. W. and Shivas, R.G. (2014) The co-evolution of wild rice and its pathogens, especially Pyriculaira spp. Science Exchange 2014, Plant Biosecurity Cooperative Research Centre, 11th–14th May 2014, Twin Waters, Queensland, Australia. Khemmuk, W., Geering, A. D. W. and Shivas, R.G. (2015) The co-evolution of wild rice and its pathogens, especially Pyriculaira spp. Science Exchange 2015, Plant Biosecurity Cooperative Research Centre, 11th–14th August 2015, Twin Waters, Queensland, Australia. Khemmuk, W., Geering, A. D. W., Robert, J. Henry and Shivas, R.G. (2015) Wild rice pathogens in northern Queensland. Tropical Agriculture Conference 2015, 16th–18th November 2015, Brisbane Convention and Exhibition Center, Brisbane, Australia. 5 Publications included in this thesis Khemmuk, W., Geering, A. D. W. and Shivas, R.G. 2016. Wongia gen. nov. (Papulosaceae, Sordariomycetes), a new generic name for two root-infecting fungi from Australia. IMA Fungus 7(2), 247–252. — incorporated as Chapter 3 Contributor Statement of contribution Wanporn Khemmuk - Designed experiments (50%) - Performed the experiments, analysed and interpreted data (80%) - Discussion of taxonomy and phylogeny (50%) - Wrote the paper (50%) Andrew D.W. Geering - Designed experiments (20%) - Discussion of taxonomy and phylogeny (10%) - Critically reviewed and edited the manuscript (20%) Roger G. Shivas - Designed experiments (20%) - Discussion of taxonomy and phylogeny (20%) - Critically reviewed and edited the manuscript (80%) 6 Wong, P.T.W., Khemmuk, W., Geering, A. D. W. and Shivas, R.G. (2015). Budhanggurabania
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