Characterization of Pyrenophora teres isolates and mapping of virulence genes

Pyrenophora teres is the causative agent of net blotch of barley and one of the most economically important fungal pathogens affecting the Australian barley industry. The estimated yield losses due to net blotch exceed $300 million annually. It is a foliar pathogen that exists as two forms: P. teres f. teres (Ptt) and P. teres f. maculata (Ptm), causing net form-net blotch (NFNB) and spot form-net blotch (SFNB), respectively. In order to address some of the research gaps in our understanding of the P. teres-barley pathosystem, this project was designed to: 1. genetically characterise Pyrenophora teres f. teres populations collected from different continents; 2. investigate the mating preference between the two forms of P. teres and its hybrids; and 3. identify QTL (quantitative trait loci)/genes associated with virulence of P. teres using a hybrid population developed by crossing the two forms. The genetic characterization of a pathogen population is important in understanding the genetic variation existing within a pathogen population. In this study we characterized the most geographically diverse Ptt population investigated in a single study using the genome-wide marker system DArTseq. Results obtained from different cluster analyses revealed that an Australian Ptt population shared more admixture with a Republic of South Africa (RSA) population than a Hungarian Ptt population. Neighbor-joining dendrogram analysis and form-specific PCR amplifications detected two field collected hybrids from Hungary (H-919) and Japan (CBS 281.31). CBS 281.31 was a historical isolate which was previously authenticated as P. japonica. Evidence for recent/ongoing gene migration among different continents was observed, which highlights the importance of practicing biosafety measures to prevent the introduction of a pathogenic gene pool from one geographical region to another. After establishing that hybridization between the two forms of P. teres are more frequent than previously assumed, we investigated whether Ptt and Ptm have a mating preference for the same form over the opposite form in vitro when they were given the opportunity to mate with both forms. Results revealed that Ptt isolates preferred Ptt isolates at the early reproduction stage, however, later they did not have any preference but hybridized with Ptm. Ptm isolates did not have any preference toward isolates from the same form but underwent hybridization with Ptt isolates. Results also showed that Ptt isolates had greater reproduction vigour than Ptm isolates under the given laboratory conditions. Progeny arising from a hybrid cross could have devastating effects on the barley industry in the absence of suitable resistant barely varieties as these hybrids may acquire combined virulence from both Ptt and Ptm. Hence, comprehensive knowledge of hybrids and the P. teres-barley pathogen system would allow barley breeders to develop novel barley germplasms to withstand potential future outbreaks caused by hybrids. Therefore, as a part of this study we identified QTL/genomic regions associated with virulence and leaf symptoms of net blotch. Nine QTL associated with virulence and leaf symptoms across five linkage groups/chromosomes were identified. Phenotyping of selected highly virulent progeny isolates on net blotch-resistant barley genotypes revealed that some progeny isolates are highly virulent across all of the 20 tested current widely used net blotch-resistant barley varieties. In conclusion, results obtained from this study give an insight into the P. teres-barley pathogen system, which can aid future development of disease resistant barley varieties. Future studies on cloning and gene expression of the identified genomic regions would improve our knowledge of the P. teres-barley pathosystem. Determining the mating preference of Ptt and Ptm under different environmental conditions would allow us to understand what environmental conditions favour hybrid production and to have control measures in place to prevent or reduce the occurrence of hybrids