Assessing the biology of wheat-infecting Botryosphaeriaceae spp.

Abstract

In 1999, a novel wheat disease was observed in Queensland, Australia. This disease induced grain shrivelling and was described as white grain disorder (WGD). WGD is now a documented disease across all mainland wheat-growing States. It was unknown where the causal fungus originated, how it was able to infect wheat, and whether there were any other non-grass hosts. Using molecular techniques and morphological analyses to examine fungi from infected grain, I determined that the causal agent of WGD was three distinct species of fungi of the genus Eutiarosporella, in the Botryosphaeriaceae family. I classified these as Eutiarosporella darliae, E. pseudodarliae, and E. tritici-australis. The three species’ genomes were sequenced to gain a better understanding of their lifestyle. My primary focus became analysing secondary metabolite (SM) clusters, particularly polyketide synthases (PKS) and non-ribosomal peptide synthases (NRPS). I discovered that the WGD-Eutiarosporella spp. harbour modular PKS genes (mPKSs). mPKSs have previously only been observed in bacteria and protist species, not in fungi. Through phylogenetic analyses, I concluded that these mPKSs were horizontally acquired from an unknown species. I also identified an SM cluster with three co-localised hybrid PKS-NRPS genes in E. darliae and E. pseudodarliae, but absent in E. tritici-australis. A comparison of syntenic genes among all three species revealed that this cluster was once present in E. tritici-australis, but was lost. Genes homologous to one of the PKS-NRPSs are linked to disease in woody plants in other fungi. Subsequent inoculation of Hakea leaves with E. darliae and E. pseudodarliae induced necrotic disease symptoms, 9 whereas E. tritici-australis did not. I concluded that the WGD-Eutiarosporella species were likely once primarily woody plant pathogens and speculate that such species may act as reservoirs for the fungi. Another aspect of the life-style of the WGD-Eutiarosporella that I sought to elucidate was a mechanism of sexual reproduction. Sexual spores have been observed in the field for the WGD-Eutiarosporella spp.. Consequently, I analysed their genomes to determine the composition of mating type genes. E. darliae and E. pseudodarliae each possessed a copy of each mating type gene, MAT1-1-1 and MAT1-2-1, which indicates that these species are homothallic (self-fertile). However, MAT1-1-1 was located distal to the MAT-locus, within the middle of an unrelated gene. Only MAT1-2-1 could be identified in the sequenced E. tritici- australis isolate. However, additional screened isolates harboured either MAT1-1-1 or MAT1-2-1, indicating that E. tritici-australis is heterothallic (non-self-fertile). I concluded that E. darliae and E. pseudodarliae’s ancestor transitioned from heterothallism to homothallism via a cryptic DNA integration event.