Phenotype-genotype distinctions among clones of the pine pitch canker pathogen Fusarium circinatum

Abstract

The population of Fusarium circinatum in South Africa is large and diverse, yet relatively few isolates have sequenced genomes or have been genotyped using microsatellites. Indeed, only one strain so far has a draft genome that is fully annotated. However, there has been significant progress in studies to better understand the population biology of this fungus in order to develop effective disease control measures for it in the local pine-based forestry industry. This study established a foundation for guiding subsequent full characterisation of the entire F. circinatum population using vegetative incompatibility. The literature review included here outlines and summarizes what is currently known about F. circinatum populations and the knowledge gap. The first research chapter (Chapter Two) focused on the phenotypic traits of F. circinatum isolates that were considered clonal based on ten unlinked microsatellite loci. The results showed that isolates thought to be clonal based on microsatellites differed significantly on a phenotypic level. This implies that we might be dealing with a more diverse population sharing the same multilocus genotypes. Two possible concluding hypotheses were drawn: previous genotyping methods were either not sensitive enough to delineate true clones or the phenotypic differences observed were due to differential gene regulation. Therefore, a subsequent study should use genomic and transcriptomic approaches to uncover the genetic identity of these isolates. For this purpose, after the South African population of F. circinatum isolates is sequenced, vegetative compatibility groups-based genotyping could be performed using both bioinformatics and laboratory-based techniques. The second research chapter (Chapter Three) lays the groundwork for understanding the molecular basis of vegetative/heterokaryon incompatibility in F. circinatum. This was accomplished by characterizing the known het/vic genes from N. crassa, P. anserina, C. parasitica, and A. oryzae in the F. circinatum reference strain (FSP34). The findings revealed that most of these known genes have at least one putative homolog in F. circinatum. Phylogenetic analyses also revealed that 13 of the 14 known genes had orthologs in F. circinatum. The only exceptions were C. parasitica's vic-6 and P. anserina's HNWD genes, both of which had multiple co-orthologs in F. circinatum. Almost all orthologs encoded similar functional domains, apart from the P. anserina co-orthologs in F. circinatum, which did not encode a HET domain but rather a NACHT-N. In addition, two of the HNWD genes in F. circinatum had an extra domain. Finally, in addition to the HET domain, F. circinatum orthologs of the AO404 and pin-C genes encoded an ankyrin repeat domain. This dissertation thus adds valuable information into the population structure of F. circinatum. Future studies can focus on the molecular validation of the genes involved in vegetative incompatibility by production of knock-out mutants and the bioinformatic analysis of het/vic loci of more isolates. In addition, transcriptional and genomic characterization of isolates from the same multilocus genotype will reveal the basis for their phenotypic variation.