Population biology of the tan spot pathogen of pyrethrum

Tan spot is one of the most significant foliar diseases of pyrethrum (Tanacetum cinerariifolium) in Tasmania, Australia. It is associated with tan coloured, necrotic lesions on leaves, stems and flower buds which result in a loss of green leaf area. Severe outbreaks can lead to plant death and termination of crops. Phylogenetic and morphological analysis of the causal pathogen, originally described as Microsphaeropsis tanaceti, resulted in reclassification of the pathogen as two closely related species of Didymella, namely D. tanaceti and D. rosea. Molecular differences between the two species were evident for four of the five genome regions examined. Furthermore, morphological differences in culture pigmentation and conidial size allowed rapid identification of the two species. To elucidate explanations for the shift from a minor disease in 2001 to one of the most frequent and severe diseases of pyrethrum, the reproductive strategy and population structure of the dominant tan spot pathogen, D. tanaceti were investigated. Two first harvest fields in each of two regions, were intensively sampled in July/August 2012, using two 50 m transects in each field, to provide field populations of D. tanaceti for analysis. Tan spot incidence in these fields was high, with 325 isolates obtained from 800 sampling units. Analysis of the structure and arrangement of mating-type (MAT) genes identified a putative heterothallic mode of reproduction for D. tanaceti, with either a single MAT1-1-1 or MAT1-2-1 gene occurring in D. tanaceti isolates. A multiplex MAT-specific PCR assay was developed and validated. This assay was utilised to quantify the number of D. tanaceti isolates of each MAT gene within the field populations. Isolates with a MAT1-1-1 gene occurred in equal frequencies with isolates containing a MAT1-2-1 gene. Significant spatial structure of isolates of each MAT gene in the fields was absent. Additionally, two haplotypes of the MAT1-1-1 gene, sharing 99.6% sequence homology, were identified. Isolates of the two haplotypes were differentiated using a restriction enzyme digest of the MAT1-1-1 amplicon. Within the field populations, haplotype I was dominant, occurring in 95.6% of isolates with a MAT1-1-1 gene. These results suggested that the occurrence of a cryptic sexual reproduction cycle in Tasmanian pyrethrum fields could not be dismissed. However, in vitro crosses between compatible isolates failed to produce ascospores, suggesting that specific environmental conditions and temporal requirements were necessary. To assess the genetic structure of D. tanaceti, a set of polymorphic microsatellite (SSR) markers were identified based on sequence data obtained from genomic sequencing. The D. tanaceti field populations were genotyped using eight SSR markers, with alleles determined by high resolution melt analysis. Within the 317 isolates genotyped, 127 multilocus genotypes were identified, with 82 represented by a single isolate. Furthermore, high average genetic and genotypic diversity were identified within the field populations. However, genotypic spatial structure between regions, fields and within transects in each field were absent. Similar frequencies of alleles were observed for each marker in each of the fields. Evidence of high levels of genotypic migration between fields was also detected. These results suggested that individuals within these populations may have originated from a common genetic source, such as an alternative host or seed crop. Linkage disequilibrium was detected within fields. Thus, long distance dispersal of inoculum via ascospores may have only minimally contributed to disease incidence in these fields. Control of tan spot has relied, mostly, on the use of boscalid, a succinate dehydrogenase inhibitor fungicide. Boscalid has been used widely in pyrethrum for control of multiple pathogens. To initiate the characterisation of the molecular mechanisms associated with the observed boscalid resistant phenotypes, the succinate dehydrogenase subunit B (SDHB) gene of D. tanaceti was sequenced. The SDHB gene sequences of isolates with varied in vitro growth responses to boscalid (different resistant phenotypes) were compared. The results revealed that a decreased sensitivity to boscalid was associated with the substitution of a highly conserved histidine residue at codon 277 with either tyrosine (H277Y) or arginine (H277R). These two substitutions have been shown to cause boscalid resistance in other fungal species. In addition, an isoleucine to valine (I279V) substitution occurred at codon 279, but was not correlated with a decreased sensitivity to boscalid. Both the H277Y and H277R substitutions were associated with isolates exhibiting moderately resistant (EC₅₀ 0.5 ‒ 5.0 μg a.i/mL), resistant (EC₅₀ 5.0 ‒ 50.0 μg a.i/mL) and highly resistant (EC₅₀ 50.0 ‒ 250.0 μg a.i/mL) phenotypes. No isolates with a boscalid susceptible phenotype (EC₅₀ 0.0 ‒ 0.5 μg a.i/mL) were associated with these substitutions. However, the association of isolates with a WT SDHB gene for each of the moderately resistant, resistant and highly resistant phenotypes restricted the ability to correlate the H277Y and H277R substitutions with the resistant phenotypes. This indicated that mutations in the succinate dehydrogenase subunit C (SDHC) and D (SDHD) genes, or other regions may also occur in D. tanaceti. To evaluate the extent of SDHB gene mutations in the 2012 field populations, a high resolution melt analysis assay was developed and its ability to identify mutations in codons 277 and 279 of the SDHB gene verified. The majority of D. tanaceti isolates within the field populations contained a mutation in the SDHB gene. The H277Y substitution was the most dominant, occurring in 52.3% of isolates, while the H277R substitution occurred in 9.3%. Overall this thesis has identified and characterised the pathogens associated with tan spot of pyrethrum in Australia; establishing them as two species (D. tanaceti and D. rosea) from the genus Didymella. It has developed a collection of SSR markers and assays for the rapid identification of mating-type for use in future studies. Furthermore, it has initiated the characterisation of the molecular mechanisms associated with boscalid resistance and developed an assay for the rapid identification of SDHB alleles. Moreover, it has provided a greater understanding of population biology and structure of the dominant tan spot pathogen; D. tanaceti by the characterisation of intensively sampled field populations. While the specific reasons for the rapid increase in tan spot incidence and severity remain unclear, this study has identified possible factors which could be associated with it. Despite evidence against a frequent sexual cycle in the field populations, the high genotypic diversity within populations suggests D. tanaceti individuals have a high adaptive ability. The adaptive ability of the pathogen population was demonstrated by the development of insensitivity to the fungicide boscalid. However, it may have also provided D. tanaceti individuals with a competitive advantage (e.g. increased virulence) over other pyrethrum pathogens. Furthermore, the decreased efficiency of disease control from boscalid, due to the moderately high incidence of fungicide resistance, has undoubtedly played a significant role in increasing disease incidence. Thus, efficient long term control of the disease will need to be delivered from an integrated approach, incorporating methods to decrease inoculum loads and strategies based on alternation of fungicides in different resistance groupings with regular evaluation of the pathogen population for fungicide resistance