Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans.

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

Independent breakdown events of the Brassica napus Rlm7 resistance gene including via the off-target impact of a dual-specificity avirulence interaction.

Protection of many crops is achieved through the use of genetic resistance. Leptosphaeria maculans, the causal agent of blackleg disease of Brassica napus, has emerged as a model for understanding gene-for-gene interactions that occur between plants and pathogens. Whilst many of the characterized avirulence effector genes interact with a single resistance gene in the host, the AvrLm4-7 avirulence gene is recognized by two resistance genes, Rlm4 and Rlm7. Here, we report the "breakdown" of the Rlm7 resistance gene in Australia, under two different field conditions. The first, and more typical, breakdown probably resulted from widescale use of Rlm7-containing cultivars whereby selection has led to an increase of individuals in the L. maculans population that have undergone repeat-induced point (RIP) mutations at the AvrLm4-7 locus. This has rendered the AvrLm4-7 gene ineffective and therefore these isolates have become virulent towards both Rlm4 and Rlm7. The second, more atypical, situation was the widescale use of Rlm4 cultivars. Whilst a single-nucleotide polymorphism is the more common mechanism of virulence towards Rlm4, in this field situation, RIP mutations have been selected leading to the breakdown of resistance for both Rlm4 and Rlm7. This is an example of a resistance gene being rendered ineffective without having grown cultivars with the corresponding resistance gene due to the dual specificity of the avirulence gene. These findings highlight the value of pathogen surveillance in the context of expanded knowledge about potential complexities for Avr-R interactions for the deployment of appropriate resistance gene strategies.

Evolution of virulence in fungal plant pathogens: exploiting fungal genomics to control plant disease.

The propensity of a fungal pathogen to evolve virulence depends on features of its biology (e.g. mode of reproduction) and of its genome (e.g. amount of repetitive DNA). Populations of Leptosphaeria maculans, a pathogen of Brassica napus (canola), can evolve and overcome disease resistance bred into canola within three years of commercial release of a cultivar. Avirulence effector genes are key fungal genes that are complementary to resistance genes. In L. maculans these genes are embedded within inactivated transposable elements in genomic regions where they are readily mutated or deleted. The risk of resistance breakdown in the field can be minimised by monitoring disease severity of canola cultivars and virulence of fungal populations using high throughput molecular assays and by sowing canola cultivars with different resistance genes in subsequent years. This strategy has been exploited to avert yield losses due to blackleg disease in Australia.