Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum

Abstract

Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50–100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment, and when repeated, only one isolate had higher EC50 while most isolates showed no difference. Results of this support the hypothesis that sublethal fungicide stress increases mutation rates in a largely clonal plant pathogen under in vitro conditions. Collectively, this work will aid our understanding how non-lethal fungicide exposure may affect genomic variation, which may be an important mechanism of novel trait emergence, adaptation, and evolution for clonal organisms.

Temporal Genetic Differentiation ofCercospora beticolaPopulations in New York Table Beet Fields

“…A study of the pathogen Sclerotinia sclerotiorum demonstrated that with a sublethal fungicide selection pressure, estimated mutation rates in six microsatellite markers were 1.7-to 60-fold higher than expected under neutral conditions (Amaradasa and Everhart 2016). Field 2 was maintained using conventional practices (fungicidetreated seed, in-furrow fungicide applications for pocket rot control, and two to three foliar fungicides applications [Reiners et al 2017]) and it may be expected that a subset of the field population would be exposed to sublethal fungicide levels.…”

Identification and application of exogenous dsRNA confers plant protection against Sclerotinia sclerotiorum and Botrytis cinerea

“…Current methods to control S . sclerotiorum infection involve the application of broad-spectrum fungicides, which have been attributed to the rise of chemical resistance and may have deleterious or unwanted effects on the surrounding agro-ecological landscape, if managed poorly 3 – 5 . Other practices, such as crop rotations, can be ineffective due to the formation of overwintering structures, termed sclerotia, which can remain in the soil for several years 6 .…”

Candida albicansgenetic background influences mean and heterogeneity of drug responses and genome stability during evolution to fluconazole

“…Here, consistent patterns among strains are lacking: of the five strains backgrounds that were exposed to a subinhibitory level of fluconazole, no significant changes were observed among replicates from three of them (A1, A12, A18), MIC 50 decreased in all replicates from one replicate (A20) , and variable, yet numerically significant, changes occured in replicates of another strain (A5). This variability in phenotypic outcomes is reminiscent of the case in Sclerotinia sclerotiorum, a plant pathogenic fungus, where both MIC increases and decreases (and no change) were observed and no consistent relationship was found between the change in resistance and exposure to sublethal concentrations of five different antifungal drugs (67) . Fungal strains exposed to subinhibitory drug levels thus seem less likely than bacteria to gain an advantage at higher drug levels.…”