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.
Highlights
Fungicide resistance in populations of fungal plant pathogens has three phases: emergence, selection, and adjustment [1]
The objectives of the present study were to: (i) assess genomic variation of S. sclerotiorum isolates exposed to long-term sublethal doses of fungicides in vitro using simple sequence repeat (SSR) and amplified fragment length polymorphisms (AFLPs) markers, (ii) estimate the affect of sublethal stress on mutation rates at SSR loci, and (iii) determine in vitro trends of effective concentration of fungicides required for 50% growth inhibition (EC50) over time in S. sclerotiorum isolates exposed to sublethal doses of fungicides
All control isolates were from the first experiment and depicted as ‘Con’ followed by isolate name and generation (G0 or G12). 4A) Original number of alleles detected for each isolate. 4B) Number of alleles present after censoring AFLP data by removing loci polymorphic from G0 to G12 in the control for each isolate, resulting in the same multilocus genotype for the non-exposed control of each isolate
Summary
Fungicide resistance in populations of fungal plant pathogens has three phases: emergence, selection, and adjustment [1]. Once the resistant population has reached an intermediate level in the adjustment phase, a change in fungicide dose or mode of action is needed in order to achieve disease control. Research on bacterial pathogens of humans and livestock animals suggests sublethal antibiotic exposure plays an important role in antibiotic resistance emergence via horizontal gene transfer, recombination, and both random and non-random mutations [4,5]. A clear picture is emerging that exposure to sublethal fungicides may increase mutation rates, a pre-requisite to resistance emergence, and may serve as heretofore unexplored source of population genetic variation of importance for primarily clonal fungal plant pathogens
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
