Screening for fungicide resistance in orchard fruit: Biochemical and molecular analyses

Kacie Louise Quello, Purdue University

Abstract

Management of tree fruit pathogens in Indiana relies upon the application of effective fungicides that may be compromised by the development of fungicide resistance, resulting in a significant economic impact to orchard fruit production. Testing for fungicide resistance is traditionally accomplished through the use of mycelial growth assays of the pathogen and compares the growth of isolates on fungicide-amended medium relative to the growth on non-amended medium. Although this assay can conclusively identify resistance to the fungicide in question, the results cannot be quickly determined and may take several weeks for recalcitrant fungi. Such delays in the determination of fungicide sensitivity prevent growers from identifying appropriate chemical management options within the same season. Furthermore, these tests are often costly in terms of material and labor such that, when they are offered to growers as a service, they are prohibitively expensive. The purpose of this study was to develop a faster method of testing for fungicide resistance in two common pathogens of fruit crops, Monilinia fructicola that causes brown rot of stone fruit and the causal agent of apple scab, Venturia inaequalis. To test for fenbuconazole fungicide resistance in M. fructicola, we modified a medically standardized biochemical assay that uses Alamar blue, a chromogenic substrate that can be used as an indicator of respiration, in a 96-well microtiter format. We compared the AB method to traditional mycelial growth assays for resistance screening to fenbuconazole. Alamar blue can be used as a colorimetric test for fungicide resistance or quantitative tested with use of a plate reader. At this level, the isolates could be classified as resistant, shifted, or susceptible in as little as 24 hours. The situation is quite different with V. inaequalis, which cannot be readily cultured late in the field season. Thus, if fungicide resistance is identified in the summer, determination of resistance may not occur until the following year. To circumvent the problem of pathogen isolation, we developed a screen that utilizes PCR in situ to detect Topsin-M® (thiophanate-methyl) resistance. To develop this screen, V. inaequalis isolates collected from Indiana were first screened with mycelium assays for thiophanate-methyl resistance. Isolates were found to range from sensitive (no growth at 0.5 μg active ingredient (a.i) thiophanate-methyl/mL) to low resistance (growth at 0.5 μg a.i./mL but not 5 μg a.i./mL) to medium resistance (growth at 5 μg a.i./mL but not at 50 μg a.i./mL) to very high resistance (rapid growth at 50 μg a.i./mL). Concordance between known mutations in the β-tubulin gene and phenotype was determined to test the accuracy of a molecular assay. DNA was extracted from pure cultures and the β-tubulin gene was amplified and digested with restriction enzyme BstUI to verify a restriction fragment length polymorphism (RFLP) at codon 198 that corresponded to very high fungicide resistance. 69% of resistant isolates were positive for the polymorphism. Of the remaining 15 isolates that were scored as resistant but lacking the known RFLP, upon sequence analysis, we found that six of these isolates possessed a point mutation at codon 240 in the β-tubulin gene. This mutation can be differentiated by PCR-RFLP using Cac 8I. Thus, all resistant isolates could be identified with the two restriction enzyme digests. These two PCR-based RFLP detection methods could be used to rapidly detect thiophanate-methyl resistant isolates of V. inaequalis .

Degree

M.S.

Advisors

Beckerman, Purdue University.

Subject Area

Horticulture|Plant sciences

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