Management of brown rot of stone fruit relies upon the application of effective fungicides that may be compromised by the development of fungicide resistance. We evaluated fungicide resistance in the brown rot pathogen, Monilinia fructicola, using Alamar blue (AB) dye, or resazurin, 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 using 10 isolates of M. fructicola that represented a range of sensitivities to fenbuconazole. Using traditional mycelial growth assays, isolate sensitivity ranged from 17.7 to 115.3% growth on medium amended with fenbuconazole at 0.03 μg/ml relative to that on nonamended medium. Concordant results between both assays were obtained (R2 = 0.9943, P < 0.0001), but the AB method provided results within 24 h, as opposed to the 3- to 5-day period required for mycelial growth assays. We found that sensitive isolates reduced AB less than resistant isolates in the presence of fungicide. Spore density influenced the reduction of AB by M. fructicola; spectrophotometric discrimination of fungicide sensitivity was best achieved at a density of 105 spores/ml.

Venturia inaequalis, the causal agent of apple scab, infects both commercial apples and ornamental crabapples. We found four classes of benzimidazole fungicide sensitivity in the Indiana population: sensitive (S) isolates unable to grow on 0.5 μg active ingredient (a.i.)/ml; low resistant (LR) isolates that grew at 0.5 μg a.i./ml, but not at 5 μg a.i./ml; moderately resistant (MR) isolates that grew at 5 μg a.i./ml, but not at 50 μg a.i./ml; and very highly resistant (VHR) isolates that grew rapidly at 50 μg a.i./ml. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of the β-tubulin gene with two restriction enzymes, BstUI and Cac8I, enabled us to rapidly identify benzimidazole resistance among all tested isolates. Sixty-nine percent of the resistant isolates tested possessed the BstUI RFLP at codon 198 that corresponds to VHR, and the remaining LR and MR isolates possessed the Cac8I RFLP corresponding to a newly identified resistance allele at codon L240F. Combined, PCR-RFLP correctly identified the resistance status of all isolates tested to date. The preponderance of benzimidazole-resistant isolates from commercial apple orchards and their absence in the landscape on ornamental crabapple suggests that two distinct populations of V. inaequalis coexist in Indiana.