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Laboratory Evaluation of Three Rapid, Agar-Based Assays to Assess Fungicide Sensitivity in Monilinia fructicola

Three rapid, agar-based assays were compared with a traditional petri dish method for assessing the sensitivity of Monilinia fructicola to propiconazole (0.3 and 2.0 μg/ml), thiophanate-methyl (1.0 and 50 μg/ml), and azoxystrobin (1.0 and 35 μg/ml) in the laboratory. The three assays were based on mycelial growth inhibition on agar disks sliced from lipbalm tubes filled with fungicide-amended potato dextrose agar (PDA), on PDA-coated cotton swabs, or in PDA-filled microcentrifuge tubes. Mycelial growth inhibition of eight previously characterized isolates (two resistant to propiconazole, two highly resistant to thiophanate-methyl, two with low levels of resistance to thiophanate-methyl, and two sensitive to all three fungicides) was determined visually 24, 48, and 72 h after inoculation. The 48-h time point was the earliest suitable time to collect data for all methods because insufficient growth was recorded in the petri dish and tube assays after 24 h. With the exception of the swab assay, all methods classified the isolates previously determined to be fungicide sensitive correctly (i.e., no fungal growth was observed for these isolates). For propiconazole-resistant isolates, the lipbalm assay resulted in levels of growth inhibition very similar to the petri dish method, whereas the swab assay and the tube assay overestimated and underestimated, respectively, the level of resistance. Both the lipbalm and the swab assays classified isolates correctly as being thiophanate-methyl resistant, and both were able to discriminate the isolates previously classified as having low versus high levels of resistance when treated with this fungicide at 50 μg/ml, as was the petri dish method. None of the eight isolates which previously were determined to be azoxystrobin sensitive grew on azoxystrobin-amended media, regardless of the assay type. Overall, the average percentage of correct isolate classifications (relative to their previously determined resistance status) on propiconazole- and thiophanate-methyl-amended media after 48 h ranged from 87.5 to 100, 85.3 to 100, 63.2 to 94.5, and 50.5 to 81.0% for the petri dish, lipbalm, swab, and tube assays, respectively. The lipbalm assay provided the most accurate assessments (85.3 to 100%) after only 24 h of incubation, supporting its use as a rapid and simple tool to monitor resistance levels in M. fructicola field populations.