Resistance to Thiophanate-Methyl in Botrytis cinerea Isolates From Californian Vineyards and Pistachio and Pomegranate Orchards

Abstract

In this study, a mycelial growth assay was used to evaluate the sensitivity to thiophanate-methyl of 144 Botrytis cinerea isolates (collection A) from Californian vineyards and pistachio and pomegranate orchards. Based on the effective concentration that inhibits 50% of growth (EC50) values for mycelial growth inhibition on fungicide-amended media, 3, 28, 10, and 58% of the isolates showed sensitivity (SS; EC50 < 1 µg/ml), low resistance (LR; 1 < EC50 < 10 µg/ml), weak resistance (WR; 10 < EC50 < 50 µg/ml), and high resistance (HR; EC50 > 100 µg/ml) toward thiophanate-methyl, respectively. The LR and HR phenotypes were observed in pistachio and pomegranate orchards, even though pomegranate was not sprayed with thiophanate-methyl. Sensitivity to thiophanate-methyl of a historical collection of 257 B. cinerea isolates (collection B) isolated from pistachio orchards in 1992, 2005, and 2006 was assessed on potato dextrose agar amended with thiophanate-methyl at the discriminatory concentration of 10 µg/ml. Average percentages of thiophanate-methyl–resistant isolates were 50, 72, and 64% in the orchards in 1992, 2005, and 2006, respectively. A study of fitness components of selected thiophanate-methyl–resistant (LR, WR, and HR) and –sensitive (SS) isolates from collection A did not reveal any significant difference between them with respect to mycelial growth on fungicide-free media and pathogenicity on cultivar Crimson Seedless berries. Comparison of β-tubulin sequences from resistant and sensitive phenotypes revealed that a glutamic acid at position 198 was changed to alanine in all HR isolates and three LR isolates. The occurrence of thiophanate-methyl resistance in B. cinerea populations should be considered when designing spray programs against blossom and shoot blight of pistachio and gray mold of grape.

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“…The selected isolates exhibited EC50 values ranging from 0.012 to over 800 ppm for carbendazim and 0.948 to over 1280 ppm for thiophanate-methyl. Previous studies have also reported the existence of isolates with high EC50 values that can withstand high fungicide concentrations [6,10,17,18]. The mutation in the β-tubulin gene has been found to be responsible for resistance to MBC fungicides, and the effects of this mutation have been examined by several researchers [11,16,19,20].…”

“…Different levels of resistance were found in the isolates towards carbendazim and thiophanate-methyl. Avenot et al [10] reported that mutations in β-tubulin gene regions associated with high levels of Botrytis resistance to thiophanate-methyl were not related to low levels of resistance. In the present study, isolates with low levels of resistance to thiophanate-methyl in the EC50 test exhibited the pattern of sensitive isolates in PCR-RFLP.…”

“…The colony diameter was measured after a 4-day incubation in darkness at 22˚C, and the percentage of mycelial growth inhibition was calculated for each isolate. The B. cinerea isolates were then categorized as highly resistant (EC50 > 100 μg/ml), moderately resistant (50 < EC50 < 100 μg/ml), weakly resistant (10 < EC50 < 50 μg/ml), low resistant (1 < EC50 < 10 μg/ml), and sensitive (EC50 < 1 μg/ml) based on the calculated EC50 values [10].…”

“…MBC fungicides, including carbendazim, fuberidazole, benomyl, thiabendazole, and thiophanatemethyl, were some of the earliest systemic single-site specific fungicides used to control B. cinerea in the 1970s [9]. These fungicides work by binding to the β-tubulin subunit and disrupting the fungal cytoskeleton, thus inhibiting germ tube elongation, mycelial growth, and nuclear division during mitosis [9,10]. However, resistance to these fungicides emerged soon after their introduction [9].…”