A loss of fungicide efficacy, particularly for carbendazim, was noted in soybean fields in Thailand and was considered to be due to the development of Colletotrichum truncatum resistance. The carbendazim sensitivity of C. truncatum populations isolated from various soybean fields in Thailand was thus evaluated with in vitro sensitivity assays and molecular characterization of mutations in the sequences of the ß2‐tubulin (TUB2) gene that confer carbendazim resistance in the pathogen. Among 52 isolates, 46 isolates were classified as highly resistant (HR) to carbendazim (EC50 > 1,000 µg/ml). All HR isolates grew on PDA amended with carbendazim at 1,000 µg/ml. Six isolates were classified as carbendazim sensitive (S) (EC50 < 1 µg/ml). Mycelial growth on PDA amended with 1 µg/ml carbendazim was inhibited by over 50% compared with growth on PDA alone. When a partial TUB2 gene from the isolates was amplified and analysed using predicted amino acid sequences, an alteration from glutamic acid to alanine at codon 198 (E198A) was found in 45 HR isolates for which the EC50 was higher than 2000 µg/ml. This mutation resulted from a nucleotide substitution from adenine to cytosine (GAG → GCG). The other HR isolate, CtPhS_1, with EC50 of 1,127 µg/ml, had an alteration at codon 200 (F200Y) (TTC → TAC).
In Thailand, four systemic fungicides, carbendazim (Car), azoxystrobin (Azo), difenoconazole (Dif), and penthiopyrad (Pen), are commonly used to control soybean anthracnose caused by Colletotrichum truncatum, but the pathogen has developed resistance. From 2019 to 2020, fungicide resistance in C. truncatum from fields in Chiang Rai and Chiang Mai was monitored. In tests of 85 C. truncatum isolates for resistance to multiple fungicides, 15.3% were CarRAzoR, 34.1% were triple- (CarRAzoRDifR or CarRAzoRPenR), and 50.6% were CarRAzoRDifRPenR. Surprisingly, all isolates tested had lost their sensitivity to one or more of the fungicides tested. The carbendazim-resistant isolates carried a point mutation in the β-tubulin (TUB2) gene at codon 198 (E198A) or 200 (F200Y), and all azoxystrobin-resistant isolates had a mutation in the cytochrome b (cytb) gene at codon 143 (G143A) or 129 (F129L). Moreover, a novel mutation at codon 208 (S208Y) in the gene encoding succinate dehydrogenase subunit B (sdhB) was detected in all of the isolates highly resistant to penthiopyrad. No mutation linked with difenoconazole resistance was detected in the genes encoding cytochrome P450 sterol 14-demethylase (CYP51A and B). To the best of our knowledge, this is the first report of C. truncatum isolates resistant to multiple fungicides and serves as a warning to take measures to prevent the occurrence and distribution of these multiple-fungicide-resistant populations in soybean fields.
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