Development of colorimetric loop-mediated isothermal amplification (LAMP) assay for detecting the azoxystrobin-resistant isolates in Colletotrichum truncatum

Anthracnose, mainly caused by Colletotrichum truncatum, is a serious threat to soybean production worldwide. Azoxystrobin, a quinone outside inhibitor (QoI) fungicide, has long been frequently used in Thailand. As expected, its performance has decreased as the pathogen has developed fungicide resistance, mainly due to mutations in the target cytochrome b (cytb) gene. The substitution from glycine to alanine at codon 143 (G143A) is related to the azoxystrobin-completely resistant phenotype of C. truncatum. We thus used the G143A mutation as the reaction marker to develop a loop-mediated isothermal amplification (LAMP) method combined with hydroxynaphthol blue (HNB) for visual, simple, and rapid detection of azoxystrobin-resistant isolates of C. truncatum. Among four sets of primers designed, primer sets 1, 2, and 4 distinguished the isolate carrying the G143A mutation from wild-type isolate. Optimum conditions for the LAMP assay using primer set 4 were 63 °C for 80 min with a visible detection limit of 1 ng. Moreover, the LAMP assay was highly specific and accurate in tests of 125 isolates of C. truncatum from 13 fields in northern Thailand. This LAMP assay is highly efficient, simple, accurate for rapid field monitoring of azoxystrobin resistance in C. truncatum and implementing effective control measures.