Diversity and Resistance to Thiophanate-Methyl of Colletotrichum spp. in Strawberry Nursery and the Development of Rapid Detection Using LAMP Method

Abstract

Anthracnose is a devastating fungal disease in strawberry nurseries. Multiple Colletotrichum species are responsible for strawberry anthracnose. In this study, 105 Colletotrichum isolates were obtained from strawberry seedlings with anthracnose symptoms in fifteen nurseries located in Zhejiang province, China, and were classified based on multilocus sequence and morphological characteristic analyses. Analysis of ITS, ACT, CAL, CHS-1, and GAPDH revealed that four species within C. gloeosporioides species complex, including C. siamense (56 isolates, 53.3%), C. fructicola (37 isolates, 35.2%), C. gloeosporioides (7 isolates, 6.7%), and C. aenigma (5 isolates, 4.8%), were detected in diseased seedlings. Thiophanate-methyl is one of the benzimidazole fungicides, and has long been used to control strawberry anthracnose in China. Here, thiophanate-methyl resistance of Colletotrichum isolates was determined by the minimum inhibitory concentration (MIC) method. Our results indicated that the resistance frequency was up to 96.2%, containing 94.3% of highly resistant isolates. Only four sensitive isolates (two C. fructicola, one C. gloeosporioides, and one C. siamense isolates) and two moderately resistant isolates (one C. aenigma isolate and one C. siamense isolate) were detected. Our data indicated that the high resistance was mainly caused by the E198A mutation in the β-tubulin protein. In addition, F200Y (TTC→TAC) in the β-tubulin protein were detected in two moderately resistant isolates. Based on the point mutation at codon 198 (GAG→GCG) in the β-tubulin gene of Colletotrichum isolates, we developed a loop-mediated isothermal amplification (LAMP) assay to rapidly detect the E198A mutants. Collectively, our study indicated that four species within the C. gloeosporioides species complex were associated with anthracnose symptoms in strawberry nurseries in Zhejiang province, and serious resistance was widespread in each Colletotrichum species.

Species of the Colletotrichum spp., the Causal Agents of Leaf Spot on European Hornbeam (Carpinus betulus)

“…Five conventional genes (ACT, CHS-1, GAPDH, ITS, and TUB2), four specific genes (ApMat, CAL, and GS), and one additional specific gene (APN2) were used in this study, and four species in this study were separated from the C. gloeosporioides species complex. The combined phylogenetic tree was consistent with trees presented in other studies [61,[70][71][72].…”

Multi-Locus Phylogenetic Analysis Revealed the Association of Six Colletotrichum Species with Anthracnose Disease of Coffee (Coffea arabica L.) in Saudi Arabia

“…In contrast, only a single isolate of C. aeschynomenes was recovered. The presence of six species of Colletotrichum associated with anthracnose disease on coffee indicates that more than one Colletotrichum species can colonize a single host, which is consistent with the conclusion of previous studies [16,19,25,27,55]. The compositions of Colletotrichum species from coffee appeared to differ according to the geographical origin, host, and species complex.…”

Development of a colorimetric loop‐mediated isothermal amplification (LAMP) assay to facilitate monitoring of carbendazim‐resistant isolates of Colletotrichum truncatum based on the β‐tubulin E198A mutation

“…The newly developed LAMP assay detected the highly resistant (HR) isolates after 40 min incubation at 67°C, excluding the process of extracting the DNA and preparing the reaction mixture. Compared with the previously developed LAMP assays, the assay developed in this study is quicker by at least 10 min (Duan et al., 2015; Fan et al., 2019; Liu et al., 2019; Vielba‐Fernández et al., 2019; Wu et al., 2022; Yuan et al., 2022). The difference is caused by the additional loop primers (LF and LB), which hybridize to the stem‐loops of the LAMP products, except for the loops that are hybridized by the inner primers (FIP and BIP) (Nagamine et al., 2002).…”

“…The emergence of fungicide-resistant isolates in fungal populations resulting in ineffective disease control is one of the most significant problems in crop production (Deising et al, 2008). Such a problem not only directly causes crop losses but also increases costs for Fan et al, 2019;Liu et al, 2019;Vielba-Fernández et al, 2019;Wu et al, 2022;Yuan et al, 2022). The difference is caused by the additional loop primers (LF and LB), which hybridize to the stem-loops of the LAMP products, except for the loops that are hybridized by the inner primers (FIP and BIP) (Nagamine et al, 2002).…”