A real-time PCR for detection of pathogens of anthracnose on Chinese fir using TaqMan probe targeting ApMat gene

BACKGROUND Anthracnose is one of the most widespread and destructive diseases on Chinese fir. Colletotrichum cangyuanense, Colletotrichum fructicola, Colletotrichum gloeosporioides, and Colletotrichum siamense are the causal agents of anthracnose on Chinese fir. A rapid and accurate diagnosis of different pathogens is critical for the disease management. RESULTS Phylogenetic tree and sequence similarity analysis showed that the single-locus ApMat provides superior phylogenetic information and is an appropriate target to distinguish C. cangyuanense, C. fructicola, C. gloeosporioides, and C. siamense. The real-time PCR assays with the primer sets of MQ-F/R, 1#C-F/R, YK-F/R, and WZ-F/R, and corresponding TaqMan probes of MQ-P, 1#C-P, YK-P, and WZ-P were specific for C. cangyuanense, C. fructicola, C. gloeosporioides, and C. siamense, respectively. The sensitivity tests showed that the lowest amount of gDNA that the PCRs can detect was 1 ng of genomic DNA. The validity of the assays was confirmed by directly detecting the pathogens from both the fungal culture and infected Chinese fir. CONCLUSION These results demonstrated the potential of the TaqMan real-time PCR targeting the ApMat gene to provide rapid, specific, and reliable molecular detection of C. fructicola, C. gloeosporioides, C. siamense, and C. cangyuanense, respectively. The data also provided a reference solution for the identification of species within Colletotrichum gloeosporioides species complex (CGSC), which share similar morphological characteristics. (c) 2022 Society of Chemical Industry.

Automated summary

The TaqMan real-time PCR targeting the ApMat gene has the potential to provide rapid, specific, and reliable molecular detection of C. fructicola, C. gloeosporioides, C. siamense, and C. cangyuanense. This study also offers a reference solution for identifying species within the Colletotrichum gloeosporioides species complex (CGSC) that share similar morphological characteristics.