Detection and identification of six Monilinia spp. causing brown rot using TaqMan real-time PCR from pure cultures and infected apple fruit

Authors: WANG, JING-RU - China Agricultural University; GUO, LI-YUN - China Agricultural University; Xiao, Chang-Lin; ZHU, XIAO-QIONG - China Agricultural University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/12/2018
Publication Date: 6/25/2018
Citation: Wang, J., Guo, L., Xiao, C., Zhu, X. 2018. Detection and identification of six Monilinia spp. causing brown rot using TaqMan real-time PCR from pure cultures and infected apple fruit. Plant Disease. 102(8):1527-1533. https://doi.org/10.1094/PDIS-10-17-1662-RE.
DOI: https://doi.org/10.1094/PDIS-10-17-1662-RE

Interpretive Summary: Brown rot is a major fungal disease of stone fruits and pome fruits, resulting in pre- and postharvest losses. This disease is caused by six closely related Monilinia species. The distribution of these six species is geographically dependent, and as a result, some of these species are quarantine pathogens in certain countries. Accurate detection and identification of these fungi is critical to international trade. Traditional isolation and morphology-based identification of these fungi is time consuming and requires specialized microbiological skills. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. These DNA-based molecular assays can accurately detect target fungal pathogens from infected fruits and identify themto the species level.

Technical Abstract: Brown rot is a severe disease affecting stone and pome fruits. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, Monilinia laxa, Monilinia fructigena, Monilia polystroma, Monilia mumecola and Monilia yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for M. fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase 2 genes. Additionally, according to the sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limit of 10–100 fg DNA, which is equivalent to 1–7 conidia. The assays were also able to detect the target pathogens in the mixed DNA samples comprising all six Monilinia spp. and related species. Moreover, the real-time PCR assays accurately detected target fungi from infected apple fruits. Furthermore, the results were consistent with those of the traditional identification method.