Simultaneous Detection of Colletotrichum acutatum and C. gloeosporioides from Quiescently Infected Strawberry Foliage by Real-Time PCR Based on High Resolution Melt Curve Analysis

Abstract

Anthracnose of strawberry, caused primarily by the fungal pathogens belonging to Colletotrichum acutatum species complex (CASC) and C. gloeosporioides species complex (CGSC) is an economically important disease in the Southeast United States. Quiescently infected (QI) planting stock is one of the most important sources of inoculum in the fruiting field that can only be reliably detected by highly sensitive real time quantitative PCR (q-PCR) assay. In this study, a q-PCR assay was developed and optimized that can discriminate anthracnose fruit rot (AFR) and anthracnose crown rot (ACR) causing species based on the difference in post PCR melting temperatures of amplicons. Controlled environment grown plants artificially inoculated with different levels of CASC and CGSC showed a significant (P < 0.001) correlation with levels of quantification expressed by Ct values in q-PCR from petioles and leaf blades. The leaf blade was a significantly larger reservoir of QI than that of the petiole. Both TaqMan and SYBR Green assay showed similar sensitivity and specificity. Detection of QI on leaves at young middle and older stages from inoculation with same number of conidia indicated that middle aged leaves were the best for assessing QI. Quantification of QI from middle aged leaf samples from a strawberry fruiting field that has been planted with pre-inoculated plants at both ends of rows and let inoculum spread showed higher sensitivity and precision by q-PCR compared to that of a traditional paraquat assay. The assay developed and validated in this study offers a new tool for evaluating planting stocks for QI to make decision on preventative control for strawberry anthracnose.