A SYBR Green qPCR Method for Detecting and Quantifying Spores of Colletotrichum acutatum and C. gloeosporioides Species Complexes Causing Ripe Rot of Grape

Abstract

Four species belonging to Colletotrichum acutatum and Colletotrichum gloeosporioides species complexes, including C. aenigma, C. fioriniae, C. fructicola, and C. nymphaeae, were identified from ripe rot symptomatic fruit from Mid-Atlantic vineyards. A quantitative real-time PCR (qPCR) SYBR Green method was developed to detect and quantify conidia of the two Colletotrichum complexes to better understand the distribution and the extent of Colletotrichum inoculum. Primers were designed to specifically target the β-tubulin gene of the C. acutatum complex or the C. gloeosporioides complex and were found to be specific to the desired complex, not amplifying DNA of other prevalent fungi from vineyards. The sensitivity of the primers was also assessed, and the C. acutatum complex-specific and C. gloeosporioides complex-specific primers were able to quantify as little as 50 C. fioriniae conidia and 100 C. aenigma conidia, respectively. Therefore, this would be a cost-effective, specific, and sensitive detection and quantification method for these two destructive species complexes in vineyards and many other crops through spore trapping applications.