Simultaneous Detection of <i>Colletotrichum acutatum</i> and <i>C. gloeosporioides</i> 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 C t 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 How to cite this paper:

A SYBR Green qPCR assay for specific detection of Colletotrichum ocimi, which causes black spot of basil

“…Schena et al (2017) developed a duplex qPCR TaqMan method to detect and quantify C. godetiae and C. acutatum sensu stricto in olive tissues. An assay for the simultaneous detection of C. acutatum and C. gloeosporioides from infected strawberry leaves by real-time PCR was developed by Rahman et al (2019). Du et al (2021) elaborated a real-time PCR method to detect, quantify and monitor C. siamense infecting rubber trees, and Kamber et al (2021) developed a specific quantitative real-time TaqMan PCR assay that allowed rapid and reliable detection and quantification of C. lupini in infected seeds and plant material.…”

Colletotrichum abscissum: Detection on symptomless citrus leaves and symptomatic citrus flowers using high‐resolution melting analysis

“…acutatum and C . gloeosporioides from strawberry plants without the requirement of using a probe for pathogen detection, in contrast to qPCR (Forcelini et al, 2018; Rahman et al, 2019; Wang et al, 2021).…”

“…The highresolution melting (HRM) technique was introduced in 2002 by a collaboration between the University of Utah and industry (Reed et al, 2007) and, since then, it has been used for genetic studies in humans. In agricultural sciences, this high-throughput method was initially used for various planting breeding programmes (Lee et al, 2012(Lee et al, , 2017Taheri et al, 2017), and recently has been deployed to detect, among other species, C. acutatum and C. gloeosporioides from strawberry plants without the requirement of using a probe for pathogen detection, in contrast to qPCR (Forcelini et al, 2018;Rahman et al, 2019;Wang et al, 2021).…”