TITLE:
Simultaneous Detection of Colletotrichum acutatum and C. gloeosporioides from Quiescently Infected Strawberry Foliage by Real-Time PCR Based on High Resolution Melt Curve Analysis
AUTHORS:
Mahfuz Rahman, Tofazzal Islam, Rosemary Schwegel, Frank J. Louws
KEYWORDS:
Anthracnose, q-PCR, Quiescent Infection, Disease Prevention, Diagnostics
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.10 No.3,
March
11,
2019
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.