Quantitative detection and monitoring of Colletotrichum acutatum in strawberry leaves using real‐time PCR

Abstract

Real-time PCR assays for Colletotrichum acutatum , one of the most important pathogens of strawberry worldwide, were developed using primers designed to the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) and the β -tubulin 2 gene. Using TaqMan technology, the ITS-based assay could reliably detect as little as 50 fg genomic DNA, 100 copies of target DNA, or 25 conidia. The β -tubulin-based assay was c . 66 times less sensitive, and therefore less suitable for detection purposes. The TaqMan-ITS assay recognized all C. acutatum isolates tested from various intraspecific molecular groups, while no amplification was observed with several other Colletotrichum species or other strawberry pathogens, indicating the specificity of this assay. Detection and quantification of C. acutatum was demonstrated in artificially and naturally infected strawberry leaves. First, C. acutatum was detected in plant mixes of which only 0·001% of the tissue was infected by C. acutatum . Secondly, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allowed monitoring of growth progression of C. acutatum . This real-time PCR-mediated monitoring of the pathogen was well-correlated with microscopic data, and confirmed that leaf age may play a role in the extent of C. acutatum infection. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material.

The Colletotrichum acutatum species complex

“…The closest match in a blastn search with the TUB2 sequence of CBS 607.94 (with 97 % identity, 13 bp differences) was Ga. acutata isolate PCF 459 (EU635504) from strawberry in Belgium (Debode et al 2009). With the GAPDH sequence of CBS 607.94 no match closer than 87 % identity was found.…”

Use of QuantitativePCRDetection Methods to Study Biocontrol Agents and Phytopathogenic Fungi and Oomycetes in Environmental Samples

“…Similarly, Colletotrichum acutatum was detected by qPCR in strawberry leaves 2 h after inoculation, whereas the first symptoms of the disease appeared after 96 h (Debode et al. ). Phytophthora cryptogea was detected by qPCR 4 days earlier than cPCR and 6 days before the appearance of disease symptoms on gerbera plants (Li et al.…”

Effect of fungicides on epiphytic yeasts associated with strawberry

“…Management of this disease is greatly hindered by the difficulty in detection and control of this fungus during symptomless infections on strawberry leaves and unripe fruit (Debode et al. ; Van Hemelrijck et al. ; Guidarelli et al.…”