A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of Colletotrichum capsici

Abstract

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.

Current status of Colletotrichum capsici strains, causal agents of Brown blotch disease of cowpea in Burkina Faso

“…showed that all the strains were C. capsici (Table 2). Among the strains, 92% contained the specific sequence of C. capsici (Torres-Calzada et al, 2011) and were designated as variant 1. In total, four variants of C. capsici were identified on the basis of the sequence analysis of species (Table 3).…”

“…PCR detection assay was conducted using the genus Colletotrichum specific primer CC1F1 5'ACCTAACTGTTGCTTCGGCG-3' (Chandra et al, 2009) and the species C. capsici specific primer CcapR 5'-CCCAATGCGAGACGAAATG3' (Torres-Calzada et al, 2011), which gave a specific band size of 425 bp. The PCR mixture of 25 µl contained 1 FTA disk, 5 µl of Buffer (Flexer) 5X, 0.5 µl of dNTP (10 nM), 18.42 µl of distilled H2O, 0.5 µl of each primer (5 µm), and 0.08 µl of DNA Go Taq (Promega).…”

“…In a worldwide perspective, C. capsici pathogen causes a disease commonly known as anthracnose on a wide range of *Corresponding author. E-mail: gilthiol@yahoo.fr Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License plants, including legumes, vegetables, and small fruits (Sérémé, 1999;Banerjee et al, 2007;Torres-Calzada et al, 2011). Recently, strains of C. capsici and Colletotrichum dematium f. truncatum have been described as the same species (Hyde et al, 2009) and are now considered to be synonym of Colletotrichum truncatum (Damm et al, 2009).…”

“…The internal transcribed spacer (ITS) markers have also been successfully used for detection of many species including plants, fungi and bacteria (Cros et al, 1993;Shivaprakash et al, 2011). Some ITS markers notably C.capR/C.capF and CC1F1/CC2R2 have been used for C. capsici species detection and phylogenetic analysis (Banerjee et al, 2007;Chandra et al, 2009;Torres-Calzada et al, 2011).…”

Molecular Tools for Detection of Plant Pathogenic Fungi and Fungicide Resistance

“…As recent examples, PCR methods for identification of Sclerotium rolfsii (Jeeva et al, 2010) and Colletotrichum capsici (Torres-Calzada et al, 2011) have been developed based in specific sequences of the ITS region. Improved variants of PCR have emerged with higher sensitivity, specificity and throughput and allowing the quantification of fungi in infected plants or environment.…”