Species diversity in Colletotrichum causing anthracnose on Lamiaceae and SYBR Green qPCR assay for the species-specific detection of C. ocimi.

Species of Colletotrichum are considered important plant pathogens, saprobes, and endophytes on a wide range of host plants. In this study, the occurrence, diversity and pathogenicity of Colletotrichum spp. associated with aromatic and ornamental plants belonging to the Lamiaceae family was investigated. A total of 19 Colletotrichum isolates were collected from symptomatic leaves and seeds of Ocimum basilicum (basil), Origanum vulgare (oregano), Salvia leucantha, S. nemorosa and S. greggii. A multi-locus phylogeny was established based on four genomic loci (ITS, gapdh, act and tub2) and the virulence of representative isolates was tested. Colletotrichum fioriniae, C. bryonicola and C. fructicola were found in association with Salvia leucantha, S. nemorosa and S. greggii, respectively. Colletotrichum nigrum was isolated from S. greggii. Colletotrichum fioriniae and C. ocimi were responsible for causing leaf anthracnose of oregano and basil, respectively. All the tested isolates were pathogenic and C. ocimi revealed the most virulent species along with C. fioriniae. Considering the results obtained and the economic importance of basil in the Mediterranean area, a SYBR Green real-time PCR assay was developed to detect Colletotrichum ocimi in basil leaves and seeds, based on the partial β-tubulin (tub2) gene sequence. The real-time PCR assay was validated for analytical specificity, sensitivity, selectivity, repeatability and reproducibility. The assay was specific for C. ocimi with respect to ten Colletotrichum spp. and to further 12 fungal pathogens of basil. Sensitivity was 1 pg μL-1 of genomic fungal DNA and amplification analyses were not influenced by basil genomic DNA. The assay detected and quantified C. ocimi in artificially inoculated basil leaves and seeds. The pathogen detection in seeds was achieved, however an optimization of the extraction method in necessary. Specific primers to detect C. ocimi were obtained and the validated tool was demonstrated as specific for its target organism.