Development and validation of a direct PCR based assay for the detection of Colletotrichum species on chili seeds

Abstract

An investigation was executed to detect Colletotrichum truncatum (synonymous C. capsici) and C. coccodes in solo or as a disease complex through direct PCR (dPCR) in anthracnose-infected chili seeds. Direct PCR was performed with C. coccodes and C. truncatum-s specific markers and Tris-EDTA buffer aliquots (obtained from infected seeds soaked up to five hours) as source of template DNA. This method efficiently and specifically detected the respective species in seeds with minimum 2.5% infection, yielding species-specific ∼500 bp (C. truncatum) and ∼340 bp (C. coccodes) fragments without any non-specific amplification with other mycoflora. Further, the seeds used in the experiment were tested for their germination efficiency along with a complete set of dried seeds as control. Among the soaked seeds, germination frequency ranged between 50 (infected seeds) to 100% (healthy seeds) without any significant loss in germination, confirming the sustainability of the current protocol. We recommend the use of direct PCR from soaked seeds without prior DNA extraction as a cost-effective and quick method for detecting the pathogen directly from infected seeds in fields.