Development of a new methodology for the detection of Colletotrichum truncatum and Fusarium sp. in bell pepper seed

Abstract

This study was carried out towards the development of rapid PCR-based assays for detection and identification of Colletotrichum truncatum, C. gloeosporioides sensu lato and Fusarium sp. infecting green and red bell pepper seeds in Trinidad using DNA templates produced by automated grinding and robotic paramagnetic particle capture. DNA was extracted from freshly collected symptomatic green and red bell pepper seeds using Promega’s Maxwell® 16 robotic system. The PCR assay of 220 seeds harvested from field-infected bell pepper fruit compared different optimization parameters including seeds that were symptomatic and asymptomatic of infection, the relative success of two different DNA polymerase enzymes, genus-specific and species-specific primers that were pre-existing in the literature in addition to primers that were newly designed for this study and duplex PCR for the simultaneous detection of DNA templates belonging to two different fungal species. The findings indicated that seeds carried dual infections of C. truncatum and Fusarium sp. Compared to other primers that were screened, our newly designed primers, ITS219-F/ITS419-R with GoTaq® Green Master Hot Start Taq DNA Polymerase, detected C. truncatum in the highest number of symptomatic seeds of infected green and red bell pepper fruit as well as in asymptomatic seeds of infected red bell pepper fruit. Fusarium sp. was detected only in red bell pepper seeds. Duplex PCR using ITS219-F/ITS419-R and Fa/Ra primers successfully simultaneously detected and identified C. truncatum and Fusarium sp. in symptomatic seeds of infected red bell pepper fruit. None of the seeds were infected with C. gloeosporioides sensu lato which is currently considered to be a minor pathogen in bell pepper in Trinidad. PCR-based detection methods are key tools in the detection and identification of fungal plant pathogens especially where seeds are an important inoculum source. The molecular approach as optimized in this study, is faster, more sensitive and the presence of single or dual fungal infections can be assessed in one assay.