Multiplex PCR-based detection of potential fumonisin-producing Fusarium in traditional African vegetables

Culture-independent methods employed in fungal genetic studies using in vitro amplification (PCR) and analysis of specific genes or gene fragments have proved to be useful for detection, identification, and molecular taxonomy of various plant pathogens including Fusarium spp. This approach may be faster than culture-dependent methods, and could especially be of value for the rapid detection of slow-growing toxin-producing species in food samples. The present study was aimed at the development and evaluation of multiplex PCR-based methods for the detection and identification of potential fumonisin-producing Fusarium spp. in traditional morogo-leafy vegetables supplementing the maize-based staple diet of rural communities in South Africa. In these rural subsistence settings, some morogo plants grow as weeds in maize fields where they might become contaminated with potential fumonisin-producing Fusarium strains before being collected for consumption. Substances released by senescent vegetables could induce toxin production during storage. Using fumonisin-positive MRC Fusarium verticillioides strains as reference cultures, the following primer sets for the detection of specific gene fragments in fumonisin-positive Fusarium spp. were evaluated: (i) the conserved transcription elongation factor gene (EF-1), (ii) the FUM1 gene encoding polyketide synthase for fumonisin B1 production, and (iii) 18S rRNA gene. Preliminary results indicated that, these DNA fragments were amplified from MRC reference strains as well as Fusarium spp. isolated from morogo. The annealing temperature for the multiplex PCR was 55 degrees C and each reaction contained 25 pmol of each of the primer sets EF and FUM1 and 12.5 pmol of the 18S primer set. The detection limit of the individual primers was up to 1 ng and for the multiplex up to 10 ng. This demonstrates the potential of this method for the detection of potential fumonisin-positive strains.