The evaluation of multiplex PCR and DNA profiling methods (DGGE and SSCP) for the detection of mycotoxigenic Fusarium species

Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2008.

The genus Fusarium is an important plant pathogen that is responsible for severe yield losses of economically important plant species. In addition, some Fusarium species also produces mycotoxins. There is thus a need for the early detection and identification of Fusarium to prevent yield losses and mycotoxin contamination and consumption. The present study investigated the potential of a multiplex PCR-DGGE and SSCP method for the detection, identification and differentiation of Fusarium species. Two extraction methods, i.e. the CTAB-PVP and E.Z.N.A. Fungal DNA Mini Kit were tested for the isolation of fungal DNA. The DNA Mini Kit showed the best results to successfully isolate DNA from the various Fusarium species that was amplifiable with the beta-tubulin, EF-la, 18S and FUM primer sets. Optimized amplification conditions were tested and applied for each primer set. EF-la, FUM and 18S primer sets were combined in multiplex PCR because they yielded amplification products of distinct sizes. However, preferential amplification of the 18S rDNA region occurred with this combination. Therefore, multiplex PCR was performed with the EF-la and FUM primer sets which permitted the detection of fumonisin positive Fusarium species. Subsequent DGGE and SSCP analysis of the EF-la fragments from the multiplex PCR showed that DGGE was not sufficient in discriminating between the Fusarium isolates while SSCP permitted clear differentiation. However, multiple banding patterns for a single species were observed with both profiling methods. This can impede interpretation of results and may also lead to wrong conclusions. Limits of detection were also determined for fumonisin producing Fusarium species individually and in combination with non-Fusarium species through conventional and real-time PCR. The real-time PCR method proved to be more sensitive in detecting small amounts of fungal DNA than conventional PCR. The sensitivity and accuracy of this method would allow the quantification of toxigenic Fusarium species in contaminated soil and plant tissues. As a result, proper control and management strategies can be executed in time to prevent the occurrence of devastating diseases and yield losses. Sequences of the beta-tubulin and EF-la genes were analyzed to determine phylogenetic relationships between various the Fusarium isolates. Sequencing of the amplified fragments indicated conflict between GenBank and MRC/PPRI identities for several Fusarium isolates. This conflict was observed for both protein-coding genes. Phylogenetic relationships between the various Fusarium isolates were more accurate with the EF-la gene sequences than the beta-tubulin sequences. This study demonstrated the potential of a multiplex PCR-SSCP method to detect and identify Fusarium species. With further careful optimization, this technique can be applied to contaminated food and feed samples to assess Fusarium diversity.

Masters