Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/19/2008
Publication Date: 4/2/2008
Citation: Baird, R., Abbas, H.K., Windham, G.L., Williams, W.P., Baird, S., Ma, P., Kelley, R., Hawkins, L.K., Scruggs, M. 2008. Identification of select fumonisin forming Fusarium spp. using PCR applications of the polyketide syntase gene and its relationship to fumonisin production in vitro. International Journal of Molecular Sciences. 9:554-570. Interpretive Summary: Fusarium verticilliodes is a fungus commonly found inside stalks and ears of corn grown in the United States. This fungus may or may not cause visible damage to corn plants. However, under certain conditions, this fungus can produce a group of toxins named fumonisins which cause a variety of health problems in humans and livestock. To determine if a specific strain of this fungus can produce fumonisins, it must be grown in culture media for 7-10 days. Our study was conducted to develop a rapid diagnostic technique that could be used to determine if a specific strain of F. verticilliodes produces fumonisins. Techniques were evaluated that could identify fumonisin-producing fungi by looking at their DNA. A technique was found that could accurately identify fungi that produce fumonisins over a 24 hour period. This technique will help researchers study fungal movement and growth plants. It will also help researchers develop corn plants that prevent development of fumonisins.
Technical Abstract: A polymerase chain reaction (PCR)-based diagnostic assay was used to develop markers for detection of Fusarium verticillioides, a fumonisin forming fungus in maize tissues. Species-specific primers were designed based on sequence data from the polyketide synthase (PKS) gene (FUM5) responsible for fumonisin production in fungi. Four sets of oligonucleotide primers were tested for their specificity using 27 isolates of F. verticillioides (=F. moniliforme), 13 F. proliferatum, and 16 other Fusarium spp. In addition, 14 species of related taxa or ones from the same niche as F. verticillioides were tested as negative controls. Among the four sets, primer set B consistently amplified a 419-bp fragment from the DNA for almost all isolates of F. verticillioides, 69% of F. proliferatum, and one isolate of F. sporotrichioides. All other fungi tested were negative for the PCR amplification product. Furthermore, only 38% of the F. verticillioides isolates tested formed fumonisin in culture on a defined medium which enhances toxin production. Fumonisin production in culture did not correspond to positive PCR amplification using primer set B. PCR detection of as little as 100 pg/µl was consistent, but at 10 pg/µl results varied. The system described in this study requires only 1 day for completion compared to 10 days for cultural work and morphological determination. Based on results of this study, future research will now be conducted to determine variety resistance or movement of F. verticillioides within the maize plants.