Identification of Select Fumonisin Forming Fusarium Species Using PCR Applications of the Polyketide Synthase Gene and its Relationship to Fumonisin Production in vitro

Abstract

A polymerase chain reaction (PCR) based diagnostic assay was used to develop markers for detection of Fusarium verticillioides (=F. moniliforme), a fumonisin producing fungus in maize tissues. Species-specific primers were designed based on sequence data from the polyketide synthase (PKS) gene (FUM1-previously FUM5) responsible for fumonisin production in fungi. Four sets of oligonucleotide primers were tested for their specificity using 24 strains of F. verticillioides, 10 F. proliferatum, and 12 of other Fusarium species. In addition, 13 species of other fungal genera, from four phyla, were tested as negative controls. Among the four sets, primer set B consistently amplified a 419-bp fragment from the DNA 96% of all F. verticillioides strains and 83% of F. proliferatum. All other fungi tested were negative using primer set B. A total of 38% of the F. verticillioides strains grown on a selective liquid medium produced fumonisin and 92% formed the toxin on standard rice medium. When fumonisin formed in culture, PCR assay using primer set B detected every strain of F. verticillioides, but only amplified 80% of F. proliferatum strains that produced the toxin. PCR detection was consistent at 100 pg/µl concentration of genomic DNA from 4 F. verticillioides strains, but varied at 10 pg/µl. Two duplicate greenhouse tests using artificially inoculated maize plants, had greater levels of F. Int. J. Mol. Sci. 2008, 9555 verticillioides detected after re-evaluting using primer set B than from culturing of the tissues. The molecular protocols described in this study requires only 1 day for completion compared to approximately 10 days for cultural work and morphological determination. In conclusion, conventional PCR assay using primer set B provides a sensitive and accurate detection assay that can be used as a primary or secondary confirmation method for identification and occurrence of F. verticillioides within the maize tissues. However, studies using primer set B for fumonisin production determined by strains of F. verticillioides and F. proliferatum will require further verification.

Population Structure of the Gibberella fujikuroi Species Complex Associated with Rice and Corn in Korea

“…The presence of FUM1, by PCR amplification, was used to narrow down the fungal isolates to be studied here from the Fusarium collections initially selected based only on colony characteristics. However, it cannot validate the species identification within the G. fujikuroi species complex because no species-specific amplification of FUM1 has been achieved (Baird et al, 2008;Jurado et al, 2010). Moreover, the usefulness of the species-specific primers sets used in this study was limited because they showed no specificity in some isolates (data not shown), as described previously (Kang et al, 2011).…”

Fusarium spp. Contamination of Wheat, Maize, Soybean, and Pea Grain in Croatia

“…Theoretically, Fusarium mycotoxins can be produced on every plant and all plant parts infested by toxigenic Fusarium species. Over the last ten years, detection of mycotoxins and toxigenic Fusarium species has advanced signifi cantly with the development of PCR-based techniques (13)(14)(15)(16). However these techniques are still not widely used because they are expensive.…”

Isolation and characterization of Fusarium verticillioides from maize in eastern China

“…PCR using species-specific primers ( Table 1) was used to identify the principal Fusarium species of maize (e.g., F. verticillioides, F. proliferatum, F. subglutinans, and F. graminearum). PCR using the FUM5F and FUM6R primer pair was performed to determine whether or not the FUM1 gene was present (Baird et al 2008). PCR, using the Gfmat1a/Gfmat1b and Gfmat2c/Gfmat2d primer pairs, was used to identify the mating type (MAT-1 or −2) of F. verticillioides strains (Steenkamp et al 2000).…”