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Figure 3. PCR amplification of F. verticillioides and F. proliferatum isolates by using primers designed to bind to one of the conserved regions of fuml. Lanes 1 and 3, fumonisin non-producing isolates of F. verticillioides Gf11 and Gf8 respectively (20); lanes 2, 6 and 7 fu-monisin-producing F. verticillioides isolates Gf20, ITEM 2142 (FGSC 7600, A-0149) and ITEM 2143 (FGSC 7603, A-0999) and lanes 4, 5 and 8, fumonisin-producing isolates of F. proliferatum ITEM 2287, ITEM 2400 and ITEM 2148 (FGSC 7614, D-4853).

Figure 3. PCR amplification of F. verticillioides and F. proliferatum isolates by using primers designed to bind to one of the conserved regions of fuml. Lanes 1 and 3, fumonisin non-producing isolates of F. verticillioides Gf11 and Gf8 respectively (20); lanes 2, 6 and 7 fu-monisin-producing F. verticillioides isolates Gf20, ITEM 2142 (FGSC 7600, A-0149) and ITEM 2143 (FGSC 7603, A-0999) and lanes 4, 5 and 8, fumonisin-producing isolates of F. proliferatum ITEM 2287, ITEM 2400 and ITEM 2148 (FGSC 7614, D-4853).

Figure 4. Schematic representation of the regions flanking the fumonisin gene cluster in fu-monisin-producing isolates of F. verticillioides and F. proliferatum and the corresponding region from some non-producers of F. verticillioides. The genomic sequences of F. graminearum and F. verticillioides (Broad Institute, 2005) were used to align the gene clusters. Synteny in the flanking regions of both species extends for > 300 kb (data not shown).

Figure 4. Schematic representation of the regions flanking the fumonisin gene cluster in fu-monisin-producing isolates of F. verticillioides and F. proliferatum and the corresponding region from some non-producers of F. verticillioides. The genomic sequences of F. graminearum and F. verticillioides (Broad Institute, 2005) were used to align the gene clusters. Synteny in the flanking regions of both species extends for > 300 kb (data not shown).

strains that can and cannot produce fumonisins, unlike those based on sequences not directly related to the biosynthetic gene cluster (Mirete et al., 2003; Bluhm et al., 2004).

The structure and organization of the gene cluster in both species is very similar except for the flanking regions (Waalwijk et al., 2004b). In F. verticillioides, the gene cluster is flanked by stretches of DNA that are contiguous in F. graminearum. In F. proliferatum, the gene cluster is adjacent to a region that resides in a very different part of the F. graminea-rum genome (Fig. 4). These differences suggest that the fumonisin gene cluster might be (part of) a mobile genetic element that can insert in and excise from various locations in the genome. Fumonisin nonproducing isolates of F. verticillioides could have lost the entire fumonisin gene cluster. We tested this hypothesis with primers designed to amplify the flanking regions with complete homology in F. graminearum. In F. graminearum these primers produced an amplicon of 5.8 kb and in some non-fumonisin producing isolates of F. verticillioides the fragment is 12 kb in length. The distance between the primers in fumoni-sin-producing strains of F. verticillioides, however, is > 55 kb which prevents amplification by PCR, and in these strains no amplification product results.

Homologous sequences of the fuml gene were identified by comparing the published sequences of the F. verticillioides and F. proliferatum genes with the corresponding regions in F. globosum and F. nygamai. These sequences were used to design a set of primers and a fluorescent probe for a Taqman assay that was then tested on a series of Fusarium species known to occur on maize. All isolates of F. verticillioides, F. proliferatum, F. globosum and F. nygamai were detected in this assay, but no signal was obtained for any of the tested strains of F. equiseti, F. graminearum, F. oxysporum, F. semitectum or F. subglutinans, suggesting that this PCR is diagnostic for fumonisin-producing Fusarium species.

Conclusions

Multiplex PCR is a powerful method to characterize the composition of disease complexes such as the FHB complex of wheat, and pink or red ear rot of maize. The emergence of new species in a complex can be identified easily as isolates that do not have fragments from the previously known species. The only requirement for including a species in the multiplex system seems to be the design of a species-specific amplicon of unique size, since combining multiple primers has not yet been limiting. Quantitative PCR allows the study of population dynamics of plant pathogens during the growing season, their survival on crop residues, and the competition between species within a disease complex, as well as monitoring the effects of agronomic measures such as fungicide treatments.

Acknowledgments

We thank Dr. M.-T. Gonzalez-Jaen (University of Madrid, Spain) for providing the non-producing isolates of F. verticillioides, Prof. W. F. O. Marasas (PROMEC, Capetown, South Africa) for providing strains of F. globosum and F. nygamai, and Drs. K. O'Donnell and T. Ward (USDA, Peoria, IL, USA) for sequence analyses of the Chinese, Dutch and French isolates of F. graminearum sensu lato. Xiude Xu was supported by a grant from the China Scholarship Council.

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