So

0 500 1000 1500 2000

DON (pph'l

Figure 2. Top. Linear correlation (y = 0.71x + 147; r2 = 0.82) between the concentration of DON and the total amount of DNA of DON producing species (F. graminearum and F. cul-morum). Bottom. The correlation (y = 19.3ln(x) - 89.7; r2 = 0.82) between the DON concentration in field samples and the number of DON-producing colonies among 100 kernels from the corresponding samples.

0 500 1000 1500 2000

DON (pph'l

Figure 2. Top. Linear correlation (y = 0.71x + 147; r2 = 0.82) between the concentration of DON and the total amount of DNA of DON producing species (F. graminearum and F. cul-morum). Bottom. The correlation (y = 19.3ln(x) - 89.7; r2 = 0.82) between the DON concentration in field samples and the number of DON-producing colonies among 100 kernels from the corresponding samples.

primers and internal probes. The assay also includes an internal standard that provides a control for the presence of inhibitors of the PCR reaction. Different probes are labeled with fluorescent dyes that fluoresce at different wave lengths, and the amount of fluorescence observed is a measure of the amount of DNA replication that has occurred. A serial dilution of DNA from a pure culture of F. graminearum had a high correlation (r2 = 0.99) with the threshold cycle, and a dynamic range of four orders of magnitude (Waalwijk et al., 2004a). Similar values were obtained for the other species and we could quantify accurately the amount of DNA from F. graminearum, F. culmorum, F. avenaceum, F. poae, F. prolifera-tum and M. nivale var. majus between 0.9 pg and 9000 pg per reaction. In some cases we could detect the subsequent dilution of 0.09 pg, but these results were not consistent. The genome size of the PH-1 sequenced isolate of F. graminearum is estimated as 36 Mb (Broad Institute, 2005), which equals 0.04 pg, so the detection limit of the F. graminearum TaqMan assay we developed is ~ 5 genome equivalents (Waalwijk et al., 2004a).

These quantitative PCRs were used to study fungal population dynamics during the growing season in different geographic areas in the Netherlands. Although there were regional differences, the lower leaves contained primarily M. nivale var. majus at levels ranging from zero to 40 pg/mg sample (dry weight). However, in wheat heads from the same fields collected at growth stages 78 through 89 M. nivale var. majus was hardly detectable (< 3 pg/mg sample). Instead, F. graminearum dominated with F. avenaceum and/or F. culmorum present in some of the samples. The dominance of F. graminearum was even more striking at harvest with some samples containing up to 300 pg/mg sample. We validated the methodology by making similar analyses in the Netherlands in 2002, when most of the fields had a much broader spectrum of Fusarium species. In some fields nearly equal amounts of all five species were found at levels between 50 and 150 pg/mg of sample. In samples from France, the situation was dramatically different as F. graminearum was present almost exclusively in most samples at levels up to 22,000 pg/mg of sample (Waalwijk et al., 2004a). The DON concentrations of a series of field samples also were compared with the combined quantities in these samples of F. graminearum and F. culmorum (as these are the DON producers in the FHB complex). The combined DNA quantities for F. graminearum and F. culmorum were correlated r = 0.82) with DON concentration (Fig. 2). A similar correlation (r = 0.82) was observed between DON concentration and the total number of F. graminearum and F. culmorum colonies, as determined by plating 100 kernels per sample and morphological characterization of the colonies present (Fig. 2).

Recently, we also used quantitative PCR to evaluate the survival of species from the FHB complex in the field after harvest. The population dynamics of Fusarium spp. in crop residues of wheat left in the field after harvest was monitored by sampling at regular intervals, from harvest until mid-anthesis of the next season. We found that the fungal biomass of each of the species decreased significantly in nodes and internodes, but not in residues of stem bases, suggesting that stem bases are a major source of inoculum for the succeeding crop. At harvest, only 14% and 1% of the biomass of F. avenaceum or F. graminearum, respectively, found in haulm tissues was present in stem bases (Köhl et al., unpublished). After field exposure for ten months, more than 85% of the biomass of the two species present in the haulm residues was found in the stem bases. Such knowledge of population dynamics of the head blight pathogens in different plant tissues is being used to develop preventive agronomic measures such as stubble treatments or soil tillage aimed at the reduction of inoculum sources of head blight pathogens.

Quantitative detection of Fusarium species in maize

Some of the Fusarium species that occur in maize consist in part of species also found on wheat (Table 1) and the quantitative tools we developed for these species can be directly employed in maize and maize products. Both F. graminearum and, to a lesser extent, F. culmorum can occur on maize (Munkvold et al., 1998), but the greatest concern in maize is the presence of F. verticillioides and F. proliferatum because of their capacity to produce fumonisins primarily fumonisin B1. These mycotoxins are associated with high incidences of esophageal cancer in Southern Africa, certain provinces in China (Chu and Li, 1994) and some areas in Italy and Iran (Shephard et al., 2000). In most maize growing areas F. verti-cillioides is by far the most common species and the fungus may be living as an endophyte in maize (Miller, 2001). The subtle balance between plant and fungus can be disturbed by biotic and/or abiotic stresses, including insect damage and drought stress.

We developed a TaqMan PCR assay quantification for fumonisin-producing fungi that is based on one of the genes in the toxin biosynthetic pathway. Fumonisin biosynthesis requires at least 15 genes that are organized in a gene cluster found in the genome of both F. verticillioides and F. proliferatum (Proctor et al., 2003; Waalwijk et al., 2004b). Unlike the trichothecene toxins produced by F. graminearum on wheat, disruption of the ability to produce fumonisins does not reduce the virulence of the nonproducing strains (Desjardins and Plattner, 2000). A few naturally occurring nonproducing strains have been identified, and we included them in our analyses. The first gene in the fumonisin biosynthetic pathway is fum1, which encodes a polyketide synthase that is member of a family of PKS proteins. A conserved domain in this protein was used to design primers that can amplify DNA from both F. verticillioides and F. proliferatum. Fumonisin nonproducing isolates of F. verticillioides however, are negative in this test (Fig. 3). This PCR can discriminate between

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