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are hard to distinguish by morphology even though the ability of these species to produce fumonisins differs by several orders of magnitude. We developed a diagnostic PCR that rapidly identifies the most common species occurring on wheat (Waalwijk et al., 2003). Primer sets that specifically amplify target sequences from one of F. graminearum, F. cul-morum, F. avenaceum, F. poae, F. proliferatum, and the non-toxigenic species M. nivale var. majus and Microdochium nivale var. nivale were combined into a multiplex PCR containing 14 primers. We used this primer mixture to survey five geographic regions in the Netherlands in 2000 and 2001 (Waalwijk et al, 2003; Table 2).

Fusarium graminearum was clearly the dominant species. In surveys made in the 1980s and 1990s, F. culmorum was the most common species, with occasionally high levels of F. avenaceum detected as well (de Nijs et al., 1997). This difference suggests that a dramatic shift has occurred in the composition of the FHB complex in The Netherlands during the last decade. There is no clear cause for this shift, but increased production of maize may play an important role. As F. graminearum also is a pathogen on maize, rotations of maize with wheat allow the fungus to cycle from one host to the other. F. culmorum also can be found on maize in temperate climates (Munkvold et al., 1998) but inoculum of F. culmorum is less likely to increase in the absence of wheat. Alternatively, Gibbe-rella zeae, the teleomorph of F. graminearum, could affect inoculum pressure since it produces ascospores that are capable of traveling long distances (Fernando et al., 1997; Mal-donado-Ramirez and Bergstrom, 2001). Indeed, mutants of F. graminearum that cannot produce ascospores also cause less disease (Brown et al., 2001b). Whether this results from a direct effect of the disruption on the mating capacity remains to be elucidated, since disruption of the mating type gene mat1-2 in F. verticillioides also affects the expression of many genes (Waalwijk et al., 2006; Keszthelyi et al., 2007). A third possibility is that F. graminearum has a higher temperature optimum for growth and sporulation than does F. culmorum (Doohan et al., 2003). Thus, the increased incidence of F. graminearum might be an indicator of climate change occurring in Europe. However, in a comparative study of Dutch and Italian strains of both species both F. culmorum and F. graminearum had the same temperature requirements (Köhl et al., unpublished).

The diagnostic multiplex PCR described above also was used for an inventory of isolates collected from wheat in China and France in 2002. All the French isolates had a PCR fragment unique for F. graminearum, but none of the Chinese isolates had this fragment. Instead 156/172 isolates had a fragment that was ~ 30 bp longer. This fragment was pre-

NL - 2000 France-2002

NL - 2000 France-2002

Figure 1. Distribution of chemotypes of F. graminearum lineage 6 in a single wheat field in Hubei province in China and F. graminearum lineage 7 in France and in two consecutive years in The Netherlands. Sizes of the circles are proportional to the size of the samples.

viously identified from Chinese isolates (Waalwijk et al., 2003) that belong to F. graminearum lineage 6. The remaining 16 Chinese isolates produced the diagnostic M. nivale var. majus fragment (Waalwijk et al., 2003).

Individual F. graminearum isolates produce either deoxynivalenol (DON) or nivalenol (NIV). The molecular basis for the production of these related trichothecenes has been studied extensively and the ability to produce either DON or NIV depends upon a mutation in one of the genes involved in the toxin biosynthetic pathway (Brown et al., 2001a, 2002; Lee et al., 2001, 2002). The tri7 and tri13 genes have been used by several groups to differentiate between DON- and NIV-producing strains of F. graminearum (Jennings et al., 2004a,b; Waalwijk et al., 2003). In the Netherlands we observed 76% and 68% DON producers in F. graminearum in 2000 and 2001, respectively. DON producers could be subdivided into those that produce 15-AcDON or 3-AcDON in an ~ 4:1 ratio (Fig. 1). A similar survey in the United Kingdom, that used specific primers to identify NIV, 15-AcDON and 3-AcDON found that 25% of the F. graminearum isolates and 43% of the F. culmorum isolates had the NIV chemotype (Jennings et al., 2004a,b). The regular increase in the propor tion of NIV producers observed in the United Kingdom, is consistent with a hypothesis that NIV producers might be becoming more dominant (Waalwijk et al., 2003).

Quantitative detection of the Fusarium Head Blight complex

The multiplex detection tool is very useful for screening isolates recovered from the field, but it is still too time consuming for large scale epidemiological studies. We developed a quantitative real-time PCR assay (Waalwijk et al., 2004a) based on TaqMan technology. For each of the common members of the FHB species complex, the fragments from the multiplex analysis were sequenced and these sequences used for the design of additional

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0 SOD 1(100 1500 2000

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