Quantitative Detection of Fusarium Species in Wheat Using TaqMan

Abstract

Fusarium head blight (FHB) of wheat and other small-grain cereals is a disease complex caused by several fungal species. To monitor and quantify the major species in the FHB complex during the growing season, real-time PCR was developed. TaqMan primers and probes were designed that showed high specificity for Fusarium avenaceum, F. culmorum, F. graminearum, F. poae and Microdochium nivale var. majus. Inclusion of an internal PCR control and serial dilutions of pure genomic DNAs allowed accurate determination of the concentration of fungal DNA for each of these species in leaves, ears as well as harvested grains of winter wheat. The DNA concentration of F. graminearum in grain samples correlated (r 2 = 0.7917) with the incidence of this species on the grain as determined by isolation from individual kernels. Application of the TaqMan technology to field samples collected in 40 wheat crops in the Netherlands during the growing season of 2001 revealed that M. nivale var. majus predominated on leaves early in the season (GS 45–65). Ears and harvested grains from the same fields, however, showed F. graminearum as the major species. In 2002, grain samples from 40 Dutch fields showed a much wider range of species, whereas in ears from 29 wheat crops in France, F. graminearum was the predominant species. The concentration of DON correlated equally well with the incidence of the DON-producing species F. culmorum and F. graminearum in the grain samples (r 2 = 0.8232) as well as with total DNA of both these species (r 2 = 0.8259). The Fusarium TaqMan technology is an important tool to quantify and monitor the dynamics of individual species of the complex causing FHB in cereals during the growing season. This versatile tool has been applied in a comparison of different genotypes, but can also be applied to other disease management systems, e.g. fungicide treatments.