A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels

摘要

Aims: To develop a real-time PCR assay to quantify Fusarium graminearum biomass in blighted wheat kernels.Methods and Results: Primers designed to amplify a gene in the trichothecene biosynthetic cluster (TRI6) were evaluated for sensitivity and specificity. Primer pair Tri6_10F/Tri6_4R specifically and consistently amplified a 245-bp DNA fragment from F. graminearum. A workflow was developed and validated to extract DNA from infested grain. The assay detected as little as 10 mu g of F. graminearum mycelia in 1 g of ground wheat grain with a high correlation between fungal biomass and cycle threshold values (R-2 = 0.9912; P = 0.004). In field-inoculated grain, qPCR measurements of biomass correlated closely with deoxynivalenol levels (R = 0.82, P < 0.0001) and two visual techniques to assess grain quality (R = 0.88, P < 0.0001 and R = 0.81, P < 0.0001).Conclusions: The qPCR assay provided accurate and precise assessments of the amount of F. graminearum biomass in blighted wheat kernels. This method represents a technical advance over other approaches to quantify kernel colonization and real-time PCR detection methodologies for F. graminearum that do not correlate quantification of fungal genomic DNA to biomass.Significance and Impact of the Study: Quantifying F. graminearum biomass, especially low levels of growth associated with kernels that are visually asymptomatic, represents a new approach to screen for resistance to kernel infection, an understudied yet potentially important avenue to reduce the impact of head blight.