Quantification of Fusarium culmorum in crown rot resistant and susceptible wheat varieties using a tri5 gene-based real-time PCR assay

The tri5 gene of F. culmorum encodes trichodiene synthase, a product that is 5necessary for trichothecene synthesis. The tri5 gene can be used in a real-time PCR assay to quantify the amount of F. culmorum infection in plant tissue. The objective of this research was to quantify the amount of F. culmorum infection in resistant and susceptible wheat varieties to establish a procedure for quantification of infection for future microarray studies. Seedlings of Australian wheat varieties with varying levels of resistance to F. pseudo-graminearum '2-49','Sunco', and 'Gala', and the highly susceptible variety 'Puseas' were inoculated with a single germinating spore of F. culmorum isolate NRRL 3288. Total genomic DNA was extracted from control and inoculated seedling leaf tissue after seven days and assayed using real-time PCR with Gzeae87T primers, Taqman registered probe, and the Cepheid SmartCycler registered II system. Results of the real-time assay showed that the tri5 gene sequence could be detected in both resistant and susceptible inoculated wheat seedlings, but not in control seedlings.