Development and validation of a quantitative PCR assay method to assess relative resistance of winter wheat to dwarf bunt at early growth stages

Dwarf bunt, caused by Tilletia controversa, is a major biotic constraint and grain contaminant in winter wheat (Triticum aestivum L.) production. The conventional approach for evaluating dwarf bunt resistance in wheat cannot be conducted until maturity. Hence, there is a need to develop a method to determine host resistance at an earlier growth stage. A quantitative polymerase chain reaction (qPCR) assay was developed for the quantification of T. controversa biomass in wheat plants that correlates fungal DNA (fDNA) content in the host tissue with host resistance. A previously developed pathogen primer–probe set and host primer pairs as well as a new host probe were used in this study. The respective primer–probe sets were specific to T. controversa and wheat, respectively. The qPCR assay amplified as little as 0.05 pg of fDNA. The assay was validated in field evaluations conducted in a dwarf bunt nursery in Logan, UT, using susceptible and resistant wheat varieties. The assay detected fDNA in both susceptible varieties at all growth stages. In the resistant varieties, fDNA was detected in the first leaves of all varieties, but only a single plant of the resistant variety Juniper exhibited fDNA at the third leaf stage. There was no fDNA detection in plants beyond the third leaf in any of the resistant varieties. These results established the proof of concept that the qPCR technique is rapid, highly sensitive, and easily applicable for the evaluation of dwarf bunt resistance in wheat at an earlier growth stage and may significantly reduce the time required to develop resistant varieties compared to the conventional method.