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.

中文翻译：

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开发和验证定量 PCR 检测方法，以评估冬小麦在生长早期对矮秆短矮秆短稚的相对抗性


由 Tilletia controversa 引起的矮小短鸫是冬小麦 （Triticum aestivum L.） 生产中的主要生物限制和谷物污染物。评估小麦矮矮短纤维抗性的常规方法在成熟之前不能进行。因此，需要开发一种方法来确定早期生长阶段的宿主抗性。开发了一种定量聚合酶链反应 （qPCR） 测定法，用于定量小麦植株中的 T. controversa 生物量，该测定法将宿主组织中的真菌 DNA （fDNA） 含量与宿主抗性相关联。本研究使用了先前开发的病原体引物-探针组和宿主引物对以及新的宿主探针。相应的引物-探针组分别对 T. controversa 和小麦具有特异性。qPCR 检测扩增的 fDNA 低至 0.05 pg。该测定在犹他州洛根的矮小短鸡苗圃中使用易感和抗性小麦品种进行的田间评估中得到了验证。该测定在所有生长阶段都检测到两种易感品种的 fDNA。在抗性品种中，在所有品种的第一片叶子中检测到 fDNA，但只有一株抗性品种杜松在第三叶期表现出 fDNA。在任何抗性品种中，第三片叶子以外的植物中均未检测到 fDNA。这些结果证明了 qPCR 技术快速、高灵敏度且易于适用于评估小麦早期生长阶段的矮缩短棍抗性，与传统方法相比，可能显着减少开发抗性品种所需的时间。