Steps to improve molecular detection of barley loose smut (Ustilago nuda) in seedlings

Agricultural policies encourage a reduction of synthetic chemical plant protection products (PPPs). Cereal seeds are often prophylactically treated to prevent seed-borne diseases, including the internally located barley loose smut (Ustilago nuda). Cultivar resistance or biological seed treatments can contribute to chemical PPP reduction, but evaluating the effectiveness of new varieties or treatments can be cumbersome. U. nuda infections are symptomless in seeds and plants until its teliospores develop at emergence. Previously described detection methods are laborious and/or yield a non-target product. We investigated steps to improve U. nuda detection by optimizing seedling growth and qPCR conditions, including using filter paper as a substrate, growing the seedling without light, shortening the growth time, and testing new primers. The protocol alterations were tested on seedlings with either a low or high U. nuda infection level. Untreated, infected seed was directly used for the high infection rate. To reduce the infection, the same seed was treated with warm water, an effective U. nuda treatment. The less laborious seedling growth conditions, including on filter paper and without light, seems to yield more U. nuda DNA than growth in soil. Preliminary results suggest that the seedling growth period can be reduced. The newly designed COX3 primers appeared to be more sensitive than the ITS primers. Using the optimized seedling growth procedures and COX3 as the target gene can potentially streamline the molecular detection of U. nuda in seedlings, which in turn, can aid in the evaluation of new seed treatments or varieties.