Abstract
Seed health tests are performed to prevent sowing untreated seeds with problematic pathogen levels. The detection of internal seedborne pathogens like Ustilago nuda, causing loose smut in barley (Hordeum vulgare), is challenging because symptoms only appear when teliospores replace barley inflorescences and smutted ears develop. Currently, U. nuda seed infection levels are determined from the visual inspections of extracted embryos or fields of plants used for seed production, both of which are laborious and can be unreliable. To improve U. nuda detection, we developed a multiplex qPCR method targeting U. nuda and H. vulgare DNA. Naturally infected seed lots were tested using this qPCR method and the visual analysis of embryos. We grew the same seed lots in the field over two seasons and used the observed smutted ears as the reference infection level. The qPCR results, U. nuda DNA normalized to H. vulgare DNA, better correlated with observed field infections than the number of infected embryos. Our qPCR method more accurately distinguished seed lots with infections above and below the field tolerance threshold. It offers a reliable alternative to the visual analysis of extracted embryos. The integration of our qPCR method with field observations can enhance U. nuda management and reduce unnecessary prophylactic seed treatments, thereby bolstering integrated pest management strategies.