Design of species-specific PCR method for the detection of pathogen Embellisia astragali in standing milk vetch seeds.

UNLABELLED: Embellisia astragali is an important seedborne pathogenic fungus responsible for yellow stunt and root rot of standing milk vetch (Astragalus adsurgens). The current detection methods that are based on culture and morphological identification are time-consuming, laborious and not always reliable. A PCR-based diagnostic method was developed with the species-specific primer pairs AatpF and AatpR designed from the sequence of the plasma membrane ATPase gene of E. astragali. The specificity of the primers was verified by PCR analysis of DNA from three strains of E. astragali and 19 isolates of nontarget fungi from other genera. A single 135-bp amplicon was detected only from E. astragali isolates, and no cross-reactions were observed with any other tested isolates. The detection limit of this new method was 5 pg of template DNA. The specific primers enabled the detection of E. astragali from the seed of standing milk vetch. This species-specific PCR method provides quick, simple, powerful and reliable detection of E. astragali. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-based detection is fast, convenient, precise and sensitive compared to the traditional methods of pathogen detection. This study develops the first PCR method for the detection of Embellisia astragali in standing milk vetch seeds. The species-specific primer set designed from the plasma membrane ATPase gene of E. astragali can detect the pathogen. This assay could be applied in the standing milk vetch seed industry.