P-208 - Development of a quantitative PCR assay for the detection and quantification of Xylaria necrophora infesting soybean

Abstract Text: Xylaria necrophora (Xn) is a pathogenic fungus that causes taproot decline (TRD), a disease of increasing importance in soybean. The fungus can infect soybean all season, causing symptoms ranging from seed rot and seedling death to yellow chlorotic foliar symptoms, stunting, and significant necrosis of roots. Current identification relies primarily on symptom, culture morphology, and phylogenetic characterization. These methods are time-consuming, enforcing the need for an improved diagnostic assay. This study developed a TaqMan-based qPCR assay targeting the Xn ITS region for quantification of fungal DNA in soybean roots. Primers and probe design were based on comparisons of 227 publicly available ITS sequences within the Xylariaceae family. The detection limit of this assay was determined to be 10 fg/μl of Xn genomic DNA, both alone and when multiplexed with a soybean internal control. The assay showed no cross-reactivity with 12 other soybean root pathogens, nor with seven tested Xylariaceae species. Using this assay, we successfully detected Xn in eight cover crop varieties following inoculation experiments. Additionally, we confirmed the successful detection of the pathogen in soybean root field samples. We are currently cross-validating the assay with other research and diagnostic labs. This assay provides a rapid and reliable diagnostic tool for detecting Xn in planta and may help determine better strategies to manage TRD.