Development of a translation elongation factor 1-alpha (TEF) based TaqMan qPCR assay for Diaporthe humulicola, the causal agent of halo blight of hop.

Halo blight of hop, caused by Diaporthe humulicola, was first described in 2018 and is a major concern for growers in the eastern United States and Canada. This pathogen can cause quality and yield losses by desiccating hop cones, leading to shatter. However, traditional disease diagnosis is time-consuming, with morphological features taking up to 30 days to develop in culture. To address this issue, a quantitative polymerase chain reaction (qPCR) assay based on the translation elongation factor 1-alpha (TEF) gene was developed. We assessed capabilities and limitations of this assay for detection of D. humulicola in plant tissue and investigated aspects of the disease through: (1) testing of hop rhizomes for the presence of fungal pathogens; (2) determining the time required to detect D. humulicola in detached hop leaves; and (3) comparing plating methods with the qPCR assay to monitor D. humulicola in a hop yard. The limit of detection for the assay was 100 fg/µl of DNA. The assay showed no cross-reactivity with other hop pathogens, endophytes, or other Diaporthe species tested. Detection of D. humulicola occurred one day after inoculation. The assay detected D. humulicola in both asymptomatic and symptomatic rhizome tissue, but further investigation is required to determine the cause of the observed symptoms. The assay successfully detected the pathogen in individual hop cones and inflorescences throughout the season, with higher positive identification rates than culture-based assays. This assay will provide time-limited diagnosticians a tool for detection of D. humulicola.