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

Authors: HATLEN, ROSS - Michigan State University; SZYMANSKI, SHAY - Michigan State University; Adair, Nanci; FAN, QIURONG - Michigan State University; PANWAR, POOJA - Virginia Tech; SYSAK, ROGER - Michigan State University; MILES, LAURA - Michigan State University; HIGGINS, DOUG - Virginia Tech; ROJAS, J - (NCE, CECR)networks Of Centres Of Exellence Of Canada, Centres Of Excellence For Commercilization A; Gent, David; MILES, TIMOTHY - Michigan State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/30/2025
Publication Date: 5/5/2025
Citation: Hatlen, R., Szymanski, S.L., Adair, N.L., Fan, Q., Panwar, P., Sysak, R., Miles, L.A., Higgins, D.S., Rojas, J.A., Gent, D.H., Miles, T.D. 2025. Development of a translation elongation factor 1-alpha (TEF) based TaqMan qPCR assay for Diaporthe humulicola, the causal agent of halo blight on hop. Plant Disease. https://doi.org/10.1094/PDIS-11-24-2331-SR.
DOI: https://doi.org/10.1094/PDIS-11-24-2331-SR

Interpretive Summary: Halo blight is a potentially damaging disease of hop that was recently described in eastern North America. Current methods for diagnosing halo blight are time consuming and cumbersome. In this research, we developed a molecular diagnostic assay for the halo blight fungus. The assay is quick to apply, detected all isolates of the halo blight fungus that were tested, and is more sensitive than culture-based methods. Testing of the assay in multiple laboratories found that it had acceptable performance characteristics when transferred to other users. This assay will help to speed diagnosis of and research on halo blight.

Technical Abstract: Halo blight of hop, caused by the fungus 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 culture-based morphological features taking up to 30 days to develop, thus reducing the amount of time growers have to make management decisions. To address this issue, a quantitative polymerase chain reaction (qPCR) assay based on the translation elongation factor 1-alpha gene was developed. We assessed this assay for direct detection of D. humulicola in plant tissue and investigated aspects of the disease cycle through three distinct experiments: 1) detection of D. humulicola in hop rhizomes to determine the colonization range of the pathogen, 2) determining how quickly can D. humulicola be detected in hop leaves post inoculation, and 3) monitoring the presence of D. humulicola in cones in a hop yard and comparing isolation methods and the assay. The limit of detection for the assay was 100 fg/µl of DNA. The assay showed no cross-reactivity with other hop pathogens or endophytes, nor with other Diaporthe species tested. Detection of D. humulicola occurred one day after inoculation. The assay detected D. humulicola in both apparently healthy and diseased 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, surpassing the culture-based method in positive identification rates. This assay will provide time-limited diagnosticians a tool for detection of D. humulicola.