A Multiplex RT-PCR for the Detection of Three Viruses and One Viroid Infecting Hemp

Author:

Grunwald Derrick J.1,MacWilliams Jacob2,Hackenberg Laine2,Stroschein Sydney1,Rioux Renee3,Nachappa Punya2ORCID,Ellison Shelby1ORCID

Affiliation:

1. Department of Plant and Agroecosystem Sciences, University of Wisconsin, Madison, Madison, WI 53705

2. Department of Agricultural Biology, Colorado State University, Fort Collins, CO 80523

3. Department of Plant Pathology, University of Wisconsin, Madison, Madison, WI 53705

Abstract

Hemp ( Cannabis sativa L.) is an emerging industrial crop in the United States. In recent surveys of hemp growers, disease identification and management were determined to be significant priority areas for future research. From previous studies on disease identification in Western U.S. hemp production systems, hop latent viroid (HLVd), beet curly top virus (BCTV), Cannabis sativa mitovirus 1 (CasaMV1), and citrus yellow vein associated virus (CYVaV) were detected at high incidences. In the present study, a multiplex reverse transcription PCR protocol was developed to detect this viroid and viruses from hemp leaf samples simultaneously. Using the developed protocol, the multiplex reverse transcription PCR could detect each virus and viroid specifically in 10−1-diluted cDNA. The protocol was validated on 6 hemp metavirome leaf samples from field-grown hemp grown in 2019 and 23 uncharacterized leaf samples in 2021 from Colorado. Of the samples tested, 95% tested positive for CasaMV1 in 2021. This was greater than infections of BCTV (4%), HLVd (0%), and CYVaV (0%). Coinfections accounted for 4% of samples in 2021. This method offers sensitive, specific, and fast simultaneous detection of three viruses and one viroid from hemp, contributing to large-scale pathogen-free hemp certification schemes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Funder

USDA National Institute of Food and Agriculture

U.S. Department of Agriculture

Foundation for Food and Agriculture Research

Publisher

Scientific Societies

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