A multiplex RT-PCR for the detection of three viruses and one viroid infecting hemp

Abstract

Hemp (Cannabis sativa L.) is an emerging industrial crop in the US. 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 US 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-polymerase chain reaction (RT-PCR) protocol was developed to detect these viruses and viroid from hemp leaf samples simultaneously. Using the developed protocol, the multiplex RT-PCR could detect each virus and viroid specifically in 10-1-diluted cDNA. The protocol was validated on six 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.