Hemp (Cannabis sativa L.) is a multifaceted crop, sourcing communities with food, fiber and medicinal properties (Schluttenhofer and Yuan, 2017). With the passage of the 2014 Farm Bill, hemp is no longer a controlled substance and is now a legal agricultural crop in the U.S. Section 7606 (Legitimacy of Industrial Hemp Research) provided a formal definition of the crop as “the plant Cannabis sativa L. and any part of such plant, whether growing or not, with a delta-9 tetrahydrocannabinol concentration of not more than 0.3% on a dry weight basis.” Currently, 46 U.S. states have passed laws to define the crop and remove barriers to its production. In 2020, hemp was produced on 336,655 acres with 13,475 grower licenses across 34 states, and 46 states with active hemp program and 41 tribes with approved USDA plans, according to “U.S. Hemp Report,” by the organization Vote Hemp (www.votehemp.com). This is a more than 300% increase since 2018 licensed acreage. Colorado, the leading state in hemp production in the new era, increased production from 4,873 licensed acres in 2017 to 36,225 licensed acres in 2020. With the current resurgence of hemp as a crop to be produced within the U.S. there are many challenges with associated pests and diseases that are essentially undescribed, as are the management strategies. Disease identification and management is an increasing challenge for hemp farmers across the country. As production increases, the crop diversifies, and in turn legitimizes, the emergence of viral diseases and their spread is imminent (Fike, 2016).

There are over 100 pathogens that affect hemp with potential to cause economic damage. These include fungal, bacterial, viral, and nematode species that affect hemp during production (Mcpartland, 1994, 1996, 1999; Mcpartland and Cubeta, 1997; Mcpartland et al., 2000). A recent review summarizes important pathogens affecting the cannabis and industrial hemp production in North America (Punja, 2021). The earliest reports of viral syndromes affecting hemp were described as hemp streak virus (HSV) (Röder, 1941) and hemp mosaic virus (HMV) described in 1958 (Ceapoiu, 1958); however, the causal agents of these diseases have yet to be isolated and characterized. Other viruses that are known to infect hemp are alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), potato virus X (PVX), tomato ringspot virus (TomRSV), potato virus Y (PVY), broad bean wilt virus (BBWV), arabis mosaic virus (ArMV), and raspberry ringspot virus (RpRSV) (Kegler and Spaar, 1997). More recently, cannabis cryptic virus (CanCV) was isolated from hemp plants (Ziegler et al., 2012; Righetti et al., 2018). In addition, hemp was found to be infected with hop latent viroid (HLVd) in California (Bektaş et al., 2019; Warren et al., 2019). A survey of cannabis farms in Israel detected the presence of lettuce chlorosis virus (LCV) in plants showing leaf yellowing, interveinal chlorosis, that are typically associated with general nutrient deficiency (Hadad et al., 2019). Diagnosis of hemp viruses is challenging due to the lack of research that characterizes symptomology and transmission mechanisms of known viruses and novel viruses. Next generation sequencing (NGS) technologies circumvent many of these problems and allows for the characterization of complete genomes from known or novel viruses (Villamor et al., 2019). Indeed a diversity of virus and viroid communities were identified in hemp that were previously unknown using NGS (Nachappa et al., 2020).

In 2019, beet curly top virus (BCTV) was found infecting hemp plants in Colorado (Giladi et al., 2020) and the virus was also detected in Arizona (Hu et al., 2021). Beet curly top virus is a type member of the genus Curtovirus in the family Geminiviridae (Chen and Gilbertson, 2016). Curly top disease is one of the most economically-important diseases for sugar beet production in the western United States; in addition, BCTV has resulted in yield losses in vegetable crops such as tomato, pepper, spinach, cucurbits and common bean (Chen and Gilbertson, 2016). The virus is transmitted only by the beet leafhopper, Circulifer tenellus Baker (Hemiptera: Cicadellidae) in a circulative non-propagative manner (Bennett, 1971) (reviewed in Chen and Gilbertson, 2016). There are 11 strains of BCTV [California/Logan (CA/Logan), Colorado (CO), Kimberly1 (Kim1), Leafhopper 71 (LH71), Mild (Mld), Pepper curly top (PeCT), Pepper yellow dwarf (PeYD), Severe (Svr), Severe pepper (SvrPep), Spinach curly top (SpCT), and Worland (Wor)] and recent isolates sequenced from sugar beet and tomato added the Kim1 and LH71 strains (reviewed in Chen and Gilbertson, 2016; Strausbaugh et al., 2017). The occurrence of the various strains of BCTV varies over time by geographic region and are often observed as co-infections (Strausbaugh et al., 2017; Creamer, 2020).

The objective of this study is to understand diversity and distribution of BCTV, and to analyze the presence of established and emerging virus/viroid communities in hemp in Colorado using NGS analysis. The information obtained in this study will aid in the development of accurate detection methods and effective virus and vector management strategies to minimize disease incidence and spread.

One of biggest challenges facing the hemp industry is detection and management of viral diseases (Nachappa et al., 2020; Punja, 2021). As hemp acreage increases across the U.S., across the U.S., additional viruses and viroids will likely be found in hemp that can potentially impact the crop, but also serve as a reservoir for disease spread to other economically important crops in the vicinity. In the current study, we assessed the diversity and distribution of BCTV affecting hemp in Colorado using PCR analysis. As a further attempt to identify previously known viruses/viroids in hemp, we also performed NGS analysis.

A survey of symptomatic hemp tissue samples from different stages from outdoor productions across 12 counties revealed high incidence (81%) of BCTV in Colorado. Growers reported disease incidence levels above 90% in some cases and disease severity reaching >50% of leaf area in infected plants. This led to diminished crop yield, including low quality of the flower and overall stunted growth (John House, hemp grower personal communication). The symptoms observed were variable among plants, and disease progression differed across farms (Nachappa et al., 2020). Given the broad host range of the virus and the vector (reviewed in Chen and Gilbertson, 2016), it is possible that curly top disease may become of the most serious disease affecting hemp production. There are 11 BCTV strains and the presence of the strains in a particular location may change over time and are often found as mixed infections (Strausbaugh et al., 2017; Creamer, 2020). For instance, a survey of BCTV strains from Idaho and Oregon showed a reduction in the prevalence of the Svr strain and the number of mixed infections from 2006–2007 (87% Svr and 60% Wor-like; 59% mixed infections) to 2012–2015 (2% Svr and 87% Wor-like; 16% mixed infections) (Strausbaugh et al., 2017). During this time frame a significant shift in crop management occurred when neonicotinoid seed treatments became labeled for use on sugar beet in 2008. Thus, beet leafhoppers coming from the desert in the spring were likely limited in their ability to feed on sugar beet (influence of neonicotinoids lasts at least 77 days) and had to survive on alternate host plants such as common bean and weeds. These alternate hosts may have favored the Wor and CO strains over Svr. Another possibility might have been the addition of strain specific resistance to commercial sugar beet cultivars but the combination of genes in these cultivars is not publicly known. In addition, new strains can emerge likely due to recombination among strains during mixed infections (Strausbaugh et al., 2008; Chen et al., 2010; Bach and Jeske, 2014).

We identified the mild strains, BCTV-CO and BCTV-Wor present as a single or mixed infection in hemp leaf samples in Colorado using strain-specific primers. In contrast, we did not detect the BCTV-Svr in any of the samples. The DNA fragments from Sanger sequencing and whole genomes from NGS revealed high nt similarity to BCTV genotype previously from Colorado (Giladi et al., 2020) and Arizona (Hu et al., 2021). Reports suggest that mild strains (strains that produce mild symptoms such as slight leaf curling, stunting, and vein thickening) are more effective in infecting alternative and weed hosts than severe strains (Chen and Gilbertson, 2009). All the beet leafhoppers collected in the current study also carried BCTV-Wor and CO strains. This complements the findings that CO and Wor are the predominant strains in hemp. The beet leafhopper transmits BCTV in a circulative manner (Bennett, 1971). The leafhopper acquires the virus in as little as 1–2 min, but maximum accumulation occurs after acquisition access period (AAP) of 24–48 h (Soto and Gilbertson, 2003). There is a 4-h latent period before the insect can transmit the virus. The longer the AAP the higher the rate of transmission. The virus does not replicate in the insects (Soto and Gilbertson, 2003). Leafhoppers can transmit the virus by feeding for 15 min and the virus is retained for days to weeks, but there is no transstadial transmission (Bennett, 1971).

There is limited information about curly top epidemiology in Colorado. Beet leafhoppers are most found in the Western Slope of Colorado where outbreaks of this disease have been reported on many crops such as tomato, bean, squash, sugar beets, spinach (Cranshaw, 2020). It is thought that leafhoppers migrate from their overwintering sites in southern states including Arizona and New Mexico to Colorado in spring. The insect survives the winter on various kinds of weedy plants, particularly mustard-family (Bennett, 1971). Hence, the abundance of winter host plants in the southern breeding areas could be an important factor in the number of beet leafhoppers that appear in Colorado in spring. We hypothesize that in winter 2019 moisture conditions were very favorable in the southern breeding areas to support the large population of overwintering plants on which beet leafhopper could develop resulting in large number of migrants that moved into Colorado causing an outbreak in hemp, which is one of the most widely grown crops in the Western slope.

Virome analysis revealed a diversity of viruses and one viroid pathogen infecting hemp in Colorado. The number of identified viruses and viroid in each location ranged from 2 to 5. Beet curly top virus, CasaMV1, and HLVd were commonly present in several locations, whereas other viruses were unique to specific locations. Cannabis sativa mitovirus 1 was detected in hemp from all counties sampled with the exception of Larimer County, where tissues were collected from indoor hemp production. Mitoviruses are capsidless viruses known for their ability to infect eukaryotic mitochondria (Shahi et al., 2019). Several mitovirus sequences have been recovered during the transcriptome analysis of a variety of invertebrates across different phyla (Shi et al., 2016). Complete plant mitovirus genomes were recovered from publicly available transcriptome data of 10 different plant species including hemp, hops and sugar beet (Nibert et al., 2018). These viruses are generally considered to be cryptic viruses and there is no information on the impact on plant hosts.

A new genotype of TSV which is only 81–83% identical to the closest TSV sequence in GenBank was identified from hemp samples from Conejos County. Tobacco streak virus has a wide host range (Brunt et al., 1996) and is transmitted by thrips and pollen (Sdoodee and Teakle, 1988; Sharman et al., 2015) and by seed transmission (Sharman et al., 2009). The virus was first reported to infect hemp in 1971 and had described symptoms of stunting and mosaic patterning (Hartowicz et al., 1971). TSV is a non-enveloped, quasi-spherical virion with tripartite (RNA1, RNA2, and RNA3) segmented linear (+) sense RNA genome and all 3 segments were retrieved in the hemp virome dataset. Further characterization of these sequences is challenging because the leaf samples from number of plants were pooled and/or mixed infection of viruses in these plants.

Cannabis cryptic virus was only detected in the indoor hemp sample from Larimer County and had high nt sequence similarity (99%) to the RdRP gene of previously reported CanCV sequence (Righetti et al., 2018). Historically, interveinal chlorosis and leaf margin wrinkling in hemp was attributed to the so-called hemp streak virus (HSV) (Röder, 1941). In 2012, Ziegler et al. (2012) first identified CanCV accidently while using hemp as a host for a hop latent virus (Carlavirus) and found that the virus was seed-transmissible. More recently, Righetti et al. (2018) tested hemp samples with typical hemp streak syndrome and identified CanCV in all tested samples irrespective of presence and severity of symptoms. This calls into question the role of CanCV in symptomology.

Citrus yellow vein-associated virus and opuntia umbra-like virus were identified from Delta and Rio Blanco counties. Citrus yellow-vein disease (CYVD) was first reported Dr. L. G. Weathers in California in 1957 (Weathers, 1957) resulting in typical yellow vein symptoms. Recently, Kwon et al. (2021) demonstrated that CYVD is associated with a virus-like agent, tentatively named CYVaV and is transmitted via grafting to virtually all citrus varieties. The virus appears to be closely related to unclassified virus-like RNAs in the family Tombusviridae specifically OULV (Kwon et al., 2021). Indeed, phylogenetic analysis of the RdRp and ORF1-3genes placed the CYVaV and OULV from the current study were in a well-supported cluster. Umbraviruses lack a capsid protein (CP) gene, which makes them dependent on a “helper” virus, usually from the Luteoviridae, for replication, encapsidation and cell-to-cell movement (Syller, 2003). However, no helper virus was identified using NGS in this dataset. It is possible that a helper virus too distant to be recognized was missed by the analysis. Indeed, the first report of OULV from Opuntia ficus indica fruit cactus plants with symptoms of pad swelling disease was not associated with Luteoviridae (Felker et al., 2019). The authors hypothesized that as Umbraviruses occur throughout the plant, but Luteoviruses only occur in the phloem, low concentrations of Luteovirus can be expected in the sample. To our knowledge, this is the first report of CYVaV and OULV in hemp.

Hop latent viroid has been previously identified in hemp from symptomatic and asymptomatic plants (Bektaş et al., 2019) and from symptomatic plants (Warren et al., 2019). Viroids are small non-encapsulated infectious pathogens, comprised of closed single stranded RNA molecules and biological resources to drive host specificity (Flores et al., 2005). There was high nt sequence identity (99–100%) between the HLVd genomes retrieved from hemp. Typical symptoms of HLVd include stunting, malformation or chlorosis of leaves, brittle stems, and reduction in yields. Indeed, some of the samples collected from Delta County demonstrated typical HLVd symptoms.

Our study identified a diversity of viruses and viroids in hemp in Colorado using NGS. The number of identified viruses and viroid in each location ranged from 2 to 5. Cannabis sativa mitovirus, BCTV and HLVd were commonly present in several locations, whereas other viruses were unique to specific locations. We identified a divergent virus with 81% sequence identity to TSV. Future research should focus on surveying hemp viruses across multiple hemp genotypes, locations, and stages of the crop. Overall, outcomes of this study and future research will result in the identification of unique target sequences for the development of rapid and accurate nucleic acid-based detection tools for viruses and viroids and a rich characterization of hemp-associated pathogens. The viruses/viroids identified in the study were mechanically transmitted, seed-transmitted or insect-transmitted making management of these viruses challenging (Mcpartland et al., 2000; Nachappa et al., 2020; Punja, 2021). Planting certified disease-free materials will be critical to minimize disease spread. Future investigations should explore vector metagenomics to determine potential insect vectors of viruses/viroids associated with hemp with the goal of identifying anticipated threats and developing early prevention tactics. Once the insect vector community is identified, a deeper exploration of timing of insect flights, reproduction and feeding behaviors can be targeted for pest management and interruption of transmission cycles. Lastly, identifying potential sources of resistance to insect pests and viruses is an especially important disease control strategy, as commercial insecticides that are effective against insect vectors in other crops are restricted on hemp.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.

PN and JC conceived and designed the experiments and wrote the original draft of the manuscript. JC, KL, AF, and WC collected samples. JC, KL, JM, and TA performed experiments and phylogenetic analyses. MK and MS performed library preparation and bioinformatic analyses. JC, JM, MS, and PN reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by USDA-NIFA Critical Agricultural Research and Extension (CARE) 2020-04954 and the Foundation for Food and Agriculture Research (FFAR)-Rapid Outcomes from Agricultural Research (ROAR) program.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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