The earliest observations of Cannabis sativa viral disease are reported in the 40s and 50s of the XX century in Europe [1,2] (Figure 1) when fiber and/or seed crops were widely cultivated. These first reports indicate two symptomatologic conditions that were correlated to supposed viral infections, never formally demonstrated: the hemp streak virus (HSV) and the hemp mosaic virus (HMV). However, these reports were purely descriptive of symptomatology since no pathogenicity test was ever performed, and the viral etiology was never confirmed. A comprehensive analysis of these conditions has never been conducted, probably because hemp cultivation faced a period of regression due to the development of synthetic fibers and the use of other plants as a source for vegetable oil production in later years. In 1970 Schmidt and Karl [3] identified three viruses able to infect C. sativa plants and cause symptoms: the cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV), and hemp mottle virus. While CMV and AMV were confirmed as infectious and pathogenic in later studies, the latter was never reported again. In the 1970s, Hartowicz et al. (1971) [4] performed viral mechanical transmission in C. sativa using isolates of twenty-two different viruses, and half were proven to infect inoculated plants. While this work was presented at the “North Central Weed Control Conference”, to our knowledge, a comprehensive and peer-reviewed paper reporting the methodology alongside the results obtained has never been published. A later publication [5] mentions this work and lists those viruses that resulted in symptomatologies in inoculated plants: tobacco ringspot virus (TRSV), tomato ringspot virus (TomRSV), tobacco streak virus (TSV), and CMV have been associated with mosaic symptoms and dwarfism, while AMV and the so-called “eunoymous ringspot virus (ERSV)” were only associated with mosaic. Of these, only TSV, CMV, and AMV were reported again in later works on viruses that infect C. sativa, while the others were no longer mentioned.

No new manuscripts on the topic were then published until 1997, when Kegler and Sparr [6] performed inoculations of different viruses in several European industrial varieties for seeds and fiber production (hemp) in a controlled environment. After six weeks from the first inoculations, systemic leaves of infected hemp plants were used for re-inoculations on test plants (Nicotiana spp. and Chenopodium spp.) using serological tests to verify viral infections. In this way, it was possible to identify the viruses described in Table 1, including CMV and AMV, and describe a range of symptom severity in hemp (Table 2).

More than ten years passed until a new virus infecting hemp was accidentally identified in 2012 [9]. The new Partitiviridae identified was proven to be widely endemic since it was detected in almost all varieties tested, but no correlation with symptoms was observed, so it was reported as cannabis cryptic virus (CanCV) (Table 1). In a follow-up study, characterizing the virus in other hemp varieties, Righetti et al. (2018) [10] confirmed that the virus is not associated with any symptoms (Table 2) and described a variable viral load depending on the plant analyzed. Vertical transmission has been demonstrated crossing both infected male plants and healthy females and healthy males and infected females. The resulting progenies were found to be 100% positive. Otherwise, mechanical transmission in Chenopodium quinoa and Nicotiana benthamiana test plants was proven to be ineffective as was the transmission via cross-grafting between rootstocks and scions from infected and not-infected C. sativa plants. In fact, members of the Betapartitivirus genus have no movement proteins so, in plants, their tropism occurs only with cell division [16].

The hop latent viroid (HLVd) has recently been increasingly associated with severe cannabis symptoms [14,15,17] (Table 2) that impair seed and fiber production in industrial varieties and cannabinoid contents in medical varieties. Infected medical cannabis plants can suffer vigor loss and a 50% up to 70% reduction in THC content. Furthermore, a survey carried out in California on 200,000 samples verified the presence of HLVd in 90% of cannabis-growing facilities. The potential estimated losses can therefore reach up to USD 4 billion loss per year in the United States of America alone [17]. Infected mother plants for agamic propagation act as a reservoir for the viroid that easily spreads to the clonal progeny. Moreover, HLVd diffusion is higher than field inspections would indicate because symptoms develop only in the late stages of infection.

Where the decriminalization and legalization of C. sativa for medical (Israel) and recreational (in some USA states) use have taken place, cultivated areas have increased, leading to favorable conditions for the spread of new viral infections. Lettuce chlorosis virus (LCV) was identified as a causal agent of leaves yellowing and stunted growth in Israel [11] (Table 2), symptoms that were previously only associated with nutrition deficiencies. In the USA, apical leaf deformations have been correlated with the presence of beet curly top virus (BCTV) [12,13] (Table 2).

Recently, Pitt et al. (2022) [8] confirmed that potato virus Y (PVY) infects C. sativa and demonstrated that it can be transmitted by aphids in a non-persistent manner. This makes PVY-infected cannabis fields potentially dangerous from a phytosanitary point of view because of the possible spread of the virus to other crops.

C. sativa virome investigation has recently found a new boost thanks to high-throughput sequencing (HTS) techniques. Chiginsky et al. (2021) [7] performed an Illumina NextSeq500 sequencing of several C. sativa plants showing virus-like symptoms. While aiming to characterize different BCTV strains and describe their spread among symptomatic plants in Colorado (USA), several viruses have been identified (Table 1), some even never reported in C. sativa. The genome of a new strain of TSV was partially reconstructed, and grapevine line pattern virus (GLPV), citrus yellow vein-associated virus (CYVaV), and opuntia umbra-like virus (OULV) were identified and reported for the first time infecting cannabis. Recently, Jarugula et al. (2023) [18] reported co-infections of BCTV along with HLVd and CYVaV after canonical reverse transcription–PCR analysis and HTS in Washington State (USA).

All accession numbers of viruses that have been detected through HTS in cannabis are listed in Table S1 in the Supplementary material.

In recent years, in addition to expanding knowledge of the cannabis virome, cannabis virology has focused on exploiting viral vectors to compensate for the lack of established tools for in vivo functional genomic analysis. With the expansion of genomic and transcriptomic resources, significant advances have been made in the understanding of cannabis genetics allowing the identification of regulatory genes responsible for valuable medical or agronomical phenotypic traits [19,20,21,22,23]. However, reverse genetics studies are required to validate bioinformatic predictions or to conduct molecular characterizations of protein effectors. The basic requirement of reverse genetics is to possess tools for targeted gene editing to vary or abolish the expression of a particular gene or to obtain specific mutations to its product. The use of sequence-targeted DNA endonucleases underlies genome-editing technologies such as CRISPR/Cas9, which are widely used in other crops of agricultural interest [24,25,26]. The repair of DNA double-strand breaks by non-homologous end joining or homology-directed repair (in the occurrence of a donor template) makes it possible to modify a single DNA base pair, larger genomic portions, or the regulation of gene expression [27]. For this to occur, the CRISPR/Cas9 complex must be delivered or expressed in callus-forming cells (e.g., by Agrobacterium tumefaciens-mediated transformation), which will then be regenerated in an adult plant. However, cannabis plants are described as recalcitrant to callus regeneration even though they are effectively transformed by A. tumefaciens [28]. Indeed, the production of transgenic plants is still not easy to achieve [15], and despite their significant potential, transgenesis and genome editing have been successfully employed in only a few works [29,30]. In this context, virus-induced gene silencing (VIGS) represents an alternative system to genome editing for functional genomics studies because it allows the down expression of target genes through post-transcriptional gene silencing (PTGS). Viral vectors engineered from cotton leaf crumple virus (CLCrV) [31] and tobacco rattle virus (TRV) [32] have been successfully used for the purpose of harboring portions of C. sativa gene sequences in their genome and promoting the silencing of visible markers such as phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI). These promising results encourage the use of VIGS for rapid reverse genetic screening by silencing specific genes involved in the regulation of specialized metabolites, disease resistance, plant development, and abiotic stress in cannabis.