First Report of Tobacco Streak Virus in Cannabis sativa in New York

Authors: GRUNWALD, DERRICK - University Of Wisconsin; WIJESINGHEGE, CHATHURA - University Of Wisconsin; Gordon, Tyler; Stansell, Zachary; ELLISON, SHELBY - University Of Wisconsin

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/14/2023
Publication Date: 4/23/2024
Citation: Grunwald, D., Wijesinghege, C.W., Gordon, T.C., Stansell, Z.J., Ellison, S. 2024. First report of Tobacco Streak Virus in Cannabis sativa in New York. Plant Disease. https://doi.org/10.1094/PDIS-09-23-1810-PDN.
DOI: https://doi.org/10.1094/PDIS-09-23-1810-PDN

Interpretive Summary: Tobacco streak virus was identified and verified using serological and PCR methods in a low percentage of hemp plants in the state of New York for the first time. TSV has a wide host range and can cause stunting, leaf mosaics, and deformed growing tips in infected plants. In hemp plants that tested positive for TSV in New York, leaves were slightly curled, and the plants were stunted compared with plants from the same accessions that were found to be uninfected. Hemp can act as a host to TSV and exhibits symptoms that may impact essential oil, fiber, and seed production.

Technical Abstract: In September 2022 less than 1% of plants in a field of diverse hemp (Cannabis sativa L.) germplasm in Ontario County, New York were stunted with curled leaves (Figure 1). Fifteen symptomatic plants representing nine accessions were tested for 12 viruses and viroids through Agdia Testing Services (Elkhart, IN). Of these, eight plants representing five accessions including: G 33204 21UO SD (‘Cherry Wine S1’), G 33211 21UO SD (‘Wife’), G 33225 22CL01 CL (‘Candida #2’), G 33270 22UO SD (‘Falkowski CBD Mix’), and G 33365 22UO SD (‘Queen Dream’), were positive for tTobacco streak virus (TSV), a type of Ilarvirus in the Bromoviridae family,) confirmed through enzyme-linked immunosorbent assay testing. TSV is a positive-sense, single-stranded RNA virus with a wide host range that can be transmitted by thrips, mechanical injury, seed, and pollen (Zambrana-Echevarría et al. 2021). To confirm the presence of TSV, two putatively TSV-infected samples were subjected to RNA-Seq analysis. RNA-Seq data was trimmed using the fastp program with default parameters to remove adapter sequences and low-quality bases (Chen et al. 2018). After quality filtering, 49,696,041 and 56,126,804 paired-end reads were retained from Wife and Falkowski CBD Mix samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. Wife and Falkowski CBD Mix samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. Wife and Falkowski CBD Mix samples, 153 and 139 reads were mapped to the above-mentioned TSV reference genome. To further validate the presence of TSV reads in the RNA-seq data, we analyzed RNA-Seq data using the Kkraken2 pipeline (Wood et al. 2019). Using the Kraken2 (Wood et al. 2019) virus database, we identified reads associated with TSV (NCBI taxonomy ID: 12317). This analysis identified 172 and 151 TSV readings from samples. Wife and Falkowski CBD Mix, respectively. Higher numbers of reads identified using the Kraken2 analysis is due to the more permissive k-mer matching approach implemented in Kraken2. Furthermore, we identified several other virus taxa in the samples. Of note, both samples had a higher number of reads associated with Amazon lily mild mottle virus with. 254,493 and 116,150 reads from Wife and Falkowski CBD Mix, respectively. Among other virus species belongs to Ilarviruses, we detected Cassava Ivorian bacilliform virus and Cowpea chlorotic mottle viruses from both samples to further validate infection by TSV, samples from both ELISA-positive and ELISA-negative plant samples were subjected to PCR using the primers and protocol described in Zambrana-Echevarría et al. 2021 (Figure 2). Amplification of an approximately 700 base-pair product was observed in the putatively ELISA-positive samples, but not in the ELISA-negative samples. The amplicons were further cloned into the pGEM-T Easy vector using the manufacturer’s protocol and sequenced using M13 forward and M13 reverse primers. Sequencing results indicated considerable similarity to TSV genomes available in GenBank, between 88 % and 99 %. Our results indicate that hemp can host TSV, and should be considered a potential source of TSV inoculum. Raw sequence data generated from this study is deposited in NCBI under the bioproject ID PRJNA1009441.