First report of ‘Candidatus Phytoplasma trifolii’ and Spiroplasma citri in Cannabis sativa in Washington State, U.S.A

Authors: WAGSTAFF, CAMILLE - Washington State University; GORMAN, CHRISTOPHER - Washington State University; CROWDER, DAVID - Washington State University; Swisher Grimm, Kylie

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/25/2026
Publication Date: 4/1/2026
Citation: Wagstaff, C., Gorman, C., Crowder, D., Swisher Grimm, K.D. 2026. First report of ‘Candidatus Phytoplasma trifolii’ and Spiroplasma citri in Cannabis sativa in Washington State, U.S.A. Plant Disease. 110(4). Article 1471. https://doi.org/10.1094/PDIS-08-25-1736-PDN.
DOI: https://doi.org/10.1094/PDIS-08-25-1736-PDN

Interpretive Summary: The beet leafhopper, (Neoaliturus tenellus) transmits several pathogens to vegetable and seed crops across the western U.S., causing yield or quality losses of economic importance. In collaboration with Washington State University, researchers with the USDA ARS in Wapato, Washington, identified hemp plants at three research farms in Washington State with symptoms often associated with beet leafhopper-transmitted pathogens in a variety of crops: stunting, leaf curling, chlorosis, darkening or purpling of leaves, and stem fasciation. Initial screening of these plants identified three beet leafhopper transmitted pathogens present in the symptomatic tissue, including 'Candidatus Phytoplasma trifolii' and Spiroplasma citri which previously were not reported in hemp plants in Washington State. Further analysis targeted key genes of each pathogen for sequencing analysis to confirm their presence in the symptomatic tissue. It is currently unknown how hemp yield and quality are impacted by these pathogens, but this finding suggests that integrated pest management strategies must be developed to reduce beet leafhopper transmission in hemp and other important vegetable and seed crops grown across Washington State.

Technical Abstract: In 2022 and 2024, field surveys were conducted in the Columbia Basin of Washington State, U.S.A., for pathogens transmitted by the beet leafhopper, Circulifer tenellus (recently reclassified as Neoaliturus tenellus), including beet curly top virus (BCTV), ‘Candidatus Phytoplasma trifolii’ (CPt), and Spiroplasma citri. For these pathogens, an association of symptoms with a causal agent is often complicated by coinfections and the inability to culture CPt (Hu 2021; Rivedal et al. 2022, 2024; Schoener and Wang 2023). In 2022, 35 hemp (Cannabis sativa) plants were observed at the Washington State University (WSU) Research Station in Othello, WA, with typical symptoms of BCTV (a finding published by Jarugula et al. 2023), and we further tested these samples for C. tenellus-associated bacteria. In 2024, 23 and 15 hemp plants were observed in Prosser, WA, from the WSU Irrigated Agriculture Research Center and WSU Roza Farm, respectively, with symptoms of CPt infection, including stem fasciation and purpling. Leaf tissue from each plant was ground in liquid nitrogen, DNA was extracted using the DNeasy Plant Mini Kit (Qiagen), and a multiplex real-time PCR protocol was used to detect the C. tenellus-associated pathogens (Swisher Grimm et al. 2023). CPt was identified in 8.6 and 65.8% of plants in 2022 and 2024, respectively, while S. citri was not detected in 2022 but was found in 15.8% of plants in 2024. Coinfections were common. Nested PCR (primers P1/P7 and R16F2n/R16R2, Crosslin et al. 2006) and conventional PCR (primers Trif-SecY-163F, 5'-AGCAGCTAAAAAAGTTAGAAAAAACCTC-3'/Trif-SecY-1040R, 5'-AAATCTAGCGAAAATGATTTTTTGTTTTCA-3') targeted the CPt 16S rRNA and secY genes, respectively. S. citri infection was confirmed by PCR targeting the spiralin gene (primers SpiralinF/R, Yokomi et al. 2008). All targets were amplified using PrimeSTAR HS DNA Polymerase (Takara Bio). Thermal cycle conditions for CPt 16S rRNA and S. citri spiralin genes consisted of 30 or 40 cycles, respectively, at 98°C for 10 s, 55°C for 15 s, and 72°C for 10 s, followed by a final elongation at 72°C for 5 min. Conditions for CPt secY consisted of 35 cycles at 98°C for 15 s, 55°C for 30 s, and 72°C for 10 s, followed by a final elongation at 72°C for 5 min. Amplicons of size 1,250 bp, 877 bp, and 675 bp for the 16S rRNA, secY, and spiralin genes, respectively, were visualized on an agarose gel stained with GelRed (Sigma-Aldrich) or ethidium bromide. For CPt, sequencing of 16S rRNA from seven infected samples (2 from 2022, 5 from 2024) revealed identical sequences (GenBank accession PV983691) with 100% identity to CPt in U.S. hemp (OQ597521), and sequencing of secY in one sample from 2022 and one from 2024 revealed identical sequences (PX725980), with 100% identity to CPt in U.S. periwinkle (GU004317). For S. citri, two sequenced samples had identical spiralin sequences (PV955037), matching 100 and 99.85% with S. citri found in Oregon cabbage (PV099668) and hemp (OQ969984), respectively. This is the first confirmed report of CPt and S. citri in C. sativa in Washington State. These findings highlight the need to evaluate effects of mollicutes on hemp and underscore the need to develop integrated pest management strategies to reduce vector transmission. In addition, these findings suggest that hemp could serve as a reservoir of the C. tenellus-transmitted pathogens, leading to higher pathogen prevalence across the region and negatively impacting economically important vegetable and seed crops grown in Washington State that are susceptible to these pathogens.