First Report of ‘Candidatus Phytoplasma trifolii’ Associated with a Witches’ Broom Disease in Cannabis sativa in Nevada, U.S.A.

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In September 2018, symptoms including leaf curling, mottling, chlorosis, witches’ broom, stunting, and node shortening were detected in Cannabis sativa L. plants at two growing sites in Central and Southern Nevada, U.S.A., respectively. Incidence of the disease varied between 5 and 20% at the growing site in Central Nevada, and 30% of plants were affected at the growing site in Southern Nevada. Symptomatic leaves showed “green islands” that were constrained by the veins on the upper leaf surface, and interveinal discoloration on the lower surface, forming a pale “pustule-like” appearance. One infected plant with 80% of leaves exhibiting symptoms was sampled from the Central Nevada site. One infected plant with 50% of leaves exhibiting symptoms was sampled from the Southern Nevada site. Two nonsymptomatic plants were provided by a local grower in California. Total DNA was extracted from the petioles of the most symptomatic leaves. DNA extracted from nonsymptomatic plants was used as the negative control. A diagnostic TaqMan real-time PCR test was conducted using the universal primer pair JH-F/JH-R (Hodgetts et al. 2009) targeting the 23S rRNA gene. Positive amplification signals were generated only from the symptomatic plants indicating the presence of Phytoplasma species. To identify the species, the 16S ribosomal RNA gene was amplified with the universal primer pairs P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), P1/Tint (Smart et al. 1996), followed by nested PCR primer pair R16F2/R2 (Lee et al. 1993). The anticipated amplicons (1.8, 1.6, and 1.2 kb, respectively) were gel purified and subjected to Sanger sequencing (UC Davis DNA Sequencing Facility). The sequence data were deposited in GenBank (accession no. MK377248). BLASTn results suggested a 99% sequence similarity with ‘Candidatus Phytoplasma trifolii’ (accession no. KF178706.1), a member of 16SrVI group associated with alfalfa witches’ broom disease. iPhyClassifier analysis (Zhao et al. 2013) was conducted to generate a restriction fragment length polymorphism profile using the amplified 16S rRNA gene sequence, revealing the most similarity (98.8%) to ‘C. P. trifolii’. Phytoplasmas of elm yellows group (16SrV) (Zhao et al. 2007) and aster yellows group (16SrI) (Raj et al. 2008), both associated with witches’ broom disease on Cannabis sp., have been reported in China and India, respectively. To our knowledge, this is the first report of 16SrVI group ‘Ca. P. trifolii’ infecting C. sativa in the United States. The identified pathogen may pose a significant threat to the production of C. sativa in the United States.