First report of 'Candidatus Phytoplasma pruni' associated with X-disease on sweet cherry (Prunus avium L.) in Canada.

British Columbia (BC) is the lead producer of sweet cherries in Canada with more than 2,000 ha in production and a farm gate value of over CAD$100 million annually. Since 2010, an outbreak of little cherry disease caused by Little cherry virus 1 (LChV1) and Little cherry virus 2 (LChV2), as well as X-disease (XD) caused by 'Candidatus Phytoplasma pruni' has caused significant economic losses in neighboring Washington State (WA), USA. LChV1 and LChV2 have long been known to occur in BC (Theilmann et al. 2002); however, 'Ca. P. pruni' has not yet been reported in BC. Due to its geographical proximity to WA State, the BC cherry industry expressed significant concerns about the possible presence of the phytoplasma in cherry orchards. Accordingly, the main objective of this study was to survey cherry orchards to determine whether 'Ca. P. pruni' was present in symptomatic trees in BC. A total of 118 samples of leaves and fruit stems from individual symptomatic trees were collected prior to harvest from nine cherry orchards and one nectarine orchard in the Okanagan and Similkameen Valleys in BC. Characteristic symptoms included small and misshapen fruit with poor color development. Samples were submitted to AGNEMA, LLC (Pasco, WA) for testing using qPCR TaqMan assays for LChV1 (Katsiani et al. 2018), LChV2 (Shires et al. 2022) and 'Ca. P. pruni' (Kogej et al. 2020). Test results showed 21 samples (17.8%) from three cherry orchards positive for LChV2 and 2 samples (1.7%) from one cherry orchard positive for 'Ca. P. pruni'. In order to confirm the identification of 'Ca. P. pruni', part of the 16S ribosomal RNA gene was amplified by nested PCR using the P1/P7 followed by R16F2n/R2 primer sets (Gundersen and Lee 1996) and Sanger sequenced. BC-XD-Pa-1 (GenBank Acc. No. OR539920) and BC-XD-Pa-2 (OR537699) were identical to one another and showed 99.92% identity to the 'Ca. P. pruni' reference strain CX-95 (JQ044397). Analysis using iPhyClassifier (Zhou et al. 2009) indicated that they were 16SrIII-A strains. Interestingly, the two partial 16S sequences showed 100% nucleotide identity to strain 10324 (MH810016) and others from WA. For additional confirmation, partial secA (Hodgetts et al. 2008) and secY (Lee et al. 2010) translocases were amplified and sequenced. As with the 16S sequences, secY sequences (OR542980, OR542981) showed 99.92% nucleotide identity to strain CX-95 (JQ268249), and 100% to strain 10324 (MH810035). The secA sequences (OR542978, OR542979) had nucleotide identities of 99.77% to strain CX (MW547067), and 100% to the Green Valley strain from California (EU168733). Accordingly, 'Ca. P. Pruni' was confirmed to be present in sweet cherry samples from BC. 'Ca. P. Pruni'-related strains have been previously reported to occur in Canada in commercial poinsettias (Euphorbia pulcherrima) (Arocha-Rosete et al. 2021). To our knowledge, this is the first report of 'Ca. P. Pruni' in sweet cherry in Canada. Due to the important economic value of sweet cherries in BC, these findings are highly significant and represent the first steps towards the development of a surveillance system for early detection of XD, and consequent implementation of management strategies, including vector control. As required by federal and provincial regulations, cherry trees infected with LChV2 and 'Ca. P. Pruni' found in the survey were removed by the growers.