Recurrent epiphytotics of X-disease, caused by ‘Candidatus Phytoplasma pruni,’ have inflicted significant losses on commercial cherry and peach production across North America in the last century. During this period, there have been multiple studies reporting different disease phenotypes and, more recently, identifying different strains through sequencing core genes, but the symptoms have not, to date, been linked with genotype. Therefore, in this study we collected and assessed differing disease phenotypes from multiple U.S. states and conducted multilocus sequence analysis on these strains. We identified a total of five lineages associated with the induction of X-disease on commercial Prunus species and two lineages that were associated with wild P. virginiana. Despite a century of interstate plant movement, there were regional trends in terms of lineages present, and lineage-specific symptoms were observed on P. avium, P. cerasus, and P. virginiana, but not on P. persica. Cumulatively, these data have allowed us to define “true” X-disease–inducing strains of concern to the stone fruit industry across North America, as well as potential sources of infection that exist in the extraorchard environment.

X-disease phytoplasma and little cherry virus 1 and 2 have resulted in substantial loss of stone fruit acreage in Washington and Oregon. The extent and financial impact on the industry was not previously known. A Washington and Oregon State University survey documented 238,856 trees equivalent to 974 acres of sweet cherries removed due to X-disease and little cherry disease between 2015 and 2020. Removed trees reduced revenue to the industry by an estimated $30 million in 2020 and $65 million between 2015 and 2020. Over the seven-year re-establishment period estimated lost revenue and establishment costs to growers is an estimated $115 million.

Little cherry disease (LCD) and X-disease have critically impacted the Pacific Northwest sweet cherry (Prunus avium) industry. Current detection methods rely on laborious visual scouting or molecular analyses. This study evaluates the suitability of field asymmetric ion mobility spectrometry (FAIMS) for rapid detection of LCD and X-disease infection in three sweet cherry cultivars (‘Benton’, ‘Cristalina’, and ‘Tieton’) at the post-harvest stage. Stem cuttings with leaves were collected from commercial orchards and greenhouse trees. FAIMS operated at 1.5 L/min and 50 kPa, was used for headspace analysis. Molecular analyses confirmed symptomatic and asymptomatic samples. FAIMS data were processed for ion current sum (I_sum), maximum ion current (I_max), and area under the curve (I_AUC). Symptomatic samples showed higher ion currents in specific FAIMS regions (p < 0.05), with clear differences between symptomatic and asymptomatic samples across compensation voltage and dispersion field ranges. Cultivar-specific variation was also observed in the data. FAIMS spectra for LCD/X-disease symptomatic samples differed from those for asymptomatic samples in other Prunus species, such as peach and nectarines. These findings support FAIMS as a potential diagnostic tool for LCD/X disease. Further studies with controlled variables and key growth stages are recommended to realize early-stage detection.

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.

Candidatus Phytoplasma pruni is the causative agent of X-disease on peach (Prunus persica) trees. Infected trees exhibit premature yellowing, leaf necrosis causing a shot-hole appearance, limb dieb...

Chokecherry (Prunus virginiana L.) is a native shrub that is widely found across North America and is a host of ‘Candidatus Phytoplasma pruni’ associated with X-disease of stone fruits. This pathogen is of major concern to U.S. cherry and peach growers, with the most recent and ongoing outbreak in the Pacific Northwest causing major economic harm. To investigate the role of chokecherry as a potential reservoir and source of orchard-associated strains, we surveyed wild chokecherry trees across 12 U.S. states, and compared the strains present to those in commercially cultivated Prunus species. Approximately 47% of chokecherry sampled tested positive for a ‘Ca. P. pruni’-related strain and phylogenetic analysis revealed high strain diversity, including six distinct clades and four newly identified lineages. In contrast, ‘Ca. P. pruni’ strains isolated from cultivated cherry and peach trees exhibited lower diversity and were infected by known disease outbreak-associated strains such as NWX. These data suggest limited transmission between wild and cultivated hosts in the western U.S., with patterns indicating spillover from orchards into nearby chokecherry populations rather than the reverse. However, in states east of the Rocky Mountains, chokecherry likely functions as a reservoir for orchard infections.