Hoskins, Mason C.

Big Basin Sagebrush (Artemisia tridentata ssp. tridentata) is a common shrub found across high desert and arid regions of North America. Although its natural distribution has been reduced over the past century due to farm and urban expansion it remains a keystone species in much of the high desert and is a host for native arthropod species. However, there have been few studies that examined the effect of phytopathogens on A. tridentata (Allen & West 1987; Welch & Nelson 1995). During a survey for alternate hosts of the X-disease phytoplasma (‘Candidatus Phytoplasma pruni’) (Shires et al. 2025) we observed phytoplasma-like foliar chlorosis and dieback-like symptoms on A. tridentata in uncultivated areas of Chelan County, WA. Approximately 30-40% of plants were symptomatic at these sites, and symptom progression was seasonal, with the emergence of enlarged, pale-yellow chlorotic leaves between late spring (May-June) and summer (July-August). Chlorosis began at the leaf apex and extended towards the petiole, with tip necrosis then leaf drop occurring in late August (Figure 1). Dieback of shoot apices and limbs was also observed on individual plants. Leaf and stem tissues were collected from 19 symptomatic and 63 asymptomatic sagebrush at three sites in Chelan Co., DNA extracted using the Qiagen DNeasy plant mini kit (Qiagen Corp. Germantown, MD) per the manufacturer’s instructions, and tested for 16SrIII phytoplasmas on the basis of proximity to orchards known to be infected with ‘Ca. P. pruni’ by qPCR as per Shires et al. (2025). All symptomatic plants tested positive, while asymptomatic plants did not. For identification, the partial 16S rRNA gene was amplified from seven samples by nested PCR using the P1/P7 (Deng & Hiruki 1991) then R16F2n/R2 primers (Gundersen & Lee 1996); resulting products were Sanger-sequenced (Eton Biosciences, San Diego, CA) and deposited in GenBank (PV994682-PV994688). Examination using iPhyClassifier (Zhao et al. 2012) indicated that these were a ‘Ca. P. pruni’-related strain with 99.66% identity to reference sequence JQ0443393. BLASTn indicated a 100% nucleotide identity match to Lilac decline phytoplasma strain LlcDec1 (KP877578) (Davis et al. 1996). For further identification, the partial secY (849 bp), secA (490 bp), Ef-Tu (793 bp), and imp (457 bp) genes were amplified and sequenced as per Molnar et al. (2024). Of these secY (PX023655-PX023661), secA (PX023662-PX023668) and Ef-Tu (PX023669-PX023675) had 99.8%, 98.9% and 98.5% identity to X-disease strains PX11CT1 (JQ268254), 20-15 (OR67122), and 22-2470 (OR670177) respectively, whereas imp (PX023676-PX023682) had 96.9 % identity to PoIBI strain “Annette Hegg Maxi” (AB636364) (100% coverage for all fragments). Neighbor-joining phylogenetic analysis of the concatenated marker regions showed that this phytoplasma forms a separate clade from other 16SrIII strains, and whose closest relative is PoiBI strain JR1 (ASM30946v1) with 98.9% identity across the aligned region (Figure 2). Cumulatively these data suggest the Sagebrush yellows-associated phytoplasma is a ‘Ca. P. pruni’-related strain that is novel and distinct from previously described members of the 16SrIII group, based on sequence identity and the host on which it causes disease. This find is significant as it is the first report of a novel phytoplasma infecting a keystone species in western North America and an indicator of where 16SrIII phytoplasmas infecting cultivated plants may have originated.

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.