First report of 'Candidatus Phytoplasma fraxini'-related strain associated with peach yellows in Pennsylvania

Authors: Costanzo, Stefano; JONES, TAMMY - Pennsylvania State University; KARI, PETER - Pennsylvania State University; NIKOLAEVA, EKATERINA - Pennsylvania Department Of Agriculture

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/22/2025
Publication Date: 9/2/2025
Citation: Costanzo, S., Jones, T., Kari, P., Nikolaeva, E. 2025. First report of Candidatus Phytoplasma fraxini-related strain associated with peach yellows in Pennsylvania. Plant Disease. https://doi.org/10.1094/PDIS-06-25-1240-PDN.
DOI: https://doi.org/10.1094/PDIS-06-25-1240-PDN

Interpretive Summary: Peach production plays a significant role in Pennsylvania’s agricultural landscape, contributing both economically and culturally to the state’s fruit industry. During disease surveys in 2015 and 2016, peach trees in a Pennsylvania orchard exhibited unusual symptoms such as leaf yellowing, reddening, and distortion suggesting a possible phytoplasma infection. Phytoplasmas are a group of bacteria known to cause serious decline in fruit trees, but the identity and origin of the pathogen involved were unknown. The Pennsylvania Department of Agriculture collected leaf and stem samples from symptomatic and healthy trees. Scientists from ARS in Beltsville, MD performed molecular analyses and sequencing of housekeeping genetic markers from the collected samples. The research team compared the sequences obtained from the infected samples with known phytoplasma strains using tools like iPhyClassifier, the USDA ARS curated sequence database for phytoplasma classification. The analyses revealed that the strains are related to ‘Candidatus Phytoplasma fraxini’ but represent a genetically distinct lineage. These findings suggest the presence of a novel, peach-associated phytoplasma strain belonging to the Ash yellows group that differ substantially from any known phytoplasma species documented to date. This is the first report of a ‘Ca. P. fraxini’-related strain infecting peach trees in Pennsylvania, raising concerns for the stone fruit industry in the region and beyond. Early detection and accurate identification are critical for growers, plant disease diagnosticians, and extension personnel for managing the spread of emerging phytoplasma diseases. The findings underscore the importance of ongoing surveillance and genetic characterization efforts to protect agricultural production and biosecurity.

Technical Abstract: In the fall of 2015 and 2016, during annual disease surveys conducted by the Pennsylvania Department of Agriculture, several peach trees (Prunus persica (L.) Batsch) from a commercial orchard in Berks County, PA, were observed exhibiting symptoms consistent with phytoplasma infection. Symptoms included premature leaf yellowing and reddening, foliar epinasty, shotholes and vein reddening. Approximatively one percent of the peach trees in the orchard displayed these symptoms and were ultimately removed. Leaf and stem samples were collected from seven symptomatic trees as well as from several asymptomatic ones for further analysis. Total DNA was extracted from 100 mg of leaf midvein tissue using the Qiagen DNeasy Plant Kit. All DNA samples were initially screened using a phytoplasma-specific real-time PCR assay (Hodgetts et al., 2009). Positive samples were subsequently used in conventional nested PCR targeting the 16S rRNA gene, using primer pairs P1/16S-SR followed by P1A/16S-SR (Deng & Hiruki, 1991; Lee et al., 2004). PCR products were purified, cloned, and sequenced, and four representative nucleotide sequences were deposited in the GenBank database under accession numbers PV770936 to PV770939. Sequence analysis using BLASTn (NCBI) and iPhyClassifier (Zhao et al., 2009) revealed that the phytoplasma strains, designated as PYP-PA03, -PA04, -PA06 and -PA07, shared between 97.6% and 97.73% sequence identity with the ‘Candidatus Phytoplasma fraxini’ reference strain (GenBank accession AF092209), suggesting that the peach associated phytoplasma is a ‘Ca. P. fraxini’-related strain. However, virtual RFLP analysis of the 16S rDNA F2nR2 fragments revealed patterns distinct from all previously established 16Sr groups and subgroups. The closest similarity was observed with members of the 16Sr group VI, but the resulting similarity coefficients were below the accepted subgroup threshold (0.97), supporting the distinctiveness of these strains. To further characterize the PYP-PA phytoplasma, partial sequences of the tuf and secA genes were PCR-amplified, cloned, and sequenced following protocols by Makarova et al. (2012) and Dickinson & Hodgetts (2013). The resulting sequences were identical for each gene and one sequence was deposited in GenBank under accession numbers PV769898 (tuf gene) and PV769899 (secA gene). BLASTn analysis of the tuf gene revealed 93.25% and 90.67% sequence identity with the homologous gene in the Argentinian alfalfa witches’-broom phytoplasma isolate ArAWB-2021 (CP131022) and the ‘Ca. P. fraxini’ isolate AshY1 (CP146843), respectively. Similarly, the secA gene shared 89.16% and 88.30% sequence identity with the same isolates ArAWB-2021 (CP131022) and AshY1 (CP146843), respectively. These collective results suggest that the PYP-PA phytoplasma is phylogenetically related to members of the Ash yellows group (16SrVII). In 2010, Zunnoon-Khan et al., identified a phytoplasma strain of group 16SrVII-A associated with peach trees in Ontario, Canada. To our knowledge, this study is the first to report of a ‘Ca. P. fraxini’-related strain associated with peach trees in Pennsylvania. These findings highlight the need for further research to fully characterize this potentially novel phytoplasma, which constitute a distinct lineage from the one identified in Canada and underscore the importance of continued surveillance and molecular investigation of emerging phytoplasma-associated diseases in the region.