Nikolaeva, Ekaterina V.

In the fall of 2015 and 2016, during annual disease surveys conducted by the Pennsylvania Department of Agriculture, seven peach trees (Prunus persica) 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. Seven out of 700 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 five asymptomatic ones for further analysis. Total DNA was extracted using the Qiagen DNeasy Plant Kit. All DNA samples were initially screened using a phytoplasma-specific real-time PCR assay (Hodgetts et al., 2009). Phytoplasma was detected in all symptomatic plants, but in none of the five asymptomatic plants. 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 cloned, and sequenced. Four representative sequences were deposited in GenBank 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, 04, 06, and 07, shared between 97.5% (1,482/1,520 bp) and 97.63% (1,483/1519 bp) sequence identity with the ‘Candidatus Phytoplasma fraxini’ reference strain 16SrVII-A (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/subgroups. The closest similarity was observed with members of the 16Sr group VI, but 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% (470/504 bp) and 90.67% (457/504 bp) sequence identity with the homologous gene in the Argentinian alfalfa witches’-broom phytoplasma isolate ArAWB-2021 16SrVII-C (CP131022) and the ‘Ca. P. fraxini’ isolate AshY1 16SrVII-A (CP146843), respectively. Similarly, the secA gene shared 89.16% (691/775 bp) and 88.30% (679/769 bp) 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 PRU0430 of group 16SrVII associated with peach trees in Ontario, Canada (GU223903). To our knowledge, the present study is the first to report a ‘Ca. P. fraxini’-related strain associated with peach trees in the United States. These findings highlight the need for further research to fully characterize this potentially novel phytoplasma, which constitutes 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.

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.