First Report of ‘<i>Candidatus</i> Phytoplasma pruni’-Related Strain and ‘<i>Candidatus</i> Phytoplasma asteris’-Related Strain Associated with North American Grapevine Yellows of Cultivated Grapevines in Minnesota

Surveys for exotic plant pests conducted during July and August of 2023 and 2024 across 20 vineyards in 12 counties throughout Minnesota, USA, revealed that less than 2% of the approximatively 3000 vines inspected (<i>Vitis</i> spp., hybrid grape varieties) exhibited symptoms suggestive of phytoplasma yellows disease. Observed symptoms included yellowing of leaf lamina, downward rolling of leaf margins, and necrosis of leaf margins. To investigate a potential association between these symptoms and phytoplasmas, two symptomatic plants were selected in August of 2024 from two distinct vineyards for further analysis. Petiole tissue DNA extracts were initially tested using a phytoplasma-specific real-time PCR assay (Hodgetts et al. 2009), which confirmed the presence of phytoplasma DNA in both symptomatic plants. Subsequently, the samples underwent semi-nested PCR amplification targeting the phytoplasma 16S rRNA gene, using primers P1/16S-SR followed by P1A/16S-SR (Deng and Hiruki 1991; Lee et al. 2004). The resulting amplicons were cloned and sequenced. No amplicon was produced from an asymptomatic grapevine DNA sample included as a test control. Analysis of the 16S rRNA gene sequences revealed that each plant was infected by one of two distinct phytoplasma strains, designated MN450 and MN466. Representative sequences obtained from the two samples exhibiting interoperon heterogeneity were deposited in the GenBank database under accession numbers: PQ889195 (<i>rrnA</i>) and PQ889196 (<i>rrnB</i>) for strain MN450, and PQ889197 (<i>rrnA</i>) and PQ889198 (<i>rrnB</i>), for strain MN466. Sequence analysis using the online phytoplasma classification tool <i>i</i>PhyClassifier (Zhao et al. 2009) showed that the 16S rRNA gene sequences from MN450 shared 1491/1495 bp (99.73%) and 1492/1495 bp (99.80%) identity (<i>rrnA</i> and <i>rrnB</i>, respectively) with that of the ''<i>Ca</i>. Phytoplasma pruni' rrnA' reference strain (GenBank JQ044393), identifying MN450 as a ''<i>Ca</i>. Phytoplasma pruni' rrnA'-related strain and possibly representing a new subgroup within the 16Sr group III. For MN466, the 16S rRNA gene sequences shared 1471/1482 bp (99.26%) and 1469/1482 bp (99.12%) identity (<i>rrnA</i> and <i>rrnB</i>, respectively) with that of the '<i>Ca</i>. Phytoplasma asteris' reference strain (GenBank M30790), and identifying the phytoplasma as a '<i>Ca</i>. P. asteris'-related strain belonging to subgroup 16SrI-A. To further characterize the two phytoplasma strains, the <i>secY</i> gene was PCR amplified from the DNA extracts as described by Lee et al. (2010). The obtained amplicons were cloned and sequenced, and representative <i>secY</i> gene sequences were deposited in GenBank under accession numbers PQ899621 (MN450) and PQ899622 (MN466). BLASTn searches against the NCBI core nucleotide database revealed that the <i>secY</i> gene sequence for MN450 exhibited 100% identity (1614/1614 bp) with '<i>Ca</i>. P. pruni' (GenBank KM268860). Similarly, the sequence for MN466 showed 100% identity (1287/1287 bp) with '<i>Ca</i>. P. asteris' (GenBank CP000061). In North America, grapevine yellows disease associated with strains related to '<i>Ca</i>. P. asteris' and '<i>Ca</i>. P. pruni' has previously been reported in various states and referred to as North American grapevine yellows (NAGY) by Davis et al. (2015). To our knowledge, this is the first report of NAGY in Minnesota, confirming that the two phytoplasma species detected are consistently associated with the disease in U.S. vineyards. While further research is needed to evaluate the potential impacts of NAGY in Minnesota, this study highlights the broader distribution of the disease across the United States.