First Report of North American Grapevine Yellows (NAGY) in Kansas: Detection of ‘Candidatus Phytoplasma pruni’- and ‘Ca. Phytoplasma asteris’-Related Strains in Diseased Plants

Abstract

During surveillance for possible presence of exotic phytoplasma pathogens in the United States, plants of cultivated grapevine (Vitis vinifera L.) exhibiting symptoms suggestive of grapevine yellows disease were observed in vineyards in Kansas during the 2013 and 2014 growing seasons. Symptoms observed in <1% of plants examined consisted of leaf yellowing, downward rolling of leaf margins, and shriveling of fruit clusters. Symptomatic plants of cultivars Riesling (designated KS-JO48) and Chardonnay (designated KS-DG12) were selected for further work. To detect and identify the suspected phytoplasmal pathogen strain(s), DNA was extracted from veins excised from symptomatic leaves and used as template in polymerase chain reactions (PCRs) for amplification and cloning of phytoplasma rRNA (rDNA) and secY gene sequences as previously described (Davis et al. 2015; Lee et al. 2010). Nucleotide sequences of the PCR products were deposited in the GenBank database under accession numbers MH085064 and MH085065 (for cloned rrnA and rrnB gene sequences, respectively, reflecting interoperon heterogeneity) and MG992477 (secY) for KS-JO48; and MH085066 (rrnA), MH085067 (rrnB), and MG992478 (secY) for KS-DG12. In silico analysis of 16S rRNA gene sequences, using iPhyClassifier (Zhao et al. 2009), revealed that plant KS-JO48 was infected by a ‘Candidatus Phytoplasma asteris’-related phytoplasma belonging to subgroup 16SrI-A (I-A), and that plant KS-DG12 was infected by a ‘Ca. P. pruni’-related phytoplasma belonging to subgroup variant 16SrIII-A* (III-A*). Nucleotide sequence alignment of the strain KS-DG12 16S rRNA gene sequence with those of North American grapevine yellows (NAGY) strains previously reported (Davis et al. 2015) revealed that strain KS-DG12 is a NAGYIIIβ sequevar corresponding to sequevar NAGYIIIβ strains found in Maryland, Virginia, New York, and Pennsylvania (Davis et al. 2015). Alignments and phylogenetic analyses of secY gene sequences confirmed the close relatedness of the Kansas subgroup 16SrI-A strain KS-JO48 and subgroup 16SrIII-A* (NAGYIIIβ sequevar) strain KS-DG12 phytoplasmas with the reference strains of ‘Ca. P. asteris’ and ‘Ca. P. pruni’, respectively. The results provide the first evidence for the presence of NAGY in the state of Kansas, while revealing that the two NAGY phytoplasma strain lineages reported here are closely related to NAGY phytoplasma strains detected in other U.S. states. Although further work will be necessary to assess the impacts of NAGY in Kansas, the findings reported here expand the known geographic range of NAGY disease and support the concept that the geographic distribution of NAGY in the United States is greater than currently realized.