Phytoplasmas associated with grapevine yellows in Virginia belong to group 16SrI, subgroup A (tomato big bud phytoplasma subgroup), and group 16SrIII, new subgroup I

Grapevine yellows disease in Virginia closely resembles flavescence doree and other grapevine yellows diseases, but the phytoplasmas infecting grapevines in Virginia are distinct from other grapevine yellows pathogens. RFLP analysis of PCR-amplified 16S rDNA indicated that a Virginia grapevine yellows phytoplasma, designated VGYIII was distinct from all other phytoplasmas studied, but was most closely related to spirea stunt (SP1), walnut witches' broom (WWB), and poinsettia branch-inducing (PoiB 1) phytoplasmas in subgroups E, G, and H, respectively, of 16S I RNA group 16SrIII. RFLP analysis also indicated the existence of sequence heterogeneity between the two rRNA operons in the genomes of SP 1 and WWB. Based on the results from RFLP and sequence comparisons with other group 16SrIII phytoplasmas, the VGYIII phytoplasma was classified in a new subgroup, designated 16SrIII-I. A second phytoplasma (VGYI) was detected in cultivated grapevines(Vitis vinifera L.) and in wild grapevines(V. riparia Michx.) and identified as a member of subgroup 16SrI-A. There was no evidence of flavescence doree, bois noir, or Australian grapevine yellows phytoplasmas in Virginia

Ancient, recurrent phage attacks and recombination shaped dynamic sequence-variable mosaics at the root of phytoplasma genome evolution

Mobile genetic elements have impacted biological evolution across all studied organisms, but evidence for a role in evolutionary emergence of an entire phylogenetic clade has not been forthcoming. We suggest that mobile element predation played a formative role in emergence of the phytoplasma clade. Phytoplasmas are cell wall-less bacteria that cause numerous diseases in plants. Phylogenetic analyses indicate that these transkingdom parasites descended from Gram-positive walled bacteria, but events giving rise to the first phytoplasma have remained unknown. Previously we discovered a unique feature of phytoplasmal genome architecture, genes clustered in sequence-variable mosaics (SVMs), and suggested that such structures formed through recurrent, targeted attacks by mobile elements. In the present study, we discovered that cryptic prophage remnants, originating from phages in the order Caudovirales, formed SVMs and comprised exceptionally large percentages of the chromosomes of ‘Candidatus Phytoplasma asteris’-related strains OYM and AYWB, occupying nearly all major nonsyntenic sections, and accounting for most of the size difference between the two genomes. The clustered phage remnants formed genomic islands exhibiting distinct DNA physical signatures, such as dinucleotide relative abundance and codon position GC values. Phytoplasma strain-specific genes identified as phage morons were located in hypervariable regions within individual SVMs, indicating that prophage remnants played important roles in generating phytoplasma genetic diversity. Because no SVM-like structures could be identified in genomes of ancestral relatives including Acholeplasma spp., we hypothesize that ancient phage attacks leading to SVM formation occurred after divergence of phytoplasmas from acholeplasmas, triggering evolution of the phytoplasma clade