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

Differential amplification of sequence heterogeneous ribosomal RNA genes and classification of the ‘Fragaria multicipita’ phytoplasma

Ribosomal (r) RNA interoperon sequence heterogeneity in the ‘Fragaria multicipita’ phytoplasma, a member of group 16SrVI, was initially observed in RFLP patterns of rDNA amplified in the polymerase chain reaction (PCR), and was confirmed through sequence analysis of cloned rDNA. Sequences from operons rrnA and rrnB were amplified in PCR primed by primer pair P1/P7 but from only rrnA in PCR primed by primer pair R16mF2/R16mR1. Preferential amplification of DNAfrom operon rrnA was explained by base mismatches between the R16mF2/R16mR1 primers and primer annealing sites in rrnB. The results revealed potential for classification of a phytoplasma into two different subgroups within a 16S rRNA group, if the phytoplasma’s 16S rRNA gene sequences are independently characterized. It is suggested that the rRNA operon containing species-specific signature sequence(s) should be specified, and where possible sequences from both 16S rRNA genes should be included, in descriptions of new ‘Candidatus Phytoplasma species’

Cryptic plasmid pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an adhesin and components of a type IV translocation-related conjugation system

A cryptic plasmid of the wall-less plant pathogenic mollicute, Spiroplasma kunkelii CR2-3X, was cloned and its sequence analyzed. The 14,615 bp plasmid, designated pSKU146, has a nucleotide content of 28 mol% G + C, and contains 18 potential protein-coding regions (open reading frames, ORFs), of which six encode proteins that exhibit similarity to virulence-associated proteins involved in cell-to-cell adhesion or conjugal DNA transfer. One ORF encodes a 96 kDa protein, SkARP1, that is highly similar to SARP1 adhesin involved in attachment of Spiroplasma citri to insect vector gut membrane. Five ORFs encode proteins similar to TraE and Mob in walled bacteria, and to ORFs found in the integrative, conjugative element (ICEF) of Mycoplasma fermentans, respectively. Presence of domains similar to proteins of the Type IV secretion system in pathogenic bacteria suggests that spiroplasma possesses a related translocation system. Plasmid pSKU146 also contains two identical oriT regions each containing a nick sequence characteristic of the IncP conjugative plasmid family, as well as a 58 bp palindromic sequence, palSK1. Features in pSKU146 suggest that the plasmid functions as a mobile genetic element in conjugative transmission of spiroplasma pathogenicity-related genes

Complete Genome Sequence of Spiroplasma kunkelii Strain CR2-3x, Causal Agent of Corn Stunt Disease in Zea mays L.

Spiroplasma kunkelii causes corn stunt disease of Zea mays L. in the Americas. Here, we report the nucleotide sequence of the 1,463,926-bp circular chromosome and four plasmids of strain CR2-3x. This information will facilitate studies of Spiroplasma pathogenicity and evolutionary adaptations to transkingdom parasitism in plants and insect vectors