The phytoplasma associated with ash yellows and lilac witches'-broom: 'Candidatus Phytoplasma fraxini'

Phytoplasmas associated with the plant diseases ash yellows (AshY, occurrin g in Fraxinus) and lilac witches'-broom (LWB, occurring in Syringa) represe nt a putative species-level taxon. Phytoplasmal DNA from 19 ash or lilac so urces across the known geographic range of AshY (71-113 degrees W) was exam ined to determine if AshY and LWB phytoplasmas are a coherent group, if var iability exists in both conserved and anonymous DNA, and if variability in 16S rDNA is related to host or geographic origin. The 16S rRNA gene and the 16S-23S spacer were amplified using primer pair P1/P7 and analysed using 1 5 restriction enzymes. RFLPs were detected in digests obtained with Alul, H hal or Taql, for a total of four RFLP profile types. Sequencing of the ampl imers from strains AshY1(T), AshY3, AshY5 and LWB3 (which represent the fou r 16S rDNA RFLP profile types) revealed only three positions in the 16S rRN A gene and one position in the 16S-23S spacer at which differences occurred ; these were single nucleotide substitutions. Sequence homology between any two strains was > 99.8%. A portion of a ribosomal protein operon, amplifie d with primer pair rpF1/R1 from each of the four strains noted above, was a nalysed with six restriction enzymes, resulting in the detection of two RFL P profiles with Msel. Southern analysis, utilizing two non-specific probes from other phytoplasma groups, revealed three RFLP profile types in anonymo us chromosomal DNA of strains representing the four 16S rDNA genotypes. Two strains, AshY3 and LWB had unique combinations of characters in the variou s assays. On the basis of RFLP profiles, the strains from the other plants sampled comprised two groups. The grouping was not clearly related to host or geographic origin. The genome size of strain AshY3 was estimated from PF GE data to be 645 kbp. Phylogenetic analysis of a 1423 bp 16S rDNA sequence from strains AshY1(T), AshY3, AshY5 and LWB3, together with sequences from 14 other mollicutes archived in GenBank, produced a tree on which the AshY and LWB strains clustered as a discrete group, consistent with previous an alyses utilizing only type strain AshY1T. Thus, the AshY phytoplasma group is coherent but heterogeneous. The name 'Candidatus Phytoplasma fraxini' is proposed for this group.