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1 Department of Plant Pathology, Cornell University, 334 Plant Science Building, Ithaca, NY 14853, USA
2 Department of Plant Pathology, University of California, Davis, CA 95616, USA
3 Molecular Plant Pathology Laboratory, USDA Agricultural Research Service, Beltsville, MD 20705, USA
Author for correspondence: Helen M. Griffiths. Tel: +1 607 255 5052. Fax: +1 607 255 4471. e-mail: hmg1{at}cornell.edu
ABSTRACT
Phytoplasmas associated with the plant diseases ash yellows (AshY, occurring in Fraxinus) and lilac witches'-broom (LWB, occurring in Syringa) represent a putative species-level taxon. Phytoplasmal DNA from 19 ash or lilac sources across the known geographic range of AshY (71–113 °W) was examined to determine if AshY and LWB phytoplasmas are a coherent group, if variability 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 15 restriction enzymes. RFLPs were detected in digests obtained with Alul, Hhal or Taql, for a total of four RFLP profile types. Sequencing of the amplimers from strains AshY1T, AshY3, AshY5 and LWB3 (which represent the four 16S rDNA RFLP profile types) revealed only three positions in the 16S rRNA 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, amplified with primer pair rpF1/R1 from each of the four strains noted above, was analysed with six restriction enzymes, resulting in the detection of two RFLP profiles with Msel. Southern analysis, utilizing two non-specific probes from other phytoplasma groups, revealed three RFLP profile types in anonymous chromosomal DNA of strains representing the four 16S rDNA genotypes. Two strains, AshY3 and LWB3, had unique combinations of characters in the various 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 PFGE data to be 645 kbp. Phylogenetic analysis of a 1423 bp 16S rDNA sequence from strains AshY1T, 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 analyses utilizing only type strain AshY1T. Thus, the AshY phytoplasma group is coherent but heterogeneous. The name ‘Candidatus Phytoplasma fraxini’ is proposed for this group.
Key Words: 16S rRNA • plant disease • Fraxinus • Syringa • ‘Candidatus Phytoplasma fraxinin’
Present address: Department of Plant Pathology, Cornell University, 334 Plant Science Building, Ithaca, NY 14853, USA.
The GenBank accession numbers for the P1/P7 amplimer (the 16S rRNA gene and the 16S-23S spacer) of phytoplasma strains AshY1T, AshY3, AshY5 and LWB3 are AF092209, AF105315 and AF105316, AF105317, respectively.
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