GH LACY and VK STROMBERG

Phytobacteriology Section, Laboratory for Molecular Biology of Plant Stress, Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0330, USA

Background and objectives
DNA similarity groups are the basis for the current classification of xanthomonad pathovars and species [1]. These data were based on 790 pairwise tests among 183 strains. Because these comparisons were collected from smaller studies scattered in the literature, they lack coherence and are difficult to interpret. Our objective was to provide a more comprehensive basis for xanthomonad phylogenetic relationships. To this end, we constructed a matrix including 2002 pairwise DNA similarity tests among 122 strains including Xanthomonas albilineans, X. axonopodis, X. fragariae, X. oryzae, Stenotrophomonas maltophilia (ex X. maltophilia), and 73 pathovars often identified with X. campestris [2]. To confirm the groupings determined from the DNA similarity analyses, 56 strains were selected for RFLP analyses [3].

Materials and methods
DNAs were provided from the American Type Culture Collection as cleared lysates. DNA purified was prepared as high molecular mass DNA for RFLP analyses or sheared to 400 to 600 ; bp and denatured to ssDNA for DNA similarity studies. For DNA similarity analyses, probes were prepared by labelling sheared ssDNA with ¹²⁵I. Probe DNA was hybridized in liquid with 100- to 1000-fold excess non-labelled tester ssDNA. Hybridized DNAs were digested with S1 nuclease to remove excess ssDNA and single-stranded regions from annealed DNAs. Digested DNAs were precipitated and washed on glass fiber membranes. Per cent DNA similarity (%S) was determined by comparing gamma emissions remaining in heterologous hybridizations with those retained in homologous reannealed samples.

For the RFLP analyses, probes were prepared using PCR- amplified 16S rRNA genes from X. campestris pv. campestris ATCC 33913. DNAs were digested with restriction endonucleases BamHI, EcoRI, or PstI; electrophoresed; denatured; transferred to membrane support; hybridized with probe ssDNA; and profiles were developed on X-ray film with chemical luminescence.

Results and conclusions
DNA similarity analyses indicated that stenotrophomonads are quite separate from xanthomonads. Nine major groups of xanthomonads with about 20% S were identified: Albilineans, Badrii, Campestris, Fragariae, Hyacynthi, Juglandis, Malvacearum, Oryzae, and Pisi. Among these groups, 25 strain clusters reassociated at species-level (70% S). RFLP analyses confirmed these major groups and that X. albilineans shares as little similarity with xanthomonads and stenotrophomonads as stenotrophomonads share with xanthomonads. Both DNA similarity and RFLP data indicated that X. axonopodis and the pathovars currently grouped within this species (DNA group 9 [1]) comprise at least five species-level clusters.

References
1. Vauterin L, Hoste B, Kersters K. Swings J, 1995. International Journal of Systematic Bacteriology 45, 472-89.
2. Lacy GH, Gherna RL, Stromberg VK, Toth J, Cote R, Johnson JL, 1997. Phytopathology 87, S55.
3. Lacy GH, Stromberg VK, Toth J, Gherna RL, Cote R, Johnson JL, 1997. Phytopathology 87, S56.