XANTHOMONAD PHYLOGENETICS: CORRELATIONS AMONG DNA
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 .
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 . To confirm the groupings determined from the DNA similarity analyses, 56
strains were selected for RFLP analyses .
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
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 ) comprise at least five
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