International Journal of Systematic and Evolutionary Microbiology (2007), 57, 2881–2887 DOI 10.1099/ijs.0.

65220-0

Phylogenetic analysis of Xanthomonas species by

comparison of partial gyrase B gene sequences
Neil Parkinson,1 Valentine Aritua,2 John Heeney,1 Claire Cowie,1
Janice Bew1 and David Stead1
Correspondence 1
Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK
Neil Parkinson 2
National Agricultural Biotechnology Centre, Kawanda Agricultural Research Institute,
n.parkinson@csl.gov.uk
PO Box 7065, Kampala, Uganda

The genus Xanthomonas currently comprises 27 species with validly published names that are
important crop and horticultural pathogens. We have constructed a phylogram from alignment of
gyrase B (gyrB) sequences for all xanthomonad species, both to indicate inter-species
relatedness and as an aid for rapid and accurate species-level identification. The phylogeny
indicated a monophyletic group, with X. albilineans and X. sacchari as the most ancestral species.
Three species, X. hyacinthi, X. translucens and X. theicola, formed an early-branching group.
Three clades were supported by high bootstrap values: group 1 comprised X. cucurbitae, X.
cassavae and X. codiaei; group 2 comprised X. arboricola, X. campestris, X. populi, X. hortorum, X.
gardneri and X. cynarae; group 3 contained the remaining species, within which two further
clades, supported by a 100 % bootstrap value, were identified. Group 3A comprised X.
axonopodis, X. euvesicatoria, X. perforans and X. melonis, together with X. alfalfae, X. citri and X.
fuscans, whose names were recently validly published. Group 3B contained the monocot
pathogens X. vasicola and X. oryzae. Two recently identified species, X. cynarae and X. gardneri,
were poorly discriminated and were related closely to X. hortorum. Three species, X. perforans, X.
euvesicatoria and X. alfalfae, had identical gyrB sequences. Partial sequencing of a further five
genes from these species found only minor sequence differences that confirmed their close
relatedness. Although branch lengths between species varied, indicating different degrees of
genetic distinctiveness, the majority (n521) were well-differentiated, indicating the utility of the
method as an identification tool, and we now use this method for routine diagnosis of
xanthomonad species.

INTRODUCTION The complexity of the genus and resource requirements for

comprehensive pairwise analyses required for DNA–DNA
Classification of species within the genus Xanthomonas,
hybridization analysis make this technique unsuitable for
which was hitherto based on biochemical characteristics
routine identification in most diagnostic laboratories. A
(Van den Mooter & Swings, 1990), underwent major revision
rapid characterization method based on analysing cell-wall
after a comprehensive spectrophotometric DNA–DNA
fatty acids using GC was developed and applied to the
hybridization study of the genus (Vauterin et al., 1995),
identification of plant pathogens (Stead, 1989, 1992).
which resulted in the recognition of 20 constituent species.
Whilst this method can be very useful for typing of some
Following this study, names of seven further species have
xanthomonads, it does not provide the necessary resolu-
been validly published: X. cynarae (Trébaol et al., 2000), X.
tion or reproducibility to be useful for all species. More
euvesicatoria, X. perforans, X. gardneri (Jones et al., 2004;
recently, a major and comprehensive analysis of the genus
Euzéby, 2006), X. citri, X. fuscans and X. alfalfae (Gabriel
was completed (Rademaker et al., 2005) that compared
et al., 1989; Euzéby, 2007; Schaad et al., 2005, 2006, 2007).
amplicon profiles produced by using PCR primers
designed to repeated sequences dispersed in bacterial
The GenBank/EMBL/DDBJ accession numbers for the gyrB sequences
of xanthomonad species determined in this study are EU007516–
genomes (Martin et al., 1992; Louws et al., 1994;
EU007542 and DQ676938. Thwaites et al., 1999). This study utilized all three
commonly used repetitive PCR methods (REP, BOX and
Supplementary tables showing type strains of all xanthomonad species
used in the study, PCR and sequencing primers and a similarity matrix ERIC) to produce a high-resolution identification scheme,
indicating all inter-species percentage nucleotide identities are available and encompassed many of the pathovars of X. campestris
with the online version of this paper. that could not be resolved in the earlier study by Vauterin

65220 G 2007 Crown copyright Printed in Great Britain 2881

N. Parkinson and others
et al. (1995) using DNA–DNA hybridization. Comparison
of gel profiles, however, still presents problems outside
reference laboratories, and inter-laboratory standardization
can be problematic. DNA sequencing studies of the 16S
rRNA gene and the 16S–23S intergenic region of
Xanthomonas species (Hauben et al., 1997; Gonçalves &
Rosato, 2002) have indicated the phylogenetic position of
the genus within the Gammaproteobacteria. Whilst the
16S–23S phylogeny had greater resolution than that
produced from the 16S study, most of the species within
the genus fell into a single group, and neither method
could be used reliably to differentiate the majority of
species.
Recently, DNA sequencing of genes encoding conserved
proteins involved in essential cell processes that constitute
the ‘core genome’ has been used to produce phylogenies
that are more resolving than 16S and 16S–23S studies.
Increasingly, this approach is being adopted for bacterial
identification, as the method provides a convenient and
rapid means of providing information on the relatedness of
species and strains. This study aimed to produce a
xanthomonad phylogeny based on partial sequence align-
ments of the gyrB gene, both to indicate xanthomonad
relatedness and as a simple and reliable means of species-
level identification.
As published DNA–DNA hybridization values are available
for species comparisons (Vauterin et al., 1995), it was
possible to relate percentage gyrB sequence identities to
these values for several species comparisons, and to
produce a scatter graph to clarify the relationship between
the two methods for quantifying DNA sequence similarity.
During the study, it was found that X. euvesicatoria, X.
perforans and X. alfalfae had identical gyrB sequences. To
address this anomaly and to clarify relatedness between
these species, partial sequences from an additional five
genes were analysed. Concatenated sequences from these
loci were produced and compared in a phylogeny along
with similar sequences derived from genome sequences for
X. axonopodis pv. citri, X. oryzae and X. campestris.
METHODS
Bacterial strains. Type strains of all xanthomonad species used in
the study were obtained from international culture collections
(mostly the National Collection of Plant Pathogenic Bacteria;
NCPPB) and their reference numbers, together with alternative strain
references from other collections, are indicated in Supplementary
Table S1, available in IJSEM Online. Species are as indicated in the
phylogram (Fig. 1).
gyrB locus and PCR amplification. Primers were designed from
xanthomonad alignments of gyrB sequences available from GenBank
and corresponded to the C-terminal region of the protein. In relation
to the gyrB gene from X. euvesicatoria (strain 85-10; GenBank
accession no. CAJ23349), the region amplified corresponded to
aa 603–778, of a total of 832 residues for this protein. DNA, prepared
from suspensions of freshly grown xanthomonad cultures, in water
was adjusted to an A650 of 0.1 using a spectrophotometer, and then
heated in a microfuge tube for 6 min at the highest temperature
2882
setting (100 uC) in a dry block heater. After cooling, the prepared
DNA suspensions were frozen at 220 uC prior to use, when 1 ml
preparation was added to 24 ml PCR reagent mix. Centrifuging of the
boiled cells or other purification treatment was not required.
As xanthomonads can have very high G+C contents, a blend of Taq
polymerase and a proof-reading polymerase was used for all
amplifications (Long PCR Enzyme mix; Fermentas). The PCR buffer
included magnesium and was as supplied by the enzyme supplier. The
reaction components comprised: forward primer (10 pmol ml21),
0.75 ml; reverse primer (10 pmol ml21), 0.75 ml; 106 PCR buffer
(including 15 mM MgCl2), 2.5 ml; dNTP mix (2.5 mM each), 2.0 ml;
Long PCR Enzyme mix, 0.125 ml; prepared DNA, 1.0 ml; water,
17.9 ml. The final concentrations of reagents were: MgCl2, 1.5 mM;
dNTPs, 0.2 mM; primers, 0.3 mM each; enzyme mix, 0.75 units (all
per 25 ml reaction volume). PCR and sequencing primers are shown
in Supplementary Table S2, available in IJSEM Online. We routinely
used standard primers that produced large yields of PCR product for
all species except X. albilineans and related xanthomonads; for these
species, the alternative conserved primer set was used. The conserved
primers were designed from accrued gyrB alignments from this study
and can be used for gyrB amplification from all species. The PCR
cycling conditions were 2.5 min at 94 uC, then 30 s at 94 uC, 45 s at
50 uC and 1 min at 68 uC for 34 cycles, then 7 min at 68 uC to end.
Purity and yield of PCR product for sequencing were checked by
running 5 ml reaction mixture on a 1.5 % agarose gel and post-
staining using ethidium bromide, according to standard protocols
(Thwaites et al., 1999). The remaining mixture was purified by using a
commercial kit to remove residual primers and other reaction
components (Wizard PCR Clean-up kit; Promega). After elution of
bound DNA in 50 ml water, the equivalent of approximately 12 mg
DNA (usually 5–10 ml purified product) was tranferred to a fresh
microfuge tube and lyophilized prior to sequencing from a
commercial service (MWG Ltd), using a Sanger sequencing-based
fluorescent-label PCR method. The sequences, provided in FASTA
format files, were cropped further to provide a 530 nt sequence,
corresponding to nt 1807–2336 in the gyrB sequence from X.
euvesicatoria strain 85-10. The cropped sequences all start with
ATCACCGG; the end of the sequence finishes (with some variants)
with AGCTGTG.
Sequencing of X. euvesicatoria, X. perforans and X. alfalfae
using additional gene loci. PCR amplification and sequencing
primers for a further five loci [sigma factor 70 (rpoD), aconitate
hydratase B (acnB), phosphoglucoisomerase (pgi), glyceraldehyde-3-
phosphate dehydrogenase (gap) and phosphofructokinase (pfkA)]
were designed from the X. euvesicatoria genome sequence (strain 85-
10) available from GenBank (accession no. CAJ23349) (Thieme et al.,
2005). PCR conditions, cycling parameters and sequencing protocol
were the same as used above for the gyrB analysis. PCR and
sequencing primers for multi-locus sequence typing of X. euvesica-
toria, X. perforans and X. alfalfae are shown in Supplementary Table
S3, available in IJSEM Online.
Concatenated sequences were compared with the same loci derived
from GenBank for X. campestris and X. axonopodis pv. citri (da Silva
et al., 2002), as well as for X. oryzae (Lee et al., 2005).
Sequence alignment and phylogenetic analysis. All loci were
sequenced using both forward and reverse primers. Unprocessed gyrB
sequences were cropped to a common start and finish sequence to
produce 530 nt sequences that were aligned by using the CLUSTAL W
program (Thompson et al., 1994), available at http://www.ebi.ac.uk/
clustalw. Phylogenies were rooted by using the gyrB sequence from a
laboratory isolate that, by 16S rRNA gene sequencing, was related
most closely to the genus Stenotrophomonas. The TREECON program
(Van de Peer & De Wachter, 1993) was used for phylogenetic analysis.
International Journal of Systematic and Evolutionary Microbiology 57
 Xanthomonas phylogeny

Fig. 1. gyrB phylogeny of the genus Xanthomonas produced by using the neighbourhood-joining method. Sequences were
analysed by using the method of Jukes & Cantor (1969). Percentage bootstrap values .50 % from 500 samplings are
indicated. All species are type strains, except for X. hortorum pv. pelargonii. The phylogeny was rooted by using a laboratory
isolate that, by 16S rRNA gene sequencing, was related most closely to the genus Stenotrophomonas. Bar, 0.1 substitutions
per site.

Distance estimation was done by using two methods available in the
program (Jukes & Cantor, 1969; Tajima & Nei, 1984). Tree
construction used the neighbour-joining method (Saitou & Nei,
1987).
RESULTS
Xanthomonas gyrB phylogeny
The phylogeny indicates a monophyletic classification, in
agreement with previous studies comparing 16S rRNA
gene and 16S–23S intergenic sequences (Fig. 1). Sequence
alignments revealed the absence of any insertions or
deletions, consistent with a single origin for the genus.
Of 530 nucleotide positions, 55.3 % were invariant. Inter-
species sequence similarities ranged between 75 and 100 %;
a similarity matrix indicating all inter-species percentage
nucleotide identities is provided in Supplementary Table
S4, available in IJSEM Online.
The two most ancestral species within the phylogeny were
X. albilineans and X. sacchari. The relative phylogenetic
positions of these species, and of the clade containing X.
theicola, X. translucens and X. hyacinthi, were supported by
low bootstrap values. To account for this ambiguity in the
relationship between these basal species, which occurs
often in the most ancestral regions of phylogenies (Sarker
& Guttman, 2004), they are designated ‘early-branching
species’ in the phylogram. In contrast, three major clades
representing the remaining species were supported by high
bootstrap values (.77 %). Group 1 comprised X. cassavae,
X. codiaei and X. cucurbitae. Group 2 comprised X.
arboricola, X. campestris, X. populi, X. hortorum, X. gardneri
and X. cynarae, which are all species indigenous to Europe.
Group 3 comprises a large group of the remaining species,

http://ijs.sgmjournals.org 2883
N. Parkinson and others

and contained two further clades (3A and 3B) that were
supported by a high bootstrap value (100 %). Clade 3A
comprised X. perforans, X. euvesicatoria, X. alfalfae, X.
melonis, X. axonopodis, X. fuscans, X. citri and X. bromi,
whilst clade 3B comprised the monocot pathogens X.
vasicola and X. oryzae.
Most of the reference type species (n521) were distin-
guished clearly according to branch length. Exceptions
were X. perforans, X. euvesicatoria and X. alfalfae, which
had identical sequences. The relatedness of these species
was investigated further by partial sequencing of an
additional five loci and these results are described below.
The type strains of X. hortorum (pv. hederae), X. gardneri
and X. cynarae were distinguished only by very short
branch lengths compared with other species. The pelargonii
pathovar of X. hortorum, which was used in DNA–DNA Fig. 2. Relationship between DNA–DNA hybridization and gyrB
hybridization studies to support X. cynarae and X. gardneri sequence. Mean DNA–DNA hybridization values for species
as distinct species, was separated from the type strain of X. comparisons (Vauterin et al., 1995) are plotted against corres-
hortorum with a branch length similar to those distinguish- ponding comparisons using percentage gyrB identities. The paired
ing other species of Xanthomonas, and was supported by a species comparisons were: X. populi vs X. hortorum; X. populi vs
high bootstrap value (97 %). X. cassavae; X. populi vs X. codiaei; X. populi vs X. hyacinthi; X.
populi vs X. sacchari; X. codiaei vs X. cassavae; X. codiaei vs X.
bromi; X. codiaei vs X. vesicatoria; X. codiaei vs X. hyacinthi; X.
Relationship between gyrB sequences and codiaei vs X. sacchari; X. vesicatoria vs X. hyacinthi; X. vesicatoria
reported DNA–DNA hybridization values for vs X. sacchari; X. hyacinthi vs X. sacchari; X. bromi vs X. cassavae;
paired species comparisons X. oryzae vs X. cassavae; X. vasicola vs X. cassavae; X. populi vs X.
fragariae; X. codiaei vs X. fragariae; X. populi vs X. translucens; X.
The percentage gyrB sequence similarity and published
hortorum vs X. cucurbitae; X. codiaei vs X. cucurbitae; X. oryzae vs
DNA–DNA hybridization values (Vauterin et al., 1995) for
X. vasicola.
24 species comparisons (Fig. 2) were selected for plotting
on a scatter graph. As the reported hybridization values
were means from groups of strains, those species
comparison values with high standard errors (SEM) were DISCUSSION
avoided. The paired sequence identity and DNA–DNA
Until recently, phylogenetic analysis of plant-pathogenic
hybridization values were plotted as a scatter plot by using
bacteria based on protein-encoding genes has been used to
the SigmaPlot software package. The scatter plot (Fig. 2)
analyse diversity within a species. Endoglucanase and hrpB
indicates a clear correlation between the published DNA–
genes have provided a detailed description of sequevar
DNA hybridization data and gyrB percentage sequence
diversity within Ralstonia solanacearum, which identified
identity for the same-paired species comparisons, sup-
five major phylotypes associated with distinct geographical
ported by a Pearson correlation coefficient of 0.755.
regions (Poussier et al., 2000; Fegan & Prior, 2005). Seven
loci were analysed in a large multi-locus sequence study of
Relatedness between X. euvesicatoria, Pseudomonas syringae (Sarkar & Guttman, 2004; Sarkar
X. perforans and X. alfalfae, determined by using et al., 2006) that provided high-resolution analyses of
additional gene loci lineages within five major clades identified within this
species. Sequence analysis revealed that recombination
Concatenated rpoD, acnB, pgi, gap and pfkA sequences were
accounted for a very small proportion of nucleotide
cropped to a total length of 2720 nt. The CLUSTAL W
divergence compared with other bacteria. This was
sequence alignment indicated scores of 99 % sequence
suggested to be attributable to adaptation to specific host
identity for all three comparisons of X. euvesicatoria, X.
plants, which restricted contact with other lineages and
perforans and X. alfalfae. The unrooted phylogeny
minimized genetic exchange. This feature may have
produced from the alignment (Fig. 3) indicated that these
contributed to a phylogeny that reflects very accurately
three species were most similar to X. citri, in agreement
the evolutionary history of this species. As multiple loci
with the gyrB phylogeny. Branch lengths between the three
were analysed, it was possible to compare phylogenies that
species were very short, indicating that none of the
revealed close agreement between six of the seven loci
additional five loci sequenced produced a species-level
examined.
differentiation. The close sequence similarity found for all
five additional loci confirms a close level of core-genome Most recently, phylogenies have been developed that
relatedness for X. euvesicatoria, X. perforans and X. alfalfae. encompass species within higher-level taxa, including the

2884 International Journal of Systematic and Evolutionary Microbiology 57


genus Pseudomonas and the family Enterobacteriaceae (Ait
Tayeb et al., 2005; Paradis et al., 2005). Both of these
studies revealed good correlation between established
reference species and their phylogenetic identification.
The two genes used to study the family Enterobacteriaceae
(Paradis et al., 2005), elongation factor Tu (tuf) and F-
ATPase b-subunit (atpD), produced phylogenies of similar
resolution. However, comparison of the two loci used for
analysis of the genus Pseudomonas (Ait Tayeb et al., 2005)
revealed that the rpoB sequences produced a phylogeny of
approximately three times higher resolution than that
produced by using the alternative rrs gene, indicating that
choice of locus can affect the degree of strain discrimina-
tion afforded by a phylogeny.
The gyrB sequences used in this study to produce the
phylogeny (Fig. 1) were sufficiently distinct to discriminate
the majority of established species in the genus
Xanthomonas. The phylogram indicates that the genus is
a monophyletic group derived from a common ancestor
that acquired the ability to exploit plant tissue as an
environmental niche. Four of the five species that branch at
the base of the phylogeny, and which probably represent
early-evolving species, are pathogens of monocot hosts, i.e.
X. albilineans, X. sacchari, X. hyacinthi and X. translucens,
and it is possible that the genus first arose as a monocot
pathogen. Earlier studies using the 16S or 16S–23S loci
(Hauben et al., 1997; Gonçalves & Rosato, 2002) also found
X. albilineans, X. hyacinthi, X. theicola, X. translucens and
X. sacchari to be the most ancestral species. The gyrB
phylogeny identifies three major inter-species groups that
are well supported by bootstrap analysis (groups 1–3),
whilst a fourth comprises early-branching species of
uncertain relatedness. Extensive species diversification has
occurred within groups 2 and 3, which constitute the
majority of the species within the genus. A bootstrap-value
threshold of 70 % is often used as a guideline to indicate a
high level of confidence in phylogram branches, and the
separation of groups 2 and 3 is well supported with a
bootstrap value of 78 %. Group 3 is the largest species-level
group, and the most basal members of this clade comprised
X. fragariae, X. pisi and the tomato pathogen X. vesicatoria.
Clades 3A and 3B were supported by a 100 % bootstrap
http://ijs.sgmjournals.org
Xanthomonas phylogeny
Fig. 3. Core-genome relatedness of X. perfor-
ans, X. euvesicatoria and X. alfalfae. The
unrooted neighbour-joining phylogram was
produced from concatenated sequences
derived from the rpoD, acnB, pgi, gap and
pfkA loci. Sequences from the same loci for X.
axonopodis pv. citri (strain 306), X. oryzae
(KACC 10331) and X. campestris (ATCC
33913T) were derived from genome
sequences (da Silva et al., 2002; Lee et al.,
2005; Thieme et al., 2005). Bar, 0.05 sub-
stitutions per site.
value. Clade 3A comprises both monocot and eudicot
pathogens, including economically important pathogens of
bean, citrus, tomato and pepper. Clade 3B comprises the
sorghum and rice pathogens X. vasicola and X. oryzae.
DNA–DNA hybridization data are considered to be the
‘gold standard’ to measure relatedness between species. The
scatter graph clearly indicates a correlation between DNA
hybridization and the gyrB phylogeny, which is supported
by the high Pearson correlation coefficient (0.755), and this
forms the basis for the general agreement between the
two methods of distinguishing xanthomonad species. The
deviation from the calculated trend line for many
comparisons may be attributable to experimental error,
inherent in DNA–DNA hybridization studies. The presence
of genetic elements associated with the flexible genome,
especially those of large size or that occur as multiple
copies, including phage, transposable elements, insertion
sequences and plasmids, could all potentially affect DNA
reassociation kinetics and hybridization values. These
elements are a further potential source of deviation
between DNA–DNA hybridization- and nucleotide
sequence-based measures of relatedness.
The gyrB phylogeny clearly offers greater resolution than
those produced previously from the 16S rRNA gene or
16S–23S intergenic loci (Hauben et al., 1997; Gonçalves &
Rosato, 2002), and has resolved X. citri and X. fuscans,
which were previously classified as pathovars of X.
axonopodis. These two species, whose names were recently
validly published, were identified by using a DNA–DNA
hybridization technique (Schaad et al., 2005) that may be
more discriminating than the spectrophotometric assay
used previously (Vauterin et al., 1995). The gyrB phylogeny
supports the distinctiveness of these two species.
The inability of the gyrB phylogeny to discriminate X.
euvesicatoria, X. perforans and X. alfalfae was investigated
by partial sequencing of a further five genes. None of these
five loci was able to distinguish any of the three species
with branch lengths consistent with species-level differ-
entiation, confirming their close core-genome relatedness
(Fig. 3). Similarity between X. euvesicatoria and X. alfalfae
has been found recently in the REP-PCR study reported by
2885
N. Parkinson and others
Rademaker et al. (2005), which placed both species in the
same group, designated 9.2. Relatively high DNA–DNA
hybridization values (58 %) between X. perforans and X.
euvesicatoria have been reported (Jones et al., 2004). DNA–
DNA hybridization data differ from sequence analysis in
that the former are a measure of whole-genome DNA
similarity, which may include elements of the flexible
genome, e.g. phage, transposable elements and plasmids,
that are often variable within strains of a species. These
components could affect DNA reassociation kinetics and
lead to differential DNA–DNA hybridization values. A
large 190 or 155 kb plasmid has been reported in 75 % of
X. euvesicatoria strains (Wichmann et al., 2005). The
genome-sequenced strain of X. euvesicatoria, 85-10, con-
tains a large plasmid and three others, totalling 241 686 bp
and representing 4.66 % of the genome, which, taking into
account that most plasmids are present in more than a
single copy, constitutes a significant proportion of the
genome. Whilst the plasmid contents of X. perforans and X.
alfalfae are unknown, it is possible that the presence of
plasmids in X. euvesicatoria may have contributed to
DNA–DNA hybridization values and this may explain the
disparity between the DNA hybridization and gyrB
sequence data. The close core-genome relatedness of these
three species that infect pepper, tomato and alfalfa suggests
that evolution of the pathogens to enable utilization of
different hosts may be proceeding relatively rapidly, before
substantial substitutions in the core genome have had time
to accumulate.
Two other species, X. cynarae and X. gardneri, could not be
distinguished from each other or from the type strain of X.
hortorum by using the gyrB phylogeny. The reference strain
of X. hortorum used for comparison in the DNA–DNA
hybridization study to discriminate X. cynarae from X.
hortorum (Trébaol et al., 2000) was the pathotype reference
strain of X. hortorum pv. pelargonii, rather than the species
type strain, which belongs to X. hortorum pv. hederae. The
study by Vauterin et al. (1995) recognized heterogeneity
within X. hortorum and reported DNA–DNA hybridization
values of 77 % between these two pathovars. The gyrB
phylogeny placed X. hortorum pv. pelargonii as a distinct
lineage with a branch length close to that differentiating
other species, indicating that this pathotype was a poor
choice to represent the species in the DNA–DNA
hybridization studies. Consequently, X. cynarae may not
have been differentiated adequately from X. hortorum by
using DNA–DNA hybridization. The same X. hortorum pv.
pelargonii reference strain was also used to distinguish X.
gardneri as a separate species from X. hederae (Jones et al.,
2004). We could not find reported DNA–DNA hybridiza-
tion data comparing X. cynarae and X. gardneri. The
phylogeny illustrates how the greater resolution of the gyrB
phylogeny compared with DNA–DNA hybridization can
clarify xanthomonad relatedness.
One issue that may need to be addressed in the future, if a
phylogenetic approach is used for classification, is to decide
what level of sequence divergence should be used to
2886
delineate a species. The phylogeny could distinguish 24 of
the currently accepted species and clearly there is excellent
potential for gyrB sequencing as a convenient and accurate
means of indicating xanthomonad relatedness at the
species and, possibly, strain or pathovar level, and we
now use the method for routine xanthomonad identifica-
tion. We are currently sequencing the gyrB locus for all of
the xanthomonad pathovar type strains to provide a
comprehensive phylogeny of the genus Xanthomonas.
ACKNOWLEDGEMENTS
This work was supported by the Plant Health Division of DEFRA. The
licence number for working with the quarantine bacteria is PHL
251C/ 5574 (02/2007).
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