APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2009, p. 1173–1184
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 4
From Local Surveys to Global Surveillance: Three High-Throughput
Genotyping Methods for Epidemiological Monitoring of
Xanthomonas citri pv. citri Pathotypes?†
Lan Bui Thi Ngoc,1Christian Vernie `re,1Philippe Jarne,2Sylvain Brisse,3Fabien Gue ´rin,1
Se ´bastien Boutry,1Lionel Gagnevin,1and Olivier Pruvost1*
CIRAD, UMR Peuplements Ve ´ge ´taux et Bioagresseurs en Milieu Tropical CIRAD-Universite ´ de la Re ´union, Po ˆle de Protection des
Plantes, 7 chemin de l’Irat, 97410 Saint Pierre, La Re ´union, France1; Centre d’Ecologie Fonctionnelle et Evolutive, UMR 5175,
campus CNRS, 1919 route de Mende, 34293 Montpellier Cedex 5, France2; and Unite ´ Biodiversite ´ des Bacte ´ries Pathoge `nes Emergentes,
Institut Pasteur, 25-28 rue du Dr Roux, 75724 Paris Cedex 15, France3
Received 30 September 2008/Accepted 10 December 2008
Asiatic citrus canker is a major disease worldwide, and its causal agent, Xanthomonas citri pv. citri, is listed
as a quarantine organism in many countries. Analysis of the molecular epidemiology of this bacterium is
hindered by a lack of molecular typing techniques suitable for surveillance and outbreak investigation. We
report a comparative evaluation of three typing techniques, amplified fragment length polymorphism (AFLP)
analysis, insertion sequence ligation-mediated PCR (IS-LM-PCR) typing, and multilocus variable-number
tandem-repeat analysis (MLVA), with 234 strains originating from Asia, the likely center of origin of the
pathogen, and reference strains of pathotypes A, A*, and Aw, which differ in host range. The typing techniques
were congruent in describing the diversity of this strain collection, suggesting that the evolution pattern of the
bacterium may be clonal. Based on a hierarchical analysis of molecular variance, the AFLP method best
described the genetic variation found among pathotypes whereas MLVA best described the variation found
among individual strains from the same countries or groups of neighboring countries. IS-LM-PCR data
suggested that the transposition of insertion sequences in the genome of X. citri pv. citri occurs rarely enough
not to disturb the phylogenetic signal. This technique may be useful for the global surveillance of non-
epidemiologically related strains. Although pathological characteristics of strains could be most often pre-
dicted from genotyping data, we report the occurrence in the Indian peninsula of strains genetically related to
pathotype A* strains but with a host range similar to that of pathotype A, which makes the classification of this
bacterium even more complicated.
The definition of host range is a central parameter for the
understanding and, ultimately, the control of infectious dis-
eases in general and bacterial plant diseases in particular. In
phytobacteriology, host range is an important aspect of
pathogenicity. Control of diseases can be achieved with re-
sistance genes which reduce host range (50, 64). Epidemio-
logical characteristics are highly dependent on host range,
and the emergence of new diseases is sometimes correlated
with broadened host ranges (74). Xanthomonads have the
particularity of an extremely narrow host range (sometimes
reduced to a single plant genus), although a very large
number of plant families can be hosts when all members of
the genus are considered (33), which led plant pathologists
to create the concept of pathovar at an infrasubspecific
level. Pathovars were defined as groups of strains sharing
several pathological characteristics, such as their host range
and the disease facies they cause (18). Based on molecular
data, strains classified as a single pathovar usually form a
discrete monomorphic or weakly polymorphic cluster, sug-
gesting that strains of a pathovar have a common ancestral
origin (3, 56). Xanthomonas citri pv. citri is the causal agent
of Asiatic canker, a severe disease infecting most commer-
cial citrus cultivars and some genera in the Rutaceae family
in many citrus-producing areas worldwide (6, 60, 61). This
pathovar has two types of strains, which differ in their host
ranges: pathotype A has a wide host range and a worldwide
distribution and is a permanent threat for citriculture (29);
in contrast, the more recently characterized pathotype A*
causes citrus canker on Mexican lime (Citrus aurantifolia)
and has a much less severe impact on citriculture (72).
Strains of this pathotype were considered to belong to the
pathovar citri because of their phenotypic and genetic re-
latedness to pathotype A. Their distribution was initially
reported to include Saudi Arabia, Oman, Iran, and India
and was recently found to extend to southeast Asia, with
reports of these strains in Thailand (10) and Cambodia (11).
Finally, strains genetically related to pathotypes A and A*
but able to infect Mexican lime and Citrus macrophylla nat-
urally were recently detected in Florida and classified as a
pathotype designated Aw(68). The molecular basis of the
specific interaction of X. citri pv. citri pathotypes A* and Aw
with a restricted range of citrus hosts is not known (5). An
interaction between a host resistance gene and an avr gene
product from the pathogen inducing host-pathogen incom-
* Corresponding author. Mailing address: CIRAD, UMR Peuple-
ments Ve ´ge ´taux et Bioagresseurs en Milieu Tropical CIRAD-Univer-
site ´ de la Re ´union, Po ˆle de Protection des Plantes, 7 chemin de l’Irat,
97410 Saint Pierre, La Re ´union, France. Phone: 262 262 49 92 20. Fax:
262 262 49 92 93. E-mail: email@example.com.
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 16 December 2008.
patibility has not yet been demonstrated for the X. citri pv.
citri-citrus pathosystem, as it has been previously for other
plant pathogenic bacteria (43).
No assumption can be made about whether the apparent
contemporary emergence of pathotype A* strains is due to a
change in virulence or to environmental or human factors.
Host range shifts have sometimes been related to modifica-
tions in the repertoire of virulence genes by horizontal gene
transfer or intragenomic recombinations or mutations (20, 32,
75). A clear understanding of the evolutionary relationships
among pathotypes A, A*, and Awand of the diversity among
strains of each pathotype would be helpful for assessing these
Due to the extreme difficulty and cost of the complete erad-
ication of Asiatic citrus canker, several canker-threatened cit-
rus-producing regions rely on integrated pest management
strategies for control (30). Data derived from the huge effort
put into the molecular typing of human bacterial pathogens
(46, 63, 67, 69) suggest that an extensive knowledge of popu-
lations of plant pathogenic bacteria may improve our under-
standing of epidemic situations.
The tools most often used for the molecular epidemiology of
citrus canker have been repetitive-element-based PCR (rep-
PCR) and pulsed-field gel electrophoresis (PFGE) (13, 16, 19,
28, 68). The lack of discriminatory power of rep-PCR and the
high labor requirement for PFGE make it difficult to use these
techniques extensively for outbreak investigations or regional
or global surveillance (67). Therefore, alternative high-resolu-
tion and high-throughput molecular typing systems for X. citri
pv. citri should be developed. Amplified fragment length poly-
morphism (AFLP) analysis of an Iranian collection of strains
causing Asiatic citrus canker suggested previously that this
technique has better discriminatory power than the rep-PCR
method (39). AFLP has the advantage of generating a large
number of randomly located markers over the whole genome.
The detected polymorphism may arise from point mutations at
the targeted restriction sites or from insertions and/or dele-
tions in the amplified region (73). The determination of the
complete sequence of X. citri pv. citri strain 306 (17) should
facilitate the development of molecular typing tools well-suited
for deciphering taxonomy, evolution, and/or epidemiology. For
instance, it gave access to specific primers associated with
transposable elements present in this bacterium (45), which
were used for typing DNA from herbarium specimens showing
canker-like symptoms and originating from different geograph-
ical origins. This technique revealed an unexpectedly high de-
gree of genetic diversity. However, this typing scheme requires
more than 50 PCRs for the full analysis of unknown DNA. A
new insertion sequence ligation-mediated PCR (IS-LM-PCR)
scheme (9) also revealed considerable diversity and is less
labor-intensive. This technique amplifies DNA fragments be-
tween an insertion sequence element and a selected restriction
site (9). We also recently developed a multilocus variable-
number tandem-repeat analysis (MLVA) approach for this
bacterium, a promising technique targeting tandem repeats
(minisatellite-like loci) for fine-scale epidemiology with dis-
tinctive advantages, such as high discriminatory power, maxi-
mal reproducibility of results, and portability of equipment
(12). The characteristics of these newly developed techniques
need to be subjected to a comparative evaluation in order to
determine which methods would be most useful for global
surveillance and molecular epidemiology on small spatial
scales. In this study, we compared the AFLP, MLVA, and
IS-LM-PCR techniques to explore the genetic diversity of a
collection of pathotype A, A*, and Awstrains originating from
Asia. Furthermore, we sought to determine the genetic diver-
sity and structure of X. citri pv. citri strains, including a large
collection of pathotype A* strains for which no extensive char-
acterization study is available at the moment, from the area of
origin of the pathogen.
MATERIALS AND METHODS
Bacterial strains and DNA extraction. A total of 234 bacterial strains isolated
from citrus canker lesions and collected from 26 countries in Asia were used in
this study, together with reference pathotype A, A*, and Awstrains. All strains
were assigned to a pathogenicity group on the basis of results from detached-leaf
inoculation assays performed on Mexican lime (C. aurantifolia), C. macrophylla,
and grapefruit (C. paradisi) (72). Strains producing canker-like lesions on the
three host species were classified as pathotype A strains. Both pathotype A* (n ?
59) and Aw(n ? 6) strains produced canker-like lesions on C. aurantifolia and C.
macrophylla but not on C. paradisi. Thus, the two types were pathogenically
indistinguishable (see Table S1 in the supplemental material for information on
each strain, including the geographical origin, host species, date of collection,
and pathogenicity group). Single colonies were subcultured on plates containing
YPGA (yeast extract, 7 g liter?1; peptone, 7 g liter?1; glucose, 7 g liter?1; agar,
18 g liter?1; and propiconazole, 20 mg liter?1) for 24 h at 28°C. These subcul-
tures were used to inoculate tubes containing 4 ml of YP broth (yeast extract, 7 g
liter?1; peptone, 7 g liter?1; pH 7.2), and the tubes were incubated at 28°C on an
orbital shaker for 16 to 18 h. These suspensions were used for DNA extraction
with the DNeasy tissue kit according to the instructions of the manufacturer
(Qiagen, Courtaboeuf, France). DNA concentrations were estimated by flu-
orometry with a TKO 100 fluorometer (Hoefer, San Francisco, CA).
AFLP analysis. AFLP fingerprinting was performed mainly according to the
original protocol by Vos et al. (73) as described previously (2). In brief, 25 ng of
DNA was digested with MspI and SacI restriction enzymes as recommended by
the manufacturer (New England Biolabs/Ozyme, Saint Quentin en Yvelines,
France). Then 2.5-?l aliquots of the digested products were added to 22.5-?l
ligation mixes containing 2 ?M MspI adaptor (see Table S2 in the supplemental
material), 0.2 ?M SacI adaptor (Applied Biosystems, Courtaboeuf, France) (see
Table S2 in the supplemental material), and 2 U of T4 DNA ligase (New England
Biolabs/Ozyme, Saint Quentin en Yvelines, France) in 1? T4 DNA ligation
buffer. Ligations were performed for 3 h at 37°C before enzyme inactivation at
65°C for 10 min. For preselective PCR, 10-fold-diluted ligation products were
used as a template in a mix containing 5 mM MgCl2, 0.23 ?M (each) MspI and
SacI primers (see Table S2 in the supplemental material), 0.45 mM (each)
deoxynucleoside triphosphates (New England Biolabs/Ozyme, Saint Quentin en
Yvelines, France), and 0.5 U of Taq DNA polymerase (Goldstar red; Eurogen-
tec, Seraing, Belgium) in 1? Goldstar buffer. The following PCR conditions were
used: initial extension to ligate the second strand of the adaptors at 72°C for 2
min; a denaturation step at 94°C for 2 min; 25 cycles at 94°C for 30 s, 56°C for
30 s, and 72°C for 2 min; and a final extension step at 72°C for 10 min. Tenfold-
dilutions of PCR products were used as templates for selective amplifications.
The selective amplifications using the unlabeled MspI?A, MspI?C, MspI?T, or
MspI?G primer and the SacI?C primer labeled with one of four different
fluorochromes (Applied Biosystems, Courtaboeuf, France) (see Table S2 in the
supplemental material) were performed under the same conditions as the pre-
selective PCR, except that the SacI?C primer concentration was 0.12 ?M. The
following PCR conditions were used: initial denaturation at 94°C for 2 min; 37
cycles of 94°C for 30 s, annealing for 30 s at 65°C in the first cycle, at tempera-
tures decreasing by 0.7°C per cycle for the next 12 cycles, and then at 56°C for the
last 24 cycles, and extension at 72°C for 2 min; and a final extension step at 72°C
for 10 min. Samples were then prepared for capillary electrophoresis by adding
1 ?l of the final PCR product to a mixture of 18.7 ?l of formamide and 0.3 ?l of
a GeneScan 500 LIZ DNA ladder (Applied Biosystems, Courtaboeuf, France) as
an internal standard. The samples were then denatured for 5 min at 95°C and
placed on ice for at least 5 min. Electrophoresis was performed in an ABI
PRISM 3100 genetic analyzer (Applied Biosystems, Courtaboeuf, France) using
a performance-optimized polymer, POP-4, at 15,000 V for about 20 min at 60°C,
with an initial injection of 66 s. The AFLP fingerprints were analyzed visually
using the software GeneScan 3.7 (Applied Biosystems, Courtaboeuf, France). To
1174 BUI THI NGOC ET AL.APPL. ENVIRON. MICROBIOL.
test the reproducibility of the results from the AFLP technique, two independent
DNA extractions were used for all strains and strain 306 of X. citri pv. citri (17)
was used as a control in each AFLP experiment.
IS-LM-PCR analysis. IS-LM-PCR fingerprinting was performed as described
previously (9). In brief, aliquots of bacterial genomic DNA were subjected to
restriction enzyme and ligated to the adaptor by incubation for 3 h at 37°C in
total volumes of 20 ?l containing 2 ng of DNA, 9 U of MspI (New England
Biolabs/Ozyme, Saint Quentin en Yvelines, France), 50 U of T4 DNA ligase
(New England Biolabs/Ozyme, Saint Quentin en Yvelines, France), 50 mM
NaCl, 1 ?M MspI adaptor (Applied Biosystems, Courtaboeuf, France) (see
Table S3 in the supplemental material), and 1? bovine serum albumin in 1? T4
DNA ligation buffer, followed by enzyme inactivation at 65°C for 10 min. Tenfold
dilutions were used as template DNA in 20 ?l of a PCR mix which contained 1
mM (each) deoxynucleoside triphosphates, 5 mM MgCl2, 0.25 ?M unlabeled
MspI primer (Applied Biosystems, Courtaboeuf, France) (see Table S3 in the
supplemental material), 0.25 ?M 5?-end-labeled insertion sequence-specific
primer (Applied Biosystems, Courtaboeuf, France) (see Table S3 in the supple-
mental material), and 0.5 U of Taq DNA polymerase (Goldstar red; Eurogentec,
Seraing, Belgium) in 1? Taq Goldstar buffer. The following PCR conditions
were used: initial extension to ligate the second strand of the adaptors at 72°C for
2 min; a denaturation step at 94°C for 2 min; 35 cycles at 94°C for 45 s, 60°C for
60 s, and 72°C for 60 s; and a final extension step at 72°C for 10 min. Samples
were prepared and subjected to capillary electrophoresis as explained above. To
test the reproducibility of results from the IS-LM-PCR technique, two indepen-
dent DNA extractions were used for all strains and strain 306 of X. citri pv. citri
(17) was used as a control in each experiment.
MLVA scheme. Fourteen primer pairs targeting single-locus alleles designed
from the full sequence of X. citri pv. citri strain 306 (17) were used in a multiplex
PCR format with a PCR kit from Qiagen (Courtaboeuf, France) (12). Briefly, 2
to 5 ng of genomic DNA was used as a template in mixes containing 0.2 ?M
(each) primers (one of which was marked with one of the fluorescent dyes
6-carboxyfluorescein, NED, PET, and VIC [Applied Biosystems]), 1? Qiagen
multiplex mastermix (containing a hot-start Taq DNA polymerase), 0.5? Q-
solution (Qiagen, Courtaboeuf, France), and RNase-free water to yield a volume
of 15 ?l. PCR amplifications were performed in a GeneAmp PCR system 9700
thermocycler (Applied Biosystems) under the following conditions: 15 min at
95°C for hot-start activation; 25 cycles of 94°C for 30 s, annealing at temperatures
ranging from 64 to 70°C for 90 s, and 72°C for 90 s; and a final extension step at
72°C for 30 min (12). Aliquots of 1 ?l of amplified products diluted 1/50 to 1/200
were mixed with 10.7 ?l of Hi-Di formamide and 0.3 ?l of a GeneScan 500 LIZ
internal lane size standard (Applied Biosystems). Capillary electrophoresis was
performed in an ABI PRISM 3130xl genetic analyzer (Applied Biosystems). To
test the reproducibility of results from the MLVA technique, two independent
DNA extractions were used for all strains and strain 306 of X. citri pv. citri (17)
was used as a control in each experiment.
Data scoring and exploration. For the AFLP and IS-LM-PCR techniques, the
presence and absence of fragments were scored as a binary matrix and analyzed
with the software R (version 2.6.1; R Development Core Team, Vienna, Aus-
tria). The size of each fragment in the range of 50 to 500 bp was determined.
Fragments with fluorescence above a threshold set to 500 relative fluorescence
units were scored. This threshold was found to be suitable for minimizing scoring
discrepancies among DNA replicates in earlier studies (2, 53). Only fragments
detected for both DNA replicates were scored as positive in the data matrix. Dice
dissimilarities were used as distances to construct a weighted neighbor-joining
(NJ) tree (26, 59) with the software R. The robustness of the tree was assessed
by bootstrap analysis (1,000 resamplings). Metric multidimensional scaling
(MDS) was used to represent distances between strains based on a Dice dissim-
ilarity matrix. MDS transforms a distance matrix (which cannot be analyzed by
eigendecomposition) into a cross-product matrix and then solves the eigenvector
problem to find the coordinates of individuals so that distortions in the distance
matrix are minimized. As in principal component analysis, individuals are pro-
jected into n dimensions (1). MDS was performed using the cmd-scale function
in the R software.
For MLVA, integer numbers of tandem repeats were used as input data.
Manhattan distances were calculated and used to build NJ trees with the R
software (version 2.6.1; R Development Core Team, Vienna, Austria) using
“cluster” and “ape” packages. The robustness of trees was assessed by bootstrap
analysis (1,000 resamplings). MDS was also performed as described above, based
on the Manhattan distance matrix. The identification of MLVA types (i.e.,
groups of strains differing by one to three variable-number tandem-repeat
[VNTR] loci) was performed with eBURST, version 3 (25), available at http:
//eburst.mlst.net/. Whether MLVA loci evolve following a stepwise mutation
model (SMM), i.e., preferentially by the addition or loss of a single repeat, was
explored separately for each locus. For this purpose, the difference in the number
of repeats for each pair of haplotypes along the evolutionary path inferred by
eBURST analysis was calculated. The occurrence of each value of repeat differ-
ence was recorded for each group (defined as a collection of strains each with a
maximum of three allelic mismatches with at least one other member of the
collection), and values from all eBURST groups were pooled. This analysis was
performed using multilocus analyzer software (S. Brisse, unpublished data),
which is an independent implementation (coded in Python) of the eBURST
algorithm, to which the SMM test function was added.
Predictive in silico analyses of AFLP, IS-LM-PCR, and MLVA techniques. A
predictive analysis of the AFLP, IS-LM-PCR, and MLVA methods was per-
formed with the genome sequence of X. citri pv. citri strain 306 (17) to determine
the accuracy and reproducibility of the results from each system (7). In the AFLP
analysis, the lengths of predicted fragments corresponded to the lengths of the
restriction fragments produced by simulating digestion with SacI and MspI and
then selecting restriction fragments based on selective nucleotides present on
selective AFLP primers, plus 24 bp corresponding to the length of adaptors. In
the IS-LM-PCR analysis, the lengths of the predicted fragments were calculated
as the size of the fragment bordered by each primer pair from a selection of MspI
restriction fragments containing the targeted insertion sequences. The predicted
fragment size for MLVA corresponded to the length of the PCR product for
each primer pair. A total of 31, 41, and 82 fingerprints using strain 306 were
analyzed for the AFLP, IS-LM-PCR, and MLVA techniques, respectively.
Genetic diversity and population structure. The discriminatory power of each
typing system was calculated using Hunter’s single numerical index of discrimi-
nation (D) (34). This analysis was performed on our collection (n ? 234) typed
by the AFLP, IS-LM-PCR, and MLVA methods and on a subcollection (n ? 34)
including strains studied by Cubero and Graham (16). The correlations between
distance matrices were tested pairwise using the Mantel test (48). All Mantel
tests were performed using GenAlEx, version 6.1, with 9,999 permutations (52).
Nei’s unbiased estimates of genetic diversity (HE) for MLVA data were calcu-
lated using FSTAT 2.9.3 (http://www2.unil.ch/popgen/softwares/fstat.htm). For
biallelic data (AFLP and IS-LM-PCR results), AFLP-SURV software, version
1.0 (71) (http://www.ulb.ac.be/sciences/lagev/aflp-surv.html), was used for com-
puting (i) allelic frequencies from observed frequencies of fragments according
to the method of Lynch and Milligan for haploid species (47) and (ii) HE.
The allelic richness of our two sample sets composed of strains of pathotypes
A and A*/Awwas estimated by a rarefaction method (36) producing unbiased
estimates due to uneven sample sizes. The rarefaction method was performed
with HP-RARE, version 1.0 (37). The degree of linkage disequilibrium was
determined using the index of association (IA) (49) with the software GenAlEx,
version 6.1 (52). IAis calculated by comparing the observed variance (VO) in the
distribution of allelic mismatches in all pairwise comparisons of the allelic pro-
files with the expected variance (VE) in the freely recombining population, as
follows: IA? (VO/VE) ? 1. Significant linkage disequilibrium is established if the
variance observed in the MLVA allele profiles is greater than the maximum
variance observed in 1,000 randomized allele profiles (P ? 0.001) (49).
We described the structure of the strain collection by using different ap-
proaches. Populations were defined at the geographical level when at least 10
strains of pathotype A or A*/Aworiginated from the same country. When fewer
than 10 strains per pathotype-country combination were present, we grouped
strains from neighboring countries with no natural geographical barriers and/or
with a common past history, e.g., those of the Indian peninsula; otherwise, such
countries were not included in the analyses. A total of eight (10- to 27-strain) and
three (10- to 24-strain) populations of pathotype A and A* strains, respectively,
were defined (see Table S1 in the supplemental material). Genetic differentiation
among populations was examined using different approaches. For MLVA data,
pairwise population differentiation was assessed by Fisher exact tests (57) and
tested for significance by the Markov chain Monte Carlo method using Arlequin,
version 3.1 (22). For biallelic data, the results of exact tests were computed using
the software TFPGA (Tools for Population Genetic Analyses), version 1.3 (42).
A hierarchical analysis of molecular variance (AMOVA) was conducted with
Arlequin software, version 3.1 (22). Significance was tested using a nonparamet-
ric approach. Partitioning between data sets for pathotypes A and A* and among
data sets for strains of each type from different countries and from the same
country was conducted to evaluate the different contributions of these sources of
variation for each genotyping technique. Estimates for Wright’s fixation index of
genetic differentiation (FST) and Slatkin’s FSTanalogue RST, which takes into
account the size difference among alleles and is more appropriate than FSTwhen
the loci studied evolve under the basic SMM (66), were obtained from the three
sets of data (AFLP, IS-LM-PCR, and MLVA results) and from MLVA data,
respectively. Estimates of FSTfor MLVA data and their respective P values for
population pairs and all populations were calculated by using FSTAT 2.9.3. In
VOL. 75, 2009GENETIC DIVERSITY OF X. CITRI PV. CITRI IN ASIA1175
the same way, RSTestimates were calculated using the software RST CALC,
version 2.2 (27). For AFLP and IS-LM-PCR data, FSTwas calculated using the
software AFLP-SURV, version 1.0.
Isolation by distance among the eight populations of pathotype A was tested
(58). The correlation between the logarithm of the geographical distances, re-
taining a central point for each population area, as defined above, and FST/(1 ?
FST) was evaluated with a Mantel test using the software GenAIEx 6.1 for each
data set. Statistical testing was conducted using random permutation (n ? 9,999).
The Bayesian clustering approach implemented in the software STRUCTURE,
version 2.2.3, was used to infer population structure and assign individuals to groups
characterized by distinct allele frequencies (54). The method estimates a probability
of ancestry for each individual from each of the groups. Individuals are assigned to
one of the groups or populations or jointly to two or more populations if their
genotypes indicate that they are admixed. Twenty independent runs of
STRUCTURE were performed by setting the number of subpopulations or groups
(K) from 1 to 10, with 20,000 burn-in replicates and a run length of 105replicates to
decide which value of K best fit the data. The selection of K was done by examining
the estimates of the posterior probability of the data for a given value of K, Pr(X K)
(where X represents the number of genotypes in the sample), as a guide and by
estimating the modal value of the distribution of ?K [calculated from the
STRUCTURE output Pr(X K)], which is a good indicator of the real K (21). We
examined the clustering of strains of X. citri pv. citri for the inferred number of
groups. We used different ancestry models in STRUCTURE according to the data
sets. The linkage model (24) was used for MLVA data. For the biallelic markers
(AFLP and IS-LM-PCR data), we used the admixture model and performed clus-
that the allele frequencies in the populations are correlated (23).
Predictive in silico AFLP, IS-LM-PCR, and MLVA typing.
In the AFLP analysis, 111 of 147 predicted fragments (76%)
for the sequenced X. citri pv. citri strain 306 were always re-
corded and 8 predicted fragments were amplified in an irre-
producible way. AFLP markers were scattered throughout the
X. citri pv. citri genome. In the IS-LM-PCR analysis, 34 of 39
predicted fragments (87%) were always recorded, 3 predicted
fragments were occasionally amplified, and 4 unpredicted frag-
ments were reproducibly recorded. In MLVA, all predicted
fragments were observed.
AFLP analysis. Although fingerprints obtained for DNA
replicates sometimes differed in fragment intensity, the scoring
system allowed overall good reproducibility of AFLP results,
with ?95% of the corresponding fragments in replicates being
identically assigned. A total of 182 fragments, 100 (55%) of
which were polymorphic, were scored. Ninety-four haplotypes
were identified within the strain collection (n ? 234) (Table 1).
Based on the NJ tree (see Fig. S1A in the supplemental ma-
terial) and the MDS plot (Fig. 1A), a clear-cut separation
between pathotype A and A*/Awstrains was most often ob-
served. A noticeable exception concerned a set of pathotype A
strains from Bangladesh and India that were genetically close
to pathotype A* strains but produced canker-like lesions on all
assayed Citrus species (data not shown). The two graphical
representations also suggested greater polymorphism within
the A*/Awgroup, an indication supported by the calculation of
Nei’s unbiased total gene diversity index (HT; 0.13 for patho-
type A*/Awstrains versus 0.03 for pathotype A strains) (Table
1). Although pathotype Awstrains formed a subclade sup-
ported by a maximal bootstrap value, these strains were closely
related to pathotype A* strains from India, with which they
formed a cluster with a bootstrap value of 97%. The A*/Aw
group formed a total of eight robust clusters supported by
bootstrap values of ?84% (see Fig. S1A in the supplemental
material). Some A*/Awclusters contained strains from a single
origin (e.g., India or Thailand), whereas other clusters con-
tained strains from more than one country (e.g., Saudi Arabia
and Iran or Saudi Arabia, Oman, and India). In contrast,
pathotype A strains did not generally form robust clusters (see
Fig. S1A in the supplemental material). In rare cases, patho-
type A strains grouped according to their countries of origin
(e.g., strains from the Philippines and Bangladesh), but most
often, closely related strains originated from different coun-
tries. Some haplotypes of pathotype A strains were identified
in several countries. For example, haplotype 1 (24 strains) was
detected in China, South Korea, Japan, the Philippines, Tai-
wan, and Thailand. Similarly, haplotype 5 (19 strains) was
detected in China, Japan, Malaysia, the Philippines, and Tai-
wan (see Table S1 in the supplemental material).
IS-LM-PCR analysis. The overall reproducibility of IS-LM-
PCR results was similar to that described for AFLP results.
The global diversity (HT) ranged from 0.06 (for insertion ele-
ment ISXac2) to 0.13 (for ISXac1) (Table 1). Based on Mantel
test results, all distance matrices derived from a single primer
pair were significantly correlated (P ? 0.001). Pooling data
ensured that the NJ tree had a robust structure, as indicated by
bootstrap values (data not shown). When data derived from
the four insertion sequence primer pairs were pooled, a total of
336 markers, 329 (98%) of which were polymorphic, were
scored. The relationships among the 146 haplotypes that were
identified within the strain collection (n ? 234) are shown by
an NJ tree (see Fig. S1B in the supplemental material) and the
MDS plot in Fig. 1B. The two first MDS axes described 85.8%
of the total variation. A clear-cut separation between patho-
type A and A*/Awstrains was observed, which is consistent
with AFLP results (Fig. 1A and B). Pathotype Awstrains
formed a subclade supported by a bootstrap value of 87% but
were closely related to pathotype A* strains from India, form-
ing a cluster with a maximal bootstrap value. The A*/Awgroup
formed a total of 10 robust clusters supported by maximal
bootstrap values (see Fig. S1B in the supplemental material).
This high level of diversity was confirmed by calculating the
genotypic diversity. The gene diversity (Nei’s unbiased gene
diversity index [HT]) calculated for pathotype A*/Awstrains
(HT? 0.20) was greater than that calculated for pathotype A
strains (HT? 0.05). The strain compositions of A*/Awclusters
derived from AFLP and IS-LM-PCR techniques were highly
consistent. Pathotype A strains did not generally form robust
clusters (see Fig. S1 in the supplemental material), which is
consistent with AFLP data. Due to the greater discriminatory
power of IS-LM-PCR, haplotypes that included a large num-
ber of strains were rare. Nevertheless, as in the AFLP analysis,
it was possible to identify a few haplotypes of pathotype A
strains that were found in several countries. For example, hap-
lotype 10 (seven strains) was detected in China, Japan, and
Taiwan. Pathotype A strains that showed the greatest genetic
relatedness to pathotype A* strains originated from western
Asia (Bangladesh, India, and the Maldives), which is consistent
with AFLP data.
MLVA scheme. A total of 209 haplotypes among the 234
strains were detected when data from the 14 loci were
pooled, with allele numbers per locus ranging from 6 (for
locus XL11) to 29 (for locus XL2). Five pathotype Aw
strains and two pathotype A* strains from India did not
produce amplicons for the XL5 locus. The mean numbers of
1176BUI THI NGOC ET AL.APPL. ENVIRON. MICROBIOL.
observed alleles for types A and A* were 12.3 and 9.8,
respectively, revealing a slightly higher degree of allelic rich-
ness in pathotype A strains (Table 1). Strains with the same
allelic profile were primarily those isolated from the same
site during the same year. In a few cases, strains sharing the
same allelic profile were isolated from sites several miles
apart in the same year or from the same site over two years.
Similarly, each MLVA type originated from a single coun-
try. The relationships among the haplotypes that were iden-
tified within the strain collection are shown by an NJ tree
(see Fig. S1C in the supplemental material) and the MDS
plot in Fig. 1C. An eBURST analysis of the 209 MLVA
types yielded 32 groups and 108 singletons, which is consis-
tent with AFLP and IS-LM-PCR data. In order to determine
whether tandem repeats evolve by following an SMM, we
computed all the differences in the number of repeats along
the evolutionary path deduced by the eBURST analysis
within each eBURST group. Generally, the distribution of
occurrences showed a single mode centered around zero.
For most loci where at least four changes occurred in total
(e.g., loci XL1, XL3, XL7, and XL11 to XL14), the most
frequent change was either ?1 or ?1 repeat unit and the
symmetric change was generally the second most frequent.
Two noticeable exceptions were locus XL10, with more ?2
TABLE 1. Genetic diversity parameters for X. citri pv. citri strains estimated by using MLVA (14 VNTR loci), AFLP (4 conditions), and IS-
LM-PCR (4 conditions)a
Method Locus(i) or primer combination(s)
Total no. of
All X. citri loci
Loci from pathotype A strains
Loci from pathotype A* strains
137.6 0.79 0.480.54*** 0.92***
AFLP analysis SacI?C/MspI?A
Combinations for pathotype A
strains (167 strains)
Combinations for pathotype A*
strains (65 strains)g
Combinations for pathotype A
strains (167 strains)
Combinations for pathotype A*
strains (65 strains)g
a???, P ? 0.001; ??, P ? 0.01; ?, P ? 0;05;NS, not significant (P ? 0.05).
bNumber of alleles observed at each locus. Allelic richness for both pathotypes was calculated using the rarefaction method (n [corresponding to the number of
individuals in the smallest group] ? 65).
cHW, Nei’s mean index of gene diversity within populations.
dFSTestimates were calculated for eight and three geographical groups (with 10 or more members) composed of pathotype A and A* strains, respectively.
eRSTestimates from VNTR data were calculated for eight and three geographical groups (with 10 or more members) composed of pathotype A and A* strains,
fParameters were calculated for 225 strains, as 7 pathotype A*/Awstrains did not produce any amplicon for the XL5 locus.
gStrains originally described as pathotype Aw(68) were included in the pathotype A* group for data derived from all three genotyping techniques.
VOL. 75, 2009 GENETIC DIVERSITY OF X. CITRI PV. CITRI IN ASIA 1177
changes (n ? 6) than ?1 (n ? 3) and ?1 (n ? 3) changes,
and locus XL9, with more ?2 changes (n ? 4) than ?1 (n ?
3) or ?1 (n ? 1) changes. However, the numbers were small
in these two cases.
The IAcalculated for the whole strain collection or for each
pathotype separately showed that there was significant linkage
disequilibrium (P ? 0.001) between loci.
Genetic diversity and population structure. The discrimina-
tory abilities of the AFLP, IS-LM-PCR, and MLVA tech-
niques was determined and compared by calculating D for 234
strains typed by all three methods. MLVA differentiated 209
strains and showed the best level of discrimination, with a D
value of 0.998. IS-LM-PCR and AFLP markers distinguished
146 strains (D ? 0.991) and 94 strains (D ? 0.970), respectively
(Table 2). The combination of MLVA and IS-LM-PCR data
improved the discrimination among the strains to a D value of
0.999 (218 of 234 strains were discriminated). The three meth-
ods were more discriminative than rep-PCR based on the data
from a collection of 34 strains (Table 2).
Mantel test results suggested that data derived from the
three typing techniques were congruent (P ? 0.001) (Table 3).
The highest values of correlation between individual genetic
distances were observed for AFLP and IS-LM-PCR data (r ?
0.866; P ? 0.001). Lower but highly significant values of cor-
relation between MLVA and IS-LM-PCR data (r ? 0.618, P ?
0.001) and MLVA and AFLP data (r ? 0.599; P ? 0.001) were
found. Correlations for individual pairs of genetic distances
were also highly significant when determined by considering
pathotype A and A*/Awstrains separately (Table 3).
Hierarchical AMOVA revealed highly significant genetic
variation between pathotypes (P ? 0.01), as well as between
populations or within populations, whatever the molecular
markers used (Table 4). The patterns of partitioning of the
FIG. 1. MDSrepresentationofthedistancesamong234strainsofX.citri
pv. citri based on AFLP (A), IS-LM-PCR (B), and MLVA (C) data. X. citri
pv. citri pathotypes are represented by different symbols, including symbols
indicating the geographical origins of pathotype A strains, as follows: E,
pathotype A from eastern and southern Asia; R, pathotype A from western
Asia; f, pathotype A*; and Œ, pathotype Aw. Numbers indicate the geo-
graphical origins of pathotype A* strains, as follows: 1, Saudi Arabia; 2,
Florida; 3, the Indian peninsula; 4, Iran; 5, Oman; and 6, Thailand or Cam-
bodia. The two first MDS axes described 86.2% (A), 85.8% (B), and 83.4%
(C) of the total variation.
TABLE 2. Discriminatory powers of AFLP, IS-LM-PCR, and MLVA
methods based on results for a collection of 234 strains of X. citri pv.
citri and a subset of 34 strains typed by rep-PCRa
(n ? 234)
(n ? 34)
aRep-PCR data are from Cubero and Graham (16).
bND, not determined.
TABLE 3. Mantel test results for pairwise correlations between
genetic distances among individual strains for the different
typing methods and pairwise population
Pathotype A strains
Pathotype A* strains
All populations0.8660.599 0.618
arA, AFLP versus IS-LM-PCR data; rB, AFLP versus MLVA data; and rC,
IS-LM-PCR versus MLVA data. P, ?0.001 for all values.
1178BUI THI NGOC ET AL.APPL. ENVIRON. MICROBIOL.
total variance differed among the different data sets. For
AFLP, most of the total genetic variation (66.5%) was found
between pathotypes and the remainder was distributed evenly
among or within populations. Most of the genetic variation
indicated by IS-LM-PCR was also between pathotypes, but to
a lesser extent (52.5%), and the second source of variation was
found at the population level. In contrast, analysis of the
MLVA data set revealed that most of the total genetic variance
was found among individual strains within populations (65.5%)
and that the genetic variation between pathotypes accounted
for only 12.2% of the total variation (Table 4).
For the eight defined X. citri pv. citri pathotype A popula-
tions, significant correlation was found between the logarith-
mic geographical distances and FST/(1 ? FST), as estimated
from IS-LM-PCR and MLVA data sets (P ? 0.05) but not
from the AFLP data set, with correlation values of 0.536, 0.507,
and 0.371, respectively.
Fisher exact tests revealed little differentiation among the
eight groups of pathotype A strains. The Indian peninsula
group was differentiated from the group defined for Thailand
(P ? 0.05; Fisher exact test) and, with some significance, from
those defined for China and the Philippines (P ? 0.10), what-
ever the genotyping method (Table 5). Genetic differentiation
between pathotype A strains from Thailand and the groups
from China and the Philippines (P ? 0.05) was also observed,
but only for the MLVA data set. Unlike pathotype A strains,
the three defined groups of pathotype A* strains, i.e., those
from Saudi Arabia, Iran, and Thailand, were found to be ge-
netically differentiated, whatever the typing technique (P ?
0.01; Fisher exact test). Based on FSTanalogues, when patho-
types were analyzed separately, larger differentiation estimates
were obtained for pathotype A* strains than for pathotype A
strains, whatever the typing technique (Table 1). Furthermore,
genetic differentiation was observed in pairwise population
analyses performed with biallelic markers, with AFLP-based
and IS-LM-PCR-based FSTestimates showing 27 of 28 and 28
of 28 pairs, respectively, to be significantly different (P ? 0.01).
FSTestimates, when significant, varied from 0.07 (Taiwan/
Thailand) to 0.62 (South Korea/the Maldives) and from 0.059
(Japan/Taiwan) to 0.67 (Japan/the Maldives) for AFLP and
IS-LM-PCR, respectively. The level of genetic differentiation
revealed by the MLVA-based FSTvalues and RSTvalues was
lower, with 24 of 28 and 26 of 28 pairs, respectively, being
significantly different (P ? 0.01). Differentiation estimates var-
ied from 0.07 (Philippines/Thailand) to 0.31 (Japan/the
Maldives) and from 0.07 (Philippines/Thailand) to 0.81
(China/the Maldives) for MLVA-based FSTand RSTestimates,
respectively. All the analyzed pairs of pathotype A* strains
(n ? 3) revealed genetic differentiation (P ? 0.01) when FST
and RSTwere estimated from biallelic markers or MLVA data.
FSTestimates were between 0.47 and 0.87, 0.78 and 0.93, and
0.46 and 0.63 for AFLP, IS-LM-PCR, and MLVA data, re-
spectively. MLVA-based RST estimates for comparisons
among pathogen A* groups varied from 0.90 to 0.95.
Bayesian clustering was used to analyze multilocus haplotypes
to infer the genetic ancestry of the individual strains from both
pathotypes separately. When pathotype A strains (n ? 167) were
considered, the analyses showed a pattern typical of an unstruc-
tured population, whatever the typing technique. No plateau in
the estimates of the log likelihoods was reached, and no consis-
TABLE 4. Hierarchical AMOVA of X. citri pv. citri populations (eight pathotype A and three pathotype A* groups) by using AFLP, IS-LM-
PCR, and VNTR markers
Level at which variation
Variance by: % Total variation by:
Among populations within
173 2.24 5.19 4.0418.7718.6465.54 0.81***0.81*** 0.34***
adf, degrees of freedom.
b??, P ? 0.01; ???, P ? 0.001. Populations/geographical groups with 10 or more strains were considered.
TABLE 5. Genetic differentiation between X. citri pv. citri pathotype A subpopulations as estimated by Fisher exact testsa
P value for comparison with population from:
aP values for the AFLP/IS-LM-PCR data sets (below the diagonal) and for the MLVA data set (above the diagonal) are given.
VOL. 75, 2009GENETIC DIVERSITY OF X. CITRI PV. CITRI IN ASIA 1179
tency among typing techniques for the compositions of putative
populations was found. On the contrary, STRUCTURE identi-
fied two to four ancestral groups within the collection of patho-
type A* strains, depending on the typing technique. Observa-
tions of plateaus in the estimates of ln [Pr(X K)] and a clear
modal value in the distribution of ?K indicated values of K of
2, 3, and 4 for the IS-LM-PCR, AFLP, and MLVA data sets,
respectively (Fig. 2). The assignments of individual pathotype
A* strains to ancestral clusters were consistent among the
different data sets and correlated primarily with the strains’
geographical origins. Strains from Florida, together with two
strains from the Indian peninsula, were identified as a single
population, whatever the typing technique. Two populations of
strains from Saudi Arabia were consistently identified. One of
the populations identified shared a common ancestor with
strains from Iran, based on AFLP and IS-LM-PCR results, but
the latter strains were unique based on MLVA data (Fig. 2).
The second identified population of strains from Saudi Arabia
shared a common ancestor with strains from India, Oman,
Thailand, and Cambodia, whatever the genotyping technique
The availability of the complete genome of a Brazilian
strain of X. citri pv. citri with a wide host range among citrus
plants (pathotype A) (17) made it easier to design appro-
priate markers and predict the DNA fragments produced by
different PCR-based techniques (9, 12, 45). This genome is
relatively rich in insertion sequence elements and tandem
repeats (17, 51). We evaluated the potentials of AFLP,
IS-LM-PCR, and MLVA techniques as genotyping methods
for molecular epidemiology studies of X. citri pv. citri. The
analysis of the genetic diversity of large collections or pop-
ulations requires discriminative, high-throughput tech-
niques able to identify strain types and provide reproducible
results. Despite the worldwide distribution and the major
economic importance of this quarantine organism, no com-
parative evaluation of different molecular typing techniques
for population structure analyses based on large strain col-
lections has been conducted previously. Earlier studies con-
sidered primarily the usefulness of PFGE, AFLP, or rep-
PCR markers in terms of their abilities to determine genetic
relationships between Xanthomonas pathovars that are
pathogenic to citrus and X. citri pv. citri pathological vari-
ants (16, 19, 39, 44, 68).
All three techniques used in this study typed all strains,
except in the case of the XL5 VNTR locus, for which no
amplicon was obtained from a limited number of strains within
pathotype A*. MLVA revealed the greatest genetic diversity
(based on Nei’s and Hunter’s indices), followed by IS-LM-
PCR and AFLP analyses. For instance, pathotype A* strains
from Saudi Arabia and Iran were not differentiated using
AFLP, while IS-LM-PCR analysis and MLVA clearly sepa-
rated strains from the two origins. Furthermore, MLVA iden-
tified each Iranian strain as a haplotype. The discriminatory
powers of these methods were markedly greater than that of
rep-PCR, based on results for a subset of 34 strains used in our
study and a previous study by Cubero and Graham (16).
VNTRs are known to exhibit much higher levels of mutation
than other parts of the genome (35). The discriminatory power
of MLVA was indeed found to be higher than or similar to
those of insertion sequence-based typing techniques in popu-
FIG. 2. Population structure among the collection of X. citri pv. citri pathotype Awstrains and pathotype A* strains from the six geographical
origins as inferred by STRUCTURE and assignment of individuals to the K ancestral groups. Numbers on the x axis indicate the geographical
origins of strains, as follows: 1, Saudi Arabia; 2, Florida; 3, the Indian peninsula; 4, Iran; 5, Oman; and 6, Thailand or Cambodia. Each strain is
represented by a vertical segment. Inside each segment, the length of each color section (y axis) indicates the estimated probability of assignment
to each of the defined K ancestral groups. The use of the same color suggests that strains have a common ancestral group.
1180BUI THI NGOC ET AL.APPL. ENVIRON. MICROBIOL.
lation studies of Mycobacterium bovis and M. tuberculosis (4,
The comparison of experimental data with in silico data
derived from the complete sequence of X. citri pv. citri strain
306 (17) indicated that the MLVA scheme was 100% accurate,
whereas not all predicted fragments were amplified in the
IS-LM-PCR and AFLP analyses, leading to accuracy values of
87 and 75%, respectively. This finding suggests that digestion
and/or ligation may be deficient at some sites. The level of
accuracy obtained by the AFLP method for X. citri pv. citri
strain 306 was between those obtained previously for a strain of
Escherichia coli (92%) and two strains of M. tuberculosis (55
and 66%) (7, 65). Although the overall intralaboratory repro-
ducibility of AFLP and IS-LM-PCR data was good (95 to
97%), it did not reach that obtained for MLVA results (100%).
Consistent with our data, previous interlaboratory compari-
sons of DNA typing methods for M. tuberculosis indicated
better reproducibility of MLVA results than IS-LM-PCR and
AFLP results (40).
Based on results from Mantel tests of dissimilarities, AFLP,
IS-LM-PCR, and MLVA data were significantly congruent.
Congruency among the data from the different methods was
further observed for the parameters of genetic diversity. Patho-
type A* strains were always clearly separated from the large
majority of pathotype A strains. However, a few pathotype A
strains were consistently shown to be related to pathotype A*
strains. These strains originated from Bangladesh and India.
Interestingly, they were all isolated from lime and displayed
pathogenicity patterns typical of pathotype A. These observa-
tions suggest that variations in host range may have occurred in
populations of X. citri pv. citri that originated from the Indian
peninsula. Populations of the pathogen from this region
display the greatest genetic diversity revealed to date, with the
presence of two groups of pathotype A* strains and with
pathotype A strains that were genetically related either to
pathotype A strains present in southeast Asia or to pathotype
A* strains. It is likely that undescribed genetic and/or patho-
logical variants of the pathogen are yet to be discovered.
Clusters identified in relation to the geographical origins of
the strains were consistently reproduced among X. citri pv. citri
pathotype A* strains, whatever the typing technique. A patho-
type A* strain was detected in Cambodia in 2007 (11), and this
strain was closely related to pathotype A* strains from Thai-
land (10). Similarly, pathotype Awstrains originating from
Florida were closely related to some pathotype A* strains
isolated in India: these strains belonged to a single cluster with
high bootstrap values, whatever the typing technique, and none
of them could be typed using the XL5 VNTR locus. In addi-
tion, STRUCTURE suggested a common ancestry for patho-
type Awstrains and the pathotype A* strains isolated in India.
Thus, our molecular data fully support the results of investi-
gations in Florida, which revealed that pathotype Awstrains
had been isolated from Mexican lime in the yard of a family
that had recently arrived from India and did not reveal the
source of the diseased plant (62). The distribution area of
pathotype A* strains in Asia is larger than previously reported
and confirms the epidemiological significance of this narrow-
Interestingly, the level of genetic diversity among pathotype
A* strains was higher than that among pathotype A strains,
whatever the typing technique. This finding suggests that
pathotype A* may have a longer evolutionary history. Li et al.
(45) analyzed a few lime herbarium specimens collected in
western Asia during the first half of the 20th century, and no
conclusion could be drawn. Genetic differentiation analyses
indicated that pathotype A* strain populations are well-struc-
tured. The number of ancestral populations computed by
STRUCTURE was genotyping technique dependent, but
pathotype A* strains could originate from at least two ances-
tral populations. Pathotype A* strains from Saudi Arabia were
consistently classified into two populations, whatever the typ-
ing technique, which may suggest at least two different inci-
dents involving the introduction of the pathogen into this
Based on NJ trees and MDS, no clear geographically in-
ferred structure among the pathotype A strains was revealed.
This result is consistent with those of previous studies targeting
diversity by using rep-PCR or transposable elements (16, 45).
Significant patterns of isolation by distance were observed at
the level of the Asian continent. The low degree of global
genetic diversity and the significant isolation by distance sug-
gest a spatially limited gene flow for pathotype A strains and/or
a rather short evolutionary history.
The different levels of structuring were also reflected in
the different values of differentiation estimates for subpopu-
lations of each pathotype. The elevated RSTvalues com-
pared to FSTestimates calculated from the MLVA data set
suggested that the geographical subpopulations of X. citri
pv. citri or of each pathotype differed not only in allele
frequencies but also significantly in the evolutionary dis-
tance between alleles. Different estimates of FSTfrom each
molecular marker data set revealed lower values for MLVA
than for the two biallelic marker systems. MLVA revealed
the highest discriminatory power, as shown in this study by
the identification of 209 different haplotypes among 234
strains. Estimations of FSTdepend partly on the level of
within-population diversity, and estimates derived from
highly mutable loci should be considered with caution (14).
Gene flow between populations homogenizes allele frequen-
cies and tends to decrease genetic differentiation. Further-
more, the differentiation level is not affected strictly by the
mutation potential of the observed loci but also by the ratio
between mutation and migration (8). This situation makes
FSTsensitive to the mutation rates for pathogens such as X.
citri pv. citri with limited long-distance migration (31). The
RSTanalogue values from the MLVA data set are indepen-
dent of the mutation rate and are more appropriate esti-
mates than FSTvalues, given that tandem repeats in our
scheme can be considered as evolving by the progressive
gain or loss of single repeat units. RSTvalues were in accor-
dance with the estimates of genetic differentiation obtained
from the two biallelic data sets. It is likely that the signifi-
cance of the long-distance movement of infected plant ma-
terial, although globally limited, is pathotype dependent.
Pathotype A strains can be transported by most of the citrus
species, primarily through propagative material, providing
much greater potential for the exchange of pathotype A
strains than for that of pathotype A* strains, which are
hosted mainly by limes and, to a lesser extent, C. macro-
phylla. In many Asiatic countries, lime trees are produced
VOL. 75, 2009 GENETIC DIVERSITY OF X. CITRI PV. CITRI IN ASIA1181
from seed and C. macrophylla is used mainly as rootstock,
and the pathogen is not seed borne (15). These host char-
acteristics drastically decrease the movement of budwood (a
major source of long-distance spread). The low level of
migration of pathotype A* strains, together with their nar-
row host range, minimizes gene flow, which can explain the
greater genetic differentiation observed within A* subpopu-
lations. This observation may also be the result of the
greater genetic diversity observed within X. citri pv. citri
Significant levels of linkage disequilibrium among the
VNTR loci were observed using a multilocus estimate, which
revealed the absence of frequent genetic exchanges. Given our
strain collection, however, this finding should be considered
with caution because specific ecological niche and/or geo-
graphical barriers may explain the limited DNA exchange ob-
served (49). Nevertheless, these results, together with the sig-
nificant congruence among data derived from the three
independent genotyping techniques, suggested that both
pathotypes of X. citri pv. citri are clonal. Testing for nonran-
dom association of loci on small scales should confirm the level
of clonality, which may vary among populations. Extensive
sampling of pathotype A* strains in the Indian peninsula, for
which admixtures within some individual strains suggest differ-
ent origins, may reveal a population structure which differs
from that in other geographical areas.
The various discriminative powers of the three genotyping
techniques made it possible to conduct molecular epidemiol-
ogy analyses on different spatial scales and for different pur-
poses. Highly variable markers, such as VNTRs, are not
adapted for international long-term strain screening (70). Al-
though the concomitant use of several typing techniques
strengthens analyses, the increased discriminatory power of the
IS-LM-PCR method compared to those of rep-PCR (16) and
AFLP (this study) analyses makes this technique most appro-
priate for the global surveillance of non-epidemiologically re-
lated strains of X. citri pv. citri. IS-LM-PCR targets transpos-
able elements, similar to the technique developed by Li et al.
(45). IS-LM-PCR seems superior because of the smaller num-
ber of PCR amplifications required. Furthermore, in contrast
with the method used by Li et al. (45), IS-LM-PCR was proved
to provide results highly congruent with those of the AFLP
method, a technique well-suited for analyzing phylogenetic
relationships among xanthomonads (55). These findings sug-
gest that the transposition of insertion sequences in the ge-
nome of X. citri pv. citri occurs so rarely that it does not disturb
the phylogenetic signal. In previous interlaboratory tests in
which nine typing techniques were compared, two IS-LM-
PCR-based techniques were the best alternative to MLVA for
M. tuberculosis in terms of reproducibility (40). This observa-
tion suggests that our IS-LM-PCR protocol may be amenable
to an efficient interlaboratory typing system.
VNTR loci display a high degree of polymorphism and are
well-adapted for the typing of epidemiologically related iso-
lates. Among the three typing techniques used in this study,
MLVA best described the X. citri pv. citri intrapopulation
genetic variation. MLVA may be useful for tracing haplotypes
during epidemics on small spatial scales and for investigating
inoculum sources associated with outbreaks. MLVA was
proved previously to be very useful for the discrimination of
anthrax-causing bacterial populations with a low level of ge-
netic diversity (38). We are currently evaluating MLVA for the
molecular epidemiology of X. citri on different spatial scales
and in different epidemiological contexts (such as integrated
management and eradication).
We thank E. L. Civerolo for helpful discussion and K. Vital, C.
Boyer, and V. Ledoux for their technical expertise.
The European Union (FEOGA and FEDER), Conseil Re ´gional de
La Re ´union, and CIRAD provided financial support. The platform
“Genotyping of Pathogens and Public Health” acknowledges financial
support from the Institut Pasteur (Paris, France) and the Institut de
Veille Sanitaire (Saint-Maurice, France).
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