Investigating the population structure of Xanthomonas citri pv. citri in Vietnam. Which molecular markers to discrimi-

nate among low polymorphic bacterial populations ?

Xanthomonas citri • pv. citri (Xcc)

causes Asiatic citrus canker, a ma- -jor threat of citrus worldwidetwo main pathotypes with a broad -(Xcc-A) and a restricted (Xcc-A*) host rangesa quarantine organism in many countries. -low genetic diversity (rep-PCR, PFGE) -

Analyses of large collections or populations require •highly discriminant, typeable, reproducible and hi-gh-throughput techniques.

Investigating the population structure is valuable to understand : •

epidemic situations at small geographical or temporal scales (identification of -inoculum sources and pathway)evolution of pathogenic populations and identification of evolutive factors -

Material & methods

557 strains isolated in 2006 in Vietnam, an ende- •mic situation

IS-LM-PCR : targets regions between insertion se- •quences present as several copies in the genome of Xcc-strain 306 and a Msp site

MLVA : 14 primer pairs targeting single-VNTR locus •designed from the strain 306 sequence and used in multiplex PCR format

Population structure

Both markers produced a •similar population structure and individuals assignment as given by STRUCTURE

Two “ancestral” populations •constituted the Vietname-se X. citri pv. citri contem-porary strains (based on Structure analyse). Circles present the average pro-portion of ancestral popu-lations by province as in-ferred by structure (I for IS-LM-PCR and M for MLVA data)

Discriminatory power of IS-LM-PCR and MLVA

markerSimpson

indexnumber of haplotypes

polymorphism

IS-LM-PCR 0.959 190 54%

MLVA 0.998 463 100%

Hierarchical AMOVA of Xcc from 9 province collections

variance of components

% total variation F statistics

marker IS-LM-PCR MLVA IS-LM-PCR MLVA IS-LM-PCR MLVA

among regions (N-S) 0.012 0.40 3 7 0.028*** 0.072***

among provinces 0.050 0.63 11 12 0.108*** 0.120***

within provinces 0.415 4.38 87 81 0.133*** 0.183***

Genetic differentiationHa Noi Ha Tinh Hung Yen Nghe An Can Tho Dong Thap Lam Dong Long An Tien Giang

Ha Noi 0.361 0.003NS 0.099 0.331 0.453 0.356 0.274 0.296Ha Tinh 0.176 0.419 0.472 0.529 0.626 0.567 0.441 0.521Hung Yen 0.013NS 0.220 0.128 0.369 0.505 0.391 0.297 0.335Nghe An 0.073 0.227 0.102 0.319 0.426 0.360 0.274 0.295Can Tho 0.127 0.143 0.181 0.100 0.129 0.100 0.145 0.040Dong Thap 0.213 0.229 0.256 0.172 0.093 0.317 0.324 0.215Lam Dong 0.226 0.202 0.278 0.199 0.097 0.160 0.116 0.054Long An 0.164 0.158 0.223 0.120 0.049 0.112 0.079 0.106Tien Giang 0.152 0.155 0.210 0.126 0.036 0.100 0.076 0.035FST values for pairwise comparisons of 9 province collections based on IS-LM-PCR data (above) and MLVA data (be-low). Pairwise comparisons of populations within a same region have the same color NORTH & SOUTH

Highly significant differentiation between northern and southern pro- •vinces

Low to moderate differentiation between southern populations •

Variable differentiation between some northern provinces •

Congruence (Mantel test) between:

Distance matrices from IS markers • (P<0.001)

Distance matrices from both techniques •(P<0.001)

Conclusions

IS-LM-PCR MLVADiscriminatory power + ++Intra-laboratory reproducibility + ++Labor intensive technique + -Ease of data scoring + ++

Both markers produced congruent results •

MLVA markers: great interest in molecular epidemiology •of Xcc at small scale and population structure analysis

IS-LM-PCR: more appropriate for a global surveilance sys- •tem of X. citri pv. citri

Lan Bui Thi Ngoc1,2, Christian Verniere2,Karine Vital2 and Olivier Pruvost2

1Southern Horticultural Research Institute (SOFRI) – Long Dinh, Tien Giang, VietNam 2CIRAD- UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, Saint-Pierre, La Réunion, France

Objectives

Evaluation of two typing techniques, insertion sequence ligation-mediated PCR (IS-LM-PCR) and multilocus variable number tandem repeat analysis (MLVA) for epidemiological and popula-tion structure analysis based on a large collection from nine provinces in Viet Nam

Multidimensional scaling of the distances between 554 strains from 12 provinces based on (A) IS-LM-PCR and (B) MLVA

population 2

population 1

IS-LM-PCR:ISXac1, ISXac2, ISXac3

336 markers

genome of Xcc 306

VNTR:14 loci

Funding :

➀ Ha Noi➁ Ha Tinh➂ Hung Yen➃ Nghe An➄ Phu Tho

➊ Can Tho➋ Dong Nai➌ Dong Thap➍ Lam Dong➎ Long An➏ Tien Giang➐ Vinh Long

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➀ Ha Noi➁ Ha Tinh➂ Hung Yen➃ Nghe An➄ Phu Tho➊ Can Tho➋ Dong Nai➌ Dong Thap➍ Lam Dong➎ Long An➏ Tien Giang➐ Vinh Long

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