Lan Bui Thi Ngoc

Amplified fragment length polymorphism and multilocus sequence analysis-based genotypic relatedness among pathogenic variants of Xanthomonas citri pv. citri and Xanthomonas campestris pv. bilvae

Three pathogenic variants (i.e. pathotypes) have been described within Xanthomonas citri pv. citri, the causal agent of Asiatic citrus canker. Pathotype A strains naturally infect a wide range of Citrus species and members of some related genera. In contrast, pathotypes A* and A(w) have narrow host ranges within the genus Citrus and have been isolated from Mexican lime (Citrus aurantifolia L.) and from Mexican lime and alemow (Citrus macrophylla L.), respectively. We used amplified fragment length polymorphism (AFLP) and multilocus sequence analysis (MLSA) based on four partial housekeeping gene sequences (atpD, dnaK, efp and gyrB ) for the genotypic classification of Xanthomonas citri pv. citri and the poorly characterized citrus pathogen Xanthomonas campestris pv. bilvae. A Mantel test showed that genetic distances derived from AFLP and MLSA were highly correlated. X. campestris pv. bilvae showed a close relatedness to the type strain of X. citri, indicating that this pathovar should be reclassified as X. citri pv. bilvae. All pathotype A* and A(w) strains were most closely related to X. citri pv. citri strains with a wide host range (pathotype A), confirming previous DNA-DNA hybridization data. Pathotype A(w) should be considered a junior synonym of pathotype A* on the basis of pathogenicity tests, AFLP, MLSA and PCR using pathovar-specific primers. Evolutionary genome divergences computed from AFLP data suggested that pathotype A* (including A(w) strains) is a group of strains that shows a wider genetic diversity than pathotype A.

From local surveys to global surveillance: three high-throughput genotyping methods for epidemiological monitoring of Xanthomonas citri pv. citri pathotypes.

ABSTRACT 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 predicted 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.

Highly polymorphic markers reveal the establishment of an invasive lineage of the citrus bacterial pathogen Xanthomonas citri pv. citri in its area of origin

Investigating the population biology of plant pathogens in their native areas is essential to understand the factors that shape their population structure and favour their spread. Monomorphic pathogens dispatch extremely low genetic diversity in invaded areas, and native areas constitute a major reservoir for future emerging strains. One of these, the gammaproteobacterium Xanthomonas citri pv. citri, causes Asiatic canker and is a considerable threat to citrus worldwide. We studied its population genetic structure by genotyping 555 strains from 12 Vietnam provinces at 14 tandem repeat loci and insertion sequences. Discriminant analysis of principal components identified six clusters. Five of them were composed of endemic strains distributed heterogeneously across sampled provinces. A sixth cluster, VN6, displayed a much lower diversity and a clonal expansion structure, suggesting recent epidemic spread. No differences in aggressiveness on citrus or resistance to bactericides were detected between VN6 and other strains. VN6 likely represents a case of bioinvasion following introduction in a native area likely through contaminated plant propagative material. Highly polymorphic markers are useful for revealing migration patterns of recently introduced populations of a monomorphic bacterial plant pathogen.

Ligation-mediated PCR, a fast and reliable technique for insertion sequence-based typing of Xanthomonas citri pv. citri

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a major disease threatening citrus crops throughout the world. The most common methods for strain differentiation of this pathogen are repetitive element sequence-based PCR (rep-PCR) and pulsed field gel electrophoresis (PFGE), using rare-cutting restriction enzyme analysis. We developed a ligation-mediated PCR targeting three insertion sequences (IS-LM-PCR) present as several copies in the genome of the fully sequenced strain 306 of X. citri pv. citri. This technique amplifies DNA fragments between an insertion sequence element and an MspI restriction site. The analysis of strains can be conducted within 24 h, starting from very small amounts of bacterial DNA, which makes IS-LM-PCR much less labor-intensive than PFGE. We used IS-LM-PCR to analyze a collection of 66 strains of X. citri pv. citri from around the world. The overall reproducibility of IS-LM-PCR reached 98% in this data set and its discriminatory power was markedly superior than rep-PCR. We suggest that IS-LM-PCR could be used for the global surveillance of non-epidemiologically related strains of X. citri pv. citri.