From Local Surveys to Global Surveillance: Three High-Throughput Genotyping Methods for Epidemiological Monitoring of Xanthomonas citri pv. citri Pathotypes

CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical CIRAD-Université de la Réunion, Pôle de Protection des Plantes, 7 chemin de l'Irat, 97410 Saint Pierre, La Réunion, France.
Applied and Environmental Microbiology (Impact Factor: 3.67). 02/2009; 75(4):1173-84. DOI: 10.1128/AEM.02245-08
Source: PubMed


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 A(w), 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.

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    • "Given that Xau B and C have evolved to be citrus canker-causing pathogens from a separate lineage from Xac strains (Mhedbi-Hajri et al., 2013), analysis of how these Xanthomonas pathogens have evolved to specialize on citrus and associated relatives to cause citrus canker disease via convergent evolution may reveal common mechanisms underlying this disease. The evolution of Xac has only been examined using multilocus sequence analysis or genotyping (Ngoc et al., 2009; Mhedbi-Hajri et al., 2013). To investigate the evolutionary history and potential of Xac, we sequenced 21 representative Xac strains from North America and Asia. "
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    ABSTRACT: Understanding the evolutionary history and potential of bacterial pathogens is critical to prevent the emergence of new infectious bacterial diseases. Xanthomonas axonopodis subsp. citri (Xac) (synonym X. citri subsp. citri), which causes citrus canker, is one of the hardest-fought plant bacterial pathogens in US history. Here, we sequenced 21 Xac strains (14 XacA, 3 XacA* and 4 XacA(w)) with different host ranges from North America and Asia and conducted comparative genomic and evolutionary analyses. Our analyses suggest that acquisition of beneficial genes and loss of detrimental genes most likely allowed XacA to infect a broader range of hosts as compared with XacA(w) and XacA*. Recombination was found to have occurred frequently on the relative ancient branches, but rarely on the young branches of the clonal genealogy. The ratio of recombination/mutation ρ/θ was 0.0790±0.0005, implying that the Xac population was clonal in structure. Positive selection has affected 14% (395 out of 2822) of core genes of the citrus canker-causing Xanthomonas. The genes affected are enriched in 'carbohydrate transport and metabolism' and 'DNA replication, recombination and repair' genes (P<0.05). Many genes related to virulence, especially genes involved in the type III secretion system and effectors, are affected by positive selection, further highlighting the contribution of positive selection to the evolution of citrus canker-causing Xanthomonas. Our results suggest that both metabolism and virulence genes provide advantages to endow XacA with higher virulence and a wider host range. Our analysis advances our understanding of the genomic basis of specialization by positive selection in bacterial evolution.
    The ISME Journal 02/2015; 9(10). DOI:10.1038/ismej.2015.15 · 9.30 Impact Factor
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    • "The MLVA analysis was subsequently adopted by microbiologists to study population structure of human pathogens such as Mycobacterium tuberculosis (Mazars et al., 2001), Yersinia pestis (Klevytska et al., 2001) and Staphylococcus aureus (Malachowa et al., 2005). Later, genotype information was linked with geographical information to study how bacteria behave within smaller geographical regions or even single outbreaks (Girard et al., 2004; U'Ren et al., 2007; Bui Thi Ngoc et al., 2009; Stratilo and Bader, 2012). "
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    ABSTRACT: Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on Variable Number of Tandem Repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools and VNTR markers were developed and validated. A Multiple Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region.
    Environmental Microbiology 09/2013; 16(7). DOI:10.1111/1462-2920.12289 · 6.20 Impact Factor
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    • "To date, several MLVA schemes have been published on plant pathogens such as Xanthomonas citri pv. citri[31], X. oryzae pv. oryzicola[26], Pseudomonas syringae pv. "
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    ABSTRACT: Background Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato. Cmm is present nearly in all European countries. During the last three years several local outbreaks were detected in Belgium. The lack of a convenient high-resolution strain-typing method has hampered the study of the routes of transmission of Cmm and epidemiology in tomato cultivation. In this study the genetic relatedness among a worldwide collection of Cmm strains and their relatives was approached by gyrB and dnaA gene sequencing. Further, we developed and applied a multilocus variable number of tandem repeats analysis (MLVA) scheme to discriminate among Cmm strains. Results A phylogenetic analysis of gyrB and dnaA gene sequences of 56 Cmm strains demonstrated that Belgian Cmm strains from recent outbreaks of 2010–2012 form a genetically uniform group within the Cmm clade, and Cmm is phylogenetically distinct from other Clavibacter subspecies and from non-pathogenic Clavibacter-like strains. MLVA conducted with eight minisatellite loci detected 25 haplotypes within Cmm. All strains from Belgian outbreaks, isolated between 2010 and 2012, together with two French strains from 2010 seem to form one monomorphic group. Regardless of the isolation year, location or tomato cultivar, Belgian strains from recent outbreaks belonged to the same haplotype. On the contrary, strains from diverse geographical locations or isolated over longer periods of time formed mostly singletons. Conclusions We hypothesise that the introduction might have originated from one lot of seeds or contaminated tomato seedlings that was the source of the outbreak in 2010 and that these Cmm strains persisted and induced infection in 2011 and 2012. Our results demonstrate that MLVA is a promising typing technique for a local surveillance and outbreaks investigation in epidemiological studies of Cmm.
    BMC Microbiology 06/2013; 13(1):126. DOI:10.1186/1471-2180-13-126 · 2.73 Impact Factor
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