Christian Vernière

Polyphasic characterization of xanthomonads pathogenic to members of the #Anacardiaceae# and their relatedness to species of #Xanthomonas#

We have used amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA) and DNA-DNA hybridization for genotypic classification of Xanthomonas pathovars associated with the plant family Anacardiaceae. AFLP and MLSA results showed congruent phylogenetic relationships of the pathovar mangiferaeindicae (responsible for mango bacterial canker) with strains of Xanthomonas axonopodis subgroup 9.5. This subgroup includes X. axonopodis pv. citri (synonym Xanthomonas citri). Similarly, the pathovar anacardii, which causes cashew bacterial spot in Brazil, was included in X. axonopodis subgroup 9.6 (synonym Xanthomonas fuscans). Based on the thermal stability of DNA reassociation, consistent with the AFLP and MLSA data, the two pathovars share a level of similarity consistent with their being members of the same species. The recent proposal to elevate X. axonopodis pv. citri to species level as X. citri is supported by our data. Therefore, the causal agents of mango bacterial canker and cashew bacterial spot should be classified as pathovars of X. citri, namely X. citri pv. mangiferaeindicae (pathotype strain CFBP 1716) and X. citri pv. anacardii (pathotype strain CFBP 2913), respectively. Xanthomonas fuscans should be considered to be a later heterotypic synonym of Xanthomonas citri.

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.

Disease Development and Symptom Expression of Xanthomonas axonopodis pv. citri in Various Citrus Plant Tissues

ABSTRACT Experimental inoculations of Xanthomonas axonopodis pv. citri in different tissues of Tahiti lime and Pineapple sweet orange were conducted monthly under natural conditions on Réunion Island. The interactions between a set of environmental and epidemic variables associated with disease expression and 184 different factor combinations were investigated to determine the parameters needed to explain Asiatic citrus canker (ACC) disease expression. Area under the disease progress curve (AUDPC), inoculation date (Id), fruit and leaf age ratings (FAR and LAR), and number of days during the first 2 weeks postinoculation for which the temperature was less than 14 degrees C (T(min)) or more than 28 degrees C (T(max)) were retained by principal component analysis and canonical correlation analysis as the most meaningful epidemic and environmental variables, respectively. AUDPC as the strongest dependent variable and combinations of the environmental variables as independent variables were used in multiple regression analyses. Tissue age rating at the time of infection was a good predictor for disease resulting from spray inoculation on fruits and leaves and also on fruits following a wound inoculation. Temperature, as expressed by T(min) or T(max), was also a significant factor in determining disease development described by AUDPC. Mature green stems were highly susceptible after wounding, similarly to leaves, but buds and leaf scars expressed the lowest susceptibility. These variations in disease expression according to the tissues will have different impacts on ACC epidemiology.

Genome sequence of Xanthomonas fuscans subsp. fuscans strain 4834-R reveals that flagellar motility is not a general feature of xanthomonads

BackgroundXanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences.ResultsComparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations.ConclusionsThis work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

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.

Using Ecology, Physiology, and Genomics to Understand Host Specificity in Xanthomonas†

How pathogens coevolve with and adapt to their hosts are critical to understanding how host jumps and/or acquisition of novel traits can lead to new disease emergences. The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria that collectively infect a broad range of crops and wild plant species. However, individual Xanthomonas strains usually cause disease on only a few plant species and are highly adapted to their hosts, making them pertinent models to study host specificity. This review summarizes our current understanding of the molecular basis of host specificity in the Xanthomonas genus, with a particular focus on the ecology, physiology, and pathogenicity of the bacterium. Despite our limited understanding of the basis of host specificity, type III effectors, microbe-associated molecular patterns, lipopolysaccharides, transcriptional regulators, and chemotactic sensors emerge as key determinants for shaping host specificity.

Variations in type III effector repertoires, pathological phenotypes and host range of Xanthomonas citri pv. citri pathotypes

The mechanisms determining the host range of Xanthomonas are still undeciphered, despite much interest in their potential roles in the evolution and emergence of plant pathogenic bacteria. Xanthomonas citri pv. citri (Xci) is an interesting model of host specialization because of its pathogenic variants: pathotype A strains infect a wide range of Rutaceous species, whereas pathotype A*/A(W) strains have a host range restricted to Mexican lime (Citrus aurantifolia) and alemow (Citrus macrophylla). Based on a collection of 55 strains representative of Xci worldwide diversity assessed by amplified fragment length polymorphism (AFLP), we investigated the distribution of type III effectors (T3Es) in relation to host range. We examined the presence of 66 T3Es from xanthomonads in Xci and identified a repertoire of 28 effectors, 26 of which were shared by all Xci strains, whereas two (xopAG and xopC1) were present only in some A*/A(W) strains. We found that xopAG (=avrGf1) was present in all A(W) strains, but also in three A* strains genetically distant from A(W) , and that all xopAG-containing strains induced the hypersensitive response (HR) on grapefruit and sweet orange. The analysis of xopAD and xopAG suggested horizontal transfer between X. citri pv. bilvae, another citrus pathogen, and some Xci strains. A strains were genetically less diverse, induced identical phenotypic responses and possessed indistinguishable T3E repertoires. Conversely, A*/A(W) strains exhibited a wider genetic diversity in which clades correlated with geographical origin and T3E repertoire, but not with pathogenicity, according to T3E deletion experiments. Our data outline the importance of taking into account the heterogeneity of Xci A*/A(W) strains when analysing the mechanisms of host specialization.

A MLVA genotyping scheme for global surveillance of the citrus pathogen Xanthomonas citri pv. citri suggests a worldwide geographical expansion of a single genetic lineage.

MultiLocus Variable number of tandem repeat Analysis (MLVA) has been extensively used to examine epidemiological and evolutionary issues on monomorphic human pathogenic bacteria, but not on bacterial plant pathogens of agricultural importance albeit such tools would improve our understanding of their epidemiology, as well as of the history of epidemics on a global scale. Xanthomonas citri pv. citri is a quarantine organism in several countries and a major threat for the citrus industry worldwide. We screened the genomes of Xanthomonas citri pv. citri strain IAPAR 306 and of phylogenetically related xanthomonads for tandem repeats. From these in silico data, an optimized MLVA scheme was developed to assess the global diversity of this monomorphic bacterium. Thirty-one minisatellite loci (MLVA-31) were selected to assess the genetic structure of 129 strains representative of the worldwide pathological and genetic diversity of X. citri pv. citri. Based on Discriminant Analysis of Principal Components (DAPC), four pathotype-specific clusters were defined. DAPC cluster 1 comprised strains that were implicated in the major geographical expansion of X. citri pv. citri during the 20th century. A subset of 12 loci (MLVA-12) resolved 89% of the total diversity and matched the genetic structure revealed by MLVA-31. MLVA-12 is proposed for routine epidemiological identification of X. citri pv. citri, whereas MLVA-31 is proposed for phylogenetic and population genetics studies. MLVA-31 represents an opportunity for international X. citri pv. citri genotyping and data sharing. The MLVA-31 data generated in this study was deposited in the Xanthomonas citri genotyping database (http://www.biopred.net/MLVA/).

Genomic Survey of Pathogenicity Determinants and VNTR Markers in the Cassava Bacterial Pathogen Xanthomonas axonopodis pv. Manihotis Strain CIO151

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.

Development of 14 minisatellite markers for the citrus canker bacterium, Xanthomonas citri pv. citri.

We screened the genome of Xanthomonas citri pv. citri strain 306 for tandem repeats. A multiplex polymerase chain reaction protocol was used to assess the genetic diversity of 239 strains of X. citri pv. citri from Asia. The total number of alleles per locus ranged from three to 20. Using pooled data sets, 223 different haplotypes were identified. Successful amplifications were obtained at most loci for seven other X. citri pathovars. This typing scheme is expected to be useful at different spatial scales for population studies of pathovars of X. citri, several of which cause plant diseases of economic importance.