Genomic variability of the Xanthomonas pathovar mangiferaeindica, agent of mango bacterial black spot

The genetic diversity of 138 strains of the #Xanthomonas# pathovar mangiferaeindicae, which were isolated from three different hosts (mango, ambarella, and pepper tree) in 14 different countries, was assessed with restriction fragment length polymorphism markers. An analysis of patterns obtained by hybridization with an #hrp# cluster probe from #Xanthomonas oryzae# pv. oryzae separated 11 of the strains from all of the other strains, which suggested that these 11 strains may not be #Xanthomonas# pv. mangiferaeindicae strains. Hybridization with an avirulence gene from #X. oryzae# pv. oryzae and a repetitive DNA fragment from #Xanthomonas# pv. mangiferaeindicae separated the remaining 127 strains into four groups that were consistent with both geographic and host origins. The group with the greatest diversity consisted of strains from Southeast Asia, where mango originated. Other groups and subgroups contained strains that were either from widely separated countries, which suggested that wide dissemination from a single site occurred, or from localized areas, which suggested that evolution of separate lineages of strains occurred. One group of strains contained only strains isolated from pepper trees in Réunion, indicating that pepper tree may not be an alternate host for #Xanthomonas# pv. magiferaeindicae strains. (Résumé d'auteur

Collaboration for diagnosis of Xanthomonas citri pv. mangiferaeindicae causing mango bacterial canker on Mangifera indica in Myanmar : [P2-10]

During 2006-2008, mango disease survey training for pest Iist development was supported under the ASEAN Australia Development Co-operation Program (AADCP) Program Stream: Strengthening ASEAN Plant Health Capacity Project (1). The project involved regional training workshops and practical experience in surveying and disease diagnostics in selected ASEAN countries, in partnership with Australian mango pest and disease specialists. The surveying also provided an opportunity for extending collaboration with CIRAD and for strengthening CIRAD-ASEAN links, when specialist expertise in bacterial disease diagnostics was required. Bacterial canker of mango (or bacterial black spot) caused by #Xanthomonas citri pv. Mangiferaeindicae# (2) is a disease of economic importance in tropical and subtropical producing areas. #X. citri pv. Mangiferaeindicae# can cause severe infection in a wide range of mango cultivars and induces raised, angular, black leaf lesions, sometimes with a chlorotic halo. Suspected leaf lesions of bacterial canker were collected from mango nursery stock cv. Yin Kwe at a nursery in Yangon, Myanmar during March 2007. Sub-samples of representative accessions were dispatched by air-courier to 2CIRAD UMR PVBMT, La Réunion, with additional reference material retained in the plant disease herbarium of'PPD. In tests at CIRAD UMR PVBMT2, nonpigmented Xanthomonas-like bacterial colonies were isolated on KC and NCTM3 semiselective agar media (4,7). Amplified fragment length polymorphism analysis was performed on three isolates from Myanmar and additional reference isolates of xanthomonads originating from Anacardiaceae (#X. citri pv. Anacardii#, #X. citri pv. Mangiferaeindicae#, #X. axonopodis pv. Spondiae#, and #X. translucens# strains from pistachio) (2, 4). On the basis of multidimensional scaling (2), the Myanmar isolates were identified as #X. citri pv. Mangiferaeindicae# and were most closely related to group B strains that were isolated from mango in India and Eastern Asia (5). Mango cv. Maison Rouge leaves, inoculated as previously reported (3) with the Myanmar isolates, showed typical symptoms of bacterial canker 1 week after inoculation. One month after inoculation, mean #X. citri pv. Mangiferaeindicae# population sizes ranging from 5 x 106 to 8 x 106 CFU per lesion were recovered from leaf lesions, typical of a compatible interaction (3). #Mangifera indica# L. probably evolved in the area that includes northwestern Myanmar (6) and to our knowledge, this is the first confirmed detection of #X. citri pv. Mangiferaeindicae# from Myanmar. Further surveys and strain collection will be necessary to evaluate its geographic distribution and prevalence in the country (4). The diagnosis and confirmation of bacterial spot on mango from Myanmar (4) has assisted in the development of Myanmar's mango pest list, and enabled Myanmar partners to gain experience in international collaboration in plant disease specimen dispatch and diagnostics. (Résumé d'auteur

Host range and population structure of Xanthomonas citri pv. mangiferaeindicae : [P4-55]

#Xanthomonas citri# pv. #mangiferaeindicae# (Xcm) is a bacterium attacking two plant species of the anacardiaceae family : mango (#Mangifera indica#) and pepper tree (#Schinus terebinthifolius#), which is a pest frequently found bordering mango orchards. Cross-inoculation indicated that there is a host specialization: strains isolated on mango are weakly pathogenic when artificially inoculated on pepper tree and strains from pepper tree are weakly pathogenic on mango. A strong host specialization can lead to a sympatric speciation, therefore we wondered what are the consequences of the observed specialization onto natural populations of the pathogen. What are the evolutionary and epidemiological relationships between these populations? What tools may help to describe evolution at a very small geographic and probably time scale? Can this example help to understand host shifting in xanthomonads? To address such questions we analysed the genetic diversity of populations of Xcm isolated on mango and pepper tree in three places. A MultiLocus Vntr Analysis (MLVA) approach was used because of its high discriminating power and because it allows to make precise evolutionary hypotheses. Twelve minisatellite sequences were identified based on the complete genomic sequence of #X. citri# pv. #citri#. The average genetic diversity is higher for populations isolated in mango orchards than on pepper tree. Genetic differentiation indices and clonal complexes indicate that differentiation is stronger between populations isolated on different hosts than between population isolated on the same host in different places. Genetic distances between strains are low, confirming the evolutionary relatedness of the two types of strains and their classification as a single pathovar. Populations are structured as a genetic continuum, with some strains isolated from mango more closely related to some strains isolated on pepper tree than from some of the mango strains. Host specialization of Xcm is probably a recent event which did not (yet) result in speciation. (Résumé d'auteur

New powerful tools for epidemiological analyses of Xanthomonas citri pv. citri with a strong evolutionary aftertaste

The aggravation of epidemic situations for bacterial diseases is often correlated with the emergence of new groups of strains, either because changes in the environment have favored such groups or because the strains themselves have evolved to become more fit to their environment (including the host). To understand the parameters of these emergences plant bacteriologists must improve their knowledge of the populations dynamics (ie epidemiology) as well as adaptation mechanisms (ie evolution). They also need to be able to efficiently monitor changes while they happen. For pathogens such as Xanthomonas citri pv. citri (Xcc), the causal agent of Asiatic citrus canker, common genotyping tools are often not discriminant enough to address questions at rather small time or spatial scales. The sequence of the complete genome of Xcc proved a goldmine of new appropriate markers. In order to delve further into the structure of the populations of Xcc at a large (Asia) or smaller (Vietnam) scale, genotyping schemes involving insertion sequences (IS-LM-PCR) and variable number of tandem repeats (MLVA) were developed and used in addition to a reference technique, AFLP. The data were analyzed by classical population genetics methods, as well as by Bayesian and phylogeographical approaches. The three types of markers were highly congruent in describing the genetic diversity and population structure. Besides a clear identification of the Xcc pathotypes, these genotyping techniques allowed to make assumption s on the relationships between them, to describe the distribution of Xcc in Asia and to understand the population dynamics and genetics of Xcc at a smaller regional scale. The higher genetic diversity of pathotype A* suggests that it may have a longer evolutionary history than pathotype A. In Vietnam, two differentiated pathotype A populations were identified with characteristics suggesting a recent and massive dissemination of a new population, likely through propagative material. (Texte intégral

Test de détection de Xanthomonas axonopodis pv dieffenbachiae

La présente invention concerne une séquence nucleotidique présente chez toutes les souches de Xanthomonas axonopodis pv. dieffenbachiae pathogènes pour I'anthurium et un procédé de dépistage de Xanthomonas axonopodis pv. dieffenbachiae utilisant cette séquence. (Résumé d'auteur

Population structure and molecular epidemiology of Xanthomonas citri pv. citri refine citrus canker epidemiology : [O4-34, Abstract]

The genetic characterization of isolates and structure analysis of pathogenic bacterial populations are useful to investigate their epidemiology. Short-term epidemiological questions such as the identification of inoculum sources or the interconnection of different outbreaks could be approached by molecular tracing. #Xanthomonas citri# pv. #citri# (Xcc), the causal agent of Asiatic citrus canker, is a bacterium displaying little genetic diversity, based on genotyping tools such as MLST or rep-PCR. It presents low levels of recombination, which result in clonal populations. For pathogens such as Xcc, common genotyping tools are often not discriminant enough to address questions at a rather small time or spatial scale. Variable Number of Tandem Repeats targeting 14 loci were developed to detect variations and to discriminate between isolates within a strain collection from Vietnam, where Asiatic citrus canker is endemic with no current control, and from Brazil, where the pathogen is currently under containment in Saõ Paulo state. Four hundred and sixty four haplotypes out of 557 Vietnamese Xcc isolates from twelve provinces were identified. The Structure model-based clustering algorithm allows inference on genetic ancestry and identified two populations, V1 and V2, that have distinctive allelic frequencies and a Nei diversity index of 0.77 and 0.21, respectively. V1 was distributed in all provinces whereas V2 was limited in three northern provinces. A clonal complex analysis by eBurst produced 35 complexes, where all haplotypes are a single locus variant (SLV) of at least one other haplotype in the group, and 313 singletons. Nine complexes were distributed among different provinces. The largest clonal complex included 108 Xcc-V2 strains from three provinces with the founder located in a national citrus propagation center. Two hundred and twenty seven haplotypes were identified out of the 308 isolates in our Brazilian collection. Two ancestral populations were most significantly inferred by the Structure Bayesian approach. Population B1 was only found in the two southern states of Rio Grande do Sul (RS) and Santa Catarina (SC). Population B2 was inferred in the states of Parana (PR), Saõ Paulo (SP) and RS. Twenty-six clonal complexes, all within SP state, and 147 singletons were identified. Most clonal complexes were composed of strains from a single location and year of isolation, indicative of epidemic clonality. Allelic richness calculated by the rarefaction method and diversity index were a little higher for the group of strains from SC, RS and PR than for SP strains which are under eradication, with values of 6.22 / 0.67 and 5.25 / 0.54, respectively. At a larger level, allelic richness and diversity index were greater in Vietnam than in Brazil with values of 12.9 / 0.74 and 8.4 / 0.60, respectively. Molecular epidemiology and population's genetics may contribute to improve and refine our knowledge in the epidemiology of plant pathogenic bacteria even in the case of genetically monomorphic pathogens. (Résumé d'auteur

Variations in type III effector repertoires do not correlate with differences in pathological phenotypes and host range observed for Xanthomonas citri pv. citri pathotypes

Xanthomonas citri pv. citri (Xac) is a quarantine bacterium causing Asiatic citrus canker. Strains of Xac are classified as pathogenic variants i.e. pathotypes, according to their host range: strains of pathotype A infect a wide range of rutaceous species, whereas strains of pathotype A*/Aw infect a restricted host range consisting of Mexican lime (C. aurantifolia) and alemow (C. macrophylla). Based on a collection of 55 strains we investigated the role of type III effectors (T3E) in host specialization. By PCR we screened 56 Xanthomonas T3Es and showed that Xac possesses a repertoire of 28 effectors, 24 of which are shared by all strains, while 4 (xopAI, xopAD, xopAG and xopC1) are present only in some A*/ Aw strains. However, their distribution could not account for host specialization. XopAG is present in all Aw strains, but also in three A* strains genetically distant from Aw , and all xopAG-containing strains induced HR-like reactions on grapefruit and sweet orange. A strains are genetically less diverse, induce identical phenotypic responses, and share exactly the same T3Es. Conversely, A*/ Aw strains exhibited a wider genetic diversity in which clades correlated to geographical origin and T3Es repertoire but not to pathogenicity. A*/Aw strains showed a broad range of reactions on several Citrus, but genetically related strains did not share phenotypic responses. Our results showed that A*/Aw strains are more variable (genetically and pathogenetically) than initially expected and that this variability should not be ignored when trying to describe mechanisms involved in the pathogen evolution and host specialization. (Texte intégral

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

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. (Résumé d'auteur

Genetic and pathological diversity among Xanthomonas strains responsible for bacterial spot on tomato and pepper in the South West Indian Ocean region : [P4-94]

Bacterial spot of tomato and pepper (BSTP), a major problem in tropical and subtropical climates, can be caused by several #Xanthomonas genospecies#, #X. euvesicatoria#, #X. vesicatoria#, #X. perforans#, and #X. gardneri.# #X. campestris# pv. #raphani# strains primarily pathogenic to #Brassicaceae# have also been associated with outbreaks of BSTP on tomato. BSTP has been observed in the South West Indian Ocean (SWIO) region but no data is available about the status of #Xanthomonas# species associated with BSTP in this region. A total of 72 strains collected from Comoros, Madagascar, Mauritius, Réunion and the Seychelles were characterized using AFLP (Amplified Fragment Length Polymorphism) and MLSA (MultiLocus Sequence Analysis) based on four housekeeping genes. AFLP and MLSA consistently assigned strains at the species level. All strains were unambiguously classified in one species, namely: #X. euvesicatoria#, #X. vesicatoria#, #X. perforans# and #X. gardneri#. MLSA identified two to three sequence types per genospecies. No #X. campestris# pv. #raphani# strains were identified in the collection. Most of the strains were identified as #X. euvesicatoria# (65 %). MLSA data suggested that sequence variations primarily consisted of synonymous mutations, although a recombination event spanning several hundreds nucleotides was detected for some strains of #X. euvesicatoria# on the atpD gene coding for the F1-F0-ATPase ß subunit. Pathogenicity tests were also performed with pathogenicity profiles of strains consistent with the classification in genospecies. Some pathological variations were primarily observed among strains identified as #X. euvesicatoria#. This study provides for the first time a comprehensive description of the status in the SWIO region of #Xanthomonas# species causing BSTP. (Résumé d'auteur

A new minisatellite-based scheme for the global surveillance of Xanthomonas citri pv. citri, the causal agent of Asiatic Citrus Canker. : S12O12

Xanthomonas citri pv. citri (Xcc), the causal agent of Asiatic Citrus Canker, is an internationally major bacterial pathogen currently not reported from countries in the Mediterranean basin, including the EU. Some pathological diversity among strains was reported, leading to the description in Xcc of pathotypes differing in host range and aggressiveness. Xcc is recognized as a monomorphic pathogen and the very low intrapathovar diversity at housekeeping genes precludes the use of the increasingly popular MLST (MultiLocus Sequence Typing) for the global molecular surveillance of Xcc. We developed a new MLVA (MultiLocus Variable number of tandem repeats Analysis) scheme. It targeted 51 minisatellite loci dispersed over the Xcc genome with tandem repeat size ranging from 10 to 217-bp, thus allowing the use of standard agarose gel electrophoresis in laboratories that are not equipped with a genotyper. Among these loci, 34 were found to be polymorphic within a strain collection composed of strains from all continents where Xcc has been recorded, including recent outbreaks in Africa where the bacterium is reemerging. This typing scheme displayed a maximal typeability and repeatability. The produced data support the usefulness of this new MLVA scheme for global surveillance of Xcc. (Texte integral