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

Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method

Le plasmide pFL1 a été obtenu après clonage d'un fragment d'ADN plasmidique isolé de Xanthomonas campestris pv. citri XC62 dans le vecteur pUC9. La séquence nucléotidique de pFL1 a été déterminée en vue d'appliquer la technique PCR pour la détection de Xanthomonas campestris pv. citri, l'agent causal du chancre bactérien des agrumes. 7 amorces de 18 pb ont été testées pour amplifier de l'ADN de X. c. pv. citri et de X. campestris associé au "citrus bacterial spot". 4 paires d'amorces ont permis d'amplifier l'ADN cible de X. c. pv. citri mais pas des souches de X. campestris associé au "citrus bacterial spot". La paire d'amorces 2-3 a permis l'amplification spécifique de l'ADN cible des souches du pathotype A, mais aucun signal n'a été obtenu après amplification lorsque de l'ADN de souches des autres pathotypes a été testé. L'utilisation d'un tampon pH 9,0 contenant 1 % de triton X100 et 0,1 % de gélatine est absolument nécessaire pour que l'amplification de l'ADN cible, qui a un ratio G+C de 61 % se produise. Cette technique a permis de détecter 25 pg d'ADN purifié ou environ 10 bactéries lorsque des "Southern blots" ont été réalisés après l'électrophorèse. Ces niveaux de détection représentent une augmentation de sensibilité près de 100 fois supérieure à une hybridation utilisant la même sonde dans un format "dot blot

A multiplex quantitative real time PCR to detect Xanthomonas axonopodis pv.allii from onion seeds : [P2-26]

Bacterial blight of onion is a seed-borne emerging disease threatening world onion production, and causing damage to other Allium crops. Its causal agent, #Xanthomonas axonopodis pv. Allii# (Xaa) is listed in quarantine European and Mediterranean Protection Plant Organization (EPPO) A1 list since 2009. The development of a reliable tool is necessary to manage Xaa spreading via international seed trade. We developed a triplex quantitative real-time PCR capable of detecting Xaa using the Taqman® technology. This quantitative PCR targets two markers specific from Xaa (1) and an internal control chosen in NADH dehydrogenase from Alliaceae. The multiplex real-time PCR was assayed on a large collection of Xaa strains isolated worldwide and pathogenic to onion or to other Allium species. Xaa strains were detected by the amplification of one or both of the two specific markers. In case of poor or no amplification of these Xaa markers, the internal control signal validates both the extraction process and the reaction itself. Specificity was assayed on 80 Xaa strains, and 120 non target strains belonging to other #X. axonopodis# pathovars including strains from the same #X. axonopodis# subgroup 9.2 sensu Rademaker, other species, other genera, and saprophytic strains isolated from onion seed and plants. We obtained standard curves with high correlation coefficients (r2> 0,99) and amplification efficiencies of more than 90% on bacterial suspension (107 to 103 CFU/ml), and efficiencies of more than 80% from seed macerate, allowing the detection of 5.103 to 5.107 CFU/g for seed artificially inoculated with Xaa strains. We successfully detected both bacterial DNA and internal control DNA from plant by performing two successive steps: homogenization of a seed macerate with a stomacher®, followed by DNA extraction using DNeasy Plant minikit (Qiagen). We are currently validating our assay on naturally contaminated seed lots. This tool would be useful for the international sanitary surveillance of seed exchanges. (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

Assessing plant health risk in relation to Xanthomonas citri strains causing citrus bacterial canker and evaluating measures for managing this risk : S12P22

In the frame of a project funded by the European Food Safety Authority (Prima Phacie), effort was put into identifying and testing qualitative plant-pest risk assessment schemes for their suitability in supporting risk management decisions for the European Union. Five schemes were tested, two largely based on the EPPO scheme and three adapted from schemes used in non-European countries. We report the results from the application of these schemes as applied to Xanthomonas citri strains causing Citrus Bacterial Canker, in regard to the risk of its entry, establishment and spread, as well as its potential impact. For this pathogen, three entry pathways into the EU risk assessment area were considered: a) import of fresh citrus fruits, b) import of ornamental rutaceous plants or plant parts, and c) illegal entry of plant propagative material. With the current EU measures in place, of the five schemes tested, two indicated path (c) as that of the highest risk, whereas the other three suggested path (a) as such. This discrepancy is due to the different level of details the components of the risk of entry are considered in each scheme. Most schemes suggested that the establishment potential lay around the mid-range of possible values. All schemes indicated a high rate for potential spread (primarily through human activities) and a medium to high rate for impact potential. The effectiveness of risk management measures was evaluated by comparing results of assessments with and without management measures in place. (Texte integral

Investigating the population structure of Xanthomonas citri pv. citri. Which molecular markers to use to distinguish between low polymorphic bacterial populations? : [P-96]

Comprehensive knowledge of pathogen population structures is crucial to understand the epidemiology and history of infectious diseases, but such data is largely unavailable for plant pathogenic bacteria. This constitutes a challenge for genetically monomorphic bacteria. #Xanthomonas citri# pv. #citri#, which causes asiatic citrus canker, a major disease and potential threat of citrus worldwide, 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 two typing techniques, insertion sequence ligation-mediated PCR (IS-LM-PCR) typing and multilocus variable-number tandem-repeat analysis (MLVA), in terms of the information derived from the techniques and their effectiveness for analysis of genetic and population structure of 557 strains of #X. citri# pv. #citri# originating from Vietnam. The results were as follows: (1) a higher level of polymorphism was observed for MLVA as shown by the greater number of haplotypes and the higher value of Simpson's index of diversity for MLVA data; (2) Pairwise correlations between genetic distances among individual strains or pairwise population genetic differentiation were highly significant for two typing methods; (3) The two molecular markers yielded similar phylogenetic trees and population structure of #X. citri# pv. #citri# in Vietnam. These results provide guidance for the effective use of these molecular methods in the genetic analysis and epidemiology of #Xanthomonas citri# pv. #citri# at different temporal or spatial scales. (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

PCR-based assays for detecting Xanthomonas axonopodis pv. allii in onion seed

Bacterial blight of onion is an emerging disease threatening world onion production, and causing damage to other Allium crops. The causal agent, Xanthomonas axonopodis pv. allii (Xaa) has been listed on the EPPO A1 list of pests recommended for regulation as quarantine pests, since 2009. A duplex nested-PCR assay, targeting two markers specific to Xaa has been recently developed (Robène-Soustrade et al., 2010). A triplex quantitative real-time PCR assay (Taqman® technology) was developed targeting the same Xaa-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Xaa strains were detected by the amplification of one or both of the two specific markers. The internal control signal validates both the extraction process and the reaction itself. Several successive steps have to be performed before detection from seed: seed maceration for 48h at 4°C, followed by homogenization of the seed macerate with a stomacher® and DNA extraction using DNeasy® Plant mini kit (Qiagen). This assay is currently being validated following the European standard EN ISO 16140: 2003 and the EPPO standard PM7/98 (1). The performance of Nested-PCR and real-time PCR assays are discussed. These PCR-based tools could be useful for the international sanitary surveillance of seed exchanges. (Résumé d'auteur