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Chabirand A.,ANSES Plant Health Laboratory | Jouen E.,University of Reunion Island | Pruvost O.,University of Reunion Island | Chiroleu F.,University of Reunion Island | And 17 more authors.
Plant Pathology | Year: 2014

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires sensitive and reliable diagnostic tools. The European standard EN ISO 16140:2003 has been followed to compare a nested PCR assay (N-PCR) to a reference method (isolation and serological identification of bacterial colonies) and to other alternative serological detection methods. The evaluation was performed in two steps: a comparative study and a collaborative study involving 15 European laboratories. Although inclusivity was maximal (100%) for all methods, a maximal exclusivity was obtained only with N-PCR followed by an enzymatic restriction digestion of the amplicons. Exclusivity indices of 90·6, 88·7 and 47·2% were found for indirect ELISA, immunofluorescence and double antibody sandwich ELISA, respectively. An exclusivity of 92·5% was obtained with the reference method, further increased to 100% if pathogenicity tests were performed as a supplemental assay. The best level of sensitivity (relative detection level) was obtained with the reference method followed by the N-PCR assay. The N-PCR performance in terms of relative accuracy, accordance and concordance was very similar to that of the reference method. Moreover, N-PCR had undeniable advantages compared to the reference method (less labour-intensive and less time-consuming). In addition, post-test probabilities of infection were calculated to select the most appropriate detection scheme related to the prevalence of the pathogen. The N-PCR assay has since been included in a revised version of the EPPO detection protocol. © 2013 British Society for Plant Pathology.

Ailloud F.,CIRAD | Ailloud F.,ANSES Plant Health Laboratory | Lowe T.,University of Wisconsin - Madison | Cellier G.,ANSES Plant Health Laboratory | And 4 more authors.
BMC Genomics | Year: 2015

Background: Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains. Results: These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences. Conclusions: This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation. © Ailloud et al.

Robene I.,University of Reunion Island | Perret M.,University of Reunion Island | Jouen E.,University of Reunion Island | Escalon A.,University of Reunion Island | And 6 more authors.
Journal of Microbiological Methods | Year: 2015

Bacterial blight of onion is an emerging disease threatening world onion production. The causal agent Xanthomonas axonopodis pv. allii is seed transmitted and a reliable and sensitive tool is needed to monitor seed exchanges. A triplex quantitative real-time PCR assay was developed targeting two X. axonopodis pv. allii-specific markers and an internal control chosen in 5.8S rRNA gene from Alliaceae. Amplification of at least one marker indicates the presence of the bacterium in seed extracts. This real-time PCR assay detected all the 79 X. axonopodis pv. allii strains tested and excluded 85.2% of the 135 non-target strains and particularly all 39 saprophytic and pathogenic bacteria associated with onion. Cross-reactions were mainly obtained for strains assigned to nine phylogenetically related X. axonopodis pathovars. The cycle cut-off was estimated statistically at 36.3 considering a risk of false positive of 1%. The limit of detection obtained in at least 95% of the time (LOD 95%) was 5×103CFU/g (colony forming unit/g). The sensitivity threshold was found to be 1 infected seed in 32,790 seeds. This real-time PCR assay should be useful for preventing the long-distance spread of X. axonopodis pv. allii via contaminated seed lots and determining the epidemiology of the bacterium. © 2015 Elsevier B.V.

Chabirand A.,ANSES Plant Health Laboratory | Anthoine G.,ANSES Plant Health Laboratory | Pierson O.,Anses | Hostachy B.,ANSES Plant Health Laboratory
Accreditation and Quality Assurance | Year: 2014

The use of methods of analysis capable of producing reliable analytical results is a prerequisite to the effective control of quarantine plant pathogens. Proficiency testing is considered to be one of the most reliable ways to verify and coordinate analytical results. As a French national reference laboratory in plant pathology, the Anses Plant Health Laboratory organizes proficiency tests in order to ensure that officially approved laboratories (certified by government services) are capable of producing reliable analytical results for the detection of plant pathogens. Proficiency tests in plant pathology have a number of notable features including the processing of qualitative results. This paper presents the experience of the Anses Plant Health Laboratory's Unit for Tropical Pests and Diseases (LSV-RAPT) as an organizer of proficiency tests in plant pathology. The LSV-RAPT has gained recognition for the methodology it has developed in the form of accreditation as a proficiency testing provider according to the ISO/IEC 17043. The methodology can be applied to many other disciplines that use qualitative detection methods. © 2014 The Author(s).

Mathis R.,British Petroleum | Fricot C.,British Petroleum | Larenaudie M.,British Petroleum | Quillevere A.,British Petroleum | And 7 more authors.
Acta Horticulturae | Year: 2015

Clavibacter michiganensis subsp. michiganensis (Cmm) is one of the most important seed-transmitted pathogens in tomato. In recent years, the number of outbreaks has increased. Due to the absence of chemical controls, the most effective way to prevent the spread of this disease is through the use of clean seed, requiring effective seed health testing. A French collaborative program sponsored by the Ministry of Agriculture was launched to study Cmm and improve detection methods. The protocols used routinely in laboratories for seed testing are not highly satisfactory. A program to improve the existing detection/identification methods was implemented; method performance was compared using a collection of 213 isolates (including 141 Cmm, saprophytes, other Clavibacter subspp. and other bacteria pathogenic to tomato). The methods were immunofluorescence, media plating, PCR and a pathogenicity test. Comparison of bacterial growth on two different semi selective media showed that slow and fast growing isolates should be taken into consideration for seed health analysis. The ability of PCR methods to detect the pathogen directly from a seed soaking or after a media growth step was evaluated. A new direct PCR protocol gave the best results. Moreover, efficiency of detection on treated vs. untreated contaminated seed lots was studied and sensitivity of each method was determined. Addition of inhibition controls is suggested to prevent false negative results. This study contributed to the validation of new protocols based on media growth, IF and PCR by the International Seed Federation and the European and Mediterranean Plant Protection Organization.

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