Aude Chabirand

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements

Copper‐based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid‐encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite‐based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper‐resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper‐resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper‐resistant Xanthomonas and Stenotrophomonas strains using long‐read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance–nodulation–division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3‐like transposons and included in a conjugative plasmid. This indicates the systems great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

Data processing of qualitative results from an interlaboratory comparison for the detection of “Flavescence dorée” phytoplasma: How the use of statistics can improve the reliability of the method validation process in plant pathology

A working group established in the framework of the EUPHRESCO European collaborative project aimed to compare and validate diagnostic protocols for the detection of “Flavescence dorée” (FD) phytoplasma in grapevines. Seven molecular protocols were compared in an interlaboratory test performance study where each laboratory had to analyze the same panel of samples consisting of DNA extracts prepared by the organizing laboratory. The tested molecular methods consisted of universal and group-specific real-time and end-point nested PCR tests. Different statistical approaches were applied to this collaborative study. Firstly, there was the standard statistical approach consisting in analyzing samples which are known to be positive and samples which are known to be negative and reporting the proportion of false-positive and false-negative results to respectively calculate diagnostic specificity and sensitivity. This approach was supplemented by the calculation of repeatability and reproducibility for qualitative methods based on the notions of accordance and concordance. Other new approaches were also implemented, based, on the one hand, on the probability of detection model, and, on the other hand, on Bayes’ theorem. These various statistical approaches are complementary and give consistent results. Their combination, and in particular, the introduction of new statistical approaches give overall information on the performance and limitations of the different methods, and are particularly useful for selecting the most appropriate detection scheme with regards to the prevalence of the pathogen. Three real-time PCR protocols (methods M4, M5 and M6 respectively developed by Hren (2007), Pelletier (2009) and under patent oligonucleotides) achieved the highest levels of performance for FD phytoplasma detection. This paper also addresses the issue of indeterminate results and the identification of outlier results. The statistical tools presented in this paper and their combination can be applied to many other studies concerning plant pathogens and other disciplines that use qualitative detection methods.

Development and validation of a real-time quantitative PCR assay to detect Xanthomonas axonopodis pv. allii from onion seed.

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 5×10(3) CFU/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.

A new semi-selective medium for the isolation of Xanthomonas axonopodis pv. dieffenbachiae, the etiological agent of anthurium bacterial blight

Aims:  Xanthomonas axonopodis pv. dieffenbachiae causes anthurium blight, which is regarded as the most threatening disease for the anthurium industry worldwide. The bacterium is listed as a quarantine pathogen in several regions, including Europe. We evaluated the use of Neomycin‐Cephalexin‐Trimethoprime‐pivMecillinam 4 (NCTM4) medium for its isolation.

First Report of Xanthomonas citri pv. citri Pathotype A Causing Asiatic Citrus Canker in Martinique, France

Asiatic canker, caused by Xanthomonas citri pv. citri, is a major threat to worldwide citriculture. Three pathotypes differing in host range and hypersensitive reactions toward citrus species have been defined. Whereas pathotypes Aw and A* have a restricted host range, X. citri pv. citri pathotype A infects a broader range including most commercial citrus species and hybrids and can cause important economic losses in tropical and subtropical areas. Pathotype A strains, especially those assigned to lineage 1, were implicated in the major geographical expansion of X. citri pv. citri during the 20th century from their native area, Asia (Pruvost et al. 2014). X. citri pv. citri is listed as a quarantine pathogen in the European Union (EU) – Directive 2000/29/EC annex II A1. Martinique (France) is an outermost region of the EU in the eastern Caribbean Sea. Canker lesions were first observed at Morne Rouge, Martinique in June 2014 on grapefruit (Citrus paradisi), mandarin (C. reticulata), Tahiti lime (C. latifolia), and Valencia and Washington Navel oranges (C. sinensis). Official diagnostics, including bacterial isolations on YPGA or KC semiselective medium (Pruvost et al. 2005), PCR-based identification with 4/7 primers (Hartung et al. 1993), and pathogenicity tests, were performed following the EPPO standard PM7/44 (www.eppo.int) and identified isolates as X. citri pv. citri. Three strains isolated in Martinique in 2014 from grapefruit or Tahiti lime were further characterized (LL074-4, LL077-2, and LL079). Multilocus sequence analysis (MLSA) targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (Almeida et al. 2010; Bui Thi Ngoc et al. 2010) identified Martinique strains as X. citri pv. citri with 100% sequence identity to the type strain LMG 9322. Using MLVA-31 targeting 31 minisatellites, Martinique strains were assigned to lineage 1 composed of pathotype A strains (Pruvost et al. 2014). All strains were inoculated by a detached leaf assay onto Mexican lime SRA 140 (C. aurantifolia), sweet orange Washington Navel SRA 102, and grapefruit Henderson SRA 336 (Bui Thi Ngoc et al. 2010). All inoculated leaves produced typical erumpent, callus-like tissue at wound sites. Xanthomonas-like colonies were reisolated from lesions that had developed. Boiled suspensions were assayed by PCR with 4/7 primers and produced the expected amplicon, fulfilling Kochs postulates. No lesions developed on the negative control consisting of sterile 0.01M tris buffer pH 7.2. This is the first report of X. citri pv. citri in Martinique and to our knowledge in the Caribbean region. Surprisingly, all strains collected to date in Martinique grew on YPGA supplemented with 300 mg liter−1 copper sulfate even when no extensive copper spray programs have been used, suggesting that copper-resistant strains may have been introduced. Disease is contained by tree removal and burning and the situation is presently under apparent control although positive trees were sporadically detected in 2015 in backyards or small orchards at Le Lorrain and Saint Pierre. An extensive surveillance program is currently implemented in Martinique for quarantine pathogens of citrus. (Texte integral)

Performance of diagnostic tests for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) from woody samples

The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an inter-laboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and real-time PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave false-positive results. The use of the most reliable detection test is suggested for routine analyses, but when Psa-free status needs to be accurately assessed, it is recommended that at least two detection tests are used. This work provides a wide comparison of the available diagnostic methods, giving new information for a possible revision of the official diagnostic protocols (e.g. European and Mediterranean Plant Protection Organization (EPPO) protocol PM7/120 for the detection of Psa).