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Copyright of Canadian Journal of Plant Pathology is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

• Opines are biomarkers specifically produced by agrobacteria-infected plants. • A general, specific and sensitive UHPLC-ESI-MS QTOF method for opines was developed. • Detection selectivity of opines was validated in pure solution and plant extracts. • A powerful tool with easy sample preparation for crown gall/hairy roots diagnosis. • Nopaline, octopine and mannopine were accurately quantified in plant extracts. Opines are low-molecular-weight metabolites specifically biosynthesized by agrobacteria-transformed plant cells when plants are struck by crown gall and hairy root diseases, which cause uncontrolled tissue overgrowth. Transferred DNA is sustainably incorporated into the genomes of the transformed plant cells, so that opines constitute a persistent biomarker of plant infection by pathogenic agrobacteria and can be targeted for crown gall/hairy root disease diagnosis. We developed a general, rapid, specific and sensitive analytical method for overall opine detection using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-MS-QTOF), with easy preparation of samples. Based on MS, MS/MS and chromatography data, the detection selectivity of a wide range of standard opines was validated in pure solution and in different plant extracts. The method was successfully used to detect different structural types of opines, including opines for which standard compounds are unavailable, in tumors or hairy roots induced by pathogenic strains. As the method can detect a wide range of opines in a single run, it represents a powerful tool for plant gall analysis and crown gall/hairy root disease diagnosis. Using an appropriate dilution of plant extract and a matrix-based calibration curve, the quantification ability of the method was validated for three opines belonging to different families (nopaline, octopine, mannopine), which were accurately quantified in plant tissue extracts. [ABSTRACT FROM AUTHOR]

Tumor-inducing (Ti) and root-inducing (Ri) plasmids of Agrobacterium that display a large diversity are involved in crown gall and hairy root plant diseases. Their phylogenetic relationships were inferred from an exhaustive set of Ti and Ri plasmids (including 36 new complete Ti plasmids) by focusing on T-DNA and virulence regions. The opine synthase gene content of T-DNAs revealed 13 opine types corresponding to former classifications based on opines detected in diseased plants, while the T-DNA gene content more finely separate opine types in 18 T-DNA organizations. This classification was supported by the phylogeny of T-DNA oncogenes of Ti plasmids. The five gene organizations found in Ti/Ri vir regions was supported by the phylogeny of common vir genes. The vir organization was found to be likely an ancestral plasmid trait separating "classic" Ti plasmids (with one or two T-DNAs) and "Ri and vine-Ti" plasmids. A scenario generally supported by the repABC phylogeny. T-DNAs likely evolved later with the acquisition of opine characteristics as last steps in the Ti/Ri plasmid evolution. This novel evolutionary classification of Ti/Ri plasmids was found to be relevant for accurate crown gall and hairy root epidemiology., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Strains of Agrobacterium genomospecies 3 (i.e., genomovar G3 of the Agrobacterium tumefaciens species complex) have been previously isolated from diverse environments, including in association with plant roots, with algae, as part of a lignocellulose degrading community, from a hospital environment, as a human opportunistic pathogen, or as reported in this study, from a surface within the International Space Station. Polyphasic taxonomic methods revealed the relationship of Agrobacterium G3 strains to other Agrobacterium spp., which supports the description of a novel species. The G3 strains tested ( n = 9) were phenotypically distinguishable among the strains from other genomospecies of the genus Agrobacterium. Phylogenetic analyses of the 16S rRNA gene, gyr B gene, multi-locus sequence analysis, and 1,089-gene core-genome gene concatenate concur that tested G3 strains belong to the Agrobacterium genus and they form a clade distinct from other validly described Agrobacterium species. The distinctiveness of this clade was confirmed by average nucleotide identity (ANI) and in silico digital DNA-DNA hybridization (dDDH) comparisons between the G3 tested strains and all known Agrobacterium species type strains, since obtained values were considerably below the 95% (ANI) and 70% (dDDH) thresholds used for the species delineation. According to the core-genome phylogeny and ANI comparisons, the closest relatives of G3 strains were Agrobacterium sp. strains UGM030330-04 and K599, members of a novel genomospecies we propose to call genomovar G21. Using this polyphasic approach, we characterized the phenotypic and genotypic synapomorphies of Agrobacterium G3, showing it is a bona fide bacterial species, well separated from previously named Agrobacterium species or other recognized genomic species. We thus propose the name Agrobacterium tomkonis for this species previously referred to as Agrobacterium genomospecies 3. The type strain of A. tomkonis is IIF1SW-B1 T (= LMG 32164 = NRRL B-65602). Comparative genomic analysis show A. tomkonis strains have species-specific genes associated with secretion of secondary metabolites, including an exopolysaccharide and putative adhesins and resistance to copper. A. tomkonis specific gene functions notably relate to surface adhesion and could be involved to colonize nutrient-poor and harsh habitats. The A. tomkonis strains from the ISS showed presence of a 40-kbp plasmid and several other potential mobile genetic elements detected that could also be part of conjugative elements or integrated prophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Singh, Lavire, Nesme, Vial, Nesme, Mason, Lassalle and Venkateswaran.)

Agrobacterium fabrum C58 is a plant-associated bacterium that is able to denitrify under anoxic conditions. The cluster of denitrification genes harbored by this strain has been well characterized. It includes nir and nor operons encoding nitrite and nitric oxide reductases, respectively. However, the reductase involved in nitrate reduction has not yet been studied and little information is available on denitrification regulators in A. fabrum C58. In this study, we aimed to (i) characterize the nitrate reductase, (ii) determine its role in A. fabrum C58 fitness and root colonization and (ii) reveal the contribution of small RNA on denitrification regulation. By constructing a mutant strain defective for napA, we demonstrated that the reduction of nitrate to nitrite was catalyzed by the periplasmic nitrate reductase, NapA. We evidenced a positive role of NapA in A. fabrum C58 fitness and suggested that A. fabrum C58 is able to use components exuded by plant roots to respire anaerobically. Here, we showed that NorR small RNA increased the level of norCBQ mRNA and a decrease of NorR is correlated with a decrease in N2O emission. Together, our results underscore the importance of understanding the denitrification pathway at the strain level in order to develop strategies to mitigate N2O production at the microbial community level., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Assessment of the taxonomy and diversity of Xanthomonas strains causing bacterial leaf spot of lettuce (BLSL), commonly referred to as Xanthomonas campestris pv. vitians, has been a long-lasting issue which held back the global efforts made to understand this pathogen. In order to provide a sound basis essential to its study, we conducted a polyphasic approach on strains obtained through sampling campaigns or acquired from collections. Results of a multilocus sequence analysis crossed with phenotypic assays revealed that the pathotype strain does not match the description of the nomenspecies provided by Brown in 1918. However, strain LMG 938=CFBP 8686 does fit this description. Therefore, we propose that it replaces LMG 937=CFBP 2538 as pathotype strain of X. campestris pv. vitians. Then, whole-genome based phylogenies and overall genome relatedness indices calculated on taxonomically relevant strains exhibited the intermediate position of X. campestris pv. vitians between closely related species Xanthomonas hortorum and Xanthomonas cynarae. Phenotypic profiles characterized using Biolog microplates did not reveal stable diagnostic traits legitimizing their distinction. Therefore, we propose that X. cynarae Trébaol et al. 2000 emend. Timilsina et al. 2019 is a later heterotypic synonym of X. hortorum, to reclassify X. campestris pv. vitians as X. hortorum pv. vitians comb. nov. and to transfer X. cynarae pathovars in X. hortorum as X. hortorum pv. cynarae comb. nov. and X. hortorum pv. gardneri comb. nov. An emended description of X. hortorum is provided, making this extended species a promising model for the study of Xanthomonas quick adaptation to different hosts., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

In this review, we synthesise current models and recent comparative genomic studies describing how bacterial species may emerge through adaptation to a new ecological niche and maintain themselves in the same niche over long time periods. We notably consider the impact of genetic exchange with phylogenetically close relatives living in sympatry and how this leads to the heterogeneous evolution of different genes within the bacterial genome. This heterogeneity provides landmarks to recognise genes that determine adaptation to the ecological niche, and we present reverse ecology strategies to unravel ecological properties of bacterial populations., (Crown Copyright © 2015. Published by Elsevier Masson SAS. All rights reserved.)

The analysis of housekeeping recA gene sequences from 138 strains from 13 species or genomic species of Agrobacterium, nine being biovar 1 genomospecies, and the others Agrobacterium larrymoorei, Agrobacterium rubi, Agrobacterium sp. NCPPB 1650, and Agrobacterium vitis and one "former" Agrobacterium species, Rhizobium rhizogenes, led to the identification of 50 different recA alleles and to a clear delineation of the 14 species or genomospecies entirely consistent with that obtained by amplified fragment length polymorphism (AFLP) analysis. The relevance of a recA sequencing approach for epidemiological analyses was next assessed on agrobacterial Tunisian isolates. All Tunisian isolates were found to belong to the Agrobacterium tumefaciens/biovar 1 species complex by both biochemical tests and rrs sequencing. recA sequence analysis further permitted their unambiguous assignment to A. tumefaciens genomospecies G4, G6, G7, and G8 in total agreement with the results of an AFLP-based analysis. At subspecific level, several Tunisian recA alleles were novel, indicating the power and accuracy of recA-based typing for studies of Agrobacterium spp.

According to current taxonomical rules, a bona fide bacterial species is a genomic species characterized by the genomic similarity of its members. It has been proposed that the genomic cohesion of such clusters may be related to sexual isolation, which limits gene flow between too divergent bacteria. Homologous recombination is one of the most studied mechanisms responsible for this genetic isolation. Previous studies on several bacterial models showed that recombination frequencies decreased exponentially with increasing DNA sequence divergence. In the present study, we investigated this relationship in the Agrobacterium tumefaciens species complex, which allowed us to focus on sequence divergence in the vicinity of the genetic boundaries of genomic species. We observed that the sensitivity of the recombination frequency to DNA divergence fitted a log-linear function until approximately 10% sequence divergence. The results clearly revealed that there was no sharp drop in recombination frequencies at the point where the sequence divergence distribution showed a "gap" delineating genomic species. The ratio of the recombination frequency in homogamic conditions relative to this frequency in heterogamic conditions, that is, sexual isolation, was found to decrease from 8 between the most distant strains within a species to 9 between the most closely related species, for respective increases from 4.3% to 6.4% mismatches in the marker gene chvA. This means that there was only a 1.13-fold decrease in recombination frequencies for recombination events at both edges of the species border. Hence, from the findings of this investigation, we conclude that--at least in this taxon--sexual isolation based on homologous recombination is likely not high enough to strongly hamper gene flow between species as compared with gene flow between distantly related members of the same species. The 70% relative binding ratio cutoff used to define bacterial species is likely correlated to only minor declines in homologous recombination frequencies. Consequently, the sequence diversity, as a mechanistic factor for the efficiency of recombination (as assayed in the laboratory), appears to play little role in the genetic cohesion of bacterial species, and thus, the genomic species definition for prokaryotes is definitively not reconcilable with the biological species concept for eukaryotes.

Ralstonia solanacearum is a widely distributed phytopathogenic bacterium that is known to invade more than 200 host species, mainly in tropical areas. Reference strain GMI1000 is naturally transformable at in vitro and also in planta conditions and thus has the ability to acquire free exogenous DNA. We tested the ubiquity and variability of natural transformation in the four phylotypes of this species complex using 55 strains isolated from different hosts and geographical regions. Eighty per cent of strains distributed in all the phylotypes were naturally transformable by plasmids and/or genomic DNA. Transformability can be considered as a ubiquitous physiological trait in the R. solanacearum species complex. Transformation performed with two independent DNA donors showed that multiple integration events occurred simultaneously in two distant genomic regions. We also engineered a fourfold-resistant R. solanacearum GMI1000 mutant RS28 to evaluate the size of DNA exchanged during natural transformation. The results demonstrated that this bacterium was able to exchange large DNA fragments ranging from 30 to 90 kb by DNA replacement. The combination of these findings indicated that the natural transformation mechanism could be the main driving force of genetic diversification of the R. solanacearum species complex.

Bacterial diversity is central to ecosystem sustainability and soil biological function, for which the role of roots is important. The high-throughput analysis potential of taxonomic microarray should match the breadth of bacterial diversity. Here, the power of this technology was evidenced through methodological verifications and analysis of maize rhizosphere effect based on a 16S rRNA-based microarray developed from the prototype of H. Sanguin et al. (Environ. Microbiol. 8:289-307, 2006). The current probe set was composed of 170 probes (41 new probes in this work) that targeted essentially the Proteobacteria. Cloning and sequencing of 16S rRNA amplicons were carried out on maize rhizosphere and bulk soil DNA. All tested clones that had a perfect match with corresponding probes were positive in the hybridization experiment. The hierarchically nested probes were reliable, but the level of taxonomic identification was variable, depending on the probe set specificity. The comparison of experimental and theoretical hybridizations revealed 0.91% false positives and 0.81% false negatives. The microarray detection threshold was estimated at 0.03% of a given DNA type based on DNA spiking experiments. A comparison of the maize rhizosphere and bulk soil hybridization results showed a significant rhizosphere effect, with a higher predominance of Agrobacterium spp. in the rhizosphere, as well as a lower prevalence of Acidobacteria, Bacteroidetes, Verrucomicrobia, and Planctomycetes, a new taxon of interest in soil. In addition, well-known taxonomic groups such as Sphingomonas spp., Rhizobiaceae, and Actinobacteria were identified in both microbial habitats with strong hybridization signals. The taxonomic microarray developed in the present study was able to discriminate and characterize bacterial community composition in related biological samples, offering extensive possibilities for systematic exploration of bacterial diversity in ecosystems.