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The bacterial species Xanthomonas arboricola contains plant pathogenic and nonpathogenic strains. It includes the pathogen X. arboricola pv. juglandis, causing the bacterial blight of Juglans regia. The emergence of a new bacterial disease of J. regia in France called vertical oozing canker (VOC) was previously described and the causal agent was identified as a distinct genetic lineage within the pathovar juglandis. Symptoms on walnut leaves and fruits are similar to those of a bacterial blight but VOC includes also cankers on trunk and branches. In this work, we used comparative genomics and physiological tests to detect differences between four X. arboricola strains isolated from walnut tree: strain CFBP 2528 causing walnut blight (WB), strain CFBP 7179 causing VOC and two nonpathogenic strains, CFBP 7634 and CFBP 7651, isolated from healthy walnut buds. Whole genome sequence comparisons revealed that pathogenic strains possess a larger and wider range of mobile genetic elements than nonpathogenic strains. One pathogenic strain, CFBP 7179, possessed a specific integrative and conjugative element (ICE) of 95 kb encoding genes involved in copper resistance, transport and regulation. The type three effector repertoire was larger in pathogenic strains than in nonpathogenic strains. Moreover, CFBP 7634 strain lacked the type three secretion system encoding genes. The flagellar system appeared incomplete and nonfunctional in the pathogenic strain CFBP 2528. Differential sets of chemoreceptor and different repertoires of genes coding adhesins were identified between pathogenic and nonpathogenic strains. Besides these differences, some strain-specific differences were also observed. Altogether, this study provides valuable insights to highlight the mechanisms involved in ecology, environment perception, plant adhesion and interaction, leading to the emergence of new strains in a dynamic environment.

Xanthomonas campestris pv. campestris is the causal agent of black rot on Brassicaceae . The draft genome sequences of strains CFBP 1869 and CFBP 5817 have been determined and are the first ones corresponding to race 1 and race 4 strains, which have a predominant agronomic and economic impact on cabbage cultures worldwide.

International audience; Lactobacillus sanfranciscensis is the predominant key lactic acid bacterium in traditionally fermented sourdoughs. Despite its prevalence, sourdough and their related breads could be different regarding their physicochemical and sensorial characteristics. The intraspecific diversity of L. sanfranciscensis might explain these observations. Fifty-nine strains isolated from French sourdoughs were typed by a polyphasic approach including Multilocus Sequence Typing (MLST) and Pulsed-field Gel Electrophoresis (PFGE), in order to study their genotypic diversity. MLST scheme can be reduced from six to four gene fragments (gdh, gyrA, nox and pta) without a major loss of discrimination between strains. The genes mapA and pgmA are not good candidates for inclusion in an MLST scheme to type L. sanfranciscensis strains, as they could not be amplified for a set of 18 strains among the 59 studied. This method revealed 20 sequence types (STs). Of these, 19 STs were grouped in one clonal complex, showing a strong relatedness between these strains. PFGE using SmaI discriminated 41 pulsotypes and so distinguished isolates better than the MLST scheme. Both genotypic methods indicate a low diversity between strains isolated from the same sourdough and a higher diversity between strains isolated from different sourdoughs, suggesting an influence of baker practices and/or environmental conditions on the selection of strains. The use of these two methods targeting genetic variations gives an optimal genotypic characterization of L. sanfranciscensis strains.

Seeds have evolved in association with diverse microbial assemblages that may influence plant growth and health. However, little is known about the composition of seed-associated microbial assemblages and the ecological processes shaping their structures. In this work, we monitored the relative influence of the host genotypes and terroir on the structure of the seed microbiota through metabarcoding analysis of different microbial assemblages associated to five different bean cultivars harvested in two distinct farms. Overall, few bacterial and fungal operational taxonomic units (OTUs) were conserved across all seed samples. The lack of shared OTUs between samples is explained by a significant effect of the farm site on the structure of microbial assemblage, which explained 12.2% and 39.7% of variance in bacterial and fungal diversity across samples. This site-specific effect is reflected by the significant enrichment of 70 OTUs in Brittany and 88 OTUs in Luxembourg that lead to differences in co-occurrence patterns. In contrast, variance in microbial assemblage structure was not explained by host genotype. Altogether, these results suggest that seed-associated microbial assemblage is determined by niche-based processes and that the terroir is a key driver of these selective forces.

Oligonucleotides used in this study. (PDF 865 kb)

Seeds carry complex microbial communities, which may exert beneficial or deleterious effects on plant growth and plant health. To date, the composition of microbial communities associated with seeds has been explored mainly through culture-based diversity studies and therefore remains largely unknown. In this work, we analyzed the structures of the seed microbiotas of different plants from the family Brassicaceae and their dynamics during germination and emergence through sequencing of three molecular markers: the ITS1 region of the fungal internal transcribed spacer, the V4 region of 16S rRNA gene, and a species-specific bacterial marker based on a fragment of gyrB . Sequence analyses revealed important variations in microbial community composition between seed samples. Moreover, we found that emergence strongly influences the structure of the microbiota, with a marked reduction of bacterial and fungal diversity. This shift in the microbial community composition is mostly due to an increase in the relative abundance of some bacterial and fungal taxa possessing fast-growing abilities. Altogether, our results provide an estimation of the role of the seed as a source of inoculum for the seedling, which is crucial for practical applications in developing new strategies of inoculation for disease prevention.

The bacterial species Xanthomonas campestris infects a wide range of Brassicaceae. Specific pathovars of this species cause black rot (pv. campestris), bacterial blight of stock (pv. incanae) or bacterial leaf spot (pv. raphani)., In this study, we extended the genomic coverage of the species by sequencing and annotating the genomes of strains from pathovar incanae (CFBP 1606R and CFBP 2527R), pathovar raphani (CFBP 5828R) and a pathovar formerly named barbareae (CFBP 5825R). While comparative analyses identified a large core ORFeome at the species level, the core type III effectome was limited to only three putative type III effectors (XopP, XopF1 and XopAL1). In Xanthomonas, these effector proteins are injected inside the plant cells by the type III secretion system and contribute collectively to virulence. A deep and strand-specific RNA sequencing strategy was adopted in order to experimentally refine genome annotation for strain CFBP 5828R. This approach also allowed the experimental definition of novel ORFs and non-coding RNA transcripts. Using a constitutively active allele of hrpG, a master regulator of the type III secretion system, a HrpG-dependent regulon of 141 genes co-regulated with the type III secretion system was identified. Importantly, all these genes but seven are positively regulated by HrpG and 56 of those encode components of the Hrp type III secretion system and putative effector proteins., This dataset is an important resource to mine for novel type III effector proteins as well as for bacterial genes which could contribute to pathogenicity of X. campestris.

Xanthomonas translucens pv. cerealis is the causal agent of bacterial leaf streak on true grasses. The genome of the pathotype strain CFBP 2541 was sequenced in order to decipher mechanisms that provoke disease and to elucidate the role of transcription activator-like (TAL) type III effectors in pathogenicity.

Deep and comprehensive knowledge of the genetic structure of pathogenic species is the cornerstone on which the design of precise molecular diagnostic tools is built. Xanthomonas arboricola is divided into pathovars, some of which are classified as quarantine organisms in many countries and are responsible for diseases on nut and stone fruit trees that have emerged worldwide. Recent taxonomic studies of the genus Xanthomonas showed that strains isolated from other hosts should be classified in X. arboricola , extending the host range of the species. To investigate the genetic structure of X. arboricola and the genetic relationships between highly pathogenic strains and strains apparently not relevant to plant health, we conducted multilocus sequence analyses on a collection of strains representative of the known diversity of the species. Most of the pathovars were clustered in separate monophyletic groups. The pathovars pruni, corylina, and juglandis, responsible for pandemics in specific hosts, were highly phylogenetically related and clustered in three distinct clonal complexes. In contrast, strains with no or uncertain pathogenicity were represented by numerous unrelated singletons scattered in the phylogenic tree. Depending on the pathovar, intra- and interspecies recombination played contrasting roles in generating nucleotide polymorphism. This work provides a population genetics framework for molecular epidemiological surveys of emerging plant pathogens within X. arboricola . Based on our results, we propose to reclassify three former pathovars of Xanthomonas campestris as X. arboricola pv. arracaciae comb. nov., X. arboricola pv. guizotiae comb. nov., and X. arboricola pv. zantedeschiae comb. nov. An emended description of X. arboricola Vauterin et al. 1995 is provided.

Xanthomonas arboricola is conventionally known as a taxon of plant-pathogenic bacteria that includes seven pathovars. This study showed that X. arboricola also encompasses nonpathogenic bacteria that cause no apparent disease symptoms on their hosts. The aim of this study was to assess the X. arboricola population structure associated with walnut, including nonpathogenic strains, in order to gain a better understanding of the role of nonpathogenic xanthomonads in walnut microbiota. A multilocus sequence analysis (MLSA) was performed on a collection of 100 X. arboricola strains, including 27 nonpathogenic strains isolated from walnut. Nonpathogenic strains grouped outside clusters defined by pathovars and formed separate genetic lineages. A multilocus variable-number tandem-repeat analysis (MLVA) conducted on a collection of X. arboricola strains isolated from walnut showed that nonpathogenic strains clustered separately from clonal complexes containing Xanthomonas arboricola pv. juglandis strains. Some nonpathogenic strains of X. arboricola did not contain the canonical type III secretion system (T3SS) and harbored only one to three type III effector (T3E) genes. In the nonpathogenic strains CFBP 7640 and CFBP 7653, neither T3SS genes nor any of the analyzed T3E genes were detected. This finding raises a question about the origin of nonpathogenic strains and the evolution of plant pathogenicity in X. arboricola . T3E genes that were not detected in any nonpathogenic isolates studied represent excellent candidates to be those responsible for pathogenicity in X. arboricola .

The first outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 were detected in France in 2010. P. syringae pv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine. P. syringae pv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains of P. syringae pv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains of P. syringae pv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes of P. syringae pv. actinidiae biovar 3 ICMP 18744 and of P. syringae pv. actinidifoliorum ICMP 18807. MLVA enabled the structuring of P. syringae pv. actinidiae biovar 3 and P. syringae pv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct from P. syringae pv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed for P. syringae pv. actinidifoliorum. We developed an MLVA scheme to explore the diversity within P. syringae pv. actinidiae biovar 3 and to trace the dispersal routes of epidemic P. syringae pv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes of P. syringae pv. actinidiae at a global level.

International audience; Plant pathogenic bacteria disseminate and survive mainly in association with seeds. This study addresses whether seeds are passive carriers or engage a molecular dialogue with pathogens during their development. We developed two pathosystems using Medicago truncatula with Xanthomonas alfalfae subsp. alfalfae (Xaa), the natural Medicago sp. pathogen and Xanthomonas campestris pv. campestris (Xcc), a Brassicaceae pathogen. Three days after flower inoculation, the transcriptome of Xcc-infected pods showed activation of an innate immune response that was strongly limited in Xcc mutated in the type three secretion system, demonstrating an incompatible interaction of Xcc with the reproductive structures. In contrast, the presence of Xaa did not result in an activation of defence genes. Transcriptome profiling during development of infected seeds exhibited time-dependent and differential responses to Xcc and Xaa. Gene network analysis revealed that the transcriptome of Xcc-infected seeds was mainly affected during seed filling whereas that of Xaa-infected seeds responded during late maturation. The Xcc-infected seed transcriptome exhibited an activation of defence response and a repression of targeted seed maturation pathways. Fifty-one percent of putative ABSCISIC ACID INSENSITIVE3 targets were deregulated by Xcc, including oleosin, cupin, legumin and chlorophyll degradation genes. At maturity, these seeds displayed decreased weight and increased chlorophyll content. In contrast, these traits were not affected by Xaa infection. These findings demonstrate the existence of a complex molecular dialogue between xanthomonads and developing seeds and provides insights into a previously unexplored trade-off between seed development and pathogen defence.

Scatter plots of the normalized expression levels of the 141 hrpG -regulated genes of strain CFBP 5828R of X. campestris pv. raphani by RNA sequencing (Table 4 , Fig. 4a ). (PDF 635 kb)

We report here the draft genome sequence of Xanthomonas axonopodis pv. allii strain CFBP 6369, the causal agent of bacterial blight of onion. The draft genome has a size of 5,425,942 bp and a G+C content of 64.4%.

Xanthomonas arboricola is an important bacterial species, the pathovars of which are responsible for bacterial blight diseases on stone fruit, hazelnut, Persian walnut, poplar, strawberry, poinsettia and banana. In this study, we evaluated variable number tandem repeats (VNTR) as a molecular typing tool for assessing the genetic diversity within pathovars of X. arboricola. Screening of the X. arboricola pv. pruni genome sequence (CFBP5530 strain) predicted 51 candidate VNTR loci. Primer pairs for polymerase chain reaction (PCR) amplification of all 51 loci were designed, and their discriminatory power was initially evaluated with a core collection of 8 X. arboricola strains representative of the different pathovars. Next, the 26 polymorphic VNTR loci present in all strains were used for genotyping a collection of 61 strains. MLVA is a typing method that clearly differentiates X. arboricola strains. The MLVA scheme described in this study is a rapid and reliable molecular typing tool that can be used for further epidemiological studies of bacterial diseases caused by X. arboricola pathovars. (C) 2014 Elsevier B.V. All rights reserved.

We report the draft genome sequence of the flagellated strain CFBP 4884 of Xanthomonas fuscans subsp. fuscans , which was isolated in an outbreak of common bacterial blight of beans along with non-flagellated strains. Comparative genomics will allow one to decipher the genomic diversity of strains cohabiting in epidemics.

We report the high-quality draft genome sequence of Xanthomonas alfalfae subsp. alfalfae strain CFBP 3836, the causal agent of bacterial leaf and stem spot in lucerne ( Medicago sativa ). Comparative genomics will help to decipher the mechanisms provoking disease and triggering the defense responses of this pathogen of the model legume Medicago truncatula .

l pasado 21 de abril de 2009 la Honorable Consiliatura dio su aprobación al proyecto denominado Escuela de Medicina y Ciencias de la Salud Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.

Background: In order to select for quantitative plant resistance to pathogens, high throughput approaches that can precisely quantify disease severity are needed. Automation and use of calibrated image analysis should provide more accurate, objective and faster analyses than visual assessments. In contrast to conventional visible imaging, chlorophyll fluorescence imaging is not sensitive to environmental light variations and provides single-channel images prone to a segmentation analysis by simple thresholding approaches. Among the various parameters used in chlorophyll fluorescence imaging, the maximum quantum yield of photosystem II photochemistry (Fv/Fm) is well adapted to phenotyping disease severity. Fv/Fm is an indicator of plant stress that displays a robust contrast between infected and healthy tissues. In the present paper, we aimed at the segmentation of Fv/Fm images to quantify disease severity., Results: Based on the Fv/Fm values of each pixel of the image, a thresholding approach was developed to delimit diseased areas. A first step consisted in setting up thresholds to reproduce visual observations by trained raters of symptoms caused by Xanthomonas fuscans subsp. fuscans (Xff) CFBP4834-R on Phaseolus vulgaris cv. Flavert. In order to develop a thresholding approach valuable on any cultivars or species, a second step was based on modeling pixel-wise Fv/Fm-distributions as mixtures of Gaussian distributions. Such a modeling may discriminate various stages of the symptom development but over-weights artifacts that can occur on mock-inoculated samples. Therefore, we developed a thresholding approach based on the probability of misclassification of a healthy pixel. Then, a clustering step is performed on the diseased areas to discriminate between various stages of alteration of plant tissues. Notably, the use of chlorophyll fluorescence imaging could detect pre-symptomatic area. The interest of this image analysis procedure for assessing the levels of quantitative resistance is illustrated with the quantitation of disease severity on five commercial varieties of bean inoculated with Xff CFBP4834-R., Conclusions: In this paper, we describe an image analysis procedure for quantifying the leaf area impacted by the pathogen. In a perspective of high throughput phenotyping, the procedure was automated with the software R downloadable at http://www.r-project.org/. The R script is available at http://lisa.univ-angers.fr/PHENOTIC/telechargements.html.

Pathovars of Xanthomonas are interesting working models for the analysis of mechanisms of adaptation and specialization to hosts, plant tissues, niches and environments. The understanding of such mechanisms is important on practical and academic points of view since they can help explain how emergence of new diseases or of diseases in new situations may occur. Four research groups belonging to the French Network on Xanthomonas (FNX) have recently focused on genome and transcriptome sequencing of several pathovars of Xanthomonas chosen for their adaptation particularities, in a project called “Xanthomix”. Xanthomonas strains attacking Anacardiaceae, citrus, crucifers, legumes or rice were selected to help understand adaptation processes: genetic structure revealing either convergence or divergence for causing a specific disease on a specific host ; host tissue specialization (xylem vs. mesophyll) ; virulence variation (broad vs. narrow host range); contrasted epidemic situation (endemic vs. epidemic). RNAseq experiments were performed on a large set of strains to determine the extent and the diversity of the hrp regulon. In addition, the RNAseq dataset is also being used to improve Xanthomonas genome annotation and gene prediction. Analysis of transcripts allowed us to scan for unknown specific and non-specific effectors as well as small non-coding RNAs and hypothesize new mechanisms that could be involved in adaptation to host and environment. (Texte integral)

We report here the high-quality draft genome sequences of two strains of Xanthomonas axonopodis pv. glycines, the causal agent of bacterial pustule on soybeans. Comparison of these genomes with those of phylogenetically closely related pathovars of Xanthomonas spp. will help to understand the mechanisms involved in host specificity and adaptation to host plants.

Background Xanthomonads are plant-associated bacteria responsible for diseases on economically important crops. Xanthomonas fuscans subsp. fuscans (Xff) is one of the causal agents of common bacterial blight of bean. In this study, the complete genome sequence of strain Xff 4834-R was determined and compared to other Xanthomonas genome sequences. Results Comparative genomics analyses revealed core characteristics shared between Xff 4834-R and other xanthomonads including chemotaxis elements, two-component systems, TonB-dependent transporters, secretion systems (from T1SS to T6SS) and multiple effectors. For instance a repertoire of 29 Type 3 Effectors (T3Es) with two Transcription Activator-Like Effectors was predicted. Mobile elements were associated with major modifications in the genome structure and gene content in comparison to other Xanthomonas genomes. Notably, a deletion of 33 kbp affects flagellum biosynthesis in Xff 4834-R. The presence of a complete flagellar cluster was assessed in a collection of more than 300 strains representing different species and pathovars of Xanthomonas. Five percent of the tested strains presented a deletion in the flagellar cluster and were non-motile. Moreover, half of the Xff strains isolated from the same epidemic than 4834-R was non-motile and this ratio was conserved in the strains colonizing the next bean seed generations. Conclusions This work describes the first genome of a Xanthomonas strain pathogenic on bean and reports the existence of non-motile xanthomonads belonging to different species and pathovars. Isolation of such Xff variants from a natural epidemic may suggest that flagellar motility is not a key function for in planta fitness.

We report high-quality draft genome sequences of two strains (race 18 and 20) of Xanthomonas citri pv. malvacearum, the causal agent of bacterial blight of cotton. Comparative genomics will help to decipher mechanisms provoking disease and triggering defense responses and to develop new molecular tools for epidemiological surveillance.

We report the draft genome sequence of the Xanthomonas cassavae type strain CFBP 4642, the causal agent of bacterial necrosis on cassava plants. These data will allow the comparison of this nonvascular pathogen with the vascular pathogen Xanthomonas axonopodis pv. manihotis, both infecting the same host, which will facilitate the development of diagnostic tools.

Xylan is a major structural component of plant cell wall and the second most abundant plant polysaccharide in nature. Here, by combining genomic and functional analyses, we provide a comprehensive picture of xylan utilization by Xanthomonas campestris pv campestris (Xcc) and highlight its role in the adaptation of this epiphytic phytopathogen to the phyllosphere. The xylanolytic activity of Xcc depends on xylan-deconstruction enzymes but also on transporters, including two TonB-dependent outer membrane transporters (TBDTs) which belong to operons necessary for efficient growth in the presence of xylo-oligosaccharides and for optimal survival on plant leaves. Genes of this xylan utilization system are specifically induced by xylo-oligosaccharides and repressed by a LacI-family regulator named XylR. Part of the xylanolytic machinery of Xcc, including TBDT genes, displays a high degree of conservation with the xylose-regulon of the oligotrophic aquatic bacterium Caulobacter crescentus. Moreover, it shares common features, including the presence of TBDTs, with the xylan utilization systems of Bacteroides ovatus and Prevotella bryantii, two gut symbionts. These similarities and our results support an important role for TBDTs and xylan utilization systems for bacterial adaptation in the phyllosphere, oligotrophic environments and animal guts.

International audience; In this study we developed an algorithm to screen for all exact molecular signatures of the quarantine pathogen Xanthomonas axonopodis pv. phaseoli (Xap), based on available data of the presence or absence of virulence-associated genes. The simultaneous presence of genes avrBsT and xopL is specific to Xap. Therefore we developed a multiplex PCR assay targeting avrBsT and xopL for the molecular identification of Xap. The specificity of this multiplex was validated by comparison to that of other molecular identification assays aimed at Xap, on a wide collection of reference strains. This multiplex was further validated on a blind collection of Xanthomonas isolates for which pathogenicity was assayed by stem wounding and by dipping leaves into calibrated inocula. This multiplex was combined to the previously described X4c/X4e molecular identification assay for Xap. Such a combination enables the molecular identification of all strains of Xanthomonas pathogenic on bean. Results also show that assay by stem wounding does not give reliable results in the case of Xap, and that pathogenicity assays by dipping should be preferred. © 2012 Elsevier B.V.

The family Xanthomonadaceae is a wide-spread family of bacteria belonging to the gamma subdivision of the Gram-negative proteobacteria, including the two plant-pathogenic genera Xanthomonas and Xylella, and the related genus Stenotrophomonas. Adhesion is a widely conserved virulence mechanism among Gram-negative bacteria, no matter whether they are human, animal or plant pathogens, since attachment to the host tissue is one of the key early steps of the bacterial infection process. Bacterial attachment to surfaces is mediated by surface structures that are anchored in the bacterial outer membrane and cover a broad group of fimbrial and non-fimbrial structures, commonly known as adhesins. In this chapter, we discuss recent findings on candidate adhesins of plant-pathogenic Xanthomonadaceae, including polysaccharidic (lipopolysaccharides, exopolysaccharides) and proteineous structures (chaperone/usher pili, type IV pili, autotransporters, two-partner-secreted and other outer membrane adhesins), their involvement in the formation of biofilms and their mode of regulation via quorum sensing. We then compare the arsenals of adhesins among different Xanthomonas strains and evaluate their mode of selection. Finally, we summarize the sparse knowledge on specific adhesin receptors in plants and the possible role of RGD motifs in binding to integrin-like plant molecules.

International audience; The family Xanthomonadaceae is a wide-spread family of bacteria belonging to the gamma subdivision of the Gram-negative proteobacteria, including the two plant-pathogenic genera Xanthomonas and Xylella, and the related genus Stenotrophomonas. Adhesion is a widely conserved virulence mechanism among Gram-negative bacteria, no matter whether they are human, animal or plant pathogens, since attachment to the host tissue is one of the key early steps of the bacterial infection process. Bacterial attachment to surfaces is mediated by surface structures that are anchored in the bacterial outer membrane and cover a broad group of fimbrial and non-fimbrial structures, commonly known as adhesins. In this chapter, we discuss recent findings on candidate adhesins of plant-pathogenic Xanthomonadaceae, including polysaccharidic (lipopolysaccharides, exopolysaccharides) and proteineous structures (chaperone/usher pili, type IV pili, autotransporters, two-partner-secreted and other outer membrane adhesins), their involvement in the formation of biofilms and their mode of regulation via quorum sensing. We then compare the arsenals of adhesins among different Xanthomonas strains and evaluate their mode of selection. Finally, we summarize the sparse knowledge on specific adhesin receptors in plants and the possible role of RGD motifs in binding to integrin-like plant molecules.

International audience; Xanthomonad-like bacteria that are associated with common bacterial blight of bean in Iran were identified on the basis of their colonial morphology, biochemical and serological properties, presence of a specific DNA fragment using PCR primers and pathogenicity on bean. Xanthomonas axonopodis pv. phaseoli (Xap) strains were further characterized using rep-PCR and restriction fragment length polymorphism (RFLP). RFLP profiles generated by the restriction endonucleases RsaI, TaqI, HaeIII and Sau96I and rep-PCR analysis revealed that Iranian strains were relatively genetically homogenous. The similarity coefficients among the strains ranged from 0.87 to 1. The genetic diversity coefficients among strains from three infected provinces, Isfahan, Markazi and Lorestan, were 0.019, 0.072 and 0.033, respectively. The low overall level of polymorphism within Xap isolates collected from the three Iranian infected regions could suggest that few initial inoculum introductions might have distributed among these different bean-growing areas in Iran.

An understanding of the mechanisms involved in the different steps of bacterial disease epidemiology is essential to develop new control strategies. Seeds are the passive carriers of a diversified microbial cohort likely to affect seedling physiology. Among seed-borne plant-pathogenic bacteria, seed carriage in compatible situations is well evidenced. The aims of our work are to determine the efficiency of pathogen transmission to seeds of a nonhost plant and to evaluate bacterial and plant behaviors at emergence. Bacterial transmission from flowers to seeds and from seeds to seedlings was measured for Xanthomonas campestris pv. campestris in incompatible interactions with bean. Transmissions from seeds to seedlings were compared for X. campestris pv. campestris, for Xanthomonas citri pv. phaseoli var. fuscans in compatible interactions with bean, and for Escherichia coli , a human pathogen, in null interactions with bean. The induction of defense responses was monitored by using reverse transcription and quantitative PCR (RT-qPCR) of genes representing the main signaling pathways and assaying defense-related enzymatic activities. Flower inoculations resulted in a high level of bean seed contamination by X. campestris pv. campestris, which transmitted efficiently to seedlings. Whatever the type of interaction tested, dynamics of bacterial population sizes were similar on seedlings, and no defense responses were induced evidencing bacterial colonization of seedlings without any associated defense response induction. Bacteria associated with the spermosphere multiply in this rich environment, suggesting that the colonization of seedlings relies mostly on commensalism. The transmission of plant-pathogenic bacteria to and by nonhost seeds suggests a probable role of seeds of nonhost plants as an inoculum source.

International audience; Deciphering the mechanisms enabling plant-pathogenic bacteria to disperse, colonize, and survive on their hosts provides the necessary basis to set up new control methods. We evaluated the role of bacterial attachment and biofilm formation in host colonization processes for Xanthomonas fuscans subsp. fuscans on its host. This bacterium is responsible for the common bacterial blight of bean (Phaseolus vulgaris), a seedborne disease. The five adhesin genes (pilA, fhab, xadA1, xadA2, and yapH) identified in X. fuscans subsp. fuscans CFBP4834-R strain were mutated. All mutants were altered in their abilities to adhere to polypropylene or seed. PilA was involved in adhesion and transmission to seed, and mutation of pilA led to lower pathogenicity on bean. YapH was required for adhesion to seed, leaves, and abiotic surfaces but not for in planta transmission to seed or aggressiveness on leaves. Transmission to seed through floral structures did not require any of the known adhesins. Conversely, all mutants tested, except in yapH, were altered in their vascular transmission to seed. In conclusion, we showed that adhesins are implicated in the various processes leading to host phyllosphere colonization and transmission to seed by plant-pathogenic bacteria.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Epiphytic survival of several Xanthomonas pathovars has been reported, but most studies failed to determine whether such populations were resident epiphytes, resulting from latent infections, or casual epiphytes. This study aimed at understanding the nature of Xanthomonas citri pv. mangiferaeindicae populations associated with asymptomatic leaves. When spray-inoculated on mango leaves cv. Maison Rouge, the pathogen multiplied markedly in association with juvenile leaves, but was most often detected as low population sizes (

Background The Xanthomonadaceae family contains two xylem-limited plant pathogenic bacterial species, Xanthomonas albilineans and Xylella fastidiosa. X. fastidiosa was the first completely sequenced plant pathogen. It is insect-vectored, has a reduced genome and does not possess hrp genes which encode a Type III secretion system found in most plant pathogenic bacteria. X. fastidiosa was excluded from the Xanthomonas group based on phylogenetic analyses with rRNA sequences. Results The complete genome of X. albilineans was sequenced and annotated. X. albilineans, which is not known to be insect-vectored, also has a reduced genome and does not possess hrp genes. Phylogenetic analysis using X. albilineans genomic sequences showed that X. fastidiosa belongs to the Xanthomonas group. Order of divergence of the Xanthomonadaceae revealed that X. albilineans and X. fastidiosa experienced a convergent reductive genome evolution during their descent from the progenitor of the Xanthomonas genus. Reductive genome evolutions of the two xylem-limited Xanthomonadaceae were compared in light of their genome characteristics and those of obligate animal symbionts and pathogens. Conclusion The two xylem-limited Xanthomonadaceae, during their descent from a common ancestral parent, experienced a convergent reductive genome evolution. Adaptation to the nutrient-poor xylem elements and to the cloistered environmental niche of xylem vessels probably favoured this convergent evolution. However, genome characteristics of X. albilineans differ from those of X. fastidiosa and obligate animal symbionts and pathogens, indicating that a distinctive process was responsible for the reductive genome evolution in this pathogen. The possible role in genome reduction of the unique toxin albicidin, produced by X. albilineans, is discussed.

International audience; At present, much attention is being given to the potential of plant pathogens, including plant-pathogenic bacteria. as biological weapons/bioterror weapons. These two terms are sometimes used interchangeably and there is need for care in their application. It has been claimed that clandestine introduction of certain plant-pathogenic bacteria could cause such crop losses as to impact so significantly on a national economy and thus constitute a threat to national security. As a separate outcome, it is suggested that they could cause serious public alarm, perhaps constituting a source of terror. Legislation is now in place to regulate selected plant-pathogenic bacteria as potential weapons. However, we consider it highly doubtful that any plant-pathogenic bacterium has the requisite capabilities to justify such a classification. Even if they were so capable, the differentiation of pathogens into a special category with regulations that are even more restrictive than those currently applied in quarantine legislation of most juridictions offers no obvious benefit. Moreover, we believe that such regulations are disadvantageous insofar as they limit research on precisely those pathogens most in need of study. Whereas some human and animal pathogens may have potential as biological or bioterror weapons, we conclude that it is unlikely that any plant-pathogenic bacterium realistically falls into this category

Understanding the survival, multiplication, and transmission to seeds of plant pathogenic bacteria is central to study their pathogenesis. We hypothesized that the type III secretion system (T3SS), encoded by hrp genes, could have a role in host colonization by plant pathogenic bacteria. The seed-borne pathogen Xanthomonas fuscans subsp. fuscans causes common bacterial blight of bean ( Phaseolus vulgaris ). Directed mutagenesis in strain CFBP4834-R of X. fuscans subsp. fuscans and bacterial population density monitoring on bean leaves showed that strains with mutations in the hrp regulatory genes, hrpG and hrpX , were impaired in their phyllospheric growth, as in the null interaction with Escherichia coli C600 and bean. In the compatible interaction, CFBP4834-R reached high phyllospheric population densities and was transmitted to seeds at high frequencies with high densities. Strains with mutations in structural hrp genes maintained the same constant epiphytic population densities (1 × 10 5 CFU g −1 of fresh weight) as in the incompatible interaction with Xanthomonas campestris pv. campestris ATCC 33913 and the bean. Low frequencies of transmission to seeds and low bacterial concentrations were recorded for CFBP4834-R hrp mutants and for ATCC 33913, whereas E. coli C600 was not transmitted. Moreover, unlike the wild-type strain, strains with mutations in hrp genes were not transmitted to seeds by vascular pathway. Transmission to seeds by floral structures remained possible for both. This study revealed the involvement of the X. fuscans subsp. fuscans T3SS in phyllospheric multiplication and systemic colonization of bean, leading to transmission to seeds. Our findings suggest a major contribution of hrp regulatory genes in host colonization processes.

International audience; Xanthomonas axonopodis pv. phaseoli and its variant fuscans are the causal agents of common bacterial blight of bean. Production of seeds is recommended in arid climates with the use of pathogen-free seeds. However, contamination of seeds still occurs in these seed production areas. To verify if low contamination levels of sown seeds could explain these field contaminations, we used seeds that were naturally contaminated with CFBP4834-R, a rifamycin-resistant X. axonopodis pv. phaseoli fuscous strain, to contaminate field plots at different rates. We also inoculated seeds to verify some parameters of plant colonization and seed transmission. In growth chambers, seedling contamination was always successful from seeds contaminated with CFBP4834-R having population sizes greater than 1 x 10(3) CFU seed(-1) and were not successful below 1 x 10(2) CFU seed(-1). In the greenhouse, the efficiency of contamination of seeds was not significantly different between contaminated plants ! that had a low or a high CFBP4834-R population size and reached between 40% and 52% whatever the origin of the inoculum (aerial or seed-borne). In field experiments, under low relative humidity, plots with 0.1-0.003% contamination rates or plots sown with seeds that were inoculated with low CFBP4834-R population sizes (1 x 10(2) and 1 x 10(4) CFU seed(-1)) led to an asymptomatic colonization of bean during the entire growing season with low CFBP4834-R population sizes. Seeds were contaminated both in primary and secondary foci. The contamination of seeds without symptom expression during the growing season represents a risk for eventual disease outbreaks

The occurrence of “ Xanthomonas axonopodis pv. phaseoli var. fuscans” (proposed name) populations as biofilms on bean leaves was investigated during three field experiments on plots established with naturally contaminated bean seeds. Behavior of aggregated versus solitary populations was determined by quantification of culturable cells in different fractions of the epiphytic population separated by particle size. X. axonopodis pv. phaseoli var. fuscans population dynamic studies confirmed an asymptomatic and epiphytic colonization of the bean phyllosphere. For all years of experiment and cultivars tested, biofilms and solitary components of the populations were always detected. Biofilm population sizes remained stable throughout the growing season (around 10 5 CFU/g of fresh weight) while solitary population sizes were more abundant and varied with climate. According to enterobacterial repetitive intergenic consensus fingerprinting, aggregated bacterial isolates were not different from solitary isolates. In controlled conditions, application of a hydric stress resulted in a decrease of the solitary populations on the leaf surface while the biofilm fraction remained stable. Suppression of the hydric stress allowed solitary bacterial populations to increase again. Aggregation in biofilms on leaf surfaces provides protection to the bacterial cells against hydric stress.

The discovery that biofilms are ubiquitous among the epiphytic microflora of leaves has prompted research about the impact of biofilms on the ecology of epiphytic microorganisms and on the efficiency of strategies to manage these populations for disease control and to ensure food safety. Biofilms are likely to influence the microenvironment and phenotype of the microorganisms they harbor. However, it is also important to determine whether there are differences in the types of bacteria within biofilms compared to those outside of biofilms so as to better target microorganisms via disease control strategies. Broad-leaved endive (Cichorium endivia var. latifolia) harbors biofilms containing fluorescent pseudomonads. These bacteria can cause considerable post-harvest losses when this plant is used for manufacturing minimally processed salads. To determine whether the population structure of the fluorescent pseudomonads in biofilms is different from that outside of biofilms on the same leaves, bacteria were isolated quantitatively from the biofilm and solitary components of the epiphytic population on leaves of field-grown broad-leaved endive. Population structure was determined in terms of taxonomic identities of the bacteria isolated, in terms of genotypic profiles, and in terms of phenotypic traits related to surface colonization and biofilm formation. The results illustrate that there are no systematic differences in the composition and structure of biofilm and solitary populations of fluorescent pseudomonads, in terms of either genotypic profiles or phenotypic profiles of the strains. However, Gram-positive bacteria tended to occur more frequently within biofilms than outside of biofilms. We suggest that leaf colonization by fluorescent pseudomonads involves a flux of cells between biofilm and solitary states. This would allow bacteria to exploit the advantages of these two types of existence; biofilms would favor resistance to stressful conditions, whereas solitary cells could foster spread of bacteria to newly colonizable sites on leaves as environmental conditions fluctuate.

The presence of microbial biofilms in the phyllosphere of terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities. This technique is based on removal of bacteria from leaves by gentle washing, separation of biofilm and solitary bacteria by filtration, and disintegration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solitary bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here were also evaluated. Isolation and quantification of bacteria by this technique is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.

International audience; Epifluorescence microscopy, scanning electron microscopy, and confocal laser scanning microscopy were used to observe microbial biofilms directly on leaf surfaces, Biofilms were observed on leaves of all species sampled (spinach, lettuce, Chinese cabbage, celery, leeks, basil, parsley, and broad-leaved endive), although the epifluorescent images were dearest when pale green tissue or cuticle pieces were used, With these techniques, biofilms were observed that were about 20 mu m in depth and up to I mm in length and that contained copious exopolymeric matrices, diverse morphotypes of microorganisms, and debris, The epifluorescence techniques described here can be used to rapidly determine the abundance and localization of biofilms on leaves, An additional technique was developed to recover individual biofilms or portions of single biofilms from leaves and to disintegrate them for isolation of the culturable microorganisms they contained, Nineteen biofilms from broad-leaved endive, spinach, parsley, and olive leaves were thus isolated and characterized to illustrate the applications of this technique.

6. International Symposium on the Microbiology of aerial plant surfaces, Bandol (France),(1995/09/11-15); International audience; Epiphytic microbial communities of terrestrial plants are non uniformly distributed in space and in time on leaf surfaces. The size and composition of microbial populations vary under the influence of biotic and abiotic factors related to the micro-organisms themselves (traits conferring epiphytic fitness, nutritional resource utilisation, abilities to compete for space, resistance or production of toxic compounds), to the host (its genotype, the age and the position of the leaves), and to the environmental conditions (micro- and macro-climate, activity of vectors and pathogens, application of pesticides and other chemicals). Moreover, the microbial population dynamics results from four processes -immigration, emigration, multiplication and death of cells as discussed in a previous chapter (Lindow, this volume) — which are under the influence of the previously mentioned factors.

International audience; Total, fluorescent, and pectolytic epiphytic bacterial population sizes were quantified on leaves of different age groups of broad-leaved endive during field cultivation from leaf emergence until harvest, Greater bacterial population densities (log(10) CPU per square centimeter) were observed on outer leaves than on inner leaves of the plants throughout the growing season, These differences were statistically significant for total bacterial populations at all sampling times and were often significant for fluorescent and pectolytic bacterial populations. At harvest, a linear gradient of decreasing densities of epiphytic bacteria from outer (older) to inner (younger) leaves of the head was significant, Leaf age influenced the frequency distribution and variability of bacterial population sizes associated with leaves of broad-leaved endive. Total bacterial population sizes were greater at leaf emergence for leaves emerging during the second half of the cultivation period than for leaves emerging earlier. The size of fluorescent and pectolytic bacterial populations on newly emerged leaves increased throughout the season as plants aged, To assess the importance of plant age on bacterial immigration at leaf emergence, bacterial densities were quantified on leaves emerging simultaneously on plants of different ages, In two of the three experiments, greater bacterial population sizes were observed on leaves emerging on younger plants, This indicates that factors other than an increase in concentration of airborne bacteria can lead to increases in population sizes at leaf emergence as plants age in the field. Results of leaf pruning experiments suggested that adjacent leaves may act as a barrier for immigration of fluorescent bacteria on newly emerged leaves, Survival of an inoculated strain of Pseudomonas fluorescens on newly emerged leaves generally did not vary with the age of plants, However, these effects were not consistent among experiments, suggesting that interactions among micro- and macroenvironmental conditions, physiological condition of leaves, and accessibility of leaves to airborne bacteria are important in controlling epiphytic bacterial population sizes.

Background Bacterial plant pathogens belonging to the Xanthomonas genus are tightly adapted to their host plants and are not known to colonise other environments. The host range of each strain is usually restricted to a few host plant species. Bacterial strains responsible for the same type of symptoms on the same host range cluster in a pathovar. The phyllosphere is a highly stressful environment, but it provides a selective habitat and a source of substrates for these bacteria. Xanthomonads colonise host phylloplane before entering leaf tissues and engaging in an invasive pathogenic phase. Hence, these bacteria are likely to have evolved strategies to adapt to life in this environment. We hypothesised that determinants responsible for bacterial host adaptation are expressed starting from the establishment of chemotactic attraction and adhesion on host tissue. Results We established the distribution of 70 genes coding sensors and adhesins in a large collection of xanthomonad strains. These 173 strains belong to different pathovars of Xanthomonas spp and display different host ranges. Candidate genes are involved in chemotactic attraction (25 genes), chemical environment sensing (35 genes), and adhesion (10 genes). Our study revealed that candidate gene repertoires comprised core and variable gene suites that likely have distinct roles in host adaptation. Most pathovars were characterized by unique repertoires of candidate genes, highlighting a correspondence between pathovar clustering and repertoires of sensors and adhesins. To further challenge our hypothesis, we tested for molecular signatures of selection on candidate genes extracted from sequenced genomes of strains belonging to different pathovars. We found strong evidence of adaptive divergence acting on most candidate genes. Conclusions These data provide insight into the potential role played by sensors and adhesins in the adaptation of xanthomonads to their host plants. The correspondence between repertoires of sensor and adhesin genes and pathovars and the rapid evolution of sensors and adhesins shows that, for plant pathogenic xanthomonads, events leading to host specificity may occur as early as chemotactic attraction by host and adhesion to tissues.

International audience; Xylella fastidiosa (Xf) is an insect-borne bacterium confined to the xylem vessels of plants. This plant pathogen has a broad host range estimated to 560 plant species. Five subspecies of the pathogen with different but overlapping host ranges have been described, but only three subspecies are widely accepted, namely subspecies fastidiosa, multiplex, and pauca. Initially limited to the Americas, Xf has been detected in Europe since 2013. As management of X. fastidiosa outbreaks in Europe depends on the identification of the subspecies, accurate determination of the subspecies in infected plants as early as possible is of major interest. Thus, we developed various tetraplex and triplex quantitative PCR (qPCR) assays for X. fastidiosa detection and subspecies identification in planta in a single reaction. We designed primers and probes using SkIf, a bioinformatics tool based on k-mers, to detect specific signatures of the species and subspecies from a data set of 58 genome sequences representative of X. fastidiosa diversity. We tested the qPCR assays on 39 target and 30 non-target strains, as well as on 13 different plant species spiked with strains of the different subspecies of X. fastidiosa, and on samples from various environmental and inoculated host plants. Sensitivity of simplex assays was equal or slightly better than the reference protocol on purified DNA. Tetraplex qPCR assays had the same sensitivity than the reference protocol and allowed X. fastidiosa detection in all spiked matrices up to 10(3) cells.ml(-1). Moreover, mix infections of two to three subspecies could be detected in the same sample with tetraplex assays. In environmental plant samples, the tetraplex qPCR assays allowed subspecies identification when the current method based on multilocus sequence typing failed. The qPCR assays described here are robust and modular tools that are efficient for differentiating X. fastidiosa subspecies directly in plant samples

Pseudomonas syringae pv. actinidifoliorum causes necrotic spots on the leaves of Actinidia deliciosa and Actinidia chinensis. P. syringae pv. actinidifoliorum has been detected in New Zealand, Australia, France and Spain. Four lineages were previously identified within the P. syringae pv. actinidifoliorum species group. Here, we report the draft genome sequences of five strains of P. syringae pv. actinidifoliorum representative of lineages 1, 2 and 4, isolated in France. The whole genomes of strains isolated in New Zealand, representative of P. syringae pv. actinidifoliorum lineages 1 and 3, were previously sequenced. The availability of supplementary P. syringae pv. actinidifoliorum genome sequences will be useful for developing molecular tools for pathogen detection and for performing comparative genomic analyses to study the relationship between P. syringae pv. actinidifoliorum and other kiwifruit pathogens, such as P. syringae pv. actinidiae.

Background Xanthomonas albilineans causes leaf scald, a lethal disease of sugarcane. X. albilineans exhibits distinctive pathogenic mechanisms, ecology and taxonomy compared to other species of Xanthomonas. For example, this species produces a potent DNA gyrase inhibitor called albicidin that is largely responsible for inducing disease symptoms; its habitat is limited to xylem; and the species exhibits large variability. A first manuscript on the complete genome sequence of the highly pathogenic X. albilineans strain GPE PC73 focused exclusively on distinctive genomic features shared with Xylella fastidiosa—another xylem-limited Xanthomonadaceae. The present manuscript on the same genome sequence aims to describe all other pathogenicity-related genomic features of X. albilineans, and to compare, using suppression subtractive hybridization (SSH), genomic features of two strains differing in pathogenicity. Results Comparative genomic analyses showed that most of the known pathogenicity factors from other Xanthomonas species are conserved in X. albilineans, with the notable absence of two major determinants of the “artillery” of other plant pathogenic species of Xanthomonas: the xanthan gum biosynthesis gene cluster, and the type III secretion system Hrp (hypersensitive response and pathogenicity). Genomic features specific to X. albilineans that may contribute to specific adaptation of this pathogen to sugarcane xylem vessels were also revealed. SSH experiments led to the identification of 20 genes common to three highly pathogenic strains but missing in a less pathogenic strain. These 20 genes, which include four ABC transporter genes, a methyl-accepting chemotaxis protein gene and an oxidoreductase gene, could play a key role in pathogenicity. With the exception of hypothetical proteins revealed by our comparative genomic analyses and SSH experiments, no genes potentially involved in any offensive or counter-defensive mechanism specific to X. albilineans were identified, supposing that X. albilineans has a reduced artillery compared to other pathogenic Xanthomonas species. Particular attention has therefore been given to genomic features specific to X. albilineans making it more capable of evading sugarcane surveillance systems or resisting sugarcane defense systems. Conclusions This study confirms that X. albilineans is a highly distinctive species within the genus Xanthomonas, and opens new perpectives towards a greater understanding of the pathogenicity of this destructive sugarcane pathogen.