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Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue-cultured plantlets grown in vitro. Six mutants of strain XaFL07-1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly-β-hydroxybutyrate than the wild-type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non-ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild-type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves. (Resume d'auteur)

International audience; A loop-mediated isothermal amplification (LAMP) assay was developed and compared to polymerase chain reaction (PCR), nested PCR, and selective isolation assays for detection of Xanthomonas albilineans, the causal agent of sugarcane leaf scald. The pathogen was isolated on selective medium from 44 out of 45 (98%) samples taken from symptomatic stalks, and from 44 out of 70 (63%) samples from asymptomatic stalks that were collected from plots with symptomatic stalks. Forty-two (93%), 41 (91%), and 42 (93%) symptomatic samples tested positive by LAMP, PCR and nested PCR, respectively. The pathogen was detected in 19 (27%), 8 (11%), and 25 (36%) of the 70 asymptomatic samples by LAMP, PCR and nested PCR, respectively. Symptomatic stalks were mainly, but not always, associated with high populations of the pathogen (10(7)-10(9) CFU/ml), and asymptomatic stalks with low populations (< 10(3) CFU/ml) or no bacteria. Although our LAMP and nested PCR methods detected 10 CFU/ml of X. albilineans in suspensions prepared with pure culture, they sometimes failed to detect the pathogen in samples with low pathogen populations. Isolation on selective medium along with another method should therefore be used for detection of the pathogen in asymptomatic stalks, especially in quarantine programs.

The BKBT strain of Helicoverpa armigera was derived from a susceptible BK77 strain (collected from Bouake, Cote D'Ivoire in 1977) through 30 generations of selection with activated Bacillus thuringiensis d-endotoxin Cry1Ac. Unlike recessive inheritance of Cry1Ac resistance in H. armigera from previous reports, resistance to activated Cry1Ac in the BKBT strain is dominant. A backcross approach was used to map dominant resistance to Cry1Ac in the BKBT strain. One hundred and forty-seven informative amplified fragment length polymorphism (AFLP) DNA markers covered all 31 linkage groups of H. armigera. Five AFLP markers linked to Cry1Ac resistance in the BKBT strain were on the same autosomal linkage group, which is the only linkage group contributing dominant Cry1Ac resistance in the BKBT strain of H. armigera. (Resume d'auteur)

International audience; How pathogens coevolve with and adapt to their hosts are critical to understanding how host jumps and/or acquisition of novel traits can lead to new disease emergences. The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria that collectively infect a broad range of crops and wild plant species. However, individual Xanthomonas strains usually cause disease on only a few plant species and are highly adapted to their hosts, making them pertinent models to study host specificity. This review summarizes our current understanding of the molecular basis of host specificity in the Xanthomonas genus, with a particular focus on the ecology, physiology, and pathogenicity of the bacterium. Despite our limited understanding of the basis of host specificity, type III effectors, microbe-associated molecular patterns, lipopolysaccharides, transcriptional regulators, and chemotactic sensors emerge as key determinants for shaping host specificity.

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue‐cultured plantlets grown in vitro. Six mutants of strain XaFL07‐1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly‐β‐hydroxybutyrate than the wild‐type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non‐ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild‐type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.

Xanthomonas albilineans is the bacterium responsible for leaf scald, a lethal disease of sugarcane. Within the Xanthomonas genus, X. albilineans exhibits distinctive genomic characteristics including the presence of significant genome erosion, a non-ribosomal peptide synthesis (NRPS) locus involved in albicidin biosynthesis, and a type 3 secretion system (T3SS) of the Salmonella pathogenicity island-1 (SPI-1) family. We sequenced two X. albilineans-like strains isolated from unusual environments, i.e., from dew droplets on sugarcane leaves and from the wild grass Paspalum dilatatum, and compared these genomes sequences with those of two strains of X. albilineans and three of Xanthomonas sacchari. Average nucleotide identity (ANI) and multi-locus sequence analysis (MLSA) showed that both X. albilineans-like strains belong to a new species close to X. albilineans that we have named “Xanthomonas pseudalbilineans”. X. albilineans and “X. pseudalbilineans” share many genomic features including (i) the lack of genes encoding a hypersensitive response and pathogenicity type 3 secretion system (Hrp-T3SS), and (ii) genome erosion that probably occurred in a common progenitor of both species. Our comparative analyses also revealed specific genomic features that may help X. albilineans interact with sugarcane, e.g., a PglA endoglucanase, three TonB-dependent transporters and a glycogen metabolism gene cluster. Other specific genomic features found in the “X. pseudalbilineans” genome may contribute to its fitness and specific ecological niche.

We report the high-quality draft genome sequence of Xanthomonas sacchari strain LMG 476, isolated from sugarcane. The genome comparison of this strain with a previously sequenced X. sacchari strain isolated from a distinct environmental source should provide further insights into the adaptation of this species to different habitats and its evolution.

Xanthomonas albilineans causes leaf scald, a lethal disease of sugarcane. Compared to other species of Xanthomonas, X. albilineans exhibits distinctive pathogenic mechanisms, ecology and taxonomy. Its genome, which has experienced significant erosion, has unique genomic features. It lacks two loci required for pathogenicity in other plant pathogenic species of Xanthomonas: the xanthan gum biosynthesis and the Hrp-T3SS (hypersensitive response and pathogenicity-type three secretion system) gene clusters. Instead, X. albilineans harbours in its genome an SPI-1 (Salmonella pathogenicity island-1) T3SS gene cluster usually found in animal pathogens. X. albilineans produces a potent DNA gyrase inhibitor called albicidin, which blocks chloroplast differentiation, resulting in the characteristic white foliar stripe symptoms. The antibacterial activity of albicidin also confers on X. albilineans a competitive advantage against rival bacteria during sugarcane colonization. Recent chemical studies have uncovered the unique structure of albicidin and allowed us to partially elucidate its fascinating biosynthesis apparatus, which involves an enigmatic hybrid PKS/NRPS (polyketide synthase/non-ribosomal peptide synthetase) machinery.

Albicidin is a potent DNA gyrase inhibitor produced by the sugarcane pathogenic bacterium Xanthomonas albilineans. Here we report the elucidation of the hitherto unknown structure of albicidin, revealing a unique polyaromatic oligopeptide mainly composed of p-aminobenzoic acids. In vitro studies provide further insights into the biosynthetic machinery of albicidin. These findings will enable structural investigations on the inhibition mechanism of albicidin and its assessment as a highly effective antibacterial drug.

The first linkage map established by Lanaud et al. (1995) was used as a starting point to produce a high-density molecular linkage map. A mapping population of 181 progenies resulting from a cross between two heterozygous genotypes, a Forastero and a Trinitario (hybrid between Forastero and Criollo), was used for the linkage analysis. A new DNA isolation protocol was established, which allows enough good quality DNA to construct a genetic map with PCR-based markers. The map comprises 424 markers with an average spacing between markers of 2.1 cM. The marker types used were five isozymes, six loci from known function genes, 65 genomic RFLPs, 104 cDNA RFLPs, three telomeric probes, 30 RAPDs, 191 AFLPs and 20 microsatellites. The use of new marker types, AFLP and microsatellites, did not disturb the original order of the RFLP loci used on the previous map. The genetic markers were distributed over ten linkage groups and cover 885.4 cM. The maximum distance observed between adjacent markers was 16.2 cM, and 9.4% of all loci showed skewed segregation.

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.

Background Various bacteria can use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in silico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant species. To date, the only known small molecule synthesized by NRPS in this genus is albicidin produced by Xanthomonas albilineans. This study aims to estimate the biosynthetic potential of Xanthomonas spp. by in silico analyses of NRPS genes with unknown function recently identified in the sequenced genomes of X. albilineans and related species of Xanthomonas. Results We performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv. oryzae strain BAI3 and Xanthomonas spp. strain XaS3. These two latter strains, together with X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains X8-1A and X11-5A, possess novel NRPS gene clusters and share related NRPS-associated genes such as those required for the biosynthesis of non-proteinogenic amino acids or the secretion of peptides. In silico prediction of peptide structures according to NRPS architecture suggests eight different peptides, each specific to its producing strain. Interestingly, these eight peptides cannot be assigned to any known gene cluster or related to known compounds from natural product databases. PCR screening of a collection of 94 plant pathogenic bacteria indicates that these novel NRPS gene clusters are specific to the genus Xanthomonas and are also present in Xanthomonas translucens and X. oryzae pv. oryzicola. Further genome mining revealed other novel NRPS genes specific to X. oryzae pv. oryzicola or Xanthomonas sacchari. Conclusions This study revealed the significant potential of the genus Xanthomonas to produce new non-ribosomally synthesized peptides. Interestingly, this biosynthetic potential seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement of non-ribosomally synthesized peptides in plant-bacteria interactions.

Xanthomonas albilineans is the causal agent of sugarcane leaf scald. Interestingly, this bacterium, which is not known to be insect or animal associated, possesses a type III secretion system (T3SS) belonging to the injectisome family Salmonella pathogenicity island 1 (SPI-1). The T3SS SPI-1 of X. albilineans shares only low similarity with other available T3SS SPI-1 sequences. Screening of a collection of 128 plant-pathogenic bacteria revealed that this T3SS SPI-1 is present in only two species of Xanthomonas: X. albilineans and X. axonopodis pv. phaseoli. Inoculation of sugarcane with knockout mutants showed that this system is not required by X. albilineans to spread within xylem vessels and to cause disease symptoms. This result was confirmed by the absence of this T3SS SPI-1 in an X. albilineans strain isolated from diseased sugarcane. To investigate the importance of the T3SS SPI-1 during the life cycle of X. albilineans, we analyzed T3SS SPI-1 sequences from 11 strains spanning the genetic diversity of this species. No nonsense mutations or frameshifting indels were observed in any of these strains, suggesting that the T3SS SPI-1 system is maintained within the species X. albilineans. Evolutionary features of T3SS SPI-1 based on phylogenetic, recombination, and selection analyses are discussed in the context of the possible functional importance of T3SS SPI-1 in the ecology of X. albilineans.

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.

The phytotoxin and polyketide antibiotic albicidin produced by Xanthomonas albilineans is a highly potent DNA gyrase inhibitor. Low yields of albicidin production have slowed studies of its chemical structure. Heterologous expression of albicidin biosynthetic genes in X. axonopodis pv. vesicatoria resulted in a sixfold increase in albicidin production, offering promising strategies for engineering overproduction.

Pathogenicity of 75 strains of Xanthomonas albilineans from Guadeloupe was assessed by inoculation of sugarcane cv. B69566, which is susceptible to leaf scald, and 19 of the strains were selected as representative of the variation in pathogenicity observed based on stalk colonization. In vitro production of albicidin varied among these 19 strains, but the restriction fragment length polymorphism pattern of their albicidin biosynthesis genes was identical. Similarly, no genomic variation was found among strains by pulsed-field gel electrophoresis. Some variation among strains was found by amplified fragment length polymorphism, but no relationship between this genetic variation and variation in pathogenicity was found. Only 3 (pilB, rpfA, and xpsE) of 40 genes involved in pathogenicity of bacterial species closely related to X. albilineans could be amplified by polymerase chain reaction from total genomic DNA of all nine strains tested of X. albilineans differing in pathogenicity in Guadeloupe. Nucleotide sequences of these genes were 100% identical among strains, and a phylogenetic study with these genes and housekeeping genes efp and ihfA suggested that X. albilineans is on an evolutionary road between the X. campestris group and Xylella fastidiosa, another vascular plant pathogen. Sequencing of the complete genome of Xanthomonas albilineans could be the next step in deciphering molecular mechanisms involved in pathogenicity of X. albilineans.

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, produces a highly potent polyketide-peptide antibiotic and phytotoxin called albicidin. Previous studies established the involvement of a large cluster of genes in the biosynthesis of this toxin. We report here the sub-cloning and sequencing of an additional gene outside of the main cluster and essential for albicidin biosynthesis. This gene encodes a 634-amino-acid protein that shows high identity with the Escherichia coli heat shock protein HtpG. Complementation studies of X. albilineans Tox- mutants confirmed the requirement of htpG for albicidin biosynthesis and revealed functional interchangeability between E. coli and X. albilineans htpG genes. HtpG was co-localised with albicidin in the cellular membrane, i.e., the cellular fraction where the toxin is most probably biosynthesised. Here we show the requirement of an HtpG protein for the biosynthesis of a polyketide-peptide antibiotic.

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin called albicidin. We report here sequencing and homology analysis of the major gene cluster, XALB1 (55,839 bp), and a second, smaller region, XALB2 (2,986 bp), involved in albicidin biosynthesis. XALB1 contains 20 open reading frames, including i) three large genes with a modular architecture characteristic of polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and ii) several putative modifying, regulatory, and resistance genes. Sequencing and complementation studies of six albicidin-defective mutants enabled us to confirm the involvement of the three PKS and NRPS genes encoded by XALB1 in albicidin production. XALB2 contains only one gene that is required for post-translational activation of PKS and NRPS enzymes, confirming the involvement of these enzymes in albicidin biosynthesis. In silico analysis of these three PKS or NRPS enzymes allowed us to propose a model for the albicidin backbone assembly and to gain insight into the structural features of this pathotoxin. This is the first description of a complete mixed PKS—NRPS gene cluster for toxin production in the genus Xanthomonas.

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS −like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.

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.