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The genus Xanthomonas has been primarily studied for pathogenic interactions with plants. However, besides host and tissue-specific pathogenic strains, this genus also comprises nonpathogenic strains isolated from a broad range of hosts, sometimes in association with pathogenic strains, and other environments, including rainwater. Based on their incapacity or limited capacity to cause symptoms on the host of isolation, nonpathogenic xanthomonads can be further characterized as commensal and weakly pathogenic. This study aimed to understand the diversity and evolution of nonpathogenic xanthomonads compared to their pathogenic counterparts based on their cooccurrence and phylogenetic relationship and to identify genomic traits that form the basis of a life history framework that groups xanthomonads by ecological strategies. We sequenced genomes of 83 strains spanning the genus phylogeny and identified eight novel species, indicating unexplored diversity. While some nonpathogenic species have experienced a recent loss of a type III secretion system, specifically the hrp2 cluster, we observed an apparent lack of association of the hrp2 cluster with lifestyles of diverse species. We performed association analysis on a large data set of 337 Xanthomonas strains to explain how xanthomonads may have established association with the plants across the continuum of lifestyles from commensals to weak pathogens to pathogens. Presence of distinct transcriptional regulators, distinct nutrient utilization and assimilation genes, transcriptional regulators, and chemotaxis genes may explain lifestyle-specific adaptations of xanthomonads., Competing Interests: Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Historically, Xanthomonas species are primarily known for their pathogenicity against plants, but recently, there have been more findings of non-pathogenic xanthomonads. In the present study, we report isolates from healthy rice seeds that belong to a new species, Xanthomonas protegens, a protector of the rice plants against a serious pathogenic counterpart, i.e. X. oryzae pv. oryzae upon leaf clip co-inoculation. The new member species is non-pathogenic to rice and lacks a type III secretion system. The pangenome investigation revealed a large number of unique genes, including a novel lipopolysaccharide biosynthetic gene cluster, that might be important in its adaptation. The phylo-taxonogenomic analysis revealed that X. protegens is a taxonomic outlier species of X. sontii, a core, vertically transmitted rice seed endophyte with numerous probiotic properties. Interestingly, X. sontii is also reported as a keystone species of healthy rice seed microbiome. The findings and resources will help in the development of unique gene markers and evolutionary studies of X. sontii as a successful symbiont and X. oryzae as a serious pathogen. Here, we propose X. protegens sp. nov. as a novel species of the genus Xanthomonas with PPL118 = MTCC 13396 = CFBP 9164 = ICMP 25181 as the type strain. PPL117, PPL124, PPL125, and PPL126 are other strains of the species., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Aim: To investigate the genetic and pathogenic variability of Xanthomonas oryzae pv. oryzae causing bacterial blight in rice on the remote Andaman Islands, India., Methods and Results: A total of 27 yellow-pigmented bacterial isolates representing rice fields of Andaman Islands incited blight on the susceptible-rice cultivar, C14-8. Phenotypic, pathogenic traits and 16S rRNA gene sequences revealed their identity as X. oryzae pv. oryzae. Virulence profiling indicated the prevalence of seven pathotypes of X. oryzae pv. oryzae on the Islands. Pathotypes-VI and -VII were highly virulent, whereas the pathotype-I was less virulent. Multilocus sequence typing based on nucleotide sequence polymorphism in nine housekeeping genes dnaK; fyuA; gyrB (two loci): rpoD; fusA; gapA; gltA and lepA clustered 27 isolates into 17 sequence types (STs) segregated into two clonal-complexes (CC). While CC-I comprised of isolates from Andaman Island, the CC-II is a mixture of isolates representing mainland India and Andaman Island. The data revealed trans-boundary pathogen introduction and a consequent intra-regional diversification on these islands due to the deployment of different rice cultivars in different regions., Conclusions: Genotyping and pathotyping of sland isolates revealed seven pathotypes distributed in two clonal complexes with strong indications for trans-boundary movement and consequent diversification of the bacterial pathogen. Highly virulent pathotypes of X. oryzae pv. oryzae that could overcome combinations of R-genes, xa13+Xa21 as well as xa5+xa13 were found prevalent in the Andaman Islands SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic and virulence analysis of X. oryzae pv. oryzae in the Andaman Islands revealed introduction and host-mediated regional diversification and local adaptation of X oryzae pv. oryzae. The study calls for the need of multi-gene pyramiding for durable disease resistance and establishing stringent quarantine measures for safeguarding island agricultural practices in the future., (© 2020 The Society for Applied Microbiology.)

Transcription activator-like effectors (TALEs) are secreted by plant-pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

We announce the genome sequence for Xanthomonas species strain Nyagatare, isolated from beans showing unusual disease symptoms in Rwanda. This strain represents the first sequenced genome belonging to an as-yet undescribed Xanthomonas species known as species-level clade 1. It has at least 100 kb of genomic sequence that shows little or no sequence similarity to other xanthomonads, including a unique lipopolysaccharide synthesis gene cluster. At least one genomic region appears to have been acquired from relatives of Agrobacterium or Rhizobium species. The genome encodes homologues of only three known type-three secretion system effectors: AvrBs2, XopF1 and AvrXv4. Availability of the genome sequence will facilitate development of molecular tools for detection and diagnostics for this newly discovered pathogen of beans and facilitate epidemiological investigations of a potential causal link between this pathogen and the disease outbreak., (© FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: To establish protocols for the simultaneous detection and identification of Xanthomonas species causing tomato bacterial spot., Methods and Results: We verified the specificity and sensitivity of the previously reported sets of primers designed for strains of the four species of Brazilian tomato bacterial spot xanthomonads, consisting of 30 of Xanthomonas euvesicatoria, 30 of X. vesicatoria, 50 of X. perforans and 50 of X. gardneri. Furthermore, we tested a multiplex PCR protocol for the purpose of concurrent species identification. The possibility of direct detection of the pathogens in diseased leaf samples was also verified. The primers were highly specific, amplifying only target DNA. The sensitivity of the primers in conventional PCR was 50 pg μl(-1) for purified DNA and ranged from 5 × 10(2) to 5 × 10(4) CFU ml(-1) when bacterial suspensions were analysed. The multiplex PCR was suitable for the detection of all four species and showed similar sensitivity to conventional PCR when tested on purified DNA. When using bacterial suspensions, its sensitivity was similar to conventional PCR only when a biological amplification step (Bio-PCR) was included. Both methods were able to detect the pathogens in symptomatic tomato leaves., Conclusions: Brazilian Xanthomonas strains causing tomato bacterial spot can be differentiated and identified at species level by a PCR-based method and by a multiplex PCR., Significance and Impact of the Study: This protocol may be a feasible alternative tool for the identification and detection of these pathogens in plant material and may be used for routine diagnostic purposes in plant pathology laboratories., (© 2012 The Society for Applied Microbiology.)

Banana Xanthomonas wilt is a newly emerging disease that is currently threatening the livelihoods of millions of farmers in East Africa. The causative agent is Xanthomonas campestris pathovar musacearum (Xcm), but previous work suggests that this pathogen is much more closely related to species Xanthomonas vasicola than to X. campestris. We have generated draft genome sequences for a banana-pathogenic strain of Xcm isolated in Uganda and for a very closely related strain of X. vasicola pathovar vasculorum, originally isolated from sugarcane, that is nonpathogenic on banana. The draft sequences revealed overlapping but distinct repertoires of candidate virulence effectors in the two strains. Both strains encode homologues of the Pseudomonas syringae effectors HopW, HopAF1 and RipT from Ralstonia solanacearum. The banana-pathogenic and non-banana-pathogenic strains also differed with respect to lipopolysaccharide synthesis and type-IV pili, and in at least several thousand single-nucleotide polymorphisms in the core conserved genome. We found evidence of horizontal transfer between X. vasicola and very distantly related bacteria, including members of other divisions of the Proteobacteria. The availability of these draft genomes will be an invaluable tool for further studies aimed at understanding and combating this important disease.

The integron platform and the gene cassette arrays of 34 Xanthomonas arboricola pv. juglandis and of 47 Xanthomonas arboricola pv. pruni strains isolated from different geographical areas were screened to check their variability. Genetic variability of the strains was also tested by means of BOX-PCR. For two representative strains of the two pathovars, the integrase gene intI and part of the flanking gene ilvD were also cloned and sequenced. Whereas X. a. pv. pruni strains did not show relevant variability, six X. a. pv. juglandis strains isolated in Australia showed some differences in the gene sequences. The CLUSTALW algorithm indicated that the majority of the X. a. pv. juglandis strains are closely related to X. a. pv. pruni, whereas the X. a. pv. juglandis strains isolated in Australia were more similar to Xanthomonas hortorum pv. pelargonii. Similarly, the gene cassette array pattern of the Australian strains, as well as that of the oldest strain maintained in culture, was different from the other strains. Also, three X. a. pv. pruni strains showed a different cassette array pattern when compared with the majority of other strains but no relationships with geographical area of isolation or host plant was revealed. This study confirmed that in addition to species, integrons may generate diversity also within two X. arboricola pathovars.

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a major disease threatening citrus crops throughout the world. The most common methods for strain differentiation of this pathogen are repetitive element sequence-based PCR (rep-PCR) and pulsed field gel electrophoresis (PFGE), using rare-cutting restriction enzyme analysis. We developed a ligation-mediated PCR targeting three insertion sequences (IS-LM-PCR) present as several copies in the genome of the fully sequenced strain 306 of X. citri pv. citri. This technique amplifies DNA fragments between an insertion sequence element and an MspI restriction site. The analysis of strains can be conducted within 24 h, starting from very small amounts of bacterial DNA, which makes IS-LM-PCR much less labor-intensive than PFGE. We used IS-LM-PCR to analyze a collection of 66 strains of X. citri pv. citri from around the world. The overall reproducibility of IS-LM-PCR reached 98% in this data set and its discriminatory power was markedly superior than rep-PCR. We suggest that IS-LM-PCR could be used for the global surveillance of non-epidemiologically related strains of X. citri pv. citri.

The genetic characterization of Xanthomonas species remains a challenge. Several DNA-based techniques have been previously employed, including the analysis of the 16S rRNA and 16S-23S rRNA genes in order to differentiate and classify the Xanthomonas species. However, several species could not be distinguished in these studies, due to the high degree of conservation of these molecular markers. In order to obtain more efficient markers, and to better understand the phylogenetic relationships between the Xanthomonas species, two genes commonly found in the different species have been analyzed. The genes rpfB and atpD involved in the regulation of pathogenicity factors and in the synthesis of ATP, respectively, were amplified in Xanthomonas species and further analyzed by PCR-restriction fragment length polymorphism. Dendrograms with the data sets of the rpfB and atpD analyzed separately and combined were constructed. The results obtained revealed that several Xanthomonas species, previously grouped together, could be successfully distinguished using these markers. The results obtained herein provide an alternative method for the distinction of the Xanthomonas species and contribute to a better understanding of the genetic and phylogenetic relationships of Xanthomonas.

Two DNA fragments from Xanthomonas albilineans were used as probes to study the molecular diversity among strains of this pathogen. Two serologically distinct groups, serovars I and II, could be differentiated by hybridization to the probes. These probes, designated 830 and 838, were cloned after subtractive DNA hybridization of common sequences of Xanthomonas campestris pv. vasculorum from a serovar I strain of X. albilineans. They did not hybridize to the DNA of several other xanthomonads or to sugarcane DNA under the conditions of hybridization used. Faint bands were observed upon hybridization of probe 830 with one strain of X. campestris pv. phaseoli. The same banding patterns were obtained with a strain of X. albilineans from Burkina Faso and the serovar II strains of Mauritius. The serovar I strains from Mauritius and two other strains each from Reunion and South Africa had similar pattern.

The O antigen polymer recovered from the reference strain for Stenotrophomonas (Xanthomonas or Pseudomonas) maltophilia serogroup O7, after mild acid hydrolysis of the lipopolysaccharide, was constructed from D-rhamnose. By means of chemical degradations and NMR studies, the repeating unit of the polymer was shown to be a linear trisaccharide with the structure -->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-R hap-(1-->. The same repeating unit is present in the common antigen of Pseudomonas aeruginosa and in O antigens from some pathovars of Pseudomonas syringae and a strain of Burkholderia (Pseudomonas) cepacia.

Carbohydrate utilization profiles by means of the API (Appareils et Procédés d'Identification) system and sensitivity to antibiotics and heavy metal salts of 68 Xanthomonas sp. mangiferaeindicae strains isolated in nine countries from mango (Mangifera indica L.) and other genera of the Anacardiaceae were examined to assess the variability of the taxon. The strains could be separated into 10 groups according to Ward clustering. Apigmented strains isolated from the pepper tree [syn. Brazilian pepper] (Schinus terebenthifolius Raddi) could not be clearly differentiated from most apigmented strains isolated from mango. Yellow-pigmented strains isolated from mango in Brazil and Reunion Island, apigmented strains isolated from mango in Brazil and from ambarella in the French West Indies, clustered in distinct groups. The results are consistent with those of other studies, based on isozyme analysis of esterase, phosphoglucomutase and superoxide dismutase, and hrp-RFLP analysis; they indicate the need for a comprehensive taxonomic evaluation of xanthomonads associated with Anacardiaceae.

The nearly complete, PCR-amplified, 16S rRNA gene sequences have been determined from the representative type strains of eight xanthomonad phena, including six validly described species of the genus Xanthomonas and Stenotrophomonas maltophilia. Pairwise sequence comparisons and phylogenetic analysis demonstrated that the xanthomonads comprise a monophyletic lineage-within the gamma-subclass of the Proteobacteria. Although the genus Xanthomonas was observed to comprise a cluster of very closely related species, the observed species-specific primary sequence differences were confirmed through sequencing additional strains belonging to the respective species.

Five strains of Xanthomonas albilineans, causal agent of leaf scald disease in sugarcane from various geographical regions, were compared using random amplification of polymorphic DNA (RAPD) to determine whether they could be differentiated at the DNA level. CsCl-purified genomic DNA from these strains were amplified by the polymerase chain reaction (PCR) using arbitrary 10-mer primers according to standard RAPD conditions and the amplification product profiles analysed by conventional agarose gel electrophoresis. Although most RAPD markers were common to all five strains, unique profiles for each strain were discernible using four 10-mer arbitrary primers individually. Reproducible DNA fingerprints indicate that RAPD analysis can be used to identify and differentiate the X. albilineans strains. This technique has the potential for use in monitoring the appearance of foreign strains of X. albilineans in various geographical regions and could be used for the construction of phylogenetic trees.