Genetic relationship among worldwide strains of Xanthomonas causing canker in citrus species and design of new primers for their identification by PCR

Partial sequence analysis of the ribosomal operon in Xanthomonas axonopodis allowed discrimination among strains causing the A, B, and C types of citrus bacterial canker (CBC) and quantification of the relationship of these organisms with other species and pathovars in the same genus. Sets of primers based on sequence differences in the internally transcribed spacer and on a sequence from the plasmid gene pthA involved in virulence were designed for specific identification of xanthomonads causing CBC diseases. The two sets were validated with a collection of Xanthomonas strains associated with citrus species. The primer set based on ribosomal sequences had a high level of specificity for X. axonopodis pv. citri, whereas the set based on the pthA gene was universal for all types of CBC organisms. Moreover, the relationships among worldwide Xanthomonas strains causing CBC were analyzed by amplification of repetitive sequences (enterobacterial repetitive intergenic consensus and BOX elements). Under specific conditions, pathotypes of these Xanthomonas strains could be discerned, and subgroups of the pathotypes were identified. Subgroups of strains were associated with certain geographic areas of the world, and on this basis the origin of type A strains introduced into Florida could be inferred.     

Genomic Insights into the Evolutionary Origin of Xanthomonas axonopodis pv. citri and Its Ecological Relatives

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker (CBC) and is a serious problem worldwide. Like CBC, several important diseases in other fruits, such as mango, pomegranate, and grape, are also caused by Xanthomonas pathovars that display remarkable specificity toward their hosts. While citrus and mango diseases were documented more than 100 years ago, the pomegranate and grape diseases have been known only since the 1950s and 1970s, respectively. Interestingly, diseases caused by all these pathovars were noted first in India. Our genome-based phylogenetic studies suggest that these diverse pathogens belong to a single species and these pathovars may be just a group of rapidly evolving strains. Furthermore, the recently reported pathovars, such as those infecting grape and pomegranate, form independent clonal lineages, while the citrus and mango pathovars that have been known for a long time form one clonal lineage. Such an understanding of their phylogenomic relationship has further allowed us to understand major and unique variations in the lineages that give rise to these pathovars. Whole-genome sequencing studies including ecological relatives from their putative country of origin has allowed us to understand the evolutionary history of Xac and other pathovars that infect fruits.     

Global analysis of the eukaryotic pathways and networks regulated by Salmonella typhimurium in mouse intestinal infection in vivo

Background

Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.

Results

We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.

Conclusion

We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.     

Molecular characterisation of Xanthomonas strains isolated from aroids in Mauritius

Mauritius is one of the largest world producers of Anthurium cut flowers but outbreaks of bacterial blight have never been reported on the island. This work was about the characterisation and identification of bacterial strains isolated from Anthurium andreanum, Dieffenbachia maculata and Aglaonema simplex in Mauritius. Fifteen strains, that showed the morphological properties of Xanthomonas on conventional media, were tested on two semi-selective media (Esculin-trehalose and cellobiose-starch). ELISA tests using a panel of monoclonal antibodies were carried out and three out of 15 strains reacted with a Xanthomonas-specific monoclonal antibody (MAb XII). Analysis using four sets of ribosomal primers revealed that the same three Mauritius strains shared conserved PCR products with reference xanthomonads including virulent strains of Xanthomonas axonopodis pv. dieffenbachiae (Xad). BIOLOG tests and the Sherlock Microbial Identification system (MIDI) identified these three new strains at the species level as X. axonopodis. The complementary tests that were carried out clearly confirmed that the three strains are xanthomonads and, moreover, a DNA probe which showed specificity to Xad strains suggested that the three Mauritius strains are non-virulent forms of the pathogen causing Anthurium blight.     

Characterization of Bacteriophages Cp1 and Cp2, the Strain-Typing Agents for Xanthomonas axonopodis pv. citri

The strains of Xanthomonas axonopodis pv. citri, the causative agent of citrus canker, are historically classified based on bacteriophage (phage) sensitivity. Nearly all X. axonopodis pv. citri strains isolated from different regions in Japan are lysed by either phage Cp1 or Cp2; Cp1-sensitive (Cp1^s) strains have been observed to be resistant to Cp2 (Cp2^r) and vice versa. In this study, genomic and molecular characterization was performed for the typing agents Cp1 and Cp2. Morphologically, Cp1 belongs to the Siphoviridae. Genomic analysis revealed that its genome comprises 43,870-bp double-stranded DNA (dsDNA), with 10-bp 3′-extruding cohesive ends, and contains 48 open reading frames. The genomic organization was similar to that of Xanthomonas phage phiL7, but it lacked a group I intron in the DNA polymerase gene. Cp2 resembles morphologically Escherichia coli T7-like phages of Podoviridae. The 42,963-bp linear dsDNA genome of Cp2 contained terminal repeats. The Cp2 genomic sequence has 40 open reading frames, many of which did not show detectable homologs in the current databases. By proteomic analysis, a gene cluster encoding structural proteins corresponding to the class III module of T7-like phages was identified on the Cp2 genome. Therefore, Cp1 and Cp2 were found to belong to completely different virus groups. In addition, we found that Cp1 and Cp2 use different molecules on the host cell surface as phage receptors and that host selection of X. axonopodis pv. citri strains by Cp1 and Cp2 is not determined at the initial stage by binding to receptors.     

In Planta Horizontal Transfer of a Major Pathogenicity Effector Gene

Xanthomonas citri pv. citri is a clonal group of strains that causes citrus canker disease and appears to have originated in Asia. A phylogenetically distinct clonal group that causes identical disease symptoms on susceptible citrus, X. citri pv. aurantifolii, arose more recently in South America. Genomes of X. citri pv. aurantifolii strains carry two DNA fragments that hybridize to pthA, an X. citri pv. citri gene which encodes a major type III pathogenicity effector protein that is absolutely required to cause citrus canker. Marker interruption mutagenesis and complementation revealed that X. citri pv. aurantifolii strain B69 carried one functional pthA homolog, designated pthB, that was required to cause cankers on citrus. Gene pthB was found among 38 open reading frames on a 37,106-bp plasmid, designated pXcB, which was sequenced and annotated. No additional pathogenicity effectors were found on pXcB, but 11 out of 38 open reading frames appeared to encode a type IV transfer system. pXcB transferred horizontally in planta, without added selection, from B69 to a nonpathogenic X. citri pv. citri (pthA::Tn5) mutant strain, fully restoring canker. In planta transfer efficiencies were very high (>0.1%/recipient) and equivalent to those observed for agar medium with antibiotic selection, indicating that pthB conferred a strong selective advantage to the recipient strain. A single pathogenicity effector that can confer a distinct selective advantage in planta may both facilitate plasmid survival following horizontal gene transfer and account for the origination of phylogenetically distinct groups of strains causing identical disease symptoms.     

Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease

Citrus canker provoked by Xanthomonas axonopodis pv. citri is a bacterial disease causing severe losses in all citrus-producing areas around the world. Xanthomonas infection is considered as an endemic disease in Northeast and Northwest Argentina, affecting as much as 10% of commercial citrus plantations. There is not known natural resistance neither in orange varieties nor in rootstocks used for grafting of commercial cultivars. To introduce resistance to this disease, plants of Pineapple sweet orange were transformed with a genetic construct allowing constitutive accumulation of dermaseptin. In comparison with non-transformed plants, transgenic plants showed symptom reduction levels of up to 50% in in planta assays performed under controlled conditions.     

Genetic Diversity and Pathogenicity of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> Strains Inducing Citrus Canker Disease in Iran and South Korea

For the first time in 1989 citrus bacterial canker disease has seen on Citrus aurantiifolia in southern Iran. A total of 43 strains from affected citrus trees, ten strains from South Korea and representative from all known five pathotypes of Xanthomonas axonopodis pathogenic on citrus trees were used in this study. Isolated strains from Iran were indistinguishable by phenotypic, FAMEs, and SDS-PAGE analyses but showed different host range. First group were pathogenic on all tested citrus seedlings including C. aurantiifolia, C. limettioides, C. limon, C. jambhiri, Poncirus trifoliata X C. paradisi, C. aurantium, C. paradise, C. medica, P. trifoliate, C. grandis, C. sinensis, C. reticulate and C. sinensis X P. trifoliate. Pathogenicity of the second group were limited to C. aurantiifolia, C. limettioides, C. limon, C. jambhiri, P. trifoliata X C. paradis, and C. aurantium. Among the strains studied by AFLP fingerprinting six clusters were found. These clusters were: (1) strains of pathotype C; (2) strains of pathotypes B and D; (3) strains of pathotype A together with the main group of the Iranian strains; (4) strains isolated from Korea; (5) strains of pathotype E; and (6) seven strains from Iran which made a completely separate cluster. Strains from pathotypes B and D could not be differentiated by AFLP. The tested Iranian strains belongs to the two different groups and strains from Korea grouped as a subcluster from main cluster of Iranian strains belong to the pathotype A.     

Use of 16S rDNA Sequences as Signature Characters to Identify Xylella fastidiosa

The nucleotide sequences of 16S rDNAs (coding for the small subunit ribosomal RNAs) were used to identify Xylella fastidiosa, a nutritionally fastidious plant pathogenic bacterium. The near-complete 16S rDNAs from nine strains of Xyl. fastidiosa, including seven pathotypes and one strain of Xanthomonas campestris pv. campestris, were amplified through PCR with two conserved primers (forward primer 5′-AGA GTT TGA TCC TGG CTC AG-3′ and reverse primer 5′-AAG GAG GTG ATC CAG CC-3′) and sequenced. The 16S sequences were compared with all eukaryote and prokaryote DNA entries in GenBank database. A Xyl. fastidiosa 16S rDNA sequence, M26601, was determined to be the most similar to all the near-complete (1537 bp) and partial 5′ end sequences from Xyl. fastidiosa, but not those from the Xanthomonas strain. A 20-bp oligonucleotide (5′-TTG GTA GTA ATA CCA TGG GT-3′) was found to be highly characteristic of Xyl. fastidiosa. Since the 16S rDNA of Xyl. fastidiosa strains are highly homologous and characteristically different from other bacteria, including the most closely related Xanthomonas, 16S rDNA sequences can be used as signature characters to identify this bacterium. Received: 22 June 1999 / Accepted: 2 August 1999     

Phylogenetic relatedness of Xanthomonas axonopodis pv. punicae, the causal agent of bacterial blight of pomegranate based on two loci, 16S rRNA and gyrB

Bacterial blight caused by Xanthomonas axonopodis pv. punicae (Xap) is a major disease in pomegranate (Punica granatum) cultivation in India. The Xap strains from three distinct geographical origins, Delhi, Maharashtra and Andhra Pradesh were studied for their genetic variability and phylogenetic relationship with other Xanthomonads targeting two important loci 16S rRNA and gyrB. All Xap strains showed 100 % sequence conservation in both the loci, suggesting that geographical origin does not necessarily reflect variation to genetic make-up of the Xap. Phylogeny derived from 16S rRNA gene revealed that two Xanthomonas species, Xanthomonas citri subsp. malvacearum DSM 3849 T and X. axonopodis pv. manihotis NCPPB1834 formed a single cluster along with Xap. Further, analysis in the gyrB locus indicated that X. citri subsp. malvacearum shared 99.4 % identity while pathovars X. axonopodis pv. manihotis shared only 95 % identity with the Xap strains. Thus, we established that gyrB was the preferred locus over 16S rRNA gene to discriminate the Xap strains from closely related Xanthomonas species type strains. Nevertheless, our study demonstrated for the first time that pomegranate bacterial blight pathogen is phylogenetically very close to Xanthomonas citri subsp. malvacearum infecting cotton.     

Evolutionary History of the Plant Pathogenic Bacterium Xanthomonas axonopodis

Deciphering mechanisms shaping bacterial diversity should help to build tools to predict the emergence of infectious diseases. Xanthomonads are plant pathogenic bacteria found worldwide. Xanthomonas axonopodis is a genetically heterogeneous species clustering, into six groups, strains that are collectively pathogenic on a large number of plants. However, each strain displays a narrow host range. We address the question of the nature of the evolutionary processes – geographical and ecological speciation – that shaped this diversity. We assembled a large collection of X. axonopodis strains that were isolated over a long period, over continents, and from various hosts. Based on the sequence analysis of seven housekeeping genes, we found that recombination occurred as frequently as point mutation in the evolutionary history of X. axonopodis. However, the impact of recombination was about three times greater than the impact of mutation on the diversity observed in the whole dataset. We then reconstructed the clonal genealogy of the strains using coalescent and genealogy approaches and we studied the diversification of the pathogen using a model of divergence with migration. The suggested scenario involves a first step of generalist diversification that spanned over the last 25 000 years. A second step of ecology-driven specialization occurred during the past two centuries. Eventually, secondary contacts between host-specialized strains probably occurred as a result of agricultural development and intensification, allowing genetic exchanges of virulence-associated genes. These transfers may have favored the emergence of novel pathotypes. Finally, we argue that the largest ecological entity within X. axonopodis is the pathovar.     

Genomic Survey of Pathogenicity Determinants and VNTR Markers in the Cassava Bacterial Pathogen Xanthomonas axonopodis pv. Manihotis Strain CIO151

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.     

Positive selection is the main driving force for evolution of citrus canker-causing Xanthomonas

Understanding the evolutionary history and potential of bacterial pathogens is critical to prevent the emergence of new infectious bacterial diseases. Xanthomonas axonopodis subsp. citri (Xac) (synonym X. citri subsp. citri), which causes citrus canker, is one of the hardest-fought plant bacterial pathogens in US history. Here, we sequenced 21 Xac strains (14 XacA, 3 XacA* and 4 XacA^w) with different host ranges from North America and Asia and conducted comparative genomic and evolutionary analyses. Our analyses suggest that acquisition of beneficial genes and loss of detrimental genes most likely allowed XacA to infect a broader range of hosts as compared with XacA^w and XacA*. Recombination was found to have occurred frequently on the relative ancient branches, but rarely on the young branches of the clonal genealogy. The ratio of recombination/mutation ρ/θ was 0.0790±0.0005, implying that the Xac population was clonal in structure. Positive selection has affected 14% (395 out of 2822) of core genes of the citrus canker-causing Xanthomonas. The genes affected are enriched in ‘carbohydrate transport and metabolism'' and ‘DNA replication, recombination and repair'' genes (P<0.05). Many genes related to virulence, especially genes involved in the type III secretion system and effectors, are affected by positive selection, further highlighting the contribution of positive selection to the evolution of citrus canker-causing Xanthomonas. Our results suggest that both metabolism and virulence genes provide advantages to endow XacA with higher virulence and a wider host range. Our analysis advances our understanding of the genomic basis of specialization by positive selection in bacterial evolution.     

Genetic Structure and Population Dynamics of Xanthomonas axonopodis pv. manihotis in Colombia from 1995 to 1999

Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different X. axonopodis pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the X. axonopodis pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of X. axonopodis pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of X. axonopodis pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.     

Molecular Typing and Genetic Characterization of Pathogenic Variants of Xanthomonas axonopodis pv. citri in Taiwan

Molecular typing was applied and optimized for genetic characterization for three pathogenic variants of Xanthomonas axonopodis pv. citri (Xac) from Taiwan. These three novel variants of atypical symptom–producing X. axonopodis pv. citri were designated as Xac‐A^f, Xac‐A^p and Xac‐A^r. Based on polymerase chain reaction (PCR) with primers specific to X. axonopodis pv. citri, leucine‐responsive regulatory protein (lrp) gene assay and DNA fingerprintings generated by repetitive‐sequence PCR (rep‐PCR) and amplified fragment length polymorphism (AFLP) were used to compare strains including the three types of atypical symptom–producing strains Xac‐A^f, Xac‐A^p and Xac‐A^r, and additional reference strains from pathotypes Xac‐A, Xac‐A*, Xac‐A^w, X. axonopodis pv. auruantifolii and X. axonopodis pv. citrumelo. These three types of X. axonopodis pv. citri variants can be detected with six sets of primer specific for X. axonopodis pv. citri. Cluster analyses by lrp sequence assay, AFLP and combing the band patterns of rep‐PCR clearly grouped the atypical symptom–producing variants in types Xac‐ A^f, Xac‐A^r and Xac‐A^p into the same cluster with typical symptom‐producing strains in pathotype Xac‐A. These three types of X. axonopodis pv. citri variants could be excluded from strains of Xac‐A* and Xac‐A^w in these genotypic analyses. Strains of Xac‐A* and Xac‐A^w were closely related to Xac‐A strains in our results. No Taiwan isolate was related to X. axonopodis pv. auruantifolii or X. axonopodis pv. citrumelo. The results further confirmed the atypical symptom–producing variants of X. axonopodis pv. citri in Taiwan belong to pathotype Xac‐A.     

Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa

A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierce's disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.     

Acquisition and Evolution of Plant Pathogenesis–Associated Gene Clusters and Candidate Determinants of Tissue-Specificity in Xanthomonas

Background

Xanthomonas is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown.

Methodology/Principal Findings

To assess potential contributions to host- and tissue-specificity, pathogenesis-associated gene clusters were compared across genomes of eight Xanthomonas strains representing vascular or non-vascular pathogens of rice, brassicas, pepper and tomato, and citrus. The gum cluster for extracellular polysaccharide is conserved except for gumN and sequences downstream. The xcs and xps clusters for type II secretion are conserved, except in the rice pathogens, in which xcs is missing. In the otherwise conserved hrp cluster, sequences flanking the core genes for type III secretion vary with respect to insertion sequence element and putative effector gene content. Variation at the rpf (regulation of pathogenicity factors) cluster is more pronounced, though genes with established functional relevance are conserved. A cluster for synthesis of lipopolysaccharide varies highly, suggesting multiple horizontal gene transfers and reassortments, but this variation does not correlate with host- or tissue-specificity. Phylogenetic trees based on amino acid alignments of gum, xps, xcs, hrp, and rpf cluster products generally reflect strain phylogeny. However, amino acid residues at four positions correlate with tissue specificity, revealing hpaA and xpsD as candidate determinants. Examination of genome sequences of xanthomonads Xylella fastidiosa and Stenotrophomonas maltophilia revealed that the hrp, gum, and xcs clusters are recent acquisitions in the Xanthomonas lineage.

Conclusions/Significance

Our results provide insight into the ancestral Xanthomonas genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates.     

16S rRNA gene sequence analyses and inter- and intrageneric relationships of Xanthomonas species and Stenotrophomonas maltophilia

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 γ-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.     

Biocontrol of citrus bacterial canker caused by Xanthomonas citri subsp. citri by Bacillus velezensis

Microorganisms with biocontrol capabilities against plant pathogens are considered as one of the most promising approaches for healthy crop management. In this study, ethyl acetate extracts of 25 Bacillus strains were investigated for their antagonistic effect on Xanthomonas citri subsp. citri (Xcc), which causes the citrus bacterial canker (CBC) disease. Among them, 21 strains exerted antibacterial activity against wild-type Xcc strains. Based on the strength of the antibacterial activity, nine Bacillus strains were selected for 16S rRNA analysis. 16S rRNA sequence homology revealed that several strains were closely related to B. velezensis, where strains with no antibacterial activity grouped as the soil-associated community of B. amyloliquefaciens. B. velezensis Bv-21 exhibited the highest antibacterial activity against wild type and streptomycin resistant Xcc with inhibition zones of 22.91 ± 0.45 and 20.28 ± 0.53, respectively. Furthermore, B. velezensis Bv-21 strain was tested for biocontrol activity against a streptomycin-resistant XccM4 in detached susceptible citrus leaves. The strain reduced the incidence of CBC by 26.30% and pathogen density of XccM4 by 81.68% over control. The results of the study strongly suggest that B. velezensis can be used as an effective and eco-friendly biocontrol agent either by itself or as an active compound, against both, the wild-type and streptomycin-resistant Xcc.     

Characterization of ISXax1, a Novel Insertion Sequence Restricted to Xanthomonas axonopodis pv. phaseoli (Variants fuscans and non-fuscans) and Xanthomonas axonopodis pv. vesicatoria

ISXax1 is a novel insertion sequence belonging to the IS256 and Mutator families. Dot blot, Southern blot, and PCR analyses revealed that ISXax1 is restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and X. axonopodis pv. vesicatoria strains. Directed AFLP also showed that a high degree of polymorphism is associated with ISXax1 insertion in these strains.