Virulence and type III effector diversities of Xanthomonas citri pv. fuscans and X. phaseoli pv. phaseoli in Brazil

Common bacterial blight (CBB) is caused by four genetic lineages belonging to two species of Xanthomonas, namely Xanthomonas citri pv. fuscans (includes fuscans, NF2 and NF3 lineages) and X. phaseoli pv. phaseoli (lineage NF1). A collection of 117 strains of Xanthomonas isolated from common bean plants grown in several producing regions of Brazil, between 2007 and 2016 was established. For species and lineage identification, the following tests were performed: multiplex PCR with a set of four specific primer pairs, pathogenicity tests on susceptible cultivar BRS Artico and phylogenetic analysis based on housekeeping gene sequences. The presence of the two species were confirmed among the 117 strains, being 62 non-fuscans strains (NF1, NF2 and NF3) and 55 fuscans strains of X. citri pv. fuscans. To select a set of representative strains for the virulence assay, a PCR-based analysis of effector diversity was performed with 42 strains belonging to the two species. PCR with primers for xopL, avrBsT, xopE2 and xopE1 genes were positive for all strains, while for the other six effectors there was variation. Six distinct effector profiles were detected, and one strain representing each type was inoculated in 15 common bean cultivars with varying levels of resistance to CBB. The fuscans strains showed uniformity in their effector profiles and were the most virulent. The phylogenetic analyses of our strain collection revealed that all genetic variants of CBB pathogens (NF1, NF2, NF3 and fuscans) are present in Brazil, with significant variability in virulence to common bean cultivars.     

Identification of the flagellar chaperone FlgN in the phytopathogen <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar <Emphasis Type="Italic">citri</Emphasis> by its interaction with hook-associated FlgK

Genome annotation of the plant pathogen Xanthomonas axonopodis pv. citri (Xac), identified flagellar genes in a 15.7 kb gene cluster. However, FlgN, a secretion chaperone for hook-associated proteins FlgK and FlgL, was not identified. We performed extensive screening of the X. axonopodis pv. citri genome with the yeast two-hybrid system to identify a protein with the characteristics of the flagellar chaperone FlgN. We found a candidate (XAC1990) encoded by an operon for components of the flagellum apparatus that interacted with FlgK. In order to further support this finding, Xac FlgK and XAC1990 were cloned, expressed, and purified. The recombinant proteins were characterized by spectroscopic methods and their interaction in vitro confirmed by pull-down assays. We, therefore, conclude that XAC1990 and its homologs in other Xanthomonas species are, in fact, FlgN proteins. These observations extend the sequence diversity covered by this family of proteins.     

Genetic and phenotypic characterization of Xanthomonas axonopodis pv. maculifoliigardeniae causing bacterial leaf spot of Ixora in Taiwan

Ixora spp. are Rubiaceae plants commonly planted as hedges or potted flower. Recently, incidents of bacterial leaf spot of Ixora were observed in central parts of Taiwan. Previous research on the disease has been scarce and focused mainly on its diagnosis. Therefore, many characteristics of the causal agent remain unclear. The present study aims to improve our understanding of this lesser-characterized pathogen and provide information useful for its identification and management. Bacterial strains Ixo1, Ixo2 and Ixo3 were isolated from infected Ixora x westii. All three isolates were able to grow and induce leaf spot symptoms on Ixora. They also exhibited morphological and physiological characteristics typical of Xanthomonads. Biolog analysis indicated that Ixo1 to Ixo3 have metabolic fingerprints similar to X. axonopodis pv. poinsettiicola. Multilocus sequence analysis and inoculation assays identified Ixo1 to Ixo3 as X. axonopodis pv. maculifoliigardeniae, albeit their gene sequences were very similar to other species/pathovars belonging to the X. euvesicatoria species complex; members of this species complex have different plant hosts, yet share similar housekeeping gene sequences. A semi-specific PCR assay evaluated in this work was able to differentiate Ixo1 to Ixo3 from bacteria not belonging to the X. euvesicatoria species complex, suggesting that the assay may be used in diagnosing bacterial leaf spot of ixoras. Finally, the sensitivity of the isolated pathogen to multiple commercial pesticides was tested, and the results showed that the bacterium is sensitive to streptomycin + tetracycline (10％ SP), thiophanate methyl + streptomycin (68.8％ WP) and oxolinic acid (20% WP), but more tolerant against copper-based chemicals. Overall, the findings from this work may facilitate the identification and management of X. axonopodis pv. maculifoliigardeniae.     

Structural-functional characterization and physiological significance of ferredoxin-NADP reductase from Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP⁺ reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of ^•O₂ ^-. Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762.     

Variations in type III effector repertoires,pathological phenotypes and host range of Xanthomonas citri pv. citri pathotypes

The mechanisms determining the host range of Xanthomonas are still undeciphered, despite much interest in their potential roles in the evolution and emergence of plant pathogenic bacteria. Xanthomonas citri pv. citri (Xci) is an interesting model of host specialization because of its pathogenic variants: pathotype A strains infect a wide range of Rutaceous species, whereas pathotype A*/A^W strains have a host range restricted to Mexican lime (Citrus aurantifolia) and alemow (Citrus macrophylla). Based on a collection of 55 strains representative of Xci worldwide diversity assessed by amplified fragment length polymorphism (AFLP), we investigated the distribution of type III effectors (T3Es) in relation to host range. We examined the presence of 66 T3Es from xanthomonads in Xci and identified a repertoire of 28 effectors, 26 of which were shared by all Xci strains, whereas two (xopAG and xopC1) were present only in some A*/A^W strains. We found that xopAG (=avrGf1) was present in all A^W strains, but also in three A* strains genetically distant from A^W, and that all xopAG‐containing strains induced the hypersensitive response (HR) on grapefruit and sweet orange. The analysis of xopAD and xopAG suggested horizontal transfer between X. citri pv. bilvae, another citrus pathogen, and some Xci strains. A strains were genetically less diverse, induced identical phenotypic responses and possessed indistinguishable T3E repertoires. Conversely, A*/A^W strains exhibited a wider genetic diversity in which clades correlated with geographical origin and T3E repertoire, but not with pathogenicity, according to T3E deletion experiments. Our data outline the importance of taking into account the heterogeneity of Xci A*/A^W strains when analysing the mechanisms of host specialization.     

Appraisal of plant extracts and streptomycin sulfate against citrus canker disease

Abstract

Plant extracts and streptomycin sulfate were evaluated against Xanthomonas axonopodis pv. citri. In first experiment, Azadirachta indica, Allium cepa, Catharanthus roseus, Allium sativum, Zingiber officinale (Roscoe) were investigated in vitro through inhibition zone technique against the growth of X. a. pv. citri. Results indicated that A. indica exhibited statistically significant inhibition (4?cm) zone over control. In second experiment, A. indica and streptomycin sulfate disjointedly and in amalgamation were evaluated in vitro. Streptomycin alone and in permutation with A. indica articulated significant inhibition of the bacterium. In third study, streptomycin sulfate and A. indica (S) and in combination were evaluated against citrus canker disease in green house. Results showed that streptomycin sulfate reduced disease significantly than control. In fourth experiment, streptomycin sulfate, A. indica, in combination and their interaction with days were evaluated under field condition. Streptomycin sulfate proved to be most effective and reduced the disease severity as compared to control.     

Detection, Survival and Transmission of Xanthomonas axonopodis pv. manihotis and X. axonopodis pv. vignicola, Causal Agents of Cassava and Cowpea Bacterial Blight, respectively, in/by Insect Vectors

Populations of Xanthomonas axonopodis pv. manihotis and X. axonopodis pv. vignicola, causal agents of cassava and cowpea bacterial blight, respectively, were quantified in insects. The pathogens were found in the faeces, the intestines, and on the legs and mandibles of Zonocerusvariegatus. Additionally, X. axonopodis pv. manihotis was localized in the insect gut by immunofluorescence microscopy. Xanthomonas axonopodis pv. manihotis survived at least 1 week in the insect intestines and at least 5 weeks in faeces kept under controlled conditions, while survival in faeces exposed to sunlight was <2 weeks. Five percentage [e.g. 5.8 × 10⁷ colony‐forming units (CFU)/g faeces] of the fed population of X. axonopodis pv. manihotis in cassava leaves were recovered viable in the faeces after passage through the insect. The transmission of cassava bacterial blight by pathogen‐contaminated insect faeces to intact, healthy cassava leaves was demonstrated for the first time. Xanthomonas axonopodis pv. vignicola was isolated from organs and faeces of the grasshopper Pyrgomorpha cognata, the Senegalese grasshopper (Oedaleus senegalensis), bee (Apis mellifera) and three Coleoptera (Ootheca mutabilis, Mylabris spp., Exochomus troberti) collected in bacterial blight‐infected cowpea fields. Cowpea belonged to the diet of 19 grasshopper species collected in cowpea fields as demonstrated by residues in their faeces. Pathogen‐contaminated Z. variegatus initiated an epiphytic population of 8.9 × 10⁴ CFU/g on healthy cowpea leaves. Spraying cassava and cowpea leaves with 10² and 10⁴ CFU/ml of their respective pathogen was sufficient to evoke symptoms. A possible role of insects in the transmission of X. axonopodis pvs. vignicola and manihotis is discussed.     

Pseudomonas protegens CS1 from the lemon phyllosphere as a candidate for citrus canker biocontrol agent

• Citrus canker is a worldwide‐distributed disease caused by Xanthomonas citri subsp. citri. One of the most used strategies to control the disease is centred on copper‐based compounds that cause environmental problems. Therefore, it is of interest to develop new strategies to manage the disease. Previously, we reported the ability of the siderophore pyochelin, produced by the opportunistic human pathogen Pseudomonas aeruginosa, to inhibit in vitro several bacterial species, including X. citri subsp. citri. The action mechanism, addressed with the model bacterium Escherichia coli, was connected to the generation of reactive oxygen species (ROS). This work aimed to find a non‐pathogenic strain from the lemon phyllosphere that would produce pyochelin and therefore serve in canker biocontrol.
• An isolate that retained its capacity to colonise the lemon phyllosphere and inhibit X. citri subsp. citri was selected and characterised as Pseudomonas protegens CS1. From a liquid culture of this strain, the active compound was purified and identified as the pyochelin enantiomer, enantio‐pyochelin.
• Using the producing strain and the pure compound, both in vitro and in vivo, we determined that the action mechanism of X. citri subsp. citri inhibition also involved the generation of ROS. Finally, the potential application of P. protegens CS1 was evaluated by spraying the bacterium in a model that mimics the natural X. citri subsp. citri infection.
• The ability of P. protegens CS1 to reduce canker formation makes this strain an interesting candidate as a biocontrol agent.

     

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.     

The Oligopeptide Permease (Opp) of the Plant Pathogen <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

The oligopeptide permease (Opp), a protein-dependent ABC transporter, has been found in the genome of Xanthomonas axonopodis pv. citri (Xac), but not in Xanthomonas campestris pv. campestris (Xcc). Sequence analysis indicated that 4 opp genes (oppA, oppB, oppC, oppD/F), located in a 33.8-kbp DNA fragment present only in the Xac genome, are arranged in an operon-like structure and share highest sequence similarities with Streptomyces roseofulvus orthologs. Nonetheless, analyses of the GC content, codon usage, and transposon positioning suggested that the Xac opp operon does not have an exogenous origin. The presence of a stop codon at one of the ATP-binding domains of OppD/F would render the uptake system nonfunctional, but detection of a single polycistronic mRNA and periplasmic OppA in actively growing bacteria suggests that the Opp permease is active and could contribute to the distinct nutritional requirements and host specificities of the two Xanthomonas species.     

Occurrence of a New Orange Variant of Curtobacterium flaccumfaciens pv. flaccumfaciens,Causing Common Bean Wilt in Iran

A new orange variant of Curtobacterium flaccumfaciens pv. flaccumfaciens was isolated from seeds of common bean cv. Daneshkadeh and Dehghan stored in the seed banks in Khomein Bean Research Station, and field plants (cv. Local Khomein) in Arak, Iran. The pathogenicity of the isolates was confirmed on 5‐ to 7‐day‐old seedlings of cv. Daneshkadeh. Marginal necrosis and interveinal chlorosis on first trifoliate leaves were observed 10–15 days after inoculation. Amplification of 306 bp fragment of orange‐pigmented strains using CffFOR2‐ and CffREV4‐specific prime pair characterized them as C. flaccumfaciens pv. flaccumfaciens. Although the yellow‐pigmented variant of the causal agent was previously reported on cowpea, this is the first report of orange variant of C. flaccumfaciens pv. flaccumfaciens causing bacterial wilt on common bean in Iran.     

Specific Detection of Xanthomonas axonopodis pv. dieffenbachiae in Anthurium (Anthurium andreanum) Tissues by Nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10³ CFU ml⁻¹) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

A Novel Two-Component Response Regulator Links rpf with Biofilm Formation and Virulence of Xanthomonas axonopodis pv. citri

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a serious disease that impacts citrus production worldwide, and X. axonopodis pv. citri is listed as a quarantine pest in certain countries. Biofilm formation is important for the successful development of a pathogenic relationship between various bacteria and their host(s). To understand the mechanisms of biofilm formation by X. axonopodis pv. citri strain XW19, the strain was subjected to transposon mutagenesis. One mutant with a mutation in a two-component response regulator gene that was deficient in biofilm formation on a polystyrene microplate was selected for further study. The protein was designated as BfdR for biofilm formation defective regulator. BfdR from strain XW19 shares 100% amino acid sequence identity with XAC1284 of X. axonopodis pv. citri strain 306 and 30–100% identity with two-component response regulators in various pathogens and environmental microorganisms. The bfdR mutant strain exhibited significantly decreased biofilm formation on the leaf surfaces of Mexican lime compared with the wild type strain. The bfdR mutant was also compromised in its ability to cause canker lesions. The wild-type phenotype was restored by providing pbfdR in trans in the bfdR mutant. Our data indicated that BfdR did not regulate the production of virulence-related extracellular enzymes including amylase, lipase, protease, and lecithinase or the expression of hrpG, rfbC, and katE; however, BfdR controlled the expression of rpfF in XVM2 medium, which mimics cytoplasmic fluids in planta. In conclusion, biofilm formation on leaf surfaces of citrus is important for canker development in X. axonopodis pv. citri XW19. The process is controlled by the two-component response regulator BfdR via regulation of rpfF, which is required for the biosynthesis of a diffusible signal factor.     

Bacteria causing important diseases of citrus utilise distinct modes of pathogenesis to attack a common host

In this review, we summarise the current knowledge on three pathogens that exhibit distinct tissue specificity and modes of pathogenesis in citrus plants. Xanthomonas axonopodis pv. citri causes canker disease and invades the host leaf mesophyll tissue through natural openings and can also survive as an epiphyte. Xylella fastidiosa and Candidatus Liberibacter are vectored by insects and proliferate in the vascular system of the host, either in the phloem (Candidatus Liberibacter) or xylem (X. fastidiosa) causing variegated chlorosis and huanglongbing diseases, respectively. Candidatus Liberibacter can be found within host cells and is thus unique as an intracellular phytopathogenic bacterium. Genome sequence comparisons have identified groups of species-specific genes that may be associated with the particular lifestyle, mode of transmission or symptoms produced by each phytopathogen. In addition, components that are conserved amongst bacteria may have diverse regulatory actions underpinning the different bacterial lifestyles; one example is the divergent role of the Rpf/DSF cell–cell signalling system in X. citri and X. fastidiosa. Biofilm plays a key role in epiphytic fitness and canker development in X. citri and in the symptoms produced by X. fastidiosa. Bacterial aggregation may be associated with vascular occlusion of the xylem vessels and symptomatology of variegated chlorosis.     

In vitro antimicrobial activity of zimmu ( Allium sativum L. ‘ Allium cepa L.) leaf extract

The fungitoxic effect of various medicinal plants belonging to different families was evaluated in vitro on Rhizoctonia solani, the rice sheath blight pathogen. Of the various plant extracts, the leaf extract of zimmu (Allium cepa × Allium sativum) showed the maximum antifungal activity against R. solani and recorded an inhibition zone of 12?mm. The leaf extract of zimmu was also effective in inhibiting the growth of other agronomically important fungal and bacterial pathogens viz., Aspergillus flavus, Curvularia lunata, Alternaria solani, Xanthomonas oryzae pv. oryzae, Xanthomonas campestris pv. malvacearum and Xanthomonas axonopodis pv. citri. The antimicrobial compound was dissoluble in methanol and the methanolic extract showed the absorption maxima at 210?nm and 230?nm. Phenolic compounds were present in greater amounts in methanol extract of zimmu. TLC analysis showed the appearance of two blue spots at R _f ?=?0.65 and R _f ?=?0.90. The compounds eluted at R _f ?=?0.65 and R _f ?=?0.90 by preparative TLC exhibited strong antifungal activity against R. solani.     

Increasing l‐isoleucine production in Corynebacterium glutamicum by overexpressing global regulator Lrp and two‐component export system BrnFE

Aims

To increase the l ‐isoleucine production in Corynebacterium glutamicum by overexpressing the global regulator Lrp and the two‐component export system BrnFE.

Methods and Results

The brnFE operon and the lrp gene were cloned into the shuttle vector pDXW‐8 individually or in combination. The constructed plasmids were transformed into an l ‐isoleucine‐producing strain C. glutamicum JHI3‐156, and the l ‐isoleucine production in these different strains was analysed and compared. More l ‐isoleucine was produced when only Lrp was expressed than when only BrnFE was expressed. Significant increase in l ‐isoleucine production was observed when Lrp and BrnFE were expressed in combination. Compared to the control strain, l ‐isoleucine production in JHI3‐156/pDXW‐8‐lrp‐brnFE increased 63% in flask cultivation, and the specific yield of l ‐isoleucine increased 72% in fed‐batch fermentation.

Conclusions

Both Lrp and BrnFE are important to enhance the l ‐isoleucine production in C. glutamicum.

Significance and Impact of the Study

The results provide useful information to enhance l ‐isoleucine or other branched‐chain amino acid production in C. glutamicum.     

A Simple Method for In Vivo Expression Studies of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pv. <Emphasis Type="Italic">citri</Emphasis>

A major problem in studying bacterial plant pathogens is obtaining the microorganism directly from the plant tissue to perform in vivo expression (protein or mRNA) analyses. Here we report an easy and fast protocol to isolate Xanthomonas axonopodis pv. citri directly from the host plant, in sufficient amounts to perform protein fingerprinting by 2-D gel electrophoresis as well as RNA expression assays. The protein profile obtained was very similar to that of X. axonopodis pv. citri grown in the presence of a leaf extract of Citrus sinensis; however, some differential proteins expressed in vivo were observed. Total RNA extraction revealed typical 16S and 23S bands in the agarose gel, and RT-PCR reactions using primers specific for genes of the bacterium confirmed the quality of the RNA preparation. Also, RT-PCR reactions using plant ribosomal primers were employed, and no amplification product was obtained, indicating that plant RNA is not present in the bacterium RNA sample.     

Genotypic Characterization of the Common Bean Bacterial Blight Pathogens, Xanthomonas axonopodis pv. phaseoli and Xanthomonas axonopodis pv. phaseoli var. fuscans by rep-PCR and PCR–RFLP of the Ribosomal Genes

Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli and X. axonopodis pv. phaseoli var. fuscans is one of the most destructive diseases of common bean worldwide. The interrelatedness, genetic diversity and geographical distribution of the CBB pathogens was assessed using restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction amplified 16S ribosomal gene, including the 16S–23S intergenic spacer region and repetitive element PCR (rep‐PCR). RFLP profiles generated by the restriction endonucleases MboI, RsaI and HaeIII differentiated X. axonopodis pv. phaseoli from X. axonopodis pv. phaseoli var. fuscans and non‐pathogenic Xanthomonas species associated with common bean. Cluster analysis of rep‐PCR profiles revealed a high level of genetic differentiation (G_ST = 0.56) between the two CBB pathogens, showing that they are genetically distinct. Significant levels of genetic diversity were observed within each strain, indicating that the two bacteria are not clonal. More genetic diversity was observed in X. axonopodis pv. phaseoli (H = 0.134; I = 0.223) than X. axonopodis pv. phaseoli var. fuscans (H = 0.108; I = 0.184). However, no geographical differentiation was evident for either X. axonopodis pv. phaseoli var. fuscans (GST = 0.013) or X. axonopodis pv. phaseoli (G_ST = 0.017). This lack of geographical differentiation has important practical implications, as available host resistance genes are likely to be effective in controlling the disease in diverse geographical areas.