Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.     

Genomic Relatedness of Xanthomonas campestris Strains Causing Diseases of Citrus

Xanthomonas campestris strains that cause disease in citrus were compared by restriction endonuclease analysis of DNA fragments separated by pulsed-field gel electrophoresis and by DNA reassociation. Strains of X. campestris pv. citrumelo, which cause citrus bacterial spot, were, on average, 88% related to each other by DNA reassociation, although these strains exhibited diverse restriction digest patterns. In contrast, strains of X. campestris pv. citri groups A and B, which cause canker A and canker B, respectively, had relatively homogeneous restriction digest patterns. The groups of strains causing these three different citrus diseases were examined by DNA reassociation and were found to be from 55 to 63% related to one another. Several pathovars of X. campestris, previously shown to cause weakly aggressive symptoms on citrus, ranged from 83 to 90% similar to X. campestris pv. citrumelo by DNA reassociation. The type strain of X. campestris pv. campestris ranged from 30 to 40% similar in DNA reassociation experiments to strains of X. campestris pv. citrumelo and X. campestris pv. citri groups A and B. Whereas DNA reassociation quantified the difference between relatively unrelated groups of bacterial strains, restriction endonuclease analysis distinguished between closely related strains.     

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.     

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.     

The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf.     

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.     

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.     

The pilA gene of Xanthomonas campestris pv. citri is required for infection by the filamentous phage cf

 Host factors that are important for infection of Xanthomonas campestris pv. citri by the filamentous bacteriophage cf were investigated by transposon mutagenesis with Tn5tac1. A mutant, XT501, that was resistant to cf infection was recovered, showing that the gene inactivated by the transposon is required for infection by the phage but not for cf replication or assembly. A 1.7-kb SacI-ApaI DNA fragment from XT501 containing the bacterial DNA flanking one end of the transposon was cloned and shown to be required for cf infection. Nucleotide sequence analysis of the 1.7-kb fragment reveals the presence of an ORF that encodes a protein of 146 amino acids. This protein shows 42% identity to the type 4 prepilin encoded by the pilA genes of other bacteria. The pilA gene of X. campestris pv. citri is thus essential for infection by the bacteriophage cf. Received: 30 November 1998 / Accepted: 21 April 1999     

Mutation in the xpsD gene of Xanthomonas axonopodis pv. citri affects cellulose degradation and virulence

The Gram-negative bacterium Xanthomonas axonopodis pv. citri, the causal agent of citrus canker, is a major threat to the citrus industry worldwide. Although this is a leaf spot pathogen, it bears genes highly related to degradation of plant cell walls, which are typically found in plant pathogens that cause symptoms of tissue maceration. Little is known on Xac capacity to cause disease and hydrolyze cellulose. We investigated the contribution of various open reading frames on degradation of a cellulose compound by means of a global mutational assay to selectively screen for a defect in carboxymethyl cellulase (CMCase) secretion in X. axonopodis pv. citri. Screening on CMC agar revealed one mutant clone defective in extracellular glycanase activity, out of nearly 3,000 clones. The insertion was located in the xpsD gene, a component of the type II secretion system (T2SS) showing an influence in the ability of Xac to colonize tissues and hydrolyze cellulose. In summary, these data show for the first time, that X. axonopodis pv. citri is capable of hydrolyzing cellulose in a T2SS-dependent process. Furthermore, it was demonstrated that the ability to degrade cellulose contributes to the infection process as a whole.     

A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.     

DNA polymorphisms and biocontrol of bacillus antagonistic to citrus bacterial canker with indication of the interference of phyllosphere biofilms

Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri is a devastating disease resulting in significant crop losses in various citrus cultivars worldwide. A biocontrol agent has not been recommended for this disease. To explore the potential of bacilli native to Taiwan to control this disease, Bacillus species with a broad spectrum of antagonistic activity against various phytopathogens were isolated from plant potting mixes, organic compost and the rhizosphere soil. Seven strains TKS1-1, OF3-16, SP4-17, HSP1, WG6-14, TLB7-7, and WP8-12 showing superior antagonistic activity were chosen for biopesticide development. The genetic identity based on 16S rDNA sequences indicated that all seven native strains were close relatives of the B. subtilis group and appeared to be discrete from the B. cereus group. DNA polymorphisms in strains WG6-14, SP4-17, TKS1-1, and WP8-12, as revealed by repetitive sequence-based PCR with the BOXA1R primers were similar to each other, but different from those of the respective Bacillus type strains. However, molecular typing of the strains using either tDNA-intergenic spacer regions or 16S-23S intergenic transcribed spacer regions was unable to differentiate the strains at the species level. Strains TKS1-1 and WG6-14 attenuated symptom development of citrus bacterial canker, which was found to be correlated with a reduction in colonization and biofilm formation by X. axonopodis pv. citri on leaf surfaces. The application of a Bacillus strain TKS1-1 endospore formulation to the leaf surfaces of citrus reduced the incidence of citrus bacterial canker and could prevent development of the disease.     

Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method.

pFL1 is a pUC9 derivative that contains a 572-bp EcoRI insert cloned from plasmid DNA of Xanthomonas campestris pv. citri XC62. The nucleotide sequence of pFL1 was determined, and the sequence information was used to design primers for application of the polymerase chain reaction (PCR) to the detection of X. campestris pv. citri, the causal agent of citrus bacterial canker disease. Seven 18-bp oligonucleotide primers were designed and tested with DNA from X. campestris pv. citri strains and other strains of X. campestris associated with Citrus spp. as templates in the PCR. Four primer pairs directed the amplification of target DNA from X. campestris pv. citri strains but not from strains of X. campestris associated with a different disease, citrus bacterial spot. Primer pair 2-3 directed the specific amplification of target DNA from pathotype A but not other pathotypes of X. campestris pv. citri. A pH 9.0 buffer that contained 1% Triton X-100 and 0.1% gelatin was absolutely required for the successful amplification of the target DNA, which was 61% G+C. Limits of detection after amplification and gel electrophoresis were 25 pg of purified target DNA and about 10 cells when Southern blots were made after gel electrophoresis and probed with biotinylated pFL1. This level of detection represents an increase in sensitivity of about 100-fold over that of dot blotting with the same hybridization probe. PCR products of the expected sizes were amplified from DNA extracted from 7-month-old lesions from which viable bacteria could not be isolated. These products were confirmed to be specific for X. campestris pv. citri by Southern blotting. This PCR-based detection protocol will be a useful addition to current methods of detection of this pathogen, which is currently the target of international quarantine measures.     

柑桔溃疡病生防细菌Bt8的研究

柑桔溃疡病是中国柑桔的重要病害。从南宁柑桔园土壤中分离到1株对柑桔溃疡病菌具有强抑制力的细菌Bt8。根据Bt8菌株的形态1、6S rDNA序列分析以及生理生化特性,将其鉴定为鲍氏不动杆菌。Bt8菌株的抑菌效果受温度、pH及培养基等环境因素的影响。在温室条件下将该细菌悬浮液喷施到柑桔叶片上,获得了55.2%的病斑抑制效果。研究结果揭示了鲍氏不动杆菌在柑桔溃疡病田间防治上的潜能。     

HrpG and HrpX play global roles in coordinating different virulence traits of Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri is the causal agent of citrus canker, which is one of the most serious diseases of citrus. To understand the virulence mechanisms of X. axonopodis pv. citri, we designed and conducted genome-wide microarray analyses to characterize the HrpG and HrpX regulons, which are critical for the pathogenicity of X. axonopodis pv. citri. Our analyses revealed that 232 and 181 genes belonged to the HrpG and HrpX regulons, respectively. In total, 123 genes were overlapped in the two regulons at any of the three selected timepoints representing three growth stages of X. axonopodis pv. citri in XVM2 medium. Our results showed that HrpG and HrpX regulated all 24 type III secretion system genes, 23 type III secretion system effector genes, and 29 type II secretion system substrate genes. Our data revealed that X. axonopodis pv. citri regulates multiple cellular activities responding to the host environment, such as amino acid biosynthesis; oxidative phosphorylation; pentose-phosphate pathway; transport of sugar, iron, and potassium; and phenolic catabolism, through HrpX and HrpG. We found that 124 and 90 unknown genes were controlled by HrpG and HrpX, respectively. Our results suggest that HrpG and HrpX interplay with a global signaling network and co-ordinate the expression of multiple virulence factors for modification and adaption of host environment during X. axonopodis pv. citri infection.     

The Occurrence of Citrus Canker Disease in United Arab Emirates (U.A.E.)

Citrus canker disease caused by Xanthomonas campestris pv, citri is reported on lime in the present study for the first time in United Arab Emirates. The disease was found only on lime in 32 orchards out of 4456 citrus orchards inspected during 1984–85. The citrus canker organism in U.A.E. has a host range similar to “A” strain. Copper hydroxide was significantly superior to all other treatments in decreasing the incidence of citrus canker disease that developed on inoculated lime seedlings. Streptocycline, Kasumin, copper oxychloride and Bordeaux mixture were not effective in reducing the incidence of the disease.     

Simple Deletion: a vector- and marker-free method to generate and isolate site-directed deletion mutants

We have designed a new vector- and marker-free site-directed deletion system for gram-negative bacteria. In this system, a specific DNA fragment is amplified from a parental strain by using polymerase chain reaction (PCR), then circularized and introduced back into the parental strain for homologous recombination. The recombinant mutant is then detected and isolated by PCR-based sib selection. Unlike conventional methods, our Simple Deletion method requires no cloning procedures, and no foreign genes such as antibiotic-resistance genes are introduced as selection markers. The resulting mutant is, therefore, the same as the parental strain except for the lack of the target region. This method is categorized as a type of “self-cloning,” and the resulting mutant can be used for laboratory research without restrictions. Using this method, we generated a mutant of a plant pathogenic bacterium, Xanthomonas campestris pv. campestris, in which the 20.4-kb hrp gene cluster involved in the type III secretion system and in pathogenicity was deleted. In addition, we proved that this method can also be used to delete smaller DNA regions of X. campestris pv. campestris and to generate deletion mutants of the bacterium Ralstonia solanacearum.     

Primers based on the rpf gene region provide improved detection of Xanthomonas axonopodis pv. citri in naturally and artificially infected citrus plants

AIMS: To have a PCR-based detection method for Xanthomonas axonopodis pv. citri (Xac) using primers designed in a specific region of its genome. METHODS AND RESULTS: A Xac-specific region was identified inside the rpf gene cluster of strain IAPAR 306 in an analysis of its complete genomic sequence. Two primers were designed, Xac01 and Xac02, which, when used in a standard PCR assay, direct the amplification of a 581 bp fragment from DNA of strains belonging to Xac from different regions around the world including unusual American and Asian strains. This product was not observed when DNA from strains of the closely related X. a. aurantifolli and X. a. citrumelo were used as templates. Extracts prepared from 28 xanthomonads of other species, and epiphytic bacteria isolated from citrus also failed to produce products with these primers. Amplification was obtained from cells grown in vitro, from extracts of both fresh and dried citrus canker lesions and from washes of inoculated but asymptomatic leaf surfaces. In sensitivity tests, this PCR technique detected as few as 100 cells. CONCLUSIONS: Primers Xac01 and Xac02 provide specific and sensitive detection of Xac in all citrus tissues where the pathogen is found. SIGNIFICANCE AND IMPACT OF THE STUDY: This PCR-based diagnostic test is suitable for monitoring asymptomatic plants in areas where the bacteria is endemic, in plant quarantine and regulatory situations, and also for obtaining an accurate diagnosis in a very short time. These are important characteristics for any assay to be used for the management of citrus canker disease.     

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.     

Evaluation of the Biolog Substrate Utilization System To Identify and Assess Metabolic Variation among Strains of Xanthomonas campestris pv. Citri

Metabolic fingerprints of 148 strains of Xanthomonas campestris pv. citri originating from 24 countries and associated with various forms of citrus bacterial canker disease (CBCD) were obtained by using the Biolog substrate utilization system. Metabolic profiles were used to attempt strain identification. Only 6.8% of the studied strains were correctly identified when the commercial Microlog 2N data base was used alone. When the data base was supplemented with data from 54 strains of X. campestris pv. citri (40 CBCD-A strains, 8 CBCD-B strains, and 6 CBCD-C strains) and data from 43 strains of X. campestris associated with citrus bacterial spot disease, the percentage of correct identifications was 70%. Thus, it is recommended that users supplement the commercial data base with additional data prior to using the program for identification purposes. The utilization of Tween 40 in conjunction with other tests can help to differentiate strains associated with CBCD and citrus bacterial spot disease. These results confirmed the separation of X. campestris pv. citri into different subgroups (strains associated with Asiatic citrus canker [CBCD-A], cancrosis B [CBCD-B], and Mexican lime canker [CBCD-C]). The utilization of l-fucose, d-galactose, and alaninamide can be used as markers to differentiate strains associated with these groups. A single strain associated with bacteriosis of Mexican lime in Mexico (CBCD-D) was closely similar to CBCD-B strains.