Xanthan gum production and rheological behavior using different strains of Xanthomonas sp.

The proposal of the present study was to select and carry out the molecular characterization of strains of Xanthomonas sp. in order to correlate with gum production and determine possible genetic alterations during the study. The gums produced were also evaluated rheologically. Ten strains of Xanthomonas were used in the screening and the best ones in terms of productivity were Xanthomonas campestris pv. mangiferaeindicae 1230 (8.93 g/L), X. campestris pv. campestris 254 (9.49 g/L) and X. campestris pv. campestris 1078 (9.67 g/L). The gum produced by X. campestris pv. mangiferaeindicae presented the best apparent viscosity. The results for the profiles of the bands produced by RAPD showed considerable genetic variability amongst the evaluated strains, making not possible to neither group the strains according to pathovar or species, nor correlate the band profile with the productivity obtained. According to the RAPD analysis, no detectable mutations occurred in these bacteria during the study.     

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.     

Pepper Tree (Schinus terebenthifolius Radii), a New Host Plant for Xanthomonas campestris pv. mangiferaeindicae

Apigmented bacterial colonies were obtained in Reunion Island from angular leaf lesions on Pepper tree (Schinus terebenthifolius Radii), a member of Anacardiaceae. All isolates were identified as Xanthomonas campestris, using physiological and biochemical tests. These strains were reinoculated to Pepper tree leaves, and Koch postulates were verified. Furthermore, they were inoculated to mango leaves and produced lesions identical to those induced by Xanthomonas campestris pv. mangiferaeindicae, the causal agent of bacterial black spot of mangoes. Apigmented and pigmented strains of X. c. pv. mangiferaeindicae from Mango and Ambarella were pathogenic to Pepper tree. Strains isolated from Pepper tree were compared to X. c. pv. mangiferaeindicae, by means of phenotypic features (utilization of 147 carbon sources) and using a serological assay. A high homology among the strains was observed. Thus, It is concluded that strains isolated from Pepper tree belong to pv. mangiferaeindicae, and that Pepper tree is a host species for X. c. pv. mangiferaeindicae.     

AvrAC(Xcc8004), a type III effector with a leucine-rich repeat domain from Xanthomonas campestris pathovar campestris confers avirulence in vascular tissues of Arabidopsis thaliana ecotype Col-0

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC_Xcc8004, functions as an avirulence gene whose product seems to be recognized in vascular tissues.     

Microbiota Communities of Healthy and Bacterial Pustule Diseased Soybean

Soybean is an important source of protein and for a wide range of agricultural, food, and industrial applications. Soybean is being affected by Xanthomonas citri pv. glycines, a causal pathogen of bacterial pustule disease, result in a reduction in yield and quality. Diverse microbial communities of plants are involved in various plant stresses is known. Therefore, we designed to investigate the microbial community differentiation depending on the infection of X. citri pv. glycines. The microbial community’s abundance, diversity, and similarity showed a difference between infected and non-infected soybean. Microbiota community analysis, excluding X. citri pv. glycines, revealed that Pseudomonas spp. would increase the population of the infected soybean. Results of DESeq analyses suggested that energy metabolism, secondary metabolite, and TCA cycle metabolism were actively diverse in the non-infected soybeans. Additionally, Streptomyces bacillaris S8, an endophyte microbiota member, was nominated as a key microbe in the healthy soybeans. Genome analysis of S. bacillaris S8 presented that salinomycin may be the critical antibacterial metabolite. Our findings on the composition of soybean microbiota communities and the key strain information will contribute to developing biological control strategies against X. citri pv. glycines.     

Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.     

Variation in Pathogenicity and Virulence of Strains of Xanthomonas campestris pv. glycines, the Incitant of Bacterial Pustule of Soybean

Standardized methods were developed to determine the pathogenicity and the degree of virulence of Xanthomonas campestris pv. glycines (Xcg) as well as the reaction of soybean plants in the greenhouse. A glass atomizer is described which allowed uniform inoculation without damaging the leaves. Optimum bacterial concentration was determined as 6 × 10⁶ CFU/ml for the pathogenicity test and 1.3 × 10⁵ CFU/ml for the virulence test. A total of 64 isolates were tested. Forty-five strains were designated as pathogenic, six of which were considered highly virulent. It was shown, for the first time, that large differences in the virulence of Xcg strains exist. All the highly virulent strains of Xcg were fresh isolates from diseased soybean leaves collected in Thailand. On the other hand, all the “old” cultures from bacterial collections possessed a low or very low virulence. Decrease of virulence of the pathogen did not occur very fast, however, that is: not within 2 years when stored on YDC-agar slants. Therefore, the bacteria may be kept on slants at 15 °C for short time storage, but the strains should be preserved permanently as lyophilized cultures.     

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta)

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava.     

Similarity between Copper Resistance Genes from Xanthomonas campestris and Pseudomonas syringae

Plasmid-borne copper resistance genes from copper-resistant strains of Xanthomonas campestris pv. vesicatoria from California, Florida, and Oklahoma shared structural similarities. A strain of X. campestris pv. campestris also contained plasmid-borne copper resistance genes similar to the resistance genes from X. campestris pv. vesicatoria. Furthermore, a region of the copper resistance genes from X. campestris pv. vesicatoria 07882 hybridized with copA, the first gene of the copper resistance operon (cop) of Pseudomonas syringae pv. tomato. A copper-inducible protein of similar size to CopA was detected by Western blot (immunoblot) analysis from the wild-type strain 07882 and from the cloned copper resistance genes of 07882 introduced into a copper-sensitive strain of X. campestris pv. vesicatoria. A low level of hybridization was observed with chromosomal DNA from other xanthomonads when the copper resistance genes from strain 07882 were used as probes.     

Phenotypic Switching Affecting Chemotaxis, Xanthan Production, and Virulence in Xanthomonas campestris

The chemotaxis towards sucrose and yeast extract of nine strains of Xanthomonas campestris representing pathovars campestris, armoraciae, translucens, vesicatoria, and pelargonii was analyzed by using swarm plates. Unexpectedly, each of these strains formed small or reduced swarms typical of nonmotile or nonchemotactic bacteria. With time, however, chemotactic cells appeared on the swarm plates as blebs of bacteria. These cells were strongly chemotactic and were concomitantly deficient in exopolysaccharide production. The switch from the wild type (exopolysaccharide producing and nonchemotactic) to the swarmer type (exopolysaccharide deficient and chemotactic) appeared irreversible ex planta in bacteriological medium. However, in radish leaves swarmer-type strains of X. campestris pv. campestris were able to revert to the wild type. Swarmer-type derivatives of two X. campestris pv. campestris wild-type isolates showed reduced virulence and growth in the host plants cauliflower and radish. However, exocellular complementation of X. campestris pv. campestris Hrp (nonpathogenic) mutant was achieved by coinoculation with a swarmer-type strain.     

Coronatine analogues produced by Xanthomonas campestris pv Phormiicola

Liquid cultures of Xanthomonas campestris pv phormiicola were found to contain two analogues of coronatine lacking the cyclopropane ring structure, and no trace of either coronatine or norcoronatine. The two compounds were isolated and fully characterised by NMR, MS, hydrolysis and GC of hydrolysis products, as N-coronafacoyl- -valine and N-coronafacoyl- -isoleucine. A survey of 12 strains from 10 other X. campestris pathovars did not locate another source of production of these compounds, whereas all three strains of X. campestris pv phormiicola examined produced comparable levels of both compounds. This is the first report of phytotoxins biosynthetically derived from coronafacic acid outside of the genus Pseudomonas. The implications of these findings to the biosynthesis of the cyclopropane ring structure of coronatine are discussed.     

Pathovar-Specific Antigens of Xanthomonas campestris pv. begoniae and X. campestris pv. pelargonii Detected with Monoclonal Antibodies

Two monoclonal antibodies specific for lipopolysaccharide antigens of Xanthomonas campestris pv. begoniae and pv. pelargonii reacted with all of their respective pathovar strains and not with 130 strains of other xanthomonads or 89 nonxanthomonads tested. These results, as well as previous results, indicate that pathovar-specific monoclonal antibodies were readily generated to strains of X. campestris pathovars that generally infect single hosts.     

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.     

Bacteriocins and temperate phage ofXanthomonas campestris pv. glycines

Sixteen strains ofXanthomonas campestris pathovar (pv.) glycines produced bacteriocins (glycinecins) on agar media. Optimal incubation conditions were for 48 h at 20°C. In addition to strains ofX. campestris pv. glycines, bacteriocins were also inhibitory towardsX. campestris pv. phaseoli andX. campestris pv. vesicatoria. All bacteriocins were susceptible to inactivation by a nonspecific protease and resistant to ribonuclease, but they differed in their sensitivity to trypsin, deoxyribonuclease, and heat treatment. Differential heat and enzyme sensitivities also indicated that some strains ofX. campestris pv. glycines produce more than one bacteriocin. Attempts to induce bacteriocin production in liquid cultures were unsuccessful. However, temperate bacteriophage were released from cultures ofX. campestris pv. glycines strains XP175, B83, 17915, and MINN after addition of mitomycin C or nalidixic acid or after exposure to UV light.Reference to brand or firm names does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Insights into the genome of the xanthan-producing phytopathogen Xanthomonas arboricola pv. pruni 109 by comparative genomic hybridization

The phytopathogenic bacterium Xanthomonas arboricola pv. pruni is the causal agent of Prunus Bacterial Spot disease that infects cultivated Prunus species and their hybrids. Furthermore, X. arboricola pv. pruni (Xap) plays a role in biotechnology since it produces xanthan gum, an important biopolymer used mainly in the food, oil, and cosmetics industry. To gain first insights into the genome composition of this pathovar, genomic DNA of X. arboricola pv. pruni strains was compared to the genomes of reference strains X. campestris pv. campestris B100 (Xcc B100) and X. campestris pv. vesicatoria 85-10 (Xcv 85-10) applying microarray-based comparative genomic hybridizations (CGH). The results implied that X. arboricola pv. pruni 109 lacks 6.67% and 5.21% of the genes present in the reference strains Xcc B100 and Xcv 85-10, respectively. Most of the missing genes were found to be organized in clusters and do not belong to the core genome of the two reference strains. Often they encode mobile genetic elements. Furthermore, the absence of gene clusters coding for the lipopolysaccharide (LPS) O-antigens of Xcc B100 and Xcv 85-10 indicates that the structure of the O-antigen of X. arboricola pv. pruni 109 differs from that of Xcc B100 and Xcv 85-10.     

Biological Role of Xanthomonadin Pigments in Xanthomonas campestris pv. Campestris

Previous studies have indicated that the yellow pigments (xanthomonadins) produced by phytopathogenic Xanthomonas bacteria are unimportant during pathogenesis but may be important for protection against photobiological damage. We used a Xanthomonas campestris pv. campestris parent strain, single-site transposon insertion mutant strains, and chromosomally restored mutant strains to define the biological role of xanthomonadins. Although xanthomonadin mutant strains were comparable to the parent strain for survival when exposed to UV light; after their exposure to the photosensitizer toluidine blue and visible light, survival was greatly reduced. Chromosomally restored mutant strains were completely restored for survival in these conditions. Likewise, epiphytic survival of a xanthomonadin mutant strain was greatly reduced in conditions of high light intensity, whereas a chromosomally restored mutant strain was comparable to the parent strain for epiphytic survival. These results are discussed with respect to previous results, and a model for epiphytic survival of X. campestris pv. campestris is presented.     

Variability of Xanthomonas Campestris pv. vasculorum From Sugarcane and Other Gramineae in Reunion Island. Characterization of a Different Xanthomonad

Strains presumed to be of Xanthomonas campestris pv. vasculorum (Cobb.) Dye, obtained from sugarcane and other gramineae in Réunion Island, were compared in terms of cultural aspects, pathogenic and physiological reactions, fatty acid profiles and restriction fragment length polymorphism (RFLP) of genomic DNA. The strains could be divided into two separate groups (G1 and G2). The G1 strains were identical to strains described as X. campestris pv. vasculorum; they showed an important variability in their cultural characteristics and in their aggressiveness. The G2 strains did not induce the usual symptoms of gumming disease on sugarcane cultivars infected under natural conditions or inoculated in the greenhouse. The G2 strains grew faster on agar medium, their colonies were more pigmented and less fluidal and had a different morphology on agar slant. Unlike the G1 strains, G2 strains hydrolyzed starch weakly and casein strongly; they utilized L-fucose and, to a lesser extent, melibioze. The fatty acid and genomic DNA profiles differed between the groups. Differences seemed large enough to support separation of G1 and G2 strains into distinct taxonomic entities, namely G1 as Xanthomonas campestris pv. vasculorum and G2 as a different pathovar of X. campestris. The taxonomic position of G2 strains is discussed.     

Copper Resistance Gene Homologs in Pathogenic and Saprophytic Bacterial Species from Tomato

Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. In Southern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid and chromosomal DNA in X. compestris pv. vesicatoria, P. putida, and the yellow Pseudomonas sp. Homology was detected only in the chromosomal DNA of P. cichorii and P. fluorescens. Homology with cop was also detected in chromosomal DNA from copper-sensitive strains of P. cichorii, P. fluorescens, and P. syringae pv. tomato, suggesting that the cop homolog may be indigenous to certain Pseudomonas species and have some function other than copper resistance. No homology was detected in DNA from a copper-sensitive X. campestris pv. vesicatoria strain. Copper-inducible protein products were detected in each copper-resistant bacterium by immunoblot analysis with antibodies raised to the CopB protein from the cop operon. The role of the homologous DNA in copper resistance was confirmed for the X. campestris pv. vesicatoria strain by cloning and transferring the cop homolog to a copper-sensitive strain of X. campestris pv. vesicatoria. The possibility and implications of copper resistance gene exchange between different species and genera of pathogenic and saprophytic bacteria on tomato plants are discussed.     

Characterization of pXV10A, a Copper Resistance Plasmid in Xanthomonas campestris pv. vesicatoria

The efficacy of copper bactericides for control of Xanthomonas campestris pv. vesicatoria in eastern Oklahoma tomato fields was evaluated. Copper bactericides did not provide adequate control, and copper-resistant (Cu^r) strains of the pathogen were isolated. The Cu^r genes in these strains were located on a large indigenous plasmid designated pXV10A. The host range of pXV10A was investigated; this plasmid was efficiently transferred into 8 of 11 X. campestris pathovars. However, the transfer of pXV10A to other phytopathogenic genera was not detected. DNA hybridization experiments were performed to characterize the Cu^r genes on pXV10A. A probe containing subcloned Cu^r genes from X. campestris pv. vesicatoria E3C5 hybridized to pXV10A; however, a subclone containing Cu^r genes from P. syringae pv. tomato PT23 failed to hybridize to pXV10A. Further DNA hybridization experiments were performed to compare pXV10A with pXvCu plasmids, a heterogenous group of Cu^r plasmids present in strains of X. campestris pv. vesicatoria from Florida. These studies indicated that the Cu^r genes on pXV10A and pXvCu plasmids share nucleotide sequence homology and may have a common origin. Further experiments showed that these plasmids are distinctly different because pXV10A did not contain sequences homologous to IS476, an insertion sequence present on pXvCu plasmids.