Defence-related enzymatic response in cabbage to Xanthomonas campestris pv. campestris

Black rot of cabbage caused by Xanthomonas campestris pv. campestris is one of the most important diseases of crucifers worldwide. Expression of defence-related enzymes in cabbage in response to X. campestris pv. campestris was investigated in the current experiment. Among the defence-related enzymes (phynylalanine ammonia lyase, peroxidase, polyphenol oxidase, superoxide dismutase [SOD] and chitinase) and quantity of phenolic compounds studied in the present investigation, phenylalanine ammonia lyase (PAL), the key enzyme in the phenylpropanoid pathway was the first enzyme suppressed at three days after inoculation in X. campestris pv. campestris-cabbage system. Correlation analysis indicated that PAL and phenolic compounds are the two most important compounds determining the susceptibility of cabbage to X. campestris pv. campestris. Induction of peroxidase isoform-1 (Rf value: 0.059) and SOD isoform-1 (Rf value: 0.179) three days after pathogen inoculation implicated the role of these isozymes in susceptible cabbage – X. campestris pv. campestris interaction. This study demonstrates the susceptibility of cabbage to X. campestris pv. campestris is a result of declination of PAL and phenolic contents at biochemical level as a manifestation of increase in bacterial population at the cellular level within the host tissues.     

Bioherbicidal potential of Xanthomonas campestris for controlling Conyza canadensis

The effects of environmental parameters on bioherbicidal activity of the bacterium Xanthomonas campestris, against glyphosate-resistant and – susceptible Conyza canadensis (horseweed), were studied under greenhouse conditions. Rosette leaf-stage plants were more susceptible than older plants, and increasing inoculum from 10⁵ to 10⁹ cells/mL caused significantly greater plant mortality and biomass reduction of plants in both the rosette and bolting growth stages. A dew period at 25°C was required to cause an 80% and 60% mortality of plants in the rosette and bolting growth stages, respectively. Results indicate that X. campestris can infect and kill horseweed, demonstrating its bioherbicidal potential.     

Biological Control of Black Rot (Xanthomonas campestris pv. campestris) of Cabbage in Tanzania with Bacillus strains

We evaluated the biocontrol efficacy of strains of Bacillus from Tanzania against the black rot pathogen, Xanthomonas campestris pv. campestris, in cabbage and the influence of the method of application under field conditions. The incidence and severity of black rot in the foliage, stems and heads of the highly susceptible cultivar, Copenhagen Market, were significantly reduced, especially when antagonists were applied through the roots as compared to application through the seeds or foliage (cotyledons). Promising antagonists included strains of B. cereus, B. lentimorbus and B. pumilus.     

Oxidative-stress-induced production of pyocyanin by Xanthomonas campestris and its effect on the indicator target organism, Escherichia coli

Pyocyanin, a potential antimicrobial agent, was secreted by Xanthomonas campestris. Treatments with agents causing oxidative stress in the organism caused up to 4.4-fold increase in pyocyanin production. Pyocyanin added in the extracellular space did not affect growth rate of X. campestris, but decreased maximum cell concentration and specific product formation. However, the growth of Escherichia coli, the indicator target organism, was affected by pyocyanin. There was also a significant increase in the intracellular reactive oxygen species (ROS) concentration and antioxidant enzyme [catalase, superoxide dismutase (SOD)] concentrations, in the presence of pyocyanin. The intracellular ROS concentrations in E. coli formed upon exposure to pyocyanin, which is an indicator of the toxicity, was dependent on the growth phase of the organism. Studies with mutants of E. coli showed that intracellular ROS concentration was not significantly affected by the absence of the regulon OxyR, but, was significantly higher in cases when the regulon rpoS or the genes katG or katE were absent. Journal of Industrial Microbiology & Biotechnology (2000) 25, 266–272. Received 08 May 2000/ Accepted in revised form 04 August 2000     

Xanthomonas campestris pv. campestris lpsI and lpsJ genes encoding putative proteins with sequence similarity to the α- and β-subunits of 3-oxoacid CoA-transferases are involved in LPS biosynthesis

A 2.1-kb SmaI-EcoRI DNA fragment upstream of the xanA and xanB genes of Xanthomonas campestris pv. campestris carries two ORFs encoding putative proteins with sequence similarities to the α- and β-subunits of 3-oxoacid-CoA transferases. The two ORFs were termed lpsI and lpsJ because strains carrying appropriate mutations showed an autoagglutination phenotype and because lipopolysaccharides of these mutant strains were altered according to silver-stained polyacrylamide gels. The monosaccharide composition of the exopolysaccharide xanthan produced by lpsI and lpsJ mutants remained unchanged. Received: 29 March 1997 / Accepted: 21 July 1997     

Virulence analysis and genetic diversity of Xanthomonas campestris pv. campestris causing black rot of crucifers

Xanthomonas campestris pv. campestris (Xcc) is a devastating bacterium to cause black rot disease in crucifers. To study the genetic diversity and virulence analysis, 24 isolates of Xcc were collected from cole crops including cauliflower, cabbage, broccoli and knol khol from different agro-climatic regions of India ranging from temperate to subtropical climates. For virulence analysis, 24 isolates of Xcc were tested on 27 cultivars of crucifers including seven species of Brassica spp. (B. campestris, B. carinata, B. juncea, B. napus, B. nigra, B. oleracea and B. rapa), Sinapsis alba, Eruca sativa and Raphanus sativus under field conditions at IARI, New Delhi, during November 2010–March 2011. Maximum disease incidence 85.15% was found in the cultivars of crucifers caused by strains Xcc-C124, Xcc-C6, Xcc-C125, Xcc-C111 and Xcc-C131 after 15 days of inoculation and significantly increased after 30 days. Black rot severity in cultivars of crucifers varied from 0 to 6.9 and 0 to 7.9 out of 9 scale after 15 and 30 days of inoculation, respectively. But, no disease incidence was recorded on all the tested cultivars of B. juncea (Pusa Bold, Varuna, Pusa Mustard-21 and Pusa Vijay) against all the strains of Xcc after 15 days. Genetic diversity of 24 strains of Xcc was studied using REP- and BOX-PCR, indicating the existence of wide range of genetic diversity among the strains. The strains were clustered into two groups at 50% similarity coefficient and designated as Group 1 and Group 2. The majority of the strains (23 strains) were clustered under Group 1 except Xcc-C120, which formed separate group (Group 2). In the present study, genetic diversity and virulence pattern in Indian strains of Xcc were established, which will be helpful in the development of resistant genotypes against this bacterial pathogen.     

Isolation and characterization of microsatellite markers for Sabatia campestris (Gentianaceae), an Illinois state‐endangered plant

Sabatia campestris is a regionally endangered prairie plant. Eight polymorphic loci have been generated for S. campestris (Gentianaceae) using non‐enriched and enriched libraries. Polymorphism was quantified from an isolated population from central Illinois yielding mean observed and expected heterozygosities of 0.723 and 0.801, respectively. The number of alleles per locus for this population ranged from three to 25 with a median of 15.5. These genetic markers will be used to determine population genetic structure of Illinois populations as well as to estimate fine‐scale gene flow rates.     

First report of bacterial leaf blight of jute (Corchorus capsularis L.) caused by Xanthomonas campestris pv. capsularii in India

Xanthomonas campestris pv. capsularii causing blight on jute (Corchorus capsularis) leaves was reported for the first time in India. The symptom of the disease initially observed was appearance of small angular brown leaf spots and later as blighted areas on leaf lamina. The disease-causing pathogen was isolated and identified on the basis of its colony morphology, PCR, sequencing and subsequent BLASTn analysis.     

A novel 3-oxoacyl-ACP reductase (FabG3) is involved in the xanthomonadin biosynthesis of Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot in crucifers, produces a membrane-bound yellow pigment called xanthomonadin to protect against photobiological and peroxidative damage, and uses a quorum-sensing mechanism mediated by the diffusible signal factor (DSF) family signals to regulate virulence factors production. The Xcc gene XCC4003, annotated as Xcc fabG3, is located in the pig cluster, which may be responsible for xanthomonadin synthesis. We report that fabG3 expression restored the growth of the Escherichia coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrated that FabG3 catalyses the reduction of 3-oxoacyl-acyl carrier protein (ACP) intermediates in fatty acid synthetic reactions, although FabG3 had a lower activity than FabG1. Moreover, the fabG3 deletion did not affect growth or fatty acid composition. These results indicate that Xcc fabG3 encodes a 3-oxoacyl-ACP reductase, but is not essential for growth or fatty acid synthesis. However, the Xcc fabG3 knock-out mutant abolished xanthomonadin production, which could be only restored by wild-type fabG3, but not by other 3-oxoacyl-ACP reductase-encoding genes, indicating that Xcc FabG3 is specifically involved in xanthomonadin biosynthesis. Additionally, our study also shows that the Xcc fabG3-disrupted mutant affects Xcc virulence in host plants.     

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine‐specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62‐ and atpp2c26‐deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis‐related protein, chaperonin‐60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two‐dimensional isoelectric focusing sodium dodecylsulfate‐polyacrylamide gel electrophoresis (2D‐IDF‐SDS‐PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.     

Systemic Invasion of Plum Seed and Fruit by Xanthomonas campestris pv. pruni through Stalks

Many fruits on Golden King plum trees inoculated through the stalks with Xanthomonas campestris pv. pruni developed unusual lesions extending from the exocarp to the endocarp. A few uninoculated, diseased fruits had similar lesions. The pathogen was isolated from both inoculated and uninoculated stalks and from seeds inside fruits. Scanning electron microscopy of inoculated stalks and mature fruits with unusual lesions revealed that vascular channels of the stalk, seed coat, stony endo, carp, and mesocarp were filled with masses of X. campestris pv. pruni. Bacterial colonies also occurred in other tissues of these fruit parts but were apparently absent from the starchy endosperm or surface of the diseased exocarp. This is the first full report of systemic movement of X. campestris pv. pruni to seed and fruit through stalks.     

Phytochemical Investigation of Cuscuta campestris Yunck. Stem Extract and Evaluation of Its Bioherbicidal Effect on Amaranthus retroflexus L. and Portulaca oleracea L

This study focused on characterizing chemically and evaluating in vitro allelopathic and bioherbicidal potential of secondary metabolites extracted from the stem of Cuscuta campestris in seed germination, early seedling growth and early plant growth of Amaranthus retroflexus and Portulaca oleracea. The combined effects of stem extract and a reduced dose of herbicide metribuzin were also examined. Plant extract contained 17 phenolic compounds and the most abundant phenols were flavonoids: quercetin, (+)-catechin, daidzin, luteolin, and rutin. The seeds of P. oleracea were less sensitive than the seeds of A. retroflexus. The seed bioassay confirmed the inhibitory effect of stem extract on germination and early growth of both weed seedlings at concentrations of 0.75 % and 1 %, and a minor inhibitory effect in the plant bioassay. On the other hand, a synergy of C. campestris stem extract and metribuzin was revealed, as their combination achieved better results in the control of both weed species. Based on obtained data C. campestris stem extract could be a potential source of natural-based weed control molecules.     

The type III effector AvrXccB in Xanthomonas campestris pv. campestris targets putative methyltransferases and suppresses innate immunity in Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) causes black rot, one of the most important diseases of brassica crops worldwide. The type III effector inventory plays important roles in the virulence and pathogenicity of the pathogen. However, little is known about the virulence function(s) of the putative type III effector AvrXccB in Xcc. Here, we investigated the immune suppression ability of AvrXccB and the possible underlying mechanisms. AvrXccB was demonstrated to be secreted in a type III secretion system‐dependent manner. AvrXccB tagged with green fluorescent protein is localized to the plasma membrane in Arabidopsis, and the putative N‐myristoylation motif is essential for its localization. Chemical‐induced expression of AvrXccB suppresses flg22‐triggered callose deposition and the oxidative burst, and promotes the in planta growth of Xcc and Pseudomonas syringae pv. tomato in transgenic Arabidopsis plants. The putative catalytic triad and plasma membrane localization of AvrXccB are required for its immunosuppressive activity. Furthermore, it was demonstrated that AvrXccB interacts with the Arabidopsis S‐adenosyl‐l ‐methionine‐dependent methyltransferases SAM‐MT1 and SAM‐MT2. Interestingly, SAM‐MT1 is not only self‐associated, but also associated with SAM‐MT2 in vivo. SAM‐MT1 and SAM‐MT2 expression is significantly induced upon stimulation of microbe‐associated molecular patterns and bacterial infection. Collectively, these findings indicate that AvrXccB targets a putative methyltransferase complex and suppresses plant immunity.     

Direct biosynthesis of ascorbic acid from glucose by Xanthomonas campestris through induced free-radicals

Ascorbic acid (20.4 g l^-1 in 50 h) was synthesized directly from glucose by Xanthomonas campestris as an adaptive response to induced free-radicals through HOCl treatment. Identity of ascorbic acid was confirmed through IR and NMR spectroscopy.     

Use of agricultural wastes for xanthan production by Xanthomonas campestris

Four different acid-hydrolyzed wastes, from melon, watermelon, cucumber and tomato were compared for xanthan production. Growth of Xanthomonas campestris, xanthan biosynthesis, kinetics and chemical composition were investigated. Both growth and xanthan production were dependent on the acid hydrolysate concentrations and available nitrogen. Melon acid hydrolyzed waste was the best substrate for xanthan production. Exopolysaccharide obtained throughout this study was compared to commercial xanthan, showing a very similar chemical composition. Acid hydrolyzed wastes are proposed as a new carbon source for xanthan production. Received 16 July 1998/ Accepted in revised form 8 October 1998     

Production of xanthan by in-flow cultures of Xanthomonas campestris

The kinetics of xanthan formation in Xanthomonas campestris continuous and fed-batch fermentations was studied along with metabolic changes due to growth rate variation. A maximum growth rate within the range 0.11–0.12 h^–1 was obtained from the continuous culture data in defined medium, producing xanthan at rates up to 0.36 g l^–1 h^–1 corresponding to a maximum 67% glucose conversion at a dilution rate (D) of 0.05 h^–1. Comparatively, fed-batch cultivation was more efficient, producing maximum xanthan at 0.75 g l^–1 h^–1 and 63% glucose conversion at 0.1 h^–1. When reaching D=0.062 h^–1 in continuous cultures, a change was observed and the values of the specific rate of substrate consumption shifted, initiating an uncoupled growth region expressing a lack of balance of the catabolic and anabolic reactions. The deviation was not accompanied by a change in specific xanthan production indicating that xanthan metabolism was not affected by D. For fed-batch-grown X. campestris cells within the range D=0.03–0.1 h^–1, both metabolic parameters changed linearly with the growth rate showing a wide region coupled to growth. Outside that range, glucose accumulated and the specific xanthan production dropped, suggesting substrate inhibition. Correspondence to: J. C. Roseiro     

Transgenic Brassica campestris Plants Expressing the gfp Gene

A protocol has been worked out for efficient regeneration and genetic transformation of summer rape (Brassica campestris L. var. oleifera). Cotyledons of the 5-day-old seedlings were transformed with Agrobacterium tumifaciens strain AGL cells comprising a binary vector with a selectable neomycin phosphotransferase II gene and a reporter gene encoding green fluorescent protein (GFP). Explants were cultured on a regeneration MS medium supplemented with ABA, and transformants were selected on the same medium (minus ABA and plus 70 mM AgNO₃ and 15 mg/l kanamycin). The frequency of shoot regeneration on explant petioles was 30–40%. Transgenic plants were identified by GFP fluorescence and by polymerase chain reaction and Western blotting analysis. The transformation efficiency was as high as 75% of the total number of regenerated shoots.