The 9-lipoxygenase GhLOX1 gene is associated with the hypersensitive reaction of cotton Gossypium hirsutum to Xanthomonas campestris pv malvacearum.

Hypersensitive reaction (HR) cell death of cotton to the incompatible race 18 from Xanthomonas campestris pathovar malvacearum (Xcm) is associated with 9S-lipoxygenase activity (LOX) responsible for lipid peroxidation. Here, we report the cloning of cotton (Gossypium hirsutum L.) LOX gene (GhLOX1) and the sequencing of its promoter. GhLOX1 was found to be highly expressed during Xcm induced HR. Sequence analysis showed that GhLOX1 is a putative 9-LOX, and GhLOX1 promoter contains SA and JA responsive elements. Investigation on LOX signalisation on cotyledons infiltrated with salicylic acid (SA), or incubated with methyl-jasmonate (MeJA) revealed that both treatments induced LOX activity and GhLOX1 gene expression. HR-like symptoms were observed when LOX substrates were then injected in treated (MeJA and SA) cotyledons or when Xcm compatible race 20 was inoculated on MeJA treated cotyledons. Together these results support the fact that GhLOX1 encodes a 9 LOX whose activity would be involved in cell death during cotton HR.     

Water-soaking reaction inhibitory metabolite excreted byXanthomonas campestris pv.malvacearum

The total cultural filtrate (TCF) ofXanthomonas campestris pv.malvacearum (i.e. cultural filtrate after removal of bacterial cells) induced non-persistent watersoaking (WS), on infiltration, in cotton. However diethyl-ether-extracted TCF produced persistent WS reaction. Diethyl-ether-extracted metabolite (EEM) did not induce any reaction but when EEM was co-inoculated with CF (TCF after extraction with diethyl ether), the persistence of WS on cotton was reduced, indicating the presence of an inhibitory metabolite (for WS) in EEM. Analysis of EEM indicated the presence of ammonia, iron and sulfate. Apparently, NH₃, Fe and SO₄ were covalently linked with diethyl-ether-extracted extracellular matrix. Ferrous/ferric ammonium sulfate, on co-inoculation and/or preinoculation toX. campestris in cotton, was shown to inhibit both susceptible (SR) and hypersensitive (HR) reactions. Inactivation of WS inducing factor by ferric/ferrous ammonium sulfate seem to be the cause for inhibition of induction and/or persistence of SR and HR in cotton.     

Resident population of Xanthomonas campestris pv. malvacearum on cotton leaves: a source of inoculum for bacterial blight

Xanthomonas campestris pv. malvacearum was transmitted from infested seed to the cotyledons of cotton cv. Deltapine 61 seedlings at 28°C and relative humidities (RH) of 90% or 73%. A resident population was present on the first and second true leaves but not on the third true leaf of plants at either RH. There were smaller numbers of resident bacteria on fewer leaves of plants at the lower RH than on plants at the higher RH. Cotton plants grown from infested seed at 25°C and 30°C and incubated at 100% RH at different stages of growth developed bacterial blight on leaves that were in bud or partly expanded when incubated. Resident cells of this pathogen can thus invade susceptible leaves when conditions are favourable for infection. Bacterial blight developed on more plants at 30°C than at 25°C. In a field trial, X. campestris pv. malvacearum transmitted from seed was present as resident bacteria on the third leaf from the growing point during the vegetative development of the plant. Resident bacteria, which infected young leaves during rainy periods, were isolated from the bacterial blight lesions which subsequently developed.     

Effect of phenolic substances on pigmentation and pathogenicity of Xanthomonas campestris pv. malvacearum

Summary Phenols inhibited the growth as well as pigment production by Xanthomonas campestris pv. malvacearum (Xcm). Albino (white) colonies were formed at 0.005M concentration of certain phenois, which retained their white colour on successive transfers on phenol containing media indefinitely. Both, albino and yellow isolates were pathogenic and maintained their race number.     

Transformation of virulence factor among bacterial blight ofXanthomonas campestris pv.malvacearum in plants

Transformation of an avirulent strain of bacterial blight of the cotton pathogenXanthomonas campestris pv.malvacearum (Xcm) to a virulent strain was achieved in plants by using total (i. e. chromosomal and plasmid DNA) as well as chromosomal DNA, but not the plasmid DNA, of a virulent Xcm strain, thereby indicating that virulence was located in the chromosomal replica. The process of transformation began 1 d after inoculation of the DNA preparation with the avirulent strain in plant tissues and was completed within 2 d with a recombination frequency of 10⁻²−10⁻⁴.     

Population Dynamics of Virulence Genes of Xanthomonas campestris pv. malvacearum on Cotton

Population genetic principles in relation to the pathogenicity genes have been applied on the genotypes (races) of Xanthomonas campestris pv. malvacearum(Xcm) which are characterized on the basis of bacterial blight resistant host genes ( B -genes) attacked. Observed (OF) and expected (EF) frequencies were determined to predict the intensity of selection pressure operating in the pathogen population due to the introduction of particular host resistant gene(s). Race 32 (V_p, V₇ V₂ V₁₀ V_N) was the most prevalent genotype representing 41.55% of the Xcm population. Other prevalent genotypes were race 30 (11.08%, V_p V₂ V_in V_N), race 20 (8.56%, V_p V₂ V_N), race 9 (6.80%, V_p V_in) and race 8 (11.59%, V_p V₂). The OF (observed frequency) of race 32 was 41.55%, whereas EF (expected frequency) was 15.74% indicating a strong selection pressure favouring this highly virulent genotype. Whereas, race 31 (V₇ V₂ V_in V_N) also overcomes four major genes like race 32 but not the polygene complex, it was less fit and possessed low EF and OF, i.e. 0.25% and 1.18% respectively. Xcm genotypes capable of attacking 3–4 major B -genes were prevalent on G. hirsutum , while genotypes with virulence against 1–2 B -genes favoured G. barbadense cottons. High virulence level in pathogen genotypes, was maintained on resistant/tolerant host genotypes of G. arboreum and G. hirsutum whereas, it was diluted on the highly susceptible G. barbadense.     

Cloning and analysis of a 35.3-kilobase DNA region involved in exopolysaccharide production by Xanthomonas campestris pv. campestris.

Cosmid clones able to restore exopolysaccharide production in possibly insertion sequence element-induced surface mutants of Xanthomonas campestris pv. campestris were isolated. By fragment-specific Tn5-lac mutagenesis of one of the cosmids, pXCB1002, a new DNA region which is involved in exopolysaccharide biosynthesis and which is organized into at least 12 complementation groups was identified.     

Salicylic acid mediated by the oxidative burst is a key molecule in local and systemic responses of cotton challenged by an avirulent race of Xanthomonas campestris pv malvacearum

We analyzed the production of reactive oxygen species, the accumulation of salicylic acid (SA), and peroxidase activity during the incompatible interaction between cotyledons of the cotton (Gossypium hirsutum) cv Reba B50/Xanthomonas campestris pv malvacearum (Xcm) race 18. SA was detected in petioles of cotyledons 6 h after infection and 24 h post inoculation in cotyledons and untreated leaves. The first peak of SA occurred 3 h after generation of superoxide (O(2)(.-)), and was inhibited by infiltration of catalase. Peroxidase activity and accumulation of SA increased in petioles of cotyledons and leaves following H(2)O(2) infiltration of cotyledons from 0.85 to 1 mM. Infiltration of 2 mM SA increased peroxidase activity in treated cotyledons and in the first leaves, but most of the infiltrated SA was rapidly conjugated within the cotyledons. When increasing concentrations of SA were infiltrated 2. 5 h post inoculation at the beginning of the oxidative burst, the activity of the apoplastic cationic O(2)(.-)-generating peroxidase decreased in a dose-dependent manner. We have shown that during the cotton hypersensitive response to Xcm, H(2)O(2) is required for local and systemic accumulation of SA, which may locally control the generation of O(2)(.-). Detaching cotyledons at intervals after inoculation demonstrated that the signal leading to systemic accumulation of SA was emitted around 3 h post inoculation, and was associated with the oxidative burst. SA produced 6 h post infection at HR sites was not the primary mobile signal diffusing systemically from infected cotyledons.     

Characterization of Isolates of Bacterial Blight of Cotton (Xanthomonas campestris pv. malvacearum) from Nicaragua

Seeds from cotton plants infected with Xanthomonas campestris pv. malvacearum were collected in different parts of Nicaragua in 1986. When the seeds were homogenized the pathogen could not always be identified by dilution plating. Therefore, an enrichment of bacteria on the natural host was induced before isolation. The cotton seeds were shaken with water and sand for 2 days and then sown in sand for germination. Rather often the developing cotyledons showed typical water-soaked spots, from which the pathogen could be isolated easily. This new method needed more time but made it possible to detect a low level of bacterial infestation. Altogether, 42 bacterial isolates were obtained. For inoculation experiments suspensions with 5x10⁵ CFU - ml^?1 were infiltrated into cotton leaves. Incubations of inoculated plants in growth chambers, but not in greenhouses, resulted in typical and uniform disease symptoms (water-soaked leaf spots). Nine of the ten cotton differentials tested were highly susceptible to all the 42 bacterial isolates. Since only line 101-102B proved to be resistant, the Nicaraguan isolates of bacterial blight of cotton were characterized as race 18, of the pathogen. The main cotton cultivars grown in Nicaragua (H-373 and G-286) were strongly affected by the isolated bacterial strains. In order to reduce the disease incidence in Nicaragua, the cultivation of resistant cotton varieties is suggested.     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.     

Structures of the O-polysaccharide chains of the lipopolysaccharides of Xanthomonas campestris pv phaseoli var fuscans GSPB 271 and X campestris pv malvacearum GSPB 1386 and GSPB 2388

O-polysaccharides of phytopathogenic bacteria Xanthomonas campestris were isolated by mild acid degradation of the lipopolysaccharides and studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy. The following structures of the repeating units of the polysaccharides of X. campestris pv. phaseoli var. fuscans GSPB 271 (1). and X. campestris pv. malvacearum GSPB 1386 and GSPB 2388 (2). were established:The O-polysaccharides of X. campestris are structurally similar to those of some Pseudomonas syringae strains.     

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.     

Comparative proteome analysis of Xanthomonas campestris pv. campestris in the interaction with the susceptible and the resistant cultivars of Brassica oleracea

Black rot of cruciferous plants, caused by Xanthomonas campestris pv. campestris , causes severe losses in agriculture around the world. This disease affects several cultures, including cabbage and broccoli, among others. Proteome studies of this bacterium have been reported; however, most of them were performed using the bacterium grown under culture media conditions. Recently, we have analyzed the proteome of X. campestris pv. campestris during the interaction with the susceptible cultivar of Brassica oleracea and several proteins were identified. The objective of the present study was to analyze the expressed proteins of X. campestris pv. campestris during the interaction with the resistant cultivar of B. oleracea . The bacterium was infiltrated in the leaves of the resistant plant and recovered for protein extraction and two-dimensional electrophoresis. The protein profile was compared with that of the bacterium isolated from the susceptible host and the results obtained revealed a group of proteins exclusive to the resistant interaction. Among the proteins identified in this study were plant and bacterium proteins, some of which were exclusively expressed during the resistant interaction.     

Use of cloned DNA methylase genes to increase the frequency of transfer of foreign genes into Xanthomonas campestris pv. malvacearum.

In vitro-packaged cosmid libraries of DNA from the bacterium Xanthomonas campestris pv. malvacearum were restricted 200- to 1,000-fold when introduced into Mcr+ strains of Escherichia coli compared with restriction in the Mcr- strain HB101. Restriction was predominantly associated with the mcrBC+ gene in E. coli. A plasmid (pUFR052) encoding the XmaI and XmaIII DNA methylases was isolated from an X. campestris pv. malvacearum library by a screening procedure utilizing Mcr+ and Mcr- E. coli strains. Transfer of plasmids from E. coli strains to X. campestris pv. malvacearum by conjugation was enhanced by up to five orders of magnitude when the donor cells contained pUFR052 as well as the plasmid to be transferred. Subcloning of pUFR052 revealed that at least two regions of the plasmid were required for full modification activity. Use of such modifier plasmids is a simple, novel method that may allow the efficient introduction of genes into any organism in which restriction systems provide a potent barrier to such gene transfer.     

Inhibition of Seed Germination and Root Development Caused by Xanthomonas campestris pv. vesicatoria in Pepper and Tomato

Inoculation of pepper seeds with the leaf pathogen Xanthomonas campestris pv. vesicatoria inhibited pepper germination. The inhibitory effect, which was stronger in non-sterilized light textured soils, decreased with time, and after 20, days or more, there was no difference between inoculated and non-inoculated seeds. Inhibitory substance(s) within the cytoplasmatic fraction of pathogen cells inhibited the germination of non-host tomato seeds. No relationship between pathogenicity to pepper leaves and inhibition of pepper seed germination was detected. The inhibitory substance(s) was found in two out of four X. campestris pv. vesicatoria strains. Heat-killed bacteria suppressed growth of pepper but not tomato seedlings. It is, therefore, suggested that the inhibition of seed germination and the decrease in root development are different modes of X. campestris pv. vesicatoria pathogenesis toward pepper plants.