| Iron, zinc and other metal ions are essential trace elements in bacteria growth. Bacteria adjust themselves ion balance by regulating intracellular iron absorption system and exhaust system. Fur (ferric uptake regulator) and Zur (Zinc uptake regulator) are two important members of the Fur family protein, it can regulate many high-affinity iron and zinc absorption systems in G-negative bacteria. Tomatoes-peppers scab caused by X. campestris pv vesicatoria is a worldwide disease. Fur and zur gene in X. campestris pv. vesicatoriwas were cloned and obtained two mutant (fur-m, zur-m) with method of homologous recombination in this study. The functions of the two genes were more comprehensively analyzed, simultaneously, the role of Fe in the host-pathogen interaction and potentiality of fur-m preventing bacteria disease were studied. A strong antagonistic endophytic bacteria X8 was screened, control potential of X8 and fur mutant on tomato scab was studied. Obtained the following results:1. fur gene analysis According genome sequence of X. campestris pv. vesicatoria 85-10, standard strains in NCBI, as a reference, fur was composed of 136 amino acids, compared with fur in several other Xanthomonas, it amino acid homology are more than 90%. Phylogenetic analysis showed fur in Xcv as has high homology compared to other Xanthomonas spp., especially to the Xac (X.axonopodis Xac29-1), Xoo (X. oryzae pv. Oryzae), Xcr (X. campestris pv. raphani) and Xcc (X. campestris pv. campestris), homology is above 90%, which indicates that fur is highly conserved in Xanthomonas spp.2. function expression of fur phenotypes fur mutant (fur-m) exhibit abnormalities in many ways. fur-m can produce siderophores, increasing the absorption of iron. It almost can not produce biofilms. The ability to produce quorum sensing is significantly weakened. Swimming diameter in fur-m was 8.57 mm, and only was 39.5% of wild type in semi-solid medium on swimming motility essay. Swimming ability became weak. Extracellular polysaccharide content (EPS) in fur-m was 0.52 mg/mL, and only was 40.2% of wild-type. Sensitivity of fur-m to streptonigrin (SNG) significantly increased the survival number of bacteria was only Ig7.46 cfu/mL, and only was one ten thousandth of the wild type (lg11.85 cfu/mL). Resistance of mutant to hydrogen peroxide (H2O2) was significantly reduced. Inhibit diameter was 18.12 mm and survival number was Ig7.68 cfu/mL, whereas Inhibit diameter in wildtype was 14.59 mm and survival number was 104 times of mutant. Growth curves of strains essentially show "S" curve, but the growth rate in fur-m was less than half of that of wildtype. Pathogenicity of the fur-m to tomatoes reduced was significant, fur-m can not stimulate hypersensitive response (HR) in the tobacco. fur-m can absorb most iron under low iron concentration,up to 2172.2 μg/g. Iron can promote the growth of fur-m under low concentration. Different metal ions affected significantly the growth of the fur-m. The other seven ions (Zn2+、Pb2+、Cu2+、Co2+、Ni2、Cd2+、Mn2+) significantly inhibited the growth of fur-m except Mn2+. After fur-m was complemented (fur-c), above phenotypes obtain recover, being lose to the performance of the wild type. It is showed fur gene is a global regulator in Xcv.3. Function expression and genetic analysis of zur homologous genes of zur in Xcv and other Xanthomonas were higher, especially when compared with Xac and Xcc-8004, homology is up to 100% and 89%, especially, it indicates zur is also highly conserved in Xanthomonas.Some phenotypes of zur mutant (zur-m) had also changed. Siderophores produce, biofilm formation, capable of producing quorum sensing in mutant were similar compared with the wild type. Movement significantly weakened, its swimming diameter was 9.60 mm,and only was 44.3% of wild-type. Extracellular polysaccharides (EPS) had 0.70 mg/mL, only was 61.8% of the wild-type. Sensitivity of mutant to streptonigrin (SNG) is increased, the survival bacteria are lg10.70 cfu/mL, only about one-tenth of wild-type (lg 11.85 cfu/mL). Resistance to hydrogen peroxide (H2O2) was significantly reduced, inhibit diameter was 17.50 mm and survival number was Ig9.25 cfu/mL, however inhibit diameter was 14.59 mm and survival number was ten times of mutant. Growth curve also showed "S" curve, growth rate of mutants was lowed. Pathogenicity of zur mutant to tomatoes was greatly reduced with three inoculating methods. Mutants can hardly stimulate the hypersensitive response in the tobacco. Seven different metal ions (Zn2+、 Pb2+、Cu2+、Co2+、Ni+、Cd2+、Mn2+) significantly inhibited the growth of mutant, and higher the metal concentration was, the stronger the inhibition was. After the mutant was complemented (zur-c), some phenotypes can recover, be close to the wild type.4. Impact of fur, zur to the expression of other genes related fur negatively regulates bfd and Xcv935 significantly, negatively regulates copB, Xcv 1654, katE, zur, sufC, hrpG slightly, fur significantly positively regulates bfr gene, slightly positively regulates fliM, pilU, sodB, sufD gene, zur negatively regulates many genes, especially bfd and Xcv935, sufD, sufC, pilU, Xcv 1654, copB, hrpG. bfr and fliM reduce most obviously. It positively regulates bfr, fliM, katE and sodB.5. Effect of Fe and Xcv on the germination and growth of tomato Adhesion amount of zt and ft on seeds was different. Adhesion amount of Y (widetype) was 1.9x 108 strains/mL,while zt (zur mutant, zur-m) and ft (fur mutant, fur-m) were 3.29×108 and 1.10×104 strains/mL respectively. This ability of ft decreased remarked. ft and zt had some promotion on seed germination, but Y had significant inhibition. Different iron concentrations and bacteria could effect seed germination and growth of tomato, ft and low iron concentration had promotion and high concentration and Y had inhibition. strainsUnder different iron concentrations, POD activity of tomatoes was significantly increased. CAT activity of tomatoes increased inoculated by to mutant. CAT activity was highest under middle iron, was lowest low-iron. SOD activity was increased to peak at sixth day under each concentration, then changed smoothly. Under low-iron, PAL activity of tomatoes inoculated by ft was higher than that by Y. It was contrary in high-iron. Under different concentrations, MDA content of each treatments tend to rise. Tomatoes inoculated by ft and Y, its basic sequence of MDA content was low iron> CK> high iron. Under low-iron, SS content of tomatoes inoculated by Y and ft increased then decreased, the Changes of three treatments were down-up-down under middle and high iron; treatments were basically Y> CK> ft. Pro content of tomatoes inoculated by ft was higher under middle-iron, while that of Y was lower. Pro change in each treatment was drop then rise. Under each concentration, total chlorophyll changes was similar in three treatments. It was the lower at 9 d and first drop then rise. The change of carotenoid concentration was consistent, rise at 9 d and sown at 12 d significantly. Height, total fresh weight, stem diameter and leaf area were the most in middle iron concentration. All biomass of tomatoes inoculated by ft also were most. Ft promoted tomato leaves to absorb more iron. Tomato bacteria scab mitigate and disease index decreased when inoculated by ft in high iron concentration.6. Identification of biocontrol bacteria X8 and control of tomato scab Six antagonistic bacteria strains were isolated from mangy vegetables and fruit. Strains X8 has stronger antagonism to many pathogens. With the 16S rDNA sequence analysis, strain X8 shared 99% homology with Bacillus subtilis. According to morphology, physiological and biochemical characters as well as 16S rDNA sequences, strain X8 was identified as B. subtilis (GenBank accession number HQ647257). Control of X8 and ft to scab.7. Control of tomato scab In protection, control effect of X8 to tomato scab was 42.90% at 21th day, while that of ft was 50.4%%. In therapy, effect of X8 was 25.32% at 21th day, oppositely, ft has no effect. In integrated control, control effects above treatment combined with Fe were better, and those effects were closed to chemical agents’. |
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