Study On The Tomato Endophytic Bacteria And Their Biological Control Of Tomato Bacterial Wilt

Endophytic bacteria are a kind of bacteria which live in health plant tissues mainly in cell and intercellular and form compatible association relationships with their host. For stimulating plant growth, improving phytoremediation and increasing disease resistance in plant, endophytic bacteria are an important group of microorganism in microecosystem. Generally endophytic bacteria were studied on the antagonist against plant pathogens. Few study on the population and dynamics of endophytic bacteria and the relationships between control plant disease and its colonizing ability in soil and plant.Tomato bacterial wilt, caused by Ralstonia solanacearum, is a lethal soil-born plant disease. It is very difficult to control this disease recently. In this study, basis on the analysis of the population and dynamics of endophytic bacteria and the test of interaction between endophytic bacteria, an endophytic bacterial strain with plant growth promotion and inhibition to the pathogen of tomato bacterial wilt were screened. Then, the biologic character and the colonization ability in soil and within plant of this strain were studied. Finally, the biological control of tomato bacterial wilt by using this strain as biological control agent was carried out. The research result might give the scientific principle for the control of tomato bacterial wilt by using endophytic bacteria. The main result of this research as follows: 1. Population and dynamics of endophytic bacteria in tomato1.1 Groups of endophytic bacteria in tomatoThree hundred and three symptomless tomato plants were collected from Guangxi for the analysis of the population and dynamic of endophytic bacterium by means of cultural-dependent. Six hundred and twenty-four strains were obtained. These strains were identified by traditional methods. The result shown that the groups of tomato endophytic bacterium were Bacillus spp., Pseudomonas spp., Xanthomonas spp., Microbacterium spp., Corynebacterium spp., Erwinia spp., Enterobacter spp. and Agrobacterium spp..1.2 Group difference of endophytic bacteria in tomato varieties and cultivation regions The groups of endophytic bacteria in tomato varied with the varieties. By the test ofthe groups of endophytic bacteria in two varieties, Xanthomonas spp was isolated from the variety of Hong Bao Shi, but it did not be found in the variety of Mei Xing. The result suggested that endophytic bacterium in tomato might select suitable variety to live in.The groups of endophytic bacteria in tomato also varied with cultivation regions. A variety cultivated in different regions, the groups of bacteria in plant were different. By the comparison with the groups of bacteria in variety Tai Wan Sheng Nu, Microbacterium spp. could be isolated from the tomato plant cultivated in Tianyan county, but this genus of the bacteria could not lived in the plant cultivated in Yulin city. On the contrary, Xanthomonas spp. was isolated from the plant cultivated in Yulin city, this bacterium could not be found in the plant cultivated in Tianyan County. This result appeared that the groups of endophytic bacteria in tomato might be influenced by environment, such as temperature, character of soil and soil microorganisms. Although the groups of tomato endophytic bacteria varied with varieties and cultivation regions, the main groups of tomato endophytic bacteria were Bacillus spp., Pseudomonas spp. and Agrobacterium spp.; the dominated group of tomato endophytic bacterium was Bacillus spp.. 1.3 Population fluctuation of endophytic bacteria in tomatoThe communities of endophytic bacteria in different tissues of tomato plants during their growth periods were studied by means of cultural-dependent. The result shown that the total population of endophytic bacteria was the largest in roots, followed by in stems and leaves. From seedling stage to fruit stage of tomato, the population sizes of endophytic bacteria gradually increased, and then decreased from fruit stage to fructescence. The population sizes of most endophytic bacterial genera in the growth stages of tomato varied very similar with total population of endophytic bacteria, but Microbacterium spp. reduced gradually from seedling stage to fructescence. This result indicated that the population fluctuation of endophytic bacteria in tomato corresponded with the biological and biochemical activity of tomato. Meanwhile, the population fluctuation of endophytic bacteria in tomato also varied with cultivation seasons. The plants cultivated in the autumn were higher population densities than that of plant cultivated in the spring in Guangxi; it might be that the temperature influenced thebacteria activity in plant.2. Interaction among organism in microecosystem of tomato2.1 Interaction among endophytic bacteriaAccording to the result of the test for antibiosis of 26 endophytic bacterial strains against 10 strains of these 26 strains, the interaction among endophytic bacteria could be divided into two types. One was neutralism. Most of endophytic bacteria did not inhibit each other in tomato; they had formed relationships of compatible association during their evolution. Another type was antagonist. There were few bacterium genera in tomato, such as Bacillus, most of its strains inhibited to many strains of other genus. This might be why Bacillus was dominative genus among endophytic bacteria in tomato.2.2 Endophytic bacteria inhibition to the pathogen of tomato bacterial wiltTwenty-one strains, which inhibited to Ralstonia solanacearum, were screened from 624 strains of endophytic bacteria of tomato by means of inhibition ring. Of 21 strains, 16 strains belonged to Bacillus spp.; other 5 strains belonged to Agrobacterium spp. (2 strains), Pseudomonas spp. (2 strains) and Xanthomonas spp. (1 strain), respectively. Strain B47 was the most strongly inhibition to the growth of Ralstonia solanacearum in 21 strains.2.3 Tomato growth promoting activity of tomato endophytic bacteriaTwenty-one antagonistic strains were tested for stimulating the tomato growth by means of injecting tomato stems and dipping tomato roots respectively. The result of test showed that tomato growth promoting activity of endophytic bacteria could be divided into three groups: 1) some strains, such as B47and Bfll8, stimulated tomato growth significantly, both heights and leaves of plant treated with these strains were significant difference with the untreated plants (CK); 2) Some strains, such as Bsl2, Bm21 and Bfl20, could promote plant growth in heights, but the leaves of plant treated with these strain were the same as the untreated plant; 3) strain Bm25 could not promote the growth of tomato.2.4 Interaction between endophytic bacteria and soil microbesInteraction between Bacillus spp. strain B47 and soil microbes was tested with inhibition ring. According to the result of the experiment, interaction between strain B47and soil microbial strains could be divided into three types: 1) strain B47 and 21 soil microbial strains could live together, they did not inhibit each other and formed the relationship of neutralism; 2) strain B47 and soil microbial strain St3 inhibited each other; 3) Strain B47 inhibited to some soil microbial strains, such as Bl and B24, but these soil microbial strains could not inhibit to stain B47.3. Biological character of strain B473.1 Identification of strain B47Basis on the test of morphological, Physiological and biochemical characteristics and the analysis of 16S rDNA sequence, strain B47 was identified as Bacillus subtilis (Ehrenberg) Cohn.3.2 Biological character of strain B47Bacillus subtilis strain B47 could grow under the condition of higher temperature and lower pH. For the growth of strain B47, the optimal temperature and pH were 33°C and 5.8, respectively; the strain could use D-lactose, D-mannitol, glycerine, D-glucose, D-fructose, D-galactose as carbonic source and soya peptone, tryptone peptone beef extract, Yeast extract as nitrogen source. Dulcitol and rhamnose were not suitable carbonic source for its growth. This strain could not grow in the medium contained the inorganic nitrogen, such as casein, ammonium chloride, potassium nitrate, urea and ammonium oxalate.3.3 Entry host plant and colonization site in plant for strain B47Entry host plant for strain B47 were detected by means of dipping seeds, dipping roots, watering roots, injecting stems and spray. The result indicated that the bacterium penetrated into plant tissues through wounds. Detecting the plant tissue after inoculation 20 days under microscope and electron microscope, strain B47 could be seen inside vascular and cell.4. Viability of strain B47 in soil and inside plant.After irrigating soil, the numbers of strain B47 increased gradually from the 15th days to 45th day, and decreased after 45days. The viability of strain B47 in soil varied with the treatment and the type of soil. After irrigating soil 45 days, the numbers of strain B47 in steamed garden soil were 9.84 X107 cfu/g soil, however, its number in raw gardensoil were 9.91 X 105 cfu/g soil. The viability of strain B47 in laterite was the same as in garden soil, but its numbers were lower than in garden soil. This result suggested that strain B47 was a bacterium of part time living in soil, but soil types and soil microorganisms influenced over the viability of the bacterium in soil.Inoculation strain B47 by dipping root showed that the bacterium could colonize inside the roots and stems. The numbers of strain B47 in tomato plant fluctuated with the growing period of tomato. After inoculation by means of dipping root at the transplantation of tomato seedling, the numbers of strain B47 inside roots and stems increased gradually from the seedling stage to fruit stage, and then decreased after fruit stage. This result indicated that the viability of the bacterium in plant related to the biological activity of host.5. Character of antagonistic substance produced by strain B47Crude antagonistic substance was obtained by acid precipitation of the cell-free culture of strain B47 with IN HC1 and methanol extraction. It was thermostable (121°C 30 min) and resisted to protein K, pepsin and trypsin , and had inhibitory activity against many plant pathogenic fungi and bacteria, such as Pyricularia grisea, Bipolaris maydis, Fusarium oxysporum, Xanthomonas axonopodis and Ralstonia solanacearum. This antagonistic substance was purified by means of DEAE-52 anion exchange chromato-graphy and sephadex G-50 chromatography. After purification, antagonistic substance showed only one spot on silicon thin layer chromatography (ILC). Plate assay shown that the recovered substance from TLC could inhibit to the growth of Ralstonia solanacearum. This result indicated that the antagonistic substance might be purified.6. Control efficacy of Bacillus subtilis strain B47 to tomato bacterial wilt 6.1 Development rat of strain B47 and Ralstonia solanacearum in plantThere was a difference in development rat between strain B47 and Ralstonia solanacearum in tomato. The pathogen could be recovered from the stem of plant after inoculation 1 day. But strain B47 did not be recovered from the stem of the plant until after inoculation 3 days. By the comparison with the numbers of bacterium in the stem of plant, the numbers of Ralstonia solanacearum were 2.03 X 105cfu/g fw, the numbers of strain B47 were 7.00xi02cfu/g fw after inoculation 7 days. The result of the experiment...