| Tobacco is an important cash crop. Tobacco black shank, caused by Phytophthora nicotianae, is an economically important disease of tobacco world-wide. In China, it occurs frequently and commonly, which causes a huge loss at several provinces. For adapting to the industrial demand in specific quality, most of tobacco varieties to Tobacco black shank is not much strong, therefore, the main way to control the disease still keeps taking chemical pesticides. Thus, it causes the drug-resistant development, environmental pollution, and the heavy metals contents in tobacco leaves getting over-standard etc., finally impacts on the disease control effect, the ecological safety and the mankind health. For this reason, based on the thought for ecological control, the effect of endophytic bacteria on rhizosphere ecosystem, physiology and biochemistry of host plant, and pathogens causing Tobacco black shank, was studied in this paper, from the plant micro-ecosystem. The main results of the study are as follows.The impact of endophytic bacteria on tobacco rhizosphere ecosystem The absorption and utilization of soil organic matter by tobacco plants got certain difference after three kinds of treatments. The strain Itb295 showed a maximum content of soil organic matter after the treatment of seed-soaking and root-irrigation among the ten strains tested, while that of the strains Itb225, Itb220 and Itb57 was most closed to the control (CK). The soil pH value of majority of biological control strains differed slightly with the CK after three kinds of treatments, only that of the strain Itb185 got the highest level at 8.23 after seed-soaking. The pH values of strain Itb162 seed-soaking and strain Itb28 seed-soaking and root-irrigation were relatively lower in all the treatments. Furthermore, an interesting result was found that total potassium content in the soil decreased after all the tested strains treated by seed-soaking and root-irrigation. However, it was not shown all the tested strains positively impacted on the tobacco seedling taking advantage of effective potassium. Compared to CK, the effective potassium content in soil of strains Itb225 and Itb28 was much higher after three kinds of treatments. It was studied how the change of soil total phosphor contents was after ten strains treatments. Compared to CK, the soil total phosphor contents changed slightly, except that of treatments by Itb220 seed-soaking or Itb8O spraying irrigation far increased. The impact of tested strains on effective phosphor was great, varied from 2.59 to 18.36 mg/kg. The majority of strains could make the effective phosphor contents decreased in the soil after a certain treatment.There was no obviously consistent trend towards affecting of the tested strains on the activity of three kinds of enzymes (Catalase, Urease and Sucrase) in the soil related to soil fertility. The difference in the study was that a part of experimental strains raised the activity of a certain enzyme, while others preformed repressing.The effect of different treatments on soil micro-organisms showed as follows. After three kinds of treatments (seed-soaking, root-irrigation and spray irrigation) of two strains Itb57 and Itb12, in contrast with CK and the other eight strains, the number of soil fungi was 10 times lower. After two kinds of treatments (seed-soaking, root-irrigation) of strain Itb57, the number of soil bacteria was 10 times more than the CK treatment. The quantity of soil actinomyces of all strains tested was in the same quantity grade (the logarithm value ranged from 5.1 to 5.8) in three kinds of treatments. From the tendency, it was found that the majority of experimental strains had made the quantity of soil actinomyces increase. As one of functional groups of soil micro-organisms, the number of Azotobacter increased treated by ferment liquid of most strains tested, the number of Phosphate-solubilizing bacteria increased in the treatments with all the biological control strains tested, while that of Potassium-releasing bacteria in the treatments of most tested strains was much closed to the CK trial.The impact of endophytic bacteria upon tobacco growth The effect of the ferment liquid often endophytic bacteria on fresh weight and dry weight aboveground, width and length of the biggest leaves, euphylla numbers of tobacco seedlings were studied in the paper. The results indicated that strain Itb57 could promote the seedling's growing well, and followed by the strains Itb185 and Itb295. But strain Itb162 negatively affected on the dry weight aboveground and euphylla numbers of tobacco seedlings with the treatment by seed-soaking. After three kinds of treatments (seed-soaking, seed-soaking+root-irrigation, and spray irrigation) of the ten strains, seed-soaking+ root-irrigation showed a good and stabilized effect on the growth promotion of tobacco seedlings, on the other hand, the seed-soaking treatment with part of tested strains restricted the seedlings' growth.Integrated evaluation and selection of the optimal from experimental endophytic bacteria Integrated evaluation of the ten strains in the study, it was found that strain Itb57 was more excellent in accelerating the soil mineralization, promoting the absorption and utilization of nutrients for tobacco plants, and the seedlings growth, except no significant effect on three sorts of enzymes activities. Besides, strain Itb57 could reduce the amount of soil fungi, and make the numbers of soil bacteria increase, especially the three kinds of functional groups of soil micro-organisms, Azotobacter, Phosphate-solubilizing bacteria and Potassium-releasing bacteria. Therefore, strain Itb57 was taken as key objective strain in the research, and YBX1-4 was chosen as one standand strain of Phytophthora nicotianae for antagonistic measurement.Antibiotic effect and metabolism products of strain Itb57 It was found the antagonistic spectrum of endophytic strain Itb57 was broad, showing an antagonistic effect on nine tested pathogenic fungi in a certain degree. The best was observed in Alternaria alternata, A. solani, the width of restrained zone was 9.83 mm and 9.50 mm, respectively. The better repressing effect was on Cercospora personata on peanut with a zone of 9.33 mm, besides on the pathogens Colletotrichum, Botrytis, Sclerotium. The antagonistic effect on P. nicotianae showed that the hypha of pathogen became distortion, more branches, even formed a small lump and short knot in the duel culture plate. Metabolizing products was detected in strain Itb57 generated Cellulase, Proteinase and Siderophore, but no Chitinase.The taxonomy of strain Itb57 Based on properties, morphological, physiological and biochemical characteristics of colonies, and 16S rDNA (GenBank accession No.GQ153538) phylogenetic analysis, strain Itb57 was identified as Bacillus subtilis. The sequence length of 16S rDNA of Itb57 of fragments was 1397 bp.The physiological and biochemical effects of strain Itb57 on tobacco seedlings The tobacco seed germination was entirely restrained after treatments with the original bacterial suspension and the above liquid post centrifugal practice of Itb57. But the bacterial suspension with certain concentration could promote the seed germination, and the growth of tobacco buds, especially when the bacterial suspension was at the concentration of 3×10(?) cfu/mL; the effect of growth-promotion was the best. Additionally, strain Itb57 was found to be able to stimulate the growth and development of seedling rootlets, and to lift the activity of roots. The activities of three sorts of enzymes related to tobacco resistance, such as Phenylalanine Ammonium Lyase (PAL), Peroxidase (POD) and Polyphenol Oxidase (PPO), were all significantly taken up after the treatments of tobacco seedlings with the bacterial suspension, only the detailed time segment and enhancement ratio for inducing the resistance and enzymes activities varied slightly.Research on endogenesis and colonization of strain Itb57 Strain Itb57 was tagged with rifampicin, the drug-resistant strain was acquired at the level of anti-Rif 200μg/mL. The cultivation character in the medium and antagonistic activities on P. nicotianae of the re-isolated strain after inoculation was similar to the original strain of Itb57. This result adequately proved strain Itb57 was endophytic bacteria. After tobacco roots were inoculated with the bacterial suspension of tagged strain Itb57, it was isolated again in the stem and leaves, which suggested that the tagged strain could transfer upwards in tobacco plants. Moreover, the number of colony form unit in tobacco tissue was found mostly in the rootlets (3.72×104 cfu/g fresh weight), followed by the stem (2.14×104 cfu/g FW), and the leaves (0.15×104 cfu/g FW) to colonize the interior tissues of tobacco plants. The highest amount (3.72×104 cfu/g fresh weight) of the inoculated bacterium was detected in the rootlets and the lowest in the leaves of the tobacco plants. Under the condition of sterilization, after tobacco seedlings was inoculated with tagged strain Itb57, its colonization was found in the surface of rootlets, even in the cortex cells, which further provided strong evidence that strain Itb57 was endophytic bacteria.The biology characteristics of strain Itb57 and fermentation conditions The experiments for screening the culture medium indicated that was beneficial to the growth, reproduction of strain Itb57 and its producing antagonistic substances in the conditions of peanut powder as nutrition source. According to the growth curve of strain Itb57, it was found that the strain grew slowly within 0-12 hours, subsequently the growth became fast into logarithm increasing stage, at 48 hour the top abundance obtained, and then kept into a stable period. Measurement of each single factor for fermentation of strain Itb57, the results showed that the optimal conditions for the growth and production of antagonistic substances of strain Itb57, were at 28℃of fermentation temperature, the initial pH7.0 value of culturing medium,48 hour of fermentation period, the rotation speed 180 rpm of triangle bottles, and the volume ratio of 25 mL/500 mL triangle bottles for supplying oxygen. The optimized conditions by trial consequently were at 28℃of fermentation temperature, the initial pH7.0 value of culturing medium, fermentation period 72 h, the rotation speed 180 rpm of triangle bottles, and the volume ratio of 25 mL/500 mL triangle bottles.The control efficacy of strain Itb57 to tobacco black shank Green-house pot-grown tobacco plant assays showed that the control efficacy to tobacco black shank by strain Itb57 was 69.28% when the bacterium was applied by root-irrigation in advance of P. nicotianae inoculation. Further, field trials in 2007 and 2008 demonstrated that the application of strain Itb57 controlled the disease by with the efficiency of 61.25% and 72.49%, respectively. Statistical analysis indicated that the disease control efficacy was not significantly different from that obtained by treatment with the chemical (58% metalaxyl mancozeb wettable powder) (P>0.05). Measurement of the chemical components in the tested tobacco plants after treatments with strain Itb57 in plot trial, the major components changed little, compared with the control (CK). The positive effect on the reduction of sucrose-glucose variance, nitrogen-alkali ratio, and balancing glucose-alkali ratio was better than the CK after treatment with bacterial suspension of strain Itb57.
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