Screening Of Biocontrol Strains Of Bacillus Spp. And Their Mode Of Action On Plant Disease

46 bacterial strains were isolated from root-knot nematode infected tomato roots, 13 of which could inhibit the growth of Ralstonia solanacearum in vitro with the inhibition zone diameter ranging from 7．1 mm to 26．3 mm on NA plate, and 12 strains of them suppressed the growth of several plant pathogenic fungi. In the greenhouse tests, bacterial wilt disease caused by Ralstonia solanacearum was controlled effectively by the isolates EN5, EN15, EN01 and EN16 with the average control efficacy of 70．82%,89．54%,85．38% and 78．81% respectively; and root-knot disease caused by Meloidogyne incognita was reduced 49% by EN5, SW1 and SB2．The strains' ability to control the complex disease caused by two pathogens were also tested, likewise, the isolates EN5, SW1 and SB2 significantly reduced the infection percentage compared with non-treated control, among which, SW1 showed best effects on reduction of incidence of bacterial wilt disease by 56．66 and root galling index by 5．21．The effects of 7 strains were re-examined in unsteriled soils, strains EN5, EN16, SW1 and SB1 showed stable and higher control effects. Based on their physiological and biochemical characteristics, EN5, EN15, EN01, SW1, SB1 and SB2 were identified as Bacillus subtilis, while EN16 was Bacillus pumilus. The results from 16S rDNA sequence further proved strain EN5 were B. subtilis subsp endophyticus.The biocontrol mechanisms of strains EN5, EN16, SW1 and SB1 against bacterial wilt disease were studied. Results showed that population of strain EN5 in tomato were 3．60×10~4cfu/g·FW at 20 days after inoculation, and the culture filtrate show no inhibition ability against pathogen and low effects (13．56%) on bacterial wilt disease, which indicating that strong colonization in root and shoot system play a key role in its disease control. Strain SB1 showed great inhibition activity on R. solanacearum, and the biocontrol effect of culture filtrate of SB1 was 50．85 %, while low colonization ability and induced resistance abililty were detected, so the mode of action of strain SB1 was mainly dependent on inhibitory substances. The biocontrol effectiveness of culture filtrate of SW1 and EN16 were only 22．03% and 27．12 % respectively, lower than those of cells. Both of them showed good colonization ability and significantly induced resistance against bacterial wilt disease with higher induce effects and the activities of peroxidase (POD), polypenoloxidase (PPO) and phenylalanine ammonia-lyase (PAL) in tomato leaves enhancing obviously, which suggesting that induced resistance was the most important mechanism involved in the biocontrol effect of strains SW1 and EN16．SEM observation further showed that strain EN5 mainly colonized in the phloem and cell gaps of main roots and stems. Pot tests indicated that strain EN5 was able to colonize in tomato with the conditions of natural soils and steriled soils, and the colonization was stable with the inoculation methods of root-pouring, while poor colonization was detected with seed-dipping method. Besides tomato, strain EN5 could colonize in tobacco and cucumber, the colonization in tomato and tobacco were obviously larger than those in cucumber. With the treatment of strain EN5, the quantities of indigenous bacteria in tomato were reduced in different stages, while those of rhizobacteria, especially ammonifier, nitrite bacteria were reduced in initial stages, and increased significantly in late stages. Further evidence provided by ERIC-PCR showed that the inoculation of EN5 made an effect on the species of rhizobacteria.Within 2～10 days between strains EN 16 and SW1 application and challenge inoculation with R. solanacearum, two strains both induced systemic resistance against bacterial wilt disease, with the best induced effects obtained with 7～10 days intervals. The activities of POD, PPO and PAL enhanced significantly and tend to peak at 7～10 days intervals, which were consistant to the results above. Strains EN16 and SW1 were inoculated by the methods of pouring and seed-treatment, only when inoculated by pouring treatment, both strains induced tomato against bacterial wilt disease. In addition, the ability of two strains to induce resistance against Tobacco mosaic virus disease was tested; better induced protection was obtained in plants treated with strain SW1．Induced accumulation of pathogenesis-related proteins (PRs) in tobacco leaves by strain SW1, EN16 and acetylsalicylic acid (ASA) indicated that PR proteins highly induced by ASA and strain SW1 were similar in the protein molecular size and expression intensity, while those of EN16 and ASA were different in some ways.Mutant tests showed that the control effects of strain SB1 was positively correlated with the inhibitory effects of the culture filtrate. To enhance the production of antibacterial compounds, several medium with different components were used, and it was found that glucose and mineral salts promote secretion of the active compounds, which showed thermostable, UV and enzyme tolerant properties. From PD culture filtrate, an active fraction was isolated and determined as two isoforms of surfactin by silica column and high performance liquid chromatography. In addition, surfactin and iturin A synthease gene were also detected by PCR, and 98% of srfA sequence from SB1 was identical to the partial regions of known B.subtilis surfactin operon (BACSRFAB), 97．2% sequence identity were shown between ituB gene from SB1 and the corresponding region of reported iturin A operon (AB050629) . Another study was undertaken to investigate the possible mechanisms of the active compounds on R. solanacearum, the results showed that condensed cytoplasm and lysed cell were observed in treated group with TEM, a hyperchromic effect on bacterial DNA was detected by UV spectrum analysis, and the expression of two extralcelllur and two intracellular proteins were inhibited by the active compound, indicating cell morphology and the synthesis of DNA and protein were greatly influenced.Three digoxigenin-labeled probes specific to surfactin, iturins and TasA protein were prepared by PCR with three pairs of specific primers and were submitted to dot-blotting for the genes detection from 24 bacterial strains. In addition, competition ELISA was used to evaluate the yield of surfactin of the positive samples with surfactin gene. Results showed that three probes with the length of 1200 bp, 1479 bp and 790 bp were obtained with the positive plasmids as templates, the detection sensitivities of three probes were 2, 20 and 0．1 ng, respectively; and ten samples with surfactin related gene, ten samples with iturins related gene and four samples with tasA gene were detected from twenty-four bacterial strains. Competition ELISA detection indicated that surfactin was detected from the KMB cultures of ten samples with surfactin gene, among them, strain EN1 and SB1 have high yield of surfactin, with the output of 32．9 mg/L and 41．0 mg/L respectively.