| Tomato Fusarium Wilt caused by F. oxysporum and pear ring rot caused by B. berengeriana are two serious diseases in many areas of the world. Biological control on plant diseases could reduce the remainder of chemical pesticides and protect natural ecological environment. The aim of this study was to screen and evaluate the antagonistic bacteria against F. oxysporum and B. berengeriana. The results showed that:1、There were 367 strains isolated and purified from root and soil of tomato, pepper and watermelon in Ningxia, Jiangsu and Fujian province. Antagonistic examination against F. oxysporum revealed that 22 strains among all tested strains showed strong and steady antagonism and produced protease. The strain C8-8 could produced chitinase. Several strains among the above 22 strains could produce cellulose and siderophore. On pot experiment, strain PTS-394, H-70, L-1 and SJ-280.of 22 strains could control Fusarium Wilt effectively with the control effect 68.64-77.40%. The molecular identification experiments indicated the strain PTS-394, H-70, L-1 and SJ-280 were B. subtilis.2、An open reading frame that coding chiA gene putatively was cloned from the strain C8-8 genomic DNA by PCR method. Sequence alignment showed that the sequence was 97% identical with the ChiA of enzymogenes of S. marcescens. The PCR fragment was cloned into vector 28a. A protein about 60KDa, was expressed by the host strain DH3ChiA. The purified protein could form a clear hydrolyzed zone on the colloidal chitin plate and inhibit the growth of the mycelia of F. oxysporum. It showed that chiA gene from C8-8 could be utilized as a potential biological control factor.3、The strain C8-8, which could produce high-level chitinase, is an antagonistic bacterium against many pathogenic fungi. The proper cultured temperature, pH value and initial inoculum for growth were 30℃-35℃,7.0-8.0 and 0.5-1/300 of total cultured agar volume, respectively. Ventilation had no influence on the growth of the strain C8-8. Using N-Acetyl-D-glucosamine as carbonic sources could inhibit chitinase activity of strain C8-8 totally, but yeast extract powder and peptone could improve chitinase activity of strain C8-8 greatly. It could reach the highest activity,485μg GlcNAc·h-1 when strain C8-8 was cultured at 31℃, pH 7.3 for 6 days.4、The activity of phenylanine ammonia lyase (PAL) and peroxidase(POD) of tomato treated with C8-8 was increased greatly compared with that of non-treated, which implied that the potential function of resistance disease was induced by inoculating strain C8-8. A large amount of GA3 produced by C8-8 in NA medium were measured by HPLC analysis and its value reached 158mg/l. However none of IAA and GA3 was measured in essence medium, which implied that there are likely a tie between plant hormone produced by antagonistic bacterium C8-8 and promoting growth mechanism.5、Multiple interactions were involved in the combinations of antagonistic bacteria. The compatible combination of SJ-280 with PTS394 resulted in enhanced disease suppression (47.1%) against Fusarium Wilt of solanaceous vegetables as compared to the single strain SJ-280 (35.3%) or PTS394 (38.7%). The incompatible combination of C8-8 with PTS394 led to reduced disease suppression. However, increased disease suppression was also observed in the biocontrol by the incompatible combination of L-1 with PTS394.6、There were 871 strains isolated and purified from the diseased leaves, the diseased pomes, the healthy leaves, the healthy pomes, the diseased tress, the healthy tress and soil from the orchards of Nanjing, Lianyungang and Gaoyou, Jiangsu Province. Antagonistic examination against B. berengeriana revealed that 21 strains of all tested strains showed strong and steady antagonism. Strain sf 628 could inhibit the mycelia growth of B. berengeriana effectively with 45 mm inhibition zone and the inhibition rate 59.21%. The inoculating experiments on pear pomes showed that five strains of 21 tested strains could inhibit effectively necrosis spread of B. berengeriana with the inhibition rate more than 50 %, and the inhibition rate of strain sf 628 reached 60.61%. The physicochemical property and molecular (16S rRNA) identification experiments indicated the strain sf 628 was B. subtilis subsp. subtilis.7、Toxicity test of 26 fungicides on B. berengeriana showed 25% Propiconazole could inhibit effectively the mycelia growth of B. berengeriana with EC50 value 0.1522μg/ml. The mixed experiments against B. berengeriana showed that the synergistic ratios(SR) were greater than 1.5 when the mixed ratios were 1:3,1:2,1:1 and 1:2 between antagonistic bacterium sf 628 and Propiconazole and the SR was 1.071 under the mixed ratio was 3:1. The synergism under the ratio of 1:3 between antagonistic bacterium and Propiconazole was the most significant with synergistic ratio 2.942. 8、We also evaluate influence of sodium bicarbonate (SBC) on effectiveness of biocontrol agents (BA) to control ring rot on postharvest pear fruit. The results indicated that the inhibition rate against B. berengeriana was 42.30%,75.77%-85.41%, and 83.00% 90.23% in vitro when the plates were treated with 2% SBC (w/v), BA (strain 11,2,28 and sf628), and the combination, respectively. On fruit, the control effect was 23.83%,12.79%, 71.09%,48.16%,74.57%, and 57.90% when fruit were first inoculated with B. berengeriana (curative activity), then treated with BA 11 30mL, SBC 30mL, BA 11 15mL +SBC 15mL, SBC 15mL+BA 1115mL, BA 11 30mL+SBC 30mL, and SBC 30mL+BA 11 30mL, respectively. When fruit were treated with the same above, then inoculated with the pathogen (preventive activity), the control effect was 28.72%,17.31%,78.25%,51.06%, 80.91%, and 61.50%. |
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