Research On Bacterial Leaf Spot Of Tomato Leaves In Gansu Province

The investigation found that the bacteria I disease in seed production of Gansu province, and collected diseased leaves and diseased fruit from Zhangye and Jiuquan,17 strains were isolated by conventional separation method. The strains were determined through pathogenicity test, and they were identified by morphological,physiology and biochemistry, Biolog identification and 16 S r RNA sequence analysis.At the same time, other studies also had done that included toxicity determination of bactericides, screening and identification of antagonistic bacteria and optimization of fermentation conditions. The results were as following:1. Pathogenicity test of bacterial leaf spot of tomatoThere were two pathogenic strains can cause disease among the 17 strains isolated and purified from diseased leaves and fruits of tomato, the two isolates produced the same symptoms with naturally diseased leaves and the same strains were re-isolated from the inoculated tomato, it was fulfilled with Koch’s postulation, therefore, the two isolates were tomato bacterial leaf spot pathogens.2. Identification of bacterial leaf spot of tomatoNo.1 was Gram-negative bacteria, the size of 0.34~0.87 × 0.34~1.03(0.693 ×0.533) μm, No.2 was Gram-positive bacteria, the size of 0.31 ~ 0.9 × 0.61~ 2.25(0.629 × 1.378) μm. Combined morphological, cultural characteristics, physiological and biochemical, Biolog identification and 16 S r RNA gene sequence analysis, No.1pathogen was identified as Sphingobacterium multivorum and No.2 pathogen was identified as Curtobacterium flaccumfaciens.3. Research of biological characteristics and pathogenesis of tomato bacterial leaf spotThe result of biological characteristics showed that the minimum, maximum,lethal temperature and optimum growth temperature of Sphingobacterium multivorum and Curtobacterium flaccumfaciens were 2℃, 45℃, 51℃, 24-28℃ and 2℃, 45℃,52℃, 22-28℃, respectively. In addition, the two pathogenic bacteria both could growfrom p H 6 to p H 8, and could enter the logarithmic phase after 3h and 5h respectively,enter the decline phase after 33 h, 34 h respectively in NA medium.The result of the pathogenic mechanisms showed that two strains did not produce pectinase, but both could produce cellulase enzymes and cutinase, and the capacity of enzyme production of Curtobacterium flaccumfaciens was stronger than Sphingobacterium multivorum.4. Control of bacterial leaf spot of tomato and screening and identification of antagonistic bacteriaThrough indoor toxicity determination of 7 bactericides on Sphingobacterium multivorum and Curtobacterium flaccumfaciens, the results showed that there were seven kinds of bactericides could strongly inhibited the bacterium, 0.15%tetramycin had the best effectiveness, and 50% chlorobromoisocyanurate was the worst. 9 antagonistic strains of Sphingobacterium multivorum were screened, and the XSZ3 had the best inhibitory effect. There were 35 antagonistic strains of Curtobacterium flaccumfaciens, in which 262ZY2 was the best, and Ai 2 had a good antagonism to two kinds of pathogenic bacteria. Xian 4 and Ai 2 were identified respectively as Bacillus amyloliquefaciens and Bacillus methylotrophicus combined with 16 S r RNA sequence analysis, morphological, physiological and biochemical characteristics.5. Optimization of fermentation conditions of antagonistic bacteriaAccording to orthogonal test, the optimal medium of Xian 4 was maltose 10 g,ammonium nitrate 5g, Na Cl 8g, K2HPO4 2g, Mg SO4 1.5g, distilled water 1000 m L.the best medium of Ai 2 was maltose 15 g, beef extract 3g, Na Cl 4g, K2HPO4 1.75 g,Mg SO4 1.25 g, distilled water 1000 m L. The optimal inoculum of Xian 4 was 3%,and Ai 2 was 0.5%. Optimal liquid medium volume of Xian 4 and Ai 2 were 80 m L /500 m L, 100 m L / 500 m L, respectively.