Study On The Isolation,identification And Genetic Mechanism Of Drug Resistance Of Stenotrophomonas Maltophilia In Strawberry Leaves

Fragaria ananassa Duchesne have abundant nutrition,important medicinal and medical value.With strawberries planted around the world,a variety of disease that infected strawberries also appear.Common diseases just like fusariu wilt,root rot,anthracnose,gray mold caused great losses to strawberry cultivation and strawberry fruit production.In this study,an unknown pathogen(SPBWSM001)was isolated from strawberry leaves by the method of tissue separation.According to the rule of Koch,the pathogen SPBWSM001 was re-inoculated into the leaf margin,leaf surface,stem and root of strawberry to verify the pathogenicity of SPBWSM001.The results showed that SPBWSM001 was a pathogen of strawberry leaves,and it could infect plants through the waterstoma at leaf margin,the wound at leaf margin and leaf surface,and the main way of infection is the wound at leaf margin and leaf surface.Then the pathogenic temperature and humidity conditions of SPBWSM001 that infecting leaves were verified,the results showed that the strawberry leaves had the highest mobidity at the condition of 30? and 95% RH.In this paper,we observed the cell morphology of SPBWSM001,and measuring the physiological and biochemical reactions,such as carbon source utilization,anaerobic,sugar alcohol fermentation acid,salt tolerance,hydrolyzed gelatin,hydrolyzed starch,growth p H,antibiotic sensitivity ect,and analysising the phylogenetic of 16 S r RNA and rpo B gene sequences.The pathogen SPBWSM001 was identified as Stenotrophomonas maltophilia.In this experiment,the method of bacterial liquid spraying was used to determine the pathogenicity of SPBWSM001 to cucumber,thale cress and tomato plants.It was found that SPBWSM001 had no pathogenicity to plants except strawberry plants.The tomatoes were treated with different concentrations of SPBWSM001,and the growth of tomato and the dynamic changes of microbial community in different treatment groups were observed.The results showed that the undiluted and 10 times diluted SPBWSM001 bacterial solution had significant effect of promoting growth on the plant height of tomato,which was significantly higher than the control group;while the treatment of different concentration had no significant effect on root length and stem circumference.The number of microbes in tomato roots was positively correlated with the concentration of SPBWSM001 bacteria,while the microbial species increased with the presence of SPBWSM001,it can be inferred that this phenomenon is related to the colonization of SPBWSM001 in roots and soils.A large-scale insertion mutant of Stenotrophomonas maltophilia was established by the method of conjugation transfer,the kanamycin sulfate mutant was screened according to thecharacteristics that SPBWSM001 has high resistance.Then the nested PCR was used to identify the resistance gene of kanamycin sulfate of SPBWSM001,the results showed that the mutant gene was 23 S r RNA.In this paper,the effect of SPBWSM001 growth stage and shock voltage condition on the conversion efficiency were studied,the results showed that the best condition for the transformation of Stenotrophomonas maltophilia SPBWSM001 was OD600 value 0.5 and voltage 20KV/cm,and the transformation efficiency reached 406CFU/?l.