Isolation And Identification Of Cucumber Endophytic Antagonistic Bacteria Y-10 And Research On Biocontrol And Growth Promotion Characteristics

Sanya South breeding base in Hainan Province is one of the important breeding bases in China.According to the investigation,the cucumber leaf spot caused by Pseudomonas Cichorium has caused serious damage to the production and quality of Cucumber in the base.In order to better control the cucumber leaf spot,the healthy cucumber plants were collected from Hainan,Hunan and Yunnan,and 189 strains were isolated from its root,stem,leaf and fruit.And then,using the plate confrontation method,the Y-10 strain was screened to have a good antagonistic effect against the cucumber bacterial leaf spot pathogen（P.Cichorium）,with an antagonistic radius of 16mm.there was no symptom after that the Y-10 strain was inoculated into tobacco and cucumis leaves by injection,so this Y-10 strain is determined to be not a pathogenic bacteria.The antimicrobial spectrum of the Y-10strain was explored,and the results showed that the Y-10 strain have effects on cucumis diseases like cucumber root rot fungus（Fusarium solani）,cucumber wilt fungus（Fusarium oxysporum f.sp.cucumerinum,FOC）,and wheat head blight（Fusarium solani f.sp.cucurbitae）.Fusarium graminearum,Neoscytalidium dimidiatum,Cryphonectria japonica,Alternaria solani,Corynespora cassiicola,Colletotrichum gloeosporioides（Penz.）Sac.),banana Fusarium wilt（Fusarium oxysporum f.sp.cubense,FOC）and other crops have antagonistic effects,and the inhibition radius to F.oxysporum,F.solani and Corynespora leaf spot reached 0.87cm,0.65 cm and 0.73 cm independently.It was carried out to determine the taxonomic status of Y-10 which contains morphological identification,physiological and biochemical charateristics determination,16S r DNA and rpo A sequence joint anlysis.The Gram Staining were positive and KOH rapid identification tests of the Y-10 strain were negative,and it could see the blue-green oval spores obviously through spore staining.Citrate utilization,contact enzyme reaction,V.P.Are the tests and the methyl red test,starch hydrolysis,gelatin liquefaction and other physical and chemical characteristics of Y-10 is positive.It Can’t produce indole,and can make the use of 8 carbon sources such as sucrose,raffinose,maltose,ammonium nitrate,ammonium chloride,peptone,and 4 kinds of nitrogen sources including urea.The 16S r DNA and rpo A sequence analysis showed that the Y-10 strain had the highest similarity with Bacillus subtilis,and the Analysis of joint establishment of two genes showed that the Y-10 strain and B.subtilis clustered in the same branch,and the homology was 100%.In summary,the endophytic antagonistic bacterium Y-10 was finally identified as endophytic B.subtilis.Rifampicin induced marker Y-10 was used to observe its colonization in cucumber plants.The results showed that the three treatment methods,including seed soaking method,root irrigation method and foliar spraying method,can all inoculate the Y-10 strain into cucumber plants.Among this three treatment,the seed soaking inoculation is better than others,and 3 days after inoculation,the colonization number of this treatment was reached to the largest,which concentration is about5.90×10⁶CFU·g^-1,and the number of root irrigation inoculation remain stable at 5days after inoculation,and the colonization in the root was about 7.9×10⁶CFU·g^-1,The amount of colonization in leaves is about 6.4×10⁵CFU·g^-1.under the treatment of Foliar spraying,the colonization of endophytes in the leaves and roots of cucumber seedlings showed a continuous downward trend,and reached the maximum colonization at 1 day after inoculation,and the colonization in leaves was about4.39×10⁵CFU·g^-1,while the root colonization amount is about 0.13×10⁵CFU·g^-1.The results of disease control effect of detached leaves and potted plants showed that Y-10 strain had good control effect on cucumber bacterial leaf spot.On the potting experiment,its effect of Y-10 on cucumber leaf spot reached 64.30%.The activities of defense enzymes in cucumber plants were determined by ultraviolet absorption method.The activities of catalase（CAT）and peroxidase（POD）in cucumber plants were compared with those in the control The activities of defense related enzymes such as superoxide dismutase（POD）,polyphenol oxidase（PPO）,phenylalanine ammonia lyase（PAL）were changed.The results showed that when Y-10 strain was inoculated only and Y-10 strain was inoculated only,pal,pod The activities of cat,SOD,pod,PPO and pal in cucumber leaves were increased by 2.6,1.9,1.3,13.77 and 1.3 times respectively when Y-10 strain was inoculated,while the activities of cat,SOD,pod,PPO and pal in cucumber leaves were increased by 3.8,2.9,1.9,16.58 and 1.2 times respectively when Y-10 strain was inoculated The activities of PPO and pod in Y-10 and pathogen treatments were increased by 14.5times and 19.0 times,respectively.Endophytic antagonistic bacteria Y-10 can improve the changes of defense related enzyme activities of cucumber plants,and improve the disease resistance of cucumber plants.The results of Y-10 strain on cucumber growth promotion showed that the seed germination of Y-10 solution was inhibited,and the seed germination rate of Y-10solution was increased by 6.01%compared with the control group.Compared with the control,the seedling growth was significantly promoted by soaking seeds and irrigating roots with Y-10.Among them,the concentration of seed soaking was 4×10⁷cfu·ml^-1,the root length,shoot height,root fresh weight,stem fresh weight,root dry weight and stem dry weight of cucumber seedlings increased by 40.71%,7.48%,33.59%,35.21%,2.33%and 58.18%,respectively,compared with CK group.The best growth promoting effect was obtained when the dilution gradient of bacterial solution was 100,that is,the bacterial solution concentration was 4×10⁹cfu·ml^-1,cucumber seedling root length increased by 11.49%,shoot height increased by23.57%,root fresh weight increased by 19.57%,stem fresh weight increased by77.25%,root dry weight increased by 37.84%,stem dry weight increased by 47.15%,the best growth promotion effect.The results of phosphorus and potassium solubilization test,nitrogen fixation test,IAA and ferritin production test showed that Y-10 strain had the functions of nitrogen fixation and ferritin production.