Study On Primary Biological Functions Of Endophytic Bacteria In Preponderant Grasses On Alpine Grassland

This study is about biological functions of primary endophytic bacteria in six species of preponderant grasses on Alpine grassland in the East Qilian Mountains. Traditional microbiological methods and molecular methods were combined to analysis the endophytic bacteria's isolation and culture conditions, and screened some endophytic bacteria with biological functions, which includes nitrogen fixing, phosphate solubilizing, cellulose decomposing, IAA producing and antagonistic effect towards pathogenic fungi.And phylogenetic study has been studied by sequencing the 16S rDNA of some endophytic bacteria with biological functions. The results of the researches showed as follows:1. Isolation of endophytic bacteria in preponderant grass on alpine grasslandSixty-four strains of endophytic bacteria were isolated from six species of preponderant grasses on Alpine grassland, and two strains were isolated from Stipa capillata Linn., thirteen strains were isolated from Elymus dahuricus Turcz., nine strains were isolated from Deschampsia cespitosa., thirty-one strains were isolated from Koeleria cristata, four strains were isolated from Kobresia capillifolia, five strains were isolated from Leymus secalinus (Georgi) Tzvel..And the result showed that the species and amount of endophytic bacteria were not same in different tissues of the same plant. Twenty-eight species of endophytic bacteria were isolated from the roots of the tested plant, thirty-six species of endophytic bacteria were isolated from the stem and leaves.2. Screening of endophytic bacteria with ability of cellulose decomposingDiameter ratio between cellulose decomposing circle and bacterial colony was measured by inoculating tested strains on flat of sodium carboxymethyl cellulose, determined cellulose decomposing ability of cellulolytic endophytic bacteria preliminarily. Seven cellulose decomposing endophytic bacteria strains were screened in this test(ZG2,PB1,QG9,XC1,LB3,PC3 and LXA10), accounted for 10.6% of total number.3. Study of endophytic bacteria with ability of IAA producingTesting germinability, germination percentage , radicle length,etc. of four species of plant seeds which soaked in dilute culture solution which made up with endophytic bacteria that has strong ability of IAA producing. The test showed that strains promoted germination of tested seeds, the average increased percentage was 5%^-15%.?Obviously,QS3 and QG3 strains have better effect than QS15 strain on promoting the weight and radicle length of four species of plant seeds(P<0.05).4. Study on phosphate dissolving endophytic bacteriaThe phosphate-solubilizing ability of endophytic bacteria was measured by qualitative and quantitive method. The study content includes several aspects below: the effect to strains dissolve phosphate rock in medium with different component; the requirements of different carbon and nitrogen sources for strain's phosphate solubilization; ability of strains dissolving phosphate rockⅣ;ability of strains dissolving different phosphate and effect to ability of phosphorus-solubilizing strains in different cultivate condition. The result showed that different carbon and nitrogen source can affect phosphate-solubilizing ability of endophytic bacteria significantly, and when glucose works as the sole carbon source, the ability of dissolving phosphate of QS4 strain was best, and with which the efficiency of dissolving phosphate was 5.832μg·mL^-1. Cane sugar was invested as the best carbon source for QC2 strain, with which the efficiency of dissolving phosphate was 5.692μg·mL^-1. In addition, the same strain has different ability to dissolve the different inorganic phosphate source. QC2 strain has better ability to dissolve Ca₃(PO₄)₂ than phosphate rockⅣ,with which the efficiency of dissolving phosphate was 5.692μg·mL^-1 and 0.279μg·mL^-1 respectively. For QC2 strain,the orthogonal design was employed in testing the optimum composition of medium composed of 10g·L^-1 cane sugar, 0.05 g·L^-1 (NH₄)₂SO₄, pH 8, 0.5 g·L^-1 NaCl.5. Determine of antifungal effect about endophytic bacteria with antagonistic functionThree strains of endophytic bacteria with antagonistic function were screened on coculture plates(LXA10,QS12 and ZHA10). These three strains can be researched further since they have broad antifungal spectrum and strong antifungal ability to eleven common plant disease, such as Fusarium oxysporum f.sp.cucumerinum,Rhizoctonia solani(pepper strain)uhn Schl,Bipolaria sorokiniana,Alternaria solani (Ell&Mart) Sorauer,Botrytis cinema,Sclerotinia sclerotiorum (egg plant strain) (Lib) De Bary,Stysanus stemonitis,Colletotrichum coccodes,Fusarium sulphureum,Fusarium spp.2 and Fusarium solani (mart.)Sacc. In addition, the average inhibition rate of ZHA10 strain was high, and this strain has the best antifungal effect to Fusarium solani (mart.)Sacc, with which the inhibition rate was 83.22%.Strain LXA10 has the best antifungal effect to Sclerotinia sclerotiorum (egg plant strain) (Lib) De Bary, with which the inhibition rate was 92.57%. Strain QS12 has the best antifungal effect to Rhizoctonia solani(pepper strain)uhn Schl, with which the inhibition rate was 72.94%.6. Screening of endophytic bacteria with ability of nitrogen fixingTwo strains of endophytic bacteria with ability of nitrogen fixing were screened in this study(QB3,FB3),and QB3 strain was identified as one strain with function of associative nitrogen fixation.7. Identification of some functional endophytic bacteria by 16SrDNASome functional endophytic bacteria strains were identified by molecular biological method 16SrDNA, 16SrDNA genes of strains QG3,QB3,LXA10 and QS14 are phy- logenetically belong to the genus of Ochrobactrum sp.,Agrobacterium tumefaciens, Bacillus subtilis and Sphingobacterium sp. with the significance 99%.According to the results of the analysis, the tested strain QG3,QB3,LXA10 and QS14 which were pre- liminarily determinated.