Identification And Diversity Function Study Of Endophytic Bacterias From Polygonum Viviparum In The Eastern Qilian Mountains

Endophytic bacteria reside in plant hosts with out causing obvious diseases symptom. Polygonum viviparum ,the dominant species of polygonum grassland in the eastern Qilian mountains in Gansu province were selected for systematic studies on the endophytic bacteria of their different tissues. Traditional microbiological methods and molecular methods were combined to analysis the endophytic bacterias.The bacterias had the functions of nitrogen fixation, phosphorus-solubilizing , IAA secretion and so on were isolatied and selected,to identified the dominant multi-function bacteria's use 16SrDNA techniques and there ability of colonizations.The results of the researches showed as follows:1. Isolation of endophytic bacteria from Polygonum viviparumThe optimal condition were washed each step with the sterilized water three to four times after different tissues of plants immersed in 0.1% SDS for 15 min, 3% NaClO for 3 min, 0.1% mercuric chloride for 10 min, 75% alcohol for 1 ~ 2 min respectively.The endophytic bacteria were isolated after aseptic grinding them and diluted to 10-3 and using TSA culture medium cultured for 5 to 7 days in 28℃. We had obtained 35 strains of endophytic bacteria with different size ,shape and color in different tissues of Polygonum viviparum..2. Screening of multi-function bacteriasAntagonism of 35 endophytic bacterias isolated from different tissues of healthy Polygonum viviparum. were evaluated from the ablity of nitrogen fixation, phosphorus-solubilizing , IAA secretion and decomposition of cellulose. 6 of the tested strains had the ablity of nitrogen fixation, accounted for 17.14%, the strain Z10 had the highest ability of nitrogen fixation for it can be seen nicer growing at the medium with no nitrogen source at 3 days; 6 of the tested strains had the ablity of phosphorus-solubilizing, accounted for 17.14%, the strain Z10 was the highest(1111.15mg/L) and the others were between 150.42 mg/L -1091.88 mg/L. 7 strains had the ablity of IAA secretion, accounted for 20%, for the medium with no Try the strain Z1 was the highest(17.01μg/mL) and Z12 was the smallest(4.26μg/mL), the medium with Try the strain Z5 was the highest(70.24μg/mL) and Z7 was the smallest (0.87μg/mL). 10 strains had the ability of cellulose decomposition, accounted for 28.27%. The strain Z4 had the four ability tested, the strains, the strain Z10 had the abilities of nitrogen fixation, phosphorus-solubilizing and cellulose decomposition. the strains Z1,Z5,Z7 and Z12 had the abilities of nitrogen fixation, IAA secretion and cellulose decomposition, the strains Z16 and ZA6 had the abilities of phosphorus-solubilizing, IAA secretion and cellulose decomposition, the strains Z14 and ZC3 had the abilities of phosphorus-solubilizing and cellulose decomposition, That the endophytic bacterias with a variety of physiological functions were selected, provided a basis for further development.3. 16S rDNA sequence analysis of antagonistic bacteriaThe 16S rDNA sequence were 1456bp and 1462bp for Z1 and Z5,1443bp for Z10,1488 bp for Z12,1476 bp for Z14,1458 bp for Z16,1454 bp for ZC3,1472 bp forZ4 and Z7 respectively.The 16S rDNA sequence of the strains Z1,Z5 and Z16 were found to be homogeneous at the rate of 99% to the members of Bacillus subtilis(Accession number were AB201120 and AB440269 in GenBank), and Z4 were found to be homogeneous at the rate of 99% to the members of Pseudomonas fluorescens(Accession number was AJ278813 in GenBank),Z7 and Z12 16S rDNA were found to be homogeneous at the rate of 99% to the members of Bacillus cereus(Accession number were AJ969106 and AF176322 in GenBank), Z10 were found to be homogeneous at the rate of 99% to the members of Serratia plymuthica(Accession number was EF064206 in GenBank), Z14 were found to be homogeneous at the rate of 99% to the members of Bacillus pumilus(Accession number was EU855220 in GenBank), ZC3 were found to be homogeneous at the rate of 99% to the members of Stenotrophomonas maltophilia(Accession number was X95924 in GenBank), which all conformed with the results of gram stain.Therefore,the position of 9 strains in bacterial phylogeny were established. The strains Z1,Z5 and Z16 were identified as Bacillus subtilis.The strain Z4 was identified as Pseudomonas fluorescens. The strains Z7 and Z12 were identified as Bacillus cereus. The strain Z10 was identified as Serratia plymuthica. The strain Z14 was identified as Bacillus pumilus . The strain ZC3 was identified as Stenotrophomonas maltophilia.4 colonisation research of endophytic bacteriaThrough natural resistance screening to the 50 strains, selected 17 strains and determined which two kinds of antibiotics between streptomycin, kanamycin, ampicillin and rifampicin antibiotics were used to mark up the 17 endophytic bacterias selected. There were only four strains R1,X4, Z10, ZB2 marked to the antibiotic concentration 70μg/mL after marked. To the plant source, marked endophytic bacteria R1, X4 could be isolated from anaphalis lacteal and kobresia capillifolia respectively. the technology of DGGE were used to detected the mutants of R1 and X4 to make sure that R1 and X4 couled be coloniazted to the plant source . To the host range ,R1, X4, Z10, ZB2 were colonizated to Avena sativa, Poa, Lolium, festuca arundinacea, trifolium repens,there were no endophytic bacterias isloted from these plants at the antibiotic concentration of 70μg/mL. the result showed that these four strains had a narrow host range.