Isolation And Identification Of Endophytic Bacteria Of Alpine Pastures In The Eastern Qilian Mountains And The Studying Of Its Biological Functions

Endophytic bacteria reside in plant hosts without causing obvious diseases symptom. It has been paid attention by researchers in many disciplines for their biological effects, such as promoting plant growth , improving resistance to diseases , insects penetration and the phosphate solubilizing power.Five alpine pastures reside in three different sites of alpine 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 bacteria's isolation and culture conditions, antiagonistic bacteria's screening and its interactions and biological characteristics, antifungal substances and the 16SrDNA identification. The results of the researches showed as follows:1. Isolation and culture conditions of endophytic bacteriaThe Anaphalis Lactea Maxin's root and leaf were selected to screen the conditions of endophytic bacteria,such as disinfectant,diluted times and medium.The 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 315 strains of endophytic bacteria with different size ,shape and color in different tissues of five alpine pastures.2. Screening of antiagonistic bacteriaAntagonism of 315 endophytic bacteria isolated from different tissues of five healthy alpine pastures were evaluated against ten pathogenic fungi,through dual culture in Petri dish,such as Rhizoctonia solani(pepper strain),Sclerotinia sclerotiorum (rape strain),Rhizoctonia solani (cotton strain),Sclerotinia sclerotiorum (egg plant strain),Botrytis cinerea,Fusarium oxysporum (schl) f.sp cucumerinum,Exserohilum turcicum,Bipolaria maydis,Bipolaria.sorokiniana and Fusarium oxysporum. 69 of the tested strains had antagonistic effect for more than one tested fungi,accounted for 21.9%; among them 47 of the tested strains had antagonistic effect for more than two tested fungi,accounted for 14.9% and 17 strains were showed broad antagonistic spectra.to ten tested fungi,just accounted for 5.4%.3.Iteractions between antagonistic bacteria The results of interactions showed that multiple interactions were involved in the combinations of antagonistic bacteria. Whether the strain X4 was the test strain or tested strain, it was compatible with the strain Z10,Q3 and R1.It was also compatible and incompatible with the strain ZHC10 as tested strain and test strain respectively. When strain ZHC10 as a test strain, it was compatible with the strains :Z10,Q3,R1,X4 and ZB2,but incompatible with the strain Q3 and R1 as a tested strain.Different combination of antagonistic bacteria had different suppresive effects against Sclerotihia sclerotiorum and Bipolaria.sorokiniana. The disease suppression showed that different combination of antagonistic bacteria increased or decreased ,which interaction was compatible and incompatible. X4,Q3,R1 and Z10 were compatible each other and the inhibition rate of combination was higher than the single strain to Bipolaiar. sorokiniana.The combination of R1 with Z10 had enhanced the inhibition rate against Bipolaria.sorokiniana compared to the single strain,which increased to 8.3%.The inhibition rate of combination X4 and Q3 was also increased to 6.5% Q3 and Z10 were incompatible, inhibition rate of combinations was increased 9.3% for Sclerotihia sclerotiorum and 7.3% for Bipolaria.sorokiniana. The inhibition rate of combinations ZHC10 and R1was decreased to 18.1% for Sclerotihia sclerotiorum .4. Biological effects of antagonistic bacteriaFor ZHC10 and Z10, the time when the OD600 reach the peak is 72h and 60h respectively.The strains Q3,R1,X4 and Z10, the time when the OD600 reach the peak is 48h.. The results showed that the optimal pH for Z10,Q3 and R1, ZHC10,ZB2 and X4 were 4～6 and 6～8 respectively The range of NaCl concentration for 6 strains was 0～11%. ZHC10 and ZB2 grow slowly in 9% and 7% respectively.6 strains had ability to produced IAA,the strain Z10 was the highest(34.80μg/mL) and R1 was the smallest(0.58μg/mL).The strains Q3,R1,X4 and Z10 have the phosphate solubilizing ability, but the strains ZHC10 and ZB2 were contrary to them.The strain X4 has the highest ability to solubilize inorganic phosphate,the D/d value was 1.75. The strain Q3 was smallest and the D/d value was 1.48.The D/d value showed no significant difference at P<0.01.To organic phosphate,the biggest D/d value was Q3 and the smallest was Z10.The D/d value showed significant difference at P<0.01 for Q3,X4,R1 and Z10.5. Preliminary analysis of antifungal subtancesThree strains(ZB2,X4,ZHC10) were showed stable suppression to different pathogens after cultured in 15 times. The antifungal substances were unstable and the suppressive eff- ects reduced about 50% after 60℃to 80℃tretement respectively. The suppressive effects were stable when the pH was 6～8. The inhibition effect on the growth of Sclerotihia sclerotiorum and Bipolaria.sorokiniana was studied.The result showed that antifungal substances of 3 strains had strong antagonistic activity against the hyphae growth.The mycelia showed distortion, protoplasm concentration, swelling with the shape of sphericity or ellipse, and some vesicles were found.Its antifungal substances were identified as protein.6. 16S rDNA sequence analysis of antagonistic bacteriaThe 16S rDNA sequence were 1465bp and 1471bp for Q3 and R1,1456bp for ZB2,X4 and ZHC10 respectively.The 16S rDNA sequence of the strains Q3 and R1 were found to be homogeneous at the rate of 99% to the members of Serratia plymthica(Accession number were AJ233433 and EU266583 in GenBank), and X4 were found to be homogeneous at the rate of 99% to the members of Serratia sp.(Accession number was DQ988935 in GenBank),ZHC10 and ZB2 16S rDNA were found to be homogeneous at the rate of 99% to the members of Bacillus sp.(Accession number were EU082292 and EF61- 2722 in GenBank), which all conformed with the results of gram stain.Therefore,the posit- ion of 5 strains in bacterial phylogeny were established. The strain X4 was identified as Serratia sp.The strains Q3 and R1 were identified as Serratia plymuthica. The strains ZHC10 and ZB2 were identified as Bacillus spp.