| Populus euphratica is the only perennial tree plant that can grow in extremely arid deserts and semi-desert environments.Populus euphratica forests in Xin Jiang account for about 90%of the total area of same forestland in China.In order to clarify the bacterial diversity and community dynamics of different parts of Populus euphratica in different distribution areas of northern Xinjiang,we isolated the endophytic bacteria and sap bacteria from Populus euphratica,and further screened out the bacteria which can promote the growth of plants and improve the crop production from these isolated microorganisms.In this study,Populus euphratica forest from the Ebinur Lake Wetland Nature Reserve and Mori county were taken as the sampling target,respectively.The isolation and purification of culturable bacteria,from the storage liquids in Populus euphratic、s stem and Populus euphratic、s sap,was performed by tissue dilution culture method and five culture media(MA,TSB,BPA,PEA,R2A).Based on the 16S rRNA gene sequence analysis and phylogenetic analysis,the diversity and population structure of the endophytes and sap、s bacteria of Populus euphratica have been demonstrated.Two potential new species isolated in this work,were determined it’s exact taxonomic position by uzing polyphasic approach.Evaluation and analyzing on the plant growth-promoting function of the bacterial isolates,was performed through the Petri dish cultivation approach combined with the germination and potting methods.Finally,three bacteria with outstanding functions of plant growth-promoting,also have been discussed.The obtained main results are listed below:1.In this study,a total of 434 bacterial isolates have been obtained;in which includes 258 strains of endophytic and 176 trains of sap、s bacteria.Among them 113 strains of endophytic and 109 trains of sap、s bacteria isolated from original Populus euphratica forest of Mori county;whereas 145 strains of endophytic and 67 trains of sap、s bacteria were isolated from Populus euphratica at Ebinur Lake Wetland Nature Reserve.2.Phylogenetic analysis of the 16S rRNA gene sequence revealed that 23 strains among the 113 endophytic bacteria isolated from storage liquid of Populus euphratica at Mori original Populus euphratica forest are belonged to Actinobacteria＿c,accounting for 20.4%;49 strains are belonged to Gammaproteobacteria,accounting for 43.4%;3 strains are belonged to Alphaproteobacteria,accounting for 2.6%;38 strains are belonged to Firmicutes Bacilli,accounting for 33.6%.All the 113 strains could be classified into 3 phyla,4 classes,9 orders,17 families and 19 genera.In which the Bacillus was the most numerical genus,and the Brenneria Salicis was a most dominant species,represented by 22 bacterial isolates.3.The 109 strains isolated from sap of Populus euphratica at Mori original Populus euphratica forest were classified into 3 phyla(Gammaproteobacteria,Firmicutes,Actinobacteria),6 orders,8 families and 14 genera.Of these,74 isolates belonged to Firmicutes Bacilli,accounting for 67.8%,and 6 belonged to Gammaproteobacteria,accounting for 5.5%.The genus with the highest frequency of isolation was Bacillus,a total of 45 strains belonging to Bacillus;followed by the genus Nesterenkonia with 23 strains.The dominant species were the Bacillus zhangzhouensis and Nesterenkonia populi.4.The 145 endophytes isolated from storage liquid of Populus euphratica at Ebinur Lake Wetland Nature Reserve were classified as Gammaproteobacteria(40%),Firmicutes Bacilli(48%),Actinobacteria＿c(9.7%),Alphaproteobacteria(1.4%),Betaproteobacteria(accounting for 0.7%)3 phyla,5 classes,8 orders,15 families and 19 genera.The predominant genera are Bacillus with 44 strains,followed by Halomonas with 41 strains.The dominant species are Bacillus zhangzhouensis and Halomonas songnenensis.5.The 67 strains isolated from sap of Populus euphratica at Ebinur Lake Wetland Nature Reserve were classified into four groups:Gammaproteobacteria(19.4%),Firmicutes Bacilli(59%),Actinobacteria＿c(16.4%),and Alphaproteobacteria(4.5%).There are 3 phyla,4 classes,6 orders,8 families,and 10 genera.The dominant genus is Bacillus,accounting for 53%;followed dominant species is Bacillus zhangzhouensis,accounting for 17.9%.6.The isolation effect of the medium:69 strains were isolated from the TSB medium,144 strains were isolated from the MA medium,52 strains were isolated from the R2A medium,71 strains were isolated from the PEA medium,and 98 strains were isolated from the BPA medium;The bacteria isolated from MA medium were most numerical,and the 144 isolates belonged to 25 genera and 54 species.However,the isolation effect data of used medium showed that the R2A have a highest dominance index of 0.576,by using it 30 species were obtained from 52 isolates.The followed dominance index was PEA culture medium supplemented with Populus euphratica stem extract with a value of 0.408.7.The bacterium MD2 isolated from sap of Populus euphratica in the Ebinur Lake Wetland Nature Reserve,as a potential novel species,it’s exact taxonomic position was determined by using polyphasic approach and proposed that the strain MD2~T is a new species within Nesteenkonia,and named it as Nesterenkonia Ebinurense,(MD2T=KCTC52999T=MCCC1K03343T).8.The bacteria PA5,TA6 and TA22 isolated from the Populus euphratica、s sap from original Populus euphratica forest of Mori county,the 3 strains as potential novel species,it’s exact taxonomic position was determined by using polyphasic approach and demonstrated that the 3 bacterial isolates were belonged to the same novel species of the genus Halomonas.For that the name Halomonas populi,(TA22T=MCCC1K03439T=KCTC62169T)have been suggested.9.Using the plate-to-culture method and screening with a specific medium,158 isolates of functional bacteria which can potentially promote the plant growth,were obtained.From these,61 strains had the ability to dissolve phosphorus,80 strains had the ability to dissolve potassium,32 strains had the ability to fix nitrogen,and 151 strains had the ability to use iron and ammonia salts.The germination and potting studies on maize seeds showed that the germination rate,plant height,and seedling dry weight of maize seedlings under the activation of bacterial strains BB33-1,TC10 and RC6,were all significantly greater than those of the control group.10.(1)Strain BB33-1 has the ability of phosphate-removing and nitrogen-fixing,and it’s16Sr DNA sequence similarity to Bacillus zhangzhouensis DW5-4T is 99.93%.Gram staining is positive,colonies are yellow,the growth temperature range is 8-45℃,the growth salt concentration range is 0-12%,the growth pH range is 5-11.(2)The strain TC10 has the ability of dissolving phosphorus and using ferric ammonium salt.The 16Sr DNA similarity to Halomonas olivaria C17＿DQ645593T is 98.39%.Gram stain is negative,flagellate,dark yellow,opaque single Colonies.The growth temperature range is 4℃-50℃,the growth salt concentration range is 0-25%,and the growth pH range is 5-10.(3)The strain RC6 has potassium-releasing and nitrogen-fixing abilities.The 16Sr DNA sequence similarity to the Bacillus siamensis KCTC 13613T is 99.93%,Gram-positive,flagellum,translucent,mucus-loaded,Milky white colonies.Its growth temperature range is 4℃-55℃,growth pH range is4.5-9,and growth salt concentration range is 0-14%. |
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