| Paris polypholla is a traditional Chinese medicine plant,however its wild resource is scarce.Aritificially cultivated P.polypholla has become the main source of its market.However,there is a problem that quality degradation frequently occurs in artificially cultivated P.polypholla.Endophytes,one of the key factors affecting the quality of medicine plants,have become a new direction and technology to ensure and improve the quality of traditional medicine plants.In this study,some of five-year-old artificially cultivated P.polypholla were collected from one of the representative planting area of Sichuan province,and the diversity,richness,composition and distribution characteristics of total endophytic bacteria and cultivable endophytic bacteria in their rhizomes,stems and leaves were investigated by the high-throughput sequencing technology and traditional microbial culture method,respectively.The contents of Polyphyllin(PP)in the rhizomes,stems,and leaves were detected by HPLC method,and the correlations between the total endophytic bacteria and cultivable endophytic bacteria with the Polyphyllin contents were analyzed,respectively.Subsequently,growth-promoting bacterial strains were screend through co-culturation between wheat seedlings and these cultivable endophytic bacteria with positive correlation to PP contenes.Further,the co-culturation experients between Astragalus membranaceus seedlings and two growth-promoting bacteria were performed,respectively,and the changes in the growth and physiological indexes of co-cultured A.membranaceus seedlings at different time points were analyzed to preliminarily explore the growth-promoting mechanism of the two endophytic bacteria on A.membranaceus seedlings.These results laid a foundation for further research on the improvement of the quality of aritificially cultivated A.membranaceus and P.polypholla using endophytic bacteria.The main results of this study were as follows:1)Total endophytic bacteria in the rhizomes,stems and leaves of five-year-old P.polypholla cultivated in one representative plantation of Sichuan privence were investigated for the first time by using the Illumina Miseq high-throughput sequencing technology.A total of 346730 valid sequences were obtained and integrated into 1844 OTUs.Based on OTU classification,total endophytic bacteria were classified into 21 phyla,58 classes,143 orders,245 families and 502 genera.The dominant genus of the total endophytic bacteria in the rhizomes,stems and leaves was Sphingobium(8.29%),Cupriavidus(14.03%)and Methylobacterium(28.14%),respectively.The richness,diversity and uniformity of total endophytic bacteria in the rhizomes were higher than those in the stems and leaves,and the composition of total endophytic bacteria in the rhizomes and stems was more similar.The function prediction of ‘metabolism’ showed that the highest relative abundance of ‘amino acid metabolism’ in the rhizomes,while the‘energy metabolism’ in the stems and leaves.The content of PPI,II,VI and VII in the rhizomes,stems and leaves were detected,and four kinds of PPs were found in all the three tissues.The content of PPI(1.496 mg/g),II(1.122 mg/g)and VII(0.785 mg/g)were the highest in the rhizomes,and that of PPVI(0.360 mg/g)was the highest in the leaves.Through correlation analysis,it was found that 33 genera of total endophytic bacteria were significantly positively correlated with the PP contents.Among them,23 genera had the highest relative abundance in the rhizomes.The ‘synergy’ relationship(53.21%)among total endophytes was more than the ‘competition’ relationship(46.79%).2)A total of 784 cultivable endophytic bacteria were isolated from 0.1 g each of the rhizomes,stems and leaves of P.polypholla by the traditional microbial culture method.The 16 S r DNA sequences of 748 cultivable endophytic bacteria were amplified and analyzed.These cultivable endophytic bacteria were classified into 4 phyla,7 classes,16 orders,26 families and 43 genera.The dominant genus of cultivable endophytic bacteria in the rhizomes,stems and leaves was Acinetobacter(40.59%),Pseudomonas(37.08%),Bacillus(41.18%),respectively.The diversity and richness of cultivable endophytic bacteria in the rhizomes were higher than those in the stems and leaves.A total of 20 genera of culturable endophytic bacteria were found to have a significantly positive correlation to the PP contents.3)A total of 250 cultivable endophytic bacteria from those genera having significantly correlation to the PP contents were selected to co-culture with wheat seedlings,respectively.As a result,7 of cultivable endophytic bacteria with growthpromoting effect were obtained,and were classified into 4 genera.Among them,SCR21026 had the most obvious growth-promoting effect on wheat seedlings by rescreening verification,and the growth rate of plant height and root length of the cocultured wheat seedlings(15th day)reached 52% and 36% when compared with the control,respectively.Subsequently,SCR21026 and HBR31044(obtained in our previous experiments)were selected to co-culture with Astragalus membranaceus seedlings,respectively.The two endophytic bacteria exhibited obviously growth-promoting effects on A.membranaceus seedlings,and the growth rate of the plant height and root length of A.membranaceus seedlings co-cultured with SCR21026 and HBR31044 were 13.46% and11.59%,53% and 49% when compared to the control,respectively(5th day).4)SCR21026 strain could induce the increase of the contents of soluble sugar,amylum and malondialdehyde(MDA)as well as the activity of peroxidase(POD),meanwhile the decrease of gibberellins(GAs)and soluble protein contents,of A.membranaceus seedlings.The contents of GAs as well as the POD activity of A.membranaceus seedlings co-cultured with SCR21026 on the 5th day had a significant deference(P<0.01)with the control,which were 0.66 and 3.20 folds those of the control,respectively;the contents of soluble sugar,MDA and soluble protein on the 10 th day had a significant deference(P<0.01 or P<0.05)with the control,which were 2.52,1.46 and 0.86 folds those of the control,respectively;meanwhile,the amylum content on the 15 th day had a significant deference(P<0.05)with the control,which was 2.14 folds that of the control.HBR31044 strain could induce the increase of the contents of GAs,Zeatin(ZT),amylum and MDA as well as the activity of ethylene biosynthesis enzymes and POD,meanwhile the decrease of the contents of abscisic acid(ABA)and soluble sugar,of A.membranaceus seedlings.The contents of GAs,ZT,amylum and MDA as well as the ethylene biosynthesis enzyme activity of A.membranaceus seedlings co-cultured with HBR31044 on the 10 th day had a significant deference(P<0.01 or P<0.05)with the control,which were 1.13,1.42,1.80,1.56 and 1.30 folds those of the control,respectively;the POD activity and soluble sugar content on the 5th day had a significant deference(P<0.01 or P<0.05)with the control,which were 2.73 and 0.74 folds those of the control,respectively;meanwhile,the ABA content on the 15 th day had a significant deference(P<0.05)with the control,which was 0.85 folds that of the control.These results preliminery uncover the physiologically growth-promoting mechanism of SCR21026 and HBR31044 on A.membranaceus seedlings. |
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