| Paris polypholla(also name Chong Lou)is a rare medicinal plant included in the Chinese Pharmacopoeia and has a large market demand.Artificially cultivated of P.polypholla has become one main source of its market.However,there are several problems,such as reduction of the disease resistance and the Polyphyllin contents,in the artificially cultivated P.polypholla.In this study,some of five-year-old,artificially cultivated P.polypholla were collected from one of the representative planting areas of Hubei,and used as objects for subsequently research.The diversity and composition characteristics of total endophytic bacteria and cultivable endophytic bacteria in the roots,stems and leaves of artificially cultivated P.polypholla were investigated through high-throughput sequencing technology and traditional microbial culture methods,respectively.The Polyphyllin contents of the roots,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.Some strains of dominant bacterial communities significantly related to the Polyphyllin contents were further selected to co-cultivate with wheat seedlings for screening of growth-promoting bacteria,which laid a foundation for improvement of the artificial cultivation technology of P.polypholla using endophytic bacteria.The main results of this study were as follows:1)The total endophytic bacteria in the roots,stems and leaves of P.polypholla planted in Hubei were investigated for the first time by using the Illumina Miseq high-throughput sequencing technology.At a similarity level of 97%,the obtained334878 valid sequences were integrated into 1129 OTUs.Total endophytic bacteria were classified into 20 phyla,39 classes,96 orders,181 families and 340 genera.The most dominant genus of the total endophytic bacteria in the roots,stems and leaves was Cupriavidus(20.57%),Cupriavidus(29.93%)and Rhodococcus(25.63%),respectively.The distribution and composition of total endophytic bacteria were different in the three tissues.The richness,diversity and uniformity of total endophytic bacteria in the roots were higher than those in the stems and leaves,and the composition of total endophytic bacteria in roots was more similar to that in stems than that in leaves.The analysis of the species interaction network showed that 250pairs(65.96%)of total endophytic bacteria were positively correlated,129 pairs(34.03%)of them were negatively correlated,and the cooperative relationship was in the majority.2)A total of 1090 cultivable endophytic bacteria were isolated from 0.1 g each of the roots,stems and leaves of P.polypholla planted in Hubei using two kinds of bacterial media.Among them,the 16 S r DNA sequences of 927 cultivable endophytic bacteria were amplified and analyzed.These cultivable endophytic bacteria were classified into 4 phyla,7 classes,17 orders,26 families and 50 genera.The most dominant genus of cultivable endophytic bacteria in roots,stems and leaves was Bacillus(44.49%),Pseudomonas(21.22%)and Acinelobacter(47.17%),respectively.The distribution and composition of cultivable endophytic bacteria were different in the three tissues.The number of cultivable endophytic bacteria in the roots was the largest(589 strains),followed by stems(331 strains)and leaves(170 strains).The similarity coefficients about Sorenson from high to low were: roots/stems(42.11%)>stems/ leaves(22.64%)> roots/leaves(16.67%),and the composition similarity of the cultivable endophytic bacteria in the roots and stems was highest.The diversity and richness of cultivable endophytic bacteria in roots and stems were higher than those in leaves.The dominant genera of cultivable endophytic bacteria and total endophytic bacteria in the three tissues were inconsistent,but tissue difference of cultivable endophytic bacteria and total endophytic bacteria in the three tissues was basically the same.3)Polyphyllins VI and VII were found in the three tissues by detecting the contents of Polyphyllins I,II,VI and VII in the roots,stems and leaves,respectively.The contents of Polyphyllins I(36.22 mg/g),II(28.38 mg/g)and VI(0.99 mg/g)were the most abundant in the roots,and of the Polyphyllin VII(6.51 mg/g)content was highest in the leaves.By analyzing the correlation between the total endophytic bacteria and cultivable endophytic bacteria with the Polyphyllin contents in the three tissues,it was found that the OTUs numbers and diversity index of the total endophytic bacteria were significantly positively correlated to the Polyphyllin contents,and the diversity and richness of cultivable endophytic bacteria were significantly positively correlated to the Polyphyllin contents.A total of 23 genera of the total endophytic bacteria were significantly positively correlated to the contents of four Polyphyllins,and 20 genera of the cultivable endophytic bacteria were significantly positively correlated to the contents of four Polyphyllins.Among the total endophytic bacteria that were significantly positively correlated to the Polyphyllin contents,a total of 19 genera were not isolated in the study of cultivable endophytic bacteria.4)A total of 258 cultivable endophytic bacteria of 20 genera that were significantly positively correlated to the content of four Polyphyllins,were selected to co-culture with wheat seedlings,respectively.As a result,56 of cultivable endophytic bacteria with growth-promoting effect were obtained,and were classified into 14 genera.Furthermore,the HBR21038(Agrobacterium)strain with the most obvious growth promoting effect was verified.The HBR21038 strain had a very significant promotion effect on the root length of wheat seedlings,which was increased by 100%,126.09%,96.30% and 46.51% compared to the control on co-culturation days of 5,10,15 and 20,respectively.The HBR21038 strain also had a significant promotion effect on the plant height of wheat seedlings,which was increased by 31.58%,37.07%,68.70% and 10.45% compared to the control on co-culturation days of 5,10,15 and20,respectively. |
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