| Paris polyphylla var.yunnanensis is a traditional medicinal plant and its wild resource is scarce.Artificial cultivation of P.polyphylla var.yunnanensis is an effective way to solve the shortage of its medicine resources,however the degradation of medicinal quality in many artificial planting products is a general problem.Using endophytes to safeguard and improve the quality of medicine plants is one of current research hotspots.In this study,some of 5-year-old and artificially planted P.polyphylla var.yunnanensis were collected,and the diversity and flora characteristics of total endophytic fungi and cultivable endophytic fungi of their rhizomes,stems and leaves were investigated using Illumina Miseq sequencing technology and microbial culture technology,respectively.The correlation between the endophytic fungi(genus)and polyphyllin contents were analyzed.Subsequently,growth-promoting bacterial strains were screened through cocultivation between endophytic bacteria with wheat seedlings.Further,the effects of growth-promoting bacteria on the growth and physiological indexes of Astragalus membranaceus seedlings were analyzed in order to reveal the physiologically growthpromoting mechanism of endophytic bacteria on A.membranaceus.These results provided a reference for further research to safeguard and improve the quality of artificially cultivated P.polyphylla var.yunnanensis using endophytic bacteria.The main results of this study were as follows:(1)Based on the ITS sequences,the diversity and composition of total endophytic fungi in the rhizomes,stems and leaves of the 5-year-old and artificially planted P.polyphylla var.yunnanensis were analyzed.At a sequence similarity level of 97%,388253 effective sequences were categorized into 722 OTUs,which are further classified into 5 phyla,12 classes,30 orders,44 families,and 50 genera.The abundance of total endophytic fungi in the rhizomes was higher than that in the stems and the leaves.The common endophytic fungi within the three tissues were classified to 6 genera,and the tissue-specific fungi of the rhizomes,stems and leaves were belonged to 11,5 and 4genera,respectively.The dominant genera of total endophytic fungi within the rhizomes,stems and leaves were Acremonium(5.8%),Papiliotrema(1.1%)and Microidium(0.4%),respectively.The ‘cooperative’ relationship(62.5%)among the total endophytic fungi was more than the ‘competitive’relationship.The correlation analysis results showed that6,3,3,and 7 fungal genera were significantly positively correlated with the contents of polyphyllin Ⅰ,polyphyllin Ⅱ,polyphyllin Ⅵ,and polyphyllin Ⅶ,respectively.(2)The composition and distribution of cultivable endophytic fungi of the rhizomes,stems and leaves were investigated using microbial culture technology.A total of 199 cultivable endophytic fungi(71,65 and 63 from the stems,rhizomes and leaves,respectively)were isolated,and their 18 S r DNA sequences were analyzed.The diversity from high to low of cultivable endophytic fungi was within the rhizomes,stems,and leaves.The dominant genus of cultivable endophytic fungi in the rhizome,stem,and leaf samples was Aspergillus,with relative abundances of 63%,49%,and 68%,respectively.The composition of cultivable endophytic fungi in the rhizomes and the stems was most similar.The culturable endophytic fungi common to the three tissues were classified into4 genera,and the culturable endophytic fungi specific to the rhizomes,stems and leaves were classified into 4,1 and 1 genera,respectively.A total of 6 genera and 2 genera of the cultivable endophytic fungi were positively and negatively correlated to the contents of three kinds of polyphyllins,respectively.(3)Through co-cultivation with wheat seedlings,18 bacterial strains with growthpromoting effect were selected from 300 endophytic bacteria.The YNR32041 and YNR32058 strains with obvious growth-promoting effects were selected to co-culture with A.membranaceus seedlings,and it was found that these two endophytic bacteria strains could promote the growth of A.membranaceus seedlings.The A.membranaceus seedlings co-cultured with YNR32041 showed rapid growth of plant height and root length on the 10 th day,with growth rates of 118.8% and 113.6% respectively,meanwhile the promotion rate of fresh weight was most significant on the 15 th day and was 206.2%.The plant height and root length of A.membranaceus seedlings co-cultured with YNR32058 grew rapidly on the 10 th day with a growth rate of 190.4% and 257%,respectively,and the promotion rate of their fresh weight was most significant on the 15 th day and was 380%.(4)A total of 10 physiological and biochemical indexes of the A.membranaceus seedlings co-cultured with YNR32041 and YNR32058 at different time points were detected,respectively.Within the investigated time points,the starch content,soluble sugar content,soluble protein content,ZT content,and ACC activity of the A.membranaceus seedlings co-cultured with YNR32041 were significantly changed on the10 th day compared with the control,and the growth rates were 62%,57.1%,20.8%,28.9%,and 25.6%,respectively,meanwhile the MDA content was increased by 227%and the ABA content was significantly reduced by 43.5% on the 15 th day,and other physiological indexes had no significant differences to the control.Compared with the control,the ZT content and ACC activity of A.membranaceus seedlings co-cultured with YNR32058 showed significant changes on the 10 th day,with growth rates of 21.1% and21.5%,respectively,while the growth rate of MDA content on the 5th day was 25.2%,the starch content was significantly reduced by 71% on the 10 th day,the soluble sugar content was significantly reduced by 88% on the 15 th day,and other physiological indexes had no significant differences to the control.These results preliminarily revealed the physiological mechanism of two endophytic bacteria strains promoting the growth of A.membranaceus seedlings. |
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