| Psammosilene tunicoides, a China-endemic and single-species-genus plant, belongs to Psammosilene genus, Caryophyllaceae family. The roots of this plant can be used not only as original and precious materials of Chinese herbal medicines, but also as one of main components in some famous traditional Chinese herbal medicines, e.g. Yunnan Baiyao and Guizhou Jingulian capsules etc. Due to the narrow distribution of wild resources of Psammosilene tunicoides, the raw materials of many Chinese herbal medicine companies mainly depended on its wild resources and the unrestrained exploitation measures were not adopted in the long term, which led to its quantity decreasing rapidly. Psammosilene tunicoides is considered as a national rare and endangered protected plant. Both endophytes and rhizospheric microorganisms with special characteristics have been considered as good and important alternatives at home and abroad so far, which can replace some medicines and new active substances. In this study, the roots and rhizospheric soil were utilized as experimental materials, the community diversity and activities of endophytic and rhizospheric actinobacteria were analyzed by using the pure culture, and the community change of prokaryotic microorganisms from the roots, rhizospheric soil and marginal soil of Psammosilene tunicoides cultivated both in red soil and in sandy soil, and the roots of Psammosilene tunicoides in vitro were investigated and analyzed systematically. The main results were as following:Total 182 actinobacterial strains were obtained from the roots of Psammosilene tunicoides gathered from four places, which were assigned to 21 genera and one potential new genus,13 families and 8 suborders. Among them, Streptomyces was a dominant genus, followed by genera Nocardia, Mycobacterium and Micromonospora. Total 488 actinobacterial strains were obtained from the rhizospheric soil of Psammosilene tunicoides gathered from six places, which were assigned to 40 genera, 17 families and 10 suborders. Among them, the dominant actinobacteria was also Streptomyces, followed by genera Kribbella and Micromonospora. The richest diversity of endophytes was Lijiang while the poorest was Shangri-La, and the richest diversity of rhizospheric microorganisms was Shangri-La while the poorest was Ernandui. Total 435 actinobacterial strains were obtained from the roots and rhizospheric soil of Psammosilene tunicoides gathered from four same places, which were assigned to 38 genera and 10 suborders. All the eight suborders of actinobacteria from the roots were included in the suborders from the rhizospheric soil, and the genera found simultaneously both in the roots and in the rhizosperic soil took up 34.2% among all the genera of actinobacteria.On the basis of Miseq high throughout sequencing analysis,330 genera and 44 families were screened from 54 samples of red soil collected from six places. Among them,212 genera and 26 phyla were isolated from the roots,212 genera and 26 phyla from the rhizospheric soil and 238 genera and 37 phyla from the marginal soil respectively. As for the unique phylum, no one existed in the roots while four in the rhizospheric soil and one in the marginal soil. As for the unique genus, there were 42 special genera and 19 special dominant genera in the roots,31 and 4 in the rhizospheric soil, and 24 and one in the marginal soil respectively.291 genera and 43 phyla were identified from 36 samples of sandy soil gathered from four places. Among them, there existed 195 genera and 29 phyla in the roots,213 genera and 38 phyla in the rhizospheric soil and 215 genera and 36 phyla in the marginal soil respectively. As for the unique phyla, two existed in the roots while one in the rhizospheric soil and five in the marginal soil. As for the unique genera, there were 44 special genera and 9 special dominant genera in the roots,16 and one in the rhizospheric soil, and 20 and 4 in the marginal soil respectively. There existed significant difference in the species of actinobacteria between red and sandy soil, which were 337 and 372 species respectively, on the basis of above-mentioned results, the richness of microorganisms from the roots of Psammosilene tunicoides were less than those from the soil, while the units of special genera from the roots were more than those from the soil. It indicated that the plant played an important role in the richness of actinobacteria. The diversity of microorganisms from wild resources of Psammosilene tunicoides were richer than that from the cultivated, which suggested that the distribution of microorganisms existed some certain host specialty. Meanwhile, it's found that the species of microorganisms were rich in the roots of Psammosilene tunicoides in vitro, and 14 families and 112 genera were identified from this material; that there existed significant difference in the species of microorganisms from the roots between in vitro and in the wild environment.Based on the comprehensive and comparative analysis, there existed big difference in the genera of microorganisms between red and sandy soil while there were similar in the phyla of microorganisms; that the most dominant phylum both in red soil and in sandy soil was Proteobacteria. In the roots, the second most dominant phylum was Firmicutes and followed by Actinobacteria, while Actinobacteria and Acidobacteria both in the rhizosperic soil and in the marginal soil. The dominant genera from the roots of red and sandy soil were followed by Siaphylococcus, Bacillus and Rhodoplanes respectively. In the microorganisms from the soil, the proportion of main dominant genera from the soil was significant less than that from the roots, moreover, there existed great difference in the dominant genera from red and sandy soil. Among them, the dominant genera from red soil were followed by Rhodoplanes, DA101 and Candidatus Xiphinematobacter, while Rhodoplanes and Kaistobacter in sandy soil.On the basis of the results of pure culture and free culture, both the richness and diversity of microorganisms in the soil were much higher than those in the roots, there existed some certain correlation in the microorganisms between in the roots and in the rhizospheric soil, and their distribution of microorganisms existed some certain heterogeneity. Relatively, both the quantity of actinobacteria and its diversity from the free culture were more than those from the pure culture, while all the microorganisms in the pure culture were not detected by the free culture, among which about 40% genera from the pure culture were not found in the free culture. In the pure culture, the most dominant genus was Streptomyces; while the most dominant genus was Pseudonocardia from the roots and Mycobacterium from the rhizospheric soil in the free culture.The rich function genes were predicted in all the samples, whose relative richness from the microorganisms of soil was totally higher than that from the microorganisms of roots. Many genes (e.g. farnesyl diphosphate synthase, squalene monooxygenase and cytochrome 450 monooxygenase etc.) of some important enzymes of pentacyclic triterpenoid saponins biosynthesis in Psammosilene tunicoides were detected, which testified that some microorganism species from the roots and rhizospheric soil of Psammosilene tunicoides had the potential abilities of promoting the biosynthesis of pentacyclic triterpenoid saponins. There existed different certain correlation between 10 factors of soil physical and chemical characteristics and microorganism population. On the basis of their correlation and comprehensive analysis, among which both Ca2+ and pH value had positive correlation with the distribution of microorganisms in the soil.The polyphasic approach to 3 new candidates were carried out to confirm their accurate taxonomic positions. Among them, strain YIM TG2190, named Nocardia zhihengii sp. nov., was a novel species of Nocardia; strain YIM DR.1091, named Nocardioides intraradicalis sp. nov., was a new species of Nocardioides, and strain YIM DR4008, named Allostreptomyces gen. nov., was a novel genus of the family Streptomycetaceae, in which the typical species was Allostreptomyces psammosilenae sp. nov..After detecting their antimicrobial abilities and enzymatic activities of 92 endophytic strains and 111 rhizospheric strains from the roots and rhizospheric soil, some actinobacteria strains with high enzyme activity and strong ability of inhibiting pathogens were screened.In summary, it manifested that there were rich and numerous resources of actinobacteria and prokaryotic microorganisms in the roots and rhizospheric soil of Psammosilene tunicoides; that there existed some certain correlation in the microorganism population among the roots, rhizospheric soil and marginal soil. In addition, both the rich species of actinobacteria found in the pure culture and functional strains screened in this study not only provided some valuable microorganism resources for agricultural production and industrial application in the future, but also exerted some certain theoretical instruction and practical application values for the cultivation and protection of Psammosilene tunicoides. |
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