| In this work, nested PCR-DGGE, clone sequence, RT-PCR, BIOLOG and chlorophyll fluorescence techniques were employed to explore the arbuscular mycorrhizal fungal and dark septate endophytes colonization status, arbuscular mycorrhizal fungal (AMF) community structure diversity and glomalin content associated with the Lycium barbarum from arid and semi-arid Loess Plateau, China. Whilst, we investigated the soil particle size distributions and diversity of soil microbial communities in the rhizosphere of L. barbarum growing in different ecological environmental conditions. We also isolated several DSE strains from the roots of L. barbarum, identified these fungal species by molecular method as rDNA-ITS, and determined the effects of carbon and nitrogen sources on the growth of three dark septate endophytes. After that, we examined the responses of DSE under drought stress conditions. Finally, we examined the effects of inoculation with AMF and DSE on the growth and drought stress alleviation of L. barbarum seedlings by determination of leaf and root structures as well as photosynthetic parameters. The main points of this dissertation are as follows:1. Arbuscular mycorrhizas and dark septate endophytes colonization status in L. barbarum L. growing on Loess Plateau. The AM and DSE fungal colonization status in three different L. barbarum cultivars (NQ-1, NQ-5, NQC-1) in arid northwestern China were investigated. The results showed that the three cultivars were simultaneously colonized by Paris-type AM and DSE associations. The highest colonization by AM was found in L. barbarum Ningqi No.1. The significant"month"and"cultivar"indicates that the AM colonization varied among the months within the cultivar. Meanwhile, roots of the three cultivars were heavily colonized by DSE fungi. Melanized hyphae were frequently observed. Microsclerotia of varied shapes were also found in the cortex cells of L. barbarum. Hyaline hyphae were most abundant in August but their occurrence decreased in December. Lipid contents were abundant in hyaline hyphae, and changed with month. Lipid may act as energy material reserves to sustain the DSE-host symbioses under drought conditions.2. Arbuscular mycorrhizal fungal community and glomalin content associated with the rhizosphere of L. barbarum. We aimed to examine the AM fungal community diversity using nested PCR-DGGE and clone sequencing techniques, and to quantify the glomalin content associated with different cultivars of L. barbarum. The results showed that the significant"month"and"cultivar"indicates that easily extractable glomalin content varied among the months within the cultivar, the highest content was found in August, while the lowest was in December. The significant positive corelation was observed between total glomalin and easily extractable glomalin content. According to the DGGE fingerprints, the significant"month"and"cultivar"indicates that Species richness (S) and Shannon's diversity (H) index of AMF community varied among the months within the cultivar, the Species richness (S) in December was lower than August and October, the interaction effect was no significant. Redundancy analysis (RDA) showed the significant relationship between AMF community diversity and soil environmental factors. The results of clone sequence showed that 17 sequences were belonged to Glomus.3. The soil particle size distributions and diversity of soil microbial communities associated with the rhizosphere of L. barbarum from different ecological conditions were investigated. The results showed that the soils from Guyuan, Tongxin, Yinchuan, and Zhongning were most distributed from 20~50μm, the percentages were 37.21±0.21%, 33.25±0.21%, 38.35±0.18%, and 42.33±0.45%, respectively. In Huinong area, there was 40.21±0.26% soils distributed from 100~200μm. Nested PCR-DGGE suggested that the bacterial species richness of the five ecological regions was ordered as: Tongxin>Zhongning>Guyuan=Yinchuan>Huinong, whilst, fungal species richness was ordered as: Yinchuan>Tongxin>Huinong>Zhongning>Guyuan. There was no significant correlation between bacterial and fungal species richness diversity. Principle Component Analyses (PCA) revealed that bacterial community in Yinchuan was instability, because the community variation was lowest in Guyuan and highest in Yinchuan, no significant fungal community diversity was observed between Guyuan and Tongxin regions. The results of clone sequencing suggested that the bacterial and fungal bands were belonged to uncultured microbial community, respectively.4. Isolation, identification, and biological characteristics of dark septate endophytes isolates were determined. The results showed that three DSE isolates were belonged to Phoma chrysanthemicola,Cladosporium cladosporioides LBF3, and C. cladosporioides LBF6 based on rDNA-ITS analyses. There were different responses of P. chrysanthemicola, C. cladosporioides LBF3, and C. cladosporioides LBF6 grown on the same carbon and nitrogen medium, and differences in patterns of growth on the different substrates were also found. Glucose, mannitol and xylose were the best carbon sources; glycine and proline were the best nitrogen sources for the growth of P. chrysanthemicola, C. cladosporioides LBF3 and C. cladosporioides LBF6, respectively. Clone and mycelial structures, and protein expression were changed when P. chrysanthemicola exposed to water stress, whilst, fourier transform infrared spectroscopy showed that protein structues were changed under drought stress conditions.5. To explore the effects of dark septate endophytes inoculation on microecosystem associated with the rhizosphere of L. barbarum under pot culture conditions, bacterial abundance, functional and genetic diversity were determined. The results showed that bacterial abundance, functional and genetic diversity were affected by dark septate endophytes inoculation. DSE inoculations have higher bacterial metabolic activity (BIOLOG), and metabolic diversity than that of control, and varied among different DSE isolates. Principle Component Analyses (PCA) of BIOLOG data revealed that the application of DSE significantly in?uenced bacterial functional diversity in the rhizosphere of L. barbarum seedlings. A wider range of sole carbon sources were utilized by the bacterial community in the rhizosphere of inoculated seedlings. Whilst, nested PCR-DGGE showed that inoculation with DSE influenced bacterial genetic community diversity in the rhizosphere of L. barbarum seedlings.6. To explore the mechanism of the interaction between DSE and L. barbarum. Effect of a dark septate endophyte fungus on the growth of L. barbarum seedlings were examined under the lab conditions. LBF-2, isolated from the roots of L. barbarum, was inoculated onto the roots of plants, which were grown for 35 d. The results showed that melanized septate hyphae and microsclerotia were observed in the root cortical cells of L. barbarum. Nested PCR-denaturing gradient gel electrophoresis showed, for the first time, that LBF-2 colonizes L. barbarum systemically, with LBF-2 being detected in the root, stem and leaf tissues of L. barbarum. Inoculation with LBF-2 increased the total biomass by 28% and also enhanced chlorophyll fluorescence. Inoculation increased the concentration of total chlorophyll was by 19% and of chlorophyll a by 16%, relative to uninoculated controls. LBF-2 also increased the flavonoid concentration of L. barbarum seedlings by 26%. These data indicate that LBF-2 might be used to facilitate the cultivation of L. barbarum, which has medicinal applications.7. To examine the response of L. barbarum seedlings to inoculation with AMF and DSE under drought stress conditions, the pot cultured experiment was assayed. The results showed that root and leaf structues were changed with inoculation process. AM and DSE inoculation increased stomatal conductance. Inoculation with symbiotic fungal species was effective for L. barbarum seedlings as an acceptable and ecofriendly technology to improve root and leaf performance and development, and alleviate the damage of drought stress by improving stomatal conductance and efficiency of photosystem II.
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