蒙古沙冬青深色有隔内生真菌（DSE）多样性研究
发布时间：2019-06-26 12:24
【摘要】：蒙古沙冬青(Ammopiptanthus mongolicus)是我国西北荒漠地区生长的一种超旱生常绿阔叶豆科灌木,属国家三级濒危保护植物,是荒漠地区防风固沙的优良树种。深色有隔内生真菌(Dark septate endophyte,DSE)是泛指定殖于植物根细胞或间隙的小型土壤真菌,不会在根组织内形成病原菌所引起的病理学特征,具有广泛的生态分布和类似菌根的潜在生态功能。本研究以蒙古沙冬青与DSE真菌共生关系为基础,选择内蒙古乌海、阿拉善左旗和乌拉特后旗3样地,系统研究了蒙古沙冬青根系DSE真菌物种多样性和定殖时空异质性及其与土壤因子的关系,并对部分典型DSE菌株生长特性及化学成分含量进行了分析。旨在探明荒漠地区蒙古沙冬青DSE真菌物种多样性和生态分布规律,为充分利用DSE真菌资源,促进荒漠植被恢复和生态重建提供依据。主要研究结果如下:1.碱解离酸性品红染色法证实DSE菌丝在所选样地沙冬青根系组织普遍侵染,胞间或胞内定殖,并存在深色有隔菌丝和 微菌核‖特化结构。随海拔从低到高,DSE真菌定殖形态出现显著性变化规律,即菌丝变粗,颜色加深,横隔膜变短。微菌核由分散颗粒状逐渐聚集成簇,并出现迷宫状形态。2.样地生态条件和采样深度对DSE分布和活动有显著影响,不同样地DSE真菌菌丝和微菌核最大定殖率出现在0-20cm浅土层,并随土层加深显著减少。随海拔升高,菌丝、微菌核和总定殖率呈现显著性规律。即高海拔的阿拉善左旗样地显著高于低海拔的乌拉特后旗和乌海样地。土壤有机C、磷酸酶和碱解N与DSE菌丝和微菌核定殖率极显著正相关(P0.01),土壤脲酶与有机C、酸性磷酸酶极显著正相关(P0.01),与速效P显著正相关(P0.05);土壤p H与速效P和酸性磷酸酶正相关(P0.05)。主成分分析表明,土壤有机C、磷酸酶和碱解N是影响DSE真菌活动和生态分布的主要因子。3.三样地5大类土壤微生物数量及分布与蒙古沙冬青及样地生态条件显著相关,不同样地不同土层微生物数量差异显著,垂直动态分布明显。3样地均是细菌芽孢菌放线菌固氮菌真菌。0-20cm土层显著高于其它土层,并随土层加深,数量呈现减少趋势。同一土层,乌海的土壤细菌、芽孢菌、固氮菌、放线菌显著高于乌拉特后旗和阿拉善左旗;乌拉特后旗的真菌显著高于乌海和阿拉善左旗。相关性分析表明:土壤细菌、真菌和放线菌均与土壤有机C、碱解N和碱性磷酸酶极显著正相关。4.三样地蒙古沙冬青(A.mongolicus)根围共分离纯化DSE菌株274株,利用系统形态学和分子生物学方法相结合对菌株进行鉴定。划分为12种,分属9属。既有较为少见的Leptodontidium orchidicola、基利恩帚枝霉菌(Sarocladium kiliense)和互隔交链孢霉(Alternaria alternata)等;也有比较常见的甘瓶霉(Phialophora mustea)、菊异茎点霉(Paraphoma chrysanthemicola)和枝孢菌属(Cladosporium sp.)、Phoma、Exophiala和Phialophora类群。甘瓶霉(Phialophora mustea)为乌海和阿拉善左旗共有属,异茎点霉属(Paraphoma sp.)为乌海和乌拉特后旗共有常见属种,枝孢菌(Cladosporium)为阿拉善左旗和乌拉特后旗共有常见属。三样地分离得到的12种DSE真菌有10种产生孢子,占80%以上,与参阅的多种文献研究观点不同。究其原因主要由于生态条件及目标植物差异,DSE真菌产生大量孢子以抵御不良环境。5.利用真菌r DNA通用引物ITS4和ITS5进行PCR扩增,目的基因片段长度为525bp-627 bp。利用在线BLAST工具进行序列比对,12种DSE菌株与亲缘关系最近菌株序列相似性均在97%以上。6.三样地DSE真菌种丰度低是导致香农-维纳指数较低的主要原因。不同样地环境异质性增加了DSE定殖类群。Simpson指数和Pielou指数相近,说明三样地DSE类群分布比较均匀。7.初步确定了三样地沙冬青根围3个典型DSE菌株最适固体培养基及相关培养条件。即使用PDA培养基,并添加0.4%蛋白胨;25℃,p H7,黑暗培养。通过优化前后菌落形态及孢子菌丝微观形态比较,差异显著。8.乌拉特后旗样地,WLTHQ-2 Cladosporium cladosporioides黑色素含量显著高于WH(4)10-20 Phialophora mustea和ALS(3)30-40 Alternaria alternate;另外,WLTHQ-2Cladosporium cladosporioides微量元素锰、锌和钾含量也呈现显著性,与DSE真菌黑色素含量变化规律基本一致。
[Abstract]:Ammopiptanus mongol is a kind of super-arid evergreen broad-leaf leguminous shrub growing in the desert area of the northwest of China, and belongs to the three-level endangered plant of the state. It is an excellent tree species for the prevention of wind and sand fixation in the desert area. Dark septa endophyte (DSE) is a small soil fungus that is the root cell or gap of a plant, does not form the pathological characteristics of the pathogenic bacteria in the root tissue, and has a wide ecological distribution and a potential ecological function similar to the mycorrhizal. Based on the symbiotic relationship between the Ammopiptanthus mongolicus and the DSE, the species diversity and the spatial and temporal heterogeneity of the DSE fungi in the root system of Ammopiptanthus mongolicus and their relationship with the soil factors were studied. The growth and chemical composition of some typical DSE strains were analyzed. The purpose of this study is to find out the species diversity and the ecological distribution of the DSE fungi in the mongolian mongolicus in the desert area, and to provide the basis for the full use of the DSE fungus resources and promoting the restoration of desert vegetation and the ecological reconstruction. The main results are as follows:1. The alkali-dissociative acid fuchsin staining method confirmed that the DSE hyphae were generally infected by the root tissue of the selected sample of Ammopiptanthus mongolicus, and the cells were colonized in the cell or in the cell, and there was a specific structure of the dark-colored hyphae and the microsclerotium. There was a significant change in the colonization of the DSE fungi as the altitude decreased from low to high, that is, the hyphae became thicker, the color deepened, and the transverse membrane became shorter. The micro-sclerotium is gradually clustered into clusters by the dispersed particles, and a labyrinth-like form is present. The site ecological condition and sampling depth have a significant effect on the DSE distribution and activity, and the maximum colonization rate of the DSE fungal hyphae and the microsclerotium occurs in the 0-20 cm shallow soil layer, and the depth of the soil layer is obviously reduced. With the elevation of altitude, the hyphae, the micro-sclerotium and the total colonization rate were found to be significant. That is, the high-altitude Alashan left flag is significantly higher than the low-altitude Urazzo flag and the Wuhai-like land. Soil organic C, phosphatase and alkaline solution N were significantly positively correlated with the strains of DSE and micro-organisms (P0.01), and the soil enzyme was positively correlated with organic C and acid phosphatase (P0.01), and was significantly positively correlated with quick-acting P (P0.05). The p-H of the soil was positively correlated with the quick-acting P and the acid phosphatase (P0.05). The principal component analysis indicated that soil organic C, phosphatase and alkaline solution N are the main factors that affect the activity and ecological distribution of DSE fungi. The number and distribution of micro-organisms in the five major categories of soil in three samples were significantly related to the ecological conditions of the Ammopiptanthus mongolicus and the samples in Mongolia, and the number of microorganisms in different soil layers was significantly different, and the vertical dynamic distribution was obvious. And with the depth of the soil, the number presents a tendency to decrease. In the same soil layer, the soil bacteria, the bacillus, the azotobacter and the actinomycetes of the Usea were significantly higher than that of the Urater flag and the Alashan left flag; and the fungus of the Urateh flag was significantly higher than that of the Wuhai and Alashan left flag. The correlation analysis showed that soil bacteria, fungi and actinomycetes were positively correlated with soil organic C, alkaline solution N and alkaline phosphatase. A total of 274 strains of DSE were isolated and purified from the root of Ammopiptanthus mongolicus (A. mongol), and the strains were identified by the methods of system morphology and molecular biology. It is divided into 12 species, belonging to 9 genera. It is not only rare in Leptodontidium orchidicola, Sarcoladium kiliense, and Alternaria alternata. It is also a common species of Phytophthora muguea, Paraphoma chrysum and Cladosporium sp. ), Phoma, Exophila and Philospora. Phytophthora mustanea is a common genus of the genus Usea and Alxa, and is a species of Parapoma sp. ) Cladosporium is a common genus of the post-flag of the Uhai and Urazzo, and is a common genus of the flag of Alashan and the post-flag of Urateh. The 12 DSE fungi obtained by three-sample separation have ten producing spores, accounting for more than 80%. The reason is mainly due to the difference of ecological conditions and target plants, and the DSE fungi produce a large amount of spores to resist the adverse environment. PCR was carried out by using the general primers ITS4 and ITS5 of the fungus r DNA, and the length of the target gene fragment was 525bp-627bp. The sequence similarity of the 12 DSE strains and the nearest strain was 97% or more by using the on-line BLAST tool. The low abundance of the three-like DSE fungi is the main reason for the lower index of the Shannon-Wiener index. The DSE colonizing group was not increased in the same environmental heterogeneity. The Simpson index and the Pielou index are similar to those of the Pielou index. The optimum solid culture medium and related culture conditions of three typical DSE strains were determined. I. e. using a pda culture medium and adding 0.4% protein solution;25 c, p h7, dark culture. The morphology of the colony and the micromorphology of the spores of the spores were optimized before and after the optimization, and the difference was significant. The content of the melanin in WTHQ-2 Cladosporium cladosporioides was significantly higher than that of WH (4)10-20, and the content of Zn and K in WTHQ-2 Cladosporium cladosporioides was significantly higher than that of WH (4)10-20, and the content of Zn and K in WTHQ-2 Cladosporium cladosporioides was also significant.
【学位授予单位】：河北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93
本文编号：2506173
[Abstract]:Ammopiptanus mongol is a kind of super-arid evergreen broad-leaf leguminous shrub growing in the desert area of the northwest of China, and belongs to the three-level endangered plant of the state. It is an excellent tree species for the prevention of wind and sand fixation in the desert area. Dark septa endophyte (DSE) is a small soil fungus that is the root cell or gap of a plant, does not form the pathological characteristics of the pathogenic bacteria in the root tissue, and has a wide ecological distribution and a potential ecological function similar to the mycorrhizal. Based on the symbiotic relationship between the Ammopiptanthus mongolicus and the DSE, the species diversity and the spatial and temporal heterogeneity of the DSE fungi in the root system of Ammopiptanthus mongolicus and their relationship with the soil factors were studied. The growth and chemical composition of some typical DSE strains were analyzed. The purpose of this study is to find out the species diversity and the ecological distribution of the DSE fungi in the mongolian mongolicus in the desert area, and to provide the basis for the full use of the DSE fungus resources and promoting the restoration of desert vegetation and the ecological reconstruction. The main results are as follows:1. The alkali-dissociative acid fuchsin staining method confirmed that the DSE hyphae were generally infected by the root tissue of the selected sample of Ammopiptanthus mongolicus, and the cells were colonized in the cell or in the cell, and there was a specific structure of the dark-colored hyphae and the microsclerotium. There was a significant change in the colonization of the DSE fungi as the altitude decreased from low to high, that is, the hyphae became thicker, the color deepened, and the transverse membrane became shorter. The micro-sclerotium is gradually clustered into clusters by the dispersed particles, and a labyrinth-like form is present. The site ecological condition and sampling depth have a significant effect on the DSE distribution and activity, and the maximum colonization rate of the DSE fungal hyphae and the microsclerotium occurs in the 0-20 cm shallow soil layer, and the depth of the soil layer is obviously reduced. With the elevation of altitude, the hyphae, the micro-sclerotium and the total colonization rate were found to be significant. That is, the high-altitude Alashan left flag is significantly higher than the low-altitude Urazzo flag and the Wuhai-like land. Soil organic C, phosphatase and alkaline solution N were significantly positively correlated with the strains of DSE and micro-organisms (P0.01), and the soil enzyme was positively correlated with organic C and acid phosphatase (P0.01), and was significantly positively correlated with quick-acting P (P0.05). The p-H of the soil was positively correlated with the quick-acting P and the acid phosphatase (P0.05). The principal component analysis indicated that soil organic C, phosphatase and alkaline solution N are the main factors that affect the activity and ecological distribution of DSE fungi. The number and distribution of micro-organisms in the five major categories of soil in three samples were significantly related to the ecological conditions of the Ammopiptanthus mongolicus and the samples in Mongolia, and the number of microorganisms in different soil layers was significantly different, and the vertical dynamic distribution was obvious. And with the depth of the soil, the number presents a tendency to decrease. In the same soil layer, the soil bacteria, the bacillus, the azotobacter and the actinomycetes of the Usea were significantly higher than that of the Urater flag and the Alashan left flag; and the fungus of the Urateh flag was significantly higher than that of the Wuhai and Alashan left flag. The correlation analysis showed that soil bacteria, fungi and actinomycetes were positively correlated with soil organic C, alkaline solution N and alkaline phosphatase. A total of 274 strains of DSE were isolated and purified from the root of Ammopiptanthus mongolicus (A. mongol), and the strains were identified by the methods of system morphology and molecular biology. It is divided into 12 species, belonging to 9 genera. It is not only rare in Leptodontidium orchidicola, Sarcoladium kiliense, and Alternaria alternata. It is also a common species of Phytophthora muguea, Paraphoma chrysum and Cladosporium sp. ), Phoma, Exophila and Philospora. Phytophthora mustanea is a common genus of the genus Usea and Alxa, and is a species of Parapoma sp. ) Cladosporium is a common genus of the post-flag of the Uhai and Urazzo, and is a common genus of the flag of Alashan and the post-flag of Urateh. The 12 DSE fungi obtained by three-sample separation have ten producing spores, accounting for more than 80%. The reason is mainly due to the difference of ecological conditions and target plants, and the DSE fungi produce a large amount of spores to resist the adverse environment. PCR was carried out by using the general primers ITS4 and ITS5 of the fungus r DNA, and the length of the target gene fragment was 525bp-627bp. The sequence similarity of the 12 DSE strains and the nearest strain was 97% or more by using the on-line BLAST tool. The low abundance of the three-like DSE fungi is the main reason for the lower index of the Shannon-Wiener index. The DSE colonizing group was not increased in the same environmental heterogeneity. The Simpson index and the Pielou index are similar to those of the Pielou index. The optimum solid culture medium and related culture conditions of three typical DSE strains were determined. I. e. using a pda culture medium and adding 0.4% protein solution;25 c, p h7, dark culture. The morphology of the colony and the micromorphology of the spores of the spores were optimized before and after the optimization, and the difference was significant. The content of the melanin in WTHQ-2 Cladosporium cladosporioides was significantly higher than that of WH (4)10-20, and the content of Zn and K in WTHQ-2 Cladosporium cladosporioides was significantly higher than that of WH (4)10-20, and the content of Zn and K in WTHQ-2 Cladosporium cladosporioides was also significant.
【学位授予单位】：河北大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93
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