【摘要】：蒙古沙冬青(Ammopiptanthus mongolicus)是我國(guó)西北荒漠地區(qū)生長(zhǎng)的一種超旱生常綠闊葉豆科灌木,屬國(guó)家三級(jí)瀕危保護(hù)植物,是荒漠地區(qū)防風(fēng)固沙的優(yōu)良樹(shù)種。深色有隔內(nèi)生真菌(Dark septate endophyte,DSE)是泛指定殖于植物根細(xì)胞或間隙的小型土壤真菌,不會(huì)在根組織內(nèi)形成病原菌所引起的病理學(xué)特征,具有廣泛的生態(tài)分布和類似菌根的潛在生態(tài)功能。本研究以蒙古沙冬青與DSE真菌共生關(guān)系為基礎(chǔ),選擇內(nèi)蒙古烏海、阿拉善左旗和烏拉特后旗3樣地,系統(tǒng)研究了蒙古沙冬青根系DSE真菌物種多樣性和定殖時(shí)空異質(zhì)性及其與土壤因子的關(guān)系,并對(duì)部分典型DSE菌株生長(zhǎng)特性及化學(xué)成分含量進(jìn)行了分析。旨在探明荒漠地區(qū)蒙古沙冬青DSE真菌物種多樣性和生態(tài)分布規(guī)律,為充分利用DSE真菌資源,促進(jìn)荒漠植被恢復(fù)和生態(tài)重建提供依據(jù)。主要研究結(jié)果如下:1.堿解離酸性品紅染色法證實(shí)DSE菌絲在所選樣地沙冬青根系組織普遍侵染,胞間或胞內(nèi)定殖,并存在深色有隔菌絲和 微菌核‖特化結(jié)構(gòu)。隨海拔從低到高,DSE真菌定殖形態(tài)出現(xiàn)顯著性變化規(guī)律,即菌絲變粗,顏色加深,橫隔膜變短。微菌核由分散顆粒狀逐漸聚集成簇,并出現(xiàn)迷宮狀形態(tài)。2.樣地生態(tài)條件和采樣深度對(duì)DSE分布和活動(dòng)有顯著影響,不同樣地DSE真菌菌絲和微菌核最大定殖率出現(xiàn)在0-20cm淺土層,并隨土層加深顯著減少。隨海拔升高,菌絲、微菌核和總定殖率呈現(xiàn)顯著性規(guī)律。即高海拔的阿拉善左旗樣地顯著高于低海拔的烏拉特后旗和烏海樣地。土壤有機(jī)C、磷酸酶和堿解N與DSE菌絲和微菌核定殖率極顯著正相關(guān)(P0.01),土壤脲酶與有機(jī)C、酸性磷酸酶極顯著正相關(guān)(P0.01),與速效P顯著正相關(guān)(P0.05);土壤p H與速效P和酸性磷酸酶正相關(guān)(P0.05)。主成分分析表明,土壤有機(jī)C、磷酸酶和堿解N是影響DSE真菌活動(dòng)和生態(tài)分布的主要因子。3.三樣地5大類土壤微生物數(shù)量及分布與蒙古沙冬青及樣地生態(tài)條件顯著相關(guān),不同樣地不同土層微生物數(shù)量差異顯著,垂直動(dòng)態(tài)分布明顯。3樣地均是細(xì)菌芽孢菌放線菌固氮菌真菌。0-20cm土層顯著高于其它土層,并隨土層加深,數(shù)量呈現(xiàn)減少趨勢(shì)。同一土層,烏海的土壤細(xì)菌、芽孢菌、固氮菌、放線菌顯著高于烏拉特后旗和阿拉善左旗;烏拉特后旗的真菌顯著高于烏海和阿拉善左旗。相關(guān)性分析表明:土壤細(xì)菌、真菌和放線菌均與土壤有機(jī)C、堿解N和堿性磷酸酶極顯著正相關(guān)。4.三樣地蒙古沙冬青(A.mongolicus)根圍共分離純化DSE菌株274株,利用系統(tǒng)形態(tài)學(xué)和分子生物學(xué)方法相結(jié)合對(duì)菌株進(jìn)行鑒定。劃分為12種,分屬9屬。既有較為少見(jiàn)的Leptodontidium orchidicola、基利恩帚枝霉菌(Sarocladium kiliense)和互隔交鏈孢霉(Alternaria alternata)等;也有比較常見(jiàn)的甘瓶霉(Phialophora mustea)、菊異莖點(diǎn)霉(Paraphoma chrysanthemicola)和枝孢菌屬(Cladosporium sp.)、Phoma、Exophiala和Phialophora類群。甘瓶霉(Phialophora mustea)為烏海和阿拉善左旗共有屬,異莖點(diǎn)霉屬(Paraphoma sp.)為烏海和烏拉特后旗共有常見(jiàn)屬種,枝孢菌(Cladosporium)為阿拉善左旗和烏拉特后旗共有常見(jiàn)屬。三樣地分離得到的12種DSE真菌有10種產(chǎn)生孢子,占80%以上,與參閱的多種文獻(xiàn)研究觀點(diǎn)不同。究其原因主要由于生態(tài)條件及目標(biāo)植物差異,DSE真菌產(chǎn)生大量孢子以抵御不良環(huán)境。5.利用真菌r DNA通用引物ITS4和ITS5進(jìn)行PCR擴(kuò)增,目的基因片段長(zhǎng)度為525bp-627 bp。利用在線BLAST工具進(jìn)行序列比對(duì),12種DSE菌株與親緣關(guān)系最近菌株序列相似性均在97%以上。6.三樣地DSE真菌種豐度低是導(dǎo)致香農(nóng)-維納指數(shù)較低的主要原因。不同樣地環(huán)境異質(zhì)性增加了DSE定殖類群。Simpson指數(shù)和Pielou指數(shù)相近,說(shuō)明三樣地DSE類群分布比較均勻。7.初步確定了三樣地沙冬青根圍3個(gè)典型DSE菌株最適固體培養(yǎng)基及相關(guān)培養(yǎng)條件。即使用PDA培養(yǎng)基,并添加0.4%蛋白胨;25℃,p H7,黑暗培養(yǎng)。通過(guò)優(yōu)化前后菌落形態(tài)及孢子菌絲微觀形態(tài)比較,差異顯著。8.烏拉特后旗樣地,WLTHQ-2 Cladosporium cladosporioides黑色素含量顯著高于WH(4)10-20 Phialophora mustea和ALS(3)30-40 Alternaria alternate;另外,WLTHQ-2Cladosporium cladosporioides微量元素錳、鋅和鉀含量也呈現(xiàn)顯著性,與DSE真菌黑色素含量變化規(guī)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.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q93

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本文編號(hào)：2506173