【作者】 张俊忠；

【导师】 陈秀蓉；

【作者基本信息】 甘肃农业大学 ， 草地生物多样性， 2010， 博士

【摘要】 土壤微生物是草地农业生态系统的重要组成部分,在能量流动、物质循环以及土壤的形成与熟化过程中均起重要作用。同时也是反应环境变化的敏感指示生物,其种群数量和组成是评价草地土壤环境质量的重要参数。研究土壤微生物的数量、种群多样性、生态分布以及生理功能群对提高草地生态系统的生产力,使之持续稳定发展具有重要意义。真菌是一类种类繁多、分布广泛的真核微生物,真菌构成了土壤的大部分微生物生物量,具有分解有机质,为植物提供养分的功能,是生态系统健康的指示物,真菌多样性在维持生物圈生态平衡和为人类提供大量未开发的生物资源方面起到了重要作用。因此,研究东祁连山高寒草地土壤真菌的种类、数量组成和生态分布等,并对该区的真菌群落进行多样性比较与分析,旨在揭示该区域草地土壤环境中真菌群落的基本特征、分布状况及物种多样性等,以期为东祁连山高寒草地的有效保护和综合开发提供依据。本文选取东祁连山七类具有代表性的高寒草地植被类型作为试验样地,即杜鹃灌丛(Rhododendron fruticosa shrub land),高山柳灌丛(Salix cupularis fruticosa shrub land),金露梅灌丛(Dasiphoru fruticosa shrub land),珠芽蓼草地(Polygonum grassland),禾草草地(Grass grassland),沼泽草地(Swamp grassland)和嵩草草地(Kobresia grassland)。选用PDA培养基、PSA培养基、玉米粉琼脂培养基和马丁氏-孟加拉红培养基分离土壤真菌;对土壤中的真菌采用表型鉴定和基于ITS rDNA基因序列的系统发育分析鉴定;并根据鉴定的土壤真菌和各测度指数的特点及取样数据的类型,应用生态学评价方法对可培养真菌多样性进行分析;把DGGE和Nested PCR运用到东祁连山高寒草地土壤微生物多样性的研究中;对土壤中的优势真菌——被孢霉,选择其中的具有代表性的3个高山被孢霉株进行了生物学特性研究。主要研究成果如下:1对不同培养条件下土壤真菌的分离数量进行测定分析可知,同一样品在不同pH、温度和培养基下培养后,分离出的菌群种类和数量都差异较大,马丁培养基较PDA、PSA和玉米粉培养基测得的数量、种类较多,相对适合土壤真菌的分离培养。通过比较采用的几种培养方案,真菌的最适分离温度为20℃,最适分离pH值为6.5,最适分离培养基为马丁氏培养基。2对分离的可培养土壤优势真菌应用表型鉴定和ITS rDNA序列分析进行分类,通过大规模的分离培养方法,综合表型鉴定和序列分析结果可以得出,东祁连山高寒草地土壤中主要的可培养真菌为:被孢霉属(Mortierella spp.)、柔束霉属(Doratomyces spp.)、小球腔菌属(Leptosphaeria spp.)、枝孢属(Cladosporium spp.)、轮枝菌属(Verticillium spp.)、毛霉属(Mucor spp.)、弯颈霉属(Tolypocladium spp.)、白僵菌属(Beauveria spp.)、肉座菌属(Hypocrea spp.)、截盘多毛孢属(Truncatella spp.)、虫草属(Cordyceps spp.)、木霉属(Trichoderma spp.)、丛赤壳属(Nectria spp.)、亚隔孢壳属(Didymella spp.)、青霉属(Penicillium spp.)、生赤壳属(Bionectria spp.)、链格孢属(Alternaria spp.)、曲霉属(Aspergillus spp.)、地丝菌属(Geomyces spp.)、镰孢菌属(Fusarium spp.)、帚霉属(Scopulariopsis spp.)、暗球腔菌属(Phaeosphaeria spp.),鉴定所得菌株可分属于22个属,绝大多数属于半知菌亚门和接合菌亚门真菌。另外,有大于10%的分离菌种暂时无法确定其分类地位,极可能是新种。3本试验也对可培养土壤真菌的多样性及群落生态特征进行了研究,结果表明,物种的丰富度(S)、Shannon- Wiener多样性指数(H)、Simpson优势度(D)和Pielou均匀度指数(J)变化范围分别为15～18,2.47～2.81,0.89～0.93,0.91～0.97;各指数在各类型草地中波动较为平缓,反映出基本一致的变化趋势,多样性指数的大小顺序为:嵩草草地>珠芽蓼草地>金露梅灌丛>高山柳灌丛>禾草草地>杜鹃灌丛>沼泽草地,除Pielou指数外其他指数均以嵩草草地较高,沼泽草地较低;东祁连山高寒草地土壤多样性较丰富,其多样性和草地类型的特异性有着密切的关系。4四不同草地土壤真菌的DGGE分析研究,采用Nested-PCR技术扩增土壤真菌ITS1 rDNA区域,利用该产物优化土壤真菌DGGE条件,应用DGGE图谱分析技术,并结合切胶测序及系统发育分析对土壤真菌菌群结构进行研究。结果表明Nested-PCR其具有较高的灵敏性,可有效的从微量DNA中扩增出约250 bp的ITS1 rDNA目的片段。DGGE条带测序及图谱分析表明,4种类型草地土壤DGGE带谱在条带的数量和亮度方面均存在较大差异,和可培养真菌的变化趋势基本一致。5三个高山被孢霉菌株的生物学特性的研究表明它们之间差异显著,M. ap27、M.ap60和M.ap9的最适氮源均为蛋白胨,M.ap27的最适碳源为淀粉,M.ap60的最适碳源为蔗糖,M.ap9的最适碳源为葡萄糖;3个高山被孢霉菌株在5～30℃的温度处理下都能生长,M.ap27的最适生长温度为30℃,M.ap60和M.ap9的最适生长温度均为5℃;3个高山被孢霉菌株在pH值4～9范围内均能生长,适宜pH值为7。更多还原

【Abstract】 As a main part of the agricultural ecosystem of grassland, soil microorganisms play vital roles in many processes, such as energy flowing and subtance conversion & cycling as well as soil developing and maturation. It is a sensitive index of any variation in environmental conditions as well. Constitutions and quantities of population are two key parameters for evaluating the soil quality. Therefore, it is very significative to study quantity of soil microbial population, diversity of population, ecological distribution characteristics and physiological fuction groups enhancing productivity of grassland ecosystem for sustainable development. The fungi are a kind of extensive eukaryotes microorganism, The fungi form the most microorganism amount of soil, resolve the organic matter, offer nutrient for plant and are healthy instruction thing for ecosystem. Fungal diversity has played important function in maintain the biosphere ecological balance and offer the undeveloped living resources to mankind in a large amounting, therefore, the study on soil fungal species, quantitative composition, ecological distribution et al, in order to reveal the fungal community, species diversity and to provide evidence for effective protection and comprehensive development of alpine grassland in Eastern Qilian mountains.In this study, seven different representative vegetation types of alpine grassland in Eastern Qilian Mountains, which were Rhododendron fruticosa shrub land, Salix cupularis fruticosa shrub land, Dasiphoru fruticosa shrub land, Polygonum grassland, Grass steppe, Swamp grassland and Kobresia grassland were selected to be experimental samples. The soil fungi were isolated by PDA, PSA, Maize Powder Media and Rose Bengal Medium, which were analyzed by ITS rDNA sequence analysis phenotype identification. According to the identificated fungi, the characteristics of each measure index and types of sampling data, the fungi diversity were analyzed by ecology-evaluating methods. The DGGE and Nested PCR were used to study the fungi diversity; The biological characteristics of three Mortierella were study, which were seclected from dominated fungi, the main results were as follows:1 The quantity of soil fungi were determined in the different incubation conditions, the same sample have different species and quantities in different pH, temperature and media different incubation conditions, Rose Bengal Medium could get more species and quantities than PDA、PSA and corn powders, which was suitable for isolating fungi from soil. By comparing, the optimal incubation temperature, pH and respectively were 20℃, 6.5 and Rose Bengal Medium, respectively.2 The soil fungi were classfied by ITS rDNA sequence analysis and phenotype identification, through large-scale isolation methord, Synthetic ITS rDNA sequence analysis and phenotype identification, the following cultured fungi in the alpine grassland of Eastern Qilian Mountains can be drawn: Mortierella spp., Doratomyces spp., Leptosphaeria spp., Cladosporium spp., Verticillium spp., Mucor spp., Tolypocladium spp., Beauveria spp., Hypocrea spp., Truncatella spp., Cordyceps spp., Trichoderma spp., Nectria spp., Didymella spp., Penicillium spp., Bionectria spp., Alternaria spp., Aspergillus spp., Geomyces spp., Fusarium spp., Scopulariopsis spp. and Phaeosphaeria spp. The strains belong to 22 genera, most of them belong to imperfect fungi and zygomycetes. In addition, more than 10 % fungi strains could not clarify the taxonomic status temporarily, which might be new species.3 The diversity and Ecological Characteristics of cultured soil fungi. Species abundance (S), Shannon-Wiener index (H), Simpson dominance index (D) and Pielou evenness index (J) range were 15-18, 2.47-2.81, 0.89-0.93, 0.91-0.97, respectively. Each index fluctuated slowly, and revealed a similar pattern. The Shannon-Wiener index was: Kobresia grassland> Polygonum grassland> Dasiphoru fruticosa shrub land> Salix cupularis fruticosa shrub land> Grass grassland> Rhododendron fruticosa shrub land> Swamp grassland. Except for Pielou index, the others index relatively high in Kobresia grassland and relatively low in Swamp grassland; The results indicate that the diversity of soil fungi were very abundant in the alpine grassland of Eastern Qilian Mountains, and there exist close corelation between fungal diversity and grassland types.4 DGGE analysis of rDNA in 4 types soil fungi, Nested PCR amplification of the ITS1 rDNA, under DGGE optimized condition, soil fungi community structure were analyzed by sequencing and phylogenetic analysis. The results showed, Nested- PCR has high sensitivity, ITS1 rDNA were amplified from micro amounts of nucleic acids, which was about 250 bp in length. The DGGE band sequencing and mapping analysis demonstrated that the DGGE band has big difference in quantity and brightness, and it has same trend as the cultured soil fungi in 4 types grassland.5 The biological characteristics of three Mortierella alpina isolated from alpine grassland in the Eastern Qilian mountains were discussed. The results showed that there were significant differences on their biological characteristics.The optimum nitrogen sources of three M. alpina was Peptone, while the optimum carbon sources of M.alpina 27 was Starch, the optimum carbon sources of M. alpina 60 was Sucros and Gcose was the optimum carbon sources of M.alpina 9. The suitable growth temperature for three M.alpinas was 5～30℃and the optimum of M.alpina 27 was 30℃, while that for M.alpina 60 and 9 was 5℃. All of them can grow at pH 4～9 and pH 7 was the optimum .更多还原