【作者】 来航线；

【导师】 王益权；

【作者基本信息】 西北农林科技大学 ， 土壤学， 2008， 博士

【摘要】 盐渍化土壤改良与利用是世界范围内所面临的难题，长期以来在盐渍化土壤生态学研究领域内，人们以实现生物改良与利用为主要目标，开展了许多盐生植物生理生态的科学研究。由于受盐渍化土壤是“生命禁区”思想的长期禁锢，也由于缺乏适宜于盐渍化土壤环境条件下微生物分离、培养方法的系统研究，对盐渍化极端生态环境下土壤微生物类群情况研究至今仍然很少有人问津，极端盐渍环境下微生物资源的开发与利用价值研究更属于空白。为了探索极端盐渍环境条件下土壤微生物生态这个重大科学领域内所蕴藏的秘密，开发特殊生境中微生物资源在植病防治、渔业以及动物养殖等病害防治方面的利用价值，本研究以干旱地区的陕西北部以及宁夏不同类型盐渍化土壤为研究对象，开展了土壤微生物资源与微生物生态的系统研究，探求了适宜于盐渍环境微生物分离、培养的方法，并开展相关盐渍环境中放线菌多相分类鉴定以及在生产实际中应用研究。本研究在盐碱极端生态环境条件下微生物资源研究和放线菌资源开发应用方面迈出了坚实的一步，将为极端生态环境微生物资源研究提供重要的理论基础。本论文从不同典型生态系统中盐渍土的性状研究入手，分析了微生物及放线菌区系构成规律、土壤酶活性与盐渍土的盐分含量、盐分离子组成等因子间的关系；探讨了DGGE法在微生物生态研究中的应用，建立了盐渍极端环境条件下放线菌分离的方法，从中获取了许多放线菌资源；同时以农业生产中常见的危害较为严重的植物和动物病原菌为靶标菌进行拮抗放线菌资源的应用研究，旨在获得具有应用价值的拮抗放线菌；同时采用多相分类技术对形态特殊、生理特征特殊和优良拮抗菌株进行分类研究，确定其分类地位，并获得了几株潜在新物种。通过研究得出以下主要结论：1．获得了盐渍化极端生态环境条件下土壤微生物生态分布规律研究对盐渍化极端生态环境条件下土壤微生物生态分布规律研究得出，盐渍土壤微生物生态分布受盐渍化程度、盐分离子组成及土壤其它理化性质的影响很大。其中细菌和真菌的数量表现出与土壤养分含量、[CO₃^2-+HCO₃^-]／[Cl^-+SO₄^2-]显著的正相关关系，与pH和水溶性总盐含量显著负相关关系；而放线菌却除了与养分含量显著正相关外，与盐渍土的其它因子相关性不强，说明放线菌对土壤盐分含量和pH具有很强的耐受性。不同盐碱极端环境条件下土壤放线菌均具有相对较高的比例分布，进一步揭示了从极端盐碱环境获得放线菌资源的重要性。土壤盐渍化程度不同其中耐盐性放线菌的相对比例明显不同。总体上呈现出重盐化土壤中分布的耐盐放线菌最多，其次为中度盐渍化、轻度盐渍化土壤。根据放线菌生长状况对盐分的依赖关系的进一步研究得出，盐渍土壤中放线菌可以分为具有仅能在盐渍环境条件下才能良好生长的嗜盐性放线菌和对盐渍化的程度具有不同敏感度的耐盐性放线菌。对极端环境中放线菌生态分布及耐盐、嗜盐规律的进一步研究为西北地区其它极端盐渍环境放线菌资源研究提供了理论支撑和科学依据。2．探讨了DGGE法在微生物生态研究中的应用采用DGGE免培养法对宁夏5个典型盐渍土中微生物的多样性进行了研究。通过富集培养后间接提取总DNA与从土壤样品直接提取总DNA，利用DGGE方法进行比较，也与传统的平板计数法进行比较，结果表明富集培养对环境中微生物多样性的分析均优于土样直接提取法和平板计数法，而且从环境盐碱环境中直接提取总DNA分析微生物多样性，应注意DNA的多种提取方法的组合。应用DGGE免培养法可以有效地揭示盐碱环境中微生物物种的多样性，也为进一步发掘盐渍极端环境放线菌资源、研究盐渍化土壤基因特征提供了理论依据。3．建立了适宜于盐碱极端环境条件下放线菌分离的方法体系本次研究探讨了样品的预处理、激活剂、富集培养、培养基设计、盐种类及浓度梯度设计、不同pH设计等对放线菌分离效果的影响，获得了不同盐渍环境样品的预处理方法和适宜的分离培养基，建立了盐碱环境放线菌的分离方法体系，对进一步获得更多的盐渍环境中放线菌的纯培养具有重要的指导意义，也为研究该环境放线菌资源提供了良好的技术支撑。4．分析了盐渍土中放线菌区系组成，筛选出部分具有特殊生理特征的菌株较为系统地分析了盐渍环境中放线菌与其他生态因子的关系，探明了盐渍土中放线菌在不同盐碱环境的分布规律性，盐渍土中放线菌的种属数与土壤pH、[CO₃^2-]、多酚氧化酶活性均为极显著正相关关系；荒地中的放线菌组成比耕地复杂，盐土和龟裂碱土中放线菌组成比其它土壤类型复杂。该地区放线菌生态分布规律为进一步研究盐碱环境放线菌资源提供了良好的理论依据。同时确定了33株形态较特殊的稀有放线菌和22株耐在盐含量高达18％以上的耐盐放线菌菌株，为进一步的通过多相分类技术获得新的分类单元提供了供试菌株。5．获得了盐渍化环境放线菌对9种动植物病原菌的抗菌谱特征，从中筛选出了一批对动物病原菌具有良好拮抗效果的菌株，为特殊生境放线菌资源的进一步开发与利用奠定了基础。。获得了盐碱环境放线菌对农业生产中及畜牧养殖中危害较大的9种动植物病原菌的抗菌谱特征，获得了一批对动物病原菌具有良好拮抗效果的菌株，为畜牧养殖业健康发展中重大疾病的生物防治提供了优良的出发菌株。通过初筛和复筛试验得到了3株优良广谱拮抗放线菌02D01、43A04和13H06。通过生物试验确定出一株发酵液对于引发鱼细菌性肠炎的肠型点状气单胞菌有良好抑制作用的菌株02D01。用药量在2 mg/kg·d就能达到很好的治疗效果，其发酵液对生物无急性毒性，具有较好的温度和酸稳定性。通过发酵工艺优化，确定了02D01菌株产生抑菌活性物质的最佳培养基配方为可溶性淀粉2％，鱼粉2％，NaCl 2％，K₂HPO₄ 0．05％，MgSO₄．7H₂0 0．05％，CaCO₃ 0．1％；在种龄为48 h，接种量5％，25℃，初始pH 9，发酵时间120 h发酵液的抑菌活性为最大，发酵液效价可达到34197．9μg／mL，比初始效价提高27倍。6．完善了适宜于盐碱极端环境条件下放线菌的多相分类技术，初步确定了部分特殊放线菌株的分类学地位本研究采用了形态学、生理生化特征、细胞化学、以及16S rDNA序列分析等多相分类的技术方法对分离筛选的供试放线菌进行了系统的分类研究。确定产特殊抑菌活性物质菌株02d01、13h06、14j18、43a04和56h02为链霉菌（Streptomyces），形态特殊菌株09j02为拟无枝酸菌（Amycolatopsis），耐盐菌株59h05为诺卡氏菌（Nocardia），其他形态特殊菌株和耐盐菌株均为拟诺卡氏菌（Nocardiopsis）。经构建供试菌株与拟诺卡氏菌属各种间的16S rDNA序列系统进化树分析，菌株09j03和47d01与N．synnemataformans，菌株04d01与N．synnemataformans，菌株68d14与N．dassonvillei subsp．Albirubida、菌株20f03和22f02与N．exhalans可能为同种，但有待于DNA-DNA杂交来证实。菌株07j03和21f15极可能给予新种的分类地位，但若想确定到种的水平同样需要借助DNA-DNA同源性分析即进一步确定。本研究较为系统的采用的多相分类技术，并依据盐碱极端环境的生态特征，对很多方法进行了修改，建立了一套较为系统的、适应于盐碱环境放线菌分类鉴定的多相分类技术体系。为该环境的放线菌系统分类奠定了良好的理论基础。更多还原

【Abstract】 The melioration of saline and alkali soils is a difficult topic faced in the world. For long time, in the study filed of saline and alkali soil ecology, the research has been focused on plant physiology and bio-ecology. However, the research on microorganism in saline and alkali extreme environments is little because there are some old ideas that saline and alkali soils were life forbidden zone and shortage of the suitable methods for microorganism isolation and culture. Therefore, the exploitation and application of microorganism resources in saline and alkali extreme environments are very scarce. In order to investigate the secret and mechanisms of microorganism-ecological disciplines and exploitation and application of microorganism resources in plant, fishing and animal feeding for disease preventing and killing, saline and alkali soils in Ningxia, Shaanxi was used in this research for systematically studying the microorganism ecology and resource, and detecting the isolation and culture methods which are suitable for microorganism in saline-alkali extreme environment. At the same time, polyphasec techniques were used for classification and identification of microorganism in extreme environments and applied the techniques to practice. These will push the study on microorganism resource in saline-alkali extreme eco-environment advanced and forward the exploitation of actinomycetes resource. All that will be the solid foundation for the research of microorganism resource in saline-alkali extreme environment.This dissertation started with the studies on characteristics of saline and alkali soils in different typical ecology environments, analyzed the composition and population of microorganism and actinomycetes, and the relationships between them to enzyme activity, the primary properties of saline and alkali soils, salt ions constituent and other factors. DGGE as a method was employed in microorganism-ecological studies. The isolation methods of actinomycetes in saline-alkali extreme environments were built so as to obtaining a lot of actinomycetes. Meanwhile, application research of antagonistic were done by adopting the most common and dangerous botanical and animal pathogens in agricultural production as target pathogens in order to get some antagonistic actinomycetes with application values; At the same time, polyphasec techniques were used for classification of actinomycetes with special morphology or physiological characteristics and strains with high antagonistic function. The classification status of the actinomycetes was determined and several potential new species were obtained. The main results are as follows:1. The distribution principle of microbial ecology in saline and alkali extreme environments was obtainedBased on this research, the results showed that the distribution of microbial ecology in soil samples was greatly affected by soil types, salt ions constituent and soil physical and chemical properties. The bacterial and fungi amounts have significant positive correlation with nutrient content and [CO₃^2-+HCO^3-]/[Cl^-+SO₄^2-], and it was significant negative correlation with pH value and water-soluble salt content. However, actinomycetes was significant positive correlation with nutrient content, but was not correlated with the other factors. These indicate that actinomycetes have strong toleration to salt content and pH value. There was large population proportion of actinomycetes in different saline-alkali soils. This further revealed the importance of obtaining actinomycetes resources from extreme environments.The population distribution of shalotolerant actinomycete was different from various salinization degrees of soils. As a whole, population of shalotolerant actinomycete distributed in heavy salinization soil was the most, and followed by in moderate salinization soil and in light salinization soil. Based on the development of actinomycetes depended on salt content, actinomycetes can be divided into halophilic actinomycete developing well only in saline-alkali soil and halotolerant actinomycete with different susceptivity to salt content. The population distribution of actinomycete in extreme environment and the characteristic of halophilic and halotolerant actinomycete will provide the theoretical support and science foundation for the studies on actinomycete resources.2. Detection of the application of DGGE method in microbial ecology researchSoil microbial diversity in five typical saline-alkali soils was studied with the DGGE free-culture method. The total DNA indirectly extracted from the enrichment culture and the total DNA extracted directly from soil samples using DGGE method were compared. And it was also compared with the traditional plate count method. The results showed that the enrichment culture of microbial diversity analysis was better than directly extraction from soil samples and plate count. DNA direct extraction from environment access to the population and types of micro-organisms had obvious advantages, but the methods and effects of DNA extraction should be noted. This approach effectively reveals the microbial species diversity in saline environment, and also provides a theoretical basis for further explore the actinomycetes resources in extreme environments.3. Establishment of the isolation method system of actinomycetes in salinized extreme environmentsThis study investigated the effects of pretreatment of samples, activators, enrichment culture, medium design, salt types and concentrations design, different pH and others on the isolation effects of actinomycetes. The pretreatment methods to different saline samples and feasible isolation mediums were obtained. The isolation method systems of actinomycetes in saline environment were preliminarily established. These are very important for obtaining more actinomyceres in saline environment as well as for providing great technical support for the actinomyceres research.4. Analysis the components and population of actinomycetes flora in saline soil and selection of some strains with special physiological characteristicsThe relationshiip between actinomycetes and other ecological factors in saline soil was analyzed systematically and the distribution of actinomycetes in different environments was studied. The relationship between actinomycetes population and soil pH, [CO₃^2-], polyphenol oxidase activity are very significant positive correlated. Aactinomycetes composition in waste land is more complex than in arable land. In saline soil or in alkali soil, actinomycetes composition is more complex than other soil type. The ecological distribution of actinomycetes provides good theoretical basis for further studies on actinomycetes resources in saline-alkali soil. Simultaneously, 33 rare actinomycetes with specific form and 22 actinomycetes with salt-tolerant more than 18 percent salt content were identified, which providing a strain for the further study by the multi-phase classification technologies in order to obtain new taxonomy system.5. Determination of the characteristics of antibacterial spectrum of habitat salinity actinomycetes to nine kind animal and plant pathogens and Selection of some strains which had good antagonism.It was obtained that the characteristics of antibacterial spectrum of habitat salinity actinomycetes to nine kind animal and plant pathogens. Some strains with good antagonistic effects on animal pathogens were screened, which could be better strains for the biological prevention of major disease in animal husbandry. Three strains of actinomycetes 02D01, 43A04 and 13H06 with good antagonism spectrum were selected according to screening and re-screening.A strain 02D01 was found that its fermentation solution had good Inhibition effect on Point Aeromonas causing Fish bacterial enteritis. Dosage of 2 mg /kg·d could reach good treatment, and the fermentation solution had no acute toxicity but good temperature and acid stability.Through fermentation technology optimization, the best culture contained 2% soluble starch, 2% fish powder, 2% NaCl, 0.05% K₂HPO₄, 0.05% MgSO₄·7H₂O, 0.1% CaCO₃, in which the strain could grow and produce antibacterial substances, and the activity were best at the seed age of 48 h, inoculum of 5%, temperature of 25℃, initial pH of 9, fermentation time for 120 h, fermentation solution titer could reach 34197.94μg/ml, improved 27 times, which could be basis for deep production and application.6. The multiple taxonomy technology of actinomycetes habitated in salinity was perfected and classification status of some special actinomycetes was determined.In the study, morphology, physiological and biochemical characteristics, cytochemistry and 16S rDNA sequence and other multiple taxonomy technologies were used for the classification of actinomycetes isolated and screened from saline-alkali soils. The strains 02d01、13h06、14j18、43a04 and 56h02 producing antibacterial substances were identified as Streptomyces, and strain09j02 with special morphology as Amycolatopsis, salinity-resistance strain 59h05 as Nocardia, other strains all as Nocardiopsis.Strain 09j03, 47d01 and N.synnemataformans, 04d01 and N.synnemataformans, 68d14 and N.dassonvillei subsp. Albirubida, 20f03, 22f02 and N.exhalans may be the same species. Strain 07j03 and 21f15 may be a new species, but need be confirmed through DNA-DNA hybrid.Based on revising many methods and ecological characteristics of salinity environment a multiple taxonomy technology system was founded, which was adapted to the classification and identification of actinomycetes in salinity environment. This technology system set up the foundation for the classification of actinomycetes in extreme environments.更多还原