基于磷脂脂肪酸（PLFA）分析技术的土壤微生物群落结构多样性的研究
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摘要
土壤微生物是生态系统的分解者，对于维持生态系统的平衡起着至关重要的作用。然而由于土壤微生物种类的多样性、数量的庞大性和土壤环境的复杂性，人们对于土壤微生物的认识还非常有限。本论文以磷脂脂肪酸(PLFA)技术为切入点，以土壤微生物群落结构为主线，探讨了土壤微生物群落的分布规律，及其多样性对于生态功能的影响，获得以下主要成果：
(1)在国内首次确定了应用PLFA技术对土壤纯培养细菌菌株鉴定的方法和土壤PLFA的提取测定方法，并分别对这两种试验方法的试验条件进行了优化。
(2)采集了我国东部自北而南的14个典型地带性土壤，对土壤微生物PLFAs进行了提取和测定。首次发现我国地带性土壤中微生物如同动植物一样，具有地带性分布规律，表现在北方温带土壤中含有较多的革兰氏阳性菌PLFAs，而南方热带及亚热带土壤中真菌和革兰氏阴性菌PLFAs含量较高，年降水量和土壤pH为影响微生物地带性分布的最主要因子。
(3)以黑龙江黑土、江苏黄棕壤和广西红壤为对象，研究了温度和水分对土壤微生物生物量和群落结构分布的影响，结果表明，土壤微生物对于温度的响应与采样地气候条件有关，表现在黑土土壤微生物群落结构对较高温度(35℃)的响应明显，而红壤中微生物群落结构的变化不大；在40-80％WHC水分范围内，土壤微生物生物量和群落结构没有明显变化。
(4)采集了英国洛桑研究所Highfield长期定位试验上具有约60年不同管理措施的休闲地、农耕地和草地土壤，研究了这三种土壤对于两种不同类型底物(较容易被利用的酵母菌提取物和较难被利用的黑麦草粉末)的矿化作用及土壤微生物生物量和群落结构的变化。结果表明，三种长期不同管理措施的土壤在微生物生物量和群落结构上有显著差异，但外源添加的底物在这些土壤中的矿化以及相对应的土壤微生物生物量的响应差异不大。土壤有机质的矿化主要取决于底物类型。土壤微生物生物量和群落结构与土壤微生物代谢土壤有机质的生态功能并没有直接联系。
(5)以Highfield的农耕地和草地土壤为研究对象，采用DNA-DGGE技术研究了氯仿熏蒸作用对于土壤微生物的影响。结果表明，氯仿熏蒸杀死大部分土壤微生物后，熏蒸土壤对土壤有机质的矿化能力并未减弱，土壤微生物生物量以外的其它因素影响土壤有机质的矿化；熏蒸土壤与仅含耐热孢子体筛选土壤具有相似的DNA-DGGE谱图，土壤微生物可能以孢子体形态在氯仿熏蒸中幸存。
Soil microbes are the decomposers of ecosystem, play an important role in keepingecosystem balance. However, as there are large range of soil microbes and the habitatin which they live is very complicated, our knowledge about this versatile microbialcommunity is very limeted. Thus, the thesis is stringed along soil microbialcommunity structure to study the distribution of soil microbes and the relationshipbetween soil microbial diversity and ecosystem function, based on the phospholipidfatty acid(PLFA) analysis. The following main results were obtained:
The methods about using PLFAs to identify pure soil bacteria strains, extractingand quantifying PLFAs from soils were built first time in China. Moreover, weoptimized the soil PLFAs extraction method, which is an important complementary tosoil PLFA extraction in the world and the first integrated, easy handling PLFAextracting methods in China.
We collected 14 typical climate zonal soils from north to south of China andmeasured the soil microbial PLFAs. The results showed that soil microbes are just likeplants and animals, distributing regularly from north to south. The northern soilscontain more gram positive bacterial PLFAs, while the southern soils have more gramnegative and fungi PLFAs. Mean annual rainfall and soil pH are the two majorcontributors to the distribution of soil microbes in this study.
Besides, Heilongjiang, Jiangsu and Guangxi soils were used to determine theresponses of soil microbes under incubation at different temperature and water content.The results showed that the response of soil microbes to different temperature wascorrelated with the climate history of the sampling site. The microbial biomass andcommunity structure of Heilongjiang soil(from the coldest temperature region)changed most when incubated at 35℃. Guangxi soil from the hottest region changedleast. Both soil microbial biomass and community structure did not change very muchwhen soils were incubated at 40-80% WHC for up to 56 days..
Three very contrasting managements: permanent fallow, permanent arable andpermanent grassland, which have been applied from the same soil for at least 60 years,were used to determine the relationships of community structure and ecosystemfunction(mineralizing labile substrates(yeast extract) and more complex substrate (ryegrass)). The results showed that although the three soils have quite differentmicrobial biomass and community structures, the metabolism of the substrates and theefficiencies of microbial biosynthesis were quite similar. The ability of mineralizationwas mainly determined by the type of substrates. Soil microbial community structuresmeasured by PLFA analysis responded differently to different substrates, mainlydetermined by the history of substrates utilization.
Arable and grassland soils collected from Highfield Ley-Arable experiment wereused to determine the response of soil microbes to chloroform fumigation. Thecommunity structure was indexed by DNA-DGGE method. Chloroform fumigationkilled most but not all microbes. However, the respiration rate did not decrease in thechloroform fumigated soils. Soil microbial community structures after chloroformfumigation were quite similar to that in the heat-resistant spore forming treated soils.Spores may be the predominant microbes left after chloroform fumigation.
(1)在国内首次确定了应用PLFA技术对土壤纯培养细菌菌株鉴定的方法和土壤PLFA的提取测定方法，并分别对这两种试验方法的试验条件进行了优化。
(2)采集了我国东部自北而南的14个典型地带性土壤，对土壤微生物PLFAs进行了提取和测定。首次发现我国地带性土壤中微生物如同动植物一样，具有地带性分布规律，表现在北方温带土壤中含有较多的革兰氏阳性菌PLFAs，而南方热带及亚热带土壤中真菌和革兰氏阴性菌PLFAs含量较高，年降水量和土壤pH为影响微生物地带性分布的最主要因子。
(3)以黑龙江黑土、江苏黄棕壤和广西红壤为对象，研究了温度和水分对土壤微生物生物量和群落结构分布的影响，结果表明，土壤微生物对于温度的响应与采样地气候条件有关，表现在黑土土壤微生物群落结构对较高温度(35℃)的响应明显，而红壤中微生物群落结构的变化不大；在40-80％WHC水分范围内，土壤微生物生物量和群落结构没有明显变化。
(4)采集了英国洛桑研究所Highfield长期定位试验上具有约60年不同管理措施的休闲地、农耕地和草地土壤，研究了这三种土壤对于两种不同类型底物(较容易被利用的酵母菌提取物和较难被利用的黑麦草粉末)的矿化作用及土壤微生物生物量和群落结构的变化。结果表明，三种长期不同管理措施的土壤在微生物生物量和群落结构上有显著差异，但外源添加的底物在这些土壤中的矿化以及相对应的土壤微生物生物量的响应差异不大。土壤有机质的矿化主要取决于底物类型。土壤微生物生物量和群落结构与土壤微生物代谢土壤有机质的生态功能并没有直接联系。
(5)以Highfield的农耕地和草地土壤为研究对象，采用DNA-DGGE技术研究了氯仿熏蒸作用对于土壤微生物的影响。结果表明，氯仿熏蒸杀死大部分土壤微生物后，熏蒸土壤对土壤有机质的矿化能力并未减弱，土壤微生物生物量以外的其它因素影响土壤有机质的矿化；熏蒸土壤与仅含耐热孢子体筛选土壤具有相似的DNA-DGGE谱图，土壤微生物可能以孢子体形态在氯仿熏蒸中幸存。
Soil microbes are the decomposers of ecosystem, play an important role in keepingecosystem balance. However, as there are large range of soil microbes and the habitatin which they live is very complicated, our knowledge about this versatile microbialcommunity is very limeted. Thus, the thesis is stringed along soil microbialcommunity structure to study the distribution of soil microbes and the relationshipbetween soil microbial diversity and ecosystem function, based on the phospholipidfatty acid(PLFA) analysis. The following main results were obtained:
The methods about using PLFAs to identify pure soil bacteria strains, extractingand quantifying PLFAs from soils were built first time in China. Moreover, weoptimized the soil PLFAs extraction method, which is an important complementary tosoil PLFA extraction in the world and the first integrated, easy handling PLFAextracting methods in China.
We collected 14 typical climate zonal soils from north to south of China andmeasured the soil microbial PLFAs. The results showed that soil microbes are just likeplants and animals, distributing regularly from north to south. The northern soilscontain more gram positive bacterial PLFAs, while the southern soils have more gramnegative and fungi PLFAs. Mean annual rainfall and soil pH are the two majorcontributors to the distribution of soil microbes in this study.
Besides, Heilongjiang, Jiangsu and Guangxi soils were used to determine theresponses of soil microbes under incubation at different temperature and water content.The results showed that the response of soil microbes to different temperature wascorrelated with the climate history of the sampling site. The microbial biomass andcommunity structure of Heilongjiang soil(from the coldest temperature region)changed most when incubated at 35℃. Guangxi soil from the hottest region changedleast. Both soil microbial biomass and community structure did not change very muchwhen soils were incubated at 40-80% WHC for up to 56 days..
Three very contrasting managements: permanent fallow, permanent arable andpermanent grassland, which have been applied from the same soil for at least 60 years,were used to determine the relationships of community structure and ecosystemfunction(mineralizing labile substrates(yeast extract) and more complex substrate (ryegrass)). The results showed that although the three soils have quite differentmicrobial biomass and community structures, the metabolism of the substrates and theefficiencies of microbial biosynthesis were quite similar. The ability of mineralizationwas mainly determined by the type of substrates. Soil microbial community structuresmeasured by PLFA analysis responded differently to different substrates, mainlydetermined by the history of substrates utilization.
Arable and grassland soils collected from Highfield Ley-Arable experiment wereused to determine the response of soil microbes to chloroform fumigation. Thecommunity structure was indexed by DNA-DGGE method. Chloroform fumigationkilled most but not all microbes. However, the respiration rate did not decrease in thechloroform fumigated soils. Soil microbial community structures after chloroformfumigation were quite similar to that in the heat-resistant spore forming treated soils.Spores may be the predominant microbes left after chloroform fumigation.
引文
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