土壤微生物多样性研究的DGGE/TGGE技术

分子生物学技术比传统的培养方法可得到土壤微生物种群多样性更全面的信息。变性梯度凝胶电泳(Denaturing gradient gel electrophoresis, DGGE)和温度梯度凝胶电泳(Temperature gradient gel electrophoresis, TGGE)可分离PCR扩增的DNA片段,已成为研究土壤微生物群落多样性的重要手段。本文综述了DGGE/TGGE技术在土壤微生物多样性研究中的应用进展,分析了该方法的主要影响因素及其优点和存在的问题。     

指纹图谱技术在动物肠道微生物多样性研究中的应用

随着分子生物技术的发展,不可培养微生物多样性研究的难题得到了解决。肠道微生物处于特殊的生态环境条件下,分子生物学技术的应用使得肠道微生物多样性的研究进入了一个崭新的阶段。本文主要介绍了基于16S rRNA基因片段的一些肠道微生物研究工作中常用的分子生物学分析方法,主要包括变性梯度凝胶电泳(DGGE),温度梯度凝胶电泳(TGGE),单链构象多态性(SSCP),限制性片段长度多态性(RFLP),放大片断长度多态性(AFLP)和随机扩增多态性DNA(RAPD)等指纹图谱技术。     

三种土壤微生物总DNA提取方法的比较

本文对3种常用的土壤微生物总DNA提取方法Martin法、高盐改进法及试剂盒法进行了比较,并通过DNA得率、纯度及16S rDNA V3可变区的PCR扩增结合DGGE法（denatumg gradient gel electrophoresis）,分别对3种方法进行评价。结果表明,3种方法提取的DNA均能满足土壤微生物多样性分析的要求。其中试剂盒方法操作简单,提取的DNA质量较高,但DNA得率较低且成本昂贵。Martin法和高盐改进法用时较长,DNA得率较高,纯度较低,但对后续PCR扩增和DGGE分析没有明显影响,且成本低廉。     

变性梯度凝胶电泳(DGGE)技术在土壤微生物多样性研究中的应用

应用变性梯度凝胶电泳(DGGE)谱图分析技术对土壤微生物多样性进行描述,可以克服传统微生物分离纯化培养方法和显微技术的局限性.本文介绍了变性梯度凝胶电泳指纹图谱分析方法及其在土壤微生物多样性中的应用,包括对土壤特殊微生物生理类群,自然环境条件下微生物多样性变化,污染土壤微生物的多样性,不同种植制度下的土壤微生物多样性,转基因植物、微生物介入的土壤微生物多样性等方面的研究.     

不同年限毛竹林土壤固氮菌群落结构和丰度的演变

应用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和荧光定量PCR(real time fluorescent quantitative PCR,q PCR)方法研究了不同年限毛竹林土壤固氮菌群落结构和丰度的变化。结果表明:土壤p H、有机质、有效磷、速效钾和铵态氮含量在马尾松林改造成毛竹林5 a后明显提高,而后逐渐降低,并趋于稳定;土壤固氮菌多样性和nif H基因丰度也呈现相似的趋势。条带测序分析表明,毛竹林土壤固氮菌均为不可培养的固氮菌,与慢生根瘤菌(Bradyrhizobium sp.)具有较高的相似度。冗余分析结果表明,不同栽培年限毛竹林地土壤固氮菌群落组成发生了明显变化,长期栽培毛竹林引起的土壤养分变化对土壤固氮菌多样性具有重要影响。     

土壤微生物群落多样性研究方法及进展

微生物多样性是指群落中的微生物种群类型和数量、种的丰度和均度以及种的分布情况。研究土壤微生物群落多样性的方法包括传统的以生化技术为基础的方法(直接平板计数、单碳源利用模式等)和以现代分子生物技术为基础的方法(从土壤中提取DNA,进行G C%含量的分析,或杂交分析,或进行PCR,产物再进行DGGE/TGGE等分析)。现代生物技术与传统微生物研究方法的结合使用,为更全面地理解土壤微生物群落的多样性和生态功能提供了良好的前景。     

等碳投入的有机肥和生物炭对红壤微生物多样性和土壤呼吸的影响

施用有机肥是快速培育瘠薄土壤的一个重要措施。针对中亚热带第四纪红黏土发育的红壤旱地,建立了玉米和花生单作系统等碳量投入有机肥和生物炭的田间试验,利用聚合酶链式反应—变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)方法研究了土壤细菌和真菌群落组成和多样性的变化,分析了土壤呼吸速率(CO2通量)的变化及其与微生物多样性的关系。两年的试验表明,不同施肥方式导致微生物群落结构显著分异,施用有机肥和生物炭显著增加了细菌多样性,但施肥第二年真菌多样性有下降趋势。秸秆和猪粪配施显著增加了土壤呼吸速率,土壤呼吸速率与细菌和真菌多样性呈显著正相关,细菌多样性对土壤呼吸的影响(相对贡献率为71%)显著高于真菌(29%)。土壤磷素(全磷和速效磷)含量的变化是驱动红壤微生物多样性变化的主导因素,其对细菌和真菌多样性的相对贡献率分别为44.8%和47.4%。因此,合理配施秸秆和猪粪可以快速提高瘠薄红壤的生物功能。     

我国中亚热带毛竹林土壤微生物群落的空间分布特征及其影响因素

针对我国中亚热带毛竹林主要分布区,在福建、浙江、湖南、江西沿经度和纬度设置2个采样带,从5个县(市)采集了15个表层(0~20 cm)土样和15个土壤剖面(0~60 cm),利用磷脂脂肪酸(phospholipid fatty acids,PLFAs)和聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)方法研究毛竹林土壤微生物群落空间分布特征与剖面分布特征。结果表明:毛竹林表层土壤微生物生物量和细菌α多样性指数的地带性变化趋势不显著,但不同地点的土壤微生物群落结构存在显著差异;气候因子和土壤理化性质共同影响了土壤微生物的群落结构,但气候因子的影响随土壤剖面深度增加而减弱。毛竹林土壤细菌β多样性与距离之间存在显著的衰减关系,表层(0~20 cm)土壤细菌群落结构相似度(β多样性)随空间距离的衰减速率低于亚表层(20~40 cm)土壤,这可能与毛竹林根系的影响有关。总体上,环境选择和扩散限制共同影响了毛竹林土壤微生物的空间分布状况。     

土壤微生物群落结构研究方法进展

分离培养技术在土壤微生物的研究中有很大的局限性，因为土壤中的微生物大部分还处于不能被分离培养状态。随着微生物研究技术的发展尤其是分子生物学技术的发展，一系列不依赖于培养的技术在土壤微生物研究中得到广泛应用。本文介绍了生物标志物法、SCSU、DNA复性分析、DGGE、TGGE、ARDRA、T-RFLP、SSCP、PAPD和ERIC—PCR图谱分析等方法在土壤微生物群落结构研究中的应用。     

广谱抗真菌蛋白转基因水稻秸秆模拟还田对土壤真菌群落结构的影响

为探讨广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响,本文在室温条件下进行田间秸秆还田模拟试验,设不添加秸秆(S)、添加转基因水稻‘转品1’秸秆(S-Z1)、添加转基因水稻‘转品8’秸秆(S-Z8)、添加非转基因水稻‘七丝软粘’秸秆(S-CK)4个土壤处理,采用传统的平板计数法和变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)技术,分析广谱抗真菌蛋白转基因水稻秸秆模拟还田过程中土壤可培养真菌数和土壤真菌群落的变化情况。平板计数结果表明,在秸秆降解的第40 d,转基因水稻秸秆处理(S-Z1、S-Z8)与非转基因水稻秸秆处理(S-CK)土壤之间的可培养真菌数差异显著,但秸秆降解中后期(50~90 d),S-Z1、S-Z8和S-CK之间土壤可培养真菌数的差异均不显著。真菌18S r RNA的PCR-DGGE图谱显示,S-Z1、S-Z8和S-CK在秸秆降解过程中没有显著不同的条带出现,仅有个别条带在亮度上存在差异。DGGE图谱条带多样性分析结果表明,在秸秆降解的个别时间段,S-Z1、S-Z8和S-CK之间在丰富度和Shannon-Wiener多样性指数上存在显著差异,而在秸秆降解的整个过程均匀度指数差异均不显著。对DGGE主要条带和差异性条带进行克隆测序后发现,子囊菌占最大比重,其次为担子菌、壶菌,而在转基因和非转基因土壤处理间亮度上存在差异的条带属于子囊菌。以上研究结果表明,广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响是短暂的、不持续的。     

氯氰菊酯与铜复合污染对土壤微生物群落的影响

采用常规手段研究了土壤在受氯氰菊酯、铜及二者复合污染后土壤呼吸率及微生物量碳的变化,采用了分离效果较好的双变性梯度凝胶电泳（DG—DGGE）技术研究微生物群落的变化。结果表明,低浓度的铜与高浓度的氯氰菊酯复合污染更能促进微生物量碳及土壤呼吸率的增加,微生物的群落结构也会受到明显影响。而两种污染物分别单独作用时,铜对微生物的胁迫更大,有铜组和无铜组在DGGE条带上差异较大,Shannon指数上也有明显不同。当铜的浓度较高时,加入高浓度的氯氰菊酯在较长的时间后（60d）对土壤呼吸作用、微生物量碳有一定影响,可能高浓度氯氰菊酯的加入在一定程度上减弱了高浓度铜对微生物的胁迫,而微生物群落并无显著的变化。     

现代气候变化情景下土壤微生物活性和碳动态研究进展

Microbial activities are affected by a myriad of factors with end points involved in nutrient cycling and carbon sequestration issues. Because of their prominent role in the global carbon balance and their possible role in carbon sequestration, soil microbes are very important organisms in relation to global climate changes. This review focuses mainly on the responses of soil microbes to climate changes and subsequent effects on soil carbon dynamics. An overview table regarding extracellular enzyme activities (EAA) with all relevant literature data summarizes the effects of different ecosystems under various experimental treatments on EAA. Increasing temperature, altered soil moisture regimes, and elevated carbon dioxide significantly affect directly or indirectly soil microbial activities. High temperature regimes can increase the microbial activities which can provide positive feedback to climate change, whereas lower moisture condition in pedosystem can negate the increase, although the interactive effects still remain unanswered. Shifts in soil microbial community in response to climate change have been determined by gene probing, phospholipid fatty acid analysis (PLFA), terminal restriction length polymorphism (TRFLP), and denaturing gradient gel electrophoresis (DGGE), but in a recent investigations, omic technological interventions have enabled determination of the shift in soil microbe community at a taxa level, which can provide very important inputs for modeling C sequestration process. The intricacy and diversity of the soil microbial population and how it responds to climate change are big challenges, but new molecular and stable isotope probing tools are being developed for linking fluctuations in microbial diversity to ecosystem function.     

Distribution of microbial communities in a forest soil profile investigated by microbial biomass, soil respiration and DGGE of total and extracellular DNA

We studied the distribution of the indigenous bacterial and fungal communities in a forest soil profile. The composition of bacterial and fungal communities was assessed by denaturing gradient gel electrophoresis (DGGE) of total and extracellular DNA extracted from all the soil horizons. Microbial biomass C and basal respiration were also measured to assess changes in both microbial biomass and activity throughout the soil profile. The 16S rDNA-DGGE revealed composite banding patterns reflecting the high bacterial diversity as expected for a forest soil, whereas 18S rDNA-DGGE analysis showed a certain stability and a lower diversity in the fungal communities. The banding patterns of the different horizons reflected changes in the microbial community structure with increasing depth. In particular, the DGGE analysis evidenced complex banding patterns for the upper A1 and A2 horizons, and a less diverse microflora in the deeper horizons. The low diversity and the presence of specific microbial communities in the B horizons, and in particular in the deeper ones, can be attributed to the selective environment represented by this portion of the soil profile. The eubacterial profiles obtained from the extracellular DNA revealed the presence of some bands not present in the total DNA patterns. This could be interpreted as the remainders of bacteria not any more present in the soil because of changes of edaphic conditions and consequent shifting in the microbial composition. These characteristic bands, present in all the horizons with the exception of the A1, should support the concept that the extracellular DNA is able to persist within the soil. Furthermore, the comparison between the total and extracellular 16S rDNA-DGGE profiles suggested a downwards movement of the extracellular DNA.     

Effect of pruned material,extracts, and polyphenols of tea on enzyme activities and microbial community structure in soil

Pruning is a regular management practice performed in tea fields, but its effects on soil properties and microbial community structures have not been well explored. Effects of tea pruned material, tea extracts, and tea polyphenols application on soil properties and microbial characteristics, including pH, soil enzyme activities, culturable microbial population, and bacterial community, were investigated in a pot-incubation experiment lasting for 10 weeks. Results indicated that returning of tea pruned material or its extracts significantly increased the soil pH as compared to control. The soil invertase and urease activities, as well as the culturable microbial population treated with pruned material were much higher than those in control, while polyphenol oxidase activity was similar among different treatments. Denaturing gradient gel electrophoresis analysis of bacterial community indicated that the Shannon’s diversity index (H) of all nine treatments receiving additives slightly decreased as compared to control. Pruning returning specifically induced several beneficial bacterial species, including Brevibacterium sp. and Burkholderia sp. In conclusion, pruning returning alleviated soil acidification, induced soil enzyme activities, stimulated culturable microbial population, and reshaped the microbial community. This research suggests that this management could be applied as an alternative agricultural practice in tea cultivation.     

Response of soil microbial abundance and diversity in Sacha Inchi (Plukenetia volubilis L.) farms with different land-use histories in a tropical area of Southwestern China

Land-use conversion affects the soil community and microbial abundance, which are essential dynamic indicators of soil quality and sustainability. However, little to no work has been performed to analyse the impact from different land-use histories (i.e. fallow, tea, rice, banana, and maize) on the microbial abundance and diversity in the soil of sacha inchi (Plukenetia volubilis L.). Real-time quantitative PCR (qPCR) was performed to quantify soil bacterial and fungal abundance. Denaturing gradient gel electrophoresis (DGGE) combined with cloning and sequencing was used to assess the microbial communities. Our results showed that the bacterial and fungal abundance in fallow land-use conversion soils was significantly lower than that in the other four land-use conversion soils (tea, rice, banana, and maize). Moreover, the highest abundance of bacteria and fungi was detected in the soils converted from maize to sacha inchicultivation. In addition, canonical correspondence analysis (CCA) showed that the total N and pH were significantly related to bacterial and fungal community structures. These results suggest that land-use conversion from maize fields to sacha inchi farms is an effective way to maintain the soil microbial quantity and hence the sustainability of the soil.     

Bacterial communities are more dependent on soil type than fertilizer type, but the reverse is true for fungal communities

The soil microbial community is strongly influenced by a wide variety of factors, such as soil characteristics and field management systems. In order to use biological indicators based on microbial community structure, it is very important to know whether or not these factors can be controlled. The present study aimed to determine whether soil type or fertilization has a greater influence on the soil microbial community based on denaturing gradient gel electrophoresis (DGGE) analysis of 12 experimental field plots containing four different soil types, Cumulic Andosol, Low-humic Andosol, Yellow Soil and Gray Lowland Soil, kept under three different fertilizer management systems since 2001 (the application of chemical fertilizer, the application of rice husk and cow manure, and the application of pig manure). Bacterial DGGE analysis using 16S rRNA genes and fungal DGGE analysis using 18S rRNA genes revealed that the bacterial community was related to the soil type more than the fertilization; however, the fungal community was related to the fertilization more than the soil type. These results might suggest that the fungal community is easier to control by fertilization than the bacterial community. Thus, we propose that indicators based on the fungal community might be more suitable as microbial indicators for soil quality.     

抑制烟草青枯病型生物有机肥的田间防效研究

【目的】烟草青枯病是影响烟叶生产最主要的土传病害之一,生物防控烟草青枯病是近年来的研究热点。为验证抑制烟草青枯病型生物有机肥对生防青枯病的影响,进行2年田间试验研究其防效及其对土壤微生物的影响。【方法】本试验采用从烟草根际原位土壤分离得到的烟草青枯病拮抗菌株L-25和L-9,利用有机肥二次固体发酵技术,制成烟草青枯病拮抗生物有机肥。连续两年在安徽进行田间试验,分别在烟草移栽后50天和105天调查生物防控率,探求生物有机肥对青枯病的田间防效和对烟叶产量的影响;利用平板计数法、Biolog特征性碳源法和梯度变性凝胶电泳法摸索生物有机肥施用后根际土壤微生物数量、群落功能多样性和结构多样性的变化,揭示生物有机肥对青枯病的防控机理。【结果】 1）第一年和第二年施用生物有机肥处理移栽50天后烟草青枯病的生物防控率分别为82.2%和96.2%,105天后烟草青枯病的生物防控率分别达到75.2%和95.4%; 2）生物有机肥处理第一年和第二年烟叶产量分别为2212.5 kg/hm2、1475.5 kg/hm2,是对照的2.4和2.6倍; 3）两年生物有机肥处理的根际土壤可培养细菌、放线菌数量均显著高于对照,真菌数量显著低于对照（P＜0.05）,其中第一年生物有机肥处理50天和105天拮抗菌数量分别为对照的241.8倍和13.4倍,病原菌数量仅为对照的19.7%和56.6%,第二年生物有机肥处理50天和105天拮抗菌数量分别是对照的111.0倍和26.7倍,病原菌数量仅为对照的9.1%和31.4%; 4）两年生物有机肥处理50天和105天根际土壤的微生物群落功能多样性,即Shannon指数、Simpson指数和Mclntosh指数均显著高于对照（P＜0.05）; 5）生物有机肥处理与对照土壤微生物群落结构也不相同,细菌和真菌梯度变性凝胶电泳图谱明显不同,分别属于不同的聚簇,生物有机肥处理的细菌种类较对照有所增加,同时真菌的种类有所减少。【结论】在烟草青枯病发病较为严重的烟田施用生物有机肥,可以显著降低青枯病发病率,增加烟草产量。生物有机肥可以显著提高拮抗菌数量,抑制根际土壤病原菌的数量,提高土壤微生物群落功能多样性,改善微生物种群和组成,丰富土壤微生物群落结构,使土壤保持健康的微生物生态平衡。     

Gaseous carbon emission in relation to soil carbon fractions and microbial diversities as affected by organic amendments in tropical rice soil

The interactive effects of moisture and organic amendments (farmyard manure (FYM), crop residue (CR) and green manure (GM) (Sesbania aculeata) on gaseous carbon (C) emission, soil labile C fractions, enzymatic activities and microbial diversity in tropical, flooded rice soil were investigated. The amendments were applied on equal C basis in two moisture regimes, that is, aerobic and submergence conditions. The CO₂ production was significantly higher by 22% in aerobic than in submergence condition; on the contrary, the CH₄ production was 27% higher under submergence condition. The labile C fractions were significantly higher in GM by 26% under aerobic and 30% under submergence conditions, respectively, than control (without any kind of fertilizer or amendments). Eubacterial diversity identified by PCR-DGGE method (polymerase chain reaction coupled with denaturant gradient gel electrophoresis) was higher under GM followed by FYM, CR, and control and it is pronounced in submerged condition. GM favored the labile C accumulation and biological activities under both submergence and aerobic conditions, which makes it most active for soil–plant interactions compared to other organic amendments. Considering environmental sustainability, the use of GM is the better adoptable option, which could enhance labile C pools, microbial diversities in soil and keep soil biologically more active.