镉胁迫下稻田土壤微生物基因多样性的DGGE分子指纹分析

采用现代分子生物学技术,避开传统的分离培养过程,探讨重金属镉污染条件下稻田土壤微生物种群的基因多样性.经过直接从土壤中抽提总DNA,并对总DNA中16S rDNA及其中 V3可变区序列作PCR扩增、变性梯度凝胶电泳(DGGE)分析等,对镉胁迫下稻田土壤总DNA、微生物种类分布进行了初步的研究,发现不同浓度镉胁迫下稻田土壤间的菌种有明显差异,DGGE技术可以用于污染环境下微生物多样性研究.同时对DGGE分子指纹图谱的生物信息学分析作了初步尝试,为污染环境下土壤微生物多样性研究提供了新的实验方法与依据.     

莠去津对野生植物群落下土壤微生物功能多样性的影响

为探明亚致死剂量农药对土壤微生物多样性影响,基于大田控制试验,采用Biolog-ECO微平板技术分析喷施亚致死剂量的莠去津对野生植物群落下土壤微生物群落功能多样性的影响.结果表明:① 莠去津的喷施浓度对土壤微生物群落利用全部碳源的能力无显著影响（P>0.05）,但与喷施处理后第30天相比,处理后第60天的土壤微生物群落利用全部碳源的能力有所降低,其中处理Ⅰ〔喷施浓度（以质量浓度计）为1 200 g/hm2〕、处理Ⅲ（喷施浓度为300 g/hm2）显著下降（P < 0.05）,而CK（对照组）、处理Ⅱ（喷施浓度为600 g/hm2）降低程度不显著（P>0.05）,并且其各处理间土壤微生物群落对全部碳源的利用能力大小为CK >处理Ⅲ >处理Ⅱ >处理Ⅰ.② 喷施处理后第30天土壤微生物群落对碳水化合物类碳源的利用能力最强,对羧酸类碳源的利用能力相对较弱;喷施处理后第60天土壤微生物群落对碳水化合物类碳源的利用能力降幅最大,处理Ⅰ、处理Ⅱ、处理Ⅲ分别下降了0.63、0.78、0.85、0.76.③ 喷施除草剂后第30天处理Ⅱ的Shannon-Wiener指数和Simpson指数显著低于其他处理（P < 0.05）,而第60天各处理间多样性指数无显著差异;与喷施处理后第30天相比,第60天各处理多样性指数都有所下降,但没有显著差异（P>0.05）.④ 对土壤微生物群落碳源利用能力进行主成分分析结果显示,提取的与土壤微生物碳源利用相关的主成分累积贡献率为86.6%,主成分1能够区分喷施处理后两个时期的土壤微生物群落特征.⑤ 冗余分析（RDA）表明,pH是土壤微生物利用碳源能力差异的重要影响因素之一.研究显示,除草剂莠去津的施用可在一定程度上降低野生植物群落下土壤微生物群落功能多样性.      

除草剂硝磺草酮对土壤微生物生态效应研究

采用室内培养试验,研究了硝磺草酮对土壤酶活性和微生物群落功能多样性的影响.结果表明,土壤过氧化氢酶和蔗糖酶活性随硝磺草酮浓度增加呈现先增加后降低的趋势,但比对照处理都有所升高,分别增加了22.6%~41.0%和3.4%~54.2%,且在硝磺草酮浓度为50mg/kg时达到最大.与之相反,尿酶活性则降低了12.0%~18.6%,但差异性不明显(P > 0.05).施用一定浓度的硝磺草酮激活了土壤微生物活性,微生物群落丰富度、均匀性和多样性都呈增长趋势,单孔的平均颜色变化率(AWCD)值和利用速率均随硝磺草酮浓度增加而增大,微生物群落对碳水化合物类、氨基酸类、多聚物类、羧酸类、胺类和酚酸类利用率整体上均有所提高,与对照相比,最大增幅分别达到5.3、1.0、4.4、3.2、0.2和6.8倍,但不同浓度硝磺草酮处理下土壤微生物在利用碳源的类型上是存在一定的差异.     

Effects of plant species coexistence on soil enzyme activities and soil microbial community structure under Cd and Pb combined pollution

The relationship between plant species coexistence and soil microbial communities under heavy metal pollution has attracted much attention in ecology. However, whether plant species coexistence could o set the impacts of heavy metal combined pollution on soil microbial community structure and soil enzymes activities is not well studied. The modified ecological dose model and PCR-RAPD method were used to assess the e ects of two plant species coexistence on soil microbial community and enzymes activities subjected to Cd and Pb combined stress. The results indicated that monoculture and mixed culture would increased microbe populations under Cd and Pb combined stress, and the order of sensitivity of microbial community responding to heavy metal stress was: actinomycetes > bacteria > fungi. The respirations were significantly higher in planted soil than that in unplanted soil. The plant species coexistence could enhance soil enzyme activities under Cd and Pb combined. Furthermore, planted soil would be helpful to enhance soil genetic polymorphisms, but Cd and Pb pollution would cause a decrease on soil genetic polymorphisms. Mixed culture would increase the ecological dose 50% (ED50) values, and the ED50 values for soil enzyme activities decreased with increasing culture time. The dehydrogenase was most sensitive to metal addition and easily loses activity under low dose of heavy metal. However, it was di cult to fully inhibit the phoshpatase activity, and urease responded similarly with phosphatase.     

Effect of heavy metals on soil microbial activity and diversity in a reclaimed mining wasteland of red soil area

The microbial biomass, basal respiration and substrate utilization pattern in copper mining wasteland of red soil area, southern China, were investigated. The results indicated that soil microflora were obviously different compared with that of the non-mine soil. Microbial biomass and basal respiration were negatively affected by the elevated heavy metal levels. Two important microbial ecophysiological parameters, namely, the ratio of microbial biomass C（ Cmic ）/organic C（ Corg ） and metabolic quotient（qCO2 ） were closely correlated to heavy metal stress. There was a significant decrease in the Cmic/Corg ratio and an increase in the metabolic quotient with increasing metal concentration. Multivariate analysis of Biolog data for sole carbon source utilization pattern demonstrated that heavy metal pollution had a significant impact on microbial community structure and functional diversity. All the results showed that soil microbiological parameters had great potential to become the early sensitive, effective and liable indicators of the stresses or perturbations in soils of mining ecosystems.     

重金属复合污染农田土壤的微生物群落遗传多样性研究

采集了浙江省富阳市环山乡某冶炼厂小高炉附近受铜、锌、铅、镉不同程度复合污染的4个农田土壤样品，首先扩增土壤总DNA中的16S rDNA，然后进行变性梯度凝胶电泳(Denaturing Gradient Gel Electrophoresis)，分析了长期受重金属复合污染的农田土壤的微生物群落遗传多样性变化．结果表明，不同程度的重金属复合污染明显改变了农田土壤的微生物群落遗传多样性，但与多样性的改变不是简单的负相关关系，最大的多样性指数出现在中等污染程度的土壤中．     

三叶鬼针草幼苗对镉污染的耐性及其吸收积累特征研究

通过盆栽实验研究了镉(Cd)胁迫下三叶鬼针草(Bidens pilosaL.)幼苗的耐性能力(包括生长反应和生理生化特性)以及对Cd的吸收积累特征.结果表明,当土壤中投加的Cd浓度为≤32 mg/kg时,三叶鬼针草植物的生长和发育没有受到抑制,与对照相比,地上部和根部生物量分别增加了32.4%~44%和29.1%~57.6%,其中,当Cd处理浓度为8 mg/kg时,地上部干重达到最大值,为0.22 g/pot.在不同Cd处理下,叶绿素(Chl)和可溶性蛋白(SP)含量下降,分别比对照处理降低了23.3%和41.5%;超氧化物歧化酶(SOD)活性随着Cd浓度增加先有所抑制,后逐渐增大;过氧化物酶(POD)活性和丙二醛(MDA)含量随Cd处理浓度增加而增大,分别比对照增加了1.2~6.6和1.1~1.5倍.然而,当Cd浓度为50~100 mg/kg时,显示出对三叶鬼针草幼苗的生长发育和生理生化特征都产生了一定的负效应,说明三叶鬼针草对Cd污染的耐性能力具有一定的阈值.在梯度实验中,植物体内Cd的富集系数和转移系数都大于1.0.当土壤中Cd含量达到100 mg/kg时,地上部Cd含量达到119.1 mg/kg,具备...     

In situ stabilization remediation of cadmium contaminated soils of wastewater irrigation region using sepiolite

The effects of immobilization remediation of Cd-contaminated soils using sepiolite on soil pH, enzyme activities and microbial communities, TCLP-Cd (toxicity characteristic leaching procedure-Cd) concentration, and spinach (Spinacia oleracea) growth and Cd uptake and accumulation were investigated. Results showed that the addition of sepiolite could increase soil pH, while the TCLP-Cd concentration in soil was decreased with increasing sepiolite. The changes of soil enzyme activities and bacteria number indicated that a certain metabolic recovery occurred after the sepiolite treatments, and spinach shoot biomass increased by 58.5%-65.5% in comparison with the control group when the concentration of sepiolite was ≤qslant 10 g/kg. However, the Cd concentrations in the shoots and roots of spinach decreased with an increase in the rate of sepiolite, experiencing 38.4%-59.1% and 12.6%-43.6% reduction, respectively, in contrast to the control. The results indicated that sepiolite has the potential for success on a field scale in reducing Cd entry into the food chain.     

种植绿肥对作物产量和细菌群落稳定性的影响

为深入研究青海高原地区长期种植绿肥,减施化肥条件下小麦/油菜产量及土壤理化性状及微生物群落的变化,通过2011年建立的定位试验,设GF0（毛苕子作为绿肥,不施用化肥）、GF60（毛叶苕子配施60%化肥）、GF70（毛叶苕子配施70%化肥）、GF80（毛叶苕子配施80%化肥）、GF90（毛叶苕子配施90%化肥）、GF100（毛叶苕子配施100%化肥）、F0（休耕并且不施化肥）、F100（休耕并且施用100%化肥）共8个处理,利用高通量测序等技术,研究了种植利用绿肥减施化肥条件下小麦和油菜的产量、土壤性质和微生物群落结构的变化特征.结果表明,在保证小麦、油菜不减产的基础上,种植绿肥后茬作物减施化肥达到30%左右.绿肥配施减量化肥对土壤肥力有显著的提升作用,特别是土壤有机碳和全氮含量有明显的提高,分别提高1.34%~7.46%、2.16%~7.48%.化肥与绿肥配施增强了土壤微生物群落多样性,其中酸杆菌门和变形菌门是本研究土壤中的优势菌群.共生网络分析表明,绿肥处理提高了细菌群落的稳定性和抗干扰能力,同时绿肥应用增加了细菌群落中的关键物种.本研究表明,不同土壤微生物丰度受种植模式、施肥量影响很大,长期种植绿肥、减施化肥提高了土壤微生物丰度和多样性,增强微生物群落的稳定性.种植绿肥条件下化肥减肥量30%左右保证作物稳产可为当地的施肥管理提供参考.     

生物质炭对Cd污染土壤团聚体酶活性的影响

为探究生物质炭对Cd污染土壤团聚体酶活性的影响,采用盆栽试验,向模拟Cd污染土壤中添加生物质炭并测定水稻根际土壤团聚体碳循环酶与氧化还原酶活性.结果表明:Cd污染土壤团聚体酶活性对添加量为2.5％的生物质炭响应明显,氧化还原酶活性指数值介于0.522～0.792之间.在2.5mg/kg的外源Cd条件下,2.5％的生物质...     

邻苯二甲酸酯复合污染对土壤微生态的影响

在实验室模拟条件下,通过测定土壤呼吸,w(PAEs)和微生物RAPD条带数,研究了邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)和邻苯二甲酸二辛酯(DOP)3种PAEs复合污染对农田土壤基础呼吸、微生物多样性的影响. 结果表明,PAEs污染初期的土壤基础呼吸被激活,但这种激活作用随着培养时间的延长而减弱,400 mg/kg组土壤基础呼吸最高;3种PAEs含量均有一定程度的下降,其中w(DEP)显著降低;试验终止时各组多样性条带的比例较预培养后有所增加(平均增加13%);不同处理组土壤微生物群落DNA序列Shannon-Weaver指数顺序为0 mg/kg>50 mg/kg>100 mg/kg>200 mg/kg>400 mg/kg. 可见,PAEs复合污染提高了土壤基础呼吸,但降低了土壤微生物多样性.      

Structural and metabolic responses of microbial community to sewage-borne chlorpyrifos in constructed wetlands

Long-term use of chlorpyrifos poses a potential threat to the environment that cannot be ignored, yet little is known about the succession of substrate microbial communities in constructed wetlands(CWs) under chlorpyrifos stress. Six pilot-scale CW systems receiving artificial wastewater containing 1 mg/L chlorpyrifos were established to investigate the effects of chlorpyrifos and wetland vegetation on the microbial metabolism pattern of carbon sources and community structure, using BIOLOG and denaturing gradient gel electrophoresis(DGGE) approaches. Based on our samples, BIOLOG showed that Shannon diversity(HV) and richness(S) values distinctly increased after 30 days when chlorpyrifos was added. At the same time, differences between the vegetated and the non-vegetated systems disappeared. DGGE profiles indicated that H Vand S had no significant differences among four different treatments. The effect of chlorpyrifos on the microbial community was mainly reflected at the physiological level. Principal component analysis(PCA) of both BIOLOG and DGGE showed that added chlorpyrifos made a difference on test results.Meanwhile, there was no difference between the vegetation and no-vegetation treatments after addition of chlorpyrifos at the physiological level. Moreover, the vegetation had no significant effect on the microbial community at the genetic level. Comparisons were made between bacteria in this experiment and other known chlorpyrifos-degrading bacteria. The potential chlorpyrifos-degrading ability of bacteria in situ may be considerable.     

鄱阳湖湿地土壤酶及微生物生物量的剖面分布特征

为探明淡水沼泽湿地土壤微生物功能活性随土壤剖面深度的变化,以鄱阳湖区内典型的苔草湿地土壤为研究对象,选择剖面深度为1 m共5层(0~20、>20~40、>40~60、>60~80、>80~100 cm)的土壤,对土壤酶[Bglu(β-葡萄糖苷酶)、NAG(乙酰氨基葡萄糖苷酶)、Bxyl(β-木糖苷酶)、Phos(酸性磷酸酶)、Phox(酚氧化酶)、Pero(过氧化物酶)]活性、w(MBC)(微生物生物量碳含量)和w(MBN)(微生物生物量氮含量)及土壤理化性质进行研究.结果表明,代表微生物功能活性的土壤酶和w(MBC)、w(MBN)均随着剖面深度的增加而逐渐降低,表层(0~20 cm)土壤中的酶活性和w(MBC)、w(MBN)均显著高于深层土壤.值得注意的是,不同土壤酶活性随着剖面深度的变化规律不一,但总体酶活性在土壤深度为>40~60 cm时达到稳定,并且仍都具有较高活性;表层土壤中w(MBC)为65.58~161.90 mg/kg,w(MBN)为7.39~16.28 mg/kg,二者占所研究剖面土壤总微生物碳氮含量的51%~69%.进一步的相关性分析发现,土壤微生物功能特性与有机质AFDM(去灰分干重)、w(TOC)、w(TN)、含水量及pH存在显著的相关性,其中土壤w(TOC)、w(TN)是影响鄱阳湖湿地土壤微生物数量和活性的最主要因素.研究显示,土壤深度对湿地土壤微生物功能特性及土壤性质具有显著影响,表层土壤中微生物功能活性最高,但湿地深层土壤中仍存在大量的微生物,由微生物参与的代谢活动仍然活跃,深层土壤微生物功能特性不容忽视.      

不同森林土壤微生物群落对Biolog-GN板碳源的利用

应用Biolog方法研究了不同森林恢复类型（人工恢复的湿地松林、杉木林、油茶林和自然恢复的天然次生林）土壤微生物群落对Biolog-GN板不同类型碳源的利用情况,结果表明,自然恢复的天然次生林土壤微生物群落的碳源代谢能力比3种人工林强,其次是油茶林.4种森林类型土壤微生物群落比较偏好、利用率较高的3类碳源是糖类、羧酸类和氨基酸类.对4类森林恢复类型土壤微生物群落具有分异作用的主要碳源类型亦为糖类、羧酸类和氨基酸类,这3类碳源是研究森林恢复后导致土壤微生物群落变化的敏感碳源.     

Effects of Cd（Ⅱ） and Cu（Ⅱ） on microbial characteristics in 2-chlorophenol-degradation anaerobic bioreactors

The effects of Cd^2＋ and Cu^2＋ at 300 mg/L on anaerobic microbial communities that degrade 2-cholorophenol （2-CP） were examined. Based on the polymerase chain reaction （PCR） of 16S rDNA, bacterial community diversity and archaeal community structure were analyzed with denaturing gradient gel electrophoresis （DGGE） and cloning, respectively. Degradation capabilities of the anaerobic microbial community were drastically abated and the degradation efficiency of 2-CP was reduced to 60% after shock by Cu^2＋ and Cd^2＋, respectively. The bacterial community structure was disturbed and the biodiversity was reduced after shock by Cu^2＋ and Cd^2＋ for 3 d. Some new metal-resistant microbes which could cope with the new condition appeared. The sequence analysis showed that there existed common Archaea species in control sludge and systems when treated with Cu^2＋ and Cd^2＋, such as Methanothrix soehngenii, Methanosaeta concilii, uncultured euryarchaeote, and so on. Both the abundance and diversity of archaeal species were altered with addition of Cd^2＋ and Cu^2＋ at high concentration. Although the abundance of the predominant archaeal species decreased with Cd^2＋ and Cu^2＋ addition for 3 d, they recovered to some extent after 10 d. The diversity of archaeal species was remarkably reduced after recovery for 10 d and the shift in archaeal composition seemed to be irreversible. The 2-CP-degradation anaerobic system was more sensitive to Cu^2＋ than Cd^2＋.     

Do land utilization patterns affect methanotrophic communities in a Chinese upland red soil?

Soil samples were collected from three plots under different land utilization patterns including degradation,farming,and restoration.The abundances of methanotrophs were quantified using real-time polymerase chain reaction(PCR)based on the pmoA and 16S rRNA genes,and the community fingerprint was analyzed using denaturing gradient gel electrophoresis(DGGE)aiming at pmoA gene.Significantly lower 16S rRNA and pmoA genes copies were found in the degradation treatment than in farming and restoration.Higher abundances of Type Ⅰ than those of Type Ⅱ methanotrophs were detected in all treatments.The treatment of farming was clearly separated from degradation and restoration according to the DGGE profile by cluster analysis.The lowest diversity indices were observed in the F(farming plot),suggesting that the community structure was strongly affected by farming activities.There were significantly positive correlations between the copy numbers of pmoA also Type Ⅱ-related 16S rRNA genes and soil available K content.Strong negative and positive correlations were found between Type Ⅰ and soil pH,and available P content,respectively.We concluded that the vegetation cover or not,soil characteristics including pH and nutrients of P and K as a result of anthropogenic disturbance may be key factors affecting methanotrophic communities in upland soil.     

稻草还田对土壤微生物群落功能多样性的影响

为了探讨稻草还田的微生物学过程与效应,借助平板涂抹法和BIOLOG检测法,比较研究了免耕条件下3种不同稻草还田量(33%、67%和100%,以稻草不还田土壤为对照)对晚稻田土壤微生物群落功能多样性的影响.结果表明:①土壤细菌数量差异显著,以67%稻草还田处理为最高,稻草不还田处理最低,稻草还田提高了土壤细菌数量;②碳源平均颜色变化率(AWCD法)在培养早期各处理间的差异不大,但随着培养时间的延长,67%和100%稻草还田处理土壤微生物的AWCD值显著大于对照和33%稻草还田处理组;③土壤微生物代谢多样性指数(丰富度和多样性)在3种稻草还田量处理土壤中也表现出明显差异,67%和100%稻草还田处理的土壤中微生物功能多样性显著大于33%稻草还田处理和对照;④67%和100%稻草还田处理显著改善了土壤微生物的功能多样性.以67%稻草还田处理改善土壤微生物群落结构和功能的效果最为显著.     

Bacterial diversity in soils around a lead and zinc mine

Five samples of soil collected from a lead and zinc mine were used to assess the effect of combined contamination of heavy metals on soil bacterial communities using a polyphasic approach including characterization of isolates by culture method, community level catabolic profiling in BIOLOG GN microplates, and genetic community fingerprinting by denaturing gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from community DNA （PCR-DGGE）. The structure of the bacterial community was affected to a certain extent by heavy metals. The PCR-DGGE analysis of 16S rRNA genes showed that there were significant differences in the structure of the microbial community among the soil samples, which were related to the contamination levels. The number of bacteria and the number of denaturing gradient gel electrophoresis （DGGE） bands in the soils increased with increasing distance from the lead and zinc mine tailing, whereas the concentration of lead （Pb） and cadmium （Cd） was decreased. Heavily polluted soils could be characterized by a community that differs from those of lightly polluted soils in richness and structure of dominating bacterial populations. The clustering analysis of the DGGE profiles showed that the bacteria in all the five samples of soil belonged to three clusters. The data from the BIOLOG analysis also showed the same result. This study showed that heavy metal contamination decreased both the biomass and diversity of the bacterial community in soil.     

PCR-DGGE技术解析生物制氢反应器微生物多样性

为了揭示发酵法生物制氢反应器厌氧活性污泥的微生物种群多样性 ,从运行不同时期取厌氧活性污泥 ,通过细胞裂解直接提取活性污泥的基因组DNA .以细菌 16SrRNA基因通用引物F338GC/R5 34进行V3高变异区域PCR扩增 ,长约 200bp的PCR产物经变性梯度凝胶电泳 (DGGE)分离后 ,获得微生物群落的特征DNA指纹图谱 .研究表明 ,不同时期的厌氧活性污泥中存在共同种属和各自的特异种属 ,群落结构和优势种群数量具有时序动态性 ,微生物多样性呈现出协同变化的特征 .微生物多样性由强化到减弱 ,群落结构之间的相似性逐渐升高 ,演替速度由快速到缓慢 .优势种群经历了动态演替过程 ,最终形成特定种群构成的顶级群落 .     

Do land utilization patterns a ect methanotrophic communities in a Chinese upland red soil?

Soil samples were collected from three plots under di erent land utilization patterns including degradation, farming, and restoration. The abundances of methanotrophs were quantified using real-time polymerase chain reaction (PCR) based on the pmoA and 16S rRNA genes, and the community fingerprint was analyzed using denaturing gradient gel electrophoresis (DGGE) aiming at pmoA gene. Significantly lower 16S rRNA and pmoA genes copies were found in the degradation treatment than in farming and restoration. Higher abundances of Type I than those of Type II methanotrophs were detected in all treatments. The treatment of farming was clearly separated from degradation and restoration according to the DGGE profile by cluster analysis. The lowest diversity indices were observed in the F (farming plot), suggesting that the community structure was strongly a ected by farming activities. There were significantly positive correlations between the copy numbers of pmoA also Type II-related 16S rRNA genes and soil available K content. Strong negative and positive correlations were found between Type I and soil pH, and available P content, respectively. We concluded that the vegetation cover or not, soil characteristics including pH and nutrients of P and K as a result of anthropogenic disturbance may be key factors a ecting methanotrophic communities in upland soil.