设施种植模式对土壤细菌多样性及群落结构的影响

为了研究有机和常规设施种植模式及轮作对土壤细菌多样性和群落结构的影响,本研究采用Illumina平台Hiseq 2500高通量测序技术,于2016年6月(作物处于收获期)对北京市顺义区不同设施种植模式(分别为有机设施种植模式和常规设施种植模式下的叶菜连作、茄果连作和叶茄轮作)下土壤细菌进行16S r RNA测序。测序质控后共获得17 278个操作分类单元(operational taxonomic units,OTUs),共计318 851条有效序列。比较不同种植模式和轮作下土壤细菌多样性、细菌群落结构组成、相对丰度及土壤理化性质与细菌群落多样性关系的差异性。结果表明:土壤微生物群落结构在有机和常规设施种植模式下差异明显,有机设施种植土壤细菌多样性高于常规设施种植;有机设施种植下轮作与连作土壤细菌群落结构表现出明显差异,而常规设施种植下,两者没有明显差异;有机种植模式下,轮作土壤细菌群落多样性高于连作土壤;设施种植土壤细菌群落主要属于鞘氨醇单胞菌属(Sphingomonas,5.05%)和芽孢杆菌属(Bacillus,4.84%),相对丰度大于0.5%的共有14个属。有机设施种植土壤含有较多促进植物生长、有机质分解的细菌,常规设施种植土壤中降解化学杀虫剂、防治土壤病害、促进硝化过程的细菌较多。RDA分析结果显示土壤细菌群落主要受全磷、速效磷、有机质的影响。Tumebacillus、Candidatus Solibacter和Acidothermus都是分解有机质、利用碳源的细菌属,与土壤有机质含量呈正相关关系。由此可见,设施条件下,有机和常规种植土壤微生物群落结构的差异性主要源于肥料使用、有害生物防治措施和管理方式的不同。有机设施种植模式下,轮作更有利于发挥其改良土壤营养循环和防治土壤病虫害的作用。上述结果为在微生物水平上研究设施条件下不同种植模式的土壤生态质量差异提供了参考。     

有机和常规生产模式下菜田土壤酶活性差异研究

通过对露地及温室环境下有机和常规蔬菜栽培土壤采样,测定分析了5种参与土壤碳氮循环的酶活性,及其与土壤相关理化性质之间的关系。结果显示:温室及露地土壤EC值在有机生产中相应低于常规生产12%和16%;有机生产土壤微生物碳氮含量显著高于常规生产;不同生产模式下土壤酶活性差异显著,有机生产土壤中的蛋白酶、脲酶、脱氢酶、β-葡糖苷酶活性高于常规生产,而硝酸还原酶活性较常规生产低;有机与常规栽培对蛋白酶活性影响极显著(P=0.006 8),对脲酶活性影响程度达显著水平(P=0.012 4)。除脱氢酶以外,不同栽培模式环境对土壤中另外4种酶活性均有显著影响,温室栽培环境中的蛋白酶、脲酶和硝酸还原酶活性高于露地。除硝酸还原酶外,其他4种酶活性与可溶性全氮、微生物碳、微生物氮相关系数达到显著水平。分析表明,土壤酶活性受到栽培方式以及环境的影响,并且有机生产能够提高参与土壤碳氮循环的酶活性。土壤蛋白酶、脲酶、脱氢酶和β-葡糖苷酶活性能够作为表征土壤碳氮循环以及微生物活性的指标。     

高通量测序技术下连作植烟土壤细菌群落与土壤环境因子的耦合分析

为了揭示连作条件下植烟土壤细菌群落结构及其与土壤环境间的响应关系,本研究采用了Illumina平台Hiseq2500高通量测序技术,对不同施肥处理(常规施肥,蚯蚓粪肥,微生物菌肥以及蚯蚓粪和微生物菌肥混合)的漯河烟区连作植烟土壤细菌进行16S rRNA V4区测序,结合冗余分析(redundancyanalysis,RDA)研究土壤细菌微生物的群落结构组成、多样性以及与土壤环境间的相关关系.结果表明,测序质控后共获得25 203个操作分类单元(operational taxonomic units,OTUs),计1 600 239条读数.分层聚类图显示,不同施肥处理的连作植烟土壤细菌群落有较大差异,但这种差异不体现在结构多样性上.多样性指数分析表明,连作植烟土壤细菌群落易受环境变化的影响,体现出一定的时间差异性;烤烟成熟期土壤丰度指数明显升高,微生物菌肥和蚯蚓粪肥处理下土壤细菌群落丰度变化较大.主成分分析表明,不同土壤环境因子间有很强的相关关系,可以将原有的11个土壤环境因子按照强正相关关系划分为4类;RDA结果表明,土壤pH既影响土壤细菌群落的多样性,又影响土壤细菌群落的丰度;而有机质主要对土壤细菌群落丰度有积极影响.研究结果为在微生物水平上研究连作植烟障碍的形成机理提供了依据.     

土壤与基质栽培系统对生菜(Lactuca sativa)根际细菌群落的影响

以生菜(Lactuca sativa)"申选5号"与"罗莎红"为材料,采用PCR-DGGE和Real-Time PCR技术,分析了土壤栽培系统和基质栽培系统根际细菌群落的差异。Real-Time PCR检测结果表明,基质栽培的两个生菜品种根际细菌数量均显著高于土壤(P0.05);PCR-DGGE图谱条带结果表明,根际细菌群落多样性基质高于土壤。栽培系统是造成多样性差异的主要原因,但也与品种有关:"申选5号"基质的Shannon-Wiener指数(H),Simpson指数(D)和均匀度(E)均显著高于土壤(P0.05);"罗莎红"基质的H显著高于土壤,而D和E无显著差异。结合土壤和基质理化性质的RDA分析结果,土壤和基质具有不同的细菌群落,p H值与硝态氮是塑造根际细菌群落的主要因子,含水量、碳氮比和有效磷与细菌群落的形成呈正相关。     

典型设施菜地中土壤微生物代谢功能多样性

土壤微生物代谢功能多样性是维持土壤生态系统健康的关键。为评价设施蔬菜种植对土壤微生物代谢功能多样性的影响,采用Biolog-Eco微平板法,研究了2个典型设施蔬菜种植市、不同种植年限设施菜地中土壤微生物代谢功能多样性,分析了与碳源利用相关的细菌群落及影响因子。结果表明：两地设施菜地土壤中平均颜色变化率（Average well color development,AWCD）、Shannon指数、Simpson指数和McIntosh指数随着种植年限的增加而降低,但安丘种植14 a和寿光种植10 a菜地土壤不符合此规律。主成分分析发现,两地之间土壤微生物对碳源利用的差异大于不同种植年限之间的差异。相关分析发现,在安丘土壤中,11种碳源（分属糖类、氨基酸类、羧酸类和聚合物类）与不同门细菌显著相关（P<0.05）;Firmicutes是与碳源相关种类最多的细菌,与9种碳源显著相关（P<0.05）。在寿光土壤中,16种碳源（分属糖类、氨基酸类、羧酸类、聚合物类、酚类和胺类）与不同门细菌显著相关（P<0.05）;Latescibacteria是与碳源相关种类最多的细菌,与4种碳源显著相关（P<0.05）。冗余分析发现,Cd是影响安丘土壤微生物碳源利用的最强环境因子,其负作用显著（P<0.01）,有机质（OM）的正作用为显著（P<0.01）。Zn、OM、Cd对寿光土壤微生物碳源利用的负作用显著（P<0.05）,As、pH值的正作用显著（P<0.05）。设施蔬菜长期种植导致土壤微生物代谢功能多样性持续降低,土壤生物质量退化,亟需采取有效耕作措施改善土壤微生态环境,保障设施菜地土壤健康。     

岷江流域不同土地利用方式下的土壤微生物特征及其与土壤养分的关系

采用经典统计分析与排序分析,研究了2014—2018年岷江流域不同土地利用方式下(撂荒地、次生林、灌丛、果园和耕地)土壤微生物群落多样性变化特征,并探讨了其与土壤养分和土壤微生物数量之间的关系。结果表明:(1)土壤pH值变化范围为6.13～7.02,其大小依次表现为撂荒地次生林灌丛果园耕地,其中耕地和果园差异不显著(p0.05),耕地最高(p0.05);土壤有机碳、全氮、全钾、有效磷和有效钾大小依次表现为撂荒地次生林灌丛果园耕地,不同土地利用土壤有效磷和有效钾差异均显著(p0.05),而不同土地利用土壤全磷差异不显著(p0.05)。(2)土壤微生物以细菌数目最多,占到90%以上,土壤细菌、真菌、放线菌和微生物总数目大小依次表现为撂荒地次生林灌丛果园耕地。(3)根据培养第100小时的AWCD值计算土壤微生物群落的物种丰富度指数(H)、均匀度指数(E)、优势度指数(Ds)和碳源利用丰富度指数(S)。土壤微生物多样性指数大小依次表现为撂荒地次生林灌丛果园耕地,不同土地利用均匀度指数(E)、优势度指数(Ds)差异不显著(p0.05)。(4)相关性分析表明土壤养分、土壤微生物数量均与土壤微生物群落多样性具有显著的相关性(p0.05),其中,土壤微生物数量对微生物群落多样性的贡献最大(其相关系数绝对值最大)。(5)冗余分析表明土壤微生物群落多样性与土壤养分含量均呈正相关(除了pH);有机碳和全氮与丰富度指数相关性最大,由此可知,有机碳和全氮是影响该区土壤微生物群落多样性分布的主要因子。     

不同栽培方式对菜地土壤养分和生物学特性的影响

通过田间小区试验,以芹菜、西兰花、生菜和胡萝卜为供试作物,分析比较了有机栽培、安全环境质量栽培、特别栽培和常规栽培对土壤养分和生物学特性的影响.结果表明,有机栽培、安全环境质量栽培和特别栽培下土壤有机质、全氮、碱解氮、速效磷和速效钾含量比常规栽培均不同程度地增加.有机栽培、安全环境质量栽培和特别栽培均显著提高了土壤微生物量碳含量,比常规栽培分别提高48.6％、42.9％和26.6％,并达到显著(P＜0.05)或极显著(P＜0.01)差异.土壤过氧化氢酶和脲酶活性在有机栽培、安全环境质量栽培和特别栽培下比常规栽培显著提高17.4％ ～38.8％,且过氧化氢酶活性达到显著(P＜0.05)或极显著(P＜0.01)差异.有机栽培、安全环境质量栽培下土壤转化酶活性比常规栽培有所提高,但是没有达到显著差异.不同栽培方式对土壤养分和生物学特性影响作用的大小为有机栽培＞安全环境质量栽培＞特别栽培＞常规栽培.     

有机栽培和常规栽培水稻体系土壤酶及微生物量的比较研究

本研究探讨了有机栽培与常规栽培体系下水稻土微生物量及脲酶、酸性磷酸酶和过氧化氢酶的动态变化过程,以及有机栽培体系不同肥料调控措施对上述指标的影响.结果表明:与常规栽培水稻体系相比,有机栽培水稻有利于土壤微生物的生长和繁衍;水稻生长不同时期有机栽培方式的土壤微生物生物量碳、脲酶、酸性磷酸酶和过氧化氢酶活性均高于常规栽培体系.就水稻全生育期而言,土壤微生物生物量碳高于常规栽培7.3%～9.1%;脲酶、酸性磷酸酶和过氧化氢酶活性分别高于常规栽培7.3%～14.5%、5.2%～6.5%和12.5%～29.2%;有机水稻栽培体系下配施生态肥,在有机肥施用量减半时,土壤微生物生物量碳含量及土壤脲酶、酸性磷酸酶和过氧化氢酶活性较单施有机肥平均分别提高1.6%、6.8%、1.3%和14.8%,在水稻生长前期和中期该增加作用尤为显著.     

沼液对种植狼尾草的山地红壤可溶性有机氮和土壤微生物特征的影响

通过沼液浇灌人工草地定位试验,研究浇灌不同浓度梯度沼液下土壤可溶性无机氮、可溶性有机氮、可溶性有机碳、微生物量碳(SMBC)、微生物量氮(SMBN)、细菌16SrDNA基因、真菌SSU rDNA基因、固氮菌nifH基因丰度及细菌、真菌、固氮菌群落多样性的变化。结果表明,人工草地土壤可溶性氮以有机氮为主,占可溶性总氮含量的77.87%~88.88%,沼液浇灌使人工草地土壤可溶性有机氮含量呈增加趋势,但这种变化未达到显著水平;沼液浇灌能显著提高SMBN、SMBC和MQ,还能显著增加细菌16SrDNA基因和真菌SSU rDNA基因的丰度,降低固氮菌nifH基因的丰度,但对细菌、真菌和固氮菌多样性无显著影响,表明沼液短期浇灌可促进土壤微生物的繁殖,但对微生物群落构成无显著影响;沼液与SMBC、细菌16SrDNA基因丰度、真菌SSU rDNA基因丰度之间显著正相关,SON与土壤真菌SSUrDNA基因丰度显著正相关,与固氮菌nifH基因丰度显著负相关(p0.05)。     

不同栽培模式对中小型土壤动物多样性的影响

土壤动物是土壤中重要的生物群落,在分解凋落物、土壤有机质以及维护生态系统平衡等方面中扮演重要角色。为了研究不同栽培模式下中小型土壤动物多样性特征,探究有利于中小型土壤动物的种植方式,本文研究了有机与常规栽培、大棚与露天、茄果与叶菜等不同栽培模式对中小型土壤动物种群数量、组成和多样性等特征的影响。结果表明：1）利用Tullgren法在18个样地共获得中小型土壤动物3 869只,隶属于2门14目30科（亚目）。辐螨亚目、甲螨亚目以及等节跳科构成了研究区土壤动物的主要部分。2）露天条件下,有机栽培使5~10 cm和10~15 cm土层中小型土壤动物数量显著高于常规栽培;但大棚条件下,有机栽培使0~5 cm和5~10 cm土层中小型土壤动物数量显著低于常规栽培。3）有机栽培下,大棚内0~5 cm和10~15 cm土层中小型土壤动物数量显著低于露天,且棚内10~15 cm层中小型土壤动物丰富度显著高于常规栽培;而常规栽培下,大棚内5~10 cm土层中小型土壤动物数量著高于露天。无论有机和常规栽培,棚内0~5 cm层中小型土壤动物Shannon-Weiner多样性高于露天。4）有机栽培使叶菜作物10~15 cm土层中小型土壤动物数量显著低于常规栽培,且0~5 cm土层中小型土壤动物均匀度指数显著高于常规栽培;常规栽培使叶菜作物0~5 cm和5~10 cm土层中小型土壤动物数量显著高于茄果作物。因此,在设施条件下采用有机栽培和管理,且利用作物轮作可能更有利于土壤动物群落数量和多样性的增加。     

Community-level responses of metabolically-active soil microorganisms to the quantity and quality of substrate inputs

The community fingerprints of both the prevalent and the metabolically active microbial community were related to a quantitative estimation of microbial biomass in an arable soil, revealed by substrate-induced-respiration (SIR). Two concentrations of glucose or l-asparagine, representing those used in the SIR measurement or equivalent to those released in root exudates, were studied. Respiration rates and changes in community structure fingerprints were followed for 48 h. Bacterial and fungal community fingerprints were obtained using both reverse transcribed 16S and 18S ribosomal RNA (rRNA) regions and the corresponding rDNA as a template in PCR. Samples were then analysed by denaturing gradient gel electrophoresis (DGGE). Low concentrations of substrate amendments resulted in minor changes in rRNA or rDNA-based bacterial and fungal banding patterns during the whole 48 h incubation. High concentrations of substrates, especially l-asparagine, increased respiration rates and induced significant changes in both 16S rRNA and rDNA-community fingerprints. The prominent rRNA and rDNA bacterial community sequence types were common to all treatments, but in general the bacterial rDNA fingerprints had fewer bands than the corresponding rRNA profiles that assess the active fraction of the community. In contrast, there was little difference between fungal 18S rRNA and rDNA patterns. The number of fungal ribosomal sequence types in DGGE fingerprints was lower than the number of bacterial types. This study indicated that there was a rapid respiration response by the whole microbial community during SIR estimates in soil, but that community structure did not change during the conventional incubation period. In an extended (8-48 h) incubation with high amounts of l-asparagine increased respiration was associated with growth of the microbial community.     

Alphaproteobacteria communities depend more on soil types than land managements

Soil properties and agricultural practices take a joint effect on the communities of soil bacteria. The aim of the present study was to survey Alphaproteobacterial communities as possible indicators of soil quality considering clay, loamy and sandy soils under conventional and organic farming. Alphaproteobacteria community composition were analysed by 16S rRNA gene with nested-PCR (polymerase chain reaction) and denaturing gradient gelelectrophoresis (DGGE). Sequencing of partial 16S rRNA gene from the DGGE bands were performed. Conventional and organic farming resulted in significant differences in chemical properties of soils. According to the results community fingerprints were separated into groups depending on soil types and farming systems. This separation can be attributed mostly to soil pH, AL-P₂O₅,-K₂O. The analysed sequences were identified as soil bacteria which could play the main role in nitrogen fixing, mineralisation and denitrification. The highest diversity index was revealed from the organic farming at sandy texture site, where mainly Mesorhizobium sp. and Rhizobium sp. were detected. The soil type and actual crop could have a stronger impact on the soil bacterial composition than the management.     

DNA- versus RNA-based denaturing gradient gel electrophoresis profiles of a bacterial community during replenishment after soil fumigation

We compared the responsiveness and sensitivity to soil fumigation of DNA- and RNA-based analyses of a bacterial community. We first established an improved RNA extraction method using DNA as an adsorption competitor, because it is extremely difficult to extract nucleic acids from clay-rich volcanic ash soil (Andisol), which adsorbs nucleic acids. This novel method facilitated RNA extraction from 500 mg of Andisol for molecular analyses. Then we monitored 16S rDNA PCR and 16S rRNA RT-PCR denaturing gradient gel electrophoresis (DGGE) profiles of samples collected from a chloropicrin (CP)-treated field over 2 months. The difference between untreated control and CP-treated plots was detected clearly both in DNA- and RNA-based DGGE profiles after treatment. The temporal changes in DGGE profiles, however, differed between DNA- and RNA-based analyses in CP-treated plots. RNA-based DGGE showed quicker and greater changes in the bacterial community after CP treatment than did DNA-based DGGE, which showed similar trends to RNA-based DGGE but with a time lag. The extent of decrease in the diversity index (H′) and the change in principal response curves was larger in RNA-based analyses. These results indicate that the rDNA PCR-DGGE method also detects DNA of microbes no longer alive after fumigation, and that rRNA provides a more responsive biomarker than rDNA.     

Effect of organic mulches on soil bacterial communities one year after application

The application of organic mulches as a soil cover is effective in improving the quality of soil. However, very little information is available on the effect of mulches on the soil microbial community. In this study, we investigated the effect of various organic mulches on soil dehydrogenase activity (DHA) and microbial community structures in the top 1 cm and 5 cm below the soil surface 1 year after application of the mulches. DHA was stimulated at both depths in plots mulched with grass clippings (GC), but was not significantly different from the control for the other mulch treatments. Fatty acid methyl ester (FAME) analysis and denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified 16S rDNA fragments were used to assess changes in the soil microbial community structure. Cluster analysis and principle component analysis of FAME profiles showed that only soil mulched with pine chips distinctively clustered from the other treatments. At the soil surface, bacterial DGGE profiles revealed that distinct shifts in several bacterial populations occurred in soils mulched with GC and eucalyptus yardwaste (EY), while DGGE profiles from soil at the 5 cm depth revealed no distinct changes. Changes in bacterial diversity at the soil surface under different mulches were calculated based on the number of bands in the DGGE profile using the Shannon-Weaver index of diversity ( H). Compared to the control ( H =0.9), the GC- and EY-treated soils showed slightly increased bacterial diversity, with an H of 1.1 and 1.0, respectively. These results indicate that the long-term effect of organic mulches on the soil microbial activity and community structure is highly dependent upon the type of mulch and is mostly exerted in the top few centimeters of the soil profile.     

DGGE法与常规培养法对稻田蓝细菌多态性分析结果比较

研究运用蓝细菌和硅藻16SrDNA特异引物,将晚季水稻生长后期稻田土壤中提取的总DNA进行PCR扩增后,以DGGE技术对PCR产物进行分析结果表明,14条DGGE带经克隆测序,经NCBI基因库比对得晚季水稻生长后期存在10种蓝细菌,包括4种Leptolyngbya、1种Chamaesiphon、1种Nostoc、1种Oscillatoria、2种Syne-chococcus和1种Chroococcidiopsis。同层不同位置土壤中蓝细菌种群亦有所不同,但每个取样点都有一些特有的蓝细菌种类。用常规方法对同一稻田土壤样品进行分离培养,根据蓝细菌鉴定图谱观察到类似Lyngbya、Oscillatori-a、Chroococcidiopsis及Nostoc的蓝细菌,但显微镜下无法准确分类。比较结果表明采用DGGE法比常规培养法能更准确进行蓝细菌多态性鉴定。     

Soil aggregation and bacterial community structure as affected by tillage and cover cropping in the Brazilian Cerrados

Microbial-based indicators of soil quality are believed to be more dynamic than those based on physical and chemical properties. Recent developments in molecular biology based techniques have led to rapid and reliable tools to characterize microbial community structures. We determined the effects of conventional and no-tillage in cropping systems with and without cover crops on bacterial community structure, total organic carbon (TOC) and soil aggregation. Tillage and rotation did not affect TOC from bulk soil. However, TOC was greater in the largest aggregate size class (7.98–19 mm), and had greater mean-weight diameter under no-tillage than under conventional tillage in the 0–5 cm soil layer. Soil bacterial community structure, based on denaturing gradient gel electrophoresis of polymerase chain reaction amplified DNA (PCR/DGGE) using two different genes as biomarkers, 16S rRNA and rpoB genes, indicated different populations in response to cultivation, tillage and depth, but not due to cover cropping. Soil bacterial community structure and meanweight diameter of soil aggregates indicated alterations in soil conditions due to tillage system.     

利用PCR-DGGE技术研究长期自然恢复、种植作物和裸地条件下黑土土壤细菌群落结构变化

Soil microbial biomass and community structures are commonly used as indicators for soil quality and fertility. A investigation was performed to study the effects of long-term natural restoration, cropping, and bare fallow managements on the soil microbial biomass and bacterial community structures in depths of 0--10, 20--30, and 40--50 cm in a black soil (Mollisol). Microbial biomass was estimated from chloroform fumigation-extraction, and bacterial community structures were determined by analysis of 16S rDNA using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Experimental results showed that microbial biomass significantly declined with soil depth in the managements of restoration and cropping, but not in the bare fallow. DGGE profiles indicated that the band number in top 0--10 cm soils was less than that in depth of 20--30 or 40--50 cm. These suggested that the microbial population was high but the bacterial community structure was simple in the topsoil. Cluster and principle component analysis based on DGGE banding patterns showed that the bacterial community structure was affected by soil depth more primarily than by managements, and the succession of bacterial community as increase of soil depth has a similar tendency in the three managements. Fourteen predominating DGGE bands were excised and sequenced, in which 6 bands were identified as the taxa of Verrucomicrobia, 2 bands as Actinobacteria, 2 bands as α-Proteobacteria, and the other 4 bands as δ-Proteobacteria, Acidobacteria, Nitrospira, and unclassified bacteria. In addition, the sequences of 11 DGGE bands were closely related to uncultured bacteria. Thus, the bacterial community structure in black soil was stable, and the predominating bacterial groups were uncultured.     

DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.