基于整合高通量绝对定量法的土壤微生物多样性分析

目前,常用的高通量测序技术只能得到微生物群落结构的物种种类和相对丰度,而整合高通量绝对定量法（iHAAQ）结合了高通量测序技术和qPCR技术,可以进一步计算得到微生物群落结构的绝对含量。相对丰度和绝对含量均是描述微生物群落结构的必备指标,其中相对丰度适合描述和评价微生物群落结构在单个样本内微生物之间的关系,而绝对含量更适于描述和评价微生物群落结构真实的数量变化以及在样本间微生物之间的关系。本研究基于发表的香蕉土传病害、再造沙地农业生态系统和微生物抑制剂作用3篇文献的高通量测序和qPCR数据,通过整合高通量绝对定量法获得3篇文献中古菌域、细菌域和真核生物域的真菌三类微生物群落结构的绝对含量数据,并进一步计算出这三类微生物整体（简称三域微生物）的绝对含量和相对丰度,从而对土壤微生物群落结构不同物种的相对丰度和绝对含量进行分析,旨在更加深入、准确地揭示土壤微生物群落及其生态功能。结果表明：（1）土壤中细菌的物种丰度和绝对含量高于古菌和真菌,在三域微生物中占据主导地位,缺乏绝对含量指标可能造成对微生物群落结构变化理解的偏差;（2）基于相对丰度和绝对含量数据分析,微生物群落结构α多样性指数结果相同,但β多样性指数结果不同;（3）在香蕉土传病害和再造沙地农业生态系统研究中,三域微生物的PCoA结果与细菌的结果较为相似,表明这两项研究中三域微生物群落结构主要受细菌的影响,但在微生物抑制剂的研究中未发现类似结果。整合高通量绝对定量法可以应用于所有同时进行高通量测序和qPCR测序的研究,在未来的土壤微生物群落结构研究中,采用高通量绝对定量法开展三域微生物群落的物种种类、相对丰度和绝对含量的整体评价,具有重要的生态学意义。     

局域尺度麦田土壤微生物群落构建过程及其驱动因素

为探究局域尺度麦田土壤微生物群落构建过程，本研究采集河南省济源市小麦种植户麦田土壤，应用高通量测序技术，研究局域尺度下麦田土壤细菌群落结构、群落构建过程及其影响因素。结果表明：①土壤细菌群落中优势菌门是酸杆菌门（Acidobacteria）和变形菌门（Proteobacteria）。通过曼特尔检验以及冗余分析发现有机质和全氮对细菌群落结构影响显著（P<0.05）。②细菌分子生态网络中不同细菌类群之间以合作关系为主，关键物种来自拟杆菌门（Bacteroidetes）、酸杆菌门和变形菌门。③细菌群落在系统发育上聚类，有机质和有效磷对细菌群落系统发育影响显著（P<0.05）。④确定性过程和随机性过程在细菌群落构建中的贡献率分别为74.59%和25.41%。⑤曼特尔检验发现最近物种指数（βNTI）值与有机质、全氮、全磷含量变化呈显著正相关（P<0.05）。有机质、全氮、全磷含量差异的增大导致细菌群落从同质性选择以及均质扩散向异质性选择过程的转变。综上所述，本研究发现确定性过程在局域尺度麦田土壤细菌群落构建中占主导地位，土壤有机质、全氮、全磷是影响细菌群落构建的关键环境因素。有机质和全氮还对细菌群落结构有显著影响。本研究有利于对局域尺度下麦田生态系统土壤微生物多样性形成机制的认识。     

复合菌剂防控香蕉枯萎病的效果及其微生物学机制

由病原菌尖孢镰刀菌古巴专化型（Fusarium oxysporum f. sp. cubense (Foc)）侵染引起的香蕉枯萎病对全世界香蕉产业带来了毁灭性的影响,且目前尚无广泛采用的防治方法。研究复合生防真菌制剂对香蕉枯萎病的防治效果,以期为大田香蕉枯萎病的防治提供依据。设置3组不同的菌剂处理,分别为对照组CK、复合菌剂NFP、复合菌剂NFPT,通过两季的盆栽试验,研究复合菌剂对香蕉枯萎病的防治效果及其对土壤微生物多样性的影响;利用 Illumina Miseq 高通量测序平台对细菌16S rRNA基因和真菌ITS区域进行测序分析,采用实时荧光qPCR定量分析各处理病原菌的数量。结果表明：（1）复合菌剂处理（NFP和NFPT）对香蕉枯萎病有较好的防治效果,其防效分别为43%和48%。（2）施用复合菌剂增加了细菌和真菌群落丰富度和多样性。基于Bray-curtis距离矩阵的主坐标分析（PCoA）结果表明NFP和NFPT改变了细菌和真菌群落结构。NFP和NFPT处理增加了潜在有益微生物中与香蕉枯萎病病情指数呈显著负相关的大理石雕菌属Marmoricola、类诺卡氏菌属Nocardioides、野野村式菌属Nonomuraea、 norank_c__Acidobacteria、DS-100属和norank_f__Blastocatellaceae__Subgroup_4属的相对丰度,显著减少了病原菌尖孢镰刀菌的数量,重塑了土壤微生物结构和功能,增强其抗病性。     

毛乌素沙地苔藓结皮对沙化土壤性质和细菌群落的影响

我国沙化土地占国土面积的17.93%,对北方地区经济和社会的发展、人们的生活以及生态平衡影响巨大。土壤生物结皮能有效防风固沙,微生物在其形成中起着重要作用。之前在对生物结皮微生物群落研究方面,缺乏对生物结皮层与同厚度裸沙表层之间以及它们各自下方土壤之间的比较与分析。采用高通量测序技术,对毛乌素沙地苔藓结皮层与裸沙表层,结皮下方土壤与裸沙下层土壤细菌群落结构进行了分析。结合土壤理化因子数据,分析了苔藓结皮对沙地细菌群落多样性的影响。结果表明：相比于裸沙表层苔藓结皮显著提高了土壤速效养分、全氮和有机质的水平,结皮下方土壤中速效养分、全磷和有机质含量均高于裸沙下层。结皮层及其下方土壤中粗粉砂和粉粒的含量显著高于裸沙表层和下层,表明苔藓结皮显著改善了沙地土壤的理化性质。速效磷、速效钾、速效氮、黏粒、粉粒、粗砂是影响沙地土壤细菌群落组成的重要环境因子。多样性分析显示,结皮下方土壤中细菌多样性最高。在苔藓结皮层中unclassifiled_f__Micromonosporaceae（小单孢菌科的未知属）,norank_c__Cyanobacteria（蓝藻纲的未知属）和Pseudonocardia（假诺卡氏菌属）的相对丰度都显著高于裸沙表层;在结皮下方土壤中norank_c__Acidobacteria（酸杆菌纲的未知属）和Rubrobacter（红色杆菌属）的相对丰度均显著高于裸沙下层,这些差异显著的物种对稳定沙地土壤结构具有重要作用。在研究生物结皮的过程中,对照沙地也要分层取样,微生物群落对比分析才更准确。因此,苔藓结皮的形成对沙地原有表层及下层土壤的细菌群落产生显著影响,利于生物固沙。研究结果为风沙治理和荒漠生态恢复提供了重要的微生物学理论依据。     

复合微生物菌剂与氨基酸水溶肥组合施用对香蕉土壤理化性质及微生物群落的影响

为了研究复合微生物菌剂和氨基酸水溶肥组合施用后香蕉土壤性质及微生物群落结构变化,在海南省东方市开展香蕉大田试验,以组合施用复合微生物菌剂与氨基酸水溶肥为处理组,在香蕉的苗期、中期、孕蕾期各处理一次,以滴灌清水为对照组,全部处理结束30 d后进行土壤取样,测定土壤理化性状,并提取土壤DNA进行土壤微生物高通量测序,对比研究土壤性质及菌群的变化情况。结果如下:(1)处理组有效地改善了土壤养分,土壤有效磷和速效钾含量极显著上升(P0.01);碱解氮显著增加(P0.05);根结线虫数量显著下降(P0.05)。(2)细菌测序结果表明,在目水平上,对照组土壤中细菌相对丰度≥2%的优势菌目在处理组土壤中都呈下降趋势;在属水平上,物种丰度≥1%的菌属中,处理组土壤的Rhodanobacter(罗丹杆菌属)、Sphingomonas(鞘氨醇单胞菌属)、Dyella(分类未定)的物种丰度都明显升高,其中Sphingomonas呈显著性升高。(3)真菌测序结果显示,处理组土壤中Colletotrichum(炭疽菌属)的丰度明显下降,但Penicillium(青霉菌属)的丰度有所升高。综上,采用复合微生物菌剂和氨基酸水溶肥组合处理,对土壤理化性状和微生物具有明显的改良和改善作用,有利于减少香蕉的病害发生。     

不同类型水稻土微生物群落结构特征及其影响因素

选取基于我国土壤地理发生分类的不同类型土壤发育的四种水稻土,利用¹⁵N₂气体示踪法测定生物固氮速率,采用实时荧光定量PCR（Real-time PCR）技术测定细菌丰度,通过16S rRNA基因高通量测序分析微生物群落组成和多样性。结果表明：变形菌门（Proteobacteria）、酸杆菌门（Acidobacteria）、绿弯菌门（Chloroflexi）和蓝藻门（Cyanobacteria）是水稻土中优势微生物类群。四种类型土壤发育的水稻土细菌群落结构差异显著（Stress<0.001）,群落结构分异（NMDS1）与土壤pH存在极显著正相关关系（P<0.01）。土壤有机碳和碱解氮含量显著影响水稻土中细菌丰度和群落多样性（P<0.01）。红壤发育的水稻土细菌16S rRNA基因拷贝数显著高于其他三种类型水稻土,但OTU数量、Chao1指数和PD指数均低于其他三种类型水稻土。土壤pH对水稻土生物固氮速率有显著影响（P<0.01）,紫色土发育的水稻土具有最高的生物固氮速率（3.2±0.7 mg×kg^-1×d^-1）,其中优势类群细鞘丝藻属（Leptolyngbya）可能是生物固氮的主要贡献者。研究结果丰富了对水稻土微生物多样性的认识,为通过调控土壤pH和微生物群落组成来提高稻田生物固氮潜力提供了理论依据。     

毛竹纯林土壤微生物多样性高于杉木纯林

杉木连作障碍现象普遍发生,同为人工林的毛竹则很少发生。作为土壤健康的重要指标,微生物对土壤肥力具有不可忽视的作用。采用高通量测序法对阔叶、毛竹和杉木三种林分的土壤细菌和真菌群落进行研究,结果表明,毛竹林土壤细菌和真菌Shannon多样性和Invsimpson均匀度指数均显著高于杉木林,甚至高于阔叶林;而优势物种多样性Berger-Parker指数则是杉木显著高于毛竹林;毛竹林土壤Actinobacteria门细菌相对丰度高于阔叶林和杉木林、Basidiomycota门真菌相对丰度显著高于阔叶林和杉木林,杉木林土壤Chloroflexi门细菌相对丰度和Mortierellomycota门真菌相对丰度显著高于毛竹林和阔叶林。结合土壤理化性质分析表明,杉木林土壤养分贫瘠是形成其特殊微生物群落的原因之一,而毛竹林土壤丰富的养分和高pH有利于形成良好微生物群落结构。与杉木相比,毛竹林土壤细菌和真菌特征以及土壤理化性质与阔叶的相似度更高。     

黄土高原旱地麦田26年免耕覆盖对土壤肥力及原核微生物群落多样性的影响

为明确黄土高原旱作麦田长期保护性耕作对土壤肥力和土壤原核微生物的效应,以位于山西省临汾市实施保护性耕作26年的小麦田为试验基地,采用Illumina Hiseq 2500高通量测序等手段,开展了不同耕作措施[免耕覆盖（NTS）、深松免耕覆盖（SNTS）和传统耕作（TT1）]对土壤理化性质和土壤原核微生物多样性的影响分析。研究结果表明：1）NTS和SNTS处理比TT1处理显著提高了土壤全氮、碱解氮、速效磷及速效钾的含量,降低了土壤pH,提高了土壤贮水能力和水分含量,降低了0~10 cm土层的土壤容重,但提高了10~20 cm土层的土壤容重;同时,SNTS处理显著增加了土壤的有机质含量。2）Illumina Hiseq高通量16S rRNA基因V4区测序结果表明：NTS和SNTS处理比TT1处理显著降低了绿弯菌门的相对丰度;NTS比SNTS处理显著降低了土壤中疣微菌门和绿弯菌门的相对丰度;NTS处理显著增加了土壤原核微生物群落的多样性,但未显著改变原核微生物群落的丰度;SNTS对原核微生物群落的多样性和丰富度均未有显著改变;NTS处理的显著性差异物种（Biomarker）高于其他2个处理;其他原核微生物门的相对丰度,在3个处理间尚未有明显差异。3）聚类分析可见：NTS和SNTS处理与TT1处理的微生物群落结构差异较大;NTS处理与SNTS处理间的微生物群落结构差异较小。4）CCA分析可知：土壤pH、有机质、速效氮、速效磷、速效钾含量对土壤原核微生物群落遗传多样性的变化起着重要作用;与TT1处理相比,NTS和SNTS处理在一定程度上改变了土壤原核微生物群落结构,但仍存在结构的相似性。综上所述,长期进行NTS和SNTS处理对黄土高原旱地麦田土壤微生物多样性、丰富度以及土壤肥力因子等的正效作用明显。     

毛乌素沙地人工林恢复对土壤剖面化学性质和细菌群落的影响

明确贫瘠沙地人工林恢复过程中土壤化学性质和微生物群落的动态变化可为风沙区人工林的健康经营和土壤肥力培育提供理论依据。以毛乌素沙地广泛分布的不同年限樟子松林为研究对象,运用高通量扩增子测序和FAPROTAX功能预测技术,解析沙地人工林恢复过程中土壤剖面（0~100 cm）化学性质和细菌群落的分布格局及其变化特征。结果表明：（1）人工林恢复提高了土壤有机碳（SOC）、全氮（TN）及全磷（TP）含量,但降低了土壤有效氮（AN）、有效磷（AP）含量和pH;随土层深度增加,TN含量呈显著下降趋势,而AP含量和pH则相反,TP含量无显著变化。（2）人工林恢复显著改变了变形菌门（Proteobacteria）、酸杆菌门（Acidobacteria）和绿弯菌门（Chloroflexi）的相对丰度,增加了土壤细菌群落基于多度覆盖的物种估计量（ACE指数）;随林分发育,细菌群落的垂直空间变异减小。（3）功能预测表明：人工林恢复降低了土壤细菌对有机物质的分解潜力,造林15年后纤维素分解和芳香族化合物降解过程与未造林样点相比分别下降54.65%和72.18%;但增强了氮的固定及反硝化过程,分别增加99.26%和100.5%。（4）冗余分析和相关性分析表明：SOC和pH是影响细菌群落及其潜在生态功能的重要环境因子。综上可知,沙地人工林恢复虽然增加了土壤碳氮磷总量,但人工林恢复至15年时土壤有效氮磷养分仍显著下降,并显著改变了土壤细菌群落多样性与功能。因此,人为调控措施（如调控凋落物分解与养分释放）对提升干旱和贫瘠生境中沙化土壤养分有效性和微生物多样性与功能具有重要作用,今后在风沙区人工林生态恢复工作中应着重关注林下凋落物的就地留存和腐解。     

基于高通量测序的两种植被恢复类型根际土壤细菌多样性研究

为研究库布齐沙地生态恢复过程中不同植被恢复类型土壤微生物细菌群落结构、多样性的变化特征,以流动沙地为对照,运用高通量测序技术,对自然恢复的油蒿群落、人工种植的中间锦鸡儿群落根际和非根际土壤细菌多样性进行了研究,并分析了土壤理化性质对其分布的影响。结果表明:(1)与流沙对照相比,两种植被恢复类型对细菌多样性产生了正效应,细菌群落丰度、多样性和均匀度明显增加。其中,自然恢复的油蒿群落土壤细菌丰度高于人工种植的中间锦鸡儿群落;(2)变形菌门、酸杆菌门和放线菌门为研究区土壤中的优势细菌类群,其中变形菌门在各样地丰度比例最高,变形菌的4个亚群变化趋势一致,α-变形菌相对含量在油蒿和中间锦鸡儿群落根际土壤中明显增加,尤其是自然恢复的油蒿群落根际土壤中α-变形菌得到了很好的恢复;(3)土壤有机质、全氮、速效氮、速效钾含量和土壤含水量是影响土壤细菌群落丰度和多样性的主要土壤因子,典型相关分析表明土壤有机质、全氮、全钾、速效钾、速效氮含量对于研究区土壤细菌群落遗传多样性的变化起着重要作用。     

The impacts of roads and other infrastructure on mammal and bird populations: A meta-analysis

Biodiversity is being lost at an increased rate as a result of human activities. One of the major threats to biodiversity is infrastructural development. We used meta-analyses to study the effects of infrastructure proximity on mammal and bird populations. Data were gathered from 49 studies on 234 mammal and bird species. The main response by mammals and birds in the vicinity of infrastructure was either avoidance or a reduced population density. The mean species abundance, relative to non-disturbed distances (MSA), was used as the effect size measure. The impact of infrastructure distance on MSA was studied using meta-analyses. Possible sources of heterogeneity in the results of the meta-analysis were explored with meta-regression.Mammal and bird population densities declined with their proximity to infrastructure. The effect of infrastructure on bird populations extended over distances up to about 1 km, and for mammal populations up to about 5 km. Mammals and birds seemed to avoid infrastructure in open areas over larger distances compared to forested areas, which could be related to the reduced visibility of the infrastructure in forested areas. We did not find a significant effect of traffic intensity on the MSA of birds. Species varied in their response to infrastructure. Raptors were found to be more abundant in the proximity of infrastructure whereas other bird taxa tended to avoid it. Abundances were affected at variable distances from infrastructure: within a few meters for small-sized mammals and up to several hundred meters for large-sized mammals.Our findings show the importance of minimizing infrastructure development for wildlife conservation in relatively undisturbed areas. By combining actual species distributions with the effect distance functions we developed, regions sensitive to infrastructure development may be identified. Additionally, the effect distance functions can be used in models in support of decision making on infrastructure planning.     

土壤多样性: 中国山东省基于1:100万比例尺的SOTER的实例研究

The diversity of soils derived from different parent materials and developed on different terrains inShandong Province, China, was analyzed with the theory of pedodiversity using a 1:1000000 scale soilsand terrain digital database (SOTER) of Shandong Province in a geographical information system (GIS)environment. Diversity index of soils derived from different parent materials followed the sequence of igneousrock ＞ sedimentary rock ＞ metamorphic rock ＞ unconsolidated material. Diversity index of soils developedon different terrains followed the sequence of plain ＞ medium-gradient mountain ＞ medium-gradient hill＞ valley floor ＞ medium-gradient escarpment zone ＞ depression ＞ high-gradient mountain. Abundancedistributions of these soil groups matched the logarithmic normal distributions.     

Fungal abundance and distribution as influenced by clearing and land use in a vertic soil of Argentina

The influence of native vegetation clearing and different further soil managements on fungal propagule population diversity was studied in the present work. In each of the 3 years (1998, 1999, and 2000), soil samples were collected at the depth of 0–7.5 cm from sites under native vegetation (V0); naturalized prairie, cleared in 1982 (P16); conventional tillage, cleared in 1972 (T26); and direct drilling, cleared in 1958 (D40). Fungal population size and relative abundance of fungal genera were studied by plate counts and further identification of isolates on potato dextrose agar. The undisturbed site and the other sites with increasing time elapsed since native vegetation clearing and different management history showed a distinctive distribution of fungal genera. There were significant differences (p<0.05) among the sites in the abundance of fungal genera analyzed in all the 3 years. Principal component analysis based on relative fungal genus abundance differentiated the sites with 75% variance explained by the first and second components. Diversity and abundance of isolated fungal genera were increased as density of Penicillium spp. decreased, suggesting a competitive effect of this fungal genus. The largest diversity was found in the site under no-till management. The different distribution and relative abundance of the fungal genera studied seemed to be influenced strongly by the management and the presence of surface residue in the no-tilled site.     

用单路三束质谱法测定生物样品的~(15)N丰度

对 Ｆｉｎ ｎｉｇａｎ Ｍ Ａ Ｔ ２５１ 质谱计的数据采集和处理系统进行了改造,使其能用单路三束法测定生物样品的１５ Ｎ 丰度。就测定方法的精密度、准确度和所用样品氮量及记忆效应问题做了试验,证实该测定方法精确可靠。     

Composition and abundance of birds in Andean alder (Alnus acuminata) patches with past and present harvest in Bolivia

We compared species richness and abundance of birds between five patches under selective Alnus exploitation and five patches that have not been harvested for at least 10 years prior to our study, during the early dry season (April-July 2001), in Cotapata National Park. Using “point counts” we recorded birds and their distribution in two (<1.5 m and >1.5 m) forest layers. Simultaneously we evaluated the floristic structure (size [dbh] distribution, basal area, tree density, tree height, and vegetation cover) and composition (diversity) on three transects placed within each Alnus patch. Both bird diversity and vegetation cover were significantly higher in not presently used patches but only for the higher layer of the forest, whereas plant diversity was higher in presently used patches. Lack of differences between the two types of Alnus patches in any of the vegetation parameters measured in the lower layer was coupled with an indistinct avifauna. Small changes in habitat characteristics following a perturbation like selective logging have the potential to affect richness and abundance of birds, at least within the habitats directly affected by the perturbation.     

Monitoring the recovery of smalltooth sawfish, Pristis pectinata, using standardized relative indices of abundance

The US population of smalltooth sawfish, Pristis pectinata, is currently listed as endangered under the Endangered Species Act. An important component of monitoring the recovery of this species is establishing long-term baseline trends in abundance. In the absence of scientific survey data, assessing and monitoring the status of some marine species has required the utilization of fishery-dependent data. Using voluntary dockside interviews of sport fishers collected in Everglades National Park, a standardized index of abundance was created for smalltooth sawfish using the delta method. The index was developed as the product of separate generalized linear models of the proportion of positive trips and the positive catch rates on successful trips. Development of the final model included testing factors that were expected to influence the catch of smalltooth sawfish. The final model assumed a binomial distribution for the proportion of positive trips and a lognormal distribution for positive catch rates. Year was significant as a main effect in the binomial model whereas year and skill level of the fishing party were significant in the lognormal model. The relative abundance index shows a small increase in abundance at an average rate of about 5% per year since 1989. These results indicate that the population of smalltooth sawfish in Everglades National Park has at least stabilized and may be increasing. However, additional data and analyses from multiple sources are required before definitive conclusions on the recovery of smalltooth sawfish can be established.     

福建将石自然保护区鸟类物种相对多度模型的拟合研究

应用对数级数分布模型、对数正态分布模型、几何级数分布模型和分割线段模型对福建将石自然保护区鸟类物种相对多度分布格局对比研究结果表明 ,对数级数分布模型、对数正态分布模型能较好地描述将石自然保护区鸟类多样性 ,而几何级数分布模型和分割线段模型则不适于描述该自然保护区鸟类多样性 ;“多度 /频度”图解可较好地反映对数级数分布模型拟合效果 ,丘陵山地鸟类群落物种相对多度不遵从对数级数分布 ,各生境群落百分率相似性指数说明各生境群落百分率相似性较低。     

用~(15)N天然丰度法估测结瘤作物的共生固N_2量

测定了位于北京西北郊的本所农场耕地及附近几个土样的土壤总Nδ15N值,农场耕地的δ15N值为6.39±0.42‰,本所庭园土壤为5.03±0.26‰,圆明园土壤为1.66±0.49‰。农场耕地土壤总Nδ15N的垂直变化为6.09±0.77～7.71±0.67‰,水平变化为6.52±0.55～7.13±0.73‰。 无N营养液砂培大豆的测定结果表明,根瘤富含15N,δ15N值为8.32±0.16‰和10.54±0.30‰,地上部贫化15N,δ15N为—2.25±0.48～—4.16±0.75‰,说明在固N2过程中和固N2产物输送转化过程中发生了N同位素的分馏,固N2分馏因数β=1.0023～1.0042。 用15N天然丰度法估计不同大豆品种的％Ndfa表明,不同大豆品种的N素自给能力不同。固N2植物的δ15N值明显低于非固N2植物的δ15N值,一般相差3～5δ15N,说明用15N天然丰度法评价这些植物的固N2状况是可能的。然而,不固N02植物也有较低的δ15N值,即便在同一地块上,非固N2植物的δ15N值也很不相同,有待进一步研究。