长期不同施肥制度对水稻土氨氧化微生物数量和群落结构的影响

由氨氧化微生物驱动的氨氧化过程是硝化作用的限速步骤,在土壤氮素循环过程中扮演着重要角色.以湖南省宁乡县长达30 a定位试验水稻土壤为研究对象,采用荧光定量PCR和Illumina MiSeq高通量测序分析方法,以amoA基因为靶标,研究了4种施肥制度[不施肥(CK)、化肥(CF)、70%化肥+30%有机肥(CFM₁)和40%化肥+60%有机肥(CFM₂)]水稻土壤氨氧化微生物的数量和群落结构变化.结果表明: 不同施肥处理氨氧化古菌(AOA)和氨氧化细菌(AOB) amoA基因拷贝数分别为3.09×10⁷～8.37×10⁷和1.04×10⁷～7.03×10⁷ copies·g^-1干土.施肥显著提高了AOA和AOB数量,但处理CFM₂中AOB数量与CK差异不显著.有机肥配施比例对AOB群落α多样性指数的影响强于AOA,处理CFM₁中AOA群落的多样性指数(Shannon)和AOB群落的丰富度指数(ACE和Chao1)均显著高于CK.奇古菌门和泉古菌门是AOA群落的优势门类群,占AOA amoA基因总序列的83.4%;亚硝化螺菌属、environmental_samples_norank、Bacteria_unclassified和Nitrosomonadales_unclassified是AOB群落的优势属类群,占AOB amoA基因总序列的97.8%.维恩分析结果显示,有机肥配施比例对AOB群落操作分类单元(OTU)数量的影响强于AOA,但对各处理共有AOA和AOB amoA基因序列条数的影响均较小.冗余分析结果显示,不同施肥处理AOB群落结构差异强于AOA,且所有土壤理化性质均与AOA和AOB群落结构存在显著相关关系.综上可知:有机肥配施比例显著改变了AOA和AOB数量、多样性和群落结构,配施30%有机肥时,AOA群落的Shannon指数最高,AOB群落数量、ACE和Chao1指数均最高.研究结果可为进一步探讨农业系统中氨氧化微生物对不同施肥制度的响应机制及其在氮素转化中的作用提供科学依据.     

施氮肥对华北平原土壤氨氧化细菌和古菌数量及群落结构的影响

利用荧光定量PCR、末端限制性片段长度多样性(T-RFLP)和基因克隆文库技术,比较了4种施氮水平(不施氮肥,0 kg N/hm~2,CK;施低水平氮肥,75 kg N/hm~2,N1;施中水平氮肥,150 kg N/hm~2,N2;施高水平氮肥,225 kg N/hm~2,N3)下华北平原地区小麦季表层(0—20 cm)土壤总细菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的丰度和群落结构。结果表明,土壤总细菌、AOB和AOA数量分别在每克干土5.74×10~9—7.50×10~9、8.89×10~6—2.66×10~7和3.83×10~8—7.78×10~8之间。不同施氮量土壤AOA数量均高于AOB数量,AOA/AOB值在81.72—14.38之间。增施氮肥显著显著提高AOB数量(P0.05),对总细菌和AOA数量的影响不显著(P0.05)。与CK相比,处理N1、N2和N3中AOB数量分别提高了0.64、1.50和1.99倍。增施氮肥显著改变了AOB和AOA的群落结构,且不同施氮量处理中AOB群落结构差异更大。系统进化分析显示,施氮肥小麦土壤AOB主要为Nitrosospira属类群,分布在Cluster 3的两个分支中;AOA分布在Cluster S的4个分支中。相关性分析显示,AOB数量与全氮和铵态氮含量呈显著正相关关系,与土壤pH和碳氮比呈显著负相关关系(P0.05);AOA数量与硝态氮含量和土壤pH呈显著正相关关系,与铵态氮含量呈显著负相关关系(P0.05)。研究结果表明:增施氮肥可显著改变华北平原地区碱性土壤AOB数量与群落结构,该地区小麦土壤中AOB比AOA对氮肥响应更敏感。     

长期施加氮肥及氧化钙调节对酸性土壤硝化作用及氨氧化微生物的影响

以长期施加氮肥及添加氧化钙调节的酸性土壤为研究对象,运用定量PCR和DGGE技术,探讨了土壤氨氧化微生物及硝化作用对不同施肥处理及氧化钙调节的响应。长期施化学氮肥导致酸性土壤p H(KCl)值(3.35—3.47)和硝化潜势(0.02—0.14μg NO-2-N g-1土壤h-1)进一步降低,而添加Ca O后土壤酸化得以缓解(p H值4.10—4.46),土壤硝化潜势(0.22—0.34μg NO-2-N g-1土h-1)显著增加。同时,添加Ca O处理对氨氧化古菌(AOA)的群落结构无明显影响,但明显提高了各施肥处理土壤中氨氧化细菌(AOB)的群落多样性,加Ca O处理的土壤中,AOA的数量降低而AOB的数量增加。这些结果表明虽然酸性土壤中AOA在数量和活性上占主导优势,AOB在功能上冗余,但当添加Ca O后,AOA和AOB对环境变化迅速作出响应,并根据其不同的生态位需求重新分配优势地位,二者交替作用共同驱动酸性土壤硝化作用。     

长期不同施肥对玉米根茬生物量及养分累积量的影响

以黄土高原南部两个长期定位试验（分别开始于1990和2003年）为研究对象,探讨了不同肥料处理对玉米根茬生物产量和养分累积的影响.于2011年10月玉米收获后采集0～20 cm土层不同施肥处理玉米根茬.结果表明：与不施肥及偏施N、NK、PK化肥相比,氮磷配施（NP）、氮磷钾平衡施肥（NPK）、有机无机配施（M₁NPK、M₂NPK）及化肥配合秸秆（SNPK）处理均显著提高了玉米根茬干质量.根茬固碳量及氮、磷、钾养分累积量在NP、NPK、M₁NPK、M₂NPK、SNPK处理显著高于不施肥和偏施N、NK、PK化肥处理,其中以有机无机配施处理效果最好.与不施氮肥（N₀）相比,施氮120 kg N·hm^-2（N₁₂₀）和240 kg N·hm^-2（N₂₄₀）处理根茬干质量分别提高38%和45%,高量氮肥对根茬增量效果不显著.施用氮肥也显著提高了根茬碳、氮、磷、钾累积量.根茬可溶性有机碳、可溶性总氮含量在NP、NPK、M₁NPK、M₂NPK、SNPK及N₁₂₀和N240处理中较高.氮磷钾平衡施肥、有机无机配施以及秸秆还田处理降低了根茬的纤维素、木质素含量.根茬C/N、木质素/N在CK、PK、N₀处理间显著高于其他施肥处理.因此,氮磷配施、氮磷钾平衡施肥、有机无机配施及秸秆还田处理能够促进玉米根生长,提高营养成分含量,有利于土壤培肥和固碳.     

施肥方式对干旱半干旱地区土壤氨氧化微生物数量和群落的影响

氨氧化是硝化作用的限速步骤,也是评估土壤氮循环和提高氮肥利用效率的重要指标。以内蒙古农牧业科学院旱作实验站长期定位实验为基础,通过实时荧光定量PCR和末端限制性片段长度多态性分析,研究了5种施肥方式（单施氮肥、单施有机肥、氮磷钾配施、有机无机配施和不施肥）对土壤氨氧化古菌（AOA）和氨氧化细菌（AOB）群落丰度、结构和活性的影响。结果表明：单施氮肥、氮磷钾肥配施以及有机无机肥配施均能显著提高AOB的丰度以及土壤硝化潜势。Nitrosospiria cluster 3a.1是不施肥土壤中主要的AOB种群,而施用氮肥后优势种群转变为Nitrosospiria cluster 3a.2。Nitrosospiria cluster 3b的比例在施用有机肥处理土壤中显著升高。在干旱半干旱地区,土壤pH和含水量是解释AOB群落结构变化的关键环境因子。AOA的丰度在单独施用氮肥处理中显著升高,但不同施肥方式对AOA的群落结构没有显著影响。     

氮肥减施与配施有机肥对冬小麦土壤线虫群落结构的影响

土壤线虫是指示土壤健康的典型生物之一,为了揭示氮肥减施对土壤健康的影响,以冬小麦土壤为对象,研究了氮肥减施和配施有机肥对拔节期冬小麦土壤线虫群落结构的影响。共设置了6个施肥处理:CF(315 kg N·hm^-2,常规施肥量)、N₂₄₀(240 kg N·hm^-2)、N₂₁₀(210 kg N·hm^-2)、N₁₈₀(180 kg N·hm^-2)、F₁₅₀(180 kg N·hm^-2+150 kg·hm^-2黄腐酸)、F₂₂₅(180 kg N·hm^-2+225 kg·hm^-2黄腐酸)。结果表明: 1)氮肥减施会降低土壤线虫数量,降幅为15.3%～68.5%;2)各处理均以原杆属为优势属(19.6%～50.4%)。氮肥减施提高了食真菌类、植食类和捕食/杂食类线虫的丰度,食细菌类线虫的丰度先降低后升高。配施有机肥后,食细菌类和食真菌类线虫的丰度降低,植食类和捕食/杂食类线虫丰度升高;3)N₂₄₀和F₂₂₅处理分别使线虫多样性指数H提高了48.1%和58.5%。N₂₄₀处理线虫成熟度指数MI最高(1.95)。N₁₈₀处理线虫结构指数SI最低(43.33),配施有机肥的F₂₂₅线虫结构指数SI达到62.72,但线虫富集指数EI最低(80.82)。说明减施氮肥并配施有机肥可以提高土壤线虫的多样性,使食物网向复杂稳定方向发展,对农田土壤生态系统有积极意义。     

不同利用方式对红壤坡地微生物多样性和硝化势的影响

采集了中国科学院桃源农业生态试验站红壤坡地农田、自然恢复林和茶园土壤样品,采用末端限制性酶切片段长度多态性分析(T.RFLP)技术分析土壤细菌、古菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的多样性,采用好气培养法测定不同土壤的硝化势,研究不同土地利用方式对微生物多样性和硝化势的影响.结果表明:土壤AOB和AOA多样性指数差异不显著,且在3种不同土地利用方式中呈现相同的趋势,均为农田=茶园>自然恢复林;通过RDA分析发现,不同利用方式造成土壤理化性状的改变是影响土壤AOA和AOB群落结构的主要原因;好气培养法测得不同土壤硝化势农田最高,茶园次之而自然恢复林最低;相关性分析显示,硝化势与细菌16S rRNA、AOA和AOB amoA基因多样性指数呈显著正相关,其中与AOA amoA基因关系最为密切;总体来说,红壤坡地不同利用方式改变了土壤细菌、古菌、AOA和AOB的多样性,土壤AOB和AOA积极参与了土壤的硝化过程,且AOA在氨氧化微生物群落生态功能中占有重要地位,AOA比AOB与硝化势的关系更为密切.     

Responses of soil N2O emissions to experimental warming regulated by soil moisture in an alpine steppe

土壤氧化亚氮(N₂O)排放是大气N₂O不可忽视的来源。然而, 目前学术界在气候变暖对土壤N₂O排放影响方面的认识仍存在较大争议, 且调控土壤N₂O排放的微生物机制尚不明确。为此, 该研究以青藏高原高寒草原生态系统为研究对象, 使用透明开顶箱(OTCs)模拟气候变暖, 并基于静态箱法测定了2014和2015年生长季(5-10月)的土壤N₂O通量, 同时利用定量PCR技术测定了表层(0-10 cm)土壤中氨氧化古菌(AOA)和氨氧化细菌(AOB)的基因丰度。结果显示: 增温处理导致2014和2015年生长季表层(0-10 cm)土壤温度分别升高了1.7 ℃和1.6 ℃, 土壤体积含水量下降了2.5%和3.3%, 其他的土壤理化性质没有发生显著变化。土壤N₂O通量呈现年际差异, 2014和2015年生长季的平均值分别为3.23和1.47 μg·m ^-2·h ^-1, 然而, 增温处理并没有显著改变土壤N₂O通量。2014年生长季主导硝化作用的AOA和AOB的基因丰度分别为5.0 × 10 ⁷和4.7 × 10 ⁵拷贝·g ^-1, 2015年为15.2 × 10 ⁷和10.0 × 10 ⁵拷贝·g ^-1。尽管基因丰度存在显著的年际差异, 但在两年中与对照相比并未发生显著变化。在生长季尺度上, 增温导致的土壤N₂O变化量与其引起的土壤水分变化量之间显著正相关, 而与土壤温度的变化量之间没有显著相关关系。以上结果表明, 增温导致的土壤干旱会抑制土壤N₂O通量对增温的响应, 意味着未来评估气候变暖情景下土壤N₂O排放量时需考虑增温引发的土壤干旱等间接效应。     

减量施氮下基肥后移对南方冬小麦产量和氮素利用效率的影响

过量施用氮肥导致氮肥利用率降低,环境风险加大.合理降低施氮量、优化氮肥运筹对于小麦高产高效栽培具有重要意义.本研究采用大田试验,以常规施氮方式(240 kg N·hm^-2, 基肥∶拔节肥∶孕穗肥=5∶3∶2)为对照,研究了不同施氮量(240、180、150 kg N·hm^-2,分别用N₂₄₀、N₁₈₀、N₁₅₀表示)及基苗肥施用时期(基施、4叶期施、6叶期施,分别用L₀、L₄、L₆表示)对小麦产量和氮素利用效率的影响.结果表明: 小麦籽粒产量随施氮量的降低而降低,但N₁₈₀与N₂₄₀处理相比无显著差异,而N₁₅₀处理显著降低;氮肥农学效率和吸收效率均以N₁₈₀处理最高.不同施肥时期间,L₄处理的籽粒产量和氮肥利用率最高.N₁₈₀四叶施肥(N₁₈₀L₄)处理的产量与对照无显著差异,但氮肥利用率显著提高.N₁₈₀L₄处理叶面积指数、旗叶光合速率、叶片氮含量、旗叶硝酸还原酶和谷氨酰胺合成酶活性、拔节后干物质和氮素积累量较对照未显著降低.适量降低氮肥用量配合基肥后移能够提高生育后期光合生产能力和氮素吸收同化能力,在保持高产的条件下实现氮素利用效率的同步提高.     

秸秆还田对玉米根际氨氧化微生物群落及红壤硝化潜势的影响

为探讨酸性红壤根际氨氧化微生物群落以及硝化作用对不同秸秆还田处理的响应,基于中国科学院鹰潭红壤生态实验站设置的秸秆还田长期试验平台(9年),采用荧光定量PCR和高通量测序技术,研究不同秸秆还田处理(不施肥(CK);氮磷钾肥(NPK);氮磷钾肥+秸秆(NPKS);氮磷钾肥+秸秆猪粪配施(NPKSM);氮磷钾肥+秸秆生物炭(NPKB))下玉米根际土壤氨氧化古菌(ammonia-oxidizing archaea, AOA)和细菌(ammonia-oxidizing bacteria, AOB)丰度和群落结构的变化,揭示了秸秆还田对根际氨氧化微生物群落结构和硝化潜势(potential nitrification activity, PNA)的影响机制。结果发现：相比CK和NPK处理,秸秆还田显著提高了土壤养分含量和硝化潜势,其中有机碳(SOC)、全氮(TN)、全磷(TP)、速效磷(AP)、速效钾(AK)、硝态氮(NO~-₃-N)和铵态氮(NH~+₄-N)含量显著增加,NPKSM处理对土壤肥力提升效果最佳。AOA的硝化潜势显著高于AOB,表明AOA...     

间作对马铃薯种植土壤硝化作用和硝态氮供应的影响

土壤硝态氮供应对满足作物氮素需求至关重要,但间作如何影响土壤硝态氮供应及其作用机制尚不清楚。本研究基于4个氮水平(N₀, 0 kg·hm^-2; N₁, 62.5 kg·hm^-2; N₂, 125 kg·hm^-2; N₃, 187.5 kg·hm^-2)的马铃薯单作、马铃薯与玉米间作小区试验,分析土壤硝态氮含量与强度、硝化势和氨氧化功能基因丰度的差异,探讨间作影响土壤硝态氮供应和氮调控的机理。结果表明: 土壤硝态氮含量和强度随施氮量增加而升高,但同一施氮水平下间作均低于单作。施氮提高了土壤硝化势,且单作的响应高于间作。土壤中氨氧化细菌(AOB)的amoA基因丰度大于氨氧化古菌(AOA),二者在间作时均随施氮量增加呈现先增加后降低的趋势;相同施氮量下,间作的AOA和AOB基因丰度(除N₂外)均低于单作。相关分析、回归分析和主成分分析显示,马铃薯间作后,土壤AOB、AOA的amoA基因丰度下降,硝化势减弱,导致土壤硝态氮含量和强度降低。因此,间作导致土壤硝态氮供应降低与土壤氮转化的微生物过程有关,间作条件下的马铃薯种植应注意保障土壤氮素供应。     

小兴安岭酸性泥炭土硝化作用的类型及其驱动因子

以小兴安岭酸性森林泥炭土为研究对象,通过加入10 mL·L^-1乙炔及不同浓度的外源硫酸铵(0、1.2、6.0 mmol N·kg^-1)进行硝化培养试验,探究酸性泥炭土中硝化作用类型及主要驱动因子.结果表明:无论有无外加氮源,酸性泥炭土均存在较强的矿化作用(0.9～1.4 mg N·kg^-1·d^-1),经过2周的培养均发生了硝化作用(0.4～0.6 mg N·kg^-1·d^-1),且不同浓度硫酸铵处理之间无显著差异;而乙炔处理虽有较强的矿化作用(0.8 mg N·kg^-1·d^-1),但未发生明显的硝化作用(0 mg N·kg^-1·d^-1),说明该酸性泥炭土以自养硝化为主,外源无机氮源浓度对硝化作用无显著影响,硝化底物NH₃的主要来源不是外源硫酸铵,更可能来源于土壤中有机氮的矿化.培养0～14 d,无论有无外加氮源,酸性泥炭土氨氧化细菌(AOB)和古菌(AOA)丰度均显著增加,但不同浓度硫酸铵处理间无显著差异,表明外源无机氮浓度对氨氧化微生物的生长无显著促进作用.与不加乙炔的对照相比,乙炔处理AOB和AOA丰度随时间均无显著变化,推测AOA与AOB在该酸性泥炭土的硝化过程中都可能起一定的作用.     

Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices

The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were investigated by using quantitative real-time polymerase chain reaction, cloning and sequencing approaches based on amoA genes. The soil, classified as agri-udic ferrosols with pH (H(2)O) ranging from 3.7 to 6.0, was sampled in summer and winter from long-term field experimental plots which had received 16 years continuous fertilization treatments, including fallow (CK0), control without fertilizers (CK) and those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): N, NP, NK, PK, NPK and NPK plus organic manure (OM). Population sizes of AOB and AOA changed greatly in response to the different fertilization treatments. The NPK + OM treatment had the highest copy numbers of AOB and AOA amoA genes among the treatments that received mineral fertilizers, whereas the lowest copy numbers were recorded in the N treatment. Ammonia-oxidizing archaea were more abundant than AOB in all the corresponding treatments, with AOA to AOB ratios ranging from 1.02 to 12.36. Significant positive correlations were observed among the population sizes of AOB and AOA, soil pH and potential nitrification rates, indicating that both AOB and AOA played an important role in ammonia oxidation in the soil. Phylogenetic analyses of the amoA gene fragments showed that all AOB sequences from different treatments were affiliated with Nitrosospira or Nitrosospira-like species and grouped into cluster 3, and little difference in AOB community composition was recorded among different treatments. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). Cluster M dominated exclusively in the N, NP, NK and PK treatments, indicating a pronounced difference in the community composition of AOA in response to the long-term fertilization treatments. These findings could be fundamental to improve our understanding of the importance of both AOB and AOA in the cycling of nitrogen and other nutrients in terrestrial ecosystems.     

Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam

The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) communities under different long-term (17 years) fertilization practices were investigated using real-time polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE). A sandy loam with pH (H(2)O) ranging from 8.3 to 8.7 was sampled in years 2006 and 2007, including seven fertilization treatments of control without fertilizers (CK), those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): NP, NK, PK and NPK, half chemical fertilizers NPK plus half organic manure (1/2OMN) and organic manure (OM). The highest bacterial amoA gene copy numbers were found in those treatments receiving N fertilizer. The archaeal amoA gene copy numbers ranging from 1.54 x 10(7) to 4.25 x 10(7) per gram of dry soil were significantly higher than those of bacterial amoA genes, ranging from 1.24 x 10(5) to 2.79 x 10(6) per gram of dry soil, which indicated a potential role of AOA in nitrification. Ammonia-oxidizing bacteria abundance had significant correlations with soil pH and potential nitrification rates. Denaturing gradient gel electrophoresis patterns revealed that the fertilization resulted in an obvious change of the AOB community, while no significant change of the AOA community was observed among different treatments. Phylogenetic analysis showed a dominance of Nitrosospira-like sequences, while three bands were affiliated with the Nitrosomonas genus. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). These results suggest that long-term fertilization had a significant impact on AOB abundance and composition, while minimal on AOA in the alkaline soil.     

Impacts of organic and inorganic fertilizers on nitrification in a cold climate soil are linked to the bacterial ammonia oxidizer community

The microbiology underpinning soil nitrogen cycling in northeast China remains poorly understood. These agricultural systems are typified by widely contrasting temperature, ranging from −40 to 38°C. In a long-term site in this region, the impacts of mineral and organic fertilizer amendments on potential nitrification rate (PNR) were determined. PNR was found to be suppressed by long-term mineral fertilizer treatment but enhanced by manure treatment. The abundance and structure of ammonia-oxidizing bacterial (AOB) and archaeal (AOA) communities were assessed using quantitative polymerase chain reaction and denaturing gradient gel electrophoresis techniques. The abundance of AOA was reduced by all fertilizer treatments, while the opposite response was measured for AOB, leading to a six- to 60-fold reduction in AOA/AOB ratio. The community structure of AOA exhibited little variation across fertilization treatments, whereas the structure of the AOB community was highly responsive. PNR was correlated with community structure of AOB rather than that of AOA. Variation in the community structure of AOB was linked to soil pH, total carbon, and nitrogen contents induced by different long-term fertilization regimes. The results suggest that manure amendment establishes conditions which select for an AOB community type which recovers mineral fertilizer-suppressed soil nitrification.