长期施肥对酸性土壤氨氧化微生物群落的影响

【目的】 长期施肥显著影响着酸性土壤的pH,研究由此引起的土壤中氨氧化古菌 (ammonia-oxidizing archaea,AOA) 和氨氧化细菌 (ammonia-oxidizing bacteria,AOB) 的变化,为土壤培肥提供理论依据。 【方法】 供试土壤为27年长期定位施肥试验的红壤,供试作物为玉米。选择不施肥对照 (CK)、氮肥120 kg/(hm2·a)(N)、氮磷钾肥 (NPK) 和猪粪2000 kg/(hm2·a)(OM) 4个处理采集土壤样品,测定了土壤基本理化性状;利用qPCR、PCR-DGGE方法,分析土壤AOA和AOB群落丰度与组成。 【结果】 1) 长期定位施肥导致土壤pH值发生显著变化,N处理的土壤pH值最低,仅为4.03,其次是NPK和CK处理的土壤,OM处理土壤pH值最高,接近中性达6.40。2) 与CK相比,长期施肥提高了土壤有机质、全氮、铵态氮和硝态氮含量。3)OM处理显著提高了土壤NH₃浓度,而其它处理对NH₃浓度无显著影响。4) 施肥显著增加了土壤AOA的丰度,OM处理提升幅度最大;AOA丰度与土壤有机质碳、全氮呈极显著正相关 (P < 0.01),与铵态氮、土壤NH ₃浓度呈显著正相关 (P < 0.05),与土壤pH、硝态氮关系不显著 ( P > 0.05);施肥改变了AOA的群落结构,CK、N、NPK处理的群落结构差异不显著,OM处理与另外三个处理差别较大。主要AOA类群是Group 1.1b,少数属于Group 1.1a-associated。RDA分析表明,土壤pH值、有机质、总氮、铵态氮、土壤中NH ₃浓度是导致AOA群落变化的主要环境因子。5) 仅OM处理对AOB丰度和群落产生了显著影响,主要类群是Nitrosospira Cluster 3,少数属于Nitrosospira Cluster 9。AOB丰度与土壤NH₃浓度呈极显著正相关 (P < 0.01),与有机质碳、全氮呈显著正相关关系 ( P < 0.05),与土壤pH、铵态氮、硝态氮关系均不显著 ( P > 0.05)。 【结论】 长期施用不同肥料对酸性土壤的理化性质影响差异大,AOA和AOB的丰度和群落结构也发生了明显变化,尤其是施加有机肥之后。来自不同处理的大部分AOA属于Group 1.1b类群,少数属于Group 1.1a-associated类群。仅在OM处理中检测到AOB类群,大部分属于Nitrosospira Cluster 3,少数属于Nitrosospira Cluster 9。      

稻虾共作模式下稻田土壤氨氧化微生物丰度和群落结构的特征

【目的】研究稻虾共作模式下土壤氨氧化微生物数量、群落多样性及群落结构,深入了解该模式下的土壤微生态环境的演变。【方法】试验点在湖北省荆州市长江大学农学院基地,设置稻虾共作模式 (CR) 与常规中稻种植模式 (MR),借助荧光定量PCR技术与Illumina Miseq高通量测序平台,分析了土壤氨氧化细菌 (AOB) 与古菌 (AOA) 丰度、多样性及群落结构。【结果】与MR模式相比,CR模式显著提高了土壤硝态氮、总碳及总氮含量,对土壤pH、碱解氮及土壤碳氮比无显著影响。CR模式土壤AOA与AOB amoA基因拷贝数为3.13 × 105和7.01 × 105 copies/g干土,MR模式土壤AOA、AOB amoA基因拷贝数为1.41 × 105和3.87 × 105 copies/g干土,两个模式土壤AOB的数量均显著高于AOA,CR模式土壤AOA、AOB的数量均显著高于MR模式 (P < 0.05)。α群落多样性指数表明,相比MR,CR模式显著降低了土壤AOA群落多样性,对AOB群落多样性无显著影响。Venn结果分析,CR模式增加了AOA amoA基因的物种,改变了AOB amoA基因的物种组成,且AOB amoA物种数量下降。在属水平上,norank_c_environmental_samples_p_Thaumarchaeota、unclassified_k_norank_d_Archaea、norank_c_environmental_samples_p_Crenarchaeota、norank_p_environmental_samples_k_norank为AOA的优势类群,相对丰度占AOA amoA基因总序列的99.25%～99.46%,CR模式显著提高了norank_c_environmental_samples_p_Crenarchaeota在AOA群落属水平的相对丰度;unclassified_k_norank_d_Bacteria、norank_f_environmental_samples、norank_o_environmental_samples_c_Betaproteobacteria、unclassified_o_Nitrosomonadales为AOB的优势类群,相对丰度共占97.78%～98.49%,且CR模式显著增加了norank_o_environmental_samples_c_Betaproteobacteria与unclassified_o_Nitrosomonadales在AOB群落属水平的相对丰度。冗余分析 (RDA) 结果显示,土壤基本理化性质对于土壤AOA、AOB群落结构影响有着相似的趋势,其中对AOA、AOB群落结构影响最大的因子是硝态氮,其次分别为总碳、铵态氮、碱解氮、pH。根据RDA投影距离分析,稻虾共作模式对土壤AOA群落结构的影响大于AOB,且MR与稻虾共作模式土壤AOB的群落结构具有一定的相似度。【结论】稻虾共作模式显著降低了AOA群落多样性,而对AOB群落无显著影响;稻虾共作模式显著增加了AOA与AOB的丰度并显著影响了群落结构组成。土壤硝态氮、总碳、铵态氮、碱解氮、pH含量是导致土壤微生物数量、多样性及群落结构变化的主要原因。     

氨氧化基因丰度和潜在氨氧化速率对加拿大一枝黄花入侵和土壤类型的响应

  【目的】  土壤类型和植物入侵影响土壤微生物群落结构和功能，基于此，我们研究加拿大一枝黄花 (Solidago canadensis) 入侵两种类型土壤后，土壤中氨氧化古细菌 (AOA) 和氨氧化细菌 (AOB) 的基因丰度和潜在氨氧化速率 (PAOR) 的变化规律及影响机理。  【方法】  云南和浙江是加拿大一枝黄花入侵的重点地区，本研究在云南省滇池周边的海东湿地公园、捞鱼河湿地公园和安乐村 (土壤类型均为冲积土) 以及浙江省东部的杭州湾湿地公园、临海上盘镇和路桥镇峰江村 (土壤类型均为黄泥田土)，分别选择一块采样地，面积为100～150 m2。在每块采样地加拿大一枝黄花入侵 (Mono) 和未入侵 (Nat) 之处，划出0.5 m × 0.5 m，采集土壤和植物样品。Mono和Nat地块相距10～20 m，每个地块重复3次。采用qPCR和室内培养技术分析土壤中AOA、AOB基因丰度和PAOR，用植物生态和土壤化学方法分析植物生物量和土壤化学性状。  【结果】  加拿大一枝黄花入侵后，冲积土中AOA丰度和PAOR显著下降，黄泥田土中AOA丰度和PAOR却显著提高，而AOB丰度在两种类型土壤中均显著提高。Nat冲积土中AOA丰度和PAOR大于黄泥田土，而Mono条件下冲积土中AOA丰度和PAOR小于黄泥田土，AOB丰度在两种土壤类型中均变化较小。Mono样地植被地上部分和地下部分生物量、土壤有机质含量和pH是影响冲积土和黄泥田土AOA丰度和PAOR的重要因素。与AOA不同，冲积土AOB丰度仅受植物地下部分生物量的影响，而黄泥田土AOB丰度同时受植物地下部分生物量、土壤总磷含量和pH影响。  【结论】  加拿大一枝黄花入侵大大增加了植被地上部和地下部生物量，进而为微生物提供了大量的碳源，同时提高了土壤pH，因此，氨氧化细菌AOB的丰度显著增加。土壤类型仅影响AOA的丰度，对AOB和氨氧化潜力没有显著影响，而加拿大一枝黄花入侵显著影响两类土壤中的AOA、AOB丰度以及氨氧化潜力。     

脲酶抑制剂与硝化抑制剂对稻田土壤硝化、反硝化功能菌的影响

[目的]在农业生产中,脲酶抑制剂(urease inhibitor,UI)与硝化抑制剂(nitrification inhibitor,NI)常作为氮肥增效剂来提高肥料利用率。本文研究了在我国南方红壤稻田施用脲酶抑制剂与硝化抑制剂后,土壤中氨氧化细菌(ammonia oxidizing bacteria,AOB)、氨氧化古菌(ammonia-oxidizing archaea,AOA)以及反硝化细菌的丰度以及群落结构的变化特征,旨在揭示抑制剂的作用机理及其对土壤环境的影响。[方法]试验在我国南方红壤稻田进行,共设5个处理:1)不施氮肥(CK);2)尿素(U);3)尿素+脲酶抑制剂(U+UI);4)尿素+硝化抑制剂(U+NI);5)尿素+脲酶抑制剂+硝化抑制剂(U+UI+NI),3次重复。脲酶抑制剂与硝化抑制剂分别为NBPT[N-(n-butyl)thiophosphrictriamide,N-丁基硫代磷酰三胺]和DMPP(3,4-dimethylpyrazole phosphate,3,4-二甲基吡唑磷酸盐)。通过荧光定量PCR(Real-time PCR)研究水稻分蘖期与孕穗期抑制剂对三类微生物标记基因拷贝数的影响,并分析土壤铵态氮、硝态氮与三种菌群丰度的相关性;利用变性梯度凝胶电泳(DenaturingGradient Gel Electrophoresis,DGGE)分析抑制剂对土壤AOB、AOA以及反硝化细菌群落结构的影响,并对优势菌群进行系统发育分析。[结果]1)荧光定量PCR结果表明,施用氮肥对两个时期土壤中AOB的amoA基因与反硝化细菌nirK基因的拷贝数均有显著提高,而对AOA的amoA基因始终没有明显影响;AOB与nirK反硝化细菌的丰度与两个时期的铵态氮含量、分蘖期的硝态氮含量呈极显著正相关,与孕穗期的硝态氮含量相关性不显著;DMPP仅在分蘖期显著减少了AOB的amoA基因拷贝数,表明DMPP主要通过限制AOB的生长来抑制稻田土壤硝化过程;NBPT对三类微生物的丰度无明显影响;2)DGGE图谱表明,在分蘖期与孕穗期,施用氮肥均明显增加了图谱中AOB的条带数,而对AOA却没有明显影响;氮肥明显增加了孕穗期反硝化细菌的条带数;与氮肥的影响相比,抑制剂NBPT与DMPP对AOA、AOB以及反硝化菌的群落结构影响甚微;系统发育分析结果表明,与土壤中AOB的优势菌群序列较为接近的有亚硝化单胞菌和亚硝化螺菌。[结论]在南方红壤稻田中,施入氮肥可显著提高AOB与反硝化细菌的丰度,明显影响两种菌群的群落结构,而AOA较为稳定;NBPT对三类微生物的群落结构丰度无明显影响;硝化抑制剂DMPP可抑制AOB的生长但仅表现在分蘖期,这可能是其缓解硝化反应的主要途径;这也说明二者对土壤生态环境均安全可靠。     

长期施肥对棕壤氨氧化细菌和古菌丰度的影响

【目的】氨氧化是氮转化过程的限速步骤,其由氨氧化微生物所驱动。本研究旨在探明 37 年玉米–大豆轮作施肥条件下影响棕壤氨氧化微生物丰度的主要影响因子及变化规律。【方法】以沈阳农业大学棕壤肥料长期定位试验耕层土壤 (0—20 cm) 为材料,选取其中 9 个施肥处理进行取样分析:不施肥 (CK)、低量氮肥 (N₁)、高量氮肥 (N₂)、氮磷肥 (N₁P)、氮磷钾肥 (N₁PK)、高量有机肥 (M₂)、高量有机肥 + 低量氮肥 (M₂N₁)、高量有机肥 + 氮磷肥 (M₂N₁P)、高量有机肥 + 氮磷钾肥 (M₂N₁PK)。采用实时荧光定量 PCR 技术测定其氨氧化微生物丰度,通过对土壤基本化学性质和氨氧化微生物丰度的冗余分析找出影响氨氧化微生物丰度的主要因素。【结果】施用有机肥处理的土壤 pH、有机质、全氮、碱解氮、速效钾、速效磷、铵态氮、硝态氮含量明显高于不施肥和单施化肥处理。各施肥处理土壤有机质、全氮、碱解氮、速效钾、速效磷的含量总体呈现有机肥处理 > 化肥处理 > CK;与不施肥处理 (CK) 相比,单施化肥处理显著降低了土壤 pH 值,施用有机肥处理显著提高了土壤 pH 值,其中 N₂ 处理的土壤 pH 最低,M₂ 处理的土壤 pH 最高。不同施肥处理氨氧化细菌 (AOB) 的丰度为 0.94 × 106～5.77 × 106 copies/g 干土,氨氧化古菌 (AOA) 的丰度为 3.56 × 106～1.22 × 107 copies/g 干土;施用有机肥处理 AOB 和 AOA 丰度显著高于不施肥和单施化肥处理,其中 M₂ 处理的 AOB 和 AOA 丰度最高,单施氮肥处理的 AOB 和 AOA 丰度最低。冗余分析 (RDA) 表明,影响棕壤 AOB 和 AOA 丰度的主要环境因子有土壤 pH、有机质、全氮、碱解氮、速效磷、速效钾,且与 AOB 和 AOA 丰度呈正相关关系。【结论】长期轮作施肥显著改变了棕壤的化学性质,从而对氨氧化微生物的丰度产生了显著影响。长期施用有机肥显著提高了土壤养分含量及 AOB 和 AOA 的丰度,对维持土壤氨氧化微生物的数量起到十分重要的作用;同时试验结果也为今后通过改变土壤 pH、有机质、全氮、碱解氮、速效磷、速效钾等性质对 AOB 和 AOA 进行调节提供了依据。     

长期施用含氯化肥对棕壤硝化作用及氨氧化微生物的影响

【目的】氨氧化微生物是氨氧化过程的主要驱动者,氨氧化过程作为硝化作用的限速步骤对氮循环具有重要作用。本研究以沈阳农业大学棕壤含氯化肥长期定位试验的土壤为研究对象,探讨了连续34年施用高氯和低氯化肥对棕壤硝化作用及氨氧化微生物的影响。【方法】该长期试验在等量氮、磷、钾条件下,设置高氯和低氯处理,共8个处理:T1(不施肥);T2(单施尿素);T3(尿素+氯化钾);T4(尿素+过磷酸钙);T5(尿素+过磷酸钙+氯化钾);T6(尿素+磷酸一铵+氯化钾);T7(尿素+氯磷铵+氯化钾);T8(硝酸磷肥+过磷酸钙+氯化钾),T7为高氯处理。采集0—20cm土壤样品,利用荧光定量PCR技术测定氨氧化细菌(AOB)和古菌(AOA)丰度,并结合土壤硝化潜势和基本化学性质,分析长期施用含氯化肥对棕壤硝化作用及氨氧化微生物丰度的影响及影响氨氧化微生物丰度的主要环境因素。【结果】长期施肥降低了土壤pH值,高氯处理降低得最多,显著低于其他处理;高氯处理的土壤硝化潜势也显著低于其他处理,且除高氯处理外,配施磷肥的处理土壤硝化潜势显著高于不施磷处理。各处理土壤中AOA丰度均显著高于AOB,高氯处理土壤中AOA、AOB丰度均显著低于其他处理,土壤硝化潜势与AOA和AOB均呈显著正相关关系。【结论】连续施用高氯化肥34年显著降低了棕壤AOA和AOB丰度,抑制了硝化潜势。该结果可为通过含氯化肥的合理施用来调节土壤AOA和AOB,进而调控土壤氮素循环提供参考。     

添加生物炭改善菜地土壤氨氧化细菌群落并提高净硝化率

  【目的】  氨氧化过程是硝化作用的限速步骤，对氮循环有着重要影响。本研究通过分析生物炭输入下土壤氨氧化微生物群落的变化，揭示其影响土壤硝化作用的生物学机制。  【方法】  以华北潮土区设施菜地土壤为对象，设置生物炭梯度 (C₀、C_0.5、C_1.5、C_4.0) 土壤培养试验，结合PCR和T-RFLP等分析技术，观测生物炭输入下土壤氨氧化细菌群落变化动态，解析生物炭、土壤硝化作用与氨氧化细菌群落之间的关系。  【结果】  添加生物炭明显改变了土壤氨氧化微生物群落结构及氮素硝化过程。与未添加生物炭处理相比，生物炭添加处理培养前期土壤氨氧化细菌群落Shannon、Evenness指数分别升高5.4%～18.8%、26.2%～33.8%，后期Shannon指数降低20.7%～34.2%。生物炭输入对AOA群落没有明显影响，AOB群落256、58 bp代表物种丰度分别增加61.4%～56.0%、60.6%～78.6%，488 bp代表物种丰度降低22.8%～26.9%。21 bp代表物种丰度前期增加后期降低，与491 bp代表物种丰度变化相反。添加生物炭土壤AOB amoA基因丰度增加48.9%～53.2%。土壤NO₃–-N含量提高1.7%～25.6%，NH₄+-N含量下降13.4%～31.1%，土壤净硝化速率提高21.8%～70.2%。  【结论】  生物炭的输入可以改善以AOB为主的土壤氨氧化微生物群落结构，提高amoA酶活性，但是对氨氧化古菌微生物群落结构未产生明显影响。因此，生物炭提高土壤净硝化速率的作用与其对土壤氨氧化细菌群落和组成的影响密切相关。     

氮肥施加条件下增温对硝化菌活性和丰度的影响

温度在多种生物地球化学过程中起到关键的调节作用,是影响土壤硝化作用和微生物分布的重要因素之一。硝化过程的第1个步骤由氨氧化细菌(AOB)和氨氧化古菌(AOA)催化,然而,不同施氮量下,增温对硝化菌活性和丰度的影响尚不清楚。本研究基于2008年10月起设立于太行山山前平原的长期增温试验平台(高于地表2m的红外加热器使土壤温度升高1.5℃),于2018年5月对不施氮(N0)和施氮[N1,240kg(N)·hm-2·a-1]下增温分别对0～10 cm和10～20 cm土壤硝化潜势(PNR)、AOA和AOB丰度的影响进行了研究。硝态氮(NO3--N和铵态氮(NH4+-N)含量用分光光度法测量,应用缓冲液培养法测定土壤PNR,提取土壤DNA后用实时荧光定量PCR技术测定功能基因AOA和AOB的丰度。结果表明:温度升高显著增加N1条件下PNR和NO3--N含量(P0.05),降低了N0条件下PNR和NO3--N含量,但差异不显著。N1条件下,增温土壤AOB丰度显著提高(P0.05); N0条件下,增温土壤AOA丰度显著降低(P0.05)。与N0相比, N1条件下的AOA/AOB比值明显降低,表明增温加氮肥处理对AOB的生长刺激更强烈。在增温加施氮条件下,细菌(AOB)表现显著的正反应,在增温不施氮条件下,古菌(AOA)和AOB表现显著的负反应。本研究结果可为全球增温背景下进一步了解硝化活性和氨氧化微生物对增温和氮有效性的响应提供科学依据。     

亚硝化球菌属(Nitrososphaera)可能是酸性土壤硝化作用的重要驱动者

选择初始pH相近的两个酸性土壤(JX-3和JX-7)样品进行培养试验,探讨了氨氧化古菌(ammonia-oxidizing archaea,AOA)和氨氧化细菌(ammonia-oxidizing bacteria,AOB)在酸性土壤硝化过程中所发挥的作用。结果显示,经过50 d的培养,JX-7样品硝化速率显著高于JX-3,且明显降低土壤pH。培养后,两个土壤样品AOB丰度均增加,但样品间没有显著差异;JX-7土壤AOA丰度显著增加,而JX-3无显著变化。两个土壤样品AOA群落组成本身存在分异,但对于同一样品培养前后均无显著分异;AOB群落组成在两土壤间没有分异,但培养前后分别有分异。培养后,JX-7样品中AOA优势属Nitrososphaera和某些未知微生物的个别OTUs绝对丰度显著增加,而两样品AOB中Nitrosospira属的一些OTUs的绝对丰度均显著增加。因此,所研究的酸性土壤样品中AOA是硝化作用的主要贡献者,而且AOA主要通过提高Nitrososphaera属中个别OTUs的丰度,而不是整个群落来调控硝化作用。     

土壤氨氧化古菌及其可能代谢类型

将氨氧化成亚硝态氮的氨氧化过程是土壤硝化作用的关键步骤,主要由氨氧化细菌(ammonia-oxidizing bacteria,AOB)和氨氧化古菌(ammonia-oxidizing archaea,AOA)驱动。其中,AOA具有丰度高、硝化能力强及特殊生态环境偏好等特征,在土壤生态系统的氮素转化过程中发挥重要作用。AOA基因组序列中氨单加氧酶基因(amo A)的发现、纯培养物的分离及其不同生境的赋存状况,为土壤古菌的氨氧化研究提供了新思路。近年研究表明,氨氧化微生物的代谢类型多为化能自养型,而复杂土壤环境中的AOA,其代谢类型呈多样化,除营化能自养生长外,还可能营化能异养和混合营养代谢。其不同代谢方式在氨氧化过程中的响应机制仍需进一步研究。本文概述了土壤中AOA的发现与分布,重点介绍了其可能的代谢途径,并探究其在环境中发挥重要作用的机理。     

Responses of bacterial and archaeal ammonia oxidizers to soil organic and fertilizer amendments under long-term management

Ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) co-exist in soil, but their relative distribution may vary depending on the environmental conditions. Effects of changes in soil organic matter and nutrient content on the AOB and AOA are poorly understood. Our aim was to compare effects of long-term soil organic matter depletion and amendments with labile (straw) and more recalcitrant (peat) organic matter, with and without easily plant-available nitrogen, on the activities, abundances and community structures of AOB and AOA. Soil was sampled from a long-term field site in Sweden that was established in 1956. The potential ammonia oxidation rates, the AOB and AOA amoA gene abundances and the community structures of both groups based on T-RFLP of amoA genes were determined. Straw amendment during 50 years had not altered any of the measured soil parameters, while the addition of peat resulted in a significant increase of soil organic carbon as well as a decrease in pH. Nitrogen fertilization alone resulted in a small decrease in soil pH, organic carbon and total nitrogen, but an increase in primary production. Type and amount of organic matter had an impact on the AOB and AOA community structures and the AOA abundance. Our findings confirmed that AOA are abundant in soil, but showed that under certain conditions the AOB dominate, suggesting niche differentiation between the two groups at the field site. The large differences in potential rates between treatments correlated to the AOA community size, indicating that they were functionally more important in the nitrification process than the AOB. The AOA abundance was positively related to addition of labile organic carbon, which supports the idea that AOA could have alternative growth strategies using organic carbon. The AOB community size varied little in contrast to that of the AOA. This indicates that the bacterial ammonia oxidizers as a group have a greater ecophysiological diversity and potentially cover a broader range of habitats.     

Abundance and community structure of ammonia-oxidizing archaea and bacteria in an acid paddy soil

Nitrification is essential to the nitrogen cycle in paddy soils. However, it is still not clear which group of ammonia-oxidizing microorganisms plays more important roles in nitrification in the paddy soils. The changes in the abundance and composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were investigated by real-time PCR, terminal restriction fragment length polymorphism, and clone library approaches in an acid red paddy soil subjected to long-term fertilization treatments, including treatment without fertilizers (CT); chemical fertilizer nitrogen (N); N and potassium (NK); N and phosphorus (NP); N, P, and K (NPK); and NPK plus recycled crop residues (NPK+C). The AOA population size in NPK+C was higher than those in CT, while minor changes in AOB population sizes were detected among the treatments. There were also some changes in AOA community composition responding to different fertilization treatments. Still few differences were detected in AOB community composition among the treatments. Phylogenetic analysis showed that the AOA sequences fell into two main clusters: cluster A and cluster soil/sediment. The AOB composition in this paddy soil was dominated by Nitrosospira cluster 12. These results suggested that the AOA were more sensitive than AOB to different fertilization treatments in the acid red paddy soil.     

长期施肥对水稻土剖面氨氧化古菌和细菌丰度及组成的影响

长期施肥影响稻田土壤理化性质和硝化微生物群落,但长期施肥对稻田不同土层氨氧化古菌(AOA)和氨氧化细菌(AOB)群落结构的影响尚不明确.以湖南宁乡稻田不同施肥制度长期定位试验为平台,选取不施肥(CK)、施秸秆有机肥(ST)、有机-无机肥配施(OM)和施全量化肥(NPK)4个处理,采用实时荧光定量PCR和Illumina...     

Different roles of rhizosphere effect and long-term fertilization in the activity and community structure of ammonia oxidizers in a calcareous fluvo-aquic soil

Ammonia oxidation is a critical step in the soil nitrogen (N) cycle and can be affected by the application of mineral fertilizers or organic manure. However, little is known about the rhizosphere effect on the function and structure of ammonia-oxidizing bacterial (AOB) and archaeal (AOA) communities, the most important organisms responsible for ammonia oxidation in agricultural ecosystems. Here, the potential nitrification activity (PNA), population size and composition of AOB and AOA communities in both the rhizosphere and bulk soil from a long-term (31-year) fertilizer field experiment conducted during two seasons (wheat and maize) were investigated using the shaken slurry method, quantitative real-time polymerase chain reaction and denaturing gradient gel electrophoresis. N fertilization greatly enhanced PNA and AOB abundance, while manure application increased AOA abundance. The community structure of AOB exhibited more obvious shifts than that of AOA after long-term fertilization, resulting in more abundant AOB phylotypes similar to Nitrosospira clusters 3 and 4 in the N-fertilized treatments. Moreover, PNA was closely correlated with the abundance and community structure of AOB rather than that of AOA among soils during both seasons, indicating that AOB play an active role in ammonia oxidation. Conversely, the PNA and population sizes of AOB and AOA were typically higher in the rhizosphere than the bulk soil, implying a significant rhizosphere effect on ammonia oxidation. Cluster and redundancy analyses further showed that this rhizosphere effect played a more important role in shaping AOA community structure than long-term fertilization. Overall, the results indicate that AOB rather than AOA functionally dominate ammonia oxidation in the calcareous fluvo-aquic soil, and that rhizosphere effect and fertilization regime play different roles in the activity and community structures of AOB and AOA.     

Responses of bacterial and archaeal ammonia oxidisers of an acidic luvisols soil to different nitrogen fertilization rates after 9?years

It is still not clear which group of ammonia-oxidizing microorganisms plays the most important roles in nitrification in soils. Change in abundances and community compositions of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) under long-term different nitrogen (N) fertilization rates were investigated in an acidic luvisols soil using real-time polymerase chain reaction and denaturing gradient gel electrophoresis, respectively, based on the ammonia monooxygenase a-subunit gene. The experimental plan included the following treatments: control without N fertilization (N_CK), low N fertilization rate, middle N fertilization rate, and high N fertilization rate as 0, 100, 150, and 250?kg urea-N?ha^?1, respectively. Long-term different N fertilization rates did not significantly alter the total C and N contents of soil while it significantly decreased soil pH, which ranged from 5.60 to 5.20. The AOB abundance was more abundant in the N fertilization treatments than the N_CK treatment; the AOA abundance decreased by the increasing N fertilization rates, as did the ratios of AOA/AOB. The large differences in the potential nitrification rates among four treatments depended on the changes in AOA abundance but not to changes in AOB abundance. Phylogenetic analysis showed that the AOB communities were dominated by Nitrosospira clusters 1, 3, and 9 while all AOA sequences were grouped into soil/sediment cluster except for one sequence. Taken together, these results indicated that AOB and AOA preferred different soil N conditions and AOA were functionally more important in the nitrification than AOB in the acidic luvisols soil.     

长期施肥对稻田土壤微生物群落结构及氮循环功能微生物数量的影响

  【目的】   稻田是陆生生态系统中重要的氮库之一，在氮素生物地球化学循环中具有重要地位。研究不同施肥处理对稻田土壤微生物群落结构及其功能的影响具有重要意义。   【方法】   田间试验位于江苏省金坛市，在取样时试验已进行了6年。施肥处理包括:不施肥对照 (CK)、施化肥 (CF)、化肥+猪粪混施 (CMF)、化肥+秸秆混施 (CSF)。采用高通量测序和定量PCR方法测定稻田土壤微生物群落结构及氮循环相关功能微生物数量。   【结果】   在施用肥料6年后，土壤全碳、可溶性有机碳、全氮、铵态氮和硝态氮含量均不同程度地提高。与CF相比，CSF和CMF处理土壤pH升高，全碳、可溶性有机碳与养分含量升高。CK与施肥处理的土壤细菌群落结构差异明显，不同施肥处理的细菌群落结构之间有明显差别。聚类结果显示，CK与CMF处理细菌群落聚类更接近，CF处理和CSF处理细菌群落结构更为接近；与CK相比，CF、CMF、CSF处理土壤中氨氧化细菌 (AOB) 和铁氨氧化微生物Feammox A6的丰度显著提高，其中Feammox A6分别增长87.6%、158%和157%。冗余分析结果表明，施肥过程及其对土壤化学性质的改变显著影响土壤细菌群落的组成和分布。   【结论】   施肥导致的反应底物 (NH₄+、NO₃–含量) 及土壤理化性质的差异，是土壤微生物群落结构和功能微生物数量响应的主要决定因素。不施肥与化肥配施猪粪的土壤细菌群落聚类更接近，施化肥与化肥配施秸秆的细菌群落结构更为接近。施肥对氨氧化细菌AOA数量影响不明显，但显著提高氨氧化古菌AOB和厌氧铁氨氧化功能微生物Feammox A6的数量，特别是有机肥 (猪粪、秸秆) 提高Feammox A6数量的效果大于化肥。长期单施化肥土壤中厌氧氨氧化细菌丰度显著降低，反硝化功能基因nirK、nosZ丰度显著增高；化肥配施猪粪土壤中的厌氧氨氧化细菌丰度变化不明显，反硝化功能基因narG、nirK、nosZ丰度显著增高；化肥配施秸秆处理厌氧氨氧化细菌丰度变化不明显，反硝化功能基因nirK、nosZ丰度显著增高。     

两种农田土壤中的氨氧化细菌和氨氧化古菌对硝化抑制剂DCD和DMPP的响应-盆栽试验

Taking two important agricultural soils with different pH, brown soil （Hap-Udic Luvisol） and cinnamon soil （Hap-Ustic Luvisol）, from Northeast China, a pot culture experiment with spring maize （Zea mays L.） was conducted to study the dynamic changes in the abundance and diversity of soil ammonia-oxidizing bacteria （AOB） and ammonia-oxidizing archaea （AOA） populations during maize growth period in response to the additions of nitrification inhibitors dicyandiamide （DCD） and 3,4-dimethylpyrazole phosphate （DMPP） by the methods of real-time polymerase chain reaction （PCR） assay, PCR-denaturing gradient gel electrophoresis （DGGE）, and construction of clone library targeting the amoA gene. Four treatments were established, i.e., no urea （control）, urea, urea plus DCD, and urea plus DMPP. Both DCD and DMPP inhibited growth of AOB significantly, compared to applying urea alone. Soil bacterial amoA gene copies had a significant positive linear correlation with soil nitrate content, but soil archaeal amoA gene copies did not. In both soils, all AOB sequences fell within Nitrosospira or Nitrosospira-like groups, and all AOA sequences belonged to group 1.1b crenaxchaea. With the application of DCD or DMPP, community composition of AOB and AOA in the two soils had less change except that the AOB community composition in Hap-Udic Luvisol changed at the last two growth stages of maize under the application of DCD. AOB rather than AOA likely dominated soil ammonia oxidation in these two agricultural soils.