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。     

O_3浓度升高对麦田土壤氨氧化细菌、氨氧化古菌和硝化细菌数量的影响

硝化作用在氮循环过程中至关重要,包括氨氧化作用和亚硝酸盐氧化作用,通过氨氧化反应和亚硝酸盐氧化反应将N素转化为植物可利用的NO;形态。利用开顶式臭氧气室（OTCs,open-topchambers）试验平台,通过大田模拟熏气试验,结合Real-timePCR探讨大气O,浓度升高对麦田土壤氨氧化细菌（AOB）、氨氧化古菌（AOA）及硝化细菌（NOB）数量的影响。结果表明,AOB、AOA和NOB对O,胁迫的反应不一样,AOB基因拷贝数基本上随着O,浓度的升高呈现出降低的趋势,而AOA和NOB基因拷贝数随O_3浓度的升高变化不明显。冬小麦拔节期,当O_3浓度为40、80、120nmol·mol。时,20-40em土层的AOB基因拷贝数分别比对照处理降低39．8％、51．2％和59．4％。AOB和NOB基因拷贝数灌浆期多于收获期,0-10cm土层多于10-20em。AOA基因拷贝数随季节的变化不大。O_3胁迫可通过影响AOB、AOA和NOB的数量和活性来影响土壤的硝化反应,从而影响土壤的氮素循环过程。     

三种硝化抑制剂抑制土壤硝化作用比较及用量研究

【目的】硝化抑制剂是调控土壤氮素转化与硝化作用微生物群落结构的有效途径。本文通过室内模拟试验对3种硝化抑制剂在不同剂量下的硝化抑制效果进行研究,旨在筛选出效果最佳的剂型与剂量,为石灰性土壤硝化抑制剂的合理应用提供依据。 【方法】培养试验在生长箱内进行,25℃黑暗条件培养;盆栽试验在温室内进行。供试硝化抑制剂为双氰胺（DCD）、3,4-二甲基吡唑磷酸盐（DMPP）和2-氯-6-三氯甲基吡啶（Nitrapyrin）,DCD和DMPP用量均设定为纯氮（N）量的0（CK）、1.0%、2.0%、3.0%、3.5%、4.0%、4.5%、5.0%、6.0%和7.0%;Nitrapyrin用量分别为纯氮量的0、0.1%、0.125%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%和0.5%,三种硝化抑制剂均设10个水平,每个水平3次重复。盆栽试验氮加入量为每公斤风干土0.50 g,三种硝化抑制剂用量分别为纯氮用量的5%、1%、0.648%。调查比较了三者的硝化抑制效果及对土壤氮素转化的影响及其对小青菜鲜重的生物学效应;采用变性梯度凝胶电泳（DGGE）法分析了不同硝化抑制剂对土壤AOA、AOB群落结构的影响。 【结果】DCD、DMPP、Nitrapyrin均可显著抑制土壤硝化作用（P＜0.05）,各硝化抑制剂处理土壤的NH₄+-N含量分别较对照提高了46.2~256.1 mg/kg、291.8~376.7 mg/kg、3.68~372.9 mg/kg。DCD与DMPP处理的硝化抑制率分别为49.3%~79.4%和96.4%~99.4%,DCD表现出明显的剂量效应,但DMPP在1%~7%浓度范围内的剂量效应不明显。Nitrapyrin在0.1%~0.2%浓度范围内有明显的剂量效应。0.25%~0.5% Nitrapyrin的硝化抑制率为98.9%~99.9%,其硝化抑制效果与DMPP处理相同。DCD、DMPP、Nitrapyrin处理的小青菜地上部分鲜重分别比氮肥处理（ASN）提高了12.7%、11.1%、17.6%。施用硝化抑制剂可改变土壤AOA和AOB群落结构,且对AOA群落结构的影响大于AOB,不同硝化抑制剂之间对AOA和AOB群落结构的影响无差异。 【结论】3种硝化抑制剂的硝化抑制效果表现为Nitrapyrin≥DMPP>DCD,均对AOA与AOB群落结构产生明显影响。各硝化抑制剂处理均可提高小青菜地上部鲜重、叶片Vc含量及可显著提高小青菜叶片氨基酸含量（P＜0.05）。综合比较,Nitrapyrin硝化抑制效果好于DMPP,DCD效果最差,推荐用量为基于纯氮0.25%的Nitrapyrin添加量。     

不同品种水稻土壤氨氧化细菌和氨氧化古菌群落结构组成

本研究通过提取土壤总DNA,利用特异引物进行PCR扩增和变性梯度凝胶电泳(DGGE),研究了不同品种水稻对稻田土壤氨氧化细菌和氨氧化古菌群落结构组成的影响.结果显示:稻田土壤具有丰富的氨氧化细菌和氨氧化古菌,且氨氧化古菌种类更多;不同品种水稻氨氧化细菌群落结构组成差异较大,其中以"天A/Km71"、"闽优1027"和"Km62/1027"3个品种相似性较高,且明显不同于其余3个品种:而氨氧化古菌群落结构组成在不同土层间表现出较大差异,其中以"天A/Km71"和"Km62/1027"的表土与根际土氨氧化古菌群落组成差异最大.研究表明不同水稻品种及土壤层次对氨氧化微生物群落结构组成具有一定影响,证明氨氧化微生物尤其是氨氧化古菌在稻田土壤生态系统中占有重要地位.     

两个水稻品种根际土壤细菌和氨氧化细菌的群落结构差异

通过根盒试验比较了籼稻汕优63和粳稻武运粳7号苗期不同采样期根际土和土体土壤的硝化强度以及氨氧化细菌数量的差异,并且采用16S rDNA PCR-DGGE(Denaturing gradient gel electrophoresis)指纹图谱技术比较分析了上述两种水稻苗期不同采样期根际和土体土壤中细菌及其氨氧化细菌的群落结构变化。结果表明,两个水稻品种根际土壤中硝化强度和氨氧化细菌的数量随着生育期的延长均表现出一定的正相关性,汕优63籼稻根部土壤中的细菌和氨氧化细菌的丰富度和群落变化特征随着水稻生育时期的延长较武运7号粳稻的变化更为多样,说明籼稻品种根系和根际硝化作用更强,在其根系附近会产生更多的硝态氮。这种差异性严重影响水稻植株对氮素的利用效率。     

两种硝化抑制剂对不同土壤中氮素转化的影响

采用实验室人工气候箱培养的方法,研究了两种硝化抑制剂双氰胺和硫代硫酸钾在两种碱性土壤中对土壤pH值变化、氨挥发特性及铵态氮转化的影响.实验结果表明,各处理在两种碱性土壤中的pH值都是先上升到一个峰值,然后下降,且速率先快后慢.硫代硫酸钾处理和对照处理的pH值约在实验第4天出现峰值,双氰胺处理pH值出现峰值的时间较硫代硫酸钾处理及对照处理土壤推迟了3天左右.整个实验期间,双氰胺处理的pH值一直处于较高水平,硫代硫酸钾处理的次之,对照处理的最小.氨挥发强度与土壤pH值同步,各处理氨的挥发量一般在第7天达到最大值,此时双氰胺处理氨的挥发量最大,硫代硫酸钾处理的次之,对照处理的最少.在晋城菜园土中,双氰胺和硫代硫酸钾处理的土壤比未添加硝化抑制剂的对照土壤中氨挥发的总量分别增加523.0%,33.6%,在北京菜园土中,双氰胺和硫代硫酸钾处理的土壤比未添加硝化抑制剂的对照土壤中氨挥发的总量分别增加575.8%,125.0%.土壤中铵态氮含量与土壤pH值的变化趋势相似,先快速上升到一个峰值,然后开始缓慢下降.硝化抑制剂的添加能使两种碱性土壤中铵态氮的释放时间延长3天左右.     

南方典型母质发育稻田土壤氨氧化细菌和古菌群落结构特征

依托中国科学院桃源农业生态试验站水稻土易地置土长期定位试验,以广东英利(YL,发育于玄武岩风化物)、江西鹰潭(YT,发育于第四纪红黏土)和湖南桃源古市(TYG,发育于河流冲积物)3种母质发育稻田土壤为供试土壤,采用常规土壤农化分析方法测得3种土壤的理化性质、硝化速率,并结合实时荧光定量PCR技术和Illumina MiSeq测序技术,分析了土壤氨氧化古菌(AOA)和氨氧化细菌(AOB)种群丰度及群落结构的异同。结果表明：土壤硝化速率除分蘖期外,均表现为：YT>YL>TYG。YL与YT土壤中AOA数量占主导地位,AOA/AOB比值比值分别在0.72 ~ 3.05、0.98 ~ 1.52;TYG土壤中AOB数量占主导地位,AOA/AOB比值在0.21 ~ 0.75。群落组成方面,不同母质土壤间AOA、AOB群落结构差异显著,3种土壤AOA优势菌属各不相同,AOB优势菌属均为Nitrosospira。冗余分析表明,pH和可溶性有机碳是造成AOA、AOB群落结构差异的核心因子。综上,在环境背景均统一的前提下,不同母质发育稻田土壤理化性质之间依旧存在差异,这种差异影响着氨氧化微生物AOA、AOB的丰度以及群落结构。     

硝化抑制剂对毛竹林土壤N_2O排放和氨氧化微生物的影响

为了探索硝化抑制剂在毛竹生产中的施用技术,通过培养试验研究3,4-二甲基吡唑磷酸盐(DMPP)和双氰胺(DCD)两种硝化抑制剂对毛竹林施用尿素后土壤N2O排放、氮素转化和相关氨氧化细菌(AOB)、氨氧化古菌(AOA)群落结构和丰度的影响。试验设(1)对照(CK)、(2)单施尿素(Urea)、(3)尿素+1%DMPP(DMPP占总N的1%,下同);(4)尿素+1.5%DMPP;(5)尿素+10%DCD;(6)尿素+15%DCD等6个处理,测定N2O的排放动态以及气体排放转折点时的土壤特征指标。结果表明:与单施尿素相比,160 d的时间内两种DMPP用量处理的土壤N2O累积排放减排幅度均为54%,而10%DCD和15%DCD处理的土壤分别减少28%和41%。DMPP和DCD处理50 d和90 d时土壤的NH4+-N含量均显著高于(p0.05)单施尿素处理,而NO3--N含量和表观硝化率则恰好相反,但两种抑制剂间无差异。DMPP处理的AOB群落结构的变化从10 d开始显现,至50 d和90 d时仍保持明显的抑制状态,而DCD处理则至90 d时抑制作用基本消失。单施尿素AOB功能基因(amo A)的丰度均显著高于硝化抑制剂处理(90 d时尿素+10%DCD处理除外);在整个培养期内,尿素和对照土壤的AOA群落结构相似,硝化抑制剂反而增加了AOA功能基因的丰度,表明硝化抑制剂对AOA丰度无明显抑制作用。即两种硝化抑制剂主要通过抑制AOB起作用;调节土壤p H至中性范围,并在1%DMPP施用条件下,硝化抑制剂的抑制效果最显著。     

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。     

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。     

Contributions of ammonia-oxidizing archaea and bacteria to nitrification in Oregon forest soils

Ammonia oxidation, the first step of nitrification, is mediated by both ammonia-oxidizing archaea (AOA) and bacteria (AOB); however, the relative contributions of AOA and AOB to soil nitrification are not well understood. In this study we used 1-octyne to discriminate between AOA- and AOB-supported nitrification determined both in soil-water slurries and in unsaturated whole soil at field moisture. Soils were collected from stands of red alder (Alnus rubra Bong.) and Douglas-fir (Pseudotsuga menziesii Mirb. Franco) at three sites (Cascade Head, the H.J. Andrews, and McDonald Forest) on acidic soils (pH 3.9–5.7) in Oregon, USA. The abundances of AOA and AOB were measured using quantitative PCR by targeting the amoA gene, which encodes subunit A of ammonia monooxygenase. Total and AOA-specific (octyne-resistant) nitrification activities in soil slurries were significantly higher at Cascade Head (the most acidic soils, pH < 5) than at either the H.J. Andrews or McDonald Forest, and greater in red alder compared with Douglas-fir soils. The fraction of octyne-resistant nitrification varied among sites (21–74%) and was highest at Cascade Head than at the other two locations. Net nitrification rates of whole soil without NH₄⁺ amendment ranged from 0.4 to 3.3 mg N kg⁻¹ soil d⁻¹. Overall, net nitrification rates of whole soil were stimulated 2- to 8-fold by addition of 140 mg NH₄⁺-N kg⁻¹ soil; this was significant for red alder at Cascade Head and the H.J. Andrews. Red alder at Cascade Head was unique in that the majority of NH₄⁺-stimulated nitrifying activity was octyne-resistant (73%). At all other sites, NH₄⁺-stimulated nitrification was octyne-sensitive (68–90%). The octyne-sensitive activity—presumably AOB—was affected more by soil pH whereas the octyne-resistant (AOA) activity was more strongly related to N availability.     

沿海滩涂土壤中反硝化细菌群落随盐分梯度的更迭

To better understand the effect of salinity on denitrification communities,soils along a salinity gradient (ranging from 7.32 to 1.70 mS cm 1) in a wetland along the Yellow Sea coastline in Jiangsu Province,China,were studied using both culture-dependent and-independent methods.Culture efforts yielded 82 isolates in total,81.7% of which were close relatives of Bacillus sp.based on partial sequences of their 16S rRNA genes.Denaturing gradient gel electrophoresis (DGGE) analysis based on 16S rRNA sequences suggested possible existence of bacterial community succession along the salinity gradient.Clone library analysis based on nosZ gene sequences (coding nitrous oxide reductase) showed that operational taxonomic units (OTUs) associated with α-proteobacteria dominated in all three soils,whereas those associated with β-and γ-subdivisions showed a clear succession.In the high salinity soil,only the OTUs associated with α-subdivision were found.In the medium salinity soil,small proportions of β-(6.5%) and γ-associated (19.6%) OTUs were found.In the low salinity soil,the proportions were further increased to 33% and 25% for β-and γ-Proteobacteria,respectively.Statistic analysis using Unifrac P test showed that nosZ-communities in different saline soils were significantly different from each other.It could be concluded that α-subdivision of nosZ-community tended to be sustained in high salinity environments whereas β-and γ-subdivisions,especially the former,tended to be sustained in low salinity environments.Salinity was the key determinant of nosZ-community composition in the environment.     

cry1Ac/cpti转基因大米水稻土中微生物群落的多样性研究

Two types of cry1Ac/cpti transgenic rice(GM1 and GM2)and their parental non-cry1Ac/cpti rice(CK1 and CK2)were planted in the field at Wufeng,Fujian Province,China for four years to investigate the influence of genetically modified rice on diversity of bacterial and fungal community in the paddy soil.The community composition and abundance of bacteria or fungi in the paddy soil were assessed at different growth stages of rice by denaturing gradient gel electrophoresis and real-time polymerase chain reaction based on 16S rRNA gene or SSU rRNA gene in the 4th year after the experimental establishment.The composition of bacterial or fungal community changed during rice growth,while no significant differences were observed between the fields cultivated with GM1and CK1,or between the fields cultivated with GM2 and CK2 in either bacterial or fungal community composition.The copy numbers of bacterial 16S rRNA gene in the soils with CK1,CK2,GM1 and GM2 ranged from 5.64×1011to 6.89×1011copies g-1dry soil at rice growth stages,and those of fungal SSU rRNA gene from 5.24×108to 8.68×108copies g-1dry soil.There were no marked differences in the copies of bacterial 16S rRNA gene or fungal SSU rRNA gene between CK1 and GM1 or between CK2 and GM2at any growth stage of rice.Planting cry1Ac/cpti transgenic rice had no significant effect on composition and abundance of bacterial and fungal community in paddy soil during the rice growing season at least in the short term.     

Long-term fertilization regimes affect bacterial community structure and diversity of an agricultural soil in northern China

Background, Aims, and Scope  Knowledge about shifts of microbial community structure and diversity following different agricultural management practices could improve our understanding of soil processes and thus help us to develop sound management strategies. A long-term fertilization experiment was established in 1989 at Fengqiu (35°00′N, 114°24′E) in northern China. The soil (sandy loam) is classified as aquic inceptisols and has received continuous fertilization treatments since then. The fertilization treatments included control (CK, no fertilizer), chemical fertilizers nitrogen (N) and potassium (K) (NK), phosphorous (P) and K (PK), NP, NPK, organic manure (OM), and half chemical fertilizers NPK plus half organic manure (1/2NPKOM). The objective of this study was to examine if the microbial community structure and diversity were affected by the long-term fertilization regimes. Materials and Methods  Soil samples were collected from the long-term experimental plots with seven treatments and four replications in April 2006. Microbial DNAs were extracted from the soil samples and the 16S rRNA genes were PCR amplified. The PCR products were analyzed by DGGE, cloning and sequencing. The bacterial community structures and diversity were assessed using the DGGE profiles and the clone libraries constructed from the excised DGGE bands. Results  The bacterial community structure of the OM and PK treatments were significantly different from those of all other treatments. The bacterial community structures of the four Ncontaining treatments (NK, NP, NPK and 1/2NPKOM), as well as CK, were more similar to each other. The changes in bacterial community structures of the OM and PK treatments showed higher richness and diversity. Phylogenetic analyses indicated that Proteobacteria (30.5%) was the dominant taxonomic group of the soil, followed by Acidobacteria (15.3%), Gemmatimonadetes (12.7%), etc. Discussion  Irrespective of the two fertilization treatments of OM and PK, the cluster analysis showed that bacterial communities of the remaining five treatments of CK, NK, NP, NPK and 1/2NPKOM seemed to be more similar to each other, which indicated the relatively weak effects of the four N-containing treatments on soil bacterial communities. N fertilizer may be considered as a key factor to counteract the effects of other fertilizers on microbial communities. Conclusions  Our results show that long-term fertilization regimes can affect bacterial community structure and diversity of the agricultural soil. The OM and PK treatments showed a trend towards distinct community structures, higher richness and diversity when compared to the other treatments. Contrasting to the positive effects of OM and PK treatments on the bacterial communities, N fertilizer could be considered as a key factor in the soil to counteract the effects of other fertilizers on soil microbial communities. Recommendations and Perspectives  Because of the extremely high abundance and diversity of microorganisms in soil and the high heterogeneity of the soil, it is necessary to further examine the effects of fertilization regimes on microbial community and diversity in different type soils for comprehensively understanding their effects through the appropriate combination of molecular approaches. ESS-Submission Editor: Chengrong Chen, PhD (c.chen@griffith.edu.au)     

三种硝化抑制剂在石灰性土壤中的应用效果比较

在人工气候室内采用25℃黑暗培养法研究双氰胺(DCD)、3,4-二甲基吡唑磷酸(DMPP)及2-氯-6-三氯甲基吡啶(Nitrapyrin)在石灰性土壤中的硝化抑制效果。结果表明:施用DCD、DMPP、Nitrapyrin的土壤NH4+-N含量较单施硫酸铵的土壤(对照)分别提高228.45~244.85 mg/kg(砂土)、209.75~254.79 mg/kg(黏土),NO3--N含量较对照分别降低93.85%~94.99%(砂土)、91.82%~95.38%(黏土)。表观硝化率随培养进程增加缓慢,培养期间只增加了1.28%~2.09%(砂土)、2.72%~8.40%(黏土),而对照增加了86.00%(砂土)、80.89%(黏土)。3种硝化抑制剂均显著抑制了石灰性土壤中硫酸铵水解铵硝化作用的进行,并且在砂土中的硝化抑制率高于黏土,硝化抑制效果最好的为DMPP处理,0.54%Nitrapyrin处理次之但用量最小,0.27%Nitrapyrin和10.8%DCD处理抑制效果相对较弱。     

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.     

Effect of application rate of a nitrification inhibitor,dicyandiamide (DCD), on nitrification rate,and ammonia-oxidizing bacteria and archaea growth in a grazed pasture soil: An incubation study

Purpose

Dicyandiamide (DCD) has been used commercially in New Zealand to reduce nitrate leaching and N₂O emissions in grazed pastures. However, there is a lack of information in the literature on the optimum rate of DCD to achieve the environmental benefits while at the same time reducing the cost of the technology. The objective of this study was to determine the effect of DCD application rate on its effectiveness to inhibit ammonia oxidizer growth and nitrification rate in a grazed pasture soil.

Materials and methods

The soil was a Templeton silt loam (Immature Pallic Soil; Udic Haplustepts) collected from Lincoln University Research Dairy Farm with a mixed pasture consisting of perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) and was incubated alone (control) or with cow urine at 700 kg N/ha with 6 rates of DCD [0, 2.5, 5, 7.5, 10 (applied twice), 15 and 20 kg/ha] in incubation vessels. The incubation vessels were placed randomly in an incubator with a constant temperature of 12 °C. During 112 days of incubation, soil subsamples were taken at different time intervals to measure the concentrations of NO₃ ^?-N and NH₄ ⁺-N and the amoA gene copy numbers of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA).

Results and discussion

DCD applied at all the different rates inhibited nitrification in urine-treated soils, but the effectiveness increased with DCD application rate. In addition, AOB growth and the amounts of nitrate-N in the soil were significantly related to the application rate of DCD. However, AOA population abundance showed no relationship to the application rate of DCD. The DCD rate at which the AOB growth rate and nitrate-N concentration were halved (effective dosage that causes 50 % reduction in nitrification rate, or ED₅₀) was about 10 kg DCD/ha.

Conclusions

These results suggest that DCD applied at relatively low rates still slowed down the nitrification rate, and the current recommended rate of 10 kg DCD/ha for DCD use in New Zealand grazed pastures would result in a 50 % reduction in nitrification rate in this soil. The actual rate of DCD application used would depend on the cost of the product and the environmental and agronomic benefits that would result from its use.     

Responses of ureolytic and nitrifying microbes to urease and nitrification inhibitors in selected agricultural soils in Victoria,Australia

Journal of Soils and Sediments - Urease inhibitors (UIs) such as N-(n-butyl)thiophosphoric triamide (NBPT) and nitrification inhibitors (NIs) such as 3,4-dimethylpyrazole phosphate (DMPP) have been...     

Long-term submergence of non-methanogenic oxic upland field soils helps to develop the methanogenic archaeal community as revealed by pot and field experiments

The community structure of methanogenic archaea is relatively stable,i.e.,it is sustained at a high abundance with minimal changes in composition,in paddy field soils irrespective of submergence and drainage.In contrast,the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils.This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments.In the pot experiment,a soil sample obtained from an upland field was incubated under submerged conditions for 275 d.Soil samples periodically collected were subjected to culture-dependent most probable number(MPN)enumeration,polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)analysis of archaeal 16 S r RNA gene,and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene(mcr A)of methanogenic archaea.The abundance of methanogenic archaea increased from 102 to 103 cells g-1 dry soil and 104 to 107 copies of mcr A gene g-1 dry soil after submergence.Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis,members from Methanocellales,Methanosarcinaceae,and Methanosaetaceae proliferated in the soils,and the community structure was relatively stable once established.In the field experiment,the number of viable methanogenic archaea in a rice paddy field converted from meadow(reclaimed paddy field)was monitored by MPN enumeration over five annual cycles of field operations.Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer’s paddy field was dressed onto the meadow(dressed paddy field)and an upland crop field converted from the meadow(reclaimed upland field).The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field.Then,the number gradually increased over five years and finally reached 103–104 cells g-1 dry soil,which was comparable to that in the dressed paddy field.These findings showed that the low abundance of autochthonous methanogenic archaea in the non-methanogenic oxic upland field soils steadily proliferated,and the community structure was developed following repeated and long-term submergence.These results suggest that habitats suitable for methanogenic archaea were established in soil following repeated and long-term submergence.     

Responses of ammonia-oxidizing bacteria and archaea to nitrogen fertilization and precipitation increment in a typical temperate steppe in Inner Mongolia

As the first and rate-limiting step of nitrification, ammonia oxidation can be realized either by ammonia-oxidizing bacteria (AOB) or archaea (AOA). However, the key factors driving the abundance, community structure and activity of ammonia oxidizers are still unclear, and the relative importance of AOA and AOB in ammonia oxidation is unresolved. In the present study, we examined the effects of long-term (6 years) nitrogen (N) addition and simulated precipitation increment on the abundance and community composition of AOA and AOB based on a field trial in a typical temperate steppe of northern China. We used combined approaches of quantitative PCR, terminal-restriction fragment length polymorphism (T-RFLP) and clone library analyses of amoA genes. The study objective was to determine (1) AOA and AOB diversity and activity in response to N addition and increased precipitation and (2) the relative contributions of AOA and AOB to soil ammonia oxidation in the typical temperate steppe. The results showed that the potential nitrification rate (PNR) increased with N addition, but decreased with increased precipitation. Both N addition and increased precipitation significantly increased AOB but not AOA abundance, and a significant correlation was only observed between PNR and AOB amoA gene copies. The T-RFLP analysis showed that both N and precipitation were key factors in shaping the composition of AOB, while AOA were only marginally influenced. Phylogenetic analysis indicated that all AOA clones fell within the soil and sediment lineage while all AOB clones fell within the Nitrosospira. The study suggested that AOA and AOB had distinct physiological characteristics and ecological niches. AOB were shown to be more sensitive to N and precipitation than AOA, and the ammonia oxidation process was therefore supposed to be mainly driven by AOB in this temperate steppe.