东北黑土nirS型反硝化细菌群落和网络结构对长期施用化肥的响应

【目的】探明长期施用氮磷钾化肥对东北黑土农田土壤nirS型反硝化细菌群落和网络结构的影响,为更加合理的肥料配施提供理论依据。【方法】基于农业农村部黑龙江耕地保育与农业环境科学观测实验站平台,选取不施肥(NoF)、氮肥(N)、磷肥(P)、钾肥(K)、氮钾肥(NK)、氮磷肥(NP)、磷钾肥(PK)、氮磷钾肥(NPK)8个施肥处理,借助荧光定量PCR、Illumina MiSeq高通量测序和分子生态网络技术,分析东北黑土nirS型反硝化细菌丰度、群落及网络结构,及影响反硝化细菌群落变异的主要环境因子。【结果】1)长期施用氮肥均在显著增加nirS基因拷贝数的同时,降低了nirS型反硝化细菌的群落多样性,而磷、钾肥对其影响并不显著。2)Proteobacteria是所有处理中的优势反硝化细菌门,相对丰度为16.96%~27.34%,且氮肥的施用促进了隶属于该菌门中Bradyrhizobium的生长。3)PCoA分析结果显示,8个施肥处理nirS型反硝化细菌群落主要分成施氮肥和不施氮肥两组,说明长期氮肥的施用显著改变了东北黑土反硝化细菌的群落结构。结合Mantel test的结果可知,土壤pH是影响nirS型反硝化细菌群落改变的主要因素。4)分别构建施氮肥和不施氮肥的反硝化细菌分子生态网络,发现施氮肥和不施氮肥网络结构存在很大差异,长期施用氮肥明显简化了nirS型反硝化细菌的网络结构,同时使其网络结构稳定性降低,易受外界环境扰动。【结论】尽管施用化学氮肥有利于土壤养分的增加,但其土壤nirS型反硝化细菌群落及网络结构发生了较大改变,而磷、钾肥的施用对反硝化细菌群落无显著影响。本试验结果为进一步研究东北黑土区农田土壤反硝化微生物对不同施肥管理的响应机制提供了重要科学依据。     

稻田土壤nirS型反硝化细菌群落对氮肥水平的响应

【目的】基于水稻田间定位试验,利用nirS功能基因研究不同氮肥水平对稻田土壤反硝化细菌群落多样性的影响。【方法】运用PCR-DGGE（聚合酶链反应-变性梯度凝胶电泳）结合DNA克隆测序和荧光定量PCR（Real-time PCR）技术对反硝化细菌nirS基因进行检测,分析田间定位试验第4年不同氮肥水平下稻田nirS型反硝化细菌群落结构和丰度的变化。【结果】依据DGGE图谱计算的群落多样性指数显示,与不施肥对照处理（CK）比较,施用氮肥处理（N1：75 kg N•hm-2,N2：150 kg N•hm-2和N3：225 kg N•hm-2）可促进稻田土壤nirS型反硝化细菌群落多样性指数提高,尤其在水稻生长的齐穗期和成熟期后者均显著高于前者（P＜0.05）。但群落多样性指数在N1、N2 和N3处理间的差异主要表现在水稻分蘖期和齐穗期的表层土壤中,N3可显著高于N1（P＜0.05）。冗余分析结果显示水稻生育时期对稻田土壤nirS型反硝化细菌群落结构的影响较大,表层和根层土壤的群落结构都与生育时期存在显著相关性（P=0.002,0.002）;而不同氮肥水平对群落结构的显著性影响仅表现在稻田表层土壤中（P=0.002）。荧光定量PCR结果显示氮肥水平提高可促进稻田土壤nirS型反硝化细菌丰度增加,在水稻分蘖期和齐穗期内表层和根层土壤的nirS基因拷贝数均存在CK＜N1＜N2＜N3的趋势,且以齐穗期时在表层土壤中的差异最大,不同处理间的差异都达到显著水平（P＜0.05）。同时,本研究获得稻田反硝化细菌nirS基因片段序列8条登录GenBank（登录号：JX997923、JX997924、JX997926—JX997931）。此外,本试验中N1、N2和N3处理比CK处理分别增产59%、92%和107%,表层和根层土壤中NO3--N含量也随氮肥用量提高而增加。【结论】氮肥用量增加促进了稻田土壤nirS型反硝化细菌丰度及群落多样性指数的提高,尤其在稻田表层土壤中。氮肥水平提高还可改变稻田表层土壤nirS型反硝化细菌的群落结构。综合分析表明稻田表层土壤的nirS型反硝化细菌群落对氮肥水平提高的响应程度更明显。     

氯化苦熏蒸对土壤反硝化作用及nirS型反硝化细菌群落结构的影响

探究氯化苦熏蒸对土壤反硝化作用的影响及机制。采用理化分析和DGGE方法分析了氯化苦熏蒸后土壤反硝化及nirS型反硝化细菌群落结构。不同浓度氯化苦熏蒸对土壤nirS型反硝化细菌群落均匀度无影响,而对nirS型反硝化细菌多样性和丰富度指数的影响为高浓度中浓度低浓度对照。对土壤反硝化作用的影响表现为,经500 mg/kg氯化苦熏蒸的土壤在培养初期(0 d)反硝化率比对照降低10.55%,土壤反硝化作用被显著抑制。2周后3种浓度氯化苦均抑制土壤反硝化作用, 4周后土壤的反硝化作用缓慢恢复。500 mg/kg浓度氯化苦对土壤反硝化强度的影响与nirS型反硝化细菌群落结构变化不同,说明土壤反硝化作用的强弱并非完全决定于土壤nirS型反硝化细菌。     

撂荒地亚硝酸还原酶基因nirK和nirS丰度动态

使用荧光定量PCR法测定亚硝酸还原酶基因(nirK和nirS)遗传特异性片段,研究了草原撂荒地土壤反硝化微生物丰度随着撂荒时间的变化。结果表明,轻度放牧草原和3种不同撂荒时间地的土壤中nirK基因型反硝化微生物丰度都显著高于nirS基因型反硝化微生物丰度(P0.05)。土壤nirK及nirS基因型反硝化微生物丰度撂荒地和轻度放牧地之间均有显著差异(P0.05),但3种撂荒地均未发生显著变化(P0.05)。此外,这两种基因型反硝化微生物丰度之间有极显著线性负相关关系(P0.001)。以上结果说明,nirK和nirS基因型反硝化微生物对环境因子的响应有差异。     

Advantages of a diluted nutrient broth medium for isolating N2-producing denitrifying bacteria of α-Proteobacteria in surface and subsurface upland soils

This research aimed to determine whether a diluted nutrient broth (DNB) medium was different from a conventional nutrient broth (NB) medium when counting and isolating denitrifying bacteria in surface and subsurface upland soils. To this end, we investigated populations of denitrifying bacteria isolated from the surface to a depth of 4 m of subsurface upland soil that had received slurry. The DNB medium gave higher viable counts of denitrifying bacteria than the NB medium and a higher isolation ratio of denitrifying bacterial isolates. In total, 74 isolates from the DNB medium (D-isolates) and 26 isolates from the NB medium (N-isolates) were collected. We characterized their denitrifying activity and analyzed the diversity of 16S rDNA and denitrifying-related genes. Seventy-three percent of the D-isolates were oligotrophic denitrifying bacteria. The N₂-producing, oligotrophic denitrifying bacteria, largely of α-Proteobacteria, increased in the D-isolates. The D-isolates and the N-isolates had some taxonomic overlapping on a phylogenetic tree based on 16S rDNA. It was not possible to identify the denitrification phenotype (N₂-producing or N₂O-producing) on the phylogenetic tree. Phylogenetic groups of isolates corresponded to nirK groups, except in some isolates in which horizontal gene transfer might have occurred. The terminal gas emission of the isolates was consistent with the existence of the nosZ gene. The DNB medium may be very useful in isolating N₂-producing denitrifying α-Proteobacteria. Its use highlights the ecological significance of oligotrophic isolates and the different viable counts resulting from the selectivity of conventional and diluted media.     

夏玉米根系密集区与行间N2O浓度及与氨氧化细菌和反硝化细菌数量的关系

采用硅胶管法和微生物分子生态学方法对华北平原典型旱地土壤夏玉米根系密集区与行间土壤产生的N2O及与氨氧化细菌和反硝化细菌数量的关系进行了研究。结果表明:夏玉米三叶期和十叶期的施肥灌水措施以及超过15 mm左右的降雨均显著促进了土壤N2O的产生。夏玉米行间产生的N2O略高于根系密集区,但二者未达显著差异。相关分析表明影响土壤N2O产生的主要环境因子为土壤空隙含水量（WFPS）及土壤NO3--N含量,二者决定系数分别为0.79和0.51。N2O浓度与氨氧化细菌amoA基因拷贝数及反硝化细菌nirS基因拷贝数之间的显著正相关关系（决定系数分别达0.55和0.62）表明土壤中N2O的产生与氨氧化细菌和nirS基因反硝化细菌数量的增加亦有关。表明在该试验条件下,夏玉米根系生长对根系密集区土壤中氮和水的消耗以及氨氧化细菌和反硝化细菌数量的变化并未引起根系密集区和玉米行间N2O浓度的显著差异,即在田间布设N2O观测箱时,采样箱是否覆盖作物根系或采样箱覆盖作物根系的多少对N2O排放量的测定结果没有显著性影响。     

连续施氮对马铃薯根际细菌群落结构及反硝化作用的影响

为了揭示连续施用不同氮量对半干旱地区马铃薯根际细菌群落结构及反硝化作用的影响。通过大田试验设置了6个不同施氮量处理:N₀(对照,不施氮)、N₇₅(施氮量75 kg·hm^-2)、N₁₅₀(施氮量150 kg·hm^-2)、N₂₂₅(施氮量225 kg·hm^-2)、N₃₀₀(施氮量300 kg·hm^-2)和N₃₇₅(施氮量375 kg·hm^-2),研究了连续5年不同施氮量对半干旱地区马铃薯根际细菌群落结构及反硝化作用过程中编码亚硝酸还原酶的关键酶基因nirK和nirS丰度的影响。结果显示:各处理土壤中$NO_{3}^{-}$ -N含量随施氮量的增加显著增加,最高施氮量处理(N₃₇₅)土壤中$NO_{3}^{-}$-N含量高达80.41 mg·kg^-1,分别比其他处理增加了86.87%~856.12%;土壤亚硝酸还原酶活性随着施氮量的增加随之增强,高氮处理显著高于低氮处理。高通量测序结果显示,大量施氮降低了细菌群落的Alpha多样性,显著改变了土壤细菌群落的物种组成;与N₀处理相比,其他各施氮处理根际土壤中变形菌门和厚壁菌门的相对丰度增加了1.66%~26.53%和22.59%~85.52%,放线菌门和酸杆菌门的相对丰度降低了3.48%~34.63%和4.21%~37.12%,高氮处理显著低于不施氮及低氮处理;高量施氮显著提高了根际土壤中Lactococcus、Bacillus和Lysobacter的相对丰度。根际土壤中nirK和nirS相对丰度随施氮量的增加随之增加,高氮处理中nirK和nirS相对丰度分别比N₀处理增加了116.26%、101.49%和28.24%、8.47%。Person相关性分析表明,nirK相对丰度与土壤$NO_{3}^{-}$-N含量呈显著正相关关系,pH与nirK和nirS的丰度呈显著负相关关系。大量施氮造成土壤剖面中$NO_{3}^{-}$-N积累量的增加以及pH值降低是引起根际细菌群落结构以及nirK和nirS丰度变化的主要原因。     

复合垂直流人工湿地基质理化性质对nirS和nirK型反硝化菌多样性的影响

采用Illumina高通测序技术,探讨喀斯特地区复合垂直流人工湿地基质理化性质对nirS和nirK型反硝化菌多样性的影响。结果表明,复合垂直流人工湿地中4个池子（即一级垂直流下行池、一级垂直流上行池、二级垂直流下行池、二级垂直流上行池）的基质理化性质存在显著差异。其中,pH在中4个池子中均呈中性,DO呈先下降后升高趋势,下行池的基质电导率EC均比上行池要高;TN、TP含量随着人工湿地梯级呈显著下降趋势,未出现厌氧现象;NO3--N和NH4+-N含量在二级垂直流下行池中大量积累并达到峰值。在AlpHa和Beta多样性分析中,nirS、nirK型两类反硝化细菌丰度随着人工湿地梯级呈先降低后升高的趋势,多样性随人工湿地级数逐渐降低,且下行池丰度多样性显著高于上行池。基质中nirS型反硝化细菌鉴定为5门9属,nirK型为6门14属,变形菌门是nirS、nirK型反硝化菌群的共同优势菌门,相对丰度为55.36%~65.72%、30.06%~45.66%。其中,优势菌属为红杆菌属（Rhodobacter）、脱氯单胞菌属（Dechloromonas）、红假单胞菌属（Rhodopseudomonas）、慢生根瘤菌属（Bradyrhizobium）和氏菌属（Bosea）。红杆菌属和脱氯单胞菌属相对丰度在空间上随着人工湿地级数升高总体呈上升趋势,红假单胞菌属、慢生根瘤菌属和氏菌属在空间上随人工湿地级数升高总体呈下降趋势,此2类优势菌属在空间上存在显著差异（P<0.01）。韦恩图表明nirS型反硝化菌群丰度明显高于nirK型,且各级池中反硝化菌群相似度较高。冗余分析表明基质中nirS和nirK反硝化菌群的构建主要受pH、NH4+-N、DO、TP、TN影响,且nirS型对pH,以及nirK型对NH4+-N的响应更为强烈。综上,本文研究了人工湿地nirS、nirK型反硝化菌多样性特征及其与环境因子的关系,为复合垂直流人工湿地高效脱氮提供理论依据。     

连作与轮作大豆土壤反硝化细菌多样性与组成结构(英文)

应用PCR-RFLP方法对连作与轮作大豆土壤中反硝化微生物种群和分类进行研究。结果 2种土壤样品中均可检测到反硝化微生物存在。从2种土壤中获取268个nirS基因克隆,46种基因型,其中连作土壤中有29种基因型,轮作土壤中有34种基因型,分析结果显示2种土壤样品中均有优势菌群存在,并且连作土壤样品中反硝化细菌的优势菌群略多于轮作土壤样品。序列同源性分析显示nirS基因克隆序列与已知反硝化微生物具有较远的亲缘关系。结果提示:与轮作大豆相比,连作大豆土壤中反硝化微生物的遗传多样性和组成有所改变,类群的结构组成易受耕作方式影响。     

The mitigation potential of hippuric acid on N2O emissions from urine patches: An in situ determination of its effect

Previous laboratory studies have demonstrated that hippuric acid, a ruminant urine constituent, can mitigate nitrous oxide (N₂O) emissions from simulated urine patches. Hippuric acid has the potential to be a N₂O mitigation tool because animal diets can be manipulated to adjust its concentration in the urine. This study was conducted to determine if the effect observed in previous laboratory studies would also occur in situ under field conditions. In our field study, plots were treated with unadulterated bovine urine (56 mM hippuric acid), the same bovine urine amended with either benzoic acid (34 mM), dicyandiamide (DCD) or varying rates of hippuric acid (up to 90 mM). Soil inorganic-N, N₂O fluxes, and plant N responses were monitored over a 78 d period. Effects on microbial communities were monitored by determining the size and structure of nitrite oxidizer (nxrA) and nitrite reducer (nirS) bacterial populations using real-time PCR and denaturing gradient gel electrophoresis (DGGE), respectively. Decreases in N₂O emissions, with increasing hippuric and benzoic acid concentrations, were only seen on Day two of the trial. With the exception of the DCD treatment (0.60% of N applied) the amended urine treatments did not significantly affect emissions of N₂O as a percentage of N applied (1.28-1.65%). Soil inorganic-N and plant response were not affected by urinary amendment, except in the DCD treatment where nitrification inhibition occurred. Nitrite oxidizer community structures shifted and increased approximately 5.4-fold in size over 48 d in response to urine, although no specific response to elevated hippuric acid or benzoic acid was observed. No treatment effects were observed on community structure of the nitrite reducing bacteria but averaged over time the highest rate of hippuric acid significantly decreased nirS gene copy numbers g⁻¹ soil. We concluded that under the conditions of this field study, increasing hippuric or benzoic acid concentrations in bovine urine had no effect on N₂O mitigation in situ. We argue that the discrepancy with previous laboratory studies may be related to differences in soil pH, microbial communities and the presence of vegetation. Further research is needed to determine the potential for hippuric acid as a tool to mitigate N₂O emissions, and its effect(s) on resident N cycling microorganisms.