陈 娜,刘 毅,黎 娟,袁 婧,葛体达,吴金水,孙志龙,徐华勤*



长期施肥对稻田不同土层反硝化细菌丰度的影响

陈 娜1,2,刘 毅2,黎 娟1,袁 婧2,葛体达2,吴金水2,孙志龙3,徐华勤1*

(1.湖南农业大学农学院,湖南 长沙 410128；2.中国科学院亚热带农业生态研究所亚热带农业生态过程重点实验室,湖南 长沙 410125；3.湖南省宁乡市回龙铺镇农业综合服务中心,湖南 宁乡 410606)

为了探讨长期施肥对稻田不同土层关键反硝化功能种群丰度的影响及核心驱动因子,以湖南宁乡长期施肥定位试验田为平台,选取不施肥(CK)、全量化肥(NPK)和秸秆还田(ST)3个处理,结合实时荧光定量PCR(qPCR)技术,系统分析了稻田不同土层(0~10,10~20,20~30,30~40cm)关键反硝化功能基因(和)的丰度及其与土壤理化性质的内在联系.结果表明,相比于不施肥处理(CK),施肥处理(NPK和ST)在0~40cm土层土壤SOC、TN、NO3--N、NH4+-N和Olsen-P分别显著增加了2.2%~83.6%,3.5%~58.3%,70.8%~222.1%,0.9%~83.7%和16.5%~94.5%,pH值下降了0.31~0.67个单位;长期施用化肥和秸秆使和基因丰度分别增加0.75~7.18倍,1.57~3.02倍和0.53~3.81倍,其中秸秆还田对反硝化细菌数量的影响比单施化肥更显著;稻田、和反硝化型细菌的丰度随土层深度增加而逐渐降低,具有明显的垂直分布特征;RDA分析结果显示,土壤养分如SOC和TN是影响水稻土、和反硝化型细菌垂直分布的关键因子,而pH值是调控反硝化细菌在稻田底土分布的核心驱动因子.研究结果可为提升稻田土壤肥力和减少稻田氮素损失和温室气体排放提供理论依据.

水稻土；长期施肥；不同土层；反硝化细菌；种群丰度

农田土壤为N2O排放的主要人为源,年均排放量约为4.2Tg,占全球总释放量的70%[1].我国是世界上最大的水稻生产国,稻田耕种面积占世界水稻生产面积的27%.因此,我国稻田N2O的排放日益受到关注[3].稻田土壤N2O的排放主要由土壤微生物参与的硝化和反硝化作用所驱动[2].由于稻田土壤长期处于淹水厌氧环境,因此反硝化作用被认为是稻田N2O产生的主要途径[3].其中,基因编码的硝酸还原酶是反硝化过程的第一步反应酶,和基因编码的亚硝酸还原酶控制着关键的限速步骤,因此,、和常用作标记基因用以研究土壤反硝化细菌[4-5].大量研究结果表明[6-8],反硝化功能种群丰度的增加会导致反硝化作用的增强,进而引起N2O排放增多.因此,明确稻田土壤关键反硝化功能种群的丰度变化规律,对于减少稻田土壤氮素损失及N2O排放具有重要的意义.

近年来,由于稻田增值高产的需要,施肥成为保障粮食安全和产量的关键措施.但研究发现,长期不同施肥会引起稻田土壤理化性质出现差异,进而影响反硝化微生物,从而影响稻田土壤反硝化作用及N2O的排放[9-10].因此,关于稻田土壤反硝化微生物对长期施肥响应的研究逐渐成为国内外学者关注的热点.研究发现[11-12],施用化肥和有机肥均显著提高反硝化细菌丰度,且施用有机肥处理与化肥处理间反硝化细菌丰度和群落结构差异显著.这些研究表明不同施肥制度显著影响反硝化微生物,然而到目前为止,关于长期施肥对稻田土壤反硝化微生物分布影响的研究主要集中在耕作层(0~20cm)土壤,而深层土壤反硝化微生物对长期不同施肥制度的响应的研究少有报道.因此,非常有必要针对长期施肥对稻田土壤剖面不同深度反硝化微生物的分布特征开展研究.

为了探究长期施肥对稻田不同土层关键反硝化功能种群丰度的影响,本研究依托湖南省宁乡县长期定位试验田,选取无肥对照、全化肥、秸秆还田3种不同施肥处理,采集0~10,10~20,20~30,30~40cm土层土壤样品,运用荧光定量PCR(qPCR)技术,系统分析了和等3种关键反硝化功能基因在不同施肥处理中沿土壤剖面的丰度变化特征及其核心驱动因子.研究结果可为提高稻田氮素利用效率和温室气体减排提供理论依据.

1 材料与方法

1.1 供试样点及样品采集

采样地点位于湖南省宁乡县农业技术推广中心,地理位置为113°00′20″E,北纬28°25′16″N,海拔36.1m,年平均气温为17.5℃,年均降雨量为1300mm,年均无霜期在274d左右,年日照为1663h.该地区为典型双季稻产区,于1986年设立田间试验,为长期定位施肥实验田.本研究采用其中3个施肥处理:①无肥对照(CK):不施加任何肥料;②全化肥处理(NPK):仅施氮磷钾化肥,按每公顷施60kg氮((NH4)2SO4)、30kg磷(P2O5)和60kg钾(K2O)的比例进行施肥;③秸秆还田处理(ST):施用上一季度收割的水稻秸秆为主(早季稻秸秆还田量为2775.0kg/hm2,晚季稻秸秆还田量为3600.0 kg/hm2),若与处理②比,总氮磷钾量不足则用化肥补足.试验小区面积为28.22m2,每个处理3次重复,随机区组排列.

土壤样品采集于2017年5月中旬,在每个小区采用五点法采集,土壤剖面0~40cm范围内按10cm间距分段采样.采样时将鲜土分为2份:一份用锡箔纸包好立即用液氮速冻,存放于-80℃冰箱用于分子生物学实验;另一部分室内剔除石块和植物残体后,存于4℃冰箱,进行理化指标的测定.土壤的基本理化性质见表1.

表1 供试土壤基本理化性质 Table 1 Characteristics of the soil in this study

1.2 样品理化指标测定及方法

土壤pH值按水土比2.5:1用Mettler- toledo320 pH计测定;土壤有机碳(SOC)和全氮(TN)采用碳氮元素分析仪(VARIO MAX C/N,德国)测定(干烧法);土壤速效磷(Olsen-P)含量采用Olsen法;硝态氮(NO3--N)和铵态氮(NH4+-N)用连续流动分析仪(Flastar 5000Analyzer)测定.

1.3 土壤DNA的提取和实时荧光定量PCR

土壤DNA的提取方法参考陈哲[13]方法,并稍作修改:取0.5g经液氮速冻的土壤.DNA浓度测定使用核酸蛋白测定仪(Nanodrop ND-1000UV-Vis分光光度计),并取2μL用1%琼脂糖胶进行电泳检测,DNA样品保存于-20℃冰箱.

实时荧光定量PCR所用仪器为ABI 7900 (Applied Biosystem),标准曲线的建立参照陈哲[13]的方法:、和PCR反应体系均为10μL,含有5μL SYBR GREEN Ⅰ (Takara),0.7μL ROX (Takara),0.3μL引物,1μL浓度为5ng的DNA模板,加水补至10μL.基因引物对为-571F-773R[13],引物序列为CCGATYCCGGCVAT- GTCSAT/GGNACGTTNGADCCCCA,反应条件为95℃预变性30s,95℃变性5s,60℃退火30s,共40个循环;基因引物对为-1aCuF/K-3CuR[14],引物序列为ATCATGCTSCTGCCGCG/GCCTCGA- TCAGRTTGTGGTT,反应条件为95℃预变性30s, 95℃变性5s,56℃退火30s,共40个循环;基因引物对为-cd3aF/-R3cd[15],引物序列为GTSAACGTSAAGGARACSGG/GASTTCGGRTGSGTCTTGA,反应条件为95℃预变性30s,95℃变性5s,56℃退火20s,72℃延伸30s,共40个循环.溶解曲线均为95℃ 15s,60℃ 15s,90℃ 15s.

1.4 数据处理与分析

qPCR数据的导出采用SDS 2.3软件,数据处理与统计分析采用Microsoft Excel 2016和SPSS 20.0,图形绘制采用ORIGIN 9.0.不同处理显著性用One-way ANOVA(单因素方差分析)进行检验,采用Duncan多重比较分析组间差异.冗余分析(RDA)用Canoco 5.0实现.

2 结果与讨论

2.1 长期施肥对稻田土壤基本理化性质的影响

如图1所示,经长期施肥后,各处理间0~40cm土层土壤SOC、TN、NO3--N、NH4+-N、Olsen-P含量和pH值差异显著.同时多重比较(表2)结果发现,施肥和土壤深度及其交互作用均对土壤SOC、TN、NO3--N、NH4+-N和Olsen-P含量及pH值有极显著影响(<0.01).相比于CK处理,施肥处理(NPK和ST)在0~40cm土层土壤SOC、TN、NO3-- N、NH4+-N和Olsen-P分别增加了2.2%~83.6%, 3.5%~58.3%,70.8%~222.1%,0.9%~83.7%和16.5%~ 94.5%, pH值下降了0.31~0.67个单位;相比于全化肥处理,秸秆还田使0~30cm土层土壤SOC、TN、NO3--N和NH4+-N含量分别增加了20.3%~26.6%、18.1%~25.0%、3.2%~85.3%和22.8%~78.4%,pH值下降0~0.16个单位,使20~40cm土层土壤Olsen-P含量增加6.0%~34.4%.长期施肥增加土壤养分,降低pH值,这一结论在施肥试验中具有普遍性,如Zhao等[16]在稻麦种植制度的施肥试验中发现所有施肥处理包括化肥及化肥配施秸秆处理土壤有机质、有效磷和全氮含量均增加;孟红旗等[17]和于冰等[18]的研究表明施用化肥及秸秆配施化肥处理较不施肥处理pH值降低,秸秆配施化肥处理的有机质、TN、NH4+-N、NO3--N和有效磷含量高于其他处理.

表2 施肥处理和土壤深度对土壤理化性质的多重比较Table 2 Effects of fertilization and soil depth on soil properties

注:数值及*号为值和显著水平.**为极显著(<0.01).

由于耕作层土壤养分的向下迁移,使得土壤养分具有垂直分布特征.图1中土壤C、N、P含量均在0~10cm土层中最高,与土层深度呈显著负相关.杜林森等[19]通过38a的长期培肥试验发现,土壤碳氮含量随土壤深度增加而逐渐降低,且施用秸秆和化肥处理是不施肥处理的1.1~13.7倍,单艳红等[20]研究发现0~30cm土层土壤Olsen-P含量与土层深度呈负相关,这与本研究结果一致.长期施肥稻田土壤养分垂直分布在水稻生产上具有双重意义[21]:一方面,养分适度下移,可以丰富稻田底土养分含量,对于培育稻田土壤肥力极为有利;另一方面,当养分下移超过水稻根系所能吸收的范围,将造成养分的淋失,进而造成地下水的污染.由此可见,长期施化肥或秸秆秸秆对提高稻田土壤肥力和促进稻田可持续性具有显著的效果,同时需对施肥可能导致的环境问题引起注意.

2.2 不同施肥处理对土壤narG、nirK和nirS型反硝化型细菌丰度的影响

、和型反硝化细菌基因拷贝数如图2所示,、和基因丰度分别在1.20´107~5.02´109,5.10´106~3.36´109,4.71´106~2.75´109拷贝数/g干土,其在不同土壤深度土层的分布对不同施肥的响应趋势较相似.长期施肥提高了土壤、和型反硝化细菌基因丰度,相比较于不施肥处理,施用秸秆和化肥使基因丰度增加0.75~7.18倍,基因丰度增加1.57~3.02倍,基因丰度增加0.53~3.81倍.这与之前的研究结论基本一致,如解开治等[22]指出无机肥和无机有机肥配施均能显著提高稻田土壤和基因丰度;靳振江等[26]也发现施用化肥和化肥配施秸秆处理的基因丰度较无肥处理提高了0.51倍和0.80倍.其原因为,反硝化微生物主要为异养厌氧型微生物,在稻田土壤长期的淹水厌氧环境下,土壤养分的增加能极大地满足反硝化微生物生长繁殖对养分的需求,从而刺激反硝化微生物的大量繁殖.同时,从图中可得,秸秆还田处理中、和基因丰度较仅施用化肥高,表明长期施用秸秆对稻田反硝化微生物数量的促进作用更显著.这与很多国内外研究结果相似[23-24],如尹昌等[25]研究表明施用有机肥显著促进型反硝化细菌的生长,而施用化肥需配施有机肥才促进反硝化微生物的生长;Chen[30]等发现有机肥处理、和反硝化细菌数量显著高于无机肥处理.其原因为,首先,秸秆有机肥的长期输入为反硝化细菌和其他微生物提供了大量生物有效性碳源,促进微生物的大量繁殖,微生物活性的提高反过来又促进有机物的降解,从而增加土壤中速效养分,为微生物的生长繁殖提供适宜的环境[26];其次,NO3--N是反硝化作用的底物,NO3--N显著增加(图1)为其提供了丰富的反应底物[27];最后,有机质增加刺激了土壤微生物活性,呼吸作用强,加速了厌氧环境的形成[28].化肥虽然也能为反硝化微生物直接快速地提供NO3--N底物,但并不能直接增加土壤碳养分,而是通过促进植株根系生长和产生根系分泌物来保障土壤反硝化微生物对碳源的需求[29].

图2 不同施肥处理下不同土壤深度反硝化细菌基因丰度 Fig.2 Gene Abundance of denitrifying bacteria in soil profile with different fertilizer application

之前有报道指出,土壤养分的垂直分布也会引起土壤微生物具有垂直分布特征[30].如Taylor等[31]发现农田土壤表层细菌数量远高于底层;Fierer等[32]指出草地土壤微生物数量随土壤深度的增加而减少.图2中随着土壤深度的增加,不同施肥处理、和基因丰度均显著降低,具有明显的垂直分布特征, 类似结果在森林土壤中也有发现[33].造成这种变化趋势的主要原因可能为土壤表层受耕作等影响较大,肥料和凋落物等直接施入表土,土壤表层养分如有机碳、硝态氮和速效磷等含量较高(图1),因而土壤表层微生物更易于获取足够的养分和底物;随着深度增加,土壤养分含量逐渐减少(图1),微生物生长受限,微生物数量减少.另外,本研究表明,相对于施用化肥处理,秸秆还田处理中不同土层反硝化微生物的丰度都要更高(图2),类似试验结果在长期定位施肥的旱地(菜地)土壤中也有所发现[34].水稻田不同于旱地土,其土壤养分向下迁移速率和渗漏损失量相对较小,大多沉积于底底土中,因此不同施肥处理所引起的稻田耕作层养分含量的差异,其类似特征也能反映在底土上.正是因为不同施肥处理间土壤养分垂直分布的差异,导致了、和反硝化型细菌在不同施肥处理中垂直分布存在差异.

2.3 narG、nirK和nirS型反硝化型细菌丰度与土壤理化性质的关系

RDA排序结果(图3)表明,第一和第二排序轴占总特征值的72.76%,表明这6项环境因子能解释大部分反硝化细菌基因丰度的变化.土壤SOC、TN、NH4+-N、NO3--N、Olsen-P和pH值均极显著影响反硝化功能种群丰度(蒙特卡罗检验值分别为=0.002,0.002,0.002,0.002,0.006,0.002),其中土壤SOC和TN在第一排序轴上的投影长度较长, NH4+-N、NO3--N和pH值次之,Olsen-P最短,表明在6项理化因子中SOC和TN对土壤反硝化基因丰度的影响较大.因此,土壤养分如SOC和TN是影响稻田土壤、和反硝化型细菌垂直分布的关键因子,尤其对稻田耕作层的影响更为显著(图3).类似的结果在其他生态系统也有发现,如Fierer等[32]认为土壤微生物数量随土壤深度的增加而减少主要是由于土壤碳的有效性降低;Liu等[35]认为在森林土壤中,土壤DOC、DON、NH4+和NO3-垂直分布是、和型反硝化细菌呈垂直分布特征的关键驱动因子.同时RDA结果显示,pH值与20~30cm和30~40cm土层反硝化细菌的丰度紧密相关,暗示pH值可能是驱动、和型反硝化细菌在稻田底土分布的核心驱动因子. pH值对反硝化细菌具有选择效应,因而在不同的土壤环境中反硝化细菌对pH值的响应不同[36]. Dandie等[37]发现,农田土壤中pH值是限制型反硝化细菌群落结构的唯一影响因子;而王亚男等[43]在设施菜地中的研究和Enwall[38]在稻田的研究发现pH值与反硝化细菌丰度并没有相关性.本研究pH值显著影响反硝化基因丰度,与前者研究结果相似.0~20cm施肥处理、10~ 20cm、20~30cm和30~40cm土层处理基因丰度在二维排序图中彼此分离,表明土壤深度影响反硝化基因丰度.在0~ 30cm土层中,全化肥处理、秸秆还田处理与不施肥处理彼此分离,表明施用化肥及秸秆还田显著影响土壤反硝化细菌基因丰度.

图3 影响反硝化细菌基因丰度因素的RDA分析 Fig.3 RDA analysis of influencing factors to gene abundance of denitrifying bacteria

3 结论

3.1 长期施肥显著提升水稻土不同土层碳、氮、磷等含量,降低土壤pH值.

3.2 稻田、和反硝化型细菌的丰度随土层深度增加而逐渐降低.长期施肥显著增加水稻土不同土层、和反硝化型细菌的丰度,其中秸秆还田对反硝化细菌数量的影响比施用化肥更显著.

3.3 土壤养分如SOC和TN是影响水稻土、和反硝化型细菌垂直分布的关键因子,而pH值是调控反硝化细菌在稻田底土分布的核心驱动因子.

[1] IPCC, Climate Change 2001: A scientific basis, intergovernmental panel on climate change [R]. Cambridge University Press, Cambridge, UK, 2001.

[2] Conrad R. Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, CO2, N2O, and NO) [J]. Microbiological Reviews, 1996,60(4):609-640.

[3] Baggs E M. A review of stable isotope techniques for N2O source partitioning in soils: recent progress, remaining challenges and future considerations [J]. Rapid Communications in Mass Spectrometry, 2008,22(11):1664-1672.

[4] Levy-Booth D J, Prescott C E, Grayston S J. Microbial functional genes involved in nitrogen fixation, nitrification and denitrification in forest ecosystems [J]. Soil Biology & Biochemistry, 2014,75:11-25.

[5] Braker G, Zhou J, Wu L, et al. Nitrite reductase genes (and) as functional markers to investigate diversity of denitrifying bacteria in Pacific Northwest marine sediment Communities [J]. Applied and Environmental Microbiology, 2000,66(5):2096-2104.

[6] Philippot L, Čuhel J, Saby N P A, et al. Mapping field-scale spatial patterns of size and activity of the denitrifier community [J]. Environmental Microbiology, 2009,11(6):1518–1526.

[7] Morales S E, Cosart T, Holben W E. Bacterial gene abundances as indicators of greenhouse gas emission in soils [J]. Isme Journal, 2010,4(6):799-808.

[8] Regan K, Kammann C, Hartung K,et al. Can differences in microbial abundances help explain enhanced N2O emissions in a permanent grassland under elevated atmospheric CO2[J]. Global Change Biology, 2011,17(10):3176–3186.

[9] 李勇先.稻田土壤中氧化亚氮的释放机制及控制[D]. 杭州:浙江大学, 2003. Li Y X. Study on relationship between CH4and N2O emission and the control of CH4and N2O from paddy soil [D]. Hangzhou: Zhejiang University, 2003.

[10] Chaparro J M, Badri D V, Vivanco J M. Rhizosphere microbiome assemblage is affected by plant development [J]. Isme Journal, 2014, 8(4):790-803.

[11] 陈 哲,袁红朝,吴金水,等.长期施肥制度对稻田土壤反硝化细菌群落活性和结构的影响[J]. 生态学报, 2009,29(11):5923-5929. Chen Z, Yuan H Z, Wu J S, et al. Activity and composition of denitrifying bacterial community respond differently to long-term fertilization [J]. Acta Ecologica Sinica, 2009,29(11):5923-5929.

[12] Enwall K, Philippot L, Hallin S. Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization [J]. Applied and Environmental Microbiology, 2005,7(1): 8335-8343.

[13] 陈 哲.长期施肥对水稻土反硝化作用和反硝化功能微生物的影响机理[D]. 北京:中国科学院研究生院, 2010. Chen Z. Effects of long-term fertilization on denitrification and denitrifying microorganisms in paddy soil [D]. Beijing: Chinese academy of science, 2010.

[14] Hallin S, Lindgren P E. PCR Detection of genes encoding nitrite reductase in denitrifying bacteria PCR detection of genes encoding nitrite reductase in denitrifying bacteria [J]. Applied & Environmental Microbiology, 1999,65(4):1652-1657.

[15] Michotey V, Méjean V, Bonin P. Comparison of Methods for Quantification of Cytochrome, cd1-Denitrifying Bacteria in Environmental Marine Samples [J]. Applied & Environmental Microbiology, 2000,66(4):1564-1571.

[16] Zhao J, Ni T, Li Y,. Responses of bacterial communities in arable soils in a rice-wheat cropping system to different fertilizer regimes and sampling times [J]. PloS one, 2014,9(1):e85301.

[17] 孟红旗,刘 景,徐明岗,等.长期施肥下我国典型农田耕层土壤的pH演变[J]. 土壤学报, 2013,50(6):1109-1116. Meng H Q, Liu J, Xu M G,et al. Evolution of pH in topsoils of typical Chinese croplands under long-term fertilization [J]. Acta Pedologica Sinica, 2013,50(6):1109-1116

[18] 于 冰,宋阿琳,李冬初,等.长期施用有机和无机肥对红壤微生物群落特征及功能的影响[J]. 中国土壤与肥料, 2017,(6):58-65. Yu B, Song A L, Li D C, et al. Influences of long-term application organic and inorganic fertilizers on the structure and function of microbial community in red soil [J]. Soil and Fertilization Science in China, 2017,(6):58-65.

[19] 杜林森,唐美铃,祝贞科,等.长期施肥对不同深度稻田土壤碳氮水解酶活性的影响特征[J]. 环境科学, 2018,39(8):3901-3909. Du L S, Tang M L, Zhu Z K, et al. Effects of long-term fertilization on enzyme activities in profile of paddy soil profiles [J]. Environmental Science, 2018,39(8):3901-3909.

[20] 单艳红,杨林章,沈明星,等.长期不同施肥处理水稻土磷素在剖面的分布与移动[J]. 土壤学报, 2005,42(6):970-976. Shan Y H, Yang L Z, Shen M X, et al. Accumulation and downward transport of phosphorus in paddy soil in long-term fertilization experiments [J]. Acta Pedologica Sinica, 2005,42(6):970-976.

[21] 鲁如坤,时正元,赖庆旺.红壤长期施肥养分的下移特征[J]. 土壤, 2000,32(1):27-29. Lu R K, Shi Z Y, Lai Q W. Characteristics of nutrient decline of long-term fertilization in red soil [J]. Soils, 2000,32(1):27-29.

[22] 解开治,徐培智,蒋瑞萍,等.有机无机肥配施提升冷浸田土壤氮转化相关微生物丰度和水稻产量[J]. 植物营养与肥料学报, 2016, 22(5):1267-1277. Xie K Z, Xu P Z, Jiang R P,et al. Combined application of inorganic and organic fertilizers improve rice yield and the abundance of soil nitrogen-cycling microbes in cold waterlogged paddy fields [J]. Journal of Plant Nutrition and Fertilizer, 2016,22(5):1267-1277.

[23] Marschner P, Kandeler E, Marschner B. Structure and function of the soil microbial community in a long-term fertilizer experiment [J]. Soil Biology & Biochemistry, 2003,35(3):453-461.

[24] 徐一兰,唐海明,李益锋,等.长期施肥大麦生育期双季稻田土壤微生物和酶活性动态变化特征[J]. 中国农学通报, 2017,33(13):12-20. Xu Y L, Tang H M, Li Y F,et al. Dynamic changes of soil microbe and soil enzyme activities during barley main growth stages under different long-term fertilizer treatments [J]. Chinese Agricultural Science Bulletin, 2017,33(13):12-20.

[25] 尹 昌,范分良,李兆君,等.长期施用有机和无机肥对黑土型反硝化菌种群结构和丰度的影响[J]. 环境科学, 2012,33(11):3967-3975. Yin C, Fan F L, Li Z J, et al. Influences of long-term application of organic and inorganic fertilizers on the composition and abundance of-type denitrifiers in black soil [J]. Environmental Science, 2012,33(11):3967-3975.

[26] 龚 伟,颜晓元,王景燕.长期施肥对土壤肥力的影响[J]. 土壤, 2011,43(3):336-342. Gong W, Yan X Y, Wang J Y. Effect of long-term fertilization on soil fertility [J]. Soils, 2011,43(3):336-342.

[27] Liu E K, Yan C R, Mei X R,. Long-term effect of chemical fertilizer, straw, and manure on soil chemical and biological properties in northwest China [J]. Geoderma, 2010,158(3):173-180.

[28] Klemedtsson L, Svensson B H, Rosswall T. Relationship between soil moisture content and nitrous oxide production during nitrification and denitrification [J]. Biology & Fertility of Soils, 1988,6(2):106-111.

[29] 乔云发,苗淑杰,韩晓增.长期施肥条件下黑土有机碳和氮的动态变化[J]. 土壤通报, 2008,39(3):545-548. Qiao Y F, Miao S J, Han X Z. Dynamics of soil organic carbon and nitrogen in black soil under a long-term application of fertilizers [J]. Chinese Journal of Soil Science, 2008,39(3):545-548.

[30] 李晨华,张彩霞,唐立松,等.长期施肥土壤微生物群落的剖面变化及其与土壤性质的关系[J]. 微生物学报, 2014,54(3):319-329. Li C H, Zhang C X, Tang L S, et al. Effects of long-term fertilizing regime on soil microbial diversity and soil property [J]. Acta Microbiologica Sinica, 2014,54(3):319-329.

[31] Taylor J P, Wilson B, Mills M S, et al. Comparison of microbial numbers and enzymatic activities in surface soils and subsoils using various techniques [J]. Soil Biology & Biochemistry, 2002,34(3):387-401.

[32] Fierer N, Schimel J P, Holden P A. Variations in microbial community composition through two soil depth profiles [J]. Soil Biology & Biochemistry, 2003,35(1):167-176.

[33] Xian L, Chen C, Wang W, et al. Vertical distribution of soil denitrifying communities in a wet sclerophyll forest under long-term repeated burning [J]. Microbial Ecology, 2015,70(4):993.

[34] 曾希柏,王亚男,王玉忠,等.施肥对设施菜地型反硝化细菌群落结构和丰度的影响[J]. 应用生态学报, 2014,25(2):505-514. Zeng X B, Wang Y N, Wang Y Z, et al. Effects of different fertilization regimes on abundance and community structure of the-type denitrifying bacteria in greenhouse vegetable soils [J]. Chinese Journal of Applied Ecology, 2014,25(2):505-514.

[35] Liu S, Chen Y X, Sun H, et al. Temporal dynamics of DOC in forest soil along an elevational gradient of subalpine-alpine in the southwestern China [J]. Journal of Northwest Forestry University, 2015.

[36] Palmer K, Drake H L, Horn A M A. Association of novel and highly diverse acid‐tolerant denitrifiers with N2O fluxes of an acidic fen [J]. Applied & Environmental Microbiology, 2010,76(4):1125-1134.

[37] Dandie C E, Wertz S, Leclair C L, et al. Abundance, diversity and functional gene expression of denitrifier communities in adjacent riparian and agricultural zones [J]. Fems Microbiology Ecology, 2011, 77(1):69-82.

[38] Enwall K, Throback I N, Stenberg M, et al. Soil resources influence spatial patterns of denitrifying communities at scales compatible with land management [J]. Applied & Environmental Microbiology, 2010, 76(7):2243-2250.

Effects of long-term fertilization on the abundance of the key denitrifiers in profile of paddy soil profiles.

CHEN Na1,2, LIU Yi2, LI Juan1, YUAN Jing2, GE Ti-da2, WU Jin-shui2, SUN Zhi-long3, XU Hua-qin1*

(1.College of Agronomy, Hunan Agriculture University, Changsha 410128, China；2.Key Laboratory of Subtropical Agriculture Ecology, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China；3.Integrated Service for Agriculture Ningxiang County Huilongpu Town, Ningxiang 410606, China)., 2019,39(5)：2154~2160

The aims of this study were to explore the effect of long-term fertilization on the abundance of the key denitrifiers in paddy soil profiles (0~40cm), and the core factors driving denitrifiers. Soils with non-fertilization (CK), inorganic fertilizer (NPK) and organic fertilizer (ST) were collected in Ning xiang County, Hunan Province, and real-time fluorescent quantitative PCR technology was used to analyze the abundance of-,- and-containing communities in paddy soil profile (0~10cm, 10~20cm, 20~30cm, 30~40cm) and their relationship with soil properties. The results showed that compared with CK, SOC、TN、NO3--N、NH4+-N and Olsen-P in soil profile under NPK and ST increased by 2.2%~83.6%、3.5%~58.3%、70.8%~222.1%、0.9%~ 83.7% and 16.5%~94.5% respectively, and pH decreased by 0.31~0.67 units. Long-term application of inorganic fertilizer and organic fertilizer increased,, andgene abundance by 0.75~7.18 times, 1.57~3.02 times, and 0.53~3.81 times, respectively. And the effect of organic fertilizer on the abundance of denitrifiers was more significant than that of inorganic fertilizer application; The abundance of-,- and-containing communities decreased gradually with soil depth increasing, which presented an obvious vertical distribution; RDA analysis showed that soil nutrients such as SOC and TN were the core factors affecting the vertical distribution of-,- and-containing populations in paddy soil, especially in the cultivated horizon, while pH was the core driving factor regulating the distribution of denitrifying bacteria in paddy field subsoil. The results can provide theoretical basis for improving soil fertility and reducing nitrogen loss and greenhouse gas emission in paddy soils.

paddy soil；long-term fertilization；soil profile；denitrifying bacteria；abundance

X172

A

1000-6923(2019)05-2154-07

陈 娜(1994-),女,湖南娄底人,湖南农业大学硕士研究生,主要从事环境生态修复.发表论文1篇.

2018-10-14

国家自然基金资助项目(41771300,41301274)

*责任作者, 副教授, xu7541@163.com