Soil Bacterial Communities Under Different Long-Term Fertilization Regimes in Three Locations Across the Black Soil Region of Northeast China

Although soil bacteria play critical roles in agro-ecosystems, the knowledge of their response to long-term fertilization across the black soil region of Northeast China is limited. In this study, we sequenced 16 S rRNA genes to assess the effects of four long-term fertilization regimes—non-fertilization(NoF), chemical fertilizer(CF), manure(M), and chemical fertilizer plus manure(CFM)—on soil properties and bacterial communities in three locations, the northern, middle, and southern parts, across the black soil region. Results showed that the influence of fertilization regimes on soil properties varied significantly among the three locations. Manure fertilization significantly increased microbial biomass carbon and relative abundance of copiotrophic bacteria. Principal component analysis(PCA)showed that the total bacterial communities were separated into three groups according to the sampling location despite long-term fertilization, and that soil pH was the most important factor in shifting bacterial communities. In addition, similar fertilization regimes resulted in different influences on bacterial community composition, and the most influential soil properties varied among the three locations. Our results highlighted that geographical separation was a more dominant factor affecting bacterial communities than fertilization, and that long-term similar fertilization regimes did not induce consistent changes in bacterial community composition in the black soil region.     

Effects of Rhizosphere and Long-Term Fertilization Practices on the Activity and Community Structure of Denitrifiers Under Double-Cropping Rice Field

The soil physicochemical properties, soil denitrification rates (PDR), denitrifiers via nitrite reductases (nirK and nirS) and nitrous oxide reductase (nosZ), abundance and community composition of denitrifiers in both the rhizosphere and bulk soil from a long-term (32 year) fertilizer field experiment conducted during late rice season were investigated by using the MiSeq sequencing, quantitative PCR, terminal restriction fragment polymorphism (T-RFLP). The experiment including four treatments: without fertilizer input (CK), chemical fertilizer alone (MF), rice straw residue and chemical fertilizer (RF), and organic manure and chemical fertilizer (OM). The results showed that the application of rice straw residue and organic manure increased soil organic carbon (C), total nitrogen (N), and NH₄⁺-N contents. The nirK, nirS, and nosZ copy numbers with OM and RF treatments were significant higher than that of the MF and CK treatments in the rhizosphere and bulk soil (p < 0.05). The principal coordinate analysis (PCoA) analysis showed that the different parts of root zone are the most important factors for the variation of denitrifying bacteria community, and the different fertilization treatments is the second important factors for the variation of denitrifying bacteria community. The MiSeq sequencing result showed that nirK, nirS and nosZ-type denitrifiers communities within bulk soil had lower species diversity compared with rhizosphere soil, and were dominated by Rhizobiales, Rhodobacterales, Burkholderiales, and Pseudomonadales. As a result, the application of fertilization practices had significant effects on soil N and PDR levels, and affected the abundance and community composition of N-functional microbes.     

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。     

Manure contaminated with the antibiotic sulfadiazine impairs the abundance of nirK- and nirS-type denitrifiers in the gut of the earthworm Eisenia fetida

The antibiotic sulfadiazine (SDZ) can affect denitrifying bacteria in soil. However, effects on denitrifiers in the gut of earthworms have not been described so far. Therefore, the influence of SDZ-contaminated manure applied to soil on denitrifiers in the gut of the earthworm Eisenia fetida was assessed by quantitative polymerase chain reaction targeting genes coding for nirK- and nirS-type nitrite reductases of denitrifiers. Gut contents of Eisenia fetida contained 2.5 × 10⁶ and 5.1 × 10⁵ gene copies of nirK and nirS, respectively, after 2 weeks in soils amended with manure only. Copy numbers of nirK and nirS in gut contents from manure treatments with SDZ were up to ten times less. Overall, the data indicate a negative impact of SDZ on denitrifiers in the gut of earthworms.     

Identifying response groups of soil nitrifiers and denitrifiers to grazing and associated soil environmental drivers in Tibetan alpine meadows

Defining response groups within N-related microbial communities is needed to predict land management effect on soil N dynamics, but information on such response groups and associated environmental drivers is scarce. We investigated the abundance and major populations of ammonia-oxidizing archaea (AOA) and bacteria (AOB), and nirS- and nirK-harboring denitrifiers under different grazing managements in Tibetan alpine meadow soils. Grazing increased AOB and AOA abundances up to 42 fold and 3.7 fold, respectively, and increased the percentage of AOB within total ammonia oxidizers from 3.1% to 10.8%. The abundance of nirK-like denitrifiers increased with grazing intensity, while the abundance of nirS-like denitrifiers tended to decrease. However, sub-groups within each of these broad groups of (de)nitrifiers responded differently to grazing. Soil nitrate was the main driver of the abundance of denitrifier sub-groups (nirK or nirS) positively responding to grazing, while soil moisture and carbon concentration were the main drivers of the abundance of denitrifier sub-groups negatively responding to grazing. AOB and nirK-harboring denitrifiers thus generally responded more positively to grazing than AOA and nirS-harboring denitrifiers, but significant functional diversity existed within each group. Our approach demonstrates the usefulness of the concept of response groups to better characterize and understand (de)nitrifier response to grazing.     

氮肥对稻田土壤反硝化细菌群落结构和丰度的影响

以氮肥田间定位试验为研究对象,利用PCR-DGGE(聚合酶链反应变性梯度凝胶电泳)和荧光定量PCR(real-time PCR)技术,通过对反硝化细菌nirS基因的检测,分析了定位试验第2年稻田反硝化细菌群落结构和丰度的变化。DGGE图谱及依据其条带位置和亮度数字化数值进行的主成分分析(PCA)结果均显示:在氮肥定位试验第2年,与不施肥对照(CK)比较,在水稻各个生育期(分蘖期、齐穗期和成熟期)内,施用氮肥[150kg(N)·hm-2]的稻田根层土或表土中的反硝化细菌群落结构均无明显变化;且稻田根层土或表土中的反硝化细菌群落结构在水稻各个生育期间也均无明显差异。荧光定量PCR结果显示,在水稻生长发育过程中,施用氮肥的稻田根层土或表土中的反硝化细菌nirS基因拷贝数始终显著(P<0.05)高于其对应的不施肥对照。此外,无论施用氮肥与否,根层土中的反硝化细菌nirS基因拷贝数在水稻成熟期时都会显著(P<0.05)降低;但表土中的nirS基因拷贝数在水稻各生育期间无明显变化;且水稻成熟期时施用氮肥和不施肥的稻田表土中nirS基因拷贝数都显著(P<0.05)高于根层土。同时,与对照比较施用氮肥可促进水稻增产44%。研究表明,短期定位试验中施用氮肥能够显著提高稻田土壤反硝化细菌的丰度,但对其群落结构没有明显影响。     

Molecular analysis of the major capsid genes (<Emphasis Type="Italic">g23</Emphasis>) of T4-type bacteriophages in an upland black soil in Northeast China

Bacteriophages (phages) are the most abundant biological entities on the planet and are important as the greatest genomic reservoirs in both marine and terrestrial environments. In this study, we analysed T4-type phage communities in an upland black soil by monitoring g23 clones in DNA extracted from seasonal soil samples with no fertilizer, chemical fertilizers, chemical fertilizers plus manure, and natural restoration treatments. PCR products with degenerate primers MZIA1bis and MZIA6 were subjected to denaturing gradient gel electrophoresis. In total, 46 clones with different g23 sequences were obtained. Phylogenetic analyses indicated that T4-type phage communities in the upland black soil were distinctly different from those in marine environments and in an Antarctic lake, which strongly suggested that T4-type phage communities in soil differed from those in aquatic environments. Among 46 clones, 18 clones formed clusters with the clones from rice field soils, 14 clones formed three new clusters, and 13 clones were left as ungrouped, which indicated that T4-type phage communities in the upland black soil were relatively similar to those in rice field soils but that specific communities also inhabit in the upland black soil exclusively.     

长期施肥对黑土农田土壤微生物群落的影响

基于中国科学院海伦农业生态试验站长期定位试验区,应用实时荧光定量PCR(Real-time PCR)和变性梯度凝胶电泳(DGGE)技术研究了无施肥(NF)、单施N、P化肥(NP)以及化肥配施有机猪粪肥(NPM)等3种长期施肥措施对黑土区玉米田土壤微生物群落密度和结构的影响.Real-time PCR方法定量NF、NP及NPM措施土壤细菌群落基因组DNA质量分别为381、1 351和1 773 ng g-1干土,真菌群落基因组DNA质量分别113.3、127.3和20.6 ng g-1干土,真菌与细菌的比率分别为0.31、0.09和0.01,NPM措施显著低于另两种施肥方式(p＜0.05).DGGE方法研究表明,NP和NPM措施不能改善土壤细菌和真菌群落的多样性、均匀性及优势菌优势程度;但主成分分析结果显示NP和NPM措施均可改变土壤细菌和真菌群落的构成,且真菌群落的变化更为显著;聚类分析结果显示NP和NPM措施下细菌群落结构较相近,其相似系数为0.89,真菌群落中NP措施与NF措施相近,相似系数为0.63,高于NP与NPM措施的相似系数0.51.上述结果表明有机猪粪肥的长期施用可以显著降低黑土农田土壤真菌与细菌的比率,且明显地改变土壤细菌和真菌群落的结构.     

Bacterial communities in paddy soils changed by milk vetch as green manure: A study conducted across six provinces in South China

The use of green manures contributes to sustainable soil and nutrient management in agriculture; however, the responses of soil microbial communities to different fertilization regimes at the regional scale are uncertain. A study was undertaken across multiple sites and years in Hunan, Jiangxi, Anhui, Henan, Hubei, and Fujian provinces of South China to investigate the effects of green manuring on the structure and function of soil bacterial communities in rice-green manure cropping systems. The study included four treatments:winter fallow with no chemical fertilizer as a control (NF), milk vetch as green manure without chemical fertilizer (GM), winter fallow and chemical fertilizer (CF), and a combination of chemical fertilizer and milk vetch (GMCF). Significant differences were found in the responses of soil microbial communities at different sites, with sampling sites explaining 72.33% (F=36.59, P=0.001) of the community composition variation. The bacterial communities in the soils from Anhui, Henan, and Hubei were broadly similar, while those from Hunan were distinctly different from other locations. The analysis of Weighted UniFrac distances showed that milk vetch changed soil microbial communities compared with winter fallow. Proteobacteria and Chloroflexi predominated in these paddy soils; however, the application of green manures increased the relative abundance of Actinobacteria. There was evidence showing that the functional microbes which play important roles in the cycling of soil carbon, nitrogen (N), and sulfur (S) changed after several years of milk vetch utilization (linear discriminant analysis score > 2). The abundance of methane-oxidizing bacteria and S-reducing bacteria increased, and microbes involved in N fixation, nitrification, and denitrification also increased in some provinces. We concluded that the application of milk vetch changed the bacterial community structure and affected the functional groups related to nutrient transformation in soils at a regional scale.     

长期施肥下黑土有机肥替代率变化特征

探索长期施肥下黑土有机肥替代率与土壤肥力提升的关系,可为农田土壤培肥和有机替代提供理论依据。对吉林省公主岭黑土32年的长期肥力试验定位观测数据进行系统分析,基于作物氮素吸收量和土壤氮素供需方程探讨高产条件下施用不同量有机肥的黑土有机肥替代率的变化特征。研究表明,作物产量随着有机肥施用年限增加逐渐提高,32年的持续施肥,施用有机肥的作物产量趋同甚至高于NPK化肥处理的作物产量。基于作物氮素吸收量,高产条件下有机肥替代率与施肥年限呈极显著线性正相关(P0.01),高量有机肥处理(M2)的有机肥替代率高于常量有机肥处理(M1);且有机肥施用29年后,高量有机肥处理(M2)的有机肥替代率达到100%,并保持稳定不变。基于土壤氮素供需方程估测的常量和高量有机肥处理(M1和M2)的有机肥替代率与基于作物氮素吸收量得到的有机肥替代率相关系数(R2)达到0.78和0.84(P0.01),相对均方根误差(RMSE)均小于15%(分别为10.4%和14.6%),表明土壤氮素供需方程可以较好地估测土壤有机肥替代率。基于作物氮素吸收量和土壤氮素供需方程能够准确反映长期有机培肥下黑土有机肥替代率的变化规律。本研究结果表明,基于作物氮素吸收量和土壤氮素供需方程两种方法验证,高产条件下有机肥替代率是土壤肥力的函数;随着有机肥施肥年限的增加,土壤肥力提升,有机肥替代率逐渐增加。     

Spatial patterns of microbial denitrification genes change in response to poultry litter placement and cover crop species in an agricultural soil

Subsurface-banding manure and winter cover cropping are farming techniques designed to reduce N loss. Little is known, however, about the effects of these management tools on denitrifying microbial communities and the greenhouse gases they produce. Abundances of bacterial (16S), fungal (ITS), and denitrification genes (nirK, nirS, nosZ-I, and nosZ-II) were measured in soil samples collected from a field experiment testing the combination of cereal rye and hairy vetch cover cropping with either surface-broadcasted or subsurface-banded poultry litter. The spatial distribution of genes was mapped to identify potential denitrifier hotspots. Spatial distribution maps showed increased 16S rRNA genes around the manure band, but no denitrifier hotspots. Soil depth and nitrate concentration were the strongest drivers of gene abundance, but bacterial gene abundance also differed by gene, soil characteristics, and management methods. Gene copy number of nirK was higher under cereal rye than hairy vetch and positively associated with soil moisture, while nirS gene copies did not differ between cover crop species. The nirS gene copies increased when manure was surface broadcasted compared to subsurface banded and was positively associated with pH. Soil moisture and pH were positively correlated to nosZ-II but not to nosZ-I gene copy numbers. We observed stronger correlations between nosZ-I and nirS, and nosZ-II and nirK gene copies compared to the reverse pairings. Agricultural management practices differentially affect spatial distributions of genes coding for denitrification enzymes, leading to changes in the composition of the denitrifying community.     

Specific assemblages of major capsid genes (g23) of T4-type bacteriophages isolated from upland black soils in Northeast China

We surveyed the major capsid genes (g23) of T4-type bacteriophages using the primers MZIA1bis and MZIA6 and DNA extracted from seven upland black soils in Northeast China. In total, 99 different g23 clones were obtained. Approximately half of the clones fell into paddy groups, whereas the rest belonged to one of several groups containing only clones from upland black soils or remained ungrouped, suggesting that the T4-type phage communities in the upland black soil were relatively similar to those in paddy field soils but that specific communities exclusively inhabit the upland black soil. UniFrac analysis of all of the g23 clones obtained from various environments indicated that the T4-type phage communities varied among marine, lake, paddy field soil and upland soil environments and that the T4-type phage communities in upland black soils varied by sampling location.     

Soil microbial biomass,crop yields,and bacterial community structure as affected by long-term fertilizer treatments under wheat-rice cropping

Soil microbial biomass carbon (SMBC) and nitrogen (SMBN), soil microbial community structure, and crop yields were studied in a long-term (1982–2004) fertilization experiment carried out in Suining, Sichuan province of PR China. Eight treatments included three chemical fertilizer (CF) treatments (N, NP, NPK), three CF + farmyard manure (M) treatments (NM, NPM, NPKM), M alone and no fertilizer (CK) as control. The results showed that the soil microbial biomass was higher in soil treated with CFM than in soil treated with CF alone, and that NPKM gave the highest rice and wheat yields. The SMBC and SMBN were higher after rice than those after wheat cropping. SMBC correlated closely with soil organic matter. Average yields of wheat and rice for 22 years were higher and more stable in the fertilized plots than in control plots. Bacterial community structure was analyzed by PCR-DGGE targeting eubacterial 16S rRNA genes. A higher diversity of the soil bacterial community was found in soil amended with CFM than in other fertilizer treatments. Some specific band emerged in the soil amended with M. The highest diversity of bacterial communities was found in the NPKM treated soil. The bacterial community structures differed in rice and wheat plots. Sequencing of PCR products separated in DGGE showed that some of the common and dominant bands were closely related to Aquicella lusitana and to Acidobacteria. This study demonstrated that mixed application of N, P, and K with additional M amendment increased soil microbial biomass, diversified the bacterial communities and maintained the crop production in the Calcareous Purplish Paddy soil.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

Responses of soil microbial community to nitrogen fertilizer and precipitation regimes in a semi-arid steppe

Purpose

Changes of nitrogen (N) cycle caused by N fertilization and precipitation regimes have affected the key ecosystem structure and functions in temperate steppe, which may modify the structure of soil microbial communities involved in N transformation. This paper was designated to examine the response of soil ammonia oxidizers and denitrifiers to the N fertilization and precipitation regimes in a semi-arid steppe where N and water contents are major limiting factors of the grassland productivity.

Materials and methods

This study was based on a long-term N fertilization and precipitation regimes experiment in Inner Mongolia (116° 17′ 20″ E, 42° 2′ 29″ N). The treatments including CK (control), R (reduced precipitation), W (30% increase in precipitation), N (10 g N m^?2 y^?1), RN (reduced precipitation and 10 g N m^?2 y^?1), and WN (30% increase in precipitation and 10 g N m^?2 y^?1). Soil basic chemical properties and microbial activities were analyzed. Molecular methods were applied to determine the abundance, structure and diversity of ammonia oxidizers and denitrifiers. Statistical analysis detected the main and interactive effect of treatments on soil microbial communities and revealed the relationship between soil microbial community structures and environmental factors.

Results and discussion

N fertilization significantly increased ammonia-oxidizing bacteria (AOB) abundance. Ammonia-oxidizing archaea (AOA) community structure was markedly changed in N fertilizer treatment and strongly affected by soil pH, while soil nitrate and water content correlated with AOB community structure. Soil nitrate was the key factor influencing nirK gene community structure, while soil pH and water content explained much of the variations of nosZ gene community. AOB-amoA and nosZ gene community diversities were influenced by precipitation regimes and interaction of N fertilization and precipitation regimes, respectively.

Conclusions

N fertilization and precipitation regimes had significant influences on the changes of soil properties and microbial functional communities. Soil nitrification was mainly driven by AOB in the semi-arid grassland. Changes of substrate content and soil pH were the key factors in shifting functional microbial communities. The non-synergistic effects of N fertilization and precipitation regimes on the microbial functional groups indicated that the negative effect of lower pH induced by N fertilization would be alleviated by precipitation regimes, which should be well considered in grassland restoration.

     

Soil Total Nitrogen and Natural 15Nitrogen in Response to Long-Term Fertilizer Management of a Maize–Wheat Cropping System in Northern China

The Fengqiu long-term field experiment was established to examine effects of organic manure and mineral fertilizers on soil total nitrogen (N) and natural ¹⁵N abundance. Fertilizer regimes include organic manure (OM), one-half N from organic manure plus one-half N from mineral N fertilizer (1/2OMN), mineral fertilizers [N–phosphorus (P)–potassium (K), NP, NK, PK], and a control. Organic manure (OM and 1/2OMN) significantly increased soil total N and δ¹⁵N, which was expected as a great amount of the N applied remained in soils. Mineral NPK fertilizer and mineral NP fertilizer significantly increased total N and slightly increaed δ¹⁵N. Phosphorus-deficient fertilization (NK) and N-deficient fertilization (PK) had no effect on soil total N. Significantly greater δ¹⁵N was observed in the NK treatment as compared to the control, suggesting that considerable N was lost by ammonia (NH₃) voltalization and denitrification in this P-deficiency fertilization regime.     

Season and management related changes in the diversity of nitrifying and denitrifying bacteria over winter and spring

This study assessed the effects that season and tillage practices have on the diversity of nitrous oxide producing bacteria (nitrifiers and denitrifiers) and to relate this to measured N₂O fluxes at our field site. Large-scale field plots (1.5 ha) were established in Elora, Ontario in 2000, and managed using conventional tillage (CT) or no-tillage (NT). Each field plot was instrumented with micrometeorological equipment to determine N₂O fluxes on a field scale. Soil samples were taken at four time points between the fall of 2005 and the spring of 2006. The diversity of the nitrifier and denitrifier communities was assessed by PCR–denaturing gradient gel electrophoresis (DGGE) using primer pairs targeting the amoA, nirS and nirK gene. Seasonal variation (a combination of soil temperature, available soil moisture, nutrient levels and other potential factors) had the largest influence on the diversity of nitrifier and denitrifier populations; while tillage practice also influenced the diversity of the microbial community at certain time periods. Tillage significantly affected all communities in March and affected denitrifiers on all other dates except for the nirS community in February. Further statistical analysis revealed that diversity of the nitrifying and denitrifying populations was the lowest in February, in frozen soils, and rapidly increased in March, corresponding with spring thaw N₂O emissions. Long-term soil nutrient, temperature and N₂O data taken at this site added additional information on the dynamics of the nitrogen cycle.     

长期不同施肥处理下有机质含量变化对土壤中多环芳烃降解的影响——以太湖地区黄泥土长期试验为例

采集太湖地区一个水稻土长期肥料试验定位监测田【化肥与秸秆配施（CFS）、化肥与猪粪配施（CFM）、单施化肥（CF）和不施肥（NF）】表层（0．15cm）土壤样品,用高效液相色谱测定了原土中16种多环芳烃（PAHs）含量和培养下外加芘的含量变化。结果显示,无论是PAHs的土壤本底含量还是培养下外加芘残留量,在不同施肥处理间均呈现显著差异;相对于单施化肥（CF）,化肥与有机肥配施下PAHs土壤降解能力显著提高。统计分析表明,增强的有机污染物降解能力与土壤有机质含量变化及其带来的微生物活性的变化有关。因此,长期实行有机无机配施有利于增强土壤的环境功能,促进生态系统健康。