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。     

稻虾共作对稻田土壤nirK反硝化微生物群落结构和多样性的影响

稻虾共作是水稻种植与克氏螯虾共作形成的互利共生的稻田种养复合生态模式。目前对稻虾共作模式稻田反硝化微生物多样性和群落结构的影响尚不清楚。本研究以江汉平原常规中稻模式(MR)为对照,设置连续3年(2014—2016年)稻虾共作模式(CR)为处理,通过特异引物提取中稻抽穗期稻田土壤nirK基因,采用Illumina Miseq高通量测序技术,探讨稻虾共作模式对稻田土壤nirK反硝化微生物多样性和群落结构的影响。结果表明:稻虾共作模式显著提升水稻抽穗期稻田土壤中硝态氮、全氮及全碳的含量,对土壤碳氮比、碱解氮和铵态氮含量没有显著影响。稻虾共作模式显著增加稻田土壤nirK基因微生物的丰富度指数,但对nirK基因微生物的多样性指数影响不显著。稻虾共作模式改变了nirK基因微生物在目、科、属、种水平的群落组成,较常规中稻模式,稻虾共作模式在各分类水平组成类群均减少;稻虾共作模式较常规中稻模式改变了目的种类,对共有目相对丰度没有显著性改变。RDA分析表明稻虾共作模式对土壤nirK基因菌群的群落结构有一定的改变,但稻虾共作模式与常规中稻模式在群落结构上仍保留着一定的相似性。硝态氮含量是影响nirK反硝化细菌群落结构的主效因子。可见,稻虾共作模式对微生物多样性指数没有显著影响,但显著增加了微生物丰富度指数,改变了稻田土壤nirK反硝化微生物在目、科、属、种的群落结构。     

Overwintering management on upland pasture causes shifts in an abundance of denitrifying microbial communities, their activity and N2O-reducing ability

Pasture soils used for cattle overwintering may represent significant sources of N₂O emissions from soils. Therefore, the long-term effect of cattle overwintering on the abundance and activity of a denitrifying community was explored. The study was performed at a cattle overwintering area in South Bohemia (Czech Republic), where three sites differing in the degree of animal impact were selected: severely impacted (SI) and moderately impacted (MI), as well as a control site with no impact (NI). N₂O flux measurement and soil sampling were performed in spring and fall of 2005. The activity was measured in terms of potential denitrification activity. Bacterial nirK, nirS and nosZ genes were used as functional markers of the denitrifying communities; abundance was analyzed using a real-time PCR assay. Surprisingly, in situ N₂O emissions were the highest in spring at MI and significantly differed from those at SI and NI, while in autumn, rates of emissions generally decreased. In contrast potential denitrification rates were highest at SI, followed by MI, and the lowest at NI. An overall significant shift in N₂O/N₂ molar ratio was shown in cattle impacted sites. The highest abundance of all genes measured at both sampling times was found at site SI, whereas at site MI increased numbers were observed only in spring. Our results indicate a strong influence of cattle on the abundance as well as the activity of microbes involved in denitrification.     

The effects of silicon fertilizer on denitrification potential and associated genes abundance in paddy soil

This study evaluated the effect of silicate fertilizer on denitrification and associated gene abundance in a paddy soil. A consecutive trial from 2013 to 2015 was conducted including the following treatments: control (CK), mineral fertilizer (NPK), NPK plus sodium metasilicate (NPK + MSF), and NPK plus slag-based silicate fertilizer (NPK + SSF). Real-time quantitative PCR (qPCR) was used to analyze the abundances of nirS, nirK, and nosZ genes. Potential N₂O emissions and ammonium and nitrate concentrations were related to the nirS and nirK gene abundance. Compared with the NPK treatments, the addition of a Si fertilizer decreased N₂O emission rates and denitrification potential by 32.4–66.6 and 22.0–59.2%, respectively, which were probably related to increased rice productivity, soil Fe availability, and soil N depletion. The abundances of nirS and nirK genes were decreased by 17.7–35.8% and 21.1–43.5% with addition of silicate fertilizers, respectively. Rates of total N₂O and N₂O from denitrification (DeN₂O) emission were positively correlated with the nirS and nirK gene abundance. Nitrate, exchangeable NH₄ ⁺, and Fe concentrations were the main factors regulating the nirS and nirK gene abundance. Silicate fertilization during rice growth may serve as an effective approach to decreasing N₂O emissions.     

不同生物硝化抑制剂对红壤性水稻土N2O排放的影响及其机制

为揭示不同生物硝化抑制剂(BNIs)对红壤性水稻土N₂O排放的影响差异及作用机制,通过21 d的土柱淹水培养试验,比较了三种BNIs 1,9-癸二醇(1,9-D)、亚麻酸(LN)和3-(4-羟基苯基)丙酸甲酯(MHPP)与化学合成硝化抑制剂双氰胺(DCD)对土壤N₂O排放及相关硝化、反硝化功能基因的影响。结果表明:不同BNIs(1,9-D、LN、MHPP)可以显著平均降低土壤N₂O日排放峰值40.1%;1,9-D和MHPP可分别抑制N₂O排放总量44.5%和43.9%,而DCD和LN对N₂O排放总量没有显著影响。1,9-D和MHPP对AOA(氨氧化古菌)、AOB(氨氧化细菌)硝化菌和nirS、nirK型反硝化菌的调控均有所不同,1,9-D可以同时抑制AOA、AOB和nirS微生物的生长;MHPP仅可以抑制AOA的生长;其中,AOA-amoA和nirS基因丰度与土壤N₂O的排放呈显著正相关关系。同时,1,9-D和MHPP均增加了nosZ基因丰度及其与AOA-...     

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.     

Incongruous variation of denitrifying bacterial communities as soil N level rises in Canadian canola fields

Soil N fertilization stimulates the activity of the soil bacterial species specialized in performing the different steps of the denitrification processes. Different responses of these bacterial denitrifiers to soil N management could alter the efficiency of reduction of the greenhouse gas N₂O into N₂ gas in cultivated fields. We used next generation sequencing to show how raising the soil N fertility of Canadian canola fields differentially modifies the diversity and composition of nitrite reductase (nirK and nirS) and nitrous oxide reductase (nosZ) gene-carrying denitrifying bacterial communities, based on a randomized complete blocks field experiment. Raising soil N levels increased up to 60% the ratio of the nirK to nirS genes, the two nitrite reductase coding genes, in the Brown soil and up to 300% in the Black soil, but this ratio was unaffected in the Dark Brown soil. Raising soil N levels also increased the diversity of the bacteria carrying the nitrite reductase gene nirK (Simpson index, P = 0.0417 and Shannon index, 0.0181), and changed the proportions of the six dominant phyla hosting nirK, nirS, and nosZ gene-carrying bacteria. The level of soil copper (Cu) and the abundance of nirK gene, which codes for a Cu-dependent nitrite reductase, were positively related in the Brown (P = 0.0060, R² = 0.48) and Dark Brown (0.0199, R² = 0.59) soils, but not in the Black soil. The level of total diversity of the denitrifying communities tended to remain constant as N fertilization induced shifts in the composition of these denitrifying communities. Together, our results indicate that higher N fertilizer rate increases the potential risk of nitrous oxide (N₂O) emission from canola fields by promoting the proliferation of the mostly adaptive N₂O-producing over the less adaptive N₂O-reducing bacterial community.     

Comparison of g23 gene sequence diversity between Novosphingobium and Sphingomonas phages and phage communities in the floodwater of a Japanese paddy field

Our previous study indicated that the diversity of the major capsid gene (g23) of T4-type bacteriophages (phages) of Novosphingobium and Sphingomonas strains isolated from the floodwater of a Japanese paddy field is comparable to those of the clones obtained from other Japanese paddy fields. For more strict comparison of the diversity, this study examined g23 sequences between Novosphingobium and Sphingomonas phages and phage communities in the identical floodwater of a Japanese paddy field. The clones were obtained by applying g23-specific primers to DNA extracted from the floodwaters. Many 23 clones in the floodwater were grouped into the same clusters of Paddy Groups I-VI with g23 genes of Novosphingobium/Sphingomonas phages with some clones belonging to an additional cluster. In addition, the remaining clones belonged to the clusters of marine clones and T4-type enterophages. These findings indicate that the g23 genes in the floodwater are more diversified than those of Novosphingobium/Sphingomonas phages including g23 genes closely related to the genes of enterophages and marine origins.     

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.     

氮肥水平对稻田细菌群落及N2O排放的影响

作为土壤氮素转化的驱动者,微生物群落结构关系着稻田氮素利用及温室气体N_2O排放等问题。本研究分别基于高通量测序和荧光定量PCR技术,分析了不同氮肥水平[CK(不施氮)、N(施N 180 kg·hm-2)、2/3N(施N 120 kg·hm-2)、1/3N(施N 60 kg·hm-2)]下稻田细菌群落及硝化反硝化关键微生物功能基因丰度的变化。结果显示:氮肥水平提高增加了稻田细菌物种丰富度Chao1指数和群落多样性Shannon指数,改变了细菌群落组成,其中与硝化作用相关的硝化螺菌门Nitrospirae和嗜酸的醋杆菌门Acidobacteria的相对丰度随氮肥水平提高而增加,但甲烷氧化菌Methylosinus的相对丰度随氮肥水平提高而降低。氮肥水平对稻田硝化作用关键微生物氨氧化细菌amo A基因丰度的影响较大,0~5 cm和10~20 cm深度土层中的amo A基因丰度均随氮肥用量增加而提高;反硝化作用关键微生物功能基因nir S、qno B和nos Z的丰度在不施肥处理(CK)中显著低于施肥处理(1/3N、2/3N和N)(P0.05),但1/3N、2/3N和N处理的稻田nir S基因丰度没有明显差异;0~5 cm土层中qno B和nos Z基因丰度存在随氮肥水平提高而增加的趋势,10~20 cm土层中nos Z基因丰度在2/3N和N处理下显著高于1/3N处理(P0.05)。N处理的稻田N_2O排放通量显著高于2/3N及1/3N处理(P0.05),后者又显著高于CK处理(P0.05)。相关分析结果表明稻田N_2O排放通量与0~5 cm土层中硝化螺菌门Nitrospirae相对丰度及10~20 cm土层中amo A基因丰度存在显著相关性(P0.05,n=10)。综上所述,氮肥水平提高增加了稻田细菌群落多样性,促进了稻田N_2O排放,且本研究稻田中硝化作用微生物群落及丰度变化与稻田N_2O排放的关系更为密切。     

Cr3+污染对稻田土壤的反硝化细菌种群数量及其活性影响的研究

在实验室条件下,对黄松稻田土壤、紫色稻田土壤和红壤稻田土的反硝化细菌(DenitrifyingBacteria,DNB)种群数量及其反硝化活性,以及Cr2(SO4)36H2O处理4周后Cr3+污染对DNB种群数量及其反硝化活性影响进行了研究。结果表明,紫色稻田土壤、黄松稻田土壤和红壤稻田土的DNB种群数量每克干土分别为(59.04～157.59)104cfu、(42.89～108.97)104cfu和(32.14～75.30)104cfu。在每千克干土中加入200mgCr3+时,对稻田土的DNB种群数量及其活性有明显的抑制作用,但稻田土壤还能通过自身的能力,在培养3周后完全恢复稻田土壤的DNB种群数量及其活性。随着Cr3+加入量的增加,Cr3+对DNB的毒性越来越大,稻田土壤通过自身来恢复DNB种群数量及其活性越来越困难,但可通过外界力量(如调节土壤pH)逐渐恢复稻田土壤的生物活性。     

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.     

Survey of major capsid genes (g23) of T4-type bacteriophages in Japanese paddy field soils

Many studies have shown the ecological importance of viruses as the greatest genomic reservoirs on the planet. As bacteriophages (phages) comprise the majority of viruses in the environment, we surveyed the capsid genes (g23) of T4-type phages, Myoviridae, from DNA extracts of three paddy field soils located in northern, central and southern Japan using the degenerate primers MZIA1bis and MZIA6. Denaturing gradient gel electrophoresis (DGGE) was performed to separate PCR-amplified g23 products, and 56 DGGE bands were identified as g23 fragments. Only nine clones were grouped into T-evens, PseudoT-evens and ExoT-evens, and most of the other clones were classified into Paddy Groups I-VI. No significantly different distribution of g23 clones was observed among the paddy fields at the group level, indicating that phage communities estimated from the g23 composition were common on the nationwide level. Comparison of g23 sequences showed that g23 genes in paddy fields were different from those in marine environments, and more divergence of g23 genes was estimated in the paddy fields compared to the marine environment. Two novel g23 clones with very short amino acid residues were detected, suggesting the existence of uncharacterized, novel groups of g23 genes in paddy field soils.     

Effect of P stoichiometry on the abundance of nitrogen-cycle genes in phosphorus-limited paddy soil

Previous studies have shown that phosphorus addition to P-limited soils increases gaseous N loss. A possible explanation for this phenomenon is element stoichiometry (specifically of C:N:P) modifying linked nutrient cycling, leading to enhanced nitrification and denitrification. In this study, we investigated how P stoichiometry influenced the dynamics of soil N-cycle functional genes. Rice seedlings were planted in P-poor soils and incubated with or without P application. Quantitative PCR was then applied to analyze the abundance of ammonia-oxidizing (amoA) and denitrifying (narG nirK, nirS, nosZ) genes in soil. P addition reduced bacterial amoA abundance but increased denitrifying gene abundance. We suggest this outcome is due to P-induced shifts in soil C:P and N:P ratios that limited ammonia oxidization while enhancing P availability for denitrification. Under P application, the rhizosphere effect raised ammonia-oxidizing bacterial abundance (amoA gene) and reduced nirK, nirS, and nosZ in rhizosphere soils. The change likely occurred through greater C input and O₂ release from roots, thus altering C availability and redox conditions for microbes. Our results show that P application enhances gaseous N loss potential in paddy fields mainly through stimulating denitrifier growth. We conclude that nutrient availability and elemental stoichiometry are important in regulating microbial gene responses, thereby influencing key ecosystem processes such as denitrification.

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Shifts in size,genetic structure and activity of the soil denitrifier community by nematode grazing

Bacterial-feeding nematodes represent an important driver of the soil microbial activity and diversity. This study aimed at characterizing the impact of nematode grazing on a model functional bacterial guild involved in N-cycling, the denitrifiers. Bacterial-feeding nematodes (Cephalobus pseudoparvus) were inoculated into soil microcosms whose indigenous nematofauna had previously been removed. The size, genetic structure and activity of the soil denitrifier community were characterized 15 and 45 days after nematodes inoculation using quantitative PCR of the nirK, nirS and nosZ denitrification genes, fingerprinting of the nirK and nirS genes and denitrification enzyme activity measurements, respectively. A significant impact of C. pseudoparvus was observed on genetic structure of the nirK community, mainly due to shifts in the relative abundance of the dominant populations, but not on the nirS community. The grazing pressure also tended to decrease the density of all denitrification genes as well as that of 16S rRNA genes. Despite being non-significant, the extent of this decline in gene copy numbers ranged between 60 and 80% of the control microcosm genes densities. Finally, compared to non-inoculated microcosms, denitrification activity significantly decreased by 8% in response to the nematodes inoculation. The herewith data showed that predation by a single species of bacterial-feeding nematode can affect the soil denitrifier community.     

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.     

Abundance of ammonia oxidizing microbes and denitrifiers in different soil horizons of an agricultural soil in relation to the cultivated crops

The role of subsoils and their microbial communities for the nutrient supply for plants is to a large extent unknown, especially in comparison to well investigated topsoil layers. Therefore, in this study, the influence of three different plant species with different rooting systems and different N uptake strategies on ammonium and nitrate levels and microbial communities involved in ammonia oxidation and denitrification was investigated in different soil horizons. Overall, our results show a higher genetic potential for both processes in topsoils than in subsoils independent of the present plant. Although we found accumulation of N in top and subsoils in plots with legumes, we could not observe an impact of the higher nitrate content on the genetic potential of denitrification and ammonia oxidation. However, differences in the ratios of ammonia oxidizing archaea to bacteria and also between denitrifying bacteria harboring genes for copper- (nirK) or cytochrome- (nirS) dependent nitrite reductase in top and subsoil samples reveal different ecophysiologies of microbes involved in N turnover in top and subsoil habitats.     

Survey of major capsid genes (g23) of T4-type bacteriophages in rice fields in Northeast China

We surveyed the capsid genes (g23) of T4-type bacteriophages in DNA extracted from fifteen rice field soils in Northeast China using primers MZIA1bis and MZIA6. Denaturing gradient gel electrophoresis (DGGE) was performed to separate PCR-amplified g23 products. In total, 53 DGGE bands were identified as g23 clones, nine of which belonged to a novel, Northeast China-specific group. In addition, four and six clones formed two novel groups with previously ungrouped clones obtained from Japanese rice fields. The majority of the remaining clones fell into Paddy Groups I and V, none of the clones belonged to Paddy Groups II, III, IV, and VI, indicating that phylogenetic distribution of g23 genes in rice fields in Northeast China was different from that in Japanese rice fields.