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.     

Microbial composition and diversity of an upland red soil under long-term fertilization treatments as revealed by culture-dependent and culture-independent approaches

Background, aim, and scope  Fertilization is an important agricultural practice for increasing crop yields. In order to maintain the soil sustainability, it is important to monitor the effects of fertilizer applications on the shifts of soil microorganisms, which control the cycling of many nutrients in the soil. Here, culture-dependent and culture-independent approaches were used to analyze the soil bacterial and fungal quantities and community structure under seven fertilization treatments, including Control, Manure, Return (harvested peanut straw was returned to the plot), and chemical fertilizers of NPK, NP, NK, and PK. The objective of this study was to examine the effects on soil microbial composition and diversity of long-term organic and chemical fertilizer regimes in a Chinese upland red soil. Materials and methods  Soil samples were collected from a long-term experiment station at Yingtan (28°15′N, 116°55′E), Jiangxi Province of China. The soil samples (0–20 cm) from four individual plots per treatment were collected. The total numbers of culturable bacteria and fungi were determined as colony forming units (CFUs) and selected colonies were identified on agar plates by dilution plate methods. Moreover, soil DNAs were extracted and bacterial 16S rRNA genes and fungal 18S rRNA genes were polymerase chain reaction amplified, and then analyzed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. Results  The organic fertilizers, especially manure, induced the least culturable bacterial CFUs, but the highest bacterial diversity ascertained by DGGE banding patterns. Chemical fertilizers, on the other hand, had less effect on the bacterial composition and diversity, with the NK treatment having the lowest CFUs. For the fungal community, the manure treatment had the largest CFUs but much fewer DGGE bands, also with the NK treatment having the lowest CFUs. The conventional identification of representative bacterial and fungal genera showed that long-term fertilization treatments resulted in differences in soil microbial composition and diversity. In particular, 42.4% of the identified bacterial isolates were classified into members of Arthrobacter. For fungi, Aspergillus, Penicillium, and Mucor were the most prevalent three genera, which accounted for 46.6% of the total identified fungi. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Discussion  It was evident that more representative fungal genera appeared in organic treatments than other treatments, indicating that culturable fungi were more sensitive to organic than to chemical fertilizers. A very notable finding was that fungal CFUs appeared maximal in organic manure treatments. This was quite different from the bacterial CFUs in the manure, indicating that bacteria and fungi responded differently to the fertilization. Similar to bacteria, the minimum fungal CFUs were also observed in the NK treatment. This result provided evidence that phosphorus could be a key factor for microorganisms in the soil. Thus, despite the fact that culture-dependent techniques are not ideal for studies of the composition of natural microbial communities when used alone, they provide one of the more useful means of understanding the growth habit, development, and potential function of microorganisms from soil habitats. A combination of culture-dependent and culture-independent approaches is likely to reveal more complete information regarding the composition of soil microbial communities. Conclusions  Long-term fertilization had great effects on the soil bacterial and fungal communities. Organic fertilizer applications induced the least culturable bacterial CFUs but the highest bacterial diversity, while chemical fertilizer applications had less impact on soil bacterial community. The largest fungal CFUs were obtained, but much lower diversity was detected in the manure treatment. The lowest bacterial and also fungal CFUs were observed in the NK treatment. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Phosphorus fertilizer could be considered as a key factor to control the microbial CFUs and diversity in this Chinese upland red soil. Recommendations and perspectives  Soil fungi seem to be a more sensitive indicator of soil fertility than soil bacteria. Since the major limitation of molecular methods in soil microbial studies is the lack of discrimination between the living and dead, or active and dormant microorganisms, both culture-dependent and culture-independent methods should be used to appropriately characterize soil microbial diversity.     

Effects of vegetational type and soil depth on soil microbial communities on the Loess Plateau of China

Soil microbial communities are very sensitive to changes in land use and are often used as indicators of soil fertility. We evaluated the microbial communities in the soils of four types of vegetation (cropland (CP), natural grassland (NG), broadleaf forest (BF) and coniferous forest (CF)) at depths of 0–10 and 10–20 cm on the Loess Plateau in China using phospholipid fatty acid (PLFA) profiling and denaturing gradient gel electrophoresis (DGGE) of DNA amplicons from polymerase chain reactions. The soil microbial communities were affected more by vegetation type than by soil depth. Total organic carbon, total nitrogen, soil-water content, pH, bulk density (BD) and C:N ratio were all significantly associated with the composition of the communities. Total PLFA, bacterial PLFA and fungal PLFA were significantly higher in the BF than the CP. The DGGE analyses showed that NG had the most diverse bacterial and fungal communities. These results confirmed the significant effect of vegetation type on soil microbial communities. BFs and natural grass were better than the CFs for the restoration of vegetation on the Loess Plateau.     

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.     

等碳投入的有机肥和生物炭对红壤微生物多样性和土壤呼吸的影响

施用有机肥是快速培育瘠薄土壤的一个重要措施。针对中亚热带第四纪红黏土发育的红壤旱地,建立了玉米和花生单作系统等碳量投入有机肥和生物炭的田间试验,利用聚合酶链式反应—变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)方法研究了土壤细菌和真菌群落组成和多样性的变化,分析了土壤呼吸速率(CO2通量)的变化及其与微生物多样性的关系。两年的试验表明,不同施肥方式导致微生物群落结构显著分异,施用有机肥和生物炭显著增加了细菌多样性,但施肥第二年真菌多样性有下降趋势。秸秆和猪粪配施显著增加了土壤呼吸速率,土壤呼吸速率与细菌和真菌多样性呈显著正相关,细菌多样性对土壤呼吸的影响(相对贡献率为71%)显著高于真菌(29%)。土壤磷素(全磷和速效磷)含量的变化是驱动红壤微生物多样性变化的主导因素,其对细菌和真菌多样性的相对贡献率分别为44.8%和47.4%。因此,合理配施秸秆和猪粪可以快速提高瘠薄红壤的生物功能。     

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.     

农牧交错带不同施肥措施土壤剖面生物量动态变化

通过田间试验,研究了农牧交错带不同施肥对土壤剖面微生物量的影响。结果表明:(1)有机肥及有机无机肥配施(MNP)土壤剖面的各层次微生物量碳、氮及总有机碳、全氮含量均高于有机肥(M)、无机肥配施(NP)和不施肥(CK)处理。(2)土壤有机碳和全氮剖面的变化趋势4个处理的基本一致,随土壤剖面的深度增加而下降。而土壤微生物量碳、氮含量为10-20cm0-10cm20-40cm。(3)微生物商作为长期培肥过程中土壤质量演变的评价指标有待商榷。(4)土壤微生物生物量碳、氮与土壤有机碳、氮之间存在线性相关关系,特别是土壤微生物生物量碳、氮之间关系显著(p0.05),表明土壤微生物量可以用来指示土壤肥力的水平。研究结果能够很好的反映农牧交错带不同施肥措施下土壤肥力的变化情况。     

Community-level responses of metabolically-active soil microorganisms to the quantity and quality of substrate inputs

The community fingerprints of both the prevalent and the metabolically active microbial community were related to a quantitative estimation of microbial biomass in an arable soil, revealed by substrate-induced-respiration (SIR). Two concentrations of glucose or l-asparagine, representing those used in the SIR measurement or equivalent to those released in root exudates, were studied. Respiration rates and changes in community structure fingerprints were followed for 48 h. Bacterial and fungal community fingerprints were obtained using both reverse transcribed 16S and 18S ribosomal RNA (rRNA) regions and the corresponding rDNA as a template in PCR. Samples were then analysed by denaturing gradient gel electrophoresis (DGGE). Low concentrations of substrate amendments resulted in minor changes in rRNA or rDNA-based bacterial and fungal banding patterns during the whole 48 h incubation. High concentrations of substrates, especially l-asparagine, increased respiration rates and induced significant changes in both 16S rRNA and rDNA-community fingerprints. The prominent rRNA and rDNA bacterial community sequence types were common to all treatments, but in general the bacterial rDNA fingerprints had fewer bands than the corresponding rRNA profiles that assess the active fraction of the community. In contrast, there was little difference between fungal 18S rRNA and rDNA patterns. The number of fungal ribosomal sequence types in DGGE fingerprints was lower than the number of bacterial types. This study indicated that there was a rapid respiration response by the whole microbial community during SIR estimates in soil, but that community structure did not change during the conventional incubation period. In an extended (8-48 h) incubation with high amounts of l-asparagine increased respiration was associated with growth of the microbial community.     

不同培肥模式对茶园土壤微生物活性和群落结构的影响

以闽东地区红黄壤茶园定位实验地为对象,通过测定6种不同施肥处理土壤微生物学特性,研究不同培肥对土壤微生物特性和生物化学过程的影响,阐明各指标间的相互关系.结果表明,除了单施无机肥处理外,半量化肥+半量有机肥、全量有机肥、全量化肥+豆科绿肥以及半量化肥+半量有机肥+豆科绿肥等的培肥方式均不同程度提高了土壤有机质,可培养微生物数量,微生物量碳、氮含量及土壤酶活性,尤以半量无机肥+半量有机肥+豆科牧草的培肥模式增幅更为明显,而单施无机肥不利于微生物的生长、酶活性的提高和维持生态系统的稳定性.微生物群落磷脂脂肪酸(PLFAs)标记主成分分析显示,各种不同施肥方式使微生物群落结构发生改变.相关分析表明,微生物量与可培养微生物数量、微生物磷脂脂肪酸含量之间的相关性明显高于微生物量与各种酶活性之间的相关性,说明微生物数量大小对微生物群落结构的影响大于对酶活性功能的影响.研究也表明土壤各微生物指标能从不同方面反映土壤肥力水平,所以采用各种不同的方法能更客观地评价闽东地区茶园红黄壤质量的优劣.     

A DGGE analysis shows that crop rotation systems influence the bacterial and fungal communities in soils

A better understanding of the relationships among different cropping systems, their effects on soil microbial ecology, and their effects on crop health and productivity is necessary for the development of more efficient, sustainable crop production systems. We used denaturing gradient gel electrophoresis (DGGE) to determine the impacts of crop rotations and crop types on bacterial and fungal communities in the soil. The communities of bacterial 16S rRNA genes and fungal 18S rRNA genes were analyzed in experimental field plots that were kept under 4 different crop rotation systems from 1999 to 2008 (continuous cabbage (Brassica oleracea var. capitata L.), cabbage–lettuce (Lactuca sativa L.) rotation, cabbage–radish (Raphanus sativus L. var. longipinnatus L.H. Bailey) rotation, and a 3-year crop rotation). A principal component analysis (PCA) and a canonical correspondence analysis (CCA) revealed that both the bacterial and fungal communities in bulk soils were influenced by the crop rotation systems. However, the primary factors influencing each community differed: bacterial communities were most affected by soil properties (especially carbon content), while fungal communities were influenced most strongly by rotation times. To elucidate factors that may cause differences in crop rhizosphere microbial communities, the microbial communities in the harvested cabbage rhizospheres were also analyzed. The results suggest that the fungal communities in bulk soil are related to the rhizosphere fungal communities. Our present study indicates that the microbial communities in bulk and rhizosphere soils could be managed by crop rotation systems.     

Long-term effect of plastic film mulching and fertilization on bacterial communities in a brown soil revealed by high through-put sequencing

The objective of this study was to access the effect of long-term fertilization and film mulching application on soil properties and bacterial community structure. We used 16S rRNA gene to investigate soil bacterial community composition by high through-put sequencing. The results demonstrated that predominant groups in the bacterial community were: Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria, Firmicutes, Planctomycetes, Gemmatimonadetes, Verrucomicrobia, Chloroflexi and Cyanobacteria. Long-term fertilization of combined manure and nitrogen application caused significant decrease in soil bacterial diversity and richness compared to non-fertilization control, though manure fertilization alone played a significant role in restoring bacterial diversity. Film mulching and manure fertilization significantly increased the relative abundances of soil bacterial groups mentioned above. Furthermore, film mulching played significant role in shaping the bacterial community structure regardless of fertilization. Redundancy analysis (RDA) showed that soil moisture content, pH, total N and soil organic C had significant (P < 0.05) influence on dominant bacterial groups. Altogether, plastic film mulching and manure application prevented loss of soil bacterial diversity and abundance during long-term fertilization. These findings showed the detrimental use of combined manure and nitrogen fertilization to soil microbes and the useful application of manure fertilization coupled with film mulching to soil biodiversity in long-term fertilization experiments.     

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)     

Short-term effects of mineral and combined mineral-organic fertilization in soil microbial communities

Fertilization is a widely used practice to maintain soil fertility. To unravel the rapid response patterns of soil microbial under mineral and combined mineral-organic fertilization treatments, 1-year fertilization trial was conducted in an intensive greenhouse on the Qinghai-Tibet Plateau (QTP). Higher soil nutrient concentrations and lower soil pH were detected in mineral and combined mineral-organic fertilized soils than in unfertilized soils. Both fertilized soils also showed higher bacterial richness and diversity and lower fungal diversity than unfertilized soils. Compared with the soil without fertilizer application, the soil microflora structure was significantly changed by fertilizer application; however, no differences were detected between both fertilized soils. Both short-term fertilizations recruited more abundant Proteobacteria and Ascomycota, and a smaller proportion of Acidobacteriota, Basidiomycota and Mortierellomycota. In addition, available phosphorus (AP), was most closely linked to the variations in the structures of soil bacterial and fungal communities. Our findings highlight the significance of the temporal component for fertilization effects, indicating that 1 year of mineral and combined mineral-organic fertilization only affected the diversity of some bacterial and fungal communities in the soil. Therefore, a moderately longer fertilization year based on this study should be pondered in intensive greenhouse cultivation in the QTP.     

长期施用有机肥增加黄壤稻田土壤微生物量碳氮

【目的】微生物量碳、氮是土壤中易于利用的养分库及有机物分解和矿化的动力,与土壤养分循环密切相关,其变化可反映土壤耕作制度和土壤肥力的变化。本研究旨在揭示长期施肥对贵州黄壤稻田土壤微生物生物量碳 (SMBC) 和土壤微生物生物量氮 (SMBN) 的影响,并探讨其合理培肥模式。【方法】以贵州黄壤肥力与肥效长期定位监测基地为依托,采用氯仿熏蒸—K₂SO₄提取法,重点研究不同施肥条件下土壤微生物生物量碳氮的变化及其与全量有机碳氮的关系。试验处理包括不施肥 (CK)、单施化肥 (NPK)、单施有机肥 (M)、低量有机无机肥配施 (0.5MNPK) 和高量有机无机肥配施 (MNPK)。【结果】长期不同施肥处理下,SMBC的变化范围在423.87～695.04 mg/kg之间,SMBN的变化范围在44.36～91.65 mg/kg之间。施用化肥 (NPK) 和施用有机肥及两者配施 (M、0.5MNPK和MNPK) 能增加SMBC和SMBN含量,其中MNPK处理较CK处理SMBC含量增幅最高,达64.0%,显著高于NPK和0.5MNPK处理,但与M处理差异不明显;M处理较CK处理SMBN含量增幅最高,达106.6%,显著高于NPK和0.5MNPK处理,但与MNPK处理差异不明显;长期单施化肥 (NPK) 仅对SMBN含量有显著提高作用 (44.1%),对SMBC作用不明显。SOC、TN和微生物熵 (qMB) 的变化与SMBC一致,均表现为MNPK处理最高,其次为M和0.5MNPK处理,NPK处理最低;所有施肥处理下的SMBC/SMBN无显著性差异且均低于CK处理。【结论】土壤微生物碳、氮量和微生物熵的显著提高均与土壤有机质和全氮的含量变化呈正相关,单施有机肥和高量有机无机肥配施是提高土壤微生物生物量的有效途径。     

长期不同施肥对黄泥田土壤酶活性和微生物的影响

以农业部耕地保育福建观测实验站的长期肥料试验为平台,研究了长期不同施肥对黄泥田土壤酶活性和微生物的影响。结果表明:与不施肥(CK)相比,单施NPK(NPK)、NPK配施牛粪(NPKM)及NPK配施秸秆(NPKS)的土壤酶活性均有不同程度的提高,其中NPKM可显著提高转化酶活性,NPKS可显著提高脲酶活性。施肥均不同程度地提高了土壤细菌、真菌及放线菌的数量,尤其是放线菌数量,提高幅度均达到显著性水平;NPKS可显著提高土壤细菌数量,NPKM可显著提高土壤真菌数量。施肥均会对真菌群落产生重要影响;单施化肥对土壤细菌群落的影响不大,增施有机物料会对细菌群落产生明显影响;施用牛粪会对放线菌优势群落产生影响。以上结果表明,有机无机配施更有利于提高土壤酶活性和土壤微生物数量,提升土壤生物肥力。     

长期无机有机肥配施对红壤性水稻土微生物群落多样性及酶活性的影响

【目的】长期有机与无机肥配合施用是促进农田生产力和土壤有机碳固定的重要技术途径。本文以江西省红壤研究所长期不同施肥试验田的表土(0—15 cm)为对象,探讨不同施肥措施对土壤微生物群落多样性和酶活性的影响。【方法】在水稻收获后,采集表土壤样品,提取土壤总DNA。采用聚合酶链反应结合变性梯度凝胶电泳(PCR-DGGE)的方法研究土壤微生物的群落结构多样性,并结合克隆测序研究土壤微生物的群落组成;用实时荧光定量PCR(q PCR)的方法研究土壤微生物的丰度。土壤细菌定量和群落结构分析的分子标靶基因分别为16S rRNA基因V3区和V6区片段,土壤真菌定量和群落结构分析的标靶基因均为18S rRNA基因。DGGE分析采用8%的聚丙烯酰胺凝胶分离细菌和真菌,所用变性梯度分别为35%65%和20%40%。同时采用荧光微孔板检测技术测定土壤几丁质酶、α-葡萄糖苷酶、β-葡萄糖苷酶、纤维素酶、酸性磷酸单脂酶和木聚糖酶活性;用紫外分光光度计法测定土壤过氧化物酶活性。【结果】PCR-DGGE分析表明,与不施肥对照(CK)相比,有机无机肥配施(NPKM),土壤细菌的香农指数和丰富度指数显著增大,而土壤真菌的香农指数和丰富度指数在不同施肥处理间无显著差异。DGGE图谱聚类分析显示,NPKM处理的土壤细菌和真菌的群落结构显著区别于其他3个处理。后续的切胶测序得出,土壤细菌分属于Chloroflexi(绿弯菌门),Proteobacteria(变形菌门)和Firmicutes(厚壁菌门);NPKM处理下隶属于Clostridum(梭菌属)和Anaerolineaceae(厌氧绳菌科)的两类细菌显著增加。土壤真菌主要分属于Basidiomycota(担子菌门)和Ascomycota(子囊菌门),这些真菌条带在DGGE图谱上的分布不同处理间均无明显的规律性,因而不同处理间真菌的群落分布未出现较清晰的变化。q PCR的结果显示,土壤细菌和真菌拷贝数在不同处理间无显著差异。土壤酶的检测结果表明,与CK相比,单施氮肥(N)处理的土壤几丁质酶活性显著提高,常规氮磷钾处理(NPK)处理的几丁质酶和α-葡萄糖苷酶活性显著增强,NPKM处理提高了土壤几丁质酶、纤维素酶和过氧化物酶活性;酸性磷酸单酯酶和木聚糖酶活性在各处理间无显著差异。归一化酶活性值,NPKM处理显著高于CK和其他施肥处理。【结论】长期有机无机肥配施可显著提高土壤细菌多样性,并改变土壤细菌和真菌的群落结构,提高土壤酶活性,因而提高了农田生态系统的生产力并对生态系统健康有改善作用。     

Soil microbial biomass, dehydrogenase activity, bacterial community structure in response to long-term fertilizer management

This study describes the effects of balanced versus nutrient-deficiency fertilization on soil microbial biomass, activity, and bacterial community structure in a long-term (16 years) field experiment. Long-term fertilization greatly increased soil microbial biomass C and dehydrogenase activity, except that the P-deficiency fertilization had no significant effect. Organic manure had a significantly greater (P<0.05) impact on the biomass C and the activity, compared with mineral fertilizers. Microbial metabolic activity (dehydrogenase activity per microbial biomass C) was significantly higher (P<0.05) under balanced fertilization than under nutrient-deficiency fertilization. General bacterial community structure was analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) targeting eubacterial 16S rRNA gene. Mineral fertilization did not affect the DGGE banding pattern, while specific DGGE band was observed in organic manure-fertilized soils. Phylogenetic analysis showed that the change of bacterial community in organic manure-fertilized soil might not be because of the direct influence of the bacteria in the compost, but because of the promoting effect of the compost on the growth of an indigenous Bacillus sp. in the soil. We emphasize the importance of balanced-fertilization, as well as the role of P, in maintaining soil organic matter, and promoting the biomass and activity of microorganisms.     

Long-term and legacy effects of manure application on soil microbial community composition

We analyzed soil prokaryotic and fungal community composition in soils with varying histories of cattle manure application. The manure treatments were (i) annual application for 43 years (MF), (ii) annual application for 14 years followed by 29 years without application (MF14), and (iii) annual application for 30 years followed by 13 years without application (MF30). An annual application of chemical nitrogen (N) fertilizer (CNF) and a non-amended control (Con) were also included. Soil prokaryotic evenness and diversity significantly decreased in MF relative to other treatments in fall, but were similar to the other fertilizer treatments in spring and summer. Distinct prokaryotic and fungal community composition was observed in MF compared to other treatments across fall, spring, and summer seasons. The MF treatment significantly increased the relative abundance of Firmicutes, Gammaproteobacteria, and Gemmatimonadetes, but significantly decreased the relative abundance of Acidobacteria. In fall, the soil prokaryotic and fungal community composition with MF30 was significantly different than the other fertilization treatments. Overall, the study showed that annual manure application (MF) led to a different microbial community composition than the other fertilizer treatments. Soil without manure application for 13 years (MF30) had a significantly different microbial community composition from other fertilizer treatments in fall, while the soil without manure application for 29 years (MF14) resembled a microbial community that had never received manure.     

长期施肥对旱地红壤细菌群落的影响

为探讨长期不同施肥对旱地红壤细菌群落的影响,以中国农业科学院祁阳红壤实验站的冬小麦—夏玉米定位试验为研究对象,选取不施肥(CK)、单施氮肥(N)、施化学氮磷钾肥(NPK)和化学氮磷钾+有机肥配施(NPKM)4个处理,于试验开展25年(2015年)小麦收获后采集各处理0~20 cm的土壤样品,利用Illumina MiSeq高通量测序技术对土壤细菌群落进行测定,并深入揭示影响旱地红壤细菌群落的关键因素。结果表明:(1)长期不同施肥显著改变了旱地红壤的化学性质,N和NPK处理的土壤pH显著降低至4.02和4.15,而NPKM处理的土壤pH显著上升至5.99。NPK和NPKM处理均显著改善土壤肥力,但后者效果明显优于前者,而N处理对土壤肥力的提升效果微弱。(2)长期不同施肥改变了旱地红壤优势菌的相对丰度,非度量多维度分析(NMDS)和相似性分析(ANOSIM)表明不同处理的土壤细菌群落发生显著变化。(3)与CK相比,N处理的4种多样性指数(物种丰富度、Chao1指数、系统发育多样性和香农指数)显著降低了21.4%~49.4%,而NPKM处理显著增加了7.0%~66.9%,NPK处理也会使系统发育多样性和香农指数显著降低10.3%和13.0%。(4)逐步回归分析表明土壤pH是决定优势菌相对丰度及4种多样性指数的首要因素,多元回归树分析(MRT)探明土壤pH共解释了83.1%的细菌群落变异,不同处理间细菌群落转变均由土壤pH驱动。(5)STAMP分析发现,N、NPK和NPKM处理与CK分别有11、14和8个显著差异细菌属。综上所述,长期施肥后旱地红壤细菌群落主要受土壤pH的影响,而土壤肥力的作用相对较弱,长期施用化学氮肥造成的红壤酸化的负面效应已远超肥力改善的正面效应。因此,旱地红壤施肥应以防治土壤酸化为前提,长期化肥有机肥配施是一项适宜的施肥措施。