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)     

利用PCR-DGGE技术研究长期自然恢复、种植作物和裸地条件下黑土土壤细菌群落结构变化

Soil microbial biomass and community structures are commonly used as indicators for soil quality and fertility. A investigation was performed to study the effects of long-term natural restoration, cropping, and bare fallow managements on the soil microbial biomass and bacterial community structures in depths of 0--10, 20--30, and 40--50 cm in a black soil (Mollisol). Microbial biomass was estimated from chloroform fumigation-extraction, and bacterial community structures were determined by analysis of 16S rDNA using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Experimental results showed that microbial biomass significantly declined with soil depth in the managements of restoration and cropping, but not in the bare fallow. DGGE profiles indicated that the band number in top 0--10 cm soils was less than that in depth of 20--30 or 40--50 cm. These suggested that the microbial population was high but the bacterial community structure was simple in the topsoil. Cluster and principle component analysis based on DGGE banding patterns showed that the bacterial community structure was affected by soil depth more primarily than by managements, and the succession of bacterial community as increase of soil depth has a similar tendency in the three managements. Fourteen predominating DGGE bands were excised and sequenced, in which 6 bands were identified as the taxa of Verrucomicrobia, 2 bands as Actinobacteria, 2 bands as α-Proteobacteria, and the other 4 bands as δ-Proteobacteria, Acidobacteria, Nitrospira, and unclassified bacteria. In addition, the sequences of 11 DGGE bands were closely related to uncultured bacteria. Thus, the bacterial community structure in black soil was stable, and the predominating bacterial groups were uncultured.     

Anammox反应器的启动及其菌群演变的研究

为了研究工艺条件对反应器内微生物多样性的影响,该论文采用城市污水处理厂活性污泥接种,通过培育硝化污泥,进行了启动厌氧氨氧化(Anammox)反应器的试验,并对启动过程中细菌的多样性变化作了跟踪研究。研究结果表明,以好氧活性污泥作为接种物,可成功地培育硝化生物膜;通过反应器运行条件的控制,硝化生物膜可从进行好氧氨氧化反应过渡到进行厌氧氨氧化反应。在此过程中,异养型细菌的数量大幅度下降,硝化细菌、反硝化细菌和厌氧氨氧化细菌的数量增大,推测它们都与厌氧氨氧化作用有关。运用PCR-DGGE技术证明,随着反应器运行时间的延长,菌群发生明显变化并呈现简化趋势。     

Community structure of methanogenic archaea in paddy field soil under double cropping (rice-wheat)

Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.     

Leaf litter is the main driver for changes in bacterial community structures in the rhizosphere of ash and beech

The rhizosphere and the surrounding soil harbor an enormous microbial diversity and a specific community structure, generated by the interaction between plant roots and soil bacteria. The aim of this study was to address the influences of tree species, tree species diversity and leaf litter on soil bacterial diversity and community composition. Therefore, mesocosm experiments using beech, ash, lime, maple and hornbeam were established in 2006, and sampled in October 2008 and June 2009. Mesocosms were planted with one, three or five different tree species and treated with or without litter overlay.Cluster analysis of DGGE-derived patterns revealed a clustering of 2008 sampled litter treatments in two separated clusters. The corresponding treatments sampled in 2009 showed separation in one cluster. PCA analysis based on the relative abundance of active proteobacterial classes and other phyla in beech and ash single-tree species mesocosm indicated an effect of sampling time and leaf litter on active bacterial community composition. The abundance of next-generation sequencing-derived sequences assigned to the Betaproteobacteria was higher in the litter treatments, indicating a higher activity, under these conditions. The Deltaproteobacteria, Nitrospira and Gemmatimonadetes showed an opposite trend and were more active in the mesocosms without litter. The abundance of alphaproteobacterial sequences was higher in mesocosms sampled in 2009 (P = 0.014), whereas the Acidobacteria were more active in 2008 (P = 0.014). At the family level, we found significant differences of the litter vs. non-litter treated group. Additionally, an impact of beech and ash as tree species on soil bacterial diversity was confirmed by the Shannon and Simpson indices. Our results suggest that leaf litter decomposition in pH-stable soils affect the soil bacterial composition, while tree species influence the soil bacterial diversity.     

不同年限毛竹林土壤固氮菌群落结构和丰度的演变

应用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)和荧光定量PCR(real time fluorescent quantitative PCR,q PCR)方法研究了不同年限毛竹林土壤固氮菌群落结构和丰度的变化。结果表明:土壤p H、有机质、有效磷、速效钾和铵态氮含量在马尾松林改造成毛竹林5 a后明显提高,而后逐渐降低,并趋于稳定;土壤固氮菌多样性和nif H基因丰度也呈现相似的趋势。条带测序分析表明,毛竹林土壤固氮菌均为不可培养的固氮菌,与慢生根瘤菌(Bradyrhizobium sp.)具有较高的相似度。冗余分析结果表明,不同栽培年限毛竹林地土壤固氮菌群落组成发生了明显变化,长期栽培毛竹林引起的土壤养分变化对土壤固氮菌多样性具有重要影响。     

Rapid changes in the rhizosphere bacterial community structure during re-colonization of sterilized soil

Diversity has been shown to be pivotal in ecosystem stability and resilience. It is therefore important to increase our knowledge about the development of diversity. The aim of this study was to investigate the temporal dynamics of the bacterial community structure in the rhizosphere of wheat plants growing in a soil in which the initial conditions for bacterial re-colonization were modified by mixing different amounts of sterilized with native soil at ratios of 19:1, 9:1, 4:1 and 1:1. Additional treatments comprised sterilized soil or native soil. Plant dry weight at day 20 decreased with increasing percentage of native soil in the mix. The bacterial community structure in the rhizosphere was assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) at days 3, 14 and 20 after planting. The bacterial community in the sterilized soil had a lower diversity and evenness than the native soil. Both diversity and evenness increased with time in the sterilized soil. Community structure in the different mixes changed over time and the changes were mix-specific. Principal component analyses of the DGGE banding patterns showed clear differences between the treatments particularly at day 3 and day 14 and revealed changes in community structure within a few days in a given treatment. The results of the present study show that bacterial communities rapidly re-colonize sterilized soil. During re-colonization, the community structure changes rapidly with a general trend towards higher diversity and evenness. The changes in community structure over time are also affected by the amount of sterile substrate to be re-colonized.     

Community structure of bacteria on different types of mineral particles in a sandy soil

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types.     

沿海滩涂土壤中反硝化细菌群落随盐分梯度的更迭

To better understand the effect of salinity on denitrification communities,soils along a salinity gradient (ranging from 7.32 to 1.70 mS cm 1) in a wetland along the Yellow Sea coastline in Jiangsu Province,China,were studied using both culture-dependent and-independent methods.Culture efforts yielded 82 isolates in total,81.7% of which were close relatives of Bacillus sp.based on partial sequences of their 16S rRNA genes.Denaturing gradient gel electrophoresis (DGGE) analysis based on 16S rRNA sequences suggested possible existence of bacterial community succession along the salinity gradient.Clone library analysis based on nosZ gene sequences (coding nitrous oxide reductase) showed that operational taxonomic units (OTUs) associated with α-proteobacteria dominated in all three soils,whereas those associated with β-and γ-subdivisions showed a clear succession.In the high salinity soil,only the OTUs associated with α-subdivision were found.In the medium salinity soil,small proportions of β-(6.5%) and γ-associated (19.6%) OTUs were found.In the low salinity soil,the proportions were further increased to 33% and 25% for β-and γ-Proteobacteria,respectively.Statistic analysis using Unifrac P test showed that nosZ-communities in different saline soils were significantly different from each other.It could be concluded that α-subdivision of nosZ-community tended to be sustained in high salinity environments whereas β-and γ-subdivisions,especially the former,tended to be sustained in low salinity environments.Salinity was the key determinant of nosZ-community composition in the environment.     

芦苇定居后铜尾矿细菌群落结构的变化

Soil samples were collected from both bare and vegetated mine tailings to study the changes in bacterial communities and soil chemical properties of copper mine tailings due to reed (Phragmites communis) colonization. The structures of bacterial communities were investigated using culture-independent 16S rRNA gene sequencing method. The bacterial diversity in the bare mine tailing was lower than that of the vegetated mine tailing. The former was dominated by sulfur metabolizing bacteria, whereas the latter was by nitrogen fixing bacteria. The bare mine tailing was acidic (pH = 3.78), whereas the vegetated mine tailing was near neutral (pH = 7.28). The contents of organic matter, total nitrogen, and ammonium acetate-extractable potassium in vegetated mine tailings were significantly higher than those in the bare mine tailings (P < 0.01), whereas available phosphorus and electrical conductivity were significantly lower than those in the bare mine tailings (P < 0.01). The results demonstrated that 16S rRNA gene sequencing could be successfully used to study the bacterial diversity in mine tailings. The colonization of the mine tailings by reed significantly changed the bacterial community and the chemical properties of tailings. The complex interactions between bacteria and plants deserve further investigation.     

Fire severity shapes plant colonization effects on bacterial community structure,microbial biomass,and soil enzyme activity in secondary succession of a burned forest

The increasing frequency and severity of wildfires has led to growing attention to the effects of fire disturbance on soil microbial communities and biogeochemical cycling. While many studies have examined fire impacts on plant communities, and a growing body of research is detailing the effects of fire on soil microbial communities, little attention has been paid to the interaction between plant recolonization and shifts in soil properties and microbial community structure and function. In this study, we examined the effect of a common post-fire colonizer plant species, Corydalis aurea, on soil chemistry, microbial biomass, soil enzyme activity and bacterial community structure one year after a major forest wildfire in Colorado, USA, in severely burned and lightly burned soils. Consistent with past research, we find significant differences in soil edaphic and biotic properties between severe and light burn soils. Further, our work suggests an important interaction between fire severity and plant effects by demonstrating that the recolonization of soils by C. aurea plants only has a significant effect on soil bacterial communities and biogeochemistry in severely burned soils, resulting in increases in percent nitrogen, extractable organic carbon, microbial biomass, β-glucosidase enzyme activity and shifts in bacterial community diversity. This work propounds the important role of plant colonization in succession by demonstrating a clear connection between plant colonization and bacterial community structure as well as the cycling of carbon in a post-fire landscape. This study conveys how the strength of plant–microbe interactions in secondary succession may shift based on an abiotic context, where plant effects are accentuated in harsher abiotic conditions of severe burn soils, with implications for bacterial community structure and enzyme activity.     

Overland flow systems for treatment of landfill leachates—Potential nitrification and structure of the ammonia-oxidising bacterial community during a growing season

Overland flow systems are useful for treating landfill leachates, because they provide favourable conditions for nitrification and they are easy to maintain. However, little is known about the microbial communities in such systems or the nitrification capacity of those microorganisms. In this study, seasonal variations in potential nitrification and in community composition of nitrifying bacteria were investigated in two overland flow areas receiving leachate from landfills at Korslöt and Hagby, Sweden. Samples were collected in the settling ponds sediment and at two depths in the overland flow areas (the macrophyte litter layer and the rhizosphere) in May, August and November 2003. A short-term incubation method was used to measure potential oxidation of ammonia and nitrite (designated PAO and PNO). The ammonia-oxidising bacterial (AOB) community was investigated using a 16S rRNA gene approach that included PCR amplification and analysis of PCR products by denaturing gradient gel electrophoresis (DGGE), followed by nucleotide sequencing and phylogenetic analysis.PAO was determined in the range 5-2700 (NO₂⁻+NO₃⁻)-N g⁻¹ dw d⁻¹ and PNO in the range 60-2000 μg NO₂⁻-N g⁻¹ dw d⁻¹. At Korslöt, PAO and PNO showed similar temporal variation in the different ecosystems, whereas no such relationship was noticed at Hagby. Considering both sites, there was no obvious change in the composition of the AOB community over the growing season. However, the composition did differ between the ecosystems: Nitrosomonas-like sequences were more common in the ponds, and in the litter layers they were found as often as Nitrosospira-like sequences, whereas Nitrosospira-like sequences were more common in the rhizospheres. Altogether, we found nine different AOB sequences, five Nitrosomonas-like and four Nitrosospira-like, which belonged to clusters 0, 2, 3b, 6a, 6b and 7. There was no apparent relationship between the number of AOB populations and the PAO in different soil layers and sediments.     

Single particle analysis reveals that bacterial community structures are semi-specific to the type of soil particle

Abstract

With the aim of understanding how diverse bacteria are distributed within a heterogeneous soil, we compared local bacterial communities on individual soil particles. We picked up 11 coarse sand-sized particles (quartz, whitish feldspar, yellow feldspar or unidentified brown particles) from a sandy soil, extracted DNA from each particle, and carried out partial 16S rDNA polymerase chain reaction denaturing gradient gel electrophoresis analysis. Bacterial communities located on soil particles of the same type were more similar in composition than communities located on particles of the other types. Thus, the local structure of a bacterial community is related to the type of soil particle, which suggests that a high diversity of soil bacteria emerges through a combination of local bacterial communities on different types of soil particles.     

鸡粪好氧堆肥过程中氨氧化古菌群落结构的动态变化

应用聚合酶链式反应-变性梯度凝胶电泳（PCR DGGE）技术,研究了好氧堆肥过程氨氧化古菌（ammonia oxidizing archaea, AOA）的群落结构和多样性变化。结果表明,不同堆肥时期鸡粪好氧堆肥AOA菌群的群落结构发生了明显的变化。与AOA HH 2(GU2258721)亲缘关系较近的b条带(相似性96%)和未培养泉古菌属［uncultured crenarchaeote NM 152(HQ8752251)］的m条带(相似性99%)是堆肥过程一直存在的AOA菌属。条带c、 b、 f、 i和条带m、 k、 l、 n代表的种属分属两个大类。AOA群落的Shannon Weiner指数（H）、 均匀度指数（EH）均表现为第30 d＞第5 d＞第25 d第45 d＞第3 d第12 d＞第1 d第15 d。对AOA种群数据与不同时期堆体温度、 pH、 全氮、 铵态氮、 亚硝态氮和硝态氮等环境因子的冗余分析表明,堆体温度、 全氮、 亚硝态氮和硝态氮对氨氧化古菌群落演替有着显著的影响（P0.05）,AOA的群落结构在堆肥第1 d、 第5 d、 第25 d、 第30 d、 第45 d变化较大。说明这些参数能有效控制鸡粪好氧堆肥过程中AOA的群落结构。     

cry1Ac/cpti转基因大米水稻土中微生物群落的多样性研究

Two types of cry1Ac/cpti transgenic rice(GM1 and GM2)and their parental non-cry1Ac/cpti rice(CK1 and CK2)were planted in the field at Wufeng,Fujian Province,China for four years to investigate the influence of genetically modified rice on diversity of bacterial and fungal community in the paddy soil.The community composition and abundance of bacteria or fungi in the paddy soil were assessed at different growth stages of rice by denaturing gradient gel electrophoresis and real-time polymerase chain reaction based on 16S rRNA gene or SSU rRNA gene in the 4th year after the experimental establishment.The composition of bacterial or fungal community changed during rice growth,while no significant differences were observed between the fields cultivated with GM1and CK1,or between the fields cultivated with GM2 and CK2 in either bacterial or fungal community composition.The copy numbers of bacterial 16S rRNA gene in the soils with CK1,CK2,GM1 and GM2 ranged from 5.64×1011to 6.89×1011copies g-1dry soil at rice growth stages,and those of fungal SSU rRNA gene from 5.24×108to 8.68×108copies g-1dry soil.There were no marked differences in the copies of bacterial 16S rRNA gene or fungal SSU rRNA gene between CK1 and GM1 or between CK2 and GM2at any growth stage of rice.Planting cry1Ac/cpti transgenic rice had no significant effect on composition and abundance of bacterial and fungal community in paddy soil during the rice growing season at least in the short term.     

Bacterial Communities Responsible for the Decomposition of Rice Straw Compost in a Japanese Rice Paddy Field Estimated by DGGE Analysis of Amplified 16S rDNA and 16S rRNA Fragments

To estimate the succession and phylogenetic composition of the bacterial communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analyses targeting 16S rDNA and 16S rRNA, followed by sequencing were conducted in a Japanese paddy field. The DGGE bands of the bacterial communities in the rice straw compost were significantly more numerous in the DNA samples than in the RNA samples. Although the band number of the DNA samples was almost constant throughout the period, RNA samples showed fewer DGGE bands after mid-season drainage than before it. Thus, about 81% of the bacteria present in rice straw compost were considered to be metabolically "active" before mid-season drainage and about 62% after it. The changes in the DGGE patterns of bacterial DNA and RNA before and after mid-season drainage, respectively, were also revealed by cluster analysis and principal component analysis of the DGGE patterns. These results indicated that the bacterial communities of rice straw compost incorporated into flooded paddy fields changed gradually along with the decomposition, except for the period of mid-season drainage, but that they were influenced by mid-season drainage. Members of β-, γ- and δ-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Chlorobia, Verrucomicrobia, Chloroflexi, Spirochaetes, Firmicutes (clostridia) and Actinobacteria were present during the decomposition of rice straw compost. Characteristic "active" bacteria among them were as follows: Clostridium, Acinetobacter (γ-Proteobacteria) and β-Proteobacteria before mid-season drainage, Flavobacterium, Chondromyces , Chlorflexi and δ-Proteobacteria after mid-season drainage, and Spirochaeta and myxobacteria throughout the period.     

Analysis of bacterial communities on alkaline phosphatase genes in soil supplied with organic matter

Abstract

We studied the effects of the application of organic matter (OM) and chemical fertilizer (CF) on soil alkaline phosphatase (ALP) activity and ALP-harboring bacterial communities in the rhizosphere and bulk soil in an experimental lettuce field in Hokkaido, Japan. The ALP activity was higher in soils with OM than in soils with CF, and activity was higher in the rhizosphere for OM than in the bulk soil. Biomass P and available P in the soil were positively related to the ALP activity of the soil. As a result, the P concentration of lettuce was higher in OM soil than in CF soil. We analyzed the ALP-harboring bacterial communities using polymerase chain reaction based denaturing gradient gel electrophoresis (DGGE) on the ALP genes. Numerous ALP genes were detected in the DGGE profile, regardless of sampling time, fertilizer treatment or sampled soil area, which indicated a large diversity in ALP-harboring bacteria in the soil. Several ALP gene fragments were closely related to the ALP genes of Mesorhizobium loti and Pseudomonas fluorescens. The community structures of the ALP-harboring bacteria were assessed using principal component analysis of the DGGE profiles. Fertilizer treatment and sampled soil area significantly affected the community structures of ALP-harboring bacteria. As the DGGE bands contributing to the principal component were different from sampling time, it is suggested that the major bacteria harboring the ALP gene shifted. Furthermore, there was, in part, a significant correlation between ALP activity and the community structure of the ALP-harboring bacteria. These results raise the possibility that different ALP-harboring bacteria release different amounts and/or activity of ALP, and that the structure of ALP-harboring bacterial communities may play a major role in determining overall soil ALP activity.