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.     

Rhizosphere microbiota assemblage associated with wild and cultivated soybeans grown in three types of soil suspensions

Soil microbial community composition is determined by the soil type and the plant species. By sequencing the V3-V4 region of the bacterial 16S rRNA gene amplicons, the current study assessed the bacterial community assemblage in rhizosphere and bulks soils of wild (Glycine soja) and cultivated (Glycine max) soybeans grown in the suspensions of three important soil types in China, including black, red and soda-saline-alkali soils. The alpha-diversity of the bacterial community in the rhizosphere was significantly higher than that of the bulk soils suggesting that bulk soil lacks plant nurturing effect under the current study conditions. Black and red soils were enriched with nitrifying and nitrogen-fixing bacteria but the soda-saline-alkali soil suspension had more denitrifying bacteria, which may reflect agronomic unsuitability of the latter. We also observed a high abundance of Bradyrhizobium and Pseudomonas, enriched cellulolytic bacteria, as well as a highly connected molecular ecological network in the G. soja rhizosphere soil. Taken all, the current study suggest that wild soybeans may have evolved to recruit beneficial microbes in its rhizosphere that can promote nutrients requisition, biostasis and disease-resistance, therefore ecologically more resilient than cultivated soybeans.     

Effects of pH and ionic strength on elemental sulphur oxidation in soil

Elemental S oxidation in soil is a microbially mediated process and is hypothesised to be influenced by changes to soil chemical properties such as acidity and ionic strength, which may arise from co-granulation with macronutrients or elemental S oxidation itself. Soil incubation was conducted with a sandy soil from South Australia to assess the effect of acidification and increased ionic strength on bacterial abundance and community composition and on elemental S oxidation during a 14-week incubation at 25 °C and 70% field capacity. Prior to incubation, the soil was treated with HNO₃ to bring the pH to 6.7–4.4 or with KH₂PO₄ to increase the ionic strength by 0–0.7 M. Elemental S was applied at 200 mg kg^?1 air-dried soil. Acidification or increased ionic strength had no or little effect on elemental S oxidation but decreased the abundances of 16S ribosomal deoxyribonucleic acid (rRNA) and soxB genes and changed the bacterial community composition. A second experiment with two other soils also showed that acidification did not, or only slightly, decreased elemental S oxidation, even though acidification strongly reduced 16S rRNA and soxB gene abundances in one of the soils. This study suggests that shifts in bacterial population brought about by temporary changes in pH and ionic strength, as may occur around fertiliser granules, have no or little effect on elemental S oxidation, indicating that the S-oxidising bacterial community in these agricultural soils contains functionally redundant taxa, which responded to changing conditions.     

Effects of straw and its biochar applications on the abundance and community structure of CO2-fixing bacteria in a sandy agricultural soil

Purpose

Crop straw and biochar application can potentially increase carbon sequestration and lead to changes in the microbial community in agricultural soils. Sequestration of CO₂ by autotrophic microorganisms is key to biogeochemical carbon cycling in soil ecosystems. The effects of straw and its biochar, derived from slow pyrolysis, on CO₂ fixation bacteria in sandy soils, remain unclear. Therefore, this study compared the response of abundance and community of CO₂ fixation bacteria to the two straw application methods in a sandy agricultural soil. The overall aim of the study was to achieve an efficient use of straw residues for the soil sustainablility.

Materials and methods

We investigated the soil organic carbon content and autotrophic bacteria over four consecutive years (2014–2018) in a field experiment, including the following four treatments: whole maize straw amendment (S), whole maize straw translated biochar amendment (B), half biochar and half straw amendment (BS), and control (CK) without straw or biochar amendment. The autotrophic bacterial abundance and community structure were measured using molecular methods of real-time PCR, terminal restriction fragment length polymorphisms (T-RFLP), and a clone library targeting the large subunit gene (cbbL) of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Results and discussion

The results showed that the content of soil total organic carbon (TOC), dissolved organic carbon (DOC), and microbial biomass carbon (MBC) in B, S, and BS treatments was significantly increased compared with the CK treatment. Soil TOC and available potassium (AK) in the B treatment significantly increased by 15.4% and 23.3%, respectively, but soil bulk density, DOC, and MBC significantly decreased by 8.5%, 10.6%, and 14.5%, respectively, compared with the S treatment. The abundance of the cbbL gene as well as of the bacterial 16S rRNA gene increased significantly in straw or biochar application treatments as compared to the CK treatment. The B treatment, but not the BS treatment, significantly increased the cbbL gene abundance when compared to the S treatment. No significant differences were observed in the bacterial 16S rRNA gene abundance among the three straw or biochar applications. The application of straw biochar could increase the diversity of the autotrophic bacteria, which also altered the overall microbial composition. Physicochemical properties of the soil, such as soil pH, SOC, and bulk density, can help explain the shift in soil microbial composition observed in the study.

Conclusions

Taken together, our results suggest that straw biochar, rather than straw application, leads to an increase in the abundance and diversity of CO₂-fixing bacteria, which would be advantageous for soil autotrophic CO₂ fixation.

     

Bacterial diversity of terra preta and pristine forest soil from the Western Amazon

The survey presented here describes the bacterial diversity and community structures of a pristine forest soil and an anthropogenic terra preta from the Western Amazon forest using molecular methods to identify the predominant phylogenetic groups. Bacterial community similarities and species diversity in the two soils were compared using oligonucleotide fingerprint grouping of 16S rRNA gene sequences for 1500 clones (OFRG) and by DNA sequencing. The results showed that both soils had similar bacterial community compositions over a range of phylogenetic distances, among which Acidobacteria were predominant, but that terra preta supported approximately 25% greater species richness. The survey provides the first detailed analysis of the composition and structure of bacterial communities from terra preta anthrosols using noncultured-based molecular methods.     

Shifts in bacterial community structure associated with inputs of low molecular weight carbon compounds to soil

Low molecular weight carbon (C) substrates are major drivers of bacterial activity and diversity in the soil environment. However, it is not well understood how specific low molecular weight C compounds, which are frequently found in root exudates and litter leachates, influence bacterial community structure or if there are specific groups of soil bacteria that preferentially respond to these C inputs. To address these knowledge gaps, we added three simple C substrates representative of common root exudate compounds (glucose, glycine, and citric acid) to microcosms containing three distinct soils from a grassland, hardwood forest, and coniferous forest. CO₂ production was assessed over a 24 h incubation period and, at the end of the incubation, DNA was extracted from the samples for assessment of bacterial community structure via bar-coded pyrosequencing of the 16S rRNA gene. All three C substrates significantly increased CO₂ production in all soils; however, there was no relationship between the magnitude of the increase in CO₂ production and the shift in bacterial community composition. All three substrates had significant effects on overall community structure with the changes primarily driven by relative increases in β-Proteobacteria, γ-Proteobacteria, and Actinobacteria. Citric acid additions had a particularly strong influence on bacterial communities, producing a 2-5-fold increase in the relative abundance of the β-Proteobacteria subphylum. These results suggest that although community-level responses to substrate additions vary depending on the substrate and soil in question, there are specific bacterial taxa that preferentially respond to the substrate additions across soil types.     

不同类型水稻土微生物群落结构特征及其影响因素

选取基于我国土壤地理发生分类的不同类型土壤发育的四种水稻土,利用¹⁵N₂气体示踪法测定生物固氮速率,采用实时荧光定量PCR（Real-time PCR）技术测定细菌丰度,通过16S rRNA基因高通量测序分析微生物群落组成和多样性。结果表明：变形菌门（Proteobacteria）、酸杆菌门（Acidobacteria）、绿弯菌门（Chloroflexi）和蓝藻门（Cyanobacteria）是水稻土中优势微生物类群。四种类型土壤发育的水稻土细菌群落结构差异显著（Stress<0.001）,群落结构分异（NMDS1）与土壤pH存在极显著正相关关系（P<0.01）。土壤有机碳和碱解氮含量显著影响水稻土中细菌丰度和群落多样性（P<0.01）。红壤发育的水稻土细菌16S rRNA基因拷贝数显著高于其他三种类型水稻土,但OTU数量、Chao1指数和PD指数均低于其他三种类型水稻土。土壤pH对水稻土生物固氮速率有显著影响（P<0.01）,紫色土发育的水稻土具有最高的生物固氮速率（3.2±0.7 mg×kg^-1×d^-1）,其中优势类群细鞘丝藻属（Leptolyngbya）可能是生物固氮的主要贡献者。研究结果丰富了对水稻土微生物多样性的认识,为通过调控土壤pH和微生物群落组成来提高稻田生物固氮潜力提供了理论依据。     

Decrease in diversity and shift in composition of the soil bacterial community were closely related to high available phosphorus in agricultural Fluvisols of North China

ABSTRACT

Our objectives were to investigate whether AP affects the soil bacterial community composition and diversity in high-level available phosphorus (AP) soils. The bacterial community was analysed through high-throughput sequencing using the Illumina MiSeq platform. Fifteen soils, including barren land, cropland and greenhouse soils which were sandy loam Fluvisols, were selected from different fields in Beijing, China, with AP contents ranging from 5.03 to 391.45 mg kg^?1. Statistical analyses revealed high AP (>100 mg kg^?1) decreased alpha diversity (Shannon’s index, H’) but not beta diversity of the soil bacterial community. The sequencing of 16S rRNA genes showed that Proteobacteria, Bacteroidetes and Acidobacteria were the dominant phyla in sandy loam Fluvisols. AP, soil organic carbon (SOC) and total nitrogen (TN) had synergistic influence on the shift of the bacterial community composition. Moreover, AP was the main driving factor affecting the soil bacterial community composition compared with other environmental factors. The members of the Proteobacteria, Bacteroidetes and Actinobacteria belonging to copiotrophic taxa typically increased in relative abundance in high-P soils, while oligotrophic taxa (mainly Acidobacteria) decreased in relative abundance. Our results demonstrated that the bacterial community composition would shift from oligotrophic to copiotrophic with increasing levels of AP.     

Establishment of macro-aggregates and organic matter turnover by microbial communities in long-term incubated artificial soils

The study of interactions between minerals, organic matter (OM) and microorganisms is essential for the understanding of soil functions such as OM turnover. Here, we present an interdisciplinary approach using artificial soils to study the establishment of the microbial community and the formation of macro-aggregates as a function of the mineral composition by using artificial soils. The defined composition of a model system enables to directly relate the development of microbial communities and soil structure to the presence of specific constituents. Five different artificial soil compositions were produced with two types of clay minerals (illite, montmorillonite), metal oxides (ferrihydrite, boehmite) and charcoal incubated with sterile manure and a microbial community derived from a natural soil. We used the artificial soils to analyse the response of these model soil systems to additional sterile manure supply (after 562 days). The artificial soils were subjected to a prolonged incubation period of more than two years (842 days) in order to take temporally dynamic processes into account. In our model systems with varying mineralogy, we expected a changing microbial community composition and an effect on macro-aggregation after OM addition, as the input of fresh substrate will re-activate the artificial soils. The abundance and structure of 16S rRNA gene and internal transcribed spacer (ITS) fragments amplified from total community DNA were studied by quantitative real-time PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE), respectively. The formation of macro-aggregates (>2 mm), the total organic carbon (OC) and nitrogen (N) contents, the OC and N contents in particle size fractions and the CO₂ respiration were determined. The second manure input resulted in higher CO₂ respiration rates, 16S rRNA gene and ITS copy numbers, indicating a stronger response of the microbial community in the matured soil-like system. The type of clay minerals was identified as the most important factor determining the composition of the bacterial communities established. The additional OM and longer incubation time led to a re-formation of macro-aggregates which was significantly higher when montmorillonite was present. Thus, the type of clay mineral was decisive for both microbial community composition as well as macro-aggregation, whereas the addition of other components had a minor effect. Even though different bacterial communities were established depending on the artificial soil composition, the amount and quality of the OM did not show significant differences supporting the concept of functional redundancy.     

Evaluation of soil enzyme activities and microbial communities in tomato continuous cropping soil treated with jerusalem artichoke residues

ABSTRACT

Jerusalem artichoke (JY) (Helianthus tuberosus L.) has been reported to have a strong inhibitory effect on weed growth and root knot nematodes, but little information is available on the effects on soil ecosystems, especially soil microorganisms and soil enzyme activities. Understanding the dynamics of soil microbes and soil enzyme activities in cropping systems can help determine how agricultural practices influence soil processes mediated by JY residues. This study used a pot experiment, with five-year continuous cropping soil of tomato plants as the experimental material and 2% (w/w) JY residue as the treatment material in the soil. The treatment was compared to continuously monocropped tomato soil that was not treated with JY residues. The results of 16S high-throughput sequencing showed that both fungal and bacterial community structure and composition varied significantly at each stage of JY treatment. The analysis showed that the major phyla in the soil fungal community included Ascomycota, Zygomycota and Basidiomycota. Chytridiomycota was dominant in only the JY-treated soil. At the genus level, the abundances of Mortierella, Cephaliophora, Cryptococcus and Fusarium notably changed at each stage of JY treatment. In the bacterial community in the JY-treated group, the abundance of Proteobacteria increased significantly, while that of Firmicutes decreased significantly, compared to the control group. JY enhanced the activity of soil sucrase and urease. In addition, the soil sucrase activity showed a strong negative correlation with Fusarium and Bacillus. Overall, our results revealed that JY residues changed both the soil bacterial and fungal community composition and the soil enzyme activities.     

Dynamics of extracellular DNA decomposition and bacterial community composition in soil

Microbial necromass is an important source of stabilized organic matter in soil, yet the decomposition dynamics of necromass constituents have not been adequately characterized. This includes DNA, a nutrient-rich molecule that when released into the environment as extracellular DNA (eDNA) can be readily used by soil microorganisms. However, the ecological relevance of eDNA as a nutrient source for soil microorganisms is relatively unknown. To address these deficits, we performed a laboratory experiment wherein soils were amended with ¹³C-labeled eDNA and clay minerals known to interact with DNA (kaolinite and montmorillonite). The amount of eDNA-carbon remaining in the soil declined exponentially over time. Kaolinite amendment decreased eDNA decomposition rates and, after 30 days, retained a higher fraction of eDNA-carbon (∼70% remaining) than control or montmorillonite soils (∼40% remaining), indicating that clay mineral sorption can stabilize eDNA-derived carbon in soil. Sequencing of bacterial 16S rRNA genes showed that during the incubation the relative abundance of the added eDNA's sequence decreased by 98%, 92% and 99% in the control, montmorillonite, and kaolinite amended soils respectively. These results suggest that the fraction of eDNA-carbon that remained in the soil was incorporated into microbial biomass, firmly bound to soil constituents, or fragmented and no longer amenable to sequencing. In addition, the eDNA amendment affected the composition of the bacterial community. Specifically, the relative abundance of select phyla (Planctomycetes and TM7) and genera (e.g., Arthrobacter and Nocardioides) were elevated in soils that received eDNA, suggesting these groups may be particularly effective at degrading eDNA and using it for growth. Taken together, these results indicate that while eDNA is consumed by bacteria in soil, a fraction of eDNA material is resistant to decomposition, particularly when stabilized by soil minerals, suggesting a substantial amount of recalcitrant eDNA could accumulate over time.     

Long-term no-till management affects microbial biomass but not community composition in Canadian prairie agroecosytems

No-till (NT) management greatly reduces soil physical disturbance and can result in a stratification of nutrients and organic matter in the soil profile due to the retention of crop residues on the soil surface potentially affecting the dynamics of microbial interactions in the soil. Microbial abundance and diversity can be used to assess the relative impact of management on the long-term sustainability of cropping systems. The objective of this study was to assess the impact of long-term NT vs. conventional tillage (CT) management on soil microbial community structure at four different sites on the Canadian prairies using phospholipid fatty acid analysis (PLFA) and DNA fingerprinting. Analysis of 16S and 18S rDNA using denaturing gradient gel electrophoresis revealed high inherent variability within bacterial and fungal community fingerprints among replicate field plots. Differences in bacterial and fungal phylogeny were related to depth in the soil profile but not to tillage management. Abundance of individual PLFA biomarkers were 7 to 86% greater in NT surface soils (0- to 5-cm depth), except at the Ellerslie site in 2005 where biomass was greater in CT. Responses at the 5- to 10-cm and 10- to 15-cm depths were more varied, in some cases with greater biomass in CT than NT soils. Ordination analysis of PLFA profiles showed clear community separation with depth but not tillage. Physiological stress biomarkers were correlated with simple measures of nutrient concentration and indicated that resource availability was likely the main factor determining community structure. It was concluded that tillage disturbance was not an overriding factor in determining microbial community composition in the long-term NT and CT soils studied. Further study of the interaction of cropping frequency with tillage management is needed to understand the effects of tillage disturbance on microbial turnover of plant derived residues.     

Parasitism by Cuscuta australis affects the rhizhospheric soil bacterial communities of Trifolium repens L.

ABSTRACT

The effect of parasitism on belowground microbial communities is not well understood. 16S rRNA gene amplicon sequencing was used to test the effect of Cuscuta australis parasitism on the composition and diversity of bacterial community in the rhizospheric soil of the host plant Trifolium repens. 94569 sequences were obtained from the amplicons of non-parasitised, and 97172 sequences were obtained from the parasitised rhizospheric soil bacterial community. Parasitism of C. australis significantly decreased the relative abundance of the bacterial phylum Nitrospirae, while it significantly increased that of Verrucomicrobia. Parasitism of C. australis significantly decreased the relative abundance of 10 bacterial genera, while it significantly increased those of nine genera. The Chao 1 indexes of the rhizospheric soil bacterial community of parasitised T. repens were significantly lower than those of non-parasitised T. repens. Principal coordinate analysis (based on the genus) and principal component analysis (based on the predicted gene function of bacterial communities) showed that rhizospheric bacterial communities from parasitised and non-parasitised T. repens differed and can be divided into two groups. These results suggest that infection of the holoparasitic plant could indirectly change the composition, diversity, and function of rhizospheric soil bacteria of the host plant.     

The importance of C,N and P as driver for bacterial community structure in German beech dominated forest soils

Among several environmental factors shaping soil microbial communities the impact of soil nutrients is of special interest. While continuous application mainly of N and P dramatically shifts community composition during fertilization, it remains unclear whether this effect is consistent in generic, unfertilized beech forest ecosystems of Germany, where differences in nutrient contents are mostly a result of the parental material and climatic conditions. We postulate that in such ecosystems nutrient effects are less pronounced due to the possibility of the soil microbiome to adapt to the corresponding conditions over decades and the vegetation acts as the major driver. To test this hypothesis, we investigated the bacterial community composition in five different German beech dominated forest soils, representing a natural gradient of total‐ and easily available mineral‐P. A community fingerprinting approach was performed using terminal‐Restriction Fragment Length Polymorphism analysis of the 16S rRNA gene, while abundance of bacteria was measured applying quantitative real‐time PCR. Bacterial communities at the five forest sites were distinctly separated, with strongest differences between the end‐members of the P‐gradient. However the majority of identified microbial groups (43%) were present at all sites, forming a core microbiome independent from the differences in soil chemical properties. Especially in the P‐deficient soil the abundance of unique bacterial groups was highly increased, indicating a special adaption of the community to P limitation at this site. In this regard Correspondence Analysis elucidated that exclusively soil pH significantly affected community composition at the investigated sites. In contrast soil C, N and P contents did mainly affect the overall abundance of bacteria.     

Analysis of the microbial communities in soils of different ages following volcanic eruptions

Volcanism is a primary process of land formation.It provides a model for understanding soil-forming processes and the role of pioneer bacteria and/or archaea as early colonizers in those new environments.The objective of this study was to identify the microbial communities involved in soil formation.DNA was extracted from soil samples from the Llaima volcano in Chile at sites destroyed by lava in different centuries(1640,1751,and 1957).Bacterial and archaeal 16 S r RNA genes were analyzed using quantitative polymerase chain reaction(q PCR)and Illumina Mi Seq sequencing.Results showed that microbial diversity increased with soil age,particularly between the 1751 and 1640 soils.For archaeal communities,Thaumarchaeota was detected in similar abundances in all soils,but Euryarchaeota was rare in the older soils.The analysis of bacterial 16 S r RNA genes showed high abundances of Chloroflexi(37%),Planctomycetes(18%),and Verrucomicrobia(10%)in the youngest soil.Proteobacteria and Acidobacteria were highly abundant in the older soils(16%in 1640 and 15%in 1751 for Acidobacteria;38%in 1640 and 27%in 1751 for Proteobacteria).The microbial profiles in the youngest soils were unusual,with a high abundance of bacteria belonging to the order Ktedonobacterales(Chloroflexi)in the 1957 soil(37%)compared with the 1751(18%)and 1640(7%)soils.In this study,we show that there is a gradual establishment of the microbial community in volcanic soils following an eruption and that specific microbial groups can colonize during the early stages of recovery.     

长期施用有机肥潮土细菌的多样性及功能预测

  【目的】  细菌作为土壤中最多的微生物物种,其多样性是土壤质量和土壤健康的标志。施肥不仅为土壤细菌提供了矿质养分,也为细菌提供了不同的碳源。探究长期不同施肥下潮土细菌的多样性及其功能,对于深入理解土壤养分转化的微生物驱动过程及优化施肥管理措施具有重要意义。  【方法】  选取潮土上连续37年不施肥(CK)、单施化学氮肥(N)、单施有机肥(M)、有机肥和化学氮肥配施(MN) 的4个处理,采用高通量测序方法分析土壤细菌丰度(16S rRNA基因拷贝数)、α多样性和β多样性,采用FAPROTAX功能预测的方法分析主要功能种群的丰度在处理间的差异,采用Mantel 检验分析细菌多样性等与产量、土壤养分含量间的相关关系。  【结果】  长期施用有机肥显著提高了土壤细菌丰度,MN和M处理下土壤中的16S rRNA基因拷贝数分别是CK和N处理平均值的11.8和10.7倍(P< 0.05)。土壤细菌多样性指数(Shannon index)和丰富度指数(Chao1 index)均在M处理下最高。潮土中变形菌门、酸杆菌门和放线菌门为优势菌门。与CK相比,施肥处理均提高了潮土拟杆菌门、厚壁菌门和疣微菌门的相对丰度。与N处理相比,MN处理显著提高了潮土变形菌门、厚壁菌门和拟杆菌门的丰度,降低了酸杆菌门的丰度。潮土细菌生态功能以化能异养、有氧化能异养、发酵作用、硝化作用和硝酸盐还原为主,相应种群丰度均在1%以上。MN处理下化能异养功能细菌的丰度较其他处理显著提高2.2%～4.2%。MN和M处理中起发酵作用的种群相对丰度是其他处理的3倍以上。与CK 相比,3个施肥处理均显著增加了光异作用和光异养作用功能种群的相对丰度。土壤细菌β多样性和功能结构均与土壤有机质(SOM)、全氮(TN)、硝态氮(NO₃?-N)含量及作物产量呈显著(P < 0.05)或极显著(P < 0.01)相关。  【结论】  长期施用有机肥提高了潮土中细菌的数量和多样性,提高了土壤中变形菌门、厚壁菌门和拟杆菌门的丰度,降低了酸杆菌门的丰度。有机肥和化肥配施处理中化能异养型细菌丰度较高,可能是施用有机肥后优化土壤养分循环和响应作物产量提高的主要菌群,今后可进一步深入研究其菌群组成和功能调控。     

施肥及秸秆还田对砂姜黑土细菌群落的影响

利用Illumina平台Miseq高通量测序技术对小麦分蘖期砂姜黑土耕层土壤细菌进行高通量测序,结合相关生物信息学分析,探讨了不施化肥秸秆不还田(CK)、施化肥秸秆不还田(F)以及不施化肥秸秆还田(W)3种处理土壤细菌群落组成、多样性和结构的变化。结果显示,测序共获得14 873个OTUs,计173 323条读数,平均读长439 bp。砂姜黑土细菌优势门(相对丰度10%)为变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes);优势纲(相对丰度10%)为α-变形菌纲(Alphaproteobacteria)、β-变形菌纲(Betaproteobacteria)、酸杆菌纲(Acidobacteria)、鞘脂杆菌纲(Sphingobacteriia)和γ-变形菌纲(Gammaproteobacteria);优势属(相对丰度1%)共47个,3个处理中均有分布的优势属21个,F处理的细菌优势属的种类最多,为39个。相对丰度最大的门、纲和属分别是变形菌门(38.7%~43.1%)、α-变形菌纲(14.5%~18.1%)和鞘氨醇单胞菌属(Sphingomonas)(4.6%~7.7%)。F处理细菌丰富度指数(Chao1指数和ACE指数)显著低于CK及W处理,W处理和CK处理土壤细菌丰富度指数无显著差异,与CK处理相比,F处理ACE指数降低22.8%。W处理土壤细菌Shannon多样性指数显著大于CK及F处理,W处理Shannon指数较CK处理提高4.1%,而F处理土壤细菌Shannon指数与CK处理无显著差异。F处理Simpson指数显著高于CK及W处理;F处理Simpson指数较CK处理提高38.1%,而W处理细菌Simpson指数最小,显著低于CK处理,较CK降低23.8%。分层聚类图显示在属的水平上,W处理和CK处理土壤细菌群落结构相似性较高,F处理与CK处理及W处理细菌群落结构差异较大。施化肥对土壤细菌优势类群组成、相对丰度及群落结构的影响大于秸秆还田,施化肥显著降低了土壤细菌丰富度,秸秆还田显著提高了土壤细菌的多样性。     

红壤坡地利用方式对土壤细菌群落结构的影响

利用中国科学院桃源农业生态试验站坡地不同利用方式长期定位观测试验场农田、自然恢复和茶园土壤为研究对象,直接从土壤中抽提总DNA,应用T-RFLP和RT-PCR技术研究红壤坡地利用方式对土壤细菌群落结构的影响。结果表明,红壤坡地三种土地利用方式土壤细菌多样性指数农田>茶园>自然恢复(p<0.05),但土壤细菌数量茶园>自然恢复>农田(p<0.05),茶园土壤细菌数量是农田的8.76倍。基于T-RFLP图谱的样品间相似性指数和主要限制性片段(T-RFs)定性分析均表明,农田与茶园和自然恢复土壤均存在显著的差异(p<0.05),而茶园和自然恢复土壤细菌群落比较相似。不同土地利用方式土壤有机质、有效磷和速效钾均对土壤细菌群落结构产生显著影响(p<0.05)。综合考虑经济效益和保持红壤坡地的可持续利用,茶园将成为中国南方红壤丘陵坡地可持续利用的一种有效方式。     

Bacterial diversity in soils subjected to long-term chemical fertilization can be more stably maintained with the addition of livestock manure than wheat straw

Addition of organic matter such as livestock manures and plant residues is a feasible practice to mitigate soil degradation caused by long-term application of chemical fertilizers, and the mitigation is largely mediated though activities of the soil-dwelling microorganisms. However, the roles of different kinds of organic matter in maintaining bacterial community structure have not been assessed in a comparative manner. In this study, 454 pyrosequencing of 16S rRNA gene was employed to compare the bacterial community structure among soils that had been subjected to 30 years of NPK fertilization under six treatment regimes: non-fertilization control, fertilization only, and fertilization combined with the use of pig manure, cow manure or low- and high-level of wheat straws. Consistent with expectation, long-term application of NPK chemical fertilizers caused a significant decrease of bacterial diversity in terms of species richness (i.e. number of unique operational taxonomic units (OTU)), Faith's index of phylogenetic diversity and Chao 1 index. Incorporation of wheat straw into soil produced little effects on bacterial community, whereas addition of either pig manure or cow manure restored bacterial diversity to levels that are comparable to that of the non-fertilization control. Moreover, bacterial abundance determined by quantitative PCR was positively correlated with the nutritional status of the soil (e.g., nitrate, total nitrogen, total carbon, available phosphorus); however, bacterial diversity was predominantly determined by soil pH. Together, our data implicate the role of livestock manures in preventing the loss of bacterial diversity during long-term chemical fertilization, and highlight pH as the major deterministic factor for soil bacterial community structure.     

Optimizing the indirect extraction of prokaryotic DNA from soils

The objective of this work was to develop protocols to selectively extract prokaryotic DNA from soils, representative of the whole community, amenable to high-throughput whole genome shotgun sequencing. Prokaryotic cells were extracted from soils by blending, followed by gradient centrifugation. Detergent (sodium deoxycholate) was required for complete dispersion of soil aggregates and detachment of prokaryotic cells from a broad range of soil types. Repeated extractions of a given soil sample were critical to maximize cell yield. Furthermore, cells obtained through repeated extractions captured unique prokaryotic assemblages that would otherwise have been missed in single-pass extractions. DNA was isolated from extracted cells using one of the following treatments: i) lysozyme-SDS-proteinase K (enzymatic) digestion; ii) potassium ethyl xanthogenate digestion; or iii) enzymatic digestion of cells embedded in agarose plugs. In addition, these methods were compared to a commercial bead-beating extraction kit (MoBio UltraClean). Of the indirect DNA extraction methods, plug digestion generated the largest yields (up to 70% of yields obtained by direct DNA extraction) of high-molecular weight DNA (>400 kb). Thus, plug digestion is amenable to large-insert metagenomic library construction and analysis. Comparisons of banding patterns generated by RAPD-PCR and DGGE indicated that sequence composition and inferred community composition of a given extract varied greatly with DNA isolation method. While overall diversity did not change significantly with the cell lysis method, analysis of 16S rRNA gene clone libraries revealed that each extraction procedure produced unique distributions of prokaryotic phyla within the sample population.