Evaluating the physical capture method of terminal restriction fragment length polymorphism for comparison of soil microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a popular method of comparative microbial community analysis which is normally accomplished by tagging terminal restriction fragments (T-RFs) with a fluorescent primer. Here, we evaluate an alternative method of T-RFLP where T-RFs are physically captured using a biotinylated primer and streptavidin-coated beads. This eliminates one of the primary criticisms of T-RFLP, namely that T-RFs cannot be identified by sequence analysis, and also represents an alternative method for collecting T-RFLP profiles. Microbial communities from forest, agricultural, and turf soils were investigated using several sets of primers specific for different microbial groups. The physical capture method of T-RFLP resulted in similar profiles to those generated by fluorescent T-RFLP. The relationships among ecosystem types captured by both methods and revealed by ordination were virtually identical. The total variance in the profiles that was attributed to ecosystem type was approximately equal, or greater, when generated by the physical capture method, depending on the primers used. However, physical capture T-RFLP resolved fewer T-RFs than fluorescent T-RFLP, and this may reduce the sensitivity to changes in non-dominant populations within the community. Direct cloning and sequencing of physical capture T-RFs revealed that most bands were not comprised of sequences related to those in the database that would generate T-RFs of similar size. T-RFs should therefore be identified by sequencing, rather than by comparing the sizes of T-RFs to computer digests of database sequences. Physical capture T-RFLP should be a useful tool to identify T-RFs by sequencing, and for laboratories without economical access to equipment required to perform fluorescent T-RFLP.     

转Bt基因棉花根际细菌与古菌群落结构分析

在一年内棉花的四个生长时期（苗期,蕾期,花铃期,吐絮期）分别采集转Bt基因抗虫棉GK12和非转基因亲本棉花泗棉3号根际土壤,以及未种植棉花的背景土壤,利用末端标记限制性片段长度多态性（T-RFLP）分析技术,分析三种土壤中细菌和古菌的16S rRNA基因片段多态性,结合克隆文库建立和测序,研究了土壤中细菌和古菌群落结构的变化.结果表明：在棉花生长的各个时期,背景土壤中细菌群落结构发生了明显的变化,生物多样性指数明显降低,古菌群落结构也有一定的变化,说明季节性变化对土壤中微生物群落产生了明显的影响.与背景土壤相比,棉花种植后根际土壤中细菌和古菌群落发生显著的变化.转基因棉花与非转基因棉花相比,根际土壤细菌和古菌的种类和种群大小的分布也发生了明显的改变.克隆文库和测序结果表明土壤中主体微生物为目前未培养的、功能特性未知的细菌和古菌,转基因棉花种植对这些细菌和古菌影响的原因、环境危害和生态风险目前尚不清楚.与古菌群落相比,棉花种植对细菌群落结构的影响较小.     

Glucose additions to aggregates subjected to drying/wetting cycles promote carbon sequestration and aggregate stability

Biogeochemical mechanisms at microscale regions within soil macroaggregates strengthen aggregates during repeated DW cycles. Knowledge of additional biogeochemical processes that promote the movement of dissolved organic carbon (DOC) into and throughout soil aggregates and soil aggregate stabilization are essential before we can more accurately predict maximum carbon (C) sequestration by soils subjected to best management practices. We investigated the spatial distribution of ¹³C-glucose supplied to individual soil macroaggregate surfaces and subjected to multiple drying and wetting (DW) cycles. Subsequent distribution of added glucose-C, CO₂ respiration, increased microbial community activity and concomitant changes in soil aggregate stabilization were monitored. Moist macroaggregates were treated with no DW cycles and zero glucose C (Control), 5 DW cycles and zero glucose (DW0G), and 5 DW cycles with additions of 250 μg glucose-¹³C/g soil during each cycle (DW+G). Repeated additions of glucose-C to aggregate surfaces reduced the mineralization of pre-existing soil C by an average of 45% and established concentric gradients of glucose-derived C. It is concluded these increasing gradients promoted the diffusion of soluble C into interior regions and became less available to microbial respiration. Spatial gradients of glucose-derived C within aggregates influenced a shift in the abundance of unique ribotypes spatially distributed within aggregates. Rapid decreases in the mineralization rates of glucose-C during repeated DW cycles suggested greater C sequestration by either physical restriction of microbes or chemical sorption of new C that diffused into aggregates. Aggregate stability decreased significantly following 2-3 DW cycles, when glucose-C was not added. Additions of glucose-C with each DW cycle maintained soil aggregate stability equal to the moist but not cycled control throughout the 5 DW cycles of this study. These data simulate the strengthening of soil aggregates in no tillage agroecosystems which provides continuous additions of DOC compounds generated by decomposing plant residues on the soil surface, and root exudates and decomposition, as well as the mineralization of POM materials within nondisturbed soil profiles.     

Microbial community response to soil solarization in Nepal's rice-wheat cropping system

The Indo-Gangetic Plains of South Asia support 13.5 million hectares of rice-wheat cropping systems, which currently feed over one billion people. Intensified agriculture has resulted in a more than two-fold increase in rice and wheat yields since the 1970s; however, this continuous cropping has also exacerbated weed, pest and disease problems. Soil solarization is an accessible, low-risk management practice for small-holder farmers that has ameliorated these problems in some settings and has the potential to dramatically improve yields. Field trials were conducted at two sites in Nepal to test whether soil solarization: (i) had a lasting effect on soil bacterial, fungal and nematode communities; (ii) altered the rhizosphere communities of rice nursery seedlings and (iii) improved crop growth and yield in the rice-wheat cropping system. Rice seedlings were grown in nursery plots that were solarized for 28 days or left untreated and were transplanted to field plots that were also either solarized for 28 days or not in a randomized complete block design with four replications. Rice was grown to maturity and harvested, followed by a complete wheat cropping cycle. Solarization of main field plots increased counts of fungal propagules and decreased root galling and nematode counts and decreased weed biomass. Terminal restriction fragment length polymorphism (T-RFLP) analyses of extracted soil DNA revealed significant shifts in fungal community composition following soil solarization, which was sustained throughout the entire rice cropping cycle at both field sites. The bacterial community composition was similarly affected, but at only one of the two sites. Despite the observed changes in soil microbial community composition over more than one cropping period, solarization had no impact on crop productivity at either site. Nevertheless, such changes in soil microbial communities in response to solarization may be responsible for increased yields observed at other sites with greater pathogen pressure. This practice has shown promising results in many farmers’ fields in South Asia, but further elucidation of the mechanisms by which solarization increases productivity is needed.     

Effects of Consecutively Monocultured Rehmannia glutinosa L. on Diversity of Fungal Community in Rhizospheric Soil

Continuous monoculture problems, or replanting diseases, are one of the key factors affecting productivity and quality of Chinese medicinal plants. The underlying mechanism is still being explored. Most of the studies on continuous monoculture of Rehmannia glutinosa L. are focused on plant nutritional physiology, root exudate, and its autotoxicity. However, the changes in the diversity of microflora in the rhizosphere mediated by the continuous monoculture pattern have been remained unknown. In this study, terminal restriction fragment length polymorphism (T-RFLP) technique was used for fingerprinting fungal diversity in the rhizosphere soil sampled from the fields of R. glutinosa monocultured for 1 and 2 yr. The results showed that the structure of fungal community in consecutively moncultured rhizosphere soil was different from that in control soil (no cropping soil), and varied with the consecutive monoculture years (1 and 2 yr). The comprehensive evaluation index (D) of fungal community estimated by principal component analysis of fragment number, peak area, Shannon-Weiner index, and Margalef index was higher in 1 yr monoculture soil than that in 2 yr monoculture soil, suggesting that consecutive monoculture of R. glutinosa could be a causative agent to decrease the diversity of fungal community in the rhizosphere soil.     

Methane concentrations and methanotrophic community structure influence the response of soil methane oxidation to nitrogen content in a temperate forest

Methane oxidation in forest soils removes atmospheric CH₄. Many studies have determined methane uptake rates and their controlling variables, yet the microorganisms involved have rarely been assessed simultaneously over the longer term. We measured methane uptake rates and the community structure of methanotrophic bacteria in temperate forest soil (sandy clay loam) on a monthly basis for two years in South Korea. Methane uptake rates at the field site did not show any seasonal patterns, and net uptake occurred throughout both years. In situ uptake rates and uptake potentials determined in the laboratory were 2.92 ± 4.07 mg CH₄ m⁻² day⁻¹ and 51.6 ± 45.8 ng CH₄ g⁻¹ soil day⁻¹, respectively. Contrary to results from other studies, in situ oxidation rates were positively correlated with soil nitrate concentrations. Short-term experimental nitrate addition (0.20-1.95 μg N g⁻¹ soil) significantly stimulated oxidation rates under low methane concentrations (1.7-2.0 ppmv CH₄), but significantly inhibited oxidation under high methane concentrations (300 ppmv CH₄). We analyzed the community structures of methanotrophic bacteria using a DNA-based fingerprinting method (T-RFLP). Type II methanotrophs dominated under low methane concentrations while Type I methanotrophs dominated under high methane concentrations. Nitrogen addition selectively inhibited Type I methanotrophic bacteria. Overall, the results of this study indicate that the effects of inorganic N on methane uptake depend on methane concentrations and that such a response is related to the dissimilar activation or inhibition of different types of methanotrophic bacteria.     

象山港网箱养殖对近海沉积物细菌群落的影响

微生物在有机物质分解、能量转移等生物地化循环中起着重要作用,研究养殖活动对沉积物中微生物多样性的影响对正确评价近海生态系统结构和功能具有重要意义.结合沉积物的理化分析(T、pH、Eh和SO2-),通过T-RFLP(末端限制性片段长度多态性)分析了象山港网箱养殖对近海不同深度沉积物中细菌群落结构和多样性的影响,构建了对照区0-5 cm沉积物样品的细菌16S rRNA基因克隆文库,并进行了系统发育分析.结果表明,近海网箱养殖活动不但使象山港沉积物Eh、pH值等理化性质有所改变,而且使细菌的群落结构也发生了变化,变形细菌纲细菌的相对丰度降低,细菌群落的多样性和均匀度也显著降低,致使近海沉积物细菌群落由多样性高的稳定结构向多样性降低的不稳定结构转变.     

Effects of salinity on the soil microbial community and soil fertility

Saline area is an important reserve resource of arable land, however, the effects of soil microorganisms on the soil fertility in saline coastal ecosystems remain poorly understood. The salinity effects on soil microorganisms, nutrient availabilities and their relationships were studied in soils along a salinity gradient. A total of 80 soil samples were collected from 16 sites at four salinity levels(non-saline soil, salt content1 g kg–1; low salinity soil, salt content=1–2 g kg–1; middle salinity soil, salt content=2–4 g kg–1; high salinity soil, salt content4 g kg–1). The results showed that the salinity increased soil pH and exchangeable Na percent, but decreased soil organic matter, soil exchangeable K, and soil microbial biomass. Both the abundance and community composition of soil bacteria and fungi were significantly different between the non-saline and the saline soils. The predominant genera of soil bacteria(Planctomyces and Archangium, positive for carbon fixation) and fungi(Hydropisphaera, efficient in lignin degradation) changed with the increasing soil salinity and the decreasing soil organic matter. In summary, soil salinity changed the abundances of soil bacterial, fungal, and arbuscular mycorrhizal communities and, subsequently, affected their function in saline coastal ecosystems.     

乌鲁木齐10号泉水体细菌群落结构的动态变化及响应模式初析

[目的]了解乌鲁木齐10号泉水体细菌多样性及其对水文地球化学指标的响应.[方法]利用末端限制性片段长度多态性(Terminal Restriction Fragment length polymorphism,T- RFLP),对不同日期水体细菌多样性进行分析,所获信息与水文地球化学指标进行典型相关分析.[结果]通过各样品水体细菌类群的多样性指数计算和样品间相似性分析共同显示,乌鲁木齐10号泉不同日期水体细菌呈现随机性动态变化;典型相关分析(Canonical correspondence analysis,CCA)显示水体细菌群落结构与H2S、CH4、F-三项水文地球化学指标呈显著正相关,并且受多种水文地球化学指标的共同影响.[结论]乌鲁木齐10号泉水体细菌类群表现为随机性动态变化,且可对水文地球化学元素产生响应.