Multiplex Terminal Restriction Fragment Length Research Articles

The Status of Soil Microbiome as Affected by the Application of Phosphorus Biofertilizer: Fertilizer Enriched with Beneficial Bacterial Strains.

Regarding the unfavourable changes in agroecosystems resulting from the excessive application of mineral fertilizers, biopreparations containing live microorganisms are gaining increasing attention. We assumed that the application of phosphorus mineral fertilizer enriched with strains of beneficial microorganisms contribute to favourable changes in enzymatic activity and in the genetic and functional diversity of microbial populations inhabiting degraded soils. Therefore, in field experiments conditions, the effects of phosphorus fertilizer enriched with bacterial strains on the status of soil microbiome in two chemically degraded soil types (Brunic Arenosol—BA and Abruptic Luvisol—AL) were investigated. The field experiments included treatments with an optimal dose of phosphorus fertilizer (without microorganisms—FC), optimal dose of phosphorus fertilizer enriched with microorganisms including Paenibacillus polymyxa strain CHT114AB, Bacillus amyloliquefaciens strain AF75BB and Bacillus sp. strain CZP4/4 (FA100) and a dose of phosphorus fertilizer reduced by 40% and enriched with the above-mentioned bacteria (FA60). The analyzes performed included: the determination of the activity of the soil enzymes (protease, urease, acid phosphomonoesterase, β-glucosidase), the assessment of the functional diversity of microorganisms with the application of BIOLOGTM plates and the characterization of the genetic diversity of bacteria, archaea and fungi with multiplex terminal restriction fragment length polymorphism and next generation sequencing. The obtained results indicated that the application of phosphorus fertilizer enriched with microorganisms improved enzymatic activity, and the genetic and functional diversity of the soil microbial communities, however these effects were dependent on the soil type.

Methodological Aspects of Multiplex Terminal Restriction Fragment Length Polymorphism-Technique to Describe the Genetic Diversity of Soil Bacteria, Archaea and Fungi.

The molecular fingerprinting methods used to evaluate soil microbial diversity could also be used as effective biosensors for the purposes of monitoring ecological soil status. The biodiversity of microorganisms is a relevant index of soil activity and there is a necessity to develop tools to generate reliable results for an emerging approach in the field of environmental control using microbial diversity biosensors. This work reports a method under development for determining soil microbial diversity using high efficiency Multiplex PCR-Terminal Restriction Fragment Length Polymorphism (M-T-RFLP) for the simultaneous detection of bacteria, archaea and fungi. Three different primer sets were used in the reaction and the analytical conditions were optimized. Optimal analytical conditions were achieved using 0.5 µM of primer for bacteria and 1 µM for archaea and fungi, 4 ng of soil DNA template, and HaeIII restriction enzyme. Comparative tests using the proposed analytical approach and a single analysis of each microorganism group were carried out to indicate that both genetic profiles were similar. The Jaccard similarity coefficient between single and multiplexing approach ranged from 0.773 to 0.850 for bacteria and fungi, and 0.208 to 0.905 for archaea. In conclusion, the multiplexing and pooling approaches significantly reduced the costs and time required to perform the analyses, while maintaining a proper effectiveness.

Soil microbial communities following bush removal in a Namibian savanna

Abstract. Savanna ecosystems are subject to desertification and bush encroachment, which reduce the carrying capacity for wildlife and livestock. Bush thinning is a management approach that can, at least temporarily, restore grasslands and raise the grazing value of the land. In this study we examined the soil microbial communities under bush and grass in Namibia. We analyzed the soil through a chronosequence where bush was thinned at 9, 5, or 3 years before sampling. Soil microbial biomass, the biomass of specific taxonomic groups, and overall microbial community structure was determined by phospholipid fatty acid analysis, while the community structure of Bacteria, Archaea, and fungi was determined by multiplex terminal restriction fragment length polymorphism analysis. Soil under bush had higher pH, C, N, and microbial biomass than under grass, and the microbial community structure was also altered under bush compared to grass. A major disturbance to the ecosystem, bush thinning, resulted in an altered microbial community structure compared to control plots, but the magnitude of this perturbation gradually declined with time. Community structure was primarily driven by pH, C, and N, while vegetation type, bush thinning, and time since bush thinning were of secondary importance.

Influence of geogenic factors on microbial communities in metallogenic Australian soils

Links between microbial community assemblages and geogenic factors were assessed in 187 soil samples collected from four metal-rich provinces across Australia. Field-fresh soils and soils incubated with soluble Au(III) complexes were analysed using three-domain multiplex-terminal restriction fragment length polymorphism, and phylogenetic (PhyloChip) and functional (GeoChip) microarrays. Geogenic factors of soils were determined using lithological-, geomorphological- and soil-mapping combined with analyses of 51 geochemical parameters. Microbial communities differed significantly between landforms, soil horizons, lithologies and also with the occurrence of underlying Au deposits. The strongest responses to these factors, and to amendment with soluble Au(III) complexes, was observed in bacterial communities. PhyloChip analyses revealed a greater abundance and diversity of Alphaproteobacteria (especially Sphingomonas spp.), and Firmicutes (Bacillus spp.) in Au-containing and Au(III)-amended soils. Analyses of potential function (GeoChip) revealed higher abundances of metal-resistance genes in metal-rich soils. For example, genes that hybridised with metal-resistance genes copA, chrA and czcA of a prevalent aurophillic bacterium, Cupriavidus metallidurans CH34, occurred only in auriferous soils. These data help establish key links between geogenic factors and the phylogeny and function within soil microbial communities. In particular, the landform, which is a crucial factor in determining soil geochemistry, strongly affected microbial community structures.

Differential effect of afforestation on nitrogen-fixing and denitrifying communities and potential implications for nitrogen cycling

Denitrification and nitrogen (N) fixation are two major processes which drive N transformation in ecosystems. We investigated the effect of afforestation of pasture in New Zealand on denitrifying and N-fixing communities using molecular techniques. We optimised and applied multiplex-terminal restriction fragment length polymorphism for simultaneous analysis of denitrifying and N-fixing communities. Soil samples from four pine (Pinus radiata) forests of various age and adjacent pastures were analysed for microbial communities, denitrification rate and some environmental variables. Analysis of variance revealed that the denitrifying community was mainly influenced by site while the N-fixing community was influenced by both site and land-use types. Among environmental variables, moisture and porosity had the strongest impact on denitrifying communities while C:N ratio explained most of the variability in the N-fixing community. Denitrification rate as measured by nitrous oxide (N2O) flux, was significantly lower in older forest soils compared to adjacent pasture soils. Simple and step-wise regression analyses revealed that the N2O flux rate was strongly linked to the denitrifying community composition, providing evidence for the influence of community composition on denitrification. Moreover, we explored the relationship between denitrifying and N-fixing communities using various statistical approaches. Our results for the first time showed that denitrifying and N-fixing communities are related in situ, and suggest that changes in land-use may have a significant impact on N-transformations and ecosystem processes.

Comparison of microbial community assays for the assessment of stream biofilm ecology

We investigated a range of microbiological community assays performed on scrapes of biofilms formed on artificial diffusing substrates deployed in 8 streams in eastern Scotland, with a view to using them to characterize ecological response to stream water quality. The assays considered were: Multiplex Terminal Restriction Fragment Length Polymorphism or M-TRFLP (a molecular method), Phospholipid Fatty Acid or PLFA analysis (a biochemical method) and MICRORESP ™ (a physiological method) alongside TDI, diatom species, and chlorophyll a content. Four of the streams were classified as of excellent status (3–6 μg/L Soluble Reactive Phosphorus (SRP)) with respect to soluble P content under the EU Water Framework Directive and four were of borderline good/moderate or moderate status (43–577 μg/L SRP). At each site, 3 replicates of 3 solute diffusion treatments were deployed in a Latin square design. Solute diffusion treatments were: KCl (as a control solute), N and P (to investigate the effect of nutrient enrichment), or the herbicide isoproturon (as a “high impact” control, which aimed to affect biofilm growth in a way detectable by all assays). Biofilms were sampled after 4 weeks deployment in a low flow period of early summer 2006. The chlorophyll a content of biofilms after 4 weeks was 2.0 ± 0.29 mg/m 2 (mean ± se). Dry matter content was 16.0 ± 13.1 g/m 2. The M-TRFLP was successfully used for generating community profiles of cyanobacteria, algae and bacteria and was much faster than diatom identification. The PFLA and TDI were successful after an increase in the sample size, due to low counts. The MICRORESP ™ assays were often below or near detection limit. We estimated the per-sample times for the successful assays as follows: M-TRFLP: 20 min, PLFA 40 min, TDI 90 min. Using MANOVA on the first 5 principal co-ordinates, all the assays except MICRORESP ™ showed significant differences between sites, but none of the assays showed a significant effect of either initial stream trophic status (as classified by the EU Water Framework Directive using chemical standards for soluble P), or of the diffusing solute treatment. Multiple Procrustes analysis on the ordination results showed that the diatom and M-TRFLP data sets hold distinct, though as yet unexplored, information about the ecological factors affecting stream biofilms. The diatom data were subjected to principal components analysis, to identify which taxa were more strongly influenced by site variables, trophic status or treatment effects. These were Acnanthes lanceolata, A. minutissimma, Nitzchia spp., Coccineis spp. and Navicula spp. Further experimentation and data analysis on a larger number of sites, to identify specific M-TRFLP bands that could be used as indicators linked to specific taxa, are desirable. Results highlight the need for a multifactorial approach to understanding controls on stream ecology.

Microbial Community Structure and Enzyme Activities in a Sequence of Copper-Polluted Soils

The microbial community structure and enzyme activities of seven paddy soils with different Cu concentrations were investigated in the vicinity of a Cu smelter in Fuyang County, Zhejiang Province in Southeast China. The microbial community structure was analyzed using the phospholipid fatty acid (PLFA) and multiplex-terminal restriction fragment length polymorphism (M-TRFLP) techniques. There was no clear dose-response relationship between Cu pollution and soil enzyme activity except for urease. Both PLFA and M-TRFLP methods showed that Cu contamination had a large effect on the soil microbial community structure. PLFA indicators of Gram-positive bacteria (16:0i, 15:0i) and fungi (18:2w6,9) relatively decreased with increasing Cu concentration, whereas indicators of Gram-negative bacteria (19:0cy, 16:1w7) increased. The M-TRFLP results suggested that there was a dose-dependent response between Cu pollution and bacterial community or fungal community. The fungal community was more sensitive to Cu pollution than the bacterial community. There were no significant differences in archaeal community structure between the different Cu pollution plots and archaea might be more tolerant to Cu pollution than both bacteria and fungi.

Is vegetation composition or soil chemistry the best predictor of the soil microbial community?

With the species composition and/or functioning of many ecosystems currently changing due to anthropogenic drivers it is important to understand and, ideally, predict how changes in one part of the ecosystem will affect another. Here we assess if vegetation composition or soil chemistry best predicts the soil microbial community. The above and below-ground communities and soil chemical properties along a successional gradient from dwarf shrubland (moorland) to deciduous woodland (Betula dominated) were studied. The vegetation and soil chemistry were recorded and the soil microbial community (SMC) assessed using Phospholipid Fatty Acid Extraction (PLFA) and Multiplex Terminal Restriction Fragment Length Polymorphism (M-TRFLP). Vegetation composition and soil chemistry were used to predict the SMC using Co-Correspondence analysis and Canonical Correspondence Analysis and the predictive power of the two analyses compared. The vegetation composition predicted the soil microbial community at least as well as the soil chemical data. Removing rare plant species from the data set did not improve the predictive power of the vegetation data. The predictive power of the soil chemistry improved when only selected soil variables were used, but which soil variables gave the best prediction varied between the different soil microbial communities being studied (PLFA or bacterial/fungal/archaeal TRFLP). Vegetation composition may represent a more stable ‘summary’ of the effects of multiple drivers over time and may thus be a better predictor of the soil microbial community than one-off measurements of soil properties.

Physiological, biochemical and molecular responses of the soil microbial community after afforestation of pastures with Pinus radiata

Afforestation and deforestation are key land-use changes across the world, and are considered to be dominant factors controlling ecosystem functioning and biodiversity. However, the responses of soil microbial communities to these land-use changes are not well understood. Because changes in soil microbial abundance and community structure have consequences for nutrient cycling, C-sequestration and long-term sustainability, we investigated impacts of land-use change, age of stand and soil physico-chemical properties on fungal and bacterial communities and their metabolic activities. This study was carried out at four sites in two geographical locations that were afforested on long-established pastures with Pinus radiata D. Don (pine). Two of the sites were on volcanic soils and two on non-volcanic soils and stand age ranged from 5 to 20 y. Microbial communities were analysed by biochemical (phospho-lipid fatty acids; PLFA) and molecular (multiplex-terminal restriction fragment length polymorphism; M-TRFLP) approaches. Both site and stand age influenced microbial properties, with changes being least detectable in the 5-y-old stand. Land use was a key factor influencing soil metabolic activities as measured by physiological profiling using MicroResp. Pasture soils had higher microbial biomass ( P < 0.001), and metabolic activities ( P < 0.001), and basal respiration rates were up to 2.8-times higher than in the pine soils. Microbial abundance analysis by PLFA showed that the fungal to bacterial ratio was higher in the pine soils ( P < 0.01). Community analysis suggested that soil bacterial communities were more responsive to site (principal component 1; P < 0.001) than to land use (principal component 5; P < 0.001). In contrast, the fungal community was more affected by land-use change (principal component 1; P < 0.001) than by site, although site still had some influence on fungal community structure (principal component 2; P < 0.001). Redundancy analysis also suggested that bacterial and fungal communities responded differently to various soil abiotic properties, land-use change and location of sites. Overall, our results indicate that the change in land use from pasture to P. radiata stands had a direct impact on soil fungal communities but an indirect effect, through its effects on soil abiotic properties, on bacterial communities. Most of the changes in bacterial communities could be explained by altered soil physico-chemical properties associated with afforestation of pastures.

Multiple profiling of soil microbial communities identifies potential genetic markers of metal-enriched sewage sludge

The long-term impacts of Cu- and Zn-rich sewage sludge additions on the structure of the microbial community in a field under pasture were investigated using a combination of multiplex-terminal restriction fragment length polymorphism (M-TRFLP) and T-RFLP profiling approaches. Changes in the community structure of bacteria, fungi, archaea and actinobacteria were observed in soils that had previously received Cu- (50-200 mg kg(-1) soil) and Zn- (150-450 mg kg(-1) soil) rich sewage sludge additions. Changes in the structure of all microbial groups measured were observed at Cu and Zn rates below the current EU guidelines (135 mg kg(-1) Cu and 300 mg kg(-1) Zn). The response of the fungal community, and to a lesser extent the bacterial and archaeal community, to Cu was dose dependent. The fungal community also showed a dose-dependent response to Zn, which was not observed in the other microbial groups assessed. Redundancy analysis demonstrated that individual terminal restriction fragments responded to both Cu and Zn and these may have potential as genetic markers of long-term metal effects in soil.

Microbial DNA profiling by multiplex terminal restriction fragment length polymorphism for forensic comparison of soil and the influence of sample condition

To evaluate: (i) the impact of air-drying on bacterial, archaeal and fungal soil DNA profiles and (ii) the potential use of multiplex-terminal restriction fragment length polymorphism (M-TRFLP) as a tool for forensic comparison of soil. An M-TRFLP approach was used to profile bacterial, archaeal and fungal DNA profiles from five different soil sites. Air-drying soil significantly reduced the quantity of DNA but the number of operational taxanomic units (OTU) was unaffected. The impact of air-drying on soil DNA profiles was dependent on soil site and microbial primers. Fungal profiles were altered the least by air-drying. For prokaryotic profiles, air-drying altered the relative similarity/dissimilarity between soil sites. The M-TRFLP approach was more discriminatory compared with soil colour and single-taxa profiling, but did not significantly improve resolution between two similar soils. Of those tested, soil fungi were potentially the more robust target for application to soil forensic studies as they were altered less by air-drying and provided clear discrimination of soils from different sites. The M-TRFLP method demonstrated potential to achieve greater resolution, discriminating the soil sites based on both bacterial and fungal components. Soil DNA profiling has potential as a forensic tool, but sample condition and the appropriate selection of microbial target taxa must be considered.

Long-term exposure to Zn-spiked sewage sludge alters soil community structure

An 8 year study to investigate the effects of Zn-spiked sewage sludge additions on the microbial community structure and microbial processes was carried out in a field soil under pasture. The microbial community structure was evaluated using a combination of multiplex-terminal restriction fragment length polymorphism (M-TRFLP) and T-RFLP fingerprinting approaches. Soil respiration, microbial biomass and enzymatic activities were measured as indicators of soil microbial processes. Changes in the microbial community structure, with Zn additions were evident in all the microbial groups investigated (bacteria, fungi, archaea, actinobacteria and rhizobia/agrobacteria). The fungal community showed the greatest response to Zn additions compared to the other microbial communities measured. The relative abundance of several fungal terminal restriction fragments (TRFs) significantly increased in high Zn treated treatments, at the expense of others, some of which were lost from T-RFLP profiles completely. These results indicate that metal-spiked sludge application can have long-lasting impacts on the composition of the microbial community in pasture soils. Despite notable changes in community structure there was no significant long-term impact of Zn-spiked sludge applications on microbial respiration, biomass or enzyme activities.

Multiplex-terminal restriction fragment length polymorphism

A novel method called "multiplex-terminal restriction fragment length polymorphism (M-TRFLP)" has been recently developed which can be used for simultaneous analysis of the community composition of two or more microbial taxa (up to four). This method can also be used for microbial diagnostic purposes. For M-TRFLP analysis, primers specific to different target genes are used for multiplex-PCR, with one primer for each target being labeled with a unique fluorescent dye at its 5' end. Restriction digestion of the amplified products followed by fragment size analysis on a DNA sequencer produces profiles for targeted genes, which can be distinguished from each other by the color of the terminal fragments imparted by the unique fluorescent dye used for primer labeling. In contrast to current protocols, M-TRFLP allows multiple communities or multiple targets (genes) data to be obtained in just one reaction and therefore saves time, cost and labor. This protocol can be completed in 5-8 h.

Use of Multiplex Terminal Restriction Fragment Length Polymorphism for Rapid and Simultaneous Analysis of Different Components of the Soil Microbial Community ▿

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.