Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

Quantification of the slug parasitic nematode Phasmarhabditis hermaphrodita from soil samples using real time qPCR

Phasmarhabditis hermaphrodita is a nematode parasite that infects and kills several species of slugs. The nematode is produced commercially as a biological control agent for slug pests of agriculture and horticulture. Given the difficulties of distinguishing this species from other nematode species in soil samples, very little is known about its natural ecology or its behaviour and persistence following application for biological control. Here we describe a method to quantify P. hermaphrodita in soil samples based on real time PCR. We designed primers and a dual labelled fluorescent probe that can be used to quantify numbers of P. hermaphrodita and which is capable of distinguishing this species from the morphologically identical Phasmarhabditis neopapillosa. We compared different methods whereby the entire nematode community is extracted prior to DNA extraction, and three methods to extract DNA directly from soil samples. Both nematode extraction and DNA extraction from large (10 g) samples of soil gave reliable estimates of nematode numbers, but methods which extracted DNA from small (1 g or less) soil samples substantially underestimated numbers. However, direct extraction of DNA from soils may overestimate numbers of live nematodes as DNA from dead nematodes was found to persist in soil for at least 6 days. The technique could be modified for detection and quantification of all soil borne parasitic nematodes.     

Development of a catabolically significant genetic probe for polycyclic aromatic hydrocarbon-degrading <Emphasis Type="Italic">Mycobacteria</Emphasis> in soil

A gene probe for the detection of polycyclic aromatic hydrocarbon (PAH) induced nidB and nidA dioxygenase genes has been designed from Mycobacteria JLS, KMS, and MCS. The probe detects a catabolic gene involved in the initial steps of PAH biodegradation in mycobacteria. The gene probe is comprised of three PCR primer sets designed to detect the genes that code for two subunits of the PAH induced dioxygenase enzyme within PAH-degrading mycobacteria. The probe was built by combining three primer sets with a DNA extraction procedure that was designed to lyse the gram-positive mycobacteria cells while in the soil matrix and remove PCR inhibitors. The probe was tested on PAH contaminated soils undergoing bioremediation through landfarming and uncontaminated soils from the same site. The PAH gene probe results demonstrate that the dioxygenase genes can be detected in soils. Sequencing the nidA and nidBPCR products verified that the genes were detected in soil. Comparisons of the sequences obtained from the soil probe to seven known nid gene sequences from various PAH-degrading mycobacteria showed between 97 and 99% nucleotide matches with the nidB gene and 95 and 99% matches with the nidA gene.     

Development of a gas diffusion probe for the determination of methane concentrations and diffusion characteristics in flooded paddy soil

Abstract A probe for the measurement of dissolved CH₄ in anoxic methanogenic environments was developed. The probe was based on the diffusion of dissolved CH₄ through a silicone membrane into a gas space at the end of the probe. This gas space was flushed with N₂ and analyzed gas-chromatographically for CH₄. The probe had a spatial resolution of < 1.3 mm, the detection limit was about 20 μM CH₄, the precision of the measurement was 9%, and consecutive measurements could be made every 4 min. Memory effects after analysis of high CH₄ concentrations could be avoided by flushing the probe with N₂ between each measurement. The probe was sensitive for water movement and, therefore, was calibrated in an artificial sediment of glass beads (100 μm diam.) immersed by aqueous solutions of known CH₄ concentrations. Sensitivity of the probe for changes in the sediment's porosity could not presently be excluded. The probe was used to measure vertical profiles of dissolved CH₄ in microcosms of anoxic paddy soil. The vertical CH₄ profiles measured with the probe compared fairly well with those measured after an extraction procedure. The profiles clearly showed that CH₄ was produced in deeper layers and diffused upwards to be consumed in the oxic top 2 mm soil layers. The probe was also used to determine the diffusion coefficient of CH₄ in an inactivated paddy soil microcosm using a set-up which allowed modelling of a measured CH₄ concentration profile using Fick's 2nd law.     

Analysis of the spatial variability in maize root density

In a maize field, one inter-row out of two was compacted two years down to 30-cm depth. This compacted inter-row (CIR) had a low root density down to 85-cm depth, while the soil below the row and the non compacted inter-row (NCIR) was densely rooted. Soil water status was monitored in each of these three compartments using tensiometers, neutron probe and gravimetric measurements. Both years, the rate of water extraction was about one half in the CIR compared with the row and the NCIR. As a consequence, appreciable differences in soil water potential were observed between colonized and sparsely colonized zones of each layer. These horizontal gradients were steeper than the vertical gradient between layers. This calls into question the suitability of one-dimensional models of water extraction for non-regular root systems, which are common in the field.     

Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and PCR amplification assay.

A magnetic capture-hybridization PCR technique (MCH-PCR) was developed to eliminate the inhibitory effect of humic acids and other contaminants in PCRs targeting specific soil DNA. A single-stranded DNA probe, which was complementary to an internal part of the target gene, was used to coat magnetic beads. After hybridization in a suspension of soil DNA, magnetic extraction of the beads separated the hybrid DNA from all other soil DNA, humic acids, and other interfering soil components. The MCH was followed by PCR amplification of the specific target DNA. In barley rhizosphere soil, detection of a lux gene inserted in a Pseudomonas fluorescens strain could be demonstrated in nonsterile soil samples (0.5 mg). This corresponded to a detection of fewer than 40 bacterial cells per cm of barley root. The MCH-PCR technique greatly improves the current protocols for PCR detection of specific microorganisms or genes in soil because specific target DNA sequences from very small soil samples can be extracted and determined.     

DNA extraction from soil: comparison of different methods using spore-forming bacteria and theswrAA gene as indicators

Soil microcosms seeded with spores of a tracer organism (Bacillus subtilis strain PB5332) were used to test five different DNA extraction protocols hereby indicated as A, B, C, D and E. The representativity of DNA samples obtained from each procedure was evaluated by PCR amplification of theswrAA gene, unique to PB5332 strain, followed by Southern hybridization with a gene-specific probe. A significant improvement of DNA extraction from spores was obtained using grinding under liquid N2 associated with sodium-dodecyl sulphate (SDS)-based lysis in presence of 1% hexadecyltrimethylammonium bromide (CTAB; protocol C). The same procedure was tested on soil samples from two distinct greenhouse trials carried out with genetically modified white poplars (Populus alba L) expressing theStSy gene for resveratrol production and thebar gene for Basta® tolerance, respectively. The representativity of DNA samples recovered from the greenhouse soil was assessed using three spore-forming bacteria (SFB) as tracer organisms. The tracers (SFB-1, SFB-2 and SFB-3) were previously isolated from the same trials classified as members of the genusBacillus. All the tested DNA samples produced the expected amplification products, indicating the presence at the soil level of the tracers and confirming the reliability of the optimized DNA extraction protocol.     

Development of a molecular method to detect and quantify Aphanomyces euteiches in soil

A real-time PCR assay using 136F/211R primers and 161T TaqMan probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.     

Experimental determination and modelling of the soil water extraction capacities of crops of maize,sunflower, soya bean,sorghum and wheat

The estimation of soil water reserves is essential for irrigation management. The usual way of calculating these reserves, held between the soil moisture content at field capacity and the classical limit of –1.5 MPa considered as the lower limit of available water, over the rooting depth of the crop, does not correspond with the real behaviour of crops as regards their ability to extract soil water and should be only considered as the apparent available water (AAW). Measurements of moisture profiles made using a neutron probe soil moisture meter from 1970 until 1991 on unirrigated crops at the INRA Agronomy Station at Toulouse-Auzeville, France, on a deep silty clay soil with a high water holding capacity have enabled us to define the water extraction capacities of maize ( Zea mays L.), sunflower (Helianthus annuus L.), sorghum (Sorghum bicolor L. Moench), soya bean (Glycine max L. Merr.), and winter wheat (Triticum aestivum L.). The results show, not only that all the crops can extract soil water from beyond –1.5 MPa in the surface layers to varying degrees and depths, depending on the crop, but also that deeper down, AAW is not fully used, as the moisture profile gradually returns to field capacity. Of the five crops studied, maize extracts the most water from the top 0.5 m, removing 150% of AAW. This amount falls rapidly lower down, reaching nil at 1.6 m. Conversely sunflower extracts less near the surface, but uses all AAW up to 1.2 m, and still extracts 85% of AAW at 1.6 m. Sorghum is somewhat comparable to sunflower, but with a lower use over the entire profile. Soya bean exhibits strong extraction to 1.0 m, and then much less at depth. As to wheat, its extraction capability is quite high near the surface, and then falls steadily with depth where it is still 30% of AAW at 1.6 m. Soil moisture measurements realised on a bare soil during several successive years were used to fix the maximum soil evaporation and to suggest the contribution of crops in soil water depletion from uppermost layers.The water extraction capacities have been modelled and introduced into the model EPICphase, a modified version of the model EPIC, adapted for irrigation management. Four parameters have been introduced to simulate: (1) the rooting pattern of the crop (parameter ), (2) the degree of involvement of deep layers (parameter p), (3) the fraction of AAW beyond which crop transpiration is affected (parameter t) and (4) the intensity of extraction beyond the limit of –1.5 MPa as a function of soil depth (parameter d). Calibrated on the basis of the driest year since 1970 for each crop, the model was then validated under unirrigated conditions, and then tested on irrigated maize plots. Under unirrigated conditions, the simulations correctly reproduced the water extraction by the five crops, both in an extremely dry year and in a wet year. The observed differences between simulations and observations were found mostly at about 0.1 m depth, and were due to lack of precision of moisture measurements with the neutron probe. From 0.2 to 0.6 m the simulations have a tendency to overestimate the extraction. These differences are explained by water fluxes which are especially high in these layers because of the processes of evaporation from the soil and plant transpiration, which are difficult to simulate with precision. Below 0.6 m, a more stable zone where water movements are of minor importance, the simulations are very precise. For irrigated maize, the results show a very good fit between simulation and measurement, indicating that these water extraction capacity figures could be used for irrigation management provided that the rules for exploitation of the water reserves are well established.     

Growth of crop roots in relation to soil moisture extraction

Growth of the roots of sugar beet, potato and barley in the field was observed through glass panels and related to changes in soil moisture measured by a neutron probe during 1969–71. The depth of observed root growth was generally related to, but 10–15 cm deeper than, the maximum depth of soil-moisture extraction. On average of three years, sugar beet, potato and barley used water from the top 23, 33 and 45 cm soil respectively by the beginning of June, and from the top 70, 68 and > 100 cm soil by the end of June. Maximum soil drying in each horizon gave an in situ measure of available water capacity, and showed that sugar beet and barley eventually extracted similar amounts of water from each horizon, but potatoes extracted less, especially from below 60 cm. Between 30 and 100 cm deep, the in situ available water capacity (per 10 cm soil) progressively decreased from 16 to 10, 15 to 5 and 16 to 8 mm under sugar beet, potato and barley respectively. The calculated soil-moisture deficit (potential evapotranspiration minus rainfall) and measured soil moisture deficit were not related early in the growing period before the crops established much leaf cover.     

Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.     

Effects on microbial activity by extraction of indigenous cells from soil slurries

Abstract: Possible effects on the physiological activity and culturability of soil microorganisms by different soil dispersion procedures, and effects on activity caused by extracting bacteria from soil, were investigated. There was no apparent difference in cfu's with dispersion of a silty loam soil and a loamy sand soil with pyrophosphate as compared to dispersion in NaCl. Substrate-induced respiration was reduced in the silty loam soil, and methanol oxidation was reduced in the loamy sand soil with dispersion in pyrophosphate, and the soil pH was irreversibly increased by the treatment. Extracted bacterial fractions had lower numbers of culturable cells as percentage of the total number of bacteria in each fraction, lower respiration rates and no methanol oxidation activity as compared to the soil slurry both before and after extraction. The physiological activity was apparently not affected by the number of cells extracted. This indicates that the increased extraction rate of indigenous soil bacteria obtained by effective disruption of aggregates and detachment of cells from surfaces, only results in increased extraction of cells that have been physiologically changed as a result of the extraction process.     

土壤微生物的分离、提取与纯化研究进展

综合评述了土壤微生物提取与纯化研究的最新进展及存在的主要问题。土壤微生物的分离提取过程一般分为土壤分散、提取与纯化3个步骤。采用过滤、离心和淘选3种方法可以成功地分离提取大部分土壤细菌;但土壤真菌的提取则相对较为困难,目前可采用的方法有旋转框技术、液相提取与滤膜检测、以及低速离心技术,这些方法可提取出部分真菌菌丝。两相分离技术可用以提取的土壤微生物进行纯化。     

Quantification of the carbazole 1,9a-dioxygenase gene by real-time competitive PCR combined with co-extraction of internal standards

The fluorogenic probe assay, competitive polymerase chain reaction (PCR) and co-extraction with internal standard cells were combined to develop a rapid, sensitive, and accurate quantification method for the copy number of a target carbazole 1,9a-dioxygenase gene (carAa) and the cell number of Pseudomonas sp. strain CA10. The internal standard DNA was modified by replacement of a 20-bp long region with one for binding a specific probe in fluorogenic PCR (TaqMan). The resultant DNA fragment was similar to the corresponding region of the intact carAa gene in terms of G+C content. When used as a competitor in the PCR reaction, the internal standard DNA was distinguishable from the target carAa gene by two specific fluorogenic probes with different fluorescence labels, and was automatically detected in a single tube using the ABI7700 sequence detection system. To minimize variations in the efficiency of cell lysis and DNA extraction between the samples, the co-extraction method was combined. A mini-transposon was used to introduce competitor DNA into the genome of other pseudomonads, and the resultant construct was used as the standard cell. After the addition of a fixed amount of the internal standard cells to soil samples, total DNA was extracted (co-extraction). Using this method, the copy number of the carAa gene and the cell number of strain CA10 in soil samples could be quantified rapidly.     

Controlled environment soil‐core microcosm unit for investigating fate,migration, and transformation of chemicals in soils

The controlled environment soil‐core microcosm unit (CESMU) methods embody a collection of techniques that began with soil sampling in the field and continued throughout the laboratory investigation of chemical fate, migration, and transformation in site‐specific soils; it was a cost‐effective investigative methodology that could be used to screen chemical materials before initiating high‐cost environmental field studies. Intact soil cores were collected in the field using a hydraulically controlled probe, delivering intact soil‐cores with minimal disturbance directly into high‐density polyethylene pipe (10.3‐cm ID). The inert polyethylene pipe was an effective hydrophobic barrier that remained an integral part of the soil‐core column, obviating subsequent transfers of soil. In the laboratory, each soil column was fitted with a porous ceramic plate and a polyethylene endcap containing fittings for teflon tubing, so that a tension could be applied at the bottom of each soil column (30–35 kPa) to mimic field conditions, thus preventing the undue buildup of water within columns that otherwise would change the chemical, physical, and biological properties of the soil. The intact soil‐cores were housed in the CESMU chamber, a controlled temperature unit with sufficient capacity for maintaining constant temperature within entire soil‐cores. Synthetic rain was added twice a week by peristaltic pump at rates simulating rainfall. Leachates were collected under tension via teflon tubing into flasks in darkness and kept at soil column temperature inside CESMU until harvested for analyses. Soil columns were harvested at intervals for sectioning by depth, extraction, and soil analyses. CESMU methods are applicable to investigations of water movement, soil chemistry, solute transport/transformation, and effects on plants.     

Vacuum‐enhanced recovery of water and NAPL: Concept and field test

Many hydrocarbon‐contaminated soils contain nonaqueous phase liquid (NAPL) following releases from facilities such as underground storage tanks and pipelines. The recovery of free product by pumping from extraction wells or trenches is often an essential prerequisite step prior to further remedial actions. Vacuum‐enhanced NAPL recovery (sometimes referred to as dual‐phase extraction or bioslurping) has attracted recent attention because it offers a means to increase NAPL recovery rates compared with conventional methods, and to accomplish dewatering, while also facilitating vapor‐based unsaturated zone cleanup. A conceptual model is presented that recognizes the effects that vacuum‐enhanced recovery has on soil water and NAPL, with a focus on liquid residing at negative gage pressures and therefore lacking sufficient potential energy to flow into a conventional recovery well or trench. The imposition during vacuum‐enhanced recovery of subatmospheric pressures within the subsurface can reduce the required potential energy (i.e., the entry suction), allowing liquid to be extracted that hitherto had not been able to flow into the well; moreover, it induces both pneumatic and hydraulic gradients toward the vacuum source that increase the rate of water and NAPL recovery. This conceptual model was tested during a 3‐week‐long pilot study at a South Carolina industrial site at which diesel fuel had been discovered in a saprolite formation. During Phase 1 of the pilot study, conventional recovery (liquid only) was carried out from a well screened at the water table, while during Phase 2 dual‐phase extraction was performed at the same well. The application of 27 kPa vacuum resulted in an increase in NAPL recovery from negligible (Phase 1) to approximately 6.6 l/d (Phase 2), with a concurrent increase in water recovery from approximately 190 to 760 l/ d. Neutron moisture probe observations revealed that vadose‐zone liquids underwent redistribution toward the extraction well in response to the onset of Phase 2, also in accordance with the conceptual model. An understanding of soil physical relationships is crucial to the successful application of these and other in situ soil remediation technologies.     

Counting and size classification of active soil bacteria by fluorescence in situ hybridization with an rRNA oligonucleotide probe

A fluorescence in situ hybridization (FISH) technique based on binding of a rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high cellular rRNA contents, were assumed to be active (and growing) in the soil. Hybridization to an rRNA probe, EUB338, for the domain Bacteria was performed with a soil slurry, and this was followed by collection of the bacteria by membrane filtration (pore size, 0.2 micrometer). A nonsense probe, NONEUB338 (which has a nucleotide sequence complementary to the nucleotide sequence of probe EUB338), was used as a control for nonspecific staining. Counting and size classification into groups of small, medium, and large bacteria were performed by fluorescence microscopy. To compensate for a difference in the relative staining intensities of the probes and for binding by the rhodamine part of the probe, control experiments in which excess unlabelled probe was added were performed. This resulted in lower counts with EUB338 but not with NONEUB338, indicating that nonspecific staining was due to binding of rhodamine to the bacteria. A value of 4.8 x 10(8) active bacteria per g of dry soil was obtained for bulk soil incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 x 10(8) active bacteria per g of dry soil was obtained for soil which had been air dried and subsequently rewetted. In both soils, the majority (68 to 77%) of actively growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 micrometer), but the active (and growing) bacteria still represented only approximately 5% of the total bacterial population determined by DAPI (4', 6-diamidino-2-phenylindole) staining. The FISH technique in which slurry hybridization is used holds great promise for use with phylogenetic probes and for automatic counting of soil bacteria.     

Towards a universally adaptable method for quantitative extraction of high-purity nucleic acids from soil

A universally adaptable protocol for quantitative extraction of high-purity nucleic acids from soil is presented. A major problem regarding the extraction of nucleic acids from soil is the presence of humic substances, which interfere with the extraction process itself and in subsequent analytical manipulations. By the approach described here, the humic compounds are precipitated prior to cell lysis with Al(2)(SO(4))(3), and thus eliminated prior to the nucleic acid extraction. The protocol allows for removing of a considerable content and range of humic acids and should therefore be applicable for a wide spectrum of soil types. Accordingly, reproducible results in analyses of different soil types are made possible, inclusively for quantitative comparisons.     

Improving soil protein extraction for metaproteome analysis and glomalin‐related soil protein detection

Soil contains low amounts of protein but high amounts of interfering substances. Current extraction methods for soil protein cannot produce high‐quality samples suitable for proteomic analysis. To resolve the problem, we devised a sequential extraction method, through sequentially extracting soil in citrate and SDS buffers, followed by phenol extraction. The method allows for obtaining applicable 1‐D and 2‐D protein profiles with various agricultural soils and detecting glomalin‐related soil protein. The method may be a valuable tool for soil proteomics.     

球囊霉素相关土壤蛋白的提取条件

球囊霉素是由丛枝菌根真菌分泌的一种含金属离子的糖蛋白,因被非专一性提取而称为球囊霉素相关土壤蛋白（GRSP）。由于提取条件能影响GRSP的测定值,因此研究了柑橘园土样前期灭菌、离心力和土样质量等3个提取条件对GRSP测定值的影响。结果表明,土样前期灭菌和较高的离心力（15,000′g）均能显著影响GRSP的测定值;在土样质量0.25－1.0g范围内,GRSP的测定值随着土样质量的提高而升高;经过3次和6次的连续提取,总球囊霉素相关土壤蛋白的累积量分别达到理论最大值的66.8%和92.1%以上。研究结果为GRSP测定的标准化提供了依据。