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Metagenomics approaches represent an important way to acquire information on the microbial communities present in complex environments like soil. However, to what extent do these approaches provide us with a true picture of soil microbial diversity? Soil is a challenging environment to work with. Its physicochemical properties affect microbial distributions inside the soil matrix, metagenome extraction and its subsequent analyses. To better understand the bias inherent to soil metagenome 'processing', we focus on soil physicochemical properties and their effects on the perceived bacterial distribution. In the light of this information, each step of soil metagenome processing is then discussed, with an emphasis on strategies for optimal soil sampling. Then, the interaction of cells and DNA with the soil matrix and the consequences for microbial DNA extraction are examined. Soil DNA extraction methods are compared and the veracity of the microbial profiles obtained is discussed. Finally, soil metagenomic sequence analysis and exploitation methods are reviewed.

In order to optimize polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) rDNA region from fungi found in black soil in North China, an orthogonal experimental design [L16(45)] was used to evaluate five factors (template, Mg2+, dNTP, Taq DNA polymerase, and primer) from four levels. Subsequently, the optimal annealing temperature, annealing time, extension time and cycle numbers were evaluated. The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer, 30 ng soil DNA template, 3.0 mmol · L−1 Mg2+, 0.2 mmol · L−1 dNTPs, 0.1 μmol · L−1 each forward and reverse primer, and 2.0 U Taq enzyme in 50 μL reaction volume. The optimal thermal cycling protocol consisted of initial melting at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 90 s, and a final extension of 72°C for 10 min.

International audience; A PCR-based molecular tool was developed to estimate the diversity of the catechol-degrading bacterial community in a coal wasteland heavily contaminated with PAHS. A degenerate primer pair specific to catA sequences was designed by multiple alignment of known sequences coding a key intermediate of the β-ketoadiapate pathway degrading catechol, namely catechol 1,2-dioxygenase. The specificity of this primer pair was assessed in 21 pure strains by PCR and sequencing. Comparison of the 16S rDNA and catA phylogenies revealed an absence of congruence between these two genes. The primer set was able to amplify catA sequences in DNA extracts from an industrial soil highly contaminated with heavy metals and polycyclic aromatic hydrocarbons (PAHs). RFLP screening of the catA library (95 clones) yielded 32 RFLP families. All of the 43 clone sequences obtained exhibited 86% identity on average to known CatA. Phylogenetic analysis revealed that these CatA sequences were related to Actinobacteria, α-, β- and γ-Proteobacteria phyla and confirmed the absence of congruence with 16S rDNA sequences, which implies horizontal gene transfer of the cat gene cluster between soil microbiota. Our results suggest that the diversity of the catA bacterial community is maintained in highly contaminated soil.

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.

Microorganisms degrading phenolic compounds play an important role in soil carbon cycling as well as in pesticide degradation. The pcaH gene encoding a key ring-cleaving enzyme of the -ketoadipate pathway was selected as a functional marker. Using a degenerate primer pair, pcaH fragments were cloned from two agricultural soils. Restriction fragment length polymorphism (RFLP) screening of 150 pcaH clones yielded 68 RFLP families. Comparison of 86 deduced amino acid sequences displayed 70% identity to known PcaH sequences. Phylogenetic analysis results in two major groups mainly related to PcaH sequences from Actinobacteria and Proteobacteria phyla. This confirms that the developed primer pair targets a wide diversity of pcaH sequences, thereby constituting a suitable molecular marker to estimate the response of the pca community to agricultural practices. Copyright © 2007 Society of Chemical Industry

linA is a gene encoding γ-hexachlorocyclohexane dehydrochlorinase identified in a γ-HCH-degrading Sphingobium japonicum UT26 that had been isolated from soil of an experimental field treated with γ-HCH. The presence of linA-homologous nucleotide sequences and their sequence diversity was investigated by linA-targeted PCR amplification of DNA obtained from different soils in the field and in Japan. linA-homologous sequences were obtained from almost all the soils some of which had never been treated with γ-HCH. Deduced amino acid sequences suggested that they can be regarded as fragments of linA that encode LinA. Some common, natural function of LinA in soil was suggested., Microbes and environments. 2007, 22(4), p.399-404

Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3′)-IIa/nptII and aph(3′)-IIIa/nptIII – frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides – was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31–856) and 85% for nptIII (1190 copies/g soil; 13–61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0–3.3%) were positive for nptIII, none for nptII (0–0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems.

Fusarium species are ubiquitous in soil. They cause plant and human diseases and can produce mycotoxins. Surveys of Fusarium species diversity in environmental samples usually rely on laborious culture-based methods. In the present study, we have developed a molecular method to analyze Fusarium diversity directly from soil DNA. We designed primers targeting the translation elongation factor 1-alpha (EF-1 alpha) gene and demonstrated their specificity toward Fusarium using a large collection of fungi. We used the specific primers to construct a clone library from three contrasting soils. Sequence analysis confirmed the specificity of the assay, with 750 clones identified as Fusarium and distributed among eight species or species complexes. The Fusarium oxysporum species complex (FOSC) was the most abundant one in the three soils, followed by the Fusarium solani species complex (FSSC). We then compared our molecular approach results with those obtained by isolating Fusarium colonies on two culture media and identifying species by sequencing part of the EF-1 alpha gene. The 750 isolates were distributed into eight species or species complexes, with the same dominant species as with the cloning method. Sequence diversity was much higher in the clone library than in the isolate collection. The molecular approach proved to be a valuable tool to assess Fusarium diversity in environmental samples. Combined with high throughput sequencing, it will allow for in-depth analysis of large numbers of samples

A real-time polymerase chain reaction (PCR) assay was developed to quantify simultaneously the biocontrol agent Colletotrichum coccodes (DAOM 183088) and soil compounds inhibitory to PCR. The external control used in this assay was spiked at known concentrations in soil DNA extracts and amplified in real-time PCR with its own primer set. A comparison between the estimated quantities of the external control and the known quantities added to the extracts allowed an estimation of the PCR efficiency on a sample per sample basis in 18 extracts of soil DNA analyzed, originating from the bioherbicide field-release trials. All 18 extracts tested positive for the presence of inhibitory compounds, but with substantial variability in the magnitude of PCR efficiency (from 12% to 82%) from 1 g of soil sample to another, even when soil DNA extracts had been diluted. This variability demonstrates quantitatively the heterogeneity of soil with regards to content in PCR-inhibitory compounds. The differences in amplificatio...

The entomopathogenic fungus Metarhizium anisopliae var. acridum is registered as a mycopesticide for acridid control in Africa and Australia. Traditionally, identification of M. anisopliae var. acridum infection in grasshoppers and locusts has relied upon development of fungal growth in infected cadavers. Conventional methods of detection of this entomopathogen in the environment and non-target organisms have been based on culture and bioassay. A PCR-based method for the detection of M. anisopliae var. acridum was developed. Sequence data from the distinct ITS rDNA regions facilitated the design of PCR primers that were used in PCR-based diagnostic assays for the detection of fungal DNA. The amplified sequence was 420 bp in length and specific to M. anisopliae var. acridum. Isolates of M. anisopliae var. anisopliae and M. flavoviride produced no PCR product with these primers. Other fungal entomopathogens, plant pathogens, mycopathogens, and soil saprophytes were also not detected by the pathogen-specific primers. The assay was also effective for the detection of M. anisopliae var. acridum DNA in the presence of soil DNA extracts and in infected grasshoppers.

Using a widely used commercial DNA extraction kit and a newly modified direct DNA extraction method proposed in this report, soil DNA was extracted from arable land in diverse geological locations in Japan and the quality and quantity of the DNA were examined. A modified direct DNA extraction method was developed, consisting of one-step extraction and two-step purification using potassium acetate and a DEAE-cellulose column, respectively, and designated as a DSPD (Differential Salt Precipitation and DEAE-cellulose) method. The total time needed to process six soil samples with this method was less than two hours, including the time taken to set up the DEAE-cellulose columns. Other advantages of our DSPD method are the use of non-hazardous reagents and running costs only 5-10% of the commercial kits currently used in soil microbiological research. This DSPD method was tested using 24 soil samples collected from diverse locations in Japan and showed that it is a reliable technique for DNA extraction from a wide range of soil types when compared with commercial DNA extraction kits. This DSPD method will therefore contribute to the molecular and genomic analyses of microbial populations and ecosystems that require numerous samples of soil DNA.

Summary Two decades of culture-independent studies have confirmed that microbial communities represent the most complex and concentrated pool of phylogenetic diversity on the planet. There remains a need for innovative molecular tools that can further our knowledge of microbial diversity and its functional implications. We present the method and application of serial analysis of ribosomal sequence tags (SARST) as a novel tool for elucidating complex microbial communities, such as those found in soils and sediments. Serial analysis of ribosomal sequence tags uses a series of enzymatic reactions to amplify and ligate ribosomal sequence tags (RSTs) from bacterial small subunit rRNA gene (SSU rDNA) V1-regions into concatemers that are cloned and sequenced. This approach offers a significant increase in throughput over traditional SSU rDNA clone libraries, as up to 20 RSTs are obtained from each sequencing reaction. To test SARST and measure the bias associated with this approach, RST libraries were prepared from a defined mixture of pure cultures and from duplicate arctic soil DNA samples. The actual RST distribution reflected the theoretical composition of the original defined mixture. Data from duplicate soil libraries (1345 and 1217 RSTs, with 525 and 505 unique RSTs, respectively) indicated that replication provides a strongly correlated RST profile (r2 = 0.80) and division-level distribution of RSTs (r2 = 0.99). Using sequence data from abundant soil RSTs, we designed specific primers that successfully amplified a larger portion of the SSU rDNA for further phylogenetic analysis. These results suggest that SARST is a powerful approach for reproducible high-throughput profiling of microbial diversity amenable to medical, industrial or environmental microbiology applications.

This paper describes a efficient screening gene nitrilase from contaminated soil from Lombok gold mine industry. DNA was extracted directly from soil using the soil DNA isolation kit based on enzymatic, chemical and mechanical lysis. The existence of nitrilase gene in soil sample can be identified by nitrilase gene amplification using H1F˗H1R primer. BLASTN analysis result revealed that the nitrilase gene fragment which was amplified by H1F˗H1R primer has a high homology with Rhodococcus rhodochrous strain tg1˗A6 nitrilase gene. These amplification and DNA fragment sequencing results indicated that nitrilase gene existence on soil sample can be identified by metagenomic approach Keywords: Metagenomics; nitrilase gene; Rhodococcus rhodochrous; soil.

The need for identification of soil microbial community mainly depends on direct extraction of DNA from soil, a multifaceted environment that is a major pool for microbial genetic diversity. The soil DNA extraction procedures usually suffer from two major problems, namely, inappropriate rupturing of cells and contamination with humic substances. In the present study, five protocols for single type of rhizospheric soil were investigated and their comparison indicated that the inclusion of 120 mM phosphate buffered saline (PBS) for washing and mannitol in the lysis buffer allowed the processing of soil sample in minimal time with no specific equipment requirement. Furthermore, DNA purity and yield were also improved, which allowed the exploitation of genetic potential of soil microbes within soil sample thereby facilitating the amplification of metagenomic DNA. The effectiveness of methods was analyzed using random amplification of polymorphic DNA. The banding patterns revealed that both the abundance and the composition of indigenous microbial community depend on the DNA recovery method.

Volume 80, no. 6, p. 1985–1994, 2014. Page 1986, column 1, paragraph 5, lines 7 to 10: “For the PowerMax soil DNA isolation kit, the sediment was vigorously shaken with autoclaved metal beads (5 mm in diameter) using a Shake Master auto (Bio Medical Science, Tokyo, Japan) operated at 1,500 rpm

DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent.

A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems.

The presence of non-indigenous microbial contaminants resulting from human faecal contamination of old and currently occupied base and field camp sites in South Victoria Land, Antarctica, was assessed by PCR amplification of extracted soil DNA using species-specific PCR primers. Positive controls (samples recovered from the environs of Scott Base, including the sewage outfall) gave strong signals with Escherichia coli primers whereas Clostridium clostridiiforme primers yielded a signal only with the sewage outfall sample. A comparison was made of PCR amplification results from samples from the abandoned Canada Glacier camp site, the Lake Fryxell summer camp site, the Cape Bird Adelie penguin colony and pristine sites from relatively inaccessible regions of the Taylor Valley. Results indicated a possible residual level of E. coli contamination in the abandoned Canada Glacier camp site, but no significant contamination of the currently occupied Lake Fryxell camp site. These data may provide indirect evidence for improved awareness and standards of waste handling and disposal over the past two decades of Dry Valley field research.

Root systems are critical for water and nutrient acquisition by crops. Current methods measuring root biomass and length are slow and labour-intensive for studying root responses to environmental stresses in the field. Here, we report the development of a method that measures changes in the root DNA concentration in soil and detects root responses to drought in controlled environment and field trials. To allow comparison of soil DNA concentrations from different wheat genotypes, we also developed a procedure for correcting genotypic differences in the copy number of the target DNA sequence. The new method eliminates the need for separation of roots from soil and permits large-scale phenotyping of root responses to drought or other environmental and disease stresses in the field.

Metagenomics is a modern and rapidly growing field of molecular genetics and ecology that studies the “collective” genome of the microbial community and is based on the analysis of environmental DNA, extracted directly from a variety of natural habitats. The advent of high-throughput sequencing techniques had opened the principally new opportunities in studies of the genetic structure of microbial communities, but at the same time highlighted significant difficulties, arising particularly during the investigation of the soil metagenome.

This review examines methods to sterilize soil or inhibit microbial activity in soil. This is often necessary for experiments involving microbial survival, enzyme measurements, soil respiration, biodegradation or as controls in direct soil DNA extraction-PCR analysis.

Analysis of physical evidence is typically a deciding factor in forensic casework by establishing what transpired at a scene or who was involved. Forensic geoscience is an emerging multi-disciplinary science that can offer significant benefits to forensic investigations. Soil is a powerful, nearly 'ideal' contact trace evidence, as it is highly individualistic, easy to characterise, has a high transfer and retention probability, and is often overlooked in attempts to conceal evidence. However, many real-life cases encounter close proximity soil samples or soils with low inorganic content, which cannot be easily discriminated based on current physical and chemical analysis techniques. The capability to improve forensic soil discrimination, and identify key indicator taxa from soil using the organic fraction is currently lacking. The development of new DNA sequencing technologies offers the ability to generate detailed genetic profiles from soils and enhance current forensic soil analyses. Here, we discuss the use of DNA metabarcoding combined with high-throughput sequencing (HTS) technology to distinguish between soils from different locations in a forensic context. Specifically, we provide recommendations for best practice, outline the potential limitations encountered in a forensic context and describe the future directions required to integrate soil DNA analysis into casework.

International audience; Impact of long-term biosolids application on soil-living micro-organisms key players of ecosystemic services is scarcely reported. Here, the impact of the 19 year-long application of farmyard manure (FM) and sewage sludge (SS) organic fertilisation regimes on the protocatechuate-degrading bacterial (pca) community was estimated by comparison to a mineral fertilisation regime (U). The structure, diversity and density of the pca community were determined using pcaH, a gene encoding the protocatechuate 3,4-dioxygenase. Ten years after the last application, the structure of the pca community in soils amended with 55100 (100 t/ha/2 years) and to a lesser extent with FM (10 t/ha/year) was still different from that in U treatment. pcaH amplicons from all treatments were cloned, screened by RFLP and sequenced. The diversity was studied by Shannon-Weiner and Simpson indexes and by rarefaction curves estimated from pcaH library analyses, showing that the pcaH community was impacted in SS10 and SS100, compared to U. The sequencing of pcaH amplicons supports the results from the RFLP analysis. Quantification of the abundance of the pca community by qPCR assays showed a significant increase in SS100 in comparison to U. FM and SS10. Overall, 10 years after the last application, the impact of 19 years' organic fertilisation on the pcaH community was still traceable, highlighting the lack of resilience of this functional community

Article en open access; International audience; CTX-M [a major type of extended-spectrum beta-lactamase (ESBU] producing Escherichia coli are increasingly involved in human infections worldwide. The aim of this study was to investigate potential reservoirs for such strains: soils, cattle, and farm environment. The prevalence of bla(CTX-M) genes was determined directly from soil DNA extracts obtained from 120 sites in Burgundy (France) using real-time PCR. bla(CTX-M) targets were found in 20% of the DNA extracts tested. Samples of cattle feces (n = 271) were collected from 182 farms in Burgundy. Thirteen ESBL-producing isolates were obtained from 12 farms and further characterized for the presence of bla genes. Of the 13 strains, five and eight strains carried bla(TEM-71) genes and bla(CTX-1) genes respectively. Ten strains of CTX-M-1 producing E. coli were isolated from cultivated and pasture soils as well as from composted manure within two of these farms. The genotypic analysis revealed that environmental and animal strains were clonally related. Our study confirms the occurrence of CTX-M producing E. coli in cattle and reports for the first time the occurrence of such strains in cultivated soils. The environmental competence of such strains has to be determined and might explain their long term survival since CTX-M isolates were recovered from a soil that was last amended with manure 1 year before sampling.

BACKGROUND: The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. METHODOLOGY/PRINCIPAL FINDINGS: The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a) elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES) approaches, and b) amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA). CONCLUSIONS: Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping.

The growth and activity of Streptomyces violaceolatus and Streptomyces lividans was studied in soil under controlled conditions. The life cycle was followed under differing nutrient regimes and the fate of plasmid- and phage-borne genes determined by direct and indirect techniques. Methods were developed for the direct monitoring of plasmid DNA extracted from soil which allowed differentiation of the cellular location of plasmid DNA between mycelium and spores. In a dynamic, nutrient-fed soil microcosm, inoculants survived poorly, but a specific stage was defined by direct and indirect methods when the inoculants were most active and this correlated with the detection of gene transfer events. Plasmid transfer, phage infection and lysogeny only occurred to a significant extent within this stage at days 15-17 during a 60-day incubation. Estimates based on plasmid DNA recovery indicated that viable counts underestimated spore and mycelial propagules by a factor of greater than 100.

PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches promise the accessibility of the genetic resources and their potential applications. Genetic resources from terrestrial environments can be accessed by exploring the soil metagenome. Soil metagenomic analyses are usually initiated by the isolation of environmental DNAs. Several methods have been described for the direct isolation of environmental DNAs from soil and sediments. Application of metagenomics largely depends on the construction of genomic DNA libraries and subsequent high-throughput sequencing or library screening. Thus, obtaining large quantities of pure cloneable DNA from the environment is a prerequisite. This review discusses the recent developments related to efficient extraction and purification of soil metagenome highlighting the considerations for various metagenomic applications.

Summary The β-ketoadipate pathway is the major route for degradation of aromatic compounds by various soil microorganisms. Protocatechuate 3,4-dioxygenase, a key enzyme of this pathway and which is encoded by pcaGH genes, catalyses the ring cleavage of protocatechuate. Microorganisms harbouring pcaGH genes are widely distributed in the environment but little is known about their relative abundance within the total microflora. Hence, this paper reports the development of a real-time PCR assay to quantify the bacterial pcaH sequence that encodes the β sub-unit of the protocatechuate 3,4-dioxygenase. This real-time PCR assay was linear over seven orders of magnitude with a calculated efficiency of 99% and sensitive up to 102 copies of the pcaH sequence per assay. Real-time PCR analyses performed on six soils with different physico-chemical properties, revealed pcaH densities ranging from 103 to 104 copies of pcaH ng−1 of soil DNA, which corresponded to approximately 0.2–10.9% of the total bacterial community. The sequencing of real-time PCR amplicons yielded 48 deduced amino acid sequences that exhibited 44–100% identity to known bacterial PcaH sequences, thereby confirming the accuracy of this real-time PCR assay.

During the last two centuries, industrialisation has led to increasing contamination of the environment by xenobiotics, notably aromatic compounds (i.e. PAHs, pesticides). These widespread pollutants are a major threat affecting soil quality and human health. In this context, numerous policies have been drawn up notably in Europe, to monitor the threats of pollution to ecosystems and to detect and implement measures to counterbalance the damage. Nevertheless, the European Commission and scientific community are still confronted with a lack of appropriate tools to monitor biodiversity in relation to environmental processes affecting global warming and soil protection. This study proposes tools to assess the biodiversity of functional bacterial communities involved in the process of natural and xenobiotic aromatics biodegradation in soil environments. The major route for the degradation of aromatic compounds is the ß-ketoadipate pathway. We developed two molecular markers i.e. two primer sets targeting two key genes (pcaH and catA) of the ß-ketoadipate pathway, encoding enzymes responsible for the ring cleavage of catechol and protocatechuate. The PCAH molecular marker targeted a sequence from the ß-subunit of 3,4-PCD encoded by the pcaH gene. PCAH was shown to amplify a wide diversity of pcaH sequences in the soil environment, thereby highlighting its interest in characterising the pca bacterial community responsible for the degradation of protocatechuate. This tool was applied to assess the structure, diversity and density of the pca bacterial community. When tested in experimental fields, it revealed changes in the structure, diversity and density of the pca bacterial community in response to organic amendments. This work showed that PCAH could constitute a tool of prime interest for assessing the impact produced by extrinsic factors such as agricultural practices on biodiversity of the pca community. The CATA molecular marker targeted a fragment of the 1,2-CTD encoding gene (catA). The wide diversity of catA sequences was revealed. At this stage in the development of pca and cat bioindicators, we can only provide tools to describe the structure, diversity and density of these bacterial communities. The use of PCR-RFLP fingerprinting and q-PCR assays is recommended to assess the impact of environmental stresses on these functional bacterial communities., Les deux derniers siècles ont été marqués par une industrialisation responsable d’une contamination accrue de l’environnement par des xénobiotiques, notamment les composés aromatiques (HAPs et pesticides). Ces composes sont très communs et présentent un risque important aussi bien pour la qualité des sols que la santé humaine. Ainsi, de nombreuses mesures politiques ont été établies, en Europe, pour la surveillance des menaces liées à la pollution des écosystèmes et la mise en place de mesure pour contrebalancer les effets notoires de telles contaminations. Néanmoins, la commission Européenne et la communauté scientifique demeurent confrontées au manque d’outil de contrôle de la biodiversité en lien avec les processus environnementaux affectant le changement climatique et la protection des sols. De ce fait, cette étude propose l’évaluation de la biodiversité fonctionnelle des communautés bactériennes impliquées dans les processus de biodégradation des polluants organiques dans le sol. La voie principale, connue pour la biodegradation de ces composés, est la voie des the ß-ketoadipate. Nous avons ainsi développés deux marqueurs moléculaires (deux couples d’amorces PCR) ciblant deux gènes clés (pcaH et catA) de la voie des ß-ketoadipate, codant pour les enzymes responsables de la fission des cycles aromatiques. Le marqueur moléculaire PCAH a pour cible la sous unité ß de la protocatéchuate 3,4-dioxygénase codée par le gène pcaH. Ce couple d’amorce permet d’amplifier des fragments de gènes provenant d’une grande diversité de bactéries issues du sol. Ceci montre l’intérêt de pouvoir caractériser la communauté bactérienne pca responsable de la biodégradation du protocatéchuate. Cet outil a été développé pour décrire ma structure, la diversité et la densité de la communauté bactérienne pca, montrant ainsi, sur une étude de cas, l’impact de l’amendement sur ces trois composantes de la biodiversité. Ce travail a montré que PCAH représente un outil pertinent pour étudier et estimer l’impact provoqué par des perturbations extrinsèques telles que les pratiques agricoles sur la biodiversité de cette communauté bactérienne. D’autre part, le marqueur moléculaire CATA a pour cible un fragment du gène codant la catéchol 1,2-dioxygénase (catA) et permet d’amplifier une diversité importante de séquences. A ce stade de l’étude, le développment de pca et cat comme bioindicateur se limite à la description de la la structure, diversité et densité de ces communautés.

Soil fungal community structures are often highly heterogeneous even among samples taken from small field plots. Sample pooling is widely used in order to overcome this heterogeneity, however, no objective criteria have yet been defined on how to determine the number of samples to be pooled for representatively profiling a field plot. In the present study PCR/RFLP and T-RFLP analysis of fungal 18S rDNA in ten soil samples obtained from a grassland plot of 400 m(2) also revealed this known heterogeneity in fungal community structures. Based on these data a three-step approach to assess representativity of fungal community profiles was established. First, soil DNA quantities needed for robust community profiling were determined. Second, profiles of single or multiple samples were theoretically averaged to test for statistically significant clustering in order to determine the minimal number of samples to be pooled to achieve representativity. Third, DNA extracts of single or multiple samples were pooled prior to profiling in order to test for statistically significant clustering. Analyses revealed robust profiles for 50 ng soil DNA but not for 5 ng. Averaged T-RFLP profiles from five or more soil samples and experimental T-RFLP profiles from pools of seven or more samples formed one significant branch. Theoretical averaging and experimental pooling revealed that five to seven samples have to be pooled for robustly representing the field plot. Our results demonstrate that representativity of soil fungal community profiles can objectively be determined for a field plot with only little deviation between theoretical and experimental approaches. This three-step approach will be of assistance for designing sampling and pooling strategies for comparative analyses of soil fungal communities in ecological studies.

This study describes the development and evaluation of a specific Legionella pneumophila Taqman duplex real-time PCR (qPCR) for fast and reliable quantification of this human pathogen in suspected man-made water systems. The qPCR assay was 100% specific for all L. pneumophila serogroups 1-15 with a sensitivity of 60 genome units/l and an amplification efficiency of 98%. Amplification inhibitors were detected via an exogenous internal positive control, which was amplified simultaneously with L. pneumophila DNA using its own primer and probe set. Mean recovery rates of the qPCR assay for tap water and cooling circuit water, spiked with a known number L. pneumophila bacteria, were 93.0% and 56.3%, respectively. Additionally, by using the Ultraclean Soil DNA isolation kit, we were able to remove amplification inhibitors ubiquitously present in cooling water. The practical value of our qPCR assay was evaluated through analysis of 30 water samples from showers, taps, eyewash stations, fire sprinklers and recirculation loops with qPCR and traditional culture. In conclusion, the described L. pneumophila Taqman duplex real-time assay proved to be specific, sensitive and reproducible. This makes it a promising method complementing the current time-consuming culture standard method.

Soil microorganisms play important roles in maintaining soil quality and ecosystem health. Development of effective methods for studying the composition, diversity, and behavior of microorganisms in soil habitats is essential for a broader understanding of soil quality. Forest management strategies and practices are of vital significance for sustainable forest production. How the different forest management measures will influence soil microbial communities is a widespread concern of forest industry and scientific communities. Only a small proportion (~0.1%) of the bacteria from natural habitats can be cultured on laboratory growth media. Direct extraction of whole-community DNA from soil, followed by polymerase chain reaction (PCR) and other analysis circumvents the problems of the culture-dependent methods and may shed light on a broader range of microbial communities in the soil. DNA-based molecular methods rely on high quality soil microbial DNA as template, and thus extraction of good quality DNA from soil samples has been a challenge because of the complex and heterogeneous nature of the soil matrix. The objectives of this research were to establish a set of DNA-based molecular methods and to apply them to investigate forest soil microbial composition and diversity. Soil samples were collected from different forest ecosystems, i.e., the natural forest (YNF) and the first rotation (~ 50 years) (Y1R) and the second rotation (~ 1 year) (Y2R) of hoop pine plantations at Yarraman, and from different forest residue management practices (the experiments had established 6.4 years before the samples were collected) at Gympie, two long-term experimental sites of the Queensland Department of Primary Industry-Forestry in subtropical Queensland, Australia. Some DNA-based molecular techniques, including DNA extraction and purification, PCR amplification, DNA screening, cloning, sequencing and phylogenetic analyses, were explored using Yarraman soil samples, which were high in organic matter, clay and iron oxide contents. A set of methods was assembled based on the recommendations of the method development experiments and applied to the investigations of the microbial composition and diversity of the Yarraman and Gympie soil samples. Four soil DNA extraction methods, including the Zhou method (Zhou et al., 1996), the Holben method (Holben, 1994), the UltraClean (Mo Bio) and FastDNA (Bio 101) soil DNA extraction kits, were explored. It was necessary to modify these methods for Yarraman soil. I designed and introduced a pre-lysis buffer washing step, to partially remove soil humic substances and promote soil dispersion. This modification greatly improved the quality of the extracted DNA, decreasing co-extracted humic substances by 31% and increasing DNA yield by 24%. The improved Holben method was recommended for fungal community studies, and the improved Zhou method for bacterial community studies. The extracted DNA was good in quality, with a consistent size of ~20 kb and a yield of 48-87 g g-1 soil, and could be successfully used for 16S (Zhou method) and 18S (Holben method) rDNA amplifications. For less difficult environmental samples, UltraClean kits could be a good option, because they are simple and fast and the extracted DNA are also of good quality. Screening of the DNA PCR products using TGGE, Heteroduplex-TGGE and SSCP was also explored. These methods were not so effective for the screening of the soil DNA PCR products, owing to the difficulty in interpretation of the results. Cloning was a necessary step to obtain a single sequence at species level in soil microbial community studies. The screening of the clone library by TGGE, Heteroduplex-TGGE and SSCP could only separate the clones into several major bands, although SSCP gave better separation. Sequencing of selected clones directly from the clone library obtained ultimate results of microbial taxonomic composition and diversity through well-established sequence analysis software packages and the databases. It was recommended that, in this project with the target of microbial community composition and diversity, soil DNA PCR products were directly cloned to construct clone libraries and a sample of clones were sequenced to achieve an estimate of the taxonomic composition of the soil. Fungal communities of the Yarraman soil samples under the natural forest (YNF) and the hoop pine plantations (YHP) were investigated using 18S rDNA based cloning and sequencing approaches. Twenty-eight clone sequences were obtained and analysed. Three fungal orders, i.e., Zygomycota, Ascomycota and Basidiomycota were detected from the YNF and YHP samples. By contrast, culture-based analyses of fungi in the literature were mostly Ascomycetes. YNF appeared to have more Ascomycota but less Zygomycota than YHP, and within the Zygomycota order, YHP had more unidentified species than YNF. Bacterial communities of Yarraman soil samples of YNF, Y1R and Y2R were investigated using 16S rDNA-based cloning and sequencing approaches. 305 16S rDNA clone sequences were analysed and showed an overall bacterial community composition of Unclassified bacteria (34.4%), Proteobacteria (22.0%), Verrucomicrobia (15.7%), Acidobacteria (10.2%), Chloroflexi (6.9%), Gemmatimonadetes (5.6%), and Actinobacteria (5.2%). There was a significant difference among YNF, Y1R and Y2R in the taxonomic group composition. YNF had a greater proportion of Acidobacteria (18.0%), Verrucomicrobia (23.0%) and Chloroflexi (9.0%) than Y1R and Y2R (corresponding to 6.3%, 12.1% and 5.9%, respectively), while Y1R and Y2R had a higher percentage of the Unclassified group (38.5% for Y1R and 46.5% for Y2R) than YNF (18.0%). For the Proteobacteria group, YNF had more Alpha-subdivision but Y1R and Y2R had more Delta-subdivision. From YNF to Y1R to Y2R, the clone sequence variable site ratios, 5% and 10% OTU numbers and Shannon's diversity index H' values tended to decrease, indicating the soil bacterial diversity decreased from the natural forest to the first and the second rotation hoop pine plantations. The large amount of unclassified clone sequences could imply a novel group of bacteria in the soil, particularly in the hoop pine soil samples. Alternatively they may result from artefacts during the PCR process. Bacterial communities of the Gympie soil under different residue management practices, i.e., residue (litter plus logging residue) removed (G0R), residue retained (G1R), and residue doubled (G2R), were also investigated using the 16S rDNA-based cloning and sequencing approaches. Acidobacteria (37.6%) and Proteobacteria (35.6%, including Alpha-subdivision of 29.9% and Gamma-subdivision of 5.7%) were dominant components of the communities, followed by Actinobacteria (14.7%), Verrucomicrobia (7.3%) and Unclassified bacteria. There was no significant difference among G0R, G1R and G2R in the bacterial community compositions and diversity. These findings provided an in-depth vision of the soil microbial communities under different forest management practices. Their combination with other soil analysis results, such as physical and chemical properties, and forest production data, could provide an improved understanding of sustainable forest management strategies.

The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78°N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced for identification. The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 °C. The two genera Methylobacter and Methylosinus were found in all localities studied. The DGGE band patterns were simple, and DNA fragments with single base differences were separated. The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon. Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.Key words: methanotrophic diversity, Svalbard, arctic wetland, denaturing gradient gel electrophoresis.

The efficiency of soil DNA extraction using a bead beater homogenizer (Biospec Products - Germany) was compared to that obtained with a hand held mixer (Moulinex - Brazil). The hand held mixer costs 100 times less and extracted seven times more crude DNA than the bead beater. A eficiência da extração de DNA do solo utilizando um homogeneizador bead beater (Biospec Products - Alemanha) foi comparada àquela obtida utilizando um homogeneizador manual (Moulinex - Brasil). O homogeneizador manual tem custo 100 vezes menor e revelou-se mais eficiente que o bead beater extraindo sete vezes mais DNA.

To the Editor:E. M. Geigl (2001xInadequate use of molecular hybridization to analyze DNA in Neanderthal fossils. Geigl, EM. Am J Hum Genet. 2001; 68: 287–290Abstract | Full Text | Full Text PDF | PubMed | Scopus (6)See all References2001) challenges the results published by Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) on the genomic differentiation of Neanderthals and modern humans, deduced from Southern hybridization. She is convinced that Scholz et al. presented artifacts resulting from coextracted DNA of soil organisms. However, I do not feel that Geigl has provided any evidence for her “contamination hypothesis.” Because of space limitations, I will mention only the most important aspects supporting my point of view, but the list is far from complete.Geigl claims that the indirect evidence for DNA survival (e.g., amino acid racemization data) given by Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) is not sufficient to prove authenticity of aDNA samples. It is certainly a very difficult task to exclude contamination of samples. For this reason, researchers usually apply a variety of methods to assess the preservation of DNA by biochemical means. However, none of these methods is able to prove conclusively the authenticity of DNA; these methods can merely indicate whether or not it is likely that a certain sample is contaminated with contemporary DNA. Surprisingly, Geigl focuses exclusively on the biochemical data and does not even mention the most convincing evidence that supports the authenticity of the ancient DNA extracts used by Scholz et al.—namely, the experiments themselves. The reindeer and mammoth controls do not yield any hybridization signal. Why should we assume that the controls are not contaminated with soil DNA when all hominid samples are? Why would contemporary human and chimpanzee DNA exclusively cross-hybridize with soil DNA coextracted from hominid fossils but not do so with soil DNA coextracted from other fossils? Contemporary human DNA extracted from blood gives basically the same hybridization pattern that the DNA extracted from a human fossil gives. Furthermore, contemporary chimpanzee DNA hybridizes exclusively to hominid ancient DNA extracts (and not to reindeer and mammoth) but yields signals that are clearly different from those obtained with human DNA. Thus, the results of the experiments make phylogenetic sense. In particular, the results of the hybridization of chimpanzee DNA to the ancient DNA extracts are crucial for the conclusions drawn by Scholz et al. It therefore remains to be resolved why Geigl claims that “chimpanzee DNA, with 99% homology to modern human DNA, fails to hybridize.” Furthermore, the electrophoretic separation of the ancient DNA extracts prior to blotting will separate DNA of high molecular weight (expected to be contaminating contemporary DNA) from DNA of low molecular weight (expected to be authentic ancient DNA). Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) did not observe any hybridization signal with high–molecular-weight DNA.Rather than discussing the evidence just mentioned, Geigl attempts to prove that the data presented by Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) result from coextracted contemporary soil DNA by mere theoretical considerations and artificial experimental set-ups. However, her calculation that under the hybridization conditions used by Scholz et al. only sequences diverging by 30%–40% would be distinguishable is meaningless. Such calculations refer to contemporary DNA and do not apply to ancient DNA, which is of low molecular weight and is highly degraded. It also seems unlikely to me that coextracted inhibitors prevent an enzymatic labeling of ancient DNA but will cause a preferential labeling of contaminating DNA. If such inhibitors are coextracted, they will certainly inhibit the labeling of all DNAs. In addition, it is not justified to conclude from hybridization signals that look similar to those of Scholz et al. (2000xGenomic differentiation of Neanderthals and anatomically modern man allows a fossil-DNA-based classification of morphologically indistinguishable hominid bones. Scholz, M, Bachmann, L, Nicholson, GJ, Bachmann, J, Giddings, I, Ruschoff-Thale, B, Czarnetzki, A, and Pusch, CM. Am J Hum Genet. 2000; 66: 1927–1932Abstract | Full Text | Full Text PDF | PubMed | Scopus (24)See all References2000) but were obtained with mixtures of fungal, bacterial, plant, and human DNA that the procedure of Scholz et al. “allows for the identification of the presence of common microorganismal DNA in the sample far better than it allows for the distinguishing of closely related species.” One can design an almost unlimited number of analogous experiments, but none of them will be a proof of Geigl's “contamination hypothesis.”To conclude, I believe that it is certainly worthwhile to exploit the power of Southern hybridizations in ancient DNA research. However, a discussion on the pros and cons should be based on appropriate scientific evidence rather than on unsupported assumptions.

This manuscript is a review of the innovative methodlogies that enable more precise evaluation of soil microbial diversity. Highlighting the molecular approach, which does not require the isolation of microorganisms and allows the inclusion of non-culturable genotypes in the analyses, the described methodologies revolutionised the environmental microbiology and opened gateways for an accurate understanding of the ecology and diversity of microorganisms. The application of techniques based on soil total DNA extraction, PCR amplification of genes or gene fragments, and sequence analysis revealed that the microbial universe in far more complex than ever imagined. Examples of applications of the molecular approach to study the diversity of soil diazotrophic bacteria are given. Made available in DSpace on 2021-10-19T02:10:11Z (GMT). No. of bitstreams: 1 Evaluating-the-microbial-diversity-of-soil-samples-1999.pdf: 6059600 bytes, checksum: 964955f3540bfc2f559198b7e24229c3 (MD5) Previous issue date: 1999

Rhizoctonia solani is an important pathogen of potatoes. The fungus is comprised of 13 anastomosis groups (AGs) of which AG3-PT (the potato subgroup) is most closely associated with potatoes (Tsror, 2010). Maize plants (cv. Ballade) and…