Search

Conservation Translocation (CT), which includes reintroduction, reinforcement and introduction, is gaining momentum, responding to serious population decline in many orchids. Orchid conservation underpinned by a greater emphasis on understanding biotic and abiotic factors of habitats is critical for successful recovery and restoration programmes. Anacamptis morio, commonly known as green-winged orchid (GWO), is a terrestrial orchid found throughout Europe, but populations in England rapidly declined in its native range in the last several decades. The current study explored the relationship between soil abiotic characteristics and the community composition of key fungal groups. Wild sites in Essex and Cumbria in England, where successful colonies are currently present, were compared to potential sites for CT in Cumbria. The Cumbrian sites managed by cattle grazing include the wild site and three potential CT sites, with two of them hosting no GWO plants. The Essex site, fOxley Meadow, where no cattle is used for grassland management, hosts the largest population of GWO in England. The aim of this study was to understand whether the community composition of fungi and soil characteristics of the potential CT sites in Cumbria are nearly compatible with that of Oxley Meadow. Oxley Meadow, with around 65,000 plants, stands out as a unique habitat compared to all Cumbria sites, as it showed low organic content. Nitrate and phosphate content were smaller for Oxley Meadow compared to other Cumbrian sites. The proportion of Basidiomycota fungi was greater in Oxley Meadow compared to all Cumbrian sites where Ascomycota dominated. The abundant fungal group found in Oxley Meadow was Agaricales. From Agariclaes, Hygrophoraceae or waxcaps fungi are considered an indicator group of fungi and were the most abundant group in Oxley Meadow. They have a negative correlation with targeted key fungal groups and abiotic parameters. More in-depth assessments using additional primers are essential to better understand the fungal diversity and how this diversity translates to the resilience of orchid habitats. This preliminary study points to future studies to assess whether sites are near-compatible to the wild site where large colonies are present using additional primers collected over different time scales. [ABSTRACT FROM AUTHOR]

Background: Sustainable agriculture seeks to optimize the application of nitrogen (N) fertilizers to reduce adverse economic and ecological effects. Crop diversification has been proposed to increase the efficiency of N fertilization. An open question is how the soil microbiome responds to these beneficial practices. Methods: In a field study we investigated the effects of mineral N fertilizer with a 0-control, a conventional amount of 150 kg N ha−1 and an excessive application of 250 kg N ha−1 on the soil microbiome within a diversified cropping system with oil radish and undersown ryegrass over a period of 2.5 years and a non-diversified control, both in rotation of potato, winter rye and maize. Results: N-fertilizations and crop rotations altered the pH, but differences were less pronounced with the diversified system. Compared to the crop species and season, N fertilization and crop diversification had less influence on the abundance of soil bacteria, archaea and fungi. The crop diversification showed a much stronger effect on archaeal than on bacterial or fungal abundances, while the microbial carbon use efficiency correlated strongly with bacterial abundance. At the end of the growing seasons, crop diversification increased prokaryotic richness and Shannon diversity in response to N addition, with a greater increase in the conventional N. At conventional N supply, prokaryotic co-occurrence networks revealed a much denser and complex structure in the diversified system. Conclusions: The diversified cropping system under conventional N application rates showed positive effects on the prokaryotic soil microbiome by increasing their richness, Shannon diversity, and promoting a more elaborated network structure. [ABSTRACT FROM AUTHOR]

The relationship between the concentration of soil DNA and microbial biomass determined by the method of substrate-induced respiration was studied in a wide range of soils differing in particle size distribution, acidity, organic carbon content, microbial biomass, and type of human activity in antiquity and at the present time. Calcaric Leptosols and Leptic Phaeozems of medieval agricultural lands in the Central Caucasus, Stratozems of agricultural terraces of the late Middle Ages—Modern times in the middle mountain zone of the Eastern Caucasus, as well as Kastanozems and Solonetzes with different grazing intensity in the dry steppe zone (Rostov region) were chosen as the key sites. It was shown that the determination of soil double-stranded DNA concentration is a reliable and simple method for determining microbial biomass in soils with a loamy texture, the organic carbon content < 2%, and the microbial biomass <700 µg C/g dry soil. The conversion factor FDNA in such soils varied in a narrow range from 5.24 to 5.41. In soils with a high content of organic carbon, an increase in FDNA (6.56 and 10.56) was observed due to the presence of recalcitrant extracellular DNA. Agristratified soil of sandy loamy texture was characterized by a lower degree of double-stranded DNA preservation, which resulted in a decrease in the determined microbial biomass (FDNA = 4.22). A reduced conversion factor FDNA (4.78) was also found in the soils of pastures in the dry steppe zone, which confirms the known limitations of using the substrate-induced respiration method in alkaline soils. Human activity did not significantly affect the relationship between the amount of soil DNA and microbial biomass. [ABSTRACT FROM AUTHOR]

Purpose: Binucleate Rhizoctonia sp. (BNR), indigenous to old olive groves of the Apulia region (Southern Italy), and its relationship with olive trees were investigated to search for beneficial soil microorganisms suitable for supporting plant growth and crop productions in degraded semi-arid soils. Methods: Binucleate Rhizoctonia sp. was first isolated from five ancient olive groves using olive tree plantlets as "living baits". The functional relationship of BNR with olive trees was estimated with in-pot growth assays, using native soil amended with an artificial inoculum from the native BNR isolates. BNR was then quantified in the olive groves by digital PCR using a TaqMan assay specifically designed on the ITS regions of the native AG-A isolates. Results: Digital PCR was able to discriminate BNR (Ceratobasidium sp.) from the phylogenetically close Basidiomycetes such as Rhizoctonia solani (Thanatephorus cucumeris). This assay detected an amount of BNR in five olive groves ranging from traces up to 105 target gene copies per µL of DNA. When artificially added to native soil samples, BNR AG-A increased olive tree growth by 50% and 25% respectively under constant optimal water availability and repeatedly induced water stress. Indole-3-acetic acid production was demonstrated for the BNR AG-A isolates, thus, in part, explaining growth promotion. Conclusion: Thanks to the absolute quantification of fungal DNA fragments in soil with digital PCR, findings suggest that new soil functional indicators, such as the growth promoting Rhizoctonia AG-A, may be identified with this research approach to be investigated as a biotic resource for enhancing soil ecosystem services. [ABSTRACT FROM AUTHOR]

Entomopathogenic fungus are important component in pests regulation in ecosystem. Isaria fumosorosea, as one of the entomopathogenic fungi, was reported to successfully control outbreaks of forest defoliators known to attack larch (Larix kaempferi) plantation in Furano, Japan and beech (Fagus crenata) forest in Hachimantai, Japan. Instead of semi-cultured methods, this paper developed and used a culture-independent method based on DNA using qPCR for specific I. fumosorosea detection and quantification directly from soil DNA extraction using specific primer. The primer IFU5821F/IFU6061R was designed and the best primer pair for I. fumosorosea. Strong relationship and good fitting with five levels (R2 = 0.989, E = 0.58) were obtained standard soil DNA extraction. I. fumosorosea could not be detected from all soil samples possibly caused by low density of the fungi. The qPCR was likely a rapid and specific method to detect the fungus from soil. [ABSTRACT FROM AUTHOR]

The analysis of 18S rRNA gene amplicons is an important tool to characterize the diversity of the eukaryotic soil microbiome. Here we analyzed two primer sets (TAReuk, EKeuk) and the impact of a newly designed antifungal peptide nucleic acid to enhance the detection of protists in silico and with soil DNA extracted from croplands and a forest. The in silico analyses showed for TAReuk pronounced specificities for protist SAR supergroup and Metazoa, while EKeuk was particularly specific for Ascomycota and Basidiomycota. In silico , the PNA matched with the majority of Ascomycota (81.3 %) and Basidiomycota (65.4 %), but with <6 % of protists. The contrasting primer specificities were confirmed with soil DNA, but the proportion of protist amplicons was similar. In contrast to in silico , effects of the PNA were not as clear with soil DNA, even though it completely inhibited the amplification of the targeted fungal sequences. PNA effects were more pronounced with TAReuk, and results with cropland and forest soil DNA were not consistent, e.g. , for cropland, PNA decreased the relative abundance of fungi but for forest it was the opposite, possibly because of different fungal diversity. The divergence between PNA in silico -predictions and results with soil DNA are likely an outcome of primer binding to <100 % complementary target sequences and a still limited DNA sequence databases for soil microbial eukaryotes. With TAReuk, the presence of PNA enhanced the detection of Conosa and, thus, could be a useful tool to study this group in the future. • A new PNA probe inhibited in silico PCR amplification of most fungal 18S rRNA genes. • With soil DNA, the PNA probe did not inhibit amplification of fungal 18S rRNA genes. • PNA effects on detection of protists varied between two 18S rRNA primer pairs. • PNA effects varied between DNA extracted from five different soils. • With the common TAReuk, protist primers PNA increased the detection of Conosa. [ABSTRACT FROM AUTHOR]

Background: Advances in DNA sequencing technologies have transformed our capacity to perform life science research, decipher the dynamics of complex soil microbial communities and exploit them for plant disease management. However, soil is a complex conglomerate, which makes functional metagenomics studies very challenging. Results: Metagenomes were assembled by long-read (PacBio, PB), short-read (Illumina, IL), and mixture of PB and IL (PI) sequencing of soil DNA samples were compared. Ortholog analyses and functional annotation revealed that the PI approach signifcantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also ofered comparable or higher species abundance than either PB or IL alone, and showed signifcant advantages for studying natural product biosynthetic genes in the soil microbiomes. Conclusion: Our results provide an efective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics. [ABSTRACT FROM AUTHOR]

Summary: The endomigratory root-lesion nematode, Pratylenchus scribneri , is one of the major plant-parasitic nematodes infecting potato. Accurate identification and quantification of this nematode are essential to develop management strategies but microscopic observations are particularly challenging and time consuming. In this study, a SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. scribneri from field soil DNA extracts. A primer set was designed from the internal transcribed spacer (ITS) region of the P. scribneri rDNA gene. Primer specificity to the target nematode was evaluated by both in silico analysis and qPCR and no detection or non-specific amplification was observed for other non-target nematode species/communities tested in this study. Standard curves were generated using DNA extracts from autoclaved soil infested with varying nematode numbers for calibration. The curves were supported by a high correlation between the P. scribneri numbers artificially added to soil or estimated from naturally infested field soils by traditional methods, and the numbers quantified using the qPCR assay. The assay was able to detect 1 out of 128 (0.0078) equivalents of the DNA of a single nematode in 0.5 g of soil. The qPCR assay developed in this study provides a specific and sensitive detection and quantification of P. scribneri from field soils and a rapid alternative to time-consuming traditional nematode identification and enumeration. [ABSTRACT FROM AUTHOR]

Belowground arthropod communities are diverse and our ability to characterize them remains logistically difficult and time consuming. Molecular metabarcoding techniques are routinely used to assess the diversity of both microbial and some ‘macrobial’ taxa across a range of environments, but the use of such techniques for characterizing soil arthropod diversity remains limited. Here we used three approaches to profile soil arthropod communities at the family level of resolution across 10 distinct sites via morphological identification, metabarcoding of DNA from the extracted arthropods, and metabarcoding directly from bulk soils. Although the three methods differed to some degree in their ability to detect some individual taxa, we found that all three methods yielded well-correlated site-level estimates of diversity (Spearman's ρ ≥ 0.63 with P < 0.05 for all correlations) and overall arthropod community composition (Mantel ρ ≥ 0.45 with P < 0.05). Of particular note is that DNA extracted directly from bulk soil yielded results comparable to analyses of DNA from extracted arthropods. Thus, DNA metabarcoding of bulk soil will likely be a useful tool for those researchers looking to incorporate multi-domain comparisons or for studies that require rapid assessments of arthropod diversity across a large number of soil samples. [ABSTRACT FROM AUTHOR]

The effects of different tillage systems on the properties and microbial diversity of an agricultural soil was investigated. In doing so, soil physical, chemical and biological properties were analysed in 2013-2015, on a long-term field experiment on a loamy sand at the IUNG-PIB Experimental Station in Grabów, Poland. Winter wheat was grown under two tillage treatments: conventional tillage using a mouldboard plough and traditional soil tillage equipment, and reduced tillage based on soil crushing-loosening equipment and a rigid-tine cultivator. Chopped wheat straw was used as a mulch on both treatments. Reduced tillage resulted in increased water content throughout the whole soil profile, in comparison with conventional tillage. Under reduced tillage, the content of readily dispersible clay was also reduced, and, therefore, soil stability was increased in the toplayers, compared with conventional tillage. In addition, the beneficial effects of reduced tillage were reflected in higher soil microbial activity as measured with dehydrogenases and hydrolysis of fluorescein diacetate, compared with conventional tillage. Moreover, the polimerase chain reaction - denaturing gradient gel electrophoresis analysis showed that soil under reduced tillage had greater diversity of microbial communities, compared with conventionally-tilled soil. Finally, reduced tillage increased organic matter content, stability in water and microbial diversity in the top layer of the soil. [ABSTRACT FROM AUTHOR]

Soil-extracted DNA is widely used to characterise the composition of microbiomes. However, there is no commonly accepted protocol or understanding of how sample preservation and laboratory pre-incubation affect the results obtained. Here, we evaluated six different pre-treatments of sieved cropland and forest topsoils from five sites in Northern Germany, including freezing, drying, fresh analyses, and pre-incubations for 1 or 14 day prior to DNA extraction. Based on PCR-amplified 16S rRNA genes and ITS sequences, we characterized the abundance and diversity of bacteria, archaea and fungi, and compared it to a reference community composition obtained from samples immediately frozen with liquid nitrogen and without further manipulation. Irrespective of the pre-treatments, soil microbiomes could be differentiated according to their land use and sampling site. For prokaryotes, Acidobacteriota , Actinobacteriota or Thermoproteota , were more affected by pre-treatments than Bacteriodota , Firmicutes or Planctomycetota. For fungi, Basidiomycota and Mucoromycota, which predominated in forest soils , were more susceptible than the cropland predominant Ascomycota , indicating that different pre-treatments have a stronger impact on assessing the composition of fungal microbiomes from forest than from cropland. Differences between the reference and pre-treatments suggest that generally soil sieving enhanced the DNA extraction efficiency, except for dried soils, but it shifted the proportionality of detected taxa, as shown by reduced Shannon diversity. Overall, oven-drying had a more detrimental effect than air drying, freezing treatments or fresh analyses. For large scale monitoring projects, where fresh analysis is not possible, the gentle freezing approach as applied in this study would be the best choice. • DNA of six pre-treatments from forest and cropland soils analysed. • Irrespective of pre-treatments, microbiomes could be differentiated by site and land use. • Soil sieving appeared to increase DNA yield of fresh and frozen, but not of dried samples. • Compared to cropland forest fungal microbiomes are more pre-treatment sensitive. • Fresh analyses and gentle freezing provided the least biased results. [ABSTRACT FROM AUTHOR]

Plant soil feedbacks ( PSFs) are thought to be important to plant growth and species coexistence, but most support for these hypotheses is derived from short-term greenhouse experiments. Here we use a seven-year, common garden experiment to measure PSFs for seven native and six nonnative species common to the western United States. We use these long-term, field-based estimates to test correlations between PSF and plant landscape abundance, species origin, functional type, and lifespan. To assess potential PSF mechanisms, we also measured soil microbial community composition, root biomass, nitrogen cycling, bulk density, penetration resistance, and shear strength. Plant abundance on the landscape and plant lifespan were positively correlated with PSFs, though this effect was due to the relationships for native plants. PSFs were correlated with indices of soil microbial community composition. Soil nutrient and physical traits and root biomass differed among species but were not correlated with PSF. While results must be taken with caution because only 13 species were examined, these species represent most of the dominant plant species in the system. Results suggest that native plant abundance is associated with the ability of long-lived plants to create positive plant-soil microbe interactions, while short-lived nonnative plants maintain dominance by avoiding soil-borne antagonists, increasing nitrogen cycling and dedicating resources to aboveground growth and reproduction rather than to belowground growth. Broadly, results suggest that PSFs are correlated with a suite of traits that determine plant abundance. [ABSTRACT FROM AUTHOR]

The ectoparasitic stubby root nematode, Paratrichodorus allius, transmits tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. A diagnostic method for direct quantification of P. allius from soil DNA using TaqMan probe and SYBR Green real-time PCR assays was developed to assist the potato industry in management of this important vector. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA of P. allius was demonstrated by in silico analysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculated P. allius individuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil. Moreover, the numbers of P. allius determined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented positive correlation relationships (R2 = 0.80 and 0.86). Although the quantification using TaqMan probe overestimated the target nematodes compared to using SYBR Green in eight out of ten field soil samples, results of the two methods correlated well (R2 = 0.92). This is the first report of P. allius quantification from soil DNA extracts using real-time PCR, providing a rapid and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification. [ABSTRACT FROM AUTHOR]

This study evaluated the effect of inorganic mercury (Hg) on bacterial community and diversity in different soils. Three soils-neutral, alkaline and acidic-were spiked with six different concentrations of Hg ranging from 0 to 200 mg kg and aged for 90 days. At the end of the ageing period, 18 samples from three different soils were investigated for bacterial community structure and soil physicochemical properties. Illumina MiSeq-based 16s ribosomal RNA (rRNA) amplicon sequencing revealed the alteration in the bacterial community between un-spiked control soils and Hg-spiked soils. Among the bacterial groups, Actinobacteria (22.65%) were the most abundant phyla in all samples followed by Proteobacteria (21.95%), Bacteroidetes (4.15%), Firmicutes (2.9%) and Acidobacteria (2.04%). However, the largest group showing increased abundance with higher Hg doses was the unclassified group (45.86%), followed by Proteobacteria. Mercury had a considerable negative impact on key soil functional bacteria such as ammonium oxidizers and nitrifiers. Canonical correspondence analysis (CCA) indicated that among the measured soil properties, Hg had a major influence on bacterial community structure. Furthermore, nonlinear regression analysis confirmed that Hg significantly decreased soil bacterial alpha diversity in lower organic carbon containing neutral and alkaline soils, whereas in acidic soil with higher organic carbon there was no significant correlation. EC values obtained by a nonlinear regression analysis indicated that Hg significantly decreased soil bacterial diversity in concentrations lower than several guideline values. [ABSTRACT FROM AUTHOR]

Soil complexity, heterogeneity and transferability make it valuable in forensic investigations to help obtain clues as to the origin of an unknown sample, or to compare samples from a suspect or object with samples collected at a crime scene. In a few countries, soil analysis is used in matters from site verification to estimates of time after death. However, up to date the application or use of soil information in criminal investigations has been limited. In particular, comparing bacterial communities in soil samples could be a useful tool for forensic science. To evaluate the relevance of this approach, a blind test was performed to determine the origin of two questioned samples (one from the mock crime scene and the other from a 50:50 mixture of the crime scene and the alibi site) compared to three control samples (soil samples from the crime scene, from a context site 25m away from the crime scene and from the alibi site which was the suspect's home). Two biological methods were used, Ribosomal Intergenic Spacer Analysis (RISA), and 16S rRNA gene sequencing with Illumina Miseq, to evaluate the discriminating power of soil bacterial communities. Both techniques discriminated well between soils from a single source, but a combination of both techniques was necessary to show that the origin was a mixture of soils. This study illustrates the potential of applying microbial ecology methodologies in soil as an evaluative forensic tool. [ABSTRACT FROM AUTHOR]

The polymorphisms of soil fungal rDNA Internal Transcribed Spacer regions were studied in Korean pine forests of various ages (10-100-year-old trees) by means of cloned libraries, and analyzed to determine the effects of the trees' developmental stage on soil fungal community structure. The obtained Shannon diversity index (H) and richness (S) indicated that the diversity of the soil fungal community increased significantly with the development of Korean pines ( P < 0.05). In addition, cluster analysis (UPGMA) showed that the soil fungal community variety associated with differently aged Korean pines was higher than 50 %. The soil fungal community diversity correlated significantly with the N content and C/N ratio of the soil ( P < 0.05). The results of this study indicate that the age of in Korean pine can affect soil fungal community by altering soil properties, which in turn could affect the nutrient cycling in the forest ecosystem. [ABSTRACT FROM AUTHOR]

We developed a method for isolation of DNA of cellulolytic microbies from soil, so that DNA sample was free from soil organic matter (SOM). The method consists of three procedures: propagation of cellulolytic microbes, separation of microbial cells from SOM, and DNA extraction using cetyl trimethyl-ammonium bromide (CTAB). Differential centrifugation with Ficoll-PM400 successfully separated the cells from soil particles and SOM. From 1 g of soil, 0.12-0.51 μg of microbial DNA with an average size of 15-20 kbp was isolated. Denaturing Gradient Gel Electrophoresis showed that DNA from a variety of cellulolytic microbes was recovered. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

For the first time, the composition of microbiomes in the biological crust (AKL) horizons of extremely arid desert soils (Aridic Calcisols) developed from saline and nonsaline alluvial deposits in the Ili Depression (eastern Kazakhstan) was analyzed. To describe the diversity of microorganisms in the soil samples, a novel method of pyrosequencing (Roche/454 Life Sciences) was applied. It was shown that bacteria from the Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, Acidobacteria, and Bacteroidetes phyla predominate in all the samples; these are typical representatives of the microbiome of soil crusts. A distinctive feature of the extremely arid soils is the high contribution of cyanobacteria (25-30%) to the total DNA. In the soils developed from saline sediments, representatives from the Rubrobacteraceae, Streptococcaceae, and Caulobacteraceae families and from the Firmicutes phylum predominated. In the soils developed from nonsaline gypsiferous deposits, bacteria from the class of Acidobacteria, subgroup Gp3, of the Methylobacteriaceae family and the class of Subdivision 3 from the Verrucomicrobia phylum predominated. [ABSTRACT FROM AUTHOR]

The objective of this study was to characterize the microbial communities attached to clay (< 2 μm), fine silt (2-20 μm), coarse silt (20-63 μm) and sand-sized fractions [> 63 μm; including particulate organic matter ( POM)] of an arable soil and analyse their response to more than 100 years of two different fertilization regimes. Mild ultrasonic dispersal, wet-sieving and centrifugation allowed the separation of soil particles with the majority of bacterial cells and DNA still attached. Fertilizations increased soil organic carbon ( SOC), total DNA and the abundance of bacterial, archaeal and fungal r RNA genes more strongly in the larger-sized fractions than in fine silt, and no effect was seen with clay, the latter representing above 70% of the total microbial populations. A highly positive correlation was found between microbial r RNA genes and the surface area provided by the particles, while the correlation with SOC was lower, indicating a particle-size-specific heterogeneous effect of SOC. The prokaryotic diversity responded more strongly to fertilization in the larger particles but not with clay. Overall, these results demonstrate that microbial responsiveness to long-term fertilization declined with smaller particle sizes and that especially clay fractions exhibit a high buffering capacity protecting microbial cells against changes even after 100 years under different agricultural management. [ABSTRACT FROM AUTHOR]

Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10 of the initial amount of O. sinensis genomic DNA ( r2 = 0.999) over 7 orders of magnitude (4 × 101 to 4 × 107 fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium ( P < 0.05). Our protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil. [ABSTRACT FROM AUTHOR]

Abstract: 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 SS100 (100t/ha/2 years) and to a lesser extent with FM (10t/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. [Copyright &y& Elsevier]

Analysing soil microbial communities is often hampered by DNA adsorption on soil organic compounds such as humic acids. However the role of humic acids in DNA adsorption and stability in soils remains controversial. To characterize DNA-humic acid interactions, we studied DNA adsorption on two commercially available humic acids and a soil humic acid extracted from an Andosol. Desorption of the adsorbed DNA using 4 different solutions-distilled water, 0.1 M NaCl, 0.1 M sodium phosphate buffer (pH 6.0), and 1% sodium dodecyl sulfate solution-was also studied to understand the mechanism of DNA adsorption on humic acids. Here, we show that humic acids play an important role in DNA adsorption to soils. DNA molecules were adsorbed on the humic acids, with adsorption increasing proportionally with the DNA concentrations in the solution. The adsorption on all humic acid samples was fitted with Freundlich equation, and the parameters obtained from the equation indicated a high affinity between the humic acids and DNA molecules. The total amount of DNA desorbed by the 4 solutions was less than 2% of the total DNA adsorbed on all the humic acids. The results demonstrate that DNA molecules are able to bind strongly to humic acids by ligand binding, hydrophobic interaction, aggregation, or precipitation. [ABSTRACT FROM AUTHOR]

Hyperarid Atacama soils are reported to contain significantly reduced numbers of microbes per gram of soil relative to soils from other environments. Molecular methods have been used to evaluate microbial populations in hyperarid Atacama soils; however, conflicting results across the various studies, possibly caused by this low number of microorganisms and consequent biomass, suggest that knowledge of expected DNA concentrations in these soils becomes important to interpreting data from any method regarding microbial concentrations and diversity. In this paper we compare the number of bacteria per gram of Atacama Desert soils determined by real-time quantitative polymerase chain reaction with the number of bacteria estimated by the standard methods of phospholipids fatty acid analysis, adenine composition (determined by liquid chromatography - time-of-flight mass spectrometry), and SYBR-green microscopy. The number determined by real-time quantitative polymerase chain reaction as implemented in this study was several orders of magnitude lower than that determined by the other three methods and probably underestimates the concentrations of soil bacteria, most likely because of soil binding during the DNA extraction methods. However, the other methods very possibly overestimate the bacteria concentrations owing to desiccated, intact organisms, which would stain positive in microscopy and preserve both adenine and phospholipid fatty acid for the other methods. [ABSTRACT FROM AUTHOR]

Abstract: The entomopathogenic fungus Metarhizium anisopliae and sister species are some of the most widely used biological control agents for insects. Availability of specific monitoring and quantification tools are essential for the investigation of environmental factors influencing their environmental distribution. Naturally occurring as well as released Metarhizium strains in the environment traditionally are monitored with cultivation-dependent techniques. However, specific detection and quantification may be limited due to the lack of a defined and reliable detection range of such methods. Cultivation-independent PCR-based detection and quantification tools offer high throughput analyses of target taxa in various environments. In this study a cultivation-independent PCR-based method was developed, which allows for specific detection and quantification of the defined Metarhizium clade 1, which is formed by the species Metarhizium majus, Metarhizium guizhouense, Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii and Metarhizium brunneum, formerly included in the M. anisopliae cryptic species complex. This method is based on the use of clade-specific primers, i.e. Ma 1763 and Ma 2097, that are positioned within the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal RNA gene cluster, respectively. BLAST similarity searches and empirical specificity tests performed on target and non-target species, as well as on bulk soil DNA samples, demonstrated specificity of this diagnostic tool for the targeted Metarhizium clade 1. Testing of the primer pair in qPCR assays validated the diagnostic method for specific quantification of Metarhizium clade 1 in complex bulk soil DNA samples that significantly correlated with cultivation-dependent quantification. The new tool will allow for highly specific and rapid detection and quantification of the targeted Metarhizium clade 1 in the environment. Habitat with high Metarhizium clade 1 densities can then be analyzed for habitat preferences in greater detail using cultivation-dependent techniques and genetic typing of isolates. [Copyright &y& Elsevier]

The conservation of rare fungal sites occurring on actively managed forest lands requires efficient site monitoring methods. In this study species-specific primers for the putatively mycorrhizal Albatrellus ellisii (Russulales) were developed so that DNA extracted from soil samples at known sites of this fungus could be tested for the presence of A. ellisii mycelium with PCR. This method was used to measure seasonal changes in the levels of A. ellisii mycelium at three study sites while the utility of this monitoring method was assessed. We found that A. ellisii maintained a constant level of soil occupancy over three seasons, except at one site where mycelium abundance increased in the fall. No reduction in abundance was seen in the summer, although all three sites experienced significant summer drought. We found species-specific genetic marker detection to be an effective and practical method for monitoring the mycelial distribution of a target fungus in soil. The ability to obtain this data from rare fungal sites advances our capability to conserve these fungi, particularly within the managed forest landscape. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Five primer/probe sets to identify the tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici ( FOL), and its three races selectively were designed based on the rDNA-intergenic spacer and avirulence genes. Real-time PCR using genomic DNA from mycelia and soil DNA with the primer/probe sets allowed the successful identification of FOL and its races. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study was to develop molecular assays for investigating the presence and diversity of pathogenicity genes from the pea footrot pathogen Nectria haematococca (anamorph Fusarium solani f.sp. pisi) in soils. Methods and Results: Polymerase chain reaction (PCR) assays were developed to amplify four N. haematococca pathogenicity genes ( PDA, PEP1, PEP3 and PEP5) from isolates and soil-DNA from five agricultural fields with a prior footrot history. A collection of 15 fungi isolated on medium selective for Fusarium spp. exhibited variation in their virulence to peas as assessed via a disease index (DI: 0–5; no virulence to the highest virulence). PCR analyses showed that three isolates in which all four pathogenicity genes were detected resulted in the highest DI (>3·88). All four pathogenicity genes were detected in soil-DNA obtained from all five fields with a footrot disease history, but were not amplified from soils, which had no footrot history. Denaturing gradient gel electrophoresis and/or sequence analysis revealed diversity amongst the pathogenicity genes. Conclusion: The PCR assays developed herein enable the specific detection of pathogenic N. haematococca in soils without recourse to culture. Significance and Impact of the Study: Molecular assays that specifically target pathogenicity genes have the capacity to assess the presence of the footrot-causing pathogen in agricultural soils. [ABSTRACT FROM AUTHOR]

Agriculture has a strong effect on soil microbial communities, but it is still unclear how different management practices drive their diversity and composition. To disentangle the effects of temporally contrasting crop management practices on soil microbial abundance and prokaryotic diversity, we analysed samples from a long-term agricultural field experiment, in which plant residues were exported (RE) or returned to soil, i.e., restituted (RR), over a period of 60 years. For 2.5 years, we followed a cropping sequence of maize, winter wheat and barley, in which, as an additional treatment, wheat cultivation was diversified once with pea intercropping. Based on soil-extracted DNA, abundances of bacteria, archaea and fungi were analysed by domain-specific qPCR and the diversity and composition of the prokaryotic community by 16S rRNA gene amplicon sequencing. The abundance of bacteria and fungi, but not for archaea, increased with the long-term restitution, but this effect was only detectable in spring due to their stabilized abundance during winter. The long-term effect of crop restitution on bacterial diversity became tangible when rare and dominant community members were differentiated, with higher sensitivity shown for the rare. In contrast, the cropping sequence equally affected members of both groups. The short-term effect of crop diversification by intercropping was much stronger in the C-depleted RE soils, than in RR soils where the C-loss was compensated, indicating that crop residue restitution increased the environmental resilience of soil microbial communities. Finally, we could confirm that rare bacterial community members, suspected to represent more oligotrophs and synergistic bacteria, formed stronger network structures to each other than the dominant, suspected to be more copiotrophic and competitive. Therefore, our results emphasize the importance to consider the response of rare microbial community members when evaluating long-term effects of agricultural management on the soil microbiome. • Impacts of crop residue soil restitutions are more pronounced in spring than in autumn. • Crop restitution increases the environmental resilience of the soil microbial community. • Rare taxa showed higher sensitivity to long-term crop residue return (restitution) to soil. • A single intercropping event had a strong, but only short-term effect on dominant and rare soil bacterial taxa. [ABSTRACT FROM AUTHOR]

• EcM fungal richness differed more between elevated N-ambient than abated N-ambient • EcM fungal richness declined with increased soil N. • EcM soil fungal richness was two-fold greater than hyphal or root-tip richness. • Exploration type: why "nitrophilic" taxa flourish in conditions limiting other taxa. Atmospheric nitrogen (N) pollution visibly and rapidly changes forest systems, for example impacting the diversity of forest ectomycorrhizal (EcM) fungi. EcM fungi are measurable by components that vary in longevity, hence, reflecting different temporal scales of presence: EcM root-tips are viable for, at most, a couple growing seasons, compared to fungal structures that may persist in soil as DNA for decades or longer. In-growth mesh bags, in contrast, typically capture a single growing season of hyphal growth. The different components might portray different diversity responses by EcM fungi to large-scale N pollution, as well as any subsequent reductions from improved environmental standards. Within an established oak forest study system, we examined the impact of sustained and recently reduced experimental N addition on EcM fungal diversity and composition, measured in three main fungal components (root-free soil, root-tips, and mesh bag hyphae). We hypothesized that elevated soil N would reduce EcM fungal diversity, and that composition would change, but with differences among fungal components related to the temporal longevity of the components. Our expectations were largely met, in that richness primarily declined with increased soil N, and all trends were most pronounced with the soil EcM fungi (the only component potentially reflective of long-term fungal presence). We discovered that abatement of the experimental N treatment did not revert fungal trends to those of the same-site plots with ambient N treatment. Instead, the stochastic nature of local-scale disturbances, related to invasive earthworms and forest stand dynamics, likely impacted N levels and, thus, EcM fungal trends. Due to the context-dependency of localized disturbance(s), assessing the effects of reduced large-scale N deposition on EcM fungi can prove to be challenging. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

γ-Hexachlorocyclohexane (γ-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS 6100 with lin genes for γ-HCH degradation in several γ-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS 6100 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. [ABSTRACT FROM AUTHOR]

Abstract: Agroforestry and grass buffers have been shown to improve soil properties and overall environmental quality. The objective of this study was to examine management and landscape effects on water stable soil aggregates (WSA), soil carbon, soil nitrogen, enzyme activity, and microbial community DNA content. Treatments were row crop (RC), grass buffer (GB), agroforestry buffer (AG), and grass waterways (GWW). A corn (Zea mays L.)–soybean (Glycine max L.) rotation under no-till management was established in a watershed in northeast Missouri in 1991; grass buffers were implemented in 1997. Grass buffers, 4.5m wide and 36.5m apart, consisted of a mixture of redtop (Agrostis gigantea Roth), brome grass (Bromus spp.), and birdsfoot trefoil (Lotus corniculatus L.) on contour within the watershed. Agroforestry buffers have pin oak (Quercus palustris Muenchh.) trees distributed down the center of the grass buffers on one half of the watershed. Soils were collected from two transects extending from the summit to lower landscape positions within the grass and agroforestry portions of the watershed in June 2006. Soil enzymes studied include: fluorescein diacetate hydrolase, β-glucosidase, glucosaminidase, and dehydrogenase. Soil DNA content was determined as an alternative for microbial biomass. WSA was significantly different among treatments and landscape positions. WSA decreased from GWW>AG>GB>RC management treatments and also decreased from lower>middle>summit landscape positions. Soil carbon and nitrogen were highest for the GWW treatment and lowest for RC. The landscape position effect was significant for RC and AG treatments. Fluorescein diacetate, β-glucosidase and glucosaminidase enzyme activities were significantly higher in buffers and GWW areas than RC areas. Dehydrogenase activity was different between grass (GB and GWW) and crop areas. The landscape effect was insignificant for enzyme activity. Although soil DNA may be a good indicator of microbial biomass, it did not appear to differentiate among management systems as selectively as other microbial parameters. Results of the study show that establishment of AG, GB, and GWW increased WSA, soil carbon, soil nitrogen, and enzyme activity. [Copyright &y& Elsevier]

Rhizosphere soils of chickpea (Cicer aeritinum) and wheat (Triticum aestivum) were analysed for the diversity of diazotrophic microorganisms by sequencing the nifH gene. A clone library of soil DNA amplified using highly degenerated nifH primers targeting the prokaryotic nitrogenase was constructed and subsequent RFLP analysis of nifH gene from clone libraries identified 28 and 25 genotypes in chickpea and wheat rhizosphere respectively. A total of 41 nifH genotypes were identified, out of which 12 were common in both rhizosphere soils. Sequencing of 41 nifH genotypes showed similarity to the corresponding genera of diazotrophs belonging to alpha, beta, gamma and delta proteobacteria. The prominent diazotrophs were Azospirillum brasilense, Bradyrhizobium spp., Azotobacter chroococcum, Methylococcus capsulatus, M. sylvestris, Sinorhizobium meliloti and Burkholderia vietnamenesis. A. brasilense was the dominant diazotroph present in the both rhizosphere soils. S. meliloti was only present in the chickpea rhizosphere, while B. vietnamanesis and Azoarcus tolulyticus were observed only in the wheat rhizosphere. The chickpea and wheat rhizospheres were diverse in diazotrophic bacterial community, as indicated by the occurrence of the nifH genotypes. [ABSTRACT FROM AUTHOR]

To evaluate the microbial diversity of Fushan forest soils, the variation of soil properties, microbial populations, and soil DNA with soil depth in three sites of different altitude were analyzed. Microbial population, moisture content, total organic carbon (Corg), and total nitrogen (Ntot) decreased with increasing soil depth. The valley site had the lowest microbial populations among the three tested sites due to the low organic matter content. Bacterial population was the highest among the microbial populations. The ratios of cellulolytic microbes to the total bacteria in organic layers were high, implying their roles in the carbon cycle. The microbial biomass carbon (Cmic) and nitrogen (Nmic) contents ranged from 130.5 to 564.1 μg g−1 and from 16.7 to 95.4 μg g−1, respectively. The valley had the lowest Cmic and Nmic. The organic layer had the highest Cmic and Nmic and decreased with soil depth. Analysis using denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplicons of 16S rDNA showed that the bacterial diversity of the three sites were very similar to each other in the major bands, and the variation was in the minor bands. However, the patterns in PCR-DGGE profile through gradient horizons were different, indicating the prevalence of specific microbes at different horizons. These results suggest that the microbial diversity in the deeper horizons is not simply the diluted analogs of the surface soils and that some microbes dominate only in the deeper horizons. Topography influenced the quantity and diversity of microbial populations. [ABSTRACT FROM AUTHOR]

Abstract: Recent advances in molecular techniques have allowed for the routine examination of nucleic acids in environmental samples. Although current methodologies are very sensitive, accurate target DNA quantification from environmental samples remains challenging. To facilitate high-throughput DNA quantification from environmental samples, we developed a novel DNA quantification method based on a non-linear curve-fitting approach to extract additional information from quantitative PCR amplification curves and used the fitted parameters to develop multiple regression standard equations for target DNA quantification. A 3-parameter sigmoidal function performed superior to a 4-parameter Weibull function for generating the multiple regression standard equations. In a verification experiment, target DNA was quantified in a series of ‘unknown’ samples in three soils using this approach and the results were compared to target DNA values determined using corrected and uncorrected Ct-based (threshold cycle) methods. For each method, the deviations from the expected target DNA content were determined. Results clearly showed that over all DNA concentrations, target DNA content determined by the non-linear curve-fitting method was more accurate and more precise than values predicted by all other methods. Analysis of variance conducted on the predicted DNA contents also revealed fewer statistical artifacts with the non-linear curve fitting method compared to the conventional Ct-based methods. The novel approach described here is accurate, inexpensive, and very amenable for automation and high-throughput applications. [Copyright &y& Elsevier]

Abstract: The impacts of tillage regime, cover crop, and nitrogen on various soil organisms inhabiting a fragile sandy soil (Brunosolic Gray Brown Luvisol) were determined. Soil samples were collected between 2000 and 2003 from a long-term tillage experiment, established in 1988 to determine the effect of tillage systems on yield of corn (Zea mays), soil quality, and weed populations. Populations of several of the soil organisms studied were significantly affected by one or more agronomic treatments. A single earthworm species, Aporrectodea turgida, was found in the experimental area. Worm populations were generally low and dominated by juveniles. Spring-sampled populations were significantly higher in no-till plots than in conventionally tilled plots. Fall-sampled populations were not affected as greatly by tillage, but were generally higher in no-till plots not receiving additional nitrogen or in plots overseeded with a rye (Secale cereale) cover crop. Soil microbial biomass, as represented by extractable soil DNA, was higher in the spring than in the fall. Populations of the soilborne stramenopile Pythium were generally higher in conventionally tilled plots, and were increased by a rye cover crop. Higher rates of nitrogen increased populations of total soil fungi but nitrogen had little effect on prostigmatid or cryptostigmatid mites; prostigmatid populations were generally higher in no-till plots. Spring populations of mesostigmatid mites were higher in plots with a rye cover crop than in plots without an overwintering plant cover. Conventional tillage stimulated populations of astigmatid mites during periods of high rainfall. Collembola populations were dominated by the families Onychiuridae and Isotomidae, but neither was greatly affected by any tillage treatment. Principal component analysis showed that populations of A. turgida and soil aggregation tended to be positively associated with one another, but that variations in populations of Onychiuridae springtails, prostigmatid mites, and Pythium tended not to be associated with changes in other variables. Overall, effects of tillage treatments on soil organisms were found to differ from previous reports in several respects, suggesting that soil type may impose conditions that over-ride the impacts of agronomic cultivation systems on populations of soil organisms. [Copyright &y& Elsevier]

Abstract: Soil solarization is a widespread, nonchemical agricultural practice for disinfesting soils, which is often used in combination with organic amendment, and whose action represents an important factor impacting on soil bacterial communities structure and population dynamics. The present study was conducted to investigate whether and to which extent a 72-day plot-scale soil solarization treatment, either combined or not with organic amendment, could stimulate compositional changes in the genetic structure of indigenous soil bacterial communities. Soil solarization with transparent polyethylene film, in combination or not with farmyard manure addition, was carried out during a summer period on a clay loam agricultural soil located in Southern Italy. Soils from a four-treatment (NS, nonsolarized control soil; S, solarized soil; MA, manure-amended nonsolarized soil; MS, manure-amended and solarized soil) plot block were sampled after 0, 8, 16, 36 and 72 days. Compositional shifts in the genetic structure of indigenous soil bacterial communities were monitored by denaturing gradient gel electrophoresis (DGGE) fingerprinting of 16S rRNA gene fragments amplified from soil-extracted community DNA using primers specific for Bacteria, Actinomycetales, α- and β-Proteobacteria. Changes in soil temperature, pH, and electrical conductivity (EC1:1) were also monitored from 0 to 72 days. Beneath the polyethylene film the average soil temperature at 8-cm depth reached 55°C compared to 35°C in nonsolarized soil. In general, without amendment both soil pH and EC1:1 were not significantly affected by solarization, whereas in manured plots either variables were greatly increased (from 7.0 to 8.0 pH and from 271 to 3021μScm−1 EC1:1), and both showed long-lasting effects due to soil solar heating. The eubacterial DGGE profiles revealed that soil solarization was the main factor inducing strong time-dependent population shifts in the community structure either in unamended or amended soils. Conversely, the addition of organic amendment resulted in an altered bacterial community, which remained rather stable over time. A similar behaviour was also observed in the DGGE patterns of β-proteobacterial and actinomycete populations, and also, albeit to a lesser extent, in the DGGE profiles of α-Proteobacteria. An increased bacterial richness was evidenced by DGGE fingerprints in 16- and 36-day samplings, followed by a decrease appearing in 72-day samplings. This could be explained, other than by a direct thermal effect on soil microflora, by solarization-induced changes in the physico-chemical properties of soil microbial habitats or by other ecological factors (e.g. decreased competitiveness of dominating bacterial species, reduced grazing pressure of microfaunal predators, increased nutrient availability). [Copyright &y& Elsevier]

We tested whether fungal communities are impacted by nitrogen deposition or increased precipitation by PCR-amplifying partial fungal rRNA genes from 24 soil and 24 root samples from a nitrogen enrichment and irrigation experiment in a tallgrass prairie at Konza Prairie Biological Station in northeastern Kansas, U.S.A. Obtained fungal sequences represented great fungal diversity that was distributed mainly in ascomycetes and basiodiomycetes; only a few zygomycetes and glomeromycetes were detected. Conservative extrapolated estimates of the fungal species richness suggest that the true richness may be at least twice as high as observed. The effects of nitrogen enrichment or irrigation on fungal community composition, diversity or clone richness could not be unambiguously assessed because of the overwhelming diversity. However, soil communities differed from root communities in diversity, richness and composition. The compositional differences were largely attributable to an abundant, soil-inhabiting group placed as a well-supported sister group to other ascomycetes. This group likely represents a novel group of fungi. We conclude that the great fungal richness in this ecosystem precluded a reliable assessment of anthropogenic impacts on soil or rhizosphere communities using the applied sampling scheme, and that detection of novel fungi in soil may be more a rule than an exception. [ABSTRACT FROM AUTHOR]

Examines the cloning of aminoglycoside-biosynthetic genes from soil DNA for accumulation of genetic information. Identification of aminotransferase genes; Phylogenetic analysis of the genes; Occurrence of horizontal gene transfer in the soil; Biosynthesis of aminoglycoside antibiotics.

Fungal oxidative exo-enzymes lacking substrate specificity play a central role in the cycling of soil organic matter. Due to their broad ecological impact and available knowledge of their gene structure, laccases appeared to be appropriate markers to monitor fungi with this kind of oxidative potential in soils. A degenerate PCR-primer pair Cu1F/Cu2R, specific for basidiomycetes, was designed to assess directly the diversity of laccase genes in soils. PCR amplification of mycelial cultures and fruit-bodies of a wide spectrum of basidiomycetes, covering all functional groups (saprophytes, symbionts, and pathogens), produced multiple DNA fragments around 200 bp. A neighbor-joining tree analysis of the PCR-amplified laccase sequences showed a clear species-specificity, but also revealed that most fungal taxa possess several laccase genes showing a large sequence divergence. This sequence diversity precluded the systematic attribution of amplified laccase of unknown origin to specific taxa. Amplification of laccase sequences from DNA, extracted from a brown (moder) forest soil, showed a specific distribution of laccase genes and of the corresponding fungal species in the various soil horizons (Oh, Ah, Bv). The most organic Oh-horizon displayed the highest gene diversity. Saprophytic fungi appeared to be less widespread through the soil horizons and displayed a higher diversity of laccase genes than the mycorrhizal ones. [Copyright &y& Elsevier]

In most studies, the distribution of the mycelia of ectomycorrhizal fungi in forest soils has been inferred from areas occupied by fruit bodies. Here, we investigated the below-ground spatio-temporal distribution of Hebeloma cylindrosporum by polymerase chain reaction quantification of a specific DNA sequence present in DNA extracted from soil. Soil samples were collected in a Pinus pinaster stand located in a campsite where fruit bodies of H. cylindrosporum had been mapped from 1990 to 2000. In samples collected underneath fruit bodies, DNA of H. cylindrosporum was always detected in large amounts. However, this DNA was often undetectable in the absence of fruit body even 1 yr after their disappearance. This result was supported by the failure to identify mycorrhizas of this species on seedlings planted in soil samples collected in places where fruit bodies were present 1 yr before sampling. This pattern suggests a recolonization of the site each year by H. cylindrosporum basidiospores in ‘receptive’ patches of ground created either by the local elimination of competitors or by local nutrient enrichments, which could frequently occur in a campsite. Our results demonstrate that an ectomycorrhizal species can be completely eliminated from the roots within 1 yr and does not necessarily contribute to the next generation of mycorrhizas. [ABSTRACT FROM AUTHOR]

In natural forest ecosystems several ectomycorrhizal fungal species cohabit on host plant root systems. To evaluate the ecological and functional impact of each species, it is necessary to appreciate the distribution and abundance of its mycelia in the soil. We developed a competitive PCR (cPCR) method for the basidiomycete Hebeloma cylindrosporum that allows quantification of its DNA in complex DNA mixtures extracted directly from soil samples. The target sequence chosen for the cPCR analysis was a 533-bp fragment of the nuclear ribosomal intergenic spacer, amplified using two species-specific primers. The detection threshold of the cPCR protocol developed was 0.03 pg of genomic DNA. This method was applied to soil samples collected from beneath and at various distances from a group of fruit bodies in a Pinus pinaster forest stand. The results revealed that H. cylindrosporum below-ground biomass was concentrated directly underneath the fruit bodies or very close to them, while no DNA of this species could be detected in soil samples collected at more than 50 cm away. In the vicinity of fruit bodies, H. cylindrosporum soil DNA concentration varied considerably (between 10 and 0.07 ng g soil−1) and decreased sharply with increased distance from the fruit bodies. This work demonstrates the potential of competitive quantitative PCR for the study of the distribution, abundance and persistence of the mycelia of an ectomycorrhizal fungal species in soil. [Copyright &y& Elsevier]

Actinomycetes play a relevant role in soil ecology and are also of important biotechnological interest as they produce several bioactive metabolites. Within the filamentous actinomycetes, it would be desirable to recognize and characterize environmental samples containing unusual genera. To this end, we have developed selective primer sets for polymerase chain reaction (PCR) amplification of 16S rDNA from the Actinomycetales families Micromonosporaceae, Streptomycetaceae, Streptosporangiaceae and Thermomonosporaceae, and from the genus Dactylosporangium. Each primer set, evaluated on genomic DNA from reference strains, showed high specificity and good sensitivity. After amplification of DNA extracted from soil samples, the sequence of the cloned PCR products confirmed the specific amplification of the desired group of sequences in at least 95% of the clones for each primer set. The application of these primers to environmental samples showed the frequent occurrence of these groups in soil samples and also revealed sequences that can be attributed to new groups of actinomycetes. [Copyright &y& Elsevier]

Knowledge of microbial numbers and activity in subsoils is essential for understanding the transformation and downward movement of natural and synthetic organics. Soil cores were taken from two soil profiles (surface textures: silty clay loam and loamy sand), and samples extracted from the 0–30 cm (surface), 1.0–1.3 m (mid) and 2.7–3.0 m (deep; clay) and 3.9–4.2 m (deep; sand) layers. A variety of soil biotic (microbial numbers, microbial biomass, enzyme activities) and abiotic properties (pH, organic C, texture, CEC) were measured. Bacterial numbers decreased with depth as indicated by viable counts and by calculations based upon biomass carbon and extracted DNA. Direct microscopic counts were the most sensitive method of enumeration and gave bacterial numbers between 37 and 442× greater than colony forming units and those calculated from DNA extracted from soil. DNA extracted from soil ranged from 1.23 (sand surface) and 1.34 (clay surface) μg g−1 d wt soil to 0.02 (sand deep) and 0.01 (clay deep) μg g−1 d wt soil. Bacterial numbers, estimated from biomass-C measurements, were comparable to direct counts. Large numbers of bacteria were recorded in the subsoils (direct counts: 5.6×108 sand, 4.5×108 clay) even though this was equivalent to only 4.7 and 1.7% of those in the surface soils. Fungi were isolated from surface and mid-depth layers of both soils but were absent from the deep soil samples. Enzymatic activities (arylsulphatase, β-glucosidase, phosphomonoesterases, urease, dehydrogenase, FDA hydrolysis), assayed with or without buffers, also decreased with depth. The exception was urease activity in the clay soil where no difference was seen between mid and deep in non-buffered assays but a 2.9-fold greater activity was exhibited in the mid than in the surface soil when buffered. Strong positive correlations (R>0.95) were observed between all enzyme activities (except with urease activity in clay soil and non-buffered phosphatase activity in sand soil) and between all methods of estimating bacterial abundance. Strong positive correlations (R>0.90) were also found between bacterial abundance and enzyme activities and between enzyme activities and organic matter content. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

We investigated the use of multiplex polymerase chain reaction (PCR) techniques coupled with Southern analysis to detect xenobiotic-degrading organisms that had been added to three soils. Two soils highly contaminated with petroleum hydrocarbons and a less contaminated control soil were amended with tenfold dilutions of Pselldomonas putida mt-2 (pWW0), P. oleovorans (OCT), and Alcaligenes eutrophus JMP134 (pJP4), or, for controls, phosphate buffer alone. Total DNA was then isolated from the soils and purified using a sequential precipitation and dissolution purification procedure. This DNA was subjected to multiplex polymerase chain reaction (PCR) using primers that amplify regions of xylM (PCR product = 631 bp), aIkB (546 bp) and tfdA (710 bp), which are found on pWW0, OCT and pJP4, respectively. The sizes of the amplified DNA fragments were designed to permit simultaneous amplification and detection of the target genes. Ethidium bromide-stained gels of the initial PCR reaction indicated detectable amplification of between 100 to 106 cells per gram soil, depending on the soil and the target gene. Southern analysis of the PCR amplified DNA improved detection limits to between 1 and 10 cells of each target species per gram of soil, and confirmed the identity of the PCR products. For some samples that were initially resistant to PCR, dilution of the environmental DNA resulted in positive PCR results. This treatment presumably overcame the inhibition of the PCR by diluting coextracted contaminants in the environmental DNA. A second PCR on an aliquot (1 pL) of the first reaction increased the ethidium bromide-based detection limits for one of the soils to six cells per gram of soil; it did not increase the detection limits for the other soils. Therefore, the DNA extraction procedure and multiplex PCR permitted the simultaneous detection of three types of biodegradative cells, at a lower detection limit of = 10 cells per gram of highly contaminated, organic soil. However, due to kinetic limitations of multiplex PCR, the amplified signals did not follow a close dose response to the numbers of added target cells. [ABSTRACT FROM AUTHOR]

Soil microbes exert critical control over ecosystem functions by regulating organic matter decomposition and nutrient cycling. Diverse methods have been adopted to estimate the soil microbial biomass carbon (C mic) and nitrogen (N mic), largely based on fumigation and respiration or biomarkers, such as phospholipids and ATP. Although soil microbial DNA is also an important biomarker, whether this biomarker can be used to estimate C mic and N mic at a large scale in arid and semi-arid regions that remains unclear. To explore this, the 124 soil samples collected from 28 different vegetation/microhabitat types in diverse ecosystems (i.e., desert, cropland, typical steppe, meadow steppe and forest) across arid and semi-arid regions in northern China; then, the strong linear relationships were developed among soil microbial DNA concentrations with the C mic and N mic contents estimated by the classic chloroform fumigation-extraction (CFE) method. The results showed that the soil microbial DNA concentrations were linearly related to the observed C mic (r = 0.99) and N mic (r = 0.97) across the diverse vegetation types, although soil microbial biomass presented an enormous fluctuation. Moreover, based on the above-mentioned coefficients of the linear relationship, it was further inferred that the mean proportions of carbon and nitrogen elements in DNA were 0.99% and 3.70% for C mic and N mic , respectively, when assuming the G-C base-pair proportion is 50% in double-stranded DNA. In conclusion, the study proved that soil microbial DNA as a powerful indicator may provide convenient and efficient estimates for C mic and N mic in arid and semi-arid systems. Although this method still needs to be further improved and examined at a larger scale, it provides a good addition to the already existing methods to determine microbial biomass. Unlabelled Image [ABSTRACT FROM AUTHOR]