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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]

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

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.

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]

Based on the new generation sequencing methods, such as pyrosequencing (Roche/454 Life Sciences), the metagenomic characteristics of typical chernozem microbiomes were determined and described for the first time in Moldova under different land use conditions. It was revealed that the community of prokaryotes is represented by both domains, particularly twelve bacterial phyla (61.4%) and one phylum Archaea (6.2%). Only nine phyla had an abundance above 1.0%: the archaeal phylum Сrenarchaeota (Thaumarchaeota) and the bacterial phyla Proteobacteria, Actinobacteriota, Bacteroidota, Acidobacteriota, Firmicutes, Verrumicrobiota, Platctomycecota, and Myxococcota. Phyla, with a share of less than 1.0%, preferred the conditions of experimental variants of crop rotations. A significant proportion were unidentified microorganisms, the abundance of which varied from 25.8 to 38.2% in spring and from 27.9 to 40.2% in summer. The influence of soil and climatic conditions on the formation of the typical chernozem microbiome was more significant than agricultural practices.

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]

Abstract 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 significantly increased the contig length of the metagenomic sequences compared to IL and enlarged the gene pool compared to PB. The PI approach also offered comparable or higher species abundance than either PB or IL alone, and showed significant advantages for studying natural product biosynthetic genes in the soil microbiomes. Conclusion Our results provide an effective strategy for combining long and short-read DNA sequencing data to explore and distill the maximum information out of soil metagenomics.

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]

Diversified cropping systems and fertilization strategies were proposed to enhance the abundance and diversity of the soil microbiome, thereby stabilizing their beneficial services for maintaining soil fertility and supporting plant growth. Here, we assessed across three different long-term field experiments in Europe (Netherlands, Belgium, Northern Germany) whether diversified cropping systems and fertilization strategies also affect their functional gene abundance. Soil DNA was analyzed by quantitative PCR for quantifying bacteria, archaea and fungi as well as functional genes related to nitrogen (N) transformations; including bacterial and archaeal nitrification (amoA -bac,arch ), three steps of the denitrification process (nirK, nirS and nosZ -cladeI,II ) and N 2 assimilation (nifH), respectively. Crop diversification and fertilization strategies generally enhanced soil total carbon (C), N and microbial abundance, but with variation between sites. Overall effects of diversified cropping systems and fertilization strategies on functional genes were much stronger than on the abundance of bacteria, archaea and fungi. The legume-based cropping systems showed great potential not only in stimulating the growth of N-fixing microorganisms but also in boosting downstream functional potentials for N cycling. The sorghum-based intercropping system suppressed soil ammonia oxidizing prokaryotes. N fertilization reduced the abundance of nitrifiers and denitrifiers except for ammonia-oxidizing bacteria, while the application of the synthetic nitrification inhibitor DMPP combined with mineral N reduced growth of both ammonia-oxidizing bacteria and archaea. In conclusion, this study demonstrates a strong impact of diversified agricultural practices on the soil microbiome and their functional potentials mediating N transformations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Truffles are ectomycorrhizal species forming edible ascocarps. The Italian white truffle (Tuber magnatum Pico) is the most famous and expensive species harvested to date; it comes exclusively from natural habitats in European countries. The annual production of T. magnatum is generally insufficient to respond to the high demands making its cultivation a research hotspot. The first attempt to cultivate T. magnatum started in the 1970s without success; only recently have mycorrhized plants been successfully produced. The aims of this study were (1) to assess the persistence of T. magnatum in the soil of plantations realized with mycorrhized plants and (2) to characterize the first T. magnatum orchard that produced ascocarps outside the known natural geographic range of this species. In 2018, five orchards were sampled in France, and T. magnatum was investigated in the soil. We confirmed that T. magnatum survived in the soil 3 to 8 years after planting. The key finding of this study was the harvest of T. magnatum ascocarps in 2019 and 2020 from one orchard. The production of ascocarps started 4.5 years after planting, and the ascocarps were harvested under different trees and during two consecutive seasons. A detailed analysis of the productive orchards (i.e., soil features, soil water availability, cultivation techniques) is presented. These results demonstrate the feasibility of T. magnatum cultivation worldwide by planting mycorrhized plants. The cultivation of T. magnatum could therefore become a real opportunity for farmers and could respond to the high demand of this high-priced food. [ABSTRACT FROM AUTHOR]

Anthrax is one of the most important diseases that can be transmitted from cattle to humans, especially in countries with a high density of cattle. Cattle are usually infected by the ingestion of Bacillus anthracis spores with food ingredients. For that reason, the adequacy of anthrax spores in the soil is considered as one of the causes of anthrax in cattle. The present study is based on the identification of anthrax spores in the soil and the development of a map based on the presence of spores, so that the people of the community can take a practical step in this regard in selecting the place for their cattle to graze. For this purpose, soil samples were collected from the studied areas. After extraction of soil DNA with DNA extraction kits, all samples were tested using a qualitative detection method. For a rapid and reliable qualitative detection system for Pathogenic B. anthracis spores isolated from soil samples, the specific primer of two plasmids, pXO1 (gene Pag), pXO2 (genes Cap), and S-layer locus (gene Sap) were used. The presence of spores was found in 7 out of 50 samples. The results of a qualitative detection method of Bacillus anthracis spores in the pastureland inserted as per their GPS coordinates. Based on the presence of pathogenic anthrax spore in a specific location, a map was then prepared and displayed in the community. So that the cattle owners can decide where their cattle will graze and be careful to prevent spore contact of their animal. Among the various effective anthrax prevention measures, rich mapping according to the presence of spores can be helpful in community level that will protect people from anthrax disease in the future. [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]

Genetic fingerprinting demonstrated in previous studies that differently sized soil particle fractions (PSFs; clay, silt, and sand with particulate organic matter (POM)) harbor microbial communities that differ in structure, functional potentials and sensitivity to environmental conditions. To elucidate whether specific bacterial or archaeal taxa exhibit preference for specific PSFs, we examined the diversity of PCR-amplified 16S rRNA genes by high-throughput sequencing using total DNA extracted from three long-term fertilization variants (unfertilized, fertilized with minerals, and fertilized with animal manure) of an agricultural loamy sand soil and their PSFs. The PSFs were obtained by gentle ultrasonic dispersion, wet sieving, and centrifugation. The abundance of bacterial taxa assigned to operational taxonomic units (OTUs) differed less than 2.7% between unfractionated soil and soil based on combined PSFs. Across the three soil variants, no archaeal OTUs, but many bacterial OTUs, the latter representing 34–56% of all amplicon sequences, showed significant preferences for specific PSFs. The sand-sized fraction with POM was the preferred site for members of Bacteroidetes and Alphaproteobacteria, while Gemmatimonadales preferred coarse silt, Actinobacteria and Nitrosospira fine silt, and Planctomycetales clay. Firmicutes were depleted in the sand-sized fraction. In contrast, archaea, which represented 0.8% of all 16S rRNA gene sequences, showed only little preference for specific PSFs. We conclude that differently sized soil particles represent distinct microenvironments that support specific bacterial taxa and that these preferences could strongly contribute to the spatial heterogeneity and bacterial diversity found in soils.

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]

Genetic fingerprinting demonstrated in previous studies that differently sized soil particle fractions (PSFs; clay, silt, and sand with particulate organic matter (POM)) harbor microbial communities that differ in structure, functional potentials and sensitivity to environmental conditions. To elucidate whether specific bacterial or archaeal taxa exhibit preference for specific PSFs, we examined the diversity of PCR-amplified 16S rRNA genes by high-throughput sequencing using total DNA extracted from three long-term fertilization variants (unfertilized, fertilized with minerals, and fertilized with animal manure) of an agricultural loamy sand soil and their PSFs. The PSFs were obtained by gentle ultrasonic dispersion, wet sieving, and centrifugation. The abundance of bacterial taxa assigned to operational taxonomic units (OTUs) differed less than 2.7% between unfractionated soil and soil based on combined PSFs. Across the three soil variants, no archaeal OTUs, but many bacterial OTUs, the latter representing 34-56% of all amplicon sequences, showed significant preferences for specific PSFs. The sand-sized fraction with POM was the preferred site for members of Bacteroidetes and Alphaproteobacteria, while Gemmatimonadales preferred coarse silt, Actinobacteria and Nitrosospira fine silt, and Planctomycetales clay. Firmicutes were depleted in the sand-sized fraction. In contrast, archaea, which represented 0.8% of all 16S rRNA gene sequences, showed only little preference for specific PSFs. We conclude that differently sized soil particles represent distinct microenvironments that support specific bacterial taxa and that these preferences could strongly contribute to the spatial heterogeneity and bacterial diversity found in soils. [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]

Rhizoctonia cerealis causes sharp eyespot in cereals and the pathogen survives as mycelia or sclerotia in soil. Real-time Polymerase Chain Reaction (qPCR) assays based on TaqMan chemistry are highly suitable for use on DNA extracted from soil. We report here the first qPCR assay for R. cerealis using TaqMan primers and a probe based on a unique Sequence Characterised Amplified Region (SCAR). The assay is highly specific and did not amplify DNA from a range of other binucleate Rhizoctonia species or isolates of anastomosis groups of Rhizoctonia solani. The high sensitivity of the assay was demonstrated in soils using a bulk DNA extraction method where 200 μg sclerotia in 50 g of soil were detected. DNA of the pathogen could also be amplified from asymptomatic wheat plants. Using the assay on soil samples from fields under different crop rotations, R. cerealis was most frequently detected in soils where wheat was grown or soil under pasture. It was detected least frequently in fields where potatoes were grown. This study demonstrates that assays derived from SCAR sequences can produce specific and sensitive qPCR assays. [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]

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils. [ABSTRACT FROM AUTHOR]

Abstract: The largely subterranean nature of the mycorrhizal fungal life form impedes efforts to monitor protected fungi and evaluate the effectiveness of mitigations in timber harvest areas. In this study we developed and tested a multiplex PCR system capable of detecting 14 listed Phaeocollybia species in soil. We then investigated the persistence of Phaeocollybia species at three sites in northwestern Oregon where Phaeocollybia attenuata and other Phaeocollybia sporocarps had been observed prior to timber harvest. The three sites had three different levels of protection for the fungi. One site was in a buffer zone next to a thinned area, one was within a thinned area, and one was in a clearcut area. The method was effective at detecting the target species in soil. We detected Phaeocollybia species in the soil at all sites, even in the clearcut after 12 yr, during which no sporocarps were observed. [Copyright &y& Elsevier]

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]

An investigation was undertaken using a combination of microbiological and DNA-based approaches to evaluate combinations of two/three/four cyanobacterial strains (BF1 Anabaena torulosa; BF2, Nostoc carneum; BF3, Nostoc piscinale; BF4, Anabaena doliolum), using a novel vermicompost-based carrier, for their promise as inoculants in rice crop. The crop was maintained under 60% WHC through intermittent irrigation. Selected combinations (BF1 + 2 + 3; BF1 + 2 + 4; BF1 + 3 + 4) showed a consistent trend regarding their superiority over other treatments in terms of plant growth promotion and soil microbiological parameters. Microscopic examination of soil enrichment cultures raised from the treatments revealed their comparative abundance over native flora. A significant enhancement in terms of soil microbial biomass carbon and humus content was also recorded in these treatments. The soil enzymatic profile of the inoculated treatments involving FDA, dehydrogenase, acid phosphates, alkaline phosphatase, aryl esterase and β-glucosidase also revealed the promise of the inoculated strains. 16SrDNA sequencing, followed by BLAST analyses provided valuable information regarding the establishment of Anabaena strains. RFLP analyses of the 16SrDNA sequences of soil DNA and phylogenetic analyses vis a vis sequences of the inoculated cyanobacterial strains revealed the promise of treatments −BF1 + 2 + 3, BF1 + 2 + 4 and BF1 + 3 + 4. Panicle weight (yield attribute) was statistically at par with the fertilizer controls 1/2N + PK and significantly higher than 1/3N + PK. To our knowledge, this represents a first report on evaluating the establishment of cyanobacterial inoculants in rice crop grown under non-flooded conditions (or intermittent irrigation) using a combination of agronomic, microbiological and soil DNA related attributes. [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]

It is unclear whether certain plant species and plant diversity could reduce the impacts of multiple heavy metal pollution on soil microbial structure and soil enzyme activities. Random amplified polymorphic DNA (RAPD) was used to analyze the genetic diversity and microbial similarity in planted and unplanted soil under combined cadmium (Cd) and lead (Pb) pollution. A metal hyperaccumulator, Brassica juncea, and a common plant, Festuca arundinacea Schreb, were used in this research. The results showed that microorganism quantity in planted soil significantly increased, compared with that in unplanted soil with Cd and Pb pollution. The order of microbial community sensitivity in response to Cd and Pb stress was as follows: actinomycetes>bacteria>fungi. Respiration, phosphatase, urease and dehydrogenase activity were significantly inhibited due to Cd and Pb stress. Compared with unplanted soil, planted soils have frequently been reported to have higher rates of microbial activity due to the presence of additional surfaces for microbial colonization and organic compounds released by the plant roots. Two coexisting plants could increase microbe population and the activity of phosphatases, dehydrogenases and, in particular, ureases. Soil enzyme activity was higher in B. juncea phytoremediated soil than in F. arundinacea planted soil in this study. Heavy metal pollution decreased the richness of the soil microbial community, but plant diversity increased DNA sequence diversity and maintained DNA sequence diversity at high levels. The genetic polymorphism under heavy metal stress was higher in B. juncea phytoremediated soil than in F. arundinacea planted soil. [ABSTRACT FROM AUTHOR]

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]