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Abstract

Knowledge of the microbial diversity in natural ecosystems has long been limited because only a minority of naturally occurring microbes can be cultured using standard techniques. Several protocols for the extraction of nucleic acids directly from the environmental matrix have been recently developed to circumvent this problem and this review covers the major extraction procedures currently used to obtain microbial DNA from environmental samples.DNA extraction procedures can involve cell extraction or direct lysis, depending on whether or not the microbial cells are isolated from their matrix. An extraction protocol generally comprises three steps: cell lysis that can be chemical, mechanical and enzymatic, removal of cell fragments and nucleic acid precipitation and purification.Direct lysis methods are more often used than cell extraction ones because they are less time consuming and give a better recovery, resulting in an extracted DNA more representative of the whole microbial community present in the sample. However, with direct lysis, contaminants are also extracted which interfere with the DNA extract. As a consequence, a more extensive purification step is required. At least four types of purification are commonly used: cesium chloride density gradient ultracentrifugation, chromatography, electrophoresis and dialysis and filtration. To remove all contaminants, it could be recommended that several purification procedures be combined, depending on the environmental matrix.The efficiency of extraction/purification depends on the properties of the environmental sample, and each step of the extraction procedure must be adjusted for each sample. Moreover, each step of the procedure suffers from shortcomings, and each additional step inevitably induces a DNA loss. Thus, the choice of a protocol must be a compromise between the recovery of DNA that will be the most representative of the microbial community and the quality of the DNA obtained that is imposed by the objectives of the work, such as detection of specific organisms or assessment of the total microbial community structure. Nevertheless, molecular techniques, that could be used in combination with cultivation techniques, are powerful methods for surveying the microbial diversity in environmental samples, although investigators must be aware that such techniques are not exempt of methodological biases.

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... It is not an easy task to extract eDNA from soil, sediments, and activated sludge since the eDNA is weakly or tightly adsorbed onto organic or inorganic particles [34]. A sodium phosphate (NaP) buffer was first applied to elute the eDNA off the solid phase due to the competition between the NaP buffer and the eDNA for binding sites on solid particles [129]. Therefore, this approach has been extensively employed with different modifications [18,72,[130][131][132]. ...
... After the extraction, the crude eDNA extracts need to be further purified. There are multiple eDNA purification methods such as ethanol precipitation, BaSO 4 precipitation, polyethylene glycol precipitation, chromatography, cetyltrimethylammonium bromide (CTAB) precipitation, and additionally, the DNA extraction kits are frequently used to purify the DNA (Figure 4) [1,5,18,129,[135][136][137][138]. mation was observed when DNaseI was added to conduct eDNA digestio ...
... It is not an easy task to extract eDNA from soil, sediments, and activat the eDNA is weakly or tightly adsorbed onto organic or inorganic particles phosphate (NaP) buffer was first applied to elute the eDNA off the solid p competition between the NaP buffer and the eDNA for binding sites on [129]. Therefore, this approach has been extensively employed with diffe tions [18,72,[130][131][132]. ...
Article
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In nature, DNA is ubiquitous, existing not only inside but also outside of the cells of organisms. Intracellular DNA (iDNA) plays an essential role in different stages of biological growth, and it is defined as the carrier of genetic information. In addition, extracellular DNA (eDNA) is not enclosed in living cells, accounting for a large proportion of total DNA in the environment. Both the lysis-dependent and lysis-independent pathways are involved in eDNA release, and the released DNA has diverse environmental functions. This review provides an insight into the origin as well as the multiple ecological functions of eDNA. Furthermore, the main research advancements of eDNA in the various ecological environments and the various model microorganisms are summarized. Furthermore, the major methods for eDNA extraction and quantification are evaluated.
... The amounts of DNA isolated from different soil types per a gram of soil range from less than one microgram to approximately 500 micrograms (Daniel, 2005). Direct lysis is preferred because it is suitable for various soil textures, less time consumption and better DNA yield (Roose-Amsaleg et al., 2001;Daniel, 2004). ...
... transformation, cloning and DNA hybridization. To remove humic substances, purification steps often require PVP (polyvinylpolypyrrolidone) (Roose-Amsaleg et al., 2001). DNA from soil extraction should be precipitated using 5% polyethylene glycol instead of absolute ethanol or isopropanol in order to remove the humic impurity. ...
... Soil DNA measurement should be done with densitometric analysis of ethidium bromide stained agarose gel instead of spectrophotometric because OD 260 indicates the levels of humic substances rather than the DNA (Arbeli and Fuentes, 2007). In general, purity of soil genomic DNA can be evaluated by absorbance ratio at 260/230 nanometers (DNA/humic acid) and 260/280 nanometers (DNA/protein) (Roose-Amsaleg et al., 2001). Absorbance at higher wavelengths (320 or 340 nanometers) have been reported that they can be used to measure the level of humic acid and give the optical density that is independent from the OD of DNA and protein contents (Rajendhran and Gunasekaran, 2008). ...
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Metagenomic studies of unculturable bacteria may discover enzymes involved in the synthesis of novel natural products, e.g. nonribosomal peptide synthase (NRPS). Soil samples collected from Klongkone mangrove, Thailand was used for soil direct DNA extraction. Soil metagenomes was then purified and screened for nrps by polymerase chain reaction using an A domain-specific primer, MTF2/MTR. one-kb amplicons were cloned and sequenced for further analysis. Sequence alignment and phylogenetic analysis of the deduced amino acid sequence of amplified A domain revealed that the genes were evolutionary related to NRPSs of cyanobacteria, actinobacteria, and proteobacteria. These implied the novelty and diversity of nrps from the mangrove soil metagenome. These metagenomes were used for metagenomic library construction in E. coli resulted in 14000 library clones divided into 95 pools. A total of 31 pools were screened for anti-Candida albicans ATCC 90028 and anti-Bacillus subtilis (chloramphenicol resistant) activities. PCR screening of pools 30-39 of the library found A domain of nrps in pools 36 and 37. Sequence alignment and phylogenetic analysis revealed the novelty of these nrps. This suggested that mangrove soil metagenomic library harbored the novel and diverse nrps genes which might potentially lead to new drug discovery.
... However, due to the abundant contents of metabolites in camphoraceae plants [17] and the complex metabolic process of endophytes, it has been difficult to explore the co-evolution process between camphoraceae plants and endophytes. Exploring the diversity and distribution of endophytes in various plant parts is an important tool that can provide valuable resources for plant growth promotion and biotransformation [18]. Although numerous studies have focused on C. camphora and its secondary metabolites, no studies have been done to characterize endophytic microorganisms in C. camphora during different sampling times. ...
... The heatmap showed that most of the bacteria of C. camphora were distributed in D3, B3, and E3 samples in October in the Nanping region. The samples from this region had the highest number of phyla (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Heat map analysis of the relative abundances and distribution of bacterial endophytes phyla showed variations across the samples (Figure 7). ...
... October in the Nanping region. The samples from this region had the highest number of phyla (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). ...
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Investigations on the density, diversity, and distribution of endophytic bacterial community associated with leaves of Cinnamomum camphora (L.) Presl. were carried out during three seasons using 16s rDNA high-throughput sequencing technology. Samples were collected from five species in Nanping (A, B, C, D, and E) and one from Fuqin (F) in the Fujian province, China in the months of April, July, and October (represented by 1, 2 and 3), indicating spring, summer, and early winter, respectively. Results from 16s rDNA sequences revealed 10,844,124 effective sequences. The highest OTUs (Operational taxonomic units) was highest in the A1 sample (1086), while the lowest was in C2 (509). Our observations showed that samples taken in October had the highest diversity of endophytes as indicated by the Shannon index (B3 = 5.3337), Chao1 (E3 = 1233.10), abundance-based coverage estimator (ACE) (A3 = 1764.72), and the Simpson indices of diversity (C3 = 0.1655) irrespective of the species. The order of the endophytes richness in the samples was April > July > October. The Ribosomal Database Project (RDP) classifier showed that the obtained sequences belonged to nine major phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Gemmatimonadetes, Acidobacteria, Planctomycetes, Chloroflexi, and Fusobacteria. Proteobacteria accounted for the highest proportion in each sample, ranging from 35.15% to 89.72%. These sequences belonged mainly to 10 orders: Rhizobiales, Clostridiales, Peseudomonadales, Burkholderiales, Bacteroidales, Enterobacteriales, Rhodocyclales, Sphingomonadales, Lactobacillales, and Bacillales. Also, other taxa with possible taxonomic statuses, which were unclassified, were present.
... Purification of nucleic acid is influenced by the soil organic content (Roose-Amsaleg et al., 2001;Sharma et al., 2007;Saleh-Lakha et al., 2011). For the purification of nucleic acid the two main contaminants are proteins and humic acid. ...
... For protein aggregation solvent extraction (Ogram et al., 1987;Smalla et al., 1993) and salting out methods (Selenska and Klingmüller, 1991) have been used. Humic acid, being polyphenolic in nature, is inhibitory as the phenols bind to proteins via hydrogen bonds resulting in altered conformation of the enzymes used in downstream analysis (Kreader, 1996;Saleh-Lakha et al., 2011) and physiochemical properties similar to those of nucleic acid (Roose-Amsaleg et al., 2001). The presence of humic acid can be marked with the brownish colouration of the extracted DNA (Roose-Amsaleg et al., 2001;Robe et al., 2003). ...
... Humic acid, being polyphenolic in nature, is inhibitory as the phenols bind to proteins via hydrogen bonds resulting in altered conformation of the enzymes used in downstream analysis (Kreader, 1996;Saleh-Lakha et al., 2011) and physiochemical properties similar to those of nucleic acid (Roose-Amsaleg et al., 2001). The presence of humic acid can be marked with the brownish colouration of the extracted DNA (Roose-Amsaleg et al., 2001;Robe et al., 2003). Tebbe and Vahjen (1993) showed that the minimum inhibitory concentration (MIC) of humic acid varies with its composition, source and the enzyme used in downstream applications. ...
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This book includes 25 contributions from vastly experienced, global experts in PGPR research in a comprehensive and influential manner, with the most recent facts and extended case studies. Also, the chapters address the current global issues in biopesticide research.
... Thus, commercial kits require additional steps to obtain a sufficient concentration of gDNA free of contamination. Vortexing with glass beads, for example, has been used for mechanical sludge lysis to allow better hydration with the extraction buffers [14], whereas CTAB was added to improve chemical lysis and help to precipitate polysaccharides and humic substances [18]. ...
... Previous studies have reported the use of a combination of mechanical and chemical lysis. For example, glass bead vortexing was used for mechanical sludge lysis to allow better hydration with the extraction buffers [14], while CTAB was added to improve chemical lysis and helped to precipitate polysaccharides and humic substances [18]. ...
Article
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Obtaining sufficient and high-quality genomic DNA from sludge samples is a fundamental issue of feasibility and comparability in genomic studies of microbial diversity. Commercial kits for soil are often used for the extraction of gDNA from sludge samples due to the lack of specific kits. However, the evaluation of the performance of commercial kits for sludge DNA extraction is scarce and optimization of these methods to obtain a high quantity and quality of DNA is necessary, especially for downstream genomic sequencing. Sequential batch reactors (SBRs) loaded with lignocellulosic biomass are used for the synthesis of renewable resources such as levulinic acid (LA), adipic acid (AA), and polyhydroxyalkanoates (PHAs), and the biochemical synthesis of these compounds is conducted through the inoculation of microbes present in the residual activated sludge (AS) obtained from a municipal wastewater treatment plant. To characterize these microbes, the extraction of DNA from residual sewage sludge was conducted with three different commercial kits: Nucleospin® Soil from Macherey-Nagel, DNEasy® PowerSoil® from Qiagen, and E.Z.N.A.® Plant DNA Kit from Omega BIO-TEK. Nevertheless, to obtain the highest load and quality of DNA for next-generation sequencing (NGS) analysis, different pretreatments and different combinations of these pretreatments were used. The pretreatments considered were an ultrasonic bath and a temperature of 80 °C, together and separately with different incubation time periods of 30, 60, and 90 min. The results obtained suggest a significant improvement in the efficiency and quality of DNA extraction with the three commercial extraction kits when used together with the ultrasonic bath and 80 °C for 60 min. Here, we were able to prove that physical pretreatments are a viable alternative to chemical lysis for DNA extraction from complex samples such as sludge.
... The bacterial DNA isolation, DNeasy kit (Qiagen) was used for extraction of DNA from the pure culture according to manufacturer's specifications. The 16S rRNA gene fragment from each isolate was amplified by PCR using 8F and 149R primers [41][42]. The PCR process was carried out by taking 50 µl reaction mixture with 2 units of Taq DNA polymerase. ...
... Denaturation process was followed at 95 ºC for 3 min; followed by 30 cycles at 95 ºC for 30 s; 50 ºC for 30 s and 72 ºC for 1 min, with a final extension at 72 ºC for 7 min using thermal cycler. Further, amplified sequence was identified by agarose gel electrophoresis and conserved sequence identified [42]. ...
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This study evaluates soil chemical profile, bacterial diversity and a total number of halophytes from three regions, Southern Gulf of Kachchh (SGK) (A, B), South coastline (SC) (C, D) and Northern Gulf of Kachchh (NGK) (E, F). Soil enzymes related to C, N, and P were performed by the chemical and zymological method; while bacterial species were identified through 16s rRNA technique. Results showed that all the sites showed moderate to high alkaline conditions and sodic nature due to high concentration of sodium and chloride ions. The greatest bacterial and halophytes diversity were observed in soil sample of SGK followed by SC and NGK. Soil components and enzymes showed carbon and the associated enzymes positive correlation obtained at all sites (P < 0.001). Except the nitrate reductase soil N showed a positive correlation with protease and urease activity in soils (P < 0.05). The correlation PCA result observed total variance 74.48 % in biplot which indicates with B and D sites highesr, A and F moderate and E and F least nutrient components obtained from a different region. The correlation between enzymes and related nutrients components disclosed maximum positive correlation for B and D (P < 0.001), negative correlation for E and F and both correlation variability at C and A sites (P < 0.001, P < 0.05). These results indicate a moderate saline condition higher number of halophytes, significant enzymatic and microbial activities were found which essential for the nutrient recycling process in the coastal ecosystem.
... The extraction of DNA from environmental samples can be performed using direct or indirect (which involves an initial cell extraction step) approaches (Gabor et al., 2003) and requires lysis through physical, chemical, and/or enzymatic methods to disrupt the cell walls and membranes of the microorganisms and release their nucleic acids into the medium; followed by the removal of cell fragments, DNA capture and purification from contaminants (Roose-Amsaleg et al., 2001). Numerous studies have compared the most important extraction methods for soils and sediments (e.g., Stach et al., 2001;Carrigg et al., 2007;_ Inceoǧlu et al., 2010;Terrat et al., 2012;Leite et al., 2014;Devi et al., 2015), aiming to obtain DNA samples of high concentration and purity and consequently generate low-biased representations of their microbial communities (Robe et al., 2003). ...
... The soil environment results from multiple interacting factors, including texture, pH, and nutrient content (Robe et al., 2003). A number of studies have revealed the importance of physicochemical properties on the structure and functioning of tropical soil microbial communities (Lammel et al., 2018;Merloti et al., 2019;Pedrinho et al., 2019), although access to this diversity can be challenging as soil characteristics (including clay and organic matter contents) influence the efficiency of microbial DNA recovery (Roose-Amsaleg et al., 2001). In this sense, the results of the physicochemical analysis demonstrated that even nearby areas within the Amazon biome provide different environments for soil microbial communities; and therefore, it is imperative to establish a unique DNA extraction protocol that takes into account all these heterogeneous properties. ...
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Studies in the Amazon are being intensified to evaluate the alterations in the microbial communities of soils and sediments in the face of increasing deforestation and land-use changes in the region. However, since these environments present highly heterogeneous physicochemical properties, including contaminants that hinder nucleic acids isolation and downstream techniques, the development of best molecular practices is crucial. This work aimed to optimize standard protocols for DNA extraction and gene quantification by quantitative real-time PCR (qPCR) based on natural and anthropogenic soils and sediments (primary forest, pasture, Amazonian Dark Earth, and v arzea, a seasonally flooded area) of the Eastern Amazon. Our modified extraction protocol increased the fluorometric DNA concentration by 48%, reaching twice the original amount for most of the pasture and v arzea samples, and the 260/280 purity ratio by 15% to values between 1.8 to 2.0, considered ideal for DNA. The addition of bovine serum albumin in the qPCR reaction improved the quantification of the 16S rRNA genes of Archaea and Bacteria and its precision among technical replicates, as well as allowed their detection in previously non-amplifiable samples. It is concluded that the changes made in the protocols improved the parameters of the DNA samples and their amplification, thus increasing the reliability of microbial communities' analysis and its ecological interpretations.
... Roose-Amsaleg et al., 2001). DNA extracted from soils usually has more inhibitors such as humic acids, lignins, carbohydrates and resin and the amount can vary depending on several factors such as soil types(Roose-Amsaleg et al., 2001;Robe et al., 2003;O'Brien et al., 2009). ...
... Roose-Amsaleg et al., 2001). DNA extracted from soils usually has more inhibitors such as humic acids, lignins, carbohydrates and resin and the amount can vary depending on several factors such as soil types(Roose-Amsaleg et al., 2001;Robe et al., 2003;O'Brien et al., 2009). Positive PCR amplification was possible by cleaning up ...
Thesis
Bud rot disease has been considered as a devastating disease of oil palm in Latin America. Severe outbreaks of this disease have been reported in Colombia, Brazil, Ecuador, Panama and Suriname. The causal agent of bud rot disease in Colombia has been identified as Phytophthora palmivora. This pathogen is known to be responsible for several tropical diseases such as black pod and stem canker disease of cocoa, especially during the rainy season. Phytophthora palmivora has also been reported to attack durian, rubber, pepper and jackfruit causing diseases in various parts of the plant such as fruit, leaves and stems. However, no outbreaks of the disease have been reported in oil palm in Malaysia or other Southeast Asian countries. Several aspects of research need to be conducted to understand why this pathogen causes problems in oil palm in South America but not in Southeast Asia. This study aimed to analyze variation between the Colombian P. palmivora isolates that cause bud rot disease in comparison with Malaysian isolates and other isolates gathered from different hosts and regions. Our hypothesis was that P. palmivora isolates from the different regions and/or hosts have different molecular characteristics and have dissimilar levels of pathogenicity. Sequence alignments of several genetic markers, the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) gene cluster, beta-tubulin (β-tubulin), translation elongation factor 1 alpha (EF-1α), cytochrome c oxidase subunit I (CoxI) and subunit II (CoxII) genes failed to distinguish between Colombian oil palm isolates and P. palmivora from different hosts and regions. It was concluded that these markers are more suitable for inter-specific studies between species but not for intra-specific evaluation within species of P. palmivora. However, a new marker named as P. palmivora hypothetical avirulence effector protein (PpHPAVR) along with analyses of amplified fragment length polymorphisms (AFLPs), separated the Malaysian and Colombian isolates into distinct clades. This indicates that there is genomic variation within P. palmivora isolates. The zoospores of P. palmivora from various hosts and demographic origin were shown to have the ability to cause infection to oil palm seedlings, durian and rubber. However, not enough evidence has been collected to confirm that pathogenicity correlates with the distinct clades observed with AFLPs and PpHPAVR. Phytophthora palmivora species-specific diagnostic using PCR and loop-mediated isothermal amplification (LAMP) have been developed based on the PpHPAVR region.
... There are different methods for the extraction of bacterial DNA including the Phenol/Chloroform separation method (Wright et al. 2017), direct cell lysis method (Roose-Amsaleg et al. 2001), extraction with guanidium thiocyanate (Pitcher et al. 1989), commercial DNA extraction kits (Mcorist et al.), etc. In this study, a boiling lysis method was employed as it is a simple, more efficient, cost-effective, and more reliable technique when compared to alternative techniques (Yamagishi et al. 2016;Dimitrakopoulou et al. 2020). ...
Article
Commercially available liquid organic fertilizers have gained popularity in agriculture due to their convenience and perceived environmental benefits. However, these fertilizers are susceptible to various contaminations, including heavy metals such as lead (Pb) and cadmium (Cd), as well as bacterial contaminants like Escherichia coli (E. coli). Monitoring these contaminants helps to identify potential health risks to consumers, prevent soil and water pollution, and maintain the quality of agricultural products. Therefore, this study involved a comprehensive analysis of Pb and Cd heavy metals and E. coli bacterial contaminants in two widely used liquid organic fertilizers in the local market. The samples were digested using an open-vessel acid digestion followed by the flame atomic absorption spectroscopic (FAAS) analysis. The limit of detection (LOD), limit of quantification (LOQ), matrix effect, and spike recovery were determined. The Pb concentration of one fertilizer was 3.45 (± 0.07) mg/kg with 92% recovery whereas the other was 2.87 (± 0.08) mg/kg with 91% recovery. In contrast, the Cd concentration was 0.09 (± 0.01) mg/kg and 0.11 (± 0.01) mg/kg with percentage recoveries of 93% and 90% for two fertilizers respectively. The Pb content exceeded the maximum permissible limit of 1.0 mg/kg set by the Sri Lanka Standard (SLS 1702:2121), while Cd levels remained within the limit of 0.5 mg/kg. Polymerase chain reaction (PCR) coupled with gel electrophoresis confirmed the presence of the pathogenic O157:H7 E. coli strain in both fertilizers. Thus, the results of this study confirmed the lead (Pb) and Escherichia coli contamination in liquid organic fertilizers in the local market of Sri Lanka.
... There are different methods for the extraction of bacterial DNA including the Phenol/Chloroform separation method (Wright et al. 2017), direct cell lysis method (Roose-Amsaleg et al. 2001), extraction with guanidium thiocyanate (Pitcher et al. 1989), commercial DNA extraction kits (Mcorist et al.), etc. In this study, a boiling lysis method was employed as it is a simple, more efficient, cost-effective, and more reliable technique when compared to alternative techniques (Yamagishi et al. 2016;Dimitrakopoulou et al. 2020). ...
Article
Full-text available
Commercially available liquid organic fertilizers have gained popularity in agriculture due to their convenience and perceived environmental benefits. However, these fertilizers are susceptible to various contaminations, including heavy metals such as lead (Pb) and cadmium (Cd), as well as bacterial contaminants like Escherichia coli (E. coli). Monitoring these contaminants helps to identify potential health risks to consumers, prevent soil and water pollution, and maintain the quality of agricultural products. Therefore, this study involved a comprehensive analysis of Pb and Cd heavy metals and E. coli bacterial contaminants in two widely used liquid organic fertilizers in the local market. The samples were digested using an open-vessel acid digestion followed by the flame atomic absorption spectroscopic (FAAS) analysis. The limit of detection (LOD), limit of quantification (LOQ), matrix effect, and spike recovery were determined. The Pb concentration of one fertilizer was 3.45 (± 0.07) mg/kg with 92% recovery whereas the other was 2.87 (± 0.08) mg/kg with 91% recovery. In contrast, the Cd concentration was 0.09 (± 0.01) mg/kg and 0.11 (± 0.01) mg/kg with percentage recoveries of 93% and 90% for two fertilizers respectively. The Pb content exceeded the maximum permissible limit of 1.0 mg/kg set by the Sri Lanka Standard (SLS 1702:2121), while Cd levels remained within the limit of 0.5 mg/kg. Polymerase chain reaction (PCR) coupled with gel electrophoresis confirmed the presence of the pathogenic O157:H7 E. coli strain in both fertilizers. Thus, the results of this study confirmed the lead (Pb) and Escherichia coli contamination in liquid organic fertilizers in the local market of Sri Lanka. Keywords: liquid organic fertilizers, open vessel acid digestion, flame atomic absorption spectrometry, polymerase chain reaction, heavy metals, Escherichia coli.
... Molecular techniques overcome the aforementioned limitations and are now commonly used in diagnostics (Sweeney et al. 2007, Pereira et al. 2023. However, PCR also has technical constraints, namely cell lysis efficiency (when direct extraction is not performed), bacterial adherence to soil particles and the presence of inhibitors in the sample (e.g., Roose-Amsaleg et al. 2001). We therefore encourage the optimization and development of better techniques with which to assess both MTC presence in the environment (Fine et al. 2011a,b, Kukielka et al. 2013) and other biological features such as quantity, viability and infectious potential. ...
... Step Caveat of samples and scale of sampling), abundance of DNA templates in a sample [17], lysis of cells, condition and recovery of DNA [18], and contaminants (summary in [19]). Study design and sampling protocols are part of most ecosystem studies, and the design issues for microbial ecological studies have been considered by a number of publications [20][21][22][23]. ...
Article
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Collectively, we have been reviewers for microbial ecology, genetics and genomics studies that include environmental DNA (eDNA), microbiome studies, and whole bacterial genome biology for Microbial Ecology and other journals for about three decades. Here, we wish to point out trends and point to areas of study that readers, especially those moving into the next generation of microbial ecology research, might learn and consider. In this communication, we are not saying the work currently being accomplished in microbial ecology and restoration biology is inadequate. What we are saying is that a significant milestone in microbial ecology has been reached, and approaches that may have been overlooked or were unable to be completed before should be reconsidered in moving forward into a new more ecological era where restoration of the ecological trajectory of systems has become critical. It is our hope that this introduction, along with the papers that make up this special issue, will address the sense of immediacy and focus needed to move into the next generation of microbial ecology study.
... The extraction of microbial DNA from sub-seafloor sediment is more challenging than that from water, soil, or marine surface sediment because of the diversity of sediment types (e.g., fine sand, silt, and clay), low biomass, complex structures (e.g., spores and cysts), and multiple potential enzyme inhibitors (e.g., heavy metals, colloids, fulvic acids, and humic acids) that exist in the deep biosphere [12]. Sediment microbial DNA can be extracted by separating the cells from the sediment particles before lysis (cell extraction method) or by direct lysis of cells in the sediment without separation (direct lysis method) [13]. 2 of 13 The cell extraction method is time-consuming, has a low recovery rate, and may introduce extraction bias for different microbial groups [14]. By contrast, several studies have shown that the direct lysis method provides a high DNA yield, ribotype richness, and diversity and is a better method than cell extraction for investigating microbial diversity in sediments [15][16][17][18][19][20]. ...
Article
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Sub-seafloor sediment is the largest microbial habitat on Earth. The study of microbes in sub-seafloor sediment is largely limited by the technical challenge of acquiring ambient microbial DNA because of sediment heterogeneity. Changes in the extraction method, even just by one step, can affect the extraction yields for complicated sediment samples. In this work, sub-seafloor sediment samples from the Baltic Sea with high organic carbon content were used to evaluate the influence of different grinding beads on DNA extraction. We found that the grinding beads can affect the DNA extraction from the organic-matter- and biosiliceous-clay-rich samples. A mixture of 0.5-mm and 0.1-mm grinding beads exhibited higher DNA yields and recovered more unique taxa than other bead combinations, such as Stenotrophomonas from Gammaproteobacteria and Leptotrichia from Fusobacteria; therefore, these beads are more suitable than the others for DNA extraction from the samples used in this study. This advantage might be magnified in samples with high biomass. On the contrary, the use of only small beads might lead to underestimation for certain Gram-positive strains. Overall, the discovery of abundant widespread deep biosphere clades in our samples indicated that our optimized DNA extraction method successfully recovered the in situ microbial community.
... Theoretically, the microbial DNA, isolated from a soil, sediment and water sample represents the collective DNA of all the indigenous environmental microorganisms [17] and [18]. In a broad range the DNA strategies are separation of cells and direct lysis [19].The main problem when isolating DNA from soil and sediment is co-precipitation of contaminating substances (humic compounds & phenolics) that interfere with downstream processes [20]. Although there are many methods published worldwide for the extraction of soil and sediment DNA and many commercial kits are available as well, the problem mentioned above is usually encountered in these protocols as physico-chemical properties of soil and sediment vary from location to location. ...
... [56]. Different techniques have been developed to purify the total DNA from environmental samples [57][58][59]. There are two types of approaches used to lyse the cells: through chemical and/or enzymatic and/or mechanical lysis [59,60]. ...
Article
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Indoor house dust is a blend of organic and inorganic materials, upon which diverse microbial communities such as viruses, bacteria and fungi reside. Adequate moisture in the indoor environment helps microbial communities multiply fast. The outdoor air and materials that are brought into the buildings by airflow, sandstorms, animals pets and house occupants endow the indoor dust particles with extra features that impact human health. Assessment of the health effects of indoor dust particles, the type of indoor microbial inoculants and the secreted enzymes by indoor insects as allergens merit detailed investigation. Here, we discuss the applications of next generation sequencing (NGS) technology which is used to assess microbial diversity and abundance of the indoor dust environments. Likewise, the applications of NGS are discussed to monitor the gene expression profiles of indoor human occupants or their surrogate cellular models when exposed to aqueous solution of collected indoor dust samples. We also highlight the detection methods of dust allergens and analytical procedures that quantify the chemical nature of indoor particulate matter with a potential impact on human health. Our review is thus unique in advocating the applications of interdisciplinary approaches that comprehensively assess the health effects due to bad air quality in built environments.
... Scientists have developed a wide range of methods to study microbial diversities and community structures in rhizosphere soils, including culture-dependent approaches (Garrido et al., 2008), Biolog microplates (Choi and Dobbs, 1999), DGGE (denaturing gradient gel electrophoresis) (Lu et al., 2012), and PLFA (phospholipid fatty acid) method (Yao et al., 2016). However, due to the complex components in soil samples, it is hard to isolate and cultivate the microorganisms from soil samples (approximately 1% can get the pure cultures) (Amann et al., 1995;Roose-Amsaleg et al., 2001). Developing new methods to study complex rhizosphere microbial diversities and community structures is urgent. ...
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Anthracnose caused by Colletotrichum spp. was widespread in recent years and resulted in great damage to strawberry production. Soil microbial communities were key contributors to host nutrition, development, and immunity; however, the difference between the microbial communities of healthy and anthracnose-infected strawberry rhizosphere soils remains unclear. In this study, the Illumina sequencing technique was used to comparatively study the prokaryotic and fungal community compositions and structures between healthy and anthracnose-infected strawberry rhizosphere soils in Yuxi, Yunnan Province. Both microbial community diversities and richness of anthracnose-infected strawberry rhizosphere soils were higher than those of healthy strawberry rhizosphere soils. A total of 2,518 prokaryotic and 556 fungal operational taxonomic units (OTUs) were obtained at the 97% similarity threshold. Proteobacteria, Thaumarchaeota, and Acidobacteria were the dominant prokaryotic phyla; Ascomycota, unclassified_k__Fungi, and Mortierellomycota were the dominant fungal phyla. The relative abundances of beneficial bacterial phyla Actinobacteria and Firmicutes, genera Streptomyces, Azospirillum , and Bacillus were significantly reduced in anthracnose-infected strawberry rhizosphere soils; the relative abundance of beneficial fungal species Trichoderma asperellum shows a similar tendency with bacterial abundance. Besides Colletotrichum , 15 other potential fungal pathogen genera and seven fungal pathogen species were identified; among the potential pathogen genera and species, eight pathogen genera and Fusarium oxysporum showed significant differences between healthy and anthracnose-infected strawberry rhizosphere soils. The results suggested that strawberry planted in this area may be infected by other fungal pathogens except for Colletotrichum spp. Our present research will provide theoretical basis and data reference for the isolation and identification of strawberry pathogens and potential probiotics in future works.
... Many studies have compared DNA extraction methods and documented the biases imposed by lysis procedures [16][17][18][19]. This has provoked proposals of different methods for DNA extraction and purification from soil over the past few decades [16,[20][21][22] in an attempt to obtain an unbiased picture of soil microbiome biodiversity [10]. Nevertheless, no method has been shown to overcome all the biases described above, and the debate on the choice of DNA extraction methods is still ongoing. ...
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Although Next-Generation Sequencing techniques have increased our access to the soil microbiome, each step of soil metagenomics presents inherent biases that prevent the accurate definition of the soil microbiome and its ecosystem function. In this study, we compared the effects of DNA extraction and sequencing depth on bacterial richness discovery from two soil samples. Four DNA extraction methods were used, and sequencing duplicates were generated for each DNA sample. The V3–V4 region of the 16S rRNA gene was sequenced to determine the taxonomical richness measured by each method at the amplicon sequence variant (ASV) level. Both the overall functional richness and antibiotic resistance gene (ARG) richness were evaluated by metagenomics sequencing. Despite variable DNA extraction methods, sequencing depth had a greater influence on bacterial richness discovery at both the taxonomical and functional levels. Sequencing duplicates from the same sample provided access to different portions of bacterial richness, and this was related to differences in the sequencing depth. Thus, the sequencing depth introduced biases in the comparison of DNA extraction methods. An optimisation of the soil metagenomics workflow is needed in order to sequence at a sufficient and equal depth. This would improve the accuracy of metagenomic comparisons and soil microbiome profiles.
... The qPCR is widely used to estimate cell abundance and to monitor harmful algal species [27][28][29][30][31]. However, the results are affected by the standard curve and potential contamination (e.g., by salts, pigments, and other substances) [32][33][34]. Unlike the qPCR, a digital PCR (dPCR) is insensitive to potential PCR inhibitors and enables the absolute quantification of a target gene by counting the number of amplified DNA fragments in reaction samples using fluorescent signals; cell abundance in field samples can be estimated by chip-based dPCRs without standard curves as references [35,36]. Recently, Lee et al. [37] quantified the cells of Margalefidinium (Cochlodinium) polykrikoides in field samples by droplet dPCRs (ddPCRs) and measured the rDNA copy numbers per cell with high accuracy. ...
Article
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Alexandrium pacificum, which produces the paralytic shellfish toxin (PST) saxitoxin (STX), is one of the causative species of paralytic shellfish poisoning outbreaks in coastal areas of Korea. In this study, we developed a chip-based digital PCR (dPCR) method for A. pacificum detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an A. pacificum strain isolated in 2017, species-specific primers targeting sxtA4 (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of A. pacificum. Cell abundance in field samples, estimated by a chip-based dPCR, was compared with the PST content, and measured using a mouse bioassay. A comparison with shellfish PST concentrations indicated that cell concentrations above 500 cells L−1, as measured using the dPCR assay, may cause shellfish PST concentrations to exceed the allowed limits for PSTs. Concordance rates between dPCR and PST results were 62.5% overall in 2018–2021, reaching a maximum of 91.7% in 2018–2019. The sensitivity of the dPCR assay was higher than that of microscopy and sxtA4-based qPCRs. Absolute quantification by chip-based dPCRs targeting sxtA4 in A. pacificum exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms.
... Therefore, though only a minor portion of the available soil space is colonized by the microbial communities (Young and Crawford, 2004), the stability and the resilience of the soil system are determined by the combination of soil physical structure, nutrient availability, microbial diversity and activity (Meuer et al., 2020). The soil microbiome is impacted by human activities like agriculture, soil sealing and industrial emissions that cause environmental pollution (Roose-Amsaleg et al., 2001;Maron et al., 2011) due to the changes that these activities induce in the soil structure. Since anthropogenic activities have decreased biodiversity in soils, the assessment of the soil microbiome can be a crucial indicator of soil quality (Lehmann et al., 2020;Vieira et al., 2022). ...
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The microbial diversity is, among soil key factors, responsible for soil fertility and nutrient biogeochemical cycles, and can be modified upon changes in main soil physicochemical properties and soil pollution. Over the years, many restoration techniques have been applied to restore degraded soils. However, the effect of these approaches on soil microbial diversity is less understood and thus requires more investigation. In this study, we analyzed the impact, on soil microbial diversity of a patented novel technology, used to restore degraded soils. Soil samples were collected from three nearby sites located in Borgotrebbia, Piacenza, Italy, and categorized as reconstituted, degraded, and agricultural soils. After total soil DNA extraction, 16S rDNA multi-amplicon sequencing was carried out using an Ion GeneStudio S5 System to compare soils’ bacterial community profiles. Sequenced reads were processed to assign taxonomy and then key microbial community differences were identified across the sampling sites. Species diversity featured significant abatement at all rank levels in the degraded soil when compared to the agricultural control. The 5 year restoration technique showed full recovery of this index at the genus level but not at the phylum level, displaying a rank-dependent gradient of restored richness. In parallel, the abundance of genes involved in the nitrogen (N) biogeochemical cycle was assessed using quantitative Real-Time PCR (qPCR). Total DNA content was significantly higher (p < 0.05) in degraded (μ = 12.69 ± 2.58 μg g⁻¹) and reconstituted (μ = 11.73 ± 1.65 μg g⁻¹) soil samples when compared to the agricultural soil samples (μ = 2.39 ± 0.50 μg g⁻¹). The taxonomic diversity of each soil site was significantly different, with some instances unique of the agricultural soil even at the phylum level. The analysis of N functional genes showed that the relative abundance of bacterial amoA (p < 0.05) and nosZ (p < 0.01) genes were significantly lower in the agricultural than in the reconstituted and degraded soils. We concluded that the application of the soil reconstitution technique appears to enhance the active microbial community, with distinct diversity and functionality towards genes involved in N biogeochemical cycle, as compared to both the degraded and the agricultural soil.
... The direct extraction of nucleic acids from sediments revolutionized the study of microbial diversity in benthic ecosystems, since only a very small fraction of fungi, archaea and bacteria can be cultivated using standard techniques (Roose-Amsaleg et al., 2001). Since then, metabarcoding of marine and freshwater sediment eDNA has revealed huge and largely undescribed diversity of viruses (Tangherlini et al., 2020;Zheng et al., 2020), archaea (Corinaldesi et al., 2019;Hoshino et al., 2020), bacteria (Zinger et al., 2012), fungi (Grossart et al., 2019;Manohar and Raghukumar, 2013;Panzer et al., 2015;Picard, 2017;Wurzbacher et al., 2016) and protists (Forster et al., 2016;Pawlowski et al., 2011;Scheckenbach et al., 2010). ...
Article
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Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.
... Environmental samples contain materials that are incompatible with PCR analysis, and extraction protocols involve the removal of materials that can interfere with the analysis such as heavy metal salts, phenolic polymers, DNA-binding proteins and other cell debris. The most scrupulous measures are those employing chromatography or gel electrophoresis, or combinations of the two [68,69]. Under less stringent requirements, the DNA can simply be precipitated, and recovered from a filter membrane or silica beads [70]. ...
Article
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Antimicrobial resistance (AMR) is threatening modern medicine. While the primary cost of AMR is paid in the healthcare domain, the agricultural and environmental domains are also reservoirs of resistant microorganisms and hence perpetual sources of AMR infections in humans. Consequently, the World Health Organisation and other international agencies are calling for surveillance of AMR in all three domains to guide intervention and risk reduction strategies. Technologies for detecting AMR that have been developed for healthcare settings are not immediately transferable to environmental and agricultural settings, and limited dialogue between the domains has hampered opportunities for cross-fertilisation to develop modified or new technologies. In this feature, we discuss the limitations of currently available AMR sensing technologies used in the clinic for sensing in other environments, and what is required to overcome these limitations.
... Scatter plots showing distributions of reads obtained upon mapping on LMR001 PEGs in control (X Axis) as a function of salt or PEG8000 treatments (Y Axes) ( Figure S2A,B, respectively) or M-A plots (showing the logarithmic values of mean read numbers for control and salt or control and PEG8000, as a function of the log2 ratios ( Figure S2C,D, respectively)) indicated that while salt and control showed rather centered distributions, the sample PEG8000 returned a strong bias for lowly expressed transcripts that were over-represented in this library as compared with control and salt libraries. This bias may result from the presence of PEG8000 remaining in pelleted cells as these molecules favor precipitation of nucleic acids [29,30]. To estimate whether the presence of PEG8000 in the samples also influenced the recovery of particular size fractions of the RNAs, we compared the relative abundances of read counts in the three libraries upon normalization to the total read numbers mapped to PEGs for all predicted genes and ordering based on PEG size, their mean sequencing coverage (obtained from the three libraries), and, finally, by predicted gene order, which is independent of size or coverage. ...
Article
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The recently proposed species Ensifer aridi represents an interesting model to study adaptive mechanisms explaining its maintenance under stressful pedo-climatic conditions. To get insights into functions associated with hyperosmotic stress adaptation in E. aridi, we first performed RNAseq profiling of cells grown under sub-lethal stresses applied by permeating (NaCl) and non-permeating (PEG8000) solutes that were compared to a transcriptome from unstressed bacteria. Then an a priori approach, consisting of targeted mutagenesis of the gene encoding alternative sigma factor (rpoE2), involved in the General Stress Response combined with phenotyping and promoter gfp fusion-based reporter assays of selected genes was carried out to examine the involvement of rpoE2 in symbiosis and stress response. The majority of motility and chemotaxis genes were repressed by both stresses. Results also suggest accumulation of compatible solute trehalose under stress and other metabolisms such as inositol catabolism or the methionine cycling-generating S-adenosyl methionine appears strongly induced notably under salt stress. Interestingly, many functions regulated by salt were shown to favor competitiveness for nodulation in other rhizobia, supporting a role of stress genes for proper symbiosis’ development and functioning. However, despite activation of the general stress response and identification of several genes possibly under its control, our data suggest that rpoE2 was not essential for stress tolerance and symbiosis’ development, indicating that E. aridi possesses alternative regulatory mechanisms to adapt and respond to stressful environments.
... DNA extraction method was modified from Dong et al. and Roose et al. [39,40]. Approximately one gram of 0.5 mm glass beads was added in the washing step. ...
Article
Immobilization of microorganism on material is one of the effective methods to improve the production of bio-products including biofuel such as biohydrogen and biohythane (H2+CH4). In this work, we studied the effect of immobilizing media including clay/activated carbon (CAC), clay/zeolite (CZ) and clay/mixed activated carbon and zeolite (CACZ) on microbial attachment and biogas production including biohydrogen and biohythane using shrimp pond sediment as organic resource and microbial consortium. The result showed that CAC was the most appropriate material for microbial attachment among the other materials. CAC digesters demonstrated higher H2 concentration than control reactors. Interestingly, the control reactors of using only shrimp pond sediment without CAC showed the ability of biohythane production. From 454-pyrosequencing results, we found that Alcaligenaceae family, Tissierella Soehngenia sp. and Planomicrobium sp. are the predominant species in shrimp pond sediment. Meanwhile, the study of microbial species on CAC after digestion process by Illumina MiSeq platform represented Clostridium cellobioparum, Tolumonas auensis and Microbacter margulisiae for bacteria and Methanobacterium aggregans for archaea as predominant species. It was shown that the immobilized media (CAC) favored some types of microbes mainly hydrogen producing microbes and hydrogenotrophic methanogen. This research suggests the CAC as an alternative immobilizing media for bioenergy production especially biohydrogen and biohythane.
... low-copy eDNA) (Renshaw et al., 2014;Natarajan et al., 2016). It is also noted that different methods and adaptations to protocols, which is common for groups using commercial kits, can produce differences in eDNA yield and species detectability (Roose-Amsaleg, Garnier-Sillam & Harry, 2001;Deiner et al., 2015;Eichmiller, Best & Sorensen, 2016;Eichmiller, Miller & Sorensen, 2016;Djurhuus et al., 2017;Deiner et al., 2018). As a result, the importance of testing and optimizing workflows for the study objective has been emphasized (Kumar, Eble & Gaither, 2020). ...
Article
• Pressures on coastal ecosystems are increasing and aquatic species that are restricted to these habitats are facing the threat of extinction. However, the true extent of many threatened and rare aquatic species, especially elasmobranchs, remains unclear due to high levels of data deficiency and poor efficacy of traditional survey methods. Sawfishes (Pristidae), a family of shark-like rays, are among the most threatened and rare elasmobranch species and are difficult to detect in turbid, coastal habitats. Reliable cost-effective tools to detect these species are urgently needed to increase their conservation potential. • Characterization of environmental DNA (eDNA) extracted from water samples has garnered significant appeal for detection of rare and threatened species. To assist conservation and monitoring efforts for sawfishes using eDNA, species-specific TaqMan quantitative polymerase chain reaction assays were developed and validated to detect 1.25–5 copies of a 12S rRNA gene fragment. Filter samples were collected in Northern Territory, Australia to assess the utility of the developed eDNA assays and compare the efficacy of preservation and extraction workflows for detecting rare species. • Dwarf sawfish (Pristis clavata) were detected in three of 20 sites, and there was a significant effect of preservation and extraction workflow on total eDNA yield and subsequent detection success. Longmire's preserved samples extracted using glycogen-aided precipitation yielded a significantly higher concentration of total eDNA (n = 60; β = 1.27, t(95) = 8.172, P < 0.0001) and yielded positive P. clavata eDNA detections compared to ethanol preserved samples extracted using QIAGEN DNeasy kit, which did not yield any positive detections. • The optimized eDNA assays were developed to support monitoring efforts for endangered sawfishes. Importantly, this study demonstrates that choice of preservation and extraction workflow requires careful consideration, especially when detection of rare or threatened species can have important management and conservation outcomes.
... Le terme « d'ADN environnemental » émerge pour la première fois dans la littérature scientifique en 1987, dans des études de microbiologie visant à identifier l'ADN d'organismes microbiens spécifiques à partir de sédiments (Ogram G.S, 1987). Quelques années plus tard, en 1990, une équipe publiera la première étude généraliste visant à analyser la diversité génétique de bactérioplanctons à partir d'ARN ribosomique par des approches de metabarcoding sur des échantillons d'eau de mer (Giovannoni, Britschgi, Moyer, & Field, 1990;Taberlet et al., 2018). ...
Thesis
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Les changements globaux, qu’ils soient d’origine climatique ou anthropique ont diverses conséquences en santé humaine et animale, mais aussi sur les écosystèmes mondiaux. L’une des plus importantes est la modification des aires de répartitions géographiques des espèces et de celle des pathogènes qui leurs sont associés. C’est dans ce contexte que nous assistons ces dernières années à une recrudescence des cas d’émergences et de réémergences de maladies infectieuses dans le monde. Alors que les efforts de recherche menés dans ce domaine se focalisent principalement sur les maladies virales, les maladies transmises par les mollusques d’eau douce, qui affectent plus d’un milliard d’individus dans le monde, sont également sujettes à ces évènements d’émergences devenus fréquents. Cependant, l’étude de la dynamique des parasites associés à ces maladies se focalisent essentiellement sur le diagnostic et le traitement des hôtes définitifs, en particulier l’Homme. Toutefois, une telle approche ne permet pas de prévenir de la transmission de ces parasites à l’Homme et encore moins de prévenir d’un évènement d’émergence, et les outils actuels utilisés pour le suivi de ces parasites dans l’environnement sont difficilement applicables à large échelle. Ce travail de thèse se propose donc d’apporter une vision plus environnementale de la dynamique de ces maladies. Avec l’exemple de l’émergence de bilharziose urogénitale en Corse, nous avons analysé cette émergence en intégrant l’étude des traits d’histoire de vie du parasite tropical en cause, notamment sa thermo tolérance, ainsi que le rôle des hôtes intermédiaires mollusques et des hôtes définitifs sauvages et domestiques dans le maintien local du cycle parasitaire. Dans un second temps nous avons développé des outils de diagnostic par ADN environnemental pour la détection de mollusques hôtes dans l’environnement afin d’identifier les zones à risque d’émergence, ainsi que des outils de détection intramolluscal de schistosomes pour identifier les sites de transmission actif, et donc permettre un suivi environnemental des acteurs de ces maladies. Pour compléter ces approches, nous avons développé un outil plus généraliste de metabarcoding environnemental pour caractériser les communautés de mollusques d’eau douce, et initié le développement d’un outil similaire pour la caractérisation des communautés de trématodes, ceci afin d’étudier les interactions entre ces organismes. Enfin nous discutons de l’intégrations de tous ces éléments dans de nouvelles stratégies de contrôle à l’encontre de maladies transmises par les mollusques d’eau douce.
... Additionally, it is known that certain compounds-polysaccharides, humic acids, may affect PCR reactions Rådström et al., 2004, requiring the need for additional DNA clean-up. It has been established that DNA losses occur during the purification steps (Roose-Amsaleg, Garnier-Sillam & Harry, 2001), including when using commercial column methods (Howeler, Ghiorse & Walker, 2003;Lloyd et al., 2013), and phenol-chloroform (Ogram, Sayler & Barkay, 1987). Interestingly, we found similar losses when using the magnetic bead clean-up, whereas the ethanol precipitation method was inefficient compared to the phenol-chloroform protocol. ...
Article
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Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.
... The cell lysis step can be performed by two methods: (1) an indirect DNA extraction, which consists of first removing cells from soil and subsequently lysing them, or (2) a direct DNA extraction where cells are lysed directly within the soil. The direct method is often used because it is faster and has a higher DNAyield (Roose-Amsaleg et al. 2001). The treatment used during lysis must be as efficient as possible to obtain the maximum of the DNA. ...
... (Schmeisser, Steele, & Streit, 2007). Two strategies have been popular for metagenomic DNA isolation, and they are the cell recovery method and the direct lysis method (Roose-Amsaleg, Garnier-Sillam, & Harry, 2001). The cell recovery method isolates intact organisms from the gut content before cell lysis, and the cell isolation is achieved either by repeated homogenization and differential centrifugation (Hopkins, Macnaughton, & Odonnell, 1991) or by gradient centrifugation (Robe, Nalin, Capellano, Vogel, & Simonet, 2003). ...
Chapter
Microbes through its symbiotic relationship between the host and their invaded organisms, owes a major part in nature as well as the evolutionary processes. Insects are the most diversified fauna with stable surveillance in innumerable ecological niches. Many among those act as secondary symbionts as well. These symbiotic connotations originated more recently and can be ephemeral. In some cases, the direct contact with the host also is not needed in a population for these organisms to survive. Because when the primary symbionts transfer the genes directly from mother to progeny, the secondary symbionts do it through several means. This chapter reviews the association between insect gut microbiomes and their interactions with the environment. Recent insights and potential applications of the insect gut microbiome will provide a metagenomic thorough knowledge of the microbial census in the insect gut.
... To investigate the phylogeny of the CB discovered in Yaquina Bay, genomic DNA was extracted from three sediment plug samples and from two separated filamentous biomass samples using a MoBio PowerSoil DNA Extraction Kit. To avoid insufficient cell lysis, all samples went through five to seven freeze-thaw cycles before the use of the extraction kit (Roose-Amsaleg et al., 2001). Bacterial 16S rRNA genes were amplified by polymerase chain reaction (PCR) with random primers 357wF (5 -CCTACGGGNGGCWGCAG-3 ) and 785R (5 -GACTACHVGGGTATCTAATCC-3 ). ...
Article
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Cable bacteria (CB) are multicellular, filamentous bacteria within the family of Desulfobulbaceae that transfer electrons longitudinally from cell to cell to couple sulfide oxidation and oxygen reduction in surficial aquatic sediments. In the present study, electrochemical reactors that contain natural sediments are introduced as a tool for investigating the growth of CB on electrodes poised at an oxidizing potential. Our experiments utilized sediments from Yaquina Bay, Oregon, USA, and we include new phylogenetic analyses of separated filaments to confirm that CB from this marine location cluster with the genus “Candidatus Electrothrix”. These CB may belong to a distinctive lineage, however, because their filaments contain smaller cells and a lower number of longitudinal ridges compared to cables described from other locales. The results of a 135 d bioelectrochemical reactor experiment confirmed that these CB can migrate out of reducing sediments and grow on oxidatively poised electrodes suspended in anaerobic seawater. CB filaments and several other morphologies of Desulfobulbaceae cells were observed by scanning electron microscopy and fluorescence in situ hybridization on electrode surfaces, albeit in low densities and often obscured by mineral precipitation. These findings provide new information to suggest what kinds of conditions will induce CB to perform electron donation to an electrode surface, further informing future experiments to culture CB outside of a sediment matrix.
... To investigate the phylogeny of the cable bacteria discovered in Yaquina Bay, genomic DNA was extracted from 155 washed sediment samples and from tangles of bacterial filaments separated from sediments using a MoBio PowerSoil DNA Extraction Kit. To avoid insufficient cell lysis, all samples went through 5-7 freeze-thaw cycles before the use of the extraction kit (Roose-Amsaleg et al., 2001). Bacterial 16S rRNA genes were amplified by PCR with random primers 357wF (5′-CCTACGGGNGGCWGCAG-3′) and 785R (5′-GACTACHVGGGTATCTAATCC-3′). ...
Article
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The scope of the present study is to introduce electrochemical reactors as a tool for investigating the growth of novel filamentous cable bacteria and their unique extracellular electron transfer ability. New evidence that cable bacteria are widely distributed in sediments throughout an estuarine system connected to the NE Pacific Ocean is also presented. Cable bacteria found within Yaquina Bay, Oregon, USA, appear to cluster with the genus, Candidatus Electrothrix. Results of a 135-day bioelectrochemical reactor experiment confirm a previous observation that cable bacteria can grow on oxidatively poised electrodes suspended in anaerobic seawater above reducing sediments. However, several diverse morphologies of Desulfobulbaceae filaments, cells, and colonies were observed on the carbon fibers of the suspended electrodes including encrusted chains of cells. These observations provide new information to suggest what conditions will induce cable bacteria to perform electron donation to an electrode surface, further informing future experiments to culture cable bacteria apart from a sediment matrix.
... As reported elsewhere, spores and mycelia could not always be cracked by extraction methods due to their complex and sturdy cell wall [60]. The bead beating method failed partially as described in other studies [35,56], even if we could also see its usefulness for gaining an increased DNA yield as shown by Roose-Amsaleg et al. and Lakay et al. [30,47]. In addition, fungal nucleases, polysaccharides, and pigments could also contribute to difficulties in isolating DNA [35]. ...
Article
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The original version of this article contained a mistake in the co-author's conflict of interest statement. Erika von Mutius wishes to add the following disclosures: "E. von Mutius is listed as an inventor on the following patents: publication number EP 1411977, composition containing bacterial antigens used for the prophylaxis and the treatment of allergic diseases, granted on 18 April 2007; publication number EP1637147, stable dust extract for allergy protection, granted on 10 December 2008; publication number EP 1964570, pharmaceutical compound to protect against allergies and inflammatory diseases, granted on 21 November 2012. E. von Mutius is listed as inventor and has received royalties on the following patent: publication number EP2361632, specific environmental bacteria for the protection from and/or the treatment of allergic, chronic inflammatory and/or autoimmune disorders, granted on 19 March 2014".
... One bog PowerSoil sample, and one fen CTAB sample, had unusually high A260:280 ratios, suggesting the presence of leftover extraction reagents in the sample. Soil microbial metagenome protocols commonly include further DNA clean-up after extraction to remove inhibitory substances commonly seen in soil (summarized in Roose-Amsaleg, Garnier-Sillam & Harry, 2001;Roslan, Mohamad & Omar, 2017), therefore we evaluated the potential improvement in viral DNA purity from clean-up by AMPure beads. Purity (measured via A260:280) improved significantly in the bog PowerSoil + AMPure samples and was best in the CTAB + AMPure samples, while in the fen, onlyPowerSoil Although DNA extract yield and purity metrics are useful indicators of extract quality, the goal is successful library preparation and sequencing. ...
Article
Full-text available
Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ∼1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1-2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified How to cite this article Trubl G, Roux S, Solonenko N, Li Y-F, Bolduc B, Rodríguez-Ramos J, Eloe-Fadrosh EA, Rich VI, Sullivan MB. 2019. Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils. PeerJ 7:e7265 http://doi.org/10.7717/peerj.7265 compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ∼4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates 'living' protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.
... Possible explanations including: 1) P LLOD provides information about the smallest amount of fecal matter rather than the number of DNA molecules; 2) Bacteroidales only makes up 20% to 40% of the amount of total fecal bacteria ( Stephen and Cummings, 1980;Vandeputte et al., 2017); 3) DNA recovery from filtration process and DNA extraction is inherently variable and inefficient, resulting in an increase in LLOD due to target loss. Previous research has shown that DNA recovery from the DNA extraction protocol varies from 16.5% to around 91%, and the filtration protocol only recovers 79% of the DNA target ( Staley et al., 2012;Robe et al., 2003;Roose-Amsaleg et al., 2001;Zhou et al., 1996; unpublished results from our group). Other factors such as the decay of Bacteroidales and any inhibitors present in the fecal samples would also adversely affect P LLOD ( Bell et al., 2009;Rogers et al., 2011). ...
Preprint
Identification of fecal contamination sources in surface water has become heavily dependent on quantitative PCR (qPCR) because this technique allows for the rapid enumeration of fecal indicator bacteria as well as the detection and quantification of fecal source-associated genetic markers in the environment. Identification of contamination sources in impaired waters is a prerequisite for developing best management practices to reduce future pollution. Proper management decisions rely on the quality and interpretation of qPCR data. In this study, we developed a method to determine analytical and process lower limits of detection (LLOD) and quantification (LLOQ) using two cattle-associated genetic markers targeting Bacteroidales. Analytical LLOD (A LLOD) for both CowM2 and CowM3 genetic markers in the qPCR assay were five gene copies per reaction. Using composite fecal DNA, the analytical LLOQ (A LLOQ) determined for CowM2 and CowM3 were 78 and 195 gene copies/reaction, respectively. When plasmid DNA was used, the A LLOQ for CowM2 and CowM3 were 46 and 20 gene copies/reaction, respectively. The process LLOD (P LLOD) for CowM2 and CowM3 were 0.4 and 0.02 mg feces/filter (wet weight), respectively. Using the standard deviation value of 0.25 as a cutoff point for LLOQ in regression analysis, the process LLOQ (P LLOQ) for CowM2 and CowM3 were 3.2 and 0.3 mg feces/filter, respectively. These results indicate that CowM3 exhibited superior performance characteristics compared with CowM2 for fecal samples collected from our geographical region. Moreover, the method for calculating LLOD and LLOQ developed here can be applied to other microbial source tracking studies.
... One bog PowerSoil sample, and one fen CTAB sample, had unusually high A260:280 ratios, suggesting the presence of leftover extraction reagents in the sample. Soil microbial metagenome protocols commonly include further DNA clean-up after extraction to remove inhibitory substances commonly seen in soil (summarized in Roose-Amsaleg, Garnier-Sillam & Harry, 2001;Roslan, Mohamad & Omar, 2017), therefore we evaluated the potential improvement in viral DNA purity from clean-up by AMPure beads. Purity (measured via A260:280) improved significantly in the bog PowerSoil + AMPure samples and was best in the CTAB + AMPure samples, while in the fen, onlyPowerSoil Although DNA extract yield and purity metrics are useful indicators of extract quality, the goal is successful library preparation and sequencing. ...
Preprint
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Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ~1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1–2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ~4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates ‘living’ protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.
... One of the advantages of the qPCR method is its relative simplicity and high-throughput capacity, allowing simultaneous quantification of cysts across different samples. However, phenolic substances and humic acids commonly found in sediment samples could inhibit amplification reactions (Roose-Amsaleg et al., 2001), lowering the efficiency of the qPCR reaction. This is shown by the lower standard curve efficiency when used to quantify diploid cysts compare to haploid planktonic cells (Figures 3C, 4). ...
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Paralytic shellfish toxin producing dinoflagellates have negatively impacted the shellfish aquaculture industry worldwide, including in Australia and New Zealand. Morphologically identical cryptic species of dinoflagellates that may differ in toxicity, in particular, species of the former Alexandrium tamarense species complex, co-occur in Australia, as they do in multiple regions in Asia and Europe. To understand the dynamics and the ecological drivers of the growth of each species in the field, accurate quantification at the species level is crucial. We have developed the first quantitative polymerase chain reaction (qPCR) primers for A. australiense, and new primers targeting A. ostenfeldii, A. catenella, and A. pacificum. We showed that our new primers for A. pacificum are more specific than previously published primer pairs. These assays can be used to quantify planktonic cells and cysts in the water column and in sediment samples with limits of detection of 2 cells/L for the A. catenella and A. australiense assays, 2 cells/L and 1 cyst/mg sediment for the A. pacificum assay, and 1 cells/L for the A. ostenfeldii assay, and efficiencies of >90%. We utilized these assays to discriminate and quantify co-occurring A. catenella, A. pacificum, and A. australiense in samples from the east coast of Tasmania, Australia.
... 8,9 These days commercial kits are available which quickly isolate DNA for various PCR based applications. 10,11 PCR REACTION Isolated DNA alongwith other components, Taq DNA Polymerse, buffers, nucleoside triphosphates and DNA primers is placed in thin walled PCR tubes and these tubes are then placed in the PCR Thermal cycler. The primers are 15 -30 nucleotides long and are complimentary in sequence to the ends of target DNA. ...
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To fully understand cellular processes, scientists often examine events at the level of nucleic acids and protein molecules. These studies are complicated by the fact that cells have miniscule amounts of molecules of interest which are too small to be seen. So we require molecular tools to visualize these molecules. Polymerase chain reaction is one such technique widely used in molecular biology and produce quantities that are sufficient to study and visualize. With the advancement in this technique, it has revolutionized the field of research and diagnosis. In this article, we present a review on the principle, basic technique, applications, limitations and recent advances of Polymerase Chain Reaction.
Chapter
The steadily increasing presence of both natural and anthropogenic pollutants in our environment poses a considerable challenge, given the recalcitrance of many of these pollutants. Microbial bioremediation presents a promising and sustainable strategy that harnesses a diverse array of microorganisms, operating either concurrently or sequentially, to eliminate or mitigate the presence of pollutants within the environment. Recent years have witnessed the application of multiomics techniques to the study of biodegradation and bioremediation, yielding an abundance of novel data that enrich our comprehension of pivotal pathways and offer fresh perspectives on the adaptability of organisms amidst shifting environmental conditions. This book brings together recent progress in microbial bioremediation, emphasizing the emerging field of multiomics technologies. It serves as a valuable reference for microbiologists exploring multiomics applications and environmental scientists seeking innovative remediation solutions.
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During the 20th century, antibiotic therapy has drastically reduced the mortality caused by bacterial pathogens. However, during the last few decades, the human community is continuously facing new cases of antibiotic resistance in clinics, which may result in antibiotic therapy failure. Antibiotics and antibiotic resistance predate the discovery of antibiotics by thousands of years. However, the industrialization and extensive use of antibiotics in humans and animals has imposed a selective pressure without precedents on bacterial communities, accelerating the development of antibiotic resistance at a global scale. Antibiotics are used worldwide not only to treat diseases caused by human pathogens, but also with therapeutic and growth promotion purposes in farms, aquacultures and crops. As a consequence of anthropogenic activities, residual concentrations of antibiotics, antibiotic resistance genes (ARG) and antibiotic resistant bacteria (ARB) may reach the environment. This may result in the development and selection of antibiotic resistance in environmental settings and the dissemination of ARB and ARG from the environment to animal and human microbiomes. Nonetheless, the scope of this phenomenon remains unclear. The goal of this thesis was to evaluate the response of the environmental microbiome and resistome to chemical pollution with antibiotics, as well as to biological pollution caused by anthropogenic activities. Two microcosm studies were carried out to evaluate the effects of antibiotic pollution on the microbiome and resistome of an agricultural soil and of water from the Rhône river using a combination of culture-based and metagenomics/qPCR approaches. Gentamicin sub-inhibitory and inhibitory concentrations in soil and water bacteria enriched in culture media were determined by evaluating bacterial growth. Soil microcosms were polluted with a range of inhibitory concentrations of gentamicin, whereas water microcosms were polluted with two sub-inhibitory concentrations and an inhibitory concentration, in order to establish links between gentamicin dose and the magnitude of the response in the environmental microbiome and resistome at different exposure times. These two studies illustrate how the effects of the same antibiotic on different environments are strongly dependent of environmental factors and physicochemical properties. Our findings support the concern that sub-inhibitory concentrations of antibiotics may select for resistance in the environment. In addition, this research underlines the limitations of the terms “sub-inhibitory” and “inhibitory” in complex environments and the importance of microcosm and field studies for the evaluation of the effects of antibiotic pollution on the environmental resistome. Finally, a field study was carried out in snow samples from the Sudety Mountains (Poland) with a range of exposure to human activities and surrounding vegetation in order to evaluate the impact of both anthropogenic and environmental factors on the snow microbiome and resistome using a metagenomics and qPCR approach. This research supports the hypothesis that both environmental and anthropogenic factors impact snow ecology and induce changes in the snow microbiome and antibiotic resistome by providing bacterial communities with higher levels of organic carbon and other nutrients. This would support the growth of a more abundant bacterial community, which in turn increases the abundance of the antibiotic resistome and could stimulate competition and ARG proliferation in snow. This organic pollution could stimulate the development of antibiotic resistance in the snow microbiome that might be subsequently disseminated through the atmosphere or snow melting. The research carried out in this study highlights the need for survey of antibiotic resistance development in anthropogenic polluted sites and the consideration of organic sources of pollution in addition to biological pollutants (ARB and ARG).
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Cell lysis is an essential step in many studies related to biology and medicine. Based on the scale and medium that cell lysis is carried out, there are three main types of the cell lysis: (1) lysis of the cells in the surrounding environment, (2) lysis of the isolated or cultured cells, and (3) single cell lysis. Conventionally, several cell lysis methods have been developed, such as freeze‐thawing, bead beating, incursion in liquid nitrogen, sonication, and enzymatic and chemical‐based approaches. In recent years, various novel technologies have been employed to develop new methods of cell lysis. The aim of studies in this field is to introduce more precise and efficient tools or to reduce the costs of cell lysis procedures. Nanostructure‐based lysis methods, acoustic oscillation, electrical current, irradiation, bacteria‐mediated cell lysis, magnetic ionic liquids, bacteriophage genes, monolith columns, hydraulic forces, and steam explosion are some examples of newly developed cell lysis methods. Besides the significant advances in this field, there are still many challenges and tools must be further improved.
Chapter
Insects, the most successful groups in animal kingdom, harbor diverse groups of microbes, such as bacteria, archaea, fungi, protozoa, and viruses, which profoundly influence their survival and adaptations over a wide range of ecological niches. These microbes are associated with their host insects permanently or transiently and such associations may be beneficial or harmful to the host insect under various instances. Attempts were made earlier to characterize insect microbiome by isolation and cultivation techniques and polymerase chain reaction (PCR)-based cloning methods that resulted in identification of a few groups of microbes. The metagenomic approaches under the next-generation sequencing platforms provide unparallel opportunities to understand the composition of the microbiome and their functional role in the biology of the insects, thus expanding our understanding from a single microbial species to the whole community. These approaches provide an ample opportunity to understand the components of the microbiome that can potentially and collectively affect the behavior and physiological traits of insects through genetic and metabolic interactions. For instance, endosymbionts (i.e., microbes that live inside host cells or tissues) depend on the insect hosts for obtaining nutrients, provide fitness advantages to their insect hosts in terms of the breakdown of plant cell wall components, viz, cellulose, lignocelluloses, and xylan, supplying essential amino acids and vitamins to host insects, thereby upgrading the nutrient status of their diet, detoxification of lethal insecticide molecules, plant defensive compounds such as phenolics, and production of anti-microbial peptides against insect pathogens. However, in some instances, the microbes may also be pathogenic to the insect hosts by producing insecticidal toxins, which reduce viability and cause morbidity.
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Most commonly, NGS‐based microbiome studies are performed on the total DNA (totDNA) pool that, however, consists of extracellular‐ (exDNA) and intracellular (iDNA) DNA fractions. By investigating the microbiomes of different anaerobic digesters over time, we found that totDNA suggested lower species richness considering all and/or only common species and yielded fewer unique reads as compared to iDNA. Additionally, exDNA‐derived sequences were more similar to those from totDNA than from iDNA and finally, iDNA showed the best performance of tracking temporal changes in microbial communities. We postulate that abundant sequences present within the exDNA fraction are masking the overall results of totDNA and provide evidence that exDNA has the potential to qualitatively bias microbiome studies at least in the anaerobic digester environment as it contains information about cells that were lysed hours or days ago. iDNA, however, was found more appropriate to gain reliable genetic information about potentially alive as well as rare microbes within the target habitat.
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The knowledge about biochemical and physiological mechanisms by microorganisms in soils are required for a complete description of microbial diversity, lately different molecular techniques have been developed to study this feature (qPCR, DGGE, T- RFLP, RAPD). DNA extraction techniques ensure the description of the microbial diversity success, according the soil samples characteristics. Generally, DNA extraction protocols used for separation of microorganism of matrix soil before DNA extraction by physical and chemical lysis. Other protocol is direct extraction of microbial DNA from soil samples, humic acids and phenolic substances affect the quality of DNA, which affect the development of subsequent molecular studies. The purpose of this study was to establish pretreatment procedures for different kind of soil samples (frank, sandy and clay ey) in order to describe richness and bacterial diversity by PCR DGGE. In this sense, we determined the addition of CaCO3 in frank soils samples allows the identification of greater diversity and bacterial richness (10 bands) than the other method. Besides, PVPP pretreatment is no only useful to obtain bacterial diversity in sandy soil (8 bands), but also in clayey soils (3 bands) soils by PCR-DGGE method. This indicates that the pretreatment procedures with CaCO3 and PVPP are specific for soil microbial community’s isolation.
Thesis
Harmful algal blooms that produce Paralytic Shellfish Toxins (PSTs) are prevalent and affect shellfish harvesting areas worldwide. PSTs have caused shellfish harvesting closures and product recalls, resulting in economic losses, as well as brand damage and damage to the wider economy including the tourism industry. In Tasmania, it is known that four PST producing species co-occur, comprising 𝘈𝘭𝘦𝘹𝘢𝘯𝘥𝘳𝘪𝘶𝘮 𝘤𝘢𝘵𝘦𝘯𝘦𝘭𝘭𝘢, 𝘈. 𝘱𝘢𝘤𝘪𝘧𝘪𝘤𝘶𝘮, 𝘈. 𝘢𝘶𝘴𝘵𝘳𝘢𝘭𝘪𝘦𝘯𝘴𝘦 and 𝘎𝘺𝘮𝘯𝘰𝘥𝘪𝘯𝘪𝘶𝘮 𝘤𝘢𝘵𝘦𝘯𝘢𝘵𝘶𝘮. In particular, of these, three species are morphologically almost identical, the species of the former 𝘈𝘭𝘦𝘹𝘢𝘯𝘥𝘳𝘪𝘶𝘮 𝘵𝘢𝘮𝘢𝘳𝘦𝘯𝘴𝘦 species complex (𝘈. 𝘤𝘢𝘵𝘦𝘯𝘦𝘭𝘭𝘢, 𝘈. 𝘱𝘢𝘤𝘪𝘧𝘪𝘤𝘶𝘮, and 𝘈. 𝘢𝘶𝘴𝘵𝘳𝘢𝘭𝘪𝘦𝘯𝘴𝘦), which cannot be differentiated using light microscopy. Therefore, phytoplankton monitoring using light microscopy and total PST in shellfish using High Performance Liquid Chromatography (HPLC) may not be sufficient to allow for an early warning with enough time to take appropriate shellfish harvesting management decisions. In this thesis, quantitative Polymerase Chain Reaction (qPCR) assays are investigated as an in-field early warning system, as well as a tool for long-term risk assessment of PST-associated harmful algal blooms. A commercial on-farm pipeline based on the collection and filtration of water samples using a custom designed gravity filter, a cell lysis, and a qPCR assay based on 𝘴𝘹𝘵𝘈4 was also developed and validated. QPCR assays based on ribosomal DNA (rDNA) ‘barcoding’ regions and an assay based on a gene associated with PST biosynthesis (𝘴𝘹𝘵𝘈4) were found to be generally specific, sensitive and efficient. The efficacy of an rDNA-based assay for cyst quantification was demonstrated, showing potential for its use as a long-term risk assessment tool for a new harvest area. However, qPCR assays based on rDNA gene regions were found to overestimate cell abundances. An analysis of rDNA copy number variation among strains of species of 𝘈𝘭𝘦𝘹𝘢𝘯𝘥𝘳𝘪𝘶𝘮 showed a variation of up to 3-5 orders of magnitude within a species, and was correlated significantly with genome size, which also varied within a species. An analysis of the variation in genomic copies of 𝘴𝘹𝘵𝘈4 genes showed variation as well, however this was of a lesser degree, of up to one order of magnitude. A positive correlation was found between 𝘴𝘹𝘵𝘈4 copies per cell and the total PST produced per cell, showing that the dosage effect may contribute to the regulation of PST biosynthesis.
Article
Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (<359 µm), indicating more total DNA amount in the largest nematodes. Soil pre-treatments showed that autoclaved field soil had significantly reduced DNA amount and quality. The air- or oven-dried soil yielded a lower amount of DNA with similar purity, compared with natural field soil. PCR inhibitors were detected in soil DNA substrates targeting pBluescript II SK(+)-plasmid DNA. Al(NH4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA.
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Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ~1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1–2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ~4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates ‘living’ protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.
Chapter
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Microbial ecology will undoubtedly take a great leap forward by adopting molecular-based techniques for monitoring specific populations in natural habitats. It seems that in the near future, researchers will be able to detect and possibly enumerate DNA sequences which are specific to a species, a phylogenetic or functional group of organisms. The methods also give a new dimension to microscopy, since fluorescent dyes can be coupled to specific oligonucleotides, thus making it possible to observe and enumerate specific genotypes in soil (Amann et al. 1992). A third dimension offered is the use of reporter genes to detect specific events (such as nutrient limitations) in the natural habitat. The direct application of these molecular genetic methods on soil bacteria is often hampered by the large amounts of soil colloids. Hence extraction of bacterial cells from soil may have to precede analysis of their genetic material.
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We developed a protocol which yields purified bacterial DNA from the soil bacterial community. The bacteria were first dispersed and separated from soil particles in the presence of polyvinylpolypyrrolidone, which removes humic acid contaminants by adsorption to this insoluble polymer. The soil bacteria were then collected by centrifugation and lysed by using a comprehensive protocol designed to maximize disruption of the various types of bacteria present. Total bacterial DNA was purified from the cell lysate and remaining soil contaminants by using equilibrium density gradients. The isolated DNA was essentially pure as determined by UV spectral analysis, was at least 48 kilobases long, and was not subject to degradation, which indicated that there was no contaminating nuclease activity. The isolated DNA was readily digested by exogenously added restriction endonucleases and successfully analyzed by slot blot and Southern blot hybridization. Using single-stranded, ³²P-labeled DNA probes, we could detect and quantitate the presence of a specific microbial population in the natural soil community on the basis of the presence of a DNA sequence unique to that organism. The sensitivity of our methodology was sufficient to detect Bradyrhizobium japonicum at densities as low as 4.3 x 10⁴ cells per g (dry weight) of soil, which corresponds to about 0.2 pg of hybridizable DNA in a 1..mu..g DNA sample.
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In this study, we test the use of the RAPD (Random Amplified Polymorphic DNA) molecular markers as a way to estimate the similarity of the microbial communities in various termite mounds and soils. In tropical ecosystems, termite activities induce changes in the chemical and physical properties of soil. The question then arises as to whether or not termites affect the presence of natural microbial communities. Successful 16S rDNA amplifications provided evidence of the occurrence of bacterial DNA in termite constructions including both soil feeder and fungus grower materials. A phenetic dendrogram using the similarity distance calculated from pairwise data including 88 polymorphic RAPD markers was reconstructed and bootstrap scores mapped. The microbial communities of the mounds of the four soil-feeding termites were clustered in the same clade, while those of the mounds of the fungus-growing species were distinct like those of control soils. Microbial changes in nests result from termite building behavior, depending on whether they include feces in their constructions for soil-feeders or use saliva as particle cement for fungus-growers. It is argued that RAPDs are useful markers to detect differences in microbial community structure not only between termitaries and control soils but also between mounds of soil-feeders.
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A limitation of the use of the Polymerase Chain Reaction (PCR) for soil DNA analysis is the contamination by humic substances. Numerous studies have been devoted to the elaboration of an effective purification method but none appears universal. During our investigations of soil bacterial changes induced by soil-feeding termites, we found that humic acid content usually impede DNA purification. Indeed, humic acids and nucleic acids share similar physicochemical properties. Here, we tested eight purification procedures including electrophoretical and chromatographical methods. The results show for DNA extracted from humic rich samples, e.g. termite mounds, that only the combination of two methods gave a DNA sufficiently pure to perform successful amplifications.
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Three clones of Folsomia candida from different locations in Europe were compared in four experiments investigating genetic and phenotypic correlations between life-history traits. The first three experiments focused on the effects of food type, clone and temperature on traits associated with the first clutch. Differences in clutch size between clones and treatments were almost completely attributable to body size. Clones differed in length of the juvenile period, but the difference decreased at low temperatures. Age and weight at first reproduction were negatively correlated in the food type experiment and positively correlated in the temperature experiment, an often-encountered result for which no general explanation is as yet available. In the temperature experiment egg size variation was considerable, and was highest at low temperatures. The fourth experiment, with two clones at two feeding levels, aimed at finding trade-offs, in particular between reproduction and survival. It was hypothesized that higher fecundity led to increased scenescence through a higher metabolic rate. The trade-off was clearly present among the clones: one combined fast growth, late reproduction and high lifetime fecundity with lower survival, while in the other the relation between these traits was opposite. The proposed mechanism, however, was not confirmed, as no difference in metabolic rate was found. The effect of food level was too small to result in significant differences in the life-history traits in either of the clones.
Article
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
Chapter
The 16S and 23S rRNA genes have been utilized for phylogenetic analysis of both prokaryotic and eukaryotic organisms (see Section 3). In addition to direct comparison of the nucleic acid sequences [9], numerous groups have used the rapid method of polymerase chain reaction (PCR) amplification of this gene [6] as well as the complete rRNA locus [3, 4, 8] for a simple method for identification of bacterial genera and species. In these latter procedures, the amplified ribosomal gene (rONA) is subjected to restriction endonuclease digestion; this has been termed ARDRA (Amplified Ribosomal DNA Restriction Analysis [8]). The resulting restriction fragment pattern is then used as a fingerprint for the identification of bacterial genomes. This method is based on the principle that the restriction sites on the RNA operon are conserved according to phylogenetic patterns.
Chapter
Soils are colonized by numerous microorganisms reaching up to 1011 bacterial cells per g soil (Torsvik et al. 1990). They also can be considered as a reservoir for biodiversity which remains largely unknown, since only 10%–20% of the species living in soil have been isolated and characterized (Ward et al. 1990). This includes nitrogen-fixing bacteria such as Rhizobium, Frankia or Azospirillum, plant pathogens such as Agrobacterium and various fungi, and free-living bacteria involved in various biological processes. Concern has also been voiced about the fate of genetically engineered microorganisms (GMO) released in soils. Consequently, soil microbial populations as well as their in situ activities have remained largely unknown, indicating the need for new techniques which can provide detection tests with high levels of specificity and sensitivity regardless of physical and chemical characteristics of the environment.
Article
The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail.
Chapter
The life-history of an organism is its lifetime pattern of growth, differentiation, storage and reproduction (Begon et al., 1990). These characteristics together determine the population growth, and energy spent on each of these may vary depending on many environmental factors. A change in one of the environmental factors will therefore result in a response of the population and hence may change the occurrence of the species (Underwood, 1989)
Chapter
Deep marine sediments remain poorly studied with respect to bacterial communities inhabiting them. Sediments up to 500 m below the sea floor (mbsf) in the Japan Sea have been laid down over the last 3 million years and therefore represent the early stages of sediment diagenesis. These sediments contain substantial bacterial populations (1.1 × 107 to 1.4 × 109 bacteria/g wet weight) with surprisingly high activity (Cragg et al. 1992; Getliff et al. 1992). Better knowledge of the types and diversity of bacteria which are dominant in these sediments, particularly those predominantly involved in important biogeochemical and biochemical processes, will lead to increased understanding of the diagenesis and evolution of organic matter. This unique habitat might also contain bacteria not found elsewhere in the biosphere.
Article
Specific nif sequences of Enterobacter agglomerans plasmid pEA9 were detected in total DNA recovered from soil 70 days after its inoculation with the bacteria, when these were no longer culturable on agar medium. For this, a modified method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was necessary.
Article
Survivorship data can be effectively summarized using the shape and scale parameters of the Weibull frequency distribution. The shape parameter controls the rate of change of the age-specific mortality rate and, therefore, the general form of the survivorship curve. Estimates of shape and scale parameters and their confidence intervals can be easily calculated and used to compare survivorship curves of different populations.
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Five herbicides, atrazine, dalapon, monuron, TCA, and 2,4-D, were applied once to grassland to evaluate their effects for at least two summers after application on the numbers of wireworms, Agriotes spp., springtails (Collembola suborder Arthropleona), mites (Acarina), earthworms (Lumbricidae), and millipedes (Diplopoda) in the soil. In plots treated with atrazine, wireworms, earthworms, and springtails declined in numbers. Dalapon increased the numbers of millipedes, springtails, and mites. Monuron reduced the numbers of wireworms, millipedes, earthworms, springtails, and mites. TCA increased the numbers of millipedes, springtails, and mites and decreased the number of earthworms. Treatment with 2,4-D did not affect the numbers of wireworms, springtails, or mites. The data suggest that faunal fluctuations were chiefly caused indirectly by the herbicides altering the floristic composition of the grassland.The results show that certain herbicides may diminish or increase the numbers of both beneficial and ...
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The responses of three different soil microbial communities to the experimental application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2, 4-D, and one had no prior direct application of any herbicide. Dominant 2, 4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2, 4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2, 4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.
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Methods for introducing and recovering GEMs in and from the environment, mainly soil and sediment, have been discussed. A major difficulty to studies on the fate of GEMs and the heterologous DNA is the difficulty of obtaining good, representative samples from the environment both quantitatively and qualitatively, as a result of the presence of nonculturable cells and the difficulties in quantitative cell and DNA extractions primarily from soils and sediments. In addition, samples from soils and sediments suffer from persistent impurities, mainly because of the presence of humic matter and clay particles. The use of molecular biological techniques in environmental research has been discussed, illustrating the potential of these techniques to solve hitherto recalcitrant questions: e.g., presence of (specific) nonculturable cells and of the specific DNA sequences present can now be assessed with the application of total community DNA extraction protocols followed by DNA enhancement using the polymerization chain reaction. Although these developments are just starting, the combination of traditional microbial ecological techniques with molecular biology techniques offers excellent possibilities for future research.
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Microarthropods have a variable but generally significant effect on litter disappearance and therefore on detrital standing crops. An average increase in litter decomposition rates of 23% was noted for 15 studies that ranged from 9-30 months in duration. Increased mineralization of N, P and K was seen in only about half of those studies reporting microarthropod effects on nutrient dynamics. Mass loss is more affected than are nutrients owing to stimulation of microbial respiration by microarthropods. This respiration results in carbon mineralization but converts other elements into microbial tissues, most of which is recycled within the detritus. Mineralization of N, P and cations will probably increase in older litter as a result of microarthropod feeding activities. Directions for future research are indicated.-from Author
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The degradation and leaching potential of the herbicide [14C]glufosinate-ammonium [the ammonium salt of dl-homoalanin-4-yl(methyl)phosphinic acid] in a northern Ontario forest environment were examined. The DT50 and maximum leaching depth were 4.3 days and 10 cm (humic layer), respectively. [14C]MPPA-3 (3-methylphosphinylpropionic acid) and [14C]MPAA-2 (2-methylphosphinylacetic acid), the two main metabolites of glufosinate-ammonium, also did not leach beyond 10 cm. At 32 days postapplication, approximately 10−20% of the parent and metabolites remained in the soil, but by the following season (day 295) residue levels had declined to near zero. Keywords: Glufosinate-ammonium; forestry; persistence; leaching potential
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Soil thin-layer chromatographic analysis revealed that [14C]glufosinate ammonium was slightly more mobile than amitrole less mobile than picloram in Fox sandy loam and Guelph loam soils. Two glufosinate ammonium metabolites, 3-(methylphosphinyl)propionic acid (MPPA-3) and 2-(methylphosphinyl)acetic acid (MPAA-2), were more mobile than the parent herbicide in the Fox sandy loam. In the Guelph loam, the MPAA-3 metabolite was again more mobile than the parent herbicide. However, the MPAA-2 metabolite was significantly less mobile than the parent herbicide. The adsorption coefficients for [14C]glufosinate ammonium in the Guelph loam and Fox sandy loam soils were 0.37 and 0.21, respectively. Laboratory degradation studies indicated that [14C]glufosinate ammonium was rapidly degraded to its metabolites, MPPA-3 and MPAA-2, and eventually to 14CO2. In all studies conducted, the half-life of glufosinate was between 3 and 7 days.
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The herbicide 2,4,5‐T is commonly used against Prunus serotina in forests and nature reserves. To evaluate the possibility of side effects laboratory experiments have been carried out. When exposed continuously to an agar/soil substrate with different doses of the herbicide (0.003–0.05 ml/dm ² ) adults of the springtail Onychiurus quadriocellatus were not affected negatively at low doses (0.003 and 0.006 ml/dm ² ), but there was about 40 days later, when reproduction started) a decrease in the number of eggs produced. The herbicide also affected the food consumption. Depending on the type of food the consumption decreased (the algae Pleurococcus spp., needles of Larix leptolepis ) or increased (pellets of isopods were fed with sprayed or unsprayed leaves of Prunus serotina ). Consumption of Pleurococcus shortened the lifespan of the springtails. Moreover the herbicide, whether taken in with food or from the substrate, did increase the moulting frequency. A possible explanation for this increase may be the particular way of excretion in soringtails, that is by means of ecdysis of the gut epithelium during moulting. It is postulated that intake of the herbicide causes a more rapid increase in amount of waste products incorporated in the gut epithelium and that this induces more frequent moulting. Zusammenfassung Nebenwirkungen des Herbizids 2,4,5‐T auf die Reproduktion, Ernährung und Häutung von Onychiurus quadriocellatus (Collembola) Die Verwendung des Unkrautbekämpfungsmittels 2,4,5‐T gegen Prunus serotina und Betula spp. in Wäldern und Naturreservaten hat in den Niederlanden mehr und mehr zugenommen. Zur Prüfung der evtl. Einwirkungen dieses Mittels auf das Bodenleben wurden Laborversuche mit Collembolen durchgeführt. Insbesondere wurde die Lebensweise erwachsener Tiere des Collembolus Onychiurus quadriocellatus Gisin auf einer mit 2,4,5‐T vergiftete Agar/Boden‐Mischung beobachtet. Es stellte sich heraus, daß Dosen von 0,003 und 0,006 ml/dm ² anfänglich keine merkbaren schädlichen Einwirkungen auf die Versuchstiere hatten, daß aber nach 40 Tagen, d. h. zu Beginn der Fortpflanzung, die Anzahl produzierter Eier niedriger war als bei den Kontrolltieren. Weiterhin wurde ein Zusammenhang zwischen der Anwendung des Herbizids und der Ernährung der Versuchstiere festgestellt. Tiere, die mit Algen (Pleurococcus spp.) und Nadeln von Larix leptolepis ernährt wurden, hatten eine niedrigere Nahrungsaufnahme als Kontrolltiere. Dagegen konnte eine bedeutende Zunahme der Nahrungsmenge festgestellt werden, wenn die Tiere mit Kotballen der Assel Philoscia muscorum und mit Blättern von Prunus serotina ernährt wurden, die entweder mit dem Herbizid gespritzt oder nicht gespritzt waren. Weiterhin wurde bei Ernährung mit Pleurococcus spp. auch eine bedeutende Kürzung der Lebensdauer der Springschwänze beobachtet. Die Aufnahme des Herbizids mit der Nahrung oder durch Berührung mit der Agar/Boden‐Mischung ergab eine erhöhte Frequenz der Häutung des Mitteldarmepithels. Es ist anzunehmen, daß die Zersetzung des Unkrautbekämpfungsmittels die Produktion von Exkretstoffen veranlassen kann und daraus eine häufigere Häutung des Mitteldarmepithels resultiert.
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From cultures of Streptomyces viridochromogenes a new antibiotic, phosphinothricylalanyl-alanine, has been isolated. The new amino acid phosphinothricin is 2-amino-4-methyl-phosphino-butyric acid, its structure is confirmed by synthesis. The tripeptide is highly active against Gram-positive and Gram-negative bacteria and against the fungus Botrytis cinerea. Phosphinothricin is an active glutamine synthetase inhibitor.
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Soil properties are a major influence on the bioavailability and toxicity of metals and represent one of the important factors that complicate the extrapolation of results from laboratory tests to field situations. The influence of soil characteristics and way of contamination on the bioaccumulation and toxicity of zinc was investigated for the springtail Folsomia candida, and the applicability of chemical extraction techniques for the prediction of zinc uptake and toxicity was evaluated. Bioaccumulation of zinc in F. candida was related to water-soluble zinc concentrations, and uptake was dependent on the test soil used. Effects of zinc for F. candida could not be fully explained by bioaccumulation. This indicates that the existence of a fixed internal threshold concentration of zinc above which physiological functions are impaired is not likely for F. candida. In freshly contaminated soils, zinc toxicity was related to organic matter and clay content of the soil; however, the use of these soils overestimated the effects of zinc for F. candida by a factor of 5 to 8 compared to a test soil that was subjected to ageing under field conditions for 1.5 years. Equilibration of the zinc contamination by percolating the soils with water before use in the toxicity experiment strongly reduced the difference in zinc toxicity between laboratory-treated and aged soils. Water-soluble concentrations are most appropriate to predict effects of zinc on reproduction of F. candida in soils with unknown contamination histories. For laboratory toxicity tests, it is recommended to percolate soils with water after contamination and to include an equilibration period prior to use to achieve a more realistic exposure situation.