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We investigated bacterial community dynamics in response to used motor oil contamination and perennial crop cultivation by 16S rRNA gene amplicon sequencing in a 4-year field study. Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes were the major bacterial phyla, and Rhodococcus was the most abundant genus. Initially, oil contamination decreased the overall bacterial diversity. Actinobacteria, Betaproteobacteria, and Gammaproteobacteria were sensitive to oil contamination, exhibiting clear succession with time. However, bacterial communities changed over time, regardless of oil contamination and crop cultivation. The abundance difference of most OTUs between oil-contaminated and non-contaminated plots remained the same in later sampling years after the initial abundance difference induced by oil spike. The abundances of three oil-favored actinobacteria (Lysinimonas, Microbacteriaceae, and Marmoricola) and one betaproteobacterium (Aquabacterium) changed in different manner over time in oil-contaminated and non-contaminated soil. We propose that these taxa are potential bio-indicators for monitoring recovery from motor oil contamination in boreal soil. The effect of crop cultivation on bacterial communities became significant only after the crops achieved stable growth, likely associated with plant material decomposition by Bacteroidetes, Armatimonadetes and Fibrobacteres.

The widespread use of motor oil makes it a notable risk factor to cause scattered contamination in soil. The monitoring of microbial community dynamics can serve as a comprehensive tool to assess the ecological impact of contaminants and their disappearance in the ecosystem. Hence, a field study was conducted to monitor the ecological impact of used motor oil under different perennial cropping systems (fodder galega, brome grass, galega-brome grass mixture and bare fallow) in a boreal climate zone. Length heterogeneity PCR characterized a successional pattern in bacterial community following oil contamination over a four-year bioremediation period. Soil pH and electrical conductivity were associated with the shifts in bacterial community composition. Crops had no detectable effect on bacterial community composition or complexity. However, the legume fodder galega increased soil microbial biomass, expressed as soil total DNA. Oil contamination induced an abrupt change in bacterial community composition at the early stage, yet the effect did not last as long as the oil in soil. The successional variation in bacterial community composition can serve as a sensitive ecological indicator of oil contamination and remediation in situ., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Accumulation of pesticides in the environment causes serious issues of contamination and toxicity. Bioremediation is an ecologically sound method to manage soil pollution, but the bottleneck here, is the successful scale-up of lab-scale experiments to field applications. This study demonstrates pilot-scale bioremediation in tropical soil using atrazine as model pollutant. Mimicking field conditions, three different bioremediation strategies for atrazine degradation were explored. 100 kg soil mesocosms were set-up, with or without atrazine application history. Natural attenuation and enhanced bioremediation were tested, where augmentation with an atrazine degrading consortium demonstrated best pollutant removal. 90% atrazine degradation was observed in six days in soil previously exposed to atrazine, while soil without history of atrazine use, needed 15 days to remove the same amount of amended atrazine. The bacterial consortium comprised of 3 novel bacterial strains with different genetic atrazine degrading potential. The progress of bioremediation was monitored by measuring the levels of atrazine and its intermediate, cyanuric acid. Genes from the atrazine degradation pathway, namely, atzA, atzB, atzD, trzN and trzD were quantified in all mesocosms for 60 days. The highest abundance of all target genes was observed on the 6th day of treatment. trzD was observed in the bioaugmented mesocosms only. The bacterial community profile in all mesocosms was monitored by LH-PCR over a period of two months. Results indicate that the communities changed rapidly after inoculation, but there was no drastic change in microbial community profile after 1 month. Results indicated that efficient bioremediation of atrazine using a microbial consortium could be successfully up-scaled to pilot scale., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Actinobacteria are a prolific source of antibiotics. Since the rate of discovery of novel antibiotics is decreasing, actinobacteria from unique environments need to be explored. In particular, actinobacterial biocontrol strains from medicinal plants need to be studied as they can be a source of potent antibiotics. We combined culture-dependent and culture-independent methods in analyzing the actinobacterial diversity in the rhizosphere of seven traditional medicinal plant species from Panxi, China, and assessed the antimicrobial activity of the isolates. Each of the plant species hosted a unique set of actinobacterial strains. Out of the 64 morphologically distinct isolates, half were Streptomyces sp., eight were Micromonospora sp., and the rest were members of 18 actinobacterial genera. In particular, Ainsliaea henryi Diels. hosted a diverse selection of actinobacteria, although the 16S ribosomal RNA (rRNA) sequence identity ranges of the isolates and of the 16S rRNA gene clone library were not congruent. In the clone library, 40% of the sequences were related to uncultured actinobacteria, emphasizing the need to develop isolation methods to assess the full potential of the actinobacteria. All Streptomyces isolates showed antimicrobial activity. While the antimicrobial activities of the rare actinobacteria were limited, the growth of Escherichia coli, Verticillium dahliae, and Fusarium oxysporum were inhibited only by rare actinobacteria, and strains related to Saccharopolyspora shandongensis and Streptosporangium roseum showed broad antimicrobial activity.

Nucleic acid-based community fingerprinting methods are valuable tools in microbial ecology, as they offer rapid and robust means to compare large series of replicates and references. To avoid the time-consuming and potentially subjective procedures of peak-based examination, we assessed the possibility to apply direct curve-based data analysis on community fingerprints produced with bacterial length heterogeneity PCR (LH-PCR). The dataset comprised 180 profiles from a 21-week rhizoremediation greenhouse experiment with three treatments and 10 sampling times. Curve-based analysis quantified the progressive effect of the plant (Galega orientalis) and the reversible effect of the contaminant (fuel oil) on bacterial succession. The major observed community shifts were assigned to changes in plant biomass and contamination level by canonical correlation analysis. A novel method to extract relative abundance data from the fingerprint curves for Shannon diversity index revealed contamination to reversibly decrease community complexity. By cloning and sequencing the fragment lengths, recognized to change in time in the averaged LH-PCR profiles, we identified Aquabacterium (Betaproteobacteria) as the putative r-strategic fuel oil degrader, and K-strategic Alphaproteobacteria growing in abundance later in succession. Curve-based community fingerprint analysis can be used for rapid data prescreening or as a robust alternative for the more heavily parameterized peak-based analysis., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Extracting DNA directly from micro-organisms living in soil is a crucial step for the molecular analysis of soil microbial communities. However, the use of a plethora of different soil DNA extraction protocols, each with its own bias, makes accurate data comparison difficult. To overcome this problem, a method for soil DNA extraction was proposed to the International Organization for Standardization (ISO) in 2006. This method was evaluated by 13 independent European laboratories actively participating in national and international ring tests. The reproducibility of the standardized method for molecular analyses was evaluated by comparing the amount of DNA extracted, as well as the abundance and genetic structure of the total bacterial community in the DNA extracted from 12 different soils by the 13 laboratories. High quality DNA was successfully extracted from all 12 soils, despite different physical and chemical characteristics and a range of origins from arable soils, through forests to industrial sites. Quantification of the 16S rRNA gene abundances by real time PCR and analysis of the total bacterial community structure by automated ribosomal intergenic spacer analysis (A-RISA) showed acceptable to good levels of reproducibility. Based on the results of both ring-tests, the method was unanimously approved by the ISO as an international standard method and the normative protocol will now be disseminated within the scientific community. Standardization of a soil DNA extraction method will improve data comparison, facilitating our understanding of soil microbial diversity and soil quality monitoring., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Symbiotic nitrogen fixation is the main route for sustainable input of nitrogen into ecosystems. Nitrogen fixation in agriculture can be improved by inoculation of legume crops with suitable rhizobia. Knowledge of the biodiversity of rhizobia and of local populations is important for the design of successful inoculation strategies. Soybeans are major nitrogen-fixing crops in many parts of the world. Bradyrhizobial inoculants for soybean are very diverse, yet classification and characterization of strains have long been difficult. Recent genetic characterization methods permit more reliable identification and will improve our knowledge of local populations. Forage legumes form another group of agronomically important legumes. Research and extension policies valorizing rhizobial germplasm diversity and preservation, farmer training for proper inoculant use and legal enforcement of commercial inoculant quality have proved a successful approach to promoting the use of forage legumes while enhancing biological N(2) fixation. It is worth noting that taxonomically important strains may not necessarily be important reference strains for other uses such as legume inoculation and genomics due to specialization of the different fields. This article points out both current knowledge and gaps remaining to be filled for further interaction and improvement of a rhizobial commons., ((c) 2010. Published by Elsevier SAS.)

Thirty-three rhizobial strains isolated from the root nodules of Astragalus luteolus and Astragalus ernestii growing on the west plateau at two different altitudes in Sichuan province, China, were characterized by amplified rDNA restriction analysis (ARDRA), amplified fragment length polymorphism (AFLP), and by sequencing of rrs, glnA, glnII and nifH. The ARDRA analysis revealed considerable genomic diversity. In AFLP analysis, 20 of 33 Astragalus rhizobia formed three distinct clades, with others dispersed into different groups with the reference strains. Phylogenetic analysis of the rrs gene of six representative strains showed that the isolates were members of the genus Mesorhizobium. Three of the isolates formed a sister clade to Mesorhizobium loti and Mesorhizobium ciceri, whereas the other three formed a sister clade to a clade harboring the species Mesorhizobium huakuii, Mesorhizobium plurifarum, Mesorhizobium septentrionale and Mesorhizobium amorphae, indicating the existence of two new species. Phylogenetic analysis of glnA and glnII confirmed the rrs phylogenies for four strains, but the trees were incongruent. The nifH sequences of the strains formed a monophyletic clade and were typical of those of mesorhizobia forming symbioses with inverted repeat lacking clade legume species. The incongruent phylogenies of the genes studied suggest that horizontal gene transfer and recombination shape mesorhizobial populations in the gene center of the host plants.

Knowledge about the presence and diversity of arbuscular mycorrhizal fungi (AMF) in a specific area is an essential first step for utilizing these fungi in any application. The community composition of AMF in intensively managed agricultural soil in the Sichuan Province of southwest China currently is unknown. In one set of samples, AMF were trapped in pot cultures from 40 fields growing legumes in the Panxi region, southeast Sichuan. In a second set of samples, the MPN method with four-fold dilutions and maize as host was used to estimate infective propagules in soil from another 50 agricultural sites throughout the province. Soil types were heterogeneous and were classified as purple, yellow, paddy and red. Crops at each site were either maize, wheat or sweet orange. From this set of soil, AMF spores were also extracted and identified. Including all ninety soils, thirty glomeromycotan species in Glomus (20 species), Acaulospora (four species), Scutellospora (three species), Ambispora (one species), Archaeospora (one species) and Paraglomus (one species) were identified. Yellow, red and purple soils yielded similar numbers of AMF species, while AMF species diversity was clearly lower in paddy soil. In trap culture soils, the most frequent species were Glomus aggregatum or Glomus intraradices, Glomus claroideum and Glomus etunicatum. The species Acaulospora capsicula, Acaulospora delicata, G. aggregatum (or intraradices), G. claroideum, Glomus epigaeum, G. etunicatum, Glomus luteum, Glomus monosporum, Glomus mosseae and Glomus proliferum were successfully cultured as single-species pot cultures in Plantago lanceolata. The three most frequent species in field soils were G. mosseae, Glomus caledonium and Glomus constrictum. MPN values varied between 17 and 3334 propagules 100 g soil(-1) among the fifty field sites sampled. Regression analysis, including host&soil, log(P) and pH as explanatory variables explained 59% of the variation in log(MPN). The highest MPN estimates were found in purple soil cropped with maize and citrus, 324 and 278 propagules 100 g soil(-1), respectively. The lowest MPN value, 54 propagules 100 g soil(-1), was measured in wheat in purple and yellow soil. Despite intensive agricultural management that can include often repeated tillage, our examination of 90 agricultural sites revealed that soils of the Sichuan region have moderate to high numbers of infective AMF propagules as well as a high AMF species diversity. This opens possibilities for further studies and utilization of AMF in agriculture and horticulture in the Sichuan province, People's Republic of China.

A collection of 50 indigenous meta-toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16S rDNA partial sequencing showed the presence of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. Only one-fifth of the strains that tolerated m-toluate also degraded m-toluate. The inoculum Pseudomonas putida PaW85 was not found in the rhizosphere samples. The ability to degrade m-toluate by the TOL plasmid was detected only in species of the genus Pseudomonas. However, a few Rhodococcus erythropolis strains were found which were able to degrade m-toluate. A new finding was that Pseudomonas migulae strains and a few P. oryzihabitans strains were able to grow on m-toluate and most likely contained the TOL plasmid. Because strain specific differences in degradation abilities were found for P. oryzihabitans, separation at the strain level was important. For strain specific separation (GTG)5 fingerprinting was the best method. A combination of the single locus ribotyping and the whole genomic fingerprinting techniques with the selective partial sequencing formed a practical molecular toolbox for studying genetic diversity of culturable bacteria in oil-contaminated rhizosphere.

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.

We report here the results of phylogenetic analysis of archaeal 16S rRNA gene sequences amplified by PCR with Archaea-specific primers with mixed-population DNA extracted directly from forest soil used as a template. Nucleotide signature and phylogenetic analyses show that the sequences obtained belong to the domain Archaea and form a new cluster. Its phylogenetic position suggests that sequences are from a previously undescribed terrestrial group within the kingdom Crenarchaeota.

Procedures based on DNA hybridization and PCR were developed for quality control of Rhizobium inoculants. Inoculants for pea and goat's rue were produced by Elomestari Ltd., Juva, Finland, in sterile dry fine peat by the standard procedure used by the company. The inoculants contained Rhizobium galegae HAMBI 1174 and HAMBI 1207 and an R. leguminosarum biovar vicia strain, 16HSA, either solely or in combinations of two or three strains. DNA was isolated from 1-g samples of each peat inoculant and analyzed by nonradioactive DNA-DNA hybridization and by PCR. The hybridization probes were total DNAs from pure cultures of R. galegae HAMBI 1207 and R. leguminosarum biovar viciae 16HSA and a 264-bp strain-specific fragment from the genome of R. galegae HAMBI 1174. The total DNA probes distinguished inoculants containing R. galegae or R. leguminosarum, and the strain-specific probe distinguished inoculants containing R. galegae HAMBI 1174. The hybridization results for R. galegae were verified in a PCR experiment by amplifying an R. galegae species-specific fragment and an R. galegae HAMBI 1174 strain-specific fragment in the same reaction. When suitable probes and primers are available, the methods described here offer promising alternatives for the quality control of peat-based inoculants.

Abstract: The aim of this study was to evaluate the long-term influence of contrasting rural land use types on the level, plot-scale variation and horizontal spatial structure of decomposition activities and the bacterial community in soil. Experimental data were collected in the southern boreal zone from topsoil layers of adjacent spruce forest, unmanaged meadow (former field) and organically cultivated field that all shared the same soil origin. The forest soil was sampled separately for the organic and mineral layers. A geostatistical design comprising 50 sampling points per plot area of 10 × 10 m2 was used. The measured microbiological characteristics included eight different hydrolytic soil enzyme activities involved in C, P and S cycles, bacterial 16S rDNA length heterogeneity profiles (LH-PCR) and total DNA yield as a relative estimate of microbial biomass. Effects of land use were pronounced on both the bacterial community structure and soil enzyme activities. Soil organic matter (SOM) content predicted well the major differences in soil enzyme activities and microbial biomass. Highest enzyme activities were generally found in the forest organic soil whereas the underlying mineral soil showed significantly lower activities with a pattern similar to those of the other mineral soils, especially the cultivated field. Bacterial LH-PCR fingerprints were distinct but at the same time remarkably similar between field and meadow soils whereas the forest organic layer differed clearly from the mineral soils. Within-plot variation of soil microbiological characteristics was best explained by the variation of SOM. Relative standard deviations of soil microbiological characteristics typically decreased in the order: forest organic layer ≈ forest mineral layer > meadow > field. However, bacterial fingerprints showed lowest variation within the meadow. Most of the microbiological variables studied showed no or only weak spatial structure at the scale sampled. [Copyright &y& Elsevier]