Microbial biomass and activity dynamics in restored lands in a metal contaminated region.

Soil microbial communities are important for biogeochemical processes, along with the cycling of nutrients in an ecosystem. Their enzymatic activities are key indicators of their responses to stress. The objective of this research was to assess the effect of land reclamation on microbial biomass and activities in soils impacted by metal contamination. Phospholipid fatty acid analysis (PLFA) (PLFA) results revealed a significant increase in total microbial biomass, fungi, actinomycetes, and bacteria when limed soils were compared to unlimed samples. This change in microbial biomass was associated with a significant increase of pH. The overall level of the ß-glucosidase (BG), cellobiohydrolase (CBH), and aryl sulfatase (AS) activities was significantly higher in the dolomitic limestone treated soils than in the untreated samples. However, the activity of glycine aminopeptidase (GAP) was significantly lower in the limed soil than in unlimed samples used as reference. No significant differences (P ≥ 0.05) were observed between the two types of lands (limed vs unlimed) for other enzymes tested, which includes ß-N-acetylglucosaminidase (NAGase), acid phosphatase (AP), alkaline phosphatase (ALP), leucine aminopeptidase (LAP), and peroxidase (PER). The levels of enzymatic responses also varied among sites. Overall, this study revealed for the first time the effects of liming on soil microbial activities in recently reclaimed sites damaged by metals.

Advances in monitoring soil microbial community dynamic and function.

Microorganisms are vital to the overall ecosystem functioning, stability, and sustainability. Soil fertility and health depend on chemical composition and also on the qualitative and quantitative nature of microorganisms inhabiting it. Historically, denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE), single-strand conformation polymorphism, DNA amplification fingerprinting, amplified ribosomal DNA restriction analysis, terminal restriction fragment length polymorphism, length heterogeneity PCR, and ribosomal intergenic spacer analysis were used to assess soil microbial community structure (SMCS), abundance, and diversity. However, these methods had significant shortcomings and limitations for application in land reclamation monitoring. SMCS has been primarily determined by phospholipid fatty acid (PLFA) analysis. This method provides a direct measure of viable biomass in addition to a biochemical profile of the microbial community. PLFA has limitations such as overlap in the composition of microorganisms and the specificity of PLFAs signature. In recent years, high-throughput next-generation sequencing has dramatically increased the resolution and detectable spectrum of diverse microbial phylotypes from environmental samples and it plays a significant role in microbial ecology studies. Next-generation sequencings using 454, Illumina, SOLiD, and Ion Torrent platforms are rapid and flexible. The two methods, PLFA and next-generation sequencing, are useful in detecting changes in microbial community diversity and structure in different ecosystems. Single-molecule real-time (SMRT) and nanopore sequencing technologies represent third-generation sequencing (TGS) platforms that have been developed to address the shortcomings of second-generation sequencing (SGS). Enzymatic and soil respiration analyses are performed to further determine soil quality and microbial activities. Other valuable methods that are being recently applied to microbial function and structures include NanoSIM, GeoChip, and DNA stable staple isotope probing (DNA-SIP) technologies. They are powerful metagenomics tool for analyzing microbial communities, including their structure, metabolic potential, diversity, and their impact on ecosystem functions. This review is a critical analysis of current methods used in monitoring soil microbial community dynamic and functions.

Evidence of prokaryote like protein associated with nickel resistance in higher plants: horizontal transfer of TonB-dependent receptor/protein in Betula genus or de novo mechanisms?

Mechanisms of metal resistance have been reported in many plants but knowledge in woody species is scarce. The TonB-dependent receptors family (TBDTs) is a large group of proteins that facilitate the transport of molecules across the membrane of Gram-negative bacteria. Some evidence exists that TBDTs are involved in metal stress. The existence of a TonB-like mechanism in non-prokaryotes has not been established. The recent development of the Betula papyrifera (white birch) transcriptome has allowed the discovery of genes involved in plant adaptation to stress. The main objective of the present study was to identify novel genes associated with nickel resistance in B. papyrifera. Our results from next generation sequencing and RT-qPCR analyses show that genes involved in transport activities are upregulated in nickel-resistant genotypes compared with susceptible forms. Detailed analysis of gene expression and genome analysis shows for the first time the existence of a TonB-dependent receptor and TonB-like family protein in non-prokaryotes. In addition, we have found that these proteins are associated with nickel resistance in B. papyrifera. Our experiments suggest that the TonB-dependent receptor may be exclusive to the Betula genus, suggesting that Betula species may have acquired the gene via horizontal gene transfer from prokaryotes or fungi.

Karyotype evolution in the Pinaceae: implication with molecular phylogeny.

The family Pinaceae is made up mostly of diploid species (2n = 24). Systematization of karyotype analysis was developed to make comparison of intra- and interspecific karyotypes among the Pinaceae more accurate and reliable. Considering all parameters, the genera Pseudotsuga and Pseudolarix have the "most derived" (or advanced) and asymmetric karyotypes in the Pinaceae, followed by Larix, Picea, Abies, and Cedrus. The genus Pinus was the "least derived" (or ancestral) of all the genera of the Pinaceae analyzed. Differences in karyotype formulae and asymmetry indices were found among species within the same genera, suggesting that structural changes may have contributed to the diversification of the genus. This review is a detailed analysis of comparative karyotyping based on similar parameters, including numeric data and cytogenetic information. Telomeric sequence repeats and rDNA distribution in the Pinaceae were surveyed. The role of transposition in rDNA chromosome distribution is analyzed. Cytogenetic implications of hybridization between related species are reported. Likewise, the relationships between molecular phylogenetic and karyotype evolution is discussed in light of several reports. Within many genera, chromosomal organization was conserved despite independent molecular divergence and adaptation through the evolutionary history of the species of the Pinaceae.

Comparative soil metal analyses in Sudbury (Ontario, Canada) and Lubumbashi (Katanga, DR-Congo).

DR-Congo is a main world producer of copper (Cu) and cobalt (Co). Several hydrometallurgical plants and smelters also produced zinc, arsenic, and cadmium as by-products. In Sudbury (Canada), the production of nickel, copper and other metals has been maintained at high levels while industrial SO(2) emissions have been reduced by approximately 90% through combination of industrial technological developments and legislated controls. Metal analysis in the present study revealed that the levels of copper and cobalt in soils from mining sites in the Lubumbashi (DR-Congo) were up to 200 fold higher compared to contaminated Sudbury sites and tailings. Zinc content in soil samples from some mining areas in Lubumbashi was at least 70 times higher compared to samples from the Sudbury area. Nickel content in soil samples from Lubumbashi were much lower compared to the Sudbury Region samples. Overall, this study confirms that the African Copper belt region is among the ten most polluted areas in the world.

Molecular cytogenetic and agronomic characterization of advanced generations of wheat x triticale hybrids resistant to Diuraphis noxia (Mordvilko): application of GISH and microsatellite markers.

The PI 386148 triticale from Russia is among the highest resistant line to the Russian wheat aphid (RWA) (Diuraphis noxia (Mordvilko)). This triticale line was used as the male parent in crosses with Lamar wheat (Triticum aestivum L.). The F1 plants were backcrossed to Lamar wheat. The progenies were tested for RWA biotype 1 reaction for at least eight backcross and selfing generations. Five lines from these selections were identified for their resistance to the RWA and their seeds were increased for agronomic and other characterizations. Molecular and cytological analyses of these lines were performed using genomic in situ hybridization and rye chromosome-specific microsatellites markers. Three lines were cytologically stable and carried a pair of rye (Secale strictum (C. Presl) C. Presl) chromosomes as disomic addition lines of 1R. One line was unstable and showed a moderate level of mixoploidy with monosomic additions of 1R. Duplication of rye chromosome 1R was also identified. No wheat-rye chromosome interchange was detected, suggesting little homology between S. strictum and T. aestivum chromosomes. Specific microsatellite primers were used to identify the rye chromosomes present in each line. One rye chromosome, 1R, from the donor species contains genes for RWA resistance. Grain yield and test weight of three of the lines were similar to some adapted released wheat varieties under stress conditions.

Metal content in soil and black spruce (Picea mariana) trees in the Sudbury region (Ontario, Canada): low concentration of arsenic, cadmium, and nickel detected near smelter sources.

Several studies have reported high metal concentrations in soil within the vicinity of smelters in the Sudbury (Ontario) region. Continued investigation and monitoring of soil and vegetation are essential to the understanding of ecosystem recovery following the reduction of emissions from smelters and the establishment of a reforestation program. The concentrations of Cd, Co, Cu, Fe, Ni, and Zn, found in the present study were within the limits set by Ontario Ministry of Environment and Energy (OMEE) guidelines even in sites within the vicinity of the Falconbridge Smelters. The levels of these elements in black spruce (Picea mariana) tissues were much lower and far below the toxic levels for vegetation. This is the first documented report of metal content in black spruce populations in the Sudbury region.

Characterization of the D18S535 STR locus in northern Ontario European and Aboriginal populations for forensic purposes.

Genetic characterization of one European and three aboriginal populations from northern Ontario was undertaken to assess the utility of the D18S535 short tandem repeat locus (STR) as a genetic marker for forensic DNA typing in the region. The D18S535 locus was amplified using monoplex polymerase chain reaction (PCR), separated by denaturing polyacrylamide gel electrophoresis (PAGE), and visualized using the silver-stain detection method. The generated population data demonstrated that the D18S535 locus is highly polymorphic with a heterozygosity of > or = 0.75. The exact test showed violations of Hardy-Weinberg equilibrium in two of the aboriginal populations. Pairwise comparisons of allele-frequency distributions indicated that the four northern Ontario populations were significantly different from each other. This test also revealed that the northern Ontario populations differed significantly from ten European populations (from Germany, Spain, and Croatia) and one population from South America (from Argentina). Forensic parameters showed that the D18S535 locus is highly discriminating (power of discrimination > or = 0.85, chance of exclusion > or = 0.51); however, the lack of Hardy-Weinberg equilibrium in some of the populations must be taken into account in the application of these results to northern Ontario forensic casework.

Application of ISSR, RAPD, and cytological markers to the certification of Picea mariana, P. glauca, and P. engelmannii trees, and their putative hybrids.

Picea glauca (white spruce) and P. engelmannii (Engelmann spruce) are so similar and integrated that it is impossible to distinguish between them and their hybrids using morphological characteristics. Although natural hybrids between P. glauca and P. mariana (black spruce) do not generally occur, even though the 2 species are sympatric in North America, a first-generation hybrid, called the Rosendahl spruce, has been reported in the literature. In this study, several inter-simple sequence repeat (ISSR) markers were developed, as were randomly amplified polymorphic DNA (RAPD) markers, to certify spruce trees and their hybrids. ISSR fingerprinting was more efficient than RAPD assay; it detected 70% polymorphic DNA markers among the spruce species analyzed, whereas RAPD fingerprinting detected only 53%. Species-diagnostic ISSR and RAPD markers differentiating P. glauca from P. engelmannii and P. mariana were cloned and sequenced. Molecular certification of the spruce samples analyzed confirmed that all the seeds from interior spruce populations were true hybrids of P. glauca and P. engelmannii. But the analysis of seeds derived from the putative Rosendahl spruce indicated that this tree is likely a pure P. glauca genotype, rather than a hybrid of P. glauca and P. mariana. These data were confirmed by cytological analyses. Further analysis, using a more sensitive DNA amplification method with designed primers flanking the species-diagnostic ISSR and RAPD markers, revealed that such sequences are not generally species-specific because they are present in other spruce species.

Detection and physical mapping of the 18S-5.8S-26S rDNA and the pKFJ660 probe with microsatellite sequences derived from the rice blast fungus (Magnaporthe grisea) in conifer species.

Sequences homologous to the pKFJ660 probe, a fragment of DNA derived from the rice blast fungus (Magnaporthe grisea) carrying TC/AG repeat microsatellite sequences and 30 bp direct repeats were identified in the genome of Picea (spruce) and Pinus (pine) species by fluorescence in situ hybridization (FISH) and slot blot analyses. Slot blot analysis using the pKFJ660 probe revealed hybridization signals with genomic DNAs from various pine and spruce species. Further analyses indicated that the copy number of the (AG)30 motif was higher than 5 x 10(4) per plant genome for all plant samples tested, but the copy number of the sequences homologous to the whole pKFJ660 probe varies considerably among the 25 plant species tested. In situ hybridization of metaphase chromosomes from Pinus resinosa, P. banksiana and P. strobus showed the presence of sequences homologous to this probe on several chromosomes in a dispersed pattern. Major signals were observed on a few chromosomes indicating that some of these sequences are clustered in specific genomic locations. The locations of these repeats were compared to those of 18S-5.8S-26S rDNA in pine species. Chromosomal distribution of 18S-5.8S-26S rDNA varied among the three pine species (P. resinosa, P. banksiana and P. strobus) studied. Ribosomal DNA (rDNA) sites were identified on 14 to 20 chromosomes in these pine species.

Identification and characterization of RAPD markers inferring genetic relationships among Pine species.

Total genomic DNAs were extracted from several populations of pine species and amplified using oligonucleotides of random sequences. Polymorphism in random amplified polymorphic DNA (RAPD) markers was high and sufficient in distinguishing each of the species. Genetic relationships among eight pine species (Pinus sylvestris, Pinus strobus, Pinus rigida, Pinus resinosa, Pinus nigra, Pinus contorta, Pinus monticola, and Pinus banksiana) from different provenances were analyzed. The degree of band sharing was used to evaluate genetic distance between species and to construct a phylogenetic tree. In general, the dendrogram corroborated the description of relationships based on morphological characteristics and crossability, but also provided new insights into pine taxonomy. RAPD markers specific to some pine species were cloned and sequenced. PCR amplifications using pairs of designed specific primers revealed that all the cloned sequences were likely genus specific because they were not found in spruce or larch. True species-specific sequences were identified using designed primers flanking cloned RAPD fragments. The analysis of RAPD fragment sequences confirmed the genetic relationships among species. A 2281-bp RAPD band called PI-Mt-Stb-23 from P. strobus was used as a probe in restriction fragment length polymorphism (RFLP) analysis and produced distinct banding patterns for each species examined, consistent with the highly polymorphic character of DNA-fingerprinting probes.

Molecular and cytological analyses of Deschampsia cespitosa populations from Northern Ontario (Canada).

Deschampsia cespitosa is widely dispersed around the globe, particularly in the northern hemisphere. A high tolerance to adverse environmental conditions allows D. cespitosa to colonize and dominate plots of land that are uninhabitable by other plants. The main objective of the present study was to determine the degree of genetic variation and relatedness among D. cespitosa populations from heavy metal contaminated sites and uncontaminated sites in Northern Ontario, using RAPD markers. Genomic DNA samples from individual plants were analyzed using 35 oligonucleotides of random sequence. Twenty-eight of these primers allowed amplification of random polymorphic DNA (RAPD) loci. Overall, 90% of RAPD bands were polymorphic. Analysis of molecular variance revealed that 72% of the variation could be attributed to individual differences within each of the populations. The within- and among-region variations accounted for 14 and 15% of the total molecular variance, respectively. Population-specific RAPD markers were identified. RAPD markers specific to D. cespitosa were isolated, cloned, and characterized. Cytogenetic analysis revealed a high level of aneuploidy in all the populations from Northern Ontario, with chromosome numbers varying from 2n = 18 to 2n = 26.

Cytological and molecular relationships between Larix decidua, L. leptolepis and Larix x eurolepis: identification of species-specific Chromosoms and synchronization of mitotic cells.

The effects of different concentrations of hydroxyurea (HU) and aphidicolin (APH) on the mitotic index (MI) were compared in cells of embryogenic cultures of Larix decidua, L. leptolepis, and L. decidua x L. leptolepis (Larix x eurolepis). The highest enhancement of the MI was obtained with HU at 1.25 mM and 0.6% colchicine. In general the MI decreased with an increase of HU or APH concentration (over 1.25 mM for HU and 5 µM for APH). Detailed karyotype analyses were made on the somatic complement of L. decidua, L. leptolepis, and their hybrid. These karyotypes were asymmetrical and advanced, with the smaller chromosomes being more submedian than the larger ones. The topography of chromosome 7 of L. decidua and chromosome 9 of L. leptolepis was found to be the most significant cytotaxonomic characteristic in differentiating these two species. Cytological data indicate that Japanese larch (L. leptolepis) is phylogenetically closer to European larch (L. decidua) than the Siberian larch group (L. sibirica and L. sukaczewii). Chromosomes with unusually long kinetochores were found in both species and the hybrid. Hyperploid cells (2n = 25) were observed in the hybrid (Larix x eurolepis) material analyzed. A genomic L. decidua probe hybridized strongly to dots of DNA from L. leptolepis indicating that there is high sequence homology between these two species.

An optimized fluorescence in situ hybridization procedure for detecting rye chromosomes in wheat.

In situ hybridization with an interspersed repeat clone from rye, pSc119, was shown to be useful for detecting rye chromosomes introduced into wheat. However, since pSc119 also shows strong hybridization to a few sites in certain wheat chromosomes, small rye chromosome segments added to wheat may be difficult to detect. In this study, detection of rye chromosomes present in triticale and triticale X wheat hybrids was accomplished with the use of a subfragment from pSc119 (pSc119.1) whose sequence is dispersed throughout the rye chromosomes and only weakly cross-hybridizes to a few telomeric and centromeric regions of wheat. The in situ hybridization conditions were optimized to readily distinguish rye chromosomes from wheat chromosomes without the need for intensive analysis of hybridization patterns. Rye chromosomes were readily detected using fluorescence in situ hybridization. Fluorescence detection provided increased sensitivity over enzymatic detection and allowed signals to be amplified with repeated use of biotinylated anti-avidin antibody and avidin-FITC. Detection of rye chromatin was further optimized by doubling the probe concentration. Finally, double exposure photography of the same cell with two different filters provided another means to further increase the contrast between rye and wheat chromosomes.