Published papers:

1. Borneman, J., P. W. Skroch, K. M. OSullivan, J. A. Palus, N. G. Rumjanek, J. L. Jansen, J. Nienhuis, and E. W. Triplett. 1996. Molecular microbial diversity of an agricultural soil in Wisconsin.
Applied and Environmental Microbiology 62:1935-1943.
Keywords: RIBOSOMAL-RNA SEQUENCES/16S RDNA ANALYSIS/
ENVIRONMENT/BACTERIA/MICROORGANISMS/AMPLIFICATION/COMMUNITY
Abstract: A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA), A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed, Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil, The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced, The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined, Of the 124 clones sequenced, 98.4% were from the domain Bacteria, Two of the rDNA sequences were derived from eukaryotic organelles, Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence, No sequences of the domain Archaea were found, Within the domain Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low-G+C-content gram-positive group (21.8%), Some kingdoms, such as the Thermotogales, the green nonsulfur group, the Fusobacteria, and the Spirochaetes, were absent, A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al, (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol, 176:1-6, 1994), From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.

2. Chandler, D. P., S. M. Li, C. M. Spadoni, G. R. Drake, D. L. Balkwill, J. K. Fredrickson, and F. J. Brockman. 1997. A molecular comparison of culturable aerobic heterotrophic bacteria and 16S rDNA clones derived from a deep subsurface sediment. Fems Microbiology Ecology 23:131-144.
Keywords: BACTERIA/16S rRNA/PCR/subsurface/sediment/RIBOSOMAL-RNA GENES/COMMUNITY STRUCTURE/PCR AMPLIFICATION/DIVERSITY/ENVIRONMENT/
SEQUENCES/ENUMERATION/ALLELES/PROBES/SOIL
Abstract: Culture-based techniques have traditionally been the primary tools utilized for studying the microbiology of terrestrial subsurface environments. Recently, nucleic acid-based methods have been employed to further characterize the microbial diversity in subsurface sediments and rocks, but the results have not been related to individual bacteria cultivated from the same environment. Restriction fragment length profiles of 16S rRNA genes derived from bulk community DNA or bacterial isolates were compared to determine the efficacy of PCR-based methods for studying microbial diversity and phylogeny in a deep (188 m) subsurface environment. The phylogenetic relatedness between 16S rRNA genes of enrichment cultures and individual clones was also determined through DNA sequence analysis of 16S rRNA genes. Restriction fragment length profiles from PCR clone libraries accounted for 64% of recovered isolates and 55% of the estimated culturable diversity based upon their 16S rDNA RFLP signatures. DNA sequence comparisons between the 16S rDNA of the most commonly occurring isolates and clones confirmed that similar DNA. sequences were contained within the RFLP groups used to categorize the isolates and clones. For 7 of 8 RFLP groups for which DNA sequences were obtained, nearest neighbor assignments corresponded at the genus level but suggested that 16S rDNA sequences from multiple genera were contained within single RFLP profiles. Phylogenetic analysis of 16S rRNA sequences supported the nearest neighbor inferences and indicated that 16S rDNA clones derived from bulk sediment were specifically related to isolates recovered on enrichment plates. This study has shown that a majority of the cultivated aerobic heterotrophic bacteria in a subsurface sediment could be described by 16S rDNA clones obtained from directly extracted DNA, but that PCR-based methods cannot account for all organisms from a given sample. Consequently, a more comprehensive assessment of microbial diversity in subsurface (and probably other) environments can be obtained by using a combination of culture- and molecular-based techniques than by using either method alone.

3. Chelius, M. K. and J. E. Lepo. 1999. Restriction fragment length polymorphism analysis of PCR-amplified nifH sequences from wetland plant rhizosphere communities. Environmental Technology 20:883-889.
Keywords: microbial diversity/nitrogen fixation/RFLP/nitrogen cycle/bioremediation/ecosystem stress/NITROGEN-FIXATION/SALT-MARSH/SPARTINA-ALTERNIFLORA/GENE-SEQUENCES/MICROBIAL DNA/
SEDIMENTS/AMPLIFICATION/extraction/SOIL/POLYMERASE
Abstract: We describe a method to assess the community structure of N-2-fixing bacteria in the rhizosphere. Total DNA was extracted from the macrophytic plants Spartina alterniflora and Sesbania macrocarpa root zones by bead beating and was purified by CsCl-EtBr gradient centrifugation. The average DNA yield was 5.5 mu g g' of soil and was of sufficient purity for PCR amplification of nifH. [alpha-P-32]dCTP was incorporated into the PCR reaction and nifH PCR products were restriction digested. Restriction Fragment Length polymorphism (RFLP) analysis of the amplified sequences revealed differences in the community structure of N-2-fixing rhizobacteria of the field collected salt marsh plant, Spartina alterniflora, and of a laboratory cultured Sesbania macrocarpa. Soil inoculation experiments were used to determine the efficiency of the methods, and amplified nifH DNA could be detected when 10(4) cells each of-Vibrio natriegens and Azotobacter vinelandii were added per gram of soil. Restriction patterns produced by each species were detected at 10(6) cells g(-1) soil. These results indicate that RFLP analysis of amplified nifH sequences from rhizosphere communities may provide information on species composition and reveal shifts in diversify. By examining population shifts within functional microbial groups, such as the nitrogen-fixing bacteria, the method should have particular utility in assessing stress effects on polluted ecosystems or on those undergoing bioremedial treatments.

4. Dalmastri, C., L. Chiarini, C. Cantale, A. Bevivino, and S. Tabacchioni. 1999. Soil type and maize cultivar affect the genetic diversity of maize root-associated Burkholderia cepacia populations. Microbial Ecology 38:273-284.
Keywords: microbial diversity/PSEUDOMONAS-CEPACIA/NATURAL-POPULATIONS/rhizosphere/PLANT/BACTERIA/SUPPRESSION
Abstract: Burkholderia cepacia populations associated with the Zea mays root system were investigated to assess the influence of soil type, maize cultivar, and root localization on the degree of their genetic diversity. A total of 180 B. cepacia isolates were identified by restriction analysis of the amplified 16S rDNA (ARDRA technique). The genetic diversity among B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique, using the 10-mer primer AP5. The analysis of molecular variance (AMOVA) method was applied to estimate the variance components for the RAPD patterns. The results indicated that, among the factors studied, the soil was clearly the dominant one in affecting the genetic diversity of maize root-associated B. cepacia populations. In fact, the percentage of variation among populations was significantly higher between B. cepacia populations recovered from maize planted in different soils than between B. cepacia populations isolated from different maize cultivars and from distinct root compartments such as rhizoplane and rhizosphere. The analysis of the genetic relationships among B. cepacia isolates resulted in dendrograms showing bacterial populations with frequent recombinations and a nonclonal genetic structure. The dendrograms were also in agreement with the AMOVA results. We were able to group strains obtained from distinct soils on the basis of their origin, confirming that soil type had the major effect on the degree of genetic diversity of the maize root-associated B. cepacia populations analyzed. On the other hand, strains isolated from distinct root compartments exhibited a random distribution which confirmed that the rhizosphere and rhizoplane populations analyzed did not significantly differ in their genetic structure.

5. Dunbar, J., L. O. Ticknor, and C. R. Kuske. 2000. Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Applied and Environmental Microbiology 66:2943-2950.
Keywords: GRADIENT GEL-ELECTROPHORESIS/POLYMERASE CHAIN-REACTION/RIBOSOMAL-RNA SEQUENCES/ENRICHMENT CULTURES/DNA FRAGMENTS/COMMUNITIES/AMPLIFICATION/BACTERIOPLANKTON/
POPULATIONS/BACTERIA
Abstract: The ability of terminal restriction fragment (T-RFLP or TRF) profiles of 165 rRNA genes to provide useful information about the relative diversity of complex microbial communities was investigated by comparison with other methods. Four soil communities representing two pinyon rhizosphere and two between-tree (interspace) soil environments were compared by analysis of 16S rRNA gene clone libraries and culture collections (Dunbar et al., Appl. Environ. Microbiol. 65:1662-1669, 1998) and by analysis of 16S rDNA TRF profiles of community DNA. The TRF method was able to differentiate the four communities in a manner consistent with previous comparisons of the communities by analysis of 16S rDNA clone libraries. TRF profiles were not useful for calculating and comparing traditional community richness or evenness values among the four soil environments. Statistics calculated from RsaI, NhaI, HaeIII, and MspI profiles of each community were inconsistent, and the combined data were not significantly different between samples. The detection sensitivity of the method was tested. In standard PCRs, a seeded population comprising 0.1 to 1% of the total community could be detected. The combined results demonstrate that TRF analysis is an excellent method for rapidly comparing the relationships between bacterial communities in environmental samples. However, for highly complex communities, the method appears unable to provide classical measures of relative community diversity.

6. Fredslund, L., F. Ekelund, C. S. Jacobsen, and K. Johnsen. 2001. Development and application of a most-probable-number-PCR assay to quantify flagellate populations in soil samples. Applied and Environmental Microbiology 67:1613-1618.
Keywords: RIBOSOMAL-RNA GENES/QUANTITATIVE PCR/microbial diversity/AGRICULTURAL SOIL/DNA EXTRACTION/
NAKED AMEBAS/BACTERIA/PROTOZOA/AMPLIFICATION/GROWTH
Abstract: This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

7. Harry, M., B. Gambier, and E. Garnier-Sillam. 2000. Soil conservation for DNA preservation for bacterial molecular studies. European Journal of Soil Biology 36:51-55.
Keywords: SOIL/conservation/DNA recovery/BACTERIA/PCR/POLYMERASE CHAIN-REACTION/RAPID METHOD/microbial diversity/extraction/SEDIMENTS/purification/
COMMUNITY/MICROORGANISMS/AMPLIFICATION/RECOVERY
Abstract: The aim of this study was to select a method for preserving bacterial DNA in soil samples in cases where there are no possibilities of using freezing or cooling methods. To overcome this difficulty, we hypothesized that adding absolute ethanol to soil samples could be as successful to preserve bacteria as it is to preserve insect or tissue samples for molecular studies. Ln an attempt to test this assumption, we compared four conservation conditions. After inoculation of soil samples with Escherichia coli, they were either kept at 28 degrees C, stored in the cold (4 degrees C), dried at 60 degrees C, or treated with absolute ethanol. The relative effectiveness of the methods was evaluated by using both DNA recoveries and bacterial 16S rDNA amplification as criteria. Two kinds of soils, i.e. sandy clay and clayey soil, were used. Soil conservation was tested for seven time periods ranging from 2 d to 1 year after bacterial inoculation. Results showed that cold conservation or addition of absolute ethanol to the samples yielded similar DNA recoveries. For these treatments, successful amplifications are still obtained after one year of conservation. The ethanol conservation of soil samples appears to be the easiest method to preserve the bacterial DNA because of its reliability and field convenience. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

8. Hu, H. Y., B. R. Lim, N. Goto, and K. Fujie. 2001. Analytical precision and repeatability of respiratory quinones for quantitative study of microbial community structure in environmental samples. Journal of Microbiological Methods 47:17-24.
Keywords: analytical method/microbial quinones/microbial community/microbial diversity/
population dynamics/quinone profile/WASTE-WATER TREATMENT/ACTIVATED-SLUDGE/BACTERIA/SOIL
Abstract: Microbial community structure is one of the important factors controlling the pollutant-degrading capacity of ecosystems. The analysis of microbial quinones has gained increased recognition as a simple and useful approach for studying microbial structure in environmental samples. The analytical precision of quinone characterization using high performance liquid chromatograph (HPLC) with a UV-detector was studied in this study. Activated sludge was used as a typical mixed culture. The coefficient of variation of quinone content was lower than 6%, and that of microbial diversity calculated from the composition of quinones was as low as 3%. Statistical analyses on the analytical precision of quinones demonstrated that the critical value of dissimilarity between two quinone profiles of activated sludge, which is used to make a judgement whether the two quinone profiles are different or not, is 0.1 for the analytical method used in this study. The values of minimum biomass required for quinone analysis to have a reliable analytical result of microbial quinones were 2 mg-dry-cell for activated sludge. (C) 2001 Elsevier Science B.V. All rights reserved.

9. Korner, J. and E. Laczko. 1992. A New Method for Assessing Soil Microorganism Diversity and Evidence of Vitamin Deficiency in Low Diversity Communities. Biology and Fertility of Soils 13:58-60.
Keywords: SOIL MICROORGANISM DIVERSITY/PHOSPHOLIPID FATTY ACIDS/SOIL RESPIRATION/VITAMIN DEFICIENCY/
LIPID EXTRACTION/BACTERIAL COMMUNITIES
Abstract: A proposed new method for assessing the diversity of a soil microbial community is based on the species-typical ester-linked phospholipid fatty acids in the membranes of living cells. Soils that support only a few dominant species (bacteria, fungi, protozoa or algae) are expected to show few dominant fatty acids and vice versa. The phospholipid fatty-acid diversity in nine soils from Central Switzerland was calculated using Shannon's formula. By means of a respiration test, it was further established that the low-diversity soils responded significantly and positively (respiration increase) to small additions of a vitamin mixture containing thiamin, pyridoxin, calpan, folic acid, and biotin. The results indicate a connection between microbial diversity and a yet unspecified vitamin deficiency within the population. Whether the vitamin deficiency is the cause or the effect of the reduced diversity remains to be established.

10. Leung, K., L. S. England, M. B. Cassidy, J. T. Trevors, and S. Weir. 1994. Microbial Diversity in Soil - Effect of Releasing Genetically-Engineered Microorganisms. Molecular Ecology 3:413-422.
Keywords: BACTERIA/DIVERSITY/GENETICALLY ENGINEERED MICROORGANISMS/SOIL/POLYMERASE CHAIN-REACTION/
MERCURY RESISTANCE GENES/RIBOSOMAL-RNA SEQUENCES/BACTERIAL COMMUNITIES/MESSENGER-RNA/RAPID METHOD/ENVIRONMENTAL-SAMPLES/
ERWINIA-CAROTOVORA/DIRECT EXTRACTION/POPULATIONS
Abstract: This review examines the potential for change in microbial diversity, with the emphasis on bacteria, in soil resulting from the introduction of genetically engineered microorganisms (GEMs). With the advent of GEMs came the impetus for new technologies to recover these micro-organisms from soil and to assess their effects on microbial diversity. This review also presents general aspects of and genetic approaches to accessing bacterial diversity in the environment.

11. Lupwayi, N. Z., M. A. Arshad, W. A. Rice, and G. W. Clayton. 2001. Bacterial diversity in water-stable aggregates of soils under conventional and zero tillage management. Applied Soil Ecology 16:251-261.
Keywords: BIOLOG method/community level physiological profile conservation tillage/
metabolic potential/soil biological quality/soil structure/ORGANIC-MATTER/MICROBIAL BIOMASS/STRUCTURAL STABILITY/
NORTHWESTERN CANADA/CROP-ROTATION/CARBON/COMMUNITIES/MECHANISMS/DYNAMICS/WHEAT
Abstract: Reduced tillage of agricultural soils has been shown to result in greater macroaggregation, microbial biomass and microbial diversity. While it has been shown that macroaggregates contain more microbial biomass per unit soil mass than microaggregates, it is unclear how microbial diversity varies with soil aggregation. We investigated the functional diversity (catabolic potential) of bacteria, evaluated by calculating Shannon's diversity index (H'), substrate richness (S) and substrate evenness (E) from potential substrate utilization patterns, in whole soil (i.e. not separated into different aggregate sizes) and aggregates of different sizes (2-4, 1-2, 0.5-1, 0.25-0.5, and 0.1-0.25 mm diameter) in loam and silt loam soils grown to barley and managed for 6 years under conventional tillage (CT) or zero tillage (ZT) systems in northern British Columbia. There were no significant tillage effects on bacterial diversity in whole soils. In soil aggregates, H' and E were significantly higher under CT than under ZT on the loam at barley planting time, with no significant aggregate size effects. However, at barley-heading stage, all diversity indices in both soils were significantly higher under ZT than under CT, and they tended to increase with increasing aggregate size. Cluster analysis and principal component analysis of substrate utilization patterns also revealed differences in bacterial community structures between CT and ZT,but the substrates that were utilized differently between the two tillage systems were not the same between soil types or sampling times. The results during the cropping cycle imply that deterioration of soil structure is probably one factor that explains the adverse effects of soil tillage on soil microbial biomass and diversity. (C) 2001 Elsevier Science B.V. All rights reserved.

12. Macrae, A., C. M. M. Lucon, D. L. Rimmer, and A. G. O'Donnell. 2001. Sampling DNA from the rhizosphere of Brassica napus to investigate rhizobacterial community structure. Plant and Soil 233:223-230.
Keywords: 16S rDNA/actinomycetes/bacilli/ecology/rhizosphere/
MOLECULAR MICROBIAL DIVERSITY/BACTERIAL DIVERSITY/RIBOSOMAL DNA/SOIL/SEQUENCE/PCR/MICROORGANISMS/PHYLOGENIES/ALIGNMENT/DYNAMICS
Abstract: We have compared, using DNA analysis, the classical 'pull and shake' method for sampling rhizosphere soil with a model rhizosphere system in which the soil was sampled at increasing distances (0.5 mm) from the root mat of Brassica napus. These studies showed that more DNA was extracted from the soil slices closest to the roots. Whilst the gradient in DNA recovered cannot be attributed solely to differences in microbial biomass, it clearly illustrates the lack of spatial resolution characteristic of the 'pull and shake' method of sampling from the rhizosphere. The DNA-rich fraction (rhizosphere soil) was compared with bulk soil DNA by PCR using actinomycete 16S rDNA primers and 16S rDNA sequencing. Phylogenetic analyses of 16S rDNA sequences showed that these primers were not specific for actinomycetes but also amplified Gram positive low G+C bacterial sequences. Comparative 16S rDNA analysis of these sequences showed an enrichment for Gram positive low G+C bacteria in the rhizosphere of Brassica napus.

13. Nakatsu, C. H., V. Torsvik, and L. Ovreas. 2000. Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Science Society of America Journal 64:1382-1388.
Keywords: GRADIENT GEL-ELECTROPHORESIS/MOLECULAR MICROBIAL DIVERSITY/RIBOSOMAL-RNA SEQUENCES/
PHYLOGENETIC ANALYSIS/AGRICULTURAL SOIL/KINGDOM CRENARCHAEOTA/DEFINED POPULATIONS/DNA FRAGMENTS/DEEP-SEA/BACTERIA
Abstract: Separation of polymerase chain reaction (PCR)-amplified 16S rDNA products using denaturing gradient gel electrophoresis (DGGE) was tested as a means to study microbial community composition in bulk soil samples. DNA was extracted from six soils from agroecosystems in Norway and the USA under different agronomic treatments (crop, rotation, and tillage); one soil is contaminated with polyaromatic hydrocarbons (PAH, 700 mg kg(-1)). Two sets of primers specific for Bacteria (V3 and the V6/V9 regions of 16S rRNA) and another for Archaea (V3 region of 16S rRNA) were used to determine the contribution of each domain to the microbial community. Reproducible, characteristic profiles of the communities were obtained by DGGE separation of the PCR amplification products. The number of fragments resolved by DGGE indicated bacterial diversity was far greater than that of the Archaea in the agricultural soils examined. Only the soil contaminated with PAHs had reduced bacterial diversity, evidenced by a distinct DGGE profile. The results showed that the method is useful as an initial step to discriminate among communities because it is rapid and multiple samples can be easily screened. There are some limitations, but under highly selective conditions it is possible to distinguish communities from different soils and to indicate the presence of numerically dominant populations.

14. Picard, C., F. Di Cello, M. Ventura, R. Fani, and A. Guckert. 2000. Frequency and biodiversity of 2,4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth. Applied and Environmental Microbiology 66:948-955.
Keywords: DNA-RESTRICTION ANALYSIS/FRAGMENT-LENGTH-POLYMORPHISM/PREEMERGENCE DAMPING-OFF/GRAMINIS VAR TRITICI/
FLUORESCENT PSEUDOMONADS/BIOLOGICAL-CONTROL/ENTEROBACTER-CLOACAE/16S rDNA/ANTIBIOTIC 2,4-DIACETYLPHLOROGLUCINOL/RAPID IDENTIFICATION
Abstract: A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG, DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates, The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique, An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype, These DAPG producers could be exploited in future screening programs for biocontrol agents.

15. Ranjard, L., F. Poly, J. C. Lata, C. Mougel, J. Thioulouse, and S. Nazaret. 2001. Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: Biological and methodological variability. Applied and Environmental Microbiology 67:4479-4487.
Keywords: MOLECULAR MICROBIAL DIVERSITY/GRADIENT GEL-ELECTROPHORESIS/SOUTHWESTERN UNITED-STATES/
AGRICULTURAL SOIL/RNA GENES/PCR/DNA/IDENTIFICATION/HETEROGENEITY/SUCCESSION
Abstract: Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 by were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (interand intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334-5339, 2000) and fungi.

16. Santosa, D. A. 2001. Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies. Molecular Biotechnology 17:59-64. Keywords: environmental DNA/extraction/purification/molecular diversity/molecular cloning/biotechnological potential/
POLYMERASE CHAIN-REACTION/SOIL/MICROORGANISMS/BACTERIA/AMPLIFICATION/COMMUNITY/SEDIMENTS
Abstract: A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-I-Z solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-IIZ solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASH((R))II Vector and Gigapack((R))III XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.

17. Trevors, J. T. 1998. Bacterial biodiversity in soil with an emphasis on chemically-contaminated soils. Water Air and Soil Pollution 101:45-67.
Keywords: BACTERIA/biodiversity/chemically-contaminated/ecology/MICROORGANISMS/molecular/PCR/SOIL/
toxic/POLYMERASE CHAIN-REACTION/GENETICALLY-ENGINEERED MICROORGANISMS/PSEUDOMONAS-FLUORESCENS STRAIN/
MICROBIAL COMMUNITY DIVERSITY/MERCURY RESISTANCE GENES/RIBOSOMAL-RNA SEQUENCES/RAPID METHOD/
CENTRIFUGATION TECHNIQUE/HERBICIDE-GLYPHOSATE/DIRECT EXTRACTION
Abstract: Microorganisms isolated from soil are sources of known and new microorganisms and genetic material. This review examines general principles of soil bacterial biodiversity, limitations in sampling soils, and examples of bacterial diversity in chemically-contaminated soils. Both conventional and molecular methods used to assess microbial biodiversity in soils will be addressed as well as selected examples of the effects of organic and inorganic pollutants on soil microbial diversity.

18. Yu, Z. T. and W. W. Mohn. 1999. Killing two birds with one stone: simultaneous extraction of DNA and RNA from activated sludge biomass. Canadian Journal of Microbiology 45:269-272.
Keywords: activated sludge/DNA/extraction/PCR/RNA/microbial diversity/RNA/DNA RATIO/RIBOSOMAL-RNA/BACTERIA/SEWAGE
Abstract: DNA and RNA are usually extracted from activated sludge samples using two separate methods developed for soil and sediment samples. However, activated sludge differs from soil and sediment in at least three aspects: high biomass density, low humic acid content, and the presence of bacterial aggregate flocs. Taking these characteristics into consideration, we developed a simple and rapid method allowing simultaneous DNA and RNA extraction from activated sludge samples. This method combines (i) mini-bead beating, which is most efficient in breaking bacterial aggregate flocs and cells, (ii) protection of RNA with diethyl pyrocarbonate, and (iii) precipitation of impurities with ammonium acetate. Phenol/chloroform extraction and column purification are not necessary. The resulting DNA and RNA are suitable for PCR and reverse transcriptase - PCR, respectively. The efficiencies of cell lysis and nucleic acid recovery were high enough to permit detection by PCR of 10(2) cells/mL of mixed liquor. By simultaneously extracting both DNA and RNA from a single sample, this method eliminates variability in cell lysis between extraction of DNA and RNA using two different methods. This extraction method is rapid, and within 1 h, one person can process four or more samples. This simple method makes it easier to analyze a large number of activated sludge samples.

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More methods:

Soil microbial diversity and community structure under wheat as influenced by tillage and crop rotation. SOURCE: Soil-biol-biochem. Oxford : Elsevier Science Ltd. Nov 1998. v. 30 (13) p. 1733-1741.

Development and application of a selective PCR-denaturing gradient gel electrophoresis approach to detect a recently cultivated Bacillus group predominant in soil AUTHOR, EDITOR, INVENTOR: Tzeneva,-Vesela-A [Author,-Reprint-Author]; Li,-Youguo [Author]; Felske,-Andreas-D-M [Author]; de-Vos,-Willem-M [Author]; Akkermans,-Antoon-D-L [Author]; Vaughan,-Elaine-E [Author]; Smidt,-Hauke [Author] SOURCE: Applied-and-Environmental-Microbiology. 2004; 70(10): 5801-5809.

New strategies for cultivation and detection of previously uncultured microbes AUTHOR, EDITOR, INVENTOR: Stevenson,-Bradley-S [Author,-Reprint-Author]; Eichorst,-Stephanie-A [Author]; Wertz,-John-T [Author]; Schmidt,-Thomas-M [Author]; Breznak,-John-A [Author] SOURCE: Applied-and-Environmental-Microbiology. 2004; 70(8): 4748-4755.

Use of polyclonal antibodies to detect and quantify the NOR protein of nitrite oxidizers in complex environments. AUTHOR, EDITOR, INVENTOR: Maron,-Pierre-Alain [Author,-Reprint-Author]; Coeur,-Caroline [Author]; Pink,-Claudia [Author]; Clays-Josserand,-Annie [Author]; Lensi,-Robert [Author]; Richaume,-Agnes [Author]; Potier,-Patrick [Author] SOURCE: Journal-of-Microbiological-Methods. 2003; 53(1): 87-95.

Effects of agronomical measures on the microbial diversity of soils as related to the suppression of soil-borne plant pathogens AUTHOR, EDITOR, INVENTOR: van-Elsas,-Jan-Dirk [Reprint-author]; Garbeva,-Paolina [Reprint-author]; Salles,-Joana [Reprint-author] SOURCE: Biodegradation-. 2002; 13(1): 29-40.

Comparison of pmoA PCR primer sets as tools for investigating methanotroph diversity in three Danish soils AUTHOR, EDITOR, INVENTOR: Bourne,-David-G [Author]; McDonald,-Ian-R [Author]; Murrell,-J-Colin [Reprint-author] SOURCE: Applied-and-Environmental-Microbiology. 2001; 67(9): 3802-3809.

Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia AUTHOR, EDITOR, INVENTOR: Niemi,-R-Maarit [Reprint-author]; Heiskanen,-Ilse [Author]; Wallenius,-Kaisa [Author]; Lindstrom,-Kristina [Author] SOURCE: Journal-of-Microbiological-Methods. 2001; 45(3): 155-165.

DNA extraction from soils: Old bias for new microbial diversity analysis methods AUTHOR, EDITOR, INVENTOR: Martin-Laurent,-F [Reprint-author]; Philippot,-L [Author]; Hallet,-S [Author]; Chaussod,-R [Author]; Germon,-J-C [Author]; Soulas,-G [Author]; Catroux,-G [Author] SOURCE: Applied-and-Environmental-Microbiology. 2001; 67(5): 2354-2359.

Identification and characterisation of bacterial populations of an in-use metal-working fluid by phenotypic and genotypic methodology AUTHOR, EDITOR, INVENTOR: van-der-Gast,-C-J [Author]; Knowles,-C-J [Author]; Wright,-M-A [Author]; Thompson,-I-P [Reprint-author] SOURCE: International-Biodeterioration-and-Biodegradation. 2001; 47(2): 113-123.

Small-scale DNA sample preparation method for field PCR detection of microbial cells and spores in soil AUTHOR, EDITOR, INVENTOR: Kuske,-Cheryl-R [Reprint-author]; Banton,-Kaysie-L [Author]; Adorada,-Dante-L [Author]; Stark,-Peter-C [Author]; Hill,-Karen-K [Author]; Jackson,-Paul-J [Author] SOURCE: Applied-and-Environmental-Microbiology. 1998; 64(7): 2463-2472.

Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers AUTHOR, EDITOR, INVENTOR: Boschker,-H-T-S [Reprint-author]; Nold,-S-C [Author]; Wellsbury,-P [Author]; Bos,-D [Author]; De-Graaf,-W [Author]; Pel,-R [Author]; Parkees,-R-J [Author]; Cappenberg,-T-E [Author] SOURCE: Nature-(London). 1998; 392(6678): 801-805.

Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients AUTHOR, EDITOR, INVENTOR: Heuer,-Holger [Author]; Krsek,-Martin [Author]; Baker,-Paul [Author]; Smalla,-Kornelia [Reprint-author]; Wellington,-Elizabeth-M-H [Author] SOURCE: Applied-and-Environmental-Microbiology. 1997; 63(8): 3233-3241.