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  • 1
    Language: English
    In: Journal of Microbiological Methods, 2010, Vol.80(1), pp.63-69
    Description: and its teleomorphic stage play a key role for ecosystem functioning in terrestrial habitats. However, little is known about the ecology of the fungus. In this study we developed a novel -specific primer pair for diversity analysis. Based on a broad range master alignment, specific primers (ITS F/ITS R) were designed that comprise an approximate 650 bp fragment of the internal transcribed spacer region from all taxonomic clades of the genus This amplicon is suitable for identification with Key and BLAST. Moreover, this primer system was successfully applied to study the communities in the rhizosphere of different potato genotypes grown at two field sites in Germany. Cloning and sequencing confirmed the specificity of the primer and revealed a site-dependent composition. Based on the new primer system a semi-nested approach was used to generate amplicons suitable for denaturing gradient gel electrophoresis (DGGE) analysis and applied to analyse communities in the rhizosphere of potatoes. High field heterogeneity of communities was revealed by both DGGE. Furthermore, qPCR showed significantly different copy numbers between the sites
    Keywords: Trichoderma ; Its Region ; Diversity ; Rhizosphere ; Biology
    ISSN: 0167-7012
    E-ISSN: 1872-8359
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  • 2
    Language: English
    In: Journal of Microbiological Methods, 2011, Vol.84(3), pp.454-460
    Description: 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...
    Keywords: Life Sciences ; Toxicology ; Ecotoxicology ; Environmental Sciences ; Inter-Laboratory Assay ; Standardization ; Soil DNA Extraction ; DNA Fingerprint ; Qpcr ; Environmental Sciences ; Biology
    ISSN: 0167-7012
    E-ISSN: 1872-8359
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  • 3
    Language: English
    In: Journal of Microbiological Methods, 2006, Vol.65(1), pp.63-75
    Description: The analysis of soil fungal communities by molecular fingerprinting and subsequent identification of the underlying populations require the amplification of a phylogenetically informative gene fragment. In this study we tested the reliability and suitability of the previously published fungal primer combination (NS1/FR1-GC) that amplifies almost the entire 18S gene for the DGGE analysis of fungal communities in soil samples from 36 sites. This direct PCR system failed to amplify the fragment of interest from the total DNA extracted from most of the soils tested. Thus, we developed a new semi-nested PCR system based on the initial amplification of over 1700 bp of the 18S gene with a new primer combination, followed by a subsequent amplification with NS1/FR1-GC. By means of the PCR approach developed in this study distinct 18S gene amplicons could be reproducibly generated for all soil samples. Amplification tests with 101 soil fungal isolates showed that with the new semi-nested system 18S gene fragments could be obtained from more fungi than with the direct approach. The subsequent DGGE separation of community amplicons resulted in a high resolution and revealed reproducible complex soil fungal communities specific for each site, despite a minor variability between replicates of the same sample. The semi-nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a robust, reliable and sensitive tool for the analysis of soil fungal community structure.
    Keywords: 18s Rrna Gene ; Dgge ; Semi-Nested Pcr ; Soil Fungal Communities ; Biology
    ISSN: 0167-7012
    E-ISSN: 1872-8359
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  • 4
    Language: English
    In: Journal of Microbiological Methods, 1997, Vol.30(1), pp.49-61
    Description: The method of comparing microbial communities by their sole-carbon-source utilization profiles using BIOLOG GN microplates was evaluated for the potato phyllosphere. Reproducibility, sensitivity to detect phyllosphere community shifts, and the bias by the unequal impact of different potato phyllosphere populations on the catabolic profiles were investigated. A reproducible set of carbon-sources was found to be frequently utilized by microbial communities from potato leaves. About 20 of the 95 substrates in the BIOLOG GN microplates varied in being utilized. The variability of the BIOLOG pattern was mainly caused by phyllosphere heterogeneity and not by the method. By comparison of microbial communities from slightly different phyllospheres using a new multivariate test in combination with principal component analysis, the sensitivity to detect community differences was evaluated. Significant quantitative differences were shown in phyllosphere communities of potato and sugar beet, two potato varieties, and leaves of the upper and lower half of a potato plant. No significant differences between catabolic profiles of microbial communities from leaves of T4-lysozyme expressing transgenic potatoes and nontransgenic potatoes could be detected. Nevertheless, significantly fewer gram-positive bacteria were isolated from leaves of the lysozyme-potatoes. In various experiments evidence was found that the gram-positive fraction of the community rarely contributed to the patterns. These data explain why effects of T4-lysozyme on gram-positive community members were not detected by BIOLOG catabolic profiling.
    Keywords: Biolog ; Community-Level ; Catabolic Profiling ; Microbial Community ; Phyllosphere ; Potato ; Biology
    ISSN: 0167-7012
    E-ISSN: 1872-8359
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  • 5
    Language: English
    In: Journal of Microbiological Methods, 2004, Vol.57(2), pp.187-195
    Description: Modern electrophoresis techniques are often applied to investigate complex microbial communities. Analysis systems like GelCompare transform the optical pattern of the electrophoresis lanes into high-dimensional observation vectors and calculate measures of difference or similarity between pairs of lanes. Usually, these measures are applied in cluster analyses. Here, we apply permutation tests for the comparison of groups of lanes based on these pairwise measures together with some extensions for a combined analysis of several electrophoresis gels. The procedures are available as a computer program. An example is given for the comparison of bacterial soil communities, testing the effect of different crop plants. Each community was represented by amplified ribosomal gene fragments separated in a denaturing gradient gel.
    Keywords: Permutation Test ; Electrophoretic Fingerprints ; Pairwise Distance Measures ; Block Design ; Biology
    ISSN: 0167-7012
    E-ISSN: 1872-8359
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  • 6
    Language: English
    In: Journal of Microbiological Methods, 1997, Vol.30(1), pp.71-80
    Description: Comparative catabolic profiling using microtiterplates with multiple sole-carbon-sources has become a popular tool for the comparison of microbial communities with respect to their functional potential. However, the statistical treatment of such data has only been partially satisfactory so far. In this paper, a new multivariate approach developed by Läuter and coworkers [J. Läuter, Exact T and F tests for analysing studies with multiple endpoints, Biometrics 52 (1996) 964–970; J. Läuter, E. Glimm, S. Kropf, New multivariate tests for data with an inherent structure, Biometrical Journal 38 (1996) 5–23.] is applied to this problem. Using replicate observations from every community in question, this approach allows testing for significant differences between communities. It is advantageous with respect to power and maintains the α -level. Discriminating carbon-sources are found according to weighted factor loadings and univariate tests. Exemplarily, changes in microbial soil communities due to rotting of plants are analyzed. Furthermore, other statistical approaches from the literature are discussed.
    Keywords: Biolog System ; Catabolic Profiling ; Microbial Community ; Multivariate Statistics ; Biology
    ISSN: 0167-7012
    E-ISSN: 1872-8359
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  • 7
    Language: English
    In: Journal of Microbiological Methods, 2007, Vol.69(3), pp.470-479
    Description: Bacterial communities of four arable soils – pelosol, gley, para brown soil, and podsol brown soil – were analysed by fingerprinting of 16S rRNA gene fragments amplified from total DNA of four replicate samples for each soil type. Fingerprints were generated in parallel by denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP) to test whether these commonly applied techniques are interchangeable. PCR amplicons could be separated with all three methods resulting in complex ribotype patterns. Although the fragments amplified comprised different variable regions and lengths, DGGE, T-RFLP and SSCP analyses led to similar findings: (a) a clustering of fingerprints which correlated with soil physico-chemical properties, (b) little variability between the four replicates of the same soil, (c) the patterns of the two brown soils were more similar to each other than to those of the other two soils, and (d) the fingerprints of the different soil types revealed significant differences in a permutation test, which was recently developed for this purpose.
    Keywords: 16s Rrna Gene ; Dgge ; Soil Bacterial Diversity ; Sscp ; Total Community DNA ; T-Rflp ; Biology
    ISSN: 0167-7012
    E-ISSN: 1872-8359
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