Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost

We investigated the fungal communities inhabiting decaying logs in a seminatural boreal forest stand in relation to host tree species, stage of decay, density, diameter, moisture, C to N ratio, Klason lignin content, and water- and ethanol-soluble extractives. Communities were profiled using denaturing gradient gel electrophoresis fingerprinting of the rDNA ITS1 region coupled with sequencing of fungal DNA extracted directly from the wood. In addition, polypore fruit bodies were inventoried. Logs from different tree species had different fungal communities and different physicochemical properties (e.g., C to N ratio, density, ethanol extractives, and diameter). Ascomycetes comprised a larger portion of communities inhabiting deciduous birch ( Betula spp.) and European aspen ( Populus tremula L.) logs compared with those living on coniferous Norway spruce ( Picea abies (L.) Karst.) and Scots pine ( Pinus sylvestris L.). A relationship between mycelial community structure and density of decaying spruce logs suggested a succession of fungi with mass loss of wood. The fruit body inventory underestimated fungal diversity in comparison with the culture-free denaturing gradient gel electrophoresis analysis that also detected inconspicuous but important species inhabiting decaying wood.

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Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6801-6807.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6801-6807 ◽

Cited By ~ 124

Author(s):

Isabel Lopez ◽

Fernanda Ruiz-Larrea ◽

Luca Cocolin ◽

Erica Orr ◽

Trevor Phister ◽

...

Keyword(s):

Lactic Acid ◽

16S Rdna ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Species ◽

Pcr Primers ◽

Fungal Species ◽

Bacterial Populations ◽

Gradient Gel Electrophoresis ◽

Primer Sets

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

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Characterization of bacterial and fungal communities in composted biosolids over a 2 year period using denaturing gradient gel electrophoresis

Canadian Journal of Microbiology ◽

10.1139/w08-152 ◽

2009 ◽

Vol 55 (4) ◽

pp. 375-387 ◽

Cited By ~ 22

Author(s):

Amy Novinscak ◽

Nadine J. DeCoste ◽

Céline Surette ◽

Martin Filion

Keyword(s):

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Species ◽

Fungal Species ◽

Fungal Communities ◽

Whole Process ◽

Agricultural Applications ◽

Gradient Gel Electrophoresis ◽

Microbial Groups ◽

Key Microorganisms

Composting is a microbial process that converts organic waste into a nutrient-rich end product used in horticultural and agricultural applications. The diversity and long-term succession of microorganisms found in composted biosolids has been less characterized than other composts. In this study, bacterial and fungal communities found in composted biosolids aging from 1 to 24 months were studied using denaturing gradient gel electrophoresis (DGGE) and sequencing. The results revealed high levels of diversity, where 53 bacterial species belonging to 10 phyla and 21 fungal species belonging to 4 phyla were identified. Significant differences were observed when comparing the bacterial DGGE patterns of young compost samples, whereas no differences were observed in samples over 8 months. For fungal patterns, no significant differences were observed during the first 4 months of composting, but the diversity then significantly shifted until 24 months. The results indicate that patterns of bacterial species vary during the first few months of composting, whereas fungal patterns generally vary throughout the whole process, except during early stages. The description of the main microbial groups found in composted biosolids could find various applications, including the discovery of biotechnologically relevant microorganisms and the development of novel markers allowing quantitative monitoring of key microorganisms.

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