Analysis of Fungal Diversity in the Composting by Sequencing of Cloned PCR-Amplified 18S rDNA and Denaturing Gradient Gel Electrophoresis

2011 ◽  
Vol 356-360 ◽  
pp. 1747-1751
Author(s):  
Wan Li ◽  
Xiu Hong Xu ◽  
Jia Lei Xiao ◽  
Bi Xian Zhang ◽  
Hong Tao Li ◽  
...  
Keyword(s):  
Fungal Community ◽  
18S Rdna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Limited Information ◽  
Rdna Sequences ◽  
Microbiological Techniques ◽  
Gradient Gel Electrophoresis ◽  
Realistic View ◽  
Specific Amplification

Like bacteria, fungi play an important role in the composting process as major decomposers of organic substances. As only a small fraction of the fungi present in composting can be cultured because conventional microbiological techniques limited information on the composition of fungal communities in composting. Molecular methods are expected to give a more realistic view of species richness and distribution. For this purpose, we selected universal PCR primer set that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences. DNA was extracted from composting samples, and 18S rDNA genes were amplified by EF4/Fung5 (0.6kb) and EF4/NS2-GC (0.4kb). DGGE analysis of the fungal community in the composting of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. Clear banding patterns were obtained with amplified production. 13 different bands excised from the DGGE gel were sequenced and compared with genbank. Sequencing showed that some could not be cultured; some were efficient cellulose-degrading strains. The results showed that diversity and composition of the fungal community in the composting can be analyzed by the combination of 18S rDNA PCR amplification and DGGE.

scholarly journals Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis

1999 ◽  
Vol 65 (6) ◽  
pp. 2614-2621 ◽  
Author(s):  
Eric Smit ◽  
Paula Leeflang ◽  
Boet Glandorf ◽  
Jan Dirk van Elsas ◽  
Karel Wernars
Keyword(s):  
Temperature Gradient ◽  
Fungal Community ◽  
18S Rdna ◽  
Gel Electrophoresis ◽  
Ribosomal Database Project ◽  
Soil Dna ◽  
Wheat Rhizosphere ◽  
Rdna Sequences ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

ABSTRACT Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

Comparative denaturing gradient gel electrophoresis analysis of fungal communities associated with whole plant corn silage

2001 ◽  
Vol 47 (9) ◽  
pp. 829-841 ◽  
Author(s):  
Lisa A May ◽  
Brenda Smiley ◽  
Michael G Schmidt
Keyword(s):  
Fungal Community ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Direct Sequencing ◽  
Corn Silage ◽  
Positive Effects ◽  
Operational Taxonomic Units ◽  
Whole Plant ◽  
Lost Productivity ◽  
Gradient Gel Electrophoresis

Significant portions of grain produced for livestock consumption are converted into ensiled forage. Silage producers have long recognized the positive effects of using an inoculant to insure the proper transformation of forage into a palatable and digestible feedstuff. When silage is fed from a storage structure, exposure to air stimulates the growth of epiphytic aerobes that may result in the loss of up to 50% of the dry matter. Moreover, fungi have been found to be associated with ensiled forage, but their growth is normally suppressed by the anaerobic conditions. However, the introduction of oxygen results in a fungal bloom, and the fungi and the associated metabolites may result in lost productivity in the livestock consuming the contaminated forage. In this study, we report on the diversity of the fungal community associated with whole plant corn silage during the ensiling process, and the effect of two different bacterial inoculants as compared with the uninoculated natural epiphytic fermentation on the distribution of the fungi associated with the silage. The fungal community from duplicate mini-silo packages of the same treatment was analyzed by denaturing gradient gel electrophoresis and direct sequencing of the resulting operational taxonomic units. This method proved useful in analyzing the complex microbial communities associated with the forage in that it was possible to determine that one inoculant dramatically influenced the fungal community associated with whole plant corn silage.Key words: fungi, silage, DGGE, OTU.

scholarly journals Application of Sequence-Dependent Electrophoresis Fingerprinting in Exploring Biodiversity and Population Dynamics of Human Intestinal Microbiota: What Can Be Revealed?

2008 ◽  
Vol 2008 ◽  
pp. 1-26 ◽  
Author(s):  
Geert Huys ◽  
Tom Vanhoutte ◽  
Peter Vandamme
Keyword(s):  
Population Dynamics ◽  
Intestinal Microbiota ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Therapeutic Interventions ◽  
Rrna Gene ◽  
Sequence Dependent ◽  
Human Intestinal Microbiota ◽  
Gradient Gel Electrophoresis ◽  
Community Fingerprinting

Sequence-dependent electrophoresis (SDE) fingerprinting techniques such as denaturing gradient gel electrophoresis (DGGE) have become commonplace in the field of molecular microbial ecology. The success of the SDE technology lays in the fact that it allows visualization of the predominant members of complex microbial ecosystems independent of their culturability and without prior knowledge on the complexity and diversity of the ecosystem. Mainly using the prokaryotic 16S rRNA gene as PCR amplification target, SDE-based community fingerprinting turned into one of the leading molecular tools to unravel the diversity and population dynamics of human intestinal microbiota. The first part of this review covers the methodological concept of SDE fingerprinting and the technical hurdles for analyzing intestinal samples. Subsequently, the current state-of-the-art of DGGE and related techniques to analyze human intestinal microbiota from healthy individuals and from patients with intestinal disorders is surveyed. In addition, the applicability of SDE analysis to monitor intestinal population changes upon nutritional or therapeutic interventions is critically evaluated.

Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA

2003 ◽  
Vol 35 (9) ◽  
pp. 1165-1173 ◽  
Author(s):  
Ian S. Waite ◽  
Anthony G. O'Donnell ◽  
Andrew Harrison ◽  
John T. Davies ◽  
Stephanie R. Colvan ◽  
...  
Keyword(s):  
18S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Soil Community ◽  
Gradient Gel Electrophoresis

Changes in the structure and diversity of bacterial communities during the process of adaptation to organic wastewater

2010 ◽  
Vol 56 (4) ◽  
pp. 352-355 ◽  
Author(s):  
Junmin Li ◽  
Zexin Jin ◽  
Binbin Yu
Keyword(s):  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Similarity Index ◽  
Pcr Amplification ◽  
Genotypic Diversity ◽  
Diversity Indices ◽  
Inhibitory Effect ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gradient Gel Electrophoresis

To explore changes in the structure and diversity of activated sludge-derived microbial communities during adaptation to gradual increases in the concentration of wastewater, RAPD–PCR and the combination of PCR amplification of 16S rRNA genes with denaturing gradient gel electrophoresis (DGGE) analysis were used. In bacterial communities exposed to 0%, 5%, 10%, 20%, or 40% wastewater, there were 27, 25, 18, 17 and 16 bands, respectively, based on DGGE data, while there were 69, 83, 97, 86, and 88 bands, respectively, based on RAPD data. The community similarity index among bacterial communities during the process of adaptation to different concentrations of wastewater was different based on DGGE and RAPD data. Based on DGGE and RAPD profiles, the Shannon–Weiner and Simpson’s diversity indices decreased sharply upon exposure to 10% wastewater, indicating that 10% wastewater might be a critical point at which the growth of bacteria could be significantly inhibited and the genotypic diversity could change. This indicated that changes in structure and diversity might have an inhibitory effect on the toxicity of organic matter and that selection and adaptation could play important roles in the changes.

scholarly journals Distribution and Diversity of Archaea Corresponding to the Limnological Cycle of a Hypersaline Stratified Lake (Solar Lake, Sinai, Egypt)

2000 ◽  
Vol 66 (8) ◽  
pp. 3269-3276 ◽  
Author(s):  
Eddie Cytryn ◽  
Dror Minz ◽  
Ronald S. Oremland ◽  
Yehuda Cohen
Keyword(s):  
Phylogenetic Analysis ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Archaeal Community ◽  
Seasonal Distribution ◽  
Stratified Lake ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Gradient Gel Electrophoresis ◽  
Solar Lake

ABSTRACT The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to theHalobacteriaceae family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the generaMethanobacterium and Methanococcus. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genusThermoplasma. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.

scholarly journals Molecular Monitoring of Succession of Bacterial Communities in Human Neonates

2002 ◽  
Vol 68 (1) ◽  
pp. 219-226 ◽  
Author(s):  
Christine F. Favier ◽  
Elaine E. Vaughan ◽  
Willem M. De Vos ◽  
Antoon D. L. Akkermans
Keyword(s):  
Sequence Analysis ◽  
16S Rdna ◽  
Bacterial Communities ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bacterial Colonization ◽  
Molecular Monitoring ◽  
Fecal Samples ◽  
16S Rdna Sequence Analysis ◽  
Rdna Sequences ◽  
Gradient Gel Electrophoresis

ABSTRACT The establishment of bacterial communities in two healthy babies was examined for more than the first 10 months of life by monitoring 16S ribosomal DNA (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE) and by analyzing the sequences of the major ribotypes. DGGE profiles of the dominant populations in the intestines of the infants were obtained by analyzing daily or weekly fecal samples. After delivery, the germfree infant gastrointestinal tracts were rapidly colonized, and the succession of bacteria in each ecosystem was monitored. During the first few days of life the profiles were simple, but they became more complex as the bacterial diversity increased with time in both babies. Clone libraries of amplified 16S rDNA fragments from baby feces were constructed, and these libraries allowed identification of the bacterial types by comparative DNA sequence analysis; the bacteria identified included members of the genera Bifidobacterium, Ruminococcus, Enterococcus, Clostridium, and Enterobacter. Species most closely related to the genera Bifidobacterium and Ruminococcus in particular dominated the intestinal microbiota based on the stability over time and the numbers, as estimated by the intensities of the bands. However, 19 of the 34 cloned rDNA sequences exhibited less than 97% identity with sequences of known bacteria or cloned sequences in databases. This study showed that using PCR-DGGE and 16S rDNA sequence analysis together resulted in a dynamic description of bacterial colonization in the infant intestinal ecosystem and allowed visualization of bacteria that are difficult to cultivate or to detect by other methods.

scholarly journals Competition for Light between Toxic and Nontoxic Strains of the Harmful Cyanobacterium Microcystis

2007 ◽  
Vol 73 (9) ◽  
pp. 2939-2946 ◽  
Author(s):  
W. Edwin A. Kardinaal ◽  
Linda Tonk ◽  
Ingmar Janse ◽  
Suzanne Hol ◽  
Pieter Slot ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Pcr Amplification ◽  
Internal Transcribed Spacer Region ◽  
Gradient Gel Electrophoresis ◽  
Competition For Light ◽  
Low Levels ◽  
Allelopathic Interactions ◽  
Light Absorbance ◽  
Absorbance Spectra

ABSTRACT The cyanobacterium Microcystis can produce microcystins, a family of toxins that are of major concern in water management. In several lakes, the average microcystin content per cell gradually declines from high levels at the onset of Microcystis blooms to low levels at the height of the bloom. Such seasonal dynamics might result from a succession of toxic to nontoxic strains. To investigate this hypothesis, we ran competition experiments with two toxic and two nontoxic Microcystis strains using light-limited chemostats. The population dynamics of these closely related strains were monitored by means of characteristic changes in light absorbance spectra and by PCR amplification of the rRNA internal transcribed spacer region in combination with denaturing gradient gel electrophoresis, which allowed identification and semiquantification of the competing strains. In all experiments, the toxic strains lost competition for light from nontoxic strains. As a consequence, the total microcystin concentrations in the competition experiments gradually declined. We did not find evidence for allelopathic interactions, as nontoxic strains became dominant even when toxic strains were given a major initial advantage. These findings show that, in our experiments, nontoxic strains of Microcystis were better competitors for light than toxic strains. The generality of this finding deserves further investigation with other Microcystis strains. The competitive replacement of toxic by nontoxic strains offers a plausible explanation for the gradual decrease in average toxicity per cell during the development of dense Microcystis blooms.

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