scholarly journals Synthetic Consolidants Attacked by Melanin-Producing Fungi: Case Study of the Biodeterioration of Milan (Italy) Cathedral Marble Treated with Acrylics

2006 ◽  
Vol 73 (1) ◽  
pp. 271-277 ◽  
Author(s):  
Francesca Cappitelli ◽  
Joshua D. Nosanchuk ◽  
Arturo Casadevall ◽  
Lucia Toniolo ◽  
Lorenzo Brusetti ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
18S Rrna Gene ◽  
Morphological Characterization ◽  
Rrna Gene ◽  
Acrylic Resins ◽  
Gradient Gel Electrophoresis ◽  
Previously Treated ◽  
Characteristic Features ◽  
Rrna Gene Sequencing ◽  
Meristematic Fungi

ABSTRACT Monuments and artistic stone surfaces are often consolidated and protected with synthetic polymers, in particular, acrylics. Although it is generally thought that acrylic polymers are resistant to biodeterioration, we report for the first time the systematic occurrence of dematiaceous meristematic fungi on many marble samples of the cathedral in Milan (Italy) previously treated with this material. Fourier transform infrared spectroscopy applied to the Milan cathedral stone samples revealed characteristic features of biodeteriorated synthetic resins that differentiated them from the aged but nonbiodeteriorated samples. Samples showing biological colonization were analyzed for the presence of fungi. Cultivation and morphological characterization and methods independent from cultivation, such as denaturing gradient gel electrophoresis coupled with partial 18S rRNA gene sequencing and immunofluorescence staining with melanin-binding antibodies, showed that melanin-producing species are heavily present on stone surfaces protected with acrylic resins. This observation raises the question of the effectiveness of acrylics in protecting stone artworks.

Quantification and genotyping of Cryptosporidium spp. in river water by quenching probe PCR and denaturing gradient gel electrophoresis

2006 ◽  
Vol 54 (3) ◽  
pp. 119-126 ◽  
Author(s):  
Y. Masago ◽  
K. Oguma ◽  
H. Katayama ◽  
S. Ohgaki
Keyword(s):  
River Water ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Detection Method ◽  
Geometric Mean ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Cryptosporidium Spp ◽  
Gradient Gel Electrophoresis ◽  
Quenching Probe

A new detection method was developed for the simultaneous quantification and genotyping of Cryptosporidium spp. in river water. Several modifications made to the US EPA Method 1623 enabled high and stable recovery of Cryptosporidium from 40 L of river water (geometric mean =35%, standard deviation =8.7%). Quenching probe PCR (QProbe PCR) was used to quantify the 18S rRNA gene of Cryptosporidium spp. This method could successfully detect single oocysts in a sample, and the lower quantitation limit was as low as 2.5 oocysts/sample. In addition, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing was used to identify the genotypes. These methods were applied to detect Cryptosporidium spp. in the Koyama River, Japan. The positive ratio was 69% (11/16) with the maximum concentration of 59 oocysts/100 L. Seven genotypes including two novel ones were identified. These results showed that this detection method could provide valuable information on Cryptosporidium in river water, both in the concentration and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.

scholarly journals Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques

2001 ◽  
Vol 67 (7) ◽  
pp. 2942-2951 ◽  
Author(s):  
Beatriz Dı́ez ◽  
Carlos Pedr�s-Ali� ◽  
Terence L. Marsh ◽  
Ramon Massana
Keyword(s):  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Phylogenetic Groups ◽  
Gradient Gel Electrophoresis ◽  
Phylogenetic Identification ◽  
Phylogenetic Composition ◽  
Primer Sets

ABSTRACT We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

scholarly journals Microscopic and Molecular Studies of the Diversity of Free-Living Protozoa in Meat-Cutting Plants

2008 ◽  
Vol 74 (18) ◽  
pp. 5741-5749 ◽  
Author(s):  
Mario J. M. Vaerewijck ◽  
Koen Sabbe ◽  
Julie Baré ◽  
Kurt Houf
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Screening Method ◽  
18S Rrna Gene ◽  
Hot Water ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Free Living ◽  
Protozoan Community ◽  
Food Borne ◽  
Gradient Gel Electrophoresis

ABSTRACT The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

scholarly journals PCR-Denaturing Gradient Gel Electrophoresis Profiling of Inter- and Intraspecies 18S rRNA Gene Sequence Heterogeneity Is an Accurate and Sensitive Method To Assess Species Diversity of Arbuscular Mycorrhizal Fungi of the Genus Gigaspora

2004 ◽  
Vol 70 (3) ◽  
pp. 1413-1424 ◽  
Author(s):  
Francisco A. de Souza ◽  
George A. Kowalchuk ◽  
Paula Leeflang ◽  
Johannes A. van Veen ◽  
Eric Smit
Keyword(s):  
Arbuscular Mycorrhizal Fungi ◽  
Mycorrhizal Fungi ◽  
Gel Electrophoresis ◽  
18S Rrna ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Arbuscular Mycorrhizal ◽  
18S Rrna Gene ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gradient Gel Electrophoresis

ABSTRACT Despite the importance of arbuscular mycorrhizal fungi in the majority of terrestrial ecosystems, their ecology, genetics, and evolution are poorly understood, partly due to difficulties associated with detecting and identifying species. We explored the inter- and intraspecies variations of the 18S rRNA genes of the genus Gigaspora to assess the use of this marker for the discrimination of Gigaspora isolates and of Gigasporaceae populations from environmental samples. Screening of 48 Gigaspora isolates by PCR-denaturing gradient gel electrophoresis (DGGE) revealed that the V3-V4 region of the 18S rRNA gene contained insufficient variation to discriminate between different Gigaspora species. In contrast, the patterns of 18S ribosomal DNA (rDNA) heterogeneity within the V9 region of this marker could be used for reliable identification of all recognized species within this genus. PCR-DGGE patterns provided insight into some putative misidentifications and could be used to differentiate geographic isolates of G. albida, G. gigantea, and G. margarita but not G. rosea. Two major clusters were apparent based upon PCR-DGGE ribotype patterns, one containing G. albida, G. candida, G. ramisporophora, and G. rosea and the other containing G. decipiens and G. margarita. Dissection of the DGGE patterns by cloning, DGGE screening, and sequencing confirmed these groupings and revealed that some ribotypes were shared across species boundaries. Of the 48 isolates examined, only two displayed any spore-to-spore variation, and these exceptions may be indicative of coisolation of more than one species or subspecies within these cultures. Two Brazilian agricultural soils were also analyzed with a Gigasporaceae-specific nested PCR approach, revealing a dominance of G. margarita within this family.

scholarly journals Influence of Particle Size on Bacterial Community Structure in Aquatic Sediments as Revealed by 16S rRNA Gene Sequence Analysis

2008 ◽  
Vol 74 (16) ◽  
pp. 5237-5240 ◽  
Author(s):  
Colin R. Jackson ◽  
Andrew Q. Weeks
Keyword(s):  
Particle Size ◽  
Community Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Gene Sequence Analysis ◽  
Gradient Gel Electrophoresis ◽  
Rrna Gene Sequencing

ABSTRACT Bacterial communities associated with sediment particles were examined using PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing. Particle size influenced community structure, with attached bacterial assemblages separating into 63- to 125-, 125- to 1,000-, and 1,000- to 2,000-μm fractions. Differences were particularly pronounced for the Verrucomicrobia-Planctomycetes, whose numbers were significantly reduced on coarser particles.

The identification and genetic diversity of endophytic bacteria isolated from selected crops

2018 ◽  
Vol 156 (4) ◽  
pp. 547-556 ◽  
Author(s):  
M. Woźniak ◽  
A. Gałązka ◽  
J. Grządziel ◽  
M. Głodowska
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Agricultural Soils ◽  
16S Rrna Gene Sequencing ◽  
Genomic Diversity ◽  
Wild Plants ◽  
Rrna Gene ◽  
Bacterial Isolates ◽  
Gradient Gel Electrophoresis ◽  
Rrna Gene Sequencing ◽  
Degree Of Differentiation

AbstractA collection of 45 isolates was created based on bacteria isolated from maize, broad bean, wheat, rye and wild plants such as horsetail and burdock. The aim of the current study was to isolate the bacteria, and then identify and assess the degree of genomic diversity. The molecular identification of microsymbionts isolated from the endosphere (root and stem) of plants grown in agricultural soils was performed using 16S rRNA gene sequencing. To evaluate the genomic diversity between strains that occurred in multiple host plants, 18 bacterial isolates representing four species were subjected to denaturing gradient gel electrophoresis. The 16S rDNA analysis assigned all bacterial isolates to ten genera, from whichRhizobiumwas represented by 19 isolates,Delftiaby 11,Agrobacteriumby five,Stenotrophomonasby three,Brevundimonasby two andNovosphingobium,Variovorax, Collimonas, AchromobacterandComamonasby only one isolate. Furthermore, the genomic diversity of the 11 isolates ofDelftiasp. was assessed using the BOX – polymerase chain reaction (BOX-PCR) and enterobacterial repetitive intergenic consensus – PCR (ERIC-PCR) methods. Typing patterns and analysis using BOX-PCR and ERIC-PCR data demonstrated similarities among the tested isolates. In general, the results obtained with BOX-PCR and ERIC-PCR were in good agreement. However, a greater degree of differentiation patterns of the genomic DNA was obtained in the ERIC-PCR method.

scholarly journals Succession of Selected Strains of Acetobacter pasteurianus and Other Acetic Acid Bacteria in Traditional Balsamic Vinegar

2009 ◽  
Vol 75 (8) ◽  
pp. 2585-2589 ◽  
Author(s):  
Maria Gullo ◽  
Luciana De Vero ◽  
Paolo Giudici
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Acetic Acid Bacteria ◽  
Rrna Gene ◽  
Enterobacterial Repetitive Intergenic Consensus ◽  
Acetobacter Pasteurianus ◽  
Gradient Gel Electrophoresis ◽  
Balsamic Vinegar ◽  
Rrna Gene Sequencing

ABSTRACT The application of a selected Acetobacter pasteurianus strain for traditional balsamic vinegar production was assessed. Genomic DNA was extracted from biofilms after enrichment cultures on GYC medium (10% glucose, 1.0% yeast extract, 2.0% calcium carbonate) and used for PCR/denaturing gradient gel electrophoresis, 16S rRNA gene sequencing, and enterobacterial repetitive intergenic consensus/PCR sequencing. Results suggested that double-culture fermentation is suitable for traditional balsamic vinegar acetification.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

scholarly journals Differences in Microbial Activity and Microbial Populations of Peat Associated with Suppression of Damping-Off Disease Caused by Pythium sylvaticum

2006 ◽  
Vol 72 (10) ◽  
pp. 6452-6460 ◽  
Author(s):  
Paul J. Hunter ◽  
Geoff M. Petch ◽  
Leo A. Calvo-Bado ◽  
Tim R. Pettitt ◽  
Nick R. Parsons ◽  
...  
Keyword(s):  
Microbial Activity ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Small Subunit ◽  
Disease Suppression ◽  
Rrna Gene ◽  
Damping Off ◽  
Small Subunit Rrna ◽  
Pythium Sylvaticum ◽  
Gradient Gel Electrophoresis ◽  
Microbial Groups

ABSTRACT The microbiological characteristics associated with disease-suppressive peats are unclear. We used a bioassay for Pythium sylvaticum-induced damping-off of cress seedlings to identify conducive and suppressive peats. Microbial activity in unconditioned peats was negatively correlated with the counts of P. sylvaticum at the end of the bioassay. Denaturing gradient gel electrophoresis (DGGE) profiling and clone library analyses of small-subunit rRNA gene sequences from two suppressive and two conducive peats differed in the bacterial profiles generated and the diversity of sequence populations. There were also significant differences between bacterial sequence populations from suppressive and conducive peats. The frequencies of a number of microbial groups, including the Rhizobium-Agrobacterium group (specifically sequences similar to those for the genera Ochrobactrum and Zoogloea) and the Acidobacteria, increased specifically in the suppressive peats, although no single bacterial group was associated with disease suppression. Fungal DGGE profiles varied little over the course of the bioassay; however, two bands associated specifically with suppressive samples were detected. Sequences from these bands corresponded to Basidiomycete yeast genera. Although the DGGE profiles were similar, fungal sequence diversity also increased during the bioassay. Sequences highly similar to those of Cryptococcus increased in relative abundance during the bioassay, particularly in the suppressive samples. This study highlights the importance of using complementary approaches to molecular profiling of complex populations and provides the first report that basidiomycetous yeasts may be associated with the suppression of Pythium-induced diseases in peats.

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