Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens

2012 ◽  
Vol 58 (5) ◽  
pp. 563-571 ◽  
Author(s):  
Robert A. Quinn ◽  
Roselynn M.W. Stevenson
Keyword(s):  
Organ Culture ◽  
Gel Electrophoresis ◽  
Lake Trout ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Coho Salmon ◽  
Simultaneous Detection ◽  
Aeromonas Salmonicida ◽  
Band Pattern ◽  
Yersinia Ruckeri ◽  
Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon ( Oncorhynchus kisutch ) with endemic A. salmonicida and from cultured lake trout ( Salvelinus namaycush ) were tested by PCR–DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR–DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR–DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens , Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR–DGGE detection of this bacterium from mucus was possible. PCR–DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.

scholarly journals Assessment of Genotypic Diversity of Antibiotic-Producing Pseudomonas Species in the Rhizosphere by Denaturing Gradient Gel Electrophoresis

2005 ◽  
Vol 71 (2) ◽  
pp. 993-1003 ◽  
Author(s):  
M. Bergsma-Vlami ◽  
M. E. Prins ◽  
M. Staats ◽  
J. M. Raaijmakers
Keyword(s):  
Sugar Beet ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Phylogenetic Analyses ◽  
Simultaneous Detection ◽  
Genotypic Diversity ◽  
Plant Diseases ◽  
Potato Plants ◽  
Dgge Analysis ◽  
Gradient Gel Electrophoresis

ABSTRACT The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD + reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD + genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD + genotypes directly in rhizosphere samples with a detection limit of approximately 5 × 103 CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD + genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD + isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD + genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD + genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.

Allelopathic and Medicinal plant. 26. Millettia speciosa

2020 ◽  
Vol 51 (2) ◽  
pp. 125-146
Author(s):  
Nasiruddin Nasiruddin ◽  
Yu Zhangxin ◽  
Ting Zhao Chen Guangying ◽  
Minghui Ji
Keyword(s):  
Community Structure ◽  
Medicinal Plant ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Wiener Index ◽  
Pseudomonas Spp ◽  
Evenness Index ◽  
Gradient Gel Electrophoresis ◽  
Rhizosphere Pseudomonas

We grew cucumber in pots in greenhouse for 9-successive cropping cycles and analyzed the rhizosphere Pseudomonas spp. community structure and abundance by PCR-denaturing gradient gel electrophoresis and quantitative PCR. Results showed that continuous monocropping changed the cucumber rhizosphere Pseudomonas spp. community. The number of DGGE bands, Shannon-Wiener index and Evenness index decreased during the 3rd cropping and thereafter, increased up to the 7th cropping, however, however, afterwards they decreased again. The abundance of Pseudomonas spp. increased up to the 5th successive cropping and then decreased gradually. These findings indicated that the structure and abundance of Pseudomonas spp. community changed with long-term cucumber monocropping, which might be linked to soil sickness caused by its continuous monocropping.

scholarly journals Impact of Intensive Land-Based Fish Culture in Qingdao, China, on the Bacterial Communities in Surrounding Marine Waters and Sediments

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Qiufen Li ◽  
Yan Zhang ◽  
David Juck ◽  
Nathalie Fortin ◽  
Charles W. Greer
Keyword(s):  
Bacterial Communities ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Fish Culture ◽  
Fish Ponds ◽  
Marine Area ◽  
Gradient Gel Electrophoresis ◽  
Reducing Bacteria ◽  
The Impact ◽  
Nitrate Reducing Bacteria

The impact of intensive land-based fish culture in Qingdao, China, on the bacterial communities in surrounding marine environment was analyzed. Culture-based studies showed that the highest counts of heterotrophic, ammonium-oxidizing, nitrifying, and nitrate-reducing bacteria were found in fish ponds and the effluent channel, with lower counts in the adjacent marine area and the lowest counts in the samples taken from 500 m off the effluent channel. Denaturing gradient gel electrophoresis (DGGE) analysis was used to assess total bacterial diversity. Fewer bands were observed from the samples taken from near the effluent channel compared with more distant sediment samples, suggesting that excess nutrients from the aquaculture facility may be reducing the diversity of bacterial communities in nearby sediments. Phylogenetic analysis of the sequenced DGGE bands indicated that the bacteria community of fish-culture-associated environments was mainly composed of Flavobacteriaceae, gamma- and deltaproteobacteria, including generaGelidibacter, Psychroserpen, Lacinutrix,andCroceimarina.

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.

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