Bacterial endophytes of the wildflowerCrocus albiflorusanalyzed by characterization of isolates and by a cultivation-independent approach

The presence and taxonomy of endophytic bacteria of the entire aerial parts of crocus (Crocus albiflorus), a wildflower native in the Alps, were investigated. A combination of plating of plant macerates, isolation and sequence identification of isolates, and direct 16S rDNA PCR amplification followed by whole-community fingerprinting (T-RFLP) and by construction of a bacterial clone library was used. The results clearly indicated that a wide range of bacteria from diverse phylogenetic affiliation, mainly γ-Proteobacteria and Firmicutes, live in association with plants of C. albiflorus. The community composition of the culturable component of the microflora was remarkably different from that of the clone library. Only three bacterial divisions were found in the culture collection, which represented 17 phylotypes, whereas six divisions were identified in the clonal analysis comprising 38 phylotypes. The predominant group in the culture collection was the low G + C Gram-positive group, whereas in the clone library, the γ-Proteobacteria predominated. Interestingly, the most prominent bacterium within the uncultured bacterial community was a pseudo monad closely related to a cold-tolerant Pseudomonas marginalis strain. The results suggest that Crocus supports a diverse bacterial microflora resembling the microbial communities that have been described for other plants and containing species that have not been described in association with plants.Key words: crocus, endophytes, 16S rRNA, 16S rDNA clone library, T-RFLP analysis, community analysis.

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A homologous comparison between mycoplasma and phytoplasma using 16S rDNA PCR amplification and RFLP analysis

Biodiversity Science ◽

10.17520/biods.2000014 ◽

2000 ◽

Vol 08 (1) ◽

pp. 103-105

Author(s):

HU Qin-Xue ◽

DENG Zhao-Qun ◽

LIU Sheng ◽

WANG Yao ◽

LIN Mu-Lan ◽

...

Keyword(s):

16S Rdna ◽

Pcr Amplification ◽

Rflp Analysis ◽

16S Rdna Pcr

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A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

Journal of Microbiological Methods ◽

10.1016/s0167-7012(03)00162-3 ◽

2003 ◽

Vol 55 (1) ◽

pp. 295-302 ◽

Cited By ~ 24

Author(s):

Jichan Jang ◽

Bongjoon Kim ◽

Jongho Lee ◽

Hongui Han

Keyword(s):

16S Rdna ◽

Rapid Method ◽

Rflp Analysis ◽

16S Rdna Pcr ◽

Pcr Rflp

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Identification of the Dominant Microbial Species of Spoiled Crisp Grass Carp (Ctenopharyngodon idellus C. et V.) and Grass Carp (Ctenopharyngodon idellus) Fillets during Cold Storage by Culture-Independent 16S rDNA Sequence Analysis

Journal of Food Protection ◽

10.4315/0362-028x.jfp-17-141 ◽

2017 ◽

Vol 81 (1) ◽

pp. 84-92 ◽

Cited By ~ 2

Author(s):

Lin Li ◽

Ziqiang Pan ◽

Zhihua Shen

Keyword(s):

16S Rdna ◽

Clone Library ◽

Grass Carp ◽

Rflp Analysis ◽

Ctenopharyngodon Idellus ◽

Total Volatile ◽

16S Rdna Clone Library ◽

Pseudomonas Spp ◽

Rdna Sequencing ◽

Rdna Clone

ABSTRACT A new freshwater cultivation species, crisp grass carp (CGC; Ctenopharyngodon idellus C. et V.) has a special texture and is popular with consumers; thus, we should pay close attention to its storage conditions and bacterial degradation. CGC and grass carp (GC; Ctenopharyngodon idellus) were commercialized as fillets and subsequently stored at 4 and 8°C. Microbial growth parameters (total viable counts, psychrotrophic bacteria, and Pseudomonas spp.), physicochemical data (pH and total volatile base nitrogen), and sensory analysis were monitored during the storage period. Dominant microorganisms were identified using a 16S rDNA clone library and restriction fragment length polymorphism (RFLP) analysis after the fillets had spoiled completely. The results showed that Pseudomonas spp. lagged behind the psychrotrophic population and the total viable counts initially and increased more rapidly after storage for 2 days. Total volatile base nitrogen contents were found to increase with storage time in both species, coinciding with ongoing microbial change. The pH results obtained for both species during storage showed an overall increase at the end of storage. Sensory evaluation showed a shelf life of 3 and 6 days for both species at 8 and 4°C, respectively. RFLP analysis of the 16S rDNA clone library revealed that there were seven and five distinct RFLP pattern groups in the microflora of the spoiled CGC and GC fillets, respectively. Through RFLP patterns and 16S rDNA sequencing from the clones, it was determined that CGC fillets stored at 4°C were dominated by Pseudomonas spp. at the point of sensory rejection, whereas GC fillets were dominated by populations affiliated with Pseudomonas sp., Acinetobacter sp., and Aeromonas sp.

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Molecular identification of Helicobacter DNA present in human colorectal adenocarcinomas by 16S rDNA PCR amplification and pyrosequencing analysis

Journal of Medical Microbiology ◽

10.1099/jmm.0.46122-0 ◽

2005 ◽

Vol 54 (11) ◽

pp. 1031-1035 ◽

Cited By ~ 31

Author(s):

Niclas Grahn ◽

Mounira Hmani-Aifa ◽

Karin Fransén ◽

Peter Söderkvist ◽

Hans-Jürg Monstein

Keyword(s):

16S Rdna ◽

Dna Sequences ◽

Pcr Amplification ◽

Limited Information ◽

Rdna Sequences ◽

Biopsy Specimens ◽

16S Rdna Pcr ◽

H Pylori ◽

Species Specific ◽

Pyrosequencing Analysis

Seroepidemiological studies have indicated that Helicobacter pylori infection might be a possible risk factor for colorectal adenocarcinoma (CRC) development. However, limited information is available as to whether or not Helicobacter species are present in CRC tissues. In this study the presence of Helicobacter DNA in 77 CRC biopsies was investigated by means of a Helicobacter species-specific 16S rDNA PCR assay and real-time DNA pyrosequencing of the 16S rDNA variable V3 region. Pyrosequencing revealed the presence of Helicobacter DNA sequences in 21 of 77 biopsy specimens (27 %). 16S rDNA sequences corresponding to H. pylori 26695 and H. pylori J99 were most commonly found. Intriguingly, one sequence belonged to Helicobacter mustelae, previously identified in ferrets. No significant correlations were found in the prevalence of Helicobacter DNA between colon and rectum tumour biopsies (P = 0.815), nor between Dukes’ classes A/B and C/D (P = 0.262). 16S rDNA PCR amplification combined with pyrosequencing analysis of 16S rDNA variable V3 regions provides a powerful molecular tool to identify Helicobacter species in human biopsy specimens.

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rpoB-Based Microbial Community Analysis Avoids Limitations Inherent in 16S rRNA Gene Intraspecies Heterogeneity

Applied and Environmental Microbiology ◽

10.1128/aem.66.8.3376-3380.2000 ◽

2000 ◽

Vol 66 (8) ◽

pp. 3376-3380 ◽

Cited By ~ 299

Author(s):

Ingela Dahllöf ◽

Harriet Baillie ◽

Staffan Kjelleberg

Keyword(s):

Microbial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

16S Rdna ◽

Pattern Analysis ◽

Community Analysis ◽

Microbial Community Analysis ◽

Rrna Gene ◽

Community Pattern ◽

16S Rdna Pcr

ABSTRACT Contemporary microbial community analysis frequently involves PCR-amplified sequences of the 16S rRNA gene (rDNA). However, this technology carries the inherent problem of heterogeneity between copies of the 16S rDNA in many species. As an alternative to 16S rDNA sequences in community analysis, we employed the gene for the RNA polymerase beta subunit (rpoB), which appears to exist in one copy only in bacteria. In the present study, the frequency of 16S rDNA heterogeneity in bacteria isolated from the marine environment was assessed using bacterial isolates from the red alga Delisea pulchra and from the surface of a marine rock. Ten strains commonly used in our laboratory were also assessed for the degree of heterogeneity between the copies of 16S rDNA and were used to illustrate the effect of this heterogeneity on microbial community pattern analysis. The rock isolates and the laboratory strains were also used to confirm nonheterogeneity of rpoB, as well as to investigate the versatility of the primers. In addition, a comparison between 16S rDNA and rpoB PCR-DGGE (denaturing gradient gel electrophoresis)-based community analyses was performed using a DNA mixture of nine isolates from D. pulchra. Eight out of 14 isolates from D. pulchra, all rock isolates, and 6 of 10 laboratory strains displayed multiple bands for 16S rDNA when analyzed by DGGE. There was no indication of heterogeneity for either the rock isolates or the laboratory strains when rpoB was used for PCR-DGGE analysis. Microbial community pattern analysis using 16S rDNA PCR-DGGE showed an overestimation of the number of laboratory strains in the sample, while some strains were not represented. Therefore, the 16S rDNA PCR-DGGE-based community analysis was proven to be severely limited by 16S rDNA heterogeneity. The mixture of isolates from D. pulchra proved to be more accurately described using rpoB, compared to the 16S rDNA-based PCR-DGGE.

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