A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments

Rastogi, Gurdeep; Tech, Jan J.; Coaker, Gitta; Leveau, Johan H. J.

doi:10.1016/j.mimet.2010.08.006

Cited by 91 publications

(63 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Nutrient broth (NB) was also tested for cultivation of the isolated bacteria, and a recovery rate similar to that achieved with the R2A medium was obtained, further proving that a high recovery rate was possible with the described method. This high percentage of cultivable bacteria with individual isolation in separate compartments was in contrast to plating of dilution series on R2A medium, which resulted in a percentage of cultivable bacteria of about 10% compared to total cell counts using a Thoma chamber and was consistent with other studies describing a similarly low percentage of cultivable bacteria from the phyllosphere of 0.1 to 10% (53,54).…”

Section: Resultssupporting

confidence: 91%

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

Stiefel

Zambelli

Vorholt

2013

Appl Environ Microbiol

View full text Add to dashboard Cite

In their natural environment, bacteria often behave differently than they do under laboratory conditions. To gain insight into the physiology of bacteria in situ, dedicated approaches are required to monitor their adaptations and specific behaviors under environmental conditions. Optical microscopy is crucial for the observation of fundamental characteristics of bacteria, such as cell shape, size, and marker gene expression. Here, fluidic force microscopy (FluidFM) was exploited to isolate optically selected bacteria for subsequent identification and characterization. In this study, bacteriochlorophyll-producing bacteria, which can be visualized due to their characteristic fluorescence in the infrared range, were isolated from leaf washes. Bacterial communities from the phyllosphere were investigated because they harbor genes indicative of aerobic anoxygenic photosynthesis. Our data show that different species of Methylobacterium express their photosystem in planta, and they show a distinct pattern of bacteriochlorophyll production under laboratory conditions that is dependent on supplied carbon sources.

show abstract

Section: Resultssupporting

confidence: 91%

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

Stiefel

Zambelli

Vorholt

2013

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…The contamination of phyllosphere derived microbial DNA with plant DNA is well known (Delmotte et al 2009;Rastogi et al 2010). While primers have been designed to overcome this problem for clone library construction (Chelius and Triplett 2001;Rastogi et al 2010), they resulted in our study in fewer and fainter DGGE bands and were thus not considered further. As expected, the rosette washings of cabinet grown A. thaliana plants showed a reduced DGGE profile in comparison to soil samples and were enriched in comparison to plants growing under sterile conditions (Suppl.…”

Section: Microbial Community Analysis On a Thaliana Leaf Surfacesmentioning

confidence: 99%

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves

Reisberg

Hildebrandt

Riederer

et al. 2011

Antonie van Leeuwenhoek

View full text Add to dashboard Cite

This study aimed to investigate whether the presence of trichomes as conspicuous physical attributes of the leaf surface affects the microbial community composition on Arabidopsis thaliana leaves. The A. thaliana ecotype Col-0 and its trichomeless gl1 mutant were grown in growth cabinets under climate-controlled conditions. The gl1 mutant showed a similar wax composition as the Col-0 wild type with slightly reduced amounts of C(29), C(31) and C(33) alkanes by GC/MS and GC/FID analyses. 120 bacterial isolates representing 39 bacterial genera were obtained from A. thaliana Col-0 leaf surfaces. Phylogenetic analysis of nearly full-length 16S rRNA sequences from 29 selected isolates confirmed their affiliation to the Proteobacteria (Alpha-, Beta-, Gamma-), Actinobacteria, Bacteroidetes and Firmicutes. The bacterial diversity on A. thaliana ecotype Col-0 and its gl1 mutant, devoid of trichomes, were further compared by denaturing gradient gel electrophoresis (DGGE). Banding patterns and sequencing of representative DGGE bands revealed the presence of phylotypes related to Sphingomonas (Alphaproteobacteria), Methylophilus (Betaproteobacteria) and Dyadobacter (Bacteroidetes) which are common phyllosphere inhabitants. Furthermore, wildtype and trichomeless mutant plants were exposed to outdoor conditions for 4-5 weeks. The DGGE gels showed only minor differences between the two plant lines, thus suggesting that trichomes per se do not affect bacterial diversity on Arabidopsis leaves under the experimental conditions tested.

show abstract

“…In present study, 16S rRNA genes were amplified from plant material and the choice of primer was made to avoid amplification of chloroplast 16S rRNA gene 23,24 . There are a wide variety of other primers available that vary in term of the product length, taxonomic power and usefulness 25,26 .…”

Section: Discussionmentioning

confidence: 99%

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Sanschagrin

Yergeau

2014

JoVE

View full text Add to dashboard Cite

One of the major questions in microbial ecology is "who is there?" This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of nextgeneration sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

show abstract

A PCR-based toolbox for the culture-independent quantification of total bacterial abundances in plant environments

Cited by 91 publications

References 26 publications

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

Isolation of Optically Targeted Single Bacteria by Application of Fluidic Force Microscopy to Aerobic Anoxygenic Phototrophs from the Phyllosphere

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Contact Info

Product

Resources

About