Online citations, reference lists, and bibliographies.

A PCR-based Toolbox For The Culture-independent Quantification Of Total Bacterial Abundances In Plant Environments.

Gurdeep Rastogi, J. J. Tech, G. Coaker, J. Leveau
Published 2010 · Medicine, Biology
Cite This
Download PDF
Analyze on Scholarcy
A major obstacle in the culture-independent estimation of the abundance of bacteria associated with plants is contamination with plant organelles, which precludes the use of universal rRNA bacterial primers in quantitative PCR applications. We present here a PCR-based method that allows a priori determination of the degree of chloroplast and mitochondrial contamination in DNA samples from plant environments. It is based on differential digestibility of chloroplast, mitochondrial and bacterial small subunit rRNA gene amplicons with the restriction enzymes AfeI and BbvCI. Using this method, we demonstrated for field-grown lettuce plants that even a gentle washing protocol, designed to recover the microbial community and its metagenome from the leaf surface, resulted in substantial contamination with chloroplast DNA. This finding cautions against the use of universal primer pairs that do not exclude chloroplast DNA from amplification, because they risk overestimation of bacterial population sizes. In contrast, contamination with mitochondrial 18S rRNA was minor in the lettuce phyllosphere. These findings were confirmed by real-time PCR using primer sets specific for small subunit rRNA genes from bacteria, chloroplasts, and mitochondria. Based on these results, we propose two primer pairs (534f/783r and mito1345f/mito1430r) which between them offer an indirect means of faithfully estimating bacterial abundances on plants, by deduction of the mito1345f/mito1430r-based mitochondrial count from that obtained with 534f/783r, which amplifies both bacterial and mitochondrial DNA but excludes chloroplast. In this manner, we estimated the number of total bacteria on most leaves of field-grown lettuce to be between 10(5) and 10(6) g(-1) of leaf, which was 1-3 orders of magnitudes higher than the number of colony-forming units that were retrieved from the same leaf surfaces on agar plates.
This paper references
The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves.
Amanda J. Redford (2010)
Improved group-specific PCR primers for denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities
Martin Mühling (2008)
Molecular Cloning: A Laboratory Manual
J. Sambrook (1983)
Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes
Jeremy A. Frank (2008)
Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots.
M. Sakai (2004)
16S/23S rRNA sequencing
D. J. Lane (1991)
Why do chloroplasts and mitochondria contain so many copies of their genome?
A. Bendich (1987)
The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L.
M. Chelius (2001)
Endophytic Bacterial Diversity in Rice (Oryza sativa L.) Roots Estimated by 16S rDNA Sequence Analysis
L. Sun (2007)
Profiling complex microbial populations by denaturing gradient gel electrophoresis analysis of polym
Gerard Muyzer (1993)
Cultivating the uncultured: limits, advances and future challenges
Karine Alain (2009)
Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes.
A. Neef (1998)
Assessment of Soil Microbial Community Structure by Use of Taxon-Specific Quantitative PCR Assays
N. Fierer (2005)
Bacterial Succession on the Leaf Surface: A Novel System for Studying Successional Dynamics
Amanda J. Redford (2009)
Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing
Uma Shankar Sagaram (2009)
Nucleic acid techniques in bacterial systematics
E. Stackebrandt (1991)
DGGE and real-time PCR analysis of lactic acid bacteria in bacterial communities of the phyllosphere of lettuce.
J. Zwielehner (2008)
Prevalence of the Chloroflexi-Related SAR202 Bacterioplankton Cluster throughout the Mesopelagic Zone and Deep Ocean
R. M. Morris (2004)
Characterization of the dynamics of initial bacterial colonization of nonconserved forage in the bovine rumen.
J. Edwards (2007)
Bacterial Diversity in Tree Canopies of the Atlantic Forest
M. R. Lambais (2006)
Isolation of maize soil and rhizosphere bacteria with antagonistic activity against Aspergillus flavus and Fusarium verticillioides.
J. Palumbo (2007)
Changes in the phyllosphere community of the resurrection fern, Polypodium polypodioides, associated with rainfall and wetting.
Evelyn F. Jackson (2006)
Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.
Cindy J. Smith (2009)
Frequency, Size, and Localization of Bacterial Aggregates on Bean Leaf Surfaces
J. Monier (2004)
Ultraviolet Radiation Alters Maize Phyllosphere Bacterial Diversity
H. Kadivar (2003)
A revised medium for the growth and bioassay with tobacco tissue culture
T. Murashige (1962)
Microbial phyllosphere populations are more complex than previously realized
C. Yang (2001)
Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica
Michael Fürnkranz (2008)
Evolutionary relationships among cyanobacteria and green chloroplasts.
S. Giovannoni (1988)

This paper is referenced by
Characterization of Bacterial and Fungal Communities in Soils under Different Farming Systems. The Cacao Plantation in Sulawesi Island—Indonesia
I. N. Suwastika (2019)
A Duplex PCR-Based Assay for Measuring the Amount of Bacterial Contamination in a Nucleic Acid Extract from a Culture of Free-Living Protists
Alan O Marron (2013)
Geographic and Host-Associated Variations in Bacterial Communities on the Floret Surfaces of Field-Grown Broccoli
Min-Soo Kim (2018)
Design and application of specific 16S rDNA-targeted primers for assessing endophytic diversity in Dendrobium officinale using nested PCR-DGGE
Jie Yu (2013)
Leaf microbiota in an agroecosystem: spatiotemporal variation in bacterial community composition on field-grown lettuce
Gurdeep Rastogi (2012)
Progress in cultivation-independent phyllosphere microbiology
Thomas Müller (2014)
Impact of solar radiation exposure on phyllosphere bacterial community of red-pigmented baby leaf lettuce.
Pilar Truchado (2017)
Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves
Eva E. Reisberg (2011)
Plant Species and Heavy Metals Affect Biodiversity of Microbial Communities Associated With Metal-Tolerant Plants in Metalliferous Soils
Sławomir Borymski (2018)
Response of Microbial Populations on the Creeping Bentgrass Phyllosphere to Periodic Fungicide Applications
Joseph R. Doherty (2017)
Omics approaches on fresh-cut lettuce reveal global molecular responses to sodium hypochlorite and peracetic acid treatment.
Loretta Daddiego (2018)
Dynamic and Assembly of Epiphyte and Endophyte Lactic Acid Bacteria During the Life Cycle of Origanum vulgare L.
Erica Pontonio (2018)
Litter traits and rainfall reduction alter microbial litter decomposers: the evidence from three Mediterranean forests.
S. Pereira (2019)
Scales of bacterial interactions on the leaf surface
Daniel S Esser (2017)
Bacteria-mediated effects of antibiotics on Daphnia nutrition.
E. Gorokhova (2015)
Leaf-litter-associated fungi and bacteria along temporal and environmental gradients in boreal streams
Jenny Bergfur (2014)
Life of Microbes on Aerial Plant Parts
Johan H J Leveau (2015)
Artificial Surfaces in Phyllosphere Microbiology.
Hung Kim Doan (2015)
Identification of the barley phyllosphere and the characterisation of manipulation means of the bacteriome against leaf scald and powdery mildew
C. Gravouil (2012)
16S rRNA Gene Primer Validation for Bacterial Diversity Analysis of Vegetable Products.
Miyo Nakano (2018)
Development of a simple root model to study the effects of single exudates on the development of bacterial community structure.
Maren Ziegler (2013)
Assessment of Rice Root‐Associated Bacteria
Pablo Rodrigo Hardoim (2013)
Salinity and macrophyte drive the biogeography of the sedimentary bacterial communities in a brackish water tropical coastal lagoon.
Pratiksha Behera (2017)
Microbiome analysis of a microalgal mass culture growing in municipal wastewater in a prototype OMEGA photobioreactor.
Laura T. Carney (2014)
Bacterial communities protect the alga Microchloropsis salina from grazing by the rotifer Brachionus plicatilis
Carolyn L. Fisher (2019)
Diversidade de cianobactérias na filosfera da Mata Atlântica do estado de São Paulo
Escola Superior de Agricultura (2011)
Caterpillars lack a resident gut microbiome
Tobin J. Hammer (2017)
Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid (LNA) oligonucleotide-PCR clamping and 454 pyrosequencing
Yu Zhen-hua (2016)
Plant Genotype Influences Physicochemical Properties of Substrate as well as Bacterial and Fungal Assemblages in the Rhizosphere of Balsam Poplar
Karelle Rheault (2020)
Single-step treatment of primary effluent by Galdieria sulphuraria: Removal of biochemical oxygen demand, nutrients, and pathogens
D. Tchinda (2019)
Season, Irrigation, Leaf Age, and Escherichia coli Inoculation Influence the Bacterial Diversity in the Lettuce Phyllosphere
Thomas Rhys Williams (2013)
Effects of Systemic Pesticides Imidacloprid and Metalaxyl on the Phyllosphere of Pepper Plants
Constantinos Moulas (2013)
See more
Semantic Scholar Logo Some data provided by SemanticScholar