PNNL-13501 - Pacific Northwest National Laboratory

Study Control Number: PN99064/1392
Role of Unculturable Bacteria in the Wheat Rhizosphere
Fred J. Brockman, Harvy Bolton
An improved understanding of plant-microbe interactions in the rhizosphere is important to DOE programs aimed at
understanding how plant-soil systems may be manipulated to increase productivity of crops (improved nutrient supply,
increased control of pathogens) and potentially increase carbon sequestration in soils.
Project Description
The objective of this project was to develop and employ
nucleic acid approaches for rapid detailed analysis of
microbial community structure and function in the
rhizosphere. Because only a very small percentage of
microorganisms are successfully cultured, improved
approaches are needed to answer these critical research
questions: What rhizosphere microorganisms are
dominant in the community but uncultured? How does
rhizosphere community structure vary with soil
management practices (e.g., high versus low fertilization
rate, no-till versus conventional tillage)? This project
extracted DNA and RNA from the microbial communities
in rhizosphere soil and soil without plants under four soil
management regimes, amplified the corresponding 16S
targets, analyzed the community nucleic acids by terminal
restriction fragment analysis, and statistically analyzed
the resulting profiles by heirarchical cluster analysis. The
results indicate that plant effects were greater than tillage
effects and that nitrogen level had little to no effect.
Terminal restriction fragment analysis is a rapid and
robust means of fingerprinting the set of dominant and
largely uncultured microorganisms in the microbial
community.
Introduction
The rhizosphere is the region in and around roots that is
influenced by the plant. Approximately 10 to 25% of the
total carbon fixed by the plant is released into the
rhizosphere, and sloughing root cells also provide high
levels of other microbial nutrients. The rhizosphere
microbial community is important in plant health and
productivity, including nitrogen fixation, mycorrhizae,
biocontrol of plant pathogens, and plant-growth
promoting bacteria. However, the composition of these
microbial communities and the factors that determine
community structure and metabolism (plant and microbial
signaling, physical and geochemical heterogeneity) are
largely unknown. As with other microbial ecosystems,
fewer than 1 to 5% of resident microorganisms can be
cultured in the laboratory. Hence, only a small fraction of
the total rhizosphere microbial community has been
adequately described. This leaves a considerable gap in
our knowledge of rhizosphere structure and function.
Approach
A growth chamber study was conducted with soil
(homogenized from 0 to 10 cm) from no-till and
conventionally tilled fields. Each soil was supplemented
with a low (8 kg nitrogen/hectare) and a high (80 kg
nitrogen/hectare) fertilization rate. Winter wheat
(12 seeds) was planted to 5 replicate pots of each of the 4
treatments and grown with spring photoperiod and
temperature regimes. Pots without wheat plants were
maintained under identical moisture, photoperiod, and
temperature regimes to provide a bulk soil microbial
community for comparison to the rhizosphere soil
microbial community. Six weeks after germination,
rhizosphere soil was isolated from each pot containing
wheat plants and soil was sampled from pots without
wheat. Soil from each pot was plated to isolate all
morphotypes of culturable aerobic heterotrophic bacteria,
and DNA and RNA was extracted and purified from soil
in each pot.
Extracted DNA was amplified using the polymerase chain
reaction (PCR) and eubacterial 16S ribosomal DNA
(rDNA) primers with one primer labeled with a fluor
(Figure 1). The amplified rDNA was subjected to a
restriction enzyme and the labeled 16S rDNA terminal
fragments separated on a DNA sequencing gel. This
approach was used to characterize each cultured isolate
and the community as a whole. For the community, the
resulting electropherogram shows the size of each
fragment in base pairs for the dominant bacterial
ribotypes in the sample. This approach, terminal
restriction fragment analysis, was selected over
construction, analysis, and sequencing of clone libraries
because it is much faster and because fingerprints of
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