PNNL-13501 - Pacific Northwest National Laboratory

Extraction of community total RNA
RT-PCR of 16S rRNA
(1-3 ng template)
Restriction digest with HhaI
Automated sequencer sizes
terminal fragments
of dominant 16S rRNAs
Analysis of electropherogram
Similarity dendrogram
to compare treatments
Detector
Figure 1. Analysis of microbial communities using terminal
restriction fragment analysis
microbial community structure were deemed more
important than specific phylogenetic identifications for
this project. Terminal restriction fragment analysis
typically identifies approximately 30 to 60 of the
dominant ribotypes in the microbial community of a
particular sample.
In fiscal year 2000, we focused our analysis on ribosomal
RNA (rRNA) to determine if greater differences could be
observed between the experimental treatments. rRNA
levels in bacteria vary depending on how metabolically
active the cells are, whereas rDNA levels per cell are
invariant. Reverse transcriptase polymerase chain
reaction (RT-PCR) is more sensitive to inhibition by soil
humic acids, so effort was devoted to maximizing rRNA
purity in order to obtain robust rRNA terminal restriction
fragment analysis profiles. In addition, terminal
restriction fragment analysis was performed on each end
of the rRNA molecule (versus one end of the rDNA
molecule in fiscal year 1999) to improve assay
robustness. Software was used to simultaneously
compare the edited electropherograms and construct
similarity dendrograms using heirarchical cluster analysis.
Results and Accomplishments
The following major results were found when analyzing
rDNA. Approximately 80% of the rhizosphere soil
ribotypes were not detected in isolates from the
rhizosphere soil, demonstrating that most of the
microorganisms that most heavily colonize the
rhizosphere were not cultured and were only detected by
molecular analysis. rDNA terminal restriction fragment
98 FY 2000 Laboratory Directed Research and Development Annual Report
analysis profiles in rhizosphere soil were nearly identical
regardles of tillage or N level (Figure 2, panel A).
Terminal restriction fragment analysis profiles in soil
without wheat plants were more variable, with a greater
effect from tillage than from nitrogen level. The highest
order difference was between rhizosphere and no-plant
soils.
A
B
Conv till, low N
Conv till, high N
No-till, high N
No-till, low N
Conv till, low N
No-till, high N
Conv till, high N
No-till, low N
Conv till, low N
Conv till, high N
No-till, high N
No-till, low N
Conv till, low N
Conv till, high N
No-till, low N
No-till, high N
}
}
}
}
Soil without wheat
Rhizosphere soil
Soil without wheat
Rhizosphere soil
Figure 2. Representative dendrograms derived from
statistical analysis of terminal restriction fragment analysis
profiles showing the relationship of microbial communities
in rhizosphere soil and soil without wheat plants under four
soil management practices. Two soils that are horizontally
connected by shorter lines are more similar than two soils
horizontally connected by longer lines. Panel A, using
rDNA; panel B, using rRNA. Conv, conventional; N,
nitrogen.
These results suggested that the wheat root influences the
structure of the microbial population and does so to a
greater degree than soil management practices (that were
examined).
In rhizosphere soil, profiles showed more variability
between the four treatments than profiles generated from
rDNA. In contrast, in soil without wheat, the rRNA
terminal restriction fragment analysis profiles differed
little from those seen using rDNA terminal restriction
fragment analysis and there was less variability than in
the rhizosphere soils. In both types of soil, tillage showed
a greater effect than nitrogen level. rRNA also yielded a
greater difference between rhizosphere and no-plant soils,
as evidenced by the more horizontal profile in Figure 2.
As in the rDNA terminal restriction fragment analysis
analysis, the highest order difference was between
rhizosphere and no-plant soils. Overall, the rRNA
terminal restriction fragment analysis results confirm the
general conclusions from the rDNA analysis: the wheat
root strongly influences the microbial population structure
and overrides the soil management practices that were
examined.