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Applied and Environmental Microbiology, February 1999, p. 374-380, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Analysis of nifH Gene Pool Complexity in
Soil and Litter at a Douglas Fir Forest Site in the Oregon
Cascade Mountain Range
F.
Widmer,1,*
B. T.
Shaffer,2
L. A.
Porteous,3 and
R.
J.
Seidler3
National Research
Council,1
Dynamac
Corp.,2 and
National Health and
Environmental Effects Research Laboratory, Western Ecology Division,
U.S. Environmental Protection Agency,3
Corvallis, Oregon 97333-4902
Received 29 June 1998/Accepted 28 October 1998
Nitrogen-fixing microbial populations in a Douglas fir forest on
the western slope of the Oregon Cascade Mountain Range were analyzed.
The complexity of the nifH gene pool (nifH is
the marker gene which encodes nitrogenase reductase) was assessed by
performing nested PCR with bulk DNA extracted from plant litter and
soil. The restriction fragment length polymorphisms (RFLPs) of PCR
products obtained from litter were reproducibly different than the
RFLPs of PCR products obtained from the underlying soil. The
characteristic differences were found during the entire sampling period
between May and September. RFLP analyses of cloned nifH PCR
products also revealed characteristic patterns for each sample type.
Among 42 nifH clones obtained from a forest litter library
nine different RFLP patterns were found, and among 64 nifH
clones obtained from forest soil libraries 13 different patterns were
found. Only two of the patterns were found in both the litter and the
soil, indicating that there were major differences between the
nitrogen-fixing microbial populations. A sequence analysis of clones
representing the 20 distinct patterns revealed that 19 of the patterns
had a proteobacterial origin. All of the nifH sequences
obtained from the Douglas fir forest litter localized in a distinct
phylogenetic cluster characterized by the nifH sequences of
members of the genera Rhizobium, Sinorhizobium,
and Azospirillum. The nifH sequences obtained
from soil were found in two additional clusters, one characterized by
sequences of members of the genera Bradyrhizobium, Azorhizobium, Herbaspirillum, and
Thiobacillus and the other, represented by a single
nifH clone, located between the gram-positive bacteria and
the cyanobacteria. Our results revealed the distinctness of the
nitrogen-fixing microbial populations in litter and soil in a Douglas
fir forest; the differences may be related to special requirements for
degradation and mineralization processes in the plant litter.
*
Corresponding author. Present address: Swiss Federal
Institute of Technology (ETH-Zürich), Institute of Terrestrial
Ecology, Soil Biology, Grabenstrasse 3, CH-8952, Schlieren,
Switzerland. Phone: (41) 1 633-6042. Fax: (41) 1 633-1122. E-mail:
franco.widmer{at}ito.umnw.ethz.ch.
Applied and Environmental Microbiology, February 1999, p. 374-380, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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