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Applied and Environmental Microbiology, December 1998, p. 5020-5022, Vol. 64, No. 12
Department of Agronomy, Center for the Study
of Nitrogen Fixation, University of Wisconsin-Madison, Madison,
Wisconsin 53706
Received 29 June 1998/Accepted 13 September 1998
The effects of antibiotic production on rhizosphere microbial
communities of field-grown Phaseolus vulgaris were assessed by using ribosomal intergenic spacer analysis. Inoculum strains of
Rhizobium etli CE3 differing only in trifolitoxin
production were used. Trifolitoxin production dramatically reduced the
diversity of trifolitoxin-sensitive members of the The great majority of microorganisms
in the environment remain unidentified because they are not culturable
with standard techniques (2, 14, 15, 23). The phylogenetic
diversity of soil bacteria observed by culture-independent methods is
enormous (4, 5, 17, 22). Ribosomal intergenic spacer
analysis (RISA) is used to assess microbial diversity in complex
systems, as well as estimate effects of disturbance on diversity
(5), such as the introduction of an antibiotic-producing
bacterium into soil. To date, nothing is known about the ability of
antibiotic production to affect microbial diversity in the environment
from a culture-independent perspective. In this study, we assessed microbial diversity changes in the rhizosphere of bean plants following
inoculation with bacterial strains that differ only in the ability to
produce a narrow-spectrum peptide antibiotic, trifolitoxin (TFX).
The Rhizobium TFX production phenotype reduces the number of
TFX-sensitive rhizobia in bean rhizospheres and enhances the ability of
a strain to limit root nodulation by TFX-sensitive Rhizobium
strains (12, 13, 18-20). By using the nearly isogenic strains of Rhizobium etli available that differ only in the
presence or absence of the TFX production phenotype, we assessed the
ability of the TFX production phenotype to alter microbial diversity in the rhizosphere by using RISA. As the taxonomic range of in vitro TFX
sensitivity is restricted to a specific group of members of the The inoculum strains used are derivatives of R. etli CE3.
CE3(pT2TFXK) and CE3(pT2TX3K) are nearly isogenic, TFX-producing and
-nonproducing strains, respectively (13). Culture
conditions, inoculation methods, antibiotic concentrations, seed
coating, bean cultivar, soil properties, and experimental design and
location were described previously (12).
To amplify RISA PCR products specific to the taxonomic group of
bacteria sensitive to TFX, a forward 16S ribosomal DNA (rDNA) primer
referred to as RB1 (5'-TGGTGACAGTGGGCAGCG-3') was designed. The specificity of RB1 for the TFX-sensitive bacteria was confirmed by
PCR analysis with template DNA of organisms within and outside this
group by using TR8 as the reverse primer (3, 11). Further evidence of RB1 specificity was obtained by using the Check Probe program from the Ribosomal Database Project (9).
Sixty-three days after planting, root sections from four plants per
plot were removed from soil, placed in 40 ml of sterile water, and
shaken for 30 min at 250 rpm at room temperature. Rhizosphere DNA was
extracted from 1 ml of root washes by using a soil DNA extraction kit
(Bio 101) and then further purified with Wizard columns (Promega).
RISA-PCR was performed as described previously (5). To
amplify rDNA from The RISA products obtained by using universal rDNA primers suggest that
the microbial diversity of rhizosphere soil is immense (5)
(Fig. 1). Many bands were observed in the
size range of 300 to 1,400 bp. Very few differences in the overall
microbial diversity were observed, regardless of the inoculum strain
used (Fig. 1). However, a significant difference between the RISA
patterns obtained with the uninoculated treatment and the two
inoculated treatments was observed. The RISA pattern from the
uninoculated rhizosphere shows a 450-bp fragment that is not present in
patterns from the rhizospheres inoculated with either CE3(pT2TFXK) or
CE3(pT2TX3K). This suggests that inoculation with R. etli
reduces the population of the microorganism(s) represented by the
450-bp fragment, while the TFX production phenotype has little
detectable effect on the overall microbial diversity.
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Bacterial Antibiotic Production on
Rhizosphere Microbial Communities from a Culture-Independent
Perspective
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ABSTRACT
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subdivision of
the class Proteobacteria with little apparent effect on
most microbes.
TEXT
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References
subdivision of the class Proteobacteria
(-Proteobacteria) (21), the specificity of the
TFX action on rhizosphere microbes was estimated.
-Proteobacteria sensitive to TFX, the
RB1 forward primer was used. The PCR conditions used to amplify rDNA
from TFX-sensitive -Proteobacteria were 94°C for
60 s; 30 cycles of 94°C for 15 s, 58°C for 15 s, and
72°C for 25 s; and extension at 72°C for 60 s. Fragments
common or specific to treatments were excised from the gel, extracted
in water, and reamplified by using the RB1 and TR8 primers. The
resulting 230 to 250-bp fragments were sequenced by dye termination
(5). Sequences from the RISA bands were compared with all
sequences in the databases by using BLAST (1).
View larger version (85K):
[in a new window]
FIG. 1.
Intergenic 16S to 23S rDNA patterns of dominant
microbial populations in the rhizosphere of beans inoculated with
R. etli CE3 strains that differ in TFX production in
Arlington, Wis., 1997. Lanes: 1, pure culture of R. etli
CE3; 2 and 3, rDNA bands from rhizosphere of uninoculated plants; 4 and
5, rDNA bands from rhizospheres inoculated with CE3(pT2TFXK) (the
TFX-producing strain); 6 and 7, rDNA bands from rhizospheres inoculated
with CE3(pT2TX3K) (the TFX-nonproducing strain); 8, molecular size
markers. The arrow indicates a 450-bp fragment absent in R. etli treatments. Each lane represents an independent replicate.
The values on the right are molecular sizes in base pairs.
When RB1 was used for amplification, the RISA pattern was considerably less complex than that obtained with 1406F (5) (Fig. 2). The complexity decreased even more dramatically when the rhizosphere was inoculated with CE3(pT2TFXK). There was very little difference between the patterns obtained with the uninoculated treatment and the treatment inoculated with CE3(pT2TX3K) (Fig. 2).
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A few bands from the -proteobacterial RISA gel were excised and
sequenced. Some bands were chosen that were common to all of the
treatments. Others were chosen because they were not found in the
rhizosphere of plants inoculated with CE3(pT2TFXK). All sequences
showed very high homology to the TFX-sensitive
-Proteobacteria, consistent with the specificity of the
forward primer used to amplify these fragments (Table
1).
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Previous studies have examined the culturable microbial community in the rhizosphere upon inoculation with an antibiotic-producing strain (7, 8, 10). All of these studies examined only the culturable microorganisms, which meant that only a small portion of the rhizosphere microbial community was examined. In addition, the effects observed on the cultured communities could not be attributed to antibiotic production, since undefined chemical mutants were used for comparison with the wild type (7, 8).
Thus, a culture-independent approach was used here to examine changes
in the bean rhizosphere after inoculation with two Rhizobium strains that differ only in antibiotic production. An uninoculated treatment was also included to determine the effects of
Rhizobium inoculation on the rhizosphere microbial
community. This approach also examined the effects on the taxonomically
defined group of TFX-sensitive microorganisms. Our results are
consistent with the taxonomic range of activity of TFX, since the
-proteobacterial RISA profile was considerably altered by the
TFX-producing strain, while the total microorganism RISA profile was
not substantially affected (Fig. 1 and 2).
Thus, TFX production has a significant effect on TFX-sensitive bacteria
in the rhizosphere of beans under agricultural conditions. This effect
is not noticeable with the total microbial RISA profile, as expected,
given the inability of TFX to inhibit most bacteria and given that the
-Proteobacteria are not common in soil (4, 5).
Although the culture bias has been removed from this work, PCR and
primer biases remain (6, 16). However, despite these potential biases, this report strongly suggests that a reduction in the
diversity of -Proteobacteria has occurred with TFX production.
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ACKNOWLEDGMENTS |
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Funds for this work were provided by USDA NRI grant 94-37-050767, USDA Risk Assessment Program grant 94-33120-0433, USEPA Risk Assessment Cooperative Agreement CR-822882-01-0, and University of Wisconsin-Madison College of Agricultural and Life Sciences Hatch project 5201.
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FOOTNOTES |
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* Corresponding author. Mailing address: University of Wisconsin-Madison, Department of Agronomy, 1575 Linden Drive, Madison, WI 53706. Phone: (608) 262-9824. Fax: (608) 262-5217. E-mail: triplett{at}facstaff.wisc.edu.
Present address: University of California-Riverside, Department of
Plant Pathology, Riverside, CA 92521.
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