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Applied and Environmental Microbiology, December 1998, p. 4944-4949, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection and Isolation of Novel
Rhizopine-Catabolizing Bacteria from the Environment
Brian B. McSpadden
Gardener1,2,3 and
Frans J.
de
Bruijn2,3,4,*
Department of Botany and Plant
Pathology,1
MSU-DOE Plant Research
Laboratory,2
Department of
Microbiology,4 and
NSF Center for
Microbial Ecology,3 Michigan State
University, East Lansing, Michigan 48824
Received 1 June 1998/Accepted 27 September 1998
 |
ABSTRACT |
Microbial rhizopine-catabolizing (Moc) activity was detected in
serial dilutions of soil and rhizosphere washes. The activity observed
generally ranged between 106 and 107 catabolic
units per g, and the numbers of nonspecific culture-forming units were
found to be approximately 10 times higher. A diverse set of 37 isolates
was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to
contain DNA sequences homologous to the known mocA,
mocB, and mocC genes of Sinorhizobium
meliloti L5-30. Twenty-one of the isolates could utilize an SI
preparation as the sole carbon and nitrogen source for growth. Partial
sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains
indicated that five distinct bacterial genera
(Arthrobacter, Sinorhizobium,
Pseudomonas, Aeromonas, and
Alcaligenes) were represented in this set. Only 6 of these
21 isolates could catabolize
3-O-methyl-scyllo-inosamine under standard
assay conditions. Two of these, strains D1 and R3, were found to have
16S rDNA sequences very similar to those of Sinorhizobium
meliloti. However, these strains are not symbiotically effective
on Medicago sativa, and DNA sequences homologous to the
nodB and nodC genes were not detected in
strains D1 and R3 by Southern hybridization analysis.
| |
INTRODUCTION |
Rhizopines are inositol derivatives
synthesized in legume nodules induced by specific members of the
Rhizobiaceae (14, 31). The first rhizopine was
isolated from alfalfa nodules infected with Sinorhizobium
meliloti L5-30 (28). The structure of this compound was
determined to be 3-O-methyl-scyllo-inosamine
(MSI) (13). Genes on the large symbiotic plasmid of strain
L5-30 were determined to be involved in the synthesis and catabolism of
this compound (12). Transposon mutagenesis and subsequent
DNA sequence analysis of the rhizopine catabolism (moc)
locus from this strain revealed the presence of four open reading
frames (ORFs) involved in the catabolism of MSI (24). Three
of these ORFs, mocA, mocB, and mocC,
have been found to be sufficient to confer microbial rhizopine-catabolizing (Moc) activity onto otherwise Moc
strains of S. meliloti (25). DNA sequences
homologous to these moc genes were not observed in a broad
screen of known soil and rhizosphere bacteria (24), and only
members of the Rhizobiaceae have been reported to be
Moc+ (14).
Some organic molecules may be used to specifically promote the growth
and metabolic activities of soil and rhizosphere bacteria capable of
utilizing them as growth substrates (5, 6, 13, 18, 23-25).
Several recent reports have shown that these specific nutritional
mediators can enrich for bacteria capable of utilizing them as growth
substrates in soil and on plant leaves and roots (3, 4, 7, 10, 20,
27, 29, 33-35). However, the potential of nutritional mediators
to promote the activities of target microbial populations may be
limited by a variety of factors, including the relative abundance of
nontarget microbes capable of catabolizing the compound. While several
reports have indicated that other proposed nutritional mediators can be
catabolized by a variety of indigenous bacteria (5, 15, 16,
19), no previous study has directly investigated rhizopine
catabolism in the environment. Here we report the detection, isolation,
and enumeration of previously unknown rhizopine-catabolizing bacteria from the environment and discuss the implications of their existence for the use of rhizopine as a selective nutritional mediator.
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MATERIALS AND METHODS |
Soil and rhizosphere sampling.
Samples of a Michigan sandy
loam were obtained from a followed plot on the Michigan State
University Crop and Soil Sciences Research Farm, East Lansing. The plot
had been left fallow for 5 years prior to these experiments and was
covered by a variety of annual and perennial plants, including
nodulated alfalfa. In September of 1995, approximately 50 kg was
removed from the top 12 in. of the soil and transported to a research
greenhouse for mixing and storage. Rocks and wooden debris were removed
as the soil was homogenized in a rotary mixer. This soil was allowed to
air dry and was stored at room temperature until use.
For the initial detection and quantification of rhizopine catabolic
activities, aliquots of this soil were placed in plastic pots,
moistened, planted with Medicago sativa var. Cardinal, and incubated in a growth chamber (12 h in the light, 12 h in the dark, 22°C). Soil and rhizosphere samples (<100 mg each) were taken
after 1 and 3 weeks of incubation. Samples were placed in 5 ml of
distilled water in 15-ml Sarstedt tubes. Bacteria were dislodged by
alternating 10-s treatments as follows: vortexing, sonication (tube
placed in Ultrasonik cleaning bath; NEY, Inc., Bloomfield,
Conn.), vortexing, sonication, and final vortexing. They were serially
diluted in distilled water, and 50-µl aliquots were inoculated into 1 ml of the catabolism assay mixtures.
Preparation of nodule extracts and catabolism assays.
Extracts containing MSI were obtained from alfalfa nodules induced by
S. meliloti L5-30 under gnotobiotic conditions, as described previously (23). Five grams of nodules was crushed with a
mortar and pestle in 25 ml of distilled water. The suspension was
centrifuged at 20,000 × g for 20 min to clarify the
solution. The supernatant was filter sterilized, aliquoted, and kept
frozen at 
20°C. The total solute concentration of these 1×
extracts was determined gravimetrically to be 0.5% (wt/vol), and the
concentration of MSI in the extracts was estimated to be 0.01%
(wt/vol) by high-voltage paper electrophoresis (HVPE) analysis (see
below). Preparations containing synthetic scyllo-inosamine
(SI) were provided by R. Hollingsworth (Michigan State University
Department of Biochemistry). myo-Inositol was obtained
commercially (Sigma Chemical Co., St. Louis, Mo.).
The rhizopine catabolism assay used to define Moc activity was similar
to that described previously (
23). Assay mixtures
contained
0.3× nodule extract in 1× BGTS (25 mM KH
2PO
4
[pH 7.3],
10 mM NaCl, MgSO
4 · 7H
2O, at
25 ppm, CaCl
2 at 1.25 ppm, FeCl
3 · 6H
2O at 0.27 ppm, Na
2MoO
4 · 2H
2O at 0.242 ppm, H
3BO
3 at 3 ppm,
NaSO
4 · H
2O at 1.83 ppm,
ZnSO
4 · 7H
2O at 0.287 ppm,
CuSO
4 · 5H
2O
at 0.125 ppm,
CoSO
4 · 6H
2O at 0.119 ppm). Assay
mixtures were
incubated for 5 days in a rotary incubator at 28°C and
200 rpm.
The mixtures were centrifuged for 2 min at 13,000 ×
g to remove
cell debris. The decanted supernatants were
concentrated 10-fold
by evaporation in a rotary Speed-Vac and
resuspended in small
volumes of sterile distilled water. Samples were
stored at
20°C
prior to analysis by HVPE. Ten microliters of each
sample was
loaded onto 3MM Whatman paper and air dried. Electrophoresis
was
performed at 3,000 V in 1.1 M acetic acid-0.7 M formic acid
buffer.
Visualization of
-diol-containing compounds, including MSI,
resulted
from staining with alkaline AgNO
3 (
5).
The rhizopine was detected
as a dark spot running at a characteristic
distance relative to
the position of the reference dye orange G
(
0.9).
Catabolic units (CUs) were defined as the dilution factor of the most
dilute suspension within which catabolism of rhizopine
was observed by
HVPE analysis after 5 days of incubation in the
standard assay
mixtures. In these assay mixtures, bacterial growth,
as indicated by
visible turbidity, was used to define the number
of culture-forming
units (CUFUs). Catabolism of SI and
myo-inositol
was scored
in similar assay mixtures, where either 0.2% SI or
0.2%
myo-inositol plus 0.1%
(NH
4)
2SO
4 was added in lieu of the
nodule extract. Growth was scored by visual inspection for turbidity.
In such instances, CUs were defined as the dilution factor of
the most
dilute suspension within which turbid growth was observed
by eye after
5 days of incubation and were equivalent, by definition,
to
CUFUs.
Isolation and maintenance of bacteria.
To increase the
diversity of isolates obtained, bacteria were isolated from the soil
described above in several different ways. For the liquid enrichment
cultures, samples included (i) air-dried soil (soil D), (ii) soil that
had been saturated and kept at approximately field capacity for just
over 5 months (soil W), and (iii) the crown rhizospheres of two
separate 5-month-old alfalfa plants grown in this saturated soil
(rhizospheres R and S). Samples (
5 g each) were placed in 25 ml of
distilled water, vigorously shaken for 15 s, and then diluted to
the equivalent of 0.01 g/ml (wt/vol) in sterile distilled water.
Seventy-five microliters of each mixture was separately inoculated into
1 ml of SI-containing medium (0.05% SI in 1× BGTS) and incubated at 28°C. After 3 days, the cultures were diluted and spread onto 0.1×
tryptic soy agar (TSA) medium (3 g of tryptic soy extract per liter,
15 g of agar per liter) for isolation of individual colonies.
Seventeen isolates representing several distinct morphotypes from each
of the samples were selected for further analysis. Isolates were also
obtained by selection of organisms growing on solid medium containing
SI. Two soil samples (soils W2a and W2b) were obtained from the same
pots as described above for soil W 2 months later. Additionally, four
soil samples (soils Aa, Ab, Ba, and Bb) were obtained in late April
1996 from within 5 meters of the original sampling site in the field
plot described above. Samples (4 g each) were diluted to 0.1 g/ml in
1× BGTS, mixed vigorously, serially diluted, and plated onto solid
media [1% agarose with or without 0.2% SI with or without 0.1%
(NH4)2SO4 or 0.1× TSA]. Plate
counts were determined after 2, 4, and 8 days of incubation at 28°C.
At the 4-day time point, 20 strains representing distinct morphotypes
of the most abundant colonies were selected from each of the
SI-containing plates and purified on 0.1× TSA medium. For all
subsequent work, cultures were grown at 28°C on 0.1× tryptic soy
broth (TSB; 3 g of tryptic soy extract per liter) or 0.1× TSA.
Frozen stocks of purified isolates were kept in 15% glycerol at
70°C.
Characterization of bacterial isolates.
MagnaGraph nylon
membranes (MSI Scientific, Westboro, Mass.) and random-primed
digoxigenin (DIG)-labeled probes prepared with a Genius I kit
(Boehringer Mannheim Biotechnologies, Indianapolis, Ind.) were used in
all hybridization experiments according to the manufacturers'
instructions. When the isolates were screened for moc-like
sequences, 10-µl samples of overnight cultures were spot inoculated
onto 0.1× TSA and incubated for 24 h at 28°C prior to colony
lifts. Probe templates consisted of the full-length ORFs of the
mocA, mocB, and mocC genes from
S. meliloti L5-30 carried by plasmids pSR8610 and pSR8611
(22, 23). For the characterization of strains D1, R3, and
L5-30, genomic DNAs were isolated, digested, and transferred to nylon
membranes according to standard procedures (26).
Hybridizations were performed overnight at 68°C, and blots were
subjected to two 10-min washes (0.1× SSC [1× SSC is 0.15 M NaCl plus
0.015 M sodium citrate], 0.1% sodium dodecyl sulfate; 
37°C).
Genomic fingerprinting with the BOX primer was performed with whole
cells, as described elsewhere (21).
In the nodulation assays, large test tubes containing 15 ml of a
nitrogen-free mineral salts medium (
1) and wicks of 3MM
Whatman filter paper were autoclaved and allowed to cool to room
temperature. Seeds of
M. sativa var. Cardinal were surface
sterilized
by immersion in 3% hydrogen peroxide for 10 min, followed
by thorough
rinsing in sterile distilled water. Two seeds were
asceptically
transferred onto the wick in each tube. Liquid cultures of
the
three bacterial strains were grown to saturation, pelleted by
centrifugation, and washed twice with sterile distilled water.
One
hundred fifty-microliter samples (containing
10
7 cells)
of these washed culture were individually used to inoculate
test tubes.
Four replicate tubes were prepared for each strain
and for the
uninoculated
control.
Statistics.
The nonparametric sign test (17) was
used for all comparisons. Differences in relative population sizes were
conservatively estimated by finding the maximum factor which still
yielded significant results in each of the comparisons. Tests were
performed with SchoolStat software (David Darby, WhiteAnt Occasional
Publishing, Victoria, Australia). All P values less than or
equal to 0.15 are reported.
| |
RESULTS |
Detection and quantification of Moc activity in the
environment.
Moc activity was detected in serial dilutions of soil
and rhizosphere washes after 5 days of incubation in standard assay mixtures (Fig. 1). Since it could not be
assumed that the breakdown of MSI in these assays was due to the
activities of individual organisms, the enumeration of Moc activity in
these assays is referred to in terms of CUs, which are analogous to
CUFUs. The median Moc activity was observed to be 106 to
107 CUs per g (n = 6) in both soil and
rhizosphere samples. The median myo-inositol-catabolizing
activity was observed to be approximately fivefold higher, but this
difference was not statistically significant. These data contrast with
the median level of bacterial growth in these assays
(P < 0.04), which was observed to be
approximately 108 CUFUs per g. Bacterial growth in the
assay mixtures increased with time and correlated well with the
disappearance of uncharged organic compounds from the nodule extract as
visualized by HVPE analysis. Catabolism of the uncharged compounds
generally preceded catabolism of MSI (data not shown).
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FIG. 1.
Detection of Moc activity in the environment. MSI in
assay mixtures is detected as a positively staining spot
( ) with an electrophoretic
mobility relative to that of orange G. Uncharged -diol-containing
organic compounds remain at the origin
( ). Moc activity is scored as the
absence of a detectable signal comigrating with MSI after 5 days of
incubation in the standard assay mix. To quantitate the observed
activity, 10-fold serial dilutions (lanes 3 to 8) of soil and
rhizosphere samples were assayed and compared to an uninoculated
control (lane C). In this example, 104 CUs per g of Moc
activity were detected.
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The number of bacteria growing on the crude nodule extract was
estimated to be just under 10 times that growing on
myo-inositol
as the sole carbon source (
P < 0.11). Likewise, the number of
CUFUs was approximately 10 times
the number of units of Moc activity
present in the assay mixtures
(
P < 0.11), though this ratio was
measured to be
100
in half of the assays. Similarly, the number
of colonies growing on
SI-containing medium was less than the
number growing on 0.1× TSA
(
P < 0.005). When the SI medium was
supplemented with
0.1% (wt/vol) (NH
4)
2SO
4, the
number of colonies
observed was intermediate between the numbers
observed on SI medium
and 0.1× TSA medium (
P < 0.04).
Again, the numbers of bacteria
growing on SI-containing media were
estimated to be approximately
10-fold less than the number growing on
0.1× TSA (
P < 0.11).
Isolation and characterization of Moc+ bacteria.
Liquid and solid media containing SI were used to enrich for
Moc+ bacteria from soil and rhizosphere samples.
Thirty-seven isolates were selected for further investigation based on
the type of enrichment used, the sample source, and the colony
morphology of the isolate. All of these isolates were screened by
colony hybridization for the presence of DNA sequences homologous to
three of the known moc genes from S. meliloti
L5-30 (Fig. 2). However, no hybridization was observed for any of the isolates.
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FIG. 2.
Colony hybridization of bacterial isolates with
moc gene probes. DIG-labeled probes corresponding to the
mocA, mocB, and mocC genes of S. meliloti L5-30 were hybridized to blots of colonies lifted from
replica plates such as those shown. All 37 of the isolates obtained
from liquid (a) and solid (b) media containing SI were examined. L5-30
(L) was included twice on all of the plates as a positive control.
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Twenty-one of the isolates were found to be capable of growing on
minimal medium containing the SI preparation as the sole
carbon and
nitrogen source (data not shown). Each of these strains
was
characterized by partial 16S ribosomal DNA (rDNA) sequencing.
DNA
sequence analysis revealed that several distinct phylogenetic
groups
were represented, including members of the gram-negative
,
, and
subdivisions of the class
Proteobacteria and a
gram-positive
phylum (Table
1). Amplified
rDNA restriction analysis of representatives
of each of these groups,
with
MspI and
RsaI in single restriction
digests,
confirmed these designations (data not shown). The 16S
rDNA sequences
of strains that were determined to belong to the
same genus were found
to be
98% identical over the region analyzed.
However, repetitive
extragenic palindromic-PCR genomic fingerprinting
of these strains with
the BOX primer revealed that most of the
isolates were genotypically
distinct (Fig.
3). Seventeen distinct
patterns were observed in the set of 21 genomic fingerprints.
Seven
strains, all belonging to the
proteobacteria based on
their 16S
rDNA sequences, were found to fall into three distinct
groups based on
their BOX-PCR-generated genomic fingerprints (isolates
D2 and W2 and
isolate D1). Additionally, some similarities were
noted in the patterns
generated from two sets of putative
Arthrobacter strains
(i.e. isolates R4 and S3 and isolates 1 and 1N). Interestingly,
the
putative
S. meliloti strains D1 and R3 displayed BOX-genomic
fingerprints that were distinctly different from that of
S. meliloti L5-30.
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TABLE 1.
Characterization of isolates capable of growing on the SI
mix as the sole carbon and nitrogen source by partial sequencing of
their 16 rDNAsa
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FIG. 3.
BOX-PCR-generated genomic fingerprints of isolates
capable of growing on SI as the sole carbon and nitrogen source.
Seventeen distinct patterns can be observed in the set of 21 genomic
fingerprints obtained from the rhizopine-catabolizing isolates.
Three sets of strains with very similar patterns were observed: D2 and
W2 (lanes 2 and 6) and D1 (lanes 7, 14, and 18). Additionally, strains
D1 and R3 had distinct BOX-genomic fingerprints from S. meliloti L5-30 (L).
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Of the 21 isolates, only 6 were designated Moc
+ based on
their performance in the standard catabolism assay (Fig.
4). Four
of these isolates, R4, S3, 1, and 1N, appeared to belong to the
genus
Arthrobacter, based
on their 16S rDNA sequences. The other
two strains, D1 and R3, appeared
to be most closely related to
S. meliloti, the species from
which the
moc genes were originally
isolated (
12,
23).
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FIG. 4.
Determination of the Moc phenotypes of isolates.
Twenty-one isolates capable of growth on SI were obtained from liquid
(a) and solid (b) media and assayed for their ability to catabolize MSI
() in nodule extracts. L5-30
(lanes L) was included as a positive control for catabolism. An
uninoculated assay mixture containing the MSI rhizopine from nodule
extract (lanes E) was used as a negative control.
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Because the
moc gene probes did not hybridize to genomic
DNAs from strains D1 and R3, these two putative
S. meliloti
strains
were further analyzed. Attempts to amplify portions of the
nifHDK and
nodBC loci with conserved primers were
unsuccessful despite
amplification of appropriately sized fragments
from
S. meliloti L5-30 (data not shown). Additionally, no
hybridization to a
nodBC probe generated from the L5-30
sequence was observed (Fig.
5).
Strains
D1 and R3 also failed to nodulate alfalfa under gnotobiotic
conditions.
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FIG. 5.
Southern blot analysis of D1, R3, and L5-30 for the
presence of nod gene sequences. (a) Ethidium-stained agarose
gel containing 1 µg of genomic DNA from each of the three strains
digested with either EcoRI (E lanes) or BamHI (B
lanes). (b) Southern blot of the same gel hybridized with a DIG-labeled
probe specific to 1 kb spanning the nodB and nodC
genes of S. meliloti.
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| |
DISCUSSION |
We have detected and characterized rhizopine-catabolizing activity
in soil and rhizosphere environments, and we isolated a collection of
novel rhizopine-catabolizing bacteria. Amounts of catabolic activity
were enumerated by serial dilution of soil and rhizosphere washes (see
Materials and Methods). Twenty-one bacterial strains capable of growing
on synthetic SI were identified, six of which were found to be able to
catabolize MSI under standard assay conditions. The abundance of two of
these Moc+ strains, 1 and 1N, approximated the total number
of rhizopine-catabolizing units in the soil, because they were
representatives of the dominant morphotypes cultured on SI-containing
solid medium (data not shown). Thus, while it is likely that we
isolated only a subset of the entire diversity of
rhizopine-catabolizing microbes from these samples, we have identified
at least some of the more abundant strains.
DNA sequences similar to the known moc genes were not
detected in any of the isolated organisms in dot blot and, in selected cases, Southern hybridization experiments. This was particularly surprising with regard to isolates D1 and R3, which were identified as
S. meliloti based on the sequences of their 16S rRNA genes. In addition, previous reports have indicated that Moc+
S. meliloti strains contain moc genes that are
very similar to those from L5-30 (24, 30, 31). Since it has
been shown that the known moc genes are located on the large
symbiotic plasmid in S. meliloti (12), D1 and R3
may be lacking all or part of that plasmid, as indicated by their
nonsymbiotic phenotype on M. sativa. Thus, alternative genes
responsible for MSI catabolism may be present in some strains of
rhizobia, although it cannot be ruled out that highly diverse
moc-like genes are present in these isolates.
One of the motivations for performing this study was to explore the
potential impact of indigenous catabolizers on a
"biased-rhizosphere" system based on rhizopine as the nutritional
mediator. Rhizopines were hypothesized to be good candidates for
rhizosphere nutritional mediation because only beneficial soil bacteria
(i.e., nitrogen-fixing rhizobia) were known to catabolize these
compounds (13, 23, 25). Additionally, some evidence
indicated that catabolism of MSI may play a role in competitiveness of
certain strains for nodule occupancy (9). Other compounds
present in the root exudates of legumes may also act as nutritional
mediators that stimulate nodulation by Rhizobium (8,
11). That nutritional mediators can stimulate other
beneficial plant-microbe interactions has also been shown. The
biological control of plant pathogens has been enhanced by the
concurrent application of salicylate and bacteria capable of
catabolizing it (2, 32). Thus, it seems possible to promote
the beneficial activities of plant-associated microbes capable of
catabolizing nutritional mediators.
It is reasonable to assume that nontarget microbial populations capable
of catabolizing any given nutrient exist, and their response to that
nutrient may complicate the predicted outcome of efforts to bias
microbial communities with nutritional mediators. Ideally, the
abundance and diversity of microbes capable of using a selected
nutritional mediator should be minimal. Some evidence indicates that
opine-catabolizing bacteria represent a relatively small fraction of
the total number of culturable bacterial heterotrophs in soil and
rhizosphere environments (20). However, we have observed
that the numbers of soil bacteria capable of growing on mannopine,
nopaline, or octopine as the sole carbon and nitrogen source were
similar to those reported here for SI and myo-inositol plus
(NH4)2SO4 (data not shown). This
discrepancy may reflect differences in media composition and/or
differences in the levels of abundance of indigenous catabolizers in
different soils. Diverse types of bacteria, including members of
Arthrobacter and Pseudomonas, have been reported
to catabolize mannityl opines (15, 16, 19). The coincidental
isolation of rhizopine catabolizers from these genera likely reflects
the metabolic diversity that is known to be present in these two
genera. Of course, plant-pathogenic Agrobacterium can also
catabolize opines (5, 6). Because of this, opine-based
nutritional mediators may not be suitable for biasing rhizosphere
microbial populations in agricultural settings (19).
Other factors may also play significant roles in determining the
effectiveness of nutritional mediators in the complex milieu of the
rhizosphere environment. The relative numbers of target and nontarget
catabolizers, their relative efficiencies in utilizing the nutritional
mediator, and the consequences of their enrichment on microbial ecology
in and around the plant roots may all be factors. In our catabolism
assays, we have noticed that the catabolism of MSI appears to follow
the catabolism of other -diol-containing compounds present in the
nodule extracts, both in the serial dilutions of environmental samples
and in the Moc+ isolates (data not shown). This finding may
indicate that MSI is not a preferred metabolic substrate for the
microorganisms capable of catabolizing it. Additionally, the growth
rate of the Moc+ isolates generally exceeded that of L5-30
both in the nodule extracts and in 0.1× TSB. To what extent these
observations relate to a rhizopine-based biased rhizosphere in the
field remains an open question.
| |
ACKNOWLEDGMENTS |
We thank Josiemeer Mattei and Baptiste Nault for their technical
assistance in these experiments and related work. We also thank Mark
Wilson and Silvia Rossbach for their critical reviews of the manuscript
and many helpful discussions and Rawle Hollingsworth for providing SI preparation.
This work was supported in part by a grant from the USDA NRICGP
(9501182) to M. Wilson, Auburn University, and by a STAR graduate fellowship from the U.S. Environmental Protection Agency to B.B.M.G.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: MSU-DOE Plant
Research Laboratory, Michigan State University, East Lansing, MI 48824. Phone: (517) 353-2229. Fax: (517) 355-9614. E-mail:
debruijn{at}pilot.msu.edu.
| |
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Applied and Environmental Microbiology, December 1998, p. 4944-4949, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
