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Applied and Environmental Microbiology, November 1999, p. 4914-4920, Vol. 65, No. 11
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Relationships of Bradyrhizobia from the Legumes
Apios americana and Desmodium
glutinosum
Matthew A.
Parker*
Department of Biological Sciences, State
University of New York, Binghamton, New York 13902
Received 10 May 1999/Accepted 24 August 1999
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ABSTRACT |
Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and
nearly full-length 16S rRNA sequences all indicated high genetic
similarity among root-nodule bacteria associated with Apios
americana, Desmodium glutinosum, and
Amphicarpaea bracteata, three common herbaceous legumes
whose native geographic ranges in eastern North America overlap
extensively. A total of 19 distinct multilocus genotypes
(electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata
populations, or differed at only one locus. Within both 23S and 16S
rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata
isolates or showed only single nucleotide differences. Growth rates and
nitrogenase activities of A. bracteata plants inoculated
with isolates from D. glutinosum were equivalent to levels
found with native A. bracteata bacterial isolates, but none
of the three A. americana isolates tested had high
symbiotic effectiveness on A. bracteata. Phylogenetic
analysis of both 23S and 16S rRNA sequences indicated that both
A. americana and D. glutinosum harbored rare
bacterial genotypes similar to Bradyrhizobium japonicum
USDA 110. However, the predominant root nodule bacteria on both legumes
were closely related to Bradyrhizobium elkanii.
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INTRODUCTION |
In recent years, much progress has
been made in clarifying the phylogenetic affinities of root nodule
bacteria from various taxa of host legumes (1, 6, 8, 9, 13, 14,
28-30, 32, 33). However, the precise relationships of symbiotic
bacteria from the vast majority of legume species in natural plant
communities remain quite poorly known. Moreover, there is a particular
need for studies of bacteria from different legumes that coexist
regionally (8), to understand patterns of host specificity
and the potential for ecological linkages among legume taxa that may
arise from overlap in bacterial symbiont utilization.
In this study, I analyze the extent to which Bradyrhizobium
sp. symbionts are shared by three common papilionoid legumes indigenous to eastern North America: Amphicarpaea bracteata,
Apios americana, and Desmodium glutinosum. Past
studies of bacteria associated with A. bracteata provide a
provisional framework for understanding diversity and relationships.
Analyses of partial nod, 16S rRNA, and 23S rRNA sequences
show that bacteria associated with A. bracteata form a
monophyletic group related to Bradyrhizobium elkanii
(25). However, multilocus enzyme electrophoresis (MLEE)
(21) of 270 isolates sampled from a 1,000-km region
indicated that the bacterial associates of A. bracteata are
fairly diverse. Three basic lineages (designated A, B, and C) exist
that differ at >50% of the enzyme loci surveyed (17), with
all three lineages present in each geographic area sampled. These
bacterial lineages are heterogeneous in nodulation specificity toward
different A. bracteata genotypes. All lineage C isolates
display unspecialized symbiotic phenotypes, forming nodules on all
A. bracteata genotypes tested. By contrast, most lineage A
isolates are specialized on one host subgroup (termed plant lineage Ia
and defined by isozyme markers) and form few or no nodules on other
A. bracteata genotypes (19). Lineage B isolates
are heterogeneous: some display specialized phenotypes identical to
lineage A bacteria and some are effective, broad host-range symbionts
(like lineage C isolates). Other lineage B isolates form nodules on all
A. bracteata genotypes yet are completely ineffective with
respect to nitrogen fixation (35).
Since many root nodule bacteria have host ranges that naturally
encompass several legume genera (for examples, see references 37 and 39), it is unlikely that
the diverse bacteria found in A. bracteata populations all
evolved strictly in association with this plant. The goal of the
present study was therefore to survey bacteria from two related legumes
that sometimes cooccur with A. bracteata, to determine the
extent to which bacterial genotypes are shared across host taxa.
Bacteria were sampled from A. americana, a perennial vine in
the subtribe Erythrininae of the tribe Phaseoleae, and from D. glutinosum, a herbaceous perennial in the tribe Desmodieae (for
brevity, I hereafter refer to the three host taxa by their generic
names only). Amphicarpaea is a herbaceous annual vine in the
Phaseoleae subtribe Glycininae (4). While traditional
classifications have placed Desmodium in a separate tribe
from the other two genera, phylogenetic analyses of chloroplast DNA
sequences have shown that Desmodium is actually more closely
related to certain legumes in the subtribes Erythrininae and Glycininae
that these are to other subtribes within the Phaseoleae (5).
In addition, the results of cross-inoculation experiments published 60 years ago showed that certain bacterial isolates from Apios
and Desmodium can form nodules on Amphicarpaea
and that bacteria from Amphicarpaea and Apios can
nodulate some species of Desmodium (37). However,
since these studies provided no information about the genetic
relationships of the bacteria tested or about their capacity for
nitrogen fixation, it is important to reanalyze the root nodule
bacteria associated with these legume taxa.
This study addressed three specific questions. First, using MLEE, is
there evidence that isolates from Apios or
Desmodium are related to those previously detected in
Amphicarpaea populations (17)? Second, do
bacterial isolates from the three legume taxa display similar length
variants and nucleotide sequences in the 5' portion of the 23S rRNA
gene (22) and in 16S rRNA? Finally, can bacteria that are
associated with Apios and Desmodium function as
effective nitrogen-fixing symbionts on Amphicarpaea plants, as evidenced by plant growth and nodule nitrogenase activity?
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MATERIALS AND METHODS |
Isolate sampling.
Root nodule bacteria were collected from
one Apios and one Desmodium population both in
Broome County and in Tompkins County, New York (Table
1). The Apios and
Desmodium sites in Tompkins County were 0.8 km apart in the
Fall Creek watershed, while the two Broome County sites were 18 km
apart. Approximately 65 km separated the Broome County and Tompkins
County sites. These four sample sites were chosen because they are each
within 2 km of A. bracteata populations where bradyrhizobial
diversity had previously been analyzed (17). At each site,
15 to 18 nodules were sampled haphazardly (each from a different
individual plant), and a single bacterial isolate was purified from
each nodule as previously described (24).
Enzyme electrophoresis.
Bacterial isolates were grown in
yeast-mannitol broth (31), and enzymes were obtained from
sonicated cells (24). Isolates were characterized by starch
gel electrophoresis at the following 20 enzyme loci as previously
described (24): acid phosphatase (ACP), alanine
dehydrogenase (ALA), butyrate esterase (EST), 
-hydroxybutyrate dehydrogenase (HBD), diaphorase (DIA), fumarase (FUM),
fructose-1,6-diphosphatase (F16), glucose-6-phosphate dehydrogenase
(G-6), glutamic-oxalacetic transaminase (GOT),
glyceraldehyde-3-phosphate dehydrogenase type 1 (GP1), isocitrate
dehydrogenase (IDH), indophenol oxidase (IPO), leucine aminopeptidase
(LAP), leucine tyrosine peptidase (PEP), malate dehydrogenase
(MDH), malic enzyme (ME), phosphoglucose isomerase (PGI),
phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6PGD), and
shikimate dehydrogenase (SDH). Each isolate was characterized by its
allelic profile for the 20 enzymes, and each unique multilocus genotype
was designated an electrophoretic type (ET). On each gel, two different
standards representing ETs from Amphicarpaea were included
with the Apios and Desmodium isolates. Two loci
were monomorphic across all isolates (IPO and MDH) and were therefore
omitted from the allele profile summary (Table 2). Pairwise genetic
distances between ETs were estimated by the proportion of enzyme loci
at which allelic differences occurred. ETs were then clustered by the
unweighted pair group method with arithmetic means (23).
DNA amplification and sequencing.
DNA was purified from five
Desmodium isolates and four Apios isolates by a
cetyltrimethylammonium bromide protocol (38). For reference,
Bradyrhizobium japonicum USDA 110, B. elkanii
USDA 94, and Amphicarpaea isolate jwc91-2 (lineage A) were
also analyzed. A 5' portion of 23S rRNA that is highly polymorphic
within the Rhizobiaceae (22) was amplified with
primers 23Sup115 and 23SrIII (25). These primers yield a
260-bp DNA fragment in B. japonicum USDA 110 (GenBank
accession no. Z35330). For five selected isolates, a larger segment of
DNA spanning this region was then sequenced on both strands, beginning
62 sites from the 5' end of the 23S rRNA gene using primers 23Sup6n and
23SrII (25) (these amplify 496 bp in B. japonicum
USDA 110). For five isolates, nearly full-length 16S rRNA was amplified
using primers fD1d (5'-GAGAGTTTGATCCTGGCTCAGA) and rPla
(5'-CTACGGCTACCTTGTTACGACTT). This fragment was sequenced on
both strands using the following internal sequencing primers (numbers
in brackets indicate primer position relative to the Escherichia
coli rrnB gene [2]): m5if
(5'-CATGCCGCGTGAGTGATGAAGG [bp 395 to 416]), 5v2ir
(5'-AAAGAGCTTTACAACCCTA [bp 420 to 438]), 16smidf (bp 774 to 795) (25), al4r (5'-GAGTTTTAATCTTGCGACCGTA [bp 890 to 911]), 16cup (5'-TCGTGTCGTGAGATGTTGGGTTA
[bp 1,069 to 1,091]), and m3ir (5'-GACTTGACGTCATCCCCACCTT
[bp 1,178 to 1,199]).
PCR used 25-µl reaction mixtures containing 10 mM Tris buffer with
0.1% Triton X-100, 50 mM KCl, 1.5 mM MgCl
2, 0.2 mM
concentrations
of each deoxynucleoside triphosphate, 0.5 µM
concentrations of
each primer, 0.5 µl of genomic DNA, and 0.5 U of
Taq polymerase.
Tubes were incubated for 70 s at 94°
and then subjected to 35
cycles of 94° (20 s), 58° (50 s), and
72° (50 s), with a final
extension of 4 min at 72°. Five
microliters of PCR product was
run on a 1.9% agarose gel with a DNA
size standard to analyze
length variation. PCR-amplified DNA was
sequenced using an Applied
Biosystems Model 310 automated sequencing
system with dye terminator
chemistry, following protocols recommended
by the
manufacturer.
Phylogenetic analyses.
Trees were constructed by maximum
parsimony using the PAUP software, version 4.0b1 (D. L. Swofford,
Smithsonian Institution, Washington, D.C.). To determine the degree of
statistical support for branches in the phylogeny (7), 1,000 bootstrap replicates of each data set were analyzed. For the 23S rRNA
region, data from Apios and Desmodium isolates
were compared to reference sequences available for B. japonicum USDA 110, Bradyrhizobium sp.
(Lupinus) strain DSM 30140 (X87283), B. elkanii
USDA 94 (AF081266), and Bradyrhizobium sp.
(Amphicarpaea) strains jwc91-2, bfs1b, and th-b2,
representing MLEE lineages A, B and C, respectively (AF081262,
AF081263, and AF081265). Rhodopseudomonas palustris (X71839)
was used as the outgroup (25). For 16S rRNA, data from
Apios, Desmodium, and Amphicarpaea
isolates were compared to the following Bradyrhizobium
reference sequences (strains without formal names are identified by
host legume genus in parentheses): B. japonicum strains USDA
6 (GenBank accession no. U69638) and USDA 110 (Z35330), strain DSM
30140 (Lupinus) (X87273), B. elkanii strains USDA
76 (U35000) and USDA 94 (D13429), strain 129 (Stylosanthes)
(D14508) (14), Bradyrhizobium genomic species B
(Bossiaea) (Z94812) (8), LMG 9966 (Acacia) (X70403) (6). Several related genera of
the alpha subgroup of the class Proteobacteria were also
included in the analysis: Azorhizobium caulinodans (X67221),
Paracoccus denitrificans (X69159), Rhodobacter
sphaeroides (D16424), and R. palustris (D25312). A. caulinodans was chosen as the outgroup (6, 36,
40).
Sequences were first aligned using CLUSTAL W (
27), which
revealed that three single-nucleotide insertion-deletion polymorphisms
(indels), together with two longer gaps of 12 and 16 bp, existed
in the
23S rRNA region. Several small indels (1 to 3 bp) were
also evident in
the aligned 16S rRNA sequence. Since specific
indels were commonly
shared across taxa and appeared to provide
useful information about
relationships, gaps were included in
the phylogenetic analysis by using
the "gapmode = newstate" option
in PAUP. However, to avoid
counting large gaps as multiple independent
characters, all but the
first position within the two long 23S
rRNA gaps were recoded as
missing data. This weighted each gap
as a single event regardless of
its
length.
Plant growth rate and nitrogenase activity.
Three
Apios and four Desmodium isolates representing
different genotypes revealed by MLEE and rRNA sequence analyses were used to inoculate Amphicarpaea plants according to
previously described procedures (35). Two lineage A isolates
(DesT1 and ApT2, representing ET1 and ET2, respectively), two lineage B
isolates (ApB2 and ApB5, representing ET8 and ET10), two lineage C
isolates (DesB1 and DesB3, representing ET13 and ET12), and the sole
lineage E isolate (DesT10, representing ET19) were tested. Two lineages of Amphicarpaea plants were inoculated with each isolate:
plants from a lineage Ia population in Tompkins County, N.Y., and
plants from a lineage Ib population in Broome County, N.Y.
(15). These Amphicarpaea populations are close to
two of the collection locations for the bacterial isolates. It was not
possible to perform reciprocal inoculations to test how plants of
Apios and Desmodium interacted with bacterial
isolates from Amphicarpaea due to a lack of seeds from these
taxa (natural populations of D. glutinosum commonly have low
seed productivity, and A. americana populations in this region are sterile triploids [3]).
Amphicarpaea seedlings were germinated under aseptic
conditions and then planted individually in 240-cm
3
containers using a
Bradyrhizobium-free mixture of sand,
perlite,
and potting soil. For each bacterial isolate, 12 seedlings of
each plant lineage were inoculated with approximately 10
9
cells grown in yeast-mannitol broth (
31). Plants were grown
in a greenhouse for 48 days with precautions to avoid bacterial
contamination across inoculation treatments (
35). Twelve
uninoculated
controls of each plant lineage were grown simultaneously
in the
same room as contamination checks. No nodules developed on any
of these plants. To compare
Apios and
Desmodium
isolates with
bacteria endemic to
Amphicarpaea populations,
12 plants per lineage
were also inoculated with a
Bradyrhizobium strain native to the
lineage Ib
A. bracteata population used in the experiment (th-b2)
(Table
1).
Starting 14 days after inoculation, all plants were
fertilized weekly
with a nitrogen-free nutrient solution (
18).
At harvest,
total plant dry mass and nodule numbers were recorded
for each plant,
and a subsample of two to three plants from each
group was analyzed for
acetylene reduction activity using a Hewlett
Packard 5890 Series II gas
chromatograph as described (
24).
Nucleotide sequence accession numbers.
The five distinct 23S
rRNA sequences found among Desmodium and Apios
isolates have been placed in GenBank under accession no. AF146820
through AF146824. The five nearly full-length 16S rRNA sequences
obtained have been assigned accession no. AF178434 through AF178438,
and the partial 16S rRNA sequence from isolate DesT10 has been assigned
accession no. AF146827.
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RESULTS |
Diversity and relationships inferred from MLEE.
Among the 68 isolates sampled from Apios and Desmodium,
variation was detected at 18 of the 20 enzyme loci examined, with a
mean of 4.4 alleles per polymorphic locus (range, two to nine alleles)
(Table 2). A total of 19 distinct
multilocus genotypes (ETs) were detected. Most ETs were recovered more
than once among different isolates from the same population. However,
only two ETs (ET2 and ET8) occurred in more than one population (Table 2). These ETs were found in both Apios populations sampled
and represented 71% of all isolates obtained from that host. No ETs were shared by Apios and Desmodium populations,
nor were any ETs shared by the two Desmodium populations
sampled.
An average linkage cluster analysis revealed the presence of five
divergent bacterial lineages (designated A through E) (Fig.
1). These differed at 57 to 69% of the
20 enzyme loci analyzed.
All
Apios isolates fell into
lineages A, B, and D. One
Desmodium population (DesB)
contained only ETs in lineage C. The other
Desmodium population was dominated by lineage A, but also had one isolate
each
from lineages B and E.
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FIG. 1.
Genetic relationships among 19 multilocus genotypes
(ETs) of Bradyrhizobium isolated from Apios and
Desmodium.
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Most of the ETs detected in
Apios and
Desmodium
populations (Table
2 and Fig.
1) were strongly similar to bacterial
genotypes
associated with
Amphicarpaea. In an isozyme survey
of 270
Bradyrhizobium isolates from 24
Amphicarpaea populations (
17), all bacteria
were
found to cluster into three groups that corresponded exactly
to
lineages A, B, and C of Fig.
1, and numerous isolates from
the three
host taxa had identical multilocus genotypes. For example,
the two most
common ETs in lineage A of Fig.
1 (ET1 and ET2) were
indistinguishable
from the two most common ETs in this lineage
among bacteria associated
with
Amphicarpaea (
17). The four most
common
lineage C ETs in
Amphicarpaea populations were identical
to
ET12, ET13, ET14, and ET16 of Fig.
1. The five lineage B ETs
from
Apios and
Desmodium did not match any
Amphicarpaea ETs. However,
ET10 differed at only one locus
from an
Amphicarpaea isolate,
and ET7 and ET9 also matched
lineage B
Amphicarpaea ETs at all
but two loci. Overall, 6 of the 19
Apios and
Desmodium ETs (representing
68% of all isolates) were identical to ETs detected in
Amphicarpaea populations, and another six ETs (representing
an additional 10%
of isolates) differed at only one locus from
corresponding
Amphicarpaea ETs.
At a finer scale, bacteria from the two
Desmodium sites each
showed a striking resemblance to those in nearby
Amphicarpaea populations. DesB had ETs only from bacterial
lineage C, while
DesT was dominated by bacterial lineage A (Table
2 and
Fig.
1).
The nearest
Amphicarpaea population to DesB was
40 m away, and
it was exclusively occupied by bacteria clustered
into lineage
C (
17). The DesT population was 1.2 km from an
Amphicarpaea site where 90% of the bacteria fell into
lineage A and 10% grouped
into lineage B (
17). Thus, within
each host legume species,
populations in these two areas had no ETs in
common. However,
bacteria from the
Desmodium population at
each site overlapped
extensively with those in the nearby
Amphicarpaea population.
A quantitative index of similarity
can be defined by PS =
min(
piv,
pjv), where
piv = the
frequency of ET
v in population
i
(
20).
Proportional similarity ranges from zero (when no ETs
are shared
in common) to one (when two populations have the same ETs at
exactly
the same frequency). Adjacent
Desmodium-Amphicarpaea
population
pairs had PS values of 0.6 to 0.7, while PS values of
bacterial
populations within each host species were
zero.
23S rRNA variation.
Bacterial lineages D and E (Fig. 1) were
uncommon in the Apios and Desmodium samples, and
no bacteria resembling these groups have been detected among 270 isolates from Amphicarpaea populations (17). To
further characterize the relationships of these divergent genotypes,
primers flanking a region in the 5' portion of 23S rRNA that commonly
shows length variation among taxa of Rhizobiaceae (22) were
used to amplify DNA from representative isolates (Fig. 2). The lineage D and E isolates (Fig. 2,
lanes 2 and 3) both displayed a single band approximately identical in
size to that of B. japonicum USDA 110 (lane 1). By contrast,
all of the lineage A, B, and C isolates from Apios and
Desmodium (lanes 5 to 11) had smaller fragments matching
those seen in Amphicarpaea lineage A (lane 4) and B. elkanii USDA 94 (lane 12) isolates.
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FIG. 2.
PCR amplification products from a 5' segment of 23S
rRNA. Lane 1, B. japonicum USDA 110; lane 2, DesT10; lane 3, ApB16; lane 4, Bradyrhizobium sp. (Amphicarpaea)
isolate jwc91-2; lane 5, DesT1; lane 6, ApT2; lane 7, ApB2; lane 8, ApB5; lane 9, DesB1; lane 10, DesB2; lane 11, DesB3; lane 12, B. elkanii USDA 94. Marker at right is HaeIII-digested
 X174 DNA.
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A larger portion of 23S rRNA spanning this region was sequenced in two
lineage A isolates (DesT1 and ApT2), one lineage C
isolate (DesB1), one
lineage D isolate (ApB16), and the sole lineage
E isolate (DesT10).
This confirmed that the 5' 23S rRNA regions
of the lineage D and E
isolates were identical in length and that
the lineage A and C isolates
had a 27-bp-shorter variant that
matched the size of
Amphicarpaea lineage A, B, and C isolates
documented
previously (
25). The
Desmodium and
Apios lineage
A isolates each differed from the sequence of
Amphicarpaea lineage
A isolates at only one nucleotide
position and showed two nucleotide
differences relative to each other.
The lineage C isolate from
Desmodium had a sequence which
was identical to those of several
Amphicarpaea lineage C
isolates. The lineage D and E isolates
both showed a number of
substitutions relative to other published
Bradyrhizobium 23S
rRNA sequences. Parsimony analysis (Fig.
3)
suggested a relationship between the
lineage E isolate (Des10)
and
B. japonicum USDA 110. The
lineage D isolate (ApB16) represented
a more basally branching member
of a clade including both
B. japonicum USDA 110 and an
isolate from
Lupinus (
11).
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FIG. 3.
Parsimony tree for five partial 23S rRNA sequences from
Desmodium and Apios (shown in bold). Numbers
above branches are bootstrap percentages (n = 1,000
replicates). GenBank accession no.: B. japonicum USDA 110, Z35330; DesT10, AF146823; Bradyrhizobium sp.
(Lupinus) strain DSM 30140, X87283; ApB16, AF146824;
B. elkanii USDA 94, AF081266; ApT2, AF146821; DesT1,
AF146820; Amphicarpaea lineage A (strain jwc91-2), AF081262;
Amphicarpaea lineage B (strain bfs1b), AF081263; DesB1,
AF146822; Amphicarpaea lineage C (strain th-b2), AF081265;
and R. palustris, X71839.
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16S rRNA variation.
Nearly full-length sequences were obtained
for isolates DesT1 (lineage A [ET1]), DesB1 (lineage C [ET13]),
ApB16 (lineage D [ET17]), and Bradyrhizobium sp.
(Amphicarpaea) strains jwc91-2 and th-b2 (representing
isozyme lineages A and C, respectively). The lineage C isolates from
Amphicarpaea and Desmodium were identical at all
1,412 bp sequenced. Both lineage A isolates shared a single nucleotide
substitution relative to this lineage C 16S rRNA sequence, and DesT1
differed at one additional site from the other lineage A and C
isolates. By contrast, ApB16 differed from the other isolates at 37 to
39 positions.
A parsimony tree indicated that these taxa of
Bradyrhizobium
fell into two distinct clades each with relatively high bootstrap
support (Fig.
4). Isolate ApB16 had a
clear relationship to the
lineage that included
B. japonicum. The other
Bradyrhizobium clade
encompassed a
set of isolates related to
B. elkanii.
Desmodium isolates from MLEE lineages A and C clustered with this group,
as did
the
Amphicarpaea isolates.
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FIG. 4.
Phylogenetic relationships of three
Bradyrhizobium isolates from Desmodium and
Apios (shown in bold) based on parsimony analysis of 16S
rRNA sequences. Numbers above branches are bootstrap percentages
(n = 1,000 replicates). GenBank accession no.: B. japonicum USDA 110, Z35330; strain 129 (Stylosanthes),
D14508; Bradyrhizobium genomic species B
(Bossiaea), Z94812; ApB16, AF178434; B. japonicum
USDA 6, U69638; DSM 30140 (Lupinus), X87273; R. palustris, D25312; B. elkanii USDA 76, U35000; LMG 9966 (Acacia) X70403; DesB1, AF178436; lineage C
(Amphicarpaea), AF178438; DesT1, AF178435; lineage A
(Amphicarpaea), AF178437; B. elkanii USDA 94, D13429; R. sphaeroides, D16424; P. denitrificans,
X69159; and A. caulinodans, X67221.
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A partial 16S rRNA sequence (383 bp, homologous to positions 795 to
1177 of the
E. coli rrnB gene [
2]) was also
obtained
for the sole lineage E from
Desmodium (DesT10).
This sequence
was identical to that of
B. japonicum USDA 6 and matched ApB16
at all but two sites. Therefore, this isolate also
appears to
be a member of the
B. japonicum clade.
Symbiotic performance of Amphicarpaea with
Apios and Desmodium bacteria.
Two
representative isolates from lineages A, B, and C, and the sole lineage
E isolate (Fig. 1) were used to inoculate Amphicarpaea plants. Because this legume is polymorphic at a locus that controls nodulation specificity, two plant genotypes were tested. Lineage Ia
plants are homozygous for a recessive allele that allows nodulation with lineage A bacteria, while lineage Ib plants have a dominant gene
that almost completely prevents nodule formation with the lineage A
bacteria found in many Amphicarpaea populations
(19).
Nodules developed abundantly on most
Amphicarpaea plants
inoculated with isolates from
Apios and
Desmodium. Lineage Ib plants
inoculated with the lineage A
isolate DesT1 mostly lacked nodules
(mean = 0.5 nodules per
plant), and lineage Ia plants inoculated
with the lineage E isolate had
a mean of only 20 nodules per plant.
Nodule numbers averaged 44 to 207 per plant in most combinations,
which resembles the range seen with
native
Amphicarpaea bacterial
isolates (
19).
However, lineage Ib plants inoculated with the
other lineage A
bacterial isolate (ApT2) developed numerous very
tiny nodules
(mean = 258 per plant). This contrasts with the lack
of nodule
formation by these plants with the lineage A bacteria
that are endemic
to
Amphicarpaea populations (
19,
35).
Only a few isolates significantly enhanced plant growth relative to
uninoculated control plants. For lineage Ia hosts (Fig.
5, top panel), Student-Newman-Keuls
multiple range tests showed
that only isolate DesT1 significantly
improved plant growth (using
an experiment-wise error rate of
= 0.05). For lineage Ib hosts
(Fig.
5, bottom panel), only isolates
DesB1, DesB3, and DesT10
caused growth significantly higher than that
seen in the controls.
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FIG. 5.
Symbiotic performance of two A. bracteata
plant lineages (Ia, top panel; Ib, bottom panel) inoculated with
Bradyrhizobium isolates from Apios and
Desmodium. Bars indicate plant biomass (mean ± 1 standard error), and above each bar is the relative nitrogenase
activity of root nodules analyzed by acetylene reduction assays (+,
0.04 to 0.16 µmol of ethylene min 1
plant1; ++, 0.17 to 0.64 µmol of ethylene
min1 plant1; +++, 0.65 to 2.56 µmol of
ethylene min1 plant1; ++++, >2.56 µmol
of ethylene min1 plant1).
|
|
There was a 1,000-fold variation in nitrogenase activity (measured by
acetylene reduction) among different combinations of
plants and
bacteria. However, since only two to three plants were
assayed per
combination, results are only presented qualitatively
in Fig.
5. The
main overall pattern was that all combinations
where bacterial
inoculation significantly increased plant growth
also showed high
nitrogenase activity. However, not all plants
displaying high
nitrogenase activity showed significant improvements
in growth relative
to the uninoculated controls. For example,
lineage Ia hosts inoculated
with lineage E bacteria had relatively
high nitrogenase activity. Yet
their mean biomass was 23% below
that of uninoculated plants, implying
that lineage E bacteria
were poorly adapted symbionts for these hosts.
Plants inoculated
with lineage B isolates from
Apios
consistently showed minimal
biomass gain, and nitrogenase activity also
tended to be low (0.08
to 0.24 µmol of ethylene min
1
plant
1). Interestingly, lineage B isolates are common in
many
Amphicarpaea populations, and these often tend to be
ineffective symbionts
as well (
35).
No lineage D isolates were tested for effects on plant biomass.
However, a small-scale inoculation test with the ET17 isolate
(Fig.
1)
indicated that it caused nodule development in the normal
range on
Amphicarpaea lineage Ib plants (mean = 114 nodules per
plant) but formed very few nodules on Ia hosts (mean = four
nodules
per plant). Nitrogenase assays revealed that activity was quite
low on Ia hosts (0.05 µmol of ethylene min
1
plant
1) and moderately high on Ib hosts (1.34 µmol of
ethylene min
1 plant
1).
| |
DISCUSSION |
The main finding of this study is that there is extensive overlap
among the bradyrhizobial symbionts associated with A. bracteata, A. americana, and D. glutinosum.
Most Desmodium and Apios isolates had multilocus
enzyme allele profiles that were identical to various ETs sampled from
Amphicarpaea. Nucleotide sequence analysis of portions of
23S and 16S rRNA also indicated a close relationship between isolates
from the three legume genera (Fig. 3 and 4). Desmodium and
Amphicarpaea, in particular, appear to share a common pool
of symbiotic bacteria, because Amphicarpaea plants
inoculated with certain Desmodium isolates had biomass gains
and nitrogenase activities that were as good as or better than those
achieved with native Bradyrhizobium isolates (Fig. 5). None
of the three isolates tested from Apios were very good
symbionts for either type of Amphicarpaea host (Fig. 5).
Thus, it would be desirable to test additional isolates from
Apios to better understand whether these two legume genera
currently interact with a common set of bacterial mutualists.
Nevertheless, the close genetic similarity of some bacteria from
Amphicarpaea and Apios suggests a historical relationship of Bradyrhizobium populations associated with
these legumes.
The vast majority of Apios and Desmodium isolates
fell into MLEE lineages A, B, and C (64 of 68 isolates [94%]). All
Amphicarpaea bacteria also cluster into these same groups
(17). The current study supports previous work
(25) indicating a phylogenetic relationship between these
bacteria and the soybean symbiont B. elkanii (Fig. 3 and 4).
For example, 16S rRNA sequences from these bacteria were >98% similar
to B. elkanii USDA 94 and were >99% similar to B. elkanii USDA 76. However, despite their 16S rRNA similarity, these
bacteria are distinct from B. elkanii in terms of isozyme
alleles, nod gene sequences, and symbiotic behavior (12, 25). Because bacterial lineages A, B, and C are also quite divergent from each other for MLEE phenotypes (Table 2) and
nodulation host range (19, 35), they possibly represent three distinct species. These bacterial groups are widely distributed across eastern North America (17). The observation that they predominate in nodule samples from three common taxa of native papilionoid legumes raises the question of how many additional legume
species in this geographic region (or elsewhere) may also be primarily
nodulated by these groups. Future studies of other legumes are planned
to resolve the host relationships and systematic status of these bacteria.
Four isolates from Apios and Desmodium (MLEE
lineages D and E) were unlike any bradyrhizobia observed in extensive
samples from Amphicarpaea. These isolates showed a 23S rRNA
length variant similar to B. japonicum USDA 110 (Fig. 2),
and parsimony analysis of both 23S and 16S rRNA sequence variation
indicated a close relationship to B. japonicum and allied
taxa. For both Apios and Desmodium, the
simultaneous presence of divergent bradyrhizobial lineages with
affinities to B. japonicum and B. elkanii (Fig. 3) emphasizes the high diversity of root nodule bacteria that may be
present within even a single local population. Similar results have
been observed in other systems (for examples, see references
8 and 9).
Bacterial symbiont sharing across legume taxa is ecologically
significant for a number of reasons. First, if plants cause local
proliferation of symbiotic bacteria in their vicinity (26, 34), then a site occupied by one plant may become a favorable microhabitat for invasion by a second host. Thus, symbiont sharing across host taxa can make bacterial mutualist partners more predictably available as plants disperse and colonize new habitats. However, since
the optimal bacterial partner is not likely to be identical among all
cooccurring host taxa, modification of bacterial population composition
by some legumes may potentially have a negative effect on certain other
plants. A possible example warranting further study is provided by the
lineage B isolates harbored by Apios populations (Table 2
and Fig. 1). These bacteria were poor-quality symbionts for both types
of Amphicarpaea (Fig. 5), yet isolates resembling these
genotypes appear to be prevalent in many Amphicarpaea populations (17, 35). Thus, one legume may potentially
create an ecological burden for a second species by serving as a source for bacteria that are inferior-quality symbionts.
Genotypes within a single legume species can also show differential
performance with specific strains of rhizobia (10, 16, 35).
Each legume may thus experience spatially heterogeneous natural
selection arising from effects of various other legume taxa on
bacterial population composition. The impact of different legumes on
the genetic structure of bacterial populations within natural
environments remains very poorly understood. Understanding the spatial
scale and temporal dynamics of these processes are key problems that
must be addressed by future ecological research on legume-bacterial symbioses.
| |
ACKNOWLEDGMENTS |
I am grateful to J. Doyle for suggesting population locations, to
J. Pfeil for assistance with sequencing, and to L. D. Kuykendall for providing bacterial isolates.
Financial support was provided by NSF grant DEB-9707697.
| |
FOOTNOTES |
*
Mailing address: Department of Biological Sciences,
State University of New York, Binghamton, NY 13902. Phone: (607)
777-6283. Fax: (607) 777-6521. E-mail:
mparker{at}binghamton.edu.
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Top
Abstract
Introduction
