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Applied and Environmental Microbiology, October 1998, p. 3860-3868, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Comparison of Paenibacillus azotofixans Strains
Isolated from Rhizoplane, Rhizosphere, and Non-Root-Associated Soil
from Maize Planted in Two Different Brazilian Soils
Lucy
Seldin,1,*
Alexandre Soares
Rosado,1
Davi
William
da Cruz,1
Alberto
Nobrega,1
Jan Dirk
van Elsas,2 and
Edilson
Paiva3
Instituto de Microbiologia Prof. Paulo de
Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro,
RJ,1 and
EMBRAPA/CNPMS, Sete Lagoas,
MG,3 Brazil, and
IPO-DLO,
Wageningen, The Netherlands2
Received 31 October 1997/Accepted 28 July 1998
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ABSTRACT |
Paenibacillus azotofixans is a nitrogen-fixing
bacterium often found in soil and in the rhizospheres of different
grasses. In this study, two Brazilian clay soils were planted with
cross-hybrid maize (BR-201) and four stages of plant growth were
analyzed to characterize the P. azotofixans populations
present in the rhizoplanes, rhizospheres, and non-root-associated soils
(herein called nonrhizospheres). A total of 106 strains were isolated
and identified as P. azotofixans with an API 50CH kit, by
classical biochemical tests, and via the use of specific primers based
on the 16S rRNA gene in PCRs. To compare the isolated strains,
phenotypic characteristics were determined and three different probes
were used in hybridization experiments: two nif probes and
one probe comprising a 0.58-kb fragment cloned from the P. azotofixans C3L4 genome. These results were used to construct a
dendrogram, in which two main clusters could be observed. One cluster
contained exclusively strains from Várzea soil, and the other
contained the majority of strains from Cerrado soil. The 60 strains
from Várzea soil and the 46 strains from Cerrado soil were
further analyzed with REP and BOX primers, respectively. Based on the
patterns obtained, it was possible to identify 21 different groups
among strains from Várzea soil and 4 different groups among
strains from Cerrado soil. These different patterns were tested by
multivariate analysis of variance, and differences in the populations
of P. azotofixans during the four stages of plant growth
were demonstrated. Moreover, strains isolated from the rhizoplanes,
rhizospheres, and nonrhizospheres of maize planted in Cerrado and
Várzea soils were shown to be statistically different; the
diversity of P. azotofixans strains was affected by
the soil type.
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INTRODUCTION |
In recent years, the interest in
soil microorganisms has increased because they play an important role
in the maintenance of soil fertility. A major challenge for the
development of sustainable agriculture is the use of nitrogen-fixing
bacteria which are able to assimilate gaseous N2 from the
atmosphere. Many different N2-fixing bacteria, including
symbionts, such as root-nodulating Rhizobium spp. (14,
26) and different free-living rhizobacteria, such as
Azospirillum spp. (16), Bacillus spp.
(8, 9, 36), and Paenibacillus spp.
(46), have already been described. When used as seed
inoculants, some of these free-living N2-fixing
bacteria show beneficial effects on plant growth, and hence they are
called plant growth-promoting rhizobacteria (11, 23).
Strains belonging to the species Paenibacillus azotofixans
were shown to be efficient nitrogen fixers prevalent in the
rhizospheres of maize, sorghum, sugarcane, wheat, banana, and
forage grasses (38, 44, 46, 48). Some strains are able
to produce antimicrobial substances (50) and
solubilize organic phosphates (38). These characteristics
can be considered to be very important for the establishment of
P. azotofixans in plant rhizospheres.
Growth promotion may occur when a plant is inoculated with a bacterium
with which it coexisted previously (7, 8), and thus the
diversity among populations of P. azotofixans
associated with a variety of different gramineous plants was
investigated (38). The results showed that the plants
studied did not select a specific phenotypic or genotypic subpopulation
of P. azotofixans. However, more data are needed to
elucidate the diversity of the populations in individual plant samples
during different stages of plant growth and in different soil types.
In this study, we aimed to determine the diversity among populations of
P. azotofixans associated with maize by their
phenotypic and genetic characteristics. Bacterial populations isolated
from three different compartments (rhizosphere, rhizoplane, and
nonrhizosphere [i.e., non-root-associated soil]) in two
agriculturally important Brazilian soils were compared. Moreover, the
influence of plant development on the diversity of the P. azotofixans populations was investigated. These approaches will
help to elucidate whether arbitrary diversity exists among strains or
whether a plant selects specific bacterial populations to coexist with
it.
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MATERIALS AND METHODS |
Maize genotype, soils, and experimental conditions.
Two
soils (Cerrado and Várzea) were planted with maize (Zea
mays L. BR-201, single cross-hybrid) in Sete Lagoas, Brazil. Cerrado soil is present over 1.8 million km2, corresponding
to about 20% of the territory of Brazil (12); it is a
dark-red distrophic latosol with a clayey texture. Várzea soil is
another dominant soil in Brazil; it is a low-humus eutrophic gley with
a clayey texture. Both soils are representatives of soils commonly
cultivated with maize and were supplied with 80 kg of nitrogen per ha
40 days after being sown with nontreated maize seeds.
Strains of P. azotofixans were isolated from the
rhizoplanes and the rhizospheres of maize plantlets 10, 30, 60, and 90 days after being sown in these soils, by using a modification of
the method described by Baldani and Döbereiner (2).
Five plantlets were harvested, and their roots were shaken to remove
the loosely attached soil. One gram of the adhering soil, considered
the rhizosphere, was mixed with 9 ml of distilled water. Roots
were washed 5 to 10 times by gentle shaking with 100 ml of distilled
water and weighed, and 1 g was macerated in 9 ml of water. Also,
1 g of non-root-associated soil (from an area 0 to 10 cm in depth)
used for the isolation procedure was collected from a maize plant. The
different suspensions were pasteurized (10 min, 80°C), and twofold
serial dilutions were plated onto thiamine-biotin agar (45)
and incubated anaerobically in GasPak jars (80% N2, 10% CO2, 10% H2) for 3 days. Further isolation and
purification were performed as described before (45).
Identification and maintenance of P. azotofixans
isolates.
All presumptive P. azotofixans strains
(white, convex, and mucous colonies isolated in thiamine-biotin solid
medium, after incubation under anaerobic conditions) were identified by
the biochemical tests proposed by Gordon et al. (19).
Cellular morphology, forms and positions of spores, and swelling of the
sporangia were observed by microscopy of crystal violet-stained smears.
Three other carbohydrates (sorbitol, dulcitol, and starch) were used to
group the strains into one of five groups based on fermentation patterns described previously (38, 48). Whenever necessary, the API 50CH microtube system (bioMérieux, Marcy l'Etoile,
France) was used in addition to conventional tests. The API test
galleries were prepared and read as described in the work of Seldin and Penido (48). In addition to these tests, a molecular method for identification of P. azotofixans was used. It
consisted of PCR amplification of part of the variable regions V1 to V4
of the 16S rRNA gene with two primers, which were BAZO1 and BAZO2, followed by hybridization with a specific 18-bp oligonucleotide probe
homologous to part of the intervening region. The PCR product generated
with P. azotofixans strains was 565 bp long and was specifically detected by a P. azotofixans-specific
probe, BAZOP (37). Strains identified as P. azotofixans were stored aerobically at room temperature on GB agar
slants (45) supplemented with 1% CaCO3
(wt/vol).
Nitrogen fixation.
Acetylene reduction was measured by
assessing the ethylene production of cultures in 18-ml vials as
described previously (46).
Solubilization of organic phosphate.
Aliquots (10 µl) of
young cultures of P. azotofixans were spot inoculated
onto calcium phytate agar plates (38) and incubated for 5 days at 30°C. A positive result was considered the formation of a
clear zone around the growth spot.
Detection of inhibitory substances.
The activities of
P. azotofixans strains inhibiting a strain of
Corynebacterium fimi were assayed by the lawn-spotting
technique described by Seldin and Penido (50).
Resistance to heavy metals and to antibiotics.
Resistance to
the heavy metals and antibiotics listed here was determined by spot
inoculating 18-h-old cultures of P. azotofixans on TBN
agar plates (48) supplemented with CuSO4 (250 µg/ml), NiSO4 (263 µg/ml), HgCl2 (20 µg/ml), CoSO4 (240 µg/ml), chloramphenicol (10 µg/ml), erythromycin (10 µg/ml), or rifampin (10 µg/ml).
Preparation of genomic DNAs.
The DNAs were isolated by a
modification of the method described by Marmur (27). Cells
from 30-ml cultures were concentrated in 2 ml of Tris-EDTA-NaCl buffer
(47) and treated with 1 mg of lysozyme per ml for 30 min at
37°C and with sodium dodecyl sulfate (final concentration, 1%). The
preparations were incubated for 10 min at 65°C. Purification steps
were done as described in the work of Seldin and Dubnau
(47). DNAs were quantified spectrophotometrically (GeneQuant
apparatus; Pharmacia Biotech, Uppsala, Sweden). Chromosomal DNA was
routinely digested with 10 to 20 U of EcoRI per µg of DNA
for 20 h at 37°C. Agarose gel electrophoresis of restricted DNA
samples was performed with 0. 8% agarose (wt/vol) in Tris-borate-EDTA
buffer (42) at 2 V cm
1 for 16 h at room
temperature. Bacteriophage lambda DNA digested with
HindIII and labeled with digoxigenin (DIG; Boehringer
Mannheim Biochemicals [BMB], Mannheim, Germany) was routinely used as
the molecular weight marker.
Cloning and nucleotide sequencing of the 0.58-kb fragment from
the strain C3L4 genome and cloning of part of the nifH gene
of strain P3L5T.
The degenerate primers described by
Zehr and McReynolds (57) were used to amplify
nifH DNA from P. azotofixans
P3L5T for the cloning experiments. Furthermore, a fragment
of 0.58 kb from the P. azotofixans C3L4 genome was
obtained by PCR with the random primer P5 (38). The PCR
products were gel purified with a GeneClean II kit (Bio 101, La Jolla,
Calif.) and cloned into the cloning vector pCRII (Invitrogen, Leek, The
Netherlands), after which competent Escherichia coli
Inv-alfa cells were transformed (TA cloning kit) according to the
protocol of the manufacturer (Invitrogen). Plasmids with the 0.58-kb
insert, to be used as templates in sequencing reactions, were purified
by using Wizard resin spin columns (Promega, Madison, Wis.), and a
Thermo Sequenase fluorescently labeled-primer cycle sequencing kit,
with 7-deaza-dGTP (Amersham Life Science, Inc., Arlington Heights,
Ill.), was used with this DNA. Both strands of the cloned amplification
products were sequenced with an automatic sequence analyzer (ALF DNA
sequencer; Pharmacia). The DNA sequence data were analyzed by using the
sequence analysis software package developed by the University of
Wisconsin's Genetics Computer Group (for its Wisconsin Package) via
the CAOS/CAMM Center (University of Nijmegen, Nijmegen, The
Netherlands).
Isolation of plasmid DNAs.
E. coli strains harboring
different plasmids were maintained at 
20°C in Luria-Bertani broth
(43) with 20% glycerol. Whenever necessary, this broth was
supplemented with 10 µg of ampicillin per ml (i.e., for plasmids
pCRII nifH or pCRII 0.58). Plasmids pSA30 (Klebsiella
pneumoniae nifKDH gene cluster cloned into pACYC184) (6), pCRII nifH (38), and pCRII 0.58 (this work) were isolated by the alkaline lysis method of Birnboim and
Doly (5). The preparations were purified in CsCl-ethidium
bromide gradients as described by Sambrook et al. (42).
Preparation of plasmid DNA probes, blotting, and hybridization
procedures.
The probes of the three plasmids were random-primer
labeled with DIG by using a DIG labeling kit (BMB). Chromosomal DNAs
digested with EcoRI were blotted from gels to positively
charged nylon membranes (BMB) as described by Sambrook et al.
(42). Prehybridization and hybridization conditions with
DIG-labeled probes were those described by the BMB manual for the DIG
nucleic acid detection kit.
PCR amplifications.
Amplification reactions with primers
BOXA1R (25) and REPI and REPII (54) were
performed in the following mix: 50 ng of target DNA; 2.5 µl of 10×
PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl); 4 mM
MgCl2; 250 µM concentrations of each deoxynucleoside triphosphate; 2 µmol of the primer BOXA1R or REPI-REPII; and 1.25 U
of Taq polymerase (Gibco BRL, Gaithersburg, Md.) in a
25-µl final volume. The reaction mixtures were overlaid with 20 µl
of mineral oil (Sigma, St. Louis, Mo.). The cycle applied was 1 course of 90 s at 94°C, 3 min at 47°C, and 3 min at 72°C; 35 courses of 30 s at 94°C, 30 s at 53°C (with BOX primers)
or 47°C (with REP primers), and 1 min at 72°C; and 10 min at
72°C. With the primers BAZO1 and BAZO2, the PCR mixtures were run for
1 course of 1 5 min at 94°C; 35 courses of 1 min at 94°C, 1 min at
55°C, and 2 min at 72°C; and 1 course of 10 min at 72°C. The mix
used contained 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 µM
concentrations of each deoxynucleoside triphosphate, 3 mM
MgCl2, 0.2 µM concentrations of each primer, 100 ng of
target DNA, and 1.25 U of Taq polymerase. Agarose gel
electrophoresis of PCR products was performed with 1.6% agarose in
Tris-borate-EDTA buffer at 10 V/cm for 2 h at room temperature.
Statistical analyses.
The results of phenotypic and
hybridization analyses were collected into a matrix indicating the
presence or absence (scored as 1 or 0, respectively) of the parameters
studied. A dendrogram was obtained on the basis of the data, following
cluster analysis with minimum-variance criteria (Ward) and Euclidean
distance (35). The data of the matrix were analyzed for
principal components with Euclidean distance. The computational
calculations of principal components and cluster analysis were done
with routines written by A. Nobrega with the software MATHEMATICA
(Wolfram Research Inc., Champaign, Ill.). Data from hybridization
experiments were clustered according to similarities between individual
isolates taken in pairs. Simple matching coefficients were calculated
with the NTSYS software package for personal computer (version 1.8; Exeter Software, Setauket, N.Y.). Multivariate analysis of variance (MANOVA) of data from rep-PCR genomic fingerprinting was done with the
statistical package NTSYS.
Nucleotide sequence accession number.
The sequence obtained
in this study was deposited in the EMBL database with the accession no.
AJ005254.
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RESULTS |
Isolation and identification of P. azotofixans
strains.
Sixty strains identified as P. azotofixans were isolated from nonrhizospheres (MSV
strains), rhizospheres (PV or LV strains), or rhizoplanes
(MRV strains) of maize planted in Várzea soil. Forty-six strains
were isolated from nonrhizospheres (CSM strains) or
rhizospheres (CRiP or CRiL strains) of maize planted in
Cerrado soil. Strains were isolated 10, 30, 60, and 90 days following maize sowing, and they were numbered based on when they were isolated: 10 days (strains with numbers 1 to 49), 30 days (strains with numbers
50 to 99), 60 days (strains with numbers 100 to 149), and 90 days
(strains with numbers 150 to 200). A total of 106 strains (Table
1) isolated in this study were analyzed
for their phenotypic and genetic characteristics.
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TABLE 1.
Designations of P. azotofixans strains
isolated from maize plantings at different stages of growth in Cerrado
and Várzea soils
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All strains were identified as
P. azotofixans based on
the characteristics previously described for the species (
38,
46,
48) and with specific primers and a probe, which were constructed
based on three variable regions of the 16S rRNA gene sequence
of
P. azotofixans (
37). The PCR product
generated with all target
strains was 565 bp long and was specifically
detected with the
18-mer probe (data not shown). All strains were
facultatively
anaerobic, were catalase positive and oxidase negative,
were Voges-Proskauer
test positive, and did not reduce nitrate, as
evidenced by the
methods described by Gordon et al. (
19).
Also, all strains isolated
here effectively reduced acetylene to
ethylene in assays for acetylene-reducing
activity and were able to
solubilize calcium phytate (organic
phosphate). When
P. azotofixans strains were tested by using the
API system (API
50CH), they all produced the pattern previously
described for the
species (
38,
48). Abilities to ferment three
carbohydrates
(starch, sorbitol, and dulcitol) varied among strains,
and these
results could be used to divide strains into groups,
as proposed in the
work of Rosado et al. (
38). Strains isolated
from Cerrado
soil could be divided into three groups of related
strains (Table
2). The largest group (61%) was formed
by strains
not able to use any of these three carbohydrates. Strains
isolated
from Várzea soil formed four different groups of related
strains
(Table
2). The largest group (70%) was made up of strains able
to metabolize starch but not sorbitol or dulcitol. Only one
Várzea
soil strain (MSV158) showed the same phenotype as the
majority
of strains from Cerrado soil (inability to metabolize
starch,
sorbitol, and dulcitol). Also, only one strain (MSV6) was able
to metabolize dulcitol.
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TABLE 2.
Phenotypic characteristics of strains of
P. azotofixans isolated from maize planted in Cerrado
and Várzea soils
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Strains were also tested for their capacity to produce antimicrobial
substances and for their resistance to different antibiotics
and heavy
metals. Among the Várzea soil isolates, 21 strains
(35%) were
able to inhibit the growth of the indicator strain,
and among the
Cerrado soil isolates, 18 strains (39%) showed this
characteristic.
None of the strains tested was able to grow in
media containing
antibiotics (chloramphenicol, erythromycin, and
rifampin) or heavy
metals (CuSO
4, NiSO
4, HgCl
2, and
CoSO
4).
When the phenotypic characteristics (fermentation of carbohydrates,
antimicrobial substance production, and solubilization
of phosphate)
were considered, six groups of strains could be
formed with the Cerrado
soil isolates and with the Várzea soil
isolates (Table
2).
However, if we consider all strains together,
seven groups of strains
could be formed.
Sequence analysis of a 0.58-kb random fragment from strain
C3L4.
A fragment of 0.58 kb was obtained from strain C3L4 by
randomly amplified polymorphic DNA-PCR with primer P5 (38),
cloned, and sequenced. This fragment was shown to be present in 34% of the P. azotofixans strains tested, and this fragment
was proposed for use as a probe to detect this subgroup of strains
directly in soil or to identify them in taxonomic studies
(38). The DNA sequence of the 0.58-kb fragment of C3L4 was
compared with sequences present in the GenBank and EMBL databases, by
using FASTA (18) or sequence alignments and by determining
percentages of homology. The results obtained showed that the sequence
has a low level of similarity to any one of the sequences present in
the databases. The best scores occurred with a 518-bp sequence that
overlaps a fragment of Bacillus subtilis chromosomal DNA
corresponding to a replication origin (63.3% identity) and with an
ATPase from B. subtilis Marburg 168.
DNA homology to nifKDH, to part of nifH,
and to a fragment of 0.58 kb from P. azotofixans
C3L4.
All strains were tested for the presence of homology to the
K. pneumoniae nifKDH gene cluster (nitrogenase structural
genes cloned in pACYC184-originating plasmid pSA30) and to part of the nifH gene from P. azotofixans
P3L5T (cloned in pCRII) by Southern hybridization.
Total DNA from all strains (digested with EcoRI) showed the
same pattern of homology to plasmid pSA30 as to plasmid pCRII
nifH. These hybridizations allowed the separation of strains
from Várzea soil into 15 different groups and strains from
Cerrado soil into only 2 groups of related strains (Table
3). Among the strains from Várzea
soil, 53.3% showed a hybridization pattern similar to that shown by
strains from Cerrado soil (Table 3; Fig.
1).
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TABLE 3.
Hybridization of DNAs from
P. azotofixans strains isolated from maize in
different stages of plant growth with K. pneumoniae
nifKDH or part of nifH from
P. azotofixans P3L5T
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FIG. 1.
Southern hybridization of DNAs from P. azotofixans strains isolated from maize planted in Cerrado and
Várzea soils with the following probes: nifKDH from
K. pneumoniae or part of nifH from P. azotofixans (A) and the 0.58-kb fragment from P. azotofixans C3L4 (B). Only one representative strain of each major
group is presented here. Lanes: 1, pattern of 1-kb ladder (Gibco BRL);
2, pattern observed in 58.7% of strains from Cerrado soil and 26.7%
of strains from Várzea soil; 3, pattern observed in 41.3% of
strains from Cerrado soil and 26.7% of strains from Várzea soil;
4, pattern observed in 100% of strains from Cerrado soil and 58.3% of
strains from Várzea soil.
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Strains were also tested for the presence of DNA homology to the
0.58-kb fragment from
P. azotofixans C3L4 cloned in
pCRII
and sequenced in this study. Genomic DNAs of all strains
digested
with
EcoRI showed homology to plasmid pCRII
0.58 but not to the
cloning vector. Five different patterns of
hybridization could
be observed in the strains from Várzea soil.
One of these groups
is made up of 58.3% of the strains. A 5.1-kb
EcoRI fragment was
detected in all strains isolated from
Cerrado soil, and this pattern
was the same as that observed for the
prevalent group of strains
from Várzea soil (Table
4; Fig.
1).
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TABLE 4.
Hybridization of DNAs from
P. azotofixans strains isolated from maize in
different stages of plant growth with a genomic fragment of 0.58 kb
from P. azotofixans C3L4 cloned in pCRII
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Cluster analysis based on phenotypic and hybridization data.
Simple matching coefficients were calculated with hybridization data
and are presented in Tables 3 and 4.
Phenotypic and hybridization results were used in a matrix indicating
the presence or absence (scored as 1 or 0, respectively)
of the
parameters studied. The data were clustered according to
minimum-variance criteria (Ward) with Euclidean distance. The
dendrogram obtained is shown in Fig.
2.
Two main clusters, denoted
A and B, were observed. Cluster A was made
up predominantly of
strains from Cerrado soil, while cluster B was made
up exclusively
of strains from Várzea soil. Cluster A comprised
16 groups harboring
82 strains, while cluster B comprised 13 groups
harboring 24 strains.
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FIG. 2.
Dendrogram based on data from phenotypic characteristics
and from hybridization studies, followed by cluster analysis with
minimum-variance criteria (Ward) and Euclidean distance. Strains in
each group are as follows: group 1, CRiP57, CRiL56, CRiP21, PV21,
MSV23, and CSM56; group 2, PV150; group 3, LV17 and LV10; group 4, CRiL151, CRiP165, MSV66, MSV54, CSM116, and LV106; group 5, CSM159 and
CSM100; group 6, CRiL123, CRiP129, CRiP128, CRiP127, CRiP115, CRiP105,
CRiP104, CRiP103, CSM64, CRiP9, CSM163, CSM157, and CSM154; group 7, PV63; group 8, CRiP126; group 9, CSM27, CRiP15, CRiP53, CRiL125, and
CSM63; group 10, MSV6; group 11, CSM108, CSM107, CSM106, CRiP125,
CRiP58, CRiL36, CRiL35, and CRiL33; group 12, LV114, MRV111, MSV159,
MSV157, PV151, CSM53, PV56, PV55, PV54, MSV15, PV23, PV18,
MSV107, and MSV101; group 13, CSM50, MSV62, CSM169, CSM165, and CSM152;
group 14, MRV117, MRV108, MRV106, MRV105, MRV100, CRiL124, LV115,
LV107, CSM121, CRiP158, CRiP153, CRiP151, PV156, PV155, and PV104;
group 15, PV163; group 16, PV159; group 17, MSV63 and MSV55; group 18, MSV68; group 19, MSV166, MSV156, MSV155, and MSV153; group 20, MSV7 and
LV12; group 21, MSV158; group 22, MSV32; group 23, LV16 and LV15; group
24, MSV25; group 25, MSV108, LV56, LV53, and MSV5; group 26, MSV151;
group 27, PV19 and PV9; group 28, MRV156 and MRV154; group 29, MRV153.
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Data from phenotypic characteristics and hybridization experiments were
also subjected to principal-component analysis (Fig.
3). Factors 1 and 2 were found to explain
56% of the total variance.
The remaining variance was more evenly
distributed among factors,
with factors 3 to 9 representing 36% of
variance. The division
of the data into two main clusters, as shown in
Fig.
3, was again
present in the factorial plane. The dichotomy was
caused mainly
by the distribution of the data in the first factor. The
contribution
of each variable to the first factor is shown in Fig.
3.
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FIG. 3.
Principal-component analysis of phenotypic and
hybridization data from the 106 P. azotofixans strains
(Cerrado and Várzea soils). Factors 1 and 2 reflect 56% of the
total variance. Numbers correspond to the groups defined in Fig. 2.
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PCR analyses.
Primers corresponding to conserved DNA sequences
of rep elements, BOXA1R and REPI-REPII, when used with P. azotofixans genomic DNA, generated different fingerprints. The
fingerprints obtained from Várzea soil strains with the BOX
primer and those from Cerrado soil strains with the REP primers could
not be used in this study because of the excessive number of bands
present in the fingerprints. With the optimized parameters for rep-PCR
described previously (38), amplification of template DNA of
each of the 60 strains from Várzea soil and of the 46 strains
from Cerrado soil was achieved with the REP primers and the BOX primer,
respectively. Comparison of the sizes of the amplification products
with molecular size markers via gel electrophoresis allowed for an
estimation of their lengths. Figure 4
shows the different P. azotofixans groups based on
these PCR patterns. From the variation in the numbers and sizes of
bands, it was possible to identify similarities among strains,
resulting in 21 different groups being identified among strains from
Várzea soil and in 4 different groups being identified among
strains from Cerrado soil.
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FIG. 4.
Grouping of P. azotofixans strains from
maize planted in Várzea and Cerrado soils by their amplification
patterns by PCR with REP and BOX primers, respectively. (A)
Várzea soil strains. Strains in each group are as follows: group
1, MSV5, MSV7, LV10, LV15, LV16, LV17, PV9, and PV19; group
2, MSV6; group 3, LV12; group 4, MSV15, MSV23,
PV18, PV21, PV23, PV54, PV55, PV56, MRV111, MSV101, MSV107,
LV114, MSV157, MSV159, PV151, and PV163; group 5, MSV25;
group 6, MSV32; group 7, MSV55 and MSV63; group
8, MSV54 and MSV62; group 9, MRV153; group 10, MRV154 and MRV156; group 11, MSV151; group 12, MSV153, MSV155, and MSV166; group 13, MSV156;
group 14, MRV100, MRV105, MRV106, MRV108, MRV117, LV107,
LV115, PV104, PV155, and PV156; group 15, PV150; group 16, PV159; group 17, MSV66; group 18, MSV68; group 19, LV106; group 20, LV53, LV56, and MSV108; and group 21, PV63 and
MSV158. (B) Cerrado soil strains. Strains in each group are as follows:
group 1, CSM50, CRiP53, CSM63, CRiL124, CRiL125, CSM107,
and CSM152; group 2, CSM53, CRiP58, CSM100, CSM106, CSM108,
CRiP103, CRiP105, CRiP115, CRiP125, CRiP127, CRiP128, CRiP129, and
CRiL123; group 3, CRiP9, CRiP15, CRiP21, CRiL33, CRiL35, CRiL36, CSM27,
CRiL56, CRiP57, CSM56, CSM64, CRiP104, CRiP126, CSM116,
CRiL151, CRiP165, CSM163, and CSM169; and group 4, (not shown) CSM121,
CSM157, CSM159, CSM165, CSM154, CRiP151, CRiP153, and CRiP158. The
underlined strains correspond to the samples on the gel. Lane numbers
correspond to group numbers. Lane a contains a 1-kb ladder (Gibco
BRL).
|
|
All
P. azotofixans strains isolated in this study were
classified into groups according to different criteria: (i)
P. azotofixans populations isolated from rhizoplanes,
rhizospheres, and nonrhizospheres
and (ii)
P. azotofixans populations obtained from four different
samplings carried out 10, 30, 60, and 90 days after planting (sampling
1 [S1], S2, S3, and S4, respectively). Patterns obtained by rep-PCR
of the different groups were tested for identity by MANOVA. The
resulting scores of comparisons between groups are shown in Table
5. The data showed the following. (i)
Strains from Várzea soil
isolated from rhizoplanes,
rhizospheres, and nonrhizospheres differed
significantly (
P = 0.006). Two-by-two group tests also
were performed,
and strains isolated from rhizoplanes and from
nonrhizospheres
showed the most significant difference
(
P = 0.019) when compared
to strains isolated from
rhizoplanes and rhizospheres (
P = 0.813)
and
those isolated from rhizospheres and nonrhizospheres
(
P =
0.189). (ii) Strains from Cerrado soil isolated
from rhizospheres
and nonrhizospheres were not
significantly different (
P = 0.938).
(iii) Strains from
S1, S2, S3, and S4 isolated from Várzea soil
were statistically
distinct with high significancy (
P < 0.002).
(iv)
Strains from S1, S2, S3, and S4 isolated from Cerrado soil
were also
statistically distinct, with high significance (
P =
0.007). MANOVA was also done for all the possible combinations
of two
by two and three by three with S1, S2, S3, and S4 strains.
The results
showed that, among strains from Várzea soil, all
three-by-three
groupings that included S1 strains were very significantly
different by
MANOVA (
P < 0.02), suggesting that the S1 group of
strains deviates from the other groups. Among strains from Cerrado
soil, the tests that included combinations of two and three groups
showed that S3 and S4 strains contribute more than S1 and S2 strains
to
the heterogeneity of the whole population.
View this table:
[in this window]
[in a new window]
|
TABLE 5.
MANOVA of 106 P. azotofixans strains
from Várzea and Cerrado soils with REP and BOX
patterns, respectively
|
|
| |
DISCUSSION |
The potential for bacterial adaptation to highly heterogeneous and
fluctuating environments like the rhizoplane, rhizosphere, or
nonrhizosphere depends on their genetic diversity. A
combination of phenotypic and genetic measures is known to provide a
good estimate of soil bacterial diversity (51); however,
studies of dominant diazotrophs or plant growth-promoting rhizobacteria associated with maize roots and rhizospheres have been
performed only with isolates of Azospirillum spp. (15,
52, 55, 56), Enterobacter cloacae (34),
Azotobacter spp. (22), Bacillus circulans, Klebsiella terrigena, Rahnella
aquatilis (4), and Burkholderia cepacia
(13). Strains of P. azotofixans in the maize
rhizosphere have also been described, but no data were provided on the putative selection of a specific phenotypic or genotypic subpopulation of P. azotofixans in this environment
(38). This fact may be explained by the approach used, as
the populations in individual plant samples during the different stages
of plant growth were not assessed. Therefore, in this study, two
dominant Brazilian clay soils were planted with maize and four stages
of plant growth were analyzed to characterize the P. azotofixans populations. The analysis performed on the phenotypic
features of 60 strains from Várzea soil and 46 strains from
Cerrado soil showed that their identification as P. azotofixans was unambiguous and that they could be divided into
six groups for each soil (Table 2). The major group of Várzea
soil isolates (70% of the strains), and also 12 strains from Cerrado
soil, were capable of metabolizing starch. Hence, starch metabolism
might play an important role in the establishment of rhizobacteria in
the vicinity of maize roots, as proposed by Mavingui et al.
(28) for sorbitol metabolism in wheat roots. Other sugars
(glucose, galactose, inositol, arabinose, and fucose) were shown to be
present in the exudates of several plants (1), but some of
them are not metabolized by P. azotofixans strains
(48).
The method developed by Pace et al. (32) to determine
species diversity with ribosomal DNA was extended by applying it to a
functionally important gene, nifH, to determine the
importance and diversity of nitrogen-fixing bacteria associated with
rice roots (53). DNA probes containing K. pneumoniae
nifKDH have been used to identify related sequences from several
nitrogen-fixing organisms (31, 33, 41, 49). Different
hybridization patterns have been reported for different strains. In
this study, 15 different hybridization patterns were observed in 60 P. azotofixans strains from Várzea soil and only
2 patterns were observed in 46 strains from Cerrado soil when the
plasmids pSA30 and pCRII nifH were used as probes (Table
3). All strains isolated from Cerrado soil showed a characteristic
fragment of 4.0 kb, demonstrating their great homogeneity with respect
to nif structural genes. Previous studies have also
demonstrated the presence of various hybridization patterns in
P. azotofixans strains (38, 49).
Data from phenotypic and hybridization analyses were used to construct
a dendrogram. Although this dendrogram does not imply phylogenetic
relationships, it is potentially useful for defining whether a
population is clonal or not, as suggested by different authors
(13, 29). All strains could be distributed in 29 groups. Some groups contained more than one strain, indicating that they are
genetically closely related and that they may have originated from a
common ancestor. The strains isolated from Cerrado soil were less
heterogeneous than the strains from Várzea soil, and a possible
reason for this major difference may be the soil type. Although both
soils have a clay texture, Cerrado soil is a dark-red latosol
distrophic soil and Várzea is a low-humus eutrophic gley. However, it is still unknown whether the difference in the
constitutions of the soils used in this study is responsible for the
differences demonstrated by the heterogeneity between Cerrado and
Várzea soil populations. Berge et al. (4) have also
shown that the compositions of the populations of B. circulans varied in the rhizospheres of maize depending on
the soil in which the maize was planted.
Total DNA from all strains from Várzea soil was amplified with
the primers REPI and REPII, and total DNA from all strains from
Cerrado soil was amplified with the primer BOXA1R. The use of
rep-PCR has previously been described (3, 24, 25, 40). Only
four PCR fingerprints were observed in Cerrado soil isolates with the
BOX primer, suggesting a considerable degree of DNA conservation over
the genomes of all 46 strains. Figure 4 shows these similar amplification patterns, with consistent bands appearing in the different groups. By PCR with the REP primers, the strains isolated from Várzea soil could be separated into 21 groups.
Data from rep-PCR analyses were used for MANOVA. Our study showed that
plant development significantly affected the diversity of P. azotofixans populations associated with maize planted in Várzea and Cerrado soils. Particularly for Várzea soil, it
was found that strains isolated during the first stage of maize growth may play an important role in the difference observed in P. azotofixans populations during the maize life cycle. This
difference could be explained by the unstable ecosystem of young plants
compared to the more stable ecosystem of mature plants. It is well
known that production and diffusion of root exudates, which represent nutritional sources for rhizosphere microorganisms, are
affected by plant development (10, 39). Hence, it is
expected that the microflora changes while the exudation patterns of
roots vary as plants grow (20). Di Cello et al.
(13) and McArthur et al. (30) working with
Burkholderia cepacia isolated from maize also found that the
Burkholderia cepacia populations changed while plants grew.
Concerning the populations of P. azotofixans isolated
from rhizoplanes, rhizospheres, and
nonrhizospheres during the four stages of maize growth,
it can be observed that among strains from Várzea soil the most
significant difference is observed between strains isolated from
rhizoplanes and those isolated from nonrhizospheres. This
suggests that a subpopulation of P. azotofixans may
have been selected by the plant. Similar results were described by
Mavingui et al. (28) for wheat roots with
Paenibacillus polymyxa isolates. No significant difference
was observed between strains isolated from Cerrado soil
rhizospheres and nonrhizospheres. Comparisons among rhizoplanes, rhizospheres, and
nonrhizospheres from Cerrado soil could not be done because
P. azotofixans strains were not isolated from
rhizoplanes. This inability to isolate P. azotofixans might have been due to a problem with sampling the rhizoplanes or to a
weak rhizosphere effect, influenced by the soil type. A similar
situation was previously reported by Hekman et al. (21) for
Burkholderia cepacia.
In this study, differences in the populations of P. azotofixans during four stages of maize growth were demonstrated.
Also, it was shown that the degree of diversity of P. azotofixans isolates was affected by the soil type and that
populations from rhizoplanes, rhizospheres, and
nonrhizospheres of Várzea soil were statistically different. These observations should be taken into account in the
selection of P. azotofixans strains for use as
maize inoculants. Furthermore, since Garcia de Salomone et al.
(17) suggested that the populations of
Azospirillum spp. in maize are dependent on the plant
genotype, additional data which consider the populations of different
cultivars of maize samples, cultivated in Várzea and Cerrado
soils, are needed in our work.
| |
ACKNOWLEDGMENTS |
This work was supported by FINEP and by the National Research
Council of Brazil (CNPq).
Thanks are due to colleagues from EMBRAPA-CNPMS who provided maize
samples.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Laboratório de Genética Microbiana, Depto. Microbiologia
Geral, Instituto de Microbiologia, Universidade Federal do Rio de
Janeiro, CCS, Bloco I, Ilha do Fundão, CEP 21941-590, Rio de
Janeiro, RJ, Brazil. Phone: 55-21-590 30 93. Fax: 55-21-259 99 57. E-mail: immgsel{at}microbio.ufrj.br.
| |
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55:2522-2526[Abstract/Free Full Text].
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Applied and Environmental Microbiology, October 1998, p. 3860-3868, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
