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Journal of Bacteriology, April 2001, p. 2634-2645, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2634-2645.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Endophytic Colonization of Rice by a Diazotrophic
Strain of Serratia marcescens
Prasad
Gyaneshwar,1
Euan K.
James,2
Natarajan
Mathan,1
Pallavolu M.
Reddy,1
Barbara
Reinhold-Hurek,3 and
Jagdish
K.
Ladha1,*
International Rice Research Institute, Los
Baños, Philippines1; Department of
Biological Sciences, University of Dundee, Dundee DD1 4HN, United
Kingdom2; and University of Bremen,
Faculty of Biology and Chemistry, Institute of General
Microbiology, D-28334 Bremen, Germany3
Received 14 August 2000/Accepted 8 January 2001
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ABSTRACT |
Six closely related N2-fixing bacterial strains were
isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia
marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen
for further analysis showed acetylene reduction activity (ARA) only
when inoculated into media containing low levels of fixed nitrogen
(yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of
15N2 incorporation and by sequence analysis of
the PCR-amplified fragment of nifH. To examine its
interaction with rice, strain IRBG500 was marked with gusA
fused to a constitutive promoter, and the marked strain was inoculated
onto rice seedlings under axenic conditions. At 3 days after
inoculation, the roots showed blue staining, which was most intense at
the points of lateral root emergence and at the root tip. At 6 days,
the blue precipitate also appeared in the leaves and stems. More
detailed studies using light and transmission electron microscopy
combined with immunogold labeling confirmed that IRBG500 was
endophytically established within roots, stems, and leaves. Large
numbers of bacteria were observed within intercellular spaces,
senescing root cortical cells, aerenchyma, and xylem vessels. They were
not observed within intact host cells. Inoculation of IRBG500 resulted
in a significant increase in root length and root dry weight but not in
total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was
added to the rooting medium.
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INTRODUCTION |
Rice (Oryza sativa) is
arguably the most important cereal crop in the world, feeding more than
50% of the world's population (18, 38). However, the
population is growing at a rapid rate; therefore, rice yields will need
to be enhanced to match the increased consumption. Achieving these
higher yields by 2020 will require at least double the amount of N
fertilizers currently being used because, after water, N is the most
limiting nutrient for rice growth (22). An alternative to
the increased use of chemical fertilizers is to explore and improve the
ability of rice to obtain N from biological N2 fixation
(BNF) (37, 69).
It has long been known that rice can form natural associations with
various N2-fixing bacteria, both phototrophs and
heterotrophs (3, 4, 42, 53, 70). All or some of these may
be responsible for supplying the plants with fixed N (27,
37). Moreover, in addition to the culturable diazotrophs
associated with rice, a substantial molecular diversity of
N2-fixing bacteria has been detected in field-grown rice
based on retrieval of nifH or nifD gene fragments
from root DNA (12, 63, 64). However, the contribution of
the bacteria externally associated with rice is insufficient to sustain
a high yield (39). It has been suggested that bacteria
colonizing the plant interior might interact more closely with the
host, with less competition for carbon sources and a more protected
environment for N2 fixation (49, 51), such as
that occurring in the relatively efficient N2-fixing
symbioses between rhizobia and legumes (45).
In view of the above, a global frontier project which aims to transfer
an N2 fixation capability to rice has begun
(38). One of the approaches toward this goal is the use of
natural N2-fixing endophytic bacteria associated with rice.
It has been suggested that endophytic N2-fixing bacteria,
particularly Acetobacter diazotrophicus and
Herbaspirillum spp. (8, 26), may be responsible
for the significant BNF observed in some Brazilian varieties of
sugarcane (Saccharum spp.) (65). Similarly,
Azoarcus spp. might be responsible for N2
fixation in Kallar grass (Leptochloa fusca) (42, 50, 51). Therefore, it is possible that rice varieties showing
potential for N2 fixation (41, 57) could also
harbor effective endophytic diazotrophs. Indeed, recent studies have
shown that numerous and varied diazotrophic bacteria can be isolated
from wetland rice after surface sterilization, thus indicating a
potential endophytic location for them (6, 60). Surface
sterilization in itself, however, is insufficient evidence to ascribe
an endophytic location for these bacteria, and it is now considered
that only direct localization using microscopy can provide such
evidence (23, 26, 50). In view of the above, this study
aimed to characterize the predominant endophytic diazotrophic bacteria
that are present within various rice varieties grown under greenhouse
conditions. Using light and electron microscopy, the colonization of
rice seedlings by a selected isolate (Serratia marcescens
IRBG500) was examined in detail. To the best of our knowledge, this is the first detailed ultrastructural study of a naturally occurring diazotrophic endophyte in rice.
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MATERIALS AND METHODS |
Isolation of endophytic bacteria and determination of
diazotrophy.
Roots and stems of seven different rice varieties
(Table 1) growing in nonsterile flooded
soil in a greenhouse were collected and washed with tap water, blotted,
and weighed. The roots were surface sterilized with 70% ethanol for 5 min and then treated with 0.2% mercuric chloride for 30 s. The stems
were cut into small (approximately 5-cm) pieces and surface sterilized
by dipping in 95% ethanol and flaming. Approximately 1 cm was then
removed from each end. The root and the stem were checked for the
efficacy of sterilization by rolling them on 0.1% tryptic soy agar
(TSA) plates. They were then homogenized, under sterile conditions, with a mortar and pestle in phosphate-buffered saline, and different dilutions were placed on TSA plates to determine the total
heterotrophic bacterial population. Serially diluted homogenate was
also inoculated into tubes containing a semisolid N-free medium
consisting of (per liter) malic acid (5 g),
K2HPO4 (0.5 g), MgSO4 · 7H2O (0.2 g), NaCl (0.1 g), CaCl2 (0.02 g), and
0.5% bromothymol blue in 0.2 N KOH (2 ml), 1.64% Fe-EDTA solution (4 ml), and agar (2 g) (33). The final pH was adjusted to 7.0 by KOH. The medium was modified by adding yeast extract (0.02 g), as it
is known that a trace amount of fixed nitrogen is required for the
isolation of most diazotrophs from the rhizosphere of rice
(67). The bacteria from the acetylene reduction activity
(ARA)-positive tubes were further streaked onto agar plates (1.5%
[wt/vol]) with the same medium containing 0.1 mM NH4Cl to
obtain pure colonies.
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TABLE 1.
Isolation of putative endophytic bacteria from seven
varieties of rice grown under greenhouse conditions
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Analysis of strain diversity and identification of diazotrophic
bacteria in various rice varieties.
Diazotrophic bacteria isolated
from different parts of rice were analyzed for diversity by
fingerprinting using BOX-PCR amplification fragment length polymorphism
as described by Verslovic et al. (66). A BOX A1R primer
(5'-CTACGGCAAGGCGACGCTGACG-3') was used at 50 pmol with 100 ng of template DNA in a 25-µl PCR mixture containing 1.25 mM each
deoxynucleoside triphosphate and 2 U of AmpliTaq DNA polymerase
(Perkin-Elmer) in a reaction buffer with 10% (vol/vol) dimethyl
sulfoxide; 5× reaction buffer stock contained 83 mM ammonium acetate,
335 mM Tris-HCl, 33.5 mM MgCl2, 33.5 µM EDTA, 150 mM
-mercaptoethanol, and 850 µg of bovine serum albumin ml
1 (pH 8.8). PCR amplification was performed in a
BIOMETRA Uno-Thermocycler with an initial denaturation (95°C, 7 min)
followed by 30 cycles of denaturation (95°C, 30 s; 95°C, 1 min), annealing (52°C, 1 min), and extension (65°C, 8 min), with a
single final extension (65°C, 16 min). After the reaction, the
samples were separated on 1.5% agarose gels. The resulting
amplification patterns were analyzed, and the strains were grouped
according to their fingerprints.
The strains showing similar fingerprints were further identified by
direct sequencing of PCR-amplified 16S rRNA gene (rDNA)
fragments as
described earlier (
21). The PCR primers 25f
(5'-AACTKAAGAGTTTGATCCTGGCTC-3')
and 1492r
(5'-ACGGYTACCTTGTTACGACTT-3') were used at 50 pmol each
with
1 to 2 ng of template DNA in PCR buffer containing 20 mM
Tris-HCl (pH
8.7), 10 mM KCl, 0.005% Tween 20, 100 µg of nuclease-free
bovine
serum albumin ml
1, 7.5 mM MgCl
2, 1.25 mM each
deoxynucleoside triphosphate, and
2 U of
Taq polymerase
(Beckman Instruments, Munich, Germany),
in a total volume of 25 µl.
The DNA was amplified in a Techne
Progene thermocycler (Thermo-Dux,
Wertheim, Germany) after initial
denaturation at 95°C with 27 cycles
of 1 min of denaturation at
95°C, 2 min of annealing at 59°C, and 2 min of extension at 72°C,
with a final extension step of 4 min at
72°C. The amplification
product was treated with exonuclease I (10 U)
and shrimp alkaline
phosphatase (2 U; Amersham International, Little
Chalfont, United
Kingdom) for 15 min at 37°C and then for 15 min at
80°C. The DNA
template was dialyzed against distilled water for 15 min before
direct sequencing with 7-deaza-dGTP (catalog no. 2438;
Amersham
International) according to the manufacturer's instructions.
A
Techne Cyclogene thermocycler (Thermo-Dux) was used with 25 cycles
of
denaturation at 95°C for 30 s and annealing and extension at
60°C for 30 s, after an initial denaturation at 97°C for 1 min.
Sequencing primers were labeled at the 5' end with the fluorescent
dye Cy-5 in order to allow automated sequencing with an
ALF
express apparatus (Pharmacia). Primers used were 35fC
(5'-CTKAAGAGTTTGATCMTGGCTCAGATTGAACG-3'),
342fC
(5'-CTCCTACGGGAGGCAGCAG-3'), and 530mfC
(5'-CTACGTGCCAGCMGCCGCGG-3').
The resulting sequences were
compared with known sequences by
using the BLAST
program.
Diazotrophy of S. marcescens IRBG500.
Diazotrophy of S. marcescens IRBG500 and S. marcescens IRBG500-gusA was determined on the basis of
ARA (5) and 15N2 incorporation
(2, 32) and by sequence analysis of the nifH
fragment. As glucose is the preferred carbon source for growth of
diazotrophic strains of enterobacteriaceae (10), nitrogen fixation was characterized using nitrogen-free dextrose medium (10), which was modified by addition of yeast extract (100 mg liter1), with or without 10 mM NH4Cl as an
additional N source. The bacteria were grown in Luria broth to
stationary phase, and then 200 µl of each culture was inoculated into
10 ml of nitrogen-free dextrose medium. The cells were incubated on a
orbital shaker (100 rpm) at 30°C overnight, and then 10% (vol/vol)
of the headspace was replaced with 15N2 (99.5%
15N; Monsanto Research Corporation, Miamisburg, Ohio)
before being incubated for another 3 days. After this time, 1 ml of
acetylene was injected into the flasks, which were then incubated for a further 4 to 6 hours at 30°C. Ethylene produced by the bacteria was
detected on a Hitachi 164F gas chromatograph. After determination of
the ARA, a subsample was removed for protein estimations by the
dye-binding method of Bradford (9), and the remainder of the cultures were freeze-dried. A 10-mg fine subsample of the freeze-dried material was used for analysis of 15N by a
mass spectrometer (VG model 903) equipped with a Dumas elemental
analyzer (Roboprep-CN 7001; Europa Scientific Ltd., Crewe, United
Kingdom) at the Analytical Services Laboratory, International Rice
Research Institute (IRRI). Uninoculated flasks served as controls. For
evaluation in the context of related bacteria, the nitrogenase activity
(ARA) of IRBG500 was compared with those of the type strains of
S. marcescens (LMG2792) and S. rubidaea (LMG5019), as well as two strains of diazotrophic enterobacteria previously isolated from the rhizosphere of rice (35),
Klebsiella planticola and Enterobacter cloacae.
The presence of
nif genes in IRBG500 was confirmed by
amplification of
nifH gene segments by PCR. Plasmid DNA was
prepared
according to the method of Kado and Liu (
30), and
1 to 5 ng
of this DNA was used as the template for amplification of
nifH by PCR with primers 19B
(5'-CGGGATCCGCIWTYTAYGGIAARGGIGG-3') and
407B
(5'-CGGGATCCAAICCRCCRCAIACIACRTC-3'). PCR was carried out
in
a BIOMETRA Uno-Thermocycler with an initial denaturation of
4 min, then
35 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min
at 72°C,
with a final extension of 4 min at 72°C (
20), using
PCR
buffer as described above for BOX-PCR analysis. The amplified
DNA
fragment was sequenced by the dideoxy-chain termination method
using an
automated DNA sequencer (model 377; Applied Biosystems).
Both strands
were sequenced
entirely.
Marking of a rice endophytic diazotroph (IRBG500) with
gusA.
Escherichia coli S17.1
containing transposon-based -glucuronidase (GUS) marker
pCAM120 (Tn5ssgusA20), which has the gusA gene under the
control of a constitutive kanamycin resistance gene promoter
(68), was maintained on Luria agar plates with 100 µg of
ampicillin ml1. pCAM120 was transferred to IRBG500 by
conjugation using a filter-mating technique (58). Because
the E. coli strains used in conjugation are auxotrophs, the
transconjugants were selected on minimal medium (M9) (56)
plates containing 10 mM glucose, 10 mM potassium nitrate, and 0.2 mg of
spectinomycin ml1. Color reagent
5-bromo-4-chloro-3-indolyl--D-glucuronate (X-Gluc; Biosynth AG) was added to the medium at 40 µg ml1, and
the blue colonies were selected for further analysis.
Inoculation of rice with IRBG500 marked with
gusA.
Dehulled seeds of rice variety IR72 were surface
sterilized with 70% ethanol followed by 0.2% mercuric chloride for
30 s and were washed thrice with sterile water. The seeds were
germinated on 0.1% TSA plates, and seedlings free of visual bacterial
and fungal contamination were used for inoculation with the
gusA-marked strain. The latter were grown in Luria broth
supplemented with spectinomycin (100 µg ml1) until they
reached an optical density of 0.6. The cells were then harvested by
centrifugation, washed twice with normal saline, and resuspended in
phosphate-buffered saline. Surface-sterilized seedlings were grown in
80-ml glass tubes with 20 ml of Fahraeus medium without nitrogen
(13). They were inoculated with a bacterial suspension
containing approximately 107 bacteria and grown in a growth
chamber (14-h light/10-h dark cycle) at 27°C (day) and 25°C
(night). Uninoculated plants served as controls.
Enumeration of IRBG500 colonizing rice roots and stems.
Plants were sampled at various times, from 1 to 14 days after
inoculation (DAI). Loosely attached bacteria were removed by washing
the roots in excess sterile water, and the roots were then immersed in
sterile water and vortexed for 30 s. The resulting solution was
serially diluted and placed on Luria broth agar plates containing
spectinomycin (100 µg ml1) and X-Gluc (40 µg
ml1). Bacterial colonies showing blue coloration were
then counted, and these counts were assumed to be those bacteria that
were closely associated with the root surface. In another set, the
roots were surface sterilized by immersion in 95% ethanol for 5 min
followed by treatment with 3% calcium hypochlorite containing 0.1%
sodium dodecyl sulfate for 1 min. After three washes with sterile
distilled water followed by maceration in saline, the homogenate was
serially diluted and plated on Luria agar as described above. The
saline solution before maceration was plated to determine the
efficiency of surface sterilization, and the number of bacteria present
in this solution, if any, was subtracted from the total count after maceration.
Preparation and specificity of a polyclonal antibody raised
against IRBG500.
A polyclonal antibody was raised against IRBG500
as described by Hurek et al. (19). Briefly, the bacteria
were grown in Luria broth to log phase, centrifuged, and washed in
normal saline. The washed, centrifuged cells were then resuspended in 3 volumes of normal saline and were killed by treatment with UV for 30 min before injection into rabbits. The cross-reaction of the antibody against various bacteria commonly isolated from the rhizosphere or
interior of rice (27) (Azoarcus communis,
Azospirillum spp., Enterobacter spp., K. planticola, E. coli, Pseudomonas spp., Rhizobium strain
IRBG74, and Herbaspirillum seropedicae Z67) was determined by enzyme-linked immunosorbent assay (ELISA) (1). Briefly, log-phase cultures of the bacteria were normalized according to protein
concentration, and various dilutions were loaded into 96-well polyvinyl
chloride microtiter plates (Nunc, Roskilde, Denmark) and then probed
with the anti-IRBG500 antibody. Anti-rabbit antibody conjugated to
alkaline phosphatase was used as the secondary antibody, and
p-nitrophenyl phosphate (Sigma Chemical Co., St. Louis, Mo.)
was the substrate. The hydrolyzed p-nitrophenol was monitored using a plate reader (Minireader II; Dynatech Laboratories Inc., Chantilly, Va.) at a wavelength of 405 nm (1).
Microscopic studies of infection and colonization by
IRBG500.
At least three seedlings from three independent
inoculations were collected at 2, 5, and 7 DAI and stained for GUS
activity in 50 mM potassium phosphate buffer (pH 7.0) containing 400 µg of X-Gluc ml1 for 4 to 6 h as described by
Jefferson et al. (28). Roots and shoots showing blue
coloration were cut into small (1- to 2-mm) pieces and fixed in 4%
glutaraldehyde (in 50 mM phosphate buffer [pH 7.0] containing 0.1%
[vol/vol] Triton X-100) (61) under vacuum for 30 min and
then at atmospheric pressure overnight. The fixed samples were prepared
for light and transmission electron microscopy (TEM) by being rinsed in
50 mM phosphate buffer, dehydrated in an ethanol series, and embedded
in LR white acrylic resin (London Resin Company, London, United
Kingdom) (24).
Sections for light microscopy and TEM were immunogold labeled with an
antibody raised against IRBG500 (see above), which was
diluted 1:800
with blocking (immunogold labeling [IGL]) buffer
(
24).
To reduce cross-reactions with plant material, the diluted
IRBG500
antibody was first cross-absorbed overnight with a rice
seedling
homogenate prepared as described by da Rocha et al. (
12).
The homogenate was prepared by grinding surface-sterilized seedlings
in
liquid nitrogen and then washing with acetone to remove the
chlorophyll.
Semithin (1- to 2-µm) sections for light microscopy were collected on
gelatin-coated glass slides and incubated for 1 h in
IGL buffer
and then for 1 h in the cross-absorbed antibody. After
being
washed, the slides were incubated for 1 h in a 1:50 dilution
of
5-nm gold-labeled goat anti-rabbit antibody (BioCell, Cardiff,
United
Kingdom) in IGL buffer. The gold labeling was then visualized
for light
microscopy using a BioCell silver enhancement kit. Toluidine
blue
(0.01%) was used to lightly counterstain the gold-labeled
sections. In
parallel with the sections used for immunogold silver
enhancement,
serial sections were collected on uncoated slides
and stained with 1%
toluidine blue. The protocol for immunogold
labeling ultrathin (50- to
70-nm) sections for TEM was as described
above except that the sections
were collected on pioloform-coated
nickel grids and were labeled with
15-nm gold-labeled goat anti-rabbit
antibody (Amersham International).
The sections for light microscopy
were viewed under a Zeiss Axiophot 2 optical microscope, and the
ultrathin sections were viewed by TEM using
a JEOL 1200
EX.
In addition to immunogold labeling with the IRBG500 antibody, serial
sections were also labeled with polyclonal antibodies
raised against
GUS (Clontech, Basingstoke, United Kingdom) or
against the Fe protein
of the nitrogenase enzyme complex originally
isolated from
Rhodospirillum rubrum (a gift from P. W. Ludden,
Madison, Wis.). Both antibodies were diluted 1:100 in IGL
buffer.
For each immunogold assay, the following controls were performed on
serial sections: (i) omission of the primary antibody
and (ii)
replacement of the primary antibody with either preimmune
or normal
serum (Sigma) diluted appropriately (1:800 or 1:100)
in IGL
buffer.
Nitrogenase activity of rice inoculated with IRBG500.
The
plants were taken from the tubes at 20 DAI and washed with sterile
distilled water three times to remove the loosely associated bacteria.
They were then transferred to fresh Fahraeus liquid medium without N
and containing 10 mM malate, sucrose, or citrate as a carbon source.
The tubes were incubated in a 14-h light/10-h dark cycle as described
earlier, and ARA was determined at 12 and 14 h after injection of
acetylene. Uninoculated plants served as controls. After measurement of
nitrogenase activity, the number of bacteria in the tubes was
determined by culturable counts on Luria agar plates.
Plant growth-promoting activity of IRBG500.
The plant
growth-promoting activity of IRBG500 was determined by measuring the
length of roots and shoots as well as by comparing the dry weights of
the inoculated plants with those of the uninoculated control plants at
20 DAI. Total N content of the plants was estimated by a Perkin-Elmer
2400 CHN analyzer (Perkin-Elmer Corp., Norwalk, Conn.) as described
earlier (29). For statistical analysis, the data were
subjected to analysis of variance and the means (± standard deviations
[SD]) were compared using Duncan's multiple range test.
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RESULTS |
Isolation and enumeration of diazotrophic endophytes from
rice.
Diazotrophic bacteria were isolated from surface-sterilized
roots and stems of five of the seven varieties of rice (Table 1). The
population of the diazotrophic endophytes was in the range of
102 to 103 g (fresh weight)1 of
the roots and the stems, whereas the total heterotrophic bacteria recovered were on the order of 105 to 106 g
(fresh weight)1 (Table 1). After further purification of
the diazotrophic isolates, one predominant morphotype was obtained in
four of the varieties tested. BOX-PCR analysis showed that all four of
these isolates gave similar fingerprints (data not shown). One of these
isolates, designated IRBG500, was chosen for further studies. A 400-bp
16S rDNA fragment was amplified from IRBG500 (accession no. AF286867), and the sequence was aligned with reference sequences from the GenBank
data library. The sequence showed highest identity of 99.5% to
S. marcescens (accession no. M59160).
Nitrogenase activity of IRBG500.
Diazotrophy in IRBG500 and
its gusA-linked transformant was confirmed on the basis of
ARA and incorporation of 15N2 (Table
2). The presence of small amounts of
fixed nitrogen (100 mg liter of yeast extract1) was found
to be essential to induce ARA by IRBG500, although activity (both ARA
and 15N2 incorporation) was abolished or
inhibited in the presence of 10 mM ammonium chloride (Table 2). The
nitrogenase activity (ARA) of IRBG500 was less than half of that of
K. planticola IRBG185 and E. cloacae IRBG236
(Table 2). No nitrogenase activity was shown by the type strains of
S. marcescens (LMG2792) and S. rubidaea (LMG5019)
(Table 2).
IRBG500 showed the presence of high-molecular-weight extrachromosomal
elements when extracted with the method of Kado and
Liu
(
30) (Fig.
1, lane 2). Using
primers designed to amplify
the fragment between positions 19 and 407 of the
nifH gene of
Azotobacter vinelandii M20568
(
20), a product of approximately
400 bp was amplified with
plasmid DNA isolated from IRBG500 (Fig.
1, lane 4). No such fragment
was amplified from the type strain
of
S. marcescens (Fig.
1,
lane 5). The nucleotide sequence of
the amplified 390-bp fragment
showed 60 to 73% homology with the
other
nifH sequences
from the DNA database.
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FIG. 1.
Isolation of high-molecular-weight plasmids from
S. marcescens IRBG500 and amplification of the
nifH fragment. Lane 1,  -HindIII molecular
weight marker; lane 2, DNA extract showing two high-molecular-weight
DNA bands (arrows) (the intense and diffuse band is the chromosomal
DNA); lane 3, 100-bp molecular weight marker; lane 4, PCR product from
IRBG500; lane 5, PCR product from S. marcescensT; lane 6, PCR product from the
nifH gene of Azoarcus sp. strain BH72.
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Specificity of the IRBG500 antibody.
In ELISAs, the polyclonal
antibody showed strong reaction with IRBG500 and its
gusA-marked derivative up to a dilution of 1:15,000 (data
not shown) but did not show a reaction at any dilutions with any of the
other bacteria tested. There were also no reactions observed with any
of the bacteria, including IRBG500, when the ELISAs were performed with
preimmune serum used as the primary antibody (data not shown).
Colonization of rice seedlings by gusA-marked
IRBG500.
Transconjugants containing gusA were obtained
at a frequency of 106 and showed a growth rate similar to
that of the wild type, IRBG500. IRBG500-gusA colonized the
exterior and interior of roots, stems, and leaves of rice variety IR72,
as suggested by reisolation of the inoculated strain. Both the external
and internal populations, particularly of the roots, increased up to 14 DAI (Table 3). The bacterial population
then remained stable for a further 20 days of observation (data not
shown). Although the bacteria could be reisolated from
surface-sterilized roots and stems at 3 DAI, they could be isolated
from leaves only after 7 DAI, and numbers in the leaves remained
relatively low throughout the experiment (Table 3). No bacteria could
be isolated from the control plants under the test conditions.
Throughout the study, both control and inoculated plants were without
any visible disease symptoms.
At 5 DAI, uninoculated control plants did not show any blue coloration
with X-Gluc (Fig.
2A). However, roots and
the basal
part of the stem of the inoculated plants showed blue
coloration
when stained for GUS activity (Fig.
2B), with that on the
lateral
roots being most intense (Fig.
2C). Blue coloration could also
be observed on the lower part of the stem as well as on leaves
at 7 DAI
(Fig.
2D).
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FIG. 2.
Light micrographs of GUS-stained roots, stems, and
leaves of rice variety IR72 at 5 days after inoculation with S. marcescens IRBG500 marked with gusA. Control
uninoculated plants showed no GUS activity (A). GUS activity was
observed on all roots (B), with the most intense color development on
the lateral roots (C). GUS staining was also observed on the stems (D).
Bars: 2 mm (A and B) and 0.5 mm (C and D).
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More detailed studies of root material sectioned for light microscopy
confirmed that there was no internal or surface colonization
of
uninoculated control plants (Fig.
3a).
However, light and electron
microscopy of inoculated plants showed that
by 5 DAI, in addition
to the surface colonization (Fig.
2b to d) there
was also considerable
internal colonization of the roots, stems, and
leaves. Transverse
sections of roots showing GUS activity
revealed bacteria within
many of the cortical intercellular spaces next
to the stele and
within the aerenchyma (Fig.
3b and c), although there
was no evidence
that the bacteria had penetrated the endodermis to
colonize the
root vascular system (Fig.
3b and c). Interestingly, many
of the
root cortical cells contained bacteria (Fig.
3c), and by TEM,
both intracellular bacteria (Fig.
3d) and those in the intercellular
spaces (Fig.
3e) were healthy in appearance and were immunogold
labeled
with the antibody raised against IRBG500 (Fig.
3e). On
the other hand,
the host plant cells containing IRBG500 appeared
not to have intact
cytoplasm (Fig.
3d), and the intercellular
bacteria were often
surrounded by electron-dense material (Fig.
3e). In addition to
reacting to the IRBG500-specific antibody
(Fig.
3e), the bacteria in
the roots also reacted with an anti-GUS
antibody (Fig.
3f), but there
was no reaction when serial sections
were incubated in nonimmune serum
(Fig.
3g).
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FIG. 3.
(a) Light micrograph of a toluidine blue
(1%)-stained transverse section of a root from an uninoculated control
plant. No bacteria are visible within the intercellular spaces
(arrows), aerenchyma (A), or vascular cylinder (*). Light (b to c)
and transmission electron (d to g) micrographs show sections of rice
roots at 5 days after inoculation with S. marcescens strain
IRBG500. Transverse sections of infected roots were stained with 1%
toluidine blue (b to c). (b) Bacteria have colonized intercellular
spaces and aerenchyma (small arrows), sometimes very densely (large
arrow), but none have penetrated the endodermis (E) to colonize the
vascular cylinder (*). (c) Higher magnification showing bacteria
within the cortical cells (small arrows), as well as a dense colony of
intercellular bacteria (large arrow). (d) Bacteria (arrowheads)
colonizing a cortical cell (see panel c). Note that the host cytoplasm
is no longer intact and exists only as fragments (small arrows). (e)
Bacteria colonizing an intercellular space in the cortex (see panel c).
The bacteria (arrows) were immunogold labeled using a primary antibody
raised against S. marcescens strain IRBG500 and a secondary
goat anti-rabbit antibody conjugated to 15-nm gold particles. Note that
electron-dense material has accumulated close to the bacteria (*). (f
and g) Transmission electron micrographs of serial sections to panels d
and e. The bacteria in these sections (arrows) were immunogold labeled
with a primary antibody raised against GUS (f) or with normal rabbit
serum (g), followed by a secondary goat anti-rabbit antibody conjugated
to 15-nm gold particles. (f) Bacteria showing internal immunogold
labeling (arrows), indicating the expression of GUS protein. w, plant
cell wall. (g) Bacteria with no significant internal immunogold
labeling (arrows). Bars: 10 µm (a to c), 2 µm (d), 1 µm (e), and
100 nm (f, g).
|
|
In the stems, large numbers of bacteria were observed within the
aerenchyma and intercellular spaces and in the gaps between
the leaf
sheaths (Fig.
4a to c). Again, these
bacteria were recognized
by immunogold labeling with an antibody raised
against IRBG500
(Fig.
4b). Unlike in the roots, high numbers of
bacteria were
also observed within some of the xylem vessels (Fig.
4a,
c, and
d), and these appeared not to have provoked any obvious host
defense
response. In the leaves, although the bacteria were fewer in
number,
some had readily colonized the aerenchyma and the intercellular
spaces (Fig.
5). They were also
occasionally seen within the xylem
(not shown). The bacteria in the
aerenchyma often showed high
levels of immunogold labeling, with a
dense halo of gold particles
around individual bacteria or around
bacterial microcolonies (Fig.
5c and d). These bacteria appeared to be
attached to the host
cell walls via the gold-labeled material that
surrounded them.
In contrast to the bacteria in the aerenchyma, those
within the
intercellular spaces were much less densely gold labeled
(Fig.
5b).
View larger version (112K):
[in this window]
[in a new window]
|
FIG. 4.
Longitudinal (a to b) and transverse (c to d)
sections of rice stems at 5 days after inoculation with S. marcescens strain IRBG500. (a) Infected stem stained with 1%
toluidine blue. Two leaf sheaths are visible, and bacteria (small
arrows) can be seen in the gaps (G) between them. Note that the xylem
in one of the leaf sheaths is particularly densely colonized (large
arrows). (b) Bacteria colonizing the stem aerenchyma (A). The bacterial
colonies (arrows) were immunogold labeled as for Fig. 3e (followed by
silver enhancement). (c) Infected stem stained with 1% toluidine blue.
A vascular bundle in one of the leaf sheaths is shown, and one of the
metaxylem vessels is heavily infected with bacteria (large arrow). As
in panel b, bacteria (small arrows) can also be seen within the gaps
(G) between the leaf sheaths. (d) Transmission electron micrograph of a
stem protoxylem vessel containing numerous bacteria. Note that there is
no obvious host defense material surrounding the bacteria. W, cell
wall. Bars: 20 µm (a), 10 µm (b to c), and 250 nm (d).
|
|
View larger version (97K):
[in this window]
[in a new window]
|
FIG. 5.
Light (a) and transmission electron (b to e)
micrographs of rice leaves at 7 days after inoculation with S. marcescens strain IRBG500. (a) Transverse section of bacteria
(arrows) colonizing the leaf aerenchyma (A) and intercellular spaces.
Some bacteria can also be seen on the leaf surface (small arrows). The
bacteria were immunogold labeled (followed by silver enhancement) as
for Fig. 4b. (b) Bacteria (arrows) within an intercellular space. The
section was immunogold labeled as for Fig. 4b. (c) A bacterium attached
to the cell wall (W) of the aerenchyma by a stalk of electron-dense
material (large arrow). The section was immunogold labeled as for Fig.
4b; note that the bacterium is very densely gold labeled compared to
the intercellular bacteria in panel b, and also that the majority of
the labeling is of extracellular material surrounding it (small arrow).
(d) Microcolony of five bacteria attached to the cell wall (W) of the
aerenchyma. The section was immunogold labeled as for Fig. 4b. Again,
note that the extracellular material surrounding the bacteria is very
densely labeled (arrow). (e) Microcolony of bacteria attached to the
cell wall (W) of the aerenchyma. This section was treated as for Fig.
4b except that the primary antibody was omitted. There is no gold
labeling visible on the bacteria or on the material surrounding them
(*). Bars: 20 µm (a) and 200 nm (b to e).
|
|
Control sections incubated in nonimmune serum (Fig.
3g) or with the
primary antibody omitted (Fig.
5e) showed little or no
gold labeling.
Similarly, none of the bacteria in the roots, stems,
or leaves reacted
significantly with an antibody raised against
the Fe protein of
nitrogenase (data not
shown).
Nitrogenase activity of plants inoculated with IRBG500.
Significant nitrogenase activity (ARA) by the inoculated plants could
be detected at 20 DAI, although only when carbon sources were added to
the medium, with organic acids, such as citrate and malate, giving the
maximum activity (12.5 ± 1.5 and 14.8 ± 2.8 nmol of
C2H4 h1 106
bacteria1, respectively, versus 5.5 ± 1.5 with
sucrose). ARA was abolished when the roots were surface sterilized
(data not shown).
Growth promotion of IR72 by IRBG500.
Inoculation of IR72 with
IRBG500 resulted in increased root length and dry weight of these
plants compared with the uninoculated control plants (Table
4). There was, however, no significant difference in the total N of the inoculated plants compared to uninoculated seedlings, it typically being 0.2 to 0.3 mg
plant1 (data not shown).
| |
DISCUSSION |
Isolation and characterization of predominant endophytic
diazotrophs.
Sterilization by treating with ethanol and mild
flaming was effective in removing most of the microbes from the plant
surfaces. Diverse diazotrophic bacteria were then isolated from the
surface-sterilized roots and stems of five rice varieties growing in
nonsterile soil in greenhouse conditions. Interestingly, no diazotrophs
could be isolated from two other varieties examined (IR387 and
Palawan). Within the five varieties that contained diazotrophs (Table
1), the total diazotrophic bacteria present in the roots and stems were
only 5 to 10% of the total heterotrophic bacteria that could be
cultured, consistent with the earlier findings of Barraquio et al.
(6).
A group of N
2-fixing bacteria isolated from the
surface-sterilized stems and roots of these five varieties showed
similar
fingerprints with BOX-PCR and were identified as
S. marcescens based on a 400-bp 16S rDNA sequence. Although
S. marcescens is
generally regarded as a potential human and animal
pathogen, various
species of
Serratia have been isolated
from cotton (
Gossypium hirsutum) and sweet corn (
Zea
mays) (
43), as well as the rhizosphere
of rice
(
54) and rice seeds (
44). Indeed, nonclinical
isolates
of
S. marcescens have been used as agricultural
biocontrol agents
due to their chitinase activity (
31,
43,
54) and ability
to induce systemic resistance in plants
(
48).
Although this is the first report of diazotrophy by
S. marcescens, Krishnapillai and Postgate (
34)
previously transferred
Klebsiella genes involved in
N
2 fixation to
S. marcescens and
showed that
they were functional. The only other report of diazotrophy
in the genus
Serratia is that of an
S. rubidea strain isolated
from the endorhizosphere of wheat (
Triticum aestivum) and
Ammophila arenaria (
55).
The diazotrophy of IRBG500 was confirmed via the significant
incorporation of
15N
2, but the
15N
atom percent excess was approximately half of that reported
for other
plant-associated diazotrophs, such as
Agrobacterium tumefaciens (
32) and
Herbaspirillum spp.
(
2). Both ARA incorporation
and
15N
2 incorporation were inhibited by the
presence of ammonium, indicating
that the regulation of nitrogenase of
IRBG500 was similar to that
of many other diazotrophs
(
17). Finally, the
S. marcescens strains
in the
present study (including IRBG500) were observed to have
reduced
nitrogenase activity (ARA) as a result of repeated subculturing
(data
not shown). The reason for this is not known but is currently
under
investigation at IRRI. It is possible that the N
2-fixing
ability of IRBG500 is localized on extrachromosomal elements present.
Such extrachromosomal nitrogenase has been detected in other enteric
bacteria such as
Enterobacter agglomerans (
59)
and
Rahnella aquatilis (
7). Although an
expected fragment of 400 bp was
amplified when the plasmid DNA was used
as the template, it remains
to be confirmed if the nitrogenase genes
are localized on the
plasmid because the plasmid DNA isolated also had
contaminating
genomic DNA (Fig.
1, lane
2).
Infection and colonization of rice seedlings by S. marcescens IRBG500.
To study the colonization of rice by
diazotrophic S. marcescens, strain IRBG500 was marked with
gusA and inoculated onto rice seedlings under gnotobiotic
conditions. The gusA-marked strain could be reisolated from
surface-sterilized roots and stems of the inoculated seedlings,
indicating internal colonization and systemic spreading within the
plants. Staining of the whole roots showed that the GUS activity was
most intense on some of the younger lateral roots, and it is possible
that the bacteria entered these at their junctions with the primary
roots. This type of infection has been observed with other diazotrophic
endophytes, such as A. diazotrophicus in sugarcane
(24), Azoarcus spp. in rice and Kallar grass
(28), and Herbaspirillum spp. in sugarcane and rice (25-27).
A major drawback of in situ GUS staining is that the presence of blue
color does not unequivocally confirm the location, or
even the
presence, of the GUS-labeled bacteria because the color
can diffuse
into bacterium-free plant material (
19,
51). Therefore,
to
prove that
S. marcescens strain IRBG500 is genuinely
endophytic
in rice, tissues stained for GUS activity were fixed with
glutaraldehyde,
embedded in resin, and sectioned for optical and
electron microscopy.
This also reduced the possibility that the
bacteria observed within
the plants had accidentally moved into them
from the external
population (
27,
50). The bacteria in the
sections reacted
well with a polyclonal antibody raised against
IRBG500, which
was specific to IRBG500 because it did not react with
other bacteria
that are commonly isolated from the interior of wetland
rice.
The bacteria also showed reaction with an anti-GUS antibody,
confirming
that the bacteria inside rice tissues were the inoculated
strain
of
gusA-marked IRBG500 (Fig.
3f).
Inside the plant, bacteria were mainly localized within the aerenchyma
and the intercellular spaces. These apoplastic locations
seem to be the
preferred sites of the few endophytic diazotrophs
so far examined in
rice. For example, Hurek et al. (
19) and
Egener et al.
(
13) have shown dense colonies of
Azoarcus in
the aerenchyma of rice (and Kallar grass) roots, and James et
al.
(
27) have presented micrographs showing
H. seropedicae extensively
colonizing the intercellular spaces and
aerenchyma of roots, stems,
and leaves. In addition to the apoplastic
localization, the cortical
cells in some of the roots also showed
intracellular bacteria.
However, TEM studies showed that these cortical
cells did not
have intact cytoplasm. It is possible that the
bacterium-colonized
cortical cells were preprogramed to senesce as part
of the processes
involved in the formation of the lysigenous aerenchyma
that characterizes
the roots of wetland rice (
50), and
hence, these cells may already
have been dead or dying prior to their
colonization by the bacteria.
In contrast to the root cortical and
aerenchyma cells, the stems
and leaves showed little or no sign of
degradation associated
with bacterial colonization, even though
bacterial numbers were
high in the localized areas that were observed
microscopically
(Fig.
4 and
5). A possible reason for the lack of an
obvious defense
response from the stems and leaves could be that the
bacteria
were primarily localized within intercellular spaces or within
already dead cells, such as xylem and aerenchyma, and were not
observed
penetrating intact host
cells.
In the present study, the bacteria were not found within the xylem of
the roots, although the xylem of the stem was extensively
colonized.
This contrasts with work on
Azoarcus and
H. seropedicae that has shown colonization of root xylem vessels,
albeit at a
relatively low frequency (
19,
27). In the
stems, the bacteria
were present in the xylem vessels in fairly high
concentrations,
and apparently unoccluded xylem may have allowed
spreading of
the bacteria through the stems to the leaves via the
transpiration
stream. This has also been suggested for rice infected
with
Azoarcus (
19) and
H. seropedicae (
27). Colonization of xylem vessels
by
endophytic diazotrophs, such as
A. diazotrophicus and
Herbaspirillum spp., is commonly reported in other tropical
grasses, particularly
sugarcane and sorghum (
23-25,
47).
James and Olivares (
26)
have suggested that xylem could be
a suitable nonnodular niche
for N
2 fixation because it can
provide the low pO
2 required for
the expression and
function of nitrogenase and also allow for
the exchange of fixed
nitrogen. Indeed, James et al. (
25) have
shown that the
phytopathogen
H. rubrisubalbicans can express nitrogenase
protein within sorghum leaf protoxylem. On the other hand,
N
2 fixation is an energetically demanding process, and
carbon sources
tend to be low in xylem (
26). Although
IRBG500 colonized stem
xylem vessels in high numbers, it could not be
labeled with an
antinitrogenase antibody, suggesting that conditions in
the xylem
were not conducive for N
2 fixation by
IRBG500.
Effect of S. marcescens IRBG500 on growth, nitrogenase
activity, and total N content of infected rice seedlings.
Although
IRBG500 could colonize both the roots and the stems, ARA could not be
detected in the plants inoculated with IRBG500, and the bacteria within
the rice seedlings also showed no immunogold reaction with an antibody
against the Fe protein of the nitrogenase complex. When carbon was
added to the rooting medium, however, significant ARA could be
detected, suggesting that the bacteria associated with the rice
seedlings were limited by the low availability of carbon and energy.
This is consistent with recent reports that supplementation of carbon
was essential for nitrogenase activity of Azorhizobium
caulinodans in association with rice (46) and also
for expression of dinitrogenase reductase by Klebsiella
pneumoniae in association with maize (11). However,
an additional factor causing the carbon limitation on rice-associated
BNF in the present study could be that the plants analyzed were in the
early stages of growth (20 DAI). In field-grown rice, significant ARA
was obtained only after 40 days of transplantation (36).
Although IRBG500 did not show ARA in association with rice, it promoted
the growth of the inoculated seedlings but led to no enhancement of
their N content, suggesting that the growth promotion was probably due to mechanisms other than N2 fixation.
The results presented here show that
S. marcescens IRBG500
could become endophytically established in roots, stems, and leaves
and
could also increase the root length and root dry weight of
the
inoculated plants. Although endophytes have been confirmed
and
localized in various plants and are postulated to play an
important
role in sustainable crop production (
62), the mechanisms
of their endophytic establishment are not well known. It has been
suggested that the bacteria could enter through the fissures created
by
the emergence of lateral roots or could actively dissolve the
cell wall
components to gain entry (
26,
50). Therefore, it
would be
interesting to determine the biochemical and molecular
mechanisms of
entry into rice by
S. marcescens IRBG500, especially
as some
strains possess hydrolytic enzymes, such as chitinase
(
14), lipase (
40), and ligninase
(
52). Finally, IRBG500
could not be isolated from two of
the seven rice varieties examined,
which indicates that there may be
some host specificity in the
interaction between IRBG500 and rice; this
also warrants further
exploration.
| |
ACKNOWLEDGMENTS |
We are thankful to W. L. Barraquio for useful discussions
and to J. I. Sprent for use of TEM facilities at the University of Dundee.
This work was supported by grants from the German Agency for Technical
Cooperation (GTZ) to IRRI in collaboration with the Max Planck
Institute, Marburg, Germany, and the University of Bremen, Bremen, Germany.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Crop, Soil and
Water Sciences Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines. Phone: 63-2-845-0563, ext. 737. Fax: 63-2-891-1292. E-mail: j.k.ladha{at}cgiar.org.
| |
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Journal of Bacteriology, April 2001, p. 2634-2645, Vol. 183, No. 8
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.8.2634-2645.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
