全 文 ：Received 23 Dec. 2003 Accepted 11 Jun. 2004
Supported by the State Key Basic Research and Development Plan of China (2001CB108903).
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Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1065-1070
Migration of Azospirillum brasilense Yu62 from Root to Stem and
Leaves Inside Rice and Tobacco Plants
CHI Feng1, 2, SHEN Shi-Hua1, CHEN San-Feng3, JING Yu-Xiang1*
(1. Center for Photosynthesis and Environmental Molecular Biology, Institute of Botany, The Chinese
Academy of Sciences, Beijing 100093, China;
2. Graduate School, The Chinese Academy of Sciences, Beijing 100039, China;
3. Biological College, China Agricultural University, Beijing 100094, China)
Abstract: Azospirillum brasilense Tarrand, Krieg et Döbereiner is one of the important plant growth-
promotion endophytes. A. brasilense Yu62 tagged with gfp gene was inoculated into roots of rice and
tobacco seedlings, which were then, cultured in gnotobiotic condition. At a certain days after inoculation
the different portions of the seedling were observed under laser confocal microscope, resulting in that A.
brasilense Yu62 bacteria were colonized in epidermal and cortical cells, intercellular spaces and vascular
system of stem and leaf tissue interiors besides in roots. Higher populations of the bacteria isolated from
roots, stems and leaves indicated that A. brasilense Yu62 bacteria could ascend themselves from roots to
stems and leaves of rice and tobacco. This observation lays down the foundation for ecology and cell
morphology of bacterial migration inside plants, interaction between A. brasilense Yu62 bacteria and host
cells as well as the plant-growth promotion, provides scientific basis for further application, and is of
importance in science and practice.
Key words: Azospirillum brasilense ; endophyte; rice; tobacco; movement in plant interior
There are many plant growth-promoting bacteria in soil.
One of them is Azospirillum brasilense (Baldani et al., 1986;
Burdman et al., 2000; Romero et al., 2003), which has the
characteristics of chemotaxis, closing to roots, attaching
on root surface and aggregation through polysaccharides
on bacterial surfaces (Broek et al., 1998; Hauwaerts et al.,
2002). It has been demonstrated that A. brasilense is able
to colonize roots of grasses, establishes the ecological as-
sociation with wheat as an endophyte (Ramos et al., 2002).
A. brasilense is a free-living diazotroph, which fixes nitro-
gen from atmosphere in the deficiency of fixed nitrogen in
soil for plant use (Yang et al., 1984), and secretes phyto-
hormone-like indole-3-acidic acid (IAA) (Lambrecht et al.,
2000). Therefore, it can promote plant growth and increase
the yield. Recently, it has been reported that A. brasilense
has the ability to resist plant diseases （Bashan and De-
Bashan, 2002; Romero et al., 2003）. A. brasilense has been
paid the great attention to for a long time and many studies
has been deeply done on its nitrogen fixation gene (nif)
expression and regulation, nitrogenase activity modulation,
IAA gene cloning and expression, as well as application
（Ma and Li, 1997; 1999; Steenhoudt and Vanderleyden,
2000）.
During recent years, A. brasilense has been tagged with
gfp gene, which encodes a protein with green fluorescence,
to investigate its association with plants and its localiza-
tion in root region, revealing that it is mainly colonized in
intercellular spaces of root cortex （Ramos et al., 2002;
Liu et al., 2003）. The questions whether Azospirillum
bacteria are immobilized in the intercellular spaces, or they
are continuously active as well as where they are going to,
are still unknown. Using the gfp-tagged A. brasilense Yu62
to pursue its destiny in root interior is a powerful tool. Here
we report this result and discuss its significance in science
and practice.
1 Materials and Methods
1.1 Azospirillum brasilense Tarrand, Krieg et Döbereiner
and gfp-plasmid construction
A. brasilense Yu62 (Ampr, Nxr), construction of plasmid
pVK101 (Tcr) containing gfp gene and its transformation
into bacteria (Yu62/pVK101) refer to Liu et al. (2003) de-
scribed in detail.
1.2 Plant materials and their culturing
Rice (Oryza sativa L. cv. Zhonghua 8) was obtained
from Chinese Academy of Agriculture and tobacco
(Nicotiana tabacum L. cv. Honghua Dajinyuan) was kept
in our laboratory.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041066
1.2.1 Surface-sterilization of rice seeds and culturing of
seedlings After rice seeds were de-hulked, they were
surface-sterilized in 70% ethanol for 10 min and washed in
sterile distilled water three times, then in 0.1% HgCl2 for 10
min and washed in the same water three times. After
sterilization, they were germinated on LB medium at 28 ℃
for 48 h. Detection of bacterial colony on the medium was
conducted to identify if the sterilization was thoroughly
clean. The glass tubes (4 cm in diameter, 25 cm in height)
with vermiculite wetted with water were sterilized at 121 ℃
for 20 min. Four to five sterilized rice seeds were transferred
into the tubes, each of which was covered with a transpar-
ent paper, and cultured at 25 ℃ and 14 h light per day till
the seedlings growing up.
1.2.2 Tobacco seed sterilization and plant culture
Tobacco seeds were soaked in 95% ethanol for 30 s and
washed in sterile water three times each 10 min, then in 1%
AgNO3 for 3-5 min and washed three times each 10 min.
The sterilized seeds were germinated in the sterile Watman
filter for a week and transferred into jars (7.5 cm in diameter,
11.5 cm in height) with vermiculite 130 cm3 and 90 mL of
water. Ten germinated seeds were in a jar and cultured at 25
℃ and 14 h light per day.
1.3 Culturing of A. brasilense Yu62 and plant inocula-
tion
The A. brasilense Yu62 bacteria tagged with gfp gene
were cultured in LD medium (10 g tryptone, 5 g yeast ex-
tract and 5 g/L NaCl) with 25 mg/mL Amp, 12.5 mg/mL Tc to
keep selected pressure and make gfp gene-plasmid trans-
mit stably at 30 ℃ for 36 h. The bacteria then were har-
vested at 5 000 r/min and suspended in PBS buffer (pH 7.4)
twice for washing and resuspended in the buffer. The OD
value of the suspension was determined at 660 nm wave
length and the concentration of bacteria was calculated.
One mL of 109 cells/mL treated as above was carefully
dropped into rhizosphere of each rice seedling in the glass
tube in order to avoid the bacteria attachment and contami-
nation of seedling above its taproots. Then, the rice seed-
lings were cultured in gnotobiotic condition. The tobacco
seedlings grown in the jars with sterilized vermiculite were
kept for one to two weeks, at which time the seedlings were
about 2 cm in length, each of them was inoculated with 1
mL of 108/mL bacterial suspension in PBS buffer by care-
fully and equably dropping it into rhizosphere and then
cultured in the gnotobiotic condition. The seedlings with
no inoculation were used as control.
1.4 Laser confocal microscopic observation of the inocu-
lated seedlings
The plants grown in the gnotobiotic condition were
taken out and their root surfaces were rinsed cleanly with
sterile water and the tissues of roots, stems and leaves
were excised, sectioned transversely and longitudinally by
hand, respectively. All the sections were examined under
the Bio-Rad MRC 1024 laser confocal microscope. Images
of GFP-labeled bacteria and host cells of plant tissues were
obtained by using a set of filters consisting of a 488-nm
bandpass exciters to emit the green fluorescence for bacte-
ria and the green autofluorescent for rice host cells and
transmitted blue color as background for tobacco host cells.
The images were recorded using Nikon E800 scanner and
converted into computer files by using digital camera. The
images of bacteria and host cells were merged using confo-
cal assistant software to generate composite images for
publication.
1.5 Isolation of Azospirillum brasilense Yu62 and calcu-
lation of its population
1.5.1 Plant samples The roots and stem of each plant
were carefully separated. The roots after getting rid of ver-
miculite were put into water, vortexed for 1 min, washed
three times (each time for 15 min). The washed roots were
divided into two parts, one of them was surface-sterilized.
The bacteria in root interior were isolated and the popula-
tion was calculated. The other part of the washed roots
was not surface-sterilized and the bacteria were directly
isolated and recorded. The obtained bacteria in this case
included those in root interior and on the root surface.
1.5.2 Root surface sterilization The washed roots were
immerged into disinfection solution (1 ´ PBS, 1% SDS,
0.2% Tween 20)（Dong et al., 2003）and vortexed for 1
min, then washed four times with sterile water. The steril-
ized roots were placed on LB medium with Amp and Tc
antibiotics and cultured at 28 ℃ for 48 h in order to check
whether there is any bacterium on the root surface.
1.5.3 Examination of bacteria on seedling surface After
rice leaf sheaths and leaves, tobacco stems and leaves (the
first and second, third and fourth leaf ) were surface-steril-
ized and examined with no bacteria, they were cut into small
fragments, respectively, to isolate the bacteria.
1.5.4 Isolation of Azospirillum brasilense Yu62 and cal-
culating its population The samples treated as above,
after the water on their surface was wiped slightly, were
weighted and homogenized in mortar and pestle. The PBS
solution with 20% glycerol was dropped into each homog-
enized sample for suspension till 5 mL, then 100 mL suspen-
sion after 5 mL of each sample was diluted in series, was
spread onto TY medium with Amp and Tc and cultured at
28 ℃ for 48-72 h. The bacteria were recorded and the popu-
lation was calculated as Log CFU/g FW. The plants in a
CHI Feng et al.: Migration of Azospirillum brasilense Yu62 from Root to Stem and Leaves Inside Rice and Tobacco Plants 1067
glass tube or a jar were regarded as one sample to isolate
the bacteria as one datum. Each datum was repeated three
times.
2 Results
2.1 Laser confocal microscopic observation of
Azospirillum brasilense Yu62 in rice leaf sheaths and
leaves, tobacco stems and leaves after inoculation in their
rhizospheres
The rice roots at 18 day after inoculation (DAI) were
sectioned transversely and the sections were observed
under laser confocal microscope. The results demonstrated
that the Azospirillum brasilense Yu62 bacteria with gfp gene
were distributed in the epidermal and cortical cells,
parenchyma, aerenchyma and vascular system, and are
consistent with the figures that the A. brasilense Yu62 was
inoculated into maize rhizosphere (Figures not shown). The
rice leaf sheaths at 18 DAI and the rice leaves at 30 DAI
were transversely sectioned, showing that the gfp-tagged
A. brasilense Yu62 bacteria entered into intercellular spaces
and vascular system of these tissues (Figs. 1, 2). This re-
sult indicated that the bacteria after inoculation could as-
cend to rice leaf sheaths and leaf tissues. The intact to-
bacco roots at 18 DAI were longitudinally observed by
Figs.1-4. Observation of Azospirillum brasilense Yu62 bacteria in rice leaf sheaths and leaves, tobacco stems and leaves after 18 day
after inoculation (DAI) by using laser confocal microscopy. 1. The transverse section of rice leaf sheaths indicating the parts of three leaf
sheaths. The fluorescent spots (arrows) of A. brasilense Yu62 bacteria are distributed in cell interior beneath the epidermis of rice leaf
sheath, intercellular spaces and near vascular system. 2. Rice leaf vein and parts of its flanked leaf at 30 DAI. Arrows show the fluorescent
spots of A. brasilense Yu62 bacteria that they are distributed in the cell beneath the epidermis, intercellular space and near vascular
system. 3. The observation of tobacco stem at 70 DAI by longitudinally scanning, indicating the cells of vascular system. The vessel with
spiral pore (short arrow) and sieve tube cell with sieve pore (long arrow) are observed. The fluorescent spots are distributed in cells and
intercellular spaces. 4. The scanning observation of tobacco leaf epidermis at 70 DAI. The contour of each leaf epidermal cell is clear and
the bacteria are almost distributed in intercellular spaces, only a few fluorescent spots within cells. Abbreviations: Ae, aerenchyma; Lb,
leaf body; S, stomata of leaf; V, vessel; Vs, vascular system.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041068
scanning under laser confocal microscope, indicating that
the bacteria entered into root interior through “lateral crack”
and spread in the epidermal and cortical cells and intercel-
lular spaces, a few in vascular system (Figures not shown).
The tobacco stems at 40 DAI were longitudinally scanned,
observing that the bacteria were distributed in cortical cells
and intercellular spaces, xylem vessel and sieve tube and
their intercellular spaces (Fig.3). The same result were ob-
tained that A. brasilense Yu62 bacteria were spread in inter-
cellular spaces of leaf epidermis on a large scale, some in
epidermal cells (Fig.4). The control materials that the rice
and tobacco seedlings were not inoculated, did not dem-
onstrate any gfp-tagged bacteria existing in their roots,
stems and leaves (Figures not shown).
2.2 Populations of A. brasilense Yu62 bacteria in rice
leaf sheaths and leaves and tobacco stems and leaves
The roots, leaf sheaths and leaves of rice were excised,
respectively, and isolation of the bacteria was carried on
according to the description as in Materials and Methods.
Figure 5 shows the populations of A. brasilense Yu62 bac-
teria in the roots, leaf sheaths and leaves of rice. The amount
of the bacteria from the roots of non-surface sterilization
included those colonized on root surface and within root
and that just representing the amount inside the roots after
root surface-sterilization. The population of the former and
the later was 106.2 and 105.8 CFU/g FW, respectively, show-
ing the difference between them, but no significance of
both in magnitude, from which it was illustrated that the
amounts of the bacteria colonized in rhizosphere and in-
side the roots were not significant. The population of the
bacteria in leaf sheath was 105.2 CFU/g FW and that in leaves
was only 102.7 CFU/g FW. The reason why the small popu-
lation was in leaves, might be that the time of isolation at 18
DAI was so short that the bacteria could not move to the
leaves on a large scale.
At 40 DAI were separated the roots, stems and leaves
of tobacco, from which the populations of the bacteria were
isolated, respectively. Figure 6 indicates that the popula-
tion densities in the roots without and with surface-steril-
ization were in great significance, the former was 107
CFU/g FW, and the later was 104.7 CFU/g FW, illuminating
that a large amount of the bacteria was colonized in
rhizosphere. The population density in tobacco stems was
106.0 CFU/g FW, in the 1st and 2nd leaf was 107.2 CFU/g
FW, and those in the 3rd and 4th leaf were 106.6 CFU/g FW
and 106.0 CFU/g FW, respectively. The above data showed
that the population densities in stems and leaves were very
high, even equal to that of the roots with no sterilization.
This may be explained that the stems and leaves of tobacco
were suitable to the bacteria to be colonized. In general, the
population densities of the bacteria were gradually de-
creased with ascending from the 1st-2nd to the 3rd and
4th leaf. The reason probably is that the bacterial densities
were relatively diluted due to the expansion of leaf area and
increase in leaf weight.
In the control experiments that the rice and tobacco
plants were not inoculated with gfp-tagged bacteria, there
was no any bacterium isolated from their tissues of the
roots, leaf sheaths, stems and leaves.
3 Discussion
The endophytic bacteria which promote plant-growth
have been isolated during last decade. Since the endophytes
were tagged with the genes of LacZ, gus and gfp and in-
oculated into the rhizosphere of plants, they have been
observed to penetrate the roots in “lateral root crack” man-
ner （O’Callaghan et al., 1997; Broek et al., 1998; Prayitno
et al., 1999; Ramos et al., 2002）. However, a large amount
Fig.5. The population densities of Azospirillum brasilense
Yu62 bacteria in rice roots, leaf sheaths and leaves after inoculation.
CFU, colony forming unit; RN, the roots without surface
sterilization; RS, the roots with surface-sterilization; LS, leaf
sheath; L, leaf.
Fig.6. The population densities of Azospirillum brasilense
Yu62 bacteria in tobacco roots, stems and leaves after inoculation.
CFU, colony forming unit; RN, the roots with no surface
sterilization; RS, the roots with surface-sterilization; S, Stem; L1-
2, L3 and L4 represent the 1st-2nd, 3rd and 4th leaf, respectively.
CHI Feng et al.: Migration of Azospirillum brasilense Yu62 from Root to Stem and Leaves Inside Rice and Tobacco Plants 1069
of the beneficial bacteria in soil or the endophytes belong-
ing to different species and genera are isolated from roots,
stem and leaves, even seeds, all of which are surface-
sterilized（Mundt and Hinkle, 1976; Stoltzfus et al., 1997;
Elbeltagy et al., 2001; Verma et al., 2001）. The question
where the bacteria in plant stems, leaves, even in seeds are
from, is still unknown. There is no more lucubrated research
and report to be systematically shown. The A. brasilense
is an endophyte in grasses. We tagged the A. brasilense
Yu62 bacteria with gfp gene, and observed that they pen-
etrated maize root interior (Liu et al., 2003). In this
presentation, we used the tagged bacteria to inoculate rice
and tobacco, not only observed that they entered into the
rice roots, but also into dicot tobacco. Furthermore, we
pursued where they are going after entrance of the roots.
We found that the bacteria could continuously ascend to
rice leaf sheaths and leaves, tobacco stems and leaves.
This is a finding that in the gnotobiotic condition the A.
brasilense Yu62 bacteria after their colonization of the root
interior could further migrate up to the parts of plants above
the taproots probably through intercellular spaces and vas-
cular system, simultaneously, some of them enter the cells
of different tissues. It is inferred from this that the other
beneficial bacteria or endophytes originally exist in soil af-
ter entrance of host plant roots would also have the as-
cending action.
The isolation of the bacteria indicates that there is a
considerably large population density of the bacteria, re-
spectively in roots, stem and leaves. The bacterial popula-
tion density in rice leaves at 18 DAI is lower than that
expected. If the isolation time is later, the population den-
sity could be further increased as that showing almost the
same population densities in tobacco stems and leaves iso-
lated at 40 DAI. The result is that the population of the
bacteria after inoculation of the roots of plants exists in
their stems and leaves, further indicates that the A.
brasilense Yu62 bacteria should migrate from the roots up
to the stems and leaves.
This study provides the ecological and cell morpho-
logical relationship between A. brasilense Yu62 and rice or
tobacco at the level of intact plant body. The migration
phenomenon of A. brasilense Yu62 bacteria in host plant
interior narrates that the endophytes in host plants exist
dynamically. The translocation and quantitative increase
of the bacteria make known that they are able to migrate
and duplicate themselves, and should have to make cell
communication and molecular interaction with the host plant.
To work on the migration mechanism is of the importance
in science that the molecular association between the
endophytes and the host plants could be ferreted out.
A. brasilense is one of the free-living diazotrophs which
provides plants with fixed nitrogen, simultaneously, secrete
IAA, both of which make plant growth-promotion
（Bashan and De-Bashan, 2002）and increase the yields.
In recent years, A. brasilense bacteria have shown the effi-
ciency of resistant plant diseases regardless in the case
that they are inoculated in the rhizosphere or leaves of
plants prior to or after the inoculation of pathogenic bacteria,
or the mixture（Bashan and De-Bashan, 2002; Romero
et al., 2003）. Our research that the migration of A.
brasilense bacteria inside plants and considerable high
population densities in plant stems and leaves provides
the scientific data for plant growth-promotion and increas-
ing yield, no doubt, is of an important significance in
practice.
Acknowledgements: We thank Dr. LIANG Yu for his
suggestion to revise the manuscript and his assistance to
make photos. We are grateful to Professor LI Ji-Lun, who is
the academician, The Chinese Academy of Sciences and
working at China Agricultural University, for his review of
this manuscript.
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