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of
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PIant Prod. Sci.4(2),]26-130

Grain Yield
of

(2001)

Related PhysiologicalCharacteristics
Rice Plants (Oryza
sativa
L) Inoculated
with
Free-Living Rhizobacteria
and

Md. Samiul Alam, ZongljunCui',

Scheel
(Graduate
ofAgricuUuralandLijla
Abstract

TohruYamagishi

and

Ryuichi Ishii'*

Sa'ences,
TneUiiiversdyqf Tblyo, Thdyo 113-865Z .14Pan)

Experiments were conduc[ed

to elucidate the effects ef the inoculationof a mixture
of several
free-living
Acetobactcr,
Bacillus,
Entf,'robacter
and
XLinthobacter
which
were
coliected
and
screened
for the nitrogenfixing
ability in China,on nitrogen
accumulation,
growth and grain yieLd of rice plants,The inoculationof several
bacterialspecies significantly
increased
acetylene-reducing
activity in the roots ofrice plants.The total dry matter
yield,
:

rhizobacteria;

increased
by the bacterial
inoculation
by 6 to I3%, 15 to 18%, and 10
24%,
inthe experirnents of 1997and l998.The increase
of grain yieldwas associated
with the increase
of root length and
leafarea, and also with the increaseof chlorophyll
content
and
photosyntheticrate duringthe
rhizobacteria
to rice plants
leclto increase
grain-Mlingperiod, Itwas hypothesizeclthat the inoculationof free-living
grain yieldand
te

nitrogen

accumulation

were

respectively,

accumulation
in the plants,stimulation
of the Ieafphotosynthesisparticularly
in the grain-filling
stage
in the increased dry matter and grain yieLd.

of nitrogen
resulting

Key

words

Free-Iiving
rhizobacteria,

Grainyield,Nitrogenfixation,Ocycasativa L.,Photosynthesis,
Rice.

In recent years,much attention has been drawn to

Iow-inputsustainable
crop production,becausemany
people

are

worricd

fertilizers
use would

that

expanding

cause

various

arnount
negative

of chemical

impact

on

the

affect the interaction

between the bacteriaand
and Brown, 1974;Tien et al,, l979),
plant (Barea
For successful inoculation eflbcts, many
authors
have
recommended
the use of bacterial
strains
isolatedfrom
the same
crop
species
(Boddeyet al,, 1986),Bacterial
inoculation
may
not always
result in persistentresponses,
becauseof varying ecological factors
and environmental
conditions
1990).The effects of the inoculation
(Lynch,
of a single
bacterialspecies have been exarnined
on
many
crops. However, only a few studies have been
could

locallevel.
global
Severalspecies of frec-living
bacteriaare tbund in the
rhizosphere
to be in close association with the roots in
some crop species, and hence itis considered
that the
association
ofthe
bacteriawith the roots could contribute
to the growth and yield of the crops. The eflects of the
inoculation of several bacterial
regarding
of several
species of the genus of
conducted,
the eflbcts of a mixture
Acospin71um, Acotobacter, Pseudomonas, KllabsieUa,
1990; Garcia et al,, 1996), Cui
Entero- species bacteria(Fayez,
bacter,
on the crop
isolated
rhizobacteria
species
from diflbr200 free-living
growth and yield,have beenexamined
in various pasture grasses and cereal crops (Kapulnik
et
ent
regions
of China, and
tested them
for acetyleneal,,1981; Baldaniet al., 1987; Garcia et al., 1996) and
reducing
activity
singly
and
in several combinations.
Cui
mixture
of
diffbrent
bacterial
also in rice
and
Lin, 1984; Yoo et aL, I986; et al. (1996)
found that a
(Watanabe
Fujiiet aL, 1987), and obvious
bencficial
cflbcts
wcre
strains
showed
significantly higher acetylene-reclucing
observed in some crop species, However, the mechanism
activity than any single strain, The presentexperiment
of the beneficialeffects
of these diazotrophs on the crop
was
designed to elucidate
the beneficial
eflects
of the
is
not
well
understood.
inoculation
of
a
mixture
of
several
bacteria
species
in
plants
total
dry
matter
The beneficial
free-living
bacteriahave a functionof
terms of the accumulation
of nitrogen,
sativa L.).
atmospheric
nitrogen fixation,
but little
isknown about
yieldand grainyieldin rice plants(Ocvza
the amounts
of
fixed nitrogen
in these interactions
Materials and Methods
becauseof the methodological
difficulties
in thc measurement
1987), O'Hara et al,(1981) suggested
The first
experiment
was
made
in 1996 to examine
the
(Boddey,
that the amount
of fixednitrogen
in these interactions etTects of bacterial
inoculation
on
nitrogenase
activity
in
rnade
was
small,
which
ledto the ideathat free-living
bacteria the excised roots, and the second experiment was
in 1997 and 1998 to elucidate
the beneficial
efi'ects of
had some beneficial
functionsto promote plant growth
other
than nitrogen
tlxation.Sorne researchers
reported
bacterial inoculation on nitrogen
accumulation,
dry
rice
that plant growth horrnonesproclucedby the bacteria matter
and
of
plants.
production
grainyield
environment

at

Received 3
362e).
'Present
"Present

the

or

July2000. Accepted 2

addrcss/
address/

November

2000. Correspondingauthor,

Ryuichi Ishii

fax+8I-0466-84{r-ishii@ra5.so-net.ne,jp,

Collegeof Agronomy, China Agricultural

University,
Beijing100094, China.
Fujisawa, 252-8510,Japan.
Collegeof BioresourceSciences,Nihon University

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ALam

In all

et al.Yield

of

The soil fbr

the pots was taken from the top (O-20crn)
layerof a field
where
rice plants had been previously
planted.Acidityof
the soil was in the range of pH 6.7 to 6.9.The soil was
air-dried,
crushed
to pass a 2mm sieve and poured into
without
adding
any
fertilizer,
at the rate
of
plasticpots,
3,5kgper pot in the experiment of 1996,and 10kg per
pot in the experiments of 1997 and 1998.The soil was
muddled
in the pot,then kept under
floodedconditions
for one week before planting,The experiments
were
carried
out in a completely
randomized
block design with
three replicate
potsfbreach treatment, Allthe data were
potted plants were

experiments,

127

Rice Inoculatedwith Rhizobacteria

used.

different
parts of the plants were excised forthe measurement
of dry weight
and
nitrogen
content.
Dry matter
weights
of different
plant parts were determined after
drying at 800C for72 h in an air-forced oven. Nitrogen
rneasured
as
content
was
by the Kjeldahl method
rnodified
by Muraki et aL (1992).
Experiment in 1998
Twenty-eight-day-oldseedlings

of

rice

plants (cv.

Koshihikari) were transplanted to each pot on 17 May

1998, and they were inoculated with a rnixture
of four
differentbacteria:Aeotebacter nign'cans, A. armeniacus,

(ANOVA) to

Bacilius
and
B, magaterium,
Inocuiationprocesphaen'ctts,
dure and growth conditions
were
the same
as those in
1997,
Afterheading,the CO, exchange
rate was
determined
parison of the means.
in the fieldat 7 d intervalwith a portablegas-exchangeExperimentin 1996
measuring
system
PP Systems,Stotfbld,
UK).
(CIRAS-1
A mixturc of six difTerent
bacterial
inocula:
Acotebacter The measurernent
was
conducted
fbrthe flagleafunder
nigricans,
A, armeniacus,
Bacr'Uus
B. magaten'um,
saturating
irradiance(1500
was
sphaericus,
#mol m-2 s-i) which
Enterobacter,
and
IYZinthobacter,
was inoculated.
The first obtained
by supplementary
lightfrom a halogenlamp.
inoculation
was
conducted
by dippingthe roots of the 29
Chlorophyllcontent was determinedin the flagleafwith
-days old seedlings
of rice plants (Ocyza
satieva L. cv.
a spectrophotometer
DU-640, USA) accord(Beckman
Nipponbare) intothe suspension
culture
of a mixture
of
ing to Schmid (1971),
At the harveston 29 Sept,1998,
different
kindsof bacteria(about
IOScells mL-i)
fbr24
root Iength was
measured
with
a root scanner
(Comair
h. Autoclaved culture suspension was used as the conRoot Length Scanner, Commonwealth aircraft Corp.
trol. Seedlings
were
transplanted
on 2 July
1996,at the
Ltd,Australia)
,Nitrogencontent in different
plantparts
rate of three seedlings
hill-i
and
they
were
was
deterrnined
using
an automatic
highlysensitive N-C
potLi,
grown
in the fieldunder a plasticreof to avoid the disturbance analyzer (SUMIGRAPH NC-90),
by the rainfall. Inoculation
was
repeated
at 30 and
60 d
Results
after
rranspianting,
by injecting10mL culture suspension of bacteria
or autoclaved
suspension
with
a syringe
The nitrogenase
activity
was
in the roots
deterrnined
intothe soil around
the plant roots in the pot.At 50, 70
inoculatedwith the mixed
bacteriaby measuring
the
and 90 d after transplanting,the plantswere taken out
acetylene-reducing
activity
1).
Nitrogenase
(Table
from the soil, and the roots were shaken
to remove
the
activity in the inoculatedplant was
15, 75 and 59%
soil and
used
for immediate assay of acetylene-reducing
higher than in the control plant, at 50, 70 and 90 days
activity.
For the assay of acetylene-reducing
activity,
after
transplanting,
respectively,
Leaf area was also sigisolated
roots
were
in
the
test
tube,
and
incubated
at
nificantly
higher
11,9%)
in
the inoculated plants
put
(by
300C in the air with 1O% C,H,, After18 h of incubation, than in the control at 45 days after transplanting (Table
the C2H, productionwas determined by gas chromato2),and the root lengrhof inoculatedplantswas signifiShimadzu Co. Ltd,Japan).
After the
cantly longer (by28.4%) than that of the control plants
graphy (Ga-14A,
assay of acetylene-reducing
activity,
the dryweight of the
at the harvest
time (Table
3).
root sample
was determincd to exprcss
the acetyleneThe effect of bacterial
inoculationon the nitrogen
reducing rate per unit weight
of the root.
accumulation
was
examined
in 1997 and
I998. The
to

subjected

an

analysis

of

variance

determine
the effects of the treatments. Fisher's
protected
leastsignificant difTerence
(LSD)was used forthe com-

Experiment in 1997
Twenty-eight-day-oldseedlings of rice plants (cv,
Koshihikari)to which the first
inoculation was conducted by the same proceduresas in 1996 experiment,
were
on

15 May

1997, at the rate ef three seedlingshill-l

Thirty
and
60 d after the transplanting,
pot-1,

inoculationwas

repeated

transplanted

culture

suspension

way

in 1996.

as

Leaf area

was

leaf area

meter

or

by injecting20mL bacteria
suspension
in the same

inoculation

after

with

of

excised

root

of

rice

bacteria(1996).

(nmol C2H, g root'i h'i)

50DAIrt
70 DA[i7
90 Dsc
Contro1Inoculated13,3a15.3a
14.4b25.2a 40.0b63,6
a
'DAT,

measured

at

ptants

Acetylene-redueingactivity

autoclaved

45 d after transplanting by
AAM-7,
Hayashi Denko Co.
(Model
Ltd,Japan).
Plants were harvestedon 26 Sept.
1997, and
a

Table l,Nitrogcnaseactivity

days after

transpLanting.

Values accompanied
by ditl'erent
lettersin each column
are
significantly diflerent
at 59b level
by Fisher's
Ieast
protected
significant

difference

(LSD).

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128

PIantProductionScienceVot, 4,200I

Table 2. Et'fects
ot' baeterial
inoculation
on Ieaf'
area
ofricc
u[
45
days
after
transplanting
plants
(1997).

Leafarea
.1
2

45403530252015105o

% increasein

dwA,g:;HRg8z.Etg8

ControlInoculated
3215b3596
11.9

Valuesaccompanied

by different
letters
ineach column
arc
difflerent
at 5% level by Fisher'sprotected lcast

signil'ic/antly

significant

(LSD).

c]ifTerence

inoculatien
Table 3. Effects of bacterial
rice
plantsat harvest(1998).

Root Iength

on

% increasein

"!ggtigIggU!h

ControlInoculated95 b122

b>,clif'ferenL letters
ineach column arc
difTlerent
at 59S lc/vcl
by Fishc/r's
protected Ieast
diffcrcnce(LSD).

significant

Table 4. Ei'fects
of' bactc/rial
inoculation
on
mulation
in rice plants at harvcst.

Nitrogencontent

Shoot

nitrogen

(mgN
Grain

Root

Fig.1 EffLc[s
of
fiag leaf ol'
of
thc

[fbtal

1614

12
108

542 b594
a

l76 b207

os

335 b416

o.-P

o=pq

en
op

osm

oo

grain of the inoculated

higher
plantswere 11.4,23.7 and 6.S%, respectively,
than thc control
in 1997. In 1998, significantly
greater
observed
than in ]997;
effects of the inoculaLion were
the increaseof

17.6%

over

nitrogen

control,

and

accumulatien

in the

shoot,

was
root,

and

27.S,52.4and
grain,respec-

tively.

Leaf chiorophyll contcnt was also determined in thc

tlag leafat 8-day intervals after heading during the
led to the eviinoculation
grain-fiIIingperiod. Baczcrial
dent increasein chlorophyll eontent, particularlyin the
1).The photosynthetic
laststage of the grain fi11ing
(Fig.
rate in the fiaglcafmeasured
at 7-day intervalsshowed
a change

and

similar
the

showing

control

2).
(Fig.

to that

of

ot'

ov

root

errer

dwoO6a"v

dcmonstrated that Lhe content of total

nitrogen was significantly higherin the inoculated
plants
4) .The nitrogen
than in the control in both years(Table
shoot,

the

mean

ineans,

10

clearly

in the

tbe

pot'i)

lettersin each column are

Val"es accompaniad
by differcnt
signifiuantly
dil'ferent
at 5% level
by Fishe's
protcctedleast
significant
difference
(LSD).

contents

in

content

rice

enop8

139 b177 21b32

results

chlorophyll

50

accu-

1998Contro1Inoculated
a

bacterial
inoculation
on

40
30
heading

plants,1998. Each value represents

three measuremcnts.
Bars represent the standard

Ar

l99ZControlInoculated
149 a166 59 b73 334 a355
a

20

Daysafter

accoinpanied

significantly

10

28,4

a
Valuus

lengrhuf

root

total

chlorophyll

conient,

also

the

40

Fig.2 Ei't'ects
of bacterial
inoculation
on the photosynthetic rate
thc mean
flagleaf ef rice plants, 1998.Each value represents
Bars
represent
the
standard
error
of three nieasuremellts.
the

in
of

means,

grain yield.The total dry weight was incrcasedby 6% in

5),The inoculation
l997 and by 13% in 1998
increased grainyieldsignificantl>,
by I5% and 18% in
Analysis of the yield compo1997 and l998,respectively.
nents
in beth yearsindicated that inoculated
plantshad

(Table

highcr fllled
grain percentageover the
that the increase
in the
It
was
considered
plants.
higher
filled
led
to
the
significantly
percentage of
grains
grain yicldsin the inoculatedplants,
a significantly
control

differencebetween the inoculated

last few days before harvest
at
the
plants

In the lastboth in 1997 and

increasedthe total dry matter productionand

30

Days after heading

maximum

I998, the inoculation

20

Discussion
The
a

with

in 1997 and 1998 showed

conducted
increase in grain yieldby thc inoculation

experiments

signiticant
a

rnixture

of

various

which
bacteria.

was

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AIam

et al,Yield

ot' Rice

Inocu!atedwith Rhizobacteria

on
Table 5. Effectsof bucterial
inoculazion
dry matter
and
component
of rice
production,grain yield
yield

plants,

Total dry Filled

grain 1000 grain Grain
weight
weight
percentage
yield

199ZCentrolInoculated

129

of the plants (O'Hara

et aL, 1981;
gen requirements
Smith et ai,, 1984). In this paper, the amount of total
nitrogen
was
increasedby the inocuiation
with bacteria
in both years, and this suggests that free-living

have some
eflects
on
the nitrogen
in the inoculated plants.Cohen et al.

rhizobacteria

may

accumulation

(1980)

an inocuiation
experirnent
with
sweet
foxtail
millet,
and
a
similar
result,
presented
a
a
a
a
and
hypothesis.
The exact rnechanisms
involvedin these
1998ControlInoculated
effects
of the inocuLation
of rhizobacteria
on
62.0b70.1 82b90
23.5 a23.7 20.6 b24.3 stimulatory
nitrogen
accumulation
are
unclear,
There
are
several
yet
a
a
a
a
mechanisrns
other
than atmospheric
N,-fixation
possible
Values accompanied
by ditl'erent
letters
in each column are
for
the beneficial
eflects
of bacteriaon
the growth of
signilicantly difl'ercnt
at 5% levelusing Fisher's
protected
inoculatedpLants.One may be that the bacteriaproduce
Seastsignillcant
difference(I,SD),
plant growth horrnonessuch as gibberellicacid, indole-3
-aceticacid, and cytokinin, which
promote plantgrowth
with
other
reports
in rice (Watanabe
and
Lin,1984)and
and Brown, 1974; Tien et aL, 1979), Glick
et al.
(Barea
in wheat
and Hess, I99S),Watanabe and Lin
suggested
that
rhizobacteria
synthesize
(Bhattarai
also
the
(1994b)
reported
that the incrcasc
in grain yield obtained
some
low-molecularcompounds
or enzymes
that are not
(1984)
by the bacterial inoculation was derived from the
well characterized
and
these can modulate
plantgrowth
increasein the percentage of fi11cd
reported
that plant
grains.The increased and development,Lin et al, (1983)
effects
of
nutrient
uptake
can
be enhanced
by the inoculation
with
percentagemay be due to the stimulating
bacteriain the grain-fi11ing
In
the
study,
free-living
bacteria.
Moreover,
Brown
suggested
period,
prcsent
(1974)
we observed
incrcased leaf photosynthesis during the
anothcr mechanism
that diseases
could
be suppressed
by
and
this
bacterial
inoculation,which in turn, stimulate
grain-fi11ing
period due to inoculation (Fig.2),
growth of
increased leafphotosynthesis was associated with the
plant, Growth and yieldof rice plantsmight, therefore,
increase in leafchlorophyll
content
in that period (Fig. be afTk]cted by one or more, of these mechanisms,
1).Therefbre,the increasedLeafphotosynthesis in the
This study showed that a mixture of several strains
inoculatcdplants was attributed partly to the increaseof
of free-living
rhizobacteria
become inoculated
within the
leafchlorophyU content due to bacterial
inoculation,
and
rhizosphere
of rice improved
the plantyield.
However,
this increaseof leafphotosynthesis
might
be responsible
the potentialityof the mixture
of free-ltving
rhizobacteria
to the increasedpercentage of fiLledgrains.
Sincethe leaf of the establishment
and activity of the bacteria
and their
area in the inoculatedplants was largerthan that in the
association with rice require
furtherresearch.
control
at 45 days after transplanting
(Table2), and
Acknowledgements
during the grain-fiIIing
not
shown),
we
period (data
considered
that along with the increased
This study was supported
in part by the Grant-in-Aid
photosynthetic
activity,
the signiticant
increasein leaf area could also
from the Ministryof Education,Science,
Sports and
contribute
to the increasedtotal photosynthetic
carbon
Cultureto R. Ishii(NO.10306002)
assimilation
during the grain-tillingperiod.An increase
References
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inoculation was observed
in
sorghum
et al,, 1988), Inoculation of bacteria
(Sarig
Baldani,V.L.D.,Baldani,J.I.
and D6bereiner,
J.1987. Inoculation
could also stimulate
the root elongation
of field-grown
wheat
aestivum)
with
Azospin71um
spp.
and enlargement
(Tliticum
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surface
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1993; Khawas and
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of plant
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itiscertain
regulating
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Appl.
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grourth
J.
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inoculationcan stimulate
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82.8 a88,1

73b81

21.6 a21.9 26.6 b30.6

conducted

corn

and

vegetative

organs,

the

although

precise mechanism

of the

promotion by bacterialinoculationis still unknown.

Acetylene-reducing
activity
measured
in the present
experiment
showed
highervalues in thc plantsinoculated with bacteria
than in the control plants.The same
has
beenreported by others (Cohenet al., 1980;Watanabe
and
Lin, 1984). However, several papers in which the
growth

acetylene-reducing
the

amount

low, and

of
could

activity

not

was

measured,

suggested

that

fixedby the free-livingbacteriais

nitrogen

contribute

significantly

to the

nitro-

BhattaTai,T,

Hess, D. 1993. Yield responses

of
Nepalese
L,) culLivars
to inoculation
with Acospin'Uum
spp,
of Nepaleseorigin.
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whcat.
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spring

and

wheat

aestivum
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130
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weight

and

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total

content
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Cui,ZJ,, Su, B.L.,Shen, D,L, and Ishii,

R. 1996,Stuclies
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uptake

by

<rea

mays

and

Sorghum

roots
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bicolor
45: 1775-

A4ospin'Uum brasilense,
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1779,

ahichesJ.M,1990. The Rhizosphere. Wiley-Interscience,

functionof nitrogen fixingbacteriain the paddy field,1,
ter, England. 1-10.
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effbcts of nitregen
fixingactivity by mixture
of
Muraki,H,,Higuchi,
K,,Sasaki,
M., Kerenaga, T. and Toei,K,
difTbrent
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