influence of composite inoculations of phosphate solubilizing organisms and an arbuscular...
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Influence of composite inoculations ofphosphate solubilizing organisms andan arbuscular mycorrhizal fungus onyield, grain protein and phosphorus andnitrogen uptake by greengramMohammad Saghir Khan a & Almas Zaidi aa Department of Agricultural Microbiology, Faculty of AgriculturalSciences , Aligarh Muslim University , Uttar Pradesh, IndiaPublished online: 25 Jan 2007.
To cite this article: Mohammad Saghir Khan & Almas Zaidi (2006) Influence of compositeinoculations of phosphate solubilizing organisms and an arbuscular mycorrhizal fungus on yield,grain protein and phosphorus and nitrogen uptake by greengram, Archives of Agronomy and SoilScience, 52:5, 579-590, DOI: 10.1080/03650340600861857
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Influence of composite inoculations of phosphatesolubilizing organisms and an arbuscular mycorrhizalfungus on yield, grain protein and phosphorus and nitrogenuptake by greengram
(Einfluss einer kombinierten Inokulation von Phosphormobilisierenden Organismen und eines arbuskularenPilzes auf den Ertrag, Proteingehalt im Korn sowie diePhosphor- und Stickstoffaufnahme von Greengram)
MOHAMMAD SAGHIR KHAN & ALMAS ZAIDI
Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim University,
Uttar Pradesh, India
(Received 23 September 2005; accepted 12 June 2006)
AbstractThe agronomic efficiency of nitrogen (N) fixing and phosphate solubilizing microorganisms and anarbuscular mycorrhizal (AM) fungus on vigour, photosynthetic pigments, seed yield, grain proteinand nutrient uptake of greengram plants, were assessed in soils, deficient in phosphorous (P). Thetripartite inoculation of Glomus fasciculatumþBradyrhizobium sp. (vigna)þBacillus subtilis, signifi-cantly increased dry matter, chlorophyll content and nutrient uptake of greengram plants. Generally,the number of nodules formed per plant was more at flowering stage, which decreased at podfillstage of plant growth. Seed yield increased significantly by 27% due to inoculation withBradyrhizobium sp. (vigna)þB. subtilisþG. fasciculatum, relative to the control. Grain proteinranged from 17% (P. variabile) to 28% (Bradyrhizobium sp. (vigna)þB. subtilisþG. fasciculatum) ininoculated greengram. A negative effect occurred on some of the measured parameters whenP. variabile was used alone or in combination treatments. The N and P contents in measured plantparts (e.g., roots, shoots, straw and grain) differed considerably among treatments. The populationsof PSM, percentage of root infection and density of the AM fungal spore improved in some of thetreatments.
Keywords: Greengram, phosphate solubilizing microorganisms, arbuscular mycorrhizal fungi, grainprotein, nutrient uptake
Correspondence: Mohammad Saghir Khan, Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh
Muslim University, Aligarh 202002, Uttar Pradesh, India. E-mail: [email protected]
Archives of Agronomy and Soil Science
October 2006; 52(5): 579 – 590
ISSN 0365-0340 print/ISSN 1476-3567 online � 2006 Taylor & Francis
DOI: 10.1080/03650340600861857
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Introduction
The dependence of fertilizer production on fossil energy source and the prospects of
diminishing availability of costly input of fertilizer production in years to come have obviously
brought the subject of mineral phosphate solubilization (MPS) and biological nitrogen
fixation (BNF) to the forefront. The microbial system can siphon out appreciable amounts of
nutrients from the natural reservoir and enrich the soil with the important but scarce
nutrients. The crop microbial ecosystem can thus, be energized in sustainable agriculture with
considerable ecological stability and environmental quality. In this regard, the rhizospheric
microorganisms including N2 fixing bacteria, phosphate solubilizing microorganisms (PSM)
and arbuscular mycorrhizal (AM) fungi possess a greater agronomic utility.
Phosphorus (P) promotes N2 fixation in legume crops and is essential for photosynthesis,
energy and production of sugars (Saber et al. 2005). The majority of the agronomic soils
contains large reserves of P, a considerable part of which has accumulated as a consequence of
excessive application of P fertilizers. A large proportion of soluble inorganic P added to soil is
however, rapidly fixed soon after its application and becomes unavailable to plants. For
instance, in acid soils, free oxides and hydroxides of Aluminium (Al) and iron (Fe) fix P
(Norrish & Rosser 1983) while in alkaline soils it is fixed by Calcium (Ca) (Lindsay et al.
1989) causing low efficiency of soluble P fertilizers. To circumvent the P deficiency in soils,
the viable option to augment the availability of P in an easily available form to plants in a more
environmentally friendly and sustainable manner is the use of PSM. In this regard, the
beneficial effects of inoculation with PSM to many crop plants have been described (Khan
et al. 1998; Zaidi 1999). An alternative approach for the use of PSM as a microbial inoculant
is the use of mixed cultures or co-inoculation with other microorganisms. In this context, N2
fixers and PSM when inoculated together colonized the rhizosphere and enhanced the growth
of legumes by providing it with Nitrogen (N) and P, respectively (Gull et al. 2004). The N2
fixing organisms, not only provide N to the plants but also improve N status of soil, alone or
in combination with PSM. Accordingly, the application of PS bacteria (Pseudomonas striata)
and nodule bacteria gave significantly higher yield in greengram (Khan et al. 1997) and
chickpea (Zaidi et al. 2003) than obtained by the use of Rhizobium alone. Furthermore,
Rhizobium and PS fungi (Aspergillus awamori) when used as seed inoculant, increased the
grain yield of chickpea under field conditions (Dudeja et al. 1981).
Mycorrhizal fungi are ubiquitous soil inhabitants and form symbioses with terrestrial plants
(Jeffries 1987). However, in association with N2 fixers, the AM fungi increase N and P
nutrients of the plants, especially in P deficient soil. Combined inoculation of Rhizobium and
Glomus etunicatum and application of rock P or PSM and AM fungus gave greatest yield and
had variable effects on nodulation in clovers (Leopold & Hofner 1991), mungbean (Satpal &
Kapoor 1998; Zaidi et al. 2004), cowpea (Thiagarajan and Ahmad 1993) and chickpea (Poi
et al. 1989). The experiments have revealed that the establishment of AM fungus on to the
root system can alter the rhizospheric microbial populations (Ames et al. 1984) which, in
turn, affects the competitive interaction between introduced and native rhizobia for
nodulation sites. However, the studies concerning the effect of rhizobial strains on the
development of AM fungus and, inversely of AM fungus on nodulation is relatively scarce.
Greengram [Vigna radiata (L.) wilczek] is a major food legume and important source of
protein in many countries. In addition, it is also widely used as green manure. In India,
greengram occupies an area of 3 million hectares with an annual production of one million
tonnes of grain. Greengram seeds contain 25% protein, 62.6% carbohydrate, 1.15% fat, and
3.32% ash (Sharma 2000) and forms a specific symbiosis with nodule bacterium like other
legume crops. Agronomists therefore, prefer to adopt an environmentally benign approach for
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nutrient management during greengram cultivation. Therefore, identifying microbial
combinations for raising the productivity of greengram will be of paramount importance.
There have been studies on the effects of microbial inoculations on nodulation and N2
fixation in greengram (Sahar et al. 2002). However, the effects of composite inoculation of N2
fixer, PSM and AM fungus on greengram are rare. This investigation was thus undertaken to
evaluate the effect of root nodule bacterium (Bradyrhizobium sp. vigna), PS bacterium
(Bacillus subtilis), PS fungus (Penicillium variabile) and AM fungus (Glomus fasciculatum),
singly or in combination, on vigour, yield and nutrient uptake of greengram plants.
Materials and methods
Microorganisms
The phosphate solubilizing bacterium, Bacillus subtilis (MTCC 121) was procured from the
Institute of Microbial Technology (IMTECH), Chandigarh, India, while Glomus fasciculatum
was obtained from the Indian Agricultural Research Institute (IARI), New Delhi, India. The
PS fungus Penicillium variabile capable of dissolving rock phosphate (RP) was isolated from
local soil and cultivated in our laboratory. The N2 fixing bacterium Bradyrhizobium sp. (vigna)
was isolated from nodules produced on root systems of greengram plants using standard
method (Vincent 1970). Bradyrhizobium sp. (vigna) was grown in a rotary shaker at 150 rpm
at 288C for five days in 250 ml flasks containing 100 ml of yeast extract mannitol broth
(g l71: mannitol 10, KH2PO4 0.5, MgSO4 � 7H2O 0.2, NaCl 0.1, yeast extract 1.0, pH 7.4) to
a cell density of 26108 cells ml71. Bacillus subtilis and P. variabile were grown in National
Botanical Research Institute Phosphate (NBRIP) growth medium (Nautiyal 1999) containing
[g l71 – glucose 10; (Ca3PO4)2 5; MgCl2 � 6H2O 5; MgSO4 � 7H2O 0.25; KCl 0.2; and
(NH4)2 SO4 0.1] for 5 and 4 days, respectively, at 28þ 28C to a cell density of 36108 and
56105 cells ml71, respectively. Glomus fasciculatum was multiplied on Chloris gayana Kunth
by the open pot culture method (Gilmore 1968).
Inoculation and plant culture
A sterilized soil experiment was conducted during the kharif season on sandy clay loam soils
containing sand 667 g kg71, silt 190 g kg71, clay 143 g kg71, organic C 0.4%, pH 7.4,
WHC 0.44 ml g71, Olsen P 16 mg kg71 and Kjeldahl N 0.75 g kg71. Undamaged, clean
and uniform sized seeds of greengram var. Pant moong 1 were surface sterilised (70%
ethanol, 3 min; 3% sodium hypochlorite, 3 min). Seeds were rinsed five times with sterile
water and shade dried. The surface sterilized seeds were then inoculated by soaking the seeds
in liquid culture medium of each organism for 1 h using 10% gum arabic as sticker to deliver
108 cells/seed (Bradyrhizobium), and 107 cells/seed (Bacillus subtilis). The N2 fixer, PSM and
AM fungus were assayed as single inocula or dual or triple N fixer-PSM-AM fungal
combinations. For combined inoculations, the liquid cultures of each organism were mixed in
equal proportion in which the seeds were then soaked. In combined treatments with G.
fasciculatum, inoculated seeds were sown in soils having 80 g of the mycorrhizal inoculum
(infected roots and spores). The spore suspension (5 ml) of 26104 per ml Penicillium variabile
was added to soils 24 h before sowing. The treatment without microbial inoculation served as
control treatment for comparison. Mussoorie rock phosphate (23.12% P) was added at
20 mg kg71 to the soil as P source, before sowing, and was common in all treatments, except
control, which had 20 and 40 mg kg71 N (urea) and P (super-phosphate), respectively. There
were 13 inoculation treatments plus one control treatment without any inoculation.
Synergistic effects of rhizospheric microorganisms on productivity of greengram 581
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Coated seeds were sown in clay pots (30 cm high and 28 cm internal diameter) having
12.5 kg autoclaved soils. The sowing was done on 16 March 2002 and repeated with the same
treatment and growth conditions on 20 March 2003. The experiment was conducted in a
randomized complete block design and maintained in a greenhouse at 22þ 28C and 60%
relative humidity. There were nine replicates per treatment and four plants per pot were
maintained seven days after emergence (DAE). The plants were watered on a daily basis
with tap water. The treatments were: (i) Bradyrhizobium sp. (vigna) (ii) Penicillium variabile
(iii) Glomus fasciculatum (iv) Bacillus subtilis (v) Bradyrhizobium sp. (vigna)þP. variabile
(vi) Bradyrhizobium sp. (vigna)þG. fasciculatum (vii) Bradyrhizobium sp. (vigna)þB. subtilis
(viii) P. variabileþG. fasciculatum (ix) P. variabileþB. subtilis (x) G. fasciculatumþB. subtilis
(xi) Bradyrhizobium sp. (vigna)þP. variabileþG. fasciculatum (xii) Bradyrhizobium sp.þG. fasciculatumþB. subtilis (xiii) P. variabileþB. subtilisþG. Fasciculatum, and (xiv) Un-
inoculated control (N20P40).
Plant and nutrient analysis
All plants in three pots were uprooted each at 45 (flowering stage) and 60 days (pod fill stage)
after sowing (DAS) and the adhering soil particles were carefully removed. Nodules were
detached from the root system, counted, oven dried (808C) and weighed. Plants removed at
flowering and at harvest (80 DAS) were also used for the measurement of root and shoot
length and oven dried (808C) before the weights of roots and shoots and total plant biomass
were determined. Remaining pots (three pots for each treatment) were maintained until
harvest. Seed mass and grain protein (N66.25) were recorded at 80 DAS. Total chlorophyll
contents in foliage were determined at flowering stage (Mechenny 1941). Total N contents in
tissues (e.g., roots, shoots and straw) and P content in grain and straw was measured at 80
DAS as suggested by Iswaran and Marwah (1980) and vanadomolybdo-yellow colour method
of Jackson (1958), respectively. Total N and residual P in soils were measured at harvest using
the modified micro-Kjeldahl and sodium bicarbonate extraction method (Olsen et al. 1954).
Bacterial quantification and mycorrhizal colonization
Populations of PSM in the rhizospheric soil were determined at 60 and 80 DAS by dilution
plate technique using NBRIP growth medium. Each plate was replicated three times,
incubated for five days at 288C and colonies showing a clear halo indicating P solubilization
were counted. At 45 and 60 DAS, the mycorrhizal infection was assayed in the root by stain
method (Phillips & Hayman 1970) while the AM spores was counted using wet sieving and
the decanting method (Gerdemann & Nicolson 1963). The data on measured parameters of
two year trials were pooled together and differences between treatments were determined
using ANOVA and the significance difference among treatments was tested at p� 0.05.
Results
Plant growth and chlorophyll content
Growth of greengram plants in sterilized soil pot experiments following microbial inoculations
varied considerably (Table I). In general, no significant effect on the length of plant parts
(e.g., roots and shoots) was observed with any of the single inoculation treatments at
flowering (45 DAS) or at harvest (80 DAS) of plant growth except P. variabile that
significantly reduced the measured length only at harvest. In contrast, the dual inoculation of
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Tab
leI.
Eff
ect
of
rhiz
otr
op
hic
mic
roo
rgan
ism
so
ngro
wth
char
acte
rat
45
day
saf
ter
seed
ing
and
ath
arve
stan
dch
loro
ph
yll
con
ten
tat
flo
wer
ing
stag
ein
gre
engra
m.
Mea
nle
ngth
(cm
)M
ean
dry
mas
s(g
pla
nt7
1)
To
tal
pla
nt
mas
s
Roo
tS
ho
ot
Ro
ot
Sh
oo
t(g
pla
nt7
1)
Ch
loro
ph
yll
Tre
atm
ent
45
dH
arve
st4
5d
Har
vest
45
dH
arve
st4
5d
Har
vest
45
dH
arve
st(m
gp
lan
t71)
Bra
dyr
hiz
obiu
msp
.(v
ign
a)1
1.6
14
.82
5.2*
29
.5*
0.6*
0.8*
1.5*
1.0*
2.1*
2.6*
2.3
Pen
icillium
vari
abi
le8
.91
0.6*
15
.21
6.4*
0.2
0.3
0.2
0.3
0.4*
0.6
1.3
Glo
mus
fasc
icula
tum
11
.21
2.6
19
.2*
23
.10
.8*
1.1*
0.6
1.2*
1.4*
2.3*
1.9
Baci
llus
subt
ilis
10
.61
3.2
15
.62
0.5
0.3
0.4
0.3
0.4
0.6
0.8
2.0
Bra
dyr
hiz
obiu
msp
.
(vig
na)þ
P.
vari
abi
le
9.6
11
.51
7.4
19
.20
.20
.40
.20
.30
.4*
0.7*
1.5
Bra
dyr
hiz
obiu
msp
.
(vig
na)þ
G.
fasc
icula
tum
12
.21
4.5
26
.5*
29
.8*
1.2*
1.5*
1.4*
1.7*
2.6*
2.6*
2.6*
Bra
dyr
hiz
obiu
msp
.
(vig
na)þ
B.
subt
ilis
11
.81
3.3
23
.6*
28
.3*
0.3
0.4
0.5
0.6
0.8
1.0
2.4
P.
vari
abi
leþ
G.
fasc
icula
tum
10
.21
2.4
19
.6*
27
.2*
0.2
0.4
0.3
0.4
0.5*
0.8
1.8
P.
vari
abi
leþ
B.
subt
ilis
11
.51
2.6
17
.51
9.4
0.1
0.3
0.2
0.4
0.3*
0.7*
1.6
G.
fasc
icula
tumþ
B.
subt
ilis
13
.1*
14
.61
9.2*
23
.10
.7*
0.9*
0.6
0.9*
1.3*
1.8*
2.8*
Bra
dyr
hiz
obiu
msp
.(v
ign
a)
þP
.vari
abi
leþ
G.
fasc
icula
tum
12
.41
3.7
18
.72
3.4
0.3
0.4
0.4
0.5
0.7
0.9
2.6*
Bra
dyr
hiz
obiu
msp
.(v
ign
a)
þG
.fa
scic
ula
tumþ
B.
subt
ilis
14
.2*
16
.4*
28
.3*
30
.7*
1.2*
1.6*
1.8*
1.8*
3.0*
3.4*
3.2*
P.
vari
abi
leþ
B.
subt
ilis
þG
.fa
scic
ula
tum
13
.5*
15
.72
6.7*
30
.3*
0.3
0.4
0.4
0.6
0.7
1.0
2.9*
Co
ntr
ol
(N2
0P
40)
10
.81
3.6
15
.22
1.7
0.5
0.6
0.5
0.6
1.0
1.2
1.9
LS
D(P
0.0
5)a
2.2
22
.60
3.8
05
.06
0.1
20
.16
0.1
80
.24
0.3
0.4
0.7
Val
ues
are
mea
ns
of
thre
ere
plica
tes
wh
ere
each
rep
lica
teco
nst
itu
ted
fou
rp
lan
ts/p
ot;*S
ign
ifica
nt
dif
fere
nce
ove
rco
ntr
ol
atp�
0.0
5.
aL
SD
isth
eL
east
Sig
nifi
can
tD
iffe
ren
ce.
Synergistic effects of rhizospheric microorganisms on productivity of greengram 583
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G. fasciculatumþB. subtilis and P. variabileþG. fasciculatumþB. subtilis significantly
(p� 0.05) increased the root length at flowering stage only. Among all the treatments, the
tripartite combination of G. fasciculatumþB. subtilisþG. fasciculatum dramatically enhanced
the length of measured plant parts both at 45 and 80 DAS, relative to the control. The dry
matter accumulations in plant parts (e.g., roots and shoots) and total plant biomass at 45 DAS
and at 80 DAS differed considerably among treatments (Table I). Single inoculations of
Bradyrhizobium sp. (vigna) significantly (p� 0.05) enhanced the total dry matter production
by 110 and 117% at flowering and at harvest, respectively, which was followed by 40
(flowering) and 92 (harvest) % increase due to inoculation of G. fasciculatum relative to the
control. In comparison, the dual inoculation of Bradyrhizobium sp. (vigna)þG. fasciculatum
increased the total dry matter production by 117% each at flowering and at harvest stage
compared to the control. The combined inoculation of Bradyrhizobium sp. (vigna)þB. subtilis
significantly enhanced the dry matter accumulation in shoots at flowering and at harvest. Among
all the treatments, the performance of Bradyrhizobium sp. (vigna)þG. fasciculatumþB. subtilis
was superior and increased the total dry matter production significantly by 200 and 183%
at flowering and at harvest stage, respectively, compared to control. Generally, the inoculation
of P. variabile either singly or in combination treatments, reduced the total plant biomass
compared to the control treatment. Chlorophyll contents in foliage increased by 37 and 47%,
with Bradyrhizobium sp. (vigna)þG. fasciculatum and G. fasciculatumþB. subtilis, respectively,
at flowering stage. Cholorophyll content increased even further by 68 and 53% in composite
inoculation of Bradyrhizobium sp. (vigna) þG. fasciculatumþB. subtilis and P. variableþB. subtilisþG. fasciculatum, respectively (Table I).
Symbiotic traits, seed yield, grain protein and nutrient uptake
Nodules on the root systems of greengram plants inoculated with Bradyrhizobium sp. (vigna)
alone or in combination treatments having Bradyrhizobium sp. (vigna), were invariably
produced at flowering and pod fill (60 DAS) stages of plant (Table II). A significantly
(p� 0.05) greater number of nodules per plant was recorded in dual inoculation treatments of
Bradyrhizobium sp. (vigna)þG. fasciculatum and Bradyrhizobium sp. (vigna)þB. subtilis (25
nodules plant71 in both treatments) compared to Bradyrhizobium sp. (vigna) alone, at
flowering stage. No significant difference in number of nodules was observed among single
inoculation treatments compared to the control. The nodulation however, improved even
further when G. fasciculatum fungus was added to the combination of Bradyrhizobium sp.
(vigna)þB. subtilis (34 nodules plant71) and was significantly higher at both flowering and
pod fill stage compared to all other experimental treatments. Generally, the inoculation effects
on nodulation were more profound at flowering stage compared to pod fill stage. P. variabile
when used along with Bradyrhizobium sp. (vigna) or with the combination of Bradyrhizobium
sp. (vigna)þG. fasciculatum, either adversely affected the number of nodules or showed little
increase in nodulation at the two stages of plant growth. The effects of microbial inoculations
on nodule dry mass occurred in a manner similar to those observed for nodule numbers per
plant. The tripartite cultures of Bradyrhizobium sp. (vigna)þG. fasciculatumþB. subtilis and
dual inoculation of Bradyrhizobium sp. (vigna)þB. subtilis increased the seed mass by 27 and
25%, respectively, compared to un-inoculated control. In comparison, the single (except the
Bradyrhizobium sp. (vigna) alone) or other dual inoculation treatments, showed little or no
significant effect on seed yield in greengram. The protein content in seeds increased
significantly (p� 0.05) by 30% due to inoculation of Bradyrhizobium alone, Bradyrhizobium
sp. (vigna)þG. fasciculatum and triple inoculation of P. variableþB. subtilisþG. fasciculatum
compared to N20P40 control. In comparison, the protein contents increased even further by
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Tab
leII
.E
ffec
to
frh
izo
tro
ph
icm
icro
org
anis
ms
on
no
du
lati
on
atfl
ow
erin
gan
dp
od
fill
stag
e,an
dyi
eld
,gra
inp
rote
in,
Nan
dP
con
ten
tat
har
vest
ingre
engra
m.
No
du
les
No
.p
lan
t71
Dry
mas
s
(mg
pla
nt7
1)
Yie
ld
See
dm
ass
Gra
in
pro
tein
Nco
nte
nt
(mg
pla
nt7
1)
Pco
nte
nt
(mg
pla
nt7
1)
Tre
atm
ent
45
d6
0d
45
d6
0d
(g/1
00
0se
ed)
(%)
Ro
ot
Sh
oo
tS
traw
Gra
inS
traw
Bra
dyr
hiz
obiu
msp
.(v
ign
a)1
81
72
016
35
.2*
26*
14
.630
.1*
22
.64.9
2.8*
Pen
icillium
vari
abi
le–
––
–2
5.6
17
10
.1*
15
.2*
18
.8*
3.2
2.2
Glo
mus
fasc
icula
tum
––
––
32
.61
91
1.7*
13
.5*
14
.6*
4.8
2.3
Baci
llus
subt
ilis
––
––
29
.21
91
2.2*
14
.6*
16
.5*
3.6
1.9
Bra
dyr
hiz
obiu
msp
.
(vig
na)þ
P.
vari
abi
le
16
15
10*
10*
26
.42
11
6.5
32
.6*
24
.65.9*
3.2*
Bra
dyr
hiz
obiu
msp
.
(vig
na)þ
G.
fasc
icula
tum
25*
19
24
21*
33
.62
6*
18
.232
.8*
24
.46.1*
3.3*
Bra
dyr
hiz
obiu
msp
.
(vig
na)þ
B.
subt
ilis
25*
20
28*
26*
35
.6*
25
18
.640
.2*
25
.45.4*
2.9*
P.
vari
abi
leþ
G.
fasc
icula
tum
––
––
29
.41
3*
13
.2*
22
.62
4.2
4.8
2.8*
P.
vari
abi
leþ
B.
subt
ilis
––
––
28
.61
91
6.4
18
.61
4.6*
4.2
2.3
G.
fasc
icula
tumþ
B.
subt
ilis
––
––
34
.2*
21
19
.522
.62
1.6
6.1*
3.0*
Bra
dyr
hiz
obiu
msp
.
(vig
na)þ
P.
vari
abi
le
þG
.fa
scic
ula
tum
20
17
12*
12*
29
.22
52
0.4*
25
.6*
20
.65.4*
3.1*
Bra
dyr
hiz
obiu
msp
.(v
ign
a)
þG
.fa
scic
ula
tumþ
B.
subt
ilis
34*
25*
31*
30*
36
.4*
28*
26
.4*
29
.8*
29
.2*
7.2*
4.5*
P.
vari
abi
leþ
B.
subt
ilis
þG
.fa
scic
ula
tum
––
–3
0.3*
26*
18
.624
.62
4.4
6.9*
3.4*
Co
ntr
ol
(N2
0P
40)
––
–2
8.6
20
16
.620
.32
3.5
4.2
2.2
LS
D(P
0.0
5)a
2.9
3.3
4.1
3.8
5.6
5.4
3.3
4.7
4.4
1.0
0.6
–¼
Inth
isan
dsu
bse
qu
ent
Tab
lein
dic
ates
neg
ativ
ere
sult
s;*S
ign
ifica
nt
dif
fere
nce
ove
rco
ntr
ol
atp�
0.0
5.
aL
SD
isth
eL
east
Sig
nifi
can
tD
iffe
ren
ce.
Synergistic effects of rhizospheric microorganisms on productivity of greengram 585
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40 and 30%, following the triple inoculation of Bradyrhizobium sp. (vigna)þB. subtilisþG. fasciculatum relative to the control. Protein content declined by 35% when P. variabile was
used in combination with G. fasciculatum.
The nutrient uptake by greengram plants following co-inoculation of N2 fixing, PSM
and AM fungus varied considerably among treatments (Table II). The application of
P. variabile, G. fasciculatum and B. subtilis alone, significantly decline N contents in roots,
shoots and straw compared with un-inoculated control. In contrast, the single inoculation of
Bradyrhizobium sp. (vigna) and dual inoculations of Bradyrhizobium sp. (vigna)þP. variabile, Bradyrhizobium sp. (vigna)þG. fasciculatum and Bradyrhizobium sp. (vigna)þB. subtilis significantly enhanced the N content in shoots of greengram plant. The increase
in N contents was highest (98%) in shoots of plants inoculated with Bradyrhizobium sp.
(vigna)þB. subtilis. Similarly, the triple inoculation of Bradyrhizobium sp. (vigna)þB. subtilisþG. fasciculatum improved the N contents by 59, 47 and 24% in roots, shoots
and straw, respectively. In contrast, the triple factor of Bradyrhizobium sp. (vigna)þP. variabileþG. fasciculatum increased the N content in roots by 23% while in shoot, it was
26%, compared to control. Generally, N contents were more in shoots compared to roots
or straw of greengram plants (Table II). Total average P uptake in grain and straw was
significantly increased with dual and triple inoculation treatments, maximum being in grain
(7.2 mg plant71) and straw (4.5 mg plant71) with Bradyrhizobium sp. (vigna)þG. fasci-
culatumþB. subtilis.
Available P and N contents, populations of PSM, mycorrhizal colonization and AM spores
The triple inoculation of Bradyrhizobium sp. (vigna)þG. fasciculatumþB. subtilis and
P. variabileþB. subtilisþG. fasciculatum significantly (p� 0.05) improved the P content of
soils at harvest relative to the control. Penicillium variabile alone, and G. fasciculatum either
singly or in combination with Bradyrhizobium sp. (vigna) significantly augmented the P
content of soils at harvest. The total residual N in soils, however, did not change appreciably
with any of the treatments (Table III). The quantification of PSM in soils, at 60 DAS and 80
DAS, revealed that the population of PS organisms were increased at 80 DAS compared to 60
DAS. Populations of PSM in combined inoculations increased many folds and was found
maximum at 60 DAS (976104 cfu g71) and 80 DAS (996104 cfu g71) in the rhizospheric
soils having Bradyrhizobium sp. (vigna)þG. fasciculatumþB. subtilis. Among the single or
dual inoculation treatments, the number of PSM were more in G. fasciculatumþB. subtilis
treatment at 60 DAS (716104 cfu g71) and 80 DAS (666104 cfu g71) of plant growth.
Microscopic examinations of stained roots showed that only AM-fungus inoculated plants
were colonized by G. fasciculatum. The percentage of root infection and number of
mycorrhizal spores were found maximum in triple inoculation (Bradyrhizobium sp.þG. fasciculatumþB. subtilis) treatment at the tested stages of plant growth. Among the dual
inoculation treatments, G. fasciculatumþB. subtilis showed maximum root colonization while
Bradyrhizobium sp (vigna)þG. fasciculatum revealed highest number of AM spores both at
flowering (92 spores/g soil) and at podfill stage (126 spores/g soil). In general, the percentage
of root infection and number of AM-fungal spores were higher at pod fill stage compared to
flowering stage.
Discussion
The complexity of inoculation effects of rhizotrophic organisms on legume crops arise from
variations in the specific functionality of microorganisms, differences in plant – microbe
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interaction and to variations in microbe – microbe interactions and soils which, in turn, have
led to many contradictions in the literature. Yet the increase in plant vitality, symbiotic traits
and yield of crop plants following inoculation with N2 fixing, PS organisms or AM fungi
either alone or in combinations have been reported (Patterson et al. 1990; Sahar et al. 2002).
Indeed, the results from this experiment have clearly indicated enhancement of plant growth,
nodulation, yield and nutrient uptake of greengram plants, in response to microbial
inoculation, especially in the presence of rock phosphate. In the present study, when nodule
bacteria, PS organisms and AM fungus were used together, a high level of plant growth
promotion and nutrient uptake was maintained with the added benefit of greater yield under
sterilized soil conditions. It is generally believed that growth regulating substances, e.g., auxin
and giberellins released by PS bacteria (Sattar & Gaur 1987), which improve the growth of
plants also stimulate the microbial activity in the rhizosphere (Khurana & Sharma 2000).
From the results of a two-year trial it seems that the additive effect of growth factors and
enhanced availability of nutrients, e.g., N by Bradyrhizobium sp. (vigna), P by PS organisms
and mobilization and transport of P by AM fungus to the plants may have accounted for
positively synergistic effect on greengram productivity. In contrast, the single action of PSM
or AM fungus along with RP was not sufficient to raise yield of greengram. These results thus
consolidate the involvement of microbial interaction subsequently leading to the enhance-
ment in yield.
Table III. Effect of rhizotrophic microorganisms on P and N content at harvest and populations of PSM at 60 DAS
and at harvest and root infection and AM fungal spores at flowering and pod fill stage of plant growth.
Available
nutrient in soil
(mg kg71)
Populations of
PSM
(6104 g71) soil
% Root
infection
No. of AM
fungal spores
(g71 of soil)
Treatment P N 60 d Harvest 45 d 60 d 45 d 60 d
Bradyrhizobium sp. (vigna) 17.6 0.34 – – – – – –
Penicillium variabile 22.6* 0.30 12 13 – – – –
Glomus fasciculatum 24.6* 0.35 – – 28 56 86 108
Bacillus subtilis 22.2 0.32 27 29 – – – –
Bradyrhizobium sp.
(vigna)þP. variabile
20.6 0.34 17 21 – – – –
Bradyrhizobium sp.
(vigna)þG. fasciculatum
22.8* 0.39 – – 33 56 92 126
Bradyrhizobium sp.
(vigna)þB. subtilis
17.2 0.30 53 49 – – – –
P. variabileþG. fasciculatum 19.4 0.34 17 19 27 48 67 90
P. variabileþB. subtilis 16.4 0.30 67 53 – – – –
G. fasciculatumþB. subtilis 17.5 0.36 71 66 56 82 87 94
Bradyrhizobium sp. (vigna)
þP. variabile
þG. fasciculatum
21.9 0.34 82 92 59 80 68 77.3
Bradyrhizobium sp. (vigna)
þG. fasciculatum
þB. subtilis
30.2* 0.40 97 99 60 82 180 240
P. variabileþB. subtilis
þG. fasciculatum
26.2* 0.33 45 51 56 69 162 220
Control (N20P40) 18.0 0.36 – – – – – –
LSD (P0.05)a 4.5 0.06 – – – – – –
*Significant difference over control at p� 0.05.aLSD is the Least Significant Difference.
Synergistic effects of rhizospheric microorganisms on productivity of greengram 587
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The combination of Bradyrhizobium sp. (vigna), P. variabile and G. fasciculatum was,
however, found inferior relative to the combination of Bradyrhizobium sp. (vigna), B. subtilis
and G. fasciculatum, which could be due to a negative interaction between PS fungus and PS
bacterium/Bradyrhizobium, as observed between MesorhizobiumþPseudomonasþAM fungus
(Zaidi et al. 2003). The inhibitory effect of P. variabile on the associative partner could be due
to the release of toxins in the growing environment (Aziz et al. 1998), which might have
adversely affected the functional symbiosis between Bradyrhizobium and greengram plants. In
addition, the solubilization of RP occurs through the production of organic acids (Maliha
et al. 2004) by PSM. In this context, PS fungi release comparatively more organic acids
(Venkateswarlu et al. 1984) than PS bacteria. However, rhizobia in general, require neutral or
alkaline conditions for developing effective symbiosis. The increased acidity as a result of PS
activity of P. variabile might have changed the soil environment and consequently adversely
affected the establishment of functional symbiosis (Downey & Kessel 1990) leading to the
depletion in N supply to the plants.
Simultaneous application of PSM and AM fungus has been shown to stimulate plant
growth more than inoculation of either organism alone in certain situations when the soil is P
deficient. The composite application of PSM solubilizing RP and AM fungus helps the plant
root to utilize the sparingly soluble P and consequently making more P available to the
developing crop. Accordingly, the plant growth, yield and uptake of nutrients were increased
in greengram plants. This study thus indicated that there existed a strong interaction between
B. subtilis, Bradyrhizobium and AM fungus. In addition, the nutrient effect on proliferation of
the root, which appears to provide more sites for AM infection, consequently increased the
spore number. The fact that plant growth and nutrient uptake increased in the presence of
AM fungi suggested a strong synergistic relationship between root colonization, P uptake and
growth promotion (Abdel Fattah 1997). The enhanced P concentration was due to more
solubilization from RP in the presence of PS organisms and thus indicated the greater utility
of PSM inoculation with RP. In agreement with these findings, Zaidi et al. (2003) observed
that in low P soils, plant growth and nutrient uptake in chickpea were greater after inoculation
with tripartite culture of Mesorhizobium, PSB and G. fasciculatum than after inoculation with
either organism used alone. Further, the better nodulation in case of composite inoculation at
flowering stage appears to be a result of favourable effects of PSM in making more P soluble
and available to the plants (Saber et al. 2005), which consequently promoted the root
development. In comparison, some of the treatments marginally augmented the dry matter
production or yield of greengram crop relative to the control. The variation in the
effectiveness of certain microbial combinations in the present study could probably be due to
the variation in the functionality of the tested microbial strains, differences in the survivability
and colonization efficiency of the introduced cultures in the soil or strong competition
among introduced organisms for limited nutrients leading to the exclusion of organisms
from the rhizosphere. Moreover, the differential rhizosphere effect of crops in harbouring a
target microbial strain (Pal 1998) or even the modulation of the PS capacity by specific
root exudates (Goldstein et al. 1999) may account for the observed differences among
treatments.
In the present study, the N contents in roots, shoots and straw and P contents in grain and
straw at harvest were dramatically greater in some of the single, dual or triple inoculation
treatments. The increase in N contents in plant parts could be due to increase in translocation
of soil N to the plants that was possibly mediated by AM fungus (Read & Perez-Moreno
2003). Obviously these organisms, except Bradyrhizobium, are unable to fix N by themselves.
The improvement in soil P following PSM application could possibly be due to the
solubilization of fixed or added RP (Kang et al. 2002). Thus the PSM inoculation along with
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RP had a marked influence on the residual P and also suggested the establishment of PSM in
the root zones of plants. As expected, the addition of AM fungus to soils either singly or in
combination, in general, increased root colonization and consequently the number of spores.
The results obtained from this study suggested that there existed a positive relationship
between the test organisms. These results agree with those of Islam et al. (1981) who also
reported a higher percentage of root infection in field plots.
Conclusions
From our study it can be concluded that the composite application of RP and Bradyrhizobium
sp. (vigna)þB. subtilisþG. fasciculatum was potentially more effective than other inoculation
treatments and can be used for raising the greengram productivity. Further, the present
findings suggest that by co-inoculation of these cultures with RP, there is a greater possibility
of saving considerable amounts of N and P and the entire super phosphate can be replaced by
RP and PS bacteria and AM fungus. However, field trials to test the performance of the
inocula under real conditions are advisable since the efficiency of the inoculation varies with
the soil type, P content of the soils and other environmental variables.
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