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Production of indole acetic acid by Pseudomonas sp.: Effect of coinoculation with Mesorhizobium sp. Cicer on nodulation and plant growth of chickpea (Cicer arietinum)

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Pseudomonas isolates obtained from the rhizosphere of chickpea (Cicer arietinum L.) and green gram (Vigna radiata) were found to produce significant amount of indole acetic acid (IAA) when grown in a LB medium broth supplemented with L-tryptophan. Seed bacterization of chickpea cultivar C235 with different Pseudomonas isolates showed stunting effect on the development of root and shoot at 5 and 10 days of seedling growth except the strains MPS79 and MPS90 that showed stimulation of root growth, and strains MPS104 and MRS13 that showed shoot growth stimulation at 10 days. Exogenous treatment of seeds with IAA at 0.5 and 1.0 μM concentration caused similar stunting effects on root and shoot growth compared to untreated control both at 5 and 10 days of observation, whereas higher concentration of IAA (10.0 μM) inhibited the growth of seedlings. Coinoculation of chickpea with IAA-producing Pseudomonas strains increased nodule number and nodule biomass by Mesorhizobium sp. Cicer strain Ca181. The plant dry weights of coinoculated treatments showed 1.10 to 1.28 times increase in comparison to Mesorhizobium-inoculated plants alone and 3.62 to 4.50 times over uninoculated controls at 100 days of plant growth. The results indicated the potential usefulness of allelopathic rhizosphere bacteria and growth-mediating IAA in enhancement of nodulation and stimulation of plant growth in chickpea.
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RESEARCH ARTICLE
Production of indole acetic acid by Pseudomonas sp.: effect
of coinoculation with Mesorhizobium sp. Cicer on nodulation
and plant growth of chickpea (Cicer arietinum)
Deepak K. Malik &Satyavir S. Sindhu
Published online: 13 January 2011
#Prof. H.S. Srivastava Foundation for Science and Society 2011
Abstract Pseudomonas isolates obtained from the rhizo-
sphere of chickpea (Cicer arietinum L.) and green gram
(Vigna radiata) were found to produce significant amount of
indole acetic acid (IAA) when grown in a LB medium broth
supplemented with L-tryptophan. Seed bacterization of
chickpea cultivar C235 with different Pseudomonas isolates
showed stunting effect on the development of root and shoot
at 5 and 10 days of seedling growth except the strains
MPS79 and MPS90 that showed stimulation of root growth,
and strains MPS104 and MRS13 that showed shoot growth
stimulation at 10 days. Exogenous treatment of seeds with
IAA at 0.5 and 1.0 μM concentration caused similar stunting
effects on root and shoot growth compared to untreated
control both at 5 and 10 days of observation, whereas higher
concentration of IAA (10.0 μM) inhibited the growth of
seedlings. Coinoculation of chickpea with IAA-producing
Pseudomonas strains increased nodule number and nodule
biomass by Mesorhizobium sp. Cicer strain Ca181. The plant
dry weights of coinoculated treatments showed 1.10 to 1.28
times increase in comparison to Mesorhizobium-inoculated
plants alone and 3.62 to 4.50 times over uninoculated
controls at 100 days of plant growth. The results indicated
the potential usefulness of allelopathic rhizosphere bacteria
and growth-mediating IAA in enhancement of nodulation
and stimulation of plant growth in chickpea.
Keywords Indole-acetic acid (IAA) .Pseudomonas sp. .
Mesorhizobium sp. Cicer .Seedling growth .Nodulation .
Chickpea
Introduction
A rich diversity of microorganisms, varying from patho-
gens to beneficial, continuously interact with higher plants
in soil ecosystem and influences the development of plant
root in the soil (Ahmad et al.2008; Taghavi et al.2009).
Potential to exploit beneficial plant-microbe interactions to
enhance plant growth and nutrient uptake has been well
documented by inoculation of plant growth promoting
rhizobacteria (PGPR). These PGPR strains improve plant
growth and soil quality by different mechanisms, including
increased mobilization of insoluble nutrients (Lifshitz et al.
1987; Ahmad et al.2008), biocontrol of phytopathogenic
organisms (Weller 2007) and/or by production of phyto-
hormones (Dubeikovsky et al. 1993; Spaepen et al.2007).
However, yield reductions have also been reported follow-
ing continuous cultivation of a single crop species on the
same area for long periods (Alstrom 1992; Kirkegaard et al.
1993).
In the rhizosphere of some crops, the accumulation of
undesirable groups of microorganisms and production of
phytotoxic allelochemicals has been found to cause soil
sickness (Schippers et al.1987; Karen et al.2001). Their
metabolites negatively influence the enzymatic activity,
plant physiological processes and reduce the availability of
some plant nutrients and their uptake by crop plants. Wide
ranges of allelochemicals have been isolated from plant
growth-mediating rhizosphere bacteria and even a single
species of bacteria could produce several allelochemicals,
e.g., geldanamycin, nigericin and hydanthocidin are pro-
duced by Streptomyces hygroscopicus. These allelochem-
icals inhibited radicle growth of Lepidium sativum by 50%
in Petri dishes at 1 to 2 ppm (Heisey and Putnam 1986).
Similarly, Pseudomonas syringae strain 3366 produced the
metabolite phenazine-1-carboxylic acid that suppressed
D. K. Malik :S. S. Sindhu (*)
Department of Microbiology,
CCS Haryana Agricultural University,
Hisar 125004, India
e-mail: sindhuss@hau.ernet.in
Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532
DOI 10.1007/s12298-010-0041-7
germination of seeds and reduced root and shoot growth of
winter wheat and other weeds in agar diffusion assays
(Gealy et al.1996). Other allelochemicals such as succinic
acid and lactic acid were produced from Pseudomonas
putida (Yoshikawa et al.1993), cyanide by Pseudomonas
spp. (Bakker and Schippers 1987) and indole acetic acid
by Pseudomonas fluorescens (Dubeikovsky et al. 1993;
Barazani and Friedman 1999).
IAA biosynthesis has been correlated with stimulation
of root proliferation by rhizosphere bacteria (Persello-
Cartieaux et al.2003; Spaepen et al.2007), which
enhanced uptake of nutrients by the associated plants
(Lifshitz et al. 1987). The effect of IAA has been found to
depend on the concentration, that is, low concentrations of
exogenous IAA can promote, whereas high concentrations
can inhibit root growth (Arshad and Frankenberger 1992).
Moreover, inoculation with an Azospirillum brasilense Sp245
mutant strain, strongly reduced in auxin biosynthesis or
addition of increasing concentrations of exogenous auxin to
the plant growth medium, indicated that the differential
response to A. brasilense Sp245 among the common bean
(Phaseolus vulgaris L.) genotypes is related to the bacterial
produced auxin (Remans et al.2008). For various PGPR, the
promotion of plant growth after inoculation with rhizobac-
teria has been attributed to biosynthesis and secretion of IAA
in case of Azospirillum brasilense (Okon and Vanderleyden
1997), Rhizobium species (Hirsch and Fang 1994)aswellas
in Xanthomonas and Pseudomonas (Patten and Glick 1996;
Zhang et al.1997).
On the other hand, the inhibitory effect of some
deleterious rhizobacteria through IAA secretion has been
related to various bacterial species including Enterobacter
taylorae, Klebsiella planticola, Alcaligenes faecalis, Xan-
thomonas maltophila, Pseudomonas sp.and Flavobacte-
rium sp. (Sarwar and Kremmer 1995; Suzuki et al.2003).
Mutants of Pseudomonas putida that produced high levels
of IAA inhibited root growth of seedlings of canola
(Brassica campestris) by ca. 33% (Xie et al.1996). Thus,
ambiguity about effect of IAA on growth of root, shoot and
rate of seedling emergence has been reported (de Freitas
and Germida 1990; Sarwar and Kremmer 1995; Barazani
and Friedman 1999). Despite the potential of allelopathic
bacteria and growth-mediating allelochemicals in agricul-
ture, it is one of the poorly understood areas of plant-
microbe interactions. Further work is needed to characterize
bacteria and allelochemicals from the rhizosphere soil and
to study their effect on the crop plants.
Chickpea (Cicer arietinum L.), the worlds third most
important food legume, is grown during the winter season
in arid and semi-arid zones in Asia. South and South-East
Asia region contributes about 80% to the global chickpea
production and India is the principal chickpea producing
country with production of 5,970,000 tonnes (83% share in
the region) in 2008. The present studies showed that IAA-
producing Pseudomonas strains showed stunting effect on
the development of root and shoot at 5 and 10 days of
seedling growth in chickpea (Cicer arietinum L.). However,
Coinoculation of IAA-producing Pseudomonas strains with
Mesorhizobium sp. Cicer strain in chickpea increased
nodule number and plant dry weights in comparison to
Mesorhizobium-inoculated plants and uninoculated plants.
Materials and methods
Bacterial cultures and seeds
Standard strains of Pseudomonas sp. MRS13 and effective
Mesorhizobium sp. Ca181 used in the present studies were
taken from the Department of Microbiology, CCS Haryana
Agricultural University, Hisar and maintained on Luria-
Bertani (LB) medium (Sambrook et al. 1989) and yeast-
extract mannitol agar (YEMA) medium (Vincent 1970),
respectively by periodic transfers. Seeds of chickpea (Cicer
arietinum L.) cv. C235 were obtained from Pulses Section,
Department of Plant Breeding, CCS Haryana Agricultural
University, Hisar.
Isolation of Pseudomonas strains from rhizosphere
Soil samples from the rhizosphere of chickpea and green
gram were collected from different locations at pre-
flowering stage of plant growth. From each location, 10-
15 healthy plants were uprooted along with adhered soil
and brought to the laboratory in polythene bags. From
pooled samples of each location, 10 g adhered rhizosphere
soil along with roots (cut to 2-3 cm pieces) was used for
serial dilutions and 10
-3
to 10
-5
dilutions were plated on
solidified Kings B (KB) agar medium plates. The plates
were incubated at 28±1°C for 3-4 days. Pseudomonas
colonies giving fluorescence against reflected light were
picked up and purified further by streaking on the KB
medium plates. Selected isolates were identified upto the
genus level by morphological and biochemical character-
istics (Palleroni 1984) and were transferred on KB medium
slopes.
Determination of IAA production
IAA production by different Pseudomonas isolates was
determined using Salkowskis reagent (Gordon and Weber
1951; Mayer 1958). The purified and freshly grown
cultures on Luria-Bertani (LB) medium slopes were
transferred into tubes containing 5 ml LB broth supple-
mented with 100 μgml
-1
L-tryptophan and were incubated
at 28±1°C for 2 and 4 days. The broth was then centrifuged
26 Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532
for 5 min at 10,000 rpm and in the supernatant equal
volume of Salkowskis reagent was added. The contents
were mixed and allowed to stand at room temperature for
30 min to develop colour. The optical density was then
recorded at 500 nm. Uninoculated broth served as control.
Standard curve was prepared with 5-100 μgml
-1
of IAA
(Sigma Chemicals) for quantification.
Effect of Pseudomonas strains on seedling growth
Healthy seeds of chickpea cv. C235 were surface sterilized
with acidic alcohol (H
2
SO
4
: ethanol, 7:3, v/v) for 3 min
followed by thorough washing with repeated changes of
sterilized distilled water (Sindhu et al. 1999). The surface
sterilized seeds were inoculated with broth culture of
different Pseudomonas isolates and allowed to be adsorbed
for 30 min. Inoculated seeds were germinated on water agar
germination plates (10 g agar L
-1
distilled water) at 28±1°C
in a BOD incubator. Uninoculated seeds treated with LB
broth alone were sown as control. The root and shoot
lengths were measured at 5 and 10 days after sowing.
Effect of exogenous IAA on root and shoot growth
StocksolutionofIAAwaspreparedinethanolata
concentration of 0.1 M and subsequently different concen-
trations of IAA (0.5, 1.0 and 10.0 μM) were made in
distilled water. Surface sterilized seeds of chickpea were
treated with different concentrations for 30 min. The IAA-
treated seeds were germinated on water agar plates at 28 ±
1°C. Root and shoot lengths of the germinated seedlings
were measured at 5 and 10 days for comparison with
bacterial treatments.
Coinoculation of Pseudomonas strains with Mesorhizobium
sp. Cicer strain Ca181 under chillum jar conditions
For preparing chillum jar assemblies, thoroughly washed
and dried coarse river sand was used to fill the upper
assembly while the lower assembly was filled with quarter-
strength of Sloger's nitrogen-free mineral salt solution
(Sloger 1969). The whole assembly was then autoclaved
at 15 lbs pressure for 3 h. Surface-sterilized seeds of
chickpea cv. C235 were inoculated with broth culture of
Mesorhizobium sp. Cicer strain Ca181 alone or as
coinoculants with Pseudomonas isolates by mixing the
broth of the two in a ratio of 1:1 (v/v). Two ml of the mixed
inoculum was inoculated on 15 seeds and left for 30 min
for adsorption. In case of strain Ca181 alone, 1 ml of broth
and 1 ml water was added to have relatively the same level
of inoculum. Seeds were then sown in sterilized chillum jar
assemblies. Uninoculated seeds were sown as control,
keeping 9 replications for each treatment. In each chillum
jar, 5 seeds were sown and after germination, 3 seedlings
were kept. The jars were kept in a net house under day light
conditions. Quarter strength Sloger's nitrogen-free mineral
salt solution was used for watering as and when required.
The plants were uprooted after 60, 80 and 100 days of
sowing and observations were taken for nodule number,
nodule fresh weight, nitrogenase activity and plant dry
weights.
Nitrogenase assay
Nitrogenase activity in nodules was determined by measur-
ing acetylene reduction activity (ARA) at different stages of
plant growth (Hardy et al. 1968). The plants from chillum
jars were uprooted and the adhered soil was removed by
shaking the plants gently. The root and shoot portions were
separated. Roots along with nodules were transferred to
250-ml conical flasks fitted with B24 joint and serum
stoppers. In each flask, 10% of the air was replaced with
freshly prepared acetylene and the flasks were incubated at
28±1°C for 1 h. Ethylene formed was determined by Gas
Liquid Chromatograph (Nucon Aimil 5700, New Delhi,
India) using dual flame ionization detector (FID) and
Porapak N columns (2 M length x 2 mm diameter).
Nitrogen at a flow rate of 40 ml min
-1
was used as a
carrier gas and hydrogen at a flow rate of 20 ml min
-1
was
used as fuel gas. Oven, detector and injector temperatures
were kept at 105, 110 and 110°C, respectively. Standard
ethylene was used for calibration and nitrogenase activity
was expressed as μM of acetylene reduced h
-1
plant
-1
.
Nodule fresh weight and plant dry weight
The nodules were detached from the roots after determina-
tion of nitrogenase activity, washed with water and blotted
in folds of filter paper. The nodules were counted and fresh
weight was taken. Shoot portion was dried in oven at 90°C
for 24 h and weighed.
Results
Screening of Pseudomonas isolates for IAA production
in broth
Out of 40 Pseudomonas isolates obtained from the
rhizosphere of chickpea and green gram, only 11 isolates
produced indole-acetic acid in LB broth (Table 1). The
amount of IAA produced varied from 10.2 to 31.2 μgml
-1
of supernatant in different isolates. Isolates CPS59, CPS63,
CPS67, CPS72, MPS77, MPS78 and MPS94 produced
significant amount of IAA (18.131.2 μgml
-1
) at 2 days of
growth. At 4 days of culture growth, the amount of IAA
Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532 27
released in the supernatant increased. Isolates CPS59,
CPS63, CPS72, MPS77, MPS78 and MPS94 excreted
22.2 to 40.6 μgml
-1
of IAA. Maximum amount of IAA
production was observed in Pseudomonas isolates CPS72
and MPS77.
Effect of seed inoculation and exogenous application
of IAA on root and shoot growth
All the IAA producing Pseudomonas isolates showed
stunting effect on root and shoot growth at 5 days
(Table 2). Maximum stunting effect on root and shoot
growth was observed with isolate MPS77 followed by
MRS13, CPS59 and MPS94. Isolates CPS67, MPS79 and
MPS90showedmaximumstuntingeffectonshootatboth
5 and 10 days. However, all the isolates comparatively had
elongation effect on root growth at 10 days in comparison
to 5 days observation. A little shoot elongation effect was
observed with isolates MRS13 and MPS104 at 10 days.
The exogenous application of IAA at 0.5 μM on seeds
showed stunting effect on both root and shoot as compared
to untreated control seedlings at 5 and 10 days of
observation (Table 3). The taproot growth was inhibited
but it caused lateral root development. At higher levels of
IAA (10 μM), there was complete inhibition of root growth
at 5 days but little root growth was observed at 10 days. At
10 μM concentration, shoot growth was completely
inhibited even at 10 days of observation.
Effect of coinoculation of Pseudomonas
with Mesorhizobium sp. Cicer strain Ca181
Seed inoculation of chickpea with Mesorhizobium sp. Cicer
Ca181 alone or on coinoculation with Pseudomonas isolates
increased the plant dry weights in comparison to uninocu-
lated controls under chillum jar conditions at all the stages of
plant growth (Table 4). The plant dry weight gains varied
from 1.14 to 1.80 times to those of Mesorhizobium-
inoculated treatments and 4.50 to 7.10 times to those of
uninoculated controls at 60 days of plant growth. Five
Pseudomonas isolates i.e., CPS10, CPS67, MPS77, MPS78
and MPS104 caused maximum gain in plant dry weight
ratios i.e., 1.48, 1.77, 1.80, 1.49 and 1.61 times to those of
Mesorhizobium-inoculated plants, respectively. The nodule
promoting effect was evident with only five Pseudomonas
isolates CPS67, MPS77, MPS94, MPS104 and MRS13, and
coinoculation resulted in increased nodule weight. Pseudo-
monas strain-dependent variations in acetylene reduction
activity were observed in nodules of various treatments.
Table 1 Production of indole acetic acid by different Pseudomonas
isolates. Control treatment contains LB broth and the Salkowskis
reagent. IAA production was calculated on the basis of equal 1.0
optical density of bacterial growth suspension
Pseudomonas isolate IAA production (μgml
-1
supernatant)
2 days 4 days
Control 0.0 0.0
CPS10 10.2 10.8
CPS59 18.2 22.8
CPS63 18.1 22.2
CPS67 18.4 19.7
CPS72 27.7 30.3
MPS77 31.2 40.6
MPS78 19.2 24.0
MPS79 11.4 16.8
MPS90 14.0 20.0
MPS94 19.7 25.1
MPS104 16.7 19.8
MRS13 13.2 19.5
Table 2 Effect of Pseudomonas isolates on seedling growth of
chickpea. Means of five replications ± SE
Treatment Root length (cm) Shoot length (cm)
5 days 10 days 5 days 10 days
Control 14.35±0.44 17.12± 0.82 8.02 ± 0.44 10.40 ± 1.1
CPS10 12.46± 0.50 16.96 ± 0.50 7.88 ± 0.41 9.38± 0.85
CPS59 9.45± 0.51 15.90 ± 0.75 6.30 ± 0.33 10.30 ± 1.1
CPS63 9.86± 0.51 15.70 ± 0.51 6.96 ± 0.56 10.40 ± 1.9
CPS67 9.50± 0.55 14.24 ± 0.05 7.64 ± 0.30 7.94±1.0
CPS72 11.52± 0.33 16.60 ± 0.29 8.10 ± 0.29 9.66± 1.7
MPS77 8.95± 0.36 16.20 ± 0.62 6.12 ±0.62 10.40±1.6
MPS78 11.24± 0.43 16.20 ± 0.19 7.46 ±0.49 9.14± 1.8
MPS79 11.35± 0.72 17.70 ± 0.71 7.50 ±0.20 8.30± 1.1
MPS90 9.85± 0.18 17.40 ± 0.28 7.20 ± 0.40 8.80± 0.3
MPS94 9.50± 0.50 11.30 ± 0.05 6.40 ±0.19 9.25± 2.1
MPS104 10.26± 0.88 12.10 ± 0.89 7.26 ± 0.80 11.00 ± 1.7
MRS13 9.04± 0.45 16.74 ± 0.20 6.20 ± 0.20 11.14± 2.2
Table 3 Effect of exogenously applied IAA on root and shoot growth
of chickpea seedlings on water agar germination plates. Means of five
replications± SE
Treatment Root length (cm) Shoot length (cm)
5 days 10 days 5 days 10 days
Control 10.85± 0.44 18.7 ± 0.69 7.5±0.24 15.0±0.37
0.5 μM 8.4 ± 0.58 7.2±0.27 4.3± 0.14 5.1± 0.12
1.0 μM 2.0 ± 0.40 4.9±0.48 2.5± 0.05 2.9± 0.08
10.0 μM 0.0 1.7± 0.06 0.0 0.0
28 Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532
At 80 days of plant growth, plant dry weight ratio of
coinoculated plants varied from 3.06 to 5.0 times over
uninoculated control and 1.19 to 1.93 times in comparison
to Mesorhizobium-inoculated plants (Table 4). More plant
growth enhancement was observed on coinoculation with
Pseudomonas cultures CPS10, CPS67, CPS72, MPS77,
MPS79, MPS94 and MPS104. Coinoculation with Pseudo-
monas cultures CPS10, CPS59, CPS72, MPS77, MPS90,
MPS94 and MRS13 significantly increased nodule biomass
and nodule number, indicating stimulation of nodulation by
Mesorhizobium on coinoculation with Pseudomonas
strains.
At later stages of plant growth (100 days), coinoculation
with six Pseudomonas strains CPS10, CPS59, CPS72,
MPS77, MPS79 and MRS13 increased the plant dry
weights of chickpea 3.62 to 4.50 times to control (Table 5).
Three Pseudomonas strains MPS78, MPS90 and MPS94
showed very little (1.10 to 1.28 times) increase in the
symbiotic effectiveness. Most of the Pseudomonas strains
promoted nodulation by Mesorhizobium sp. Cicer strain
Ca181 except the strain MPS104. The acetylene reduction
activity (ARA) in nodules at 100
th
day declined in most of
the coinoculation treatments as compared to ARA observed
at 80 days of plant growth.
Table 4 Effect of coinoculation of chickpea with Pseudomonas strains and Mesorhizobium strain Ca181 on symbiotic parameters at 60 and 80 days
of plant growth under sterile conditions. Data are average values of three plants
Treatments Plant
growth (days)
Nodule
number (plant
-1
)
Nodule
fresh weight (mg plant
-1
)
Nitrogenase
activity (μMC
2
H
2
reduced
plant
-1
h
-1
)
Plant dry weight
(mg plant
-1
)
Control 60 –– – 102
80 –– – 205
CPS63 60 –– – 86
80 –– – 188
MPS94 60 –– – 78
80 –– – 214
Mesorhizobium strain Ca181 60 25 462 3.17 407
80 35 1,208 3.52 536
Ca181+ CPS10 60 22 565 3.79 604
80 59 1,662 3.48 1,035
Ca181+ CPS59 60 20 286 4.35 534
80 43 1,432 3.97 732
Ca181+ CPS63 60 18 364 3.12 562
80 37 1,164 4.14 768
C181+ CPS67 60 30 765 2.59 724
80 34 1,035 3.70 964
Ca181+ CPS72 60 21 508 2.62 532
80 45 1,456 4.61 972
Ca181-MPS77 60 36 802 3.48 735
80 40 1,318 2.85 946
Ca181+ MPS78 60 21 468 3.78 609
80 39 1,285 4.18 756
Ca181+ MPS79 60 24 402 2.71 568
80 36 1,202 1.36 954
Ca181+ MPS90 60 24 406 2.01 504
80 41 1,342 3.86 628
Ca181+ MPS94 60 27 537 4.36 465
80 48 1,472 3.96 936
Ca181+ MPS104 60 28 564 3.24 656
80 34 956 2.43 962
Ca181+ MRS13 60 33 712 3.92 564
80 60 1,664 3.87 907
Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532 29
Discussion
The plant rhizosphere is a dynamic ecological environment
in soil for plant-microbe interactions (Benizri et al.2001;
Somers et al.2004). Beneficial microbial allelopathies in
the root zone are a key agent of change in soil ecosystems
and affect crop health, yield and soil quality (Sturz and
Christie 2003; Taghavi et al.2009). The release of
allelochemicals such as phenolic acids, phytotoxins, cya-
nide, phenazine-1-carboxylic acid and excess amount of
IAA by rhizosphere bacteria were found to suppress
germination of seeds and reduced root as well as shoot
growth in different crops (Bakker and Schippers 1987;
Gealy et al.1996; Karen et al.2001). Production of
allelochemicals, particularly IAA has been considered as
an important attribute of PGPR strains that can affect plant
growth in diverse ways, varying from pathogenesis and
growth inhibition to plant growth stimulation (Prikryl et al.
1985; Somers et al.2004; Spaepen et al.2007).
In the present study, out of 40 Pseudomonas isolates
obtained from chickpea and green gram rhizosphere, only
11 isolates were found to produce IAA. The amount of IAA
produced varied from 10.2 to 31.2 μgml
-1
of supernatant in
different Pseudomonas isolates at 2 days and from 10.8 to
40.6 μgml
-1
at 4 days of bacterial growth (Table 1). The
production of phytohormones in chemically defined media
has also been reported in other PGPR strains including
Azotobacter chroococcum (Muller et al.1989), Azospir-
illum (Bar and Okon 1992; Remans et al.2008), Rhizobium
species (Hirsch and Fang 1994), Bacillus polymyxa (Holl et
al.1988), Pseudomonas fluorescens (Dubeikovsky et al.
1993) and Pseudomonas putida (Taghavi et al.2009).
Inoculation of IAA-producing Pseudomonas isolates on
seeds of chickpea showed initial stunting effect on root and
shoot growth except in a few cases where little stimulation
of root and shoot growth was observed. Maximum stunting
effect on root as well as shoot growth was observed at
5 days with the strain MPS77 followed by strains MRS13,
CPS59 and MPS94 (Table 2). Pseudomonas isolates
CPS67, MPS79 and MPS90 showed maximum stunting
effect on shoot at both the stages of observations. All the
Pseudomonas strains comparatively increased the root
growth at 10 days than to 5 days observation. The initial
stunting effect on seedlings could be due to contact of
bacterial cell with legume seeds, due to synthesis or
secretion of excessive amount of IAA and/or some inhibitory
agent produced by the bacterium grown in synthetic medium
(Loper and Schroth 1986; Bolton and Elliott 1989). Similar
results were obtained when cuttings of sour cherry (Prunus
cerasus) and black-currant (Ribes nigrum) were inoculated
with a recombinant strain of Pseudomonas fluorescens that
produced increased amount of IAA. A high density of
bacterium inoculum on the roots of cherry cuttings inhibited
root growth, whereas lower densities on black-currant
promoted growth (Dubeikovsky et al.1993).
Exogenous application of IAA was made on chickpea
seeds to correlate the inhibitory or growth promoting effects
on seedling growth with IAA production in defined
medium. The exogenous application of IAA at 0.5 μM
concentration showed stunting effect on both root and shoot
growth of chickpea seedlings in comparison to untreated
seeds (Table 3). The tap root growth was inhibited but it
caused lateral root formation. At higher concentrations of
IAA (10.0 μM), there was complete inhibition of root
growth. Similarly, high concentrations of IAA caused
stunting of shoot and inhibited shoot emergence even at
10 days. Arshad and Frankenberger (1992) also reported
that the effect of IAA is concentration dependent, that is,
low concentrations of exogenous IAA can promote,
whereas high concentration can inhibit root growth. Loper
and Schroth (1986) observed a significant linear relation-
ship between IAA accumulation of the rhizobacterial strains
and decreased root elongation of sugar beet seedlings.
Similarly, inoculation of canola (Brassica campestris) seeds
with Pseudomonas putida GR12-2, which produced low
level of IAA, resulted in 2 to 4-fold increase in the length of
seedling roots, whereas an IAA over producing mutant
inhibited root growth of seedlings by 33% (Xie et al.1996).
In contrast, Astrom et al.(1993) reported that treatment
Tab l e 5 Effect of coinoculation of Pseudomonas strains and
Mesorhizobium sp. Cicer strain Ca181 on symbiotic parameters of
chickpea at 100 days of plant growth. Nitrogenase activity (μMC
2
H
2
reduced plant
-1
h
-1
). Data are average values of three plants
Treatments Nodule
number
(plant
-1
)
Nodule
fresh
weight
(mg plant
-1
)
Nitrogenase
activity (μM)
Plant dry
weight
(mg
plant
-1
)
Control 5 94 432
Mesorhizobium
strain Ca181
43 1,362 3.68 938
CPS63 ––465
MPS94 ––462
Ca181+ CPS10 80 2,875 3.60 1,948
Ca181+ CPS59 48 1,512 2.58 1,564
Ca181+ CPS63 54 1,768 3.35 1,262
Ca181+ CPS67 51 1,632 4.36 1,305
Ca181+ CPS72 74 2,704 3.17 1,764
Ca181+ MPS77 55 1,864 2.29 1,608
Ca181+ MPS78 48 1,566 3.43 1,035
Ca181+ MPS79 65 2,536 2.91 1,936
Ca181+ MPS90 50 1,608 2.78 1,124
Ca181+ MPS94 52 1,674 3.64 1,205
Ca181+
MPS104
41 1,335 2.84 1,266
Ca181+ MRS13 64 2,508 2.04 1,705
30 Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532
with a cell-free culture filtrate of P. fluorescens caused a
strong inhibitory effect on root elongation of wheat seed-
lings. In other studies also, inhibitory effect of some
deleterious rhizobacteria (DRB) was related to the high
amount of IAA excretion (Sarwar and Kremmer 1995;
Barazani and Friedman 1999; Suzuki et al.2003).
Inoculation of legumes and cereal plants with PGPR
strains has been found to show a wide range of effects on
plant growth that varied among different strains of PGPR.
Chickpea plants inoculated with Pseudomonas strains i.e.,
CPS10, CPS67, MPS77, MPS78 and MPS104 caused
increase in plant dry weight ratios i.e., 1.14 to 1.80 times
to those of Mesorhizobium-inoculated plants, respectively
at 60 days of plant growth. Plant dry weight ratio of
coinoculated plants varied from 3.06 to 5.0 over control and
1.19 to 1.93 times in comparison to Mesorhizobium-
inoculated plants at 80 days of plant growth (Table 4). At
later stages of plant growth (100 days), coinoculation with
Pseudomonas strains CPS10, CPS59, CPS72, MPS77,
MPS79 and MRS13 increased the plant dry weights of
chickpea 3.62 to 4.50 times over the uninoculated control
(Table 5). Similar effect of coinoculation of rhizobacteria
with Rhizobium on symbiotic parameters have been
reported in other legumes like alfalfa (Knight and
Langston-Unkeffer 1988), chickpea (Parmar and Dadarwal
1999), green gram (Sindhu et al. 1999), pea (Bolton et al.
1990; Berggren et al.2001) and soybean (Dashti et al.
1997).
Coinoculation with most of the IAA-producing Pseudo-
monas strains with Mesorhizobium sp. Cicer strain Ca181
also resulted in increased nodule number and nodule fresh
weight (Table 4,5), indicating stimulation of nodulation by
Mesorhizobium sp. on coinoculation. Rhizobacteria as well
as mycorrhizal fungi have been found to enhance the
production of flavonoid-like compounds or phytoalexins in
roots of several crop plants (Parmar and Dadarwal 1999;
Goel et al.2001) that induce the transcription of rhizobial
nodulation (nod) genes. The localized plant hormone
auxins have also been shown to participate in the
fundamental responses of nodule morphogenesis. Similar
nodule-promoting effects of Pseudomonas sp. on coinocu-
lation with Rhizobium strains have been reported in
soybean (Nishijima et al.1988; Zhang et al. 1996) and
green gram (Sindhu et al.1999).
The initial stunting effects of Pseudomonas strains on
root and shoot growth under controlled conditions, however,
did not show adverse effect on nodulation and plant growth
in these studies when these bacteria were used as coinocu-
lants with Mesorhizobium. For example, coinoculation with
Pseudomonas isolates MPS79 and CPS10, which showed
maximum stunting effect on shoot under cultural conditions
at 10 days (Table 2), resulted in significant gain in shoot dry
weight at 100 days of plant growth (Table 5) in comparison
to the high IAA producer Pseudomonas isolate MPS77. It
is, therefore, apparent that IAA production by the rhizobac-
teria beyond a critical limit may not be desired for plant
growth promotion. Because the relative concentration of
microbial allelochemicals may result in different response of
higher plants, therefore, inoculation tests under field con-
ditions are essential for evaluating the allelopathic impact of
soil-borne microorganisms.
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