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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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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 .
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.
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
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
to 10
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
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
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
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
: 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
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
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
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
was used as a
carrier gas and hydrogen at a flow rate of 20 ml min
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
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.
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
of supernatant in different isolates. Isolates CPS59, CPS63,
CPS67, CPS72, MPS77, MPS78 and MPS94 produced
significant amount of IAA (18.131.2 μgml
) 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
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
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
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
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
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)
number (plant
fresh weight (mg plant
activity (μMC
Plant dry weight
(mg plant
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
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
of supernatant in
different Pseudomonas isolates at 2 days and from 10.8 to
40.6 μgml
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
reduced plant
). Data are average values of three plants
Treatments Nodule
(mg plant
activity (μM)
Plant dry
Control 5 94 432
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
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.
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.
Ahmad F, Ahmad I, Khan MS (2008) Screening of free-living
rhizosphere bacteria for their multiple plant growth promoting
activities. Microbiol Res 163:173181
Alstrom S (1992) Saprophytic soil microflora in relation to yield
reductions in soil repeatedly cropped with barley (Hordeum
vulgare L.). Biol Fertil Soils 14:145150
Arshad M, Frankenberger WT Jr (1992) Microbial production of plant
growth regulators. In: Metting FB Jr (ed) Soil microbial ecology,
applications in agricultural and environmental management.
Dekker, New York, pp 2732
Astrom B, Gustafsson A, Gerhardson B (1993) Characteristics of a
plant deleterious rhizosphere pseudomonad and its inhibitory
metabolite(s). J Appl Bacteriol 74:2028
Bakker AW, Schippers B (1987) Microbial cyanide production in the
rhizosphere in relation to potato yield reduction and Pseudomo-
nas spp.-mediated plant growth stimulation. Soil Biol Biochem
Bar T, Okon Y (1992) Induction of indole-3-acetic acid synthesis and
possible toxicity of tryptophan in Azospirillum brasilense Sp7.
Symbiosis 13:191198
Barazani O, Friedman J (1999) Is IAA the major growth factor secreted
from plant growth mediating bacteria? J Chem Ecol 25:23972406
Benizri E, Boudoin E, Guckert A (2001) Root colonization by
inoculated plant growth-promoting rhizobacteria. Biocontrol Sci
Technol 11:557574
Berggren I, van Vuurde JWL, Martensson AM (2001) Factors
influencing the effect of deleterious Pseudomonas putida
rhizobacteria on initial infection of pea roots by Rhizobium
leguminosarum bv. viciae. Appl Soil Ecol 17:97106
Bolton H Jr, Elliott LF (1989) Toxin production by a rhizobacterial
Pseudomonas sp. that inhibits wheat root growth. Plant Soil
Bolton H Jr, Elliott LF, Turco RF, Kennedy AC (1990) Rhizoplane
colonization of pea seedlings by Rhizobium leguminosarum and a
deleterious root colonizing Pseudomonas sp. and effects on plant
growth. Plant Soil 123:121124
Dashti N, Zhang F, Hynes RK, Smith DL (1997) Application of plant
growth promoting rhizobacteria to soybean [Glycine max (L.)
Merr.] increases protein and dry matter yield under short season
conditions. Plant Soil 188:3341
de Freitas JR, Germida JJ (1990) Plant growth promoting rhizobac-
teria for winter wheat. Can J Microbiol 36:265272
Dubeikovsky AN, Mordukhova EA, Kochetkov VV, Polikarpova FY,
Boronin AM (1993) Growth promotion of blackcurrant softwood
cuttings by recombinant strain Pseudomonas fluorescens BSP53a
synthesizing an increased amount of indole-3-acetic acid. Soil
Biol Biochem 25:12771281
Gealy DR, Gurusiddaiah S, Ogg AG Jr (1996) Isolation and
characterization of metabolites from Pseudomonas syringae
strain and their phytotoxicity against certain weed and crop
species. Weed Sci 44:383392
Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532 31
Goel AK, Sindhu SS, Dadarwal KR (2001) Seed bacterization with
fluorescent Pseudomonas enhances the synthesis of flavonoid-
like compounds in chickpea (Cicer arietinum L.). Physiol Mol
Biol Plants 6:195198
Gordon SA, Weber RP (1951) Colorimetric estimation of indole acetic
acid. Plant Physiol 26:192195
Hardy RWF, Holsten RD, Jackson EK, Burns RC (1968) The
acetylene-ethylene assay for N
fixation: laboratory and field
evaluation. Plant Physiol 43:11851205
Heisey RM, Putnam AR (1986) Herbicidal effects of geldamycin and
nigericin, antibiotics from Streptomyces hygroscopicus. J Nat
Prod 49:859865
Hirsch AM, Fang Y (1994) Plant hormones and nodulation: what is
the connection? Plant Mol Biol 26:59
Holl FB, Chanway CP, Turkington R, Radley RA (1988) Response of
crested wheatgrass (Agropyron cristatum L.), perennial ryegrass
(Lolium perenne) and white clover (Trifolium repens L.) to
inoculation with Bacillus polymyxa. Soil Biol Biochem 20:1924
Karen S, Udo B, Frank L, Dominique R (2001) Can simultaneous
inhibition of seedling growth and stimulation of rhizosphere
bacterial populations provide evidence for phytotoxin transfer
from plant residues in the bulk soil to the rhizosphere of sensitive
species? J Chem Ecol 27:807829
Kirkegaard JW, Munns R, James RA, Gardener PA, Angus JF (1993)
Reduced growth and yield of wheat with conservation cropping.
II. Soil biological factors limit under direct drilling. Aust J Agric
Res 46:7588
Knight TJ, Langston-Unkeffer PJ (1988) Enhancement of symbiotic
dinitrogen fixation by a toxin-releasing plant pathogen. Science
Lifshitz R, Kloepper JW, Kozlowski M (1987) Growth promotion of
canola (rapeseed) seedlings by a strain of Pseudomonas putida
under gnotobiotic conditions. Can J Microbiol 33:390395
Loper JE, Schroth MN (1986) Influence of bacterial sources of indole-
3-acetic acid on root elongation of sugar beet. Plant Pathol
Mayer AM (1958) Determination of indole acetic acid by the
Salkowsky reaction. Nature 182:16701671
Muller F, Deigele C, Ziegler H (1989) Hormonal interactions in the
rhizosphere of maize (Zea mays L.) and their effects on plant
development. Zournal Pflanzen Bordennk 152:247254
Nishijima F, Evans WR, Vesper SJ (1988) Enhanced nodulation of
soybean by Bradyrhizobium in the presence of Pseudomonas
fluorescens. Plant Soil 111:149150
Okon Y, Vanderleyden J (1997) Root associated Azospirillum species
can stimulate plants. Am Soc Microbiol News 63:366370
Palleroni NJ (1984) Family 1. Pseudomonadaceae. In: Krieg NR, Holt
JG (eds) Bergeys manual of systemic bacteriology. Williams and
Wilkins, Baltimore, pp 143213
Parmar N, Dadarwal KR (1999) Stimulation of nitrogen fixation and
induction of flavonoid-like compounds by rhizobacteria. J Appl
Microbiol 86:3644
Patten CL, Glick BR (1996) Bacterial biosynthesis of indole-3-acetic
acid. Can J Microbiol 42:207220
Persello-Cartieaux F, Nussaume L, Robaglia C (2003) Tales from the
underground: molecular plant-rhizobacterial interactions. Plant
Cell Environ 26:189199
Prikryl Z, Vancura V, Wurst M (1985) Auxin formation by rhizosphere
bacteria as a factor of root growth. Biol Plant 27:159163
Remans R, Bebee S, Manrique MB, Tovar E, Rao I, Croonenborghs
A, Torres-Gutierrez R, El-Howeity M, Michiels J, Vanderleyden
J (2008) Physiological and genetic analysis of root responsive-
ness to auxin-producing plant growth-promoting bacteria in
common bean (Phaseolus vulgaris L.). Plant Soil 302:149161
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A
Laboratory Manual, Cold Spring Harbor
Sarwar M, Kremmer RJ (1995) Enhanced suppression of plant growth
through production of L-tryptophan compounds by deleterious
rhizobacteria. Plant Soil 172:261269
Schippers AB, Bakker AW, Bakker PAHM (1987) Interaction of
deleterious and beneficial microorganism and effect on cropping
practices. Annu Rev Phytopathol 25:339358
Sindhu SS, Gupta SK, Dadarwal KR (1999) Antagonistic effect of
Pseudomonas spp. on pathogenic fungi and enhancement of
plant growth in green gram (Vigna radiata). Biol Fertil Soils
Sloger C (1969) Symbiotic effectiveness and nitrogen fixation in
nodulated soybean. Plant Physiol 44:16661668
Somers E, Vanderleyden J, Srinivasan M (2004) Rhizosphere bacterial
signaling: a love parade beneath our feet. Crit Rev Microbiol
Spaepen S, Vanderleyden J, Remans R (2007) Indole-3-acetic acid in
microbial and microorganism-plant signaling. FEMS Microbiol
Rev 31:425448
Sturz AV, Christie BR (2003) Beneficial microbial allelopathies in the
root zone: the management of soil quality and plant disease with
rhizobacteria. Soil Tillage Res 72:107123
Suzuki S, Yuxi H, Oyaizu H, He Y (2003) Indole-3-acetic acid
production in Pseudomonas fluorescens HP72 and its association
with suppression of creeping bentgrass brown patch. Curr
Microbiol 47:138143
Taghavi S, Garafola C, Monchy S, Newman L, Hoffman A, Weyens
N, Barac T, Vangronsveld J, van der Lelie D (2009) Genome
survey and characterization of endophytic bacteria exhibiting a
beneficial effect on growth and development of poplar trees.
Appl Environ Microbiol 75:748757
Vincent JM (1970) A manual for the practical study of root nodule
bacteria. International biological programme handbook 15.
Blackwell Scientific Publisher, Oxford
Weller DM (2007) Pseudomonas biological control agents of
soilborne pathogens: Looking back over 30 years. Phytopathol-
ogy 97:250256
Xie H, Pasternack JJ, Glick BR (1996) Isolation and characterization
of mutants of plant growth promoting rhizobacterium Pseudo-
monas putida GR12-2 that overproduce indole acetic acid. Curr
Microbiol 32:6771
Yoshikawa M, Hirai N, Wakabayashi K, Sugizaki H, Iwamura H
(1993) Succinic and lactic acids as plant growth promoting
compounds produced by rhizospheric Pseudomonas putida. Can
J Microbiol 39:11501154
Zhang F, Dashti N, Hynes RK, Smith DL (1996) Plant growth-
promoting rhizobacteria and soybean [Glycine max (L.) Merr.]
nodulation and nitrogen fixation at suboptimal root zone temper-
atures. Ann Bot 77:453459
Zhang F, Dashti N, Hynes RK, Smith DL (1997) Plant growth-
promoting rhizobacteria and soybean [Glycine max (L.) Merr.]
growth and physiology at suboptimal root zone temperatures.
Ann Bot 79:243249
32 Physiol Mol Biol Plants (JanuaryMarch 2011) 17(1):2532
... A determination of indole-3-acetic acid (IAA) production was made using the Salkowski reagent. [27]. The purified and freshly grown cultures on the average slopes of Luria-Bertani (LB) were transferred into tubes containing 5 mL of LB broth supplemented by 1 mg mL¯1 of L-tryptophan (L-TRP). ...
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Cadmium (Cd) stress is an obstacle for crop production, quality crops, and sustainable agriculture. An important role is played by the application of eco-friendly approaches to improve plant growth and stress tolerance. In the current study, a pre-sowing seed treatment with Rhizobium leguminosarum strains, isolated from the leguminous plants Phaseolus vulgaris (strain Pvu5), Vicia sylvatica (strain VSy12), Trifolium hybridium (strain Thy2), and T. pratense (strain TPr4), demonstrated different effects on wheat (Triticum aestivum L.) plant growth under normal conditions. Among all tested strains, Thy2 significantly increased seed germination, seedling length, fresh and dry biomass, and leaf chlorophyll (Chl) content. Further analysis showed that Thy2 was capable of producing indole-3-acetic acid and siderophores and fixing nitrogen. Under Cd stress, Thy2 reduced the negative effect of Cd on wheat growth and photosynthesis and had a protective effect on the antioxidant system. This was expressed in the additional accumulation of glutathione and proline and the activation of glutathione reductase. In addition, Thy2 led to a significant reduction in oxidative stress, which was evidenced by the data on the stabilization of the ascorbate content and the activity of ascorbate peroxidase. In addition, Thy2 markedly reduced Cd-induced membrane lipid peroxidation and electrolyte leakage in the plants. Thus, the findings demonstrated the ability of the R. leguminosarum strain Thy2, isolated from T. hybridium nodules, to exert a growth-promoting and anti-stress effect on wheat plants. These results suggest that the Thy2 strain may enhance wheat plant growth by mitigating Cd stress, particularly through improving photosynthesis and antioxidant capacity and reducing the severity of oxidative damage. This may provide a basic and biological approach to use the Thy2 strain as a promising, eco-friendly candidate to combat Cd stress in wheat production.
... In previous studies, a significant growthpromoting effect has been reported by inoculating the plants with IAA and GA producing bacterial endophytes. 20,57,58 In another instance, 59 11 bacterial endophytes produced IAA in a considerable amount ranging from 10.8 to 40.6 μg mL −1 . IAA plays an important role in cell division and differentiation thus improving the plant root growth and development and providing more access to nutrients in the soil and water uptake. ...
Background: Bacillus species synthesize antifungal lipopeptides (LPs) making them a sustainable and eco-friendly management option to combat Fusarium wilt of chickpea. Results: In this study, 18 endophytic Bacillus strains were assessed for their antifungal activity against Fusarium oxysporum f. sp. ciceris (FOC) associated with Fusarium wilt of chickpea. Among them, 13 strains produced significant inhibition zones in a direct antifungal assay while five strains failed to produce the inhibition of FOC. Bacillus thuringiensis CHGP12 exhibited the highest inhibition 3.45 cm of FOC. The LPs extracted from CHGP12 showed significant inhibition of the pathogen. Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed that CHGP12 possessed the ability to produce fengycin, surfactin, iturin, bacillaene, bacillibactin, plantazolicin, and bacilysin. In an in vitro qualitative assay CHGP12 exhibited the ability to produce lipase, amylase, cellulase, protease, siderophores, and indole 3-acetic acid (IAA). IAA and gibberellic acid (GA) were quantified using ultra-performance liquid chromatography (UPLC) with 370 and 770 ng mL-1 concentrations of IAA and GA respectively. Furthermore, the disease severity showed a 40% decrease over control in CHGP12 treated plants compared to the control in a glasshouse experiment. Moreover, CHGP12 also exhibited a significant increase in total biomass of the plants namely, root and shoot growth parameters, stomatal conductance, and photosynthesis rate. Conclusion: In conclusion, our findings suggest that B. thuringiensis CHGP12 is a promising strain with high antagonistic and growth-promoting potential against Fusarium wilt of chickpea. © 2022 Society of Chemical Industry.
... Some of the PGPR strains were also reported to induce systemic tolerance and immunity in plants to counter abiotic stress Tzipilevich et al. 2021). Different mechanisms are employed by PGPR strains to mitigate abiotic stresses including the formation of biofilm, chemotaxis, synthesis of hormones, exopolysaccharides (EPS) and ACC deaminase (Glick 1995(Glick , 2005Malik and Sindhu 2011;Ahluwalia et al. 2021;Koza et al. 2022). In addition, biotic stress imposed on plants by infection of disease-causing pathogens, i.e. bacteria, nematodes, fungi and viruses is also alleviated by inoculation of certain antagonistic bacteria, which suppress various diseases on plants Sharma et al. 2018;Sehrawat and Sindhu 2019;Yin et al. 2021;Sehrawat et al. 2022). ...
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Main Conclusion The responses of plants to different abiotic stresses and mechanisms involved in their mitigation are discussed. Production of osmoprotectants, antioxidants, enzymes and other metabolites by beneficial microorganisms and their bioengineering ameliorates environmental stresses to improve food production. Abstract Progressive intensification of global agriculture, injudicious use of agrochemicals and change in climate conditions have deteriorated soil health, diminished the microbial biodiversity and resulted in environment pollution along with increase in biotic and abiotic stresses. Extreme weather conditions and erratic rains have further imposed additional stress for the growth and development of plants. Dominant abiotic stresses comprise drought, temperature, increased salinity, acidity, metal toxicity and nutrient starvation in soil, which severely limit crop production. For promoting sustainable crop production in environmentally challenging environments, use of beneficial microbes has emerged as a safer and sustainable means for mitigation of abiotic stresses resulting in improved crop productivity. These stress-tolerant microorganisms play an effective role against abiotic stresses by enhancing the antioxidant potential, improving nutrient acquisition, regulating the production of plant hormones, ACC deaminase, siderophore and exopolysaccharides and accumulating osmoprotectants and, thus, stimulating plant biomass and crop yield. In addition, bioengineering of beneficial microorganisms provides an innovative approach to enhance stress tolerance in plants. The use of genetically engineered stress-tolerant microbes as inoculants of crop plants may facilitate their use for enhanced nutrient cycling along with amelioration of abiotic stresses to improve food production for the ever-increasing population. In this chapter, an overview is provided about the current understanding of plant–bacterial interactions that help in alleviating abiotic stress in different crop systems in the face of climate change. This review largely focuses on the importance and need of sustainable and environmentally friendly approaches using beneficial microbes for ameliorating the environmental stresses in our agricultural systems.
... In our study, five of the 13 studied isolates could produce IAA and KV-5 showed maximum production of indole-3-acetic acid (89.28 µg/ ml). Malik et al (2011) [25] . have reported a maximum of 40.6 µg/ml of IAA production by their Pseudomonas isolate MPS77, upon 4 days of incubation. ...
In our study, a total of 13 different bacterial endophytes (KV-1 to KV-13) isolated from Gloriosa superba during a previous study, were tested for different plant growth promoting properties like IAA (Indole-3-acetic acid) production by the method of Gordon and Weber; phosphate solubilisation on Pikovskaya's medium; siderophore production on Chrome azurol S (CAS) medium; Nitrogen fixation on Jensen's Nitrogen-free medium; ammonia production using Nessler's reagent; and Hydrocyanic acid (HCN) production on Nutrient Agar supplemented with 4.4 g/L glycine, having Whatmann No.1 filter paper strip soaked in 2% sodium carbonate in 0.5% picric acid held above it. Two promising isolates (KV-5 and KV-11) among the 13, were positive for all plant growth promoting traits tested except HCN production, whereas KV-13 was positive for phosphate solubilization, siderophore production and nitrogen fixation indicating their importance as potential candidates for the development of bioinoculants.
... Previous studies have reported positive effects of dual inoculation with IAA-producing bacteria and rhizobia on leguminous crops nodulation. However, these studies do not necessary reflect the direct involvement of auxin in interactions between IAA-producing bacteria and nodulation as these bacteria show siderophore production and phosphate solubilization activity simultaneously (Fox, O'Hara, & Bräu, 2011;Malik & Sindhu, 2011;Srinivasan, Holl, & Petersen, 1996). ...
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Beneficial fungal and rhizobial symbioses share commonalities in phytohormones responses, especially in auxin signalling. Mutualistic fungus Phomopsis liquidambari effectively increases sym-biotic efficiency of legume peanut (Arachis hypogaea L.) with another microsymbiont, bradyrhizobium, but the underlying mechanisms are not well understood. We quantified and manipulated the IAA accumulation in ternary P. liquidambari-peanut-bradyrhizobial interactions to uncover its role between distinct symbioses. We found that auxin signalling is both locally and systemically induced by the colonization of P. liquidambari with peanut and further confirmed by Arabidopsis harbouring auxin-responsive reporter, DR5:GUS, and that auxin action, including auxin transport, is required to maintain fungal symbiotic behaviours and beneficial traits of plant during the symbiosis. Complementation and action inhibition experiments reveal that auxin signalling is involved in P. liquidambari-mediated nodule development and N 2-fixation enhancement and symbiotic gene activation. Further analyses showed that blocking of auxin action compromised the P. liquidambari-induced nodule phenotype and physiology changes, including vascular bundle development, symbiosome and bacteroids density, and malate concentrations, while induced the accumulation of starch granules in P. liquidambari-inoculated nodules. Collectively, our study demonstrated that auxin signalling activated by P. liquidambari symbiosis is recruited by peanut for bradyrhizobial symbiosis via symbiotic signalling pathway activation and nodule carbon metabolism enhancement. KEYWORDS carbon metabolism, peanut, symbiotic signalling pathway
The need for alternative sources of renewable fuel was felt in 1970–1980 with research being directed toward exploring algae as the starter material for biofuel production. Algal varieties are enormous and its potential for biofuel production is immense and much higher than any land plant-based product. However, there are limitations in terms of making them commercially viable. Extensive research has been conducted to address each step to make the application economically viable, such as the energy requirement mainly in the harvesting step, water requirement for cultivation during the time of fresh water scarcity, and algal growth medium requirement for cultivation. This chapter reports a case study of selective treatment of ammonia-rich dairy wastewater using a consortium of bacteria and microalgae, revealing the potential of the technology in dairy wastewater treatment with lipid-rich algal biomass production for biofuel extraction. Such approach cuts down on the use of fresh water and algal growth medium for algae cultivation and saves energy by alleviating the need for harvesting the biofilm-based algal biomass. The reclaimed water could be reused for secondary (non-potable) applications. In this way the waste could be substituted for feed and water for algal growth, while biofilm-based growth ensured energy savings and a rapid treatment within 48 h ensured more efficient treatment and biomass production compared to conventionally reported algal consortium.
Plant growth-promoting endophyte (PGPE) is a prerequisite for plant productivity, health, ecosystem functions and community organization. The endophytic relationship of microbes with the host plants is important for potential plant growth enhancement. These endophytes originate from interior tissues of roots and aerial plant parts of various host plants. The endophytic plant–microbe relationship is initiated with the colonization of root surfaces and invasion of the interior root structure, followed by the movement of endophytes into aerial tissues of the host plants. The ability of some endophytic diazotrophs in fixing N2 provides a promising source of N input which can substitute chemical nitrogen fertilizers for the host plants. Other than N, endophytes are also capable of solubilizing inorganic phosphate, potassium and promoting P and K uptake. Additionally, endophytes are also known as producers for indole-3-acetic acid (IAA) which is among the vital phytohormones for plants. They are also capable of expressing 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower the levels of plant ethylene produced when they are exposed to stress and subsequently enhance plant growth. Most endophytes can also synthesize iron-chelating siderophore that solubilizes insoluble iron in soil and facilitate in reducing heavy metals stress. In addition, with the current development of the next-generation sequencing technology, fundamental knowledge on genomic information of endophytes has improved the understanding of gene functions of selected plant-associated PGPE. This chapter discusses the importance of the application of selected beneficial endophytes as biostimulants for the promotion of sustainable crop production.
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Seeds are a vector of genetic progress and, as such, they play a significant role in the sustainability of the agri-food system. The current global seed market is worth USD 60 billion that is expected to reach USD 80 billion by 2025. Seeds are most often treated before their planting with both chemical and biological agents/products to secure good seed quality and high yield by reducing or preventing losses caused by diseases. There is increasing interest in biological seed treatments as alternatives to chemical seed treatments as the latter have several negative human health and environmental impacts. However, no study has yet quantified the effectiveness of biological seed treatments to enhance crop performance and yield. Our meta-analysis encompassing 396 studies worldwide reveals for the first time that biological seed treatments significantly improve seed germination (7±6%), seedling emergence (91±5%), plant biomass (53±5%), disease control (55±1%), and crop yield (21±2%) compared to untreated seeds across contrasted crop groups, target pathogens, climatic regions, and experimental conditions. We conclude that biological seed treatments may represent a sustainable solution to feed the increasing global populations while avoiding negative effects on human health and ensuring environmental sustainability.
Recent application of modern agricultural biotechnologies and use of high-yielding crop varieties provided sufficient crop yields and food for human population. But, the major constraint to enhance crop yield and food production is the availability of various nutrients in the soil, which include nitrogen, phosphorus, potassium, sulphur, zinc and other micronutrients. Currently, chemical fertilizers and plant growth regulators are widely utilized for obtaining high crop yields. However, the injudicious application of fertilizers along with other agrochemicals has resulted in environmental pollution along with deleterious effects on beneficial microflora and fauna. Recently, soil-inhabiting beneficial microorganisms are screened and exploited for use as biofertilizers to enhance soil fertility and crop yield with reduced application of chemical fertilizers. Thus, the use of microbial inoculants has emerged as novel agrobiotechnology for attaining sustainable agricultural production systems. These beneficial microorganisms contribute immensely towards management of plant nutrients in the soil by way of nitrogen fixation, solubilization of phosphorus, potassium and zinc along with other nutrients. Other beneficial characteristics of microbial inoculants include phytohormones production, inhibition of phytopathogens’ growth, bioremediation of pollutants and heavy metals, and amelioration of abiotic as well as biotic stresses. However, the efficacies of microbial inoculants in improving crop yield under field conditions remain variable under varied farming situations in different agro-ecosystems. Recently, genetic engineering techniques are being employed to improve the beneficial traits in plants and microorganisms to improve nutrient availability, soil fertility and crop yield. In addition, identification of effective microbial inoculants and their persistence in soil and quality of these inoculants is a never-ending process for harnessing desirable impacts on crop productivity. Considering the importance of beneficial microorganisms in biogeochemical cycling of nutrients, various mechanisms involved in improving nutrients availability are reviewed for increasing food production.
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Bacillus circulans E9 (now known as Niallia circulans) promotes plant growth-producing indole-3-acetic acid (IAA), showing potential for use as a biofertilizer. In this work, the use of a low-cost medium containing industrial substrates, soybean, pea flour, Solulys, Pharmamedia, yeast extract, and sodium chloride (NaCl), was evaluated as a substitute for microbiological Luria Broth (LB) medium for the growth of B. circulans E9 and the production of IAA. In Erlenmeyer flasks with pea fluor medium (PYM), the maximum production of IAA was 7.81 ± 0.16 μg mL⁻¹, while in microbiological LB medium, it was 3.73 ± 0.15 μg mL⁻¹. In addition, an oxygen transfer rate (OTR) of 1.04 kg O2 m⁻³ d⁻¹ allowed the highest bacterial growth (19.3 ± 2.18 × 10¹⁰ CFU mL⁻¹) and IAA production (10.7 μg mL⁻¹). Consequently, the OTR value from the flask experiments was used to define the conditions for the operation of a 1 L stirred tank bioreactor. The growth and IAA production of B. circulans cultured in a bioreactor with PYM medium were higher (8 and 1.6 times, respectively) than those of bacteria cultured in Erlenmeyer flasks. IAA produced in a bioreactor by B. circulans was shown to induce the root system in Arabidopsis thaliana, similar to synthetic IAA. The results of this study demonstrate that PYM medium may be able to be used for the mass production of B. circulans E9 in bioreactors, increasing both bacterial growth and IAA production. This low-cost medium has the potential to be employed to grow other IAA-producing bacterial species.
Intended as a textbook, and including material of relevance to less developed countries, the volume comprises 22 chapters on: structure and physiological ecology of soil microbial communities: microbial ecology of the rhisozphere; soil organic matter dynamics and crop residue management; significance and potential uses of soil enzymes; monitoring recombinant DNA microorganisms and viruses in soil; genetics and improvement of biological nitrogen fixation; molecular biology of plant-parasite relations; rhizobial ecology and technology; nitrogen fixation in forestry and agroforestry; plant growth promoting rhizobacteria as biological control agents; biological control and fungi; microbial production of plant growth regulators; inoculum production and inoculation strategies for vesicular-arbuscular mycorrhizal fungi; biology and application of ectomycorrhizal fungi; microbiological management of wetland rice fields; microalgal biotechnology and applications in agriculture; bioremediation of contaminated soil; composting as a process based on the control of ecologically selective factors; utilization of municipal wastes; composting and organic waste management in China and India; microbial inoculant production and formulation; and commercialization of soil microbial technologies. Twelve papers are abstracted separately in Ecological Abstracts. -J.W.Cooper
Phytotoxic effects of metabolites from a naturally occurring rhizobacterial isolate, Pseudomonas syringae strain 3366, were determined on downy brome and 'Hill 81' winter wheat, along with 10 other weed and crop species. Centrifuged supernatant and concentrated ethyl acetate extracts from aerobic shake cultures of strain 3366 suppressed germination of seeds and reduced root and shoot growth in agar diffusion assays, soil assays, and under field conditions. Generally, root growth was inhibited more than shoot growth. Strain 3366 metabolites applied in soil inhibited all species tested. Crude ethyl acetate extracts in soil inhibited downy brome at concentrations that had little effect on winter wheat. Inhibitory activity was greater in Palouse silt loam (pH 5.8, 3.6% organic matter) than in Shano silt loam (pH 9.0, 0.8% organic matter). Activity of extracted metabolites decreased rapidly in wet soil but remained high in dry soil. Active metabolites were isolated and purified from the ethyl acetate extract using column chromatography, thin-layer chromatography, and crystallization. Chemical analysis revealed the presence of phenazine-1-carboxylic acid, 2- amino phenoxazone, and 2-amino phenol. Activity of these metabolites against downy brome was confirmed in agar assays. Phenazine-1-carboxylic acid, the major identifiable metabolite present in ethyl acetate extracts (20% by weight), inhibited downy brome root growth by 99% at concentrations of 5.7 mg L-1. Production of these metabolites in field soil by live bacteria of strain 3366 was confirmed with thin-layer chromatography.
The influence of fluorescent Pseudomonas strain MRS13 on induction of flavonoid-like compounds in chickpea was investigated. Flavonoid-like compounds were extracted with ethylacetate from roots of 6 d old inoculated and uninoculated plants grown under aseptic conditions. Methanolic solution of the extract showed absorption maxima at 258 mm. There was a 61.1% and 35.1% increased accumulation of flavonoid-like compounds in inoculated plants of chickpea cultivars C235 and H8618 respectively in comparison to uninoculated control. The results indicate that enhanced production of flavonoid-like compounds in the seedling roots on bacterization could play a role in induction of systemic resistance as well as improving nodulation.
ABSTRACT Colonization of the rhizosphere by micro-organisms results in modifications in plant growth and development. This review examines the mechanisms involved in growth promotion by plant growth-promoting rhizobacteria which are divided into indirect and direct effects. Direct effects include enhanced provision of nutrients and the production of phytohormones. Indirect effects involve aspects of biological control: the production of antibiotics and iron-chelating siderophores and the induction of plant resistance mechanisms. The study of the molecular basis of growth promotion demonstrated the important role of bacterial traits (motility, adhesion and growth rate) for colonization. New research areas emerge from the discovery that molecular signalling occurs through plant perception of eubacterial flagellins. Recent perspectives in the molecular genetics of cross-talking mechanisms governing plant–rhizobacteria interactions are also discussed.
Application of plant growth-promoting rhizobacteria (PGPR) has been shown to increase legume growth and development under optimal temperature conditions, and specifically to increase nodulation and nitrogen fixation of soybean [Glycine max(L.) Merr.] over a range of root zone temperatures (RZTs). Nine rhizobacteria applied into soybean rooting media were tested for their ability to reduce the negative effects of low RZT on soybean growth and development by improving the physiological status of the plant. Three RZTs were tested: 25, 17.5, and 15 °C. At each temperature some PGPR strains increased plant growth and development, but the stimulatory strains varied with temperature. The strains that were most stimulatory at each temperatures were as follows: 15 °C—Serratia proteamaculans1–102; 17.5 °C—Aeromonas hydrophilaP73, and 25 °C—Serratia liquefaciens2–68. Because enhancement of plant physiological activities were detected before the onset of nitrogen fixation, these stimulatory effects can be attributed to direct stimulation of the plant by the PGPR rather than stimulation of plant growth via improvement of the nitrogen fixation symbiosis.