Studies on Rhizosphere Bacteria mediated

VEGETOS

Vol. 27 (1) : 158-169 (2014)

DOI: 10.5958/j.2229-4473.27.1.025

Studies on Rhizosphere-Bacteria mediated Biotic and Abiotic
stress tolerance in Chickpea (Cicer arietinum L.)
Ankita Sarkar, Jai Singh Patel1, Sudheer Yadav1, Birinchi K. Sarma*, Jai Singh Srivastava and
Harikesh B. Singh
Received: 01 March 2013 / Revised: 29 Nov 2013 / Accepted: 09 Jan 2014 / Published online: 30 April 2014
This article is published with open access at www.vegetosindia.org

Abstract Rhizospheric bacteria promote plant
health and combat with pathogenic microorganisms. Available reports indicate the activity
of PGPR are in protection of plant under abiotic stresses. In the present work we have
compared the growth promotion and biochemical responses of plants influenced
bybacteria isolated from the rhizosphere of
different plants. Two Pseudomonas strains S1
(P. putida) and Cgr (P. aeruginosa) were isolated from chickpea and congress grass, respectively, and their antimicrobial activity was tested against Sclerotinia sclerotiorum. Both
strains are tested for HCN, IAA and ammonia

production. Their surviving ability in salt stress
was evaluated and compatibility test was performed. We have got some interesting results
that plant defense enzymes and phenolic substances were accumulated in higher concentrations in plants that were treated with the
two bacterial strains (Cgr and S1) either individually or in combination when challenged
with biotic (Sclerotinia sclerotiorum) and abiotic stress (NaCl salt stress) compared to the
non-bacterized plants but exposed to biotic as
well as abiotic stresses. These results indicates
that the Cgr and S1 have potential to be used
as biocontrol agents that can help chickpea to
combat attack of S. sclerotiorum as well as
thrive under salt stress. Moreover, the results
also indicated a common pattern of defense
response by chickpea against both the biotic
and abiotic stress when they are bacterized by
the two bacterial strains.
Keywords: Pseudomonas putida, Pseudomonas aeruginosa, chickpea, PAL activity, NaCl
stress, Sclerotinia sclerotiorum
Introduction
Chickpea (Cicer arietinum L.) produc-

tion is plagued by various diseases of fungal,
bacterial, viral and nematode origin and also
very sensitive to abiotic stresses like salinity.
Most of the susceptible genotypes die in 25
mM NaCl and the resistant genotypes also do
not survive even in 100 mM NaCl under hydroponics condition (Flowers et al. 2010). Sclerotinia sclerotiorum (Lib.) de Bary is among
the most devastating, nonspecific, omnivorous
and cosmopolitan plant pathogen. Plants susceptible to this pathogen encompass 64 families, 225 genera, and 361 species (Purdy 1979).
The pathogen causes cottony rot, watery soft
rot, stem rot, drop, crown rot, blossom blight
and, perhaps most common, white mould
(Bolton et al. 1990).
Fluorescent Pseudomonads are used
as biocontrol agents in agricultural crops as
they have very high adaptive potential
(Lugtenberg et al. 2004, Jain et al. 2012). Pseudomonas fluorescens has been reported to be
an effective biocontrol agent against different
fungal pathogens by various workers (Meyer
et al. 1992, Singh et al. 2006). Mixtures of fluorescent Pseudomonads were used by Pierson
and Weller (1994) to suppress “Take All Disease of Wheat” and improve the growth of

wheat. Induced systemic resistance (ISR) in
plants is a mechanism adopted by fluorescent
Pseudomonads for plant disease management
(Van Loon et al. 1998; Ramamoorthy et al.
2001). ISR may be effectively used as a strategy for biological control. In ISR the existing
plant defense is only activated by several inciting agents, whereas in direct biological control it relies completely on direct actions of
the biocontrol agent like production of antibiotics, siderophore, HCN as well as competition
for nutrients and space. ISR activates multiple
defense mechanisms like synthesis and activa-

Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University,
Varanasi 221 005 India
1
Department of Botany, Faculty of Science, Banaras Hindu University, Varanasi 221005
*Corresponding author E-mail: birinchi_ks@yahoo.com

158

Studies on Rhizosphere-Bacteria mediated Biotic and Abiotic stress tolerance in Chickpea

tion of Chitinase, β 1,3- Glucanases, Peroxidases, other PR proteins (Lawton and Lamb.,
1987), synthesis and accumulation of phytoalexins (Kuć and Rush 1985), formation and
deposition of lignin, callose and other Hydroxyproline rich glycoproteins (Hammerschmidt
and Kuć 1979). ISR in plants is triggered by
strains of P. fluorescens which belong to plant
growth-promoting rhizobacteria (PGPR) (Van
Peer et al. 1991, Chen et al. 2000). ISR is activated against several fungal, bacterial and viral diseases (Liu et al., 1995; Maurhofer et al.
1998). Systemic suppression of soil born pathogen Pythium aphanidermatum in cucumber
root was obtained by use of Pseudomonas
corrugata strain B and Pseudomonas aureofaciens strain 63-28 (Chen et al. 2000). Two
strains of Pseudomonas fluorescens Pf1 and
FP7 were found to give maximum inhibition of
Rhizoctonia solani and enhanced the vigour of
seedlings in vitro conditions (Vidyasekaran
and Muthamilan 1999). Mixing of two or more
strains of Pseudomonas sp. helped in increasing their biocontrol efficacy and PGPR activity
(Pierson and Weller. 1994, Singh et al. 1999).
An isolate of P. fluorescens Pf1 was used to
treat seeds of mungbean and the treated
seeds when challenge inoculated with Macrophomina phaseolina under glass house conditions, increased accumulation of phenylalanine ammonia lyase (PAL), peroxidase (POx),

polyphenol oxidase (PPO), chitinase and β-1-3glucanase were observed (Saravanakumar et
al. 2007). Lipopeptide was found to be essential for biological control of S. sclerotiorum by
Pseudomonas sp. strain DF41 (Berry et al.
2010). Pseudomonas chlororaphis PA-23, Bacillus amyloliquefaciens BS6, Pseudomonas sp.
DF41 and Bacillus amyloliquefaciens E16
showed antagonistic activity in vitro against S.
sclerotiorum. Double application of PA-23 on
canola followed by challenge inoculation with
S. sclerotiorum increased levels of the hydrolytic enzymes chitinase, β-1,3-glucanase and
PR-3 in canola (Fernando et al. 2007).
The first committed step of phenylpropanoid synthesis is catalyzed by phenylalanine
ammonia lyase (PAL) (Jones 1984) and this is
the rate limiting enzyme in production of phenyl propanoid compounds (Bate et al. 1994).
PAL is involved in phytoalexin or phenolics
biosynthesis and hence this enzymes has been
correlated with defense against pathogens
(Binutu and Cordell 2000). Activity of PAL is
stimulated by microbial infections and this

leads to synthesis of lignin like wall bound

phenolic materials and phenyl propanoid derived phytoalexin antibiotics (Jones 1984). Biotic and Abiotic stress induces synthesis of
phenylpropanoid compounds in plants and
these phenyl propanoid compounds do play
an important role in plant defense (Dixon and
Paiva 1995). An important first line in plant
defense against infection is provided by the
very rapid synthesis of phenolics and their
polymerization in the cell wall. Plant secondary metabolites especially phenolic compounds play a role in mechanisms of plant
resistance (Friend 1977). A possible control of
Sclerotium rolfsii infection by enhancing the
levels of phenolic compounds in host tissues
was suggested by Punja (1985). Many plant
phenols possess antimicrobial activities. Phenols with antimicrobial activities have the ability to denature proteins and are classified as
surface active agents (Sousa 2006). Phenols
have the capacity to change the composition
of microflora in any environment in which these compounds are applied and/or induced in
a proper kind and concentration (PuupponenPimiä et al. 2001). Production of phenolics
with antimicrobial activities gives rise to resistance (Luzzatto et al. 2007). Phenols play
role mainly in plant protection by contributing
to structural integrity, photosynthesis and nutrient uptake among other functions in vascular plants.

Soil salinity in arid regions is frequently an important limiting factor for cultivating
agricultural crops. Nowadays special interest is
put on research directed towards development of inoculants with beneficial bacteria
(PGPR) for environmentally friendly agricultural management. The main purpose of these
biofertilizers is to stimulate the plant’s defensive metabolism that will allow crop cultivation in areas with a high pathogen incidence,
or on degraded or saline soils or soils subject
to water restriction conditions. Reclamation of
soils by the use of arbuscular-mycorrhizal fungus, and/or PGPR such as Azospirillum, Agrobacterium, Pseudomonas and several Bacillus
species is considered to be an environmentfriendly, energy efficient and economically viable approach. This treatment also increases
biomass production (Mayak et al., 2004; Rabie
and Almadini, 2005). The expression of some
genes in defense response has been related
with genes expressed in salt stress situations
by various workers (Cheong et al. 2002, Tim159

Ankita Sarkar et al.
Table 1. Growth of Pseudomonas aeruginosa Cgr and
Pseudomonas putida S1 at different NaCl concentrations

Salt Concentrations

Control (Broth without NaCl)
2% (0.34 M) NaCl
3% (0.51 M) NaCl
4% (0.68 M) NaCl
5% (0.85 M) NaCl
6% (1.02 M) NaCl
7% (1.2 M) NaCl

Cfu/ml
S1
1.7 × 1010

Cgr
1.9 × 1012

8.6 × 108
2.2 × 108
4.8 × 107
1.2 × 107
1.5 × 106

-

4.95 ×1010
4.2 × 1011
4.76 ×1011
4.66 × 109
6 × 107
2.8 × 102

musk et al. 1999). Thus it may be said that
plants inoculated with proper biological agent
may also show increased adaptability to salt
stress and drought stress situations (Timmusk
et al. 1999). Some PGPR elicit Induced Systemic Resistance (ISR) and this ISR elicited by
PGPR suppresses disease resistance in both
green house and field conditions (Kloepper et
al. 2004). Use of mixtures of PGPR strains were
also tried under glass house and field conditions to study the alleviation of abiotic stress
in plants by these strains. Lettuce was coinoculated with PGPR (Pseudomonas mendocina) and arbuscular mycorrhizal fungi
(Glomus intraradicus or Glomus mosseae), an

increase in catalase activity under severe
drought conditions was observed, thus suggesting that these strains may be used as inoculants to alleviate the oxidative damage
elicited by drought (Kohler et al. 2008).
Materials and Methods
Two bacterial cultures S1
(Pseudomonas putida) and Cgr (Pseudomonas
aeruginosa) were isolated and used in the experiment. The pathogen Sclerotinia sclerotiorum was also isolated from infected chickpea
plants and used in the experiment. Chickpea
seeds of the cv. ‘Avrodhi’ were used for the

experiments.
Isolation of Pseudomonas
Pseudomonas strain Cgr was isolated
from the rhizospheric soil of Congress Grass
(Parthenium sp.) growing around the Agricultural Research Farm of Banaras Hindu University. Pour plate technique was used to isolate
bacteria from the suspensions. One ml of suspension from each dilution of 10-4, 10-5 and 10
-6
was poured in Petri plates separately and
the plates were then transferred with 20 ml of
King’s B (KB) agar (King et al., 1954). The

plates were incubated at 28 ± 1 0C for two
days for development of colonies. S1 was isolated in a similar way from chickpea rhizosphere.
When visible colonies appeared, single
colonies were carefully picked up by an inoculation loop and streaked in another previously
poured plate containing King’s B medium for
isolation of single cells. These plates were further incubated in an incubator at 28±1 0C.
Colonies developed from single cells were finally picked up and streaked on slants of
King’s B Medium for further experimentation.
Isolation of Sclerotinia sclerotiorum (Lib.)
de Bary
The pathogen was isolated by picking
up individual sclerotia from infected chickpea
plants. The sclerotia were surface sterilized
with 0.1% HgCl2 and sterilized sclerotia were
then placed in plates of Potato Dextrose Agar
medium (PDA). These plates were incubated at
25±2 0C for a few days till the mycelia grew
actively. The cultures were purified by placing
mycelia blocks in PDA slants taken from the
growing edges of the growing culture.
Characterization of the Pseudomonas
strains
Phosphate solubilization
Phosphate solubilisation test of the
Pseudomonas strains was done by using the
NBRI-BPB medium (Mehta and Nautiyal 2001).
Cyanogenesis or HCN production

Table 2. Growth parameters of chickpea treated with two Pseudomonas strains S1 and Cgr
Treatments
Cgr
S1
Cgr+S1
Control

Length (cm)
Shoot
Root
20.13a ±
44.8a ± 9.45
1.25
18.33a± 1.18 33.00a ±
2.83
31.6a ±
17.67a,b ±
0.09
11.47
12.00b ±
27.5a ± 1.78
1.89

Fresh Weight (g)
Shoot
Root t
0.53a± 0.06
0.22a± 0.02

Dry Weight (g)
Shoot
Root
0.14a± 0.01
0.13a± 0.01

0.46a,b± 0.11

0.14a,b± 0.03

0.96a,b± 0.01

0.07b± 0.01

0.38a,b± 0.14

0.15a,b± 0.04

0.98a,b± 0.03

0.08a,b± 0.02

0.16b± 0.07

0.10b± 0.02

0.50b± 0.02

0.05b± 0.01
160

Table 3. PAL activity, total phenol accumulation and polyphenol oxidase activities in chickpea treated with two Pseudomonas strains S1 and Cgr
PAL activity (µg TCA g-1 fresh weight of
leaves)
24 h
Cgr
S1

22.1

b,c

20.1

b,c

± 0.06
± 0.05

48 h
f

50.2 ± 0.12
39.2

g,h

± 0.04

72 h
b,c

101.5 ±0.03
82.6

d,e

Total Phenol (mg per g fresh weight of
leaves)
24 h
a

3.2 ± 0.09
m

48 h
2.7

g,h

± 0.01

j

72 h
c,d

2.5

± 0.04

j

PPO activity (change in absorbance per minute per micro gram of protein)
24 h

48 h

72 h

l

l

h

4.5 ± 0.01

l

0.2 ± 0.03
k

3.9 ± 0.05

± 0.04

1.1 ± 0.00

1.5 ± 0.01

1.1 ± 0.15

0.5 ± 0.07

3.7 ± 0.03

8.6h ± 0.04

Cgr+S1

17.7b,c,d±0.04

43.6g ± 0.09

87.4d ± 0.12

2.3f,g ± 0.01

2.9a ± 0.01

1.6h ± 0.02

1.1i,j ± 0.01

2.2n ± 0.02

23.3g ± 0.13

Cgr+P

9.8f,g ± 0.05

39.5g,h ± 0.01

80.8d,e ± 0.04

2.2g ± 0.01

2.9m ± 0.04

2.3e,d ± 0.01

1.1j ± 0.04

3.7l ± 0.01

5.8h ± 0.32

S1+P

15.6c,d,e,f±0.06

34.7k,l ± 0.09

98.0b,c ± 0.15

2.3e,f ± 0.04

2.5l ± 0.01

2.1f ± 0.01

3.9g ± 0.01

4.2l ± 0.01

21.1g ± 0.07

Cgr+S1+P

10.6e,f,g ±0.05

80.2b ± 0.12

96.5c ± 0.08

2.4e,d ± 0.02

2.6k ± 0.01

2.5c,d ± 0.01

9.4c ± 0.09

9.7i ± 0.02

46.6f ± 0.04

Cgr+N50

12.8d,e,f,g±0.07

31.8i,j ± 0.34

69.8g ± 0.06

1.4l ± 0.01

1.5g ± 0.04

1.8h ± 0.03

2.7h± 0.08

3.7l ± 0.01

48.0f ± 0.05

Cgr+N100

15.7c,d,e,f±0.03

79.6b,c ± 0.06

82.3d,e ± 0.13

1.3m ± 0.01

2.1e ± 0.01

2.9b ± 0.02

8.3e ± 0.05

14.6g ± 0.01

61.3e ± 0.01

Cgr+N150

20.8b,c ± 0.05

71.7d ± 0.04

86.6d ± 0.06

1.7j,k ± 0.02

2.4c ± 0.01

3.1a ± 0.01

9.0d ± 0.00

28.0d ± 0.09

77.6b,c ± 0.05

S1+N50

21.1b,c ± 0.06

27.7j,k ± 0.02

101.0b,c±0.09

2.8c ± 0.01

1.6i ±0.01

2.3e ± 0.01

4.0g ± 0.02

11.7h ± 0.03

51.3f ± 0.08

S1+N100

35.2a ± 0.04

55.1e ± 0.09

84.1d,e ± 0.35

2.8c ± 0.01

1.9f ± 0.03

2.5c ± 0.06

8.3e ± 0.23

22.7f ± 0.07

65.3d,e ± 0.04

S1+N150

32.6a ± 0.01

56.9e ± 0.01

103.9b± 0.08

3.1b ± 0.30

1.6i ± 0.02

2.4e,d,c ± 0.06

9.0d ± 0.07

25.0e ± 0.08

72.3c,d ± 0.12

Cgr+S1+N50

8.9f,g ± 0.24

53.0e,f ± 0.07

73.6f,g ± 0.05

1.8h,i ± 0.03

2.0f ± 0.01

1.0j ± 0.01

9.0d ± 0.01

31.3c ± 0.01

84.7b ± 0.30

Cgr+S1+N100

17.43b,c,d

75.2c,d ± 0.05

110.4a ± 0.14

2.2f,g ± 0.01

2.3d ± 0.01

2.0f ± 0.01

10.7b ± 0.01

38.0b ± 0.09

85.00b ± 0.40

±0.13

161

Cgr+S1+N150

32.2a ± 0.01

86.6a ± 0.12

116.5a ± 0.02

2.7c ± 0.16

2.8b ± 0.01

2.5b ± 0.18

12.6a ± 0.07

44.3a ± 0.10

99.7a ± 0.09

N50

23.9b ± 0.04

39.2g,h ± 0.03

34.7i ±0.06

1.7i,j ± 0.01

1.5j ± 0.05

1.2i ± 0.03

4.0g ± 0.02

5.3k ± 0.09

06.7h ± 0.01

N100

20.2b,c ± 0.06

38.8g,h ± 0.01

46.0h ± 0.17

2.1g ± 0.01

1.7h ± 0.01

1.8g ± 0.06

6.0f ± 0.01

6.7j ± 0.01

08.3h ± 0.02

N150

7.8g ± 0.12

35.8h,i ± 0.02

78.5e,f ± 0.02

1.9h ± 0.02

1.1l ± 0.01

1.8g± 0.03

4.0g ± 0.08

7.0j ± 0.08

7.6h ± 0.03

P

11.8d,e,f,g±0.12

79.1b,c ± 0.05

72.3f,g ± 0.18

1.5d ± 0.01

1.5j ±0.01

1.4h±0.01

1.3i ± 0.04

3.0m ± 0.02

8.0h ± 0.05

Control

11.8d,e,f,g±0.13

21.2l ± 0.12

12.8j ± 0.01

1.1k ± 0.03

1.0g ± 0.02

1.1f± 0.01

0.2l ± 0.01

00.2o ± 0.06

00.3h ± 0.05

†P = Sclerotinia sclerotiorum; N = NaCl

Studies on Rhizosphere-Bacteria mediated Biotic and Abiotic stress tolerance in Chickpea

Treatments†

Ankita Sarkar et al.

Bacterilal isolates were screened for
production of HCN by using the protocol proposed by Baker and Schipper (1987). King’s B
medium amended with Glycine (4.4 g/L) was
prepared and sterilized. About 25ml of the
prepared medium was poured in the plates
sterilized previously. The poured medium was
allowed to solidify and then streaking with the
bacterial isolate was done. Streaking was done
with an inoculating needle in a laminar air
flow. Single strains were streaked in each
plate. Whatman No. 1 filter papers were cut
according to the size of the Petri plate, sterilized in an autoclave and dipped in freshly
prepared solution of 0.5% picric acid in 2%
Na2CO3. After soaking the filter papers were
attached to lid of Perti plates containing the
streaked cultures. Control plates contained
the amended medium as well as soaked filter
papers attached with the lid of Petri plates
without inoculation with the bacterial cultures.
The plates were sealed with paraffin and incubated in an incubator at 28 ± 1 0C for 3 to 4
days.
Bacterial isolates were tested for production of Ammonia by using the protocol
proposed by Dye (1962). For testing the production of Indole Acetic Acid bacterial isolate
was grown in broth of Luria Bertani for 48 h,
there after the culture was centrifuged at 3000
rpm for 30 minutes. Supernatant was collected, 2 ml of supernatant two drops of orthophosphoric acid and 4 ml of Salkowski reagent (50 ml, 35% per chloric acid and 1 ml of
0.5 M FeCl3 solution) was added. Antagonistic
potential of the bacterial strains were tested
against the pathogen S. sclerotiorum, a soil
borne pathogen, by using the dual culture
technique. The two strains were checked for
compatibility by spot inoculating them separately in two plates followed by spraying with
the other bacterial strain grown in broth. First,
Cgr was spot inoculated in centre followed by
spraying with S1 grown in broth , next in second Petri plate S1 was spot inoculated followed by spraying with Cgr grown in broth.
The PAL assay was carried out according to the method described by Ross and Sederoff (1992). Polyphenol oxidase activity was
determined following Mayer et al. (2001). Total Phenolic Content (TPC) was assayed according to Sarma et al. (2002).
Identification of strains Cgr and S1
16S rDNA sequence analysis was done

for identification of the strains of fluorescent
Pseudomonads.
Results
Identification of bacterial strains
Sequencing of 16S rDNA region confirmed the identity of S1 as Pseudomonas
putida (GenBank Accession: JN020962) and
Cgr as Pseudomonas aeruginosa (JN128893).
Evaluation of bacterial isolates for salt
tolerance
Both the strains Pseudomonas aeruginosa Cgr and Pseudomonas putida S1 were
able to tolerate high concentrations of salt
and showed growth upto 6% (1.02 M) NaCl
concentration in King’s B broth. The visible
turbidity of the bacterial growth in broths
amended with different concentrations of
NaCl was decreased with increase in the concentration of the salt (Plate: K) and (Plate: L).
Turbidity in broths inoculated by Cgr and S1
and amended by different concentrations of
salt was clearly visible and distinguishable
from the control up to 5% (0.85 M). However,
at 6% (1.02 M) turbidity was visible but clearly
indistinguishable from the control tubes. At
7% (1.2 M) salt concentration no such turbidity
was visible in medium, baring just a thin
(almost negligible) biofilm in the top most
layer. Colony counting of the two strains in
salt amended broths showed gradual decline
in cfu/ml of broth and the strain S1 failed to
grow at 7% NaCl. However, growth of Cgr
was recorded at 7% NaCl but significantly less
cfu/ml was obtained (Table 1).
Compatibility of the strains
The two strains were found to be fully
compatible with one another as indicated by
absence of any zone of demarcation between
the two strains and the two strains were found
to grow comfortably with one another.
Plant growth promotion activities
In presence of the bacterial strains individually or in combination, change observed
in most of the growth parameters. Maximum
root length, shoot length, root fresh weight,
shoot fresh weight, root dry weight and shoot
dry weight was obtained in the plants treated
with the strain Cgr. The results are summarized below:
Root Length: Cgr> S1> Cgr+S1> Control
Shoot Length: Cgr> S1> Cgr+S1> Control
Fresh weight of Roots: Cgr> Cgr+S1> S1>Control
Fresh weight of Shoots: Cgr> S1> Cgr+S1>Control
Dry weight of Roots: Cgr> Cgr+S1> S1> Control
Dry weight of Shoots: Cgr> Cgr+S1> S1> Control
162

Studies on Rhizosphere-Bacteria mediated Biotic and Abiotic stress tolerance in Chickpea

Statistical analysis using the data from
the CRD experiments subjected to Duncan’s
Multiple Range Test (DMRT) shows that
growth of root and shoot was highest in Cgr
treated plants followed by the combination of
both Cgr and S1, S1 alone and control. However in case of shoot growth despite the highest growth was recorded in Cgr treated plants
the next best growth was obtained in S1 treated plants compared to their combination. This
is in contrast to the results of the root growth
characteristics (Table 2).
Biochemical defense responses PAL activity
A higher induction of PAL was observed in plants treated with the two strains
Cgr and S1 individually as well as in combination (Cgr+S1) compared to control plants
without any salt stress. The amount of PAL induced was found to increase with time. After
48 h, induction of PAL was maximum in plants
treated with both the strains and challenge
inoculated with the pathogen S. sclerotiorum
(Cgr+S1+P) compared to plants treated with
the strains (Cgr and S1) individually and challenge inoculated with the pathogen (Cgr+P
and S1+P). However, increment in level of PAL
was observed till 72 h in all the plants that
were treated with the two strains, either alone
or in combination and this trend was observed
irrespective of the fact that the treated plants
were exposed to the pathogen or not. An
equal amount of PAL activity was observed in
Cgr treated as well as in the bacterial combination challenged against the pathogen. However in control plants that were not treated
with either of the strains but challenge inoculated with the pathogen (C+P), PAL activity
was found to be very low when compared to
plants that were treated with the strains (alone
or in combination and challenged with the
pathogen) (Table 3).
Salt stress also induced PAL activity in
all the treatments irrespective of the application of the bacterial strains till 72 h in most of
the treatments. However, the level of PAL induced in bacterized plants (Cgr, S1 or Cgr+S1)
was much more compared to that of the nontreated plants under salt stress and control.
PAL activity increased with salt concentrations
(50, 100 and 150mM) and time and recorded
highest activity at 72 h. The PAL activity was
higher in the salt stressed plants treated with
both the strains compared to their application
in non-stressed plants. However, highest PAL
activity was recorded in the salt stressed

plants (100 and 150 mM) that were treated
with both the bacterial strains. Between the
two strains S1 was more potent than Cgr in
inducing PAL activity in the salt stressed chickpea plants when they were applied alone.
Higher PAL activity in the plants under salt
stress and treated with both the strains thus
shows the synergistic activity of the two bacterial strains.
Total Phenol Content (TPC)
TPC was increased in all the treatments
compared to control plants not subjected to
any stress. However, the plants subjected to
both the salt and pathogen stress without
bacterizing the seeds showed little increase in
TPC especially after 24 h. The increased TPC
was declined after 24 h. Similarly, the plants
treated with the bacterial strains have also
showed highest TPC in 24h and declined
thereafter. Between the two strains Cgr induced more TPC than S1. But when the seed
bacterized plants were challenged with the
pathogen in all the treatments involved seed
bacterization the TPC was highest at 48 h and
its content reduced at 72 h. Cgr when applied
alone induced TPC maximum than S1 or in
combination with S1. However, the sustained
effect of the combination was better than their
individual effect as its content was highest at
72 h among the three treatments (Cgr, S1 and
Cgr+S1) (Table 4). When the plants were exposed to salt stress they showed a different
trend compared to the plants exposed to biotic stress. When the plants were subjected to
only salt stress in absence of the bacterial
strains highest TPC was observed at 24 h
which then declined thereafter. However, the
TPC content was higher than the unstressed
control plants showing short duration accumulation of TPC under salt stress condition. A
similar trend was observed in the plants raised
from seed bacterization with S1 and subjected
to salt stress. But the amount of TPC was significantly high than the non bacterized plants.
The TPC increased with increase in salt concentration and was highest with the highest
salt concentration (150 mM). In contrast, highest TPC was observed at 48 h in the plants
treated with Cgr and subjected to salt stress.
The TPC was then declined at 72 h. The results
thus showed a role of the bacterial strains in
inducing TPC in higher quantity under salt
stress conditions (Table 3).
Polyphenol oxidase (PPO) activity
Polyphenol oxidase (PPO) activity in all
163

Ankita Sarkar et al.

the treatments increased in the pathogen and
salt stressed plants treated with the bacterial
strains either single or in combination. All the
treatments showed a gradual increase in PPO
activity and recorded its highest activity at 72
h. The PPO activity in general was relatively
higher in salt stressed plants treated with the
bacterial strains compared to the pathogen
stressed plants treated with the bacterial
strains whether singly or in combination. However, the PPO activity was recorded highest in
the treatments where the bacterial strains
were applied in combination and the plants
were subjected to salt stress compared to
their single application. The PPO activity increased with the increase in salt concentration
thereby showing the active role of the microbes under salt stress conditions (Table 3).
Discussion
Biotic stress
The bacterial strains used in the present investigation have shown the potentiality
to induce defense responses in chickpea
against both S. sclerotiorum and NaCl stress.
The plants under both the stress conditions
have shown higher defense activities especially when the strains were applied in combination. Assay of defense related enzymes and
phenol was done in Pseudomonas fluorescens
1-94 bacterized chickpea plants by Saikia et al.
(2004) after 24 h of challenge inoculation with
the pathogen Fusarium oxysporium f.sp. ciceri.
The plants were then analysed for phenol, PAL
and PR proteins and it was observed that at
least 1 day was needed after inoculation for
induction of resistance response. In the present investigation it was observed that a
steady and gradual increase in PAL activity
was observed up to 3 days. The concentration
of PAL was more in plants that were bacterized and either inoculated or not inoculated
with pathogen, as compared to the control
ones. These observations were in agreement
with the results of PAL activity in the experiment conducted by Meena et al. (2000) where
they used Pseudomonas fluorescens for bacterization of groundnut seeds followed by
challenge inoculation with Cercosporidium
personatum. The activity of PAL was much
more in plants treated with the Pseudomonas
species and then challenge inoculated compared to the untreated controls. In the present
investigation, in case of Cgr the concentration
of PAL was more in plants that were bacter-

ized but not challenge inoculated compared
to the plants that were bacterized and then
challenge inoculated with the pathogen. A
similar phenomenon was observed with S1 as
well and the results thus indicate a strong
Pseudomonas-Sclerotinia interaction during
this period and therefore the host elicitation
of PAL activity was relatively decreased. However, the PAL activity was still higher compared to the non-bacterized plants challenged
with the pathogen. It shows the role of the
bacterial strain in the elicitation of PAL activity. A similar phenomenon was reported by
Ramamoorthy et al. (2002) while working with
the P. fluorescens strain Pf1 used to treat tomato plants against the pathogen Fusarium
osysporum f.sp. lycopersici. They observed
highest activity of PAL on 4th day whereas in
the control plants its activity declined after 2nd
day. Another similar observation was also reported by Nakkeeran et al. (2006). They found
that when hot pepper seedlings were treated
with Pseudomonas chlororaphsis PA-23 and
then challenge inoculated with Pythium aphanidermatum an increase in activity of PAL was
observed reaching maximum in 12 days and
declining thereafter whereas in control plants
activity of PAL was found to increase up to 4
days and declined thereafter. Moreover, its
activity was much lower as compared to that
of PA-23 treated plants. In another finding reported by Sangeetha et al. (2010) showed that
when three beneficial rhizospheric bacteria,
viz., non-fluorescent Pseudomonas (NFP6),
Pseudomonas fluorescens (Pf3a) and Bacillus
subtilis along with Azospirillum (AS1) and
Azotobacter (AZ1) were used in banana and
challenge inoculated with the pathogen Lasiodiplodia theobromae and Colletotrichum
musae, there was increase in defense related
enzymes up to 4 fold compared to that of
control. The defense related enzymes had increased from 1st day onwards and reached a
peak on 5th day after treatment.
Similarly, in case of TPC an increase in
the level of phenolics was observed upto 3
days in most of the treatments at varied
amount. Highest TPC accumulation in some
treatments especially in the bacterized plants
under pathogen stress was observed at 48 h
after pathogen inoculation which declined
thereafter. A similar, effect was seen in the
plants treated with the bacterial mixture without pathogen challenge although in this case
the amount of TPC was lower than the patho164

Studies on Rhizosphere-Bacteria mediated Biotic and Abiotic stress tolerance in Chickpea

gen inoculated treatments. In contrast, the
single application of bacterized plants without
pathogen challenge showed highest TPC at 24
h which then declined gradually thereafter.
Raamamoorthy et al. (2002) while working
with Pseudomonas fluorescens Pf1 in tomato
plants showed that elicitation of the phenylpropanoid metabolites takes place after the
first day of challenge inoculation with the
pathogen Fusarium oxysporum f. sp. lycopersici and their concentration reached peak levels on 5th day. These results are in conformity
with the results of the present investigation
showing an appropriate role of the Pseudomonas species in triggering the phenylpropanoid biosynthesis in plants under pathogenic stress. Similar accumulation of the phenylpropanoid metabolites over a period of
time in the plants under pathogen challenge
were also reported by several other workers
(Sangeetha et al. 2010, Karthikeyan et al. 2005,
Nakkeeran et al. 2006). Singh et al. (2002) and
Sarma et al. (2002) also reported higher accumulation of TPC in pea against Erysiphe pisi
and chickpea against Sclerotium rolfsii infection, respectively after the plants were bacterized by fluorescent Pseudomonas species.
PGPR-mediated ISR induction was also reported by several workers earlier (Bakker and
Schipper 1987, Singh et al. 2003).
In case of Polyphenoloxidase (PPO)
activity in the plants treated with the bacterial
species and challenged with the pathogen, it
was observed that a there was a consistent
and steady increase in levels of PPO up to 3
days. The accumulation of PPO was more in
plants that were bacterized and challenge inoculated with the pathogen compared to
those that were bacterized but not challenge
inoculated. Increase of PPO was also reported
in gladiolus plants treated with rhizobacterial
strains (S2B C Bacillus atrop haesus ,
S2BC2+TEPF- SUNGAL, Burlkholderia cepacia).
When the bacterized plants were challenged
inoculated with the pathogen Fusarium oxysporium f. sp. gladioli, it was observed that
induction of PPO was more in treated and
challenge inoculated plants compared to the
control plants (Shanmugam et al. 2011). Similar, findings were also demonstrated by Ramamoorthty et al. (2002a). They reported that
pretreatment of tomato and hot pepper seedlings with P. fluorescens Pf1 and challenge
inoculation with the pathogen showed rapid
and higher activity of PPO compared to the

non-inoculated ones. In the present investigation plants that were not bacterized but challenge inoculated with the pathogen also
showed PPO activity but the activity was much
lesser in those cases. Least PPO activity was
observed in plants that neither bacterized nor
challenge inoculated. Activity of PPO was
highest in the plants where the strainal mixture was applied and subjected to the pathogen challenge demonstrating the synergistic
activity of the bacterial strains in inducing the
defense response. PGPR-mediated increase
PPO activity was also reported by Nakkeeran
et al. (2006) in hot pepper and in banana by
Sangeetha et al. (2010).
Abiotic stress
Salinity stress caused increase in PAL
activity in chickpea plants at varied levels. PAL
activity was gradually and constantly increased up to 72 h in all the treatments comprising the bacterial strains and maximum activity was observed in plants treated with the
mixture of the bacterial strains and exposed to
high salinity stress. Higher PAL activity in the
mixed bacterial inoculation compared to their
single application shows the synergistic ability
of the bacterial strains in inducing the defense
response against the salinity stress. PAL is recognized as an indicator of environmental
stresses in plants (MacDonald and D Cunha
2007). According to them Jatropha seedlings
when exposed to different salinity levels (0, 50,
100, 150 and 200 mM) an increment in PAL
activity with increment in salinity levels was
observed. They proposed that the increased
PAL activity may be a response to increased
cellular damage due to high salinity stress and
changes in PAL activity thus reflects a role of
the enzyme in helping plants to respond towards salt stress. Gholizadeh and Kohnehrouz
(2010) while working with two inbreds of
maize (A-180 and A-619) reported that when
the inbreds were first exposed to salinity
stress and thereafter removal of the salt stress
caused activation of various defense responses including PAL. Analysis of PAL activity suggested that phenylpropanoid compounds synthesized by Phenylpropanoid pathway may be
a component of key importance in salt induced maize antioxidative system. One of the
early responses of plants to various stresses is
the production of Reactive Oxygen Species
(ROS) (Alscher et al. 1997, Hernandez et al.
2001, Abel et al. 2003). An enhanced generation of ROS is used as a strategy by plants to
165

Ankita Sarkar et al.

mediate the salt effect on plants cells (Asada
1994; Gossett et al., 1994). Effective removal of
ROS in salt tolerant plants is done by using an
efficient antioxidative system (Rout and Shaw
2001, Saleh and Plieth 2009). Increase in PAL
activity with increase in salt stress in the present investigation has also showed a similar
trend with the trend of total antioxidation pattern in maize plants (Gholizadeh and
Kohnehrouz 2010).
PGPR
strains
having
1aminocyclopropane-1-carboxylate (ACC) deaminase activity were reported to alleviate salt
stress in tomato plants and increased plant
growth under salt stress conditions (Mayak et
al. 2004). Bano and Fatima (2009) used Rhizobium and Pseudomonas species to treat two
cultivars of maize for alleviation of salt stress.
They found that co-inoculation of the bacterial strains helped maize plants to adapt in a
better way under salinity stress. The rhizobacterial strains also increased the osmotic potential of leaves under stress condition compared to that of control. Co-inoculation of
Pseudomonas and Rhizobium was also found
to significantly increase the proline content of
leaves as compared to untreated control
plants under salt stress. Rhizobium was found
to perform better than Pseudomonas under
unstressed conditions, but under salt stress
Pseudomonas was found to perform better in
terms of stimulation of growth and biochemical content of leaves. A similar effect was also
seen in the present investigation where the co
-inoculation of the bacterial strains (S1 and
Cgr) induced more defense responses than
their single treatments.
TPC was increased in plants subjected
to salt stress irrespective of the plants treated
with the bacterial strains. However, TPC content in seed bacterized plants was more compared to non-bacterized plants. An interesting
observation was noted in the TPC in the present investigation. The plants when treated
with the bacterial strain S1 and exposed to
salt stress accumulated highest phenolic content in 24 h compared to the plants treated
with Cgr showed highest accumulation in 72
h. However, in contrast to these observations
the TPC in co-inoculation of the bacterial
strains was highest in 48 h and the amount
was less compared to the TPC content in S1
treated plants at 24 h or Cgr treated plants at
72 h. This may be due to difference in the
mechanism of elicitation of TPC by the bacte-

rial strains under abiotic stress conditions.
Phenols are reported to accumulate in plants
under different abiotic stresses (Beckman
2000). Polyphenolic compounds such as phenolic acids, flavonoids, and anthocyanins are
reported to play an important role in scavenging free radicals produced during salt stress in
plants (Parida et al. 2004, Ksouri et al. 2007,
Hichem et al. 2009). The redox properties impart antioxidant activity to the phenolic compounds, and this allows them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (Beckman 2000). Lettuce plants
were treated with VAM 510 or Pseudomonas
and exposed to drought stress, the amount of
phenols like ferulic, caffeic, coumaric and cinnamic acids (precursors of lignin) were found
to accumulate in higher concentrations in
stressed plants pre-treated with VAM 510 or
Pseudomonas (Leinhos and Bergmann 1995).
These results are in conformity with the results
in the present investigation.
Polyphenol oxidase activity was also
increased with increase in salinity level and
time. Its activity in plants under salt stress was
highest in co-inoculation of the bacterial
strains and at 72 h compared to the single application of the bacterial strains. Higher PPO
activity in maize cultivars ‘Hack and Zan’ was
also reported by Aghaleh and Nikam (2009). A
similar observation was also reported by
Niknam et al. (2006) who reported salt stress
induced PPO activity in calli and seedlings of
Trigonella aphanoneura and Trigonella foenum-graecum. The present investigation thus
confirms the role of microbial induced PPO
activity in chickpea under salt stress conditions and the bacterial strains S1 and Cgr elicits PPO activity more under consortium mode.
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