Inoculation of Rhizobium Alleviates Salinity Stress Through Modulation of Growth Characteristics, Physiological and Biochemical Attributes, Stomatal Activities and Antioxidant Defence in Cicer arietinum L.

Abstract

Rhizobium is a plant growth-promoting bacteria, generally involved in nitrogen fixation and promotes growth in plants under abiotic-stressed conditions such as salinity. The present study investigates the significance of Rhizobium application in alleviation of salt stress in chickpea by increasing cell viability, stomatal movement, photosynthetic pigment and protein content, nitrate reductase, carbonic anhydrase as well as enzymatic and non-enzymatic antioxidant activities. Healthy and viable seeds were inoculated with Rhizobium before sowing. Salt treatment was given in terms of NaCl (50 or 150 mM) to the plants through soil at 20 days after sowing. High NaCl level (150 mM) reduced the growth attributes, pigment as well as soluble protein content, altered stomatal behaviour, reduced cell viability and enhanced the formation of superoxide radicals and other reactive oxygen species in foliage. Moreover, Rhizobium inoculation improved the mineral uptake, reduced electrolyte leakage which directly influences photosynthesis and improved yield attributes in the salt-treated chickpea plants. Therefore, Rhizobium could be applied to chickpea plants for efficient growth under salt stress.

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Vol.:(0123456789)
1 3
Journal of Plant Growth Regulation
https://doi.org/10.1007/s00344-020-10267-1
Inoculation ofRhizobium Alleviates Salinity Stress Through
Modulation ofGrowth Characteristics, Physiological andBiochemical
Attributes, Stomatal Activities andAntioxidant Defence inCicer
arietinum L.
ZeenatMushtaq1· ShahlaFaizan1· BasitGulzar2· KhalidRehmanHakeem3
Received: 1 May 2020 / Accepted: 3 November 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Rhizobium is a plant growth-promoting bacteria, generally involved in nitrogen fixation and promotes growth in plants
under abiotic-stressed conditions such as salinity. The present study investigates the significance of Rhizobium application
in alleviation of salt stress in chickpea by increasing cell viability, stomatal movement, photosynthetic pigment and protein
content, nitrate reductase, carbonic anhydrase as well as enzymatic and non-enzymatic antioxidant activities. Healthy and
viable seeds were inoculated with Rhizobium before sowing. Salt treatment was given in terms of NaCl (50 or 150mM) to
the plants through soil at 20days after sowing. High NaCl level (150mM) reduced the growth attributes, pigment as well
as soluble protein content, altered stomatal behaviour, reduced cell viability and enhanced the formation of superoxide radi-
cals and other reactive oxygen species in foliage. Moreover, Rhizobium inoculation improved the mineral uptake, reduced
electrolyte leakage which directly influences photosynthesis and improved yield attributes in the salt-treated chickpea plants.
Therefore, Rhizobium could be applied to chickpea plants for efficient growth under salt stress.
Keywords Salt stress, Rhizobium· Cell viability· Reactive oxygen species· Chickpea· Antioxidants
Abbreviations
PGPB Plant growth-promoting bacteria
ACC Aminocyclopropane-1-carboxylate
DDA Double distilled water
IARI Indian agriculture research institute
CA Carbonic anhydrase
NR Nitrate reductase
TCA Trichloroacetic acid
TBA Thiobarbituric acid
EDTA Ethylenediaminetetraacetic acid
PMSF Phenylmethanesulfonylflouride
SOD Superoxide dismutase
POD Peroxidase
CAT Catalase
NBT Nitro-blue tetrazolium
OD Optical density
SEM Scanning electron microscope
ANOVA Analysis of variance
SPSS Statistical package for the social sciences
MDA Malondialdehyde
DAS Days after sowing
EL Electrolyte leakage
ABA Abscisic acid
Introduction
Soil salinisation is considered as the main agro-ecosystem
problem globally. A large number of soluble salts, for exam-
ple, sodium, calcium, chloride, magnesium, carbonate ions,
sulphates and potassium, are present in soil, and such soil is
supposed as saline soils. At adequate amounts, these salts are
Handling Editor: Parvaiz Ahmad.
* Khalid Rehman Hakeem
kur.hakeem@gmail.com
1 Environmental Physiology Laboratory, Department
ofBotany, Aligarh Muslim University, Aligarh202002,
India
2 Cellular Differentiation andMolecular Genetics Section,
Department ofBotany, Jamia Hamdard, NewDelhi110062,
India
3 Department ofBiological Sciences, Faculty ofScience, King
Abdulaziz University, Jeddah, SaudiArabia

Journal of Plant Growth Regulation
1 3
essential for plants but in excessive quantities, they proved
very harmful (Porcel etal. 2016). Among all salts present
in soil, sodium chloride is present in abundant percentage
(3.5%), showed very deleterious effects for growth and
development of plants as well as soil structure, texture, aera-
tion and permeability especially in arid and semi-arid areas
of world (Candrabarata 2011). Salt stress would continue to
increase day by day globally due to the abating of freshwater
resources (Falkenmark 2013), by use of saline water for irri-
gating crop fields (Wang and Jia 2012; Hasanuzzaman etal.
2018) and other anthropogenic activities. Currently, due to
salt stress almost 30% of irrigated land had been affected
globally, and about 32 million ha of dry land (Parihar etal.
2015). The increased concentration of sodium and chloride
ions in the soil leads the specific ion effects in a plant cell,
changes the permeability of plant cells by replacing other
ions present in the plasma membrane, increases electrolyte
leakage (Kordrostami etal. 2017), reduces the capability of
uptaking the essential elements that are important for plant
growth, development and yield production (Nadeem etal.
2016; Acosta-Motos etal. 2017), disorganizes structure of
functional proteins, deactivated enzyme activity, declines
photosynthetic rate (Silva etal. 2011) and increases the ROS
production. The high concentrations of sodium and chlo-
ride ions in plant tissues also disturb cellular functions, the
activity of many enzymes involved in photosynthesis and
cell signalling processes. Moreover, NaCl ions compete for
uptake of potassium ion, induce K+ deficiency in plant cell
organelles and ultimately prevent the growth of the plant
as it plays a crucial part in the maintenance of cell turgor,
membrane potentials and enzymatic activities. In response
to salinity stress, plants have developed special and complex
strategies involving morphological, physiological and cel-
lular changes to alleviate the salt stress. One of the mecha-
nisms for stress tolerance is the use of plant growth-promot-
ing bacteria (PGPB) due to their protruding role in plants.
PGPB are the group of microbes that are present in soils
either freely in the root zone of a plant called as rhizos-
phere or in association with their host through symbiosis.
These microbes were found very important due to their
environmental-friendly nature, promote plant growth by
the mechanism of biological nitrogen fixation, improve
nutrient and water uptake of roots, acting as phytostimu-
lants, microbial biocontrol agents and provide the stress
tolerance such as salinity, temperature, heavy metal and
drought in plants (Kang etal. 2014; Olubukola etal. 2012).
Azotobacter, Rhizobium, Bacillus, Azospirillum and Pseu-
domonas are examples of few soil microbes that play role
in the promotion of growth and development of plants
under stressed conditions. (van Veen etal. 1997). Role of
microbes in the improvement of plant growth and develop-
ment under salt stress had been reported in various plants
such as red pepper, mung bean, tomato (Tank and Saraf.
2010; Siddikee etal. 2011; Ahmad etal. 2013a, b) etc. These
microbes can improve growth and development of plants
under salinity stress by producing siderophore, solubilizing
of mineral phosphates, production of ACC deaminase and
phytohormones.
Rhizobium is one of the important symbiotic soil
microbes that fix atmospheric nitrogen in root nodules of
leguminous plants and nitrogen to legumes. The symbi-
otic nitrogen-fixing property of Rhizobium with legumes
provides a base for the improvement of soil fertility and
eventually enhances plant biomass productivity (Peoples
etal. 1995) Plants inoculated with Rhizobium produce light
molecular weight organic solutes such as glycine betaine,
proline, polyamines and ectoines, and these solutes protect
the plant cells by stabilizing the structure and conformation
of proteins as well as cell membranes from water stress, des-
iccation; increase photosynthetic and biomass productivity
and also play role in the enhancement of the activity of vari-
ous enzymatic and non-enzymatic antioxidants (Santos and
da Costa 2002; Reina-Bueno etal. 2012; Wdowiak-Wrobel
etal. 2013; Lunn etal. 2014; Yurgel etal. 2013). Inoculation
of seeds with Rhizobium improved abiotic stress tolerance
like salinity, heavy metal, drought in plants; is attributed to
more efficient uptake of nutrients, initiates a wide response
in various physiological activities like mitigates ionic imbal-
ance and facilitation of water uptake; improves leaf water
status and decreases the generation of reactive oxygen spe-
cies (Thrall etal. 2008; Franzini etal. 2013).
Current progressions in the interaction of soil microbes
have represented a worthwhile part of plant growth-pro-
moting rhizobacteria in the regulation of salinity stress.
But petite info is available about the role of Rhizobium in
mitigating the salt stress in plants. So the current work was
conducted with the objective how inoculation of Rhizobium
could enhance the root growth, regulate stomatal behaviour,
increase cell viability and photosynthetic attributes, reduce
the generation of reactive oxygen species and enhance the
growth and yield characteristics as well as antioxidant
enzyme activities in Cicer- arietinum.
The aim of the study was performed with the objective
of how inoculation of Rhizobium could increase the growth,
photosynthetic attributes and yield in Cicer- arietinum.
Materials andMethods
Plant Material
Chickpea (Cicer- arietinum, var. Pusa-BG5023) seeds were
brought from the National seed corporation Ltd., Pusa, New
Delhi, India. Healthy seeds, similar in shape and size, were
disinfected with the help of 0.01% of mercuric chloride solu-
tion and followed by washing with DDA at least three times.

Journal of Plant Growth Regulation
1 3
Rhizobium Culture Preparation
The viable and certified Rhizobium culture specific for chick-
pea was acquired from the Indian Agriculture Research Insti-
tute (IARI) New Delhi. Rhizobium culture was mixed with
sugar and DDW water. Surface sterilized seeds were inocu-
lated with this mixture and dried in shade before sowing.
Experimental Design andTreatment Pattern
Inoculated and non-inoculated seeds were sown in earthen
pots of about 20cm width containing sandy loam soil and
manure, placed in the natural condition in net house of the
Department of Botany, Aligarh Muslim University, Aligarh
(India). On the 15days of sowing, thinning was done, and
for each pot, four plants were retained. The experiment was
conducted in a randomized block design. Thirty earthen
pots were arranged into 6 sets of five replicates (pots) cor-
responding to each treatment. At 20th day of sowing, the
seedlings were subjected to salt dose, given as NaCl (50 and
150mM NaCl) except the control plants that were treated
with DDW through the soil in equal quantity and then
allowed to grow. The morphological parameters, physiologi-
cal and biochemical attributes of plants were accessed after
harvesting the plants at 45days of sowing. The remaining
plants were allowed to grow up to yield and were harvested
at 120 DAS to read the yield characteristics.
Growth Characteristics
Chickpea plants from each treatment were removed from the
pots with full precaution and rinsed to eliminate the adhering
soil. The length of root and shoot was measured in centi-
metres using metric scale. Fresh root and shoot biomass of
chickpea plants from each treatment were assessed with the
help of an electronic weighing machine (CY204, Scalteo
Ins., Germany). For the observation of dry biomass, the root
and shoot samples were placed in an oven throughout 72h
at a temperature of 80°C. Leaf area was recorded with the
help of leaf area metre (LA211, Systronics, Ahmedabad,
India). The number of nodules was calculated by simply
counting the nodule present on each freshly harvested root
from every treatment.
Physiological Attributes
Litchtenthaler and Buschmann (2001) protocol was fol-
lowed for the estimation of total chlorophyll and carote-
noid contents in fresh. Fresh leaf samples of about 100mg
taken from intervened areas were cleaned with double
distilled water, crushed in 10ml of 80% acetone using
mortar pestle, poured and centrifuged. The absorbance of
a supernatent-containing pigment was measured at 663nm
and 645nm for the estimation chlorophyll and at 480nm
and 510nm for carotenoid estimation using UV-Visible
spectrophotometer. The following equation was used for
calculating the total chlorophyll and carotenoid content:
Leaf Electrolyte Leakage Estimation
Calculation of overall ions leaked from the leaves was
performed by the method proposed by Sullivan and Ross
(1979). About 30 leaf fragments were kept in a boiling test
tube containing 15ml of deionized water, of which elec-
tron conductivity was measured (ECa). The samples were
warmed at 45°C and 55°C in a water bath for 30min,
and the electron conductivity was calculated again (ECb).
Afterwards, samples were again boiled at 100°C for a time
period of 10min, and electron conductivity was again esti-
mated (ECc). The leaf electrolyte leakage was determined
by the formula:
Determination ofCarbonic Anhydrase (CA)
andNitrate Reductase (NR) Activity
The activity of carbonic anhydrase was calculated by the
method of Dwivedi and Randhawa (1974). Equal-sized
and small fragments of leaves were kept in test tubes con-
taining cysteine hydrochloride solution and incubated for
20min. After incubation, samples were filtered, and phos-
phate buffer, sodium bicarbonate solution and bromothy-
mol blue indicators were added and again incubated at
4°C for 20min. The samples were titrated with methyl
blue indicator, and activity of the enzyme was noted down
on per gramme fresh mass (FM) basis.
The activity of nitrate reductase was quantified with the
help of protocol given by Jaworski (1971). Neat and fresh
leaf samples were chopped and shifted to plastic vials hav-
ing phosphate buffer (pH7.5), isopropanol and KNO3. The
reaction mixture was incubated at 30°C for 2h. After
the incubation, reaction mixture was supplemented with
sulphanilamide and N-1-naphthylethylenediamine hydro-
chlorides solutions. Pink colour intensity developed after
Total Chlorophyll content =20.2 (OD 645)+8.02 (OD 663)
×V
W×1000
mg g−1FW.
Carotenoid content =7.6(OD 480)–1.49 (OD 510)
×V
d×W×1000
mg g−1
FW.
EL
(%)=
[
(ECb–ECa)∕(ECc)
]
×
100.

Journal of Plant Growth Regulation
1 3
some time was read on a spectrophotometer at a wave-
length of 540nm.
Estimation ofTotal Protein Content
Estimation of total soluble protein content was performed
by the method of Bradford (1976). The leaf material was
homogenized and centrifuged, and 200μl supernatant was
collected in test tubes to which 4ml of Bradford reagent was
added and incubated at 25°C for 10min. The absorbance of
the solution was recorded on a spectrophotometer at 595nm.
Estimation ofMalondialdehyde (MDA) Content
Lipid peroxidation was observed by the protocol as pro-
posed by Cakmak and Horst (1991). Leaf samples were
homogenized in 0.1% of trichloroacetic acid (TCA) and
centrifuged, and the supernatant was collected in test tubes
followed by addition of 0.5% thiobarbituric acid (TBA). The
reaction mixture was heated to 100°C in a water bath for
30min, then immediately cooled in an ice bath and again
centrifuged. The supernatant was collected for calculating
the MDA content at a wavelength of 523nm and 600nm
using spectrophotometrically using 155mM−1cm−1 extinc-
tion coefficient.
Estimation ofProline Content
The estimation of proline content in leaves was observed
as per the method proposed by Bates etal. (1973). Sample
extraction was done using sulphosalicylic acid followed by
the addition of glacial acetic acid and ninhydrin solutions in
equal quantity. After that, the samples were heated at 100°C
for 20min and consequently cooled in an ice bath, and then
5ml of toluene was added to every test tube. The uppermost
layer of solution was separated for calculating its wavelength
on a spectrophotometer at 520nm.
Activities ofAntioxidant Enzymes
For calculating the activities of enzymatic antioxidant,
fresh leaves (1000mg) from every treatment were ground
in chilled motor and pestle using extraction buffer containing
phosphate buffer, ethylenediaminetetraacetic acid (EDTA),
phenylmethanesulfonyl fluoride (PMSF), polyvinyl pyrro-
lidone and Triton X-100. The extract was centrifuged, and
supernatant obtained was collected for estimation of antioxi-
dant defence enzymes (SOD, POD and CAT).
Estimation of SOD activity was observed by protocol of
Beauchamp and Fridovich (1971). About 40μl of enzyme
extract was transferred in test tubes to which 50mM phos-
phate buffer (pH7.8), 55μM NBT, 9.9mML- methionine,
2mM EDTA and 0.02% Triton X-100 were added. To this
reaction mixture, riboflavin was added at last in complete
dark condition. The activity of SOD depends upon its capa-
bility to decrease the photochemical reduction of nitro-blue
tetrazolium, by reading the absorbance at 560nm for 2min
at 25°C.
Enzymatic activity of peroxidase (POD) was calculated
with the help of Sanchez etal. (1995) protocol. 100μl
of enzyme extract was transferred in test tubes following
the addition of 50mM phosphate buffer (pH 7.0), H2O2
(15mM), guaiacol (20mM). However, in control set of test
tubes, all the above-mentioned solutions excluding enzyme
extract were added. Then the POD activity was recorded at
wavelength of 436nm for 1min at 25°C.
Enzymatic activity of catalase (CAT) was measured fol-
lowing the method of Aebi (1984) by calculating the deser-
tion of H2O2 at the beginning of the reaction. 100μl of
enzyme extract was kept in test tubes following the addition
of 50mM phosphate buffer (pH7.0) and H2O2 (15mM).
The OD of the reaction mixture was recorded at 240nm
for 2min.
Estimation ofNa Accumulation inRoots andShoots
Shoots as well as roots of plant samples were harvested from
all treatments as well as from control plants, rinsed with
distilled water in order to remove the adhered soil as well as
extracellular sodium particles and then placed in an oven at
80°C for 48h. After that, oven-dried material was ground
for determination of sodium content by digesting the mate-
rial in nitric acid:perchloric acid (3:1, v:v). The Na content
was recorded by the atomic absorption spectrophotometer
(GBC, 932 plus; GBC Scientific Instrument, Braeside, Aus-
tralia), expressed in terms of μg g-1 dry mass.
Estimation ofN andP Content
For the analysis of N and P contents in leaf tissues, fresh leaf
samples were dehydrated at 80°C for 48h in hot-air oven.
Oven-dried samples were pulverized with the help of mortar
pestle and sieved for analysis. For estimation of N and P,
powdered samples were digested according to the protocol
given by Lindner (1944). For analysis of nitrogen content,
optical density was recorded at 525nm, and for phosphorus,
OD was read on a spectrophotometer at 620nm.
Stomatal Studies Using Scanning Electron
Microscopy andCompound Microscopy
Leaves from each treatment were cut into equal-sized small
fragments, fixed in glutaraldehyde buffer, paraformalde-
hyde, sodium cacodylate buffer (pH7.3) for 2h and then
again fixed in osmium oxide following the dehydration pro-
cess with graded series of ethanol (50%, 70%, 80%, 90%

Journal of Plant Growth Regulation
1 3
and 100%). The samples were coated with gold-palladium
and studied under JEOL JSM-JSM 6510 scanning electron
microscope.
For the study of compound microscopy, fresh leaf sam-
ples were dissected, and the epidermal layer of the lower
surface was peeled off and carefully transferred on a clean
glass slide for its observation under compound microscope
set with NIKON digital camera.
Study ofConfocal Microscopy forCell Viability
Chickpea roots were harvested and washed thoroughly
with tap water. Clean roots were cut with a sharp blade and
stained by immersed them into propidium iodide dye (5μM)
for half an hour. Stained root segments were then placed on a
clean glass slide, covered with a coverslip and finally studied
under the confocal microscope.
Statistical Analysis
Data were statistically evaluated using SPSS, 17.0 software
(SPSS, Chicago, IL, USA). Analysis of variance (ANOVA)
and standard error were operated on the data to assess the
Duncan test between treatments at 5% level of probability.
All the data were the mean of five replicates.
Results
Growth Indicators
Chickpea plants revealed a different reaction to the applied
NaCl concentrations. Plants treated with a lower concen-
tration of NaCl showed a slight reduction in all the studied
growth characteristics, while the higher concentration of
NaCl (150mM) caused a remarkable decline in the length
of root and shoot by 23.16% and 23.25%, root and shoot
fresh biomass by 48.68% and 45.93%, root and shoot dry
mass by 31.28%, and 24.65%, leaf area by 21.19% and
several nodules per root system by 34.17% in comparison
to their controls, respectively. However, plants inoculated
with Rhizobium were found less affected due to salt stress
and showed increased growth characteristics compared
to salt-treated ones (Figs.1a–d and 2a–d). Moreover,
Fig. 1 Effect of Rhizobium inoculation against 0, 50 and 150mM NaCl on (a) root length, (b) shoot length, (c) root fresh weight and (d) shoot
fresh weight of chickpea variety Pusa-BG 5023 at 45days after sowing

Journal of Plant Growth Regulation
1 3
non-stressed plants were inoculated with Rhizobium, and
the maximum increase in all growth parameters was found
(Fig.2).
Electrolyte Leakage
Plants treated with 150 mMNaCl showed more electro-
lyte leakage of 26.19% in comparison to control plants
(Fig. 3c). Control plants inoculated with Rhizobium
showed 13.57% decrease in electrolyte leakage; how-
ever, prominent reduction in electrolyte leakage was also
observed in stressed plants (150mM) inoculated with
Rhizobium compared to non-inoculated plants treated with
the same dose.
Physiological Parameters
Salt stress (50 or 150mM) decreased the total chlorophyll
and carotenoid in chickpea. Effect of NaCl was more promi-
nent at 150mM of NaCl compared to control and decreased
the total chlorophyll and carotenoid content by 29.36% and
37.89% at 45 DAS. Rhizobium inoculation alleviated the
toxic effect of 50 and 150mM NaCl in chickpea plants and
increased the total chlorophyll content 13.52% and carot-
enoid content by 20.38%at 50mM of salt, respectively, com-
pared to control (Fig.3a–b).
Lipid Peroxidation
Lipid peroxidation was estimated in terms of MDA content
present in foliage. Application of 50 and 150mM of salt
increased the MDA content of plants; however, among the
doses applied to plants through soil, 150mM of NaCl proved
to be more deleterious and increased the MDA content by
34.19% at 45 DAS concerning its control (Fig.3d). Inocula-
tion of Rhizobium almost nullifies the toxic effect of salt and
showed a significant decrease in MDA content. Rhizobium
inoculated plants supplemented with 150 mMNaCl showed a
19.6% increase in MDA content compared to non-inoculated
plants treated with the same concentration of salt (Fig.4).
Carbonic Anhydrase andNitrate Reductase Activity
The activity of carbonic anhydrase and nitrate reduc-
tase showed significant decrease due to salt treatment
Fig. 2 Effect of Rhizobium inoculation against 0, 50 and 150mM NaCl on (a) root dry weight, (b) shoot dry weight, (c) leaf area and (d) nod-
ules per root system of chickpea variety Pusa-BG 5023 at 45days after sowing

Journal of Plant Growth Regulation
1 3
(Fig.5a–b). Plants were supplemented with 150mM dose of
salt that showed 34.85% and 27.56% activity of CA and NR,
respectively, compared to untreated control plants. However,
Rhizobium inoculation overcomes the toxicity caused by salt
stress (50mM), thus enhancing the CA and NR activity by
19.75% and 15.39% at 45 DAS and also less decrease was
found in the activity of these two enzymes in plants treated
with 150mM NaCl as well inoculated with Rhizobium.
Thus, an appropriate and significant rise in the activity of
CA and NR was found in chickpea plants inoculated with
Rhizobium.
Proline Content
Salinity amplified the proline level in chickpea plants as
compared to control plants shown in Fig.4a. Maximum and
significant rise in proline content was observed in plants
inoculated with Rhizobium. Plants supplemented with both
Rhizobium and salt (150mM) showed 57.81% increase in
proline content in comparison to un-inoculated plants treated
with the same concentration of salt at 45 DAS.
Total Protein, Nitrogen andPhosphorous Contents
Total protein, N and P contents decreased significantly
with the increase in the level of NaCl, but the decrease was
more pronounced in plants treated with 150mM of salt
and showed a decline of 30.19%, 26.15% and 31.17% in
total protein, N and P contents (Fig.6a-c). Moreover, less
decrease of 17.64%,14.15% and 20.08% was found in total
protein content, N and P content in plants supplemented
with both Rhizobium and sodium chloride (150mM) in
comparison to non-inoculated plants treated with the same
dose (150mM) of salt at 45 DAS.
The Activity ofEnzymatic Antioxidants
Salt treatment to chickpea plants up-regulates the activities
of enzymatic antioxidants (SOD, POD and CAT) in com-
parison to control plants. An elevation of 34.95% in SOD,
33.81% in POD and 31.07% in CAT was recorded in plants
supplied with 150mM NaCl over control plants. The activ-
ity of SOD, POD and CAT showed an increase of 64.02%
in SOD, 63.12% in POD and 51.48% in CAT concerning
its control at 45 DAS when inoculated with Rhizobium
Fig. 3 Effect of Rhizobium inoculation against 0, 50 and 150mM NaCl on (a) total chlorophyll content, (b) carotenoid, (c) electrolyte leakage
and (d) malondialdehyde content of chickpea variety Pusa-BG 5023 at 45days after sowing

Journal of Plant Growth Regulation
1 3
and supplemented with 150mM of salt (Fig.4b–d). Thus,
inoculation of Rhizobium to salt-stressed plants as well as
unstressed plants enhanced the activity of these enzymes
significantly.
Na Accumulation inRoot andShoot
The accretion of Na content was greater in roots of chick-
pea in comparison to the shoots (Fig.5c–d). On the other
hand, inoculation of Rhizobium decreases Na accumulation
in roots and shoots, and the greatest decline was recorded
in plants supplemented with a low concentration of NaCl
against control plants (Fig.7).
Production ofSuperoxide Radicals
The level of superoxide radicals in tissues was represented
by blue-coloured spots. Leaf tissues from NaCl-treated
plants displayed more spots than control plants. However,
the leaf tissues from Rhizobium inoculated plants exhibited
fewer spots as compared to salt-stressed plants (Fig.8).
Confocal Studies
Salt stress decreases the cell viability of roots. Propidium
iodide dye is used to check the viability of cells. It stains the
genetic material of the dead cell and makes the nucleic acid
visible inside the departed cells as red fluorescent spots. In
our investigation, inoculation of Rhizobium increased the
cell viability of chickpea roots, whereas the plants treated
with 150mM NaCl showed less number of viable cells as
a large number of red fluorescent spots are evident in these
cells (Fig.11) compared to control plants.
Yield Characteristics
Plants treated with a low dose of NaCl showed a slight reduc-
tion in yield attributes (number of pods per plants, 100 seeds
weight), while the higher concentration of NaCl (150mM)
caused a remarkable decline in the number of pods per plants
and 100 seeds weight by 26.87% and 23.15%. Moreover,
plants free from salt stress and subjected to Rhizobium had
remarkably higher number of pods and 100 seeds weight in
comparison to control plants at harvest (Fig.7a–b) and the
increase was 25.89% and 17.95%, respectively.
Fig. 4 Effect of Rhizobium inoculation against 0, 50 and 150mM NaCl on (a) proline content, (b) superoxide dismutase activity, (c) peroxidase
activity and (d) catalase activity of chickpea variety Pusa-BG 5023 at 45days after sowing

Journal of Plant Growth Regulation
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Fig. 5 Effect of Rhizobium inoculation against 0, 50 and 150mM NaCl on (a) carbonic anhydrase, (b) nitrate reductase activity, (c) root Na con-
tent and (d) shoot Na content of chickpea variety Pusa-BG 5023 at 45days after sowing
Fig. 6 Effect of Rhizobium inoculation against 0, 50 and 150mM NaCl on (a) Protein content, (b) Nitrogen content and (c) Phosphorus content
of chickpea variety Pusa-BG 5023 at 45days after sowing

Journal of Plant Growth Regulation
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Fig. 7 Effect of Rhizobium inoculation against 0, 50 and 150mM NaCl on (a) No. of pods per plant and (b) 100 seed weight of chickpea variety
Pusa-BG 5023 at 45days after sowing
Fig. 8 Localization of superoxide ions produced by tetrazolium chlo-
ride (NBT) in 45days old leaves of C. arietinum is depicting as blue
spots on leaf surface. Image (a–c) represents control, 50mM NaCl,
150 mM NaCl-treated leaves and (d–f) represents control + Rhizo-
bium, 50mM NaCl + Rhizobium, 150mM NaCl + Rhizobium-treated
leaves of variety C- arietinum variety Pusa-BG5023

Journal of Plant Growth Regulation
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Compound Microscopy andSEM Imaging
Rhizobial inoculation in the absence of salt treatment
increased the breadth of stomatal aperture, and guard cells
were normal in shape compared to salt-treated ones (Figs.9
and 10). Moreover, the plants treated with salt showed
deformed guard cell with partially opened stomata (Fig.11).
Discussion
The present investigation aimed to observe the interaction
of Rhizobium inoculated into C. arietinum grown under salt
stress. Salt stress has a negative influence on all the growth
characteristics of plants such as germination, noticeable
deterioration in root colonization surface, plant length, bio-
mass as well as crop production, and also disturbs physio-
logical and biochemical attributes of plants (Shrivastava and
Kumar 2015). Moreover, inoculation of beneficial microbes
plays an imperative part in the amelioration of salt stress in
plants (Egamberdieva etal. 2016).
The current study demonstrated that plants treated with
less concentration of NaCl (50mM) were least affected and
showed a minimum reduction in all growth traits. But higher
concentration (150mM of NaCl) proved to be very harmful
and caused a maximum reduction in the growth parameters.
Fig. 9 Response of stomatal aperture of 45-day-old C. arietinum
variety Pusa-BG5023 leaves to the treatment was studied under com-
pound microscope. (a, b) represent control and 150mM NaCl and (c,
d) represent control + Rhizobium and 150mM NaCl at 40X magnifi-
cation, respectively
Fig. 10 Response of stoma-
tal aperture of 45-day-old C.
arietinum variety Pusa-BG5023
leaves to the treatment was
studied using scanning electron
microscope (SEM). (a, b) repre-
sent control and 150mM NaCl
and (c, d) represent control +
Rhizobium and 150mM NaCl at
3000X magnification, respec-
tively

Journal of Plant Growth Regulation
1 3
The reason behind the decline in growth characteristics in
plants may be due to salt stress imposed toxic effects of
ions and disruption in up taking of essential mineral nutri-
ents, osmotic imbalance and reduction in cyclin level which
restricts the cell division as well as the elongation (Latef
and Chaoxing 2011; Gupta and Huang 2014; Duan etal.
2015). The results we obtained are in consensus with studies
of Taffouo etal. (2009) and Kapoor and Srivastava (2010)
who also found inhibition in growth characteristics in Vigna
unguiculata L. and Vigna mungo L. due to salt stress. Fur-
thermore, inoculation of Rhizobium to salt-stressed chick-
pea plants enhanced the growth and development and also
alleviated the harmful effects of salt stress. Rhizobium being
symbiotic nitrogen-fixing bacteria promotes the growth of
plants by overcoming the nitrogen deficiency to legumes
involving a process of biological nitrogen fixation, improv-
ing uptake of water as well as mineral nutrients, maintenance
of cell turgor, etc. Rhizobial inoculation increases the nodu-
lation process and plant biomass productivity in plants under
controlled or saline conditions. Earlier studies have reported
that Rhizobium confers defence, enhances nodule number
and promotes growth biomarkers in mung bean, Vicia faba
and maize (Ahmad etal. 2013a, b; Benidire etal. 2017 and
Fukami etal. 2018) under salt stress.
Our results confirmed that NaCl (150mM) in soil reduced
the photosynthetic pigment contents (total chlorophyll and
carotenoid) of plants significantly. However, a little or insig-
nificant reduction was observed at low dose of salt (50mM).
Our results regarding the decrease in total chlorophyll and
carotenoid content due to the increase of salt stress agree
with results obtained by Abdul Qados (2011) in Vicia faba.
Sodium accumulation resulted in degradation of chloro-
phyll and carotenoid content in leaf tissues and reduces
the photosynthetic activity of plants; moreover, the effect
was more pronounced in older leaves than young leaves.
It was observed that salt stress suppresses the biosynthesis
of chlorophyll as it diminishes the mineral uptake such as
magnesium, which is the main component of the chlorophyll
molecule (Sheng etal. 2008). The decrease in photosynthetic
activity could be either through the change in the activities
as well as biosynthesis of photosynthetic enzymes that are
accountable for the production of these pigments and altera-
tion in structure and functioning of photosystem II (PS II)
(Aragao etal. 2005; Saleem etal. 2011).
Our current work illustrated that Rhizobium inocula-
tion showed positive influence on total chlorophyll as well
as carotenoid content among both salt-treated and control
plants which showed a significant increase of photosyn-
thetic pigments and activity. Our results are in agreement
with results obtained by Abd-Alla etal. (2019) in chickpea.
Microbial inoculation improved plant growth and pigment
content under salt conditions by producing the plant growth
regulators in the rhizosphere, thus improving the develop-
ment of root system and acquisition of essential mineral
nutrients (Li and Jiang 2017).
NaCl-induced electrolyte leakage and MDA content
were deceptive in our study, and future plants exposed to
salt stress showed abundant content of MDA and high EL
percentage, which results in oxidation of membrane lipids,
makes the cell membranes more permeable and enhances the
Fig. 11 Cell viability test was
performed on 45-day-old roots
of C. arietinum and confo-
cal microscopic images were
obtained. The large number of
stained nuclei indicates less
cell viability. (a) Control, (b)
150mM NaCl, (c) control +
Rhizobium and (d) 150mM
NaCl + Rhizobium are confocal
images of variety Pusa-BG5023

Journal of Plant Growth Regulation
1 3
electrolyte leakage and ultimately caused wilting of plants.
Malondialdehyde being the product of polyunsaturated
fatty acid peroxidation and is considered as the main stress
marker in plants. Other studies also confirmed that salinity
enhanced the MDA content in Brassica juncea (Ahmad etal.
2012) and Vicia faba (Azooz etal. 2011). Kordrostami etal.
(2017) also reported the same result in rice plants under
salt stress. However, the inoculation with rhizobium signifi-
cantly reduced the MDA and EL in NaCl-stressed chickpea
plants (Fig.3c–d). Similarly, other results also proved that
microbial inoculation improved salt resistance in plants by
declining the deleterious effects induced by reactive oxy-
gen species and maintains the structure and stability of cells
from the damage of salt stress (Singh and Jha 2017; Yasin
etal. 2018).
Our results indicated that NaCl treatment supplied to
chickpea plants caused a decline in total protein content. At
less concentration of salt (50mM of NaCl), less decrease
was observed while at an increased concentration of salt
(150mM of NaCl), a significant decline in total protein con-
tent was found. The decline in protein content of plants is
due to the excessive production of ROS that destroys the pro-
teins and enzymes under salinity stress (Perez-Lopez etal.
2009). Moreover, use of microbes enhanced soluble protein
content in stressed as well as non-stressed plants (Fig.6a),
ensured the maintenance of protein structure, stability and
regulation of enzymes accountable for the synthesis of pro-
teins (Hmaeid etal. 2014).
CA is the main enzyme that improves the photosynthetic
efficiency of plants by making inorganic carbon available to
plant cell. In C4 plants, carbonic anhydrase provides bicar-
bonate ions to phosphoenolpyruvate carboxylase and pro-
duces the oxaloacetic acid. In C3 plants, it affects stomatal
conductance and movement of guard cells (Hu etal. 2010).
A marked decrease was observed in the activity of CA in
chickpea under salt stress. The decline in CA activity due
to NaCl toxicity might be as a result of reduced amount of
internal CO2 concentration that could be happened by the
partial opening or complete closure of stomata; decreases
partial CO2 pressure, inactivation of enzyme Rubisco and
dysfunctioning of metabolic proteins and enzymes. The
results we obtained are in agreement with those of Talaat
and Shawk (2012) who also reported the same results in
Triticum aestivum. On the other hand, suitable escalation
in the activity of CA has been detected in chickpea plants
that were subjected to NaCl and inoculated with Rhizobium.
The increase in the activity of this enzyme could be due
to the positive role of Rhizobium on functioning and meta-
bolic state of proteins and enzymes involved in the assimila-
tion and uptake of carbon dioxide as well as activation of
Rubisco. Similar findings were observed by Talaat (2019) in
Phaseolus vulgaris treated with beneficial microbes under
salt stress.
Nitrate reductase is the main enzyme of the nitrate assim-
ilation pathway in plants. In the current study, a remark-
able decrease was observed in the activity of NR due to salt
stress. The decrease in NR activity may be due to change in
the function of enzymes involved in the pathway of NO3
_
assimilation under salt stress (Flores etal. 2004; Debouba
etal. 2006, 2007). Salinity decreases the activity of NR very
quickly as salt stress inhibits the uptake of NO3
_ through
roots system and declines the concentration of nitrates in the
cytoplasm which is the main substrate of nitrate reductase.
The results that we obtained agree with results obtained by
Abd el Baki etal. 2000) in maize and sugar beet (Ghoulam
etal. 2002) after exposure of high salt concentration. How-
ever, the salt-stressed or non-stressed chickpea plants inocu-
lated with Rhizobium showed the significant enhancement
in NR activity (Fig.9b). Microbial inoculation improved
the uptaking capability of NO3
_ which is the key substrate
of NR, water and other mineral nutrient uptake (Abiala etal.
2013).
Salinity affects the acquisition and absorption of N and P,
in plants, decreases their uptake through the root system and
increases the Na uptake. Our results suggested that high salt
concentration (150mM of NaCl) decreases the nitrogen and
phosphorous contents in plants while as enormous content
of sodium was found in salt-treated plants. It was observed
that increased concentration of Na in plants decreases the
accumulation of other elements such as nitrogen, phospho-
rous as well as potassium, creates competition in absorption,
passage or dispersal and changes the cationic and anionic
ratio, for example, Na+/K+ and Cl−/NO3
−. Na+ creates harm-
ful effects on plants by competing with K+at protein-binding
sites and thus hinders the activities of enzymes (Amtmann
and Sanders 1998). Under saline conditions, the decrease
of nitrogen absorption in plants may be due to the interac-
tion between sodium and ammonia or between chlorine and
nitrate as well as toxicity lead due to some ions like Na, S,
Cl which ultimately diminishes the absorption and accre-
tion of other essential nutrients (Jouyban 2012). Salt stress
also increased Na content in cell cytoplasm which replaces
the cytosolic potassium and leads to an upsurge in Na+/K
ratio. Our results also proved that salinity also decreases
the quantity of phosphates in soil by decreasing its adsorp-
tion processes as well as solubility of phosphate-containing
minerals (Prapaga etal. 2015). Our results are in consen-
sus with the findings of Kordrostami etal. (2016) in rice
plants under salt stress. On contrary, stressed as well as non-
stressed chickpea plants inoculated with Rhizobium showed
significant decrease in Na content in roots and shoots, and
at the same time, increased concentration of N and P was
observed. The reason behind our findings is that Rhizobium
is directly involved in promotion of growth by fulfilling the
N requirement of plant by the process of biological nitrogen
fixation, increases root growth and nodule number, improves

Journal of Plant Growth Regulation
1 3
nutrient uptake and reduces the membrane potential of root
system. Low Na+ accumulation in plants inoculated with
Rhizobium and salt was also found by Franzini etal. (2019)
and Benidire etal. (2017) in bean plants and Vicia faba.
In current study, plants supplement with NaCl and
Rhizobium revealed large accretion of proline leading
to higher water potential and maintenance of enzymatic
activity that could have declined oxidative damage cre-
ated due to salt stress. Proline is considered as compatible
osmolyte involved in the alleviation of oxidative stress
by playing the role of ROS scavenger in plants thereby
protecting the intracellular macromolecules from dehy-
dration (Hayat etal. 2012; Kordrostami etal. 2017). Non-
stressed and stressed plants showed less increase in proline
content, while an as-significant escalation was found in
plants inoculated with Rhizobium. The enhancement of
proline accumulation in plants inoculated with microbes
resulted due to the maintenance of relative water content
and uptake of nutrient elements. Our results agree with the
results obtained by Hahm etal. (2017) in pepper-inocu-
lated growth-promoting bacteria under salt stress.
In this study, we assessed the activity of enzymatic anti-
oxidants (SOD, POD and CAT) in Rhizobium inoculated
and non-inoculated chickpea plants grown under salt stress.
Salt stress induced the free radical such as H2O2, OH, and
O2
− formation which causes oxidative damage to plants
(Mushtaq etal. 2020; Islam etal. 2020). Plants have the
antioxidant defence system involving different enzymes such
as catalase, superoxide dismutase, peroxidase, etc. that safe-
guards the plant from oxidative damage, as these enzymes
scavenge the harmful radicals or converting them into less
reactive form (Zhou etal. 2018). Superoxide dismutase dis-
simulates the superoxide radicals and converts it into H2O2
and antioxidant enzymes CAT as well as POD detoxifies the
accumulated H2O2 and converted it into H2O. In the cur-
rent study, activities of enzymatic antioxidants in chickpea
plants subjected to salt were higher than the control plants.
However, prominent increases in activities of SOD, POD
and CAT were observed in plants inoculated with Rhizobium
as well as subjected to high salt concentration. Our findings
are supported by those of Etesami and Beattie (2018) and
Santos etal. (2018), who also observed that the activities of
antioxidants showed a significant increase in different types
of crop plants inoculated by microbes under salt conditions.
Our results suggested that inoculation of Rhizobium reduced
the formation of reactive oxygen species in plants treated
with salt and prevented the plant from oxidative damages.
Stomata play an imperative part in the regulation of gas
exchange in plants. Excess salt content also changed stoma-
tal response in plants. In our investigations, high salt-treated
(150mM NaCl) plants had relatively closed stomata com-
pared to control plants (Figs.9 and 10). Salt stress increases
the level of ABA in shoots and reduces the flow of water
which decreases the leaf water content and endorses clo-
sure of stomata. Salinity also damages the stomatal aper-
ture and reduces the stomatal width in plants. Same results
were found by AzevedoNeto etal. (2004). On the other hand,
plants inoculated with Rhizobiumas as well as supplemented
with salt showed open and normal stomata. Therefore, it
could be hypothesized that the normal size of stomata may
be because microbes maintain the water status of plants and
reduce the root hydraulic conductivity.
Chickpea plants supplemented with a high dose of NaCl
showed a decline in yield characteristics (number of pods
per plant, 100 seed weight and seed yield per plant). A simi-
lar reduction in yield was observed in rice under salt stress
by Zeng and Shannon (2000). The reduced yield in stressed
chickpea plants could be due to the reduction in growth, leaf
area, restricted mineral acquisition and low rate of photosyn-
thesis due to salt stress. Moreover, Rhizobium inoculation
enhanced the yield parameters in stressed as well as non-
stressed plants which may be due to the better growth and
other physiological processes.
Conclusion
The current study revealed that high dose of NaCl (150mM
NaCl) provided to chickpea reduced the growth characteris-
tics, decreased root cell viability and changed the stomatal
action and diminished the physiological and biochemical
characteristics. Inoculation of Rhizobium to chickpea seeds
before sowing is proved to be very advantageous in rela-
tions to growth-enhanced photosynthetic contents, minimum
production of reactive oxygen species, increased cell viabil-
ity and improved yield attributes in chickpea plants. Thus,
Rhizobium inoculation could help promote the growth of
chickpea under salt stress as it reduced the salt-lead damages
in it that were proved also through the microscopic analysis.
Author Contributions ZM, SF and BG have performed the experiments
and written the manuscript. KRH has analysed the data and critically
edited the manuscript.
Compliance with Ethical Standards
Conflict of interest The authors declare no conflict of interest.
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