This regions. The increasing levels of salinity of

 

 

This study was aimed to identify plant growth-promoting rhizobacterial
isolates and to evaluate their ability to improve plant growth and salt
tolerance by different plant growth promoting activities. Among the sixty
rhizobacterial isolates, two bacterial isolates HMM57 and JMM15 were able to
enhance mustard growth at the different
level of salinity. The rhizobacterial isolate HMM57, JMM15 were identified as Pseudomonas
argentinensis and Pseudomonas azotoformans, respectively, and their plant
growth promoting activities like aminolevulinic
acid, indole acetic acid, 1-aminocyclopropane-1-carboxylate deaminase
production, potassium and phosphorus solubilization may contribute the salt
tolerance and crop productivity in mustard. During evaluation in pot house
condition at 80 days of plant growth, inoculation of two bacterial isolates
HMM57 and JMM15 caused 139 to 291% increase in root and shoot dry weight of
mustard even at 12 dS/m. Thus,
these plant growth promoting rhizospheric bacterial isolates are promising for crop productivity under saline conditions
and could be used for their subsequent application as biofertilizer.

Keywords: Mustard, Rhizosphere bacteria, Salt tolerant,
Plant growth promoting activities

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____________

 * Corresponding
author:  Dr. Manisha Phour

E-mail
address:
[email protected]

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Introduction

           Indian mustard Brassica juncea (L.) is an important
winter crop grown across the Northern Indian plains which occupies third
place among the various oilseed species due to its considerable economic and
nutritional value. The maximum distribution area is centered in the North-West
agro-climatic zone, where the majorities of ground
water sources are highly saline and have medium to high sodicity
problems. Salinity is one of the major constraints in this crop throughout
the world which greatly reduces its agricultural productivity in both irrigated
and non-irrigated regions (Narusaka et al., 2003; Zhang et al.,
2010; Ashraf, 2010; Wakeel et al., 2011). For getting the optimum productivity potential of mustard, irrigation
is very important but equally important is the quality of irrigation water in
irrigated regions. The increasing levels of salinity of the irrigation water
applied at presowing and flower initiation greatly reduces seed germination
(Flower, 1991), seedling growth (Almansouri et
al., 2001), plant height (root/shoot elongation), the branching pattern and
the pod formation (Chauhan and Singh, 2004; Shekhawat et al., 2012).
Irrigation with saline water (12 and 16 dS/m) decreased the dry matter yield
significantly when applied at pre-sowing or later stage (Mishra and Anju,
1996). The irrigation with saline water at the pre-flowering stage or later
reduced the grain yield by 50% and 70%, respectively. The magnitude of the
effect of salinity varied with the plant species, type and level of salinity. Salt and osmotic stresses are responsible for
inhibition and delayed seed germination, and also for seedling establishment
(Almansouri et al., 2001). Therefore,
the growth, yield and oil production of mustard are markedly reduced due to
salinity.  This salt-induced growth
reduction could be improved by a number of means including application of growth regulating substances
(Ashraf and Foolad, 2007; Zhang et al., 2008), by improving the
nutritional status or by inoculation of salt
tolerant bacteria and mycorrhizal fungi (Plaut et al., 2013). PGPR strains are widely applied on crops as a means
of improving productivity, increasing stress resistance and regulating plant
growth (Ramesh and Kumar, 1975; Watanabe et
al., 2000; Zhang et al., 2006; Ghanbari et al., 2013; Nunkaew et
al., 2014).
Moreover, treatments with plant growth promoting rhizobacterial strains has been found to enhance crop productivity at different level of salinity. Therefore, there
are immense possibilities for developing bacterial biofertilizers using
rhizobacterial strains that could make the plant to grow under salinity
conditions.

Therefore, present
study was carried out to characterize rhizobacterial isolates for their salt
tolerance and plant growth promoting activities that could improve the mustard
growth under salt stess. Results of this study are suggested that seed
inoculation of bacterial isolate HMM57 and JMM15 rhizobacterial strain were
found to stimulate the growth of mustard up to 12dS/m. This is the recent
report establishing that some rhizobacterial isolates especially Pseudomonas
species have the potential to act as effective biofertilizer under the salt
stress conditions.

 

Material
and methods

2.1. Isolation of rhizobacteria

           Ninety four rhizobacterial isolates
were isolated from the mustard rhizosphere. For a
collection of soil, samples were collected randomly from the rhizosphere
of mustard from five different locations of mustard fields. From each location, samples were collected from six
different sites. Isolated rhizobacterial colonies were selected based on
typical morphological and biochemical characteristics.

2.2. Host species

Seeds of Brassica
juncea L. variety 749 were obtained from Department of Agronomy, C.C.S.
Haryana Agricultural University, Hisar, India.

2.3. Salt tolerance among rhizobacterial isolates at different salt
concentration                                             

All the rhizobacterial isolates
were checked for their ability to grow at different concentrations of NaCl,
i.e., 1, 2, 4, 6 and 8% (w/v), on Nutrient Agar (NA) medium containing 20 mM
HEPES (N-2-hydroxyethane-sulphonic acid)
buffer (Marsudi et al., 1999).  NA plates were spotted with a 20 ?l growth
suspension of different rhizobacterial isolates later incubated for 3-4 days at
28 ± 2°C in a B.O.D. incubator. The susceptibility or tolerance to NaCl by
different rhizobacterial was recorded by
observing the growth as a positive or negative result and their growth (colony
size) was observed at different salt concentrations.

2.4. Evaluations of different plant growth promoting
activities

Selected rhizobacterial isolates were studied for
the production of various growth promoting substances.

2.4.1. Utilization of
1-aminocyclopropane-1-carboxylate (ACC) by rhizobacteria

           Rhizobacterial
isolates were spotted on medium plates containing minimal medium (Dworkin and
Foster, 1958) supplemented with 3 mM ACC (Penrose and Glick, 2003) and minimal
medium plates containing ammonium sulfate as nitrogen sources were used as
control plate for growth comparison of different bacterial isolates. The growth of rhizobacterial isolates on ACC
supplemented medium plates were recorded after 5 days of incubation at 28±2 ?.

2.4.2. Production of ?-aminolevulinic
acid (ALA)

Rhizobacterial isolates were tested
for their ability to produce ?-aminolevulinic acid by the method as described
by Mauzerall and Garnick (1955). Cultures were inoculated in duplicate in 10 ml
LB broth supplemented with 15 mM glycine and succinate, and were incubated at
30ºC for 48 hrs under stationary conditions of growth. Culture samples were
withdrawn and centrifuged at 10,000 rpm for 15 min (Remi Instruments, Mumbai,
India). To 0.5
ml of culture supernatant, 50 µl of acetylacetone and 0.5 ml of 1M sodium
acetate buffer were added and then tubes were boiled in a water bath for 15
min. After cooling, 3.5 ml of modified Ehrlich reagent were added and the
absorbance of the mixture was measured at 556 nm after 20 min at room
temperature. The concentration of ALA in the culture supernatant was determined
by comparison with standard curve.

2.4.3. Indole acetic acid (IAA) production by rhizobacterial isolates

           Rhizobacterial
isolates were also checked for their ability to produce indole acetic acid. Isolates
were inoculated in duplicate in 30 ml of LB broth supplemented with
DL-tryptophan @ 100 µg ml-1 (Hartman et al., 1983) and were incubated at 28±2?
for 72 hrs under stationary conditions of growth and then after 72 hrs.,
centrifuged at 10,000 rpm for 15 min (Remi Instruments, Mumbai, India). Quantitative
estimation of IAA was determined in the culture supernatant by the method as
described by Gordon and Weber (1951). 

2.4.4. Phosphorous solubilization by rhizobacterial
isolates

           Phosphorous
solubilization ability of rhizobacterial isolates was determined by the spot test method (Sindhu et al., 1999) on
Pikovskaya medium (Pikovskaya, 1948) plates having tricalcium phosphate. Rhizobacterial
isolates of 48hrs old growth were spotted on above-prepared
plates and incubated at 28±2°C for 2-3 days. Presence of potassium
solubilization by rhizobacterial isolates was
based upon the ability of solubilization zone formation.

2.4.5. Potassium solubilization by rhizobacterial
isolates

           Potassium
solubilization by rhizobacterial isolates was also studied by the spot test
method (Sindhu et al., 1999) on Aleksandrov medium (Aleksandrov, 1967) plates
having mica powder (an insoluble form of
potassium) and acidic dye bromothymol blue. A loopful of 48-hour old growth of
the rhizobacterial isolate/strain was spotted on above-prepared plates and incubated at 28±2°C for 2-3 days. Presence
of potassium solubilization activity in tested rhizobacterial isolates was based upon the ability of solubilization
zone formation and change of color from greenish blue to yellow.

2.5. Efficacy of inoculated rhizobacteria on growth
of mustard under salinity conditions

2.5.1. Agar plate experiment

For evaluating the effect
of salt concentration on seed germination of mustard, selected rhizobacterial
isolates were studied for germination on water agar (0.8%) plates having
different concentrations of NaCl to obtain an electrical conductivity (EC) up to 20 dS/m. Inoculated seeds were grown at
28±2°C in the plant incubator and growth of seedlings was recorded on 5th
and 10th days after sowing. The observations for retardation or stimulation
of root and shoot growth of mustard seedlings (with or without salt) were
recorded.

2.5.2. Pot house experiment

Efficient
rhizobacterial isolates have selected the
basis of different plant growth promoting activities, their salt tolerance
ability and a stimulatory effect on
seedling growth. Their effectiveness on the growth
of mustard was studied under pot house
conditions during the month of October to January. Seeds were inoculated with 20 ml growth suspension of selected bacterial
strains according to the treatment schedule and grown in the respective earthen
pots containing saline soil of varying salt concentration. The same volume of bacterial suspension was added after one week of sowing in
respective pots according to treatment schedule to check combined effect on weed and plant growth. The
plants were grown in the pot house under daylight conditions and each treatment
had three replications. Each replication was used to observe different plant
growth parameters after 40 days and 80 days of sowing.

Identification

Selected rhizobacterial
isolates were identified by the 16S rRNA sequence analysis. Genomic DNA
extraction of these isolates was isolated according to the method described by
Pitcher et al. (1989) and extracted DNA was amplified by PCR using the
universal primers 8-27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′).
Purification of the product was done as described previously (Pandey et al.,
2002). The amplified 16S rRNA gene was sequenced by the dideoxy chain
terminator method using a Big Dye terminator kit followed by capillary
electrophoresis on an ABI 310 Genetic Analyzer (Applied Biosystems). The
partial 16S rRNA gene sequence of the selected bacterial isolates were then
used as a query to search for similar sequences in GenBank database.

3. Results

3.1. Salt tolerance of rhizobacterial isolates

Rhizobacterial isolates were tested in the basis of
their salt tolerance capacity at 1, 2, 4, 6 and 8% NaCl concentrations. The
result showed that out of ninety four rhizobacterioal isolates, HMM6, HMM13,
HMM34, HMM39, HMM44, HMM57, HMM65, HMM88, HMM92, JMM15 and JMM42 showed larger
colony size ranging from 10.1-20.0 mm colony at the 8% NaCl concentration
whereas six bacterial isolates HMM15, HMM18, HMM40, HMM46, HMM69 and HMM81 did
not show any growth. Other isolates shows colony size ranging from 0.5 to10mm
at up to 6 % salt concentration (Graph 1). To conform their tolerance, these
eleven rhizobacterial isolates were further tested by LB () broth containing 8%
NaCl concentration. These isolates were able to grow well. Therefore, these
isolates could be classified as halo-tolerant (Willey et al. 2009).

3.2. Different Plant growth promoting activities

In addition to salt tolerant, their PGPR properties
were also studied. ACC utilization were observed on the basis of bacterial
growth observed after 2 days of incubation on the minimal medium (Dworkin and
Foster, 1958) plates supplemented with 3 mM ACC. Rhizobacterial isolates i.e., HMM39,
HMM57, HMM88, HMM92, JMM15 showed significant growth on ACC supplemented plates
and two bacterial isolates HMM65 and HMM13 showed moderate growth. Observations
were taken for their ALA producing ability in the culture supernatant
(Mauzerall and Garnick, 1955) after inoculating a loopful of 48 hrs old-culture
growth of bacterial isolates in 5ml LB broth after 48 hrs of growth. ALA
production (> 15 µg/ml) was observed in bacterial isolates HMM92 and JMM15. Only
one bacterial isolate JMM15 produced more than 10 µg/ml IAA and four bacterial
isolates HMM39, HMM57, HMM88 and JMM42 produced IAA ranging from 1.0-5.0 µg/ml. Two isolates
HMM92 and JMM15 showed P solubilization zone varying between 1.51 to 2.0 mm on
tricalcium phosphate containing medium plates after 2 days of growth. Three
bacterial isolates HMM13, HMM39, JMM42 showed P solubilization zone ranging
from 1.01 to 1.50 mm, whereas two bacterial isolates HMM57 and HMM65 showed
little zone of phosphorus solubilization wheras four bacterial isolates did not
solubilize phosphorus. Isolate JMM15 showed maximum zone of P solubilization.
Results were taken after spotting a loopful of 48 hrs old-culture growth of
bacterial isolates on modified Aleksandrov medium plates containing mica and
bromothymol blue dye. After 3 days of growth, rhizobacterial isolates were
scored depending on the ability of bacteria to form zone of solubilization. Out
of eleven rhizobacterial isolates tested, four isolates formed zone of K
solubilization on mica containing modified Aleksandrov medium plates.
Rhizobacterial isolates JMM15 showed highest potassium-solubilizing index
(K-SI) more than 5.0. Other rhizobacterial isolates did not cause K
solubilization on mica containing plates.

3.3. Effect of
inoculated rhizobacteria on growth of mustard under salinity conditions

Selected
eight rhizobacterial isolates HMM39, HMM44, HMM55, HMM57, HMM65, HMM73, HMM92
and JMM15 were tested for seed germination percentage at different salt
concentrations i.e., 0, 4, 8, 16 and 20 dS/m. Observations were taken after ten
days of seedling growth. Increase in germination percentage was observed by
inoculation of all selected bacterial isolates up to 20 dS/m as compared to
control. With the increase of salt concentration, germination declined and only
four isolates i.e., HMM39, HMM44, HMM57 and HMM65 showed 100% germination at 8
dS/m. Three bacterial isolate HMM39, HMM44 and HMM57 showed 70-80% germination
at 20 dS/m. At 8 and 16 dS/m salt concentration, all the isolates showed more
germination percentage in comparison of control. These rhizobacterial isolates was
showed significant growth stimulation effects on seedling growth of mustard in
comparison to uninoculated control at both 5 and 10 days of growth even at
higher salt concentrations i.e., 16 and 20 dS/m (Table1). All the
bacterial isolates exhibited stimulation of root and shoot growth, at both 5
and 10 days of growth seedlings (Table1, 2).  More specifically, HMM57 and JMM15 received
paramount attention because of its plant growth promoting activities that could
help mustard plants withstand varied biotic and abiotic stresses. Therefore,
these two rhizobacterial isolates were tested further for their effect on
growth of mustard growth in the soil at EC 0, 8 and 12 dS/m in pot house condition.
Various plant growth parameters were recorded at 40 and 80 days after sowing.  Inoculation effect of rhizobacterial isolates
HMM57 and JMM15 on mustard were investigated in pots. Increase salinity 0 to 12
dS/m which causes progressive and significant reductions in mustard growth in
comparison of control (uninoculated). The result shows that inoculation of rhizobacterial isolate HMM57 and
JMM15 stimulated growth of mustard at 0, 8 and 12 dS/m salinity under pot house
conditions. At 80 days after sowing, inoculation of bacterial isolate HMM57
showed 119.7, 135 and 139% increase in root dry weight and 40.4, 22.5 and 200%
increase in shoot dry weight of mustard at 0, 8 and 12 dS/m respectively.
Similarly, inoculation of bacterial isolate JMM15 showed 118, 77.6 and 168%
increase in root dry weight and 83, 42 and 290.5% increase in shoot dry weight
of mustard at 0, 8 and 12 dS/m respectively. Tolerance
index (Ti) of rhizobacterial isolates HMM39, HMM57 and JMM15 was calculated at
8 and 12 dS/m. Maximum tolerance index was observed by inoculation of
rhizobacterial isolate JMM15 followed by isolate HMM57 at 8 dS/m EC after 40
days of sowing. Whereas, at 12 dS/m EC, maximum tolerance index was obtained by
inoculation of bacterial isolate JMM15 followed by isolate HMM39. Tolerance
index (Ti) of rhizobacterial isolates HMM39, HMM57 and JMM15 was calculated at
8 and 12 dS/m. Maximum tolerance index at 8 ds/m salinity level was observed by
inoculation of bacterial isolate HMM57, followed by isolates HMM39 and JMM15.
At 12dS/m salinity level, maximum tolerance index was obtained by inoculation
of bacterial isolate HMM57, followed by isolates JMM15 and HMM39. Therefore, inoculation of HMM57 and JMM15
bacterial isolates could decrease the salt stress and positively stimulate the
seed germination and plant growth. The rhizobacterial isolate HMM57, JMM15 were
identified as Pseudomonas
argentinensis and Pseudomonas azotoformans, respectively by using universal primers
8-27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′).

Discussion

In the present investigation, ninety four rhizobacterial
isolates were screened from mustard rhizosphere on the basis of morphological
and biochemical characteristics. Some bacteria have natural ability to tolerate
salinity as it was identified in a bacterial community isolated from the
rhizosphere of salt affected crop (Tilak et al. 2005). The salt tolerant
root-colonising rhizobacteria were isolated and also identified as Pseudomonas
putida, P. extremorientalis, P. chlororaphis and P. aurantiaca.
which were very efficient in causing a significant increase in the shoot, root
and dry matter of wheat, and were able to survive in saline soil (Egamberdieva
and Kucharova, 2009). Growth of the rhizobacterial isolates was much affected
by the concentration of the salt when it was increased from 1-8%. The results
shows out of 94 rhizobacterial isolates, eleven isolates i.e., HMM6, HMM13, HMM34, HMM39, HMM44, HMM57, HMM65,
HMM88, HMM92, JMM15 and JMM42 have salt tolerance capacity up to 8% NaCl
concentration, Upadhyay et al. (2012) also
observed 33% of bacterial isolates survived upto 8% NaCl (w/v) and only 19%
isolates showed PGP attributes at higher NaCl concentration.

Some bacterial strains
producing different plant growth promoting activities i.e., aminolevulinic acid,
IAA production, ACC utilization etc. have been found to stimulate plant growth
under salt stress condition (Kausar and Shahzad, 2006; Hynes et al., 2008; Fu
et al., 2014, 2015; Manafi et al., 2015; Kantha et al., 2015; Kosar et al.,
2015). These bacterial isolates may enhance the survival of some seedlings,
especially during the first few days after the seeds are planted. Eight
rhizobacterial isolates were tested on salt incorporated plates to study their
inoculation effect on mustard seedlings on 0.8% water agar plates having
different salt concentrations up to 20dS/m. It was observed that increasing
NaCl concentration decreased the growth of mustard seedling and the effect of
rhizobacterial isolates on seedling growth and germination percentage varied with
different bacterial isolates but salt tolerant rhizobacterial isolates
stimulate/enhance the mustard growth up to 20dS/m in comparison of control. Similar
variation in growth stimulation of seedling growth at different salt
concentrations has been reported in other crops. Stimulation in root and shoot
length of maize under 6 dS/m NaCl stress after inoculation of PGPR was observed
by Nadeem et al. (2006). Naeem et al. (2011) also reported that application of
ALA improved the relative growth rate of root and shoot, and leaf water
relations (osmotic potential and relative water content) of Brassica napus plants under different NaCl (100, 200 mM)
concentrations. JMM15, HMM39 and HMM57 were found to have many plant growth
promoting activities like aminolevulinic acid, IAA production, P&K
solubilization, ACC utilization etc. which could help
mustard plants withstand extreme salt stresses. It has been earlier reported
that PGPR having a potential of solubilizing phosphorus, calcium and potassium
could protect the plants against sodium toxicity (Grichko and Glick, 2001;
Egamberdiyeva and Hoflich, 2003). Under salt stress, plant growth promoting
bacteria have shown positive effects in plants on parameters like germination
rate, tolerance to drought, weight of shoots and roots, yield and plant growth
(Raju et al., 1999). In an era of sustainable agricultural production, the
interactions in the rhizosphere by soil microorganisms with soil and plant play
a vital role in mobilization of nutrients from the limited pool (Mantelin and
Touraine, 2004). The combination of IAA production ability, phosphorous
solubilization (Gyaneshwar et al., 1998) and siderophore production (Duffy,
1994) of rhizosphere bacteria help the plant rhizosphere in enhancing the
nutrient absorption potential under stress environment for enabling economic
production of agricultural and horticultural crops (Damodaran et al., 2013).
Results shows inoculation of HMM57 and JMM15 rhizobacterial isolates caused 139
to 291% increase in root and shoot dry weight of mustard even at 12 dS/m.
Similarly, Siddikee et al. (2010) showed that inoculation of the 14
halotolerant bacterial strains to ameliorate salt stress (150 mM NaCl) in
canola plants caused an increase in root length of between 5.2 and 47.8%, and
enhanced dry weight of canola between 16.2 and 43%, in comparison with the
uninoculated positive controls. Thus, salt tolerant (up to 8% NaCl) and plant
growth promoting rhizobacterial isolates, were found to be very effective for
their stimulatory effect on growth of mustard seedling at different salt
concentrations.