Introduction
Various environmental factors, such as temperature, salinity, pathogens and
drought, seriously influence agricultural productivity by reducing the growth
and development of crops. It is estimated that area of salt affected soils has
increased from 45 million to 62 million hectares since the 1990s making
salinity the main factor in plant growth and productivity (Chele et al. 2021). Salinity is more
apparent in the coastal agri-ecosystems because of the continuous entrance of
seawater, mishandling of coastal irrigation land, and weak farming practices.
Excessive salts in soil modify cellular metabolism initiating many
physiological, morphological, biochemical, and molecular variations in plants.
Excessive salts in soil adversely affect plant growth and development, causing
osmotic stress. Salinity directly affects water accessibility, accumulation of
toxic ions like Na+ and Cl– in the cells, nutrient
disparity, and oxidative stress (Munns and
Tester 2008). Excessive accumulation of Na+ in plant cells
may create metabolic disorders in functions where high K+ or Ca+2
and low Na+ are essential for perfect functioning (Tester and Davenport 2003).
The application of plant growth-promoting rhizobacteria (PGPR) is one of
the most encouraging techniques used in the plant to in plants to reduce the
harmful effects of salinity and improve growth (Shameer
and Prasad 2018). Microorganisms in the soil substantially lessen plant
salt stress, subsequently increasing crop production (Etesami and Beattie 2018). Furthermore, rhizobacteria having
various PGP properties can improve the growth and development of plants. For
example, Bacillus spp.
capable to produce various phytohormones could increase plant resistance
against salinity stress and improve plant growth (Rajendran et al. 2008). Moreover, it has been confirmed
that applying PGPR enhanced plant growth under salinity stress conditions (Han and Lee 2005).
A significant portion of the total P in the soil exists
in fixed forms like Ca3(PO4)2, AlPO4,
FePO4 and organic phosphorus, which are unavailable for plants. The
availability of P becomes reduced in saline soil, and saline ions inhibit the
amount of P taken up by the plant (Rojas-Tapias et
al. 2012). Phosphate-solubilizing rhizobacteria can solubilize the
insoluble P by manufacturing various organic acids (Chen and Liu 2019). Thus, phosphorus becomes available to the
plants, and there is a sustainable decrease in the pH of rhizosphere soil.
Phosphorus solubilizing rhizobacteria can be a valuable bio inoculant for
plants to reduce the effects of salinity and recover the quantitative and
qualitative characteristics associated with plant and soil efficiency.
Indole acetic acid (IAA)
is one of the most important plant hormones that have direct effects on
increasing plant growth. The PGPR helps plants overcome the harmful effects of
abiotic stresses by producing IAA, which directly improves plant growth, even
in other growth-inhibiting chemicals (Bianco and
Defez 2009). The amino acid tryptophan produced as root exudates are
converted into IAA by PGPR in rhizoplane and is taken up by the root cells and
stimulate auxin signal transduction pathway and different auxin responding
factors (Shameer and Prasad 2018). The
harmful effects of salinity may be minimized and plant growth can be improved
by applying IAA-producing rhizobacteria.
Siderophores are low molecular weight organic compounds
generated by rhizobacteria under low iron environments (Ahmad et al. 2016). The chief role of such compounds is to
chelate iron and make it accessible for microbial and plant cells.
Siderophore-producing rhizobacteria also play an essential role against
phytopathogens. The Fe present in the soil is firmly bound with the siderophores
produced by rhizobacteria and it becomes unavailable to plant pathogens,
consequently hindering phytopathogen growth (Beneduzi
et al. 2012; Ahmed and Holmström 2014).
The production of ammonia (NH3) is a critical
plant growth-promoting property of PGPR. The application of ammonia-producing
PGPR provided the plant's nitrogen and significantly improved plant growth and
biomass accumulation (Marques et al.
2010).
Salinity can boost ethylene synthesis via raised levels of 1-Amino
cyclopropane-1-carboxylic acid (ACC). Any constraint on increased ethylene
produced in plants can promote the plant's growth in saline soils. The
ACC-deaminase yielded by various PGPR enhances the scarcity of ACC, decreasing
the harmful concentration of ethylene in the plants under salt-stressed
conditions. ACC produced in plant tissues is immediately cleaved into
α-ketobutyrate and ammonia by ACC-deaminase (Hontzeas et al. 2004). The injection of
ACC-deaminase-producing PGPR can facilitate plant growth in stressed
environmental circumstances, including salinity, flooding, drought, heavy metal
contamination and phytopathogens.
Exopolysaccharides (EPS) are high-molecular-weight
compounds that contain sugar residues and widely vary in structure and role.
The impact of EPS producing PGPR on the amalgamation of root clinging soils has
been described by Alami et al. (2000). EPS producing PGPR can effectively increase the volume
of soil macropores and soil aggregation, as a result, water and fertilizer
accessibility to the plant increases. EPS-producing PGPR can also fix cations,
including Na+ (Upadhyay et al.
2011). Thus, the increased population of EPS-producing rhizobacteria in
the rhizosphere is likely to reduce the concentration of Na+
available for plant absorption and thus lessen the salt stress on plants grown
in saline environments.
In the current study, four salt-resistant KSB (A. pittii, R. pusense, C. oxalaticus and O. ciceri) isolated from salt-affected rice fields in the coastal
area of Kuala Muda, State Kedah, Malaysia, were evaluated for phosphorus
solubilizing ability, IAA production, siderophores, ammonia, ACC deaminase and
exopolysaccharides under salinity stress. The objective of study was to find
the strain which could show the good plant growth promoting properties under
salinity stress.
Materials and Methods
Four potential potassium
solubilizing rhizobacteria A. pittii, R. pusense, C.
oxalaticus and O. ciceri were subjected to evaluate their capability for other
plant growth-promoting properties under NaCl stress. The strains were taken
from the microbiological lab at The School of Biological Sciences, Universiti
Sains Malaysia isolated and identified by Ashfaq
et al. (2020).
Phosphorus
solubilizations by KSB under NaCl stress
Four potential potassium
solubilization rhizobacteria were subjected to evaluate their capability of
phosphorus solubilization under NaCl stress. For phosphorus solubilization,
National Botanical Research Institute's phosphate (NBRIP) broth has (g/L)
glucose (10 g); Ca3(PO4)2 (5 g); MgCl2.
6H2O (5 g); MgSO4. 7H2O (0.25 g); KCl (0.2 g)
and (NH4)2SO4 (0.1 g) was prepared (Scervino et al. 2011). The NBRIP broth
was amended with 3, 5 and 7% NaCl to provide salinity stress compared with control without NaCl. One hundred mL NBRIP
was taken in 250 mL flasks and the broth was sterilized at 121°C for 15 min.
One mL of freshly grown bacterial cultures with optical density (OD) 0.5 was
inoculated into 250 mL flasks containing 100 mL NBRIP broth. The flasks were
put on a rotary shaker (180 rpm) for eight days at 28 ± 2°C. At 4 and 8 days
after incubation (DAI) the bacterial culture was drawn from flasks and filtered
through Whatman paper no. 1. The filtered culture was centrifuged at 10000 rpm
for 10 min and available phosphorus was measured using the molybdenum
blue method (Murphy
and Riley 1962).
After mixing with the Murphy-Riley reagent, the samples were kept for thirty
minutes to develop blue color, and absorbance was recorded at 712 nm using a
spectrophotometer (Kowalenko and Babuin 2007).
The experiment was conducted in three replicates. The quantity of phosphorus
solubilized was determined from the standard curve of phosphorus prepared from
the 2 µg/mL solution of KH2PO4.
Indole acetic acid production by KSB under NaCl stress
Indole acetic acid
production by KSB strains was evaluated according to Gordon and Weber (1951). The nutrient broth having L-tryptophan @
0.2 mg/mL (Bharucha et al. 2013) was amended with 3, 5 and 7% NaCl. After adjusting OD
(0.5), bacterial cultures were inoculated into 250 mL flasks containing 100 mL
of nutrient broth. The flasks were placed
on a rotary shaker (180 rpm) for 5 days at 28 ± 2°C. IAA production by
rhizobacteria was recorded using the Salkowski reagent (2% 0.5 M FeCl3 in 35% perchloric
acid) (Ehmann 1977). At the 3rd
and 5th DAI, the samples were drawn to measure IAA quantity. The
culture was centrifuged at 10000 rpm for 15 min. One mL of cell-free supernatant was added with two mL of Salkowski reagent and left
at room temperature in the dark for
twenty minutes (Gordon and Weber 1951) to
develop the color. The IAA produced was measured using a spectrophotometer at
535 nm wavelength. The experiment was conducted in three replicates. The
standard solution of 100 µg/mL indole
acetic acid was prepared in deionized water to calculate the quantity of IAA.
This standard solution was diluted to 5, 10, 15, 20, 25 and 30 µg/mL to get working solutions and
standard curve was drawn.
Siderophores production by KSB under NaCl stress
Siderophores production
was evaluated by Chrome Azurol S (CAS) Shuttle Assay (Schwyn and Neilands 1987). Succinate medium containing (g/L), K2HPO4
(6 g); KH2PO4 (3 g); (NH4)2SO4
(1 g); MgSO4.7H2O (0.2 g); and Succinic acid, 4 g amended
with 3, 5 and 7% and without NaCl. Isolates were grown in a succinate medium
and incubated under shaking conditions (180 rpm) for four days at 28 ± 2°C.
After incubation every 48 h, the bacterial
culture was taken out from the flasks and
centrifuged at 10000 rpm for 10 min. Five hundred microliters of the
supernatant were added to 0.5 mL of the CAS solution. After twenty minutes of
incubation, OD was recorded at 630 nm by using a spectrophotometer. A reference
sample containing 0.5 mL of CAS solution and 0.5 mL of uninoculated succinate
medium was also prepared. The experiment was conducted in three
replicates. The percentage of siderophores units produced by KSB strains was
calculated by using this formula:
%20246-256_files/image002.png){kind=small}
Where, Ar is Absorbance of reference sample and As is
Absorbance of inoculated sample.
Ammonia production by
KSB under NaCl stress
The KSB strains were
grown in peptone water modified with an additional 3, 5 and 7% NaCl (Desale et al. 2014) and without NaCl to determine the ammonia production.
Autoclaved flasks containing 100 mL peptone water were inoculated with 1 mL of
fresh-grown bacterial cultures and placed in a rotary shaker (180 rpm) for 96 h
at 28 ± 2°C (Hansda et al. 2017). Two mL of peptone water with bacterial culture was
drawn and centrifuged at 10000 rpm for 10 min. One mL of the supernatant was
mixed with 1 mL of Nessler's reagent. The optical density was recorded at 450
nm using a spectrophotometer (Goswami et al. 2014). The trial was conducted in three replicates. The
quantity of ammonia was calculated using the standard curve prepared from the
standard solution of ammonium sulfate (Hansda et al. 2017).
ACC deaminase
activity by KSB under NaCl stress
The bacterial strains
were grown in 15 mL tryptic soy broth (TSB) for 24 h. After 24 h, the contents
of the tubes were centrifuged at 8000 × g for 10 min at 4°C. The supernatant
was removed and the pellets were washed with 5 mL DF salts in a minimal medium
without ACC and centrifuged for 10 min. The supernatant was removed and
suspended the cell pellets in 5 mL of 0.1 M
Tris-HCl with a pH of 7.6. Again, centrifuged at 8000×g at 4°C for 10 min and
discarded the supernatant. The washing procedure was repeated twice. The pellet
was suspended in 7.5 mL Dworkin-Foster (DF) salts minimal medium amended with 3, 5 and 7% concentrations of NaCl in a fresh culture
tube and control having no NaCl. As a sole nitrogen source, 45 μL of 0.5 M ACC solution was added to each tube. The inoculated culture was
incubated on a rotary shaker (180 rpm) at 28 ± 2°C for 24 h. After 24 h of
shaking, the tubes have centrifuged the tubes at 8000 × g for 10 min at 4°C.
The supernatant was removed and suspended in the pellet in 5 mL of 0.1 M Tris-HCl (pH 7.6). Again, the tubes
were centrifuged at 8000 × g at 4°C for 10 min and discarded the supernatant.
The washing procedure was repeated twice. The bacterial pellets were
resuspended in 1 mL of 0.1 M Tris-HCl
with pH 8.5 and centrifuged at 13000 rpm for 5 min. The supernatant was
discarded and suspended cell pellets in 600 μL
0.1 M Tris HCl with pH 8.5, then
added 30 μL of toluene and
vortexed for 30 s. Two hundred µL of colonized cells were transferred into a fresh 1.5 mL
centrifuge tube and 20 µL of 0.5 M ACC was added to each tube. After a
brief vortex, the tubes were incubated at 28°C for 15 min. After incubation, 1
mL of 0.56 M HCl was added to the
tubes and the tubes were vortexed and centrifuged for 5 min at 13000 rpm at
room temperature.
One mL of supernatant was transferred to the glass tube
and the supernatant was mixed with 800 µL
of 0.56 M HCl. Furthermore, 300 μL of the 2, 4-dinitrophenylhydrazine
was also poured into the tubes. The tubes were vortexed and then incubated at
28°C for 30 min. After incubation, 2 mL 2 N NaOH was mixed. The absorbance was
measured using a spectrophotometer at 540 nm along with the standard solutions.
The ACC deaminase activity of KSB strains was
calculated from the
standard curve of α-ketobutyrate prepared from 10, 20, 30, 40 and 50 µM solutions. The experiment was
conducted in three replicates.
Exopolysaccharides
production by KSB under NaCl stress
The exopolysaccharides
production by bacterial strains was evaluated using ATCC No. 14 broth (Mu’minah et al. 2015). ATCC No. 14 broth amended with 3, 5 and 7% NaCl and
with NaCl was autoclaved at 121°C for 15 min and cooled at room temperature.
One mL of freshly grown bacterial cultures was inoculated into 250 mL flasks
containing 100 mL of ATCC No. 14 broth. The flasks were put in a rotary shaker
(180 rpm) for eight days at 28 ± 2°C. Three mL of bacterial culture was drawn
at 4 and 8 DAI. The cell culture was centrifuged at 10000 rpm for 20 min. Three
volumes of chilled acetone were mixed with 1 mL of supernatant for the
precipitation of exopolysaccharides. The cell-free supernatant and acetone
mixture was stored at 4°C overnight. The solution was centrifuged at 8000 rpm
for 10 min. The precipitated exopolysaccharides were collected and resuspended
in 1 mL of distilled water. Three volumes of acetone were again used to
reprecipitate the dissolved exopolysaccharides. The dissolved
exopolysaccharides were used to estimate EPS using glucose as a standard by the
phenol-sulfuric acid method (Do et al. 2009). One mL of aqueous phenol and 5 mL of concentrated H2SO4
were added to the test tubes containing 1 mL of exopolysaccharides solution.
After vigorous shaking, they were allowed to stand for 20 min. The absorbance
was recorded at 490 nm wavelength. The exopolysaccharides were calculated from
the standard curve obtained from the standard solution of glucose.
Statistical analysis
The data about
the quantitative determination of PGP properties were subjected to analysis of
variance (ANOVA), following Duncan’s test. The statistical studies were
conducted using IBM SPSS Statistics v. 25. The experiments were conducted in
three replications.
Results
Phosphorus solubilizations by KSB under NaCl stress
With the increased NaCl stress, the quantity of
phosphorus solubilization decreased; however, all four tested strains could
solubilize phosphorus under the highest NaCl stress (7%). On the 4th
and 8th days after incubation, the highest phosphorus (36.42 and
41.92 µg/mL, respectively)
solubilization was noted in 0% NaCl, significantly higher than all NaCl
concentrations (Table 1). Statistically, the lowest phosphorus (9.40 and 10.89 µg/mL, respectively) was measured at 7%
NaCl concentration followed by 5% NaCl stress (16.14 and 19.17 µg/mL, respectively).
At 4th DAI,
the highest phosphorus solubilization was recorded in A. pittii (41.68,
21.96 and 11.73 µg/mL, respectively)
in the medium having 0, 5 and 7% NaCl whereas the O. ciceri solubilized
the highest phosphorus in the medium having 3% NaCl (Table 1). The lowest
phosphorus (28.22 and 16.97 µg/mL,
respectively) solubilization was recorded in R. pusense under treatment
having no and 3% NaCl. With a 5% concentration of NaCl, the lowest phosphorus
(13.35 µg/mL) was recorded in O.
ciceri. At 8th
DAI, A. pittii solubilized the highest phosphorus (45.85, 24.40 and
13.10 µg/mL, respectively) in the
medium having 0, 5 and 7% NaCl whereas O. ciceri solubilized the highest
phosphorus (44.71 µg/mL) in the
medium having 3% NaCl. The R. pusense solubilized the lowest phosphorus
(35.08, 28.10 and 9.11 µg/mL,
respectively) in the medium with 0, 3 and 7% NaCl whereas C. oxalaticus
solubilized the lowest phosphorus (16.12 µg/mL) in the medium with 5% NaCl. The P solubilization by
different KSB strains varied according to the potential. On average, at 4th
and 8th DAI maximum phosphorus (26.57 and 30.18 µg/mL, respectively), solubilization was observed in A. pittii, whereas the lowest phosphorus solubilization was
recorded in R. pusense (16.74 and 22.41 µg/mL, respectively) (Fig. 1).
Indole acetic acid production by KSB under NaCl stress
The salinity stress significantly affected IAA
production by bacterial strains. Regardless of KSB strains, at both 3rd
and 5th DAI the highest IAA (35.38 and 39.09 µg/mL, respectively) was produced under control treatment without
NaCl, whereas the lowest was in 7% NaCl (8.77 and 11.23 µg/mL, respectively) (Table 1).
In the medium having no
NaCl, significantly higher production of IAA was recorded at 3rd and
5th DAI by O. ciceri (78.78 and 85.60 µg/mL, respectively) followed by A. pittii (31.70 and 37.56 µg/mL, respectively) (Table 1). The
lowest IAA production was recorded by the C. oxalaticus KSB strain.
Results with 3% concentrations of NaCl at 3rd and 5th DAI
showed that significantly higher IAA (58.44 and 60.09 μg/mL, respectively) production was recorded in O. ciceri.
In the case of a 5% concentration of NaCl, significantly higher IAA (43.47 and
53.41 μg/mL, respectively)
production was recorded with O. ciceri, followed by A. pittii
(1.67 and 2.93 μg/mL,
respectively). The highest IAA (33.94 and 43.09 μg/mL, respectively) was produced by O. ciceri under 7%
NaCl stress at 3rd and 5th DAI, respectively.
The quantity of IAA
produced by KSB strains varied according to the efficiency of the strains. On
an average basis, O. ciceri produced a significantly higher quantity of %20246-256_files/image004.png)
Fig. 1: Average phosphorus (P) solubilization (µg/mL) by 4 strains of KSB under NaCl stress
(0, 3, 5 and 7%) on the 4th and 8th day
IAA (53.66 and 60.55 µg/mL, respectively), followed by A.
pittii (12.70 and 17.30 µg/mL,
respectively) at 3rd and 5th DAI whereas the lowest IAA
was recorded in C. oxalaticus (1.28 and 1.67 µg/mL, respectively) (Fig. 2).
Table 1: Phosphorus
solubilization and IAA (µg/mL)
production by four KSB strains under different NaCl concentrations
|
|
Strain ID |
NaCl level |
|||||||
|
0% |
3% |
5% |
7% |
||||||
|
4th Day |
8th Day |
4th Day |
8th Day |
4th Day |
8th Day |
4th Day |
8th Day |
||
|
(P) |
A. pittii |
41.68 ± 2.4a |
45.85 ± 0.70a |
30.91 ± 0.69b |
37.37 ± 0.9b |
21.96 ± 1.61a |
24.40 ± 0.7a |
11.73 ± 0.11a |
13.10 ± 0.4a |
|
|
R. pusense |
28.22 ± 1.26b |
35.08 ± 1.6b |
16.97 ± 2.72c |
28.10 ± 0.4c |
14.17 ± 2.41b |
17.36 ± 0.4b |
7.62 ± 1.16b |
9.11 ± 0.9b |
|
|
C. oxalaticus |
37.34 ± 1.58a |
44.09 ± 0.9a |
18.95 ± 0.53c |
36.84 ± 2.0b |
15.10 ± 0.53b |
16.12 ± 1.2b |
7.32 ± 1.48b |
9.11 ± 1.0b |
|
|
O. cicero |
38.44 ± 1.34a |
42.66 ± 1.0a |
37.17 ± 1.09a |
44.71 ± 0.9a |
13.35 ± 0.87b |
18.80 ± 2.1b |
10.96 ± 0.48a |
12.27 ± 0.1a |
|
|
Mean |
36.42 ± 1.67a |
41.92 ± 1.32a |
26.00 ± 2.62b |
36.75 ± 1.72b |
16.14 ± 1.22c |
19.17 ± 1.20c |
9.40 ± 0.69d |
10.89 ± 0.60d |
|
(IAA) |
|
3rd Day |
5th Day |
3rd Day |
5th Day |
3rd Day |
5th Day |
3rd Day |
5th Day |
|
|
A. pittii |
31.70 ± 0.78b |
37.56 ± 0.68b |
16.90 ± 01.14b |
28.14 ± 0.73b |
1.67 ± 0.30b |
2.93 ± 0.67b |
0.54 ± 0.07b |
0.59 ± 0.31b |
|
|
R. pusense |
26.54 ± 0.66c |
28.47 ± 0.58c |
5.31 ± 0.56c |
6.04 ± 0.73c |
0.98 ± 0.03bc |
2.13 ± 0.13b |
0.61 ± 1.02b |
0.92 ± 0.01b |
|
|
C. oxalaticus |
4.51 ± 0.29d |
4.76 ± 0.29d |
0.60 ± 0.03d |
0.98 ± 0.05d |
0.02 ± 0.00c |
0.62 ± 0.02c |
0.00 ± 0.00b |
0.35 ± 0.02b |
|
|
O. cicero |
78.78 ± 0.41a |
85.60 ± 1.55a |
58.44 ± 0.48a |
60.09 ± 0.54a |
43.47 ± 0.68a |
53.41 ± 0.40a |
33.94 ± 0.48a |
43.09 ± 0.19a |
|
|
Mean |
35.38 ± 8.16a |
39.09 ± 8.91a |
20.31 ± 6.88b |
23.81 ± 7.02b |
11.53 ± 5.56c |
14.77 ± 6.73c |
8.77 ± 4.38d |
11.23 ± 5.55d |
Mean ± standard error. Values sharing same letters
differ non-significantly (P > 0.05)
%20246-256_files/image006.png)
Fig. 2: Average Indole Acetic Acid (IAA) production (µg/mL) by 4 strains of KSB under NaCl
stress (0%, 3%, 5%, and 7%) on the 3rd and 5th day
Siderophores production by
KSB under NaCl stress
The number of siderophores increased with NaCl stress up
to 3%; however, further increases in NaCl stress (5 and 7% NaCl) decreased the
quantity of siderophore production. Significantly higher siderophores were
produced in the medium at 2nd and 4th DAI, having 3% NaCl
(48.93 and 71.76%, respectively) followed by 0% NaCl (3.62 and 34.08%,
respectively). The lowest siderophores (8.22 and 15.25%, respectively) were
recorded in the medium with 7% NaCl stress at the 2nd and 4th
DAI.
At 2nd DAI, the
highest siderophores (72.69 and 12.35%) were produced by O. ciceri in
the medium having 0 and 7% NaCl, respectively (Table 2). The highest
siderophores (54.32%) were measured in C. oxalaticus with 3% NaCl stress
and in A. pittii (12.38%) with 5% NaCl stress., The lowest
siderophores (12.44%) were produced by C. oxalaticus in the medium with
0% NaCl and R. pusense (42.46, 4.24 and 3.98%, respectively) in the
medium with 3, 5 and 7% NaCl stress. At 4th DAI, the highest
siderophores (73.43, 79.21, 33.56 and 18.25%) were recorded in O. ciceri
in the medium with 0, 3, 5 and 7% NaCl stress, respectively. The lowest
siderophores (15.52%) were produced by C. oxalaticus in the medium
without NaCl whereas in the medium having 3 5 and 7% NaCl, the lowest
siderophores (60.29, 23.33 and 12.30%, respectively).
The siderophores
produced varied according to the efficacy of potassium solubilizing
rhizobacteria. On an average basis, at the 2nd DAI, O. ciceri
produced the highest quantity (37.62%) of siderophores, followed by A.
pittii (21.25%) (Fig. 3). The lowest siderophores (15.93%) were produced by
R. pusense (19.97%). At 4th DAI, the highest quantity of
siderophores (51.11%) was produced by O. ciceri, followed by A.
pittii (34.63%), which is at par with C. oxalaticus (33.87%). The
lowest siderophores were produced by R. pusense (31.28%).
%20246-256_files/image008.png)
Fig. 3: Average siderophores production (%) by 4 strains of KSB
under NaCl stress (0, 3, 5 and 7%) on the 2nd and 4th day
Table 2: Siderophores (%) and
ammonia (µg/mL) production by four
KSB strains under different NaCl concentrations
|
|
Strain ID |
NaCl level |
|||||||
|
0% |
3% |
5% |
7% |
||||||
|
2nd Day |
4th Day |
2nd Day |
4th Day |
2nd Day |
4th Day |
2nd Day |
4th Day |
||
|
(SID.) |
A. pittii |
17.72 ± 0.89c |
18.34 ± 0.71c |
44.77 ± 0.67b |
72.62 ± 0.75b |
12.38 ± 0.50a |
31.51 ± 0.30b |
10.14 ± 0.63b |
16.04 ± 0.15b |
|
|
R. pusense |
29.22 ± 1.34b |
29.03 ± 1.03b |
42.46 ± 0.63c |
60.29 ± 1.50c |
4.24 ± 0.15c |
23.33 ± 0.54d |
3.98 ± 0.89d |
12.30 ± 0.66d |
|
|
C. oxalaticus |
12.44 ± 0.78d |
15.52 ± 0.44c |
54.32 ± 0.63a |
74.92 ± 1.24b |
6.78 ± 0.38b |
30.63 ± 0.60b |
6.40 ± 0.17c |
14.42 ± 0.32c |
|
|
O. ciceri |
72.69 ± 0.99a |
73.43 ± 1.48a |
54.18 ± 0.49a |
79.21 ± 0.59a |
11.25 ± 0.68a |
33.56 ± 0.95a |
12.35 ± 0.93 |
18.25 ± 0.46a |
|
|
Mean |
33.62 ± 7.15b |
34.08 ± 7.02b |
48.93 ± 1.64a |
71.76 ± 0.59a |
8.66 ± 1.01c |
29.76 ± 1.19c |
8.22 ± 1.02c |
15.25 ± 0.68c |
|
(AMM) |
|
2nd Day |
4th Day |
2nd Day |
4th Day |
2nd Day |
4th Day |
2nd Day |
4th Day |
|
|
A. pittii |
15.99 ± 0.37b |
20.35 ± 0.91a |
17.58 ± 0.18a |
18.03 ± 0.12a |
14.92 ± 0.37a |
15.98 ± 0.19b |
9.63 ± 0.37ab |
10.32 ± 0.11a |
|
|
R. pusense |
18.17 ± 0.37a |
18.36 ± 0.51b |
14.37 ± 0.76c |
17.57 ± 0.78a |
11.02 ± 0.44b |
16.63 ± 0.30a |
8.22 ± 0.37b |
9.60 ± 0.73a |
|
|
C. oxalaticus |
14.89 ± 0.37b |
16.72 ± 0.59c |
14.80 ± 0.71ab |
16.42 ± 0.81ab |
10.51 ± 0.50b |
15.15 ± 0.68ab |
8.66 ± 0.33b |
9.82 ± 0.44a |
|
|
O. ciceri |
18.00 ± 0.33a |
18.63 ± 0.99b |
15.21 ± 020b |
14.52 ± 0.71b |
14.17 ± 0.27a |
13.62 ± 1.05b |
11.65 ± 0.33a |
8.47 ± 0.64a |
|
|
Mean |
16.76 ± 0.33a |
18.51 ± 0.44a |
15.49 ± 0.44b |
16.63 ± 0.49b |
12.65 ± 0.53c |
15.34 ± 0.43c |
9.54 ± 0.48d |
9.55 ± 0.40d |
Mean ± standard error. Values sharing same letters
differ non-significantly (P > 0.05)
%20246-256_files/image010.png)
Fig. 4: Average ammonia production (µg/mL) by 4 strains of KSB under NaCl stress (0, 3, 5 and 7%) on
the 2nd and 4th day
Ammonia production by KSB under
NaCl stress
Regardless of
the KSB strain, an increase in salinity stress reduced ammonia production.
Without NaCl concentration at 2nd and 4th DAI, the
highest ammonia (16.76 and 18.51 µg/mL, respectively) was produced followed by 3% NaCl stress (15.14 and
16.63 µg/mL, respectively) (Table 2).
Conversely at 2nd and 4th DAI, the lowest ammonia (9.54
and 9.55 µg/mL, respectively) was produced at 7% NaCl, followed by 5% NaCl
concentration (12.65 and 15.38 µg/mL, respectively).
At 2nd DAI, R. pusense produced the highest ammonia (18.17 µg/mL) in the medium without NaCl. Whereas the lowest ammonia
(14.89 µg/mL) was produced by C. oxalaticus. The highest ammonia (17.58 and 14.92 µg/mL) was produced by A. pittii in the
medium amended with 3% and 5% NaCl, respectively. At a 7% NaCl concentration, O. ciceri produced the highest ammonia
(11.65 µg/mL); however, all other
four KSB are at par for ammonia production. At 4th DAI, the highest
ammonia (20.35, 18.03 and 10.32 µg/mL)
was produced by A. pittii in the medium amended with 0, 3 and 7% NaCl stress,
respectively. The R.
pusense produced the highest ammonia (16.63 µg/mL) in the medium amended with 5% NaCl. The lowest ammonia was
produced by C. oxalaticus (16.72 µg/mL) in the medium
without NaCl whereas O. ciceri produced the lowest ammonia (14.52,
13.62 and 8.47 µg/mL) in the medium amended with 3, 5 and 7%
NaCl, respectively.
The quantity of ammonia under NaCl varied according to
the efficacy of rhizobacteria (Fig. 4). On average, under all treatments, at 2nd
DAI, the highest ammonia (14.75 µg/mL)
was produced by O. ciceri, followed
by A. pittii (14.53 µg/mL). In
contrast, the lowest ammonia was recorded in C. oxalaticus (12.21 µg/mL).
At the 4th DAI, the highest ammonia (16.17 µg/mL) was produced by Acinetobacter pittii, followed by R. pusense (15.54 µg/mL).
The O. ciceri produced the lowest
ammonia (13.84 µg/mL), followed by C. oxalaticus (14.78 µg/mL).
%20246-256_files/image012.png)
Fig. 5: ACC deaminase activity (µmol α-ketobutyrate/mg/h) by 4 strains of KSB under NaCl
stress (0, 3, 5 and 7%)
Table 3: EPS production (µg/mL) and ACC deaminase activity (µmol α-ketobutyrate/mg/h) by four
KSB strains under different NaCl concentrations
|
(EPS) |
Strain ID |
NaCl level |
|||||||
|
0% |
3% |
5% |
7% |
||||||
|
4th Day |
8th Day |
4th Day |
8th Day |
4th Day |
8th Day |
4th Day |
8th Day |
||
|
|
A. pittii |
130.00 ± 3.21a |
256.66 ± 1.85a |
138.66 ± 3.90a |
266.33 ± 1.85a |
199.66 ± 0.33a |
285.66 ± 0.33a |
116.00 ± 7.50a |
193.66 ± 1.20a |
|
|
R. pusense |
75.66 ± 1.76b |
157.33 ± 6.33b |
111.00 ± 3.05b |
203.00 ± 2.08b |
158.33 ± 5.92b |
192.33 ± 8.19b |
58.66 ± 3.84b |
152.66 ± 7.21b |
|
|
C. oxalaticus |
84.00 ± 2.88b |
86.00 ± 5.13d |
145.00 ± 1.52a |
173.33 ± 3.17c |
194.33 ± 5.66a |
205.33 ± 3.48b |
56.00 ± 3.21b |
75.33 ± 4.91c |
|
|
O. ciceri |
58.66 ± 3.92c |
113.00 ± 0.57c |
96.33 ± 6.96b |
135.33 ± 6.74d |
129.33 ± 6.88c |
273.66 ± 4.09a |
37.00 ± 3.05c |
153.00 ± 6.11b |
|
|
Mean |
87.08 ± 8.06c |
153.25 ± 19.63c |
122.75 ± 6.89b |
194.50 ± 14.54b |
170.41 ± 8.90a |
239.16 ± 12.48a |
66.91 ± 9.13d |
143.66 ± 13.11d |
|
(ACC) |
|
0%NaCl |
3%NaCl |
5%NaCl |
7%NaCl |
||||
|
|
A. pittii |
2.366 ± 0.14a |
2.456 ± 0.11a |
2.123 ± 0.42a |
1.623 ± 0.25a |
||||
|
|
R. pusense |
1.733 ± 0.11b |
1.383 ± 0.01c |
1.060 ± 0.01b |
0.823 ± 0.08b |
||||
|
|
C. oxalaticus |
2.103 ± 0.03a |
1.760 ± 0.05b |
1.070 ± 0.05b |
0.803 ± 0.11b |
||||
|
|
O. ciceri |
2.400 ± 0.11a |
1.886 ± 0.02b |
1.376 ± 0.12b |
1.050 ± 0.13b |
||||
|
|
Mean |
2.151 ± 0.09a |
1.871 ± 0.12b |
1.407 ± 0.16c |
1.074 ± 0.12d |
||||
Mean ± standard error. Values sharing same letters
differ non-significantly (P > 0.05)
ACC deaminase activity by KSB under NaCl stress
The
concentration of NaCl in the DF minimal salt medium significantly affected the
ACC deaminase activity of KSB strains (Table 3). The highest ACC deaminase
activity (2.151 µmol
α-ketobutyrate/mg/h) was noted in the medium having no NaCl followed with
3% NaCl concentration (1.871 µmol
α-ketobutyrate/mg/h). The lowest ACC deaminase activity (1.074 µmol/mg/h) was observed under 7% NaCl
stress.
Without NaCl stress, the highest ACC deaminase activity
(2.400 µmol/mg/h) was observed in O. ciceri whereas the lowest in R.
pusense (1.733 µmol
α-ketobutyrate/mg/h). Under 3, 5 and 7% NaCl the highest ACC deaminase
activity (2.456 µmol
α-ketobutyrate/mg/h, 2.123 µmol
α-ketobutyrate/mg/h and 1.623 µmol α-ketobutyrate/mg/h) was recorded
in Acinetobacter pittii. The lowest ACC deaminase activity
(1.383 µmol α-ketobutyrate/mg/h
and 1.060 µmol
α-ketobutyrate/mg/h) was recorded in R.
pusense in the medium amended with
3 and 5% NaCl whereas in C. oxalaticus (0.803 µmol
α-ketobutyrate/mg/h) under 7% NaCl stress.
The efficacy of ACC deaminase activity varied according
to their potential (Fig. 5). On an average basis under all treatments, the
highest ACC deaminase activity (2.142 µmol
α-ketobutyrate/mg/h) was recorded in A. pittii, followed by O. ciceri (1.678 µmol
α-ketobutyrate/mg/h). The lowest ACC deaminase activity (1.250 µmol α-ketobutyrate/mg/h) was
measured by R. pusense.
Exopolysaccharides production by KSB
under NaCl stress
The
exopolysaccharides production increased to 5% NaCl concentration; however,
further increase in NaCl stress decreased the production of exopolysaccharides.
At 4th and 8th DAI, the highest exopolysaccharides
(170.41 and 239.16 µg/mL,
respectively) were produced at 5% NaCl, followed by 3% NaCl stress (122.75 and
194.54 µg/mL, respectively) (Table
3). The lowest exopolysaccharides (66.91 and 143.66 µg/mL, respectively) were produced in the medium, having 7% NaCl,
followed by the medium with 0% NaCl concentration (87.08 and 153.25 µg/mL, respectively).
At 4th DAI, A. pittii produced the highest exopolysaccharides
(130.00, 199.66 and 116.00 µg/mL) in the
medium amended with 0, 5 and 7% NaCl whereas under 3% NaCl stress, C. oxalaticus produced the highest exopolysaccharides (145.00 µg/mL). when it was inoculated in the
medium having no NaCl. In contrast, O.
ciceri produced the lowest exopolysaccharides (58.66, 96.33, 129.33 and
37.00 µg/mL, respectively) under 0,
3, 5 and 7% NaCl stress. At 8th DAI, A. pittii produced the highest exopolysaccharides
(256.66, 266.33, 285.66 and 193.66 %20246-256_files/image014.png)
Fig. 6: Exopolysaccharides (µg/mL)
production by 4 strains of KSB under NaCl stress (0, 3, 5 and 7%) 4th
and 8th day
g/mL, respectively) in the medium amended with 0, 3, 5
and 7% NaCl. The lowest exopolysaccharides (86.00 and 75.33 µg/mL, respectively) were produced by C. oxalaticus under 0 and 7% NaCl stress. Under 3% NaCl stress, Ochrobactrum ciceri (135.33 µg/mL) produced the lowest exopolysaccharides,
whereas R. pusense produced the
lowest exopolysaccharides (192.33 µg/mL)
in the medium containing 5% NaCl concentration.
The quantity of exopolysaccharides production varied
according to the efficacy of rhizobacteria. On an average basis, at 4th
and 8th DAI, A. pittii
produced the highest exopolysaccharides (146.08 and 250.58 µg/mL, respectively) (Fig. 6). In contrast, the lowest
exopolysaccharides were recorded in O.
ciceri (80.33 µg/mL) at 4th
DAI and in C.
oxalaticus (135.00 µg/mL) at 8th DAI.
Discussion
High salinity
decreases the available phosphorus and saline ions (Ca++, Na+,
Cl- etc.) control phosphorus absorption by plant roots (Beji et al. 2017). Adopting
salt-resistant phosphorus solubilizing rhizobacteria is a successful technique
to increase phosphorus availability and minimize the effects of salinity on
plant growth. All the strains were from the saline rhizosphere, and the
interaction effects between isolates and NaCl stress were substantial. The NaCl
stress in the medium significantly affected the phosphorus solubilization;
however, all the strains could solubilize phosphorus up to 7% NaCl stress.
There was a significant reduction in P solubilization with increasing NaCl
stress; this might be because NaCl stress adversely affects cell growth and
propagation, which causes lesser P solubilization. The A. pittii had the
highest phosphorus solubilization among these strains, followed by O. ciceri. Jiang et al. (2020) isolated 23 phosphorus solubilizing
bacteria, including Bacillus, Acinetobacter, Pseudomonas, Brevibacillus,
Gordonia, Chryseobacterium, Ensifer and Paenibacillus,
from saline soils. All PSB in this study could solubilize tricalcium phosphate
Ca3 (PO4)2, ranging from 65 to 496 mgL−1.
Likewise, Nautiyal et al. (2000)
isolated NBRI0603, NBRI2601, NBRI3246, and NBRI4003 phosphorus solubilizing
strains and were subjected to growth and phosphorus solubilization in the
presence of NaCl (2.5, 5, 7.5 and 10% NaCl). All the strains could solubilize
phosphorus up to 10% NaCl stress. Srinivasan et
al. (2012) isolated 12 PSB from saline soil, which could solubilize
mineral phosphorus up to 2 M NaCl
stress.
Indole acetic acid (IAA) has been considered the most
dominant, physiologically active, naturally occurring auxin, produced in larger
quantities than any other related compounds (Harikrishnan
et al. 2014). IAA is one of the essential auxins which enhances
early root growth. It promotes lateral and adventitious root formation,
enabling the plants to develop more root surface area and absorb more nutrients
from the soil (Chaiharn and Lumyong 2011).
The salt-tolerant PGPR produces IAA, which is essential for root initiation,
cell enlargement, and cell division, which helps plants to manage salt stress (Egamberdieva et al. 2019). In the current
study, the strains could produce IAA under the salinity stress up to 7% NaCl;
however, the production of IAA decreased with the increased NaCl concentration (Dilfuza 2012). The O. ciceri and A. pittii are high IAA-producing strains under
saline conditions. A. pittii was also reported by Afzal et al. (2015) as high
IAA-producing rhizobacteria than other strains.
Most micronutrients, including iron (Fe), are deficient
in saline soils and plant growth is highly reduced (Rabhi et al. 2007; Yousfi et al. 2007). The growth
of plants under saline soils is adversely affected by salinity and deficiency
of Fe simultaneously. Siderophores are low molecular weight metal-chelating
mediators that plants and microorganisms produce in Fe deficient conditions (Crowley et al. 1991). In the present
study, the highest siderophores were produced at 3% NaCl concentration compared
to the siderophores produced in the medium with no stress, which is supported
by Sadeghi et al. (2012). They
also concluded that siderophores production by PGPR isolated from saline soil
increased with NaCl stress up to 300 mM.
The addition of NaCl decreased siderophores production. Argandoña et al. (2010) also reported a lesser level of
siderophore production was noted at increased salt stress. In the present
study, the highest siderophores were produced by O. ciceri under NaCl stress. Príncipe
et al. (2007) also isolated 1 M
NaCl salt resistant Ochrobactrum spp.
from saline soils of Argentina capable of siderophores production. The
application of siderophores-producing rhizobacteria may prove a promising tool
for empowering plants to handle iron deficiency in saline soils (Ferreira et al. 2019).
Ammonia production by PGPR influences plant growth
directly and indirectly. Ammonia production by rhizobacteria directly supports
plant growth by providing nitrogen. It is an essential macronutrient to
synthesize chlorophyll, proteins, enzymes, DNA and RNA (Rodrigues et al. 2016). The application of nitrogen
increases the salinity resistance of plants as nitrogen plays nutritional and
osmotic roles in saline soils (Chen et al.
2010). Ammonia production by rhizobacteria may help the plant for
nitrogen requirements and minimize the root colonization of host plants by
pathogens. In the present study, all the KSB could produce ammonia up to 7%
NaCl stress; however, ammonia production decreased with NaCl stress. The
highest ammonia was produced by O. ciceri
and A. pittii. Sachdev et al.
(2010) isolated the Acinetobacter rhizobacteria from the wheat
field, which exhibited plant growth-promoting properties such as nitrogen
fixation, phosphorus solubilization, ammonia production and siderophores
production.
Under stress conditions, ethylene production increases
due to ion toxicity and osmotic stress (Zhang et
al. 2010; Tavakkoli et al. 2011). Increased ethylene
production causes harmful effects on root growth (Belimov et al. 2009), decreasing overall plant growth due
to water and nutrient restrictions. Previous studies showed that ACC
deaminase-producing PGPR could reduce the destructive effects of ethylene on
root growth by cleaving its direct precursor ACC into ammonia and
α-ketobutyrate (Glick et al. 1998;
Mayak et al. 2004). Ammonia and α-ketobutyrate are used as
sources of nitrogen and carbon by rhizobacteria. The α-ketobutyrate is a
precursor of various amino acids, such as leucine, used in protein biosynthesis
(Glick 2014). In this study, all four KSB
strains could utilize ACC as a nitrogen source under 3, 5 and 7% NaCl stress
conditions and control. The findings are supported by the results of Bal et al. (2013), Zhou et al. (2017) and Nascimento et
al. (2018) and A. pittii had the highest ACC utilization
rate, followed by O. ciceri and C. oxalaticus, whereas the lowest ACC degradation was observed in R. pusense. In increased salinity
stress, the strains showed an expected decrease in ACC degradation, which
causes ethylene accumulation in the soil. The constant high ACC deaminase
activity of A. pittii shows its efficiency for plant growth promotion
for a wide range of adverse conditions that would result in the production of
ethylene (Gulati et al. 2009; Ahmad et
al. 2016). The results of current study are in accordance with the
findings of previous experiments (Nadeem et
al. 2010; Ahmad et al. 2011) regarding ACC-deaminase activity
under salinity stress.
Exopolysaccharide (EPS) is a composite mixture of
macromolecular electrolytes excreted as mucus on the external surface of
bacterial cells. EPS provides a physical fence around plant roots and enhances
plant growth under salinity stress (Vaishnav et
al. 2016). EPS also bind to cations, including Na+ in
saline soils (Geddie and Sutherland 1993),
thus alleviating the salt stress effect. It increases soil aggregation for
nutrients and water uptake, thus resulting in better plant growth under saline
environments (Ashraf et al. 2004).
The number of EPS produced increased with NaCl stress up to 5%; however, it
decreased with a further increase in NaCl stress. The results are supported
with findings of Qurashi and Sabri (2012).
They also reported that EPS production by rhizobacteria increased with the
increase in NaCl stress from 0 to 1 M
NaCl and decreased with further increase in NaCl stress from 1.5 to 2.5 M NaCl. Sandhya
and Ali (2015) also reported that EPS production by PGPR improved with
the increase in NaCl stress up to 1.4 M
concentration. An abundant EPS is produced in hostile environments (Bomfeti et al. 2011; Tewari and Arora 2014).
The highest quantity of EPS was produced by A. pittii under saline
environments. Bechtaoui et al. (2019)
also reported the Acinetobacter spp.
as potential EPS-producing rhizobacteria.
Conclusion
The results of this study revealed that the KSB strains
isolated from saline conditions own significant plant growth-promoting
properties that are assumed to play an essential role in saline soils. These
results demonstrated that A. pittii and O. ciceri could exhibit
excellent plant growth-promoting properties under high saline conditions and
the findings will meaningfully contribute to the pool of knowledge. The
inoculation of the above strains to plants in salinity stress would
considerably enhance plant growth.
Acknowledgements
The authors would like to acknowledge the School of
Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia, for providing
research facilities and a research environment.
Author Contributions
MA planned
Research, HMH and AHAG supervised research.
Conflicts of Interest
The authors declare that they have no conflict of
interests
Data Availability
Data
presented in this study will be available on a fair request to the
corresponding author
Ethics Approval
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