Vol. 12(17), pp.

399-404, 7 May, 2018

DOI: 10.5897/AJMR2018.8822
Article Number: 2E927E057083
ISSN 1996-0808
Copyright © 2018
Author(s) retain the copyright of this article African Journal of Microbiology Research
http://www.academicjournals.org/AJMR

Full Length Research Paper

Effectiveness of exopolysaccharides and biofilm

forming plant growth promoting rhizobacteria on
salinity tolerance of faba bean (Vicia faba L.)
Alaa Fathalla Mohammed
Department of Agric. Botany, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt.
Received 22 February, 2018; Accepted 27 March, 2018

This study aimed to investigate the production of biofilm and exopolysaccharides by plant growth
promoting rhizobacteria (PGPR) under different salt concentrations. In this study, the activity of biofilm
formation and exopolysaccharides production by 20 strains of PGPR which previously isolated and
identified from root samples of different crops were determined under different salt concentrations. Out
of 20 strains, only 12 PGPR strains have the ability to form biofilm at 0.0, 50, 100 and 150 mM NaCl
concentration. PGPR strains with the highest activity of biofilm formation and exopolysaccharides
production were selected to check them on the faba bean plants under different concentration of salt
stress. Inoculation with PGPR strains increased plant growth at higher level of salt concentrations
compared with their corresponding uninoculated ones. The strain Pseudomonas anguilliseptica SAW 24
showed the highest activity of biofilm formation and exopolysaccharides production at different NaCl
concentrations furthermore, gave the highest records of plant height (cm), fresh and dry weight (g/plant)
of faba bean plants. The activities of biofilm formation and exopolysaccharides production of plant
growth promoting bacteria enhance faba bean plants against different salt concentrations.

Key words: Biofilm, faba bean, exopolysaccharides, salinity, plant growth promoting rhizobacteria (PGPR).

INTRODUCTION

Biotic and abiotic stress factors have a major effect on leguminous crop is most salt-sensitive which severely
plants which cause major damages to crop production affected by soil salinity throughout the world, to enhance
around the world (Nemati et al., 2011). Salt stress its yield and productivity using safe biological measures
considers one of the major problems that cause a to improve the soil management and symbiotic
decrease in fertile land productivity. Salinity not only relationships (Sarker and Erskine, 2006; Fernandez-
affects in agriculture but also has other problems that Aunión et al., 2010). Beneficial bacteria that enhance
effect on biodiversity of that environment. Unfortunately, growth promotion and prime stress tolerance of plants

E-mail: [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
400 Afr. J. Microbiol. Res.

have great chance to improve crop production and Table 1. Plant growth promoting rhizobacteria strains.
environmental friendly resource management.
Aggregation of biofilm formation is commonly detected in Code Strain
bacteria. Biofilm is the aggregation of microbial cells that SAW19 Pseudomonas putida SAW19
are irreversibly connected with biotic and abiotic surface SAW1 Pseudomonas mosselii SAW1
and usually enclosed in the self-secreted extracellular SAW7 Pseudomonas entomophila SAW7
polymeric substances. SAC1 Pseudomonas corrugata AC1
Microorganisms within biofilms have a lot of SAW10 Pseudomonas plecoglossicida SAW10
advantages (Wilson, 2005; De Beer and Stoodley, 2006; SAB12 Pseudomonas putida SAB12
Vu et al., 2009; Afrasayab et al., 2010; Nawaz and SAW23 Pseudomonas corrugata SAW23
Ashraf, 2010; Asari, 2015) such as: protect the plant
SAW24 Pseudomonas anguilliseptica SAW24
from external stress, Increase adhesion to surfaces, high
SAW5 Pseudomonas entomophila SAW5
population densities, high tolerance to antimicrobial
agents and higher level of nutritional competition between SAW9 Pseudomonas argentinensis SAW9
microorganisms. SAW22 Pseudomonas putida SAW22
The main objective of this research was to check the SAB15 Pseudomonas palleroniana SAB15
biofilm formation and exopolysaccharides production by SAW15 Pseudomonas plecoglossicida SAB1
PGPR strains at different salt concentration and study the SAB1 Pseudomonas palleroniana SAB17
most efficient PGPR strains in terms of their ability to SAB17 Pseudomonas parafulva SAB14
produce biofilm and exopolysaccharides under salt stress SAB14 Pseudomonas putida SAB12
conditions on faba bean plants. SAB12 Pseudomonas plecoglossicida SAB8
SAB8 Pseudomonas putida SAW3
SAW3 Pseudomonas putida SAW3
MATERIALS AND METHODS
SAB10 Pseudomonas putida SAB10
Microorganisms and growth condition

Selection of the best biofilm forming bacteria was under different The plates were incubated at 28°C for two days. After incubation
salt concentrations (0.0, 50, 100 or 150 mM). 20 strains of plant period, content of each well was gently decanted, then washed with
growth promoting rhizobacteria (PGPR) used in this study are phosphate buffer saline (pH 7) and stained by crystal violet (0.1%).
presented in Table 1. These strains were previously isolated and Stain was removed by washing the wells with distilled water and
identified by Fathalla et al. (2015). dried. To release the crystal violet, 75% ethanol was added to the
The strains were screened for the potential of biofilm formation in wells and OD of stained biofilm was determined by using micro
nutrient broth (NB) which was prepared according to Difco (1985). ELISA auto reader at 570 nm. Experiments for each strain were
The qualitative and quantitative biofilm formation assays were carried out in triplicate and repeated three times.
carried out as follows;

Exopolysaccharide production assay

Qualitative assay for biofilm detection
For exopolysaccharide determination, flasks )250 mL( including 100
A loopfull of bacterial strains were inoculated in 10 ml of NB with mL of a medium proposed by Verhoef et al. (2003) were
different NaCl concentrations (0, 50, 100 and 150 mM) in test supplemented with different NaCl concentrations (0, 50, 100, 150
tubes. The test tubes were incubated at 28°C for two days. Then, mM) and inoculated with 24 h old prepared bacterial culture (1000
the supernatant was thrown away and the test tubes were washed µl) then incubated at 160 rpm shaker for 48 h at 28°C. To extract
with phosphate buffer saline (pH 7). The dried glass tubes were exopolysaccharide, the method of De Vuyst et al. (1998) was used.
stained with 0.1% of crystal violet for 15 min and stain was removed Bacterial cultures were centrifuged at optimized conditions (10000
by washing the tubes with distilled water. Biofilm formation in tubes rpm for 15 min). Supernatant was carefully remove, then wash the
was detected when a visible film lined the wall and the bottom of cells by 50 mM NaCl, centrifuge at 10000 x g for 5 min and
the tube. remove supernatant. Repeat washing four additional times. Re-
suspend cells in 1 ml of 50 mM EDTA, and incubated at 28°C for 60
min, centrifuged at 14,500 x g for 5 min, carefully removed the
Quantitative assay supernatant and transfer to fresh eppendorf tubes.
Exopolysaccharides were quantified in terms of total carbohydrates
The biofilm production was quantitatively assayed according to the and measured by the phenol-sulfuric acid method using glucose as
method described by Arciola et al. (2002). Single colonies of strains a standard (Dubois et al., 1956).
from nutrient agar plates were inoculated in 10 mL of NB in
separate test tubes, with different NaCl concentrations (0, 50, 100,
150 mM) through broth incubated for 2 days at 28°C and diluted (1 Effect of PGPR biofilm on salt tolerance of faba bean growth
in 100) with fresh medium. Individual wells of sterile 96 well flat
bottom polystyrene tissue culture treated plates were filled with 0.2 Healthy seeds of faba bean (Nubaria 3 variety) were obtained from
ml aliquots of the diluted cultures. Negative control wells contained Agricultural Research Center, Giza, Egypt. Faba bean seeds were
sterile broth only. disinfected using 1% sodium hypochlorite solution for 10 min,
 Mohammed 401

Table 2. Physical properties of the could produce biofilm while eight strains could not
experimental soil. produce biofilm. The results showed that the purple ring
appeared under different levels of NaCl concentrations
Particle size distribution (%) (0, 50, 100 and 150 mM).
Sand 97.65
Silt 1.51
Clay 0.84 Biofilm quantitative screening
Textural class Sand
Field capacity % 17.0 Twelve Pseudomonas were assayed for production of
pH 7.80 biofilm. The results indicated that the activity of biofilm
formation was increased with increasing NaCl
concentration. The highest significant increase was
recorded in seven strains treated with 150 mM NaCl.
rinsed thrice in sterile distilled. After surface sterilization, seeds These antagonistic bacteria were strains (SAW 1, SAB
were inoculated with selected bacterial strains. Suspension of 12, SAW 15, SAW 24, SAW 19, SAW 7 and SAW 9).
bacterial cultures (24 h old) was centrifuged for 2 min at 13400 rpm. However, no biofilm ring was formed either in the
Cells were suspended in saline solution (NaCl 1%) to get absence of NaCl in six strains (SAW 24, SAW 1, SAW 5,
bacterial suspension (OD 600 nm adjusted 108 mL-1 CFU).
Sterilized seeds were inoculated for 30 minu before sowing. For
SAW 9, SAW 23 and SAW 19).
control, seeds were steeped in sterile water for the same period of The results in Figure 1 showed the values of
time. The PGPR populations were detected by adding suitable absorbance at 570 nm as a measure of optical density
amounts of saline solution to obtain 108 CFU / 200 ml. These (OD) which reflected the activity of biofilm formation of
dilutions served as bacterial inoculum for plant experiment, 2.8 kg 12 strains in nutrient broth cultures supplemented with
of winnowed, sterilized and air dried soil was thoroughly mixed with four concentrations of NaCl (0, 50, 100 and 150 mM).
200 ml diluted bacterial suspension for 2 min. The characters and
composition of soil are presented in Table 2.
Seeds were sown in plastic pots for 45 days and irrigated
regularly by different concentrations of NaCl (0, 50,100 and 150 Exopolysaccharide production under different salt
mM) per gram weight of soil. After 45 days, the seedlings were concentrations
harvested and different growth parameters that is, plant length
(cm), dry weight (g/plant) and fresh weight (g/plant) were measured.
Fxopolysaccharides (EPS) production of PGPR strains
under different NaCl concentrations (0, 50, 100 and 150
Statistical analysis mM) was tested. The results showed that there was a
general direction of gradual increase in EPS with
Collected data were statistically analyzed using the appropriate increasing salt concentrations (Figure 1). However, at no
analysis of variance according to Steel and Torrie (1981). The salt stress or low concentration of NaCl
experiment date designated in two ways was completely
randomized with three replicates. Computer program software
exopolysaccharide production was reduced (Figure 2).
CoStat version 6.311 was used to analyses the data of experiment.
Least significant difference (LSD) at 5% level was used separately
to evaluate the response of each character. Effect of inoculation with selected PGPR on plant
growth of faba bean

RESULTS The effect of inoculation with selected PGPR (SAW 1,

SAW 24 and SAW 19) on growth of faba bean at four salt
Out of 20 strains, 12 strains had the ability to produce concentrations (0, 50, 100 and 150 mM) was studied.
biofilm. Biofilm producing strains were confirmed by The results in Figure 3 showed that the increasing salt
various methods. In the current study, qualitative and stress affect plant height, shoot fresh weight and shoot
quantitative estimation of biofilm production by PGPR dry weight. Also, results indicated that the increasing of
strains was performed. salinity stress was significantly reduced all growth
parameters of non-inoculated plants. Results denoted
that in non-inoculated plants, there was a little bit impact
Biofilm qualitative screening on NaCl stress up to 50 mM. The effect of inoculation on
dry masses of the salinity stress on faba bean is
Obtained results observed that thick film was formed represented in Figure 3.
inside the wall and bottom of the tube. Twelve strains PGPR inoculated plants showed higher accumulation
(SAW 7, SAC1, SAW 10, SAB12, SAW 23, SAW 24, SAB of shoot dry mass than their corresponding uninoculated
8, SAW 19, SAW 5, SAW 9, SAW 15 and SAW 1) had ones under salt stress. However, the inoculation with
thick film inside the wall of the tube denoting that strains SAW 24, SAW 1 and SAW 19 gave the highest value in
402 Afr. J. Microbiol. Res.

0 50 100 150

absorbance at 570 nm (OD)

0.8

0.6

0.4

0.2

Figure 1. The optical density (OD) (at 570 nm) as a measure of the activity of biofilm
formation of 12 strains in nutrient broth cultures supplemented with four concentrations of
NaCl (0.0, 50, 100 and 150 mM).

0 50 100 150

200
Glucose ( mg 100 ml-1 culture)

150

100

50

Figure 2. Effect of different concentrations of NaCl (0.0, 50, 100 and 150 mM) on glucose
content (mg 100 ml-1 culture) of bacterial strains in EPS media.

shoot fresh mass with 7.2, 6.1 and 4.8 g, respectively DISCUSSION
comparing to 3.6 g in the uninoculated stressed treatment
at 100 mM NaCl. The results show that increasing NaCl concentration
At 100 mM NaCl, the inoculation with SAW 24, SAW1 leads to the increase in exopolysaccharide production.
and SAW 19 PGPR strains enhanced the plant height by Moreover, increasing the production of
43, 40 and 36%, respectively compared to their exopolysaccharide against higher salt stress leads to
uninoculated plants. The PGPR SAW 24 and SAW1 support biofilm formation (Ishii et al., 2004; Fujishige et
strains enhanced the accumulation of dry mass of shoots al., 2006). These results are in agreement with those
by 47 and 42%, respectively at 100 mM NaCl. Maximum obtained by Arora et al. (2010), Qurashi and Sabri
increase under stress in the plant height, fresh weight (2012), Deng et al. (2015) and Kasim et al. (2016).
and dry weight of shoot was observed at 50 mM NaCl It was reported that formation of biofilm and
stress. Depending on obtained results of the present exopolysaccharide kept the viability of bacterial cells
experiment, the results showed that the strain SAW 24 under salt stress to protect them in the rhizosphere.
showed the highest activity of biofilm formation under the Previous research showed that biofilm formation and
different levels of NaCl concentrations. Also, the same exopolysaccharide production by PGPR strains
strain Pseudomonas anguilliseptica SAW 24 showed the significantly increase soil fertility and enhance plant
highest growth measurements of faba bean plants under growth (Ashraf et al., 2005; Liaqat et al., 2009).
salt stress. The present results are similar with several previous
 Mohammed 403

LSD between strains (str) =0.45

70 LSD between salinity (sal) =0.58 control
LSD between str*sal=1.16

plant height (cm/plant)

60
50 SAW19

40
SAW1
30
20 SAW24
10
0
0 50 100 150
10 LSD between strains (str) =0.35
LSD between salinity (sal) =0.47
9
shoot fresh weight (g/plant)

LSD between str*sal=0.71

8
7
control
6
5 SAW19
4 SAW1
3
SAW24
2
1
0
0 50 100 150

1.2 LSD between strains (str) =0.014

LSD between salinity (sal) =0.018
shoot dry weight ( g/plant)

1 LSD between str*sal=0.029

0.8 control

0.6 SAW19
SAW1
0.4
SAW24
0.2

0
0 50 100 150

Figure 3. Effect of the three selected PGPR strains biofilm forming and
exopolysaccharides producing on plant height, shoot fresh weight and shoot
Fig
dry weight of faba bean under different salt concentrations.

reports that, salinity stress significantly reduced under different salt stress. Also, gave the best records of
leguminous crops (Soussi et al., 1998; Chookhampaeng, plant height, fresh and dry weight of faba bean plants.
2011). However, inoculation with PGPR encouraged the
plant growth of Leucaena esculenta with and without salt
stress by releasing phytohormones which lead to CONFLICT OF INTERESTS
increase bacterial root colonization and biofilm formation
(Lugtenberg and Kamilova, 2009; Arora et al., 2010; The authors have not declared any conflict of interests.
Ahmed and Shahida, 2010).
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