Environmental Sustainability

, Volume 1, Issue 1, pp 49–59 | Cite as

Role of salicylic acid from Pseudomonas aeruginosa PF23EPS+ in growth promotion of sunflower in saline soils infested with phytopathogen Macrophomina phaseolina

  • Sakshi Tewari
  • Naveen Kumar AroraEmail author
Original Article


Exopolysaccharides (EPS) producing Pseudomonas aeruginosa PF23EPS+ and its mutant PF23EPS− (EPS deficient strain) taken from authors collection were monitored for salicylic acid (SA) production. Strain PF23EPS+ displayed SA production and biocontrol against phytopathogen Macrophomina phaseolina up to 500 mM NaCl. However, mutant was defective in EPS production and displayed significant reduction in SA production under non-stress conditions. The mutant neither displayed SA production nor biocontrol against phytopathogen at diverse salt concentrations, authenticating the relation of SA in antagonism. Pot and field trials were conducted in salinized soil infested with M. phaseolina taking sunflower as test crop. Results of field study were in agreement with the findings of pot study. The combination of strain PF23EPS+ and extracted SA obtained from it brought significant enhancement in plant growth parameters and reduction of disease incidence under saline conditions. The study reports the application of plant growth promoting (PGP) microbe P. aeruginosa PF23 and its metabolite (SA) for enhancing growth of sunflower crop in salinized soil infested with M. phaseolina.


Biocontrol Exopolysaccharides Macrophomina phaseolina Salinity Salicylic acid Sunflower 


Development and productivity of various crops are substantially affected by soil salinity throughout the globe (Kaya et al. 2002; Prıncipe et al. 2007). More than 900 million hectares of land is suffering from the constraints of salinization stress (Flowers 2004). If remedial actions are not taken, situation is going to get worse and may affect 50% of the land area by 2050 (Munns 2002; Arora et al. 2012; Arora 2015).

Salinity can affect growth of various oil producing crops and sunflower (Helianthus annuus) is one of them. All the developmental stages of sunflower are badly affected due to soil salinity (Flagella et al. 2004). Salinity can decline the growth of sunflower plants due to various effects such as, loss of water and nutrient uptake, impact on physiological and biochemical processes, and ionic imbalance. Other effects can be disruption in enzymatic activities, resulting in impact on anabolic processes, photosynthetic activity and transportation of metabolites that cause reduction in grain yield (Parvaiz and Satyawati 2008).

Sunflower is the major oilseed crop cultivated around the world. Sunflower oil is widely used around the globe because it is rich in several health benefits, while the seeds are used as animal feed (Tewari and Arora 2014a, b). At present Ukraine is the leading producer of sunflower and India ranks tenth in world (United States Department of Agriculture 2015; Tewari and Arora 2016). The area and production of sunflower in India has started declining consistently due to several abiotic and biotic stressors (Khan 2007; Khan and Panda 2008; Paul and Nair 2008; Mostafavi and Heidarian 2012). Amongst abiotic factors salinity is the major constraint that limits plant growth, whereas in biotic factors destruction caused by dreadful phytopathogens such as Macrophomina phaseolina, needs to be tackled.

M. phaseolina is a fungal opportunist that likes to take advantage of stressed sunflower plant which is cultivated in salinized regions and causes significant reduction in oil production (Ullah et al. 2011). The pathogen has a wide host range and causes destruction in more than 500 plant species (Khan et al. 2017). As the growth of M. phaseolina under salinized regions has been on the rise every year, this is a matter of serious concern, but till now no reliable remedy has been developed (Kolte 2018).

Recent research on beneficial microorganisms such as plant growth promoting rhizobacteria (PGPR) has unraveled the options not only to mitigate salinity stress but also to combat growth of dreadful pytopathogens such as M. phaseolina (Tewari and Arora 2014a, 2016). Development of bio-products utilizing helpful PGPR or their metabolites to enhance productivity of crops under stress conditions can provide the solution for this problem (Joshi et al. 2006; Arora et al. 2016; Arora and Mishra 2016).

PGP metabolites, exopolysaccharides (EPS) and salicylic acid (SA) are known to impart stress tolerance but relatively little attention has been paid on their role and utilization under saline conditions (Upadhyay et al. 2011). Combination of PGPR and microbial product(s) for enhancing plant growth particularly under salinized field conditions infested with phytopathogens is a novel approach (Arora and Mishra 2016). In the present study, saline tolerant, EPS producing fluorescent Pseudomonas PF23EPS+ and its mutant taken from author’s collection (Tewari and Arora 2014a) were monitored for SA production. Authors already have reported the role of EPS produced by PF23EPS+ in plant growth promotion and biocontrol (Tewari and Arora 2014a). However, in the present study it was further noticed that the bacterial strain PF23EPS+ was also a very good producer of SA and maybe there was a correlation between the two metabolites production under saline conditions. With this being the objective biocontrol potential of extracted SA (from PF23EPS+) was checked against phytopathogen, M. phaseolina. Field trials were conducted in the salinized soil taking sunflower as test crop taking a set of diverse treatments. The treatments included combination of EPS producing PF23EPS+ along with SA (singly or in combination) so as to elucidate the role of metabolite in combination with the PGPR in plant growth promotion and disease management under saline conditions.

Materials and methods

Microorganisms and culture conditions

EPS producing strain P. aeruginosa PF23EPS+ and its mutant PF23EPS− displaying biocontrol against M. phaseolina were routinely grown on Davis minimal medium (DMM) (g/l) supplemented with trace element solution as described by Tewari and Arora (2014a). Strains were grown at 28 °C and maintained on DMM agar slants at 4 °C.

Phytopathogen M. phaseolina ARIFCC257 causing charcoal rot was obtained from Agharkar Research Institute, Pune, India. For subsequent experiments the strain was grown at 28 °C and preserved on potato dextrose agar (PDA) at 4 °C for further use (Khare et al. 2011).

SA estimation

The qualitative and quantitative estimation of SA production by the wild strain PF23EPS+ and its mutant PF23EPS− was done according to the method described by Meyer and Hofte (1997). Qualitative analysis of the culture supernatant for SA was performed by Thin Layer Chromatography (TLC) after ethyl acetate extraction. Briefly, cells of bacterial strains were removed from 7 days old culture grown in casamino (CAS) acids medium (at 0–600 mM NaCl concentrations) by centrifugation at 7500 rpm for 15 min at 30 °C. Supernatant was adjusted to pH 2.5 by 0.1 N HCl, and further extracted with ethyl acetate in 1:1 ratio. Extracted SA was vacuum dried and solubilized in minimum amount of methanol, before spotting on pre-coated silica gel plates (Silica gel 60F 254; Merck). The solvent system used to develop chromatograms consisted of chloroform: acetic acid: ethanol in the ratio 95: 5: 2.5 (v/v). The plates were observed under UV light (256 nm) and Rf value of the isolated SA was compared to that of the standard SA (Merck India).

To quantify SA production, ethyl acetate extract was concentrated (1:3) under vacuum. SA concentration was determined by adding 5 µl of 2 M FeCl3 and 3 ml of water to 1 ml of concentrated extract (Meyer and Hofte 1997). The absorbance of iron-SA complex was measured at 527 nm and compared with standard curve of SA dissolved in ethyl acetate. The amount of SA in culture filtrate was measured in mg/ml.

HPLC analysis of SA

Standard SA and extracted SA obtained from PF23EPS+ (at different salinity concentrations from 0 to 600 mM NaCl) and and PF23EPS− (0 mM NaCl) was analyzed by HPLC (Perkin Elmer, Model Flexar LC). 10 μl of SA (at different salinity) was injected into carbonyl C18 column (50 × 2.5 mm) having particle size 3.5 μm and run under isocratic conditions for a run time of 20 min. Acetonitrile, water and orthophosphoric acid 80: 19.9:0.1(v/v) were used as mobile phase with flow rate of 1 ml per min and wavelength set at 310 nm. Retention time (RT) of SA was matched with that of standard SA, and HPLC chromatograms were compared (Toiu et al. 2011).

Antagonistic activity of SA against M. phaseolina

The antifungal activity (against M. phaseolina) of SA obtained from PF23EPS+ was checked. To check the antagonistic activity against phytopathogen 500 μl spore suspension of M. phaseolina (in 0.85% saline; OD 610 = 1.0) and 100 μl SA (6.12 mg ml-1) obtained from PF23EPS+ at 100 mM NaCl concentration was added in 50 ml of CAS broth, and incubated at 28 °C for 7 days. Similarly, antagonistic activity of SA obtained from PF23 EPS+ (200–600 mM NaCl) was checked against pathogen. CAS broth containing 500 μl of spore suspension without SA and no salt amendment (0 mM NaCl) served as control. Both treated (amended with SA) and control sets (without SA) were incubated for a week and mycelia of M. phaseolina were filtered, and oven dried. Inhibition percentage was determined according to Khare et al. (2011).

Post interaction abnormalities in fungal mycelia

Post interaction abnormalities were identified by taking fungal mycelia from the flask treated with SA (0–500 mM NaCl) and observed under scanning electron microscopy (SEM) as described by Weidenborner et al. (1989). Fungal mycelia after placing on cover glass were flooded with osmium tetraoxide (2%) for 24 h at 20 °C. The samples were transferred to copper stubs over double adhesive tape and coated with gold so as to be observed by SEM (JEOL-JSM-6490 LV) at 20 kV. Mycelia from control flask (without SA treatment at 0 mM NaCl) were also analyzed.

In vivo pot study

In vivo trials were conducted in polypropylene pots (24 × 12 × 12 cm) during the months of March-June for two consecutive years (2012 and 2013). Fungal propagules were obtained following the methodology of Khare et al. (2011). Briefly, fungi for inoculation were grown on oat grains (Avena sativa) before adding to the sterilized sandy loam soil (pH 8.0) to achieve population size of 104 propagules/gm of soil before seed sowing (05 seeds/pot). Sunflower seeds were surface sterilized and subsequently dipped in cell suspensions of PF23EPS+ and PF23EPS− (at OD 610 = 0.1) for 10 min and mixed with 1% carboxy methyl cellulose (CMC), followed by overnight air drying (Upadhyay et al. 2011). Seed pelleting was also done with SA obtained from PF23EPS+ and standard SA according to Mehran et al. (2013). The experiment was conducted with following treatments taking five replicates of each set: (1) untreated seeds (control), (2) PF23EPS+, (3) PF23EPS−, (4) M. phaseolina, (5) PF23EPS+ + M. phaseolina, (6) PF23EPS− + M. phaseolina, (7) extracted SA (6.12 mg/ml), (8) extracted SA + M. phaseolina, (9) extracted SA + PF23EPS+, (10) extracted SA + PF23EPS+ + M. phaseolina, (11) extracted SA + PF23 EPS−, (12) extracted SA + PF23 EPS− + M. phaseolina, (13) standard SA (6.12 mg/ml) and (14) standard SA + M. phaseolina. The experiment was set both under normal (only water) and saline (irrigated with 125 mM NaCl solution) conditions (Prıncipe et al. 2007). Plant growth parameters including root length, shoot length, dry weight, head diameter and seed yield were calculated according to Maheshwari et al. (2012). Disease severity index (DSI) was calculated by checking the presence of dark brown lesions on root systems. Percentage disease incidence was calculated as number of diseased plants out of the total number of plants.

Field trials

The field experiment was carried out in the semiarid regions of Kanpur Dehat, (location 26°20ʹ39.48ʹʹN and 79°58ʹ1.85ʹʹE) (soil having electrical conductivity 10 dS/m, pH 8.1), naturally infested with M. phaseolina (103 CFU/g soil) during the month of March to June for two consecutive years (2012 and 2013). The field experiments were conducted in randomized block design with five replicas of each sets in a plot size of 100 m2 (10 m × 10 m), where each block was 1.5 m × 1.5 m, with following sets of treatment (1) untreated seeds (control), (2) PF23EPS+, (3) PF23 EPS−, (4) extracted SA (6.12 mg/ml) (from PF23EPS+), (5) standard SA (6.12 mg/ml), (6) extracted SA + PF23EPS+ and (7) extracted SA + PF23EPS−. The distance between each block was 0.20 m and seed sowing was done at 30 cm row-width. Irrigation of plots was done according to the need. Seed germination (%) was recorded on the 15th day after sowing (DAS). Vegetative growth parameters including root length, shoot length, dry weight, head diameter, stem width, root adhering soil/root tissue (RAS/RT) ratio and seed yield were recorded on 120 DAS (on harvest of crop).

Statistical analysis

The data generated was checked by analysis of variance (ANOVA), and Duncan’s Multiplicity Test Range (DMRT) using the SPSS software (ver. 10.1, SPSS Inc., The significance level for all analysis was at P = 0.05.


SA production

Qualitative and quantitative analysis suggested that the EPS producing strain PF23EPS+ displayed SA production up to 500 mM NaCl. Blue bands on silica plates confirmed SA production by the strains. The blue band in the samples co-migrated with standard SA, displaying same fluorescence. The Rf value of SA produced by the PF23EPS+ strain at different NaCl concentrations was similar to the Rf value (0.61) of the standard SA. Thickness of the band obtained at 0 and 100 mM NaCl showed exact resemblance with control SA, however, further increase in salinity resulted in thinner bands (Fig. 1) and no SA production was observed beyond 500 mM by PF23EPS+. EPS defective mutant PF23EPS− demonstrated very bleached band of SA at 0 mM NaCl and further increase in salinity brought complete loss in SA production.
Fig. 1

Thin layer chromatograph showing SA production by PF23EPS+ and its mutant under different NaCl concentrations

Under control conditions (at 0 mM NaCl) bacterial strain PF23EPS+ produced 6.14 mg/ml of SA, which was significantly similar to the SA obtained at 100 mM salinity (6.12 mg/ml). However, there was 16.2, 70.5, 90 and 99.2% reduction in SA production with progressive increase in salinity levels to 200, 300, 400 and 500 mM NaCl, respectively (Table 1). EPS defective mutant PF23EPS− demonstrated very less SA production of about 0.03 mg/ml at 0 mM NaCl which diminished further by increasing salinity.
Table 1

Effect of salinity on SA (mg/ml) production and antagonistic activity against M. phaseolina

NaCl (mM)

SA (mg/ml) produced by PF23EPS+

% of Antagonism


6.14 ± 0.08e

75.12 ± 0.01e


6.12 ± 0.04e

73.40 ± 0.02e


5.28 ± 0.02d

60.31 ± 0.03d


3.60 ± 0.06c

50.82 ± 0.04c


1.11 ± 0.07b

39.10 ± 0.06b


0.05 ± 0.03a

19.01 ± 0.08a


Results are the mean ± SD (n = 5). Means in the columns following dissimilar letters designate significant difference (P = 0.05) by DMRT. Five samples were analyzed for each replicates

HPLC analysis of SA

HPLC analysis of standard SA displayed single peak at retention time of 4.502 min. Extracted SA from PF23EPS+ at 100 mM, 200 mM, 300 mM, 400 mM NaCl and 500 mM NaCl concentrations also showed nearly similar retention time of 4.511, 4.523, 4.519, 4.521 and 4.532 respectively. SA from PF23EPS+ at 0 mM NaCl also showed strong peak at 4.523 min (Fig. 2), however, no peak was observed at 600 mM NaCl. In case of mutant PF23EPS− no peak was observed under non saline or saline conditions.
Fig. 2

HPLC chromatograms of SA. a Standard SA. b Extracted SA at 0 mM NaCl

Antagonistic activity of SA against M. phaseolina

SA obtained from strain PF23EPS+ at 100, 200, 300, 400 and 500 mM NaCl inhibited M. phasolina by 73.40, 60.31, 50.82, 39.10, and 19.01% respectively, in comparison to control (Table 1). SA acquired from PF23EPS+ at 0 mM NaCl displayed strong antagonistic activity against M. phaseolina (75.02% in comparison to control). Whereas, no antagonism was shown by the mutant strain at 0 mM NaCl or with increase in salinity.

Post interaction abnormalities in fungal mycelium

Post interaction abnormalities in fungal hyphae (due to SA) were visible in the form of hyphal deformities (up to 500 mM NaCl concentration). Twisting of fungal cell wall along with shriveling and curling of hyphae is also clear from the photographs (Fig. 3). Deformities were absent in control (untreated) hyphae (Fig. 3d).
Fig. 3

Post interaction morphological changes in M. phaseolina due to extracted SA at 500 X. Deformities in fungal hyphae a twisting and curling (at 100 mM NaCl), b twisting and curling (at 300 mM NaCl), c shriveling and breakage (at 400 mM NaCl), df twisting, curling, breakage and leakage (at 500 mM NaCl), g healthy hyphae from control (without SA at 0 mM NaCl)

In vivo pot study

Best results were observed when amalgamation of PF23EPS+ along with SA (obtained from PF23EPS+) was coated on sunflower. There was significant increase in plant growth parameters observed both under saline and non-saline sets in presence or absence of pathogen when amalgam of extracted SA and PF23EPS+ was dressed on seeds in comparison to individual pelleting of SA or PF23EPS+. Seed dressing with SA along with PF23EPS+ brought enhancement in root length, shoot length, dry weight and seed yield by 116.6, 44.44, 135.8 and 100% respectively, in comparison to unbacterized seeds under saline conditions (Table 2).
Table 2

Plant growth attributes of sunflower in presence and absence of pathogen, under saline and non-saline conditions (pot study)

Non saline conditions (control)

Saline stress (125 mM)


Root length (cm)

Shoot length (cm)

Dry weight (g)

Head diameter (cm)

Seed yield/pot (g/pot)

Root length (cm)

Shoot length (cm)

Dry weight (g)

Head diameter (cm)

Seed yield/pot (g/pot)

Untreated seeds (control)

08.00 ± 0.02b

50.3 ± 0.09b

01.51 ± 0.08b

06.32 ± 0.01b

10.50 ± 0.02b

06.31 ± 0.08b

45.1 ± 0.09b

01.31 ± 0.01b

05.01 ± 0.03b

09.00 ± 0.07b


10.95 ± 0.04d

62.1 ± 0.08d

02.39 ± 0.06d

08.10 ± 0.02d

16.10 ± 0.08d

09.24 ± 0.02c

59.80 ± 0.03c

02.18 ± 0.04d

07.45 ± 0.05d

13.50 ± 0.06d


08.50 ± 0.08b

55.6 ± 0.07b

01.58 ± 0.07b

06.71 ± 0.03b

11.70 ± 0.06b

06.32 ± 0.07b

46.9 ± 0.04b

01.34 ± 0.02b

05.12 ± 0.03b

09.30 ± 0.02b

M. phaseolina

06.17 ± 0.05a

35.9 ± 0.06a

0.89 ± 0.09a

05.10 ± 0.06a

07.11 ± 0.02a

05.22 ± 0.04a

32.6 ± 0.08a

0.68 ± 0.06a

04.20 ± 0.04a

05.00 ± 0.03a

PF23EPS+ + M. phaseolina

09.94 ± 0.07c

59.9 ± 0.05c

02.01 ± 0.03c

07.50 ± 0.08c

13.70 ± 0.03c

08.54 ± 0.08c

56.2 ± 0.05c

01.82 ± 0.07c

07.01 ± 0.09c

11.90 ± 0.02c

PF23EPS− + M. phaseolina

08.30 ± 0.06b

53.1 ± 0.04b

1.53 ± 0.04b

06.50 ± 0.09b

11.30 ± 0.06b

05.32 ± 0.06a

33.7 ± 0.05a

0.72 ± 0.04a

04.50 ± 0.01a

05.30 ± 0.02a

Extracted SA

12.21 ± 0.02f

64.27 ± 0.02f

03.04 ± 0.05f

10.65 ± 0.02f

18.21 ± 0.01f

11.76 ± 0.04e

62.01 ± 0.03d

02.39 ± 0.07e

08.02 ± 0.08f

16.09 ± 0.05f

Extracted SA +M. phaseolina

11.10 ± 0.07e

60.02 ± 0.01e

02.02 ± 0.07e

09.13 ± 0.03e

15.23 ± 0.08e

10.49 ± 0.06d

59.24 ± 0.08e

01.76 ± 0.08f

07.48 ± 0.04e

14.08 ± 0.08e

Extracted SA + PF23EPS+

14.56 ± 0.05 g

66.34 ± 0.07 g

3.82 ± 0.08 g

12.56 ± 0.09 g

19.67 ± 0.04 g

13.67 ± 0.09 g

64.75 ± 0.01 g

3.09 ± 0.07 g

10.30 ± 0.09 g

17.59 ± 0.07 g

ExtractedSA + PF23EPS++M. phaseolina

14.01 ± 0.04 g

65.51 ± 0.06 g

3.20 ± 0.01 g

12.09 ± 0.02 g

19.02 ± 0.06 g

13.12 ± 0.08 g

64.01 ± 0.08 g

2.81 ± 0.07 g

10.01 ± 0.07 g

17.01 ± 0.07 g

Extracted SA + PF23EPS−

11.98 ± 0.02f

63.01 ± 0.02f

02.84 ± 0.05f

10.51 ± 0.02f

7.99 ± 0.01f

11.21 ± 0.04e

61.01 ± 0.03d

02.19 ± 0.07e

07.98 ± 0.08f

16.00 ± 0.05f

Extracted SA + PF23EPS−+M.phaseolina

11.00 ± 0.07e

60.03 ± 0.02f

01.97 ± 0.07e

09.01 ± 0.03e

15.02 ± 0.08e

10.21 ± 0.06d

59.01 ± 0.08e

01.71 ± 0.08f

07.31 ± 0.04e

14.00 ± 0.08e

Standard SA

12.54 ± 0.08f

64.42 ± 0.05f

03.12 ± 0.09f

10.82 ± 0.01f

18.43 ± 0.07f

11.91 ± 0.09e

59.45 ± 0.02e

02.48 ± 0.09e

08.76 ± 0.03f

16.19 ± 0.03f

Standard SA + M. phaseolina

11.23 ± 0.09e

60.51 ± 0.07e

02.08 ± 0.01e

09.22 ± 0.02e

15.54 ± 0.08e

10.98 ± 0.08d

62.18 ± 0.05d

01.79 ± 0.01f

07.84 ± 0.08e

14.11 ± 0.04e

Data are mean of 2 years. Results are mean ± SD (n = 10). Means in the columns following dissimilar letters designate significant difference (P = 0.05) by DMRT. Five samples were analyzed for each replication

Seed coating with PF23EPS+ cells brought 53.3% and 50% enhancement in seed yield under non-saline and saline conditions respectively, in comparison to unbacterized seeds. However, extracted SA from same strain brought enhancement in seed yield by 73.4% and 78.7%, under non-saline and saline conditions respectively, in comparison to untreated seeds. PF23EPS− displayed significantly similar results as obtained by control (untreated seeds), both under saline and non-saline conditions. Seed pelleting with extracted SA showed significantly similar enhancement in plant growth attributes in comparison to the set receiving treatment of standard SA, under both conditions, in infested as well as non-infested sets (Table 2).

M. phaseolina infested seeds caused 81 and 79% incidence of disease in saline and non-saline conditions respectively. Treatment of seeds with PF23EPS+ resulted in 71 and 63% reduction of disease incidence in comparison with non-bacterized seeds under non-saline and saline conditions respectively. Whereas seed pelleting with extracted SA brought 67 and 75% reduction in disease incidence under saline and non- saline conditions, which was significantly similar to the sets receiving treatment of standard SA. Combination of PF23EPS+ along with SA displayed best results in respect of suppressing charcoal rot incidence in sunflower. The combination showed 77 and 69% reduction of disease incidence in sunflower under non-saline and saline conditions respectively. However, EPS-defective strain could only bring reduction of disease incidence by 17% (saline) and 23% (non-saline).

Seed pelleting with PF23EPS+ resulted in 126 and 160% increment in seed yield under non-salinized and salinized conditions respectively, in comparison to M. phaseolina infested seeds. Extracted SA, in presence of pathogen, enhanced seed yield by 129 and 180% when compared to negative control under non-saline and saline environments, respectively. The combination of SA and PF23EPS+ (in presence of pathogen) brought enhancement in seed yield by 167.5 and 240% in comparison to negative control in non-saline and saline soil respectively. Increment of plant growth parameters including seed yield by extracted SA treatment was significantly similar to the set receiving treatment with standard SA. However, the mutant was ineffective in controlling the pathogen under saline conditions. EPS defective strain PF23EPS− was also ineffective in suppressing disease incidence under saline conditions.

Field trials

Results of the field study highlighted that seeds sown in saline soil naturally infested with M. phaseolina showed 74% disease incidence. Seed dressing with PF23EPS+ suppressed disease incidence by 58.7%, and SA obtained from the same strain caused 64.2% reduction of charcoal rot disease, however, the combination of PF23EPS+ along with SA brought 70.2% reduction in disease incidence. Combination of PF23EPS+ along with extracted SA gave best results in enhancing plant growth parameters and suppressing the incidence of disease while unimpressive results were obtained when seed pelleting was done with the mutant strain. Amalgamation of PF23EPS+ and SA brought significant enhancement in germination percent, root length, dry weight, head diameter, chlorophyll content, leaf area and seed yield by 49, 97, 140, 187.6, 56.4, 93.8 and 206.5% respectively, in comparison to control seeds (Table 3).
Table 3

Plant growth parameters of sunflower in presence of pathogen, under saline conditions (in field)


Germination  %

Root length (cm)

Shoot length (cm)

Dry weight (g)

Head diameter (cm)

Chlorophyll (Chl a + Chl b) (mg/g)

Stem width (cm)

Leaf area (cm2)

Seed yield (Kg/Hectare)



40 ± 0.03a

19.9 ± 0.09a

98.7 ± 0.01a

4.5 ± 0.05a

6.5 ± 0.06a

0.0140 ± 0.01a

1.95 ± 0.09a

87.01 ± 0.06a

96.1 ± 0.06a

0.678 ± 0.08a


70 ± 0.05b

30.5 ± 0.01b

114.1 ± 0.06b

6.5 ± 0.07b

9.1 ± 0.09b

0.0171 ± 0.02b

3.96 ± 0.02b

138.89 ± 0.05b

220.9 ± 0.04b

2.109 ± 0.03b


40 ± 0.02a

20.7 ± 0.03a

97.1 ± 0.04a

4.4 ± 0.05a

6.1 ± 0.06a

0.0137 ± 0.07a

1.87 ± 0.02a

86.91 ± 0.01a

95.9 ± 0.01a

0.669 ± 0.01a

Extracted SA

83 ± 0.04c

35.9 ± 0.01c

118.2 ± 0.01c

8.6 ± 0.06c

13.9 ± 0.04c

0.0213 ± 0.03c

5.34 ± 0.06c

150.1 ± 0.01c

287.9 ± 0.05c

2.301 ± 0.07c

Standard SA

84 ± 0.01c

36.1 ± 0.06c

120.1 ± 0.05c

8.9 ± 0.05c

15.2 ± 0.01c

0.0229 ± 0.06d

6.02 ± 0.02c

147.9 ± 0.03c

289.01 ± 0.02c

2.532 ± 0.05c


89 ± 0.01d

39.2 ± 0.02d

123.2 ± 0.01d

10.8 ± 0.01d

18.7 ± 0.03d

0.0249 ± 0.05d

8.98 ± 0.04d

168.7 ± 0.03d

294.6 ± 0.01d

2.611 ± 0.03d


83 ± 0.01c

35.2 ± 0.01c

117.0 ± 0.02c

8.3 ± 0.02c

13.7 ± 0.04c

0.0210 ± 0.01c

5.31 ± 0.01c

149.9 ± 0.05c

286.5 ± 0.04c

2.312 ± 0.01c

Data are mean of 2 years. Results are mean ± SD (n = 10). Means in the columns following dissimilar letters designate significant difference (P = 0.05) by DMRT. Five samples were analyzed for each replication

Blend of PF23EPS− along with extracted SA, in presence of pathogen, displayed significantly similar result to the set receiving seed pelleting with extracted SA, suggesting the ineffectiveness of mutant strain and effectiveness of extracted SA under saline conditions. Seed pelleting with extracted SA displayed significant similarity to the set receiving treatment with standard SA in field trials.


EPS producing strain PF23EPS+ and its mutant PF23EPS− taken from the author’s collection were checked for SA production ability. It was observed that the mutant strain was not only defective in EPS production but also displayed significant reduction in SA production under non-saline (0 mM NaCl) conditions, and completely lost its biocontrol activity with increase in salinity. However, wild strain PF23EPS+ displayed SA production up to 500 mM NaCl. Hence a relationship could be established in between SA and EPS production. El Oirdi et al. (2011) reported EPS producing Botrytis cinerea B191 when inoculated with 5 week old tomato plant induced SA accumulation, thereby establishing a correlation between the two. It was also found that addition of SA to the culture of Microcystis aeruginosa enhanced EPS content (Yan et al. 2014). Tanaka et al. (2015) suggested that pretreatment of tomato plant with EPS showed significant enhancement in SA level. Signaling crosstalk in Agrobacterium tumefaciens, where SA promotes EPS synthesis has been demonstrated in the past. Regulation of exo genes involved in EPS synthesis and carbon metabolism by SA has been reported earlier in diverse bacterial strains (Kunkel and Brooks 2002; Lamothe et al. 2012; Yan et al. 2014). Several workers also reported the role of EPS and SA as signal molecules (Zipfel 2009; Tewari and Arora 2013; Arora and Mishra 2016; Shahzad et al. 2017). AHL synthase gene swrI produces quorum sensing molecule, C6HSL (N-hexanoyl l-homo-serine lactone), that induces SA production in Serratia liquefaciens (Sakhabutdinova et al. 2003). Whereas, Pantoea ananatis produces AHL synthase gene eanI that produces same signal molecule, C6HSL, that regulates EPS synthesis (Morohoshi et al. 2007). Mutation in AHL synthase gene inhibits the synthesis of metabolites thereby suggesting link in between the two (Morohoshi et al. 2007; Rad et al. 2008). During iron limitation, enhanced gene expression of EPS and SA was observed in Sinorhizobium meliloti and Pseudomonas fluorescens (Chao et al. 2005). Based on earlier studies and the present one it might be concluded that SA and EPS have strong connection and may have overlapping regulatory mechanisms hinting at molecular control in between both the metabolites.

The production of SA by PF23EPS+ was detected up to 500 mM NaCl, as monitored both by qualitative and quantitative analysis, and was further authenticated by HPLC chromatogram in present study. Toiu et al. (2011) reported RT of SA at 4.8 min as obtained on HPLC chromatogram. Shanmugam and Narayanasamy (2008) reported that Rf and RT value of SA purified from Bacillus licheniformis was found to be 0.60 and 5.24 min, respectively, which is quite similar to our results. PF23EPS+ displayed maximum SA production and biocontrol against M. phaseolina under control conditions that was significantly similar to the results obtained at 100 mM NaCl. However, subsequent increase in salt concentrations brought reduction in both SA production and biocontrol potential. As the concentration of SA decreased, inhibitory activity against pathogen also got reduced. The study thus reports the role of SA in antagonism under saline conditions. The mutant strain neither displayed SA production nor antagonism against M. phaseolina, under saline conditions, which further confirmed the role of SA in biological control. The role of SA producing stress tolerating strain of B. licheniformis in biological control against M. phaseolina, Bipolaris oryzae, Pyricularia oryzae, Curvularia lunata, Fusarium oxysporum and Alternaria alternate has been reported by several workers (Nazar et al. 2011; Prıncipe et al. 2007; Validov et al. 2007; Shanmugam and Narayanasamy 2008). Nehal and Mougy (2004) reported the inhibitory activity of extracted SA on development and sporulation of Fusarium solani and Sclerotium rolfsii. They also discussed the role of SA in antagonizing bacterial pathogens like Bacillus polymyxa, Erwinia caratovora and Pseudomonas solanacearum. Though several workers discussed the role of SA in inhibiting pathogens, but its antagonistic role under saline conditions and with respect to EPS producing microbes is novel and still needs to be worked upon. In vitro (biocontrol activity) studies confirmed the active role of SA in biological control. On the other hand pot study and field trials also suggested that SA is involved in the promotion of plant growth and suppression of charcoal rot in sunflower.

Results of pot and field trials highlighted that amongst the diverse sets of treatments, best results were observed when amalgamation (PF23EPS+ and extracted SA) was applied on seeds in comparison to individual treatments. However, insignificant results were obtained when mutant was applied on the seeds. Under saline conditions there was significant reduction in all the plant growth attributes and yield in case of unprimed seeds. Reason for reduction in growth parameters might be the alteration in physiology of sunflower plant induced by salt stress that reduced nutritional and water uptake (Hu and Schmidhalter 2005). The decline of seed germination might be due to decrease of water influx, which reduced seed humectation required for metabolic reaction involved in germination processes (El Midaoui et al. 2001).

Treatment of seeds with the combination of extracted SA and PF23EPS+ enhanced germination by 49% in comparison to unprimed seeds. Role of SA and EPS has been elucidated in seed germination and seedling growth, serving as a kernel priming agent (Munns 2002; Raheleh et al. 2013). The amalgamated combination was significantly effective in increasing growth parameters of sunflower and suppressing disease incidence. Pre-sowing application of seeds with combination reduced negative impact of salinity and enhanced plant growth and seed yield under saline conditions and in presence of pathogen. SA has been previously reported as a plant growth regulator with a significant role in recuperating of physiological processes in plants (Sakhabutdinova et al. 2003). SA can elongate the plant cells and increases cell division and therefore, results in improving plant length, leaf area, stem width and chlorophyll content. Similar results of growth improvement by SA were also reported by Raheleh et al. (2013) in maize under saline conditions. SA is also known to induce antioxidative response and membrane protection thereby preparing the plant against the impact of salt stress.

The multifunctional roles of EPS in stress amelioration and in aggressive root colonization have been reported earlier. EPS produced by PGP microbes is known to intensify soil aggregation, enhance soil texture, increase water holding capacity of soil, reduce water loss during stress conditions and protect plant from pathogenic invaders (Tewari and Arora 2014a). Roberson and Firestone (1992) also reported that EPS producing strains increase root-associated soil and hydrophilization of soil leads to improved supply of nutrients that is responsible for increasing stem width and plant growth promotion. The plant hormone SA and EPS are also reported to regulate signaling networks, involved in inducing plant immunity (Leigh et al. 1985; Yuan et al. 2008; Dempsey and Klessig 2017; Jones and Dangl 2006; Upadhyay et al. 2017). El Oirdi et al. (2011) reported that EPS induced accumulation of SA conferring resistance against phytopathogen Pseudomonas syringae in tomato.

Thus amalgamation of SA and EPS might have worked synergistically to protect the plant from attack of phytopathogens, reduce the incidence of disease and enhance growth and productivity under salinized conditions. Earlier studies have reported that SA is involved in protecting plants under abiotic and biotic stresses including salinity (Amira and Qados 2015; Wang et al. 2007; Horvath et al. 2007; Dempsey and Klessig 2017). SA a metabolite reported commonly to be produced by pseudomonads (Mehran et al. 2013) and has a role in induce systemic resistance (Ran et al. 2005; Schuhegger et al. 2006; Shahzad et al. 2017). The positive role of bacterial EPS, as an elicitor, for the induction of systemic resistance against various phytopathogens of different crop species has been reported by Prashar et al. (2013), Thenmozhi and Dinakar (2014). EPS production under stress conditions act as a strategy to protect bacterial cells from salinity stress damages (Tewari and Arora 2014a; Arora et al. 2016). Both these metabolites being induced under saline conditions may have some connection in between them at biochemical or molecular level. EPS and SA (as pure metabolites or cells producing them) can thus be used to control the phytopathogens and enhance productivity under saline conditions. For sustainability in the agriculture sector, blend of microbes and microbial products or microbial metabolites can be used for cumulative effects (Arora 2015; Arora and Mishra 2016; Arora et al. 2016). Use of metabolite based formulations can be very useful in case of PGP microbes also known as potential human pathogens (Mishra and Arora 2018). It becomes clear from this study that utilization of metabolite SA along with the P. aeruginosa PF23EPS+ or without the bacteria can be successfully done for controlling M. phaseolina and enhancing yield of sunflower under saline conditions.


Use of chemicals and resistance shown by phytopathogens has resulted in loss in soil fertility, crop yields and problems such as salinity and soil pollution. The future is only for the eco-friendly measures which not only lead to sustainable and pollution free environment but also development of a market sound product instilling confidence amongst the end users. Inoculation of PGP microbes along with/or their metabolites is an upcoming technology showing promising results under in vivo conditions. In the present study amalgamation of PGP microbe P. aeruginosa PF23EPS+ and its metabolite SA brought significant enhancement in sunflower growth and production in saline habitats even in presence of phytopathogen, M. phaseolina. The amalgamation of SA and PF23EPS+ congaing multifaceted activities of stress amelioration, biological control and growth promotion could be exploited in future for developing suitable bioformulations. Development of such a versatile bio-formulation will not only remove constraints associated with the perilous chemicals but will also help in developing eco-friendly bio-formulations with much better consistency in comparison to only cell based products.



Authors are obliged to Vice Chancellor, BBA University, Lucknow, UP, India for support.


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Copyright information

© Society for Environmental Sustainability 2018

Authors and Affiliations

  1. 1.Department of Biochemical Engineering and BiotechnologyIndian Institute of Technology DelhiNew DelhiIndia
  2. 2.Department of Environmental ScienceBabasaheb Bhimrao Ambedkar UniversityLucknowIndia

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