Introduction

Agriculture is considered to be one of the most vulnerable sectors, often exposed to a plethora of stress conditions. Currently, more than 800 million hectares of land throughout the world are affected by the level of salinity stress that substantially reduces crop productivity [5, 42]. Sunflower is one of the fastest growing oilseed crop grown in India. India majorly exports sunflower cake, which increased from 5,511.33 tons (triennium ending 2002) to 18,440 tons during 2009 [59]. International demand for sunflower seeds and oil is continuously increasing with the growing health consciousness and increasing knowledge on the dietary and health benefits. Sunflower is considered one of the major oil producing crops in the world due to its high quality oil, high protein content and moderate production requirements [53]. Sunflower oil is commonly used in food as frying oil, and in cosmetic formulations as an emollient, it is popular as healthy cooking oil due to its health benefits while the meal is used in animal feed industry. Use of sunflower oil is in great demand as it trims down the incidence of cancer, hypertension, and the cholesterol in human beings [49]. However, nowadays sunflower production is affected badly by several biotic and abiotic stresses and it has been estimated that among several abiotic stressors, salinity is the major constraint that limits sunflower yield, growth, and productivity. More than 60 % loss in the production of sunflower is due to salinization [29]. Unfortunately apart from soil salinity, sunflower is also attacked by variety of fungal pathogens which affect its yield and oil quality. Macrophomina phaseolina is a fungal opportunist that likes to take advantage of stressed sunflower plant which is cultivated in salinized regions and causes up to 70 % reduction in its oil production [26, 56]. M. phaseolina has a wide host range and is responsible for causing losses on more than 500 cultivated and wild plant species [29]. As agricultural production intensified over the past few decades, producers became dependent on agrochemicals for crop protection. Continuous use of chemicals subverts the soil ecology, disrupt environment, degrade soil fertility, and consequently show harmful effects on human health along with contaminating ground water and thus creating environmental hazards [6, 28, 35]. Therefore, there is an emergent need to develop biopreparations which are ecofriendly and lead to sustainability. Microbe-based formulations that are able to suppress the growth of phytopathogens are real alternatives to hazardous chemicals. These formulations can also be used to enhance yields under environmental stress conditions. Soil-borne fluorescent pseudomonads have received particular attention as plant growth-promoting rhizobacteria (PGPR) throughout the globe because of their catabolic versatility, excellent root colonizing ability, and capacity to produce a wide range of metabolites that favor the plant to with stand under varied biotic and abiotic stresses [31]. Members of this bacterial group are versatile and able to adapt and colonize a wide variety of ecological environments throughout the world. Extracellular polysaccharides (EPS) produced by microorganisms are instrumental in imparting stress tolerance to bacterial cells [40] but relatively little attention has been paid on their utility and role under saline conditions [1].

In the present study, fluorescent pseudomonads were isolated from the rhizosphere of sunflower plants growing in saline soil. The isolates were checked for production of plant growth promotory metabolites under saline conditions. The isolates were also tested for biocontrol potential against M. phaseolina causing charcoal rot disease in sunflower, under saline stress condition.

Materials and Methods

Microorganisms and Growth Conditions

One hundred and ten fluorescent pseudomonad isolates from the rhizosphere of different plants from Kanpur and adjoining areas (20°38′E and 80°21′N; temperature maximum 48 °C and minimum 1 °C), Uttar Pradesh (northern India), were screened on the basis of salinity tolerance and EPS production. Isolates were tested for morphological, physiological, and biochemical characters according to Bergey’s Manual of Systematic Bacteriology [22]. Among all, isolate PF23 was selected as it produced highest amount of EPS on exposure to salt stress. Isolate PF23 was further identified by analysis of 1.5 kb 16S rRNA sequences. 16S gene sequence was queried for similarities with BLAST [2] and with the Ribosomal Database Project (RDP) [30, 36]. The phylogenetic tree was constructed using Neighbor Joining method [46].

Phytopathogenic strain of M. phaseolina ARIFCC257 was procured from Mycology and Plant Pathology Group, Division of Plant Sciences, Agharkar Research Institute, Pune. The strain was grown and maintained on Potato dextrose agar (PDA) (Hi Media, Mumbai) at 28 and 4 °C, respectively.

Plant Growth Promoting Attributes

PF23 was checked for plant growth promotory (PGP) attributes including phosphate solubilization, indole acetic acid (IAA) production, HCN production, siderophore production, chitinase and β-1-3 glucanase activity, nitrogen fixation, and EPS production abilities under saline and non-saline conditions. Phosphate solubilizing activity of PF23 was tested by spot inoculation on Pikovskaya’s medium [54]. IAA production was detected in culture filtrate using salkowski’s reagent (1 ml of 0.5 M FeCl3 in 50 ml of 35 % HClO4) [23]. HCN production was checked by observing the change in color of the filter paper impregnated with 0.5 % picric acid in 1 % Na2CO3 [37], whereas siderophore production was determined on Chrome-Azurol S medium according to Schwyn and Neilands [51]. Extracellular chitinase activity was determined by spot inoculation on solid chitin minimal medium (CMM), whereas β-1,3 glucanase enzyme was assayed according to Dunne et al. [14], nitrogen fixation was checked by acetlylene reduction assay [28] and EPS production was monitored by chilled ethanol precipitation method [47, 55].

Chemical Mutagenesis for Developing EPS-Defective Mutant

A loopful of PF23 cells were inoculated into 10 ml of Davis Minimal Media (DMM) broth [32] and incubated at 28 °C up to log phase. Subsequently, 100 μg ml−1 5-bromouracil was added and further incubated for 02 h. Cells were centrifuged, washed thrice with sterile water, and resuspended in 10 ml of DMM broth, and mutants were fixed by overnight incubation at 28 °C. The mutant bank was stored in 25 % glycerol at −40 °C [30]. The clones of mutant bank were screened for EPS production according to Sandhya et al. [47]. Mutagenic procedure worked effectively as EPS-defective mutant, PF23EPS− was repeatedly checked on DMM media for stability. The EPS-defective mutants were further screened for salinity tolerance assay. The wild isolate PF23 served as a control in the mutant screening test. Maximum EPS-defective mutant was marked as PF23EPS−.

Salinity Stress Assay

The log phase culture (OD610 = 0.1) of PF23 and PF23EPS− were seeded in DMM broth, amended with different concentrations of NaCl (0 to 2,200 mM) and incubated at 28 °C. Optical density was measured at 610 nm by spectrophotometer (GENESYSTM6, Model, 335908-02) up to stationary phase (120 rpm). The experiment was conducted in five replicates.

Effect of Saline Stress on EPS Production

To determine the effect of saline stress (0–2,000 mM) conditions on EPS content, selected strain PF23 and its EPS-defective mutant PF23EPS− were grown in DMM broth, with gradients of NaCl concentrations (0–2,000 mM) for seven days. Cells were harvested by centrifugation for 10 min at 11,000×g. The supernatant was filtered through 0.45-μm nitrocellulose membrane; two volumes of cold ethanol were added to culture supernatants and stored overnight at 4 °C. Precipitated material was collected by centrifugation (20 min at 2,500×g) suspended in demineralized water, and mixed with 2 volumes of cold ethanol. Samples were centrifuged (2,500×g) and the pellets were dried at 100 °C and weighed. The amount of EPS was expressed as polymer dry mass and expressed in g/l [19, 20, 48]. Total carbohydrate content was determined in the precipitated EPS according to Dubois et al. [14] and the experiment was conducted in five replicates.

In Vitro Biocontrol Activity

Inhibitory activity of isolates PF23 and PF23EPS− against M. phaseolina was checked on DMM agar amended with different concentrations of NaCl (0–2,000 mM). Briefly, 6-mm mycelium disk of fungal pathogen was centrally placed on DMM plates, and 0.5 μl of exponentially grown cultures of PF23 and PF23EPS− were spot inoculated 1 cm away from the edge of the plate. The plates were incubated at 28 °C for 6 days and percentage inhibition was determined [4]. The 48 h culture of strain PF23 and PF23EPS− in DMM broth amended with different concentrations of NaCl (0–2,000 mM) was centrifuged at 7,826×g for 15 min followed by membrane filtration (0.45 μm, Merck, India), and antifungal activity of cell-free supernatant was detected by using the well diffusion method [24]. Briefly, spore suspension of M. phaseolina in 0.85 % saline was spread on DMM plates. A 6 mm well cut in DMM plates was filled with 50 μl of supernatant then incubated at 28 °C for 48 h to observe zone of inhibition in millimeter (mm)

In Vitro Antagonistic Activity of EPS

The antifungal activity of purified EPS obtained from PF23 (at varied salt concentrations ranging from 0 to 2,000 mM NaCl) was tested against M. phaseolina in separate flasks. For this, 500 μl spore suspension (OD 610 = 1.0) of M. phaseolina in 0.85 % saline and 100 μg of EPS (obtained at different NaCl concentrations) was added in 50 ml of DMM broth, and incubated at 28 °C for 7 days. In case of control, 500 μl of spore suspension was added in 50 ml of DMM broth. After 5 days, fungal mycelia were filtered (Whatman No. 1), oven dried at 70 °C, and percentage inhibition was calculated.

$${\text{Inhibition }}\% = 1{-}\left[ {{\text{Dry weight of mycelium }}\left( {\text{sample}} \right)/{\text{Dry weight of mycelium }}\left( {\text{control}} \right)} \right] \times 100.$$

Composition of EPS

The precipitated EPS obtained from PF23 at different levels of salinity was hydrolyzed with 2 volumes of 2.5 M H2SO4 at 100 °C for 1 h. The solution was neutralized with 1 M sodium carbonate and spotted on the silica gel plate (Silica gel 60F 254; Merck). The plate was developed in a thin layer chromatography chamber using n-butanol:acetic acid:water (4:1:5 v/v) as the mobile phase at room temperature. The plate was dried, sprayed with alkaline potassium permanganate, and incubated at 100 °C for 10 min. The Rf values of the colored spots were measured and compared with those of standard carbohydrates (glucose, mannose, fructose, mannitol, arabinose, xylose, rhamnose, raffinose, galactose, and trehalose) [25].

In Planta Activity and Suppression of Charcoal Rot of Sunflower

The study was conducted to detect PGP response and charcoal rot suppression ability of EPS producing strain PF23 and negative strain PF23EPS− taking sunflower as the test crop under saline and non-saline conditions. Seeds of sunflower were surface sterilized for 2 min with 70 % ethanol followed by 2 % sodium hypochlorite (10 min). After thorough rinsing with sterile distilled water, the seeds were germinated in glass tubes of 50 ml capacity (2 seeds per tube), 1/3 filled with autoclaved plant growth media (PGM) (containing 1.2 mM K2HPO4, 0.4 mM KH2PO4, 5 mM CaCl2, 3.35 mg ferric citrate l−1, 2.5 Mm MgSO4, 2.5 mM K2SO4, 10 μM MnSO4, 20 μM H3BO3, 5 μM ZnSO4, 0.2 μM CuSO4, 1.5 μM CaSO4, 1.0 μM NaMoO4, and 1 % agar, with pH 6.8) [18] supplemented with 125 mM NaCl (as sunflower seedlings displayed germination only up to 125 mM salinity level) and without NaCl in following sets of treatments: (i) non-bacterized seeds (ii) seeds treated with PF23 (iii) seeds + M. phaseolina (iv) seeds treated with PF23 + M. phaseolina (v) seeds + PF23EPS− and (vi) seeds + PF23EPS− + M. phaseolina. Seeds were bacterized by dipping in cell suspension of PF23 and PF23EPS− (OD 610 = 0.1) for 10 min and dried overnight under aseptic conditions. M. phaseolina treatment was given by inoculating 10 μl of spore suspension in 0.85 % saline on the surface of PGM after 2 days of seed application [30]. The experiment was conducted in five replicates and germination percentage was calculated after 5 days according to Ranal and Santana [43]. The extent of infection by M. phaseolina was indicated by the presence of dark brown lesions on root systems. Percentage disease incidence was calculated as percentage of diseased plants out of the total number of plants.

In Vivo PGP Activity and Suppression of Charcoal Rot of Sunflower

Experiment was conducted in small plastic pots (24 × 12 × 12 cm) under controlled conditions (temperature 22 ± 2 °C, humidity 70–80 %, 12 h photoperiod) in plant growth chamber. Fungal inoculum was prepared on oat (Avena sativa) grains and added to the sterilized (autoclaved) sandy loam soil (pH 7.24) so as to get 104 fungal propagules/gm of soil before seed sowing (05 seeds/pot). Sunflower seeds were dipped for 10 min in a cell suspension of isolate PF23 and PF23EPS− (OD 610 = 0.1) mixed with carboxy methyl cellulose (CMC, 1 %) and dried overnight in aseptic conditions [57]. The experiment was conducted in sterilized soil in similar sets of treatments as in planta study, taking five replicates. The effect of treatments was determined under control (received only normal irrigation water) and saline (irrigated as per requirement with 125 mM NaCl solution) conditions [41]. Five plants from each set were taken randomly to determine root associated soil/root tissue ratio. Plant watering was stopped 6 days before harvesting to facilitate the separation of root-associated soil from bulk soil. Roots with adhering soil were carefully separated from bulk soil by gentle agitation for 1 min. Root-associated soil was removed from root tissue by washing them in sterile water. Root-associated soil dry mass (dm) and root dm were measured after 24 h at 105 °C, to calculate root-associated soil/root tissue. Other plant growth parameters (shoot and root length, fresh weight, dry weight, head diameter, and seed yield) and disease incidence were measured after 120 days. Shoot length, root length, and head diameter were measured in centimeter (cm) soon after the plants were uprooted. Fresh weight was taken by uprooting the plant and rinsing with tap water until all visible sand and soil particles were removed. Particles that adhered strongly to the roots were manually removed with tweezers. After blotting the excess moisture from the plant with absorbent paper, the fresh weight was measured [10]. For dry weight, plants were placed into tarred aluminum foil pouches; samples were dried in an oven at 75 ± 2 °C for 16 h and weighed subsequently [10]. Whereas seed yield was calculated after separating the seeds from the head and weighing number of seeds per plant. Percentage disease incidence was calculated as percentage of diseased plants out of the total number of plants.

Statistical Analysis

The data generated during quantitative evaluation of EPS and seed germination or plant growth promotion values were analyzed by means of ANOVA, and means were compared by the Duncans Multiplicity Test Range (DMRT) using the SPSS software (ver. 10.1, SPSS Inc., www.spss.com). The significance level for all analysis was P = 0.05.

Results

Microorganisms and Growth Conditions

Isolate PF23 was selected from the collection of pseudomonads, as it displayed maximum stress tolerance capacity and high EPS production. Isolate PF23 was fluorescent, Gram-negative, motile rod, oxidase- and catalase-positive indicating according to Bergey’s Manual of Systematic Bacteriology [22] to be a member of the Pseudomonas species. The comparison of complete 16S rRNA gene sequence (Fig. 1) of the isolate PF23 with the sequence of the other strains of the genus Pseudomonas showed 99 % relatedness to Pseudomonas aeruginosa (Gen Bank accession number AB691548.1 and KC417305.1). Based on biochemical, physiological characteristics, and nucleotide homology, isolate PF23 was identified as Pseudomonas aeruginosa PF23 and sequence data submitted to NCBI Gen Bank with accession number KF598858.

Fig. 1
figure 1

Comparative sequence analysis of 16S rRNA gene from P. aeruginosa PF23 and representative related strains from GenBank

Full size image

Plant Growth Promoting Attributes

PF23 was positive for EPS, siderophore, and IAA production but negative for phosphate solubilization, HCN production, chitinase, β-1-3 glucanase, and nitrogen fixation. The production of IAA (in supernatant) and siderophore (on CAS agar plate) got drastically reduced with increase in salt concentration and was completely inhibited at 100 mM NaCl concentration. On the other hand, an increase in EPS production was recorded with progressive increase in salinity in precipitated supernatant.

Chemical Mutagenesis for Developing EPS-Defective Mutant

Of the 100 mutant clones, 6 were identified as defective for EPS production. Two of these mutants had stable mutations. EPS-defective stable mutant PF23EPS− showed maximum reduction of 86 % in production of EPS in comparison with wild strain PF23.

Salinity Stress Assay

Isolate PF23 could tolerate salinity level up to 2,000 mM (12 %). Salt shock with 100 mM NaCl did not affect the growth, but higher osmotic stress of 500, 1,000, and 1,500 brought significant reduction in the optical density by 32, 48, and 68 %, respectively, in comparison with non-stress conditions (0 mM NaCl) (Fig. 2). No growth was observed above 2,000 mM salt concentration. On the other side EPS-defective strain PF23EPS− displayed 80 % reduction in growth in presence of 200 mM NaCl, suggesting it to be a non-salt tolerating strain.

Fig. 2
figure 2

Salinity tolerance assay of PF23. Error bars show the standard deviation of the mean values of five replicates. Five samples were analyzed for each replication, and each treatment consisted of five replications

Full size image

Effect of Saline Stress on EPS Production

Increase in osmotic stress or increase in salinity, brought increment in the EPS production up to a certain limit. In stress tolerating strain PF23, there was increase in EPS production by about 26, 38, and 66 % at salinity level of 1,000, 1,500, and 2,000, respectively (Table 1). EPS-defective mutant PF23EPS− showed maximum EPS production at 0 mM NaCl (0.109 g/l) which drastically reduced above 100 mM NaCl, and finally got diminished beyond 200 mM NaCl, respectively.

Table 1 Effect of saline stress on EPS production
Full size table

In Vitro Biocontrol and Antagonistic Activity of EPS

Strain PF23 and its cell-free culture supernatant demonstrated 90 and 88 % inhibition of M. phaseolina at 0 mm NaCl concentration, respectively. Biocontrol ability of the strain was maintained up to 500 mM (54 and 51 % inhibition by cell and cell-free culture supernatant, respectively) salinity but thereafter inhibitory activity ceased. PF23EPS− and its cell-free culture supernatant displayed antifungal activity only under unstressed (0 mM) conditions and that was about 55 and 50 %, respectively. EPS obtained from PF23 at 100, 200, 300, 400, and 500 mM NaCl displayed strong antagonistic activity against M. phaseolina and resulted in 79, 72, 64, 55, and 41 % reduction in growth, respectively, in comparison with control. EPS obtained at 0 mM (control) antagonized M. phaseolina by 84 %.

Composition of EPS

Analysis of EPS constituents by thin layer chromatography revealed differences in the sugar components of salinity tolerant strain PF23 under non-stressed and stressed conditions. Under normal conditions (0 mM NaCl) glucose (Rf 0.42) was present as the saccharide unit in the EPS hydrosylate, whereas EPS obtained under salt stress was composed of glucose (Rf 0.42), galactose (Rf 0.37), rhamnose (Rf 0.74), mannose (Rf 0.46), and trehalose (Rf 0.32) (Table 1). Carbohydrate content of EPS progressively increased from 126 to152 μg/mg−1 with increase in salinity from 0 to 2,000 mM. EPS-defective strain PF23EPS− contained only glucose as its saccharide unit both under control and maximum stress conditions (0–200 mM) and there was steep reduction in carbohydrate content from 20 μg/mg−1 to 5 μg/mg−1 under same conditions.

In Planta Activity and Suppression of Charcoal Rot of Sunflower

Seed biopriming with PF23 showed significant increase in germination % in comparison with non-primed seeds (control) both under saline (125 mM NaCl) and non-saline (0 mM) conditions. Seed biopriming brought 25 and 50 % increment in germination  % in comparison with unprimed seeds under non-saline and saline conditions, respectively. Treatment with PF23 brought increment in root length, shoot length, fresh weight, dry weight by 105, 59, 115, 212 %, respectively, in comparison with control (untreated seeds) under saline stress. Under non-saline conditions it was observed that PF23 brought 100, 31, 46, and 117 % enhancement in root length, shoot length, fresh weight, and dry weight, respectively.

Strain PF23 displayed increase in plant growth parameters of sunflower in both the absence and the presence of pathogen, under saline and non-saline conditions. In presence of M. phaseolina, PF23 brought increment in fresh weight and dry weight by 103 and 84 % in comparison with unbacterized seeds, under saline conditions. In comparison with non-bacterized control, PF23 showed 80 and 70 % reduction of disease incidence in non-saline and saline conditions, whereas EPS-defective strain PF23EPS− was significantly ineffective in suppressing disease under saline conditions. PF23EPS− displayed significantly similar results as obtained by control (untreated seeds), both under saline and non-saline conditions (Table 2).

Table 2 Influence of P. aeruginosa PF23 treatment on growth of sunflower plants in both the absence and the presence of M. phaseolina, under non-saline (control) and saline (in planta) conditions
Full size table

In Vivo PGP Activity and Suppression of Charcoal Rot of Sunflower

Results of in vivo study showed that treatment of seeds with PF23 caused significant increase in root length, shoot length, dry weight, and head diameter under saline and non-saline conditions over untreated seeds and infested seeds in the presence as well as in the absence of phytopathogen (Table 3). Biopriming of seeds with PF23 brought 53 and 50 % increase in seed yield under non-stress and stress conditions, respectively, in comparison with unbacterized seeds.

Table 3 Influence of P. aeruginosa PF23 treatment on growth of sunflower plants in both the absence and the presence of M. phaseolina, under non-saline (control) and saline (in vivo) conditions
Full size table

M. phaseolina caused 79 and 81 % incidence of disease in non-saline and saline conditions. Strain PF23 resulted in 71 and 63 % reduction of disease incidence in comparison with non-bacterized control under non-saline and saline conditions, respectively. EPS-defective strain PF23EPS− brought reduction of disease incidence by 33 and 17 % under non-saline and saline conditions. In the presence of pathogen (M. phaseolina), strain PF23 caused 93 and 138 % enhancement in seed yield under non-saline and saline conditions, respectively, in comparison with M. phaseolina-infested untreated seeds. Treatment of seeds with PF23EPS− + M. phaseolina brought significant enhancement in dry weight, head diameter, and seed yield of sunflower by 72, 28, and 59 % in comparison with M. phaseolina-infested seeds only under non-saline state. However, the mutant was ineffective in controlling the pathogen under saline conditions and there was no EPS recorded as well (Table 3).

Discussion

Pseudomonas aeruginosa PF23 displayed high salt tolerance and EPS production with progressive increase in salinity from 100 to 2,000 mM NaCl. EPS-defective mutant PF23EPS− didn’t displayed salt tolerance above 200 mM and showed 87 % reduction in EPS production in comparison with wild strain. Thus, a strong correlation could be observed between EPS production and salinity tolerance. At low salt concentration (up to 500 mM NaCl), PF23 displayed 0.901 g/l EPS that constituted mainly of glucose and galactose as its components. Whereas further increase in salinity (up to 2,000 mM NaCl) resulted in glucose, rhamnose, mannose, and trehalose as major constituents of EPS. Strain PF23, its cell-free culture supernatant and purified EPS displayed strong biocontrol potential against M. phaseolina up to 500 mM NaCl, suggesting the presence of bioactive compounds in EPS (mainly glucose and galactose) that function well at low level of salinity. Several workers reported the role of sugar analogs (2-deoxy-d-glucose) in inhibiting the growth of several yeasts and filamentous fungi [8, 9, 11, 16, 33]. Apart from it, El Ghaouth et al. [16, 17] reported the role of glucose in inhibiting the radial growth of several pathogens and causing morphological alterations in Rhizopus stolonifer and Botrytis cinerea. Although, the inhibitory activity of EPS in antagonizing, dreadful phytopathogens have been illustrated by several workers [3, 15, 38, 52] but its role under saline conditions is unknown so far.

Though, EPS actively participated in biological control of pathogens (up to 500 mM salinity), its role could also be related to stress amelioration. Addition of NaCl (above 500 mM) in the medium stimulated the mucoid growth (profuse spreading of the EPS) of the PF23 up to 2,000 mM. Increase in EPS production with increase in salinity suggests that under stress condition energy flow of the PF23 is directed toward protective mechanism, and synthesis of EPS is opted as a defensive strategy for maintaining its survivability and ameliorating salt stress [7, 44]. Discharge of EPS results in aggregation/flocculation and sheath formation around the cell, which significantly improves survival of bacterial cells in saline stress [27]. Variations in the saccharide composition with increase in salinity indicate structural differences among different polymers. EPS isolated during stressed conditions was composed of multiple saccharide units like rhamnose and trehalose. Role of mannose, rhamnose, and trehalose as stress ameliorators has been explained earlier also [13, 48]. Trehalose protects cellular enzymes by replacing water around macromolecules [58] and also stabilizes cell membranes during stress conditions [12, 13]. Under unfavorable conditions, synthesis of EPS with multiple sugars stabilizes cellular structure and offers protection from stress [21]. Thus, it may be suggested that by storing large quantities of carbon in the form of EPS during osmotic stress, PF23 cells may balance the osmotic pressure imposed by the environment and thus it (EPS) acts as a stress ameliorator or osmoprotective agent.

In planta and in vivo studies showed that seed biopriming with PF23 displayed significant increase in germination by 25 and 50 % in comparison with unprimed seeds under non-saline and saline conditions, respectively. Bioprimed seeds emerged at a more rapid rate than control. Seed biopriming with PF23 brought increment in plant growth parameters and yield under in vivo conditions, whereas EPS-defective strain did not significantly enhanced germination, plant growth parameters, and yield under saline conditions. Treatment of seeds with PF23EPS− + M. phaseolina brought significant enhancement in plant growth parameter in comparison with M. phaseolina infested seeds only under non-saline state. However, the same treatment was ineffective in controlling the pathogen under saline conditions when there was no EPS recorded. Under non-stress conditions, PF23EPS− brought increase in root length, shoot length, and seed yield in comparison with M. phaseolina-infested seeds, suggesting the role of other PGP metabolites like siderophore and IAA in growth promotion and yield enhancement, as the mutant was positive for these PGP characteristics. Whereas same mutant PF23EPS− under stressed conditions was ineffective and unimpressive in enhancing plant growth parameters suggesting the absence of PGP activity under saline stress conditions, confirming the involvement of EPS in plant growth promotion under saline stress. Apart from plant growth promotion the role of EPS in suppression of charcoal rot disease could also be established by PF23 under saline and non-saline conditions. PF23 brought 70 and 63 % reduction of disease incidence in saline conditions both in planta and in vivo conditions. Bioprimed seeds (in M. phaseolina infested and non-infested treatment) brought significant increase in plant growth parameters like root length, shoot length, fresh weight, dry weight, and seed yield in comparison with control. The most conceivable reason for such heightened yield may be due to the fact that introduction of EPS strain brought significant increase in mass of root-associated soil/root tissue in comparison with uninoculated control as was clearly observed. Increased root associated soil/root tissue upsurges adhesion of soil particles, intensify soil aggregation, enhances soil texture, increase water holding capacity of soil, reduce water loss during stress conditions, and protect them from pathogenic invaders [45]. Increased root-associated soil on PF23 application showed that hydrophilization of soil leads to improved supply of nutrients that is responsible for plant growth promotion and disease suppression [1].

Microbial EPS released by PF23 also assisted in minimizing the effect of salt stress by functioning as an ameliorating agent. PF23 when introduced in soil releases EPS that acts as slimy, mucilaginous glue or cementing adhesive and assist in soil aggregation [1]. Aggregation of soil, influences organic matter storage, soil aeration, water infiltration, and mineral supply to plant thus playing significant role in fertility recapitalization and contributes to plant growth promotion [34]. Hence, multiple roles of EPS illustrated by the study proved that at lower concentrations EPS can function as a biocontrol metabolite that possesses antagonistic activity against dreadful phytopathogen M. phaseolina. As the concentration of EPS increased with progressive increase in salinity, it started to behave more as an osmoprotective agent and thus protected PF23 and sunflower from the plethora of osmotic stresses. Pandey et al. [39] also demonstrated that the endophytic P. aeruginosa isolated from wheat roots could confer biotic and abiotic stress tolerance in cucumber by modulating stress responses. Finally, when this EPS producing PF23 was introduced in salinized soil it served as a seed biopriming agent. It brought early emergence and germination of seedling, enhanced growth, and productivity of sunflower crop by ameliorating the effect of saline stress and suppressed the incidence of disease. Sarma and Saikia [50] reported the role of P. aeruginosa in alleviation of abiotic stresses in mung bean. Present study suggests applicability of P. aeruginosa PF23 in enhancing growth and production of sunflower crop in stress-affected regions.

It has been already assessed that more than 800 million hectare land throughout the world is suffering from salinization, and is responsible for 60 % loss in sunflower yield [42]. P. aeruginosa PF23 along with an important oil seed crop can thus also be used in reclamation of barren saline soils. Increase in production and reclamation of semiarid regions utilizing EPS-producing microbes can make great contribution in enhancing yields of stressed soils around the globe.

Conclusion

Isolate PF23 identified as P. aeruginosa displayed high salt tolerance and EPS production. PF23 acted as an excellent PGPR as well as biocontrol agent due to its capacity to produce EPS at high salt concentrations. EPS not only acted as a biocontrol metabolite but also as an osmoprotectant both for the host bacteria producing it as well as for the plant (sunflower). Inoculation of PF23 can serve as useful tool for decreasing salinity stress and enhancing the yield of sunflower crop in salt-affected soils. PGPRs such as P. aeruginosa PF23 can go a long way in enhancing crop yields in salinity-affected soils thus can be involved in reclamation of such habitats. Enhanced crop productivity from barren saline soils can be a key to the food security. However, further studies are required for designing stress tolerating bioformulation from EPS producing strain PF23 for promoting plant growth, protecting plant health, strengthening plant—microbe associations in stress-affected regions, protecting plant (sunflower) from the attack of phytopathogens and reclaiming arid and semiarid areas for increasing crop productivity.