Introduction

The rhizosphere is an environment that the plant itself helps to create and where pathogenic and beneficial microorganisms constitute a major influential force on plant growth and health [13]. Many bacteria have a neutral effect on the plant, but are part of the complex food web that utilizes the large amount of carbon that is fixed by the plant and released into the rhizosphere (i.e. rhizodeposits) [10]. The bacterial community in the rhizosphere also harbours members that exert deleterious or beneficial effects on the plant. Bacteria that adversely affect plant growth and health are the pathogenic, whereas bacteria that are beneficial include nitrogen-fixing bacteria and plant growth-promoting rhizobacteria (PGPR) [34]. Pseudomonas sp. is one of the most important members of PGPRs showing all the three major group of PGPRs, that is having biofertilizer, phytostimulator and phytopathogen biocontrol activities [31]. Several Pseudomonas sp. have been extensively used for biological control against many soil borne plant pathogens [33].

It has been observed that polyaromatic hydro-carbons (PAH) degradation in soil is dominated by bacterial strains belonging to a very limited number of taxonomic groups such as Sphingomonas, Burkholderia, Pseudomonas [9]. Bacterial cell surface hydrophobicity is important in promoting cell adhesion to soil particles [23] and plays a critical role in facilitating biodegradation in situ [24]. However, the hydrophobicity and motility can potentially increase the ability of bacteria to access polycyclic aromatic hydrocarbons (PAHs) within soil [9]. Although, numerous studies have been conducted on Pseudomonas sp. and their possible role in plant growth promotion and other traits, the information regarding the association of Pseudomonas sp. in the rhizosphere niche of Calotropis plant possessing plant growth- promoting (PGP) traits, degradation abilities and hydrophobicity has not been yet been explored. Therefore, keeping in view the above constricts, the current study was designed to (i) isolate Pseudomonas sp. from the rhizosphere of Calotropis sp., growing in northern and central regions of India; (ii) screen the abilities of plant growth promoting attributes including the production of IAA hormone, siderophore, phosphate solubilization and the ability to inhibit pathogenic fungi Alternaria alternata, Fusarium oxysporum and Cladosporium oxysporum and (iii) screening with respect to ability to utilize PAHs, quantitative degradation by HPLC, growth study with varying concentration of anthracene and naphthalene and cell surface hydrophobicity of isolate. In addition, the morphological, physiological, biochemical and 16S rRNA analysis has been attempted to determine genus and species taxa of isolated strain.

Materials and Methods

Isolation

The soil used for bacterial isolation was collected from the rhizosphere of Calotropis sp. found to grow in four different zones of the northern and central India. The rhizospheric soil was collected in sterile polythene bags and stored at the 4 °C until further use. The pure culture was maintained on slants at 4 °C and in 10 % glycerol at 20 °C.

Identification and Characterization of the Bacterial Isolate

Phenotypic characterization of the isolate was carried out by subjecting the bacterial isolate to cultural (oxygen requirement), morphological (colony morphology and pigmentation), microscopic (Gram staining, cell shape, size and arrangement of cells), biochemical (utilization of different carbon sources and enzyme activity) and physiological characterization (temperature, pH, salt tolerance and antibiotic sensitivity) following standard procedures [7].

Qualitative and Quantitative Estimation of PGP Attributes

Salkowski’s reagent was used to examine the indole acetic acid (IAA) production [19]. Quantitative estimation of IAA production was carried out by the standard method [8]. The amount of IAA produced in culture medium was calculated by comparing it with standard curve in μg/ml.

The potential to solubilize relatively insoluble calcium phosphate was checked by point inoculation on Pikovaskaya’s agar and the plates were incubated at 28 °C for 4–5 days [25]. The zone was measured and phosphate-solublizing index (PSI) was calculated by means of the formula:

$$ {\text{PSI}} = {\text{Colony}}\;{\text{diameter}} + {\text{Halozone}}\;{\text{diameter}}/{\text{Colony}}\;{\text{diameter}} $$

Quantitative estimation of phosphate solubilization for selected isolate was carried out by chlorostanus-reduced molybdo-phosphoric acid blue method [29]. HCN production was checked by culturing the organism on nutrient agar medium supplemented with glycine (0.44 %). Filter paper dipped in 0.5 % picric acid was attached to the lid of culture tube. After incubation for 48 h at 28 ± 1 °C, colour change was observed for HCN production [4]. Siderophore production was detected qualitatively on Chrome-Azurol S agar (CAS) medium [21].

Growth Profile of Isolates in Anthracene or Naphthalene and HPLC Analysis

Growth profile of isolates in anthracene or naphthalene amended medium was also determined. Minimum salt basal medium was supplemented with different concentrations (0.5, 0.8 and 1.0 mg/50 ml) of anthracene or naphthalene. Mean growth rate (K) was calculated by formula given as follows:

$$ K = 3.322\;\log Z_{t} - \, Z_{0} /\Updelta T $$

where K is the mean growth rate constant, Z t is final growth at time t, Z 0 is initial growth at time 0, and ΔT is difference in time.

Residual amounts of anthracene and naphthalene were determined by high performance liquid chromatography (HPLC) analysis in culture medium for quantitative estimation of PAH degradation.

Antagonistic Properties Due to Diffusible and Volatile Compounds

For examining the antagonism due to diffusible compounds produced by the bacterial isolate, fungal culture of test fungi (F. oxysporum, A. alternata, and C. oxysporum) were individually inoculated on potato dextrose agar (PDA) plate. The inoculation of bacterial culture was made about 2 cm away from fungal inoculation. The plates were incubated in inverted position at 28 °C in the incubator. The percentage growth inhibition (PGI) was calculated using the formula:

$$ {\text{PGI}} = \left[ {\left( {R_{ 1} - \, R_{ 2} } \right)/R_{ 1} \times 100} \right], $$

where R 1 represents the radius of the test fungus in the direction with no bacterial colony, and R 2 is the radius of the fungal colony in the direction of the bacterial colony. The antagonism due to volatile compounds was evaluated by preparing a bacterial lawn on nutrient agar plates. After incubation for 24 h, the lid was replaced by a plate containing an agar block of the test fungus grown on potato carrot agar (PCA). The two plates were sealed together with parafilm. Control sets were prepared in similar manner, without bacteria in the bottom plate. Such sealed sets of petri dishes were incubated at 28 °C. The PGI was calculated using the formula:

$$ {\text{PGI}} = \left[ {\left( {R_{1} -R_{ 2} } \right)/R_{ 1} \, \times 100} \right] $$

where R 1 represents the radius of the test fungus in the control, and R 2 is the radius of the fungal colony with the bacterial colony [22].

Phylogenetic Analysis

Molecular characterization was carried out by 16S ribosomal RNA gene sequencing of the isolate. It was performed using universal eubacterial primers fD1 and rp2 [32]. The phylogenetic tree was constructed by the neighbour joining method using the distance matrix from the alignment [26].

Statistical Analysis

The data presented have been analysed statistically with the relevant standard deviation and standard error (SE) method.

Results

Isolation and Biochemical Characterization

On the basis of morphological and biochemical characterization, the bacterial isolate was found to be aerobic, pigmented (yellow), Gram-negative, rod-shaped and motile. It was also found that the isolate was positive for glucose fermentation, casein, gelatine, starch, methyl red, nitrate reduction, urea hydrolysis, catalase and oxidase and showed negative results for indole, voges proskauer, mannitol and citrate utilization test. However, it was found to be sensitive to chloramphenicol, ampicillin, tetracycline, gentamicin, ceftriaxone and erythromycin, and it showed resistance to co-trimoxazole, cefuroxime, ciprofloxacin, penicillin G, augmentin, fusidic acid and vancomycin (Table 1). The strain showed optimum growth between 25 and 30 °C, exhibited tolerance to a wide pH range between 6 and 10 and salt concentrations up to 5 % (wt/vol). On the basis of morphological, biochemical and physiological characteristics, the isolate was grouped under Pseudomonas sp. as described in Bergey’s Manual of Determinative Bacteriology.

Table 1 Morphological, biochemical and physiological characteristics of Pseudomonas sp. isolate KS51
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Plant Growth Promoting Traits of Test Isolates

After 24, 48 and 72 h incubation time, the isolate was able to produce 8, 13 and 18 μg ml−1 day−1, IAA, respectively (Table 2). It was interesting to observe that the isolate was able to retain its functional traits even after 72 h. Phosphate solubilization was evidently visible on the Pikovaskya agar plate where isolate form a clear halo zone around it. The zone of solubilization around the bacterial colony was found to increase with the increase of incubation time. Quantitative estimation of phosphate solubilization, estimated after incubation for duration ranging from 24 to 144 h, is presented in Fig. 1a. The bacteria solubilized 71 μg ml−1 of phosphorus on 24 h of incubation, and maximum solubilization (268 μg ml−1 of P) was recorded after 144 h. The pH of the phosphate solubilization in broth was found to decline because of bacterial activity and lowering of pH coincided with increase in the efficiency of phosphate-solubilizing activity. The pH was found to decline from 6.3 to 4.5 (Fig. 1b).

Table 2 Plant growth-promoting attribute of Pseudomonas sp. at different incubation time
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Fig. 1
figure 1

a Quantitative P-solubilization by Pseudomonas sp. b Lowering of pH in the broth due to P-solubilizing activity of Pseudomonas sp. (filled square KS51 and filled circle for control)

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Formation of an orange halo zone around bacterial colony on Chrome azurole S agar medium indicates the siderophore production. A moderate siderophore production was witnessed after 24 h, and strong orange halo zones were observed after 48 and 72 h. However, the test isolates was found negative for HCN production (Table 2).

Growth Profile and HPLC Analysis

It was found that the growth rate constant increased with the increase in concentration of substrate. The K values of the isolate KS51 in medium amended with 0.5, 0.8 and 1 mg/50 ml anthracene was obtained as 0.33, 0.38 and 0.46 h−1, respectively. Similar results were also obtained for 0.5, 0.8,s and 1 mg/50 ml naphthalene, where K values was found to be 0.26, 0.37 and 0.44 h−1, respectively (Fig. 2).

Fig. 2
figure 2

Growth profile of isolate KS51 in medium supplemented with different concentrations of anthracene or naphthalene. Control (filled square), 0.5 mg/50 ml Ant (filled circle), 0.8 mg/50 ml Ant (up-pointing triangle), 1 mg/50 ml Ant (down-pointing triangle), 0.5 mg/50 ml Nap (left- and right-pointing triangles), 0.8 mg/50 ml Nap (left-pointing triangles), 1 mg/50 ml Nap (right-pointing triangles)

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The isolate was found to considerably reduce PAH concentration in medium as estimated by HPLC analysis. The isolate KS51 showed 63.53 % degradation of anthracene, while 78.44 % decrease in naphthalene concentration was recorded after 6 days.

Antifungal Activity of the Test Isolates

Biocontrol activity of the isolate was checked against F. oxysporum, A. alternata and C. oxysporum. The test isolate showed good antifungal activity fungi through the production of volatile metabolite but relatively less antifungal activity was detected through the diffusible metabolite (Table 3).

Table 3 In vitro effects of diffusible and volatile metabolites of Pseudomonas sp. of F. oxysporum, A. alternata and C. oxysporum
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Phylogenetic Analysis

Based on the 16S ribosomal RNA gene sequence, isolate KS51 showed maximum similarity with Pseudomonas sp. (Accession No. FM173664.1). The phylogenetic tree (Fig. 3) constructed using 16S rRNA gene sequences of other related members of the Pseudomonad group revealed that the isolate formed a close cluster with Pseudomonas xanthomarina (Accession No. HQ202840.1).

Fig. 3
figure 3

Phylogenetic tree, based on 16S ribosomal RNA gene sequences, showing relationships between KS51 and other Pseudomonads

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Discussion

Rhizosphere is a rich source of microbes and should be explored for obtaining potential PGPRs, which might be good bio-inoculants for improving yields of crop plants. The exact mechanisms by which PGPR promote plant growth are not fully understood, but are thought to include (i) the ability to produce or change the concentration of plant growth regulators like IAA, gibberellic acid, cytokinins and ethylene; (ii) asymbiotic N2 fixation; (iii) antagonism against phytopathogenic microorganisms by production of siderophores, antibiotics and cyanide; and (iv) solubilization of mineral phosphates and other nutrients [1]. In the present investigation, isolate KS51 of Pseudomonas sp. was screened in vitro for its PGP activities. Though the results of the present investigations are in conformity with earlier recorded observations, based on several species of Pseudomonas that produce secondary metabolites, mobilize nutrients and promote plant growth [31].

Production of phytohormone like auxin (IAA) is a desirable characteristic of a PGPR [20]. In the current investigation, IAA production was detected by the test isolate. Our findings of IAA production by the Pseudomonas isolate are in agreement with other researchers [11]. There was an increase in the concentration of IAA with the increasing incubation time. Similar trend of IAA production with the increasing concentration of incubation time has also been reported [27]. Such findings may have direct practical application, although intrinsic ability of bacteria to produce IAA in the rhizosphere depends on the availability of precursors and uptake of microbial IAA by plant [3].

The ability of the isolate to solubilize tricalcium phosphate in vitro is another important means of achieving plant growth promotion [14]. From the current study, it was evident that the isolate liberated maximum 268 μg/ml of available phosphorus after 144 h and the pH declined from 6.3 to 4.5. These results draws support from an earlier study which highlights production of a variety of organic acids by ten bacterial and three fungal strains and subsequent drop in pH [16]. The mechanism(s) of microbial solubilization of insoluble phosphate has received attention, and phosphate solubilization is considered an important attribute of plant growth-promoting rhizobacteria. Detailed studies including phylogenetic relationships on strains of phosphate-solubilizing Pseudomonas sp. isolated from different ecological niches have also been carried out [17].

Siderophore is one of the biocontrol mechanisms belonging to PGPR groups, and PGPRs produce a range of siderophore which have a very high affinity for iron [31]. Therefore, the low availability of iron in the environment would suppress the growth of pathogenic organisms including plant pathogenic fungi [33]. From this study, it was found that the isolate was a very good siderophore producer while the isolate displayed no HCN production.

In plant disease management programmes, the use of a rapid method for screening efficient biocontrol agents is a prerequisite [2]. Antagonistic activity possessed by biocontrol agents is often evaluated by measuring the inhibition zones developed under in vitro agar based assays [30]. In the present study, isolate was examined for its antagonistic activities against three phytopathogenic fungi, viz., Alternaria alternata, Fusarium oxysporum and C. oxysporum, and it showed inhibition in fungal growth (40 % in F. oxysporum and 36 % in A. alternata) because of diffusible metabolites, while the maximum inhibition in fungal growth (70.37 % in A. alternata and 65 % in C. oxysporum) due to volatile metabolite was observed after 120 h of incubation. Similar results on the effectiveness of Pseudomonas corrugata against plant pathogenic fungi like A. alternata and F. oxysporum [30], Fusarium, Rhizoctonia, Sclerotium, Pythium [18] were also reported. Our results are in conformity with the studies of Trivedy et al. [30] which prove that the effect(s) of inhibitory volatile metabolite(s) produced by P. corrugata had a predominant inhibitory role in the antagonism of the test fungi, A. alternata and F. oxysporum, and the diffusible metabolite(s) played only a subsidiary role in the antagonism. In addition, some other studies are in contrast with the current study where the effect of inhibitory volatile metabolite(s) received less importance than the inhibitory diffusible metabolite(s) [28].

The growth rate of the isolate was found to increase with increase of substrate concentration. This further confirmed that anthracene and naphthalene concentrations are significant in growth physiology of the isolates and may also act as limiting nutrients in rhizosphere [6]. However, in this study, the quantitative estimation of residual amount of PAH in culture medium showed that 78.44 % of anthracene was degraded by the isolate [15] have also reported the degradation of PAH in liquid and solid medium by Pseudomonas sp. in their study. In the present study, the bacterium was isolated from the rhizosphere of Calotropis sp., found to grow in north and central part of India. They are highly prone to grow in extreme environmental conditions with temperature 0–50 °C, low rainfall 5–10 ml/annum, low relative humidity, high salinity and deposition of kankar or hardpan in the soil [12]. Therefore, it is assumed that the isolated bacterium has naturally adapted to variable extremities of temperatures and would retain its beneficial plant growth-promoting traits when inoculated in similar conditions.

The phylogenetic tree constructed on the 16S rRNA gene sequence revealed its close identity with Pseudomonas sp., a Gram-negative bacterium, which was first described from the rhizosphere of grasses [5]. The study carried by [31] support the present investigation to screen the Pseudomonas sp. isolated from rhizosphere of Soybean plant as plant growth promoter and biocontrol agent on the basis of 16S rRNA gene sequence analysis.

Thus, it can be concluded that rhizosphere of Calotropis sp. is a source of Pseudomonas sp. possessing potent PGP attributes, PAH degradation and biocontrol activities against phytopathogenic fungi. Further studies are underway to confirm their effectiveness in field conditions.