Multifunctional Pseudomonas putida strain FBKV2 from arid rhizosphere soil and its growth promotional effects on maize under drought stress

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Multifunctional Pseudomonas putida strain FBKV2 from arid rhizosphere soil and its growth promotional effects on maize under drought stress Sai Shiva Krishna Prasad Vurukonda 1, Sandhya Vardharajula, Manjari Shrivastava, Ali SkZ n,1 Department of Microbiology, Agri Biotech Foundation, PJTS Agricultural University Campus, Rajendranagar, Hyderabad, Telangana State 500030, India

ar t ic l e i nf o

a b s t r a c t

Article history: Received 23 March 2016 Accepted 21 July 2016

In the present study, rhizobacteria were isolated from the rhizosphere of maize, okra, eggplant, tomato, green gram, peanut and red gram grown in arid and semi-arid regions in India and were screened for drought tolerance in Trypticase soy broth (TSB) supplemented with different concentrations of polyethylene glycol 6000 (PEG 6000). Out of 23 isolates, three could tolerate minimal negative water potential of
1.03 MPa and were evaluated for plant growth promoting (PGP) traits under control and drought stress (
1.03 MPa) conditions. Pseudomonas spp. strain FBKV2 isolated from eggplant (Solanum melongeana L.) rhizosphere, showed multiple PGP traits under both control and drought stress conditions. The strain was identified as Pseudomonas putida by 16S rRNA sequence analysis and the sequence was submitted to GenBank under the accession number KT311002.1. The strain was evaluated for growth promotion of maize (Zea mays L.) under drought stress. Seedlings inoculated with P. putida strain FBKV2 showed better growth in terms of shoot, root length, and dry biomass. Furthermore, inoculation improved cellular metabolites and stomatal conductance in maize seedlings. Scanning electron microscopy confirmed the colonization of P. putida strain FBKV2 on the root surface of maize seedlings. The present study demonstrates that the isolation of indigenous drought tolerant P. putida strain FBKV2 from stressed ecosystems can be a very useful approach for the development of bio-inoculants for drought stress management in crops. & 2016 Elsevier B.V. All rights reserved.

Keywords: Drought tolerance Drought response Rhizosphere bacteria PGPB pseudomonad

1. Introduction Among abiotic stresses, drought is one of the serious problems limiting crop productivity in arid and semiarid regions thus affecting food security (Minakshi et al., 2013). This form of abiotic stress, affects plant water relation at cellular and whole plant level causing specific as well as non-specific reactions and damages (Ali et al., 2014). Growth reduction under drought stress has been studied in several crops such as barley (Samarah, 2005), maize (Kamara et al., 2003), rice (Lafitte et al., 2007) and wheat (Rampino et al., 2006). Furthermore, drought stress influences the availability and transport of soil nutrients, as dissolved nutrients are absorbed by roots. Drought stress, therefore, decreases nutrient diffusion and mass flow of water-soluble nutrients such as nitrate, sulfate, Ca, Mg, and Si (Selvakumar et al., 2012). Drought n

Corresponding author. E-mail address: [email protected] (A. SkZ). 1 Both authors contributed equally to this manuscript.

also induces free radicals affecting antioxidant defenses and Reactive Oxygen Species (ROS) such as superoxide radicals, hydrogen peroxide and hydroxyl radicals resulting in oxidative stress (Vurukonda et al., 2016). At high concentrations, ROS can cause damage to various cellular structures and leads to lipid peroxidation, membrane deterioration and degradation of proteins, lipids and nucleic acids in plants (Hendry, 2005; Nair et al., 2008). Drought also affects biochemical activities like nitrate reductase (NR), due to lower uptake of nitrate from the soil (Caravaca et al., 2005). It also accentuates the biosynthesis of ethylene, which inhibits plant growth through several mechanisms (Ali et al., 2014). Drought a multidimensional stress effect various subcellular cell organelles and whole plant level (Rahdari and Hoseini, 2012). Thus, drought negatively affects quantity and quality of growth in plants. Efforts have been made to develop drought resistant/tolerant plants through breeding and biotechnological approaches, but these methods are cost intensive. Rhizosphere microorganisms, being in intimate interaction with host plant are known to influence plant response to biotic and abiotic stresses.

http://dx.doi.org/10.1016/j.rhisph.2016.07.005 2452-2198/& 2016 Elsevier B.V. All rights reserved.

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The role of microorganisms in plant growth, nutrient management, and biocontrol activity is very well established. These beneficial microorganisms colonize the rhizosphere/ endo-rhizosphere of plants and promote the growth of the plants through various direct and indirect mechanisms (Grover et al., 2011). Furthermore, the role of microorganisms in the management of abiotic stresses is gaining importance. Recently, several reports have been published on microorganism- mediated abiotic stress tolerance in plants, such as drought (Mayak et al., 2004; Sandhya et al., 2009), chilling injury (Ait bakra et al., 2006), salinity (Chakraborty et al., 2011), metal toxicity (Dell’ Amico et al., 2008), and elevated temperature (Ali et al., 2009) etc. The possible explanation for the mechanism of plant drought tolerance induced by rhizobacteria include: (1) production of phytohormones like abscisic acid (ABA), gibberellic acid, cytokinins, and IAA; (2) ACC deaminase to reduce the level of ethylene in the roots; (3) induced systemic tolerance by bacterial compounds; (4) bacterial exopolysaccharides or biofilms (Glick, 2004; Yang et al., 2009; Dimkpa et al., 2009; Kim et al., 2013; Timmusk et al., 2014; Vurukonda et al., 2016). Inoculation with phytohormones producing bacteria can improve root growth and/or enhance formation of lateral roots and root hairs resulting in better water and nutrient uptake (Dimkpa et al., 2009; Egamberdieva and Kucharova, 2009). Similarly, rhizobacteria with an ability to produce ACC deaminase enzyme reduce the deleterious effect of ethylene, ameliorating plant stress and promoting plant growth under drought stress (Glick, 2005). Inoculation with rhizobacteria may also influence physiology of the host plant under stress conditions resulting in enhanced accumulation of osmolytes and tolerance to drought stress (Minakshi et al., 2013). Furthermore, EPS production by rhizobacteria has been shown to improve permeability by increasing soil aggregation and maintaining higher water potential around the roots, thereby increasing in the uptake of nutrients by plant with an increase in plant growth and protection from drought stress (Selvakumar et al., 2012). We therefore, hypothesized that rhizobacteria isolated from arid rhizosphere soil may mitigate and support plant growth under drought stress condition. To address this hypothesis, we isolated drought tolerant and ACC deaminase producing Pseudomonas spp. strain FBKV2 from eggplant arid rhizosphere soil and evaluated for growth promotion of maize seedlings under drought stress.

2. Material and methods 2.1. Isolation and screening of drought tolerant Pseudomonas spp Rhizobacteria were isolated from rhizosphere soils of maize (Zea mays L.), okra (Abelmoschus esculentus L.), eggplant (Solanum melongeana L.), tomato (Solanum lycopersicum L.), green gram (Vigna radiate L.), peanut (Arachis hypogaea L.) and red gram (Cajanus cajan L.) collected from arid and semi-arid regions in India with a precipitation range of 129–243 mm (during summer season, 2015) across the sampling sites (DES, Telangana, 2015). The crops were grown under rain-fed production system and plants at flowering stage were uprooted and the bulk soil was removed by gently shaking the plants. The root adhering soil (RAS) was collected by dipping the roots in containers containing sterile normal saline followed by shaking for 30 min. The soil suspensions were serially diluted, and the appropriate dilutions were spread plated on solid King's B medium. The plates were incubated at 28 72 °C and morphologically different colonies were picked and purified on respective media. The pure cultures were maintained on agar slants under refrigerated conditions for further experiments. In order to screen the isolates for drought stress tolerance, TSB

with different water potentials (
0.05,
0.15,
0.30,
0.49,
0.73, and
1.03 MPa) was prepared by adding appropriate concentrations of PEG 6000 (Sandhya et al., 2009) and inoculated with the overnight-grown broth cultures adjusted to optical density (OD) of 0.5 at 600 nm. Growth of the isolates at various stress levels was estimated by measuring the OD at 600 nm after incubation at 28 °C for 24 h, under shaking conditions. 2.2. Screening for plant growth promoting activities Isolates which able to grow at maximum negative water potential (
1.03 MPa) level were tested for plant growth promoting traits under control and drought stress condition. To determine phosphate solubilization under control, Pikovskaya’s broth (Himedia, India) was inoculated with 1% of overnight culture (0.5 OD at 600 nm) raised in Luria Bertani (LB) broth and for drought stress Pikovskaya’s broth with desired water potential (
1.03 MPa) was inoculated and incubated for seven days at 28 °C on an incubator shaker. The cells were harvested by centrifugation at 2655 g for 5 min and the supernatant thus obtained was used for the quantitative estimation of phosphate (Fiske and Subbarow, 1925). 2.3. Indole-3-acetic acid LB broth (control and drought stress) amended with 5 mmol tryptophan was inoculated with 1% of overnight culture (0.5 OD at 600 nm) raised in LB broth and incubated at 28 °C for 48 h on incubator shaker. Cells were harvested by centrifugation at 2655 g for 5 min and the supernatant was mixed with Salkowsky reagent, followed by incubation for 1 h at room temperature under dark conditions. The absorbance of pink color was read at 530 nm (Gordon and Weber, 1951). The concentration of proteins in the pellet was determined by Bradford method (Bradford, 1976), and the amount of IAA produced was expressed as mg/mg cell protein. 2.4. Siderophore and HCN production To determine siderophore production under control and drought stress Chrome Azurol S (CAS) broth cultures were prepared, inoculated with 1% bacterial cultures, incubated at 28 °C for five days and checked for development of orange color (Schwyn and Neilands, 1987). HCN production under control and drought stress was tested in King’s B broth amended with 0.4% glycine and Whatmann No.1 filter paper strips soaked in 0.5% picric acid in 2% sodium carbonate were hanged in test tubes, sealed with Para film and incubated at 28 °C for four days. Conversion of strips from yellow to brown color is positive for HCN production (Bakker and Schipper, 1987). 2.5. 1-Aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity For qualitative analysis bacterial isolates were grown in LB broth and cell pellets were collected by centrifugation, washed, suspended in sterile water and spot inoculated on Dworkin and Foster (DF) salt minimal medium (Dworkin and Foster, 1958) alone (negative control), DF supplemented with 3 mmol ACC as the main source of nitrogen and DF amended with (NH4)2SO4 (positive control). In order to screen ACC deaminase activity under control and drought stress selected isolates were grown individually in liquid DF minimal medium alone, DF þACC and DF þ(NH4)2SO4 and their growth were measured at 600 nm. To measure ACC deaminase activity, isolates were grown in 5 mL of LB broth at 28 °C until they reach stationary phase. To induce ACC deaminase activity under control and drought stress conditions, the cells were collected by centrifugation, washed

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twice with 0.1 mol Tris–HCl (pH 7.5), suspended in 2 mL of DF minimal medium either supplemented with 3 mmol final concentration of ACC without PEG (control) or with PEG 6000 (drought stress) and incubated at 28 °C with shaking for another 36–72 h. ACC deaminase activity was determined by measuring the production of α-ketobutyrate and ammonia generated by the cleavage of ACC by ACC deaminase according to the method of Penrose and Glick (2003). 2.6. SDS-PAGE analysis of ACC deaminase protein Induction of ACC deaminase protein under control and drought-stress was studied by Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE). Cell proteins were extracted using Qproteome Bacterial Protein Prep Kit (QIAGEN, USA). The protein samples (15 mL each) were loaded in 12% acrylamide gel using mini-gel Electrophoresis cell (BioRad, India). The gels were stained with 0.25% Coomassie brilliant blue R-250 and the protein profiles of negative control (without N-source), ACC samples (ACC as N-source) and positive control ((NH4)2SO4 as N-source) were compared. 2.7. Identification and characterization of bacterial isolates The selected bacterial isolates were subjected to microscopic, morphological and biochemical characterization according to Bergey's manual of determinative bacteriology. For molecular characterization, bacterial genomic DNA was isolated (Chen and Kuo, 1993), and the 16 S rRNA gene was amplified by polymerase chain reaction (PCR) using universal forward (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse (5′-AAGGAGGTGATCCAGCCGCA3′) primers under standard conditions (initial denaturation, 94 °C for 5 min; 30 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 40 s, extension at 72 °C for 90 s; final extension at 72 °C for 7 min). The PCR product (approx. 1500 bp) was purified and sequenced (SciGenom Labs, India). The sequence (16 S rRNA gene) obtained was compared with the existing database of 16 S rRNA gene and the sequence was submitted to GenBank. 2.8. Evaluation of selected isolate for growth promotion of maize under drought stress A pot experiment was carried out using maize (Zea mays L. var. DHM117) as a test plant under greenhouse conditions. The soil used for pot experiments belongs to the Chalkas series and has been classified as “red earths with loamy sub-soil” in the Indian soil classification system, which falls under the order Alfisols (Bhattacharyya et al., 2007). The soil was collected from the homogeneous horizon (0–20 cm) of College Farm (Field soil), PJTSAU Campus, Rajendranagar, Hyderabad, India, a semiarid region under rain-fed production system. The soil was air-dried and sieved (o 2 mm) before being analyzed for the physicochemical properties. The soil contained 73.2% sand, 5.6% silt, 21.2% clay with 1.43 Mg m
3 bulk density, 2.52 Mg m
3 particle density, 38.4% total porosity, and 39.2% water holding capacity; it had a pH of 6.8 with an electrical conductivity of 0.13 dms; the soil contain Ca2 þ , Mg2 þ , K þ and Na þ in the ratio of 3.2, 2.1, 0.31, 0.25 cmol (pþ)/kg with CEC of 16.2 cmol (p þ )/kg. The biological properties (primary consumers) of red earths with loamy sub-soil include bacteria (2  105 cfu/g soil), fungi (3  102 cfu/g soil) and actinomycetes (2  102 cfu/g soil). The organic C, total N, and total P content of soil were, 0.89 g/kg, 0.13 g/kg, and 0.09 g/kg, respectively. Seeds were surface sterilized with 0.1% HgCl2 followed by 70% ethanol for 1 min and 5–6 times of washings with sterile water and sown in plastic pots (surface-sterilized) filled with 500 g

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sterile soil. After seedling emergence, 1 ml of bacterial culture containing approximately 108 cells was applied to the soil around each seedling. Both inoculated and un-inoculated treatments were replicated 12 times, maintaining two plants per pot. Soil moisture was maintained constant during the experiment by daily sprinkling with sterile distilled water. Drying was induced in six out of 12 replicates by discontinuing watering. Water-stressed seedlings and their corresponding control seedlings were harvested on the sixth day of exposure to drying and were studied for the physiological and biochemical status of the plant. Root, shoot length, and dry biomass were recorded according to standard protocols. 2.9. Plant physiological parameters Free proline content was determined by the rapid colorimetric method according to Bates et al. (1973). Fresh leaf samples were homogenized in sulfosalicylic acid and filtered with Whatman filter no. 2. The filtrate was reacted with acid Ninhydrin reagent and glacial acetic acid in a test tube at 100 °C for 1 h. The reaction was terminated by keeping the test tubes in ice water bath. The reaction was extracted with toluene and the chromophore aspirated from the aqueous phase, and absorbance was read at 520 nm. The proline concentration was calculated from standard curve prepared by using pure proline. The sugar content was determined by extracting 1 g of leaf sample with methanol: chloroform: water (60:25:15 v/v) mixture at 60 °C for 2 h. The clear supernatant was treated with phenol sulfuric acid and the absorbance was read at 490 nm. The amount of sugar was calculated from a standard curve prepared using glucose (Dubois et al., 1956). For estimation of total chlorophyll, leaf samples were immersed in dimethyl sulfoxide (DMSO) and incubated at 70 °C for 4 h. The absorbance of the solution was then read at 645, 663, and 480 nm (Barnes et al., 1992). Starch content in the leaf sample was homogenized and the pellet was resuspended in dimethyl sulphoxide: 8 M hydrochloric acid (4:1 v/v) and incubated at 60 °C for 30 min and estimated according to the method of Ait Barka et al. (2006). For determination of total amino acids, 1 mL of leaf extract was treated with 1 mL of 0.2 M citrate buffer (pH 5), 1 mL of 80% ethanol, and 2 mL of the Ninhydrin reagent (1% Ninhydrin and 0.006% in 100 ml acetone) followed by incubation at 95 °C in a water bath for 15 min. The samples were cooled to room temperature and absorbance read at 570 nm (Chen et al., 2007). For measuring relative water content (RWC), the leaves were cut into small discs of 1.5 cm2 and fresh weight (FW) was recorded immediately, followed by hydrating the sample overnight by immersing in water and turgid weight (TW) was recorded after blotting the leaf sample gently. The samples were dried at 70 °C until constant dry weight (DW) was observed. RWC was calculated according to the formula RWC (%) ¼[(FW–DW)/(TW–DW)]  100 (Teulat et al., 2003). 2.10. Biofilm formation assay A qualitative assay for biofilm formation was investigated using a crystal violet assay as previously described (Christensen et al., 1985; Déziel et al., 2001). Briefly, bacterial cultures were grown in TSB broth under control and drought stress condition (
1.03 MPa) and incubated statically at 28 °C. After 24 h, the liquid medium was removed, and the bacterial biofilm was visualized by staining plates with 0.01% aqueous solution of crystal violet for 10 minutes at room temperature. Biofilm formation was considered positive when a visible purple color appears on the wall and bottom of the plates. Biofilms were quantified by crystal violet staining followed by ethanol solubilization and OD measurement at 600 nm (Ơ Toole and Kolter, 1998; Déziel et al., 2001).

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2.11. Root colonization Bacterial colonization on root surface was studied by scanning electron microscopy (SEM). Root samples were washed gently with sterile saline solution to remove the soil particles and were fixed in 2.5% glutaraldehyde (prepared in 0.05 M phosphate buffer, pH 7.2) for 24 h at 4 °C and post-fixed with 2% aqueous osmium tetroxide in the same buffer for 2 h. After the post fixation, samples were dehydrated in series with graded alcohol. Dried samples were mounted on the stubs and coated with gold using an automated sputter coater (JEOL JFC-1600) for about 3 min. Finally, samples were analyzed by scanning electron microscope (Model: JOELJSM 5600, JAPAN) at various magnifications at Ruska Lab, PJTSAU, Hyderabad, India. 2.12. Stomatal aperture size To determine the stomata opening, a 1 cm2 peel was removed from the lower side of the leaf (six days after exposure to drought stress) and stained with Safranin (0.25%) for 2 min and examined the stomatal aperture size under a compound light microscope. Stomatal aperture size was measured using ocular and stage micrometer. 2.13. Statistical analysis Data were statistically tested by analysis of variance (ANOVA) followed by Tukey's multiple comparison tests using SPSS software (SPSS Inc. version 20.0). For plant experiments randomized block design (RBD) was employed to investigate an error in experimentation with a 2  2 factorial arrangement that includes: two conditions (Irrigated and drought) and two treatments (control: devoid of bacterial inoculation, inoculation: strain FBKV-2). Each treatment was analyzed with six replicates and the standard deviation was calculated and data expressed as the mean 7SD of six replicates.

3. Results 3.1. Isolation and screening of drought tolerant rhizobacteria A total of 10 fluorescent pseudomonads were isolated from rhizosphere soil of different crops grown in arid and semiarid regions of India. All the isolates were screened for drought stress tolerance using PEG 6000, among 10, only three isolates FBKV-2, FGNUT-1, and MZ-3 isolated from rhizosphere of eggplant, peanut and maize were able to grow at maximum water potentials of
1.03 MPa. Isolates able to grow at maximum drought stress (
1.03 MPa) level were screened for PGP traits under control and drought stress condition. All the three isolates produced IAA, P-solubilization, siderophore and HCN production under control and drought stress condition. However, a significant reduction in PGP traits was observed under drought stress (Table 1). Isolate

FBKV-2 produced maximum amount of IAA both under control and drought stress conditions followed by MZ-3 and FGNUT-1 respectively (Table 1). The amount of P-solubilization was significantly high in FBKV-2 both under control and drought stress conditions compared to other isolates (Table 1). Siderophore production was observed in all the three isolates under control whereas, under drought stress siderophore production was observed only in FBKV-2. Furthermore, hydrogen cyanide production was also observed in strain FBKV-2 under both control and drought stress conditions (Table 1). All the three isolates utilized ACC as sole source of nitrogen however, variation in efficacy to utilize ACC was observed (Fig. 1a, b, and c; Table 2) between the isolates. FBKV-2 showed higher growth under control followed by FGNUT-1 and MZ-3. Similarly, under drought stress condition isolate FBKV-2 showed significantly higher growth whereas, no growth was observed with FGNUT-1 and MZ-3 (Table 2). The ACC deaminase activity was assayed under both control and drought stress conditions by quantifying the amount of α-ketobutyrate produced during the deamination of ACC by the enzyme ACC deaminase. Isolate FBKV-2 utilized ACC as the sole source of nitrogen by producing ACC deaminase enzyme under both control and drought stress conditions (Table 2). Biochemical assay of ACC deaminase revealed the secretion of this enzyme by FBKV-2, which was further confirmed by SDS-PAGE analysis under both control and drought stress conditions. Protein profile of FBKV-2 revealed the presence of 42 KDa polypeptide in the culture supplemented with ACC as substrate. Whereas, such protein bands were absent in culture devoid of ACC indicating ACC as an inducer for the synthesis of ACC deaminase enzyme (ACCD) (Fig. 1d and e). 3.2. Identification and characterization of FBKV2 The best isolate selected on the basis of drought stress tolerance and PGP traits production under drought stressed condition was characterized based on microscopic, morphological, and biochemical studies. Microscopic studies revealed that the strain FBKV2 was Gram negative, motile, rod-shaped bacteria. On King's B medium isolate appeared as creamy, smooth, shiny, circular, convex colonies with greenish pigmentation under ultra-violet light. The strain FBKV-2 utilized citrate, trehalose, xylose, melibiose and positive for catalase, oxidase, esculin hydrolysis, lysine, and ornithine decarboxylation. On the basis of 16s rRNA gene sequence analysis strain FBKV-2 was identified as Pseudomonas putida and the nucleotide sequence was submitted to NCBI GenBank under accession No. KT311002.1. 3.3. Evaluation of FBKV2 for plant growth promotion of maize seedlings under drought stress Effect of P. putida strain FBKV-2 on plant growth, physiological and biochemical status under drying conditions was demonstrated using maize (var. DHM117) as a test plant under controlled condition. The soil moisture content of the pots was maintained at 100% water holding capacity (WHC). Soil moisture (13.6% of dry

Table 1 Plant growth promoting traits exhibited by Pseudomonas spp. Isolate

IAA (mg/mg protein) NS

FBKV-2 MZ-3 FGNUT-1

Pi-solubilization (ppm) DS

a

37.43 7 1.53 31.00 71.90b 28.40 71.61c

NS a

23.41 71.60 19.66 72.30b 17.217 1.54c

Siderophore DS

a

5.09 7 1.54 3.46 7 0.91b 4.26 7 0.42c

a

3.007 1.07 2.317 0.46b 2.917 0.26c

HCN

NS

DS

NS

DS

þ þ þ

þ – –

þ – –

þ – –

NS, non-stress; DS, drought stress; IAA, indole acetic acid; Pi, inorganic phosphate; HCN, hydrogen cyanide production. þ, positive; -, negative. Data were analyzed by ANOVA analysis followed by Tukey's multiple comparison test. Values are means of 7 SD, n ¼6. Values with different letters are statistically significantly different at P ¼0.05.

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Fig. 1. Screening of bacterial isolates for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. (a) DF minimal medium without nitrogen source (negative control), (b) DF minimal medium with ACC as nitrogen source, (c) DF minimal medium with (NH4)2 SO4 as nitrogen source (positive control). Arrows indicate growth of isolates on DF þACC. SDS-PAGE analysis of ACC deaminase protein under non-stress (d) and drought stress (e). Arrow indicates  42 KDa additional polypeptide band in ACC supplemented cultures under both non-stress and drought stress conditions.

Table 2 Screening of bacterial isolates for 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase and enzyme activity under control and drought stress (
1.03 MPa) conditions. Isolate

Negative Control (OD at 600 nm)

ACC (OD at 600 nm)

Positive Control (NH4)2SO4 (OD at 600 nm)

ACC Deaminase Activity ACC mmol/mg protein/h α-ketobutyrate

NS

NS

NS

NS

DS a

FFBKV-2 0.22 7 0.03 MZ-3 0.25 7 0.06b FGNUT-1 0.147 0.05c

0.08 7 0.001 ND ND

DS a

DS a

1.43 7 0.25 0.692 7 0.24 2.147 0.09 0.81 7 0.10b ND 1.58 7 0.14b 0.90 7 0.12c ND 2.157 0.11c

a

0.89 7 0.12 0.417 0.10b 0.767 0.21c

DS a

3.06 70.38 1.2470.21b 1.517 0.26c

1.19 70.16 ND ND

NS, non-stress; DS, drought stress; ACC, 1-aminocyclopropane-1-carboxylic acid (ACC); ND, not detected. Data were analyzed by ANOVA analysis followed by Tukey's multiple comparison test. Values are means of 7 SD, n¼ 6. Values with different letters are statistically significantly different at P ¼0.05.

weight of soil) was maintained constant during the experiment by daily sprinkling with sterile distilled water. In un-inoculated seedlings, drying drastically affected the growth as indicated by stunted growth, reduced leaf area, less plant vigor, wilting and rolling of the leaves. However, seedlings inoculated with P. putida strain FBKV-2 showed better growth in terms of plant growth, increased chlorophyll content resulting in dark greenish leaves as compared to respective un-inoculated control treatments (Fig. 2a and b). The un-inoculated seedlings started wilting after four days of exposure to drying stress and died completely at the end of the sixth day. However, seedlings inoculated with P. putida strain FBKV-2 survived up to 10 days after exposure to drying stress and started wilting thereafter. Bacterial inoculation significantly enhanced the seedling growth in terms of root, shoot length, and dry biomass (Fig. 3) as compared to un-inoculated control under drying stress as well as under control condition. The response of maize seedlings to drying stress was studied by determining the physiological and biochemical status of the plants in terms of proline, total sugars, chlorophyll, starch, amino acids and protein content. Inoculation with P. putida strain FBKV-2 significantly improved proline accumulation in the leaves under

drying stress and control condition. Exposure of the seedlings to drying stress resulted in higher proline accumulation in leaf tissues of un-inoculated and inoculated treatments indicating that proline was produced by the plants as a response to the stress and inoculation with P. putida strain FBKV-2 further improved the proline levels than un-inoculated treatments respectively (Table 3). Similarly, under drying stress inoculated treatments showed significantly higher sugar content over un inoculated control whereas, inoculated treatments under control showed lower sugar content but statistically at par with respective uninoculated control. Furthermore, inoculation with P. putida strain FBKV-2 significantly improved amino acid concentration in leaves of maize seedlings both under control and drying conditions over un-inoculated control. A positive effect of bacterial inoculation was also observed on leaf chlorophyll content as compared to un-inoculated control under both the conditions (Table 3). A significant decrease in starch contents of leaves was observed on exposure to drying stress; however, seedlings inoculated with P. putida strain FBKV-2 showed significantly higher starch content under control and drying stress condition as compared to respective controls. Protein content also exhibited a similar trend with a significant

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Fig. 2. Maize seedlings as influenced by P. putida strain FBKV-2 inoculation under (a) non-stress and (b) drought stress. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

reduction under drying stress. However, bacterial inoculation improved protein content under control and stress conditions (Table 3). The positive influence of microbial inoculations was also observed on RWC content under both conditions. Inoculation with P. putida strain FBKV-2 significantly increased the RWC over uninoculated control under both control and drying conditions respectively (Table 3). 3.4. Biofilm formation

Fig. 3. Shoot, root length, and dry biomass of maize seedlings inoculated with P. putida strain FBKV-2 exposed to non-stress and drought stress condition. CNS, control non-stress; INS, inoculated non-stress; CDS, control drought stress; IDS, inoculated drought stress. X-axis ¼treatments; Y-axis on left ¼shoot and root length and on right (secondary axis) dry biomass. Y-axis on left indicates bars (shoot and root length) and right indicates circles (dry biomass). Data were analyzed by ANOVA analysis followed by Tukey's multiple comparisons test. Values are the mean7 SD, n ¼6. Values with different letters are significantly different at P¼ 0.05.

Biofilm formation was observed both under control and drought stress condition as a purple color structures inside the plates. Interestingly, the biofilm that continued to form under drought stress (
1.03 MPa) was found to coat the entire surface of the plate, suggesting that drought stress induced higher biofilm formation which was further, confirmed by quantification of the color formed under drought stress (OD 1.32 70.16) and controlled (OD 1.03 70.11) conditions. 3.5. Root colonization Formation of soil aggregates surrounding the roots indicates biofilm (bacteria connected by extracellular matrix) formation by P. putida strain FBKV-2 (Fig. 4a). To visualize the cellular mode of attachment of P. putida strain FBKV-2 to the root surfaces of maize seedlings, roots were viewed by scanning electron microscopy

Table 3 Effect of inoculation of P. putida strain FBKV-2 on biochemical and physiological parameters of maize seedlings. Treatment CNS INS CDS IDS

Sugars (mg/g FW) a

23.357 1.52 30.767 1.45b 28.66 71.56b 36.93 7 1.64c

Starch (mg/g FW) a

14.68 71.33 19.86 7 1.35b 17.75 7 0.53c 24.02 7 1.06d

Proline (mmol/g FW) a

1.65 70.92 2.47 70.46a 5.92 7 0.97b 11.65 71.10c

Chlorophyll (mg/g FW) a

3.15 70.87 5.10 70.45b 2.03 7 0.64c 4.55 7 0.81b

Amino Acids (mg/g FW) a

19.26 7 2.03 21.23 7 2.30a 23.737 1.52ba 28.63 71.32c

Proteins (mg/g FW) a

49.987 1.21 53.09 7 3.60a 38.41 73.37b 45.30 7 2.91c

RWC (%) 69.23 7 1.64a 74.99 7 2.23b 61.977 1.62c 68.677 2.65a

CNS, control non-stress; INS, inoculated non-stress; CDS, control drought stress; IDS, inoculated drought stress, RWC, relative water content. Data were analyzed by ANOVA analysis followed by Tukey's multiple comparison test. Values are means of 7 SD, n¼ 6. Values with different letters are statistically significantly different at P¼ 0.05.

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Fig. 4. SEM verification. (a) soil aggregates formation by inoculated P. putida strain FBKV-2, (b) and (c) SEM images showing bacterial colonization, arrows indicate P. putida strain FBKV-2, (d) control with poor soil aggregation (clean rhizoplane).

(SEM). We observed that P. putida strain FBKV-2 cells attached perpendicularly and horizontally to the root surface of maize seedlings in all the inoculated treatments (Fig. 4b and c). SEM analysis also suggested that P. putida strain FBKV-2 colonized the root surface and produced biofilm-like structures on root surface. 3.6. Stomatal aperture size Under drought stress condition stomatal apertures of maize seedlings were measured. Bacterial inoculation reduced the size of stomatal aperture (1.92 mm70.063) than un-inoculated seedlings (3.59 mm7 0.042) indicates that systemic drought tolerance significantly induced by P. putida strain FBKV-2 inoculation.

4. Discussion Plants are constantly exposed to abiotic stresses such as drought, temperature, floods, salinity etc. leading to poor performance and yield loss (Sandhya et al., 2010). Drought being a major abiotic stress may cause huge productivity losses in arid and semiarid regions where the agriculture totally depends on rains (Minakshi et al., 2013). Although the role of rhizobacteria in nutrient management and disease control is well known, their role in the management of abiotic stress is gaining importance (Yang et al., 2009; Dimkpa et al., 2009). Therefore, isolation of rhizobacteria from stressed ecosystems may result in the selection of stress tolerant strains that can enhance the growth and development of plants. In the present study, a total of 10 fluorescent pseudomonads were isolated from rhizosphere soil of different crops. All the strains were screened for drought stress tolerance using PEG 6000. Among 10, only three strains FBKV-2, FGNUT-1 and MZ-3 were able to grow at maximum negative water potentials of
1.03 MPa. Further, strains, which were able to grow at maximum drought stress were screened for PGP traits under control and drought stress conditions respectively. In our study among the three strains, P. putida strain FBKV-2 showed significantly higher IAA, phosphate solubilization, siderophore and

HCN production under both the conditions. Reports suggested that IAA producing microorganisms resulted in better root growth, helping in water and nutrient up take (Dimkpa et al., 2009), to cope with drought stress (Egamberdieva and Kucharova, 2009; Vurukonda et al., 2016). Similarly, phosphate solubilizing microorganisms alleviated drought stress in plants through accumulation of proline and chlorophyll content (Shintu and Jayaram, 2015). Further, microbial siderophore sequests iron in the root environment and enhances iron uptake (Dimkpa et al., 2009; Deshwal, 2012; Bholay et al., 2012) and HCN effectively control the growth of phytopathogens (DeCoste et al., 2010). Furthermore, PGPR that has ACC deaminase activity helps plants to withstand both biotic and abiotic stress (Glick et al., 1998; Ali et al., 2014). P. putida strain FBKV-2 showed higher ACC deaminase activity under control and drought stress conditions compared to other isolates. Protein profile of P. putida strain FBKV-2 revealed the presence of  42 KDa ACCD polypeptide under both control and drought stress condition. A similar polypeptide of 42 KDa was reported by Hotzeas et al. (2004) from P. putida UW4. Rhizobacterial efficacy is dependent on establishing an effective population density of active cells in plant rhizosphere (Martínez-Viveros et al., 2010). Rhizobacterial suspensions are prepared at densities of 108 to 109 CFU/ml for seed, root dipping and soil inoculation. After inoculation at these high densities, the cell numbers will undergo a rapid decline depending on whether or not the soil has been sterilized. In sterile soils, inoculants will typically persist at cell densities of 107 to 108 CFU/g soil for many weeks. In nonsterile soils where there is competition with the resident flora and predation by protozoa and nematodes, bacterial populations will decline rapidly by orders of magnitude per week until the population reaches equilibrium with its environment (Martínez-Viveros et al., 2010). In the present experiment P. putida strain FBKV-2 showed multiple PGP properties (IAA, HCN, siderophore and phosphate solubilization) and was selected to study the effect of inoculation on maize seedlings. P. putida strain FBKV2 inoculation resulted in increased shoot, root length and dry biomass under control and drought stress conditions compared to un-inoculated controls. The results showed that P. putida strain

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FBKV-2 inoculation had a more stimulating effect on growth and development of maize seedlings even under sterile soil conditions without other soil microbial communities. The possibility that the use of autoclaved (sterile) soil in the present experiment is to elaborate the growth promoting substances produced by the P. putida strain FBKV-2. The un-inoculated maize plants grew as well in sterile soil but, the growth promotional effect was more in P. putida strain FBKV-2 due to its multiple PGP properties. The results are in accordance with Gholami et al. (2009) where, A. brasilense DSM 1690 inoculation enhanced the maize plant growth in sterile soil similar, to A. lipoferum DSM 1691 inoculation in field soil conditions (Gholami et al., 2009). Rhizobacterial plant growth promotion is achieved by more than one PGP trait by the associated bacterium and helps plants tolerate to abiotic stress (Yang et al., 2009). Un-inoculated maize seedlings, due to the absence of rhizobacterial populations were more susceptible to drought stress effect. Moreover, a higher population of drought tolerant P. putida strain FBKV-2 on roots of inoculated seedlings may have stimulated root exudation, in turn stimulating the growth of inoculated bacteria with higher PGP activity in the rhizosphere (Sandhya et al., 2011). Biofilm formation was higher under drought stress as compared to control condition. Scanning electron micrograph revealed colonization of P. putida strain FBKV-2 on the root surface in all the inoculated treatments. Similar modes of attachment were previously reported for P. aeruginosa, P. aeruginosa strain AKM-P6, P. putida strain AKM-P7, P. putida strain GAP-P45 and Bacillus spp. on Arabidopsis, Sorghum, Wheat and Sunflower roots (Walker et al., 2004; Ali et al., 2009, 2011; Sandhya et al., 2009; Minakshi et al., 2013). Biofilm like structure was also observed on the root surface in some segments observed under scanning electron microscope. Biofilm formation on root surface can help the seedlings in moisture conservation and nutrient uptake under stress conditions (Minakshi et al., 2013). Plants adaptation to drought stress is associated with metabolic adjustments that lead to the accumulation of several compounds/ osmolytes like proline, sugars, polyamines, betaines, quaternary ammonium compounds, polyhydric alcohols and other amino acids and water stress proteins like dehydrins (Yancey et al., 1982; Close, 1996). Inoculation with P. putida strain FBKV-2 could compensate the drying effects and improve plant development through enhanced production of proline, amino acids, and soluble sugars and resulted in better absorption of water and nutrients from the soil. Seedlings treated with P. putida strain FBKV-2 showed higher accumulation of proline content under drying conditions. Inoculating plants with PGPR adds up to the existing concentrations of proline, a sizeable quantity of proline increased when maize plants were inoculated with P. fluorescens under drought stress (Ansary et al., 2012). Higher proline accumulation in inoculated plants indicates higher plant tolerance to water stress (Gusain et al., 2015). Drought tolerance of Lavandula dentate showed that PGPR B. thuringiensis (Bt) inoculation enhanced shoot proline accumulation when compared to control plants under drought stress (Armada et al., 2014). Similarly, tomato (Lycopersicon esculentum Mill) cv. Anakha treated with phosphate solubilizing bacteria (PSB) (Bacillus polymyxa) secreted excess proline to cope up with the drought condition (Shintu and Jayaram, 2015). The role of sugars in osmotic adjustment has been suggested (Mohammadkhani and Heidari, 2008). Interestingly, soluble sugar content was significantly higher in P. putida strain FBKV-2 inoculated seedlings over un-inoculated control. The accumulation of soluble sugars has been correlated with the acquisition of drought tolerance in plants (Hoekstra and Buitink, 2001). Inoculation of maize plant with PGPR P. putida GAP-P45 (Sandhya et al., 2010) and Azospirillum lipoferum (Bano et al., 2013) improved plant growth through accumulation of soluble sugars compared to

non-treated plants under drought stress. In the present study inoculation increased total starch content both under control and drying condition compared to un-inoculated control. The amino acids content has been shown to increase under drought conditions in crops like sorghum, pepper, and wheat (Yadav et al., 2005). The amino acid content in P. putida strain FBKV-2 inoculated seedlings was higher than un-inoculated seedlings under both the conditions. Increase in amino acids content is considered to be an indication of drought tolerance (Sandhya et al., 2011). Furthermore, inoculation increased total protein content under control compared to drying condition. The decrease in protein content is much higher in un-inoculated drought stressed seedlings. The chlorophyll content in the inoculated seedlings was higher as compared to respective control in both the conditions indicating the better physiological health of inoculated plants under stress conditions. High chlorophyll content has been linked with drought tolerance in many plants such as pea, maize, wheat, sorghum (Arunyanark et al., 2008; Zaeifizade and Goliov, 2009; Khayatnezhad et al., 2011; Minakshi et al., 2013). RWC is another important criterion for drought tolerance. In the present study, bacterial inoculation showed significantly high RWC as compared to un-inoculated treatment under both the conditions. Abscisic acid (ABA) accumulation is one of the most important responses of plants to drought stress. It plays an important role in plant water maintenance under drought stress conditions by inducing stomatal closure (Leung and Giraudat, 1998). In our study, bacterial inoculation decreased the stomatal conductance and increased the RWC in the leaves of maize seedlings. Results obtained are in accordance with the findings of other researchers and showed that the PGPR inoculation helps in stomatal closure resulting less transpiration and maintain RWC, and finally induce drought tolerance (Figueiredo et al., 2008; Cho et al., 2008; Sandhya et al., 2009, 2010).

5. Conclusion The present study supports that isolation of indigenous abiotic stress tolerant microorganisms from stressed ecosystems may be helpful in the development of bio-inoculants for the management of abiotic stresses in plants. P. putida strain FBKV2 isolated from eggplant arid rhizosphere soil showed multiple PGP traits under both normal and dry conditions. Further, inoculation of maize seedlings with P. putida strain FBKV2 enhanced seedlings growth in terms of shoot, root length, and dry biomass, improved cellular metabolites and stomatal conductance under dry condition compared to control. SEM analysis confirmed the colonization of P. putida strain FBKV2 on the root surface of maize seedlings. Subsequent studies under field conditions are necessary to evaluate the efficacy of this strain in natural settings, and in the presence of a complete soil food web.

Acknowledgment The authors are thankful to Science and Engineering Research Board (SERB), Govt. of India for providing the financial assistance in the form of “Start-Up Research Grant (Young-Scientist) – SB/YS/ LS-12/2014”.

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