Rhizosphere and endophytic bacteria for induction of salt tolerance in gladiolus grown in sodic soils

Abstract

Extensive development of canal irrigation system led to formation of unproductive sodic lands, which limited the cultivation of flower crop gladiolus in the basins of major rivers. The purpose of this study was to isolate native rhizospheric and endophytic bacteria from sodic environment and evaluate their growth enhancement and bio-ameloirant properties in gladiolus under sodic soils. Sixteen isolates of plant-growth-promoting rhizospheric and endophytic bacteria were isolated and screened for growth promotion potential. The promising strains were identified and evaluated for growth and production in gladiolus. The plants treated with strains CSR-G-1, CSR-B-2, and CSR-B-3 significantly produced marketable spikes and higher number of florets/spike. Also, the treated plants showed an increased activity of superoxide dismutase, phenyl alanine lyase, catalase, peroxidase, phenols, and proline than control. Further, the soil pH, total carbonates, and sodium adsorption ratio were lower in treated soils. The Na⁺/K⁺ ratio in leaves of treated plants was observed to be lower than control. In conclusion, these bio-inoculums can be used as growth enhancer and bio-ameliorant in sodic soils as an eco-friendly management strategy.

Keywords:

• rhizospheric and endophytic bacteria
• sodicity tolerance
• gladiolus cultivation

Introduction

The global trends warrant that in order to produce more food for the expanding world population, there will be an increase in the use of marginal-quality water and land resources (Bouwer 2000; Gupta & Abrol 2000). The problem of salinity and sodicity has been developed across the globe by the introduction of major irrigation schemes, which has reduced the agricultural productivity and sustainability. Over decades the northwest plains of the Indo-Gangetic basin and Yellow river basin of China have witnessed steady increase in sodic soils, which limited the production of agricultural crops (Gupta & Abrol 2000). Sodic soils are characterized by the soil with pH >8.5 and exchangeable sodium percent >15 with high concentration of free carbonates and bicarbonates. The hydraulic conductivity in such soils used to be poor and show high impedance to root growth (Qadir & Schubert 2002). These changes in soil properties affect the rhizospheric populations, thereby affecting the productivity of the commercial crops (Tank & Saraf 2010). Gladiolus is one such commercial monocotyledonous cut flower being cultivated for years along the river basins of major rivers of many countries (Wilfret 1992). It is grown well in the soils of pH 7.5–8.5 and is considered to be a highly remunerative crop for small and marginal farmers in developing countries. The development of sodicity in the river basins limited the cultivation of this crop due to its sensitivity toward sodicity (Damodaran et al. 2011). Chemical amendments such as mineral gypsum and pyrites being used in reclamation process ameliorated top 0–15-cm soil, leaving the physical properties like congestion of water and nutrients in rhizospheric soil unchanged (Damodaran et al. 2013). Many plant-growth-promoting rhizobacteria (PGPR) are known for providing protection to the plants from stressed environment (Zahir et al. 2008). Exploitation of epiphytic and endophytic bacteria in agricultural production depends on knowledge of plant–bacteria interaction and their potential to act as a beneficial micro-organism (Hallmann et al. 1997). Sometimes the interaction between the rhizospheric bacteria and plants can be unstable, where the repeatability of the good results obtained in laboratory is being limited under field condition (Cattelan et al. 1999; Zhender et al. 1999). Moreover, it is also known that the survival of micro-organism in stressed and contaminated habitat is only possible due to their ability of utilizing the resources available under that suitable niche (Madigan et al. 1998).

Therefore, with this background, the current research was undertaken to isolate the bacterial diversity associated with saline and sodic soils and evaluate their growth promotion potential, and also to identify the promising strains and assess their efficacy in the cut flower gladiolus, grown under sodic soil conditions.

Materials and methods

Collection of isolates

The soil rhizospheric and bacterial endophytes were isolated from roots, leaf, and culms of phyto-ameliorant grasses grown on the patches of undisturbed sodic soil with prominent salt efflorescence as described by Quadt-Hallmann et al. (1997). Pure cultures of endophytes were maintained on nutrient agar slants at 4°C.

Screening and selection of bacterial isolates

The rhizosphere and endophytic bacteria grown on nutrient broth with constant shaking on rotary shaker at 150 rpm for 48 h at room temperature (28 ± 2°C) were harvested by centrifugation at 6000 rpm for 15 min and bacterial cells were resuspended in phosphate buffer (PB) (0.01 M, pH 7.0). The concentration was adjusted using a spectrophotometer to approximately 10⁸ colony forming unit (CFU) (OD₅₉₅ = 0.3) and used as inoculums for treating rice seeds (Thompson et al. 1996). Plant-growth-promoting activities of bacterial strains were assessed based on the seedling vigor index by pot culture studies in saline sodic soils of pH 9.35 and electrical conductivity 4.4 dS/m², sodium (Na⁺) 18.65 meq/l, and potassium (K⁺) 0.126 meq/l. The vigor index was calculated by using the formula as described by Abdul-Baki and Anderson (1973):

Further, the promising strains were characterized and selected for microbial inoculation in gladiolus corms grown in sodic soils.

Soil analysis

The soil samples collected were air dried and ground to pass through 2-mm sieve, and a saturated extract of them was prepared. Various physicochemical parameters were determined in the extracts by adopting standard methods. The pH of the soil extract was determined potentiometrically by an ORION ion analyzer (5 star series; Thermo Orion, USA) using a pH electrode calibrated with a pH buffer of 7.0 and 10.0. Carbonate (CO₃) and bicarbonate (HCO₃) were determined by titrimetric method (acid–base titration) (Richards 1954). Calcium (Ca) and magnesium (Mg) were determined by versenate method (EDTA titration) (Cheng & Bray 1951). Sodium (Na) was determined by flame photometer (Richards 1954), while sodium adsorption ratio (SAR) was determined by the following generic equation:

Characterization of bacteria-based polymerase chain reaction amplification, restriction, and analysis of bacterial 16S rDNA

Bacterial DNA of best-performing strains were extracted according to the method given by Araujo et al. (2002), with amplification of 16S rDNA being performed in a 25-µl reaction mixture, which consisted of 25 ng genomic DNA, 10× reaction buffer with 15 mM MgCl₂, 2.5 mM each of dNTPs, 0.2 mM bacterial universal primer (27F-AGAGTTTGATCMTGGCTCAG) (1492R-GGYTACCTTGTTACGACTT), and 1 unit of Taq DNA polymerase (Bangalore Genei Pvt Ltd, India). The reaction was carried out in a thermal cycler (PTC-200, MJ Research Inc, USA). The DNA amplification program was set to a 5-minute initial denaturation at 94°C followed by 30 cycles of 1 min at 94°C for denaturation, 1 min at 54°C for annealing, 2 min at 72°C for extension, and ended with a final 10-min extension at 72°C. The reaction products were separated by running 5 µl of the polymerase chain reaction mixture in 1.2% (w/v) agarose gel and staining the bands with ethidium bromide (Sambrook et al. 1989). For amplified rDNA restriction analysis, 1 µg of amplified 16S rDNA fragment (1350 bp) was digested with Alu I, Hae III, EcoR I, and BamH I restriction enzyme (Invitrogen, USA) according to the manufacturer's recommendations and the products run in 2.5% (w/v) agarose gel and stained with ethidium bromide. The strains were identified by sequencing of partial 16S rDNA and comparing the sequence by BLAST.

Preparation of formulation and inoculation of microbial isolates

A loopful of bacterium was inoculated into the CSR patent-protected culture media (Rai et al. 2012) and incubated in a rotary shaker at 150 rpm for 48 h at room temperature (28 ± 2°C). After 48 h of incubation, the broth containing 6 × 10⁸ CFU/ml was used for treating the gladiolus corms at 0.2% for 2 h and for foliar application during four and eight leaved stages of the crop (Nandakumar et al. 2001).

Field study

The field experiment was conducted at the sodic soil experimental research farm of Central Soil Salinity Research Institute, Regional Research Station, Lucknow, India. The experiment was laid out in randomized block design (RBD) with a plot size 2 × 2 m, and each treatment consisted of 80 plants grown during two successive rabi seasons (September–December) of the years 2011 and 2012, where all agricultural practices were followed. The bulbs of gladiolus var. Nova Lux were planted in the first week of September in rows of 50 cm apart, 10 cm between, and 15 cm in depth. During the flowering period of each season, plant height (cm), number of leaves/plant, spike length (cm), number of florets/spike, and number of cormlets were recorded. The pre-experimental and post-experimental soil parameters were recorded.

Assay of stress-related enzymes and compounds

The activities of stress-related enzymes were assessed in leaves of the gladiolus plants at harvest. Peroxidase (PO) enzyme assay was carried out using the reaction mixture (3 ml) consisting of (0.25%) v/v guaicol in 10 mM potassium PB (pH 6.9) containing 10 mM hydrogen peroxide (Hammerschmidt et al. 1982). Polyphenol oxidase (PPO) was assayed using the modified method of Mayer et al. (1966) using 0.1 M PB (pH 6.5) and 0.01 N catechol along with enzyme extract. The phenyl alanine lyase (PAL) assay was conducted using 400 ml of 50 mM Tris–HCl (pH 8.8) and 600 ml of 1 mM L-phenylalanine along with enzyme extract as reaction mixture (Whetten & Sederoff 1992).

Enzyme extract was stored in a deep freezer (−20°C) until used for biochemical analysis. Superoxide dismutase (SOD) activity (EC 1.15.1.1) was determined as described by El-moshaty et al. (1993) and was expressed in units/g tissue. Total soluble phenols were estimated calorimetrically by using Folin ciocalteu reagent (AOAC 1985). Proline was estimated using sulpho salicylic acid extraction method (Bates et al. 1973).

Statistical analysis

The experiment was conducted in RBD and the data were analyzed using SAS 9.2 version. Prior to analysis of variance, the percentage values of germination were arcsine transformed. The pooled data for two seasons were subjected to significance at P < 0.05 and were compared by Duncan's multiple-range test.

Results

Plant growth promotion by bacterial strains

Sixteen rhizospheric and endophytic strains were isolated from the soil, roots, culms, and leaves of phyto-ameliorant grasses grown in barren sodic soils of pH from 9.7 to 10.37 and assessed for their efficacy as plant-growth promoters in pot culture experiment with rice crop. Among the 16 strains tested for their efficacy to improve the vigor index of rice seedlings under sodic soils of pH 9.35, 6 showed plant-growth-enhancing activity, and interestingly CSR-B-2 (Bacillus pumilus) strain showed 95% germination with a vigor index of 4566.5, while the vigor index of CSR-G-1 and CSR-B-3 strains were on par and showed 90% germination. Apart from this, the strains CSR-G-5, CSR-B-1, and CSR-G-4 also showed higher germination percentage of>70% and vigor index between 2355.2 and 3655.1. Conspicuously, some of the strains inhibited the growth of rice seedlings and those bacterial strains were discarded (Table 1) along with others which did not show any significant increase in the growth. Further, characterization using 16S rDNA for identification using partial sequences and microbial inoculation (bio-primed) in gladiolus corms was carried out with the effective bacterial strains (CSR-B-1, CSR-B-2, CSR-B-3, CSR-G-1, CSR-G-4, and CSR-G-5).

Table 1. Plant promotion activity of rhizospheric and endophytic bacteria in rice.

S. no.	Treatment	Germination (%)	Shoot length (cm)	Root length (cm)	Vigor index
1	CSR P-1	8.000^hij	14.400^fg	6.633^ab	140.9^h
2	CSR P-2	6.000^ij	13.000^g	5.833^c	105.8^h
3	CSR-B-1	74.330^d	28.383^c	8.033^ab	2762.7^d
4	CSR-B-2	95.000^a	35.133^a	7.633^ab	4566.5^a
5	CSR-B-3	90.000^b	32.967^b	8.333^a	4010.5^b
6	CSR-G-1	85.000^c	32.900^b	8.500^a	3655.1^c
7	CSR-G-2	29.667^g	14.000^fg	7.900^ab	616.6^g
8	CSR-G-3	40.000^f	18.367^e	6.533^ab	1001.0^f
9	CSR-G-4	90.000^b	34.333^ab	8.100^a	4132.5^b
10	CSR-G-5	70.000^e	25.333^d	7.033^ab	2355.2^e
11	CSR-G-6	10.000^h	15.667^f	6.233^ab	197.3^h
12	CSR-G-7	9.000^hi	14.967^f	6.133^ab	195.4^h
13	CSR-G-8	39.667^f	18.800^e	6.322^b	1005.5^f
14	CSR-G-9	5.000^jk	8.900^i	7.267^ab	61.6^i
15	CSR-G-10	6.000^ikj	10.166^h	6.027^ab	89.7^h
16	CSR-G-11	7.000^ij	14.666^f	6.158^ab	145.2^h
17	Control	2.333^k	5.667^j	6.333^ab	21.0^i

Notes: Values are the means of three replicates. Means in the columns followed by the same superscript letter are not significantly different according to Duncan's multiple-range test at P = 0.05.

Identification of promising bacterial strains

Bacterial strains isolated from the rhizosphere zone of sodic soils were found to be gram-negative type. Six strains with higher PGPR potential under sodic conditions were partially characterized by ARDA technique with Alu I, Hae III, EcoR I, and BamH I restriction enzymes, resulting in two groups. The strains were identified using 16S rDNA sequencing and validated with BLAST (ncbi.nlm.gov.blast/Blast.cgi) database. The results showed that all the strains belonged to Bacillus group where the strain CSR-B-1 was identified as Bacillus cereus, CSR-B-2 as Bacillus pumilus, CSR-B-3 as Bacillus thuringensis, CSR-G-1 as Bacillus subtilis, CSR-G-4 as Bacillus marisflavi, and CSR-G-5 as Bacillus saffensis, with accession numbers JQ768235, JQ768236, KF383226, KCA33669, KC433668, and JQ768237, respectively.

Effect of plant bio-priming

As observed, the presence of sodicity in soil reduced the growth characters of plant significantly in untreated control, while the treatments of promising bacterial strains relieved the plant rhizosphere from salt stress, resulting in improvement of growth characters (Table 2). Significant differences were observed between treatments in plant height and spike length. Plant height was maximum in CSR-G-1 (82.22 cm)-treated plants followed by CSR-B-3 (75.77 cm). Longer spikes were obtained in treatments involving CSR-B-3 (50.83 cm) followed by CSR-B-2 (48.66 cm), while the smallest (14.16 cm) being witnessed in untreated control. Application of rhizobacteria and endophytic strains increased the number of florets in treatments than the untreated control. Treatments involving the strains CSR-G-1 and CSR-B-3 recorded higher number of florets (8.111 and 8.057, respectively) than other strains, while the lowest number of florets (3.967) was observed in the untreated control. In general, a rapid and significant increase in the corm weight and corm diameter was witnessed in all the treatments compared to control. Among the treatments, plants treated with CSR-G-1 and CSR-B-3 strains recorded significantly higher corm weights of 30.220 and 29.590 g, respectively. Significantly higher corm diameter was observed in corms treated with CSR-B-3 (5.733 cm) followed by CSR-B-2 (3.200 cm).

Table 2. Efficacy of bacterial strains on morphological characters of gladiolus in sodic soils.

Treatments	Plant height (cm)	Spike length (cm)	No. of floret	Corm weight (g)	Corm diameter (cm)
CSR-G-1	82.22^a	48.66^ab	8.111^a	30.220^a	2.900^b
CSR-G-4	61.265^b	43.66^bc	6.165^ab	16.100^de	1.200^c
CSR-G-5	63.18^b	42.85^bc	6.517^ab	20.513^c	2.433^b
CSR-B-1	67.22^b	40.22^c	5.107^ab	16.582^d	2.300^b
CSR-B-2	72.44^ab	48.05^ab	6.887^ab	23.198^b	3.200^b
CSR-B-3	75.77^ab	50.83^a	8.057^a	29.59^a	5.733^a
Control	40.87^c	14.16^d	3.967^b	13.683^e	0.800^c

Notes: Values are the means of three replicates. Means in the columns followed by the same superscript letter are not significantly different according to Duncan's multiple-range test at P = 0.05.

Stress-related enzymes

The activities of the stress-related enzymes, SOD, PAL, catalase, PO, PPO, phenol, and proline, assessed in various treatments showed highly significant differences between treatments (Table 3). Invariably, the microbial inoculated plants showed nearly 2–3 times higher activity than the control. Among the six strains, CSR-G-1, CSR-B-2, and CSR-B-3 were found to be effective in inducing higher activities of these enzymes.

Table 3. Efficacy of stress-related enzymes' activity in gladiolus leaves at flowering.

Treatments	SOD (units/g) (tissue)	PAL (nmol/(min ml))	Catalase (mmol/(min g))	PO (abs/(min g))	PPO (abs/(min g))	Phenol (µg/g)	Proline (µg/g)
CSR-G-1	58.71^a	2067.33^c	30.733^b	2.054^b	1.846^a	2.263^b	254^a
CSR-G-4	37.01^e	976.50^e	12.500^e	1.643^c	1.680^a	1.583^c	202^b
CSR-G-5	41.97^d	1022.33^e	19.900^d	1.550^c	1.673^a	1.370^c	194^b
CSR-B-1	49.04^c	1277.00^d	17.333^d	1.696^c	1.564^a	1.696^c	136^c
CSR-B-2	50.81^c	2488.00^a	24.967^c	2.505^a	1.747^a	2.482^b	205^b
CSR-B-3	56.86^b	2280.00^b	43.000^a	2.382^a	1.793^a	2.693^a	244^a
Control	20.20^f	716.67^f	8.667^e	1.167^d	0.567^b	0.752^d	102^d

Notes: Values are the means of three replicates. Means in the columns followed by the same superscript letter are not significantly different according to Duncan's multiple-range test at P = 0.05.

Effect of microbial inoculation against sodicity in the field

Microbial inoculation of gladiolus corms before planting followed by foliar sprays during the critical growth periods revealed that treatments involving the strains CSR-G-1, CSR-B-2, and CSR-B-3 recorded the least symptoms of sodicity in their rhizospheric soil. A significant decrease in pH, total carbonate (CO₃ + HCO₃), and SAR between pre- and post-treatment was found. The maximum decrease in pH was noticed in CSR-B-3 (0.26) followed by CSR-B-2 (0.24) (Figure 1). The highest reduction in total carbonate was observed in CSR-B-3 (3.04)-treated rhizosphere soil followed by CSR-G-1 (1.66) (Figure 2). The decrease in SAR was the maximum in CSR-G-1 (1.46)-treated soil followed by CSR-B-3 (1.28) (Figure 3). The lowest change in the pH, total carbonate, and SAR was recorded in the rhizosphere soil of untreated control. The Na⁺/K⁺ ratio (Table 4) in the leaves of gladiolus was significantly higher in the untreated control (1.221) compared to the treated ones. Among the six strains, the lowest Na⁺/K⁺ ratio was recorded in the strain CSR-B-2 (0.437) followed by CSR-B-3 (0.507). Higher potassium contents were observed in plants treated with CSR-G-1 (2.246 meq/l) followed by CSR-B-3 (2.187).

Figure 1. Pre- and post-treatment effect of bacterial strains on pH of rhizosphere soils of gladiolus.

Figure 2. Pre- and post-treatment effect of bacterial strains on total carbonates (CO₃ + HCO₃) of rhizosphere soils of gladiolus.

Figure 3. Pre- and post-treatment effect of bacterial strains on sodium adsorption ratio of rhizosphere soils of gladiolus.

Table 4. Effect of microbial inoculation on the Na/K ratio in the gladiolus leaves.

Treatments	Na (meq/l)	K (meq/l)	Na/K
CSR-G-1	1.321^a	2.246^a	0.587^b
CSR-G-4	0.880^c	0.990^c	0.889^c
CSR-G-5	0.840^cd	0.920^c	0.913^c
CSR-B-1	0.960^c	1.070^c	0.882^c
CSR-B-2	0.847^cd	1.952^b	0.437^a
CSR-B-3	1.110^b	2.187^a	0.507^ab
Control	0.740^d	0.606^d	1.221^c

Notes: Values are the means of three replicates. Means in the columns followed by the same superscript letter are not significantly different according to Duncan's multiple-range test at P = 0.05.

Discussion

Gladiolus, a commercial cut flower, has been grown traditionally for years in the river basins of perennial rivers, and it suffered the salt injury due to the development of sodicity in its rhizosphere. The approaches of using gypsum and pyrite in reclamation of these sodic soils have not been able to change the physical and biological properties of the soil, which limited its cultivation. Furthermore, majority of the bio-products developed using microbes were restricted to function at the pH range of 7.0–8.5, which suggest the requirement of microbes to function at high pH (>8.5). Therefore, alternative biological approach of isolation of PGPR tolerant to high pH was attempted in the current study to induce salt tolerance in gladiolus grown in sodic soils. The use of endophytic bacteria for increasing the growth of plants in neutral soils (Kloepper et al. 1992; Joseph et al. 2007) and saline soils under pot experiment (Egamberdieva 2012) in tomato and cucumber has been earlier established in various studies. Yasmin and Hasnain (2001) and Chakraborty et al. (2011) also isolated salt-tolerant bacteria and tested for germination under laboratory conditions. As observed, the presence of salinity reduced the plant growth under pot culture studies in rice for vigor index, but the treatment with strains CSR-B-2, CSR-B-3, CSR-G-1, CSR-G-4, and CSR-G-5 relieved the rice plants from stress and made them to exhibit higher vigor index compared to the plants treated with other strains and untreated control. Similar results were also reported by Munns (2002), mentioning suppression of plant growth under saline stress, which may be due to water congestion in the surface soil and also the effect of sodium (Na⁺) and carbonates in the rhizosphere. Significant decrease in shoot and root length was also observed in the presence of high NaCl concentration in the root zone by Kashyap and Sharma (2006) and Nakbanpote et al. (2013).

The knowledge about the genetic relationship and its identity may be useful in determining further use of the promising strains in gladiolus for induction of salt tolerance. The identification was carried out using 16S rDNA sequencing and comparing them by BLAST database. Similar process was adopted in characterization by many earlier workers (Sturz et al. 2000; Kuklinsky-Sobral et al. 2004; Tank & Saraf 2010).

In the current study, the promising strains (CSR-B-2, CSR-B-3, CSR-G-1, CSR-G-4, and CSR-G-5) were tested for their interaction with their plant and soil in inducing salt tolerance in gladiolus, a salt-sensitive commercial crop grown in sodic conditions. High soil pH and water congestion result in poor development of underground corms and their germination. In this study, microbial inoculation of the corms enabled a favorable soil rhizospheric environment through inducing developmental and physiological changes in plants and their rhizospheric soils. Bio-priming of banana suckers of var. Pisang Awak with CSR-B-3 strain increased the tolerance of banana to sodicity (Damodaran et al. 2013). Similar result was also observed by Kavino et al. (2007) in roots of primary hardened-tissue-cultured banana plantlets for inducing tolerance to banana bunchy top virus.

Microbial inoculated gladiolus plants showed enhanced growth and yield characters like plant height, spike length, and number of florets than the untreated control. Similar influence on the growth and development was reported in horticultural crops like strawberry (Kokalis-Burelle 2003) and Prunus sp. (Bonaterra et al. 2003) and stevia (Vafadar et al. 2013). In the present study, other than floral characters, higher corm weight and corm diameter were observed in the bio-primed corms, whereas a decreased corm weight and corm diameter were found in untreated control. An overall reduction in control under salt stress was earlier observed by Soussi et al. (1998). This reduction in weight of plants under stress may be due to the inhibition or hydrolysis of reserved food and its translocation to growing shoot portion (Singh et al. 2008). The increase in root or corm character in microbial inoculated plants was witnessed earlier by Vivas et al. (2003) in lettuce inoculated with Bacillus spp. under salt stress. It may be postulated that effective rhizobacterial strain on inoculation decreases the endogenous ethylene levels because of aminocyclopropane carboxylate (ACC) deaminase activity, thereby resulting in the formation of longer roots (Nadeem et al. 2006).

The salt induced adverse effects on plants are primarily due to specific ion toxicity, production of oxidants, and so on (Ashraf 2009). Plants produce low-molecular-weight organic solutes such as proline or enzymes like SOD and PO to tolerate the effects of salt stress (Ali & Ashraf 2011). However, most plant species do not produce sufficient antioxidants to fulfill their growth requirements under salt stress. Therefore, bio-inoculation induces salt tolerance by increasing the activity of antioxidant enzymes (SOD, PO, and catalase) and organic solutes like proline (Kaya et al. 2013). The bio-inoculation also enhanced the defense mechanism of the plants due to the elevated level of the enzyme activity in the host plants (Ashraf & Foolad 2007). Inoculation of wheat seeds with Pseudomonas putida strain AKMP7 counteracted the adverse effect of heat on leaf antioxidant enzymes by lowering the reactive oxygen scavenger (ROS) generation compared to uninoculated plants (Ali et al. 2011). The microbial inoculated gladiolus plants produced more defense enzymes and proline than the untreated controls. The higher activities of PO, PPO, PAL, SOD, and proline content increased the ability of the plants to tolerate salt stress and formulate Na⁺ exclusion mechanism. Antioxidant enzymes in roots show inverse relationship with sodicity of the soil (Myrene & Devaraj 2010). Higher activity of antioxidant has been reported in mycorrhiza-treated plants of tomato in saline soils (Hajiboland et al. 2010).

The treatment with bacterial strains increased the uptake of potassium (K⁺) as compared to sodium (Na⁺), resulting in lower Na/K ratio. The treatment with bio-inoculum reduced the soil pH, total carbonates, and soil SAR in the rhizosphere soils, facilitating favorable plant growth and nutrient mobilization. The endophyte enhances the uptake of nutrients through biological processes (Handafy et al. 1995). The higher K accumulation by mycorrhizal plants in saline soils was reported to be beneficial in maintaining high K/Na ratio (Giri et al. 2007). Earlier studies on use of phyto-ameliorants attributed the secretion of higher organic acids in the rhizosphere due to the decomposition of the biomass, which helps in diluting the carbonates and also decreasing the pH of the soil (Treeby et al. 1989; Ghaly 2002).

Taken together, corm treatment and foliar application during the critical stage of growth using native rhizobacterial strains of CSR-B-3, CSR-B-2, and CSR-G-1 were significantly effective in reducing the adverse effect of salt stress (sodicity) in gladiolus under sodic soil conditions, resulting in improved production levels. These bio-inoculums may be easily incorporated into existing production systems as an eco-friendly management strategy and as a bio-growth enhancer to restore the production of commercial crops in salt-affected soils.

Acknowledgments

The authors thank National Agricultural Innovation Project, ICAR, India, and World Bank for generously funding the project under which the study was undertaken.