Art06_Jha.indd Rhizobacteria regulate paddy physiology under salinity Journal of Applied Botany and Food Quality 85, 168 - 173 (2012) 1 N.V. Patel College of pure & Applied Sciences, S.P. University, V.V. Nagar, Anand (Gujarat), India 2 BRD School of Biosciences, Sardar Patel University, V.V. Nagar, (Gujarat), India Paddy physiology and enzymes level is regulated by rhizobacteria under saline stress Yachana Jha1*, R.B. Subramanian2 (Received May 16, 2012) Summary To investigate the physiological basis of salt adaptation in paddy due to inoculation with plant growth promoting bacteria, we compared their effect in paddy under saline and non-saline condition on root length, chlorophyll content, relative water content, stomatal conductance, membrane stability index and change in ascorbate peroxidase and nitrate reductase activities. In a pot experiment, the effect of endophytic and rhizospheric bacteria was studied in a local paddy rice (Oryza sativa L.) variety GJ-17 under salt stress. Our fi ndings suggest that inoculation with Pseudomonas pseudo- alcaligenes and Bacillus pumilus resulted in change of ascorbate peroxidase, nitrate reductase activity and plant growth parameters such as root length, chlorophyll content, RWC, stomatal conductance and membrane stability index under salinity. Mixture of both Pseudomonas pseudoalcaligenes and Bacillus pumilus revealed better response in paddy against the adverse effects of salinity. Introduction One of the most widespread agricultural problems in arid and semiarid regions is soil salinity, which make fi elds unproductive and decreases crop yield. Salinity becomes a concern when an excessive amount or concentration of soluble salts occurs in the soil or water. It has been reported that salinity limits plant growth and pro ductivity (ASHAF and FOOLAD, 2007). The linkage between belowground and above ground sections of ecological system depends mainly on root system (LIANG PENG et al., 2007). High concentration of salt in the root zone (rhizosphere) reduces soil water potential and the availability of water. As a result, reduction of the water content leading to dehydration at cellular level and osmotic stress is observed. The increased amounts of Na+ and Cl- in the environment effect the uptake of many indispensable nutrients through competitive interactions and by effecting the ion selectivity of membranes. The absorption function of root system is closely related to their morphology and activity. Moreover root systems can interact with the environmental stress under the ad- verse situation, and adjust the system to build up adaptation responses in morphology and physiology to strengthen its survival chance. The effect of salinity on root (AN et al., 2003) of plants had already been re ported. Many researchers reported that with an increase in salinity there was a decrease in the de velopment of the xylem. Decrease in development of xylem means decrease in loading of Na+ and also essential nutrients, results in shunted growth of plants. Plants in saline environment can protect themselves from Na+ toxicity through restricting Na+ entry; excluding Na+ from root cells; sequestering Na+ into vacuoles; or retrieving Na+ from the transpirational xylem stream for recirculation to roots (CHINNUSAMY et al., 2006). A strategy to acquire much water is essential for plant growth under water defi cit conditions. To overcome water defi cit, plants have developed mechanisms of physiological adaptation, such as improvement of water use effi ciency by regulation of stomatal closure, development of root system to acquire water, accumulation of osmoprotectants and control of water permeability by aquaporins (JANG et al., 2004). Salinity stress also decreases photosynthetic capacity due to the osmotic stress and partial closure of stomata. Salinity also affects the chlorophyll pigment, both chlorophyll a and chlorophyll b decreased under salinity in rice seedlings (DJANAGUIRAM and RAMADASS, 2004). Plants can also suffer from membrane destabilization and general nutrient imbalance. An important feature of salinity is the generation of reactive oxygen species (ROS) and free radicals, such as superoxide anion radical (O2-), singlet oxygen, hydrogen peroxide (H2O2) and hydroxyl radical (HO-) which cause oxidative stress to plants. Salinity promotes oxidative damage not only by direct increasing of the cellular concentration of reactive oxygen species but also by the diminution of the cellular antioxidant capacity (PINTO et al., 2002). To minimize the damaging effects of ROS, aerobic organisms evolved non-enzymatic defense systems (ascorbic acid, reduced glutathione, carotenoids, tocopherols, fl avonoids, alkaloids) and enzymatic protection mechanisms (superoxide dismutase, peroxi- dases, catalase). But in the last decade, there were number of reports on the benefi cial effects of microorganisms such as Pseudomonas, Bacillus, Pantoea, Burkholderia, Rhizobium etc. in enhancing the tolerance of crops such as sunfl ower, maize, wheat, chickpea, groundnut, spices and grapes to drought, salinity, heat stress and chilling injury under controlled conditions (ARSHAD et al., 2008). Plant growth promoting bacteria are free-living soil bacteria that can either directly or indirectly facilitate rooting (MAYAK et al., 1999). With this aim, the objective of this study to comparatively investigate the role of plant growth promoting rhizobacteria (PGPR) alone and in combination in inoculated rice seedlings under different level of salinity stress. We hypothesise that inoculation with PGPR, alone or in com bination, can confer salinity tolerance to paddy and that such tolerance is correlated with changes in plant physiology as chlorophyll content, RWC, stomatal conductance and membrane stability index and activity of enzymes (nitrate reductase and ascorbate peroxidase) by considering that such bacteria help the plant under adverse condition of salinity. Materials and methods Isolation and Identifi cation of PGPR Certifi ed seeds of rice variety GJ-17 were obtained from Main Rice Research Center, Navagam, Anand, Gujarat. These seeds were planted in pots and maintained for forty days. Microorganisms were isolated from the root tissue as well as rhizospheric soil. For isolation of endophytic bacteria from roots, fresh roots of paddy were surface sterilized with 70 % alcohol and HgCl2 for 5 min each, followed by washing with sterile distilled water. The root tissues were then homogenized in a sterile 4 % sucrose solution in mortar and pestle and the extract was used for isolation of bacteria. For isolation of rhizospheric bacteria, soil adhere with root surface were * Corresponding author Rhizobacteria regulate paddy physiology under salinity 169 collected and subjected to serial dilution, then both sample were plated on YEMA (Yeast Extract Mannitol Agar) medium. Various biochemical tests were performed (data not shown here) followed by 16S RNA ribo-typing to identify the isolates. The 16S rDNA Universal primers 8 F to 1510 R were used for amplifi cation of the DNA followed by sequencing for identifi cation of isolates (JHA et al., 2011b). The isolated endophytic Pseudomonas pseudoalcaligenes and rhizospheric Bacillus pumilus have good ability for phosphate solubilization as well as other plant growth promoting abilities (data already communicated), were selected for further studies. Rice cultivation and inoculation Seeds of rice variety GJ-17 were washed thoroughly with distilled water followed by surface sterilization with 0.1 % HgCl2 solution for 4 min and 70 % ethanol for 10 min. The seeds were washed thoroughly with sterile distilled water and kept in a shaker for 5 - 6 h in autoclaved distilled water on a rotary shaker. Later the seeds were transferred to Petri dishes containing tryptone glucose yeast extract agar medium and incubated in dark at 30 °C to test for possible con- tamination. The germinated seedlings devoid of any contamination were used for inoculation experiments. To study the effect of the isolated bacteria on the physiological and biochemical parameters selected, 4 days old germinated seedlings devoid of any contamination were transferred to culture tubes con- taining 400 µl Hoagland’s nutrient medium, 400 µl micronutrients and 1 % agar in 40 ml distilled water. Before the transfer, bacterial inoculums of the isolated bacteria Pseudomonas pseudoalcaligenes and Bacillus pumilus were added with the medium at a concentration of 6 x 108 cfu ml-1. To obtain a mixture of both bacterial cultures, an equal volume of both the cultures were mixed in the medium to give a concentration of 6 x 10 8 cfu ml-1. The tubes were incubated at 27 °C in a 12 h light-dark cycle in a growth chamber. Seven days old rice plants were carefully removed from different test tubes inocu- lated with the strain of bacterium, and planted in a pot. Similarly the control plants (un-inoculated) were also transferred to a fresh pot. The quantity of the soil possessing the following physio-chemical properties; pH 7.79, electrical conductivity 1063 µS/cm, CEC:3 cmol, organic carbon: 5500 mg kg-1 available nitrogen 200 mg dm-2, available Ca: 12.1 cmol, available P205: 9.5 mg dm-2, available K20, 265 mg kg-1, Fe, 3.1 mg kg-1, Zn, 285 mg kg-1, Mn, 3.7 mg kg-1, Cu, 2.2 mg kg-1 was maintained as 5 kg per pot. Rice seedlings were planted at the rate of 4 plants per transplant and 6 transplantations per pot. Pots were watered at the time of transplantation of the rice seedlings. All experiments were carried in 5 replicates. Maintenance of saline stress condition The saline condition was maintained at fi ve different salinity levels by adding (5g, 10g, 15g, 20g, and 25g NaCl L-1) saline solution to the pots. To avoid osmotic shocks, NaCl concentration was gradually increased for four consecutive days. A plastic bag was put underneath each pot to collect excess water due to drainage. This water was reapplied to the respective pot. All seedlings were grown for 5 weeks without any fertilizer treatment. The experiment was conducted in a greenhouse at 20 to 25 °C and the relative humidity 70 to 80 %. Effect of PGPR on morphology and anatomy of plant root Plants from each treatment after 35 days of sowing the seeds were collected carefully with plant root and cross sections of roots were examined under image analyzer microscope (Carl Zeiss) to analyze the effect of salinity on xylem in inoculated as well non-inoculated plant. Effect of PGPR on growth parameters under different salinity level The observation on physical parameters i.e., root length, chlorophyll content, RWC, stomatal conductance and membrane stability index were recorded from thee replicate from each treatment after 35 days of sowing the seeds. Total Chlorophyll was extracted from fresh leaves by 80 % acetone (v/v) and its contents were determined at 663 nm and 645 nm by a Hitachi U-2000 dual length spectrophotometer (ARNON, 1949). Stomatal conductance of plants was measured using fresh leaves by Li-Cor 6400. The N concentration was determined by colorimetric methods, after the Kjeldahl digestion. Relative Water Content Fresh weight (FW) and dry weight (DW) of leaves of each plant were determined after counting the leaf number. Leaf relative water content (RWC) was measured in the second or third youngest fully expanded leaf from top of the plant, using the following equations (SCHONFELD et al., 1988). RWC (%) = (FW-DW) × 100/ (TW-DW) where, FW is leaf fresh weight, DW is leaf dry weight after 24 h drying at 70 °C, and TW is leaf turgid weight after submergence in distilled H2O for 4 h. Plant dry weight was determined after oven drying at 70 °C until they reached constant weight. Membrane stability index Membrane stability index (MSI) was estimated as per SAIRAM et al., (1997). Fresh leaves (0.1 g) were taken in 10 cm³ of double distilled water in two sets. One set was subjected to 40 °C for 30 min and its electrical conductivity was recorded using an electric conductivity meter (C1). Second set was kept in a boiling water bath (100 °C) for 10 min and its conductivity was also recorded (C2). Membrane stability index (MSI) = [1 - (C1/C2)] × 100 Leaf enzyme extraction and effect of PGPR on enzyme activity Fresh leaves (2 g) were homogenized with a mortar and pestle at 4 °C in 4 ml of ice-cold 50 mM Tris-acetate buffer pH 6.0, con- taining 0.1mM ethylene diamine tetraacetic acid (EDTA), 5 mM cysteine, 2 % (w/v) polyvinylpyrrolidone (PVP), 0.1mM phenyl methyl sulphonyl fl uoride (PMSF) and 0.2 % (v/v) Triton X-100. The homogenate was centrifuged at 14,000 × g for 20 min and the supernatant fraction was fi ltered though Sephadex G-25 columns equilibrated with the same buffer used for homogenisation. Protein concentration was determined by taking optical density at 595 nm by spectrophotometer (Hitachi-220), using bovine serum albumin as a standard (BRADFORD, 1976). Nitrate reductase activity Nitrate reductase activity in leaves, roots and nodules was determined using the method of SYM (1983). Fresh plant ma terial (0.5 g) of the plant was homogenized in 5 ml phosphate buffer (pH, 7.0). After treating the sample extracts with 1 % sulphanilamide in 3 N HCl and 0.02 % N(1-Naphthyl ethylene diamine dihydrogenchloride), the optical density was read at 542 nm on a spectrophotometer (Hitachi- 220) and the NRA was calculated from a standard curve established with NaNO2 concentrations and expressed in produced µmol NO2 h-1 g-1 FW. All tests had been carried out in triplicate. Ascorbate peroxidase activity Ascorbate peroxidase activity was estimated according to the method of NAKANO and ASADA (1981). The reaction mixture consisted of 0.05 mol L-1 Na-phosphate buffer (pH 7), 0.5 mmol L-1 ascorbate, 0.1 mmol L-1 EDTA.Na2, 1.2 mmol L-1 H2O2 and 0.1 ml enzyme 170 Y. Jha, R.B. Subramanian Rhizobacteria regulate paddy physiology under salinity extract in a fi nal assay volume of 1 ml. The increase in absorption at A290 was recorded for 3 min. The concentration of oxidized as- corbate was calculated using extinction coeffi cient (= 2.8 mM cm-1). One unit of APX was defi ned as 1 mmol ml-1 ascorbate oxidized per minute. All tests had been carried out in triplicate. Data analysis Data was subjected to analysis of variance (ANOVA) using a statistical computer package SAS to determine whether the treat- ments effects were signifi cant. The treatment and variety means were separated using the least signifi cant differences (LSD) test. Results Identifi cation of Isolated isolates Biochemical and PCR amplifi cation of 16S rDNA indicate that isolated organisms are P. pseudoalcaligenes and Bacillus pumilus respectively having NCBI data bank accession nos. EU921258 and EU921259 respectively. Effect o PGPR on plant growth parameters under salinity In control plants (without NaCl), combination of both P. pseudo- alcaligenes and B. pumilus enhanced the root length as well as root hair development (Fig. 1), but no anatomical change was observed in the xylem vessels under salinity (Fig. 2). The overall results obtained from the present study indicate that inoculation of the two PGPRs viz. P. pseudoalcaligenes and B. pumilus either alone or in combination led to recovery of the plants from the saline stress (Tab. 1). In control plants (without NaCl) the combination of both P. pseu- doalcaligenes and B. pumilus enhanced the root length to 39 %, even upto 1 % NaCl root length increased by 22-29 % compared to non- inoculated plants. Chlorophyll content under non-saline state increased by 4.3 %, but under salinity it decreased both in inoculated as well as non- inoculated plants. Under salinity it decreased from 0.4-35 % in plant inoculated with both P. pseudoalcaligenes and B. pumilus in compared to pure control plants (at non-saline and non-inoculated), while in non-inoculated plants under salinity a decrease by 72 % was observed. Stomatal conductance also increased by 57 % at non-saline state and 19 % at 0.5 % NaCl, while a marginal decrease of 5.6 % was recorded at 1 % NaCl and 65 % decreased at 2.5 % NaCl in plant inoculated with both P. pseudoalcaligenes and B. pumilus in pure control plants. The concentration of N-substance was always higher in the plants inoculated with both the PGPRs in saline condition and a gradual increase of 3 % in N2 concentration was recorded at 1 % NaCl. In non-inoculated plants a decreased of 18 % - 54 % was recorded at higher salinity state compared to plants at non-saline state. While in inoculated plants 7.6 % enhanced N2 concentration was recorded at 1 % NaCl and gradually decreased upto 46 % compared plants at non-saline state. RWC (relative water content) in inoculated plants, increased by 29- 57 % at non saline state but under salinity, it increased by 10-14 % in plant inoculated with both P. pseudoalcaligenes and B. pumilus Fig. 1: Effect of inoculation of P. pseudoalcaligenes and B. pumilus on the root morphology (A) at 1.5% salinity, (B) at non saline state and (C) in non inoculated plant after 7 days of plantation. (P = P. pseudoalcaligenes, B = B. pumilus and C = Control). Fig. 2: (a) Effect of inoculation of P. pseudoalcaligenes and B. pumilus on the root xylem (2a) at 1.5% salinity, (2b) at non-saline state, (2c) in non-inoculated at non saline state. The scale bar = 25um (micron). Images are taken under 100x and the scales are calculated according to 100x magnifi cation. Rhizobacteria regulate paddy physiology under salinity 171 compared to non-inoculated control. Membrane stability index signifi cantly increased in inoculated plant both under non-saline as well as under salinity. MSI increased by 2.5-5 % at non-saline state and 6.7-9 % under salinity in the plant inoculated with PGPR compared to non-inoculated control plants. Effect of PGPR on enzymes activity Nitrate reductase activity was signifi cantly (P ≤ 0.0001) inhibited by NaCl treatment in non-inoculated control plant and inhibition increased progressively with an increase in NaCl concentration (Fig. 3). A decrease of 10-48 % was recorded in non-inoculated plants at different salinity compared to pure control (non-inoculated plants at non-saline condition). While plants inoculated with PGPR result in increased nitrate reductase activity under saline as well as non saline condition. The highest nitrate reductase activity was Tab. 1: Effect of PGPR on the root length, chlorophyll content, stomatal conductance, RWC and membrane stability Index of paddy variety GJ-17 at fi ve different levels of salinity (n = 5). % Salinity Treatment Root Length Total Stomatal N RWC % Membrane of irrigation Chlorophyll Conductance [g kg -1] Stability water water water Content [%] [mol m-2 s-1] Index 0.0 % 1. No inoculation 0.192 cd 43.3d 0.71d 19.1 d 90.2d 82.4cd NaCl 2. Inoculation with B. pumulis 0.211c 44.1c 0.84bc 20.4c 93 bc 84.1bc Control 3. Inoculation with P. pseudoalcaligenes 0.254ab 44.9ab 0.92b 23.8ab 94.7ab 84.7b 4. Inoculation with B. pumulis+ 0.263a 45.2a 1.12a 24.2a 96.3a 86.3a P. pseudoalcaligenes 0.5 % 1.No inoculation 0.184cd 39.8cd 0.54cd 22.4cd 78.1cd 74.5cd NaCl 2. Inoculation with B. pumulis 0.197c 40.3bc 0.62bc 23.1c 81.4 bc 76.2bc 3. Inoculation with P. pseudoalcaligenes 0.229b 41.2b 0.76b 26.3b 83.1b 77.1b 4. Inoculation with B. pumulis+ 0.235a 43.1a 0.85a 27.3a 86.1a 79.3a P. pseudoalcaligenes 1.0 % 1.No inoculation 0.167d 36.1cd 0.36d 23.1cd 65.4 cd 59.1 cd NaCl 2. Inoculation with B. pumulis 0.187c 37.2c 0.45c 24.3c 67.2 bc 61.2 bc 3. Inoculation with P. pseudoalcaligenes 0.231b 38.3b 0.58b 27.2b 68.7 ab 62.1 ab 4. Inoculation with B. pumulis+ 0. 248a 39.2a 0.67a 29.4a 69.8 a 64.1 a P. pseudoalcaligenes 1.5 % 1.No inoculation 0.154cd 32.2cd 0.23cd 18.3cd 43.1 d 38.1 cd NaCl 2. Inoculation with B. pumulis 0.161c 33.3c 0.31c 19.5c 45.2 bc 39.4 bc 3. Inoculation with P. pseudoalcaligenes 0.189ab 34.1ab 0.39 b 22.3ab 46.7 b 40.7 ab 4. Inoculation with B. pumulis+ 0.203a 34.9a 0.42a 23.2a 49.1 a 41.6 a P. pseudoalcaligenes 2 % 1.No inoculation 0.145 c d 29.3cd 0.15d 14.4d 31.1 cd 31.0 cd NaCl 2. Inoculation with B. pumulis 0.151c 30.1c 0.22bc 15.3c 32.4 bc 32.4 bc 3. Inoculation with P. pseudoalcaligenes 0.179b 31.3b 0.28b 17.1ab 33.8 ab 33.6 ab 4. Inoculation with B. pumulis+ 0.187a 32.2a 0.31a 17.9a 35.4 a 34.1 a P. pseudoalcaligenes 2.5 % 1.No inoculation 0.113d 25.4cd 0.07d 10.2cd 21.2 cd 22.1 cd NaCl 2. Inoculation with B. pumulis 0.127c 26.3c 0.13bc 11.3c 22.1 bc 22.8 bc 3. Inoculation with P. pseudoalcaligenes 0.149ab 27.2ab 0.18ab 13.6b 23.6 ab 23.4 ab 4. Inoculation with B. pumulis+ 0.157a 27.9a 0.23a 14.5a 24.2 a 24.5 a P. pseudoalcaligenes For each Parameter, values in columns followed by the same letter are not signifi cantly different at (P≤0.05). Fig. 3: Effect of inoculation with B. pumilus, P. pseudoalcaligenes alone and in combination on ascorbate peroxidase activity in paddy rice variety GJ-17 at fi ve different levels of salinity (n = 5). 172 Y. Jha, R.B. Subramanian Rhizobacteria regulate paddy physiology under salinity recorded in the plants grown in soil having 2.5 % NaCl and in- oculated with both the isolates. An increase of 6-18 % was observed in the plants inoculated with the PGPR at non saline condition. The PGPR inoculated plants showed increase in nitrate reductase activity by 4-45 % in plant inoculated with B. pumilus, 5-53 % in plants inoculated with P. pseudoalcaligenes and 4-50 % in plants inoculated with both the PGPR at different salinity level compared to plants at non saline condition. Ascorbate peroxidase activity showed that there is a gradual in- crease in activity as the concentration of NaCl in the soil increased (Fig. 4). The highest ascorbate peroxidase activity was observed in leaves of non-inoculated plants exposed to 2.5 % NaCl. The highest of ascorbate peroxidase activity was recorded in the non-inoculated control plant leaves at highest salinity level of 2.5 % which was 40 % high compared the plants grown under at non-saline condition. Inoculation of plants with PGPR resulted in 15-40 % decrease in activity compared to non-inoculated plants under non-saline con- dition. Among the two PGPR applied, P. pseudoalcaligenes showed reduction of 9-27 % and B. pumilus showed reduction of 2-15 %, but combination of both showed reduction of 16-40 % ascorbate peroxidase activity in all treatment compared to non-inoculated control. In the present study, chlorophyll content was also signifi cantly higher in plants inoculated with P. pseudoalcaligenes and B. pumilus at non saline condition, and different level of salinity, because these isolates infl uence better root development which enhance water absorption and retention, fi ndings are accordance with HAN and LEE (2005). Decrease of chlorophyll content in non-inoculated plant under salinity has been considered to be a typical symptom of oxidative stress and may be the result of pigment photo-oxidation, chlorophyll degradation or lack of chlorophyll synthesis (SMIRNOFF, 1993). Inoculations of plants with PGPR always have higher N-con- centration under saline and non-saline states as present study is supported by BASHAN et al. (2004). Azospirillum could result in signifi cant changes in various growth parameters, such as increase in plant biomass, nutrient uptake, tissue N content, plant height, leaf size and root length of cereals (BASHAN et al., 2004). An interesting observation of this study was that plants inoculated with PGPR under non saline as well as at different salinity levels has greater RWC and membrane stability index which is accordance with SANDHYA et al. (2010). An increased level of leaf RWC in paddy under salinity suggests the role of osmoprotectants in preventing cell injury from salt stress-induced dehydration, as suggested by YANCY et al. (1982). Non-inoculated plants has decreased RWC and membrane stability index with increased salinity. Membranes are main loci affected under water stress conditions. The lower membrane stability index refl ects the extent of lipid peroxidation, which in turn is a consequence of higher oxidative stress due to saline stress conditions. The nitrate and nitrogen nutrition is very important for plant growth (RAAB and TERRY, 1994). Nitrate reductase is not sensitive to osmotic effect, but sensitive to Na+ ions (GOUIA et al., 1994), the nitrate reductase activity (NRA) was used as an indicator of the damaging effects of NaCl. In present study, nitrate reductase activity increased in inoculated plant may be due to high concentration of nitrogen under saline and non-saline conditions, resulted in pro- duction of high amount of NH3. Our results are accordance with the fi ndings of HAMDIA et al. (2002) showed that nitrate reductase activity decreased with increasing salt stress. Nitrate reductase activity increased the concentration of NH3, used by a-ketoglutarate to form glutamic acid (EL-KOMY et al., 2003). High glutamic acid concentration may be used as a sink for the synthesis of other amino acids and proteins (WANG et al., 1999) or perhaps it may be directly used in osmoregulation (HSU et al., 1999). In our previous study, we also observed that these PGPR help in accumulation of osmoprotectants in paddy plants under salinity (JHA et al., 2011). Ascorbate peroxidases also play a vital role in plant defense against oxidative stress like superoxide reductase. Ascorbate per- oxidases are the key enzymes for scavenging hydrogen peroxide in chloroplast and cytosol of plant cells (ASADA, 1992). They catalyze the oxidation of ascorbate by hydrogen peroxide and give monodehydroascorbate radical. In present study inoculation of PGPR decreased the ascorbate peroxidase activity under saline and non-saline state are accordance with the Sandhya, who reported that Pseudomonas spp. treated seedlings showed decrease in ascorbate peroxidase activity, glutathione peroxidase activity, and catalase activity was higher compared to un-inoculated seedlings (SANDHYA et al., 2010). The decreased ascorbate peroxidase activity indicates that plants inoculated with PGPR were more relaxed under salinity. These enzymes proportions paralleled the changes in electrolyte leakage and were accordance with membrane stability function of these enzymes. Inoculation probably protected the seedlings where leaves could reduce water transpiration by curling and stoma regulation. The present study showed that P. pseudoalcaligenes, an endophytic Discussion Water stress caused by high salinity, is one of the serious factors to limit crop productivity. Osmotic stress caused by salinity is one of the major abiotic factors limiting crop productivity because it affects almost plant’s all functions. In the present study, plants inoculated with PGPR under non saline as well as at different salinity levels have greater root length and denser root hair is supported by BASHAN et al., 2004. Bacillus is very consistent in improving different root parameters such as rooting performance, root length and dry matter content of root in mint is reported by KAYMAK et al. (2008). The presence of denser root hairs has increased the surface area of root, which enhance water as well as mineral uptake (RAVEN and EDWARDS, 2001). An improved root growth was proposed as a possible mechanism by which PGPR affects plant growth (FALLIK et al., 1994). Salinity rapidly decreases stomatal conductance, re- sulted in reduced transpiration rate. Stomata closure is known to be an effective mechanism for economical water utilization under salt stress and for limitation of the harmful salt ions uptake (HASEGAWA et al., 2000). However inoculation of PGPR increased stomatal conductance under saline and non saline state to improve leaf water potential in adverse condition, observation is supported by MIA et al. (2010). Fig. 4: The effect of inoculation with B. pumilus, P. pseudoalcaligenes alone and in combination on nitrate reductase activity in paddy rice variety GJ-17 at fi ve different levels of salinity (n = 5). 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