IntroductionTop

Soil salinity is among the most significant envi­ronmental stresses in agriculture, suppressing plant growth and productivity of crops worldwide (Hu & Schmidhalter, 2005). Under high salinity conditions, plant growth and photosynthesis are adversely affected due to the increased amount of ethylene in root, ionic imbalance and hyper-osmotic condition in plants (Niu et al., 1995; Mayak et al., 2004). Accumulation of salts in the soil decreases the osmotic potential, thereby interferes with water absorption by roots and affects cell growth and related metabolism. Additionally, high salinity levels are toxic to the plants due to the formation of reactive oxygen species (ROS) causing oxidative damage and as a result, decreasing plant development (Munns & Tester, 2008).

Treatment of plant seeds and seedlings with plant growth-promoting rhizobacteria (PGPR) is a new approach that has been developed in recent years to alleviate the adverse effects of salinity. PGPR are a group of free-living bacteria living in the plant rhizosphere, known for their beneficial impacts on growth and health of host plants (Lucy et al., 2004; Qin et al., 2011; Grönemeyer et al., 2012). These bacteria can either directly or indirectly enhance the growth of plants (Glick, 1995). In direct growth stimulation, these bacteria provide soluble phosphate, fix nitrogen, produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase and phytohormones like indole acetic acid (IAA) and increase the bioavailability of iron through bacterial siderophore for plants (Lucy et al., 2004; Glick et al., 2007a). Indirect promotion of growth occurs when these bacteria protect plants from detrimental effects of plant pathogens, e.g. by competition, antibiosis and hyperparasitism (Raaijmakers et al., 2009). PGPRs are also capable of increasing plant resistance to biotic and abiotic environmental stresses. One of the main mechanisms by which rhizobacteria exert positive effects on plants under abiotic stresses is production of ACC deaminase and regulation of ACC, a precursor to stress-induced ethylene in host plants (Glick et al., 1998; Glick et al., 2007a,b). The effectiveness of rhizobacteria with ACC deaminase activity has been reported in reducing stress ethylene, conferring beneficial effects to plants under various environmental stressors such as pathogen and insect infection, high salinity, drought and heavy metal contamination (Mayak et al., 2004; Yildirim et al., 2006; Glick et al., 2007a,b; Qin et al., 2014).

Previous research has revealed that inoculation with PGPR can alleviate the salinity stress effects in several plant species. PGPR inoculants examined on wheat, maize, tomato as well as cotton under salt stress has shown to ameliorate deleterious effects of salt stress (Mayak et al., 2004; Egamberdieva, 2007, 2009; Yao et al., 2010).

Considering the importance of salinity stress in Iran, the objectives of the present study were: (1) to study four native Pseudomonas fluorescens strains isolated from barley rhizosphere in Iran for ACC deaminase activity and tolerance to NaCl induced salinity under in vitro conditions, (2) to evaluate other bacterial plant growth-promoting activities including production of siderophore and indole acetic acid (IAA) and solubilization of phosphate, and (3) to determine the potential of PGPR strains to enhance growth and yield of five barley cultivars under different salinity levels in natural soil by conducting pot experiments.

Material and methodsTop

Microorganisms, plants, and culture conditions

Four Pseudomonas fluorescens strains (B10, B2-10, B2-11 and B4-6) isolated from the rhizosphere of barley plants in Bushehr province, Iran, were obtained from a culture collection of Biological Control Lab., Persian Gulf University, Iran. Bacteria were routinely cultivated at 27°C on Kingʼs B medium agar (KB) (peptone 20 g, K2HPO4 1.5 g, MgSO4-7 H2O 1.5 g, glycerol 20 g, and agar 15 g, pH 7.0) (King et al., 1954) and Luria-Bertani broth (LB; Difco, Detroit, MI, USA). Glycerol stock (20% w/v) of bacterial strains was prepared and stored at -80°C until further use. Seeds of barley (Hordeum vulgare L.) cvs. ‘Desert’, ‘Mid-day’, ‘Zehak’, ‘South’ and ‘Karun’, obtained from the Seed and Plant Impro­vement Institute (Karaj, Iran), were surface-disinfected in ethanol 70% (v/v), followed by 10 min in 10% (v/v) H2O2, rinsed thoroughly with sterile double-distilled water, and pre-germinated for three days on 0.85% water agar at 25°C in darkness.

ACC deaminase activity assay

The ability of bacterial strains to utilize ACC was evaluated based on Dell’Amico et al. (2005) with slight modifications. Bacteria were grown on tryptic soy broth medium (TSB) (TSB, Merck) for 48 hours. Thereafter, 50 µL of bacterial suspension was transferred to 20 mL of DF (Dworkin and Faster) minimal medium (Dworkin & Faster, 1958) containing 3 mM L−1 ACC as the selective medium, DF containing 2 g L−1 (NH4)2SO4 as positive control and DF minimal medium with no amendments (as negative control). After 48 h incubation at 27 ºC in shaker incubator with 120 rpm, an optical density at 405 nm was determined using spectrophotometer for each suspension. The ability of a strain to utilize ACC was verified by comparing the optical density (OD) of each bacterial suspension with that of negative control. The absence of growth in negative control confirmed the utilization of ACC as N source.

Salt tolerance assay

Salt tolerance test was conducted based on Qin et al. (2014) with slight modifications. Bacteria were grown on modified mineral-based nutrient agar (CaSO4 0.1 g, K2HPO4 0.2 g, peptone 1 g, MgSO4·7H2O 0.2 g, agar 15 g in 1000 mL distilled water) amended with increasing concentrations of NaCl (0–10%, w/v) at intervals of 1% at 27 °C.

Determination of other plant growth-promoting traits

Isolates were screened for siderophore and IAA production and solubilization of phosphate based on Islam et al. (2009). Siderophores were detected on Chrome Azural S (CAS) agar plates by the formation of orange halos around bacterial colonies after incubation for 24 h at 27 °C. P-solubilization activity was tested on Pikovaskaya’s agar medium containing 2.5% tricalcium phosphate. The formation of transparent halo zone around bacterial colonies after incubation for 24-48 h at 28 °C was observed. Ability to produce IAA was detected using the colorimetric method of Bric et al. (1991). The IAA secreted in the culture was determined using a calibration curve of pure IAA (Sigma-Aldrich, St. Louis, MO, USA) as a standard following the regression analysis. IAA production assay was conducted in a completely randomized design with three replications. The experimental data were analyzed statistically using SAS program and means were separated by Duncan’s Multiple Range Test (DMRT).

Plant growth-promotion activity of Pseudomonas strains under salinity stress

The impacts of four effective Pseudomonas strains were evaluated on barley yield and growth under greenhouse conditions. Bacterial strains were grown in LB broth overnight. Cultures were washed with sterile 0.9% NaCl solution and adjusted to an OD at 600 nm of 0.125 that corresponds to cell density of about 108 colony forming units (CFU) mL−1. Before performing greenhouse experiment, root tip colonization of bacterial strains on barley cultivars under salinity (50, 100 and 150 mM NaCl) was assessed under gnotobiotic conditions as described by Egamberdieva (2011) with some modifications. It was revealed that our bacterial strains were capable of colonizing the rhizosphere of barley cultivars at the highest saline conditions (150 mM) (unpublished data).

For greenhouse experiment, after soaking pre-germinated barley seeds in the bacterial suspensions for 1 h, 25 seeds were transferred to plastic pots filled with 8 kg of sterilized soil. Soil sample for the study was collected from the experimental field at Persian Gulf University, Bushehr, Iran, air-dried, sieved (2 mm, 10 mesh) and analyzed for physico-chemical characteristics. The soil was clay loam having pH 6.2, organic matter 1.15%, total nitrogen 0.1%, total P 18 mg kg−1, and electrical conductivity 5.45 mS m−1.

Plants were watered when needed after emergence, and no fertilizers were used. Salinity conditions at four-leaf stage were established by adding 50, 100 and 150 mM NaCl into the irrigation water (sterile distilled water). Electrical conductivity (EC) of these solutions was 5 dS m−1 for 50 mM NaCl, 10 dS m−1 for 100 mM NaCl and 15 dS m−1 for 150 mM NaCl. Sterile distilled water with no addition of NaCl was considered as non-stressed controls. Plants were grown in the greenhouse at 25ºC and at a 16/8 day/night regime. After 15 days, thinning was done to leave 15 uniform seedlings in each pot. Morphological parameters like plant height, peduncle length, spike length, spike weight, number of grains per spike, 1000-grain weight (MGW) and grain yield were measured at physiological maturity stage.

Survival of bacterial strains in barley rhizosphere

The effect of salinity levels on the survival of inoculated strains in the rhizosphere of barley cultivars was determined under greenhouse conditions which were described above.

At harvest, plants were removed from the pots and gently shaken to discard loosely adhering soil. Roots with tightly adhering soil were weighted, placed in 10 mL sterile saline solution (0.9%), and shaken for 20 min at 420 rpm. The number of bacteria in the resulting suspensions was determined as CFU by plating serial dilutions on KB agar plates.

Experimental setups and statistical analyses

A pot experiment was performed in a split-split plot design based on randomized complete block with three replicate pots per treatment. Main plots, subplots and sub-sub plots were consisted of salinity (at three levels of 50, 100 and 150 mM), five varieties (‘Karun’, ‘Zehak’, ‘Mid-day’, ‘Desert’ and ‘South’’) and four bacterial strains, respectively. In the greenhouse experiment, controls were consisted of plants either not watered with NaCl solutions or not inoculated with bacterial strains. Each experimental unit was a pot consisted of 15 plants. Data were subjected to statistical analysis using SAS 9.1 (SAS Inst., Inc., Cary, NY, USA). Means were subsequently compared using the DMRT.

ResultsTop

ACC deaminase production

All selected strains from the rhizosphere of barley were able to grow on DF minimal medium amended with ACC, indicating that they have the ACC deaminase activities. Optical densities of Pseudomonas sus­pensions in minimal medium without nitrogen source (negative control), amended with 2 g of (NH4)2SO4 L−1 (positive control), or with ACC (3 mM L−1) are listed in Table 1.

Characterization of PGP properties of the bacteria and their tolerance to NaCl

The bacterial strains were screened for their plant growth-promoting (PGP) traits and the results are summarized in Table 2. All the four isolates produced IAA and siderophores and also had phosphate solubilizing activity. The NaCl tolerance of bacterial strains was tested, and the results revealed that all strains could tolerate 8% of NaCl stress after which no growth was observed. Results also showed that as the salt concentration increased, the bacterial growth decreased. Since the four strains showed high tolerance to 8% NaCl, they were selected to study their effects on barley yield and growth under saline stress.

Influence of PGP strains on barley yield and growth under saline stress

Data analysis of variance showed that the main effects of salinity, variety and bacterial strains and the interaction between them were significant on most measured traits (p≤0.05) (Table 3). Generally, as salinity increased, all measured traits decreased; however, inoculation of seeds with bacterial strains ameliorated salinity stress and resulted in an increase in barley growth parameters and yield comparing to non-inoculated control.

Under normal condition and salinity of 50 mM, isolate B2-10 (with 10% increase), at 100 mM NaCl stress, all bacterial strains and at 150 mM salinity B4-6 and B2-10 elevated plant height in comparison with other treatments (Table 4). Data regarding peduncle length and spike length showed that treatment with B2-10 strain significantly promoted these traits by 26-46% and 26-30%, respectively, under normal condition and salt stress (Table 4). Similarly, inoculation with B2-10 strain caused the higher number of grains per spike (with 12-14% increase) under all salinity levels. Under normal condition and at 50 and 150 mM, B2-10 strain increased spike number (20, 19 and 12%, respectively) significantly over untreated control; however, at 100 mM, B10 and B4-6 strains performed better (Table 4). B2-10 and B4-6 were the most effective isolates which enhanced MGW up to about 14%. The maximum spike weight was obtained under normal condition following inoculation of seeds with B2-10 strain comparing to other treatments. Under normal condition and all salinity levels, B2-10 strain had the maximum impact on increasing grain yield, with no significant difference to B2-11 under normal condition and 100 mM salinity (Table 4).

Barley varieties showed variations in plant height during NaCl induced salinity stress (Table 5). Under all conditions, ‘Karun’ showed the maximum plant height comparing to other varieties. ‘South’ showed the maximum spike length and peduncle length under all salinity levels. ‘Desert’ had the highest spike number at all salinity levels which was not significantly different to ‘Mid-day’ at 50 mM. ‘Mid-day’ had the highest number of grains per spike at all salinity levels comparing to other varieties. In all salinity levels, ‘Zehak’ had the highest MGW, which was not significantly different to ‘Karun’ and ‘Mid-day’ varieties at 50 and 100 mM salinity. Considering spike number, under normal condition, ‘Karun’ had the highest spike number which was not significantly different to ‘Desert’. Under salinity levels of 50, 100 and 150 mM, ‘Desert’ showed the maximum number of spikes, which was not significantly different to ‘Mid-day’. Considering grain yield, under normal condition and salinity of 50 and 150 mM, ‘Desert’ had the highest grain yield. Under salinity level of 150 mM, ‘Mid-day’ showed the maximum grain yield, which was not significantly different to ‘Desert’ and ‘Karun’ varieties (Table 5).

Inoculation of barley varieties with tested PGPR strains caused an increase in different measured traits comparing to uninoculated control; however, varieties did not show identical response to inoculation with different strains (Table 6). Inoculation of ‘Karun’ with B2-11 resulted in the maximum plant height as compared to other treatments. Seed inoculation of ‘Zehak’ with B2-10 (with 46% increase), and ‘South’ with B4-6 and B2-10 (with 27 and 25.6% increase, respectively) increased peduncle length more significantly than the other treatments. The maximum spike length was observed following the inoculation of ‘South’ with B2-10, which was not significantly different to the treatment of ‘Desert’ with B4-6 strain. Inoculation of ‘Mid-day’ and ‘Desert’ with B2-10 resulted in the highest spike number as compared to other treatment. The highest increase in the number of grain per spike was observed in ‘Mid-day’ following seed inoculation with B2-11 strain. Inoculation of ‘Zehak’ with B10 caused the maximum MGW, which was significantly different to all treatments. The maximum increase in spike weight was seen in ‘Mid-day’ and ‘Karun’ after seed inoculation with B2-10 strain. Inoculation of ‘Karun’ with B2-10, B4-6 and B10 strains, ‘Zehak’, ‘Mid-day’ and ‘South’ with B2-10 and B2-11 and ‘Desert’ with B2-10, B2-11 and B4-6 caused the highest increase in grain yield of barley cultivars as compared to other treatments (Table 6).

Survival of bacterial strains in the barley rhizosphere

The survival of bacterial strains in the rhizosphere of five barley cultivars grown in different salinity levels was determined under greenhouse conditions. Analysis of variance revealed that the main effects of salinity, variety and bacterial strains and the interaction between them were significant on rhizosphere colonization (p≤0.05) (Table 3). All four bacterial strains were able to colonize and survive in the rhizosphere of barley cultivars. However, their colonization level was partly inhibited under salt stress. Under normal condition isolate B2-10, at 50 mM, isolates B2-10 and B2-11, at 100 mM NaCl stress, isolate B2-11 and at 150 mM salinity B4-6, B2-11 and B2-10 had higher rhizosphere colonization in comparison to other treatments (Table 4). Barley varieties showed variations in rhizosphere colonization by bacterial strains during NaCl induced salinity stress (Table 5). Under normal conditions, ‘Mid-day’ at 50 mM, ‘South’ at 100 mM and 150 mM all barley varieties, except ‘Zehak’, showed higher rhizosphere colonization. Barley genotype at the cultivar level had significant impact on rhizosphere colonization by bacterial strains under greenhouse conditions (Table 6). In ‘Karun’ the rhizosphere colonization by B2-10, and in ‘Mid-day’ the colonization by B2-11 were higher than by other strains. In ‘Zehak’, bacterial strains were not significantly different regarding rhizosphere colonization. In ‘Desert’ and ‘South’, B2-11 strain colonized the rhizosphere better than the others; however, the colonization level was not significantly different to B2-10 and B4-6 (in ‘Desert’) and B2-10 (in ‘South’).

DiscussionTop

This study demonstrates the efficacy of P. fluorescens strains containing ACC deaminase activity for inducing salt tolerance and consequently improving the growth of barley plants under salinity stress conditions. ACC deaminase activity has been found more widely present in soil bacteria belonging to several species of Pseudomonas and to the genera Alcaligenes, Bacillus, Variovorax and Rhodococcus (Belimov et al., 2005; Bal et al., 2013; Akhgar et al., 2014). Root associated bacteria possessing ACC deaminase activity assist plants to withstand biotic and abiotic stresses by decreasing the level of stress ethylene (Mayak et al., 2004; Dimkpa et al., 2009).

The four strains were also screened for their tolerance to increasing levels of NaCl. All the bacterial strains could tolerate up to 8% (w/v) NaCl concentrations on modified mineral-based NA plates. Besides, while the NaCl concentration increased, the growth of bacteria was noticed to decrease. Increase in salt concentration outside cell membrane enhances osmotic potential which is suggested to be one of the main reasons for reduced growth of PGPRs (Tank & Saraf, 2010).

The ACC deaminase producing isolates were also screened for multiple PGP traits, including solubilization of phosphate and production of IAA and siderophore. In vitro screening for characteristics commonly associated with plant growth promotion revealed that all bacterial strains were able to produce IAA in a range of 1-3.4 mg L-1, indicating variability among barley isolates for IAA production. The ability of Pseudomonas strains to produce IAA indicates their potential to use as growth hormones or growth regulators. In addition to other factors positively influence plant growth, biosynthesis of IAA by bacterial strains is suggested as a principal means of attaining growth enhancement (Deepa et al., 2010). IAA plays major roles in root initiation and cell division and growth; it increases surface area of roots, and access to soil nutrients by enhanced root formation (Gray & Smith, 2005). These results suggest that the selected strains can be beneficial in enhancing growth of barley and other host plants by providing nutrients like iron and phosphorous. IAA production is an important PGPR trait, since this phytohormone allows plant to develop extremely organized root system by which nutrient uptake becomes more efficient.

The variation in the ability of bacterial strains to produce IAA found in the current study was also reported by Qin et al. (2014) and Majeed et al. (2015). IAA production by bacteria isolated from rhizosphere of different plants such as wheat and rice had already been reported by Cakmakci et al. (2007) and Majeed et al. (2015).

In the present study, Pseudomonas strains revealed as efficient phosphate solubilizers. The beneficial influence of PGPR in maintaining sufficient levels of nutrients mainly the phosphorus in plant productivity was documented earlier by Majeed et al. (2015). The phosphate solubilizing bacteria increase the availability of phosphorus to plants through mineralization of or­ganic phosphate and conversion of inorganic phosphate into more accessible forms (Bar-Yosef et al., 1999). Solubilization of phosphate is mainly related to the production of microbial metabolites like organic acids which reduces the pH of the culture media (Shahid et al., 2012).

Present study revealed that seed treatment with PGPR strains improved barley growth and yield over the non-inoculated seeds (Table 6). Similarly, improvement in growth indices and yields-of different crop plants like rice, maize and wheat in response to inoculation with PGPR were reported earlier by Khalid et al. (2004), Gholami et al. (2009) and Bal et al. (2013).

In pot experiment, it was observed that inoculation of barley seeds with P. fluorescens strains significantly increased plant height, peduncle length, spike length, spike number, number of grains per spike, MGW, spike weight and grain yield under different salinity levels. The results of our current study confirmed the findings of earlier studies performed by other researchers who demonstrated the increased resistance to various stresses in plants treated with ACC deaminase containing PGPR (Mayak et al., 2004; Qin et al., 2014).

Inoculation of barley seeds with P. fluorescens strains under different salinity levels resulted in increase in plant height, yield and yield components in comparison to control plants. Using rhizobacteria with multiple PGP traits is believed to help improve crop productivity on a sustainable basis. All the four ACC deaminase producing strains were tested positive for several PGP traits such as production of IAA and siderophore and solubi­li­zation of phosphate. Furthermore, inocu­­la­­tion of plants with ACC deaminase and siderophore producing PGPR helps plants to conquer the effects induced by the environmental stresses as observed in the present study (Dimkpa et al., 2009; Bal et al., 2013).

In the current study, the tested bacterial isolates had the ability to produce both IAA and ACC deaminase. It is probable that ACC deaminase and IAA stimulate root growth in a coordinated manner (Glick et al., 2007b). The collection of specific PGP traits of studied strains suggests that these rhizobacteria are able to improve plant growth by more than one mechanism.

Barley cultivars showed variations in their response to saline conditions; however, salinity reduced growth parameters and yield irrespective of the cultivar. The changes in growth factors were evident at the lowest salinity level and became more pronounced at the highest level of 150 mM salinity. The better performance of a genotype under salt stress conditions appear to be determined mainly by the tolerance of the host plant.

In this study, barley varieties responded differently to inoculation with different rhizobacterial strains. The observed differences in response to inoculation with bacteria might be due to the differences in the amount and/or composition of compounds present in root exudates, which may result in a different level of rhizosphere colonization by introduced strains. Many studies have demonstrated the effect of host plant genotype and root exudates, not only on the produc­tion of biologically active substances, but also on rhizosphere colonization of root-associated bacteria (Dazzo et al., 2000; Jamali et al., 2009). It has been reported that the benefits of bacterization to plant growth can vary with plant genotype and cultural conditions as well as PGPR strains (Nowak, 1998; Khalid et al., 2004). In addition, application of PGPR appears to be a useful biological tool in agriculture for alleviation of the negative effects of salinity and improvement of the salt tolerance of crops.

In our study, four Pseudomonas strains stimulated growth of barley plants, but the activity of some strains was reduced under salinity conditions. It has been shown that the root colonization and plant growth promoting traits of PGPRs is affected by several biotic and abiotic factors including indigenous soil type, drought, salinity and temperature (Compant et al., 2010). Salt tolerant and root colonizing bacteria which are adapted to abiotic stress can survive in such severe conditions and assist plants to tolerate salinity stress (Egamberdieva et al., 2016). Egamberdieva et al. (2017) reported that Pseudomonas extremorientalis, a salt tolerant and root colonizing bacteria could increase growth of tomato plants under saline soil conditions. Salt stress has negative effects on the rhizosphere colonization of introduced bacteria; however, salt tolerant bacteria can survive in the rhizosphere of plants through their persistence and proliferation in semi-arid soils (Paul & Nair, 2008). The bacterial strains used in this study were salt tolerant (up to 8% NaCl) and showed potential root colonizing ability for barley cultivars (unpublished data). In our current stu­dy the colonization potential of bacterial strains was not inhibited by salt stress since they were able to colonize barley rhizosphere under salinity conditions. The colonization potential of introduced bacteria is mentioned as a crucial mechanism in the positive effects of introduced bacteria (Lugtenberg et al., 2001) and this potential of bacterial strains was not inhibited by salt stress in our study.

Application of bacterial strains with multiple PGP traits is expected to help increase crop productivity on a sustainable basis. The present study concluded that inoculation with the four ACC deaminase producing PGPR caused significant alleviation of salinity stress and thus enhanced the growth of barley cultivars under salinity stress conditions. The presence of various PGP traits in the strains may be the possible reason to protect the plant from the suppressive effects of salinity and consequently induce salinity tolerance in barley plants. However, further study is essential to examine the efficacy of these bacterial strains under field conditions to use them for minimizing salinity stress to the growing plants.