The combined application of rhizobial strains and plant growth promoting rhizobacteria improves growth and productivity of mung bean (Vigna radiata L.) under salt-stressed conditions

Abstract

Salinity stress induces higher levels of ethylene in plants in arid and semi-arid regions. This increased concentration of ethylene can be inhibited by using plant growth promoting rhizobacteria (PGPR) containing ACC-deaminase. A pot trial was conducted under salt-stressed conditions to evaluate the potential of combined application of Rhizobium phaseoli (M6 and M9), and PGPR (Pseudomonas syringae, Mk1; Pseudomonas fluorescens, Mk20 and Pseudomonas fluorescens Biotype G, Mk25) to improve the productivity of mung bean. The results showed that salinity stress decreased significantly mung bean growth, yield and physiological parameters but inoculation with either rhizobia or PGPR alone enhanced these parameters significantly. However, the combined application of rhizobia and PGPR was more effective for reducing the depressing effect of salinity on mung bean. Co-inoculation increased the shoot fresh weight (145%), root fresh weight (173%), number of pods plant⁻¹(150%), pod fresh weight (182%), total dry matter (269%), relative water content (19%), water use efficiency (51%), potassium concentration in leaves (33%), sodium concentration in leaves (56%) and nitrogen concentration in grains of mung bean (99%), compared with the uninoculated control. The results imply that combined application of Rhizobium and Pseudomonas strains can improve the productivity of mung bean. Thus, these strains could be evaluated in intensive field trials for developing biofertilizers to improve the productivity of mung bean under salt-affected conditions.

Introduction

Mung bean (Vigna radiata L.) is an important conventional pulse crop in Pakistan and is grown world-wide. Its importance is due not only to its high nutritional value but also because it improves soil fertility by fixing atmospheric nitrogen (Elahi et al. 2004). However, under the agro-ecological conditions of Pakistan, mung bean yields are low. One of the major factors contributing to its low yield is salinity. Salinity is a serious production problem for crops as saline conditions are known to suppress plant growth, particularly in arid and semi-arid regions (Parida and Das 2005). Pakistan is located in such an arid/semi-arid region where rainfall is low as well as irregular and, under such conditions, the most detrimental natural problems for crop growth are salinity and sodicity. The total cultivated area of Pakistan is 23.80 million ha, of which 6.18 million ha is severely affected by salinity/sodicity (Anonymous 2010). Salinity induces osmotic stress by limiting absorption of water from soil (Mayak et al. 2004) and ionic stress resulting from high concentrations of potentially toxic ions within plant cells.

Salinity induces the imbalance of mineral nutrients and their distribution within plants (Glenn et al. 1999). Salt stress also affects the morphology, anatomy, ultra-structure and metabolism of plant species (Prat and Fathi-Ettai 1990; Silveira et al. 2003), thus resulting in an overall decrease in plant growth and yield. The decreased photosynthetic rate in plants due to salinity stress leads to a decrease in the relative water content of plants (Ben-Asher et al. 2006). Salinity stress adversely affects total dry matter and plant growth, as most of the energy available is used in to make osmotic adjustments by the plant (Munns and Termaat 1986). Salinity adversely affects plant growth and yield by enhancing ethylene production. Increased production of ethylene due to exogenous application of 1-aminocyclopropane-1-carboxylic acid (ACC) or salinity can decrease root growth (Madhaiyan et al. 2007).

The survival and distribution of rhizobia in soil and the rhizosphere is affected by salinity (Tate 1995). These bacterial strains have variable ability to tolerate salt stress (Lloret et al. 1995). Rhizobial inoculation has been reported to promote plant growth and development by multiple mechanisms such as N₂ fixation, production of plant growth regulators and disease suppression (Naz et al. 2009).

The use of rhizobacteria is one of the most acceptable approaches to reduce the effect of stress-induced ethylene on plants. These plant growth promoting rhizobacteria (PGPR) contain the enzyme ACC-deaminase, which hydrolyzes ACC (immediate precursor of ethylene) to yield ammonia and α-ketobutyrate (Mayak et al. 1999). PGPR can boost plant growth, particularly under stressed conditions, by regulating accelerated ethylene production in response to a multitude of abiotic and biotic stresses (Belimov et al. 2009; Ahmad et al. 2011). Inoculation with PGPR containing ACC-deaminase can increase growth, yield, nutrient concentrations and ionic balance within the plants due to lowering of the ethylene content of these strains (Yue et al. 2007; Nadeem et al. 2009). Inoculation/co-inoculation with Rhizobium and PGPR strains containing ACC-deaminase decreased the intensity of the classical triple response by increasing the seedling length of inoculated mung bean seedlings and decreasing the stem diameter—a typical response to the dilution in a classical triple response bioassay (Ahmad et al. 2011). Different PGPR strains differ in their ability to promote plant growth due to differences in ACC deaminase activity (Shaharoona et al. 2006; Nadeem et al. 2009; Ahmad et al. 2011). This difference may also be due to the presence of other growth-promoting characters i.e., chitinase activity, phosphate solubilization, root colonization, etc., in addition to ACC-deaminase activity (Ahmad et al. 2011).

Co inoculation of legumes with rhizobacteria and rhizobia has been reported to stimulate plant dry matter and grain yield by affecting some physiological functions in different crops (Derylo and Skorupska 1993; Dashti et al. 1997). It improves plant growth by reducing ethylene levels (Shaharoona et al. 2006), directly stimulating rhizobial growth/survival in the soil, enlarging the root system by hormone production for enhanced nutrient uptake, and increasing the number of potential colonization sites by rhizobium, phosphate solubilization and pathogen suppression due to the production of antibiotics (Gull et al. 2004; Barea et al. 2005). Root elongation rate, mineral N, P and K and microelements absorption and uptake are reported to be consequently improved after Azospirillum inoculation (Dobbelaere and Okon 2007). This could result in a general better mineral nutrition of the plant phosphorous, iron and molybdenum essential for rhizobia-nodule formation and nitrogen fixation activities (Burdman et al. 1998). Co-inoculation has also been reported to increase water use efficiency (WUE) in plants under stress (Vivas et al. 2003). The impact of co-inoculation with Rhizobium and PGPR containing ACC-deaminase has been established under normal conditions. However, we have hypothesized that this approach could be used more effectively under salt-affected conditions. In a series of experiments under axenic (Ahmad et al. 2011) and natural conditions, the efficacy of three strains of PGPR (Pseudomonas syringae, Mk1; Pseudomonas fluorescens, Mk20 and Pseudomonas fluorescens Biotype G, Mk25) and two strains of Rhizobium phaseoli (M6 and M9) was studied. The present paper describes the potential of these selected strains to improve the productivity of mung bean under salt-affected conditions in pot trials under ambient conditions.

Materials and methods

Collection of bacterial strains

Three strains of PGPR (Pseudomonas syringae, Mk1; Pseudomonas fluorescens, Mk20 and Pseudomonas fluorescens Biotype G, Mk25) and two strains of Rhizobium phaseoli (M6 and M9) were isolated from the rhizosphere and nodules of mung bean growing in salt-affected conditions (EC_e = 4.1–6.7 dS m⁻¹), respectively, (Ahmad et al. 2011). These rhizobacterial strains, which contain ACC-deaminase activity, and Rhizobium were assessed for their ability to tolerate salt stress by conducting an osmoadaptation assay (data not given) and were evaluated alone and in combination for their potential to improve the productivity of mung bean under salt-stressed conditions in pot trials.

Preparation of inocula

Inocula were prepared in flasks by using yeast extract mannitol (YEM) and DF minimal salt medium (Dworkin and Foster 1958) without agar containing ACC as substrate (N source) for rhizobial and rhizobacterial strains, respectively. Each flask containing broth was inoculated with respective strains of rhizobia or rhizobacteria and incubated at 28 ± 1°C for 72 h under shaking (100 rpm) conditions. After incubation, optical density was measured at 540 nm for Rhizobium and Pseudomonas strains, and uniform population (OD₅₄₀ = 0.45; 10⁷–10⁸ CFU mL⁻¹) was achieved by dilution with sterilized water prior to seed inoculation.

Pot trial

The PGPR and Rhizobium strains were evaluated alone and in combination for their potential to improve the productivity of mung bean under salt-stressed conditions in a pot trial.

The experiment was conducted with different set of treatments, i.e., inoculation either with Rhizobium and rhizobacteria alone, or combinations of these strains in the presence of recommended levels of chemical fertilizers. The soil used in the pots was analyzed for physical and chemical characteristics (Table 1) according to standard protocols as described by Ryan et al. (2001). Mung bean seeds were inoculated with respective bacterial strains by using slurry prepared with sterilized peat, broth culture (OD₅₄₀ = 0.45; 10⁷–10⁸ CFU mL⁻¹) and sterilized sugar solution (10%) in the ratio 5:4:1. For co-inoculation, broth cultures of PGPR and Rhizobium were used in a 1:1 ratio for preparation of slurry. In the case of the un-inoculated control, seeds were coated with sterilized (autoclaved) peat treated with sterilized broth.

Table 1 Physical and chemical characteristics of soil used for pot trials

Three salinity levels (original, i.e., 1.41; 4; 6 dS m⁻¹) were used. These levels were chosen keeping in mind the salinity status of the soils in which mung bean is cultivated in Pakistan. We surveyed the area where mung bean is cultivated and collected soil and root samples for isolation of bacterial strains and the assessment of soil salinity status. These soil samples were analyzed for EC_e and it was observed that these fields had EC_e levels ranging from 4.1 to 6.7 dS m⁻¹. The pots were lined with polythene sheets before adding soil and there was no leaching provision in the pots as the only hole at the bottom was plugged with cork. Three salinity levels, 1.41 (i.e. original), 4 and 6 dS m⁻¹ were generated by using a calculated amount of NaCl salt in each pot and mixing it using a mechanical mixer. Ten inoculated seeds of mung bean were sown in each pot containing 12 kg pot⁻¹ soil. There were six replications of each treatment. Similarly, three pots (1.41, 4 and 6 dS m⁻¹) were maintained as reference pots to check the effect of irrigation water on salinity levels. EC_e was monitored regularly in the reference pots and from the experimental pots at the end. There was no significant change in the salinity levels at the end. Pots were arranged in a wire cage under ambient light and temperature according to completely randomized design. The recommended dose of P and K fertilizers (60 kg ha⁻¹ each) as diammonium phosphate and sulfate of potash, respectively, and half of the recommended dose for N (20 kg ha⁻¹) as urea was applied to each pot. All fertilizers were applied as a basal dose at the time of sowing. Plants were irrigated with good quality irrigation water meeting the irrigation quality criteria for the crop (Ayers and Westcot 1985). After germination, thinning was carried out to maintain a uniform plant population. Plants from three replications were uprooted at the flowering stage to assess nodulation while the remaining three replications were taken to maturity for harvesting. Data on growth and yield parameters were collected at harvest upon physiological maturity.

Plant analyses

After 60 days, fresh leaf samples were collected and analyzed for sodium (Na⁺) and potassium (K⁺) concentration by flame photometer as described by Ryan et al. (2001). Grain samples were digested according to the method of Wolf (1982) and nitrogen was determined by the Kjeldhal method.

Measurement of WUE

The photosynthetic (A), and transpiration (E) rates were measured by portable infra-red gas analyzer [IRGA (LCA-4; Analytical Development Company, Hoddeson, UK)]. These parameters were taken in the morning (8:00–10:00 a.m.) at a photosynthetic photon flux density of 1,200–1,400 μmol m⁻² s⁻¹ (Ben-Asher et al. 2006). Two fully expanded leaves from one plant in each pot were selected for the measurement of the data regarding above parameters. WUE is the ratio of A to E and was calculated as

$$ {\text{Water use efficiency }}\left( {{\text{A}}/{\text{E}}} \right){ } = \frac{{{\text{Photosynthetic rate }}\left( {\text{A}} \right)}}{{{\text{Transpiration rate }}\left( {\text{E}} \right)}} $$

Relative water content

The relative water content (RWC) of shoots was determined by using the following formula as described by Mayak et al. (2004).

$$ {\text{Relative water content }}\left( {\text{RWC}} \right){ } = \frac{{{\text{Fresh weight}} - {\text{Dry weight}}}}{{{\text{Fully turgid weight }} - {\text{ Dry weight}}}} $$

The fully turgid weight of leaf was taken after putting it in 100% humidity in the dark at 4 ^°C for 48 h.

Statistical analysis

Analysis of variance techniques (ANOVA) were applied to analyze the data (Steel et al. 1997) using a completely randomized design, and means were compared by Duncan’s multiple range test (Duncan 1955).

Results

The data (Table 2) revealed that salinity stress had an inhibitory effect on shoot fresh weight of mung bean. However, co-inoculation decreased the inhibitory effect of salinity on shoot fresh weight and improved it. However, sole inoculation of either Rhizobium or PGPR was non-significant in comparison with the respective un-inoculated control at all salinity levels. The maximum improvement in shoot fresh weight (145%) compared with respective un-inoculated control was observed due to co-inoculated combination Mk1 × M9 at 6 dS m⁻¹. However, the combinations Mk25 × M6 and Mk25 × M9 were at par with the respective un-inoculated control. Under normal conditions, maximum improvement in shoot fresh weight (33%) compared with the respective un-inoculated control was observed due to co-inoculation of PGPR strain Mk20 and Rhizobium strain M6. At 4 dS m⁻¹, all the treatments were statistically similar to the corresponding un-inoculated control.

Table 2 Effect of inoculation/co-inoculation on shoot and root fresh weight of mung bean under salt-stressed conditions in pot trials. Means with the same lower case letters are statistically at par at 5% level of probability; n = 3. Mk1 = Pseudomonas syringae; Mk20 = Pseudomonas fluorescens; Mk25 = Pseudomonas fluorescens Biotype G; M6 and M9 = Rhizobium phaseoli

The data in Table 2 reveal that root fresh weight improved upon inoculation with either Rhizobium or PGPR containing ACC-deaminase alone, which otherwise decreased with salinity. But co-inoculation was more effective for reducing the effect of salinity on root fresh weight. Maximum improvement in root fresh weight (173%) compared with the respective un-inoculated control was observed due to the co-inoculated combination Mk1 × M6 at 6 dS m⁻¹. However, the co-inoculated combination Mk25 × M6 was non-significant compared with the corresponding un-inoculated control. At 4 dS m⁻¹, the increase in root fresh weight due to co-inoculation ranged from 58% to 95%, while under normal conditions this improvement was up to 105% compared with un-inoculated controls. However, the combinations Mk25 × M9 and Mk1 × M6 gave a non-significant increase in root fresh weight over the corresponding un-inoculated control. Sole inoculation with PGPR containing ACC-deaminase and Rhizobium strain M9 was also effective under salinity stress. However, the effect of Rhizobium strain M6 was non-significant in comparison with the respective un-inoculated control under normal conditions as well as at 4 dS m⁻¹. The PGPR strain Mk1 showed non-significant results when compared with un-inoculated control under normal conditions as well as under salinity stress.

Inoculation/co-inoculation had a promising effect on number of pods plant⁻¹ in mung bean growing in pot trials under salt-stressed conditions (Table 3). At 6 dS m⁻¹, maximum improvement in number of pods plant⁻¹ (150%) over the respective un-inoculated control was observed in the co-inoculated combination Mk20 × M9 and Mk1 × M9 , which otherwise was decreased by 55% due to salinity. Other treatments also showed significant effects when compared with the respective un-inoculated control except Rhizobium strain M9 and co-inoculated combinations Mk25 × M6 and Mk20 × M6 which gave non-significant results when compared with un-inoculated control. Under normal conditions as well as at 4 dS m⁻¹, most of the treatments showed non-significant effects when compared with the corresponding un-inoculated control.

Table 3 Effect of inoculation/co-inoculation on number of pods plant⁻¹ and pod fresh weight plant⁻¹ of mung bean under salt-stressed conditions in pot trials. Means sharing same letters are statistically at par at 5% level of probability (n = 3)

The data in Table 3 show that salinity stress decreased pod fresh weight of mung bean but inoculation/co-inoculation improved it by reducing the inhibitory effect of salinity. Under normal conditions, the maximum pod fresh weight (9.51 g) was observed with the co-inoculated combination Mk1 × M9, which was 72% more than the un-inoculated control, followed by 52%, 44% and 42% with the Rhizobium strain M9, co-inoculated combination Mk20 × M9 and PGPR strain Mk25, respectively. However, most of the treatments were at par with the control. At 4 dS m⁻¹, all the treatments showed non-significant results compared with the corresponding un-inoculated control except PGPR strain Mk25 and co-inoculated combination Mk25 × M9. Pod fresh weight was decreased by most of the treatments in comparison with controls. Higher salinity (6 dS m⁻¹) adversely affected pod fresh weight, with decreases of up to 57%. Inoculation/co-inoculation with Rhizobium or PGPR containing ACC-deaminase reduced this inhibitory effect. The maximum improvement in pod fresh weight (182%) over un-inoculated controls was observed upon inoculation with PGPR containing ACC-deaminase Mk1 at 6 dS m⁻¹, followed by 173% and 110% due to Rhizobium strain M9 and co-inoculated combinations Mk1 × M9, respectively. There were non-significant results in case of co-inoculated combination Mk25 × M6 at higher salinity level. While the sole inoculation of either Rhizobium or PGPR containing ACC-deaminase also showed significant increase in pod fresh weight at higher salinity levels, where a 70–173% increase in pod fresh weight was recorded.

It is evident from the data regarding total dry matter (Table 4) that inoculation with Rhizobium and PGPR containing ACC-deaminase reduced the effect of salinity significantly. But co-inoculation was more pronounced in reducing the effect of salinity on total dry matter of mung bean. Maximum improvement in total dry matter (269%) compared with the respective un-inoculated control was observed upon a co-inoculated combination at the 6 dS m⁻¹ salinity level. However, the combination Mk25 × M6 gave a non-significant increase in total dry matter compared with the respective un-inoculated control at 6 dS m⁻¹ while the PGPR strain Mk1 showed non-significant results compared with the un-inoculated control at lower salinity levels but significantly higher than the corresponding un-inoculated control at 6 dS m⁻¹.

Table 4 Effect of inoculation/co-inoculation on total dry matter and grain yield of mung bean under salt-stressed conditions in pot trial. Means sharing same letters are statistically at par at 5% level of probability (n = 3)

Salinity stress impaired grain development in mung bean, with development decreasing with increasing salinity level (Table 4). There was no grain development at higher salinity levels, i.e., 6 dS m⁻¹. However, inoculation/co-inoculation decreased the inhibitory effect of salinity and grain yield improved significantly with most treatments. Separate inoculation of PGPR containing ACC-deaminase significantly improved grain yield in mung bean. The PGPR strain Mk25 showed the best results when compared with other strains at all salinity levels. Maximum improvement in grain yield was observed by Mk25 inoculation at 6 dS m⁻¹. Under normal conditions as well as at 4 dS m⁻¹, the PGPR strain Mk25 led to increases in grain yield of 27% and 52% over the un-inoculated control. The other two PGPR strains gave significantly better results at higher salinity level but they decreased the grain yield at lower salinity levels. However, this reduction was non-significant in the case of Mk20 at 4 dS m⁻¹, while Mk25 showed a non-significant decrease in grain yield at original salinity level. The Rhizobium inoculation was also effective at higher salinity level but strain M9 was non-significant at 4 dS m⁻¹, and M6 gave non-significant results at the original salinity level. Co-inoculation with Rhizobium and PGPR containing ACC-deaminase was effective at all salinity levels; however, the effect was more pronounced at higher salinity levels. At 6 dS m⁻¹, the maximum grain yield was observed by the co-inoculated combination of Mk1 × M6, followed by co-inoculated combinations Mk20 × M9 and Mk1 × M9, respectively. At 4 dS m⁻¹, the increase in grain yield over the un-inoculated control ranged from 35% to 86%, while under normal conditions this increase was 7% to 141%. However, the combinations Mk20 × M6 and Mk1 × M6 gave non-significant results compared with the corresponding un-inoculated controls.

Relative water content (RWC) was decreased significantly due to salinity stress (Table 5), while inoculation with Rhizobium and PGPR containing ACC-deaminase significantly improved RWC. However, co-inoculation with Rhizobium and PGPR containing ACC-deaminase was more effective. A variable response was observed by all combinations under normal as well as salt-stressed conditions. At 6 dS m⁻¹, maximum improvement in RWC was observed with the combination Mk1 × M9 ,where the increase in RWC compared with its un-inoculated control was 19%.

Table 5 Effect of inoculation/co-inoculation on relative water content (RWC) and water use efficiency (WUE) of mung bean under salt-stressed conditions in pot trial. Means sharing same letters are statistically at par at 5% level of probability (n = 3)

In this study, water use efficiency (WUE) decreased significantly due to salinity but inoculation/co-inoculation reduced the adverse effect of salinity and improved WUE values (Table 5). Minimum WUE was observed in un-inoculated control at 6 dS m⁻¹ salinity, where the decrease was up to 31% compared with the salinity control (1.41 dS m⁻¹). The Rhizobium and PGPR containing ACC-deaminase decreased the adverse effects of salinity on WUE at all salinity levels significantly, while the results of Rhizobium strain M6 were non-significant compared with the respective un-inoculated control at 6 dS m⁻¹. Co-inoculation was more effective in improving WUE at all salinity levels than sole inoculation of Rhizobium and PGPR containing ACC-deaminase. However, a variable response was observed with all combinations under normal as well as salt-stressed conditions. At 6 dS m⁻¹, maximum increase in WUE (51%) over the respective un-inoculated control was observed due to co-inoculated combination Mk25 × M9 where the increase was 47%. However, the combinations Mk25 × M6 and Mk20 × M6 gave non-significant results compared with un-inoculated control.

The data shown in Table 6 show that the potassium concentration in mung bean leaves decreases significantly with increased salinity levels. However, sole and combined inoculation of Rhizobium and PGPR containing ACC-deaminase significantly reduced the adverse effect of salinity, thus improving the potassium concentration. Under normal conditions, all treatments were equally effective for improving potassium concentration. The increase in potassium concentration over the un-inoculated control ranged from 21% to 32%. As observed under normal conditions, bacterial inoculation was also effective under salinity stress and all strains and their combinations significantly improved potassium concentration over the corresponding un-inoculated control. At 6 dS m⁻¹, Mk1 × M9 gave a maximum increase in potassium concentration (33%) over the un-inoculated control, and was non-significant when compared with other treatments but significantly higher than the un-inoculated control.

Table 6 Effect of inoculation/co-inoculation on potassium and sodium concentration in leaves of mung bean under salt-stressed conditions in pot trials. Means sharing same letters are statistically at par at 5% level of probability (n = 3)

Sodium competes with potassium under salinity stress; therefore, its concentration increased when the mung bean plants were subjected to salinity stress (Table 6). The maximum sodium concentration in mung bean leaves (9.5 g kg⁻¹) was observed when plants were grown at 6 dS m⁻¹ salinity. However, inoculation with Rhizobium and PGPR containing ACC-deaminase decreased the effect of salinity, hence decreasing the sodium concentration in mung bean leaves. However, co-inoculation was more effective than sole inoculation with Rhizobium and PGPR containing ACC-deaminase at all salinity levels. Under normal conditions, the reduction in sodium concentration due to inoculation/co-inoculation ranged from 10% to 33%, and the minimum sodium concentration (4.6 g kg⁻¹) was observed due to combination Mk25 × M9. Inoculation/co-inoculation was more effective for reducing the sodium concentration under salinity conditions. At 6 dS m⁻¹, maximum reduction (56% less than the un-inoculated control) in sodium concentration was observed with combination Mk20 × M9.

It is evident from the data (Table 7) that inoculation/co-inoculation significantly improved the nitrogen concentration in mung bean grains. Co-inoculation with Rhizobium and PGPR containing ACC-deaminase gave very promising results. However, the results were variable at all salinity levels. Maximum improvement in nitrogen concentration (99%) compared with the respective un-inoculated control was observed by the combination Mk1 × M9 at 6 dS m⁻¹. But the results of Mk1 × M6 were non-significant when compared with the corresponding un-inoculated control. Sole inoculation with Rhizobium and PGPR containing ACC-deaminase also significantly improved the nitrogen concentration in grains, while the results with Rhizobium strain M9 were at par with the corresponding un-inoculated control at original and 4 dS m⁻¹, and the PGPR strain Mk25 gave statistically similar results compared with the respective un-inoculated control at all salinity levels.

Table 7 Effect of inoculation/co-inoculation on nitrogen concentration in grains of mung bean under salt-stressed conditions in pot trials. Means sharing same letters are statistically at par at 5% level of probability (n = 3)

Discussion

In a series of experiments under axenic conditions (Ahmad et al. 2011), three strains of PGPR and two strains of Rhizobium phaseoli were chosen to study their efficacy under natural conditions. These strains were evaluated for their potential to improve the productivity of mung bean under salt-affected conditions in pot trial under ambient conditions.

The results of our study in pot trials revealed that salinity significantly reduced the seedling growth of mung bean, but root growth was affected relatively more than shoots. It is very likely that root metabolic activity and physiological processes were more affected than those of shoots, or, alternatively, the difference might be due to the more intimate contact of roots with salt solution as compared to shoot (Mayak et al. 2004). The decreased growth may also be attributed needing the majority of the available energy for the making osmotic adjustments by the plant, thus decreasing plant growth and total dry matter (Munns and Termaat, 1986). Increased salinity in the rhizosphere decreases the osmotic potential of the root zone soil solution (Chartzoulakis et al. 2002), resulting in reduced availability of water to plants. It has been reported that increased production of ethylene due to exogenous application of ACC or salinity decreases root growth (Madhaiyan et al. 2007).

However, in our studies, inoculation with PGPR containing ACC-deaminase increased root and shoot growth as compared to un-inoculated controls. This could be due to lowering of ethylene levels by these strains, thus improving plant growth. It is very likely that exogenous application of ethylene and/or salinity had an adverse effect on plant growth but inoculation with PGPR containing ACC-deaminase improved plant growth by decreasing the ethylene concentration (Shaharoona et al. 2006; Nadeem et al. 2009; Ahmad et al. 2011).

It was also observed in this study that PGPR strains improved the efficiency of Rhizobium for improving the growth of mung bean but with different degrees of efficacy. PGPR containing ACC-deaminase have been reported to eliminate, or at least reduce, the stress-induced ethylene-mediated negative impact on plants (Glick 2005; Safronova et al. 2006). The effect of these PGPR containing ACC deaminase in boosting plant growth, particularly under stressed conditions, has also been studied by the regulation of accelerated ethylene production in response to a multitude of abiotic and biotic stresses (Zahir et al. 2008; Belimov et al. 2009; Ahmad et al. 2011). Co-inoculation with Rhizobium and Pseudomonas strains containing ACC-deaminase increased plant height, total dry matter and pod fresh weight by 269% and 162%, respectively, compared to the un-inoculated control in our study, but in a study conducted by Nadeem et al. (2007) on salt-affected conditions in pot trials, the separate application of ACC-deaminase strain Pseudomonas fluorescens increased the biomass, cob mass and grain yield of maize by 51%, 40% and 50%, respectively, compared to the un-inoculated control. It has also been reported that inoculation/co-inoculation improved the transpiration rate and other physiological processes (Vivas et al. 2003; Gaballah and Gomaa 2005), thus decreasing the effect of salinity on plant growth, and leading to increased total dry matter.

Some combinations of rhizobial and rhizobacterial isolates could not improve growth and yield parameters compared to un-inoculated controls, which might be due to certain compounds produced by the bacteria. Bacterial strains produce certain antibiotics, which may suppress other strains (Gull et al. 2004). It has also been reported that co-inoculation with PGPR and Rhizobium decreased plant growth due to the production of antibiotics and competition for attachment sites on root surfaces (Mirza et al. 2007).

In our study, WUE and RWC were negatively affected by salinity. This might be due to stomatal closure in plants caused by salinity stress, which decreases RWC (Lawlor and Cornic 2002; Ben-Asher et al. (2006) and physiological efficiency in plants. The decrease in physiological activity results in declined plant growth leading to reduced grain yield.

In our study, inoculation/co-inoculation decreased the effect of salinity on WUE and RWC, thus improving the yield of mung bean. This is an indication of enhanced water uptake due to inoculation/co-inoculation under salinity stress. It may be due to the longer roots of plants inoculated/co-inoculated with Rhizobium and PGPR containing ACC-deaminase under stress (Ahmad et al. 2011) which might have helped the plants to uptake relatively more water from deeper soil under stressed conditions (Hamdia et al. 2004; Gaballah and Gomaa 2005).

In our study, salinity increased Na⁺ concentration significantly while decreasing K⁺ concentration within the mung bean leaves. This might be due to the fact that plants take up more Na⁺ when they are subjected to salinity in order to maintain the turgor pressure within the plants (Ashrafet al. 2004), which might have reduced K⁺ uptake. It has been reported in previous studies that uptake of Na⁺ increases with increase in the Na⁺ content in soil, which decreases the uptake of K⁺ and Ca²⁺ by plants (Pervaiz et al. 2002; Ashraf et al. 2004).

In our study, nitrogen concentration in mung bean seeds was also adversely affected by salinity. This might be due to the reduced root growth with the increase in salinity-induced ethylene production, which decreased the root area thus making it insufficient to explore soil for required nutrient uptake (Gaballah and Gomaa 2005; Nadeem et al. 2009). However, inoculation/co-inoculation reversed the pattern and improved the nutrient balance and nitrogen concentration in mung bean seeds. This might be due to a reduction in ethylene production due to inoculation/co-inoculation with Rhizobium and PGPR containing ACC-deaminase, thus reducing the inhibitory effect of ethylene on root growth leading to greater proliferation of roots. The competency of co-inoculation for reducing the effect of salinity due to the reduction in ethylene level through ACC-deaminase activity was proved by conducting the classical triple response assay (Ahmad et al. 2011). The increased root area might have facilitated the plants to explore more soil for nutrient absorption (Hamdia et al. 2004; Nadeem et al. 2009).

The strains used in this experiment varied in their ability to decrease the effect of salinity on plant growth and the maximum response was observed when Pseudomonas fluorescens (Mk20) was co-inoculated with Rhizobium phaseoli. It is very likely that PGPR strains varied in their ACC-deaminase ability along with some other characters (Ahmad et al. 2011) that contributed differently for growth promotion. This difference may also be due to the presence of other growth-promoting characters, i.e., chitinase activity, phosphate solubilization, root colonization, etc. in addition to ACC-deaminase activity (Ahmad et al. 2011). The co-inoculation of these strains with Rhizobium might have enhanced the survival efficacy and proliferation of Rhizobium, which might be due to the solubilization of indigenous phosphorus (Ahmad et al. 2011) in the soil by Pseudomonas leading to improved plant growth. The proliferated roots and healthy plant might be a source of better nutrient supply for Rhizobium, thus improving their growth and efficacy. The more root growth enhances the root exudation, the more root colonization by microbes in the nutrient-rich environment.

In our studies, co-inoculation with Rhizobium and Pseudomonas strains containing ACC-deaminase increased the shoot fresh weight, root fresh weight, number of pods plant⁻¹, pods fresh weight, total dry matter, RWC, WUE, potassium concentration in leaves, sodium concentration in leaves and nitrogen concentration in grains of mung bean by 145%, 173%, 150%, 182%, 269%, 19%, 51%, 33%, 56% and 99%, respectively, compared with the un-inoculated control, while in previous studies conducted by Nadeem et al. (2007) under salt-affected conditions in pot trials, the separate application of Pseudomonas fluorescens increased the cob mass, total dry matter and grain yield of maize by 40%, 51% and 50%, respectively. So, co-inoculation should be preferred over separate inoculation of Pseudomonas strains containing ACC-deaminase for sustainable production of mung bean under salt-affected conditions. Moreover, under field conditions in Pakistan, high temperature and low rainfall results in net upward movement of salts, which results in secondary salinization, with higher levels of ethylene being produced (Mayak et al. 2004). In such circumstances, the use of PGPR containing ACC-deaminase in combination with Rhizobium should be preferred over single strain inoculation.

Thus, it can be concluded from the results of this study that combined application of Rhizobium phaseoli and Pseudomonas strains is more efficient for improving the productivity of mung bean under salt-stressed conditions when compared with separate inoculation of Rhizobium and Pseudomonas strains containing ACC-deaminase. However, it is imperative to evaluate these strains in combinations for the development of biofertilizers under field conditions to improve the productivity of mung bean under salt-affected conditions.