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Can Catalase and Exopolysaccharides Producing Rhizobia Ameliorate Drought Stress in Wheat?

Byline: Muhammad Baqir Hussain, Zahir Ahmad Zahir, Hafiz Naeem Asghar and Muhammad Asgher

Abstract

Catalase and exopolysaccharides producing ability of rhizobia increases its survival in arid and semiarid climates. Rhizobial exopolysaccharides activity increases root colonization and nutrient or water holding capacity of the rhizosphere. For the reason, a series of experiments were conducted to sort out efficient rhizobial isolates (belonging to Lens culinaris L., Vigna radiata L. and Cicer arietinum L. cultivated in Chakwal (arid) and Faisalabad (semiarid) areas which can ameliorate the impact of water deficit stress on wheat seedlings. Among 35 isolates, Rhizobium leguminosarum (LR-30), Mesorhizobium ciceri (CR-30 and CR-39), and Rhizobium phaseoli (MR-2) improved the growth, biomass and drought tolerance index of the wheat seedlings under PEG-6000 simulated drought. Selected isolates were producers of indole acetic acid, which enhanced the root length of the seedlings to dilute the drought impact.

However, isolates LR-26, MR-17 and CR-34 remained deleterious for seedling growth whether drought was applied or not. In conclusion, rhizobial capability to survive under drought and improving seedling growth under drought is a good criterion to select beneficial bacteria for rescuing wheat seedlings growth under water deficit conditions. (c) 2014 Friends Science Publishers

Keywords: Inoculation; Water deficit; Stress tolerance; Exopolysaccharides; Catalase

Introduction

Generally drought stress occurs when the available water in soil is reduced and atmospheric conditions cause continuous loss of water by transpiration or evaporation (Jaleel et al.,2009).

Up to 45% of the world's agricultural lands are subjected to continuous or frequent drought wherein 38% of the world human population resides (Bot et al., 2000). The agricultural regions that are affected by drought can experience yield loss up to 50% or more (Jenks et al., 2005). As far as Pakistan's situation is concerned, we are facing water shortage problem since decades due to global warming, low rain fall, high temperature and reduced reservoir capacity (Anonymous, 2012). Pakistan is an agriculture based economy and agriculture is the prime sector to be affected by the increased shortage and scarcity of fresh water (FAO, 2007).

Drought tolerance is the ability of plant to grow, flower and display economic yield under suboptimal water supply (Farooq et al., 2009) and wheat is known as moderately drought tolerant crop. However, manipulation of microbial cohabitants can be used to improve adaptive mechanisms and productivity of crop plant (Tikhonovich and Provorvo, 2011), where bacterial inoculation biotechnology could help these plants to induce drought tolerance or sustain the growth and productivity under stress. Certain bacteria are known as beneficial to plant growth while living in association with the roots of the plants and these are termed as plant growth promoting rhizobacteria (PGPR) (Arshad and Frankenberger, 1998). They comprise of many genera including Pseudomonads, Azospirillum, Serretia, Azotobacter and Rhizobium. Rhizobia are nodule forming bacteria in legumes where they fix atmospheric nitrogen while living in symbiosis. However, they can survive in bulk soil and also in the rhizosphere of the plants.

Therefore, they may also act as PGPR with non-nodulating plants. In such plants, although they cannot fix nitrogen but even then they can improve plant growth through certain other activities (Noel et al.,1996; Hussain et al., 2009) like phytohormones (Etesami et al., 2009), enzymes (Bajgiran et al., 2008; Zahir et al.,2008) and exopolysaccharides (Alami et al., 2000) production, nutrient solubilization (Zaidi et al., 2009), improvement in rhizosphere soil structure (Sandhya et al., 2009), and induced stress tolerance (Yang et al., 2009).

Rhizobia probably produce antioxidants (catalase), osmolytes, stress proteins, and exopolysaccharides to survive under harsh environments particularly drought (Goyal et al., 1986; Vanderlinde et al., 2010). However, rhizobia have been observed to survive under drought up to -3.5 MPa (Abolhasani et al., 2010). The ability of these rhizosphere bacteria to tolerate severe water deficit condition can be utilized to ameliorate drought impact on plants (Goyal et al., 1986). They can induce stress tolerance in plants by producing physical and chemical changes in plants (Yang et al., 2009).

Different stress ameliorating and plant growth improving mechanisms of rhizobia have been observed including production of chaperons and sugars (Berjak, 2006), synthesis of stress enzyme 1- aminocyclopropane 1-carboxylic acid (Zahir et al., 2009), exopolysaccharides production (Alami et al., 2000), production of low molecular weight organic compound like trehalose (Zahran, 1999), phosphate solubilization (Zaidi et al., 2009), improved nutrient availability (Hussain et al.,2009), production of siderophores (Arora et al., 2001), phytohormones production (Khalid et al., 2006) and enhanced root respiration (Volpin and Phillips, 1998) by influencing plant physiology.

p till now, rhizobial inoculation has been tested for improving growth and productivity of cereals (rice, maize and wheat) in irrigated conditions (Mehboob et al., 2008; 2011; Hussain et al., 2009). But rhizobial inoculation to non-legumes under stress particularly drought has rarely be studied. However, some sporadic studies on rhizobial capability for improving plant growth of non-legumes are found in the literature but comprehensive information is scanty for their potential to ameliorate the drought impact on non-legumes. Therefore we hypothesized that rhizobia having capability to produce catalase and exopolysaccharides would be less susceptible to drought stress and helpful in ameliorating the impact of drought on wheat seedlings growth by developing micro-aggregates around the plant roots, improving nutrient or water uptake and improving drought tolerance index of the seedlings under drought.

To confirm the hypothesis a series of studies was conducted and efficient plant growth promoting rhizobia (exopolysaccharides and catalase producing) were selected on the basis of their drought tolerance and ability for improving the growth of wheat seedlings under drought.

Materials and Methods

Rhizobial Isolation from Nodules

Leguminous plants lentil (Lens culinaris L.), chickpea (Cicer arietinum L.) and mung bean (Vigna radiata L.) were uprooted at flowering stage without damaging their nodules, from arid (Chakwal) and semiarid (Faisalabad) regions of the Punjab, Pakistan. These plants were transferred to the Soil Microbiology and Biochemistry Laboratory, University of Agriculture, Faisalabad in polythene bags. The plant along with roots and rhizosphere soil was left in flooded condition in a plastic bucket for 2-3 h. After the removal of soil, the roots were washed with tap water to remove the soil adhering to the roots. Nodules were cut from the roots with a sterilized razor and collected in Petri plate. These nodules were surface-disinfected by dipping in 95% ethanol for 5 seconds followed by 10 min dipping in 5% sodium hypochlorite and washing with sterilized distilled water for 4 times. Disinfected nodules were crushed in sterilized test tube with a sterilized glass rod.

The suspension attained was used to inoculate Petri plates containing sterilized yeast extract mannitol agar (YMA) media with a sterilized inoculating needle. Inoculated plates were incubated at 28 +- 1oC. Further, 3 to 4 times streaking was done to get the pure culture of the isolates. A total of 35 isolates capable to produce catalase (bubbling in response to 35% H2O2 was observed in the culture) and exopolysaccharides (mucoid growth of bacterial culture on the RCV-glucose medium) were identified qualitatively following standard protocols given by MacFaddin (1980) and Ashraf et al. (2004), respectively, selected, coded (Table 1) and preserved in 50% glycerol at - 20 +- 1oC. There were 10 isolates from lentil, 12 from chickpea and 13 from mung bean nodules.

Drought Tolerance Assay

All the isolates were tested for drought tolerance capability on the basis of their population growth at different drought levels simulated by polyethylene glycol (PEG) 6000 in yeast extract mannitol broth (YMB) media (Busse and Bottomley,1989).

Fresh inoculum of each isolate was prepared in 100 mL conical flask containing 50 mL sterilized YMB and incubated for 3 days in orbital shaking incubator at 28 +- 1oC and 100 rpm. Culture cells were harvested by centrifugation at 4000g and 4oC for 15 min. Inoculum of uniform cell density 0.5 (106-8 cells mL-1) was prepared in sterilized YMB using optical density meter. Growth media were developed for different drought levels by adding 0, 150, 250 and 350 g PEG L of YMB. Osmotic potential of these media were -0.04, -0.70, -1.67 and -2.18 MPa, respectively, measured by Cryoscopic Osmometer (OSMOMAT-030-D, Gonotec, Germany). Osmotic potential of the media containing PEG was measured before and after autoclaving to check the change in developed potential. Freshly prepared inocula (0.5 OD) of each isolate was inoculated (0.5 mL) in triplicated set of sterilized test tubes containing seven mL of YMB with different PEG concentrations and incubated in orbital shaking incubator at 28 +- 1oC and 100 rpm.

Un- inoculated control set of test tubes at each PEG concentration was also maintained with three repeats. After 4 days of incubation, OD was measured by spectrophotometer at 600 nm (0.5 OD = 10 6-8 cells mL-1).

Isolates showing more absorbance (OD) were supposed drought tolerant.

Plant Growth Promoting Activity of Rhizobial Isolates under Simulated Drought

Rhizobial isolates were further tested for their ability to promote seedling growth of wheat (Triticum aestivum L.) cultivar Sahar-2006 under PEG-6000 simulated drought. The experiment was performed by dividing isolates into three groups with respect to the host legume crop from which they were isolated. Three separate trials were conducted with rhizobial isolates of lentil, chickpea and mung bean.

Fresh inoculum was prepared for each experiment as described in drought tolerance assay. After maintaining the uniform cell density, two sterilized filter paper sheets were soaked with the respective inoculum. Three surface sterilized pre-germinated seeds were dipped in the inoculum for 10 min and placed in between the sheets soaked in respective inoculum. The filter paper sheets were rolled and put in sterilized glass jars (Asghar et al., 2002). Sterilized broth was used as control treatment. Each treatment was replicated thrice. Different drought levels (No PEG, 15 and 25% PEG having potentials -0.02, -0.62 and - 1.23 MPa, respectively) were maintained in sterilized Hoagland solution (1/2 strength) by using PEG-6000. Light and dark periods were adjusted at 10 and 14 h, respectively, and suitable temperature (20-25oC) was maintained.

After five days of germination, drought was imposed by applying PEG solutions. Plants were harvested after 15 days of drought treatment and data regarding root or shoot length, and fresh or dry biomass were recorded.

Drought Tolerance Index

Drought or stress tolerance index of the wheat seedlings was derived by dividing the shoot length of inoculated and stressed (SLIN and S) seedling with the shoot length of un- inoculated and unstressed (SLUN and US) seedling (Ahmad, 2011).

Where, SLIN = shoot length of inoculated, SLUN = shoot length of un-inoculated, S = stressed. US = unstressed.

Rhizobial isolates showing better performance for drought tolerance, plant growth promoting activity under drought and improving DTI were pronounced as prominent isolates.

Plant Growth Promoting Mechanisms and Identification of Selected Rhizobia

Selected rhizobial isolates were identified using BIOLOG(r) identification system (Microlog System release 4.2, Biolog Inc., USA) and confirmed by inoculation to their respective legumes (Lens culinaris L., Vigna radiata L. and Cicer arietinum L.) in axenic conditions where they produced nodules. Plant growth promoting mechanisms which in one or the other way supported plant growth and survival under stress were measured following their standard protocols. Bacterial aggregation ability was determined by measuring the turbidity of cell suspension (OD1) after 20 min settling of cell aggregates in test tube and second turbidity (OD2) after 1 min vortex. Using the formula: %aggregation = [(OD2 - OD1)/OD2] x 100 The aggregation ability was calculated (Madi and Henis, 1989; Burdman et al., 1998). Organic acid producing ability was identified with change in the colour of bromothymol blue dyed agar media from blue to yellow/orange (Vincent, 1970).

Indole acetic acid production with or without L-tryptophan using salkowski reagent was measured at 535 nm on spectrophotometer and IAA concentration was calculated using standard curve (Sarwar et al., 1992). Siderophores activity was recorded by observing the change in the colour of chrom azurol S (CAS) agar medium due to the inoculation of bacteria (Schwyn and Neilands, 1987).

Statistical Analysis

Data collected was analyzed statistically following completely randomized design in two factor factorial settings by two way analysis of variance (ANOVA) (Steel et al., 1996) and treatment means were compared using Tukey's test. The statistical software "Statistix" version 8.1 was used for analysis (Copy right 2005, Analytical Software, USA).

Results

Drought Tolerance of Rhizobia from Different Legumes

Rhizobia showed significant capability to withstand the simulated drought conditions (Table 1). Among the isolates from lentil, LR-29, LR-32 and LR-30 were prominent at 15, 25 and 35% PEG-6000 containing YMB, respectively. Whereas isolate LR-19 was following LR-29 at 15% PEG stress and LR-29 was following LR-32 and LR-30 at 25 and 35% PEG induced drought, respectively. Isolate MR-18 showed highest optical density among isolates followed by MR-2 at 15% PEG (Table 1). At 25% PEG, highest OD was recorded by MR-13 followed by MR-6. Whereas, in the most desiccated condition (35% PEG), MR-2 remained prominent among the isolates, whereas MR-13 was at second place. When chickpea rhizobial isolates were compared for drought tolerance, then CR-29 was fair enough to stand at 35% PEG induced drought followed by CR-26, CR-38 and CR-39 as compared to other isolates (Table 1). At 25% PEG, the isolate CR-25 showed highest OD followed by CR-29.

On the other hand, isolate CR-25 gave highest OD among the isolates followed by CR-26 at 15% PEG concentration.

Overall, rhizobial isolates from chickpea were more tolerant, keeping lentil nodular isolates at second place but the isolates from the nodules of mung bean were least tolerant to desiccation compared to isolates from chickpea and lentil.

Screening of Rhizobia having Potential to Improve Plant Growth under Drought

Rhizobium leguminosarum: Rhizobial isolates from lentil nodules showed a significant capability to reduce/ameliorate the impact of drought on wheat seedlings and improved their growth (root or shoot length and fresh or dry weight)

Table 1: Optical density (OD600) of rhizobial isolates from lentil, mung bean and chickpea nodules after 96 hours incubation in YEM without and with 15, 25 and 35% PEG (osmotic potential, -0.04, -0.70,-1.67 and -2.18 MPa, respectively)

Isolates###Lentil rhizobia (n = 3) (optical density (OD) at 600 nm)

###-0.04 MPa###-0.70 MPa###-1.67 MPa###-2.18 MPa

LR-15###0.50 +- 0.00###0.31 +- 0.01###0.17 +- 0.01###0.07 +- 0.00

LR-18###0.79 +- 0.01###0.26 +- 0.00###0.16 +- 0.00###0.09 +- 0.00

LR-19###1.11 +- 0.01###0.47 +- 0.00###0.22 +- 0.00###0.09 +- 0.01

LR-21###0.53 +- 0.01###0.25 +- 0.00###0.14 +- 0.00###0.09 +- 0.00

LR-23###0.50 +- 0.01###0.28 +- 0.01###0.13 +- 0.00###0.07 +- 0.00

LR-26###0.53 +- 0.00###0.22 +- 0.00###0.14 +- 0.00###0.10 +- 0.00

LR-29###1.02 +- 0.01###0.70 +- 0.00###0.23 +- 0.00###0.14 +- 0.00

LR-30###0.41 +- 0.01###0.31 +- 0.00###0.22 +- 0.00###0.16 +- 0.01

LR-32###0.79 +- 0.01###0.45 +- 0.00###0.26 +- 0.01###0.12 +- 0.01

LR-33###0.68 +- 0.01###0.24 +- 0.00###0.20 +- 0.00###0.08 +- 0.01

###Mung bean rhizobia (n = 3) (optical density (OD) at 600 nm)

MR-2###0.94 +- 0.00###0.37 +- 0.01###0.21 +- 0.01###0.17 +- 0.01

MR-3###0.49 +- 0.00###0.29 +- 0.01###0.22 +- 0.02###0.06 +- 0.01

MR-6###0.44 +- 0.00###0.36 +- 0.03###0.23 +- 0.03###0.05 +- 0.00

MR-8###0.44 +- 0.01###0.12 +- 0.01###0.05 +- 0.00###0.05 +- 0.00

MR-11###0.60 +- 0.00###0.20 +- 0.01###0.11 +- 0.00###0.11 +- 0.01

MR-12###0.36 +- 0.01###0.16 +- 0.00###0.15 +- 0.00###0.10 +- 0.00

MR-13###0.73 +- 0.01###0.20 +- 0.01###0.26 +- 0.00###0.12 +- 0.00

MR-16###0.60 +- 0.00###0.21 +- 0.01###0.08 +- 0.00###0.08 +- 0.01

MR-17###0.47 +- 0.00###0.13 +- 0.00###0.09 +- 0.01###0.08 +- 0.01

MR-18###0.61 +- 0.01###0.39 +- 0.00###0.12 +- 0.01###0.08 +- 0.00

MR-22###0.42 +- 0.00###0.07 +- 0.00###0.05 +- 0.00###0.05 +- 0.00

MR-42###0.50 +- 0.00###0.24 +- 0.00###0.19 +- 0.01###0.11 +- 0.00

MR-43###0.72 +- 0.01###0.18 +- 0.00###0.15 +- 0.00###0.11 +- 0.00

###Chickpea rhizobia (n = 3) (optical density (OD) at 600 nm)

CR-21###0.37 +- 0.00###0.25 +- 0.00###0.06 +- 0.00###0.05 +- 0.00

CR-24###0.51 +- 0.00###0.22 +- 0.01###0.17 +- 0.00###0.14 +- 0.00

CR-25###0.52 +- 0.01###0.33 +- 0.01###0.24 +- 0.01###0.14 +- 0.00

CR-26###0.87 +- 0.01###0.31 +- 0.00###0.17 +- 0.00###0.15 +- 0.01

CR-28###0.64 +- 0.00###0.25 +- 0.00###0.15 +- 0.00###0.07 +- 0.00

CR-29###0.31 +- 0.01###0.23 +- 0.01###0.20 +- 0.00###0.19 +- 0.01

CR-30###0.72 +- 0.00###0.25 +- 0.00###0.12 +- 0.00###0.12 +- 0.01

CR-31###0.54 +- 0.01###0.22 +- 0.00###0.14 +- 0.00###0.12 +- 0.00

CR-34###0.43 +- 0.00###0.23 +- 0.01###0.17 +- 0.00###0.11 +- 0.00

CR-35###0.86 +- 0.00###0.22 +- 0.01###0.13 +- 0.00###0.12 +- 0.01

CR-38###0.29 +- 0.01###0.19 +- 0.00###0.15 +- 0.00###0.15 +- 0.00

CR-39###0.50 +- 0.00###0.22 +- 0.00###0.18 +- 0.00###0.15 +- 0.00

Fig. 1: Dry biomass of wheat seedlings influenced by the inoculation of rhizobia isolated from lentil nodules under normal condition and drought induced by 15 and 25% PEG solutions. Vertical bars represent the standard error of each treatment and certain bars are invisible due to very small value. Different letters indicate significantly different seedling dry biomass at p [?] 0.05

in contrast to un-inoculated control (Table 2; Fig. 1). However, few isolates caused reduction in seedling growth including LR-26, LR-33 and LR-15. Root length of the wheat seedlings was significantly improved with the inoculation of LR-19, LR-23 and LR-32 as compared to un-inoculated control under un-stressed condition. When inoculated plants were irrigated with half strength Hoagland solution containing 15% PEG, LR-30 inoculated seedlings showed significant improvement (20%) in root length over respective control. However, isolate LR-15

Table 2: Plant growth promotion of wheat seedlings under drought (No PEG, 15 and 25% PEG) when inoculated with

Isolates###Root length (cm) (n = 3)###Shoot length (cm) (n = 3)

###-0.02 MPa###-0.62 MPa###-1.23 MPa###-0.02 MPa###-0.62MPa###-1.23 MPa

Control###16.1+-0.28d-i###14.8+-0.15f-k###10.3+-0.37no###19.2+-0.19e-g###16.0+-0.29kl###11.0+-0.26no

LR-15###14.8+-0.42f-j###14.0+-0.29h-m###11.3+-0.25m-o###17.7+-0.44h-j###17.2+-0.24i-k###11.8+-0.27m-o

LR-18###16.5+-0.55d-h###16.2+-0.42d-h###13.3+-0.73i-m###20.8+-0.60b-d###18.2+-0.44g-i###12.2+-0.60mn

LR-19###20.0+-0.29ab###16.8+-0.44c-g###10.0+-0.58o###21.7+-0.44b-d###20.3+-0.73b-f###11.5+-0.87m-o

LR-21###17.3+-0.44b-f###16.3+-0.44d-h###12.8+-0.44j-n###19.8+-0.44d-f###17.2+-0.60i-k###12.5+-0.29m

LR-23###19.3+-0.44a-c###15.2+-0.44e-j###12.0+-0.58k-o###21.5+-0.29b-d###17.7+-0.60h-j###7.5+-0.29p

LR-26###15.6+-0.34d-j###14.4+-0.80g-l###10.3+-0.67no###20.0+-0.76c-f###15.8+-0.38kl###11.2+-0.47m-o

LR-29###17.8+-0.44a-e###15.3+-0.60d-j###11.3+-0.60m-o###21.3+-0.60bc###16.3+-0.33j-l###15.0+-0.58l

LR-30###18.0+-0.29a-d###17.7+-0.44b-e###11.7+-0.67l-o###23.5+-0.76a###20.7+-0.33b-e###10.5+-0.76o

LR-32###20.5+-0.29a###14.7+-0.88f-k###10.5+-0.29no###24.7+-0.44a###19.0+-0.58f-h###11.8+-0.43m-o

LR-33###15.5+-0.29d-j###14.8+-0.43f-k###11.5+-0.00m-o###18.0+-0.58g-i###16.0+-0.58kl###11.5+-0.29m-o

HSD###0.48###1.44

###Root fresh weight (mg/plant) (n = 3)###Shoot fresh weight (mg/plant) (n = 3)

Control###177+-8.8cd###127+-8.8e-h###63+-3.3j-l###190+-2.9ef###153+-2.0hi###47+-1.8n

LR-15###147+-3.3c-f###120+-5.8f-i###35+-2.9kl###163+-2.0gh###114+-3.5l###37+-2.0n-p

LR-18###233+-3.3ab###173+-6.7c-e###43+-12.0j-l###217+-2.9d###170+-2.3g###30+-2.9op

LR-19###150+-5.8c-f###137+-8.8d-g###30+-5.8l###217+-2.9d###213+-3.7d###27+-1.2p

LR-21###153+-14.5c-f###120+-5.8f-i###80+-5.8h-k###167+-3.3gh###143+-1.8ij###47+-2.0n

LR-23###237+-14.5ab###140+-5.8d-f###37+-12.0kl###244+-2.1c###213+-3.3d###47+-0.9no

LR-26###120+-5.8f-i###90+-5.8g-j###40+-5.8kl###177+-3.3fg###123+-3.8kl###37+-1.5n-p

LR-29###180+-5.8cd###140+-10.0d-f###73+-8.8i-l###170+-3.5g###83+-3.3m###37+-2.4n-p

LR-30###257+-8.8a###190+-5.8bc###40+-5.8kl###267+-2.4b###187+-2.0f###40+-1.7n-p

LR-32###253+-14.5a###137+-8.8d-g###45+-8.7j-l###303+-3.3a###203+-2.4de###30+-0.6op

LR-33###135+-8.7d-g###140+-11.6d-f###45+-8.7j-l###130+-1.2jk###120+-2.3kl###30+-1.2op

HSD###8.61###14.17

Rhizobium leguminosarum isolated from lentil nodules in gnotobiotic conditions

Means sharing similar letters are statistically similar to each other at p [?] 0.05. The table represents the interaction effect of drought and rhizobial inoculation on different growth parameters of wheat seedlings under axenic conditions. HSD represents honestly significant difference among treatment means and standard error of each mean has been mentioned as (mean +- SE) after calculation on Microsoft Excel sheet reduced root growth up to 5% of the un-inoculated control at 15% PEG solution treatment but remained statistically similar to control. Application of 25% PEG solution affected root growth severely but root length of the most of the inoculated seedlings was at par with un-inoculated control except LR-18, which improved root length up to 30%. As far as, shoot length is concerned, LR-18, LR-19, LR-23, LR-29, LR-30 and LR-32 inoculation significantly improved shoot length over control in normal conditions.

But LR-15 caused non-significant decline in shoot length as compared to control in un-stressd condition (Table 2).

At 15% PEG, LR-18, LR-19, LR-23, LR-29, LR-30 and LR-32 inoculation showed significantly high shoot length over respective control. In the meantime, isolates LR-15, LR-21, LR-26, LR-29 and LR-33 were statistically similar with the respective un-inoculated control at 15% PEG. Similar trend was observed at 25% PEG simulated drought where inoculation remained at par with respective un-treated control but LR-29 and LR-21 produced significant increase of 36 and 13% in shoot length, respectively, compared to respective un-inoculated control. Rhizobial isolates LR-18, LR-23, LR-30 and LR-32 significantly improved root fresh weight of the wheat seedlings from 32-45% over control at normal condition and remained at par with each other (Table 2). Isolate LR-26 influenced root fresh weight negatively but non-significantly by reducing it up to 32% compared to control.

Isolate LR-30 inoculation gave significant improvement in the root fresh weight of the wheat seedlings as compared to un-inoculated control at 15% PEG simulated drought. The seedlings inoculated with LR-21 showed highest root fresh weight among the treatments followed by LR-29 at 25% PEG induced drought however, the differences were non-significant. At 25% PEG, inoculation of isolates from lentil nodules could not develop significant differences from the control for shoot fresh biomass (Table 2). Inoculation of rhizobial isolates LR-18, LR-19, LR-23, LR-30 and LR-32 gave significant improvement in the shoot fresh weight of the wheat seedlings under normal as well as drought induced by 15% PEG over respective un-inoculated controls. At 25% PEG induced drought, LR-21 and LR-32 increased root and shoot dry biomass up to 27 and 30%, respectively, in contrast to the respective controls followed by LR-29 (about 25%) for root and shoot dry biomass, respectively (Fig. 1).

Under 15% PEG induced drought, LR-30 showed highest root dry biomass followed by LR-21 and LR-30 again showed maximum shoot dry mass followed by LR-19. However, seedling dry biomass was maximum by LR-30 and LR-32 inoculation at 15 and 25% PEG induced drought, respectively, followed by LR-21 and LR-29. Isolate LR-26 remained non beneficial and reduced seedling dry biomass up to 37 and 20% at 15 and 25% PEG induced drought, respectively, compared to the respective controls.

Rhizobium phaseoli: Rhizobial isolates from mung bean nodules performed significantly for ameliorating the impact of drought on seedling growth of wheat (Table 3).

Isolate MR-2 gave significantly high improvement in the root length of the seedlings up to 37% compared to control under normal conditions but remained at par with other isolates. Similar trend was followed at 15 and 25% PEG solution treatment where rhizobial inoculation produced 22-42% and 29-65% increase in root length of seedlings, respectively, compared to respective controls. Maximum shoot length was recorded by MR-2 inoculation among the isolates followed by MR-13 and MR-6 compared to un- inoculated control under normal conditions (Table 3). However, MR-22 gave 49 and 78% more shoot length with respect to control at 15 and 25% PEG, respectively. The isolates MR-12 and MR-2 were following MR-22 for shoot length at 25 and 15% PEG induced water stress, respectively. Root fresh weight was significantly affected due to the inoculation of rhizobia but the results of many isolates remained indifferent from each other (Table 3).

Under highest drought level (25% PEG), isolates MR-2, MR-6, MR-12, MR-13 and MR-16 were prominent for improving root fresh weight as compared to un-inoculated control. At 15% PEG induced drought, significantly high improvement (up to 69%) in root fresh weight was observed by MR-2 inoculation over respective control followed by MR-6, MR-13 and MR-43. A marked decrease in root biomass was observed with MR-8 (26%) as compared to control under normal conditions whereas, its inoculation could not make significant differences at 15 and 25% PEG induced drought as compared to respective controls. Isolate MR-2, MR-6, MR-13 and MR-22 inoculation significantly improved the shoot fresh biomass over respective control under normal conditions. At 15% PEG induced drought, isolate MR-13 inoculation gave significant improvement in shoot fresh weight (up to 23%) followed by MR-2 and MR-6.

Inoculation of MR-2 gave highest significant increase in the shoot fresh weight up to 22 and 124% as compared to corresponding control at normal and 25% PEG stressed conditions, respectively (Table 3). Isolates MR-11 and MR-18 caused highest decrease in shoot fresh biomass compared to control under normal conditions followed by MR-16 and MR-17 at 15% PEG induced stress in contrast to respective control.

However, MR-16 caused maximum reduction in shoot fresh weight compared to respective control when irrigated with 25% PEG solution. Seedling dry biomass was improved due to rhizobial inoculation under PEG induced drought conditions where maximum biomass was recorded i.e. 31.0 and 27.5 mg due to isolate MR-2 inoculation at 15 and 25% PEG induced water deficit, respectively followed by isolates MR-3 (30.9 mg) and MR-

Table 3: Plant growth promotion of wheat seedlings under drought (No PEG, 15 and 25% PEG) when inoculated with Rhizobium phaseoli isolated from mung bean nodules in gnotobiotic conditions

Isolates###Root length (cm) (n = 3)###Shoot length (cm) (n = 3)

###-###0.02 MPa###-0.62 MPa###-1.23 MPa###-0.02 MPa###-0.62 MPa###-1.23 MPa

Control###11.1+-0.35i-k###9.9+-0.36k###7.7+-0.24l###16.2+-0.38p-t###14.1+-0.32u-w###10.6+-0.23x

MR-2###15.2+-0.22a###14.1+-0.38a-d###12.6+-0.32d-i###24.9+-0.31a###21.0+-0.36d-i###15.2+-0.25q-v

MR-3###13.5+-0.29a-g###12.80.32c-i###9.9+-0.35k###22.5+-0.29b-d###20.2+-0.35f-k###12.3+-0.35wx

MR-6###14.7+-0.22ab###13.7+-0.29a-g###11.5+-0.24h-k###23.3+-0.33a-c###20.4+-0.31e-j###15.4+-0.30q-v

MR-8###13.6+-0.37a-g###12.0+-0.39g-j###11.1+-0.22i-k###21.7+-0.31c-g###18.4+-0.32k-o###14.4+-0.19t-v

MR-11###14.5+-0.29a-c###12.9+-0.15c-h###11.2+-0.38h-k###19.6+-0.22i-m###17.8+-0.36m-p###15.0+-0.29r-v

MR-12###14.2+-0.27a-d###13.5+-0.38a-g###11.3+-0.32h-k###22.0+-0.23c-f###20.3+-0.33e-j###16.3+-0.33p-s

MR-13###14.8+-0.39ab###13.9+-0.09a-f###12.2+-0.33e-j###24.0+-0.40ab###19.6+-0.36i-m###15.2+-0.37q-v

MR-16###13.9+-0.24a-e###12.8+-0.30c-i###11.1+-0.39i-k###18.9+-0.31j-m###16.6+-0.38o-r###14.7+-0.39r-v

MR-17###14.5+-0.03a-c###12.2+-0.32e-j###10.6+-0.38jk###19.8+-0.33h-l###18.2+-0.38l-o###14.4+-0.30t-v

MR-18###14.0+-0.09a-d###12.6+-0.35d-i###11.3+-0.15h-k###20.8+-0.27d-i###17.0+-0.33n-q###14.5+-0.26s-v

MR-22###14.2+-0.27a-d###13.3+-0.36b-g###11.1+-0.34i-k###22.2+-0.33b-e###21.1+-0.38d-i###18.8+-0.22j-n

MR-42###13.9+-0.38a-f###12.2+-0.17f-j###11.2+-0.29h-k###21.5+-0.26c-h###20.0+-0.32g-l###15.8+-0.29q-u

MR-43###13.9+-0.32a-f###12.2+-0.36e-j###11.5+-0.29h-k###21.6+-0.32c-h###21.0+-0.27d-i###13.8+-0.38vw

HSD###1.74###1.84

###Root fresh weight (mg/plant) (n = 3)###Shoot fresh weight (mg/plant) (n = 3)

Control###86+-2.1cd###45+-2.6g-k###30+-1.7k###175+-3.7c-e###135+-3.7hi###45+-3.3l-o

MR-2###120+-2.9a###77+-2.0de###50+-1.7f-i###213+-2.9a###165+-2.3d-g###100+-2.3j

MR-3###80+-3.5d###57+-2.4fg###30+-1.7k###190+-1.7bc###140+-1.7hi###27+-3.3p

MR-6###107+-0.9ab###63+-3.3ef###50+-3.5f-i###197+-3.3b###160+-3.5e-g###60+-1.7kl

MR-8###63+-3.3ef###47+-3.8g-j###33+-3.3jk###170+-2.1d-f###127+-3.3i###33+-0.9op

MR-11###97+-3.3bc###60+-5.8fg###40+-0.6h-k###130+-1.2i###127+-2.4i###53+-3.3k-m

MR-12###100+-1.7bc###57+-1.8fg###47+-3.3g-j###157+-3.3fg###130+-1.7i###50+-0.6k-n

MR-13###120+-1.7a###63+-3.8ef###50+-2.9f-i###200+-2.3ab###167+-3.3d-f###63+-0.9k

MR-16###87+-3.8cd###47+-2.4g-j###47+-3.3g-j###163+-3.3e-g###103+-3.3j###30+-1.7op

MR-17###103+-3.8b###50+-0.6f-i###33+-1.8jk###173+-2.4de###103+-2.9j###40+-2.3m-p

MR-18###80+-2.3d###53+-3.3f-h###33+-3.5jk###130+-3.5i###130+-1.2i###57+-3.8kl

MR-22###103+-2.4b###50+-0.2f-i###37+-2.0i-k###197+-2.7b###150+-0.6gh###50+-2.9k-n

MR-42###87+-3.3cd###53+-2.3f-h###37+-1.8i-k###167+-3.8d-f###133+-2.0i###37+-3.3n-p

MR-43###107+-2.9ab###63+-3.3ef###37+-3.3i-k###180+-3.5cd###137+-3.3hi###53+-0.9k-m

HSD###16.20###15.55

Means sharing similar letters are statistically similar to each other at p [?] 0.05. The table represents the interaction effect of drought and rhizobial inoculation on different growth parameters of wheat seedlings under axenic conditions. HSD represents honestly significant difference among treatment means and standard error of each mean has been mentioned as (mean +- SE) after calculation on Microsoft Excel sheet

Table 4: Plant growth promotion of wheat seedlings under drought (No PEG, 15 and 25% PEG) when inoculated with Mesorhizobium ciceri isolated from chickpea nodules in gnotobiotic conditions

Isolates###Root length (cm) (n = 3)###Shoot length (cm) (n = 3)

###-0.02 MPa###-0.62 MPa###-1.23 MPa###-0.02 MPa###-0.62 MPa###-1.23 MPa

Control###10.7+-0.35r###8.6+-0.39s###6.9+-0.38t###18.0+-0.29j###15.7+-0.18k###7.6+-0.09r

CR-21###16.3+-0.29a-d###14.8+-0.29d-j###12.5+-0.25m-q###23.8+-0.32a###19.0+-0.32g-j###12.0+-0.29op

CR-24###16.8+-0.12ab###14.5+-0.25e-k###12.6+-0.23m-p###20.7+-0.27d-f###18.5+-0.20ij###12.2+-0.29n-p

CR-25###14.8+-0.36d-j###13.4+-0.23j-o###11.8+-0.29o-r###20.3+-0.35e-h###17.6+-0.33j###9.7+-0.15q

CR-26###15.1+-0.30c-j###14.3+-0.29f-l###13.6+-0.15j-n###21.9+-0.35b-d###18.4+-0.29ij###11.5+-0.18op

CR-28###15.9+-0.23a-e###13.9+-0.35h-n###11.8+-0.25p-r###19.7+-0.27f-i###17.7+-0.38j###9.7+-0.35q

CR-29###16.5+-0.29a-c###14.8+-0.39d-j###12.7+-0.15l-p###21.3+-0.31c-f###18.9+-0.35g-j###14.4+-0.38k-m

CR-30###16.2+-0.30a-d###15.4+-0.18b-h###11.8+-0.27o-r###23.0+-0.32ab###22.5+-0.29a-c###13.7+-0.33l-n

CR-31###14.0+-0.29g-m###13.7+-0.27i-n###12.4+-0.19n-q###21.8+-0.27b-e###14.5+-0.29k-m###11.3+-0.33pq

CR-34###15.2+-0.31c-i###14.5+-0.32e-k###10.9+-0.30qr###21.0+-0.29c-f###13.0+-0.29m-o###11.3+-0.35pq

CR-35###15.8+-0.24a-f###13.0+-0.26k-p###10.3+-0.33r###20.3+-0.33d-g###18.6+-0.23h-j###12.3+-0.27n-p

CR-38###17.2+-0.32a###14.6+-0.15e-j###12.4+-0.23n-q###20.6+-0.30d-f###18.2+-0.17ij###11.7+-0.37op

CR-39###16.3+-0.18a-d###15.6+-0.22b-g###12.9+-0.21l-p###22.5+-0.06a-c###18.1+-0.29ij###15.2+-0.22kl

HSD###1.57###1.65

###Root fresh weight (mg/plant) (n = 3)###Shoot fresh weight (mg/plant) (n = 3)

Control###61+-3.3e-g###51+-3.3f-i###21+-2.4l###161+-2.0d-f###126+-3.7h###25+-2.0o

CR-21###90+-2.3a-c###53+-2.9f-i###27+-2.0j-l###193+-0.9b###110+-1.2i###20+- 1.7o

CR-24###87+-3.5a-c###63+-3.3e-g###37+-3.3i-l###163+-3.3c-f###137+-3.8gh###67+- 1.5lm

CR-25###83+-3.3b-d###53+-3.3f-i###50+-0.6f-i###173+-3.8cd###133+-3.3h###40+-2.3n

CR-26###73+-3.3c-e###50+-1.7f-i###40+-2.3i-k###150+-3.5fg###100+-3.5ij###40+-3.5n

CR-28###83+-3.3b-d###63+-3.3e-g###27+-3.7j-l###153+-2.4ef###127+-2.0h###23+-2.4o

CR-29###83+-3.8b-d###67+-3.4d-f###50+-2.5f-i###157+-2.0ef###137+-1.8gh###87+-1.8jk

CR-30###90+-3.5a-c###83+-2.9b-d###50+-1.2f-i###197+-1.9ab###150+-3.1fg###70+-3.5lm

CR-31###90+-2.3a-c###50+-3.5f-i###40+-1.2i-k###210+-1.2a###57+-2.4m###27+-2.0no

CR-34###97+-3.4ab###37+-2.4i-l###23+-3.5kl###153+-3.3ef###70+-0.6lm###33+-0.9no

CR-35###87+-3.3a-c###67+-2.0d-f###47+-3.4g-i###167+-3.8c-e###130+-1.2h###57+-3.7m

CR-38###90+-3.8a-c###47+-2.2g-i###43+-2.0h-j###163+-3.5c-f###100+-0.6ij###33+-2.9no

CR-39###103+-3.3a###77+-3.3c-e###60+-1.7e-h###197+-1.8ab###177+-2.4c###77+-2.9kl

HSD###16.70###14.78

Means sharing similar letters are statistically similar to each other at p [?] 0.05. The table represents the interaction effect of drought and rhizobial inoculation on different growth parameters of wheat seedlings under axenic conditions. HSD represents honestly significant difference among treatment means and standard error of each mean has been mentioned as (mean +- SE) after calculation on Microsoft Excel sheet

Fig. 2: Dry biomass of wheat seedlings influenced by the inoculation of rhizobia isolated from mung bean nodules under normal condition and drought induced by 15 and 25% PEG solutions. Vertical bars represent the standard error of each treatment and certain bars are invisible due to very small value. Different letters indicate significantly different seedling dry biomass at p [?] 0.05

13 (26.0 mg) at respective 15 and 25% PEG induced droughts (Fig. 2). However, shoot dry biomass was maximally produced by isolate MR-6 (19.7 mg) followed by MR-3 (18.4 mg) at 15% PEG induced drought. But root dry matter at 15% PEG induced water limited condition was higher with MR-2 (13.8 mg) inoculation followed by MR-11 (13.6 mg). At 25% PEG induced drought MR-13 (11.5 mg) and MR-2 (16.7 mg) caused maximum root and shoot dry biomass production, respectively, followed by MR-2 (10.8 mg) and MR-22 (16.5 mg), respectively. Maximum decrease in seedling dry biomass was observed by MR-16 and MR-17 up to 20 and 19% at 25 and 15% PEG induced drought, respectively, in contrast to respective controls. Mesorhizobium ciceri: Isolates from the nodules of chickpea performed good for improving the seedling growth of wheat under PEG induced water deficit conditions (Table 4).

Root length of the seedlings was affected seriously due to drought but the inoculation of rhizobia prominently increased the root length at 15 and 25% PEG induced drought as well as under normal conditions. Under normal conditions and at 25% PEG induced drought, rhizobial inoculation significantly promoted the shoot length over control (Table 4). At 15% PEG induced drought, except isolates CR-31 and CR-34, all the inoculated rhizobia gave significant improvement in the shoot length of the wheat seedlings as compared to un-inoculated control.

At 25% PEG induced stress, isolate CR-39 showed maximum significant improvement in root fresh weight followed by CR-25, CR-26, CR-29, CR-30, CR-31, CR-35 and CR-38. At 15% PEG induced drought, CR-30 inoculation gave significantly high increase (63%) in the root fresh weight over control followed by CR-39 (up to 51% increase). Shoot fresh weight improvement was also significant due to CR-39 inoculation at 15% PEG induced drought with 41% increase over respective control followed by CR-30 with 19% increase. In the same lines, CR-29 showed maximum significant shoot fresh weight at 25% PEG stress followed by CR-39, CR-30, CR-24, CR-35, CR-25 and CR-26. Inoculated isolate CR-29 showed maximum seedling dry biomass followed by CR-30 at 25% PEG induced drought compared to control (Fig. 3). At 15% PEG induced drought, isolate CR-30 remained most prominent with 33.3 mg seedling dry biomass followed by CR-29 (31.5 mg) with respect to control (26.8 mg).

When only root dry matter was compared with respect to inoculated treatments then CR-29 and CR-30 inoculation produced maximum biomass (14.2 and 11.2 mg) at 15 and 25% PEG induced drought followed by CR-39 with 14 and 10.8 mg root dry biomass, respectively, as compared to the respective controls. As well as, shoot dry matter was maximally improved by CR-30 (20.1 mg) and CR-29 (16.5 mg) at 15 and 25% PEG induced drought, respectively, followed by CR-29 (17.3 mg) and CR-30 (15.2 mg) at respective drought treatments with respect to respective controls.

Drought Tolerance Index

Rhizobial inoculation helped wheat plants to sustain their growth under water deficit stress. Therefore, change in drought tolerance index (DTI) of the plants was derived to test the potential of inoculant bacteria for improving drought abiding ability in plants. Isolates of rhizobia belonging to lentil nodules, significantly improved DTI under 15 and 25% PEG induced drought in contrast to un-inoculated control (Fig. 4). Isolates LR-19 and LR-30 remained prominent for improving DTI of wheat seedling at 15% PEG induced drought (up to 27-29%) in contrast to the respective control, whereas LR-29 was showing maximum DTI under 25% PEG induced drought with 37% increase over respective control. A negative interaction of LR-23 was observed with 32% reduction in DTI of wheat seedlings as compared to respective un-inoculated control at 25% PEG induced drought.

Mung bean nodular isolates have also performed positively for enhancing DTI of wheat seedlings at all drought levels but most of the isolates were statistically at par with each other or un-inoculated control (Fig. 5). Even then MR-2, MR-22 and MR-43 improved tolerance index up to 16% followed by MR-3, MR-6 and MR-12 with 12% increase at 15% PEG induced drought over respective control. However, 6-8% decrease in DTI of seedlings was recorded due to MR-18 and MR-16 over control at -0.62 MPa (25% PEG) water potential. At -1.23 MPa, isolate MR-22 remained prominent for enhancing DTI about 31% over control, while MR-3 caused 15% decrease in tolerance index in contrast to control. Rhizobial isolate CR-30 from chickpea enhanced drought tolerance index of wheat seedlings up to 43% at 15% PEG induced drought compared to the respective control

Table 5: Identification and plant growth promoting characterization of selected rhizobial isolates

Characteristics###R. phaseoli###R. leguminosarum###Mesorhizobium ciceri

###MR-2###LR-30###CR-30###CR-39

Aggregation (%)###4.61###5.63###2.68###6.93

Catalase###+###++###++###++

Exopolysaccharides###++###++###+++###++

Siderophores###-###+###-###-

Organic acid###++###-###++###++

IAA production (mg L-1)###Without L-TRP###1.25###2.61###1.70###3.77

###With L-TRP###19.63###27.39###16.57###34.26

Average of three repeats, (+) for possession and (-) for absence of the character

Fig. 3: Dry biomass of wheat seedlings influenced by the inoculation of rhizobia isolated from chickpea nodules under normal condition and drought induced by 15 and 25% PEG solutions. Vertical bars represent the standard error of each treatment and certain bars are invisible due to very small value. Different letters indicate significantly different seedling dry biomass at p [?] 0.05

Fig. 4: Drought tolerance index of wheat seedlings on the basis of shoot length influenced by the inoculation of rhizobia isolated from lentil nodules under drought induced by 15 and 25% PEG solutions. Vertical bars represent the standard error of each treatment

Fig. 5: Drought tolerance index of wheat seedlings on the basis of shoot length influenced by the inoculation of rhizobia isolated from mung bean nodules under drought induced by 15 and 25% PEG solutions. Vertical bars represent the standard error of each treatment followed by CR-21 with 20% increase in DTI (Fig. 6). Isolates CR-34 and CR-31 caused reduction in DTI up to 18 and 9%, respectively at 15% PEG solution treatment compared to respective control. At 25% PEG induced stress CR-39 was prominent followed by CR-29 with an increment up to 100 and 90%, respectively, over un-inoculated control.

Identification of Factors Promoting Wheat Growth by Rhizobial Inoculation

Rhizobial isolates (MR-2, LR-30, CR-30 and CR-39) selected from the series of experiments were further tested for their possible mechanisms of action which could be responsible for the improved growth of seedlings under drought (Table 5). All the four isolates were capable to produce exopolysaccharides and catalase. Isolates MR-2, CR-30 and CR-39 had the ability to produce organic acids but unable to produce siderophores. On the contrary, LR-30 can produce siderophores but had no capacity to produce organic acids. When these isolates were quantified to make aggregates, CR-39 had highest aggregation capacity

Fig. 6: Drought tolerance index of wheat seedlings on the basis of shoot length influenced by the inoculation of rhizobia isolated from chickpea nodules under drought induced by 15 and 25% PEG solutions. Vertical bars represent the standard error of each treatment followed by LR-30, MR-2 and CR-30, respectively. Quantification of indole-3-acetic acid (IAA) production in the presence and absence of precursor L-tryptophan (T- TRP) showed enormous ability of CR-39 up to produce IAA. Other isolates including MR-2, LR-30 and CR-30 were following CR-39 for producing IAA in the absence and presence of L-TRP, respectively. Isolates CR-30 and CR-39 were identified as Mesorhizobium ciceri, MR-2 as Rhizobium phaseoli and LR-30 as Rhizobium leguminosarum by BIOLOG(r) identification system. Further, upon inoculation they caused nodulation to respective crops i.e. mung bean (MR-2), chickpea (CR-30, CR-39) and lentil (LR-30).

Discussion

Rhizobial capability to survive water deficit conditions has been proved by the findings of drought tolerance assay (at - 0.70, -1.67 and -2.18 MPa osmotic potential) of the isolates from the nodules of three legume species (Lens culinaris L., Vigna radiata L. and Cicer arietinum L.). Their ability to produce catalases and possibly oxidases are helpful in saving cellular membranes and nucleic acid from destruction and sustaining metabolism under stress (Goyal et al., 1986; Boumahdi et al., 1999). Rhizobia are producers of exopolysaccharides which helps in the development of biofilm, where they get protection from extremities and have the availability of water and nutrients (Vanderlinde et al., 2010).

Accumulation of osmolytes and change in cellular morphology has previously been identified as probable mechanisms to avoid damage from drought stress by rhizobia (Busse and Bottomley, 1989; Smith and Smith, 1989). One more surprising characteristics of bacteria is the siderophores production which has been reported to enhance its survivability under drought (Arzanesh et al., 2011). Amrani et al. (2010) recorded similar results, while growing rhizobial species up to 25% PEG in YMB media. Similarly, 60% of the 45 Rhizobium sullae stains were reported to tolerate drought up to -0.9MPa by Fitouri et al. (2012). These findings lead them to suggest that rhizobia can be utilized as inoculant to crops in marginal lands.

Inoculation of symbiotic bacteria to non-nodulating crop (wheat) seeds resulted into beneficial, neutral and harmful associations. Isolates producing beneficial association with wheat seedlings have improved the root or shoot lengths and fresh or dry biomass of the seedlings under water deficit conditions. These improvements could be due to their ability to colonize roots hugely (bacterial aggregation ability), production of phytohormones (IAA), exopolysaccharides or catalase production and increased mobilization of nutrients through lowering rhizosphere pH (organic acid) or binding the nutrients (siderophores production) (Table 5). Rhizobial inoculation has rescued plant growth through improving drought tolerance index of the seedlings, which could be due to the production of osmolytes and antioxidants in the rhizosphere. Findings of these experiments have been supported by the results of certain researchers where they used to inoculate non-legumes with rhizobium under water limited conditions.

Moderately drought tolerant Rhizobium sullae from the semi-arid region of Tunisia significantly improved the shoot dry biomass of inoculated plants under drought (Fitouri et al., 2012). Similarly, exopolysaccharides producing rhizobial strain YAS34 was inoculated to sunflower under reduced water system and improvement in dry biomass, nitrogen nutrition and water uptake of plants was observed (Alami et al.,2000).

Rhizobia (KYGT207) isolated from drying soils improved the soil aggregation in the rhizosphere of wheat seedlings due to exopolysaccharides production and thus rescued plant growth under stress (Kaci et al., 2005). Whereas, the auxin and cytokinin producing capability of Rhizobium and Bradyrhizobium is an important mechanism for improving dry biomass of inoculated sorghum under water deficit situation (Rashad et al., 2002).

Isolates LR-26, MR-17 and CR-34 showed negative association with the inoculated plants and resulted in the reduction of root or shoot lengths and fresh or dry biomasses either in normal or water stressed conditions. This harmful correlation could be due to the excessive production and accumulation of toxins like cyanides, growth regulators (auxins) and nitric oxides in the rhizosphere (Perrine et al., 2005; Francine et al., 2007).

On the basis of drought tolerance assay and plant growth promotion trials, isolates MR-2, LR-30, CR-30 and CR-39 were selected as proficient rhizobia for supporting wheat seedlings growth under drought stress. Overall, the rhizobial isolates from chickpea nodules performed better than the rhizobial isolates from mung bean and lentil nodules; therefore, two isolates were selected from the group of chickpea rhizobia. Selected isolates were identified as Rhizobium phaseoli (MR-2), Rhizobium leguminosarum (LR-30), and Mesorhizobium ciceri (CR-30 and CR-39)

using BIOLOG(r) identification system (Table 5).

Inoculation of the respective legumes with selected rhizobia caused nodulation to confirm their identity.

Catalase producing ability (Table 5) of the rhizobia makes them capable to stand in water deficit conditions and beneficial to host plants under stress. However, exopolysaccharides, siderophores and organic acid (Table 5) producing ability could be responsible to improve water and nutrient holding capacity and nutrient bioavailability of the rhizosphere. Auxin production (Table 5) by the selected rhizobia might have improved the root length, which enhanced the nutrient and water capturing capacity of the seedlings under stress. Microbial aggregation ability (Table 5) of the selected isolates highlights their capability to colonize the plant roots and they have good ability to live on the rhizoplane of the plants.

Inoculation of catalase and exopolysaccharides producing Rhizobium and Mesorhizobium species has proved beneficial interaction to wheat (non-legume) under water stress conditions. Further, it can be concluded that rhizobial capability to produce catalase and exopolysaccharides not only a key to their drought tolerance property but also a reliable attribute to select efficient rhizobial isolates as PGPR for non-legumes. These experiments have opened up new avenues to explore the mechanisms of rhizobia to associate and support non-legumes growth under stress environments. Anyhow, Rhizobium phaseoli (MR-2), Rhizobium leguminosarum (LR-30), and Mesorhizobium ciceri (CR-30 and CR-39) are potential rhizobia to rescue wheat growth under drought but evaluation trials are suggested to confirm their efficiency.

Acknowledgements

We are grateful to the Higher Education Commission (HEC) of Pakistan for financial assistance. Funding was provided by HEC under Indigenous 5000 PhD Fellowship Program (Batch V) for this PhD research.

References

Abolhasani, M., A. Lakzian, A. Tajabadipour and G. Haghnia, 2010. The study salt and drought tolerance of Sinorhizobium bacteria to the adaptation to alkaline condition. Aust. J. Basic Appl. Sci., 4: 882-886

Ahmad, M., 2011. Microbial ACC-deaminase may improve the efficiency of Rhizobium inoculation in mung bean under salt affected conditions. PhD Thesis, University of Agriculture, Faisalabad, Pakistan

Alami, Y., W.A. Achouak, C. Marol and T. Heulin, 2000. Rhizosphere soil aggregation and plant growth promotion of sunflower by an exopolysaccharides producing Rhizobium sp. strain isolated from sunflower roots. Appl. Environ. Microbiol., 66: 3393-3398

Amrani, S., N.E. Noureddine, T. Bhatnagar, M. Argandona, J.J. Nieto and C. Vargas, 2010. Phenotypic and genotypic characterization of rhizobia associated with Acacia saligna (Labill.) Wendl. in nurseries from Algeria. System Appl. Microbiol., 33: 44-51

Anonymous, 2012. Economic Survey of Pakistan. Government of Pakistan, Finance Division. Economic Adviser Wing, Islamabad Arora, N.K., S.C. Kang and D.K. Maheshwari. 2001. Isolation of

siderophores producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut. Curr. Sci. 8: 673-677

Arshad, M. and W.T. Jr. Frankenberger, 1998. Plant growth regulating substances in the rhizosphere: Microbial production and functions. Adv. Agron., 62: 46-51

Arzanesh, M.H., H.A. Alikhani, K. Khavazi, H.A. Rahimian and M. Miransari. 2011. Wheat (Triticum aestivum L.) growth enhancement by Azospirillum sp. under drought stress. World J. Microbiol. Biotechnol., 27: 197-205

Asghar, H.N., Z.A. Zahir, M. Arshad and A. Khaliq, 2002. Relationship between in vitro production of auxins by rhizobacteria and their growth-promoting activities in Brassica juncea L. Biol. Fert. Soils, 35: 231-237

Ashraf, M., S.H. Berge and O.T. Mahmood, 2004. Inoculating wheat seedling with exopolysaccharides producing bacteria restricts sodium uptake and stimulates plant growth under salt stress. Biol. Fert. Soils, 40: 57-162

Bajgiran, A.R., A. Lakzian and N.S. Rastin, 2008. Elongation of shoot and root in wheat by ACC deaminase of Rhizobium Spp. indigenous to soils of Iran. Int. J. Agric. Biol., 10: 481-486

Berjak, P. 2006. Unifying perspectives of some mechanisms basic to desiccation tolerance across life forms. Seed Sci. Res., 16: 1-15

Bot, A.J., F.O. Nachtergaele and A. Young, 2000. Land resource potential and constraints at regional and country levels World Soil Resources Reports90. Land and Water Development Division, FAO, Rome, Italy Boumahdi, M., P. Mary and J.P. Hornez, 1999. Influence of growth phases and desiccation on the degrees of unsaturation of fatty acids and the survival rates of rhizobia. J. Appl. Microbiol., 87: 611-619

Burdman, S., E. Jurkevitch, B. Schwartsburd, M. Hampel and Y. Okon, 1998. Aggregation in Azospirillum brasilense: effect of chemical and physical factors and involvement of extracellular components. Microbiology, 144: 1989-1999

Busse, M.D. and P.J. Bottomley, 1989. Growth and nodulation responses of Rhizobium meliloti to water stress induced by permeating and non- permeating solutes. Appl. Environ. Microbiol., 55: 2431-2436

Etesami, H., H.A. Alikhani, M. Jadidi and A. Aliakbari, 2009. Effect of superior IAA producing rhizobia on N, P, K uptake by wheat grown under greenhouse condition. World Appl. Sci. J., 6: 1629-1633

FAO, 2007. Coping with Water Scarcity: Challenge of the Twenty-first Century. Report on World Water Day, 22nd March. http://www.fao.org/nr/water/docs/escarcity.pdf (Accessed 20.8.2013) Farooq, M., A. Wahid, N. Kobayashi, D. Fujita and S. M.A. Basra, 2009. Plant drought stress: effects, mechanisms and management. Agron.Sust. Dev., 29: 185-212

Fitouri, S.D., D. Trabelsi, S. Saidi, K. Zribi, F.B. Jeddi and R. Mhamdi, 2012. Diversity of rhizobia nodulating sulla (Hedysarum coronarium L.) and selection of inoculant strains for semi-arid Tunisia. Ann. Microbiol., 62: 77-84

Francine, M.P.W., J. Prayitno, B.G. Rolfe, J.J. Weinman and C.H. Hocart, 2007. Infection process and the interaction of rice roots with rhizobia. J. Exp. Bot., 58: 3343-3350

Goyal, V., S. Chetal and H.S. Nainawatee, 1986. Alteration in Rhizobium trifolii catalase under water stress. Folia Microbiol., 31: 164-166

Hussain, M.B., I. Mehboob, Z.A. Zahir, M. Naveed and H.N. Asghar, 2009. Potential of Rhizobium spp. for improving growth and yield of rice (Oryza sativa L.). Soil Environ., 28: 49-55

Jaleel, C.A., P. Manivannan, A. Wahid, M. Farooq, H.J. Al-juburi, R. Somasundaram and R. Panneerselvam, 2009. Drought stress in plants: a review on morphological characteristics and pigments composition. Int. J. Agric. Biol., 11: 100-105

Jenks, M.A. and P.M. Hasegawa. 2005. Plant Abiotic Stress, 1st edition, p: 1 Kaci, Y., A. Heyraud, M. Barakat and T. Heulin, 2005. Isolation and identification of an EPS-producing Rhizobium strain from arid soil (Algeria): characterization of its EPS and the effect of inoculation on wheat rhizosphere soil structure. Res. Microbiol., 156: 522-531

Khalid, A., M. Arshad and Z.A. Zahir. 2006. Phytohormones: microbial production and applications. p. 207-220. In: Biological Approaches to Sustainable Soil Systems. Uphoff, N., A.S. Ball, E. Fernandes, H. Herren, O. Husson, M. Laing, C. Palm, J. Pretty, P. Sanchez, N. Sanginga and J. Thies. (eds.). Taylor and Francis/CRC Press, Boca Raton, Florida, USA

MacFaddin, J.F., 1980. Biochemical Tests for Identification of Medical Bacteria. Williams and Wilkins, Baltimore, Maryland, USA

Madi, L. and Y. Henis, 1989. Aggregation in Azospirillum brasilense Cd: conditions and factors involved in cell-to-cell adhesion. Plant Soil, 115: 89-98

Mehboob, I., Z.A. Zahir, A. Mahboob, S.M. Shahzad, A. Jawad and M. Arshad, 2008. Preliminary screening of rhizobium isolates for improving growth of maize seedlings under axenic conditions. Soil Environ., 27: 64-71

Mehboob, I., Z.A. Zahir, M. Arshad, A. Tanveer and Farooq-e-Azam, 2011. Growth promoting activities of different Rhizobium spp., in wheat. Pak. J. Bot., 43: 1643-1650

Noel, T.C., C. Cheng, C.K. Yost, R.P. Pharis and M.F. Hynes, 1996. Rhizobium leguminosarum as a plant growth promoting rhizobacterium: direct growth promotion of canola and lettuce. Can. J. Microbiol., 42: 279-283

Perrine, F.M., B.G. Rolfe, M.F. Hynes and C.H. Hocart, 2005. Plasmid associated genes in the model micro-symbiont Sinorhizobium meliloti 1021 affect the growth and yield of young rice seedlings. Environ. Microbiol., 7: 1826-1838

Rashad, M., A. Ragab and S. Salem, 2002. The influence of some Bradyrhizobium and Rhizobium strains as plant growth promoting rhizobacteria on the growth and yield of sorghum (Sorghum bicolor L.) plants under drought stress. J. Plant Nutr., 92: 664-665

Sandhya, V., S.Z. Ali, M. Grover, N. Kishore and B. Venkateswarlu, 2009. Pseudomonas sp. strain P45 protects sunflowers seedlings from drought stress through improved soil structure. J. Oilseed Res., 26: 600-601

Sarwar, M., M. Arshad, D. A. Martens and W.T. Frankenberger Jr, 1992. Tryptophan dependent biosynthesis of auxins in soil. Plant Soil, 147: 207-215

Schwyn, B. and J.B. Neilands, 1987. Universal chemical assay for the detection and determination of siderophores. Anal. Biochem., 160: 47-56

Smith, L.T. and G.M. Smith, 1989. An osmoregulated dipeptide in stressed Rhizobium meliloti. J. Bacteriol., 171: 4714-4717

Steel, R.G.D., J.H. Torrie and D.A. Dicky, 1997. Principles and Procedures of Statistics: A Biometrical Approach, 3rd edition. McGraw Hill, New York, USA

Tikhonovich, I.A. and N.A. Provorvo, 2011. Microbiology is the basis of sustainable agriculture: an opinion. Ann. Appl. Biol., 159: 155-168

Vanderlinde, E.M., J.J. Harrison, A. Muszynski, R.W. Carlson, R.J. Turner and C.K. Yost, 2010. Identification of a novel ABC-transporter required for desiccation tolerance, and biofilm formation in Rhizobium leguminosarum bv. viciae 3841. FEMS Microbiol. Ecol., 71: 327-340

Vincent, J.M., 1970. A Manual for the Practical Study of Root Nodule Bacteria, 1st edition, p: 164. Oxford Publication for the International Biological Program Volpin, H. and D.A. Phillips. 1998. Respiratory elicitors from Rhizobium meliloti affect intact alfalfa roots. Plant Physiol. 116: 777-783

Yang, J., J.W. Kloepper and C.M. Ryu. 2009. Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci., 14: 1-4

Zahir, Z.A., A. Munir, H.N. Asghar, B. Shahroona and M. Arshad, 2008. Effectiveness of rhizobacteria containing ACC-deaminase for growth promotion of peas (Pisum sativum) under drought conditions. J. Microbiol. Biotechnol., 18: 958-963

Zahir, Z.A., U. Ghani, M. Naveed, S.M. Nadeem and H.N. Asghar. 2009. Comparative effectiveness of Pseudomonas and Serratia sp. containing ACC-deaminase for improving growth and yield of wheat (Triticum aestivum L.) under salt-stressed conditions. Arch. Microbiol., 191: 415-424

Zahran, H.H. 1999. Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol. Mol. Biol. Rev., 63: 968-989

Zaidi, A., M.S. Khan, M. Ahemad and M. Oves, 2009. Plant growth promotion by phosphate solubilizing bacteria. Acta Microbiol. et Immunol. Hung., 56: 283-284

1Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad-38040, Pakistan

2Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan

For correspondence: zazahir@yahoo.com

To cite this paper: Hussain, M.B., Z.A. Zahir, H.N. Asghar and M. Asghar, 2014. Can catalase and exopolysaccharides producing rhizobia ameliorate drought stress in wheat? Int. J. Agric. Biol., 16: 3-13
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Author:Hussain, Muhammad Baqir; Zahir, Zahir Ahmad; Asghar, Hafiz Naeem; Asgher, Muhammad
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9PAKI
Date:Feb 28, 2014
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