Efficacy of aminocyclopropane-1-carboxylic acid ( ACC )-deaminase-producing rhizobacteria in ameliorating water stress in chickpea under axenic conditions

To mitigate environmental stresses, use of aminocyclopropane-1-carboxylic acid (ACC)-deaminase containing plant growth promoting rhizobacteria (PGPR) as agricultural inputs for improved crop production is required. A total of 47 bacterial isolates from different rhizospheric soils of chickpea from Punjab were biochemically characterized and found to be representatives of genus Bacillus (25) and Pseudomonas (22). Ten (10) of the isolates were able to utilize ACC as a sole source of nitrogen, maximum growth (in terms of optical density λ600) being recorded with Bacillus isolate 23-B (0.463) followed by Pseudomonas 6-P (0.317). Three isolates were P-solubilizers and their relative Psolubilization efficiency ranged from 14.6 to 21.6 mg/100 ml culture broth. All the isolates produced Indole-3-acetic acid (IAA) (4.4-22.8 μg/ml). Two PGPR’s 23-B and 6-P alone and in combination with recommended (for Punjab state) Mesorhizobium ciceris, were evaluated for water stress mitigation and plant growth promotion under axenic conditions on Cicer arietinum varieties (Kabuli L-552 and Desi GPF-2). Both the rhizobacteria significantly improved germination, root and shoot length and fresh weight of chickpea seedlings under osmotic potential of up to 0.4 MPa over uninoculated control. Proline content was considerably higher in PGPR treated varieties of chickpea under water stress. Coinoculation of 23-B with Mesorhizobium enhanced all growth parameters under water stress.


INTRODUCTION
Abiotic stresses caused by complex environmental conditions, for example, drought, salinity, high and low temperatures etc lead to substantial crop losses worldwide (Mittler et al., 2006).As a consequence of these stresses, plants typically stimulate 1-aminocyclo-propane 1-carboxylic acid (ACC) synthesis, a precursor to ethylene (Gamalero and Glick, 2012) which helps in inducing multifarious physiological changes in plants at molecular level but at higher levels is usually deleterious, as it induces defoliation, changes cellular processes leading to growth inhibition, premature senescence, restricted nodulation, all of which reduce crop yield (Lie et al., 2005).Amongst the legumes, chickpea (Cicer arietinum L.) is third most important grain legumes in the world cultivated on 11.55 million hectares with production of 10.46 million tons, India being the largest producer *Corresponding author.E-mail: veenadk@rediffmail.com.(FAO STAT, 2010).To reduce the deleterious effects of ethylene stress, plant growth-promoting rhizobacteria (PGPR) that facilitate the proliferation of plants under stress conditions are a potentially viable option (Gamalero and Glick, 2012).These beneficial rhizobacteria with ACC deaminase activity sustain plant development by lowering ethylene levels by metabolizing ACC into α-ketobutyrate and ammonia (Mehta et al., 2010).
Under stress conditions, increased proline biosynthesis has been reported in various plant species inoculated with different PGPR (Vardharajula et al., 2011).Proline acts as osmoprotectant and reactive oxygen species scavenger thus supporting plant growth under stress.ACC-deaminase activity has been widely reported in numerous species of PGPR like Azospirillum, Bacillus, Burkholderia, Pseudomonas and Rhizobium etc (Shaharoona et al., 2006).
The result of adding PGPR to plants is a significant increase in seed germination and the biomass that the plants are able to attain under otherwise stressful and inhibitory conditions (Shaharoona et al., 2012).The present work was undertaken with the objective to screen rhizobacteria producing ACC-deami-nase for plant growth promoting potential under water stressed conditions.

Isolation and characterization of bacterial strains
Soil samples were collected from the depth of 10 to 15 cm from twenty different chickpea fields of Punjab.The soil samples were serially diluted up to 10 -9 dilutions and were plated on nutrient agar (for Bacillus spp.) and King's B (Pseudomonas from spp).The plates were then incubated at 30°C for 24 h.
The colonies were picked up, sub-cultured and preserved on Nutrient agar slants at 4 to 5°C.The isolates were then characterised on the basis of colony morphology, Gram staining and biochemical tests namely: catalase production, nitrate reduction, starch hydrolysis and methyl red test (Cappuccino and Sherman, 1992).

Aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity
Bacterial isolates were grown in Luria Broth medium and cell pellet collected by centrifugation washed and resuspended in sterile water and spot inoculated on petriplates containing Dworkin and Foster (DF) salt minimal medium (Dworkin and Foster, 1958) supplemented with 3 mM ACC as a main source of Nitrogen.
In quantitative assay each selected isolate was grown individually in liquid DF minimal medium alone, DF+ACC and DF+ (NH4)2SO4 and their growth measured (OD60).ACC-deaminase producing rhizobacterial isolates were selected for evaluation of other PGP traits.

Production of Indole-3-acetic acid (IAA)
Bacteria were grown in Luria broth (72 h at 30°C) and IAA estimated by the method of Gordon and Weber (1951).

Induction of water stress by polyethylene glycol 6000 (PEG)
Seeds of two Cicer arietinum varieties (L-552 and GPF-2) obtained from Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, were surface-sterilized and soaked in rhizobacterial culture (23-B and 6-P showing relatively higher ACC-deaminase activity, P-solubilization, IAA synthesis and compatibility with Mesorhizobium ciceris) alone and in combination with Mesorhizobium culture for 30 min.The experiment was conducted in completely randomized design with three replications.
To simulate drought stress, PEG-6000 equivalent to water potential -0.2, -0.4 and -0.6 MPa (Mega Pascals) were used.Ten seeds of each genotype were germinated on sterilized filter paper lined petri dishes, moistened with 5 ml solution of PEG-6000 having appropriate osmotic pressure and kept in incubator at 22 ± 2°C with 90% humidity for duration of 10 days.Seeds germinated using distilled water only served as absolut e control.

Proline content
Samples were extracted in Methanol: Chloroform: Water (12:5:1), centrifuged and the supernatant collected and made up to 10 ml with same solvent.To this 6 ml of chloroform and 4 ml of distilled water was added and allowed to stand till the two layers got separated.The final volume of upper layer was made 10 ml with distilled water, to 5 ml of this solution 2.5 ml of acid ninhydrin was added and mixture boiled for 45 min till pink colour developed and OD at 515 nm was recorded (Bates et al., 1973).Proline was used as standard to make standard curve (Sahu and Sindhu, 2011).

Isolation and characterization of rhizobacteria
A total of 47 rhizobacteria were isolated from chickpea rhizospheric soils and out of these 22 were from Kings B medium, all showed yellowish green pigment characteristic of Pseudomonas spp whereas 25 were from nutrient agar medium showed predominantly off-white to creamish colonies, typical of the genus Bacillus (Figure 1).The predominance of Pseudomonas and Bacillus spp. in legume rhizosphere has been reported by many workers (Yadav et al., 2010)   hydrolysed starch.On the basis of these tests, the isolates were tentatively assigned to genera Bacillus (B) and Pseudomonas (P).

Indole-3-acetic acid (IAA) production
Diverse soil microorganisms are known to produce IAA, which is an important hormone for plant growth, root initiation and elongation (Yasmin et al., 2009).All the rhizobacteria produced IAA, Bacillus (11.2-22.8µg/ml) and Pseudomonas isolates (4.4-21.6 µg/ml), highest being 23-B (22.8 µg/ml) (Table 2).In the present study Bacillus spp were found to be strong IAA producers.These results are in accordance to Etesami et al. (2009) they reported significant difference in IAA production amongst the isolates.

Yellowish green pigment on
Creamish pigment on Kings B medium Nutrient Agar medium
Root growth was also greatly affected by water stress at all the three levels (Table 3).At 0.2 and 0.4 MPa maximum increase in root elongation was recorded in seeds treated with PGPR 23-B (3.6 and 2.7 cm) whereas at 0.6 MPa, isolate 6-P+R (1.2 cm) was more effective.At 0.2 MPa application of isolate 23-B+R recorded root fresh weight of 70.3 mg/seedling followed by isolates 23-B, 6-P, 6-P+R (60, 57 and 52.7 mg/seedling), at 0.4 MPa isolate 23-B alone gave maximum root fresh weight (45 mg/seedling) but at 0.6 MPa isolate 23-B+R increased root fresh weight to maximum extent (18.3 mg/seedling).Shoot traits also exhibited significant decrease under stress, seeds showed no plumule emergence at 0.6 MPa stress.At 0.2 MPa, isolate 23-B+R showed maximum shoot length (1.2 cm) followed by 23-B (1 cm), at 0.4 MPa, 23-B showed maximum shoot length (0.6 cm) Followed by 23-B+R (0.5 cm).Shoot fresh weight was maximum at 0.2 MPa when inoculated with isolate 23-B+R, followed by 23-B (24 & 22.7 mg/seedling) as compared to control (15.6 mg/seedling).At 0.4 MPa isolate 23-B showed maximum shoot fresh weight (25 mg/seedling) (Figure 3).It is clear from the data that 23-B alone and along with Mesorhizobium exhibited profound effect on chickpea growth under water stress.These results are in corroboration with results of Mayak et al. (2004) who reported that ACC-deaminase PGPR Achromobacter piechaudii ARV8 significantly increased the fresh and dry weights of tomato and pepper seedlings exposed to transient water stress.

Effect of aminocyclopropane-1-carboxylic acid (ACC)-deaminase positive rhizobacteria on germination and growth of chickpea GPF-2 under water stress
Chickpea variety GPF-2 also showed significant reduction in plant growth under water stress conditions and germination was inhibited at osmotic potential (0.6 MPa) which is considered as threshold potential (Table 4).Only radicle emergence was noticed in GPF-2 variety whereas plumules failed to emerge at all the three water stress levels.Here too, at 0.2 MPa isolates 23-B and 23-B+R showed maximum germination (63.3%) and at 0.4 MPa 23-B+R showed maximum germination (56.6%) followed by isolate 23-B (40%) over uninoculated control (Figure 4).At 0.2 MPa and 0.4 MPa seed inoculation with 6-P+R gave maximum root elongation (3.0 and 3.1 cm) over control (1.8 cm).At 0.2 MPa isolate 23-B exhibited profound increase in root fresh weight whereas, at 0.4 MPa, 6-P+R showed higher root fresh weight ((53 &32 mg/seedling) as compared to uninoculated control (18.3 mg/seedling) (Figure 5).This study shows that water stress inhibited coleoptile emergence more than the radicle which is in accordance to results of Macar et al. (2008).The ACC-deaminase containing rhizobacteria probably reduced the endogenous ethylene levels in chickpea at early stage of development thus enhanced their growth and root elongation under stress.Bacillus isolate 23-B was found to be more effective in plant growth promotion probably due to its higher ACCdeaminase activity.Treatment with ACC deaminaseproducing bacteria typically reduces ethylene levels by about 2-4 fold (Reed and Glick, 2005).It has been reported ACC deaminase-PGPR protects the plants against damage from drought, high salt and polyaromatic hydrocarbons (Mayak et al., 2004;Glick et al., 2007).

Proline accumulation
The proline content shows an increasing trend in both chickpea varieties under water stress which is in accordance to the result of Madhurendra (2009).Treatment with Bacillus isolate 23-B shows maximum proline accumulation (0.80 and 0.66 mg/g fresh weight radicle) in comparison to Pseudomonas isolate 6-P (0.32 and 0.31) in both chickpea varieties as compared to control and absolute control (Table 5).Co-inoculation of isolate 23-B with Mesorhizobium also gave similar results.These results are in corroboration with work of Singh (2004) who reported accumulation of proline in saline stress tolerant chickpea genotypes.Slow utilization of proline for protein synthesis during stress results in its accumulation.Proline is known to act as a compatible osmolyte, antioxidant and maintains cytosolic pH (Verbruggen and Hermans, 2008).

Conclusion
The present study indicates that co-inoculation of ACCdeaminase producing PGPR with Mesorhizobium significantly promoted growth of chickpea by positively influencing seed germination andother growth factors under

Figure 3 .
Figure 3.Effect of ACC-deaminase positive rhizobacteria on germination and growth of chickpea (L-552) under water stress

Figure 4 .
Figure 4. Effect of ACC-deaminase positive rhizobacteria on germination of chickpea GPF-2 seeds under water stress.

Figure 5 .
Figure 5.Effect of ACC-deaminase positive rhizobacteria on germination and growth of chickpea (GPF-2) under water stress.

Table 1 .
Comparative growth of rhizobacteria on media containing ACC/ Ammonium sulphate as sole nitrogen source.

Table 2 .
Comparative performance of rhizobacteriain terms of plant growth promoting traits.
Figure 2. Effect of ACC-deaminase positive rhizobacteriaon germination of chickpea L-552 seeds under water stress.

Table 3 .
Effect of ACC-deaminase positive rhizobacteria on root and shoot traits of chickpea L-552 under water stress.

Table 4 .
Effect of ACC-deaminase positive rhizobacteria on root traits of chickpea GPF-2 under water stress.

Table 5 .
Proline content in water stress induced radicles of chickpea.
Data recorded at 0.2 MPa PEG induced water stress.*Uninoculated seeds under water stress.**Uninoculated seeds under stress free conditions.