Plant Soil (2013) 366:93105 DOI 10.

1007/s11104-012-1402-5

REGULAR ARTICLE

Isolation of ACC deaminase producing PGPR from rice rhizosphere and evaluating their plant growth promoting activity under salt stress
Himadri Bhusan Bal & Lipika Nayak & Subhasis Das & Tapan K. Adhya

Received: 26 May 2012 / Accepted: 30 July 2012 / Published online: 15 August 2012 # Springer Science+Business Media B.V. 2012

Abstract Aims Bacteria possessing ACC deaminase activity reduce the level of stress ethylene conferring resistance and stimulating growth of plants under various biotic and abiotic stresses. The present study aims at isolating efficient ACC deaminase producing PGPR strains from the rhizosphere of rice plants grown in coastal saline soils and quantifying the effect of potent PGPR isolates on rice seed germination and seedling growth under salinity stress and ethylene production from rice seedlings inoculated with ACC deaminase containing PGPR. Methods Soils from root region of rice growing in coastal soils of varying salinity were used for isolating ACC deaminase producing bacteria and three bacterial isolates were identified following polyphasic taxonomy. Seed germination, root growth and stress ethylene
Responsible Editor: Bernard Glick. Electronic supplementary material The online version of this article (doi:10.1007/s11104-012-1402-5) contains supplementary material, which is available to authorized users. H. B. Bal : L. Nayak : S. Das : T. K. Adhya Laboratory of Soil Microbiology, Division of Crop Production, Central Rice Research Institute, Cuttack, 753006 Odisha, India Present Address: T. K. Adhya (*) School of Biotechnology, KIIT University, Bhubaneswar, 751024 Odisha, India e-mail: adhyas@yahoo.com

production in rice seedlings following inoculation with selected PGPR under salt stress were quantified. Results Inoculation with selected PGPR isolates had considerable positive impacts on different growth parameters of rice including germination percentage, shoot and root growth and chlorophyll content as compared to uninoculated control. Inoculation with the ACC deaminase producing strains reduced ethylene production under salinity stress. Conclusions This study demonstrates the effectiveness of rhizobacteria containing ACC deaminase for enhancing salt tolerance and consequently improving the growth of rice plants under salt-stress conditions. Keywords Growth promoting rhizobacteria . Coastal rice soils . ACC deaminase . Polyphasic taxonomy . Salinity stress . Ethylene production

Introduction Plant growth-promoting rhizobacteria (PGPR), freeliving soil bacteria thriving in the plant rhizosphere, have been studied as plant growth promoters for increasing agricultural productivity (Lucy et al. 2004). PGPR can either directly or indirectly facilitate growth of plants (Glick 1995). Indirect stimulation of plant growth includes mechanisms by which the bacteria prevent phytopathogens from inhibiting plant growth and development (Raaijmakers et al. 2009) while direct stimulation may include providing plants with

94

Plant Soil (2013) 366:93105

fixed nitrogen, phytohormones, iron that has been sequestered by bacterial siderophores, and soluble phosphate (Lucy et al. 2004). Many PGPRs can also increase plant resistance to biotic and abiotic stress factors. Presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity in several rhizospheric bacteria and regulation of ACC, a precursor to plant ethylene levels, is one of the principal mechanisms by which bacteria exert beneficial effects on plants under abiotic stress (Glick et al. 1998, 2007a, b; Glick 2004, 2005; Saleem et al. 2007). Earlier studies indicated that bacteria having ACC deaminase activity reduce the level of stress ethylene conferring resistance and resulting in better growth of plants under various stresses such as salt stress, flooding stress, heavy metal stress and pathogen attack (Glick et al. 2007a). Rice is a semi-aquatic plant and its ecosystem is classified into irrigated, rainfed lowland, upland, and flood-prone on the basis of hydrology. Rice growing under these field conditions is exposed to various types of biotic and abiotic stresses at various developmental stages during its life cycle. These stresses include drought, submergence, extreme temperature, high salt, presence of toxic materials and environmental contaminants and various others which affect plant growth negatively. Soil salinity is one of the important factors affecting soil microbial activities and crop productivity in most of the humid and sub-humid conventional rice growing areas of coastal Asia (Ismail et al. 2009). Limitations of major macro and micronutrients in soil and high soil salinity are the important yield reducing factors in the coastal rice ecosystem (Asch et al. 2000). Previous research has shown that inoculation with PGPR can alleviate the salt stress effects in different plant species. Enhancement of growth and salt tolerance in PGPR inoculated red pepper (Siddikee et al. 2011), tomato (Mayak et al. 2004) and Groundnut (Saravanakumar and Samiyappan 2007) have been reported. Plant growth-promoting bacteria found in association with plants grown under chronically stressful conditions, including high salinity, may have been adapted to the stress conditions, and could provide a significant benefit to the plants. Since coastal soils with salinity are natural habitats of halophilic/ halotolerant bacteria (Lichfield 2002), isolation of ACC deaminase-producing PGPR from such natural habitat, and their utilization could prove to be beneficial for mitigating the salt stress to the plants growing

in such environment. The objectives of the present study were: (1) to isolate efficient ACC deaminase producing PGPR from the rhizosphere of rice plants grown in coastal saline soils, and characterize them (2) to evaluate other plant growth promoting (PGP) activities including production of indole acetic acid (IAA) by the most promising ACC deaminase producing isolates (3) to determine the effect of potent PGPR isolates on root elongation under salinity stress; (4) to estimate ethylene from rice seedlings treated with ACC deaminase containing PGPR or chemical ethylene inhibitors.

Materials and methods Sampling and characterization of soils The root adhering soil (RAS) samples were collected from coastal rice fields from five different locations of Sundarban area (214054.0 to 214750.5 N latitude and 88142.8 to 883733.2 E longitude) of West Bengal, India during September, 2008 at the tillering stage of the monsoon season (kharif) rice. The rice plants were carefully uprooted along with the soil and brought to the laboratory in polythene bags in portable cool chambers (~4C). The non-rhizosphere soil was removed by vigorously shaking the uprooted rice hills leaving behind the rhizosphere soil strongly adhering to the roots (Ramakrishna and Sethunathan 1982). Physicochemical parameters of the soils were determined according to Spark et al. (1996) and reported in Table 1. Both the rhizosphere and the non-rhizosphere soils were used for the isolation of bacteria. Isolation of ACC-utilizing bacteria ACC-utilizing bacteria were isolated from both the rhizospheric and non-rhizospheric soil following the procedure of Penrose and Glick (2003) with some modifications. Representative soil samples (1 g each) were separately enriched for ACC-utiltizing bacteria by growing in sterile DF salts (Dworkin and Foster 1958) minimal medium containing 3 mM ACC as the nitrogen source and incubated on a rotary shaker at 200 rpm and 30 C for 24 h. Four fold dilutions of this culture were plated onto solid DF salts agar medium containing ACC (500 mol mL1) and incubated for 48 h at 30 C. Bacterial colonies were chosen based on

Plant Soil (2013) 366:93105 Table 1 Characteristics of the soil samples Properties Soil samples SB1 pH EC (dS/m) Organic Carbon (%) Total Carbon (%) Total Nitrogen (%) Available P (mg.kg1) Available K (mg.kg1) 6.94 6.88 0.15 0.062 0.567 14 298.2 SB2 5.64 5.74 0.20 0.061 0.556 20 87.6 SB3 6.54 11.32 0.24 0.077 0.661 16 66.7 SB4 6.52 6.60 0.20 0.065 0.571 13.3 298.2 SB5

95

6.85 1.03 0.31 0.085 0.839 10 311.5

their colony morphology, further purified and maintained onto the respective medium slants at 4 C and/ or in 65 % glycerol at 80 C till further use. Characterization of the bacterial isolates Morphological and biochemical characterization Morpho-physiological and biochemical characters of the bacterial isolates were examined according to the Bergeys Manual of Determinative Bacteriology (Holt et al. 1994). Individual cultures grown on NA (Nutrient Agar) medium at 30 C were examined for the colony morphological features. Motility and morphology were studied by phase contrast microscopy (Olympus BX-51, Olympus America Inc., USA). Gram staining was performed as per standard procedures with exponentially growing cultures. Salt tolerance of the bacterial isolates was tested by growing them on nutrient broth amended with different levels of NaCl. BIOLOG(R) analysis Potential carbon source utilization of the isolates was assessed by using the BIOLOG(R) GEN III MicroPlates (Biolog Inc., Hayward, CA) according to manufacturers instructions. The reactions were observed after 22 h incubation at 33C and read using automated Biolog(R) MicroStation Reader. FAME analysis The cellular fatty acids were extracted from approximately 20 mg bacterial cells using the standard extraction technique (Sasser 2001). FAME profiles were then obtained by running the samples on a gas

chromatograph (GC) (Agilent Technologies, USA) equipped with flame ionization detector (FID) and MIDI(R) Sherlock Microbial Identification System (MIDI Inc., Newark, DE, USA) software. FAMEs were identified according to their retention time, as compared to a commercial standard mixture (MIS standard calibration, Part no. 1200-A) (Sasser 2001). Sequencing of 16 S rRNA gene for identification of rhizobacterial isolates Most effective strains were identified by partial sequencing of the 16 S rRNA gene. Genomic DNA was isolated from the culture by using Genomic DNA isolation kit (Sigma, India). 16 S rRNA gene was amplified using universal forward (5 AGAGTRTGATCMTYGCTWAC-3 ) and reverse (5-CGYTAMCTTWTTACGRCT-3) primers (Hazra et al. 2011). 100 l reaction mixture containing 100 ng DNA template, 400 ng of each primer, 4 l dNTP (2.5 mM each), 10 l 10x Taq DNA polymerase assay buffer, 1 l Taq DNA polymerase (3Ul 1). PCR reactions were carried out in a thermal cycler (Model ABI 2720, Applied Biosystems International, Foster City CA, USA). The PCR cycle used for amplification was as follows: 5 min at 94 C, followed by 35 cycles of 30 s at 94 C, 30 s at 55 C, 2 min at 72 C and a final extension of 5 min at 72 C. The amplified 16 S rRNA gene was purified with a PCR purification kit (Qiaquick PCR purification kit, Qiagen, India) and outsourced for sequencing (Chromus Biotech Pvt. Ltd., Bangalore, India). The sequence data was aligned with System Software aligner and analyzed to identify the bacterium and its closest neighbors by using BLAST (NCBI, USA). The partial 16 S rRNA gene sequences were deposited in GenBank data base.

96

Plant Soil (2013) 366:93105

Phylogenetic and molecular evolutionary analyses of the 16 s rDNA sequences were conducted by using software MEGA4 and aligned using CLUSTAL-X. The pair-wise evolutionary distance matrix was generated and the evolutionary tree was inferred using the Neighbor-Joining method. The bootstrap test has been done to cluster together the associated taxa. The evolutionary distances were compared using the Maximum Composite Likelihood method (Tamura et al. 2007). Screening for plant growth promoting activities ACC deaminase activity assay ACC deaminase activity was determined by monitoring the amount of -ketobutyric acid generated from the cleavage of ACC (Penrose and Glick 2003). The ACC deaminase activity was induced by growing the bacterial cells in minimal medium containing ACC as the sole nitrogen source, after growing them in 15 ml TSB medium up to log phase. -ketobutyrate produced by the reaction was determined by comparing the absorbance at 540 nm of the sample to a standard curve of -ketobutyrate ranging between 0.1 and 1.0 mol. The ACC deaminase activity was expressed as the amount of -ketobutyrate produced per mg of protein per hour. Determination of other plant growth-promoting traits Ability to produce IAA was detected by the method of Salkowski (Glickmann and Dessaux 1995). Isolates were screened for hydrogen cyanide (HCN) production, siderophore production, ammonia production and P-solubilization following Islam et al. (2009). Production of HCN was determined on NA plates supplemented with glycine (4.4 gl1) after 3 days of incubation at 28 C. Siderophores were detected by the formation of orange halos around bacterial colonies on Chrome Azural S (CAS) agar plates after incubation for 24 h at room temperature. Ammonia production was detected by adding Nessler s reagent (0.5 ml/tube) to 48-h old bacterium grown in peptone water. P-solubilization activity was tested on Pikovaskayas agar medium containing 2 % tricalcium phosphate. The appearance of clear halo zone around bacterial colonies after incubation for 2448 h at 28 C was observed.

Plant growth-promotion activity by bacterial isolates Seed treatment and root elongation assay with rice (cv. Naveen) were performed according to Penrose and Glick (2003). The bacterial cell pellets of the selected strains were suspended in 0.5 ml sterile 0.03 M MgSO4 and the absorbance adjusted to 0.15 at 600 nm. Seeds were surface-sterilized by dipping in 95 % ethanol and in 0.2 % HgCl2 solution for 3 min followed by rinsing 5 times with sterile distilled water (Zahir et al. 2009). Seeds were incubated at room temperature for 1 h with the appropriate treatment - sterile 0.03 M MgSO4 (negative control) or bacterial suspensions in sterile 0.03 M MgSO4. Soil sample for the study was collected from the experimental fields at CRRI, Cuttack, air-dried, sieved (2-mm/10-mesh) and analyzed for physico-chemical characteristics. The soil was a typic haplaquept having pH 6.16, electrical conductivity 0.5 dS.m1, cation exchange capacity 15.0 meq.g1 soil, organic carbon 0.86 %, total nitrogen 0.09 % and contained 25.9 % clay, 21.6 % slit and 52.5 % sand. The soil was sterilized by autoclaving at 121.1 C for 1 h for three consecutive days to kill all the soil microorganisms and their spores, and 200 g portions of sterilized soil were filled in each thermocol cup (4.5cmx4.5cmx5.5 cm). Six seeds were placed in each cup aseptically and placed in a growth chamber with maximum and minimum temperatures maintained at 28 C and 20 C, respectively with 12 h day night photoperiod. Initial and final growth parameters (plant height, plant biomass and chlorophyll content) were recorded on day 5 and 15 respectively (Yoshida et al. 1976). For measuring the chlorophyll content, 100 mg of finely chopped fresh leaves were placed in a capped measuring tube containing 25 mL of 80 % acetone, and placed inside a refrigerator (4 to 8 C) for 28 h (Panda et al. 2008). The chlorophyll content was measured at 646.6 and 663.6 nm in a spectrophotometer and calculated using the equation of Porra (2002). Effect of selected isolates on seed germination under saline stress and ethylene estimation Three most efficient strains were selected to study their effect on seed germination and ethylene emission under salt stress. In an initial screening on the level of salinity, germination of rice seeds (cv. Naveen) was reduced by ~20 % at 150 mM NaCl (data not shown), and the specific salinity level was used for subsequent

Plant Soil (2013) 366:93105

97

studies on salt stress. Surface sterilized seeds were incubated for 1 h at room temperature with the appropriate treatment: sterile 0.03 M MgSO4 (negative control), 104 M L--(2-aminoethoxyvinyl) glycine hydrochloride (AVG) (Sigma, India), a known inhibitor of ethylene production, in 0.03 M MgSO4 (positive control), heat-killed or fresh bacterial suspensions in sterile 0.03 M MgSO4 (OD of 0.15 at 600 nm) (Penrose and Glick 2001). Positive control contained AVG and should completely inhibit ethylene production in the presence of salt stress while negative control contained neither AVG nor bacteria and should not inhibit ethylene production. Heat killed cells of 24 h old bacterial cultures were retained as the secondary control to verify the impact of cellular metabolites, if any, released from cells on ethylene production. Twenty five seeds were planted on sterile filter paper inside each pre-sterilized 100 ml GL 45 clear glass bottles (Schott Duran, Germany), and 5 ml of sterile half strength N-free Hoaglands solution, a hydroponic nutrient solution (Hoagland and Boyer 1936) supplemented with 150 mM NaCl, was added as nutrient solution. For preparing 1 L of Hoaglands solution, 1 M solution each of KH2PO4, MgSO4.7H2O and 0.5 M solution of K2SO4 were prepared and 2, 1 and 4 ml of each solution were added respectively. Subsequently, 1 g of CaSO4, 1 ml micronutrients solution (g/L: H3BO3, 1; MnCl2.4H2O, 1; ZnSO4.7H2O, 0.58; CuSO4.5H2O, 0.13; Na2MoO4.2H2O, 0.10) and 1 ml of 2 % (w/v) iron solution were added and the volume made to 1 l with distilled water. The experiment was conducted for 7 days and the glass bottles were placed in a growth chamber with maximum and minimum temperatures maintained at 28 and 20 C, respectively with a cycle of 12 h dark/light. The bottles were arranged in a completely randomized design with 5 replications for each treatment (Fierov et al. 2008; Sapsirisopa et al. 2009; Siddikee et al. 2011). After 7 days, germinated seeds were counted and percent of germination was calculated. Root and shoot length, fresh and dry weight of root and shoot both of five randomly selected seedlings from each replication were also recorded. Seedling vigor index (VI) was calculated using the formula: VI mean root length mean hypocotyl length % germination: For ethylene estimation, glass bottles were sealed by airtight sterile neoprene septum with inner Teflon

lining and kept in the dark for 1 h at 30 C. Headspace gas (1 ml) was drawn by airtight syringe (2 ml) and injected into GC (Model-Ceres 800 plus, Thermo Scientific, USA) packed with a Porapak-Q column (183 cm length and 0.3 cm internal diameter, 80/100 mesh) and equipped with FID. The GC was adjusted to 100 C, 300 C and 150 C for oven, injection and detection temperature. The flow rate of N2, H2 and air were 30, 30 and 300 ml min1. Under these conditions, retention time of ethylene was 2.28 min and the minimum concentration of ethylene detected was 0.1 ppm. The amount of ethylene produced was expressed as nmol of C2H4 g1 fresh weight h1 by comparing with the standard curve of pure ethylene (9.12 ppm in nitrogen, Matheson Tri-Gas) (Fierov et al. 2008; Siddikee et al. 2011). Statistical analyses All results presented are the means of five independent replicates. Data were subjected to statistical analysis (Gomez and Gomez 1984) by a statistical package (IRRISTAT version 3.1; International Rice Research Institute, Los Baos, Philippines). The mean difference comparison between the treatments was analyzed by analysis of variance (ANOVA) and subsequently by Duncans multiple range test at p <0.05. Simple correlation between specific datasets was analyzed by Microsoft Excel program on correlation and regression.

Results Isolation and characterization of bacteria A total of 17 ACC utilizing bacteria were isolated from the coastal rice field soil from five different locations and screened for their ACC deaminase metabolism. Results indicated that all the strains metabolized ACC but with variable degrees of efficacy (Table 2). Highest ACCdeaminase activity per hour was exhibited by the isolate SB1.ACC2 (2664.08 nmol -ketobutyrate mg1 h1). Eleven strains exhibiting high ACC utilization rate were further selected to screen their growth-promoting activity in rice under axenic conditions (root elongation assay). Plant growth-promotion activity by bacterial isolates Statistical analysis of data recorded 15 days after seed germination is summarized in Table 3. All the tested

98 Table 2 ACC deaminase activity (nmol -ketobutyrate mg1 protein h1) of the isolates S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Isolates SB1.ACC1 SB1.ACC2 SB1.ACC3 SB2.ACC1 SB2.ACC2 SB2.ACC3 SB2.ACC4 SB2.ACC5 SB2.ACC6 SB3.ACC1 SB3.ACC2 SB3.ACC3 SB3.ACC4 SB4.ACC1 SB4.ACC2 SB5.ACC1 SB5.ACC2 ACC deaminase activity* 894.11f 41.81 2664.08j 63.21 1468.90h 31.60 397.04d 21.16 2049.42i 52.20 295.72bc 19.09 236.05b 18.58 1145.43g 66.44 526.04e 42.65 440.51d 27.24 329.09c 28.15 919.42f 73.05 906.07f 56.02 61.29a 6.24 92.49a 8.43 62.02a 15.50 91.28a 12.16

Plant Soil (2013) 366:93105

Mean values sharing the same letter (s) in column do not differ significantly according to Duncans multiple range test (P 0 0.05) *Mean of five replicate observations SD (Standard deviation)

strains had considerable impact on different growth parameters of rice compared with the negative control. Data on root length indicate that inoculation with all the isolates enhanced root length significantly (P 0 0.05) in comparison with the control. Inoculation with SB1.ACC2 caused the maximum increase in root length (16.20 cm) which was 41.2 % higher than the control, followed by SB2.ACC6. Inoculation with all the selected bacterial isolates except SB3.ACC3 also promoted root fresh weight significantly (P 0 0.05) in comparison with the control. The maximum root fresh weight (311.48 % over control) was observed by isolate SB1.ACC2 (726.83 mg). Isolates SB2.ACC1 and SB1.ACC3 were next effective isolates that promoted root fresh weight by 281 % and 260 % respectively. Likewise, root dry weight also increased significantly (P 0 0.05) in response to inoculation with the isolates except SB3.ACC3. The maximum increase (255 % over uninoculated control) in root dry weight was observed by inoculation with SB1.ACC2 (75.68 mg). Isolate SB1.ACC3 and SB2.ACC1 were next effective isolates. Data regarding shoot length showed that treatment with seven isolates out of eleven, significantly (P 0 0.05) promoted the shoot length in comparison with the control. Strain SB1.ACC2 (43.03 cm) inoculation

Table 3 Effect of inoculation of eleven PGPR on different growth parameters of rice (cv. Naveen) after 15 days of seed germination Treatments RL (cm) RFW (mg) RDW (mg) 21.27i 35.18f 75.68 70.83 70.63 59.51 37.43 30.99 25.70 25.71
a b b

SL (cm)

SFW (mg)

SDW (mg) 33.84h 119.50e 159.94 141.12 163.82 159.96

b d a

Chl a Chl b Chl a + b (mg/gm fresh wt) (mg/gm fresh wt) (mg/gm fresh wt) 3.36h 3.73g 7.10 7.13 6.23 6.20 4.52 4.40 4.62 4.83
a a b

MgSO4

10.80g* 176.64i 632.81c 726.83 635.95 672.40 459.67 379.90 256.84 227.50 264.66
a c b a de bc

28.00fgh 155.84e 34.86e 43.03 38.89 36.99 41.54 39.40 27.25 29.12 26.86
a c d

0.88g 0.99f 1.87 1.89 1.66 1.67 1.22 1.16 1.28 1.30
a a b

4.24i 4.71h 8.98b 9.02ab 7.89c 9.04a 7.87c 5.74f 5.56g 5.90e 4.27i 6.12d 1.000

SB1.ACC1 13.54e SB1.ACC2 15.25 SB1.ACC3 13.93 SB2.ACC1 14.46 SB2.ACC5 14.05 SB2.ACC6 14.77 SB3.ACC1 13.05 SB3.ACC2 13.83 SB3.ACC4 14.20 LSD (5 %)

409.46d 712.06 711.42 811.66

c c ab

SB2.ACC2 14.49bc
cd b f de

511.14d
e f g h

61.55c
d e g h

42.42ab
b c gh f

766.20bc 155.16c 866.42 343.20 159.72 188.80 240.62

a d e e b f g h

7.14a
b e f d

1.90a
b d e c

93.86 37.14 34.60 38.46

SB3.ACC3 14.21cd
cd

192.53i
g

21.54i
h

28.53fg
h

196.18e
e

37.46g
g

3.40h
c

0.89g
c

0.430

19.940

0.993

1.214

91.500

1.720 0.050

0.023

Data are represented as average of five replicates RL root length, SL shoot length, RFW root fresh weight, SFW shoot fresh weight, RDW root dry weight, SDW shoot dry weight, LSD least significant difference *Mean values sharing the same letter (s) in column do not differ significantly according Duncans multiple range test (P 0 0.05)

Plant Soil (2013) 366:93105

99

gave maximum increase (54 % over untreated control) which was followed by isolates SB2.ACC2 and SB2.ACC5. Similarly, inoculation with the same isolates showed significant increase in shoot fresh weight as compared to the control. Maximum shoot fresh weight, obtained with SB2.ACC5 (866.42 mg), was six times than that of untreated control, and SB2.ACC1 and SB2.ACC2 were the next effective strains. The results for shoot dry weight showed that all the isolates except SB3.ACC2, significantly (P 0 0.05) increased the shoot dry weight in comparison to uninoculated control. SB2.ACC1 (163.82 mg) was the most effective isolate which enhanced shoot dry weight by 384 % over control. Total chlorophyll content significantly (P 0 0.05) increased over the uninoculated control with all the strains except SB3.ACC3. It was highest in the plants treated with the isolate SB2.ACC2 (113 % higher than control) and statistically at par with the isolate SB1.ACC3. Effect of selected isolates on seed germination under salt stress and ethylene estimation The effect of three efficient ACC deaminase producing strains, namely SB1.ACC2, SB1.ACC3 and SB2.ACC2 on seed germination, plant growth parameters and ethylene production under salt stress condition is shown in Table 4. The tested live strains

significantly enhanced the germination percentage. Germination percentage was highest (98.4 %) when rice seeds were treated with the isolates SB1ACC2 and AVG in comparison to the negative control (82 %) which was followed by SB1.ACC3 and SB2.ACC2. Inoculation with SB1.ACC2 also increased seedling vigor (1378.76) as compared to the negative control (683.73) and followed the order of SB2.ACC2>AVG >SB1.ACC3. However inoculation of seeds with the heat killed isolates had no significant effect on both germination percentage and seedling vigor, and was statistically at par with negative control. Highest amount of ethylene production was observed from the negative control (18.22 nmol C2H4 g1fw hr1) plants as compared the plants from the positive control (1.42 nmol C2H4 g1fw hr1) under the salt stress. However, inoculation with the ACC deaminase containing strains SB1.ACC2, SB1.ACC3 and SB2.ACC2 reduced ethylene production by 90.2, 81.6 and 81.5 % respectively compared to the negative control. Inoculation with heat killed SB2.ACC2 and SB1.ACC2 isolates also reduced ethylene production by 38.2 and 36.7 % and could not be explained. Strain SB1.ACC2 was found to be the most efficient in reducing stress ethylene production. Salt stressed plants inoculated with all the three live strains grew to a significantly (P 0 0.05) greater extent than the negative control (Fig. 1). Both length and fresh weight of root and shoot were increased (Table 4).

Table 4 Effect of PGPR on plant growth parameters and ethylene production of rice seedlings under salt stress conditions Isolates Germination % SVI C2H4 (nmol C2H4 gfw1 h1) 18.22c 1.42a 11.52
b a c

RL (mm)

SL (mm)

RFW (mg)

SFW (mg)

RDW (mg)

SDW (mg)

MgSO4 AVG HK SB1-ACC2 SB1-ACC2 HK SB1-ACC3 SB1-ACC3 HK SB2-ACC2 SB2-ACC2 LSD (5 %)

82.0a* 98.4c 80.4 80.4

a c a b

683.73a 1194.61b 761.16 698.80 1181.09

a c

4.01a 6.13c 4.75 6.97 4.03 5.66 6.13 0.65

b d a c

4.32a 6.01b 4.71 7.05 4.66 6.64 6.57 0.52

a d a c

5.33a 9.69c 7.27 10.97 5.29

b d a c

14.94a 18.84b 14.12 23.83 17.04 20.21 21.02

a d b c

0.47a 0.65a 0.47 1.13 0.51 1.07 1.25 0.18

a b a b

1.77a 2.10b 1.64a 3.16c 1.43a 2.93c 1.57a 2.90c 0.35

98.4 96.0 95.2

1378.76

1.78 3.35 3.36 3.31

a b

18.54

9.39 10.13

80.8a
b

694.56a 1209.68
b

11.26b
a

4.05a
c

4.56a
c

7.04b
c

14.90a
c

0.57a
b

2.95

78.72

1.02

1.80

Data are represented as average of five replicates SVI seedling vigor index, RL root length, SL shoot length, RFW root fresh weight, SFW shoot fresh weight, RDW root dry weight, SDW shoot dry weight, LSD least significant difference *Mean values sharing the same letter(s) in a column do not differ significantly according to Duncans Multiple Range Test (P 0 0.05)

100 Fig. 1 Salt stress effect on shoot and root growth of 7 day old rice seedlings exposed to salt stress (150 mM) under gnotobiotic conditions. a, Negative Control (0.03 M MgSO4); b, SB1ACC2; c, Heat killed SB1ACC2; d, SB1ACC3; e, Heat killed SB1ACC3; f, SB2ACC2; g, Heat killed SB2ACC2; h, AVG

Plant Soil (2013) 366:93105

SB1.ACC2 was the most efficient strain which enhanced both length and fresh weight of root and shoot up to 73.8, 63.2, 105.8 and 59.5 % respectively. But heat killed strains did not promote the plant growth parameters significantly. In case of root and shoot dry weight also live cells of the three strains increased the plant biomass significantly ( P 0 0.05) over the negative control whereas the heat killed strains failed to do so. SB2.ACC2 and SB1.ACC2 were the most effective isolates to enhance root and shoot dry weight by 166 and 78 % over negative control. Characterization of other plant growth promoting traits of isolates The three selected strains were screened for their other plant growth promoting activities and the result was summarized in Table 5. All the three isolates produced IAA, siderophore and ammonia and also had phosphate solubilizing activity but none of them were HCN producer. Morphological and biochemical characterization of the bacterial isolates, their identification and phylogenetic analysis Morphological and biochemical characteristics of three selected bacterial strains are given in Table 5.

Following morphological characterization, motility and gram staining, the isolates were compared with those of the standard species using Bergeys Manual of Determinative Bacteriology. The most effective PGPR isolates also exhibited different degree of tolerance to salinity (Table 5). The isolate SB1-ACC2 showed the highest salinity tolerance with normal (< 10 % growth as compared to growth in non-saline control Nutrient agar broth) growth at 1.54 M NaCl amendment followed by isolates SB1.ACC3 and SB2.ACC2. Carbon substrate utilization profile of the isolates was obtained using BIOLOG(R) system (Supplementary Table 1), which employs a redox reaction by tetrazolium dye to test the ability of isolates to utilize different carbon sources. The phenotypic fingerprint thus generated was used to identify the organisms to genus level and tentatively identified as Alcaligens sp., Bacillus sp. and Ochrobactrum sp (Table 6). The predominant FAMEs for each pure culture (Supplementary Table 2) were selected on the basis of those FAMEs that comprised greater than 5 % of the total area of FAMEs in each respective database file of FAME profiles (Sherlock Software, Microbial ID, Newark, NJ, USA) and tentatively identified (Table 6). The selected strains were further identified by 16 S rRNA gene sequence analysis to ascertain their

Plant Soil (2013) 366:93105 Table 5 Morphological and biochemical characters the isolates Characters Isolates SB1-ACC2 SB1-ACC3 SB2-ACC2 Morphological Gram Reaction Cell shape Cell Length () Colony Color Motility Biochemical MR MRVP Citrate utilization Nitrate reduction Starch hydrolysis Oxidase Catalase Tween 80 hydrolysis Urease Plant growth promoting traits IAA production (M ml1) HCN production Phosphate solubilization Siderophore Production Salt tolerance Growth in maximum salt concentration (M NaCl) + + + + + + + + + + + + + + Rod 2.00.1 White + + Rod 2.00.1 White + Rod 4.00.2 White +

101

Discussion This study demonstrates the effectiveness of rhizobacteria containing ACC deaminase for inducing salt tolerance and consequently improving the growth of rice plants under salt-stress conditions. We tested eleven high ACC deaminase producers for their growthpromoting activity without salt stress and three most promising strains, SB1.ACC2 ( Alcaligenes sp.); SB1.ACC3 (Bacillus sp.) and SB2.ACC2 (Ochrobactrum sp.) were further evaluated under salt stress condition. ACC deaminase activity was more widely present in soil bacteria belonging to the genera Alcaligenes, Variovorax, Rhodococcus, and Bacillus and to different species of Pseudomonas (Belimov et al. 2005). ACC deaminase producing ability in Ochrobactrum sp. was previously reported by Corsini (2011). An assessment of the plant growth-promoting capabilities of the isolates was based on enhanced plant parameters of 15-day old plants grown under gnotobiotic conditions from rice seeds inoculated with the eleven strains without stress. In comparison to control plants, all the plant growth parameters of the bacteria treated plants were significantly higher. The longest roots and shoots; highest root fresh weight and dry weight were observed in plants treated with Alcaligenes sp. A simple correlation analysis between in vitro ACC deaminase production by the isolates (Table 2) and plant root elongation under controlled condition (Table 3) indicated a positive correlation ( r 0 0.559, n 0 11), albeit statistically not significant, suggesting a direct impact of ACC deaminase activity on root elongation . Inoculation with rhizobacteria having ACC deaminase activity resulted in the development of a better root system, which subsequently affected shoot growth positively (Glick et al. 1998; Belimov et al. 2002). Inoculation with ACC-deaminase containing bacteria promotes root growth of developing seedlings of various crops (Zahir et al. 2003). The differences in plant growth promotion among the isolates are also attributed to their individual rhizospheric competencies and hydrolyzing the ACC synthesized in roots. PGPRs having ACC deaminase activity help plants to withstand stresses (biotic or abiotic) by reducing the level of stress ethylene (Mayak et al. 2004; Dimkpa et

Tributyrin hydrolysis +

49.562.81 45.911.79 152.372.38 + + + + + + + +

Ammonia production +

1.54

1.03

0.68

Numerical values are the mean SD of three replicate observations Cell lengths of exponential phase cultures grown in NB were recorded Colony color were recorded after growing the isolates on NA (Nutrient agar)

taxonomic positions (Table 6). The phylogenetic analysis of the isolates was done based on neighborhood joining method with 100 bootstrap sampling. The isolates SB1.ACC2, SB1.ACC3 and SB2.ACC2 showed a 99 % similarity with the 16 S rRNA gene sequence of Alcaligenes, Bacillus and Ochrobactrum respectively (Fig. 2).

102

Plant Soil (2013) 366:93105

Table 6 BIOLOG GenIII, FAME and molecular identification of isolates SB1-ACC2, SB1-ACC3 and SB2-ACC2 Isolates Biolog GEN III identification FAME identification Molecular identification Accession Number SB1-ACC2 SB1-ACC3 SB2-ACC2 Alcaligens sp. Bacillus sp. Ochrobactrum sp. Alcaligens sp. Bacillus sp. Ochrobactrum sp. JN256919 JN256920 JN256921 16 S rRNA fragment length (bp) 1430 1452 1379 Closest relatives and NCBI accession number Alcaligenes faecalis strain DZ2; HQ202537 Bacillus pumilus strain S68; FJ763649 Ochrobactrum sp. TH-N-29; AB695227 Similarity (%) 99 99 99

al. 2009; Zahir et al. 2009). We screened three most promising isolates ( Alcaligenes, Bacillus and Ochrobactrum sp.) with multiple PGP traits for their growth promoting activity under axenic conditions at 150 mM NaCl, by conducting root elongation assay on rice. Similar to other reports (Mayak et al. 2004; Zahir et al. 2009) present study also revealed that inoculation with live ACC deaminase producing bacteria reduced ethylene production significantly in
Fig. 2 Neighbor joining tree showing phylogenetic relationship between the selected PGPR from rice soil and their representative species from NCBI database. The bar represents 0.05 substitutions per site. Bootstrap values (n 0 100) were displayed at the node. a, SB1ACC2; b, SB1ACC3; SB2ACC2

comparison to negative control and heat killed bacteria treated plants (Table 4). Present study revealed that seed treatment with PGPR strains improved seed germination and seedling vigor over the non-inoculated seeds (Table 4). Similar improvement of seed germination has been reported with other cereals such as sorghum and pearl millet (Gholami et al. 2009). Seeds treated with the isolates showed a considerable increase in seedling vigor
47 55 Uncultured bacterium clone: U-3 Alcaligenes faecalis Nic-2 Alcaligenes sp. F78 Alcaligenes faecalis NBRC 14479 50 Alcaligenes faecalis AE1.16 SB1-ACC2 Alcaligenes faecalis DZ2

(A)

0.0002

51 54 48

38

Bacillus subtilis ZFJ-8 Bacillus pumilus MTCC 7514 Bacillus sp. YXC1-10 Bacillus pumilus TW3 Bacillus subtilis CCM7 Bacillus pumilus S68 SB1-ACC3

(B)
34 56

0.0002
Ochrobactrum sp. NBRC 12953 Ochrobactrum pseudogrignonense BIHB 340 Ochrobactrum sp. 10 Ochrobactrum grignonense IHB B 1375 Ochrobactrum sp. BH3 SB2-ACC2 Ochrobactrum sp. TH-N-29

56

(C)

0.0002

Plant Soil (2013) 366:93105

103

index under salt stress conditions and Alcaligenes significantly (P 0 0.05) increased vigor index compared to the negative control. These findings may be due to the increased synthesis of phytohormones like IAA, which would have triggered the activity of enzymes like -amylase that promoted early germination by bringing an increase in availability of soluble sugars from starch decomposition (Kim et al. 2006). Besides, significant increase in seedling vigor would have occurred by better synthesis of plant growth hormones (Bharathi et al. 2004). As isolates have the ability to produce both ACC deaminase and IAA they promoted root, shoot and other growth indices of rice to a greater extent. It is likely that IAA and ACC deaminase stimulate root growth in a coordinated fashion (Glick et al. 2007b). Root growth is relatively more affected compared to shoots in the presence of an inhibitory level of salt (Lin and Kao 2001). This might be due to the stressinduced ethylene disrupting the metabolic activity and physiological processes more in roots than shoots. In addition, this might be due to the roots being in more intimate contact with the salt solution with the shoots experiencing a lower salt concentration (Mayak et al. 2004). Like other studies (Mayak et al. 2004; Madhaiyan et al. 2007) treatment with AVG and ACC deaminase-producing bacteria increased root growth of rice plants as compared to the negative control plants. Simple correlation analysis between ACC deaminase production capability of isolates and the root elongation under the salt stress condition indicated a highly significant positive relationship ( r 0 0.991 at P >0.01, n 0 15) (Glick et al. 1998; Belimov et al. 2002). Inoculation with the PGPR isolates also increased the fresh and dry weight of both root and shoot. It was assumed that higher dry weight would mean longer and stronger roots and shoots as well as plants that would be able to better withstand salt stress (Mayak et al. 2004). Use of strains with multiple PGP traits is expected to help increase crop productivity on a sustainable basis. All the three ACC deaminase producing strains were tested positive for multiple PGP traits like production of IAA, siderophore and ammonia and also solubilized phosphorus. Treating plants with ACCdeaminase and siderophore producing PGPR can help the plants to overcome many of the effects caused by the environmental stresses as observed in the present study (Dimkpa et al. 2009). The assemblage of

specific PGP traits of these studied PGPR suggests that these particular organisms can promote plant growth by more than one mechanism.

Conclusions The current study concluded that inoculation with the three ACC deaminase containing PGPR caused significant alleviation of stress induced ethylene production and consequently improving the growth of rice under high salinity stress condition. The reduction in stress ethylene production and presence of multiple plant growth promoting traits of the strains may be the possible reason to protect the plant from the growth inhibitory effect of salt and thus induce salinity tolerance in rice and could be useful in coastal areas where salinity is a major constraint. However, further research is necessary to evaluate the effectiveness of these strains under actual field conditions to use them for alleviating salinity stress to the growing crop.
Acknowledgments This work was supported in part by the ICAR Networking Project, Application of Microorganisms in Agriculture and Allied Sciences (AMAAS) - theme Microbial Diversity and Identification by the Indian Council of Agricultural Research, New Delhi.

References
Asch F, Dingkuhn M, Dorffling K (2000) Salinity increases CO2 assimilation but reduces growth in field grown, irrigated rice. Plant Soil 218:110 Belimov AA, Hontzeas N, Safronova VI, Demchinskaya SV, Piluzza G, Bullitta S, Glick BR (2005) Cadmium-tolerant plant growth-promoting rhizobacteria associated with the roots of Indian mustard (Brassica juncea L. Czern). Soil Biol Biochem 37:241250 Belimov AA, Safronova VI, Mimura T (2002) Response of spring rape (Brassica napus L. var. Oleifera) to inoculation with plant growth promoting rhizobacteria containing 1aminocyclopropane-1-carboxylate deaminase depends on nutrient status of the plant. Can J Microbiol 48:189199 Bharathi R, Vivekananthan R, Harish S, Ramanathan A, Samiyappan R (2004) Rhizobacteria-based bio-formulations for the management of fruit rot infection in chillies. Crop Prot 23:835843 Corsini A (2011) Microbial arsenic transformations in contaminated soils. PhD thesis, Universita degli Studi di Milano Dimkpa C, Weinan T, Asch F (2009) Plantrhizobacteria interactions alleviate abiotic stress conditions. Plant Cell Environ 32:16821694

104 Dworkin M, Foster J (1958) Experiments with some microorganisms which utilize ethane and hydrogen. J Bacteriol 75:592601 Fierov H, Mikuov Z, Klem M (2008) Estimation of ethylene production and 1-aminocyclopropane-1-carboxylic acid content in plants by means of gas chromatography. Plant Soil Environ 54:5560 Gholami A, Shahsavani S, Nezarat S (2009) The effect of Plant Growth Promoting Rhizobacteria (PGPR) on germination, seedling growth and yield of maize. Int J Biol Life Sci 5:3540 Glick BR (1995) The enhancement of plant growth by freeliving bacteria. Can J Microbiol 41:109117 Glick BR (2004) Bacterial ACC deaminase and the alleviation of plant stress. Adv Appl Microbiol 56:291312 Glick BR (2005) Modulation of plant ethylene levels by the enzyme ACC deaminase. FEMS Microbiol Lett 251:17 Glick BR, Penrose DM, Li J (1998) A model for the lowering of plant ethylene concentration by plant growth-promoting bacteria. J Theor Biol 190:6368 Glick BR, Cheng Z, Czarny J, Duan J (2007a) Promotion of plant growth by ACC deaminase-containing soil bacteria. Eur J Plant Pathol 119:329339 Glick BR, Todorovic B, Czarny J, Cheng Z, Duan J, McConkey B (2007b) Promotion of plant growth by bacterial ACC deaminase. Crit Rev Plant Sci 26:227242 Glickmann E, Dessaux Y (1995) A critical examination of the specificity of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Appl Environ Microbiol 61:793796 Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research, 2nd edn. John Wiley and Sons Inc, Singapore Hazra C, Kundu D, Ghosh P, Joshi S, Dandi N, Chaudhari A (2011) Screening and identification of Pseudomonas aeruginosa AB4 for improved production, characterization and application of a glycolipid biosurfactant using low-cost agro-based raw materials. J Chem Technol Biotechnol 86:185198 Hoagland DR, Boyer, TC (1936) General nature of the process of salt accumulation by roots with description of experimental method. Plant Physiol 11:471507 Holt JG, Kreig NR, Sneath PHA, Staley JT, Williams ST (1994) Bergeys Manual of determinative bacteriology, 9th edn. Williams and Wilkins, Baltimore Islam MR, Madhaiyan M, Deka Boruah HP, Yim W, Lee G, Saravanan VS, Fu Q, Hu H, Sa T (2009) Characterization of plant growth-promoting traits of three-living diazotrophic bacteria and their inoculation effects on growth and nitrogen uptake of crop plants. J Microbiol Biotech 19:12131222 Ismail AM, Thapa B, Egdane J (2009) Salinity tolerance in rice: physiological bases and implications on management strategies for better crop establishment. In: Improving productivity and livelihood for fragile environments. IRRI technical bull. 13. International Rice Research Institute, Los Banos, pp 813 Kim SK, Son TK, Park SY, Lee IJ, Lee BH, Kim HY, Lee SC (2006) Influences of gibberellin and auxin on endogenous plant hormone and starch mobilization during rice seed germination under salt stress. J Environ Biol 27:181186

Plant Soil (2013) 366:93105 Lichfield CD (2002) Halotolerant bacteria. J Ind Microbiol Biotechnol 28:2122 Lin CC, Kao CH (2001) Cell wall peroxidise activity, hydrogen peroxide level and Nacl-inhibited root growth of rice seedlings. Plant Soil 230:135143 Lucy M, Reed E, Glick BR (2004) Applications of free living plant growth-promoting rhizobacteria. Antonie van Leeuwenhoek 86:125 Madhaiyan M, Kim BY, Poonguzhali S, Kwon SW, Song MH, Ryu JH, Go SJ, Koo BS, Sa TM (2007) Methylobacterium oryzae sp. nov., a novel aerobic, pink-pigmented, facultatively methylotrophic, 1-aminocyclopropane-1-carboxylate deaminase producing bacterium isolated from rice. Int J Syst Evol Microbiol 57:326331 Mayak S, Tirosh T, Glick BR (2004) Plant growth-promoting bacteria confer resistance in tomato plants to salt stress. Plant Physiol Biochem 42:565572 Panda D, Sharma SG, Sarkar RK (2008) Chlorophyll fluorescence parameters, CO2 Photosynthetic rate and regeneration capacity as a result of complete submergence and subsequent reemergence in rice (Oryza sativa L.). Aquat Bot 88:127133 Penrose DM, Glick BR (2001) Levels of 1-aminocyclopropane1-carboxylic acid (ACC) in exudates and extracts of canola seeds treated with plant growth-promoting bacteria. Can J Microbiol 47:368372 Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant 118:10 15 Porra RJ (2002) The chequered history of the development and use of simultaneous equations for accurate determination of chlorophylls a and b. Photosynth Res 73:149 156 Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Monne-Loccoz T (2009) The rhizosphere: a playground and battlefield for soil-borne pathogens and beneficial microorganisms. Plant Soil 321:341361 Ramakrishna C, Sethunathan N (1982) Stimulation of autotrophic ammonium oxidation in rice rhizosphere soil by the insecticide carbofuran. Appl Environ Microbiol 44:14 Saleem M, Arshad M, Hussain S, Bhatti AS (2007) Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture. J Ind Microbiol Biotechnol 34:635648 Sapsirisopa S, Chookietwattana K, Maneewan K and Khaengkhan P (2009) Effect of salt-tolerant Bacillus inoculum on rice KDML 105 cultivated in saline soil. Asian J Food Ag-Ind, S69S74 Saravanakumar D, Samiyappan R (2007) ACC deaminase from Pseudomonas fluorescens mediated saline resistance in groundnut (Arachis hypogea) plants. J Appl Microbiol 102:12831292 Sasser M (2001) Identification of Bacteria by Gas Chromatography of Cellular Fatty Acids. MIDI Technical Note 101 Siddikee MA, Glick BR, Chauhan PS, Yim W, Sa T (2011) Enhancement of growth and salt tolerance of red pepper seedlings ( Capsicum annuum L.) by regulating stress ethylene synthesis with halotolerant bacteria containing 1-aminocyclopropane-1-carboxylic acid deaminase activity. Plant Physiol Biochem 49:427434

Plant Soil (2013) 366:93105 Spark DL, Page AL, Summer ME, Tabatabai MA, Helmke PA (1996) Methods of soil analysis, part 3, chemical methods. American Society of Agronomy, USA Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:15961599 Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Laboratory manual for physiological studies of rice, 3rd edn. International Rice Research Institutes, Manila

105 Zahir ZA, Muhammad A, Frankenberger WT Jr (2003) Plant growth promoting rhizobacteria: applications and perspectives in agriculture. Adv Agron 81:97168 Zahir ZA, Ghani U, Naveed M, Nadeem SM, Asghar HN (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