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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: firstname.lastname@example.org
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
2″ E longitude) of West Bengal. 2004). 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. Rice growing under these field conditions is exposed to various types of biotic and abiotic stresses at various developmental stages during its life cycle. presence of toxic materials and environmental contaminants and various others which affect plant growth negatively. 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. Rice is a semi-aquatic plant and its ecosystem is classified into irrigated. flooding stress. Enhancement of growth and salt tolerance in PGPR inoculated red pepper (Siddikee et al. Physicochemical parameters of the soils were determined according to Spark et al. and soluble phosphate (Lucy et al.0″ to 21°47′50. 2004) and Groundnut (Saravanakumar and Samiyappan 2007) have been reported. and their utilization could prove to be beneficial for mitigating the salt stress to the plants growing in such environment. These stresses include drought.94 Plant Soil (2013) 366:93–105 fixed nitrogen. Saleem et al. may have been adapted to the stress conditions. rainfed lowland.5″ N latitude and 88°14′2. 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. a precursor to plant ethylene levels. The rice plants were carefully uprooted along with the soil and brought to the laboratory in polythene bags in portable cool chambers (~4°C).8″ to 88°37′33. high salt. 2000). phytohormones. 2005. Bacterial colonies were chosen based on . and flood-prone on the basis of hydrology. isolation of ACC deaminase-producing PGPR from such natural habitat. Plant growth-promoting bacteria found in association with plants grown under chronically stressful conditions. India during September. 2008 at the tillering stage of the monsoon season (kharif) rice. Four fold dilutions of this culture were plated onto solid DF salts agar medium containing ACC (500 μmol mL−1) and incubated for 48 h at 30 °C. (1996) and reported in Table 1. 2009). Previous research has shown that inoculation with PGPR can alleviate the salt stress effects in different plant species. 2007a). 2007a. iron that has been sequestered by bacterial siderophores. Glick 2004. 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. Both the rhizosphere and the non-rhizosphere soils were used for the isolation of bacteria. 2011). 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. 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 (21°40′54. extreme temperature. 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. (4) to estimate ethylene from rice seedlings treated with ACC deaminase containing PGPR or chemical ethylene inhibitors. upland. 2007). tomato (Mayak et al. 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). Since coastal soils with salinity are natural habitats of halophilic/ halotolerant bacteria (Lichfield 2002). 1998. including high salinity. and could provide a significant benefit to the plants. Many PGPRs can also increase plant resistance to biotic and abiotic stress factors. 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. b. Presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity in several rhizospheric bacteria and regulation of ACC. is one of the principal mechanisms by which bacteria exert beneficial effects on plants under abiotic stress (Glick et al. heavy metal stress and pathogen attack (Glick et al. submergence.
BIOLOG(R) analysis Potential carbon source utilization of the isolates was assessed by using the BIOLOG(R) GEN III MicroPlates (Biolog Inc.52 6.571 13. .661 16 66.. USA) equipped with flame ionization detector (FID) and MIDI(R) Sherlock Microbial Identification System (MIDI Inc. CA) according to manufacturer’s instructions.7 SB4 6.kg−1) 6. Part no. Foster City CA.. FAMEs were identified according to their retention time.31 0. Motility and morphology were studied by phase contrast microscopy (Olympus BX-51. USA). USA). Applied Biosystems International.32 0.. Salt tolerance of the bacterial isolates was tested by growing them on nutrient broth amended with different levels of NaCl. The PCR cycle used for amplification was as follows: 5 min at 94 °C. Characterization of the bacterial isolates Morphological and biochemical characterization Morpho-physiological and biochemical characters of the bacterial isolates were examined according to the Bergey’s Manual of Determinative Bacteriology (Holt et al. Ltd. PCR reactions were carried out in a thermal cycler (Model ABI 2720.2 SB5 95 6. 30 s at 55 °C. India) and outsourced for sequencing (Chromus Biotech Pvt.kg−1) Available K (mg.065 0. India). 1994). The partial 16 S rRNA gene sequences were deposited in GenBank data base.03 0.061 0. The reactions were observed after 22 h incubation at 33°C and read using automated Biolog(R) MicroStation Reader. 100 μ l reaction mixture containing 100 ng DNA template. Gram staining was performed as per standard procedures with exponentially growing cultures. 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.085 0. FAME analysis The cellular fatty acids were extracted from approximately 20 mg bacterial cells using the standard extraction technique (Sasser 2001).Plant Soil (2013) 366:93–105 Table 1 Characteristics of the soil samples Properties Soil samples SB1 pH EC (dS/m) Organic Carbon (%) Total Carbon (%) Total Nitrogen (%) Available P (mg. 1200-A) (Sasser 2001).567 14 298. further purified and maintained onto the respective medium slants at 4 °C and/ or in 65 % glycerol at −80 °C till further use. 1 μl Taq DNA polymerase (3Uμl −1). 2011).556 20 87. Individual cultures grown on NA (Nutrient Agar) medium at 30 °C were examined for the colony morphological features. DE.24 0. Hayward.5 their colony morphology.60 0.64 5.20 0.839 10 311. Newark. USA) software.54 11. Qiagen.062 0. 2 min at 72 °C and a final extension of 5 min at 72 °C. 10 μl 10x Taq DNA polymerase assay buffer.6 SB3 6. 4 μl dNTP (2. FAME profiles were then obtained by running the samples on a gas chromatograph (GC) (Agilent Technologies. as compared to a commercial standard mixture (MIS standard calibration. The amplified 16 S rRNA gene was purified with a PCR purification kit (Qiaquick PCR purification kit. followed by 35 cycles of 30 s at 94 °C. Olympus America Inc. 16 S rRNA gene was amplified using universal forward (5 ′ AGAGTRTGATCMTYGCTWAC-3 ′ ) and reverse (5′-CGYTAMCTTWTTACGRCT-3′) primers (Hazra et al. India).74 0..077 0. 400 ng of each primer.88 0.94 6. Bangalore.20 0.5 mM each).3 298. Genomic DNA was isolated from the culture by using Genomic DNA isolation kit (Sigma.85 1. USA).15 0. The sequence data was aligned with System Software aligner and analyzed to identify the bacterium and its closest neighbors by using BLAST (NCBI.2 SB2 5.
(2009). Plant growth-promotion activity by bacterial isolates Seed treatment and root elongation assay with rice (cv.5cmx4. Soil sample for the study was collected from the experimental fields at CRRI.03 M MgSO4. Cuttack. Determination of other plant growth-promoting traits Ability to produce IAA was detected by the method of Salkowski (Glickmann and Dessaux 1995).sterile 0. Seeds were surface-sterilized by dipping in 95 % ethanol and in 0. and the specific salinity level was used for subsequent . siderophore production.m−1. The soil was sterilized by autoclaving at 121. Ammonia production was detected by adding Nessler ’s reagent (0. For measuring the chlorophyll content. In an initial screening on the level of salinity. after growing them in 15 ml TSB medium up to log phase.5 ml sterile 0. Naveen) were performed according to Penrose and Glick (2003).g−1 soil.5 dS. respectively with 12 h day night photoperiod.4 gl−1) after 3 days of incubation at 28 °C.03 M MgSO4 and the absorbance adjusted to 0.5cmx5. α-ketobutyrate produced by the reaction was determined by comparing the absorbance at 540 nm of the sample to a standard curve of α-ketobutyrate ranging between 0.03 M MgSO4 (negative control) or bacterial suspensions in sterile 0. plant biomass and chlorophyll content) were recorded on day 5 and 15 respectively (Yoshida et al. The evolutionary distances were compared using the Maximum Composite Likelihood method (Tamura et al. The appearance of clear halo zone around bacterial colonies after incubation for 24–48 h at 28 °C was observed.1 °C for 1 h for three consecutive days to kill all the soil microorganisms and their spores. 2009). 21. P-solubilization activity was tested on Pikovaskaya’s agar medium containing 2 % tricalcium phosphate.6 and 663. electrical conductivity 0.6 nm in a spectrophotometer and calculated using the equation of Porra (2002).2 % HgCl2 solution for 3 min followed by rinsing 5 times with sterile distilled water (Zahir et al.16. and placed inside a refrigerator (4 to 8 °C) for 28 h (Panda et al.1 and 1.5 cm). Initial and final growth parameters (plant height.0 meq. The pair-wise evolutionary distance matrix was generated and the evolutionary tree was inferred using the Neighbor-Joining method.6 % slit and 52. Naveen) was reduced by ~20 % at 150 mM NaCl (data not shown). The bootstrap test has been done to cluster together the associated taxa. The bacterial cell pellets of the selected strains were suspended in 0. total nitrogen 0.09 % and contained 25.96 Plant Soil (2013) 366:93–105 Phylogenetic and molecular evolutionary analyses of the 16 s rDNA sequences were conducted by using software MEGA4 and aligned using CLUSTAL-X. Isolates were screened for hydrogen cyanide (HCN) production. The ACC deaminase activity was expressed as the amount of α-ketobutyrate produced per mg of protein per hour.5 ml/tube) to 48-h old bacterium grown in peptone water. 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.15 at 600 nm. 2007). Screening for plant growth promoting activities ACC deaminase activity assay ACC deaminase activity was determined by monitoring the amount of α-ketobutyric acid generated from the cleavage of ACC (Penrose and Glick 2003). 2008).86 %. 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. The ACC deaminase activity was induced by growing the bacterial cells in minimal medium containing ACC as the sole nitrogen source. organic carbon 0. 1976). Production of HCN was determined on NA plates supplemented with glycine (4.5 % sand. and 200 g portions of sterilized soil were filled in each thermocol cup (4. air-dried. ammonia production and P-solubilization following Islam et al. The soil was a typic haplaquept having pH 6. Seeds were incubated at room temperature for 1 h with the appropriate treatment .9 % clay. cation exchange capacity 15. The chlorophyll content was measured at 646.0 μmol. 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. sieved (2-mm/10-mesh) and analyzed for physico-chemical characteristics. germination of rice seeds (cv. 100 mg of finely chopped fresh leaves were placed in a capped measuring tube containing 25 mL of 80 % acetone.
0. Highest ACCdeaminase activity per hour was exhibited by the isolate SB1. if any. The amount of ethylene produced was expressed as nmol of C2H4 g−1 fresh weight h−1 by comparing with the standard curve of pure ethylene (9. All the tested . After 7 days. USA) packed with a Porapak-Q column (183 cm length and 0. was added as nutrient solution. released from cells on ethylene production.5H2O. CuSO4.1.08 nmol α-ketobutyrate mg−1 h−1). Under these conditions. 10−4 M L-α-(2-aminoethoxyvinyl) glycine hydrochloride (AVG) (Sigma. 1 and 4 ml of each solution were added respectively. heat-killed or fresh bacterial suspensions in sterile 0. 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. Surface sterilized seeds were incubated for 1 h at room temperature with the appropriate treatment: sterile 0. For preparing 1 L of Hoagland’s solution. 1. Headspace gas (1 ml) was drawn by airtight syringe (2 ml) and injected into GC (Model-Ceres 800 plus. Data were subjected to statistical analysis (Gomez and Gomez 1984) by a statistical package (IRRISTAT version 3. 1. 2011).58. The GC was adjusted to 100 °C. MgSO4. International Rice Research Institute. 0. germinated seeds were counted and percent of germination was calculated. a known inhibitor of ethylene production.7H2O. Heat killed cells of 24 h old bacterial cultures were retained as the secondary control to verify the impact of cellular metabolites. 2008.28 min and the minimum concentration of ethylene detected was 0. 1 M solution each of KH2PO4.05. and 5 ml of sterile half strength N-free Hoagland’s solution. Plant growth-promotion activity by bacterial isolates Statistical analysis of data recorded 15 days after seed germination is summarized in Table 3.3 cm internal diameter. respectively with a cycle of 12 h dark/light. The bottles were arranged in a completely randomized design with 5 replications for each treatment (Fišerová et al.1 ppm. Philippines). Root and shoot length. Los Baños. 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. Subsequently. glass bottles were sealed by airtight sterile neoprene septum with inner Teflon lining and kept in the dark for 1 h at 30 °C. 80/100 mesh) and equipped with FID. Germany).2H2O. a hydroponic nutrient solution (Hoagland and Boyer 1936) supplemented with 150 mM NaCl. The mean difference comparison between the treatments was analyzed by analysis of variance (ANOVA) and subsequently by Duncan’s multiple range test at p <0. The flow rate of N2.7H2O and 0. Simple correlation between specific datasets was analyzed by Microsoft Excel® program on correlation and regression. 2011). Matheson Tri-Gas) (Fišerová et al. in 0. Eleven strains exhibiting high ACC utilization rate were further selected to screen their growth-promoting activity in rice under axenic conditions (root elongation assay).ACC2 (2664. 0. 2009. Results indicated that all the strains metabolized ACC but with variable degrees of efficacy (Table 2). Seedling vigor index (VI) was calculated using the formula: VI ¼ ðmean root length þ mean hypocotyl lengthÞ Â % germination: For ethylene estimation. Sapsirisopa et al.10) and 1 ml of 2 % (w/v) iron solution were added and the volume made to 1 l with distilled water.03 M MgSO4 (positive control).03 M MgSO4 (negative control). Statistical analyses All results presented are the means of five independent replicates. 2008.15 at 600 nm) (Penrose and Glick 2001). 1 g of CaSO4. 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. H2 and air were 30.5 M solution of K2SO4 were prepared and 2. 30 and 300 ml min−1.Plant Soil (2013) 366:93–105 97 studies on salt stress. fresh and dry weight of root and shoot both of five randomly selected seedlings from each replication were also recorded. Na2MoO4.12 ppm in nitrogen. retention time of ethylene was 2.4H2O. 1 ml micronutrients solution (g/L: H3BO3.13. India). Siddikee et al. 300 °C and 150 °C for oven. ZnSO4.03 M MgSO4 (OD of 0. injection and detection temperature. Thermo Scientific. Twenty five seeds were planted on sterile filter paper inside each pre-sterilized 100 ml GL 45 clear glass bottles (Schott Duran. Siddikee et al. MnCl2.
No. RFW root fresh weight.80g* 176. followed by SB2. RDW root dry weight.98 Table 2 ACC deaminase activity (nmol α-ketobutyrate mg−1 protein h−1) of the isolates S.ACC4 SB2.81c 726.60 38.90e 4.46 SB2.63 59.56g 5. The maximum root fresh weight (311. SL shoot length.06 711.28 1.ACC3 13.20 295.42ab b c gh f 766.05) .02a ±15.40 27.ACC1 SB1.ACC6. The maximum increase (255 % over uninoculated control) in root dry weight was observed by inoculation with SB1.05 SB3.46g g 3.64i 632.08j ±63. Data on root length indicate that inoculation with all the isolates enhanced root length significantly (P 0 0.ACC1 14.95 672.20 cm) which was 41.40 4.ACC2 (726.02 61.21cd cd 192.40 459.25 29.66 1.20 4.ACC4 SB4.ACC1 13.ACC1 SB3. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Isolates SB1.93 SB2.98b 9.53i g 21.500 1.16 1.87c 5.05 906.ACC2 15.48 % over control) was observed by isolate SB1.81 2664.36h 3.ACC3 and SB2.20 LSD (5 %) 409.ACC3 were next effective isolates that promoted root fresh weight by 281 % and 260 % respectively.ACC2 13.90a b d e c 93.ACC3 SB3.30 a a b 4.ACC6 SB3.22 1.88g 0.46d 712.43 62. root dry weight also increased significantly (P 0 0.73g 7.16 Plant Soil (2013) 366:93–105 Mean values sharing the same letter (s) in column do not differ significantly according to Duncan’s multiple range test (P 0 0.90 256. LSD least significant difference *Mean values sharing the same letter (s) in column do not differ significantly according Duncan’s multiple range test (P 0 0.ACC1 and SB1.09 236.16 2049.23 6.ACC3 SB2.ACC1 SB2.ACC3 14.430 19.940 0.72bc ±19.21 1468.04a 7.20bc 155.89 36.68 mg).050 0.ACC2 SB1.214 91.ACC2 SB3.43g ±66.74f 5.14d e f g h 61.03 cm) inoculation Table 3 Effect of inoculation of eleven PGPR on different growth parameters of rice (cv.50e 159.ACC2 ACC deaminase activity* 894.12 26.15 919.83 SB3.03 38.12d 1.ACC3 SB2.84e 34.16c 866.65 440. Data regarding shoot length showed that treatment with seven isolates out of eleven.82 159.993 1.25 SB1.ACC1 SB4.05) in comparison with the control.94 141.49bc cd b f de 511.83 635.49a ±8.ACC1 were next effective isolates.54e SB1.58 1145.99 25. Isolate SB1.05) in response to inoculation with the isolates except SB3.71h 8.90h ±31.50 91. Naveen) after 15 days of seed germination Treatments RL (cm) RFW (mg) RDW (mg) 21.10 7.66 a c b a de bc 28.86 a c d 0.84 227.18f 75.83 a a b MgSO4 10.09c ±28.05) in comparison with the control.04e ±42.99f 1.83 70. SFW shoot fresh weight.67 379.2 % higher than the control.62 4.96 b d a Chl a Chl b Chl a + b (mg/gm fresh wt) (mg/gm fresh wt) (mg/gm fresh wt) 3.24i 4.12 163. SDW shoot dry weight.ACC2 (75.62 a d e e b f g h 7.27i 35.83 mg).68 70.05) promoted the shoot length in comparison with the control.46 SB3.86 37.54 39. Strain SB1.ACC2 caused the maximum increase in root length (16.71 a b b SL (cm) SFW (mg) SDW (mg) 33.67 1.ACC4 14.05) *Mean of five replicate observations ± SD (Standard deviation) strains had considerable impact on different growth parameters of rice compared with the negative control. Inoculation with SB1. significantly (P 0 0.11f ±41.023 Data are represented as average of five replicates RL root length.77 SB3.52 4.60 397.43 30.80 240.42 343.28a ±12.53fg h 196.55c d e g h 42.40h c 0.84h 119.00fgh 155.ACC2 SB2.ACC1 SB5.ACC3.89c 9.04d ±21.ACC2 (43.42f ±73.29a ±6.07f ±56.87 1.02ab 7. Isolates SB2.44 526.ACC5 SB2.99 41.86e 43.ACC2 14.72 188.42i ±52.42 811.18e e 37.66 c c ab SB2.51d ±27.ACC2 SB5.50 264.ACC6 14.ACC5 14.720 0.89g c 0. Likewise.70 25.ACC1 13.51 37.000 SB1.14 34.14a b e f d 1.13 6.24 329.05 SB2.54i h 28.89 1.ACC3 also promoted root fresh weight significantly (P 0 0.24 92.05b ±18.20 159.27i 6. Inoculation with all the selected bacterial isolates except SB3.
Inoculation with heat killed SB2. inoculation with the ACC deaminase containing strains SB1.2.16 698. and SB2.12 23.4 %) when rice seeds were treated with the isolates SB1ACC2 and AVG in comparison to the negative control (82 %) which was followed by SB1.80 1181.43a 2.42 mg).68 b 11.35 3. Inoculation with SB1.5 % respectively compared to the negative control.04b c 14.ACC5 (866.66 6.10b 1.ACC3.51 1. Total chlorophyll content significantly (P 0 0.94a 18.ACC2 isolates also reduced ethylene production by 38.ACC3 and SB2. It was highest in the plants treated with the isolate SB2.ACC3 and SB2.ACC3. 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 gfw−1 h−1) 18.ACC2 (113 % higher than control) and statistically at par with the isolate SB1.66 6.71 7.73a 1194.8a b 694.72 1.ACC2 and SB2.90a c 0.77a 2.18 a b a b 1.95 78.42 nmol C2H4 g−1fw hr−1) under the salt stress.ACC1 (163.0a* 98.ACC2>AVG >SB1.05) increased the shoot dry weight in comparison to uninoculated control.54 a 9.2 1378.ACC2 also increased seedling vigor (1378. was six times than that of untreated control.ACC3 and SB2. However inoculation of seeds with the heat killed isolates had no significant effect on both germination percentage and seedling vigor.83 17.64 6. The tested live strains significantly enhanced the germination percentage.22c 1.ACC3.84b 14. Salt stressed plants inoculated with all the three live strains grew to a significantly (P 0 0.05 4. However.32a 6.27 10.01a 6.ACC2 was found to be the most efficient in reducing stress ethylene production.4c 80. inoculation with the same isolates showed significant increase in shoot fresh weight as compared to the control. SB1. obtained with SB2.52 a d a c 5.13 0.97 4.93c 1.ACC2 on seed germination.56a c 7.61b 761.2 and 36.75 6. SB2.26b a 4.52 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 %) 82.4 80. and was statistically at par with negative control.76) as compared to the negative control (683.39 10.73) and followed the order of SB2. Germination percentage was highest (98. SFW shoot fresh weight. RL root length. RDW root dry weight. Highest amount of ethylene production was observed from the negative control (18.57a 2.07 1. Effect of selected isolates on seed germination under salt stress and ethylene estimation The effect of three efficient ACC deaminase producing strains.7 % and could not be explained.25 0.ACC2.64a 3.47 1. namely SB1. Maximum shoot fresh weight.65a 0. SDW shoot dry weight.76 1.ACC1 and SB2.05) .80 Data are represented as average of five replicates SVI seedling vigor index.0 95.36 3. 1).82 mg) was the most effective isolate which enhanced shoot dry weight by 384 % over control.ACC2 were the next effective strains.ACC2.02 a d b c 0.16c 1. 81. RFW root fresh weight.97 5.69c 7.4 96.4 a c a b 683.33a 9.05) increased over the uninoculated control with all the strains except SB3. Both length and fresh weight of root and shoot were increased (Table 4).04 20.ACC2 reduced ethylene production by 90.47a 0. The results for shoot dry weight showed that all the isolates except SB3. plant growth parameters and ethylene production under salt stress condition is shown in Table 4.13c 4.42a 11.57a b 2.65 b d a c 4.57 0.13 0.21 21.35 98.05a c 4.13 80.29 b d a c 14.01b 4.09 a c 4.ACC2 and SB1. SB1.03 5.ACC2.78 3.22 nmol C2H4 g−1fw hr−1) plants as compared the plants from the positive control (1.31 a b 18.6 and 81.ACC2.ACC5. significantly (P 0 0. Similarly. SL shoot length.Plant Soil (2013) 366:93–105 99 gave maximum increase (54 % over untreated control) which was followed by isolates SB2.02 1. Strain SB1.56a 1209. LSD least significant difference *Mean values sharing the same letter(s) in a column do not differ significantly according to Duncan’s Multiple Range Test (P 0 0.05) greater extent than the negative control (Fig.90c 0.
USA) and tentatively identified (Table 6). SB2. e. 105. Microbial ID. All the three isolates produced IAA. their identification and phylogenetic analysis Morphological and biochemical characteristics of three selected bacterial strains are given in Table 5. 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. NJ.ACC2 and SB1. SB1ACC2. In case of root and shoot dry weight also live cells of the three strains increased the plant biomass significantly ( P 0 0. Bacillus sp. g.ACC2 was the most efficient strain which enhanced both length and fresh weight of root and shoot up to 73.2.ACC2. motility and gram staining. Negative Control (0. which employs a redox reaction by tetrazolium dye to test the ability of isolates to utilize different carbon sources. The selected strains were further identified by 16 S rRNA gene sequence analysis to ascertain their . f. Heat killed SB2ACC2.ACC3 and SB2. 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 5.54 M NaCl amendment followed by isolates SB1. 63.100 Fig. SB1ACC3.8 and 59. siderophore and ammonia and also had phosphate solubilizing activity but none of them were HCN producer. a. h. Heat killed SB1ACC3.8. Newark. Morphological and biochemical characterization of the bacterial isolates. and Ochrobactrum sp (Table 6). AVG Plant Soil (2013) 366:93–105 SB1. d. b. the isolates were compared with those of the standard species using Bergey’s Manual of Determinative Bacteriology.ACC2 were the most effective isolates to enhance root and shoot dry weight by 166 and 78 % over negative control. c. The ‘phenotypic fingerprint’ thus generated was used to identify the organisms to genus level and tentatively identified as Alcaligens sp. 1 Salt stress effect on shoot and root growth of 7 day old rice seedlings exposed to salt stress (150 mM) under gnotobiotic conditions. SB2ACC2.05) over the negative control whereas the heat killed strains failed to do so. Carbon substrate utilization profile of the isolates was obtained using BIOLOG(R) system (Supplementary Table 1). The most effective PGPR isolates also exhibited different degree of tolerance to salinity (Table 5). But heat killed strains did not promote the plant growth parameters significantly.03 M MgSO4).. 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. Following morphological characterization.5 % respectively. Heat killed SB1ACC2.
albeit statistically not significant. was previously reported by Corsini (2011). We tested eleven high ACC deaminase producers for their growthpromoting activity without salt stress and three most promising strains.ACC2 ( Alcaligenes sp.81 45. ACC deaminase producing ability in Ochrobactrum sp. SB1. Belimov et al.37±2.38 − + + − + + + − + + + Ammonia production + 1.559.ACC3 (Bacillus sp.Plant Soil (2013) 366:93–105 Table 5 Morphological and biochemical characters the isolates Characters Isolates SB1-ACC2 SB1-ACC3 SB2-ACC2 Morphological Gram Reaction Cell shape Cell Length (μ) Colony Color Motility Biochemical MR MRVP Citrate utilization Nitrate reduction Starch hydrolysis Oxidase Catalase Tween 80 hydrolysis Urease Plant growth promoting traits IAA production (μM ml−1) HCN production Phosphate solubilization Siderophore Production Salt tolerance Growth in maximum salt concentration (M NaCl) − − − − − + + − − + − − + − + + + − − + − + + − + + + + − − Rod 2.2 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.ACC2 (Ochrobactrum sp. 2004.79 152.) and SB2. Bacillus and Ochrobactrum respectively (Fig.03 0. SB1.) were further evaluated under salt stress condition. Dimkpa et Tributyrin hydrolysis + 49. The phylogenetic analysis of the isolates was done based on neighborhood joining method with 100 bootstrap sampling. The differences in plant growth promotion among the isolates are also attributed to their individual rhizospheric competencies and hydrolyzing the ACC synthesized in roots. 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. Rhodococcus.ACC2 showed a 99 % similarity with the 16 S rRNA gene sequence of Alcaligenes.0±0. . 2003). Variovorax.1 White + − Rod 4.56±2. SB1.ACC2. Inoculation with rhizobacteria having ACC deaminase activity resulted in the development of a better root system. The isolates SB1. and Bacillus and to different species of Pseudomonas (Belimov et al. In comparison to control plants. suggesting a direct impact of ACC deaminase activity on root elongation . The longest roots and shoots. 2005). 2002).54 1. 1998. which subsequently affected shoot growth positively (Glick et al. 2).1 White + + Rod 2. Inoculation with ACC-deaminase containing bacteria promotes root growth of developing seedlings of various crops (Zahir et al.ACC3 and SB2. ACC deaminase activity was more widely present in soil bacteria belonging to the genera Alcaligenes. PGPRs having ACC deaminase activity help plants to withstand stresses (biotic or abiotic) by reducing the level of stress ethylene (Mayak et al.0±0. 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 0.68 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).91±1. all the plant growth parameters of the bacteria treated plants were significantly higher. n 0 11). highest root fresh weight and dry weight were observed in plants treated with Alcaligenes sp.0±0.).
0002 51 54 48 38 Bacillus subtilis ZFJ-8 Bacillus pumilus MTCC 7514 Bacillus sp. AB695227 Similarity (%) 99 99 99 al. Similar to other reports (Mayak et al. SB1-ACC3 and SB2-ACC2 Isolates Biolog GEN III identification FAME identification Molecular identification Accession Number SB1-ACC2 SB1-ACC3 SB2-ACC2 Alcaligens sp. 2004. F78 Alcaligenes faecalis NBRC 14479 50 Alcaligenes faecalis AE1. The bar represents 0. 2009. Bacillus and Ochrobactrum sp.0002 . 10 Ochrobactrum grignonense IHB B 1375 Ochrobactrum sp. 2 Neighbor joining tree showing phylogenetic relationship between the selected PGPR from rice soil and their representative species from NCBI database. Bacillus sp.0002 Ochrobactrum sp. 2009) present study also revealed that inoculation with live ACC deaminase producing bacteria reduced ethylene production significantly in Fig. by conducting root elongation assay on rice. Similar improvement of seed germination has been reported with other cereals such as sorghum and pearl millet (Gholami et al. BH3 SB2-ACC2 Ochrobactrum sp. SB2ACC2 comparison to negative control and heat killed bacteria treated plants (Table 4). SB1ACC2. Ochrobactrum sp. FAME and molecular identification of isolates SB1-ACC2. Alcaligens sp. Ochrobactrum sp. NBRC 12953 Ochrobactrum pseudogrignonense BIHB 340 Ochrobactrum sp. Bacillus sp. 2009). FJ763649 Ochrobactrum sp. TH-N-29 56 (C) 0. Bootstrap values (n 0 100) were displayed at the node. Zahir et al. We screened three most promising isolates ( Alcaligenes.05 substitutions per site. Zahir et al. Present study revealed that seed treatment with PGPR strains improved seed germination and seedling vigor over the non-inoculated seeds (Table 4). a. JN256919 JN256920 JN256921 16 S rRNA fragment length (bp) 1430 1452 1379 Closest relatives and NCBI accession number Alcaligenes faecalis strain DZ2.) with multiple PGP traits for their growth promoting activity under axenic conditions at 150 mM NaCl. HQ202537 Bacillus pumilus strain S68. 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. YXC1-10 Bacillus pumilus TW3 Bacillus subtilis CCM7 Bacillus pumilus S68 SB1-ACC3 (B) 34 56 0.16 SB1-ACC2 Alcaligenes faecalis DZ2 (A) 0. TH-N-29. b. SB1ACC3.102 Plant Soil (2013) 366:93–105 Table 6 BIOLOG GenIII.
Like other studies (Mayak et al. n 0 15) (Glick et al. Oleifera) to inoculation with plant growth promoting rhizobacteria containing 1aminocyclopropane-1-carboxylate deaminase depends on nutrient status of the plant. Hontzeas N. 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.theme Microbial Diversity and Identification” by the Indian Council of Agricultural Research. 2004). Crop Prot 23:835–843 Corsini A (2011) Microbial arsenic transformations in contaminated soils. 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.Plant Soil (2013) 366:93–105 103 index under salt stress conditions and Alcaligenes significantly (P 0 0. significant increase in seedling vigor would have occurred by better synthesis of plant growth hormones (Bharathi et al. Vivekananthan R. 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. In addition. Glick BR (2005) Cadmium-tolerant plant growth-promoting rhizobacteria associated with the roots of Indian mustard (Brassica juncea L. Inoculation with the PGPR isolates also increased the fresh and dry weight of both root and shoot. Harish S. 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. Can J Microbiol 48:189–199 Bharathi R. siderophore and ammonia and also solubilized phosphorus. 2002). New Delhi. shoot and other growth indices of rice to a greater extent. Acknowledgments This work was supported in part by the ICAR Networking Project. which would have triggered the activity of enzymes like α-amylase that promoted early germination by bringing an increase in availability of soluble sugars from starch decomposition (Kim et al. Asch F (2009) Plant–rhizobacteria interactions alleviate abiotic stress conditions. 2009). Safronova VI.01. 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. These findings may be due to the increased synthesis of phytohormones like IAA. 2004). Dingkuhn M. Dorffling K (2000) Salinity increases CO2 assimilation but reduces growth in field grown. 2004. As isolates have the ability to produce both ACC deaminase and IAA they promoted root. 1998.05) increased vigor index compared to the negative control. This might be due to the stressinduced ethylene disrupting the metabolic activity and physiological processes more in roots than shoots. Universita’ degli Studi di Milano Dimkpa C. Safronova VI. 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. 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. Plant Cell Environ 32:1682–1694 . Plant Soil 218:1–10 Belimov AA. 2007b). Besides. All the three ACC deaminase producing strains were tested positive for multiple PGP traits like production of IAA. Bullitta S. Soil Biol Biochem 37:241–250 Belimov AA. Use of strains with multiple PGP traits is expected to help increase crop productivity on a sustainable basis. Belimov et al. Madhaiyan et al. irrigated rice. 2006). Piluzza G. Demchinskaya SV. It is likely that IAA and ACC deaminase stimulate root growth in a coordinated fashion (Glick et al. Czern). “Application of Microorganisms in Agriculture and Allied Sciences (AMAAS) .991 at P >0. var. The assemblage of specific PGP traits of these studied PGPR suggests that these particular organisms can promote plant growth by more than one mechanism. Samiyappan R (2004) Rhizobacteria-based bio-formulations for the management of fruit rot infection in chillies. References Asch F. 2007) treatment with AVG and ACC deaminase-producing bacteria increased root growth of rice plants as compared to the negative control plants. Mimura T (2002) Response of spring rape (Brassica napus L. PhD thesis. Weinan T. Root growth is relatively more affected compared to shoots in the presence of an inhibitory level of salt (Lin and Kao 2001). Ramanathan A. 2004).
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