REGULAR ARTICLE

Isolation of ACC deaminase producing PGPR from rice rhizosphereand evaluating their plant growth promoting activity under saltstress

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

AbstractAims Bacteria possessing ACC deaminase activity re-duce the level of stress ethylene conferring resistanceand stimulating growth of plants under various bioticand abiotic stresses. The present study aims at isolat-ing efficient ACC deaminase producing PGPR strainsfrom the rhizosphere of rice plants grown in coastalsaline soils and quantifying the effect of potent PGPRisolates on rice seed germination and seedling growthunder salinity stress and ethylene production from riceseedlings inoculated with ACC deaminase containingPGPR.Methods Soils from root region of rice growing incoastal soils of varying salinity were used for isolatingACC deaminase producing bacteria and three bacterialisolates were identified following polyphasic taxono-my. Seed germination, root growth and stress ethylene

production in rice seedlings following inoculationwith selected PGPR under salt stress were quantified.Results Inoculation with selected PGPR isolates hadconsiderable positive impacts on different growthparameters of rice including germination percentage,shoot and root growth and chlorophyll content ascompared to uninoculated control. Inoculation withthe ACC deaminase producing strains reduced ethyl-ene production under salinity stress.Conclusions This study demonstrates the effective-ness of rhizobacteria containing ACC deaminase forenhancing salt tolerance and consequently improvingthe growth of rice plants under salt-stress conditions.

Keywords Growth promoting rhizobacteria . Coastalrice soils . ACC deaminase . Polyphasictaxonomy . Salinity stress . Ethylene production

Introduction

Plant growth-promoting rhizobacteria (PGPR), free-living soil bacteria thriving in the plant rhizosphere,have been studied as plant growth promoters for in-creasing agricultural productivity (Lucy et al. 2004).PGPR can either directly or indirectly facilitate growthof plants (Glick 1995). Indirect stimulation of plantgrowth includes mechanisms by which the bacteriaprevent phytopathogens from inhibiting plant growthand development (Raaijmakers et al. 2009) while di-rect stimulation may include providing plants with

Plant Soil (2013) 366:93–105DOI 10.1007/s11104-012-1402-5

Responsible Editor: Bernard Glick.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-012-1402-5) containssupplementary material, which is available to authorized users.

H. B. Bal : L. Nayak : S. Das : T. K. AdhyaLaboratory of Soil Microbiology, Division of CropProduction, Central Rice Research Institute,Cuttack, 753006 Odisha, India

Present Address:T. K. Adhya (*)School of Biotechnology, KIIT University,Bhubaneswar, 751024 Odisha, Indiae-mail: adhyas@yahoo.com

fixed nitrogen, phytohormones, iron that has beensequestered by bacterial siderophores, and solublephosphate (Lucy et al. 2004). Many PGPRs can alsoincrease plant resistance to biotic and abiotic stressfactors. Presence of 1-aminocyclopropane-1-carboxyl-ate (ACC) deaminase activity in several rhizosphericbacteria and regulation of ACC, a precursor to plantethylene levels, is one of the principal mechanisms bywhich bacteria exert beneficial effects on plants underabiotic stress (Glick et al. 1998, 2007a, b; Glick 2004,2005; Saleem et al. 2007). Earlier studies indicatedthat bacteria having ACC deaminase activity reducethe level of stress ethylene conferring resistance andresulting in better growth of plants under variousstresses such as salt stress, flooding stress, heavy metalstress and pathogen attack (Glick et al. 2007a).

Rice is a semi-aquatic plant and its ecosystem isclassified into irrigated, rainfed lowland, upland, andflood-prone on the basis of hydrology. Rice growingunder these field conditions is exposed to varioustypes of biotic and abiotic stresses at various develop-mental stages during its life cycle. These stressesinclude drought, submergence, extreme temperature,high salt, presence of toxic materials and environmen-tal contaminants and various others which affect plantgrowth negatively. Soil salinity is one of the importantfactors affecting soil microbial activities and crop pro-ductivity in most of the humid and sub-humid con-ventional rice growing areas of coastal Asia (Ismail etal. 2009). Limitations of major macro and micronu-trients in soil and high soil salinity are the importantyield reducing factors in the coastal rice ecosystem(Asch et al. 2000).

Previous research has shown that inoculation withPGPR can alleviate the salt stress effects in differentplant species. Enhancement of growth and salt toler-ance in PGPR inoculated red pepper (Siddikee et al.2011), tomato (Mayak et al. 2004) and Groundnut(Saravanakumar and Samiyappan 2007) have beenreported. Plant growth-promoting bacteria found inassociation with plants grown under chronicallystressful conditions, including high salinity, may havebeen adapted to the stress conditions, and could pro-vide a significant benefit to the plants. Since coastalsoils with salinity are natural habitats of halophilic/halotolerant bacteria (Lichfield 2002), isolation ofACC deaminase-producing PGPR from such naturalhabitat, and their utilization could prove to be benefi-cial for mitigating the salt stress to the plants growing

in such environment. The objectives of the presentstudy were: (1) to isolate efficient ACC deaminaseproducing PGPR from the rhizosphere of rice plantsgrown in coastal saline soils, and characterize them (2)to evaluate other plant growth promoting (PGP) activ-ities including production of indole acetic acid (IAA)by the most promising ACC deaminase producingisolates (3) to determine the effect of potent PGPRisolates on root elongation under salinity stress; (4) toestimate ethylene from rice seedlings treated withACC deaminase containing PGPR or chemical ethyl-ene inhibitors.

Materials and methods

Sampling and characterization of soils

The root adhering soil (RAS) samples were collectedfrom coastal rice fields from five different locations ofSundarban area (21°40′54.0″ to 21°47′50.5″ N latitudeand 88°14′2.8″ to 88°37′33.2″ E longitude) of WestBengal, India during September, 2008 at the tilleringstage of the monsoon season (kharif) rice. The riceplants were carefully uprooted along with the soil andbrought to the laboratory in polythene bags in portablecool chambers (~4°C). The non-rhizosphere soil wasremoved by vigorously shaking the uprooted rice hillsleaving behind the rhizosphere soil strongly adheringto the roots (Ramakrishna and Sethunathan 1982).Physicochemical parameters of the soils were deter-mined according to Spark et al. (1996) and reported inTable 1. Both the rhizosphere and the non-rhizospheresoils were used for the isolation of bacteria.

Isolation of ACC-utilizing bacteria

ACC-utilizing bacteria were isolated from both therhizospheric and non-rhizospheric soil following theprocedure of Penrose and Glick (2003) with somemodifications. Representative soil samples (1 g each)were separately enriched for ACC-utiltizing bacteriaby growing in sterile DF salts (Dworkin and Foster1958) minimal medium containing 3 mM ACC as thenitrogen source and incubated on a rotary shaker at200 rpm and 30 °C for 24 h. Four fold dilutions of thisculture were plated onto solid DF salts agar mediumcontaining ACC (500 μmol mL−1) and incubated for48 h at 30 °C. Bacterial colonies were chosen based on

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their colony morphology, further purified and main-tained 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 ofthe bacterial isolates were examined according to theBergey’s Manual of Determinative Bacteriology (Holtet al. 1994). Individual cultures grown on NA(Nutrient Agar) medium at 30 °C were examined forthe colony morphological features. Motility and mor-phology were studied by phase contrast microscopy(Olympus BX-51, Olympus America Inc., USA).Gram staining was performed as per standard proce-dures with exponentially growing cultures. Salt toler-ance of the bacterial isolates was tested by growing themon nutrient broth amended with different levels of NaCl.

BIOLOG(R) analysis

Potential carbon source utilization of the isolates wasassessed by using the BIOLOG(R) GEN III MicroPlates(Biolog Inc., Hayward, CA) according to manufac-turer’s instructions. The reactions were observed after22 h incubation at 33°C and read using automatedBiolog(R) MicroStation Reader.

FAME analysis

The cellular fatty acids were extracted from approxi-mately 20 mg bacterial cells using the standard extrac-tion technique (Sasser 2001). FAME profiles werethen obtained by running the samples on a gas

chromatograph (GC) (Agilent Technologies, USA)equipped with flame ionization detector (FID) andMIDI(R) Sherlock Microbial Identification System(MIDI Inc., Newark, DE, USA) software. FAMEswere identified according to their retention time, ascompared to a commercial standard mixture (MISstandard calibration, Part no. 1200-A) (Sasser 2001).

Sequencing of 16 S rRNA gene for identificationof rhizobacterial isolates

Most effective strains were identified by partial se-quencing of the 16 S rRNA gene. Genomic DNAwas isolated from the culture by using GenomicDNA isolation kit (Sigma, India). 16 S rRNA genewas amplif ied using universal forward (5 ′-AGAGTRTGATCMTYGCTWAC-3′) and reverse(5′-CGYTAMCTTWTTACGRCT-3′) primers (Hazraet al. 2011). 100 μl reaction mixture containing100 ng DNA template, 400 ng of each primer, 4 μldNTP (2.5 mM each), 10 μl 10x Taq DNA polymeraseassay buffer, 1 μl Taq DNA polymerase (3Uμl −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 ampli-fication was as follows: 5 min at 94 °C, followed by 35cycles of 30 s at 94 °C, 30 s at 55 °C, 2 min at 72 °C anda final extension of 5 min at 72 °C. The amplified 16 SrRNA gene was purified with a PCR purification kit(Qiaquick PCR purification kit, Qiagen, India) and out-sourced for sequencing (Chromus Biotech Pvt. Ltd.,Bangalore, India). The sequence data was aligned withSystem Software aligner and analyzed to identify thebacterium and its closest neighbors by using BLAST(NCBI, USA). The partial 16 S rRNA gene sequenceswere deposited in GenBank data base.

Table 1 Characteristics of thesoil samples Properties Soil samples

SB1 SB2 SB3 SB4 SB5

pH 6.94 5.64 6.54 6.52 6.85

EC (dS/m) 6.88 5.74 11.32 6.60 1.03

Organic Carbon (%) 0.15 0.20 0.24 0.20 0.31

Total Carbon (%) 0.062 0.061 0.077 0.065 0.085

Total Nitrogen (%) 0.567 0.556 0.661 0.571 0.839

Available P (mg.kg−1) 14 20 16 13.3 10

Available K (mg.kg−1) 298.2 87.6 66.7 298.2 311.5

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Phylogenetic and molecular evolutionary analysesof the 16 s rDNA sequences were conducted by usingsoftware MEGA4 and aligned using CLUSTAL-X.The pair-wise evolutionary distance matrix was gener-ated and the evolutionary tree was inferred using theNeighbor-Joining method. The bootstrap test has beendone to cluster together the associated taxa. The evolu-tionary distances were compared using the MaximumComposite Likelihood method (Tamura et al. 2007).

Screening for plant growth promoting activities

ACC deaminase activity assay

ACC deaminase activity was determined by monitor-ing the amount of α-ketobutyric acid generated fromthe cleavage of ACC (Penrose and Glick 2003). TheACC deaminase activity was induced by growing thebacterial cells in minimal medium containing ACC asthe sole nitrogen source, after growing them in 15 mlTSB medium up to log phase. α-ketobutyrate pro-duced by the reaction was determined by comparingthe absorbance at 540 nm of the sample to a standardcurve of α-ketobutyrate ranging between 0.1 and1.0 μmol. The ACC deaminase activity was expressedas the amount of α-ketobutyrate produced per mg ofprotein per hour.

Determination of other plant growth-promoting traits

Ability to produce IAAwas detected by the method ofSalkowski (Glickmann and Dessaux 1995). Isolateswere screened for hydrogen cyanide (HCN) produc-tion, siderophore production, ammonia production andP-solubilization following Islam et al. (2009).Production of HCN was determined on NA platessupplemented with glycine (4.4 gl−1) after 3 days ofincubation at 28 °C. Siderophores were detected bythe formation of orange halos around bacterial colo-nies on Chrome Azural S (CAS) agar plates afterincubation for 24 h at room temperature. Ammoniaproduction was detected by adding Nessler’s reagent(0.5 ml/tube) to 48-h old bacterium grown in peptonewater. P-solubilization activity was tested onPikovaskaya’s agar medium containing 2 % tricalciumphosphate. The appearance of clear halo zone aroundbacterial colonies after incubation for 24–48 h at 28 °Cwas 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 strainswere suspended in 0.5 ml sterile 0.03 M MgSO4 and theabsorbance adjusted to 0.15 at 600 nm. Seeds weresurface-sterilized by dipping in 95 % ethanol and in0.2 % HgCl2 solution for 3 min followed by rinsing 5times with sterile distilled water (Zahir et al. 2009). Seedswere incubated at room temperature for 1 h with theappropriate treatment - sterile 0.03 M MgSO4 (negativecontrol) or bacterial suspensions in sterile 0.03MMgSO4.

Soil sample for the study was collected from theexperimental fields at CRRI, Cuttack, air-dried, sieved(2-mm/10-mesh) and analyzed for physico-chemicalcharacteristics. The soil was a typic haplaquept havingpH 6.16, electrical conductivity 0.5 dS.m−1, cation ex-change capacity 15.0 meq.g−1 soil, organic carbon0.86 %, total nitrogen 0.09 % and contained 25.9 %clay, 21.6 % slit and 52.5% sand. The soil was sterilizedby autoclaving at 121.1 °C for 1 h for three consecutivedays to kill all the soil microorganisms and their spores,and 200 g portions of sterilized soil were filled in eachthermocol cup (4.5cmx4.5cmx5.5 cm).

Six seeds were placed in each cup aseptically andplaced in a growth chamber with maximum and mini-mum temperatures maintained at 28 °C and 20 °C,respectively with 12 h day night photoperiod. Initialand final growth parameters (plant height, plant biomassand chlorophyll content) were recorded on day 5 and 15respectively (Yoshida et al. 1976). For measuring thechlorophyll content, 100 mg of finely chopped freshleaves were placed in a capped measuring tube contain-ing 25 mL of 80 % acetone, and placed inside a refrig-erator (4 to 8 °C) for 28 h (Panda et al. 2008). Thechlorophyll content was measured at 646.6 and663.6 nm in a spectrophotometer and calculated usingthe equation of Porra (2002).

Effect of selected isolates on seed germinationunder saline stress and ethylene estimation

Three most efficient strains were selected to studytheir effect on seed germination and ethylene emissionunder salt stress. In an initial screening on the level ofsalinity, germination of rice seeds (cv. Naveen) wasreduced by ~20 % at 150 mM NaCl (data not shown),and the specific salinity level was used for subsequent

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studies on salt stress. Surface sterilized seeds were in-cubated for 1 h at room temperature with the appropriatetreatment: sterile 0.03 M MgSO4 (negative control),10−4 M L-α-(2-aminoethoxyvinyl) glycine hydrochlo-ride (AVG) (Sigma, India), a known inhibitor of ethyl-ene production, in 0.03 M MgSO4 (positive control),heat-killed or fresh bacterial suspensions in sterile0.03 M MgSO4 (OD of 0.15 at 600 nm) (Penrose andGlick 2001). Positive control contained AVG andshould completely inhibit ethylene production in thepresence of salt stress while negative control containedneither AVGnor bacteria and should not inhibit ethyleneproduction. Heat killed cells of 24 h old bacterial cul-tures were retained as the secondary control to verify theimpact of cellular metabolites, if any, released from cellson ethylene production. Twenty five seeds were plantedon sterile filter paper inside each pre-sterilized 100 mlGL 45 clear glass bottles (Schott Duran, Germany), and5 ml of sterile half strength N-free Hoagland’s solution,a hydroponic nutrient solution (Hoagland and Boyer1936) supplemented with 150 mM NaCl, was addedas nutrient solution. For preparing 1 L of Hoagland’ssolution, 1 M solution each of KH2PO4, MgSO4.7H2Oand 0.5M solution of K2SO4 were prepared and 2, 1 and4 ml of each solution were added respectively.Subsequently, 1 g of CaSO4, 1 ml micronutrients solu-tion (g/L: H3BO3, 1; MnCl2.4H2O, 1; ZnSO4.7H2O,0.58; CuSO4.5H2O, 0.13; Na2MoO4.2H2O, 0.10) and1 ml of 2 % (w/v) iron solution were added and thevolume made to 1 l with distilled water.

The experiment was conducted for 7 days and theglass bottles were placed in a growth chamber withmaximum and minimum temperatures maintained at 28and 20 °C, respectively with a cycle of 12 h dark/light.The bottles were arranged in a completely randomizeddesign with 5 replications for each treatment (Fišerová etal. 2008; Sapsirisopa et al. 2009; Siddikee et al. 2011).After 7 days, germinated seeds were counted and percentof germination was calculated. Root and shoot length,fresh and dry weight of root and shoot both of fiverandomly selected seedlings from each replication werealso recorded. Seedling vigor index (VI) was calculatedusing the formula:

VI ¼ mean root lengthþmean hypocotyl lengthð Þ�% germination:

For ethylene estimation, glass bottles were sealedby 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 injectedinto GC (Model-Ceres 800 plus, Thermo Scientific,USA) packed with a Porapak-Q column (183 cm lengthand 0.3 cm internal diameter, 80/100 mesh) and equippedwith FID. The GC was adjusted to 100 °C, 300 °C and150 °C for oven, injection and detection temperature. Theflow rate of N2, H2 and air were 30, 30 and 300 ml min

−1.Under these conditions, retention time of ethylene was2.28 min and the minimum concentration of ethylenedetected was 0.1 ppm. The amount of ethylene producedwas expressed as nmol of C2H4 g

−1 fresh weight h−1 bycomparing with the standard curve of pure ethylene(9.12 ppm in nitrogen, Matheson Tri-Gas) (Fišerová etal. 2008; Siddikee et al. 2011).

Statistical analyses

All results presented are the means of five independentreplicates. Data were subjected to statistical analysis(Gomez and Gomez 1984) by a statistical package(IRRISTAT version 3.1; International Rice ResearchInstitute, Los Baños, Philippines). The mean differencecomparison between the treatments was analyzed byanalysis of variance (ANOVA) and subsequently byDuncan’s multiple range test at p<0.05. Simple correla-tion between specific datasets was analyzed byMicrosoftExcel® program on correlation and regression.

Results

Isolation and characterization of bacteria

A total of 17 ACC utilizing bacteria were isolated fromthe coastal rice field soil from five different locations andscreened for their ACC deaminase metabolism. Resultsindicated that all the strains metabolized ACC but withvariable degrees of efficacy (Table 2). Highest ACC-deaminase activity per hour was exhibited by the isolateSB1.ACC2 (2664.08 nmol α-ketobutyrate mg−1 h−1).Eleven strains exhibiting high ACC utilization rate werefurther selected to screen their growth-promoting activityin rice under axenic conditions (root elongation assay).

Plant growth-promotion activity by bacterial isolates

Statistical analysis of data recorded 15 days after seedgermination is summarized in Table 3. All the tested

Plant Soil (2013) 366:93–105 97

strains had considerable impact on different growthparameters of rice compared with the negative control.Data on root length indicate that inoculation with allthe isolates enhanced root length significantly (P00.05) in comparison with the control. Inoculation withSB1.ACC2 caused the maximum increase in rootlength (16.20 cm) which was 41.2 % higher than thecontrol, followed by SB2.ACC6.

Inoculation with all the selected bacterial isolatesexcept SB3.ACC3 also promoted root fresh weightsignificantly (P00.05) in comparison with the control.The maximum root fresh weight (311.48 % over con-trol) was observed by isolate SB1.ACC2 (726.83 mg).Isolates SB2.ACC1 and SB1.ACC3 were next effec-tive isolates that promoted root fresh weight by 281 %and 260 % respectively. Likewise, root dry weight alsoincreased significantly (P00.05) in response to inoc-ulation with the isolates except SB3.ACC3. The max-imum increase (255 % over uninoculated control) inroot dry weight was observed by inoculation withSB1.ACC2 (75.68 mg). Isolate SB1.ACC3 andSB2.ACC1 were next effective isolates.

Data regarding shoot length showed that treatmentwith seven isolates out of eleven, significantly (P00.05) promoted the shoot length in comparison withthe control. Strain SB1.ACC2 (43.03 cm) inoculation

Table 2 ACC deaminase activity (nmol α-ketobutyrate mg−1

protein h−1) of the isolates

S. No. Isolates ACC deaminase activity*

1 SB1.ACC1 894.11f±41.81

2 SB1.ACC2 2664.08j±63.21

3 SB1.ACC3 1468.90h±31.60

4 SB2.ACC1 397.04d±21.16

5 SB2.ACC2 2049.42i±52.20

6 SB2.ACC3 295.72bc±19.09

7 SB2.ACC4 236.05b±18.58

8 SB2.ACC5 1145.43g±66.44

9 SB2.ACC6 526.04e±42.65

10 SB3.ACC1 440.51d±27.24

11 SB3.ACC2 329.09c±28.15

12 SB3.ACC3 919.42f±73.05

13 SB3.ACC4 906.07f±56.02

14 SB4.ACC1 61.29a±6.24

15 SB4.ACC2 92.49a±8.43

16 SB5.ACC1 62.02a±15.50

17 SB5.ACC2 91.28a±12.16

Mean values sharing the same letter (s) in column do not differsignificantly according to Duncan’s multiple range test (P00.05)

*Mean of five replicate observations ± SD (Standard deviation)

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)

SL(cm)

SFW(mg)

SDW(mg)

Chl a(mg/gm fresh wt)

Chl b(mg/gm fresh wt)

Chl a + b(mg/gm fresh wt)

MgSO4 10.80g* 176.64i 21.27i 28.00fgh 155.84e 33.84h 3.36h 0.88g 4.24i

SB1.ACC1 13.54e 632.81c 35.18f 34.86e 409.46d 119.50e 3.73g 0.99f 4.71h

SB1.ACC2 15.25a 726.83a 75.68a 43.03a 712.06c 159.94b 7.10a 1.87a 8.98b

SB1.ACC3 13.93de 635.95c 70.83b 38.89c 711.42c 141.12d 7.13a 1.89a 9.02ab

SB2.ACC1 14.46bc 672.40b 70.63b 36.99d 811.66ab 163.82a 6.23b 1.66b 7.89c

SB2.ACC2 14.49bc 511.14d 61.55c 42.42ab 766.20bc 155.16c 7.14a 1.90a 9.04a

SB2.ACC5 14.05cd 459.67e 59.51d 41.54b 866.42a 159.96b 6.20b 1.67b 7.87c

SB2.ACC6 14.77b 379.90f 37.43e 39.40c 343.20d 93.86f 4.52e 1.22d 5.74f

SB3.ACC1 13.05f 256.84g 30.99g 27.25gh 159.72e 37.14g 4.40f 1.16e 5.56g

SB3.ACC2 13.83de 227.50h 25.70h 29.12f 188.80e 34.60h 4.62d 1.28c 5.90e

SB3.ACC3 14.21cd 192.53i 21.54i 28.53fg 196.18e 37.46g 3.40h 0.89g 4.27i

SB3.ACC4 14.20cd 264.66g 25.71h 26.86h 240.62e 38.46g 4.83c 1.30c 6.12d

LSD (5 %) 0.430 19.940 0.993 1.214 91.500 1.720 0.050 0.023 1.000

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, LSDleast significant difference

*Mean values sharing the same letter (s) in column do not differ significantly according Duncan’s multiple range test (P00.05)

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gave maximum increase (54 % over untreated control)which was followed by isolates SB2.ACC2 andSB2.ACC5. Similarly, inoculation with the same isolatesshowed significant increase in shoot fresh weight ascompared to the control. Maximum shoot fresh weight,obtained with SB2.ACC5 (866.42 mg), was six timesthan that of untreated control, and SB2.ACC1 andSB2.ACC2 were the next effective strains. The resultsfor shoot dry weight showed that all the isolates exceptSB3.ACC2, significantly (P00.05) increased the shootdry weight in comparison to uninoculated control.SB2.ACC1 (163.82 mg) was the most effective isolatewhich enhanced shoot dry weight by 384 % overcontrol.

Total chlorophyll content significantly (P00.05)increased over the uninoculated control with all thestrains except SB3.ACC3. It was highest in the plantstreated with the isolate SB2.ACC2 (113 % higher thancontrol) and statistically at par with the isolateSB1.ACC3.

Effect of selected isolates on seed germinationunder salt stress and ethylene estimation

The effect of three efficient ACC deaminase produc-ing strains, namely SB1.ACC2, SB1.ACC3 andSB2.ACC2 on seed germination, plant growth param-eters and ethylene production under salt stress condi-tion is shown in Table 4. The tested live strains

significantly enhanced the germination percentage.Germination percentage was highest (98.4 %) whenrice seeds were treated with the isolates SB1ACC2 andAVG in comparison to the negative control (82 %)which was followed by SB1.ACC3 and SB2.ACC2.Inoculation with SB1.ACC2 also increased seedlingvigor (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 theheat killed isolates had no significant effect on bothgermination percentage and seedling vigor, and wasstatistically at par with negative control.

Highest amount of ethylene production was ob-served from the negative control (18.22 nmol C2H4

g−1fw hr−1) plants as compared the plants from thepositive control (1.42 nmol C2H4 g−1fw hr−1) underthe salt stress. However, inoculation with the ACCdeaminase containing strains SB1.ACC2, SB1.ACC3and SB2.ACC2 reduced ethylene production by 90.2,81.6 and 81.5 % respectively compared to the negativecontrol. Inoculation with heat killed SB2.ACC2 andSB1.ACC2 isolates also reduced ethylene productionby 38.2 and 36.7 % and could not be explained. StrainSB1.ACC2 was found to be the most efficient inreducing stress ethylene production.

Salt stressed plants inoculated with all the three livestrains grew to a significantly (P00.05) greater extentthan the negative control (Fig. 1). Both length and freshweight 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

gfw−1 h−1)

RL(mm)

SL(mm)

RFW(mg)

SFW(mg)

RDW(mg)

SDW(mg)

MgSO4 82.0a* 683.73a 18.22c 4.01a 4.32a 5.33a 14.94a 0.47a 1.77a

AVG 98.4c 1194.61b 1.42a 6.13c 6.01b 9.69c 18.84b 0.65a 2.10b

HK SB1-ACC2 80.4a 761.16a 11.52b 4.75b 4.71a 7.27b 14.12a 0.47a 1.64a

SB1-ACC2 98.4c 1378.76c 1.78a 6.97d 7.05d 10.97d 23.83d 1.13b 3.16c

HK SB1-ACC3 80.4a 698.80a 18.54c 4.03a 4.66a 5.29a 17.04b 0.51a 1.43a

SB1-ACC3 96.0b 1181.09b 3.35a 5.66c 6.64c 9.39c 20.21c 1.07b 2.93c

HK SB2-ACC2 80.8a 694.56a 11.26b 4.05a 4.56a 7.04b 14.90a 0.57a 1.57a

SB2-ACC2 95.2b 1209.68b 3.36a 6.13c 6.57c 10.13c 21.02c 1.25b 2.90c

LSD (5 %) 2.95 78.72 3.31 0.65 0.52 1.02 1.80 0.18 0.35

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, SDWshoot dry weight, 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 (P00.05)

Plant Soil (2013) 366:93–105 99

SB1.ACC2 was the most efficient strain whichenhanced both length and fresh weight of rootand shoot up to 73.8, 63.2, 105.8 and 59.5 %respectively. But heat killed strains did not pro-mote the plant growth parameters significantly. Incase of root and shoot dry weight also live cells ofthe three strains increased the plant biomass sig-nificantly (P00.05) over the negative controlwhereas the heat killed strains failed to do so.SB2.ACC2 and SB1.ACC2 were the most effectiveisolates to enhance root and shoot dry weight by166 and 78 % over negative control.

Characterization of other plant growth promoting traitsof isolates

The three selected strains were screened for their otherplant growth promoting activities and the result wassummarized in Table 5. All the three isolates producedIAA, siderophore and ammonia and also had phos-phate solubilizing activity but none of them were HCNproducer.

Morphological and biochemical characterizationof the bacterial isolates, their identificationand phylogenetic analysis

Morphological and biochemical characteristics ofthree selected bacterial strains are given in Table 5.

Following morphological characterization, motilityand gram staining, the isolates were compared withthose of the standard species using Bergey’s Manualof Determinative Bacteriology. The most effectivePGPR isolates also exhibited different degree of toler-ance to salinity (Table 5). The isolate SB1-ACC2showed the highest salinity tolerance with normal (<10 % growth as compared to growth in non-salinecontrol Nutrient agar broth) growth at 1.54 M NaClamendment followed by isolates SB1.ACC3 andSB2.ACC2.

Carbon substrate utilization profile of the isolates wasobtained using BIOLOG(R) system (SupplementaryTable 1), which employs a redox reaction by tet-razolium dye to test the ability of isolates toutilize different carbon sources. The ‘phenotypicfingerprint’ thus generated was used to identifythe organisms to genus level and tentatively identifiedas Alcaligens sp., Bacillus sp. and Ochrobactrum sp(Table 6).

The predominant FAMEs for each pure culture(Supplementary Table 2) were selected on the basisof those FAMEs that comprised greater than 5 %of the total area of FAMEs in each respectivedatabase file of FAME profiles (Sherlock Software,Microbial ID, Newark, NJ, USA) and tentativelyidentified (Table 6).

The selected strains were further identified by 16 SrRNA gene sequence analysis to ascertain their

Fig. 1 Salt stress effect onshoot and root growth of7 day old rice seedlings ex-posed to salt stress(150 mM) under gnotobioticconditions. a, NegativeControl (0.03 M MgSO4); b,SB1ACC2; c, Heat killedSB1ACC2; d, SB1ACC3; e,Heat killed SB1ACC3; f,SB2ACC2; g, Heat killedSB2ACC2; h, AVG

100 Plant Soil (2013) 366:93–105

taxonomic positions (Table 6). The phylogenetic anal-ysis of the isolates was done based on neighborhoodjoining method with 100 bootstrap sampling. The iso-lates SB1.ACC2, SB1.ACC3 and SB2.ACC2 showeda 99 % similarity with the 16 S rRNA gene sequenceof Alcaligenes, Bacillus and Ochrobactrum respec-tively (Fig. 2).

Discussion

This study demonstrates the effectiveness of rhizobac-teria containing ACC deaminase for inducing salttolerance and consequently improving the growth ofrice plants under salt-stress conditions. We tested elev-en high ACC deaminase producers for their growth-promoting activity without salt stress and three mostpromising strains, SB1.ACC2 (Alcaligenes sp.);SB1 .ACC3 (Bac i l l u s sp . ) and SB2 .ACC2(Ochrobactrum sp.) were further evaluated under saltstress condition. ACC deaminase activity was morewidely present in soil bacteria belonging to the generaAlcaligenes, Variovorax, Rhodococcus, and Bacillusand to different species of Pseudomonas (Belimov etal. 2005). ACC deaminase producing ability inOchrobactrum sp. was previously reported byCorsini (2011).

An assessment of the plant growth-promotingcapabilities of the isolates was based on enhancedplant parameters of 15-day old plants grown undergnotobiotic conditions from rice seeds inoculatedwith the eleven strains without stress. In comparison tocontrol plants, all the plant growth parameters of thebacteria treated plants were significantly higher. Thelongest roots and shoots; highest root fresh weight anddry weight were observed in plants treated withAlcaligenes sp.

A simple correlation analysis between in vitroACC deaminase production by the isolates(Table 2) and plant root elongation under con-trolled condition (Table 3) indicated a positivecorrelation (r00.559, n011), albeit statisticallynot significant, suggesting a direct impact ofACC deaminase activity on root elongation .Inoculation with rhizobacteria having ACC deami-nase activity resulted in the development of abetter root system, which subsequently affectedshoot growth positively (Glick et al. 1998; Belimov etal. 2002). Inoculation with ACC-deaminase con-taining bacteria promotes root growth of develop-ing seedlings of various crops (Zahir et al. 2003).The differences in plant growth promotion amongthe isolates are also attributed to their individual rhizo-spheric competencies and hydrolyzing the ACC synthe-sized in roots.

PGPRs having ACC deaminase activity help plantsto withstand stresses (biotic or abiotic) by reducing thelevel of stress ethylene (Mayak et al. 2004; Dimkpa et

Table 5 Morphological and biochemical characters the isolates

Characters Isolates

SB1-ACC2 SB1-ACC3 SB2-ACC2

Morphological

Gram Reaction − + −Cell shape Rod Rod Rod

Cell Length (μ) 2.0±0.1 2.0±0.1 4.0±0.2

Colony Color White White White

Motility + + +

Biochemical

MR − + +

MRVP − − −Citrate utilization − − +

Nitrate reduction − + +

Starch hydrolysis − − −Oxidase + + +

Catalase + + +

Tributyrin hydrolysis + + +

Tween 80 hydrolysis − − +

Urease − − −Plant growthpromoting traits

IAA production(μM ml−1)

49.56±2.81 45.91±1.79 152.37±2.38

HCN production − − −Ammonia production + + +

Phosphatesolubilization

+ + +

SiderophoreProduction

+ + +

Salt tolerance

Growth inmaximum saltconcentration (MNaCl)

1.54 1.03 0.68

Numerical values are the mean ± SD of three replicateobservations

Cell lengths of exponential phase cultures grown in NB wererecorded

Colony color were recorded after growing the isolates on NA(Nutrient agar)

Plant Soil (2013) 366:93–105 101

al. 2009; Zahir et al. 2009). We screened three mostpromising isolates (Alcaligenes, Bacillus andOchrobactrum sp.) with multiple PGP traits for theirgrowth promoting activity under axenic conditions at150 mM NaCl, by conducting root elongation assayon rice. Similar to other reports (Mayak et al. 2004;Zahir et al. 2009) present study also revealed thatinoculation with live ACC deaminase producing bac-teria reduced ethylene production significantly in

comparison to negative control and heat killed bacteriatreated plants (Table 4).

Present study revealed that seed treatment withPGPR strains improved seed germination and seedlingvigor over the non-inoculated seeds (Table 4). Similarimprovement of seed germination has been reportedwith other cereals such as sorghum and pearl millet(Gholami et al. 2009). Seeds treated with the isolatesshowed a considerable increase in seedling vigor

Table 6 BIOLOG GenIII, FAME and molecular identification of isolates SB1-ACC2, SB1-ACC3 and SB2-ACC2

Isolates Biolog GEN IIIidentification

FAMEidentification

Molecular identification

AccessionNumber

16 S rRNAfragment length (bp)

Closest relatives andNCBI accession number

Similarity(%)

SB1-ACC2 Alcaligens sp. Alcaligens sp. JN256919 1430 Alcaligenes faecalisstrain DZ2; HQ202537

99

SB1-ACC3 Bacillus sp. Bacillus sp. JN256920 1452 Bacillus pumilus strainS68; FJ763649

99

SB2-ACC2 Ochrobactrum sp. Ochrobactrum sp. JN256921 1379 Ochrobactrum sp.TH-N-29; AB695227

99

(A)

Uncultured bacterium clone: U-3

Alcaligenes faecalis Nic-2

Alcaligenes sp. F78

Alcaligenes faecalis NBRC 14479

Alcaligenes faecalis AE1.16

SB1-ACC2

Alcaligenes faecalis DZ2

50

55

47

0.0002

(B)

Bacillus subtilis ZFJ-8

Bacillus pumilus MTCC 7514

Bacillus sp. YXC1-10

Bacillus pumilus TW3

Bacillus subtilis CCM7

Bacillus pumilus S68

SB1-ACC3

5154

48

38

0.0002

0.0002

(C)

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

56

34

Fig. 2 Neighbor joiningtree showing phylogeneticrelationship between the se-lected PGPR from rice soiland their representative spe-cies from NCBI database.The bar represents 0.05substitutions per site. Boot-strap values (n0100) weredisplayed at the node. a,SB1ACC2; b, SB1ACC3;SB2ACC2

102 Plant Soil (2013) 366:93–105

index under salt stress conditions and Alcaligenessignificantly (P00.05) increased vigor index com-pared to the negative control. These findings may bedue to the increased synthesis of phytohormones likeIAA, which would have triggered the activity ofenzymes like α-amylase that promoted early germina-tion by bringing an increase in availability of solublesugars from starch decomposition (Kim et al. 2006).Besides, significant increase in seedling vigor wouldhave occurred by better synthesis of plant growthhormones (Bharathi et al. 2004).

As isolates have the ability to produce both ACCdeaminase and IAA they promoted root, shoot andother growth indices of rice to a greater extent. It islikely that IAA and ACC deaminase stimulate rootgrowth in a coordinated fashion (Glick et al. 2007b).Root growth is relatively more affected compared toshoots in the presence of an inhibitory level of salt(Lin and Kao 2001). This might be due to the stress-induced ethylene disrupting the metabolic activity andphysiological processes more in roots than shoots. Inaddition, this might be due to the roots being in moreintimate contact with the salt solution with the shootsexperiencing a lower salt concentration (Mayak et al.2004). Like other studies (Mayak et al. 2004;Madhaiyan et al. 2007) treatment with AVG andACC deaminase-producing bacteria increased rootgrowth of rice plants as compared to the negativecontrol plants. Simple correlation analysis betweenACC deaminase production capability of isolates andthe root elongation under the salt stress conditionindicated a highly significant positive relationship(r00.991 at P>0.01, n015) (Glick et al. 1998;Belimov et al. 2002). Inoculation with the PGPR iso-lates also increased the fresh and dry weight of bothroot and shoot. It was assumed that higher dry weightwould mean longer and stronger roots and shoots aswell as plants that would be able to better withstandsalt stress (Mayak et al. 2004).

Use of strains with multiple PGP traits is expectedto help increase crop productivity on a sustainablebasis. All the three ACC deaminase producing strainswere tested positive for multiple PGP traits like pro-duction of IAA, siderophore and ammonia and alsosolubilized phosphorus. Treating plants with ACC-deaminase and siderophore producing PGPR can helpthe plants to overcome many of the effects caused bythe environmental stresses as observed in the presentstudy (Dimkpa et al. 2009). The assemblage of

specific PGP traits of these studied PGPR suggeststhat these particular organisms can promote plantgrowth by more than one mechanism.

Conclusions

The current study concluded that inoculation with thethree ACC deaminase containing PGPR caused signif-icant alleviation of stress induced ethylene productionand consequently improving the growth of rice underhigh salinity stress condition. The reduction in stressethylene production and presence of multiple plantgrowth promoting traits of the strains may be thepossible reason to protect the plant from the growthinhibitory effect of salt and thus induce salinity toler-ance in rice and could be useful in coastal areas wheresalinity is a major constraint. However, further re-search is necessary to evaluate the effectiveness ofthese strains under actual field conditions to use themfor alleviating salinity stress to the growing crop.

Acknowledgments This work was supported in part by theICAR Networking Project, “Application of Microorganisms inAgriculture and Allied Sciences (AMAAS) - theme MicrobialDiversity and Identification” by the Indian Council of Agricul-tural Research, New Delhi.

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