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Applied Soil Ecology 46 (2010) 222–229

Contents lists available at ScienceDirect

Applied Soil Ecology

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timulatory effect of phosphate-solubilizing bacteria on plant growth, steviosidend rebaudioside-A contents of Stevia rebaudiana Bertoni

amtaa,b, Praveen Rahic, Vijaylata Pathaniad, Arvind Gulati c, Bikram Singhd,avinder Kumar Bhanwrae, Rupinder Tewaria,∗

Department of Biotechnology, Panjab University, Chandigarh-160014, IndiaDepartment of Environment and Vocational Studies, Panjab University, Chandigarh-160014, IndiaPlant Pathology and Microbiology Laboratory, Hill Area Tea Science Division, Institute of Himalayan Bioresource and Technology, Palampur-176061, Himachal Pradesh, IndiaDepartment of Natural Plant Products, Institute of Himalayan Bioresource and Technology, Palampur-176061, Himachal Pradesh, IndiaDepartment of Botany, Panjab University, Chandigarh-160014, India

r t i c l e i n f o

rticle history:eceived 30 January 2010eceived in revised form 6 August 2010ccepted 9 August 2010

eywords:tevia rebaudianahosphate-solubilizing bacteria (PSB)

a b s t r a c t

The effect of four phosphate-solubilizing bacteria (PSB), (Burkholderia gladioli 10216, Burkholderia gladi-oli 10217, Enterobacter aerogenes 10208 and Serratia marcescens 10238) as identified on the basis of 16SrRNA gene sequencing was evaluated on plant growth and commercially important glycosides, stevio-side (ST) and rebaudioside-A (R-A) of Stevia rebaudiana in pots containing tricalcium phosphate (TCP)supplemented soil. The PSB were isolated from the rhizosphere of S. rebaudiana plants and tested forP-solubilization ability, biocompatibility, indole acetic acid (IAA) and siderophore production. In green-house study, treatment of either individual PSB or a consortium (of PSB) resulted in increased plant

teviosideebaudioside-A

growth, ST and R-A contents. The stimulatory effect was observed with consortium treatment in plantgrowth parameters (shoot length, 22.5%; root length, 14.7%; leaf dry weight, 89.0%; stem dry weight,76.3% and shoot biomass, 82.5%) and glycoside contents (ST, 150% plant−1 and R-A, 555% plant−1) ascompared to the un-inoculated plants. Among individual PSB treatments, B. gladioli 10216 showed mostpromising response in majority of the parameters studied. The root colonization potential of PSB, assayed

ed th

by RAPD technique, show

. Introduction

Stevia rebaudiana is a perennial shrub of Asteraceae (Compositae)amily native to certain regions of South America. Its leaves produceero-calorie ent-kaurene glycosides – stevioside and rebaudiosideshich are in demand as non-nutritive and high potency sweetener

n food and beverages by the persons suffering from diabetes andbesity. The amount of active principles depends on total biomass,hich further depends on climatic features, agro-techniques, wateranagement and fertilizer applications. Recently, the integrated

pplication of microbial inoculants to agro-technologies for theultivation of medicinal plants is being promoted for improvingheir productivity in terms of biomass and biochemical constituentsLeithy et al., 2006; Jaleel et al., 2007). PSB are well known to

romote plant growth because of their ability to convert insolu-le form of P to soluble form that can be readily taken up by thelant roots. Usually the soils are supplemented with inorganic P inhe form of chemical fertilizers. A large proportion of the applied

∗ Corresponding author. Tel.: +91 172 9872216184; fax: +91 172 2541409.E-mail address: rupinder@pu.ac.in (R. Tewari).

929-1393/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.apsoil.2010.08.008

e colonization of all PSB isolates, though their extent of colonization varied.

© 2010 Elsevier B.V. All rights reserved.

P gets fixed in the soil as phosphates of iron, aluminum and cal-cium (Altomare et al., 1999). This fixed form of P is not efficientlytaken up by the plants and known to cause many environmen-tal problems like eutrophication and soil salinity (Del Campilloet al., 1999). The use of PSB as biofertilizers could decrease theenvironmental problems associated with conventional chemicalfertilizers.

In addition to P-solubilization, PSB may also improve the plantproductivity by producing other secondary metabolites. Severalevidence related to plant growth promotion by PSB through theproduction of IAA (Patten and Glick, 2002; Shahab et al., 2009) andsiderophore (Koo and Cho, 2009) make the PSB more suitable asbiofertilizers.

The effect of PSB on medicinal plants is gaining momentum, asevidenced by an increase in the number of publications. There arelimited reports (Earanna, 2007; Das et al., 2008) related to the effectof PSB on the growth of S. rebaudiana and no report has been found

on the yield enhancement of stevioside and rebaudiosides by PSBinoculation. The present study was carried out to isolate the PSBfrom the rhizosphere of Stevia plants and examine their effect onthe plant growth, availability of P in soil, P uptake by plants andyield of ST and R-A of S. rebaudiana.

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. Materials and methods

.1. Isolation of PSB

Rhizospheric soil samples of five S. rebaudiana plants growing inrganic farms (loamy soil, without any input of chemical fertilizers)ere collected in sterile plastic bags. Samples were processed on

he same day. One gram soil of each sample was suspended sepa-ately in 9.0 ml of phosphate buffer saline (PBS) of pH 7.2. The serialilutions (1:10) were made and spread on Pikovskaya’s (PVK) agarlates containing 0.5% TCP and incubated at 30 ◦C. Colonies showinghe zone of solubilization, which indicates microbial P-solubilizingbility, were streaked on nutrient agar plates to check their puritynd stored for further studies.

.2. Screening of PSB

.2.1. Quantitative estimation of phosphate solubilizationP-solubilization was estimated in a liquid medium amended

ith 0.5% TCP. The isolates were grown in a 100 ml liquidedium at 30 ◦C on a rotary shaker (130 rev min−1). The compo-

ition of the medium was (g l−1): yeast extract, 0.50; dextrose,0.0; TCP, 5.0; (NH4)2SO4, 0.50; KCl, 0.20; Mg2SO4·7H2O, 0.10;n2SO4·H2O, 0.0001; Fe2SO4·7H2O, 0.0001, pH adjusted to 7.0.

he 5.0 ml culture was taken out at regular intervals of 24 h, fordays, centrifuged (Sigma 103456 cooling centrifuge, Germany at0,000 × g for 10 min) and soluble-P content of culture supernatantas estimated by colorimetric chlorostannous reduced molybdo-hosphoric acid blue method (Jackson, 1973). The final valuesere calculated with the help of a standard curve obtained using

–2 mg l−1 KH2PO4.

.2.2. Biocompatibility assayIn order to prepare a consortium of PSB, it is essential that all

icrobes present in a consortium should be biocompatible. Bio-ompatibility among PSB isolates was determined by streaking oneSB isolate across the middle of the PVK agar plate. Other isolatesere streaked perpendicular to the above isolate and incubated

30 ◦C, 48 h). Zone of inhibition of growth at the junction of culturesas noted (Mittal et al., 2008).

.2.3. Quantitative estimation of indole acetic acid (IAA)IAA was assayed by the colorimetric method using ferric

hloride–perchloric acid reagent (FeCl3–HClO4) (Gordon and Paleg,957). PSB were inoculated in the minimal medium (g l−1):H2PO4, 1.36; Na2HPO4, 2.13; MgSO4·7H2O, 0.2, pH 7.0, amendedith 5.0 mM l-tryptophan solution (g 100 ml−1: glucose, 10; l-

ryptophan, 1.0; yeast extract 0.1; filtered through sterile 0.2 �millipore membrane filter) (Frankenberger and Poth, 1988). Flasksere incubated at 30 ◦C on a rotary shaker (130 rev min−1). Culturesere withdrawn after 48 h intervals and centrifuged (10,000 × g

or 10 min). The 2.0 ml of Salper’s reagent was added dropwise to.0 ml of culture supernatant, and samples were incubated in theark for 30 min. Development of pink color was assayed with apectrophotometer at 530 nm. The concentration of IAA in �g ml−1

as determined from a standard curve of IAA (0–10 �g ml−1).

.2.4. Quantitative estimation of siderophore productionPSB were grown in an iron deficient medium containing (g l−1):

2HPO4, 0.1; KH2PO4, 3.0; MgSO4·7H2O, 0.2; (NH4)2SO4, 1.0; suc-inic acid, 4.0 (Bharbaya and Rao, 1985) at 30 ◦C on a rotary

haker at 130 rev min−1 for 48 h. The 0.5 ml of cell free supernatantas mixed with 0.5 ml of Chrome Azurol Sulfonate (CAS) assay

olution (1.5 ml of 1 mM Fe stock solution + 7.5 ml of 2 mM CAStock solution added to 0.003 mM hexadecyltrimethylammoniumHDTMA) + 30 ml of 1.5 mM Piperazine buffer) along with 10 �l of

logy 46 (2010) 222–229 223

shuttle solution (0.2 M 5-sulfosalicylic acid) (Schwyn and Neilands,1987). Absorbance was read at 630 nm for the loss of blue color. Theactivity was recorded in percentage siderophore units calculated as[((Ar − As) × Ar−1) × 100], where ‘Ar’ is defined as absorbance of ref-erence (un-inoculated media + CAS solution) and ‘As’ is absorbanceof test (culture supernatant + CAS solution).

2.3. Physiological characterization of PSB

PSB isolates were characterized based on colony morphology,Gram staining and biochemical testing: catalase (Graham andParker, 1964), oxidase (Kovaks, 1956) and lactose fermentation(Ronald and James, 2006). The guanosine + cytosine content (mol%G + C) of the genomic DNA was determined by thermal denaturationmethod (Marmur and Doty, 1962).

2.4. Molecular identification of PSB

The genomic DNA of PSB isolates were extracted usingQiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA). Theprimers fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (3′-ACGGCTACCTTGTTACGACTT-5′) were used for amplification of 16SrRNA gene (Weisburg et al., 1991). The total PCR reaction mix-ture was 50.0 �l, comprising 200 �M dNTPs, 50 �M each primer,1× PCR buffer, 3 U Taq polymerase, and 100 ng genomic DNA. Thethermocycler conditions involved an initial denaturation at 94 ◦Cfor 4 min, followed by 35 cycles of 94 ◦C for 1 min, 52 ◦C for 1 min,and 72 ◦C for 2 min and final extension at 72 ◦C for 8 min. The 16SrRNA gene was purified from the gel and was ligated to pGEM-Teasy vector (Promega, Madison) and transformed in E. coli JM109.The sequences of the insert were determined using a Big-Dye Ter-minator Cycle Sequencer and an ABI Prism 310 Genetic Analyzer(Applied Biosystems, CA). The RNA gene sequences were analyzedusing the gapped BLASTn (http://www.ncbi.nlm.nih.gov) searchalgorithm and aligned to their nearest neighbors. The evolutionarydistances among phosphate-solubilizing isolates and their relatedtaxa were calculated using TREECON software and Kimura’s two-parameter model, after aligning the sequences with ClustalW.

2.5. Site of pot experiments

Experiments were conducted in a greenhouse (uncontrolledconditions) during March–June, 2008, in the Department of Botany,Panjab University, Chandigarh, India.

2.5.1. Preparation of inoculumEach PSB was grown separately in the nutrient broth at 30 ◦C in

an orbital shaker (150 rev min−1) for 24 h. The cultures were cen-trifuged in 50 ml sterile plastic tubes at 6000 × g for 15 min. Thepellets were re-suspended in PBS and optical density (OD) wasadjusted to have a final concentration of colony forming units, i.e.108 CFU ml−1. This liquid culture of each PSB was used for the indi-vidual inoculation in pot experiments. For making a PSB consortiuminoculum, all individual cultures of PSB of equal cell density, i.e.108 CFU ml−1 were mixed together into a 250 ml sterilized flask.This mixture was used as a consortium inoculum.

2.5.2. Soil conditions and sowing of plantletsUnsterile loamy soil (pH, 7.8; available N, 46.9 mg kg−1;

available P, 5.0 mg kg−1; available K, 14.9 mg kg−1; total Ca,6.0 mequiv. kg−1; total Mg, 1.2 mequiv. kg−1; total organic carbon,

0.10%) was thoroughly mixed and passed through 2 mm sieve toremove large particulate matter and kept under sunlight for 7days. Ethanol disinfected plastic pots (10.2 cm × 25.4 cm), werefilled with 3.5 kg of soil. Roots of tissue culture plantlets weresurface-sterilized by dipping in 2% NaOCl solution for 10 min and

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hen washed thrice with distilled water. The surface-sterilizedlantlets roots were dipped in the desired culture inoculum≈108 CFU ml−1, as mentioned in Section 2.5.1) for 15 min and thenllowed to dry in the shade for 30 min. After drying, plantlets werelanted in six different sets of treatment with three replicationsf each. These sets included: (1) soil + TCP (200 mg kg−1 soil); (2)oil + TCP + B. gladioli 10216; (3) soil + TCP + B. gladioli 10217; (4)oil + TCP + S. marcescens 10238; (5) soil + TCP + E. aerogenes 10208;6) soil + TCP + consortium of all isolates. Experiments were per-ormed in completely randomized block design.

.6. Analysis of available P content in soil and P uptake in plants

After the harvesting of plants, the P content of soil and driedlant parts were analyzed. Available P content in soil was deter-ined by colorimetric sodium bicarbonate-extractable P method

Olsen et al., 1954). P uptake in plant parts was determined usinganado–molybdo-phosphoric yellow color method (Koeing andohnson, 1942).

.7. HPLC analysis of ST and R-A content in the leaves of S.ebaudiana

The S. rebaudiana leaves were dried in an oven at 40 ◦C for 5 days.he 50 mg of completely dried and powdered samples were taken in5 ml conical flasks. Samples were extracted with 10 ml methanolor 24 h and then filtered (Whatman filter paper of size 102 �l, Sar-orius, grade 389) in 50 ml distillation flask. The extraction processas repeated again for the residue left. The filtrate was distilled

n a rotary evaporator (at 45 ◦C, 50 rev min−1 and 70 mbar vac-um pressure), until the whole methanol get evaporated. Residueas defatted two times with 20 ml hexane and air dried prop-

rly (Megeji et al., 2005). The 5.0 ml of acetonitrile:water (80:20)as added into the residue, mixed properly and filtered (0.45 �Millipore membrane filter) into HPLC vials. Samples in vials were

ut into an auto-sampler (WATER’S 717 plus) and analysis wasone by using WATER’S HPLC machine having EMPOWER software,ATER’S 600 controller, WATER’STM 600 pump, Lichrocart® 250-4

H2 column (5 �M), wavelength of 210 nm, flow rate of 1 ml min−1,ressure 800 psi, and photodiode array detector. As a mobile phasee used a mixture of acetonitrile and water (Isocratic, 50:50). ST

nd R-A of Sigma Chemicals were used as standards.

.8. Estimation of root colonization by PSB

At the end of the pot experiments plants were harvested andnalyzed for rhizosphere colonization of PSB based on their ran-om amplification of polymorphic DNA (RAPD) banding pattern

able 1elease of soluble-P (�g ml−1) by bacterial isolates for 7 days in TCP supplemented liquid

Isolate no. Soluble phosphorus (�g ml−1)

0 h 1 day 2 day 3 day

S1 0 90.0 ± 2.0 128 ± 2.0 228 ± 2S3 0 45.3 ± 1.2 79.3 ± 3.1 89.3 ± 3S6 0 24.7 ± 3.0 54.0 ± 3.5 109 ± 1S7 0 34.7 ± 4.2 50.7 ± 5.0 86.0 ± 4S8 0 23.3 ± 1.1 38.7 ± 3.1 81.3 ± 3S9 0 102 ± 4.0 76.0 ± 4.0 6.0 ± 2.S11 0 0 73.3 ± 2.3 162 ± 3S12 0 25.3 ± 1.1 71.3 ± 3.1 97.3 ±S14 0 128 ± 2.0 188 ± 2.0 316 ± 4S15 0 0 0 52.0 ± 2S17 0 64.0 ± 4.0 105 ± 3.1 94.7 ± 4S18 0 70.0 ± 2.0 102 ± 6.0 148 ± 2

esults are mean of three replicates, ± indicate standard deviation, values in bold letters

logy 46 (2010) 222–229

analysis. The rhizosphere soil adhering to the roots of harvestedplants was separated by gentle tapping and stored in sterilizedpetri plates at 4 ◦C. One gram soil of each replicate soil sampleswas serially diluted (1:10), and spread on PVK agar medium. Theplates were incubated at 30 ◦C for 5 days. The isolates showingP-solubilization were marked and counted (number of isolatesshowing different morphologies and number of isolates show-ing similar morphology). Isolated PSB were purified by repeatedstreaking of culture on PVK agar plate. The genomic DNA of PSBwas isolated using the protocol suggested by Himedia (MB505HiPurATM Bacterial and Yeast Genomic DNA Purification Spin Kit).The PSB isolates of different morphologies were analyzed for DNAbanding pattern using primers OPA-04 (5′-AATCGGGCTG-3′) andBOX A1 (5′-CTACGGCAAGGCGACGCTGACG-3′). The similarity ofband patterns was quantified by simple matching (Apostol et al.,1993). PSB isolates having similar banding pattern to inoculatedPSB confirmed their survival in the rhizosphere.

2.9. Statistical analysis

Pot experiments were arranged in completely randomized blockdesign. Statistical analysis was conducted using one-way analysisof variance (ANOVA) statistical package for social sciences (SPSS)software, Version 30. Comparisons of means were performed bythe LSD test at P ≤ 0.05.

3. Results

3.1. Isolation and screening of PSB

A total of 12 PSB, showing P-solubilizing zone greater than 5 mmon the PVK agar medium were isolated from the rhizosphere of Ste-via plants. In liquid PVK medium, The P-solubilizing ability of theseorganisms varied from 97.3 to 316 �g ml−1 and grouped as highP-solubilizers (>200 �g ml−1; S1 and S14), average P-solubilizers(100–200 �g ml−1; S3, S6, S7, S8, S9, S11, S15, S17 and S18) andlow P-solubilizers (<100 �g ml−1; S12) (Table 1).

In order to find antimicrobial substances, all the P-solubilizerswere subjected to biocompatibility assay. Based on the P-solubilizing activity and biocompatibility test, four PSB (S1, S9, S14,S18) were selected for further study. All four isolates were biocom-patible and good P-solubilizers.

The selected bacterial isolates were further tested for

siderophore and IAA production (Fig. 1). Isolates, S1 and S9 pro-duced siderophore (91.3% and 94.8%, respectively) as well asIAA (15.3 �g ml−1 and 1.5 �g ml−1, respectively). Isolate S14 pro-duced only siderophore (80.4%), whereas, S18 produced only IAA(40.8 �g ml−1).

medium.

4 day 5 day 6 day 7 day

.0 236 ± 4.0 252 ± 4.0 182 ± 2.0 158 ± 2.0.1 125 ± 5.0 56.0 ± 5.3 1.33 ± 2.3 0.1 191 ± 4.2 143 ± 3.1 64.7 ± 3.1 63.3 ± 5.0.0 142 ± 4.0 185 ± 4.2 151 ± 5.0 92.7 ± 5.0.1 132 ± 4.0 185 ± 3.1 124 ± 3.5 92.7 ± 3.1

0 2.67 ± 3.2 1.33 ± 2.3 0.67 ± 1.1 0.5 171 ± 4.2 101 ± 3.1 80.7 ± 3.1 70.7 ± 5.0

3.1 46.7 ± 3.1 37.3 ± 3.1 19.3 ± 3.1 2.67 ± 4.6.0 304 ± 4.0 236 ± 4.0 232 ± 2.0 230 ± 4.0.0 62.7 ± 3.1 81.3 ± 1.1 123 ± 3.1 103 ± 6.4.2 86.0 ± 2.0 58.7 ± 3.1 39.3 ± 5.0 25.3 ± 4.2.0 158 ± 2.0 200 ± 2.0 128 ± 2.0 150 ± 4.0

represent the maximum P-solubilization by respective bacterial isolates.

Mamta et al. / Applied Soil Eco

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observed in some of the inoculated plants. An increase in the ST

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ig. 1. Indole acetic acid (A) and siderophore (B) produced by PSB isolates. Errorars represent standard deviation.

.2. Identification of PSB

The isolates were examined for their colony morphology onutrient agar after 24 h of incubation (Table 2). All were circular,aised and smooth in appearance. The colonies of three isolatesS1, S9 and S14) were off white, whereas that of S18 was yellowishrown in appearance. The colony size of the isolates was found to be.5 mm, 1.5 mm, 2.0 mm and 2.0 mm for S9, S18, S1 and S14, respec-ively. The microscopic identification showed that all were Gramegative, small to medium sized rods. All PSB isolates were cata-

ase positive and oxidase negative. The isolates S14 and S18 wereactose fermenters while S1 and S9 were non-lactose fermenters.

he % G + C content of the PSB isolates was 67.5% (S1), 65.1% (S9),8.3% (S14) and 53.5% (S18), respectively (Table 2).

The results of BLAST search of 16S rRNA gene sequences of theour selected PSB on comparison with the available 16S rRNA gene

able 2haracterization of PSB isolates.

PSBisolate

Catalaseproduction

Oxidaseproduction

Lactosefermentation

aColony size and morphol

S1 +ve −ve NLF 2.0 mm, cream color, circuS9 +ve −ve NLF 0.5 mm, cream color, circuS14 +ve −ve LF 2.0 mm, cream color, circuS18 +ve −ve LF 1.5 mm, yellowish brown,

LF: non-lactose fermenter, LF: lactose fermenter.a The colonies were grown on nutrient agar for 24 h at 30 ◦C.

logy 46 (2010) 222–229 225

sequences in the GenBank database indicated Burkholderia gladi-oli as the closest related species to the isolates S1 and S9 whileother two isolates S14 and S18 were closely related to Serratiamarcescens and Enterobacter aerogenes, respectively. The phyloge-netic tree (Fig. 2) based on 16S rRNA gene sequences of the isolatesand representative species of closely related taxa, formed threeclearly distinguishable groups. The Burkholderia group consisted ofisolate S1 and S9 along with B. gladioli CIP105410, B. gladioli R406,B. gladioli ATCC 33664, B. vietnamiensis LMG 10929, B. cepacia ATCC53130 and Pseudomonas antimicrobica NCIMB 9898. The Serratiagroup included isolate S14, S. marcescens DSM 30121, S. marcescensstrain A3 and S. marcescens J2P3. The Enterobacter group consistedof isolate S18, E. aerogenes JCM 1235, E. dissolvens LMG 2683, E. lud-wigii EN119, E. cancerogenus LMG 2693, E. cowanii CIP 107300 andPantoea agglomerans HK14-1.

3.3. Effect of PSB on plant growth

The effect of PSB (individually and in consortium) on thegrowth of S. rebaudiana was observed in TCP (200 mg kg−1 of soil)amended soil. All four PSB showed stimulatory effect, in termsof shoot length, root length, leaf dry weight, stem dry weightand total shoot biomass when inoculated separately (Table 3).The maximal increase for majority of the parameters (root length,leaf dry weight, stem dry weight and total shoot biomass) wasshown by B. gladioli 10216 plants in comparison to un-inoculated(i.e. control) plants. The maximum increase (23.8%) in shootlength was shown by plants treated with E. aerogenes 10208.The individual treatments did not differ significantly with eachother.

The stimulatory effect of PSB was found to be more pronouncedwhen inoculated as a consortium. Compared to control plant, anincrease of 14.7%, 22.5%, 76.3%, 82.5% and 89.0% was observed inthe root length, shoot length, stem dry weight, total biomass andleaf dry weight, respectively.

3.4. Effect of PSB on the yield of stevioside and rebaudioside-Acontent

The effect of PSB on ST and R-A contents both on per gram dryleaf weight as well as per plant dry leaf weight of S. rebaudianawas observed. Based on statistic analysis, no significant change wasobserved in the ST content in the leaves of inoculated as well asun-inoculated plants (Table 4). However, a statistically significantincrease in R-A content of leaves was found in plants inoculatedwith PSB consortium (247%) (Fig. 3), S. marcescens 10238 (250%)and B. gladioli 10216 (221%). Whereas, a statistically insignificantchange was observed in plants inoculated with E. aerogenes 10208or B. gladioli 10217. When the total yield of ST and R-A plant−1

was calculated, a profound statistically significant increase was

content of 150% and 91.0% was seen in plants inoculated with con-sortium and B. gladioli 10216, respectively. Similarly, an increase inR-A content of 555%, 466% and 447% was observed in plants treatedwith the consortium, B. gladioli 10216 and S. marcescens 10238. In

ogy Gram staining and microscopicexamination

% G + Ccontent

lar, raised, smooth margins Gram negative, small rods 67.5%lar, raised, smooth margins Gram negative, small rods 65.1%lar, raised, smooth margins Gram negative, medium size rods 58.3%circular, raised, smooth margins Gram negative, small rods 53.5%

226 Mamta et al. / Applied Soil Ecology 46 (2010) 222–229

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est of the treatments, a non-significant change was observed in STnd R-A contents (Table 4).

.5. Phosphorus availability in the soil and its uptake by thelants

The available P content of soil after harvesting of plants wastudied. A significant increase was found in available P content ofoils treated with PSB in comparison to control soils (Table 5). Soilsreated with B. gladioli 10216 and consortium of all PSB showed

able 3ffect of phosphate-solubilizing bacteria on the growth of Stevia rebaudiana.

Treatment Shoot length (cm) Root length (cm)

S + TCP (control) 65.0 ± 3.9 10.2 ± 0.2S + TCP + B. gladioli (MTCC 10216) 78.0 ± 1.3 (a) a(20.0) 11.5 ± 0.3 (a) (12.7)S + TCP + B. gladioli (MTCC 10217) 80.4 ± 3.6 (a) (23.6) 11.4 ± 0.3 (a) (11.8)S + TCP + E. aerogenes (MTCC 10208) 80.5 ± 4.4 (a) (23.8) 11.5 ± 0.3 (a) (12.5)S + TCP + S. marcescens (MTCC 10238) 79.8 ± 4.2 (a) (22.8) 11.4 ± 0.1 (a) (11.5)S + TCP + consortium 79.7 ± 2.7 (a) (22.5) 11.7 ± 0.2 (a) (14.7)

alues are mean of three replications, mean values (mean ± S.D.) sharing the same letterhosphate.a Values in parenthesis represents percentage increase over control.

able 4ffect of phosphate-solubilizing bacteria on stevioside and rebaudioside-A contents of Ste

Treatment ST (mg g−1 leaves) R-A (mg g

S + TCP (control) 35.0 ± 2.6 (a, b) 5.67 ± 2.S + TCP + B. gladioli (MTCC 10216) 38.0 ± 5.2 (a. b) 18.2 ± 3.S + TCP + B. gladioli (MTCC 10217) 34.8 ± 0.7 (a, b) 3.67 ± 1.S + TCP + E. aerogenes (MTCC 10208) 26.2 ± 5.4 (b) 15.1 ± 2.S + TCP + S. marcescens (MTCC 10238) 30.2 ± 4.5 (a, b) 19.8 ± 3.S + TCP + consortium 46.2 ± 2.2 (a) 19.7 ± 2.

alues are mean of three replications, mean values (mean ± S.D.) sharing the same letterhosphate.a Values in parenthesis represents percentage increase over control.

among selected PSB isolates (shown in bold letters) and representatives of othern within brackets. Bar = 0.02 substitutions per site. The number at the node, indicate

maximal value which was ≈330% more than the control value.Other individual inoculation with PSB isolates showed 213–268%increase in P content.

The uptake of P in Stevia plants was also influenced by PSB inoc-ulation (Table 5). The individual inoculation with PSB showed an

increase in P content of leaves (59.2–102%) in comparison to con-trol. Treatment with PSB consortium showed more pronouncedincrease (165%) than individual PSB treatments. In the case of stemP uptake, an increase of (104–150%) was observed by PSB inoc-ulations in comparison to control plants, though no significant

Leaf dry weight (g) Stem dry weight (g) Total shoot biomass (g)

0.46 ± 0.4 0.48 ± 0.1 0.94 ± 0.40.80 ± 0.1(a, c) (74.5) 0.80 ± 0.4 (a) (66.9) 1.61 ± 0.1 (a, b) (70.7)0.66 ± 0.1 (b) (44.1) 0.78 ± 0.2 (a) (62.7) 1.44 ± 0.1 (a) (53.6)0.71 ± 0.1 (a b) (54.4) 0.79 ± 0.1(a) (65.6) 1.51 ± 0.1 (a) (60.1)0.71 ± 0.1 (a, b) (55.1) 0.80 ± 0.2 (a, b) (67.9) 1.52 ± 0.1 (a) (61.7)0.87 ± 0.3 (89.0) 0.84 ± 0.2 (76.3) 1.72 ± 0.1 (b) (82.5)

do not differ significantly by LSD at P ≤ 0.05, S: un-inoculated soil, TCP: tricalcium

via rebaudiana.

−1 leaves) ST (mg plant−1) R-A (mg plant−1)

1 (a) 16.2 ± 0.7 (a) 2.61 ± 0.9 (a)6 (b) a(221) 30.9 ± 2.9 (b, c) (91.0) 14.8 ± 2.4 (b) (466)1 (a) 23.2 ± 1.0 (a, b) 2.42 ± 0.6 (a)5 (a, b) 18.7 ± 3.6 (a, b) 10.8 ± 1.6 (a, b)8 (b) (250) 21.7 ± 3.0 (a, b) 14.3 ± 3.2 (b) (447)3 (b) (247) 40.5 ± 2.6 (c) (150) 17.1 ± 3.1 (b) (555)

do not differ significantly by LSD at P ≤ 0.05, S: un-inoculated soil, TCP: tricalcium

Mamta et al. / Applied Soil Ecology 46 (2010) 222–229 227

Fig. 3. Chromatogram showing stevioside and rebaudioside-A content of control plants (A) and plants inoculated with PSB consortia (B).

Table 5Effect of phosphate-solubilizing bacteria on available P content in soil and P uptake in Stevia rebaudiana.

Treatment Available P content in soil (mg kg−1) P uptake in leaves (mg plant−1) P uptake in stem (mg plant−1)

S + TCP (control) 1.78 ± 0.1 1.65 ± 0.2 0.89 ± 0.1S + TCP + B. gladioli (MTCC 10216) 7.69 ± 0.2 (a) a(331) 3.33 ± 0.1 (a) (102) 2.11 ± 0.4 (a) (137)S + TCP + B. gladioli (MTCC 10217) 5.58 ± 0.1 (213) 2.62 ± 0.3 (a) (59.2) 1.82 ± 0.1 (a) (104)S + TCP + E. aerogenes (MTCC 10208) 6.57 ± 0.2 (b) (268) 2.86 ± 0.3 (a) (73.4) 1.95 ± 0.2 (a) (119)S + TCP + S. marcescens (MTCC 10238) 6.57 ± 0.2 (b) (268) 2.86 ± 0.3 (a) (73.8) 2.03 ± 0.3 (a) (128)S + TCP + consortium 7.67 ± 0.4 (a) (330) 4.37 ± 0.1 (165) 2.22 ± 0.3 (a) (150)

V letterpa

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alues are mean of three replications, mean values (mean ± S.D.) sharing the samehosphate.Value in parenthesis represents percentage increase over control.

ifference was observed between individually inoculated and con-ortium inoculated plants.

.6. Soil analysis for inoculated PSB isolates by RAPD technique

The soil clinging tightly to the roots of the harvested Stevia plantsas examined for the colonization of PSB by analyzing DNA bandingattern (Fig. 4) of the P-solubilizing colonies obtained after plat-

ng the soil samples. The CFU were counted on dilution 10−6 and0−5. In individual PSB treatment studies, maximal colonizationas observed with B. gladioli 10216 (4.5 × 109 CFU g−1 soil at 10−6

ilution) followed by S. marcescens 10238 (4.0 × 109 CFU g−1 soil

t 10−6 dilution), B. gladioli 10217 (6.6 × 108 CFU g−1 soil at 10−5

ilution) and E. aerogenes 10208 (4.0 × 108 CFU g−1 soil at 10−5 dilu-ion). In consortium treatment, B. gladioli 10216 (2.0 × 108 CFU g−1

oil at 10−5 dilution) and S. marcescens 10238 (1.5 × 108 CFU g−1 soilt 10−5 dilution) colonization was observed. However, other inoc-

do not differ significantly by LSD at P ≤ 0.05, S: un-inoculated soil, TCP: tricalcium

ulated PSB could not be detected at this dilution (10−5), but theirpresence at lower dilutions cannot be discounted, as it was impos-sible to count the number of colonies at lower dilutions becauseindividual bacterial colonies could not be clearly seen.

4. Discussion

In the present study, four PSB isolated from the rhizosphericsoil of S. rebaudiana were characterized as B. gladioli 10216, B. glad-ioli 10217, E. aerogenes 10208 and S. marcescens 10238. This is thefirst report on the use of these microbes as P-solubilizers for Stevia,although microbes belonging to these genera have been reported as

biofertilizers for other plants also, e.g. B. gladioli for Malus domestica(Karakurt and Aslantas, 2010) and Mentha piperita (Kaymak et al.,2008); E. aerogenes for Zea mays (Nadeem et al., 2007) and Brassicajuncea (Kumar et al., 2009) and S. marcescens for Zea mays (Hameedaet al., 2008) and Cucurbita pepo (Selvakumar et al., 2008).

228 Mamta et al. / Applied Soil Ecology 46 (2010) 222–229

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ig. 4. Genotypic profiling of PSB isolates using primer OPA-04 (A) and BOX A1 (B). L0208; 5, Serratia marcescens 10238; 3, DNA standard (100–10,000 bp).

Although all PSB used in this study were good solubilizers, it isesirable that P-solubilizers have additional plant growth promot-

ng properties like IAA and siderophore production (Torres-Rubiot al., 2000). Out of four PSB, two isolates, i.e. B. gladioli 10216 and. gladioli 10217 produced IAA as well as siderophore whereas iso-

ates E. aerogenes 10208 and S. marcescens 10238 produced only IAAnd siderophore, respectively.

The present study found a significant increase in the biometricarameters of Stevia plants (total biomass, shoot length, root length,

eaf dry weight plant−1, and stem dry weight plant−1) after treat-ent with PSB. The effect was more pronounced with consortium

reatment as compared to individual PSB treatments. The vari-ble degree of stimulatory effect among individual or consortiumSB treatments on plant growth may be due to diverse interac-ions of inoculated PSB with plant roots or with native microflora,hich often results in the promotion of key processes benefitinglant growth and health (Braeken et al., 2008; Barea et al., 2005).he significantly lesser plant growth in control plants than PSBnoculated plants indicated that the native PSB did not contributeirectly towards the plant growth thorough P-solubilization. Sim-

lar observations related to growth promotion of Stevia plants bySB inoculations have been made by Earanna (2007) and Das et al.2008), though PSB used were different from the ones in the presenttudy.

Stevia plants have gained importance as sweeteners because ofheir ST and R-A contents. Until now there is no report on the effectf P-solubilizers on ST and R-A levels of Stevia. We are reportingor the first time a significant increase in ST and R-A contents ofSB treated plants as compared to control plants. Of all the PSBreatments, the maximum increase was observed in PSB consor-ium treated plants of 2.49-fold in ST content and 6.55-fold in R-Aontent on per plant basis. Among individual PSB treatments, aixed response was observed. The treatment with B. gladioli 10216

howed a significant increase in ST and R-A contents, whereas,

reatment with S. marcescens showed a significant increase in R-Aontent only.

The increase in ST content was primarily due to increase in theumber of leaves (plant−1) as not much difference was observed

n ST content of leaves based on per gram dry leaf weight between

, Burkholderia gladioli 10216; 2, Burkholderia gladioli 10217; 4, Enterobacter aerogens

PSB treated and untreated plants. However, increase in R-A contentcould be attributed to increase in (a) number of leaves per plant and(b) its biosynthesis in plants treated with either consortium or B.gladioli 10216 or S. marcescens 10238.

Rhizosphere colonization by microbial inoculants has beendescribed as a crucial factor for plant growth promotion (De Wegeret al., 1995; Lugtenberg et al., 2001). Our results are also in agree-ment with this statement. The degree of stimulatory effect of PSBon the plant growth, ST and R-A contents could be correlated withthe extent of colonization of the roots by PSB with B. gladioli 10216showing maximal colonization and maximal enhanced effect onplant growth parameters, ST and R-A contents.

The inoculation of PSB also resulted in the increased amount ofavailable P in soil. These results suggested that a subsequent cropwill reap the benefits imparted by PSB to the soil in terms of avail-able P content, physical and biological characteristics of soil (Mittalet al., 2008). The maximum amount of available P was observed insoil treated with B. gladioli 10216 showing maximum root coloniza-tion ability. These results showed the important role of inoculatedmicroorganisms for immobilizing relatively high amounts of P intheir biomass (Demetz and Insam, 1999).

The PSB inoculation of S. rebaudiana showed stimulatory effecton P uptake of plant. This might be due to better utilization of P fromthe pool of soil nutrients by the action of PSB. Maximal increasein P uptake was shown by Stevia plants treated with PSB consor-tium. In case of individual PSB treatments, maximal increase in Pcontent was shown by B. gladioli 10216 treated plants. The high-est amount of P uptake in leaves as well as in stems was observedin plants showing the highest amount of available P. These find-ings suggested that the microbially available P corresponds to plantavailable P through mineralization of complex P compounds (Yangand Jacobsen, 1990; Demetz and Insam, 1999). The increase in Puptake in some other plants like Phaseolus vulgaris by inoculat-ing Burkholderia sp. (Peix et al., 2001); in Soybean by inoculating

Serratia sp. (Han and Lee, 2005) and in Lycopersicon esculentum byinoculating Enterobacter sp. (Kirankumar et al., 2008) supports ourfindings.

Taken together, the results suggested that the treatment ofS. rebaudiana with suitable PSB has significant influence on the

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rowth and high value metabolites (ST and R-A) in pot experimentsarried out in greenhouse.

. Conclusions

The use of PSB as biofertilizers is an efficient approach to replacehemical phosphorus fertilizers for sustainable cultivation of S.ebaudiana. The inoculation of PSB significantly increased the plantrowth (shoot length, root length, leaf dry weight, stem dry weightnd biomass), available P content in soil as well as its uptake andlso the yield of commercially important ST (mg plant−1) and R-Aontents (mg plant−1 as well as mg g−1).

cknowledgements

The authors are grateful to Union Grant Commission, New Delhi,ndia for financial support and Haryali Biotech, Zirakpur (Punjab)or soil sample collection and providing tissue culture plantlets.

e are also thankful to Dr. Mohinder Kaur (Dr. Y.S. Parmar Uni-ersity of Horticulture and Forestry, Nauni, H.P., India), Dr. Vaniittal and Mr. Onkar Bal for their useful suggestions and Mr.avtej Singh (Micrographer, SAIF, Panjab University, Chandigarh)

or timely help.

eferences

ltomare, C., Norvell, W.A., Bjorkman, T., Harman, G.E., 1999. Solubilization of phos-phates and micronutrients by plant-growth promoting and biocontrol fungusTrichoderma harzianum Rifai. Appl. Environ. Microbiol. 65, 2926–2933.

postol, B.L., Black, W.C., Miller, B.R., Reiter, P., Beaty, B.J., 1993. Estimation of thenumber of full sibling families at an oviposition site using RAPD-PCR markers:applications to the mosquito Aedea aegypti. Theor. Appl. Genet. 86, 991–1000.

area, J.-M., Pozo, M.J., Azcón, R., Azcón-Aguilar, C., 2005. Microbial co-operation inthe rhizosphere. J. Exp. Bot. 56, 1761–1778.

harbaya, H.B., Rao, K.K., 1985. Production of pyoverdin, the fluorescent pigmentsof Pseudomonas aeruginosa PAO1. FEMS Microbiol. Lett. 27, 233–235.

raeken, K., Daniels, R., Ndayizeye, M., Vanderleyden, J., Michiels, J., 2008. Quorumsensing in bacteria–plant interactions. In: Nautiyal, C.S., Dion, P. (Eds.), Molecu-lar Mechanisms of Plant 265 and Microbe Coexistence. Springer–Verlag, Berlin,Heidelberg, Germany, pp. 265–289.

as, K., Dang, R., Shivananda, T.N., 2008. Influence of bio-fertilizers on the availabilityof nutrients (N, P and K) in soil in relation to growth and yield of Stevia rebaudianagrown in South India. Int. J. Appl. Res. Nat. Prod. 1, 20–24.

e Weger, L.A., Van Der Bij, A.J., Dekkers, L.C., Simons, M., Wijffelman, C.A.,Lugtenberg, B.J.J., 1995. Colonization of the rhizosphere of crop plants by plant-beneficial Pseudomonads. FEMS Microbiol. Ecol. 17, 221–228.

el Campillo, S.E., Van der Zee, S., Torrent, J., 1999. Modelling long term phosphorusleaching and changes in phosphorus fertility in excessively fertilized acid sandysoils. Eur. J. Soil. Sci. 50, 391–399.

emetz, M., Insam, H., 1999. Phosphorus availability in a forest soil determined witha respiratory assay compared to chemical methods. Geoderma 89, 259–271.

aranna, N., 2007. Response of Stevia rebaudiana to biofertilizers. Karnataka J. Agric.Sci. 20, 616–617.

rankenberger Jr., W.T., Poth, M., 1988. l-Tryptophan transaminase of a bac-terium isolated from the rhizosphere of Festuca octoflora (Gramineae). Soil Biol.Biochem. 20, 299–304.

ordon, S.A., Paleg, L.G., 1957. Observations on the quantitative determination of

indole acetic acid. Plant Physiol. 10, 39–47.

raham, P.H., Parker, C.A., 1964. Diagnostic features in characterization of the rootnodule bacteria of the legumes. Plant Soil 20, 383–396.

ameeda, B., Harini, G., Rupela, O.P., Wani, S.P., Reddy, G., 2008. Growth promo-tion of maize by phosphate-solubilizing bacteria isolated from composts andmacrofauna. Microbiol. Res. 163, 234–242.

logy 46 (2010) 222–229 229

Han, H.S., Lee, K.D., 2005. Physiological responses of soybean-inoculation ofBradyrhizobium japonicum with PGPR in saline soil conditions. Res. J. Agric. Biol.Sci. 1, 216–221.

Jackson, M.L., 1973. Methods of Chemical Analysis. Prentice Hall of India (Pvt.) Ltd.,New Delhi.

Jaleel, C.A., Manivannan, P., Sankar, B., Kishorekumar, A., Gopi, R., Somasundaram, R.,Panneerselvam, R., 2007. Pseudomonas fluorescens enhances biomass yield andajmalicine production in Catharanthus roseus under water deficit stress. ColloidSurf. B-Biointerfaces 60, 7–11.

Karakurt, H., Aslantas, R., 2010. Effects of some plant growth promoting rhizobacte-ria treated twice on flower thinning, fruit set and fruit properties on apple. Afr.J. Agric. Res. 5, 384–388.

Kaymak, H.C., Yarali, F., Guvenc, I., Donmez, M.F., 2008. The effect of inoculation withplant growth rhizobacteria (PGPR) on root formation of mint (Mentha piperitaL.) cuttings. Afr. J. Biotechnol. 7, 4479–4483.

Kirankumar, R., Jagadeesh, K.S., Krishnaraj, P.U., Patil, M.S., 2008. Enhanced growthpromotion of tomato and nutrient uptake by plant growth promoting rhizobac-terial isolates in presence of tobacco mosaic virus pathogen. Karnataka J. Agric.Sci. 21, 309–311.

Koeing, R.A., Johnson, C.R., 1942. Colorimetric determination of phosphorus in bio-logical materials. Ind. Eng. Chem. 14, 155–156.

Koo, S.-Y., Cho, K.-S., 2009. Isolation and characterization of a plant growth promot-ing rhizobacterium Serratia sp. SY5. J. Microbiol. Biotechnol. 19, 1431–1438.

Kovaks, N., 1956. Identification of Pseudomonas pyocyanea by the oxidation reaction.Nature 178, 703.

Kumar, K.V., Srivastava, S., Singh, N., Behl, H.M., 2009. Role of metal resistant plantgrowth promoting bacteria in ameliorating fly ash to the growth of Brassicajuncea. J. Hazard. Mater. 170, 51–57.

Leithy, S., El-Meseiry, T.A., Abdallah, E.F., 2006. Effect of biofertilizer, cell stabilizerand irrigation regime on Rosemary herbage oil yield and quality. J. Appl. Sci. Res.2, 773–779.

Lugtenberg, B.J.J., Dekkers, L., Bloemberg, G.V., 2001. Molecular determinants ofrhizosphere colonization by Pseudomonas. Annu. Rev. Phytopathol. 39, 461–490.

Marmur, J., Doty, P., 1962. Determination of the base composition of deoxyribonu-cleic acids from its thermal denaturation temperature. J. Mol. Biol. 5, 109–118.

Megeji, N.W., Kumar, J.K., Singh, V., Kaul, V.K., Ahuja, P.S., 2005. Introducing Steviarebaudiana, a natural zero-calorie sweetener. Curr. Sci. 88, 801–804.

Mittal, V., Singh, O., Nayyar, H., Kaur, J., Tewari, R., 2008. Stimulatory effect ofphosphate-solubilizing fungal strains (Aspergillus awamori and Penicillium cit-rinum) on the yield of chickpea (Cicer arietinum L. cv. GPF2). Soil Biol. Biochem.40, 718–727.

Nadeem, S.M., Zahir, Z.A., Naveed, M., Arshad, M., 2007. Preliminary investigationson inducing salt tolerance in maize through inoculation with rhizobacteria con-taining ACC deaminase activity. Can. J. Microbiol. 53, 1141–1149.

Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., 1954. Estimation of available phos-phorous in soils by extraction with sodium bicarbonate. USDA Circular 1, 939.

Patten, C.L., Glick, B.R., 2002. Role of Pseudomonas putida indole acetic acid in devel-opment of the host plant root system. Appl. Environ. Microbiol. 68, 3795–3801.

Peix, A., Mateos, P.F., Rodriguez-Barrueco, C., Martinez-Molina, E., Velazquez, E.,2001. Growth promotion of common bean (Phaseolus vulgaris L.) by a strain ofBurkholderia cepacia under growth chamber conditions. Soil Biol. Biochem. 33,1927–1935.

Ronald, M.A., James, W.S., 2006. Handbook of Media for Clinical Microbiology. Taylorand Francis, New York, USA, p. 73.

Schwyn, B., Neilands, J.B., 1987. Universal chemical assay for the detection anddetermination of siderophores. Anal. Biochem. 160, 47–56.

Selvakumar, G., Mohan, M., Kundu, S., Gupta, A.D., Joshi, P., Nazim, S., Gupta,H.S., 2008. Cold tolerance and plant growth promotion potential of Serratiamarcescens strain SRM (MTCC 8708) isolated from flowers of Summer squash(Cucurbita pepo). Lett. Appl. Microbiol. 46, 171–175.

Shahab, S., Ahmed, N., Khan, N.S., 2009. Indole acetic acid production and enhancedplant growth promotion by indigenous PSBs. Afr. J. Agric. Res. 4, 1312–1316.

Torres-Rubio, M.G., Valencia-Plata, S.A., Bernal-Castillo, J., Martinez-Nieto, P., 2000.Isolation of Enterobacteria, Azotobacter sp. and Pseudomonas sp., producers of

Latinoamer. Microbiol. 42, 171–176.Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J., 1991. 16S ribosomal DNA

amplification for phylogenetic study. J. Bacteriol. 173, 697–703.Yang, J.E., Jacobsen, J.S., 1990. Soil inorganic phosphorus fractions and their uptake

relationships in calcareous soils. Soil Sci. Soc. Am. J. 54, 1666–1669.