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Applied Soil Ecology 46 (2010) 222229

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Applied Soil Ecology


journal homepage: www.elsevier.com/locate/apsoil

Stimulatory effect of phosphate-solubilizing bacteria on plant growth, stevioside


and rebaudioside-A contents of Stevia rebaudiana Bertoni
Mamta a,b , Praveen Rahi c , Vijaylata Pathania d , Arvind Gulati c , Bikram Singh d ,
Ravinder Kumar Bhanwra e , Rupinder Tewari a,
a

Department of Biotechnology, Panjab University, Chandigarh-160014, India


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

a r t i c l e

i n f o

Article history:
Received 30 January 2010
Received in revised form 6 August 2010
Accepted 9 August 2010
Keywords:
Stevia rebaudiana
Phosphate-solubilizing bacteria (PSB)
Stevioside
Rebaudioside-A

a b s t r a c t
The effect of four phosphate-solubilizing bacteria (PSB), (Burkholderia gladioli 10216, Burkholderia gladioli 10217, Enterobacter aerogenes 10208 and Serratia marcescens 10238) as identied on the basis of 16S
rRNA gene sequencing was evaluated on plant growth and commercially important glycosides, stevioside (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 for
P-solubilization ability, biocompatibility, indole acetic acid (IAA) and siderophore production. In greenhouse study, treatment of either individual PSB or a consortium (of PSB) resulted in increased plant
growth, ST and R-A contents. The stimulatory effect was observed with consortium treatment in plant
growth 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% plant1 and R-A, 555% plant1 ) as
compared to the un-inoculated plants. Among individual PSB treatments, B. gladioli 10216 showed most
promising response in majority of the parameters studied. The root colonization potential of PSB, assayed
by RAPD technique, showed the colonization of all PSB isolates, though their extent of colonization varied.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Stevia rebaudiana is a perennial shrub of Asteraceae (Compositae)
family native to certain regions of South America. Its leaves produce
zero-calorie ent-kaurene glycosides stevioside and rebaudiosides
which are in demand as non-nutritive and high potency sweetener
in food and beverages by the persons suffering from diabetes and
obesity. The amount of active principles depends on total biomass,
which further depends on climatic features, agro-techniques, water
management and fertilizer applications. Recently, the integrated
application of microbial inoculants to agro-technologies for the
cultivation of medicinal plants is being promoted for improving
their productivity in terms of biomass and biochemical constituents
(Leithy et al., 2006; Jaleel et al., 2007). PSB are well known to
promote plant growth because of their ability to convert insoluble form of P to soluble form that can be readily taken up by the
plant roots. Usually the soils are supplemented with inorganic P in
the 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).
0929-1393/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2010.08.008

P gets xed in the soil as phosphates of iron, aluminum and calcium (Altomare et al., 1999). This xed form of P is not efciently
taken up by the plants and known to cause many environmental problems like eutrophication and soil salinity (Del Campillo
et al., 1999). The use of PSB as biofertilizers could decrease the
environmental problems associated with conventional chemical
fertilizers.
In addition to P-solubilization, PSB may also improve the plant
productivity by producing other secondary metabolites. Several
evidence related to plant growth promotion by PSB through the
production of IAA (Patten and Glick, 2002; Shahab et al., 2009) and
siderophore (Koo and Cho, 2009) make the PSB more suitable as
biofertilizers.
The effect of PSB on medicinal plants is gaining momentum, as
evidenced by an increase in the number of publications. There are
limited reports (Earanna, 2007; Das et al., 2008) related to the effect
of PSB on the growth of S. rebaudiana and no report has been found
on the yield enhancement of stevioside and rebaudiosides by PSB
inoculation. The present study was carried out to isolate the PSB
from the rhizosphere of Stevia plants and examine their effect on
the plant growth, availability of P in soil, P uptake by plants and
yield of ST and R-A of S. rebaudiana.

Mamta et al. / Applied Soil Ecology 46 (2010) 222229

2. Materials and methods


2.1. Isolation of PSB
Rhizospheric soil samples of ve S. rebaudiana plants growing in
organic farms (loamy soil, without any input of chemical fertilizers)
were collected in sterile plastic bags. Samples were processed on
the same day. One gram soil of each sample was suspended separately in 9.0 ml of phosphate buffer saline (PBS) of pH 7.2. The serial
dilutions (1:10) were made and spread on Pikovskayas (PVK) agar
plates containing 0.5% TCP and incubated at 30 C. Colonies showing
the zone of solubilization, which indicates microbial P-solubilizing
ability, were streaked on nutrient agar plates to check their purity
and stored for further studies.

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. The
activity was recorded in percentage siderophore units calculated as
[((Ar As) Ar1 ) 100], where Ar is dened as absorbance of reference (un-inoculated media + CAS solution) and As is absorbance
of 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 and
Parker, 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 denaturation
method (Marmur and Doty, 1962).

2.2. Screening of PSB


2.4. Molecular identication of PSB
2.2.1. Quantitative estimation of phosphate solubilization
P-solubilization was estimated in a liquid medium amended
with 0.5% TCP. The isolates were grown in a 100 ml liquid
medium at 30 C on a rotary shaker (130 rev min1 ). The composition of the medium was (g l1 ): yeast extract, 0.50; dextrose,
10.0; TCP, 5.0; (NH4 )2 SO4 , 0.50; KCl, 0.20; Mg2 SO4 7H2 O, 0.10;
Mn2 SO4 H2 O, 0.0001; Fe2 SO4 7H2 O, 0.0001, pH adjusted to 7.0.
The 5.0 ml culture was taken out at regular intervals of 24 h, for
7 days, centrifuged (Sigma 103456 cooling centrifuge, Germany at
10,000 g for 10 min) and soluble-P content of culture supernatant
was estimated by colorimetric chlorostannous reduced molybdophosphoric acid blue method (Jackson, 1973). The nal values
were calculated with the help of a standard curve obtained using
02 mg l1 KH2 PO4.
2.2.2. Biocompatibility assay
In order to prepare a consortium of PSB, it is essential that all
microbes present in a consortium should be biocompatible. Biocompatibility among PSB isolates was determined by streaking one
PSB isolate across the middle of the PVK agar plate. Other isolates
were streaked perpendicular to the above isolate and incubated
(30 C, 48 h). Zone of inhibition of growth at the junction of cultures
was noted (Mittal et al., 2008).

The genomic DNA of PSB isolates were extracted using


Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA). The
primers fD1 (5 -AGAGTTTGATCCTGGCTCAG-3 ) and rP2 (3 ACGGCTACCTTGTTACGACTT-5 ) were used for amplication of 16S
rRNA gene (Weisburg et al., 1991). The total PCR reaction mixture was 50.0 l, comprising 200 M dNTPs, 50 M each primer,
1 PCR buffer, 3 U Taq polymerase, and 100 ng genomic DNA. The
thermocycler conditions involved an initial denaturation at 94 C
for 4 min, followed by 35 cycles of 94 C for 1 min, 52 C for 1 min,
and 72 C for 2 min and nal extension at 72 C for 8 min. The 16S
rRNA gene was puried from the gel and was ligated to pGEM-T
easy vector (Promega, Madison) and transformed in E. coli JM109.
The sequences of the insert were determined using a Big-Dye Terminator Cycle Sequencer and an ABI Prism 310 Genetic Analyzer
(Applied Biosystems, CA). The RNA gene sequences were analyzed
using the gapped BLASTn (http://www.ncbi.nlm.nih.gov) search
algorithm and aligned to their nearest neighbors. The evolutionary
distances among phosphate-solubilizing isolates and their related
taxa were calculated using TREECON software and Kimuras twoparameter model, after aligning the sequences with ClustalW.
2.5. Site of pot experiments
Experiments were conducted in a greenhouse (uncontrolled
conditions) during MarchJune, 2008, in the Department of Botany,
Panjab University, Chandigarh, India.

2.2.3. Quantitative estimation of indole acetic acid (IAA)


IAA was assayed by the colorimetric method using ferric
chlorideperchloric acid reagent (FeCl3 HClO4 ) (Gordon and Paleg,
1957). PSB were inoculated in the minimal medium (g l1 ):
KH2 PO4 , 1.36; Na2 HPO4 , 2.13; MgSO4 7H2 O, 0.2, pH 7.0, amended
with 5.0 mM l-tryptophan solution (g 100 ml1 : glucose, 10; ltryptophan, 1.0; yeast extract 0.1; ltered through sterile 0.2 m
Millipore membrane lter) (Frankenberger and Poth, 1988). Flasks
were incubated at 30 C on a rotary shaker (130 rev min1 ). Cultures
were withdrawn after 48 h intervals and centrifuged (10,000 g
for 10 min). The 2.0 ml of Salpers reagent was added dropwise to
1.0 ml of culture supernatant, and samples were incubated in the
dark for 30 min. Development of pink color was assayed with a
spectrophotometer at 530 nm. The concentration of IAA in g ml1
was determined from a standard curve of IAA (010 g ml1 ).

2.5.1. Preparation of inoculum


Each PSB was grown separately in the nutrient broth at 30 C in
an orbital shaker (150 rev min1 ) for 24 h. The cultures were centrifuged in 50 ml sterile plastic tubes at 6000 g for 15 min. The
pellets were re-suspended in PBS and optical density (OD) was
adjusted to have a nal concentration of colony forming units, i.e.
108 CFU ml1 . This liquid culture of each PSB was used for the individual inoculation in pot experiments. For making a PSB consortium
inoculum, all individual cultures of PSB of equal cell density, i.e.
108 CFU ml1 were mixed together into a 250 ml sterilized ask.
This mixture was used as a consortium inoculum.

2.2.4. Quantitative estimation of siderophore production


PSB were grown in an iron decient medium containing (g l1 ):
K2 HPO4 , 0.1; KH2 PO4 , 3.0; MgSO4 7H2 O, 0.2; (NH4 )2 SO4 , 1.0; succinic acid, 4.0 (Bharbaya and Rao, 1985) at 30 C on a rotary
shaker at 130 rev min1 for 48 h. The 0.5 ml of cell free supernatant
was mixed with 0.5 ml of Chrome Azurol Sulfonate (CAS) assay
solution (1.5 ml of 1 mM Fe stock solution + 7.5 ml of 2 mM CAS
stock solution added to 0.003 mM hexadecyltrimethylammonium
(HDTMA) + 30 ml of 1.5 mM Piperazine buffer) along with 10 l of

2.5.2. Soil conditions and sowing of plantlets


Unsterile loamy soil (pH, 7.8; available N, 46.9 mg kg1 ;
available P, 5.0 mg kg1 ; available K, 14.9 mg kg1 ; total Ca,
6.0 mequiv. kg1 ; total Mg, 1.2 mequiv. kg1 ; total organic carbon,
0.10%) was thoroughly mixed and passed through 2 mm sieve to
remove large particulate matter and kept under sunlight for 7
days. Ethanol disinfected plastic pots (10.2 cm 25.4 cm), were
lled with 3.5 kg of soil. Roots of tissue culture plantlets were
surface-sterilized by dipping in 2% NaOCl solution for 10 min and

224

Mamta et al. / Applied Soil Ecology 46 (2010) 222229

then washed thrice with distilled water. The surface-sterilized


plantlets roots were dipped in the desired culture inoculum
(108 CFU ml1 , as mentioned in Section 2.5.1) for 15 min and then
allowed to dry in the shade for 30 min. After drying, plantlets were
planted in six different sets of treatment with three replications
of each. These sets included: (1) soil + TCP (200 mg kg1 soil); (2)
soil + TCP + B. gladioli 10216; (3) soil + TCP + B. gladioli 10217; (4)
soil + TCP + S. marcescens 10238; (5) soil + TCP + E. aerogenes 10208;
(6) soil + TCP + consortium of all isolates. Experiments were performed in completely randomized block design.
2.6. Analysis of available P content in soil and P uptake in plants
After the harvesting of plants, the P content of soil and dried
plant parts were analyzed. Available P content in soil was determined by colorimetric sodium bicarbonate-extractable P method
(Olsen et al., 1954). P uptake in plant parts was determined using
vanadomolybdo-phosphoric yellow color method (Koeing and
Johnson, 1942).
2.7. HPLC analysis of ST and R-A content in the leaves of S.
rebaudiana
The S. rebaudiana leaves were dried in an oven at 40 C for 5 days.
The 50 mg of completely dried and powdered samples were taken in
25 ml conical asks. Samples were extracted with 10 ml methanol
for 24 h and then ltered (Whatman lter paper of size 102 l, Sartorius, grade 389) in 50 ml distillation ask. The extraction process
was repeated again for the residue left. The ltrate was distilled
on a rotary evaporator (at 45 C, 50 rev min1 and 70 mbar vacuum pressure), until the whole methanol get evaporated. Residue
was defatted two times with 20 ml hexane and air dried properly (Megeji et al., 2005). The 5.0 ml of acetonitrile:water (80:20)
was added into the residue, mixed properly and ltered (0.45 M
Millipore membrane lter) into HPLC vials. Samples in vials were
put into an auto-sampler (WATERS 717 plus) and analysis was
done by using WATERS HPLC machine having EMPOWER software,
WATERS 600 controller, WATERSTM 600 pump, Lichrocart 250-4
NH2 column (5 M), wavelength of 210 nm, ow rate of 1 ml min1 ,
pressure 800 psi, and photodiode array detector. As a mobile phase
we used a mixture of acetonitrile and water (Isocratic, 50:50). ST
and R-A of Sigma Chemicals were used as standards.
2.8. Estimation of root colonization by PSB
At the end of the pot experiments plants were harvested and
analyzed for rhizosphere colonization of PSB based on their random amplication of polymorphic DNA (RAPD) banding pattern

analysis. The rhizosphere soil adhering to the roots of harvested


plants was separated by gentle tapping and stored in sterilized
petri plates at 4 C. One gram soil of each replicate soil samples
was serially diluted (1:10), and spread on PVK agar medium. The
plates were incubated at 30 C for 5 days. The isolates showing
P-solubilization were marked and counted (number of isolates
showing different morphologies and number of isolates showing similar morphology). Isolated PSB were puried by repeated
streaking of culture on PVK agar plate. The genomic DNA of PSB
was isolated using the protocol suggested by Himedia (MB505
HiPurATM Bacterial and Yeast Genomic DNA Purication Spin Kit).
The PSB isolates of different morphologies were analyzed for DNA
banding pattern using primers OPA-04 (5 -AATCGGGCTG-3 ) and
BOX A1 (5 -CTACGGCAAGGCGACGCTGACG-3 ). The similarity of
band patterns was quantied by simple matching (Apostol et al.,
1993). PSB isolates having similar banding pattern to inoculated
PSB conrmed their survival in the rhizosphere.
2.9. Statistical analysis
Pot experiments were arranged in completely randomized block
design. Statistical analysis was conducted using one-way analysis
of variance (ANOVA) statistical package for social sciences (SPSS)
software, Version 30. Comparisons of means were performed by
the 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 mm
on the PVK agar medium were isolated from the rhizosphere of Stevia plants. In liquid PVK medium, The P-solubilizing ability of these
organisms varied from 97.3 to 316 g ml1 and grouped as high
P-solubilizers (>200 g ml1 ; S1 and S14), average P-solubilizers
(100200 g ml1 ; S3, S6, S7, S8, S9, S11, S15, S17 and S18) and
low P-solubilizers (<100 g ml1 ; S12) (Table 1).
In order to nd antimicrobial substances, all the P-solubilizers
were subjected to biocompatibility assay. Based on the Psolubilizing activity and biocompatibility test, four PSB (S1, S9, S14,
S18) were selected for further study. All four isolates were biocompatible and good P-solubilizers.
The selected bacterial isolates were further tested for
siderophore and IAA production (Fig. 1). Isolates, S1 and S9 produced siderophore (91.3% and 94.8%, respectively) as well as
IAA (15.3 g ml1 and 1.5 g ml1 , respectively). Isolate S14 produced only siderophore (80.4%), whereas, S18 produced only IAA
(40.8 g ml1 ).

Table 1
Release of soluble-P (g ml1 ) by bacterial isolates for 7 days in TCP supplemented liquid medium.
Isolate no.

S1
S3
S6
S7
S8
S9
S11
S12
S14
S15
S17
S18

Soluble phosphorus (g ml1 )


0h

1 day

2 day

3 day

4 day

5 day

6 day

7 day

0
0
0
0
0
0
0
0
0
0
0
0

90.0 2.0
45.3 1.2
24.7 3.0
34.7 4.2
23.3 1.1
102 4.0
0
25.3 1.1
128 2.0
0
64.0 4.0
70.0 2.0

128 2.0
79.3 3.1
54.0 3.5
50.7 5.0
38.7 3.1
76.0 4.0
73.3 2.3
71.3 3.1
188 2.0
0
105 3.1
102 6.0

228 2.0
89.3 3.1
109 1.1
86.0 4.0
81.3 3.1
6.0 2.0
162 3.5
97.3 3.1
316 4.0
52.0 2.0
94.7 4.2
148 2.0

236 4.0
125 5.0
191 4.2
142 4.0
132 4.0
2.67 3.2
171 4.2
46.7 3.1
304 4.0
62.7 3.1
86.0 2.0
158 2.0

252 4.0
56.0 5.3
143 3.1
185 4.2
185 3.1
1.33 2.3
101 3.1
37.3 3.1
236 4.0
81.3 1.1
58.7 3.1
200 2.0

182 2.0
1.33 2.3
64.7 3.1
151 5.0
124 3.5
0.67 1.1
80.7 3.1
19.3 3.1
232 2.0
123 3.1
39.3 5.0
128 2.0

158 2.0
0
63.3 5.0
92.7 5.0
92.7 3.1
0
70.7 5.0
2.67 4.6
230 4.0
103 6.4
25.3 4.2
150 4.0

Results are mean of three replicates, indicate standard deviation, values in bold letters represent the maximum P-solubilization by respective bacterial isolates.

Mamta et al. / Applied Soil Ecology 46 (2010) 222229

225

sequences in the GenBank database indicated Burkholderia gladioli as the closest related species to the isolates S1 and S9 while
other two isolates S14 and S18 were closely related to Serratia
marcescens and Enterobacter aerogenes, respectively. The phylogenetic tree (Fig. 2) based on 16S rRNA gene sequences of the isolates
and representative species of closely related taxa, formed three
clearly distinguishable groups. The Burkholderia group consisted of
isolate S1 and S9 along with B. gladioli CIP105410, B. gladioli R406,
B. gladioli ATCC 33664, B. vietnamiensis LMG 10929, B. cepacia ATCC
53130 and Pseudomonas antimicrobica NCIMB 9898. The Serratia
group included isolate S14, S. marcescens DSM 30121, S. marcescens
strain A3 and S. marcescens J2P3. The Enterobacter group consisted
of isolate S18, E. aerogenes JCM 1235, E. dissolvens LMG 2683, E. ludwigii EN119, E. cancerogenus LMG 2693, E. cowanii CIP 107300 and
Pantoea agglomerans HK14-1.
3.3. Effect of PSB on plant growth
The effect of PSB (individually and in consortium) on the
growth of S. rebaudiana was observed in TCP (200 mg kg1 of soil)
amended soil. All four PSB showed stimulatory effect, in terms
of shoot length, root length, leaf dry weight, stem dry weight
and 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) was
shown by B. gladioli 10216 plants in comparison to un-inoculated
(i.e. control) plants. The maximum increase (23.8%) in shoot
length was shown by plants treated with E. aerogenes 10208.
The individual treatments did not differ signicantly with each
other.
The stimulatory effect of PSB was found to be more pronounced
when inoculated as a consortium. Compared to control plant, an
increase of 14.7%, 22.5%, 76.3%, 82.5% and 89.0% was observed in
the root length, shoot length, stem dry weight, total biomass and
leaf dry weight, respectively.
Fig. 1. Indole acetic acid (A) and siderophore (B) produced by PSB isolates. Error
bars represent standard deviation.

3.2. Identication of PSB


The isolates were examined for their colony morphology on
nutrient agar after 24 h of incubation (Table 2). All were circular,
raised and smooth in appearance. The colonies of three isolates
(S1, S9 and S14) were off white, whereas that of S18 was yellowish
brown in appearance. The colony size of the isolates was found to be
0.5 mm, 1.5 mm, 2.0 mm and 2.0 mm for S9, S18, S1 and S14, respectively. The microscopic identication showed that all were Gram
negative, small to medium sized rods. All PSB isolates were catalase positive and oxidase negative. The isolates S14 and S18 were
lactose fermenters while S1 and S9 were non-lactose fermenters.
The % G + C content of the PSB isolates was 67.5% (S1), 65.1% (S9),
58.3% (S14) and 53.5% (S18), respectively (Table 2).
The results of BLAST search of 16S rRNA gene sequences of the
four selected PSB on comparison with the available 16S rRNA gene

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


content
The effect of PSB on ST and R-A contents both on per gram dry
leaf weight as well as per plant dry leaf weight of S. rebaudiana
was observed. Based on statistic analysis, no signicant change was
observed in the ST content in the leaves of inoculated as well as
un-inoculated plants (Table 4). However, a statistically signicant
increase in R-A content of leaves was found in plants inoculated
with PSB consortium (247%) (Fig. 3), S. marcescens 10238 (250%)
and B. gladioli 10216 (221%). Whereas, a statistically insignicant
change was observed in plants inoculated with E. aerogenes 10208
or B. gladioli 10217. When the total yield of ST and R-A plant1
was calculated, a profound statistically signicant increase was
observed in some of the inoculated plants. An increase in the ST
content of 150% and 91.0% was seen in plants inoculated with consortium and B. gladioli 10216, respectively. Similarly, an increase in
R-A content of 555%, 466% and 447% was observed in plants treated
with the consortium, B. gladioli 10216 and S. marcescens 10238. In

Table 2
Characterization of PSB isolates.
PSB
isolate

Catalase
production

Oxidase
production

Lactose
fermentation

S1
S9
S14
S18

+ve
+ve
+ve
+ve

ve
ve
ve
ve

NLF
NLF
LF
LF

2.0 mm, cream color, circular, raised, smooth margins


0.5 mm, cream color, circular, raised, smooth margins
2.0 mm, cream color, circular, raised, smooth margins
1.5 mm, yellowish brown, circular, raised, smooth margins

NLF: non-lactose fermenter, LF: lactose fermenter.


a
The colonies were grown on nutrient agar for 24 h at 30 C.

Colony size and morphology

Gram staining and microscopic


examination

% G+C
content

Gram negative, small rods


Gram negative, small rods
Gram negative, medium size rods
Gram negative, small rods

67.5%
65.1%
58.3%
53.5%

226

Mamta et al. / Applied Soil Ecology 46 (2010) 222229

Fig. 2. Phylogenetic tree based on 16S rRNA gene sequences, showing the relationships among selected PSB isolates (shown in bold letters) and representatives of other
related taxa with validly published names. The 16S rRNA gene accession numbers are given within brackets. Bar = 0.02 substitutions per site. The number at the node, indicate
bootstrap percentiles from 100 replicates.

maximal value which was 330% more than the control value.
Other individual inoculation with PSB isolates showed 213268%
increase in P content.
The uptake of P in Stevia plants was also inuenced by PSB inoculation (Table 5). The individual inoculation with PSB showed an
increase in P content of leaves (59.2102%) in comparison to control. Treatment with PSB consortium showed more pronounced
increase (165%) than individual PSB treatments. In the case of stem
P uptake, an increase of (104150%) was observed by PSB inoculations in comparison to control plants, though no signicant

rest of the treatments, a non-signicant change was observed in ST


and R-A contents (Table 4).
3.5. Phosphorus availability in the soil and its uptake by the
plants
The available P content of soil after harvesting of plants was
studied. A signicant increase was found in available P content of
soils treated with PSB in comparison to control soils (Table 5). Soils
treated with B. gladioli 10216 and consortium of all PSB showed
Table 3
Effect of phosphate-solubilizing bacteria on the growth of Stevia rebaudiana.
Treatment

Shoot length (cm)

S + TCP (control)
S + TCP + B. gladioli (MTCC 10216)
S + TCP + B. gladioli (MTCC 10217)
S + TCP + E. aerogenes (MTCC 10208)
S + TCP + S. marcescens (MTCC 10238)
S + TCP + consortium

65.0
78.0
80.4
80.5
79.8
79.7

3.9
1.3 (a) a (20.0)
3.6 (a) (23.6)
4.4 (a) (23.8)
4.2 (a) (22.8)
2.7 (a) (22.5)

Root length (cm)


10.2
11.5
11.4
11.5
11.4
11.7

Leaf dry weight (g)

0.2
0.3 (a) (12.7)
0.3 (a) (11.8)
0.3 (a) (12.5)
0.1 (a) (11.5)
0.2 (a) (14.7)

0.46
0.80
0.66
0.71
0.71
0.87

Stem dry weight (g)

0.4
0.1(a, c) (74.5)
0.1 (b) (44.1)
0.1 (a b) (54.4)
0.1 (a, b) (55.1)
0.3 (89.0)

0.48
0.80
0.78
0.79
0.80
0.84

0.1
0.4 (a) (66.9)
0.2 (a) (62.7)
0.1(a) (65.6)
0.2 (a, b) (67.9)
0.2 (76.3)

Total shoot biomass (g)


0.94
1.61
1.44
1.51
1.52
1.72

0.4
0.1 (a, b) (70.7)
0.1 (a) (53.6)
0.1 (a) (60.1)
0.1 (a) (61.7)
0.1 (b) (82.5)

Values are mean of three replications, mean values (mean S.D.) sharing the same letter do not differ signicantly by LSD at P 0.05, S: un-inoculated soil, TCP: tricalcium
phosphate.
a
Values in parenthesis represents percentage increase over control.

Table 4
Effect of phosphate-solubilizing bacteria on stevioside and rebaudioside-A contents of Stevia rebaudiana.
Treatment

ST (mg g1 leaves)

S + TCP (control)
S + TCP + B. gladioli (MTCC 10216)
S + TCP + B. gladioli (MTCC 10217)
S + TCP + E. aerogenes (MTCC 10208)
S + TCP + S. marcescens (MTCC 10238)
S + TCP + consortium

35.0
38.0
34.8
26.2
30.2
46.2

2.6 (a, b)
5.2 (a. b)
0.7 (a, b)
5.4 (b)
4.5 (a, b)
2.2 (a)

R-A (mg g1 leaves)


5.67
18.2
3.67
15.1
19.8
19.7

2.1 (a)
3.6 (b) a (221)
1.1 (a)
2.5 (a, b)
3.8 (b) (250)
2.3 (b) (247)

ST (mg plant1 )
16.2
30.9
23.2
18.7
21.7
40.5

0.7 (a)
2.9 (b, c) (91.0)
1.0 (a, b)
3.6 (a, b)
3.0 (a, b)
2.6 (c) (150)

R-A (mg plant1 )


2.61
14.8
2.42
10.8
14.3
17.1

0.9 (a)
2.4 (b) (466)
0.6 (a)
1.6 (a, b)
3.2 (b) (447)
3.1 (b) (555)

Values are mean of three replications, mean values (mean S.D.) sharing the same letter do not differ signicantly by LSD at P 0.05, S: un-inoculated soil, TCP: tricalcium
phosphate.
a
Values in parenthesis represents percentage increase over control.

Mamta et al. / Applied Soil Ecology 46 (2010) 222229

227

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

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

Available P content in soil (mg kg1 )

S + TCP (control)
S + TCP + B. gladioli (MTCC 10216)
S + TCP + B. gladioli (MTCC 10217)
S + TCP + E. aerogenes (MTCC 10208)
S + TCP + S. marcescens (MTCC 10238)
S + TCP + consortium

1.78
7.69
5.58
6.57
6.57
7.67

0.1
0.2 (a) a (331)
0.1 (213)
0.2 (b) (268)
0.2 (b) (268)
0.4 (a) (330)

P uptake in leaves (mg plant1 )


1.65
3.33
2.62
2.86
2.86
4.37

0.2
0.1 (a) (102)
0.3 (a) (59.2)
0.3 (a) (73.4)
0.3 (a) (73.8)
0.1 (165)

P uptake in stem (mg plant1 )


0.89
2.11
1.82
1.95
2.03
2.22

0.1
0.4 (a) (137)
0.1 (a) (104)
0.2 (a) (119)
0.3 (a) (128)
0.3 (a) (150)

Values are mean of three replications, mean values (mean S.D.) sharing the same letter do not differ signicantly by LSD at P 0.05, S: un-inoculated soil, TCP: tricalcium
phosphate.
a
Value in parenthesis represents percentage increase over control.

difference was observed between individually inoculated and consortium inoculated plants.

ulated PSB could not be detected at this dilution (105 ), but their
presence at lower dilutions cannot be discounted, as it was impossible to count the number of colonies at lower dilutions because
individual bacterial colonies could not be clearly seen.

3.6. Soil analysis for inoculated PSB isolates by RAPD technique


The soil clinging tightly to the roots of the harvested Stevia plants
was examined for the colonization of PSB by analyzing DNA banding
pattern (Fig. 4) of the P-solubilizing colonies obtained after plating the soil samples. The CFU were counted on dilution 106 and
105 . In individual PSB treatment studies, maximal colonization
was observed with B. gladioli 10216 (4.5 109 CFU g1 soil at 106
dilution) followed by S. marcescens 10238 (4.0 109 CFU g1 soil
at 106 dilution), B. gladioli 10217 (6.6 108 CFU g1 soil at 105
dilution) and E. aerogenes 10208 (4.0 108 CFU g1 soil at 105 dilution). In consortium treatment, B. gladioli 10216 (2.0 108 CFU g1
soil at 105 dilution) and S. marcescens 10238 (1.5 108 CFU g1 soil
at 105 dilution) colonization was observed. However, other inoc-

4. Discussion
In the present study, four PSB isolated from the rhizospheric
soil of S. rebaudiana were characterized as B. gladioli 10216, B. gladioli 10217, E. aerogenes 10208 and S. marcescens 10238. This is the
rst 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 Brassica
juncea (Kumar et al., 2009) and S. marcescens for Zea mays (Hameeda
et al., 2008) and Cucurbita pepo (Selvakumar et al., 2008).

228

Mamta et al. / Applied Soil Ecology 46 (2010) 222229

Fig. 4. Genotypic proling of PSB isolates using primer OPA-04 (A) and BOX A1 (B). Lane 1, Burkholderia gladioli 10216; 2, Burkholderia gladioli 10217; 4, Enterobacter aerogens
10208; 5, Serratia marcescens 10238; 3, DNA standard (10010,000 bp).

Although all PSB used in this study were good solubilizers, it is


desirable that P-solubilizers have additional plant growth promoting properties like IAA and siderophore production (Torres-Rubio
et al., 2000). Out of four PSB, two isolates, i.e. B. gladioli 10216 and
B. gladioli 10217 produced IAA as well as siderophore whereas isolates E. aerogenes 10208 and S. marcescens 10238 produced only IAA
and siderophore, respectively.
The present study found a signicant increase in the biometric
parameters of Stevia plants (total biomass, shoot length, root length,
leaf dry weight plant1 , and stem dry weight plant1 ) after treatment with PSB. The effect was more pronounced with consortium
treatment as compared to individual PSB treatments. The variable degree of stimulatory effect among individual or consortium
PSB treatments on plant growth may be due to diverse interactions of inoculated PSB with plant roots or with native microora,
which often results in the promotion of key processes beneting
plant growth and health (Braeken et al., 2008; Barea et al., 2005).
The signicantly lesser plant growth in control plants than PSB
inoculated plants indicated that the native PSB did not contribute
directly towards the plant growth thorough P-solubilization. Similar observations related to growth promotion of Stevia plants by
PSB inoculations have been made by Earanna (2007) and Das et al.
(2008), though PSB used were different from the ones in the present
study.
Stevia plants have gained importance as sweeteners because of
their ST and R-A contents. Until now there is no report on the effect
of P-solubilizers on ST and R-A levels of Stevia. We are reporting
for the rst time a signicant increase in ST and R-A contents of
PSB treated plants as compared to control plants. Of all the PSB
treatments, the maximum increase was observed in PSB consortium treated plants of 2.49-fold in ST content and 6.55-fold in R-A
content on per plant basis. Among individual PSB treatments, a
mixed response was observed. The treatment with B. gladioli 10216
showed a signicant increase in ST and R-A contents, whereas,
treatment with S. marcescens showed a signicant increase in R-A
content only.
The increase in ST content was primarily due to increase in the
number of leaves (plant1 ) as not much difference was observed
in ST content of leaves based on per gram dry leaf weight between

PSB treated and untreated plants. However, increase in R-A content


could 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 been
described as a crucial factor for plant growth promotion (De Weger
et al., 1995; Lugtenberg et al., 2001). Our results are also in agreement with this statement. The degree of stimulatory effect of PSB
on the plant growth, ST and R-A contents could be correlated with
the extent of colonization of the roots by PSB with B. gladioli 10216
showing maximal colonization and maximal enhanced effect on
plant growth parameters, ST and R-A contents.
The inoculation of PSB also resulted in the increased amount of
available P in soil. These results suggested that a subsequent crop
will reap the benets imparted by PSB to the soil in terms of available P content, physical and biological characteristics of soil (Mittal
et al., 2008). The maximum amount of available P was observed in
soil treated with B. gladioli 10216 showing maximum root colonization ability. These results showed the important role of inoculated
microorganisms for immobilizing relatively high amounts of P in
their biomass (Demetz and Insam, 1999).
The PSB inoculation of S. rebaudiana showed stimulatory effect
on P uptake of plant. This might be due to better utilization of P from
the pool of soil nutrients by the action of PSB. Maximal increase
in P uptake was shown by Stevia plants treated with PSB consortium. In case of individual PSB treatments, maximal increase in P
content was shown by B. gladioli 10216 treated plants. The highest amount of P uptake in leaves as well as in stems was observed
in plants showing the highest amount of available P. These ndings suggested that the microbially available P corresponds to plant
available P through mineralization of complex P compounds (Yang
and Jacobsen, 1990; Demetz and Insam, 1999). The increase in P
uptake in some other plants like Phaseolus vulgaris by inoculating Burkholderia sp. (Peix et al., 2001); in Soybean by inoculating
Serratia sp. (Han and Lee, 2005) and in Lycopersicon esculentum by
inoculating Enterobacter sp. (Kirankumar et al., 2008) supports our
ndings.
Taken together, the results suggested that the treatment of
S. rebaudiana with suitable PSB has signicant inuence on the

Mamta et al. / Applied Soil Ecology 46 (2010) 222229

growth and high value metabolites (ST and R-A) in pot experiments
carried out in greenhouse.
5. Conclusions
The use of PSB as biofertilizers is an efcient approach to replace
chemical phosphorus fertilizers for sustainable cultivation of S.
rebaudiana. The inoculation of PSB signicantly increased the plant
growth (shoot length, root length, leaf dry weight, stem dry weight
and biomass), available P content in soil as well as its uptake and
also the yield of commercially important ST (mg plant1 ) and R-A
contents (mg plant1 as well as mg g1 ).
Acknowledgements
The authors are grateful to Union Grant Commission, New Delhi,
India for nancial support and Haryali Biotech, Zirakpur (Punjab)
for soil sample collection and providing tissue culture plantlets.
We are also thankful to Dr. Mohinder Kaur (Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, H.P., India), Dr. Vani
Mittal and Mr. Onkar Bal for their useful suggestions and Mr.
Navtej Singh (Micrographer, SAIF, Panjab University, Chandigarh)
for timely help.
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