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.
223
224
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
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.
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.
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
% G+C
content
67.5%
65.1%
58.3%
53.5%
226
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
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)
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
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)
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)
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)
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.
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
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)
0.2
0.1 (a) (102)
0.3 (a) (59.2)
0.3 (a) (73.4)
0.3 (a) (73.8)
0.1 (165)
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.
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
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).
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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