Journal of Plant Interactions

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Effects of plant-growth-promoting rhizobacteria

and arbuscular mycorrhizal fungus on plant
growth, stevioside, NPK, and chlorophyll content
of Stevia rebaudiana

Farinaz Vafadar, Rayhaneh Amooaghaie & Mahmoud Otroshy

To cite this article: Farinaz Vafadar, Rayhaneh Amooaghaie & Mahmoud Otroshy (2014) Effects
of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth,
stevioside, NPK, and chlorophyll content of Stevia�rebaudiana , Journal of Plant Interactions, 9:1,
128-136, DOI: 10.1080/17429145.2013.779035

To link to this article: https://doi.org/10.1080/17429145.2013.779035

© 2013 Taylor & Francis

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Journal of Plant Interactions, 2014
Vol. 9, No. 1, 128
136, http://dx.doi.org/10.1080/17429145.2013.779035

RESEARCH ARTICLE
Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant
growth, stevioside, NPK, and chlorophyll content of Stevia rebaudiana
Farinaz Vafadara, Rayhaneh Amooaghaiea* and Mahmoud Otroshyb

a
Department of Biology, Shahrekord University, Shahrekord, Iran; bDepartment of Tissue Culture,
Agricultural Biotechnology Research Institute, Isfahan, Iran
(Received 14 December 2012; accepted 19 February 2013)

Stevia rebaudiana Bertoni is a unique medicinal plant which is mostly utilized as a sugar substitute for diabetic
patients. In this research, regenerated plantlets of stevia in tissue culture is transferred to pots in greenhouse and
inoculated with plant-growth-promoting rhizobacteria (PGPRs) (Bacillus polymixa, Pseudomonas putida, and
Azotobacter chroococcum) and arbuscular mycorrhizal fungus (AMF) (Glomus intraradices). The results showed
that in comparison to control, inoculation with a single microorganism, significantly increased root and shoot
biomass as well as stevioside, chlorophyll, and NPK content in plants. However, such increased effects have been
found to be further enhanced significantly due to dual compatible mixtures of inoculants resulting from their
strong synergistic relationships among themselves. All growth parameters recorded the highest in 60-days-old
plants in the treatment of GlomusAzotobacter and followed with GlomusBacillus and Azotobacter
Pseudomonas treatments, respectively. Furthermore, high-performance liquid chromatography (HPLC) chroma-
tograms revealed that the highest stevioside content have been produced in same treatments. Triple treatments
had less positive effects compared to dual inoculations. Probably competence between microorganisms in triple
inoculations has reduced their efficiency. Thus, suitable combination of mycorrhizal fungi and PGPR as biotic
elicitors can enhance growth and stevioside content in tissue culture-regenerated plantlets of stevia.
Keywords: AMF; growth; PGPR; Stevia; stevioside

Introduction arbuscular mycorrhizal (AM) fungi (Adesemoye et al.

Stevia rebaudiana Bertoni belongs to Asteraceae
family. The plant is native to certain regions of South
America such as Paraguay and Brazil (Sivaram &
Mukundan 2003). The leaves of Stevia are the source
of diterpene glycosides such as stevioside and rebau-
dioside (Ahmed & Salahin 2007). Stevioside, the main
sweet component in the leaves of S. rebaudiana, tastes
about 300 times sweeter than sucrose (0.4% solution)
(Geuns 2003). Thus, it is considered to be a sugar
substitute and commercial sweetener both in the form
of stevioside and stevia extract. In addition to their
sweetness, stevia extract and stevioside have been used
as a traditional medicine by local people in South
America for hundreds of years (Chatsudthipong &
Muanprasat 2009). Hence, there is a need to enhance
biomass through cultural techniques, application of
manures, and fertilizers, including biofertilizers.
It has been demonstrated that chemical fertilizer
has increased crop yield, but has also caused deleter-
ious effects such as soil acidification and production
of the greenhouse gas nitrous oxide (N2O) through
denitrification on ecosystems (Biswas et al. 2000).
One potential way to decrease negative environmen-
tal impacts resulting from continued use of chemical
fertilizers is inoculation with biofertilizers such as
plant-growth-promoting rhizobacteria (PGPR) and
2009).
The PGPR are a group of rhizosphere colonizing
bacteria that produces substances to increase the
growth of plants and protect them against diseases
(Miransari 2011). The enhancement in various agro-
nomic yields by PGPR was because of the production
of growth-promoting phytohormones, phosphate
mobilization, production of siderophores and anti-
biotics, inhibition of plant ethylene synthesis, and
induction of plant systemic resistances to pathogens.
Among PGPR, N2-fixing and P-solubilizing bacteria
(PSB) are important for crop plants as they increase
nitrogen (N) and phosphorus (P) uptake and play a
crucial role as PGPR in the biofertilization (Singh
et al. 2011).
In addition, inoculation of plants with AM fungi
resulted in enhancing plant water and nutrient uptake
(Gosling et al. 2006). The most important benefit of
AM fungi is the ability to scavenge the available P
through their hyphae that have large surface areas on
which the extraradical hyphae act as a bridge between
the soil and plant roots (Liu et al. 2000; Bianciotto &
Bonfante 2002). Also, the use of AM fungi often
results in better plant growth (Wu et al. 2005) and
improves the productivity of medicinal compounds
such as organosulfides, polyphenols, phytosterols,

*Corresponding author. Email: Rayhanehamooaghaie@yahoo.com

# 2013 Taylor & Francis
 Journal of Plant Interactions
stilbenes, vitamins, lignans, and terpenoids including
carotenoids which can be important for plant toler-
ance to abiotic and biotic stresses or beneficial to
human health through their antioxidant activities
(Gianinazzi et al. 2008, 2010). These fungi can also
induce plant systemic resistance against root patho-
gens (Gosling et al. 2006). AM fungi impart other
benefits to plants, including enhanced enzymatic
production (Adriano-Anaya et al. 2006), increased
rate of photosynthesis (Wu & Xia 2006), enhance-
ment of nitrogen fixation by symbiotic or associative
N-fixing bacteria (Javaid et al. 1994; Antunes et al.
2006), and improving soil fertility (Charles et al.
2006).
Although AM fungi are important symbionts to
plants, and their symbiosis can significantly enhance
the growth of the host plant, they are also interactive
with different soil bacteria. AM hyphae are able to
produce C products in a little amount as a source of
energy for soil microbes in the mycorrhizosphere.
Bacterial communities are able to promote germina-
tion of AM fungal spores and increase the rate and
extent of root plant colonization by AM fungi. Also,
soil microbes produce plant hormones, which can
influence AM establishment as well as spore and
hyphal growth (Miransari 2011).
The major objective of this greenhouse experiment
was to evaluate the effect of single, dual, or triple
inoculation of Azotobacter chroococcum, Pseudomonas
putida, Bacillus polymixa, and AM fungus (Glomus
intraradices) as biofertilizers on the promotion of plant
growth, stevioside, NPK, and chlorophyll content of
60-days-old stevia plants.
Materials and methods
Stevia plantlet preparation
The seeds of stevia show a very low germination
percentage and genetic variability. Vegetative propa-
gation is too limited by lower number of individuals
that can be obtained from a single plant and having
the possibilities of pathogen accumulation in the
tissues. So plant tissue culture technology is the
only alternative method for rapid propagation of S.
rebaudiana plants (Debnath 2008).
Therefore, complete plantlets of S. rebaudiana
were obtained by direct organogenesis from nodal
segments (1.0
1.2 cm) on Murashige and Skoog
medium (1962), containing 3% (w/v) sucrose, 0.6%
(w/v) agar, and 0.4% (w/v) activated charcoal. In
stevia medium, 0.5 (mg/l) IBA and 1(mg/l) BAP were
used as growth regulators. All media were adjusted to
pH 5.7 with 1 N NaOH or 1 N HCl and autoclaved at
1218C for 20 min. All cultures were incubated in a
growth chamber under the controlled photoperiod of
16 h light and 8 h dark cycle with a light intensity of
3000
4000 lux provided by white fluorescent lamps
and 25928C temperature condition.
129
For acclimatization, 4-week-old rooted plantlets
were transplanted from the agar medium to plastic
bags (10 5 cm) containing sterilized peat moss. The
plastic bags were covered with clear plastic bags (for
maintenance of humidity) containing pores (for
aeration) and lied in a phytotron under the same
controlled photoperiod and temperature condition, as
mentioned above, for nodal segment culture stage.
Covers were removed after 2 weeks. In general, the
hardened plantlets were allowed to remain in the
plastic bags for 3
4 weeks before being transferred to
pots.
Inoculum preparation
In this pot experiment, N-fixer (A.chroococcum strain
PTCC 1658), P solubilizer (Pseudomonas putida strain
recorded in NCBI (accession number JX 44133)), N-
fixer and P solubilizer (Bacillus polymixa strain NCIB
8527), and AM fungus (Glomus intraradices) were
used as biofertilizers. Strains of A.chroococcum PTCC
1658 and Bacillus polymixa NCIB 8527 were pur-
chased from Iranian bacteria and fungi collection
(Persian Type Culture Collection). Strain of
Pseudomonas putida recorded in NCBI (accession
number JX 44133) was kindly received from Profes-
sor Gity Emtiazi’s bacterial collection. The AM
fungus (G. intraradices) was purchased from the
organic medical plant clinic, Hamedan, Iran.
B. polymixa and P. putida were cultured in Luria
Broth medium, and they were kept for 24 h in a
shaking incubator under 28918C and 150 rpm.
A.chroococcum was cultured in Burk’s N-free liquid
medium (Wilson & Knight 1952) and was kept for
24 h in a shaking incubator under 28918C and 150 rpm.
In the treatments containing bacterium, 3108 colony
forming units (CFU)/ml bacterial concentrations were
used, and the AM fungus inoculum used in the present
experiment consisted of 250 g sandy loam soil contain-
ing six spores of AMF and colonized roots of legume
plants.
Plant inoculation
Treatments used in this study were T1: control
(treated by liquid media applied for bacteria growth
without bacteria and sandy loam soil applied for
AMF without AMF) and; T2: A. chroococum; T3:
B. polymixa; T4: P. putida; T5: G. intraradices; T6:
A. chroococcumG. intraradices; T7: P. putida
G. intraradices; T8: B. polymixaG. intraradices; T9:
A. chroococcumB. polymixa; T10: A. chroococcum-
P. putida; T11: A. chroococcumB. polymixa
G. intraradices; T12: A. chroococcumP. putida
G. intraradices. The pots were filled with sterilized
soil and watered a day prior to inoculation of
bacteria.
Mycorrhizal treatments were inoculated with 250
g (per pot) soil inoculated with G. intraradices, by
placing a thin layer of mycorrhizal inoculum around
130 F. Vafadar et al.
the roots of plantlets below the surface of the soil
when plantlets were planted. For inoculation of
PGPR, the roots were treated with 5 ml suspension
inoculum (3 108 CFU/ml) of each bacterium. After
2 weeks, plantlets were reinoculated with 5 ml of the
same cell suspension on the soil of roots planting in
the pots. The pots were maintained at a temperature
range of 28958C, 50% humidity, and 16 h of light,
alternating with 8 h of darkness condition, and plants
were watered three times a week, at 100% water-
holding capacity of the pots (predetermined).
Plant growth measurement
The plants were harvested 60 days after transplanting.
Growth parameters such as plant height, fresh and
dry weight were recorded after harvest. Shoots were
separated from roots at 0.5 cm above the soil surface,
and the length of shoots was recorded. Roots were
separated from the soil by washing with running tap
water. Fresh weight of shoots and roots were
measured immediately, and dry weight was measured
by drying the samples in an oven at 408C for 5 days.
NPK analysis
Total N in shoots was determined calorimetrically
after Kjeldahl digestion of ground and dried samples
(Anonymous 1980). Phosphorus content of the plants
was estimated by vanadomolybdate phosphoric acid
method (Jackson 1973), and available K was deter-
mined by atomic absorption spectrometer.
Chlorophyll content analysis
Chlorophyll content also measured in an 80%
acetone extract of leaves at 60 days after transplant-
ing by Arnon method (1949) according to following
equations:
ð12:7  D663 Þ  ð2:69  D645 Þ  V
Chl:a ¼
1000  W
ð22:9  D645 Þ  ð4:93  D663 Þ  V
Chl:b ¼
1000  W
Chl: Total ¼ Chl:a þ Chl:b:
In the equations V is the volume of solutions, W is
the weight of leaf samples, D663 and D645 are the
solution absorbance at 663 and 645 nm, respectively.
Stevioside analysis
For sample preparation, 100 mg powdered leaf
samples were extracted by the modified method of
Bovanova et al. (1988). Stevioside extracted in 3 ml
distilled water, using a shaking hot-water bath for 30
min at 808C and 100 rpm. Then, they were centri-
fuged under 10,000 rpm for 5 min. The volume of
extracts was adjusted to 10 ml with distilled water. All
solvents of samples were filtered through a 0.45 mm
syringe filter (Biofil) and passed through the SPE
cartridges (C18, 3 ml) (Cronus) that were conditioned
with 3 ml methanol and 3 ml deionized water. The
samples (0.5 ml) were loaded. Then, they were washed
with 3 ml deionized water, followed with 3 ml
acetonitrile: water (20:80). Stevioside was eluted
with 1 ml acetonitrile: water (80:20).
Concentration of stevioside in the extractions of
samples was determined by high-performance liquid
chromatography (HPLC). The standard curve was
obtained after injection of three different concentra-
tion of stevioside standard (Sigma). The chromato-
graphic separations were carried out by using NH2
column (Merck). Operating conditions for HPLC
were as follows: the mobile phase, gradient acetoni-
trile: deionized water (80:20), a flow rate of the
mobile phase of 1.2 ml/min, and UV detection at
210 nm (Kolb et al. 2001; Woelwer-Rieck et al. 2010).
Experimental design and statistical analysis
The experiment was carried out in a completely
randomized design. The results were statistically
analyzed by using the SAS software (version 8), and
the means were separated by Duncan’s multiple range
test at 5% using generalized linear models (GLM).
Results
The effects of single or coinoculation with A.
chroococcum, B. polymixa, P. putida, and G.
intraradices on the growth parameters of 60-days-old
plants are shown in Table 1. All four microorganisms
showed stimulatory effect, in terms of plant height,
root length, fresh and dry weight of shoots and roots
when inoculated separately. Among the single treat-
ments, the maximal increase for majority of the
growth parameters (root length, fresh and dry weight
of shoots and roots) was shown by G. intraradices
plants in comparison to uninoculated plants. In
contrast, dual inoculation significantly enhanced the
plant height, root length, fresh and dry weight of
shoots and roots. Despite the enhanced growth
parameters of shoots and roots by biofertilizers, the
stimulatory effect of biofertilizers (in particular, dual
inoculation) on root dry weight was higher than shoot
dry weight. Inoculation with triple microorganisms
had less positive effects on plant growth of stevia in
comparison to dual inoculations (Table 1).
The maximum increase in shoot fresh weight
(53.7%), dry weight (57%), and height (37%) was
obtained in A. chroococcumG. intraradices treat-
ment. Furthermore, maximum root length was ob-
tained in dual inoculations of A. chroococcumG.
intraradices by 21.54%. A. chroococcumG.
intraradices produced almost 2.52 and 2.9 times
more fresh and dry root matter relative to the
noninoculated plants. Dual symbiotic plants were
approximately twofold larger than nonsymbiotic
plants, and the shoot growth of the dual symbiotic
plants was increased approximately 2.5 and 5-fold
 Journal of Plant Interactions 131
Table 1. Effect of biofertilizer inoculation on plant growth after 60 days of growth.A

Plant height Root length Fresh weight Dry weight Fresh weight Dry weight
Treatments (cm) (cm) shoot (g) shoot (g) root (g) root (g)

Control 46.7595.38b 16.2590.96bc 23.7992.21d 6.4990.67b 13.9590.19d 1.5390.06f

A. chroococcum 51.2592.87ab 15.2590.50c 28.9191.59abcd 7.290.39ab 17.6790.85d 2.0390.50ef
B. polymixa 48.595.01ab 15.2590.96c 25.7693.05cd 6.9891.01ab 18.1790.74d 2.7690.05de
P. putida 50.593.11ab 16.7590.96abc 27.0193.52bcd 7.190.89ab 18.0790.77d 2.6690.04de
G. intraradices 50.7592.5ab 18.591.29ab 31.0593.16abcd 7.9490.42ab 18.8890.90d 2.8590.05cde
A. chroococcum 64.2594.92a 19.7590.96a 36.5790.76a 10.2190.77a 35.2391.11a 4.3990.05a
G. intraradices
P. putidaG. 59.590.58ab 19.591.29a 33.0192.44abcd 8.8990.33ab 30.2490.92abc 3.9490.04abc
intraradices
B. polymixaG. 59.594.04ab 19.2590.96a 36.5393.50a 9.3590.49ab 31.5890.58abc 4.0990.08ab
intraradices
A. chroococcum 60.2592.06ab 16.7590.96abc 31.6393.16abcd 9.1592.04ab 27.2590.83bc 3.2290.06bcd
B. polymixa
A. chroococcum 58.7596.99ab 19.2590.96a 35.8294.20ab 9.1791.72ab 33.9790.85ab 4.1890.10ab
P. putida
A. chroococcum 53.2594.27ab 18.590.58ab 33.8096.82abc 7.7791.01ab 26.9490.88bc 3.290.08bcde
B. polymixa
G. intraradices
A. chroococcum 52.7593.20ab 15.2590.50c 32.7993.10abcd 7.7290.91ab 26.3190.50c 3.0790.08bcde
P. putidaG.
intraradices.
A
For each response variable, values (means of four replicates) not sharing a common letter differ significantly (Pb 0.05) from each other
(Duncan’s multiple range test).

compared to single inoculated or uninoculated plants,
respectively. Although slightly less pronounced, a
similar pattern was observed for root growth with
the same treatments (Table 1).
The effects of single or coinoculation with A.
chroococcum, B. polymixa, P. putida, and G.
intraradices on the chlorophyll content of 60-days-
old plants are shown in Figure 1. The maximum
chlorophyll a content was recorded in the dual
symbiotic plants with A. chroococcumG.
intraradices, B. polymixaG. intraradices, and A.
chroococcumP. putida by 3.8, 3.5, and 3.3 times
compared to the controls. The highest chlorophyll b
content was found in treated plants with A.
chroococcumG. intraradices, A. chroococcumP.
putida and A. chroococcumB. polymixa by increas-
ing 3.2, 2.6, and 2.5 times compared to the unin-
oculated plants. Results also showed that highest
total chlorophyll was found in treated plants with A.
chroococcumG. intraradices, A. chroococcumP.
putida and B. polymixaG. intraradices by 3.5, 3,
and 2.8 times compared to the controls. Among the
single inoculations, A. chroococcum showed maxi-
mum chlorophyll a, b, and total content, and the
uninoculated plants displayed the lowest chlorophyll
a, b and total content. Moreover, the results showed
that triple inoculations had more positive effects on
chlorophyll a, b, and total content than had the single
inoculation, but their effects were less than those of
dual inoculations (Figure 1).
Single inoculation treatments, in general, in-
creased the N content relative to the control (Table
2). Among the single inoculation, A. chroococcum
increased the N content significantly by 28% relative
to the control. Results also showed that dual inocu-
lations significantly stimulated the N content in
plants compared with the control. Among the dual
inoculation treatments, coinoculation of A.
chroococcumG. intraradices, A. chroococcumP.
putida showed the highest positive effect on N content
and increased the N content by 49 and 39%,
respectively, in comparison to control (Table 2).
The results also showed P content in plants
significantly increased with the inoculation of AM
fungi alone or in combination with PGPR (Table 2).
Among the single inoculation treatments, P-solubilizing
bacteria and AM fungi significantly increased P con-
tent compared to the control. However, the effect of P-
solubilizing bacteria on P content had been less than
AM fungi (Table 2). The P content was significantly
enhanced in inoculated plants with A. chroococcum-
G. intraradices, B. polymixaG. intraradices, and P.
putidaG. intraradices by 3.3, 3.09, and 3.05 times in
comparison to the control (Table 2).
Yet once again, the dual symbiotic plants had
higher K content than any of the other treatments
(Table 2). The present results also showed that among
all dually inoculated plants, plants inoculated with
AM fungi and PGPR had maximum K content. In
plantlets inoculated with B. polymixaG.
intraradices, A. chroococcumG. intraradices, and
P. putidaG. intraradices K content increased 62%,
60%, and 58% in comparison to noninoculated
plants (Table 2).
The triple inoculation of A. chroococcumB.
polymixaG. intraradices, and A. chroococcumP.
132 F. Vafadar et al.

(a) 0.45
a
0.4 b b
0.35 c

chlorophyll a
d

(mg/g.FW)
0.3 e e
0.25 f g g
0.2 h
0.15 i
0.1
0.05
0

Treatments
(b) 0.18 a
0.16
chlorophyll b (mg/g.FW)

0.14 b
b
b
0.12 c c
0.1 cd cd
d
0.08 e e
e
0.06
0.04
0.02
0

Treatments

(c) 0.6 a
b
0.5 c
d d
total chlorophyll

0.4 e e
(mg/g.FW)

f
0.3 g g
h
0.2 i

0.1

Treatments

Figure 1. Bio-fertilizer inoculation effects on chlorophyll a (a) chlorophyll b (b) and total chlorophyll (c) content after 60 days
of growth. Error bars represent standard deviation.

putidaG. intraradices significantly improved the
NPK content compared to the control. The lowest
N, P, and K uptake was detected in plants grown in
uninoculated pots (Table 2).
The results also showed that among the single
microbial inoculums, the AM fungi were better at
improving stevioside content (Table 2). Among all
inoculation treatments, dual inoculation of plants
had the highest positive effect on stevioside. Coin-
oculation of A. chroococcum with G. intraradices
increased stevioside by 83% in comparison with the
control. The results showed inoculated plantlets with
B. polymixaG. intraradices also significantly in-
creased stevioside by 53% compared to the control.
Triple inoculation had less positive effects on stevio-
side compared to dual inoculations (Table 2).
Discussion
The synergistic benefits of the dual inoculation of
stevia obviously improved the relative growth, bio-
mass, and nutrition assimilation of the host plants
 Journal of Plant Interactions 133
Table 2. Effect of biofertilizer inoculation on NPK and stevioside after 60 days of growth.A

Nitrogen Phosphorus Potassium Stevioside

Treatments (mgg 1) (mgg 1) (mgg 1) (mgg 1)

Control 2.8390.06i 4.0190.14i 243.7911.1g 48.0591.08e

A. chroococum 3.6290.04c 7.2590.33h 330.3796.22e 47.1994.22e
B. polymixa 3.3490.05efg 8.7290.37g 342.6796.39de 61.0796.92d
P. putida 3.1590.04h 9.7390.83f 353.2794.90cd 60.6297.9d
G. intraradices 3.2490.06gh 11.1390.66de 306.6398.39f 63.0193.75cd
A. chroococcumG. intraradices 4.2190.13a 13.2590.18a 391.6595.75a 87.795.01a
P. putidaG. intraradices 3.28f90.06g 12.2190.22bc 385.0794.80a 66.5193.41bcd
B. polymixaG. intraradices 3.3890.03ef 12.3790.35b 395.07913.8a 73.7191.61b
A. chroococcumB. polymixa 3.5490.07cd 10.4490.41ef 371.595.56b 61.9591.62d
A. chroococcumP. putida 3.9490.08b 11.5390.52cd 358.7393.48bc 71.2299.1bc
A. chroococcumB. polymixaG. intraradices 3.4390.03de 8.8990.22g 354.0397.51cd 67.9294.51bcd
A. chroococcumP. putidaG. intraradices 3.3790.04ef 10.4290.6ef 360.2392.6bc 64.1791.92cd
A
For each response variable, values (means of four replicates) not sharing a common letter differ significantly (Pb 0.05) from each other
(Duncan’s multiple range test).

(Tables 1 and 2). The improved growth of the dual
symbiotic plants is in agreement with the previous
study carried out by Hemavathi et al. (2006), who
reported improved growth and biomass yield of
Ocimum basilicum that was inoculated with G.
fasciculatum and PGPR. Vasanthakumar (2003) also
reported that combined inoculation of Azospirillum
(AZUS10) and PSB isolate (PSB7) produced syner-
gistic effect, resulting in increased root length, shoot
length, stem girth, number of leaves, and number of
branches in solanaceous crop plants. This improved
growth is most likely a result of improved nutrition,
leading to higher photosynthetic rates.
The increasing of chlorophyll in the dual symbio-
tic plants (Figure 1) probably resulted in higher
photosynthetic rates and thus improved plant bio-
mass. It has also been shown in the study of Aseri
et al. (2008) who reported that the highest total
chlorophyll was observed in dual inoculated plants
of Punica granatum by G. mosseae and A. brasilense
after 4 months. The increasing of chlorophyll that
could lead to higher rates of photosynthesis is
dependent on two factors: first, a greater C sink
below ground due to the two symbionts and second,
by the improved nutrition of the host plants. Inter-
estingly, despite the below-ground C sink, more C
was invested into the above-ground tissue of the dual
symbiotic plants in comparison to the other treat-
ments. This was, likely, the result of cost-benefit
effects between the C and nutrient sinks. To begin
with, the higher demand for below-ground C resulted
from having to support both symbionts may have
caused an increase in the photosynthetic rates of these
plants, as shown in the work of Kaschuk et al. (2009).
Additional evidence in favor of the argument regard-
ing the C and nutrient sinks resulting in higher above-
ground C investment is that the dual symbiotic plants
were dramatically more efficient in terms of NPK
capture than any of the other treatments; thus,
allowing more C to be available for growth in place
of nutrient assimilation. The high shoot NPK
concentrations would allow for increased shoot
growth and photosynthetic rates. The occurrence of
increased above-ground growth was also reported by
Singh et al. (2012), who found that Coleus forskohlii
plants colonized by both Pseudomonas monteilii and
Glomus fasciculatum had higher N, P, and K uptake
and greater above-ground biomass. Aseri et al. (2008)
assessed the effectiveness of PGPR (A.chroococcum
and A. brasilense) and AM fungi (G. mosseae and G.
fasciculatum) on the growth, nutrient uptake, and
biomass production of pomegranate (Punica
granatum L.) in the field experiments in India. Results
showed that dual inoculation of PGPR and AM fungi
led to higher biomass production and increase in the
uptake of N as well as P, K, Ca, and Mg in
pomegranate seedling. Aseri et al. (2008) attributed
this to the increased N, P levels, resulting from
improved biological N fixation (BNF) and phospha-
tase activity. Das et al. (2007) conducted a green-
house study to evaluate the effects of three
biofertilizers consisting of AM fungi, PSB, and
Azospirillum on the growth of S. rebaudiana. They
reported that AM fungi and PGPR inoculants
increased the growth, nutritional assimilation, and
microelements of stevia due to their ability to fix
atmospheric nitrogen and transform native soil
nutrients such as P, Zn, Cu, Fe, and sulfur from
nonuasble (fixed) to soluble form. On the other hand,
it is suggested that production of phytohormones by
PGPR led to improved root and hairy roots growth.
Thus, the levels of NPK uptake were increased from
soil (Wu et al. 2005).
The AM-inoculated plants had dramatically in-
creased levels of P in plants especially in comparison
with the non-AM plants. It is also shown that the AM
fungi plays an important role in both indirect and
direct N nutrition of host plants. The high levels of N
and K were found in single AM fungi inoculated
plants, compared to the uninoculated plants. It
indicated the direct influence of AM fungi on the
levels of N in host plants. This is in line with previous
134 F. Vafadar et al.
studies showing that AM fungi are able to directly
assimilate and provide N and K to the host plants
(Toussaint et al. 2004; Mortimer et al. 2009).
In the present study, the high level of N concen-
trations was found in the dual inoculated plant by A.
chroococcum with G. intraradices plants. It indicated
the indirect role of AM fungi in the N nutrition of the
host plants, by enhancing biological N fixation
(BNF). Mortimer et al. (2009) illustrated that in
tripartite symbiosis between legumes, AM fungi,
and nodule bacteria, the presence of AM fungi led
to increased rate of BNF and plant N concentrations.
These findings suggest that AM plays an important
role both directly through enhancing nutrition and
indirectly through enhancing synergistic benefits of
the dual colonization in order to increase the ability
of stevia to become established in soils low in N and
P. The increased levels of P in roots in dual
inoculation would ensure continued nitrogen fixation
by Azotobacter, thus providing additional N to be
used in the photosynthetic process of the host plants.
This is evident from the higher transport of N to the
shoots and subsequent increase in above-ground
biomass, as discussed earlier.
In the present study, stevioside was also signifi-
cantly increased in inoculated plants especially by
exposure to A. chroococcumG. intraradices (Table
2). Steviol glycosides are diterpenoids whose biosyn-
thetic pathways share four steps in common with
gibberellic acid formation (Brandle and Telmer 2007).
Awasthi et al. (2011) reported that while individual
inoculation with an AM fungus (G. mosseae) or
Bacillus subtilis was not effective in increasing arte-
misinin content, dual-inoculation (G. mosseaeB.
subtilis) significantly increased the content of artemi-
sinin. Banchio et al. (2010) reported that biosynthesis
of terpenoids depends on primary metabolism, photo-
synthesis, and oxidative pathways for carbon and
energy supply. Both AM fungi and PGPR are capable
of increasing the primary metabolites by improving
the photosynthesis, mineral content, and growth of
plants. They suggested that the promoting effect of
biofertilizers on secondary metabolites production
may be due to the good balance of nutrients and
water in the root zone. Besides, we believe that the
observed increases in the synthesis of stevioside,
which is a secondary metabolite, may be related to
a production of elicitors by AM fungi and PGPR.
Maybe releasing of hormones and other biologically
active molecules by microorganisms in soil acts as
elicitors which stimulated stevioside production. Re-
cently, the effect of rhizobacterial volatile organic
compounds (VOCs) on the formation of plant
secondary compounds in Mentha piperita has indi-
cated an almost twofold increase in accumulation of
essential oils in plants treated with P. fluorescens as
compared to B. subtilis, A. brasilensis, or control
(Santoro et al. 2011).
The physiological advantages associated with the
triple inoculation of stevia were less than dual
inoculations. It may be related to the fact that a
triple symbiosis would involve a larger C sink burden
than either symbionts on its own. Therefore, by
limiting symbiotic development of both AM fungus
and PGPR, the amount of C used by the respective
symbionts can be regulated. In support of this fact,
Pearson et al. (1993) found that competition for root
C could limit the extent of AM colonization. On the
other hand, it is possible that reduction effect of triple
inoculation, due to the release of inhibitory metabo-
lites in the growing environment, in turn adversely
affected the plant growth or other microorganisms.
These hypotheses must be investigated further in
future. However, these findings are contrary to other
studies showing improved growth and development
of plants under triple inoculations. Zaidi et al. (2003)
reported that in low P soils, the highest plant growth
and nutrient uptake in chickpea were obtained after
inoculation with tripartite culture of Mesorhizobium,
PSB, and G. fasciculatum. Das and Dang (2010) also
reported that the highest increase of biomass and
stevioside were recorded in combined application of
AMFPSBAzospirillum in acidic soils. The differ-
ence in the results of these authors with our data may
be due to difference in type, concentration and
combination of applied microorganisms or different
conditions of experiment performance.
Our results demonstrate that using an appropriate
combination of mycorrhizal fungi and PGPR can
significantly increase the production of leaf dry mass
and concentration of stevioside. The application of
dual inoculation with AM fungi and N-fixing bacteria
seems to be the most effective treatment combination
to improve plant growth, plant nutrient uptake, total
chlorophyll, and stevioside content. In fact, dual root
colonization usually increases the growth of host
over-and-above that of single inoculation. One reason
for this response is the improvement of the nutritional
balance of hosts, leading to increase chlorophyll
content and maintain a high rate of photosynthesis,
which compensates the loss of carbon by a large sink
of both symbionts. In addition, G. intraradices and A.
chroococcum consortium seems to provide the most
optimal mix of macronutrients for plants which in
turn reflects the maximum stevioside content as well
as yield compared to that obtained with the other
bioinoculants tested (single, triple, or other dual
combination). It is also known that P availability in
soils is important for the uptake of soil nitrogen and
its utilization in plants. Therefore, more available P
due to its solubilization by inoculated AM fungus or
PSB could lead to improved N uptake or nitrogen
fixation by Azotobacter. These aspects have caused
increased dry matter and stevioside content of S.
rebaudiana.
Despite attempts to produce stevioside in hetero-
logous systems till date, the S. rebaudiana is the only
 Journal of Plant Interactions
source of commercial stevioside in the world, and
global attempts to increase the stevioside content of
this plant, reduce its cost of production. To this end,
the use of microbial consortia as G. intraradices and
A. chroococcum is precisely aimed in this direction. In
addition, it demonstrates the possibility of replacing
chemical fertilizers with biofertilizers, which in turn
will help to mitigate the major global problem of
environmental pollution and also fetch a premium in
the agriculture market.
Acknowledgments
We thank Prof. Emtiazi for the strain of Pseudomonas
putida (accession number JX 44133). The authors are
grateful to Dr. Abdol Reza Nabinejad for his kind
cooperation and help in providing laboratory facilities for
this research, and Dr. Javad Hashemi for HPLC analysis,
and Prof. Asghari for his guidance. The work was
supported by the research assistance of Shahrekord Uni-
versity and the Agricultural Biotechnology Research In-
stitute, Central region of Iran (ABRICI), Isfahan, Iran.
References
Adesemoye AO, Torbert HA, Kloepper JW. 2009. Plant
growth-promoting rhizobacteria allow reduced appli-
cation rates of chemical fertilizers. Microb Ecol.
58:921
929.
Adriano-Anaya ML, Salvador-Figueroa M, Ocampo JA,
Garcia-Romera I. 2006. Hydrolytic enzyme activities
in maize (Zea mays) and sorghum (Sorghum bicolor)
roots inoculated with Gluconacetobacter diazotrophicus
and Glomus intraradices. Soil Biol Biochem. 38:879

886.
Ahmed MB, Salahin M. 2007. An efficient method for in
vitro clonal propagation of a newly introduced sweet-
ener plant Stevia rebaudianain in Bangladesh. Am J Sci
Res. 2:121
125.
Anonymous. 1980. Official methods of analysis. 13th ed.
Washington, DC: The Association of Official Analy-
tical Chemists.
Antunes PM, Varennes Ade, Zhang T, Goss MJ. 2006. The
tripartite symbiosis formed by indigenous arbuscular
mycorrhizal fungi, Bradyrhizobium japonicum and
soybean under field conditions. J Agron Crop Sci.
19:373
378.
Arnon DI. 1949. Copper enzymes in isolated chloroplast:
polyphenol oxidase in Beta vulgaris. Plant Physiol.
24:1
15.
Aseri GK, Jain N, Panwar J, Rao AV, Meghwal PR. 2008.
Biofertilizers improve plant growth, fruit yield, nutri-
tion, metabolism and rhizosphere enzyme activities of
Pomegranate (Punica granatum L.) in Indian Thar
desert. Scientia Hort. 117:130
135.
Awasthi A, Bharti N, Nair P, Singh R, Shukla AK, Gupta
MM, Darokar MP, Kalra A. 2011. Synergistic effect of
Glomus mosseae and nitrogen fixing Bacillus subtilis
strain Daz26 on artemisinin content in Artemisia annua
L. Appl Soil Ecol. 49:125
130.
Banchio E, Bogino PC, Santoro M, Torres L, Zygadlo J,
Giordano W. 2010. Systemic induction of monoter-
pene biosynthesis in Origanummajoricum by soil
bacteria. J Agric Food Chem. 58:650
654.
135
Bianciotto V, Bonfante P. 2002. Arbuscular mycorrhizal
fungi: a specialized niche for rhizospheric and endo-
cellular bacteria. Anton van Leeuwenhoek. 81:365

371.
Biswas JC, Ladha JK, Dazzo FB, Yanni YG, Rolfe BG.
2000. Rhizobial inoculation influences seedling vigour
and yield of rice. Agron J. 92:880
886.
Bovanova L, Brandsteterova E, Baxa S. 1988. HPLC
determination of stevioside in plant material and food
samples. Z Lebensm Unters Forsch A. 207:352
355.
Brandle JE, Telmer PG. 2007. Steviolglycoside biosynth-
esis. Phytochemistry 68:1855
1863.
Charles P, Raj ADS, Kiruba S. 2006. Arbuscular mycor-
rhizal fungi in the reclamation and restoration of soil
fertility. Mycorrhiza News. 18:13
14.
Chatsudthipong V, Muanprasat C. 2009. Stevioside and
related compounds: therapeutic benefits beyond sweet-
ness. Pharmacol Ther. 121:41
54.
Das K, Dang R. 2010. Influence of biofertilizers on
stevioside content in Stevia rebaudiana grown in acidic
soil condition. Archiv Appl Sci Res. 2:44
49.
Das K, Dang R, Shivananda T, Sekeroglu N. 2007.
Influence of bio-fertilizers on the biomass yield and
nutrient content in Stevia rebaudiana Bert. grown in
Indian subtropics. J Med Plants Res. 1:005
008.
Debnath M. 2008. Clonal propagation and antimicrobial
activity of an endemic medicinal plant Stevia
rebaudiana. J Med Plants Res. 2:045
051.
Geuns JMC. 2003. Molecules of interest stevioside. Phy-
tochemistry 64:913
921.
Gianinazzi S, Gollotte A, Binet MN, Van Tuinen D,
Redecker D, Wipf D. 2010. Agroecology: the key role
of arbuscular mycorrhizas in ecosystem services. My-
corrhiza. 20:519
530.
Gianinazzi S, Huchette O, Gianinazzi-Pearson V. 2008.
New outlooks in mycorrhiza applications. In: Baar J,
Estaun V, Ortas I, Orfanoudakis M, Alifragis D,
editors, Proceedings of the COST870meeting ‘‘Mycor-
rhiza Application in Sustainable Agriculture and
Natural Systems’’; 2008 Sept 17
19; Thessaloniki,
Greece, p. 20
22.
Gosling P, Hodge A, Goodlass G, Bending GD. 2006.
Arbuscular mycorrhizal fungi and organic farming.
Agric Ecosystems Environ. 113:17
35.
Hemavathi VN, Sivakumar BS, Suresh CK, Earanna N.
2006. Effect of Glomus fasciculatum and plant growth
promoting rhizobacteria on growth and yield of
Ocimum basilicum. Karnataka J Agric Sci. 19:17
20.
Jackson ML. 1973. Soil chemical analysis. New Delhi:
Prentice Hall, p. 239
241.
Javaid A, Iqbal SH, Hafeez FY. 1994. Effect of different
strains of Bradyrhizobium and two types of vesicular
arbuscular mycorrhizae (VAM) on biomass and nitro-
gen fixation in Vigna radiata (L.) Wilczek var. NM 20

21. Sci Inter (Lahore) 6:265
267.
Kaschuk G, Kuyper TW, Leffelaar PA, Hungaria M, Giller
KE. 2009. Are the rates of photosynthesis stimulated
by the carbon sink strength of rhizobial and arbuscular
mycorrhizal symbioses? Soil Biol Biochem. 41:1233

1244.
Kolb NJ, Herrera L, Ferreyra DJ, Uliana RF. 2001.
Improved HPLC method. J Agric Food Chem.
49:4538
4541.
Liu A, Hamel C, Hamilton RI, Ma BL, Smith DL. 2000.
Acquisition of Cu, Zn, Mn, and Fe by mycorrhizal
136 F. Vafadar et al.
maize (Zea mays L.) grown in soil at different P and
micronutrient levels. Mycorrhiza. 9:331
336.
Miransari M. 2011. Interactions between arbuscular my-
corrhizal fungi and soil bacteria. Appl Microbiol
Biotechnol. 89:917
930.
Mortimer PE, Pérez-Fernández MA, Valentine AJ. 2009.
Arbuscular mycorrhiza affects the N and C economy
of nodulated Phaseolus vulgaris (L.) during NH4
nutrition. Soil Biol Biochem. 41:2115
2121.
Murashige T, Skoog F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiol Plantarum. 15:473
497.
Pearson JN, Abbott LK, Jasper DA. 1993. Mediation of
competition between two colonizing VA mycorrhizal
fungi by the host plant. New Phytol. 123:93
98.
Santoro MV, Zygadio J, Giordano W, Banchio E. 2011.
Volatile organic compounds from rhizobacteria in-
crease biosynthesis of essential oils and growth para-
meters in peppermint (Mentha piperita). Plant Physiol
Biochem. 49:1177
1182.
Singh JS, Pandey VC, Singh DP. 2011. Efficient soil
microorganisms: a new dimension for sustainable
agriculture and environmental development. Agric
Ecosystems Environ. 140:339
353.
Singh R, Soni SK, Kalra A. 2012. Synergy between Glomus
fasciculatum and a beneficial Pseudomonas in reducing
root diseases and improving yield and forskolin con-
tent in Coleus forskohlii Briq. under organic field
conditions. Mycorrhiza. 23(1):35
44. doi:10.1007/
s00572-012-0447-x
Sivaram L, Mukundan U. 2003. In vitro culture studies on
Stevia rebaudiana. In Vitro Cell Dev Biol Plant.
39:520
523.
Toussaint JP, ST-Arnaud M, Charest C. 2004. Nitrogen
transfer and assimilation between the arbuscular
mycorrhizal fungus Glomus intraradices Schench and
Smith and Ri T-DNA roots of Daucus carota L. an in
vitro compartment system. Can J Microbiol. 50:251

260.
Vasanthakumar SK. 2003. Studies on beneficial endorhizo-
sphere bacteria in solanaceous crop plants. [M.Sc.
(Agri) thesis], Dharwad, Karnataka, India: University
of Agricultural Sciences.
Wilson PW, Knight SC. 1952. Experiments in bacterial
physiology. Minneapolis: Burguess, p. 49.
Woelwer-Rieck U, Lankes C, Wawrzun A, Wüst M. 2010.
Improved HPLC method for the evaluation of the
major steviol glycosides in leaves of Stevia rebaudiana.
Eur Food Res Technol. 231:581
588.
Wu SC, Cao ZH, Li ZG, Cheung KC, Wong MH. 2005.
Effects of biofertilizer containing N-fixer, P and K
solubilizers and AM fungi on maize growth: a green-
house trial. Geoderma. 125:155
166.
Wu OS, Xia RX. 2006. Arbuscular mycorrhizal fungi
influence growth, osmotic adjustment and photosynth-
esis of citrus under well-watered and water stress
conditions. J Plant Physiol. 163:417
425.
Zaidi A, Khan MS, Amil M. 2003. Interactive effect of
rhizotrophic microorganisms on yield and nutrient
uptake of chickpea (Cicer arietinum L.). Eur J Agron.
19:15
21.