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Contents lists available at ScienceDirect

South African Journal of Botany

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

Phytochemical response of Stevia plant to growth promoting

microorganisms under salinity stress
Arezou Forouzi, Azim Ghasemnezhad*, Reza Ghorbani Nasrabad
Gorgan University of Agricultural Science and Natural Resources, Gorgan, Iran

A R T I C L E I N F O A B S T R A C T

Article History: Stevia (Stevia rebaudiana Bertoni) is a salt-sensitive herb with sweet leaves without side effects for diabetic
Received 1 October 2019 patients. The present research was conducted to investigate the effect of endophytic fungus Piriformospora
Revised 17 March 2020 indica and two bacterial isolates, namely of Streptomyces sp. (kj152148) and (kj152149) with Stevia host
Accepted 2 April 2020
plants. These microbial isolates were utilized as growth-promoting agents for induction of salt stress toler-
Available online xxx
ance and secondary metabolite accumulation in inoculated plants. The present experiment was designed as
Edited by MG Kulkarni factorial basis experiment based on a completely randomized design with six replications undertaken in two
consecutive years (2016-2017). Treatments included three levels of salinity [0.85 (control), 1.5 and 3 dS/m]
Keywords:
and four levels of plant growth-promoting microorganisms [no-inoculation, Piriformospora indica, Kj152148
Bio-stimulant
Streptomyces sp (S1) and K152149 Streptomyces sp (S2)]. Under stress and non-stress conditions, endophytic
Elicitor
fungus and bacterial isolates had significant positive effect on the recorded biochemical and physiological
Glycoside
Medicinal plant parameters of stevia. Inoculation of fungus and bacterial isolates reduced the negative effects of salinity
Salinity stress by increasing antioxidant enzymes and free radical scavenging activity, phenol and flavonoid accumu-
lation and total glucose and proline concentrations. From the results it may be concluded that, indicated a
positive effect of applied fungus and bacterial treatments on the resistance of stevia plant to the salinity
stress and due to the relative susceptibility of stevia to salinity, pre-treatment of the plant using of the
desired microbes is recommended before planting in soil areas susceptible to salinity.
© 2020 SAAB. Published by Elsevier B.V. All rights reserved.

Introduction phenomenon of salinity and to minimize the effects of salinity and to

Stevia rebaudiana Bertoni is a herbaceous, perennial herb belongs
to the family Asteraceae (Madan et al., 2010; Barroso et al., 2016) and
native to South America, especially northeastern Paraguay (Molina-
Calle, 2016), which, due to its sweetening power, is known as "stevia"
or "honey leaves" (Ferrazzano et al., 2015). This plant grows naturally
on the Amambian Mountain near to the Mandy River (a narrow
region between Brazil and Paraguay) and is currently cultivated in
many parts of the world (Soufi et al, 2016; Porkayasta et al., 2016).
Today, the global importance of stevia is due to the presence of
sweet leaves which has traditionally been used in Japan, Korea, China
and South America. Stevia’s sweetness is due to the presence of gly-
cosides; they are soluble in water and considered 250 -300 times
sweeter than conventional sugar (Ramesh and Megeji, 2006). Stevia
plant is very sensitive to day length and needs 12 to 16 hours of sun-
light. Salinity is considered as a major problem in many arid and
semi-arid regions of the world, limiting growth, quality and perfor-
mance of horticultural and medicinal plants (Munns, 2005).
Researchers are now looking for new solutions for deal with the
* Corresponding author.
E-mail address: aghasemnajad@hotmail.com (A. Ghasemnezhad).
mitigate yield losses (Gallagher, 1985).
A possible solution to this context is the use of mycorrhizal symbi-
osis to stimulate plant growth, especially in stressful conditions
(Adya et al., 2013), and application of biotechnology and genetic anal-
ysis (Liang et al., 2019). Plants can overcome salt stress through inter-
action with soil microorganisms such as mycorrhizal fungi and plant
growth-promoting rhizobacteria (PGPR) (Smith and Read, 2008).
Plant growth-promoting rhizobacteria (PGPRs) may stimulate plant
growth through which these bacteria act as biological fertilizers,
plant stimulants and/or stress-ameliorating factors (Lugtenberg and
Kamilova, 2009). These bacteria can increase the plant resistance to
salinity stress through the production of 1-amino-cyclopropane-1-
carboxylate (ACC) deaminase enzyme (Costa et al., 2014).
In addition to that, solubilization of insoluble phosphate, nitrogen
fixation and phytohormone production are found to be involved in
salinity stress resistance induced by bacteria (Costa et al., 2014). Piri-
formospora indica is a mycorrhizal-like fungus that has the ability to
establish symbioses with the roots of different plants (Varma et al.,
2012). This fungus acts as a mycorrhizal fungus due to the conditions
stimulating plant growth (Varma et al., 2001). Unlike arbuscular
mycorrhizal fungi P. indica grows easily on artificial growth media
(Artursson, 2006).

https://doi.org/10.1016/j.sajb.2020.04.001
0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.04.001
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2 A. Forouzi et al. / South African Journal of Botany 00 (2020) 1 10
The importance of making a symbiotic relationship between P.
indica and different plant species has been reported by different
authors (Waller et al., 2005). This symbiotic relationship can affect
plant growth by stimulating plant growth and increasing plant toler-
ance to salinity, drought and pathogen (Waller et al., 2005). Stevia is
a relatively saline sensitive plant and the regeneration of it does not
recover at saline conditions (Ahmadi et al., 2007). As the relationship
between Stevia, a relatively saline sensitive plant, with grow pro-
moter microbes (bacteria and fungi) in salinity conditions did not
investigated well, the present work was carried out. Thus, according
to the problem of salinity of irrigation water, wide range of saline
soils and the importance of crop production in these conditions, the
purpose of current study was to investigate the ability of P. indica and
growth-promoting bacteria [Kj152148 Streptomyces sp (S1) and
K152149 Streptomyces sp (S2)] to improve growth, biomass yield,
secondary metabolite production and resistance of stevia plant to
salinity stress.
Materials and methods
The study was performed as pot experiment in outdoor condi-
tions, in two consecutive years of 2016-2017 from late of March to
beginning of September of each year. The stem cuttings of Stevia
rebaudiana Bertoni were collected from the research station of Gor-
gan University of Agricultural Sciences and Natural Resources. Exper-
imental pots were kept in outdoor conditions and were irrigated
with saline water in different salinity levels from two weeks after
planting till one week before harvest. Based on the time of growing
period and the year of experiments the temperature and relative
humidity of experimental location was were varied from 25-35°C
and 45-55%, respectively.
Preparation of inoculum of Piriformospora indica
Isolate of Piriformospora indica was procured from the department
of Phytopathology of Sari University of Agricultural Sciences and Nat-
ural Resources. The isolate of P. indica was grown onto Kaefer
medium (Kaefer, 1977) and incubated at 25°C for four weeks. Fungus
spores were scratched by spoon from surface of Petri dishes. Then, to
measure the concentration of suspension, a drop of sample was
placed on a hemocytometer slide and was inspected under the micro-
scope. Desirable concentration of 5 £ 107 spores per ml was obtained.
Preparation of inoculum of Streptomyces sp
The bacterial strains of used bacteria were taken from the Depart-
ment of Soil Science of Gorgan University of Agricultural Sciences
and Natural Resources. Yeast extract-malt extract (ISP2) medium was
used to growth Streptomyces isolates at 28 § 2°C for 5 days. For this
purpose the roots of experimental plants were kept in container con-
taining suspension of bacteria in the concentration of 108 CFU for one
hour and then planted in prepared pots.
Plant cultivation and treatments
The experimental plants were treated with either Piriformospora
indica or and two Streptomyces isolates [kj152148 (S1) and kj152149
(S2)] and non-inoculated plants were considered as control. To pre-
pare experimental plants, the obtained stem cuttings were planted in
medium containing sterilized perlite and cocopite (1:1) and equipped
with Mist system under greenhouse conditions. After two weeks, the
rooted cuttings were immersed in the inoculation suspension for
3 hours on the shaker (Behdad 3436, Iran) with speed of 75 rpm.
Then the treated and non-treated plants were transferred to plastic
pots. The bottom of pots was filled with coarse sand for proper drain-
age and 4 kg of soil mixture containing different parts of sand, peat
Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.04.001
[m5G;June 25, 2020;21:29]
and field soil (1:1:2, respectively). The pots were randomly arranged
and their location changed once a week. This experiment was con-
ducted as factorial in a completely randomized design with 6 pots
per treatments as 6 replications with two plants per pot. After culti-
vation, and when the plants were fully developed (two weeks after
planting), irrigation was done with water containing different levels
of salinity based on the treatments. The applications of treatments
was carried out via irrigation with saline water at three levels of
0.85 dS/m and 1.5 and 3 dS/m as non-saline (as control), mild and
severe salinity levels, respectively. In order to prevent the accumula-
tion of salt around the root, once a week pots were leached with non-
saline water. The plant biomass was then harvested in September.
The salinity levels determination were done based on Ahmadi et al.,
(2017).
Biochemical analysis
Total phenols
The total phenol content of samples was measured by Folin ciocal-
teu method (Singleton et al., 1999). First, 20 ml of the leaf methanolic
extract was mixed with 1.16 ml distilled water and 100 ml folin cio-
calteu. After 5 minutes, 300 ml of sodium carbonate (1M) was added
to the solution. Then, sample was placed in dark conditions for
20 min at room temperature. Estimation of total phenol was per-
formed at 760 nm using spectrophotometer (UNICO 2800 model,
USA). Different concentrations of gallic acid (soluble in methanol:
water 1:1) were used for drawing the calibration curve in three tech-
nical replicates. Total phenol content was reported in mg gallic acid
per gram of fresh weight (McDonald et al., 2001).
Flavonoid measurements
To measure flavonoid content, the method of aluminum chloride
was used (Yisa, 2009). In brief, 0.5 ml of methanolic extract with
1.5 ml of methanol, 0.1 ml of aluminum chloride (10% in ethanol),
0.1 ml of potassium acetate (1M) and 8.2 ml of distilled water were
mixed with each other and after 30 minutes incubation in the dark
conditions at room temperature, the spectrum absorption of sample
was read at 415 nm using spectrophotometer (UNICO model 2800,
USA). To calculate the flavonoid content of samples, the standard
curve was prepared with different concentrations of quercetin in
three replications. Total flavonoid content was expressed in terms of
mg/gr quercetin (mg/FW).
Antioxidant activity assay
Determination of the free radical scavenging (antioxidant) activity
of methanolic extract from samples was done according to Brand
William et al. (1995). One milliliter of the methanolic extract was
added to the 100 ml of 1, 1-diphenyl-2-picril hydroxyl (DPPH) solu-
tion. The mixture was incubated for 15 min at room temperature and
the antioxidant activity was measured at 517 nm using spectropho-
tometer (UNICO model 2800, USA) and reported as percentage.
Catalase (CAT) and peroxidase (POD) assessment
Catalase activity was assessed by measuring H2O2 reduction at
240 nm for one minute. The reaction mixture consisted of 100 mM
phosphate buffer (pH 7) and 15 mM H2O2 and 50 ml of enzyme solu-
tion in final volume of 3 ml. The activity of the enzyme was calculated
using an extinction coefficient of 39.4 mM/cm, and expressed as
micromole of hydrogen peroxide consumption per minute per millili-
ter of protein (Abi, 1984). The reaction mixture for measuring peroxi-
dase activity contained one milliliter of 100 mM phosphate buffer
(pH 7), 0.1 mM EDTA (250 ml), one milliliter of 5 mM guaiacol, one
milliliter of hydrogen peroxide and 50 ml of enzyme solution. The
reaction was started at room temperature, by adding the enzyme
solution via increasing the absorbance at 470 nm for one minute
(Tang and Newton, 2005).
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A. Forouzi et al. / South African Journal of Botany 00 (2020) 1 10 3

Proline content Total phenol and flavonoid contents

In this study, proline content was measured according to the Bates
method (Bates et al., 1973) as mMol/g FW. Briefly, 0.5 gram of leaf
was extracted in 3% sulphosalicylic acid. Proline concentrations of
the samples were determined by ninhydrin acid in toluene using
spectrophotometry (UNICO 2800 model) at 520 nm. Proline was used
as standard in different concentrations and each concentration was
repeated three times.
Total sugars content
Total sugars measurement was performed based on Mac Cready et
al. (1950) method expressed as mg/g DW. A volume of 0.1 ml (100
ml) of alcoholic extract was concentrated and 100 ml of distilled
water was mixed with 3 ml of anthrone and placed in a water bath
(100°C) for 20 minutes. When the temperature of samples reduced,
the absorbance was recorded at 620 nm. Different concentrations of
pure glucose were used to obtain standard curve and was reported as
mg/g fresh weight.
Reduced Sugar content
The content of total reduced sugar was assessed by the
method of Miller et al. (1959) mg/g FW. For this purpose, 1.5
milliliters of concentrated alcoholic extract with 1.5 milliliters of
dinitro-salicylic acid was placed in a water bath for 20 minutes at
90°C. Immediately and before cooling the contents of the tube,
0.5 milliliters of potassium sodium tartrate salt solution (40%)
was added to the mixture. After cooling, the spectrum of dark-
red solution was determined at 620 nm. Different concentrations
of pure glucose were used to obtain standard curve and the result
was reported as mg/g fresh weight.
Statistical analysis
This experiment was conducted as factorial in a completely ran-
domized design with 6 replications. Each replicate included one pot
with two plants per pot. Statistical analysis was performed using SAS
software (SAS Institute, 2001). Mean values were compared by LSD
test at a significance level of 0.95% (P <0.05).
Results and discussion
The effect of experimental year (Y) on the most of measured
parameters except proline content and antioxidant activity was sig-
nificant. All measured parameters were significantly influenced by
utilized microbes (M), salinity, the interaction of year and microbes,
microbes and salinity and the triple interactions of year, salinity and
microbes. Catalase activity of the plant extract was not influenced by
interaction effect of year and microbes (Table 1).
The highest amount of phenolic compounds accumulation
belonged to plants inoculated with endophytic fungi in salinity level
of 3 dS/m. It is noteworthy that application of endophytic fungi and
plant growth-promoting bacteria increased phenol content in leaves
by increasing salinity level. Increased production of phenolic com-
pounds under salinity effects has been reported in various plants
(Sarahi Nobar et al., 2010; Radi et al., 2013). Studies by other
researchers have also highlighted the effects of salinity on increasing
metabolism activity. For example, in mint, artichoke and buckwheat
the phenolic content of the cell significantly increased by increasing
the salinity level (El-Danasoury, 2010; Rezazadeh et al., 2012; Lim et
al, 2012). It has also been reported that the phenolic content of olive
roots and leaves was significantly influenced by salinity ( Petridis et
al, 2012). The total phenolic content of the plant tissue increased
with increasing salinity (Fig. 1). The overall trends in total phenolic
content were relatively similar in two consecutive years of the exper-
iment. Although, the total phenolic content increased under the influ-
ence of salinity, accumulation of phenolic compounds when plant
treated with different microbes was much higher than the non-inoc-
ulated plants. At all salinity levels, the phenol accumulation of fun-
gus inoculated plants tended to be higher than those inoculated
with bacteria. The same trends were observed in the second year of
the experiment (Fig. 1).
Considering flavonoid variations under the influence of salinity
stress, it was observed that, similar to the total phenol, the amount of
these compounds increased with increasing salt stress and improved
on the use of endophytic fungus and growth-promoting bacteria
(Fig. 2). Under severe stress conditions, the highest amount of flavo-
noid (73.47 mg/g dry weight) was recorded in plants exposed to S1
bacteria (3 dS/m) (Fig. 2). The results showed that, the phenol and fla-
vonoid accumulation of S1 contaminated plants in the second year of
experiment, decreased as compared to the first year. At high salinity
level there was no significant difference on phenolic content of the
cell between plants exposed to bacteria (S1) and fungi but plants
exposed to fungi accumulated flavonoid much lower than those
exposed to bacteria in both two consecutive years. It is obvious that,
the plants use various phenolic and antioxidant sources in order to
protect themselves in oxidative stress-inducing conditions. Increas-
ing the phenylpropanoid compounds affected by fungi and bacteria
in salinity conditions indicates that, at the presence of plant growth-
promoting microbes and under the saline conditions, plants try to
keep phenolic sources at high levels so as to mitigate salinity stress.
Recent studies indicate that fungi contribute to/or are responsible for
plant adaptation to stress (Singh et al, 2011). Symbiotic fungi and
bacteria may induce plant tolerance to different biotic and abiotic
stresses. In addition to that they promote plant growth and facilitate
nutrient acquisition (Singh et al, 2001). Plant responses to stress are
complex, involving signals reception and transduction followed by
genetic and physiological reactions (Bartels and Sunkar, 2005). For

Table 1
Analysis variance of the effect of salinity (S) and microbes (M) on biochemical properties of stevia leaves extract in two consecutive years.

Proline content Flavonoid content Antioxidant activity Phenol content Total sugar Reduced sugar Peroxidase Catalase
ns ns
Year (Y) 0.627 48.349** 10.687 7978.956** 7488222.453** 1905.541** 4870.179** 0.192**
Microbe (M) 42.518** 50.113** 2086.158** 14973.187** 238868.153** 10398.353** 2908.697** 0.189**
Salinity (S) 759.436** 129.989** 11652.866** 990.006** 837631.095** 51729.756** 10123.014** 2.849**
Y£M 3.77* 14.612** 608.429** 11471.244** 46737.947** 901.072** 472.684** 0.002ns
Y£S 10.141** 24.274** 165.13** 3140.737** 72858.755** 343.014** 2431.517** 0.313**
M£S 35.056** 10.361** 208.49** 2496.68** 26537.913** 2148.991** 814.723** 0.068**
Y£M£S 10.099** 16.249** 648.955** 1650.827** 43974.505** 1287.387** 46.343** 0.008**
CV% 17.06 0.70 2.81 0.58 0.36 0.76 2.11 2.38
* ns, and
** non-significant, Significant at the 5% and 1% levels probability, respectively

Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
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Fig. 1. Effect of NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on the total phenolic content of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and
K152149 Streptomyces sp (S2), Different letters in the same column indicate significant differences according to LSD's test (P<0.05). Big and small letters referred to first and second
experimental years, respectively.

Fig. 2. Effect of NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on the total flavonoid content of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and
K152149 Streptomyces sp (S2). Different letters in the same column indicate significant differences according to LSD's test (P<0.05). Big and small letters referred to first and second
experimental years, respectively.

example, it has been reported that tripartite interactions among Pae-
nibacillus lentimorbus, Piriformospora indica and Cicer arietinum L.
(Chick pea) enhance root nodulations and plant growth. Due to this
interaction, it has been shown that Proline, lysine, glutamine and glu-
tamic acid were greatly utilized under stress conditions (Nautiyal et
al, 2010).
Antioxidant activity
Antioxidant activity was measured by DPPH free radical capturing
procedure on plant extract. As, no significant effect of experimental
year was observed on antioxidant activity of plant extract (Table 1),
only the data of second experimental year was presented. The high-
est inhibitory activity was observed in plants inoculated with P. indica
under salinity level of 3 dS/m and the lowest was observed in control
treatments. Increasing salinity increased free radical inhibitory activ-
ity in inoculated plants compared to controls (Fig. 2). Comparing the
effect of used microorganisms on the antioxidant potency of leaf
extract of stevia, it seems that plants treated with fungi had higher
Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.04.001
potency than Streptomyces isolates. A higher inhibitory activity can
be attributed to phenylpropanoid content. The highest amount of
these compounds was found in the methanolic extract of plants inoc-
ulated with fungi and bacteria. One of the first responses of plants
against stressful conditions is the excessive production of reactive
oxygen species (ROS). The removal of ROS from plant cells, in addi-
tion to the enzyme system, in many cases, occurs with non-enzy-
matic antioxidant systems. In tomato, it has been showed that,
antioxidant activity was strongly affected by salinity stress (Al Hassan
et al., 2015). As the salinity increased from zero to 450 mM, the
malondialdehyde increased from 145 to 241 nmol/g DW. A similar
trend was observed in other antioxidant agents including total phe-
nol and total flavonoid contents. Non-enzymatic antioxidant acts as
free radical inhibitors to protect plants from oxidative damage (Rice-
Evans et al., 1996; Wu et al., 2014). Also, this kind of antioxidant sys-
tem acts as inhibitors and reduces the free radicals to protect plants
from oxidative damage (Rice-Evans et al., 1996; Wu et al., 2014).
Kumar et al. (2009) reported that inoculation of P. indica increased
the activity of corn antioxidant enzymes under the stress of fungal
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Fig. 3. Effect of NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on the radical scavenging activity of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and
K152149 Streptomyces sp (S2), Different letters in the same column indicate significant differences according to LSD's test (P<0.05).

Fig. 4. Effect NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on catalase (CAT) activity of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and K152149
Streptomyces sp (S2), Different letters in the same column indicate significant differences according to LSD's test (P<0.05). Big and small letters referred to first and second experi-
mental years, respectively.

pathogens. They also stated that by increasing the antioxidant capac-
ity of barley, P. indica increased the tolerance to Fusarium spp. In
wheat, Afridi et al, (2019) showed that, high levels of NaCl exhibited
negative effects. However, inoculation with microbes increased the
morphological traits and antioxidant activities of the plants while
decreasing the Na+ contents in all treatments compared to non-inoc-
ulated treatment. Previous studies showed that endophytic microbe
might not only degrade polycyclic aromatic hydrocarbons (PAHs)
absorbed into plant when they colonize and grow inside of the plant,
but also might promote plant growth and enhance host plant resis-
tance to stresses through improving the activities of enzymes and the
physiological and biochemical properties of the plant (Fu et al., 2020;
Duran et al., 2018; Ghosh et al., 2018).
Antioxidant enzyme activity
Analysis of variance between two consecutive years showed that,
the activities of peroxidase and catalase (CAT) enzymes in stevia leaf
were influenced by the type of microbe, salinity and the interaction
of microbe and salinity (Table 1). In both experiments, the mean
value showed that the effect of microbes and salinity on the activity
of the peroxidase and the catalase is significant and the activities of
the antioxidants enzymes increased with increasing the salinity level
(Fig. 4 and 5 ). So that, under the salinity level of 3 dS/m, the highest
activity of both catalase and peroxidase was observed in plants which
were treated with Streptomyces sp. (S2). The highest activity of anti-
oxidant enzymes was observed in plants that were treated with bac-
teria. However, at the high salinity level, the antioxidant activity of
plants treated with endophytic fungus was higher than growth-pro-
moting bacteria (Fig. 3). Studies have been suggested, that tolerance
of plants to salt stress is associated with the induction of antioxidant
enzymes (Herna
ndez et al., 2000, Sekmen et al, 2007). It has been
shown that, root colonization by P. indica increased plant growth and
reduced the NaCl-induced lipid peroxidation, reduced the flow of
metabolism and decreased the saturated fatty acid content in the
leaves of salt-sensitive cultivar of barley (Baltruschat et al., 2008).

Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
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Fig. 5. Effect of NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on peroxidase (POD) activity of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and
K152149 Streptomyces sp (S2), Different letters in the same column indicate significant differences according to LSD's test (P<0.05). Big and small letters referred to first and second
experimental years, respectively.
Kohlera et al. (2009) reported that, the inoculation of growth pro-
moter bacteria increased the level of antioxidant enzymes in
response to severe salinity. It seems that the process of endophytic
infection, by increasing the activity of antioxidant enzymes, has pro-
tective effect on the plant (Baltruschat et al., 2008, Li et al., 2012).
Plants contain a set of antioxidant enzymes such as catalase, superox-
ide dismutase and peroxidase that reduce oxidative stress (Baltru-
schat et al., 2008). Catalase enzyme can convert hydrogen peroxide to
water and oxygen, protecting the plant against stress-induced damages
(Willekens et al., 1997). Plant growth-promoting bacteria (PGPB)
increase the resistance of plants to non-polluting stress such as boron
toxicity or salinity (Khan et al., 2016). Therefore, PGPBs can use similar
strategies and increase the antioxidant activity of various enzymes.
Plant Growth-Promoting Bacteria enhanced the activity of POD and
SOD enzymes and organic acids in chickpea (Cicer arietinum) (Israr et al.,
2016). It has been showed that, Maize (Zea mays L.) plant grown in non-
saline soil without bacterial treatment expressed low enzymatic activi-
ties in their roots compared to other treatments (Singh et al., 2020). Qin
et al., (2017) reported that, inoculation of KLBMP5084 isolate signifi-
cantly increased the POD, CAT and SOD activities in Limonium sinense
leaves under salt stress conditions. Reduced salinity stress in plant
growth-promoting bacteria inoculated plants has also been reported on
various plants, including sweet pepper (Egamberdieva, Shrivastava and
Varma, 2016) and red pepper (Park et al., 2013). The beneficial effects of
endophytes on plant growth in the presence of cadmium (Zhang et al.,
2010), aluminum (Zaurov et al., 2001), zinc (Fabien et al., 2001), lead
(Gou et al., 2007), salt (Zhang et al., 2011) and drought (Swarthout et al.,
2009) stresses were also reported.
The role of P. indica on enzyme activation was previously reported
by Sherameti et al (2005) who have reported that nitrate reductase
activity can be influenced by p. indica in tobacco and Arabidopsis. The
activities of POD, CAT and SOD in the leaves of polyethylene glycol
treated Chinese cabbage increased when exposed to P. indica within
24 h (Sun et al, 2010). They also indicated that expression levels of the
drought-related genes such as DREB2A, CBL1, ANAC072 and RD29A
were upregulated in the drought-stressed leaves of colonized plants.
Proline
Proline accumulation in stressed plants is one of the most impor-
tant indicators of resistance to stress (Sarmast et al. 2015). As far, the
Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.04.001
[m5G;June 25, 2020;21:29]
varieties of a plant species are classified according to the proline pro-
duction potential. As salinity increased, proline accumulation
increased in leaf tissue. Increasing the amount of proline under the
salinity in fungus and bacteria inoculated plants compared with con-
trol, indicates the efforts of plant cells to maintain osmotic pressure
(Fig. 6). Due to the non-significant effect of year of experiment, only
the data of second year was discussed. Based on the findings of the
first and second year, the effect of growth-stimulating microorgan-
isms and salinity stress, as well as their interaction on the amount of
proline was significantly different (P<0.05) (Table 1). Increasing the
amount of proline in the tissue of plants under salt stress has been
reported before (Khan et al., 2009). Better growth of plants treated
with P. indica under salinity stress compared with control plants can
be attributed to increased carbohydrate production and proline con-
tent of leaves, which is noted in Balturoshat et al. (2008) and Hajienia
et al. (2012) reports. Balturoshat et al. (2008) reported that P. indica
via influencing the activity of antioxidant enzymes and increasing
the compatible solutes, reduces the adverse effects of salinity and
increase the plant growth under salinity stress. Inoculation of plants
with different strains of Streptomyces showed similar results of com-
patible solutes. Increasing the proline affected by bacteria might be
referring to the role of bacteria in nitrogen fixation, which increase
the amino acid production such proline (Kandovangko et al., 2009,
Giannakoula and Ilias, 2013). Proline can be included in the list of
non-enzymatic antioxidants that microbes, animals and plants need
to neutralize the ROS inhibitor effects (Gill and Tuteja, 2010). Proline
accumulation is an osmotic potential modulation mechanism that
varies among different plant species and can increase up to 100 times
in environmental stress conditions (Verbruggen, 2008). Mahmoud et
al. (2020) found that halotolerant PGP bacteria strains producing IAA
and proline mitigate salt-induced plant growth inhibition in barley
plants at the vegetative stage by controlling leaf water potential and
ion homeostasis. Niu et al., (2019) reported that soluble protein and
proline in tomato’s leaf treated with Schizophyllum sp. DL12 were
increased. Based on the results of Bagheri and Mohammad Ali Pour,
2011, with increasing salinity, soybean plants increased leaf proline
content for tolerance. Kazemeini et al., (2016) found that, increasing
the salinity level increased the proline content of millet. Similar
results were found in Mint (Asghari, Khademian and Sedaghati, 2020
2020), Wheat (Askari, 2016), Maize (Xu et al., 2016), Grizzly Tomato
(Al-Hassan et al., 2015) Apples (Jaarsma et al., 2013), as well as Stevia
JID: SAJB
ARTICLE IN PRESS [m5G;June 25, 2020;21:29]

A. Forouzi et al. / South African Journal of Botany 00 (2020) 1 10 7

Fig. 6. Effect of NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on proline content of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and K152149 Strep-
tomyces sp (S2), Different letters in the same column indicate significant differences according to LSD's test (P<0.05).

(Pandey, 2014) and Pepper (Beltrano, 2013). This increase is justified
by the role of this amino acid in modulating the negative effects of
salt stress. It has been confirmed that proline, lysine, glutamine and
glutamic acid are associated with abiotic stress tolerance (Ashraf,
2007), while lignin, chitin and cellulose are associated with providing
defense against pathogenic fungi (Cano ~ -Delgado et al., 2003). The
results of present study confirmed the findings of previous studies in
which proline as compatible solute strongly contribute in plant
stress.
Total and reducing sugars
Based on the results of analysis of variance, for two consecutive
years, total and reducing sugars were influenced by microorganisms
and salinity. The interaction of microorganisms and salinity also had
a significant effect on the concentrations of total and reducing sugars
in the second year, while total sugar was not influenced by the
interaction effect of salinity and utilized microbes in the first year of
experiment (Table 2). However, the effect of salinity stress on the
total and reducing sugars was increased in both years. The highest
amount of reducing sugar was observed in Streptomyces sp. inocu-
lated plants (179.8 and 179.22 mg/g) when were treated with 3 dS/m
salinity in the first and second years, respectively (Fig. 7). In contrast,
the highest amount of total sugars was observed in the plants treated
with fungus both in the first and second years of experiment (466.1
and 88.86 mg / g) at a salinity level of 3 dS/m (Fig. 8). Cantabella et al.
(2017) as Aghighi et al. (2017), reported that, NaCl induced stress
increasing total glucose and steviol glycosides of stevia. Tavarini et
al., (2018) reported that, compared to non-inoculated stevia plants
the amount of steviol glycosides increased in mycorrhizal inoculated
plants. Amirjani, 2010 reported that the total and reducing sugars of
the rice plants significantly increased as the salinity increased. Simi-
lar results has been reported in tomato (Amini and Ehsanpour, 2005)
and barely (Bagheri and Sadeghipour, 2009). Increasing sugar

Fig. 7. Effect of NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on the reducing sugar of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and K152149
Streptomyces sp (S2), Different letters in the same column indicate significant differences according to LSD's test (P<0.05). Big and small letters referred to first and second experi-
mental years, respectively.

Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.04.001
JID: SAJB
ARTICLE IN PRESS
8 A. Forouzi et al. / South African Journal of Botany 00 (2020) 1 10
Fig. 8. Effect of NaCl (0.85, 1.5 and 3 dS/m) and the type of utilized microbes on total sugar of Stevia rebaudiana leaf extract. Kj152148 Streptomyces sp (S1) and K152149 Streptomy-
ces sp (S2), Different letters in the same column indicate significant differences according to LSD's test (P<0.05). Big and small letters referred to first and second experimental years,
respectively.
accumulation in plant tissues is important under stress conditions for
osmotic regulation (Dhanapackiam and Ilyas, 2010). Under salinity
stress, sugar accumulation with other compatible solutes helps to sta-
bilize cell membranes and proteins (Sa
nchez et al., 1998). It has been
demonstrated that the accumulation of soluble sugars plays an
important role in osmotic regulation of plant cells and facilitates
water absorption (Pessarakli, 1991). The role of microorganisms in
the management of biotic and abiotic stresses has been studied and
reviewed by various workers (Singh et al., 2020).
Conclusion
The results of this study showed that Stevia plants increases their
antioxidant capacity by increasing the antioxidant content in order to
control the salinity stresses. Using growth-promoting microorgan-
isms under the salinity stress conditions helps to increase biomass
growth. Plants subjected to salinity have a lower antioxidant activity
compared to those treated with both salinity and growth promoter
microorganisms. Stevia is classified in the category of saline-sensitive
plants, and salt stress causes undesirable morphological, physiologi-
cal and biochemical changes. In the present experiment, the aug-
mented level of catalase and peroxidase activity contributes to
maintaining cellular homeostasis against drought stress. Under stress
and non-stress conditions, endophytic fungus and Streptomyces iso-
lates had significant positive effect on the biochemical and physiolog-
ical parameters of stevia. Inoculation of fungus and bacterial isolates
reduced the negative effects of salinity stress by increasing antioxi-
dant compounds, antioxidant enzymes and free radical scavenging
activity. The application of microorganisms by osmotic compression,
such as glucose, causes osmotic regulation, and thus stevia can toler-
ate the salinity to some degree. Results well demonstrated that the
salinity level higher than 2 dS/m is tolerable for stevia plant at the
presence of inoculated microorganisms. Totally it should be men-
tioned that, although the highest antioxidant activity and proline
accumulation are obtained in high salinity levels, the increase of
these compounds for osmotic regulation, equal with the biomass
reduction, is an important point that should be considered.
Acknowledgements
We would like to thank Gorgan University of Agricultural Sciences
and Natural Resources for this research financial support.
Please cite this article as: A. Forouzi et al., Phytochemical response of Stevia plant to growth promoting microorganisms under salinity stress,
South African Journal of Botany (2020), https://doi.org/10.1016/j.sajb.2020.04.001
[m5G;June 25, 2020;21:29]
Supplementary materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.sajb.2020.04.001.
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