Scientia Horticulturae 263 (2020) 109132

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Plant growth promoting rhizobacteria (PGPR) confer drought resistance and T

stimulate biosynthesis of secondary metabolites in pennyroyal (Mentha
pulegium L.) under water shortage condition
Behvar Asgharia, Raheleh Khademianb,*, Behnam Sedaghatic
a
Department of Horticultural Sciences Engineering, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran
b
Department of Genetic and Plant Breeding, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran
c
Department of Biotechnology, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Water deficit is one of the main devastating environmental factors limiting crops productivity. Plant growth
Plant growth promoting rhizobacteria promoting rhizobacteria (PGPR) can improve plant growth in stress conditions and enhance plant tolerance to
Non-enzymatic antioxidants drought stress. The aim of this study was to investigate the role of PGPR to protect pennyroyal (Mentha pulegium
Essential oil L.) as an important industrial and functional plant against drought damages. The greenhouse study was con-
Water deficit
ducted as a factorial experiment in a completely randomized design. The first factor was PGPR inoculation
Secondary metabolites
(Azotobacter chroococcum (Ac), Azospirillum brasilense (Ab), Ac + Ab and control (without PGPR)). The second
factor was irrigation regime that applied at three levels: field capacity (FC), 0.7 FC and 0.4 FC. Drought stress
significantly decreased chlorophyll fluorescence (Fv/Fm) and relative water content (RWC) of pennyroyal.
Conversely, antioxidant enzymes activity, osmoprotectants accumulation, membrane lipid peroxidation, abscisic
acid (ABA) and secondary metabolites including phenolic, flavonoid and essential oil contents and DPPH radical
scavenging activity of the plant extract increased due to water deficit. PGPR inoculation significantly reduced
adverse effects of water stress on physio- biochemical characteristics and secondary metabolites production in
pennyroyal. The results indicated that the bacteria were different in their ability to enhance plant investigated
characteristics, and co-inoculation of the bacteria was more effective on improving of pennyroyal physiological
and phytochemical parameters. The highest ABA, proteins and soluble sugars, phenolic, flavonoid and oxyge-
nated monoterpenes contents as well as DPPH radical scavenging activity were observed in the plants treated
with Ac + Ab under severe drought stress.

1. Introduction 2019). Plant growth-promoting rhizobacteria (PGPR) are the soil bac-
teria inhabiting on the plants root surface that can enhance their
Environmental stresses are main constraints for crop yield, food growth and protect them from biotic and abiotic stresses through a wide
quality and global food security. Drought is one of the major factors variety of mechanisms (Khademian et al., 2019). PGPR belongs to di-
affecting plant growth and productivity in the worldwide. Water scar- verse genera of rhizosphere bacteria including Arthrobacter, Azoto-
city causes alterations in the physiological, morphological, biochem- bacter, Azospirillum, Bacillus, Klebsiella, Microbacterium, Paenibacillus,
ical, and molecular attributes in plants (Cheng et al., 2018; Khademian Pseudomonas, Streptomyces and etc. (Numan et al., 2018). These bacteria
and Yaghoubian, 2018). More than half of agricultural land of the world have an important role in crop growth and development by enhancing
is located in arid and semi-arid regions, and also global climate change soil fertility, increasing nutrient uptake and improving plant resistance
is increasing the frequency of severe drought conditions (Dai, 2013). to environmental stresses. There exist many reports about beneficial
Therefore, it is very important to find an approach for tolerance en- effects of PGPR on growth and yield of plants under abiotic stress such
hancement of crops against water shortage. as drought and salinity (Paul and Lade, 2014; Bharti et al., 2016;
It has been reported that native microorganisms from arid and semi- Kaushal and Wani, 2016; Vurukonda et al., 2016).
arid regions have the drought tolerance advantage, which can live with Azotobacter spp. are free-living, aerobic and nitrogen-fixing rhizo-
plants and help host plants to cope drought stress (Shirinbayan et al., bacteria, which can stimulate plant growth through nutrient

⁎
Corresponding author.
E-mail address: r.khademian@eng.ikiu.ac.ir (R. Khademian).

https://doi.org/10.1016/j.scienta.2019.109132
Received 10 July 2019; Received in revised form 8 December 2019; Accepted 12 December 2019
Available online 26 December 2019
0304-4238/ © 2019 Elsevier B.V. All rights reserved.
B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

supplementation or production of phytohormones such as auxins, gib- the sterilized seeds were inoculated by the bacteria suspensions (the
berellins, and cytokinins (Jnawali et al., 2015). It has been reported bacterial community was equal to 108 colonies/ml) with shaking
that under environmental stress, utilization of Azotobacter spp. leads to (120 rpm) for 8 h. For Ab + Ac treatment equal mixture of the bacteria
an increase of soil mineral elements and biosynthesis suspensions were mixed and used for co-inoculation. The respective
of biologically active substances and ultimately affects plants residual bacteria suspension was poured in each treatment (5 ml pot-1).
growth and development (Delshadi et al., 2017; Rodrigues et al., 2018; Sterilized distilled water was applied to treatment of non-inoculated
Rojas-Tapias et al., 2012; Turan et al., 2017). plants (control). Ten seeds were sown in each pot (28.0 cm diameter,
Azospirillum spp., as a nitrogen-fixing bacterial genus, are wide- 22.5 cm height) having same weight of sterilized soil. The soil used
spread in nature, especially in the soils of drylands. These species are in this experiment was from the research field of Imam Khomeini
used as a common model for research on plant–bacterial interactions International University (IKIU). It was sandy loam soil with pH 7.2, EC
(Alen’kina et al., 2018). Azospirillum spp. were employed as PGPR to 0.21 dS m-1, organic carbon (0.42 %), nitrogen (0.09 %), available
improve the yield of several important industrial and medicinal plants phosphorus (19.7 ppm) and available potassium (238 ppm). Then, the
such as wheat (Vogel et al., 2014), corn (Hungria et al., 2010), rice pots were moved to the greenhouse. When plants were fully established
(Chamam et al., 2013), sugarcane (Moutia et al., 2010), jojoba (5 weeks after sowing), drought treatment was started and continued
(Gonzalez et al., 2015) and crambe (de Aquino et al., 2018). until harvest (9 weeks after sowing). The gravimetric method was used
Mentha pulegium L., commonly known as pennyroyal, is an im- for the estimation of pots field capacity according to the following
portant aromatic and medical herbs from Lamiaceae family, which is equation (Rolando et al., 2015):
native to North Africa, Europe, and Middle East and grows sponta-
Water content at field capacity = (WW – DW)/(DW – PW) × 100
neously in humid regions of Iran. Pennyroyal is a source of biologically
and pharmaceutically active molecules such as flavonoids, polyphenols
and terpenoids. It has been widely used in traditional medicine for For the obtaining of WW the pots containing water saturated soil
treatment of cold, sinusitis, food poisoning, colic and infectious diseases were weighted. Pots weights were recalculated after 24 h incubation at
(Mahboubi and Haghi, 2008; Fatiha et al., 2015). 105 °C to obtain DW. PW represented the weight of
Pennyroyal grows widely in humid areas and riverside grounds and empty pots. Drought stress was controlled by weighing pots daily, to
its biomass production is highly sensitive to water shortage (Rodrigues record the mass of water loss by evapotranspiration and adding needed
et al., 2013). Hassanpour et al. (2012) reported that growth and yield of amounts of water to bring each pot to appropriate FC (Bazargani et al.,
pennyroyal was significantly decreased by water deficit. Therefore, the 2011).
use of new approach to improve the performance of pennyroyal under
erratic water deficit conditions is of great importance. Plant-microbe 2.2. Relative water content (RWC)
interaction is an important strategy for adapting plants to drought stress
(Bakhshandeh et al., 2019). Thus, we hypothesize that pennyroyal in- For determination of RWC, fully developed leaves were harvested
oculated plant with PGPRs can withstand against damaging impacts of and weighed immediately to record fresh weight (FW) and then placed
drought via improvement and alteration of physio-biochemical char- in distilled water for 16 h, and subsequently their turgid weight (TW)
acteristics. The present experiment was established to investigate the was calculated. The leaves were dried in an oven at 65 oC for 48 h to
effects of individual and combined application of Azotobacter chroo- obtain dry weight (DW). Finally, RWC was calculated in five replica-
coccum (Ac) and Azospirillum brasilense (Ab) on physiological and bio- tions using the following formula:
chemical attributes of pennyroyal under drought stress.
RWC (%) = [(FW – DW)/(TW – DW)] × 100
2. Materials and methods

2.1. Plant materials, soil preparation, inoculation, and planting 2.3. Chlorophyll fluorescence

This study was conducted as a factorial experiment in a completely Chlorophyll fluorescence parameters in the fresh leaf samples was
randomized design, with five replications in the greenhouse at a tem- determined according to the method of Yaghoubian et al. (2016). After
perature range of 18–25 °C with 50–60 % relative humidity under dark acclimation of leaves (for 15 min), the minimum (F0) and max-
natural light conditions. The first factor was the use of PGPR at four imum (Fm) fluorescence intensity were measured in leaves using a
levels: (1) non- portable fluorometer (PAM2500, Walz, Germany). Then, maximum
inoculated with PGPR (control), (2) inoculation with Ac, (3) in- quantum yield of the photosystem II (Fv/Fm) was calculated in five
oculation with Ab, and (4) co- inoculation with Ac and Ab (Ac + Ab). replications by the following formula:
The second factor was drought stress application at three levels: (1) Fv/Fm = (Fm − F0)/Fm
Field Capacity (FC) (no drought stress), (2) 0.7 FC (moderate drought
stress) and (3) 0.4 FC (severe drought stress).
Seeds of M. pulegium were collected from the village of Ali-Qapu 2.4. Antioxidant enzyme activities
(Latitude 39o 08′ N, Longitude 47o 32′ E, Altitude 1343 m above sea
level), located in Tazeh Kand-e Angut, Ardabil Province, Iran. The seeds To determine the antioxidant enzymes activities, the leaf tissue was
were washed in running tap water and were surface sterilized by dip- homogenized in an ice bath by 5 ml phosphate buffer (50 mM, pH 7.8).
ping in 70 % (v/v) ethanol for 30 s, followed by 1 % (v/v) sodium Then, the sample was centrifuged at 12000 rpm for 20 min at 4 °C. The
hypochlorite for 15 min. The surface sterilized seeds were thoroughly supernatant was used for the determination of different enzyme activ-
washed 3 times with sterile distilled water. ities. All tests were carried out in five replications.
A. chrococcum and A. brasilense were provided from the Soil and The glutathione peroxidase (GPX) activity was assayed by spectro-
Water Research Institute, Karaj, Iran. Then, the bacteria were in- photometer according to the modified method of Drotar et al. (1985).
oculated into 100 ml of Luria-Bertani (LB) liquid medium and grown for The reaction mixture (250 μl) included 2 mM glutathione, 1 mM
3 days at 28 °C on rotary shaker (150 rpm). The bacteria culture was NADPH, 1 mM EDTA, 2 mM t-butyl hydroperoxide and 0.5 U of glu-
centrifuged at 5000 rpm at 4 °C for 5 min. Then, the supernatant was tathione reductase in 100 mM sodium phosphate buffer (pH 7.0) and
removed and the pellet was re-suspended in autoclaved distilled water 10 μg of extracted proteins. The rate of NADPH oxidation was measured
and the optical density (at 660 nm) was adjusted to 1. Before sowing, at 340 nm for 15 min. A unit of the enzyme activity was expressed as the

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B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

change in absorbance per min. 0.5 g of fresh leaves was homogenized with 3 ml 80 % ethanol and
The activity of superoxide dismutase (SOD) was measured by the then mixed with 3 ml of freshly prepared anthrone reagent. The samples
photochemical method, previously described by Giannopolitis and Ries were incubated in water bath (50 °C) for 10 min and its absorbance was
(1977). The reaction mixture contained 50 mM phosphate buffer (pH recorded at 625 nm. The soluble sugar contents were determined using
7.8), 13 μM methionine, 75 μM p-nitro blue tetrazolium chloride (NBT), a glucose standard and expressed as mg g − 1 FW of leaves. All the tests
1.3 μM riboflavin, and 40 ml of enzyme extract. The reaction was in- were performed in five replications.
cubated by placing the tubes under fluorescent light with
80 μmol m – 2 s –1 for 10 min. One unit of SOD activity was defined as 2.7. Abscisic acid (ABA) content
the amount of enzyme required to obtain a 50 % inhibition of the rate of
NBT reduction measured at 560 nm. The content of endogenous ABA was measured in five replications
The activity of catalase (CAT) was measured according to the pro- by radioimmunoassay according to Vernieri et al. (1989). Monoclonal
cedure of Chance and Maehly (1955). The reaction mixture contained antibody (DBPA1) was raised against free (S)- ABA.
15 mM H2O2, 50 mM phosphate buffer (pH 7.0), and 50 μl of enzyme
extracts. The activity of CAT was measured by monitoring the decrease 2.8. Preparation of plant extract
in absorbance at 240 nm as a consequence of H2O2 consumption. Ac-
tivity was expressed as units (μmol of H2O2 decomposed per minute) The powdered leaves of pennyroyal (20 g) were extracted succes-
per mg of protein. sively with methanol by maceration at room temperature for 48 h. The
solvent of the extracts was removed using rotary evaporator at 40 °C to
2.5. Lipid peroxidation afford crude extract.

The extent of the oxidative lipid degradation was estimated by 2.9. Determination of total phenolic content (TPC)
measuring leaf malonyldialdehyde (MDA) content and electrolyte
leakage (EL) percentage. In this experiment, the MDA value was de- TPC of the extracts was calculated using Folin-Ciocalteu method
termined according to Dhindsa et al. (1981) with some modification. (Bahadori et al., 2016). Briefly, 20 μl of extract solution in methanol
About 0.25 g of fresh leave was homogenized in 5 ml of 0.1 % tri- with 2 mg ml-1 concentration were mixed with 100 μl of 1:10 Folin-
chloroacetic acid (TCA) solution. The mixture was centrifuged at Ciocalteu reagent. After 6 min in the dark, 80 μl of sodium carbonate
10000 rpm for 5 min. Then, 1 ml aliquot of the supernatant was mixed (7.5 %) was added into the mixture. The absorbance was measured at
by 4 ml of 20 % TCA containing 0.5 % TBA. The mixture was heated at 740 nm after 2 h of incubation in the dark at the room temperature. TPC
95 °C for 30 min and then rapidly cooled in an ice bath and centrifuged of the extracts were expressed as milligrams of gallic acid equivalents
at 10000 rpm for 10 min. The absorbance of the supernatant was as- per gram of dry weight of extracts (mg GAEs g-1 DW) through the ca-
sayed at 532 nm and value for nonspecific absorbance at 600 nm was libration curve with gallic acid. The calibration curve range was
subtracted. The MDA content was calculated in five replications by 1−1000 mg l-1. All samples were analyzed in five replications.
using an extinction coefficient of 155 mM-1 cm-1.
Electrolyte leakage (EL) was measured according to Shi et al. 2.10. Determination of total flavonoid content (TFC)
(2006). The leaf segments were soaked in deionized water for 24 h at
room temperature. After determination of electrical conductivity of The aluminum chloride colorimetric method was used to determine
solution (EC1), the samples were incubated at 95 °C for 20 min and after the total content of flavonoids (Bahadori et al., 2015). A special volume
cooling the electrical conductivity (EC2) was assayed in the solution. of extracts or a standard solution (20 μl) of quercetin (1–200 μg ml−1)
The EL was calculated in five replications by the following equation: was diluted with 60 μl of methanol and 10 μl of 5 % AlCl3. Subse-
EL (%) = (EC1/EC2) × 100 quently, 10 μl of 0.5M potassium acetate was added to the mixture and
the total volume was made up to 200 μl by distilled water. The solution
2.6. Determination of proline, total soluble protein and soluble sugar was mixed well and after 30 min the absorbance was read at 415 nm.
contents All tests were carried out in five replications, and mean values of fla-
vonoid content are expressed as milligrams of quercetin equivalents per
The proline content of fresh leaf was assayed according to the pre- gram of dry weight of extracts (mg QEs g-1 DW).
vious report (Bates et al., 1973). The leaf tissue (0.5 g) was homo-
genized in 3 % (w/v) sulphosalycylic acid and then filtered using filter 2.11. Measurement of radicals scavenging capacity
paper. Ninhydrin and glacial acetic acid were mixed by sample and
heated at 100 oC for 1 h in water bath; then reaction was stopped in ice The DPPH radical scavenging activity of extracts was determined
bath. The mixture was extracted with toluene, and the absorbance of from the bleaching of purple- colored solution of 2,2-diphenyl-1-pi-
fraction with toluene aspired from liquid phase was read at 520 nm. The crylhydrazyl (DPPH) (Asghari et al., 2018b). The appropriate volume
concentration of proline was presented as μg g-1 FW. (20 μl) of samples dissolved in methanol, was mixed with 180 μl of
The amount of total soluble protein content was determined by the DPPH solution (0.1 mM). After 30 min, discoloration of the mixtures
method of Bradford (1976). Briefly, fresh leaf (0.5 g) was ground and was measured at 517 nm. Inhibition of DPPH in percent was calculated
homogenized in 10 ml of phosphate buffer (0.2 M, pH 7.5) and cen- as given below:
trifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was mixed I (%) = [(Ac – As)/Ac] × 100
with equal volume of 10 % TCA. The mixture was centrifuged at Where Ac is the absorbance of the control reaction (containing all
6000 rpm for 10 min at 4 °C and the pellet was washed twice with reagents except the test sample), and As is the absorbance of the ex-
distilled water and dissolved in NaOH (0.1 N). Then Bradford’s reagent tracts. All the assays were run in five replications and the results were
was added and the mixture was kept at room temperature for 10 min to expressed as average values with standard deviation (SD).
color development. The concentration of proteins was determined by
using a standard curve of bovine serum albumin (BSA). Absorbance was 2.12. Essential oil isolation
read at 595 nm. The concentration was expressed in mg g-1 FW of
leaves. The essential oil of the plant was extracted by hydrodistillation
Soluble sugars were estimated by the anthrone reagent method method using a Clevenger apparatus during 3 h. Anhydrous sodium
(Yemm and Willis, 1954). About sulphate was used for drying of the obtained oil. Obtained essential oil

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B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

stored at 4 °C in dark until analysis. Table 1

Effect of A. chroococcum (Ac) and A. brasilense (Ab) and their combination
(Ac + Ab) on RWC (%) and chlorophyll fluorescence (Fv/Fm) of pennyroyal
2.13. GC-FID and GC–MS analysis
leaves under various irrigation regimes. Results are represented as means of 5
replicates ± SD. Different letters show significantly different according to
GC-FID analysis was performed using Younglin instrument Duncan’s multiple-range test at P ≤ 0.05.
(YL6500) equipped with a flame ionisation detector (FID) and a DB-5
Treatments RWC Chlorophyll contents
fused silica column (30 m ×0.32 mm i.d., film thickness
0.25 μm). Nitrogen was used as carrier gas at the constant flow rate FC 74.93 ± 2.27 a 0.783 ± 0.007 a
of 1.1 ml/min. The split ratio was 1:50. Temperatures of the detector FC + Ac 74.03 ± 1.72 ab 0.756 ± 0.005 cd
and the injector were 250 and 240 °C, respectively. The column tem- FC + Ab 74.40 ± 1.87 ab 0.766 ± 0.007 bc
perature was raised from 60 to 250 °C at the rate of 5 °C/min, and was FC + Ac + Ab 74.63 ± 1.31 ab 0.773 ± 0.006 ab
0.7 FC 70.67 ± 0.81 c 0.638 ± 0.008 g
kept at 250 °C for 10 min. The injection volume was 1 μl. 0.7 FC + Ac 72.97 ± 1.64 abc 0.709 ± 0.006 e
GC–MS analysis was performed on a Younglin instrument (YL6900), 0.7 FC + Ab 72.10 ± 1.08 bc 0.688 ± 0.007 f
equipped with the same column and temperature programming as 0.7 FC + Ac + Ab 73.83 ± 0.40 ab 0.707 ± 0.007 e
mentioned for GC. The essential oil was diluted in dichloromethane and 0.4 FC 64.6 ± 1.15 e 0.564 ± 0.007 h
0.4 FC + Ac 67.77 ± 0.50 d 0.634 ± 0.009 g
0.5 μl of the solution was injected into the GC–MS apparatus with a split
0.4 FC + Ab 65.70 ± 1.11 de 0.645 ± 0.006 g
ratio of 1:100. EI-MS measurements were carried out with ionisation 0.4 FC + Ac + Ab 72.07 ± 1.45 bc 0.754 ± 0.006 d
voltage of 70 eV. Mass range was scanned from 35 to 456 amu.
Identified of essential oil constituents was carried out by calculating
their retention indices for n-alkanes (C6–C24) and the oil on a DB-5 3.3. Antioxidant enzymes activities
column under the same chromatographic conditions. Essential oil
components were identified by matching of their mass spectra with The effect of drought stress and PGPR on the antioxidant enzymes
those published in the literature and confirmed by comparison of their activities (GPX, SOD and CAT) in pennyroyal leaves are shown in Fig. 1.
retention indices with those of authentic compounds or with those re- In moderate and severe drought conditions, we observed that drought
ported in the literature (Adams, 2007). For quantification purpose, stress significantly increased the antioxidant enzymes activities of
relative area percentages obtained from GC-FID were applied. pennyroyal. Compared to control, water deficit significantly enhanced
the enzymes activity. The highest activity of GPX, SOD and CAT were
2.14. Statistical analysis obtained in severe drought stress condition and the least activity is
related to the control plants. Application of PGPR decreased the activity
All the assays were carried out in five replications. The results were of GPX. Under severe drought stress, a maximum decrease of 26.9 % in
expressed as mean ± standard deviation (SD). Data analysis was per- GPX activity was observed with Ac + Ab treatment followed by in-
formed using SPSS v. 16.0. Differences between means were de- dividual application of the bacteria showing 14.7 % and 12.5 % de-
termined by one-way analysis of variance (ANOVA) followed by crease for Ab and Ac, respectively (Fig. 1A). The SOD activity in
Duncan's Post Hoc test for multiple comparisons with control. A value moderate and severe drought stress increased by 34.7 % and 57.2 % as
of p < 0.05 was considered to indicate statistical significance. compared to the control, respectively (Fig. 1B). The highest
CAT activity was observed in the severe drought stress with the
value of 58.9 U mg-1 protein which was 2.6 fold more than control
3. Results
(22.7 U mg-1 protein). The results showed that inoculation with PGPR
had significantly reduced the antioxidant enzyme activities. The level of
3.1. RWC
CAT decreased by 19.8 % in drought treated (0.7 FC) plants inoculated
with Ab as compared to that of respective control (Fig. 1C).
Drought stress and PGPR had significant effects on RWC of the
pennyroyal leaves. As expected, RWC of the plants subjected to drought
stress was greatly reduced in comparison with the plants that were 3.4. Lipid peroxidation
cultivated under full irrigation. However, RWC was improved by PGPR
treatment under drought stress. The highest and lowest amounts of The effect of drought stress and PGPR on lipid peroxidation and
RWC were recorded in FC and 0.4FC treatments without PGPR in- membrane integrity was evaluated through determination of MDA ac-
oculation, respectively. Under severe drought stress, co-inoculation cumulation and electrolyte leakage changes in membrane lipid com-
with PGPR strains (Ac + Ab) led to increase RWC from 64.6 to 72.1 % position. Our results revealed that drought exposure led to an increase
(Table 1). of MDA content and EL percentage in pennyroyal leaves (Fig. 2).
However, bacterial inoculation significantly reduced MDA and EL le-
3.2. Chlorophyll contents vels, particularly when Ab was present either alone or co-inoculated.
The concentration of MDA was higher in severe drought when com-
The effect of drought stress and PGPR on plant photosynthesis were pared with moderate stress and control.
determined by evaluating the changes in Fv/Fm values. Fv/Fm is the
maximum efficiency at which light absorbed by PSII and is used as a 3.5. Proline, protein and soluble sugars contents
sensitive indicator of plant photosynthetic performance. Thus, a re-
duction of this parameter indicates the destructive effects of stress According to the results, when the plant was exposed to drought
factors on PSII and decline in the plant photosynthesis (Yaghoubian stress proline, protein and total soluble sugars contents in the pennyr-
et al., 2016). Drought stress treatments led to significant decrease in oyal leaves were significantly increased (Fig. 3 and 4). The results
Fv/Fm of pennyroyal. The highest amount of Fv/Fm (0.78) was found showed that bacteria have a different effect on proline content de-
in the non-inoculation treatment under well-watered condition. Under pending on water regime. In control and moderate drought stress
severe drought conditions, the amount of Fv/Fm were decreased from conditions, there was no obvious differences between non- inoculated,
0.78 to 0.56. Significant changes in Fv/Fm were observed in response to Ac and Ab treatments with respect to the proline concentration (Fig. 3).
bacterial inoculation. Under severe drought condition, inoculation with In full irrigation condition Ac + Ab treatment significantly promoted
Ac + Ab led to increase Fv/Fm from 0.56 to 0.75 (Table 1). the proline content while, under moderate drought stress, the opposite

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B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

Fig. 1. Effect of A. chroococcum (Ac) and A. brasilense (Ab) and their combination (Ac + Ab) on A) glutathione peroxidase, B) superoxide dismutase and C) catalase
activity of pennyroyal leaves under various irrigation regimes. Bars are means of 5 replicates ± SD. Bars labeled by different letters are significantly different
according to Duncan’s multiple-range test at P ≤ 0.05.

was true. Whereas the highest proline content (49.8 μg g-1 FW) was lowest amount (86.6 ppm) was observed in well-watered treatment
observed in plants inoculated with Ac under severe drought condition without bacteria (Table 2).
and inoculation with Ac + Ab significantly reduced this compound
amount (Fig. 3).
Furthermore, PGPR inoculation improved soluble protein and sugar 3.7. TPC and TFC
contents under both non- stress and stress conditions. PGPR treatment
under severe drought stress enhanced the protein and soluble sugar According to the obtained results TPC and TFC of pennyroyal were
contents as compared with respective control (Fig. 4A and C). It should significantly affected by the degree of water stress (Table 2). Significant
be mentioned that interaction between drought stress and PGPR ap- enhancement of TPC and TFC was observed at severe drought stress
plication had no significant effect on soluble protein and sugars con- (81.7 mg g-1 DW and 16.2 mg g-1 DW, respectively) as compared to the
tents. control (53.9 mg g-1 DW and 8.6 mg g-1 DW, respectively).
Exclusive Ab treatment under different water regimes, did not show
3.6. ABA content obvious effect on TPC and TFC of the plants. Individual application of
Ac had no significant effect on pennyroyal TPC and TFC at control
The finding showed that water deficit stress and PGPR inoculation plants, while under moderate and severe drought stress the same
had significant effect on ABA accumulation. ABA concentration in treatment, increased TPC (17.2 and 30.3 %, respectively) and TFC (35.9
plants under severe drought stress was on average about 1.9 times and 33.7 %, respectively) of the plants significantly. The maximum of
higher than those of respective control. An increase of ABA con- TPC under all levels of water deficit conditions was exhibited at si-
centration was observed across all water regimes in pennyroyal leaves multaneous application of Ab and Ac (Ac + Ab). This enhancement for
due to PGPR application. The highest amount of ABA (218.9 ppm) was control, 0.7 FC and 0.4 FC treatment were about 41.6, 47.4 and 55.8 %,
found in co-inoculation treatment under severe drought stress and the respectively.

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B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

moderate and severe water stress, respectively. Single and combined

bacterial inoculum
significantly increased essential oil content of pennyroyal under
well-watered condition, while there was no significant difference be-
tween single and combined bacterial treatments. In both of moderate
and severe drought stress conditions, inoculation with pure PGPR
strains did not increase essential oil content compared to respective
control. Contrarily, co-inoculation of the plant by PGPR strains
(Ac + Ab) prominently enhanced the accumulation of essential oil in
stressed condition (10.9 and 11.3 % increase for moderate and severe
drought stress, respectively) (Fig. 5).
The essential oil of pennyroyal at various degree of drought stress
and PGPR treatments had 1,8- cineole, menthone, pulegone, and pi-
peritone as the main constituents (Table 3). In control sample 1.8-ci-
neole, menthone, pulegone and piperitone contents were 6.6, 19.3. 34.6
and 3.7 %, respectively. The moderate and severe water deficit in-
creased contents of 1,8-cineole, menthone and pulegone in pennyroyal
essential oil. Also all bacterial treatments increased the amounts of the
mentioned compounds. The highest concentrations of 1,8-cineole,
menthone and pulegone (8.8, 35.1 and 42.3 %, respectively) were
found in plants cultivated in severe drought stressed condition in-
oculated with Ac + Ab. Piperitone content of the samples essential oil
was not affected by various water regimes and PGPR bacterial treat-
ments.

Fig. 2. Effect of A. chroococcum (Ac) and A. brasilense (Ab) and their combi- 4. Discussion
nation (Ac + Ab) on A) malondialdehyde (MDA) content and B) electrolyte
leakage of pennyroyal leaves under various irrigation regimes. Bars are means
Drought stress causes generation of reactive oxygen species (ROS),
of 5 replicates ± SD. Bars labeled by different letters are significantly different
which leads to induction of osmotic stress in crops. At low to modest
according to Duncan’s multiple-range test at P ≤ 0.05.
levels, ROS acts as signalling molecules and activates signal transduc-
tion processes in response to various stresses (Redza-Dutordoir and
Averill-Bates, 2016). However, excessive cellular levels of ROS can
cause lipids peroxidation, proteins oxidation, damage to nucleic acids,
enzyme inhibition, activation of programmed cell death (PCD) pathway
and ultimately leading to cell death (Sharma et al., 2012). The enzy-
matic and non- enzymatic antioxidant defense mechanisms protect
plants against ROS toxicity. Antioxidant enzymes, such as GPX, SOD
and CAT are the main line of defense mechanism for scavenging toxic
ROS. Under drought stress, high levels of antioxidant enzymes activities
are positively correlated with plant drought tolerance (Guo et al., 2018;
Kaushal and Wani, 2016). Our results showed that drought stress in-
creased the GPX, SOD and CAT activities in the pennyroyal leaves. The
enhancement of these enzymes activity, is likely be linked to over-
production of ROS that resulted from drought stress. Similar results
have been reported in safflower (Hojati et al., 2011), chickpea (Kumar
Fig. 3. Effect of A. chroococcum (Ac) and A. brasilense (Ab) and their combi-
et al., 2016), milk thistle (Zahir et al., 2014), fennel (Askari and Eh-
nation (Ac + Ab) on proline content of pennyroyal leaves under various irri-
gation regimes. Bars are means of 5 replicates ± SD. Bars labeled by different sanzadeh,
letters are significantly different according to Duncan’s multiple-range test at 2015), and maize (Saruhan et al., 2012). According to the results,
P ≤ 0.05. bacterium inoculation significantly decreased the activities of anti-
oxidant enzymes as compared to the control plants. The findings are in
agreement with previous study which showed that inoculation of
3.8. DPPH radical scavenging activity
chickpea with PGPR, reduced antioxidant enzymes activity under
drought stress (Kumar et al., 2016). Reduction of the enzymes activity
The antioxidant ability of pennyroyal extracts was measured by
due to PGPR-inoculation have been also reported in wheat (Upadhyay
DPPH free radical scavenging assay. Drought stress significantly en-
et al., 2012) and maize (Fukami et al., 2018) under salinity stress.
hanced the DPPH scavenging activity compared with respective control.
Plant secondary metabolites (PSMs) are a wide range of biologically
The extracts of inoculated plants showed significantly higher scaven-
active substances that are remarkably important for plant growth and
ging activity against DPPH than the control plants. The highest DPPH
development. Plants synthesize various PSMs under unfavorable
scavenging activity (about 90 %) was related to co-inoculated plants
growth conditions that play an essential role in protecting plants from
treated with severe drought stress (Table 2).
harmful effects of stresses (Jaleel et al., 2009). Some PSMs as non-en-
zymatic antioxidant take part in defense responses against oxidative
3.9. Essential oil content and main constituents stress. PSMs such as essential oil, phenolic and flavonoid compounds
can scavenge free radicals by donating electron or hydrogen (Asghari
The findings established that essential oil synthesis considerably et al., 2018a; Ashraf et al., 2019). In this study, PSMs increased in
increased by drought stress. Essential oil content in non-inoculated drought stressed and PGPR inoculated pennyroyal plants but more
control was 1.05 % which increased up to 1.29 and 1.51 % under enhancement was exhibited in the plants inoculated with bacteria.

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B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

Fig. 4. A) Effect of various irrigation regimes on protein content, B) Effect of various bacterial treatments on protein content, C) Effect of various irrigation regimes
on soluble sugar content, D) Effect of various bacterial treatments on soluble sugar content of pennyroyal leaves. Bars are means of 5 replicates ± SD. Bars labeled by
different letters are significantly different according to Duncan’s multiple-range test at P ≤ 0.05.

Table 2
Effect of Azotobacter chroococcum (Ac) and Azospirillum brasilense (Ab) and their combination (Ac + Ab) on abscisic acid, total phenolic and flavonoid contents and
DPPH radical scavenging activity of pennyroyal leaves under various irrigation regimes. Results are represented as means of 5 replicates ± SD. Different letters show
significantly different according to Duncan’s multiple-range test at P ≤ 0.05.
Treatments ABA (ppm) TPC (mg GAEs/g DW) TFC (mg QEs/g DW) DPPH (%)

FC 86.80 ± 2.31 j 53.87 ± 2 .85 f 8.60 ± 1.37 e 26.63 ± 2.83 i

FC + Ac 101.53 ± 3.85 i 58.47 ± 1.37 f 10.63 ± 0.80 de 30.63 ± 3.70 hi
FC + Ab 139.00 ± 3.06 f 53.13 ± 1.68 f 8.73 ± 0.61 e 33.97 ± 2.43 h
FC + Ac + Ab 142.00 ± 3.96 e 76.27 ± 2.66 d 16.30 ± 1.41 c 63.13 ± 1.72 d
0.7 FC 123.37 ± 2.96 h 57.43 ± 2.74 f 11.87 ± 1.31 d 40.27 ± 2.49 g
0.7 FC + Ac 129.13 ± 3.28 g 67.33 ± 2.85 e 16.13 ± 0.87 c 59.30 ± 2.10 d
0.7 FC + Ab 178.60 ± 3.37 b 58.27 ± 2.19 f 11.53 ± 0.96 d 48.43 ± 2.63 f
0.7 FC + Ac + Ab 180.40 ± 2.75 b 84.57 ± 3.95 c 20.07 ± 1.19 b 71.20 ± 2.45 c
0.4 FC 162.50 ± 2.29 d 81.70 ± 4.37 cd 16.20 ± 1.08 c 53.73 ± 2.44 e
0.4 FC + Ac 168.97 ± 2.81 c 106.50 ± 2.86 b 21.67 ± 1.07 b 80.27 ± 4.14 b
0.4 FC + Ab 217.47 ± 4.00 a 80.40 ± 3.72 cd 15.87 ± 2.06 c 58.43 ± 3.01 d
0.4 FC + Ac + Ab 218.93 ± 5.55 a 127.27 ± 6.09 a 29.83 ± 1.70 a 89.63 ± 1.91 a

Increment in PSMs compounds during drought stress has previously maize plants (Islam et al., 2014a, b). Bharti et al. (2014) reported that
been reported in Dendrobium moniliforme (Kleinwächter et al., 2015), PGPR inoculation cause an increase in the essential oil yield of Mentha
Rhus tripartitum and Periploca laevigata (Ncib et al., 2018). PGPR acts as arvensis. Couto et al. (2011) has also reported that application of PGPR
biotic elicitor resulting in 'induced systemic resistance' (ISR) and ex- enhances the antioxidant capacity of soybean.
pression of various genes, which trigger agglomeration of diverse plant In the present study, chlorophyll fluorescence was significantly af-
defensive bioactive molecules and secondary product in plant cells fected by drought stress and PGPR inoculation in contrasting ways. Our
(Bhattacharyya and Jha, 2012). In the present study, PGPR inoculation results indicated that drought stress impairs
significantly increased non-enzymatic antioxidants including phenols, photosynthesis by decreasing Fv/Fm ratios. These findings are in
flavonoids and oxygenated monoterpenes of essential oil of pennyroyal good agreement with previous studies (Guo et al., 2018). Water deficit
and DPPH radical scavenging capacity of the plant extract. Increasing of induce photo-inhibitory damage to PSII via oxidation of D1 and D2
non-enzymatic antioxidant contents in PGPR-inoculated plants can reaction center proteins (Simancas et al., 2016; Vass, 2012). Under
withstand against the negative effects of drought stress. It has been drought stress, bacterial inoculation improved the amount of Fv/Fm.
reported that increasing of non- enzymatic antioxidant components This enhancement may be related to reducing of photo-oxidative stress.
such as ascorbic acid and phenolic compounds is one of the resistance Similar results have been previously reported in wheat (Singh and Jha,
mechanisms against oxidative stress in PGPR-inoculated wheat and 2016), chickpea (Kumar et al., 2016), maize (Rojas-Tapias et al., 2012)

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B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

Fig. 5. GC-Ms chromatogram of pennyroyal essential oil and its main constituent's chemical structure.

Table 3
Effect of drought stress and PGPR inoculation on main constituents' percentage of pennyroyal essential oil. Results are represented as means of 5 replicates ± SD.
Different letters show significantly different according to Duncan’s multiple-range test at P ≤ 0.05.
Treatments Main components

1,8-cineole menthone pulegone piperitone

FC 6.60 ± 0.26 f 19.30 ± 0.46 h 34.63 ± 0.42 f 3.67 ± 0.12 bc

FC + Ac 6.90 ± 0.20 ef 22.13 ± 0.32 fg 34.93 ± 0.51 ef 3.43 ± 0.23 c
FC + Ab 6.53 ± 0.21 f 20.70 ± 0.53 gh 35.27 ± 0.49 def 3.57 ± 0.15 bc
FC + Ac + Ab 7.83 ± 0.12 bcd 25.43 ± 0.72 de 37.20 ± 0.26 cdef 3.73 ± 0.40 bc
0.7 FC 7.10 ± 0.36 def 23.53 ± 0.75 ef 36.77 ± 0.51 cdef 4.10 ± 0.20 ab
0.7 FC + Ac 7.40 ± 0.17 cdef 26.27 ± 0.84 d 37.67 ± 0.87 cde 4.16 ± 0.06 ab
0.7 FC + Ab 7.53 ± 0.35 cde 25.13 ± 1.36 de 37.53 ± 0.76 cde 4.06 ± 0.11 abc
0.7 FC + Ac + Ab 8.26 ± 0.30 abc 28.60 ± 0.79 bc 38.87 ± 1.80 bc 4.53 ± 0.32 a
0.4 FC 7.90 ± 0.72 bcd 27.20 ± 0.26 cd 38.06 ± 1.29 bcd 3.60 ± 0.20 bc
0.4 FC + Ac 8.57 ± 0.81 ab 29.37 ± 1.22 bc 42.33 ± 2.15 a 3.93 ± 0.47 abc
0.4 FC + Ab 8.13 ± 0.78 abc 29.93 ± 1.60 b 40.57 ± 2.63 ab 3.83 ± 0.25 bc
0.4 FC + Ac + Ab 8.83 ± 0.76 a 35.13 ± 3.30 a 42.27 ± 2.76 a 3.93 ± 0.76 abc

and finger millet (Chandra et al., 2018). contents, leading to a higher water potential gradient and thereby im-
Drought stress induces oxidative stress in pennyroyal leaves and proving the water uptake and plant growth under stress condition. Si-
consequently increases MDA and EL. MDA, final product of lipid per- milarly, Sandhya et al. (2010) have reported that inoculation of maize
oxidation, is commonly used as an important physiological index for with PGPR can improve water absorption through
determining the extent of oxidative damage. The changes in MDA and increasing of proline, amino acids and soluble sugars contents. Our
EL levels depend on the severity and duration of oxidative stress results showed that individual and combined inoculation with Ac and
(Hosseini et al., 2018). Numerous studies have shown that drought Ab had different effects on proline accumulation. Although there are
stress results in an oxidative damage that troubles the membrane several reports that PGPR inhibit proline accumulation (Naveed et al.,
system and increases MDA and EL (Boldaji et al., 2012; Jia et al., 2015). 2014; Tiwari et al., 2016), other researchers have reported that PGPR
In the present study, PGPR- inoculated plants under drought stress have a positive effect on overproduction of proline (Chandra et al.,
exhibited significantly low EL and MDA. This suggests a protective 2018; Sandhya et al., 2010). These disagreements may be due to dif-
property, induced by PGPR against adverse effect of drought stress on ferences in the bacterial species, mechanism of bacterial communica-
cell membrane. PGPR have a key role in decreasing ROS accumulation, tion with plant, interaction between bacteria and intensity of drought
eliminating MDA resulting cell peroxidation of membrane lipids and stress.
maintaining cell membrane integrity. In agreement with the present ABA, an important stress-related signalling molecule, plays a key
results, Tiwari et al. (2016) reported that PGPR-inoculation prevented role in response and tolerance of plants to environmental stresses. ABA
the accumulation of MDA in chickpea. A remarkable reduction in MDA can regulate many stress-responsive genes under osmotic stress condi-
content was also recorded in soybean plant inoculated with A. brasilense tions (Agarwal and Jha, 2010). Under drought stress, ABA has been
and A. chrococcum as compared to non-inoculated controls under both excessively produced in root cells, which acts as a chemical messenger
non-stressed and drought-stressed conditions (Zakikhani et al., 2012). to regulate water conductivity, root growth and stomatal closure
Analysis of total soluble sugar and proline contents of pennyroyal (Figueiredo et al., 2008). It has been established that the presence of
showed that drought stress significantly increased the amount of these Azospirillum increases the ABA levels under both well-watered and
molecules. The increment may be a result of starch and protein de- drought conditions and actually overproduction of ABA in plant cells is
gradation, respectively. Enhancement in the amount of osmoprotectant one of the PGPR strains mechanisms to alleviate the adverse effects of
molecules are considered to be an indication of drought tolerance drought stress (Cohen et al., 2009; Zakikhani et al., 2012; Cohen et al.,
(Sandhya et al., 2010). Askari and Ehsanzadeh (2015) found that 2015). We found that drought stress enhanced the amount of ABA
drought stress caused an increase of proline and total soluble sugar in content, while PGPR inoculation induce more accumulation of ABA in
different fennel genotypes. PGPR inoculation can affect the osmor- pennyroyal leaves. PGPR improve plant hormone status by releasing
egulation capacity by enhancing the soluble sugar, protein and proline exogenous phytohormones into plant cell or stimulating the production

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B. Asghari, et al. Scientia Horticulturae 263 (2020) 109132

of endogenous hormones in plant root cells (Dodd et al., 2010). Fujita, M., Nahar, K., Biswas, J.K. (Eds.), Advances in Rice Research for Abiotic Stress
According to the results, drought stress significantly decreased the Tolerance. Woodhead Publishing, pp. 489–504.
Askari, E., Ehsanzadeh, P., 2015. Drought stress mitigation by foliar application of sal-
RWC in pennyroyal. RWC is commonly considered as a physiological icylic acid and their interactive effects on physiological characteristics of fennel
indicator to assess the water balance of plants (Lata et al., 2011). The (Foeniculum vulgare Mill.) genotypes. Acta Physiol. Plant. 37, 4.
reduction in RWC could be related to low water availability under Bahadori, M.B., Valizadeh, H., Asghari, B., Dinparast, L., Bahadori, S., Moridi Farimani,
M., 2016. Biological activities of Salvia santolinifolia Boiss. A multifunctional med-
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observed in many crops (Cheng et al., 2018; Hodaei et al., 2018). The Bahadori, M.B., Valizadeh, H., Asghari, B., Dinparast, L., Farimani, M.M., Bahadori, S.,
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oculation induces well-developed root system, which consequently promoting microorganisms can improve germination, seedling growth and potassium
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PGPRs influence plant growth in a wide range of crops via a variety of physiological status in Mentha arvensis. Acta Physiol. Plant. 36, 45–60.
Bhattacharyya, P.N., Jha, D.K., 2012. Plant growth-promoting rhizobacteria (PGPR):
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Chandra, D., Srivastava, R., Glick, B.R., Sharma, A.K., 2018. Drought-Tolerant
Behvar Asghari: Conceptualization, Methodology, Investigation, Pseudomonas spp. improve the growth performance of finger millet (Eleusine cor-
acana (L.) Gaertn.) under non-stressed and drought-stressed conditions. Pedosphere
Writing - review & editing. Raheleh Khademian: Supervision, 28, 227–240.
Investigation, Writing - original draft. Behnam Sedaghati: Cheng, L., Han, M., Yang, L.-m., Li, Y., Sun, Z., Zhang, T., 2018. Changes in the physio-
Methodology, Investigation, Writing - review & editing. logical characteristics and baicalin biosynthesis metabolism of Scutellaria baicalensis
Georgi under drought stress. Ind. Crop. Prod. 122, 473–482.
Cohen, A.C., Bottini, R., Pontin, M., Berli, F.J., Moreno, D., Boccanlandro, H., Travaglia,
Declaration of Competing Interest C.N., Piccoli, P.N., 2015. Azospirillum brasilense ameliorates the response of
Arabidopsis thaliana to drought mainly via enhancement of ABA levels. Physiol. Plant.
153 (1), 79–90.
The authors declare that they have no known competing financial
Cohen, A.C., Travaglia, C.N., Bottini, R., Piccoli, P.N., 2009. Participation of abscisic acid
interests or personal relationships that could have appeared to influ- and gibberellins produced by endophytic Azospirillum in the alleviation of drought
ence the work reported in this paper. effects in maize. Botany 87, 455–462.
Couto, C., Silva, L.R., Valentão, P., Velázquez, E., Peix, A., Andrade, P.B., 2011. Effects
induced by the nodulation with Bradyrhizobium japonicum on Glycine max (soybean)
Acknowledgements metabolism and antioxidant potential. Food Chem. 127, 1487–1495.
Dai, A., 2013. Increasing drought under global warming in observations and models. Nat.
The authors would like to thank Dr. Ammar Maryamabadi and Dr. Clim. Change 3, 52–58.
de Aquino, G.S., Ventura, M.U., Alexandrino, R.P., Michelon, T.A., de Araujo Pescador,
Yaser Yaghoubian for their technical assistance. P.G., Nicio, T.T., Watanabe, V.S., Diniz, T.G., de Oliveira, A.L.M., Hata, F.T., 2018.
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