Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation

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Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation Rasekh Amiri, Ali Nikbakht ∗ , Nematollah Etemadi Department of Horticulture, College of Agriculture, Isfahan University of Technology, 8415683111 Isfahan, Iran

a r t i c l e

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Article history: Received 19 June 2015 Received in revised form 24 August 2015 Accepted 30 September 2015 Available online xxx Keywords: Arbuscular mycorrhizal fungi Antioxidant enzyme Drought stress Essential oil Secondary metabolites

a b s t r a c t Drought stress is a major problem that suppresses the growth and yield of plants. Certain microorganisms, including arbuscular mycorrhizal fungi (AMF), can improve plant resistance against environmental stresses. The current study was performed to investigate the effectiveness of mycorrhizal inoculation on rose geranium under different irrigation regimes. The experiment was conducted as a factorial experiment based on a completely randomized design (CRD) with three irrigation levels, including 100, 75 and 50% Field Capacity (FC). Mycorrhizal inoculation included Glomus mosseae (Gm), Glomus intraradices (Gi), a combination of both species (Gi + Gm), and a non-inoculated control (NM). The results showed that root colonization was suppressed in the severe water deficit condition (50% FC). The essential oil content significantly increased by 16% when the plants were under moderate water deficit (75% FC) compared to 100% FC. AMF inoculation improved the essential oil content and oil yield compared to the non-inoculated treatment regardless of water regime. Catalase, ascorbate peroxidase and glutathione peroxidase activities reached peak values when plants were under the moderate water deficit condition (75% FC). Regardless of the AMF inoculation treatment, the plant enzymatic defence system was significantly improved compared to that of non-inoculated plants, and all inoculations resulted in lower MDA and H2 O2 accumulation in plant tissue. In general, the results suggest that rose geranium can be successfully grown in areas with limited available water and that AMF can be employed to grow this plant under water stress conditions to alleviate the adverse effects of water stress. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Drought stress is one of the major abiotic stresses and causes a significant decline in yield and growth in most plants. Rose geranium (Pelargonium graveolens L.) is an aromatic plant, mostly cultivated for essential oil production and as a bedding plant (Eiasu et al., 2012). Because the essential oil of the plant accumulates in the leaves and stems, any decline in herbage yield as a result of drought stress can reduce oil production (Charles et al., 1990). Arbuscular mycorrhizal fungi (AMF) are microbial symbionts in the roots (endomycorrhizae). Under natural conditions, approximately 90% of plant species form a symbiotic association with AMF (Quilambo, 2000). They benefit from the carbon provided by the host plants and have numerous beneficial effects on plants,

∗ Corresponding author. Fax: +98 31 3391 3412. E-mail addresses: [email protected] (R. Amiri), [email protected] (A. Nikbakht), [email protected] (N. Etemadi).

including enhanced nutrient uptake, plant growth and resistance to abiotic stresses, such as salinity (Sheng et al., 2008), high temperature stress (Chen et al., 2013), and drought stress (Wu et al., 2006). The mechanism that enhances drought stress in AMF inoculated plants against water deficit stress is not fully described; however, mycorrhizal inoculation enhances water uptake as a result of extraradical hyphae extension and stomatal regulation (Fagbola et al., 2001). Moreover, increasing the phosphorus (P) concentration in the plant following mycorrhizal inoculation is another reason for the drought tolerance in AMF inoculated plants (Fitter, 1988). However, some reports have shown that drought stress decreases the P concentration in the plant, which negatively affects roots mycorrhizal colonization (Augé, 2001). Alleviating drought stress damage by the mycorrhizae depends both on the species of mycorrhiza and the host plant (Bezemer and Van Dam, 2005; Gupta et al., 2002). Abiotic stress leads to oxidative damage by increasing the reactive oxygen species (ROS). A physiological mechanism to mitigate the deleterious effects of ROS on plant cells is the biosynthesis of antioxidant enzymes, including catalase (CAT), superoxide

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Please cite this article in press as: Amiri, R., et al., Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.062

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dismutase (SOD), and ascorbate peroxidase (APX), which cause plant cell protection against oxidative damage (Caverzan et al., 2012; Wu et al., 2006). There are some reports indicating that mycorrhizal inoculation restricts the excessive production of ROS by enhancing antioxidant activity enzymes, such as SOD, POD and CAT (Wu et al., 2014). Wu et al. (2014) reported that mycorrhizal inoculated plants had higher antioxidant activities, including SOD, CAT and APX, and lower malondialdehyde (MDA) concentration compared to non-inoculated control plants. Promising but limited reports are available on the effect of AMF inoculation on medicinal and aromatic plants including: Farahani et al. (2008) on coriander (Coriandrum sativum), Cabello et al. (2005) on mint (Mentha piperita), Kapoor et al. (2004) on fennel (Foeniculum vulgare) and Zitterl-Eglseer et al. (2015) on angelica (Angelica archangelica L.). Nell et al. (2010) found that mycorrhiza inoculation increased the sesquiterpenic acid concentrations in valerian (Valeriana officinalis). Previous study by Eiasu et al. (2012) showed that drought stress causes a reduction in the growth and yield of pelargonium (P. graveolens). Although pelargonium is an important medicinal and aromatic plant in terms of essential oil production, the information on the effect of drought stress on this plant is limited. On the other hand, the impact and mechanisms of AMF inoculation on the alleviation of drought stress damage to the plant has not been studied. Hence, the objective of the present study was to evaluate the effectiveness of two species of AMF inoculation separately and in combination to determine the mechanisms involved in the antioxidant defence of the plant under drought stress and the production of secondary metabolites.

2.3. Mycorrhizal inoculation There were four inoculum treatments, including Glomus intraradices, G. mosseae, a combination of both and a non-inoculated control. The AM fungi were provided by Touran Biotech Company (Shahrod, Iran). The fungal inoculums were isolated from annual medic (Medicago scutellata L.). Inoculum from the culture comprised a mixture of spores (approximately 25 spores per gram) and was counted under a light microscope before the experiment. The pots were inoculated with 100 g AM fungal inoculums (sandy soil and annual medic root fragments) consisting of a mixture of 2000–3000 spores. 2.4. Irrigation treatments Plants were irrigated daily based on 100% FC after the plants were established (2 months), and then different irrigation regimes were applied. The irrigation volume was determined by estimating the evapotranspiration (ETC ; mm) of the crop as follows: ETC = ET0 × KC , where ET0 was the reference evapotranspiration (mm d−1 ) calculated by the Penman–Monteith method and KC was the crop coefficient for the plant (Allen et al., 1998). Irrigation treatments were applied as 100, 75 and 50% FC. The amounts of 100, 75 and 50% ET were applied for full irrigation, moderate and severe deficit irrigation, respectively. To monitor the soil water content, tensiometry probe tubes were inserted into the soil in the control pot around the root. Irrigation was performed whenever 40% of the available water was consumed. To calculate the amount of water necessary to bring each soil to FC, soil samples were collected and the water content was determined by drying. The experiment was conducted for 120 days.

2. Materials and methods

2.5. Root colonization

2.1. Experimental site

To determine root colonization, roots were stained as described by Phillips and Hayman (1970), and the percentage of mycorrhizal root infection was calculated according to the gridline intersection method (Giovannetti and Mosse, 1980).

This outdoor experiment was conducted during February–September 2013 and was repeated under the same conditions in 2014 at the campus facility of the department of horticulture at Isfahan University of Technology, Isfahan, Iran (32◦ 39 N, 51◦ 40 E; 1600 m). The site was characterized as having an arid climate with cold winters, an average annual rainfall of 122.8 mm, and an average annual temperature of 23.4 ◦ C and relative humidity of 35–70%.

2.2. Experimental design and treatments This study was conducted as a factorial experiment based on a complete randomized design (CRD) with two factors, AMF inoculation and irrigation level, and with three replicates (3 pots per replicate). The treatments consisted of four mycorrhizal inoculations, including Glomus intraradices (Gi), Glomus mosseae (Gm), a combination of both (Gi + Gm) and non-inoculated plants as control (NM), as well as three irrigation levels, 100% FC as control, 75% FC (moderate deficit irrigation) and 50% FC (severe deficit irrigation). Rooted cuttings (three rooted cuttings/pot) were planted into 8-L plastic pots containing soil:sand (3:1 v/v). The soil properties were: 32.8% clay, 42% silt, and 25.2% sand; pH 8.03; EC 1.79 dS m−1 ; nitrogen (N) 45, phosphorus (P) 11, potassium (K) 126, iron (Fe) 5, zinc (Zn) 2, magnesium (Mg) 112 mg kg−1 soil; and organic material 9.5 g kg−1 . Each pot was filled with 6 kg of soil up to 3 cm under the edge of the pots. A fertilizer (2 g L−1 20-5-10 Novatec Solub, Compo, Germany) was applied twice during the experiment, including an early stage of planting before initiation of the drought stress and after 2 months (middle of the stress period).

2.6. Essential oil content measurement Essential oil contents were determined using a Neo-Clevenger apparatus. A total of 100 g of sample was subjected to 500 mL water distillation and run for 5.5 h on the Neo-Clevenger apparatus. The essential oil content was calculated as a relative percentage (British Pharmacopoeia, 1980). The oil yield was calculated based on following formula: Oil yield = (essential oil × fresh weight)/100

2.7. Phenol and flavonoids content assay One germ of dry plant samples was extracted in 50 mL of methanol (80%) by using a blender and macerated at room temperature for two days. The solvent was completely removed and the extract was kept below 4 ◦ C to assay the amount of total phenol and flavonoids content. To assay the total phenol content, the diluted extract of plants (0.5 mL of 1:10 g mL−1 ) or standard phenolic compound (Gallic acid) was mixed with the Folin–Ciocalteu reagent (5 mL, 1:10 diluted with distilled water) and aqueous Na2 CO3 (4 mL, 1 M). The total phenol content was determined using a spectrophotometer (UV-160A UV–vise Recording Spectrophotometer; Shimadzu, Tokyo, Japan) at 765 nm and measured as gallic acid equivalent per gram of plant extract (mg GAE g−1 DW) (McDonald et al., 2001).

Please cite this article in press as: Amiri, R., et al., Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.062

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The flavonoids content was measured according to the method described by Zhishen et al. (1999). A 0.5 mL sample of leaf extract was mixed with 4.5 mL distilled water and 0.3 mL of 5% NaNO2 . After 6 min, 1 mL of 10% AlCl3 ·6H2 O was added, and after another 5 min, 2 mL of 1 M NaOH and distilled water were added to reach the final volume of 10 mL. The absorbance was measured immediately at 510 nm by spectrophotometer (Shimadzu UV-160A), and the flavonoids content was determined as mg rutin equivalent (RE) per gram dry weight (DW). 2.8. Enzyme activity assay In order to assay enzyme activity, half a gram of fresh leaves was ground in liquid nitrogen and then homogenized in 5 mL K-phosphate buffer (50 mM, pH 7.0, and 0.1 mM EDTA). The homogenate was centrifuged in refrigerated centrifuge at 4 ◦ C (12 000 rpm, 30 min), then the supernatant was used for enzyme activity assays. Catalase (CAT) (EC: 1.11.1.6) activity was assayed in a reaction mixture containing 50 mM K-phosphate buffer (pH 7.0), 10 mM H2 O2 and an enzyme aliquot. The decomposition of H2 O2 was followed at 240 nm (Abedi and Pakniyat, 2010). Ascorbate peroxidase APX (EC: 1.11.1.11) activity was assayed as described by Nakano and Asada (1981). The reaction (50 mM Kphosphate buffer (pH 7.0), 0.5 mM ascorbate (AsA), 0.1 mM H2 O2 , 0.1 mM EDTA, and enzyme extract in a final volume of 0.7 mL) was initiated by the addition of H2 O2 , and the activity was measured by observing the decrease in absorbance at 290 nm for 2 min using an extinction coefficient of 2.8 mM−1 cm−1 using a spectrophotometer (UV-160A UV–vis Recording Spectrophotometer; Shimadzu, Tokyo, Japan). The glutathione peroxidase (GPX) (EC: 1.11.1.9) activity was measured as described by Rao et al. (1996) using H2 O2 as a substrate. The oxidation of NADPH was recorded at 470 nm for 2 min, and the activity was calculated using the extinction coefficient of 26.6 mM−1 cm−1 . Superoxide dismutase (SOD) (EC: 1.15.1.1) was assayed using the photochemical method described by Sairam et al. (2002). The spectrophotometric absorbance of the mixture was determined at 560 nm by a UV–vis spectrophotometer (UV-160A UV–vis Recording Spectrophotometer; Shimadzu, Tokyo, Japan). One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of nitroblue tetrazolium (NBT) photoreduction. The SOD activity was expressed as enzyme activity per gram leaf fresh weight.

Fig. 1. Root colonization in rose geranium subjected to different irrigation regimes (100%, 75% and 50% Field Capacity) and arbuscular mycorrhiza fungi (AMF) species application (Gi, Glomus intraradices; Gm, G. mosseae, and a combination of both species; Gi + Gm). Data (means ± SD, n = 3) followed by different small letters above the bars indicate a significant difference at P ≤ 0.05.

(D = 0.28 ␮M−1 cm−1 ) and was expressed as micromoles per gram fresh weight. 2.10. Statistical analysis All data were subjected to one-way analysis of variance using SAS statistical software (version 9.1; SAS Institute, Cary, NC, USA). Comparisons of the means used the least significant difference (LSD) at P ≤ 0.05. 3. Results 3.1. Root colonization Mycorrhizal colonization of roots significantly decreased in 50% FC compared to 100% (by 127%) and 75% FC (by 98%); however, colonization was not affected by the mycorrhizal inoculation species (Table 1). Although root colonization was suppressed in the severe water deficit condition (50% FC) and no mycorrhizal inoculation improved the root colonization, colonization did not decline when the roots were inoculated by G. mosseae or the combination of both species at 75% FC (Fig. 1). 3.2. Essential oil, total phenol and flavonoids contents

2.9. MDA and H2 O2 assays To determine the malondialdehyde (MDA) content in the leaves, the leaf tissue (0.5 g) was homogenized in 5 ml 0.1% (w/v) trichloroacetic acid (TCA), and the homogenate was centrifuged at 11 500 × g for 10 min. The supernatant (1 mL) was mixed with 4 mL of thiobarbituric acid (TBA) reagent (0.5% of TBA in 20% TCA). The reaction mixture was heated at 95 ◦ C for 30 min in a water bath and was then quickly cooled in an ice bath and centrifuged at 11 500 × g for 15 min. The amount of MDA–TBA complex (red pigment) was measured by a spectrophotometer (UV-160A UV–vis Recording Spectrophotometer; Shimadzu, Tokyo, Japan) at 532 and 600 nm with the extinction coefficient of 155 mM−1 cm−1 and was expressed as ␮mol MDA g−1 FW (Velikova et al., 2000). Hydrogen peroxide (H2 O2 ) was assayed according to the method described by Velikova et al. (2000). The optical absorption of the supernatant was measured by a spectrophotometer (UV-160A UV–vis Recording Spectrophotometer; Shimadzu, Tokyo, Japan) at 390 nm to determine the H2 O2 content

The essential oil and total phenol contents significantly increased by 12.4% and 16%, respectively, when plants were under moderate water deficit (75% FC) compared to 100% FC. All inoculation practices elevated the essential oil content and oil yield compared to the non-inoculated treatment (Table 1). The same trend was observed for the total phenol content (Table 1). We were not able to measure the essential oil content in the severe water deficit treatment due to insufficient plant material production. In general 75% FC provided more favourable conditions for essential oil biosynthesis in plants than the full irrigation condition (Fig. 2a); however, when plants were inoculated with G. mosseae or the combination of both species, no significant difference was observed between the 75% and 100% FC conditions (Fig. 2a). The results showed that oil yield was significantly increased by all mycorrhizal inoculations in the water and drought stress conditions (Fig. 2b). Total phenol content reached a peak when plants were inoculated by G. intraradices under 75% FC. Non-inoculated plants recorded the lowest phenol content regardless of water irrigation

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Table 1 Effect of irrigation regime and mycorrhizal inoculation on root colonization, essential oil, oil yield, total phenol and flavonoid contents of rose geranium. Root colonization (%)

Essential oil content (%)

Oil yield (mL pot−1 )

Total phenol (mg GAE g−1 DW)

Flavonoid content (mg RE g−1 DW)

Irrigation regime 100% (FC) 75% (FC) 50% (FC)

53.3a 46.4b 23.4c

0.137b 0.154a N

0.454a 0.441a N

7.22b 8.35a 4.69c

9.95a 6.58b 3.24c

AMF status Non-AMF G. intraradices G. mosseae Gi + Gm

– 39.2a 41.3a 42.6a

0.132b 0.149a 0.151a 0.148a

0.348c 0.452b 0.498a 0.492ab

5.44b 7.37a 7.06a 7.14a

4.80b 6.95a 7.49a 7.12a

**

**

ns

**

**

ns ns

* ns

** ns

** ns

** ns

Treatments

Significance Irrigation regime AMF W × AMF

Means followed by the same letter within each column shows no significant differences among treatments at 0.05 level by LSD. (Gi, Glomus intraradices; Gm, G. mosseae; AMF, arbuscular mycorrhizal fungi; FC, Field Capacity; ns, not significant. *P < 0.05, **P < 0.01). N: in the present experiment, due to lack of sufficient plant material in 50% FC, were not able to measure essential oil content in this treatment.

Fig. 2. Effect of arbuscular mycorrhiza fungi (AMF) species application (Gi, Glomus intraradices; Gm, G. mosseae, a combination of both species; Gi + Gm and NM, nonmycorrhizal plants) on essential oil content (A) and oil yield (B) in rose geranium grown under different irrigation regimes (100% and 75% Field Capacity). Means followed by different small letters above the bars (represent standard deviations) indicate a significant difference at P ≤ 0.05.

Fig. 3. Interaction effect of arbuscular mycorrhiza fungi (AMF) species application (Gi, Glomus intraradices; Gm, G. mosseae, a combination of both species; Gi + Gm and NM, non-mycorrhizal plants) under different irrigation regimes (100%, 75% and 50% Field Capacity) on total phenol (A) and flavonoid (B) contents in rose geranium. Data (means ± SD, n = 3) followed by different small letters above the bars indicate a significant difference at P ≤ 0.05.

3.3. Enzyme activities regime (Fig. 3a). Table 1 shows that the flavonoids content dramatically decreased as a consequence of both water stress conditions, and the lowest amount was recorded at 50% FC (Table 1). Neither G. intraradices nor G. mosseae treatments were effective to alleviate the adverse effect of water stress on the flavonoids content (Fig. 3b). The highest flavonoids content was observed when fully irrigated plants were inoculated by mycorrhizal fungi (Fig. 3b).

CAT, APX and GPX activity showed the same trend regarding water availability. They all reached the peak when plants were under the moderate water deficit condition (75% FC) (Table 2). All arbuscular mycorrhizal fungi inoculations significantly improved the plant enzymatic defence system compared to non-inoculated plants (Table 2). The highest CAT activity was recorded in plants

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Table 2 Effect of irrigation regime and mycorrhizal inoculation on CAT (catalase), APX (ascorbate peroxidase), GPX (glutathione peroxidase) and SOD (superoxide dismutase) activities and MDA (malondialdehyde) and H2 O2 (hydrogen peroxide) contents of rose geranium. Treatments Irrigation regime 100% (FC) 75% (FC) 50% (FC) AMF status Non-AMF G. intraradices G. mosseae Gi + Gm Significance Irrigation regime AMF W × AMF

CAT (␮mol min−1 g−1 FW)

APX (␮mol min−1 g−1 FW)

GPX (␮mol min−1 g−1 FW)

SOD (Ug−1 FW)

MDA (␮mol g−1 FW)

H2 O2 (␮mol g−1 FW)

3.00b 4.05a 1.41c

0.329b 0.477a 0.370b

4.02b 7.59a 4.49b

204.6b 334.0a 147.4c

4.10c 6.79b 9.73a

16.96c 24.61b 36.00a

1.87b 2.88a

0.315b 0.393a

4.36b 5.80a

210.1b 231.6ab

8.46a 6.78b

30.93a 25.12b

3.33a 3.21a

0.438a 0.422a

5.48a 5.84a

239.0a 233.9ab

6.15b 6.12b

23.29b 24.08b

**

**

**

**

**

**

** *

** ns

* ns

ns ns

** ns

** *

Means followed by the same letter within each column shows no significant differences among treatments at 0.05 level by LSD. (Gi, Glomus intraradices; Gm, G. mosseae; AMF, arbuscular mycorrhiza fungi; FC, Field Capacity; ns, not significant. *P < 0.05, **P < 0.01).

Fig. 4. Interaction effect of arbuscular mycorrhiza fungi (AMF) species application (Gi, Glomus intraradices; Gm, G. mosseae, a combination of both species; Gi + Gm and NM, non-mycorrhizal plants) on CAT (catalase), APX (ascorbate peroxidase), GPX (glutathione peroxidase) and SOD (superoxide dismutase) activities(A–D) in rose geranium grown under different irrigation regimes (100%, 75% and 50% Field Capacity). Data (means ± SD, n = 3) followed by different small letters above the bars indicate a significant difference at P ≤ 0.05.

grown under moderate water deficit (75%) and inoculated by G. mosseae or the combination of both fungi (Fig. 4a). APX activity reached the peak when plants at 75% FC were inoculated by G. mosseae (by 59%) or the combination of both fungi (by 45%) compared to the non-inoculated plants (Fig. 4b). GPX activity increased at 75% FC compared to the plants grown at 100% FC (by 81%). All mycorrhizal inoculations significantly increased GPX activity under the moderate water condition (75% FC). Neither 100% FC nor 50% FC reached the mycorrhizal inoculation elevated GPX activity level of 75% FC (Fig. 4c).

SOD activity followed the same trend as the other antioxidant enzymes (Table 2). SOD activity increased in the 75% FC and decreased in the 50% FC compared to the 100% FC; however, G. mosseae was more effective than G. intraradices or the combination of both species (Table 2). Mycorrhizal inoculation with G. intraradices or G. mosseae significantly increased SOD activity in the plants grown under 75% FC by approximately 23% and 24%, respectively, compared to non-mycorrhizal plants (Fig. 4d).

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Fig. 5. Interaction effect of arbuscular mycorrhiza fungi (AMF) species application (Gi, Glomus intraradices; Gm, G. mosseae, a combination of both species; Gi + Gm and NM, non-mycorrhizal plants) on H2 O2 (hydrogen peroxide) and MDA (malondialdehyde) contents (A and B) in rose geranium grown under different irrigation regimes (100%, 75% and 50% Field Capacity). Data (means ± SD, n = 3) followed by different small letters above the bars indicate a significant difference at P ≤ 0.05.

3.4. MDA and H2 O2 contents Table 2 shows that water stress significantly elevated the MDA (by 137%) and H2 O2 (by 112%) contents compared to plants under the normal irrigation condition (100% FC). All mycorrhizal inoculations resulted in lower MDA and H2 O2 accumulation in plant tissue (Table 2). Fig. 5a shows that the highest H2 O2 content was accumulated under severe water stress (50% FC). The accumulation increased more in the case of non-inoculated plants. The highest MDA content was recorded when non-inoculated plants were grown under the severe water deficit condition. All mycorrhizal inoculations significantly decreased MDA content in the leaves compared to non-mycorrhizal plants by a maximum of 67% under the combination of both species (Fig. 5b). 4. Discussion Severe drought stress restricted root colonization by mycorrhizal fungi. These results agree with Gholamhoseini et al. (2013), which showed that drought stress had a negative effect on mycorrhizal colonization. Similarly, the results of Grümberg et al. (2015) showed that mycorrhizal colonization was decreased under drought stress by inoculation with Glomus aggregatum but not by Sclerotium constrictum and an AMF mixture. Shukla et al. (2013) reported that mycorrhizal colonization was reduced by low soil moisture. Under full irrigation condition, G. intraradices and the combination of both species were more successful at colonizing the roots, which may reflect the fact that the behaviour of each mycorrhizal fungi could be different, even under similar conditions

(Gholamhoseini et al., 2013), and also depends on severity and periodicity of drought stress (Entry et al., 2002). Augé (2001) stated that the optimal amount of water for plant growth may be optimal for mycorrhiza development. Moreover, lower mycorrhizal colonization may be the result of low carbon availability in the host plant under drought stress and lower spore germination (Wu et al., 2013). Rose geranium is cultivated for its high-value essential oil. The results of the present study revealed that the essential oil and phenol contents of rose geranium increased under moderate water deficiency. The increased essential oil content may be related to the reallocation of the assimilated carbon following a decrease in plant growth (De Abreu and Mazzafera, 2005). In contrast, this phenomenon was clearer when plants were inoculated by mycorrhizal fungi. Increased oil yield was associated with root colonization (r = 0.81, P < 0.01). These findings place rose geranium into the category of aromatic and medicinal plants that favour moderate drought conditions in terms of essential oil content (Eiasu et al., 2009). Another example provided by Heidari and Karami (2014) showed that the oil content of sunflower increased with increasing water stress from 75% FC to 50% FC, and mycorrhizal inoculation increased the oil content compared to the non-inoculated control plant. Alinian and Razmjoo (2014) also reported a similar trend, indicating that the essential oil content of cumin (Cuminum cyminum L.) increased under moderate drought stress. There are also some reports showing that mint (Mentha viridis) and oregano (Origanum onites) plants inoculated with Glomus lamellosum or the combination of two species (G. lamellosum and Glomus etunicatum) resulted in higher essential oil contents than non-mycorrhizal plants (Karagiannidis et al., 2011). There are also some reports indicating that mycorrhizal inoculation increased the essential oil of dill (Anethum graveolens L.), carum (Trachyspermum ammi), coriander (C. sativum) and fennel (F. vulgare) under the normal irrigation regime (Kapoor et al., 2004, 2002a,b). Karagiannidis et al. (2011) indicated that the mechanism involved in essential oil biosynthesis in plants inoculated with AMF is not known and may be related to better nutrient absorption by mycorrhizal plants. A slight increase (by 16%) in the total phenol content of rose geranium was observed at 75% FC compared to 100% FC. Phenolic compounds have strong antioxidant activity and play important roles in the plant defence system against reactive oxygen species (ROS), which are normally produced under stress conditions (Sreenivasulu et al., 2000). A similar report by Rebey et al. (2012) showed that the total phenol content of cumin (C. cyminum L.) was increased under moderate and severe drought stress by 43.7 and 15.2%, respectively, compared to control normal plants. The results suggested that cumin could be considered as a tolerant plant to drought stress. Chung et al. (2006) found that drought stress increased the phenol content of Rehmannia glutinosa. Heidari and Nazari Deljou (2014) found that mycorrhizal (G. mosseae) zinnia (Zinnia elegance) plants had higher phenol contents than non-mycorrhizal plants. Elevated phenolic compounds and flavonoids concentrations of Viola tricolor L. were observed by Zubek et al. (2015) in AMF inoculated plants. Under moderate deficit irrigation (75%) and well irrigation conditions, all mycorrhizal inoculations increased the total phenol content of rose geranium. Although the mechanisms involved in this phenomenon are not fully understood, some researchers believe that mycorrhizal inoculation increases secondary metabolites, including phenolic compounds, in hosts (Jurkiewicz et al., 2010; Rajeshkumar et al., 2008; Selvaraj et al., 2009). The total phenol content of artichoke (Cynara scolymus) was increased in the leaves and flower head of plants inoculated with mycorrhiza (Ceccarelli et al., 2010). The same result was reported in grape by all strains of inoculated mycorrhiza compared to control plants (Eftekhari et al., 2012). Chen et al. (2013) reported that the

Please cite this article in press as: Amiri, R., et al., Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.062

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polyphenol content of cucumber was increased by AMF inoculation in low temperature stress conditions. Increased flavonoids content in Medicago sativa L. was observed by Larose et al. (2002) and Catford et al. (2006) as a result of inoculation by Glomus intraradix, G. mosseae, and Gigaspora rosea. Flavonoids are one of the major secondary metabolites in rose geranium. Flavonoids can scavenge ROS to protect plants from oxidative damage (Wu et al., 2014). The flavonoids content did not follow the essential oil content trend and decreased as a consequence of water deficiency. Mycorrhizal inoculation did not stop the reduction under drought stress (Fig. 3b). Our result is not fully in agreement with Abbaspour et al. (2012), who showed that mycorrhizal inoculation of pistachio (Pistachia vera L.) caused higher flavonoids content both under full irrigation and water stress. The difference between species is the major influencing factor in the case of the flavonoids content. The rose geranium enzymatic defence system actively defended plant physiological processes when plants were under moderate water deficiency (75% FC), and mycorrhizal inoculation had an activation role for most enzymes activities. The CAT activity of rose geranium increased with mycorrhizal inoculation under moderate deficit irrigation. CAT activity was associated with root colonization (r = 0.68, P < 0.01). Our findings are in agreement with Li et al. (2012), who reported that the CAT activity of Suaeda salsa L. increased by inoculation with G. mosseae compared to non-mycorrhizal plant. Similarly, Wu et al. (2007) found that the CAT activity in leaves of Citrus tangerine seedlings increased by AMF inoculation under both the well-watered regime and drought stress. Mycorrhizal inoculation is effective in plant enzymatic defence system induction under different stresses. The findings of Wu et al. (2010) also showed that mycorrhizal inoculation increased the CAT activity of citrus under salt stress. However, there are some reports indicating that CAT activity induction by mycorrhizal inoculation under stress condition depends on the species of AMF species (Wu and Zou, 2009). Our result indicated that GPX activity was significantly increased at moderate stress (75% FC). However, like CAT and APX, GPX activity dropped under severe water deficiency stress. G. mosseae was more effective in terms of CAT and APX activity when plants were under moderate water stress. Similarly, Huang et al. (2010) observed that G. mosseae inoculation increased the APX activity of tomato leaf and root under salt stress conditions. Wu et al. (2006) found that in both well-irrigated and drought stress, mycorrhizal inoculation increased the SOD of C. tangerine plant compared to non-mycorrhizal plant. They concluded that AMF can induce SOD gene expression or enhance SOD activity against abiotic stresses. Abbaspour et al. (2012) explained that AMF plants had low oxidative damage under drought stress conditions due to the drought avoidance mechanism powered by direct water uptake and transfer to the host plant by fungal hyphae, leading to lower ROS production and accumulation in the plant. Similar results with different AM species proved the role of mycorrhizal inoculation in the reduction of H2 O2 in plants under environmental stresses, as reported by Wu et al. (2006) (Glomus versiforme), Porcel and Ruiz-Lozano (2004), Huang et al. (2010) (G. mosseae) and Hajiboland et al. (2010) (G. intraradices). A precise study by Fester and Hause (2005) showed that H2 O2 elimination was handled by vesicle production in AMF plants. Cellular observations confirmed the important role of vesicles in H2 O2 elimination. There was a negative and significant correlation between the H2 O2 content and root colonization of rose geranium (r = −0.65, P < 0.01). Our results confirmed the findings of Hajiboland et al. (2010), who demonstrated that higher antioxidant enzyme activity in mycorrhizal plants resulted in lower accumulation of H2 O2 compared to non-mycorrhizal plants. In our study, the highest MDA content was at 50% FC in the nonmycorrhizal plants, and the lowest was recorded at 100% FC in the

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combination of Gi + Gm. A negative and significant correlation was observed between the MDA content and the root colonization of rose geranium (r = −0.63, P < 0.01). The results showed that mycorrhizal inoculation reduced the MDA content in both stress and non-stress conditions. Our results are supported by the findings of Wu et al. (2006), who showed that the MDA content and H2 O2 concentration were decreased by AMF inoculation under drought stress conditions. To the best of our knowledge, no similar information has been provided for the interaction effect of mycorrhizal inoculation and irrigation regime in growing rose geranium. 5. Conclusion The results of this study demonstrated that AMF inoculation enhanced pelargonium tolerance to drought stress in terms of antioxidant enzyme activity and successfully alleviated the consequences of ROS production in rose geranium under stress. However, under severe water deficiency, no AMF inoculation allowed the plants to handle the adverse effects of drought stress. The full irrigation regime (100% FC) did not necessarily lead to the highest essential oil content, and this species favoured mild water deficiency. Mycorrhizal inoculations can be considered as a tool to maintain the essential oil production of rose geranium plants under drought conditions. Further investigations are needed to carefully study how AMF inoculation interacts with this plant under drought stress. Acknowledgement The authors gratefully acknowledge Barij Essence Company, Kashan, Iran for supporting this work. References Abbaspour, H., Saeidi-Sar, S., Afshari, H., Abdel-Wahhab, M.A., 2012. Tolerance of mycorrhiza infected pistachio (Pistacia vera L.) seedling to drought stress under glasshouse conditions. J. Plant Physiol. 169, 704–709. Abedi, T., Pakniyat, H., 2010. Antioxidant enzyme changes in response to drought stress in ten cultivars of oil seed rape (Brassica napus L.). Czech J. Genet. Plant Breed. 46, 27–34. Allen, R.G., Pereira, L.S., Raes, D., Smith, M., 1998. Crop Evapotranspiration: Guidelines for Computing Crop Water Requirements. FAO Irrigation and Drainage Paper 56, Food and Agricultural Organization of the United Nations, Rome, Italy, p. 300. Alinian, S., Razmjoo, J., 2014. Phenological, yield, essential oil yield and oil content of cumin accessions as affected by irrigation regimes. Ind. Crops Prod. 54, 167–174. Augé, R.M., 2001. Water relations, drought and vesicular–arbuscular mycorrhizal symbiosis. Mycorrhiza 11, 3–42. Bezemer, T.M., Van Dam, N.M., 2005. Linking aboveground and belowground interactions via induced plant defenses. Trends Ecol. Evol. 20, 617–624. British Pharmacopoeia, 1980. Her Majesty’s Stationery Office. Atlantic House, Holborn Viaduct, London. 1196pp. Cabello, M., Irrazabal, G., Bucsinszky, A.M., Saparrat, M., Schalamuk, S., 2005. Effect of an arbuscular mycorrhizal fungus, Glomus mosseae, and a rock–phosphate-solubilizing fungus, Penicillium thomii, on Mentha piperita growth in a soilless medium. J. Basic Microbiol. 45, 182–189. Catford, J.G., Staehelin, C., Larose, G., Piché, Y., Vierheilig, H., 2006. Systemically suppressed isoflavonoids and their stimulating effects on nodulation and mycorrhization in alfalfa split-root systems. Plant Soil 285, 257–266. Caverzan, A., Passaia, G., Rosa, S.B., Ribeiro, C.W., Lazzarotto, F., Margis-Pinheiro, M., 2012. Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection. Genet. Mol. Biol. 35, 1011–1019. Ceccarelli, N., Curadi, M., Martelloni, L., Sbrana, C., Picciarelli, P., Giovannetti, M., 2010. Mycorrhizal colonization impacts on phenolic content and antioxidant properties of artichoke leaves and flower heads two years after field transplant. Plant Soil 335, 311–323. Charles, D.J., Joly, R.J., Simon, J.E., 1990. Effects of osmotic stress on the essential oil content and composition of peppermint. Phytochemistry 29, 2837–2840. Chen, S., Jin, W., Liu, A., Zhang, S., Liu, D., Wang, F., Lin, X., He, C., 2013. Arbuscular mycorrhizal fungi (AMF) increase growth and secondary metabolism in cucumber subjected to low temperature stress. Sci. Hortic. 160, 222–229. Chung, I.M., Kim, J.J., Lim, J.D., Yu, C.Y., Kim, S.H., Hahn, S.J., 2006. Comparison of resveratrol, SOD activity, phenolic compounds and free amino acids in

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G Model HORTI-6089; No. of Pages 8 8

ARTICLE IN PRESS R. Amiri et al. / Scientia Horticulturae xxx (2015) xxx–xxx

Rehmannia glutinosa under temperature and water stress. Environ. Exp. Bot. 56, 44–53. De Abreu, I.N., Mazzafera, P., 2005. Effect of water and temperature stress on the content of active constituents of Hypericum brasiliense Choisy. Plant Physiol. Biochem. 43, 241–248. Eftekhari, M., Alizadeh, M., Ebrahimi, P., 2012. Evaluation of the total phenolics and quercetin content of foliage in mycorrhizal grape (Vitis vinifera L.) varieties and effect of postharvest drying on quercetin yield. Ind. Crops Prod. 38, 160–165. Eiasu, B.K., Steyn, J.M., Soundy, P., 2012. Physiomorphological response of rose-scented geranium (Pelargonium spp.) to irrigation frequency. S. Afr. J. Bot. 78, 96–103. Eiasu, B.K., Steyn, J.M., Soundy, P., 2009. Rose-scented geranium (Pelargonium capitatum × P. radens) growth and essential oil yield response to different soil water depletion regimes. Agric. Water Manage. 96, 991–1000. Entry, J.A., Rygiewicz, P.T., Watrud, L.S., Donnelly, P.K., 2002. Influence of adverse soil conditions on the formation and function of arbuscular mycorrhizas. Adv. Environ. Res. 7, 123–138. Fagbola, O., Osonubi, O., Mulongoy, K., Odunfa, S., 2001. Effects of drought stress and arbuscular mycorrhiza on the growth of Gliricidia sepium (Jacq.). Walp, and Leucaena leucocephala (Lam.) de Wit. in simulated eroded soil conditions. Mycorrhiza 11, 215–223. Farahani, H.A., Lebaschi, M.H., Hamidi, A., 2008. Effects of arbuscular mycorrhizal fungi, phosphorus and water stress on quantity and quality characteristics of coriander. Adv. Nat. Appl. Sci. 2, 55–59. Fester, T., Hause, G., 2005. Accumulation of reactive oxygen species in arbuscular mycorrhizal roots. Mycorrhiza 15, 373–379. Fitter, A.H., 1988. Water relations of red clover Trifolium pratense L. as affected by VA mycorrhizal infection and phosphorus supply before and during drought. J. Exp. Bot. 39, 595–603. Gholamhoseini, M., Ghalavand, A., Dolatabadian, A., Jamshidi, E., Khodaei-Joghan, A., 2013. Effects of arbuscular mycorrhizal inoculation on growth, yield, nutrient uptake and irrigation water productivity of sunflowers grown under drought stress. Agric. Water Manage. 117, 106–114. Giovannetti, M., Mosse, B., 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 84, 489–500. Grümberg, B.C., Urcelay, C., Shroeder, M.A., Vargas-Gil, S., Luna, C.M., 2015. The role of inoculum identity in drought stress mitigation by arbuscular mycorrhizal fungi in soybean. Biol. Fertil. Soils 51, 1–10. Gupta, M.L., Prasad, A., Ram, M., Kumar, S., 2002. Effect of the vesicular–arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum on the essential oil yield related characters and nutrient acquisition in the crops of different cultivars of menthol mint (Mentha arvensis) under field conditions. Bioresour. Technol. 81, 77–79. Hajiboland, R., Aliasgharzadeh, N., Laiegh, S.F., Poschenrieder, C., 2010. Colonization with arbuscular mycorrhizal fungi improves salinity tolerance of tomato (Solanum lycopersicum L.) plants. Plant Soil 331, 313–327. Heidari, M., Karami, V., 2014. Effects of different mycorrhiza species on grain yield, nutrient uptake and oil content of sunflower under water stress. J. Saudi Soc. Agric. Sci. 13, 9–13. Heidari, Z., Nazari Deljou, M.J., 2014. Improvement of morpho-physiological traits and antioxidant capacity of zinnia (Zinnia elegance Dreamland Red) by arbuscular mycorrhizal fungi (Glomus mosseae) inoculation. Int. J. Adv. Biol. Biomed. Res. 2, 2627–2631. Huang, Z., He, C.X., He, Z.Q., Zou, Z.R., Zhang, Z.B., 2010. The effects of arbuscular mycorrhizal fungi on reactive oxyradical scavenging system of tomato under salt tolerance. Agric. Sci. China 9, 1150–1159. ´ Jurkiewicz, A., Ryszka, P., Anielska, T., Waligórski, P., Białonska, D., Góralska, K., Tsimilli-Michael, M., Turnau, K., 2010. Optimization of culture conditions of Arnica montana L.: effects of mycorrhizal fungi and competing plants. Mycorrhiza 20, 293–306. Kapoor, R., Giri, B., Mukerji, K.G., 2004. Improved growth and essential oil yield and quality in Foeniculum vulgare mill on mycorrhizal inoculation supplemented with P-fertilizer. Bioresour. Technol. 93, 307–311. Kapoor, R., Giri, B., Mukerji, K.G., 2002a. Glomus macrocarpum: a potential bioinoculant to improve essential oil quality and concentration in Dill (Anethum graveolens L.) and Carum (Trachyspermum ammi (Linn.) Sprague). World J. Microbiol. Biotechnol. 18, 459–463. Kapoor, R., Giri, B., Mukerji, K.G., 2002b. Mycorrhization of coriander (Coriandrum sativum L.) to enhance the concentration and quality of essential oil. J. Sci. Food Agric. 82, 339–342. Karagiannidis, N., Thomidis, T., Lazari, D., Panou-Filotheou, E., Karagiannidou, C., 2011. Effect of three Greek arbuscular mycorrhizal fungi in improving the growth, nutrient concentration, and production of essential oils of oregano and mint plants. Sci. Hortic. 129, 329–334. Larose, G., Chênevert, R., Moutoglis, P., Gagné, S., Piché, Y., Vierheilig, H., 2002. Flavonoid levels in roots of Medicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus. J. Plant Physiol. 159, 1329–1339.

Li, T., Liu, R.J., He, X.H., Wang, B.S., 2012. Enhancement of superoxide dismutase and catalase activities and salt tolerance of euhalophyte Suaeda salsa L. by mycorrhizal fungus Glomus mosseae. Pedosphere 22, 217–224. McDonald, S., Prenzler, P.D., Antolovich, M., Robards, K., 2001. Phenolic content and antioxidant activity of olive extracts. Food Chem. 73, 73–84. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Nell, M., Wawrosch, C., Steinkellner, S., Vierheilig, H., Kopp, B., Lössl, A., Franz, C., Novak, J., Zitterl-Eglseer, K., 2010. Root colonization by symbiotic arbuscular mycorrhizal fungi increases sesquiterpenic and acid concentrations in Valeriana officinalis L. Planta Med. 76, 393–398. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular–arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55, 158–161. Porcel, R., Ruiz-Lozano, J.M., 2004. Arbuscular mycorrhizal influence on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress. J. Exp. Bot. 55, 1743–1750. Quilambo, O. 2000 Functioning of peanut (Arachis hypogaea L.) under nutrient deficiency and drought stress in relation of symbiotic associations. PhD thesis. University of Groningen, the Netherlands. Van Denderen B.V., Groningen. ISBN 903671284X. Rajeshkumar, S., Nisha, M.C., Selvaraj, T., 2008. Variability in growth, nutrition and phytochemical constituents of Plectranthus amboinicus (Lour.) Spreng. as influenced by indigenous arbuscular mycorrhizal fungi. Majeo Int. J. Sci. Tech. 2, 431–439. Rao, M.V., Paliyath, G., Ormrod, D.P., 1996. Ultraviolet-B and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol. 110, 125–136. Rebey, I.B., Jabri-Karoui, I., Hamrouni-Sellami, I., Bourgou, S., Limam, F., Marzouk, B., 2012. Effect of drought on the biochemical composition and antioxidant activities of cumin (Cuminum cyminum L.) seeds. Ind. Crops Prod. 36, 238–245. Sairam, R.K., Rao, K.V., Srivastava, G.C., 2002. Differential response of wheat genotypes to long term salinity stress in relation to oxidative stress, antioxidant activity and osmolyte concentration. Plant Sci. 163, 1037–1046. Selvaraj, T., Nisha, M.C., Rajeshkumar, S., 2009. Effect of indigenous arbuscular mycorrhizal fungi on some growth parameters and phytochemical constituents of Pogostemon patchouli Pellet. Majeo Int. J. Sci. Technol. 3, 222–234. Sheng, M., Tang, M., Chen, H., Yang, B., Zhang, F., Huang, Y., 2008. Influence of arbuscular mycorrhizae on photosynthesis and water status of maize plants under salt stress. Mycorrhiza 18, 287–296. Shukla, A., Kumar, A., Jha, A., Salunkhe, O., Vyas, D., 2013. Soil moisture levels affect mycorrhization during early stages of development of agroforestry plants. Biol. Fertil. Soils 49, 545–554. Sreenivasulu, N., Grimm, B., Wobus, U., Weschke, W., 2000. Differential response of antioxidant compounds to salinity stress in salt tolerant and salt sensitive seedlings of foxtail millet (Setaria italica). Physiol. Plant. 109, 435–442. Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Sci. 151, 59–66. Wu, Q.S., Srivastava, A.K., Zou, Y.N., 2013. AMF-induced tolerance to drought stress in citrus: a review. Sci. Hortic. 164, 77–87. Wu, Q.S., Zou, Y.N., 2009. Mycorrhiza has a direct effect on reactive oxygen metabolism of drought-stressed citrus. Plant Soil Environ. 55, 436–442. Wu, Q.S., Zou, Y.N., Abd-Allah, E.F., 2014. Mycorrhizal association and ROS in plants. In: Ahmad, P. (Ed.), Oxidative Damage to Plants Antioxid. Academic Press, pp. 53–475. Wu, Q.S., Zou, Y.N., Liu, W., Ye, X.F., Zai, H.F., Zhao, L.J., 2010. Alleviation of salt stress in citrus seedlings inoculated with mycorrhiza: changes in leaf antioxidant defense systems. Plant Soil Environ. 56, 470–475. Wu, Q.S., Zou, Y.N., Xia, R.X., 2006. Effects of water stress and arbuscular mycorrhizal fungi on reactive oxygen metabolism and antioxidant production by citrus (Citrus tangerine) roots. Eur. J. Soil Biol. 42, 166–172. Wu, Q.S., Zou, Y.N., Xia, R.X., Wang, M.Y., 2007. Five Glomus species affect water relations of Citrus tangerine during drought stress. Bot. Stud. 48, 147–154. Zhishen, J., Mengcheng, T., Jianming, W., 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559. Zitterl-Eglseer, K., Nell, M., Lamien-Meda, A., Steinkellner, S., Wawrosch, C., Kopp, B., Zitterl, W., Vierheilig, H., Novak, J., 2015. Effects of root colonization by symbiotic arbuscular mycorrhizal fungi on the yield of pharmacologically active compounds in Angelica archangelica L. Acta Physiol. Plant. 37, 1–11. Zubek, S., Rola, K., Szewczyk, A., Majewska, M.L., Turnau, K., 2015. Enhanced concentrations of elements and secondary metabolites in Viola tricolor L. induced by arbuscular mycorrhizal fungi. Plant Soil 390, 129–142.

Please cite this article in press as: Amiri, R., et al., Alleviation of drought stress on rose geranium [Pelargonium graveolens (L.) Herit.] in terms of antioxidant activity and secondary metabolites by mycorrhizal inoculation. Sci. Hortic. (2015), http://dx.doi.org/10.1016/j.scienta.2015.09.062