Essential oil content and chemical composition of Cymbopogon citratus inoculated with arbuscular mycorrhizal fungi under different levels of lead

Industrial Crops and Products 76 (2015) 734–738

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Essential oil content and chemical composition of Cymbopogon citratus inoculated with arbuscular mycorrhizal fungi under different levels of lead Caroline Lermen a , Fabrício Morelli b , Zilda Cristiani Gazim a , Adriana Pereira da Silva a , José Eduardo Gonc¸alves c , Douglas Cardoso Dragunski d , Odair Alberton a,∗ a Postgraduate Program in Biotechnology Applied to Agriculture, Universidade Paranaense – UNIPAR, Prac¸a Mascarenhas de Moraes, n◦ 4282, 87502-210, Umuarama, PR, Brazil b Universidade Paranaense – UNIPAR, Prac¸a Mascarenhas de Moraes n◦ 4282, 87502-210 Umuarama, PR, Brazil c Centro Universitário Cesumar – UniCesumar, Avenida Guedner, n◦ 1610, Jardim Aclimac¸ão, 87050-900 Maringá, PR, Brazil d Departamento de Engenharias e Ciências Exatas, Universidade Estadual do Oeste do Paraná – UNIOESTE, 85903-000 Toledo, PR, Brazil

a r t i c l e

i n f o

Article history: Received 4 March 2015 Received in revised form 21 May 2015 Accepted 9 July 2015 Keywords: Mycorrhiza Aromatic plants Lemon grass Heavy metals Citral Geranial

a b s t r a c t Lemon grass, Cymbopogon citratus Stapf, is an important aromatic plant used by industries that produce fragrances and aromas. This topic has been investigated very little in terms of arbuscular mycorrhizal fungi (AMF) associations and the effects of heavy metals, such as lead (Pb), in its metabolism. This study aimed to evaluate the essential oil (EO) content and the chemical composition obtained from lemon grass cultivated with or without AMF Rhizophagus clarus inoculums under five levels of Pb in the soil. The experiment was conducted in a greenhouse for six months and consisted of a completely randomized design. A 5 × 2 factorial was used for five levels of Pb (0, 50, 100, 500, and 1000 mg Pb kg−1 soil) and two levels of AMF inoculation (with or without R. clarus) and five replicates, totaling 50 experimental units. The EO content was determined and the chemical composition analyzed by GC and GC/MS. The secondary metabolites were affected by the presence of Pb in the soil as well as by the AMF association, which altered the content and chemical composition of EO. The levels of 500 and 1000 mg Pb kg−1 soil together with AMF association increased EO content up to 0.69%. In total, 21 components of EO were identified, and along the increasing levels of Pb, the plant metabolism changed and altered the major components of EO. Without AMF inoculation, the major constituent of EO was citral, with concentrations ranging from 36.66 to 45.08% for the lowest levels of Pb. With AMF inoculation, EO was composed mainly of geranial, with concentrations ranging from 39.27 to 58.97% for all Pb levels. Without AMF inoculation, the concentrations of geranial ranged from 43.66 to 62.95% for the highest levels of Pb. High levels of citral and geranial are of great interest to the aroma and fragrance industry, and citral is a basic substance for the synthesis of vitamin A and ionone. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Among the plant families that produce essential oils (EOs), Poaceae is one of the largest families, comprising approximately 500 genus and 8000 herb species generally known as grasses. Cymbopogon citratus Stapf originated from India and belongs to the Poaceae family (Barbosa et al., 2008). This plant is widespread throughout the world and is easily found in tropical countries such as Brazil. It is popularly known as citronella grass or lemon grass.

∗ Corresponding author. Fax: +55 44 36212830. E-mail addresses: [email protected], [email protected] (O. Alberton). http://dx.doi.org/10.1016/j.indcrop.2015.07.009 0926-6690/© 2015 Elsevier B.V. All rights reserved.

This plant has been highly valued in the pharmaceutical, aromatic, fragrance and food industries due to the high content of EO in its leaves. The major components of EOs obtained from lemon grass are the neral, citral and geranial, along with other components that are found in lower amounts. These major components show sedative, diuretic, analgesic, vermicide, insecticide, larvicide and anti-microbial effects (Barbosa et al., 2008; Andrade et al., 2009). The environment in which the plants develop can redirect the metabolic pathway, causing the biosynthesis of different compounds that may affect the EOs. These environmental changes include: biotic and abiotic factors such as plant age and development stage, luminosity, temperature, rainfalls, soil fertility, day and time of harvest, techniques of collection and processing, level of

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pollution and soil microbial interactions such as arbuscular mycorrhizal fungi (AMF) (Morais, 2009; Santos et al., 2009; Lermen et al., 2015; Urcoviche et al., 2015). Among various types of pollution, the soil contamination by heavy metals (HMs), in most cases, causes negative consequences to the growth and development of plants. Therefore, it prejudices and changes the production of active compounds (Lermen et al., 2015; Sá et al., 2015). Soil contamination caused by HMs has increased mainly as a result of mining and manufacturing activities, the use of sewage sludge and the application of fertilizers or pesticides in rural areas. In addition to reducing plant productivity, the accumulation of HMs in soils can indirectly affect both human and the animal health (Souza et al., 2012; Sá et al., 2015). The remediation process of HMs, such as lead-(Pb) contaminated soils, is significantly expensive for many industries and government bodies (Punamiya et al., 2010). In light of this, researchers have investigated alternatives that help to reverse this situation by using species of soil microorganisms like AMF, which are able to alter both the bioavailability of HMs and the plant response to their presence in soils (Audet and Charest, 2007; Wang et al., 2012). Few studies have demonstrated that AMF symbiosis can contribute to plant tolerance of HMs; however, such mechanisms are not totally elucidated (Wang et al., 2012). Two hypotheses have been proposed to explain the role of AMF symbiosis in the phytoremediation of soils containing HMs: the increased extraction of HMs by the fungal hyphae or the increased plant tolerance to the HMs due to their lower bioavailability caused by the chemical bonds that are established between fungi and metals (Audet and Charest, 2007). Lead is generally toxic HM that has low solubility in soils. The main visual indicator of Pb toxicity in plant is growth reduction followed by structural alterations that affect the content and quality of the EO (Lermen et al., 2015; Sá et al., 2015). In this context, the present study aimed to evaluate the content and chemical composition of EOs from lemon grass plants (whether inoculated or not) with AMF Rhizophagus clarus under five levels of Pb in the soil.

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of 0, 50, 100, and 500 mg Pb kg−1 (Lermen et al., 2015; Sá et al., 2015). Once weighed, the Pb(NO3 )2 was dissolved in 100 mL of deionized water (Sá et al., 2015), and this solution was mixed with the fumigated soil. The lemon grass seedlings were transplanted 15 days later, after the stabilization of Pb in the soil. Equivalent amounts of urea-nitrogen were applied to all treatments in order to avoid the undesirable effects of N as Pb(NO3 )2 . To balance NO3 in the control treatment, 292.7 mg Urea kg−1 soil was added to have the same amount of total N compared with 1000 mg Pb kg−1 soil treatment. In the other Pb treatments, we followed the same procedure to replace the comparable amount of NO3 . The AMF R. clarus soil inoculums from the Glomales bank of UNIPAR Access No. 10 (Lermen et al., 2015; Urcoviche et al., 2015) were applied to the upper third of each pot designed for the AMF treatment. For each pot, 200 g soil inoculum containing 500 spores and infective propagules was added. The control treatments used 100 mL of the filtered soil inoculums (100 g soil inoculum L−1 deionized water). Thus, we only have the effect of the inoculated AMF. All treatments were fertigated every two days with a half concentration of the solution by Hoagland and Arnon, (1950), except for N, which was already applied as Pb(NO3 )2 and urea at the beginning of the experiment.

2.2. Essential oil content At the end of the vegetative cycle, the lemon grass plants were harvested early in the morning (from 7:00 to 10:00 am) and then separated into aerial and root portions. The plants were obtained from all treatments and analyzed. From each treatment, 100 g of the fresh aerial parts of the lemon grass plants were submitted to hydrodistillation (with 1 L deionized water) in a modified Clevenger apparatus. The distillation time was 2 h. The EO was removed with hexane, filtered with anhydrous Na2 SO4 and stored in amber flasks at 4 ◦ C (Santos et al., 2009). The content (%) was obtained after the solvent evaporation.

2. Materials and methods 2.3. Chemical identification of essential oil by GC/MS 2.1. Experiment design and set up The soil for the experiment was collected at 0–20 cm depth in the experimental field of the Paranaense University—UNIPAR, Umuarama city, Paraná State, at coordinates S 23◦ 46 11.34 and WO 53◦ 16 41.78 and at 391-m height from sea level. A soil sample was subjected to chemical characterization at “Solo Fétil” laboratory (Table 1). The soil was passed through a 0.4 mm sieve, and then 15 kg soil was placed into two dark polyethylene bags for fumigation with chloroform at 10 mL (CHCl3 ) kg−1 soil (Endlweber and Scheu, 2006). After mixing the soil with chloroform, the bags were sealed and left for three days of fumigation. Then, the bags were opened in an exhaustion chamber where, they rested for one week before the experiment took place. Approximately, 20 cm seedlings of lemon grass were collected at the Medicinal Garden of the Paranaense University. The seedlings were washed in water and disinfected in 70% alcohol for 1 min, and one seedling was planted per pot. All experimental units, with or without AMF inoculation, were cultivated in 6 L pots with fumigated soil, some containing Pb (NO3 )2 and some not, according to their respective treatments. All plants were grown in a greenhouse for six months. To obtain 1000 mg Pb kg−1 of soil, we weighed 1614.4 mg Pb(NO3 )2 kg−1 of soil and used the proportional rates

The chemical identification of the EO was made by GC–MS, using an Agilent 5973 Network Mass Selective Detector. The capillary column was the DB-5 (5% phenyl–methylsiloxane, 30 m × 0.25 mm id, 0.25 ␮m). The detection system was the electronic impact on the “Split” mode 2:1 mL min−1 . The column temperature was initially programmed at 40 ◦ C, heating at 8 ◦ C min−1 to reach the final temperature of 300 ◦ C. The injector and detector temperatures were 250 ◦ C and 320 ◦ C, respectively. Helium was used as a carrier gas at flow rate of 4.8 mL min−1 . The amount of injected sample was 1 ␮L.

2.4. Statistical analyses The statistical design was completely randomized in 5 × 2 factorial: 5 levels of Pb(NO3 )2 (0, 50, 100, 500, and 1000 mg kg−1 soil) and two levels of inoculation (with and without AMF) with five replicates. The EO content data were subjected to an analysis of variance (ANOVA) using a general linear model with mixed-effects and balanced design. Prior to ANOVA, the Levene’s test was applied to data for homogeneity. Means were compared by Tukey’s test (p ≤ 0.05), using the SPSS statistical package, version 16.0 for Windows (SPSS Inc., Chicago, IL, USA).

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Table 1 Chemical characterization of the soil used in the experiment. pH (CaCl2 )

P

Soil Referencea

5.51 3.8–6.6

Al3+

C

H+ + Al3+

Ca2+

Mg2+

K+

SB

CEC

V

mg dm

g dm

Cmolc dm

Cmolc dm

Cmolc dm

Cmolc dm

Cmolc dm−3

Cmolc dm−3

Cmolc dm−3

(%)

8.26 16–24

3.19 0.8–15.9

0.0 –

2.28 0.6–5.0

0.88 0.3–7.2

0.38 0.3–3.3

0.18 0.1–0.7

1.44 –

3.72 2.2–12.5

37.86 –

−3

−3

−3

−3

−3

−3

Methods: P.K extracted by Mehlich-I; Ca. Mg and Al – extracted by KCl 1 mol L−1 ; C – dichromate /colorimetric. CEC = cation exchange capacity; SB = Sum of bases; V = Base saturation. a Source: (Sambatti et al., 2003).

0,8 0.8 a 0.7 0,7

Oil content (%)

0,6 0.6

a ab

ab

0,5 0.5

ab

ab

ab ab ab

0,4 0.4 0,3 0.3

b

0.2 0,2

0,1 0.1 0.00 0

50

100

500

1000

Pb levels (mg kg–1 of soil) Fig. 1. Essential oil content of lemon grass non-inoculated (light gray bars) and inoculated (dark gray bars) with AMF Rhizophagus clarus in soil under five levels of Pb (0, 50, 100, 500, and 1000 mg kg−1 soil). Columns followed by the same letter are not significantly different (Tukey, p ≤ 0.05). Bars = standard error.

3. Results and discussion 3.1. Essential oil content Fig. 1 demonstrates the EO content of lemon grass cultivated with or without levels of Pb and with or without AMF R. clarus inoculation. The EO content ranged from 0.21 to 0.69% with AMF inoculation. The level of 100 mg Pb kg−1 soil decreased the EO content significantly (Fig. 1). Significant changes were not observed in the EO content in plants without AMF inoculation, the values being approximately 0.5% independent of the Pb levels in the soil. Andrade et al. (2009) found that the EO content of lemon grass ranged from 1.1 to 1.3%, more than double what was found in the present study. In a previous study, Lermen et al. (2015) concluded that AMF inoculation increased plant growth, reduced the accumulation of Pb in roots and increased the P and N content in plant shoots, but under high Pb levels, AMF inoculation increased the Pb in shoots. This might influence EO content and composition. In the preset study, no significant differences were found for EO content among treatments with or without AMF inoculation and subjected to 0 and 50 mg Pb kg−1 soil. However, the level of 100 mg Pb kg−1 soil with AMF inoculation reduced EO content (0.21%), whereas, levels of 500 and 1000 mg Pb kg−1 soil with AMF inoculation increased EO content to 0.69% (Fig. 1). Probably, the lemon grass used its secondary metabolism as a defense against the high concentrations of

Pb, resulting in alterations in the metabolic pathways and causing an increased production of EO. This result can be compared with the results of Sá et al. (2015). The authors evaluated the content of EO of Mentha crispa cultivated in soil under different Pb levels and observed that the levels of Pb influenced EO synthesis. They also found that the highest EO content was obtained at a level of 3.600 mg Pb kg−1 . In another study, Urcoviche et al. (2015) found that M. crispa inoculated with AMF G. etunicatum showed the highest EO content (0.98%) for low levels of phosphorus (P) in the soil. However, when Pb was applied in the soil, EO content was reduced to 0.09%. Karagiannidis et al. (2012) observed that the AMF inoculation increased significantly the EO production in all plant species tested when compared with the non-inoculated treatment. Specifically, EO content increased to 28.75, 55.56, 56.95 and 55.24% for Santolina chamaecyparissus, Salvia officinalis, Lavandula angustifolia, Geranium dissectum and Origanum dictamnus, respectively. 3.2. Chemical identification of essential oil The GC and GC/MS analyses of the EO led to the identification of 21 compounds (Table 2), the majority of which belonged to the oxygenated monoterpene class. In all treatments, neral, geranial, linalool oxide and citral were the major components. Similar results were found in other studies with lemon grass plants in Brazil (Barbosa et al., 2008; Andrade et al., 2009).

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737

Table 2 Chemical composition of the essential oil (%) of Cymbopogon citratus under five Pb levels (0, 50, 100, 500, and 1000 mg kg−1 soil) and with/without AMF inoculation with Rhizophagus clarus. Without AMF

With AMF

IdentificationMethods

Pb (mg kg−1 soil) Peak 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

c

Component

Monoterpene hydrocarbon Myrcene Menth 1.(7). 8.diene Oxygenated monoterpene Linalool Citronellal Trans-Carveol Citronellol Neral Geraniol Geranial Linalool Oxide Citral Geranic acid Sesquiterpene hydrocarbon ␣ –Bergamotene Isoledene Farnesene (Z)-(␤) Germacrene Delta-selinene -gurjunene Delta-cadinene Aromadendrene Oxygenated sesquiterpene Muurolol Total identification (%)

a

RI

0

50

100

500

1000

0

50

100

500

1000

990 1003

1.33 0.33

1.58 1.47

t t

1.70 1.36

t 4.79

t t

2.00 t

3.36 t

2.55 t

t t

a,b

1006 1148 1215 1233 1235 1249 1264 1287 1316 1338

0.55 0.25 t 0.52 26.22 t 1.6 t 45.08 7.03

t 0.98 t t 20.26 1.18 13.31 15.68 36.66 2.54

t t t t 7.60 t 43.66 11.60 37.55 t

0.55 0.38 t 0.86 26.57 5.23 47.37 2.23 7.34 t

3.91 t t t 28.35 t 62.95 t t t

t t t t 21.81 t 58.97 19.22 t t

1.85 t t t 21.31 t 39.27 17.24 16.54 t

t t 1.29 t 26.57 2.75 43.43 9.90 10.56 t

t t t 1.53 30.88 1.92 55.51 2.07 t 1.09

t t t t 29.28 t 52.57 7.25 7.41 t

a,b

1432 1374 1440 1484 1489 1475 1522 1639

1.11 t 0.58 t 0.76 1.80 0.54 0.83

t t t t t t 1.25 1.78

t t t t t t t t

0.32 1.39 0.66 0.82 1.29 0.27 1.07 0.43

t t t t t t t t

t t t t t t t t

t t t t t 1.77 t t

t t t t t 2.14 t t

t t t t t t 2.48 t

t t t t t 3.48 t t

a,b

1640

0.66 89.19

t 96.69

t 100

t 99.84

t 100

t 100

t 99.98

t 100

0.91 98.94

t 99.99

a,b

a,b

a,b a,b a,b a,b a,b a,b a,b a,b a,b

a,b a,b a,b a,b a,b a,b a,b

t: trace. a Identification based on retention index. b Identification based on comparison of mass spectra. c Components listed in order of elution from a DB-5 column.

The influence of Pb, with or without AMF inoculation, in the chemical composition of lemon grass EO varied according to the different Pb levels used in the experiment. Non-inoculated AMF plants, as the Pb levels in the soil increased from 0, 50, 100, 500 to 1000 mg Pb kg−1 , the geranial content increased at 1.6; 13.31; 43.66; 47.37 and 62.95%, respectively (Table 2). The AMF inoculation under Pb levels did not considerably alter the chemical composition of EO in comparison with soils without AMF inoculation. This suggests that the AMF can stabilize geranial, the major compound, even under increasingly levels of Pb, as observed in Table 2, where geranial ranged from 39.27 to 58.97%. Lemon grass with AMF inoculation, though without Pb (0 mg kg−1 soil), showed an increased content of geranial (58.97%) and linalool oxide (19.22%) in comparison with the control group (without Pb and AMF), which showed a reduced content of geranial (1.60%) and non-linalool oxide (Table 2). Andrade et al. (2009) found geranial to be a major component of EO in lemon grass cultivated in Northern Brazil, with contents ranging from 40% to 50% (relatively similar to the present study). The chemical composition of EO from lemon grass plants was altered as the Pb levels increased. Without Pb and AMF inoculation, the major component of EO was citral, with contents ranging from 45.08 to trace (Table 2). However, under Pb additions in the soil, and with or without AMF, the major component of EO was geranial, with contents ranging from 1.6 to 62.95% (Table 2). This suggests that the plants manifested defense mechanisms, such as the increased production of secondary metabolites; in this case, geranial. In treatments without Pb in the soil, the AMF inoculation stimulated the production of geranial (from 1.6 to 58.97%). In the other hand, in treatments with Pb levels applied to the soil and AMF inoculation, no considerable variations were observed in the production of geranial with the increasing levels of Pb in the soil (Table 2), sug-

gesting the protection of the major component by the AMF. The EO of lemon grass plants that were inoculated with AMF presented 19.22% linalool oxide. However, under increased Pb levels in the soil, linalool oxide content decreased to 2.07% (Table 2). The citral was a major component of the EO in the absence of AMF and Pb levels in the soil (45.08%). However, the citral production decreased to trace under the increasing levels of Pb in the soils without AMF (Table 2). The AMF inoculation did not affect the production of citral. High levels of citral and geranial are economically important to the aroma and perfume industries (Barbosa et al., 2008). Moreover, the synthesis of ionone and vitamin A is also accomplished with citral (Lemos et al., 2013). Geranial has a strong lemon aroma. It is an aromatic substance widely used in the perfume industry because the citric aroma and is also used in the food industry to complement the EO from lemons. Geranial also has antimicrobial and insecticide benefits that create a pheromone effect in insects. Some industries have a high interest in this type of substance; it is worth investing in the AMF management to increase the production of citral and geranial with lemon grass (Robacker and Hendry, 1977; Onawunmi, 1989). Freitas et al. (2004) observed that M. arvensis increased the total content of EO to 88% when the plants were inoculated with AMF. The menthol content of EOs also increased at 89% in the treatments without P, in comparison with the control. However, no increase in EO or menthol was observed with AMF inoculation when increasing levels of P were applied to the soil. Little knowledge is available for understanding the influence of AMF in the synthesis of EO in aromatic plants. According to Smith and Read (2008), plants inoculated with AMF present metabolic, physiological and anatomic changes and produce various secondary metabolites as gibberellins, which accumulate in high amounts in these plants. Gibberellins belong to

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the diterpene class; thus, plants associated with AMF probably also accumulate monoterpenes and sesquiterpenes. The AMF plants present biochemical and physiological alterations that influence the synthesis of secondary metabolites, such as EOs and the active principles of seasoning and aromatic plants (Karagiannidis et al., 2013; Zeng et al., 2013; Urcoviche et al., 2015). According to Kapoor et al. (2004), when Foeniculum vulgare is inoculated with AMF, the plant biomass and EO production increase significantly. In treatments with AMF G. fasciculatum inoculums and P fertilization at 20 kg ha−1 , EO production increased 78%. Wei and Wang (1991) reported that Schizonepeta tenuifolia increased EO production, plant biomass and nutrient uptake significantly when these plants were inoculated with AMF. Garg and Aggarwal (2012) and Lermen et al. (2015) observed that AMF are potentially able to minimize plant uptake of HMs such as Pb. However, this evidence is not the general rule because some plants do not respond to the AMF association. Souza et al. (2011) evaluate the remediation potential of the AMF G. etunicatum inoculums in the Stizolobium aterrimum plants under Pb contamination, although the plants were tolerant, and the presence of AMF did not influence the Pb uptake. The present results were in agreement with an experiment conducted with M. crispa where the EO chemical composition was altered under the application of Pb to the soil, increasing carvone, the major EO component. Under Pb additions to the soil, the carvone contents ranged from 39.31% to 90.85%, indicating alterations in the plant metabolism (Sá et al., 2015). Alterations in EO chemical composition under Pb additions can be explained by the loss of specific enzymes and damages to the biosynthesis of secondary metabolites, such as EO, caused mainly by the mobility of this HM inside the plants (Nasim and Dhir, 2010). 4. Conclusion Significant changes in EO content were observed under the level of 100 mg Pb kg−1 in the soil, which reduced the EO content. However, under levels of 500 and 1000 mg Pb kg−1 and with AMF inoculation, an increase in EO content was observed. Without AMF inoculation and Pb, the major EO component was citral. However, under high Pb levels, and with AMF inoculation, the major EO component was geranial. Conflicts of interest The authors have declared no conflicts of interest. Acknowledgments The authors acknowledge UNIPAR for the financial support. Caroline Lermen thanks PROSUP/CAPES for the scholarship. Fabrício Morelli acknowledges a scholarship from the CNPq (National Council for Scientific and Technological Development). Odair Alberton acknowledges a research fellowship from the CNPq. References Andrade, E.H.A., Zoghbi, M.G.B., Lima, M.P., 2009. Chemical composition of the essential oils of Cymbopogon citratus (DC.) Stapf cultivated in North of Brazil. J. Essent. Oil Bear. Plants 12, 41–45. Audet, P., Charest, C., 2007. Dynamics of arbuscular mycorrhizal symbiosis in heavy metal phytoremediation: meta-analytical and conceptual perspectives. Environ. Pollut. 147, 609–614.

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