Effect of seasons on chemical composition and fungitoxicity of Cymbopogon citratus ( DC ) Staf essential oil

The study aimed to evaluate the chemical composition and the fungitoxicity of Cymbopogon citratus (DC) Staf essential oil, obtained in different seasons of the year. Therefore, C. citratus leaves were harvest at four seasons (summer, fall, winter and spring) and essential oil extracted by hydrodistillation of freshly harvested and dried leaves, with a total of eight samples. Yield was expressed in percentage and the fungitoxicity was evaluated in vitro on Colletotrichum gloeosporiodes (Penz.) Penz. & Sacc the causal agent of anthracnose at different concentrations (0, 0.25, 0.5, 0.75 and 1 μL mL -1 , respectively). The qualitative analysis of essential oil samples was made using gas chromatography coupled to mass spectrometry (GC-MS). The essential oil of freshly harvested leaves had a higher yield (0.25%) in the summer, whereas the essential oil of dried leaves had a better yield (0.48%) in the fall. In in vitro tests, concentrations of 0.75 and 1 μL mL -1 significantly inhibited mycelial growth and sporulation, in all samples. In the quantitative analysis, there was a variation in the levels of oil compounds observed in the different seasons. The most abundant compound in essential oil was citral (neral and geranial mixture) and myrcene. However, the variation in the compounds’ levels had no influence on the fungitoxicity of C. gloeosporiodes (Penz.) Penz. & Sacc.


INTRODUCTION
Cymbopogon citratus (DC) Staf is an important species of Poaceae Family, considered as a medicinal plant commonly known in Brazil as capim-limão, capimcidreira, capim-de-cheiro and capim-santo (lemon grass, lemon balm, smelling grass and holy grass).C. citrates has an essential oil rich in terpenoids such as neral, geranial and myrcene (Correa et al., 1998;Souza Filho and Alves, 2002;Souza and Lorenzi, 2005;Tyagi andMalik, 2012, Tyagi et al., 2014) and it is widely used in cosmetic, perfumes and pharmaceutical industries (Verma et al., 2013).The analysis of C. citratus essential oil from plants of multiple regions determines citral (neral *Corresponding author.E-mail: virlene@agronoma.eng.br. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License From rural laboratory of Maringá-Maringá, Paraná (2011).
and geranial mixture) as the main constituent (Andrade et al., 2009).This terpenes act in the plant defense and can inhibit phytopathogenic fungi growth (Martins et al., 2004;Castro et al., 2007;Adamczyk et al. 2013) presenting no toxicity to humans (Erler et al., 2006).However, the quality and quantity of essential oil compounds are often affected by environmental conditions (Martins et al., 2006;Blank et al., 2007;Nogueira et al., 2007).Gobbo -Neto and Lopes (2007), confirming that collection time of medicinal plant is one of the most important factors, since the amount and even the nature of active constituents is not constant throughout the year.
According to Costa et al. (2005), the steps of harvest and postharvest affect the chemical compounds' variation in medicinal plants, as well as, assist in obtaining a raw material with quality, resulting in the good quality of the final product.Also, according to Fennell et al. (2004), the chemical changes during postharvest periods of medicinal plants may affect essential oils or crude aqueous extracts' antimicrobial property.
The fungitoxicity of essential oils has been proven in several studies (Carnelossi et al., 2009;Benini et al., 2010;Moura et al., 2012;Sarmento-Brum et al., 2013), and according to Derbalah et al. (2012) the use of essential oils as antimicrobials is considered at low risk, because it is believed that it is difficult for a pathogen to develop resistance to the complex mixture of active components which comprise up the essential oils.
Thus, the current study aimed to assess the effect of harvest on the chemical composition and the fungitoxicity of C. citratus' essential oil harvest at different seasons of the year on Colletotrichum gloeosporiodes (Penz.)Penz.& Sacc.

MATERIALS AND METHODS
The study was conducted in an experimental field at the State University of Maringá (UEM), Maringá-Paraná, Brazil, with geographic coordinates 23°23'18"S, 51°54'59''O and 526 m of altitude.The regional climate is classified as Cfa, subtropical, with hot summers, uncommonly frosts and a tendency of rainfall concentration during the summer, yet no dry season defined, according to the Köppen's classification (Neto, 2010).Soil chemical analyzes were performed before the experiment.Samples of soil were collected at a depth of 20 to 40 cm at different points, resulting in a composed sample.The pH values and concentrations of chemical elements are found in the soil results in Table 1.The soil is characterized as eutrophic Red Oxisol (Souza and Gasparetto, 2010).

Cultivation of C. citratus seedlings
The seedlings were propagated by stem cuttings using polythene bags (10 × 20 cm), with 500 ml capacity of substrate (Red Oxisoleutroferric + sand + commercial substrate humus-based (2: 1: 1)).They remained in a greenhouse for one week before planting.Then the seedlings were removed from the greenhouse and relocated to an acclimatization area.The seedlings' transplant was conducted at the beginning of June, 2011, in single beds of 50 m 2 .The plants leaves were harvested randomly during the different seasons of the year.

Harvesting of C. citratus leaves
The harvesting of C. citratus leaves were carried out in late summer (March, 2012) nine months after transplanting the seedlings, in late fall (plants with twelve months of age), late winter (plants with fifteen months) and in late spring (plants eighteen months), with a total of four harvesting.The essential oil was extracted from both freshly harvested and dried leaves, in a total of eight extractions.For each sample was weighed 4 kg of leaves and 50% were placed for natural drying in the shade at room temperature for four days to obtain the dried leaves, during this period the leaves lost an average of 57% of its initial content.The other 50% of freshly harvested leaves remained were used for essential oil extraction.All samples were harvested in the late afternoon, between 4:30 and 5 p.m. Climate data (average temperature, relative humidity and rainfall recorded in the previous thirty days of collection) at the moment of harvest and the leaves' drying are described in Table 2.These data were obtained from the Experimental Farm of Iguatemi (FEI / UEM) meteorological station and Seeds analysis and Medicinal plants Laboratory.

Essential oil extraction
The essential oil was obtained using the extraction process by steam distillation, which consists of placing the plant material in the distiller, then by passing the plant material, the steam drags the essential oil (volatile) out of the plant; it passes the condenser where it is collected by a separatory funnel, then the immiscible liquids of different densities separated naturally; removal from the container is made through a stopcock.The extraction process had duration of four hours from the beginning of the first condensate drop (Worwood, 1995).The essential oil was stored in amber glass container in the refrigerator at a temperature of 4°C (+/-2°C).The essential oil harvested was weighed on an analytical scale for later yield calculation, which was expressed in percentage by the ratio between the amount of used distillable leaves and the amount of essential oil produced(g/g) (Furlan et al., 2010).

Essential oil qualitative analysis
Qualitative analysis was determined by gas chromatography coupled to mass spectrometry (GC-MS) and determination of their Kovats retention indices.

Gas chromatography coupled with mass spectrometry
The analyses of eight essential oils resulting from the extractions were performed using a GC-MS system in the Department of Analytical Chemistry of the State University of Maringá (UEM), Paraná, Brazil.The chromatograph used was FOCUSGC model ("Thermo Electron"), equipped with a fused silica capillary columnDB-5ms (30 mm x 0.25 mm, 0.25 µm of stationary phase) and mass spectrometer model DSQII ("Thermo Electron").The operating conditions were: injector temperature 200°C; drag gas flow (helium) 1.0 mL min -1 ; injection volume of 0.5 μL (dichloromethane solution) withdivision of flux ("split") at a ratio of1:40.The programming of oven temperature was 50°C for 1 min, elevated to 180°C at a rate of 3°C min -1 and180°C to 240°C at a rate of 10°Cmin -1 , remaining at 240°C for 5 min.Mass spectrum were obtained by ionization of electron impact 70eV, in the range of 50 to 550 mz -1 ; with an inter face temperature of 250°C and ion source of 200°C (Maia et al., 2013).

Kovats retention index
The calculation of Kovats retention index (KI) was based regarding the homologous series of n-alkanes, analyzed under the same experimental conditions.The retention indices of the compounds were determined according to the equation proposed by Van den Dool and Kratz (1963).The identification of main compounds was performed based on a comparison of its mass spectrum with those calculated from the mass spectrum of the spectral library" NIST MS Search2.0"contained in the soft ware "Xcalibur" accompanying the appliance, comparing their Kovats retention indices obtained experimentally with Kovats retention index data obtained from the literature (Boulanger et al., 1999;Beaulieu and Grimm, 2001;Siegmung et al., 2001;Jordán et al., 2003) for the same compounds analyzed using the same column.Quantitative data of compounds were obtained from percentages of the chromatogram are as through normalization.
Fragments from fruit were transferred into petri plates containing PDA growth medium (potato, dextrose, agar) which was maintained at 25°C in biochemical oxygen demand incubator (BOD) for five days.Later the plates with mycelia were transferred to a room with continuous light to stimulate sporulation.
After the formation of their productive structures (spores mass with rose color) C. gloeosporioides were transferred to new plates and incubated at 25°C in BOD.After seven days of incubation, C. gloeosporioides isolates were used in separate trials.The essential oil was tested at concentrations of 0 µL mL -1 , 0.025 µL mL -1 , 0.05 µL mL -1 , 0.075 µL mL -1 e 0.1 µL mL -1 , increased by 0.25µL mL - 1 Tween 20 (emulsifying agent), to inhibit the growth and sporulation of C. gloeosporioides.The essential oils at the different concentrations were added to PDA growth medium after sterilized and still liquefied, poured into petri plates.After PDA solidification, a mycelialdisc (7 mm diameter) of the isolated fungus was placed in the plates center, sealed with plastic wrap and kept in a growth chamber at 25°C and photoperiod of 12 h.
From the second day after replication, evaluations were performed every 24 h until one of the isolates reached the plates edge (144 h).The mycelial growth was measured by the colonies' radius in two perpendicular directions, using a digital paquimeter.With the obtained data it was determined the mycelia growth area of the isolated fungus, from this we calculated the percentage of my celial growth inhibition (MGI), using the formula of Bastos (1997).
For each treatment, five plates were replicated each plate represented a repetition in a completely randomized design.Data were subjected to analysis of variance (ANOVA) and when significant regression equations were applied, adjusted with a coefficient of 5% probability and the coefficient of determination (R 2 ).Analyses were performed using SISVAR software (Ferreira, 2011).

Essential oil qualitative and quantitative analysis
The highest yield was obtained in the extraction of freshly  2003), the essential oil yield of C. citrates is 0.28 to 0.50% of freshly harvested weight, higher than those found in this study.They observed that collection during the morning period (between 9 and 11 a.m.) showed higher oil yield compared to other periods.Similar results were observed by Marco et al. (2002).This explains (or justify) the lower values found in this study.
The yield variation between same species plants is attributed mainly to differences in harvest season, soil type, region climate and relative humidity (Burt, 2004).The seasonal variation of secondary metabolites can be caused by physiological demands as growth, defense and plant reproduction, as well as differences in the environment as water stress, light, nutrient deficiency and extreme temperatures.The study of this variation is very important as it allows knowing the exact time, approximately, in which some constituents are in greater proportion (Kumar et al., 2011).
According to Silva and Casali (2000), decreasing the amount of water in the plant, the amount of active ingredients in terms of dry mass increases.Justifying the findings of this study, where the highest yield values were observed in essential oil resulting from dried leaves extracts comparing to freshly harvested leaf extracts (Table 3).Yield was expressed by the ratio between the mass of distillable leaves used and the amount of essential oil produced (g/g), thus decrease the dried leaves mass (57%) has optimized performance values.The results also agree with Martins et al. (2002), who observed an increase in oil yield of 21% when compared to essential oil amount extracted from the dried leaves to that obtained from freshly harvested product of C. citratus.
For the variation observed in the essential oil yield of freshly harvested mass, from summer to fall harvest, it might have been caused by the change in micro climate parameters of temperature and rain fall recorded in the thirty days prior to harvest.The excess of rain during the thirty days prior to fall harvest (344 mm) provided a decrease in secondary metabolites accumulation in plants.According to the results of Koshima et al. (2006), when they analyzed the essential oil yield extracted from freshly harvested leaves of C. citratus grown with mulch and harvested in all seasons of the year, found a higher yield of essential oil in the period with lower rainfall.
In relation to dry mass to yields, fall presented extraction yield of 0.48% and spring presented the lowest yield of 0.29%.In summer and winter extraction yield was 0.43 and 0.44% respectively (Table 3).The lower essential oil yield extracted from the dried leaves during the spring compared to fall, is possible due to temperature differences.During the drying process in fall, it had mild temperature of 18°C, whereas during the spring it had a higher temperature (30°C) (Table 2).Blanco et al. (2002), studied the drying temperature effect on the essential oil yield of Mentha piperita and observed that drying temperature elevation caused a reduction in essential oil content.As explained by Calixto (2000), some plants constituents are labile when exposed to heat, thus plants containing them must be dried at low temperatures.In the drying process, water loss occurs naturally, but can also result in volatiles loss depending on the drying temperature.Also, Ortiz et al. (2002) related the essential oil yields with variations in temperature and found that the increase or decrease on it has a direct influence at essential oil yield.In chromatographic, analysis was possible to identify six mainly compounds in the essential oil of C. citratus: myrcene; linalool; verbenol; neral; geranial and 2-undecanone (Table 4).
The most abundant compound in the essential oil of C. citratus, obtained in different seasons, was the citral composed by a mixture of neral and geranial isomers, this had a higher area in the essential oil from freshly harvested leaves in spring (65.6%), followed myrcene presented an area of 21.48%.For its part, the essential oil extracted from dried leaves in winter, myrcene compound showed greater area of 41.28% and citral with an area of 44.94% (Table 3).These results agree with other authors, who claim the presence of citral as a major component of C. citratus essential oil (Martins et al., 2002;Leal et al., 2003;Costa et al., 2005;Furlan et al., 2010;Kumar et al., 2013;Pinto et al., 2015).Lima et al. (2008), found that essential oil of C. citratus presented geranial (43.8%), neral (34.5%) and myrcene (14.6%) as major compounds.Barbosa et al. (2008), reported a variation in citral levels of the essential oil marketed in Brazil from 40.7 to 75.4% and myrcene from 0.24 to 7.29%, being the myrcene levels below what is found in the essential oils studied.Early investigation of the leaf essential oil of C. citrates from Rio de Janeiro were also identified and quantified geranial with 53.2% and neral with 36.37% as the major components but the monoterpene myrcene was not observed (Pinto et al., 2015).
Still analyzing the compounds content of the essential oil from freshly harvested leaves it can be observed that, citral presented a larger area (65.56 and 65.49%) in the spring and summer seasons, respectively, when compared to fall and winter (58.15 and 61%).However, there was not just the increase of citral content in these conditions, but also the decrease of myrcene content (21.48 and 20.15%), while in fall and winter presented area of 28.9 and 25.35%, respectively (Table 4).This change may be caused by micro climate factors (Gobbo and Lopes,2007) and mostly rainfall, since in the climatologic seasons rainfall records presented median (113 and 90 mm) in the thirty days prior to harvest, while in the fall and winter the indexes present significant variation (344 and 4.8mm) respectively (Table 2).

Essential oil fungitoxicity
The essential oil extracted from freshly harvested and shaded dried leaves harvested in all the four seasons, presented significant inhibition on the mycelial growth of C. gloeosporioides with a 29% coefficient of variation (Figures 1, 2, 3 and 4).According to Pereira et al. (2011), essential oils can inhibit or reduce phytopathogens growth due to the action of substances present in its composition.Guerra et al. (2000), attribute the antimicrobial and antifungal activities of C. citrates essential oil to citral monoterpene.Guimarães et al. (2011), observed that myrcene compound has no fungi toxic activity, since its celial inhibition was equal to zero for all phytopathogens.The essential oils obtained from the eight extractions, it was observed that the variation in the chemicals levels, mainly citral (neral and geranial mixture) to which the anti fungal action is assigned, had no effect at the fungitoxicity of the essential oil on C. gloeosporioides.It was demonstrated that even the lowest citral content (39.03%), found in the essential oil from the dried leaves in spring, showed high fungi toxicity (99%) (Figure 4B).In relation to citral, Guimarães et al. ( 2011) also demonstrate the citral fitotoxicity and its importance activity in the C. citrates essential oil. Lee et al. (2008), observed that compounds as citronellal, neral, geraniol and geranial at a concentration of 28 x 10 -3 mg mL -1 of air are able to inhibit 100% mycelial growth of Phytophthora cactorum.
It was observed that the essential oil obtained in all extraction seasons demonstrated to be efficient in the control of the phytopathogen mycelial growth.The effect expressed as quadratic for the majority of samples with mycelial growth inhibition (MGI) of 102% at a 0.8 µL mL -1 concentration (Figure 1A), of 103% ata 1 µL mL -1 concentration (Figure 1B), of 113% at a 0.7µL mL -1 concentration (Figure 3A) and 99% at a 0.9 µL mL -1 concentration.The essential oil of freshly harvested leaves harvested in fall, although it had a cubic effect, also obtained MGI maximum point of 97% at a 0.9 µL mL -1 concentration and with minimum inhibition (1%) at a 0.078 µL mL -1 concentration 1 (Figure 2A).The mycelia growth rate of C. gloeosporioides in the presence of C. citrates essential oil from dried leaves, harvested in the fall and winter, and freshly harvested leaves, harvested in the spring, has expressed positively linear form depending on the essential oil doses, in other words, it presented dose-dependent effect (Figure 2B,3B and 4A).
These results confirmed the C. citratus essential oil potential in control of the phytopathogen that causes anthracnose in mango fruits.Santos et al. (2013), also verified that the C. citrates essential oil was effective in reducing the mycelia growth of Helminthosporium sp.    ).

Conclusion
The results presented in the current study, corroborated with the cited literature demonstrate that the C. citratus essential oil has effective control of C. gloeosporioides, due to the activity of chemical compounds present in this oil.

Figure 1 .
Figure 1. C. gloeosporioides mycelial growth inhibition (MGI %) in presence of C. citratus essential oil obtained from freshly harvested (A) or dried (B) leaves harvested in summer.

Figure 2 .
Figure 2. C. gloeosporioides mycelial growth inhibition (MGI %) in presence of C. citratus essential oil obtained from freshly harvested (A) or dried (B) leaves harvested in fall.

Figure 3 .
Figure 3. C. gloeosporioides mycelial growth inhibition (MGI %) in presence of C. citratus essential oil obtained from freshly harvested (A) or dried (B) leaves harvested in winter.

Figure 4 .
Figure 4. C. gloeosporioides mycelial growth inhibition (MGI %) in presence of C. citratus essential oil obtained from freshly harvested (A) or dried (B) leaves harvested in spring.

Table 1 .
The experiment results of soil chemical analysis: pH, aluminum concentration, hydrogen, macronutrients and micronutrients.
*Rainfall recorded in the previous 30 days to harvest.

Table 3 .
Cymbopogon citratus essential oil yield of freshly harvested and dried leaves harvested in different seasons(Maringá, 2012).

Table 4 .
The main components contents of C. citratus essential oil coming from the freshly harvested and dried leaves extractions, harvested at four seasons, identified by GC-MS and expressed as normalization area (%).