ANTIFUNGAL ACTIVITY OF ESSENTIAL OILS OF Myrcia ovata CHEMOTYPES AND THEIR MAJOR COMPOUNDS ON PHYTOPATHOGENIC FUNGI ATIVIDADE ANTIFÚNGICA DOS ÓLEOS ESSENCIAIS DE QUIMIOTIPOS DE Myrcia ovata E SEUS COMPOSTOS MAJORITÁRIOS SOBRE FUNGOS FITOPATOGÊNICOS

This work evaluated the antifungal activity of essential oils of Myrcia ovata chemotypes (MYRO-175, MYRO-156, MYRO-154, MYRO-165, and MYRO-015) and their major compounds (linalool, geraniol, citral, and (E)-nerolidol) on the phytopathogenic fungi Fusarium pallidoroseum (which causes melon postharvest rot) and Colletotrichum musae (which causes anthracnose in banana). The essential oils were obtained by hydrodistillation and analyzed by GCMS/FID. To evaluate the antifungal activity, the essential oils and their major compounds were tested at different concentrations (0.1; 0.3; 0.4; 0.5; 0.7; 1.0; 3.0, and 5.0 mL/L). The major compounds found in the essential oils were nerolic acid, linalool, geraniol, citral, and (E)nerolidol. The essential oils of the plants MYRO-154, MYRO-165, and MYRO-015 had the minimum inhibitory concentration (MIC) (0.3 mL/L) for F. pallidoroseum and the lowest minimum fungicidal concentration (MFC) (0.7 mL/L), for C. musae. Geraniol and citral had the lowest MFC (0.5 mL / L) for the two fungi tested. For F. pallidoroseum, the essential oils of the chemotypes were more effective than their major compounds. Conversely, the major compounds geraniol of the chemotype MYRO-156 (74.37%) and citral were more effective than their respective essential oils for C. musae. (E)-nerolidol and geraniol of the chemotype MYRO-015 (33.15%) were responsible for the antifungal activity of the essential oils of their respective chemotypes.


INTRODUCTION
Fungi diseases are one of the main causes of fruit losses at the post-harvest stage. The effects of these phytopathogens are also the main reasons for changes in appearance, odor, taste, texture, and reduction of nutritional values, leading to the depreciation of the commercial value of these products (KFOURY et al., 2016). The phytopathogen Fusarium pallidoroseum is one of the causative agents of melon postharvest rot, and Colletotrichum musae is the main responsible for anthracnose in banana. These fungi infect the plant through lesions or injuries in the cutting area of the peduncle during harvest (BOUBAKER et al., 2016).
The fungus F. pallidoroseum is commonly associated with melon postharvest rot and is found in the soil, in plant remains, in tropical and subtropical regions (LOKESH et al., 2008;GONDIM et al., 2008). The infection occurs through natural cracks in the peduncle abscission zone during harvest, and the pathogenesis develops at the post-harvest stage. C. musae is responsible for losses of up to 40% in banana production due to anthracnose, which is characterized by dark-brown to black, sunken spots on the peel, affecting commercialization and in natura consumption (PESSOA et al., 2007).
Synthetic fungicides have been used to control these fungi. However, the strong pressure of the society for certification seals and food safety has prevented the use of these chemicals in fungi control. The need to create safe and biodegradable alternatives, such as natural fungicides based on plant essential oils, have emerged due to the consumer's demand for synthetic chemical-free products. Another reason is the fact that phytopathogens develop resistance as a result of excessive use of fungicides (BAKKALI et al., 2008).
Myrcia ovata Cambess. presents excellent antifungal potential BLANK et al., 2015). The species belongs to the Myrtaceae family and is a shrub that produces essential oils with other biological activities, such as anti-inflammatory, antinociceptive, analgesic, antibacterial, and insecticide (SANTOS et al., 2014;QUINTANS-JÚNIOR et al., 2011;CANDIDO et al., 2010;LIMA et al., 2011). The phytochemical profile of its essential oils is characterized by the presence of the compounds nerolic acid, linalool, geraniol, citral, and (E)-nerolidol . Thus, this study aimed to evaluate the antifungal activity of the essential oil of M. ovata Cambess. chemotypes and their major compounds linalool, geraniol, citral, and (E)-nerolidol against the fungi F. pallidoroseum and C. musae.

Plant material and essential oil extraction
The essential oils of the chemotypes MYRO-175,  were characterized by   (Table 1). Leaves were collected in the municipality of Japaratuba, in the state of Sergipe, northeastern Brazil. Plants were manually defoliated and dried in a forced-air circulation oven at 40 ºC for five days. The essential oil was extracted by hydrodistillation in a modified Clevenger apparatus using 50 g of dry leaf for 140 minutes (EHLERT et al., 2006). The essential oils were collected and stored in amber flasks at -20 °C until chemical composition analysis. The compounds linalool, geraniol, citral, and (E)-nerolidol were purchased from Sigma-Aldrich Corporation.

Chromatographic analyses
The analysis of the chemical composition of the essential oils was carried out using a GC-MS/FID (QP2010 Ultra, Shimadzu Corporation, Kyoto, Japan), equipped with an autosampler AOC-20i (Shimadzu), as described by SAMPAIO et al. (2016).

Antifungal activity
Pure cultures of the fungi F. pallidoroseum and C. musae were obtained from the Phytopathology Laboratory of the Federal University of Sergipe. The antifungal activity of the essential oils was evaluated in a contact trial based on mycelial growth inhibition (CHANG et al., 2008).
The experiment consisted of a completely randomized design (CRD) with three replications in each treatment. The essential oils were tested at the concentrations of 0.1, 0.3, 0.4, 0.5, 0.7, and 1.0 mL/L, and the major compounds were tested at the concentrations of 0.1, 0.3, 0.4, 0.5, 0.7, 1.0, 3.0, and 5.0 mL/L. The substances were solubilized in 1% dimethyl sulfoxide (DMSO, Sigma-Aldrich) and homogenized in sterile PDA culture medium (Potato Dextrose Agar, HIMEDIA). Solutions were then poured into 9.0 cm diameter Petri dishes, and each dish was inoculated in the center with a 7.0 mm diameter disk of the fungus culture.
Petri dishes were stored in a B.O.D chamber at 22 ± 3 ºC, with a 12-hour photoperiod. Petri dishes containing only the PDA culture medium and PDA + DMSO were used as controls. The evaluations were performed by measuring the mycelial diameter (mean of two diametrically opposite measures), using a pachymeter. At 96 hours after incubation, the mycelial discs of the concentrations showing no visible growth were transferred to Petri dishes containing only the PDA culture medium and incubated for another 96 hours. At the end of the evaluations, the percentage of mycelial growth inhibition (PMGI) of the fungus of the treatments was calculated, in relation to the control containing only PDA and the fungus, by the formula: where dc = is the diameter of the control, and dt = is the diameter of the treatment. The lowest growth concentration after transferring to the medium without essential oil was considered as minimum inhibitory concentration (MIC). The lowest concentration at which no growth was observed after transferring to the medium without essential oil was considered as minimum fungicidal concentration (MFC).

Statistical analysis
The means of the percentage of mycelial growth inhibition with the respective standard error of mean were obtained with the Graph Pad Prism® software (mean ± SEM).
All the chemotypes of the essential oil of M. ovata exhibited antifungal activity against F. pallidoroseum and C. musae, highlighting the chemotypes represented by the major compounds geraniol, citral, and (E)-nerolidol (Table 3 and 5).
The percentage of mycelial growth inhibition (PMGI) ranged from 73.52 to 88.52% at the lowest essential oil concentration (0.1 mL/L) against F. pallidoroseum, reaching 100% inhibition at concentrations of 0.3 mL/L for all chemotypes, except for MYRO-156, whose PMGI ranged from 74.63 to 92.41% at concentrations of 0.1 to 0.4 mL/L, respectively (Table 3; Figure 1A). For C. musae, the PMGI ranged from 75.00 to 89.35% at the lowest essential oil concentration (0.1 mL/L), reaching 100% inhibition at concentrations of 0.3 mL/L for all chemotypes, except for MYRO-175, whose PMGI ranged from to 77.31 to 86.67% at concentrations of 0.1 and 0.3 mL/L, respectively (Table 3, Figure 1B). For the major compounds, the PMGI varied from 49.63 to 61.30% at the lowest concentration (equivalent to 0.1 mL / L of essential oil) for F. pallidoroseum. For this same fungus, at the lowest concentration (0.1 mL/L of essential oil), linalool, one of the major compounds of the essential oil of chemotype MYRO-175, showed lower toxicity (59.26%) than its essential oil (88.52%), with maximum PMGI at concentrations higher than 3.0 mL/L (Table 3 and 4).
Geraniol, found in the plants MYRO-156 (74.37%) and MYRO-015 (33.15%), caused maximum inhibition at the same concentration (equivalent to 0.4 mL/L of essential oil), against F. pallidoroseum. At the concentration of 0.3 mL/L, citral caused effective mycelial growth inhibition (68.06%), while its essential oil (MYRO-154) caused 100% inhibition. The sesquiterpene alcohol (E)-nerolidol caused 65.74%, while its essential oil (MYRO-165) led to 100% inhibition at the same concentration (Table 4). For the major compounds, against C. musae, the PMGI ranged from 49.26 to 92.96% at the lowest concentration, reaching 100% inhibition at concentrations of 0.3 mL/L, for geraniol b and citral. At the lowest concentration (0.1 mL/L), linalool had lower toxicity (49.26%) than its essential oil (77.31%), with a maximum PMGI at concentrations higher than 1.0 mL/L. Geraniol caused maximum inhibition at the concentration of 0.3 mL/L for MYRO-156 and 0.4 mL/L for MYRO-015. At the concentration of 0.1 mL/L, citral was more effective than its essential oil (MYRO-154), causing effective mycelial growth inhibition of 89.63% against 76.11% of its essential oil. (E)-nerolidol had 80.19% inhibition against 89.35% of its essential oil (MYRO-165) at the same concentration, with no great variations in PMGI values (Table 3 and (Table  5). In relation to the specificity of the essential oils and/or major compounds against the microorganisms studied, all the essential oils were more toxic than their major compounds for F. pallidoroseum, whereas the essential oil of the chemotypes MYRO-175, MYRO-165, and MYRO-015 had more effective mycelial growth inhibition on C.musae. Moreover, geraniol and citral were more efficient in the control of C.musae than their essential oil of origin (Figure 3). The use of plant essential oils as microbiological agents has confirmed the existence of significant biological activities, and some of them have received significant attention due to their use in the control of phytopathogenic fungi that cause diseases in fruit species of economic interest. Some plants of the family Myrtaceae are known for their antimicrobial activity, mainly fungicides ALVES et al., 2016).
The study of the antifungal activity of the essential oil of Warionia saharae, rich in (E)nerolidol (23.0%) and linalool (15.2%), on three apple rot fungi, Alternaria sp., Penicillium expansum, and Rhizopus stolonifer, showed mycelial growth inhibition at the concentration of 2.0 mL/L, demonstrating that the synergy between these major compounds was not the main responsible for the antifungal activity due to the low values of PMGI at high concentrations of this essential oil (ZNINI et al., 2013).
The biological study with linalool alone revealed a low percentage of mycelial growth inhibition against these microorganisms, which evidences the importance of combined studies on the other major compounds and/or minor compounds, aiming at their synergistic action (LEE et al., 2005). Marei et al. (2012) evaluated the antifungal activity using the technique of mycelial growth inhibition of four phytopathogenic fungi (Rhizoctonia solani, F. oxysporum, Penicillium Digitatum, and Aspergillus niger), where geraniol caused effective mycelial growth inhibition of 73.9%. Shin and Lim (2004) studied the effect of geraniol on Trichophyton spp., reaching MIC values from 0.3 to 1.0 mL/L and MFC from 0.5 to 2.0 mL/L. Lee et al. (2008) reported the antifungal activity of commercial essential oils of 11 species of the family Myrtaceae on the phytopathogenic fungi Phytophthora cactorum, Cryponectria parasitica, and F. circinatum and reported mycelial inhibition values ranging from 31.9 to 68.9% for citronellol, neral, and geranial, and a PMGI of 100% for geraniol on P. cactorum, at the concentration of 0.3 mL/L. Citral (3,7-dimethyl-2,6-octadienal) is a mixture of two isomeric acyclic monoterpene aldehydes, geranial (E-citral) and neral (Z-citral) (SADDIQ et al., 2010). The antifungal activity against the microorganisms evaluated in this work may be related to the high reactivity of the carbonyl grouping of the Z/E isomers. Recent studies have demonstrated the efficacy of this compound as an antifungal agent, and it has been used against the causative agent of post-harvest diseases in Citrus sp, such as green mold (Penicillium digitatum), sour rot (Geotrichum citri-aurantii), and blue mold (Penicillium italicum) (SADDIQ et al., 2010;ZHENG et al., 2015, ZHOU et al., 2014TAO et al., 2014). Sampaio et al. (2016) reported citral as one of the major compounds of the essential oil of M. ovata leaves (68.5%), which completely inhibited the fungus F. solani, causing its mortality at concentrations from 0.5 mL/L. Studies suggest that citral is responsible for the modification in the mitochondrial morphology and the cellular wall function of these phytopathogens, causing a decrease in the O2 level and respiratory rate and, consequently, leading to an increase in the permeability of the fungal membrane on the cell wall. Sampaio et al. (2016) reported the antifungal activity of the essential oil of a Myrcia ovata chemotype (MYRO-006) with 58.27% of (E)nerolidol, which showed 47.50% mycelial growth inhibition against F.solani. The alcohol (E)nerolidol, present as a major compound in the essential oil of Piper chaba Hunter (5.1%), showed activity against the fungi Rhizoctonia solani, Botrytis cinerea, F. solani, F. oxysporum, Sclerotinia sclerotiorum, and Phytophthora capsici, causative agents of plant diseases, with minimum inhibitory concentrations between 125 and 500 μL mL -1 (RAHMAN et al., 2011). This sesquiterpene alcohol was tested alone and showed biological activity against Trichophyton mentagrophytes, causing a change in the fungal morphology from the concentration of 0.4 μL mL -1 . Those results confirm that the antifungal activity of this compound is more pronounced in its isolated form (PARK et al., 2009).
The antifungal activity of the essential oils of all chemotypes evaluated in this study (except for MYRO-175, whose essential oil was more active than one of its major compounds, linalool) may be related to different combinations of the contents of their major and minor compounds. The essential oils of the chemotypes MYRO-175, MYRO-165, and MYRO-015 had nerolic acid at concentrations of 52.61%, 47.20%, and 31.65%, respectively, as one of their major compounds. Sampaio 2016 reported the isolation of nerolic acid and the antifungal activity on F.solani, F.pallidoroseum, and C.musae, proving that this biocompound is responsible for the pronounced antifungal activity of the essential oil of the chemotype MYRO-160, which presents a 69.44% of this acid.
Thus, results suggest that the combined synergic action between major and/or minor compounds in the complexity of the chemical composition of their essential oils may be related to their significant activity against the tested fungi. Therefore, the compounds found in larger and/or smaller amounts play an important role in these microorganisms, confirming the importance of synergism or antagonism between bioactive compounds of plant volatiles (LANGEVELD et al., 2014).
The essential oil of the tested chemotypes was more effective than their major compounds against F. pallidoroseum. For C. musae, the major compounds geraniol, found in the plant MYRO-156 (74.37%), and citral were more effective than their respective essential oils. Conversely, (E)-nerolidol and geraniol of the chemotype MYRO-015 (33.15%) were responsible for the antifungal activity of the essential oils of their respective chemotypes. Results propose that the mechanism of action of the samples tested against the phytopathogenic fungi sometimes acted on the principle of synergism and, in other moments, on the antagonism principle (KHAN et al., 2011).
Results indicate that the essential oil or its major compound showed different toxicity to both fungi tested. The essential oil was more toxic to C. musae, and the pure major compounds effectively inhibited F. pallidoroseum. This fact proves that the principle of action of the essential oils is different among microorganisms (bacteria, fungi, insects, mites). Thus, the study of the possible mechanisms of action of these compounds is fundamental to the development of new bioproducts.