Variability and antifungal activity of volatile compounds from Aniba rosaeodora Ducke, harvested from Central Amazonia in two different seasons

Industrial Crops & Products 123 (2018) 1–9

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Variability and antifungal activity of volatile compounds from Aniba rosaeodora Ducke, harvested from Central Amazonia in two different seasons

T

Renah B.Q. Pimentela, Diego P. Souzaa, Patricia M. Albuquerqueb, Andreia V. Fernandesa, ⁎ Alberdan S. Santosc, Sergio Duvoisin Jr.b, José F.C. Gonçalvesa, a

National Institute for Amazonian Research (MCTI-INPA), Laboratory of Plant Physiology and Biochemistry, André Araújo Ave., 2936, Aleixo, 69011-970, Manaus, AM, Brazil b Laboratory of Applied Chemistry and Technology, School of Technology, Amazonas State University (UEA), Manaus, Amazonas, 69050-020, Brazil c Federal University of Pará (UFPA), Laboratory of Systematic Investigation in Biotechnology and Molecular Biodiversity, Belém, Pará, 66075-110, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Lauraceae family Linalool Phytopathogenic fungi Seasonal variation

Finding new applications for the essential oils (EOs) of the branches and leaves of Aniba species represents a valuable strategy for the adoption of correct management of the crown and to help make Aniba plantations economically valuable. We report here the antifungal activity of the EO from Aniba rosaeodora Ducke against plant pathogenic fungi. The present study investigated the chemical variability and antifungal effect of EO from A. rosaeodora harvested during the wet and dry seasons in the Amazon region. The volatile content obtained from the aerial parts by hydro-distillation was analyzed for its chemical composition by gas chromatography–mass spectrometry (GC–MS). Furthermore, a broth and agar dilution method was used to determine the antifungal activity against phytopathogens. Quantitative and qualitative variations in composition among the EOs were detected. Linalool was a major component in the oil of leaves and branches from both periods. Quantification using an external standard showed a higher concentration of linalool in the wet season (74.4 ± 3.9% in leaves and 81.8 ± 5.7% in branches) than in the dry season (47.5 ± 2.2 in leaves and 49.2 ± 1.6% in branches). The EOs were toxic to all phytopathogens analyzed, displaying superior inhibitory activity toward Colletotrichum guaranicola, with inhibition zone diameters ranging from 15.2 ± 1.2 to 21.3 ± 1.7 mm and IC50 values of 0.578 to 2.094 μL mL−1. Interestingly, the EOs collected during the wet season were effective in reducing the vegetative growth of phytopathogens, providing evidence for the involvement of linalool in the inhibitory effect.

1. Introduction The Amazon contains a wide variety of plants producing various metabolites with proven applications (Maia and Andrade, 2009). Plants belonging to the family Lauraceae are known for high production of secondary metabolites, with applications in perfumes and cosmetics, agrofood industries and therapeutic purposes (Marques, 2001; Aprotosoaie et al., 2014). Trees belonging to the Lauraceae have been examined for bioprospecting of secondary plant metabolites and essential oils (EOs) (Alcântara et al., 2013). EOs are complex mixtures of volatile aromatic compounds produced and accumulated in undifferentiated cells, glandular trichomes, secretory ducts and resin ducts (Nakatsu et al., 2000; Morone-Fortunato et al., 2010). Terpenes, allyl phenols, alcohols, acids and esters are usually found in EOs (Prakash et al., 2015). The role of EOs in plants is associated with the attraction

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of pollinators, seed dispersal and defense mechanisms (Amaral et al., 2015). Each EO generally contains two or three major molecules; for example, major components of Aniba canelilla EO are 1-nitro-2-phenylethane (≈52%) and methyleugenol (≈39%) (Taveira et al., 2003). The chemical composition of each EO depends on the origin, chemotype, biotype, plant phenology, harvest period and environmental conditions (Furtado et al., 2014; Grulova et al., 2015; Verma et al., 2015). As described by Silva et al. (2013), the relative amounts of germacrene D and bicyclogermacrene in Porcelia macrocarpa EO showed differences, suggesting the influence of temperature and precipitation on the chemical composition of EOs. Several studies on the toxicity of EOs against bacteria (Ghabraie et al., 2016; Zengin and Baysal, 2014), viruses (Derksen et al., 2016), and fungi (Hong et al., 2015; Kumari et al., 2015) have demonstrated great potential for EOs as new microbiocides for the control of

Corresponding author. E-mail addresses: [email protected] (R.B.Q. Pimentel), [email protected] (D.P. Souza), [email protected] (P.M. Albuquerque), [email protected] (A.V. Fernandes), [email protected] (A.S. Santos), [email protected] (S. Duvoisin), [email protected] (J.F.C. Gonçalves). https://doi.org/10.1016/j.indcrop.2018.06.055 Received 29 July 2017; Received in revised form 19 May 2018; Accepted 14 June 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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2.2. Extraction of essential oil

pathogenic microorganisms (Rosas-Burgos et al., 2009; El Asbahani et al., 2015). Because they are lipophilic, EOs can penetrate membranes to the interior of cells, alter cell permeability, cause a breakdown of the proton pumps and reduce the production of ATP, resulting in cell death (Calo et al., 2015). The tree Aniba rosaeodora (Lauraceae) is widely distributed throughout the tropical regions of the planet (van der Werff and Richter, 1996). It produces an EO that has been intensively explored by the perfume industry due to the high economic value of linalool, its major component (80–90%) (Almeida et al., 2013; Galaverna et al., 2015). Due to high industrial demand and decades of overharvesting, A. rosaeodora trees are in danger of extinction (International Union for Conservation of Nature (IUCN, 2017). Aniba plantations assist with conservation of this species and provide beneficial income opportunities for the indigenous population, encouraging the sustainable development in Amazonia (Krainovic et al., 2017). A strategy for tree maintenance in Aniba plantations and in native forest is the extraction of oil from leaves and branches rather than from the entire trunk (a common practice in extracting oil from A. rosaeodora in the Amazon region). Given that the average yield, composition and percentage of linalool in EO from A. rosaeodora trunk wood (Chantraine et al., 2009) are comparable to those in EO from aerial parts (Maia et al., 2007; Sarrazin et al., 2016), it is clear that the leaves and branches can be used as an alternative and more sustainable way of obtaining A. rosaeodora EO. Many studies in vivo and/or in vitro have shown biological properties of medical interest for this EO and its main components (Gazim et al., 2010; Aprotosoaie et al., 2014; Siqueira et al., 2014). Previous studies reported antimicrobial activity of A. rosaeodora EO, especially against human diseases, including studies on the human epidermoid carcinoma cell line A431 and on immortal HaCaT cells (Hammer et al., 1999; Simić et al., 2004; Hussain et al., 2008). However, the action of A. rosaeodora EO against plant pathogenic fungi remains poorly studied. Our article addresses the potential of EOs as sources of eco-friendly antifungal biomolecules that may be an alternative to the use of chemicals that are highly toxic to animals and the environment. Accordingly, this study was undertaken to investigate the qualitative and quantitative composition of the EOs obtained from aerial parts (leaves and branches) of A. rosaeodora in two distinct seasonal periods in the Amazon and to evaluate the impact on its efficacy against phytopathogenic fungi, with the goal of identifying potential antifungal agents in order to reduce the use of chemical fungicides.

The aerial parts (300 g) were air-dried at room temperature (26 °C ± 3) for 7 days, finely minced and separately hydrodistilled for 3 h for leaves and 6 h for branches using a Clevenger-type apparatus. The oil was dried over anhydrous sodium sulfate, packed in sterilized glass vials and kept at 4 °C in the dark. The EO yield ranged from 0.7% to 1.9% dry mass (grams of oil per gram of dried leaves and branches or % dry mass). 2.3. Analysis of essential oils 2.3.1. Gas chromatography–mass spectrometry (GC–MS) analysis The chemical analysis of volatile constituents was performed using a gas chromatograph coupled to a mass spectrometry detector (Shimadzu, GCMS-QP2010 Ultra) under the following conditions: HP5MS capillary column (30 m length x 0.25 mm internal diameter; 0.25 μm film coating); EOs (10 μL mL−1 in ethyl acetate) samples of 1 μL were injected directly split mode (1:40); injector temperature, 220 °C; column temperature, 60–240 °C at a rate of 3 °C min−1; detector temperature, 250 °C; carrier gas, helium at a constant flow rate of 1 mL min−1; ionization energy 70 eV; mass scan range 30–500 amu. The relative amounts of individual components of the total EO are expressed as a percentage of peak area relative to the total peak area. 2.3.2. Identification of volatile components Identification of the components of the EOs was based on comparison of their retention indexes with those reported in the literature (Adams, 2001; Babushok et al., 2011) and of their mass spectra with those in the NIST 5.0 spectral library. 2.3.3. Quantification of linalool The quantification of linalool was performed in triplicate using an external standard method with a five-point regression curve. Linalool standard (Sigma-Aldrich, St. Louis, MO, USA) at 97% was diluted with ethyl acetate, resulting in final linalool concentrations of 0.25, 0.51, 1.02, 1.52, 3.04% (v/v). Analysis of the standards was performed with GC–MS (Shimadzu, GCMS-QP2010 Ultra) using the same parameters as for EO samples. 2.4. In vitro antifungal activities of essential oils 2.4.1. Plant pathogenic fungi Cultures of pathogenic fungi were obtained from the Microbiological Collections of the National Institute for Research in the Amazon (MCTI-INPA), Amazonas, Brazil. Fungal isolates utilized were as follows: Colletotrichum guaranicola, C. gloeosporioides, Colletotrichum sp., and Alternaria alternata. Fungi were cultured in Petri dishes (100 × 15 mm) containing 10 mL of potato dextrose agar (PDA).

2. Materials and methods 2.1. Plant material Leaves and branches (thin stems) of A. rosaeodora trees were collected from forest plantations in the city of Manaus (2°48′72″S, 59°53′32″W), Amazonas, Brazil. Aerial parts were harvested from 10 individual plants at each site during two very distinctive seasonal periods of Amazon: the dry period (September 2011) and wet season (February 2012). Each botanical sample was identified by INPA’s taxonomist and later compared with the voucher specimen at the INPA herbarium, Brazil (Voucher N°177,315). The collected material was later used for EO extraction. The climate in Manaus is type Af (tropical humid) according to the Köppen climate classification system, with a mean annual temperature of 26.7 °C and little seasonal variation, with temperatures ranging between 25.9 and 27.7 °C (wet and dry seasons, respectively). The average annual precipitation is 2420 mm, the wettest month is March (mean ≅300 mm month−1) and the driest month is August, when monthly precipitation is approximately 80 mm month−1 (Alvares et al., 2013).

2.4.2. Standardization of fungal inoculum The fungal pathogens were cultured on PDA medium in sterilized Petri dishes and incubated at 27 °C for 14 days. The conidial suspensions were obtained by flooding PDA plates with 10 mL of sterile distilled water containing 0.1% Tween 80 (v/v) and filtered through three layers of sterile cheesecloth to remove mycelial fragments. Conidia were adjusted on a Neubauer chamber to the final concentration (2 × 105 conidia mL−1) using a light microscope (Zeiss AxioLab A1). 2.4.3. Antifungal activity in solid media EOs and the linalool standard were solubilized in 0.5% v/v Tween 80 to final concentrations of 20 μL mL−1 and sterilized by filtration through 0.22 μm Millipore filters (Nalgene, UK). The inhibitory effect on fungus growth was evaluated by the well-diffusion method in dishes, where 100 μL of the fungal inoculum was equally spread on the surface of the PDA plates (95 mm in diameter). Wells (6 mm in diameter) were 2

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Table 1 Chemical composition of the essential oil obtained from leaves and branches of Aniba rosaeodora collected during the wet and dry season at Manaus, AM, Brazil. Constituents

RI

Dry Leaves

Wet Branches

Method of identification

Leaves

Branches

3.5 ± 0.6 0.8 ± 0.2 0.5 ± 0.1 – – 2.7 ± 0.5 2.8 ± 0.6 71 ± 3.2 0.6 ± 0.6 – – – – – – 0.7 ± 0.2 – 2.4 ± 2.4 1.8 ± 1.7 – – 2.2 ± 2.3 1.5 ± 1.5 – – 1.2 ± 1.2 – 1.3 ± 1.3 1.6 ± 1.6 – 1.4 76.7 4.9 8.0 3.6 94.6

3.0 ± 0.6 0.8 ± 0.4 0.7 ± 0.2 – – 2.0 ± 1.0 1.4 ± 1.0 84 ± 5.2 0.8 ± 0.2 – – 1.0 ± 0.3 – – – –

% Peak area Butanoic acid α-Pinene β-Pinene β-Phellandrene 1.8-Cineole cis-Linalool oxide (furanoid) trans-Linalool oxide (furanoid) Linalool Hotrienol cis-Linalool oxide (piranoid) trans-Linalool oxide (piranoid) α-Terpineol p-Anisaldehyde α-Copaene β-Elemene Caryophyllene β-Chamigrene β-Selinene α-Selinene Germacrene B (E)-Nerolidol Spathulenol Caryophyllene oxide Globulol Guaiol α-Eudesmol Agarospirol Bulnesol Eudes-7(11) 7-en-4-ol Benzyl benzoate Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Other % Total identified

800 934 970 1029 1033 1074 1091 1104 1106 1171 1176 1193 1256 1378 1394 1423 1479 1487 1496 1557 1566 1577 1581 1588 1602 1653 1660 1665 1693 1770

– 1.7 ± 2.5 1.5 ± 2.5 2.9 ± 2.6 1.1 ± 2.0 1.2 ± 0.2 1.5 ± 0.3 69 ± 4.2 – 0.4 ± 0.2 0.5 ± 0.2 1.0 ± 0.4 0.5 ± 0.3 0.6 ± 0.3 1.1 ± 1.6 – 1.7 ± 1.3 1.7 ± 1.4 1.7 ± 1.5 1.5 ± 1.4 1.3 ± 0.5 0.9 ± 0.1 – 1.0 ± 0.2 0.9 ± 0.2 1.4 ± 0.4 1.1 ± 0.1 1.8 ± 0.2 – 0.6 ± 2.0 5.6 75 8.4 8.4 1.2 98.6

– 1.2 ± 1.6 1.1 ± 1.5 1.5 ± 2.1 1.3 ± 0.7 1.2 ± 0.5 – 78 ± 4.2 – 0.4 ± 0.7 1.1 ± 1.2 0.6 ± 0.2 1.3 ± 0.9 0.5 ± 0.2 0.6 ± 0.3 – 0.8 ± 0.3 1.0 ± 0.2 0.7 ± 0.3 0.8 ± 0.5 0.7 ± 0.4 1.1 ± 0.4 – 1.2 ± 0.4 1.0 ± 0.2 1.4 ± 0.8 0.9 ± 0.4 0.3 ± 0.7 – 1.1 ± 4.0 3.8 82.4 4.5 8.0 1.1 99.8

a,b a,b a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b a,b,c a,b,c a,b a,b,c a,b a,b,c a,b,c a,b,c

– – – – – 1.2 ± 0.2 – – – – 1.1 ± 0.4 1.1 ± 0.2 – 1.6 89.1 – 3.4 3.0 97.7

a,b,c a,b a,b a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b,c a,b

RI, retention index calculated on HP-5MS capillary column using homologous series of n-alkanes (Kovat’s retention index). a Retention index (RI). b NIST-5 mass spectral library and the literature (Adams, 2001). c Babushok’s retention Index (Babushok et al., 2011).

by subsequent subculturing of 5 μL cultures onto PDA plates. The plates were incubated at 26 ± 1 °C for 5 days, and the MFC was defined as the lowest concentration that completely inhibited subculture growth. The percent inhibition was calculated using absorbance at 630 nm according to Rautenbach et al. (2006). A graph was plotted to compare the % inhibition of vegetative fungal growth with the log concentration of EOs. The IC50 value is the concentration of the EO required to reduce mycelium growth to 50% and was calculated via nonlinear regression using GraphPad Prism 6 software (GraphPad Software, Inc., San Diego, CA).

made on agar, and 50 μL of sample was added to each well. The positive and negative controls were 50 μL of DithaneNT (2 mg mL−1) and 50 μL of 0.5% Tween 80 (v/v), respectively. The inoculated plates were maintained for 7 days at 26 ± 1 °C, and the diameter of the inhibition growth zone (DIZ) was measured in millimeters. The experiments were run in triplicate. 2.4.4. Determination of the minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and IC50 Fungal growth inhibition was evaluated using an automated quantitative assay developed by Broekaert et al., (1990). Briefly, 10 μL of conidial suspension was incubated with 90 μL of yeast potato dextrose broth in a 96-well microtiter plate for 16 h at 26 ± 2 °C. Then, 100 μL aliquots of 2-fold-diluted samples were added to each well of a 96-well plate at 0.156–20 μL mL−1 and incubated at 26 ± 2 °C. Every 12 h until 96 h after incubation, the growth of fungi was determined by absorbance at 630 nm using an automated microplate reader (Thermo Plate Reader – Elx800, Biotek). The positive and negative controls were 2 mg mL−1 DithaneNT and 0.5% v/v Tween 80, respectively. After incubation, 10 μL of cell viability indicator (2,3-5-triphenyl tetrazolium chloride 1%) was added to each well of the microtiter plate and incubated at 37 °C for 1 h. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of EO showing no colour change. The minimal fungicidal concentration (MFC) was determined

2.4.5. Conidial germination assay For germination tests, 10 μL of A. alternata conidia suspension containing 2 × 105 conidia mL−1 were incubated with 10 μL of each EO or the linalool standard (10 μL mL−1) in sterile glass depression slides. Tween 80 (0.5% v/v) was used as negative control and 200 mM H2O2 was used as positive control. The depression slides were incubated at 25 °C for 24 h in a Petri dish containing wet filter paper. Inhibition of germination was observed under light microscopy on a Zeiss AxioLab A1 microscope. Approximately 200 spores were counted, and the percentage of germinating conidia was calculated according to the following formula: Germinating conidia (%) = number of conidia germinated/total number of conidia × 100. Three independent experiments were performed. 3

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in branches), while linalool in the dry season decreased to 47.5 ± 2.2 in leaves and 49.2 ± 1.6% in branches (Fig. 1). Despite several earlier reports on the chemical composition of EOs using GC–MS, few studies have used external standards to quantify the major compounds in EOs. Interestingly, considerable differences in linalool content in the percentage peak area (Table 1) were not observed until quantification using the external standard (Fig. 1). The finding that the highest concentration of linalool in the EOs was obtained during the wet season can be explained by the fact that some plants bloom during this period. At this time, the plants can produce substantial amounts of EOs to attract pollinators and defend against pathogens (Amaral et al., 2015). In contrast, the decrease in linalool in the dry season might be attributable to the high temperature and the partial evaporation of this constituent in plants (Hussain et al., 2008). In contrast to our observations, the linalool content in the leaves of Aniba duckei was higher in the dry season, ranging from 62.40 to 76.69%, while in the wet season, it ranged from 56.26 to 60.38% (da Cunha et al., 2011). To test whether the variation in chemical composition of EOs could influence its antifungal activity, additional analytical methods were performed

2.5. Experimental design and statistical analyses The experimental design was completely randomized for 2 × 2 × 4 factorial treatments with two seasonal periods (dry and wet seasons), two types of aerial parts (leaves and branches) and four pathogenic fungi (Colletotrichum guaranicola, C. gloeosporioides, Colletotrichum sp., and Alternaria alternate). The measurements were carried out with 10 replicates of each treatment (pathogenic fungi). The results are reported as the mean value ± SEM and were compared using analysis of variance (ANOVA) followed by Tukey’s post hoc test using GraphPad Prism 5.0 software (GraphPad Software, Inc.). All chemical analyses used were carried out in triplicate. 3. Results and discussion 3.1. Chemical composition of the essential oils Analysis by GC–MS of the EOs extracted from leaves and branches of Aniba rosaeodora collected in the wet and dry seasons showed quantitative and qualitative differences in chemical compositions (Table 1). A total of 15 compounds were identified in the EO of the leaves during the wet season (LWS), whereas EOs of the branches (BWS) contained 11 compounds. Linalool was the main constituent in both EOs (71 ± 3.2% in LWS and 84 ± 5.2% in BWS). Other compounds found in the wet season were butanoic acid (3.5 ± 0.6% in LWS and 3.0 ± 0.6% in BWS), cis-linalool oxide (furanoid) (2.7 ± 0.5 in LWS and 2.0% ± 1.0% in BWS) and trans-linalool oxide (2.8 ± 0.6% in LWS and 1.4 ± 1.0% in BWS). β-Selinene (2.4 ± 2.4%), spathulenol (2.2 ± 2.3%), α-selinene (1.8 ± 1.7%), αeudesmol (1.2 ± 1.2%) and caryophyllene (0.7 ± 0.2%) were found only in LWS, while α-terpineol (1.0 ± 0.3%) was unique in BWS (Table 1). In the dry season, 25 and 24 compounds were identified in the EOs from leaves (LDS) and branches (BDS), respectively (Table 1). Linalool corresponds to 69 ± 4.2% of LDS and 78 ± 4.2% of BDS. Other constituents found at lower concentrations were β-phellandrene (2.9 ± 2.6% in LDS and 1.5 ± 2.1% in BDS), α-pinene (1.7 ± 2.5% in LDS and 1.2 ± 1.6% in BDS) and β-pinene (1.5 ± 2.5% in LDS and 1.1 ± 1.5% in BDS) (Table 1). Previous reports on the composition of EOs from A. rosaeodora showed linalool as a main component, ranging from 68 to 92% of the peak areas, with other compounds at low concentrations (Alcântara et al., 2010; Almeida et al., 2013; Maia et al., 2007; Sarrazin et al., 2016; Simić et al., 2004). Seasonal periods can influence the chemical composition of EOs (Koundal et al., 2015; Verma et al., 2015). In EO from leaves of A. canelilla, the quantities of the main components differ between the two seasons. During the wet season, 1-nitro-2-phenylethane corresponds to 70.6% and methyleugenol to 3.4%, whereas in the dry season, these levels are reduced to 39.0 and 0.5%, respectively (Taveira et al., 2003). Carvacrol, the main constituent of Lippia origanoides EO, corresponds to 43.5% in the wet season and 41.4% in the dry season (Sarrazin et al., 2015). Seasonality, sunshine hours, temperature and relative humidity can influence plant phytochemicals (Dhouioui et al., 2016). Indeed, plants may alter biochemical pathways and physiological processes to adjust to variations in climatic conditions by regulating the metabolism and synthesis of new compounds (Woronuk et al., 2011).

3.3. Antifungal activity At 20 μL mL−1, all tested EOs exhibited inhibitory activity against phytopathogenic fungi using the agar-well diffusion method (Table 2). The intensity of inhibition was similar between the EOs obtained from leaves and branches within the same season. The largest zones of inhibition were detected for the fungus Colletotrichum guaranicola, with diameters ranging from 15.2 ± 1.2 to 21.3 ± 1.7 mm (Table 2). The antifungal activity of LWS (21.3 ± 1.7 mm) was more potent than that of LDS (16.4 ± 1.3 mm) (P = 0.0175); similarly, the BWS (19.8 ± 1.6 mm) was more effective than BDS (15.2 ± 1.2 mm) (P = 0.0264). A comparable effect was observed for Alternaria alternata; LWS inhibited the vegetative growth (15.4 ± 0.7 mm) more strongly than LDS (11.1 ± 1.2 mm) (P = 0.0296), and BWS (15.2 ± 1.6 mm) showed greater inhibition than BDS (9.3 ± 1.5 mm) (P = 0.0030) (Table 2). The EOs in this study consistently inhibited the growth of C. gloeosporioides and Colletotrichum sp., with DIZ values ranging from 11.2 ± 1.6 to 16.6 ± 3.7 mm and 9.7 ± 0.8 to 12.6 ± 0.9 mm, respectively (Table 2). However, for these species, oils from both tissues collected in the wet season showed effects similar in magnitude to those of oils collected in the dry season (P > 0.05). Antifungal activity of EO from A. rosaeodora was observed previously (Simić et al., 2004; Sousa et al., 2012), but those reports did not focus on the effect of seasonal variation on antifungal activity. In general, the major component present in EOs may be responsible for much of the biological activity presented (Khosravi et al., 2011; Kordali et al., 2008; Stupar et al., 2014; Znini et al., 2013). To investigate the contribution of linalool to the antifungal activity, synthetic linalool was tested against the same phytopathogenic fungi. Overall, synthetic linalool showed an ability to inhibit fungal growth that was similar to that of the EOs tested, producing inhibition zones ranging from 11.2 ± 3.2 to 15.6 ± 1.2 mm (Table 2). The fungicide DithaneNT showed a stronger inhibitory effect on the mycelial growth of phytopathogens than did the EOs in this work (p < 0.05), with inhibition ranging from 15.2 ± 2.5 to 22.8 ± 1.9 mm (Table 2). Linalool is an acyclic monoterpene tertiary alcohol that exhibits high antimicrobial and antioxidant activities (Hong et al., 2015; Pinto et al., 2013; Zore et al., 2011). Linalool showed antifungal activity against the plant pathogenic fungi Rhizoctonia solani, Fusarium oxysporum, Penicillium digitatum and Aspergillus niger, with EC50 values from 73.68 to 211.7 mg L−1 (Marei et al., 2012). This study determined the minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values using the broth microdilution method in 96-well microplates and subcultures in PDA plates. All fungi were more sensitive to EOs collected during wet season

3.2. Quantification of linalool The quantification of linalool in EOs was performed by plotting a calibration curve using a linalool external standard. The linear regression analysis of the calibration curve produced an equation (Fig. 1), which was used to calculate the concentration of linalool in the samples. Differences were evident in the quantity of linalool between the wet and dry seasons (P < 0.0001), while between the two tissues, the linalool content was similar. The highest amount of linalool was observed during the wet season (74.4 ± 3.9% in leaves and 81.8 ± 5.7% 4

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Fig. 1. Quantification of linalool in essential oil obtained from leaves and branches of Aniba rosaeodora collected during the wet and dry seasons at Manaus, AM, Brazil. (A) Calibration curve of the linalool external standard used to quantify the samples. (B) Percentages of linalool in essential oils. Data are presented as the mean ± SEM (n = 10). Different letters indicate significant differences (P < 0.05) according to ANOVA followed by Tukey’s post hoc test.

exhibited inhibitory activity against all tested fungi, with an IC50 ranging from 0.705 to 1.235 μL mL−1. Dithane showed strong inhibitory effects on the mycelial growth of phytopathogens, with IC50 values of 0.222 to 0.389 μL mL−1. In this study, the collection period significantly affected the antifungal activity. EOs collected during the wet season (≈74–81% linalool) showed greater harmful activity than those collected during the dry period (≈47–49% linalool), which might be attributable to a higher content of linalool. Hussain et al. (2008) also found seasonal variation in the components of Ocimum basilicum oil. The linalool levels present in the EOs were highest during winter (60.6%) and lowest in summer (56.7%) (p < 0.05); these authors also attribute the higher antimicrobial activity to the higher content of linalool. In the wet season, high levels of relative humidity and precipitation trigger the release of spores (Crandall and Gilbert, 2017; Gilbert and Reynolds, 2005), which may lead to an increased risk of fungal infection. The greater frequency of rains and the constant moisture on the leaf surface are crucial for the spore germination and subsequent infection (Gauthier and Keller, 2013; Magarey et al., 2005). Thus, the accumulation of linalool appears to provide a promising advantage for plant defense during the period of the highest incidence of fungal diseases. Moreover, the higher antifungal efficacy of EOs collected in the wet season suggested a link between secondary metabolites and plant defense mechanisms. Bacterial growth was inhibited by Coriandrum sativum EO (≈68% linalool) and by pure linalool, with MIC values varying from 0.5 to 1.0 μL mL−1. When the coriander oil and linalool were diluted to final concentrations of 50%, the activity was dramatically reduced, possibly due to the dilution of the linalool (Duarte et al., 2016). Recently, Sarrazin et al. (2016) showed that the antibacterial activity of EO of A. rosaeodora (≈88% linalool) was more efficient than the oil of A. parviflora (≈45% linalool). The results are very similar to this work: EOs

than those from the dry season (Table 3). The MIC and MFC values of the LWS (0.62–2.5 μL mL−1) and BWS (0.62–2.5 μL mL−1) were 2-fold lower than those of the LDS (2.5–5.0 μL mL−1) and BDS (2.5–5.0 μL mL−1) for all fungi analyzed. No differences in antifungal activity were found between the leaves and branches collected in the same season. Synthetic linalool was toxic to all studied fungi, with MIC and MFC values ranging from 1.25 to 2.5 μL mL−1. Some plants responded to changes in weather conditions by changing the amounts and types of compounds produced, which may reflect the magnitude of biological activity (Silva et al., 2013; Furtado et al., 2014; UsanoAlemany et al., 2012). The MFC value is normally higher than the corresponding MIC value, indicating a higher fungistatic activity than fungicidal activity. However, equal values for MIC and MFC have been reported in the literature (Khosravi et al., 2011; Nikkhah et al., 2017; Pinto et al., 2013; Stupar et al., 2014), suggesting a fungicidal effect of the EOs tested. In the current work, the EOs of A. rosaeodora showed fungicidal effects against all phytopathogens, with MFC values equal to the MIC values. Fungicidal activity (with equal values for MIC and MFC) of the EOs of A. rosaeodora was also observed against other fungal species (Simić et al., 2004). The concentrations of EOs providing 50% growth inhibition (IC50) were calculated from dose-response curves (Fig. 2). Inhibitory doseresponse curves of LWS and BWS showed stronger antifungal activities than LDS and BDS, with minor IC50 values for all fungi tested (Table 4). The two most sensitive species were C. guaranicola and A. alternata. LWS and BWS showed very strong activity against C. guaranicola, with the best IC50 (0.578 μL mL−1 and 0.813 μL mL−1, respectively), while the IC50 values of LDS and BDS were 3.2- and 2.5-fold higher. For A. alternata, the IC50 values of LWS and BWS were 0.823 and 0.784 μL mL−1, whereas the IC50 values of LDS and BDS were higher (2.063 μL mL-1 and 2.645 μL mL−1, respectively). Synthetic linalool

Table 2 Inhibitory activity of essential oil of leaves and branches from Aniba rosaeodora collected during the wet and dry season against phytopathogenic fungi. Season

Tissue

C. guaranicola

C. gloeosporioides Colletotrichum sp. Diameter of inhibition zone (mm) at 20 μL mL−1

A. alternata

Wet

Leaves Branches

21.3 ± 1.7A* 19.8 ± 1.6A

14.3 ± 2.0A 16.6 ± 3.7A

12.1 ± 1.4A 12.6 ± 0.9A

15.4 ± 0.7A 15.2 ± 1.6A

Dry

Leaves Branches

16.4 ± 1.3B 15.2 ± 1.2B

11.2 ± 1.6A 12.3 ± 2.8A

9.8 ± 1.1A 9.7 ± 0.8A

11.1 ± 0.8B 9.3 ± 1.5B

Linaool DithaneNT

15.1 ± 1.2B 22.8 ± 1.9A

14.6 ± 2.1A 22.7 ± 2.2B

11.2 ± 3.2A 21.4 ± 2.6B

14.2 ± 0.4A 15.2 ± 2.5A

* Different letter indicates significant differences (P < 0.05) according to ANOVA followed by Tukey’s post hoc test. The measurements were carried out with 10 replicates of each treatment (pathogenic fungi).

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Table 3 Fungal growth inhibition in broth media of essential oil of leaves and branches from Aniba rosaeodora collected during the wet and dry season against phytopathogenic fungi. Season

Tissue

C. gloeosporioides

Colletotrichum sp.

C. guaranicola

A. alternata

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

Wet

Leaves Branches

1.25 1.25

1.25 1.25

2.5 2.5

2.5 2.5

0.62 0.62

0.62 0.62

1.25 1.25

1.25 1.25

Dry

Leaves Branches

2.5 2.5

2.5 2.5

5.0 5.0

5.0 5.0

1.25 1.25

1.25 1.25

2.5 2.5

2.5 2.5

Linaool DithaneNT

2.5 0.3

2.5 0.3

2.5 0.3

2.5 0.3

1.25 0.3

1.25 0.3

2.5 0.3

2.5 0.3

MIC: Minimal inhibitory concentration expressed in μL mL−1 MFC: Minimal fungicidal concentration expressed in μL mL−1.

(Aprotosoaie et al., 2014). Linalool can alter membrane permeability, allowing the uncontrolled flow of substances through the cell (Sikkema et al., 1995; Turina et al., 2006). Further studies are required to elucidate the possible mechanisms of action of EOs of Aniba rosaeodora.

with different percentages of linalool exhibit antimicrobial activities of different magnitudes The germination of A. alternata conidia was markedly inhibited by all EOs at 10 μL mL−1 (Fig. 3). The control (0.5% Tween 80) did not inhibit conidial germination under the same experimental conditions. However, no differences were found in the inhibitory activity on germination of leaves and branches collected in the two seasons. Due to its lipophilic nature, linalool can interact with the phospholipid bilayer of the plasma membrane to cause structural and functional damage

4. Conclusions Quantitative and qualitative variations in the composition of the EOs were detected in samples collected during the wet and dry seasons.

Fig. 2. Representative dose-response curves of inhibitory activity of essential oils from leaves (circles) and branches (squares) of Aniba rosaeodora collected during the wet (closed blue symbols) and dry (open red symbols) seasons against different fungal species. Data are presented as the mean ± SEM (n = 3). The horizontal dotted lines represent IC50. An R2 > 0.970 was obtained for all curves fitted (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). 6

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Table 4 Antifungal activity of essential oil of leaves and branches from Aniba rosaeodora collected during the wet and dry season against phytopathogenic fungi. Season

Tissue

C. gloeosporioides

Colletotrichum sp.

C. guaranicola

A. alternata

IC50

95% CI

IC50

95% CI

IC50

95% CI

IC50

95% CI

Wet

Leaves Branches

2.074 2.244

1.985–2.166 2.141–2.351

2.396 2.509

2.241– 2.563 2.172 – 2.897

0.578 0.813

0.549– 0.609 0.753 – 0.878

0.823 0.784

0.768 – 0.882 0.693 – 0.888

Dry

Leaves Branches

5.187 6.133

4.685–5.744 5.436–6.920

5.490 5.962

5.086 – 5.926 5.627 –6.317

1.884 2.094

1.753 – 2,024 1.962 – 2.235

2.063 2.645

1.676 – 2.540 2.144 – 3.263

Linaool DithaneNT

1.025 0.318

0.902–1.163 0.286–0.354

0.705 0.222

0.652 – 0.762 0.188 – 0.261

1.174 0.371

1.077 – 1.280 0.349 – 0.394

1.235 0.389

1.100 – 1.385 0.346 – 0.436

IC50, Concentration in μL mL−1 that inhibits 50% fungal growth. 95% CI, confidence intervals, the values are considered significantly different when the 95% CI fail to overlap. IC50 values and 95% confidence intervals obtained by non-linear regression from dose-response curves from three independent experiments.

Fig. 3. Inhibition of Alternaria alternata conidial germination by essential oils obtained from leaves and branches of Aniba rosaeodora at 10 μL−1 mL−1. (A) Graph showing percentages of spore germination inhibition by essential oils. (Panels) Representative light microscopy images of the effects of essential oils on conidial germination. The arrows indicate the germinating conidia. Bars, 40 μm (200× magnification).

lower antifungal potency of pure linalool than of some EOs suggests that in addition to linalool, other oil components might display antifungal activity. The generation of synergistic inhibitory properties by the combined effect of the complex mixture contained in an EO seems to be an advantageous mechanism for plant defenses against pathogens.

Linalool is the major component of oil from leaves and branches in both periods. Higher concentrations of linalool were observed in the wet season than in the dry season. The EOs were toxic to all phytopathogens analyzed, displaying superior inhibitory activity toward Colletotrichum guaranicola. The EOs collected during the wet season were more effective in reducing the vegetative growth of phytopathogens, providing evidence for the involvement of linalool in the inhibitory effect. However, the

Conflicts of interest The authors declare that there are no conflicts of interest. 7

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Acknowledgments

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