Bioactivity of essential oils in the control of Alternaria alternata in dragon fruit (Hylocereus undatus Haw.)

Industrial Crops and Products 97 (2017) 101–109

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Bioactivity of essential oils in the control of Alternaria alternata in dragon fruit (Hylocereus undatus Haw.) Juliana Cristina Castro a , Eliana Harue Endo b , Marina Roberta de Souza c , Erica Benassi Zanqueta d , Julio Cesar Polonio e , João Alencar Pamphile e , Tânia Ueda-Nakamura d , Celso Vataru Nakamura d , Benedito Prado Dias Filho b , Benício Alves de Abreu Filho d,∗ a

Post-Graduate Program in Food Science, State University of Maringá, Av. Colombo, 5790, Maringá 87020-900, Paraná, Brazil Department of Pharmacy, State University of Maringá, Av. Colombo, 5790, Maringá 87020-900, Paraná, Brazil c Department of Chemistry, State University of Maringá, Av. Colombo, 5790, Maringá 87020-900, Paraná, Brazil d Department of Basic Health Sciences, State University of Maringá, Av. Colombo, 5790, Maringá 87020-900, Paraná, Brazil e Departament of Biotechnology, Genetics and Cell Biology, State University of Maringá, Av. Colombo, 5790, Maringá 87020-900, Paraná, Brazil b

a r t i c l e

i n f o

Article history: Received 10 August 2016 Received in revised form 25 November 2016 Accepted 7 December 2016 Keywords: Dragon fruit Alternaria alternata Essential oils Antifungal Cinnamomum zeylanicum Eugenia caryophyllus

a b s t r a c t This study isolated and identified the fungus that causes postharvest disease in dragon fruit (Hylocereus undatus Haw.). The in vitro and in vivo antifungal activity of some essential oils were evaluated against the fungus. Morphophysiological and molecular identification confirmed the fungus was Alternaria alternata. The essential oils of Cinnamomum zeylanicum, Cymbopogon flexuosus, Eucalyptus globulus, Eugenia caryophyllus, and Rosmarinus officinalis were evaluated by the microdilution broth technique, disc diffusion, scanning electron microscopy, and fluorescence microscopy. Evaluation of the composition of the essential oils by gas chromatography/mass spectrometry revealed substantial amounts of eugenol as a major constituent of E. caryophyllus and C. zeylanicum (90.50% and 80.70%, respectively). The other essential oils of R. officinalis contained ␣-pinene (24.5%) and camphor (22.0%) as major components. E. globulus contained 1,8-cineole (78.9%). C. flexuosus contained neral (35.1%) and geranial (42.6%). C. zeylanicum and E. caryophyllus were the most active against the isolated fungi at minimum inhibitory concentrations of 250 and 500 ␮g/ml, respectively, causing morphological changes in the hyphae. The in vivo assay indicated that the fruits that were treated with E. caryophyllus at concentrations of 500 and 1000 ␮g/ml exhibited a 31% reduction of mycelial growth compared with the control. These results suggest that the essential oils of C. zeylanicum and E. caryophyllus are active against A. alternata both in vitro and in vivo, which may be promising for control of the microorganism. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Hylocereus undatus (Haw.), known as dragon fruit, belongs to the Cactaceae family that originated in the Americas (Ortiz-Hernández

∗ Corresponding author at: Department of Basic Health Science, State University of Maringá, Laboratório de Análise de Água, Ambiente e Alimentos, Bloco T20, 3◦ Andar, Sala 312, Av. Colombo 5790, Campus Universitário, 87020-900 Maringá, Paraná, Brazil. E-mail addresses: [email protected] (J.C. Castro), [email protected] (E.H. Endo), [email protected] (M.R. de Souza), erica b [email protected] (E.B. Zanqueta), julioc [email protected] (J.C. Polonio), [email protected] (J.A. Pamphile), [email protected] (T. Ueda-Nakamura), [email protected] (C.V. Nakamura), bpdfi[email protected] (B.P. Dias Filho), baafi[email protected], baafi[email protected] (B.A.d. Abreu Filho). http://dx.doi.org/10.1016/j.indcrop.2016.12.007 0926-6690/© 2016 Elsevier B.V. All rights reserved.

et al., 1999; Brunini and Cardoso, 2011). The fruits of the cactaceae family in tropical and subtropical regions has a high nutritional content (Jaafar et al., 2009; Zhuang et al., 2012), an exotic form, and an attractive color, thus generating great interest by the food industry (Le Bellec and Vaillant, 2011). However, its postharvest period is relatively short because it deteriorates rapidly under environmental conditions through exposure, storage conditions, and the physiology of the fruit. Diseases that are caused by microorganisms lead to large postharvest losses. Fruit can be attacked by microorganisms, such as fungi, during both the production and postharvest stages, resulting in black spots, necrosis, rot, and deterioration, making the fruit unsuitable for consumption and resulting in disposal (OrtizHernández and Carrilo-Salazar, 2012).

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The Alternaria genus is a spoilage microorganism. Alternaria spp. is a latent fungus that develops during the cold storage of fruit. It becomes visible during the commercialization period (TroncosoRojas and Tiznado-Hernández, 2014) and is associated with the deterioration of fruit (Ostry, 2008). More than 100 plant species have been reported to be affected by Alternaria spp. (Armitage et al., 2015). To effectively treat fruit and control microorganisms, natural compounds are receiving increasing attention (Moghaddam et al., 2015). Essential oils (EOs) are natural volatile substances with a complex of compounds (Li et al., 2015). Such compounds are synthesized in various parts of the plant, such as the leaves, flowers, seeds, fruits, and roots (Bakkali et al., 2008; Texeira et al., 2013). They are widely used in various fields, including the cosmetics, pharmaceutical, and food industries (Harkat-Madouri et al., 2015). The constituents of these compounds have been shown to have antibacterial, virucidal, fungicidal, antiparasitic, insecticidal, and medicinal activity that is considered protective for plants (Bakkali et al., 2008). Although studies have reported the presence of Alternaria spp. in various fruits, little information is available on the presence of Alternaria spp. in dragon fruit and its relationship with postharvest disease. The objective of the present study was to isolate and identify the fungus that is responsible for the contamination of H. undatus (Haw.) during refrigerated storage for 25–30 days and evaluate the in vitro and in vivo antifungal activity of EOs (Cinnamomum zeylanicum, Cymbopogon flexuosus, Eucalyptus globulus, Eugenia caryophyllus, and Rosmarinus officinalis) against A. alternata.

2. Material and methods 2.1. Pathogen isolation Hylocereus undatus (Haw.) fruits were collected in Marialva, Paraná (coordinates: 23◦ 46 35.51 S, 51◦ 79 71.10 W), selected, washed, and sanitized with 1% sodium hypochlorite. The microorganism was isolated from the fruits by incubation for 25–30 days at 8 ◦ C until rot had clearly appeared in the epidermis, with typical symptoms of the Alternaria spp. fungus. Tissue of the epidermis (1 cm × 1 cm) that contained the microorganism was inoculated in potato dextrose agar (PDA) and incubated at 28 ◦ C for 7 days to allow identification according to Carvalho et al. (2011), with slight modifications.

2.2. Morphological characterization and molecular identification of isolated species based on sequencing of ITS1-5.8S-ITS2 region Macroscopic characterization was performed with microorganisms that were grown in solid PDA medium and analyzed with regard to the appearance of colonies, shape of the mycelium, color, and growth time. Microscopic characterization was performed using the micro-cultivation technique (Ribeiro and Soares, 2005). The isolates were viewed under an Olympus fluorescent microscope and photographed with an image capture system. The isolates were identified morphophysiologically (Hoog et al., 2000). The results of the macroscopic and microscopic evaluations were analyzed using identification keys. The genomic DNA of the isolated fungus was prepared and extracted using an extraction buffer (Pamphile and Azevedo, 2002) and stored at −20 ◦ C. The integrity of DNA was verified using 1% agarose gel and photographed. Amplification of the ITS1-5,8S-ITS2 region of rRNA was performed by polymerase chain reaction (PCR) using the following primers: ITS1 (5 -TCCGTAGGTGAACCTGCGG3 ) and ITS4 (5 -TCCCCGCTTATTGATATGC-3 ; White et al., 1990)

according to previously described methodology (Rhoden et al., 2012). The PCR product was purified using shrimp alkaline phosphatase and exonucleose I enzymes. The rRNA sequence was obtained using an ABI-Prism 3500 Genetic Analyzer (Applied Biosystems). The results were analyzed using BioEdit 7.2.5 software. The isolated nucleotide sequences were identified by comparisons with those in the National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov) database using the nBLAST sequence filter type tool (type strains). The sequences were aligned using MEGA 6.05 software and the “neighbor-joining” method, with “p-distance” for nucleotides with the “pairwise gap deletion” option and bootstrap with 10,000 replications to construct the phylogenetic tree. 2.3. Essential oil Essential oils (EOs) of cinnamon (Cinnamomum zeylanicum), lemon grass (Cymbopogon flexuosus), eucalyptus (Eucalyptus globulus), clove (Eugenia caryophyllus; Bio Essência, Jaú, São Paulo, Brazil), and rosemary (Rosmarinus officinalis; By Samia, São Paulo, Brazil) were obtained in Maringá, Paraná, Brazil. 2.3.1. Characterization of the essential oils The detailed chemical composition of the EOs was performed using gas chromatography-mass spectrometry (GC–MS) with an automatic injector (FOCUS GC – DSQ II, Thermo Electron Corp). The gas chromatograph-mass spectrometer was equipped with an Agilent DB-5 capillary column (5% phenyl/95% dimethyl siloxane stationary phase, 30 m length, 0.25 mm internal diameter, and 0.1 ␮m film thickness). Characterization was performed using a column temperature program that began at 70 ◦ C, followed by a temperature increase of 3 ◦ C/min to 230 ◦ C. Helium was used as the carrier gas at a flow rate of 1.0 ml/min (Kim et al., 2015). The total analysis time was 53 min. The temperature of the injector and detector was maintained at 250 ◦ C. A 1 ␮l volume of the samples was injected for chromatography in split mode (1:10). Each EO was diluted in hexane (high-performance liquid chromatography grade; 0.2 ␮l of EO to 1000 ␮l of hexane) to form the stock solution. Prior analyses indicated the necessity of diluting each solution again for injection of the sample in the chromatograph. The stock solution of EOs of C. flexuosus, E. globulus, E. caryophyllus, and R. officinalis was diluted in hexane (1/10 [v/v]). C. zeylanicum was diluted in hexane (1/20 [v/v]; Falasca et al., 2016; with modifications). Characterization was performed based on retention time (RT) and compared with the major compounds using Kovats retention rate (Skoog et al., 2006). The compounds of the EOs were identified by analyzing the retention times of the peaks that were obtained for each EO and confirmed via a standard mixture of nalkanes (C8 –C20 ; Sigma-Aldrich). The compounds of interest were confirmed (Adams, 2007) and are presented as percentages. 2.4. Antifungal activity The minimum inhibitory concentrations (MICs) of the EOs and eugenol were determined by the microdilution method (Clinical and Laboratory Standards Institute, 2008) with a conidia suspension of 5 × 104 conidia/ml using RPMI 1640 medium and l-glutamine without bicarbonate, buffered with 0.165 M morpholine propane sulfonic acid. Conidia were collected in sterile saline solution after 7 days of incubation. The EOs and isolated compounds were diluted in 1% Tween-80 and tested at concentrations of 62.5–4000 ␮g/ml. The microplates were incubated at 28 ◦ C for 72 h. The MIC was defined as the lowest concentration of the EO that inhibited the visual growth of the fungus.

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2.5. Disc diffusion method The disc diffusion method was used to evaluate the inhibition of hyphal growth. Plates with PDA were centrally inoculated with 10 ␮l of Alternaria alternata conidia suspension and incubated at 28 ◦ C for 3–5 days. Test discs were prepared with 10 ␮l of each oil at concentrations of 100, 50 and 25% and control discs with 1% Tween-80. These discs (5 mm diameter) were arranged around the colony on the plate, at a distance of 0.5 cm and incubated at 28 ◦ C for 72 h. The inhibition of hyphal growth was visually evaluated and photographed (Prasad et al., 2001). 2.6. Checkerboard method Checkerboard tests were performed to evaluate the effects of drug combinations. The assay was performed in 96-well microplates to obtain the fractional inhibitory concentration (FIC) of the EOs combined with the major compound that presented better activity against conidia of A. alternata. RPMI 1640 medium, buffered with 0.165 M MOPS, was used for the assay. A volume of 10 ␮l of the inoculum (5 × 104 conidia/ml) was added to each well, and the plates were incubated at 28 ◦ C for 72 h. The FIC index was calculated as FIC A + FIC B, where FIC A = MIC A combined/MIC A alone, and FIC B = MIC B combined/MIC B alone. The FICs of both combinations (C. zeylanicum combined with eugenol and E. caryophyllus combined with eugenol) were calculated. Eugenol (Biodinâmica, Madrid, Spanish) was obtained in Maringá, Paraná, Brazil. A FIC index ≤ 0.5 was considered synergism. A FIC index >4 was considered antagonism. A FIC Index >0.5 and ≤ 4 was considered indifferent (Odds, 2003). 2.7. Scanning electron microscopy Alternaria altermata inoculum (5 × 104 conidia/ml) was treated with EOs as described in the determination of the antifungal activity of EOs, C. zeylanicum, C. flexuosus, E. globulus, E. caryophyllus and R. officinalis at MICs concentration and negative control used suspension of the isolated fungus without application of EOs. After incubation at 28 ◦ C for 48 h, the samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, adhered on glass cover slips pre-treated with poly-l-Lysine (Sigma-Aldrich). Subsequently, the samples were dehydrated in an ascending series of ethanol, CO2 critical point dried, coated with gold and examined with a Shimadzu SS-550 Scanning Electron Microscope (Endo et al., 2010). 2.8. Fluorescence microscopy: hyphal growth inhibition Inhibitory concentrations of EOs in 500 ␮l of RPMI medium were prepared in 24-well plates containing cover slips. Wells were inoculated with 100 ␮l of conidia suspension at concentration 5 × 104 conidia/ml, and the plate was incubated at 28 ◦ C for 48 h. Cover slips with adhered hyphae were carefully removed and washed in phosphate buffered saline (PBS) pH 7.2, with manual shaking. Hyphae were stained with Calcofluor White M2R (Sigma-Aldrich) and mounted on a slide. Slides were observed in Olympus Fluorescent Microscope (Koroishi et al., 2008).

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wound. Control fruits were inoculated only the conidial suspension of A. alternata according to Feng et al. (2011), with slight modifications. The fruits were then stored at 25 ◦ C for 8 days. Lesions were visualized after 5 days, and the extent of infection of the fruit was recorded after 8 days of incubation. Four fruits were used for each treatment. The experiment was repeated twice. 2.10. Statistical analysis The in vivo experimental data were converted into the spot development rate (SDR) according to Carvalho et al. (2011). Data from the in vivo assay were analyzed using analysis of variance. Means were compared using Scott-Knott calculations (p < 0.05). The statistical analysis was conducted using SAS 9.0 software. 3. Results and discussion 3.1. Morphological characterization and molecular identification of isolated species based on sequencing of ITS1-5.8S-ITS2 region Based on macro- and micromorphological characteristics, including olive-colored reproductive hyphae and white vegetative hyphae, conidia with one or few conidial scars, rugulose with muriform septation, and unbranched chains of 10 or more, the isolated microorganism was identified as Alternaria alternata (Hoog et al., 2000). A. alternata is a filamentous and pathogenic fungus that grows on the epidermis of fruit, forming circular light-brown stains that are surrounded by a dark brown halo. The fruit’s growth and development presented irregular spots with a gray center, indicating deterioration and rot during storage. Fig. 1 shows the septate hyphal structure and conidia of the isolated strains of A. alternata. In some cases, precisely identifying the vegetative structure of the pathogen after isolation is difficult because of slight morphological differences and genetic similarities among different species (Armitage et al., 2015). Therefore, other methods are needed to confirm identification. Identification according to morphophysiological characteristics was confirmed by DNA identification of the isolated fungus. Fig. 2 shows the phylogenetic tree that was generated from the isolated fungus with other sequences that were obtained from the NCBI database. The Bipolaris sorokiniana strain CBS 110.14 was used as the outgroup. The isolate was grouped with four strains of A. alternata with a 99% bootstrap, confirming identification of the species. Different fruits are subject to attack by pathogenic fungi, such as A. alternata, which can cause spots, deterioration, and the loss of fruit quality. Alternaria spp. causes foliar and stem diseases and postharvest rot as previously described (Armitage et al., 2015). The presence of A. alternata has been reported in various crops, including mandarin (Carvalho et al., 2011; Huang et al., 2015), persimmon (Biton et al., 2014), strawberry (Zhang et al., 2015), and pistachio (Avenot and Michailides, 2015). In Hylocereus sp., Alternaria spp. has been reported to cause disease (Ortiz-Hernández and CarriloSalazar, 2012), and Alternaria spp. has been isolated from Hylocereus undatus (Haw.) Britton et Rose fruit that was imported from Vietnam (Ma et al., 2009). 3.2. Characterization of the essential oils

2.9. In vivo assay with Hylocereus undatus (Haw.) fruits H. undatus (Haw.) fruits that were free of injury were selected and wounded on the epidermis in the equatorial region with a sterile punch (2 mm depth, 5 mm width). Aliquots (20 ␮l) of each EO of C. zeylanicum (125, 250, and 500 ␮g/ml) and E. caryophyllus (250, 500, and 1000 ␮g/ml) were diluted in a solution of 1% Tween-80 and applied to each wound. After 30 min, 10 ␮l of a conidia suspension of A. alternata (5 × 104 conidia/ml) was inoculated into each

The characterization of the EOs was performed by GC–MS, allowing identification and quantification of the components of each EO. The results are presented as percentages (Table 1). The main components of the EO of C. flexuosus were geranial (42.6%) and neral (35.1%). Geraniol (5.21%), geranyl acetate (3.93%), and E-caryophyllene (2.36%) were present in lesser amounts. In the EO of E. globulus, the main component was 1,8-cineole (78.9%), followed by linomene (8.53%), ␳-cymene (5.65%), and ␣-pinene

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Fig. 1. Fungal morphological identification. Image by optical microscopy. A. Spores and hyphae and B. Spores chacterizated as Alternaria alternata, according morphophysiological identification, captured in microscope image capture system. Bar: 20 ␮m. Magnification: 100×.

Fig. 2. Fungal molecular identification. Phylogenetic tree of isolated post-harvest fungi of H. undatus with other fungi obtained from the GenBank database, constructed using the neighbor-joining method using p-distance for nucleotides with the pairwise gap deletion. The numbers on the tree indicate the percentage of the percentage of times the group on the right occured on the same node during consensus evalution (bootstrap with 10,000 replications). (䊉) Isolated fungi of H. undatus fruit. The Bipolaris sorokiniana strain was used as outgroup. Table 1 Chemical composition of C. flexuosus, E. globulus, E. caryophyllus, R. officinalis and C. zeylanicum essential oil. Compound

C. flexuosus a

Camphene ␣-Pinene ␤-Pinene 6-Methyl-5-heptene-2-one ␣-Phellandrene ı-2-Carene ␳-Cymene o-Cymene Linomene 1,8-cineole ϒ-Terpinene Linalool Camphor Borneol ␣-Terpineol Neral Geraniol Geranial Bornyl acetate Eugenol Geranyl acetate E-Caryophyllene ␣-Humulene Eugenol acetate Caryophyllene oxide Benzyl Benzoate Total of Identified compound a

RT = Retention time (min).

RT (min)

4.88 – – 5.51 – – – – – – – 8.90 – – – 14.1 14.5 15.3 – – 19.8 21.4 – – – – 93.8%

E. globulus %

a

1.18 – – 1.95 – – – – – – – 1.51 – – – 35.1 5.21 42.6 – – 3.93 2.36 – – – –

– 4.54 – – – – 6.65 – 6.78 6.91 7.61 – – – – – – – – – – – – – – – 98.2%

RT (min)

E. caryophyllus %

a

– 3.57 – – – – 5.65 – 8.53 78.9 1.56 – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – 18.8 – 21.4 22.8 – 27.8 – 99.4%

RT (min)

R. officinalis %

a

– – – – – – – – – – – – – – – – – – – 90.5 – 6.82 1.55 – 0.55 –

4.88 4.52 5.50 – – 6.22 – 5.63/6.63 6.77 6.88 – – 10.7 11.61 12.4 – – – 15.9 18.7 – 21.4 – – – – 97.7%

RT (min)

C. zeylanicum %

a

9.96 24.5 5.19 – – 0.72 – 1.36/2.79 2.62 19.4 – – 22.0 2.96 2.61 – – – 1.61 0.74 – 1.32 – – – –

– 4.53 – – 6.16 – – 6.64 – – – 8.92 – – – – – – – 18.7 – 21.4 – 25.3 – 34.8 95.0%

RT (min)

% – 2.81 – – 1.71 – – 2.00 – – – 2.55 – – – – – – – 80.7 – 2.80 – 1.38 – 1.07

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Fig. 3. Disc diffusion method. A. A. alternata control. B. A. alternata treated with C. flexuosus oil. C. A. alternata treated with E. globulus oil. D. A. alternata treated with E. caryophyllus oil. E. A. alternata treated with R. officinalis oil and F. A. alternata treated with C. zeylanicum oil. Dics numbered 1 (oil pure – 100%), 2 (oil 50%), 3 (oil 25%) and 4 (Tween 80 1%).

(3.57%). The EOs of E. caryophyllus and C. zeylanicum had the highest concentration of eugenol (90.–99.4% and 80.7–95.0% of the total components, respectively). The minor components of E. caryophyllus varied from 0.55% to 6.82%, including E-caryophyllene (6.82%) and ␣-humulene (1.55%). The minor components of C. zeylanicum varied from 1.07% to 2.81%, including ␣-pinene (2.81%), linalool (2.55%), and E-caryophyllene (2.80%). For R. officinalis, the main components were ␣-pinene (24.5%), camphor (22.0%), and 1,8cineole (19.4%), followed by camphene (9.96%), ␤-pinene (5.19%), and borneol (2.96%). The most abundant components of the EOs were eugenol (90.5% in E. caryophyllus, 80.7% in C. zeylanicum, and 0.74% in R. officinalis), E-caryophyllene (2.80% in C. zeylanicum, 2.36% in C. flexuosus, and 1.32% in R. officinalis), and ␣-pinene (24.5% in R. officinalis, 3.57% in E. globulus, and 2.81% in C. zeylanicum). In the present study, the EOs consisted of many chemical compounds, including terpenes, phenols, and alcohol, consistent with previous studies (Bakkali et al., 2008; Li et al., 2015). Many factors influence the chemical composition of the EOs of plants, such as agricultural factors, species, harvest method, part of the plant, harvest time, season, geography, extraction method, and bioactive properties (Moghaddam et al., 2015; Bakkali et al., 2008; Mejri et al., 2010). These factors can influence the identification and amount of the compounds. In the present study, the compounds that were present in the EO of C. zeylanicum differed from other studies, which reported that the main compounds in C. zeylanicum Blume (Lauraceae) were E-cinnamaldehyde (68.9%), benzaldehyde (9.94%), and E-cinnamyl acetate (7.44%). The concentrations of eugenol, linalool, and ␣-pinene were previously found in lower amounts than in the present study (Unlu et al., 2010). For C. flexuosus, substantial amounts of E-citral (60.9%) and ␣sinensal (12.3%) were reported (Kumar et al., 2009). The EO of E. globulus was reported to consist of 1,8-cineole (45.4%), limonene (17.8%), ␳-cymene (9.50%), ␥-terpinene (8.80%), ␣-pinene (4.20%), and ␣-terpineol (3.40%; Tyagi and Malik, 2011), which is consistent with the present study. Another study found that the EO of E. glob-

Table 2 Minimum inhibitory concentrations (MICs) of C. flexuosus, E. globulus, E. caryophyllus, R. officinalis, C. zeylanicum oil and isolated compound eugenol against conidial germination of A. alternata. Essential Oils and isolated

MIC (␮g/mL)a

C. flexuosus E. globulus E. caryophyllus R. officinalis C. zeylanicum Eugenol

1000 1000 500 1000 250 250

a

MIC = minimum inhibitory concentration.

ulus consisted of limonene (89.9%), ␥-terpinene (2.50%), ␳-cymene (2.20%), and ␣-pinene (2.10%; Stevic et al., 2014). The major compounds in R. officinalis were reported to be 1,8cineole (19.6%), camphor (17.0%), and ␣-pinene (15.1%; Badawy and Abdelgaleil, 2014), which is also consistent with the present study. In the present study, the EO of E. caryophyllus had substantial amounts of eugenol, which was also reported in other studies (e.g., eugenol [83.9%], eugenile acetate [10.7%], and ␤-caryophyllene [3.25%], Azizkhani et al., 2013; eugenol [77.6%], acetyl eugenol [10.9%], and ␤-caryophyllene [6.22%], Buentello-Wong et al., 2016). 3.3. Antifungal activity The MICs for the five EOs and the isolated compound eugenol are shown in Table 2. The EOs that were most effective against A. alternata were from C. zeylanicum (MIC = 250 ␮g/ml) and E. caryophyllus (MIC = 500 ␮g/ml), followed by C. flexuosus, E. globulus, and R. officinalis (MIC = 1000 ␮g/ml). The MIC for eugenol was 250 ␮g/ml. The effective EOs presented considerable amounts of eugenol (90.5% in E. caryophyllus and 80.7% in C. zeylanicum). The other EOs presented less inhibition of the A. alternata isolate, suggesting that neral and geranial in C. flexuosus, 1,8-cineole, limonene, and ␳-cymene in E. globulus, and ␣-pinene, 1,8-cineole, and cam-

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Fig. 4. Scanning electron microscopy with damage to the septate hyphae. A. A. alternata control. B. A. alternata treated with 1000 ␮g/ml of C. flexuosus oil. C. A. alternata treated with 500 ␮g/ml of E. caryophyllus oil and D. A. alternata treated with 250 ␮g/ml of C. zeylanicum essential oil. Bar: 5 ␮m. Magnification: 3,000×.

phor in R. officinalis were less effective against the isolated fungus. A MIC ≤ 500 ␮g/ml suggests strong inhibition (Aligiannis et al., 2001), reinforcing the inhibitory actions of the EOs of E. caryophyllus, C. zeylanicum, and eugenol. Several EOs have been shown to be effective in the control of microorganisms through the antifungal properties of their constituent compounds. In vitro studies have shown that EOs that contain eugenol are able to control microorganisms, such as Candida spp. and Aspergillus spp., reflected by the MIC (Pinto et al., 2009). Such EOs can be extremely effective against postharvest fungal pathogens, including A. alternata, in fruit (Combrinck et al., 2011). Eugenol was shown to significantly inhibit A. alternata PTCC 5224 and other fungi, such as Aspergillus ochraceus PTCC 5017 and Cladosporium spp. PTCC 5202 (Abbaszadeh et al., 2014). The MICs of the EO of C. flexuosus have been reported to effectively control several microorganisms, including Aspergillus spp., Penicillium spp., Trichoderma viride, Fusarium oxysporum, and A. alternata, among others (Kumar et al., 2009). C. zeylanicum is effective against A. alternata isolated from avocado fruit (Combrinck et al., 2011). R. officinalis oil has been shown to be effective against Erwinia cavotorova and Agrobacterium tummefaciens (Badawy and Abdelgaleil, 2014), and E. globulus was effective against A. alternata (Stevic et al., 2014; Combrinck et al., 2011). 3.4. Disc diffusion method The disc diffusion method was used to evaluate the growth of hyphae in the presence of EOs, visually confirming the inhibitory capacity and activity of the EOs against the isolates (Endo et al., 2015). Fig. 3 shows the results of the disc diffusion analysis of the inhibitory action of the EOs against hyphae.

The EOs of C. flexuosus (50% and 100%), E. caryophyllus (25%, 50%, and 100%), and C. zeylanicum (100%) inhibited the growth of the hyphae of A. alternata isolates. The other EOs (E. globulus and R. officinalis) did not effectively inhibit the growth of the hyphae at the concentrations tested. These results were confirmed by the MIC evaluation. Plants produce diverse secondary metabolites that protect plants and have antimicrobial activity through their biocidal properties (Bassolé and Juliani, 2012). R. officinalis extracts were shown to be effective in the control of dermatophyte fungi when evaluated by the disc diffusion method (Endo et al., 2015), but their constituent compounds are ineffective in the control of A. alternata. E. globulus and Cinnamomum had maximal activity against Aspergillus niger and Aspergillus fumigatus compared with controls (Bansod and Rai, 2008). However, a study of Eucalyptus oils with high levels of 1,8-cineole reported lower activity against many pathogens (Combrinck et al., 2011).

3.5. Checkerboard method The effects of the EO of C. zeylanicum combined with eugenol against A. alternata presented indifference, with a FIC index of 0.53. The EO of E. caryophyllus combined with eugenol presented a synergistic interaction, with a FIC index of 0.37. The MIC of E. caryophyllus decreased two-fold when combined with eugenol. Previous studies have shown that the primary activity of EOs is related to the major compounds of the essential oil, but minor compounds can also be fundamental to synergistic, antagonistic, and additive effects (Bassolé and Juliani, 2012). Therefore, minor components of the EOs may have influenced the interactions of the combined oils in the present study.

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Fig. 5. Fluorescence Microscopy for hyphae growth inhibition: A. A. alternaria control. B. A. alternata treated with 1000 ␮g/ml of C. flexuosus oil. C. A. alternata treated with 500 ␮g/ml of E. caryophyllus oil and D. A. alternata treated with 250 ␮g/ml of C. zeylanicum oil. Bar: 20 ␮m. Magnification: 100×.

3.6. Scanning electron microscopy The characteristics of the A. alternata hyphae that were treated with the EOs are shown in Fig. 4. The untreated control presented normal growth of the septate hyphae, with no visible structural changes (Fig. 4A). Treatment with C. flexuosus (Fig. 4B), E. caryophyllus (Fig. 4C), and C. zeylanicum (Fig. 4D) inhibited fungal growth and caused damage to the septate hyphae. The EOs of E. globulus and R. officinalis presented lower activity and did not cause structural modifications (not shown). The structures of the hyphae that were treated with E. globulus and R. officinalis were apparently unaltered. The EOs of C. flexuosus, C. zeylanicum, and E. caryophyllus reduced the growth of the hyphae and caused structural changes, including disruption of the hyphae and cell content extravasation (Fig. 4B and C), deformation of the integrity of the hyphae (Fig. 4C and D), and tumescence of the hyphae with the possible formation of adhesive mucilage (Fig. 4D). These alterations affected the growth and structural characteristics of the isolate, and the presence of conidia was detected, indicating an inappropriate environment for germination. A previous study evaluated the effects of various extracts (i.e., extracts of Anadenanthera colubrina [Vell.] Brenan, Artemisia annua L., and Ruta graveolens) against A. alternata that was isolated from Murcott tangor fruit, indicating significant reductions of conidia compared with the control group (Carvalho et al., 2011). However, few studies have reported the structural characteristics of A. alternata when treated with EOs. The mechanisms of action of EOs against A. alternata are not completely understood. The activity of EOs is related to hydrophobicity and interactions with cell wall lipids, the cell membrane, and mitochondria that alter the permeability and function of fun-

gal structures (Costa et al., 2011). These alterations may also be related to disturbances in the morphogenesis and growth of fungi and interference with enzymes that are responsible for cell wall synthesis that alter the integrity of hyphae, as described for A. niger (Rasooli et al., 2006).

3.7. Fluorescence microscopy: hyphal growth inhibition Hyphal growth inhibition was visualized by fluorescence microscopy (Fig. 5). A. alternata (Fig. 5A) presented greater amounts of hyphae. The EOs of C. zeylanicum, C. flexuosus, and E. caryophyllus inhibited the growth of the hyphae. The EOs of E. globulus and R. officinalis did not effectively control the fungus (not shown). These results were consistent with the scanning electron microscopy, MIC, and disc diffusion findings. Fluorescence microscopy is based on the intensity of fluorescence that is emitted by the organism when exposed to treatment. Calcofluor white is a fluorochrome that binds fungal cell wall chitin, which in turn is synthesized by enzymes that are present in the plasma membrane. Therefore, changes in the plasma membrane can affect cell wall chitin. Damage to the cell wall is reflected by a lower fluorescence intensity compared with an intact cell wall (Chaffin et al., 1998).

3.8. In vivo assay with Hylocereus undatus (Haw.) fruits Based on the in vitro evaluations (i.e., MICs and disc diffusion method), an in vivo experiment was conducted with the EOs of C. caryophyllus (125, 250, and 500 ␮g/ml) and E. zeylanicum (250, 500, and 1000 ␮g/ml). The results of the in vivo assay with H. undatus (Haw.) fruits are shown in Table 3.

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Table 3 In vivo inhinitory effects of E. caryophyllus and C. Zeylanicum EOs against A. alternata isolated from H. undatus (Haw.) fruits. Treatments

SDR* Means ± SD†

Control E. caryophyllus 250 ␮g/ml E. caryophyllus 500 ␮g/ml E. caryophyllus 1000 ␮g/ml C. zeylanicum 125 ␮g/ml C. zeylanicum 250 ␮g/ml C. zeylanicum 500 ␮g/ml CV%‡

1.00 ± 0.14a 0.73 ± 0.10b 0.69 ± 0.08b 0.69 ± 0.09b 0.74 ± 0.11b 0.70 ± 0.08b 0.70 ± 0.11b 14.1

* Data were converted into spots development rate (SDR) by dividing the mean value (diameter) for each fruit (treatment) by the mean value of control fruits, treated only with conidia suspension. † SD = Standard deviation. ‡ CV% = Coefficient of variation. Different letters signify significant diferences (p < 0.05), according to the Scott-Knott method.

All of the concentrations of the EOs inhibited mycelial growth on the epidermis of the fruit compared with untreated control fruit. Treatment with the EO of E. caryophyllus at 500 and 1000 ␮g/ml (0.69 ratios for both) reduced fungal growth by 31% compared with the untreated control fruit. The inhibition ratios for the other treatments were 0.74–0.70, with 26–30% reductions of the growth of A. alternata. In the present study, the in vivo experiments with H. undatus (Haw.) fruits revealed antifungal activity of the EOs of E. caryophyllus and C. zeylanicum, which reduced the growth of the A. alternata and corroborated the in vitro results. In vivo studies found strong antifungal effects of extracts of Anadenanthera colubrina against A. alternata in cherry tomatoes, which reduced fungal growth by 62% compared with controls (Carvalho et al., 2011). The treatment of A. alternata with thyme oil at a concentration of 500 ␮l/ml in cherry tomatoes reduced deterioration of the fruit by an a verage of 40% (Feng et al., 2011). However, no previous studies have evaluated the control of A. alternata in H. undatus (Haw.) fruit. The present study emphasizes the importance of controlling this fungus in Cactaceae, such as dragon fruit. 4. Conclusion The fungus that causes postharvest disease in H. undatus (Haw.) fruit was isolated and identified morphologically and molecularly as Alternaria alternata, and its susceptibility to various EOs was tested. The most effective EOs, both in vitro and in vivo, were Cinnamomum zeylanicum and Eugenia caryophyllus, which inhibited the growth of fungal hyphae, caused structural changes, and inhibited development of the fungus on the epidermis of the fruit. These results support the use of EOs as an economically viable alternative for the control of postharvest diseases through their antifungal action. Acknowledgments The authors would like to thank the Brazilian Federal Agency for the support and Evaluation of Graduate Education (Coordenac¸ão e Aperfeic¸oamento de Pessoal de Nível Superior, Brasil [CAPES]). References Abbaszadeh, S., Sharifzadeh, A., Shokri, H., Khosravi, A.R., Abbaszadeh, A., 2014. Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi. J. Med. Mycol. 24, 51–56. Adams, R.P., 2007. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, fourth ed.

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