ANTIFUNGAL ACTIVITY OF TEA TREE ESSENTIAL OILS (MELALEUCA ALTERNIFOLIA) AGAINST PHYTOPATHOGENIC FUNGI.

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The chemical composition and plant pathogenic antifungal activity of tea tree essential oils (Melaleuca alternifolia) collected in Long An, Vietnam was surveyed. By gas chromatography and mass spectrometry (GC-MS), the main chemical composition in
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  ISSN: 2320-5407 Int. J. Adv. Res. 7(9), 1239-1248  1239    Journal Homepage: -  www.journalijar.com   Article DOI:  10.21474/IJAR01/9778 DOI URL:  http://dx.doi.org/10.21474/IJAR01/9778 RESEARCH ARTICLE ANTIFUNGAL ACTIVITY OF TEA TREE ESSENTIAL OILS (MELALEUCA ALTERNIFOLIA) AGAINST PHYTOPATHOGENIC FUNGI. Thanh Khang LE 1 , Thi Thu Huong Nguyen 2  and Thi Thuy Tien LE 3 . 1.   Department of Biotechnology, Research Institute for Oil and Oil Plants - IOOP, Ministy for Industry and Trade, 171 - 175 Ham Nghi street, Nguyen Thai Binh ward, district 1, Ho Chi Minh city, Vietnam. 2.   Department of Pharmacology and Biochemistry, Research Center of Ginseng and Medicinal Materials, National Institute of Medicinal Materials (NIMM), 41 Dinh Tien Hoang street, Ben Nghe ward, district 1, Ho Chi Minh city, Vietnam. 3.   Department of Biotechnology, Chemical Engineering Faculty, Ho Chi Minh University of Technology, Vietnam National University - Ho Chi Minh City (VNU-HCM), 268 Ly Thuong Kiet street, district 10, Ho Chi Minh city, Vietnam. ……………………………………………………………………………………………………....   Manuscript Info Abstract …………………….   ………………………………………………………………    Manuscript History Received: 14 July 2019 Final Accepted: 16 August 2019 Published: September 2019 Key words:-   Melaleuca alternifolia, plant pathogenic antifungal activity, tea tree essential oils.   The chemical composition and plant pathogenic antifungal activity of tea tree essential oils (Melaleuca alternifolia) collected in Long An, Vietnam was surveyed. By gas chromatography and mass spectrometry (GC-MS), the main chemical composition in tea tree essential oils was identified to be eucalyptol with a concentration of 31.54%. Plant antifungal activity of tea tree essential oils was evaluated with many kinds of dissolvent to determine the minimum inhibitory concentration (MIC). The results showed that tea tree essential oils had the ability to inhibit the growth of five phytopathogenic fungal strains: Aspergillus niger, Corynespora cassiicola, Colletotrichum sp., Fusarium oxysporum and Pyricularia oryzae with the minimum inhibitory concentrations (MICs) were in the range of 6  –    8 μL mL -1 . Copy Right, IJAR, 2019,. All rights reserved. ……………………………………………………………………………………………………....   Introduction:- Melaleuca alternifolia, the English name “tea tree” has long been studied in the world as a valuable source of essential oils and medicinal herbs. In 1924, this plant was officially described by Maiden and Betche Chee (Maiden and Betche , 1924). The official description of science has set the stage for research on this plant and especially on its essential oils. Tea tree essential oils has been determined more and more specific and accurate in  both ingredients and content. From 12 substances (groups) of substances discovered in 1968 (Guenther , 1968), a decade later, about 48 components in tea tree essential oils have been discovered (Swords and Hunter, 1978). With the development of gas chromatography, Brophy and their research group investigated over 800 tea tree essential oils samples and identified about 100 components present in tea tree essential oils (Brophy et al., 1989). In general, the above studies have clarified the composition and content of tea tree essential oils. Thereby, it showed that tea tree essential oils contains terpinen-4- ol (≥ 30%); ter  -terpinene (10 - 28%); α - terpinene (5 - 13%); 1.8- cineole (≤ 15%) (Brophy et al., 1989; International Organization for Standardisation, 2004), with high content, promisingly, this is an essential oils with many biological activities and high application potential. Corresponding Author:-Thanh Khang LE. Address :- Department of Biotechnology, Research Institute for Oil and Oil Plants - IOOP, Ministy for Industry and Trade, 171 - 175 Ham Nghi street, Nguyen Thai Binh ward, district 1, Ho Chi Minh city, Vietnam.  ISSN: 2320-5407 Int. J. Adv. Res. 7(9), 1239-1248  1240 Tea tree essential oils has been proved to have antifungal activity against Alternaria spp., Aspergillus   flavus, A. fumigatus, A. niger, Blastoschizomyces capitatus, Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, Cladosporium spp., Cryptococcus neoformans,   Epidermophyton floccosum, Fusarium spp., Malassezia furfur, M. sympodialis,   Microsporum canis, M. gypseum, Penicillium spp., Rhodotorula rubra, Saccharomyces   cerevisiae, Trichophyton mentagrophytes, T. rubrum, T. tonsurans, Trichosporon spp.   (Bassett et al., 1990); (Southwell et al ., 1993; Nenoff et al., 1996; Rushton et al., 1997; Christoph et al., 2000; Griffin et al., 2000; D’  Auria et   al., 2001), antivirus Nicotiana glutinosa (Schnitzler et al., 2001). The studies in Vietnam are all in the field, though they have contributed to the planting of tea tree and adapting to the conditions of Vietnam. However, this studies have not specified the essential oils components as well as the extraction methods and directions of the application of tea tree oils, so has not yet improved the oils value of these  plant in Vietnam. Controlling plant diseases caused by fungi are mainly based on chemical substances; however, to avoid pathogens forming medicine resistant strains as well as reducing environmental pollution, ecological imbalance, it is necessary to have effective control measures. Therefore, using plant extracts is considered as a new trend to be studied and applied by scientists, especially in preventing plant diseases caused by fungi by essential oils. Controlling plant diseases caused by fungi are mainly based on chemical substances; however, to avoid pathogens forming medicine resistant strains as well as reducing environmental pollution, ecological imbalance, it is necessary to have effective control measures. Therefore, using plant extracts is considered as a new trend to be studied and applied by scientists, especially in preventing plant diseases caused by fungi. Materials and methods:- Materials Branches and leaves of tea tree were collected at tea tree garden in Long An province, Vietnam. Fresh materials were  purified and washed before extracting essential oils. All fungal strains were obtained from and provided by the Department of Biotechnology, Chemical Engineering Faculty, Ho Chi Minh University of Technology, Vietnam National University - Ho Chi Minh City (VNU-HCM) and Department of Biology, College of Natural Sciences, Can Tho University (CTU). For the present study, the following strains were used: Aspergillus niger, Corynespora cassiicola, Colletotrichum sp., Fusarium oxysporum and Pyricularia oryzae. Method of extracting essential oils The materials were directly extracted by steam distillation, with the distillation set of Clevenger essential oils, NaCl was used to support the diffusion of essential oils during the attraction of essential oils following the steam. Analysis of chemical composition of essential oils The composition and concentration of the constituents in the essential oils were analyzed by gas chromatography with mass spectrometry (GC - MS), performed on the device GC - MS of THERMO SCIENTIFIC Trace GC Ultra - ISQ, column type was the TG column - SQC (15 mx 0.25 mm x 0.25 µm), the gas carried helium. Temperature  program for gas chromatograph was set as follows: Test of antifungal activities of tea tree essential oils against phytopathogenic fungi Five fungal strains were cultured on PDA (Potato Dextrose Agar): Aspergillus niger, Corynespora cassiicola, Colletotrichum sp., Fusarium oxysporum and Pyricularia oryzae. Tea tree essential oils was diluted with Tween 80 for the concentrations of 2, 4, 6, 8 and 10 μL mL -1 . The diameter of colonies were notes after six days of incubation.  ISSN: 2320-5407 Int. J. Adv. Res. 7(9), 1239-1248  1241 The inhibitory ability (performance) of essential oils with the growth of fungi (%) was according to the following equation: H (%)   R r 100   R   R: Fungus radius developed in the control (cm). r: Fungus radius in essential oils (cm). In this study, we used carbendazim (97%) (Sigma-Aldrich, USA) (an antifungal drug) as posity control. Evaluating the morphology of mycelium  To evaluate the microstructural geometry of the mycelium, morphological characterization was carried out using a SEM scanning electron microscope (SEM). test of activity against phytopathogenic fungi in combination with carbendazim Carbendazim was mixed with tea tree essential oils at different ratios (Table 1) to test the antifungal activity. Table 1:- Ratio of tea tree oils concentration: carbendazim  Tea tree essential oils (ml) 80 160 240 320 Carbendazim (mg) 320 240 160 80 Ratio of tea tree oils concentration: carbendazim 1:4 2:3 3:2 4:1 Each experiment was conducted in triplicate. Results:-   Chemical composition of essential oils Table 2:- Main chemical composition of tea tree essential oils in Vietnam No.Retention timeIngredientsContent (%)1 2.352  –   pentanone, 4  –   hydroxyl  –   4  –   methyl -6.67 2 3.57 α - pinene2.71 3 4.54 β - pinene2 4 5.06 β - myrcene3.68 5 6.43Eucalyptol31.54 6 7.711, 4  –   cyclohexadiene, 1 - methyl  –   4  –   (1  –   methylethyl) -8.23 7 8.62Cyclohexene, 1 - methyl  –   4  –   (1  –   methylethylidene) -1.73 8 13.833  –   cyclohexen  –   1  –   ol, 4  –   methyl  –   1  –   (1  –   methylethyl) -22.47 9 14.493  –   cyclohexen  –   1  –    methanol, α, α, 4 –   trimethyl - , (S) -12.16 10 20.021H  –   cycloprop[e]azulene, 1a, 2, 3, 5, 6, 7, 7a, 7b  –   octahydro  –   1, 1, 4, 7  –   tetramethyl -, [1aR  –    (1aα, 7α, 7aβ, 7bα)] -1.61 11 20.4Napthalene, 1, 2, 3, 5, 6, 8a  –   hexahydro  –   4, 7  –   dimethyl  –   1  –   (1  –   methylethyl) -, (S  –   cis) -1.13 12 21.22Globulol1.86 13 21.341H  –   cycloprop[e]azulene  –   4  –   ol, decahydro - 1, 1, 4, 7  –   tetramethyl -, [1ar  –    (1aα, 4β, 4aβ, 7α, 7aβ, 7bα)] -1.21 Analysis results of GC - MS showed that there were 13 components in tea tree essential oils of Vietnam, belonging to seven groups of compounds which were groups etyl butanoat such as 2  –   pentanone, 4  –   hydroxyl  –    4  –   methyl -, pinene such as α  - pinene and β    –   pinene, β    –   myrcene, terpinene such as 1, 4  –    cyclohexadiene, 1 - methyl  –   4  –   (1  –   methylethyl) -, cyclohexene, 1 - methyl  –   4  –   (1  –    methylethylidene) -, eucalyptol, terpineol such as 3  –   cyclohexen  –   1  –   ol, 4  –   methyl  –   1  –   (1-methylethyl)  –   and 3  –   cyclohexen  –   1  –   methanol, α,   α,  4  –   trimethyl - , (S) -, aristolochene   such as 1H  –   cycloprop[e]azulene, 1a, 2, 3, 5, 6, 7, 7a, 7b  –   octahydro  –   1, 1, 4, 7  –   tetramethyl -, [1aR  –    (1aα,   7α,   7aβ,   7bα)]    –   and napthalene, 1, 2, 3, 5, 6, 8a  –   hexahydro  –   4, 7  –   dimethyl-1  –   (1  –   methylethyl) -, (S  –   cis)  –   and compounds ledol such as globulol and 1H  –    cycloprop[e]azulene  –   4  –   ol, decahydro - 1, 1, 4, 7  –   tetramethyl -, [1ar  –    (1aα,   4β,   4aβ,   7α,   7aβ,   7bα)]  -. The total content of all constituents accounts for more than 90% of the extracted oils. The constituents with the highest content in tea tree essential oils were eucalyptol and terpineol compounds, so tea tree essential oils had a fairly high boiling point, density and extraction, in accordance with other studies on tea tree essential oils that have been announced. These were eucalyptol and terpineol  ISSN: 2320-5407 Int. J. Adv. Res. 7(9), 1239-1248  1242 compounds that created biological activities to tea tree essential oils such as antimicrobial, antifungal, antioxidant and cytotoxic activities. (Roy and Vijayalaxmi, 2013). Eucalyptol is a natural organic compound, is a colorless liquid at room temperature. It has a ring ether and is a monotecenoid. Terpineol is a monoterpene that has been isolated from a variety of sources such as tea tree oils, pine oils and petitgrain oils. There are four isomers , α  -, β -, ᵞ  - terpineol and terpinen  –   4 - ol. β - and   ᵞ  - terpineol only different by the position of the double link. Terpineol usually a mixture of these isomers with α  - terpineol is the main ingredient. Antifungal activity against phytopathogenesis fungi With an initial concentration of 10 μL mL -1 , the tea tree essential oils was diluted with lower concentration scales, thereby determining the MIC value. Test results were presented in Table 3 and Figure 1. Table 3:- Antifungal performance of tea tree essential oils   The letters that follow the values in the same row are statistically significant at 5%. (-) is not antifungal. Aspergillus niger Corynespora cassiicola Colletotrichum sp. Fusarium oxysporum Pyricularia oryzae   I.   T ESTED FUNGI SPECIE   Active test sample Antifungal performance (%) 0 μL mL -1   2 μL mL -   4 μL mL -   6 μL  mL -   8 μL mL -   10 μL mL -1   Aspergillus niger Tea tree essential oils - 44.3 e  ± 1.24 68.6 ± 1.27 76.0 c  ± 1.78 85.4 ± 0.77 100 a  ± 0 Carbendazim - 45.5 e  ± 0.1 72.8 d  ± 1.75 79.1 c  ± 1.83 94  b  ± 0.66 100 a  ± 0 Corynespora cassiicola Tea tree essential oils - 43.8 ± 0.83 70.4 c  ± 0.82 91.4 ± 0.92 100 a  ± 0 100 a  ± 0 Carbendazim - 77.8 d  ± 1.08 82.4 c  ± 0.29 98.5  b  ± 0.37 100 a  ± 0 100 a  ± 0 Colletotrichum sp. Tea tree essential oils - 55.3 c  ± 1.02 76.7  b  ± 0.78 100 a  ± 0 100 a  ± 0 100 a  ± 0 Carbendazim - 61.1 c  ± 4.6 83.5 ± 0.11 100 a  ± 0 100 a  ± 0 100 a  ± 0 Fusarium oxysporum Tea tree essential oils - 15.0 e  ± 1.78 37.6 d  ± 1.91 52.7 c  ± 2.43 68.5  b  ± 11.83 100 a  ± 0 Carbendazim - 39.2 e  ± 4.74 70.7 ± 1.4 74.4 c  ± 2.7 90.6 ± 11.35 100 a  ± 0 Pyricularia oryzae Tea tree essential oils - 14.5 c  ± 2.32 41.5 ± 1.81 100 a  ± 0 100 a  ± 0 100 a  ± 0 Carbendazim - 20.7 c  ± 1.59 51.4  b  ± 1.04 100 a  ± 0 100 a  ± 0 100 a  ± 0   10 mm 10 mm 10 mm 10 mm 10 mm 0 µl/ml  ISSN: 2320-5407 Int. J. Adv. Res. 7(9), 1239-1248  1243 Figure 1:- Diameter of colonies at different concentrations of tea tree essential oils The results showed that the antifungal activity of tea tree essential oils was depended on concentration. Tea tree essential oils inhibited mycelial growth of fungi strains: Aspergillus niger, Corynespora cassiicola, Colletotrichum sp., Fusarium oxysporum and Pyricularia oryzae. In all the test fungi strains, complete mycelial growth inhibition was observed at a tea tree essential oils concentration of 10 μL mL -1  (Figure 1). Tea tree essential oils had a significantly (    ≤ 0.05) higher inhibitory effect on Colletotrichum sp. and Pyricularia oryzae than on Corynespora cassiicola, Aspergillus niger and Fusarium oxysporum at a concentration of 6 μL mL -1 after six days of incubation. The range of mycelial growth inhibition was between 14.5 % and 100%. At a concentration of 6 μL mL -1 , the tea tree essential oils completely inhibited the mycelial growth of Colletotrichum sp. and Pyricularia oryzae. However, complete inhibition of Corynespora cassiicola was observed at a concentration of 8 μL mL -1 ; Aspergillus niger and Fusarium oxysporum was observed at a concentration of 10 μL mL -1  (Table 3). The highest and lowest rates of mycelial growth inhibition by the tea tree essential oils at a concentration of 2 μL mL -1 were observed in Colletotrichum sp. (55.31 %) and Pyricularia oryzae (14.52 %), respectively. The inhibition of mycelial growth in the five test fungi strains at tea tree essential oils concentration of 2 µl/ml   was significantly (      10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 10 mm 2 µl/ml 4 µl/ml 6 µl/ml 8 µl/ml 10 µl/ml
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