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Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum
SAJB-01861; No of Pages 4 South African Journal of Botany xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum M. Ferdes a, F. Al Juhaimi b, M.M. Özcan c,⁎, K. Ghafoor b a b c
Faculty of Biotechnical Systems Engineering, Department of Biotechnical Systems, “Politehnica” University of Bucharest, Romania Department of Food Science & Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia Department of Food Engineering, Faculty of Agriculture, Selçuk University, 42079 Konya, Turkey
a r t i c l e
i n f o
Article history: Received 12 February 2017 Received in revised form 25 August 2017 Accepted 25 September 2017 Available online xxxx Edited by M Gryzenhout Keywords: Essential oil Inhibitory effect Aspergillus niger Aspergillus oryza Mucor pusillus and Fusarium oxysporum
a b s t r a c t The antifungal effects of sage, rosemary, anise, quinoa and savory essential oils against mycelium growth of Aspergillus niger, Aspergillus oryza, Mucor pusillus and Fusarium oxysporum were determined in in vitro conditions. While the 10 μg/ml of savory essential oil showed inhibitory effect against tested fungi, anise oil was ineffective at the same concentration. The essential oils of savory and sage showed a great inhibitor effects against M. pusillus when compared with other essential oils. The 20 μg/ml level of savory oil had effective on Aspergillus niger, Aspergillus oryza, Mucor pusillus and Fusarium oxysporum. The essential oils of sage and rosemary were effective on A. oryza, A. niger and Fusarium oxysporum at ligh concentrations (10 and 20 μg/ml). The essential oil of quinoa showed an antifungal effect at both concentrations (10 and 20 μg/ml) on all tested fungi. All the tested essential oils showed inhibitory effect against all the selected fungi. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction
2. Material and methods
In recent years, essential oils exhibited antimicrobial effect because of biological activities against several microorganisms (Carmo et al., 2008; Cosic et al., 2010; Istaianto and Emilda, 2011; Vitoratos et al., 2013; Mahilrajan et al., 2014). The essential oils of several aromatic plants exhibited fungicidal toxicity against soil borne phytopathogenic fungi in vitro (Tripathi et al., 2008). Many plant essential oil and their derivatives demonstrated antifungal activities against Aspergillus, Penicilium and Fusarium species (Carmo et al., 2008; Kim and Park, 2012; Ajayi and Okoh, 2013). Aspergillus and Fusarium species produce mycotoxins in food besides causing seedling blight, seed roxt, kernel roxt, stalk rot, wilt and stunt (Fandohan et al., 2003; Sitara et al., 2008). The objective of this study was to determine the potential inhibitory of some plant (sage (Salvia fruticosa), rosemary (Rosmarinus officinalis), anise (Pimpinella anisum), savory (Satureja hortensis) and quinoa (Quinoa)) essential oils against Mucor pusillus, Aspergillus niger, Aspergillus oryzae, Fusarium oxysporum fungi.
2.1. Material 2.1.1. Pathogen Mucor pusillus ATCC 16458, Aspergillus niger ATCC 15475, Aspergillus oryzae ATCC 20423, Fusarium oxysporum MUCL 791 were provided from microbiological herbarium of Faculty of Biotechnical Systems Engineering, Department of Biotechnical Systems“Politehnica” University of Bucharest, Romania. The fungi cultures were prepared on new PDA plate by subculture of mycelia, and incubated at 23 °C for five days. 2.1.2. Distillation of essential oils Essential oils of sage (Salvia fruticosa), rosemary (Rosmarinus officinalis), anise (Pimpinella anisum), savory (Satureja hortensis) and quinoa (Quinoa) were obtained by hydro-distillation in a Clevengertype apparatus. The essential oils were kept in sealed glass bottles at −18 °C. 2.2. Method
⁎ Corresponding author. E-mail address: [email protected] (M.M. Özcan).
2.2.1. Measurement of antifungal activity The antifungal activity was determined by disc diffusion method. In brief, each oil was diluted with tween 80 to obtain the final
https://doi.org/10.1016/j.sajb.2017.09.020 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020
2
M. Ferdes et al. / South African Journal of Botany xxx (2017) xxx–xxx
concentrations of 10 and 20 μg/ml. The fungi incubated at 30 °C for 3 days, and the fungal growth was monitored. The growth inhibition of fungal strain was calculated as the percentage of inhibition against the control group (Özcan, 1998).
Table 1 Inhibitory effect of some essential oils against the fungal strains after the 7-day incubation period (colony diameter: mm). Fungal strain
Inhibition ratio (%)
Essential oil concentration (μg/ml)
Colony diameter (mm)
Growth rate (mm/h)
Control Sage - 10 Sage - 20 Rosemary - 10 Rosemary - 20 Anise - 10 Anise - 20 Minth - 10 Minth - 20 Quinoa - 10 Quinoa - 20 Savory - 10 Savory - 20
54 ± 5.4a⁎ 24 ± 3.2b⁎⁎ –⁎⁎⁎ 14 ± 3.7b – – – 4 ± 1.3b – 53 ± 3.7b 46 ± 5.1c – –
0.32 ± 0.07a 0.14 ± 0.03b – 0.08 ± 0.01a – – – 0.02 ± 0.01b – 0.31 ± 0.03b 0.27 ± 0.03c – –
55 100 74 100 100 100 92 100 2 15 100 100
Control Sage - 10 Sage – 20 Rosemary - 10 Rosemary - 20 Anise - 10 Anise - 20 Minth – 10 Minth - 20 Quinoa – 10 Quinoa - 20 Savory – 10 Savory - 20
55 ± 4.2a 35 ± 1.6b 26 ± 1.3c 11 ± 0.35b – 6 ± 0.58b – 3 ± 0.71b – 52 ± 5.63b 52 ± 2.57b – –
0.32 ± 0.09a 0.21 ± 0.03b 0.15 ± 0.03c 0.06 ± 0.01b – 0.03 ± 0.01b – 0.02 ± 0.01b – 0.31 ± 0.05b 0.31 ± 0.07b – –
36 52 80 100 89 100 95 100 5 5 100 100
Control Sage - 10 Sage - 20 Rosemary - 10 Rosemary - 20 Anise -10 Anise - 20 Minth - 10 Minth - 20 Quinoa - 10 Quinoa - 20 Savory - 10 Savory - 20
44 ± 3.5a 19 ± 1.7b 10 ± 1.1c 20 ± 2.3b 3 ± 0.55c – – 9 ± 1.3b – 42 ± 3.7b 40 ± 2.9c – –
0.26 ± 0.05a 0.11 ± 0.03b 0.06 ± 0.01c 0.12 ± 0.03b 0.02 ± 0.01c – – 0.05 ± 0.01b – 0.25 ± 0.07b 0.24 ± 0.03c – –
57 77 54 93 100 100 79 100 5 9 100 100
Control Sage - 10 Sage - 20 Rosemary - 10 Rosemary - 20 Anise - 10 Anise - 20 Minth - 10 Minth - 20 Quinoa - 10 Quinoa - 20 Savory - 10 Savory - 20
42 ± 4.17a 30 ± 5.32b 22 ± 3.28c 22 ± 2.16b 3 ± 0.54c 10 ± 1.13b – 10 ± 2.08b – 35 ± 2.65b 35 ± 1.72b – –
0.25 ± 0.07a 0.18 ± 0.03b 0.13 ± 0.01c 0.13 ± 0.03b 0.02 ± 0.01c 0.06 ± 0.02b – 0.06 ± 0.01b – 0.21 ± 0.03b 0.21 ± 0.05b – –
29 48 48 93 76 100 76 100 17 17 100 100
Mucor pusillus ATCC 16458
Aspergillus niger ATCC 15475
2.3. Statistical analyses A complete randomized split plot block design was used, and analysis of variance (ANOVA) was performed by using JMP version 9.0 (SAS Inst. Inc., Cary, N.C.U.S.A). All analyses were carried out three times and the results are mean ± standard deviation (MSTAT C) of independent essential oil samples (Püskülcü and İkiz, 1989). 3. Results and discussion The antifungal effect of sage, rosemary, anise, quinoa and savory essential oils are given in Table 1. The concentration of 10 μg/ml savory essential oil showed inhibitory effect against fungi tested. But, anise oil was not effective at the same concentration. The oils of savory and sage have a great inhibitory effect against M. pusillus when compared with results of other essential oils. The 20 μg/ml concentration of savory essential oil was effective on the mycelium growth of Aspergillus niger, Aspergillus oryza, Mucor pusillus and Fusarium oxysporum (Figs. 1–4). All doses of anise oil were completely effective on all fungi. Both concentrations (10 and 20 μg/ml) of esential oils of sage and rosemary were effective on A. oryza, A. niger and Fusorium oxysporum (p b 0.05). The 10 μg/ml concentration of essential oil of rosemary showed inhibitory effect on A. niger and Fusarium oxysporum, and on other fungi species at high concentrations (20 μg/ml) (Figs. 2 and 4), while both concentrations (10 and 20 μg/ml) of the essential oil of quinoa shows antifungal effect on growth of all fungi tested. It was observed significantly differences between the inhibitor effects of essential oils in in vitro (p b 0.05). All low concentration (10 μg/ml) of essential oils showed weak effective against all fungi (except for savory and anise). Savory essential oil
Aspergillus oryzae ATCC 20423
Fusarium oxysporum MUCL 791
⁎ Mean ± standard deviation (n:3). ⁎⁎ Values within each column followed by different letters are significantly different (p b 0.05). ⁎⁎⁎ Non growth.
Fig. 1. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Mucor pusillus at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020
M. Ferdes et al. / South African Journal of Botany xxx (2017) xxx–xxx
3
Fig. 2. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Aspergillus niger at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
Fig. 3. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Aspergillus oryzae at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
at low concentration was sufficient to inhibit to the fungi growth. In addition, 20 μg/ml concentrations of sage, rosemary and quinoa essential oils were affective partly. All the oils exhibited antifungal activity at different degrees against A. niger, A. oryzae, F. oxysporium and Mucor pusillus (Figs. 1–4). Growth rate of Aspergillus niger ATCC 15475 at the 10 and 20 μg/ml levels of quinoa did not show significantly differences. In addition, growth rate of Aspergillus niger ATCC 15475 at the 10 μg/ml levels of rosemary, anise and minth essential oils was not found statistically important. In the current study, all essential oils exhibited antifungal effect. The results showed that five plant essential oils such as anise, savory and sage oils caused 29–100% inhibition on all fungi species at 10–20 μg/ml concentrations. But, quinoa and anise oils did not exhibit inhibition effect at the same concentrations (10–20 μg/ml). Savory oil had the most effective on the mycelium growth of A. niger, A. oryzae, F. oxysporum and Mucor pusillus with growth inhibition average of 100%, respectively. Whereas the sage oil showed minor effect on A. niger and F. oxysporium with growth inhibition average of 36–77% depending on concentrations (p b 0.05). Antifungal effect of essential oils can be probably due to the phenolic components of essential oils (Voda et al., 2003; Stevi et al., 2014). Thyme essential oil exhibited strong antimicrobial activity on the mycelial growth of D. caulivora, D. helianthi, P. viticola (Cosic et al., 2010). The results of savory are in agreement with that reported by Rasooli and Abyaneh (2004). Sukatta et al. (2008) reported that mixing of clove and cinnamon oils exhibited inhibitory effect against the postharvest decay fungi of grapes Aspergillus niger. C. citratus oil at a concentration of 1.5 μg/ml completely suppressed growth of A. flavus and A. niger (Helal et al., 2006). It is considered to be a relationship between the chemical structure of the most abundant compounds in the essential oil and the antimicrobial activity (Stupar et al., 2014). The essential oil
isolated from oregano showed antifungal activity against mycelium growth of Aspergillus, Fusarium and Penicillium (Stupar et al., 2014; Ibrahim and Al-Ebady, 2014). Paster et al. (1995) demonstrated antifungal activity of oregano essential oil on the mycelial and spores of A. niger, A. flavus and A. ochraceus. The high sensibility of filamentous fungi was observed by other authors with other essential oils (Cavaleiro et al., 2006; Tullio et al., 2007; Tavassoli et al., 2011). The result showed that the essential oils were able to suppress Fusarium oxysporum mycelial growth. Essential oil extracted from Eugenia aromatica provided the strongest suppression of F. oxysporum mycelial growth (Istaianto and Emilda, 2011). In previous study, Maskuki and Mortazavi (2004) studied on inhibitory effect of essential oils of thyme and ajowan to inhibit fungal growth of A. parasiticus on pear. Eucalyptus oil exhibited maximum antifungal activity against A. oryzae, followed by A. niger and F. solani then by A. flavus, F. moniliforme, A. terreus and A. fumigatus (Shirurkar and Wahegaonkar, 2012).
4. Conclusion In accordance with the earlier reports, the results of the present work suggested that some essential oils can be applied as mold inhibitors to prevent growth of toxigenic fungi. The results suggest the potential use of some essential oils as antimicrobial preservatives in food. Further studies on the combined effects of many local plant essential oils and components in food products are in progress in our model systems. The selected essential oils showed good inhibitory effect against all the tested organisms that are known food pathogens. Sage and rosemary essential oils were found to more potent against A. niger and F. oxysporum.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020
4
M. Ferdes et al. / South African Journal of Botany xxx (2017) xxx–xxx
Fig. 4. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Fusarium oxysporum at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
Acknowledgements The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding the Research group NO (RG-1435-049). References Ajayi, A.O., Okoh, A.I., 2013. Antimycotic activity of some medicinal plants on aquatic fungi. European Journal of Experimental Biology 3, 16–21. Carmo, E.S., Lima, E.O., Souza, E.L., 2008. The potential of Origanum vulgare L. essential oil in inhibiting the growth of some food-related Aspergillus species. Brazilian Journal of Microbiology 39, 362–367. Cavaleiro, C., Pinto, E., Goncalves, M.J., Salgueiro, L., 2006. Antifungal activity of Juniperus essential oils against dermatophyte, Aspergillus and Candida strains. Journal of Applied Microbiology 100, 1333–1338.
Cosic, J., Vrandecic, K., Postic, J., Jurkovic, D., Ravric, M., 2010. In vitro antifungal activity of essential oils on growth of phytopathogenic fungi. Poljoprivreda 16, 25–28. Fandohan, O., Hell, K., Marasas, W.F.O., Wingfield, M.J., 2003. Infection of maize by Fusarium species and contamination on with fumanisis in Africa. African Journal of Biotechnology 2, 570–579. Helal, G.A., Sarhan, M.M., Abu Shahla, A.N.K., Abou El-Khair, E.K., 2006. Antimicrobial activity of some essential oils against microorganisms deteriorating fruit juices. Microbiology 34, 219–229. Ibrahim, F.A.A., Al-Ebady, N., 2014. Evaluation of antifungal activity of some plant extracts andtheir applicability in extending the shelf life of stored tomato fruits. Food Processing and Technology 5, 340. Istaianto, M., Emilda, D., 2011. Preliminary study of the activity of some essential oils against. Journal of Fruit and Ornamental Plant Research 19, 111–121. Kim, E., Park, I.I.-K., 2012. Fumigant antifungal activity of myrtaceae essential oils and constituents from lepto Spermum petersonii against three Aspergillus species. Molecules 17, 10459–10469. Mahilrajan, S., Nandakumar, J., Kailayalingam, R., Monoharan, N.A., Srivijeindran, S., 2014. Screening the antifungal activity of essential oils against decay fungi from palmyrah leaf handicrafts. Biological Research 47, 35. Maskuki, A., Mortazavi, S.A., 2004. Antifungal activity of essential oils of Thymus vulgaris and Trachyspermum ammi against Aspergilus. Journal of Science and Technology of Agriculture and Natural Resources 8, 207–215. Özcan, M., 1998. Inhibitory effects of spice extracts on the growth of Aspergillus parasiticus NRRL 2999 strain. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 207, 253–255. Paster, V., Menashero, M., Ravid, U., Juven, B., 1995. Antifungal activity of oregano and thyme essential oils applied as fumigants against fungi attacking store grain. Journal of Food Protection 58, 81–85. Püskülcü, H., İkiz, F., 1989. Introduction to Statistic. Bilgehan Press, Bornova. İzmir, Turkey, p. p333 (in Turkish). Rasooli, I., Abyaneh, M.R., 2004. Inhibitory effects of thyme oils on growth and aflatoxin production by Aspergillus parasiticus. Food Control 15, 79–483. Shirurkar, D.D., Wahegaonkar, N.K., 2012. Antifungal activity of selected plant derived oils and some fungicides against seed borne fungi of maize. European Journal of Experimental Biology 2, 1693–1696. Sitara, U., Niaz, I., Naseem, J., Sultana, N., 2008. Antifungal effect of essential oils on in vitro growth of pathogenic fungi. Pakistan Journal of Botany 40, 409–414. Stevi, A., Beri, C., Katarina, S., Marina, S., Dejan, G., 2014. Antifungal activity of selected essential oils against fungi isolated from medicinal plant. Industrial Crop Production 55, 116–122. Stupar, M., Grbic, M.L., Dzamic, A., Unkovic, N., Ristic, M., 2014. Antifungal activity of selected essential oils and biocide benzalkonium chloride against the fungi isolated from cultural heritage objects. South African Journal of Botany 93, 118–124. Sukatta, U., Haruthaithanasan, V., Chantarapanont, W., Dilokkunanant, U., Suppakul, P., 2008. Antifungal activity of clove and cinnamon oil and their synergistic against postharvest decay fungi of grape in vitro. Kasetsart Journal (Natural Science) 42, 169–174. Tavassoli, S., Mousavi, S.M., Emam-Djomeh, Z., Razavi, S.H., 2011. Comparative study of the antimicrobial activity of Rosmarinus officinalis L. essential oil and methanolic extract. Middle-East Journal of Scientific Research 9, 467–471. Tripathi, P., Dubey, N.K., Shukla, A.K., 2008. Use of some essential oils as postharvest botanical fungicides in the management of grey mould of grapes caused by Botrytis cinerea. World Journal of Microbiology and Biotechnology 24, 39–46. Tullio, V., Nostro, A., Mandras, N., Dugo, P., Banche, G., Cannatelli, M.A., Cuffini, A.M., Alonzo, V., Carlone, N.A., 2007. Antifungal activity of essential oils against filamentous fungi determined by broth microdilution and vapour contact methods. Journal of Applied Microbiology 102, 1544–1550. Vitoratos, A., Bilalis, D., Karkanis, A., Efthimiadou, A., 2013. Antifungal activity of plant essential oils against Botrytis cinerea, Penicillium italicum and Penicillium digitatum. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 41, 86–92. Voda, K., Boh, B., Vrtacnik, M., Pohleven, F., 2003. Effect of the antifungal activity of oxygenated aromatic essential oil compounds on the white-rot Trametes versicolor and the Brown-not Coniophora puteana. International Biodeterioration and Biodegradation 51, 51–59.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020
Contents lists available at ScienceDirect
South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum M. Ferdes a, F. Al Juhaimi b, M.M. Özcan c,⁎, K. Ghafoor b a b c
Faculty of Biotechnical Systems Engineering, Department of Biotechnical Systems, “Politehnica” University of Bucharest, Romania Department of Food Science & Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia Department of Food Engineering, Faculty of Agriculture, Selçuk University, 42079 Konya, Turkey
a r t i c l e
i n f o
Article history: Received 12 February 2017 Received in revised form 25 August 2017 Accepted 25 September 2017 Available online xxxx Edited by M Gryzenhout Keywords: Essential oil Inhibitory effect Aspergillus niger Aspergillus oryza Mucor pusillus and Fusarium oxysporum
a b s t r a c t The antifungal effects of sage, rosemary, anise, quinoa and savory essential oils against mycelium growth of Aspergillus niger, Aspergillus oryza, Mucor pusillus and Fusarium oxysporum were determined in in vitro conditions. While the 10 μg/ml of savory essential oil showed inhibitory effect against tested fungi, anise oil was ineffective at the same concentration. The essential oils of savory and sage showed a great inhibitor effects against M. pusillus when compared with other essential oils. The 20 μg/ml level of savory oil had effective on Aspergillus niger, Aspergillus oryza, Mucor pusillus and Fusarium oxysporum. The essential oils of sage and rosemary were effective on A. oryza, A. niger and Fusarium oxysporum at ligh concentrations (10 and 20 μg/ml). The essential oil of quinoa showed an antifungal effect at both concentrations (10 and 20 μg/ml) on all tested fungi. All the tested essential oils showed inhibitory effect against all the selected fungi. © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction
2. Material and methods
In recent years, essential oils exhibited antimicrobial effect because of biological activities against several microorganisms (Carmo et al., 2008; Cosic et al., 2010; Istaianto and Emilda, 2011; Vitoratos et al., 2013; Mahilrajan et al., 2014). The essential oils of several aromatic plants exhibited fungicidal toxicity against soil borne phytopathogenic fungi in vitro (Tripathi et al., 2008). Many plant essential oil and their derivatives demonstrated antifungal activities against Aspergillus, Penicilium and Fusarium species (Carmo et al., 2008; Kim and Park, 2012; Ajayi and Okoh, 2013). Aspergillus and Fusarium species produce mycotoxins in food besides causing seedling blight, seed roxt, kernel roxt, stalk rot, wilt and stunt (Fandohan et al., 2003; Sitara et al., 2008). The objective of this study was to determine the potential inhibitory of some plant (sage (Salvia fruticosa), rosemary (Rosmarinus officinalis), anise (Pimpinella anisum), savory (Satureja hortensis) and quinoa (Quinoa)) essential oils against Mucor pusillus, Aspergillus niger, Aspergillus oryzae, Fusarium oxysporum fungi.
2.1. Material 2.1.1. Pathogen Mucor pusillus ATCC 16458, Aspergillus niger ATCC 15475, Aspergillus oryzae ATCC 20423, Fusarium oxysporum MUCL 791 were provided from microbiological herbarium of Faculty of Biotechnical Systems Engineering, Department of Biotechnical Systems“Politehnica” University of Bucharest, Romania. The fungi cultures were prepared on new PDA plate by subculture of mycelia, and incubated at 23 °C for five days. 2.1.2. Distillation of essential oils Essential oils of sage (Salvia fruticosa), rosemary (Rosmarinus officinalis), anise (Pimpinella anisum), savory (Satureja hortensis) and quinoa (Quinoa) were obtained by hydro-distillation in a Clevengertype apparatus. The essential oils were kept in sealed glass bottles at −18 °C. 2.2. Method
⁎ Corresponding author. E-mail address: [email protected] (M.M. Özcan).
2.2.1. Measurement of antifungal activity The antifungal activity was determined by disc diffusion method. In brief, each oil was diluted with tween 80 to obtain the final
https://doi.org/10.1016/j.sajb.2017.09.020 0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020
2
M. Ferdes et al. / South African Journal of Botany xxx (2017) xxx–xxx
concentrations of 10 and 20 μg/ml. The fungi incubated at 30 °C for 3 days, and the fungal growth was monitored. The growth inhibition of fungal strain was calculated as the percentage of inhibition against the control group (Özcan, 1998).
Table 1 Inhibitory effect of some essential oils against the fungal strains after the 7-day incubation period (colony diameter: mm). Fungal strain
Inhibition ratio (%)
Essential oil concentration (μg/ml)
Colony diameter (mm)
Growth rate (mm/h)
Control Sage - 10 Sage - 20 Rosemary - 10 Rosemary - 20 Anise - 10 Anise - 20 Minth - 10 Minth - 20 Quinoa - 10 Quinoa - 20 Savory - 10 Savory - 20
54 ± 5.4a⁎ 24 ± 3.2b⁎⁎ –⁎⁎⁎ 14 ± 3.7b – – – 4 ± 1.3b – 53 ± 3.7b 46 ± 5.1c – –
0.32 ± 0.07a 0.14 ± 0.03b – 0.08 ± 0.01a – – – 0.02 ± 0.01b – 0.31 ± 0.03b 0.27 ± 0.03c – –
55 100 74 100 100 100 92 100 2 15 100 100
Control Sage - 10 Sage – 20 Rosemary - 10 Rosemary - 20 Anise - 10 Anise - 20 Minth – 10 Minth - 20 Quinoa – 10 Quinoa - 20 Savory – 10 Savory - 20
55 ± 4.2a 35 ± 1.6b 26 ± 1.3c 11 ± 0.35b – 6 ± 0.58b – 3 ± 0.71b – 52 ± 5.63b 52 ± 2.57b – –
0.32 ± 0.09a 0.21 ± 0.03b 0.15 ± 0.03c 0.06 ± 0.01b – 0.03 ± 0.01b – 0.02 ± 0.01b – 0.31 ± 0.05b 0.31 ± 0.07b – –
36 52 80 100 89 100 95 100 5 5 100 100
Control Sage - 10 Sage - 20 Rosemary - 10 Rosemary - 20 Anise -10 Anise - 20 Minth - 10 Minth - 20 Quinoa - 10 Quinoa - 20 Savory - 10 Savory - 20
44 ± 3.5a 19 ± 1.7b 10 ± 1.1c 20 ± 2.3b 3 ± 0.55c – – 9 ± 1.3b – 42 ± 3.7b 40 ± 2.9c – –
0.26 ± 0.05a 0.11 ± 0.03b 0.06 ± 0.01c 0.12 ± 0.03b 0.02 ± 0.01c – – 0.05 ± 0.01b – 0.25 ± 0.07b 0.24 ± 0.03c – –
57 77 54 93 100 100 79 100 5 9 100 100
Control Sage - 10 Sage - 20 Rosemary - 10 Rosemary - 20 Anise - 10 Anise - 20 Minth - 10 Minth - 20 Quinoa - 10 Quinoa - 20 Savory - 10 Savory - 20
42 ± 4.17a 30 ± 5.32b 22 ± 3.28c 22 ± 2.16b 3 ± 0.54c 10 ± 1.13b – 10 ± 2.08b – 35 ± 2.65b 35 ± 1.72b – –
0.25 ± 0.07a 0.18 ± 0.03b 0.13 ± 0.01c 0.13 ± 0.03b 0.02 ± 0.01c 0.06 ± 0.02b – 0.06 ± 0.01b – 0.21 ± 0.03b 0.21 ± 0.05b – –
29 48 48 93 76 100 76 100 17 17 100 100
Mucor pusillus ATCC 16458
Aspergillus niger ATCC 15475
2.3. Statistical analyses A complete randomized split plot block design was used, and analysis of variance (ANOVA) was performed by using JMP version 9.0 (SAS Inst. Inc., Cary, N.C.U.S.A). All analyses were carried out three times and the results are mean ± standard deviation (MSTAT C) of independent essential oil samples (Püskülcü and İkiz, 1989). 3. Results and discussion The antifungal effect of sage, rosemary, anise, quinoa and savory essential oils are given in Table 1. The concentration of 10 μg/ml savory essential oil showed inhibitory effect against fungi tested. But, anise oil was not effective at the same concentration. The oils of savory and sage have a great inhibitory effect against M. pusillus when compared with results of other essential oils. The 20 μg/ml concentration of savory essential oil was effective on the mycelium growth of Aspergillus niger, Aspergillus oryza, Mucor pusillus and Fusarium oxysporum (Figs. 1–4). All doses of anise oil were completely effective on all fungi. Both concentrations (10 and 20 μg/ml) of esential oils of sage and rosemary were effective on A. oryza, A. niger and Fusorium oxysporum (p b 0.05). The 10 μg/ml concentration of essential oil of rosemary showed inhibitory effect on A. niger and Fusarium oxysporum, and on other fungi species at high concentrations (20 μg/ml) (Figs. 2 and 4), while both concentrations (10 and 20 μg/ml) of the essential oil of quinoa shows antifungal effect on growth of all fungi tested. It was observed significantly differences between the inhibitor effects of essential oils in in vitro (p b 0.05). All low concentration (10 μg/ml) of essential oils showed weak effective against all fungi (except for savory and anise). Savory essential oil
Aspergillus oryzae ATCC 20423
Fusarium oxysporum MUCL 791
⁎ Mean ± standard deviation (n:3). ⁎⁎ Values within each column followed by different letters are significantly different (p b 0.05). ⁎⁎⁎ Non growth.
Fig. 1. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Mucor pusillus at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020
M. Ferdes et al. / South African Journal of Botany xxx (2017) xxx–xxx
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Fig. 2. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Aspergillus niger at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
Fig. 3. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Aspergillus oryzae at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
at low concentration was sufficient to inhibit to the fungi growth. In addition, 20 μg/ml concentrations of sage, rosemary and quinoa essential oils were affective partly. All the oils exhibited antifungal activity at different degrees against A. niger, A. oryzae, F. oxysporium and Mucor pusillus (Figs. 1–4). Growth rate of Aspergillus niger ATCC 15475 at the 10 and 20 μg/ml levels of quinoa did not show significantly differences. In addition, growth rate of Aspergillus niger ATCC 15475 at the 10 μg/ml levels of rosemary, anise and minth essential oils was not found statistically important. In the current study, all essential oils exhibited antifungal effect. The results showed that five plant essential oils such as anise, savory and sage oils caused 29–100% inhibition on all fungi species at 10–20 μg/ml concentrations. But, quinoa and anise oils did not exhibit inhibition effect at the same concentrations (10–20 μg/ml). Savory oil had the most effective on the mycelium growth of A. niger, A. oryzae, F. oxysporum and Mucor pusillus with growth inhibition average of 100%, respectively. Whereas the sage oil showed minor effect on A. niger and F. oxysporium with growth inhibition average of 36–77% depending on concentrations (p b 0.05). Antifungal effect of essential oils can be probably due to the phenolic components of essential oils (Voda et al., 2003; Stevi et al., 2014). Thyme essential oil exhibited strong antimicrobial activity on the mycelial growth of D. caulivora, D. helianthi, P. viticola (Cosic et al., 2010). The results of savory are in agreement with that reported by Rasooli and Abyaneh (2004). Sukatta et al. (2008) reported that mixing of clove and cinnamon oils exhibited inhibitory effect against the postharvest decay fungi of grapes Aspergillus niger. C. citratus oil at a concentration of 1.5 μg/ml completely suppressed growth of A. flavus and A. niger (Helal et al., 2006). It is considered to be a relationship between the chemical structure of the most abundant compounds in the essential oil and the antimicrobial activity (Stupar et al., 2014). The essential oil
isolated from oregano showed antifungal activity against mycelium growth of Aspergillus, Fusarium and Penicillium (Stupar et al., 2014; Ibrahim and Al-Ebady, 2014). Paster et al. (1995) demonstrated antifungal activity of oregano essential oil on the mycelial and spores of A. niger, A. flavus and A. ochraceus. The high sensibility of filamentous fungi was observed by other authors with other essential oils (Cavaleiro et al., 2006; Tullio et al., 2007; Tavassoli et al., 2011). The result showed that the essential oils were able to suppress Fusarium oxysporum mycelial growth. Essential oil extracted from Eugenia aromatica provided the strongest suppression of F. oxysporum mycelial growth (Istaianto and Emilda, 2011). In previous study, Maskuki and Mortazavi (2004) studied on inhibitory effect of essential oils of thyme and ajowan to inhibit fungal growth of A. parasiticus on pear. Eucalyptus oil exhibited maximum antifungal activity against A. oryzae, followed by A. niger and F. solani then by A. flavus, F. moniliforme, A. terreus and A. fumigatus (Shirurkar and Wahegaonkar, 2012).
4. Conclusion In accordance with the earlier reports, the results of the present work suggested that some essential oils can be applied as mold inhibitors to prevent growth of toxigenic fungi. The results suggest the potential use of some essential oils as antimicrobial preservatives in food. Further studies on the combined effects of many local plant essential oils and components in food products are in progress in our model systems. The selected essential oils showed good inhibitory effect against all the tested organisms that are known food pathogens. Sage and rosemary essential oils were found to more potent against A. niger and F. oxysporum.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020
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M. Ferdes et al. / South African Journal of Botany xxx (2017) xxx–xxx
Fig. 4. Colony diameter (mm) Vs. Time (days) showing inhibitory effect of the oils against mycelial growth of Fusarium oxysporum at the different concentrations: (A) 10 μg/ml (B) 20 μg/ml.
Acknowledgements The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding the Research group NO (RG-1435-049). References Ajayi, A.O., Okoh, A.I., 2013. Antimycotic activity of some medicinal plants on aquatic fungi. European Journal of Experimental Biology 3, 16–21. Carmo, E.S., Lima, E.O., Souza, E.L., 2008. The potential of Origanum vulgare L. essential oil in inhibiting the growth of some food-related Aspergillus species. Brazilian Journal of Microbiology 39, 362–367. Cavaleiro, C., Pinto, E., Goncalves, M.J., Salgueiro, L., 2006. Antifungal activity of Juniperus essential oils against dermatophyte, Aspergillus and Candida strains. Journal of Applied Microbiology 100, 1333–1338.
Cosic, J., Vrandecic, K., Postic, J., Jurkovic, D., Ravric, M., 2010. In vitro antifungal activity of essential oils on growth of phytopathogenic fungi. Poljoprivreda 16, 25–28. Fandohan, O., Hell, K., Marasas, W.F.O., Wingfield, M.J., 2003. Infection of maize by Fusarium species and contamination on with fumanisis in Africa. African Journal of Biotechnology 2, 570–579. Helal, G.A., Sarhan, M.M., Abu Shahla, A.N.K., Abou El-Khair, E.K., 2006. Antimicrobial activity of some essential oils against microorganisms deteriorating fruit juices. Microbiology 34, 219–229. Ibrahim, F.A.A., Al-Ebady, N., 2014. Evaluation of antifungal activity of some plant extracts andtheir applicability in extending the shelf life of stored tomato fruits. Food Processing and Technology 5, 340. Istaianto, M., Emilda, D., 2011. Preliminary study of the activity of some essential oils against. Journal of Fruit and Ornamental Plant Research 19, 111–121. Kim, E., Park, I.I.-K., 2012. Fumigant antifungal activity of myrtaceae essential oils and constituents from lepto Spermum petersonii against three Aspergillus species. Molecules 17, 10459–10469. Mahilrajan, S., Nandakumar, J., Kailayalingam, R., Monoharan, N.A., Srivijeindran, S., 2014. Screening the antifungal activity of essential oils against decay fungi from palmyrah leaf handicrafts. Biological Research 47, 35. Maskuki, A., Mortazavi, S.A., 2004. Antifungal activity of essential oils of Thymus vulgaris and Trachyspermum ammi against Aspergilus. Journal of Science and Technology of Agriculture and Natural Resources 8, 207–215. Özcan, M., 1998. Inhibitory effects of spice extracts on the growth of Aspergillus parasiticus NRRL 2999 strain. Zeitschrift für Lebensmittel-Untersuchung und -Forschung 207, 253–255. Paster, V., Menashero, M., Ravid, U., Juven, B., 1995. Antifungal activity of oregano and thyme essential oils applied as fumigants against fungi attacking store grain. Journal of Food Protection 58, 81–85. Püskülcü, H., İkiz, F., 1989. Introduction to Statistic. Bilgehan Press, Bornova. İzmir, Turkey, p. p333 (in Turkish). Rasooli, I., Abyaneh, M.R., 2004. Inhibitory effects of thyme oils on growth and aflatoxin production by Aspergillus parasiticus. Food Control 15, 79–483. Shirurkar, D.D., Wahegaonkar, N.K., 2012. Antifungal activity of selected plant derived oils and some fungicides against seed borne fungi of maize. European Journal of Experimental Biology 2, 1693–1696. Sitara, U., Niaz, I., Naseem, J., Sultana, N., 2008. Antifungal effect of essential oils on in vitro growth of pathogenic fungi. Pakistan Journal of Botany 40, 409–414. Stevi, A., Beri, C., Katarina, S., Marina, S., Dejan, G., 2014. Antifungal activity of selected essential oils against fungi isolated from medicinal plant. Industrial Crop Production 55, 116–122. Stupar, M., Grbic, M.L., Dzamic, A., Unkovic, N., Ristic, M., 2014. Antifungal activity of selected essential oils and biocide benzalkonium chloride against the fungi isolated from cultural heritage objects. South African Journal of Botany 93, 118–124. Sukatta, U., Haruthaithanasan, V., Chantarapanont, W., Dilokkunanant, U., Suppakul, P., 2008. Antifungal activity of clove and cinnamon oil and their synergistic against postharvest decay fungi of grape in vitro. Kasetsart Journal (Natural Science) 42, 169–174. Tavassoli, S., Mousavi, S.M., Emam-Djomeh, Z., Razavi, S.H., 2011. Comparative study of the antimicrobial activity of Rosmarinus officinalis L. essential oil and methanolic extract. Middle-East Journal of Scientific Research 9, 467–471. Tripathi, P., Dubey, N.K., Shukla, A.K., 2008. Use of some essential oils as postharvest botanical fungicides in the management of grey mould of grapes caused by Botrytis cinerea. World Journal of Microbiology and Biotechnology 24, 39–46. Tullio, V., Nostro, A., Mandras, N., Dugo, P., Banche, G., Cannatelli, M.A., Cuffini, A.M., Alonzo, V., Carlone, N.A., 2007. Antifungal activity of essential oils against filamentous fungi determined by broth microdilution and vapour contact methods. Journal of Applied Microbiology 102, 1544–1550. Vitoratos, A., Bilalis, D., Karkanis, A., Efthimiadou, A., 2013. Antifungal activity of plant essential oils against Botrytis cinerea, Penicillium italicum and Penicillium digitatum. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 41, 86–92. Voda, K., Boh, B., Vrtacnik, M., Pohleven, F., 2003. Effect of the antifungal activity of oxygenated aromatic essential oil compounds on the white-rot Trametes versicolor and the Brown-not Coniophora puteana. International Biodeterioration and Biodegradation 51, 51–59.
Please cite this article as: Ferdes, M., et al., Inhibitory effect of some plant essential oils on growth of Aspergillus niger, Aspergillus oryzae, Mucor pusillus and Fusarium oxysporum, South African Journal of Botany (2017), https://doi.org/10.1016/j.sajb.2017.09.020