Subscribe Now Subscribe Today
Research Article

In vitro Antifungal Activity of Some Plant Essential Oils

Abdallah M. Elgorban, Ali H. Bahkali, Mohamed A. El- Metwally, Mohamed Elsheshtawi and Mohamed A. Abdel- Wahab
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail

In the present study the antimicrobial activity of essential oils of allium bulb (Allium cepa L.), black cumin seeds (Nigella sativa L.) and eucalyptus (Eucalyptus globulus Labill) were evaluated against five fungi (Fusarium oxysporum f.sp. melonis, Fusarium solani, Fusarium verticillioides, Sclerotinia sclerotiorum and Rhizoctonia solani). Allium cepa oils completely inhibited the mycelial growth of Fusarium oxysporum f.sp. melonis, Fusarium solani and Sclerotinia sclerotiorum at 500 ppm concentration. While, E. globulus oil completely inhibited the radial growth of F. solani, S. sclerotiorum R. solani. On the other hand, the percentage inhibition varied in case of Nigella sativa oil is 28.6-73.9% from (F. oxysporum f.sp. melonis, F. verticillioides) at 500 ppm concentration. Whereas, the spore germination of F. oxysporum f.sp. melonis and F. solani were completely inhibited by the application of A. cepa oil, while the oil of eucalyptus completely inhibited the spore germination of F. solani and highly effective against spore germination of F. oxysporum f.sp. melonis, F. verticillioides with 98 and 93%, respectively at 500 ppm concentration. Considering the inhibition in the growth and spore germination, it concluded that allium and eucalyptus essential oils could be used as possible bio-fungicides alternative to synthetic fungicides against phytopathogenic fungi.

Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

  How to cite this article:

Abdallah M. Elgorban, Ali H. Bahkali, Mohamed A. El- Metwally, Mohamed Elsheshtawi and Mohamed A. Abdel- Wahab, 2015. In vitro Antifungal Activity of Some Plant Essential Oils. International Journal of Pharmacology, 11: 56-61.

DOI: 10.3923/ijp.2015.56.61

Received: October 18, 2014; Accepted: December 10, 2014; Published: May 04, 2019


The prevalence of pathogens resistant to drug is one of the most serious impendences to effective treatment of microbial diseases. Over the centuries extracts and other essential plants have evoked interest as sources of natural products. They have been examined for their potential uses as alternative therapy for the treatment of many microbial infection. It is known that many aromatic plants synthesize diverse bio-active compounds such as phenylpropanoids, sesquiterpenes and monoterpenes. These secondary metabolites as mixtures of different lipophilic and volatile constituents are known to play important role in defense system of higher plants (Reichling, 1999). A large number of studies have revealed that medicinal plants can be considered as rich sources of antimicrobial agents (Ates and Erdogrul, 2003; Mahesh and Satish, 2008; Al-Taisan et al., 2014; Hatamleh et al., unpublished). Essential oils derived from medicinal plants such as Mentha arvensis (Imai et al., 2001), Nigella sativa (El-Kamali et al., 1998), Cryptomeria japonica (Cheng et al., 2005), Ziziphora clinopodioides (Sonboli et al., 2006), Thymbra capitata (Salgueiro et al., 2004), Salvia sclarea (Pitarokili et al., 2002), Pimpinella anisum (Kosalec et al., 2005), Thymus pulegioides L. (Pinto et al., 2006) and Tagetes patula (Romagnoli et al., 2005) were effective against numerous fungi.

Soil-borne pathogenic fungi i.e., Fusarium spp., Rhizoctonia, Pythium, Phoma spp., etc., that cause serious problem of sudden wilt (vine decline) are considered as hazardous organisms of minatory vegetable production in the nursery, protected agriculture and open fields, because of the decrease in plants number and quality (Gwynne et al., 1997).

These pathogens of sudden wilt especially in some crops such as Muskmelon (El-Sheshtawi et al., 2014) and watermelon (Anjorin and Mohammed, 2014) cause rotting, pre and post-emergence damping off, while the older plants are affected by wilting or bad growth during flowering or fruiting stages reflecting on plant growth and on the yield quantity and quality (Martyn and Miller, 1996). The recommended systemic or contact chemical fungicides for the control of such pathogens are extremely harmful in the short or long terms on the man health and on the environment, causing dangerous diseases i.e., cancer, kidney, liver diseases and others (Mansour, 1992). Furthermore, emergence of fungicide-resistant pathogens and increasing concerns over use of agrochemicals on storage products have highlighted the need of research for new antifungal substances derivative from several sources especially medicinal plants.

The purpose of this study was to evaluate the antifungal activity of essential oil derived from Allium bulb (Allium cepa L.), black cumin (Nigella staiva L.) and eucalyptus (Eucalyptus globulus Labill) leaves against the mycelial growth of five pathogenic fungi and spore germination of the three Fusarium spp. tested.


Isolation and identification of soil borne pathogenic fungi: Muskmelon plant roots with root rot characteristic were collected from the infected muskmelon fields. The samples were collected from Dakahliya and Demiatta Governorates, Egypt. The root parts were examined under light microscope to verify the presence of pathogens and then cut into pieces (3-4 mm and surface sterilized with 0.1% sodium hypochlorite for 30 sec). The samples were washed three times with sterilized distilled water and transferred aseptically on Potato Dextrose Agar (PDA). The plates were incubated at 25±2°C and examined daily for emergence of the fungal hyphae. Pure cultures of the pathogens were stored at 4°C on PDA tubes. Isolated pathogens were identified as described by Barnet and Hunter (1998), Booth (1977) and Kora et al. (2005).

Isolation of essential oils: Allium bulb (A. cepa), black cumin seeds (N. sativa) and Eucalyptus leaves (E. globulus) were cleaned with distilled water and air-dried at room temperature (25±2°C). These samples (150 g each), in triplicate, were subjected to hydrodistillation for 6 h using a Clevenger-type apparatus (Chang et al., 2001) followed by determination of oil contents. Leaf essential oils were stored in the refrigerator.

In vitro antifungal assay: Allium cepa, N. sativa and E. globulus oil were dissolved in 0.1% Tween 80 and added to the PDA medium which was autoclaved and cooled in a water bath to 45°C to obtain the final concentrations (10, 50, 100, 250 and 500 ppm) and 0.1% Tween 80 without essential oil was served as control. The mixed PDA medium was poured into 90 mm Petri plates with 20 mL for each plate. Mycelial disks of 5 mm diameter were cut out from the periphery of 7-day-old cultures of the pathogens. The disks were inoculated at the center of the plates. Four replicates were performed per treatment. All the pathogens were incubated at 25±2°C. The diameters of colonies were measured after five days. Experiments were performed three times.

Conidial germination: Plant Essential oils were dissolved in 0.1% Tween 80 and added into a 15 mL glass tube containing 5 mL Potato Dextrose Broth (PDB) to obtain the final concentrations (10, 50, 100, 250 and 500) and 0.1% Tween 80 without essential oil was added as control. One hundred microliters of spore (1×106 spores mL-1) of the Fusarium spp. were added into each tube. After 24 h from incubation at 25±2°C, on shaker (200 rpm), 100 conidia per replicate were observed microscopically to determine the germination rate. Four replicates were used for every treatment and experiments were performed three times.


Effect of essential oils against the radial growth and spore germination of Fusarium oxysporum f.sp. melonis: Data in Table 1 revealed that allium oil completely inhibited the mycelial growth of F. oxysporum f.sp. melonis at 500 concentration, while oil of eucalyptus reduced the mycelial growth of the pathogen with increasing concentrations reached to 67.2% inhibition at 500 ppm concentration. Black cumin oil inhibited the mycelial growth by 28.6% as related to the control. On the other hand, results showed that spore germination of F. oxysporum f.sp. melonis was completely inhibited by allium essential oil at 500 ppm concentration, this was followed by eucalyptus and black cumin essential oil that giving 98 and 72.5% reduction in spore germination at 500 ppm concentration, respectively (Table 1). The estimated LD50 in the fungus for A. cepa and E. globulus essential oil was 75.75 and 128.31 ppm L-1. While, LD50 of N. sativa was 3499.13 ppm L-1 and this value indicate a low toxicity of N. sativa to F. oxysporum f.sp. melonis.

Effect of essential oils against the radial growth and spore germination of Fusarium solani: All the three essential oils tested showed varied degree of inhibition over control in the mycelial growth of the pathogen F. solani at different concentrations (Table 2).

Table 1: Effect of essential oils against the radial growth and on spore germination of Fusarium oxysporum f.sp. melonis (Inhibition %)

Table 2: Effect of essential oils against the radial growth and on spore germination of Fusarium solani (Inhibition %)

Table 3: Effect of essential oils against the radial growth and on spore germination of Fusarium verticillioides (Inhibition %)

The maximum inhibition of the mycelial growth was recorded in E. globulus which completely inhibited the mycelial growth at 250 and 500 ppm concentrations and LD50 of this oil was 42.45 ppm L-1. Also, A. cepa essential oil was completely inhibited the mycelial growth of F. solani but at 500 ppm concentration. Reduction in percentage in spore germination increment is clear as essential oil concentration increased. Highest essential oil concentration of all tested oils caused most profound reduction of spore germination. The highest reduction in spore germination came from eucalyptus and allium oil which completely inhibited the spore germination of the fungus at 500 ppm and LD50 of both oils were 6.23 and 34.65 ppm L-1, respectively. Conversely, the essential oil of black cumin gave moderate reduction in spore germination with 68.50% at 500 ppm concentration and LD50 of this oil was 110.68 ppm L-1 (Table 2).

Effect of essential oils against the radial growth and spore germination of Fusarium verticillioides: The effects of different concentrations of the three essential oils tested on the radial growth of F. verticillioides are shown in Table 3. All three essential oils were found to inhibit F. verticillioides growth in a concentration-dependent manner. Eucalyptus essential oil showed the maximum inhibition in the maycelail growth of F. verticillioides with 91.9% when compared to control and the estimated LD50 obtained by linear regression was 55.77 ppm L-1. This was followed by N. sativa and A. cepa oil which significantly exhibited the radial growth of F. verticillioides giving 73.9 and 69.7% reduction in the mycelial growth of the pathogen and LD50 were 122.32 and 211.44 ppm L-1, respectively. Spore germination of F. verticillioides was inhibited by allium, black cumin and eucalyptus oils at all concentrations (Table 3). All three essential oils at 500 ppm concentration were highly effective against spore germination. The maximum inhibition of spore germination was recorded in E. globulus oil that giving 93% reduction in spore germination when compared with control. This was followed by black cumin and allium oils which produced 84.75 and 79.50% inhibition in spore germination of the fungus, respectively.

Effect of essential oils against the radial growth of Sclerotinia sclerotiorum: The in vitro results revealed that the growth of S. sclerotiorum was completely inhibited by the application of A. cepa and E. globulus at 500 ppm concentration and the estimated LD50 were 19.84 and 147.03 ppm L-1, respectively. While, black cumin oil showed moderate reduction in the mycelial growth of the fungus with 49.4% and LD50 was 940.61 ppm L-1 (Table 4).

Effect of essential oils against the radial growth of Rhizoctonia solani: Eucalyptus globulus oil was more effective against the pathogen tested which exhibited 100% mycelial inhibition of the fungus and LD50 of oil was 71.67 ppm L-1. This was followed by A. cepa essential that was significantly inhibited the mycelial growth of R. solani with 97.1% when compared with control and LD5 was 77.09 ppm L-1 (Table 4).

Table 4: Effect of essential oils against the radial growth of Sclerotinia sclerotiorum and Rhizoctonia solani (Inhibition %)


This study was conducted to assess the antifungal efficacy of essential oils from A. cepa bulb, N. sativa seeds and E. globulus leaves against soil borne pathogenic fungi. It was observed that A. cepa oil completely inhibited the mycelial growth F. oxysporum f.sp. melonis, F. solani and S. sclerotiorum, also significantly inhibited the mycelail growth of F. verticillioides and R. solani at 500 ppm concentration. Furthermore, this essential oil completely inhibited spore germination of F. oxysporum f.sp. melonis and F. solani.

The inhibitory activity of essential oil and extracts of Allium plants against fungi was reported by numerous authors, though, in general, essential oils are more effective inhibitors of fungi than of bacteria (Zaika, 1988; Hatamleh et al., 2014). Antifungal activity of Allium plants was stated by Yin and Tsao (1999), who observed that A. cepa showed highest antifungal activity against three Aspergillus species tested. Phay et al. (1999) reported that A. cepa exhibited marked antifungal activities against numerous fungal species particularly Penicillium roqueforti and Aspergillus oryzae which showed high sensitivity. Also, the essential oil of Allium plants showed marked antimicrobial activity against Staphylococcus aureus, Salmomella Enteritidis and three fungi, A. niger, Penicillium cyclopium and F. oxysporum (Benkeblia, 2004). Kocic-Tanackov et al. (2012) showed that the essential oil of A. cepa and A. sativum had a stronger inhibitory effect on the A. versicolor mycelial growth and sterigmatocystin production. This antifungal activity of A. cepa probably depend on the major components dimethyl-trisulfide, methyl-propyl-trisulfide, dietil-1, 2,4-tritiolan, methyl-(1-propenyl)-disulfide and methyl-(1-propenyl)-trisulfide (Kocic-Tanackov et al., 2012).

As a result, essential oil of eucalyptus inhibited the mycelial growth in all of the tested fungi after 5 days. The most important soil borne fungi infected muskmelon, F. solani, S. sclerotiorum and R. solani, had 100% complete inhibition and highly effective against the mycelail growth and spore germination of F. oxysporum f.sp. melonis and F. verticillioides. These results in agreement with those obtained by Hur et al. (2000) and Katooli et al. (2011) that showed Eucalyptus oil inhibited the mycelial growth of three phytopathogenic fungi such as Colletotrichum gloeosporioides, R. solani and Pythium spp. Also, Hatamleh et al. (2014) demonstrated that eucalyptus extract significantly inhibited the mycelial growth and spore germination of three Fusarium species and the mycelial growth of S. sclerotiorum and R. solani.

This high antifungal activity of eucalyptus oil probably related to the components such as 1,8-cineole (Saad et al., 2006), citronellol (Su et al., 2006), citronellal (Batish et al., 2006), ρ-cymene (Su et al., 2006), citronellyl acetate, limonene, eucamalol (Watanabe et al., 1993), limonene, linalool, α-pinene (Sartorelli et al., 2007), alloocimene, aromadendrene and α-terpineol (Duke, 2004; Liu et al., 2008).


The use of essential oils as natural fungicides is of immense significance in view of the environmental and toxicological implications of the indiscriminate use of synthetic fungicides and reducing the problem of increasing fungi resistance.


The researchers extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no RGP-277.

1:  Anjorin, S.T. and M. Mohammed, 2014. Effect of seed-borne fungi on germination and seedling vigour of watermelon (Citrullus lanatus thumb). Afr. J. Plant Sci., 8: 232-236.
CrossRef  |  Direct Link  |  

2:  Ates, D.A. and O.T. Erdogrul, 2003. Antimicrobial activities of various medicinal and commercial plant extracts. Turk. J. Biol., 27: 157-162.
Direct Link  |  

3:  Barnet, H.L. and B.B. Hunter, 1998. Illustrated Genera of Imperfect Fungi. 4th Edn., American Phytopathological Society Press, St. Paul, USA., ISBN-13: 978-0890541920, Pages: 240.

4:  Batish, D.R., H.P. Singh, N. Setia, S. Kaur and R.K. Kohli, 2006. Chemical composition and phytotoxicity of volatile essential oil from intact and fallen leaves of Eucalyptus citriodora. Zeitschrift Naturforschung, 61: 465-471.
PubMed  |  Direct Link  |  

5:  Benkeblia, N., 2004. Antimicrobial activity of essential oil extracts of various onions (Allium cepa) and garlic (Allium sativum). LWT-Food Sci. Technol., 37: 263-268.
CrossRef  |  Direct Link  |  

6:  Booth, C., 1977. Fusarium: Laboratory Guide Identification of the Major Species. Commonwealth Mycological Institute, Kew, ISBN-13: 9780851983837, Pages: 58.

7:  Chang, S.T., P.F. Chen and S.C. Chang, 2001. Antibacterial activity of leaf essential oils and their constituents from Cinnamomum osmophloeum. J. Ethnopharmacol., 77: 123-127.
CrossRef  |  Direct Link  |  

8:  Cheng, S.S., H.Y. Lin and S.T. Chang, 2005. Chemical composition and antifungal activity of essential oils from different tissues of Japanese cedar (Cryptomeria japonica). J. Agric. Food Chem., 53: 614-619.
CrossRef  |  PubMed  |  Direct Link  |  

9:  Duke, J., 2004. Dr. Duke's phytochemical and ethnobotanical databases.

10:  El-Kamali, H.H., A.H. Ahmed and A.A.M. Mohammed, 1998. Antibacterial properties of essential oils from Nigella sativa seeds, Cymbopogon citratus leaves and Pulicaria undulata aerial parts. Fitoterapia, 69: 77-78.
Direct Link  |  

11:  El-Sheshtawi, M., A.H. Bahkali, W.A. Al-Taisan and A.M. Elgorban, 2014. Pathogenicity of Fusarium oxysporum f.sp. melonis to melon genotypes (Cucumis melo L.) and its biocontrol. J. Pure Applied Microbiol., 8: 317-324.

12:  Saad, E.Z., R. Hussien, F. Saher and Z. Ahmed, 2006. Acaricidal activities of some essential oils and their monoterpenoidal constituents against house dust mite, Dermatophagoides pteronyssinus (Acari: Pyroglyphidae). J. Zhejiang Univ. Sci. B, 7: 957-962.
CrossRef  |  Direct Link  |  

13:  Gwynne, B.J., T.R. Gordon and R.M. Davis, 1997. A new race of Fusarium oxysporum f. sp. melonis causing Fusarium wilt of muskmelon in the central valley of California. Plant Dis., 81: 1095-1095.
CrossRef  |  

14:  Hatamleh, A.A., A.H. Bahkali, M. El-Sheshtawi, M.A. Elmetwally and A.M. Elgorban, 2014. Inhibitory influence of plant extracts on soil borne fungi infecting Muskmelon (Cucumis melo L.). Int. J. Pharmacol., 10: 322-327.
CrossRef  |  Direct Link  |  

15:  Hur, J.S., S.Y. Ahn, Y.J. Koh and C.I. Lee, 2000. Antimicrobial properties of cold-tolerant eucalyptus species against phytopathogenic fungi and food-borne bacterial pathogens. Plant Pathol. J., 16: 286-289.

16:  Imai, H., K. Osawa, H. Yasuda, H. Hamashima, T. Arai and M. Sasatsu, 2001. Inhibition by the essential oils of peppermint and spearmint of the growth of pathogenic bacteria. Microbios, 106: 31-39.
PubMed  |  

17:  Katooli, N., R. Maghsodlo and S.E. Razavi, 2011. Evaluation of eucalyptus essential oil against some plant pathogenic fungi. J. Plant Breed. Crop Sci., 3: 41-43.
Direct Link  |  

18:  Kocic-Tanackov, S., G. Dimic, J. Levic, I. Tanackov, A. Tepic, B. Vujicic and J. Gvozdanovic-Varga, 2012. Effects of onion (Allium cepa L.) and garlic (Allium sativum L.) essential oils on the Aspergillus versicolor growth and sterigmatocystin production. J. Food Sci., 77: M278-M284.
CrossRef  |  PubMed  |  Direct Link  |  

19:  Kora, C., M.R. McDonald and G.J. Boland, 2005. Epidemiology of Sclerotinia rot of carrot caused by Sclerotinia sclerotiorum. Can. J. Plant Pathol., 27: 245-258.
Direct Link  |  

20:  Kosalec, I., S. Pepeljnjak and D. Kustrak, 2005. Antifungal activity of fluid extract and essential oil from anise fruits (Pimpinella anisum L., Apiaceae). Acta Pharmaceutica, 55: 377-385.
PubMed  |  Direct Link  |  

21:  Liu, X., Q. Chen, Z. Wang, L. Xie and Z. Xu, 2008. Allelopathic effects of essential oil from Eucalyptus grandis x E. urophylla on pathogenic fungi and pest insects. Front For. China, 3: 232-236.
CrossRef  |  Direct Link  |  

22:  Mahesh, B. and S. Satish, 2008. Antimicrobial activity of some important medicinal plant against plant and human pathogens. World J. Agric. Sci., 4: 839-843.
Direct Link  |  

23:  Mansour, N., 1992. Integrated pest management and pesticide management: The future challenge in the Arab world. Proceedings of the Scientific Symposium on Pesticide Hazards: Effects on Human, Animal Health and Environmental Pollution, May 4-7, 1992, Beirut, pp: 241-266.

24:  Martyn, R.D. and M.E. Miller, 1996. Monosporascus root rot/vine decline: An emerging disease of melons worldwide. Plant Dis., 80: 716-725.

25:  Phay, N., T. Higashiyama, M. Tsuji, H. Matsuura, Y. Fukushi, A. Yokota and F. Tomita, 1999. An antifungal compound from roots of welsh onion. Phytochemistry, 52: 271-274.
CrossRef  |  Direct Link  |  

26:  Pinto, E., C. Pina-Vaz, L. Salgueiro, M.J. Goncalves and S. Costa-de-Oliveira et al., 2006. Antifungal activity of the essential oil of Thymus pulegioides on Candida, Aspergillus and dermatophyte species. J. Med. Microbiol., 55: 1367-1373.
CrossRef  |  Direct Link  |  

27:  Pitarokili, D., M. Couladis, N. Petsikos-Panayotarou and O. Tzakou, 2002. Composition and antifungal activity on soil-borne pathogens of the essential oil of Salvia sclarea from Greece. J. Agric. Food Chem., 50: 6688-6691.
CrossRef  |  PubMed  |  Direct Link  |  

28:  Reichling, J., 1999. Plant-Microbe Interaction and Secondary Metabolites with Antiviral, Antibacterial and Antifungal Properties. In: Functions of Plant Secondary Metabolites and their Exploitation in Biotechnology, Wink, M. (Ed.). Vol. 3, Taylor and Francis, Sheffield, UK., ISBN-13: 9781841270081, pp: 187-273.

29:  Romagnoli, C., R. Bruni, E. Andreotti, M.K. Rai, C.B. Vicentini and D. Mares, 2005. Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L. Protoplasma, 225: 57-65.
CrossRef  |  Direct Link  |  

30:  Salgueiro, L.R., E. Pinto, M.J. Goncalves, C. Pina-Vaz and C. Cavaleiro et al., 2004. Chemical composition and antifungal activity of the essential oil of Thymbra capitata. Planta Med., 70: 572-575.
PubMed  |  

31:  Sartorelli, P., A.D. Marquioreto, A. Amaral-Baroli, M.E.L. Lima and P.R.H. Moreno, 2007. Chemical composition and antimicrobial activity of the essential oils from two species of Eucalyptus. Phytother. Res., 21: 231-233.
CrossRef  |  Direct Link  |  

32:  Sonboli, A., M.H. Mirjalili, J. Hadian, S.N. Ebrahimi and M. Yousefzadi, 2006. Antibacterial activity and composition of the essential oil of Ziziphora clinopodioides subsp. Bungeana (Juz.) Rech. f. from Iran. Zeitschrift Naturforschung C, 61: 677-680.
PubMed  |  

33:  Su, Y.C., C.L. Ho, E.I.C. Wang and S.T. Chang, 2006. Antifungal activities and chemical compositions of essential oils from leaves of four eucalypts. Taiwan J. For. Sci., 21: 49-61.
Direct Link  |  

34:  Al-Taisan, W.A., A.H. Bahkali, A.M. Elgorban and M.A. El-Metwally, 2014. Effective influence of essential oils and microelements against Sclerotinia sclerotiorum. Int. J. Pharmacol., 10: 275-281.
CrossRef  |  Direct Link  |  

35:  Watanabe, K., Y. Shono, A. Kakimizu, A. Okada, N. Matsuo, A. Satoh and H. Nishimura, 1993. New mosquito repellent from Eucalyptus camaldulensis. J. Agric. Food Chem., 41: 2164-2166.
CrossRef  |  Direct Link  |  

36:  Yin, M.C. and S.M. Tsao, 1999. Inhibitory effect of seven Allium plants upon three Aspergillus species. Int. J. Food Microbiol., 49: 49-56.
CrossRef  |  Direct Link  |  

37:  Zaika, L.L., 1988. Spices and herbs: Their antimicrobial activity and its determination. J. Food Saf., 9: 97-118.
CrossRef  |  Direct Link  |  

©  2021 Science Alert. All Rights Reserved