Abstract: Back Ground and Objective: Medicinal plants are getting popular day by day because of their easy accessibility and reasonable costs. This study investigated the chemical composition and antifungal activity of Lavandula mairei, Lavandula dentata and Tetraclinis articulata plants essential oils against Penicillium digitatum, Penicillium italicum and Geotrichum citri-aurantii, the main post-harvest pathogens in citrus. Materials and Methods: Essential oils were obtained by hydrodistillation from areal parts of tested plants. Afterwards, they were analyzed by means of GC-MS and their antifungal efficacy was tested in vitro by using the agar plate’s method. Results: The main constituents were carvacrol for L. mairei, camphor, linalool and β-pinene for L. dentata and bornyl acetate, α-pinene, borneol and limonene for T. articulata. In the in vitro assay, the effect of essential oils on mycelial growth and spore germination varied significantly between tested plant species. Complete growth inhibition of the three pathogens was obtained by L. mairei essential oil. Also, L. mairei displayed the highest bioactivity, inhibiting completely the spore germination of the three pathogens. Moreover, this species showed fungistatic and fungicidal activity on the three fungal pathogens. Conclusion: In this study, L. mairei essential oil showed great antifungal activity which could represent a potential alternative to synthetic fungicides for the control of citrus fruit fungal pathogens.
INTRODUCTION
Post-harvest diseases render heavy losses to fruits and vegetables during storage and transit1. In the case of citrus fruit, the most common and serious diseases are green and blue moulds caused, respectively, by Penicillium digitatum (Pers.:Fr.) Sacc. and Penicillium italicum Wehmer followed in importance by sour rot caused by Geotrichum citri-aurantii Link ex Persn2.
Disease control is achieved mainly through the use of synthetic fungicides, especially imazalil (IMZ), thiabendazole (TBZ), fludioxonil and pyrimethanil3. However, the post-harvest use of these fungicides is subject to registration and permission for use in various countries. Furthermore, repeated application of synthetic fungicides has resulted in artificial selection of resistant fungi with multiple fungicide resistances, which further complicate the management of the diseases (especially Penicillium rots)4,5. In addition, these fungicides are not effective against all important pathogens. Indeed, sour rot is difficult to control with IMZ and TBZ4-6. Besides leading to an increase in the cost of post-harvest control of Penicillium rots due to development of resistance, the use of synthetic fungicides is increasingly becoming restricted owing to stringent regulation, high and acute residual toxicity, environmental pollution and growing public concern about chemical residues in fruit3,7. Therefore, the challenge is to develop safer and eco-friendly alternative strategies of controlling citrus post-harvest diseases, which pose less risk to human health and environment. Recently, natural products including plant extracts and essential oils have been proposed as potential alternatives to synthetic fungicides for the control of post-harvest citrus diseases8-10. The EOs are natural, volatile, complex compounds known for their antibacterial, antifungal, antiviral and medicinal properties11-15.
The genus Lavandula is represented in Moroccan flora by 9 species and subspecies of which 5 are endemic16. Among these endemic species, Lavandula mairei Humbert is considered as rare species17. Lavandula dentata L. is widely distributed in the Mediterranean region. These Lavandula species are widely used in traditional medicine for the treatment of various diseases such as gastrointestinal ailments, microbial infection, cough and asthma18. Tetraclinis articulata (Vahl) Masters, which belongs to the family of Cupressaceae is a well-known species used extensively throughout the Mediterranean basin in folk medicine for the treatment of a variety of ailments18,19.
Despite their medicinal properties, these plants have never been examined as potential source of antifungal compounds against the main postharvest fungal pathogens of citrus fruit. Hence, the objective of this study was to evaluate the effectiveness of EOs obtained from L. mairei, L. dentata and T. articulata plants against P. digitatum, P. italicum and G. citri-aurantii for management of post-harvest citrus diseases. As far as we know, this is the first report on the antifungal activity of L. mairei, L. dentata and T. articulata EOs against postharvest fungal pathogens of citrus.
MATERIALS AND METHODS
Plant material: Fresh plant samples of L. mairei, L. dentata and T. articulata were collected from their wild habitat, between March and May 2016, from two different locations in Morocco: L. mairei in Tafraout region (Western Anti Atlas), L. dentata in Tamri region (Western high Atlas) and T. articulata in Immouzzer region (Western high Atlas). Voucher specimens were deposited in the herbarium of the Laboratory of Biotechnology and Valorization of the Natural Resources, Faculty of Sciences, Ibn Zohr University, Agadir, Morocco. Plant samples were cleaned, air dried in the shade and stored in the dark at 4°C until use.
Extraction of essential oil: The essential oils (EOs) were obtained from dried (200 g) aerial plant materials by hydrodistillation using a Clevenger type apparatus for 4 h as recommended by European Pharmacopoeia20. The EOs obtained were dried over anhydrous sodium sulfate and stored in an amber bottle at 4°C until used.
Gas chromatography-mass spectrometry analysis: Plants essential oils compositions were determined by GC/MS analysis according to Boubaker et al.9. Identification of compounds were based on the comparison of their mass spectra with those of Wiley and NIST libraries as well as by comparison of their retention indices with those of authentic samples. Oil chemical relative composition was determined by peaks area for compounds contributing for more than 0.1% of the total composition.
Antifungal activities of essential oils
Fungal cultures: The fungi used in this study, P. digitatum, P. italicum and G. citri-aurantii were isolated from decayed citrus fruit. Single spore strains of these fungi were maintained on potato dextrose agar (PDA) plates at 5°C. A 1 week old culture of each fungus was used to inoculate the agar plates.
Determination of antifungal effects of the essential oils on mycelial growth: The agar dilution method was employed for the determination of the essential oils antifungal activity according to the method of Ameziane et al.21. All tests were performed in PDA supplemented with 0.05% (v/v) Tween 80 to enhance oil solubility22. Control consisted of unamended PDA medium supplemented with 0.05% Tween 80. The antifungal activity was expressed in terms of percentage of mycelial radial growth inhibition and calculated according to the following equation:
where, C and T represent mycelial growth diameter in control and EO treated Petri plates, respectively. Three plates were used for each treatment as replications. The experiment was repeated twice and similar results were obtained in each experiment.
Effect of essential oils on spore germination: For the germination test, plants essentials oils were used and prepared according to the method described by Boubaker et al.9. Different concentrations, ranging from 0.25-4.0 μL mL–1 of essential oils were prepared by dissolving the requisite amounts in 80 μL of sterilized (0.2 μm filter) orange juice and 0.5% (v/v) Tween 80 and transferred to sterile depression slides9. The results were expressed as percent spore germination inhibition and calculated by using the following equation:
where, Gc and Gt represent the mean number of germinated spores in control and treated slides, respectively. Each treatment included three replicates and the experiment was conducted twice.
Determination of minimum inhibitory concentration and minimum fungicidal concentration: The minimum inhibitory concentrations (MICs) of the plant EOs were determined by the agar dilution method9. The MICs were recorded by reading the lowest EO concentration that allowed no visible growth of the pathogen23. The MFCs were determined by taking agar plugs from well showing no visible mycelial growth and re-inoculating them on unamended PDA medium. The MFC was regarded as the lowest concentration of the EOs that prevented growth of the pathogen after the period of incubation. There were three replicates for each plant EO at each concentration and the experiment was conducted twice.
Statistical analysis: All data were subjected to statistical analysis of variance (ANOVA) using STATISTICA software, ver. 6 (Stat-Soft, 2001, Créteil, France). Percentage values were subjected to arcsine square root transformation before analysis of variance. Mean separation was performed following the Newman and Keuls test at p<0.05.
RESULTS
Chemical composition of the essential oils: The chemical analyses of L. mairei, L. dentata and T. articulata EOs led to the identification and quantification of 25, 28 and 24 compounds, respectively (Table 1). The major compound of L. mairei EO is carvacrol (83.2%) followed by caryophyllene oxide (3.06%), p-cymen-8-ol (2.02%), octen-3-ol (1.94%), spathulenol (1.62%) and carvacrol methyl ether (1.07%), which accounted for 92.91% of the total essential oil. While the major components (73.24% of the total oil) identified in L. dentata EO were camphor (64.43%), linalool (4.87%) and β-pinene (3.94%). Bornyl acetate (43.78%), α-pinene (20.3%), borneol (12.45%) and limonene (5.2%) were the main compounds identified in T. articulata EO.
Effects of essential oils on mycelial growth: The results of this study confirm that EOs from L. mairei, L. dentata and T. articulata plants possess antifungal activity against P. digitatum, P. italicum and G. citri-aurantii (Fig. 1). Of the plant species tested EO from L. mairei produced highest antifungal activity against the three fungi. Indeed, this plant had completely (100%) inhibited the mycelial growth of the three fungal pathogens at a concentration of 1.0 μL mL–1 (Fig. 1). For L. dentata EO, the inhibition percentages after 7 days of incubation were 24, 52 and 13% against P. digitatum, P. italicum and G. citri-aurantii, respectively. On the other hand, essential oil obtained from T. articulata were found to possess weaker antifungal activity against P. digitatum, P. italicum and G. citri-aurantii with a mycelial growth inhibition of only 17, 41 and 10%, respectively.
Effect of essential oils on spore germination: The results shown in Table 2 indicate that L. mairei EO inhibited the spore germination of P. italicum, P. digitatum and G. citri-aurantii in a dose-dependent manner. Indeed, tested at 1.0 μL mL–1, the EO inhibited the spore germination of P. digitatum, P. italicum and G. citri-aurantii by 38.0, 50.0 and 85.33%, respectively.
| Table 1: | Chemical relative composition of Lavandula mairei, L. dentata and Tetraclinis articulata essential oils |
aRI: Retention indices measured relative to n-alkanes (C-9 to C-24) on the non-polar DB-5 column. bCompounds listed in order of elution | |
| Fig. 1: | In vitro effects of L. mairei, L. dentata and T. articulata essential oils on mycelial growth of P. digitatum, P. italicum and G. citri-aurantii |
| Values followed by the same letters were not significantly different (p<0.05) according to Newman and Keuls test | |
| Table 2: | In vitro effect of L. mairei, L. dentata and T. articulata essential oils on spore germination of P. digitatum, |
Each value represents the mean of three replicates. Means followed by different letter(s) are significantly different at p<0.05. For all control treatments, which contained only Tween 80 in orange juice, spore germination was >90% | |
| Table 3: | Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) of Lavandula mairei and L. dentata essential oils |
| MIC: Concentration that was fungistatic, MFC: Concentration that was fungicidal | |
At concentrations of 2.0 and 4.0 μL mL–1, L. mairei EO inhibited by more than >94% spores germination of P. digitatum, P. italicum and G. citri-aurantii. When tested at 4.0 μL mL–1, the EO of L. dentata inhibited the germination of spores of P. digitatum, P. italicum and G. citri-aurantii by 95.33, 100 and 13.33%, respectively. Tetraclinis articulata EO reduced the germination of spores of P. digitatum, P. italicum and G. citri-aurantii by 97, 33.33 and 14.67%, respectively. For all control treatments, which contained only Tween 80 in orange juice, spore germination was >90%.
MIC and MFC: The inhibitory property of the oil obtained from L. mairei was observed within a range of 0.25 μL mL–1 for P. italicum to 0.5 μL mL–1 for P. digitatum and G. citri-aurantii (Table 3). Lavandula mairei EO was found to be fungicidal against P. digitatum, P. italicum and G. citri-aurantii at MFCs of 2.0, 1.0 and 1.0 μL mL–1, respectively. Lavandula dentata EO completely inhibited the growth of P. digitatum, P. italicum and G. citri-aurantii at MICs of 2.0, 4.0 and 4.0 μL mL–1, respectively. While the fungicidal effect of T. articulata and L. dentata EOs against the three pathogens appeared at a higher concentration >4.0 μL mL–1.
DISCUSSION
In the present study, α-pinene, camphor, borneol and bornyl acetate were found to be the principal constituents of T. articulata EO. These results are in agreement with those reported by Bourkhiss et al.24, in where it has been established that α-pinene, camphor, borneol and bornyl acetate were the main components of Moroccan T. articulata EO. Lavandula mairei EO is characterized by a high amount of carvacrol compared to that reported for others Lavandula species, Only L. multifida has been reported as carvacrol-rich specie25. The presence of carvacrol within L. mairei EO at very substantial proportions presents a special interest. Indeed, carvacrol possesses a very high antifungal activity against many post-harvest pathogens26,27 and essential oils containing a high amount of carvacrol could have many applications and effects on both human and plants pathogens. Furthermore, the safe use of medicinal herbs and their individual components has led to their current status of Generally Recognized as Safe (GRAS) food ingredients to control bacterial and fungal diseases15.
In several studies, it was pointed out that antimicrobial activity of EO obtained from T. articulata may be due, in part, to the presence of α-pinene and borneol among the major compounds of this species24-28. Prior reports described antifungal activities of plants EOs and several of their individual components against some of the pathogens examined in the present work and demonstrated that the mechanisms involved in control of these pathogens by plant EOs include restriction of their conidial germination and hyphal growth26,,27,29,30. In the present study, the high antifungal properties of L. mairei EO, against the three fungal pathogens, can be attributed to the presence of high concentration of carvacrol (83.2%). According to previous works, thymol, carvacrol, geraniol, eugenol, octanal and citral were recognized as the most active components against citrus fungal pathogens26,27,30-32.
In the current experiment, L. mairei, L. dentata and T. articulata essential oils have been shown to reduce or completely inhibit the spore germination of P. digitatum, P. italicum and G. citri-aurantii. These results are consistent with reports in the literature that describe carvacrol and/or thymol as an inhibitor of conidial germination and mycelial growth of several postharvest citrus pathogens26,27,32-34. In addition, it is also possible that the minor components such as linalool, borneol and α-terpineol might be involved in the antifungal activity with other active components of tested EOs, as also evident by the work of others Tao et al.30, Wolken et al.35 and Wuryatmo et al.36.
Among the three plants EOs tested, essential oils obtained from L. mairei showed the most potent antifungal effect against the three fungal pathogens. The MIC values obtained in this study, against P. digitatum, P. italicum and G. citri-aurantii were 0.50, 0.25 and 0.50 μL mL–1, respectively. A study by Tao et al.37 demonstrated that octanal, a pure plants EOs constituent, reduced strongly the mycelial growth of P. italicum and P. digitatum, with an MIC and MFC of 0.5 and 1.0 μL mL–1, respectively. In a recent study, essential oils obtained from four Moroccan Thymus species showed a MIC values against P. digitatum, P. italicum and G. citri-aurantii ranging from 0.5-4.0 μL mL–1 and MFC values9 from 1.0 to more than 4.0 μL mL–1. In a similar study, Regnier et al.27 reported the efficacy of different plants EOs and their major components (citral, eugenol, geraniol, carvacrol and thymol). Their results confirmed the antifungal activity of those substances for in vitro and in vivo control of post-harvest citrus pathogens27.
CONCLUSION
Lavandula mairei essential oil exhibited great antifungal which suggests that they may be considered as a potential alternative to the synthetic fungicides for the control of post-harvest citrus fungal pathogens. Further experimental research is needed to assess in vivo efficacy and commercial implementation of EOs as post-harvest botanical fungicides in citrus industry with respects to problems related to potential phytotoxicity, organoleptic aspects and compatibility with common post-harvest practices.
SIGNIFICANCE STATEMENT
This study demonstrates that plant essential oils have a high potential to control post-harvest fungal diseases of citrus fruits. Among the three plants tested, L. mairei essential oil showed high antifungal activities against the tested pathogens and will provide a starting point for discovering new compounds with better activity than chemical fungicides currently available. Such natural products therefore represent a sustainable alternative to the use of chemical fungicides.