1. Background
In Algeria, olive oil production is a developing industry. Olives are infected with several soilborne fungal pathogens such as Alternaria, Aspergillus and Penicillium (1). A. niger causes many diseases called black mold on fruits and vegetables and produces potent mycotoxins called ochratoxins that can be harmful to human beings. On the other hand, A. flavus, Alternaria spp. and Penicillium spp. are the most dominant fungal species during postharvest storage condition (2). It is known that fungal strains that occur most frequently at mild and cold temperatures affect fruits. Furthermore, many olives are attacked mainly by Bactrocera oleae insect that is considered to be a serious threat to olive production in the Mediterranean region.
Chemical fungicides are widely used to control phytopathogenic fungi; nevertheless, the use of these types of compounds represents a concern associated with the risk of exposure and environmental hazards; therefore, new alternatives are needed (3). The genus Mentha belongs to Lamiaceae family and includes 25 species of herbaceous perennials. Mints are distributed predominantly in the temperate regions of the world and have varied growth characteristics, and aromas. Many Mentha species are used in traditional folk medicine for its stimulant, carminative, antispasmodic, stomachic and diuretic proprieties (4).
2. Objectives
The main objective of this study was to assess (I) the antifungal activity of four mint essential oils against several phytopathogens responsible for olive diseases, such as A. flavus, A. niger, Alternaria spp. and Penicillium spp. and (II) insecticidal activity of these four oils against B. oleae insect responsible for olive rot.
3. Methods
3.1. Plant Material
The plant materials of M. spicata, M. pulegium, M. piperita and M. rotundifolia were collected from Tlemcen region (Algeria) in July 2014 during full bloom stage.
Each mint specimen was identified by Professor Noury Benabadji of University of Tlemcen (Algeria) and deposited in the Herbarium of the University with voucher specimens (M. spicata: MSP-0714; M. pulegium: MPU-0715; M. piperita: MPI-716 and M. rotundifulia: MRO-0716).
3.2. Essential Oils Isolation
The aerial parts were stored at 18ºC after harvest, and 550-600 g of each species was subjected to a Clevenger-type apparatus (13) for 5 h. The yields of the oils were 0.5% for M. spicata, 0.7% for M. pulegium, 0.67% for M. piperita, and 0.9% for M. rotundifulia. Before chromatographic analysis, the essential oils were dried over sodium sulfate and stored in sterilized amber glass flasks.
3.3. Gas Chromatography
The gas chromatography (GC) apparatus used for the determination of retention indices was a Perkin Elmer Clarus 600 GC equipped with two flame ionization detectors (FIDs) and two fused-silica capillary columns (60 m × 0.22 mm, film thickness 0.25 μm) with different stationary phases: Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol). Program conditions were temperature of 60 to 230ºC at 2ºC.min-1 and then held isothermal at 230ºC (30 min); the carrier gas was hydrogen (0.7 mL.min-1). Injector and detector temperatures were held at 280ºC. Injected volume was 0.1 μL.
3.4. Gas Chromatography-Mass Spectrometry
The essential oils were investigated using a Perkin Elmer TurboMass quadrupole apparatus, directly coupled with a Perkin Elmer Autosystem XL equipped with two fused-silica capillary columns (60 m × 0.22 mm, film thickness 0.25 μm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene glycol), with the same program as GC described above. Ion source temperature was 150ºC and energy ionization was 70 eV; electron ionization mass spectra were acquired with a mass range of 35 - 350 Da and scan mass of 1 s. The injected oil volume was 0.1 μL.
3.5. Component Identification
The different components of essential oils were identified by comparison of GC retention indices (RI), determined from retention time of a series of n alkanes with linear interpolation, with those of authentic compounds (14, 15). For this purpose, computer matching with commercial mass spectral libraries and comparison of the spectra with those of the in-house laboratory library were performed (16).
3.6. Pathogenic Fungi
Aspergillus flavus, A. niger, Alternaria spp. and Penicillium spp., the four fungal isolates causing olive rot, were isolated directly from rotten olive harvested from orchards of Remchi, Ain Temouchent (Algeria). The four fungal species were transferred to sterilized Petri dishes, and 20% of lactic acid was added to the middle to stop the growth of bacteria. The plates were incubated at 25 ± 2°C for eight days away from light. Strains identification was firstly based on morphological characters and secondly on microscopic observations according the following references (17, 18).
3.7. In Vitro Antifungal Activity
The radial growth technique was used for testing the antifungal activity of essential oils (18). The concentrations varying from 0.1 to 300 mL/L used in the in vitro tests were obtained from stock solutions. For this purpose, appropriate volumes of essential oils were dissolved in dimethyl sulfoxide (DMSO) and added to Potato Dextrose Agar (PDA) medium immediately before it was poured into the Petri dishes of 9.0 cm diameter at 40°C - 45°C. The controls were prepared with DMSO mixed with PDA (without essential oils). The mycelial discs were filled with plant pathogenic fungi taken from 7-day-old cultures on PDA plates, and then they were transferred aseptically to the center of Petri dishes and incubated. This process was performed in triplicate.
The treatments were incubated at 27°C in the dark. Colony growth diameter was measured after the fungal growth in the control treatments had completely covered the Petri dishes. The half maximal inhibitory concentration (IC50) and the minimum inhibitory concentration (MIC) were determined at 95% confidence intervals (19) using Probit analysis.
3.8. Fumigation Toxicity of Essential Oils Against Bactrocera oleae
To determine the fumigant toxicity of essential oils, appropriate concentrations were applied separately on filter papers (Whatman No. 1, 2 cm diameter) to achieve the concentrations of 8 to 65 mL/L air without using any solvent, and the filter papers were attached to the under surface of plastic jar lids at 50-ml volumes. The control sets received no oil. The lids were screwed tightly on the jars containing 15 insects each, all of the same age. These were kept at a temperature of 25 - 26ºC and in 80% - 85% relative humidity (19). Mortality was checked 24 h after commencement of exposure. The mortality of insects was expressed in % and calculated by using the Abbott correction formula:
Corrected mortality = (OMT - OMC/100-CM) × 100
OMT, observed mortality in treatment; OMC, observed mortality in control; CM, control mortality.
Percentage mortality = (NDI/NII) × 100
NDI, number of dead insect; NI, number of insect introduced.
3.9. Statistical Analysis
Statistical analysis was performed by ANOVA using the SAS software. The means were separated using the least significant difference test at P ≤ 0.05. All the tests were performed in triplicate.
4. Results
4.1. Chemical Composition of the Four Mint Species Essential Oils
A total of 29, 18, 35 and 47 compounds were identified in essential oils of M. spicata, M. pulegium, M. piperita and M. rotundifulia that accounted for 98.1%, 98.5%, 98.8% and 98.9% of the oils, respectively (Table 1). Components identification was performed by comparison of IR and GC-MS with pur compounds of Arômes library (Table 1). In the GC-MS analysis of M. spicata essential oil, the most prominent compounds were carvone (54.1%) and limonene (21.9%). The main compounds found in M. pulegium were pulegone (77.3%) and menthone (10.8%). The chemical composition of M. piperita essential oil was dominated by linalool (40.4%) and linalyl acetate (32.6%). Therefore, M. rotundifulia essential oil was characterized by an appreciable amount of menthone (28.5%) and neo-menthol (10.4%).
Table 1.Mentha Species Essential Oils
| Compounds | lRIa | RIa | RIp | M. spicata | M. pulegium | M. piperita | M. rotundifulia |
|---|---|---|---|---|---|---|---|
| 1. (E)-hex-3-en-1-ol | 812 | 810 | 1360 | tr | |||
| 2. Ethyl-2-methyl butyrate | 829 | 829 | 1016 | 0.1 | |||
| 3. (E)-2-hexenal | 830 | 830 | 1210 | tr | 0.1 | 0.1 | |
| 4. (Z)-hex-3-en-1-ol | 831 | 832 | 1375 | 0.1 | |||
| 5. (Z)-2-hexenol | 851 | 848 | 1400 | tr | |||
| 6. 1-hexenol | 852 | 851 | 1414 | tr | |||
| 7. α-thujene | 922 | 923 | 1021 | 0.4 | 0.1 | tr | 0.2 |
| 8. α-pinene | 931 | 932 | 1023 | 0.7 | 0.5 | 0.2 | 0.4 |
| 9. Camphene | 943 | 944 | 1066 | tr | |||
| 10. Oct-1-en-3-ol | 959 | 962 | 1440 | 0.8 | 0.5 | ||
| 11. Sabinene | 964 | 966 | 1118 | 0.2 | |||
| 12. β-pinene | 970 | 972 | 1108 | 0.7 | 0.2 | 0.3 | 0.4 |
| 13. Myrcene | 976 | 982 | 1159 | 3.3 | tr | 1.2 | 1.3 |
| 14. 3-octanol | 982 | 982 | 1350 | 0.8 | 0.2 | ||
| 15. γ-phellandrene | 997 | 998 | 1164 | 0.1 | |||
| 16. α-terpinene | 1008 | 1010 | 1175 | 0.3 | 0.1 | ||
| 17. P-cymene | 1010 | 1012 | 1259 | 0.1 | 1.0 | ||
| 18. Limonene | 1020 | 1021 | 1195 | 21.9 | 1.1 | 0.3 | |
| 19. 1,8-cineole | 1020 | 1021 | 1205 | 0.6 | 3.8 | 0.2 | |
| 20. (Z)-β-ocimene | 1024 | 1025 | 1225 | 0.4 | 0.2 | ||
| 21. (E)-β-ocimene | 1034 | 1036 | 1241 | 0.4 | 0.4 | tr | |
| 22. γ-terpinene | 1047 | 1049 | 1237 | 0.7 | 0.1 | 0.2 | 0.3 |
| 23. Trans-hydrate sabinene | 1051 | 1054 | 1444 | 1.7 | 3.0 | ||
| 24. Terpinolene | 1078 | 1080 | 1247 | 0.1 | 0.1 | 0.5 | |
| 25. Linalool | 1078 | 1075 | 1280 | 0.2 | tr | 40.4 | |
| 26. Cis-sabinene hydrate | 1083 | 1082 | 1535 | 0.5 | 0.1 | ||
| 27. 1-oct-3-enyl acetate | 1093 | 1087 | 1390 | tr | 0.1 | ||
| 28. 2-methyl-butyl isovalerate | 1098 | 1096 | 1274 | 0.4 | |||
| 29. Cis-p-menth-2-en-1-ol | 1108 | 1110 | 1600 | tr | 0.1 | ||
| 30. 3-octyl acetate | 1111 | 1110 | 1315 | 0.2 | |||
| 31. Trans-p-menth-2-en-1-ol | 1123 | 1126 | 1612 | tr | tr | ||
| 32. Menthone | 1134 | 1135 | 1456 | 10.8 | 28.5 | ||
| 33. P-menth-3-en-8-ol | 1135 | 1135 | 1590 | 3.1 | |||
| 34. Iso-menthone | 1143 | 1142 | 1490 | 0.7 | 19.0 | ||
| 35. Borneol | 1148 | 1150 | 1690 | - | 0.1 | ||
| 36. Neo-menthol | 1156 | 1157 | 1637 | 0.2 | 1.6 | 10.4 | |
| 37. Terpinene-4-ol | 1161 | 1162 | 1583 | 1.3 | - | 2.7 | |
| 38. Menthol | 1164 | 1163 | 1629 | tr | 1.4 | ||
| 39. Iso-menthol | 1174 | 1173 | 1660 | tr | 2.1 | ||
| 40. Z-dihydro carvone | 1175 | 1174 | 1601 | 2.6 | |||
| 41. Dihydro carveol | 1178 | 1174 | 1723 | tr | |||
| 42. α-terpineol | 1179 | 1177 | 1688 | tr | 6.4 | 2.9 | |
| 43. E-dihydro carvone | 1180 | 1180 | 1626 | 3.1 | |||
| 44. α-campholenol | 1186 | 1188 | 1782 | tr | |||
| 45. Nerol | 1211 | 1213 | 1799 | 1.1 | |||
| 46. Pulegone | 1213 | 1216 | 1640 | 77.3 | 0.1 | 5.6 | |
| 47. Carvone | 1222 | 1226 | 1739 | 54.1 | |||
| 48. Piperitone | 1232 | 1229 | 1727 | 0.3 | 1.3 | ||
| 49. Geraniol | 1232 | 1234 | 1844 | 2.4 | |||
| 50. Linalyl acetate | 1240 | 1237 | 1557 | tr | 32.6 | ||
| 51. Geranial | 1244 | 1243 | 1731 | 0.2 | |||
| 52. Neryl formate | 1263 | 1266 | 1647 | 0.1 | |||
| 53. Neo-menthyl acetate | 1263 | 1268 | 1548 | 0.1 | 5.0 | ||
| 54. Bornyl acetate | 1269 | 1268 | 1475 | tr | |||
| 55. Lavandulyl acetate | 1270 | 1273 | 1593 | 0.1 | |||
| 56. Menthyl acetate | 1282 | 1285 | 1578 | 2.1 | |||
| 57. Iso-menthyl acetate | 1294 | 1295 | 1594 | 0.1 | 1.8 | ||
| 58. Dihydro carvyl acetate | 1311 | 1312 | 1661 | 2.2 | |||
| 59. Piperitenone | 1315 | 1313 | 1900 | tr | 2.7 | 1.8 | |
| 60. Piperitenone oxide | 1333 | 1335 | 1945 | 0.3 | |||
| 61. α-terpenyl acetate | 1336 | 1336 | 1678 | 0.1 | 0.1 | ||
| 62. Neryl acetate | 1342 | 1345 | 1725 | 1.7 | 2.7 | ||
| 63. Geranyl acetate | 1361 | 1364 | 1725 | 2.5 | |||
| 64. α-copaene | 1379 | 1379 | 1475 | 0.1 | |||
| 65. β-bourbonene | 1385 | 1385 | 1515 | 0.3 | 0.1 | tr | |
| 66. E-β-caryophyllene | 1424 | 1418 | 1583 | 0.6 | 0.3 | 0.8 | 0.4 |
| 67. E-β-farnesene | 1448 | 1447 | 1660 | 0.1 | 0.2 | ||
| 68. α-humulene | 1456 | 1456 | 1665 | 0.2 | 0.4 | ||
| 69. γ-muurolene | 1471 | 1469 | 1679 | 0.1 | 0.2 | ||
| 70. Germacrene D | 1480 | 1474 | 1692 | 0.1 | 0.1 | 0.1 | |
| 71. α-muurolene | 1496 | 1492 | 1709 | 0.1 | |||
| 72. γ-cadinene | 1507 | 1506 | 1750 | 0.1 | tr | 0.2 | 0.2 |
| 73. Trans-calamenene | 1512 | 1510 | 1810 | 0.1 | 0.2 | 0.1 | |
| 74. δ-cadinene | 1516 | 1515 | 1748 | 0.1 | tr | 0.2 | 0.1 |
| 75. Cadina-1,4-diene | 1523 | 1520 | 1763 | 0.1 | |||
| 76. α-calacorene | 1531 | 1528 | 1890 | 0.1 | |||
| 77. α-cadinene | 1535 | 1530 | 1740 | tr | 0.1 | tr | |
| 78. β-calacorene | 1548 | 1546 | 1936 | tr | |||
| 79. Caryophyllene oxide | 1578 | 1580 | 1980 | 0.3 | |||
| 80. Globulol | 1580 | 1582 | 2074 | 0.5 | |||
| Total identification % | 98.1 | 98.5 | 98.8 | 98.9 | |||
| Hydrocarbon compounds | 2.7 | 4.8 | 6.5 | ||||
| Monoterpene hydrocarbons | 2.0 | 2.8 | 4.9 | ||||
| Sesquiterpene hydrocarbons | 0.7 | 2.0 | 1.6 | ||||
| Oxygenated compounds | 95.8 | 94.0 | 92.4 | ||||
| Oxygenated monoterpenes | 94.2 | 92.5 | 91.3 | ||||
| Oxygenated sesquiterpenes | - | 0.8 | - | ||||
| Non-terpenic oxygenated compounds | 1.6 | 0.7 | 1.1 |
4.2. In Vitro Antifungal Activity of the Four Mint Essential Oils Against Plant Fungi
Essential oils’ minimum and medium inhibitory concentrations (MIC and MIC50, respectively), as well as inhibition of the four fungi amended with the estimated MIC and MIC50 of each essential oil are presented in Table 2. All the essential oils presented antifungal activity against A. flavus, A. niger, Alternaria spp. and Penicillium spp. The lowest activity was observed with essential oils of M. piperita and M. rotundifulia with MIC50s ranging from 80 to 300 mL/L and MICs from 1.2 to 25.2 mL/L. M. spicata and M. pulegium essential oils exhibited good activities compared to M. piperita and M. rotundifulia essential oils. Essential oil of M. pulegium was active against A. flavus, A. niger, Alternaria spp. and Penicillium spp. with IC50s of 4.2, 1.1, 1.3, and 1.1 mL/L and MICs of 0.1, 0.2, 0.08, and 0.08 mL/L, respectively. However, essential oil of M. spicata was more active against Alternaria spp. and Penicillium spp. with IC50s of 1.5 and 0.8 mL/L and MICs of 0.1 and 0.08 mL/L, respectively. However, essential oil exhibited moderate activity against A. flavus and A. niger with IC50s of 45 and 50 mL/L and MICs of 0.2 and 1.2 mL/L, respectively.
Table 2.Minimum (MIC) and Medium (IC50) Inhibitory Concentration Values Against Radial Growth of Fungal Species Determined After Seven Days of Incubation on PDA + Tween Amended with the Essential Oilsa
| Treatment (mL/L) | A. flavus | A. niger | Alternaria Spp. | Penicillium Spp. | ||||
|---|---|---|---|---|---|---|---|---|
| CMI | IC50 | CMI | IC50 | CMI | IC50 | CMI | IC50 | |
| M. spicata | 0.2A | 45B | 1.2B | 50B | 0.1A | 1.5A | 0.08A | 0.8A |
| M. pulegium | 0.1A | 4.2A | 0.2A | 1.1A | 0.08A | 1.3A | 0.08A | 1.1A |
| M. piperita | 1.5B | 150D | 1.2B | 150C | 1.3B | 80B | 1.2B | 150C |
| M. rotundifulia | 1.3B | 90C | 12.5C | 250D | 25.2C | 300C | 1.2B | 100B |
a Values are means from the three experiments. Different letters within a column represent significant differences (P < 0.05).
4.3. Fumigation Toxicity
The results regarding fumigation toxicity of mint essential oils against Bactrocera oleae are summarized in Table 3. The efficacy of essential oils varied with their concentrations. At the concentration of 10 µL/mL air, the essential oils of M. pulegium, M. piperita and M. rotundifulia caused over 46% mortality after 24 h of exposure. However, M. spicata essential oil showed no efficacy at this concentration. At the highest concentration (15 µL/mL air), M. pulegium essential oil caused 100% mortality after 1.5 h of exposure (Table 3). Nonetheless, for the M. piperita and M. rotundifulia essential oils, a concentration of 25 µL/mL air was required to have 100% mortality.
Table 3.Larvicidal Efficacy of Mentha Species Essential Oils Against Bactrocera oleaea
| Concentrations (µL/mL air) | % Mortality ± SE | |||
|---|---|---|---|---|
| M. spicata | M. pulegium | M. piperita | M. rotundifulia | |
| 8 | - | 16.6 ± 1.2 | 20.2 ± 1.6 | 0.0 ± 0.0 |
| 10 | 0.0 ± 0.0 | 50.0 ± 2.1 | 66.6 ± 3.2 | 46.6 ± 3.2 |
| 15 | 40.3 ± 4.2 | 100.0 ± 0.0 | 86.5 ± 4.2 | 76.6 ± 5.6 |
| 25 | 53.3 ± 5.3 | - | 100.0 ± 0.0 | 100.0 ± 0.0 |
| 45 | 76.6 ± 3.5 | - | - | - |
| 65 | 86.6 ± 6.6 | - | - | - |
| LC50 (µL/L air) | 0.22 | 0.27 | ||
| LC90 (µL/L air) | 0.33 | 0.45 | ||
a The results are expressed as mean ± standard deviation.
5. Discussion
Chemical analysis of the four Mentha species essential oils showed that M. piperita mostly contains oxygenated monoterpenes principally dominated by monoterpene ketones such as pulegone, carvone, menthone and iso-Menthone, and appreciable amounts of monoterpene alcohols such as linalool and neo-menthol. However, the chemical composition of M. spicata essential oil was characterized by appreciable amounts of monoterpene hydrocarbons, such as limonene and myrcene.
Essential oils from plants have attracted increasing interest as ecologically safe alternatives to fungicides and insecticides. The in vitro evaluation of antifungal properties of essential oils was performed in the present study, which showed that essential oils of the four Mentha species have good antifungal activity against A. flavus, A. niger, Alternaria spp. and Penicillium spp. Furthermore, in review of the fumigant toxicity results of essential oils of the four mints, it can be noticed that oils show very interesting activities. Essential oils are complex volatile mixtures. Monoterpenes and sesquiterpenes are usually the main groups of compounds that are responsible for many of their biological activities. On the basis of these results, we suggest that antifungal activity and fumigant toxicity of Mentha essential oils was due to their major components such as linalool, carvone, pulegone, menthone and linalyl acetate with percentages exceeding 28%.
Carvone is abundantly found in cumin, dill and spearmint. It is a natural product with strong antiseptic properties used as a mosquito repellent (20). It has been demonstrated that carvone has strong fungicidal activity against different mycotoxigenic fungi involved in several plant diseases (20). Naigre et al. (21) and Flamini et al. (22) also found that pulegone, limonene, carvone and menthone showed biocidal activity. We found that M. pulegium essential oil is rich in pulegone and M. spicata is rich in carvone and that they have significant insect antifeedant (M. pulegium) and nematocidal (M. spicata) effects (11).
We demonstrated in this study that the essential oils could act as antifungal agents and fumigants against Bactrocera oleae. Thus, due to their antifungal and insecticidal effects, these essential oils could be used as in fungicides and insecticides to prevent the infestation of olive products. However, further trials are necessary to devise a method for the application of essential oils in fungicides against Bactrocera oleae.
