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Chemical Composition and Biological Activity of the Essential Oil of Origanum vulgare ssp. hirtum from Different Areas in the Southern Apennines (Italy) by Emilia Mancini a ), Ippolito Camele b ), Hazem S. Elshafie b ), Laura De Martino a ), Carlo Pellegrino a ), Daniela Grulova c ), and Vincenzo De Feo* a ) a

) Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, I-84084 Fisciano (Salerno) (phone: þ 39-089969751; fax: þ 39-089969602; email: [email protected]) b ) School of Agricultural, Forestry, Food and Environmental Sciences, University of Basilicata, Viale dellAteneo Lucano 10, I-85100 Potenza c ) Department of Ecology, Faculty of Humanities and Natural Sciences, University of Presˇov, 17 November St., SK-08116 Presˇov

The chemical composition of the essential oils of Origanum vulgare ssp. hirtum, growing wild in three different localities in the Southern Apennines, was studied by GC-FID and GC/MS analyses. In total, 103 compounds were identified. The oils were mainly composed of phenolic compounds and all oils belonged to the chemotype carvacrol/thymol. The three essential oils were evaluated for their in vitro phytotoxic activity by determining their influence on the germination and initial radicle elongation of Sinapis arvensis L., Phalaris canariensis L., Lepidium sativum L., and Raphanus sativus L. The seed germination and radicle growth were affected in various degrees. Moreover, the antifungal activity of the three essential oils was assayed against three species causing pre- and postharvest fruit decay (Monilinia laxa, M. fructigena, and M. fructicola). At 1000 ppm, the three oils completely inhibited fungal growth. The hemolytic activity of the oils was assayed and showed no effect on the cell membranes of bovine erythrocytes.

Introduction. – A large number of different species, usually known as oregano or origanum, are of economic interest, even though they belong to different botanical families and genera. Four main groups commonly used as culinary herbs can be distinguished, i.e., Greek oregano (Origanum vulgare L. ssp. hirtum (Link) Ietsw.), Spanish oregano (Coridothymus capitatus (L.) Hoffmanns. & Link), Turkish oregano (O. onites L.), and Mexican oregano (Lippia graveolens Kunth) [1 – 3]. In Europe and, in general, all over the world, the most commonly found oregano species belong to the botanical genus Origanum, a genus of the Lamiaceae family. Before 1980, O. vulgare L. referred indifferently some subspecies, while Ietswaart [4] proposed a new classification, with six different subspecies within O. vulgare. In the Italian Flora, four different subspecies were present, i.e., O. vulgare L. ssp. hirtum (Link) Ietsw., O. vulgare L. ssp. gracile (C.Koch) Ietsw., O. vulgare L. ssp. vulgare, and O. vulgare L. ssp. viride (Boiss.) Hayek. O. vulgare L. ssp. hirtum (Link) Ietsw. is a typical eastern Mediterranean taxon, reported only for some areas in Southern Italy [5]. However, its taxonomic discerning traits are reported as highly variable [6]. Environmentally, this species prefers warm, sunny habitats and loose, often rocky, calcareous soils, usually low in moisture content.  2014 Verlag Helvetica Chimica Acta AG, Zrich

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The morphological features of O. vulgare ssp. hirtum can be distinguished from other O. vulgare subspecies by its hairy stems, compact inflorescences, leaves and calyces densely covered with glandular structures, green bracts, which are usually as long as the calyces, and white flowers [5] [7]. Several studies on the composition of the essential oils of O. vulgare from different areas of the world, including Greece, Lithuania, India, Argentina, Croatia, Turkey, and some regions of Italy, have been reported [5] [6] [8] [9]. Moreover, the O. vulgare essential oils have been found to display biological properties, such as antioxidant, antimicrobial, and antiviral activity [10 – 12]. The essential oil of oregano and its components have also been reported to inhibit phytopathogenic fungi [13 – 16]. Adebayo et al. [17] reviewed the antimicrobial activity of oregano essential oil against a number of plant pathogens such as Aspergillus nigerTiegh., A. flavus Link, A. ochraceus K.Wilh., Fusarium oxysporum W.C.Snyder & H.N.Hansen, F. solani var. coeruleum (Mart.) Sacc., Penicillium sp. L., Pseudomonas aeruginosa J.Schr˛t. ATCC 2730, Staphylococus aureus Rosenbach ATCC 6538, Clavibacter michiganensis S., Phytophtora infestans Mont., Sclerotinia sclerotiorum Lib., and Xanthomonas vesicatoria Doidge. In addition, different species of oregano, like O. vulgare and O. syriacum L., showed antifungal activity against Botrytis cinerea Pers. [18] [19]. Khosravi et al. [20] reported the antifungal activity of O. vulgare against Candida glabrata, which demonstrated its possible utilization in the treatment of candidiasis. Only a few articles are available about the possible phytotoxic activity of O. vulgare essential oil [21 – 23]. Significant losses in fruit crops have been recorded all over the world due to the brown rot disease caused by the infection with Monilinia ssp. such as M. laxa (Aderh. & Ruhland), M. fructicola (G.Winter) Honey, and M. fructigena (Aderh. & Ruhland) [24]. In particular, M. fructicola has been enclosed in the EPPO A2 List for quarantine organisms in Europe [25]. Further spread in Europe would lead to increased costs of control and may develop the resistance to fungicides [26]. As a continuation of our studies on the oils of Lamiaceae growing wild in the Southern Apennines [5], the chemical composition of the essential oils of three O. vulgare ssp. hirtum populations growing wild in Campania and Basilicata (Southern Apennines) was investigated and their possible in vitro phytotoxic properties, i.e., effects against germination and initial radicle elongation of Sinapis arvensis L., Phalaris canariensis L., Lepidium sativum L., and Raphanus sativus L., and antifungal activity against three fungal pathogens causing pre- and postharvest fruit decay (Monilinia laxa, M. fructicola, and M. fructigena) was evaluated. Moreover, the possible hemolytic activity of the studied essential oils was assessed. Results and Discussion. – Essential-Oil Yield. Hydrodistillation of the aerial parts of O. vulgare ssp. hirtum from three areas of the Southern Apennines, i.e., Marconia di Pisticci (MP), Mandia (M), and San Giovanni a Piro (SGP), gave yellow-reddish essential oils characterized by a typical odor, with yields of 2.7, 1.0, and 1.0% (v/w, on the fresh weight basis) for the samples from MP, M, and SGP, respectively. Essential-Oil Composition. Table 1 shows the chemical composition of the three essential oils; the compounds are listed according to their elution order on a HP-5 MS capillary column. Altogether, 103 compounds were identified, 79 for O. vulgare from MP, 80 for O. vulgare from M, and 60 for O. vulgare from SGP, accounting for 91.8, 92.0,

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and 91.9% of the total oil compositions, respectively. In the oil from MP, the total phenols represented 85.8% of the oil with similar contents of carvacrol and thymol. The oil from M contained a phenolic fraction constituting 77.4% of the oil, while in the oil from SGP, the phenolic fraction made up 67.1% of the oil. In total, 21 oxygenated monoterpenes were identified, with contents ranging from 0.9% (MP) to 3.5% (SGP). Furthermore, 26 sesquiterpene hydrocarbons, with contents ranging from 2.9% (MP) to 17.6% (SGP), were detected. In all oils, germacrene A (0.8 – 4.0%) and transcaryophyllene (1.5 – 3.2%) were the most abundant compounds of this fraction, the other sesquiterpene hydrocarbons being either present in low amounts and traces or absent. The essential-oil compositions of the three O. vulgare populations appeared similar, and the oils belonged to the same chemotype. Indeed, the three oils were characterized by high percentages of phenols and can be classified as oils possessing a carvacrol/thymol chemotype. Kokkini et al. [27] reported that Origanum taxa can be divided in three groups: i) a linalool, terpinen-4-ol, and sabinene hydrate group (Group A), ii) a carvacrol and/or thymol group (Group B), and iii) a sesquiterpene group (Group C). Russo et al. [3] reported four chemotypes for O. vulgare growing in Calabria (Southern Italy), on the basis of their phenolic content, i.e., a thymol, a carvacrol, a thymol/carvacrol, and a carvacrol/thymol chemotype, with the majority of samples belonging to the thymol chemotype. Tuttolomondo et al. [28] reported that wild Sicilian oregano, O. vulgare ssp. hirtum, is a thymol-chemotype species, a feature which distinguishes Sicilian oregano from the same species growing in Greece, characterized, conversely, by carvacrol [29]. Adebayo et al. [17] reported carvacrol as the main compound in the oregano essential oil. The obtained results agree with Lopez-Reyes et al. [30], who reported a-pinene, pcymene, carvacrol, and thymol as the main components of O. vulgare. Moreover, Glsoy [7] studied the relationship between Origanum taxa by means of T-rex data analysis, and the results showed that the chemotaxonomic order of Origanum taxa based on the essential-oil data was congruent with the taxonomic order of the same taxa based on Ietswaarts classification [4]. In particular, O. vulgare ssp. hirtum was placed distantly from other Origanum species; this may be because this species is herbaceous, while others are semi-shrubs. For this reason, the results reported here may help to throw light on the apparently complex chemotaxonomy of the genus Origanum and to clarify the relationship between the chemical composition of essential oils and biotypes and/or chemotypes in this species [5]. Phytotoxic Activity of the Essential Oils. The three essential oils were evaluated for their phytotoxic activity against germination and radicle elongation of Raphanus sativus L. cv. Saxa and Lepidium sativum L., two species frequently utilized in biological assays, and of Sinapis arvensis L. and Phalaris canariensis L., two weed species (Tables 2 – 4). The oils affected the germination and radicle elongation in a different way. At doses of 0.625 and 0.125 mg/ml, the O. vulgare essential oil from MP significantly inhibited the germination of R. sativus seeds (Table 2). At 0.250 mg/ml, the same oil significantly inhibited the germination of L. sativum seeds. At doses of 2.5 and 0.625 mg/ml, the O. vulgare essential oil from M significantly inhibited the germination of P. canariensis. At different doses, the same oil significantly inhibited the radicle elongation of P.

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Table 1. Chemical Composition of the Essential Oils Isolated from the Aerial Parts of Origanum vulgare Collected in Marconia di Pisticci (MP ), Mandia ( M ), and San Giovanni a Piro ( SGP) Compound name and class

RIa a )

RIp a )

MP a-Pinene Sabinene Oct-1-en-3-ol Octan-3-one Myrcene Octan-3-ol a-Phellandrene a-Terpinene p-Cymene Limonene 1,8-Cineole ( Z )-b-Ocimene ( E )-b-Ocimene g-Terpinene cis-Sabinene hydrate Terpinolene Methyl benzoate trans-Sabinene hydrate Linalool p-Mentha-1,3,8-triene Dehydrosabinaketone endo-Fenchol Alloocimene cis-p-Mentha-2,8-dien-1-ol trans-Verbenol Thujan-3-ol Borneol Terpinen-4-ol Myrtenal g-Terpineol 3-( Methyl)sulfanylhexanol trans-Carveol cis-Carveol Thymol methyl ether Carvacrol methyl ether Linalyl acetate cis-Carvone oxide trans-Carvone oxide Thymol and Carvacrol b-Dehydroelsholtziaketone d-Elemene Piperitenone a-Cubebene Thymol acetate Eugenol Piperitenone oxide Cyclosativene

921 966 974 981 986 990 997 1010 1018 1022 1024 1034 1044 1054 1062 1083 1089 1093 1096 1105 1108 1115 1125 1131 1146 1155 1160 1173 1194 1209 1210 1214 1223 1231 1241 1253 1264 1280 1283 1330 1337 1345 1349 1354 1358 1365 1370

1032 1132 1154 1253 1162 1393 1150 1189 1269 1205 1213 1243 1262 1256 1556 1265 1474 1553

1120 1638 1683 1719 1611 1648 1718 1845 1878 1607 1975 1665

2198, 2239 1476 1948 1466 1867 2186 1983 1492

Identification b )

Content [%]

c

– ) – 0.1 tr tr tr tr tr 0.4 tr 0.1 tr tr 0.4 0.3 tr tr tr 0.1 tr tr tr tr tr tr tr 0.2 0.1 tr 0.1 – tr tr – 0.4 tr – tr 84.7 tr – tr – tr tr – –

M d

tr ) tr 0.1 tr – tr tr tr tr – 0.2 – tr tr 0.3 tr tr tr 1.3 – – 0.1 – – – tr tr 0.8 – 0.7 0.1 tr – 0.4 1.9 tr – tr 75.1 – 0.1 – tr tr tr tr tr

SGP – – 0.2 0.1 tr tr tr tr 0.1 tr tr tr – 0.7 0.3 tr – tr 2.6 – – tr 0.1 – – – 0.1 0.2 – – – – – – 1.3 – 0.2 – 65.3 – 0.3 – 0.1 – 0.1 – –

RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,

MS, Co-I MS MS MS MS, Co-I MS MS, Co-I MS, Co-I MS, Co-I MS, Co-I MS, Co-I MS MS MS, Co-I MS MS MS MS MS, Co-I MS MS MS MS MS MS MS MS, Co-I MS, Co-I MS MS, Co-I MS MS MS MS MS MS MS MS MS, Co-I MS MS MS MS MS MS, MS MS

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Table 1 (cont.) Compound name and class

Carvacrol acetate a-Ylangene b-Bourbonene b-Elemene ( E )-b-Damascone trans-Caryophyllene b-Copaene trans-a-Bergamotene a-Guaiene Thymodihydroquinone Aromadendrene a-Humulene Alloaromadendrene cis-Cadina-1(6),4-diene g-Gurjunene g-Muurolene Valencene a-Muurolene Germacrene A d-Amorphene d-Cadinene Zonarene a-Cadinene a-Calacorene Selina-3,7(11)-diene Occidentalol Thymohydroquinone b-Calacorene Germacrene D-4-ol Spathulenol Caryophyllene oxide b-Atlantol Caryophylla-4(12),8(13)-dien-5a-ol a-Muurolol Cubenol a-Cadinol Selin-11-en-4a-ol 14-Hydroxy-9-epi-[( E )-caryophyllene] a-Bisabolol Eudesma-4(15),7-dien-1b-ol Oplopanone ( Z )-Lanceol Benzyl benzoate 2a-Acetoxyamorpha-4,7(11)-diene Khusinol acetate Cyclopentadecadenolide a-Chenopodiol Cedrane-8,13-diol

RIa a )

1372 1375 1384 1390 1413 1419 1428 1434 1437 1439 1442 1453 1458 1462 1474 1480 1493 1498 1505 1511 1523 1530 1535 1540 1545 1550 1552 1561 1573 1574 1581 1606 1635 1640 1645 1653 1657 1669 1682 1685 1734 1759 1760 1803 1815 1840 1853 1896

RIp a )

1890 1493 1535 1598 1830 1612 1568

1628 1689 1661 1687 1704 1741 1740 1499 1773 1729 1743 1942

1942 2069 2150 2008

2080 2255

2219 2568

Identification b )

Content [%] MP

M

SGP

0.1 tr tr – tr 1.5 tr tr – 0.6 – 0.2 tr tr tr – 0.1 tr 0.8 0.1 0.2 tr tr tr – – tr tr – 0.1 0.7 0.1 tr tr tr tr tr 0.1 tr tr – – tr – – tr – 0.1

– tr tr tr tr 2.7 0.5 tr tr – 0.2 0.6 tr 0.1 0.4 0.4 0.2 0.1 2.0 0.2 1.0 0.1 0.1 0.1 tr tr – tr 0.1 0.3 0.3 0.1 – 0.2 0.1 0.4 – tr – 0.1 tr 0.1 – tr tr tr – 0.2

0.4 0.4 tr tr – 3.2 1.2 – – – 0.4 0.4 0.2 0.1 2.0 1.0 1.0 – 4.0 0.8 2.1 0.2 0.1 0.1 – tr – tr 0.5 0.1 0.1 tr – 0.7 – 1.1 – – – 0.1 tr tr – – tr tr tr tr

RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,

MS MS MS MS MS MS, Co-I MS MS MS MS MS MS, Co-I MS, Co-I MS MS MS MS, Co-I MS MS MS MS MS MS MS MS MS MS MS MS MS, Co-I MS, Co-I MS MS MS MS MS MS MS MS, Co-I MS MS MS MS MS MS MS MS MS

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Table 1 (cont.) Compound name and class

Columellarin Ethyl hexadecanoate Methyl linoleate Nezukol Sandaracopimarinal cis-Totarol methyl ether Larixol n-Tricosane Total identified Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Phenolic compounds Oxygenated diterpenes Other compounds

RIa a )

1948 1989 2106 2122 2187 2204 2255 2291

RIp a )

2300

Identification b )

Content [%] MP

M

SGP

– – tr 0.2 tr – tr tr

tr tr tr tr – 0.1 0.2 –

– – – tr – – – –

91.8 0.8 0.9 2.9 1.1 85.8 0.2 0.1

92.0 – 3.4 8.8 1.9 77.4 0.3 0.2

91.9 0.9 3.4 17.6 2.6 67.1 – 0.3

RI, RI, RI, RI, RI, RI, RI, RI,

MS MS MS MS MS MS MS MS, Co-I

a ) RIa and RIp are the Kovats retention indices determined relative to a series of n-alkanes (C10 – C35 ) on the apolar HP-5 MS and the polar HP Innowax capillary columns, respectively. b ) Identification method: RI, comparison of Kovats retention indices with published data; MS, comparison of mass spectra with those listed in the NIST 02 and Wiley 275 libraries and with published data; Co-I, coinjection with authentic compound. c ) –: Not detected. d ) tr: Trace ( < 0.1%).

canariensis (Table 3). At doses of 2.5 and 0.625 mg/ml, the O. vulgare essential oil from SGP significantly inhibited the germination of P. canariensis seeds. At 2.5 and 0.062 mg/ ml, the same oil significantly promoted the radicle elongation of R. sativus (Table 4). Yilar et al. [23] reported the bioherbicidal effects of essential oils isolated from Thymus fallax Fisch. & C. A.Mey., Mentha dumetorum Schult., and O. vulgare. The main components of O. vulgare were thymol (50.41%) and carvacrol (12.96%). This oil inhibited the seed germination and the root and shoot growth of Avena sterilis L., Datura stramonium L., Cucumis sativus L., and Lactuca sativa L. In the literature, it has been documented that some essential oils and the isolated phenolic compounds carvacrol and thymol possessed potent herbicidal effects on weed germination and seedling growth of various plant species [21]. Moreover, several essential oils have been evaluated for their allelopathic properties [22]. In particular, the available literature reported Origanum vulgare ssp. hirtum as an allelopathic plant, evaluating its effects against the biosensor plants Avena sativa and Lemna minor. Drastic effects of these aqueous oregano extracts, showing a substantial herbicidal activity, have been demonstrated [31]. In this context, the ecological role of volatile terpenes in multiple ecological functions and in the phenomenon of allelopathy was confirmed [32] [33]. Antifungal Activity of the Essential Oils. The in vitro activity of the studied essential oils against the three fungal pathogens, registered five days after incubation at 228, is summarized in Table 5. The three essential oils exhibited a high inhibitory

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Table 2. Effect of Increasing Doses of the Essential Oil ( EO ) Isolated from the Aerial Parts of Origanum vulgare Collected in Marconia di Pisticci on the Seed Germination and Radicle Elongation of Four Test Species 120 h after Sowing Germinated seeds a )

Radicle elongation [cm] a )

Test species

EO Dose [mg/ml]

Sinapis arvensis

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

8.9  0.9 9.7  0.6 8.3  0.6 9.7  0.6 9.0  1.0 8.7  0.6 8.7  0.6

1.9  1.1 1.8  1.1 2.2  1.2 2.4  1.2 2.3  1.0 2.0  1.1 1.5  0.7*

Phalaris canariensis

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

9.9  0.3 8.7  0.6** 10.0  0.0 9.3  0.6 9.3  1.2 9.7  0.6 9.3  0.6

3.7  1.1 3.5  1.2 3.9  0.8 3.4  1.4 3.8  1.0 3.8  1.4 3.4  1.3

Lepidium sativum

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

8.2  1.2 8.0  1.7 6.3  1.5 6.3  0.6* 7.3  1.5 7.3  2.1 7.3  1.2

1.1  1.0 1.4  0.9 0.9  0.4 0.6  0.3 1.1  0.9 1.2  1.2 1.2  0.7

Raphanus sativus

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

6.9  1.3 5.3  2.5 4.7  1.2* 7.7  0.6 4.3  1.5* 5.3  1.2 5.3  1.5

2.6  1.4 2.6  1.7 1.6  1.0* 2.0  1.2 2.0  1.0 2.1  0.7 2.1  1.1

a ) Values are means  standard deviations (n ¼ 3); significant differences compared to control: p < 0.05 (*), p < 0.01 (**).

activity against the three target pathogenic fungi, especially at the highest dose of 1000 ppm. In particular, the O. vulgare essential oil from SGP was highly effective against all three fungi, especially against M. laxa, even at the lower dose of 500 ppm. The obtained results agree with those reported by Adebayo et al. [17], who found that O. vulgare essential oil was able to inhibit the mycelial growth of Botrytis cinerea in a dosedependent manner and to achieve complete growth inhibition at higher concentrations of 150 and 200 mg/ml. On the other hand, the results obtained in this study are comparable with the in vitro antimicrobial activity demonstrated by the tested essential oils or their main constituents against several postharvest pathogens, including Monilinia ssp. [34]. In fact, the in vitro antifungal activity of several commercial essential oils, including

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Table 3. Effect of Increasing Doses of the Essential Oil ( EO ) Isolated from the Aerial Parts of Origanum vulgare Collected in Mandia on the Seed Germination and Radicle Elongation of Four Test Species 120 h after Sowing Germinated seeds a )

Radicle elongation [cm] a )

Test species

EO Dose [mg/ml]

Sinapis arvensis

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

8.9  0.9 8.0  1.7 9.7  0.6 9.3  0.6 9.7  0.6 9.3  0.6 9.0  1.7

1.9  1.1 1.6  0.9 1.2  0.8*** 2.5  1.3* 1.8  1.1 2.2  1.4 2.1  1.3

Phalaris canariensis

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

9.9  0.3 10.0  0.0 10.0  0.0 9.3  1.2 9.0  1.0* 9.3  0.6 9.0  1.0*

3.7  1.1 3.0  1.2** 3.1  1.2* 3.1  1.0* 2.7  1.0*** 3.8  1.0 3.1  1.2*

Lepidium sativum

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

8.2  1.2 7.3  1.2 6.0  1.0* 7.3  2.5 7.3  1.5 7.3  1.2 7.7  1.5

1.1  1.0 1.1  0.8 0.8  0.5 1.1  1.1 0.9  0.6 1.0  0.9 0.9  0.7

Raphanus sativus

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

6.9  1.3 6.7  0.6 7.0  2.0 6.3  1.2 4.7  2.5 7.0  1.0 7.3  0.6

2.6  1.4 2.1  0.8 2.6  1.0 2.4  1.4 2.9  1.6 2.2  0.9 2.0  0.9

a ) Values are means  standard deviations (n ¼ 3); significant differences compared to control: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

Australian lemon myrtle, cinnamon bark, oregano, thyme, clove bud, valerian, and Australian tea tree oil, on the mycelium growth and spore germination of M. fructicola was studied, and the results showed that M. fructicola exhibited a different level of sensitivity to each tested essential oil, showing an excellent control of the pathogens with respect to mycelium growth and spore germination at very low concentrations. However, higher concentrations are needed to reduce the fungal growth significantly. Hence, the fungicidal effect of the studied essential oils depended on the specific toxicity of their main active constituents or on synergic effects of several components [35]. The present results are in agreement with previous data showing that the mycelium growth of serious phytopathogens such as Phytophthora citrophthora, Rhizopus stolonifer, Botrytis cinerea, and Penicillium italicum were completely

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Table 4. Effect of Increasing Doses of the Essential Oil ( EO ) Isolated from the Aerial Parts of Origanum vulgare Collected in San Giovanni a Piro on the Seed Germination and Radicle Elongation of Four Test Species 120 h after Sowing Test species

EO Dose [mg/ml]

Germinated seeds a )

Radicle elongation [cm] a )

Sinapis arvensis

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

8.9  0.9 9.7  0.6 10.0  0.0 8.3  0.6 9.3  0.6 8.7  0.6 9.7  0.6

1.9  1.1 1.5  0.9* 1.8  1.1 1.8  1.1 2.0  0.9 2.0  1.1 1.7  0.8

Phalaris canariensis

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

9.9  0.3 10.0  0.0 9.7  0.6 10.0  0.0 9.0  1.0* 9.3  0.6 9.0  1.0*

3.7  1.1 3.5  1.1 3.6  1.3 3.4  1.4 3.2  1.2 3.6  1.2 3.4  1.4

Lepidium sativum

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

8.2  1.2 8.3  0.6 7.3  1.5 8.7  1.5 8.7  0.6 8.3  0.6 7.3  1.5

1.1  1.0 0.8  0.9 1.3  1.3 0.8  1.0 1.0  1.0 0.9  0.9 0.6  0.7*

Raphanus sativus

0.000 (Control) 0.062 0.125 0.250 0.625 1.250 2.500

6.9  1.3 7.0  2.6 6.7  1.5 6.7  1.5 8.0  1.0 7.3  0.6 7.0  1.0

2.6  1.4 4.1  1.8*** 2.3  1.2 2.7  1.7 3.1  1.7 2.6  1.6 3.5  1.3*

a ) Values are means  standard deviations (n ¼ 3); significant differences compared to control: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

inhibited by some main bioactive components of O. vulgare oil, such as citral, thymol, and carvacrol [15]. Hemolytic Activity of the Essential Oils. No hemolytic activity was found for the tested oils, indicating the possibility of using these oils and their individual components as safe and natural biopesticides. Possibly, they might also be used by the pharmaceutical industry to develop potential treatments of human diseases. The absence of any hemolytic effect of the studied essential oils indicates the possibility of their large-scale use to either control postharvest diseases or in open field against some serious phytopathogens without causing any sanitation effect to farmers, workers, and consumers. Indeed, these results indicate the absence of hemolytic effects of the residues and sediments after oil applications on stored fruits.

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Table 5. Antifungal Activity of the Essential Oils ( EOs) Isolated from the Aerial Parts of Origanum vulgare Collected in Mandia ( M ), Marconia di Pisticci (MP ), and San Giovanni a Piro ( SGP) Sample

EO Dose [ppm]

Percentage of growth inhibition ( PGI ) [%] a ) Monilinia laxa

M. fructigena

M. fructicola

O. vulgare ( M )

1000 500 250

100.0  0.00 (a) 79.6  2.21 (b) 67.9  1.10 (c)

100.0  0.00 (a) 69.2  1.67 (c) 58.0  0.83 (d)

100.0  0.00 (a) 54.3  2.65 (c) 14.3  6.19 (e)

O. vulgare ( MP)

1000 500 250

100.0  0.00 (a) 74.2  3.31 (c) 48.4  2.21 (d)

100.0  0.00 (a) 62.1  3.35 (cd) 38.4  3.35 (e)

100.0  0.00 (a) 45.6  2.65 (c) 20.6  2.65 (de)

O. vulgare ( SGP)

1000 500 250

100.0  0.00 (a) 92.9  1.10 (a) 46.8  4.42 (d)

100.0  0.00 (a) 86.9  1.68 (b) 43.1  3.34 (e)

100.0  0.00 (a) 85.0  1.77 (b) 25.6  0.88 (d)

a ) Values are means  standard deviations (n ¼ 3); means followed by the same letter in parentheses within the same column are not significantly different by the Tukey test (p < 0.05).

Conclusions. – The essential oil of three populations of O. vulgare were characterized according to their geographic origin. The three oils showed moderate phytotoxic activity, probably due to the presence of a substantial amount of phenolic compounds, in particular of thymol and carvacrol. The three essential oils also exhibited antifungal activity against three Monilinia species. At a dose of 1000 ppm, they completely inhibited the fungal growth. Moreover, these oils showed no hemolytic toxicity. The results obtained in this study contribute to the elucidation of the importance of essential oils as ecological and chemical mediators in biochemical interactions among higher plants and suggest the possibility of using O. vulgare essential oil as safe alternative to synthetic fungicides for the control of Monilinia species. Their antifungal activity could further be screened to develop novel types of selective fungicides for the eco-sustainable agriculture. The development of such natural antimicrobials and biopesticides would help in decreasing the harmful impact of synthetic pesticides such as deposits, resistance, and environmental pollution. Experimental Part Plant Material. The aerial parts of Origanum vulgare L. ssp. hirtum (Link) Ietsw. were collected in June 2012 from populations growing wild in different areas of the Southern Apennines: Marconia di Pisticci (MP), 106 m.s.l.; Mandia (M), 600 m.s.l.; and San Giovanni a Piro (SGP), 450 m.s.l. The representative homogeneous samples of each population were collected during the balsamic time, corresponding to the flowering stage. The plants were identified by V. D. F., and voucher specimens (DFE203/2012, DFE218/012, and DFE301/2012 for MP, M, and SGP, resp.) have been deposited with the Herbarium of the Medical Botany Chair of the University of Salerno. Isolation of the Essential Oils. Portions of 100 g of fresh aerial parts of each sample were ground in a Waring blender and then subjected to hydrodistillation for 3 h, according to the standard procedure described in the European Pharmacopoeia [36]. The oils were solubilized in hexane, dried (Na2SO4 ), and stored under N2 at 48 in the dark until tested and analyzed. The calculated essential oil yield was expressed in % (v/w), based on the weight of the fresh plant material. All extractions were done in triplicate.

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GC-FID Analysis. The GC-FID analyses were carried out with a Perkin-Elmer Sigma-115 gas chromatograph equipped with a flame ionization detector (FID), a data handling processor, and two fused-silica cap. columns, an apolar HP-5 MS (30 m  0.25 mm i.d., film thickness 0.25 mm) and a polar HP Innowax (polyethylenglycol; 50 m  0.20 mm i.d., film thickness 0.25 mm) column. The oven temp. was programmed isothermal at 408 for 5 min, then rising from 50 to 2708 at 28/min, and finally held isothermal at 2708 for 20 min; injector temp., 2508; detector temp., 2908; carrier gas, He (1.0 ml/min); injection mode, splitless; injection volume, 1 ml (0.1% essential-oil soln. in pentane). The relative essential-oil contents of the components were obtained by peak area normalization, without calculating response factors. GC/MS Analysis. The GC/MS analyses were performed with an Agilent 6850 Ser. II apparatus equipped with an Agilent 5973 mass selective detector (MSD) and a DB-5 fused-silica cap. column (30 m  0.25 mm i.d., film thickness 0.33 mm). The GC conditions were as described above (cf. GC-FID Analysis); transfer-line temp., 2958; ionization energy, 70 eV; electron multiplier energy, 2000 V; mass range, 40 – 500 amu; scan speed, 5 scans/s. Identification of Essential-Oil Components. The identification of the essential-oil constituents was based on the comparison of their Kovats retention indices (RIs), determined rel. to the tR values of nalkanes (C10 – C35 ) on both cap. columns, with those in [37 – 40] and their mass spectra with those of authentic compounds available in our laboratories or those listed in the NIST 02 and Wiley 275 mass spectral libraries [41]. For some compounds, the identification was confirmed by coinjection with an authentic sample (cf. Table 1). Phytotoxic Activity. The phytotoxic effects of the three essential oils were tested with a bioassay based on the evaluation of the seed germination and subsequent radicle growth of Raphanus sativus L. cv. Saxa (radish), Lepidium sativum L. (garden cress), and the two weed species Sinapis arvensis L. (wild mustard) and Phalaris canariensis L. (canary grass). The seeds of radish and garden cress were purchased from Blumen SRL (Piacenza, Italy), while those of mustard and canary grass were collected from wild plants. The seeds were surface sterilized in 95% EtOH for 15 s and sown in Petri dishes (90 mm diameter) containing five layers of Whatman filter paper impregnated with 7 ml of either the negative control soln. (dist. H2O or dist. H2O/acetone 99.5 : 0.5) or the essential oil solns. at the different assayed doses. The essential oils were dissolved in and diluted to the desired doses (2.5, 1.25, 0.625, 0.25, 0.125, and 0.062 mg/ ml) with dist. H2O/acetone 99.5 : 0.5. The germination conditions were 20  18 and natural photoperiods. Controls performed in dist. H2O/acetone 99.5 : 0.5 showed no significant differences in comparison to the controls in dist. H2O. The seed germination was observed directly in the Petri dishes, each 24 h. A seed was considered germinated, when the protrusion of the root became evident [42]. After 120 h (on the 5th day), the effects on the radicle elongation were measured and expressed in cm. Each determination was repeated three times, using Petri dishes containing ten seeds each. Data were expressed as mean  standard deviation (SD) for both germination and radicle elongation. Data were ordered in homogeneous sets, and the Students t-test of independence was applied [43]. Antifungal Activity. Fungal Isolates. The plant pathogenic fungi tested were stored as pure cultures maintained in the Mycotheca of the School of Food, Forestry and Environmental Science, Basilicata University (Potenza, Italy). The fungal species were maintained on potato dextrose agar (PDA) at 48. The micromycetes used in the experiments were the following: Monilinia laxa (isolate number 1517 from pear), Monilinia fructicola (isolate number 1561 from plum), and Monilinia fructigena (isolate number 1521 from apple). Identification of Studied Pathogenic Fungi. The fungi were identified according to traditional methods using light microscopy observations. Molecular methods based on polymerase chain reaction (PCR) were also used to confirm the identification of the fungal isolates. The total nucleic acids were extracted from the pure culture with a commercial kit (Dneasy Plant Mini Kit, Qiagen), according to the manufacturers instructions. The DNA was amplified using the universal primer pair ITS4/ITS5 [44]. The amplicons obtained were directly sequenced and the resulting sequences were compared with those available in the GenBank using BLAST software [45]. Gene sequencing of the ITS region confirmed the identification done by traditional methods. The sequences obtained were deposited with the GenBank (accession codes HF678387 for M. laxa, HF678388 for M. fructicola, and HF678389 for M. fructigena).

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Antifungal Test. The possible antifungal activity of the three O. vulgare essential oils (M, MP, and SGP) was determined as follows. The essential oils were dissolved in 1 ml of a 0.2% soln. of Tween 20  in H2O and then added into 14 ml of potato dextrose agar (PDA) to obtain final concentrations of 250, 500, and 1000 ppm of each essential oil under study. The mixture was poured in a Petri dish, and after complete drying under laminar flow, mycelia disks of 5 mm in diameter from 96-h fresh fungal cultures were inoculated in the center of each Petri dish. All dishes were incubated at 228 for 96 h in the dark, and the diameter of fungal mycelium growth was measured in mm. Dishes containing only PDA (without oils) or PDA and a 0.2% soln. of Tween 20  in H2O were inoculated with fungal disks as control. The antifungal activity was expressed as percentage of growth inhibition (PGI) calculated according to Zygadlo et al. [46] (Eqn. 1): PGI [%] ¼ (GC  GT)/GC  100

(1)

where GC and GT represent the average diameter of fungal mycelia grown in PDA (control) and in PDA containing the essential oil, resp. Hemolytic Activity (cell-membrane hemolysis of red blood cells). The three essential oils were evaluated for their ability to hemolyze the cell membrane of red blood cells (RBCs) according to the method of Munsch and Alatossava [47]. Fresh cattle blood treated with heparin (25 ml of heparin 1000U in 5 ml of blood) was washed three times in Tris buffer (0.72 g Tris-HCl, 1.16 g NaCl, 0.07 g EDTA, pH 7) and then centrifuged (20000g, 3 min) at r.t. to obtain RBCs. Bacto blood agar base (BAB; Difco) was added to the RBCs (0.25%) and the mixture was poured in Petri dishes. Aliquots of 20 ml of essential oils (diluted 1 : 1 and 1 : 2 (v/v) in a 0.2% soln. of Tween 20 in H2O) were added in the center of the Petri dishes. After 48 h of incubation of the dishes at 228, the hemolytic activity was determined by observing the formation of hemolysis areas in the dishes. The test was repeated twice with three replicates. Data were subjected to analysis of variance (ANOVA) and to Tukeys post-hoc multiple comparison test using the SPSS statistical software package version 13.0 (2004) to detect the significance of the bioactivity of the studied essential oils.

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