Chemical Composition and Antifungal Effects of Vitex agnus-castus L . and Myrtus communis L . Plants

The purpose of this study was to assess the effectiveness of essential plant oils from Vitex agnus-castus L. (VAC) and Myrtus communis L. against the plant pathogens, Fusarium oxysporum f. sp. radicis-lycopersici (Sacc.) W.C. Synder & H.N. Hans, Rhizoctonia solani J.G. Kühn., Sclerotinia sclerotiorum (Lib.) de Bary and Verticillium dahliae Kleb., and to determine the chemical composition of the compounds in these essential oils. GC/MS analysis was identified 25 different compounds in VAC essential oil, while the main compounds were determined as Eucalyptol (17.75%), β-Caryophyllene (13.21%) and Spathulenol (10.41%). On the other hand, the essential oil of M. communis, consisted of 16 different compounds which were Eucalyptol (49.15%), Myrtenol (19.49%) and α-Pinene (8.38%) being its main compounds. An assessment of antifungal activity was performed under in vitro conditions. Plant pathogens were inoculated onto Petri dishes (60 mm) containing PDA medium (10 mL/Petri), and plant essential oils were applied at concentrations of 0.5, 1, 1.5, 2, 5 and 10 (μL/Petri) into the 5 mm diameter wells opened on the Petri dish surface. After that, the Petri dishes incubated at 22±2 °C. The results of this study, the essential oil of M. communis, at a dose of 10 μL/ Petri, inhibited the 100% mycelium growth of V. dahliae, S. sclerotiorum and R. solani. The highest dose of VAC essential oil was also 100% inhibited V. dahliae and S. sclerotiorum. The LC50 and LC90 values of M. communis and VAC essential oil calculated for V. dahliae, FORL, S. sclerotiorum and R. solani. This plant extracts were shown by in vitro conditions to be potential antifungal agents.


Introduction
Although compounds with antimicrobial activity have been identified in over 1,340 plant species; about 60 are mentioned by Nychas (1995) and Beuchat (1994) (Pillai and Ramaswamy, 2012).Previous studies have demonstrated that the essential oils and plant extracts of many plant species exhibit various antibacterial, antifungal, insecticidal and antioxidant activities against plant pathogens and insects (Burt, 2004;Kordali et al., 2005, Polatoğlu et al., 2013).Some of these essential oils are being used in cancer treatment (Sylvestre et al., 2005), while others are being used for food preservation and in the cosmetics industry.Essential oil compounds offer a rich source in terms of biological activity (Prabuseenivasan et al., 2006).
Vitex agnus-castus L. (VAC) is an evergreen shrub plant of the Verbennaceae family, commonly known by names such as chaste tree, chasteberry, or monk's pepper.VAC is naturally distributed in many provinces of Turkey, including Amasya, Antalya, Bursa, Çanakkale, Muğla and Trabzon (Davis, 1972a).The essential oils and plant extracts of VAC has been reported to have antioxidant, antimicrobial and antifungal functions.Essential oils obtained from VAC seeds have demonstrated strong antifungal activity on Candida species (Asdadi et al., 2014).However, there are only a few scientific studies investigating whether or not these essential oils are effective against plant pathogens.
Myrtus communis L. is a bush-like perennial plant, naturally distributed in many provinces across Turkey, such as Antalya, Hatay, Istanbul, Ordu and Sinop (Davis, 1972b).There are various studies in the literature investigating the chemical composition of essential oils of M. communis (Zomorodian et al., 2013;Hennia et al., 2015).The essential oil of M. communis has been investigated efficiency of antimicrobial, Gas chromatography/mass spectrometry (GC/MS) analysis Compound analysis was performed through 7890 A model GC system with automatic autosampler system, 5975C inert MSD with Triple-Axis Detector.The samples were diluted with hexane in the 1:10 ratio and they were injected in the mode of split (10:1) as HP-5 (5% Phenyl Methyl Siloxan) 1 µL for distinction of compound.The internal pressure of helium used as the carrier gas was set to 5 psi.The temperatures of both injector and detector planned as 250 ℃.FID detector used for quantitative values.Clone's starting temperature was 60 ℃, its final temperature was 240 ℃ and it was programmed to increase 4 ℃ per minute.
For GC/MS distinction, electron ionization system with 70 eV ionization powered was used.The flow rate of helium that used as the carrier gas was 1.0 mL per minute, the clone was HP-5Ms (30m × 0.25mm × 0.25µm film), and the beginning and the final temperatures and works programme were the same with GC.Injector and MS's transfer temperatures were set to be respectively 230 ℃ and 250 ℃.
As in the gas chromatography, 1.0 µ L split/splitles (10:1) of the sample diluted with hexane and transferred to the clone.Identification of oil compounds was accomplished by comparison of their mass spectral fragmentation patterns with available mass library (WILLEY and NIST).

In vitro antifungal effect of the essential oils
The antifungal activities of the compounds were determined by the agar well diffusion method (Tepe et al., 2005).The PDA was autoclaved and then cooled to 40 o C, after which they were transferred to 60 mm diameter Petri dishes (10 ml Petri -1 ) and then, 5 mm diameter wells were opened on the PDA within the Petri dishes.The plant essential oils were added to the wells at concentrations of 0.5, 1, 1.5, 2, 5 and 10 μl/ Petri.Mycelium discs of 5 mm were placed at equal distances to these wells.The Petri dishes were incubated at 22 ± 2 ℃.The Petri dishes were evaluated by based on the growth observed in the control group.The inhibition zone between the wells and the mycelium discs were measured by using a compass.The measured values were compared with the controls, and the percent (%) inhibition was calculated by using the formula below (Pandey et al., 1982)
Plant pathogens cause significant yield losses in both Turkey and all around the world.Among these pathogens, Sclerotinia sclerotiorum (Lib.) de Bary is to cause white mold disease in 408 plant species belonging to 275 different varieties (Boland and Hall, 1994).Another, Verticilium dahliae is a soilborne pathogen that causes Verticillium wilt disease, which affects over 200 plant species, including the tomato (Fradin and Thomma, 2006).R. solani is a pathogen that causes various diseases, affecting the roots and tubers of different plant species (Carling et al., 1989), and induces to a significant loss of potato crops (Yanar et al., 2005).Fusarium oxysporum f. sp.radicislycopersici (Sacc.)W.C. Synder & H.N. Hans (FORL) is one of the most important and destructive tomato pathogens (Benhamou et al., 1994).Although numerous studies have conducted over the years into the control and management of plant pathogens, they continue to be a significant cause of crop loss.The persistence of these pathogens has also lead to a growing resistance to commercial synthetic pesticides.For this reason, research is increasingly focusing on new active substances and control methods that could serve as alternatives to commercial fungicides.

Plant material
The Vitex agnus-castus and Myrtus communis were collected from Demre district of Antalya province (Turkey) in 2014.

Fungus cultures
The plant pathogenic fungi have been isolated from different host plants of Fusarium oxysporum f. sp.radicislycopersici (the cause of Fusarium wilt in tomato), Rhizoctonia solani (the cause of root rot in potato), Sclerotinia sclerotiorum (the cause of white mold in cucumber) and Verticillium dahliae (the cause of Verticillium wilt in tomato).The plant pathogenic fungi were growth in the 90 mm of Petri plates including 20 ml potato dextrose agar (PDA) at 22±2 ℃ for 7 days and later used in the study.

Extraction of essential oils
The plant parts of V. agnus-castus (seeds) and M. communis (leaf) (100 g) were collected through hydro-distillation using a Schilcher device.After that, the plant samples were weighted, distilled water added to them at a ratio of 1:10 w/v, and boiled for two hours.The obtained essential oils were stored at + 4 ℃ until used.essential oils are shown in Tables 1-2 and Figs.1-2.The essential oil of M. communis consisted of 16 compounds.The main compounds of this essential oil were Eucalyptol (49.15% RT: 15.990), Myrtenol (19.49%RT: 21.823) and α-Pinene (8.38% RT: 12.727) (Table 1).
Similarly, 25 compounds were identified in VAC essential oil collected in province of Antalya, Turkey, while the main compounds were determined as Eucalyptol (17.75% RT: 16.005), β-Caryophyllene (13.21%RT: 29.508) and Spathulenol (10.41%RT: 34.322) (Table 2).Comparisons were drawn with previous studies determining the chemical content of VAC-derived essential oil.Stojkovic et al. (2011) identified 46 compounds in VAC essential oil, while 34 were identified in the leaves of VAC growing in the North-Central region of Nigeria, and 32 were identified in a study conducted by Katiraee et al. (2015).The main compounds in VAC essential oil were determined as α-Pinene (19.48%),Cyclohexene, 1-methyl-4-(1-methylethenyl) (13.37%) and Sabinene (6.89%) (Katiraee et al., 2015).In essential oil of VAC seeds, the main compounds were determined as caryophyllene oxide (24.9%), n-hexadecane (12.5%) and αterpinyl acetate (11.6%) (Ghannadi et al., 2012).The main compounds in essential oil from VAC growing in the Northern Brazil were determined as Eucalyptol, trans-βfarnesene, sabinene, α-pinene, α-terpenyl acetate, βcaryophyllene and bicyclogermacrene (Zoghbi et al., 1999).These differences stem from various ecological characteristics, such as the part of the plant being analyzed, the region where the plants are collected, and the soil.It has reported that the essential oil composition may also differ, according to the flowering period of the plants, the region where they are cultivated and climatic factors (Senatore et al., 1997).

In vitro antifungal activity
The results regarding the antifungal activity of VAC essential oil are shown in Table 3. VAC essential oil exhibited a high degree of fungicidal activity against the tested fungi.Compared with the control, VAC essential oil at a dose of 10 µL/ Petri reduced the mycelium growth of V. dahliae and S. sclerotiorum by 100%, the mycelium growth of FORL by 70.70%, and the mycelium growth of R. solani by 65.35% (Table 3).According to the dose-effect experiments, the LC50 and LC90 values of VAC essential oil were 1.063 and 7.313 µL/Petri for V. dahliae; 0.738 and 11.026 µL/ Petri for FORL; 3.322 and 9.729 µL/ Petri for S. sclerotiorum; 2.355 and 7.864 µL/Petri for R. solani, respectively (Table 5).Katiraee et al. (2015) reported that VAC essential oil exhibits antifungal effects against Alternaria spp., Penicillium spp., Aspergillus niger and Aspergillus flavus, and that its MIC values against these pathogenic fungi were 6.3, 12.5, 0.8 and 12.5 µL/Petri dish respectively.Studies have also demonstrated that VAC methanol extract exhibits a strong antifungal effect against Pythium ultimum, a tomato pathogen, under both in vivo and in vitro conditions (Svecova et al., 2013) and that essential oil of VAC fruits and leaves reveals antifungal and antibacterial effects in vitro, while also being effective against Aspergillus niger on apple fruits under in vivo conditions (Stojkovic et al., 2011).
The essential oil of M. communis displayed a strong fungicidal effect against the pathogens used in the experiment.Results concerning this fungicidal effect summarized in Table 4.The effect of M. communis essential oil on mycelium growth varies depending on the targeted pathogen and increasing dose.The dose of 10 µL/Petri dish, M. communis essential oil inhibited 100% mycelium growth of V. dahliae, S. sclerotiorum and R. solani, and inhibiting FORL mycelium growth by 93.00% compared with the control.
The values of LC50 and LC90 were calculated for plant diseases.According to the dose-effect experiments, the LC50 and LC90 values of essential oil of M. communis were 0.607 and 3.302 µL/Petri dish for V. dahliae; 0.344 and 8.691 µL/Petri dish for F. lycopersici; 3.207 and 8.269 µL/Petri dish for S. sclerotiorum and 1.072 and 9.765 µL/Petri dish for R. solani respectively (Table 5).
There are various studies in the literature investigating the biological activity of M. communis essential oils and extracts.It has reported that essential oil of M. communis exhibits antibacterial effects against pathogenic bacteria, such as Staphylococcus aureus, Proteus mirabilis and Kleibsiella pneumonia (Hennia et al., 2015).Cannas et al. (2014), found   the antifungal effects of M. communis essential oil against Candida species.It has also reported that M. communis essential oil at a dose of 1600 ppm inhibits the growth of the R. solani pathogen by 60% (Curini et al., 2003).In the present study, mycelium growth of R. solani was inhibited 65.35% by 10 ml dose of VAC essential oil, and 100% by the essential oil of M. communis.
In this study, antifungal activity of essential oils of Myrtus communis and Vitex agnus-castus on Fusarium oxysporum f. sp.radicis-lycopersici, Rhizoctonia solani, Sclerotinia sclerotiorum and Verticillium dahliae were investigated.The essential oils were showed antifungal activity against phytopathogenic fungi.Inconclusion, according to the results, essential oils of Myrtus communis and Vitex agnus-castus present a potential alternative to commercial fungicides for the management of plant pathogen fungi.

Table 1 .
Chemical composition of Myrtus communis L. essential oil

Table 2 .
Chemical composition of the Vitex agnus-castus L. essential oil

Table 5 .
Results of the dose-effect experiments between plant essential oils and plant pathogen fungi