Antifungal activity of six plant essential oils from Serbia against Trichoderma aggressivum f . europaeum

Six essential oils (EOs) extracted from plants originating in Serbia were assayed for inhibitory and fungicidal activity against a major fungal pathogen of button mushroom causing green mould disease, Trichoderma agressivum f. europaeum. The strongest activity was demonstrated by the oils of basil (Ocimum basilicum L.) and peppermint (Mentha piperita L.). Medium antifungal activity of St. John's wort (Hypericum perforatum L.) and walnut [Juglans regia (F)] oils was also recorded. Oils extracted from yarrow (Achillea millepholium L.) and juniper (Juniperus communis L.) exhibited the lowest activity. Peppermint oil showed fungicidal effect on the pathogen, having a minimum fungicidal concentration of 0.64 μl ml-1. The main components of peppermint essential oil were menthone (37.02%), menthol (29.57%) and isomenthone (9.06%).


INTRODUCTION
Production of button mushroom [Agaricus bisporus (Lange) Imbach] is affected by several diseases, resulting in lower yield and quality of marketable fruiting bodies.The major fungal pathogen, T. aggressivum f. europaeum Samuels & W. Gams, has been transmitted from the British Isles to many European countries including Serbia (Kosanović et al., 2013).The pathogen causes green mould disease, accounting for losses ranging between 30% and 100% (Grogan, 2008).Green mould disease of mushrooms is characterized by dense white mycelia of fast-growing colonies on casing or compost that changes colour into green after extensive sporulation.Spots on fruiting bodies of A. bisporus are early and accompanying symptoms.In serious outbreaks, no fruiting bodies are produced (Seaby, 1996).
A common method of disease control in mushroom farms is treatment of casing soil with disinfectants and fungicides.Many pathogens develop resistance to fungicides, and ongoing EU pesticide reviews have resulted in withdrawal of approval for many chemicals, mainly in the group of benzimidazoles (Fletcher et al., 1989;Grogan, 2008).As a result, only a few chemicals are currently available, and effective alternatives are required.The fungicides that are officially recommended in mushroom industry are: prochloraz in the EU countries, and chlorothalonil, thiabendazol and tiophanate-methyl in North America (Beyer & Kremser, 2004;Grogan, 2008).Initially, benzimidazole fungicides applied to spawn in the US had provided good control of T. aggressivum f. aggressivum, but the problem of occurring resistant isolates has emerged nevertheless, leaving improved hygiene as the only option for control of this pathogen (Romaine et al., 2005).The most effective fungicide in mushroom disease control is prochloraz (Grogan, 2008).Prochloraz is still effective against fungal pathogens in Serbia (Potočnik et al. 2008(Potočnik et al. , 2009(Potočnik et al. , 2010;;Kosanović et al., 2013), though decreased sensitivity of pathogenic fungi of the genera Lecanicillium and Cladobotryum has been already reported in Spain and Great Britain (Gea et al., 2005;Grogan, 2008).
In the course of evaluation of alternative means, antimicrobial properties of essential oils (EOs), as well as their components, have been demonstrated.Oils with very strong activity could be proved as promising agents in future extensive research and in vivo examination.Oils of oregano (Origanum vulgare L.) and common thyme (Thymus vulgaris L.) have shown very high in vitro activity against T. aggressivum f. europaeum, T. harzianum Rifai, and T. atroviride P. Karsten, while oils and components of peppermint (Mentha piperitaL.)have been found to inhibit the growth of T. viride Tul.(Soković & van Griensven, 2006).Addition of tea tree oil [Melaleuca alternifolia (Maiden & Betche) Cheel] to oyster mushroom substrate or button mushroom casing has resulted in considerable in vivo inhibition of T. harzianum (Angelini et al., 2008;Kosanović et al., 2013).
Peppermint, basil, yarrow, St. John's wort, walnut, and juniper have been acknowledged as herbs with a plenty of pharmacological properties.These plants have been used in herbal medicine, and some of them for their flavours and as antimicrobial agents (Soković & van Griensven, 2006).Therefore, these plants growing in Serbia have been chosen for in vitro testing of antimicrobial activities of their oils against T. aggressivum f. europaeum in an attempt to promote their use as alternative products for disease control.All plant material was air-dried in shade at room temperature for 20 days, and then subjected to hydrodistillation for 2.5 h, in a Clevenger type apparatus.The obtained essential oils were dried over anhydrous sodium sulphate and preserved in sealed vials at 4°C until further analysis.

Test organism and inoculum preparation
The isolate of Trichoderma aggressivum f. europaeum T77 used in the study was obtained from an A. bisporus substrate with green mould disease symptoms collected from a Serbian mushroom farm (locality Barajevo-Lisovići) in 2010 and previously identified (Kosanović et al., 2013).The isolate was maintained on Potato Dextrose Agar (PDA) medium at 20 o C for 72 hours.Conidia were harvested by flooding the plates with 10 ml of sterile distilled water and Tween 20 (v/v 0.01%), followed by filtration through a double layer of cheesecloth.Conidial suspension was prepared daily in sterile saline and adjusted to a concentration of approximately 10 6 conidia ml -1 .

Antifungal activity of essential oils in vitro
Antifungal activity was tested on PDA medium in glass Petri plates (R=100 mm).The medium was inoculated with the investigated fungi by pipetting 20 µl of conidial suspension into each well cut at the centre of the plate (R=10 mm).Inoculum was then exposed to the volatile phase of EOs for 48 h at 20 o C. The oils were applied as a single drop onto the inner side of each plate cover at concentrations of 0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 μlml -1 of air inside Petri plates using a micropipette.The bottom of each plate was immediately placed upon the cover.The plates were sealed with parafilm to prevent gas exchange with the outside environment.Oil concentrations that completely inhibited bacterial growth after two-day-exposure at 20ºC were considered to be fungistatic and the lowest of these concentrations was determined as Minimum Inhibitory Concentration (MIC).The plates were then opened and ventilated in a laminar flow hood for 30 min in order to remove volatiles and determine fungicidal effect.Oil concentrations were considered fungicidal if no fungal growth was observed two days after ventilation.The lowest concentration that had fungicidal effect was defined as Minimum Fungicidal Concentration (MFC) (Tanović et al., 2006).Four replicates per treatment were used and the experiment was repeated twice.

Essential oil analysis
Analyses of all essential oils were performed by gas chromatography (GC) using two detector types.An Agilent GC (7890A model) was equipped with a split/ splitless injector, a flame ionization detector (FID) and a HP-5 capillary column (30 m, 0.32 mm i.d., 0.25 μm film thickness).Injector and detector temperatures were set to 250 and 300 0 C, respectively, while the nitrogen flow rate was 1 ml/min.Column temperature was linearly programmed from 50 to 250 0 C at 4 0 C/min and held for 10 minutes.Analyses with a mass spectrometer (MS) as the detecting device were conducted using Varian CP-3800/Saturn 2200 model.Both the injector temperature and column temperature programmes were the same as for GC-FID analysis, while separation was performed using Agilent DB-5MS column (30 m, 0.25 mm i.d., 0.25 μm film thickness).Helium was used as the carrier gas (1 ml/ min), and the ion trap and transferline temperatures were set to 250 0 C and 280 0 C, respectively.The mass detector was operated in the electron impact (EI) mode (70 eV; 40-600 m/z range).In both cases, essential oil solutions in n-hexane (1%) were injected in split mode (1:20).
In order to determine the retention indices (RI), a mixture of n-alkanes (C 6 -C 28 ) was analysed by both GC-FID and GC-MS under the same conditions as essential oils.
Identification of essential oil components was performed using both Wiley 7.0 mass spectral library and the obtained RI data, while quantitative data were expressed as area percent obtained by GC-FID analysis.
As only peppermint oil exhibited the lethal effect on the pathogen (Table 1), it was chosen for chemical composition analysis (Table 2).As a result of that analysis, thirty two components of peppermint essential oil were identified, and they accounted for 99.07%(v/w) of total oil mass.T. harzianum and P. tolaasii (Soković & van Griensven, 2006;Soković et al., 2009).Bouchra et al. (2003) had found that M. pulegium exhibited moderate activity against Botrytis cinerea, and inhibition of mycelial growth of 58.5% at 250 µl ml -1 .We also found a remarkable activity of peppermint oil against T. aggressivum, having both fungicidal and inhibitory effects.Sweet basil Ocimum basilicum L (Lamiaceae) is usually used in herbal medicine.A previous study in Iran had shown only low activity of sweet basil EO, with 42.5% inhibition of Botrytis cinerea mycelial growth at 0.5 µl ml -1 using the macrodilution method (Abdolahi et al., 2010).However, we found that basil oil from Serbia inhibited the growth of T. aggressivum 100% at 0.02 µl ml -1 .
Yarrow (Achillea mylefollium L., Asteraceae) is a native herb in Serbia and has been used as one of the principal constituents of traditional cures and remedies.It has been used in medicine because of its anti-inflammatory, spasmolytic, hemostatic and digestive effects.Interestingly, no antimicrobial activity of yarrow extracts or oils has been observed yet, although the EO of two other species of this genus, namely A. clavennae and A. alpine, respectively, have been recognized to have some antibacterial properties (Woods-Panzaru et al., 2009).In our study, only a weak inhibitory effect of yarrow oil to the pathogen was found (MIC value of 0.64 µl ml -1 ).
Persian or common walnut, Juglans regia L. ( Juglandaceae) is an important deciduous tree commercially cultivated in Serbia.Green walnuts, shells, kernels and seeds, bark and leaves have been used in pharmaceutical and cosmetic industries.Walnut leaves are considered a source of healthcare compounds and have been intensely used in traditional medicine for treatment of venous insufficiency and haemorrhoidal symptomatology and for its anti-diarrhetic and antihelminthic properties.Antibacterial activities of leaf EO of walnut from India had been previously reported by Rather et al. (2012).We found a mild activity of walnut oil from Serbia against the pathogen (MIC=0.16µl ml -1 ).
The juniper (Juniperus communis L., Cupressaceae) is widespread in Serbia and it grows in temperate regions of Europe, Asia and North America.The EO of juniper female cones has diuretic and antiseptic properties and it is a gastrointestinal irritant.Examined by the diffusion method, a juniper oil from Croatia has shown strong fungicidal activity against yeast, yeast-like fungi and dermatophytes with MIC values below 10% (v/v) (Pepeljnjak et al., 2005).The juniper oil from Serbia only inhibited growth of the pathogen by 83.68% at 0.64 µl ml -1 (Figure 1).
Among the six essential oils analyzed, those of peppermint and basil demonstrated the strongest antifungal activity against T. aggressivum f. europaeum.Only peppermint oil exhibited lethal effect on the pathogen.The weakest inhibitory effect was displayed by yarrow oil.The oil of juniper had no antifungal effect at all.These results show that peppermint oil has a potential for further in vivo experiments against T. aggressivum f. europaeum.