Acta Botanica 1-2017 - za web.indd 64 ACTA BOT. CROAT. 76 (1), 2017 Acta Bot. Croat. 76 (1), 64–71, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0044 ISSN 0365-0588 eISSN 1847-8476 Antifungal potential of thyme essential oil as a preservative for storage of wheat seeds Sabina Anžlovar, Matevž Likar, Jasna Dolenc Koce* Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia Abstract – Plant essential oils are potential food preservatives due to their inhibitory effects on bacterial and fungal growth. Antifungal activities of common thyme (Thymus vulgaris) essential oil were tested against endophytic fungi grown from wheat (Triticum aestivum) grain, molecularly identifi ed as Alternaria alternata, Alternaria infectoria, Aspergillus fl avus, Epicoccum nigrum and Fusarium poae. Their susceptibility to thyme essential oil was tested in vitro, and ranged from fungicidal to fungistatic. Treatment combinations of prior grain surface sterilization with hypochlorite and direct/indirect treatment with the essential oil were used, which showed strong effects on infection incidence and germination. Direct soaking of the wheat grain in the essential oil was particularly effective, but inhibited both fungal growth and seed germination. In contrast, indirect treatment of the grain with the essential oil (i.e., fumigation) inhibited fungal growth without nega- tive effects on seed germination. In combination with grain surface sterilization with hypochlorite, indirect treatment with thyme essential oil reduced these fungal infections even more. Since thyme essential oil is safe for plants and consumers, in the form of fumigation it could be used as a protectant of storage containers for wheat grain intended for sowing and for food production. Keywords: antifungal activity, essential oil, seed spoilage, Thymus vulgaris, wheat * Corresponding author, e-mail: jasna.dolenc.koce@bf.uni-lj.si Introduction Wheat is an important cereal crop that can be attacked by a number of fungi. From seed germination to harvest, soil-borne and seed-borne diseases can reduce the vigour and yield of wheat plants (Soliman and Badeaa 2002). Sev- eral wheat-associated fungi are carried from the fi eld with the harvested grain, and can spread further during post-har- vest storage, and can reduce their later germination (Özer 2005, Perelló and Larrán 2013, Rajput et al. 2005). Among these fungi, Fusarium spp., Aspergillus spp. and Penicilli- um spp. are known to have negative impacts on grain dur- ing storage, which can result in serious risk for animal and human health through fungal production of a range of my- cotoxins (D'Mello et al. 1998). Furthermore, infected grain and grain transmission represent a primary source of infec- tion in the fi eld, from which these fungi can spread to broader areas, which can potentially lead to fungal epidem- ics (Perelló and Larrán 2013). Treatment of cereal plants with synthetic fungicides in the fi eld before harvest has resulted in fungal resistance to antifungal agents, and it can often be problematic due to the high toxicity of fungicide residues to mammals (Chen et al. 2008). Post-harvest synthetic fungicide treatment of stored grain can infl uence the quality of cereals and can involve serious health hazards for consumers (Osman and Abdul- rahman 2003). Therefore considerable interest has devel- oped in recent years in the preservation of grain using more consumer- and nature-friendly protectants. Plants contain a broad spectrum of antimicrobially ac- tive compounds that fi t into this category and show effec- tive inhibition of fungal growth. Essential oils are posed to become an important seed-decontamination alternative to synthetic seed preservatives, because in addition to strong fungicidal effects (Krisch et al. 2011) they are biodegrada- ble and show low toxicity to humans and animals (Krisch et al. 2011, Sivakumar and Bautista-Baños 2014) In recent years, essential oils from different plants have been used to prevent fungal growth and the consequent mycotoxin accu- mulation in cereals (Batish et al. 2006, Sumalan et al. 2013). In particular, oils from aromatic and spice plants have been applied, because of their safety and their com- mon use in the food industry for centuries. Many studies have documented the antimicrobial activi- ties of Thymus spp. essential oils and extracts (Bouzidi et al. 2013, Gonçalves et al. 2010, Soković et al. 2009, Solo- makos et al. 2008). In a previous study, we reported that the ANTIFUNGAL POTENTIAL OF THYME ESSENTIAL OIL ACTA BOT. CROAT. 76 (1), 2017 65 essential oil from common thyme (Thymus vulgaris) shows promising antibacterial and antifungal activities against foodborne isolates of a Fusarium sp. and Armillaria mellea, as model fungi from Basidiomycetes and Ascomycetes, re- spectively (Anžlovar et al. 2014). The main aim of the present study was to determine the antifungal activities and potential use of common thyme es- sential oil as a disinfectant against fungi grown on wheat grain to be used for sowing or germination. We hypothe- sised that treatment with thyme essential oil will decrease the infection rate of the stored wheat grain, although high concentrations might also have negative impact on germi- nation of the treated grain. To test our hypothesis, we: (i) isolated fungal endophytes from wheat grain and tested the effectiveness of thyme essential oil on their radial growth in vitro; (ii) evaluated the effectiveness of the thyme essential oil on fungal infection rates of wheat grain; and (iii) tested for potential negative impact of the essential oil treatments on germination of the treated wheat grain. Different con- centrations of thyme essential oil were tested, as well as in combination with initial surface sterilization of the grain with hypochlorite, to defi ne the best possible antifungal treatments for the wheat grain, for effective real-life treat- ments. Materials and methods Preparation of thyme essential oil Seedlings of common thyme (Thymus vulgaris L. cv. ‘Deutcher winter’) were grown from seeds under green- house conditions and transplanted to the experimental fi eld of the Biotechnical Faculty (University of Ljubljana, Slove- nia: 46°2’53.7’’N, 14°28’30.47’’E; altitude, 292 m a.s.l.) in March 2010. The plantation was regularly cultivated and was grown in the experimental fi eld until October 2010, when the non-fl owering shoots were harvested and pro- cessed to extract the essential oil. The thyme essential oil was isolated by water-steam distillation using a Clevenger ap- paratus (Anžlovar et al. 2014), following the procedures of the European Pharmacopoeia monograph on Thymi herba. Isolation and molecular identifi cation of fungi from wheat grain Wheat grain with developed fungal colonies on their surface were left in the dark at 20 °C until a single colony was large enough for transfer to fresh potato dextrose agar (PDA) medium (Biolife). The cultures are deposited in the fungal bank of the Plant Physiology Laboratory at the De- partment of Biology (Biotechnical Faculty, University in Ljubljana, Slovenia), under accession numbers KP271953- KP271958. The fungal mycelia were ground in liquid nitrogen and the DNA was extracted using GeneElute Plant Genomic DNA miniprep kits (Sigma), following the manufacturer in- structions. All of the PCR reactions were carried out in a thermal cycler (MJ Research) using Taq DNA polymerase (Promega). The 25-μl reaction mixture contained: 2.5 μl 10× PCR buffer, 2.5 mM MgCl2, 200 μM of each nucleo- tide, 500 nM of each primer, 0.75 U DNA polymerase, and 12.5 μl 100-fold–diluted template. The PCR conditions for the ITS1F-ITS4 primer pairs (Gardes and Bruns, 1993, White et al., 1990) were: 1 min at 94 °C, followed by 35 cycles of 45 s denaturation at 94 °C, followed by 53 s an- nealing at 55 °C, and 30 s elongation at 72 °C. The elonga- tion step was at 72 °C for 10 min. The PCR amplicons were cleaned and sequenced by Macrogen (The Netherlands), using the ITS1F and ITS4 primers. The nucleotide sequenc- es obtained were subjected to BLAST analysis, to deter- mine their homology with other sequences available in the GenBank database. Fungal growth inhibition assay The inhibitory effect of the thyme essential oil on radial growth of fungal mycelia was tested using the agar dilution method, as described by Zabka et al. (2009). The concen- trated essential oil was added to the autoclaved PDA medi- um (medium temperature, ~50 °C) to prepare fi nal essential oil concentrations of 0.05%, 0.01%, 0.005% and 0.0025% (Vessential oil/Vmedium). Fungal mycelia disks (diameter, 5 mm) were cut from the margin of the 7-day-old original cultures and aseptically inoculated by placing them in the centre of the medium with the essential oil in a Petri dish (diameter, 90 mm). The control samples were prepared at the same time, but with- out the essential oil. The fungal colonies were incubated at 23 °C in the dark for 7 days, each experiment being carried out twice. Inhibition of fungal radial growth was calculated ac- cording to the following formula: Inhibition (%) = (DC – DT) / DC × 100 where DC is the diameter of the control colonies, and DT is the diameter of the treated colonies. To evaluate the re- sponses of the fungal isolates to the thyme essential oil and to calculate the 50% effective concentration (EC50) and 90% effective concentration (EC90), the DRC software package, version 2.3, was used (Ritz and Streibig 2005). To determine the nature of the essential oil toxicity, the inhibited fungal disks grown on the medium with 0.05% thyme essential oil were re-inoculated onto fresh medium without essential oil, and the revival of the fungal growth was assessed after 7 days. The essential oil toxicity was designated as fungistatic when the fungal colonies grew again, and fungicidal when there was no growth of the fun- gal colonies (Kumar et al. 2008). Essential oil treatment of wheat grain In the present study, two types of wheat (Triticum aes- tivum L.) grain were used: conventionally grown grain obtained from the Agricultural Institute of Slovenia (T. aes- tivum cv. ‘Savinja’; designated as ‘conventional grain’ here- after), and grain from the ecological farm Vila Natura (Slo- venia, Prlekija, Vučja vas; designated as ‘eco-grain’ hereafter). Each of the grain types was divided in two groups: one group was left without any disinfection of the grain (non- sterile grain), while the other group was sterilized by soak- ANŽLOVAR S., LIKAR M., DOLENC KOCE J. 66 ACTA BOT. CROAT. 76 (1), 2017 ing in 3% hypochlorite for 3 min, to eliminate saprophytic microorganisms on the grain surface, followed by washing four times in sterilized distilled water (sterile grain). The non-sterile and sterile wheat grains (10 g) were treated with the thyme essential oil, with each treatment re- peated twice. Three concentrations of the thyme essential oil were prepared, as 2%, 0.2% and 0.05% by dilution of the essential oil in 10% dimethylsulphoxide (DMSO) in distilled water. The grain was treated with the thyme essen- tial oil according to two protocols: (i) direct treatment, where the grain was submerged and incubated in the essen- tial oil; and (ii) indirect treatment, where sterile Whatman fi lter paper was placed into the inner side of the top of the Petri dishes and impregnated with 4 ml of essential oil sus- pension, according to concentration (i.e., exposure to the essential oil vapour only). The grains were distributed on the bottom of each Petri dish, which were then sealed with parafi lm. For these essential oil treatments, the grains were incubated under shaking (50 rpm) for 24 h at 25 °C. The control grains were soaked in the vehicle, as 10% DMSO, or exposed to the 10% DMSO vapours, for direct and indi- rect exposure controls, respectively. Germination and fungal contamination of wheat grain The assessments of the fungal infection and seed germi- nation were performed by the direct plating method (Suma- lan et al. 2013). Ten subsamples (where each subsample contained 10 wheat grains) from each previously described treatment were placed on 2% PDA in Petri dishes (dia- meter, 90 mm) and incubated at 25±2 °C in the dark. After 72 h, the development of fungal colonies on the surface of the wheat grain was determined, with the seed contamina- tion index calculated according to Doolotkeldieva (2010). At the same time, the germination of the wheat grain was determined by counting the number of germinated grains, expressed as a percentage of all grains used under each con- dition. Statistical analysis All of the statistical analyses were performed with the software package R 3.1.2 (http://cran.r-project.org). The concentration/response analysis for growth of fungal iso- lates was performed using the drc package (v2.5-12) and a four-parameter log-logistic function. The effects of the seed type, prior sterilization, essential oil treatments, and essen- tial oil concentrations on the fungal infection rates and the germination rates of the wheat grain were tested using mul- tiway-ANOVA, with the level of signifi cance set at p<0.05. All post-hoc comparisons were performed using Holm-Si- dak post-hoc tests, with the level of signifi cance set at p<0.05. Results Fungal isolation and growth inhibition tests Five fungal species were isolated from the wheat grain: Alternaria alternata (2 strains), Alternaria infectoria (=Le- wia infectoria), Aspergillus fl avus, Epicoccum nigrum, and Fusarium poae (Tab. 1). The majority of these fungal endo- phytes were saprophytes, although F. poae is considered a plant pathogen. All of these fungal species were isolated from conventional grain as well as eco-grain, although the level of fungal infection was higher for eco-grain. The potential of the thyme essential oil as an antifungal preservative was tested against all of the isolated fungal en- dophytes (Fig. 1). The thyme essential oil showed in vivo fungitoxicity against all of the tested fungi, with A. infecto- ria, E. nigrum and F. poae showing the lowest EC50 and EC90 values. The highest tolerance to the thyme essential oil was for A. fl avus, with an EC50 of 0.010% and an EC90 of 0.018% (Vessential oil/Vmedium). After re-inoculation, the nature of the essential oil toxic- ity was determined as fungicidal for A. alternaria, E. nigrum and F. poae, and fungistatic for A. infectoria and A. fl avus (Tab. 1). Infection and germination of wheat grain after essential oil treatments The conventional grain and eco-grain were subjected to four treatment combinations, without and with surface ster- ilization with 3% hypochlorite, and direct and indirect ex- posure to the thyme essential oil, which yielded similar in- hibitory effects on the fungal infection rates and different effects on the germination of the wheat grain (Fig. 2, Tab. 2). The thyme essential oil signifi cantly reduced the fungal infection of the wheat grain under all of the treatment com- binations (Fig. 2A). Several factors affected the infection rates of the wheat grain (Tab. 3), including the source of the grain (conventional grain vs. eco-grain: F=59288, p<0.0001). Tab. 1. Details of the fungal endophytes isolated from the wheat grain. E-value – expectation value. End – endophyte, Sapro – sapro- phyte, Path – pathogen. Isolate No. Accession No. Nearest match E-value Maximum identity (%) Putative ecological niche Essential oil toxicity 9C KP271953 KJ739880 Alternaria alternata 0.0 99 End/Sapro Fungicidal 11C KP271955 JF440581 Alternaria alternata 0.0 100 End/Sapro Fungicidal 13C KP271957 GU584953 Alternaria infectoria 0.0 99 End/Sapro Fungistatic 12C KP271956 JX164075 Aspergillus fl avus 0.0 99 Sapro/Path Fungistatic 10C KP271954 JQ781728 Epicoccum nigrum 0.0 100 Sapro Fungicidal 14C KP271958 JQ912669 Fusarium poae 0.0 99 Path Fungicidal ANTIFUNGAL POTENTIAL OF THYME ESSENTIAL OIL ACTA BOT. CROAT. 76 (1), 2017 67 Fig. 1. Fungal growth–response curves in the presence of thyme essential oil (EO) in the growth media. Data are means±standard errors of two independent experiments (● experiment 1, ▲ experiment 2) with three individual measurements (n=6). A 95% confi dence interval is shown in grey. Fig. 2. Infection rates (A) and germination rates (B) of the wheat grain according to the concentrations and treatments with thyme essen- tial oil (EO). Data are means±standard errors (n=40). Data with different letters are signifi cantly different between treatments (Holm- Sidak post hoc tests, p<0.05). ANŽLOVAR S., LIKAR M., DOLENC KOCE J. 68 ACTA BOT. CROAT. 76 (1), 2017 In addition, the surface sterilization of the seeds prior to es- sential oil exposure also reduced the infection rates of the seeds (non-sterile vs. sterile: F=111096, p<0.0001). The in- hibition of fungal growth depended on the essential oil con- centrations (0.05% vs. 0.2% vs. 2%; F=53974, p<0.0001) and the concentrations of the essential oil showed interac- tions with the treatment methods (direct vs. indirect: F= 102801, p<0.0001), with the direct essential oil treatment showing greater reduction of the infection rate than the in- direct essential oil treatment (Tab. 2). On the other hand, the germination rates of the grain showed different susceptibility to these essential oil treat- ments. Direct treatment with the essential oil (i.e., submer- gence of grain in the essential oil) signifi cantly reduced the germination rate of the grain, while the indirect treatment did not affect the germination rate, which remained at the same level as for the control treatment (Fig. 2B). When compared to the above-described fungal infection rates, more of the factors affected the germination rates of the seeds (Tab. 4). In addition to the essential oil treatments (i.e., method and concentrations), the source of seeds was also an important factor (conventional grain vs. eco-grain; F=4212, p=0.041). As previously indicated, prior surface sterilization sig- nifi cantly contributed to the reduction of the fungal infec- tion rates (Tab. 2). Both the conventional grain and the eco- grain were similarly contaminated with fungi when no sterilization was applied (99%, 100%, respectively) (Tab. 2). The surface sterilization without the essential oil treat- ments signifi cantly reduced the fungal contamination (non- sterile vs. sterile; F=111096, p<0.0001), which on average dropped to 58% (Tab. 2). The additional direct and indirect treatments with thyme essential oil further reduced the fun- gal contamination of the wheat grain (Tab. 2). The direct treatment was generally more effective than the indirect Tab 2. Infection and germination rates of wheat grain after combi- nations of seed sources, surface sterilization and essential oil (EO) treatments. Data are means±standard errors (n=10). G ra in s ou rc e Pr io r g ra in st er ili za tio n E O tr ea tm en t EO concentration (%) Infection rate (%) Germination rate (%) C on ve nt io na l N on -s te ri le D ir ec t 0 100±0.00 97±1.53 0.05 27±4.73 52±6.63 0.2 15±4.53 0±0.00 In di re ct 0 99±1.00 100±0.00 0.2 38±7.57 99±1.00 2 31±5.26 98±1.33 St er ile D ir ec t 0 60±13.74 99±1.00 0.05 3±1.53 2±1.33 0.2 7±2.13 0±0.00 In di re ct 0 37±4.23 97±1.53 0.2 11±3.15 99±1.00 2 2±1.47 97±1.47 E co lo gi ca l N on -s te ri le D ir ec t 0 100±0.00 70±3.33 0.05 85±6.37 83±3.67 0.2 47±5.78 3±1.53 In di re ct 0 100±0.00 96±2.21 0.2 69±4.07 98±1.33 2 61±4.82 98±1.33 St er ile D ir ec t 0 72±8.00 86±3.71 0.05 57±13.75 65±9.69 0.2 11±5.04 0±0.00 In di re ct 0 63±6.33 97±1.53 0.2 32±2.91 99±1.00 2 23±3.67 95±3.07 Tab. 3. ANOVA for the differences in the infection rates among the grain sources, sterilization, and essential oil treatments and concentra- tions. Df – degrees of freedom, SS – sum of squares, MS – mean square. Factor Df SS MS F-ratio p Source 1.00 34031 34031 59288 <0.0001 Sterilization 1.00 63769 63769 111096 <0.0001 Treatment (essential oil) 1.00 108 108 0.19 0.666 Concentration (essential oil) 1.00 30981 30981 53974 <0.0001 Source: sterilization 1.00 77 77 0.14 0.714 Source: treatment 1.00 368 368 0.64 0.424 Sterilization: treatment 1.00 1835 1835 3197 0.075 Source: concentration 1.00 307 307 0.53 0.466 Sterilization: concentration 1.00 559 559 0.97 0.325 Treatment: concentration 1.00 59008 59008 102801 <0.0001 Source: sterilization: treatment 1.00 285 285 0.50 0.482 Source: sterilization: concentration 1.00 830 830 1445 0.231 Source: treatment: concentration 1.00 53 53 0.09 0.761 Sterilization: treatment: concentration 1.00 490 490 0.85 0.357 Source: sterilization: treatment: concentration 1.00 1752 1752 3053 0.082 Residuals 223 128001 574 ANTIFUNGAL POTENTIAL OF THYME ESSENTIAL OIL ACTA BOT. CROAT. 76 (1), 2017 69 treatment, and the highest fungal inhibition was seen for 0.2% thyme essential oil directly applied to the wheat grain. Further comparisons of these treatments showed that the in- direct essential oil treatment had similar antifungal effects to the surface sterilization with hypochlorite (Tab. 2). With the conventional grain, the fungal infection rate was 31% when the grain were fumigated (i.e., indirect treatment) with 2% essential oil, and 37% when only surface steriliza- tion was used. For the eco-seeds, the corresponding values were 61% and 63% (Tab. 2). Also, direct essential oil treat- ments resulted in range of fungal inhibition comparable to indirect essential oil treatments but at ~one-tenth of the concentrations used (e.g., 7% infection with direct treat- ment of the grain with 0.2% essential oil, and 2% infection with indirect treatment of the grain with 2% essential oil). The combined effects of the surface sterilization with hypochlorite and the indirect treatment with the thyme es- sential oil had no effects on seed germination, but provided greatly decreased fungal infection, thus retaining the bene- fi ts of both of these treatments (Fig. 3). Tab. 4. ANOVA for the evaluation of the differences in the germination rates according to the grain sources, sterilisation, and essential oil treatments and concentrations. Df – degrees of freedom, SS – sum of squares, MS – mean square. Factor Df SS MS F-ratio p Source 1 1123 1123 4212 0.041 Sterilization 1 1507 1507 5655 0.018 Treatment (essential oil) 1 157609 157609 591348 <0.0001 Concentration (essential oil) 1 1703 1703 6390 0.012 Source: sterilization 1 894 894 3353 0.068 Source: treatment 1 1692 1692 6349 0.012 Sterilization: treatment 1 959 959 3597 0.059 Source: concentration 1 2 2 0.01 0.931 Sterilization: concentration 1 14 14 0.05 0.820 Treatment: concentration 1 145536 145536 546050 <0.0001 Source: sterilization: treatment 1 687 687 2576 0.110 Source: sterilization: concentration 1 93 93 0.35 0.555 Source: treatment: concentration 1 35 35 0.13 0.719 Sterilization: treatment: concentration 1 23 23 0.09 0.771 Source: sterilization: treatment: concentration 1 727 727 2726 0.100 Residuals 223 59435 267 Fig. 3. Correlations between infection and germination rates for the non-sterile (A) and sterile (B) wheat grain according to the concentra- tions and treatments with thyme essential oil (EO). The sizes of the symbols depict the essential oil concentrations. Black squares denote direct essential oil treatment; white diamonds denote indirect essential oil treatment. ANŽLOVAR S., LIKAR M., DOLENC KOCE J. 70 ACTA BOT. CROAT. 76 (1), 2017 Discussion During plant growth as well as after harvest and during storage, wheat and other cereal grains are exposed to fungal colonisation. The use of such colonised seed for future sow- ing only exacerbates the problems of fungal pathogens and requires the application of fungicides. As essential oils from spice herbs are known to be safe for animal and human health, they represent a possible source for use in the de- contamination and storage of grain that is intended not only for sowing, but also for food production (e.g., fl our and its products, sprouts, dietary supplements). In the present study, fi ve indigenous fungal species were isolated from wheat grain and identifi ed using molecular methods: A. alternata, A. infectoria, A. fl avus, E. nigrum and F. poae were isolated from the grain from wheat grown in conventionally managed fi elds and in fi elds with sustain- able eco-management. With the exception of A. fl avus, these species appear to be common colonisers of wheat grain (Nicolaisen et al. 2014). However, in contrast to Nicolaisen et al. (2014), who identifi ed the genera Phae- osphearia and Microdochium as the core operational taxo- nomic units in 99% of wheat grain samples, we did not iso- late any fungi from these genera. In addition to Fusarium spp. and Alternaria spp., which are both known as soil fun- gi, the so-called storage fungi of Penicillium spp., Aspergil- lus spp. and Rhizopus spp. have also been reported for wheat grain (Bensassi et al. 2011, Bottalico and Perrone 2002, Gohari et al. 2007). In the present study, only A. fl a- vus was isolated, which suggests the potential contamina- tion of the grain with afl atoxins, which would negatively impact on the usability of the grain for animal and human consumption. Accordingly, the fungitoxic effects of thyme essential oil on the growth of A. fl avus might prove to be extremely benefi cial, as it would reduce the spoilage of food due to contamination with fungal toxins. As post-harvest synthetic fungicide treatments of stored products can infl uence the quality of the grain (Osman and Abdulrahman 2003), natural essential oils have advantages over synthetic agents due to their biodegradability and low toxicity. In the present study, in vitro thyme essential oil showed a broad spectrum of fungitoxicity against all of the fungi isolated from the wheat grain. The EC90 of the thyme essential oil was < 0.02% for all of these tested fungi, with a mainly fungicidal nature of the toxicity. The EC90 for thyme essential oil against the fi ve fungi in this study fi t well within ranges of values reported in other studies, where the fungitoxicity of thyme essential oil has been giv- en as between 0.025% (Soliman et al. 2002) and 0.07% (Kumar et al. 2008), although Zabka et al. (2009) reported a higher MIC for A. fl avus (0.23%) and a Fusarium sp. (~0.15%). The antifungal effects of thyme essential oil can be explained by modifi cations of fungal morphogenesis and growth through interference of the essential oil components with the fungal enzymes that are responsible for cell wall synthesis, which will lead to changes in hyphae integrity and to plasma membrane disruption and mitochondrial de- struction (Rassoli et al. 2006). The antifungal activities of essential oils are generally strongly associated with monot- erpenic phenols, and especially thymol, carvacrol and eug- enol (Bluma et al. 2008). Both thymol and carvacrol are also present in thyme essential oil and they are character- ised by their high antimicrobial activity (Bouzidi et al. 2013, and references therein, Gonçalves et al. 2010, So ko- vič et al. 2009, Šegvić Klarić et al. 2007). Chemical analy- sis of the thyme essential oil used in the present study has shown that the major constituent is indeed thymol (68.9%), whereas carvacrol constitutes only 1.6% (An žlo var et al. 2014). Due to the possible negative effects of essential oils on seed germination, we examined the effects of this thyme es- sential oil treatment on seed germination. Our results show that treatment with thyme essential oil is effective for the reduction of fungal contamination of wheat grain, although it can also affect the germination of the treated grain, which is an important aspect for the potential use of these treated grains. Direct treatment with thyme essential oil signifi cant- ly inhibited seed germination, and so despite the good re- duction in infection seen, this treatment is less appropriate as a prevention method for stored grain. In contrast, the in- direct essential oil treatment had no effects on the germi- nation of these wheat grains, but did successfully inhibit fungal growth, which was even more pronounced in combi- nation with the prior surface hypochlorite sterilization that was also used here. Our results are in agreement with ob- servations from several studies that have reported negative effects of direct essential oil treatments on seed germination (Kordali et al. 2008, Kotan et al. 2013, Paudel and Gupta 2008). In contrast, Kedia et al. (2014) demonstrated that wheat grain that has been indirectly fumigated with cumin seed essential oil retain viability even 12 months after stor- age. Accordingly, in combination with other studies, our re- sults indicate that indirect treatment (i.e., fumigation) with thyme essential oil has great potential as a bioprotection technique. We also show that the combination of surface sterilization and indirect thyme essential oil treatment fur- ther diminishes fungal infection without negative effects on the germination of the treated wheat grain. To conclude, fumigation with thyme essential oil shows a good in vivo effi cacy for the protection of wheat grain from fungal colonisers. Indirect treatment with thyme es- sential oil in combination with prior surface sterilization of wheat grain provided an even more effi cient treatment that signifi cantly reduced fungal infection and spread on these grain and was accompanied by retention of high germina- tion rates of the treated seeds. For the sake of safety for plants and consumers, thyme essential oil can be applied as a protection agent in storage containers for grain intended for sowing and food production. Acknowledgments This study was fi nancially supported by the Slovenian Research Agency, grant no. P1-0212. The authors are thank- ful to Tjaša Pršin for technical help and Christopher Berrie for language editing. ANTIFUNGAL POTENTIAL OF THYME ESSENTIAL OIL ACTA BOT. CROAT. 76 (1), 2017 71 References Anžlovar, S., Baričevič, D., Ambrožič Avguštin, J., Dolenc Koce, J., 2014: Essential oil of common thyme as a natural antimi- crobial food additive. Food Technology and Biotechnology 52, 263–268. Batish, D. R., Singh, H. P., Setia, N., Kaur, S., Kohli, R. K., 2006: Chemical composition and phytotoxicity of volatile essential oil from intact and fallen leaves of Eucalyptus citriodora. Ver- lag der Zeitschrift für Naturforschung C 61, 465–471. Bensassi, F., Mahdi, C., Bacha, H., Hajlaoui, M. R., 2011: Survey of the mycobiota of freshly harvested wheat grains in the main production areas of Tunisia. African Journal of Food Science 5, 292–298. Bluma, R., Amaiden, M. R., Etcheverry, M., 2008: Screening of Argentine plant extracts: Impact of growth parameters and af- latoxin B1 accumulation by Aspergillus section Flavi. Interna- tional Journal of Food Microbiology 122, 114–125. Bottalico, A., Perrone, G., 2002: Toxigenic Fusarium species and my cotoxins associated with head blight in small-grain cereals in Europe. European Journal of Plant Pathology 108, 611–624. Bouzidi, L. E., Jamali, C. A., Bekkouche, K., Hassani, L., Wohl- muth, H., Leach, D., Abbad, A., 2013: Chemical composition, antioxidant and antimicrobial activities of essential oils ob- tained from wild and cultivated Moroccan Thymus species. Industiral Crops and Products 43, 450–456. Chen, P. J., Moore, T., Nesnow, S., 2008: Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes. Toxicology in Vitro 22, 1476–1483. D’Mello, J. P. F., Macdonald, A. M. C., Postel, D., Dijksma, W. T. P., Dujardin, A., Placinta, C. M., 1998: Pesticide use and my- cotoxin production in Fusarium and Aspergillus phytopatho- gens. European Journal of Plant Pathology 104, 741–751. Doolotkeldieva, T. D., 2010: Microbiological control of fl our- manufacture: dissemination of mycotoxins producing fungi in cereal products. Microbiology Insights 3, 1–15. Gardes, M., Bruns, T. D., 1993: ITS primers with enhanced spe- cifi city of basidiomycetes: application to the identifi cation of mycorrhizae and rusts. Molecular Ecology 2, 113–118. Gohari, A. M., Sedaghat, N., Nikkhah, M. J., Saber-Riseh, R., 2007: Mycofl ora of wheat grains in the main production area in kerman province. International Journal of Agriculture and Biology 9, 635–637. Gonçalves, M. J., Cruz, M. T., Cavaliero, C., Lopes, M. C., Sal- gueiro, L., 2010: Chemical, antifungal and cytotoxic evalua- tion of the essential oil of Thymus zygis subsp. sylvestris. In- dustrial crops and products 32, 70–75 Kedia, A., Prakash, B., Mishra, P. K., Dubey, N. K., 2014: Anti- fungal and antiafl atoxigenic properties of Cuminum cyminum (L.) seed essential oil and its effi cacy as a preservative in stored commodities. International Journal of Food Microbiol- ogy 168–169, 1–7. Kordali, S., Cakir, A., Ozer, H., Cakmakci, R., Kesdek, M., Mete, E., 2008: Antifungal and insecticidal properties of essential oil isolated from turkish Origanum acutidens and its three com- ponents, carvacrol, thymol and p-cymene. Bioresource Tech- nology 99, 8788–8795. Kotan, R., Dadasoğlu, F., Karagoz, K., Cakir, A., Ozer, H., Korda- li, S., Cakmakci, R., Dikbas, N., 2013: Antibacterial activity of the essential oil and extracts of Satureja hortensis against plant pathogenic bacteria and their potential use as seed disin- fectants. Scientia Horticulturae 153, 34–41. Krisch, J., Tserennadmid, R., Vagvölgyi, C., 2011: Essential oils against yeasts and moulds spoilage. In: Mendez-Vilas, A. (ed.), Science against microbial pathogens: communicating current research and technological advances 2, 1135–1142. Formatex Research Center. Kumar, A., Shukla, R., Singh, P., Prasad, C. S., Dubey, N. K., 2008: Assessment of Thymus vulgaris L. Essential oil as a safe botanical preservative against post harvest fungal infestation of food commodities. Innovative Food Science and Emerging Technologies 9, 575–580. Nicolaisen, M., Justesen, A.F., Knorr, K., Wang, J., Pinnschmidt, H.O., 2014: Fungal communities in wheat grain show signifi - cant co-existence patterns among species. Fungal Ecology 11, 145–153. Osman, K. A., Abdulrahman, H. T., 2003: Risk assesment of pesti- cide to human and the environment. Saudi Journal of Biologi- cal Sciences 10, 81–106. Özer, N., 2005: Determination of the fungi responsible for black point in bread wheat and effects of the disease on emergence and seedling vigour. Trakya University Journal of Science 6, 35–40. Paudel, V. R., Gupta, V. N. P., 2008: Effect of some essential oils on seed germination and seedling length of Parthenium hys- terophorous L. Ecoprint 15, 69–73. Perelló, A. E., Larrán, S., 2013: Nature and effect of Alternaria spp. complex from wheat grain on germination and disease transmission. Pakistan Journal of Botany 45: 1817–1824. Rajput, M. A., Pathan, M. A., Lodhi, A. M., Shah G. S., Khanzada, K. A., 2005: Studies on seed-borne fungi of wheat in Sindh Province and their effect on seed germination. Pakistan Jour- nal of Botany 37, 181–185. Rassoli, I., Rezaei, M. B., Allameh, A., 2006: Growth inhibition and morphological alterations of Aspergillus niger by essen- tial oils from Thymus eriocalyx and Thymus x porlock. Food Control 17, 359–364. Ritz, C., Streibig, J. C., 2005: Bioassay analysis using R. Journal of Statistical Software 12, 1–22. Sivakumar, D., Bautista-Baños, S., 2014: A review on the use of essential oils for postharvest decay control and maintenance of fruit quality during storage. Crop Protection, 64, 27–37. Soković, M. D., Vukojević, J., Marin, P. D., Brkić, D. D., Vajs, V., van Griensven, L. J. L. D., 2009: Chemical composition of es- sential oils of Thymus and Mentha species and their antifungal activities, Molecules 14, 238–249. Soliman, K. M., Badeaa, R. I., 2002: Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi. Food and Chemical Toxicology 40, 1669–1675. Solomakos, N., Govaris, A., Koidis, P., Botsoglou, N., 2008: The antimicrobial effect of thyme essential oil, nisin and their combination against Escherichia coli O157:H7 in minced beef during refrigerated storage. Meat Science 80, 159–166. Sumalan, R.-M., Alexa, E., Poiana, M.-A., 2013: Assessment of inhibitory potential of essential oils on natural mycofl ora and Fusarium mycotoxins production in wheat. Chemistry Central Journal 7: 32. Šegvić Klarić, M., Kosalec, I., Mastelić, J., Pieckova, E., Pe- peljnak, S., 2007: Antifungal activity of thyme (Thymus vul- garis L.) essential oil and thymol against moulds from damp dwellings. Letters in Applied Microbiology 44, 36–42. White, T. J., Bruns, T., Lee, S., Taylor, J., 1990: Amplifi cation and direct sequencing of fungal ribosomal RNA genes for phylo- genetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (eds.), PCR-protocols and applications-a laboratory man- ual, 315–322. Academic, London. Zabka, M., Pavela, R., Slezakova, L., 2009: Antifungal effect of Pimenta dioica essential oil against dangerous pathogenic and toxinogenic fungi. Industrial Crops and Products 30, 250–253.