untitled ACTA BOT. CROAT. 75 (1), 2016 121 Acta Bot. Croat. 75 (1), 121–128, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0011 ISSN 0365-0588 eISSN 1847-8476 Comparison of anti-Aspergillus activity of Origanum vulgare L. essential oil and commercial biocide based on silver ions and hydrogen peroxide Željko D. Savković, Miloš Č. Stupar*, Milica V. Ljaljević Grbić, Jelena B. Vukojević University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Studentski Trg 16, 11000 Belgrade, Serbia Abstract – The antifungal activities of Origanum vulgare essential oil (EO) and of a biocide based on silver and hydrogen peroxide (Sanosil S003) against seven Aspergillus species isolated from different substrata (stone, brick, silk and paper) of cultural heritage objects in Serbia were evaluated. Microdilution, agar dilu- tion and microatmosphere methods were used to determine minimal fungistatic and minimal fungicidal con- centrations (MIC and MFC), and light microscopy to determine structural abnormalities. MIC and MFC val- ues for O. vulgare EO ranged from 0.2 to 5 mg mL−1 and for Sanosil S003 from 5 to 250 mg mL−1. Aspergillus sp. sect. fumigati was the most susceptible isolate, where MIC and MFC values were achieved at 0.5 mg mL−1 for O. vulgare EO, while MIC and MFC values for Sanosil S003 were achieved at 5 and 10 mg mL−1, respec- tively. Morpho-physiological changes were documented in all isolates, including lack of sporulation, depi- gmentation of conidiogenous apparatus and conidia, and presence of aberrant fungal structures. O. vulgare EO exhibited stronger anti-Aspergillus activity than Sanosil S003, as demonstrated by the higher MIC and MFC values and fewer morpho-physiological changes observed in the tested Sanosil S003 concentrations. O. vulgare EO could be an excellent alternative to commercial biocides, with high potential in the fi eld of cul- tural heritage conservation. Keywords: antifungal activity, Aspergllus spp., cultural heritage, essential oil, Origanum vulgare, Sanosil S003 Abbreviations: CYA – Czapek yeast extract agar, EO – essential oil, MEA – malt extract agar, MEB – malt extract broth, MIC – minimal inhibitory concentration, MFC – minimal fungicidal concentration * Corresponding author, e-mail: smilos@bio.bg.ac.rs Introduction Aspergillus is a genus of fungi, including more than 180 recognized species with worldwide distribution. Several species have attracted attention as human and animal patho- gens or because of their ability to produce various myco- toxins (Samson et al. 2010). Species of this genus are con- sidered one of the most frequent infestation agents of cultural heritage objects (Florian 2002, Hu et al. 2013) and are capable of producing masses of airborne conidia and can grow and reproduce on many different carbon sources, including a variety of materials of which cultural heritage artifacts are composed (paper, textile, frescoes etc.) (Garg et al. 1995, Florian 2002). They are also involved in the degradation of a broad range of organic substrata (Goldman and Osmani 2007) due to their proteolytic, cellulolytic and amylolytic activity (Borrego et al. 2010, 2012a, Eida et al. 2011). A vast number of microfungi, including Aspergillus species, are able to produce different organic and inorganic acids during their metabolic activities on monument sur- faces (Farooq et al. 2015). This leads to biodeterioration of stone substrata (such as sandstone, limestone, marble, mor- tar, brick etc.). Furthermore, Aspergilli are able to produce different pigments and contribute to the formation of bio- fi lms, which diminish the esthetic value of cultural heritage artifacts and accelerate their biodeterioration (Vivar et al. 2013). Prevention of mold growth is nowadays a signifi cant challenge for restorers, conservators and architects since chemical treatments must be non-destructive and non-toxic (Sterfl inger 2010). In the fi eld of the safeguarding of the cultural heritage, both synthetic and natural biocides are applied. Although substances with biocidal activities extracted from plants are commonly used in medicine and food preservation, their SAVKOVIĆ Ž. D., STUPAR M. Č., LJALJEVIĆ GRBIĆ M. V., VUKOJEVIĆ J. B. 122 ACTA BOT. CROAT. 75 (1), 2016 use in the control of mold growth on cultural heritage ob- jects is little reported (De Saravia et al. 2008). Oregano (Origanum vulgare L., Lamiaceae) is a widely known aro- matic plant that has been used in agricultural, pharmaceuti- cal and cosmetic industries (Şahin et al. 2004). Essential oil (EO) of this plant is recognized as an excellent natural source of substances with biological activities, such as ter- pens and phenolic compounds (Sivropoulou et al. 1996). The antimicrobial, antioxidative and cytotoxic activities of O. vulgare EO have already been demonstrated by Sivro- poulou et al. (1996) and Şahin et al. (2004), while its anti- fungal activity has been documented in numerous studies (e.g. Adam et al. 1998, Clef et al. 2013, Delić et al. 2013) along with several studies concerning anti-Aspergillus ac- tivity (e.g. Viuda Martos et al. 2007, Carmo et al. 2008, Souza et al. 2010). Commercial biocides include numerous organic com- pounds, metals, oxidizing agents and various synthetic products that have been widely used in the fi eld of cultural heritage conservation (Warscheid and Braams 2000). Silver and its compounds are known to be one of the most effec- tive antimicrobial agents (Russel 1994). Hydrogen perox- ide is an oxidizing agent capable of transforming numerous organic molecules (Petri et al. 2011). It has been document- ed that this compound exhibits antibacterial and antifungal properties (Baldry 1983). There are several studies con- cerning the antifungal activity of biocides based on silver ions and hydrogen peroxide (Tasić 2009, Abdel-Mageed et al. 2012) but, to our knowledge, very few regarding Asper- gillus species (e.g. Nabizadeh et al. 2008). The aim of this study was to evaluate the in vitro anti- fungal activity of Origano vulgare EO, a natural product, and also of a synthetic biocide based on silver ions and hy- drogen peroxide, on selected Aspergillus species isolated from cultural heritage objects in Serbia. Materials and methods Essential oil Origanum vulgare L. EO (Frey+Lau, Ulzburg, Germany), a commercial sample from the collection of the Institute for Medicinal Plant Research “Dr Josif Pančić” in Belgrade, was used in the experiment. The chemical composition of the tested EO was reported by Stupar et al. (2014). The main components were carvarcrol (64.06%), linalool (17.56%), p-cymene (4.44%) and thymol (3.86%). According to the manufacturer’s guidelines, the quality of the tested EOs corresponds to European Pharmacopeia 6 (2007). Commercial biocide The commercial biocide Sanosil S003 (Sanosil Ltd.) was obtained, as a water solution of fi nal concentration 2.7% (silver nitrate, 0.2%; and hydrogen peroxide, 2.5%), from the Institute for Protection of Cultural Monuments in Serbia. Aspergillus isolates Seven Aspergillus species used in this study were iso- lated from different substrata of cultural heritage objects in Serbia: Aspergillus sp. 1 sect. Flavi (isolated from stone sculpture in the Museum of Contemporary Art, Belgrade); Aspergillus sp. 2 sect. Flavi (brick wall of Arača Church, Novi Bečej); Aspergillus sp. sect. Circumdati and Aspergil- lus sp. sect. Nigri (silk icon from Central Institute for Con- servation in Belgrade); Aspergillus sp. sect. Terrei and As- pergillus sp. sect. Fumigati (archive paper from Central Institute for Conservation in Belgrade); Aspergillus sp. sect. Nidulantes (stone wall, Church of the Stara Pavlica Monas- tery, Raška). All tested isolates were identifi ed to section level using identifi cation keys: Raper and Fennell (1965) and Samson et al. (2010). Isolated fungi were deposited in the Mycothe- ca of the Department for Algology, Mycology and Li- chenology, Institute of Botany, Faculty of Biology, Univer- sity of Belgrade. Isolates were maintained on malt extract agar (MEA), and Czapek yeast extract agar (CYA), stored at 4 °C and subcultured once a month. Antifungal assays The antifungal activities of the selected EO and the bio- cide Sanosil S003 were investigated using three different methods: microdilution, agar dilution and microatmosphere methods. Microdilution and microatmosphere methods were used for testing the antifungal activity of EO, while the biocide Sanosil S003 was tested using microdilution and agar dilution methods. Microatmosphere method The test was performed in sterile Petri plates (85 mm dia.) containing 20 mL of MEA (Maruzzella and Sicurella 1960). Tested Aspergilli were inoculated at the center of the MEA medium, using a sterile needle under a stereomicro- scope (Stemi DV4, Zeiss), after which the Petri plates were turned over. A sterilized fi lter paper disc, soaked with vari- ous concentrations of O. vulgare EO, was placed in the cen- ter of the Petri plate lid interior. Concentrations of tested oil ranged from 0.1 to 5 mg mL−1. Plates were incubated for 21 days at 25 °C, during which the fungal growth was moni- tored weekly. After cultivation period, minimal inhibitory concentrations (MICs), defi ned as the lowest concentration of added EO with no visible fungal growth on MEA, were determined. Minimal fungicidal concentrations (MFCs) were determined by re-inoculation of treated inoculums onto sterile MEA. The lowest concentrations of EO giving no visible growth after re-inoculation were regarded as MFCs. Agar dilution method The modifi ed mycelia growth assay with MEA was used to investigate the antifungal activity of the biocide Sanosil S003 (Ishii 1995). The stock solution of biocide (2.7%) was further diluted in melted MEA in Petri plates to make fi nal concentrations of the biocide ranging from 10 to 250 mg mL−1. The fungi were inoculated at the center of the MEA. Plates were incubated for 21 days at 25 °C. MIC and MFC values were determined in the same manner as de- scribed above. ANTIFUNGAL ACTIVITY OF ORIGANUM VULGARE L. ESSENTIAL OIL AND SANOSIL S003 ACTA BOT. CROAT. 75 (1), 2016 123 Microdilution method To determine the antifungal activity of O. vulgare EO and the commercial biocide, the modifi ed microdilution technique was used (Hanel and Raether 1998, Daouk et al. 1995). Conidial suspensions of selected Aspergilli were prepared by washing conidia from the surface of the 7 days old MEA slants with sterile saline (NaCl 0.85%, Hemo- farmhospitaLogica) containing 0.1% Tween 20 (Sinex Lab- oratory). Using a hemocytometer (Reichert, Warner-Lam- bert Technologies) conidia were counted on a 1 mm2 surface and the concentration of conidia in the suspensions were calculated per formula: number of conidia / mm2 × 104 × dilution The conidia suspension was adjusted to a concentration of approximately 1.0 × 107 in a fi nal volume of 100 μL per each well. The inocula were stored at –20 °C for further use. Dilutions of all Aspergilli inocula were transferred onto solid MEA to verify contamination absence and in or- der to check the validity of the inocula. Determination of the MICs was performed by a serial dilution technique using 96-well microtiter plates. Different volumes of investigated EO and biocide Sanosil S003 were dissolved in malt extract broth (MEB) medium with Asper- gilli inoculums (10 μL) to make the same fi nal concentra- tions, as those used in microatmosphere and agar dilution methods. The microtiter plates were incubated for 72 h at 28 °C. The lowest concentrations of tested biocides without visible growth under a binocular microscope were defi ned as the concentrations that completely inhibited Aspergilli growth (MICs). The minimum fungicidal concentrations (MFCs) were determined by serial subcultivation of inocula (2 μL) into microtiter plates containing 100 μL of MEB me- dium. The lowest concentration with no visible growth was defi ned as the MFC, indicating 99.5% killing of the original inoculum. Microscopic analysis After the incubation period, a sample of mycelium was taken from the periphery of a colony grown on MEA en- closed with evaporated EO (microatmosphere method) or on MEA enriched with different concentrations of Sanosil S003 (agar dilution method). A sample of mycelium was also taken from microwell of a microtiter plate. The sam- ples were dyed and fi xed with glycerol and observed under a light microscope (Zeiss Axio Imager M.1, with AxioVi- sion Release 4.6 software) to examine structural abnormali- ties. Samples from the control plate without oil were also stained and observed. Additional observation was done 7 days after the incubation period in order to assess sustain- ability of morphological changes. Results Fungal susceptibility to essential oil High fungistatic and fungicidal activity of O. vulgare EO was demonstrated with low MIC and MFC values. MIC values ranged between 0.2 and 2.5 mg mL−1 and MFC val- ues varied between 0.5 and 5 mg mL−1, obtained in both methods used (Fig. 1). Aspergillus sp. sect. Fumigati was the most susceptible isolate to EO treatments and fungicidal effect was achieved at 0.5 mg mL−1. The most resistant iso- lates were Aspergillus sp. sect. Nigri and Aspergillus sp. sect. Terrei. For these isolates, fungicidal effect in microdi- lution method was achieved only with the highest tested EO concentration (5 mg mL−1). Furthermore, lower MIC and MFC values were obtained in microatmosphere method for most isolates. Fungal susceptibility to commercial biocide The biocide Sanosil S003 exhibited moderate antifungal activity, signifi cantly lower than O. vulgare EO. MIC val- ues from 5 to 250 mg mL−1 and MFC values from 10 to 250 mg mL−1 were obtained in both methods used (Fig. 1). As- pergillus sp. sect. Fumigati was the most susceptible isolate to biocide treatments and fungicidal effect was achieved at 10 mg mL−1. In the agar dilution method, fungicidal effect for both isolates from Flavi section and for Aspergillus sp. sect. Circumdati was obtained only with the highest tested biocide concentration (250 mg mL−1). Lower MIC and MFC values were obtained in the microdilution method for most isolates. Fig. 1. Minimal inhibitory concentration (MIC) and minimal fun- gicidal concentration (MFC) values of Origanum vulgare essen- tial oil and Sanosil S003 on tested Aspergillus isolates obtained by: a) microatmosphere and agar dilution methods, b) microdilu- tion method. Values in y axis are shown in logarithmic scale. SAVKOVIĆ Ž. D., STUPAR M. Č., LJALJEVIĆ GRBIĆ M. V., VUKOJEVIĆ J. B. 124 ACTA BOT. CROAT. 75 (1), 2016 Morpho-physiological changes Morpho-physiological changes, such as depigmentation of conidiogenous apparatus and conidia, lack of sporulation and presence of aberrant fungal structures, in addition to slower mycelial growth, were observed at EO concentra- tions lower than MICs obtained for each isolate (Tab. 1). Aberrant formation of conidial heads, including changes in shape and color, was observed in Aspergillus sp. sect. Nigri colonies grown at EO concentration of 0.1 mg mL−1 (Fig. 2, Tab. 1). Vesicles had a squashed appearance and were sometimes depigmented. Metulae were not developed – phialides were directly formed on vesicles in contrast to the biseriate conidial heads observed in control sample. Addi- tionally, hyphae of Aspergillus sp. sect. Nigri had swellings and apical buddings (On-line Suppl. Fig. 1). Pigmentation loss and shape change in reproductive structures were ob- served in Aspergillus sp. 1 sect. Flavi colonies grown at EO concentration of 2.5 mg mL−1 (On-line Suppl. Fig. 2). In biocide concentrations lower than the MICs obtained in the agar dilution and microdilution methods, fewer mor- pho-physiological changes were documented in the tested isolates (Tab. 1). These included lack of sporulation and de- pigmentation of conidiogenous apparatus and conidia. No morpho-physiological changes were documented in any of the tested concentrations of the biocide in the agar dilution method, although slower mycelial growth was observed in concentrations lower than the MICs obtained for each iso- late. Tab. 1. Observed morpho-physiological changes in tested Aspergillus isolates at different concentrations of Origanum vulgare essential oil and Sanosil S003, in microdilution method (after 72 h) and in microatmosphere and agar dilution method (after 21 day). Legend: ma – microatmosphere method, md – microdilution method, ad – agar dilution method, (+) – growth without morpho-physiological changes, (–) – no growth, DP – depigmentation of conidiogenous apparatus, LS – lack of sporulation, AC – aberrant conidiogenous apparatus de- velopment Isolates Concentrations of O. vulgare EO (mg mL−1) Concentrations of biocide Sanosil S003 (mg mL−1) 0.1 0.2 0.5 1 5 10 20 40 ma md ma md ma md ma md ad md ad md ad md ad md Aspergillus sp. 1 sect. Flavi + DP, LS + DP, LS DP – – – + + + + + DP, LS + – Aspergillus sp. 2 sect. Flavi DP DP, LS DP DP, LS DP – – – + + + + + + + DP, LS Aspergillus sp. sect. Circumdati + DP, LS DP, LS DP, LS – DP, LS – – + DP + DP + DP, LS + – Aspergillus sp. sect. Nigri DP, AC LS – DP, LS – – – – + + + + + + + – Aspergillus sp. sect. Terrei + DP, LS DP, LS DP, LS DP DP, LS – DP, LS + DP, LS + DP, LS + DP, LS + – Aspergillus sp. sect. Fumigati + DP, LS DP, LS DP, LS – – – – + – + – + – + – Aspergillus sp. sect. Nidulantes + DP, LS – DP, LS – – – – + DP, LS + DP, LS + – + – Fig. 2. Morphological changes of Aspergillus sp. sect. Nigri repro- ductive structures observed in microatmosphere method (essential oil in concentration of 0.1 mg mL−1): a) normal conidiogenous ap- paratus (control); b–d) aberrant conidiogenous apparatus. Scale bars = 10 μm. ANTIFUNGAL ACTIVITY OF ORIGANUM VULGARE L. ESSENTIAL OIL AND SANOSIL S003 ACTA BOT. CROAT. 75 (1), 2016 125 Discussion The results of the microdilution and microatmosphere methods showed that various concentrations of O. vulgare EO exhibited relatively strong fungistatic and fungicidal activity against the tested Aspergillus isolates. The antifun- gal potential of this EO was demonstrated in several other studies. Antifungal activity of O. vulgare ssp. vulgare EO against 15 fungal isolates was documented, including A. fl avus and A. variecolor (Şahin et al. 2004) and strong anti- fungal activity of the same EO against A. niger and A. fl a- vus was shown by Viuda-Martos et al. (2007). The latter authors showed that O. vulgare EO exhibited stronger anti- fungal activity than the EOs of Thymus vulgaris and Syzigi- um aromaticum. The antifungal activity of O. vulgare EO against 6 Aspergillus species was demonstrated by Carmo et al. (2008), who noted that some concentrations of EO ex- hibited stronger antifungal activity than the tested antifun- gicals (amphotericine B and ketoconasole). Some authors documented the antifungal activity of O. vulgare EO against several fungi isolated from different substrates of cultural heritage objects, including A. niger and A. ochra- ceus (Stupar et al. 2014). These authors pointed out that O. vulgare EO exhibited antifungal activity similar to that of the tested biocide benzalkonium chloride but stronger com- pared than the EOs of Rosmarinus offi cinalis and Lavandu- la angustifolia. The strong antifungal activity of O. vulgare essential oil can be attributed to its high content of some phenolic com- pounds, mostly carvacrol and thymol (Viuda-Martos et al. 2007, Clef et al. 2013). Although the thymol content in the EO used in this study was relatively low (3.86%), carvacrol was the main component (64.06%). Phenolic compounds, in appropriate concentrations, are reported to be effective against some microorganisms. Their mechanism of antimi- crobial activity is related to disruption of microbial cell membrane and precipitation of cellular proteins (Burt 2004). It is also suggested that presence of an aromatic nu- cleus with OH group is responsible for making hydrogen bonds with active sites of target enzymes (Velluti et al. 2003). Therefore, it is possible to suppose that these groups are responsible for antimicrobial activity. It has been shown that EOs are able to cause morpho- physiological changes in Aspergillus species including loss of sporulation and pigmentation and aberrant development of both hyphae and reproductive structures (De Billerbeck et al. 2001). Morpho-physiological changes were docu- mented in all tested isolates in our study, such as lack of sporulation, depigmentation of conidiogenous apparatus and conidia, terminal and intercalar swellings and apical buddings. As a result of O. vulgare EO activity, morpho- physiological changes in A. fl avus were showed by Souza et al. (2010). Authors reported loss of cytoplasm content, de- pigmentation and distorted development of hyphae with swellings and apical budding. Furthermore, the absence of conidiation was noted. Some authors used four different EOs in an antimicrobial vapor assay and documented mor- pho-physiological alterations of several fungal isolates, in- cluding A. niger (Ferdeş and Ungueranu 2012). They re- ported absence of phialides on vesicles and reduced conidial formation. Degenerative changes in hyphal morphology were reported as well. Sporulation is an essential part of the fungal life cycle and melanin is responsible for the survival and endurance of fungal spores (Wheeler and Bell 1988). Depigmentation of spores and conidial heads is probably due to the inhibition of melanin synthesis. Melanin is an important virulence factor for pathogenic fungi (Tsai et al. 1999) and, therefore, its loss can signifi cantly reduce patho- genicity. This may be of great importance when Aspergillus species are considered, since some of them are well known human and animal pathogens (e.g. A. fumigatus), and pro- ducers of mycotoxins (e.g. A. fl avus, A. parasiticus, A. ochraceus) (Samson et al. 2010). It is reported that applica- tion of EOs may result in retraction of cytoplasm and inter- action of EO components with fungal cell wall (Carmo et al. 2008). There also may be interference in enzymatic re- actions of cell wall synthesis, which affects fungal growth and morphogenesis (De Billerbeck et al. 2001, Souza et al. 2010). The fact that structural changes were observed in all fungal isolates tested in this study indicates that O. vulgare EO can affect the morphological development of different Aspergillus species and probably other fungi as well. Results of the microdilution and agar dilution method showed that different concentrations of the biocide Sanosil S003 exhibited moderate fungistatic and fungicidal activity. Antifungal studies of biocides based on silver ions and hy- drogen peroxide are scarce and there are only few studies concerning anti-Aspergillus activity. According to the man- ufacturer (Sanosil Ltd), antifungal properties of the biocide have been proven against numerous microorganisms, in- cluding A. niger as one of the tested fungal species, but there is no information about MIC or MFC values. It was shown that the biocide Sanosil Super 25 in a concentration of 40 ppm destroys the blastospores of Candida albicans (Tasić 2009). The same biocide also inhibits mycelial growth and sclerotal formation of Botrytis cinerea and Sclerotinia sclerotiorum (Abdel-Mageed et al. 2012). Anti- fungal activity of the biocide based on silver and hydrogen peroxide (Nanosil) was investigated by Nabizadeh et al. (2008). According to these authors, bacterial species were more sensitive to tested biocide than fungal species, where A. niger was the most resistant tested microorganism in the study. Antifungal activity of Sanosil S003 could be ascribed to synergistic activity of silver ions and hydrogen peroxide. Hydrogen peroxide is an inorganic compound that belongs to the group of reactive oxygen species (ROS). This mole- cule is relatively stable under physiological conditions and can oxidize lipids, proteins, DNA and other organic mole- cules (Bienert et al. 2007). It is known that silver ions are highly reactive and can interfere with numerous biological processes in microorganisms, including structural and func- tional alterations of cell membrane and cell wall (Jo et al. 2009, Rai et al. 2012). Silver ions can inhibit replication by binding to DNA molecule (Landsdown 2002, Castellano et al. 2007). Moreover, silver ions have the capacity to react with thiol groups of proteins which leads to the inactivation of enzymes (Feng et al. 2000) and they can also cause pro- SAVKOVIĆ Ž. D., STUPAR M. Č., LJALJEVIĆ GRBIĆ M. V., VUKOJEVIĆ J. B. 126 ACTA BOT. CROAT. 75 (1), 2016 tein inactivation by binding to their functional groups (Son- di and Salopek-Sondi 2004). In this way, silver ions block aquaporin-mediated water diffusion in Saccharomyces cerevisiae cells (Bienert et al. 2007). Since silver ions can interfere with structural components of the cell, cellular metabolism and reproduction, it is clear that they possess strong antifungal activity. It has been shown that combina- tion of silver ions and hydrogen peroxide exhibits a syner- gistic mode of action that can sometimes be a thousand-fold higher than the sum of the separate agents (Pedahzur et al. 2000, Tasić 2009). The aim of this study was to compare the antifungal ac- tivity of O. vulgare EO, which is a mixture of various or- ganic compounds, and Sanosil S003, a commercial product that is a solution of inorganic compounds. According to the results presented in this study, it is evident that O. vulgare EO exhibits stronger antifungal activity than Sanosil S003 on the tested Aspergillus isolates. In some cases, the MIC and MFC values obtained with Sanosil S003 were several hundred times higher than those obtained with O. vulgare EO. Weaker antifungal activity of Sanosil S003 was con- fi rmed with fewer observed morpho-physiological changes in the microdilution method, while none were observed in the agar dilution method. The global commercial supply of conventional biocides is well established. However, biocides containing EOs are typically used in small niche product applications due to limited effi cacy and therefore are only sold in small amounts (Browne et al. 2012). Additionally, EOs have only recently been recognized as a special class of biocides by EU biocide legislation (since 2011). EOs have seldom been tested in vitro against fungi isolated from cultural heritage objects. Examples include isolates from the documentary heritage (Borrego et al. 2012a), royal tomb paintings (Sakr et al. 2012), stone and wooden artifacts (Stupar et al. 2014). Reports regarding the implementation of natural products in the fi eld of conservation of cultural heritage are scarce. However, Rakotonirayni et al. (2005) demonstrated preven- tive and curative action of linalool, as a main component of some EOs, on inoculated books in a glass test chamber. Gatenby and Townley (2003) showed the effi ciency of Me- laleuca alternifolia EO, in vapor form, in reducing spore number in Castle Hill Museum (Sydney) storage rooms. According to Chung et al. (2001), volatile extracts of some EOs have no negative effects on organic materials and as such can be used as an alternative source in control of bio- deterioration of cultural heritage. Also, plant products with antimicrobial activities, such as EOs, have little negative impact on the environment or human health (Borrego et al. 2012b). Since O. vulgare EO demonstrated stronger anti- fungal activity than a commercial biocide against Aspergil- lus species isolated from cultural heritage objects the high potential of EOs as alternative agents in conservation of cultural heritage is indicated. Further studies are required to develop appropriate methods to apply EOs in the conserva- tion of cultural heritage objects. Conclusions The results of this study show the effi cacies of O. vul- gare EO and of the commercial biocide Sanosil S003. The anti-Aspergillus potential of O. vulgare EO was higher than that of Sansoil S003. This claim was substantiated by the observed morpho-physiological changes in all tested iso- lates. This study presents O. vulgare EO as an excellent al- ternative to commercial biocides and suggests it can be use- fully applied in the fi eld of the conservation of cultural heritage. 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ANTIFUNGAL ACTIVITY OF ORIGANUM VULGARE L. ESSENTIAL OIL AND SANOSIL S003 ACTA BOT. CROAT. 75 (1), 2016 1 On-line Suppl. Fig. 1. Morpho-physiological changes of Asper- gillus niger hyphae observed in microatmosphere method (essen- tial oil in concentration 0.1 μL mL−1): ab – apical budding; sw – swelling; scale bar = 10 μm. On-line Suppl. Fig. 2. Morpho-physiological change s of Aspergillus fl avus reproductive structures observed in microatmosphere method (essential oil in concentration 2.5 μL mL−1): a) normal conidiogenous apparatus (control), b) depigmented conidiogenous apparatus. Scale bars = 10 μm.