ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 21 (2004) 649–656 www.elsevier.nl/locate/jnlabr/yfmic Initial screening for inhibitory activity of essential oils on growth of Fusarium verticillioides, F proliferatum and F graminearum on maize-based agar media Andrea Velluti, Sonia Mar!ın, Pilar Gonzalez, Antonio J Ramos, Vicente Sanchis* Food Technology Department, Lleida University, CeRTA-UTPV, Rovira Roure 191, Lleida 25198, Spain Received August 2003; accepted March 2004 Abstract An in vitro initial screening of a range of 37 essential oils on inhibition of mycelial growth of Fusarium verticillioides, F proliferatum and F graminearum under different temperature (20–30 C) and water activity (aw ) (0.95–0.995) conditions was made The basic medium was a 3% maize meal extract agar The maize meal agar was modified with glycerol to a range of water activity conditions and the essential oils were incorporated at different concentrations (0, 500, 1000 mg mlÀ1) Cinnamon leaf, clove, lemongrass, oregano and palmarosa oils were the products tested suitable for being used as novel preservatives in the control of the three Fusarium species studied Although water activity was determinant for the growth of the isolates, in general, the preservative effects of the oils were not linked to aw : However, a trend to a higher inhibition by the oils when aw was low was observed Temperature had a minor importance in the inhibitory effect of the preservatives In vivo studies may be required to confirm the usefulness of the results obtained r 2004 Elsevier Ltd All rights reserved Keywords: Fusarium; Essential oil; Growth; Inhibition Introduction Many species of fungi are capable of producing mycotoxins Fusarium species cause seedling blight and root, stalk, and ear rot in corn as well as many of them produce mycotoxins The most frequently isolated species from corn in temperate climates are Fusarium verticillioides, F proliferatum and F graminearum followed by F subglutinans, F culmorum and F equiseti (Sanchis et al., 1995; Sohn et al., 1999) Fungi-inhibiting chemicals (mainly low-molecular-weight organic acids) have been used for the preservation of stored grain However, many disadvantages are associated with the use of acids (Christensen and Sauer, 1982) and there is a world trend towards reducing their use in grain and foodstuffs Natural plant extracts may provide an alter*Corresponding author Tel.: +34-973-702535; fax: +34-973702596 E-mail address: vsanchis@tecal.udl.es (V Sanchis) 0740-0020/$ - see front matter r 2004 Elsevier Ltd All rights reserved doi:10.1016/j.fm.2004.03.009 native to these preservatives Several other studies have examined the effect of compounds isolated from oils on fungi to search for natural fungicides and a number of these oil constituents have shown to be inhibitory (Chao et al., 2000) Mishra and Dubey (1994) screened 14 different essential oils for their antifungal activities against Aspergillus flavus and found that only the oil from lemongrass possessed fungitoxicity against A flavus In general, the response to the different essential oils depends on the fungal species tested, and ranges from a lack of inhibition (resistance) to various degrees of susceptibility (Pattnaik et al., 1996) Little is known about the effect of essential oils on phytopathogenic Fusarium species from corn and the impact of environmental conditions on this effect The aim of this work was to make an in vitro initial screening of a range of 37 essential oils on inhibition of mycelial growth of F verticillioides, F proliferatum and F graminearum at different water activity (aw ) and temperature conditions ARTICLE IN PRESS A Velluti et al / Food Microbiology 21 (2004) 649–656 650 Valencia, Spain) The remaining strains were retrieved from the Food Technology Department fungi collection of the University of Lleida, Spain Material and methods 2.1 Fungal isolates 2.2 Experimental design The fungal species used in this study were F verticillioides, F proliferatum and F graminearum For each species three different strains from different sources were used Strains 1111, 1097 and G2SG1 of F verticillioides, F proliferatum and F graminearum, respectively, were kindly provided by Dr Fannelli (Department of Biology, University of Roma ‘‘La Sapienza’’, Rome, Italy), while strain ITEM223 of F graminearum was provided by Dr Chulze (Universidad Nacional de Rio Cuarto, Argentina), and strain CECT 2150 was retrieved from the Spanish Type Culture ! Espan˜ola de Cultivos Tipo, Collection (Coleccion An in vitro initial screening of a range of 37 essential oils on inhibition of mycelial growth of F verticillioides, F proliferatum and F graminearum under different temperatures (20–30 C) and aw (0.95–0.995) conditions was made For the first 20 essential oils studied, the experiment was designed as a screening using a statistical design program (MODDE version 5.0, UMETRI AB, Umea, Sweden) Screening designs are used at the first stage of an investigation, where information is sought about the effects of many factors The factors and levels were aw (0.95–0.995); temperature (20–30 C); concentrations of oils (0–1000 mg mlÀ1) and three different strains of each species Two different sets of experiments with 48 runs each were required, as the program did not allow for more than 10 levels of a qualitative factor at a time For the remaining essential oils, plus the ones that showed good abilities in the first part, the experiments were designed as a response surface model (RSM) using the same statistical program RSM designs are used at a later stage of investigation, with the purpose of studying major variables in more detail This design resulted in 126 runs for each set of essential oils The factors and levels were the same as described above Table shows the essential oils studied in each experimental design Table Sets of essential oils in the different experimental designs Designed as response surface model (RSM) 1st set 1st set Aniseed Cinnamon leaf Eucalyptus Grapefruit Lemon Lemongrass Lime Mandarin Orange Spearmint 2nd set Basil Bay Clove Ginger Peppermint Pine sylvestris Rosemary Sage Sweet fennel Thyme 2nd set a Bay leaf Bircha Cinnamon leaf Clove Cypressa Incensea Lemongrass Marjorama Mossa Petitgraina Cedara Citronellaa Lavender ab Lavender ba Minta Nutmega Oreganoa Palmarosaa Tea treeb Ylang-ylangb 2.3 Inoculation, incubation and growth assessment All essential oils are from F D Copelans & Sons Ltd London a Essential oils are from Ravetllat Aromatics, S.L Barcelona b Essential oils are from Laboratorios Dicana S.L Barcelona bay leaf*conc marjoram*conc 0.30 The basic agar medium was a 3% maize meal agar (Mar!ın et al., 1995) The maize meal agar was modified incense*conc concentration petitgrain*conc 0.20 cinnamon leaf*conc *conc 0.10 responses Designed as screening model moss*conc birch*conc clove*conc temperature Fv Fv Fv Fp Fp Fv Fp Fp Fp Fv Fp Fg Fv Fp Fg Fg Fv Fv Fp petitgrain aFg w Fg Fg Fg Fg incense bay leaf temp*incense temp*birch marjoram temp*petitgrain moss temp***marjoram ttemp*moss temp*bay leaf aw* petitgrain aw*moss lemongrass temp*cinnamon leaf temp*clove aw*incense aw *lemongrass aw*bay leaf temp*lemongrass marjoram aw*marjoram aw*clove birch cinnamon leaf aw*birch aw*cinnamon innamon leaf lemongrass*conc ] wc 3] aw*temperature 0.00 -0.10 -0.20 clove -0.30 conc*conc -0.20 -0.10 0.00 wc [2] 0.10 0.20 0.30 Fig Loadings plot of PCs and from partial least-squares (PLS) regression showing the relationship of aw ; temperature, concentration, essential oils (’) and cross-terms (K) on colony diameters (m, mm) of three isolates each of F verticillioides (Fv), F proliferatum (Fp) and F graminearum (Fg) ARTICLE IN PRESS A Velluti et al / Food Microbiology 21 (2004) 649–656 with glycerol to a range of aw conditions The essential oils were incorporated into the agar after autoclaving to give final concentrations of 500 and 1000 mg mlÀ1 medium Tween-80 (0.005% v/v) was used as an emulsifying agent Tween-80 (0.005% v/v) with no oil was used as a positive growth control The media were poured in 9-cm Petri plates (20 ml plateÀ1) A mm diameter agar disk was taken from the margin of a 5-days old growing colony of each isolate on the basic maize meal agar and transferred to the center of each test plate The plates enclosed in sealed plastic bags were incubated at 20 C or 30 C for 28 days in an inverted position, and two diameters at right angles of the growing colonies were measured every days 651 Injected volume: ml Interface GC-MS temperature: 280 C Results For the first 20 essential oils, two different sets of 10 essential oils included in a screening design were analysed separately by PLS Firstly, for the first 10 essential oils assayed, PLS was applied separately for each species Then, as there were no significant intraspecific differences (P > 0:05), all three species and days of measurement were included in the same analysis as different responses A model of nine principal components (PC) was produced with a R2 ¼ 0:90 and 2.4 Data analysis The data were analysed by using MODDE 5.0 program Partial Least-Squares Projections to Latent Structures (PLS) was applied Different species, or strains, and days of measurement were included in the analyses as different responses The results were interpreted through loading plots and model coefficients PLS coefficients make the model interpretation less laborious and time-consuming when there are several components (>4–5) in a PLS model On the other hand, their big advantage is that the analyst obtains only one vector of concise model information per response, instead of several vectors of weights (Eriksson et al., 1999) On the other hand, the disadvantage is that a list of coefficients is given for each response (different days and species in our case) For this last reason loadings plots are shown in the results section instead of coefficients tables It is important to note, that the single essential oil factor represents the mean global effect of this essential oil at all concentrations levels (0, 500 and 1000 mg mlÀ1), while the cross-term essential oilÃconcentration, shows the effect of different concentrations for each individual essential oil; the program then evaluates the differences between the effect of the positive control (0 mg mlÀ1) and 500 and 1000 mg mlÀ1 concentrations 2.5 Essential oils, component analyses Those essential oils resulting in an antimicrobial activity against Fusarium spp were analysed by GC The analyses of the main components of each essential oil were run on a Hewlett Packard GC-MS system (GC HP6890; MSD HP5973, Hewlett Packard, Vienna, Austria) by the Consejo Superior de Investigaciones Cient!ıficas (CSIC, Instituto de Qu!ımica Orga! nica General, Madrid, Spain) Polyethylene glycol 20 m column (50 Â 0.25 Â 0.25 mm) Program temperature: from 70 C to 180 C (4 C minÀ1), plus 30 at 180 C Carrier gas: helium at ca 0.8 ml minÀ1 Injector temperature: 250 C Fig Contour plots based on models from PLS regression showing colony diameters (mm) of F verticillioides, F proliferatum and F graminearum growth after 12 incubation days at 25 C as affected by aw and concentration of cinnamon oil (mg mlÀ1) ARTICLE IN PRESS 652 A Velluti et al / Food Microbiology 21 (2004) 649–656 Q2 ¼ 0:72; and all responses were correlated to some extent (data not shown) PC are composite variables, i.e linear functions of the original variables, estimated to contain, in decreasing order, the main structured information in the data The first PC explained 50% of the data variability, while the second PC explained 27% aw had a positive effect on growth rates of the species studied (P > 0:05); temperature also had a positive effect on growth except for F graminearum where no significant effect was found (P > 0:05) None of the 10 essential oils studied had a significant effect (P > 0:05) on growth rates, only a slight inhibition trend was observed for lemongrass and cinnamon leaf essential oils For the second set of essential oils, all responses were correlated to some extent, too A model of nine PCs was produced with a R2 ¼ 0:89 and Q2 ¼ 0:64 (data not shown) The first PC explained 42% of the data variability, while the second PC explained a 33% The aw was the only factor that had a significant positive effect (P > 0:05) on the responses, as far as coefficients are concerned None of the 10 essential oils studied had significant effect on growth rates (P > 0:05), only a slight inhibition trend was observed for clove oil For the other 20 essential oils, two different sets of 10 essential oils designed by RSM were analysed separately by PLS, too The essential oils that had been observed to have a slightly inhibitory effect in the previous screening design (clove, cinnamon, and lemongrass) were included in this design Fig Contour plots based on models from PLS regression showing colony diameters (mm) of F verticillioides, F proliferatum and F graminearum growth after 12 incubation days at 25 C as affected by aw and concentration of lemongrass oil (mg mlÀ1) Fig Contour plots based on models from PLS regression showing colony diameters (mm) of F verticillioides, F proliferatum and F graminearum growth after 12 incubation days at 25 C as affected by aw and concentration of clove oil (mg mlÀ1) ARTICLE IN PRESS A Velluti et al / Food Microbiology 21 (2004) 649–656 For the first 10 essential oils of the RSM design, a model of nine PCs was produced with R2 ¼ 0:83 and Q2 ¼ 0:67: The first PC explained 42% of the data variability, the second explained 19% and the third explained 8% The first PC was mainly dominated by aw and concentrations of essential oils, showing that increasing aw ; growth rates increased Since the other factors had a minor importance on this PC, a loadings plot for PCs and was better to explain their importance (Fig 1) The second PC was dominated in the negative axes by cinnamon and lemongrass effects and to a lesser extent by clove and birch oils The third PC was dominated for concentrations and bay leaf, marjoram and incense oils An increase in concentrations of these oils reduced the growth rates However, only cinnamon leaf, clove and lemongrass essential oils were always significant inhibitors (Po0:05) for almost all days and species studied Cinnamon oil leaf was useful at controlling growth of Fusarium spp under all aw levels (Fig 2) In general, the preservative effect of the oil was quite independent of aw : Colony diameters were in general reduced by more than 80% when cinnamon oil concentration was increased from to 1000 mg mlÀ1 for all the species There was an inhibitory effect in growth rates of the three species due to lemongrass oil presence (Fig 3) In this case, the percentage of reduction of colony diameters was about 45–60% at 0.995aw and 75–95% at 0.95aw when lemongrass oil concentration was increased from to 1000 mg mlÀ1 for all the species studied For clove essential oil colony diameters were reduced more than 65% at 0.995aw and more than 92% at 0.95aw when clove oil concentration was increased wc [2] from to 1000 mg mlÀ1 for all the species tested (Fig 4) Similar results were shown at the different temperature levels and incubation times for the three essential oils For the second set of 10 oils, a model of 11 PCs was produced A high percentage of variation in the response was explained by the model (R2 ¼ 0:80) On the other hand, the predictive power of the model (Q2 ) is 0.48 The loadings plot of growth data for PCs and is shown in Fig The plot shows that aw ; oregano oil, concentration of oil (in general), citronella and palmarosa oils were the most important factors describing growth variables The first PC, which explains 29% of the total variation in the growth rate, was dominated by these factors The increase of aw made growth rates increase, while an increase of oil concentrations and presence of oregano, citronella and palmarosa oils, reduced growth rates The second PC (R2 ¼ 0:27) was mainly dominated by aw : It is important to point out that the coefficients for citronella oil were never significant for almost all days and species (P > 0:05), while the coefficients for oregano and palmarosa oils were always significant (Po0:05) for almost all days and species There was a negative correlation between concentrations of palmarosa oil and growth rate for all aw studied (Fig 6) For F verticillioides and F proliferatum, it was observed a high effect of this oil at low aw : The percentages of reduction of colony diameter were more than 35% at 0.995aw and more than 62% at 0.95aw when palmarosa oil concentration was increased from to 1000 mg mlÀ1 for all the species This trend was not observed for F graminearum, with a percentage of reduction of about 67% at all aw : There was a negative FpFpFp FgFpFvFvFpFpFp Fp Fv Fg Fg FvFvFv Fg Fg Fp FvFvFgFgFp Fg Fp Fg Fg Fg lavendera*conc lavender cedar* conc aw *oregano nutmeg* conc aw*cedar aw*citronella lavenderb*conc a *lavender a 0.20 tea tree*conc ylang ylang-ylang*conc w citronella conc aw *nutmeg citronella* aw *ylangylang palmarosa*conc aw* tea tree *palmarosa aw*lavender b aw*palmarosa temp temp*cedar 0.10 temp*mint temp* tea tree lavender b oregano* conc temp*lavender a temp*ylangylang temp*oregano temp*nutmeg conc*conc temp*lavender b temp*citronella 0.00 tea tree lavender a aw **aw ylang -ylang cedar aw *temperature nutmeg 0.30 -0.10 653 aw responses citronella concentration oregano -0.20 palmarosa -0.30 -0.20 -0.10 0.00 0.10 0.20 wc [1] 0.30 0.40 0.50 0.60 Fig Loadings plot of PCs and from partial least square (PLS) regression showing the relationship of aw ; temperature, concentration, essential oils (’) and cross-terms (K) on colony diameters (m, mm) of three isolates each of F verticillioides (Fv), F proliferatum (Fp) and F graminearum (Fg) ARTICLE IN PRESS 654 A Velluti et al / Food Microbiology 21 (2004) 649–656 Fig Contour plots based on models from PLS regression showing colony diameters (mm) of F verticillioides, F proliferatum and F graminearum growth after 12 incubation days at 25 C as affected by aw and concentration of palmarosa oil (mg mlÀ1) correlation between concentrations of oregano oil and growth rates for all aw studied (Fig 7) At 0.995aw the percentage of reduction of growth was more that 33% and at 0.95aw was more than 68% when oregano oil concentration was increased from to 1000 mg mlÀ1 for all the species For both essential oils, similar results were shown at different temperature levels and incubation times The composition of these essential oils is given in Table Both cinnamon leaf and clove oils had eugenol as major component, while geraniol and carvacrol were the major components of oregano and palmarosa oils, respectively Lemongrass essential oil contained high percentages of neral and geranial Fig Contour plots based on models from PLS regression showing colony diameters (mm) of F verticillioides, F proliferatum and F graminearum growth after 12 incubation days at 25 C as affected by aw and concentration of oregano oil (mg mlÀ1) Discussion Fusarium spp mainly colonize cereal grains before harvest but they may occur in stored grain when aw is high (Ramakrishna et al., 1996) Various publications have documented the antimicrobial activity of essential oils including lemongrass, cinnamon leaf, clove, palmarosa and oregano oils (Farag et al., 1989; Salmeron and Pozo, 1991; Patkar et al., 1993; Sinha et al., 1993; Mishra and Dubey, 1994; Pattnaik et al., 1996; Inouye et al., 1998; Hammer et al., 1999; Chao et al., 2000) against different microbial species In this study the ability of 37 essential oils to inhibit some of the most common Fusarium species in corn in temperate climates ARTICLE IN PRESS A Velluti et al / Food Microbiology 21 (2004) 649–656 655 Table Essential oils tested: the main components and their relative contents (%) Compounds Cinnamon leaf Clove Lemongrass Palmarosa Oregano a-Cubebene Linalool Caryophyllene Eugenol Cinnamaldehyde 2-Propenyl benzodioxol Eugenyl acetate Cinnamyl alcohol acetate Humulene Caryophyllene oxide Limonene Neral Geranial Geraniol Geranyl acetate 3-Carene g-Terpinene p-Cymene Thymol Carvacrol Phenyl-methyl-benzoate o1.0 2.6 4.7 78.5 1.1 1.1 1.7 1.0 — — — — — — — — — — — — — — — 12.6 83.9 — — — — 1.4 o1.0 — — — — — — — — — — — — — 4.1 — — — — — — — 3.8 29.4 50.5 5.0 1.6 — — — — — — — 3.8 2.5 — — — — — — — — — — 87.6 1.1 1.5 — — — — — — 2.1 2.1 — — — — — — — — — — — — — 1.8 16.7 0.2 70.0 1.3 under different laboratory conditions was evaluated Five essential oils (lemongrass, cinnamon, clove, palmarosa and oregano) showed antifungal activity Our results indicate that lemongrass oil could be an effective inhibitor of the three Fusarium species studied in corn In vivo studies should be performed to fully understand the overall processes when lemongrass oil is added to corn Similarly, Mishra and Dubey (1994) reported that this oil showed 90% and 100% of F verticillioides inhibition at 500 and 1000 mg mlÀ1, respectively They also reported that lemongrass oil is an effective post-harvest fungi toxicant of higher-order plant origin potentially suitable for protection of foodstuffs from storage fungi Under laboratory conditions, they found that lemongrass oil exhibited a broad spectrum of fungitoxicity by inhibiting completely the growth of 35, 45, and 47 fungal species at 500, 1000, and 1500 mg mlÀ1, respectively Its potency remained unaltered for 210 days of storage, after which it started to decline We found that cinnamon leaf and clove oils reduced F verticillioides, F proliferatum and F graminearum colony growth under all conditions tested; 1000 mg mlÀ1 of clove and cinnamon oils reduced colony growth about 62% and 80%, respectively The inhibition of growth and aflatoxin production by A flavus by clove and cinnamon oils has been reported (Bullerman et al., 1977; Sinha et al., 1993; Montes-Belmont and Carvajal, 1998) Mahmound (1994) observed that 1000 mg mlÀ1 of cinnamaldehyde completely suppressed growth of A flavus and consequently prevented aflatoxin formation (65–75% of cinnamon essential oil is cinnamaldehyde; Salmeron and Pozo, 1991) The minimum inhibitory concentration (MIC) revealed that cinnamaldehyde was highly effective at 200 mg mlÀ1 (Mahmound, 1994) Even though oregano and palmarosa oils were less effective in controlling Fusarium species growth than lemongrass, clove and cinnamon oils, they significantly inhibited growth under the different conditions tested The ability of oregano oil to inhibit A flavus, A ochraceus and A niger has been previously evaluated (Paster et al., 1990, 1995) The results of the in vitro experiments showed clearly that 2000 mg mlÀ1 of oregano oil had fungicidal activity in controlling mycelia of A flavus, A ochraceus and A niger (Paster et al., 1995) In vivo studies by the same authors showed that oregano oil was highly effective in controlling the internal wheat fungi (Paster et al., 1995) On the other hand, Pattnaik et al (1996) reported that palmarosa oil had the same inhibitory activity against twelve fungi tested; the response ranged from various degrees of susceptibility to total inhibition The present work showed a species dependent response to this essential oil, too The variation of temperature showed very little effect on the activity of the essential oils The aw did not show a significant effect in the effectiveness of these oils, but a slight increase in their activity at the lowest aw tested was observed As in general, grain is stored at low aw ; this may suggest that essential oils could be good candidates for post-harvest grain preservation It is important to note that there were no significant interspecific differences in the responses to the essential oils; this suggests that the essential oils may be a good alternative with a broad spectrum of application In ARTICLE IN PRESS 656 A Velluti et al / Food Microbiology 21 (2004) 649–656 both outdoor and storage environments they occur together with and compete with other species of fungi (Magan and Lacey, 1984) It is important to find essential oils with antimycotic effect against the most common species of fungi isolated from maize grain Three out of the five best essential oils, oregano, clove and cinnamon oil, have aromatic compounds among their major components Eugenol (the main component of clove and cinnamon oils) and carvacrol (from oregano oil) are phenols The antimicrobial activity of these oils can be attributed to the presence of an aromatic nucleus and a phenolic OH group that is known to be reactive and to form hydrogen bonds with active sites of target enzymes (Farag et al., 1989) The main component of palmarosa oil is geraniol, an aliphatic alcohol, and geranial and neral, aliphatic aldehydes, the main components of lemongrass oil Essential oils containing aliphatic alcohols and phenols, exhibited significant action against A aegyptiaceus, Penicillium cyclopium and Trichoderma viride (Megalla et al., 1980) To sum up, our data confirm that lemongrass, clove, cinnamon, oregano and palmarosa oils possess in vitro antifungal activity against the species of Fusarium studied In vivo studies may be required to confirm the usefulness of the results obtained Acknowledgements This work was supported by European Union (QLRT ! Inter1999-00996) and Spanish Government (Comision ! ministerial de Ciencia y Tecnologıa, CICIYT ALI980509-C04-01) References Bullerman, L.B., Lieu, F.Y., Seire, A.S., 1977 Inhibition of growth and aflatoxin production by cinnamon and clove oils, cinnamic aldehyde and eugenol J Food Sci 42, 1107–1116 Chao, S.C., Young, D.G., Oberg, C.J., 2000 Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses J Essent Oil Res 12, 639–649 Christensen, C.M., Sauer, D.B., 1982 Microflora In: Christensen, C.M (Ed.), Storage of Cereal Grains and Their Products American Association of Cereal Chemists, St Paul, MN, pp 210–241 Eriksson, L., Johansson, E., Kettaneh-Wold, N., Wold, S., 1999 Introduction to Multi- and Megavariate Data Analysis using Projection Methods (PCA & PLS) Umetrics AB, Sweden Farag, R.S., Daw, Z.Y., Abo-Raya, S.H., 1989 Influence of some spice essential oils on Aspergillus parasiticus growth and production of aflatoxins in a synthetic medium J Food Sci 54, 74–76 Hammer, K.A., Carson, C.F., Riley, T.V., 1999 Antimicrobial activity of essential oils and other plant extracts J Appl Microbiol 86, 985–990 Inouye, S., Watabable, M., Nishiyama, Y., Takeo, K., Akao, M., Yamaguchi, H., 1998 Antisporulating and respiration-inhibitory effects of essential oils on filamentous fungi Mycoses 41, 403–410 Magan, N., Lacey, J., 1984 Effect of water activity, temperature and substrate on interactions between field and storage fungi Trans Br Mycol Soc 82, 83–93 Mahmound, A.L.E., 1994 Antifungal action and antiaflatoxigenic properties of some essential oil constituents Lett Appl Microbiol 19, 110–113 Mar!ın, S., Sanchis, V., Magan, N., 1995 Water activity, temperature and pH effects on growth of Fusarium moniliforme and F proliferatum isolates from maize Can J Microbiol 41, 1063–1070 Megalla, S.E., El-Keltawi, N.E.M., Ross, S.A., 1980 A study of antimicrobial action of some essential oil constituents Herba Pol 3, 181–186 Mishra, A.K., Dubey, N.K., 1994 Evaluation of some essential oils for their toxicity against fungi causing deterioration of stored food commodities Appl Environ Microbiol 60, 1101–1105 Montes-Belmont, R., Carvajal, M., 1998 Control of Aspergillus flavus in maize with plant essential oils and their components J Food Prot 61, 616–619 Paster, N., Juven, B.J., Shaaya, E., Menasherov, M., Nitzan, R., Weisslowicz, H., Ravid, U., 1990 Inhibition effect of oregano and thyme essential oils on moulds and foodborne bacteria Lett Appl Microbiol 11, 33–37 Paster, N., Menasherov, M., Ravid, U., Juven, B., 1995 Antifungal activity of oregano and thyme essential oils applied as fumigants against fungi attacking stored grain J Food Prot 58, 81–85 Patkar, K.L., Usha, C.M., Shetty, H.S., Paster, N., Lacey, J., 1993 Effect of spice essential oils on growth and aflatoxin B1 production by Aspergillus flavus Lett Appl Microbiol 17, 4–51 Pattnaik, S., Subramanyam, V.R., Kole, C., 1996 Antibacterial and antifungal activity of ten essential oils in vitro Microbios 86, 237–246 Ramakrishna, N., Lacey, J., Smith, J.E., 1996 The effects of fungal competition on colonization of barley grain by Fusarium sporotrichioides on T-2 toxin formation Food Addit Contam 13, 939– 948 Salmeron, J., Pozo, R., 1991 Effect of cinnamon (Cinnamomum zeylanicum) and clove (Eugenia caryophyllus) on growth and toxigenesis of Aspergillus gr flavus Microbiol.-Aliments-Nutr 9, 83–87 Sanchis, V., Abadias, M., Oncins, L., Nuria, S., Vin˜as, I., Canela, R., 1995 Fumonisins B1 and B2 and toxigenic Fusarium strains in feeds from the Spanish market Int J Food Microbiol 27, 37–44 Sinha, K.K., Sinha, A.K., Prasad, G., 1993 The effect of clove and cinnamon oils on growth of and aflatoxin production by Aspergillus flavus Lett Appl Microbiol 16, 114–117 Sohn, H., Seo, J., Lee, Y., 1999 Co-occurrence of Fusarium mycotoxins in mouldy and healthy corn from Korea Food Addit Contam 16, 153–158 ... coefficients for citronella oil were never significant for almost all days and species (P > 0:05), while the coefficients for oregano and palmarosa oils were always significant (Po0:05) for almost... aldehyde and eugenol J Food Sci 42, 1107–1116 Chao, S.C., Young, D.G., Oberg, C.J., 2000 Screening for inhibitory activity of essential oils on selected bacteria, fungi and viruses J Essent Oil Res... from to 1000 mg mlÀ1 for all the species tested (Fig 4) Similar results were shown at the different temperature levels and incubation times for the three essential oils For the second set of