Impact of essential oils on growth rate, zearalenone and deoxynivalenol production by Fusarium graminearum under different temperature and water activity conditions in maize grain

Authors


Sonia Marín, Food Technology Department, Lleida University, Rovira Roure 191, 25198 Lleida, Spain (e-mail: smarin@tecal.udl.es).

Abstract

Aims:  The effect of five essential oils (oregano, cinnamon, lemongrass, clove and palmarose) on growth rate, zearalenone (ZEA) and deoxynivalenol (DON) production by Fusarium graminearum strains was assessed.

Methods and Results:  The influence of the essential oils was tested on irradiated maize at two concentrations (500 and 1000 mg kg−1), at different water activity (aw) (0·95 and 0·995) and temperature (20 and 30°C) levels. At 0·995 aw all essential oils tested had an inhibitory effect on growth rate of F. graminearum at both temperatures studied. At this aw level, DON production in general was inhibited by all essential oils at 30°C and, although palmarose and clove were the only essential oils with statistically significant inhibitory effect on ZEA production, an inhibitory trend was observed when cinnamon and oregano oils were added to maize grain.

Conclusions:  Antifungal and antimycotoxigenic activity of the essential oils assayed was shown to depend on environmental conditions.

Significance and Impact of the Study:  It is apparent that essential oils should be considered as alternative preharvest natural fungicides. Further investigation on natural maize grain might be useful to study the effectiveness of these essential oils in the presence of natural mycoflora of maize grain.

Introduction

Fungi cause significant destruction of grain during storage, rendering it unfit for human consumption by decreasing their nutritive value and sometimes by producing mycotoxins. Fusarium is a ubiquitous genus, and several fungal species are important pathogens of cereal crops (Booth 1977). One such fungus, Fusarium graminearum Schw. [Gibberella zeae (Schw.) Petch.] causes gibberella ear rot in corn which is also called pink ear rot and contaminates the kernels with mycotoxins (Miller et al. 1983; Bilgrami and Choudhary 1998) such as zearalenone (ZEA) and trichothecenes such as deoxynivalenol (DON). ZEA occurs frequently in maize used as a raw material in feedstuffs and produces a well-known hyperestrogenic syndrome (Scott 1989). DON, a cytotoxic trichothecene, has been associated with liver disease, oestrogenic disorders, oesophageal cancer and immunotoxic effects (Marasas et al. 1984; Lou et al. 1990).

Storage fungi are commonly controlled by synthetic chemicals; however, most of the fungicides of this group create several side-effects in the form of carcinogenicity, teratogenicity and residual toxicity (Mishra and Dubey 1994).

Many spices and herbs and their extracts possess antimicrobial activity, which is almost invariably because of their essential oil fraction (Paster et al. 1990). Thus the antimicrobial effects of spice and herb essential oils are of interest regarding their possible use as alternatives to food preservatives currently in use. Several studies have examined the effect of compounds isolated from oils to search for natural fungicides and a number of these oil constituents have been shown to be inhibitory (Chao and Young 2000). Essential oils containing aliphatic alcohol and phenols exhibit significant action against Aspergillus aegyptiaceus, Penicillium cyclopium and Trichoderma viride (Megalla et al. 1980). Bullerman et al. (1977), working with cinnamon and clove oils and their two major constituents, cinnamic aldehyde and eugenol, found these materials to be inhibitors of mould growth. A later report evaluated the ability of oregano and thyme essential oils and of two of their major constituents (carvacrol and thymol) to inhibit internal wheat fungi. They showed that oregano oil was highly effective, while thyme oil and the constituents carvacrol and thymol were less effective (Paster et al. 1995). However, little is known about the effect of essential oils on growth and mycotoxin production by phytopathogenic Fusarium species from corn and about the impact of environmental conditions on this effect. Velluti et al. (2002a) screened 37 essential oils for their inhibitory activity on growth of F. verticillioides, F. proliferatum and F. graminearum in maize meal extract agar. They found that cinnamon, clove, lemongrass, palmarose and oregano oils were the best oils tested.

The objectives of the present investigation were to determine the efficacy of cinnamon, clove, lemongrass, palmarose and oregano oils on growth rate and ZEA and DON production by F. graminearum in irradiated maize grain at different aw and temperature conditions.

Materials and methods

Culture material

Three different isolates of F. graminearum were used for these studies: isolate CECT 2150 provided by the Spanish Type Culture Collection (Colección Española de Cultivos Tipo, Valencia, Spain), isolate ITEM 223 provided by Dr S. Chulze (National University of Río Cuarto, Argentina) and isolate G2 SG1, provided by Dr C. Fannelli (Department of Biology, University of Roma ‘La Sapienza’, Rome, Italy).

Essential oils

The essential oils used were from cinnamon, clove, lemongrass, oregano and palmarose (Ravetllat Aromatics. S.L., Barcelona, Spain). The analyses of the main components of each essential oil (Table 1) were run on a Hewlett Packard GC-MS system (GC 6890; MSD 5973, Hewlett Packard, Vienna, Austria) by the Consejo Superior de Investigaciones Científicas (CSIC; Instituto de Química Orgánica General, Madrid, Spain).

Table 1.  Essential oils tested: the main components and their relative contents (%)
CompoundsCinnamonCloveLemongrassPalmarosaOregano
α-Cubebene<1·0
Linalool1·83·82·1
Caryophyllene4·68·22·52·1
Eugenol82·388·2
Cinnamaldehyde1·0
2-Propenyl benzodioxol1·0
Eugenyl acetate2·1
Cinnamyl alcohol acetate1·0
Humulene1·5
Caryophyllene oxide<1·0
Limonene5·4
Methyl heptanone1·4
Neral28·0
Geranial52·0
Geraniol2·087·6
Geranyl acetate3·61·1
3-Carene1·5
γ-Terpinene1·8
p-Cymene16·7
Thymol0·2
Carvacrol70·0
Phenyl-methyl-benzoate1·3

Stock solutions of each essential oil were prepared. Tween 80 (10%) was used as an emulsifying agent. Each essential oil was added to the irradiated maize to give final concentrations of 500 and 1000 mg essential oil kg−1 of maize.

Grain preparation at different water activities (aw)

Spanish dent maize grain was irradiated with 12 k Grays of gamma irradiation and stored aseptically at 4°C. In this way, the grain contained no fungal infection or contamination but had retained ability for germination. The initial water content of the grain was 13·9 % (0·71 aw). For all experiments, irradiated maize was weighed into sterile flasks and rehydrated to the desired treatment aw levels (0·95 and 0·995) by addition of sterile distilled water and essential oil solutions. The amount of water added was calculated from the moisture adsorption curve of the grain. The grain treatments were allowed to equilibrate at 7°C for 48 h, with periodic shaking. Finally, the aw values were confirmed by using a water activity meter (AquaLab, Pullman, WA, USA).

Inoculation, incubation and growth assessment

Rehydrated maize was placed in sterile Petri plates (ca 20 g per plate) forming a single layer of grain. A 5-mm diameter agar disk was taken from the margin of a 5-day-old growing colony on maize meal agar (MMEA) at 25°C of each isolate and transferred to the centre of each plate. Plates containing grain at the same aw level and the same essential oil were placed in containers along with beakers containing glycerol/water solutions of the same aw as the plates in order to maintain the atmosphere at the same equilibrium relative humidity as the substrate. Containers were incubated at 20 and 30°C. All treatments were repeated twice. Diameters of growing colonies were measured every day with the aid of a binocular magnifier. Two diameters were obtained from each colony and growth rates expressed as mm day−1 were calculated by linear regression of the colony radius against time for each strain at each set of conditions tested. After 28 days, the grains were frozen at −20°C for later ZEA and DON analyses.

Zearalenone analyses

For ZEA quantification the AOAC method for α-zearalenol and zearalenone by HPLC in maize was followed (AOAC 1995).

Deoxynivalenol analyses

The protocol recommended by Romer Labs, Inc. (Union, MO, USA; method no. LC1003·2) was followed. Its limit of detection is 0·1 mg kg−1. DON was extracted with 20 ml of acetonitrile/water (84 : 16) added to 5 g of ground sample, followed by shaking on a magnetic stirrer for 1 h. The extract was filtered through Whatman no. 1 filter paper. Purification of the crude extract was carried out by using a one-step cleanup column (MycoSepTM no. 227, Romer Labs Inc., Union, MO, USA). Two millilitres of purified extract were transferred to a vial and evaporated to dryness in a 60°C water bath under vacuum and dissolved in 1 ml of mobile phase, acetonitrile/methanol/water (4 : 4 : 92) for HPLC analysis. A 125 μl aliquot of solution was injected into the HPLC system. The flow rate for the mobile phase was 1·0 ml min−1. The wavelength of the absorbance detector was set at 220 nm. The reference standard was purchased from Sigma (St Louis, MO, USA). A standard calibration curve was constructed in the range 0·25–50 mg kg−1, and sample extracts were diluted when necessary in order to interpolate in that curve.

Dry matter determination

The dry matter content of each sample was determined by drying subsamples of ca 10 g at 105°C for 17 h (ISTA 1976). Thus, all results are presented on a dry weight basis.

Statistical analyses of the data

A full factorial design was used. The factors were aw (0·95 and 0·995), temperature (20 and 30°C), isolates (CECT 2150 and ITEM 223, G2 SG1) and concentrations of essential oils (500 and 1000 mg kg−1) and the responses were radii of growing colonies and DON and ZEA concentration. Analysis of variance was performed for colony radii and ZEA and DON concentrations using SAS version 8·02 (SAS Institute, Inc., Cary, NC, USA). Statistical significance was judged at the 5 % level.

Results

Water activity was the major factor affecting results. Growth rate, ZEA and DON production were higher at 0·995 than at 0·95 aw,regardless of temperature, in the absence of essential oils.

Growth rate

Water activity and concentration of essential oils, as well as their interaction had the major significant effect on growth of F. graminearum (Table 2). For some essential oils differences in growth were found between temperature levels and strains used. Although isolate 2150 grew slower than the others, their response to essential oils, water activity and temperature was almost identical.

Table 2.  Analysis of covariance of the effect of different concentrations (c) of essential oils on growth rate of Fusarium graminearum isolates (i) at different aw and temperature (t) levels
 DFCinnamon oilClove oilLemongrass oilOregano oilPalmarose oil
MSFMSFMSFMSFMSF
  1. *P < 0·05 and **P < 0·01.

t1589·28·4*36·80·52844·340·2**37·70·43534·550·6**
aw158287·8831·2**56354·4778·3**59799·5845·2**45350·6509·4**54720·2783·2**
i21513·621·6**1505·620·9**1198·316·9**122·41·4800·911·5**
c2668·79·5**3941·954·4**1905·526·9**15689·3176·2**2184·931·3**
i × aw2898·112·8**718·09·9**393·65·6*47·30·5341·74·9*
c × aw2403·15·8*1315·118·2**263·33·7*5854·765·8**365·45·2*
t × aw1119·81·715·70·23873·954·8**6·40·12431·134·8**
c × i4146·32·1113·51·6145·62·151·30·679·91·1
t × i234·30·5157·12·2226·43·2*87·81·0292·94·2*
t × c21624·323·2**982·213·6**18·60·313·40·253·40·8
i × aw × c4220·53·1*189·02·6*219·73·1*47·50·5191·12·7*
aw × i × t25·20·1144·02·0222·13·1*53·20·622·10·3
c × aw × t21512·321·6**1062·414·7**16·80·263·90·7317·54·6*
i × c × t4239·23·4*182·82·5*100·11·419·10·223·90·3
c × i × aw × t4316·94·5*214·03·0*61·00·98·31·0135·22·0

At 0·995 aw.

A significant inhibitory effect on the three strains tested was observed with 1000 mg cinnamon and palmarose essential oils kg−1 (Fig. 1). Both 500 and 1000 mg clove oil kg−1 had inhibitory effect for CECT 2150 strain while only 1000 mg clove oil/kg was effective for strains ITEM 223 and G2 SG1. Lemongrass and oregano essential oils significantly inhibited growth of the three strains tested; inhibition was significantly higher at 1000 mg oil kg−1 than at 500 mg kg−1. At this aw level, no significant differences were observed in the results obtained for both temperature levels.

Figure 1.

Effect of essential oils on growth rate of Fusarium graminearum CECT 2150 under different water activity and temperature conditions

At 0·95 aw.

Cinnamon and clove essential oils effect was dependent of temperature; at 20°C, growth of the three strains studied was significantly inhibited but not at 30°C. Lemongrass essential oil significantly inhibited growth at 1000 mg kg−1, while oregano and palmarose did so at both 500 and 1000 mg kg−1. The inhibitory effect of palmarose oil also was more evident at 1000 than at 500 mg kg−1 of maize.

Zearalenone production

In general, water activity was the only factor affecting zearalenone accumulation by the isolates tested (Table 3), with very low concentrations detected at 0·95 aw (Fig. 2). No significant effect was found for cinnamon, clove, lemongrass, and palmarose essential oils. When analysing the results separately for both aw levels, it was found that at 0·995 aw, clove and palmarose essential oils significantly inhibited zearalenone production at 30°C for all three isolates.

Table 3.  Analysis of variance of the effect of different concentrations (c) of essential oils on zearalenone production by Fusarium graminearum isolates (i) at different aw and temperature (t) levels
 DFCinnamon oilClove oilLemongrass oilOregano oilPalmarose oil
MSFMSFMSFMSFMSF
  1. *P < 0·05 and **P < 0·01.

c2MSF1364·32·2145·20·11348·03·7*2312·53·8*
i21644·72·731·10·16661·92·4588·01·672·40·1
aw1731·41·210356·017·0**32806·212·0*4542·912·4*6786·311·1*
t110271·516·7**44·20·110509·93·81635·74·5*311·80·5
i × aw2209·90·335·70·16477·02·4624·81·771·20·1
c × aw2730·71·21499·12·5118·20·01285·73·5*2280·03·7*
t × aw11612·42·6100·70·210908·14·01754·24·8*334·80·6
c × i4221·70·4680·41·1814·50·31015·32·8*549·80·9
t × i2349·80·61161·91·95972·32·21001·32·7784·61·3
t × c2712·41·21732·72·9208·40·11755·44·8*859·21·4
i × aw × c4954·81·6663·21·1751·00·31034·22·8*549·20·9
aw × i × t2352·50·61231·62·06145·62·2967·82·7787·91·3
c × aw × t2715·81·21920·23·2232·40·11797·94·9*876·81·4
i × c × t4981·51·6281·30·5167·40·1991·02·7*368·90·6
c × i × aw × t4361·40·6264·40·4194·00·1986·62·7*369·30·6
Figure 2.

Effect of essential oils on zearalenone production by Fusarium graminearum G2 SG1 (a) and CECT 2150 (b) under different water activity and temperature conditions

For oregano essential oil, most of the single factors (aw, temperature, concentrations) as well as almost all two- and three-way interactions had a significant effect on ZEA production by F. graminearum (Table 3). The statistical analysis showed that the inhibition only took place at 0·95 aw where most of the times no zearalenone could be detected when adding oregano essential oil.

Deoxynivalenol production

All single factors (aw, temperature, essential oils concentrations and strains) as well as all two- and three-way interactions had a significant effect on DON production by F. graminearum (Table 4).

Table 4.  Analysis of variance of the effect of different concentrations (c) of essential oils on deoxynivalenol production by Fusarium graminearum isolates (i) at different aw and temperature (t) levels
 DFCinnamon oilClove oilLemongrass oilOregano oilPalmarose oil
MSFMSFMSFMSFMSF
  1. *P < 0·05 and **P < 0·01.

c221251·026·4**26728·933·8**16603·214· 6**1591·257·4**21033·214·6**
i28879·811·0*9978·712·6**13072·611·5**379·113·7**16352·211·4**
aw156401·570·1**40538·751·2**75946·866·6**2621·894·6**59479·341·3**
t136936·145·9**26934·534·0**47612·641·8**1331·948·1**33458·423·2**
i × aw28951·811·1*10031·612·7**12691·611·1*473·117·1**16471·311·4**
c × aw221313·626·5**26774·033·8**16487·814·5**1643·859·3**20298·914·1**
t × aw138946·748·4**26194·533·1**50058·543·9**1159·941·9**36156·125·1**
c × i410046·112·5**8553·110·8**7980·97·0*465·216·8**6775·84·7*
t × i210791·013·4**12814·316·2**15220·113·4**292·410·6*21144·314·7**
t × c212924·016·1**16187·820·5**10074·08·8*615·92622·2**14060·49·8*
i × aw × c49870·712·3**8488·710·7**8012·97·0*405·114·6**6653·14·6*
aw × i × t210473·013·0**12256·615·5**15705·013·8**348·712·6**20839·014·5**
c × aw × t213011·716·2**17322·021·9**10317·29·1*697·025·2**13977·49·7*
i × c × t412379·215·4**10583·013·4**10039·38·8**319·311·5**8033·55·6*
c × i × aw × t412609·915·7**10901·913·89810·28·6**327·811·8**8125·75·7*

At 0·995 aw/30°C DON production by strains G2 SG1 and CECT 2150 was inhibited by both concentrations of cinnamon, clove, lemongrass and palmarose essential oils used (Fig. 3b), moreover the three strains studied were also inhibited by clove essential oil at 20°C. No significant effect of these essential oils was found at 0·95 aw.

Figure 3.

Effect of essential oils on deoxynivalenol production by Fusarium graminearum ITEM223 (a) and G2 SG1 (b) under different water activity and temperature conditions

At 0·995 aw DON production by ITEM 223 and CECT 2150 strains was significantly inhibited at 30°C by 500 and 1000 mg oregano oil kg−1. At 20°C, oregano oil had an inhibitory effect only on the ITEM 223 strain. At 0·95 aw significant inhibitory effect only was found at 20°C for the ITEM 223 strain (Fig. 3a).

Discussion

This study has shown that potential exists for controlling predominantly growth and deoxynivalenol production by F. graminearum using essential oils over a range of aw and temperature conditions. Our results indicate that the essential oils tested were more effective at high aw. At 0·95 aw the inhibitory effect was not as clear, the low ZEA and DON production and the high differences between replicates could be the reason why almost none of the essential oils had a statistically significant effect on the mycotoxin production by F. graminearum.

At 0·995 aw all essential oils tested had inhibitory effects on growth of F. graminearum at both temperatures studied. At this aw level, DON production in general was inhibited by all essential oils at 30°C and, although clove and palmarose were the only essential oils with statistically significant inhibitory effect on ZEA production, an inhibitory trend was also observed when cinnamon and oregano oils were added to maize grain. Recent studies have demonstrated that oregano, clove and lemongrass oils were effective in inhibiting growth of F. verticillioides and F. proliferatum at 0·995 aw in irradiated maize. Moreover, oregano, cinnamon and palmarose oils significantly reduced fumonisin B1 at 0·995 aw and 30°C (Velluti et al. 2003). These results together with the results of the present study indicate that the efficacy of the five essential oils studied is quite similar for the isolates of the three Fusarium species tested; the higher the aw of the grain, the better the inhibitory effect of essential oils. It might be assumed that the penetration of the oils into the internal parts of the grain is improved in the presence of water, and therefore pathogens could be more easily controlled in the inner parts of moist grain (Paster et al. 1995). Weather factors favourable for production and dispersal of spores on the host surface and infection must coincide with the time when the corn is very receptive to F. graminearum. Optimum temperatures are between 25 and 32°C (Bilgrami and Choudhary 1998). Gibberella ear rot epidemics are usually completed in one cycle because corn ears remain highly receptive for only 10–20 days following silking, at this time moisture content of the kernel is ca 30% (≅0·98–0·99 aw) (Sutton 1982). Therefore, the essential oils tested could be effective in controlling mainly growth and DON production by F. graminearum in maize under preharvest conditions.

At present, information on the mechanism of action of the essential oils on Fusarium species is limited. It has been suggested that antimicrobial activity of essential oils depends not only on their components but also on the chemical structure of these components (Megalla et al. 1980). It has been reported that the antioxidant effect of aromatic plants is because of the presence of hydroxyl groups in their phenolic compounds (Shahidi et al. 1992). Three of the five essential oils tested, oregano, clove and cinnamon oils, 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). It was described earlier that the hydroxyl group (bound to a benzene ring) is important for the activities of some antimicrobial compounds and that these activities are enhanced by the presence of αβ double bonds (Ultee et al. 2002). Several authors have pointed out the antimicrobial activity of carvacrol; it has been suggested that it interacts with the cell membrane, disrupting it (Kim et al. 1995; Thompson 1996). However, the main component of palmarose oil is geraniol, an aliphatic alcohol, while the aliphatic aldehydes, geranial and neral, are the main components of lemongrass oil. It has been reported that, geraniol, nerol and citronellol (aliphatic alcohol) completely suppressed growth of A. flavus and consequently prevented formation of aflatoxin (Mahmoud 1994).

From the results obtained in this investigation, it is apparent that essential oils should be considered as alternative preharvest natural fungicides. Further investigations on natural maize grain might be useful to study the effectiveness of these essential oils in presence of natural mycoflora of maize grain.

Acknowledgements

This work was supported by EC, Quality of Life Programme (QoL), Key Action 1 (KA1) on Food Nutrition and Health, (QLRT 1999-00996) and Spanish Government (Comisión Interministerial de Ciencia y Tecnología, CICYT ALI98-0509-C04-01). The authors are grateful to Montse Prim for her technical assistance.

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