ORIGINAL ARTICLE/ARTICLE ORIGINAL
Assessment of growth-inhibiting effect of some plant essential oils on different Fusarium isolates
´ valuation de l’activite E ´ antifongique de quelques huiles essentielles de plantes sur divers isolats de Fusarium
A. Naeini
a,* , T. Ziglari
b, H. Shokri
b, A.R. Khosravi
baDepartment of Mycology, Faculty of Medicine and Medicinal Plants Research Center, Shahed University, Keshavarz Boulevard, P.O. Box 14155-7435, Tehran, Iran
bMycology Research Center, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
Received 16 March 2010; received in revised form 18 May 2010; accepted 24 May 2010 Available online 13 August 2010
KEYWORDS Fusarium;
Essential oils;
Fungistatic activity
Summary
Objective.— Essential oils (EO) of five medicinal plants were tested for anti-Fusariumactivity against 10 non-toxigenic (Fusarium solaniandFusarium oxysporum) and 11 toxigenic (Fusarium verticillioides,Fusarium poaeandFusarium equiseti) isolates.
Materials and methods.— Different dilutions of EO were prepared and 5103conidia of each fungal suspension were inoculated into Sabouraud’s glucose broth tubes. The cultures were incubated at 288C for 4 days.
Results.— Mean concentrations of the EO of Zataria multiflora (165.4 and 88.9mg/ml), Cuminum cyminum (159 and 185.3mg/ml), Foeniculum vulgare (496.4 and 532.9mg/ml), Pinaceae (869.7 and 852.43mg/ml) and Heracleum persicum (753.5 and 1492.6mg/ml) completely inhibit all the non-toxigenic and toxigenic isolates, respectively. Z. multiflora andH. persicumshowed the highest and lowest activity against toxigenicFusariumisolates, whereasC. cyminum andH. persicum had the highest and lowest effect on non-toxigenic isolates, respectively. However,F. vulgareandPinaceaehad moderate effects against tested fungi.
Conclusion.— The results indicated that non-toxigenic and toxigenicFusariumisolates were sensitive to the five EO, particularly sensitive toC. cyminumandZ. multiflora, respectively.
#2010 Published by Elsevier Masson SAS.
MOTS CLÉS Fusarium;
Résumé
Objectif.— Les huiles essentielles (essential oils [EO]) de cinq plantes médicinales ont été testées pour leur activité antifongique contre diverses souches deFusarium: dix non toxigènes
* Corresponding author.
E-mail addresses:[email protected],[email protected](A. Naeini).
1156-5233/$—see front matter#2010 Published by Elsevier Masson SAS.
doi:10.1016/j.mycmed.2010.05.005
Huiles essentielles ; Activité fongistatique
(Fusarium solanietFusarium oxysporum) et 11 toxinogènes (Fusarium verticillioides,Fusarium poaeandFusarium equiseti).
Mate´riels et me´thodes.— Les dilutions différentes d’EO ont été préparées et 5103conidies de chaque suspension fongique a été ensemençées dans les tubes de bouillon de glucose de Sabouraud contenant les EO. Les cultures ont été incubé à 288C pendant quatre jours.
Re´sultats.— Les concentrations moyennes de l’EO deZataria multiflora(165,4 et 88,9mg/ml), Cuminum cyminum(159 et 185,3mg/ml),Foeniculum vulgare(496,4 et 532,9mg/ml),Pinaceae (869,7 et 852,43mg/ml),Heracleum persicum(753,5 et 1492,6mg/ml) inhibent complètement tous les isolats toxigènes et non toxigènes.Z. multifloraetH. persicumont montré respective- ment la plus forte et la plus faible activité contre les isolats toxigènes deFusarium, tandis que C. cyminumetH. persicumont eu respectivement la plus forte et la plus basse activité sur les isolats non toxigènes. Cependant,F. vulgareetPinaceae ont eu un effet modéré contre les mycètes essayés.
Conclusion.— Les résultats ont indiqué que les isolats deFusariumtoxigènes et non toxigènes testés étaient sensibles au cinq EO, et notamment sensibles à celles de C. cyminum et de Z. multiflora.
#2010 Publié par Elsevier Masson SAS.
Introduction
Many species of fungi are capable of producing fungal infec- tions and mycotoxins in food and feeding. Among them Fusariumspecies cause seedling blight and root, stalk, and ear rot in maize as well as many of them produce mycotoxins.
The most frequently isolated species from maize in tempe- rate climates areFusarium verticillioides,Fusarium prolif- eratum and Fusarium graminearum followed by Fusarium subglutinans, Fusarium culmorum and Fusarium equiseti [22].
On the other hand, food-borne diseases are major dilemma in the developing countries, and even in developed nations[23]. Consumption of foods contaminated with some microorganisms represents a serious health risk to humans and animals. The subsistence and growth of fungi in foods may lead to spoilage and formation of toxins. These fungal metabolites are structurally diverse compounds, which represent the most important category of biologically pro- duced natural toxins relative to human health and economic impact worldwide[7].
On the other hand, the widespread indiscriminate use of chemical preservatives has resulted in the emergence of resistant microorganisms leading to fungal diseases[1]. To reduce this problem, there is a need to adopt strategies that are accessible, simple in application, and nontoxic to humans and animals, and that have sustainable broad-spec- trum fungitoxicity.
Among natural substances, the essential oils (EO) and herbal extracts have attracted a great deal of scientific interest due to their potential as a source of natural anti- oxidants and biologically active compounds[4,28,30]. The antimicrobial and antioxidant activities of EO have formed the basis of many applications including fresh and processed food preservation, pharmaceuticals, alternative medicine and natural therapies[7,18,24].
Data concerning the effects of EO against different myco- toxins production in inoculated wheat are limited. A previous study in examination of grain-based foods indicated the food preservative ability of EO (thyme, cinnamon, anise and
spearmint) at different concentrations in inhibiting myco- toxins production[25].
In this study, we examined the anti-Fusariumactivity of herbs such Zataria multiflora, Heraclum persicum, Pina- ceae, Cuminum cyminum and Foeniculum vulgare, which have long been used as spices or important medicinal sources in Iran[13,16]. The objective of this study was to examine the effect of different herb oils on someFusariumisolates such as Fusarium solani, Fusarium oxysporum, Fusarium verticillioides,Fusarium poaeandF. equisetiby determina- tion of the minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC).
Materials and methods
Isolation ofFusariumspecies and culture
Ten non-toxigenic Fusarium species (F. solani and F. oxysporum) isolated from clinical specimens and maize and 11 toxigenic Fusarium species (F. verticillioides, F. poaeandF. equiseti) isolated from maize were included in this study. The selective criteria of toxigenic Fusarium isolates were based on the production of fumonisin B1 by F. verticillioides, T2 toxin by F. poaeand zearalenone by F. equiseti. On the other hand, the production of above- mentioned toxins in non-toxigenicFusariumspecies such as F. solaniandF. oxysporumwas non-detectable. Fungal iso- lates were cultured onto Sabouraud’s glucose agar (Merck Co., Darmstadt, Germany) slant at 288C for 7 days. Unless other- wise indicated, all the chemicals were purchased from Sigma Chemical Co. (St., MO, USA).
Preparation of the conidial suspension
Conidia were harvested from 7-day-old cultures by pouring a sterile 0.1% aqueous solution of Tween 80 onto the culture plates and scraping the plate surface with a bent glass rod to facilitate the release of conidia. The number of conidia in the suspension was adjusted to approximately 1105conidia/
ml using a haemocytometer slide.
Essential oils
StandardZ. multiflora(Labiatae),H. persicum(Apiaceae), C. cyminum (Apiaceae), Pinaceae (Pine) and F. vulgare (Apiaceae) EO were obtained from Barij Essence Pharma- ceutical Company (Kashan, Iran).
Antifungal assay
The MIC and MFC values of EO for differentFusariumisolates were determined by the broth dilution method using the serially diluted plant oils as described[14]. Briefly, different EO concentrations (0.03—8%) diluted with DMSO 2% was incorporated in Sabouraud’s glucose broth (Merck Co., Darm- stadt, Germany). Fungal conidia (5103conidia) was added to each tube and incubated at 288C for 4 days. Positive control tubes containing only broth media and fungal suspen- sion as well as negative control tubes containing broth media accompanied by each EO and DMSO were prepared and incubated at the same conditions. The lowest EO concentra- tion that did not permit any visible fungal growth was taken as the MIC. The tubes that did not show visible fungal growth were sub-cultured onto oil-free Sabouraud’s glucose agar (Merck Co., Darmstadt, Germany) to determine if the inhibi- tion was reversible. The MFC was the lowest concentration that did not permit growth on the plates.
Statistical analyses
The Mann-Whitney, Kruskal-Wallis andt-Student tests were used. Probabilities of 5% were taken to be statistically sign- ificant.
Results
Table 1show the inhibitory and fungicidal activities on the growth of different non-toxigenicFusariumspecies including F. solaniandF. oxysporum. The results show that the mini- mum inhibitory and fungicidal concentrations of different EO were determined to be 164.9 to 1103.8mg/ml and 166.9 to 1248.7mg/ml, respectively.C. cyminum(MIC: 164.9mg/ml and MFC: 166.9mg/ml) andH. persicum(MIC: 1103.8mg/ml and MFC: 1248.7mg/ml) had the highest and the lowest activity.
In addition, the MIC and MFC of different EO were assayed against toxigenic Fusarium species; F. verticillioides, F. poaeandF. equiseti.Z. multiflorahad the highest activity (MIC: 175.2mg/ml and MFC: 203mg/ml), whereas H. persicum showed the lowest activity (MIC and MFC:
2307.7mg/ml) on fungi tested (Table 1).
Means of MIC/MFC between EO demonstrated significant differences betweenZ. multifloraandH. persicumas well as H. persicum and C. cyminum against Fusarium species
Table 1 Antifungal activity of different essential oils of medicinal plants against variousFusariumisolates.
Activite´ antifongique de diverses huiles essentielles de plantes me´ dicinales contre diffe´ rents isolats deFusarium.
Samples
Essences F1 F2 F3 F4 F5 F6 F7 F8 F9 F10
Z MIC 153 77 153 306 612 97 183 123 76 66
MFC 153 77 153 306 612 111 183 123 76 66
H MIC 1125 2250 70 1125 2250 895 753 1151 675 743
MFC 1125 2250 70 1125 2250 895 753 1151 998 743
P MIC 147 147 147 1172 2344 113 142 913 102 743
MFC 147 147 293 2344 2344 113 142 913 102 743
C MIC 145 145 72 578 289 103 70 122 69 59
MFC 145 145 72 578 289 102.5 89 122 69 59
F MIC 77 77 77 616 1232 94 81 72 78 82
MFC 77 77 77 616 1232 112 81 149 78 82
Samples
Essences F11 F12 F13 F14 F15 F16 F17 F18 F19 F20 F21
Z MIC 77 306 306 306 77 184 219 99 145 99 113
MFC 306 306 306 306 153 184 219 99 145 99 113
H MIC 2250 4500 225 4500 4500 1412 1984 952 1180 795 1062
MFC 2250 4500 2250 4500 4500 1412 1984 952 1180 795 1062
P MIC 293 147 586 2344 2344 152 175 142 163 176 130
MFC 586 586 586 2344 2344 152 115 148 163 176 130
C MIC 145 73 289 578 578 202 185 96 75 99 172
MFC 145 73 289 578 578 202 185 96 75 99 172
F MIC 77 77 1232 1232 1232 151 96 73 69 63 64
MFC 154 77 1232 1232 1232 151 96 73 69 63 64
Z:Zataria multiflora; H:Heracleum persicum; P:Pinaceae; C:Cuminum cyminum; minimum inhibitory concentrations (MIC) (w/v)mg/ml;
minimum fungicidal concentrations (MFC) (w/v)mg/ml; F:Foeniculum vulgare; F1, F2, F6, F7and F9:Fusarium solaniisolates; F3, F4, F5, F8
and F10:Fusarium oxysporumisolates; F11, F12, F13, F14, F15, F16and F17:Fusarium verticillioidesisolates; F18and F21:Fusarium poae isolates; F19and F20:Fusarium equisetiisolates.
(P<0.05). Although significant difference was not observed between the MIC and MFC of the EO onFusarium isolates understudy, this difference was demonstrated only on H. persicum (P<0.05). Overall, varying sensitivities to anti-Fusariumproperties were observed in five EO emphasiz- ing the variability in the antifungal effects against different Fusarium isolates. Although toxigenic Fusarium isolates were more resistance than non-toxigenicFusariumisolates in this study, no significant difference was observed between them according to EO sensitivity.
Discussion
Fusariumspecies mainly colonize cereal grains before har- vest and cause fungal infections in human and animals[19].
Various publications have documented the antimicrobial activity of EO including lemongrass, cinnamon leaf, clove, palmarosa and oregano oils against different microbial spe- cies[8,11,12,17]. In an old great medical Persian book, ‘‘Al- Aghraz al — Tibbia val Mabahess al — Alaiia’’ by Sayyed Esma’il Jorjani (1105 BH), more than half of the book has been established to plant pharmacology and pharmacoki- netics, which some of his ideas and speculations are accepted in the modern medicine. In fact, pure EO fight infections and are known to exhibit antimicrobial activities, and many applications for controlling the growth of food- borne pathogens have been developed using these EO as natural food preservatives[31].
Expression of different level of antifungal activities may be due to the differences in the content of known antimi- crobial compounds in each EO as earlier determined by Amvam Zollo et al.[2]and Tassou et al.[27]. Higher anti- Fusariumactivity was found in the EO fromC. cyminumand Z. multiflora against non-toxigenic (F. solani and F. oxysporum) and toxigenic (F. verticillioides, F. poae and F. equiseti) isolates studied, respectively. Moderate activity was observed for the EO from Pinaceae and F. vulgarewhile the EO fromH. persicumwas less inhibitory.
Similar antifungal activities of EO from Z. multiflora and C. cyminum were not reported on Fusarium species. In general, the antimicrobial activity of EO is mostly due to the presence of phenols such as thymol, carvacrol and aldehydes such as geranial, citronellal as well as alcohols such as geraniol, linalool, citronellol and lavandulol [6,20,26].Z. multiflorashowed the highest activity against toxigenicFusariumspecies, which is consistent with several studies that showed EO of the phenol type such as thymol and carvacrol possess antimicrobial activity [3,5]. The EO of C. cyminumwas another most effective of the EO against non-toxigenic species and main components, such as pinene, cineole and linalool exhibited strong activity onF. solaniand F. oxysporum[21]. Previous studies demonstrated that EO containing aliphatic alcohols and phenols have significant action against Aspergillus aegyptiaceus,Penicillium cyclo- pium,Trichoderma virideandCandida albicans[15,16]. Main constituents of C. cyminum oil were pinene, cineole and linalool, while the main components found inF. vulgarewere anethole, limonene, fenchone, and inH. persicumoil were anethole, terpinolene[16]. Although the three EO including H. persicum,C. cyminumandF. vulgarebelong to Apiaceae family, the presence of higher contents of alcoholic
compounds and different antimicrobial components in C. cyminumwas attributed to its higher activity. This may explain the general superiority of the EO fromC. cyminum than that of other EO belonged to Apiaceae family in the present study. The superiority of the EO fromC. cyminum againstF. solaniandF. oxysporumin this work is in agree- ment with the findings of Naeini et al. [16]. Overall, the antifungal activity of the EO is related to the respective composition of the herbal EO, the structural configuration of the constituent components and their functional groups and possible synergistic interactions between components[10].
It is necessary to mention that there is a direct relation- ship exists between inhibitory effects of EO on fungal growth and fusariotoxins production [9]. In a study conducted by Velluti et al.[29], all EO tested had an inhibitory effect on growth rate of zearalenone and deoxynivalenol produced by Fusariumspecies. Therefore, EO can also serve as alterna- tive means to prevent or control fungal attack and the presence of mycotoxin in stored corn and upon foodstuff.
In conclusion, our data confirmed that Z. multiflora and C. cyminumpossessed higher than in vitro antifungal activity against the species ofFusariumstudied.
Conflict of interest statement
The authors have not declared any conflict of interest.
Acknowledgement
This work was supported by the Research Council of Uni- versity of Tehran. The author would like to thank all autho- rities for invaluable kind help.
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