atholog P y & nt a M Oukhouia et al., J Plant Pathol Microbiol 2017, 8:10 l i P c f r o o b DOI: 10.4172/2157-7471.1000423 l i Journal of a o l n o r g u y o J ISSN: 2157-7471 Plant Pathology & Microbiology ReviewResearch Article Article OpenOpen Access Access In-vitro Study of Anti-Fusarium Effect of Thymol, Carvacrol, Eugenol and Menthol Oukhouia M1, Sennouni CI1, Jabeur I1, Hamdani H1 and Remmal A2* 1Faculty of Science Dhar El-Mahraz, Atlas-Fez, University Sidi Mohammed Ben Abdellah, Fez, Morocco 2Faculty of Science Dhar El-Mahraz, Atlas-Fez, University Sidi Mohammed Ben Abdellah and Industrial Laboratory of Veterinary Alternatives (LIAV, LLC) Fez, Morocco Abstract This study aims at shedding light on the anti-fusarium action of some Major Compounds (MCs) of Essential oils (EOs). To this end, Fusarium oxysporum f. sp. dianthi (Fod) was used as fungal model. A screening of four MCs was conducted in order to identify the most active one with the highest anti-Fusarium effect, using both agar and broth dilution methods. The obtained data revealed that thymol, with its minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values was the most effective, with concentrations between 0.25 mg ml-1 and 1 mg ml-1. Among the four compounds tested, thymol and carvacrol showed anti-Fusarium activity by inhibiting the germination and destroying Fod conidia. As a preliminary test, we have also tested the applicability of thymol on soil disinfection, with very promising results. The antifungal activity of the four MCs used in this study involves inhibition of germination and destruction of Fod conidia. Thymol, the most effective MC, has shown great effect on soil disinfection. This work is a preliminary contribution aiming at developing an alternative product, environmental friendly, safe for both the workers and consumers, with high anti-Fusarium activity. Keywords: Major compounds; Essential oils; Fusarium oxysporum f. Culture media sp. dianthi; Fungicidal action; Soil disinfection Potato dextrose agar (PDA) (Biokar, France) was used for fungus Introduction identification [9]. Sabouraud dextrose agar (SDA), Sabouraud dextrose broth (SDB) (Biokar, France) and Malt extract broth (MEB) were used Fusarium wilt in carnations is a vascular disease caused by the for fungus culture and antifungal test [10]. fungus Fusarium oxysporum f. sp. dianthi (Fod). It is the most common and serious fungal disease that attacks carnations worldwide [1]. The Preparation of conidial suspension fungus can cause big financial losses to the floriculture industry. To Conidia of fod were harvested from 7-day-old cultures in PDA control the Fusarium wilt, growers use systemic fungicides like methyl by pouring a sterile 0.9% aqueous solution of NaCl onto the culture bromide [1], chloropicrin and benomyl [2]. Beside the fact that these plates and scraping the plate surface with a bent glass rod to facilitate chemicals are toxic to humans and the environment [3], they can also the release of conidia. The number of conidia cells was adjusted to 106 cause selection for resistant mutated pathogen [4]. ml-1 and was determined by transferring 10 µl of the sample suspension of Fod conidia to a Malassez chamber for microscopic examination Severe epidemics of Fusarium wilt in the 1980s and 1990s have and counting. Fod conidia were counted in 10 different fields utilizing caused a strong reduction in the acreage devoted to the carnation standard techniques [11]. industry in some areas of Southern Europe, such as Italy, France, and Spain [5]. Fusarium has obliged the industry players to leave Europe Anti-fungal agents and the United States towards other countries such as Colombia and Four MCs were used in this study: Thymol; Carvacrol; Eugenol and Morocco [5]. Despite the availability of cultivars that are relatively Menthol, all were obtained from Sigma-Aldrich (Steinheim, Germany). resistant to some strains of Fod, Fusarium wilt remains a threatening Each MC was dispersed in a suspension containing 0.2% agar in sterile disease for this crop wherever it is grown [5]. distillated water in order to maintain viscosity to disperse MCs without adding solvent or detergent [12]. A stock suspension of 100 mg ml-1 for Several publications from our laboratory have previously reported each MC was then prepared. in vitro and in vivo antifungal activity of some EOs and their MCs, especially the phenolic compounds such as thymol, carvacrol and Determination of anti-Fusarium effect of MCs in agar medium eugenol [6-8]. The MCs were added to the medium at the concentrations of (0; The major aim of the present work was to elucidate the in vitro antifungal activity of some MCs of EOs on the growth of Fod in order to develop an alternative to chemical fungicides. *Corresponding author: Remmal A, Faculty of Science Dhar El-Mahraz, P.O. Box 1796, Atlas-Fez, University Sidi Mohammed Ben Abdellah and Materials and Methods Industrial Laboratory of Veterinary Alternatives (LIAV, LLC) Fez, Morocco, Tel: (+212) 661532398; E-mail: [email protected] Fungal strain Received October 04, 2017; Accepted October 19, 2017; Published October 23, 2017 The Fod strain used in this study was isolated in our laboratory from a diseased carnation plant collected from a greenhouse of Prim’Rose Citation: Oukhouia M, Sennouni CI, Jabeur I, Hamdani H, Remmal A (2017) In-vitro Study of Anti-Fusarium Effect of Thymol, Carvacrol, Eugenol and Menthol. J Plant Company (Azemmour, Morocco). Pathol Microbiol 8: 423. doi: 10.4172/2157-7471.1000423 The Fod strain identification was done using a conventional method Copyright: © 2017 Oukhouia M, et al. This is an open-access article distributed based on the colony’s macroscopic characteristics and the microscopic under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original appearance of conidia [9]. author and source are credited. J Plant Pathol Microbiol, an open access journal ISSN: 2157-7471 Volume 8 • Issue 10 • 1000423 Citation: Oukhouia M, Sennouni CI, Jabeur I, Hamdani H, Remmal A (2017) In-vitro Study of Anti-Fusarium Effect of Thymol, Carvacrol, Eugenol and Menthol. J Plant Pathol Microbiol 8: 423. doi: 10.4172/2157-7471.1000423 Page 2 of 6 0.125; 0.25; 0.5; 1; 2 and 4 mg ml-1) from a stock suspension and poured The Fod conidial destruction assay in sterile Petri dishes (90 × 16 mm). The determination of anti-Fusarium Each MC was tested in increasing concentrations i.e., 0; 0.0625; effect of each MC in SDA medium was carried out by the inoculation of 0.125; 0.25; 0.5 and 1 mg ml-1. 10 µl calibrated drop (104 conidia) in agar plates containing increasing concentrations of MCs. Three Petri dishes were considered for each 100 µl of freshly prepared conidia suspension (106 ml-1) was put in concentration, each petri dish contained three calibrated drops of Fod direct contact with 900 µl of MC at various concentrations in sterile and the petri dish were incubated for 5 days at 27°C. 0.9% aqueous solution of NaCl containing 0.2% agar. The diameter of mycelial colony of Fod was measured in centimeters The Fusarium conidia destruction assay was evaluated after an for each concentration of all the MCs used and was compared to the incubation at 27°C for 24 h. control agar plate. As control tube, we used a 900 µl aqueous solution of sterile 0.9% 6 -1 The minimal inhibitory concentration (MIC) was determined as the of NaCl containing 0.2% agar and 100 µl of 10 ml freshly prepared lowest antifungal agent concentration at which no mycelial growth was conidia suspension. visually observed. The number of conidia was determined in a Malassez chamber (as previously described) after 1 h, 3 h, 6 h and 24 h of incubation at 27°C The anti-fusarium effect of MCs was expressed by the percentage of and the percentage of conidial destruction was calculated using the growth inhibition of Fod and calculated using the following formula [13]: following equation [14]: I(%) = (Dc-Di)/Dc × 100 I(%) = (Nc-Ni)/Nc × 100 Where Dc and Di represent respectively the mycelial growth Where Nc and Ni represent respectively the number of conidia in the diameters in control and in treated petri dishes. control and the treated tubes. Determination of anti-Fusarium effect of MCs in liquid Fusarium density in the soil medium 18 aliquots of 100 g sterilized soil were placed in sterile 400 ml The determination of anti-fusarium effect of MCs in liquid medium plastic boxes. Fod was incorporated into sterilized soil (previously sifted (MEB) was conducted in triplicate using sterile 96 well microplates by through a 2-mm sieve, and then was autoclaved 30 min at 121°C) at an adding 20 µl of conidial suspension of Fod (2 × 104 conidia ml-1) and 70 inoculum density of 106 spores per gram of soil. µl of increasing concentrations of MCs (0; 0.0625; 0.125; 0.25; 0.5; 1; 2 Experimental treatments were: (1) uninfested and untreated and 4 mg ml-1) to 160 µl of MEB medium in order to have a final volume soil, only water added as a check for residual populations of Fod (not of 250 µl in each well. After 5 days of incubation at 27°C, the mycelial included in analysis); (2) infested and untreated soil, only water added; growth was assessed by measuring optical density (OD) at 600 nm using (3) infested and treated soil with thymol. Four concentrations of thymol spectrophotometer (J.P. Selecta). were used for treatments (0.25; 0.5; 1 and 2 mg g-1 of soil) and each The anti-Fusarium effect of MCs was expressed by the percentage of concentration was calculated per gram of soil.