Optimization of the Antifungal Activity of Essential Oil Isolated from Aerial Parts of Thymus Boiss

: Although utilization of synthetic chemicals is inevitable for management of economically detrimental agents, numerous side-effects such as environmental contaminations and effects of non-target organisms associated with them. Plant essential oils with low/without toxicity on mammals and as bio-degradable natural materials have been considered for different pests and fungi management in the recent years. In the present study, the essential oil of Thymus kotschyanus isolated by a Clevenger apparatus and its mycelial growth inhibition was measured against two phytopathogenic fungi Botrytis cinerea and Fusarium graminearum . The best models for predicting of antifungal effects were quadratic models. The essential oil showed a prospective mycelial growth inhibition against both phytopathogenic fungi. Optimization of the antifungal effects indicated that 206.207 ppm of the essential oil caused 50% mycelial growth inhibition of B. cinerea after 89.651 h. This value was 85.600 ppm for F. graminearum within 117.194 h. Results of the present study designated a great potential

Fusarium graminearum is a cause of Fusarium head blight (FHB) of wheat and other cereals throughout the world (Suga et al., 2008). Due to this fungus activity, a poor quality product with wrinkled grains is produced and the weight of the seeds is reduced. Along with the reduction of product quality, it also creates some mycotoxins including Nivalenol, Deoxynivalenol, and Zearalenone which pose serious health risks for humans and animals (Windels, 2000). Botrytis cinerea with a very wide range of hosts is one of the other dangerous fungi that infect many plants before and after harvest. In terms of scientific and economic importance, this fungus has the second place among the 10 important fungi (Dean et al., 2012). B. cinerea causes gray mold disease in many fruits, vegetables and ornamental plants (Williamson et al., 2007;Romanazzi, 2013).
The use of synthetic chemicals is a typical method for plant protection against fungal infections. However, the overuse of such compounds has led to several negative effects, such as adverse effects on the environment, human health threats, and the emergence of resistant populations (Pimentel et al., 2008;Damalas and Eleftherohorinos, 2011;Cavalcanti et al., 2010). Therefore, the search and introduction of natural and bio-degradable agents are critical.
Plant essential oils in the form of secondary metabolites play an important role in plant protection against fungi and phytophagous insects. These bioagents act as signaling molecules and show an evolutionary relationship with their functional role in the plants (Tholl, 2006). In recent years, several investigations have been accomplished on the use of essential oils in managing pathogenic fungi (Bakkali et al., 2008;Pinto et al., 2013). Thymus kotschyanus Boiss and Hohen is one of the popular medicinal plants, which is used in traditional medicine and herbal tea. Further, some biological effects including antibacterial, antifungal, and antioxidant activities of T. kotschyanus essential oil were recognized recently (Afshari et al., 2016;Sevindik et al., 2016).
The main goals of the present study were the evaluation of the antifungal effect of T. kotschyanus essential oil against two phytopathogenic fungi Botrytis cinerea and Fusarium graminearum. Further, development of mathematical models to find the optimized conditions for these bio-effects was the other objective.

MATERIALS AND METHODS
Optimization of the Antifungal Activity of…..

EBADOLLAHI, A; TAGHINEZHAD, E; DAVARI M
Extraction of essential oil: Aerial parts of and Thymus kotschyanus were collected, respectfully, from Hassan Baroog and Sardabeh regions (Ardabil province, Iran). After drying at room temperature, the specimens were chopped with an electric grinder. Extraction of essential oil was done using a Clevenger apparatus with 100 g of each plant sample and 1500 ml distilled water within 3 hours. Obtained essential oil was dewatered using anhydrous sulfate sodium and kept in a refrigerator at 4 ° C until use.

Antifungal
activity of the essential oil: Phytopathogenic fungi Fusarium graminearum and Botrytis cinerea, which were isolated respectfully from infected wheat and strawberry fruits, were considered for mycelial growth inhibition activity of T. kotschyanus essential oil. The fungal isolates were identified and stored in Mycology Laboratory of the Department of Plant Protection, University of Mohaghegh Ardabili, Iran. Both isolated were stored on PDA medium (Potato Dextrose Agar) at 25 °C.
Mycelial growth inhibitory of T. kotschyanus essential oil was investigated by mixing of the essential oil concentration with culture medium. Concentrations of 75, 150, 300, 600 and 1000 ppm were prepared in 0.05% aqueous Tween-80. Flasks (250 ml) containing a PDA medium were placed at room temperature to reduce the temperature to 45-42 °C after autoclaving and the concentrations were added to them. The flasks stirred to form a uniform emulsion and the resulting media was immediately poured into 9 cm Petri dishes and allowed to become solid. The fungal discs were then prepared from the young cultures of the pathogenic fungi and positioned in the middle of the Petri dishes. Mycelial growth of each fungus was measured daily until the surfaces of the culture media in Petri dishes were fully absorbed. Aqueous Tween-80 without any essential oil concentration was used for control groups and three replicates were considered for each treatment. Inhibition Percentage (IP) of different concentrations of essential oil was determined using the following formula: Where C is the mean diameter of the mycelial disc in the control group and T is the mean diameter of the mycelial disc in the treatments.
Modeling of insecticidal and antifungal effects of the essential oil and statistical analysis: The data were analyzed by Design Expert versions 7.0.0 (2007, Stat-Ease company, USA). The coded independent variables are the essential oil concentrations (X1) and time (X2) in 5 and 3 levels, respectively, and 3

replications. Mathematical models between the independent variables [Concentration (ppm) and time (h)] and dependent variables [Inhibition Percentage
(%)] evaluated by means of multiple linear regression analysis in the following form: where β0, βi, βii, βij are constant coefficients of regression, Xi and Xj are the independent variables, Y is the dependent variable, n is a number of independent variables and e is the random error term. The relationships between the responses were checked by correlation coefficients of determination (R 2 ), adjusted R 2 , and predicted R 2 . A good model will have a large predicted R 2 and a low PRESS. The significance was analyzed with a confidence level of 95% (P < 0.05).

RESULTS AND DISCUSSION
The results showed that the studied concentrations of T. kotschyanus essential oil had pleasing antifungal activity on F. graminearum and B. cinerea fungi and the mycelial growth of pathogenic fungi has decreased with increasing time (Figure 1). The results of analysis of variance for mycelial growth inhibitory of T. kotschyanus essential oil shows that the effect of exposure time (A) and essential oil concentration (B) along with AB, A 2 and B 2 were significant F. graminearum and B. cinerea fungi. The greatest inhibition percentages on the mycelial growth of fungi were obtained by essential oil concentration factor (59% and 85%, respectively), which indicate that this factor is very important as compared to other factors (Table 1).
According to the models presented in Table 2 and the coefficient of variations, it can be concluded that these are the best models for estimating of the antifungal effects of T. kotschyanus essential oil. The coefficients of the independent variables (concentration and time) are positive: an increase in each of the variables is an incremental effect on the response variable. The negative sign of the variables in each model indicates the decreasing effect of the variable on the amount of the mycelial growth inhibition percentage of fungi.  (Table 3).
Antifungal effects of the essential oils of Thymus kotschyanus, Ocimum basilicum L. and Rosmarinus officinalis L. were ascertained on two major phytopathogenic fungi Penicillium expansum and B. cinerea (Jalili-Marandi et al., 2011). In the other study, antifungal effects of the essential oils isolated from some medicinal plants were investigated against phytopathogenic fungi Fusarium oxysporum, Aspergillus flavus and Alternaria alternate (Mohammadi et al., 2014). It was found that the essential oils of T. kotschyanus, Stachys pubescens Ten. and Bupleurum falcatum L. exhibited an appropriate antifungal activity against these fungi. According to the susceptibility of B. cinerea and Fusarium oxysporum fungi to the essential oil of T. kotschyanus, results of mentioned works are in parallel with results of the present study. However, the optimization and modeling of these antifungal effects were measured in our work for the first time. Conclusion: F. graminearum and B. cinerea are among the most damaging agents in the many countries, which have economic losses on many agricultural products. Management of such destructive agents is principally carried out by synthetic agrochemicals but the application of these chemicals prompted several environmentally adverse effects. Therefore, the use of eco-friendly natural compounds in the management of harmful agents is required. In the present study, the mycelial growth inhibitory effect of T. kotschyanus essential oil was confirmed on F. graminearum and B. cinerea. Furthermore, results of the present study were announced the response surface methodology (RSM) was efficaciously applicable to the optimization of T. kotschyanus essential oil bioeffects.