## 4 Discussion  
In the present work, thyme EO has been applied on _A. alternata_ classified among the \"indoor fungi\" able to produce toxic and/or allergenic secondary metabolites on the spore surface [30]. Among the different EO, known for their antifungal activity, _Thymus vulgaris_ has been selected considering its several effects, both at the level of cell membrane/wall and within the microbial cell [31]. Moreover, thymol, \(p\)-cymene, and \(\gamma\)-terpinene, main compounds present in thyme EO, are Generally Recognized as Safe (GRAS) [32]. In scientific literature, different methods (i.e., direct contact and vapor phase) have been applied to evaluate the antimicrobial effect of thyme EO on _A. alternata_ as reported below. In the work of [PERSON] et al. [21], thyme EO inhibition on mycelial radial growth of _A. alternata_ was tested by the agar dilution method: MIC50 and MIC100 values were 0.077 and 0.146 \(\upmu\)L/mL, respectively. [PERSON] et al. [33] reported that thyme EO from leaves and thymol had MIC of 500 and 250 \(\upmu\)mL\({}^{-1}\), respectively, lower than the value (1250 \(\upmu\)g mL\({}^{-1}\)) obtained with a commercial fungicide. A MIC of 26.7 \(\upmu\)g/1 and a 20% reduction in fungal biomass were obtained for _A. alternata_ exposed to vapors of commercial thyme EO (72 h, at 25 \({}^{\circ}\)C) [32]. In the work of [PERSON] et al. [34], the mycelial growth inhibition was proportional to thyme EO concentration: 48.0% and 100%, were obtained with 1 and 5 \(\upmu\)L.  
In the present study, the effect of thyme EO on _A. alternata_ growth was evaluated with MAM in Petri dishes. Results clearly showed the influence of applied EO volume on the lag-phase duration, see Figure 1A. A linear relationship (R-Square = 0,987) was observed between the pure EO volume applied and the inhibition period of _A. alternata_ growth, see Figure A2a. Otherwise, for the diluted EO, the influence of DMSO on oil evaporation was observed (see Figure A2b), as previously reported by [PERSON] et al. [35]. Also, for MGI% (see Figure 1B), the inhibition was proportional to EO concentration, according to results of [PERSON] et al. [34]. The low influence on the maximum growth rate value (see Figure 2) indicates a mycostatic effect of EO, depending on the applied volume. For a given EO volume (10 \(\upmu\)L), the exposure time delays the achievement of the maximum growth rate (see Figure 3B) and influences the conidiogenesis. After 6 h (0.25 day) exposure, it is possible to observe the conidiogenesis at the same time of the control (see Figure 4), while prolonging the exposure for at least 1 day, an inhibition of conidiogenesis is evident, as previously reported for the mycotoxigenic _A. flavus_[36; 37; 38]. Concerning the EO effect on metabolism, in [39] many targets and mechanisms are reported such as cell wall damage or changes in hyphal morphology which even led to the inhibition of conidiogenesis. The scale-up of the process in GAC (EO volumes of 210 and 420 \(\upmu\)L) proved that the evaporation rate influences the starting point of the inhibition; in fact, in tests with MEA medium, at low evaporation rate (Figure 5), 14 days were necessary to reach the MID of the EO active components in the vapor phase, independently from the EO volume applied, after that the fungal growth was inhibited till to 42 days. Once the EO treatment was set-up in GAC with CZ\({}_{\text{mod}}\) medium, with the lowest EO volume (210 \(\upmu\)L) and applying the disc volatilization method, one day was enough to inhibit the _A. alternata_ growth, independently from the number of treated colonies. In this case, the higher contact area between the oil and the environment reduced the time necessary to reach the MID in the vapor phase (see Figure 7A). In the work of [PERSON] [10], a fast vaporization method of thyme EO in indoor air was used, the effectiveness was monitored for 1 day reporting a 48.7% reduction in fungal colonies.