## 4 Discussion
### Antibacterial and Antifungal Activity of the EO of Marigold  
Based on the experimental results obtained, the hypothesis that this EO could have a bactericidal effect against _P. carotovorum_ is not excluded, and a correlation is observed between the inhibitory effect and the concentration used. The recorded bacterial growth resulted from the colonization and expansion of colonies only at the periphery of the EO spots. No isolated colonies were observed on the EO footprint, supporting the hypothesis that bacteria that came into direct contact with the EO lost their viability and could not proliferate further. EOs contain various active compounds that can disrupt multiple targets in bacterial cells, and one of the most important is the cytoplasmic membrane [38]. Some compounds in EOs increase the permeability of the cell membrane, leading to its loss of viability, a phenomenon associated with ion homeostasis and the electron transport chain [39].  
The results obtained in the tests performed on _P. marginalis_ (bacteriostatic effect) confirm a series of experimental findings. Although after 24 h of incubation good inhibitory efficiency was observed, in line with the increase in essential oil concentration, after 7 days of incubation, it was noticed that in the variants where a solvent was used, the colony density was lower in the previously clear area. Colonies developed in that region were rarer compared to plates where undiluted EO was tested. Colony density, however, could not be assessed differentially between test dilutions. Marigold EO used undiluted maintained a clear area of inhibition of bacterial growth even after 7 days of incubation. This aspect can be justified by the fact that this oil, being denser, may have evaporated more slowly compared to the others. However, taking into account the fact that after 7 days bacterial colonies were also observed in the initially clear area (after 24 h), the EO effect was bacteriostatic. Tests conducted by [40], showed that hyssof essential oil was almost inactive against certain Gram-negative bacteria (_P. aeruginosa, E. coli, Salmonella typhi_). [PERSON] et al. [41] extracted the EO from dried _T. patula_ flowers and investigated its antifungal effect on _Penicillium digitatum_ and _B. cinerea_ strains. The EO showed remarkable activity in both fungi, reaching 100% inhibition, even at the lowest concentrations. Flower extracts of _T. patula_ exhibited toxicity against soil-borne fungus _F. oxysporum_ f.sp. _Iyocpersici_, causing wilt disease in tomato plant [41].  
[PERSON] et al. [42] used the aerial parts from _T. minuta_ against isolates from four fungi species of agricultural and clinical importance: _F. verticillioides, F. proliferatum, Aspergillus flavus_, and _A. parasiticus_. The extraction solvents used were hexane, dichloromethane, methanol, and water. The concentration of the extracts was 10 mg/mL. The drug amphoteric B and the agricultural fungicide _Cantus_ were used as positive controls.  
Despite the promising in vitro results, in-depth studies are required to understand the mechanisms of action of EOs obtained from _Tagetes_ spp. for their potential use in biotechnology [43; 44]. Compounds in these EOs, especially terpenoids (dihydrotagetones, tagetones, ocimenones), are responsible for the identified antimicrobial activity [43]. In the future, the goal is to identify the active compounds in these EOs by fractionating them and determining the antimicrobial activity for each compound individually, considering both synergistic and antagonistic antimicrobial interactions [45].