Cellular display of the effects of prothioconazole on Fusarium reference strains

In the context of this master thesis, the effects of the azole fungicide active ingredient prothioconazole on F. graminearum PH1 and F. culmorum UK99 reference strains causing diseases in various plant species were investigated at the cellular level (oxidative stress, apoptosis, cell viability and mycotoxin production). The minimum inhibitory concentrations (MIC) of prothioconazole were determined as 1,2 μg/ml and 1 μg/ml for PH1 and UK99, respectively. In the experiments, reference cultures grown on PDA medium were represented as control group, while cultures grown on PDA containing acetone, the solvent of prothioconazole, were used as normalization group. The IC25 and IC50 doses of prothioconazole were calculated as 0,3 μg/ml and 0,6 μg/ml for PH1, 0,25 μg/ml and 0,5 μg/ml for UK99, respectively. These doses were determined to reduce radial growth rates in both references (p<0,001). The effect of this active ingredient on spore production was observed in UK99 reference grown on czapek dox broth (CDB) medium. The IC50 dose caused an increase in macroconidia size (%66) and diameter (%123) compared to the control (p<0,001). Oxidative stress and apoptotic effects triggered by protioconazole were visualized at cellular level under inverted fluorescence microscope using 2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA) staining and acridine orange/ethidium bromide (ao/etbr) dual staining.. It was determined that catalase (for IC25: %105; for IC50: %278) and superoxide dismutase (for IC25: %48; for IC50: %103) activities increased in PH1 due to the increase in the concentration of prothioconazole (p<0,001). Similarly, it was determined that these two prothioconazole doses caused an increase in superoxide dismutase (for IC25: %38; for IC50: %84) activity in UK99 (p <0,001). The IC25 dose caused a significant increase in catalase activity (%52) in this reference strain (p<0,001). The effect of prothioconazole on cell viability were detected by using WST-1. For PH1 reference, IC25 and IC50 protioconazole doses reduced cell viability at the rate of 40.2% and 53.82%, respectively. IC25 and IC50 doses of proticonazole also reduced the viable cell count of UK99 by 25.33% and 41.93% (p <0.001). In addition, protioconazole has been shown in thin layer chromatography (TLC) to reduce the amount of deoxynivalenol (DON) production in UK99 reference strain. Within the scope of the master's thesis, demonstration of the influences of prothioconazole on F. graminearum PH1 and F. culmorum UK99 references in cellular level revealed that different mechanisms activated by fungicidal chemical compounds can be monitored in a cell, in real time. In addition, the findings that protioconazole inhibits the DON production are important in terms of demonstrating the importance of the presence of an agent that suppresses mycotoxin production in the composition of fungicidal active ingredients to be used in the fight against Fusarium phytopathogens in protecting public health.