Cellular and molecular display of the effect of tebuconazole on Fusarium reference strains

In the master thesis, the effect of tebuconazole active substance, which is frequently used in agricultural fields, on F. graminearum PH-1 and F. culmorum FcUK99 reference strains were investigated at molecular and cellular levels. In this context, the effective concentration (EC50) value, which reduces the growth by 50%, was determined by applying tebuconazole to reference strains with agar dilution technique. The EC50 value was calculated 0.94µg/mL for the PH-1 strain while the EC50 value for FcUK99 was detected 0.8µg/mL. After the presence of the cyp51A gene was determined and sequenced in both isolates, the relationship between the sequence variations and the mechanism of action of tebuconazole was examined. After the presence and sequence of the cyp51A gene were sequenced in both isolates and the relationship between sequence variation and mechanism of action was investigated. According to EC50 value, the effect of active substance on cell viability on Fusarium species was investigated by WST-1 analysis. It was observed that cell viability was suppressed at a rate of 57.43% in PH-1 and 64.24% in FcUK99. In fluorescence microscopy analysis, the effect of tebuconazole on the apoptotic process was revealed by performing with acridine orange/ethidium bromide (AO/EB) dual staining and on oxidative stress with 2', 7'-dichlorofluorescin diacetate (DCF-DA) single staining. Moreover, it was determined that catalase activity increased 3.076 and 4.062 times in F. culmorum and F. graminearum, respectively. Changes in expression of cyp51A, which is the target gene of the active substance, and mgv1, which is an essential for Fusarium species, were detected by quantitative polymerase chain reaction (qPCR). The average E value was calculated as 2.04 ± 0.03. It was calculated that cyp51A gene expression increased 7.426 ± 0.48 fold in FcUK99 and 15.47 ± 0.75 fold in PH-1 strains. At the same time, increase in the expression of the mgv1 was also detected. Values were found as 3.056 ± 0.16 fold for FcUK99 and 2.113 ± 0.04 fold for PH-1. The findings revealed that tebucanozole was effective on the viability of Fusarium species both by activating the apoptotic process and by causing oxidative stress. It also enabled us to understand the cellular and molecular mechanisms of this active compound beside its known inhibition effect. Therefore, the data obtained from the master's thesis has a high potential to be used in the fight against Fusarium pathogens in plant pathology field.