Cellular display of the effects of linalool on Fusarium graminearum

In this master thesis, the effects of linalool, which is a terpene alcohol phytochemical of volatile essential oil (EO) components on Fusarium graminearum PH-1 reference strain at the cellular level (associated with oxidative stress, apoptosis processes, cell viability and mycotoxin production). The minimum inhibitory concentration (MIC) of linalool on the F. graminearum PH-1 reference strain was found as 52,2 mg/L. In the experiments, cultures without linalool addittion was grown on potato dextrose agar (PDA) medium called as a control group. The IC25, IC50 and IC75 doses of linalool corresponding to 25%, 50% and 75% of the concentration that maximally inhibits radial growth were calculated as 13,05 mg/L; 26,1 mg/L; and 37,15 mg/L. It was shown that these doses decreased the radial growth rate and the decrease was statistically significant (p<0,0001). Effect of linalool on cell viability was determined via WST-1 test. IC25, IC50 and IC75 doses reduced number of viable cells in the ratio of 33,09%; 76,44% and 86.03% (respectively) compared to the control group (p<0,0001). The effect of linalool on lipid peroxidation was studied via TBARS test. It was measured that IC25, IC50 and IC75 doses increased lipid peroxidation 64,68%; 145,16% and 392,47% (respectively) compared to the control group (p<0,0001). Intracellular oxidation of linalool at IC25, IC50 and IC75 doses was demonstrated by spectrophotometric method, which increased by 467%, 1477% and 3523%, respectively, compared to the control group (p<0,0001). It was shown that peroxidase (POD) enzyme activity increased by 83,6% for IC25; 213,7% for IC50 and 407,7% for IC75 depending on the increase in linalool dose. Catalase (KAT) enzyme activity value increases were calculated as 180% (IC25), 409% (IC50) and 1180% (IC75), respectively. Superoxide dismutase (SOD) activity increases were also determined as 847% (IC25), 1840% (IC50) and 2340% (IC75) (p<0,0001). It was thought that the severe increase in SOD activity was due to the fact that linalool increased the H2O2 concentration in the cell due to increasing oxidative stress. The fact that POD and CAT activity were lower than SOD and the increase in CAT activity at IC75 suggested that POD had reached the saturation point in terms of H2O2. In the light microscopy examination, differences at the cellular level were observed in the reference strain PH-1 treated with linalool. The increase in linalool concentration caused swelling in the cells and organelles of the experimental groups compared to the control, and thickened the mycelium structure. In addition, chlamidospore-like structure formations were observed. The stress effect suppressed spore formation. Oxidative stress and apoptotic effects induced by linalool were visualized at the cellular level in fluorescence microscopy using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) staining and acridine orange/ethidium bromide (At/EtBr) dual staining, respectively. The increase in oxidative stress due to the increase in linalool concentration was revealed by increasing green light intensity as a result of H2DCF-DA staining. In addition, the effect of linalool on the amount of deoxynivalenol (DON) mycotoxin production was demonstrated via thin layer chromatography (TLC). No spot was observed at an Rf value of 0,28; where DON mycotoxin was examined. In the context of the master thesis, it was demostrated that linalool; which have flower aroma, and naturally exist in aromatic plant species, is a compound of monoterpene EOs, inhibates fungal growth at very low concentrations on F. graminearum PH-1 reference strain. By simultaneously examined that the effects of linalool on cells due to oxidative stress, it was revealed thet the potential of using hydrophobic EOs as a potent antifungal agent in the fight against pathogenic fungal species.