Detection of the effects of fungicides on the cell membrane by proton nuclear magnetic resonance spectroscopy

Abstract

The observed spin-spin relaxation time ( T 2) of intracellular water protons reflects the membrane water permeability. Effects of various types of fungicides on the membrane were investigated by using the T 2 of the water protons in the mycelial cells of Botrytis cinerea. The treatments of the cells with nystatin and dehydroabietylamine, ion channel attackers, remarkably increase the membrane water permeability within 1 hr, which suggests that the functions of the ion channels relate closely to the water exchange through the membrane. Edifenphos and iprobenfos, phosphatidylcholine biosynthesis inhibitors, slightly increase the membrane water permeability. Triadimefon and triflumizole, ergosterol biosynthesis inhibitors, also slightly increase the membrane water permeability only after a 15-hr treatment. These results indicate that the phospholipid biosynthesis inhibitors and the ergosterol biosynthesis inhibitors do not attack the water channels in the membrane directly but have some secondary effects on them through the change in the membrane structure. Among the SH and NH 2 inhibitors, captan, captafol, chlorothalonil, and HgCl 2 increase the membrane water permeability probably by binding to the proteins at the ion channels. On the other hand, maneb, mancozeb, thiram, and ziram have no effect on the permeability, suggesting that the SH and NH 2 inhibition is not the primary action of them. Benomyl, thiophanate methyl, procymidone, iprodione, blasticidin S, and polyoxin B were also tested, but these fungicides do not change the membrane water permeability, which confirms that the action sites of these fungicides are not on the membrane. In addition, the relation between the change in the T 2 and growth inhibition is different among ion channel attackers, membrane biosynthesis inhibitors and SH inhibitors. Therefore, this NMR technique is useful in fungicide pharmacology.