Microscopic, Biochemical and Physiological Assessment of the Effect of Methylene Bisthiocyanate on the Sapstain Fungus Ophiostoma floccosum

In vitro effects of methylene bisthiocyanate (MBT) on hyphal morphology and ultra-structure of Ophiostoma floccosum were examined using differential interference contrast, epifluorescence and transmission electron microscopy (TEM). To understand the mode of action of MBT, experiments were undertaken to measure potassium ion (K⁺) leakage from cells, oxygen consumption, glucose and ATP levels. Differential interference contrast microscopy indicated that MBT caused rapid changes in O. floccosum hyphae resulting in extensive vaculoation and accumulation of granular materials within the cytoplasm. Epifluorescence microscopy provided evidence that MBT treatment causes a loss in the permeability properties of the plasma membrane. TEM showed retraction of the plasma membrane from the cell wall, aggregation of cytoplasmic contents, vesiculation of membranous components, a dramatic increase in vacuolation, and eventually a complete loss in the integrity of organelles. There was a rapid efflux of intracellular K⁺ ions from cells, a substantial loss in K⁺ ions occurring within the first 5 min of MBT treatment. The rate of K⁺ leakage was MBT concentration treatment-time dependent. The study also showed that the effect of lower concentrations of MBT (0.01 and 0.1 mM) on respiratory activity was negligible. However, at the same concentrations, glucose consumption and ATP production were affected. Taken together, these observations suggest that the target site of MBT in O. floccosum alters membrane properties and uncouples oxidative phosphorylation from the respiratory chain.