Mode of action of methylene bisthiocyanate on sapstain fungi of Pinus radiata

Methylene bisthiocyanate (MBT) is the active ingredient of Sentry™, a common antisapstain product used for protection of export radiata pine logs. This research was carried out to advance the understanding of the mode of action of MBT against wood inhabiting fungi. The germination of propagules of Ophiostoma floccosum, O. piceae, O. piliferum, Leptographium procerum, Sphaeropsis sapinea, Trichoderma viride and Phlebiopsis gigantea was studied on both agar medium and on radiata pine wafers. Generally, the fungi showed slightly slower onset of spore germination on wood than on agar but all fungi formed germ-tubes within 20 h of incubation. There were significant differences between species of fungi, in the number of spores reaching different developmental stages of spore germination (P≤0.05) with L. porcerum the slowest compared to the other test fungi. The minimum inhibitory concentration of MBT for O. floccosum, L. procerum, S. sapinea and T. viride spores or mycelial fragments was between 0.00001 - 0.0005% w/v in liquid culture and 0.04 - 0.64% w/v on wood wafers, with T. viride having the highest tolerance to MBT (P≤0.05). To determine penetration and concentration of MBT at different wood depths, NMR and ICP-ASE analyses were performed. Subsequently bioassays using stereomicroscopy and CLSM were used to determine the effect of actual wood MBT concentrations on growth of O. floccosum. Quantitative data obtained from ICP-ASE showed that within 7 days of incubation, MBT penetrated from surface to pith, however, the concentration of MBT was significantly higher on surface wood compared to pith wood (P≤0.05). Furthermore, data obtained from ICP-ASE and CLSM analysis indicated that MBT concentration greater than 55 µg/g of air-dried wood was required to inhibit the growth of O. floccosum. The toxicity of MBT to both propagules and established mycelium of S. sapinea and O. floccosum was determined using a double wood wafer assay. The characteristic of MBT, diffusion vs. vapour activity, was also evaluated. The results demonstrated that fungicide tolerance was higher for established hyphae than propagules and that O. floccosum could tolerate considerably higher levels of fungicide than S. sapinea. The present study provided the first record of the release of MBT vapour from MBT treated wood using GC-MS. In addition to a diffusion effect, vapour action of MBT played an important role in fungal growth inhibition. However, the combined effect of MBT diffusion and vapour action was more pronounced than vapour action by itself. In vitro effects of MBT on hyphal morphology and ultrastructure of O. floccosum were examined using different microscopic techniques. Differential interference contrast microscopy showed that MBT caused rapid changes to the morphology of O. floccosum, resulting in excessive formation of vacuoles and granules within the cytoplasm of the hyphal tip and epifluorescence microscopy revealed damage to the plasma membrane in the MBT treated, but not control, hyphae. Under TEM, the exposure of O. floccosum to MBT appeared to cause different levels of damage depending on the concentration used; including retraction of the plasma membrane from the cell wall, cytoplasmic aggregation, vacuolation and complete disintegration of cytoplasmic materials. Microscopy indicated that the primary site of MBT action was at the plasma membrane. The extent of K⁺ leakage from the treated cells provided confirmation that the integrity of the plasma membrane was affected by MBT. No intracellular K⁺ leakage was measured from untreated and 0.01 mM treated hyphae. However, significant leakage occurred with 0.1 mM treated hyphae and K⁺ leakage increased with increasing concentrations up to 10 mM MBT. The leakage of K⁺ was rapid and significant, occurring within the first 5 min of exposure to the fungus for all effective MBT concentrations. There was a delayed effect of MBT on fungal respiration when treated O. floccosum was monitored for oxygen consumption and glucose depletion. However, the synthesis of ATP was severely affected, suggesting that MBT is acting as an energy uncoupler affecting either oxidative or substrate level phosphorylation