Detection of hydroxyl radicals produced by wood-decomposing fungi

The hydroxyl radical ((*)OH) is believed to act as the small non-enzymatic agent involved in the brown-rot decay of wood. However, knowledge about the relation between hydroxyl radical production and the activity of wood-decomposing fungi in wood or about the significance of these radicals during interactions with other organisms is limited due to a lack of reliable methods for detecting the radicals. A sensitive and specific fluorescence method was developed in this study to detect the production of (*)OH by wood-decomposing fungal species. The method was based on the hydroxylation of coumarin-3-carboxylic acid, which produces 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA), a fluorescent, stable and specific product. Wood discs colonized by fungi were placed on water agar containing coumarin-3-carboxylic acid, where the formation of 7-OHCCA occurred and the fluorescence could be measured. The production of (*)OH was above the detection limit for eight of the 10 fungal species. The highest level, 8-25 times that of the detection limit, was produced by the brown-rot fungus Antrodia vaillantii. In interaction experiments where A. vaillantii had established contact with the antagonistic bacterium Pseudomonas fluorescens, the production of (*)OH increased, whereas contact with Bacillus subtilis did not change the amount of (*)OH generated compared with controls containing only the fungus. In contrast, the production of (*)OH increased above the control level when the fungus Coniophora puteana was in contact with any of these bacteria. The method was also tested in soil, with the result that 40% of 7-OHCCA added to the soil could be recovered with K2HPO4 buffer.