Detection of hydroxyl radicals produced by wood-decomposing fungi

Abstract

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 K 2HPO 4 buffer.