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.
Saprotrophic fungi producing cellulolytic and lignolytic enzymes contribute significantly to the decomposition of dead plant remains. The most important organisms in the decomposition of wood are the white- and brown-rot fungi, whose activities result in a tremendous loss of resources in, for example, the timber industry. White-rot fungi degrade all components of lignocellulose, while brown-rot fungi mineralize polysaccharides but are unable to depolymerize lignin .
The early stages of wood degradation by brown-rot fungi take place in the inner layer (S2 layer) of the wood cell walls, while the layer closer to the hyphae (S3 layer) remains unchanged [2,3]. Enzymes are too large to penetrate the S3 layer of wood cell walls and therefore a small diffusible oxidative agent is believed to be responsible for the initial degradation of the S2 layer. Several studies have proposed that hydroxyl radicals (•OH) are the small non-enzymatic agents involved in brown-rot decay [4–8], and to a lesser extent in the decay by white-rot fungi . Hydroxyl radicals, which are the most potent oxidizing agents in aqueous systems, can be produced via the Fenton reaction: Fe2++H2O2→Fe3++•OH+HO−. However, the evidence for Fenton chemistry involvement in wood decay is scarce. This is partly due to a lack of reliable methods for measuring either the reagent, H2O2, or the product, •OH . However, a semiquantitative colorimetric assay for H2O2 in liquid media was recently presented . For a reliable detection of •OH, Backa et al.  used a chemiluminescence method to test the growth media of different brown-rot fungi and found that •OH were generated at surprisingly high yields after an incubation period. The mechanisms generating the Fenton reagent in fungi are not yet clearly understood, although several pathways have been proposed [12–15].
Wood-decomposing fungi often produce the enzymes involved in lignin degradation during interaction with other organisms, especially other fungi [16–18]. Laccase production has been demonstrated in interactions between fungi and bacteria . It is unknown whether hydroxyl radicals are produced in a similar way in interactions with bacteria, although this could have important ecological implications.
The objective of this study was to develop an alternative method for hydroxyl radical detection in fungal growth systems and to investigate the potential use of the method in different environments, including soil. The aims were to use a substrate that is specific and is not used as a nutrient source by the fungi. In addition, the product should be stable. Hydroxylation of coumarin-3-carboxylic acid (3-CCA) produces one major fluorescent substance, 7-hydroxy-coumarin-carboxylic acid (7-OHCCA) (Fig. 1), which has previously been used for the specific detection of hydroxyl radicals generated chemically and by γ-radiation . We used this reaction for fungal growth systems since the fluorescence reaction is specific and sensitive. In addition, our objective was to investigate whether the production of hydroxyl radicals is influenced by interactions with bacteria with or without known anti-fungal activity.
2Materials and methods
2.1Organisms and materials
Ten fungal species, each represented by one strain, were obtained from the culture collection at the Division of Microbial Ecology, Department of Ecology, Lund University, Lund, Sweden: Antrodia vaillantii (DeCandolle: Fr.) (72086–2), Collybia maculata (Albertini and Schweinitz: Fr.) Quélet (FV9509–2), Coniophora puteana (Fr.) Karsten (C54), Crucibulum laeve (Hudson: Persoon) Kambly (FB9509), Hygrophoropsis aurantiaca (Wulfen: Fr.) Maire (FA9509–5), Hypholoma fasciculare (Hudson: Fr.) Kummer (FC9509–3), Phanerochaete chrysosporium Burdsall (P), Pholiota spumosa (Fr.: Fr.) Singer (FA9509–10), Serpula lacrimans (Wulfen: Fr.) Schroeter (C113) and Recinicium bicolor (Albertini et Schweinitz: Fr.) Parmeter (M1). Cultures were maintained on 1% (w/v) malt agar (MA: 10 g malt extract, 10 g low mineral bacteriological agar No. 1, both from Oxoid, Hampshire, UK), except for H. aurantiaca, which was grown on a medium containing 3.0 g glucose, 0.4 g NaNO3, 0.2 g K2HPO4, 0.1 g KCl, 0.0048 g Fe(EDTA), 0.0005 g thiamine, trace elements and 1 l MilliQ water.
The bacterial cultures of Pseudomonas fluorescens (PS7) with known anti-fungal activity and of Bacillus subtilis with no known anti-fungal activity were kindly provided by Tommy H. Nielsen at the Section of Genetics and Microbiology, KVL, Copenhagen, Denmark  and Claes von Wachenfeldt, Department of Microbiology, Lund University, Sweden, respectively.
Veneer discs of birch (1 mm thick with diameters of 50 or 100 mm) were autoclaved and used as the fungal substrate in all experiments. The wood discs were inoculated by placing them on top of mycelia growing on malt agar and incubating them for different periods of time. Wood functioned both as the inoculum medium and as the growth substrate for the fungi.
A stock solution (5 mM) of 3-CCA (99%, Sigma-Aldrich, Stockholm, Sweden) was prepared by dissolving 3-CCA in K2HPO4 buffer (40 mM, pH 9) at 50°C overnight. Purchased 7-OHCCA (Molecular Probes, Leiden, The Netherlands) was dissolved in K2HPO4 buffer (40 mM, pH 9). The relative fluorescence was measured with a Perkin-Elmer LS 50B luminescence spectrometer with a microtiter plate reader (Perkin-Elmer Beaconsfield, UK). The resulting values are presented in this study as arbitrary fluorescence units (AFU).
The fungal tolerance to 3-CCA was tested by allowing fungal cultures to grow on 1% malt agar Petri dishes (diameter 90 mm) with the addition of 3-CCA (0, 0.1 or 0.5 mM), and then measuring the radial growth. In addition, we tested the optimal time for the incubation of the fungal wood discs on 0.1 mM 3-CCA agar by measuring the fluorescence after 1, 2, 3, 4 and 7 days of incubation. The highest level of relative fluorescence was detected after 4 days, and this was then used as the incubation time in all subsequent experiments.
2.2The effect of pH on the 3-CCA hydroxylation reaction
Buffers with pH levels between 3 and 9 were made by using different ratios of (20 mM) K2HPO4 and KH2PO4 (replaced by 2 mM citric phosphate buffer for pH levels below 4). The buffering capacity was not considered critical and the buffers were only used to set the pH to a specific value. The 3-CCA (100 μM) was hydroxylated in a 96-microwell plate by γ-radiation (30 Gy) (12 replicates at each pH). Irradiation was performed using 6 MV photons from a linear accelerator (Elekta Sli, Crawley, UK). The fluorescence of the product of the hydroxylation reaction, 7-OHCCA, is significantly higher at high pH , thus, different mixtures of 0.7 M K2HPO4+0.13 M NaOH and MilliQ H2O (Millipore, Lund, Sweden) were added before fluorescence measurements in order to obtain a final pH of 10 in all samples. The experiment was performed twice.
2.3Hydroxylation reaction in agar
In a standard procedure, 0.1 mM 3-CCA was added to the medium after autoclaving low mineral bacteriological agar (1%) and water. The agar was poured into plates of relevant size, and wood discs colonized by fungi were placed on top of the solid agar and incubated for 4 days, unless indicated otherwise. The wood discs were then removed from the agar and K2HPO4 buffer (40 mM, pH 9) was poured onto the agar. The samples were then incubated for 2 h in order to increase the pH before the fluorescence was recorded.
Water agar (3 ml) at four different concentrations of 3-CCA (0.01, 0.1, 0.5, 1 mM) was poured into separate wells of a 12-well microtiter plate (Life Technologies, Stockholm, Sweden). Wood discs colonized with A. vaillantii were placed on top of the solid agar in all wells (three wells for each 3-CCA concentration) and then removed. Subsequently, 3 ml K2HPO4 buffer were poured into each well.
Hydroxyl radical production by 10 different fungal species was tested with wood discs that had been previously colonized by mycelia for 3, 30, 60 or 90 days. The incubated wood discs were placed on 0.1 mM 3-CCA agar in the 12-well plates, with one well left without a wood disc as control (two replicate plates for each inoculum of each age). The detection limit was determined as: control mean+3×S.D. .
To test the degradation of 7-OHCCA by fungi, wood discs that had been colonized by different fungi for 1 month were placed on agar containing 670 nM 7-OHCCA (three replicates). Wells without wood discs and sterile wood discs were used as controls.
2.4Hydroxyl radical production from interactions between fungi and bacteria
Inoculation streaks of the bacterial species P. fluorescens or B. subtilis were made on 0.1 mM 3-CCA/potato dextrose agar (PDA) (Difco, Le Pont de Claix, France) (0.39 g l−1) in 5-cm Petri dishes. After 4 days, pure cultures of the three fungi A. vaillantii, C. puteana and S. lacrimans that had been growing on wood discs for 17 days were inoculated onto the agar to form different combinations with the bacterial species. The samples were thereafter incubated in the dark until the hyphal front was in contact with the bacterial colony. The wood discs were then removed from the plates and the agar was soaked in 4 ml K2HPO4 buffer (40 mM, pH 9) for 30 min. After the buffer had been removed, the plates were turned upside down and an area of 55×55 mm was scanned for fluorescence (1-mm intervals between each pixel in both x- and y-directions) with a Perkin-Elmer LS 50B luminescence spectrometer with a microtiter plate reader.
The extraction efficiency of purchased 7-OHCCA was investigated in several ways. The 7-OHCCA, which is a weak acid, was added to sandy soil (80 nmol g−1 soil) and the extraction efficiencies of both the acid and its conjugate base were tested. We tested acid extraction of 7-OHCCA by using 28 combinations of different acidification procedures and extractions with different solvents. The soil was acidified in four different ways: 0.1 M HCl (pH 1.3), 80:20 ratio of 20 mM citric acid and 40 mM K2HPO4 (pH 3.3), 40 mM KH2PO4 (pH 4.5), and 50:50 ratio of 40 mM K2HPO4 and 40 mM KH2PO4 (pH 6.7). The extractions were tested with the following seven solvents: cyclohexane, n-hexane, chloroform, isobutanol, 2-propanol, methanol, and K2HPO4 (40 mM, pH 9). The conjugate base extraction was tested by increasing the pH in the soil by adding 0.1 M NaOH and extracting with either methanol or a salt at a high concentration (1 M): KCl, NaNO3, K2HPO4. Three other extraction techniques were also tested. The first one involved boiling the soil in 1 M HCl for 60 min and increasing the pH before fluorescence measurement with K2HPO4 buffer (40 mM, pH 9). The second technique used Tris–malate buffer (50 mM, pH 5) and increased pH with 2.5 M Tris buffer  and the third one extraction with K2HPO4 buffer (40 mM, pH 9).
For fungal experiments, soil samples were prepared by dissolving 3-CCA in water and mixing it with dried soil. The water was then evaporated to give a 10% water content and the final 3-CCA concentrations of 1 or 10 mol l−1 in the soil solution. Six colonized wood discs (A. vaillantii and H. fasciculare) were placed on top of 5 g 3-CCA-spiked soil (0.5 mM) in the Petri dishes. Controls with non-inoculated wood discs and soil without 3-CCA were also prepared. After 2–4 weeks, the 7-OHCCA was extracted by vortexing (30 s) 0.5 g soil in 4 ml of K2HPO4 buffer followed by centrifugation at 900×g for 5 min. One ml of the supernatant was centrifuged at 16 000×g for 10 min and 200 μl were transferred to a microwell plate where the fluorescence was measured. One well contained 200 nM purchased 7-OHCCA.
3.1Emission and excitation maxima and stability of 7-OHCCA
The purchased 7-OHCCA in a K2HPO4 buffer (40 mM, pH 9) exhibited an emission maximum at 444 nm and an excitation maximum at 388 nm. Thus, the emission was measured at 444 nm in all experiments. The fluorescence of purchased 7-OHCCA kept in light decreased with time, while temperature did not affect the fluorescence (Fig. 2). In samples that were kept in the dark at 8°C the fluorescence intensity was constant for up to 10 months (not shown).
3.2Effect of pH on the 3-CCA hydroxylation reaction
The highest fluorescence was found when the hydroxylation reaction occurred in buffers with a pH around 6–7. At pH 4, the fluorescence was approximately 50 AFU, while the maximum at pH 6–7 was 240 AFU. At pH 8, the fluorescence yield was approximately 170 AFU.
3.33-CCA tolerance by fungi
White-rot fungi were found to tolerate higher 3-CCA concentrations than brown-rot fungi. For example, after 7 days of incubation, the radial growth of the brown-rot fungus A. vaillantii was 26 mm on 0.1 mM 3-CCA agar, while it had grown only a few mm on 0.5 mM agar (Fig. 3). The 3-CCA tolerance of S. lacrimans and C. puteana was very similar to that of A. vaillantii. On the other hand, the growth of the white-rot fungus H. fasciculare was almost the same on 0, 0.1 and 0.5 mM 3-CCA over a period of 7 days (Fig. 3). The other white-rot fungi, C. laeve, R. bicolor, P. spumosa, and P. chrysosporium, showed similar tolerance levels to that of H. fasciculare.
3.4Fluorescence measurements in agar
The fluorescence that developed by incubation with A. vaillantii was higher when the 3-CCA concentration in the agar was increased from 0.01 to 0.5 mM. However, at 1 mM 3-CCA less fluorescence developed (Fig. 4). The repeated experiment showed comparable results as well as a low fluorescence (<10 AFU) that developed at an even higher 3-CCA concentration (10 mM).
3.4.1Hydroxyl radical production by different fungi
A control water agar plate generated a fluorescence of 16±1.9 AFU. If three standard deviations are accepted as a valid difference from the mean, a signal of 22 AFU is the lower limit of detection for the production of •OH. In eight out of 10 species the hydroxyl radical production exceeded the detection limit in at least one sample (Table 1). Especially A. vaillantii formed relatively high amounts of •OH, i.e. 10–25 times that of the detection limit. C. puteana and S. lacrimans reached •OH levels of 2 and 1.5 times the detection limit, respectively. The other fungal samples produced relatively low amounts of •OH (Table 1).
Table 1. Hydroxyl radical productiona of fungal species incubated on wood discs for different periods of time
aPrior to fluorescence measurement, incubated wood discs were placed on water agar with 0.1 mM 3-CCA, removed after 4 h and the pH was increased in the agar with addition of 40 mM K2HPO4 (pH 9). Fungal species are ordered in groups of brown-rot fungi (b-r-f) and white-rot fungi (w-r-f). Means (n=2) and S.D. are given. Detection limit was determined to 22 relative AFU, background mean+3×S.D. (16+3×1.9).
Relative fluorescence units (AFU) after an incubation period of
200 nM 7-OHCCA
The production of •OH was three times higher when the wood discs had been colonized with A. vaillantii for 65 or 95 days compared to 7 days of colonization. This effect of inoculum age could not be seen clearly for any of the other isolates (Table 1).
We also investigated whether fungi themselves could affect the results by degrading 7-OHCCA. Therefore, colonized wood discs were incubated on water agar containing 7-OHCCA for 3 weeks. Fig. 5 illustrates that 30% of the 7-OHCCA had disappeared when sterile wood discs were placed on the agar. In the presence of colonizing fungi, up to 70% of the 7-OHCCA had disappeared.
3.4.2Hydroxyl radical production from interactions between fungi and bacteria
The background values (25×26 pixel values in the middle of the plate) from a non-inoculated PDA-CCA plate had a mean of 29 AFU and a standard deviation of 2.9. Hydroxyl radical production in monocultures of A. vaillantii reached levels of 75 AFU (Fig. 6). The hydroxyl radical production during interactions between A. vaillantii and the anti-fungal P. fluorescens increased to levels up to 100–120 AFU, while the interaction with B. subtilis did not affect the level of radicals produced (maximum level 60 AFU). The production of •OH by C. puteana alone, 30–40 AFU, was not much higher than the background values. In interactions between C. puteana and the anti-fungal P. fluorescens the maximum fluorescence levels were 70 and 100 AFU, and with B. subtilis fluorescence reached levels up to 110–150 AFU. S. lacrimans did not reach fluorescence levels above the background in any sample (data not shown).
3.57-OHCCA extraction from soil
The best extraction efficiencies of 7-OHCCA from soil were obtained when using 40 mM (pH 9) K2HPO4 buffer (40% recovery) and 50 mM Tris buffer (30% recovery).
Several separate experiments were performed with fungi-inoculated soil spiked with 3-CCA in order to detect the formation of the hydroxylation product. We observed 7-OHCCA formation in some experiments, yet the biological variability seemed to be too high to obtain conclusive results.
A new method for the detection of hydroxyl radical production by wood-decomposing fungi on solid materials is presented in this paper. In addition, the method gave interesting results in biological experiments.
Manevich et al.  showed that the intensity of the fluorescence from 7-OHCCA when excited by UV radiation is pH-dependent. In this study, it has been shown that the reaction between 3-CCA and •OH is also pH-dependent. The buffers used to obtain the lowest pH in this study contained citric acid, which may act as a hydroxyl radical quencher. Nevertheless, the 7-OHCCA formation at pH 5.2 was slower and, as a consequence, the developed fluorescence was less than that at pH 5.6 and 6.8, and therefore pH between 6 and 7 is still assumed to be optimal for the reaction. According to Manevich et al. , it does not matter if the pH is increased before or after the reaction in order to generate the maximal fluorescence. Together with our observations, it is obvious that the fluorescence of the product 7-OHCCA is considerably more sensitive to pH than the 3-CCA hydroxylation.
To our knowledge the only study that has previously given reliable evidence of hydroxyl radical production in fungi is that by Backa et al. . They measured the chemiluminescence (CL) from 3-hydroxyphthalic hydrazide formed after attack by •OH on the aromatic ring of phthalic hydrazide. In their critical review they concluded that the CL method was pH-dependent, specific to •OH and sensitive enough to measure amounts in the pico- to nanomol range , much the same as the present fluorescence method. They also stated that the product, 3-hydroxyphthalic hydrazide, was stable at pH 12 for at least 1 day. The present study shows that the product 7-OHCCA remains stable for months in 40 mM K2HPO4 buffer (pH 9) and in the dark independent of the temperature but, in contrast, is degraded by light.
The advantage of the present fluorescence method, compared with the CL method of Backa et al.  where a sample of the reaction mixture is withdrawn and mixed with a solution before CL measurement, is that measurements can be made directly at the site where •OH production takes place. The direct measurement is useful when spatial distribution of the production of •OH is of interest, for example, when studying interactions.
In this study, the production of •OH by wood-decomposing fungi was tested in environments where wood functioned as both the inoculum medium and the growth substrate. We used solid material since it is a natural growth medium for fungi. In addition, brown-rot fungi typically lower the local pH and the gradient formed outside the hyphae would, according to Hyde and Wood , enable both reagents in the Fenton reaction to be formed at a distance from the hyphae where damage to the fungus would be avoided. The gradient cannot be obtained in liquid solution but only in solid material, such as wood. In preliminary studies it was seen that A. vaillantii produced less hydroxyl radicals when growing on malt agar as a substrate compared to growth on wood. This could be due to enhanced radical production by the fungi on a natural substrate such as wood.
Ten fungal species were tested for hydroxyl radical production after fungal wood discs had been incubated on 3-CCA agar. Many of the fungal samples exhibited greater variation than the control samples, which indicates that there is biological activity in the fungal samples. There was generally a higher fluorescence yield from the brown-rot fungi, e.g. A. vaillantii, C. puteana, and S. lacrimans, than from the white-rot fungi like H. fasciculare, P. chrysosporium, and C. laeve. In the present study, the hydroxyl radical formation by A. vaillantii was much greater than that of the other fungi.
Another interesting observation was that incubation time seemed to affect the amount of hydroxyl radical formed by A. vaillantii. The longer the fungus had colonized a wood disc before being placed for 4 days on the 3-CCA agar, the more •OH were detected in the agar under the disc. This could be due to the larger fungal biomass in the wood disc that had been colonized for 65 days or due to depletion of the substrate.
The reason why white-rot fungi tolerated higher concentrations of 3-CCA is not known. However, it could be that the lignolytic activities of white-rot fungi degrade some 3-CCA. The amounts of hydroxyl radicals were probably not considerably affected by the fungal degradation of 7-OHCCA, since the samples were measured after 4 days of incubation. In the test for fungal degradation of 7-OHCCA, the samples were investigated after 3 weeks of incubation. However, care should be taken when interpreting the fluorescence results in terms of •OH quantities.
This paper also demonstrates that the production of •OH can change when fungal hyphae come into contact with bacteria. In samples with C. puteana, which produced low levels of •OH on its own, the production of •OH increased two- or threefold when hyphae were in contact with either of the two bacterial species tested. In samples with A. vaillantii, the generation of •OH was more specific and almost doubled when contact was made with the anti-fungal P. fluorescens, while it was unchanged together with B. subtilis. This increase in •OH-production could be a defense mechanism, like the involvement of reactive oxygen species in plant defense responses . Another suggestion is that •OH are generated by the fungi in order to attack the bacterial colonies. Previous studies have shown that wood-decomposing fungi attack and use bacteria as a substrate [26,27].
The method described in the present study has worked well on agar and can be used to provide answers to questions about hydroxyl radical production by fungi in such systems. It also has a potential for soil studies, since the highest extraction recovery from soil was 40%.
We are grateful to Eva Englund and Per Nilsson (Department of Radiation Physics, Lund University) for performing the γ-irradiations. This research was supported by the Swedish Environmental Protection Agency.