OXYGEN FREE RADICAL DETECTION IN WOOD COLONIZED BY THE BROWN-ROT FUNGUS, POSTIA PLACENTA

Transcription

1 BIODETERIORATION RESEARCH 2: Plenum Press, New York, 1989 OXYGEN FREE RADICAL DETECTION IN WOOD COLONIZED BY THE BROWN-ROT FUNGUS, POSTIA PLACENTA BARBARA L. ILLMAN*, DORE C. MEINHOLTZ, and TERRY L. HIGHLEY, USDA, Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI , USA INTRODUCTION Rapid depolymerization of cellulose occurs shortly after brown-rot fungi colonize wood. The chemical agent responsible for this initial depolymerization is most likely a low molecular weight compound (Cowling, 1961; Cowling and Brown, 1969) that diffuses through the crystalline microfibrils of cellulose, degrading the amorphous non-crystalline regions (Cowling and Brown, 1969; Highley, Palmer, Murmanis, 1983). It is important to identify the depolymerizing agent(s) produced by brown-rot fungi, because such information could serve as a foundation for the development of new methods to prevent wood decay. A possible role for oxygen free radicals has been proposed for brown-rot depolymerization of cellulose (Cowling and Brown, 1969; Koenigs, 1974; Highley, 1982; Schmidt et al., 1981). Schmidt et al. (1981) outlined a hypothetical scheme for hydroxyl radical (OH ) oxidative fragmentation of cellulose by brown-rot fungi. In this scheme, the oxalic acid secreted by the fungi (Takao, 1965) reduces the Fe(III) normally found in wood to Fe(II). Hydrogen peroxide (H 2O 2), that is reported to be secreted by some brown-rot fungi (Koenigs, 1972; Highley, 1987), would react with Fe(II) to produce OH. 497

2 This hypothetical scheme is consistent with data from at least three studies on radical oxidation. Free radicals are known to degrade some of the chemical components of wood. Chemically generated oxygen free radicals have been reported to fragment cotton cellulose and perhaps initiate a chain reaction that propagates through the amorphous regions of the fibers (Thompson and Corbett, 1985). Free radicals can be formed when wood surfaces are exposed to strong light (Hon et al., 1982). The radicals cause deterioration (photooxidation) of the ultrastructure of wood cell walls (Hon et al., 1980; Hon et al., 1985). Free radical mechanisms are also involved in the enzymatic degradation of the lignin component of wood by the white-rot fungus, Phanerochaete chrysosporium (Hammel et al., 1985; Kersten et al., 1984). Direct evidence of oxygen free radical involvement in brown-rot degradation of wood has not been obtained. A better understanding of the mechanisms of decay can be gained by determining if the fungi produce oxygen radicals under physiological conditions. Culture filtrates of the fungi have been shown to bleach p-nitrosodimethylaniline, suggesting that OH radicals were present in the filtrates (Highley, 1982). Bleaching of p-nitrosodimethylaniline, however, is not a specific test for the presence of OH as organic radicals and easily auto-oxidizable compounds can produce the same reactions (Bors et al., 1979). Electron spin resonance spectroscopy (ESR) is a sensitive and specific technique that can detect the presence of very low concentrations of free radicals (Halliwell and Gutteridge, 1986). The highly reactive hydroxyl radical is detected by allowing it to react with a more stable compound, a spin trap, to produce a long-lived radical. The resulting spin trap-radical adduct gives an ESR spectrum that is highly specific and allows immediate identification of the free radical component of the adduct. The major objective of the present report was to test the hypothetical scheme for a free radical mechanism involved in brown-rot decompostion of wood. Postia placenta (Fr.) M. Lars. et Lomb. was selected as the model fungus for the test. Production of hydrogen peroxide by P. placenta has not been well established due to difficulties inherent in culture procedures and in H 2O 2-indicator chromogens (Highley, 1987; Highley, 1982). The experimental approach in this report was (1) to test for the production of H 2O 2 by P. placenta in liquid culture media containing variable sources and concentrations of carbohydrate and nitrogen, (2) to use ESR to determine if P. placenta produces the hydroxyl radical (a) in liquid culture medium and (b) during the colonization of wood. 498

3 MATERIALS AND METHODS Materials Postia placenta (MAD-698) was obtained from the culture collection maintained at the Forest Products Laboratory, Madison, Wisconsin. The chromogen, 2.2'-azino-di-(3-ethylbenzthiazole-6-sulphonic acid) (ABTS) and horseradish peroxidase were obtained from Sigma Chemical Company (St. Louis, MO, USA). Mineral salts were obtained from Sigma or Baker Scientific Company (Phillipsburg, N.J.). The nitrone spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), was used to trap the hydroxyl radical in media and wood inoculated with P. placenta. DMPO and diethylenetriaminepentaacetic acid (DETAPAC) were obtained from Sigma. All other chemicals for the ESR study were of the purest commercial grade available. Hydrogen Peroxide Cultural Conditions The basic liquid medium contained 11 mm H 2PO 4, 2 mm MgSO 4.7Hp, 0.6 mm Cal 2.2H 2O, 9 mm H 3BO mm MnCl 2.4H 20, 1.1mM ZnSO 4.7H 2O, 0.2 mm CuSO 4.5H 2O mm ammonium tartrate, and 2.9 X 10 3 mm thiamine hydrochloride at a ph of 5.5. The basic medium was amended with or without 25 mm NH 4NO 3 and with the following carbohydrates (0.01, 0.1, or 1%): cellobiose, glucose, galactose, arabinose, and mannose. After autoclaving at 121 C for 15 min, three replicates of each medium was cooled to 50 C and an aqueous stock solution of horseradish peroxidase and ABTS were added aseptically to the medium according to published procedures (Muller, 1984; Highley, 1987). Media were dispensed in 5 ml aliquots into 25 ml Erlenmeyer flasks, inoculated with an aqueous suspension of P. placenta hyphae, and maintained in the dark at 27 C and 70% relative humidity. The experiment was repeated two times. Development of a greenish-blue color in the initially colorless medium was recorded as a positive test for H 2O 2 production. OH Cultural Conditions - Liquid Cultures P. placenta was allowed to grow for four weeks in stationary liquid culture containing the basal medium listed above for the hydrogen peroxide culture medium. The medium was amended with either 0.1% cellobiose or 0.1% glucose. Three replicates were prepared for each carbohydrate source. Cultures were inoculated with an aqueous hyphal suspension, incubated in the dark at 27 C and 70% relative humidity. Three aliquotes from each replicate were tested for hydroxyl radical production 0, 4, 10 and 17 days after inoculation. Medium without fungus was the biological control. 499

4 OH Cultural Conditions - Wood Slivers Wood samples of Douglas-fir, Pseudotsugamenziesii, and white fir. Abiesconcolor, were taken from the longitudinal axis of board interiors in order to avoid any surface changes that may have been caused by prior exposure to ultraviolate light. Wood slivers, approximately 3.0 cm x 0.1 cm x 0.1 cm, were inoculated with P. placenta by incubating them for four to five weeks on 'feeder strip' wood blocks decayed by the fungus. The feeder strips were prepared by incubating them on P. placenta malt agar plates for at least three weeks. Control slivers were prepared in an identical manner by incubation on feeder-type strips without fungus. Electron Spin Resonance ESR spectra were recorded using a Varian E-3 ESR system operating at a frequency of 9.00 to 9.01 GHz (X-band) and power of 5.00 mw. The instrument settings used were time constant at 0.1 second, field set at 3250 G, scan range at 50 or 100 G and receiver gain at 10 x 10 4 or 10 x Hyperfine splittings were measured directly from magnetic field separations. Spin Trapping Procedure Commercial DMPO powder was dissolved in deionized water and tested for impurities with the ESR. At least three paramagnetic signals were detected; two of these signals were probably from decomposition of the products of the reaction of hydroxamic acid with light. A purification step to remove DMPO contaminants was followed (Buettner, 1985) in which the aqueous DMPO was mixed with activated charcoal, then filtered. The concentration of DMPO was determined by dilution of small aliquots of DMPO into 95% ethanol, reading the absorbance at 234 nm and using an extinction coefficient of 7700 M 1 cm 1 (Thornalley and Bannister, 1985). Aliquots of the aqueous solution were separately frozen in light-tight containers and removed from the freezer directly before use. ESR tubes and capillaries were acid washed and oven dried to remove trace metals. The metal chelating agent, DETFPAC, was used to help reduce the presence of trace metals in the experimental system. Care was taken throughout the experiments to thoroughly mix aqueous samples with DETAPAC before addition of the DMPO and to avoid exposing all chemicals and fungal filtrates to strong light. Liquid samples were prepared by mixing fungal hyphae and filtrate to give final concentrations of 1 mm DETAPAC and 180, 200, or 300 mm DMPO in 100 microliters. Samples were drawn into an open-ended quartz ESR capillary tube approximately 15.0 cm x 0.2 cm o.d. x 0.1 cm i.d. 500

5 The bottom of the capillary tube was sealed with plastic film and placed in the spectrometer for tuning. Measurements were completed within 20 minutes after addition of DMPO to the sample. Spin trapping procedures for wood slivers were as follows: silvers were individually removed from inoculation dishes, soaked for 2.5 minutes in an aqueous solution of 1 mm DETAPAC and 200 or 300 mm DMPO, then transferred to a quartz ESR tube approximately 13.5 cm x 3.0 cm o.d. x 2.0 cm i.d. The bottom of the ESR tube was sealed and the tube inserted into the sample cavity of the spectrometer. Measurements were taken immediately after the instrument was tuned and completed within 20 minutes after the DMPO came into contact with the wood. RESULTS Hydrogen Peroxide Experiments Hydrogen peroxide was consistently detected by ABTS in all nitrogen-limited cultures of P. placenta. The cultures exhibited the green-colored positive test for H 2O 2. The color was a result of the reaction of the chromogen, ABTS, with H 2O 2 in the presence of horseradish peroxidase (Muller, 1984). When nitrogen was added to the culture medium, the color development was affected by carbohydrate, varying with carbohydrate source and concentration (Table 1). No color change developed in the media containing a high concentration (1%) of the carbohydrates. The green color developed in 0.1 and 0.01 % galactose and arabinose. A distinctively dark color developed in arabinose media. A positive H 2O 2 test was only detected with the lowest concentration of cellobiose. glucose and mannose (Table 1). ESR Experiments Reaction of OH with DMPO produces a fairly stable nitroxide radical that has a reported half-life of about 2.5 hours (Halliwell and Gutteridge, 1986). The DMPO-OH adduct radical was detected by ESR in liquid cultures of P. placenta containing cellobiose or glucose as the carbohydrate source. A representative spectrum is shown in Figure 1. A free radical is identified by the hyperfine structure, g-value, and line shape of its ESR spectrum. The hyperfine structure refers to the number and relative intensity of lines in the spectrum. The g-value or line splitting factor is usually around (the g-value for a free electron) for almost all biologically important free radicals (Halliwell & Gutteridge, 1986). TheDMPO-OHspectraseen in the present study was consistent with the reported ESR signal for this adduct. The DMPO-OH 501

6 Table 1. Effect of Carbohydrate and Nitrogen on Hydrogen Peroxide a Production by Postia placenta in Liquid Medium. b Nitrogen (%) Carbohydrate Present Absent Cellobiose Glucose Galactose Arabinose Mannose C d a Hydrogen peroxide detected with ABTS. b 25 mm NH4NO 3. C No ABTS coloration by H2O 2. d ABTS coloration by H2O

7 signal (Figures 1 and 2) was a four line signal with a 1:2:2:1 intensity ratio, 15 G line splitting, and g-factor of about The weakness of the ESR signal in most liquid samples of P. placenta required a modulation amplitude of 5 G, which prevents the absolute measurement and comparison of line widths and line shapes for these narrow lines. Accurate splittings could be measured, however. and relative comparisons of equally distorted lines is legitimate. The appearance of the DMPO-OH signal in both glucose and cellobiose was time dependent. It was not found in either medium on the 4th day after inoculation with the fungus. The adduct appeared on the 10th and subsequent days in glucose and cellobiose media. The four line DMPO-OH ESR spectrum was also detected in wood slivers of Douglas-fir and white fir. A spectrum, representative of the signal found in white fir slivers, is given in Figure 2. The ESR signal appeared on a shoulder of one of the hyperfine lines of the paramagnetic manganese signal reported elsewhere for P. placenta infected wood (Illman et al., 1988b). The DMPO-OH signal was strong enough in all cases to be readily apparent at a modulation amplitude of 1 G. The signal was considerably stronger in white fir than in Douglas-fir which may indicate a species difference. No DMPO-OH ESR signal was detected in culture medium or wood slivers without the fungus (see Figure 1 for background signals in control medium). DISCUSSION This study supports the hypothesis that oxygen free radicals are involved in wood decomposition by brown-rot fungi. Hydrogen peroxide and the hydroxyl radical were detected in liquid culture medium inoculated with Postia placenta. The hydroxyl radical was detected in woodsamples. Nitrogen and carbohydrate metabolism influence the production of hydrogen peroxide by P. placenta. The in vitro production of H 2O 2 was repressed by nitrogen in the culture mediun. Nitrogen-limited medium gave a positive-abts reaction with all carbohydrates (cellobiose, glucose, galactose, arabinose, mannose) used in the study. Hydrogen peroxide detection in nitrogen-amended medium was dependent upon carbohydrate concentration. Hydrogen peroxide production was repressesd by the highest and enhanced by the lowest concentration of carbohydrate. The hydroxyl radical was detected by ESR in wood samples and nitrogen-limited liquid cultures inoculated with the brown-rot fungus, P. placenta. A relatively more stable DMPO-OH signal was detected in 503

8 Figure 1. Electron Spin Resonance (ESR) Spectra of Paramagnetic Manganese in Douglas-Fir; A, Without P. placenta ; B, With P. placenta. Figure 2. Electron Spin Resonance (ESR) Spectra of Paramagnetic Manganese in Wood, With and Without the Brown-RotFungus, Postia placenta. Microwave frequency at GHz, microwavepower10mw, modulation amplitude of 10 G. 504

9 wood slivers than the adduct signal in liquid culture. The shorter-lived signal in cultures may be due to the accessability of more oxidizing chemicals in the liquid. A stronger DMPO-OH signal was detected in white fir than in Douglas-fir. This may reflect a species difference in (1) the action of the fungus on the wood, (2) the ability of wood components to compete with DMPO for the free radicals, or (3) the rapid decomposition of the adduct by the wood components. The exact chemical source of the hydroxyl radical was not determined in our study, but our working hypothesis at this time is that the chemical reaction(s) producing OH is the same in cultures and in wood. A posssible source of OH is the reaction of hydrogen peroxide with a transition metal, such as iron, Fe(II). In discussing the potential role of H 2O 2 and ferrous salts ('Fenton's reagent') in brown-rot degradation, Cowling and Brown (1969) referred to Halliwell's (1965) use of H 2O 2 and ferrous salts to depolymerize cotton cellulose. In support of H 2O 2 as the source of OH, Koenigs (1974) reported that brown-rot fungi produce H 2O 2 and that the change in depolymerization of pine and sweetgum woods by Fe(II) + H 2O 2 is similar to that produced by acid hydrolysis and brown-rot decay. Schmidt et al (1981) found that oxalic acid, which is secreted by brown-rot fungi (Takao, 1965), reduces Fe(III) to Fe(I1). Production of OH detected by ESR in our study may follow a H 2O 2 chemical pathway. Partially characterized brown-rotted cellulose has been found to be oxidized and depolymerized in a manner similar to, but not identical to, H 2O 2 and Fe(II) (Ivanov et al., 1952; Highley, 1977). In parallel experiments (Illman et al., 1988a; Illman et al., 1988b). we detected increases of paramagnetic manganese (MnII) in P. placenta colonized wood slivers of Douglas-fir and white fir. Results were more dramatic in white fir, with a 300 fold increase in ESR detectable Mn(II) in infected vs non-infected slivers in 39 days. Detection of changes in Mn occurred within 13 days after the wood came into contact with the fungus. No ESR signals for low or high spin iron were detected at room temperature. ESR signals for Mn(II) increased in fungal colonized wood at approximately the same time that the hydroxyl radical was detected. It is possible that the two occurrences are related. Mn(III) typically found in wood (Young and Carpenter, 1967) could be catalyzing the hydrogen peroxide generation of OH. Alternatively, Mn(II) ESR-detectable signals could be masked by chemical 505

10 binding that is changed during fungal colonization. The chemical changes could result in OH production. Other potential sources of OH are enzymatic processes in which one-electron transfer from one compound to another produces radical intermediates. Eriksson (1981) found that cellobiose oxidase from Sporotrichum pulverulentum and several wood-rotting fungi produce the superoxide radical. The superoxide radical has been shown to reduce Fe(III) to Fe(II), resulting in the superoxide-driven Fenton reaction (Cohen, 1985). DMPO reacts with the superoxide radical to form an unstable adduct, DMPO-OOH, that can decompose to form DMPO-OH. Part or all of the DMPO-OH signal detected in cultures and wood inoculated with P. placenta could be from direct reaction of the spin trap with OH or from a decomposition product of the superoxide radical. Studies are being made to use free radical scavengers such as superoxide dismutase, flavonoids and ethanol to help interpret the chemistry responsible for the ESR data presented here. In conclusion, oxygen free radical intermediates do appear to be involved in brown-rot interaction with wood and with fungal growth in culture media. Hydrogen peroxide is a possible free radical source. Increases in ESR signals of Mn(II) in a parallel study lends support to a manganese mechanism of radical generation in brown-rot decomposition of wood. SUMMARY Hydrogen peroxide and the hydroxyl radical were detected in Postia placenta liquid nitrogen-limited cultures. The hydroxyl radical was detected in wood samples. This supports the hypothesis that brown-rot fungi decompose wood by an oxygen free radical mechanism. In an electron spin resonance (ESR)survey of various liquid cultures and wood slivers inoculated with the brown-rot fungus, Postia placenta, the spin trap 5.5-dimethyl-1-pyrroline N-oxide (DMPO) was used to detect the presence of the hydroxyl radical. The ESR spectra for the paramagnetic DMPO-hydroxyl radical adduct was observed in (1) nitrogen-limited, liquid cultures having 0.1% glucose or 0.1% cellobiose as the carbohydrate source, and (2) fungal-infected wood slivers of Douglas-fir (Pseudotsuga menziesii ) and white fir (Abies concolor). The 4-line ESR signal had a 1:2:2:1 intensity ratio, 15 G line splitting, and a g-factor of The chromogen, ABTS, was used to detect P. placenta generated H 2O 2 in liquid medium. Hydrogen peroxide production was 506

11 repressed in the presence of nitrogen by carbohydrates at levels above 0.01% for cellobiose, glucose and mannose and above 0.1% for arabinose and galactose. In the absence of added nitrogen the repression does not occur. REFERENCES Bors, W., Michel, D. and Saran, M. (1979). On the nature of biochemically generated hydroxyl radicals. Studies using the bleaching of p-nitroso-dimethyaniline as a direct assay method. Eur. J. Biochem., 95, Buettner, G.R. (1985). Spin trapping of hydroxyl radical. In: Handbook of Methods for Oxygen Radical Research. pp (R.A. Greenwald, ed.) CRC Press, Roca Raton, USA. Cohen, G. (1985). The Fenton reaction. In: Handbook of Methods for Oxygen Radical Research. pp (R.A. Greenwald, ed.) CRC Press, Boca Raton, USA. Cowling, E.B. (1961) Comparative biochemistry of the decay of sweetgum sapwood by white-rot and brown-rot fungi. USDA Tech. Bull. 1258, Washington, DC, 79 p. Cowling, E.B. and Brown W. (1969) Structural features of cellulosic materials in relation to enzymatic hydrolysis. In: Advances in Chemistry Series 95 Cellulases and Their Applications, pp (J. Hajny and E.T. Reese, eds.) American Chemical Society, Washington, DC. Eriksson, K.-E. (1981). Microbial degradation of cellulose and lignin. In: Proc. of the International Symposium Wood Pulping Chem. (Stockholm), 3, Halliwell, G. (1965). Catalytic decomposition of cellulose under biological conditions. Biochem. J., 95, Halliwell, B. and Gutteridge, J.M.C. (1986). Free Radicals in Biology and Medicine, pp Clarendon Press, Oxford. Hammel, K.E., Tien, M., Kalyanaraman, B. and Kirk, T.K. (1985). Mechanism of oxidative C -C cleavage of a lignin model dimer by Phanerochaete chrysosporium ligninase. J. Biol. Chem., 260(14), Highley, T.L. (1977). Requirements for cellulose degradation by a brown-rot fungus. Mater. und Org., 12, Highley, T.L. (1982). Is extracellular hydrogen peroxide involved in cellulose degradation by brown-rot fungi? Mater. und Org,. 7(3),

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13 Takao, S. (1965). Organic acid production by basidiomycetes. Appl. Microbiol., 13, Thompson, N.S. and Corbett, H.M. (1985). The effect of potassium superoxide on cellulose. Tappi, 68(1), Thornalley, P.J. and Bannister, J.V. (1985). The spin trapping of superoxide radicals. IN CRC Handbook of Methods for Oxygen Radical Research, (R.A. Greenwald, ed.), CRC Press Inc., Boca Raton, FL. Young, H.E. and Carpenter, P.N. (1967). Weight, nutrient element, and productivity studies of seedlings of eight tree species in natural ecosystems. Maine Agric. Exp. Sta. Tech. Bull. 28. Acknowledgments The authors wish to thank Dr. George H. Reed, Biochemistry Department, The University of Wisconsin, Madison, for the use of the ESR; Les Ferge for technical assistance; Richard Paynter for graphics; and Beverly Crapp for clerical assistance. Present address of Dr. D. C. Meinholtz is Chemistry Department, The Ohio State University, 140 W. 18th Avenue, Columbus, Ohio In: O'Rear, Charles E.; Llewellyn, Gerald C., eds. Biodeterioration research 2-General biodeterioration, degradation, mycotoxins, biotoxins, and wood decay: Proceedings, 2d meeting, Pan American Biodeterioration Society; 1989 July 28-31; Washington, DC. New York: Plenum Press; 1989: