BIODETERIORATION RESEARCH 2: 485-496
Plenum Press, New York, 1989
MANGANESE AS A PROBE OF FUNGAL DEGRADATION OF WOOD
BARBARA L. ILLMAN*, DORE C. MEINHOLTZ, and TERRY L. HIGHLEY, USDA, Forest Service, Forest Products Laboratory, One Gifford Pinchot Drive, Madison, WI 53705-2398,USA INTRODUCTION
Transition state metals, such as manganese (Mn) and iron (Fe), have been reported to be involved in fungal degradation of wood (Ellis, 1959; Shortle and Shigo. 1973; Blanchette, 1984; Glenn et al., 1986).
Manganese is also involved in the enzymatic degradation of lignin model compounds by the white-rot fungus Phanaerochaete chrysosporium Burds. (Tien and Kirk, 1984; Glenn et al., 1986).
Iron is believed to be involved in the oxidative degradation of cellulose by brown-rot fungi (Cowling and Brown, 1969; Koenigs, 1974; Schmidt et al., 1981). Current techniques that are typically used for determining the metal content of decayed wood are time-consuming, laborious. and nonspecific (Hunter and Coleman, 1960).
A quicker, more specific technique is needed for detecting metals during the wood decay process.
Such a technique could be used (1) to obtain knowledge that could serve as a foundation for the development of new methods to prevent wood decay, and (2) as a diagnostic tool to detect the early stages of decay in living trees and in wood in use. The chemical
reactions that involve
transition metals during the degradation of wood could change the electronic characteristics of the metals.
Although the reactions proposed for iron and manganese in the decay process are different, both involve a one electron transfer step that results in changes in the paramagnetic state of the metals.
The paramagnetic state of a metal is characterized by unpaired electrons, i.e. electrons unpaired with an oppositely directed spin of another electron.
It is possible that these electronic changes could be exploited to detect the chemical nature of the metals during wood decay. 485 Electron spin resonance (ESR) spectroscopy is a technique that can be used to detect and characterize paramagnetic metals.
The ESR spectrometer is highly selective for unpaired electrons and has been used to detect free radicals in wood decayed by Postia placenta (Fr.)
M.Lars. et Lomb. (Illman et al.. 1988a; Illman et al., 1988b) and in wood surfaces damaged by light This highly specialized technology may serve to (Kalnins et al., 1966).
identify microelement components in fungal degradation of wood. The objective of the present study was to determine if ESR could be used
to detect any changes that might occur in the spin orientation of transition
metals during wood decay.
A model system was developed in which ESR was used
to scan for paramagnetic iron and manganese in cottonwood, southern yellow
pine, Douglas-fir, white fir, and redwood after inoculation with the brown-rot
fungus, Postia placenta.
EXPERIMENTAL PROCEDURE Preparation of samples:
Quartz tubes approximately 13.5 cm x 0.3 cm o.d. x 0.2 cm i.d. were custom made to accommodate wood samples.
The tubes served a dual function as incubation vessels for fungal inoculated wood slivers and as ESR tubes that could be inserted directly into the sample cavity of the spectrometer.
The tubes were acid washed to remove any trace elements that might contaminate the system. Wood slivers, approximately 3 cm x 0.1 cm x 0.1 cm, were cut from
cottonwood (Populus deltoides Bartr.), southern yellow pine (Pinus sp.).
Douglas-fir (Pseudotsuga menziesii). white fir (Abies concolor). and redwood
(Sequoia sempervirens.
interiors of boards.
Wood was taken on the longitudinal axis from the
Unexposed regions of boards were used in order to avoid
any degradative surface changes that could be caused by incident illumination
(Feist and Hon, 1984).
During preparation of the wood slivers, care was taken
to avoid contact of the slivers with light and with fungal culture medium.
Slivers were inoculated with Postia (formerly Poria) placenta (Mad-698) by placing them on fungal infected wood blocks (feeder strips).
The feeder strips had been previously prepared by incubating them on malt agar culture plates of the fungus for at least three weeks.
After a three day incubation on the feeder strips, ESR slivers were transferred to sterile quartz tubes fitted with foam plugs.
Wood slivers for control experiments were prepared in an identical manner by incubating them on feeder-type strips without fungi. All tubes were incubated in the dark at 27°C in zip-lock plastic bags for moisture retention.
ESR scans were made 10, 21, and 36 days after they had been placed in the quartz tubes. In order to determine any fungal contribution of metals to the wood decay system in the ESR tubes, aerial hyphae of P. placenta were tested for the 486 Hyphae were removed from culture presence of paramagnetic iron and manganese.
flasks with a glass rod, placed in quartz ESR tubes, sealed with plastic film, placed in the ESR sample cavity, and scanned in the iron and manganese regions of the magnetic field. Care was taken to omit any contact of hyphae and glassware with the culture medium. ESR spectra were obtained with a Electron spin resonance measurements:
Varian E-3 spectrometer at microwave frequency of 9.00-9.02GHz (X-band). The ESR system was operated at time constant 0.1 seconds, 10 G modulation amplitude and a microwave power of 10 milliwatts.
room temperature using an 8 minute scan time.
Measurements were made at Wood samples were scanned in the low field and high field regions of the magnetic spectrum for iron and manganese, respectively.
Measurements of hyperfine splittings were taken directly from spectra of magnetic field separations. The most distinguishing feature of
the manganese spectra is
the characteristic six-line splitting of the ESR signal which is due to Mn(II) The six-line spectra is highly specific for paramagnetic hyperfine coupling.
Mn and has been used as an internal standard for g values in mammalian ESR studies (Foster, 1984).
The term amplitude is used in the present study to denote height (cm) of the spectral lines and, therefore, the amount of the paramagneticmetal. Measurements were made of Douglas-fir and white fir manganese signals.
The amplitude of the sixth line was used to compare the paramagnetic manganese
in wood, with and without P. placenta.
Sixth-lineamplitude is least affected
by any signals from underlying impurities.
The ratio of the sixth-line
amplitude of fungal infected to noninfected white fir was used to determine
the
degree
of
paramagnetic
manganese
accumulation during
the
36 day
experiment.
RESULTS Cultures of P. placenta on wood slivers were readily established in the quartz tubes by "feeder strip" inoculation.
Wood slivers of Douglas-fir, redwood, and white fir colonized by P. placenta had a well-resolved six-line manganese signal.
A representative ESR spectrum of manganese exhibited by wood slivers of Douglas-fir, with and without fungus, is given in Figure 1. The perturbations of the six-line spectrum were unique for each species of wood. Assignment of the six-line spectrum to manganese is justified on the basis of the signal's isotropic g factor of 2, the equal intensity of the six lines, line separation of about 100 G, lack of signal saturation at a power of 80 mW as compared to 10 mW, and signal observation at room temperature.
The degree of resolution of the spectra was reflected in the smoothness of all 487 lines and in the peak-to-peakwidths of the fifth and sixth hyperfine lines. In all cases, resolution of the spectra was greater for wood inoculated with fungus than without.
The widths varied over time, but were generally between 20 and 30 gauss in decayed wood. Although it is only strictly correct to 2
compare amplitude and width directly as measures of intensity, rough conclusions can be legitimately drawn from the data when the amplitudes refer to lines of similar line shape and width. The Mn(II) signal in uninoculated wood was unique for each wood species. The six-linespectra was resolvedincottonwood, white fir and southern yellow pine silvers without P. placenta.
A powderey ESR manganese signal was detected in wood slivers of Douglas-fir (Figure 1) and redwood (Figure 2) that had not been inoculated with P. placenta. This relatively weak, rough, partially resolved six-line ESR signal was found at about g=2, the region where paramagnetic manganese would be expected. Amplitudes for spectra of Douglas-firand white fir, with and without P. placenta, are given in Table 1.
A very rough signal at about g=2 was apparent in spectra of Douglas-firwithout P. placenta (see Figure 1 and Table 1). Table 1. Amplitudes of the sixth line ESR spectra of paramagnetic manganese
in Douglas-fir and white fir, with and without P. placenta.
Douglas-fir
Time (days)
- P. placenta
10
-
21
-
36
-
+ P. placenta
white fir - P. placenta
+ P. placenta
-
31.8
12.7
7.20
45.0
16.0
8.70
43.1
9.90
This signal was often too small and undefined to be measured and blanks appear in the data.
In all cases where the signal was measured, the amplitude was smaller than in wood colonized by the fungus. The amplitude ratio of the white fir signal from fungal infected and noninfected slivers was calculated from data in Table 1.
On the 10th day after slivers were placed in ESR tubes, the amplitude of noninfected wood could not be measured while fungal infected wood had an amplitude of 31.8. The ratios for day 21 and 36 were 332 and 373. respectively.
The rapid increase in the white fir manganese signal during the first 21 days had almost leveled off by the 36th day and remained over 300 times higher than noninfected wood.
The manganese signal from fungal infected Douglas-fir did not increase as rapidly as white fir, but did continue to steadily increase by 488 Figure 1. ESR Spectra of Paramagnetic Manganese
A, without P. placenta;
in Douglas-fir;
B, with P. placenta.
489 the 36th day.
The amplitude of Douglas-fir increased by a factor of 1.3 from day 10 to day 21 and from day 21 to day 37, whereas white fir increased by a factor of 1.4 and 1.
The signal from P. placenta-colonized wood of both species was always higher than from noncolonized wood. No significant signal for low- or high-spin iron was observed in any of
the wood slivers, with or without P. placenta.
Aerial hyphae of 50% of the P.
placenta exhibited a single line at a g of approximately 6-7, and an apparent
powder-type signal at g=2.5, 3.0 and 2.
The field values were not well
resolved and do not justify the identification of these signals at 6, 2.8, 2.2
and 1.7 g which are the locations of high- and low-spin iron (Smith and
Pilbrow. 1980).
Aerial hyphae did not exhibit any paramagnetic manganese
signals.
The Mn(II) ESR signals for cottonwood, southern yellow pine, white fir, Douglas-fir and redwood, with and without P. placenta, are given in Figure 2. The ESR signals are representative spectra from scans made on the samples 36 days after the wood was inoculated with the fungus.
The Mn(II) ESR signal exhibited by cottonwood and southern yellow pine colonized by P. placenta was approximately ten times larger than the signal in wood without the fungus (Figure 2).
The signal in white fir without fungus was approximately four times larger than the ones with P. placenta. The rough signals in Douglas-fir and redwood were resolved at g = 2 when P. placenta colonized the wood. DISCUSSION Electron spin resonance spectroscopy was successfully used to identify changes in paramagnetic manganese in wood during brown-rot decay.
This technique proved to be a very specific, rapid, and relatively simple detection method that can be used to scan whole piecies of wood without complicated extraction procedures.
To our knowledge, this is the first report of direct detection of manganese changes in wood that are caused by brown-rot fungi. ESR shows promise as a diagnostic tool to detect wood decay in living trees and in wood in use.
The early stages and subsequent development of decay may be analyzed by scanning wood samples for paramagnetic manganese. Changes in Mn(II) were detected in this study by ESR within the first 13 days after P. placenta came into contact with the wood.
Early ESR detection of and increases in Mn(II) signal may not be unique to wood inoculated with brown-rot fungi.
Because Mn(II) and Mn(III) are known to be involved. in lignin degradation by P. chrysosporium and Trametes versicolor (Glenn et al., 1986; Tien and Kirk, 1984; and Johansson and Nyman, 1987, respectively), it is likely that ESR can be employed to detect and study white-rot decay.
In addition to studies on white rot, current projects in our laboratory include 490 Figure 2. ESR Spectra of Paramagnetic Manganese in Wood, with and without the
Brown-rot fungus, Postia placenta. Microwave frequency at 9.003
GHz, microwave power 10 mW, modulation amplitude of 10 G. 491 the use of ESR
to detect changes
in other
transition metals
during biodegradation of wood. The increased Mn(II) ESR signal of fungal colonized cottonwood, southern yellow pine, white fir, Douglas-firand redwood (Figure 2) correlates with the susceptibility of the wood. species to decay.
Cottonwood is classified as a wood species that is nonresistant to decay; Douglas-fir as moderately resistant; and redwood as resistant (FPL-0153, 1967).
The powdery Mn(II) signal in decay resistant redwood without P. placenta was resolved in fungal colonized wood.
This is in contrast to the Mn(II) signal exhibited by cottonwood, a species that is nonresistant to decay.
Mn(II) signal in cottonwood with P. placenta was approximately ten times larger than cottonwood without the fungus.
These data have two outstanding implications: (1)Mn(II) changes during decay are most likely species-specific; (1) Mn(II) has the potential to be used as an ESR probe for wood decay studies. The source of Mn(II) responsible for the increased ESR signal in P. placenta colonized wood is most likely from the manganese originating in the wood.
Manganese is ubiquitous in trees and has been shown to be the most abundant transition metal in wood of deciduous trees in northeastern United States (Young and Carpenter, 1967).
In the present study, a small amount of Mn(II) was detected by ESR in noninfected white fir, cottonwood and southern yellow pine, and an unresolved Mn(II)-like signal was detected in Douglas-fir and redwood.
Fungal inoculated slivers were placed in ESR tubes without any culture medium and sealed as a closed system during the experiment.
P. placenta hyphae did not give a Mn(II) signal and the fungal inoculum was probably too small to contain the amounts of the metal that would be required for the increased Mn(II) signal.
The origin of manganese from the structure of wood is significant to the understanding of the fungal mechanisms of wood decay. The chemical basis of manganese changes during brown-rot is not known.
A fungal mechanism(s) is acting in some manner to resolve the orientation dependence of the zero field splitting interaction and/or ligand field interaction as reflected in the smaller line widths and the absence of orientation-dependent shoulders and extraneous structure to the hyperfine lines.
The extreme case is seen in Douglas-fir and redwood which have no measurable signal until inoculated with P. placenta. Following inoculation, a smooth sextet signal appears at g=2.
The amplitude of the manganese signal may be increasing as a result of ligand interaction as Mn(II) forms complexes with many complexing agents (Reed, 1986), coordination state and electronic state of manganese, or to a combination of these changes during fungal growth on the wood. 492 A number of reactions could account for the increase in the paramagnetic manganese signal in decayed wood.
It is possible, but not probable with the small Mn(II) ESR signal in non-infected wood, that aqueous manganese ions accumulated in the wood, resulting in the sextet ESR signal.
Alternatively, the explanation for the increased Mn(II) may best be understood by considering the hypothetical role of iron during brown rot. The current hypothesis for the role of iron in brown-rot decay is that Fe supplies a one electron transfer in the conversion of hydrogen peroxide in a classic Fenton reaction (Cowling and Brown, 1969; Koenigs, 1972 and 1974). Schmidt et al. (1981) proposed a mechanism in which the oxalic acid, known to be produced by brown-rot fungi (Takao, 1965), reduces wood Fe(III) to Fe(II). The Fe(II) would react with hydrogen peroxide, believed to be produced by the fungi (Koenigs, 1974). to produce the highly reactive hydroxyl free radical which could initiate the oxidative depolymerization of cellulose.
If these reactions take place, Fe(III) conversion to paramagnetic Fe(II) would result in a characteristic ESR spectrum for Fe(II). Paramagnetic iron was not detected by ESR in the present system using P. placenta-infected Douglas-fir, white fir, cottonwood, southern yellow pine or redwood.
The possiblility that iron changes were occurring cannot be ruled out at this time, because the ESR scans were made at room temperature and iron may be in a state that requires ESR detection at lower temperatures.
The hydroxyl radical was detected by ESR in a parallel study with P. placenta inoculated white fir and Douglas-fir (Illman et al., 1988a).
The radical was detected
manganese
during
occurring.
the
approximate
time
that
changes
in
were The simultaneous appearance of an oxygen free radical and the reduction of a wood transition metal may be related. Our working hypothesis at this time for the ESR detection of the hydroxyl radical and increases in the ESR signal of manganese, but not iron, is that a low molecular weight, fungal compound, converts wood Mn(III) to Mn(II) or changes the chemical bonding, hence the spin orientation of wood Mn(II).
The metal changes could be part of a chain reaction that generates the hydroxyl radical,
initiating
the
rapid
depolymerization
characteristic of brown-rot fungi.
of
cellulose
that
is Depolymerization of cellulose would predispose the wood to degradation by fungal enzymes. This explanation is consistent with the recent report by Takagi (1987) that hydrogen peroxide pretreatment of lignocellulosic materials enhances susceptibility to enzymatic saccharification with the addition of manganese but not with iron. In conclusion, ESR was successfully employed to detect changes of a transition metal during wood decay in a model system where five wood species were scanned for paramagnetic manganese and iron.
ESR has promise as a 493 diagnostic tool to detect early stages of wood decay.
The use of ESR promises to lead to more knowledge about mechanisms of fungal decay. SUMMARY Electron spin resonance (ESR) spectrometry was used to examine wood decay by the brown-rot fungus, Postia placenta. Wood slivers of Douglas-fir, white fir, redwood, cottonwood and southern yellow pine were incubated for 4 weeks in custom-made quartz ESR tubes with or without P. placenta.
In Douglas-fir
and redwood without fungus, a weak, partially resolved signal (about g=2, presumably due to manganese) wasdetected.
in aerial hyphae of the fungus.
No manganese-likesignal was found All wood slivers with fungus had a smooth, well-resolved manganese signal with a larger amplitude than wood without fungus. The ratio of amplitudes for slivers with and without fungus increased with
incubation
time.
reflecting
paramagnetic manganese during decay.
an
accumulation
of
ESR-detectable Due to the nature of this closed system without culture media, the increase in the manganese ESR signal is most likely due to the change of wood manganese by the fungus. REFERENCES Blanchette, R.A.
(1984). Manganese accumulation in wood decayed by white-rot fungi. Phytopathology,74(6):725-730. 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, (G.J. Hajny and E.T. Reese, eds.) American Chemical Society, Washington. DC. Ellis, E.L. (1959). The effects of environment and decay on mineral
composition of grand
fir.
In: Marine Boring and Fouling Organisms.
pp. 477-513 (D.L. Ray, ed.) Univ. Wash. Press, Seattle, USA.
Feist, W.C. and Hon, D.N.S. (1984). Chemistry of weathering and protection. The Chemistry of Solid Wood, Am. Chem. Soc., pp. 401-451. FPL-0153. (1967). Comparative decay resistance of heartwood of native species.
U.S. Department of Agriculture, Forest Service, Forest Products Laboratory, Madison, WI Foster, M.A. (1984). Magnetic Resonance in Medicine and Biology.
Pergamon
Press, New York.
Glenn, J.K., Akileswaran, L. and Gold, M.H. (1986). Mn(II) oxidation is the
principal function of the extracellular Mn-peroxidase from Phanerochaete
chrysosporium. Arch. Biochem. Biophys., 251(2), 688-696.
Hunter, A.H. and Coleman, N.T. (1960). Ion-exchangeseparations in the determination of some polyvalent metal ions in plant tissue.
214-218. 494 Soil Sci. 90, Illman, B.L., Meinholtz, D.C. and Highley, T.L. (1988a). Generation of hydroxyl radical by the brown-rot fungus, Postia placenta. The Intl.Res. Group on Wood Preser ., Doc. IRG WP-1360. Illman, B.L., Meinholtz, D.C. and Highley, T.L. (1988b). Oxygen free radical detection in wood colonized by the brown-rot fungus, Postia placenta.
In: Biodeterioration Research II. (C.E. O'Rear and G.C. Llewellyn, eds.), Plenum Press, New York. Johansson, T. and Nyman, P.O. (1987). A manganese(II)-dependent extracellular peroxidase from the white-rot fungus Trametes versicolor .
Acta Chem. Scand. B, 41, 762-765. Kalnins, M.A., Steelink, C. and Tarkow, H. (1966). Light-induced free radicals in wood,
Res. Pap. FPL-58. U.S. Department of Agriculture, Forest Service, Forest Products Laboratory, Madison, Wis. Koenigs, J.W. (1972).
Effects of hydrogen peroxide on cellulose and on its susceptibility of cellulase.
Koenigs, J.W. (1974).
Mat. und Organ. 7, 133-147. Hydrogen peroxide and iron: A proposed system for decomposition of wood by brown-rot basidiomycetes.
Wood and Fiber, 6, 66-79. Reed, G.H. (1986). Manganese: An overview of chemical properties. In: Manganese in Metabolism and Enzyme Function, pp. 313-325 (R. Schramm and F.C. Wedler, eds.), Academic Press, Inc., N.Y. Schmidt, C.J.. Whitten, B.K. and Nicholas, D.D. (1981). A proposed role for oxalic acid in non-enzymatic wood decay by brown-rot fungi.
Proc. Am. Wood Preser. Assoc., 77, 157-164. Shortle, W.C. and Shigo, A.L. (1973). Concentrations of manganese and microorganisms in discolored and decayed wood in sugar maple.
Res., 3(3), 354-358. Smith, T.D. and Pilbrow, J.R. (1980).
ESR of iron proteins.
Can. J. For. In: Biological Magnetic Resonance , pp. 85-168 (L.H. Berliner and F. Reuben, eds.)
Plenum Press, N.Y. Takagi, M. (1986). Pretreatment of lignocellulosic materials with hydrogen peroxide in presence of manganese compounds.
Biotech . Bioengin., 29, 165-170. Takao, S. (1965). Organic acid production by basidiomycetes. Appl. Microbiol., 13, 732-737. Tien, M. and Kirk, T.K. (1984). Lignin-degrading enzyme from Phanerochaete chrysosporium:
Purification, characterization and catalytic properties of a unique hydrogen peroxide-requiring oxygenase.
Proc. Natl. Acad. Sci. 81, 2280-2284. 495 Young, H.E. and Carpenter, P.N. (1967).
Weight, nutrient element, and productivity studies of seedlings and saplings of eight tree species in naturalecosystems.
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. The present address of the collaborating author, Dr. D. C. Meinholtz, is Department of Chemistry, 140 W. 18th Avenue, Columbus, Ohio 43210-1173. 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: 485-496.
496