Influence of Tropolone on Poria placenta Wood Degradation

Fenton reactions are believed to play important roles in wooddegradation by brown rot fungi. In this context, the effectof tropolone (2-hydroxycyclohepta-2,4,6-trienone), a metal chelator,on wood degradation by Poria placenta was investigated. Tropolone(50 µM) strongly inhibits fungal growth on malt agar,but this inhibition could be relieved by adding iron salts.With an experimental system containing two separate parts, onesupplemented with tropolone (100 µM) and the other not,it was shown that the fungus is able to reallocate essentialminerals from the area where they are available and also togrow in these conditions on malt-agar in the presence of tropolone.Nevertheless, even in the presence of an external source ofmetals, P. placenta is not able to attack pine blocks impregnatedwith tropolone (5 mM). This wood degradation inhibition is relatedto the presence of the tropolone hydroxyl group, as shown bythe use of analogs (cyclohepta-2,4,6-trienone and 2-methoxycyclohepta-2,4,6-trienone).Furthermore, tropolone possesses both weak antioxidative andweak radical-scavenging properties and a strong affinity forferric ion and is able to inhibit ferric iron reduction by catecholates,lowering the redox potential of the iron couple. These dataare consistent with the hypothesis that tropolone inhibits wooddegradation by P. placenta by chelating iron present in wood,thus avoiding initiation of the Fenton reaction. This studydemonstrates that iron chelators such as tropolone could bealso involved in novel and more environmentally benign preservativesystems.

INTRODUCTION

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Introduction

The use of traditional wood preservative chemicals is increasinglycoming under scrutiny, as questions concerning the environmentalacceptability of biocides in general become more prominent bothsocially and politically (18). There is a strong general desireto develop wood protection systems with improved environmentalacceptability as alternatives to traditional methods (28, 29).

Brown rot caused by fungi is one of the most common and destructivetypes of decay in wooden structures in the northern hemisphere.These basidiomycetes are unusual in that they rapidly depolymerizecellulose in wood without removing the surrounding lignin thatnormally prevents microbial attack (10).

Mechanisms of brown rot action have been studied extensively,particularly with Gloeophyllum trabeum. Recent data suggestthat this fungus uses an extracellular Fenton system (Fe²⁺ H₂O₂)to generate hydroxyl radicals, powerful oxidants that degradewood (16, 20, 25, 32). Biochelators produced by wood-degradingfungi might function to mediate this reaction, reducing Fe(III)to Fe(II), the iron being found essentially in the ferric statein an aerobic environment (12, 25). Indeed, these chelators,like catechols, not only sequester metals, but are also knownto possess reductive capabilities to reduce transition metalspecies. This capability would be important for the productionof the reduced iron required to generate hydroxyl radicals viathe Fenton reaction.

Tropolone (2-hydroxycyclohepta-2,4,6-trienone) is an analogof hinokitiol, a naturally occurring terpene responsible forresistance to fungal decay and insect attack on heartwood ofseveral cupressaceous trees (1, 19, 24). The metal-chelatingproperties of tropolone have been used in several experimentsto create metal-limiting conditions (9, 27) or as an inhibitorof copper enzymes (8, 13).

In a previous study, we have shown that tropolone strongly inhibitsthe growth of several wood rot fungi on malt-agar, the antifungalactivity of tropolone being quite similar to that of commerciallyavailable fungicides in current use as wood preservatives (4).In order to understand the effects of tropolone on P. placentawood degradation, we tested in this study whether tropoloneproduced its effects by acting as a chelator, an antioxidant,a scavenger of chemical radicals, or some combination of these.Furthermore, we tested whether the observed tropolone inhibitionof wood decay was due to an iron starvation of the fungus orto inhibition of a specific extracellular reaction involvedin wood degradation.

MATERIALS AND METHODS

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Materials and Methods

Chemicals.
Commercially available products, including 4-tert-butylcatechol,2,3-dihydrobenzoic acid, and tropolone, were purchased fromAldrich (St. Louis, Mo.) or Lancaster (Morecambe, England).

Synthesis of tropolone and 2-methoxycyclohepta-2,4,6-trienone.
Tropolone and 2-methoxycyclohepta-2,4,6-trienone were synthesizedas previously described (4, 26).

Organism.
The brown rot fungus used in this study was P. placenta strainFPRL 280 (4). Fungal stock cultures were maintained on malt-agarslants (Difco), which were kept at 4°C before use.

Growth inhibition.
Mycelia were grown in 10-cm petri dishes filled with 20 ml ofmalt-agar medium containing different concentrations of growthinhibitors, which were added aseptically from a filter-sterilized1 M ethanolic solution. Plates were inoculated by placing a10-mm-diameter plug cut from the edge of a starting colony growingon malt-agar medium. The cultures (two replicates for each experiment)were incubated for 15 days in a growth chamber at 25°C.Growth was evaluated every 2 or 3 days by measuring two perpendiculardiameters of the colony and expressed as a percentage of thespace available for growth, i.e., the diameter of the dish.

In some experiments, 25 ml of agar medium was supplemented withvarious concentrations of FeCl₃ or FeSO₄ (0 to 100 µM).All experiments were performed in triplicate.

Growth on separate areas.
Petri dishes were filled with 20 ml of malt-agar medium. Onehalf of the medium was aseptically removed after solidification,and the empty space was filled with malt-agar medium supplementedwith 100 µM tropolone. After solidification, a 0.5-cm-wideborderline band of the medium was aseptically removed, leadingto two separate areas, one with and one without tropolone. Plateswere inoculated by placing a 10-mm-diameter plug, cut from theedge of a starting colony growing on malt-agar medium, in thecenter of the agar devoid of tropolone. The cultures (two replicatesfor each experiment) were incubated for 50 days at 25°C.Growth was evaluated every 2 or 3 days by measuring the colonizedsurface of the tropolone-treated zone and expressed as a percentageof the space available for growth.

Chelator impregnation and wood block test.
Weighed (m_i), oven-dried pine blocks (Pinus sylvestris) (15by 20 by 50 mm, radial by tangential by longitudinal) were usedfor biological trials (5, 6). Four dried blocks were placedin a beaker inside a desiccator equipped with a two-way valveand subjected to a 4-mbar vacuum for 1 h. Blocks were then impregnatedby suction and covered with 5 mM aqueous solutions of each compounddissolved in the minimal amount of ethanol, and the pressurewas returned to the atmospheric level. After 3 h of soaking,the blocks were placed under air and dried at 80°C for 2days. Treated (two replicates) and untreated (two replicates)blocks were placed in petri dishes on sterile culture medium(15 g of malt, 15 g of gelose, 1 liter of distilled water) previouslyexposed to P. placenta for 2 weeks. The incubation was carriedout for 10 weeks at 25°C under 70% relative humidity. Theblocks were then extricated from the mycelium, dried at 80°Cfor 2 days, and weighed (m_f). The fungicidal efficiency of thetreatment was estimated according to the weight loss (WL), calculatedaccording to the following formula:

The tropolone concentration in pine wood was estimated by thedifference between the tropolone concentration in the impregnationsolution before and after the treatment. gas chromatographyanalysis were performed on a Fisons GC-8000 series instrumentwith a DB1 capillary column (J & W Scientific; length, 10m; internal diameter, 0.32 mm; filter, 0.25 µm). The temperatureprogram started at 50°C for 1 min, raised at 25°C/minto 150°C, and held at 150°C for 1 min. Nitrogen wasused as the carrier gas. The tropolone concentration was determinedwith phenol as the internal standard. The retention time was2.01 and 3.22 min for phenol and tropolone, respectively.

Oxidation experiment.
The inhibition of oxygen uptake with methyl linoleate (LH) asthe substrate was used (3, 10). L· free radicals aregenerated by the initiator AIBN (2,2'-azobis[2-methylpropionitrile]),and oxidation of methyl linoleate is a chain reaction propagatedby the processes:

In the presence of an inhibitor having a labile hydrogen andcalled AH, chain carriers easily abstract an H atom from AH(called a chain-breaking antioxidant), giving an unreactivefree radical A^·:

and so inhibitingchain propagation. Oxidation was monitored by measuring theoxygen pressure for the reaction.

The solvent used throughout was 1-butanol. Oxidation of methyllinoleate (0.4 M) was performed in a closed borosilicate glassapparatus with 9 x 10^–3 M AIBN as an initiator (11). Thedouble-shell reactor was thermostated at 60°C by an externalheating bath. Oxygen (154 Torr) was bubbled by a gas-tight oscillatingpump with a magnetically coupled motor. A small condensor at5°C was inserted in the gas circulation just after the reactorto ensure condensation of the solvent. The volumes of the liquidand gas phases were 4 ml and 100 ml, respectively. Oxygen uptakewas monitored continuously with a pressure transducer (Viatronmodel 104). Tropolone or reference antioxidant concentrationswere generally 5 x 10^–4 M.

Radical-scavenging activity.
Methanolic solutions of 2,2-diphenyl-1-picrylhydrazyl free radicaland tropolone or reference compounds were rapidly mixed in therapid kinetic accessory SFA-11 (Hi-Tech Scientific, Salisbury,United Kingdom), so that their concentration was 100 µM(31). Absorbance was measured at 517 nm (a wavelength at whichonly 2,2-diphenyl-1-picrylhydrazyl absorbs) for 30 min witha CaryWin UV spectrophotometer (Varian, Zug, Switzerland).

Reduction of Fe(III) by 4-tert-butylcatechol.
Fe(II) production was determined by the ferrozine assay (30).The test was performed in 50 mM acetate buffer (pH 4.8) containingferrozine (10 mM), FeCl₃ (0.2 mM), and various concentrationsof tropolone from 0 to 1.2 mM. The mixture was incubated for5 min at room temperature, and the reaction was initiated byaddition of 4-tert-butylcatechol (1 mM). Fe(II) complex formationwas measured by spectrophotometry at 562 nm after 15 min witha CaryWin UV spectrophotometer (Varian, Zug, Switzerland). Theblank was the reaction mixture without 4-tert-butylcatechol.The same experimental procedure was also performed with 2,3-dihydroxybenzoicacid.

RESULTS

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Results

Effects of iron solution on tropolone growth inhibition.
Growth of P. placenta was monitored on malt-agar medium containingtropolone (100 µM) and various FeCl₃ concentrations (25,50, and 100 µM) (Fig. 1). At these concentrations, ferricions have no effect on fungal growth (data not shown). As wereported previously (4), tropolone at this concentration stronglyinhibited P. placenta growth. Tropolone growth inhibition waspartially removed with 25 and 50 µM FeCl₃ and completelysuppressed with 100 µM, suggesting that tropolone stronglyreduces the availability of iron (or other metals) present initiallyas traces in the medium, leading to inhibition of fungal growth.The same result was obtained when Fe(SO₄)₃ was added at thesame concentrations.

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FIG. 1. Effects of ferric ion on P. placenta growth inhibition by tropolone. Growth of P. placenta was followed for 8 days in malt-agar plates supplemented with • 100 µM tropolone; 100 µM tropolone and ${blacktriangleup}$ 25 µM FeCl₃; ${circ}$ 50 µM FeCl₃; and ${blacksquare}$ 100 µM FeCl₃; X, control without any addition. Each point is the mean ± t standard deviation (SD), indicated by an error bar, for four different cultures.

Metal reallocation.
The inoculum was placed in the tropolone-free area, and thecolonization area was measured as a function of time in thesecond area supplemented or not with 100 µM tropolone(Fig. 2). P. placenta was able to colonize malt-agar mediumsupplemented with 100 µM tropolone when the inoculationwas performed on a separate tropolone-free area. Nevertheless,the growth rate observed (0.8 cm² per day) and the colonizedarea were clearly reduced when the inhibitor was present, incomparison with control experiments performed without addedtropolone (4.5 cm² per day). Despite these differences, theresults suggest that the fungus is able to overcome the inhibitoryeffects of tropolone under the conditions tested.

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FIG. 2. Effects of external metal sources on P. placenta growth inhibition by tropolone. Petri dishes were filled with malt-agar medium in order to obtain two separate areas. The first part did not contain tropolone and was inoculated. The second part of the petri dish was supplemented ${diamondsuit}$ or not ${blacksquare}$ with 50 µM tropolone. The fungal colonization of the second part was monitored for 45 days. Each point is the mean ± standard deviation for six different cultures.

With the same experimental system (i.e., two split areas ina petri dish, one corresponding to the inoculum, the other containingthe wood block), pine blocks treated or not with 5 mM tropolonesolution were subjected to P. placenta attack. The iron concentrationof the pine wood used in these experiments was of 0.31 µmol/g,measured by atomic absorption spectrometry. The tropolone concentrationin pine wood after treatment was estimated at about 6 µmol/g.This treatment strongly inhibited growth of P. placenta on pineblocks because no colonization was observed in the 5 mM treatmentand no weight loss was detected. At the same time, about 25%of mass loss was measured in the untreated pine blocks, whichwere wholly colonized.

Importance of tropolone hydroxyl group in wood protection.
P. placenta attack was monitored on pine sapwood blocks impregnatedwith either 5 mM tropolone (2-hydroxycyclohepta-2,4,6-trienone),cyclohepta-2,4,6-trienone (tropone) (5 mM), or 2-methoxycyclohepta-2,4,6-trienone(5 mM). About 20% mass loss was measured in untreated pine blocksor cyclohepta-2,4,6-trienone- (5 mM) and 2-methoxycyclohepta-2,4,6-trienone-(5 mM) impregnated pine blocks incubated in the presence ofthe fungus for 10 weeks (Fig. 3). However, treatment with 5mM tropolone completely inhibited the growth of P. placenta,as demonstrated by the low weight loss observed. The 2-methoxycyclohepta-2,4,6-trienoneand cyclohepta-2,4,6-trienone solutions also had no effect atthis concentration. These results confirm the importance ofthe hydroxyl group of tropolone, since when it is replaced bya methoxy group, the derivative is inactive as a wood preservative.

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FIG. 3. Effects of tropolone and tropolone analogs on P. placenta wood degradation. Pine sapwood blocks were subjected to P. placenta degradation after different treatments: A, none; B, impregnated with 5 mM tropolone; C, impregnated with 5 mM cyclohepta-2,4,6-trienone; or D, impregnated with 5 mM 2-methoxycyclohepta-2,4,6-trienone. Each point is the mean ± standard deviation for four different cultures.

Tropolone as antioxidant.
Since wood degradation by brown rot fungi is believed to beassociated with the production of free radicals, the antioxidantactivity of tropolone was tested and compared to that of compoundsknown for their antioxidant properties, i.e., ${alpha}$ -tocopherol, 2,6-di-tert-butyl-4-methylphenol,and (+)-catechin. As shown in Fig. 4, oxidation of methyl linoleatewas strongly inhibited by the reference compounds at a concentrationof 5 x 10^–4 M, but was only weakly inhibited by tropoloneat the same concentration. Thus, the wood protection inducedby tropolone cannot be explained by its very weak antioxidantproperties.

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FIG. 4. Antioxidant properties of tropolone. Oxidation of methyl linoleate (0.4 M) induced by AIBN (9 x 10^–3 M) was followed by measuring oxygen pressure in the presence of either 5 mM tropolone, 2,6-di-tert-butyl-4-methylphenol (BHT), (+)-catechin, ${alpha}$ -tocopherol, or no addition at 60°C. The reaction was inhibited in the presence of antioxidant molecules, e.g., 2,6-di-tert-butyl-4-methylphenol, (+)-catechin, and ${alpha}$ -tocopherol used as positive controls.

On the other hand, we tested the antiradical activity of tropoloneby the 2,2-diphenyl-1-picrylhydrazyl method. As shown in Fig.5, only 14% of 2,2-diphenyl-1-picrylhydrazyl reacted with tropoloneafter 30 min, whereas more than 90% reacted with tocopheroland catechin and 80% with 2,6-di-tert-butyl-4-methylphenol.These results demonstrated that tropolone is a poor radicalscavenger. They were in good agreement with data previouslyreported which suggest that the scavenging activity of ß-thujaplicinagainst active oxygen species was due to suppression of hydroxyradical formation, such as the Fenton reaction, by the formationof ß-thujaplicin-Fe complex (2, 33).

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FIG. 5. Antiradical properties of tropolone. The consumption of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (100 µM) for its reaction with either tropolone, 2,6-di-tert-butyl-4-methylphenol (BHT), (+)-catechin, or ${alpha}$ -tocopherol at 100 µM, the latter three being used as positive controls.

Tropolone as iron chelator.
Since iron is believed to play an essential role in wood degradationby brown rot fungi (14), we investigated iron-tropolone interactions.A mixture of ferric ion (0.7 mM) and tropolone (21 mM) led instantaneouslyto the formation of an orange-red precipitate. A precipitatewas also obtained when tropolone (21 mM) was mixed with ferrousion (0.7 mM). Thermogravimetric analysis indicated that theprecipitates obtained involve three equivalents of tropolonefor one equivalent of ferric ion [Fe(trop)₃] and two equivalentsof tropolone for one equivalent of ferrous ion [Fe(trop)₂] (datanot shown). Furthermore, the solubility constants (pKs) of Fe(trop)₃and Fe(trop)₂ were estimated by pH metric methods to be 30 and13.5, respectively (C. Rapin, unpublished results).

Inhibition of Fe(III) reduction.
Since catecholate siderophores are involved in the first stepsof wood degradation by brown rot fungi (16), we investigatedthe effects of tropolone on Fe(III) reduction by catecholate.Figure 6 shows that reduction of Fe(III) occurred in the presenceof 4-tert-butylcatechol. This reduction has also been observedin the presence of 2,3-dihydroxybenzoic acid (data not shown)(25). The production of an Fe(II)-ferrozine complex was stronglyinhibited in the presence of tropolone, since no complex formationwas observed when 0.7 mM tropolone was added to the system.When the accumulation of Fe(II)-ferrozine complex was followedwith 2,3-dihydroxybenzoic acid, the same pattern of inhibitionby tropolone was observed (data not shown).

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FIG. 6. Reduction of Fe(III) by 4-tert-butylcatechol in the presence of tropolone. Fe(III) (0.2 mM) reduction by 4-tert-butylcatechol (1 mM) was measured by monitoring the absorbance of Fe(II)-ferrozine complex at 562 nm. Each point is the mean ± standard deviation for four different experiments.

DISCUSSION

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Discussion

In this study, we have shown that tropolone inhibits the growthof P. placenta on malt-agar, but this inhibition could be relievedby adding iron salts. These data are consistent with the hypothesisthat P. placenta growth inhibition is the result of tropolonemetal chelation, leading to limitation of available iron (orother essential minerals). Although P. placenta, like otherwood rot fungi, synthesizes extracellular iron-binding compounds(14, 22, 23), it is not able to overcome this inhibition, demonstratingthe high affinity of tropolone for iron. Similar microbe-inhibitoryeffects of tropolone have been reported previously on the growthof Tuber borchii (27), Listeria monocytogenes (9), Aureobasidiumpullulans, Penicillium glabrum, and Trichoderma harzianum (17).Nevertheless, P. placenta is able to overcome the inhibitoryeffects of tropolone on malt-agar medium when colonization startedfrom a tropolone-free area.

These data, correlated with tropolone inhibition of P. placentagrowth, suggest that the fungus is able to mobilize iron (orother essential minerals) and to transport it to areas of hightropolone concentration. It is well known that fungi are ableto reallocate nutrients between different parts of their mycelium(5, 7, 21). However, descriptions of the translocation of essentialminerals are not as detailed as they are for N, S, and P, andthe biochemical mechanisms remain largely unknown. The modeldeveloped in this study with tropolone and P. placenta couldbe useful for studying such phenomena.

Interestingly, P. placenta is not able to degrade tropolone-treatedwood even when colonization is started from tropolone-free areas.This phenomenon could also be related to the wood degradationmechanisms rather than to metabolism inhibition, since the fungusseems to be able to reallocate essential minerals and to growin the presence of tropolone. In this study, we also demonstratedthat wood protection induced by tropolone could not be explainedby its very weak antioxidant properties. Since the presenceof the tropolone hydroxyl group is important, the chelatingproperties of this compound are probably involved in the observedwood degradation inhibition.

Iron, hydrogen peroxide, and biochelators such as catecholatecompounds are believed to play essential roles in wood degradationby brown rot fungi. Iron is probably present in wood in itsinsoluble, oxidized form (32). In the model described by Goodelland coauthors, oxalate lowers the pH of the decay environmentand sequesters iron from cellulose. At the appropriate pH, ironis sequestered from this complex by biochelators (catecholates)and is reduced to Fe(II), allowing the Fenton reaction to proceedin the presence of hydrogen peroxide. Maximal degradation inthis system occurs at approximately pH 4 (16, 32). The oxidationof catecholates by Fe³⁺ is thermodynamically possible betweenpH 6 and pH 1.

The E-pH diagram calculated from constants determined in thisstudy shows that iron sequestration by tropolone thermodynamicallyinhibits this reaction. Indeed, for instance, at pH 5, the redoxpotential of Fe(Trop)_3/Fe²⁺ couple is about 0 mV, while thatof the Fe(OH)_3/Fe²⁺ couple is about 700 mV. In the same conditions,the redox potential of the 4-tert-butyl-o-benzoquinone/4-tert-butylcatecholcouple chosen as the catecholate example is about 600 mV (15).Complex formation between tropolone and Fe³⁺ strongly lowersthe redox potential of the iron couple, thermodynamically preventingits reaction with potential reductants such as catecholates.This is consistent with our experimental results showing thatthe presence of tropolone strongly inhibits Fe³⁺ reduction byeither 4-tert-butylcatechol or 2,3-dihydroxybenzoic acid (Fig.6).

All the data presented in this study are consistent with themodel proposed by Goodell and coworkers, showing indirectlythe importance of iron in wood degradation by brown rot fungi.

Since the Fenton reaction is important for the initial attackby brown rot fungi, it follows that breakdown of polymeric materialshould be inhibited by a chelating agent that is unable to reducethe Fe(III) complex to Fe(II) under physiological conditions(this is the case with tropolone) or in which the Fe(II) complexhas a negligible rate constant for HO^. formation. Furthermore,in this study, we demonstrated that putative metal translocationoccurring in the fungus is not able to overcome tropolone effectson wood degradation, this property being important for the useof tropolone as a wood preservative. Taking into account theessential role of iron in wood degradation by brown rot fungi,iron chelators such as tropolone could also be involved in noveland more environmentally benign preservative systems. Nevertheless,further work, particularly in the field, will be necessary todetermine the applicability of this method.

FOOTNOTES

* Corresponding author. Mailing address: UMR INRA 1136, Equipe Interactions Arbres Micro-organismes, Université Henri Poincaré Nancy I, Faculté des Sciences, BP 239, 54506 Vandoeuvre-lès-Nancy, France. Phone: 33 383 68 42 27. Fax: 33 383 91 25 64. E-mail: gelhaye{at}lcb.uhp-nancy.fr.

REFERENCES

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