Induction of oxalic by and nitrogen sources in the brown-rot fungus Postia placenta

By JESSIE A. MICALES

USDA Forest Service, Forest Products Laboratory1 Madison. Wisconsin, USA

K e y w o r d s: Wood decay, brown-rot fungi, oxalic acid, basidiomy- cetes, cellulose, hemicellulose, Postia placenta

1. Introduction

The role of oxalic acid in brown-rot decay is currently a topic of increased interest and controversy among researchers. Brown rot is char- acterized by the rapid depolymerization of cellulose early in the decay process (E. COWLING, 1961). The depolymerization of cellulose associated with incipient decay is generally accepted to be a nonenzymatic process, catalyzed by low molecular weight compounds that diffuse rapidly. Enzymes are too large to penetrate the fine pore structure of wood (D. FLOURNOY et al., 1991; E. SREBOTNIK and K. MESSNER , 1991; T. HIGHLEY et al., 1989). Many researchers agree that oxalic acid is a key component in brown- rot decay. Both direct and indirect roles have been proposed for oxalic acid in the depolymerization process. This topic has been reviewed by F. GREEN III et al. (1991). M. SHIMADA et al. (1991, 1992) proposed that oxalic acid could initiate the depolymerization of amorphous cellulose directly. Most researchers assign a more indirect role to oxalic acid. It has been associated with the Fenton reaction (J. KOENIGS, 1974, 1975; C. SCHMIDT et al., 1981) where it is thought to reduce ferric ions to the more active ferrous form. The ferrous ions then react with hydrogen peroxide

Received: 3 May 1994. 1 The Forest Products Laboratory is maintained in cooperation with the Uni- versity of Wisconsin. This article was written and prepared by U.S. Government employees on official time, and it is therefore in the public domain and not sub- ject to copyright. 198 Jessie A. Micales

to form extremely reactive hydroxyl radicals that depolymerize cellulose. In an alternate hypothesis (E. ESPEJO and E. AGOSIN, 1991), the fungus Gloeophyllum trabeum may use an extracellular acid oxidase to form hydrogen peroxide, thus generating hydroxyl radicals. It has also been demonstrated that acid hydrolyzes the hemicellulose in wood, thus making the cellulose fibers more accessible to cellulases (J. BECH- A NDERSON, 1987). F. GREEN III et al. (1991, 1992) expanded upon that hypothesis and suggested that the acidic conditions in wood, initiated by oxalic acid production, are responsible for early acid hydrolysis and depolymerization of hemicellulose and amorphous cellulose, thus increasing the wood porosity. Enzymes and other degrading agents in the hyphal sheath would then have access to the remaining cellulose and cause its final removal. Despite its importance, little work has been done on the physiology of oxalic acid in wood-decay fungi. Oxalic acid is known to accumulate in liquid cultures of brown-rot fungi (S. TAKAO, 1965). It does not normally accumulate in white-rot cultures as a result of the activity of an oxalate decarboxylase in the medium (H. SHIMAZANO, 1955). The pathways involved with oxalic acid production by wood-decay fungi are just beginning to be explored (Y. AKAMATSU et al., 1992). M. SHIMADA et al. (1992) have proposed that oxalic acid production is a means by which wood-decay fungi decrease the large pool of organic that accumu- lates from the degradation of wood . The objective of this study was to study the in vitro induction of oxalic acid by various carbon and nitrogen sources in the brown-rot fungus Postia placenta.

2. Materials and methods

2.1 Isolate storage

All work in this study was done with isolate MAD698 of Postia placenta (Fr.) M. Lars. et Lomb., a standard test isolate used at the Forest Products Laboratory. The isolate was stored on 2% malt-extract agar slants at 4°C for the duration of the study.

2.2 Oxalic acid assay

The amount of oxalic acid in culture filtrates was determined using a microtiter plate adaptation of a diagnostic test kit for oxalic acid (Sigma Chemical Com- pany, St. Louis, MO).2 This is a spectrophotometric assay based on the conversion 2 The use of trade or firm names in this publication is for reader information and does not imply endorsement by the U.S. Department of Agriculture of any product or service. Induction of oxalic acid in Postia placenta 199 of oxalic acid to hydrogen peroxide by oxalate oxidase. Hydrogen peroxide then reacts with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethyl- amino) benzoic acid (DMAB) to form a colored indamine dye in the presence of horseradish peroxidase. The initial chelating step was eliminated since experimen- tal results showed that the assay was stable within the pH range found in this study. All other steps were decreased in volume proportionally so that the reagents could fit into standard ELISA microtiter plate wells. The test was in- cubated at room temperature for 5 minutes and then read in a Dynatech MR 5000 microplate reader. Oxalic acid concentration was measured in micrograms per milliliter based on a standard curve that was calculated using known concentra- tions of oxalic acid (Sigma oxalic acid standards).

2.3 Induction of oxalic acid by different carbon sources

Strain MAD 696 was grown in 0.028 M gluconic acid (equivalent to 0.5% glu- cose), a carbon source that does not induce significant levels of oxalic acid (J. MICALES, 1992) in 0.05 M ammonium nitrate and a basal salts solution (T. HIGHLEY, 1973) at 27°C. On day 10 (when mycelial growth was observed in all flasks), sup- plemental carbohydrate was added to the cultures in the form of sterile, concen- trated stock solutions (three replications per treatment). Final concentration of the added carbohydrate in the growth medium was 0.028 M. The cultures were allowed to grow for an additional 10 days at 27°C. Mycelial dry weight, culture filtrate pH, and oxalic acid concentration were then determined. In an additional experiment, increasing concentrations of were added to 0.028 M glyoxylate, another carbon source that does not induce significant concen- trations of oxalic acid (J. MICALES, 1992), in 0.05 M ammonium phosphate and basal salts (T. HIGHLEY, 1973). The final concentrations of glucose in the media were 1, 5, 10, and 28 mM. The control flask contained no additional glucose. There were five replicate cultures per treatment. Cultures were incubated for 14 days at 27°C. Mycelial dry weight, culture filtrate PH, and oxalic acid concentration were then determined. The effect of time on glucose induction of oxalic acid production was deter- mined by growing MAD 698 in 0.028 M glyoxylate or 0.028 M furnarate, carbon sources that did not induce significant levels of oxalic acid (J. MICALES. 1992), in 0.05 M ammonium phosphate and basal salts (T. HIGHLEY, 1973). The growth me- dium was supplemented (before autoclaving) with 0.028 M glucose or 0.014 M cello- biose. Control cultures were not supplemented with additional carbohydrate. Cul- tures were incubated at 27°C. Aseptic, 5-µL aliquots were removed from each of three replicate cultures per treatment daily for 7 days. Oxalic acid concentration was then determined. The production of oxalic acid by MAD 698 when was the sole car- bon source was also determined. Isolate MAD 698 was grown in 0.055 M and 0.028 M acetic acid + 0.05 M ammonium phosphate in basal salts (T. HIGHLEY, 1973) with three replicate cultures per treatment. The cultures were incubated at 27°C for 14 days; oxalic acid concentration was then determined.

14 Material und Organismen 28/3 200 Jessie A. Micales

2.4 Induction of oxalic acid by different nitrogen sources

Strain MAD 698 was grown in 0.055 M glucuronic acid and basal salts (T. HIGH- LEY, 1973) containing one of the following nitrogen sources: 0.05 M ammonium sul- fate, potassium nitrate, ammonium chloride, ammonium phosphate, or sodium nitrate: 0.025 M ammonium nitrate or ammonium tartrate; or 0.5% (w/v) peptone. Control flasks contained no nitrogen source. Three replicate cultures were made for each treatment. The cultures were incubated for 14 days at 27°C. Mycelial dry weight, culture filtrate pH, and oxalic acid concentration were determined.

3. Results

The ability of various carbon sources to induce oxalic acid production is shown in Table 1. Glucose (and certain of its polymers), mannose, and the uronic acids significantly increased the amount of oxalic acid in the culture filtrate. With the exception of carboxymethylcellulose, these carbohydrates also significantly reduced the pH of the culture filtrate, probably due to the accumulation of oxalic acid. Other carbon sources, including galactose, , xylose, lichenan, galactomannan, and guar gum, did not significantly increase oxalic acid production but did significantly lower the pH of the culture filtrate. This was probably achieved through the production of other organic acids. Acetic acid did not significantly increase oxalic acid production in this experiment, but it did when acetic acid was the sole carbon source. An average oxalic acid concentration of 357.50 µg/rnl was detected in the culture filtrates of MAD 698 grown in 0.028 M acetic acid + 0.05 M ammo- nium phosphate in basal salts after 14 days growth at 27°C. This effect was highly dependent on concentration. Cultures containing 0.055 M acetic acid failed to produce any detectable oxalic acid in the culture filtrate. Adding acetic acid on day 10 to actively growing hyphae (as described in Table 1) may have resulted in a dramatic pH drop, which may have damaged the fungi and prevented oxalic acid formation. The optimum concentration of glucose for oxalic acid induction when the noninducer glyoxylate was used as the principal carbon source was 5 mM (Table 2). Higher concentrations of glucose resulted in higher abso- lute levels of oxalic acid, but this merely reflected increased amounts of mycelial growth. The culture filtrates from treatments containing glucose had significantly higher pH levels than those that did not, despite the elevated levels of oxalic acid. Other organic acids produced by P. pla- centa must also influence the pH of the culture filtrate. The substitution of 0.014 M cellobiose for 0.028 M glucose resulted in slightly higher levels of oxalic acid production when MAD 698 was grown in fumarate (Fig. 1). Induction of oxalic acid in Postia placenta 201

The reason for this is not known but may involve the activity of ß-D- glucosidase, the enzyme that cleaves cellobiose into glucose. Glucose and cellobiose induced similar quantities of oxalic acid when the fungus was

14* 202 Jessie A. Micales

grown in glyoxylate (Fig. 2). In both cases, oxalic acid production was biphasic. This may represent an attempt by the fungus to stabiIize the pH of the culture filtrate. The effect of different nitrogen sources on growth, pH, and oxalic acid production by MAD 698 is shown in Table 3. Glucuronic acid (0.055 M) was chosen as the carbon source because of its ability to induce oxalic acid. The highest level of oxalic acid production per milligram of myce- lium was formed under conditions of low nitrogen (i. e., when no nitro- gen source was added to the medium). Absolute levels of oxalic acid were higher when ammonium phosphate (monobasic) and peptone were the nitrogen sources because of the greater amount of mycelia formed.

4. Discussion

Oxalic acid production in the brown-rot fungus P. placenta, growing in a noninducing medium, was stimulated by the addition of glucose and certain glucose polymers, mannose, glucuronic, and galacturonic acid. Acetic acid was also a powerful inducer of oxalic acid formation if pre- sent in the proper concentration. Induction of oxalic acid in Postia placenta 203 204 Jessie A. Micales

Oxalic acid accumulation was also dependent on the nitrogen source used in the medium. The greatest amount of oxalic acid per milligram of mycelium was formed under conditions of low nitrogen. Wood is very low in nitrogen content (R. LAIDLAW and G. SMITH, 1965; W. MERRILL and E. COWLING, 1966), so low nitrogen conditions may further induce oxalic acid production in wood. Absolute levels of oxalic acid were higher when ammonium phosphate (monobasic) and peptone were used because of the greater amount of mycelial growth. Ammonium nitrogen has been reported to inhibit oxalic acid accumulation as a result of inhibition of glyoxylate dehydrogenase, the enzyme that forms oxalic acid from gly- oxylate via the glyoxyIate pathway (G. KRITZMAN et al., 1977; F. LAPEYRIE et al., 1987). Such inhibition does not always occur. Ammonium phos- phate was reported to stimulate oxalic acid accumulation in Sclerotium rolfsii (Z. PUNJA and S. JENKINS, 1984). Other pathways besides the glyoxylate shunt may be responsible for oxalic acid production, or the phosphate may have a powerful buffering effect that overrides any in- hibition by ammonium (Z. PUNJA and S. JENKINS , 1984).

Although it is difficult to extrapolate the results from liquid culture to in vivo activity in wood (C. CLAUSEN et al., 1994), this study raised some interesting possibilities about the mechanisms of decay by brown-rot fungi. High levels of oxalic acid were induced in the presence of man- nose, glucuronic, and galacturonic acid. Acetic acid at the proper con- centration also greatly stimulated oxalic acid production by MAD 698. F. GREEN III (1992) reported the release of oxidized products, including uro- nic and acetic acids, during the interaction of oxalic acid with cotton cellulose. Mannose is also a component of hemicellulose and would be released upon its hydrolysis. If mannose, glucuronic, galacturonic, and acetic acid are released early in the decay process by the acid-catalysed hydrolysis of hemicellose, the production of oxalic acid by the decay fun- gus would be greatly stimulated, thus rapidly lowering the pH of the wood and causing all the effects described by other researchers (J. BECH- ANDERSON , 1987; E. ESPEJO and E. AGOSIN, 1991; F. GREEN III et al., 1991; J. KOENIGS, 1974, 1975; M. SHIMADA et al., 1991). Oxalic acid production would also be enhanced by the low levels of nitrogen in the wood. As the reaction proceeds and cellulose is hydrolyzed, oxalic acid production would continue to be induced by the release of glucose, thus maintaining the acidic conditions in the wood. Because oxalic acid is continuously broken down in wood (E. ESPEJO and E. AGOSIN, 1991; C. SCHMIDT et al., 1981), its continued production may be necessary to maintain the low pH associated with brown-rot decay (F. GREEN III et al., 1991).

If oxalic acid is essential to the development of brown-rot decay, it may be possible to prevent or inhibit its production or accumulation. Induction of oxalic acid in Postia placenta 205

Current studies are examining oxalic acid metabolism and accumulation in low decay isolates of P. placenta.

5. Summary

Brown-rot wood decay fungi rapidly depolymerize the cellulose of wood, lead- ing to strength losses early in the decay process. The exact role of oxalic acid in this process is unclear. One hypothesis is that oxalic acid rapidly reduces the pH of sound wood during incipient decay, leading to early acid hydrolysis and depo- lymerization of hemicellulose and amorphous cellulose. The resultant increase in wood porosity allows enzymes and other degrading agents access to the cellulose. In this study, under in vitro conditions, oxalic acid production was stimulated by low nitrogen conditions and the presence of glucose, certain glucose polymers, mannose, acetic acid, and certain uronic acids. The release of glucomannan, acetic and uronic acids from hemicellulose early in the decay process, combined with the low nitrogen conditions found in wood, should greatly stimulate the production of oxalic acid by the fungus, thus lowering the pH of the wood rapidly in a self- potentiating system. As decay progressed, oxalic acid would continue to be induced by the release of glucose from cellulose.

Zusammenfassung

Bildung von Oxalsäure durch Kohlenhydrat- und Stickstoffquellen in Laborkulturen des Braunfäulepilzes Postia placenta

Braunfäulepilze sind in der Lage, Holzzellulose schnell abzubauen und dadurch erhebliche Festigkeitsverluste bereits in einem frühen Stadium des Holzangriffs zu verursachen. Die Rolle, die Oxalsäure in diesem Prozeß spielt, ist noch nicht ein- deutig geklärt. Eine Hypothese besagt, daß die Oxalsäure den pH-Wert des gesun- den Holzes sehr schnell herabsetzt und so zu einer schnellen Hydrolyse und Entpolymerisation von Hemizellulosen und amorpher Zellulose führt. Die dadurch entstehende größere Porosität des Holzes gestattet es Enzymen und anderen Abbaumechanismen, die Zellulose anzugreifen. In dieser Untersuchung wurde die Bildung von Oxalsäure unter Laborbedingungen durch geringe Stickstoffmengen und das Vorhandensein von Glukose, gewissen Glukose-Polymeren, Mannose, Essigsäure und gewissen Harnsäuren gefördert. Die Abgabe von Glukomannan, Essig- und Harnsäure aus der Hemizellulose zu Beginn des Holzbefalls zusammen mit dem geringen Stickstoffgehalt sollte die Bildung von Oxalsäure durch den Pilz erheblich erhöhen und gleichzeitig den pH-Wert des Holzes in einem wechselseiti- gen Prozeß senken. Bei fortschreitender Holzzerstörung, würde sich die Menge der Oxalsäure durch die Abgabe von Glukose aus der Zellulose weiter erhöhen.

Résumé

Induction de l’acide oxalique par les sources de carbone et d’azote chez Postia placenta, champignon des pourritures brunes

Les champignons des pourritures brunes dépolymérisent la cellulose du bois, ce qui entraìne une perte de resistance tôt dans le processus de pourrituze. Le rôle de 206 Jessie A. Micales

l’acide oxalique clans ce processus n’est pas clairement établi. Une des hypothèses est que l’acide oxalique diminue le pH du bois sain au cours du pourrissement, ce qui entraîne une hydrolyse et une dépolymérisation de l’hémicellulose et de la cel- lulose amorphe. Le résultat est une plus grande porosité du bols qui permet l’accès de la cellulose aux enzymes et à d’autres agents de dégradation. Dans ce travail realise in vitro, la production d’acide oxalique est stimulée par de basse concentrations d’azote et la présence de glucose, de certains polymères de glucose, de mannose, d’acide acétique, et de certains acldes uroniques. La basses concen- trations d’azote clans le bois combine avec le rejet de glucomannane, d’acides acé- tique et uronique à partir de l’hemicellulose au debut du processus de pourriture, doivent fortement stimulés la production d’aclde oxalique par le champignon, ce qui diminue le pH du bois rapidement. Avec la progression de la pourriture, l’acide oxalique continue à être induit par le rejet de glucose a partir de la cellu- lose.

References Induction of oxalic acid in Postia placenta 207

Address of the author: Dr. JESSE A. MICALES USDA Forest Service Forest Products Laboratory One Gifford Pinchot Drive Madison, WI 53705-2398 U.S.A.

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