Microbiol. Res. (1998) 153,71-80

© Gustav Fischer Verlag

Differential phenol-induced laccase activity and total oxidative capacity of the Sand P intersterility groups of the conifer root pathogen Heterobasidion annosum

Martin Johansson I, Lennart Lundgren2, Frederick O. Asiegbu 1

I Department of Forest Mycology and Pathology 2 Department of Chemistry, Swedish University of Agricultural Sciences, S-7S007 Uppsala, Sweden

Accepted: December 8, 1997

Abstract

A number of aromatic compounds, related to conifer wood and bark, were compared in terms of their effects on the laccase activity of strains of the Sand P intersterility groups (lGs) of Heterobasidion annosum. Under most conditions - in both liquid and solid media - the laccase activity of P strains, meas­ured in relation to biomass production, was significantly higher compared with that of S strains. P strains released mostly significantly higher amounts of proteins in an early growth phase, which somewhat reduced the difference in lac­case activity between the IGs, in terms of specific activity. Laccase activity in both IGs was stimulated by a pine bark ex­tract and phenolic acids - stilbenes as well as flavanoids and proanthocyanidines - and some other low-molecular weight phenolic compounds. However, only nonpolar compounds were active. The degree of stimulation of specific laccase ac­tivity by phenolics was generally higher in P strains: signifi­cant with pinosylvin, piceid, taxifolin, catechin, ferulic acid and cinnamic aldehyde. The oxidative capacity of culture filtrates of P strains, as determined with an oxygen electrode, was significantly higher than that of S strains with astringin, isorhapontin, taxifolin, catechin and proanthocyanidines, and with water-soluble extractives of Scots pine wood and bark. The pathological significance of these results in relation to the host preferences and the differential aggressiveness of Sand P types is discussed.

Key words: polar/nonpolar phenolics - stilbenes - flavanoids - phenylpropanoids - conifer bark and wood extractives

Corresponding author: M. Johansson

Introduction

Extracellular laccase (EC 1.10.3.2; benzenediol: oxy­gen oxidoreductase (Webb 1984» produced by white­rot fungi is generally considered to be active in the bio­degradation of lignin (Eriksson et al. 1990; Thurston 1994), a major cell wall component that may block pathogen penetration at sites of infection. In addition to lignin, a diverse array of phenolic compounds, including lignans, phenylpropanoids, stilbenes, flavanoids and proanthocyanidines, are constitutively present in living bark and woody tissues of trees or synthesized following wounding and fungal attack (Lyr 1962 a, b; Loman 1970; Hart 1981). Several of these aromatic compounds are, along with resins, presumed, to be important factors contributing to the resistance of coniferous trees to in­fection by Heterobasidion annosum (Fr.) Bref., the main cause of root and butt rot in conifers in the Northern Hemisphere (Hodges 1969). The oxidizing enzyme lac­case produced by Heterobasidion has been shown to play a role in detoxifying host defence substances (Popoff et al. 1975; Haars and Htittermann 1980). Therefore, the ability of Heterobasidion species to det­oxify such compounds could be an important compo­nent of the mechanisms of pathogenicity.

Heterobasidion annosum (Fr.) Bref., investigated in this study, consists of two intersterility groups (IGs), S and P, with somewhat different host preferences (Kor­honen 1978; Stenlid and Swedjemark 1988). The S-type is confined to Norway and Sitka spruces and seedlings of Scots pine, whereas the P-type is able to attack, decay and kill spruce and pine species of all ages, as well as other conifer and broadleaf tree species (Korhonen

Microbiol. Res. 153 (1998) 1 71

1978; Piri et al. 1991; Swedjemark and Stenlid 1995). One reason for this difference in specificity may be a differential ability of the fungi to detoxify constitutive and induced phenolic compounds in bark, cambium and wood (Johansson, Lundgren and Asiegbu, unpubl.). Dif­ferences found in pectic enzymes and pectic isozyme patterns between Sand P groups (Johansson 1988; Karlsson and Stenlid 1991) are also of pathogenic sig­nificance.

The aims of the present study were to assess the in­fluence of bark- and wood-related phenolic compounds on laccase production by Sand P strains of H. annosum and to determine whether differences in the induction and/or substrate specificity of laccases could be of im­portance in explaining host preferences.

Materials and methods

Fungal strains. In total, about 20 strains of each inter­sterility group (S and P) of Heterobasidion annosum (Fr.) Bref. were used in this investigation. They were provided by Dr. Jan Stenlid, Uppsala, Sweden, and Dr. Kari Korhonen, Vantaa, Finland, who isolated the strains and determined them to IG. Isolates of the P type ori­ginated from Pinus sylvestris L., Picea abies (L.) Karst.,

Juniperus communis L., Pinus nigra Arnold, and Betula sp. S strains were isolated from Picea abies and Pinus sylvestris. The fungal cultures were maintained on Hagem agar (Stenlid 1985).

Media for laccase production. Laccase production by H. annosum IGs was studied using the following media and cultivation methods:

- Norkrans' defined glucose-asparagine-mineralliquid medium (NM) (Norkrans 1963)

- NM + phenolic compounds, dissolved in ethanol - NM + 1% agar - NM + 1 % agar + ethanol-dissolved phenolic com-

pounds.

In liquid cultures, 25 ml of the medium was added to 100-ml Erlenmeyer flasks, autoclaved, and inoculated with pieces of mycelium. The cultures were incubated stationarily at 22°e. Phenolic compounds, dissolved in 70% ethanol, were added to the autoclaved medium, while controls received the same amount of ethanol but without any dissolved compounds. Agar plates were in­oculated with conidial suspensions of the strains. After 4 days of incubation, 3-mm-diam wells were made in the agar and filled with 5 llmoles of the phenolic in 1511L 70% ethanol, whereupon the cultures were incubated for another 7 days.

Table 1. Phenolic compounds used as stimulators of laccase activity and as substrates for oxidizing enzymes.

Compounds

Benzoic acids, benzaldehydes

Salicylic acid (o-hydroxy-benzoic acid) p-Hydroxybenzoic acid p-Hydroxybenzaldehyde Vanillic acid

Phenylpropanoids

Cinnamic alcohol Cinnamic aldehyde 3-Phenyl-l-propanol Coniferin (Dihydroconiferin) p-Coumaric acid Ferulic acid ChI orogenic acid

Stilbenes

Pinosylvin Piceid Astringin Isorhapontin

Flavanoids and procyanidines

Catechin Taxifolin Procyanidines Bland B3

72 Microbiol. Res. 153 (1998)

Presence

Low conc. in plants

Bark

Conifer cambium Pine bark Spruce bark Most plants Most plants

Pine heartwood Spruce bark Spruce bark Spruce bark

Pine and spruce bark Pine and spruce bark Pine and spruce bark

References

Harborne and Simmonds (1974)

Pan and Lundgren (1995)

Erdtman (1939) Pan and Lundgren (1995)

Pan and Lundgren (1995, 1996)

Aromatic compounds, bark and wood extractives. Phe­nolic compounds occurring in conifer bark and wood, and some other structurally related phenolic compounds were selected with the aim of ensuring that most of the common phenolic structural types were represented (Table 1). In addition, Scots pine stem bark and sap­wood, including both fresh samples and samples inocu­lated with H. annosum (P) 7 days earlier, were extracted with 96% ethanol in an Ultra Turrax. After evaporation, water- and petroleum ether-soluble compounds were separated, whereupon the solvent was evaporated, and the extractives were weighed. Only the water-soluble part (mainly phenolics) was retained for analysis.

Sampling and estimation of laccase and total oxidizing activity. From the liquid cultures, samples were asepti­cally taken after various periods of incubation. Guaiacol (2-methoxyphenol) was used as substrate for laccase (Mayer 1987; Thurston 1994; Dean and Eriksson 1994). After centrifugation (15 min at 5°C and 10000g, rav 10 cm), 0.5 ml of the cell-free supernatant (culture filtrate) was added to 2 ml of 0.016 M guaiacol in 0.05 M citrate-phosphate buffer, pH 4.8 (CP) and in­cubated at 22°C. Laccase activity was estimated as dA480nm min-I, ml-I culture filtrate or dA480nm min-I, mg-I protein (specific activity) or dA480nm min-I, mg-1

dry mycelium. Protein content was estimated according to Bradford (1976). From cultures on solid media, agar samples were taken in the mycelial front around the wells 7 days after phenolics had been added. The samples were extracted in equivalent volumes of CP for 24 h at 4°C, followed by centrifugation (10 min at 10 000 g, rav 10 cm). We used one part of the extract for measuring protein content and another part for measuring laccase activity, estimated and expressed as above. The oxidizing activities of Sand P culture fil­trates (NM) were estimated with various phenolic compounds as substrates, using an oxygen electrode (PHA934 P02 Module, PHM72 Mk2, Radiometer, Co­penhagen). The cellfree culture filtrates were dialyzed (M w cutoff 12 -14 kD) against distilled water for 24 h in the cold. Using guaiacol as substrate, oxygen consump­tion (dp02 min-I) by the Sand P enzyme preparations was adjusted to be similar. The phenolic compounds were dissolved in 70% ethanol, and 0.1 ml was added to 1.4 ml CP and 0.5 ml dialysed culture filtrate, giving a final phenolic concentration of about 5 mM. The linear change in p02 was followed for 5-10min at 22°C and estimated as dp02 min-I at maximum rate. The values were estimated as % of those with 5 mM guaiacol as substrate. Using the same method, the relative oxidizing capacities of Sand P culture filtrates on water-soluble extractives from infected and non-infected bark and sap­wood of stems of Scots pine (Pinus sylvestris L.) were estimated. The extractives were dissolved in the reaction

mixture to a concentration of 10% of that in bark and wood, respectively.

The identity of laccase produced by Sand P strains of H. annosum has been confirmed by comparing oxi­dation of guaiacol and syringaldazine (Johansson et al. in prep.)

Differences were analyzed statistically using Stu­dent's t-test.

Results

Effect of phenolic compounds on laccase activity

1. In liquid media. A preliminary test showed that cin­namic alcohol significantly increased the specific lac­case activity of P strains, compared with its effects on S strains. Addition of cinnamic alcohol, salicylic acid (Malamy and Kleissig 1992), pinosylvin and astringin in four concentrations (O.l, 0.3, l.0 and 5.0mM) to 7-day­old Sand P cultures often increased protein release sig­nificantly, mainly in P cultures (Fig. 1 A).

Ethanol caused a significant increase in specific laccase activity of S cultures, which was further en­hanced only with high concentrations of pinosylvin (Fig. 1 B). With exception for one concentration of pinosylvin (0.3 mM), P strains accomplished a higher specific laccase activity than S strains in the presence of cinnamic alcohol, salicylic acid and pinosylvin. Calculated as activityibiomass or ml-J filtrate the differences were still more significant (not shown). Astringin did not affect P laccase at any of the con­centrations tested, but it inhibited the release of S laccase (P<O.OI).

The higher laccase activity of the P strains was also observed in the cultures with NM + 1 mmole of catechin, isorhapontin or procyanidines (B 1 + B3), respectively (Table 2). 3-Phenyl-l-propanol, earlier observed to be a very potent laccase stimulator without being oxidized by the enzyme (Johansson, unpubl.), stimulated specific laccase activity significantly (P<O.OOl) and to similar levels in the two IGs, but did not cause an increase in protein release (Tables 2 and 3).

Pectin, added to NM together with phenolic com­pounds, did not further increase laccase activity (not shown).

2. Agar media. In agar media, totally 17 compounds were tested (Table 3). After 10 days of incubation with phenolics, the width of inhibition zones, with no growth, around the wells was 2-30 mm with both the Sand P strains. As the incubation continued, the inhibition zones were successively overgrown. P-strains accounted for most of the growth in these zones, indicating that they were more tolerant or more able to oxidize/detoxify the compounds. This was particularly the case with

Microbial. Res. 153 (1998) 1 73

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74 Microbial. Res. 153 (1998)

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Table 2. Protein release and laccase activity in NM liquid medium, with I mM concentration of various phenolic compounds. Results after 11 days' incubation with Sand P strains H. annosum.

Compound SIP Protein, Laccase activity Ilg ml- I

A B

Control S 4.5 ± 1.2 6.8 ± 1.7 0.03 P 7.5 ± 1.9 59.7 ± 15.9** 0.45***

Catechin S 8.9 ± 2.1 4.0 ± 1.1 0.04 P 11.6 ± 2.0 31.3±11.2* 0.36***

Isorhapontin S 26±4 1.2 ± 0.5 0.03 P 42± 8 2.2 ± 1.0 0.09**

Pinosylvin S 30±5 2.5 ± 1.5 0.08 P 45 ±4 6.1±1.9 0.27***

B 1 + B 3 (proanthocyanidines) S 18.3 ± 3.2 7.8 ± 1.0 0.l4 P 22.3 ± 4.1 40.8 ± 8.1 ** 0.91 ***

3-Phenyl-l-propanol S 3.2 ± 1.1 199 ± 56 0.65 P 4.6± 1.4 151 ± 52 0.69

Protein release: Ilg ml-' culture filtrate. Laccase activity: A: dA480nm mg-I protein, min-I; B: dA480nm ml- ' culture filtrate, min-I Results are the mean ± S. D. of two independent experiments, each including 5 Sand 5 P strains; three replicates. Difference between Sand P strains in laccase activity: t-test: *p <0.05; **p <0.01; ***p<O.OOI.

p-hydroxybenzaldehyde (P < 0.01), cinnamic alcohol, chlorogenic acid, ferulic acid and pinosylvin.

After 11 days of incubation, the laccase activity and protein content of extracts from the agar in the mycelial front around the wells were as shown in Table 3. Some compounds stimulated protein release, and most of them stimulated laccase activity. The generally higher specific laccase activity of P strains was only signifi­cant with some compounds, but when calculated as activity ml- ' extract it was significantly higher with most of them, compared with S strains. It is interesting to note that astringin and isorhapontin (common stilbene glucosides in Norway spruce bark) had no effects, whereas pinosylvin did. The polarity of the compounds influences the laccase stimulation. The most polar phe­nolic glucosides and polyhydroxyphenols did not stimu­late laccase activity.

Oxidation of phenolic compounds and extractives from pine bark and wood in relation to the oxidation of guaiacol

Estimated as oxygen consumption, stilbenes, flavanoids and procyanidines, occurring in bark, were significantly more oxidized by dialyzed culture filtrates of P strains than by those of S strains, cultivated in phenol-free NM

(Table 4). This was also the case with chi orogenic acid and p-phenylene diamine. These compounds are polar, having o-dihydroxy groups (except p-phenylene dia­mine), whereas those compounds being oxidized to the same extent by both IGs are unpolar. However, S strains also oxidized these compounds, as well as ferulic acid, more rapidly than they oxidized guaiacol.

The comparison between amounts of extractives in fresh bark and sapwood of Scots pine and amounts in bark and sapwood that had been infected 7 days earlier with a P strain showed that infection leads to a rapid decrease in concentrations of water-soluble phenolics (Table 5). Using dialyzed culture filtrates (NM) from S and P strains, it was found that the oxidation rates of ex­tractives from noninfected and infected bark and wood were significantly higher in P strains compared with S strains. This was particularly valid for bark extractives (Table 6).

Discussion

The results of both the present and earlier studies (Jo­hansson 1988; Karlsson and Stenlid 1991) indicate that the Sand PIGs of Heterobasidion annosum differ in

Fig. I.A. Effect of a series of concentrations (0.1, 0.3, 1.0 and 5.0mM) of cinnamic alcohol, salicylic acid, pinosylvin and astringin on protein release (Ilg ml-1) in cultures of Sand P strains of H. annosum in Norkrans' defined medium (NM). Phenolic compounds added after 4 d incubation and sampling after another 7 d. The results are the mean of two independent experiments, each including 5 Sand 5 P strains; three replicates. Significance of difference between Sand P: * P<0.05; ** P<O.Ol, *** P < 0.001. S strains: blank 0, P strains: block /§8, C: control, Et: ethanol control. Fig. lB. Laccase activity (dA480nm mg-I protein, min-I) in cultures of Sand P strains according to A.

Microbiol. Res. 153 (1998) 75

Table 3. Protein release and specific laccase activity in NM agar cultures of Sand P strains of H. annosum 7 days after addi-tion of 5 /l moles of aromatic compounds to 3 mm-diam. wells in the agar 4 days after inoculation with conidia

Compound SIP Protein, Laccase activity /lg ml-I

A B

Control S 4.9±0.6 4.2 ± 0.7 0.02 P 8.9 ± l.3* 5.8 ± l.2 0.05

p-Hydroxybenza1dehyde S 5.4 ± 1.2 48 ± 6.2 0.26 P 11 ± 1.7* 73 ± 5.9* 0.80**

p-Hydroxybensoic acid S 6.8 ± 1.5 59 ± 18 0.40 P 12 ± 2.2 96±24 1.15*

Vanillic acid S 5.2 ± 1.5 17 ± 3.8 0.09 P 8.2 ± 2.0 19 ± 4.2 0.l6

Cinnamic alcohol S 4.4 ± 1.3 89 ± 12 0.39 P 10 ± 2.4* 116 ± 22 1.16

Cinnamic aldehyde S 5.l ± 1.8 38 ± 11 0.19 P 13±3.l* 100 ± 16* l.30**

3-Pheny1-1-propano1 S 3.7 ± 1.1 67 ± 12 0.25 P 5.6 ± 1.6 98 ± 18 0.58*

Ferulic acid S 7.1 ± 2.0 19 ± 3.3 0.14 P 18 ± 2.3* 102 ± 9*** 1.84***

Chi orogenic acid S 7.1±1.8 16±4 0.11 P 13.l ± 3.3* 28 ± 8 0.37***

Pinosylvin S 24.2 ± 5.3 29±4 0.70 P 36.8 ± 7.0 81 ± 12** 3.00***

Piceid S 4.2± 0.6 6.0 ± 0.5 0.03 P 7.7 ± 1.3 17.3 ± 2.4** 0.13**

Astringin S 4.9± 0.7 6.l ± 1.3 0.03 P 5.7 ± 0.6 7.8 ± 1.7 0.04

Isorhapontin S 5.4 ± 0.6 4.6 ± 1.0 0.02 P 7.5 ± 1.1 7.0 ± l.5 0.05

Catechin S 4.l ± 0.4 4.3 ± 0.2 0.02 P 6.0 ± 0.4* 17.5 ± 0.6*** 0.l1 ***

Gallocatechin S 3.3 ± 0.3 6.1 ±0.4 0.02 P 4.7 ± 0.6 11.1 ± 3.3 0.05

B 1 + B 3 (proanthocyanidines) S 3.5 ± 0.3 5.4 ± 0.7 0.02 P 5.6 ± 1.0 8.5 ± 2.2 0.05

Taxifolin S 9.0 ± 1.3 2.3 ± 0.7 0.07 P 10.5 ± l.6 7.5 ± 1.5*** 0.07**

Samples were taken in the mycelial front around the wells and extracted in equivalent amounts of CP (w/v). Protein release: /lg ml-I. Laccase activity: A: dA480nm mg-I protein, min-I; B: dA480nm ml-I culture filtrate, min- i , Results are the mean ± S. D. of two independent experiments, each including 5 Sand 5 P isolates; three replicates. Difference in protein release and laccase activity between Sand P strains: t-test: *p < 0.05; **p < 0.01 ; ***p < 0.001.

several aspects of protein synthesis, here documented as total amounts of proteins released and their oxidizing capacity. The hypothesis tested was that the documented differences between the lOs in aggressiveness and host specificity (Korhonen 1978; Stenlid and Swedjemark 1988; Swedjemark and Stenlid 1995) are partly due to differences in their enzymatic response to phenolic compounds in bark and/or wood. Similar comparative studies concerning pectolytic enzymes showed that the lOs differed significantly in terms of both enzyme ac-

76 Microbiol. Res. 153 (1998) 1

tivities and isozyme patterns (Johansson 1988; Karlsson and Stenlid 1991). Earlier investigations on the laccase of H. annosum only included one strain, which, although it was not identified, most probably belonged to the S group (Haars et al. 1981, 1983; Haars and Htittermann 1983). The present study is the first in which lOs have been compared regarding laccase activity. The results confirm earlier findings that the extent of laccase ac­tivity is strongly dependent on stimulatinglinducing agents and may involve the combined effects of carbo-

Table 4. Oxidative capacity of dialyzed culture filtrates (from NM) of Sand P strains of H. annosum with bark and wood phe­nolics as substrates.

Compound S strains P strains t-test

p-Hydroxybensoic acid 8±3 4±1 n.s. Vanillic acid 36±7 38 ± 5 n.s. 3-phenyl-l-propanol 65 ± 18 86 ± 19 n.s. Coniferin 15 ± 4 13±3 n.s. p-Coumaric acid 14± 9 21 ± 10 n.s. Ferulic acid 134 ± 15 lOCh 11 n.s. Pinosylvin 66± 8 28 ± 12 Astringin 120± 9 262 ± 71 P<0.05 Chi orogenic acid 129 ± 17 197 ± 10 P<0.02 Isorhapontin 140± 16 222±44 P<0.05 Taxifolin 132 ± 25 244± 40 P<0.02 Proanthocyanidines llO± 9 252 ± 15 P<O.OOI Catechin 113 ± 12 271 ±7 P<O.OOI p-Phenylene diamine 97 ± 12 197 ± 10 P<0.02

Assay: 1 ml filtrate + 0.5 ml CP buffer (pH 5), 0.1 ml 70% ethanol, containing 3 mg phenolic compound. Oxygen consumption measured as dp02 min-I at 22°C in % of that obtained with 5 mM guaiacol as substrate. Results are the mean of two indepen­dent experiments, including a total of II Sand 11 P strains; three replicates. Difference between Sand P strains: t-test.

Table S. Concentrations of extractives, obtained from uninfec­ted Scots pine bark and sapwood and corresponding samples that had been infected with a P strain of H. annosum seven days earlier.

Extract from

Non-infected wood Water soluble Petr. ether soluble

Infected wood Water soluble Pet.-ether soluble

Non-infected bark, total

Infected bark, total

mg g-l fresh material

3.33 8.10

2.04 3l.2

142

100

hydrates and phenols (Fahraeus and Lindeberg 1953; Fahraeus 1952; 1954; Grabbe et al. 1968; Johansson et al. 1976; Leonowicz and Trojanowski 1975; Wood 1980; Haars et at. 1981; 1983; Haars and Htittermann 1983). Marbach etal. (1985) were able to enhance lac­case activity by adding pectin to a phenol-containing medium. This was not the case in our trials (not shown),

even though P strains are known to degrade pectin to oligomers (Johansson 1988) which may stimulate lac­case activity.

Results of additional studies by us indicate that under most conditions P strains grow more slowly than S strains (Johansson et al. in prep.). In spite ofthat the lac­case activity of the P strains almost always exceeds that of the S strains in a given substrate irrespective of the type of additive. The often strong induction of laccase activity - even by very low amounts of various phenolic

compounds (less than 1 % of those used by Haars et at. (1981)) - may be caused by de novo synthesis of the en­zyme, as shown by Haars and Hiittermann (1983). Also, the P strains seemed to be more sensitive than S strains to such induction. Our finding that intracellular laccase was not influenced by stimulating agents (not shown) is in agreement with the results of these investigators. In terms of specific laccase activity, the difference between the IGs was somewhat less because of the generally higher protein release of the P strains. Furthermore, the production and release of proteins were significantly stimulated by phenolic compounds. In P strains, even

Table 6. Oxidation of water-soluble extractives (cf. Table 5) of non-infected Scots pine bark and wood and corresponding samples infected with H. annosum 7 days earlier. Enzyme source: dialyzed culture filtrates (NM) from Sand P strains of H. annosum.

Strains

S-strains P-strains PIS

Control bark

67 ± 9 237 ± 31 3.5 (P<O.OOI)

Inf. bark

73 ± 19 205 ± 17 2.8 (P < 0.00 I)

Control wood

36±5 63 ±6 1.8 (P<O.Ol)

lnf. wood

30±3 55 ± 7 1.8 (P < 0.01)

Oxygen consumption measured as dp02 min-1 at 22°C in % of that obtained with 5 mM guaiacol as substrate. Results are the mean ± S. D. of three independent experiments, each including 5 Sand 5 P strains; two replicates. Difference between Sand P strains: t -test.

Microbiol. Res. 153 (1998) 1 77

micromolar concentrations increased protein release, whereas S strains were less sensitive. It might be worth­while to study the function of proteins other than lacca­ses with the aim of explaining the wider host range (Korhonen 1978) and stronger wood-decaying proper­ties of P strains (Daniel et al. in press). Both infection and decomposition processes are dependent on other enzymes (Kirk and Farrel 1987; Eriksson et ai. 1990; Evans et al. 1991). However, reports of the differential production of enzymes other than laccase and pectinases implicated in wood decay by Sand P strains are lacking. The stronger phenol oxidizing capacity of P strains, in combination with cellobiose quinone oxidoreductase, may be important for their higher cellulose decaying activity (Muller et al. 1988)

The chemistry of bark and wood phenolics of Norway spruce and Scots pine is rather well known (Pan and Lundgren 1955, 1996). However, the changes occurring successively - or very rapidly - after wounding and infection have yet to be thoroughly investigated (Shain 1971; Shain and Hillis 1971; Popoff etai. 1975; Jo­hansson et ai. 1976; Lindberg and Johansson 1991; Lindberg et al. 1992). We obtained significant differ­ences between clones of Norway spruce in the concen­tration of astringin, taxifolin, piceid and isorhapontin (Lundgren and Johansson, unpublished). Hart (1981) reviewed the role of phytostilbenes in decay and di­sease resistance. Stilbenes are produced in the sapwood under stressful conditions. Infection with H. annosum induces pinosylvin and pinosylvinmonomethyl ether very rapidly in the sapwood of Scots pine (Johansson et al. unpubl.). The stimulation of laccase activity by low-molecular aromatic compounds and by bark and wood phenolics (stilbenes, etc.), which is generally more pronounced for P strains, may reflect the induction of certain isozymes specialized for degrading the re­spective compounds.

However, using dialyzed culture filtrates from de­fined substrates as an enzyme source (without added laccase-stimulating phenolics), several bark and wood phenolics were oxidized to a greater extent than guaia­col, and in these cases P strains were mostly superior. The superiority of P strains was also pronounced in terms oftheir ability to oxidize water-soluble extractives of bark and wood of Scots pine. This indicates a consti­tutive difference in laccase (oxidative) activity between the lOs. Although p-hydroxybensoic acid strongly sti­mulated laccase activity with 2,6-dimethoxyphenol as substrate (Haars et al. 1981), it was barely oxidized under our experimental conditions. Thus it would ap­pear that the induction/stimulation of laccase activity by a certain phenol is no guarantee that the compound is oxidized by that enzyme. Similarly, 3-phenyl-1-pro­panol, a strong inducer oflaccase in both Sand P strains, is, at most, only moderately oxidized by this enzyme

78 Microbial. Res. 153 (1998) 1

(Table 2-4 and Johansson unpubl.). Astringin did not stimulate laccase activity, but it was significantly oxid­ized by the "non-stimulated" enzyme source (NM). Al­though vanillic acid stimulated laccase activity, it was an inferior substrate for oxidative enzymes. The rapid sti­mulation of laccase activity by low-molecular weight phenols may, however, indicate adaptability to succes­sive chemical and structural changes during wood com­position. From Tables 2 and 3 is found that nonpolar phenolic compounds stimulate laccase activity, whereas polar compounds (glucosides and polyhydroxyphenols) have no effect. Even though laccases have a broad range of substrates, many of which have yet to be identified (Thurston 1994; Dean and Eriksson 1994), a culture filtrate may contain other enzymes, such as manganese peroxidase, able to interfere in the oxidative processes. The higher protein release and decaying ability of P strains may have some interesting correlations. In addi­tion to the role of laccases in lignin degradation (Eriks­son et ai. 1990) it is assumed that pathogens use them to polymerize and detoxify phenolic compounds formed and released during pathogenesis (Popoff et ai. 1975; Haars and Hiittermann 1980; 1983; Haars et ai. 1981; Johansson and Stenlid 1985; Bollag et al. 1988; Cwie­long and Hiittermann 1988; Cwielong et ai. 1994; Binz and Canevascini 1996). Johansson and Hagerby (1974) found that the addition of phenols blocked essential functions in metabolic pathways of H. annosum but stimulated laccase activity. The fact that laccases detoxify phenolic compounds indicates that they are es­sential in the detoxification process and for the survival of the fungus in the wood. Hart (1981) suggested that the detoxification process consists of several steps. Laccase activity may be mediated by the composition of isozymic forms which are successively induced by certain phenols (Haars et ai. 1981; 1983). The induction of various iso­zymes of 1accases, which is promoted by oxidized phe­nols (Haars et al. 1981), is probably an essential element in the survival of the fungus in necrotic bark/wood where changes in phenolic metabolism occur rapidly.

Acknowledgement

This work was supported by grants from the Swedish Council for Forestry and Agricultural Research (SJFR). We thank Dr. Jan Stenlid and Dr. Kari Korhonen for Sand P isolates of Heterobasidion annosum.

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