Eur.J. For. Path. 25 (1995) 169-178© 1995 Blackwell Wissenschafts-Verl.ig, BerlinISSN 0300-1237

Department oj Forest Mycology and Pathology and Department of Forest Products,

Swedish University of Agricultural Sciences, Uppsala, Sweden

Immunocytochemical localization of pathogenesis-relatedproteins in roots of Norway spruce infected with

Heterobasidion annosum

By F. O. ASIEGBU, M. DENEKAMP, G. DANIEL and M. JOHANSSON

Summary

The oecurrenee and .lecumulation of /J-1, 3-glucanase and chitinase in seedling roots of spruce {Piceaahies) following challenge by the root-rot pathogen Heterobasidion annosum were studied. Chitinaseactivity inereased 2-3 fold following inoculation, whereas no signifieant inerease in the activity levelsof glucanasc was recorded during infection. With TEM immunogold labelling, the enzymes werelocalized in protein aggregates in host tissues and in the cell walls of intercellular hyphae. Gold particleswere sparse and irregularly distributed within host-cell walls. Only minor labelling was observed onhyphaf walls coated with electron-dense materials. The labelling intensity increased with infeetion timeand was always higher than in non-infected seedling roots. Wlien this experiment was repeated usingroot samples inoeulated with the saprophyte Phlebiopsis gigantea, a similar labelling pattern wasobsei-ved. The cross reactivity of antisera raised .against sugar-beet ehitinase and glucanase with spruce-root enzyme extracts was demonstrated using dot-blot assays and ELISA.

1 Introduction

Plants respond to infection or abiotic stress by secretion of several acid-soluble proteins.These hydrolytic enzymes, termed pathogenesis-related (PR) proteins, have been widelyimplicated in plant defence reactions and arc mainly located in intercellular spaces (ABELESet al. 1971; WARGO 1975; VAN LoON 1985; RiGDENS and COUETS 1988; CARR and KEESSIG1989; BOL et al. 1990; LlNTHORST 1991). FlNK et al. (1988) argued that, since PR proteinsare predominantly located in the vacuolar space, their intervention in pathogenesis couldnave occurred too late to resist fungal invasion.

Of all PR proteins secreted by photosynthetic plants, only chitinases and glucanase,which possess a hydrolytic ability against the fungal cell wall, have been fully characterized(KAUEFMAN et al. 1987; LEGRAND et al. 1987; KOMBRINK et al. 1988; SIMMONS 1994).Previous studies (WARGO 1975; SoCK et al. 1990) have noted that, since some fungalplant pathogens contain chititi and /i-1,3 glucans, enzymes which affect these cell-wallcomponents may be necessary during active disease-resistance reactions of plants againstinfection.

Angiosperms, unlike gymnosperms, have been thoroughly stucfied in this respect. Interms of their evolution, they are thought to he quite distinct groups and are considered tohave separated from each other several hundred million years ago (JANSSON and Gus-TAFSSON 1990). In a recent paper, SHARMA et al. (1993) reported that, in spite of theirseparation, PR proteins secreted by gymnosperms (spruce) are similar to those describedfor angiosperm plants. They recorded eight different isoforms of chitinase and two isozymesof [i-l, 3 glucanase in roots of spruce seedlings challenged in vitro by Pythium species.

U. S. Copyright Clearance Center Code Statement: 0300-^1237/95/2503-0169 $11.00/0

170 F. O. Asiegbu, M. Denekamp, G. Daniel and M. ]ohansson

However, they concluded that the secretion of these two PR proteins by spruce roots is anon-specific response, as Pythium sp. is not known to contain chitin. In a related paper,NSOEOMO and WOODWARD (1994) demonstrated a significant increase in chitinase but not/i-l,3-glucanase levels in three pine species infected with Heterobasidion annosum, thussupporting earlier results with Norway spruce (AsiEGBU et al. 1993; DENEKAMP et al.1994). Earlier studies with angiosperms have shown increased anti-chitinase and anti-glucanase labelling of cell walls of a compatible Fusarium strain during infection of tomatoplants (WUBBEN et al. 1992). However, virtually nothing is known about the cellularlocation of these antifungal enzymes in gymnosperm systems, particularly conifers.

The aims of this paper were: 1. To study the occurrence and sites of accumulation of anti-fungal hydrolases in Norway-spruce roots following fungal infection; and 2. To assess thedifference, if any, in the accumulation or labelling patterns of such antifungal productssecreted during challenge by either the saprophyte Phlebiopsis gigantea (Fr.: Fr.) Julich orthe parasite Heterobasidion annosum (Fr.) Bref.

2 Materials and methods

2.1 Fungi

H. annosum S-type, (FaS6) and P. gigantea were maintained on Hagem-agar medium(STENLID 1985). Conidiospores from H. annosum were recovered from cultures grown for3-4 weeks in the dark. After several washes with sterile distilled water, conidiospores weresuspended in 0.5% w/v malt extract and left to germinate overnight at room temperature.Following further washing, the spores were resuspended in sterile distilled water (10''germinating spores per ml) and used for inoculation. Oidiospores of P. gigantea were usedwithout pre-germination.

2.2 Plant material and inoculation

Spruce seeds were surface sterilized with 30% H2O2 for 15 min, sown in sterile 1% w/vwater agar, and left to germinate at 22°C. Sprouting seedhngs (6-11 days old) were usedfor inoculation experiments. Inoculation conditions were the same as those used by AsiEGBUet al. (1993), except for P. gigantea, where ungerminated oidiospores (10'" spores/ml) wereused to inoculate the seedling roots.

2.3 Polyclonal antibodies

Antibodies raised against sugar-beet chitinase and glucanase were obtained from DaniscoLaboratories, Copenhagen, Denmark.

2.4 Dot blotting and ELISA

The method used for dot-blot assays was as previously described by AsiEGBU et al. (1994).Enzyme-linked immunosorbent assay (ELISA) was used for demonstration of cross reac-tivity of the antibodies with spruce-root extracts, using the procedure illustrated by DANIEEand NILSSON (1991).

Pathogenesis-related proteins in Norway-spruce roots 171

2.5 Tissue preparation for light microscopy (LM) and transmission-electronmicroscopy (TEM)

Samples were harvested at defined time intervals, fixed in glutaraldehyde, and embedded asdescribed by AsiEGBU et al. (1993, 1994) in Technovit (Kulter, Cermany; for LM) orLondon Resin (Hampshire, UK; for TEM).

2.6 Immunocytochemical labelling

The procedure adopted was the same as that described by AsiEGBU et al. (1994), except thatultrathin sections were neither pretreated with hydrogen peroxide nor incubated in O.IMglycine. Briefly, four immunocytochemical-labelling sectors were incubated, in succession,in phosphate-buffered saline (PBS), in normal goat serum, and in antichitinase or anti-glucanase, then labelled with gold antirabbit IgG conjugate and stained with uranyl acetate.The labelling specificity was demonstrated by the following controls: 1. Incubation withrabbit pre-immune sera; and 2. Incubation of sections by omitting the primary-antibodystep.

2.7 Estimation of chitinase and /i-l,3-glucanase

The procedures used for chitinase and /i-l,3-glucanase determination in root-tissue extractsand culture filtrates were the same as those described by AsiEGBU et al. (1993) andDENEKAMP et al. (1994), respectively.

3 Results

3.1 Effect of infection on chitinase and /i-l,3-glucanase secretion

Protein extracts from fine roots of spruce exhibited both glucanase and chitinase activitythroughout development. The activity of chitinase enzymes increased 2 days after inocu-lation with H. annosum spores. For at least 3 further days, the activity remained at a higherlevel in infected roots than in the control roots (Fig. 1). To find out if the response was age

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• — ControlInfected

10 12

Period after inoculation (days)

Fig. 1. Chitinase activity of Norway-spruce fine roots following infection by Heterobasidion annosum.Values arc the average of three replicates

172 F. O. Asiegbu, M. Denekamp, G. Daniel and M. Johansson

dependent, the effect of inoculation on chitinase activity when inoculation occurred at alater developmental stage (27-day-old roots) was determined. The results indicated that theolder roots react in a similar way to the 7-day-old roots (data not shown).

In contrast to chitinase produetion, no statistically significant increase in the /i-1,3-glucanase activities of infected seedling roots was recorded (data not shown).

3.2 Imniuno-dot assay

The cross reactivity of polyclonal antibodies raised against sugar-beet ehitinase and glu-canase by extracts from seedling roots and fungi was demonstrated using lmmuno-dotassays. The polyclonal antibodies cross-reacted positively with extracts from spruce roots(data not shown), hut no detectable cross reactivity of anti-chitmase/anti-glucanase withthe culture filtrates (20-100 /(g protein/ml) of these fungi was observed. By contrast, a weakcross-reaction was observed between anti-glucanase and hyphal extracts of H. annosum(data not shown). The cross reactivity of polyclonal antibody with spruce-root extracts wasfurther confirmed using ELISA (data not shown).

3.3 Immuno-gold labelling of non-inoculated roots

Only minor labelling was recorded in root tissues of non-infected seedlings. Such labellingwas sometimes associated with the membrane layer (plasmalemma) of host-cell walls.Usually, the gold particles were irregularly distributed and did not follow any consistentdefined pattern (Fig. 2a).

3.4 Immuno-gold labelling of infeeted roots

3.4.1 ji-1,3-glucanase

Only very scanty labelling of root tissues was recorded 24-48 h post inoculation (p.i).During this period, no hyphal material was detected within host tissues. By 3-5 days p.i,hyphal materials were observed in host cells, which coincided with the increased numberof gold particles reeorded.

Strong labelling was recorded at 7-25 days p.i. on hyphal cell walls (Fig. 2b). No labellingwas observed on electron-dense deposits encasing hyphae. Similarly, there were fewer goldparticles on hyphal cell walls coated with electron-dense materials (Fig. 2c). Inereasedlabelling of cell walls of some severely degraded hyphae (7 days p.i.; Fig. 2d) was apparent,in contrast to those of relatively new invading hyphae (3 days p.i.; Fig. 3a). The membranelayer of host cell walls in contact with invading hyphae was equally labelled (Fig. 3b). H.annosum organelles were labelled (Fig. 3a), and, similarly, protein aggregates inside infeetedhost cells were strongly labelled (Fig. 3c).

When this experiment was repeated using seedling roots inoeulated with the saprophyteP. gigantea, a similar labelling pattern was observed. The major difference was in thefrequency of gold particles, which was higher in H. annosum infected roots than thoseinfected by Phlebiopsis (data not shown). As in H. annosum, P. gigantea hyphal materialswere also labelled.

3.4.2 Chitinases

At 7-25 days p.i., increased labelling of protein aggregates withm infected roots wasobserved, as was labelling of hyphal walls and tips (Fig. 3d). Electron-dense materialssurrounding invading hyphae were not labelled (Fig. 4a), but minor labelling was recorded

Pathogenesis-related proteins in Norway-spruce roots 173

Fig. 2. Localization of glucanasc in Norway-spruce seedling roots infected with H. annosum. a. Cell-wall (CW) regions of non-inoculated root was almost unlabelled (arrow), as were the intercellularspaces of the middle lamellar eell-eorner region (MR); b. Strong labelling (white arrows) of cell wallsof invading hyphae. Host-cell wall (star) was not labelled, c. labelling was nearly absent on hyphal (H)materials surrounded by electron-dense deposits (arrow). The electron-dense bodies were not labelled.Some gold particles were observed on host-cell-regions (CW); d. Strong labelling on cell walls (triangles)

of an agemg or degraded hypha (H) at later stages of infection. Bars: a, d = 0.1 /ini; b, c = 0.5 /(m

on hyphal materials surrounded by such opaque deposits. No labelling was observed ontranslucent areas ahead of invading hyphae, nor on fungal organelles (data not shown).Gold particles were recorded within the intercellular air spaces of middle lamellar cell-corner regions of host tissues (Fig. 4b). Host-cell-wall regions in direct contact with invadinghyphae were labelled. Similar observations were made for sectiotis from roots infected withP. gigantea (Fig 4c, d).

3.5 Imniunocytochemical controls

No labelling was observed in sections incubated with pre-immune sera, or when the primaryantibody step was omitted (data not shown). It was found that background labelling could

174 F. O. Asieghu, M. Denekamp, G. Daniel and M. Johansson

H

Fig. 3. Localization of glucanase (a-c) and chitinase (d) in Norway-spruce seedling roots infected withH. annosum. a. Sparse labelling of hyphal walls of a relatively young hypha (H). Cellular organells(endoplasmic reticulum; triangles) of the fungus were labelled. Host-cell wall (cw) was almost free oflabelling; b. Inner-membrane layer (arrow) of host-cell wall (star) in contact with invading hyphae (H)was labelled; c. Protein aggregates (arrows) within the vacuolar spaces of host tissues \vere labelled; d.Scanty labelling of hyphal walls (triangles). Some labelling of protein aggregates and hyphal (H) material

within intercellular spaces (star) of host wall with anti-chitinase. Bars (a-d) = 0.5 /im

be minimized by adopting a prolonged blocking time, using a primary antibody titre of1:500 and an incubation time of 3-5 h.

4 Discussion

As an experimental model, the infection trials were carried out using seedlings grown underuniform sterile eonditions. Although a more practical approach would have been to use theroot systems of standing conifers, the infection conditions were developed to optimize thereproducibility of host responses and minimize exogenous-stress influences. The PR pro-teins (chitinase and /?-l, 3-glucanase) investigated in this study are known to contribute tothe partial protection of forest trees from fungal pathogens (WARGO 1975). However, therelative importance of each in the defence mechanisms of plants remains largely unresolved(SIMMONS 1994). Similarly, other authors have questioned the specificity of chitinases inplant-defence reactions (WYATT et al. 1991; SHARMA et al. 1993), since sorne stress-relatedtreatments are known to increase secretion of these enzymes.

Pathogenesis-related proteins in Norway-spruee roots 175

Fig. 4. Localization of chitinase in spruce-seedling roots inoculated with H. annosum (a,b) or P. gigantea(c,d). a. Number of gold particles was greater on hyphal tip (TP) but no lahelling was observed onelectron-dense materials (arrow) encircling hyphae (H); b. Labelling of protein aggregates (arrows)within the intereellular spaces of the middle lamellar cell-corner regions (star); c. Lahelling of hyphal(H) tips (arrow) and host-membrane layer (triangles) at sites of intercellular penetration; d. Labellingof protein aggregates (triangles) within host tissues. Host-cell wall (CW) was not labelled. Bars: a,

b = 0.5 /(m; c, d = 0.1 /im

In this study, increases in extracellular chitinase activity were observed in the seedlingroots of spruce infeeted with the pathogen. This suggests that the enzyme might be involvedin the host-parasite interactions of conifer roots, as previously reported for most angio-sperms tested (MAUCH et al. 1988). An increase in the chitinase activity of the rootsfollowing challenge with boiled hyphal extracts had also been observed (F. O. AsiEGBU etal. unpubl. data). NSOLOMO and WOODWARD (1994) implicated chitinase in the resistancereactions of pine species to infection, and SAUTER and HAGER (1989) in mutualistic(mycorrhiza) interactions.

The spectrophotometric assays of protein extracts in this study revealed no increase in /?-1, 3-glucanase activities following infection of spruce and pine seedlings with H. annosum. A

176 F. O. Asiegbu, M. Denekamp, G. Daniel and M. Johansson

similar observation was made by NsOLOMO and WOODWARD (1994) using Pinus sylvestris.Those authors demonstrated increased levels of chitinase in pine embryos but no significantincreases in glucanase activity. These spectrophotometric results contrast with previousreports emphasizing a correlation between /f-l,3-glueanase activity and some degree ofdefence in many angiosperm crop species against fungal pathogens. However, at the eellularlevel, increased labelling of hyphal walls within infected roots using antiglucanase antibodywas recorded in this study. This probably indicates that specific isozymic forms of thisenzyme could be involved in the initial interaction of spruce roots with the pathogen. Thisexplanation is supported by the work of SHARMA et al. (1993) on Norway spruce, followinginfection with Pythium. The significantly low levels of this enzyme detected under theexperimental conditions in this study could imply that the enzyme had formed complexeswith polyphenols (A. LONNEBORG pers. comm.).

Furthermore, the precise role of /?-l, 3-glucanase in host-pathogen interactions is furthercomplicated by the ability of some fungal pathogens to secrete the enzyme. In an earlierstudy from this laboratory, no glucanase activity was detected in extracellular culturefiltrates of H. annosum (M. DENEKAMP, unpubl. data). However, in this study, a weakcross reactivity between the hyphal extracts and antibodies against sugar-beet glucanase wasnoted. This may indicate an intracellular presence of the enzyme in the hyphae. The minorlabelling of the enzyme in the endoplasmic reticulum (Fig. 3a) of this fungus further confirmsthat this organism may be expressing intracellularly bound glucanases. On the other hand,WYATT et al. (1991) noted that /i-1, 3-glucanase and chitinase can act synergistically, andthis was considered essential for their optimal funetion in inhibiting growth of fungi thatcontain chitin (MAUCH et al. 1988; VOGELEI et al. 1988).

Chitinase and /i-1, 3-glucanase of spruce-root extracts were deteeted on immuno-dotsusing polyclonal antibodies raised against sugar-beet chitinase and glucanase. This suggestsa possible serological relationship between angiospertn (sugar beet) glucanase and chitinaseand those of gymnosperms (spruce). The absence of labelling observed for al! cytochemicalcontrols confirms the specificity of the antisera. The higher labelling of infected tissues ascompared to non-infected cells perhaps indicates a role of the enzyme in host-pathogeninteractions. However, the scanty labelling of host-cell walls of infected roots could alsoindicate that much of the enzyme has been secreted into vacuolar spaces. In this study, onlyvacuolar proteins present as protein aggregates were localized.

The lower density of labelling at early stages of infection in contrast to strong labellingat late stages could be explained differently. It is possible that, at the initial stage of infection,the enzyme accumulation was much lower, or that enzyme binding sites on pathogen-cellwalls 'were jnasked by electron-dense materials deposited on invading hyphae. Alternatively,It IS possible that, after prolonged infection, increased accumulation of these enzymesconcomitant with depolymerization of electron-dense deposit led to inereased labelling ofexposed hyphal walls. To determine whether these enzymes were secreted as a specificresponse to host-cell-wall invasion by a parasitic fungus, the experiment was repeated usinga saprophytic wood-decay fungus {P. gigantea). The results showed that there was nodistinct difference between accumulation and response of host antifungal products to cell-wall materials from these nutritionally disparate fungal species. It was concluded that thesecretion of these proteins and their possible involvement in recognition processes werelargely non-selective. Consequently, this must be taken into consideration when rootsystems from standing trees are used for such assays.

Acknowledgments

This study was supported hy grants from the Swedish Council for Forestry and Agricultural Research(SJFR). The authors thank E)r J.D. MlKKELSEN of Daniseo Laboratories, Copenhagen, Denmark, forsupplying polyclonal antibodies.

Pathogenesis-related proteins in Norway-spruce roots 177

Resume

Localisation immunocytochimique des PR protcines dans les raeines de Picea abies infectces par Hetero-basidion annosum

L'existenee et l'accumulation de /i-1,3-glucanase et de chitinase dans les raeines de semis d'cpiccas a lasuite de la confrontation avec H. annosum, ont etc ctudices. L'activite chitinase etait multipliee par 2ou 3 a la suite de I'lnoculation alors qu'aucune augmentation des niveaux d'activite de la glucanaseii'etait observee durant l'infeetion. Par mieroseopie eleetronique apres marquage immunologique a l'or,les enzymes ont c'te loealisees dans des agregats proteiques dans les tissus de l'hote et dans les paroiscellulaires des hyphes intereellulaires. Les particules d'or etaient reparties de fa^on disseminee etnre^uliere dans les parois eellulaires de l'hote. Un faible marquage seulement a etc' observe sur les paroisdesliyphes rccouverts par des materiaux denses aux eleetrons. L'intensitc du marquage augmentait avecla duree d'infection et ctait toujours plus forte qtie dans les raeines des semis non infeetes. Quand l'essaia etc repetc" en utilisant des cehantiilons de raeines lnoculees avec le saprophyte Phlebiopsis gigantea,un marquage semblable a ete observe. La reaction croisee de I'antiserum ohtenu eontre la glucanase etla chitinase de la betteiave a Sucre, avec des extraits d'enzymes dc raeines d'epieea, a etc montree enutilisant des tests Dot Blot et par ELISA.

Zusanimenfassung

Immunocytochemische Lokalisierung von PR-Proteinen in mit Hcterobasidion annosum infiziertenFiehtenwurzeln

In Wurzeln von Fichtensamlmgen wtirden das Vorkommen und die Akkumulation von /i-l,3-Gluea-nase und Chitinase naeh einem Angriff durch den Wurzelfauleerreger H. annosum untcrsucht. Nachder Inokulation erhohte sich die Chitinaseaktivitat um das Zwei- bis Dreifache. Bei der Glucanaseakti-vitat wurde hingegen keine signifikante Zunahme festgestellt. Mit Hilfe von Immunogoldmarkierungwurden die Enzyme im Transmissionselektronenmikroskop in Proteinaggregaten des Wirtsgewebesund in den Zellwanden interzellularer Hyphen lokalisiert. Im Bereieh der Pflanzenzellwande kamendie Coldpartikel nur sparlich und in unregelmalSiger Verteilung vor. Auf Hyphenzcllwanden, die miteinem elektronendichtcn Material ubcrzogen waren, wurde nur eine geringe Markierung beobaehtet.Die Intensitat der Markierung nahni im Verlauf der Infektion zu imd war stets holier als in den niehtinfizierten Wurzeln. Inokulationen mit dem saprophytischen Pilz Phlebiopsis gigantea fiihrten zu einemahnlichen Markierungsmuster. Mit Dot Blots und ELISA wurde naehgewiesen, da(5 Antiseren gegenChitinase und Glueanase atis Zuckerriiben mit den Enzymextrakten aus Fiehtenwurzeln reagicrten.

References

ABELES, F . B.; BOSSHART, R. P.; FORRENCE, L . E.; HABIG, W . H . , 1971: Preparation of glucanase andchitinase from bean leaves. Plant Physiol. 47, 129-134.

ASIEGBU, F . O . ; DANIEL, G.; JOHANSSON, M. , 1993: Studies on the infection of Norway spruee rootsby Heterobasidion annosum. Can. J. Bot. 71, 1552-156 L

—; —; —, 1994: Defence related reactions of seedling roots of spruce to infection hy Heterobasidionannosum. Physiol. Mol. Plant PathoL 45, 1-19.

BoL, J. F.; LiNTHORST, H. J.; COMEEISSEN, B. J., 1990: Plant pathogenesis related proteins induced byvirus infection. Annu. Rev. Phytopathol. 28, 113-138.

CARR, J. P.; KEILSSIG, D . G. , 1989: The pathogenesis related proteins of plants. In: Genetic Engineering.Principles and Methods II . Ed. by SETTOW, J. K. New York: Plenum Press, pp. 65-109.

DANIEL, G.; NII .SSON, T., 1991: Antiserum to the fungus Phialophora mutabdis and its use in enzymelinked imniunosorbent assays for detection of soft rot in preservative treated and untreated wood.Phytopathol. 81, 1320-1325.

DENEKAMP, M.; ASIEGBU, F.; JOHANSSON, M. , 1994: /J-l,3-i;lucanases in fine roots of Picea abies (L.)and Pinus sylvestris (L.) induced by infection with Heterobasidion annosum. In: Proc. 8th Int. Conf.Root and Butt Rots. Ed. by JOHANSSON, M.; STENLID, J. Uppsala: SLU Info. pp. 117-125.

FlNK, W.; LlEflAND, M.; M E N D G E N , K., 1988: Chitinases and /i-l,3-glucanases in the apoplasticcompartment of oat leaves. Plant Physiol. 88, 270-275.

JANSSON, S.; GUSTAFSSON, P., 1990: Type I and type II genes for the chlorophyll a/b binding proteinsin the gymnosperm Pinus sylvestris (Scots pine): cDNA cloning and sequence analysis. Plant Mol.Biol. 14, 2S7-296.

KAUFFMAN, S.; LI 'GRAND, M. ; GEOITROY, P.; FRITIG, B., 1987: Biologieal function of pathogenesis

178 F. O. Asiegbu, M. Denekamp, G. Daniel and M. Johansson

related proteins: four PR proteins of tobacco have /i-1,3-glucanase activity. The EMBO Journal 6,3209-3212.

KOMBRINK, E.; SCHROER, M.; HAHLBROCK, K., 1988: Several pathogenesis related proteins are /i-1,3-glueanases and ehitinases. Proe. Nat. Aead. Sei., USA 85, 782-786

LEGRAND, M.; KAUFFMAN, S., GEOFFROY, P.; FRITIG, B., 1987: Biological functions of pathogenesisrelated proteins: 4 tobacco pathogenesis related proteins are ehitinases. Proc. Nat. Acad. of Sci. USA84, 6750-6754.

LiNTHORST, H. J. M., 1991: Pathogenesis Related Proteins of Plants. Crit. Rev. Plant Sei. 10, 123-150.VAN LOON, L. C , 1985: Pathogenesis related proteins. Plant Mol. Biol. 4, 111-116.MAUGH, F.; MAUGH-MANT, B.; BOLLER, T., 1988: Antifungal hydrolases in pea tissue. II. Inhihition

of fungal growth by eomhination of chitinase and /i-1,3-glucanase. Plant Physiol. 88, 936-942.NsOLOMO, V. R.; WOODWARD, S., 1994: Glucanohydrolase enzyme activity in embryos of Seots pine,

Corsiean and lodgepole pines infected in vitro with Heterobasidion annosum. Eur. J. For. Path. 24,144-154.

RiGDENS, J.; COULTS, R., 1988: Pathogenesis related proteins in plants. Trends in Genetics 4, 87-89.SAUTER, M.; HAGER, A., 1989: The myeorrhizal fungus Amanita muscaria induees chitinase activity

in roots and suspension cultured eells of its host Picea abies. Planta 179, 61-66.SHARMA, P.; BORJA, D.; STOUGAARD, P.; LONNEUORG, A., 1993: PR-proteins aeeumulating in spruee

roots infected with a pathogenic Pytbium sp. isolate include chitinases, chitosaiiases and /i-1,3-glucanases. Physiol. Mol. Plant PathoL 43, 57-67.

SIMMONS, R., 1994: The physiology and molecular hiology of plant l,3-/i-D-glucanases and l,3;l,4-/i-D-glueanases. Crit. Rev. Plant Sei. 13, 325-387.

SOCK, J.; ROHRINGER, R.; KANG, Z., 1990: Extracellular /i-1, 3-glucanase in stem rust affected andabiotically stressed wheat leaves. Plant Physiol. 94, 1376-1389.

STENLID, J., 1985: Population structure of Heterobasidion annosum as determined by soinatie incom-patibility and isozyme patterns. Can. J. Bot. 63, 2268-2273.

VOGELLI, ij .; MEINS, F.; BOLLER, T., 1988: Co-ordinated regulation of ehitinase and /i-l,3-glucanasein bean leaves. Planta 174, 364-372.

WARGO, P. M., 1975: Lysis of the cell wall oi Armillaria mellea by enzymes from forest trees. Pysiol.Plant Pathol. 5, 99-105

WUBBEN, J. P.; JooSTEN, M. H. A. J.; VAN KAN, J. A. L.; DE WIT, P . J. M., 1992: Subcellular localizationof plant chitinases and /i-l,3-glueanases in Gladosporium fulvum (syn.fulviafulva) infected tomatoleaves. Physiol. Mol. Plant Pathol. 41, 23-32.

WYATT, S. E.; PAN, S. Q.; KUC, J., 1991: /i-1, 3-glueanase, chitinase and peroxidase activities in tobaccotissues resistant and suseeptible to blue mould as related to flowering, age and sueker development.Physiol. Mol. Plant Pathol. 39, 433-440.

Authors'addresses: F. O. AsiEGBU, M. DENEKAMP and M. JOHANSSON (for correspondenee). Depart-ment of Forest Myeology and Pathology, Swedish University of AgrieulturalSciences, S-750 07, Uppsala, Sweden; G. DANIEL, Department of Forest Products,Swedish University of Agricultural Scienees, S-750 07, Uppsala, Sweden

Received: 23.11.1994; accepted: 28.1.1995