Defence related reactions of seedling roots of Norway spruce to infection by Heterobasidion annosum (Fr.) Bref.

Physiological

and Molecular

Plant Pathology

( 1994)

1

45, 1-19

Defence related reactions of seedling spruce to infection by Heterobasidion Bref. F.O.

ASIEGBU*,

G.DANIEL~ and M.

JOHANSSON*$

* Department of Forest Mycology and Pathology and t Department Agricultural Sciences, S-750 07 Uppsala, Sweden (Accepled for publication

March

roots of Norway annosum (Fr.)

of Fores! Producti,

Swedish lJnivers@

of

1994)

Norway spruce seedlings were cultivated in sterile conditions. Roots were infected with a concentration series of germinating conidiospores of Heterobasidion annosum (IO’-10s ml-‘). In other experiments, roots were treated with either mycelial preparations ofH. annosum, other wood inhabiting fungi, with protein fractions ofculture filtrates ofH. annosum, or with chemical elicitors. Successive steps observed during infection were necrosis, formation of phenolics and increasing lignification ofcortex and endodermis, colonization ofmeristem and finally of the stele. High spore concentrations caused necrosis and invasion within 48-72 h; these processes were delayed at low spore concentrations. Lyophilized culture filtrates ofH. annosum caused a greater hypersensitive response than protein fractions but less than NaCI, Polygalacturonic acid or ethephon. Mycelial homogenate from nine other wood inhabiting fungi (saprophytes/parasites) induced a hypersensitive response to various extents but this was not correlated to their degree of cross-reactivity with a polyclonal antibody to H. anuosum [enzyme-linked immunosorbent assay (ELISA)]. Peroxidase activity increased (twothreefold) in roots infected with H. annosum and one acidic isozyme was considered responsible for the increase in peroxidase. Using immunohistochemical and enzyme staining, peroxidase was found mainly in the cortical/endodermal regions ofroots. Cytochemical labelling using anti-peroxidase and immunogold demonstrated increased peroxidase activity in cell walls, papillae and uninvaded middle lamellar cell corners of infected roots.

INTRODUCTION

Root and butt rot of conifers causesannual economic lossesof 500-1000 milljon crowns in Sweden. Much is known about the biology [19,21,24] and spread of the fungi causing the diseaseand also about the effect of silvicultural, chemical and biological control methods [I& 311, but less about the mechanisms determining variation in resistance between trees. The most important of these root rot- fungi is Heterobasidion annosum.’ Plant-pathogen interactions have been studied almost exclusively in annual plants, most of them agricultural crops. Some of the frequently observed events are rapid cell $To whom correspondence should be addressed. Abbreviations used in text: BSA, bovine serum albumin; HRP, horse radish peroxidase; linked immunosorbent assay; PAP, peroxidase-anti-peroxidase; PGA, polygalacturonic inoculation; TBS, tris buffered saline; TTBS, TBS containing Tweets-PO. 0885-5765/94/070001+ I

19 808.00/O

0

1994 Academic

ELISA, acid;

enaymep.i., post

Press Limited MPP45

F. 0. Asiegbu

2

et al.

death, papilla formation and the synthesisof defence compounds such asphytoalexins, phenolics, lignin, suberin and pathogenesis-related proteins [3,8]. One of the proteins often induced upon infection [S, 12,161 or wounding [34]’ is peroxidase. As a polymerizing enzyme, peroxidase plays a role in lignin and suberin synthesis [2.5,34] two groups of compounds which may block penetration at the sites of infection. Furthermore, peroxidase may be involved in defence by cross-linking phenolic compounds into papillae containing callose [IO]. Peroxidase may alsoenhance defence by the production of toxic radicals [16,26,29]. Recent cytological and biochemical techniques may help towards a better underst&ding of the mechanismswhich regulate such interactions. The outcome may involve a series of molecular and cellular recognition processesbetween the plant and pathogen [23,28]. H. annosum normally attacks living tissues in conifer root bark and sapwood including: the phelloderm, phloem, cambium and ray cells [21,24]. The present work is part of a joint project aimed at investigating the infection processof H. annosum and conifer roots at various stagesof development in order to characterize age-dependent changes in host defence mechanisms and resistance [2,17, 18,21,24]. We have examined, (1) whether defence responsesymptoms such as hypersensitive reactions induced on seedling roots of Norway spruce are due to specific recognition of H. annosum cellular fragments or extracellular metabolites; (2) papillae formation in roots ofspruce in responseto cortical invasion; and (3) changesin peroxidase activity in respdnseto infection by H. annosum and its localization in seedling roots of spruce.

MATERIALS

AND METHODS

Fungal strains Parasites. H. annosum FaS6; H. annosum FaP8; Armillaria borealis 3 16A (Marxmuller & Korhonen); A. gallica (Marxmuller & Romagn.); A. ostoyae (Romagn.) ; Phaeolus schweinittii Cl 79 (Fr.) Pat. ; Fusarium sp. Saprophytes. Fomitopispinicola (Sow. ex. Fr.) Karst. C76; Phlebia gigantea (Fr. : Fr.) Julich C125; Resinicium bicolor (AIb & Schw.:Fr.) Parm. C18. Facultative saprophyte. Stereum sanguinolentum (AIb. & Schw. ex. Fr.) Fr. C19.

Chemicals

Ethephon (2-chloroethylphosphonic acid: C-0143), polygalacturonic acid (PGA: P1879), chitin (C-3132) and pectin were obtained from Sigma Chemical Co., Sweden.

Seedling preparation

Spruce seedswere surface sterilized with 30% H,O, for 15 min, sown on sterile 1y0 w/v water agar and left to germinate in the dark. Sprouting seedlings(6-l 1 days old) were aseptically transferred into a second set of Petri dishescontaining the same agar. Half the Petri dish was overlaid with moist sterile filter paper prior to transfer of seedlings,with the root region placed on the filter paper. A second moist sterile filter paper was laid over the roots after fungal inoculation. The root region (half the Petri

Norway

spruce infection

3

by H. annosum

dish) was covered with aluminium foil and the edges of the Petri dish sealed with parafilm. The seedlings were then left under a photoperiod of 16 h at 20 “C (200 pE m-* s-l). Spore inoculunl

Conidiospores of H. annos~m(S-strain, FaS6) were recovered from Petri dishes (Hagem agar medium [30]), washed twice with sterile water, recovered by centrifugation, resuspendedin sterile 0.5% w/v malt extract and left to germinate overnight in the dark at room temperature. Germinating sporeswere washed, serially diluted in sterile distilled water (lo’-10’ ml-‘) and used for inoculations. Roots used for enzyme measurements were inoculated with lo6 sporesml-‘. ‘Treatments of seedling roots for hjpersensitiue

response

For testson the hypersensitive responseofseedling roots, nine parasitic and saprophytic fungal species were grown in stationary culture for 21 days in Norkrans [27] liquid medium. The hyphae together with culture filtrate (50 ml) were asceptically homogenized and the homogenate (1 ml) was added to the roots. To obtain culture filtrate preparations from H. annown (FaSG), the fungus was grown for 28 days as described above. The culture supernatant was collected and lyophilized (fraction A) or proteins were precipitated by adding ammonium sulphate to the indicated concentrations (fraction B). The precipitates were resuspended in water, dialysed. against water at 4 “C (cutoff point ILI, 10000) and lyophilized. The lyophilized fractions were reconstituted in water to a protein concentration of 25 pg ml-‘. Fraction A, polygalacturonic acid (PGA), pectin and chitin were sterilized by autoclaving for 15 min at 121 “C; fraction B, ethephon and NaCl were filter sterilized by membrane filtration (0,22 pm filters) before addition to the roots. All treatments were given as 1 ml additions per Petri dish containing seedling roots. Enzyme extraction front rools

Roots (from 10 seedlings) were homogenized using a mortar and pestle in 3 ml 0.1 M Tris-HCI buffer pH 7.6, containing 1O
Peroxidase was measured spectrophotometrically at room temperature, using as substrate 0.08 M phosphate buffer (pH 6.0) containing 0.03 M guaiacol and @3% H,O,. The enzyme-substrate mixture was vortexed and changes in absorbance were measured at 470 nm against substrate blank (dA 470 nm min-‘). Protein

The procedure described by Bradford [9] was usedfor all protein determinations, using bovine serum albumin (BSA) as standard. l-2

4

F. 0. Asiegbu

et al.

Peroxidaseantibody Commercially available horseradish peroxidase (HRP) antibody (Sigma no. P7899) was used. Gel electrophoresis of peroxidaseproteins Electrophoresis under native conditions wasperformed with a Phast system (Pharmacia, Stieden) and polyacrylamide isoelectrofocusiig gels (IEF 3-9) for detection and separation of isoforms of spruce peroxidase. The method was similar to that described by Holden & Rohringer [ZO] except that the substrate solution in addition to 0.06% 4-chloro- 1-naphthol and 0.03 y0 H,O,, contained 20% ethanol in 805 M phosphate buffer (pH 6.0). Antibody Polyclonal antibody against hyphal extracts of H. annosumwas raised in New Zealand rabbits as described previously [2]. ELISA ELISA procedures used were the same as described previously [11] except that primary and secondary antibody titres were 1: 1000, and the reaction was terminated after 15 min. Briefly, the antigenic material (hyphal extracts) used was obtained from fungi previously grown for 30 days in Norkrans liquid medium. Extracts were washed with several changes of sterile distilled water and freeze dried. Equal weights of lyophilized mycelia from each fungus was homogenized with 1 ml carbonate buffer (pH 9.6). The mixture was centrifuged and 100 ~1 of supernatant used for coating purposes. Westernblots Fungal and spruce root extracts were applied to nitrocellulose membranes (previously equilibrated in Tris buffered saline [TBS (pH 7*5)] and blocked for 2 h using TBS-3 y. w/v gelatin. Thereafter membranes were washed with TBS (2 x 5 min) containing @05o/oTween-20 (TTBS) and probed with anti-peroxidase (1: 1000 diluted in TTBS1y. w/v gelatin). Following overnight incubation at room temperature under gentle agitation, membranes were washed in TTBS (2 x 5 min) and treated with protein A HRP (1: 1000 diluted in TTBS-1 y. w/v gelatin) for 2 h. Membranes were then washed with TTBS (2 x 5 min) and TBS (2 x 5 min) and colour developed according to manufacturer’s instructions (BioRad, Sweden). Tissuepreparationfor light microscopyand immunohistochemistry Samples(approx. 5 roots per sample) were collected 1,3,7, 10, 15,20 and 25 days post inoculation (p.i.). Root regions (first and second 3 mm) were fixed for 3.5 h in 0.1 M sodium phosphate buffer (pH 7.2), containing 3 o/ov/v glutaraldehyde, dehydrated in ethanol, infiltrated using a seriesof ethanol:Technovit mixtures (2: 1, 1: 1, 1: 2, 0 : 1) and finally embedded in a mixture of 15 ml Technovit and 1 ml hardner II. Sections

Norway

spruce

infection

by H. annosum

5

(4 pm) were cut using an ultratome (LKB 4804). For assessmentof lignin or phenolic deposition, sections were stained with either aqueous toluidine blue 0 (10 mg ml-‘) or 3 y0 phloroglucinol in 95 y0 ethanol (2-3 drops) followed by 1 drop of concentrated HCI. Experiments were repeated three or four times. (PAP) labelling for light microscopy The procedure employed was the same as that described byjohnstone & Thorpe [.72] except that BSA was substituted for normal goat serum and sectionswere mounted and viewed using Normarski optics. Peroxidase-anti-peroxidase

of samples for transmission electron microscopy Samples(first and second3 mm region ofroots) were fixed in 4 y0 v/v paraformaldehyde, 1 y0 v/v glutaraldehyde in.O.1 M sodium cacodylate buffer (pH 7.2) for 4 h at 4 “C, and then washed in several changes of buffer. Thereafter the sampleswere dehydrated in an ethanol seriesand embedded in London resin (London Resin Co. Basingstoke, UK). Selected sampleswere sectioned using a Reichert FCD ultramicrotome (Buffalo, NY). Ultrathin sections were collected on nickel grids, stained with uranyl acetate and observed using a Philips CM 12 transmission electron microscope operated at 60 kV.

Preparation

Immunocytochemical

labelling

Ultrathin sections were pre-treated with 10% H,O, for 2 min, washed in water and incubated with O-1M glycine (30 min) to quench aldehyde groups induced during fixation. Sections were then washed in phosphate buffered saline (PBS) and incubated in drops of normal goat serum (1: 30 in PBS) for 30 min and subsequently incubated with anti-HRP antibody (1:500) in PBS-l y0 (w/v) BSA-O-05 y0 Tween-20. After overnight incubation at 4 “C, sections were washed in PBS-O.1o/oBSA-0.05 o/oTween20 for 10 min and again in Tris-HCl-@l y. BSA-Tween-20 (pH 7.2) for 10 min. The sectionswere then incubated with a gold labelled anti-rabbit IgG conjugate (Au 15 nm) diluted 1: 50 in Tris HCI-1 oh BSA-Tween-20 (pH 8.4) for 1 h at room temperature. Sections were washed thoroughly in Tris-HCl-1 y. BSA-Tween-20 (pH 8.4) ( 10 min), then in Tris-HCl (pH 8.4) ( 10 min) and finally in distilled water. Controls included (a) incubation of antibody with excessof commercial HRP enzyme (P8250) ; (2) incubation of antibody with excessof extracts from roots; and (3) incubation with rabbit pre-immune serum. Samples were observed as above. R ES ULTS Hypersensitive

response

Pre-germinated sporesor blended mycelial cultures of H. annosz.-m induced browning of roots 48 to 72 h p.i. The intensity of the browning reactions increased with time pathogenicity after inoculation (Fig. 1). At 5 days p.i., dense opaque areas appeared on the root cap region. With prolonged incubation browning symptoms extended to the shoot. Challenge of seedling roots with homogenates of other fungi and with nonspecific elicitors showed that the browning symptoms appeared irrespective of the pathogenicity of the fungi. Also there was no apparent corre1atio.n between browning symptoms and the cross-reactivity of the fungal extracts with antibodies against

F. 0. Asiegbu

6

FIG. 1. Light microscopy of roots of Norway spruce Transparent region (TN) of uninfected root (control). response as dense opaque background (OP) in infected with toluidine blue 0. Bars = I@0 pm.

et al.

infected with Heterobaridion annosum. (A) (B) Presence of strong hypersensitive (H) root. (C) Papillae (arrows) stained

H. annosum (Table 1). Inoculation with H. annosum caused an earlier appearance and development of much darker areas on roots than inoculation with other species,such asFusarium sp. or Phlebia gigantea (results not shown). Thus the browning responsewas relatively non-specific. This conclusion was further substantiated by treatment of the seedling roots with various substances(e.g. NaCl, PGA, pectin, chitin and ethephon) known to induce stresssymptoms (Table 2). As shown in Table 2, H. annosum culture filtrates also caused browning reactions. Using the same protein concentration, the preparation containing lyophilized total culture filtrate was a more potent inducer of the responsethan fractions precipitated with ammonium sulphate. Since the latter had been dialysed, this suggeststhat low molecular weight metabolite(s) in the culture filtrate were capable of inducing browning reactions. This was further supported by the

Norway

spruce

infection

7

by H. annosum TABLE

1

The effect of treatments with mycelial homogenatesfrom fungal strains on Norway reactivity of hyphal extracts with antibodies against Heterobasidion

Extracts

Browning relative

from

Heterobasidion annosum Pa.95 Heterobasidion annosum FaP8 Fomitopsis pinicola CT6 Phlebia giganteat Phaeolus schweinitziit Armillaria gallica Annillaria ostoyae Resinicium bicolort Stereum sanguinolentumt Fusarium sp. Control: Water

response degree*

spruce roots and the crossannosum Cross-reactivity A,,,,s ELISA test

+++ ++ +++ +++ + + + +++ ++ +++ 0

1.8 1.6 0.5 1.3 0.6 1.7 1.7 0.2 0.7 nd 0

*Samples were examined 5 days post inoculation by eye. The browning response graded as follows: 0 = not detected; + = slight; + + = moderate; + + + = strong. tsaprophyte. ELISA, enzyme-linked immunoasorbent assay; nd, not determined.

TABLE

The effects of culturejillrate

Preparation

fractions

and potential

was

?

elicitors on seedling roots Browning relative

or compound

Heterobasidion annosum culture filtrate preparation Total 60 o/0 Ammonium sulphate precipitate 100/60°/0 Ammonium sulphate precipitate @5 M NaCl @2% Polygalacturonic acid (w/v) 0.2% Pectin (w/v) @2% Chitin (w/v) @05 M Ethephon *Samples were examined 5 days post inoculation graded as follows: 0 = not detected; + = slight; .+ + + + = very strong.

of Norway spruce

response degree*

++ 0 + ++++ +++ + ++ +++ by eye. The browning response was + + = moderate; + + + = strong;

fact that no browning reactions were observed on root surfaceswithin the 5-day study period when uninoculated nutrient media were used (data not shown). E$ect of time and spore concentration tissues

on cortical penetration

and disintegration

of varcular

The effect of spore concentration on the infection process,is shown in Table 3. When an inoculum density of lo3 sporesml-’ or higher was used,, reactions such as lignification and accumulation of phenol& were seen as early as S days p.i. At

F. 0. Asiegbu et al.

8 3

TABLE

.

Time-courseof ~WU.Jin seedlingroot tissues after challenge with d$rent concentraliov of pre-germinated spores of Heterobasidion annosum Period (days)

Spore concentration no. ml-’

7

3

25

15

NF NCP NCP

NF NCP NCP, SL

.NF NCP CC, SP, MND, SIS

IO5

NCP, SP, SL

NCP, SP, SL

CC, MD, SI, HL

IO4

NCP, SP, SL

IO5

CC, DP, SL, SI

CC, SP, SL, MND, SI CC, HP, MND, SI

10s

CC, SL, HP

CC, MD, SI

CC, HP, MD, SI, HL CC, HP, MD, SD, HL CC, HP, MD, SD, HL

. H,O lOI 10*

NF, SP*, SLt NCP CC, SP, MND, SI, HLt CC, HP*, MD, SI, HL CC, MD, SDS, LOP CC, MD, SD, LOP CC, MD, SD, LOP

NF, no infection; NCP, no cortical penetration; CC, colonization ofcortical tissues; SP, low accumulation ofphenolics; SL, slight lignification ofcortex and endodermis; MND, meristem infected but not destroyed; MD, meristem infected and destroyed; SI, stele not invaded or infected

(i.e. no hyphae);

SD,

stele invaded

and

destroyed;

HP,

high

accumulation

of

phenolics; HL, highly lignified cortex and endodermal walls; LOP, loss of phenolics. *No. of cortical cells (L.S) that stained densely bluish green with toluidine per field of view: SP = (> 40 < 80 cells) ; HP = (> 80 cells). tNo. of cells (L.S) that stained intensely reddish brown and retained their typical cell outline after phloroglucinol stain per field ofview: SL = ( > 15 < 30 cells) ; HL = ( > 30 cells).

SObservations of vascular tissues were recorded from the second 3 mm region only. 15 days p.i., a strong lignification and high accumulation ofphenolic compounds was observed in cortical and endodermal regions. At a much later stage of infection the accumulation of phenolics decreased. The first signs of infection and cortical penetration were observed within 72 h p.i. when spore inoculations of lo5 or 10’ spores ml-l were used, but with lower inoculum densities (10’ or lo3 spores ml-‘) cortical penetration was delayed until 15 days p.i. The meristematic region was destroyed 7 days p.i. using the highest spore concentration ( lo6 spbres ml-‘), but at 10’ spores ml-’ this region was not destroyed within the time period studied (25 days; Table 3). During this period, the stele was not infected unless a spore inoculum of lo4 spores or higher was used. The fungus might have gained entry into the stele either through the meristem, a very susceptible region to attack by this fungus or through the endodermal region (data not shown). Localization of papillae and accumulation of phenolics Papillae were usually observed in cortical and endodermal regions points of intercellular penetration by invading hyphae (Figs 1 and all hyphae in this region were restricted by papillae [Fig. 2(b)] and infection, fungal hyphae were able to penetrate through papillae and

(data not shown). In meristematic and vascular

tissues, papillae

were rarely

observed,

and mostly at the 2). However, not at a later stage of phenolic barriers possibly

because

Norway

spruce

infection

by H. annosum

. FIG. 2. Transmission electron microscopy of roots of Norway spruce infected nnnosctm. (A) Papilla (P) overlying sites of fungal (H) invasion into cortical cells (H) penetration of cortical region (CT) without restriction by papillae. (C) hyphae (H) within middle lamellar cell corner regions (MR). (D) Deposition around invading hyphae (H). Bars A, C and D = I.0 pti; B = 2.0 pm.-

with Heferobmidion (CT). (B) Hyphal Growth of fungal of phenolics (PH)

oft he rapid destruction of these regions once infected. Papillae were also rarely see]n in the infected middle lamellar cell corners. After penetration, growth of the fungus wit :hin the middle lamellar region often occurred [Fig. 2(c)]. Electron dense materials Mrere fou nd deposited on invading hyphae [Fig. 2(d)] an d were in somecasesassociatedvvith desId hyphae.

F. 0. Asiegbu

I.0

et al.

Resistance of seedling root tissues

The different root cell regions examined varied in their degree of resistance to H. annosum infection, with root cap and endodermal cells being most resistant, whereas meristematic and vascular cells were very susceptible (Fig. 3). Furthermore, despite

Vascular

region (R )

FIG. 3. Degree of resistance of different cell regions of spruce roots to infection by Hettrobasidion annosum. Degree of resistance increases with number of R. Resistance in this context is defined as the ability to resist total disintegration of cellular and structural tissues. (Modified from R. Scott Russel (1977) : Plant Root System.r. New York: McGraw-Hill.

extensive colonization ofroot cap regionsasobserved with scanning electron microscopy (data not shown), the cells were not easily penetrated and disintegrated as compared to meristematic regions. Effect of inoculation

with H. annosum spores on peroxidase activity The highest observed increase in peroxidase activity was two to three orders of magnitude higher in the infected than in mock inoculated roots 6 days p.i. (Fig. 4). This increase in peroxidase activity appeared in the same period as the manifestation of diseasesymptoms (Table 3) observed with histochemical studies. Such physiological and morphological changes gave an indication of the seedlingsresponseto infectibn. Therefore to find out if distinct differences in peroxidase accumulation existed with infection, a more critical investigation at the cellular level was carried out as shown below. Peroxidase isofom

with infection

Root extracts were subjected to electrophoretic separation to determine if specific isozymic forms were associated with defence reactions. One major finding was increasedintensity of one slowly migrating acidic isozyme (Fig. 5; lane 2 lowest arrow).

Norway

spruce infection 90

11

by H. annosum

60 70 -

0

2

4

6

6

10

;

,

Period (days) FIG. 4. Peroxidase (-•-),

Control;

FIG. 5. Peroxidase 11; lane 2, infected,

activity (-•+-),

of seedling infected.

roots

following

infection

by Heterobusidion

isozymes of Norway spruce before and after infection. day 11; lane 3, control, day 13; lane 4, infected, day

Lane 13.

annosum.

1, control,

day

F. 0. Asiegbu

et al.

FIG. 6. Localizations of peroxidase activity within uninfected and infected roots of Norway spruce. (A) Guaiacol peroxidase staining of fresh tissues. Note uniform levels of peroxidase reaction in epidermis (E), cortex (C) and stele (S) of control root. (B) Guaiacol peroxidase staining of fresh tissues. Note strong peroxidase stain (arrows) in epidermis and cortex (C) of infected root. (C) Peroxidascanti-peroxidase (PAP) reaction; note faint staining of endodermal (E), cytoplasm and cell walls of cortical (C) cells of control root. (D) PAP reaction; note strong peroxidase reaction in endodermis (E) and cortex (C) of infected root. Bars = 10.0 pm.

Norway

spruce

infection

13

by H. annosum

Sometimes varying result patterns were obtained (Fig. 5; lanes 3 and 4), assome of the rapidly migrating acidic isozymes (4-5) were either missing or appeared as weaker bands in the infected samples. Cjtochemical

localization

of peroxidase

Using guaiacol in the presence of H,O, on longitudinal sections of fresh tissues, peroxidase activity was found to be uniformly distributed within epidermal, cortical and stelar regions [Fig. 6(a)]. However following inoculation, increased concentrations of peroxidase was detected in epidermal and cortical regions [Fig. 6(b)]. No staining was observed in control roots when H,O, was omitted. The commercial polyclonal HRP antibody used in this work cross-reacted with peroxidase from fungal extracts and with all peroxidase isozymes from roots, shoots and spruce. This was demonstrated using dot (Fig. 7) or Western needles of Norway

2

.-. .. ;

: !, -:.;r

FIG. 7. Cross-reactivity ofhorseradish polyclonal antibody to fungal and root extracts. Lane 1, root (IO, 5, 3 and 1 f.tl extract); lane 2, root control (pm-immune sera); lane 3, culture filtrate (0.06, 0.03, 0.018 and 0.006 pg protein); lane 4, culture filtrate control; lane 5, fresh hyphal extract (2.5 g ml-‘) (10, 5, 3 and 1 111); lane 6, hyphal extract control; lane 7, 80% (NH,),SO, culture filtrate precipitate (1.6, 0.8, 0.5 and @I6 pg extract); lane 8, culture filtrate precipitate control.

blotting and ELISA (data not shown). With the PAP technique and 3,3diaminobenzidine as substrate, peroxidase activity was observed with the light microscope both in the cell wails and in cytoplasm of root tissues[Fig. 6(c) and (d)]. In infected tissues, reactions were generally strong in cortical/endodermal regions compared to control roots, and faint in vascular cells [Fig. 6(c) and (d)]. Under the electron microscope labelled immunogold ultrathin section with HRP antibody showed strong labelling of primary and secondary cell walls of cortical, endodermal and stelar cells [Fig. 8(a)]. A n increased deposition of gold particles

F. 0. Asiegbu

et al.

of peroxidase in roots of Norway spruce infected with Hefmbaridion FIG. 8. Localization nnnosam. (A) Peroxidase labelling of vascular cells are noticeable but some cell wall areas are intensely labelled (arrows) in infected samples. (B) Surrounding matrix of middle lamellar cell corner (MR) is intensely labelled prior to pathogen invasion. At advanced stage ofinfection (IF) cell walls of MR are disintegrated and are marked by presence ofscanty gold labelling. (C) Gold particles are predominantly associated with apposition structure (papillae) at the site [endodermis, (ED)] of attempted pathogen invasion (PP). Scanty gold particles are also found scattered over fungal hyphae (H). Bars = 1.0 pm.

Norway

spruce

infection

by H. annosum

15

occurred over middle lamellar cell corners; but no,labelling was observed in fungal invaded intercellular spaces [Fig. 8(b)]. Labelling of cytoplasmic material also occurred in cortical and endodermal regions [Fig. 8(c)]. Papillae were strongly labelled in all infected samplesexamined {Fig. 8(c)] and some labelling of invading hyphal cell walls was apparent [Fig. 9(a)]. Preferential deposition of gold particles was noted in

FIG. 9. Localization ofperoxidase in roots ofNorway spruce infected with Heferobasidion annosum. (A) Dense labelling of middle lamellar (MR) and cell wall (arrows) regions overlying sites of attempted pathogen invasion. Also note concentration of gold particles in pre-forming papilla (arrows). (B) Constitutive peroxidase formation as marked by presence and uniform distribution ofgold particles in cell walls ofendodermal cells (ED) of uninfected control root. Bars = 1-O pm.

host areasin the vicinity of invading hyphae or at sitesof intercellular penetration [Fig. 9(a)]. However, in control samplesgold particles were randomly distributed acrosscell walls without any sign of preferential accumulation [Fig. 9(b)].

DlSCtiSSlON model for H. annosum infection This study is part of an investigation of changes in ‘the responsimechanisms of conifer roots of different ages. During development the conifer root undergoes several morphological and physiological transformations. Non-suberized seedling roots, mainly containing epidermis, cortex, endodermis, meristem and stele are successivelysuberized, the cortex disappears and the secondary xylem in the vascular region (stele) becomes dominant. Later the epidermis, cortex and endodermis are replaced by the rhytidome, phelloderm and phloem layers. The living parts of the xylem consist mainly of Seedling roots as an experimental

F. 0. Asiegbu

16

et al.

cambium, rays and resin parenchyma cells. These successivemorphological changes occur concomittantly with constitutive and inducible defence mechanisms.H. annosum is one of the very few fungal pathogens able to infect conifer roots of all ages. For our experimental model, seedlingswere grown under uniform sterile conditions and usedat an agewhen their genetic differences are only weakly expressed(Sandberg, pers. comm.). Inoculum potential

and disease outcome

Increasing amounts of inoculum may affect the infection rate [33] : the sporesmay act independently, they may compete for susceptible sites, they may act antagonistically (i.e. prevent germination) or synergistically (i.e. stimulate germination and counteract defenceinduction). Using germinating spores,asin the present study, the growth of the mycelium is the critical factor in the establishment ofinfection on roots and formation of infection structures. For breaking the successively formed peripheral barriers at an early stage, a certain density of hyphae may be needed. With lower initial amounts of hyphae this density is not obtained until induced barriers are strong enough to prevent infection. The results obtained indicate that the cellular responsesof seedling roots of spruce are similar to those of other plants infected by pathogens, i.e. an integrated set of resistance mechanismsare activated during infection [28,32,35]. In the present investigation initial responsesinclude localized cell death (hypersensitive response), papilla formation, physiological thickening of cell walls (lignification), and increased peroxidase activity. Keen [23] noted that such responses,when specific, can be effective in limiting the spread and development of the pathogen. To determine the specificity of hypersensitive responsesin respect of H. annosum and roots of spruce, nine other fungi, chemical elicitors and culture filtrates were assessed. The major conclusion was that the reactions were non-specific and could be related more to stress than to specific recognition of H. annorum cellular components. It is difficult to estimate the extent to which these responsereactions contribute to limiting pathogen penetration acrossepidermal walls. Similar reports on acquired physiological resistance to diseaseby angiosperm plants exposed to chemicals and glycoprotein elicitors have been reported [I, 131. The cross-reactivity of the polyclonal antibody raised against H. annosum hyphal extracts with necrosis-inducing mycelial preparations from other fungi suggeststhat H. annosum mycelia share significant homology with these fungi. The fact that some of the fungi which showed low cross-reactivity (Table 1) elicited an equally strong hypersensitive response(and vice versawith Armillaria spp.), suggeststhat other extraneous agents may also contribute in inducing host defence signal recognition. Apart from hypersensitivity reactions (browning), Signification and papilla formation were alsomajor morphological responsesobserved. They have often been suggestedas diseaseresistancefactors in plants [7,34]. In this study we identified the indeterminate and random nature of papilla formation. Papilla were observed in some cells but not in others during pathogen invasion. It seemsthat the processof papilla formation is regulated by cellular and genetic factors which can vary even between similar cells. It is suggestedthat the restriction of pathogen invasion by papillae and ligmfication may be weakened in host cells exposed to multiple infection and. high inoculum concentrations. This is basedon the finding that cortical penetration was delayed by 15

Norway

spruce

infection

17

by H. annosum

days with an inoculum concentration of 102-lo3 sporesml-‘, whereas cortical invasion was achieved within 3 days p.i. using 105-10’ sporesml-‘. The ability of papillae to restrict fungal development or the pathogen to penetrate structural and chemical barriers may be determined in part by the physiological state of the seedlings. In some seedlings, invasion by the pathogen was arrested in cortical and endodermal regions and no penetration into the stele was recorded. The absence of such resistance may account for the lossof root turgor and wilting observed in some seedlings.Bone110et al. [7] made similar observations on pine seedlingsinfected with Cylindrocarpondestructanr.Our results also revealed variations in the degree of resistance to H. annosuminfection by different cell regions of the root. Although our results on regional resistancewere based on microscopic observation after histochemistry, a more appropriate approach would have been to estimate numbers of dead cells. However, it is sometimes ambiguous as to what constitutes cell death. A major dilemma is whether cells reacting hypersensitively to stressor infection should be classified as dead. We considered that using cell death as a measure of disease resistance could be subjective, asour results and those of other workers [14] have shown that hypersensitive cell death may be incapable of arresting fungal growth. The slow progression ofinfection from the cortex into the stele after 5-7 days was largely attributable to suberization of endodermal regions. It was supposed that deposition of callose and inorganic matter in the root cap may also account for strong resistance in this region. The relationship between peroxidase activity and diseaseresistancehas been studied in angiosperm systems mainly [IO, 331 and not much is known concerning these processesin the gymnosperms. In this study, the increase in plant peroxidase activity ‘upon infection correlates with cellular penetration and the manifestation of initial defence responsesymptoms. The long period required for the induction of peroxidase activity results in the activity being too late for activation of defence mechanisms that would have prevented disease development in the seedling roots. However, when analysing peroxidase activity in infected roots we have to consider a possible contribution by fungal peroxidase. We did find peroxidase activity in fungal culture filtrates, although this activity was very low and could only be detected in concentrated preparations (1.4 x 10m4nanokatal mg-’ protein) of culture filtrate (i.e. < 0.01 y0 of the activity in host protein (unpublished observations)). Moreover since the isozyme analysis of root extracts did not reveal any new peroxidase bands in infected roots, we believe that the activity measured primarily reflects plant peroxidase activities. This is further supported by the fact that the infection-related isoperoxidase band was seen weakly in extracts from uninoculated roots. The isozyme analysis suggestedthat one specific acidic isozyme may be responsible for the increase in spruce peroxidase on infection. On the other hand, since the effect of other factors (elicitors) on isozyme pattern were not analysed, no conclusive * statement can be made on the specificity of this isoperoxidase enzyme in relation to resistance. Van Loon [34] noted that in systemically resistant tobacco leaves, no additional peroxidase isozyme was present after infection, but Cadema-Gomez & Nicholson [IO] found two new peroxidase isozymes following infection of maize with Colletotrichum gramincola. Holden & Rohringer [20] were unable to detect peroxidase isozyme differences between treated and untreated wheat leaves.. The localization studies showed that the increased peroxidase in infected tissue was 2

MPP

45

18

F. 0. Asiegbu

et al.

seenmainly in cell walls and papillae, which indicates that it is probably involved in the formation of defence compounds such as lignin, suberin and .phenolic polymers. This is supported by Fig. 1(c) and earlier histochemical data’on the presenceof lignified depositson papillae [Z, 101. In spite of the cross-reactivity of fungal peroxidase with the HRP antibody, only minor labelling of hyphal cell walls was observed, supporting spectrophotometric results. Similar localization of pathogenesis-related proteins (chitinase, P14, peroxidase) has been reported for other plant tissuesfollowing infection [4,.5,10]. However, the question as to the role ofperoxidase in defence mechanismsof gymnosperms and their specificity against root rot fungi (e.g. H. annosum P and S strains) deservesfurther investigation together with other pathogenesis-related enzymes involved in lignin biosynthesis. This work was supported by grants from the Swedish Council for Forestry and Agricultural Research (SJFR) . We thank Dr Lisbeth Jonssonfor useful discussionsand for critically reading the manuscript. REFERENCES 1. Anderson AJ. 1987. The biology of glycoproteins as elicitors. In: Kosuge T & Nester EW eds. Plant-Microbe Interactions: Molecular and Genetic Perspectives. Vol. 3. New York: McGraw-Hill. 2. Asiegbu FO, Daniel G, Johansson M. 1993. Studies on the infection of Norway spruce roots by Hettrobasidion annosum : Canadian journal of Botany, 71: 1552-156 1. 3. Beckman JS, Siedow JN. 1983. Bactericidal agents generated by peroxidase catalysed oxidation of para-hydroquinones. Journal of Biological Chemistry 260: 1460414609. 4. Benhamou N, Jooste MHA, De Wit PGM. 1990. Subcellular localization of chitinase and of its potential substrate in tomato tissues infected by Fusarium oxysporium sp mdicti-lycopersici. Plant Physiology 92: 1108-I 120. 5. Benhamou N, Grenier J, Asseln A. 1991. Immunogold localization of pathogenesis related protein P14 in tomato root cells infected by Fusarium oxysporium tsp. radicis ~copeki. Physiological and Molecular Plant Pathology 38: 237-253. 6. Birecka H, Catalfamo JL, Urban P. 1975. Cell wall protoplast isoperoxidases in tobacco plants in relation to virus. Plant Physiology 55: 611-619. 7. Bonello P, Pearce RB, Watt F, Grime GW. 1991. An induced papilla response in primary roots of Scats pine challenged in vitro with Cylindrocarpon destructans. Physiological and Molecular Plant Pathology 39: 213-228. 8. Bostock RM, Stermer BA. 1989. A perspective in wound healing in resistance to pathogens. Annual Reoiew of Phytopathology 27: 343-371. 9. Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein dye binding. Analyt&l Biochemistry 72: 248-254. 10. Cadena-Gomez G, Nicholson RL. 1987. Papilla formation and associated peroxidase activity: A nonspecific response to attempted fungal penetration of maize. Physiological and Molecular Plant Pathology 31: 51-67. 11. Daniel G, NiIsson T. 1991. Antiserum to the fungus Phialophora mutabilis and its use in enzyme linked immunosorbent assays for detection of soft rot in preservative treated and untreated wood. Pl$opathology 81: 1319-1325. 12. Espelie K, Kolattukudy PE. 1985. Purification and characterization of an abscisic acid-inducible anionic peroxidase associated with suberization in potato (Solarium tuberosum). Archives of Biochemistry atid Biophysics 240: 539-545. 13. Gisninazzi S. 1984. Genetic and molecular aspects ofresistance induced by infections or chemicals. In: Nester EW % Kosuge T, eds. Plant-Microbe Interactions-Molecular and Genetic Perspectives, Vol. 1. New York: Macmillan, 321-342. 14. Gorg R, Hollrichei K, Schulze-lefert P. 1993. Functional analyses of the MLG resistance locus in Barley. In: Fritig B % Legrand M, eds. Mechanisms of Plant Defence Responses. Netherlands: Kluwer Academic Publishers, 45-48. 15. Gteig BJW. 1978. Chemical, biological and silvicultural control of Fames aunosk. Proceedings of the 5th International Conftrmcc on Problems of Root and Butt Rot in Conijk, 76-81.

Norway

spruce

infection

19

by H. annosum

16. Hammerschmidt R, NuckIes EM, Kuc J. 1982. Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagennrium. Physiological Plant Pathology 20: 73-82. 17. Heneen WK, Gustafsson M, Karlsson G, Brismar K, Jobansson M. 1994. Interactions between Norway spruce (Picea abies) and Heterobasidion annosum. I: Infection of non-suberized and young suberized roots. Canadian Journal of Botany (in press). 18. Heneen WK, Gustafsson M, Karlsson G, Brismar K, Johansson M. 1994. Interactions between Norway spruce and Heterobasidion annosum. II: Infection of woody roots. Canadian Journal of Botany (in press). 19. Hodges CS. 1969. Modes of infection and spread of Fomes annosus. Annual Review of Phytopathology 7: 247-266. 20. Holden DW, Rohringer R. 1985. Peroxidases and glycosides in intercellular fluids from noninoculated and rust affected wheat leaves. Plant Physiology 79: 820-824. 21. Johansson M, Stenlid J. 1985. Infection of roots of Norway spruce (Picea abies) by Heterobasidion annosum. 1. Initial reactions in sapwood by wounding and infection. European Journal of Forest Pathology 15: 32-45. 22. Johnstone A, Thorpe R. 1987. Immunochemistry in Practice. 2nd edn. London: Blackwell Scientific Publications, 261-289. 23. Keen NT. 1990. Gene-for-gene complementarity in plant-pathogen interactions. Annual Review of Genetics 24: 447463. 24. Lindberg M, Johansson M. 1992. Growth ofHeterobasidion annosum through bark ofPicea abies. European Journal of Forest Pathology 21: 377-388. 25. Mader M, Fussi R. 1982. Role ofperoxidase in lignification of tobacco cells II. Regulation by phenolic compounds. Plant Physiology 70: 1132-l 134. 26. Milder M, Ungemach J, Schloss P. 1980. The role of peroxidase isozyme groups of Kcotiana tabacum in hydrogen peroxide formation. Planta 147: 467-470. 27. Norkrans B. 1963. Influence of some cultural conditions on fungal cellulase production. Physiologia Plantarum 16: 1 l-19. 28. Slusarenko AJ, Croft RP, Voisey CR. 1991. Biochemical and molecular events in the hypersensitive response of bean to Pseudomonas syringae pv. phaseolicola. In: Smith CJ, ed. Biochemishy and Molecular Biology of Host-Pathogen Interactions. Oxford: Oxford University Press, 126-143. ‘29. Stich K, Ebermann R. 1984. Investigation of hydrogen peroxide formation in plants. Phytochemistry 23: 2719-2722. 30. Stenlid, J. 1985. Population structure oftieterobasidion annosum as determined by somatic incompatibility, sexual incompatibility and isoenzyme patterns. Canadian Journal of Botany 63: 2268-2273. 31. Stenlid J. 1987. Controlling and predicting the spread ofHeterobasidion annosum from infected stumps and trees of Picea abies. Scandinavian Journal of Forest Research 5: 59-67. 32. Vance CP, Kirk TK, Sherwood RT. 1980. Lignification as a mechanism of disease resistance. Annual Review of Phytopathology 18: 259-288. 33. Van der Plank, JE. 1975. Principles of Plant Infection. London: Academic Press, 57-63. 34. Van Loon LC. 1985. The significance of changes in peroxidase in diseased plants. In: Greppin H, Penel C & Gasper T, eds. Molecular and Physiological Aspects of Plant Peroxidase. Universite de Geneve Centre de Botanique, 405-408. 35. Ward ER, Uknes S, Williams S, Dincher S, Wiederhold D, Alexander D, Ahl-Coy P, Metraux J-P, Ryals J. 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. The Plant Cell 3: 1085-1094.

2-2