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Preprint of: Krekling, T., Franceschi, V. R., Krokene, P., & Solheim, H. (2004). Differential anatomical response of Norway spruce stem tissues to sterile and fungus infected inoculations. Trees - Structure and Function, 18(1), 1–9. doi:10.1007/s00468-003-0266-y The final publication is available at: http://link.springer.com/article/10.1007%2Fs00468-003-0266-y

Differential anatomical response of Norway spruce stem

tissues to sterile and fungus infected inoculations

T. Krekling Institute of Chemistry and Biotechnology, EM, Agricultural University of Norway, N-1432 Ås, Norway

V.R. Franceschi School of Biological Sciences, Washington State University, Pullman, WA 99164, USA

P. Krokene ⋅ H. Solheim () Division of Forest Ecology, Norwegian Forest Research Institute, Høgskoleveien 12, N-1432 Ås, Norway

Email: halvor.solheim@skogforsk.no; Tel.: +47- 64 94 90 26, Fax.: +47- 64 94 29 80.

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Abstract

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of

different resistance to pathogenic fungi were characterized over time and distance from

small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion

annosum. Common responses for both treatments included division of ray parenchyma and

other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma

cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts

(TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells,

or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered

by a signal, which propagates a developmental wave in the axial direction at about 2.5 cm

per day. TDs are formed at least 30 cm above single inoculations within 16-36 days after

inoculation. The size and number of TDs is attenuated further away from the inoculation

site, indicating a dose-dependent activity leading to TD development. Compared to sterile

wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple

rows of TDs in weak clones. Fungal inoculation also induced the formation of more new

PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to

100%. TD and PP cell formation was greater in susceptible compared to resistant clones

and after fungal versus sterile inoculation. Potential mechanisms responsible for this

variable response are discussed

Key words Norway spruce . Resin ducts . Parenchyma . Phenolics . Heterobasidion

annosum

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Introduction

Bark beetles and pathogenic fungi represent major threats to conifer forests (Schowalter

and Filip 1993; Paine et al. 1997). To meet these challenges, conifers have evolved

sophisticated constitutive and inducible defense mechanisms that reside in both bark and

sapwood. Constitutive defenses include suberized/lignified derivatives, sclerified cells

(Wainhouse et al. 1990), cells containing calcium oxalate crystals (Srivastava 1963;

Kartuch et al. 1991), bark parenchyma cells containing phenolics (Srivastava 1963;

Cheniclet et al. 1988; Franceschi et al. 1998; 2000; Krekling et al. 2000), and resin filled

ducts in both bark and sapwood (Wu and Hu 1997). However, as the attacking organisms

may overcome these constituents that provide an immediate resistance to invasion of the

bark, conifers have also evolved inducible defense mechanisms. Challenges can induce

changes in cell metabolism, such as synthesis of secondary resin (Raffa and Berryman

1982; Croteau et al. 1987; Klepzig et al. 1995) and production of new phenolics, leading to

changes in chemical composition (Brignolas et al. 1995a, b; Klepzig et al. 1995) or the

appearance of phenolic bodies in tissues where they normally are absent (Nagy et al.

2000). Challenges can also induce changes in the activity or patterns of cell division, and

in cell differentiation, resulting in the formation of new structures such as wound periderm

(Oven and Torelli 1994; Fahn 1990; Franceschi et al. 2000) and traumatic resin ducts (Reid

et al. 1967; Berryman 1969; Werner and Illman 1994; Alfaro 1995; Tomlin et al. 1998;

Christiansen et al. 1999a, Nagy et al. 2000, Krokene et al. 2003). Changes in cell

metabolism might be viewed as rapid defense responses geared to deal with the immediate

threat, while changes involving cell division and differentiation are slow, taking weeks to

months to be completed, and are aimed at containing the threat and enhancing the

constitutive defense of the plant against further attack.

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In a series of experiments we have studied defense mechanisms of conifer stems

using clonal stands of Norway spruce [Picea abies (L.) Karsten] as a model system. In this

system bark beetle attacks are simulated by wounding and inoculating with Ceratocystis

polonica (Siem.) C. Moreau, a virulent blue stain fungus that is associated with the spruce

bark beetle Ips typographus L. (Furniss et al. 1990; Krokene and Solheim 1998).

Biochemical (Brignolas et al. 1995a, b), physiological (Krokene et al. 1999, 2003) and

anatomical defense responses (Franceschi et al. 1998; 2000), can be examined under

controlled conditions in this system. In the phloem, changes in the activity of phloem

parenchyma cells, called polyphenolic parenchyma cells (PP cells) due to their phenolic

content (Krekling et al. 2000), are a major response to experimental challenges and bark

beetle attack. (Franceschi et al. 1998; 2000; Krekling et al. 2000). In the cambial zone

there is induction of traumatic resin duct (TD) formation, and these ducts eventually end

up in the sapwood (Christiansen et al. 1999a; Franceschi et al. 2000; Nagy et al. 2000).

The purpose of this study was to provide a more quantitative analysis of inducible

anatomical based defense reactions observed in the phloem and sapwood of Norway

spruce. Specifically, we wanted to describe how these responses vary with respect to the

type of challenge received, the genotype of the plant relative to resistance to fungi, and

over time and distance from the site of a challenge. The results are examined and analyzed

within the context of signaling pathways, based on anatomical properties of the living cells

of the phloem, cambium and sapwood. The observations presented provide further insight

into anatomically based defense reactions in conifers.

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Material and methods

Plant material

Experiments were carried out in June to September 1997 using 29-year-old Norway spruce

trees from a clonal stand at the Hoxmark plantation of the Norwegian Forest Research

Institute, Ås, Norway (the stand is described in Franceschi et al. 1998). Four clones, out of

15 clones tested for resistance by mass inoculation with C. polonica, were used. Clones

579 and 265 were considered more resistant, whereas clones 53 and 267 were considered

susceptible (H. Solheim, unpublished data). All trees were growing close to each other and

thus experiencing almost identical environmental conditions.

Experimental treatments

Three trees per clone were used in this experiment, one served as control while the stem of

the other two was experimentally wounded into the cambium by removing a plug of bark

(5 mm in diameter) with a cork borer (Wright, 1935). The wounds were inoculated with

malt agar (hereafter called agar) either sterile or colonized by the root rot fungus

Heterobasidion annosum (Fr. Bref.). Inoculations were made on June 1, 1997, in two rings

encircling the stem at 1.5 and 3 meters above ground with five inoculations per ring.

Sampling

To follow temporal and spatial events in inoculated trees, samples containing bark and

sapwood were collected 0, 2, 6, 16, 36, and 85 days after inoculation (DAI). Each

inoculation point was sampled once. On 0 DAI, a single sample (standard dimensions: 1.6

cm width, height and depth) was collected before inoculation. On 2-85 DAI, two samples,

one for each treatment, (dimensions: 1.6 cm width, 5 cm height and 1.6 cm depth)

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containing the upper half of the inoculation hole were collected. On 16-85 DAI, additional

samples (standard dimensions) were collected 10, 20 and 30 cm above the inoculation site.

Control trees were sampled only at 0 and 114 DAI by removing single samples (standard

dimensions) from opposite sides of the stem 1.5 meters above ground. As an internal

control in inoculated trees, one sample of fresh bark was collected 1.5 meters above the

uppermost ring of inoculations at 114 DAI. All samples were immediately placed in

fixative (2% paraformaldehyde and 1.25% glutaraldehyde in 50 mM L-piperazine-N-N’-

bis (2-ethane sulfonic) acid buffer, pH 7.2). In the laboratory, sub samples (4 mm width, 6

mm height, 6 mm depth) for light microscopy (LM) were cut with a fresh razor blade while

the samples were immersed in fixative, ensuring that all samples contained phloem,

cambium and at least one annual ring of xylem cells. Subsequently, all sub samples were

placed in fresh fixative at 20oC over night, then rinsed in the same buffer used in the

fixative, dehydrated with an ethanol series, infiltrated with L. R. White acrylic resin

(TAAB Laboratories, England), and polymerized at 60oC over night.

Microscopy

Sections (1µm thick) for light microscopy (LM), were cut from L.R. White-embedded

material with a diamond knife, and dried onto gelatin-coated slides. Half of the sections

were stained with Stevenel’s blue (Del Cerro et al. 1980) for conventional LM, while the

other half was left unstained for fluorescence microscopy. Immersion oil was used as a

mounting medium. The stained sections were examined and photographed with a Leitz

Aristoplan photomicroscope using phase contrast or bright field optics. Unstained sections

as well as the block faces from where the sections had been cut were also studied with a

confocal system (BioRad MCR 1024, ArKr-laser) attached to a Nikon TE 300 inverted

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microscope. Immersion oil was used to mount the L.R. White blocks onto a cover glass

with their sectioned face oriented down when using the inverted microscope.

Analysis of anatomical features

Various measurements were made in order to study temporal and spatial anatomical

changes throughout the experiment. A circumferential layer of PP cells is formed annually

and provides a marker for age of the phloem layers (Krekling et al. 2000), while the

interface of late wood to new sapwood provides a marker for cambial activity in the

treatment year (1997). To assess cambial activity, we counted the number of cell cross

sections per lane of cells (radial orientation), starting with the annual row of PP cells

formed in 1995 (PP95), including the cambium and tracheids produced in 1997 and 1996,

when present. The extent of TD production was expressed by the percentage of tracheid

lanes that contained ducts across the full tangential width of each section. To compare the

timing of TD initiation at different sampling positions, we counted the number of cell cross

sections per tracheid lane formed between last year’s growth ring and the row of TDs. In

the phloem, the number of mature PP cells (i.e. cells containing phenolic bodies) within 20

consecutive lanes of sieve cells was counted. We differentiated between PP cells found

within, and extra PP cells (PPex) (Krekling et al. 2000) produced between the annual

layers of PP cells.

Results

Anatomy at the start of the experiment

Phloem and sapwood anatomy for all clones was similar to previously examined fresh

samples from the same time in the growth season (Franceschi et al. 1998; Krekling et al.

2000). The phloem, containing circumferential layers of PP cells, blocks of sieve cells, and

8

radial rays, was separated from the sapwood by a narrow vascular cambium zone, 8-10 cell

rows wide. Four rows of (early) sieve cells and the current annual layer of PP cells (PP97,

one cell wide and containing phenolic inclusions) had already formed at 0 DAI, while

mature (late) sieve cells centripetal to PP97 were absent (Figure 1a). A few extra PP cells

were occasionally found scattered between the last eight annual layers of PP cells. The

cambium together with the newly formed sapwood was approximately 27 cell rows wide.

Scattered axial resin ducts were occasionally seen in the secondary xylem of untreated

Norway spruce samples, mainly within the last 1-5 rows of tracheids formed at the end of

the previous growth season (Figure 1a). Axial resin ducts were not found in the developing

sapwood or in the cambial zone at the start of the experiment.

Growth pattern

The growth rate varied both with time and tissue (Figure 2), but there were only small

differences between clones and between fungus and sterile inoculated material (data not

shown). On an average, approximately 0.06 rows of (late) sieve cells and 0.5 rows of

tracheids were produced per day, giving a ratio of phloem to xylem production of

approximately 1:8 during the experimental period. The number of sieve cell rows between

the annual PP cell layers was similar across years and clones (data not shown, but see

Krekling et al. 2000).

Early responses close to the inoculation site

The first visible effect of inoculation was a pronounced swelling of the cells in the cambial

zone, 4 mm above the inoculation point by 6 DAI (Figure 1b). For ray parenchyma cells,

the swelling was accompanied by anticline and pericline divisions and by the appearance

of phenolic bodies, not only within the cambial zone but also in ray parenchyma cells both

9

in the phloem and the xylem (not shown). PP cells also showed swelling which was

accompanied by compression of sieve cells (Franceschi et al, 1998, 2000). During later

stages the PP cells at this location lost their phenolic content and collapsed. This resulted in

a totally destroyed phloem mainly consisting of compressed sieve cell walls with orange

auto fluorescence (Figure 3a) compared to the green auto fluorescence of living phloem

seen at 0 DAI and at locations further away from the inoculation site at later sampling

dates (Figures 3b-c). Fungal hyphae could be observed in this region, but not 10 cm or

further away from the inoculation site. As the secondary cambium was destroyed close to

the inoculation site only data from locations 10, 20 and 30 cm above the inoculation site is

included in the following description.

Traumatic resin duct formation

Developmental features of TD formation were consistent for all trees and clones, and

observations from one clone (clone 053) will therefore be used for the description that

follows. By 16 DAI, tangentially oriented rows of TDs with secreting epithelial cells had

appeared in the xylem (Figures 4a-c). With time, there was an increase in the cross

sectional area of these TDs, reflecting the maturation of duct epithelial cells, expansion of

individual duct lumen and fusion of ducts. By 85 DAI, many of the epithelial cells had

developed thick lignified walls (Figure 3c). Developing TDs were frequently associated

with cells containing polyphenolics, and even some secretory epithelial cells accumulated

phenolics. Another significant observation was the development of a secondary row of

TDs close to the cambium (Figure 3b), primarily observed in the samples taken 10 cm

above the inoculation site in weak clones inoculated with fungus.

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Timing of traumatic resin duct initiation

Ten cm above the inoculation site the number of tracheid rows (28.5) formed between the

growth ring produced the year prior to inoculation and the first row of TDs was

approximately equal to the total number of cell rows (27, including cambial cells and

newly formed tracheids) found at the start of the experiment (Figure 2b). This indicates

that TD initiation took place in a row of cells positioned within the cambial zone of xylem

mother cells. There appeared to be a small delay in initiation of TD formation with

increasing distance from the inoculation site (Figures 4a-c). The number of tracheid rows

produced before the appearance of TDs increased from 28.5 rows 10 cm above the

inoculation site to 31.2 and 33.4 rows 20 and 30 cm above the inoculation site, respectively

(average values for fungal and sterile inoculations on 16, 36 and 85 DAI), giving an

increase of 0.2 rows/cm in the axial direction from the inoculation. Assuming an average

growth rate of 0.5 tracheid rows/day in the time interval 0 - 85 days (see Figure. 2b), this is

consistent with a TD developmental “wave” traveling in the axial direction with a velocity

of approximately 2.5 cm/day.

The number of tracheids produced before TD initiation was slightly lower after

fungal inoculations compared to sterile agar inoculations. Assuming similar growth rates in

the xylem for different trees and sampling positions, this implies that TD-formation was

initiated sooner after fungal than sterile inoculations. There appeared to be no difference in

the speed of the response of resistant and susceptible clones.

Traumatic resin duct quantity

The percentage of sapwood circumference that was covered by TDs decreased with

increasing distance from the inoculation site (Figures 4a-c and 5a). This was true for both

treatments and for all clones. Although both treatments induced extensive TD formation in

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all clones, inoculation with fungus led to an enhanced response compared to sterile

inoculations (Figures 5a and 6a). This was evident from an increase both in the number of

ducts and in the size of their lumens. The susceptible clones 053 and 267 appeared to

produce more TDs than the resistant clones 265 and 579 (Figure 6a), and, as mentioned

above, a secondary layer of TDs was frequently observed in susceptible clones at positions

close to the cambium in fungus-infected material at 85 DAI (Figure 3b).

Polyphenolic phloem parenchyma cell (PP cells) quantity

All clones produced a similar number of PP cells in the annual layers produced before

inoculation. The mean number of PP cells across 20 lanes of sieve cells in the tangential

direction varied little between clones (19.1-21.3), and was also very similar across years

(19.5-20.5).

Inoculation induced formation of extra PP cells within the layers of late sieve cells

produced after the formation of the annual layer of PP cells (Figures 4d-f). Inoculations

with H. annosum gave a stronger response than inoculations with sterile agar (Figure 5b),

and susceptible clones responded stronger than resistant clones (Figure 6b). Clone 267

responded much stronger to sterile inoculations than the other clones (Figure 6d), and

produced more extra PP cells in the years before the inoculation as well as in controls (data

not shown). Extra PP cells were absent in controls from all other clones. As for TDs, the

production of extra PP cells diminished with increased distance from the inoculation site

(Figures 4d-f and 5b). Unlike TDs and the annual rows of PP cells, however, the extra PP

cells did not form a tangential row, but were scattered among the late sieve cells (Figures

4d-f). This was true for both H. annosum and sterile agar inoculations.

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Discussion

This study demonstrates that both the TDs of the xylem and the PP cells of the phloem are

under dynamic regulation with respect to inducible defense responses to wounding and

fungal infection of Norway spruce stems. Monitoring the growth patterns of phloem and

xylem allowed us to reveal temporal and spatial characteristics of the development of these

defenses. Our data also indicate that these two anatomical based defense responses might

be induced by separate mechanisms.

The mechanism of TD induction and development is not well understood, and this

report provides new information on timing and spatial distribution, and demonstrates that

there is a differential response to wounding and pathogenic fungi, as well as a genetic

component to the intensity of the response. In this study, TDs always occurred as well-

defined tangentially oriented rows. Thus, all the ducts at the same distance from the

inoculation site are initiated synchronously within a tangential row of cells. By comparing

the number of cambial and tracheid cell rows at the start of the experiment with the number

of tracheid rows formed before the development of TDs we conclude that TDs are induced

in a narrow row of cells located within the cambial zone of xylem mother cells. This

indicates that a subset of xylem mother cells or their derivatives form a functional domain

that can be induced to synchronously form TDs.

Previous experiments have shown that TDs are formed both circumferentially and

centripetal to (Franceschi et al. 2000) and above inoculation sites (Nagy et al. 2000), but

these studies did not determine the strength of the response at different positions. This

study demonstrates that the amount of TDs and the degree of TD development decreased

with increasing distance from the inoculation site. Thus, the inductive stimulus for TD

formation arrives sooner and/or is stronger closer to the inoculation point, resulting not

only in more rapid development but also in the formation of more TDs closer to the origin

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of the stimulus. This spatial/temporal gradient suggests that some transportable signaling

agent mediates TD initiation, and propagates a developmental wave traveling at a speed of

approximately 2.5 cm/day in the axial direction.

The parameter (development of TDs) we are using to assess “signal transport” is

cellular differentiation, and thus cannot be strictly related to transport rate of a diffusible

substance. However, given that TD development appears to be restricted to

“symplasmically-coupled domains”, it seems likely that symplasmic transport of some

signaling agent takes place along the files of TD-competent cells in the cambium from the

initial point of induction. The absolute requirement for developmental coordination of a

subset of cells in a tightly restricted layer is consistent with a specialized symplasmic

domain within the cambial zone.

Formation of multiple TD layers supports our hypothesis of the involvement of a “TD-

competent” symplasmic domain. Normal cambial activity is usually restored after

formation of the first row of TDs, and it appears that a certain number of cambial zone

cells must be produced before a new symplasmic domain capable of TD formation is

produced. Multiple rows of TDs located close to the wound have previously been observed

within growth rings formed at the time of bark beetle attack and in at least three

consecutive years following the attack (Franceschi et al. 2000). In this experiment the

formation of a second layer of TDs was only observed close to fungus infected

inoculations in weak clones. Why and what causes formation of such secondary rows of

TDs is not clear, but we speculate it is related to continued signal production either caused

by fungal activity in the wound site or by multiple wounding introduced by repetitive

sampling.

The signaling agent responsible for induction of TD formation has not been

established. However, we have recently found that exogenous application of methyl

jasmonate (MJ) in the absence of wounding can induce TD formation in mature and young

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Norway spruce trees (Franceschi et al. 2002). Application of MJ resulted in TD formation

some distance from the site of application, and so we propose that jasmonate or some

compound induced by jasmonates, are the signaling agents responsible for TD induction. It

is possible that a methyl jasmonate derivative acts as a transportable signaling agent

inducing TD formation in the cambial region. This is also consistent with the attenuation of

TD development further away from the inoculation site as seen here, or away from the MJ

application zone as seen by Franceschi et al. (2002). Further studies needs to be done to

determine the actual signal transported from the site of initiation of the defense response.

Resin production is only one component of the defense systems in Norway spruce. As

soon as 6 days after inoculation phenolic bodies appeared in the ray parenchyma cells of

the phloem, the cambium and the xylem close (4 mm) to the wound. Subsequently, the PP

cells in the phloem at this location lost their content, and fluorescence microscopy

indicated that it was released to the cell walls of the surrounding collapsed sieve cells. This

induction of formation and mobilization of phenolic compounds may represent a strategy

to contain an attack locally, and may be similar to a hypersensitive response in the

immediate vicinity of a wound.

Previous experiments have highlighted PP cells as an important site of constitutive and

inducible defenses (Franceschi et al. 1998; 2000; Krekling et al. 2000). Careful spatial and

temporal analysis of PP cells in this study provided a new and surprising observation on PP

cells and induced defenses: the appearance of a large number of extra PP cells amongst the

sieve cells formed after inoculation. These extra PP cells were evident at a considerable

distance from the treatment site. As seen for TD formation, the induction of extra PP cell

formation showed a spatial gradient but required a much longer time (85 days) to become

apparent. The extra PP cells that formed after fungal inoculation may represent a strategy

of enhancing long-term resistance. Alternatively, they can be the result of disruption of the

15

normal developmental program of the cambium relative to phloem formation after TDs are

produced.

Our observations indicate that TDs and extra PP cells might be induced by separate

mechanisms. First, the time of formation is on completely different scales. TDs were

already formed 16 days after inoculation, while the extra PP cells were mainly observed

after 85 days. This might reflect the slower growth rate of phloem compared to xylem

(approx. 1:8), since the extra PP cells arise in the new phloem produced by the cambium

that is reorganized after TD initiation. And second, while TDs always appear in rows

parallel to the cambium, the extra PP cells are scattered randomly among the rows of late

sieve cells.

Inoculation with H. annosum gave a stronger response than sterile inoculation with

regard to both TD induction and formation of extra PP cells. This might reflect a simple

dose-response relationship with respect to wounding, as continuous fungal growth

obviously will increase the size of the wounded area (Christiansen et al. 1999b, Krokene et

al. 2001). However, it is also possible that the fungus releases, or causes the stem cells to

release, an inducible substance. In a dose-response experiment we have shown that pre-

inoculation with fungus results in local enhancement of resistance to subsequent

inoculations (Christiansen et al. 1999b, Krokene et al. 1999). While the mechanisms

behind this resistance have not been fully characterized, they seem to be related to PP cell

activation and TD induction, since resistance was seen in a time frame when induction of

these defenses were completed (Krokene et al. 2003).

The present study demonstrates that clonal genetic differences are reflected in the

degree of anatomical changes induced by inoculation, though not in a simple manner.

Quite surprisingly, susceptible clones produced more TDs and more extra PP cells in

response to fungal inoculations than resistant clones. The weakest clone (053) also showed

pronounced production of extra rows of TDs close to the cambium in H. annosum-

16

inoculated material. One possible explanation for the enhanced production of TDs and PP

cells in weak clones is that the pathogenic fungus was able to grow more extensively in

these clones, thus increasing the amount of wounding compared to the more resistant

clones.

In summary, this study provides new information on the temporal and spatial

induction of anatomical-based defenses in Norway spruce. The results provide some

insight into transport of the inductive stimulus and indicate that symplasmic domains are

involved in TD induction and formation. Differences in the response to sterile and fungal

inoculations and between relatively resistant and susceptible clones were documented and

discussed relative to defense capacity of spruce trees. Additional PP cells were produced in

the phloem after inoculation, but in a time frame more related to enhancing future

resistance than as an immediate response to the current attack. These results are likely

applicable to many species in the Pinacea where PP cells and axial resin ducts are

commonly observed (Srivastava 1962; Wu and Hu 1997). Our artificial inoculations on

clonal trees provide a good experimental system to further elaborate the mechanisms

responsible for resistance of conifer bark against invading organisms.

Acknowledgements This work was supported by grant no. 104023/110 from the Research

Council of Norway to H.S. The work is part of an international scientific effort on conifer

defense mechanisms (CONDEF group). The excellent technical assistance of Elisabeth

Reed Eng and Martin A. Krekling is kindly appreciated.

17

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Figures

Fig. 1 Normal anatomy of Norway spruce sapwood/phloem and early responses to inoculations (cross

sections). A Anatomy of the xylem, cambial zone and phloem on the day of inoculation. The current annual

circumferential layer of phloem parenchyma cells (PP97) had already been formed (annual layers produced in

the two previous years, PP96-95, are also shown). Scattered axial resin ducts (*) were occasionally seen within

the last rows of tracheids formed in the xylem in the previous growth season (X96). B Induction of cell

divisions in the cambial zone 6 days after inoculation. Close to (4 mm above) the inoculation site the ray

parenchyma cells (R) were swollen, and showed mitotic activity (arrow) and evidence of having undergone

anti- and pericline division (arrowheads). V - vacuole lined with darkly stained polyphenolic material, C -

cambium, S - sieve cell. (Bars 100 µm.)

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Fig. 2 Growth of Norway spruce trees over the course of the experiment expressed as the number of cell rows

produced in A phloem and B cambium and xylem. (Data points represent averages for all clones, sampling

positions and treatments, n=8. Bars indicate ± 1 SE).

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Fig. 3 Auto fluorescence in Norway spruce stems tissues in a susceptible clone (clone 53) 85 days after

inoculation with Heterobasidion annosum (cross sections). A The cambium is destroyed close to (4 mm

above) the inoculation site. The phloem cells are collapsed and the phloem polyphenolic parenchyma (PP)

cells have released their content, resulting in an orange-red fluorescence of the collapsed cells walls. R - ray

parenchyma cells, S - sieve cell. B Numerous extra polyphenolic parenchyma cells (*) were produced

centripetal to the annual layer of phloem polyphenolic parenchyma cells formed in 1997 (PP97). A second

row of immature, developing traumatic ducts (TD2) was frequently found close to the cambium 10 cm above

the inoculation site. C A well-developed tangential row of traumatic ducts (TD) interrupted the lanes of

tracheids further down in the new-formed sapwood. These ducts had thick lignified cell walls and were

associated with cells containing polyphenolic inclusions (arrows) having orange-red auto fluorescence in blue

light.

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Fig. 4 Formation of traumatic resin ducts (TDs) and extra polyphenolic parenchyma (PPex) cells at various

distances above the inoculation site (cross section). A-C In the xylem, a tangential row of TDs had developed

16 days after inoculation. This row was formed earlier 10 cm from the inoculation site (A) compared to 20

(B) and 30 (C) cm, where the ducts were fewer and less well developed (circled area in C) and located closer

to the cambium. D-F In the phloem, a similar gradient concerning the number of PPex was seen 85 days after

inoculation. The PPex cells were scattered among the late sieve cells produced after the formation of the

annual layer of polyphenolic parenchyma (PP97) cells laid down at the beginning of the growth season. Extra

PP cells were most numerous 10 cm above the inoculation site (D) and became less abundant further away (E

and F). PP96 – annual layer of PP cells produced in 1996, Camb. – cambium.

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Fig. 5 Defense responses in Norway spruce trees 85 days after inoculation with Heterobasidion annosum

(grey bars) or sterile wounding (white bars) (n = 8). A TD formation (expressed as the percentage of tracheid

lanes interrupted by TDs across the sections) and B formation of extra polyphenolic parenchyma (PPex) cells

(expressed as the number of PPex produced per 20 lanes of late sieve cell) decreased with distance from the

inoculation sites.

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Fig. 6 Formation of A traumatic resin ducts (TDs) and B extra polyphenolic parenchyma (PPex) cells in

Norway spruce clones 85 days after inoculation with Heterobasidion annosum (grey bars) or sterile

wounding (white bars). Clone 53 and 267 were susceptible to fungal infection, whereas clone 265 and 579

were more resistant. Data are averages for 3 sampling positions, 10, 20 and 30 cm above the inoculation site,

in two trees per clone (n = 2). Error bars are ± 1 SE.