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Mycorrhiza (1999) 8 :229–240 Q Springer-Verlag 1999
H.B. Massicotte (Y)
University of Northern British Columbia,
College of Science and Management,
Faculty of Natural Resources and Environmental Studies,
Prince George, BC, Canada V2N 4Z9,
e-mail: hugues6unbc.ca
L.H. Melville 7 R.L. Peterson
Department of Botany, University of Guelph,
Guelph, Ontario, Canada, N1G 2W1
T. Unestam
Swedish University of Agricultural Sciences, Department of
Forest Mycology and Pathology, Box 7026,
S-750 07 Uppsala, Sweden
ORIGINAL PAPER
H.B. Massicotte 7 L.H. Melville 7 R.L. Peterson
T. Unestam
Comparative studies of ectomycorrhiza formation in
Alnus glutinosa
and
Pinus resinosa
with
Paxillus involutus
Accepted: 22 October 1998
Abstract Mycorrhiza ontogeny and details of Hartig
net and mantle structure were compared in ectomycorr-
hizas synthesized in growth pouches between the broad
host range fungus Paxillus involutus and the tree spe-
cies European black alder (Alnus glutinosa) and red
pine (Pinus resinosa). In Alnus glutinosa, a paraepider-
mal Hartig net was restricted to the proximal (basal)
portion of first-order laterals; the hypodermal layer ap-
peared to be a barrier to fungal penetration. Phi-thick-
enings were present in some cortical cells but these
were not related to lack of fungal ingress into the cor-
tex. The mantle was often present close to the root
apex but in many roots it was loosely organized and
patchy. In several instances, the mantle formed around
the root apex was only temporary; renewed root
growth occurred without the formation of a mantle. In
Pinus resinosa, the Hartig net developed between corti-
cal cell layers of monopodial and dichotomously
branched first–order laterals. Fungal hyphae in the
Hartig net exhibited a complex labyrinthine mode of
growth. The mantle had a pseudoparenchymatous
structure and covered the root, including apices of di-
chotomously branched roots. The Paxillus–Pinus resi-
nosa interaction had all the characteristics of a compati-
ble ectomycorrhizal association. The Paxillus–Alnus
glutinosa interaction, however, showed only aspects of
superficial ectomycorrhizas, including the presence of a
minimal (sometimes absent) and mostly proximal Har-
tig net and variable mantle development. Sclerotia
were produced in the extraradical mycelium of Paxillus
involutus when associated with either Alnus glutinosa
or Pinus resinosa.
Key words Black alder 7 Red pine 7 Structure 7
Hartig net 7 Compatibility 7 Mycorrhiza
Introduction
Paxillus involutus, a fungus species most frequently
found in temperate zones of the Northern Hemisphere
(Laiho 1970), is regarded as a wide-host-range ectomy-
corrhizal symbiont associating with both angiosperm
and conifer hosts (Trappe 1962; Laiho 1970). Some re-
ports, however, have suggested that Paxillus involutus
is a facultative symbiont, based on observations that
sporocarps occur following trenching of host trees or in
the absence of host trees, although this was questioned
by Laiho (1970). Paxillus involutus is of interest as an
ectomycorrhizal symbiont because of its potential use
in biological control of various pathogenic fungus spe-
cies (Duchesne et al. 1988, 1989) and its ability to de-
grade lignin (Haselwandter et al. 1990), cellulose, and
proteins (Maijala et al. 1991). In addition, this species
forms sclerotia (Grenville et al. 1985a; Moore et al.
1991) that can be induced in vitro by a cold tempera-
ture treatment (Moore and Peterson 1992). Sclerotia
are of potential use as inocula and as a means of con-
serving fungus genotypes (Grenville et al. 1985b).
Paxillus involutus forms typical ectomycorrhizas
with various host tree genera including Pinus (Laiho
1970; Grenville et al. 1985b; Pargney and Gourp 1991;
Turnau et al. 1994; Shaw et al. 1995), Betula (Gaie
1977a; Brun et al. 1995), Salix (Gaie 1977b), Picea (Kif-
fer 1974; Marschner and Godbold 1995) and Quercus
(Branzanti and Zambonelli 1989). Reports on the asso-
ciation between Paxillus involutus and the genus Alnus,
however, have been variable. Laiho (1970) did not find
230
231
Figs. 1–4 Pinus resinosa roots colonized with Paxillus involutus
Fig. 1 Portion of a growth pouch, 17 days after inoculation,
showing numerous second-order mycorrhizal roots (arrowheads).
Inoculum plugs (*) are present; scale mm
Fig. 2 Portion of a root system, 34 days after inoculation, show-
ing monopodial (arrowhead) ectomycorrhizas and young dichoto-
mous second-order mycorrhizal roots (double arrowheads). Roots
are mostly colonized in the apical portions; scale mm
Fig. 3 Portion of a root system, 34 days after inoculation, show-
ing well-developed dichotomous second-order mycorrhizal roots
(arrowheads) as well as extensive colonization of the first-order
root (*). Note the patchy texture of the mantle on this first-order
root. A sclerotium (S) has developed; bar 1mm
Fig. 4 Portion of a root system, 34 days after inoculation, show-
ing older second-order dichotomous mycorrhizal roots that have
dichotomized once again. Many apices have grown out of their
mantle (arrowheads). Rhizomorphs (double arrowheads) are evi-
dent; bar 1mm
Paxillus involutus – Alnus ectomycorrhizas in nature,
and synthesis studies with Alnus glutinosa (L.) Gaertn.
and Alnus incana (L.) Moench failed to result in my-
corrhiza formation. Molina (1979, 1981), however, was
able to synthesize ectomycorrhizas between Paxillus in-
volutus and Alnus rubra Bong, Alnus glutinosa (L.)
Gaertn., Alnus incana (L.) Moench, Alnus sinuata (Re-
gel) Rydb., and Alnus rhombifolia Nutt. but all resulted
in very poor Hartig net formation. Godbout and Fortin
(1983), using the growth pouch method, obtained ecto-
mycorrhizas with good paraepidermal Hartig net devel-
opment when A. rugosa var. americana (Regel) Fern.
and A. crispa (Ait.) Pursh. seedlings, inoculated with
Frankia to induce nodule formation, were subsequently
inoculated with Paxillus involutus. Alnus serrulata
(Ait.) Willd. inoculated with Paxillus involutus formed
ectomycorrhizas with sporadic Hartig net development
(Murphy and Miller 1994). Several of the 16 field-col-
lected morphotypes of Alnus glutinosa recently charac-
terized in Germany exhibited a paraepidermal Hartig
net (Pritsch et al. 1997) but none appeared to belong to
Paxillus involutus.
Ectomycorrhiza formation between Paxillus involu-
tus and Alnus species appears to be variable, but no de-
tailed anatomical studies have been done. Differing re-
sults with Alnus glutinosa (Laiho 1970; Molina 1981),
and the dependency of ectomycorrhiza formation on
numerous abiotic and biotic factors (Smith and Read
1997), make it a good choice for a comparative anatom-
ical study. Pinus resinosa, known to form ectomycorrhi-
zas with Paxillus involutus (Grenville et al. 1985b) was
included for comparison.
Materials and methods
Plant material and ectomycorrhiza synthesis
Red pine (Pinus resinosa Ait.) seeds, obtained from Petawawa,
Ontario (45724b N, 75733b W, 70 m) and European black alder
[Alnus glutinosa (L.) Gaertn.] seeds, obtained from Turkey (posi-
tional data unrecorded), were germinated as described for Alnus
crispa by Godbout and Fortin (1983), with the exception that
H
2
O
2
surface-sterilization was performed for 40 and 20 min, re-
spectively.
Seedlings of Pinus resinosa were transferred, 10 days after ger-
mination, into growth pouches containing 10 ml of modified Me-
lin-Norkrans (MMN) solution without glucose (Marx and Bryan
1975). The mycobiont was grown and introduced as plugs into the
pouches as described previously (Massicotte et al. 1986). Forty-
two days after germination, seedlings were inoculated with Paxil-
lus involutus (Batsch.) Fr. using the strain CG-9 (CRBF, Univer-
sity Laval, Québec, Canada) isolated in 1980 in the vicinity of
Populus tremuloides Michx. (Godbout and Fortin 1983). Seed-
lings of Alnus glutinosa were transferred 8 days after germination
into growth pouches containing 10 ml of modified Crone’s miner-
al solution without glucose (Lalonde and Fortin 1972). Seedlings
were then inoculated with Frankia 14 days after germination and
with Paxillus involutus 32 days after germination. Alnus glutinosa
and Pinus resinosa were also separately inoculated with a second
strain of Paxillus involutus (CRBF-0262), isolated in the vicinity
of Larix laricina in 1980. Differences in timing for inoculation be-
tween the two hosts depended on the degree of root development
in the pouches.
Approximately 100 pouches of Alnus glutinosa out of 150 and
30 pouches of Pinus resinosa out of 50 were successfully colonized
in four different experiments over 3 years.
Growth conditions
Seedlings were grown under light (130 mE/m
–2
sec
–1
) on a 16-h
light–8-h dark cycle at 24 7C day–18 7C night temperatures. High
levels of humidity (60–80% RH) were maintained with a humidif-
ier. Additional nutrient solution was added to pouches as
needed.
External morphology and light microscopy
The external morphology of roots and ectomycorrhizas was ex-
amined with a Zeiss DR photodissecting microscope at intervals
of 2–3 days after inoculation. Samples were collected from a peri-
od up to 2 weeks after the appearance of a woolly mantle on Al-
nus glutinosa and up to 8 weeks on Pinus resinosa. Tissue was
fixed according to a procedure described previously (Massicotte
et al. 1986), then dehydrated in a graded ethanol series and em-
bedded in LR White Resin (London Resin Co.). Sections
(1–1.5 mm) were cut with glass knives and stained for light micros-
copy with 0.05% toluidine blue O in 1% sodium borate. More
than 20 samples of each ectomycorrhizal association were exam-
ined. The samples were collected from two separate sets of ex-
perimental syntheses for Pinus and from four separate experi-
ments in the case of Alnus.
Scanning electron microscopy
Samples were fixed and dehydrated as above, critical point-dried,
sputter coated with gold-palladium and viewed at 25 kV with a
JEOL 35C scanning electron microscope.
Results
Strain variation
Only strain CG-9 was successful in establishing some
level of root colonization on Alnus glutinosa. None of
the pouches inoculated with strain CRFB-262 were suc-
cessful. Both strains established mycorrhizas with Pinus
232
resinosa. We, therefore, focus the following descrip-
tions using the CG-9 strain only.
External morphology
Seedlings of Pinus resinosa grew moderately well in
plastic pouches and produced many first- and second-
order lateral roots, the majority of which became my-
corrhizal (Fig. 1). Typically, thin mantles were detected
as early as 4 days after inoculation and well-developed
mantles within 6–10 days after inoculation. Monopodial
and dichotomous second-order laterals were often my-
corrhizal within 1–2 weeks (Fig. 2). As the root system
developed, once-dichotomized (Fig. 3) and twice-dicho-
233
Figs. 5–12 Alnus glutinosa roots inoculated with Frankia and colo-
nized with Paxillus involutus
Fig. 5 Portion of a growth pouch showing Frankia-induced root
nodules (double arrowhead), first-order and second-order my-
corrhizal roots (arrowheads), 35 days after inoculation. An inocu-
lum plug (*), approx. 6 mm in diameter, and sclerotium (triple
arrowhead) are present
Fig. 6 Portion of a root system, 35 days after inoculation, show-
ing a well-colonized first-order mycorrhizal root (arrowhead) ad-
jacent to a non-mycorrhizal apex (double arrowhead) and a Fran-
kia-induced nodule (*); bar 0.5 mm
Fig. 7 Scanning electron micrograph (SEM) of root similar to
that indicated by the arrowhead in Figure 6. The mantle (*) adja-
cent to the root surface is compact and numerous loosely ar-
ranged hyphae (arrowheads) are attached to this; bar 50 mm
Fig. 8 A young mycorrhizal root, 13 days after inoculation, that
has barely formed a mantle at the root apex (arrowheads); bar
100 mm
Fig. 9 SEM of root similar to that indicated in Figure 8 showing
the interwoven mantle hyphae (arrowheads) confined to the root
apex. Portions of rhizomorphs (double arrowheads) are evident;
bar 25 mm
Fig. 10 A young mycorrhizal root, 13 days after inoculation, with
a thin, patchy mantle covering most of the root. The pigmented
apex (arrowhead) is still visible; bar 100 mm
Fig. 11 A young mycorrhizal root, 13 days after inoculation, with
a pigmented apex (arrowhead) that had just started to grow out of
a well-formed mantle; bar 100 mm
Fig. 12 An older mycorrhizal root with an apex that has grown
out of its mantle (*). Root hairs (arrowhead) have formed on the
exposed root tip; bar 250 mm
tomized (Fig. 4) mycorrhizal roots soon appeared. On
older non-mycorrhizal portions of the roots adjacent to
mycorrhizal roots (Fig. 3), hyphal growth on the root
often appeared patchy. Mycorrhizal roots often ap-
peared to have a fluffy, well-developed basal mantle
and an apex free of hyphae (Figs. 3, 4).
Seedlings of Alnus glutinosa grew more rapidly than
seedlings of Pinus resinosa under similar conditions,
and produced numerous first-, second-, and third-order
laterals, some of which became mycorrhizal (Fig. 5).
Nodules formed in more proximal regions of the prima-
ry root (Fig. 5), and thin mantles were detected as early
as 5 days following inoculation. On mycorrhizal roots,
well-developed fluffy mantles surrounded the apex
within 7–14 days (Fig. 6). SEM images revealed com-
pact hyphae on the root surface and loose mantle hy-
phae at the periphery (Fig. 7). Fungus colonization pat-
tern varied on different root tips (Fig. 8–12). Some
young mycorrhizal tips were colonized at the apex
(Fig. 8) and had a mantle of interwoven hyphae
(Fig. 9). Other roots were completely surrounded by a
patchy mantle (Fig. 10), or displayed renewed growth
of an apparently uncolonized root apex from a root
with a basal mantle (Fig. 11). Renewed apical root
growth was often followed by root hair initiation
(Fig. 12). Alnus seedlings did not always form mycorr-
hizas when mycelium was present on the root system
and a general darkening of the root system was often
observed.
Light microscopy
In Pinus resinosa, colonized lateral roots formed ecto-
myccorhizas with varying degrees of mantle and Hartig
net development (Figs. 13–15). Hartig net hyphae
showing characteristic labyrinthic growth occurred up
to the collapsed and “phenolized” endodermis
(Fig. 16). The mantle was unequal in thickness and oft-
en incorporated dark, collapsed, root cap cells (Fig. 16).
Paradermal sections revealed a pseudoparenchymatous
layer consisting of wide-diameter hyphae in the inner
mantle (Fig. 17), and the labyrinthic growth of hyphae
in the cortex (Fig. 18). Many ectomycorrhizal roots di-
chotomized to produce two apical meristems (Fig. 19).
Extension growth of the two axes occurred and each
had a well-developed mantle and intercellular penetra-
tion of Hartig net hyphae behind the apex (Figs. 20, 21)
in all cortical cell layers up to the collapsed endodermal
layer (Fig. 22). Septa and nuclei were present in Hartig
net hyphae (Fig. 22), and the inner mantle often incor-
porated densely staining material, presumably col-
lapsed root cap cells (Fig. 22).
Alnus glutinosa first-order mycorrhizal roots showed
varying degrees of mantle development, ranging from
patchy (Fig. 23) to complete (Fig. 24). In sub-apical re-
gions, the mantle was apposed against epidermal cells,
some of which had intensely staining walls (Fig. 24, 25).
In more basal portions, intercellular penetration of
Hartig net hyphae was sporadic and confined to the
epidermis (Fig. 26). In the sub-apical regions (Fig. 27),
the hyphae did not penetrate between epidermal cells,
the outer tangential wall of epidermal cells stained in-
tensely, the hypodermal layer showed shrinkage, and
thickenings (Phi-thickenings) occurred in the radial
walls in the second row of cortical cells (Fig. 30). More
proximally, the mantle was still apposed to epidermal
cells, some of which differentiated into root hairs, and
the Phi-thickenings were thinner and fewer in number
(Fig. 28). A still more proximal section revealed a com-
pact mantle and a well-developed intercellular epider-
mal Hartig net that showed both paraepidermal and
periepidermal characteristics (Fig. 29). Higher magnifi-
cation also indicated a darkening of tangential walls be-
tween the second and third layer of cells (Fig. 31) and a
mucilaginous matrix embedding the hyphae in the man-
tle (Fig. 31).
Discussion
Isolate CG-9 of Paxillus involutus used in this study
formed ectomycorrhizas with a full mantle and an Har-
tig net around epidermal and cortical cells in roots of
Pinus resinosa seedlings. This is consistent with results
obtained with a different Paxillus involutus isolate on
this tree species (Grenville et al. 1985b). Cortical cell
walls did not show thickening and vacuolar deposits
were not synthesized in response to colonization by the
fungus. These results, along with work on other Pinus
234
235
236
237
Figs. 13–18 Light microscopy of toluidine blue O (TBO)-stained
sections of Pinus resinosa monopodial second-order mycorrhizal
roots colonized with Paxillus involutus
Fig. 13 Longitudinal section of partially colonized root. A por-
tion of the mantle (double arrowhead) and intercellular penetra-
tion (arrowheads) can be seen on one side of the root. A few hy-
phae are also present on the first-order root (*); bar 100 mm
Fig. 14 Higher magnification of an adjacent section of the same
root as in Figure 13 showing a localized, partially-formed mantle
(*) and intercellular penetration (arrowheads); bar 25 mm
Fig. 15 A well-colonized longer root with a mantle (arrowheads)
enveloping most of the root and a well-developed Hartig net
mainly in the proximal region (double arrowhead); bar 100 mm
Fig. 16 A higher magnification of a proximal portion of Figure 15
showing intercellular penetration of the Hartig net up to the col-
lapsed endodermis (arrowhead). Note the labyrinthic mode of
growth (double arrowheads). Sloughed cells with dense material
(*) are present among mantle hyphae; bar 25 mm
Fig. 17 A paradermal section (at the inner mantle level) of a root
similar to the one shown in Figure 15. Hyphae have started to
swell and have formed a compact pseudoparenchymatous layer.
Nuclei (arrowheads) are obvious in some hyphae and dark mate-
rial, likely sloughed root cap cells, are included within the mantle
(*); bar 25 mm
Fig. 18 A paradermal section of a root similar to the one shown
in Figure 15, taken at the epidermal level. The labyrinthic growth
(arrowheads) of the Hartig net is obvious between the epidermal
cells. Note the compact portion of the mantle (*); bar 25 mm
Figs. 19–22 Light microscopy of TBO-stained sections of Pinus
resinosa second-order mycorrhizal roots colonized with Paxillus
involutus
Fig. 19 A root that has dichotomized showing two apical meris-
tems (*). The root is well covered with mantle (double arrow-
heads) and has intercellular hyphal penetration (arrowheads)
present up to the level of root splitting; bar 100 mm
Fig. 20 Older dichotomous root with elongated branches. A
mantle (arrowheads) surrounds both branches and the un-
branched base. Two meristems (double arrowheads) are present;
bar 250 mm
Fig. 21 Higher magnification of an elongated branch shown to
the left in Figure 20 with a well-developed meristem (*). The
mantle (double arrowheads) envelopes the branch and Hartig net
hyphae (arrowheads) are present close to the meristem; bar
100 mm
Fig. 22 Higher magnification of a portion of root indicated in
Figure 21 showing Hartig net up to the collapsed endodermis (*).
Nuclei (arrowheads) and septa (double arrowhead) are present in
branched Hartig net hyphae; bar 25 mm
Figs. 23–26 Light microscopy of longitudinal TBO-stained sec-
tions of Alnus glutinosa first-order mycorrhizal roots colonized
with Paxillus involutus
Fig. 23 Long root showing a sporadically-developed mantle (ar-
rowheads), without detectable intercellular penetration; bar
100 mm
Fig. 24 Shorter root showing a well-developed mantle (double
arrowheads) covering the root apex. The root apical meristem (*)
is obvious. Sporadic intercellular penetration (arrowheads) is
present in the basal portion of the root; bar 50 mm
Fig. 25 Higher magnification in the sub-apical portion of a root
similar to the one shown in Figure 24 showing the mantle (*) ap-
posed on epidermal (E) cells. Hyphal tips (arrowheads) have
started to penetrate between epidermal cells; bar 10 mm
Fig. 26 Higher magnification in basal portion of a root similar to
the one shown in Figure 24 showing intercellular penetration up
to the hypodermis (*) and some labyrinthic growth (arrowheads)
of Hartig net hyphae; bar 10 mm
and Picea species (Kiffer 1974; Marschner and Godbold
1995), indicate that this fungus forms typical ectomy-
corrhiza features, and presumably is an effective sym-
biont with these conifer genera. The situation is similar
to that with several angiosperm genera including Betula
(Gaie 1977a; Brun et al. 1995), Salix (Gaie 1977b) and
Quercus (Branzanti and Zambonelli 1989), and con-
firms the broad host range typical of Paxillus involutus
(Laiho 1970).
In specific conditions tested in the synthesis experi-
ments (MMN without glucose), we observed two differ-
ent outcomes with Paxillus involutus CG-9 and Alnus
glutinosa. The first outcome was observed repeatedly in
several pouches: the entire root system was colonized
by a thin covering of hyphae, but root tips were not col-
onized to form an ectomycorrhiza structure. The sec-
ond outcome was the formation of more typical ecto-
mycorrhizal rootlets, with a patchy and variable mantle.
In some cases, the appearance of stable ectomycorrhiza
was very transitory, as the apex would often grow
through the mantle. These mycorrhizas usually had a
sporadic paraepidermal Hartig net in the basal (proxi-
mal) portions of the rootlet. This is in agreement with
previous work (Godbout and Fortin 1983; Massicotte et
al. 1986, 1989a,b; Pritsch et al. 1997), even though deep-
er Hartig net penetration has been reported on Alnus
rubra (Miller et al. 1991) and Alnus sinuata (Helm et al.
1996). Pritsch et al. (1997) also documented an unusual
instance of intracellular penetration in epidermal and
cortical cells in a Lactarius lilacinus morphotype. More
observations are required from field and lab synthesis
material to clarify the pattern of root colonization on
alder.
The mantles we observed were often patchy and
very loosely organized. Molina (1981) described the
mantle formed between these symbionts as ‘irregular’.
Where the mantle interfaced with epidermal cells, the
latter often developed intensely staining walls, a feature
indicated as well in the micrograph of an ectomycorrhi-
za between Paxillus involutus and Alnus serrulata
(Murphy and Miller 1994) and in written descriptions
of ectomycorrhizas between Paxillus involutus and a
number of Alnus species (Molina 1981). Duddridge
(1986) reported similar results in interactions between
Suillus grevillei (Klotzsch) Sing. and seedlings of both
Pseudotsuga menziesii (Mirb.) Franco and Pinus sylves-
tris L. Deposition of phenolic compounds in plant cell
walls and vacuoles frequently indicates an incompatible
interaction between ectomycorrhizal fungi and host
roots (Nylund and Unestam 1982; Malajczuk et al.
1984; Duddridge 1986). It is noteworthy that several at-
tempts in processing our mycorrhizal alder roots from
this experiment, using either Spurr’s or Epon as an em-
bedding medium, failed. Only protocols using LR
White resin were successful for this recalcitrant materi-
al, perhaps because of the presence of phenol-like com-
pounds in the epidermis. In spite of the deposition of
intensely staining material in epidermal and cortical
cell walls, some hyphae were able to penetrate between
238
239
Figs. 27–31 Light microscopy of transverse TBO-stained sections
of Alnus glutinosa first-order mycorrhizal roots colonized with
Paxillus involutus
Fig. 27 Sub-apical portion of a root similar to the one shown in
Figure 24 showing a well-developed mantle (double arrowheads)
apposed against the epidermis (E). Note the maturing protoxylem
elements (arrowheads) in the stele and Phi-thickenings (arrows)
along cortical cell walls; bar 50 mm
Fig. 28 Section taken more proximally than the one in Figure 27
showing the loose mantle (*) apposed to epidermal (E) cells,
some of which have developed into root hairs (arrowheads). No
obvious intercellular penetration is present at this level. Phi-thick-
enings are sporadic (arrows); bar 50 mm
Fig. 29 Section taken more proximally than the one in Figure 28
showing a compact mantle (*) and obvious intercellular penetra-
tion of Hartig net hyphae (arrowheads) up to the hypodermis
(H). Note the differentiated metaxylem elements (double arrow-
heads) in the stele. Root hairs and Phi-thickenings are not present
at this level; bar 50 mm
Fig. 30 Higher magnification of Figure 27 showing the loose hy-
phal structure of the mantle (*), hyphae that had started to pene-
trate between epidermal cells (E), collapsed cells (likely due to
suberin walls) of the hypodermis (H), a third layer of cells with
Phi-thickenings (double arrowheads). Note the deposition of ex-
tracellular material (arrowheads) between the epidermis and pen-
etrating hyphae; bar 10 mm
Fig. 31 Higher magnification of Figure 29 showing the intercellu-
lar penetration of Hartig net hyphae (arrowheads) up to the sec-
ond layer of root cells, and a thick and compact mantle (*) sur-
rounding the root. Tangential walls between the second and third
layer of cells stain intensely; bar 10 mm
epidermal cells to form a limited Hartig net; wall appo-
sitions, a frequent incompatible response (Nylund et al.
1982), were not formed. The Hartig net found in some
roots was never very extensive and rarely showed laby-
rinthic branching, in agreement with the observations
of Molina (1981) for these same symbionts.
In the Paxillus involutus–Alnus glutinosa mycorrhi-
zas synthesized in this study, epidermal cells did not
show marked radial enlargement, a feature typical of
some Alnus species colonized by other fungus sym-
bionts e.g. Alnus crispa colonized by Alpova diploph-
loeus (Godbout and Fortin 1983; Massicotte et al.
1986), and of most angiosperm species forming ectomy-
corrhizas (Smith and Read 1997).
In a recent descriptive study of Alnus glutinosa field
morphotypes (Pritsch et al. 1997), photographic evi-
dence suggests that for almost all morphotypes, a pa-
raepidermal Hartig net develops, and radial enlarge-
ment of epidermal cells is variable among morpho-
types. A comparison of other fungus symbionts of Al-
nus glutinosa for this feature might determine if radial
elongation of epidermal cells can be used as a good in-
dicator for Alnus--fungus symbiont compatibility. Also,
monitoring the formation of extracellular fibrillar mate-
rial that bridges hyphae and the root surface during
contact (Lei et al. 1991) could help determine the com-
patibility of symbioses with Alnus species. In the pres-
ent study of root apices of Alnus glutinosa, the forma-
tion of what appeared to be transient ectomycorrhizas,
i.e. the apex was able to grow out of the mantle and
initiate root hairs from the protoderm, may indicate a
certain degree of incompatibility. The observations
made in the present study support the view that ecto-
mycorrhiza formation in the genus Alnus is more fun-
gus-specific than with many tree species (Molina
1981).
In previous synthesis experiments with Alpova di-
plophloeus, a reputedly genus-specific fungus for Alnus
spp. (Molina et al. 1992), ontogenetic analysis revealed
that the fungus colonized the root readily and initiated
the paraepidermal Hartig net in close proximity to the
apex, both on Alnus crispa (Massicotte et al. 1986) and
Alnus rubra (Massicotte et al. 1989a,b). In the experi-
ments reported here on Alnus glutinosa, the location of
the minimally developed Hartig net was confined to
proximal (basal) regions of the root. Godbout and For-
tin (1983) reported similar observations with Alnus
crispa and Alnus rugosa colonized by Paxillus involu-
tus. The colonization pattern within the root between
the broad-host-range Paxillus involutus and the genus-
specific Alpova diplophloeus is different, and seems to
be independent of the host tested.
Evidence that a functional relationship can be estab-
lished between Paxillus involutus and Alnus glutinosa is
provided by the work of Arnebrant et al. (1993) in
which nitrogen fixed by the actinorrhizal species Alnus
glutinosa was translocated to Pinus contorta Doug. Ex
Loud seedlings via an interconnecting mycelium. How-
ever, it was demonstrated recently that the net transfer
of nitrogen between Alnus incana and Pinus sylvestris
is dependent upon the nutritional status of pine and
that mycorrhiza-mediated nitrogen transfer is low and
may not be significant to the fitness of the “receiver”
plant (Ekblad and Huss-Danell 1995).
In the present study, the formation of sclerotia in the
extensive extraradical mycelium network associated
with both Alnus glutinosa and Pinus resinosa might be
indirect evidence that Paxillus involutus acquires car-
bon from these hosts. However, labelling experiments
would be required to confirm this because this fungus is
known to be able to degrade lignin (Haselwandter et al.
1990) and cellulose (Maijala et al. 1991), which are
components of the paper wick in the growth pouches.
Paxillus involutus, a broad host range ectomycorrhi-
zal fungal species, exhibits properties such as sclero-
tium production (Grenville et al. 1985a), potential use
in biocontrol of pathogenic fungal species (Duchesne et
al. 1988, 1989), and the ability to sequester heavy me-
tals (Turnau et al. 1994). These features, and the differ-
ent patterns of colonization between gymnosperms and
angiosperms such as Alnus spp. make it an important
fungus species for further work in the context of bio-
chemical and genetical responses between host and
fungus.
Acknowledgements This work was partially funded by a stipend
from the Swedish Institute and from the Skogs-och Jordbrukets
ForskningsRåd (SJFR) to H.B.M, and by a Natural Sciences and
Engineering Research Council of Canada operating grants to
R.L.P. and H.B.M.
240
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