Tree Physiology 20, 123–129
© 2000 Heron Publishing—Victoria, Canada
Dynamics of symbiotic establishment between an IAA-overproducing
mutant of the ectomycorrhizal fungus Hebeloma cylindrosporum and
Pinus pinaster
1
1
1
2
H. TRANVAN, Y. HABRICOT, E. JEANNETTE, G. GAY and B. SOTTA
1,3
1
Université P. et M. Curie PARIS VI, UMR CNRS 7632, Laboratoire de Physiologie du Développement des Plantes, 4 place Jussieu, T 53 E5, casier
156, 75252 Paris cedex 05, France
2
Université Claude-Bernard LYON 1, UMR CNRS 5557, Laboratoire d’Ecologie Microbienne du Sol, 43 Boulevard du 11 Novembre 1918, 69622
Villeurbanne cedex, France
3
Author to whom correspondence should be addressed
Received November 26, 1998
Summary To clarify the early steps of symbiotic establishment, we studied the dynamics of Pinus pinaster (Ait.) Sol. tap
root colonization and mycorrhiza formation by an IAA-overproducing mutant of the ectomycorrhizal fungus Hebeloma
cylindrosporum Romagnesi and by the corresponding wild
type strain. Differences between wild type and mutant strains
were quantitative rather than qualitative and were detected two
days after inoculation. Both fungal strains established a typical
Hartig net when they colonized the tap roots. Consequently,
colonized tap roots exhibited features of a true mycorrhiza and
fungal colonization enhanced plant growth. Fungal colonization and Hartig net formation were more rapid with the mutant
than with the wild type. Colonization, especially with the mutant strain, increased rhizogenesis and the production of
mycorrhizas. The mutant formed a hypertrophic Hartig net in
tap roots and mycorrhizal short roots and we obtained evidence
that the process of short root transformation into mycorrhiza
started before their emergence from the tap root. Hyphae of the
Hartig net in the tap root penetrated the cortex of young lateral
roots at the beginning of their elongation, after the endodermis
layer broke under the pressure of the elongating lateral root.
Colonization was inhibited when triiodobenzoic acid was
added to the culture medium, providing circumstantial evidence that auxin is involved in mycorrhiza formation.
Keywords: auxin overproducer mutant, growth, Hartig net,
mycorrhiza, rhizogenesis, triiodobenzoic acid.
Introduction
In temperate forests, most trees are symbiotically associated
with ectomycorrhizal fungi. Although this symbiosis, which
improves the growth of the trees, is widespread, the mechanisms involved in its formation are poorly understood (Martin
et al. 1997). Several theories have been suggested but none of
them have been experimentally assessed. The hormonal theory of mycorrhiza formation (Slankis 1973) stipulates that
auxin released by the fungal associate is responsible for the
typical morphology of ectomycorrhizas and is necessary for
mycorrhiza formation. According to this theory, environmental conditions that favor fungal auxin production would also
favor mycorrhiza formation. This theory has never been rigorously tested (see reviews by Harley and Smith 1983, Gogala
1991, Beyrle 1995). To help clarify the role of fungal auxin in
mycorrhizal development, Durand et al. (1992) selected and
studied auxin-overproducing mutants of the ectomycorrhizal
fungus Hebeloma cylindrosporum Romagnesi. These mutants
overproduce tryptophan and metabolize part of it to IAA. Gay
et al. (1994) isolated mono- and dikaryotic progeny of these
mutants and observed that mean mycorrhizal activity was
higher in IAA-overproducing mutants than in wild type
strains. From these results, Gay et al. (1995) concluded that,
although mycorrhiza formation is under polygenic control,
auxin overproduction gives the mutants an advantage for mycorrhiza formation. Karabaghli-Degron et al. (1998) reported
further evidence supporting the role of auxin in mycorrhiza
formation when they demonstrated that the IAA transport inhibitor triiodobenzoic acid inhibited auxin-induced lateral
root formation and mycorrhizal development in the Picea
abies (L.) Karst–Laccaria bicolor (Maire) Orton symbiotic
association.
IAA-overproducing mutants of Hebeloma cylindrosporum
are useful in determining the role of fungal IAA in the establishment of mycorrhizal associations, and their high
infectivity makes them a suitable tool for studying the early
steps of mycorrhiza formation (Gea et al. 1994). A detailed description of mycorrhiza infection processes is a prerequisite
for determining the mechanisms involved in mycorrhiza formation (Harley and Smith 1983). Although many
ectomycorrhizal associations have been described, several aspects of the process remain unclear because the sequence of
events during root colonization depends on the type of ectomycorrhizal association. For instance, according to Horan et
al. (1988), mycorrhiza formation in Eucalyptus globulus
subsp. bicostata (Maid. et al.) by Pisolithus tinctorius Coker
124
TRANVAN, HABRICOT, JEANNETTE, GAY AND SOTTA
and Couch or Paxillus involutus (Batsch ex Fr.) Fr. starts with
chemical modification of root cap cells before the mycelium is
attached to the root apices. This modification is followed by
formation of the sheath, swelling of the apices of lateral roots,
an intercellular penetration of hyphae between root cap and
epidermal cells, and the formation of a Hartig net. Although
Brun et al. (1995) observed a similar process in the P.
involutus–Betula pendula (Roth.) association, Nylund and
Unestam (1982) reported that the Hartig net appeared before
the fungal mantle in Picea. Recently, Karabaghli-Degron et al.
(1998) found colonization in the cortex of the main root in the
P. abies–L. bicolor association, and similar features have been
reported in other symbiotic associations (Robertson 1954,
Wilcox 1968, Wong et al. 1989) and in the ectendomycorrhiza
Pinus resinosa Ait.–Wilcoxina mikolae (Yang and Wilcox)
Yan and Krof association (Piché et al. 1986). However, it is
not known whether hyphae present in the cortex of a colonized
tap root can cause mycorrhizal development in lateral roots.
We studied the dynamics of the plant–fungus interaction
from fungal inoculation of the tap root to mature mycorrhiza
formation. We took advantage of the high infectivity of an
IAA-overproducing mutant of H. cylindrosporum and its ability to form a hypertrophic Hartig net when in association with
Pinus pinaster (Ait.) Sol. (Gea et al. 1994), to identify the
steps leading to the transformation of a normal root to a mycorrhiza. To assess the validity of our observations, we compared mycorrhiza formation of the mutant strain of
H. cylindrosporum with that of the corresponding wild-type.
We found evidence that infestation of the tap root by mycelium of the ectomycorrhizal fungi is followed by
mycorrhization of lateral roots prior to their emergence. Our
results corroborate the premise that auxin is a key factor in colonization.
tilled water and germinated in 90-mm petri dishes on sterile
water agar (10 g l –1), supplemented with 5.5 mM glucose to
facilitate detection of contaminants. The petri dishes were incubated in the dark for 15 days, at 18 ± 1 °C, then sealed with
parafilm (American National Can Co., Greenwich, CT) and
placed in a growth chamber (18 ± 1 ° C, 16-h photoperiod and
35 µmol m –2 s –1). Four weeks after germination, the plants
had 3–4 cm-long tap roots and were used for ectomycorrhizal
syntheses.
Ectomycorrhizal syntheses
Ectomycorrhizal syntheses were performed as described by
Gay et al. (1994) with some modifications. The plants were
placed in 140-mm diameter petri dishes on cellophane-covered sterile Melin-Norkrans medium, to prevent the penetration of roots into the medium. When necessary, the medium
was supplemented with triiodobenzoic acid (TIBA,
5 × 10 –5 M) before autoclaving. Plants were inoculated by
covering the entire tap root with a strip of mycelium-covered
cellophane. One strip was placed upside down on each root
and the cellophane was removed, leaving the roots covered
with the mycelium. Petri dishes containing inoculated plants
were sealed with parafilm, partially covered with aluminum
foil to protect the roots from direct illumination, and placed at
an angle of 45° in a growth chamber (22 ± 1 °C, 16-h
photoperiod and 75 µmol m –2 s –1). For each experiment, 12
plants were treated with each fungal strain. Three weeks after
inoculation, the length of the tap root was measured, and the
numbers of lateral roots and mycorrhizas were counted with
the aid of a lens. The mycelium was then removed with forceps and the roots and aerial parts of the plants were weighed.
Each experiment was repeated twice.
Histological observations
Materials and methods
Fungal strains and precultures
We used two strains of the ectomycorrhizal fungus
H. cylindrosporum. A wild type monokaryotic strain (h1) obtained from the in vitro fruiting dikaryon HC1 (Debaud and
Gay 1987) and an IAA-overproducing monokaryotic mutant,
h1 FIR4 F1 331 (abridged as 331 in this work), obtained from
the monokaryon h1 (Gay et al. 1994). Both strains were maintained on Oddoux’s medium (1957) at 4 °C, in the dark. Mycelium used to inoculate root systems was grown in petri dishes,
on a sterile sheet of cellophane covering Melin-Norkrans agar
(10 g l –1) medium (Norkrans 1949). Each petri dish was inoculated with an agar plug obtained from the margin of a
fast-growing culture on Oddoux’s medium. After a 4-week
culture period, two mycelium-bearing cellophane strips (80 ±
10 mm) were cut tangentially at the edges of the fungal culture
and used as inoculum.
At Days 1 and 3 and Weeks 1 and 3 after inoculation, tap roots
were cut into 5-mm segments, from the neck to the apex. The
samples were fixed with Navaschine’s fixative (1% chromic
acid:40% formaldehyde:acetic acid, 10/4/1, v/v) for 24 h,
washed in running tap water for 24 h, dehydrated in an ethanol
series, embedded in paraffin, and cut in serial sections (10 µm
thick) with a steel-knife-equipped microtome (Minot, Paris,
France). The sections were dewaxed and stained with either
Trypan blue (0.1% in lactic acid) or Cotton blue (1% in lactic
acid) for 30 min, then counterstained for 5 min with 0.5%
Congo red in ethanol. For more precise observations,
semi-thin sections were prepared from about 3-mm-long segments cut from mycorrhiza-bearing parts of tap roots, embedded in LR White resin, cut in 2 µm sections with a Diatome
Histo ultramicrotome (Leica Microsystems GmbH, Nussloch,
Germany), and stained with methylene blue:Azur blue
A:Safranine (Warmke and Lee 1976).
Results
Host plant cultivation
Half-sib seeds of P. pinaster were soaked in water for 48 h at
4 °C, and sterilized with 0.2% HgCl2 for 5 min, followed by
30% H2O2 for 15 min. Seeds were then rinsed in sterile dis-
Fungal effects on plant growth and development
Both the H. cylindrosporum wild-type and mutant strains
stimulated elongation of the tap root and increased rhizogenic
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SYMBIOSIS DYNAMICS BETWEEN AN ECTOMYCORRHIZAL FUNGUS AND CLUSTER PINE
activity of P. pinaster seedlings. Irrespective of the fungal associate, inoculated seedlings formed about three times more
short roots than uninoculated controls, three weeks after inoculation (Table 1). The IAA-overproducing mutant formed
about three times more mycorrhizas than the corresponding
wild type (9.4 versus 3.6 mycorrhizas per seedling). Both the
wild type and the IAA-overproducing mutant fungal strains
stimulated seedling growth by about 30%. Shoot growth was
only increased about 20% (Table 1), but biomass of inoculated
root systems was about twice that of uninoculated root
systems. ynamics of root system colonization and mycorrhiza
formation
One day after inoculation with the IAA-overproducing mutant, fungal hyphae were observed growing from the inoculum
to the tap root (Figure 1a). One day later, hyphae entered the
epidermis through cracks and reached the cortex of the root,
near the cracks (Figure 1b). On Day 3 after inoculation, a
Hartig net structure appeared below the entrance points (Figure 2a). On Day 7 after inoculation, discontinuous patches of
well-developed, multilayered Hartig net could be seen in the
cortex of the tap root (Figure 2b). The Hartig net seemed to develop radially under entrance points rather than tangentially
and almost reached the endodermis. No wound tissue appeared below the breaks in the epidermis. Three weeks after
inoculation, the tap root was completely colonized by a
well-developed regularly multilayered Hartig net that completely surrounded each cortical cell and always reached the
endodermis (Figure 2c).
The dynamics of tap root colonization by the wild type
monokaryon h1 were comparable with those observed with
the mutant, except that colonization was much slower. Three
days after inoculation, no fungal hyphae could be seen inside
tap roots (Figure 2d), even near cracks (not shown). The first
stages of fungal colonization were observed only on Day 7 after inoculation (Figure 2e). As observed with the mutant, the
fungus preferentially penetrated through cracks and spread in
the cortex mostly in the radial direction. The extension of
Hartig net on Day 7 was comparable with that observed with
the mutant on Day 3 after inoculation (see Figure 2a). Three
weeks after inoculation, fungal hyphae of the wild type
monokaryon had colonized the whole cortex of tap roots,
forming a typical Hartig net, and had reached the endodermis
(Figure 2f). Compared with the mutant (see Figure 2c), the
wild type strain formed a poorly developed Hartig net. Although the Hartig net was well developed in the intercellular
spaces, it generally did not completely surround each cortical
125
Figure 1. Cross sections through Pinus pinaster tap roots inoculated
with the IAA-overproducing mutant strain 331 of Hebeloma
cylindrosporum. (a) Fungal hyphae from the inoculum at the root surface, 1 day after inoculation. (b) Hyphae in a crack in the epidermis
(arrow) and cortex of the root near the crack, 2 days after inoculation.
Abbreviations: CC = cortex cell; and H = hyphae. Bar = 50 µm.
cell.
We compared the early stages of short root formation in tap
roots inoculated with the IAA-overproducing mutant and
showing either a well-developed Hartig net (Figure 3) or having no Hartig net (see Figure 5). Lateral roots always originated from the division of a pericycle cell in front of a
protoxylem pole. In plants inoculated with the IAA-overproducing mutant, lateral roots appeared in a part of the tap root
having a well-developed Hartig net (Figure 3a). Once initiated, but before growing out of the vascular cylinder, lateral
roots quickly acquired the typical shape of a short root; i.e.,
they had a rounded apex covered by a single-layered cap.
When elongating, the short root pushed the endodermis into
the cortex (Figure 3b). Before breakage of the endodermis, the
lateral roots growing through a cortex with a well-developed
Hartig net were completely devoid of fungal colonization.
Under the pressure of the elongating young root, the
endodermis of the tap root broke at the basal part of the lateral
root, just behind the root cap (Figure 3c), and a new connection was immediately established between the endodermis of
the tap root (cells E) and the endodermis of the lateral root
(cells E′). Breaking the endodermis of the tap root allowed
fungal hyphae of the Hartig net of the tap root to colonize the
lateral root cortex (Figure 3c). Finally, short roots formed on a
tap root having a Hartig net were colonized and could therefore be considered to be engaged in a mycorrhizal process
even before emerging from the tap root. When the short root
Table 1. Rhizogenesis, mycorrhizal development, and growth of Pinus pinaster plants after 3 weeks of co-culture with the IAA-overproducing
monocaryotic mutant 331 of Hebeloma cylindrosporum, the corresponding wild type strain h1 of Hebeloma cylindrosporum, or no mycorrhizal
fungus (control). Values represent means (± confidence limits, Student’s t-test, α = 0.01, n = 24). Differences between means followed by an asterisk (*) are not significant (α = 0.01).
Control
WT h1
Mutant 331
Number of
short roots
Number of
mycorrhizas
Tap root length
(mm)
Plant fresh
weight (mg)
Shoot fresh
weight (mg)
Root fresh
weight (mg)
12.7 ± 5.7
33.5 ± 7.0 *
27.9 ± 9.1 *
0
3.6 ± 1.2
9.4 ± 2.6
57.6 ± 5.1
81.0 ± 6.3
100.1 ± 10.0
219.5 ± 22.3
291.9 ± 19.6 *
289 ± 18.8 *
186.6 ± 18.0
227.5 ± 14.2 *
219.9 ± 14.3 *
32.9 ± 5.4
64.4 ± 7.6 *
69.1 ± 9.2 *
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TRANVAN, HABRICOT, JEANNETTE, GAY AND SOTTA
Figure 2. Cross sections through Pinus pinaster tap roots colonized
by the the IAA-overproducing mutant 331 of Hebeloma cylindrosporum (a–c) or by the corresponding wild-type strain h1 (d–f). (a)
Typical Hartig net structure near the cracks in the cortex of plants 3
days after inoculation with the mutant strain. (b) Multilayered Hartig
net growing radially toward the endodermis, 7 days after inoculation
with the mutant strain. (c) Typical mycorrhiza structure with well-developed Hartig net in the cortex of the tap root and a thin mantle covering the root 3 weeks after inoculation with the mutant strain. (d) Tap
root devoid of fungal hyphae 3 days after inoculation with the
wild-type strain. (e) Hyphae in a crack in the epidermis (arrow) and
Hartig net in the cortex of the tap root 7 days after inoculation with the
wild-type strain. (f) A Hartig net in the cortex of the tap root 3 weeks
after inoculation with the wild-type strain. Abbreviations: CC = cortex cell; E = endodermis layer; HN = Hartig net; and arrows indicate
cracks in the epidermis. Bar = 50 µm.
emerged from the tap root under these conditions (Figure 4a),
it already presented a typical Hartig net (arrows) in the outer
cortex of its basal part. At that time, the emerging part of the
mycorrhiza was completely covered by a root cap (star) and by
remnants of the tap root, which prevented any colonization by
extramatrical hyphae. Three weeks after inoculation, the short
root had formed dichotomously branched mycorrhizas that
had a typically multilayered Hartig net, particularly in its basal
part, inside the cortex of the tap root (Figure 4b). By comparison with mycorrhizas formed by the wild type strain, mutant
mycorrhizas were much shorter and they had a larger diameter
and were swollen (Figure 4c). Both wild type and mutant ma-
Figure 3. Sections of tap roots and elongating short roots of Pinus
pinaster inoculated with the IAA-overproducing mutant strain 331 of
Hebeloma cylindrosporum showing endogenous colonization of a lateral root by hyphae from the Hartig net in the tap roots. (a) Cross section of a short root primordium with a round-shaped apex covered by
a single layered cap (white star). (b) Cross section of an elongating
short root pushing against the endodermis and breaking through at the
basal part of the lateral root. (c) Magnification of the rectangle highlighted in (b), showing a cross section of the basal part of the lateral
root where it breaks through the tap root endodermis (E), connects
with the endodermis of the lateral root via the cells labeled E′, and the
cortex of the lateral root is continuous with the cortex of the tap root.
Abbreviations: CC = tap root cortical cell; E = tap root endodermis; E′
= lateral root endodermis; H = hyphae; HN = Hartig net; and white
star indicates root cap. Bar = 50 µm.
ture mycorrhizas presented a poorly developed fungal sheath
comprising a few layers of hyphae (Figures 4b and c), similar
to the mycorrhizal tap roots (Figures 2c and f).
Short roots were also initiated in parts of a tap root not colonized by the fungus. In this case, the very early stages of root
growth were identical to those described above. Typical short
roots having a rounded tip covered by a uniseriate root cap
could be observed in the absence of fungal colonization (Figure 5a). Because the tap root had no Hartig net, breakage of the
endodermis (Figure 5b) did not lead to an early fungal colonization of the short root. However, as soon as the short root was
no longer completely covered by its root cap as it grew out of
the tap root, it was colonized by extramatrical hyphae that rapidly formed a well-developed Hartig net (Figure 5c). The
Hartig net developed basipetally, inside the tap root, as well as
acropetally so that mature mycorrhizas formed under these
conditions had the same morphology and anatomy as those de-
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SYMBIOSIS DYNAMICS BETWEEN AN ECTOMYCORRHIZAL FUNGUS AND CLUSTER PINE
Figure 4. Sections of tap roots and elongating short roots of Pinus
pinaster inoculated with the IAA-overproducing mutant strain 331 of
Hebeloma cylindrosporum (a and b) and the corresponding wild-type
strain h1 (c). (a) Longitudinal section of a short root emerging from
the tap root with Hartig net in its basal part but no colonization by
extramatrical hyphae on its root cap (white star). (b) Longitudinal
section of mature mycorrhiza on tap root showing a hypertrophic
Hartig net, 3 weeks after inoculation with the mutant strain. (c) Longitudinal section through short root 3 weeks after inoculation with the
wild type strain. Abbreviations: CC = cortex cell; E = endodermis
layer; H = hyphae; HN = Hartig net; white star = root cap. Bar = 50
µm.
127
Figure 5. Cross sections of an uncolonized portion of the apical part of
Pinus pinaster tap roots inoculated with the IAA-overproducing mutant strain 331 of Hebeloma cylindrosporum. (a) Short root primordia
with a round-shaped apex covered by a single layer cap (white star).
(b) Elongating short root pushing against the endodermis and breaking through at the basal part of the lateral root. (c) Short root emerging
from tap root with extramatrical hyphae colonizing short root to form
Hartig net. Abbreviations: CC = cortex cell; E = endodermis layer; H
= hyphae; HN = Hartig net; and white star indicates root cap. Bar = 50
µm.
Discussion
scribed in Figure 4b.
The addition of TIBA to the culture medium resulted in a total inhibition of tap root colonization by the fungus. After
3 weeks of co-cultivation of the seedlings with the mutant of
H. cylindrosporum, no hyphae were observed in the cortex of
tap roots, even in the cracks in the epidermis (Figure 6). The
addition of TIBA to the culture medium stimulated cell division in the pericycle and vascular bundles. However, hyphae
never penetrated through cracks in the epidermis and the first
layers of cortical cells (cf. Figure 6 with Figures 1 and 2).
Both wild type and IAA-overproducing mutant strains of
H. cylindrosporum colonized tap roots of P. pinaster seedlings where they formed a typical Hartig net (cf. Gea et al.
1994 and Gay et al. 1994). This observation suggests that the
mechanisms involved in Hartig net formation are similar in
tap roots and short roots. The overdevelopment of Hartig net
formation in tap roots by the mutant supports the hypothesis
that fungal IAA facilitates Hartig net establishment (Gay et al.
1995).
Both fungal strains formed a very thin mantle around
mycorrhizas and tap roots. Poor fungal sheath development
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128
TRANVAN, HABRICOT, JEANNETTE, GAY AND SOTTA
Figure 6. Cross section of a Pinus pinaster tap root, co-cultivated for
3 weeks with the IAA-overproducing mutant strain 331 of Hebeloma
cylindrosporum on a medium supplemented with TIBA (10 –5 M),
showing a thin mantle is present over the root but there is no penetration of hyphae (H) even through cracks (arrows) in the epidermis and
no Hartig net formation in the cortex. Abbreviations: CC = cortex
cell; E = endodermis layer; pC = pericycle cell; and c = resin canal.
Bar = 50 µm.
seems to be a characteristic of H. cylindrosporum–P. pinaster
symbiosis (cf. Debaud et al. 1988, Gay et al. 1994 and Gea et
al. 1994).
Our results suggest that the early fungal colonization of tap
roots in the H. cylindrosporum–P. pinaster symbiotic system
is very similar to that in the P. tinctorius–E. globulus symbiotic system (see Martin and Tagu 1995). In both systems, the
tap root is colonized by the fungus and exhibits typical
mycorrhizal features before true mycorrhiza synthesis takes
place. The formation of a typical Hartig net in long roots of
gymnosperm seedlings inoculated with ectomycorrhizal fungi
has been reported in several symbiotic systems (Robertson
1954, Wilcox 1968, and Wong et al. 1989). Colonization of
tap roots of P. abies seedlings co-cultivated with L. bicolor has
also been described (Karabaghli-Degron et al. 1998). All of
these observations indicate that a tap root having a Hartig net
and a fungal sheath can be considered to be a mycorrhiza, both
anatomically and functionally. Moreover, it has been demonstrated in laboratory studies that the Hartig net in P. pinaster
tap roots inoculated with wild type dikaryotic strains of
H. cylindrosporum is functional (P. Jargeat and R. Marmeisse,
University Lyon I, France, pers. comm.). The presence of a
functional Hartig net in seedling tap roots could play a significant role in the early survival of seedlings. Hartig net development in pine tap roots was comparable with that observed by
Gea et al. (1994) and Gay et al. (1994). The difference between the two strains was quantitative rather than qualitative
and the difference was detectable within two days after inoculation. The rates of fungal colonization and Hartig net formation were about twice as high with the mutant than with the
wild type (Figures 1 and 2), indicating that IAA-overproducing mutants have an increased infectivity (cf. Gay et al. 1994,
1995). The IAA-overproducing strain formed a hypertrophic
Hartig net in tap roots (Figure 2c) as well as in short lateral
roots (Figure 4b), suggesting that the mechanisms involved in
Hartig net formation are similar in tap roots and in short roots.
Because most mycorrhizas did not appear until more than
one week after inoculation, we conclude that the hyphae grew
through the tap root cortex, creating a Hartig net. If this conclusion is correct, it follows that the hyphae of the tap root
Hartig net represent a mechanism for colonization of young
short roots (Figure 3). As a consequence of this endogenous
colonization, short roots have typical Hartig net development
before emerging from the tap root. Similar observations have
been reported by Melville et al. (1987) who described the ontogeny of H. cylindrosporum–Dryas integrifolia (Vahl)
mycorrhizas. The difference between D. integrifolia and
P. pinaster is that lateral root colonization by hyphae emanating from the Hartig net in the primary root is more rapid with
P. pinaster than with D. integrifolia, indicating that
H. cylindrosporum behaves in a similar manner in both angiosperms and gymnosperms. Our findings also corroborate
those of Robertson (1954), Wilcox (1968), Ashton (1976) and
Nylund and Unestam (1982) who demonstrated that colonization of pine lateral roots and Hartig net formation occurred
from the existing Hartig net of parental roots. However, this is
not the only process of colonization. Several studies have
shown that fungal penetration in the cortical layer occurred
only after short roots emerged from the tap root and after a
mantle surrounded them (Chilvers and Gust 1982, Kottke and
Oberwinkler 1986, Massicotte et al. 1987a, 1987b, Brunner
and Scheidegger 1992). Similarly, we observed that P. pinaster short roots emerging through a zone of tap root devoid of
Hartig net were colonized by extramatrical hyphae (Figure 5).
Thus, colonization by both endogenous and exogenous means
can occur in an individual plant, with the colonization process
depending on the presence of a Hartig net in the tap root.
When the medium was supplemented with TIBA, an IAA
transport inhibitor, tap root colonization by the IAAoverproducer mutant was blocked and a dramatic swelling of
the central cylinder occurred (Figure 6). We believe that cell
proliferation in the central cylinder was caused by a local accumulation of auxins produced by the plant. The absence of
colonization may be a result of TIBA blocking the transport of
fungal auxin. However, Karabaghli-Degron et al. (1998) reported that TIBA does not affect the capacity of L. bicolor to
excrete IAA. Therefore, even if fungal IAA is involved in mycorrhiza formation, its mechanism of action remains unclear.
TREE PHYSIOLOGY VOLUME 20, 2000
SYMBIOSIS DYNAMICS BETWEEN AN ECTOMYCORRHIZAL FUNGUS AND CLUSTER PINE
Acknowledgments
This work was supported by a European research contract (AIR 3 CT
93-1742). The authors are thankful to Dr. M. Ahmad for critical reading of the manuscript.
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