iVezo Phytol. (1994), 129, 83-95
Effects of high nitrogen concentrations on
ectomycorrhizal structure and growth of
seedlings of Picea abies (L.) Karst.
BY I V A N 0 B R U N N E R
AND
CHRISTOPH SCHEIDEGGER
Swiss Federal Institute for Fovest, S n o w and Landscape Research ( W S L ) ,C H - 8 9 0 3
Birmensdorf, Switzerland
(Received 29 November 1993 ;accepted 2 0 M a y 1994)
SUMMARY
High nitrogen concentrations are known to affect ectomycorrhizas and ectornycorrhizal fungi in field and
laboratory experiments. Using NH,+ as a nitrogen source in the growth pouch system, a variety of structural
modifications were documented on first order lateral roots of Picea abies (L.) Karst. seedlings. Root cells increased
in size and number at high levels of NH4+, resulting in a hypertrophic appearance of roots in both uncolonized
seedlings and in those inoculated with mycelia of the ectornycorrhizal fungus Hebeloma crustuliniforrne (Bull. ex
St. Amans) QuCl. A fungal mantle surrounded short roots of inoculated seedlings, and the outer walls of epidermal
and cortical cells were often thickened when in contact with fungal hyphae. N o Hartig net developed, although
intracellular penetration of fungal hyphae into epidermal and cortical cells frequently occurred. At moderate N H 4 +
concentrations, Hartig net development was incomplete, but occasional intracellular penetration of Hartig net
hyphae into epidermal and cortical cells occurred. T h e addition of high levels of NH,+ after ectornycorrhizal
development resulted in ectomycorrhizas with distinctly altered apical structures. None of the various NH,+
concentrations resulted in significantly different plant dry weights after two months of exposure, either with or
without fungal inoculation. After four months, dry weight, root length and branching indices were higher in both
inoculated plants and those exposed to high nitrogen. T h e root/shoot ratio and number of short roots per seedling
were high with fungal inoculation. Shoot nitrogen levels after two and four months and the Ca levels after 4
months were higher with increasing NH,+ in the nutrient solution. Other mineral elements were not influenced
by nitrogen concentrations and fungal inoculation.
Key words: Ectomycorrhizas, nitrogen, anatomy, intracellular penetration, plant growth.
INTRODUCTION
Elevated nitrogen inputs in the form of oxides of
nitrogen (NO,) or ammonia (NH,) over long periods
into forest ecosystems in Central Europe are a cause
of manifold ecological changes including negative
impacts on tree health (Nihlglrd, 1985). Excess
nitrogen not only has a fertilizing effect but also
alters soil chemistry by leaching several plant
nutrients, and increases acidification resulting in
high concentrations of A1 in the soil solution (Papke
et al., 1987). Trees experience nutrient stress both
due to growth stimulation, which leads to a higher
demand for Mg, K and P, and reduced nutrient
uptake caused by A1 damage to fine roots.
Air pollution, including deposition of NO, and
NH,, causes reductions in growth of extramatrical
hyphae, leading to reductions in the frequency and
diversity of ectornycorrhizal roots and fungi (Dighton
& Jansen, 1991). Meyer (1985) observed a dramatic
reduction in ectornycorrhizal development after
nitrogen fertilization in pot experiments. Following
nitrogen fertilization of forest stands, an increase in
the ratio of uncolonized to ectornycorrhizal short
roots was seen, but the diversity of ectomycorrhizal
types did not change (Haug, Pritsch & Oberwinkler,
1992).
Little is known about the effect of high levels of
nitrogen on the cellular processes related to ectomycorrhizal development and ectornycorrhizal function. Under natural conditions, lateral roots of most
tree species in the northern hemisphere establish a
fungal mantle and a system of intercellular fungal
hyphae called the Hartig net. T h e Hartig net is
believed to play an important role in the transport
and exchange of nutrients within this mutualistic
symbiosis (Harley & Smith, 1983). I n Picea abies
(L.) Karst. colonized by Pisolitlzus tinctorius (Pers.)
84
I . Brunner and C. Scheidegger
Figures 1-4. For legend see opposite.
Nitrogen and ectomycorrhizas
Coker & Couch., Hartig net development is suppressed and intracellular penetration by the fungus
occurs at 'elevated nitrogen concentrations whether
the source of nitrogen is nitrate (NO,-) and ammonium (NH,+) (Haug et al., 1992).
The goals of the present study were to determine
which structural modifications occur at elevated
nitrogen concentrations in the P . abies-Hebeloma
crustuliniforme (Bull. ex St. Amans) QuCl. system,
and to evaluate the consequences of structural
alterations on various growth parameters and shoot
mineral element concentration.
MATERIALS AND METHODS
Synthesis experiments
Autoclaved polyethylene pouches, 13 x 16 cm, longitudinally divided into two chambers with a welding
seam and containing two activated charcoal filter
papers, were moistened with 5 ml per chamber of
modified Melin-Norkrans nutrient solution (MMN)
without glucose (Brunner & Scheidegger, 1992).
Seeds of Picea abies were surface-sterilized for
30 min in 30 % H,O,, germinated on water agar, and
one seedling was inserted into each chamber. In one
experiment (Expt 3), two surface sterilized seeds
were inserted directly into each chamber of the
growth pouches and reduced to one seedling after
germination. After 2-3 months, four to six inoculum
discs with mycelia of Hebeloma crustuliniforme were
placed within 3 mm of the lateral roots and another
5 ml M M N (including 5 g 1-I glucose) were added.
Additional nitrogen was added with the M M N in the
form of NH,Cl either simultaneously with inoculation (Expts l , 2 and 3) or 2 months after inoculation
(Expt 4, with another 5 ml M M N minus glucose),
leading to various final NH,+ concentrations (Expt
1 : 7.6, 15.0, 30.0, 44.9 mM; Expt 2: 7.6, 44.9, 82.3,
119.7, 157.1 mM; Expt 3 : 7.6, 44.9 mM; Expt 4: 11.3,
33.8, 48.7, 86.1, 123.5 and 160.9 m n ~ )T. h e pH of the
solutions at all NH,+ concentrations were 6.1-6.2
after autoclaving, and was 6.5-6.8 after 2 months,
irrespective of fungal inoculation. A strip of foam
(1 x 1 x 3 cm) was placed beside each root system to
provide air space. Sterile distilled water was added as
needed. Synthesis experiments were carried out in a
growth chamber with a 16 h day period (PAR:
100 pmol m-2 s-l) at 20 "C and 70 % relative hu-
85
midity. Two (Expts 1, 2 and 4) or 4 months after
inoculation (Expt 3) plants were harvested and
ectomycorrhizal roots were analysed. Six (Expts 1, 2
and 4) or 19 (Expt 3) replications per treatment were
used.
Structural analyses of lateral roots
Uncolonized and ectomycorrhizal first order lateral
roots were fixed in 6 % formaldehyde for light
microscopy, dehydrated in ethanol, embedded in
glycol-methacrylate,
longitudinally
sectioned
(1.5 pm), and stained with Giemsa for chitinoid
material (Clarli, 1981). Microphotographs were
talien with a Leitz Aristoplan photomicroscope.
Outer cortical and epidermal cell walls were viewed
in unstained sections with a Leitz Aristoplan photomicroscope with epifluorescence optics using ultraviolet light excitation (BP 340-380).
Ectomycorrhizal first order lateral roots were
slightly degased in water for low-temperature scanning electron microscopy (LTSEM), mounted in
water between copper plates (Scheidegger &
Brunner, 1992), and plunge-frozen in liquid propane
with T F D 010 (Bal-Tec). T h e sandwiches were then
introduced into a double replica device and transferred to the preparation chamber of the cryopreparation unit SCU 020 (Bal-Tec) (Miiller et al.,
1991 ; Scheidegger et al., 1991). T h e double replica
device was opened under high vacuum ( P <
2.10-"a)
and etched for 1 min at -90 "C. Platinum
sputter coating and specimen transfer onto the cold
stage of a Philips SEM 515 was performed as
described in Scheidegger & Brunner (1993).
Analyses of plant material
Dry weights of roots and shoots was measured after
drying for 3 d at 60 "C. Root length (Expt 3),
number of ectomycorrhizas (Expts 3 and 4) and
uncolonized short roots (Expt 3), and branching
index (number of uncolonized short roots/root
length; Expt 3) were estimated before drying.
Statistical analyses for each treatment were carried
out using standard analysis of variance. Least
significant differences were calculated at P < 0.05
using Fisher's P L S D test.
Figures 1-4. Light micrographs of longitudinal sections of uncolonized and ectomycorrhizal Picea abiesHebeloma crustulinifor~nelateral roots grown for 2 months at concentrations of 7.6 and 44.9 mM NH,+.
Figure 1. Uncolonized lateral root grown at a concentration of 7.6 mn4 NH,+. Root hairs (R) are present and
epidermal (E) and cortical cells (C) have a 'normal' growth. Scale bar = 0.1 m m . Figure 2. Uncolonized lateral
root grown at a concentration of 44.9 mM hTH,t.Root hairs are absent and epidermal (E) and cortical cells (C)
have a hypertrophic growth. Scale bar = 0.1 m m . Figure 3. Ectomycorrhizal lateral root grown at a
concentration of 7.6 mM NH,+. A mantle (M) and a Hartig net (arrowheads) are present and epidermal (E) and
cortical cells (C) have a 'normal' growth. Scale bar = 0.1 m m . Figure 4. Ectomycorrhizal lateral root grown
at a concentration of 44.9 mM NH,+. A mantle (M) is present, but the Hartig net is absent, and epidermal (E)
and cortical cells (C) show hypertrophic growth. Scale bar
=
0.1 m m .
86
I. Brunner and C. Sclzeidegger
Figures 5-10. For legend see opposite.
Nitrogen and ectomycorrhizas
For the mineral element analyses, dried shoots of
replicate pouches were pooled, ground and stored in
1 ml Eppendorf tubes. Between 100 mg and 250 mg
of dried plant material was ashed at 480 OC for 12 h,
boiled in 1 ml HNO, (10 Oj,)/2 ml HC1 (30 O/,) for
30 min, diluted to 10 ml with H,O, and filtered.
Determination and quantification of cations were
made using a Bausch & Lomb ARL 3580 inductively
coupled plasma atomic-emission spectrometer (ICPAES). For quantification of nitrogen, 6-8 mg of
dried plant material was burned at 1020 "C, evolved
NO, was reduced to N,, and measured by gas
chromatography using a Carlo Erba NA1500 analyser. Nitrogen analyses were performed twice,
analyses of other cations were performed once due to
limited material.
Dead plants or ones which had grown poorly (less
than five green needles present at the end of the
experiments) were not included in the analyses.
RESULTS
Light microscopy
Structural features of uncolonized short roots from
seedlings grown at low (7.6 mM, Fig. 1) v s high levels
of NH,+ (44.9 mM, Fig. 2) showed distinct
differences after two months. At high NH,+ levels,
root hair formation was suppressed, and the cortical
cells were roundish rather than elongated as at low
NH,+ levels. T h e number of cell rows of the apical
meristem, the central cylinder, and the cortex
increased, and cortical cells were rounded, giving the
root a hypertrophic appearance. Ectomycorrhizal
short roots grown for 2 months at low levels of NH,+
(7.6 m ~resembled
)
uncolonized short roots in terms
of cell size and number of cell rows (Fig. 3).
However, a fungal mantle replaced root hairs and a
Hartig net occurred between cortical cells up to the
subapical region. T h e mantle was two-layered with
the inner layer pseudoparenchymatous and the outer
layer plectenchymatous, and Hartig net hyphae
penetrated to the endodermis (Fig. 3). At high levels
of NH,+ (44.9 mnx), roots appeared hypertrophic
with rounded cortical cells as in the uncolonized
87
roots (Fig. 4). In addition, a fungal mantle surrounded the short roots but no Hartig net developed
(Fig. 4).
Detailed structural analyses showed that at mod) 2
erate NH,+ concentrations (15.0 and 30.0 m ~after
months a two-layered fungal mantle was present but
Hartig net development was incomplete (Fig. 5-7).
In addition, Hartig net hyphae occasionally penetrated into epidermal and cortical cells (Figs 5, 6),
and a few host cells were completely filled with
fungal hyphae (Fig. 7). At high levels of NH,+
(44.9 miv or more) outer walls of epidermal and/or
cortical cells were often thickened (Figs. 8, 9), and
showed autofluorescence under ultra-violet light (not
shown). However, intracellular penetrations occurred frequently in epidermal and cortical cells (Fig.
8). Intracellular fungal hyphae had distinct, apically
dominated growth (Fig. lo), significantly different
from the multi-lobed mode of growth typical for
Hartig net hyphae. Living ectomycorrhizas (with
intact meristematic root cells) at very high levels of
NH,+ (85 mM and higher) had no additional distinctive features compared to ectomycorrhizas grown
at 44.9 mM.
If supplementary nitrogen was added 2 months
after fungal inoculation, ectomycorrhizal first order
lateral roots did not alter their morphology due to
the additional nitrogen if their growth had stopped
before the nitrogen was added. However, if growth
of ectomycorrhizal roots continued after the addition
of nitrogen, the morphology of the newly grown root
tips was distinctly altered, producing hybrid types of
ectomycorrhizal short roots (Fig. 11). A metacutislike transitional zone arose in these roots preventing
the longitudinal spread of the Hartig net, and cortical
cells assumed a more rounded appearance (Fig. 12).
In addition, intracellular penetrations of fungal
hyphae occurred in the epidermal and cortical zone
behind the apex (Fig. 13).
Low-temperature scanning electron microscopy
Complementary freeze-fractures of ectomycorrhizal
first order lateral roots grown at low levels of NH,+
Figures 5-10. L i g h t micrographs o f longitudinal sections o f ectomycorrhizal Picea abies-Hebeloma
csustulinifor~nelateral roots g r o w n for 2 m o n t h s at concentrations o f 30.0, 44.9 or 82.3 miv NH,+.
Figure 5. Ectomycorrhizal lateral root g r o w n at a concentration o f 30.0 miv N H 4 + .Incomplete Hartig net ( H )
w i t h a fungal h y p h a (arrowhead) entering i n t o a cortical cell ( C ) . Scale bar = 50 p m . Figure 6. Ectomycorrhizal
lateral root grown at a concentration o f 30.0 m M NH,+. Incomplete Hartig n e t ( H ) w i t h fungal h y p h a e
(arrowheads) i n epidermal ( E ) and cortical cells ( C ) . Scale bar = 50 pm. Figure 7. Ectomycorrhizal lateral root
grown at a concentration o f 30.0 m M NH,+. Incomplete Hartig net ( H ) w i t h fungal h y p h a e (arrowhead) have
filled o u t a cortical cell ( C ) completely. Scale bar = 50 ,urn. Figure 8. Ectomycorrhizal lateral root g r o w n at a
concentration o f 44.9 m M N H 4 + . Fungal h y p h a e (arrowheads) are present i n epidermal ( E ) and cortical cells
( C ) ,b u t n o Hartig n e t has developed. Cells w i t h thicker outer walls are present (*). Scale bar = 50 p m . Figure
9. Ectomvcorrhizal lateral root c r o w n at a concentration o f 44.9 m M N H . + . T h i c k e n e d outer walls o f t h e
epidermal cells (arrowheads) are present, probably preventing h y p h a e f r o m entering intercellularly t o f o r m a
Hartig n e t . Scale bar = 50,um. Figure 10. Ectomycorrhizal lateral root g r o w n at a concentration o f
82.3 m n t N H 4 + .Fungal h y p h a e (arrowheads) have penetrated a cell wall and have colonized t h e cortical cell ( C ) .
A Hartig net has n o t developed. Scale bar = 50 pm.
-
88
I. Brunner and C . Scheidegger
Figures 11-13. Light micrographs of longitudinal sections of a ectomycorrhizal Picea abies-Hebeloma
crustulinifortne lateral root grown for 2 months at a concentration of 11.3 miv and for additional 2 months under
123.5 miu NH,'.
Nitrogen and ectomycorrhizas
(7.6 m ~ demonstrated
)
a well developed fungal
mantle and a Hartig net growing radially from the
inner mantle towards the endodermis (Fig. 14).
Plasmic and exoplasmic fracture faces of the multibranched Hartig net hyphae show intimate contact
with epidermal and cortical cells in the interfacial
zone of the symbiosis (Fig. 14). Complementary
freeze-fractures of ectomycorrhizal first order lateral
roots grown at high levels of NH,' (44.9 mM)
demonstrated neither a well developed fungal mantle
nor a Hartig net (Fig. 15). Instead, intracellular
penetration of apically dominant fungal hyphae with
clamp connections was observed within host cells
that were probably dead (Fig. 15).
Growth analyses
Very high levels of NH,' (above 70 m ~ often
) led to
a browning of needles. Dead or poorly grown plants
occurred in Expt 2 with fungal inoculation at 119.7
(50 0/, dead or poorly grown plants) and 157.1
(100q.b) mM NH,', without fungal inoculation at
(400/,),
119.7
(830/,),
and
157.1
82.3
123.5
(83 %) mM NH,', and in Expt 4 at 86.1 (83
(83 %), and 160.9 (83 0/,) mM NH,'. Therefore, plant
growth parameters and ectomycorrhizal colonization
at 85 mM NH,+ and above were not considered in the
analyses.
Neither NH,' concentration (7.6-44.9 mM) nor
fungal inoculation resulted in significantly different
values for plant dry weight after two months of
exposure (Table 1). However, after 4 months, plant
dry weight was significantly enhanced by both high
nitrogen and fungal inoculation (Table 1). Higher
NH4+concentrations (82.3 m ~ led
) to delayed plant
growth, resulting in significantly lower dry weights
in both uncolonized and ectomycorrhizal treatments
(Table 1). Root and shoot dry weights of plants were
significantly greater at higher NH,' concentrations
(44.9 mM) and in inoculated plants after 4 months
(Table 2). T h e root-shoot ratio and numbers of short
roots were significantly influenced by fungal inoculation, while root length and branching index
were influenced significantly by both NH4+ concentration and fungal inoculation (Table 3). T h e
number of ectomycorrhizas per seedling was not
dependent on nitrogen treatment (Table 3). T h e
elevated NH,+ concentrations two months after
fungal inoculation led to a significant increase in
plant dry weight but not in number of ectomycorrhizas (Table 4).
x),
Shoot mineral element concentrations
I n all experiments, shoot nitrogen values increased
2-3 fold after 2 months with increasing NH,+
concentrations from 7.6 to 44.9 miLI, except in Expt 3
with a four-month duration, where nitrogen levels
did not increase (Table 5). No influence of fungal
inoculation on nitrogen levels could be detected.
After two months of exposure to various NH,+
concentrations, shoot Ca levels did not differ, except
in Expt 3 with a four-month duration, where the Ca
values were more than doubled at high NH4+
concentrations, independent of fungal inoculation
(Table 5). Conversely, 2-3-fold lower Mg levels
were seen after four months exposure, also independent of the nitrogen treatment or fungal
inoculation. Other macronutrient concentrations did
not vary in response to NH,' concentration, fungal
inoculation, or experiment duration (Table 5).
Micronutrient concentrations (Fe, M n , Zn, Cu, B)
did not display any clear behaviour in relation to
nitrogen treatment, fungal inoculation, or experiment duration (not shown).
DISCUSSION
A variety of structural modifications occurred on
first order lateral roots of Picea abies at high nitrogen
concentrations. These included larger apical meristems and more rows of cortical cells. Warren Wilson
& Harley (1983) observed that the size of the
meristematic region in Fagus is not only dependent
on the stage of root development (extensive in the
first stage, small in the second stage), but that large
apical meristems are correlated with greater extensions of root elongation and root diameter. High
nitrogen has been shown to induce greater root
diameters in Pinus and Quercus seedlings (Termorshuizen & Ket, 1991 ; Herrmann et al., 1992). I n the
present work, high nitrogen concentration may be
the reason for the enlarged meristematic regions in
both uncolonized and ectomycorrhizal first order
lateral roots. Herrmann et al. (1992) observed both
an increase in cortical cell number and enlarged
intercellular spaces at high nitrogen treatments.
High concentrations of nitrogen may generally
promote larger plant cells, and promote an increase
in the ratio of parenchyma to sclerechyma, resulting
in a soft and spongy tissue (Bergmann, 1993).
Comparable structural alterations were seen in this
study in uncolonized and in ectomycorrhizal first
Figure 11. Longitudinal section s h o w i n g a developed Hartig n e t (arrowheads) o n l y i n t h e basal portion g r o w n
at a concentration o f 11.3 mn? NH,', b u t n o t i n t h e apical portion g r o w n at 123.5 miv NH,'. N o t e t h e
h y p e r t r o p h i c g r o w t h o f t h e apical portion o f t h e root. Scale bar = 0.1 mm. Figure 12. Transitional z o n e o f t h e
root (detail o f Fig. 11). A metacutis-like structure (arrowheads) h a s f o r m e d after increasing t h e nitrogen
concentration, preventing t h e longitudinal spread o f t h e Hartig n e t ( d o u b l e arrowheads). Scale bar = 5 0 p m .
Figure 13. Apical z o n e o f t h e root (detail o f Fig. 1 1 ) . Intracellular penetrations o f fungal h y p h a e (arrowheads)
are present at h i g h nitrogen concentration. Scale bar = 5 0 p m .
90
I . Brunner and C . Scheidegger
Figure 14. Complementary L T S E M micrographs of a longitudinal freeze-fracture of an ectomycorrhizal Picea
abies-Hebeloma crz~stz~liniforme
lateral root grown at a concentration of 7.6 mM NH,'. A mantle (M) and a Hartig
net are well developed. Note the plasmic ( P F ) and the exoplasmic fracture faces (EF) of plasmalemma of
multibranches Hartig net hyphae. Scale bar = 10 p m .
Figure 15. Complementary L T S E M micrographs of a longitudinal freeze-fracture of an ectomycorrhizal Picea
abies-Hebeloma crustulinijorme lateral root grown at a concentration of 44.9 tnnl NH,+. A ~ n a n t l e(M) is present,
but a Hartig net is absent between epidermal (E) and cortical (C) cell walls (arrowheads). Intracellular fungal
hyphae (double arrowheads) with apical dominance are present in an air-filled epidermal cell. Note the clamp
connection (*) at an intracellular hypha. Scale bar = 10 p m .
order lateral roots, with cortical cells being more
rounded at high nitrogen concentrations, giving the
root a spongy and hypertrophic appearance.
Thickenings of outer epidermal and cortical cell
walls of these roots at high nitrogen concentrations
occurred only in the presence of ectomycorrhizal
fungal hyphae. Wall thickenings, which appear
electron dense using transmission electron microscopy, have also been observed in the Picea-Pisolithus
association (Haug et a l . , 1992). These wall thickenings or appositions may be compared with the
formation of papillae, pathogen-induced mechanical
91
Nitrogen and ectomycorrhizas
Table 1. Effect of N and ectomycorrhizal colonization on the plant biomass (mg d.wt & S E ) after 2 or 4 months
of simulthneous fungal inoculation and N treatment
Expt 1
NH,+ conc.
(mhl)
Expt 2
NM*
Months: 2
n: 6
Expt 3
M*
2
6
7.6
15.0
30.0
44.9
82.3
Probability
N treatment
Fungus (F)
NxF
Different letters within columns indicate a significant difference ( P < 0.05).
For the analysis of variance: ns, not significant; *, P < 0.05; **, P < 0.01 ; ***, P < 0.001 ; ****, P < 04.l001.
only 5 replicates. 2only 3 replicates were living.
* N M , non-mycorrhizal; M, m~corrhizal(and in subsequent Tables).
Table 2. Effect of N and ectomycorrhizal colonization on the root and shoot d r y weight and the root-shoot ratio
after 4 months of simultaneous fungal inoculation and N treatment ( E x p t 3 ) ( n = 19)
Root f
NH,+ conc.
(mnl)
SE
(mg)
Shoot f
SE
(mg)
Root-shoot ratio
-
7.6
44.9
Probability
N treatment
Fungus (F)
NxF
+
SE
NM
M
NM
M
NM
M
21.3 f l.3a
25.8 f 2.2 a
38.32 1.0b
48.3 f2.8 a
33.3 2 2.3 a
40.3 f 3.7 a
43.3 f 1.5b
62.2 f 5.8 a
0.66 k 0.02 a
0.66 0.04a
0.89 _f 0.02 a
0.84 i0.05 a
****
***
****
****
ns
ns
ns
*I*
Different letters within columns indicate a significant difference ( P < 0.05)
For the analysis of variance: ns, not significant; *, P < 0.05 ; **, P < 0.01 ;
ns
***, P < 0.001, ****, P < 0.0001.
Table 3. Effect of N and ectomycorrhizal colonization on the number of short roots, root length, branching index
and numbers of ectomycorrhizas after 4 months of simultaneous fungal inoculation and N treatment ( E x p t 3)
( n = 19)
NH,+ conc.
(mnl)
Short roots
f SE (n)
NM
Root length
iSE (cm)
M
NM
Ectomycorrhizas
- SE (n)
NM
M
M
3.68 f 0.22a
3,2620.20a
3.17 f 0.24a
2.52f0.19b
49.5 f 3.1 a
39.0k4.9a
+
M
+
Branching index
- SE (n. cm-l)
7.6
133.2 f 13.9a 197.2 k 12.0~1 35.6 2.3 a 63.7 _f 2.9a
44.9
129.6f10.2a 172.4f13.0a 41.2f3.0a 69.6k3.0a
Probability
N treatment
ns
X:
Fungus (F)
*x**
*1*1
NxF
ns
ns
*
**
ns
Different letters within columns indicate a significant difference ( P < 0.5).
For the analysis of variance: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001;
barriers of hosts cells against colonization and
penetration by pathogens (Goodman, Kiraly &
Wood, 1986). I n ectomycorrhizal systems, cell-wall
thickenings have been observed in certain compatible
species combinations in presence of exogenous
+
ns
-
****, P ,< 0.0001
carbohydrates and in some incompatible species
combinations in the absence of exogenous carbohydrates (Duddridge, 1986 a , b ) . Papilla formation
has also been observed in senescent and dead cortical
cells of Pinus sylvestris L. inoculated with Piloderma
92
I. Brunner and C . Scheidegger
Table 4. Biomass and number of ectomycorrhizas 2
mortths after N addition to plants which h a d been
inoculated 2 months previously ( E x p t 4 ) ( n = 6 )
fungal invasion, and both may be influenced by
environmental conditions (Jorns, 1 9 8 8 ; Thomson,
Matthes-Sears & Peterson, 1 9 9 0 ) . NIetacutis layers
are built up by suberization of cells walls, accumulation of polyphenols in root cap cells, and
interconnecting small cells between endodermis and
.
root cap (Kottke & Oberwinkler, 1 9 8 6 ~ )Although
wall thickenings may form in a similar manner, they
do not characterize the dormant stage of the rootlets
as they are formed during growth and development.
Cell wall thickenings are distinct from cell wall
ingrowths primarily known from pisonioid mycorrhizas, as these ingrowth probably have a transfer
cell function in the rapid absorption and storage of
nutrients (Ashford & Allaway, 1982 ; 1 9 8 5 ) . Cell wall
ingrowth have also been observed with P. abies in
association with A m a n i t a muscaria (L. : Fr.) Hooker
(Kottke & Oberwinkler, 1986 b ) and Hebeloma crustuliniforme (Scheidegger & Brunner, 1993).
Herrmann et al. ( 1 9 9 2 ) felt that the structural
alterations in short roots grown at high nitrogen
concentrations would not allow colonization by
Ectomycorrhizas
+ (mg) + (n)
97.5 + 7.3 b
40.8 f8.5 a
143,5+ 14.9a
61.0+ 11.4a
117.7 + 7.7a,b
40.8 7.3 a
NH,+ conc. (mra)
D.wt
11.3
33.8
48.7
Probability
N treatment
*
SE
SE
ns
Different letters within columns indicate a significant
difference ( P < 0.05).
For the analysis of variance: ns, not significant; *, P <
0.05.
croceum Erikss. & Hjortst. (Nylund, Kasimir &
Strandberg Arveby, 1 9 8 2 ) . Wall thickenings in the
present work are comparable in function to the
metacutis described by Wilcox ( 1 9 5 4 ) as both
probably protect the cortex and meristem from
Table 5. Effect of N and ectomycorrhizal colonization o n the concentration ( m g g-' d.wt) of mineral elements i n
shoots
NH,+ conc.
( m ~ )
Expt 1
(2 months)
Expt 2
(2 months)
NM
NWI
" Concentrations in Expt 4.
M
M
Expt 3
(4 months)
NWI
' Not measured (N.m.) due to little plant material for ICP-analysis.
M
Expt 4
(2 2 months)
+
M
Nitrogen and ectornycorrhizas
ectornycorrhizal fungi. As in this work, Haug et al.
(1992) also observed the absence of Hartig net
development at high nitrogen concentrations. I t is
likely that recognition between potential ectomycorrhizal partners or establishment of the symbiosis can
be disturbed by environmental factors and/or altered
physiological conditions of the root cells, resulting in
plant reaction against colonization with cell wall
thickenings or papilla formations, resulting in the
absence of a Hartig net. Classical ectomycorrhizal
theories are aware of suppression or termination of
the symbiosis at high nitrogen concentrations.
Bjorkrnan's carbohydrate theory (Bjorkman, 1942)
suggests that low concentrations of carbohydrate in
the roots of well-fed seedlings are responsible for the
lack of ectomycorrhizal development, while Slankis's
hormonal theory (Slankis, 1973) suggests that the
inhibition or termination of ectomycorrhizal infection at high nitrogen concentrations is due to the
inhibition of fungal auxin synthesis. After a reexamination of this phenomenon by Nylund (1988))
Wallander & Nylund (1991), and Gogala (1991),
neither hypothesis could conclusively explain the
inhibition of ectomycorrhizal development, although
alternative mechanisms were not proposed.
T h e observations of Harley & Smith (1983) on
'perirhizic mycorrhizae' are not comparable as the
authors mentioned them as a time- and stagedependent phenomenon of long roots of Fagus,
Nothofagus and Eucalyptus, or in connection with
the pisonioid mycorrhiza of Pisonia grandis R. Br.
(Ashford & Allaway, 1982). As both multibranched
hyphae of the Hartig net and the inner mantle are
structurally different from those of the extramatrical
hyphae or hyphae of the outer mantle (Scheidegger
& Brunner, 1992; Scheidegger & Brunner, 1993), it
can be hypothesized that the multibranched hyphal
organization is adapted to facilitate nutrient exchange between host and fungus (Kottke & Oberwinkler, 1987).
Hebelorna crustuliniforrne in the present study
exhibited growth characteristic of a pathogenic or
saprobic organism, as intracellular penetrations into
epidermal and cortical cells occurred frequently at
high nitrogen concentrations. Haug et al. (1992) and
Holopainen & Heinonen-Tanski (1993)) using high
nitrogen treatments, and Jentschke (1990) using
A1(N03), observed similar intracellular penetrations
of ectomycorrhizal fungi into spruce roots with
Pisolithus tinctorius and Lactarius theiogalus (Bull.)
Fr., respectively. Intracellular penetrations have also
been observed in natural ectomycorrhizas in disturbed or polluted forest stands (Meyer, 1984;
Blaschke, 1986). However, Jentschke (1990) suggests
that intracellular hyphae in cortical cells are not
necessarily a pathological aberration as they have
often been observed within senescent phases of
ectomycorrhizas. It is unusual that in Jentschke's
studies (1990) cortical senescence occurred in the
93
developmental stage of the pre-Hartig net zone.
Perhaps, under stress conditions, a shortage of easy
utilized carbohydrates occurred and pathogenic
characteristics of the ectomycorrhizal fungi increased
to make new carbohydrate sources available
(Jentschke, 1990).
T w o months after inoculation, shoot dry weights
did not differ significantly between the nitrogen
treatments, suggesting that the symbioses needed
further time to develop, as has been demonstrated in
earlier studies (Brunner & Scheidegger, 1992). Four
months after inoculation, an increase in dry matter
production with fungal inoculation at both low and
high nitrogen concentrations compared to uncolonized control plants could be detected. Similar
results have been obtained by Alexander & Fairley
(1986) and Bledsoe & Zasoski (1983). At high
nitrogen concentrations, the increase in shoot dry
weight in ectomycorrhizal plants is seemingly contradictory to the absence of Hartig net and presence
of wall thickenings, suggestive of pathogenesis, in
these ectornycorrhizal seedlings. A possible explanation for this result is the involvement of fungalproduced growth hormones which stimulate plant
growth without a functional interface for nutrient
transfer. Auxin production has been demonstrated
in many ectomycorrhizal fungi, including H . crustuliniforrne (Ek, Ljungquist & Stenstrom, 1983), and it
is known to enhance rooting and dichotomy in host
plants (Slankis, 1973). This explanation is supported
by the present data with highly significant increases
in the root-shoot ratio, number of short roots, root
length, and root branching index due to the presence
of H . crustuliniforrne but not by high nitrogen
concentrations. Rudawaska (1983) showed that auxin
production at high nitrogen levels is possible for
some ectomycorrhizal species but not others.
T h e present results contradict the shortage of
certain mineral elements in the shoots (mainly Mg,
K, P, B) observed by Nihlglrd (1985). Instead, our
observations of higher shoot nitrogen and Ca agree
with those of Haug et al. (1992). I n addition, shoot
concentrations of mineral elements were not dependent on whether the plant roots were uncolonized
or ectomycorrhizal. This result is surprising for low
nitrogen concentrations, as several studies have
reported an increase of certain mineral elements
(mainly N and P) in plants following ectomycorrhizal
formation (Reid, Kidd & Ekwebelam, 1983;
Rygiewicz, Bledsoe & Zasoski, 1984; Boxman &
Roelofs, 1986).
Our findings suggest that recognition of ectomycorrhizal partners or establishment of ectomycorrhizas may be inhibited by certain environmental
factors such as high nitrogen concentration. Plants
respond to surface colonization with wall thickenings
or appositions, which are similar to the changes
induced by pathogenic fungi or to features of
incompatible ectornycorrhizal associations. It is
I. Brunner and C. Scheidegger
94
therefore important, when studying the functioning
of ectomycorrhizal associations, to understand the
structural basis of the plant-fungus relationship, as
the structure, as shown in the present study, is not an
invariable parameter.
ACKNOWLEDGEMENTS
We thank B. WIilakovic, B. Schneider, and WI. Zollinger
for technical assistance, D . Pezzotta and his group for
mineral element analysis, M. J . Sieber for her corrections
of the English text, and D r . C. G r u h n for her thoughtful
review of various stages of this manuscript.
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