Research Paper
Abstract: Tropospheric ozone (O3) triggers physiological
changes in leaves that affect carbon source strength leading to
decreased carbon allocation below-ground, thus affecting roots
and root symbionts. The effects of O3 depend on the maturity-
related physiological state of the plant, therefore adult and
young forest trees might react differently. To test the applicabil-
ity of young beech plants for studying the effects of O3 on forest
trees and forest stands, beech seedlings were planted in con-
tainers and exposed for two years in the Kranzberg forest FACOS
experiment (Free-Air Canopy O3 Exposure System, http://www.
casiroz.de) to enhanced ozone concentration regime (ambient
[control] and double ambient concentration, not exceeding
150 ppb) under different light conditions (sun and shade). After
two growing seasons the biomass of the above- and below-
ground parts, beech roots (using WinRhizo programme), ana-
tomical and molecular (ITS-RFLP and sequencing) identification
of ectomycorrhizal types and nutrient concentrations were as-
sessed. The mycorrhization of beech seedlings was very low
(ca. 5% in shade, 10% in sun-grown plants), no trends were ob-
served in mycorrhization (%) due to ozone treatment. The num-
ber of Cenococcum geophilum type of ectomycorrhiza, as an indi-
cator of stress in the forest stands, was not significantly differ-
ent under different ozone treatments. It was predominantly oc-
curring in sun-exposed plants, while its majority share was re-
placed by Genea hispidula in shade-grown plants. Different light
regimes significantly influenced all parameters except shoot/
root ratio and number of ectomycorrhizal types. In the ozone fu-
migated plants the number of types, number of root tips per
length of 1 to 2mm root diameter, root length density per vol-
ume of soil and concentration of Mg were significantly lower
than in control plants. Trends to a decrease were found in root,
shoot, leaf, and total dry weights, total number of root tips,
number of vital mycorrhizal root tips, fine root (mass) density,
root tip density per surface, root area index, concentration of
Zn, and Ca/Al ratio. Due to the general reduction in root growth
indices and nutrient cycling in ozone-fumigated plants, alter-
ations in soil carbon pools could be predicted.
Key words: Ozone fumigation, light exposure, beech, fine root
growth, biomass, ectomycorrhizal types, nutrient concentration.
Introduction
Tropospheric ozone (O3), a secondary atmospheric pollutant,
generated from nitrogen oxides and volatile organic com-
pounds reacting in the presence of sunlight, has been recog-
nized as an increasingly damaging agent to plants (Karnosky
et al., 2005). The effects of O3 on plant growth and develop-
ment at different structural and functional levels of the above-
ground parts have been extensively studied (Matyssek and
Sandermann, 2003), while the relative inaccessibility of plant
roots has hampered efforts to understand effects of O3 below-
ground (Andersen, 2003; Schloter et al., 2005). O3 triggers
physiological changes in leaves that affect carbon source
strength, i.e., the amount of carbon available for allocation to
sink tissues. Decreased carbon assimilation, increased meta-
bolic costs for repairmechanisms, and decreased phloem load-
ing, all lead to decreased carbon allocation belowground, thus
affecting roots, root symbionts, rhizodeposition, litter quality
and quantity, and, consequently, the whole soil food web (An-
dersen, 2003).
Belowground carbon allocation links the soil ecosystem and
foodweb with the forest canopy, providing a flow of organic
carbon to the soil (Giardina et al., 2005). The annual flux be-
lowground is the largest sink for gross primary production,
with most of this gross carbon flux occurring in ecosystems
with trees. Carbon allocation in the root and rhizosphere sys-
tem depends on the complex bio-soil environment, and is sub-
ject to changes in different stress conditions. Belowground
process rates depend on tree species, age-related physiology,
nutrients, water supply, temperature, and on above- and be-
lowground species composition, among which mycorrhizal
fungi have been recognized as “key species” in determination
of ecosystem functioning (Nielsen and Winding, 2002; Read
et al., 2004; Giardina et al., 2005; Read and Perez-Moreno,
2003; Taylor and Alexander, 2005).
Belowground net primary production (BNPP) has been defined
as the mass of roots produced plus any root mortality occur-
ring over a specified period of time, including all carbon allo-
cated belowground by plants and not used for autotrophic res-
piration (Giardina et al., 2005). Therefore, the change in BNPP
equals detritus generated (root and mycorrhizal turnover),
change in biomass (coarse and fine roots) measured over time,
losses due to herbivory and exudation, and carbon flow to my-
corrhizae. Among these BNPP constituents, fine roots have re-
CASIROZ: Root Parameters and Types of Ectomycorrhiza of Young
Beech Plants Exposed to Different Ozone and Light Regimes
P. Zˇeleznik1, M. Hrenko1, C. Then2, N. Koch3, T. Grebenc1, T. Levanicˇ1, and H. Kraigher1
1 Slovenian Forestry Institute, Vecˇna pot 2, 1000 Ljubljana, Slovenia
2 Department of Forest Tree Physiology, Federal Research and Training Centre for Forests, Natural Hazards and Landscape,
Rennweg 1, 6020 Innsbruck, Austria
3 Ecophysiology of Plants, Department of Ecology, Technische Universität München, Am Hochanger 13, 85354 Freising, Germany
Received: February 28, 2006; Accepted: November 8, 2006
Plant Biol. 9 (2007): 298–308
© Georg Thieme Verlag KG Stuttgart · New York
DOI 10.1055/s-2006-955916
ISSN 1435-8603
298

ceived most attention because their entire mass may be re-
placed (turnover) within one year or less, which constitutes
an important fraction in BNPP. Furthermore, most fine roots
of stand-forming broadleaves and conifers in temperate and
boreal forests in Europe are ectomycorrhizal (Kraigher, 1999;
Taylor et al., 2000), which have been shown to contribute up
to 60% of total carbon allocated belowground (Rygiewicz and
Andersen, 1994).
Ozone fumigation was shown to reduce total biomass of 3-
year-old sugar maple seedlings, the species which is described
as an intermediate among ozone-tolerant species, grown un-
der different light conditions (Topa et al., 2004), while a
change in physiology of seedlings regarding the response to
ozone fumigation in biomass productionwas reported to occur
between year three and four (Karnosky et al., 2005). Root plas-
ticity has been proposed as the major mechanism by which
plants cope with the naturally occurring heterogeneous sup-
plies of nutrients in soil (Hodge, 2004), and species-specific
differences have been predicted using several specific root in-
dices, such as specific root length (SRL, root length per unit
mass). Thinner roots were reported to proliferate, i.e., show a
fast response to a change in the environmental nutrient con-
ditions (ibid). Carbon source-sink relationships or functional
balance of roots and shoots were reported as primary actors
in continuous adjustments between root and shoot growth
(Tingey et al., 1996), possibly acting through root to shoot sig-
nalling, including hormonal regulation of root proliferation.
Cytokinins have been studied in adult beech trees within this
project (Grebenc et al., 2005) with respect to mycorrhizal-in-
duced changes in cytokinin concentrations in the host plants
(Kraigher et al., 1991, 1993).
The aims of our study were to assess light regime- and ozone
fumigation-derived changes in root parameters and mycorrhi-
zal characteristics of young beech plants in the common ex-
perimental setup in Kranzberg forest. It was hypothesized that
ozone would: i) reduce mycorrhization and ectomycorrhizal-
type diversity; ii) change ectomycorrhizal community struc-
ture in the differently exposed containers; iii) reduce root
growth (biomass, number of root tips, root volume, specific
root indices); and iv) change allocation of biomass between
belowground and aboveground parts. We also expected that
light regime and ozone treatment interactions might be hard
to establish since the light regime was set at extreme condi-
tions, while ozone fumigation was limited to two-fold (not ex-
ceeding 150 ppb) ambient ozone concentration.
Materials and Methods
In the Kranzberg forest (near Freising, Germany; Pretzsch et
al., 1998) Free-Air Canopy Ozone Exposure System (FACOS),
adult beech trees were fumigated with double-ambient ozone
concentration (Werner and Fabian, 2002; www.casiroz.de).
Young beech trees were tested in the same experimental setup
(Herbinger et al., 2005). Two-year-old, 20–40 cm high beech
seedlings were planted into 20 containers, 6 plants per 30-l
container. The seedlings (category selected, origin unknown)
originated from the forest tree nursery “Forstgarten Laufen”,
Bavaria. According to Brand (1991), a few sporocarps of ecto-
mycorrhizal fungi were determined in the nursery: Tuber pu-
berulum, Laccaria tortilis, Laccaria laccata, Hebeloma meso-
phaeum, Sphaerosporella brunnea, Tarzetta cupularis, and two
Tomentella spp. He had also identified several ectomycorrhizal
types on beech seedlings from the same nursery: Tuber puber-
ulum was present on more than 90% of seedlings, Type B sim-
ilar to Laccaria tortilis on about 90% seedlings, Hebeloma mes-
ophaeum on about 20%, and Type A similar to Genea hispidula
on about 50% of seedlings in small containers, while in the
neighbouring beech forests, Fagirhiza cystidiophora (Tuber sp.)
was also present. Before planting into containers in the Kranz-
berg Forest experiment, beech seedlings were not inspected
for mycorrhization.
The containers were filled with soils from Höglwald (luvisol
derived from loess over tertiary sediments, Kreutzer and Bit-
tersohl,1986). The important data on the top soil mixture (pro-
vided by dr. A. Göttlein, pers. comm.) were: pH (CaCl2): 3.76 ±
0.02, CEC (1m NH4Cl extract): 77.12 ± 2.05 μeq/g; base satura-
tion: 27.06 ± 1.24%. A 5-cm Ah layer was filled over a 30-cm
B layer and 1–2 cm of leaf litter (from Kranzberg Forest) was
added on the top (Grams et al., 2002). The bottom 3 cm were
filled with inert draining material, the deepest layer was an in-
ert plastic mesh, ca. 0.3 cm thick. There was no soil treatments
or any fertilization in order to keep the containers as natural as
possible.
The containers were exposed for two vegetation periods in the
sun part of crowns (on towers, SUN) or in the shade (at the
ground level, SHA), and exposed to ozone fumigation (not ex-
ceeding 150 ppb, 2 × O3) or to the ambient ozone level (control,
1 × O3). The containers were not weeded, so that grass and oth-
er perennial plants were found abundantly in sun-exposed
containers, while in containers from the shade some grasses,
Oxalis acetosella, single germlings of Picea abies, and a few oth-
er plants were noticed. The SUN containers were watered by
hand and kept moist throughout the experiment (in 2003 up
to 1.5 l daily, in 2004 1.0–2.0 l per week were used). The shad-
ed containers were not watered at all since they were always
moist. Leaf water potential was measured in the previous (pi-
lot) container experiment with same-aged beech seedlings,
soil, container, planting, and exposure conditions, establishing
the mean values for SUN at – 2.4 ± 0.3MPa and for SHA at
– 1.3 ± 0.2MPa (Then et al., 2004).
To precisely characterize the microclimatic conditions for the
container seedlings, we used a 2-channel miniature datalogger
(Hobo Pro Series, Syntech, Germany) to record air temperature
and relative air humidity (rH) in the different light regimes. In
2003, a clear air temperature and air humidity gradient was
measured between light regimes (Koch, 2005): On the forest
floor, air temperature was lower maximally by 3.4 8C (in June)
and air humidity was higher by a maximum of 19.3% (in July).
In 2003, the temperature was at most 5.1 8C higher than in
2004 (in June) and precipitation in 2003 reached only 39% of
precipitations in 2004 (in August) (Koch, 2005).
A sensor above the forest canopy of the adult trees quantified
solar irradiation. It was also used to quantify light reduction
under the shading device which was installed above the tow-
ers containing the seedlings to protect the light-sensitive
young beech plants from excess sunlight. The summed irradi-
ation from May to October 2003 above the forest canopy was
6.2 kmol m–2 (representing 100% of irradiation). Just 1–3% of
the irradiance (0.1 ± 0.01 to 0.2 ± 0.01 kmol m–2) reached the
shade crown and the forest floor with SHA containers. The
CASIROZ: Root Parameters and Types of Ectomycorrhiza of Young Beech Plants Plant Biology 9 (2007) 299