Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol16.1996:
Tree Physiology 16, 705--711
© 1996 Heron Publishing----Victoria, Canada
Influence of ectomycorrhization and cesium/potassium ratio on uptake
and localization of cesium in Norway spruce seedlings
IVANO BRUNNER,1 BEAT FREY1 and THOMAS K. RIESEN2
1
Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), CH-8903 Birmensdorf, Switzerland
2
Paul Scherrer Institute (PSI), CH-5232 Villigen, Switzerland
Received August 8, 1995
Summary Norway spruce (Picea abies (L.) Karst.) seedlings
growing in a growth pouch system were used to investigate the
effects of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex St. Amans) Quél. and various Cs/K ratios on
the uptake of 134Cs, expressed as a percentage of the total
amount of 134Cs supplied. The amount of 134Cs taken up by
seedlings increased with increasing Cs/K ratio. At a Cs/K ratio
of 0.1, uptake of 134Cs ranged between 7.2 and 7.3% and was
independent of ectomycorrhizal status, whereas at Cs/K ratios
≥ 1 uptake of 134Cs varied from 8.1 to 11.1% for ectomycorrhizal and from 10.4 to 14.4% for non-inoculated plants. Ectomycorrhizal seedlings contained a lower concentration of 134Cs
than non-inoculated seedlings. Among plant parts, the amount
of 134Cs was significantly lower in needles and lateral roots of
ectomycorrhizal seedlings compared with non-inoculated
seedlings. Among fungal and seedling tissues, highest X-ray
net counts of 133Cs were measured in fungal hyphae of ectomycorrhizal mantles. X-Ray net counts of 133Cs in lateral roots of
ectomycorrhizal and non-inoculated plants were similar, but 5
to 10 times higher than in main roots and needles, suggesting
an accumulation of 133Cs in lateral roots and slow translocation
to other plant parts. In contrast, X-ray net counts of K indicated
that K was readily mobilized from lateral roots to main roots
and needles. Elemental mapping showed a relatively homogeneous distribution of 133Cs within the root.
Keywords: gamma spectrometry, growth pouch, Hebeloma
crustuliniforme, low-temperature scanning electron microscopy, Picea abies, X-ray microanalysis.
Introduction
Radiocesium (137Cs) is a major radionuclide that has been
deposited on the Earth’s surface as fallout from nuclear weapons testing during the 1950s and 1960s and has entered the
biogeochemical cycles. The main sources of 134Cs and 137Cs in
Europe, however, are depositions from the Chernobyl nuclear
reactor accident in 1986. The amount of radioactive Cs in
Swiss forest soils differs widely (from 0 to 1900 Bq kg −1) from
area to area and even from site to site (Riesen et al. 1995). Most
of the Cs isotopes in contaminated forest soils are fixed in the
top layers; however, because Cs isotopes may be bound in
organic matter complexes, adsorbed by clay particles, or precipitated on surfaces of micaceous minerals, the concentration
of Cs in the soil solution is low and migration in the soil profile
is slow (Myttenaere et al. 1993).
Although Cs ions in the soil solution can be readily taken up
by plant roots, this uptake is competitive with that of K,
resulting in markedly reduced content of Cs in plants grown in
the presence of high concentrations of K (Coughtrey and
Thorne 1983). Thus, the Cs/K ratio may be a key factor in
determining Cs uptake by plant roots. Moreover, it is possible
that plants are able to discriminate between K and Cs, because
of the larger atomic weight of Cs (Coughtrey and Thorne
1983). It has been assumed that roots mobilize Cs ions passively as a consequence of the mobilization and uptake of K
from clay particles (Schaller et al. 1990). If this assumption is
correct, then plants grown in K-impoverished soil should contain more Cs than plants grown in a K-enriched soil.
The Cs content of forest soils is largely a reflection of
biological activity in the soil layers (Schaller et al. 1990,
Guillitte et al. 1994). Fungi, especially basidiomycetes which
are capable of forming ectomycorrhizae, are efficient accumulators of radiocesium (Dighton and Horrill 1988, Olsen et al.
1990, Römmelt et al. 1990, Haselwandter and Berreck 1994).
Schaller et al. (1990) suggested that Cs transport into the plant
is facilitated by ectomycorrhizal fungi that accumulate Cs
during microbial decomposition of the litter. However, it is not
known to what extent high Cs contents within fungal mycelia
are transferred to the host plants (Shaw and Bell 1994).
We investigated the effects of ectomycorrhizae and various
Cs/K ratios on the uptake of 134Cs by Norway spruce seedlings
raised in a growth pouch plant--fungus system (Brunner and
Scheidegger 1992, 1995). In addition, gamma-spectrometry
and cryo-scanning X-ray microanalysis (Stelzer and Lehmann
1993) were used to study the distribution and localization of
Cs in various plant parts.
706
BRUNNER, FREY AND RIESEN
Materials and methods
Plant and fungal material
Seeds of Norway spruce (Picea abies (L.) Karst.) were collected from a single tree growing near Tägerwilen in the
canton of Thurgau, Switzerland. Seeds were surface sterilized
in 30% H2O2 for 20 min. Mycelia of the ectomycorrhizal
fungal partner Hebeloma crustuliniforme (Bull. ex St. Amans)
Quél. were isolated from a fruiting body collected in 1990 in a
Norway spruce forest near Plaffeien in the canton of Freiburg,
Switzerland. Small tramal pieces were cut from the cap under
sterile conditions and cultured in petri dishes on modified
Melin-Norkrans nutrient agar (Marx and Bryan 1975). Growing mycelia, which were subcultured at regular intervals, were
identified as No. 6.2 of the culture collection of the Swiss
Federal Institute for Forest, Snow and Landscape Research,
Birmensdorf, Switzerland.
Growth of seedlings and fungal inoculation
Fifty seven autoclaved polyamide/polypropylene pouches
(Cellpack), measuring 13 × 16 cm, and divided longitudinally
into two chambers that were lined with two activated charcoal
filter papers (5 × 9 cm), were moistened with 5 ml per chamber
of modified Melin-Norkrans nutrient solution without glucose
(Brunner and Scheidegger 1995). Four surface-sterilized Norway spruce seeds were inserted in each chamber of the growth
pouches. Pouches were suspended on glass rods and randomly
distributed among four plastic buckets. The buckets were
placed in a growth chamber at 20 °C, 70% relative humidity
and a 16-h photoperiod (PAR = 100 µmol m −2 s −1). After seed
germination, seedlings were reduced to one per pouch chamber to give a total of 114 plants.
After 10 weeks, when lateral roots had developed, another
5 ml of modified Melin-Norkrans nutrient solution containing
5 g l −1 glucose was added to each pouch chamber. To obtain
ectomycorrhizal plants, approximately 60 seedlings were inoculated with mycelia of H. crustuliniforme by placing four to
six agar squares (4 × 4 mm) within 3 mm of the lateral roots of
each seedling. The remaining seedlings were not inoculated
with fungal mycelia. The pH of the nutrient solutions was 6.1
to 6.2 after autoclaving, and was 6.5 to 6.8 after two months,
irrespective of fungal inoculation (Brunner and Scheidegger
1995). One strip of foam (1 × 1 × 3 cm) was placed beside each
root to provide air space. Sterile distilled water was added as
needed. Plants that became visibly contaminated were discarded.
Application and analysis of radioactive 134Cs
Nine weeks after inoculation, 40 plants each of the inoculated
and the non-inoculated treatments were selected, and the nutrient solutions in the pouches were washed out twice with 10
ml of sterile water. Two ml of sterile water was then added to
the bottom of the pouches (without contacting the charcoal
paper) to provide the roots reaching the bottom of the pouches
with water during the experiment. After two days, Cs (133Cs +
134
Cs) and K were applied continuously as CsCl and KH2PO4
to the activated charcoal filter paper in daily portions of
50--250 µl to keep the charcoal paper moist. The Cs and K
were added to maintain Cs/K ratios of 100, 10, 1, or 0.1 for
each of 10 plants in the inoculated and the non-inoculated
treatments. The concentration of Cs was kept constant,
whereas the K concentration was varied from 367 µM to 367
mM. Over the 23-day experimental period, a total of 2.65 ml
of a 36.7 mM CsCl solution containing 270 µl radioactive 134Cs
was applied, resulting in a total of 26,474 Bq per plant with a
133
Cs/134Cs (stable/radioactive) ratio of 23,791/1.
One day after the last application of Cs and K, plants were
harvested, washed three times with 0.1 N HCl, separated into
needles, stems, main roots (diameter 1--2 mm), and lateral
roots (diameter < 1 mm). Roots grown on the activated charcoal filter paper were freed of adhering charcoal paper with a
pair of tweezers. Plant parts were dried at 100 °C for 3 days,
weighed, and dissolved in 2.5 ml of 65% HNO3 and 1.5 ml of
30% H2O2 in a high pressure microwave (Milestone MLS 1200
Mega, Microwave Laboratory Systems) for 10 min at 250 W,
6 min at 500 W, 2 min at 0 W, 6 min at 450 W, and 5 min of
ventilation. Radioactivity was measured with a gamma-spectrometer equipped with a Ge-detector connected to a multichannel analyzer. The dry weight data were subjected to a
one-factor analysis of variance to test for inoculation. The
Cs/K ratio was analyzed only as a second treatment factor and
did not influence growth over the 23-day exposure period. All
other dependent data were subjected to a two-factor analysis
of variance to test for the variable factors inoculation and Cs/K
ratio.
Application and analysis of stable 133Cs
Nine weeks after inoculation, 10 plants each from the inoculated and the non-inoculated treatments were selected and
treated as described above for the treatment with a Cs/K ratio
of 100, except that 133Cs was substituted for radioactive 134Cs.
One day after the last application, plant material was washed
in deionized water and dried with blotting-paper. Needles,
main roots, and lateral roots were cut into pieces approximately 5--10 mm in length with a razor blade. The samples
were set vertically in holes on aluminum stubs and held in
place with cryo-adhesive (Scheidegger and Brunner 1993) and
frozen in liquid nitrogen, or mounted in water between copper
platelets (Brunner and Scheidegger 1995) and frozen in liquid
propane. For cryo-scanning electron microscopy, the frozen
specimens were transferred to the preparation chamber (Balzers SCU 020) of a scanning electron microscope (Philips 515),
partially freeze-dried for 10 min at −80 °C in a high vacuum at
< 2 × 10 − 4 Pa (Müller et al. 1991), freeze-fractured with a
rotating microtome at − 90 °C, and immediately sputter-coated
with platinum (5 nm). The specimens were then transferred to
the cold stage of the scanning electron microscope with the
temperature kept below − 120 °C.
Elemental analyses of freeze-fractured samples were carried
out at different sites in the samples with a Tracor Northern
energy dispersive X-ray microanalysis system. Electron-induced X-rays were detected by a Pioneer Si(Li) light element
analytical detector (30 mm2 Microtrace) with a take-off angle
of 15°. The microscope was operated at an acceleration voltage
CESIUM IN NORWAY SPRUCE
707
of 18 kV with a beam current of 80 µA and a working distance
of 12 mm. The scan raster was a 4 µm2 square with a maximum
magnification of 10,000. In total, eight non-inoculated and
eight ectomycorrhizal plants were analyzed. For each plant,
one sample each of needles, main roots, and lateral roots was
taken. Within the lateral roots of the ectomycorrhizal plants,
only ectomycorrhizae were considered. Four spectra per cell
type within the various plant parts were acquired for analysis
with the live-time set for 120 s and the dead-time for approximately 20%. Spectra were processed to calculate net counts of
Cs and K using the Voyager software package (Noran Instruments Inc., Middleton, WI) which included an automatic peak
identification system. The values were not converted to concentrations because of the problems associated with obtaining
quantitative results from bulk-frozen hydrated samples (Van
Steveninck and Van Steveninck 1991).
Elemental mapping of the distribution of Cs in lateral roots
was done by energy window mapping using the Voyager software package. X-Rays characteristic for Cs were collected at
Lα = 4.29 keV across the cryosection area of interest. The
spatial resolution was 128 × 128 pixels for X-rays with a dwell
time of 0.1 s per pixel.
Results
Root colonization by the ectomycorrhizal fungus
Within 9 weeks of fungal inoculation, mycelial colonization of
the roots on the activated charcoal filter paper by the ectomycorrhizal fungus was approximately 100% and resulted in
abundant formation of ectomycorrhizae (Figure 1). The root
systems below the charcoal paper were not colonized by mycelia because these roots were submerged.
Uptake and distribution of 134Cs
Ectomycorrhization had no significant effect on dry weights of
whole plants or plant parts during the experimental period
(Table 1). However, concentrations of 134Cs in total plants were
Figure 1. Two, 5-month-old Norway spruce seedlings in a growth
pouch system showing roots on activated charcoal filter papers thoroughly colonized by mycelia of the ectomycorrhizal fungus Hebeloma
crustuliniforme and with ectomycorrhizae (arrowheads). Grid = 1 cm.
significantly reduced by fungal inoculation, whereas 134Cs
concentrations increased significantly with increasing Cs/K
ratio (Table 1). Needles and lateral roots exhibited the greatest
increase in 134Cs concentration with increasing Cs/K ratio,
Table 1. Effect of ectomycorrhization on dry weight and effects of ectomycorrhization and Cs/K ratio on the concentration of 134Cs of total plants
and plant parts. Values are means of 10 replicates. For the one- and two-factor analyses of variance: NS = not significant; * = significant at P ≤ 0.05;
** = significant at P ≤ 0.01; *** = significant at P ≤ 0.001; **** = significant at P ≤ 0.0001.
Cs/K ratio
Non-inoculated
0.1
Ectomycorrhizal
Probability
1
10
100
0.1
1
10
100
Fungus
Cs/K
86.1
49.4
4.9
8.6
23.2
109.3
60.0
5.9
10.1
33.3
94.3
51.8
6.5
9.1
26.9
82.4
44.8
6.7
6.7
24.2
87.0
43.6
5.2
10.0
28.2
92.5
49.3
5.2
8.3
29.7
95.6
54.4
6.6
8.4
26.2
NS
NS
NS
NS
NS
------
Concentration of 134Cs (Bq.mg −1)
Total plant
21.8
32.3
Needles
13.6
18.5
Stem
42.4
40.2
Main roots
37.5
41.3
Lateral roots
25.6
55.3
31.1
20.6
32.6
40.7
47.5
43.2
28.1
53.4
47.0
65.9
23.4
12.0
44.5
45.2
35.0
24.9
13.3
31.6
46.5
36.1
27.8
16.4
46.0
32.1
40.5
31.0
18.9
52.3
49.3
43.4
*
**
NS
NS
**
***
**
NS
NS
****
Dry weight (mg)
Total plant
88.7
Needles
48.3
Stem
5.6
Main roots
9.7
Lateral roots
25.1
708
BRUNNER, FREY AND RIESEN
whereas 134Cs concentrations of stems and main roots were not
significantly influenced by the Cs/K treatments (Table 1). At a
Cs/K ratio of 0.1, ectomycorrhization had almost no effect on
the concentration of 134Cs in total plants and needles, whereas
at a Cs/K ratio of 100, ectomycorrhization led to a significant
reduction in the concentration of 134Cs in total plants and
needles (Table 1). At a Cs/K ratio of 0.1, lateral roots of
non-inoculated plants had a lower concentration of 134Cs than
lateral roots of ectomycorrhizal plants, whereas at Cs/K ratios
of ≥ 1 the concentration of 134Cs was higher in ectomycorrhizal
plants than in non-inoculated plants and increased with increasing Cs/K ratio (Table 1).
Between 7.2 and 14.4% of the applied 134Cs was taken up by
the plants (Table 2). At a Cs/K ratio of 0.1 and independently
of fungal inoculation, 7.2--7.3% of the applied 134Cs was taken
up by the plants. In non-inoculated plants, uptake of 134Cs was
doubled by increasing the Cs/K ratio from 0.1 to 100, whereas
in ectomycorrhizal plants uptake was only enhanced by a
factor of 1.5 (Table 2). Uptake of 134Cs by stems and main roots
was not affected by any of the treatments. In contrast, uptake
of 134Cs by needles and lateral roots was significantly reduced
by the inoculation treatment but it increased significantly with
increasing Cs/K ratios (Table 2).
There was a linear dependency of 134Cs uptake by plants for
log-transformed Cs/K ratios, with ectomycorrhizal plants absorbing significantly less 134Cs than nonmycorrhizal plants
(Figure 2). Ectomycorrhization led to a 21 to 27% decrease in
Cs uptake in treatments in which the Cs concentration was
equal to or higher than the K concentration in the application
solution.
The distribution of 134Cs in different plant parts is given in
Table 2. Most of the 134Cs was detected in lateral roots (38.3 to
48.3%) and needles (25.9 to 36.0%), and only 5.6 to 19.4% of
the total 134Cs in the plants was located in stems and main roots.
Although the Cs/K ratio treatments had no effect on the distribution of 134Cs in needles of nonmycorrhizal plants, 134Cs in
needles of ectomycorrhizal plants increased with increasing
Cs/K ratio. The Cs/K ratio treatments had no significant effects
Figure 2. Effect of ectomycorrhization on the uptake of 134Cs in
non-inoculated and ectomycorrhizal plants. Values are means of 10
replicates.
on the distribution of 134Cs in stems and lateral roots (Table 2).
Averaged across the Cs/K ratio treatments, ectomycorrhization
significantly decreased 134Cs distribution in needles (30 versus
33.6%), significantly increased the amount of 134Cs in stems
(10.9 versus 7.7%), and had no effect on the amounts of 134Cs
in main roots and lateral roots (14.3 versus 13.9% and 44.8
versus 44.8%, respectively).
Localization of 133Cs
The X-ray net counts revealed that the largest amounts of 133Cs
were in fungal hyphae of the ectomycorrhizal mantle (Table 3),
whereas less 133Cs was evident in the Hartig net. The amounts
of 133Cs in the cortex and stele of lateral roots were similar and
independent of fungal inoculation. Cells of main roots and
needles contained 5 to 10 times less 133Cs than cells of lateral
roots. No differences in the distribution of 133Cs were evident
between epidermal and mesophyll cells of needles, or between
apoplastic and symplastic cells of the cortex and stele (Ta-
Table 2. Effects of ectomycorrhization and Cs/K ratio on uptake (percent recovered from the application solution) and distribution of recovered
134
Cs in total plants and plant parts. Values are means of 10 replicates. For the two-factor analysis of variance: NS = not significant; * = significant
at P ≤ 0.05; ** = significant at P ≤ 0.01; *** = significant at P ≤ 0.001; **** = significant at P ≤ 0.0001.
Cs/K ratio
Non-inoculated
0.1
Ectomycorrhizal
1
10
100
7.3
2.5
0.8
1.5
2.5
10.4
3.4
0.9
1.4
4.7
12.5
4.5
0.7
1.4
5.9
14.4
5.0
1.4
1.7
6.4
7.2
2.0
1.1
1.1
3.1
8.1
2.2
0.6
1.6
3.8
Distribution (%)
Needles
33.4
Stem
8.9
Main roots
19.4
Lateral roots
38.3
31.7
7.1
12.9
48.3
36.0
5.6
11.4
47.0
33.2
9.2
12.0
45.4
28.2
13.5
14.4
43.9
25.9
7.3
19.1
47.8
Uptake (%)
Total plant
Needles
Stem
Main roots
Lateral roots
0.1
1
Probability
10
100
Fungus
Cs/K
9.5
3.0
1.0
0.9
4.5
11.1
3.9
1.4
1.6
4.3
**
**
NS
NS
***
****
****
NS
NS
****
31.6
10.4
10.0
48.1
34.5
12.4
13.8
39.3
*
*
NS
NS
*
NS
*
NS
CESIUM IN NORWAY SPRUCE
709
Table 3. X-Ray net counts of Cs and K in various cell types of freeze-fractured plant parts (lateral roots, main roots, needles) of non-inoculated
and ectomycorrhizal plants. Within the lateral roots of the ectomycorrhizal plants, only ectomycorrhizae were considered for analysis. Values are
means ± SD of eight non-inoculated and eight ectomycorrhizal samples and of four spectra of each cell type. ND = not detected.
Cell type
Lateral roots
Main roots
Needles
Cs
K
Cs
K
Cs
K
Non-inoculated
Cortex (Symplasm)
Cortex (Apoplasm)
Stele (Symplasm)
Stele (Apoplasm)
Epidermis (Symplasm)
Mesophyll (Symplasm)
1604 ± 424
1367 ± 322
1398 ± 245
1276 ± 403
---
573 ± 131
555 ± 84
2044 ± 483
1308 ± 478
---
213 ± 102
ND
155 ± 76
ND
---
942 ± 256
271 ± 107
612 ± 318
165 ± 38
---
----211 ± 208
287 ± 144
----1272 ± 590
1953 ± 771
Ectomycorrhizal
Mantle hyphae (Symplasm)
Hartig net hyphae (Symplasm)
Cortex (Symplasm)
Cortex (Apoplasm)
Stele (Symplasm)
Stele (Apoplasm)
Epidermis (Symplasm)
Mesophyll (Symplasm)
3511 ± 817
1964 ± 521
1327 ± 315
1547 ± 432
1033 ± 341
1270 ± 298
---
670 ± 210
903 ± 341
349 ± 127
843 ± 236
1450 ± 211
2068 ± 467
---
--241 ± 69
ND
128 ± 45
ND
---
--879 ± 244
175 ± 86
427 ± 187
211 ± 103
---
------280 ± 212
293 ± 256
------2381 ± 403
2567 ± 831
ble 3). The distribution pattern of K was more or less the
inverse of that of Cs. Low amounts of K and high amounts of
Cs were present in mantle and Hartig net hyphae and in cortical
cells of lateral roots. High amounts of K and low amounts of
Cs were present in epidermal and mesophyll cells of needles.
However, large amounts of both K and Cs were found in the
stele of the lateral roots, and low amounts of both elements
were measured in all types of cells of main roots (Table 3).
The elemental mapping of Cs in a longitudinal fracture of a
frozen-hydrated ectomycorrhiza is shown in Figure 3. The map
shows that Cs was distributed relatively homogeneously
within the root fracture, but with a tendency for higher net
counts in the fungal mantle than in the Hartig net or in the
epidermal and cortical cells.
Discussion
Ectomycorrhizal fungi did not increase seedling biomass during the first two months after inoculation. Similar observations
have been made by Brunner and Scheidegger (1992, 1995)
who showed that although ectomycorrhizal development occurs during the first two months after inoculation, the increase
in nutrient uptake that accompanies ectomycorrhization does
not result in an increase of plant biomass for at least two
months. Uptake of 134Cs by Norway spruce seedlings was
significantly affected by the K concentration in the application
solution. Lowering the K concentration by a factor of 1000 by
increasing the Cs/K ratio from 0.1 to 100 enhanced the uptake
of Cs by a factor of two in non-inoculated seedlings and by a
factor of 1.5 in ectomycorrhizal seedlings. The mechanism by
Figure 3. Low-temperature scanning electron micrograph (A)
and Cs mapping with an X-ray
microanalysis system (B) of a
longitudinal freeze-fracture of an
ectomycorrhiza. M = fungal mantle, E = epidermal root cell, C =
cortical root cell, arrowheads =
Hartig net. Bar = 10 µm.
710
BRUNNER, FREY AND RIESEN
which plants and fungi discriminate between Cs and K is not
known nor is the identity of the uptake mechanism (Shaw and
Bell 1994).
Under conditions similar to those used in our study, Haselwandter and Berreck (1994) found significantly less 137Cs in
shoots of endomycorrhizal Festuca than in nonmycorrhizal
controls. Clint and Dighton (1992) measured short-term uptake of 137Cs by Calluna and found that the apparent influx of
137
Cs into mycorrhizal plants was less than into nonmycorrhizal controls. In a study using the growth pouch system, Riesen
and Brunner (unpublished data) measured less 134Cs in ectomycorrhizal Picea seedlings under low ammonium conditions than under high ammonium conditions. In contrast to
these studies, increased amounts of 137Cs in endomycorrhizal
Melilotus and Paspalum were observed by Rogers and Williams (1986) and McGraw et al. (1979), respectively. In addition, Entry et al. (1994) observed a significantly higher uptake
of 90Sr in two ectomycorrhizal Pinus species than in non-inoculated controls. Although it is not known whether mycorrhizae generally reduce plant uptake of radionuclides
(Haselwandter and Berreck 1994), it is evident that harvest
time, fungal and plant species, and the test systems used can
all influence radionuclide accumulation in plants (Haselwandter and Berreck 1994).
Based on our observation of lower uptake of Cs by ectomycorrhizal plants compared to non-inoculated controls, we conclude that Cs is trapped by the hyphae of the fungal mantles
and by the extramatrical mycelium. Our finding of high net
counts of Cs in the hyphae of the fungal mantles supports the
hypothesis that fungi have the potential to accumulate large
amounts of radionuclides and heavy metals (Colpaert and Van
Assche 1987, Galli et al. 1994, Haselwandter and Berreck
1994, Haselwandter et al. 1994). Furthermore, the potential is
higher for ectomycorrhizal fungi than for saprophytic fungi
(Römmelt et al. 1990). In the present study, however, hyphae
of the extramatrical mycelium, growing throughout the charcoal paper, could not be recovered and measured for their Cs
concentration, making the Cs balance impossible. Although
the mechanism by which Cs accumulates in filamentous fungi
is unknown, binding to cell walls components, polyphosphate
granules, or sulfhydryl compounds can probably be excluded
because Cs has similar characteristics to those of K (Galli et al.
1994, Haselwandter and Berreck 1994). In fungal fruiting
bodies of the ectomycorrhizal species Xerocomus, Cs is bound
in large amounts to the pigments of the cap (Aumann et al.
1989).
We used an X-ray microanalysis method to determine the
distribution of Cs in frozen-hydrated, freeze-fractured roots
and needles of an ectomycorrhizal plant. We were able to
demonstrate the localization of stable Cs in various plant
tissues and to compare patterns of 133Cs distribution between
cells of the cortex and stele, the inner and outer cortex and the
apoplasm and symplasm. Analysis of Cs within the root-fractures revealed a relatively homogeneous distribution, which is
similar to that of Sr (Frey et al. unpublished data) but different
from that of La which is mainly localized in apoplasts of
cortical cells and Hartig net hyphae, but is absent from symplasts (Scheidegger and Brunner 1995). X-Ray microanalysis
showed that the distribution patterns of Cs in the various plant
tissues were more or less the inverse of those of K. Thus, high
amounts of Cs and low amounts of K were found in the outer
parts of roots, whereas high amounts of K and low amounts of
Cs were found in the inner parts of roots. Although Cs and K
are similar in mobility and uptake, the different distribution
patterns indicate a strong preference by the fungus for K
uptake over Cs uptake. The mechanisms that regulate the
transfer of K and Cs ions between the ectomycorrhizal fungus
and its host are unknown (Haselwandter et al. 1994).
There are technical difficulties associated with quantification of ion concentrations in bulk-frozen hydrated and freezefractured plant tissues including the uncertain etching process,
the uneven fracture planes, and the lack of available standards
(Stelzer et al. 1988, Van Steveninck and Van Steveninck 1991).
However, cryosectioning and freeze-drying under controlled
conditions in an EM provides a more reliable method (Zierold
1988).
Acknowledgments
This work was supported by the Swiss Federal Nuclear Safety Inspectorate and the Federal Office of Energy. We thank A. Wolf for technical
assistance, C. Scheidegger and D. Rigling for critical reading, and S.
Ortloff for correcting the English text.
References
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© 1996 Heron Publishing----Victoria, Canada
Influence of ectomycorrhization and cesium/potassium ratio on uptake
and localization of cesium in Norway spruce seedlings
IVANO BRUNNER,1 BEAT FREY1 and THOMAS K. RIESEN2
1
Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), CH-8903 Birmensdorf, Switzerland
2
Paul Scherrer Institute (PSI), CH-5232 Villigen, Switzerland
Received August 8, 1995
Summary Norway spruce (Picea abies (L.) Karst.) seedlings
growing in a growth pouch system were used to investigate the
effects of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex St. Amans) Quél. and various Cs/K ratios on
the uptake of 134Cs, expressed as a percentage of the total
amount of 134Cs supplied. The amount of 134Cs taken up by
seedlings increased with increasing Cs/K ratio. At a Cs/K ratio
of 0.1, uptake of 134Cs ranged between 7.2 and 7.3% and was
independent of ectomycorrhizal status, whereas at Cs/K ratios
≥ 1 uptake of 134Cs varied from 8.1 to 11.1% for ectomycorrhizal and from 10.4 to 14.4% for non-inoculated plants. Ectomycorrhizal seedlings contained a lower concentration of 134Cs
than non-inoculated seedlings. Among plant parts, the amount
of 134Cs was significantly lower in needles and lateral roots of
ectomycorrhizal seedlings compared with non-inoculated
seedlings. Among fungal and seedling tissues, highest X-ray
net counts of 133Cs were measured in fungal hyphae of ectomycorrhizal mantles. X-Ray net counts of 133Cs in lateral roots of
ectomycorrhizal and non-inoculated plants were similar, but 5
to 10 times higher than in main roots and needles, suggesting
an accumulation of 133Cs in lateral roots and slow translocation
to other plant parts. In contrast, X-ray net counts of K indicated
that K was readily mobilized from lateral roots to main roots
and needles. Elemental mapping showed a relatively homogeneous distribution of 133Cs within the root.
Keywords: gamma spectrometry, growth pouch, Hebeloma
crustuliniforme, low-temperature scanning electron microscopy, Picea abies, X-ray microanalysis.
Introduction
Radiocesium (137Cs) is a major radionuclide that has been
deposited on the Earth’s surface as fallout from nuclear weapons testing during the 1950s and 1960s and has entered the
biogeochemical cycles. The main sources of 134Cs and 137Cs in
Europe, however, are depositions from the Chernobyl nuclear
reactor accident in 1986. The amount of radioactive Cs in
Swiss forest soils differs widely (from 0 to 1900 Bq kg −1) from
area to area and even from site to site (Riesen et al. 1995). Most
of the Cs isotopes in contaminated forest soils are fixed in the
top layers; however, because Cs isotopes may be bound in
organic matter complexes, adsorbed by clay particles, or precipitated on surfaces of micaceous minerals, the concentration
of Cs in the soil solution is low and migration in the soil profile
is slow (Myttenaere et al. 1993).
Although Cs ions in the soil solution can be readily taken up
by plant roots, this uptake is competitive with that of K,
resulting in markedly reduced content of Cs in plants grown in
the presence of high concentrations of K (Coughtrey and
Thorne 1983). Thus, the Cs/K ratio may be a key factor in
determining Cs uptake by plant roots. Moreover, it is possible
that plants are able to discriminate between K and Cs, because
of the larger atomic weight of Cs (Coughtrey and Thorne
1983). It has been assumed that roots mobilize Cs ions passively as a consequence of the mobilization and uptake of K
from clay particles (Schaller et al. 1990). If this assumption is
correct, then plants grown in K-impoverished soil should contain more Cs than plants grown in a K-enriched soil.
The Cs content of forest soils is largely a reflection of
biological activity in the soil layers (Schaller et al. 1990,
Guillitte et al. 1994). Fungi, especially basidiomycetes which
are capable of forming ectomycorrhizae, are efficient accumulators of radiocesium (Dighton and Horrill 1988, Olsen et al.
1990, Römmelt et al. 1990, Haselwandter and Berreck 1994).
Schaller et al. (1990) suggested that Cs transport into the plant
is facilitated by ectomycorrhizal fungi that accumulate Cs
during microbial decomposition of the litter. However, it is not
known to what extent high Cs contents within fungal mycelia
are transferred to the host plants (Shaw and Bell 1994).
We investigated the effects of ectomycorrhizae and various
Cs/K ratios on the uptake of 134Cs by Norway spruce seedlings
raised in a growth pouch plant--fungus system (Brunner and
Scheidegger 1992, 1995). In addition, gamma-spectrometry
and cryo-scanning X-ray microanalysis (Stelzer and Lehmann
1993) were used to study the distribution and localization of
Cs in various plant parts.
706
BRUNNER, FREY AND RIESEN
Materials and methods
Plant and fungal material
Seeds of Norway spruce (Picea abies (L.) Karst.) were collected from a single tree growing near Tägerwilen in the
canton of Thurgau, Switzerland. Seeds were surface sterilized
in 30% H2O2 for 20 min. Mycelia of the ectomycorrhizal
fungal partner Hebeloma crustuliniforme (Bull. ex St. Amans)
Quél. were isolated from a fruiting body collected in 1990 in a
Norway spruce forest near Plaffeien in the canton of Freiburg,
Switzerland. Small tramal pieces were cut from the cap under
sterile conditions and cultured in petri dishes on modified
Melin-Norkrans nutrient agar (Marx and Bryan 1975). Growing mycelia, which were subcultured at regular intervals, were
identified as No. 6.2 of the culture collection of the Swiss
Federal Institute for Forest, Snow and Landscape Research,
Birmensdorf, Switzerland.
Growth of seedlings and fungal inoculation
Fifty seven autoclaved polyamide/polypropylene pouches
(Cellpack), measuring 13 × 16 cm, and divided longitudinally
into two chambers that were lined with two activated charcoal
filter papers (5 × 9 cm), were moistened with 5 ml per chamber
of modified Melin-Norkrans nutrient solution without glucose
(Brunner and Scheidegger 1995). Four surface-sterilized Norway spruce seeds were inserted in each chamber of the growth
pouches. Pouches were suspended on glass rods and randomly
distributed among four plastic buckets. The buckets were
placed in a growth chamber at 20 °C, 70% relative humidity
and a 16-h photoperiod (PAR = 100 µmol m −2 s −1). After seed
germination, seedlings were reduced to one per pouch chamber to give a total of 114 plants.
After 10 weeks, when lateral roots had developed, another
5 ml of modified Melin-Norkrans nutrient solution containing
5 g l −1 glucose was added to each pouch chamber. To obtain
ectomycorrhizal plants, approximately 60 seedlings were inoculated with mycelia of H. crustuliniforme by placing four to
six agar squares (4 × 4 mm) within 3 mm of the lateral roots of
each seedling. The remaining seedlings were not inoculated
with fungal mycelia. The pH of the nutrient solutions was 6.1
to 6.2 after autoclaving, and was 6.5 to 6.8 after two months,
irrespective of fungal inoculation (Brunner and Scheidegger
1995). One strip of foam (1 × 1 × 3 cm) was placed beside each
root to provide air space. Sterile distilled water was added as
needed. Plants that became visibly contaminated were discarded.
Application and analysis of radioactive 134Cs
Nine weeks after inoculation, 40 plants each of the inoculated
and the non-inoculated treatments were selected, and the nutrient solutions in the pouches were washed out twice with 10
ml of sterile water. Two ml of sterile water was then added to
the bottom of the pouches (without contacting the charcoal
paper) to provide the roots reaching the bottom of the pouches
with water during the experiment. After two days, Cs (133Cs +
134
Cs) and K were applied continuously as CsCl and KH2PO4
to the activated charcoal filter paper in daily portions of
50--250 µl to keep the charcoal paper moist. The Cs and K
were added to maintain Cs/K ratios of 100, 10, 1, or 0.1 for
each of 10 plants in the inoculated and the non-inoculated
treatments. The concentration of Cs was kept constant,
whereas the K concentration was varied from 367 µM to 367
mM. Over the 23-day experimental period, a total of 2.65 ml
of a 36.7 mM CsCl solution containing 270 µl radioactive 134Cs
was applied, resulting in a total of 26,474 Bq per plant with a
133
Cs/134Cs (stable/radioactive) ratio of 23,791/1.
One day after the last application of Cs and K, plants were
harvested, washed three times with 0.1 N HCl, separated into
needles, stems, main roots (diameter 1--2 mm), and lateral
roots (diameter < 1 mm). Roots grown on the activated charcoal filter paper were freed of adhering charcoal paper with a
pair of tweezers. Plant parts were dried at 100 °C for 3 days,
weighed, and dissolved in 2.5 ml of 65% HNO3 and 1.5 ml of
30% H2O2 in a high pressure microwave (Milestone MLS 1200
Mega, Microwave Laboratory Systems) for 10 min at 250 W,
6 min at 500 W, 2 min at 0 W, 6 min at 450 W, and 5 min of
ventilation. Radioactivity was measured with a gamma-spectrometer equipped with a Ge-detector connected to a multichannel analyzer. The dry weight data were subjected to a
one-factor analysis of variance to test for inoculation. The
Cs/K ratio was analyzed only as a second treatment factor and
did not influence growth over the 23-day exposure period. All
other dependent data were subjected to a two-factor analysis
of variance to test for the variable factors inoculation and Cs/K
ratio.
Application and analysis of stable 133Cs
Nine weeks after inoculation, 10 plants each from the inoculated and the non-inoculated treatments were selected and
treated as described above for the treatment with a Cs/K ratio
of 100, except that 133Cs was substituted for radioactive 134Cs.
One day after the last application, plant material was washed
in deionized water and dried with blotting-paper. Needles,
main roots, and lateral roots were cut into pieces approximately 5--10 mm in length with a razor blade. The samples
were set vertically in holes on aluminum stubs and held in
place with cryo-adhesive (Scheidegger and Brunner 1993) and
frozen in liquid nitrogen, or mounted in water between copper
platelets (Brunner and Scheidegger 1995) and frozen in liquid
propane. For cryo-scanning electron microscopy, the frozen
specimens were transferred to the preparation chamber (Balzers SCU 020) of a scanning electron microscope (Philips 515),
partially freeze-dried for 10 min at −80 °C in a high vacuum at
< 2 × 10 − 4 Pa (Müller et al. 1991), freeze-fractured with a
rotating microtome at − 90 °C, and immediately sputter-coated
with platinum (5 nm). The specimens were then transferred to
the cold stage of the scanning electron microscope with the
temperature kept below − 120 °C.
Elemental analyses of freeze-fractured samples were carried
out at different sites in the samples with a Tracor Northern
energy dispersive X-ray microanalysis system. Electron-induced X-rays were detected by a Pioneer Si(Li) light element
analytical detector (30 mm2 Microtrace) with a take-off angle
of 15°. The microscope was operated at an acceleration voltage
CESIUM IN NORWAY SPRUCE
707
of 18 kV with a beam current of 80 µA and a working distance
of 12 mm. The scan raster was a 4 µm2 square with a maximum
magnification of 10,000. In total, eight non-inoculated and
eight ectomycorrhizal plants were analyzed. For each plant,
one sample each of needles, main roots, and lateral roots was
taken. Within the lateral roots of the ectomycorrhizal plants,
only ectomycorrhizae were considered. Four spectra per cell
type within the various plant parts were acquired for analysis
with the live-time set for 120 s and the dead-time for approximately 20%. Spectra were processed to calculate net counts of
Cs and K using the Voyager software package (Noran Instruments Inc., Middleton, WI) which included an automatic peak
identification system. The values were not converted to concentrations because of the problems associated with obtaining
quantitative results from bulk-frozen hydrated samples (Van
Steveninck and Van Steveninck 1991).
Elemental mapping of the distribution of Cs in lateral roots
was done by energy window mapping using the Voyager software package. X-Rays characteristic for Cs were collected at
Lα = 4.29 keV across the cryosection area of interest. The
spatial resolution was 128 × 128 pixels for X-rays with a dwell
time of 0.1 s per pixel.
Results
Root colonization by the ectomycorrhizal fungus
Within 9 weeks of fungal inoculation, mycelial colonization of
the roots on the activated charcoal filter paper by the ectomycorrhizal fungus was approximately 100% and resulted in
abundant formation of ectomycorrhizae (Figure 1). The root
systems below the charcoal paper were not colonized by mycelia because these roots were submerged.
Uptake and distribution of 134Cs
Ectomycorrhization had no significant effect on dry weights of
whole plants or plant parts during the experimental period
(Table 1). However, concentrations of 134Cs in total plants were
Figure 1. Two, 5-month-old Norway spruce seedlings in a growth
pouch system showing roots on activated charcoal filter papers thoroughly colonized by mycelia of the ectomycorrhizal fungus Hebeloma
crustuliniforme and with ectomycorrhizae (arrowheads). Grid = 1 cm.
significantly reduced by fungal inoculation, whereas 134Cs
concentrations increased significantly with increasing Cs/K
ratio (Table 1). Needles and lateral roots exhibited the greatest
increase in 134Cs concentration with increasing Cs/K ratio,
Table 1. Effect of ectomycorrhization on dry weight and effects of ectomycorrhization and Cs/K ratio on the concentration of 134Cs of total plants
and plant parts. Values are means of 10 replicates. For the one- and two-factor analyses of variance: NS = not significant; * = significant at P ≤ 0.05;
** = significant at P ≤ 0.01; *** = significant at P ≤ 0.001; **** = significant at P ≤ 0.0001.
Cs/K ratio
Non-inoculated
0.1
Ectomycorrhizal
Probability
1
10
100
0.1
1
10
100
Fungus
Cs/K
86.1
49.4
4.9
8.6
23.2
109.3
60.0
5.9
10.1
33.3
94.3
51.8
6.5
9.1
26.9
82.4
44.8
6.7
6.7
24.2
87.0
43.6
5.2
10.0
28.2
92.5
49.3
5.2
8.3
29.7
95.6
54.4
6.6
8.4
26.2
NS
NS
NS
NS
NS
------
Concentration of 134Cs (Bq.mg −1)
Total plant
21.8
32.3
Needles
13.6
18.5
Stem
42.4
40.2
Main roots
37.5
41.3
Lateral roots
25.6
55.3
31.1
20.6
32.6
40.7
47.5
43.2
28.1
53.4
47.0
65.9
23.4
12.0
44.5
45.2
35.0
24.9
13.3
31.6
46.5
36.1
27.8
16.4
46.0
32.1
40.5
31.0
18.9
52.3
49.3
43.4
*
**
NS
NS
**
***
**
NS
NS
****
Dry weight (mg)
Total plant
88.7
Needles
48.3
Stem
5.6
Main roots
9.7
Lateral roots
25.1
708
BRUNNER, FREY AND RIESEN
whereas 134Cs concentrations of stems and main roots were not
significantly influenced by the Cs/K treatments (Table 1). At a
Cs/K ratio of 0.1, ectomycorrhization had almost no effect on
the concentration of 134Cs in total plants and needles, whereas
at a Cs/K ratio of 100, ectomycorrhization led to a significant
reduction in the concentration of 134Cs in total plants and
needles (Table 1). At a Cs/K ratio of 0.1, lateral roots of
non-inoculated plants had a lower concentration of 134Cs than
lateral roots of ectomycorrhizal plants, whereas at Cs/K ratios
of ≥ 1 the concentration of 134Cs was higher in ectomycorrhizal
plants than in non-inoculated plants and increased with increasing Cs/K ratio (Table 1).
Between 7.2 and 14.4% of the applied 134Cs was taken up by
the plants (Table 2). At a Cs/K ratio of 0.1 and independently
of fungal inoculation, 7.2--7.3% of the applied 134Cs was taken
up by the plants. In non-inoculated plants, uptake of 134Cs was
doubled by increasing the Cs/K ratio from 0.1 to 100, whereas
in ectomycorrhizal plants uptake was only enhanced by a
factor of 1.5 (Table 2). Uptake of 134Cs by stems and main roots
was not affected by any of the treatments. In contrast, uptake
of 134Cs by needles and lateral roots was significantly reduced
by the inoculation treatment but it increased significantly with
increasing Cs/K ratios (Table 2).
There was a linear dependency of 134Cs uptake by plants for
log-transformed Cs/K ratios, with ectomycorrhizal plants absorbing significantly less 134Cs than nonmycorrhizal plants
(Figure 2). Ectomycorrhization led to a 21 to 27% decrease in
Cs uptake in treatments in which the Cs concentration was
equal to or higher than the K concentration in the application
solution.
The distribution of 134Cs in different plant parts is given in
Table 2. Most of the 134Cs was detected in lateral roots (38.3 to
48.3%) and needles (25.9 to 36.0%), and only 5.6 to 19.4% of
the total 134Cs in the plants was located in stems and main roots.
Although the Cs/K ratio treatments had no effect on the distribution of 134Cs in needles of nonmycorrhizal plants, 134Cs in
needles of ectomycorrhizal plants increased with increasing
Cs/K ratio. The Cs/K ratio treatments had no significant effects
Figure 2. Effect of ectomycorrhization on the uptake of 134Cs in
non-inoculated and ectomycorrhizal plants. Values are means of 10
replicates.
on the distribution of 134Cs in stems and lateral roots (Table 2).
Averaged across the Cs/K ratio treatments, ectomycorrhization
significantly decreased 134Cs distribution in needles (30 versus
33.6%), significantly increased the amount of 134Cs in stems
(10.9 versus 7.7%), and had no effect on the amounts of 134Cs
in main roots and lateral roots (14.3 versus 13.9% and 44.8
versus 44.8%, respectively).
Localization of 133Cs
The X-ray net counts revealed that the largest amounts of 133Cs
were in fungal hyphae of the ectomycorrhizal mantle (Table 3),
whereas less 133Cs was evident in the Hartig net. The amounts
of 133Cs in the cortex and stele of lateral roots were similar and
independent of fungal inoculation. Cells of main roots and
needles contained 5 to 10 times less 133Cs than cells of lateral
roots. No differences in the distribution of 133Cs were evident
between epidermal and mesophyll cells of needles, or between
apoplastic and symplastic cells of the cortex and stele (Ta-
Table 2. Effects of ectomycorrhization and Cs/K ratio on uptake (percent recovered from the application solution) and distribution of recovered
134
Cs in total plants and plant parts. Values are means of 10 replicates. For the two-factor analysis of variance: NS = not significant; * = significant
at P ≤ 0.05; ** = significant at P ≤ 0.01; *** = significant at P ≤ 0.001; **** = significant at P ≤ 0.0001.
Cs/K ratio
Non-inoculated
0.1
Ectomycorrhizal
1
10
100
7.3
2.5
0.8
1.5
2.5
10.4
3.4
0.9
1.4
4.7
12.5
4.5
0.7
1.4
5.9
14.4
5.0
1.4
1.7
6.4
7.2
2.0
1.1
1.1
3.1
8.1
2.2
0.6
1.6
3.8
Distribution (%)
Needles
33.4
Stem
8.9
Main roots
19.4
Lateral roots
38.3
31.7
7.1
12.9
48.3
36.0
5.6
11.4
47.0
33.2
9.2
12.0
45.4
28.2
13.5
14.4
43.9
25.9
7.3
19.1
47.8
Uptake (%)
Total plant
Needles
Stem
Main roots
Lateral roots
0.1
1
Probability
10
100
Fungus
Cs/K
9.5
3.0
1.0
0.9
4.5
11.1
3.9
1.4
1.6
4.3
**
**
NS
NS
***
****
****
NS
NS
****
31.6
10.4
10.0
48.1
34.5
12.4
13.8
39.3
*
*
NS
NS
*
NS
*
NS
CESIUM IN NORWAY SPRUCE
709
Table 3. X-Ray net counts of Cs and K in various cell types of freeze-fractured plant parts (lateral roots, main roots, needles) of non-inoculated
and ectomycorrhizal plants. Within the lateral roots of the ectomycorrhizal plants, only ectomycorrhizae were considered for analysis. Values are
means ± SD of eight non-inoculated and eight ectomycorrhizal samples and of four spectra of each cell type. ND = not detected.
Cell type
Lateral roots
Main roots
Needles
Cs
K
Cs
K
Cs
K
Non-inoculated
Cortex (Symplasm)
Cortex (Apoplasm)
Stele (Symplasm)
Stele (Apoplasm)
Epidermis (Symplasm)
Mesophyll (Symplasm)
1604 ± 424
1367 ± 322
1398 ± 245
1276 ± 403
---
573 ± 131
555 ± 84
2044 ± 483
1308 ± 478
---
213 ± 102
ND
155 ± 76
ND
---
942 ± 256
271 ± 107
612 ± 318
165 ± 38
---
----211 ± 208
287 ± 144
----1272 ± 590
1953 ± 771
Ectomycorrhizal
Mantle hyphae (Symplasm)
Hartig net hyphae (Symplasm)
Cortex (Symplasm)
Cortex (Apoplasm)
Stele (Symplasm)
Stele (Apoplasm)
Epidermis (Symplasm)
Mesophyll (Symplasm)
3511 ± 817
1964 ± 521
1327 ± 315
1547 ± 432
1033 ± 341
1270 ± 298
---
670 ± 210
903 ± 341
349 ± 127
843 ± 236
1450 ± 211
2068 ± 467
---
--241 ± 69
ND
128 ± 45
ND
---
--879 ± 244
175 ± 86
427 ± 187
211 ± 103
---
------280 ± 212
293 ± 256
------2381 ± 403
2567 ± 831
ble 3). The distribution pattern of K was more or less the
inverse of that of Cs. Low amounts of K and high amounts of
Cs were present in mantle and Hartig net hyphae and in cortical
cells of lateral roots. High amounts of K and low amounts of
Cs were present in epidermal and mesophyll cells of needles.
However, large amounts of both K and Cs were found in the
stele of the lateral roots, and low amounts of both elements
were measured in all types of cells of main roots (Table 3).
The elemental mapping of Cs in a longitudinal fracture of a
frozen-hydrated ectomycorrhiza is shown in Figure 3. The map
shows that Cs was distributed relatively homogeneously
within the root fracture, but with a tendency for higher net
counts in the fungal mantle than in the Hartig net or in the
epidermal and cortical cells.
Discussion
Ectomycorrhizal fungi did not increase seedling biomass during the first two months after inoculation. Similar observations
have been made by Brunner and Scheidegger (1992, 1995)
who showed that although ectomycorrhizal development occurs during the first two months after inoculation, the increase
in nutrient uptake that accompanies ectomycorrhization does
not result in an increase of plant biomass for at least two
months. Uptake of 134Cs by Norway spruce seedlings was
significantly affected by the K concentration in the application
solution. Lowering the K concentration by a factor of 1000 by
increasing the Cs/K ratio from 0.1 to 100 enhanced the uptake
of Cs by a factor of two in non-inoculated seedlings and by a
factor of 1.5 in ectomycorrhizal seedlings. The mechanism by
Figure 3. Low-temperature scanning electron micrograph (A)
and Cs mapping with an X-ray
microanalysis system (B) of a
longitudinal freeze-fracture of an
ectomycorrhiza. M = fungal mantle, E = epidermal root cell, C =
cortical root cell, arrowheads =
Hartig net. Bar = 10 µm.
710
BRUNNER, FREY AND RIESEN
which plants and fungi discriminate between Cs and K is not
known nor is the identity of the uptake mechanism (Shaw and
Bell 1994).
Under conditions similar to those used in our study, Haselwandter and Berreck (1994) found significantly less 137Cs in
shoots of endomycorrhizal Festuca than in nonmycorrhizal
controls. Clint and Dighton (1992) measured short-term uptake of 137Cs by Calluna and found that the apparent influx of
137
Cs into mycorrhizal plants was less than into nonmycorrhizal controls. In a study using the growth pouch system, Riesen
and Brunner (unpublished data) measured less 134Cs in ectomycorrhizal Picea seedlings under low ammonium conditions than under high ammonium conditions. In contrast to
these studies, increased amounts of 137Cs in endomycorrhizal
Melilotus and Paspalum were observed by Rogers and Williams (1986) and McGraw et al. (1979), respectively. In addition, Entry et al. (1994) observed a significantly higher uptake
of 90Sr in two ectomycorrhizal Pinus species than in non-inoculated controls. Although it is not known whether mycorrhizae generally reduce plant uptake of radionuclides
(Haselwandter and Berreck 1994), it is evident that harvest
time, fungal and plant species, and the test systems used can
all influence radionuclide accumulation in plants (Haselwandter and Berreck 1994).
Based on our observation of lower uptake of Cs by ectomycorrhizal plants compared to non-inoculated controls, we conclude that Cs is trapped by the hyphae of the fungal mantles
and by the extramatrical mycelium. Our finding of high net
counts of Cs in the hyphae of the fungal mantles supports the
hypothesis that fungi have the potential to accumulate large
amounts of radionuclides and heavy metals (Colpaert and Van
Assche 1987, Galli et al. 1994, Haselwandter and Berreck
1994, Haselwandter et al. 1994). Furthermore, the potential is
higher for ectomycorrhizal fungi than for saprophytic fungi
(Römmelt et al. 1990). In the present study, however, hyphae
of the extramatrical mycelium, growing throughout the charcoal paper, could not be recovered and measured for their Cs
concentration, making the Cs balance impossible. Although
the mechanism by which Cs accumulates in filamentous fungi
is unknown, binding to cell walls components, polyphosphate
granules, or sulfhydryl compounds can probably be excluded
because Cs has similar characteristics to those of K (Galli et al.
1994, Haselwandter and Berreck 1994). In fungal fruiting
bodies of the ectomycorrhizal species Xerocomus, Cs is bound
in large amounts to the pigments of the cap (Aumann et al.
1989).
We used an X-ray microanalysis method to determine the
distribution of Cs in frozen-hydrated, freeze-fractured roots
and needles of an ectomycorrhizal plant. We were able to
demonstrate the localization of stable Cs in various plant
tissues and to compare patterns of 133Cs distribution between
cells of the cortex and stele, the inner and outer cortex and the
apoplasm and symplasm. Analysis of Cs within the root-fractures revealed a relatively homogeneous distribution, which is
similar to that of Sr (Frey et al. unpublished data) but different
from that of La which is mainly localized in apoplasts of
cortical cells and Hartig net hyphae, but is absent from symplasts (Scheidegger and Brunner 1995). X-Ray microanalysis
showed that the distribution patterns of Cs in the various plant
tissues were more or less the inverse of those of K. Thus, high
amounts of Cs and low amounts of K were found in the outer
parts of roots, whereas high amounts of K and low amounts of
Cs were found in the inner parts of roots. Although Cs and K
are similar in mobility and uptake, the different distribution
patterns indicate a strong preference by the fungus for K
uptake over Cs uptake. The mechanisms that regulate the
transfer of K and Cs ions between the ectomycorrhizal fungus
and its host are unknown (Haselwandter et al. 1994).
There are technical difficulties associated with quantification of ion concentrations in bulk-frozen hydrated and freezefractured plant tissues including the uncertain etching process,
the uneven fracture planes, and the lack of available standards
(Stelzer et al. 1988, Van Steveninck and Van Steveninck 1991).
However, cryosectioning and freeze-drying under controlled
conditions in an EM provides a more reliable method (Zierold
1988).
Acknowledgments
This work was supported by the Swiss Federal Nuclear Safety Inspectorate and the Federal Office of Energy. We thank A. Wolf for technical
assistance, C. Scheidegger and D. Rigling for critical reading, and S.
Ortloff for correcting the English text.
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