202 B. Enkhtuya et al. Applied Soil Ecology 14 2000 201–211
Substrates in these waste disposal sites show high concentrations of toxic heavy metals such as Mn, Fe
and Al, high salinity and pH fluctuations. Successful survival and growth of plants in soils
degraded by industrial activity is greatly dependent not only upon the abiotic properties of the soil but
also on the activity of microbial populations Visser, 1985. The presence of arbuscular mycorrhizal fungi
AMF may reduce negative effects of stresses caused by lack of nutrients or organic matter, by adverse soil
structure, extreme pH or by pathogens Sylvia and Williams, 1992. AMF can also enhance the resis-
tance of plants to drought stress and high salinity due to the increased absorption zone of mycorrhizal roots
Hardie, 1985. An important feature of AMF might be a protective role of mycorrhiza against stress in-
duced by high concentrations of heavy metals Galli et al., 1994. Schuepp et al. 1987 have postulated
that AMF can serve as a filtration barrier against trans- fer of heavy metals to the plant shoots. Better P nu-
trition and increase in plant biomass have also been proposed as possible reasons for a higher tolerance to
heavy metals Haselwandter et al., 1994. However, different populations or geographical isolates of AMF
were found to show high variability in their tolerance to heavy metals and associated stress Leyval et al.,
1991; Weissenhorn et al., 1993; Bartolome-Esteban and Schenck, 1994.
Elimination of AMF populations leads to prob- lems with plant establishment and survival Pfleger
et al., 1994. Even if AMF are ubiquitous in terres- trial ecosystems, mechanical or chemical disturbance
of the soil can substantially reduce AMF population vigor and functioning Sylvia and Williams, 1992.
Numbers of spores and root colonization are often reduced by soil disturbance Waaland and Allen,
1987, but AMF isolates adapted to local soil con- ditions are still able to stimulate plant growth at
that site compared with non-indigenous isolates. It seems probable that such AMF ecotypes result from
long-term adaptation to soils with extreme properties Sylvia and Williams, 1992. Isolation of indigenous
and presumably stress-adapted AMF is a potential biotechnological tool for inoculation of plants in dis-
turbed ecosystems Dodd and Thompson, 1994. The isolation and study of these ‘stress-tolerant’ isolates
might contribute to knowledge of the ecophysiology of AMF under stress conditions.
The aim of the present study was to study the effec- tiveness of indigenous AMF isolates from disturbed
soils and non-indigenous isolates from undisturbed soils in symbiosis with maize a model universal host
plant for AMF growing in disturbed soils.
2. Materials and methods
2.1. Site characteristics 2.1.1. Sedimentation ponds
The Chvaletice CHV and Opatovice OPA sedi- mentation ponds are located in the eastern part of the
Labe river basin 50
◦
02
′
28
′′
N, 15
◦
26
′
39
′′
E, altitude 204 m, and 50
◦
04
′
00
′′
N, 15
◦
50
′
00
′′
E, altitude 208 m. The Tusimice TUS sedimentation pond is located
in the North Bohemia 50
◦
04
′
00
′′
N, 15
◦
50
′
00
′′
E, alti- tude 208 m. Waste from a smelter factory processing
pyrite raw materials has been stored in the Chvalet- ice sedimentation pond. The OPA and TUS ponds
contain fly ash from a power station burning brown coal. The OPA and CHV ponds were abandoned in
the 1980s, whereas TUS is still being used for fly ash storage. Vegetation spontaneously developed on
the CHV pond, dominated by Calamagrostis epigejos with small hardwoods such as birch, poplar and wil-
low. The OPA and TUS ponds have been vegetated by a mixture of grasses Festuca rubra, Poa pratensis,
but C. epigejos has become gradually dominant over the sown grass species. Soil from the CHV pond is
characterized by a high content of sulfides, and there- fore a low pH. Weathering of the soil causes strong
acidification followed by an increase in salinity. The soil also shows high concentrations of heavy metals,
mainly Mn, Fe and Al Rauch, 1996. Fly ash mixtures from the OPA sedimentation pond are acid, whereas
those from TUS are alkaline and both are rich in the soluble elements, Ca, Mg, K and Na, which influences
the toxicity of other elements and compounds. Both soils are very vulnerable to erosion and drought.
2.1.2. Spoil banks Thousands of hectares of spoil banks in the Most
coal basin northwest Bohemia have been created following the mining of brown coal. The major part
of the spoil banks consists of gray Miocene clay. Three spoil banks at different stages of succession
B. Enkhtuya et al. Applied Soil Ecology 14 2000 201–211 203
were selected: 1 the Albrechtice ALB spoil bank 50
◦
33
′
31
′′
N, 13
◦
31
′
58
′′
E, altitude 250 m, 31-year old, with spontaneous plant succession. Initial plant
colonization occurred 1 year after mining and the majority of plants are annuals. After about 15 years,
the site was completely covered by herbaceous veg- etation, although, the establishment of hardwoods is
rather limited with scarce occurrence of birch and willow Prach, 1987; 2 the Brezno BRE spoil
bank 50
◦
28
′
03
′′
N, 13
◦
32
′
56
′′
E, altitude 250 m, with a 2-year old plantations of Acer pseudoplatanus and
3 the Velebudice VEL spoil bank 50
◦
28
′
33
′′
N 13
◦
38
′
23
′′
E, altitude 270 m, with an 8-year old plantations of A. pseudoplatanus and Fraxinus
excelsior. 2.1.3. Acid rain polluted site
The Lesna LES site is located in a clear-cut area in the Krusne hory Mountains 50
◦
34
′
02
′′
N, 13
◦
26
′
12
′′
E, altitude 890 m where the original spruce forest died off due to acid rain pollution. The site is
a 30-year old plantation of Sorbus aucuparia with ground cover of Calamagrostis villosa. As the pH of
the soils exposed to acid rain decreased, the acidify- ing process induced Al toxicity and led to a decrease
in nutrient availability.
2.2. Soil analysis The pH was determined on 50 g air-dried soil
sub-samples extracted by distilled water pH actual or by 0.1 M KCl pH exchangeable and stirred for
10 min, using a Radiometer TT2 pH meter Kub´ıková, 1972.
Other 5 g soil sub-samples were ground to a pow- der of maximum particle size 0.1 mm, weighed into
the Al containers and analysed for C and N by the CHN-Rapid Heraeus Elemental Analyser. After com-
bustion at 950
◦
C and reduction of NO
x
the contents of C and N were determined by thermo-conductibility
detection Monar, 1972. Soil sub-samples 50 g were air dried and ground
to a maximum particle size of 2 mm and extracted with 0.5 M NaHCO
3
pH 8.5 for extractable P determination. The content of P–PO
3
was deter- mined by a photometric method with ammonium
molybdenate-sulfuric acid reagent Olsen, 1954, using the UV–VIS Spectrometer Unicam UV4-200.
For exchangeable ions Ca, Mg, K, Na, Mn, Zn determination, 50 g soil sub-samples were air dried
and ground to the maximum particle size of 2 mm and extracted with 1 M ammonium acetate pH 7 for
Fe with pH 4.8. In the resulting solution, Ca, K and Na content was determined by flame atomic emission
spectroscopy, Mg and heavy metals content was de- termined using the AAS Spectrometer Unicam 9200X
Moore and Chapman, 1986.
2.3. Experimental design The indigenous AMF were isolated during 2 years
of successive trapping in pots with various host plants cultivated in a greenhouse. Pure cultures of AMF
were established from multiple spore sub-cultures and identified on the basis of spore morphology
Walker, 1992 and isozyme analysis of spores Dodd et al., 1996. Sixty-four treatments were included in a
two-factorial design. The first factor was inoculation: seven AM fungi indigenous isolates: G. mosseae —
spoil bank ALB; G. fistulosum — CHV sedimentation pond; G. etunicatum — TUS sedimentation pond;
G. intraradices — OPA sedimentation pond; the non-indigenous isolates from the Bank of European
Glomales BEG: G. mosseae, BEG25; G. fistulosum, BEG23; G. geosporum, BEG11 and non-inoculated
control. The second factor was growing substrate: seven soils collected from the sites and sand as an in-
ert substrate Table 1. There were five replicates for each treatment. Maize Zea mays L. as the universal
host plant for AMF was used to compare effective- ness of mycorrhizal symbiosis. Plant growth response
to inoculation and development of mycorrhizal colo- nization was measured after a 14-week period of cul-
turing in a greenhouse with no additional fertilization and no supplementary light.
Shoot dry mass was assessed after drying in an oven at 80
◦
C for 48 h. The root system was cut into 1 cm segments and the segments were then mixed with the
soil from the pot. A sub-sample 50 g was taken and wet sieved on a 0.036 mm sieve and root length was de-
termined by the gridline intersect method Giovannetti and Mosse, 1980. The washed root segments were
stained with Trypan Blue in lacto-glycerine modified from Philips and Hayman, 1970 and mycorrhizal
colonization was quantified on 30 root segments, ran- domly sampled from each root system, by the modi-
204 B. Enkhtuya et al. Applied Soil Ecology 14 2000 201–211
Table 1 Characteristics of substrates and soils used in the experiment
a
Substrate SAN
ALB VEL
BRE OPA
TUS LES
CHV pH
H
2
O 7.0
6.7 6.6
7.3 7.5
6.2 3.6
7.0 KCl
6.2 6.0
6.2 7.0
7.5 5.8
3.1 6.9
C, N C
0.3 2.9
2.9 1.2
2.2 3.0
13.8 1.7
N 0.0
0.2 0.2
0.04 0.04
0.1 0.7
0.1 CN
– 12.5
19.1 28.1
57.8 47.7
21.3 31.7
Macroelements mg kg
− 1
P 30
6.0 7.7
0.7 9.0
5.9 2.9
16.0 Ca
416 2506
2774 713
2401 785
194 10356
Mg 37
1597 1484
318 44
101 29
383 K
55 536
361 136
416 218
119 155
Na 5
106 76
52 72
170 3
Heavy metals mg kg
− 1
Fe 0.7
0.1 0.4
0.4 0.1
0.6 25.8
1.1 Mn
2.6 6.0
1.3 1.7
0.8 1.1
1.9 186
Zn 0.1
0.3 0.1
0.1 0.1
0.1 0.7
0.2 Cu
0.1 0.3
0.1 0.1
0.1 0.1
0.7 0.2
Cd 0.03
0.2 0.2
0.6 0.3
0.1 0.03
0.03 Ni
0.1 0.3
0.2 0.1
0.2 0.4
0.1 0.1
Pb 0.03
0.03 0.03
0.03 0.1
0.2 9.4
0.03
a
SAN — sand; ALB — Albrechtice spoil bank; VEL — Velebudice spoil bank; OPA — Opatovice fly ash disposal site; BRE — Brezno spoil bank; TUS — Tusimice fly ash disposal site; LES — Lesna acid rain polluted site; CHV — Chvaletice pyrite waste disposal site.
fied gridline intersect method Giovannetti and Mosse, 1980 using an ocular grid at 100× magnification.
The length and NADH diaphorase activity of the ex- traradical mycelium ERM were estimated. A 15 ml
core was removed from the middle part of each pot, homogenized by hand in a dish and a 5 g sub-sample
was mixed in a blender. The suspension 1 ml was pipetted onto a membrane filter 24 mm in diameter
and 0.45 mm pore size and vacuum filtered. The mem- brane filter was then placed on a microscope slide
and stained with 0.05 Trypan Blue solution in lac- toglycerine. The remaining content of the blender was
sieved through two sieves 0.25 and 0.036 mm. The ERM clusters from the finer sieve were collected us-
ing sharp tweezers and put into an Eppendorf micro- tube with 300 ml of the NADH diaphorase staining
solution Sylvia, 1988. Staining solution for NADH diaphorase Sylvia, 1988 was prepared by dissolving
1 mg ml
− 1
of iodonitrotetrazolium in 0.5 ml of 100 ethanol and vortexing for 5 min in an Eppendorf tube.
Then 3 mg ml
− 1
NADH were added to 0.2 M Tris buffer pH 7.4 and the final solution was then stirred
for 1 h on a magnetic stirrer. The microtubes were in- cubated at 28
◦
C for 14 h in the dark. The total length of mycelium was evaluated under an Olympus BX60
microscope using a grid inside the eyepiece at 100× magnification Brundrett et al., 1994. The results were
expressed as centimeters of mycelium in 1 g of dry soil. The percent proportion of mycelium length which
contained red precipitate NADH diaphorase activity was measured after mounting mycelium clusters from
Eppendorf tubes in glycerol on the microscope slides at magnification of 400×.
2.4. Statistical analysis of data Statistical analysis was carried out with SOLO
4 BMDP Statistical Software. Data showing nor- mal distribution were analyzed by two-way ANOVA
followed by the Duncan Multiple Range test. Data with non-normal distribution were logarithmi-
cally transformed and analyzed by non-parametric Kruskal–Wallis and Connover tests. Relationships
between all measured parameters were tested using correlation analysis.
3. Results
