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
3.1. Plant growth Significant effects of growth substrate on all mea-
sured parameters were found at p0.001, while the
B. Enkhtuya et al. Applied Soil Ecology 14 2000 201–211 205
effect of AMF was not significant. Maize shoot dry mass was not reduced in disturbed soils in any AMF
treatment as compared to plants growing in sand Table 2. Non-inoculated host plants had higher
shoot dry mass in soils from the VEL spoil bank and the CHV sedimentation pond than in sand, BRE
spoil bank and the LES site. Host plants growing in soil from the ALB spoil bank had higher shoot dry
mass than plants growing in most other soils in all mycorrhizal treatments. In contrast, in the acidified
substrate from LES shoot dry mass was very low irrespective of inoculation with AMF. Maize root
lengths were not reduced in disturbed soils in any inoculation treatment in comparison with root lengths
of plants growing in sand Table 2. Plants growing in soils from the ALB and VEL spoil banks had higher
root length than plants growing in most other soils in all mycorrhizal treatments.
Shoot dry mass of maize was positively influenced by AMF only in soil from ALB with the exceptions
of G. geosporum BEG11 and G. mosseae indigenous isolates. In substrate from CHV, inoculation of host
plants with G. intraradices, G. fistulosum BEG23,
Table 2 Shoot dry mass and root length of maize grown in sand and seven soils from the disturbed ecosystems and man-made habitats listed in
Table 1, uninoculated or inoculated with indigenous and non-indigenous isolates of arbuscular mycorrhizal fungi
a
AMF Control
G. intraradices G. fistulosum
G. etunicatum G. geosporum
G. etunicatum G. mosseae
G. mosseae substrate
isolate BEG23
isolate BEG11
isolate BEG25
isolate Shoot dry mass g
SAN 1.2 bcd k
1.3 bcd k 1.6 bcd k
1.7 bcd k 1.3 cd k
1.3 cd k 1.6 bc k
1.2 bcd k ALB
2.4 ab m 4.5 a kl
4.6 a kl 6.6 a k
3.5 a lm 4.6 a kl
4.7 a kl 3.2 a lm
VEL 2.5 a k
2.2 ab k 2.3 ab k
2.8 ab k 2.7 ab k
2.4 ab k 1.7 b k
2.5 ab k BRE
1.0 cd k 0.9 cd k
1.5 bcd k 1.4 cd k
1.5 cd k 1.2 cd k
1.5 bc k 1.1 cd k
OPA 1.8 abc k
1.5 bc k 1.6 bc k
1.8 bc k 1.9 abc k
1.5 bc k 1.4 bc k
1.3 abc k TUS
1.6 abc k 1.6 bc k
1.9 abc k 2.2 abc k
1.8 abc k 1.9 abc k
2.1 ab k 1.4 abc k
LES 0.4 d k
0.5 d k 0.6 d k
0.6 d k 0.5 d k
0.5 d k 0.7 c k
0.4 d k CHV
2.7 a k 1.3 bcd l
1.3 cd l 1.8 bcd kl
1.6 bc kl 1.3 cd l
2.2 ab l 1.2 bcd l
Root length cm g
− 1
SAN 7.9 abc k
7.3 bc k 6.7 bc k
5.5 cd k 5.6 bcd k
5.0 cd k 6.2 bc k
8.1 bc k ALB
12.0 a m 22.7 a k
11.0 a m 22.6 a kl
12.1 ab lm 11.5 a m
23.2 a k 15.8 a klm
VEL 10.6 ab k
10.9 ab k 8.4 ab k
11.5 a k 13.4 a k
12.1 a k 10.7 ab k
13.4 ab k BRE
9.8 ab k 8.1 bc k
6.6 bc k 8.5 abc k
6.0 abc k 5.5 bcd k
7.0 bc k 4.8 c k
OPA 11.6 ab k
6.7 bc k 10.2 ab k
10.0 ab k 11.3 ab k
9.4 abc k 10.5 ab k
12.7 ab k TUS
6.5 bc lm 10.6 ab k
9.2ab klm 5.6 cd m
5.9abc lm 11.2 ab kl
5.9 bc m 8.3 abc klm
LES 3.3 c l
2.9 c l 6.0 bc kl
3.3 d l 2.9 d l
8.6 abcd k 9.8 ab k
4.3 c kl CHV
7.7 abc k 4.4 c lm
3.5 c m 6.1bcd kl
3.9 cd m 4.1 d lm
4.4 c lm 6.1 c kl
a
Means followed by the same letter a–d within columns comparisons between soils and within rows k–n comparisons between AMF are not significantly different according to Duncan’s Multiple Range test p0.05, n=5.
G. geosporum and G. mosseae had negative effects on shoot dry mass and root length.
3.2. Development of arbuscular mycorrhizal symbiosis
Mycorrhizal colonization was significantly influ- enced by both substrate and fungus p0.001. Sub-
strate and AMF isolate interactions were significant for mycorrhizal colonization p0.01, and for length
of ERM p0.001. The development of mycorrhizal colonization for most AMF isolates was higher in the
soils from spoil banks and the OPA fly ash disposal site than in soils with more adverse chemical properties
the acidified LES soil and the CHV and TUS sedi- mentation ponds Fig. 1. The most successful isolate
with respect to ability to colonize the roots was the in- digenous isolate of G. intraradices, originating from
the OPA sedimentation pond Table 3. G. intraradices showed higher colonization compared to other AMF
in most treatments except in the Lesna soil. In the sub- strates from OPA and TUS sedimentation ponds and
in the sand, the plants inoculated with this isolate had
206 B. Enkhtuya et al. Applied Soil Ecology 14 2000 201–211
Fig. 1. Mycorrhizal colonization of maize roots grown in sand and seven soils from disturbed ecosystems and man-made habitats, and inoculated with indigenous and non-indigenous isolates of arbuscular mycorrhizal fungi. 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. Bars indicated by the same letter are not significantly different according
to Duncan’s Multiple Range test p0.05, n=5.
B. Enkhtuya et al. Applied Soil Ecology 14 2000 201–211 207
Table 3 Comparison of mycorrhizal colonization of indigenous isolate G.
intraradices with other AMF isolates associated with maize grown in sand and seven soils from disturbed ecosystems and man-made
habitats
a
AMFSubstrate SAN ALB VEL OPA BRE TUS LES CHV
G. fistulosum BEG23 n.s. n.s.
n.s. n.s. G. fistulosum isolate
n.s. n.s. n.s.
G. geosporum BEG11 n.s.
G. etunicatum isolate n.s.
n.s. G. mosseae BEG25
n.s. n.s.
G. mosseae isolate n.s.
n.s.
a
Substrate abbreviations are as in Table 1. Asterisks indi- cate significantly higher colonization in G. intraradices; n.s.:
non-significant according to Duncan’s Multiple Range test p0.05.
significantly higher percentages of mycorrhizal colo- nization compared to treatments inoculated with all
other isolates. ERM length and NADH-diaphorase activity were
significantly influenced only by substrate Table 4. Length of ERM was highest in soil from the ALB
spoil bank for most of the AMF isolates. In substrates with more unfavorable chemical characteristics from
Table 4 NADH-diaphorase activity and total length of extraradical mycelium associated with roots of maize grown in sand and seven soils from
disturbed ecosystems and man-made habitats listed in Table 1, inoculated with indigenous and non-indigenous isolates of arbuscular mycorrhizal fungi
a
AMF G. intraradices
G. fistulosum G. fistulosum
G. geosporum G. etunicatum
G. mosseae G. mosseae
substrate isolate
BEG23 isolate
BEG11 isolate
BEG25 isolate
NADH-diaphorase activity SAN
54 bc k 44 bcd k
65 ab k 50 bcd k
51 bcd k 46 bc k
52 bcd k ALB
63 ab l 80 a k
72 a kl 57 abc l
71 a kl 74 a k
70 a kl VEL
55 bc k 53 bc k
51 bcd k 61 ab k
63 abc k 51 bc k
48 cd k BRE
78 a k 62 ab l
68 ab kl 64 ab l
67 abc kl 70 a kl
65 abc l OPA
58 abc lm 56 ab m
67 ab kl 66 a kl
71 a k 60 ab lm
72 a k TUS
58 abc kl 53 bc lmn
55 bcd klm 13 cd mn
0 d n 57 ab klm
68 ab k LES
0 d k 0 d k
0 d k 12 d k
0 d k 11 d k
14 d k CHV
41 cd k 37 cd k
43 d k 41 cd k
45 cd k 38 bc k
50 cd k ERM total length cm g
− 1
SAN 20 b lmn
29 b lm 103 a k
54 ab l 34 ab l
4 b a 29 bc lm
ALB 94 a k
81 a k 101 a k
64 a lm 49 a m
81 a k 94 a k
VEL 17 b k
12 bc k 14 b k
27 cd k 8 c k
14 b k 21 bc k
BRE 21 b k
14 bc k 6 b k
20 cd k 22 bc k
22 b k 20 bc k
OPA 17 b k
26 bc k 13 b k
30 bc k 20 bc k
19 b k 32 b k
TUS 14 b k
7 bc k 14 b k
3 de k 2 c k
7 b k 3 c k
LES 0 b k
2 c k 0 b k
1 e k 0 c k
0 b k 0 c k
CHV 3 b k
7 bc k 1 b k
11 cde k 2 c k
2 b k 0.4 c k
a
Substrate abbreviations and methods of comparing treatment means are as for Table 2.
LES and CHV sites, there was a tendency for reduced growth of ERM as compared to other soils. Some iso-
lates, which were very successful in some substrates, had very low ERM length in others e.g. G. fistulosum
in sand and in the soil from the ALB spoil bank ver- sus the same isolate in the OPA sedimentation pond
and BRE spoil bank. The AMF growing in soil from ALB had higher NADH-diaphorase activity in com-
parison with most other substrates, the lowest one was found in acidified soil from the LES site. Some AMF
isolates showed no NADH-diaphorase activity in the most adverse substrates G. intraradices, G. fistulosum
BEG23, G. fistulosum, G. etunicatum in soil from the LES acidified forest site and the indigenous isolate
G. etunicatum in substrate from TUS.
Indigenous AMF isolated from degraded ecosys- tems or man-made habitats when grown in symbiosis
with maize in the soils of their origin or other disturbed soils did not show, in most soils, better development
than the isolates from undisturbed soils Table 5. Results of correlation analysis showed that in most
substrates there were no significant relationships between plant growth and mycorrhizal parameters
Table 6. On the other hand, in some substrates,
208 B. Enkhtuya et al. Applied Soil Ecology 14 2000 201–211
Table 5 Comparison of development of native isolates from disturbed and
man-made habitats G. intraradices, G. mosseae, G. etunicatum and G. fistulosum and from undisturbed soils G. mosseae BEG25,
G. geosporum BEG11 and G. fistulosum BEG23 associated with maize grown in sand and seven soils from disturbed ecosystems
and man-made habitats
a
Substrate Mycorrhizal
ERM NADH-diaphorase
colonization length
activity of ERM SAN
n.s. ALB
n.s. n.s.
n.s. VEL
n.s. n.s.
n.s. BRE
n.s. n.s.
OPA n.s.
n.s. TUS
n.s. n.s.
n.s. LES
n.s. n.s.
n.s. CHV
n.s. n.s.
n.s.
a
Substrate abbreviation are as in Table 1. Asterisks indicate higher values of measured parameters for AMF isolates indige-
nous in disturbed soils; n.s.: non-significant according to Duncan’s Multiple Range test p0.05.
mycorrhizal parameters such as mycorrhizal colo- nization and NADH-diaphorase were correlated with
each other.
4. Discussion
