J. Jansa, M. Vos´atka Applied Soil Ecology 15 2000 125–136 127
2. Material and methods
2.1. Isolation of ericoid mycorrhizal fungi The roots of different ericaceous plants were
sampled in nature from a range of habitats from Rhododendrons cultivated in botanical gardens to
Vaccinium grown in high altitude mountain area.
The roots were stained to confirm presence of fun- gal structures according to the following protocol:
maceration of the roots in 10 KOH in autoclave at 121
◦
C for 1 h, cleaning with 5 hydrogen peroxide for 15 min, washing under tap water, acidifying with
1 HCl and staining for 2 h in 0.1 Trypan Blue in lactoglycerol lactic acid:glycerol:water — 1:1:1 at
80
◦
C and leaving the roots in the staining solution at room temperature overnight. After this, the roots
were destained in water for 24 h and observed under compound microscope at the magnification of 400×.
The technique for isolation of the ERM fungi from root samples was modified from Pearson and Read
1973a. The root samples were washed with tap water and surface sterilized by six times repeatedly
shaking in sterile tap water to protect plasmolysis of the fungal hyphae, which usually occurs while using
distilled water. This was followed by sterilization step with active chlorine. The roots were submerged
in 5 solution of SAVO bleaching solution 10 NaOCl for 3 min, then the roots were transferred into
5 hydrogen peroxide after washing the previous solution out, followed by another 5 min in 5 SAVO
bleaching solution. All the sterilization solutions used contained a drop of detergent BRIJ 35 to support
appropriate wetting of the root surface. Roots were cut under sterile conditions using scissors into pieces
approximately 5 mm long and they were put onto the surface of the isolation medium containing strepto-
mycin, to prevent the growth of bacteria. Sixteen root pieces were equally distributed over the surface of
9 cm Petri dishes. Root segments were cultivated for 3 weeks on both nutrient 1 malt extract and water
agar at pH 4.5 according to Leake and Miles, 1996 at room temperature 25
◦
C in dark. 2.2. Cultivation and maintenance of ericoid
mycorrhizal fungi Particular isolates of the ERM fungi growing from
the roots were transferred on the specific medium after Dalpe 1990 and on malt extract agar MEA.
The cultures of isolated ERM fungi strains were main- tained on the medium in both Petri dishes and vials
with skewed agar plates. The vials were closed with a Parafilm foil, sealed over the stopper, to ensure good
viability of the isolates cultures up to 1 year under storage at 4
◦
C. For preparing inocula of ERM fungi, liquid cultures were used. Ten 9 cm Petri dishes con-
taining 25 ml of cultivation media 4 malt extract, pH 4.5 were used for each isolate. The cultures were
left to grow for 4 weeks in dark at 20
◦
C. Then the mycelia were collected on a paper filter, homogenized
by blending at high speed by Waring blender in 200 ml sterile water for 5 s and diluted in sterile water to final
volume of 500 ml.
2.3. Inoculation in vitro Experiment 1: Azalea cv. AK 504 were grown in
vitro on modified 0.5 agar medium after Anderson 1984 without phytohormones, amended with 2 g of
charcoal per 1l of media, pH 5.2. Re-establishment of mycorrhiza was performed in sterile 200 ml Erlen-
meyer flasks, filled with 30 ml of Lignocel substrate, enriched with 12 g Osmocote per 1 l. Inoculation was
made using mycelium plugs from vial cultures af- ter 3 weeks acclimation period following transfer of
plantlets from nutrient medium. Ten strains of the ERM fungi were used for inoculation and the plants
were grown for 12 weeks. For evaluation of presence of mycorrhizal structures, plant roots were processed
as described above.
2.4. Inoculation experiments, post vitro Experiment 2: In vitro propagated seedlings of
Rhododendron cv. Belle-Heller were used in post vitro inoculation experiments. Regenerants from
stem-induction medium were transplanted onto root- ing medium Lignocel peat, prepared from tree bark,
enriched with coconut fiber: 500 g of Lignocel per 4.5 l of distilled water — pH 4.8 amended with
3 g of Osmocote slow release fertilizer 5–6 months release time per 1 l of substrate. After 8 weeks,
plantlets were transplanted into 3.5 l plastic boxes with Lignocel enriched with 3 g Osmocote per 1 l.
For inoculation of one plant, about 1.3 mg dry weight of mycelia was used, pipetted under each plant. Also
128 J. Jansa, M. Vos´atka Applied Soil Ecology 15 2000 125–136
the root system of the seedlings was dipped into the mycelial suspension before planting into inoculated
container. Each experimental container represented a unit infected with one of the 12 selected ERM fun-
gal isolates; 15 plants were grown in each container. Mortality, biomass of shoots and roots, leaf area
Fig. 1. a Intraradical colonization of Calluna vulgaris from natural habitat Jesen´ıky Mts. by an ericoid mycorrhizal fungus bar represents 10 mm. b Detail of hyphal coils of an ericoid mycorrhizal fungus within cortical cells of a root of Rhododendron sp. from natural habitat
Jesen´ıky Mts. bar represents 10 mm.
were evaluated after 12 weeks of cultivation in the greenhouse.
Experiment 3: This experiment was set in a similar way as the previous one except that 20 different strains
of ericoid mycorrhiza isolates were used for inocula- tion. In this case, the cultivation period was 16 weeks.
J. Jansa, M. Vos´atka Applied Soil Ecology 15 2000 125–136 129
2.5. Statistical evaluation of the data One-way ANOVA was used for evaluation of the
effect of different fungal isolates on plant growth. G
-test was used only to estimate significant differences in mortality rates. The statistical evaluation was per-
formed using SOLO statistical package BMDP Soft- ware, Los Angeles, CA, 1991. G-test was calculated
using CoStat package.
3. Results
Kategori