2.4. Pot experiment
The seeds of rape (Brassica napus L. subsp. napus) and fescue
(Festuca ovinia L.) were surface-sterilized with a solution of 1.5%
(v/v) sodium hypochlorite for 10 min and washed thoroughly with
sterile water. Sterile seeds of fescue and rape were sown in plastic
tubes filled with approximately 100 g (dry weight) of autoclaved
soil originated from a zinc smelter area (Table 1). In this study
there were used three inoculation methods: Method 1 the germination
of seeds for a 48 h on plates with bacteria suspension (bacteria
pellet, suspended in sterile water), Method 2 the seed inoculation
with bacterial suspension just after seeding, Method 3 inoculation
of plants after two weeks of growth. The same amount of bacteria
was used for the three inoculation methods. Plants (fescue and
rape; 5 units/tube; six repetitions) were inoculated with 48 h suspension
of selected bacteria isolates in the amount of 1 ml (density
2 × 106 CFU g−1) per 100 g of soil. Then bacterial suspension (sole
isolates) was used for seed inoculation while the uninoculated
control treatment received water treatment. A plant growth experiment
was conducted in a growth chamber for rape 8 weeks and
for fescue 12 weeks. The plants were grown under artificial light
(350 mol m−2 s−1). The temperature was set to 20 ◦C and 14 ◦C by
night, with the relative humidity was maintained at 70%. During
the growing season, fertiliser was not applied, and irrigation with
deionised water was applied as needed. After growth period plants
were harvested. The roots were gently separated from the soil and
rinsed under running water and air dried. Stems and roots of the
plants were weighed for biomass determination and length of the
plants was also measured.
2.5. Statistical analysis
Results were expressed as means + standard errors. Differences
between means were determined by one-way ANOVA with posthoc
t-Tukey’s test (Origin Pro8) with the level of significance
established at p < 0.05. To confirm interaction between 2 plants and
3 inoculation methods the two-way ANOVA was used.