146 M. Vos´atka, M. Gryndler Applied Soil Ecology 15 2000 145–152
state McArthur and Knowles, 1992, 1993; Niemira et al., 1995. Micropropagated plants form the rhizo-
sphere during post-vitro establishment and there is a free niche and space for interaction with introduced
soil microflora. During the step of post-vitro trans- plantation, there is a unique possibility to influence the
composition of newly formed rhizosphere by preinoc- ulation of the cultivation substrate with selected mi-
croorganisms Vestberg, 1992. After dual inoculation with AMF and an appropriate bacterial partner, a syn-
ergistic effect on plant growth and nutrition can be observed Meyer and Lindermann, 1984; Barea and
Azcón-Aguilar, 1986; Vosatka et al., 1992.
The aim of this study was to investigate the effects of dual inoculation with different AMF and bacteria on
two varieties of micropropagated potatoes Solanum tuberosum transplanted to a peat-based substrate in
three different cultivation systems. In addition, the ef- fect of Zeolite clinoptinolite clay used as substrate
conditioner amendment on the mycorrhization and growth response of potato plants was tested.
2. Materials and methods
2.1. Experiment 1 Potato plants of two varieties — Karin and Krista
were micropropagated and transplanted to multiple trays with 90 cell compartments, each 38 ml, filled
with a peat-based substrate mixture of peat and Per- lite 5:2. Each compartment was inoculated with 0.2 g
of AMF inoculum consisting of Perlite-sand substrate, fragments of colonized roots, mycelium and spores
from the AMF pot cultures grown for 4 months on maize. The fungal isolates used were Glomus etu-
nicatum isolate S 329 from University of Florida, Gainesville, Glomus fasciculatum isolate from the
Experimental Station Rothamsted, and Glomus fis- tulosum BEG23. Bacteria B1 — isolate of Bacillus
subtilis and M11 — isolate of Agrobacterium ra- diobacter, both from the rhizosphere of wheat were
inoculated as 1 ml suspension of bacterial culture cultivated for 48 h on Taylor media Taylor, 1951.
Factorial combination of two AMF and bacterized control with two bacteria and uninoculated control
12 treatments involved six replicate blocks each with eight plants, in total 576 plants of each variety.
After 6 weeks of cultivation in the greenhouse, small seedlings of the potato were transplanted from mul-
tiple trays to the 0.5 l plastic pots with the same peat substratum. Pots were randomly placed on the table
in temperated greenhouse and grown for 12 weeks and irrigated daily as needed. Plants were harvested,
the minitubers were counted, total fresh weight of minitubers TFW was measured and means of fresh
weight of minitubers per plant and per eight plants in the experimental block replicate were calculated.
Roots from eight plants in each replicate were sam- pled and stained according to Phillips and Hayman
1970 except that lactoglycerol was used instead of lactophenol. Mycorrhizal colonization MC was
evaluated under a binocular microscope at the mag- nification of 120× by grid-line intersect method
according to Giovannetti and Mosse 1980.
2.2. Experiment 2 Preinoculated plants of two varieties as in experi-
ment 1 were transplanted to the greenhouse table beds 100 cm×40 cm made from black plastic foil. The ex-
perimental design, AMF isolates and bacterial strains were the same as in experiment 1, but 12 replicates
were involved. One bed was a replicate and contained eight plants. Plants were cultivated for 12 weeks and
the way of harvest and evaluation of growth parame- ters and MC were the same as in experiment 1.
2.3. Experiment 3 Potato plants of Karin variety were micropropa-
gated and transplanted from tissue cultures directly to the 0.5 l pots with peat-based substrate. The ef-
fects of microorganisms on plants in this experiment were studied using the factorial design involving
three factors: amendment of Zeolite into the sub- strate, inoculation with AMF and inoculation with
bacteria. In half of the treatments, the substrate was amended with clinoptinolite clay mineral Zeolite
peat substrate: Zeolite, 2:1, v:v commonly used for the nutrient sorption and improvement of nutritional
quality of growing media in horticulture, whereas other half of treatments remained with no substrate
amendment. These two treatments represented two levels of first factor. These substrates were either
left non-inoculated or they were inoculated with soil
M. Vos´atka, M. Gryndler Applied Soil Ecology 15 2000 145–152 147
inoculum of six isolates of AMF 0.2 g per plant, seven levels of the second experimental factor: G. fis-
tulosum isolate BEG23, Glomus manihotis BEG112, Glomus geosporum BEG11, Glomus deserticola
BEG73, Glomus mosseae BEG25, Scutellospora het- erogama BEG35. Latter five isolates were obtained
from The International Institute of Biotechnology, MIRCEN, Canterbury, Kent. As the third experimen-
tal factor, the plants were either left non-inoculated or they were inoculated with two bacteria three
levels of the experimental factor: isolate B1 and iso- late M30 the latter was isolated from hyphosphere
under mycorrhizal culture of maize. Bacteria were propagated and applied the same way as in previous
experiment. This factorial experiment thus involved 42 treatments 2×7×3 with eight replicates. All the
336 pots were placed randomly on a cultivation table in temperature-controlled greenhouse and the plants
were irrigated daily with tap water as needed. Plants were harvested after 11 weeks, and dry biomass of
the shoots, numbers and weight of minitubers were measured, and MC was evaluated as in experiment 1.
2.4. Experiment 4 Potato plants of Karin variety were either left
non-inoculated or they were preinoculated in multi- ple trays in the same way as in experiment 1 with
four isolates of AMF. All the inoculated treatments also received 1 ml of a mixture 1:1 of cultures of
bacterial strains M30 and B1. AMF isolates used were G. manihotis BEG112, G. mosseae BEG25, G.
deserticola BEG73, and G. etunicatum S329. The plants were transplanted to the shadowhouse beds
100 cm×40 cm. Each bed contained 30 plants. Each treatment involved nine replicates beds. The plants
were cultivated for 14 weeks and, after the harvest, the mean number of minitubers NT, total weight of
minitubers and number and weight of marketable size minitubers per plant were estimated, and the MC in
each treatment was evaluated.
2.5. Statistical evaluation of the data Data sets were checked for normal distribution and
logarithmically transformed when necessary. One-, two- or three-way ANOVA was used for evaluation
of effect of different factors and their interaction. Duncan multiple range test was used to analyze sig-
nificant differences between means. The statistical evaluation was performed using SOLO statistical
package BMDP Software, Los Angeles, CA, 1991. Control non-inoculated treatments were excluded in
all experiments from statistical analysis of MC.
3. Results
