ESTABLISHMENT AND GROWING OF TRAP AND ONE-SPECIES CULTURES OF ARBUSCULAR MYCORRHIZAL FUNGI (GLOMEROMYCOTA)
Generally, pot trap cultures were established according to Morton et al. (1993). Some modifications introduced resulted from our experience and the specificity of the investigations conducted. Many studies made by one of the authors of this website, J. Blaszkowski, regarded arbuscular fungi of dune sites.
When the root-rhizosphere soil mixtures came from non-dune sites, they were mixed with autoclaved coarse sand (1:1 v/v) and placed in 15-cm plastic pots (1350 cm3). The material from dune soils was either the only growing medium of the trap cultures (when the amount of the material collected was sufficient) or was mixed with autoclaved dune sand. The growing medium of the trap cultures was subsequently wetted and seeded. In investigations with dune soils, the plant host used was only Plantago lanceolata L. When other soils were examined, P. lanceolata or Zea mays L. were utilized. Earlier comparative investigations showed that Sorghum sudanense (Piper) Stapf and S. vulgare Pers. were not good trap plants in
our conditions. The cultures were grown in a greenhouse at 15-30oC with supplemental 8-16-h lighting provided by SON-T AGRO sodic lamps (Philips Lighting Poland S. A.) placed 1 m above pots. The maximum light intensity was 180 µE m-2s-1 at pot level. Plants were watered 2-3 times a week. Fertilization was applied only when plants showed signs of phosphorous deficiency (purpling of leaf sheaths) or nitrogen deficiency (chlorosis of young leaves). Trap cultures were harvested at approximately 1-month intervals, beginning 3 months and ending 11 months after plant emergence. Spores were extracted by wet sieving and decanting (Gerdemann and Nicolson 1963).
One-species cultures were established from about 50 to 100 newly formed spores stored before inoculation in water at 4oC for 24 h. Recently, many cultures were also initiated from single spores. Spores were collected in a pipette and transferred onto a compact layer of roots of 10-14-day-old seedlings of P. lanceolata or Z. mays placed at the bottom of a hole of ca. 1 cm wide and 4 cm deep formed in a wetted growing medium filling 8-cm plastic pots (250 cm3) or 3x12 cm plastic tubes (50 cm3). The growing medium was an autoclaved sand of maritime dunes (when fungi came from dune sites) or a mixture (3:1 v/v) of dune sand and a soil coming from young plantations with Pinus sylvestris L. The rhizosphere soil of P. sylvestris effectively remained an acid reaction of the growing medium for a long time.
Subsequently, the spores were covered with another layer of roots coming from 4-6 plants of the hosts. Finally, the roots and sandwiched spores were buried in the growing medium. The cultures were lighted by one SON-T AGRO sodic lamp and harvested after 4-12 months. Successful one-species cultures were increased in the 13x9-cm pots.
Plants of both trap and one-species cultures were watered with either tap water or tap water acidified with H2S04 to decrease a pH to 6.2.
The trap and single-species cultures were maintained in two greenhouses. Unfortunately, the greenhouses are also utilized by scientists of other departments of the Agricultural University. Both greenhouses are located ca. 100 m from our laboratories. In one greenhouse, a growth room was build, in which only one-species cultures are maintained. It is accessible only for us. The air temperature in both greenhouses is automatically regulated. All works regarding conservation of greenhouse devices and protection of plants against pests and diseases are made by the greenhouse personnel.
Gerdemann J. W., Nicolson T. H. 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Brit. Mycol. Soc. 46, 235-244.
Morton J. B., Bentivenga S. P., Wheeler W. W. 1993. Germ plasm in the International Collection of Arbuscular and Vesicular-arbuscular Mycorrhizal Fungi (INVAM) and procedures for culture development, documentation and storage. Mycotaxon 48, 491-528.