Trees (1991) 5:90-94 9 Springer-Verlag 1991 Comparative studies on ectomycorrhizae synthesized with various in vitro techniques using Picea abies and two Hebeloma species Ivano Brunner Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), 8903 Birmensdorf, Switzerland Received November 9, 1990/Accepted February 6, 1991 Summary. Various in vitro synthesis techniques with Picea abies and two Hebeloma species showed that struc- tures of the mantle and Hartig net of synthesized ectomy- corrhizae within the given two fungus species are stable. However, thickness of mantle, and penetration depth and number of hyphal cell rows between cortical cells of the Hartig net are dependent on techniques and substrates. Porous glass balls as substrate in the Erlenrneyer technique seem to suppress or delay mantle and Hartig net formation. With the other techniques (growth pouch, open cuvette, Erlenmeyer with a vermiculite-peat moss mixture) development of the mantle is simultaneous with or shortly in advance of Hartig net formation. The ectomycorrhizae of the two tested Hebeloma species are similar and cannot be morphologically differentiated by the in vitro tech- niques used. Key words: Picea abies - Hebeloma crustuliniforme - Hebeloma cylindrosporum - Ectomycorrhizal syntheses - Characteristic-stability Introduction There is a long lasting dispute as to whether in vitro synthe- sized and naturally occurring ectomycorrhizae can be com- pared and, if so whether the methods used for synthesizing symbioses influence morphologically important features under certain cultural conditions (Harley and Smith 1983). Only recently Kottke (1986) showed for a few Picea- and Larix-ectomycorrhizae that naturally and synthesized sym- bioses do not differ significantly from each other and that syntheses can be used to identify ectomycorrhizae. How- ever, Melin's cultural method, which uses aseptic Eden- meyer flasks, was applied. However, the question as to whether various synthesis methods influence the mor- phology and anatomy of ectomycorrhizae remains to be answered. Synthesis methods range from aseptic, semiaseptic to nonsterile cultural conditions. Erlenmeyer flask or glass tube methods are commonly used to synthesize ectomy- corrhizae in an aseptic environment to describe the ultra- structure and morphology of symbioses (e.g. Kottke and Oberwinkler 1986 a). The semiaseptic growth pouch tech- nique is commonly used to describe the ontogeny of ecto- mycorrhizae (e.g. Melville et al. 1987). Both methods, however, are suitable for evaluating ability to form ecto- mycorrhizae with a given putative host-plant (e.g. Brunner 1987; Brunner et al. 1990). The growth pouch method is often criticized as being too artificial due to the lack of a soil-like matrix that can act as a morphogenetic agent (Har- ley and Smith 1983). Thus, the resulting ectomycorrhizae may not be relevant for comparing naturally occurring with in vitro synthesized symbioses. Important morphological features of ectomycorrhizae are Hartig net, mantle, emanating hyphae and cystidia, and rhizomorphs (Agerer et al. 1986; Kottke and Oberwinkler 1986b). Anatomically, the structure of the Hartig net, the structure of tangential longitudinal sections of the hyphal mantle, and rhizomorphs can vary. Agerer et al. (1986) observed three types of Hartig net and Haug (1987) re- ported 12 different types of tangential longitudinal sections of the hyphal mantle. Rhizomorphs may vary from single aggregated hyphae to highly complex and differentiated cords with a central transport xylem similar to those of higher plants (Agerer et al. 1986). The objectives of this study were to synthesize ectomy- corrhizae with various methods or various substrates, to describe the arising symbioses and to distinguish between morphologically and anatomically stable and variable characters of ectomycorrhizae. Materials and methods Plant material Seeds of Picea abies (L.) Karst. originated from Dielsdorf (canton of Ztirich, Switzerland) and were collected in 1977. For germination, the seeds were surface sterilized for 30 rain in 30% H202 and placed on