Myca/. Res. 95 (8): 905-914 (1991) Printed in Great Britain Cell wall structure and composition of cultured mycobionts from the lichens Cladonia macrophylla, Cladonia caespiticia, and Physcia stellaris (Lecanorales, Ascomycetes) ROSMARIE HONEGGER' AND SALOMON BARTNICKI-GARCIA' 1 Institut fur Pjlanzenbiologie, University of Zurich, Switzerland 2 Department of Plant Pathology, University of California, Riverside, CA 92521, US.A. 905 The mycobionts of three ascomycetous (lecanoralean) lichens (Cladonia macrophylla, C caespiticia and Physcia stellaris) were isolated and cultured in vitro. Cell walls of the three mycobionts were purified and analyzed by chemical techniques and X-ray diffractometry. Light microscopy of mycobiont colonies revealed hyphae with a smooth surface at the growing edges; the centre of the colonies contained filamentous and swollen cells, both of which were darkly pigmented and with a verrucose wall surface. The cell walls of all three mycobionts are made of polysaccharides, proteins and lipids. Glucose was the most abundant polysaccharide monomer; mannose, galactose, and glucosamine were also detected. By X-ray diffraction, a-chitin was demonstrated in the cell walls of all three mycobionts; the mycobiont of C caespiticia contained the most crystalline chitin. Electron microscopy of cell wall replicas revealed micro fibrils, presumably chitin, embedded in an amorphous matrix on the inner wall surface. Fewer fibrils were seen in the walls of hyphae, but a prominent microfibrillar meshwork was evident in the walls of swollen cells. We concluded that the cell walls of the three lichen mycobionts were similar in structure and composition to the walls of non-lichenized euascomycetes - they all belong in the same category. Lichenized ascomycetes, a very heterogenous group of fungi containing members of different not closely related orders, make up about 47% of all known ascomycetes (Hawksworth et at 1983). Biochemists and chemotaxonomists have been greatly interested in the secondary metabolites of lichens, but many aspects of their primary metabolism are still poorly understood. This is especially true of the composition of the mycobiont cell walL Due to technical difficulties in separating pure mycobiont fractions from whole lichen thalli, com- pounded by the very slow growth rates of lichen-forming fungi in pure culture, a quantitative analysis of the cell walls of either symbiotic or aposymbiotic lichen mycobionts seemed hitherto impossible. Although some review articles (Mosbach, 1973; Hale, 1974; Santesson, 1974) state that the cell walls of lichen mycobionts are mainly built from the glucans lichenin and isolichenin, neither of these linear ,3-1,4 glucans has ever been positively identified as a cell wall component of a lichen mycobiont. Lichenin and isolichenin were isolated not from purified cell walls, but from a thallus homogenate of the officinal 'Iceland moss' Cetraria islandica, which contained wall material. storage and other products of myco- and photobiont origin. Furthermore, the recovery was low: 6 % of thallus dry weight of lichenin, and 1% of isolichenin (Peat et al., 1957, 1961). Various highly branched polysaccharides containing D- glucose, D-galactose and D-mannose were identified in alkali- soluble extracts of whole thallus homogenates of lecanoralean and peltigeralean species (Aspinall et aI., 1955; Gorin & Iacomini, 1984, 1985; Iacomini et aI., 1985). By comparing extracts of whole thallus homogenates with those of the aposymbiotically cultured mycobionts and photobionts, Takahashi et al. (1979) demonstrated that most but not all water-soluble polysaccharides are produced by the fungal partner. Polysaccharides soluble in hot water or alkali comprised 68 and 84 %, respectively, of the cell walls prepared from homogenates of whole thallus of Peltigera canina and Lasallia pustulata (Boissiere, 1987). Chitin, an important cell wall component of the mycelial higher fungi (Bartnicki-Garcia, 1968), is not always mentioned as a component of lichenized ascomycetes (e.g. Mosbach, 1973; Hale, 1974; Santesson, 1974). On the basis of colour tests applied to a large series of protists and fungi, van Wisselingh (1898) detected various amounts of chitin in the vegetative hyphae of several. distantly related lichen mycobionts. He found very little or no chitin in the symbiotic phenotype of members of the Lecanorales, Pertusariales, Caliciales, Craphidiales, and Opegraphales, but large amounts in members of the Peltigerales. Similar results were obtained by the Boissieres (1967, 1968, 1970) and by Schlarmann (1987) and Schlarmann et al. (1990) in comparative histo- chemical and cytochemical investigations of different distantly related lichens. Cell walls prepared from thallus homogenates of Lasallia pustulata and Peltigera can ina (Boissiere, 1987) contained, respectively, 5 and 12 % hexosamine (presumably chitin). By two microscopic criteria, autoradiographic localiz- ation of the incorporation of [3H1N-acetylglucosamine and binding of wheat germ agglutinin, Calun et al. (1976) provided additional evidence for the presence of chitin in three cultured lecanoralean mycobionts. Precise qualitative and quantitative knowledge of