Notes and brief articles ENDOGONE LACTIFLUA FORMING ECTOMYCORRHIZAS WITH PINUS CONTORTA BY CHRISTOPHER WALKER Forestry Commission, Northern Research Station, Roslin, Midlothian EH25 9SY A mycorrhizal association between Pinus contorta (lodgepole pine) and Endogone lactiflua is described. 353 Lodgepole pine plants (Pinus contorta Dougl. ex Loud.) that had been grown in a seedbed for 18 months and then transplanted to another bed for 18 months in Newton Nursery, Morayshire, Scotland, were transferred to root-observation boxes with transparentfronts, and placedin a greenhouse (18 h day, day 21°, night 17°C) so that mycorrhizal development could be observed. The boxes were filled with sphagnum peat and covered with black polythene, which could be removed to allow observations to be made. After 2 months, the roots appeared to be non-mycorrhizal as there was little bifurcation or other morphological modification of short roots (Fig. 1). However, there were few root hairs on the short roots, and it was surmised that ectendomycorrhizas similar to those reported on red pine (P. resinosa Ait.) in the United States (Wilcox, 1971), and on pine (presumably P. sylvestris L.) in Finland (Mikola, 1966) might be present. Roots were therefore sampled and micro- scopically examined both as whole mounts cleared and stained in aniline blue and gently squashed under a coverglass (Walker, 1979), and as sections cut on a freezing stage and stained in polyvinyl- alcohollactophenol (PVL) with aniline blue. This latter method provides a rapid and easy way of examining the anatomy of root tips, resulting in a semi-permanent preparation when the mountant hardens on drying. Fungal material stains deep blue, whereas cortical cells remain unstained, or are only lightly stained. Althoughthe roots did not appearmacroscopically to be mycorrhizal, observation of squashes showed a loose weft of deeply-stained thick-walled coeno- cytic mycelium ramifying over the surface. There was no definite sheath of the type described in Harley (1969), but the hyphae had a characteristic palmate appearance (Fig. 2). Individual hyphal elements, not visible to the unaided eye, extended away from the root surface. These exploratory hyphae could be seen when gently washed roots were examined at 40 x under a dissecting micro- scope with reflected light. Examination of sections showed a well-developed Hartig net that usually penetrated almost to the endodermis (Fig. 3). Root hair formation was suppressed, and the outer layer of cells was filled with a brown deposit. Attempts to isolate the causal fungus on a variety of media yielded only sterile or yeast-like cultures that subsequently failed in attempts to synthesize mycorrhizas with lodgepole pine. After three months, fruiting bodies of Endogone lactiflua Berk. & Br. appeared on and just beneath the surface of the peat in which the plants were growing. Hyphae still could not be observed macroscopically, but under microscopic examin- ation a connexion by fine, hyaline hyphae between the base of the fruiting body and the mycorrhiza could be traced (Fig. 4). The fruiting bodies continued to appear in the boxes for three seasons. Attempts to isolate E. lactiflua in pure culture, either from mycorrhizas or from fruiting bodies, failed. In the absence of a typical mycorrhizal mor- phology, the anatomy and staining characteristics of the roots were examined. The Lugol blue staining technique (Fassi, Fontana & Trappe, 1969) was used on freshly cut sections from the freezing microtome. The hyphae stained the characteristic violet believed to be specific for Endogone spp. (Fassi et al., 1969). Sections cut from roots attached to the fruiting bodies revealed the characteristic anatomy described above, and palmate hyphae were observed not only on the root surface but also in the Hartig net. The appearance, staining characteristics and anatomy of the mycorrhizas were identical to those reported as formed between E.lactifiua and P. strobus L. (Fassi, 1965), although the illustrations of sporocarps there, and the comments of Trappe & Gerdemann (1972) suggest that the species of fungus on that occasion was E. fiammicorona Trappe & Gerd. and not E. lactifiua. Further specimens of lodgepole pine from the Newton Nursery were examined to find if the fungus originated in the nursery, or had come from the unsterilised peat used as a growth medium in the boxes. Sections and squashes of roots from the nursery possessed the distinctive palmate hyphae which became violet in Lugol blue. Seeds of lodgepole pine were surface disinfested by immer- sion in 100 vol. H 2 0 2 for 30 min, followed by three washes of 15 min each in sterile distilled water. The treated seeds were then germinated on agar containing 5 gl-1 sucrose in sterile jars. Seedlings Trans. Br. mycol. Soc. 84 (2), (1985) Printed in Great Britain 12-2