The association of Morchella rotunda (Pers.) Boudier with roots of Picea abies (L.) Karst.

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1 New Phytol. (1990), 116, The association of Morchella rotunda (Pers.) Boudier with roots of Picea abies (L.) Karst. BY F. BUSCOTAND I. KOTTKE Universitdt Tubingen, Institut fur Botanik, Spezielle Botanik, Mykologie, Auf der Morgenstelle 1, D 7400 Tubingen, Germany (Received 5 February 1990; accepted 9 July 1990) SUMMARY A histo-cytological study of Norway spruce [Picea abies (L.) Karst.] roots connected to ascocarps of Morchella rotunda (Pers.) Boudier revealed that hyphae of morel were associated with all parts of the root system. The main suberized roots were surrounded by a mycelial sheath. The finer rootlets were enveloped by a cottony mycelium and their absorbing root tips were found to be ectomycorrhizal. Morel hyphae also invaded the oldest parts of a heterobasidiomycete ectomycorrhiza. Key words: Morchella rotunda, Picea abies, ectomycorrhiza. INTRODUCTION For 100 yr, there has been controversy about the trophic status of morels. Field observations showing that the base of ascocarps adheres to roots or rhizomes of plants initially led to speculation that morels were parasitic (Robert, 1865; Condamy & Cornu, 1878; Roze, 1882, 1883) or ectomycorrhizal (Matruchot, 1909). Later, the abundant and rapid growth of the mycelium in pure culture on a wide range of synthetic culture media (Molliard, 1904; Fron, 1905; Melin & Gunhild, 1941; Brock, 1951; William, Trzcinski & William-Engels, 1956; Impens, 1972; Kaul, 1977; Wassom & Holden, 1977) indicated the potential of morel mycelium for saprophytism. The abundant ascocarp production that follows plant destruction by fire or week-killers (Kaul, 1975; Carpenter, Trappe & Ammirati, 1987; Turnau, 1987) also indicates saprophytic nutrition. This has been confirmed experimentally, as ascocarps were recently obtained in culture (Ower, 1982; Ower, Mills & Malachowski, 1986). The associations of ascocarps of three Morchellaceae with several herbs and trees have been recently reassessed (Buscot, 1987; Buscot & Roux, 1987). The ascocarps were connected by rhizomorphs to main suberized roots, around which the fungus formed a compact mycelial sheath. This structure is formed during summer and autumn, survives over winter, and may provide nutrients for vernal fruiting (Buscot, 1989). The cytological studies reported here demonstrate ectomycorrhizal association between Morchella rotunda (Pers.) Boudier and Picea abies (L.) Karst. The role of this association in the life-cycle of morels is discussed. MATERIALS AND METHODS Two ascocarps of M. rotunda were collected at their ' maturation stage' (fully developed but asci not yet mature; Buscot, 1989) at the edge of a mature plantation of P. abies in a Rhine forest near Strasbourg. About 3 dm^ of the soil surrounding the base of the ascocarps to a depth of 30 cm were collected at the same time. Washing revealed a tight association with roots of spruce, which was analysed by light and electron microscopy. Three kinds of roots were sampled: (i) main suberized roots (2-10 mm diam), 10 samples; (ii) conducting rootlets (0 5-2 mm diam), 10 samples; (iii) root tips ( < 0 5 mm diam), 20 samples. The samples were fixed in 2% glutaraldehyde in a cacodylate buffer, post-fixed in osmium tetroxide in the same buffer, and embedded in ERL plastic (Spurr, 1969). Semithin sections (0-5 /tm) were cut on glass knives with a Reichert ultramicrotome, and stained with toluidine blue. Ultrathin sections (70 nm) were cut with a diamond knife, contrasted with uranyl acetate and lead citrate following standard procedures, and mounted on formvar slotted copper grids. Transmission electron microscopical observations were performed with Zeiss EM 109 and Zeiss EM 9-S2.

2 426 F. Buscot and I. Kottke RESULTS Morphology As previously reported (Buscot & Roux, 1987; Buscot, 1989), the ascocarps were connected by rhizomorphs to compact mycelial sheaths surrounding several of the suberized main roots along sectors about 10 cm in length. Fine rootlets emerged from the mycelial sheaths. They were surrounded by a thin loosely adhering, cottony, hyphal envelope. The root tips were irregularly pinnate mycorrhizas, about 3 mm in length, with cottony, brownish-yellow mantle. These mycorrhizas were formed by two different fungi. CB CB OP W 1b y ^ W -.. OP V,;;. ; dpi -...'../ 2b PI I 3;..... _. :.. : ;. _ ^. Figures 1 5. Cytological features. Figures 1-3. Ultrastructure of septal pores in hyphae oi Morchella rotunda. Scale bars, 1 pbvsx. Figure 1. (a, V) Occluded pores (OP) with crystal-like bodies (CB) and Woronin bodies (W) in ascogenous hyphae. Figure 2. (a) Occluded pore with crystal-like bodies and vesicles (V) in hyphae of the mycelial sheath surrounding the main suberized roots of Picea abies. {b) Occluded pore with Woronin bodies and vesicles in hyphae of the fine cottony envelope that surrounds rootlets of the spruce. Figure 3. Free pore (FP) in the hyphal mantle of the morel mycorrhiza. Figure 4. Plasmalemmasome (PI) in the morel mycorrhiza. Scale bar, 0-5 /im. Figure 5. Ultrastructure of a dolipore with continuous parenthesome (P) in the basidiomycete mycorrhiza. Scale bar, 0-5

3 Morchella rotunda on spruce roots 427 Identification of the partners involved in the association The bordered pits observed on the sections of the roots confirmed that the association was with P. abies. The great majority of hyphae in association with these roots could be identified as morel hyphae, and about the distal third of the mycorrhizas in the samples appeared to be formed by the morel as the cytological features of the hyphae were similar to those of ascocarp hyphae. Morel hyphae have a variable diameter (2-20/*m), and each hyphal cell includes many small ovoi'd nuclei (600x300nm). The hyphae have simple septal pores (0-5-1 /*m diam) typical of ascomycetes. Two kinds of inclusions were associated with the pores: oblong, electron dense, crystal-like bodies (100 x 1000 nm), and typical Woronin bodies (600 nm diam). Several cytological features of the morel mycelium varied slightly, depending on their location. In ascogenous hyphae the septal pores were more or less always occluded with amorphous electron dense material, which also covered the Woronin bodies and the crystal-like bodies (Fig. 1 a, b). This can be interpreted as a character of senescence. In the stipe and the mycelial sheath, the hyphae also had this senescent feature. Electron translucent vesicles were located at the margin of the occluded pores, and the Woronin bodies became the predominant inclusions (Fig. 2 a, b). In the fine envelope around the rootlets and in mycorrhizas, the septal pores were mostly open, and only clear, well-delimited, Woronin bodies were observed (Figs 3, 7). Additionally, the presence of numerous plasmalemmasomes, in particular on septa, was a constant and common feature in morel hyphae in contact with the roots, specially in the mycorrhizas. These plasmalemmasomes included vesicles with double membranes (Fig. 4). The other mycorrhizal type was formed by a basidiomycete. Its hyphae were generally of smaller diameter (2-5 /im), and eacb hyphal cell only included two large nuclei (1-5 /im diam) except in the inner part of the mantle and in the Hartig net, which were coenocytic. The septa were dolipores with unperforated parenthesomes (Fig. 5). Description of the mycorrhizas P. abies M. rotunda. The hyphal mantle was {im thick (Fig. 6), changing gradually from a loose external prosenchyma with hyphae 2-3 /jbm diam, to an irregular and compact internal synenchyma with hyphae 3-6 pum diam. Distally, the mantle was homogeneous. Proximally, it included several layers of collapsed cortical cells containing an electron opaque residue (Figs 6, 7). The Hartig net surrounded only one layer of cortical cells, but its hyphae (1-5 /tm diam) were highly branched, in 6 Figure 6. Semi-thin longitudinal section of the ectomycorrhiza Morchella rotunda-picea abies. Scale bar, 10 //m. The hyphal mantle (HM) distally contains layers of collapsed cortical cells (LC). The Hartig net (HN) only surrounds one layer of cortical cells. intimate juxtaposition, unseptate, and multinucleate (Figs 6, 8), a condition that is typical and efficient according to Blasius et al. (1986). P. abies and a heterobasidiomycete. The hyphal

4 428 F. Buscot and I. Kottke 8 Figure 7,8. Longitudinal section in the hyphal mantle, showing the morel hyphae with Woronin bodies (W), the layers of collapsed cortical cells (CCC) with residual tannin, and the beginning of the Hartig net (HN), which surrounds living cortical cells (CC). Figure 8. Tangential section in the Hartig net, which is formed of branched, intimately juxtaposed, non septate hyphae with numerous nuclei (N). mantle was 40 /im thick, changing gradually from an external loose prosenchyma with mostly dead hyphae 2 plxn diam, to an internal prosenchyma with hyphae 5 /«m diam (Fig. 9). The hyphae of the Hartig net were smaller (2 /^m diam), and surrounded up to four layers of cortical cells. Only two ectomycorrhizas of spruce with continuous dolipores have been described (types 8 and 10; Haug & Oberwinkler,

5 Morchella rotunda on spruce roots Figures Picea abies mycorrhizas Figure 9. Ultrathin longitudinal section of the ectomycorrhiza of Picea abies with a heterobasidiomycete, showing the mantle with hyphae containing large nuclei (N) and dolipores (D), and a part of the Hartig net (HN). Figure 10. Transverse semi-thin section of the proximal senescent part of the same myeorrhiza, the partially collapsed Hartig net (CHN) is invaded intercellulary by morel hyphae (MO). Figure 11. As in Figure 10 but more distally, showing the thin envelope of morel hyphae. 1987). They are assumed to be formed by heterobasidiomycetes. Our sample had common cytological features with type 8, but differed in colour and texture of the mantle. At the proximal part of the basidiomycete mycorrhizas a Hartig net with partially collapsed cells remained, but the hyphal mantle was progressively replaced by a thin envelope of morel hyphae, which also grew intercellulary between the cortical cells and the senescent Hartig net (Figs 10, 11). There was no obvious interaction between the two fungi. The senescent state of the basidiomycete and the juvenile state of the morel hyphae indicate that this could be a secondary development, occurring after the formation of the basidiomycete mycorrhizas. DISCUSSION Our observations demonstrate that morels can ectomycorrhizas with spruce in nature. The identification of morel hyphae was based upon the morphology of the septa and of their associated inclusions. Differences exist between the septa in the ascocarp hyphae and in the mycorrhizas, but they appear gradually along the mycelium associated to the root system, and only reflect different physiological states. The morphology of the septa and their

6 430 F. Buscot and I. Kottke variations are consistent with those described in other Pezizales (Kimbrough & Curry, 1986). Another cytological factor which confirms the identification is the multinucleate state in the mycelium associated with the roots, which is a typical feature of the Morchellaceae (Berthet, 1964). Our attempts to synthesize mycorrhizas of spruce with M. rotunda under sterile conditions have not been successful. Taken with the field observations, this may indicate that morels only occasionally form ectomycorrhizas, or do so only when the mycelium is in a specific physiological state, which does not occur in pure culture. The infection induces no formation of mycorrhizal clusters. In other fungi a similar lack of morphogenetic change in mycorrhizal roots has been taken to indicate a low production of phytohormones by the mycelium, and a lower ability to form mycorrhizas (Gay & Debaud, 1987). The Hartig net is restricted to one cortical layer. The layers of collapsed cortical cells in the mantle are a common feature in mycorrhizas of Tuber melanosporum Vitt. and Quercus robur L., which have been taken to indicate that the hyphal mantle may be interpreted as a secondary structure formed by a degenerate Hartig net (J. Dexheimer, personal communication). Studies of such associations could improve our understanding of ectomycorrhizas. What is the role of M. rotunda mycorrhizas in the life-cycle of the fungus? The occluded septal pores in the mycelial sheath indicate that this structure, which may provide nutrients for fructification, was at the end of its function when the material was collected. In contrast, the hyphae in the mycorrhizas often had open septal pores, which indicates continuing physiological activity. It is possible that, at the beginning of spring, the mycelial sheath gives rise both to the ascocarps (Buscot, 1989), and the mycorrhizas. These mycorrhizas could be the starting point for the next cycle, which leads to production of ascocarps a few meters from the previous location (Buscot, 1987). Morels do not produce resting propagules able to persist for a long time in the soil, and to germinate when conditions become favourable (Schmidt, 1983). Association with roots and the formation of ectomycorrhizas are a possible way for morels to survive in forest ecosystems, after their massive but ephemeral fruiting on recently disturbed soils (Buscot, 1987). This growth of morels in forest is weak but probably essential for the taxon to survive. ACKNOWLEDGEMENTS We are indebted to the Alexander von Humboldt Foundation for support and to Professor Dr Oberwinkler for discussion of this paper. REFERENCES s, D., FEIL, W., KOTTKE, I. & OBERWINKLER, F. (1986), Hartig net structure and formation in fully ensheathed ectomycorrhizas. Nordic Journal of Botany 6, BROCK, T. D. (1951). 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