Building a basidiocarp: a case study of Laccaria spp. fruitbodies in the extraradical mycelium of Pinus ectomycorrhizas

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1 . British Mycological Society Printed in the United Kingdom. DOI: /S XO Building a basidiocarp: a case study of Laccaria spp. fruitbodies in the extraradical mycelium of Pinus ectomycorrhizas H. B. MASSICOTTE 1, L. H. MELVILLE 2 & R. L. PETERSON 2 1 Ecosystem Science and Management Program, College of Science and Management, The University of Northern British Columbia, Prince George, B.C. V2N 4Z9, Canada Tel (250) hugues@unbc.ca. Fax (250) Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada The extraradical mycelium of ectomycorrhizas is comprised of a network of hyphae that may initiate rhizomorphs, sclerotia and sexual reproductive structures. The development of these structures requires photosynthates produced by host trees. In this study, the initiation and early development of Laccaria bicolor (Maire) Orton fruitbodies (basidiocarps) were studied. Seedlings of Pinus resinosa Ait. and Pinus sylvestris L. were colonized by L. bicolor, a broad host epigeous ectomycorrhizal basidiomycetous fungal species, in growth pouches. Ectomycorrhizas with an extensive extraradical mycelium formed on short roots within 7-12 days after fungal inoculum was introduced. Numerous sites of hyphal aggregation, many of which subsequently developed into basidiocarp primordia, were initiated in the extraradical mycelium. Initial changes in aggregating hyphae included swelling and branching followed by growth of hyphae perpendicular to the paper wick in growth pouches. A stipe and a pileus primordium developed but none of these basidiocarp primordia matured. Distinct regions were evident in the stipe and pileus when sections were stained by various methods. Loose hyphae at the apex and periphery of the pileus were separated by mucilage. In Petri dishes with vermiculite as the substrate, basidiocarps of Laccaria laccata formed in ectomycorrhizal associations with Pinus sylvestris, developed a mature pileus with an hymenium and mature basidiospores. Keywords: Laccaria, Pinus, Ectomycorrhizas, Basidiocarps, Microscopy Introduction The extraradical mycelium is an integral component of ectomycorrhizas forming an extensive network (mycelial fans) in the substrate that plays a major role in nutrient exchange between the plant and fungal partners (Finlay & Read, 1986a,b). In many ectomycorrhizal associations, this network consists of aggregates of hyphae (rhizomorphs) in addition to single hyphae (Smith and Read, 1997). Considerable information is available concerning the initiation and morphology of rhizomorphs (Agerer, 1992, 1995) and their importance in transport of nutrients and water (Smith & Read, 1997). Several ectomycorrhizal fungal species also form sclerotia that are initiated in the extraradical mycelium often in association with rhizomorphs (Grenville, Peterson & Piché, 1985a,b). These authors have studied the initiation and histology of sclerotia in ectomycorrhizas synthesized in growth pouches between Pisolithus tinctorius (Pers.) Coker and Couch and Paxillus involutus (Batsch) Fr. and two pine species, Pinus strobus L. and Pinus resinosa. These studies, and that of Moore, Ashford and Peterson (1991), have shown that mature sclerotia store large amounts of polysaccharides, lipids, and protein and are obviously sinks for photosynthates. Periodically, the extraradical mycelium is also involved in initiating fungal reproductive structures (basidiocarps or ascocarps) and, as has been shown under greenhouse conditions for Pinus strobus - Laccaria bicolor ectomycorrhizal associations, basidiocarp formation is dependent upon current photosynthates from the host tree (Lamhamedi, Godbout & Fortin, 1994) and can be affected by temperature (Godbout and Fortin, 1990). Although basidiocarps of 141

2 Figs 3-7 Mycorrhizal roots of Pinus resinosa and developing basidiocarps of Laccaria bicolor. Fig 3 Portion of root system of a seedling showing monopodial (arrows) and dichotomously branched (arrowheads) mycorrhizas days post inoculation. Note the obvious purple hue on the apical portions of the roots due to fungal hyphae. Scale bar = 1.0 mm. Fig 4 Monopodial ectomycorrhiza (arrowhead) and an adjacent developing basidiocarp (arrow). Scale bar = 1.0 mm. Fig 5 Later stage of basidiocarp formation located close to an inoculum plug. (*). Scale bar = 1.0 mm. Fig 6 Mature basidiocarp grown in association with Pinus sylvestris in a Petri dish system. Scale bar = 1.0 cm. Fig 7 Portion of a gill from a mature basidiocarp with mature basdiospores (arrowhead) attached to a basidium. Scale bar = 20µm. 142

3 ectomycorrhizal fungal species have been obtained under laboratory and greenhouse conditions (e.g. Hacskaylo & Bruchet,1972; Debaud, Pepin & Bruchet,1981; Debaud & Gay,1987; Oh, Melville & Peterson, 1994), there has not been a developmental study of these structures and they have not been produced in a system that lends itself to experimental manipulation. To date, most developmental studies of basidiocarp formation have involved saprophytic fungi grown in vitro (Heckman et al., 1989; Godbout and Fortin, 1990). Although the Petri dish system used by Unestam and Stenström (1989) to establish ectomycorrhizas between Laccaria laccata and Pinus sylvestris led to the formation of mature basidiocarps (see Fig. 6), the vermiculite substrate used did not allow early stages in basidiocarp formation to be studied. The objective of this study, therefore, was to determine the ontogeny of basidiocarps of Laccaria bicolor in ectomycorrhizal association with host tree species using growth pouches in which the extraradical mycelium can be easily visualized (Fortin et al., 1980). Materials and methods Plant material and growth conditions Pinus resinosa seeds, obtained from Petawawa, Ontario (45 58' X 77 19', 120m) were surface sterilized in 30% H 2 O 2 for 30 min, washed with sterile distilled water and germinated on filter paper moistened with sterile distilled water in parafilm-sealed Petri dishes. Seedlings were transferred 10 d post-germination into growth pouches that consist of a transparent plastic bag enclosing an absorbent paper wick (Fortin, Piché & Lalonde, 1980). These were purchased from Northrup King Co., PO Box 959, Minneapolis, MN 55440, USA. The paper wick in each pouch was moistened with 10 ml sterile distilled water. Two seedlings were placed in each pouch and 50 pouches were set up. Modified Melin-Norkrans (MMN) nutrient solution (Marx & Bryan, 1975) was added weekly to the pouches beginning 25 d post-germination and continuing for eight weeks. Sterile distilled water was added as needed between nutrient treatments and for the last 12 weeks of growth. Seedlings were grown under light (mixed incandescent/fluorescence) providing 130µE/m -2 s -1 at seedling level on a 16h light / 8h dark cycle at 24 C day/18 night temperature cycle. High levels of humidity (60-80%) were maintained using a humidifier. Pinus sylvestris seedlings were placed in a Petri dish system with fungal inoculum of Laccaria laccata as described in Unestam and Stenström (1989). Mycorrhiza synthesis Forty-five day old seedlings in growth pouches were inoculated with Laccaria bicolor using the strain CRBF originally obtained from A. Fortin, Université Laval, Québec, Canada. Cultures were maintained on MMN nutrient agar and introduced into the pouches as plugs taken from the actively growing mycelium front. Scanning electron microscopy Pouches were observed periodically with a stereobinocular microscope to monitor the formation of mycorrhizas, extraradical mycelium and stages in basidiocarp initiation. Small pieces of the paper wick substrate containing either mycorrhizal roots or basidiocarp primordia were collected between 16 and 20 weeks post-inoculation, fixed in 2.5% glutaraldehyde in 0.1M HEPES buffer, rinsed in buffer, post-fixed in OSO 4, dehydrated in an ethanol series, critical point dried and mounted on metal stubs. Samples were coated with gold-palladium and viewed at 12 KeV using a JEOL JSM-35C scanning electron microscope. Numerous paper wick pieces were examined to generate the series in the ontogeny of basidiocarps. Light microscopy Basidiocarp primordia were removed from the paper wick substrate, fixed and dehydrated as for scanning electron microscopy and embedded in LR White resin (London Resin Company Ltd.). Serial thick sections ( µm) were cut with glass knives and heat-fixed to microscope slides. A series of slides for each primordium was prepared in order that adjacent sections could be used for various histochemical procedures. Sections for general structural features were stained with 1.0% methylene blue/0.1 azure B / 1.0% sodium borate in distilled water. These were rinsed and then counterstained with 0.5% aqueous basic fuchsin. Some sections were stained with acriflavin hydrochloride (Culling, 1974) to localize polysaccharides; these sections were viewed with an epifluorescence microscope using UV light. Results Growth pouches Pinus resinosa seedlings produced a prominent primary root with laterals (Fig. 1) and with time the heterorhizic nature of the root system became evident in that first order laterals generally elongated considerably more than second order laterals (Fig. 2). Mycorrhizal laterals 143

4 formed within 7-12 days post inoculation and by 20 days dichotomously-branched laterals, many with full mantles, were apparent (Figs. 2, 3). At about 50 days post-inoculation, basidiocarp primordia began to appear on the paper wick surface either adjacent to roots (Fig. 4) or to inoculum plugs (Fig. 5). Although mature basidiocarps did not develop in growth pouches, they did in the Petri dish system (Fig. 6). Basidia with basidiospores (Fig. 7) were formed along the gills of these basidiocarps. Details of the ontogeny of basidiocarp primordia were obtained by scanning electron microscopy. The earliest stage consisted of small rings of hyphae, some with evident clamp connections and some showing an increased diameter (Fig. 8). Hyphae adjacent to the initial ring branched and became very irregular in appearance (Fig. 9); this continued and hyphae became inter-twined with many hyphal apices directed towards the center of the mass (Fig. 10). Clamp connections were prevalent along these hyphae (Figs.9, 10). A central core of closely appressed hyphae (Fig. 10) was formed from which the stipe primordium was initiated (Fig. 11). These latter stages (Figs. 10, 11) involved growth of hyphae away from the paper substrate which was 90 from the vertical since the pouches were hanging vertically. Figs 1 and 2 Pinus resinosa seedlings colonized by Laccaria bicolor. Scale bars = 10 mm. Fig 1 Growth pouch with seedlings showing primary roots (arrows), colonized first order laterals (arrowheads), and plugs of fungal inoculum (*). Fig 2 Heterorhizic root system of an older seedling 50 days post inoculation showing monopodial (arrow) and dichotomously branched (triple arrowheads) mycorrhizal laterals, hyphal aggregations (arrowheads), basidiocarp primordia (double arrowheads) and plugs of fungal inoculum (*). 144

5 The majority of basidiocarp primordia were initiated close to the seedling root system (Fig. 2), and were connected to the mantle of adjacent ectomycorrhizas by clamped hyphae (Fig.12). Hyphae also emanated from the mantle and grew towards developing basidiocarps (Fig. 13). The pileus primordium was initiated as a center of growth at the distal end of the stipe primordium (Figs. Figs 8-11 Scanning electron microscopy showing stages in development of basidiocarps in the extraradical mycelium of Laccaria bicolor in growth pouches with seedlings of Pinus resinosa. All scale bars = 50 µm. Fig 8 Ring of clamp connectionbearing hyphae (arrows) on the surface of the paper wick (*). Figs 9 and 10 Stages in the aggregation and branching of hyphae. A central core of closely appressed hyphae from which the stipe primordium develops is evident in Fig. 10 (arrowheads). Fig 11 Stipe primordium consisting of hyphae that have grown away from the surface of the paper wick. 145

6 Figs Scanning electron microscopy of developing basidiocarps of Laccaria bicolor adjacent to mycorrhizal roots of Pinus resinosa in growth pouches. All scale bars = 100 µm. Fig 12 A basidiocarp primordium (arrow) adjacent to a dichotomously branched ectomycorrhiza. Hyphae (arrowheads) connect the basidiocarp to the mantle of the ectomycorrhizal root. Fig 13 Enlargement of the area of connecting hyphae shown in Fig. 12. Hyphae (arrowheads) have originated from the mantle surface and have grown toward the basidiocarp primordium. Fig 14 A developing basidiocarp showing the initiation of a pileus (arrow) at the distal end of the primordium. Loose hyphae (arrowheads) are evident at the base of the stipe (Reprinted with permission from Peterson, Massicotte and Melville (2004)). Fig 15 A top view of a developing basidiocarp showing the size of the pileus in relation to the stipe. Hyphae at the apex of the pileus are loosely organized. 146

7 * Figures 16-21: Sections of Laccaria bicolor basidiocarp primordia embedded in LR White resin and stained with either methylene blue/azure B/basic fuchsin (Figs. 16, 20, 21) and viewed with white light or stained with acriflavine hydrochloride (Figs ) and viewed with blue light. Fig 16 Longitudinal section of basidiocarp at a stage similar to that illustrated in Fig. 14. The central core of the stipe (zone A) and the base of the developing pileus (zone C) consist of closely appressed hyphae whereas the apex of the pileus (zone D) and the periphery of the stipe (arrowheads) consist of more loosely organized hyphae. Scale bar = 100 µm. Fig 17 Basal region of the stipe including zone A and anchoring hyphae (not shown in Fig. 16). The latter consists of enlarged, vacuolated hyphae (*) whereas hyphae in zone A are more compact (arrow). Scale bar = 50 µm. Fig 18 Section of the stipe in zone B. Hyphae are of varying diameter and orientation. Scale bar = 50µm. Fig 19 The pileus medulla (zone C), consisting of hyphae of small diameter interspersed with interhyphal spaces. Scale bar = 50 µm. Fig 20 The apex of the pileus (zone D), consisting of small diameter hyphae, some radiating out from the apex, with interhyphal mucilage. Scale bar = 50 µm. Fig 21 The protohymenial region (zone E) consisting of tightly packed hyphae radiating laterally from the pileus. Scale bar = 50 µm. 147

8 14, 15); this was the most advanced stage of basidiocarp formation observed in growth pouches. The loose arrangement of hyphae at the pileus apex is evident in Figure 15. Although few basidiocarp primordia in each pouch reached the stage shown in Fig. 14, many (perhaps hundreds) formed to the stage of hyphal aggregation (Figs. 9,10). Light microscopy Longitudinal sections of a basidiocarp primordium similar to that in Figure 14 showed various regions of the stipe and pileus (Fig. 16). Hyphae at the apex of the pileus were more loosely arranged than hyphae at its base and in the stipe (Fig. 16). Hyphae in the basal region of the stipe were more compactly arranged and of smaller diameter than those in the region anchoring the primordium to the substrate (Fig. 17). The diameter and degree of vacuolation of hyphae varied from the mid-region of the stipe to the base of the pileus (Fig. 18). The medulla region of the pileus consisted of densely packed hyphae interspersed with interhyphal spaces (Fig. 19). The apex (Fig. 20) and peripheral region of the pileus (Fig. 21) has loosely arranged hyphae separated by mucilage. Discussion Although numerous L. bicolor basidiocarp primordia were initiated in the extraradical mycelium associated with roots of P. resinosa seedlings grown in plastic growth pouches, none matured under the environmental conditions of the experiment. Since photosynthate supply from the host is required for basidiocarp formation in ectomycorrhizal fungal species (Last & Fleming, 1985; Lamhamedi et al., 1994) it is probable that photosynthate supply was limiting, particularly since an extensive extraradical mycelium network was formed in growth pouches and this would be a sink for photosynthates. Although the environmental stimulus involved in the initiation of basidiocarps of L. bicolor in growth pouches is not known, the numerous primordia formed allowed a detailed morphological and anatomical analysis of these structures. The structural features described for L. bicolor basidiocarp primordia (hyphal aggregation, hyphal swelling and branching) are similar to those reported for the non-mycorrhizal basidiomycetes, Flammulina velutipes (M.A. Curtis: Fr.) Singer (Williams et al., 1985), Coprinus lagopus Fr (Matthews and Niederpruem, 1972) and the economically important mushroom, Agaricus bisporus (Lange) Imbach (Heckman et al., 1989). The formation of the stipe primordium in L. bicolor was very similar to that reported for F. velutipes in that hyphae continued to be added to the periphery of the stipe as elongation proceeded (Williams et al., 1985). The initiation of the precursor to a pileus observed here was very similar to that reported as the exogenous type reported by Burnett (1976). The extracellular matrix material present in the growing front of the pileus primordium may contain hydrophobins, cell wall surfactant proteins known to be involved in the aggregation of hyphae during the formation of basidiocarps in a number of nonmycorrhizal fungal genera (Wessels, 1997). Although hydrophobins have not been identified in reproductive structures of ectomycorrhizal fungi, they have been isolated and identified from hyphae of Pisolithus tinctorius, an ectomycorrhizal basidiomycete (Tagu, Kottke & Martin, 1998; Martin et al. 1999) and have been implicated in the aggregation of hyphae during mantle formation of ectomycorrhizas (Tagu et al., 2002). The system described here in which numerous stages in basidiocarp primordia can be obtained could perhaps be useful in examining the role of hydrophobins during the entire morphogenetic sequence in basidiocarp development. This system of basidiocarp initiation may also be useful to study resource allocation to these structures during their development in the extraradical mycelium of ectomycorrhizas. Acknowledgement We are grateful to Elna Stenström and Torgny Unestam for providing Figs. 6 & 7. We thank the Natural Sciences and Engineering Research Council of Canada for financial support and Ryan Geil for help with the plates. References Agerer, R. (1992). Ectomycorrhizal rhizomorphs: organs of contact. In Mycorrhizas in ecosystems. (edited by D. J. Read, D. H. Lewis, A H Fitter & I. J. Alexander), pp Wallingford, UK: C.A.B. International. Agerer, R. (1995). Anatomical characteristics of identified ectomycorrhizas: an attempt towards a natural classification. In Mycorrhiza Structure, Function, Molecular Biology and Biotechnology (edited by A.K. Varma & B. Hock), pp Berlin: Springer-Verlag. Burnett, J. H. (1976). Fundamentals of mycology. 2 nd edition. Arnold, London Culling C. F. A. (1974). Modern microscopy: elemental theory and practice. London: Butterworths. Debaud, J. C. & Gay, G. (1987). In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum associated with Pinus pinaster. New Phytologist 105:

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