Russula foetens (Pers.: Fr.) Fr.

Agerer R, Danielson RM, Egli S, Ingleby K, Luoma D, Treu R (eds) DESCRIPTIONS OF ECTOMYCORRHIZAE 5 : 163-168 (2001)

Russula foetens (Pers.: Fr.) Fr. + Fagus sylvatica L. LUDWIG BEENKEN, Section Mykologie, Institut fiir Systematische Botanik, Universitiit Miinchen, Menzinger Straj3e 67, D-80638 Miinchen, Germany.

Short description Ectomycorrhizae irregularly monopodial-pinnate, light brown, with velvety surface, all mantle layers plectenchymatous, all septa simple, outer and middle mantle layers with gelatinous matrix, mantle bearing thick-walled, bottle-shaped (20-45 x 8-12 J!m) cystidia with 1 thick-walled knob, contents of some becoming dark brown in sulphovanillin; rhizomorphs running along roots, with a peripheral layer of thick-walled hyphae, bearing slender cystidia and with an inner part containing vessel-like hyphae, ladder-like hyphae lacking. Morphological characters (Fig. 1) : Mycorrhizal systems up to 5 Jlm long, simple to irregularly monopodial-pinnate. -Main axes 0.3-0.4 Jlm diam. - Unramified ends straight to slightly bent, up to 3 Jlm long and 0.3-0.4 J.lm diam.; ochre to light brown, partly silvery from included air, very tips lighter- Surface of wzramified ends velvety to short spiny, bearing soil particles. - Rlzizomorplzs infrequent (not all mycorrhizal systems with one), mostly running along the thicker roots of the host tree, appressed onto the root with their flattened underside, up to 0.2 J.lm thick and 0.3 Jlm broad, up to 2 em long, branched. connection to the ectomycorrhiza by thin hypha! bundles along the colonized root; surface of rhizomorphs similar to that of the mantle. Anatomical characters of mantle in plan views (Figs. 2c-e): Mantle plectenchymatous throughout; hypha! walls hyaline, colourless to yellowish, smooth; clamps lacking. - Outer mantle layers (Fig. 2c) loosely plectenchymatous, with gelatinous matrix between the hyphae, hyphae net-like arranged, bearing the cystidia, (mantle type C, D, AGERER 1987-1998, 1991, 1995); hyphae abundantly branched, cells straight to bent, 10-30 Jlm long, 2-4 J.lm diam.; walls ca. 0.3 J.lm thick; anastomoses without septa. - Middle mantle layers (Fig. 2d) densely plectenchymatous, with gelatinous matrix, hyphae mostly arranged in parallel bundles, cells 4-30 x 2-4 J.lm; walls ca. 0.3 J.lm thick. -Inner mantle layers (Fig. 2e) densely plectenchymatous, palmetti-like hypha! structures on the inner surface, cells straight, 4-30 J.lm long, 2-6 IJm diam.; walls ca. 0.3 IJm thick . - Very tip with no differences in mantle features. Anatomical characters of emanating elements (Figs. 2a-c, 3): Rlzizomorplzs (Fig. 3): thicker rhizomorphs on roots highly organized (russuloid rhizomorph type according to AGERER 1999), differentiated into a colourless inner part with simple and vessel-like hyphae contacting the root cortex and a brownish peripheral layer with cystidia, in cross-section (Fig. 3c) semicircular. In longitudinal section (Fig. 3a): peripheral layer: ca. 20-30 J.lm thick, with dense, gelatinous matrix and soil particles between the hyphae, hyphae 2-4 J.lm diam., with simple septa, walls 0.3 J.lm thick, smooth and yellowish; cystidia of the rhizomorphs different from mantle cystidia, more slender (2,5-4,5 J.lm at base), fusiforn or similar to hypha! ends, walls 0.3-0.5 J.lm, knobs ovate, 3-5 x 2-2.5 J.lm, with 0.3 J.lm thick walls; inner part up to 100 J.lm thick, with a gelatinous matrix and few soil particles between the hyphae, the two ty163

pes of hyphae not arranged in special pattern, contents colourless, hypha! walls smooth and colourless; simple hyphae 2-20(30) x 2.5-6 J.lm, walls 0.3 J.lm thick, septa simple; vessel-like hyphae 10-18 J.lm diam., walls 0.5-1 ( 1.5) J.lm, hyphae branched and with open anastomoses, mostly constricted at septa, distance between septa ca. 100-400 J.lm, simple, with enlarged pores, frequently walls around the pores thickened (Fig. 3b), infrequently with remnants of dissolved septa; ladder-like hyphae not found. -Emanating hyphae 2-4 J.lm diam., anastomoses without septa; walls colourless to yellowish, smooth and 0.3 J.lm thick. - Cystidia of the mantle (Figs. 2a-c) of only one type, formed terminally, laterally or intercalary; fusiform to bottleshaped, noncapitate, (15)20-45(50) J.lm long, basally (4)8-12 J.lm thick, neck 3-4(5) J.lm thick, walls smooth, hyaline, colourless to yellowish, 0.5-1.5(2) J.lm thick; mostly with one, central, apical knob, infrequently 2 knobs in a row, knobs globose to subglobose, 2-3.5 x 2-3 J.lm, walls up to 1.5(2) J.lm thick, lumina of knobs connected to that of cystidial body, when knobs broken off, thin pores at tips visible, cystidiallumina mostly clear, infrequently light brownish or with small droplets; strongly tortuous, ramified hypha! ends not found.

Anatomical characters, longitudinal section: Mantle (without cystidia) 25-40 J.lm thick, at the very tip ca. 25 J.lm, without distinct layers, loosely to densely arranged, hypha! cells tangentially 2-30 J.lm and radially 1.5-6 J.lm; remnants of calyptra cells I 0-20 x 1-5( I 0) J.lm. Tannin cells lacking. -Epidermal cells obliquely oriented to the surface of the root, obliquely. tangentially 8-15 J.lm and obliquely-radially 20-45 J.lm; haustoria (Fig. 2f) originating from the Hartig net into the lumina, tortuous, frequently branched, 1.5-2(3) J.lm diam. - Hartig net paraepidermal, in section hyphae in I row, cells 2-3 J.lm thick and rectangular; Hartig net in plan view of palmetti-type, lobes 1-2 J.lm broad. Colour reaction with different reagents: Preparation of mantle: Acid fuchsin aiter rinsing with H20: walls slightly pink; cotton-blue-lactic-acid: walls blue, walls of cystidia dark-blue; ethanol 70%: n. r. (=no reaction); FeS04: n. r.; guaiac: n. r.; KOH: n. r.; lactic acid: n. r.; Melzer's reagent: n. r.; sulpha-vanillin: walls pink, contents of a few cystidia becoming dark brown. Autofluorescence: Wlzole mycorrlziza: UV 254 nm: lacking; UV 366 nm: lacking.- Mantle in section: UV-filter 340-380 nm: yellow-green throughout; blue filter 450-490 nm: yellow throughout; green filter 530-560 nm: red throughout.

DNA-Analysis: ITS regions from ribosomal DNA of the fruitbody and of adjacent ectomycorrhiza and rhizomorph, which are amplified by PCR using ITS I and ITS 4 primers (WHITE ct al. 1990, GARDES & BRUNS 1993), show equal total base-pair lengths: 710 bp. Coincident patterns of bands appear in gel electrophoresis of the three amplified DNAs after restriction digestions by Alu 1: 505 bp, 210 bp; EcoRI: 360 bp, 320 bp; Hinf 1: 350 bp, 335 bp; Taq 1: 350 bp, 240 bp. Intraspecific variability not checked. (Base-pair lengths rounded to the nearest 5 bp, all data with a tolerance of 3-5 %. Applied methods described by AGERER et al. 1996). Reference specimen for Fagus ectomycorrhiza: Germany, Bayem, Bez. Oberbayem, Lkr. Miinchen, Gmd. Pullach, GroBhesselohe, on medow under Fagus sylvatica L. 48°04'N, ll 0 32'E, alt. 580 m, MTB Nr. 7935. 14. 7. 1999, fruitbody leg. et del. and soil core exc. and myc. isol. L. Beenken, mycorrhizae isolated from clayey, mineral soil over limestone; fruitbody LB 1131 and mycorrhiza MLB 1131 (in M).- Fruitbody determined with ROMAGNESI ( 1967). -Identification of mycorrhiza and rhizomorph by PCR amplification and restriction digest (see above).

Discussion: Russula foe tens belongs to Russula sub g. lngratula Romagn. 1987 sect. Foetentinae Melzer-Zvara (ROMAGNESI 1967, 1985, 1987). Two ectomycorrhizae of this group were described to date, i.e. Russula illata Romagn. by BRAND (1991a, b) and R. amoeno/ens Romagn. by JAKUCS & BEENKEN (1999). Common characters are fusiform to bottle-shaped cy164

stidia forming only a single apical knob and plectenchymatous mantles. The ectomycorrhizae of R. faetens are very similar to those of R. illata. Both form middle mantle layers with bundles of parallel hyphae, but R. illata has more slender, 15-40 Jlm long and basally 5-8 Jlm thick cystidia with up to 1 Jlm thick walls. Ectomycorrhizae of R. amaenalens differ in both features. They have middle mantle layers of inflated cells without any parallel pattern and smaller cystidia (maximally 35 x 6 Jlm) with up to 0.5 Jlm thick walls. In this group highly differentiated rhizomorphs are known only from R. faetens. They have peripheral layers with cystidia like those of Russula subg. Campactae (Fr.) Bon and R. subg. Heteraphyllidia Romagn. 1987 (BEENKEN 2000). Ladder-like hyphae are lacking. Vessel-like hyphae forming septa with enlarged pores and thickened walls occur. The haustoria in epidermal cells of the present ectomycorrhizae originate from Hartig net cells (Fig. 2f), which are not distinguishable from the normal Hartig net cells. Similar mycorrhizal haustoria of Russula emetica (Schaeff.: Fr.) Pers. and of R. mairei Sing. were described and discussed by BRAND (1991 a). An additional comparison of Russula ectomycorrhizae is given by BEENKEN (2001). Acknowledgements: I thank very much Mrs. Fatma Medjebeur-Thrun and Mr. Wolfgang U. Thrun (Munich) for showing the very rich collecting area. References: AGERER R, ed (1987-1998) Colour atlas of ectomycorrhizae. 1st-11th del., Einhorn, Schwtibisch Gmiind. - AGERER R (1991) Characterization of ectomycorrhiza. In Norris JR, Read OJ, Varma AK (eds.): Techniques for the study of mycorrhiza: Methods in Microbiology 23: 25-73.- AGERER R (1995) Anatomical characteristics of identified ectomycorrhizas: An attempt towards a natural classification. In Varma A, Hock B (eds.): Mycorrhiza: structure, function, molecular biology and biotechnology, pp. 685-734, Springer, Berlin. - AGERER R ( 1999) Never change a functionally successful principle: The evolution of Boletales s. I. (Hymenomycetes, Basidiomycota) as seen from below-ground features. Sendtnera 6: 5-91. - AGERER R, MOLLER WR, BAHNWEG G ( 1996) Ectomycorrhiza of Rhizopogon subcaerulescens on T.mga heterophylla. Nova Hedwigia 63: 397-415.- BRAND F (1991a) Ektomycorrhizen an Fagus sylvatica. Charakterisierung und Identifizierung, iikologische Kennzeichnung und unsterile Kultivierung. Libri Botanici 2: 1-229.- BRAND F (1991b) Russula il/ota. In AGERER R (ed.) Colour atlas of Ectomycorrhizae. plate 64, Einhorn-Verlag, Schwiibisch Gmiind.- BEENKEN L (2001) Russula densifolia Seer. ex Gill.+ Fagus sylvatica L. Oeser Ectomyc 5: 147-155.- GARDES MT, BRUNS TO (1993) ITS primers with enhanced specificity for basidiomycetes, application to the identification of mycorrhizae and rusts. Molec Ecol 2: 113-118. - JAKUCs E, BEENKEN L ( 1999) Russula amoenolens Romagn. + Populus alba L. Oeser Ectomyc 4: 115-119. - RoMAGNESI H (1967) Les Russules d' Europe et d' Afrique du Nord, Bordas, Paris. - RoMAGNESI H ( 1985) Supplement of Les Russules d'Europe et d' Afrique du Nord. Cramer, Vaduz: 991030. - RoMAGNESI H ( 1987) Status et Noms nouveaux pour les taxa infrageneriques dans le Genere Russula. Doc. mycol. Tome XVIII, Fascicule 69, Novitates 3: 39-40.- WHITE TJ, BRUNS T, LEES, TAYLOR] (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: A guide to methods and applications: 315-322, Academic Press Inc.

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Captions: Fig. 1- Habit of ectomycorrhizae.- Fig. 2- a-e. Mantle layers in plan view.a. Closely packed cystidia. - b. Unusually shaped, intercalary cystidium. -c. Outer mantle layer, with gelatinous matrix. -d. Middle mantle layer. -e. Inner mantle layer. - f Tree-like haustoria originating from the Hartig net into the lumina of epidermal cells (in longitudinalsection). -Fig. 3- a. Rhizomorph in longitudinal section of 30 J.lm by cryotome: above gelatinous outer layer bearing cystidia, with soil particles and remnant of root cortex; in the middle inner part with undifferentiated and vessel-like hyphae; at the bottom root cortex as substrate (black). -b. Constricted septum of vessel-like hypha with thickened wall around the enlarged pore. -c. Schematic cross-section of rhizomorph (black = substrate, white with circles = inner part with vessel-like hyphae, squared = peripheral layer). All jigs. from MLB I 131.

Fig. 1 - Russula foetens + Fagus sylvatica 166

Fig. 2 - Russula joete11s + Fagus sylvatica 167

Fig. 3 - Russula foetens + Fagus syll'atica 168