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Instituto Multidisciplinario de Biología Vegetal (CONICET), C.C. 495, 5000. Córdoba, Argentina
Ludwig Beenken
Department Biology I and GeoBio-Center, Ludwig Maximilian University, Systematic Mycology, Menzinger Straße 67, D-80638 München, Germany
Karin Pritsch
Institute of Soil Ecology (GSF), Department of Ecology, Technische Universität München (TUM), Ingolstaedter Landstrasse 1, D-85764 Oberschleissheim, Germany
Graciela Daniele
Instituto Multidisciplinario de Biología Vegetal (CONICET), C.C. 495, 5000. Córdoba, Argentina
Michael Schloter
Institute of Soil Ecology, GSF-National Research Centre for Environment and Health GmbH, Ingolstaedter Landstraße 1, D-85764 Oberschleissheim, Germany
Reinhard Agerer
Department Biology I and GeoBio-Center, Ludwig Maximilian University, Systematic Mycology, Menzinger Straße 67, D-80638 München, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ectomycorrhizae (ECM) of Lactarius aff. omphaliformis Romagn., Russula alnijorullensis (Sing.) Sing. and Cortinarius tucumanensis Mos. on Andean alder (Alnus acuminata Kunth) were characterized and identified. The identification of the fungal symbionts was achieved by morpho-anatomical observations of mycorrhizae and by comparison of ITS-RFLP patterns obtained from ECM and fruitbodies. L. aff. omphaliformis ECM differed in some morphological details such as ramification and mantle type from ECM of the same species on A. glutinosa. L. aff. omphaliformis ECM show an orange to ochre mantle containing latex cells, which stain with sulpho-vanillin, emanating hyphae without clamps. R. alnijorullensis ECM represent a typical Russula-type-ECM, light yellow to pinkish, the outer mantle being composed of triangular latex-filled cells staining with sulpho-vanillin, emanating hyphae without clamps. C. tucumanensis ECM exhibit a white (silvery) to yellowish brown mantle covered with soil particles, emanating hyphae with clamps.
Key words: Alnus, ectomycorrhizal fungi, morphological characterization, PCR, RFLP
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The distribution of Alnus acuminata Kunth, an ecologically valuable tree species in South America, ranges from the north of Venezuela to the Andes (Dawson 1990), reaching its southernmost distribution in the Catamarca province of Argentina.
Alnus ectomycorrhizae have been described from North America (Neal et al 1968; Froidevaux 1973; Molina 1979; Godbout and Fortin 1983; Massicotte et al 1986, 1989, 1994, 1999; Miller et al 1988, 1991, 1992; Murphy and Miller 1994; Molina et al 1994), South America (Becerra et al 2002, 2005a), Japan (Masui 1926), New Zealand (Mejstrik and Benecke 1969) and Europe (Treu 1990; Agerer et al 1993; Airaudi et al 1994; Pritsch et al 1997a, b; Wiedmer and Senn-Irlet 1998, 1999a, b, c, d, 2001a, b; Beenken 2001).
Trappe (1962) and Horak (1963) listed a number of fungi of the families Cortinariaceae and Russulaceae associated with different Alnus species. Several species of Cortinariaceae have been reported in mycorrhizal association with alders (Stangl 1970; Godbout and Fortin 1983, 1985; Brunner et al 1990; Miller et al 1991; Moser 2001). The ectomycorrhizae of Cortinarius cf. helvelloides (Fr.) Fr. and C. cf. alneus (Mos.) Mos. associated with A. glutinosa (L.) Gaertn, C. bibulus Quél. associated with A. rubra Bong., C. pluvius (Fr.) Fr., C. alnobetulae Kühn., C. atropusillus Frave, C. badiovestitus Mos., C. kühneri Mos., C. bibulus Quél. and C. helvelloides (Fr.) Fr. associated with A. viridis (Chaix) DC. and C. helodes Moser, Matheny & Daniele associated with A. acuminata, have been described respectively by Pritsch et al (1997b), Miller et al (1991), Wiedmer and Senn-Irlet (1998, 1999a, b, c, d, 2001a, b) and Becerra et al (2005a).
Within Russulaceae, Brunner et al (1992) found Russula alnicrispa Brunner, R. subartica Brunner and Lactarius obscuratus (Lasch) Fr. associated with A. tenuifolia Nutt.. Pritsch et al (1997b) described the ectomycorrhizae of L. obscuratus, L. omphaliformis Romagn., L. lilacinus (Lasch) Fr. and R. pumila Rouzeau & Massart associated with A. glutinosa. The ECM of L. obscuratus, L. alpinus Peck and R. alnetorum were described in association with A. viridis (Airaudi et al 1994, Beenken 2001), and ectomycorrhizae of L. obscuratus Quél. in association with A. rubra (Miller et al 1991).
Two of the three species considered in this study (Russula alnijorullensis Sing., Cortinarius tucumanensis Mos.) have been reported to form fruitbodies under A. acuminata in Argentina (Moser and Horak 1975, Moser 2001).
In this study we complement reports related to A. acuminata-ECM associations (Becerra et al 2002, 2005a; Becerra 2002) by newly describing the ectomycorrhizae of Lactarius aff. omphaliformis, Russula alnijorullensis and Cortinarius tucumanensis on A. acuminata by morphological and anatomical parameters. The identification of the fungal symbionts was achieved by comparing ITS-PCR/RFLP patterns of mycorrhizae and fruitbodies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and Becerra et al (2005b). Both locations were visited in Feb, Mar, May and Nov 2000–2002. At every sampling date, blocks of 15 x 15 cm were cut from the litter layer around fruitbodies with a sharp knife. The rooted soil samples were transferred to a plastic box together with the fruitbodies, leaving the sample as undisturbed as possible, and stored at 4 C during transport to the laboratory. Fruitbodies and mycorrhizae were examined within 2 d after sampling. Root samples were soaked and washed in water following Agerer’s method (1991). Alder roots were distinguished easily from others roots by the presence of actinorhizal nodules and their morphological appearance. Hyphal connections from stipe bases to mycorrhizal tips were traced, when possible, under a Zeiss stereomicroscope at 10–40x magnification according to the method of Agerer (1991). Several tips of each morphotype were subjected to comparative anatomical studies. Photographs of ECM were taken with a Leica M420 stereomicroscope. Several chemical reagents (15% KOH, Melzer’s reagent, cotton blue, 70% ethanol, sulpho-vanillin, NH4OH and lactic acid) were used for studying specific color changes of mycorrhizae. In addition a portion of mycorrhizal roots was fixed in 70% alcohol and stored at 4 C in the dark. Description of the ectomycorrhizae follows the terminology of Agerer (1991, 1999) and Miller et al (1991). Fresh mycorrhizae were prepared for direct microscopy of the hyphal mantle (hand sections or mantle peeled off) according to Agerer (1991) or embedded in Leica Historesin kit after dehydration with series of 20, 30, 40, 50, 60, 70, 80, 90, 96, 100% ethanol. Semithin tangential and cross-sections of embedded ECM were cut with a microtome with steel knives. Total mantle views, peeled off mantles, and sections from resin-embedded mycorrhizae were examined and photographed with a Zeiss Axiophot light microscope at 200–1000x magnification. Characterization of the Hartig net follows the nomenclature of Godbout and Fortin (1983). Fruitbodies were identified according to Singer and Digilio (1951) and Moser (1978, 2001). Voucher specimen of fruitbodies and mycorrhizae were deposited in the Museo Botánico de Córdoba Herbarium (CORD) (Holmgren et al 1990).
Molecular identification.— –
Several tips of each morphotype were prepared for DNA extraction. Total DNA was extracted from small pieces of sporocarp tissue (1–4 mm3) cut from the central part of the cap and from single mycorrhizal tips using DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer’s instructions. Pelleted DNA was resuspended in 50 µL of Tris/HCl buffer (pH 8) containing 1 mM EDTA and stored at 4 C until use. DNA extraction from mycorrhizal tips was replicated at least twice. The primers ITS1 and ITS4 (White et al 1990) were used to amplify the ITS region. For PCR the protocol of Henrion et al (1994) was applied: 2 µL of DNA extract was mixed gently in 0.5 mL polypropylene tubes with 47.5 µL of the PCR-reactants at final concentrations of 1.5 mM MgCl2, 200 µM each of dATP, dCTP, dGTP, dTTP, and 20 pmol of each primer in buffer for the Taq DNA polymerase. In every series of amplification reactions without DNA extracts served as controls for contaminants in the reagents. The tubes were placed immediately in a thermocycler (T3 Thermocycler, Biometra, Germany). 2.5 U Taq DNA polymerase was added after a denaturation step for 5 min at 94 C. PCR reaction was run in 30 cycles under these conditions: (i) denaturation of DNA for 1 min at 94 C; (ii) annealing of the primers for 1 min at 50 C; 3) elongation for 1 min at 72 C. A final step of 10 min at 72 C allowed elongation of uncompleted DNA strands. For RFLP analysis, 10 µL of the amplified ITS products were digested with the restriction endonucleases TaqI, Hinf I and EcoRI, in separate reactions, following manufacturer’s recommendations. PCR and RFLP products were size-fractionated on 1.5% agarose gels (DNA Agarose, Biozym, Germany). The gels were stained with ethidium bromide and photographed under UV light. Gel images were analyzed visually by comparing RFLP patterns of mycorrhizal and fruitbody DNA on the same gel and by estimating the length of PCR products and RFLP fragments based on 100-bp ladder (MBI Fermentas) used as DNA marker.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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); surface of unramified ends smooth, covered with soil particles. Root tips blunt, covered by the mantle. Mantle not transparent; mycorrhiza not carbonizing. Rhizomorphs not observed.
Anatomical characters of mantle in plan view: mantle continuous over the root apex. Blue granules and a matrix lacking; needle-like contents after fixation present. Outer mantle layers pseudoparenchymatous with angular to epidermoid cells bearing a hyphal net (type P-Q, Agerer and Rambold 1998) (FIG. 2); mantle cells 13–23 µm long and 10–15 µm diam; walls of hyphal net faint, delicate, inconspicuous, rather thin, hyphal net not specialized. Cells of mantle membranaceously yellowish, thin-walled 0.3–0.5 µm, without clamps; septa as thick as walls; surface of cells smooth, more or less gelatinous (sticky); some cells filled with latex staining in sulpho-vanillin, shape rectangular, 9.5–28 µm long and 6.4–13 µm diam (FIG. 2). Middle mantle layer, plectenchymatous to pseudoparenchymatous without discernible pattern, 2.5–4 µm diam, hyphae without clamps (FIG. 3). Inner mantle layer, plectenchymatous without pattern; hyphae without clamps, membranaceously yellowish, 3–4 µm diam (FIG. 4); laticifers present, straight and even, 2–5 µm diam, ramification infrequent (FIG. 5).
Anatomical characters of emanating elements: emanating hyphae smooth, wavy, shape not striking, 1.5–2.4 µm diam, cell walls thin (<0.5 µm), colorless to membranaceously yellowish, elbow-like protrusions lacking; distal ends of hyphae simple, septal distances 4–18 µm, septa without clamps, cross walls as thick as remaining walls.
Anatomical characters in longitudinal section: mantle 11–30 µm thick (FIG. 6), three distinct mantle layers discernible; outer mantle layer pseudoparenchymatous, hyphae tangentially 4–20 µm, radially 2–11 µm; middle mantle layer plectenchymatous to pseudoparenchymatous, hyphae tangentially 2–20 µm, radially 2–11 µm; inner mantle layer plectenchymatous, hyphae tangentially 2–22 µm, radially 1.5–4 µm; mantle of very tip 12–19 µm. Tannin cells lacking. Epidermal cells rectangular, parallel to root axis, tangentially 13–35 µm, radially 9–20 µm. Cortical cells rectangular to cylindrical, tangentially 22–44 µm, radially 10–18 µm. Hartig net not completely paraepidermal (FIG. 6), in plan view infrequently lobed; 5–6 µm wide lobes around epidermal cells.
Color reaction with different reagents: whole ectomycorrhizae bleaching with 15% KOH and lactic acid. Sulphovanillin: walls pink, contents of mantle cells with black droplets, laticifers black. Emanating hyphae and laticifers staining slightly blue with cotton blue; no reaction with Melzer’s reagent, ethanol 70% and NH4OH.
Specimens examined.. ARGENTINA. Catamarca. Sierra de Narváez. 27° 43'S, 65° 54'W, elevation 1820 m. Ectomycorrhizae under A. acuminata, 7 Nov 2001, A. Becerra 04 (CORD); Tucumán. Quebrada del Portugués. Tafídel Valle. 26° 58'S, 65° 45'W, elevation 2187 m. Fruitbodies under A. acuminata, 11 Nov 2000, G. Daniele 191 (CORD), 16 Feb 2001, G. Daniele 203 (CORD). Catamarca. Sierra de Narváez. 27° 43'S, 65° 54'W, elevation 1820 m. Fruitbodies under A. acuminata, 7 May 2000, G. Daniele 186 (CORD), 15 Feb 2001, G. Daniele 204 (CORD), 7 Nov 2001, G. Daniele 210 (CORD), 27 May 2002, G. Daniele 215 (CORD).
Russula alnijorullensis (Sing.) Sing. FIGS. 7–12
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); orders of ramification 0–1; systems abundant, dense; unramified ends sinuous to tortuous, up to 10.4 mm long and 0.1–0.5 mm diam; entire mycorrhiza light yellow to pinkish when young, ochre, yellowish brown when older. Surface of unramified ends smooth, covered with few soil particles. Root tips blunt and covered by the mantle. Mantle not transparent; mycorrhizae not carbonizing. Rhizomorphs not observed.
Anatomical characters of mantle in plan views: mantle continuous over the root apex. Blue granules and a matrix lacking. Outer mantle layers pseudoparenchymatous with emanating hyphae on angular cells (type L, Agerer and Rambold 1998) (FIGS. 8–9); some cell mounds on the mantle, 10–21 µm long and 7–17 µm diam. Angular cells membranaceously yellowish, 5–32.2 µm long and 5–19 µm diam, thin-walled (<0.5 µm), 6–7 cells in a square of 20 x 20 µm. Cells filled with latex, staining in sulpho-vanillin; shape triangular, 6.5–26 µm long and 6.5–13 µm diam. Inner mantle layer plectenchymatous without discernible pattern; cells straight, membranaceously yellowish, without clamps, 2.5–5 µm diam (FIG. 10).
Anatomical characters of emanating elements: emanating hyphae tortuous to irregularly inflated, 2–4 µm diam, septal distances 12–32 µm, colorless, hyphae even at septa, without clamps, elbow-like protrusions present, smooth, but agglutinated with soil particles (FIG. 11). Cystidia lacking.
Anatomical characters in longitudinal section: mantle 40–60 µm thick (FIG. 12), two distinct mantle layers discernible; outer mantle layer pseudoparenchymatous, hyphae tangentially 7.3–27 µm, radially 5–10 µm; inner mantle layer plectenchymatous, hyphae tangentially 6–11 µm, radially 2–7 µm; mantle of very tip 30–35 µm. Tannin cells lacking. Epidermal cells roundish to rectangular, parallel to root axis. Cortical cells roundish to cylindrical, tangentially 25–47 µm, radially 11–22 µm. Hartig net paraepidermal, in plan view infrequently lobed with 3.5–6 µm wide lobes.
Color reaction with different reagents: whole ectomycorrhizae bleaching with 15% KOH, lactic acid, NH4OH and ethanol 70%. Sulpho-vanillin: walls red, contents of mantle cells with droplets becoming black. Emanating hyphae stain slightly blue with cotton blue; no reaction with Melzer’s reagent.
Specimens examined.. ARGENTINA. Catamarca. Sierra de Narváez. 27° 43'S, 65° 4'W, elevation 1820 m. Ectomycorrhizae under A. acuminata, 20 Mar 2002, A. Becerra 09 (CORD); Catamarca. Sierra de Narváez. 27° 43'S, 65° 54'W, elevation 1820 m. Fruitbodies under A. acuminata, 7 May 2000, G. Daniele 188 (CORD), 27 May 2001, G. Daniele 216 (CORD), 20 Mar 2002, G. Daniele 222 (CORD).
Cortinarius tucumanensis Mos. FIGS. 13–19
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). Surface of unramified ends cottony to densely cottony, covered with soil particles. Root tips blunt, covered by the mantle. Mantle not transparent; mycorrhizae not carbonizing. Rhizomorphs infrequent, up to 150 µm diam, concolorous to mantle, without specific origin, connection to the mantle fan-like, repeatedly ramified into smaller filaments with fan-like margins.
Anatomical characters of mantle in plan views: mantle continuous over the root apex. Blue granules and a matrix lacking. Outer mantle layers plectenchymatous with irregularly arranged hyphae, without any special pattern (type B, Agerer and Rambold 1998) (FIG. 14); hyphae undifferentiated, cylindric and slightly constricted at septa, colorless, 3–5 µm diam, thin-walled (<0.5 µm), always with clamps, anastomoses open (type A, Agerer 1991), septa as thick as walls. Inner mantle layer, plectenchymatous to pseudoparenchymatous without discernible pattern; inflated cells colorless, 6–15 µm diam, without clamps (FIG. 15).
Anatomical characters of emanating elements: rhizomorphs up to 150 µm diam (FIG. 16), undifferentiated; hyphae rather loosely woven and of uniform diameter (type A; Agerer and Rambold 1998), central and peripheral hyphae not specialized to irregularly sinuous, 3–5 µm diam, colorless cell walls up to 0.5 µm, septa with clamps as thick as hypha, surface of peripheral hyphae smooth and with soil particles. Emanating hyphae smooth, straight to wavy, 3.2–8 µm diam, slightly constricted at septa; cell walls thin (<0.5 µm), colorless, in few cases slightly thick-walled, with soil particles (FIG. 17), elbow-like protrusions lacking; distal ends of hyphae simple; septal distances 3–12 µm; septa with backward-oriented clamps, clamps in lateral view thinner than the hypha; secondary septa lacking; anastomoses open with a short bridge or bridge almost lacking, anastomosal bridge as thick as hyphae, with cell walls as thick as remaining walls.
Anatomical characters in longitudinal section: mantle 62–160 µm thick (FIG. 18), two distinct mantle layers discernible; outer mantle layer loosely plectenchymatous; hyphae tangentially 23–78 µm, radially 3–7 µm; inner mantle layer pseudoparenchymatous, hyphae tangentially 7–27 µm, radially 4–20 µm; mantle of very tip 30–35 µm. Tannin cells lacking. Epidermal cells rectangular, parallel to root axis. Cortical cells roundish to cylindrical, tangentially 30–45 µm, radially 13–45 µm. Hartig net periepidermal, rarely paraepidermal, in plan view of palmetti type with 1.5–4 µm wide lobes (FIG. 19); 1–2 rows around epidermal cells, roundish in cross-section.
Color reaction with different reagents: whole ectomycorrhizae dark with 15% KOH. Lactic acid: orange. Emanating hyphae stain slightly blue with cotton blue; tips of unramified ends stain black with NH4OH; no reaction with Melzer’s reagent, sulphovanillin and ethanol 70%.
Specimens examined.. ARGENTINA. Tucumán. Quebrada del Portugués. Tafídel Valle, 26° 58'S, 65° 45'W, elevation 2187 m. Ectomycorrhizae under A. acuminata, 19 Mar 2002, A. Becerra 02 (CORD); Tucumán. Quebrada del Portugués. Tafídel Valle, 26° 58'S, 65° 45'W, elevation 2187 m. Fruitbodies under A. acuminata. 19 Mar 2002, G. Daniele 221 (CORD). Catamarca. Sierra de Narváez. 27° 43'S, 65° 54'W, elevation 1820 m, 17 Feb 2000, G. Daniele 206 (CORD).
DNA analysis and restriction fragment patterns.— –
Single ITS-PCR products were obtained for all three species, with an approximate ITS length of 700 bp for Lactarius aff. omphaliformis and Russula alnijorullensis, and of 600 bp for C. tucumanensis (TABLE I). PCR/RFLP profiles revealed similar restriction fragment patterns for the three described anatomotypes with fruitbodies of L. aff. omphaliformis, R. alnijorullensis and C. tucumanensis, allowing the identification of these ECM (TABLE I).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ectomycorrhizal diversity is low in alder forests (Molina 1979, 1981; Gardner and Barrueco 1999). As other Alnus species, A. acuminata is associated with a number of ECM fungi belonging to the genera Alpova, Cortinarius, Gyrodon, Inocybe, Laccaria, Lactarius, Naucoria and Russula (Singer and Morello 1960; Raithelhuber 1988; Moser 2001; Becerra et al 2002, 2005a, Becerra et al b; Nouhra et al in press). ECM described within the present study belong to some of the genera known from fruitbodies commonly associated with A. acuminata.
L. aff. omphaliformis ectomycorrhizae on A. acuminata showed high similarity to L. obscuratus on A. rubra (Miller et al 1991) and to L. obscuratus and L. omphaliformis ectomycorrhizae on A. glutinosa (Pritsch et al 1997b) but also differed in some anatomical features. The pseudoparenchymatous mantle structure of L. aff. omphaliformis ECM differed from the plectenchymatous mantle of L. obscuratus on A. rubra (Miller et al 1991) and from the intermediate mantle type between compact plectenchymatous to irregularly pseudoparenchymatous of L. obscuratus and L. omphaliformis ectomycorrhizae on A. glutinosa (Pritsch et al 1997b).
Within the genus Russula, R. alnijorullensis belongs to a group of species with an angular pseudoparenchymatous mantle structure such as R. fellea (= Fagirhiza granulosa) on Fagus sylvatica (Brand and Agerer 1988), R. mairei on F. sylvatica (Brand 1991), R. ochroleuca on Picea abies (Agerer 1986, Gronbach 1988), R. pumila on A. glutinosa (Pritsch et al 1997b) and R. alnetorum on A. viridis (Beenken 2001). R. alnijorullensis on A. acuminata differed from other Russula ECM on Alnus: It did not exhibit hyaline, grouped dead cells of variable irregular shape on the outer mantle layer as R. pumila on A. glutinosa (Pritsch et al 1997b) or highly organized rhizomorphs and three mantle layers as R. alnetorum on A. viridis (Beenken 2001).
Cortinarius tucumanensis ECM showed similar features to C. bibulus ECM on A. rubra (Miller et al 1991) and Alnirhiza violacea on A. glutinosa (Pritsch et al 1997b), such as color (whitish) and two mantle layers (loosely plectenchymatous outer layer and a compactly arranged inner layer). C. tucumanensis ECM differed from C. cf. alneus (mantle 20–50 µm) and C. cf. hevelloides (mantle < 150 µm), both on A. glutinosa (Pritsch et al 1997b), in that these show a mycorrhizal system with simple ramification, and plectenchymatous inner and outer mantle layers. Other Cortinarius ECM can be discriminated from C. tucumanensis ECM by a completely plectenchymatous mantle, differing mantle thickness or ramification of mycorrhizal systems: C. cf. saturninus on A. tenuifolia, mantle up to 300 µm and monopodial ramification (Brunner et al 1990); C. atropusillus on A. viridis, mantle up to 120 µm and simple ramification (Wiedmer and Senn-Irlet 1999b); C. badiovestitus on A. viridis, mantle up to 160 µm and simple to irregularly pinnate ramification (Wiedmer and Senn-Irlet 1999c); C. kühneri and C. alnobetulae, both on A. viridis, mantle up to 80 and 95 µm respectively, and simple to irregularly monopodial-pinnate ramification (Wiedmer and Senn-Irlet 1999d, a respectively); C. helvelloides mantle up to 100 µm and C. pluvius, mantle up to 180 µm on A. viridis, both lacking ramification (Wiedmer and Senn-Irlet 2001b, 1998 respectively); C. bibulus on A. viridis with a mantle up to 90 µm and simple ramification (Wiedmer and Senn-Irlet 2001a). C. tucumanensis ECM also differ from C. helodes on A. acuminata (Becerra et al 2005a) with the latter forming a three layered mantle (inner pseudoparenchymatous, middle and outer plectenchymatous) of up to 200 µm thickness and a simply ramified mycorrhizal system.
ITS-PCR/RFLP analyses with the three endonucleases TaqI, HinfI and EcoRI let us identify and distinguish three anatomically distinct ectomycorrhizae, thus supporting the anatomical observations. The approximate band size estimates based on agarose gels resulted in similar values as observed in other studies. With an ITS length of ca 600 bp of C. tucumanensis, similar ITS lengths of 610 bp were obtained by Pritsch et al (1997a) for Cortinarius ectomycorrhizae. ITS lengths of approximately 700 bp of R. alnijorullensis and L. aff. omphaliformis similar to 710 bp of ITS for Russulaceae ectomycorrhizae that have been published by Pritsch et al (1997a).
The anatomical and molecular characterization of L. aff. omphaliformis, R. alnijorullensis and C. tucumanensis ECM reported here contribute to a more detailed knowledge on the symbionts of A. acuminata in native Argentinean forests.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: abecerra{at}imbiv.unc.edu.ar
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LITERATURE CITED |
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———. 1991. Characterization of ectomycorrhiza. In: Norris JR, Read DJ, Varma AK, eds. Methods in Microbiology 23. Techniques for the study of mycorrhiza. London: Academic Press. p 25–73.
———, Waller K, Treu R. 1993. Die Ektomykorrhizen und Sklerotien von Gyrodon lividus. Z Mykol 59:131–140.
———, Rambold G. 1998. DEEMY V 1.1-A Delta based system for characterization and Determination of EctoMYcorrhizae. Institute for Systematic Botany, Section Mycology, University München.
———. 1999. Anatomical characteristics of identified ectomycorrhizas: an attempt toward a natural classification. In: Varma AK, Hock B, eds. Mycorrhiza. Structure, function, molecular biology and biotechnology. 2nd ed. New York: Springer, Berlin Heidelberg. p 633–682.
Airaudi D, Filipello Marchisio V, Luppi Mosca AM. 1994. Ectomycorrhiza types in Alnus viridis (Chaix) DC. Allionia 32:65–78.
Becerra AG. 2002. Influencia de los suelos Ustorthentes sobre las ectomicorrizas y endomicorrizas en Alnus acuminata H.B.K [Master’s dissertation]. Fac. de Agronomía: Buenos Aires Univ. 190 p.
———, Daniele G, Domínguez L, Nouhra E, Horton T. 2002. Ectomycorrhizae between Alnus acuminata H.B.K. and Naucoria escharoides (Fr.:Fr.) Kummer from Argentina. Mycorrhiza 12:61–66.[Medline]
———, Nouhra E, Daniele G, Domínguez L, McKay D. 2005a. Ectomycorrhizas of Cortinarius helodes and Gyrodon monticola with Alnus acuminata from Argentina. Mycorrhiza 15:7–15.[CrossRef][Medline]
———, Pritsch K, Arrigo N, Palma M, Bartoloni N. 2005b. Ectomycorrhizal colonization of Alnus acuminata Kunth in northwestern Argentina in relation to season and soil parameters. Ann For Sci 62:325–332.[CrossRef]
Beenken L. 2001. Russula alnetorum Romagn. + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 5:115–123.
Brand F. 1991. Ektomykorrhizan an Fagus sylvatica. Charakterisierung und Identifizierung, ökologische Kennzeichnung und unsterile Kultivierung. Libri Botanici. Vol 2, IHW, Eching. Germany.
———, Agerer R. 1988. Studien an Ektomykorrhizen. XIII. Drei häufige Ektomykorrhizen der Buche (Fagus sylvatica L.). Charakterisierung und unsterile Kultivierung von Buchenektomykorrhizen. Sydowia 40:1–37.
Brunner IL, Brunner F, Miller Jr OK. 1990. Ectomycorrhizal synthesis with Alaskan Alnus tenuifolia. Can J Bot 68:761–767.
———, ———, Laursen GA. 1992. Characterization and comparison of macrofungal communities in an Alnus tenuifolia and an Alnus crispa forest in Alaska. Can J Bot 70:1247–1258.[CrossRef]
Dawson JO. 1990. Interactions among actinorhizal and associated plant species. In: Schwintzer CR, Tjepkema JD, eds. The biology of Frankia and actinorhizal plants. San Diego, California: Academic Press. p 299–316.
Froidevaux L. 1973. The ectomycorrhizal association, Alnus rubra + Lactarius obscuratus. Can J For Res 3:601–603.[CrossRef]
Gardner IC, Barrueco CR. 1999. Mycorrhizal and actinorhizal biotechnology-problems and prospects. In: Varma AK, Hock B, eds. Mycorrhiza: structure, function, molecular biology and biotechnology, 2nd ed. New York: Springer, Berlin Heidelberg. p 469–495.
Godbout C, Fortin JA. 1983. Morphological features of synthesized ectomycorrhizae of Alnus crispa and Alnus rugosa. New Phytol 94:249–262.[CrossRef]
———, ———. 1985. Synthesized ectomycorrhizae of aspen: fungal genus level of structural characterization. Can J Bot 63:601–603.
Gronbach E. 1998. Charakterisierung und Identifizierung von Ektomykorrhizaen in einem Fichtenbestand mit Untersuchung zur Merkmalsvariabilität in sauer beregneten Flächen. Bibl Mycol 125:1–216.
Henrion B, Chevalier G, Martin F. 1994. Typing truffle species by PCR amplification of the ribosomal DNA spacers. Mycol Res 98:37–43.
Holmgren PK, Holmgren NH, Barnett LC. 1990. Index Herbariorum. I: the herbaria of the world. Regnum Veg 120:1–693.
Horak VE. 1963. Pilzökologische Untersuchungen in den subalpinen Stufen (Picetum subalpinen and Rhodoreto vaccinietum) der rätischen Alpen. Mitte Schweiz Anst Forstl Versuchswes 39:1–112.
Massicotte HB, Peterson RL, Ackerley CA, Piché Y. 1986. Structure and ontogeny of Alnus crispa + Alpova diplophloeus ectomycorrhizae. Can J Bot 64:177–192.
———, Peterson RL, Melville LH. 1989. Ontogeny of Alnus rubra-Alpova diplophloeus ectomycorrhizae. I. Light microscopy and scanning electron microscopy. Can J Bot 67:191–200.
———, Molina R, Luoma DL, Smith JE. 1994. Biology of the ectomycorrhizal genus Rhizopogon. II. Patterns of hostfungus specificity following spore inoculation of diverse hosts grown in mono- and dual culture. New Pähytol 126:677–690.[CrossRef]
———, Melville LH, Peterson RL, Unestam T. 1999. Comparative studies of ectomycorrhiza formation in Alnus glutinosa and Pinus resinosa with Paxillus involutus. Mycorrhiza 8:229–240.
Masui K. 1926. A study of the ectotrophic mycorrhiza of Alnus. Mem Coll Sci, Kyoto Imp Univ. Ser B 2:189–209.
Mejstrik V, Benecke U. 1969. The ectotrophic mycorrhizas of Alnus viridis (Chaix) DC. and their significance in respect to phosphorus uptake. New Phytol 68:141–149.[CrossRef]
Miller SL, Koo CD, Molina R. 1988. An oxidative blue-bruising reaction in Alpova diplophloeus (Basidiomycetes, Rhizopogonaceae) + Alnus rubra ectomycorrhizae. Mycologia 80:576–581.[CrossRef]
———, ———, ———. 1991. Characterization of red alder ectomycorrhizae: a preface to monitoring belowground ecological responses. Can J Bot 69: 516–531.
———, ———, ———. 1992. Early colonization of red alder and Douglas-fir by ectomycorrhizal fungi and Frankia in soils from the Oregon coast range. Mycorrhiza 2:53–61.
Molina R. 1979. Pure culture synthesis and host specificity of red alder mycorrhizae. Can J Bot 57:1223–1228.[CrossRef]
———. 1981. Ectomycorrhizal specificity in the genus Alnus. Can J Bot 59:325–334.
———, Myrold D, Li CY. 1994. Root symbioses of Red Alder: technological opportunities for enhanced regeneration and soil improvement. In: Hibbs DE, DeBell DS, Tarrant RF, eds. The biology and management of red alder. Oregon State University Press. p 23–46.
Moser M, Horak E. 1975. Cortinarius Fr. Und verwandte Gattungen in Südamerika. Nova Hedw Beih 52, 628 p, 167 Taf 20 col. Tf.
———. 1978. Keys to Agarics and Boleti. Kibby G, ed. 535 p.
———. 2001. Some aspects of Cortinarius associated with Alnus. Journal des JEC 3:47–101.
Murphy JF, Miller OK Jr. 1994. Mycorrhizal syntheses with Alnus serrulata (Ait) Willd. Castanea 59:156–166.
Neal JL, Trappe JM, Lu KC, Bollen WB. 1968. Some ectotrophic mycorrhizae of Alnus rubra. In: Trappe JM, Franklin JF, Tarrant RF, Hansen GM, eds. Biology of alder. Pacific Northwest Forest and Range Experiment Station Forest Service. Portland, Oregon: USDA. p 179–184.
Nouhra E , Domínguez L, Becerra A, Trappe J. Morphology, molecular analysis and ecological aspects of the South American hypogeous fungus, Alpova austroalnicola sp. nov. Mycologia 97:598–604.
Pritsch K, Boyle H, Munch JC, Buscot F. 1997a. Characterization and identification of black alder ectomycorrhizas by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS). New Phytol 137:357–369.[CrossRef]
———, Munch JC, Buscot F. 1997b. Morphological and anatomical characterisation of black alder Alnus glutinosa (L.) Gaertn. ectomycorrhizas. Mycorrhiza 7:201–216.
Raithelhuber J. 1988. Flora Mycologica Argentina. Hongos II. MYCOSUR, ed. 1–287.
Singer R, Digilio APL. 1951. Prodromo de la Flora Agaricina Argentina. Lilloa 25:5–61.
———, Morello JH. 1960. Ectotrophic forest tree mycorrhizae and forest communities. Ecol 41:549–551.[CrossRef]
Stangl J. 1970. Das Pilzwachstum in alluvialen Schotterebenen und seine Abhängigkeit von Vegetationsge-sellschaften. Z Pilzkd 31:209–255.
Trappe JM. 1962. Fungus associates of ectotrophic mycorrhizae. Bot Rev 28:538–606.[CrossRef]
Treu R. 1990. Charakterisierung und Identifizierung von Ektomykorrhizen aus dem Nationalpark Berchtesgaden. Bibl Mycol 134:1–196.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification anddirect sequencing of fungal ribosomal RNA genes-for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, eds. PCR Protocols: a guide to methods and applications. New York: Academic Press. p 315–322.
Wiedmer E, Senn- Irlet B. 1998. Cortinarius pluvius (Fr.) Fr. + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 3:31–36.
———, ———. 1999a. Cortinarius alnobetulae Kühn + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 4:7–12.
———, ———. 1999b. Cortinarius atropusillus Frave + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 4:13–18.
———, ———. 1999c. Cortinarius basidiovestitus Mos. + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 4:19–23.
———, ———. 1999d. Cortinarius kühneri Mos. + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 4:25–29.
———, ———. 2001a. Cortinarius bibulus Quél. + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 5:23–27.
———, ———. 2001b. Cortinarius helvelloides (Fr.) Fr. + Alnus viridis (Chaix) DC. Descriptions of ectomycorrhizae 5:29–34.
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). 3. Middle mantle layer, plectenchymatous to pseudoparenchymatous, without discernible pattern. 4. Inner mantle layer, plectenchymatous without pattern. 5. Middle layer, straight laticifers. 6. Longitudinal section of the mantle with three distinct layers; outer mantle layer pseudoparenchymatous (om); middle mantle layer plectenchymatous to pseudoparenchymatous(mm); inner mantle layer plectenchymatous (im); epidermal cells rectangular (ec). Bars: 1 = 0.5 mm, 2–6 = 10 µm.
