516 Characterization of red alder ectomycorrhizae: a preface to monitoring belowground ecological responses STEVEN L. M ILLER AND C. D. Koo Department of Forest Science, Oregon State University, Corvallis, OR 97331, U.S.A. AND R ANDY MOLINA United States Department of Agriculture, Forest Service, Pacific Northwest Research Station, Forestry Sciences Laboratory, 3200 Jefferson Way, Corvallis, OR 97331, U.S.A. Received May 2, 1990 M ILLER, S. L., Koo, C. D., and M OLINA, R. 1991. Characterization of red alder ectomycorrhizae: a preface to monitoring belowground ecological responses. Can. J. Bot. 69: 516-531. Critical ecological research on belowground ecosystems has often been impeded because of the inability to adequately recognize ectomycorrhizal relationships, especially the abundance, diversity, and distribution of the fungus component, and the specificity of particular fungus—host combinations. Red alder, with its high degree of host specificity and paucity of fungal symbionts, provides an ideal model for studying these attributes. Eleven morphologically recognizable types of ecto- mycorrhizae were characterized from field-collected root material, greenhouse soil bioassays, and laboratory syntheses. Most mycobionts were basidiomycetes, as evidenced by abundant clamp connections present in the mantle and extramatrical hyphae. Seven mycobionts identified to species included Alpova diplophloeus, Thelephora terrestris, Lactarius obscuratus, Cortinarius bibulus, Laccaria laccata, Hebeloma crustuliniforme, and Paxillus involutus. Many of the ectomycorrhizae collected in the field appeared to have more than one mycobiont present in the mantle. Root tips could generally be categorized into either flexuous or succulent morphological types. The flexuous types were long, thin, indeterminate in growth, with an acute root apex, and the mantle and Hartig net in longitudinal section were not well formed near the root apex. The succulent types were short, thick, determinate in growth, with a rounded root apex, and the mantle and Hartig net in longitudinal section were well formed near the root apex. Additional characteristics important in distinguishing among red alder ecto- mycorrhizal types included color, extent of extramatrical hyphae development, mantle surface characteristics, and selected microchemical reactions. Mantle thickness was highly variable and not useful in characterization. Hartig net development was shallow, and regardless of mycorrhizal origin, rarely extended beyond one epidermal cell layer. Key words: ectomycorrhizae, Alnus, characterization, ecology, belowground. M ILLER, S. L., Koo, C. D., et MOLINA, R. 1991. Characterization of red alder ectomycorrhizae: a preface to monitoring belowground ecological responses. Can. J. Bot. 69 : 516-531. La recherche ecologique critique sur les ecosystemes souterrains a souvent ete limit par lincapacite a reconnaitre de fawn valable les relations ectomycorhiziennes, en particulier, labondance, la diversite et la distribution de la composante fongique ainsi que la specificite des combinaisons hOtes—champignons particulieres. Laulne rouge, avec une forte specificite de lhOte et une pauvrete en espêces fongiques, constitue un modele ideal pour (etude de ces proprietes. A partir de materiel racinaire recolte aux champs, dessais en serre et de syntheses au laboratoire, les auteurs ont caracterisd 11 types decto- mycorhizes reconnaissables morphologiquement. La plupart des mycobiontes sont des basidiomycetes, tel que demontre par la presence dabondantes boucles danastomose dans les hyphes des manchons et du mycelium extrammatriciel. On reconnait sept espêces de mycobiontes incluant Alpova diplophloeus, Thelephora terrestris, Lactarius obscuratus, Cortinarius bibulus, Laccaria laccata, Hebeloma crustiliniforme et Paxillus involutus. Plusieurs des mycorhizes recoltees sur le terrain semblent contenir plus dune espece de champignon symbiotique dans leur manchon. Les extremites racinaires peuvent habituellement être attribuees aux types morphologiques flexueux ou succulents. Les mycorhizes de types flexueux sont longues, minces, de croissance indeterminee, pourvues dun apex pointu et montrent en section longitudinale un manchon et un reseau de Hartig pas tres bien formes pres de lapex. Les mycorhizes de types succulents sont courtes, epaisses, de croissance deter- minee, pourvues dun apex racinaire arrondi et montrent en section longitudinale un manchon et un reseau de Hartig bien developpes jusquau voisinage de lapex racinaire. Parmi les autres caracteristiques importantes permettant de distinguer les types ectomycorhiziens de laulne, on retrouve la couleur, létendue du developpement de la phase extramatricielle, les caracteres superficiels du manchon et des reactions microchimiques selectionnees. Lepaisseur du manchon varie fortement et ne constitue pas un caractere tres utile. Les developpement du reaseau de Hartig est peu profond et, quel que soft lorigine mycorhizienne, ne setend que rarement au dela dune couche de cellule ápidermique. Mots des : ectomycorhizes, Alnus, caracterisation, ecologie, souterrain. [Traduit par la redaction] Introduction research has begun to emphasize the ecological importance of ectomycorrhizae and the mycobionts in belowground eco- Ectomycorrhizae are well known for improving survival and system processes and plant community dynamics. However, growth of forest trees. Consequently, much research has con- our inability to adequately identify or recognize mycobionts centrated on practical applications in forestry. Recently, and their ectomycorrhizae continues to hamper efforts to understand the individual contributions of diverse symbionts Present address: Botany Department. University of Wyoming. Laramie, WY 82071, U.S.A. in these ecological processes. Printed in Canada / Imprime au Canada MILLER ET AL 517 Often, the sheer number of ectomycorrhizal fungi or the growth-pouch technique (Fortin et al. 1980) were used for laboratory nonspecificity of the mycobionts makes a particular host ill- syntheses. suited for characterization of all or nearly all mycorrhizal types Ectomycorrhizae from the field and tube syntheses were carefully in a given locality. The study of red alder (Alnus rubra Bong.), washed to remove adherant soil particles. All ectomycorrhizae were on the other hand, provides a unique opportunity to evaluate described fresh. Representative ectomycorrhizal rootlets were then fixed overnight in 8% glutaraldehyde in 0.2 M phosphate buffer at a limited number of ectomycorrhizal fungi, many with a high pH 6.8 and washed three times with the same buffer. A subsample degree of host specificity. Less than 30 ectomycorrhizal fungi of rootlets was dehydrated in a 10% acetone series and flat embedded have been reported in association with alder worldwide (Neal in glycol-methacrylate resin (Historesin, available from LKB, et al. 1968; Trappe 1962; Mejstrik and Benecke 1969). In Bromma, Sweden). Ectomycorrhizae were then thick sectioned, both addition, Molina (1979, 1981) found that only 19% of all fungi in longitudinal and cross-sectional orientation, with a glass knife, tested could form ectomycorrhizae with A. rubra. Godbout stained in 1% Toluidene Blue, and anatomically characterized. Char- and Fortin (1983) found less than 22% could form mycorrhizae acterization of the Hartig net as periepidermic (the Hartig net com- with Alnus crispa or Alnus glutinosa. The ability to recognize pletely circumscribing entire epidermal cells) or paraepidermal (pen- so few fungal species and their ectomycorrhizae makes red etrating only to the depth of the transverse walls of the epidermal cell) was taken from Godbout and Fortin (1983). alder a prime choice for studying mycorrhizal and mycobiont Tissues in the various levels of the mantle and Hartig net were response to environmental manipulation. described from longitudinally tangential sections from the approxi- Several recent publications characterized ectomycorrhizae mate center of one or more layers of the mantle or scalp sections by using a variety of sophisticated techniques or special equip- taken tangentially at the interface of the Hartig net and the cortical ment such as Normarsky interference contrast microscopy, cells, as appropriate, and from crushes of fresh or fixed roots. Roots autofluorescence of hyphal wall material, scanning electron were mounted in fresh sulfovanillin reagent composed of one drop of microscopy, and a multitude of macrochemical reagents distilled water, five drops of sulfuric acid, and sufficient vanillin crys- (Agerer 1986, 1987a, 1987b, 1988; Agerer and Weiss 1989; tals to turn the solution yellow (Singer 1962). The sulfovanillin soft- ened the root tips for uniform crushing and stained the fungal material Brand and Agerer 1987; Grenville et al. 1985; Massicotte et al. in the mantle and Hartig net pale red and the laticiferous hyphae and 1987; Melville et al. 1987a) but there is no accepted, stand- dermatocystidia of type 3 mycorrhizae blackish. Tissue-type termi- ardized format. Because of the large number of ectomycor- nology in the mantle and Hartig net was adapted from Miller (1971). rhizae that must be examined in any ecological study and The descriptions reflect characteristics of both field-collected because many researchers do not have access to equipment and mycorrhizae and laboratory-synthesized mycorrhizae where appro- manpower necessary to analyze each root tip with precision, priate. The ink drawings were made directly from camera lucida a relatively simple yet accurate