516 Characterization of red 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 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 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 . 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.

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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 . 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 method is needed. tracings of cross, longitudinal, and scalp sections of representative This paper characterizes mycorrhizae encountered on red plastic-imbedded material for each mycorrhizal type. alder in the field, greenhouse soil bioassays, and laboratory syntheses in the Pacific northwestern United States over a Results and discussion 3-year period. The techniques used are relatively simple and Initially, 14 morphological types of ectomycorrhizae on red sufficiently distinguish ectomycorrhizae on alder in this region alder were described on the basis of macroscopic features so that additional ecological information may be procured. (Miller et al. 1987b). Subsequent laboratory syntheses, micro- Fungal sporocarps collected at the same time allowed identi- scopic analysis, evaluation of consistent features, and match- fication of several of the mycobionts. ing of field-collected sporocarps with associated mycorrhizae led to characterization of 11 recognizable types (Table 1). Sev- Materials and methods eral types were combined because they represented develop- mental stages of a single ectomycorrhizal type. Several times throughout the growing season 5- to 10-m sections The majority of fungal symbionts were basidiomycetes, as of red alder roots were excavated from approximately 20 sites in the Coast and Cascade ranges of Oregon, Washington, and northern Cal- evidenced by the abundant clamp connections present in the ifornia. Roots and soil were kept moist and cool in plastic bags until mantle and extramatrical hyphae. Several mycobionts respon- examined, usually within 12 h. Only roots with attached Frankia sible for particular ectomycorrhizal types on red alder were nodules were used in the study to insure that roots and mycorrhizae identified and include A. diplophloeus (Zeller Dodge) originated from red alder. Similarly, roots from red alder seedlings Trappe Smith, L. obscuratus (Lasch.) Fr., C. bibulus Quel., growing in a greenhouse soil bioassay for actinorhizal and ectomy- T. terrestris (Ehrh.) Fr., P. involutus (Fr.) Fr., and L. laccata corrhizal fungal propagules from nearby alder forests (Miller et al. (Scop.: Fr.) Cke. Other ectomycorrhizal types were suspected 1987a) and from red alder seedlings planted in the field were collected of being formed by Russula sp., Naucoria spp., lnocybe sp., and examined. and Hebeloma sp., but sporocarps were not encountered in the Red alder ectomycorrhizae were also characterized from routine field. It was impossible, therefore, to obtain cultures of these laboratory synthesis using isolates of putative ectomycorrhizal fungi fungi that could be used to effect resynthesis. Other ectomy- found in the field (SLM) and obtained from culture collections at the corrhizal types were readily recognizable, but the mycobiont Forestry Sciences Laboratory, Corvallis, OR, U.S.A. (FSL), at Laval was unknown. Table 1 summarizes the 11 most common ecto- University, Quebec, Canada (CRBF), and from Van Cotter, Virginia mycorrhizal types observed from field-collected roots. Polytechnic Institute and State University, Blacksburg, VA. U.S.A. (VC). Isolates used in this study included Alpova diplophloeus S-276 The root tips could generally be categorized into two dif- (FSL), SLM 881; Lactarius obscuratus SLM 835; Hebeloma crus- ferent morphological types: flexuous (Figs. 1-3, 11-13) and tuliniforme S-166 (FSL); Cortinarius bibulus SLM 1030; Laccaria succulent (Figs. 4-10), although there was overlap in these laccata SLM 1055; Thelephora terrestris S-142 (FSL); Cenococcum morphological designations. The flexuous tips were long, thin, geophilum A-145 (FSL), 0093 (CRBF); Paxillus involutus SLM 830 seemingly indeterminate in growth, and usually had an acute and Paxillus cf. filamentosus (VC 1092). The pure culture tube syn- root apex. In cross section the mantle was thin and the Hartig thesis technique described by Molina and Palmer (1982) and the net was paraepidermal and usually did not completely pene- 518 CAN. J. BOT. VOL. 69, 1991

TABLE 1. Morphological types of red alder ectomycorrhizae, their identification, origin and frequency of occurrence in the field Soil bioassays Laboratory synthesis Field Morphological types collected Greenhouse Plantation Tube Growth pouch Type 1 ++ x x Alpova diplophloeus -1- + x x x x Lactarius obscuratus x x Thelephora terrestris x x x x Cortinarius bibulus x Type 3 Type 2 x Type 4 Laccaria laccata x Hebeloma crustulitziforme x Paxillus involutus x Indicates relative abundance in field-collected samples of red alder roots. irate to a depth of one cell. In longitudinal section neither the culent mycorrhizae the Hartig net and mantle became well mantle nor Hartig net were well developed at the root apex. developed at the apex and suppressed continuous root growth. The flexuous mycorrhizae were formed by fungi with a broad Features of primary importance in distinguishing among red host range, such as T. terrestris, H. crustuliniforme, and alder mycorrhizal types included mantle and mycorrhiza color, P. involutus. bruising reactions of the fungal tissue or mycorrhiza, extent The succulent tips were short, thick, mostly determinate in and type of extramatrical hyphae development, mantle surface growth, and usually had a rounded root apex. In cross section characteristics, and selected microchemical reactions. The the mantle was either thin or thick and the Hartig net was either array of chemical reagents utilized by Froidevaux (1973) in paraepidermal or occasionally periepidermic. In longitudinal describing red alder mycorrhizae, and by others (summarized section the mantle and Hartig net were typically well devel- by Agerer 1986) as an aid in describing ectomycorrhizae, was oped near the root apex. The succulent ectomycorrhizae were largely unusable because some reagents such as 15% KOH and primarily formed by taxa specific to alder such as A. diplo- 10% FeSO4 caused pronounced color reactions even on non- phloeus and L. obscuratus. mycorrhizal control roots. Sulfovanillin was useful when the The flexuous types of mycorrhizae, both in the field and in mycobiont belonged to a particular taxonomic group, such as laboratory syntheses, were consistently characterized by expo- members of the Russulaceae (Singer 1962). sure of a nonmycorrhizal root apex. Nonmycorrhizal root-tip Mantle thickness was variable between field, greenhouse, exposure of an otherwise ectomycorrhizal root was only infre- and laboratory synthesized mycorrhizae, but also between quently observed in succulent mycorrhizae. Melville et al. rootlets of the same mycorrhizal type from a single locality or (1987a) described a similar phenomenon as an early stage in origin, and could not be reliably used as a stable characteristic. Dryas the ontogeny of ectomycorrhizae synthesized between Hartig net development, regardless of the origin of the mycor- integnfolia and Hebeloma cylindrosporum, which is most rhiza, was mostly peraepidermal and rarely periepidermal. similar to the succulent types of mycorrhizae in our study. This et al. was followed by additional production of cortical-cell layers Neal (1968) and Godbout and Fortin (1983) suggested that increased girth in the most well-developed mycorrhizae. that a shallow Hartig net is characteristic of A. rubra as well Longitudinal sections of red alder mycorrhizal root tips showed as other Alnus species; however, deeper paraepidermal Hartig greater development of mantle and Hartig net surrounding the net penetration was noted in A. tenuifolia and A. crispa by root apex in the succulent types than in the flexuous types, Brunner et al. (1988) and in A. crispa by Massicotte et al. although no demonstrable increase in the number of cortical- (1987). cell layers was observed. The flexuous type of mycorrhizae Many of the field-collected ectomycorrhizal types appeared may have resulted from host—mycobiont interactions that pre- to have more than one mycobiont present in the mantle. Sim- vented formation of the Hartig net and mantle near the root ilar multiple mycobiont mycorrhizae were reported by Marks apex, allowing continuous growth of the root, whereas in suc- and Foster (1967).

Tentative dichotomous key to individual ectomycorrhizal types from the Pacific Northwest I. Fresh, nonsenescent ectomycorrhizae predominantly white, creamy white, or at most pale greyish white throughout entire devel- opment 2 I. Fresh, nonsenescent ectomycorrhizae with definable color other than white, such as yellow, brown, orange, or red 5 2. Mantle surface smooth type 3 2. Mantle surface with abundant silky to wooly hyphae 3 3. Hyphae of mantle surface cylindrical to irregularly swollen, with abundant, blunted or slightly tapered cystidia; hyphal strands usually abundant and large (30-60 p.m in diameter) Thelephora type 3. Hyphae of mantle surface cylindrical, with few swollen hyphae and lacking blunted hyphal end cells; hyphal strands lacking or small (15 1.1,m in diameter) 4 MILLER ET AL 519 4. Ectomycorrhizae bruising brown where broken. with scattered to abundant intercalary and terminal cells filled with amor- phous, yellow, refractive material Paxillus type 4. Ectomycorrhizae not bruising brown where broken, hyphae with refractive yellow material lacking Hebeloma type 5. Fresh, nonsenescent ectomycorrhizae in part or wholly colored yellow, orange, or brown 6 5. Fresh, nonsenescent ectomycorrhizae colored some shade of red, pink, or reddish brown 10 6. Ectomycorrhizae flexuous, apex usually tapered to a point, frequently with nonmycorrhizal root apex exposed 7 6. Ectomycorrhizae succulent, apex usually blunted 8 7. Mantle with abundant brown, tapering, erect setaceous cystidia type 2 7. Mantle lacking erect setaceous cells but occasionally with scattered network of dark brown hyphae in older portion of mantle type 1 8. Mantle composed of tightly compacted, large isodiametrical hyphae type 4 8. Mantle composed of cylindrical hyphae 9 9. Ectomycorrhizae yellow brown to brown with scattered network of blue to bluish black hyphae when injured or exposed to the air Alpova diplophloeus type 9. Ectomycorrhizae pale orange to bright orange Lactarius obscurants type 10. Ectomycorrhizae succulent; mantle thick, cottony with abundant extramatrical hyphae; usually brightly colored pinkish, pinkish red, or brownish red Cortinarius bihulus type 10. Ectomycorrhizae flexuous; mantle thin, silky with scattered extramatrical hyphae; usually flushed dull red to dull brownish red Laccaria laccata type

Description of individual ectomycorrhizal types house contaminants and were found several times associated Type I: unknown mycobiont Figs. 1, 14A-14D with sporocarps of a small nodulose-spored Inocybe sp. and a Morphology: root tips monopodial or irregularly compound, Thelephora sp. fruiting in greenhouse containers with red with few or many lateral root tips, (0.5)1-15(20) mm long x alder. The genus Naucoria was another possible mycobiont for 0.2-0.4 mm thick; straight or slightly flexuous; the apices of type 1 mycorrhizae from the field. No isolates could be young tips are acute, usually with a short or long extension of obtained for laboratory confirmation. nonmycorrhizal root apex exposed; surface loosely felted with scattered to abundant, efferent, extramatrical hyphae and Type 2: unknown mycobiont Figs. 2, 15A-15D hyphal-end cells; young tips pallid white to pale brown, with Morphology: root tips monopodial or rarely irregularly com- exposed pink or red nonmycorrhizal root apex, older portions pound with few lateral root tips, 1-7(10) mm long x 0.2- of mantle becoming brown to dark brown; extramatrical hyphae 0.3 mm thick; straight or slightly flexuous; apices of young hyaline or pale brown, with scattered network of dark brown tips acute or subacute, frequently with a short extension of hyphae in mantle. Hyphal strands: not observed or absent. Mantle surface: rarely continuous over the root apex, of pro- nonmycorrhizal root apex exposed; surface loosely felted to synchymatous to loosely interwoven, cylindrical, mostly hya- woolly with scattered, efferent, extramatrical hyphae and line hyphae, 3-8 pm broad, forming a textura intricata, clamp abundant, stiff, erect setaceous cystidia overall or concentrated connections frequent, with long, blunted, hyaline cystidia, 40- near the apex; pale yellowish brown to dark brown depending 70 pm long, dark brown, thick-walled hyphae scattered on the density of setaceous cystidia; extramatrical hyphae hya- throughout older portions of the mantle; extramatrical hyphae line. Hyphal strands: absent or not observed. Mantle surface: scattered or abundant except near the apex, 3-8 pm broad, frequently continuous over the root apex, of prosynchymatous clamp connections frequent. Cross section: mantle 15-25 pm to loosely interwoven, cylindrical, mostly hyaline hyphae, 2- thick, prosynchymatous, composed of uniformly cylindrical, 4 pm broad, forming a textura intricata, with long, straight or repent and efferent hyphae 2-8 gam broad, and blunted hyphal slightly curving, rarely branched, tapered, dark brown, thick- end cells, 40-70 p.m long, irregularly interwoven, mostly hya- walled, setaceous hyphal-end cells, 100-130 p.m long, clamp line, thin-walled, circular to cylindrical in outline; Hartig net connections frequent; extramatrical hyphae scattered or abun- paraepidermal, hyphae isodiametric angular or elongated, dant, 2-6 pm broad, clamp connections frequent. Cross sec- uniseriate or with 2-4 cells thick between epidermal cells, 2- tion: mantle 18-40 p.m thick, prosynchymatous, composed of 6 p.m broad, prosynchymatous; epidermal cells 6-16 p.m uniformly cylindrical, repent and efferent hyphae 3-8 p.m broad, circular in outline. Longitudinal section: mantle similar broad, irregularly interwoven, mostly hyaline, thin walled, cir- in appearance to cross section, but a greater proportion of cular to cylindrical in outline, and tapered, setaceous cystidia, hyphal cells are elongated to cylindrical owing to longitudinal 100-130 p.m long; Hartig net paraepidermal, hyphae isodi- orientation; Hartig net paraepidermal, uniseriate or with 2- ametric angular or elongated, 1-4 cells thick between epider- 4 cells thick between epidermal cells, hyphae isodiametric, mal cells, 2-6 p.m broad, prosynchymatous; epidermal cells angular or elongated, 2-6 pm broad; in scalp section hyphae 8-16 p.m broad, circular in outline. Longitudinal section: isodiametric to angular or irregularly lobed and anastomosed, mantle similar in apearance to cross section; Hartig net par- forming a textura angularis to textura epidermoidea; epidermal aepidermal, typically uniseriate between epidermal cells, cells 35-45 p.m long, oval or elongated in outline, and angled hyphae isodiametric, angular or elongated, 4-18 p.m broad; in slightly periclinally to the axis of the root. scalp section hyphae angular, cylindrical, or irregularly lobed This type of ectomycorrhizae showed a high variability in and anastomosed, forming a textura angularis to textura epi- color, and there are possibly a number of mycobionts that dermoidea; epidermal cells 25-55 p.m long, oval or elongated exhibit a brown, flexuous ectomycorrhiza with pale apex on in outline, and angled slightly periclinally or parallel to the red alder. Type I ectomycorrhizae were common as green- axis of the root. 520 CAN J. ROT. VOL. 69. 1991

Fics. 1-7. Morphological types of ectomycorrhizae on A. rubra. Scale bars = 0.5 mm. Fig. I. Type 1 ectomycorrhiza from the field; mycobiont unknown. Fig. 2. Type 2 ectomycorrhiza collected from the field; mycobiont unknown. Fig. 3. Thelephora terrestris ectomycor- rhiza. Fig. 4. Alpova diplophloeus ectomycorrhiza collected from the field. Figs. 5 and 6. Lactarius obscurants ectomycorrhiza collected from the field. Fig. 7. Type 3 ectomycorrhiza collected from the field; mycobiont unknown.

No mycobiont could be connected with type 2 ectomycor- Alpova diplophloeus + Alnus rubra Figs. 4, 16A-16E rhizae, and they were rarely seen in great abundance in the Morphology: root tips monopodial, pinnate to irregularly field or in the greenhouse. The long, bristly, brown cystidia compound with few or many lateral root tips, (1-)4- protruding from the mantle readily distinguish this type from 15(-25) mm long x (0.3-)0.5-1.0 mm thick; the apices of other ectomycorrhizae on red alder, but it is also possible that young tips are subglobose, becoming cylindrical, slightly these are old or well-developed ectomycorrhizae of another swollen, or tapering toward the tip, if single and simple then mycobiont. tapering back toward the root, apex usually blunted, rarely MILLER ET AL. 521

Fins. 8-13. Morphological types of ectomycorrhizae on A. rubra. Scale bars = 0.5 mm. Fig. 8. Cortinarius bibulus ectomycorrhiza col- lected from the field. Figs. 9 and 10. Type 4 ectomycorrhiza collected from the field; mycobiont unknown. Fig. 11. Laccaria laccata ecto- mycorrhiza collected from the field. Fig. 12. Hebeloma crustuliniforme ectomycorrhiza synthesized in the laboratory. Fig. 13. PartIlus invo- lutus ectomycorrhiza, synthesized in the laboratory. subacute with a short or long extension of nonmycorrhizal root ing from the surface of mycorrhizal tips and portions of roots, apex exposed; surface nearly smooth or with scattered or abun- or present on nomycorrhizal main roots, usually most abundant dant, efferent, extramatrical hyphae and hyphal strands; pale at base of mycorrhizal tips; 15-90 vim broad, 2-10 cells thick, yellow when young becoming pale brown, golden brown or regularly and evenly interwoven or knobby and with scattered red-brown, often darkest near the base, in age uniformly dark cystidia near the surface. Mantle surface: usually continuous red-brown to brown; bruising blue near the apex or over the over the root apex, of prosynchymatous to loosely interwoven, entire surface where injured. Hyphal strands: golden brown to hyaline to pale yellow, cylindrical hyphae, 4-14 1.1111 broad, dark brown, highly branched with many efferent hyphae, aris- forming a textura intricata, clamp connections frequent, with 522 CAN. J. BOT. VOL. 69, 1991

B

Fics. 14 and 15. Line drawings of ectomycorrhizal types on A. rubra. Scale bars = 100 p.m. Fig. 14. Type 1 ectomycorrhiza collected from the field. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Scalp section of Hartig net. Fig. 15. Type 2 ectomycorrhiza collected from the field. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Scalp section of Hartig net. abundant large bulbous cystidia, 8-20 broad, smooth slightly oval in outline. Longitudinal section: mantle similar walled at first then with granular, amorphous material accu- in appearance to cross section, but a greater proportion of mulating on the surface; extramatrical hyphae scarce or abun- hyphal cells in the inner layer appear elongated due to longi- dant, often most abundant near the apex, hyphae 4-9 vim tudinal orientation; Hartig net paraepidermal, rarely periepi- broad, clamp connections frequent. Cross section: mantle 40- dermic, uniseriate or rarely 3-4 cells thick between epidermal 70 p.m thick, of two distinct layers; outer layer 18-30 p.m cells, 2-7 p.m broad: in scalp section hyphae adherent to the thick, prosynchymatous, composed of large, irregularly swol- face of epidermal cells irregularly shaped with tightly agglu- len hyphae and abundant bulbous cystidial end cells, efferent tinated and compacted irregularly oriented, stubby, fingerlike hyphae 4-14 p.m broad and hyphal strands, irregularly inter- branches and anastomoses, forming a textura epidermoidea, woven, mostly thin walled, circular to elongated in outline; hyphae in inner mantle angular, isodiametric, forming a tex- inner layer 20-35 p.m thick, prosynchymatous to pseudopar- tura angularis; epidermal cells 40-50 p.m long, oval or elon- enchymatous and partially gelatinized, composed of hyphae 2- gated in outline, and angled slightly periclinally to the axis of 8 p.m broad, circular in outline, mostly parallel, longitudinally the root. arrayed on the root tip; Hartig net paraepidermal, rarely per- The large, succulent type of mycorrhizae formed by iepidermic, hyphae isodiametric to slightly angular, uniseriate A. diplophloeus on red alder are present almost ubiquitously or rarely 3-4 cells thick between epidermal cells, 4-7(10) p.m in the field but are especially abundant in mesic areas. The broad, prosynchymatous to pseudoparenchymatous and tightly distinct blue-bruising reaction described by Miller et al. (1988) agglutinated; epidermal cells 12-20 p.m broad, circular or is striking, even in the field, and makes this type of red alder MILLER ET AL. 523

D C

FIGS. 16 and 17. Line drawings of ectomycorrhizal types on A. rubra. Scale bars = 100 p.m. Fig. 16. Alpova diplophloeus ectomycorrhiza synthesized in the laboratory. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Tissue of the inner mantle. (E) Scalp section of Hartig net. Fig. 17. Thelephora terrestris ectomycorrhiza synthesized in the laboratory. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Scalp section of Hartig net. mycorrhiza difficult to misidentify. Neal et a!. (1968) brown, and the blue-bruising reaction may be lacking or par- described a "dark-brown, clavate, red alder mycorrhiza" that tially obscurred. they could not connect with a specific mycobiont. Their cross section of the mycorrhiza, however, showed the typical bilay- Thelephora terrestris + Alnus rubra Figs. 3, 17A-17D ered mantle characteristic of A. diplophloeus + A. rubra ecto- Morphology: root tips monopodial, rarley irregularly com- mycorrhizae. The bilayered mantle is unusual and corresponds pound with few lateral root tips, 1-10(25) mm long x 0.2- to the peridium of A. diplophloeus sporocarps (Trappe 1975). 0.3 mm thick; straight or slightly flexuous, cylindrical or Although young A. diplophloeus mycorrhizae are only pale tapering to the apex; the apices are mostly subacute, occa- brown to golden brown, older rootlets are usually much darker sionally with the nonmycorrhizal root apex exposed; surface 524 CAN. .1. BOT. VOL 69. 1991 woolly to downy, with scattered to abundant, efferent, extra- exposed; surface smooth with scattered, efferent, extramatrical matrical hyphae and scattered hyphal strands; pallid white in hyphae and fine hyphal strands; pale yellowish orange to youth, becoming flushed with pale grey or yellowish grey; orange, in age brownish orange where broken; extramatrical hyphal strands pallid white, grey, or greyish yellow, highly hyphae hyaline. Hyphal strands: 5-30 jim broad, 3-5 hyphae branched with abundant efferent hyphae, arising from the sur- thick, cylindrical, branching with sparse efferent hyphae. face of mycorrhizal tips; extramatrical hyphae hyaline. Hyphal Mantle surface: usually continuous over the root apex; of pro- strands: hyphal strands abundant, creamy white, greyish yel- synchymatous, cylindrical, mostly hyaline hyphae, 3-5 p.m low or pale brown; 5-30(60) p.m broad, 3-5 cells thick, cylin- broad, with scattered cylindrical to irregularly swollen and drical to flattened with abundant efferent hyphae and cystidia contorted, occasionally branched laticiferous hyphae, 3-8 p.m at the surface. Mantle surface: frequently continuous over the broad, filled with sulfovanillin positive globules; clamp con- root apex, of prosychymatous to loosely interwoven, cylin- nections frequent, forming a textura porrecta; extramatrical drical to irregularly swollen, mostly hyaline hyphae forming hyphae scarce, 4-6 p.m broad, clamp connections frequent or a textura intricata, hyphae 2-6 p.m broad, with abundant, abundant except near the apex, 3-8 p.m broad, clamp con- blunted or slightly tapering cystidia, 18-40 p.m long with cell nections frequent. Cross section: mantle 10-16 p.m thick, pro- walls up to 1 p.m thick in the mantle and protruding from the synchymatous, composed of uniformly cylindrical, repent surface, clamp connections frequent; extramatrical hyphae hyphae, scattered efferent hyphae, 4-6 p.m broad, tightly usually abundant, cylindrical, hyaline, 2-5 p.m broad, clamp adjoining and interwoven, mostly hyaline, thin-walled, cir- connections frequent. Cross section: mantle 30-45 p.m thick, cular to cylindrical in outline; Hartig net paraepidermal and prosynchymatous, composed of cylindrical, repent and effer- frequently periepidermic, hyphae mostly circular or angular in ent hyphae 3-8 p.m broad, blunted, and cylindrical or slightly outline, 1-4 cells thick between epidermal cells, 2-6 p,m bulbous cystidia, 18-40 p.m long, mostly hyaline, thin walled, broad, prosynchymatous; epidermal cells 8-16 p.m broad, cir- circular to elongated in outline, and hyphal strands; Hartig net cular to elongate in outline. Longitudinal section: mantle sim- paraepidermal or periepidermic, hyphae isodiametric or angu- ilar in appearance to cross section; Hartig net paraepidermal lar in outline, uniseriate or with 2-4 cells thick between epi- and frequently periepidermic, 1-4 cells thick between epider- dermal cells, 2-6 p.m broad, prosynchymatous; epidermal cells mal cells, hyphae angular or elongated in outline, 2-16 p.m 8-14 p.m broad, circular to slightly elongated in outline. Lon- broad; in scalp section hyphae angular, elongated, or irregu- gitudinal section: mantle similar in appearance to cross section larly lobed and anastomosed, forming a textura epidermoidea; but with a greater portion of hyphal cells in the inner mantle epidermal cells 12-40 p.m long, oval or elongated in outline, elongate or cylindrical owing to longitudinal orientation; Har- and angled slightly periclinally or parallel to the axis of the tig net paraepidermal or periepidermic, uniseriate or with 2- root. 4 cells thick between epidermal cells, hyphae isodiametric, The large, succulent root tips, bright orange color, and sheer angular, or elongated, 4-16 p.m broad; in scalp section hyphae numbers combine to make L. obscuratus one of the most isodiametric to angular or irregularly lobed and anastomosed, noticeable types of ectomycorrhiza on red alder. Together, tightly agglutinated with thick, refractive walls and frequently A. diplophloeus, type 1 mycorrhizae, and L. obscuratus are discolored light brown or yellowish brown by amorphous pig- the most common mycorrhizae in the field. Neal et al. (1968) ment, forming a textura angularis to poorly developed textura described "pale brown, glabrous mycorrhizae" that were cir- epidermoidea; epidermal cells 15-45 p.m long, oval or elon- cumstantially confirmed as L. obscuratus + A. rubra ecto- gated in outline, and angled slightly periclinally or parallel to mycorrhizae by Froidevaux (1973). Once isolated into pure the axis of the root. culture, L. obscuratus mycorrhizae were synthesized readily Thelephora terrestris-type ectomycorrhizae were common on red alder, and the extramatrical hyphae spread rapidly to as greenhouse contaminants and were often associated with uncolonized roots in growth pouches. fruiting bodies. This type of mycorrhiza was also found fre- It should also be mentioned that few species of Lactarius quently in the field, although no fruiting bodies were observed form clamp connections in their sporocarps, and so it may be associated with red alder. unusual to find clamp connections in the mycorrhizae. Further The features of the ectomycorrhizae formed by T. terrestris laboratory synthesis with species in this genus would be on red alder compare favorably with those formed on aspen helpful. (Godbout and Fortin 1985), spruce (Agerer 1988; Agerer and Weiss 1989), and pine (Schramm 1966). Especially notable Type 3: unknown mycobiont Figs. 7, 19A-19D are the abundant, stout hyphal end cells or cystidia protruding Morphology: root tips monopodial, rarely irregularly com- from the surface of the mantle in younger tips and the outer pound, with few or many lateral root tips, 2-10 mm long x hyphae of hyphal strands that had pale brownish walls. The 0.3-0.6 mm thick; straight or slightly flexuous, mostly cylin- cystidia described by Schramm (1966) and Agerer (1988), drical, tapering slightly to the tip; the apices blunted to sub- however, were much thicker walled than those that formed on acute, rarely with the nonmycorrhizal root apex exposed; sur- red alder, and ours may in fact belong to a different species face smooth with scattered, fine, efferent, extramatrical hyphae of Thelephora. Young tips appear to be white or greyish white, and fine hyphal strands; pallid white to pale yellowish white, whereas older tips become brownish overall or in patches. in age becoming brown in patches where broken; extramatrical hyphae hyaline. Hyphal strands: hyaline, branched with sparse Lactarius obscuratus + Alnus rubra efferent hyphae, 5-30 p,m broad, 3-5 hyphae thick, cylindri- Figs. 5, 6, and 18A-18D cal. Mantle surface: usually continuous over the root apex; of Morphology: root tips monopodial or irregularly compound prosynchymatous, cylindrical to swollen, mostly hyaline with few or many lateral root tips, 1-15(20) mm long x 0.3- hyphae, 3-6 p.m broad, with scattered laticiferous hyphae, 4- 0.8(1.0) mm thick; straight or slightly flexuous, mostly cylin- 7 p.m broad, cylindrical to irregularly swollen and contorted; drical; apices blunted, rarely with the nonmycorrhizal root apex subulate to mucronate dermatocystidia, 15-30 p.m long, filled MILLER ET AL. 525

FIGS. 18 and 19. Line drawings of ectomycorrhizal types on A. rubra. Scale bars = 100 p.m. Fig. 18. Lactarius obscuratus ectomycorrhiza synthesized in the laboratory. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Scalp section of Hartig net. Fig. 19. Type 3 ectomycorrhiza collected from the field. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. Sulfovanillin positive staining hyphae and dermatocystidia are indicated by the dark shading. (D) Scalp section of Hartig net.

with sulfovanillin positive globules; clamp connections fre- of sulfovanillin-positive dermatocystidia and laticiferous quent, forming a textura porrecta to textura intracata; extra- hyphae in the mantle (Fig. 19) suggest that a Russula sp. or matrical hyphae scarce, 3-6 1..tm broad, clamp connections Lactarius sp. may be responsible. Horak (1963) and Singer scattered. Cross section: mantle 16-24 p.m thick, prosynchy- and Digilio (1952) have reported several Russula spp. to be matous, composed of cylindrical to swollen repent hyphae, associated with alder. Similarly, at least two species of Rus- scattered efferent hyphae, 3-7 p.m broad, tightly adjoining and sula were recently collected in A. crispa and A. tenuifolia interwoven, mostly hyaline, thin-walled, circular to cylindrical thickets in Alaska (Brunner 1989). in outline, and scattered to abundant subulate to mucronate dermatocystidia, 15-30 p.m long; Hartig net paraepidermal and Cortinarius bibulus + Alnus rubra Figs. 8, 20A-20E frequently periepidermic, hyphae mostly circular, angular or Morphology: root tips monopodial, rarely irregularly com- elongated in outline, 1-4 cells thick between epidermal cells, pound with few lateral root tips, (1—)4-8(-10) mm long x 2-10 p.m broad, prosynchymatous; epidermal cells 8-18 p.m (0.3—)0.5-1.0 mm thick, curving, contorted or rarely straight, broad, circular to elongate in outline. Longitudinal section: mantle similar in appearance to cross section; Hartig net par- cylindrical, slightly swollen or tapering toward the tip; apices aepidermal and frequently periepidermic, 1-4 cells thick blunted, rarely subacute, with the nonmycorrhizal root apex between epidermal cells, hyphae circular, angular or elongated exposed; surface densely woolly or downy, with abundant, in outline, 2-20 p.m broad; in scalp section hyphae adherent efferent, extramatrical hyphae and hyphal strands; when to the epidermal cells, angular, elongated, or irregularly lobed young, pinkish white, reddish grey to greyish rose, generally and anastomosed, forming a textura epidermoidea; epidermal lighter at the apex, in age pale reddish grey to greyish red or cells 15-40 p.m long, oval or elongated in outline, and angled reddish brown; hyphal strands pinkish white to reddish grey, slightly periclinally or parallel to the axis of the root. fine, highly branched, with abundant pinkish white to reddish No mycobiont could be confirmed for this mycorrhizal type. grey efferent hyphae. Hyphal strands: white, pinkish white or However, the smooth, tightly compacted mantle and presence reddish grey, 12-85 p.m broad, 2-10 cells thick, regularly and

526 CAN. J. BOT. VOL. 69, 1991

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D FIGS. 20 and 21. Line drawings of ectomycorrhizal types on A. rubra. Scale bars = 100 Fig. 20. Cortinarius bibulus ectomycorrhiza collected from the field. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Tissue of the inner mantle. (E) Scalp section of Hartig net. Fig. 21. Type 4 ectomycorrhiza collected from the field. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Scalp section of Hartig net.

evenly interwoven with abundant branching and anastomosing connections frequent; extramatrical hyphae abundant, branch- efferent hyphae. Mantle surface: mostly continuous over the ing, 5-8 p.m broad, single and multiple clamp connections root apex, of prosynchymatous to loosely interwoven, hyaline, frequent. Cross section: mantle 60-100 p.m thick, of two dis- cylindrical or irregularly swollen and contorted, branching tinct layers; outer layer 20-50 tim thick, prosynchymatous, hyphae, 6-20 p,m broad, forming a textura intricate, clamp composed of cylindrical to swollen repent and efferent hyphae MILLER ET AL. 527 6-10 p.m broad and hyphal strands, loosely to tightly inter- 15-45 p.m long, oval or elongated in outline, angled slightly woven, mostly thin-walled, circular to elongated in outline; periclinally or parallel to the axis of the root. inner layer 30-50 p.m thick, prosynchymatous, composed of Type 4 ectomycorrhizae were only collected once from along hyphae 6-20 p.m broad, circular to angular in outline; Hartig a moist creek bottom in the Coast Range of Oregon. The large, net paraepidermal, rarely periepidermic, hyphae isodiametric isodiametric cells of the mantle are peculiar to this type of or elongated, 1-3 cells thick between epidermal cells, 6- mycorrhiza. Several species of Naucoria were collected nearby 14 p.m broad, prosynchymatous to pseudoparenchymatous and and may be likely candidates for the mycobiont. tightly agglutinated; epidermal cells 8-22 p.m broad, circular, or slightly elongated in outline. Longitudinal section: mantle Hebeloma crustuliniforme + Abuts rubra similar in appearance to cross section; Hartig net paraepider- Figs. 12, 22A-22D mal, rarely periepidermic, 1-3 cells thick between epidermal Morphology: root tips monopodial, occasionally irregularly cells; hyphae in outer mantle 6-26 p.m broad, in scalp section compound with few lateral root tips, 1-5(8) mm long x 0.2- hyphae adherent to the face of epidermal cells are highly irreg- 0.4(0.8) mm thick; straight or slightly flexuous, cylindrical or ularly shaped with abundant intricate, stubby lobes and anas- slightly swollen, often tapering to the apex; apices blunted or tomoses, tightly agglutinated and compacted, 12-16 p.m subacute, frequently with the nonmycorrhizal root apex broad, forming a finely denticulate textura epidermoidea; exposed; surface woolly to downy, with abundant, efferent, hyphae in inner mantle angular, isodiametric to elongated, 10- extramatrical hyphae; pallid white, in age becoming flushed 35 p.m broad, forming a textura angularis; epidermal cells 12- with pale grey; extramatrical hyphae hyaline. Hyphal strands: 20 p.m long, oval or elongated in outline, angled slightly per- absent or not observed. Mantle surface: frequently continuous iclinally or parallel to the axis of the root. over the root apex, of prosynchymatous to loosely interwoven, Locally abundant in mesic areas, the greyish rose to greyish cylindrical, hyaline hyphae, 2-4 vim broad, forming a textura red color and tortuous morphology of these mycorrhizae are intricata, clamp connections frequent; extramatrical hyphae unmistakable. Also, the presence of a tightly compacted inner abundant, cylindrical, hyaline, 2-4 p.m broad, clamp connec- mantle and loosely interwoven outer mantle is distinctive. Sev- tions frequent. Cross section: Mantle 18-30 1.t.m thick, pro- eral other species of Cortinarius, forming white but otherwise synchymatous, composed of uniformly cylindrical, repent and similar mycorrhizae to C. bibulus, have been reported in asso- efferent hyphae, 2-4 pm broad, irregularly interwoven, hya- ciation with Alnus spp. from Quebec (Godbout and Fortin line, thin-walled, circular to elongated in outline; Hartig net 1983, 1985) and Alaska (1. L. Brunner, personal communi- paraepidermal, hyphae isodiametric to angular or elongated in cation) and could be associated with red alder in more north- outline, 1-2 cells thick between epidermal cells, erly latitudes. Ectomycorrhizae in the genera Cortinarius and 2-8(12) p.m broad, prosynchymatous to pseudoparenchyma- Dermocybe on spruce described by Agerer (1987a, 1987b) are tous; epidermal cells 6-16 p.m broad, circular to slightly elon- similar in overall morphology but possessed a slightly thinner gated in outline. Longitudinal section: mantle similar in mantle than C. bibulus + A. rubra ectomycorrhizae. appearance to cross section; Hartig net paraepidermal, unis- eriate between epidermal cells, hyphae isodiametric, angular Type 4: unknown m ycobiont Figs. 9, 10, and 21A-21D or elongated, 2-8(12) broad; in scalp section hyphae iso- Morphology: root tips monopodial or irregularly compound, diametric to angular or with abundant, intricate, stubby lobes with few lateral root tips, 1-10 mm long x 0.5-0.8(1.0) mm and anastomoses, tightly agglutinated with thick, refractive thick; flexuous or slightly contorted, cylindrical, tapering walls and frequently discolored light brown or yellowish brown slightly toward the tip; apices blunted to subacute, occasion- by amorphous pigment, forming a textura epidermoidea; epi- ally with the nonmycorrhizal root apex exposed; surface dermal cells 7-32 long, oval or elongated in outline, smooth to micaceous, with sparse, efferent, extramatrical angled slightly periclinally or parallel to the axis of the root. hyphae; brownish orange, light brown to dark brown; extra- Sporocarps of H. crustuliniforme were not found in asso- matrical hyphae hyaline. Hyphal strands: absent or not ciation with red alder, but mycorrhizae were synthesized from observed. Mantle surface: usually continuous over the root a laboratory isolate (S-166, Forestry Sciences Laboratory, apex; of prosynchymatous, tightly compacted, angular isodi- Corvallis, OR) isolated from a sporocarp associated with con- ametric, brown hyphae, 3-5 p.m broad, set at irregular levels, ifers. However, one collection of a Hebeloma sp., close to forming a textura angularis, clamp connections absent; extra- H. magnimamma (Fr.) Quel., was collected from a site near matrical hyphae scarce, 4-6 p.m broad, hyaline, clamp con- Cascade Head, Oregon, and mycorrhizae associated with the nections absent. Cross section: mantle 20-30 p.m thick, pro- sporocarps were similar to the H. crustuliniforme type. synchymatous, composed of tightly compacted, moderately Voiry (1981) and Godbout and Fortin (1985) demonstrated thick-walled, isodiametric hyphae, 5-25(60) p.m broad, angu- a generic level of mycorrhizal characterization for a number lar in outline; Hartig net paraepidermal, rarely periepidermic, of fungal species including those in Hebeloma. The features hyphae mostly circular or angular in outline, 1-3 cells thick of H. crustuliniforme + A. rubra mycorrhizae described here between epidermal cells, 2-10 p.rn broad, hyaline to pale agree with earlier descriptions of Hebeloma mycorrhizae, brown, prosynchymatous; epidermal cells 6-16 1..un broad, whether the fungi were synthesized on oak, beech, or aspen circular to elongate in outline. Longitudinal section: mantle (Voiry 1981; Godbout and Fortin 1985) or on D. integrifolia similar in appearance to cross section; Hartig net para- (Melville et al. I987a, I987b). These mycorrhizae were thin epidermal, rarely periepidermic, 1-3 cells thick between epi- in diameter, possessed a sparse, loosely interwoven and woolly dermal cells, hyphae angular or elongated in outline, 2-10 p.m mantle, and the hyphae had abundant clamp connections. In broad, hyaline to pale brown; in scalp section hyphae isodi- the Pacific northwest, however, the Hebeloma type of mycor- ametric, angular, elongated or irregularly lobed and anasto- rhizae were white and best characterized by negative features, mosed, tightly agglutinated with thick, refractive walls, form- i.e., no brown bruising or terminal cells filled with amor- ing a textura angularis to textura epidermoidea; epidermal cells phous, refractive yellow material as in the Paxillus type, no 528 C AN. J. BUT VOL. 69, 1991

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FIGS. 22 and 23. Line drawings of ectomycorrhizal types on A. rubra. Scale bars = 100 rn. Fig. 22. Hebeloma crustuliniforme ecto- mycorrhiza synthesized in the laboratory. (A) Cross section. (B) Longitudinal section. (C) Mantle surface. (D) Scalp section of Hartig net. Fig. 23. Laccaria laccata ectomycorrhiza collected from the field. (A) Cross-section. (B) Longitudinal section. (C) Mantle surface, showing gelatinization of mantle tissue. (D) Scalp section of Hartig net. blunted cystidia as in the Thelephora type, and abundant extra- dant, cylindrical, hyaline, 2-6 pAn broad, clamp connections matrical hyphae unlike type 3 mycorrhizae. frequent. Cross section: mantle 30-50 p.m thick, prosynchy- matous, near the surface, composed of uniformly cylindrical, Laccaria laccata + Alnus rubra Figs. 11, 23A-23D repent and efferent hyphae 2-6 broad, with frequent swol- Morphology: root tips monopodial, commonly irregularly len, elongate cells, 8-12 vm broad, irregularly interwoven, compound with many lateral root tips, 3-20(30) mm long x hyaline, thin walled, inner portions of mantel often gelatin- 0.2-0.6 mm thick; flexuous, contorted, or rarely straight, often ized, Hartig net paraepidermal, hyphae isodiametric angular with a beaded appearance, cylindrical but tapering to the apex; or elongated, uniseriate between epidermal cells, 2-6 1.Lnl apices mostly acute, usually with nonmycorrhizal root apex broad, prosynchymatous to pseudoparenchymatous; epidermal exposed; surface silky—downy, often with alternating localized cells 8-18 broad, circular to slightly elongated in outline. patches of thin mantle and sparse extramatrical hyphae; pallid Longitudinal section: mantle similar in appearance to cross white to pale yellowish white when young, then flushed red- section; Hartig net paraepidermal, uniseriate between epider- dish grey to dull red, in age brownish orange with darker brown mal cells, hyphae isodiametric, angular or elongated, 2-10 p rn root apex where exposed, extramatrical hyphae hyaline to pale broad; in scalp section hyphae isodiametric to angular or irreg- reddish grey. Hyphal strands: absent or not observed. Mantle ularly lobed and anastomosed, tightly agglutinated with thick, surface: fequently continuous over the root apex, of prosyn- refractive walls and frequently discolored light brown or yel- chymatous to loosely interwoven, cylindrical, mostly hyaline lowish brown by amorphous pigment, forming a textura angu- hyphae, 2-6 p.m broad, and swollen, intercalary and often laris to poorly developed textura epidermoidea; epidermal cells catenulate hyphae, 8-12 pm broad, forming a textura intri- 16-38 p.m long, oval or elongated in outline, and angled cate, clamp connections frequent; extramatrical hyphae abun- slightly periclinally or parallel to the axis of the root. MILLER ET AL. 529 Ectomycorrhizae synthesized by L. laccata on red alder were similar in morphological features to the bristly mycorrhizae of Laccaria bicolor + Populus tremuloides reported by Godbout and Fortin (1985) and Laccaria amethystina + Fagus sylva- ticus reported by Brand and Agerer (1987). The mantles of L. laccata + A. rubra mycorrhizae, however, were pallid white young and reddish grey to dull red in age and had fre- quent localized patches of thin mantle and sparce extramatrical hyphae that gave an irregular, angled appearance. Laccaria laccata also colonized the greatest length of red alder root tips of any fungus in this study. The mycorrhizae formed by L. laccata in laboratory syntheses, and those encountered in the field, show a slight incompatibility response as defined by Molina (1981). Many cells in the Hartig net and mantle were heavily coated with an amorphous, refractive, brown material, and the hyphae appeared to be gelatinous in portions of the mantle, Godbout and Fortin (1983) reported a similar response for L. laccata + Alnus spp. syntheses.

Paxillus involutus + Alnus rubra Figs. 13, 24A-24D Morphology: root tips monopodial, frequently irregularly compound with few or many lateral root tips, 1-10(15) mm long x 0.2-0.8 mm thick; straight or slightly flexuous, cylin- drical, occasionally tapering to the apex; apices blunted or sub- acute, occasionally with the nonmycorrhizal root apex exposed; surface felted to minutely downy, with scattered to abundant, efferent, extramatrical hyphae; root hairs commonly protrud- ing through well-developed mantle; pallid white, often "shim- mering" when submerged in water owing to trapped air bub- bles in the mantle, often discoloring to brown where broken or injured; extramatrical hyphae hyaline. Hyphal strands: absent or not observed. Mantle surface: frequently continuous over the root apex, of prosynchymatous to loosely interwoven, cylindrical or swollen, hyaline hyphae, 2-6 1.1,m broad, with blunted hyphal-end cells, 10-60 vim long, and scattered to abundant intercalary cells and terminal cells filled with amor- phous, yellow, refractive material, and hyphal strands, 5- 15 ti,m broad, 3-5 cells thick, cylindrical, hyaline, forming a textura intricata, clamp connections frequent; extramatrical hyphae sparce to abundant, cylindrical, hyaline, 2-6 p.m broad, clamp connections frequent. Cross section: mantle 20- 30 p.m thick, prosynchymatous, composed of uniformly cylin- FIG. 24. Line drawing of P. involutus + A. rubra ectomycorrhiza drical, repent and efferent hyphae 2-6 p.m broad, irregularly synthesized in the laboratory. Scale bar = 100 p.m. (A) Cross sec- interwoven, hyaline, thin-walled, circular to elongated in out- tion. (B) Longitudinal section showing mantle and protruding root line; root hairs commonly protruding through mantle; Hartig hairs. (C) Mantle surface. (D) Scalp section of Hartig net. net paraepidermal, hyphae isodiametric to angular or elongated in outline, uniseriate between epidermal cels, 2-8 p.m broad, that bruise brownish where injured or become brownish with prosynchymatous; epidermal cells 10-20 p.m broad, circular age. Paxillus filamentosus, a closely related species, has been to slightly elongated in outline. Longitudinal section: mantle reported widely in association with in Europe (R. similar in appearance to cross section; Hartig net paraepider- Watling, personal communication), as well as in Nepal (V. mal, uniseriate between epidermal cells, hyphae isodiametric, Cotter, personal communication) and Alaska (I. Bruner, per- angular or elongated, 2-8 vim broad; in scalp section hyphae sonal communication), but not as yet from Oregon, Washing- adherant to the epidermal cells, isodiametric to angular or ton, or California. irregularly lobed and anastomosed, tightly agglutinated with There remains a question concerning the mycorrhizal status thick, refractive walls and frequently discolored light brown of Paxillus with alder and other angiosperm hosts. The pres- or yellowish brown by amorphous pigment, forming a textura ence of root hairs protruding through a mantle-like layer of angularis to poorly developed textura epidermoidea; epidermal fungal tissue suggests that the association between P. involutus cells 15-35 p.m long, oval or elongated in outline, angled and red alder may not be that of a typical mycorrhizal sym- slightly periclinally or parallel to the axis of the root. biosis. However, the paraepidermal Hartig net is as well The P. involutus type of mycorrhizae were present but not formed as in other mycorrhizal types, and so a mycorrhizal common in the field. The diagnostic features of these mycor- description for this species was included. Similar results were rhizae are the thin mantle and creamy coloration in young tips obtained by Massicotte et al. (1987) with P. involutus + 530 CAN. J. HOT. VOL. 69, 1991 Betula alleghaniensis. Several attempts at laboratory synthesis collected and laboratory-synthesized root tips. Because high using the growth-pouch technique with isolates of P. involutus host-specificity appeared to be somewhat correlated with the (obtained from sporocarps associated with a Betula sp.) and fungi forming the succulent type of mycorrhizae and low spec- with a P. cf. filamentosus isolate (courtesy of V. Cotter, from ificity corresponded mostly to fungi forming flexuous mycor- sporocarps associated with a Napalese Alnus sp.) showed good rhizae, these designations require further study. mycelial growth but no mycorrhizal formation. Laboratory Most of the mycobionts (7 of 11) encountered on red alder synthesis was finally obtained by using the synthesis-tube tech- during the course of this study were identified to species, and nique and supplementing with glucose. others have been tentatively linked to possible teleomorphs. In contrast with other laboratory syntheses using growth These identifications, although tentative in some cases, pro- pouches (Godbout and Fortin 1985; Grenville et a!. 1985), no vide much information about the fungi that colonize red alder, sclerotia of P. involutus or P. filamentosus were observed dur- and their host specificity and ecology. ing this study. Both the lack of sclerotial formation and the Descriptions of ectomycorrhizae on red alder provided here general difficulty in synthensizing mycorrhizae may have been are the result of a 3-year study of a variety of habitats. Addi- due to isolate host specificity or cultural conditions. tional morphological types may be encountered in different years and in different microhabitats. It is now possible, how- Unconfirmed reports from the Pacific northwest Cenoccum geophilum + Alnus rubra ever, to monitor red alder ectomycorrhizal types before and Trappe (1964) reported black mycorrhizae with stiff, bristly after experimental treatments such as outplanting, harvesting, hyphae that were similar to typical C. geophilum mycorrhizae drought, addition of fertilizer, or other environmental in association with red alder. Likewise, Masui (1926) described perturbations. and illustrated "Form C" ectomycorrhizae, which undoubt- edly were Cenococcum-type mycorrhizae on Japanese Alnus Acknowledgements spp. After repeated trials with several isolates of C. geo- philum, Godbout and Fortin (1983) reported only meager suc- Special thanks to Gretchen Bracher from the Forest Research cess at laboratory synthesis using the growth-pouch technique. Laboratory, Oregon State University, Corvallis, OR, for ink- No Cenococcum-type mycorrhizae were encountered on red ing the final drawings. Thanks also to Van Cotter, Virginia alder in the field during this study. In addition, Miller et al. Polytechnic Institute and State University, Blacksburg, VA, (1987a) found that Cenococcum mycorrhizae formed abun- for supplying isolates of Paxillus cf. filamentosus from dantly on Douglas-fir in a greenhouse soil bioassay, but that Napalese alder, and to Christian Godbout, University Laval, no Cenoccocum mycorrhizae formed on red alder, even in the Sainte-Foy, Que., for providing isolates from his research on same soil. However, dark, highly ornamented hyphae similar red alder ectomycorrhizae. Funded in part by National Science to C. geophilum hyphae were observed growing along the roots Foundation grants BSR-8505972 and BSR-8805983 and the of red alder both in the field and in greenhouse experiments. USDA Forest Service, Pacific Northwest Research Station, Corvallis, OR. Summary AGERER, R. 1986. Studies on ectomycorrhizae. II. Introducing An initial step in understanding the dynamics and contri- remarks on characterization and identification. Mycotaxon, butions of different ectomycorrhizal fungi to belowground pro- 25: 473-492. cesses is to identify the mycobionts present in the ecosystem. 1987a. Studies on ectomycorrhizae. V. Mycorrhizae formed Red alder is an ideal host for such studies because there appears by Dermocybe cinnamomea and D. sanguinea on spruce. Nova to be a small number of ectomycorrhizal fungi with a high Hedwigia, 44: 69-89. degree of host specificity associated with alder in comparison 19876. Studies on ectomycorrhizae. X. Mycorrhizae formed by Cortinarius obtusus and Cortinarius venetus on spruce. with other hosts, and characterization of the types of mycor- Mycologia, 79: 524-539. rhizae and identification of the mycobionts is relatively simple. 1988. Studies on ectomycorrhizae. XVII. The ontogeny of It is important to note that many of the ectomycorrhizae col- the ectomycorrhizal rhizomorphs of Paxillus involutus and lected in the field likely contained more than one mycobiont. Thelephora terrestris (Basidiomycetes). Nova Hedwigia, In most cases, however, one mycorrhizal type appeared to 47: 311-334. dominate, at least on a visual basis. AGERER, R., and WEISS, M. 1989. Studies on ectomycorrhizae. )(X. Although a few recent publications illustrated the use of a Mycorrhizae formed by Thelephora terrestris on Norway spruce. variety of sophisticated techniques and specialty equipment Mycologia, 81: 444-453. such as Normarsky interference contrast microscopy, autofluo- BRAND, E., and AGERER, R. 1987. Studies on ectomycorrhizae. VIII. rescence of hyphal wall material, scanning electron micros- Mycorrhizae formed by Lactarius subdulcis, Lactarius vellereus copy, internal structure of hyphal strands, and a multitude of and Laccaria amethystina on Beech. Z. Mykol. 52: 287-320. macrochemical reagents to characterize ectomycorrhizae, 11 BRUNNER, I. L. 1989. Two species of Russula (Stirps Atropurpurea) types of red alder ectomycorrhizae could be distinguished on associated with Alnus crispa in Alaska. Mycologia, 81: 667-676. the basis of color, morphological features, and light-micro- BRUNNER, I. L., BRUNNER-GROSSMANN, F., and MILLER, 0. K., JR. 1988. Ectomycorrhizal studies with Alnus crispa and Alnus ten- scopic features of hyphae in the mantle. uifolia. Mycological Society of America Newsletter No. 39. Comparisons between the ectomycorrhizal types observed p. 22. on red alder from the Pacific northwest revealed that the root- FORTIN, J. A., Pipit, Y., and LALONDE, M. 1980. Technique for tips could generally be categorized into two broad groups of the observation of early morphological changes during ectomy- mycorrhizae, flexuous and succulent. There was some inter- corrhiza formation. Can. J. Bot. 58: 361-365. gradation between mycorrhizae in these two categories, but FROIDEVAUX, L. 1973. 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