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Ontogeny of Alnus rubra - Alpova diplophloeus ectomycorrhizae. I . Light microscopy and scanning electron microscopy
H . B. MASSICOTTE, R. L. PETERSON, A N D L. H. MELVILLE Department of Botany, Universiry of Glcelph, Guelph, Ont., Canada N1G 2 W l
Received February 1, 1988
MASSICOTTE, H. B., PETERSON, R. L., and MELVILLE, L. H. 1989. Ontogeny of .A l t~us rubra - Alpova diplophloelts ectomycorrhizae. I. Light microscopy and scanning electron microscopy. Can. J . Bot. 67: 191 -200.
Ectomycorrhizae synthesized between Alpova diplophloeus and Alnus rubra are of two morphological types: one with a mantle formed along the entire length of the lateral roots and the other, the clavate type, with the mantle confined to the apical portion of the laterals. The morphology of the mycorrhiza is dependent on the stage of lateral root elongation at the time of colonization by fungal hyphae. Clavate mycorrhizae form on lateral roots that have already elongated at the time of fungal colonization. Fungal hyphae interact with root hairs at the base of clavate mycorrhizae. Mantles of both types are fairly compact with few extramatrical hyphae. Hartig net hyphae, which branch profusely primarily in the radial direction, are confined to the epidermis and midway along the radial walls of the outer layer of cortical cells. Second-order lateral root primordia are initiated in the mature Hartig net zone. Cells in the outer layer of the cortex of mycorrhizal roots collapse during fixation, indicating the possible presence of a barrier in the cell wall blocking the ingress of fixative.
MASSICOTTE, H. B., PETERSON, R. L., et MELVILLE, L. H. 1989. Ontogeny of Alnus rubra - Alpova diplophloeus ectomycorrhizae. I. Light microscopy and scanning electron microscopy. Can. J . Bot. 67 : 191 -200.
Les mycorhizes synthCtistes entre Alpova diplophloeus et Altlus rubra dCveloppent deux types de morphologie : l'un avec un manteau fongique couvrant entikrement la racine IatCrale; I'autre, en forme de massue, avec un manteau couvrant unique- ment la portion apicale de la racine IatCrale. La morphologie de la mycorhize depend du dCveloppement de la racine laterale lorsque la colonisation fongique s'effectue. Les mycorhizes en forme de massue se forment sur des racines IatCrales qui sont dCjB allongCes au temps de la colonisation. Les hyphes interagissent avec les poils absorbants i la base des mycorhizes en forme de massue. Les manteaux des deux types sont compacts avec quelques hyphes extramatricielles. Les hyphes du rCseau de Hartig produisent de nombreuses parois, principalement dans la direction radiale, et pCnktrent entre les cellules Cpider- miques et B mi-chemin dans les parois radiales de la couche exttrieure de cellules corticales. Les primordia des racines latC- rales de second ordre sont initiCs dans la zone de rCseau de Hartig qui a atteint maturitC. Les cellules de la couche corticale extCrieure des mycorhizes s'affaissent durant la fixation, indiquant possiblement la prCsence d'une barrikre pariCtale arretant la progression du fixatif.
Introduction The genus Alnus is an actinorhizal angiosperm important in
forest management because of its nitrogen-fixing ability (Tarrant and Trappe 1971). The genus is also mycorrhizal with a limited number of ectomycorrhizal fungi able to colo- nize its roots (Molina 1979, 1981; Godbout and Fortin 1983). This observation may be of considerable interest in terms of recognition and compatibility phenomena. The ectornycor- rhizal fungus Alpova diplophloeus is rather unique in that it is restricted to the genus Alntts (Trappe 1975; Molina and Trappe 1982).
Details of the development of the ectomycorrhiza formed between A. diplophloeus and Alnus crispa were documented in a previous paper (Massicotte et al. 1986). Both symbionts showed striking structural modifications, particularly in the Hartig net - epidermis interface. Root epidermal cells elabo- rated wall ingrowths to become transfer cells, while contigu- ous hyphae underwent repeated branching and showed marked alterations in cytoplasmic organelles (Massicotte et al. 1986).
The objective of the present study was to determine if the specialized root-fungus interface observed in A. crispa - A. diplophloeus mycorrhizae is characteristic of ectomycor- rhizae established between other Alnus species and A. diplo- phloeus.
Materials and methods
crispa. Seedlings were transferred, 7 days after germination, into growth pouches (Fortin et al. 1980) containing 10 mL of modified (nitrogen-free) Crone's mineral solution (Lalonde and Fortin 1972). Fourteen-day-old seedlings were inoculated with Frankia HFPArl, (obtained from J. G. Torrey, Harvard University) to induce nodula- tion and were inoculated 30 days later with Alpova diplophloeus, iso- late 8017 (from Linn County, OR).
Frankia cultures were grown at 22OC in Qmod liquid medium (Lalonde 1979) and introduced into pouches with a sterile syringe. The fungus was grown at 20°C on modified Melin-Norkrans (MMN) agar medium (Marx and Bryan 1975) and introduced into the pouches as 10-mm diameter plugs (Picht and Fortin 1982).
Growth conditions Seedlings were grown under 5 klx (68 W/mZ) (130 pmol . m-2.
sec-') light on a 16 h light : 8 h dark cycle at a temperature of 24:18"C (1ight:dark). High levels of humidity (60-80% RH) were maintained using a humidifier. Additional nutrient solution was added to pouches as needed.
Developmental stages The external morphology of the roots and ectomycorrhizae was
examined daily after inoculation and the developmental stages were recorded with a Zeiss DR photodissecting microscope up to 4 weeks after inoculation.
First-order mycorrhizal lateral roots were collected for scanning electron microscopy (SEM) and sectioning for a period up to 3 weeks following the appearance of an obvious mantle. First-order lateral roots from noninoculated pouches were collected at the same time. Additional material consisting of first-order lateral roots in initial stages of colonization by the hyphal front (extramatrical or extraradi-
Plant material and mycorrhizal synthesis cal phase) were also collected for SEM. Alnus rubra Bong. seeds, obtained from the Salmon River, British
Columbia (50" 12 ' k 125'45 ', 304 m above sea level) were stratified Light tnicroscopy and germinated as described by Godbout and Fortin (1983) for Alnus Tissue was fixed and postfixed using the procedure described
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previously (Massicotte et al. 1985; Massicotte et al. 1986), rinsed and dehydrated in a graded ethanol series, and embedded in LR White resin (London Resin Company Ltd.).
Sections (1 .O- 1.5 p n ) were cut with glass knives and stained for light microscopy with 0.05% toluidine blue 0 in I % sodium borate. More than 60 roots and ectomycorrhizae were examined in three separate experiments.
SEM Small pieces of ectomycorrhizae and control roots were fixed by
the same procedure as for light microscopy, followed by treatment with thiocarbohydrazide and subsequent postfixation in 1 % osmium tetroxide (Kelley er al. 1973). Specimens were then washed in dis- tilled water, dehydrated in a graded series of ethanol, critical-point dried, mounted on aluminum stubs, and observed with a JEOL JSM-35C.
Results External tnorphology
Alnus rubra seedlings grow well in plastic pouches and produce a root system suitable for mycorrhizal synthesis over a 3- to 5-week period after nodulation with Frankia (Fig. 1). Since the Alpova isolate used grows slowly, mycorrhizal roots develop mainly in close vicinity to the inoculum plug 10- 16 days after inoculation. Most of the first-order lateral roots remain uncolonized (Fig. 1). Conspicuous Frankia-induced nodules develop along the root system (Fig. 1).
In noninoculated pouches, primary roots (Fig. 2) and first- order laterals (Fig. 3) develop a pointed and pigmented apex with an acropetal development of root hairs within approxi- mately 2 and 2.5 mm from the apex, respectively. There is no morphological difference between these and uncolonized apices from inoculated pouches.
In mycorrhizal pouches, once the hyphae emanating from the plug reach a lateral root, hyphal proliferation occurs and early root surface colonization occurs within 24-48 hours (Fig. 4). At this point, some pigmented root cap cells are still evident through the thin mantle (Fig. 4). Additional hyphal proliferation results in a more compact mantle within the next 24-48 hours (Figs. 5-8). Lateral roots can be colonized
either along the entire length (Fig. 5) or in the apical portion (Fig. 6). In the latter case there is an interaction between root hairs and hyphae in the proximal region of the mycorrhiza (Fig. 6). The length of a first-order mycorrhizal lateral can exceed 4 - 5 mm (Fig. 7). Well-developed extramatrical (extraradical) hyphae are evident on most mycorrhizal roots (Fig. 8), but in some cases they do not develop extensively (Fig. 7).
A first-order mycorrhizal lateral may exhibit a compact and well-developed mantle along its entire length (Fig. 9) and in some instances, second-order mycorrhizal laterals develop in the basal portion of the first-order rnycorrhizal lateral (Fig. 10). The outer mantle surface shows typical intertwining hyphae of variable diameter, the inner mantle hyphae being generally of wider diameter than peripheral hyphae (Fig. 11). A few clamp connections are also obvious on these hyphae (Fig. 11).
A surface view of the proximal region of a root similar to that in Fig. 6 shows hyphae forming a loose network over root hairs (Fig. 12). Hyphae grow amongst root hairs with greater concentration near the root hair tips, but they are not excluded from more basal regions (Fig. 13). Root hairs in close contact with fungal hyphae are rarely collapsed and several clamp connections are obvious on the hyphae (Fig. 14).
Light microscopy Nonmycorrhizal primary roots are pointed and have a small,
darkly stained, root cap, a conspicuous meristem, a cortex with four to six layers, a narrow epidermal cell layer, and a well-developed vascular cylinder (Fig. 15). Nonmycorrhizal first order lateral roots are also ~o in ted and exhibit similar features with a differentiating epidermis with developing root hairs, a cortex with four to five layers, and a small vascular cylinder (Fig. 16). Ectomycorrhizal first order laterals, simi- lar to that in Fig. 5, show many features of the association: an evenly thick mantle along the entire length of the root, a paraepidermal Hartig net, B small meristem and root cap, and a stele that has differentiated close to the apical meristem (Fig. 17). A first-order mycorrhizal lateral similar to Fig. 6 with only apical colonization has a thick mantle confined to this region (Fig. 18).
FIGS. 1-8. Alnus rubra - Alpova diplophloeus ectomycorrhiza development. Fig. 1. Portion of seedling root system in a growth pouch showing the distribution of lateral roots, mycorrhizal root tips (arrowheads), and nodules (arrows). Plugs of fungal inoculum (*) are evident. Fig. 2. Nonmycorrhizal primary root with a pigmented, pointed apex (arrow) and numerous root hairs (arrowheads) grown in an uninoculated growth pouch. Fig. 3. Nonmycorrhizal first-order lateral root with a pigmented root cap (arrow) overlying a pointed apex. A few root hairs (arrowheads) have developed. Fig. 4. Mycorrhizal first-order lateral root showing early mantle formation and extramatrical hyphae. Some pig- mented root cap cells are evident (arrowheads). Fig. 5. First-order lateral root with a complete mantle along the length of the root. Extramatrical hyphae (arrowheads) are present. Fig. 6. First-order lateral root with a complete mantle on the apical portion of the root (*). Some hyphae are also present in the root hair region (arrowheads) proximal to the root tip. Fig. 7. First-order lateral roots (*) showing extensive colonization along the entire length of the root (arrowheads), but with few extramatrical hyphae. Fig. 8. Enlargement of a root tip of a root similar to those in Fig. 7 showing a mantle with extensive extramatrical hyphae development (arrowheads).
FIGS. 9- 13. Scanning electron micrographs of Alnus rubra - Alpova diplophloeus mycorrhizal lateral roots. Fig. 9. Root similar to most of those shown in Fig. 7. A compact mantle (*) is present along the length of the root. Fig. 10. First-order mycorrhizal lateral with two mycor- rhizal second-order laterals (arrowheads). A compact mantle is present on all roots. Fig. 11. Portion of outer mantle from root shown in Fig. 9 with hyphae of varying diameter. Inner mantle hyphae (arrowheads) are of wider diameter than peripheral hyphae. Clamp connections (arrows) are present. Figs. 12 and 13. Surface view (Fig. 12) and sectional view (Fig. 13) of root hair zone from a root similar to that in Fig. 6. Interaction between fungal hyphae and root hairs (arrows) is evident. Many hyphae appear to interact with the apical portion of root hairs. Fig. 14. Root hair in close contact with fungal hyphae. The root hair has not collapsed. Several clamp connections (arrowheads) are evident.
FIGS. 15-18. Longitudinal sections of Alnus rubra roots. Fig. 15. Primary root similar to that in Fig. 2 showing root cap (RC), apical meristem (AM), vascular cylinder (VC), cortex (C), and epidermis (E). Fig. 16. First order lateral similar to that in Fig. 3. The root cap (RC), apical meristem (AM), vascular cylinder (VC), cortex (C), and epidermis (E) are evident. A few root hairs (arrowheads) have developed. Fig. 17. First order lateral similar to that in Fig. 5. A paraepidermal Hartig net (arrowheads) and a homogeneous mantle (M) are present. Fig. 18. First order lateral similar to that in Fig. 6 showing subapical enlargement of the root with a well-developed mantle (M) confined to this region. Cortical (C) and epidermal (E) cells in this region have enlarged in the radial direction.
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A sequence of micrographs from longitudinal sections of mycorrhizal first-order lateral roots from the root tip to the basal portion of the mycorrhiza reveals the progression of fungal penetration through the host tissues (Figs. 19-24). Inner mantle hyphae that have barely started to penetrate between adjacent cap cells are evident in the root cap region (Fig. 19). A little further proximally, inner mantle hyphae surround the cap cells and lie adjacent to epidermal cells (Fig. 20). Farther back, in the young Hartig net zone, hyphae penetrate a short distance between epide&al cells that are much more vacuolate (Fig. 21). Farther back proximally, hyphae penetrate between highly vacuolated epidermal cells (Fig. 22) and eventually reach the midportion of the first layer of cortical cells (Fig. 23). These cortical cells are always col- lapsed (Figs. 22-23). Finally, in the complete Hartig net zone, hyphae start to show increasing vacuolization. Darkly stained deposits, some of which could be remnants of root cap cells, are detectable in the inner mantle (Fig. 24). Granular,
morphologies, one consisting of an evenly thickened mantle along the length of the root and the other consisting of a mantle confined primarily to the apical segment of the root. The type formed is dependent on the stage of lateral root elongation at the time of hyphal colonization. Similar types of ectomycor- rhizae are formed between this same fungus and Altzus crispa (Massicotte et al. 1986) and Pisolithiis tinctorius and Eucalyp- tus pilularis roots (Massicotte et al. 19876). Ectomycorrhizae with evenly thickened mantles show a gradient of interactions that has been described in detail for these associations (Massi- cotte et al. 1986, 1987~) ; the clavate type has been described only briefly. In A. rubra - A. diplophloeus associations, the clavate ectomycorrhiza shows a gradient of fungus-root interaction along the length of the lateral root that differs from the evenly thickened type. At the base of the lateral root, fungal hyphae make contact with root hairs, change their orientation of growth, and partially envelope the elongating hairs. Similar fungus - root hair interactions have been
darkly stained deposits are detectable in the mantle hyphae of described recently for a number of gymnosperm and angio- the last three zones (Figs. 22, 23, 24). There are conspicuous sperm ectomycorrhizae (Massicotte et al. 1987a) and may be hyphal branchings in the later stages (Figs. 23 and 24). more common than previously realized. Since more distal por-
k sequence oftransverse sections of mycorrhizal first-order tions of A. rubra roots are surrounded completely by hyphae lateral roots from the root tip to the basal portion of the mycor- forming a thick mantle, any root hairs in this region are incor- rhiza reveals other aspects of fungal interaction with the sur- porated into the mantle. Presumably these root hairs collapse rounding tissues (Figs. 25-31). In the root cap zone, most after fungal colonization, but this needs to be documented. root cells, except for the cap cells, have few vacuoles and a Therefore, in the clavate type, two zones can be outlined: one thick mantle is already established (Fig. 25). Inner mantle similar to the evenly thickened types showing host cells with hyphae are located between the root cap cells up to adjacent mycorrhizal status and a transitory zone, proximal to the first epidermal cells (Fig. 26). Some darkening in the cortical cells one, showing cells that had undergone a certain amount of is discernible (Fig. 26). In the young Hartig net zone, the root differentiation prior to fungal colonization. In this latter zone, cap cells are collapsed and most cortical cells are vacuolated root hair interactions occur. (Fig. 27). The inner mantle hyphae have surrounded the root The basic organization of A. rubra - A. diplophloeus cap cells and have penetrated midway along the epidermal ectomycorrhizae as shown by light microscopy is similar to radial walls (Fig. 28). Farther back, in a complete Hartig net other Alnus mycorrhizae synthesized in the laboratory (Molina region, most root cells including the epidermis are vacuolated 1979, 1981; Godbout and Fortin 1983; Massicotte et al. 1986) (Fig. 29). The Hartig net hyphae have penetrated midway or collected from the field (Masui 1926; Neal et al. 1968; along the radial walls of cortical cells (Fig. 30). An obvious Froidevaux 1973). A line drawing of an A. japonica ectomy- darkening is now present in cell walls of the inner cortex corrhiza collected from the field (Masui 1926) is remarkably (Fig. 30). Farther back proximally, the vascular cylinder similar to an A. rubra - A. diplophloeus ectomycorrhiza. In shows early cambial activity and numerous dense granular A. rubra, Hartig net hyphae penetrate midway along the radial deposits, presumably polyphosphate granules, are present in walls of the first layer of cortical cells. Molina (1979, 1981) the mantle (Fig. 31). Occasionally, second-order lateral root states that for A. rubra and four other species of Alnus, the primordia develop in the region of well-developed Hartig net Hartig net formed by A. diplophloeus penetrates only the outer of first-order mycorrhizal laterals (Fig. 32). cortical cell layer. On the other hand, Godbout and Fortin
(1983) report that this fungus penetrates only between epider- mal cells b f A. crispa and-A. rugosa. Line drawings of ~ b z u s
Discussion viridis mycorrhizal roots collected from the field show the The growth pouch technique is particularly suited for the Hartig net of at least one subtype to be present between cells
growth of actinorhizal seedlings such as Alnus rubra, since in the first cortical layer (Mejstrik and Benecke 1969). there is no requirement for nitrogen in the nutrient solution However, regardless of the Altzus species, the Hartig net is and therefore contamination in the pouches is reduced. Lateral restricted to the peripheral 1 -2 layers of the root. This feature roots close to the inoculum plugs of Alpova diplophloeus emphasizes again the importance o f an ontogenetic approach, become colonized and form ectomycorrhizae of two distinct since the same mycorrhizal root has a Hartig net developing
FIGS. 19-24. Sequence of longitudinal sections of mycorrhizal first-order lateral roots from the root tip to the basal portion of the mycor- rhiza. The apical meristem is situated at the left in each figure. Fig. 19. Portion of root tip at level of root cap. Inner mantle hyphae (arrowheads) barely started to penetrate between adjacent cap cells (9). A thick mantle (M) has already formed. Fig. 20. Inner mantle hyphae (arrowheads) have surrounded root cap cells (9) and are adjacent to epidermal cells (E) with few vacuoles. Fig. 21. Hyphae (arrowheads) have penetrated a short distance between epidermal cells that have large vacuoles. Pairs of epidermal cells (arrows) and root cap cells (*) are evident. Fig. 22. Hyphae (arrowheads) have penetrated between highly vacuolated epidermal cells. Fig. 23. Hyphae (arrowheads) have penetrated to the midportion of the first layer of cortical cells (C). These cortical cells always show collapse after fixation. Hartig net hyphae show conspicu- ous branching (double arrowheads). E, epidermal cells. Fig. 24. Complete Hartig net formation. Hyphal branching (double arrowheads) and the accumulation of dense deposits (arrowheads) are evident. E, epidermal cells; C, cortical cells.
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FIGS. 31 and 32. Portions of the base of mycorrhizal roots. Fig. 31. Transverse section showing a thick mantle with dense granular deposits (arrowheads), present in the hyphae, a vacuolated epidermis (arrows), and cortex (C). The vascular cylinder shows early cambial activity (double arrowheads). Fig. 32. Longitudinal section showing a lateral root primordium (*) in the region of well-developed Hartig net (arrowheads).
from paraepidermal to semiparacortical. The restriction of fungal penetration to midway along the
radial walls of cells in the outer cortical layer as observed in this study suggests that a barrier may be present in this region. Recent work with herbaceous plant roots has shown the presence of a suberized Casparian band in this layer (Peterson et al. 1982). This needs to be investigated for Alnus species. The consistent collapse of this layer of cortical cells during fix- ation is indirect evidence that the wall of this layer in A. rubra is modified. This is similar to observations of Eucalyptus pilularis - Pisolithus tinctorius ectomycorrhizae (Massicotte et al. 19876). Another observation that requires further inves- tigation is the development of electron opacity in the walls of the second layer of cortical cells in A. rubra roots before fungal penetration to form the Hartig net. A similar wall change was noted in the first layer of cortical cells in A. crispa - A. diplophloeus ectomycorrhizae (Massicotte et al. 1986).
Hartig net hyphae in A. rubra - A. diploplzloeus ectomy- corrhizae form a highly branched finger-like system described in detail for Picea abies mycorrhizae (Blasius et al. 1986; Kottke and Oberwinkler 1986) and Dryas integrifolia - Hebe-
lorna cylindrosporum ectomycorrhizae (Melville et al. 1987). Signals to trigger this profuse branching must reach the fungus soon after hyphal contact with the epidermis because a redirec- tion of growth involving loss of apical dominance occurs in the root cap - epidermis region. It has been suggested recently (PichC et al. 1988) that this reorientation of growth may be a reliable marker to indicate a compatible interaction between fungus and host.
The overall direction of hyphal penetration through the middle lamella is radial, a feature emphasized for Picea abies (Blasius et al. 1986). However, it is likely that some branching in the tangential direction also occurs. Second-order lateral root primordia are produced in the mature Hartig net zone of A. diplophloeus - A. rubra ectomycorrhizae, a feature described in detail for Pisolithus tinctorius - Eucalyptus pilularis (Massicotte et al. 19876). The effect of lateral root primordia on the physiology of ectomycorrhizae has not been studied.
In Alnus rubra - Alpova diplophloeus mycorrhizae, numer- ous dense granular deposits (probably polyphosphate) are found in the mantle hyphae when fungal penetration has
FIGS. 25 -30. Transverse sections of mycorrhizal first-order lateral roots in sequence from the apex basipetally . Fig. 25. Root cap zone show- ing a mantle (M), root cap cells (arrowheads), epidermal cells (arrows), cortical cells (C), and the vascular cylinder (VC). Fig. 26. Enlargement of a portion of Fig. 25. Inner mantle hyphae (arrowheads) have penetrated between root cap cells (*) up to adjacent epidermal cells (E). Darken- ing (double arrowheads) of cortical cell walls is discernible. Fig. 27. Root cap cells (arrowheads) have collapsed and most cortical cells (C) are vacuolated. Fig. 28. Enlargement of root shown in Fig. 27. Inner mantle hyphae have surrounded root cap cells (q) and have penetrated midway along the epidermal cell walls (arrowheads). Fig. 29. Most root cells, including the epidermis, are vacuolated and the mantle (MI is very thick. Fig. 30. Enlargement of a portion of root in Fig. 29 showing Hartig net hyphae (arrowheads) that have penetrated midway along the radial cell walls of cortical cells. An obvious darkening of cortical cell walls has occurred (double arrowheads).
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reached the second layer of cells. These granules are still present in the mantle at the level in the root at which the cen- tral vascular cylinder shows early signs of cambial activity. This probably indicates that the hyphae in the thick mantle in this region are involved more in storage than in transfer between the symbionts.
Acknowledgements This research was supported by the Natural Sciences and
Engineering Research Council of Canada and Formation d e Chercheurs et Aide i la Recherche. W e thank Dr. Randy Molina, Corvallis, for supplying the fungus isolate and the Petawawa National Forestry Institute for supplying A. rubra seeds. We thank Carol Pratt for typing the manuscript.
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