Mycorrhizae of Alnus Rubra' Some Ectotrophic

u r s • Some ectotrophic ,,r. °VieCi"14 mycorrhizae of Alnus rubra

L. Neal, Jr., 2 J. M. Trappe, Abstract C. Lu, and W. B. Bollen Forestry Sciences Laboratory Two forms of mycorrhizae predominated on root systems of red alder Pacific Northwest Forest and (Alnus rubra Bong.) in a pure stand near the Oregon coast. Detailed morpho- Range Experiment Station logical studies, the first for this species, revealed distinct characteristic differ- Corvallis, Oregon ences between the fungal symbionts. The great abundance of these mycorrhizae and their immediate influence on rhizosphere microbes could markedly affect the incidence of root disease.

Introduction Red alder is unique among the trees of the Pacific coast because its roots participate in either one of two major kinds of symbiosis: root nodules formed with nitrogen-fixing endophytes, presumably Streptomyces, and mycorrhizae formed with certain fungi. Nodulation not only increases avail- able nitrogen but also profoundly affects other soil chemical and micro- biological properties (Tarrant, 1961; Chen, 1965). The mycorrhizal fungi increase alders nutrient-absorbing capability and affect it in other, less understood ways. Both kinds of symbiosis bear possible implications for minimizing incidence of root diseases when red alder is a component of forest stands (Zak, 1964; Li et al., 1967; Marx and Davey, 1967). Our interest in rhizosphere phenomena of red alder led us to investigate its mycorrhizae in a pure stand about 40 years old near the northern Oregon coast in the Cascade Head Experimental Forest (maintained by the U. S. Forest Service, Pacific Northwest Forest and Range Experiment Station). Vegetation, soils, and soil microbial activity in this stand are described in preceding papers of this symposium. Methods and Materials Mycorrhizae collected in spring and autumn 1965 were cleaned ultra- sonically, fixed in a chrome-acetic acid solution, paraffin-embedded, sec- tioned at 8 to 10n thickness, and stained with safranin-fast green. Fresh, whole specimens were saved for examination of gross morphology. Nonmycorrhizal rootlets, desired as a base for evaluating effects of mycorrhizal infection on rootlet anatomy, could not be found in the stand. This study %vas supported in pant by National Science Foundation Grant GB-3214. The findings resulted in part from research for a doctoral dissertation by the senior author while at Oregon State University.

2Dr. Neals present address is Research Station, Canada Department of Agriculture, Lethbridge, Alberta, Canada.

179 Accordingly, red alder seeds were surface-sterilized (Neal et al., 1967), germi- nated, and grown in pure culture to obtain infection-free roots. Rootlet Anatomy and Morphology The nonmycorrhizal rootlets had an epidermis of cells 5 to 10 x 8 to 16u in cross-sectional dimensions (Fig.IA). Root hairs 6 to 9u broad originated sporadically from this outer layer of cells (Fig. 1 B). Cortical cells underlying the epidermis ranged from 15 to 27 x 15 to 35 u in diameter, and endo- dermal cells from 3 to 7 x 7 to 12 u. The xylem was monarch. Two forms of alder mycorrhizae predominated in the collections. A third form occurred only infrequently, so it was not included in the study. We found no mycorrhizae formed with the fungus Cenococcum graniforme (Sow.) Ferd. et Winge, the only type heretofore reported for red alder (Trappe, 1964). One common form of mycorrhizae, comprising about 40 percent of those collected, was generally clavate with a dark-brown, distinctively roughened fungal mantle (Fig. 2, left) which was commonly ruptured by apical growth of the enclosed rootlet (Fig. 2, middle). This mantle sloughed off when roots were removed from the soil. Two layers of tissue comprised the mantle (Fig. 3, left), which totaled 40 to 60u in thickness. The outermost layer, 30 to 45u thick, was formed of irregular, more or less isodiametric, thick-walled cells 8 to 12 u broad (Fig. 3, left A). Irregular collapse of the peripheral cells of this layer accounts for the mycorrhizas surface roughness. The inner mantle layer, 10 to 15u thick, was composed of thin-walled hyphae 2 to 5 u in diameter, aligned predomi- nantly along the root axis (Fig. 3, left B). The Hartig net penetrated only the epidermis (Fig. 3, left C), separating epidermal cells by one layer of hyphae 1.5 to 2.5 u thick. The outermost root cells ranged from 15 to 27u x 11 to 20 u in cross- sectional diameter; cortical cells, 30 to 85 u x 30 to 40 u ; and endodermal cells, 10 to 20u . The xylem was triarch.

Figure 1. Cross section of noinnycorrhizal red alder root tip: epidermis; root hair.

180 I 14. Figure 2. (Left) Clavate red alder mycorrhiza with dark-brown fungal mantle. (Middle) Mantles of dark-brown mycorrhizae ruptured by apical growth of enclosed rootlet. (Right) Red alder mycorrhiza with pale-brown, glabrous mantle.

Septate hyphae, averaging about 5 Li in diameter and having walls about hi thick, sometimes originated from turgid cells at the mantle surface (Fig. 3, middle). Clamp connections were present at some septa of these hyphae. Therefore, the fungus was a Basidiomycete.

Figure 3. (Left) Cross section of mantle and outer root cortex of dark-brown, clavate, red alder mycorrhiza: A, outer mantle layer; B, inner mantle layer; C, Hartig net. (Middle) Longitudinal section showing hypha originating from outer mantle cell of dark-brown mycorrhiza. (Right) Cross section of mantle and outer root cortex of pale, glabrous mycorrhiza: A, single-layered mantle; B, opaque, amorphous layer; C, Hartig net.

181 The other common form of mycorrhiza (Fig. 2, right), comprising about 50 percent of those collected, was pale brown and glabrous. No mycorrhizal mantle was obviously present under low-power magnification, but side-by-side comparison with uninfected roots revealed distinct differ- ences, especially in color. The mantle, 12 to 25 u thick, constituted a single-layered prosenchyma tightly adhering to the rootlet surface and aligned predominantly with the root axis (Fig. 3, right A). Mantle hyphae were 2 to 2.5 p. in diameter. A single-layered Hartig net (Fig. 3, right C), 2 to 3 o broad, sporadically penetrated the rootlet epidermis. Consequently, we consider this mycorrhiza weakly ectotrophic, even though a true Hartig net was lacking from much of the fungus-root interface. A layer of opaque, amorphous, red-stained mate- rial, perhaps tannin, was often present between the epidermis and the mycorrhizal mantle (Fig. 3, right B). The outermost root cells varied from 10 to 30 o in diameter; cortical cells, 30 to 65u ; and endodermal cells 9 to 18 o . The xylem was triarch. We could not classify the fungus participating in this form of mycorrhiza for lack of distinctive, morphological characteristics.

Discussion The lack of root hairs on the mycorrhizae is the only anatomical change that can be attributed to mycorrhizal infection, considering that the mycorrhizae were collected from 40-year-old trees and the nonmycorrhizal rootlets from seedlings grown in pure culture. The outermost tier of root cells on the mycorrhizae, though smaller than the cortical cells, did not appear to be a typical epidermis in comparison with that of nonmycorrhizal roots. We cannot say, however, whether this difference is due to mycorrhizal infection or whether it merely reflects the difference in age and environment of the mycorrhizal versus nonmycorrhizal roots. Masui (1926) illustrated a similar phenomenon in his paper on mycorrhizae of Japanese alders, as did Klaka and Vukolov (1935) for Alnus incana (L.) Moench. No counterpart of the rough, brown mycorrhiza of red alder has been reported for other Alnus species, but the glabrous form resembles a mycorrhiza of Alnus incana in Europe (Kleeka and Vukolov, 1935). All have a Hartig net that penetrates only the outer tier of root cells as did several other forms of ectotrophic mycorrhizae described for Japanese Alnus species (Masui, 1926). This shallow penetration by the Hartig net appears to be characteristic of Alnus species, in contrast to the deeper penetration frequent in ectotrophic mycorrhizae of many other tree species. Sporocarps of fungi fruiting in the stand were collected over the course of 18 months. Of the 30 terrestrial species found, only 5 were likely to form mycorrhizae: Alpovah cinnamomeus Dodge, Hymenogaster alnicola A. H. Smith, Lactarius obscuratus (Larch) Fr., and two Inocybe species. We cannot say with certainty if any of these is the fungal symbiont of either of the mycorrhizae described. Lactarius obscuratus, however, fruits abundantly in

182 the stand and has basal hyphae similar to those forming the mantle of the glabrous mycorrhiza. Different forms of mycorrhizae can harbor populations of rhizosphere organisms which differ distinctly in certain physiological activities (Neal et al., 1964 and 1968). Undoubtedly, the two abundant forms of red alder mycorrhizae described here strongly influence rhizosphere populations and possibly the general soil microflora. Through this influence, the mycorrhizae could markedly affect the incidence of root disease.

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