Scanning Microscopy Volume 1 Number 3 Article 59 5-20-1987 Morphological Variations in Developing Ectomycorrhizae of Dryas integrifolia and Five Fungal Species L. H. Melville University of Guelph H. B. Massicotte University of Guelph R. L. Peterson University of Guelph Follow this and additional works at: https://digitalcommons.usu.edu/microscopy Part of the Life Sciences Commons Recommended Citation Melville, L. H.; Massicotte, H. B.; and Peterson, R. L. (1987) "Morphological Variations in Developing Ectomycorrhizae of Dryas integrifolia and Five Fungal Species," Scanning Microscopy: Vol. 1 : No. 3 , Article 59. Available at: https://digitalcommons.usu.edu/microscopy/vol1/iss3/59 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Scanning Microscopy, Vol. 1, No. 3, 1987 (Pages 1455-1464) 0891-7035/87$3.00+.00 Scanning Microscopy International , Chicago (AMF O'Hare), IL 60666 USA MORPHOLOGICALVARIATIONS IN DEVELOPINGECTOMYCORRHIZAE OF DRYASINTEGRIFOLIA AND FIVE FUNGALSPECIES L.H. Melville, H.B. Massicotte, and R.L. Peterson* Department of Botany, University of Guelph, Guelph, Ontario, Canada NlG 2Wl (Received for publication February 20, 1987, and in revised form May 20, 1987) Abstract Introduction A comparative study of ecto­ A mycorrhiza is a symbiotic mycorrhiza formation between the host association between a plant root and a species~ integrifolia and four fungus. In a mature ectomycorrhiza, fungal species belonging to the roots become colonized by fungal hyphae Basidiomycotina ( Laccaria bicolor, L. to form a mantle, and a Hartig net, a laccata, Hebeloma c)lindrosporum, network of fungal hyphae which develop Paxillus involutus and one species around epidermal, and in some species, in the Ascomycotina (Cenococcum cortical cells. Penetration into the geophilum) showed that patterns in root stele is impeded by the endodermis, ectomycorrhizal development were a layer of cells enveloping the vascular distinctive enough to characterize cylinder. The extent of mantle and each fungal symbiont. Several aspects Hartig net formation depends on both of hyphal growth on the plant root the fungal and plant species involved. during colonization were studied in Characterization of mantle order to demonstrate the usefulness of morphology of mature ectomycorrhizae SEM in observing the ontogeny of has been carried out using the light ectomycorrhizae. Each fungal species microscope ( 7). Al though light had a different rate of colonization, microscopy allows determination of and varying root and root hair gross morphology and mantle colo~r the interactions. As a consequence mantle limited resolution does not permit morphology and the overall appearance observation of fine detail. The scanning of mature ectomycorrhizal apices were electron microscope has been used also characteristic for each fungal frequently to study the topography of species. Results indicate that the mature ectomycorr hizae (2,3,11,12,15,20, mycobiont plays a major role in 22) that might be useful in classifying determining the morphology of mature mycorrhizal types (26). The interface ectomycorrhizae. between fungal and plant cells (l,5,8,ll,16,19,23,24) and inclusions in the plant cells (9,29) have been studied in fractures of mature mycorrhizae using SEM. Some studies have documented the early stages of ectomycorrhizal formation (4,18,19,21,28), but interactions between the fungus and the root from early ~tages through to full mantle development remain largely unstudied. Although studies of different KEYWORDS: Dryas, ectomycorrhi zae, roots, mycorrhizae formed on a single plant scanning electron microscopy. species have been carried out (1,26), SEM has not been used in such studies to follow the morphogenesis of the fungal mantle. In the following study, *Address for correspondence: ectomycorrhizae were produced between Department of Botany Dryas integrifolia, a boreal member of University of Guelph tlieRosaceae, and five fungal species. Guelph, Ontario, Canada Samples were fixed for SEM at different NlG 2Wl Phone No. (519) 824-4120, Ext. 3278 stages of lateral root development and 1455 Melville , L.H., Mass icotte , H.B. , Pete rso n, R.L. funga l colonization to ascertain the 1 h, and post fixed again in 2% Os04 role of the fungal symbiont in for 1 hat 4C. The tissue was dehydrated ectomycorrhiza formation. in a graded ethanol series, critical point dried, mounted on stubs, and coated with gold- palladium. Samples were observed with a JEOL JSM 35-C Materials and Methods scanning electron microscope at 15 kV. Some mycorrhizal roots were fractured with a Gillette blue razor blade cleaned Dryas integrifolia (Vahl.) seeds in acetone, prior to SEM. collectedin the Cambridge Bay district of the Northwest Territories, Canada, Results were surface sterilized in 10% hydrogen peroxide for 15 min, washed, and Rate of ectomycorrhizal formation germinated on filter paper in a Although~ integrifolia seed­ parafilm-sealed petri dish. Seedlings lings are very small, they grew success­ were transferred into growth pouches fully in plastic growth pouches. (6) containing 10 ml. of modified 1/4 Forty-eight days after germination, strength Crone's mineral solution (10). first order laterals had formed and Forty-eight days after germination, the root systems were ready for and after lateral roots had appeared, inoculation. Ectomycorrhizae were formed seedlings were inocuiated with the with all of the fungal species used, following fungi: but the rate of formation varied; P. Hebeloma cyl indrosporum Romagnesi involutus within three days, H. - (isolate 75-1 from J.A. Fortin's cylindrosporum within five days,!:..:_ laboratory); laccata and L. bicolor within seven Cenococcum geophilum Fr. strain days, and C.geophilum within twenty-one CRBF-00/4; days. Eachgrowth pouch contained Paxillus involutus (Batsch.) Fr mycorrhizal roots at different stages strain CG-9; of development because lateral roots Laccaria bicolor (R. Mre) Orton strain continued to be initiated and CRBF-010 ; subsequently colonized. The first Laccaria lac cata (Fr.) B.and Br. lateral roots to be colonized were strain CRBF-0241. closest to the fungal inoculum; All were previously grown on colonization then spread at varying Melin-Norkrans Modified (MNM) agar rates along the root system from the medium (12) and introduced randomly as point at which first infection occurred. 10 mm diameter plugs into the pouches Colonization in pouches was 100% for within 5 mm of lateral roots (17). all fungal species except f. geophilum, Each fungal species was introduced which was 20%. separately into ten pouches of~ Early mantle development on young seedlings, and ten pouches of seedlings laterals were kept as controls. Early infection of first and second Growth Conditions order lateral roots emerging from a Seedlings were grown under 5 klux 2 previously colonized older root resulted (42 watts/m 2 ) (105 pE/m /sec) light on in various patterns of mantle formation a 16 h light/ 8 h dark cycle with a depending on fungal species. Laccaria temperature regime of 24C/18C (D/N). laccata rapidly formed a compact, High levels of humidity (60-80% R.H.) interwoven, white mantle around the were maintained. Five ml. of 1/2 root apex at the earliest stage of strength Crone's mineral solution (10) lateral emergence, even when few hyphae were added to the pouches weekly. were present on the parent root (fig.l). Ectomycorrhizal roots were collected Colonization by L. bicolor was similar 25 days after the pouches were to that of L. laccata, but more rapid. inoculated. Control roots were collected Hebeloma cylindrosporum hyphae at the same time from non-inoculated spread gradually to the emerging lateral pouches. root in a random fashion, but did not Light microscopy form a well established mantle until Rate of ectomycorrhizal formation the lateral had grown 2-3 mm from the and mantle colour were determined using main root, even when a large number of a Zeiss MC-63 stereophoto- microscope. hyphae were present on the main root Scanning electron microscop~ (fig.2). Cenococcum geophilum followed Samples were fixed in .5% a similar developmental pattern to glutaraldehyde using 0.10 M Hepes buffer that of L. laccata, but the hyphae (pH 6.8)for 3 hat 25C, and then washed were arranged in a more regular pattern in the same buffer. Samples were post (fig.3), grew more slowly, and were fixed in 2% Os04 in Hepes buffer for 2 black. Paxillus involutus rapidly formed hat 4C, rinsed in buffer, treated a loose, superficial, brown mantle with 1% aqueous thiocarbohydrazide for around emerging lateral roots (fig.4). Dryas and five ecto mycorrhi zal fungi Figures 1- 4. Early mantle formation on Fig. 3. Cenococcum geophilum. Coarse, young lateral roots of ~ unbranched hyphae (arrowheads) are integrifolia. present on the primary and lateral root. A young mantle (arrows) has formed along Fig. 1. Laccaria laccata. A young the base of the lateral, but the apex mantle (*) has formed. Few hyphae (*) has few hyphae. (arrowheads) and root hairs (arrows) are evident on the primary root. Fi g. 4. Paxillus involutus • A dense mantle (*) has formed over the surface Fig. 2. Hebeloma cylindrosporum. The of two lateral roots. lateral root apex (*) is free of hyphae but a few hyphae are present at the base of the lateral (arrowheads). Root hairs (arrows) are evident. 1457 Melville, L.H., Massicotte, H.B., Peterson, R.L. Figures 5-9. Mantle development on are present at this stage of mantle elongated lateral roots of ~ formation. Sloughed cap cells (arrows) i ntegrifol i a. are evident. The apex is pointed. Fig. 5. Laccaria bicolor. A dense Fi g. 8. pa Xi l l us i n VOl u tu s • Hyp ha e Of pseudoparenchymatous mantle (*) has the outer mantle (arrowheads) are formed. The ectomycorrhizal apex usually oriented parallel to the root (arrowhead) is rounded.