AXENIC SYNTHESIS OF ERICOID MYCORRHIZA IN VACCINIUM ANGUSTIFOLIUM AIT. BY OIDIODENDRON SPECIES

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1 New Phytol. (1986) 13, AXENIC SYNTHESIS OF ERICOID MYCORRHIZA IN VACCINIUM ANGUSTIFOLIUM AIT. BY OIDIODENDRON SPECIES BY Y. DALPE Biosystematics Research Institute, Central Experimental Farm, Agriculture Canada, Ottawa, Canada {Accepted 31 January 1986) SUMMARY Oidiodendron griseum, O. cerealis, O. rhodogenum, O. tenuissimum and O. truncatum were tested for their endophytic ability on axenic seedlings of Vaccinium angustifotiiim grown on a synthetic nutrient medium. O. griseum, O. rhodogenum and O. cerealis produced typical intracellular hyphal coils characteristic of the ericoid infection. For these three species, the level of infection reached 9 to 21 % of the root cells after 6 d of incubation. O. truncatum and O. tenuissimutn did not infect the Vaccinium roots but the latter species stunted development of secondary roots and reduced the growth of the seedlings. Key words: Ericoid mycorrhiza, Vaccinium aiigustifoliuni, Oidiode?idroii griseum, Oidiodendron cerealis, Oidiodendron rhodogenum, Oidiodendron tenuissimum, Oidiodendron truncatum. INTRODUCTION First isolated from wood pulp and decayed logs, Oidiodendron species have been considered to be common soil-borne fungi regularly associated with a diversity of soil habitats (Domsch, Gams & Anderson, 198). Recently, Couture, Fortin & Dalpe (1983) confirmed theability of O.^mezyw Robak to form ericoid mycorrhizas with aseptically grown Vaccinium species, an ability previously suggested for the same species by Burgeff (1961) with Calluna sp. and other species of the genus Vaccinium. Only one other endophyte of ericoid mycorrhiza, Hymenoscyphus ericae (Read) Korf & Kernan (Kernan & Finocchio, 1983) = Pezizella ericae (Read, 1974) has been identified (Pearson & Read, 1973). There is also a possibility that species of Clavaria may be symbiotic with Ericaceae (Peterson, Mueller & Fnglander, 198). To explore potential associations between soil fungi and the Ericaceae and to provide data for the correct identification of fungal symbionts, the capacity of four other species of Oidiodendron to synthesize ericoid mycorrhiza in axenic cultures of Vaccinimn angustifolium Ait. was investigated. MATERIALS AND METHODS Plant and fungal material Seeds of Vaccinium angustifolium were extracted from frozen fruits and then surface-sterilized for 15 min in an aqueous Chloramine T (2%) solution with a drop of Tween 8 as a wetting agent. The seeds were placed on potato dextrose agar (PDA) in petri plates for a test of sterility and later transferred to a growing tube for germination and seedling development. The synthetic medium was slightly X/86/ $3./ 1986 The New Phytologist

2 392 Y. DALPE modified from Mitchell & Read (1981): NH^Cl, 32 mg; CaCl^ yh^o, 43-5 mg; MgSO^ 7H.,O, 1 mg; KCl, 5-5 mg; FeCla, 3-75 mg; sucrose, 2 g; KH.^PO^, 21 mg; pyridoxine, 1/^g T^; thiamine, \ fig 1"^; agar (Difco), 1 g; distilled water, 1 ml. Final ppi before autoclaving was 6-. The following isolates of Oidiodendron, each representing a difterent species, were tested for their ability to infect Vaccinium angustifolium roots in vitro: O. cerealis (Thum.) Barron, DAOM , isolated from washed organic soil particles, O. rhodogenum Robak, DAOM 75838, substrate unknown, O. tenuissimum (Peck) Hughes, DAOM b, isolated from rotting Pinus, and O. truncatum Barron, DAOM 17697, isolated from a Pinus log. Mycelium from PDA cultures of the growing fungi was inoculated onto the agar surface of the modified Mitchell & Read media approximately 5 d before the transfer of the surface-sterile seeds. Uninoculated control tubes containing only the host, and 1 replicate tubes for each species of Oidiodendron with the host were sealed with 'Parafilm' and maintained at C in a growth chamber with a daylength of 12 h at an irradiance of 1 lux. The lower portion of the tubes was painted black to prevent light reaction of the roots. Macroscopic and microscopic observations Seedlings 2, 3, 4, 9 and 12 d old were carefully removed from their tubes and their entire root system observed and measured before preparation for microscopic investigations. Roots were cleared in 1 % KOH for about 15 min, washed in 1 % HCl and water and mounted in Gurr's medium. Normarski Differential Interference Contrast permitted the detection of the presence of intracellular infection and extramatrical hyphae. Staining with Malachite Green and acid Fuchsin (Alexander, 198), resulting in pink fungal structures against pale green root cells, confirmed the fungal nature of the observed infection. The level of infection was calculated as a percentage of infected cells from observations on at least a hundred adjacent root cells. RESULTS Fungal and seedling development All five Oidiodendron isolates grew readily on PDA and produced abundant conidiophores and conidia. However, on the medium used for mycorrhiza synthesis only, vegetative filaments were formed. Mycelia rarely penetrated further than 3 mm into the solid substrate. The presence of -2% sucrose did not affect the development of the seedlings. About 75% of the seeds of V. angustifolium germinated in 12 d. Presence or absence of Oidiodendron did not affect the germination rate. Seedlings attained heights between 8 and 15 mm after 2 d of incubation. The first true leaves occurred around the 3th day with additional ones appearing every 8 to 1 d. After four months, the entire plant reached a height of 18 to 25 cm with a well developed root system made up of very fine secondary and main roots. The Vaccinium seedlings remained healthy for at least three months without showing any external morphological deformity except in the case of roots associated with O. tenuissimum (Figs 6 and 7). Root colonization Three of the five Oidiodendron isolates tested produced intracellular hyphal

3 Oidiodendron spp. and ericoid mycorrhisas s.' 4jurT. ^' 1 Figs 1 to 3. Mycorrhiza formed by Oidiodendron species on axenic cultures of Vaccinium angustifolium. Fig. 1. Oidiodendron griseum. Fig. 2. Oidiodendron rlwdogenum. Fig. 3. Oidiodendron cereahs. Fig. 4. Vaccinium angustifolium seedlings infected with Oidiodendron truiicatum after 6 d of incubation. Fig. 5. Pigmented filaments of Oidiodendron griseum surrounding infected roots. Fig. 6. Vaccinium angustifolium seedlings infected with Oidiodendron tenuissimum after 6 d of ineubation. Fig. 7. Roots of a seedling infected with Oidiodendron tenuissitnutn.

4 394 Y. DALPE coils that are considered to be the typical structure of a symbiotic association in ericoid mycorrhiza. The three endophytes were: O. griseum, O. cerealis and O. rhodogenum. Maximum infection rates were 9% for O. rhodogenum, 12% for O. cerealis and 21 % for O. griseum. Infection was first observed around the 4th day of incubation when the seedlings had three to four leaves and were about 5 mm high (Table 1). The level of infection increased until the 6th day. The ratio of infected cells then decreased from the 6th to the 12th day while the root system continued to expand. Maximum stem growth occurred between the 6th and the 12th day with an average increase in height of 2 mm each day. Table 1. Percentage of intracellular infection of the whole root system of Vaccinium angustifolium by Oidiodendron species Days of incubation griseum cerealis rhodogenum tenuissimum truncatum Pigmented hyphae of O. griseum and O. cerealis were readily detected along the roots (Fig. 5) whereas hyphae of O. rhodogenum were more difficult to observe because of their smaller size and lack of pigmentation. The fungal filaments surrounded almost the entire root system but microscopic observation showed that infection was confined to the large cortical cells of secondary roots having a diameter of not more than 12/tm (Figs 1, 2 and 3). Primary roots were never infected. In the three infective species the intraradical infection was made up of chains of rounded to ovoid cells 1 to 4 /i-m in diameter. The chains of swollen hyphae were at first appressed to the root cell wall and later grouped loosely at the middle of the cell or dispersed in almost the entire cell volume showing the characteristic hyphal coil. O. rhodogenum produced a very delicate net of intracellular swollen hyphae (1 to 2 /ira) compared to O. griseum and O. cerealis (hyphae 1-5 to 4- /im.) corresponding to the narrower and more delicate extramatrical hyphae of the former species. O. truncatum did not affect the growth or the morphology of the root system in Vaccinium (Fig. 4). The presence of O. tenuissimum gave rise to a restricted growth of the entire seedling (maximum height of 7-85 mm) (Fig. 6), particularly evident by the early abortion of secondary root development (Fig. 7). O. tenuissimum surrounded the primary roots and the short secondary branches with a hyphal mantle 2 to 5 /tm thick somewhat like that of an ectomycorrhizal fungus but without showing any intracellular structure related to parasitism or mycorrhizas. DISCUSSION O. griseum, recently described as an endophyte of ericoid mycorrhiza (Couture et al., 1983) can produce typical colonization of Vaccinium roots on a synthetic

5 Oidiodendron spp. and ericoid mycorrhizas 395 nutrient medium. This species has been considered, as have most other species in the genus, to be a soil-borne fungus and it is reported from forest soils (Barron, 1962; Morrall & Vanterpool, 1968; Singh, 1976); pine needles (Kendrick, 1963); conifer swamps (Christensen, Whittingham & Novak, 1962); humus (Tokamasu, 1976); and rhizosphere of both herbaceous and woody plants (Mangan, 1967; Parkinson & Crouch, 1969). The ability of three of the five species to produce typical ericoid mycorrhizas on Vaccinium seedlings suggests that the role of some soil fungi may differ depending on environmental factors. O. cerealis and O. griseum, for example, in addition to being root endophytes are good cellulose decomposers (Robak, 1932; Jefferys et al., 1953; Mangenot, 1952). Oidiodendron species have been identified as anamorphs of Gymnoascaceae, with teleomorphs in the genus Arachniotus (Barron & Booth, 1966), Myxotrichum aerugiiwsum (Malan, 1949) and Toxotrichum cancellatum (Orr & Kuehn, 1964). Together with the first identified ericoid endophyte, Hymenoscyphus ericae, (Helotiales), they are the only organisms known to produce typical ericoid mycorrhizas in vitr In the present experiment, the presence of growing mycelia of O. cerealis, O. griseum and O. rhodogenum appeared to have no stimulatory eflfect on development and external morphology of Vaccinium seedlings when compared to non-inoculated controls. However, the host colonization obtained with O. cerealis and O. rhodogenum was quite comparable to that seen in O. griseum in terms of the chronology and morphology of the infection. Inoculation with O. tenuissimum slowed the growth of the seedlings and altered root morphology. There appears to be no previous mention of parasitic behaviour for this hyphomycete (Domsch etal., 198). According to Haselwandter (1979), restrictive growing conditions such as low annual growing season temperature and a thin organic layer in the soil are mainly responsible for the poor level of root infection in Ericaceae from natural alpine ecosystems. Compared to the high proportion (59 to 7%) of mycorrhizal roots recorded by Read & Stribley (1975) from native plants of Calluna growing in a suitable environment, the levels of infection observed in the in vitro studies of Oidiodendron are low. However this might be expected in synthetic media because the growth of the fungus is restricted to the upper 3 mm of the medium. It would obviously be desirable to employ thinner layers of medium for future experiments on synthesis. The high level of Vaccinium seed germination and their ready adaptation to such drastic growing conditions makes the in vitro axenic seedling culture a convenient technique to verify mycorrhizal potential of fungi. It provides a reproducible and easily viewed growing environment and a clean root system suitable for a rapid microscopic survey or ultrastructural analysis. REFERENCES ALEXANDER, M. P. (198). A versatile stain for pollen, fungi, yeast and bacteria. Stain Technology, 55, BARRON, G. L. (1962). New species and new records of Oidiodendron. Canadian Journal of Botany, 4, BARRON, G. L. & BOOTH, C. (1966). A new species of Arachniotus with an Oidiodendron conidial state. Canadian Journal of Botany, 44, BURGEEF, H. (1961). Mikrobiologie des riochmoores. Fischer-Verlag, Stuttgart. CHRISTENSEN, M., WHITTINGHAM, W. F. & NOVAK, R. O. (1962). The soil microfungi of wet mesic forests in southern Wisconsin. Mycologia, 54,

6 396 Y. DALPE COUTURE, M., FORTIN, J. A. & DALPE, Y. (1983). Oidiodendron griseum Robak: an endophyte of ericoid mycorrhiza in Vaccinium spp. New Phytologist, 95, DM.SCH, K. H., GAMS, W. & ANDERSON, T. H. (198). Compendium of Soil Fungi, vol. 1, pp Academic Press, London. HASEI.WANDTER, K. (1979). Mycorrhizal status of ericaceous plants in alpine and subalpine areas. New Phytologist, ^7,, JEFFERYS, E. G., BRIAN, P. W., HEMMING, H. G. & LOWE, B. (1953). Antibiotic production by the microfungi of acid heath soils. Journal of General Microbiology, 9, KENDRICK, W. B. (1963). Fungi associated with breakdown of pine leaf litter in the organic horizon of a podzol. Mycopathologia et mycologia applicata, 19, KERNAN, M. J. & FINOCCHIO, A. F. (1983). A new discomycete associated with the roots of Monotropa uniflora (Ericaceae). Mycologia, 75, MALAN, C. E. (1949). Su un nuovo repertodi Dicyma ambigua Peyronel esulla posizionc sistematicadi questa specie. Nuovo Giornale Botanico Italiano n.s. 56, MANGAN, A. (1967). Studies on wheat rhizosphere soil fungi. Ireland Journal of Agriculture Research, 6, MANGENOT, F. (1952). Recherches methodiques sur les champignons de certains bois en decomposition. Revue generate de Botanique, 59, 115 pp. MITCHELL, D. T. & READ, D. (1981). Utilization of inorganic and organic phosphates by the mycorrhizal endophytes of Vaccinium macrocarpon and Rhododendron ponticum. Transaction.-: of the British Mycological Society, 76, MoRRALL, R. A. A. & VAN-IERPOOL, R. C. (1968). The soil microfungi of upland horeal forest at Candle Lake, Saskatchewan. Mycologia, 6, ORR, G. F. & KuEHN, H. H. (1964). A reevaluation of Myxotrichum spinosum and M. canceltatum. Mycologia, 56, PARKINSON, D. & CROUCH, R. (1969). Studies of fungi in a pine-wood soil. 5. Root mycofloras of seedlings of Pinus nigra var. larici Revue d'ecologie et de Biologie des sols, 6, PEARSON, V. & READ, D. H. (1973). The biology of mycorrhiza in the Ericaceae. 1. The isolation of the endophyte and synthesis of mycorrhizas in aseptic culture. New Phytologist, 7, PETERSON, T. A., MUELI^ER, W. C. & ENGLANDER, L. (198). Anatomy and ultrastructure of a Rhododendron root-fungus association. Canadian Journal of Botany, 58, READ, D. J. (1974). Pezizella ericae sp. nov., the perfect state of a typical mycorrhizal endophyte of Ericaceae. Transactions of the British Mycological Society, 63, READ, D. J. & STRIBLEY, D. P. (1975). Some mycological aspects of the biology of mycorrhiza in the Ericaceae. In Endomycorrhizas (Eds E. E. Sanders, B. Mosse & P. B. Tinker), pp Academic Press, London. ROBAK, 11. (1932). Investigations regarding fungi of Norwegian ground wood pulp and fungal infections at wood pulp mills. Saertrykk av nyt Mag. for naturvideeskaberne, 71, SINGH, P. (1976). Some fungi in the forest soils of Newfoundland. Mycologia, 68, ToKAMASU, S. (1976). On the natural habitat of Oidiodendron. Transactions of the Mycological Society of Japan, 17,

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