Plant and Soil 231: 225–232, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 225 Fungal associations of Danish Calluna vulgaris roots with special reference to ericoid mycorrhiza Marianne Johansson∗ Department of Mycology, Botanical Institute, University of Copenhagen, Øster Farimagsgade 2D, DK-1353 Copenhagen K, Denmark Received 13 April 2000. Accepted in revised form 14 December 2000 Key words: Calluna vulgaris, dark sterile fungi, ericoid mycorrhiza, heathland, Hymenoscyphus ericae, Oidiodendron spp. Abstract Fungi were isolated from young, serial-washed roots of Calluna sampled from a Danish heathland, Hjelm Hede. Of the 626 isolates, those that were dark, sterile and septate were divided into 13 morphological groups based on their appearance in culture on malt agar. Mycorrhizal synthesis in vitro showed that several groups formed typical ericoid mycorrhiza with seedlings of Calluna; these ericoid mycorrhizal fungi were morphologically similar to Hymenoscyphus ericae. The identities of the other dark, septate fungi are uncertain. Oidiodendron spp. were isolated in a very low frequency; these fungi also formed typical ericoid mycorrhiza. The Calluna root system on Hjelm Hede demonstrated a high morphological diversity among the associated dark, septate fungi suggesting that more than one fungus could coexist in the same host root system. Introduction Plants of the Ericales living in both northern and southern hemispheres on nutrient-poor heathland soils with slow mineralisation and accumulation of organic polymeric compounds are heavily dependent on ericoid mycorrhiza for nutrient uptake. The ericoid mycorrhizal fungi producing extracellular proteolytic enzymes provide access to organic compounds thereby enhancing nitrogen and phosphorus supply to the host plants (e.g., Smith and Read, 1997). This contributes to the potential competitive advantages of the host plants because they have access to soil organic resources not available to non-mycorrhizal ericaceous plants or other plants in the vegetation (Michelsen et al., 1996). The dark, septate fungi frequently reported from plant roots and soil in arctic, alpine and boreal heathland regions (e.g., Bissett and Parkinson, 1979; Burgeff, 1961; Read and Haselwandter, 1981) remain for the most part unidentified because they are sterile ∗ FAX No.: +35322321. E-mail: marianj@bot.ku.dk when cultured. In other studies isolates have been identified as Hymenoscyphus ericae (Read) Korf et Kernan (Kernan and Finocchio, 1983) based on cultural, morphological, biochemical or molecular characters (e.g., Pearson and Read, 1973; Perotto et al., 1996; Straker, 1996). H. ericae with its anamorph Scytallidium vaccinii Dalpé, Litten et Sigler (Egger & Sigler, 1993; Hambleton & Currah, 1997) is the most well known and well-researched ericoid mycorrhizal fungus (Read, 1983). Stoyke and Currah (1991) and Stoyke et al. (1992) identified several of the dark, septate isolates from the roots of plants of subalpine and alpine dwarf shrub heaths as Phialocephala fortinii Wang et Wilcox. P. fortinii form intracortical sclerotia rather than typical ericoid mycorrhizal coils (Currah et al., 1993). The ecological significance and functional relationship of this fungus is unclear (e.g., Jumponnen and Trappe, 1998). P. fortinii is not restricted to ericaceous host plants but rather distributed depending on edaphic factors (Hambleton and Currah, 1997). The genus Oidiodendron comprises a number of species, which like H. ericae are ericoid mycorrhizal. 226 Several of these taxa have been isolated from soil (Dalpé, 1986) and not from encaceous roots as O. griscum and O. maius (Couture et al., 1983, Hambleton and Currah, 1997; Perotto et al., 1996). The ericaceous dwarf shrub Calluna vulgaris (L.) Hull is an important dominant of the European heathland vegetations. During the past decades the deposition of nitrogenous air pollutants has affected these semi-natural heathland ecosystems (Lee and Caporn 1998); increasing nitrogen availability has reduced the competitive strength of Calluna, turning heathlands into grasslands (Bobbink et al., 1992). Johansson (2000) investigated the influence of ammonium nitrate on the ericoid mycorrhizal colonization of Calluna on the Danish heathland, Hjelm Hede, and found a considerable variability in the colonization level and the response to increased nutrient availability. Ericoid mycorrhiza is not simply an association between H. ericae and one plant order; more than one endophyte can be present in the same root system (Liu et al., 1998; Perotto et al., 1996) but the range and relative importance of the fungi involved is unclear (Straker, 1996). Ultrastructural studies of fieldcollected Calluna roots show that the coil-forming hyphae in the cortex cells have septa characteristic of Ascomycetes (Bonfante-Fasolo and GianinazziPearson, 1979) although Calluna root cells have also been reported to be colonized by Basidiomycetes (Bonfante-Fasolo, 1980). Serological (Straker, 1996) and biochemical data (Mitchell and Read, 1981) suggest that strains of endophytes similar to H. ericae may differ in growth and capability to utilize substrates. Thus, it can be hypothesized that the variable response of colonization level to changed nutrient availability (Johansson, 2000) reflects the presence of different fungi. However, very little is known about the community structure of ericoid mycorrhizal fungi in the field (Hambleton and Currah, 1997; Liu et al., 1998; Perotto et al., 1996). This knowledge is important because their presence influences ecosystem function and the competitive relationships of their host plants (Michelsen et al., 1996). This was investigated in the present study of the fungi associated with ericoid mycorrhizal Calluna roots collected from the Danish heathland, Hjelm Hede. The structure of the fungal community was assessed by the use of morphological data and by the ability to form ericoid mycorrhiza in vitro. The study on Hjelm Hede is unique because the effects of nitrogen on the ericoid colonization level and the fungal associations have been investigated at the same experimental locality (Johansson, 2000). Materials and methods Isolation of root-associated fungi Eighty soil cores (Ø= 5 cm) containing Calluna roots were sampled randomly from the mor layer (F–H horizons) in a Danish heathland, Hjelm Hede. The field area covered 400 m2 . The vegetation is an almost pure stand of even-aged, 10-year-old Calluna. A detailed description of the locality, history and soil is given by Nielsen et al., (1987). The soil cores were homogenized in tap water in a Sorwall Omnimixer for 20 s at low speed. The homogenized soil was washed with tap water over sieves with mesh sizes of 2.0 and 0.5 mm, respectively. Living fine roots of Calluna, identified by their white and almost transparent appearance, were collected from the 0.5-mm sieve. The Calluna roots were cleaned 10 times by serial washing in sterile distilled water and cut in approximately 1-mm pieces under aseptic conditions. From each soil core five randomly collected 1 mm pieces of roots were placed on 1% distilled water agar with penicillin and streptomycin (150 mg l−1 ) and incubated in the dark at 10◦ C for 5–7 days. Pure cultures of randomly emerging hyphae from the roots from each plate were made on 1% malt agar (1% malt extract and 1.5% agar). Characterization of the root-associated fungi Cultural characteristics were used to classify and identify the pure cultures of the root-associated fungi. Species of Oidiodendron were identified according to Barron (1962) and Ellis (1971 and 1976). Cultures of H. ericae from University of Alberta Microfungus Collection (UAMH 5828 and UAMH 6598, isolated from Vaccinum angustifolium, USA; UAMH 6735, isolated from C. vulgaris, UK) and P. fortinii (UAMH 5425) were used as references. Mycorrhizal resynthesis with isolated root associated fungi Test system The method used was based on Pearson and Read (1973). Soil from the mor layer was dried, sieved (2.0 mm mesh size) and autoclaved (20 min at 120◦C, twice). Distilled water agar (0.5%) was poured over small clumps of the sterilized soil in petri dishes. Three Calluna seedlings grown from surface sterilized seeds (1 min in 96% ethanol, 5 min in 5% 227 Ca-hypochlorite followed by three washes in sterile water) on 0.5% distilled water agar were transferred to each petri dish. Inoculation Each petri dish was inoculated with three agar plugs that were cut from the actively growing colony margins of isolates maintained on 1% malt agar. Ninetysix isolates of dark, septate mycelia and seven isolates of Oidiodendron spp. were tested. The inoculated seedlings were incubated in a growth chamber in constant light at 18◦ C for 10 weeks. Root colonization Roots of the Calluna seedlings were stained in 0.2% cotton-blue in lactoglycerol on a microscopic slide during a gentle heating over a flame. Ericoid mycorrhizal structures were identified. slightly swollen due to the external presence of these isolates. The frequency of fungal coils in the inoculated Calluna roots was low compared to the field-collected roots. In contrast, seedlings inoculated with Oidiodendron spp. had a very intensive colonization. Reference culture of H. ericae (UAMH 5828) formed ericoid mycorrhiza in culture in contrast to UAMH 6598 and UAMH 6735. The reference culture of P. fortinii (UAMH 5425) did not make any intracellular structures. Voucher cultures of representative strains have been deposited in the University of Alberta Microfungus Collection and Herbarium (UAMH) (www.devonian.ualberta.ca) and in the fungal collection at Department of Mycology, Botanical Institute, University of Copenhagen (Table 2). Discussion Results Stained Calluna roots from Hjelm Hede revealed typical ericoid mycorrhiza with varying percentages of colonization (Johansson, 2000). No potential teleomorphs were collected in the field. Root-associated fungi were isolated from 385 onemm Calluna root pieces resulting in 626 isolates; 76% of these isolates were dark, septate and slow-growing; 1.6% were identified as Oidiodendron spp. and the remaining as species of Penicillium, Mucor, Mortierella and several white sterile mycelia. Isolates of the dark, septate root-associated fungi (DS) were divided into 13 morphological groups based on cultural characteristics on 1% malt agar at 10 ◦ C (Table 1); under these culture conditions several isolates formed arthroconidia but none formed ascocarps. The Calluna seedlings all remained healthy with well-developed root systems during the resynthesis experiment. Isolates of Oidiodendron spp. and within the morphological groups DS 002 – DS 005 and DS 011 – DS 013 formed typical ericoid mycorrhizal coils in the cortex cells of the inoculated Calluna seedlings. In contrast DS 001 and DS 006 – DS 010 did not form mycorrhiza nor any intracellular structures. Of the dark, septate isolates 51% belonged to ericoid mycorrhizal groups (Figure 1). As a single group the DS 001 constituted 28% of the dark, septate fungi cultured (Figure 1); these fungi are likely not mycorrhizal as they never formed any coils nor other intracellular structures. It was observed that the cortex cells were The root systems of Calluna plants on Hjelm Hede were associated with fungi, which predominantly were dark, septate, sterile and slow growing, forming a mosaic of ericoid mycorrhizal and non-mycorrhizal fungi. These fungi may constitute a distinct community dominated by the ericoid mycorrhizal fungi and overlapping with fungi, such as Penicillium spp., which are common colonists within the soil (Johansson, 2001). In this study all the dark, septate root-associated fungi isolated had septa characteristic of Ascomycetes. The observed dominance of dark, septate fungi among the root-associated fungi is in agreement with several observations of ericaceous roots (e.g., Pearson and Read, 1973; Perotto et al., 1996). In contrast, Sewell (1959) isolated dark, sterile mycelia from sterile-water-washed 2-mm pieces of Calluna roots sampled from the mor layer in a very low frequency. Hambleton and Currah (1997) isolated four distinct endophytic taxa from boreal and alpine Ericaceae and concluded that their distribution depends on edaphic conditions rather than host availability. Read (1974) described typical isolates of H. ericae grown on malt agar as pale grey to smoke grey, the reverse dark to pale grey with a narrow white margin, fascicles of aerial hyphae in centre breaking up into segments. Additionally Egger and Sigler (1993) proposed that isolates of H. ericae are slow growing on all media. Egger and Sigler (1993) also concluded that isolates not forming conidia yet sharing similar colonial features, and demonstrating mycorrhizal as- 228 Table 1. Morphological groups recognised among the dark, septate root-associated fungi isolated from Calluna roots collected from Hjelm hede, Denmark Group Main characters on 1% malt agar Coils Growth Colony diameter at 10◦ C for 4 months Hyphae (µm) Conidia DS 001 Colonies black to olivaceous black becoming brownish with maturity. Aerial mycelium floccose with numerous droplets along the walls. Rarely Moderate to fast (8–9 cm) 2–5 Arthroconidia seen in two cultures DS 002 Colonies greyish black becoming brownish with maturity. Growing margin white, narrow. Aerial mycelium hyaline to brownish, scanty and unbranched becoming more intensive in the old part of the colony. Rarely Slow (3–4 cm) 2–5 Arthroconidia seen in 50% of the cultures DS 003 Colonies greyish black. Aerial mycelium scanty becoming intensive floccose in the old part of the colony. Colonies black. Aerial mycelium almost absent but seen as grey or brown hyphae in the old part of the colony. Old hyphae with oil-drops. Growing margin down in the agar. Colonies black, olivaceous black to olivaceous brown with a narrow brown or white margin. Aerial mycelium brown greyish. Colonies brownish in the central part and young hyphae hyaline. Aerial myceliums scanty with simple, erect hyphae. Colonies brownish with a wide hyaline margin. Formation of concentric rings of brown pigmented and hyaline hyphae. Aerial mycelium scanty with simple erects hyphae. Colonies brownish with a wide hyaline margin. Formation of concentric rings of brown pigmented and hyaline hyphae. Aerial mycelium branched. Colonies brownish with a brown margin. Formation of concentric rings of brown pigmented and hyaline hyphae. Aerial mycelium scanty with simple, erects hyphae. Colonies brownish with a brown margin. Formation of concentric rings of brown pigmented and hyaline hyphae. Aerial mycelium floccose. Colonies dark, brownish to vinaceous. Aerial mycelium intensive floccose. Growing margin down in the agar. Numerous Slow (3–5 cm) 2–5 None Slow (3–4 cm) 2–5 Arthroconidia seen in 50% of the cultures Arthroconidia seen in 50% of the cultures None Slow (3–4 cm) 2–5 Numerous Moderate (6–7 cm) 1–3(5) None Slow (4–5 cm) 2–5 Numerous Slow (4–5 cm) 2–5 Numerous Moderate to fast (8–9 cm) 2–5 None Moderate to fast (8–8 cm) 2–5 None Slow (3–4 cm) 2–5 None Slow (3–4 cm) 2–5 None Slow (3–5 cm) 2–5 DS 004 DS 005 DS 006 DS 007 DS 008 DS 009 DS 010 DS 011 DS 012 DS 013 Colonies olivaceous-brown to olivaceous-grey with a narrow brownish margin. Aerial mycelium brown and branched. Colonies greyish-black. Aerial mycelium floccose, hyaline and simple. Arthroconidia seen in one culture Arthroconidia seen in one culture Arthroconidia seen in 50% of the cultures Arthroconidia seen in 50% of the cultures Arthroconidia seen in 50% of the cultures 229 Figure 1. The frequency of dark, septate, root-associated fungi within the morphological groups outlined in Table 1 (measured as a percentage of the total numbers of dark, septate isolates). (1): DS 001, (2) DS 002, (3) DS 003, (4) DS 004, (5) DS 005, (6) DS 006, (7) DS 007, (8) DS 008, (9) DS 009, (10) DS 010, (11) DS 011, (12) DS 012 and (13) DS 013. Open bars: Did not form ericoid mycorrhiza. Closed bars: Ericoid mycorrhizal. sociation, may be identified as H. ericae. The majority of the Danish dark, septate isolates with the ability to form ericoid mycorrhiza may be taxonomically related to H. ericae based on the morphological descriptions by Read (1974) and Hambleton and Currah (1999). In the present study several of the dark mycelia formed arthroconidia. The uses of arthroconidia formation as a diagnostic feature is questionable, as arthroconidia are not formed constantly. The ability of single isolates to form ericoid mycorrhiza is not correlated to arthroconidia formation. In this study none of the dark, septate isolates formed ascocarps in culture nor were they ever seen in the field. Egger and Sigler (1993) and Hambleton and Currah (1997) reported formation of arthroconidia by several isolates of H. ericae and S. vaccinii. Oidiodendron spp. was isolated in surprisingly low frequencies from the field-collected Calluna roots, considering their ability to colonize when inoculated in vitro in pure culture. Although capable of being mycorrhizal, as also reported by Couture et al., (1983), Dalpé (1986) and Xiao and Berch (1992), in the prevailing soil or root environment this fungus may be a poor competitor compared to the dark, septate rootassociated groups of fungi. Hambleton and Currah (1997) also recovered low number of isolates of Oidiodendron, supporting the idea that the species does not play a significant endophytic role in the habitats studied. P. fortinii is commonly isolated from habitats where ericaceous plants dominate, or from ericaceous roots and other plants growing in soils of low pH and high organic matter. The ecological significance of this fungus remains speculative (Haselwandter and Read. 1982; Stoyke and Currah, 1993). Stoyke et al. (1992) found that the dominating endophyte in subalpine dwarf shrub heath was related to P. fortinii based on restriction fragment analysis of an amplified portion of ribosomal DNA. Thus, P. fortinii is one of several taxa that include the dark, septate rootinhabiting fungi of plants growing in cool, organic rich soils. Based upon hyphal morphology the isolates within DS 001 are considered to be P. fortinii (Currah, personal information) although no sclerotia were formed, not even in the agar medium as reported by Stoyke and Currah (1993) and Stoyke et al. (1992) nor were any conidia observed. The hyphae within the group formed characteristic balloon-like protrusions on the outer surface as noticed by Currah and Tsuneda (1993). This group – DS 001 – was isolated 230 Table 2. Department of Mycology, Botanical Institute, University of Copenhagen and University of Alberta Microfungus Collection and Herbarium (UAMH) deposition numbers for fungal strains isolated in this study Morphotype Strain DS 001 H-1-2-001-008 H-1-3-001-009 H-1-1-002-002 H-1-2-002-001 H-1-2-002-002 H-1-3-002-001 H-1-4-002-006 H-1-1-003-001 H-1-1-003-005 H-1-1-003-008 H-1-2-003-001 H-1-3-003-001 H-1-3-003-003 H-1-3-003-005 H-1-3-003-008 H-1-3-003-009 H-1-1-004-001 H-1-1-004-004 H-1-2-004-001 H-1-2-004-003 H-1-2-004-008 H-1-2-004-011 H-1-3-004-003 H-1-3-004-005 H-1-4-004-001 H-1-4-004-003 H-1-2-005-001 H-1-2-005-002 H-1-4-005-001 H-1-4-011-001 H-1-2-012-001 H-1-2-012-003 H-1-2-013-003 DS 002 DS 003 DS 004 DS 005 DS 011 DS 012 DS 013 Department of Botanical Institute number AAS 1547 AAS 1548 AAS 1549 AAS 1550 AAS 1551 AAS 1552 AAS 1553 AAS 1554 AAS 1555 AAS 1556 AAS 1557 AAS 1558 AAS 1559 AAS 1560 AAS 1561 AAS 1562 AAS 1563 AAS 1564 AAS 1565 AAS 1566 AAS 1567 AAS 1568 AAS 1569 AAS 1570 AAS 1571 AAS 1572 AAS 1573 AAS 1574 AAS 1575 AAS 1576 AAS 1577 as a single group in rather high frequencies but did not form ericoid mycorrhiza or any other intracellular structures. During the course of the study no Basidiomycete hyphae were isolated. Basidiomycetes may be secondary colonists of roots as suggested by Duddridge & Read (1982) and not mycorrhizal symbionts. It should be noticed that root cells, living or dead, can be colonized by non-mycorrhizal fungi. Mycology, University of Alberta Deposition Microfungus collection and Herbarium deposition number UAMH 7936 UAMH 7940 UAMH 7933 UAMH 7937 UAMH 7934 UAMH 7935 UAMH 7936 UAMH 7941 UAMH 7939 The roots of Calluna are apparently associated with a diverse group of fungi of which the dark, septate ericoid mycorrhizal mycelia predominate. The establishment of several morphological groups of ericoid mycorrhizal and non-mycorrhizal fungi demonstrates high diversity among these dark root associated fungi. Their simultaneous presence could explain the variable response to environmental impacts. The taxonomic positions of the majority of the dark, septate root associated isolates remains unclear, as they are 231 sterile in culture. Several isolates share morphological similarity with H. ericae indicating a close relationship. The relationship between different ericoid isolates may be far more complex than the morphological similarities of the anamorphs in culture suggest (Chambers et al., 2000; Liu et al., 1998; Perotto et al., 1995). Acknowledgements I acknowledge Ulrik Søchting for valuable support. Badria Nawabi and Else Meier Andersen are thanked for technical assistance. I thank Lynne Sigler for providing cultures from the University of Alberta Microfungus Collection and Randy Currah for identifying several of my cultures. The project was supported by a grant from The Danish Natural Science Research Council and by The Danish Environmental Research Programme 1992-96. References Barron G L 1962 New species and new records of Oidiodendron Can. J. Bot. 40, 589–607. 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