Mycol. Res. 106 (12): 1463–1467 (December 2002). f The British Mycological Society 1463 DOI: 10.1017/S0953756202006767 Printed in the United Kingdom. New perspectives on the niche and holomorph of the myxotrichoid hyphomycete, Oidiodendron maius Adrianne V. RICE* and Randolph S. CURRAH Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9 Canada. E-mail : arice@ualberta.ca Received 23 January 2002; accepted 10 September 2002. Oidiodendron maius is accepted widely as an ericoid mycorrhizal endophyte because it is isolated primarily from the roots of ericaceous plants. Reports of the species from other materials are much fewer, suggesting a limited role as a free-living saprobe. We show that assumptions concerning the prevalence of O. maius in a common substrate (i.e. peat) are affected by isolation protocols. Oidiodendron maius was observed on 99.6 % of peat samples incubated in moist chambers but only 0–9% of the peat fragments plated on different isolation media. These results, and previous studies showing ability to degrade Sphagnum, indicate that O. maius could inhabit a much broader niche than previously suspected. Sterile ascocarps with peridia morphologically similar to Myxotrichum arcticum were observed on moist incubated peat and in a series of controlled crosses. These observations provide evidence that the teleomorph of O. maius is a species of Myxotrichum. INTRODUCTION Oidiodendron maius Barron 1962 is a widely distributed hyphomycete that has been isolated from a variety of substrates (e.g. Barron 1962, Lumley, Gignac & Currah 2001, Thormann, Currah & Bayley 2001) although most isolates are from roots of ericaceous plants (e.g. Hambleton, Egger & Currah 1998, Monreal, Berch & Berbee 1999, Chambers, Liu & Cairney 2000). Consequently, it is often considered to have a niche similar to Hymenoscyphus ericae, an ascomycete well known as an ericoid mycorrhizal associate (Hambleton & Currah 1997, Monreal et al. 1999). O. maius, like some other species of the genus, has been reported to form infection units in the roots of Ericaceous plants (Douglas, Heslin & Reid 1989, Johansson 2001). In O. maius, prolific sporulation, the production of a diverse suite of enzymes, and relatively rapid growth on a variety of culture media (Rice & Currah 2001) are characteristics suggesting a saprobic niche rather than a mycorrhizal one (Hutchison 1991). In vitro, O. maius is a proficient decomposer of Sphagnum, the primary component of bog peat, causing significant mass losses (Thormann 2001, Piercey, Thormann & Currah 2002) and degrading all cell wall components (Tsuneda, Thormann & Currah 2001). The species is also enzymatically diverse, degrading cellulose, pectin, and selected phenolic compounds (Rice & Currah 2001) that * Corresponding author. comprise a large proportion of peat (Turetsky et al. 2000). Based on these observations, we hypothesized that O. maius is an abundant component of the saprobic microfungal community of bog peat and tested this by comparing several culturing methods for their efficacy in showing the presence of the species. In doing so, a sterile myxotrichoid ‘gymnothecium ’ (Novák & Galgóczy 1965) was observed among stands of O. maius conidiophores on moist incubated peat. A series of crossing trials using a range of isolates showed that these sterile gymnothecia were relatively easy to induce under some conditions. Molecular analyses had indicated previously that the teleomorph of O. maius should be expected in the genus Myxotrichum (Hambleton et al. 1998), but the distinctive gymnothecia typical of the genus remained unknown. This paper assesses the use of different culture techniques to determine the prevalence of O. maius in a series of peat samples and provides a description of the sterile myxotrichoid gymnothecia produced by this taxon. MATERIALS AND METHODS Peat samples were obtained (June–September) from three plots (minimum 2 m apart) within a Sphagnum fuscum – Picea mariana (black spruce) bog (54x 28N, 113x 16W) near Perryvale in southern boreal Alberta. The site is described in Thormann et al. (1999). New perspectives on Oidiodendron maius 1464 Table 1. Location and collection sites for the 21 strains of Oidiodendron maius used in a series of controlled crosses established on sterilized thalli of Cladonia spp. Collectors are listed as footnotes. Strain Location F-011 F-021 F-031 S1-P3-C-12 S1-P6-C-12 S2-P3-C-12 S2-P6-P-92 S3-P6-M-12 S4-P3-P-42 S4-P4-C-12 S4-P6-C-12 UAMH 15403 UAMH 65144 UAMH 70225 UAMH 84426 UAMH 85297 UAMH 89208 UAMH 89218 UAMH 89228 UAMH 89339 UAMH 974910 Empetrum nigrum, birch dominated fjell, Kevo Research Station, Finland Vaccinium myrtillus, birch dominated fjell, Kevo Research Station, Finland V. vitis-idaea, birch dominated fjell, Kevo Research Station, Finland V. myrtilloides, jack pine-aspen forest, 50 km S of Ft. McMurray, Alberta V. myrtiloides, jack pine-aspen forest, 50 km S of Ft. McMurray, Alberta V. myrtilloides, jack pine-black spruce forest, Ft. McKay, Alberta V. myrtilloides, jack pine-black spruce forest, Ft. McKay, Alberta V. myrtilloides, jack pine-lichen hilltop, Ft. McKay, Alberta V. myrtilloides, disturbed sand hill, Ft. McKay, Alberta V. myrtilloides, disturbed sand hill, Ft. McKay, Alberta V. myrtilloides, disturbed sand hill, Ft. McKay, Alberta Soil, cedar bog, Guelph, Ontario, ex-type Loiseleuria procumbens, dry alpine ridge, Jasper National Park, Alberta Gaultheria shallon, 3 yr old western hemlock site, coastal British Columbia Rhododendron sp., heath meadow, Ireland V. corymbosum, Quebec Oxycoccus quadripetalus, black spruce bog, Alberta V. myrtilloides, sand dune, Alberta V. vitis-idaea, sand dune, Alberta Phyllodoce empetriformis, alpine meadow, Alberta Decaying Sphagnum fuscum, Perryvale bog, Alberta 8 Collectors: 1 Currah; 2 Hill-Rackette; 3 Barron; 4 Stoyke & Currah; 5 Xiao & Berch; 6 Douglas, Heslin & Reid; 7 Couture, Fortin & Dalpe; Hambleton & Currah; 9 Hambleton; and 10 Thormann. One 15 cm core (10 cm diam) was taken from the surface peat in each plot. Cores, consisting of a heterogenous matrix of Sphagnum, spruce and ericaceous roots, and other debris, were cut into 2.5 cm thick cross sections, using a sterilized knife, and placed in sterile Petri plates for transport to the laboratory. Each cross section of peat was washed with distilled water and divided into ten smaller samples. Four fragments (5 cmr5 cmr0.5 cm) from each peat sample were placed into moist chambers, consisting of sterile plastic Petri plates lined with moist, sterile filter paper. Two fragments from each sample were cut into 30 5r5 mm segments, five of which were randomly selected for plating onto two replicate plates each of corn meal agar (CMA ; 1 l dH2O, 17.0 g Difco corn meal agar), CMA with benomyl (CMAB ; 1 l dH2O, 17.0 g Difco CMA, 0.1 ml (1 %) benomyl solution) and MycobioticØ agar (MYCO ; 1 l dH2O, 35.6 g Difco Mycobiotic Agar). Thus, 720 plates were prepared in total : 288 moist chambers, 144 CMA, 144 CMAB, 144 MYCO. All media were amended with oxytetracycline (0.02%) to control bacterial growth. Plates and moist chambers were incubated at room temperature in the dark for at least four months and monitored for fungal growth using dissecting and compound microscopes. Fungal identifications were based on morphological characters. Observed frequency for the most common sporulating species was based on the percentage of plates of each medium, including moist chambers, on or in which each taxon occurred. Differences in observational frequencies of the three most common taxa, i.e. Mucor spp., Penicillium spp., and Oidiodendron maius were assessed using a series of x2 tests. Because a sterile ascocarp was observed among conidiophores of O. maius on a peat fragment incubated in a moist chamber, we initiated a series of test crosses using 21 identified strains of O. maius (described in Rice & Currah 2001; Table 1), including UAMH 9749, which was isolated from the Perryvale site. Sterilized pieces of the fruticose thalli of a mixture of Cladonia mitis and C. rangiferina were placed in Petri plates with a basal layer of CMA, inoculated with all pairwise combinations of the 21 strains, and incubated at room temperature in the dark. Cladonia was used as a substrate because at least one species of Myxotrichum, M. bicolor, forms extensive interconnected mats of gymnothecia on fruticose lichen thalli (Currah 1985), and Cladonia spp. are the most abundant fruticose lichens in Perryvale bog. The fragments were monitored regularly for gymnothecia and these were examined using light microscopy. RESULTS AND DISCUSSION Assessment of Oidiodendron maius in bog peat Moist chambers are used to provide an accurate estimation of the fungal species that are active in some substrates (e.g. Bills & Polishook 1994, Richardson 2001) because plating on media is often biased toward the recovery of faster growing, highly sporulating species (Bisset & Widden 1972, Bills & Polishook 1994). Moist chambers are used regularly to study coprophilous fungi (Richardson 2001), but have been used rarely to sample the fungal community in soil or peat. A. V. Rice and R. S. Currah 1465 Table 2. Observational frequencies (expressed as a percentage of plates with sporulating cultures) of Oidiodendron maius and the most common genera isolated from bog peat using moist chambers (MC) and three isolation media: CMA, corn meal agar; CMAB, corn meal agar+benomyl; and MYCO, mycobiotic agar. Species/genus MC CMA CMAB MYCO Oidiodendron maius Mucor spp. Penicillium spp. 99.6a*** 27.8b** 14.6b** 9.0c** 94.4d 94.4d* 0.7c** 97.2d** 22.2b* 0c** 50.0e 91.0d** a Significantly different from b and c (P<0.0001). Significantly different from a and d (P<0.0001) and e (P< 0.0005). c Significantly different from a, d and e (P<0.0001). d Significantly different from b and c (P<0.0001) and e (P< 0.0005). e Significantly different from c (P<0.0001) and d (P<0.0005). * Significantly different from expected value, 0.0005<P<0.05. ** Highly significantly different from expected value, P<0.0005. *** Extremely significantly different from expected value, P< 0.0001. b O. maius was the most frequently observed species on peat incubated in the moist chambers, occurring in 99.6 % of the chambers (Table 2) and growing readily on all components of the peat matrix. It is possible that washing the peat cross sections may have dispersed O. maius conidia throughout the Sphagnum matrix but presumably this heavier load of propagules would have yielded greater amounts of O. maius on the agar media along with a higher number of other heavily sporulating species on the moist incubated peat. It is impossible to determine conclusively whether the O. maius observed on the moist incubated peat was present in the original substrate as propagules or actively growing mycelia, but its ability to grow well on the natural substrate, under in vitro conditions, indicates that it has considerable potential to grow on this substrate in situ. Faster growing species, including Mucor spp. and Penicillium spp., were observed more rarely in the moist chambers than O. maius (P<0.0001) and were less abundant than on the agar media (P<0.0005) (Table 2). Conversely, species of Mucor and Penicillium overgrew plates of all three types of media and O. maius was more restricted. The selection of media may also bias the results of surveys (e.g. Lumley, Abbott & Currah 2000). Previous work has shown that O. maius grows and sporulates readily on CMA (Hambleton & Currah 1997, Rice & Currah 2001). O. maius was observed on only 9 % of the CMA plates (Table 2) and sporulated only on peat fragments on one-third of these plates. Benomyl favours the growth of basidiomycetes by discouraging the growth of many faster growing molds. O. maius is an exception among these because it is tolerant of this compound ; yet conidiophores of O. maius were observed only on one peat fragment on one CMAB plate (Table 2). MYCO has been used previously to select for fungi with affinities to the Onygenales, Microascaceae, and Myxotrichaceae, including the sexual states of Oidiodendron spp. because these taxa are cycloheximide tolerant (Lumley et al. 2000). However, we did not Table 3. Crosses between 18 strains of Oidiodendron maius that produced sterile gymnothecia on sterilized Cladonia after 6 mo incubation. (Only a portion of the matrix is shown; crosses not yielding sterile gymnothecia are omitted.) Strains S3-P6-M-1 S4-P3-P-4 UAMH 8920 UAMH 8922 F-03 S1-P3-C-1 S1-P6-C-1 S2-P3-C-1 S2-P6-P-9 S3-P6-M-1 S4-P3-P-4 S4-P4-C-1 S4-P6-C-1 UAMH 1540 UAMH 6514 UAMH 7022 UAMH 8442 UAMH 8920 UAMH 8921 UAMH 8933 UAMH 9749 x x + x + x + x x x x x x + x x x + + + + + + x + + + + x + + + + + x + + x x + + x + x + x x x x x x x x x x x x x x + x x + x x x x x + Sterile ascomata formed. observe asexual or sexual structures associated with O. maius on any MYCO plates (Table 2). The differences in observational frequencies among the three species and genera on the three different isolation media are highly significant (x2 P<0.0001). While the widespread distribution of O. maius is recognized, it is considered uncommon except from the roots of ericaceous plants (Hambleton et al. 1998, Lacourt et al. 2001). O. maius in other habitats or substrates may be overlooked by traditional sampling methods. It is not reported frequently from bryophyte substrates (Thormann, Currah & Bayley 2001). However, our results indicate that O. maius may be more common in bog peat than previously reported and that the isolation protocols used by most researchers may limit the recovery of O. maius from natural substrates. Sterile myxotrichoid ascomata produced by Oidiodendron maius After 1 month of incubation, a single sterile ascoma of an unknown Myxotrichum sp. developed among the conidiophores of O. maius on one moist-incubated Sphagnum sample. A selection of O. maius strains (18, including one from Perryvale material), grown in pairs on Cladonia thalli, yielded many similar, but also sterile, gymnothecial structures (Table 3). These data may indicate that O. maius, were it to form fertile gymnothecia, would be heterothallic ; self-crosses never produced sterile gymnothecia. However, thallism remains a moot point because only four strains, in combination with others, produced the distinctive peridial elements and ascospores were absent from all of the gymnothecial structures. The stimulatory effect of the Cladonia substrate on the production of sterile gymnothecia by paired O. maius New perspectives on Oidiodendron maius 1466 1 2 3 4 Figs 1–2. Sterile gymnothecium produced after 4 mo by Oidiodendron maius, UAMH 9749 crossed with S4-P3-P-4, on sterilized Claonia. Fig. 1. Sterile gymnothecium. Bar=100 mm. Fig. 2. Close up of peridial hyphae. Note dichotomous branches with wide branch angles, tapered apices of the peridial hyphae. Bar=10 mm. Figs 3–4. Gymnothecium of Myxotrichum arcticum (UAMH 7565). Fig. 3. Gymnothecium. This structure is morphologically similar to the sterile gymnothecia of O. maius (UAMH 9749rS4-P3-P-4). Bar=100 mm. Fig. 4. Close up of peridial hyphae, showing dichotomous branches with wide branch angles and the tapered apices of the peridial hyphae. Bar=20 mm. strains was striking. Previous studies with other arthroconidial taxa have shown that native substrates can be essential for the production of cleistothecia or cleistothecium-like structures (e.g. feathers as keratin source in Oncocladium ; Sigler et al. 1987). In this case, peridium formation, at least, could be dependent on the presence of fungal residues in the substrate ; a feature that the moist-incubated Sphagnum and the Cladonia thalli shared. The documented chitinolytic abilities of O. maius (Rice & Currah 2001) support this supposition. The sterile gymnothecia (Figs 1–2), produced singly or in clusters of 2–3 on the Cladonia, have a loose peridium of thick-walled dematiaceous hyphae (2–6 mm thick). The peridial hyphae are smooth to asperulate with truncate to tapered ends (Fig. 2). In some instances, these structures resemble disorganized clusters of conidiophores but differ in that the conidiophores of O. maius are smooth, unbranched, and the pigmentation ends abruptly just before the conidiogenous apex. Many peridial hyphae are dichotomously branched with wide branching angles (Fig. 2). Appendages are absent. Among the species described in Myxotrichum, the peridium is morphologically most similar to M. arcticum Udagawa, Uchiyama & Kamiya 1994 (Figs 3–4). In M. arcticum, as in the sterile gymnothecia produced by O. maius, the thick-walled, darkly pigmented hyphae branch dichotomously and at broad angles (Figs 2, 4). Myxotrichum arcticum differs in that some peridial elements terminate in spine-like appendgages (Udagawa et al. 1994). The Oidiodendron anamorph of M. arcticum is superficially similar to O. maius in bearing a cluster of pale arthroconidia at the tip of a tall, dematiaceous conidiophore. 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