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Korean Collection for Type Cultures, Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, No. 52 Oun-dong, Yusong-ku, Daejon 305-333, Korea
Hack Sung Jung 1
School of Biological Sciences, Seoul National University, San 56–1 Shillim-dong, Kwanak-gu, Seoul 151-742, Korea
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Irpex, one of the most common polypore genera, is easily identified by macro- and microscopic characters. During field trips to Korea's Kangwon Province, some Irpex specimens with conspicuous morphological differences from I. lacteus were collected. Cultural characters and molecular evidence differentiated this new strain from I. lacteus, and this taxon is proposed as I. hydnoides sp. nov.
Key words: Australohydnum dregeanum, Irpex lacteus, Irpex vellereus, ITS, Polyporaceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), which is widely distributed as a saprotroph on dead wood. Irpex lacteus is a white-rot fungus of great commercial importance due to the production of endo- and exotype cellulases (Hamada et al 1999, Hoshino et al 1994), antibiotics and irpexans (Silberborth et al 2000). It also is important for the decolorization of chemically complex dyes (Novotny et al 2001) and bioremediation of hazardous organic compounds (Kim and Song 2000, Novotny et al 2000).
Irpex is one of the most common polypore genera in Korea (Jung 1994, Lim and Jung 2000, Yang et al 1987). Traditionally, it is recognized by its distinctly irpicoid (flattened-toothed) hymenophore, coriaceous texture, encrusted cystidia and simple-septate hyphae with a dimitic hyphal system (Gilbertson and Ryvarden 1986, Ryvarden and Gilbertson 1993). However, its morphology is variable depending on environmental conditions. In favorable conditions, it grows vigorously and fruiting bodies may reach up to 3 m in length (Jung 1987). Because of this morphological variability, numerous species have been recognized in Irpex. Maas Geesteranus (1974) concluded that these species names all were synonyms of I. lacteus or otherwise should be removed from Irpex to other genera, maintaining Irpex as a monotypic genus.
Australohydnum dregeanum morphologically is similar to I. lacteus and is described as possessing a hydnoid (toothed) to irpicoid hymenophore and a monomitic or dimitic hyphal system with simple-septate generative hyphae. The type species of Australohydnum Jül. is A. griseofuscescens (Reich.) Jül., but Hjortstam and Ryvarden (1990) suggested that this species is a synonym of A. dregeanum. Irpex vellereus also is treated as a synonym of A. dregeanum (Hjortstam and Ryvarden 1990).
Specimens of a new Irpex were collected from fallen branches of Quercus sp. in Kangwon Province. These differed from those of I. lacteus in basidiocarp morphology and cultural characters. The new taxon microscopically was quite similar to A. dregeanum but differed in hymenophoral configuration. To determine if these collections represent a new species, cultures were studied and DNA sequences of nuclear ITS1, ITS2 and 5.8S rRNA genes were analyzed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, using 
-naphthol and p-cresol. Basidiomata were examined both macroscopically and microscopically. Measurements and drawings were made from slide preparations stained with 1% (w/v) aqueous phloxine and 3% (w/v) potassium hydroxide (Hawksworth et al 1995). Melzer's reagent was applied to check spore amyloidity. Color designations were adopted from Kornerup and Wanscher (1978).
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with some modification (Lim and Jung 1998). Partial SSU rDNA and ITS regions containing the 5.8S rRNA gene were amplified with primers NS7 and ITS4 (White et al 1990). Amplification was performed as described by Lee et al (2000). Amplified products were purified with Wizard PCR Prep (Promega), and purified products were cycle sequenced using the chain termination method with 35S-labled ATP (Hillis et al 1996) of a TopTM DNA sequencing kit (Bioneer Corp., Korea). In addition to new sequences produced from nine strains in Table I, the ITS sequence of Bjerkandera fumosa (Pers. : Fr.) Karst. was obtained from GenBank (AJ006637) and sequences of Ceriporia viridans (Berk. & Br.) Donk and C. excelsa (Lund.) Parm.were provided by J. Mugnier (Rhône-Poulenc, France).
Phylogenetic analyses –
ITS sequences initially were aligned with Clustal X (Thompson et al 1997) and optimized with PHYDIT version 3.0 (Chun 1995). Ambiguously aligned regions were omitted from analyses. Maximum-parsimony (MP) analyses were performed with PAUP* 4.0b4a (Swofford 1999). Heuristic searches usingg tree-bisection reconnection (TBR) branch swapping and 100 random-taxon sequence additions were employed. Gaps were treated as missing data in all analyses. Support for individual branches was determined by bootstrap analysis (Hillis and Bull 1993) based on 1000 replicates (simple addition sequence, TBR swapping, and MAXTREES unrestricted). Based on the phylogenetic results of previous studies (Boidin et al 1998, Hibbett et al 1997, Hibbett and Thorn 2001), Bjerkandera fumosa was chosen as outgroup.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Basidiocarpus annuus, resupinatus vel effuso-reflexus; superficies albido, tomentoso-hirsuto; hymenium cremeo-lutea vel ochraceo, hydnoidea, 4 mm longis; systema hypharum dimiticum; hyphae generatoriae tenuitunicatae; hyphae skeletaleae crassitunicatae; cystidia incrustata, 25–45 x 6.5–11 µm; basidia clavata, 4-sterigmatibus, 28–32 x 6–7 µm; basidiosporae ellipsoideae, hyalinae, laeves, non-amyloideae, 5.5–6.5 x 3.5–4 µm. Holotypus in herbarium SNU; numero accessiono m-97121519.
Basidiocarps annual, resupinate or effused-reflexed, confluent and effused up to 25 cm or more; pilei often laterally fused, up to 5 mm wide, upper surface whitish to dingy white, densely tomentose to hirsute; hymenophore cream (4A3) to yellow (4A4–4A5), becoming ochraceous, hydnoid, teeth up to 4 mm long, mostly flattened, sometimes united at the base or laterally fused, often incised or denticulate at the apex; margin distinctly bounded; consistency coriaceous. Hyphal system dimitic; contextual generative hyphae thin-walled with frequent branching, simple-septate, 2.5–5 µm in diam; contextual skeletal hyphae hyaline, thick-walled with rare simple septa, 4–6 µm in diam. Cystidia conspicuous, abundant, thick-walled, apically heavily incrusted, incrusted part 25–45 x 6.5–11 µm, cylindrical to conical, originating from tramal skeletal hyphae in the subhymenium and projecting up to 55 µm beyond the hymenium. Basidia clavate, 4-sterigmate, 28–32 x 6–7 µm, simple-septate at the base. Basidiospores ellipsoid, hyaline, smooth, negative in Melzer's reagent, 5.5–6.5 x 3.5–4 µm.
Etymology. "hydnoides": denticulate.
Habitat. Fallen twigs of Quercus sp.
Distribution. Known only from the temperate oak-pine forests of the Taebaek Mountains, Kangwon Province, Korea.
Specimens examined. KOREA. Mount Odae, Kangwon Province, on Quercus sp., 11 Oct 1997, Y. W. Lim (SNU m-97101112); Mount Chiak, Kangwon Province, on Quercus sp., 15 Dec 1997, Y. W. Lim (HOLOTYPE: SNU m-97121519).
Cultural characteristics – Growth on MEA slow, forming mats of 11.2 mm diam in 7 d at 25 C. Advancing zone even, hyaline and slightly raised; hyphae distant, branched, 2–5.5 µm wide. Aerial mycelium at first downy, becoming thinly cottony, white, hyphae 2–3 µm wide. Reverse bleached (Fig. 2).
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Extracellular oxidase reactions.
-naphthol +; p-cresol ++.
Culture examined. SNU m-97121519 isolated from the tissue of the holotype basidiocarp.
Remarks. Irpex hydnoides is recognized by its effused-reflexed fruit bodies and conspicuous hydnoid hymenophore. It is similar to I. lacteus, and these two species often are confused in the field. Irpex hydnoides can be distinguished easily by its long pale yellow to ochraceous teeth, large basidia and basidiospores, slow growth on MEA and ITS sequences. Schizopora paradoxa (Fr.) Donk resembles I. hydnoides in basidiospore morphology and the shape and color of its basidiomata but forms a poroid hymenophore with daedaleoid-hydnoid dissepiments and clamped generative hyphae. Species of Steccherinum Gray share some characters with I. hydnoides, but the members of this genus generally possess odontioid hymenophores up to 1 mm in length and clamped generative hyphae when the teeth are longer than 1 mm.
DNA amplification and sequence analyses – The amplified products obtained with primers NS7 and ITS4 were approximately 1 kb in size and ITS and 5.8S rDNA products were estimated to be between 630 and 650 bp in length. The aligned sequences of species of Irpex were 651 bp in length. Sequences of strains of I. hydnoides were invariant but sequences of I. lacteus from Korea and North America differed by 0.3%. Sequence divergence between I. lacteus and I. hydnoides ranged from 2.15 to 2.31%. The final alignment for 12 species consisted of 612 characters of which 150 were parsimony informative.
Maximum-parsimony analyses yielded two equally most-parsimonious trees (tree length = 359 steps, consistency index = 0.7911, retention index = 0.7741, rescaled consistency index = 0.6124), one of which is presented in Fig. 3. These trees were identical except for altered positions of strains in each of I. hydnoides clade and I. lacteus clade. The two strains of I. hydnoides formed a well-supported clade (100% bootstrap support) that is closely related to strains of the type specie of Irpex, I. lacteus (Fig. 3). Irpex vellereus, a species treated as a synonym of A. dregeanum (Hjortstam and Ryvarden 1990), was not inferred as a member of the I. hydnoides–I. lacteus clade but grouped with a high level of support (100%) with the single isolate of A. dregeanum sequenced in this study. This clade was sister of the group that includes members of the genus Phanerochaete Karst.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), its hymenophore is always white to cream in color. Basidia of I. lacteus and I. hydnoides are similar, but those of I. hydnoides are larger (28–32 x 6–7 µm) than the basidia of I. lacteus (20–25 x 4–5 µm). The spores of I. lacteus are oblong to cylindric and straight to slightly curved and 5–6 x 2–3 µm, while those of I. hydnoides are ellipsoid to broadly ellipsoid and 5.5–6.5 x 3.5–4 µm (Fig. 1B).
Cultural characters, growth rate and extracellular oxidase reactions also distinguish I. hydnoides from I. lacteus. The growth rate of our isolates of I. lacteus was 4.9–5.3 mm per day (reported as 3.9–5 mm per day by Stalpers 1978), while that of I. hydnoides is 1.6 mm per day (Fig. 2). A strong color change occurred in a p-cresol test for I. hydnoides but there was no such reaction for I. lacteus.
The phylogeny inferred from the analysis of ITS sequence data indicates that the genus Irpex includes two species, I. hydnoides and I. lacteus. This result, coupled with morphological and cultural characters, supports adding I. hydnoides as a new species in the previously monotypic genus Irpex. Irpex vellereus is more closely related to A. dregeanum, a species that also possess a dimitic hyphal system, metuloids and skeletocystidia. Although A. dregeanum and I. vellereus have been treated as synonyms (Hjortstam and Ryvarden 1990), ITS sequence divergence between these taxa (10.1%) is greater than has been reported within other basidiomycete species (Anderson and Stasovski 1992, Gardes et al 1991, Vasiliauskas et al 1999). Testing the hypothesis that these morphologically similar taxa are conspecific awaits the study of a greater number of isolates, including A. griseofuscescens, the type species of Australohydnum.
The A. dregeanum–I. vellereus clade was sister of the group that included two species of Phanerochaete. These four taxa formed a well-supported clade (98%), but the relationship of this group to Ceriporia and Irpex clades was not resolved in our ITS phylogeny. A number of morphological characters also support the supposition that Australohydnum is more closely allied to Irpex than to Phanerochaete, but there are several tuberculate, raduloid to aculeate Phanerochaete and such a suggestion is not as strongly supported by some microscopic features and molecular data (Hjortstam and Ryvarden 1990, Lim 2001). Elucidating phylogenetic relationships within this clade will require sequencing of a greater number of taxa.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication December 20, 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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