Fung. Sci. 27(1): 1–8, 2012

Three new records of Auriculariales from Taiwan Roland Kirschner1* and Chee-Jen Chen2 1. Department of Life Sciences, National Central University, Jhongli City, Taoyuan 320, Taiwan 2. Department of Biotechnology, Southern Taiwan University, Yungkang City, Tainan 710, Taiwan, e-mail: [email protected] (Accepted: October 31, 2011)

ABSTRACT Basidiodendron deminutum, B. eyrei, and Myxarium grilletii, three species common in America and Europe, are newly recorded for Taiwan. Descriptions and line drawings of the characteristics of these species are provided. Keywords: heterobasidiomycetes, Hyaloriaceae, jelly fungi, wood decay fungi, Tremellales.

Introduction The orders Auriculariales and Tremellales have been confused in the past decades, because many species have in common gelatinous basidiomata and longitudinally septate basidia. Auriculariales are, however, additionally defined by the absence of a yeast stage, septum ultrastructure characterized by dolipores with continuous parenthesomes, and a saprobic lifestyle (Wells, 1994). Most species grow on dead wood; some of them have been shown to be white-rot fungi being able mainly to decompose lignin (e.g., Boddy and Rayner, 1983; Worrall et al., 1997), but detailed physiological analyses are rare in this group. Molecular data analyses confirm this concept and place the order Auriculariales among the most basal groups of the

*

Corresponding author, e-mail: [email protected]

Agaricomycotina, Basidiomycota (Weiß and Oberwinkler, 2001). About 200 species are known in this group (Kirk et al., 2008); DNA markers are available for roughly estimated less than 50% of the species, namely sequences of the LSU rDNA and ITS, other regions being extremely rarely represented in GenBank. Sequences are represented mostly for a single specimen per species. Identification of species collected in the field, therefore, largely depends on morphological study. In Taiwan, the most common species is Auricularia polytricha which is also grown commercially in mushroom farms (Chen, 2000, 2003, 2004). Previously, we reported a new anamorph in this group from Taiwan (Kirschner and Chen, 2004). Here, three species of this order known to be common in America and Europe are re-

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ported in Taiwan for the first time.

Materials and Methods Fresh basidiomata were collected on rotten wood in the field and brought to the laboratory, placed into a refrigerator and examined within three days. Fresh specimens were sectioned and squashed with freehand. Microscopic examination with 1000 × magnification was made with material mounted in 5–10% aqueous KOH and stained with 1% aqueous phloxine. Because the studied species are well known from America and Europe, no statistic treatment of measurements was performed, but only about ten spores

were measured. Sizes given in brackets represent extreme values found. The specimens were dried with an electric dryer and deposited in the herbarium of the National Museum of Natural Science (TNM).

Taxonomy Basidiodendron deminutum (Bourdot) LuckAllen, Can. J. Bot. 41: 1041, 1963. (Fig. 1) Basidiomata resupinate, flat, smooth, not gelatinous, dirty white, extremely thin. Hyphae smooth, colorless, with clamped septa, 1.5–2.5 µm diam. Gloeocystidia numerous, colorless or filled with yellow droplets, broader at base,

Fig. 1. Basidiodendron deminutum (R. Kirschner 429). Bar = 10 m.

Three new records of Auriculariales with broadly rounded apices, in some cases irregularly constricted, 15–55 × 6–10 µm. Hyphidia not found. Basidia four-celled, longitudinally septate, long-ellipsoidal, 13–17 × 7–8 µm, after emptying of cytoplasm remaining on the supporting hypha, sterigmata 3–5 µm long. Basidiospores colorless, smooth, with apiculus lateral at base, broad-ellipsoid, thinwalled, 6–7 × 4–5 µm, germinating only with hyphae observed. Specimen examined. TAIWAN, Taipei, National Taiwan University campus, 25°0' N/ 121°32' E, on rotten wood on the ground, 26 Oct. 1998, leg. R. Kirschner 429 (TNM F24983). Notes. The species is known from Europe and North to South America (Martin, 1942; McGuire, 1941; Oberwinkler, 1963), and Taiwan (new record). It differs from other species of the genus by having smaller spores (which is referred to the scientific name of the species; Luck-Allen, 1963; McGuire, 1941; Oberwinkler, 1963). Hyphidia were not found, which is in congruence with other authors (Luck-Allen, 1963; McGuire, 1941), whereas Kotiranta and Saarenoksa (2005) mentioned the presence of few hyphidia. Basidiodendron eyrei (Wakef.) Luck-Allen, Can. J. Bot. 41: 1034, 1963. (Fig. 2) Basidiomata forming a thin inconspicuous indeterminate layer, visible as a pink luster on the substrate. Hyphae colorless, smooth, with clamped septa, 2 µm diam. Hyphidia absent. Cystidia, interspersed between hyphae bearing basidia, cylindrical, slightly undulate, slightly broader at base, smooth, thin-walled, colorless, 15–20 × 3–4 µm, apex broadly rounded. Basidia arising sympodially from short, up to nearly

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20 µm long, erect, geniculate hyphae, obovoid, 10–12 × 8–9 µm, lacking stalk cell, longitudinally septate, with 4 short sterigmata, collapsing and covering the supporting hyphae after spore release. Basidiospores globose, colorless, smooth, thin-walled, 6–7(–8) µm diam., with 1 µm long apiculus, germinating with secondary ballistospores. Specimen examined. TAIWAN, Chiayi County, Alishan, 23°26' N/ 120°46' E, on rotten wood, 19 May 2000, leg. R. Kirschner 650 (TNM). Notes. Basidiodendron eyrei was reported from Central and Southeast Asia, Europe, and North to South America (Kisimova-Horovitz et al., 1997; Martin, 1952; Roberts and Spooner, 1998; Wells and Raitviir, 1975). Wells and Raitviir (1975) considered B. eyrei as a cosmopolitan and one of the most common species among related ones; they, however, applied a broad species concept by including B. deminutum as a synonym of B. eyrei. This synonymy has rarely been followed by subsequent authors. Spore shapes are sufficiently distinct to justify keeping these two species separate (LuckAllen, 1963). Basidiodendron eyrei is identified here due to the globose spores and lack of hyphidia. Luck-Allen (1963) provided identical sizes (4–7 µm diam.), though the spore sizes being slightly smaller in some other collections (Kotiranta and Saarenoksa, 2005) might indicate the presence of cryptic species. Myxarium grilletii (Boud.) D.A. Reid, Persoonia 7: 297, 1973. (Fig. 3) Basidiomata macroscopically forming whitish gray gelatinous layer on the substrate, when seen with the dissecting microscope appearing composed of many minute pustules that form a

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semitransparent layer. Hyphae 1.5–2 µm diam., smooth, thin-walled, with clamps at the septa. Cystidia lacking. Hyphidia numerous, extending above the basidia, undulate, with short lateral projections and branches, 1.5–2 µm wide. Basidia arising sympodially from erect, often geniculate hyphae, mostly conspicuously stalked, stalk 3–10 × 2 µm, rarely shorter, be-

coming separated from the broadly ellipsoid, rarely globose basidial body measuring 7–9 × 6–8 µm at maturity, longitudinally septate, with four sterigmata extending up to ca. 15–20 × 1.5–2 µm. Basidiospores cylindrical to slightly allantoid, rarely slightly sigmoid, with 1 nucleus, 6–9 × 2–3(–4) µm, germination not observed.

Fig. 2. Basidiodendron eyrei (R. Kirschner 650). Bar = 10 m.

Three new records of Auriculariales

Fig. 3. Myxarium grilletii (R. Kirschner et al. 417). Bar = 10 m.

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Specimen examined. TAIWAN, Nantou County, Lienhuachih, 23°54' N/ 120°55' E, on dead branch of Cunninghamia lanceolata, 12 Oct. 1998, leg. R. Kirschner et al. 417 (TNM F24982). Additional specimens examined. GERMANY: Baden-Württemberg, Tübingen, Schönblick, 48°32' N/9°3' E, on rotten branch of deciduous tree, 17 Feb. 1998, leg. R. Kirschner 287 (TNM F24981); Bayern, Allgäu, Oberjoch, Iseler, 47°29' N/10°25' E, ca. 1,700 m, on dead branch of Pinus mugo, leg. R. Kirschner et al. 580 (TNM F24985); TAIWAN, Taipei, Yangmingshan, Dingshan, 25°8' N/121° 35' E, on dead branch on ground, 24 Jul. 1999, leg. R. Kirschner 547 (TNM F24984). Notes. Myxarium grilletii can be differentiated from other species by the basidiomata composed of minute, spherical pustules remaining distinct even when forming a common layer (Hauerslev, 1993). This species is considered in its narrow sense here according to Hauerslev (1993) and Wells et al. (2004), not the broad sense of Stypella grilletii (Boud.) P. Roberts, because further studies (including molecular ones) appear to be required before accepting the synonymies proposed by Roberts (1998). Stypella grilletii given as “current name” in Index Fungorum (www.indexfungorum.org) and MycoBank (www.mycobank.org) cannot be accepted, because according to phylogenetic analysis Myxarium species and Hyaloria species are placed together in a well-supported clade, the Hyaloriaceae (Wells et al., 2004), whereas the type species of Stypella resides outside this clade (Weiß and Oberwinkler, 2001). Considering Myxarium as synonym of Stypella is not accepted by specialists of Auriculariales (Dueñas, 2005; Weiß and Oberwinkler, 2001;

Wells et al., 2004). In the “Dictionary of the Fungi”, Myxarium is also kept separate from Stypella (Kirk et al., 2008). Myxarium grilletii s. str. is known in Europe and America (Wojewoda, 1981).

Acknowledgements The authors thank Prof. Z.C. Chen for providing the opportunity and facilities to study the species presented here. The study was financially supported by the German Academic Exchange Service (DAAD), Ministry of Education (MoE), and National Science Council (NSC 97-2511-S-218-007-MY3) of R.O.C. (Taiwan).

References Boddy, L. and A.D.M. Rayner. 1983. Ecological roles of Basidiomycetes forming decay communities in attached oak branches. New Phytol. 93: 77–88. Chen, C.J. 2000. Mushrooms: Cultivation. In: Wiley Encyclopedia of Food Science and Technology. 2nd edition. John Wiley & Sons Inc. New York. Chen, C.J. 2003. Magical Chinese Wood-Ear (Auricularia polytricha) on Loosing Weight, Antitumor, and Antioxidant Activities. Proceedings of the 2nd International Conference on Medicinal Mushroom and the International Conference on Biodiversity and Bioactive Compounds. p. 211–217. Peach, Pattaya, Tailand. 17–19 July, 2003. (poster) Chen, C.J. 2004. Applications of Auricularia polytricha. Pp. 411–418. In: Frontiers in basidiomycote mycology. Eds., R. Agerer, Piepenbring, and M.P. Blanz. IHW-Verlag,

Three new records of Auriculariales Eching, Germany. Dueñas, M. 2005. New and interesting Iberian heterobasidiomycetous fungi. I. Nova Hedwigia 81: 177–198. Hauerslev, K. 1993. The genus Myxarium (Tremellales) in Denmark. Mycotaxon 49: 235–256. Kirk, P.M., P.F. Cannon, D.W. Minter, and J.A. Stalpers. 2008. Dictionary of the Fungi (10th ed.). Wallingford: CABI. Kirschner, R. and C.J. Chen. 2004. Helicomyxa everhartioides, a new helicosporous sporodochial hyphomycete from Taiwan with relationships to the Hyaloriaceae (Auriculariales, Basidiomycota). Stud. Mycol. 50: 337–342. Kisimova-Horovitz, L., F. Oberwinkler, and P.L.D. Gómez. 1997. Basidiomicetos resupinados de Costa Rica. Exidiaceae (Tremellales). Rev. Biol. Trop. 45: 1325– 1347. Kotiranta, H. and R. Saarenoksa. 2005. The genus Basidiodendron (Heterobasidiomycetes, Tremellales) in Finland. Ann. Bot. Fennici 42: 11–22. Luck-Allen, E.R. 1963. The genus Basidiodendron. Can. J. Bot. 41: 1025–1052. Martin, G.W. 1942. New or noteworthy tropical fungi—II. The subgenus Bourdotia in tropical America. Lloydia 5: 158–164. Martin, G.W. 1952. Revision of the North Central Tremellales. Univ. Iowa Stud. Nat. Hist. 19: 1–122. McGuire, J.M. 1941. The species of Sebacina (Tremellales) of temperate North America. Lloydia 4: 1–43. Oberwinkler, F. 1963. Niedere Basidiomyceten

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aus Südbayern III. Die Gattung Sebacina Tul. s. l. Berichte der Bayerischen Botanischen Gesellschaft 36: 41–55. Roberts, P. 1998. A revision of the genera Heterochaetella, Myxarium, Protodontia and Stypella (Heterobasidiomycetes). Mycotaxon 69: 209–248. Roberts, P.J. and B.M. Spooner. 1998. Heterobasidiomycetes from Brunei Darussalam. Kew Bulletin 53: 631–650. Weiß, M. and F. Oberwinkler. 2001. Phylogenetic relationships in Auriculariales and related groups – hypotheses derived from nuclear ribosomal DNA sequences. Mycol. Res. 105: 403–415. Wells, K. 1994. Jelly fungi, then and now! Mycologia 86: 18–48. Wells, K., R.J. Bandoni, S.R. Lim, and M.L. Berbee. 2004. Observations on some species of Myxarium and reconsideration of the Auriculariaceae and Hyaloriaceae (Auriculariales). Pp. 237–248. In: Frontiers in basidiomycote mycology. Eds., R. Agerer, Piepenbring, and M.P. Blanz. IHW-Verlag, Eching, Germany: Wojewoda, W. 1981. Basidiomycetes (Podstawczaki), Tremellales (Trz!sakowe), Auriculariales (Uszakowe), Septobasidiales (Czerwcogrzybowe). Pp. 1–408. In: Ma"a Flora Grzybów 2. Ed. S. Doma#ski. Pa#stwowe Wydawnictwo Naukowe, Warszawa, Kraków. Worrall, J.J., S.E. Anagnost, and R.A. Zabel. 1997. Comparison of wood decay among diverse lignicolous fungi. Mycologia 89: 199–219.

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傸㼡᳇䣬㗦仱䖬㑮䮾懂䣬 ᛳࠒኈ1! ങం྇2 1. ⚳䩳ᷕ⣖⣏⬠䓇␥䥹⬠䲣 2. ⋿⎘䥹㈨⣏⬠䓇䈑䥹㈨䲣

ᄢ! ! ौ 㛔㔯㍷徘 3 䧖㍉冒冢䀋䘬㛐俛䚖㕘䲨抬䧖烉Basidiodendron deminutumˣB. eyrei ⍲ Myxarium grilletiiˤ 㔯ᷕ㍸ὃ䈡⽝䔓⚾⍲妶婾℞冯䚠役䧖ᷳ䔘⎴ˤ 撚戳夜濣㛐僸却ˣ㖶䚖俛䥹ˣ䔘㑼⫸却ˣ戨俛䚖ˣ先岒却ˤ

Fung. Sci. 27(1): 9–15, 2012

New records of Hyphodontia sphaerospora in Taiwan and Vietnam Eugene Yurchenko and Sheng-Hua Wu Department of Botany, National Museum of Natural Science, Taichung, Taiwan 404, R.O.C. (Accepted: December 20, 2011)

ABSTRACT A corticioid basidiomycete Hyphodontia sphaerospora was known from Japan and South America. This species was found in this study as also distributed in Taiwan and Vietnam. Description and illustrations based on the studied collections are provided. The morphological distinction of H. sphaerospora from the most resembled species, H. arguta, is discussed. Keywords: Basidiomycota, corticioid fungi, Hyphodontia arguta, H. sphaerospora, taxonomy.

Introduction Hyphodontia sphaerospora was firstly reported from the type locality in Ehime Prefecture, Shikoku Island, South Japan (Maekawa, 1993); then it was recorded from northeast Equador, and from Venezuela (Hjortstam and Ryvarden, 2002; Hjortstam et al., 2005). Micromorphology of this species was only illustrated in the protologue (Maekawa, 1993). Hyphodontia sphaerospora was described for the first time by Maekawa (1993) as Grandinia arguta (Fr.) Jülich var. sphaerospora N. Maek. According to the protologue and other taxonomic reports (Hjortstam and Ryvarden, 2002; Hjortstam et al., 2005), it differs from G. arguta var. arguta by having globose and slightly thick-walled spores up to 4.5 m diam. Moreover, according to fig. 2 and fig. 3 in Maekawa Corresponding author, email: [email protected]

(1993), G. arguta var. sphaerospora differs from var. arguta in different morphology of capitate cystidia, which are narrower, cylindrical, and more or less flexuous in the former. In addition, lagenocystidia of var. sphaerospora are often lateral, while those in var. arguta are terminal. After studying Hyphodontia spp. deposited in herbarium of National Museum of Natural Science, Taiwan, R.O.C. (TNM), we found that H. sphaerospora is also distributed to Taiwan and Vietnam. Description of the species based on two studied specimens is provided in this paper. Morphological descriptions were based on dry basidiomata. Microscopic measurements were carried out on the material mounted in 3% KOH water solution. The sporal wall amyloid or dextrinoid reaction was checked in Melzer’s solution (Mz’s), and cyanophily was examined

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by using Cotton blue-lactophenol solution. Images of hymenial surface were captured by digital camera Nikon Coolpix P6000 via an ocular of Zeiss Stemi SV11 stereomicroscope.

Taxonomy Hyphodontia sphaerospora (N. Maek.) Hjortstam (Figs. 1, 2, 4A, & 4B) Basidioma totally effused, softmembranaceous. Hymenial surface odontioid. Aculei dark cream,ġ conical-subcylindrical, usually blunt, wart-shaped in young basidiomata, 4–8 per mm, up to 0.35–0.5 mm high, 0.05– 0.15(–0.2) mm wide at base, mostly solitary, under lens with fine bristles due to projecting lagenocystidia or almost smooth. The part between aculei paler in color, 0.03–0.1 mm thick, finely porulose-reticulate, slightly cracking. Margin paler, fairly abrupt or diffuse. Hyphal system monomitic, hyphae clamped at all primary septa. Subicular hyphae and hyphae of aculeal trama loosely arranged, moderately branched, straight to slightly flexuous, colorless, thin- to thick-walled, (1.5–)2.7–4(– 5.2) m diam, occasionally with adventitious septa. Subhymenium loose, indistinctly delimited from subiculum; subhymenial hyphae moderately branched, colorless, but yellowish in mass, thin-walled, constricted in branch sites up to 1.2–1.4 m, swollen between branch sites up to 3.3–3.7 m. Lagenocystidia numerous, with pale yellowish incrustation, occasionally with lateral protuberances, 16.5–28(–30) m long, 3.5–4.2(–5.2) m wide in basal part, the upper subulate part about 0.6 m wide without incrustation. Capitate and subcylindrical cystidia or hyphal ends variably abundant, aggregated mostly at aculeal apices, but also occurring be-

tween aculei, (13–)17–25(–40) × 3–4.5 m, with or without intercalary swellings, straight or flexuous, some with adventitious septa; capitate hyphal ends with strongly cyanophilous contents. Basidia subcylindrical, 11–14 × 3.8–4 m, colorless, thin-walled, 4-sterigmate. Basidiospores globose to broadly ellipsoid, (3.1–) 3.3–4(–4.5) × (2.8–)3–3.7(–4.1) m, colorless, with ca. 0.3 m thick walls, Mz’s-negative, slightly cyanophilous, with very small or indiscernible apiculus. Specimens examined. Hyphodontia sphaerospora. Taiwan. Nantou County, Sunlinksea, 120°47' E, 23°38' N, alt. 1,700 m, on dead corticated twig of Cryptomeria japonica D. Don, 1 cm in diam, coll. S.H. Wu, 1.VII.1992, Wu 9207-21 (TNM F24830). Vietnam. Ha Tay Province, Ba Vi National Park, 105°22' E, 21°04' N, alt. 1,200 m, on dead corticated angiosperm trunk, coll. S.H. Wu and S.Z. Chen, 3.VII.1998, Wu 9807-53 (TNM F9041). Hyphodontia arguta. Taiwan. Nantou County, Hsitou, alt. 1,200 m, on branch of Cryptomeria japonica, coll. S.H. Wu, 10.X.1991, Wu 911010-4 (TNM F24822); Sunlinksea, alt. 1,700 m, on branch of C. japonica, coll. S.H. Wu, 1.VII.1992, Wu 9207-17 (TNM F24829), Wu 9207-32 (TNM F24831), Wu 9207-40 (TNM F24724), coll. S.H. Wu, 19.IX.1992, Wu 9209-58 (TNM F24833), Wu 9209-82 (TNM F24835), Wu 9209-85 (TNM F24837); Tungpu, alt. 1,300 m, on branch of angiosperm, coll. S.H. Wu, 8.X.1992, Wu 9210-99 (TNM F24841). Taipei, Yangminshan National Park, alt. 350 m, on dead branch of angiosperm, coll. S.H. Wu and S.Z. Chen, 20.II.2001, Wu 010212 (TNM F12781). China. Yunnan, Yiliang County, Shiaotsapa, Houho, on rotten trunk of angiosperm, coll. S.H. Wu and J.Y. Tseng,

Hyphodontia sphaerospora in Taiwan and Vietnam 18.IX.1998, Wu 9809-86 (TNM F9180). U.S.A. Ohio, Butler County, on Quercus sp., coll. H.H. Burdsall, Jr., 18.VII.1977 [ex Herb. Center of Forest Mycology Research, HHB 9393; TNM

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F914 (duplicate)]. Sweden. Öland, Högstrum, on fallen Quercus, coll. Å. Strid, 7.X.1995 [ex Naturhistoriska Riksmuseet (S); TNM F4607 (duplicate)].

Fig. 1. Hyphodontia sphaerospora (TNM F9041). A. Basidioma section. B. Aculeus section. C. Subicular hyphae. D. Hyphae from aculeal trama. E. Subhymenial hyphae and lagenocystidia. F. Hyphal ends from the base of aculei, with lagenocystidia. G. Lagenocystidia. Scale bars for A = 0.5 mm, for B = 0.1 mm, for C–G = 10 m.

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Fig. 2. Hyphodontia sphaerospora (TNM F9041). A. Capitate cystidia and hyphal ends at aculeal apex. B (TNM F9041) & F (TNM F24830). Capitate cystidia. C. Basidioles and basidiospores. D. Basidia. E (TNM F9041) & G (TNM F24830). Basidiospores. Scale bars for A = 10 m, for B–G = 5 m.

Distribution. Japan, Taiwan, Vietnam, Equador, Venezuela (subtropical to warmtemperate areas of East Asia and northern South America). Remarks. In contrast with the description for H. sphaerospora (Maekawa, 1993; Hjortstam and Ryvarden, 2002), collections from Taiwan and Vietnam have denser and shorter aculei, and spores are from globose to broadlyellipsoid. The Taiwanese collection is probably young, and has wart-like to short-conical aculei,

60–80 m high and 45–90 m wide at base. The specimen from Vietnam has conicalsubcylindrical aculei, up to 0.5 mm high and up to 0.2 mm wide. After studying specimens of H. arguta from Taiwan, China, Sweden, and U.S.A., we found that this species is distinctly separated from H. sphaerospora. In H. arguta there are numerous wide, moderately thick-walled hyphal ends, constituting aculei tips and resembling tubular cystidia or even skeletal hyphae, often yellow-

Hyphodontia sphaerospora in Taiwan and Vietnam ish to reddish-yellow in mass (Fig. 3B) and rendering the upper part of aculeus fimbriate (Fig. 4D); hyphae in aculeal trama and subiculum are often thick-walled. In H. sphaerospora hyphal ends are fairly slender, thin-walled and colorless; hymenophoral aculei are generally

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smooth; hyphae are narrower and thin-walled, which gives more soft consistency of the basidioma. Besides, lagenocystidia in H. sphaerospora are smaller, thin-walled, and more abundant, than in H. arguta (Figs. 3C, D). Both species have thin- to slightly thick-walled basidio-

Fig. 3. Hyphodontia arguta (TNM F24822). A. Basidioma section. B. Hyphal ends near aculeus tip. C. Hymenium. D. Lagenocystidia from hymenium. E. Lagenocystidium at vegetative hypha. F. Capitate cystidia with exudate. G (TNM F24833) & H (TNM F914). Basidiospores. Scale bars: for A = 0.5 mm, for B–F = 10 m, for G & H = 5 m.

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Fig. 4. Hymenial surfaces. Hyphodontia sphaerospora. A. TNM F24830. B. TNM F9041. Hyphodontia arguta. C. TNM F24831 (irregularly odontioid). D. TNM F24822 (almost hydnoid). E. TNM F24837 (almost hydnoid). Scale bars = 1 mm

spores, but in H. sphaerospora they are smaller and often globose, whereas in H. arguta subglobose spores are seldom (Figs. 3G, H). Spores of Taiwanese material of H. arguta were measured as (4.2–)4.8–5.3 × (2.8–)3.4–3.7 m. Significant morphological variation in hymenophore was found in examined collections of H. arguta, from warted to minutely hydnoid, probably due to developmental stage of the basidiomata. However, H. arguta generally has thicker and longer aculei (Fig. 4).

Acknowledgements This study is supported by National Science Council of R.O.C. (Grant No. NSC 98-2621-B178-002-MY3). The first author is financially

supported by the National Science Council of R.O.C. (Grant No. NSC 100-2811-B-178-001). The authors are grateful to Ms. S.Z. Chen (National Museum of Natural Science), for the help in managing herbarium specimens.

References Hjortstam, K. and L. Ryvarden. 2002. Studies in tropical corticioid fungi (Basidiomycotina, Aphyllophorales): Alutaceodontia, Botryodontia, Hyphodontia s.s. and Kneiffiella. Synop. Fung. 15: 7–17. Hjortstam, K., L. Ryvarden, and T. Iturriaga. 2005. Studies in corticioid fungi from Venezuela II (Basidiomycotina, Aphyllophorales). Synop. Fung. 20: 42–78.

Hyphodontia sphaerospora in Taiwan and Vietnam Maekawa, N. 1993. Three new corticiaceous fungi (Basidiomycotina, Aphyllophorales)

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from Japan. Proc. Japan Acad., Ser B 69(5): 119–122.

䋁⨠䰰渐劊 (Hyphodontia sphaerospora) ◦傸㼡⊈屈∕䕂㑮壖懂 Eugene Yurchenko! ֔ᖐ๼ ⚳䩳冒䃞䥹⬠⌂䈑棐㢵䈑⬠䳬炻冢ᷕⶪ 404

ᄢ! ! ौ 䎫⬊䴚滺却 (Hyphodontia sphaerospora) 㗗ᶨ䧖忶⍣䘤䎦㕤㖍㛔⍲⋿伶㳚䘬㭤却栆ˤ㛔䞼䨞䘤䎦忁䧖ḇ 䓊㕤冢䀋⍲崲⋿ˤ㛔㔯᷎㍸ὃ䎫⬊䴚滺却䘬䈡⽝㍷徘⍲䈡⽝⚾ˤ㛔㔯᷎妶婾䎫⬊䴚滺却冯ᶨ䚠Ụ䧖炻 扛⮾䴚滺却 (H. arguta) 䘬䈡⽝ⶖ⇍ˤ 撚戳夜濣↮栆⬠ˣ䎫⬊䴚滺却 (H. sphaerospora)ˣ㭤却ˣ扛⮾䴚滺却 (Hyphodontia arguta)ˣ㑼⫸却攨ˤ

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Fung. Sci. 27(1), 2012

Fung. Sci. 27(1): 17–29, 2012

Diversity of Fusarium species associated with maize and sorghum grains grown in Karnataka, India M.Y. Sreenivasa1, Adkar Purushothama Charithraj2, Regina Sharmila Dass3, and G.R. Janardhana3* 1. Department of Microbiology, University of Mysore, Manasagangotri, Mysore- 570 006, Karnataka, India. Phone: +91-821-2419733, Fax: +91-821-2419759; E-mail: [email protected] 2. Plant Pathology laboratory, Faculty of Agriculture and Life sciences, Hirosaki University, Hirosaku, Japan. E-mail: [email protected] 3. Molecular Phytodiagnostic Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore- 570 006, Karnataka State, India. Phone: +91-821-2419763, Fax: +91-821-2419759; Email: [email protected] (Accepted: February 12, 2012)

ABSTRACT A total of 130 maize and sorghum samples from 15 districts of Karnataka state were analyzed for diversity and per cent incidence of Fusarium species by direct plating method on malachite green agar 2.5 medium. For further identification of Fusarium species, one set of genus-specific ITS primers was used to differentiate Fusarium species from closely related genera by polymerase chain reaction. The study revealed the occurrence of 10 diverse species of Fusarium in maize and sorghum samples collected from different districts of Karnataka. The species identified were F. verticillioides, F. proliferatum, F. oxysporum, F. anthophilum, F. pallidoroseum, F. sporotrichioides, F. solani, F. graminearum, F. decemcellulare, and F. lateritium respectively. All the Fusarium species tested by PCR scored positive with the ITS pair of primers with an expected 431bp PCR product. The study further revealed the occurrence of F. verticillioides (18.69%), F. proliferatum (6.08%), and F. anthophilum (4.46%) in maize samples respectively. Similarly, species of F. verticillioides (10.22%) and F. anthophilum (1.67%) were recorded in higher per cent incidence in sorghum samples. The data on the diversity and incidence of Fusarium species would be of great significance for predicting the extent of post-harvest deterioration and subsequent accumulation of toxins in maize and sorghum grains produced in Karnataka, India. Keywords: diversity, incidence, Fusarium, ITS, Maize, Sorghum.

Introduction Maize (Zea mays L.) and sorghum (Sorghum bicolor L.) are two important cereal crops cultivated as major rain fed crops in most of the *

Corresponding author, e-mail: [email protected]

semiarid regions. They are used in more ways than any other cereals by humans, as a feed grain, as a fodder crop, and for hundreds of industrial purposes (Anderson et al., 2004). Maize and sorghum grains are prone to pre and

18

Fung. Sci. 27(1), 2012

post-harvest fungal infection. Contamination of cereals by fungi is often unavoidable and is a worldwide problem (Placinta et al., 1999). Due to poor agricultural practices and intermittent rain at the time of harvest, a number of fungi colonize cereal grains. The susceptibility of maize and sorghum to various fungi has been well documented (Munkvold and Desjardins, 1997). Fungi such as Aspergillus, Penicillium, Alternaria, and Fusarium have been reported to occur in maize and sorghum grains (Bhattacharya and Raha, 2002). Of the fungi involved, Fusarium species are most commonly associated with maize and sorghum all over the world and can cause disease on a wide variety of other plants and plant derived products (Summerell et al., 2003; Morales-Rodriguez et al., 2007). The significant risk of contamination by mycotoxins is also related to the association of Fusarium species with the maize and sorghum grains (Janardhana et al., 1999). However, there is a lack of accurate data on the diversity and incidence of Fusarium species in maize and sorghum grains. Because of these reasons, it has not been possible to develop effective management strategies to prevent fungal bio-deterioration of grains and the entry of fusarial toxins in to the food chains. Studies on fungal diversity demands expertise in fungal taxonomy (Pinnoi et al., 2006). This is particularly complex in the case of the genus Fusarium because of the existence of several and often-conflicting taxonomic treatments and a large number of closely related species of this genus (Nelson et al., 1994). The current methods for identification of the genus Fusarium mostly relies on micro-morphological characteristics. PCR based detection methods have provided an alternative to conventional identifi-

cation of Fusarium species. Recently mycologists employ PCR based techniques for the detection Fusarium species (Abd-Elsalam et al., 2003; Mirete et al., 2004; Patino et al., 2006). Hence in this study, the diversity of Fusarium species and their per cent incidence in maize and sorghum grains was determined and further identification of Fusarium species was done by molecular methods such as PCR.

Materials and Methods Collection of samples A total of 86 maize samples from 15 districts and 44 sorghum samples (approximately 1 kg) from 14 districts of Karnataka were collected from markets, local stores, agricultural cooperatives and farm fields. Samples were brought to the laboratory in sterile plastic bags and kept at 4°C. All the samples were subjected to mycological analysis. Mycological analysis of samples To know the diversity of Fusarium species, maize and sorghum grains were plated on potato dextrose agar medium by agar plating method. Since general purpose medium was unsuitable for the isolation of Fusarium species, malachite green agar (MGA) 2.5 medium was used as recommended by Bragulat et al. (2004). Two hundred maize and sorghum kernels/grains from each sample were surface sterilized with 2% sodium-hypochlorite solution for 3 min. and rinsed twice with sterile distilled water. Samples were then plated on MGA 2.5 agar plates at the rate of 10 grains per plate. The plates were incubated under alternating periods of 12 h darkness and 12 h of light at 25 ± 2°C for 7 days. The fungal colonies emerging on grains

Diversity of Fusarium species in Karnataka were visualized using stereo-binocular microscope (Magnus MS24). Representative isolates of Fusarium species were transferred onto Potato Dextrose Agar (PDA) and Spezieller Nahrstoffarmer Agar (SNA) medium to study the macro and micro morphological characteristics. All the isolates of Fusarium species were identified up to the species level by using fungal keys (Booth, 1977; Leslie and Summerell, 2006) and the per cent incidence of Fusarium species was calculated (Ghiasian et al., 2004). Identification of Fusarium species by PCR DNA isolation. All fungal isolates were freshly inoculated in 500 µL Potato Dextrose Broth (PDB) in 2 mL microfuge tubes and incubated at room temperature for 4 days. The resulting mycelium was used for the DNA isolation. The mycelial mat was pelleted by centrifuging at 5000 rpm (REMI C24) for 5 min. The pellet was ground in microfuge tubes with blunt ends of disposable sterile pipette tips in 500 µL of extraction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, preheated at 65°C) and incubated at 65°C for 15 min. During incubation, the mixture was briefly vortexed 2–3 times. The samples were then treated with 500 µL of phenol : chloroform (1:1) and vortexed for one min and the supernatant was taken after centrifugation at 3,000 rpm for 5 min at 4°C. DNA was precipitated with an equal volume of ice-cold isopropanol, and incubated at –20°C for 60 min and again centrifuged at 8,000 rpm for 8 min at 4°C. The pellet obtained was rinsed with 70% ethanol, airdried, resuspended in 50 µL of nucleic acid free water and used directly for PCR. Primers and PCR amplification. One set of primer as described by Bluhm et al. (2004) was

19

used to amplify ITS region specific to Fusarium (ITS Forward- AAC TCC CAA ACC CCT GTG AAC ATA, ITS Reverse- TTT AAC GGC GTG GCC GC) and the expected size of amplicon was 431 bp. PCR was performed using Advanced Thermus 25 Thermocycler (Peqlab, Germany). PCR mixture (25 µL) contained 2 µL of DNA sample, 10× PCR buffer, 25 mM MgCl2, 2 mM dNTPs, 20 pmol of each forward and reverse primer and 0.5 µL (3 U/ L) of Taq DNA polymerase. The PCR conditions were 94°C for 4 min for initial denaturation, followed by 35 cycles of denaturation at 94°C for 1 min, primer annealing at 58°C for 1 min, primer extension at 72°C for 1 min. The final extension was set at 72°C for 10 min. 10 µL of the PCR product was electrophoresed on 1.5% agarose gel. The gel was visualized and documented in gel documentation system (UTP-Bio Doc, USA) after staining with ethidium bromide. Primers and reagents for PCR analysis were procured from Bangalore Genei.

Results A total of 86 maize and 44 sorghum samples were collected from different districts of Karnataka. When analyzed by agar plating method, the following ten different species of Fusarium such as Fusarium verticillioides, F. proliferatum, F. oxysporum, F. anthophilum, F. pallidoroseum, F. sporotrichioides, F. solani, and F. graminearum were expressed. Occurrence of F. decemcellulare was recorded only in maize samples and F. lateritium was recorded only in sorghum samples (Figs. 1 & 2). Mycological analysis of maize samples for the incidence of Fusarium species by agar plating method (MGA 2.5) revealed the occurrence

20

Fung. Sci. 27(1), 2012

Fig. 1. Colony and micro morphological characteristics of Fusarium species: F. verticillioides 1A) Obverse: Creamishpeach to vinaceous, 1B) Reverse: Pale cream to violet, 1C) & 1D) Chains of microconidia borne on monophialides, 1E) Long chains of microconidia (1C–1F under 40×); F. proliferatum 2A) Obverse: Creamish-peach, 2B) Reverse: Pale cream to salmon, 2C) Polyphialides, 2D) Short chain of microconidia borne on polyphialides & 2E) Series of polyphialides (2C– 2E under 40×); F. oxysporum 3A) Obverse: White to pale yellow, 3B) Reverse: Yellow to orange, 3C & 3D) Macroconidia borne on monophialides, 3E) Intercalary single chlamydospore (3C–3E under 40×); F. anthophilum 4A) Obverse: Creamish-peach to vinaceous, 4B) Reverse: Pale cream to violet, 4C) Microconidia on false heads, 4D) Two types of (pyriform and globose) microconidia (4C & 4D under 40×); F. pallidoroseum 5A) Obverse: Grayish white to cream, 5B) Reverse: Grayish-peach, 5C) Two conidia per phialide to give a rabbit ear appearance, 5D) Macroconidia on phialides, 5E) Slender macroconidia with 3–5 septate (5C–5E under 40×).

Diversity of Fusarium species in Karnataka

21

Fig. 2. Colony and micro morphological characteristics of Fusarium species: F. sporotrichioides 6A) Obverse: White, 6B) Reverse: Pale red, 6C) Microconidia/Mesoconidia on phialides, 6D) Falcate to almost lunate shaped macroconidia (6C & 6D under 40×); F. solani 7A) Obverse: White, 7B) Reverse: Pale to cream, 7C) Long monophialides, 7D) Falcate to lunate shaped macroconidia and Reniform/Fusiform microconidia (7C–7E under 40×); F. graminearum 8A) Obverse: White to pale orange, 8B) Reverse: Pale orange to yellow, 8C) Moderately curved to straight with the ventral surface straight macroconidia, 8D) Intercalary chlamydospores (8C & 8D under 40×); F. decemcellulare 9A) Obverse: Cream to red (Colony moist appearance), 9B) Reverse: Pale cream, 9C) Long well developed monophialides, 9D) Oval shaped microconidia in chains (9C & 9D under 40×); F. lateritium 10A) Obverse: White, 10B) Reverse: Pale yellow, 10C) Microconidia on monophialides, 10D) Curved macroconidia with 4–5 septa (10C & 10D under 40×).

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Fung. Sci. 27(1), 2012

of Fusarium species with high per cent incidence. The species of F. verticillioides (18.69%) was recorded with higher per cent incidence, followed by F. proliferatum (6.08%) and F. anthophilum (4.47%). The six other Fusarium species namely F. oxysporum (0.01%), F. pallidoroseum (0.81%), F. sporotrichioides (0.31%), F. solani (0.17%), F. graminearum (0.39%), and F. decemcellulare (0.03%) were recorded with lower per cent incidence. The species of F. verticillioides was recorded with high per cent incidence in samples collected from most of the districts followed by the species of F. proliferatum and F. anthophilum (Table 1). Further, analysis of sorghum samples for the incidence of Fusarium species revealed that, F. verticillioides (10.22%) was recorded with relatively higher per cent incidence followed by F. anthophilum (1.67%). Fusarium species such as F. proliferatum (0.45%), F. pallidoroseum (0.59%), F. sporotrichioides (0.47%), and F. solani (0.13%) were recorded with lower per cent incidence. F. oxysporum and F. graminearum were recoded with a very low per cent incidence of 0.06% and 0.01%, respectively. Whereas, F. lateritium was recorded only in sorghum sample with the per cent incidence of 0.16% (Table 2). Overall, the per cent incidence of F. verticillioides was 18.69% followed by F. proliferatum (6.08%) and F. anthophilum (4.47%) in maize samples. Similarly, the per cent incidence of F. verticillioides was 10.22% followed by F. anthophilum (1.67%) in sorghum samples. All the 10 different species of Fusarium and three species of each of Aspergillus flavus, Cladosporium cladosporioides, and Alternaria alternata were subjected to PCR analysis using ITS genus specific. All isolates of Fusarium

species were scored positive for the ITS region. The expected 431 bp amplified ITS DNA product was detected, except Aspergillus flavus, Cladosporium cladosporioides, and Alternaria alternata, which were used as controls (Fig. 3).

Discussion Karnataka with its varied agro-climatic conditions produces a variety of food crops throughout the year. Maize and sorghum are two important food crops produced annually. Contamination of cereals with Fusarium species and their toxins is a global problem and it has been reported from different parts of the world including Europe, America, Africa, Asia, and Australia (Placinta et al., 1999). Throughout the world, much attention has been given to know the diversity, incidence and management of toxigenic moulds. However, data on diversity of Fusarium species on cereal grains is very limited in Karnataka, India. Mycological examination of maize and sorghum samples in our study, revealed that the occurrence of 10 different Fusarium species. The nine species of Fusarium are F. verticillioides, F. proliferatum, F. oxysporum, F. anthophilum, F. pallidoroseum, F. sporotrichioides, F. solani, and F. graminearum respectively. All these species are isolated from both maize and sorghum samples except F. decemcellulare and F. lateritium which were isolated from respective maize and sorghum samples. These Fusarium species are the potent producers of some important mycotoxins such as fumonisins, trichothecenes, fusarins, moniliformin, DON, fusaric acids, beauvericin, fusoproliferin, T2 toxins, nivalenol, zearalenone, and others (Leslie and Summerell, 2006). The occurrence of Fusarium species in

2.3

0.0

1.3

0.4

0.3

0.0

0.0

0.0

F. proliferatum

F. oxysporum

F. anthophilum

F. pallidoroseum

F. sporotrichioides

F. solani

F. decemcellulare

F. graminearum

2

3

4

5

6

7

8

9

0.0

0.0

0.0

0.9

0.0

6.6

0.0

3.6

33.5

2

0.5

0.0

0.1

0.0

0.05

2.3

0.05

4.2

19.4

3

0.0

0.7

0.0

0.5

2.5

3.3

0.0

19.5

19.4

4

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

22.5

5

1.0

0.0

0.0

1.3

1.0

2.8

0.0

4.6

27.3

6

0.0

0.0

0.0

0.1

0.1

1.6

0.0

3.6

20.2

7

0.0

0.0

0.0

0.0

2.6

3.7

0.0

0.9

17.1

8

9

1.0

0.0

0.0

0.1

1.6

11.6

0.0

3.9

11.6

Incidence (%)*

0.0

1.6

0.05

0.5

0.4

3.2

0.05

9.7

25.0

10

0.0

1.5

0.0

0.0

0.2

15.5

0.0

9.2

6.1

11

0.5

1.8

0.0

0.0

0.0

10.5

0.0

4.5

3.8

12

0.0

0.0

0.0

0.1

0.0

0.2

0.0

14.2

2.0

13

0.0

0.0

0.0

0.5

0.0

2.0

0.0

0.0

6.6

14

0.0

0.0

0.0

0.5

0.0

2.0

0.0

0.0

26.0

15

0.03

0.39

0.17

0.31

0.81

4.47

0.01

6.08

18.69

Incidence Avg. (%)

* Per centincidence is based on the analysis of 200 maize kernels in each sample. ** 1: Bagalakote (Data based on 6 samples), 2: Belgam (Data based on 6 samples), 3: Bijapur (Data based on 9 samples), 4: Dharwad (Data based on 9 samples), 5: Gadag (Data based on 3 samples), 6: Gulbarga (Data based on 3 samples), 7: Bellary (Data based on 7 samples), 8: Chitradurga (Data based on 8 samples), 9: Davangere (Data based on 7 samples), 10: Haveri (Data based on 9 samples), 11: Koppala (Data based on 4 samples), 12: Shimoga (Data based on 3 samples), 13: Hassan (Data based on 4 samples), 14: Mysore (Data based on 6 samples), and 15: Tumkur (Data based on 2 samples).

21.2

F. verticillioides

1

1**

Fusarium spedies

Sl. No

Table 1. Per cent incidence (%) of different Fusarium species on maize kernels as analyzed by agar plating method (MGA 2.5)

Diversity of Fusarium species in Karnataka 23

0.75

0.00

2.00

0.00

1.13

1.13

0.00

0.00

F. proliferatum

F. oxysporum

F. anthophilum

F. pallidoroseum

F. sporotrichioides

F. solani

F. graminearum

F. lateritium

2

3

4

5

6

7

8

9

0.00

0.00

0.00

1.17

0.00

2.80

0.17

0.00

18.30

2

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

11.0

3

0.8

0.0

0.1

0.1

0.2

1.4

0.1

1.3

7.4

4

0.00

0.00

0.00

0.00

0.33

0.50

0.00

0.00

21.00

5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

10.0

6

1.50

0.25

0.00

0.00

0.00

1.00

0.25

1.00

3.25

7

0.00

0.00

0.00

0.17

0.00

5.17

0.33

0.00

11.00

8

Incidence (%)*

0.00

0.00

0.00

0.17

0.00

5.17

0.33

0.00

11.00

9

0.00

0.00

0.00

2.75

2.00

1.63

0.00

0.13

7.63

10

0.0

0.0

0.0

0.0

3.0

1.8

0.0

0.7

10.3

11

0.00

0.00

0.00

0.00

0.00

2.17

0.00

0.83

10.20

12

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

2.0

13

0.00

0.00

0.00

0.00

0.33

1.00

0.00

0.33

4.00

14

0.16

0.01

0.13

0.47

0.59

1.67

0.06

0.45

10.22

Incidence Avg. (%)

* Per cent incidence is based on the analysis of 200 sorghum grains in each sample. ** 1: Bagalakote (Data based on 4 samples), 2: Belgam (Data based on 3 samples), 3: Bijapur (Data based on 3 samples), 4: Dharwad (Data based on 5 samples), 5: Gadag (Data based on 3 samples), 6: Gulbarga (Data based on 2 samples), 7: Bellary (Data based on 2 samples), 8: Chitradurga (Data based on 3 samples), 9: Davangere (Data based on 3 samples), 10: Haveri (Data based on 4 samples), 11: Koppala (Data based on 5 samples), 12: Shimoga (Data based on 3 samples), 13: Hassan (Data based on 1 samples), 14: Mysore (Data based on 3 samples).

9.88

F. verticillioides

1

1**

Fusarium spedies

Sl. No

Table 2. Per cent incidence (%) of different Fusarium species on sorghum grains as analyzed by agar plating method (MGA 2.5)

24 Fung. Sci. 27(1), 2012

Diversity of Fusarium species in Karnataka

25

Fig. 3. Agarose gel (1.5%) showing amplified PCR products of ITS region from Fusarium species. Lane-M: DNA Marker (100 bp), Lane-1: F. verticillioides, Lane-2: F. proliferatum, Lane-3: F. oxysporum, Lane-4: F. anthophilum, Lane-5: F. pallidoroseum, Lane-6: F. sporotrichioides, Lane-7: F. solani, Lane-8: F. graminearum, Lane-9: F. decemcellulare, Lane-10: F. lateritium, Lane-11&12: Aspergillus flavus (control), Lane-13&14: Cladosporium cladosporioides (control), and Lane-15: Alternaria alternata (control).

cereals has also been reported from different countries. El-Maghraby et al. (1995) isolated four species of Fusarium from white hybrids of corn in Egypt. Research findings showed that, up to 17 species of Fusarium could be readily isolated from cereal grains. However, the ability to synthesize mycotoxins restricted to some of those 17 species, including the pathogenic ones (Parry et al., 1995). Gonzalez et al. (1995) reported that the occurrence and isolation of 1304 Fusarium isolates from sorghum grains. Morales-Rodriguez et al. (2007) reported the biodiversity of seven Fusarium species in Mexico associated with ear rot of maize. Studies on diversity of Fusarium species showed the occur-

rence of 70 Fusarium species on different hosts with different geo-graphical locations (Leslie and Summerell, 2006). Burgess et al. (1988) reported the occurrence of 20 different Fusarium species on maize and other cereal grains, with a few being of great importance due to their high pathogenecity, frequency and toxicigenic capabilities. Identification of species is made on the basis of genotypic differences. Further, detection of the target DNA molecules is made from complex mixtures, even when the fungal mycelia are no longer viable in the contaminated samples (Patino et al., 2006). The primers used in this assay were designed from the ITS region of

26

Fung. Sci. 27(1), 2012

ribosomal DNA. Specific ITS primers have been used successfully to differentiate closely related fungal species (Lim et al., 2005). The rDNA are highly stable and exhibit a mosaic of conserved and diverse regions within the genome (Hibbett, 1992). Abd-Elsalam et al. (2003) developed a primer based on the ITS region for the identification of the genus Fusarium and used a range of DNA samples to determine the specificity of the primers for PCR assay. Similar work has been done by Bluhm et al. (2004) for the specific detection of the genus Fusarium from other closely related fungal genera, by using the real time PCR with ITS based primers. Therefore, the ITS region represents a good choice for finding specific sequences in the genomic DNA of Fusarium species and it is highly useful for the detection of the genus Fusarium in a mixed population. The present investigation revealed that, out of 86 maize samples, 91% of the samples were tested positive, and out of 44 sorghum samples, 90% of the samples scored positive for Fusarium infection. The incidence of Fusarium species is comparatively high on maize kernels when compared to the incidence of Fusarium species on sorghum grains. It should be noted here that, F. verticillioides was isolated as a dominant Fusarium species with high incidence of 18.69 and 10.22% in maize and sorghum grains respectively. Similar results have been obtained in the State of Parana, Brazil where they detected high per cent incidence Fusarium species on corn (Ono et al., 1999). In the same region, F. moniliforme (= F. verticillioides) was the most frequently isolated fungal species, followed by F. proliferatum, F. subglutinans, and F. graminearum. F. moniliforme is considered to be the most common Fusarium species occur-

ring in tropical and subtropical climates and the most prevalent fungus associated with corn in USA (Munkvold and Desjardins, 1997). Ghiasian et al. (2004) reported a predominance of species of Fusarium (38.5%), followed by Aspergillus (8.7%), Rhizopus (4.8%), Penicillium (4.5%), Mucor (1.1%), and four other fungal genera. Totally 70 different Fusarium species have been isolated and identified from many substrates throughout the world (Leslie and Summerell, 2006). The survey conducted worldwide also showed that, F. verticillioides, F. proliferatum, and F. anthophilum are most frequently isolated species in sorghum and are able to produce fumonisins (Da Silva et al., 2004). High incidence of F. verticillioides has also been reported from maize and sorghum based animal feedstuffs and poultry feed mixtures produced in Karnataka state (Dass et al., 2007). F. verticillioides is regarded as one of the most commonly occurring fungi in maize and is associated with ear-rot of maize in corn growing countries of the world (Ghiasian et al., 2004). The data on the incidence and diversity of Fusarium species would be of great importance in this region for predicting the extent of postharvest infection, colonization and subsequent deterioration of cereals. Further, it also helps us to know the dry matter loss, nutritional changes and the extent of fusarial toxin levels during storage. Such a data is of immense value for assessing the possible health hazards in humans and animals upon consumption of such contaminated food grains. In view of all these, the data on Fusarium infection, diversity and incidence is of great significance. The high incidence of Fusarium species is of primary concern for policy makers and food experts in this region to

Diversity of Fusarium species in Karnataka reduce the economic losses caused by these fungi and also to minimize the exposure of human and animal life to the potential risks of mycotoxins. The results of this study are highly useful for further studies on toxin producing fungi and their epidemiological significance in cereal crops grown in Karnataka and elsewhere in India.

References Abd-Elsalam, K.A., N.I. Aly, A.M. Abdel-Satar, S.M. Khalil, and A.J. Verreet. 2003. PCR identification of Fusarium genus based on nuclear ribosomal-DNA sequence data. Afri. J. of Biotechnol. 2: 82–85. Anderson, P.K., A.A. Cunningham, N.G. Patel, F.J. Morales, P.R. Epstein, and P. Daszak. 2004. Emerging infectious diseases of plants: Pathogen pollution, climate change and agrotechnology drivers. Tren. Ecol. Evol. 19: 535–544. Bhattacharya, K. and S. Raha. 2002. Deteriorative changes of maize, groundnut and soybean seeds by fungi in Storage. Mycopathol. 155: 135–141. Bluhm, B.H., J.E. Flaherty, M.A. Cousin, and C.P. Woloshuk. 2004. Multiplex polymerase chain reaction assay for the differential detection of trichothecene- and fumonisinproducing species of Fusarium in cornmeal. J. Food Protect. 65: 1955–1961. Booth, C. 1977. Fusarium: Laboratory Guide to the Identification of Major Species. Commonwealth Mycological Institute. Ferry Lane. Kew, Surrey, England. pp. 1–70. Bragulat, M.R., E. Martinez, G. Castella, and F.J. Cabanes. 2004. Selective efficacy of culture media recommended for isolation and

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enumeration of Fusarium species. J. Food Protect. 67: 207–211. Burgess, L.W., P.E. Nelson, T.A. Toussoun, and G.A. Forbes. 1988. Distribution of Fusarium species in sections Roseum, Arthrosporiella, Gibbosum, and Discolor recovered from grassland, pasture, and pine nursery soils of eastern Australia. Mycologia 80: 815–824. Da Silva, J.B., P. Dilkin, H. Fonseca, and B. Correa. 2004. Production of aflatoxin by Aspergillus flavus and of fumonisin by Fusarium species isolated from Brazilian sorghum. Braz. J. Microbiol. 35: 182–186. Dass, R.S., M.Y. Sreenivasa, and G.R. Janardhana. 2007. High incidence of Fusarium verticillioides in animal and poultry feed mixtures produced in Karnataka, India. Plant Pathol. J. 6(2): 174–178. El-Maghraby, O.M.O., I.A. El-Kady, and S. Soliman. 1995. Mycoflora and Fusarium toxins of three types of corn grains in Egypt with special reference to production of trichothecine toxins. Microbiol. Res. 150: 225–232. Ghiasian, S.A., P. Kord-Bacheh, S.M. Rezayat, A.H. Maghsood, and H. Taherkhani. 2004. Mycoflora of Iranian maize harvested in the main production areas in 2000. Mycopathol. 158: 113–121. Gonzalez, H.H.L., S.L. Resnik, R.T. Boca, and W.F.O. Marasas. 1995. Mycoflora of Argentinian corn harvested in the main production area in 1990. Mycopathol. 130: 29–36. Hibbett, D.S. 1992. Ribosomal RNA and fungal systematics. Trans. Mycol. Soc. 33: 533–556. Janardhana, G.R., K.A. Raveesha, and H.S. Shetty. 1999. Mycotoxins contamination of maize grains grown in India (Karnataka). Food Chem. Toxicol. 37: 863–868. Leslie, J.F. and B.A. Summerell. 2006. The

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Fusarium Laboratory Manual. 1st edn. Blackwell Publishing Professional, U.S.A. pp. 8–240. Lim, Y.W., J.J. Kim, M. Lu, and C. Breuil. 2005. Determining fungal diversity on Dendroctonus ponderosae and Ips pini affecting lodgepole pine using cultural and molecular methods. Fungal Diversity 19: 79–94. Mirete, S., C. Vazquez, G. Mule, M. Jurando, and M.T. Gonzalez-Jaen. 2004. Differentiation of Fusarium verticillioides from banana fruits by IGS and EF-1! sequence analyses. Eur. J. Plant Pathol. 110: 515–523. Morales-Rodrõguez, I., M.J. Yanez-Morales, H.V. Silva-Rojas, G. Garcõa-de-los-Santos, and D.A. Guzman-de-Pena. 2007. Biodiversity of Fusarium species in Mexico associated with ear rot in maize, and their identification using a phylogenetic approach. Mycopathol. 163: 31–39. Munkvold, G.P. and A.E. Desjardins. 1997. Fumonisins in Maize: Can we reduce their occurrence? Plant Disease 81(6): 556–565. Nelson, P.E., M.C. Dignani, and E.J. Anaissie. 1994. Taxonomy, Biology and Clinical aspects of Fusarium species. Clin. Microbiol. Rev. 10: 479–504. Ono, E.Y.S., Y. Sugiura, M. Homechin, M. Ka-

mogae, E. Vizzoni, Y. Ueno, and E.Y. Hirooka. 1999. Effect of climatic conditions on natural mycoflora and fumonisins in freshly harvested corn of the State of Parana, Brazil. Mycopathol. 147: 139–148. Parry, D.W., P. Jenkinson and L. Mc Leod. 1995. Fusarium ear blight in small grain cereals–a review. Plant Pathol. 44: 207–238. Patino, B., S. Mirete, C. Vazquez, M. Jimenez, T.M. Rodriguez, and T. Gonzalez-Jaen. 2006. Characterization of Fusarium verticillioides strains by PCR-RFLP analysis of the intergenic spacer region of the rDNA. J. Sci. Food Agri. 86: 429–435. Pinnoi, A., S. Lumyong, K.D. Hyde, and E.B.G. Jones. 2006. Biodiversity of fungi on the palm Eleiodoxa conferta in Sirindhorn peat swamp forest, Narathiwat, Thailand. Fungal Diversity 22: 205–218. Placinta, C.F., J.P. D’Mello, and A.M. MacDonald. 1999. A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxins. Ani. Feed Sci. Technol. 78: 221–37. Summerell, B.A., B. Salleh, and J.F. Leslie. 2003. A utilitarian approach to Fusarium identification. Plant Disease 87 (2): 117–128.

Diversity of Fusarium species in Karnataka

∮ⵤ∟彡✒ 彤◮⇾ 䉇䬱⊈榖䭯㡔䭐᳈括⨠劊ᴉ⟘㣡⻥ M.Y. Sreenivasa1, Adkar Purushothama Charithraj2, Regina Sharmila Dass3, and G.R. Janardhana3 1. Department of Microbiology, University of Mysore, Manasagangotri, Mysore- 570 006, Karnataka, India. Phone: +91-821-2419733, Fax: +91-821-2419759; E-mail: [email protected] 2. Plant Pathology laboratory, Faculty of Agriculture and Life sciences, Hirosaki University, Hirosaku, Japan. E-mail: [email protected] 3. Molecular Phytodiagnostic Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri, Mysore- 570 006, Karnataka State, India. Phone: +91-821-2419763, Fax: +91-821-2419759; Email: [email protected]

ᄢ! ! ौ ⽆⌉恋⟼⃳恎ᷳ 15 ᾳ⛘⋨ᷕ㍉普Ḯ 130 ᾳ䌱䰛⍲檀䱙㧋㛔炻忚埴揖⬊却 (Fusarium) ⣂㧋⿏⍲䘤䓇柣䌯 ᷳ↮㜸ˤ䳸㝄⎗䘤䎦 10 䧖揖⬊却⬀⛐㕤䌱䰛⍲檀䱙ᷳ㤾䰺炻䁢 F. verticillioidesˣF. proliferatumˣF. oxysporumˣF. anthophilumˣF. pallidoroseumˣF. sporotrichioidesˣF. solaniˣF. graminearumˣF. decemcellulare ⍲ F. lateritiumˤ⛐䌱䰛ᶲẍ F. verticillioidesˣF. proliferatum ⍲ F. anthophilum ᷳ䘤䓇柣䌯㚨檀炻 ↮⇍䁢!18.69%ˣ6.08% ⍲!4.46%烊⛐檀䱙ᶲẍ F. verticillioides ⍲ F. anthophilum ᷳ䘤䓇柣䌯㚨檀炻↮ ⇍䁢!10.22% ⍲ 1.67%ˤ㬌⣂㧋⿏⍲䘤䓇柣䌯ᷳ婧㞍䳸㝄炻⮯㚱≑㕤枸㷔䌱䰛⍲檀䱙㤾䰺㍉㓞⼴⍿⇘揖 ⬊却⼙枧㇨忈ㆸ⃚ᷳ啷⿏僸⢆⍲℞㭺䳈ᷳ䓊䓇ˤ 撚戳夜濣䌱䰛ˣ⣂㧋⿏ˣ檀䱙ˣ䘤䓇柣䌯ˣ揖⬊却ˤ

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Fung. Sci. 27(1): 31–44, 2012

Five apothecium-producing lichenized fungi of the genus Usnea in Taiwan Yuan-Min Shen1, 2, Huan-Ju Hsieh2, Ruo-Yun Yeh3, and Ting-Hsuan Hung2* 1. Taichung District Agricultural Research and Extension Station, No. 370, Song Huai Road, Dacun Township, Changhua 51544, Taiwan 2. Department of Plant Pathology and Microbiology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan 3. Division of Wood Cellulose, Taiwan Forestry Research Institute, No. 53, Nanhai Road, Taipei 10066, Taiwan (Accepted: February 15, 2012)

ABSTRACT Most species of the genus Usnea that commonly produce apothecia in Taiwan have not been collected for a long time. We rediscovered five species, including Usnea masudana, U. orientalis, U. pseudogatae, U. shimadae, and U. sinensis, by investigating their morphology, chemistry, and molecular data. Ten internal transcribed spacer (ITS) sequences of the five species were first reported. Distributions of the species were also provided. Although no latitudinal tendency was observed among the Usnea species in Taiwan, we consider high elevation places of the subtropics and tropics to contain plenty of Usnea species that produce apothecia. Keywords: Usnea, lichens, HPLC, ecology, taxonomy.

Introduction Usnea is an ascomycetous genus in Parmeliaceae that forms fruticose lichens. The genus has a worldwide geographical distribution, containing 300 to 770 species (Clerc, 1998; Stevens, 1999; Kirk et al., 2001). Usnea, reported as a Chinese folk medicine 430 years ago, is called “song-luo” in Chinese. The genus differs from other fruticose lichens in bearing a cartilaginous axis and in producing usnic acids. Most of the individuals of Usnea exist as sterile thalli and their apothecia are rarely produced in *

Corresponding author, Email: [email protected]

nature. Some species however commonly produce apothecia. Studies of Usnea in Taiwan were begun during the period when Taiwan was under Japanese rule. Japanese biologists collected plenty of Usnea specimens from 1910s to the end of the Japanese occupation. Early Usnea records were published in The Journal of the Natural History Society of Taiwan and The Journal of Japanese Botany. Part of the specimens were forwarded to Zahlbruckner for identification (Zahlbruckner, 1930; Zahlbruckner, 1933). Asahina (1970) recognized some apothecium-producing species

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based on Taiwan’s specimens, including U. shimadae, U. pseudogatae, and U. masudana. Usnea shimadae is known to be distributed only in Taiwan and Mexico (Clerc, 2004), whereas U. pseudogatae and U. masudana are endemic to Taiwan (Asahina, 1970; Ohmura, 2001). Although an intensive study on these apotheciumproducing Usnea was conducted by Ohmura (2001), no further collections of some species have been recorded since they were first collected. For example, Usnea masudana has only been collected once in 1936 (Asahina, 1970; Ohmura, 2001). Despite numerous Usnea species have been collected in Taiwan, in the four decades, U. orientalis remained the only apothecium-producing species recorded until recently (Lin, 2007). Due to the lack of specimens of some apothecium-producing Usnea species about half a century, we attempted to rediscover those species and observe them in detail. In this article, we described five Usnea species that produce apothecia.

Materials and Methods Specimens of Usnea species were collected at mountain areas in Taiwan. Dry specimens selected were observed under a stereo microscope. For determining cortical types and microscopic characters of apothecia, sections were made by a freezing microtome and the tissues were examined using a light microscope. Terminology used in descriptions follows Ohmura (2001), Herrera-Campos et al. (1998), and Brodo et al. (2001). Some terms related to morphology of apothecia were defined in discussion of this article. Colors of the thalli follow Kornerup et al. (1978). All studied specimens were deposited at the herbarium of the National

Museum of Natural Science (TNM), Taichung, Taiwan. Lichen substances were detected by high performance liquid chromatography (HPLC) using a AGILENT system, a ASTEC reversed-phase column (C18, 5 m, 250 × 4.6 mm), and a spectrometric detector operating at 245 nm at a flow rate of 0.7 mL/min. Extraction method, solvent system, and retention indices for identification of substances followed standardized methods by Feige et al. (1993). Identified lichen substances were presented in descriptions, with “M” for macroscopic substances and “m” for microscopic substances. Others were displayed in HPLC chromatograms. Spot tests were performed by dropping reagents on medulla of lichen thalli. Potassium hydroxide (K), sodium hypochloride (CLOROX, C), and paraphenylenediamine (P) were used as reagents for spot tests. A positive reaction was indicated by a color change in the medulla. Total DNA from lichen specimens were extracted with a BIOKIT Plant Genomic DNA Purification Kit system. Amplification of internal transcribed spacers and 5.8s rRNA genes were carried out by polymerase chain reactions (PCR) with primer pairs of ITS5 (5’GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). The setting reactions were 94°C (5 min), 30 cycles of [94°C (45 sec), 52°C (45 sec), 72°C (2 min)], and 72°C (7 min). Target PCR products were cut after electrophoresis and purified by a QIAGEN QIAquick Gel Extraction Kit. The purified products were sequenced with the ITS5 primer and then blasted on NCBI (National Center for Biotechnology Information) database to compare identities of species. Obtained sequences were deposited at GenBank.

Apothecium-producing Usnea in Taiwan

Taxonomy Characters to distinguish the five Usnea species included in the study were listed in Table 1. HPLC chromatograms (Fig. 3) and distribution maps (Fig. 4) of these species were provided. Usnea masudana Asahina, J. Jpn. Bot. 45: 132. 1970. 㘼䏮㫍㘼哽(Figs. 1A–C) Thalli corticolous, erect, branched anisotomic-dichotomously, cylindrical at base, up to 6 cm long, (0.8)–1.0–(1.2) mm diam at main branches; surface glossy, yellowish-green, cracked and concolorous with thalli at base; branches gradually tapering, terete; lateral branches common, cylindrical at base; fibrils common; maculae absent; pseudocyphellae absent; papillae common, verrucose to cylindrical; cortex thick, merrillii-type plectenchymatous, without red pigments; medulla uninflated, dense, white; axis solid, pale orange; ratio of cortex, medulla, and axis (6.5)–9.1–(11.9) :

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(12.5)–22.0–(30.0) : (20.0)–37.8–(56.3). Soralia absent. Apothecia commonly produced, lateral, cup-shaped or everted, up to 8 mm, with an orange disc and a whitish rim; circum-fibrils common, simple to branched, cylindrical at base; amphithecial-fibrils common, spinose to aculeate, simple to branched; juvenile apothecia helicoknob-shaped, with one conspicuous curved fibril, reddish-orange at center; epihymenium 5–10 m thick; hymenium 50–62.5 m thick; hypothecium 45–62.5 m thick. Asci clavate, eight-spored. Ascospores hyaline, broadly ellipsoidal, 1-celled, (6.3)–10.4–(11.8) × (5.0)– 8.5–(9.8) m. Chemistry. galbinic acid (M), norstictic acid (M), salazinic acid (m), usnic acid (m). K+ bright yellow then turn bright orange, C–, P+ bright yellow. Specimens examined. Yilan county, Cuifenghu, elevation 1,818 m, on Alnus formosana, 14 May 2007, Shen, Y.-M. TNM L00004637, accession number at GenBank:

Table 1. Characters of the five studied Usnea species that produce apothecia Usnea species

characters thallus surface cortical types medulla texture

masudana

orientalis

pseudogatae

shimadae

sinensis

glossy merrillii-type dense

glossy ceratina-type loose

matt merrillii-type loose to dense

matt ceratina-type dense

glossy ceratina-type dense to compact

ratio of medulla

(12.5)–22.0–(30.0) (25.4)–32.2–(39.5) (21.2)–31.4–(36.1) (26.7)–29.6–(32.6) (15.7)–19.5–(21.6)

soralia

absent

disc color circum-fibrils juvenile apothecia ascospores sizes ( m) lichen substances

absent green to pale orange yellow common, simple to common, simple, branched rarely branched helicoknobknob-shaped shaped (6.3)–10.4–(11.8) (6.3)–9.1–(11.3) × × (5.0)–8.5–(9.8) (5.0)–7.1–(9.0) sal, gal, nor sal

absent or sparse

absent

absent

pale orange

orange

green to gray

sparse to absent, simple

common, simple to common, branched branched

knob-shaped

lip-shaped

knob-shaped

(6.3)–9.4–(14.0) × (5.0)–6.7–(8.8) sal, nor, pro, LS14

(5.3)–8.6–(10.8) × (5.0)–6.4–(7.5) sal, gal, nor

(7.0)–9.2–(10.8) × (4.5)–6.4–(8.0) nor

All five species contain usnic acid. Abbreviations of the lichen substances in the table are salazinic acid (sal), galbinic acid (gal), norstictic acid (nor), protocetraric acid (pro), and unknown lichen substance 14 (LS14).

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Fig. 1. Morphology of Usnea masudana, U. pseudogatae, and U. shimadae. A, U. masudana (L00004637); B, helicoknobshaped juvenile apothecia of U. masudana; C, mature apothecium of U. masudana; D, U. pseudogatae (L00004743); E, soralia and isidiomorphs of U. pseudogatae; F, juvenile apothecia of U. pseudogatae; G, proliferated apothecia of U. pseudogatae; H, U. shimadae (L00004691); I, lip-shaped juvenile apothecium of U. shimadae; J, wrinkled, mature apothecium of U. shimadae. Scales: A, D, & H = 1 cm; C, G, & J = 1 mm; B, E, F, & I = 0.1 mm.

FJ494939. Hsinchu county, Zhenxibao (Cinsbu tribe), elevation 1,532 m, on Mallotus japonicas, 11 Sep. 2007, Shen, Y.-M. TNM L00004689, accession number at GenBank: FJ494940. Notes. Usnea masudana is characterized by

a glossy surface, merrillii-type plectenchymatous cortex, apothecia with an orange disc, and helicoknob-shaped juvenile apothecia. It resembles U. shimadae in having an orange disc. However, U. shimadae has a matt surface, ceratina-type plectenchymatous cortex, and larger

Apothecium-producing Usnea in Taiwan apothecia. In addition, U. shimadae has lipshaped juvenile apothecia and slightly smaller ascospores. Galbinic acid and norstictic acid were the major lichen substances produced by our collections of U. masudana. The ITS sequence of U. masudana matched best with those of U. dasaea (AB051056) and U. ceratina (AB051052-55), with a 95% identity with each of them. To our knowledge, the sequences of U. masudana have not been reported previously. Usnea masudana was first reported by Asahina in 1970, based on a specimen collected in Taitung by Masuda in 1936. The specimens that we collected in general fit the descriptions of U. masudana in Asahina (1970) and Ohmura (2001) except for the uninflated medulla, thinner hymenium, and the presence of galbinic acid. Although slight differences existed between the descriptions, the current collections shared the hightest morphological similarity with U. masudana among Usnea species that we knew. The distribution of U. masudana in Taiwan is at the altitudes of 1,530–1,820 m. It has only been reported from Taiwan thus far. Usnea orientalis Motyka, Lich. Gen. Usnea Stud. Monogr. Pars Syst. 2: 547. 1938. 㘯㑷 㘼哽 (Figs. 2A–F) Thalli corticolous, erect, branched anisotomic-dichotomously, slightly attenuate at base, up to 7 cm long, (0.8)–1.4–(2.1) mm diam at main branches; surface glossy, yellowish-green, smooth and concolorous with thalli or paler than thalli at base; branches gradually tapering, terete; lateral branches common, constricted at base; fibrils common; maculae common, irregular; pseudocyphellae absent; papillae common, verrucose to cylindrical; cortex thin, ceratinatype plectenchymatous, without red pigments;

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medulla inflated, loose, white; axis solid, pale orange; ratio of cortex, medulla, and axis (3.9)– 6.7–(9.2) : (25.4)–32.2–(39.5) : (13.2)–22.3– (31.6). Soralia absent. Apothecia commonly produced, lateral, occasionally proliferated from amphithecial-fibrils, cup-shaped or everted, up to 11 mm diam with a green to pale yellow disc and a yellowish-green rim; circumfibrils common, simple, rarely branched, cylindrical to broadened at base; amphithecial-fibrils sparse, aculeate, simple or branched; juvenile apothecia knob-shaped, with or without circumfibrils, pale orange at center; epihymenium 8.8– 20 m thick; hymenium 72.5–87.5 m thick; hypothecium 37.5–55 m thick. Asci clavate, eight-spored. Ascospores hyaline, broadly ellipsoidal, 1-celled, (6.3)–9.1–(11.3) × (5.0)–7.1– (9.0) m. Chemistry. salazinic acid (M), usnic acid (M). K+ yellow then slowly turn orange, C–, P+ bright yellow. Specimens examined. Nantou county, Cuifeng, elevation 2,314 m, 6 May 2007, Shen, Y.-M. TNM L00004625, accession number at GenBank: FJ494942. Nantou county, Tatajia, elevation 2,598 m, 18 Jun. 2007, Shen, Y.-M. TNM L00004653, accession number at GenBank: FJ494943. Chiayi county, Yushan mountain-climbing trail, elevation 2,735 m, on Berberis sp., 18 Jun. 2007, Shen, Y.-M. TNM L00004669, accession number at GenBank: FJ494944. Taichung county, en route to Hsuehshan, elevation 2,270 m, on Alnus formosana, 15 Jul. 2007, Shen, Y.-M. TNM L00004673, accession number at GenBank: FJ494945. Kaohsiung county, Zhongzhiguan trail, elevation 1,995 m, 5 Nov. 2007, Shen, Y.M. TNM L00004748. Nantou county, Jundashan, elevation 2,954 m, on Pinus taiwanen-

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Fig. 2. Morphology of Usnea orientalis and U. sinensis. A, U. orientalis (L00004748); B, transverse section of branch of U. orientalis showing loose medulla; C, juvenile apothecia of U. orientalis; D, cortex breaking of juvenile apothecium of U. orientalis; E, young apothecium of U. orientalis; F, mature apothecium of U. orientalis. G, U. sinensis (L00004766); H, transverse section of branch of U. sinensis showing compact medulla; I, juvenile apothecium of U. sinensis; J, mature apothecium of U. sinensis. Scales: A & G = 1 cm; F & J = 1 mm; B–E, H, & I = 0.1 mm.

sis, 10 Dec. 2006, Shen, Y.-M. TNM L00004600. Chiayi county, Alishan, elevation 2,401 m, on Prunus campanulata, 24 Mar. 2007, Shen, Y.-M. TNM L00004608. Taichung county Daxueshan forest recreation area, on Photinia niitakayamensis, 28 Apr. 2007, Shih, H.-H. TNM

L00004620. Nantou county, Tatajia, elevation 2,507 m, on Photinia niitakayamensis, 16 Jun. 2007, Shen, Y.-M. TNM L00004646. Nantou county, Tatajia, elevation 2,530 m, 16 Jun. 2007, Shen, Y.-M. TNM L00004647. Nantou county, Tatajia, elevation 2,585 m, on Photinia

Apothecium-producing Usnea in Taiwan niitakayamensis, 18 Jun. 2007, Shen, Y.-M. TNM L00004651. Chiayi county, Yushan mountain-climbing trail, elevation 2,558 m, 18 Jun. 2007, Shen, Y.-M. TNM L00004660. Chiayi county, Yushan mountain-climbing trail, elevation 2,725 m, 18 Jun. 2007, Shen, Y.-M. TNM L00004667. Kaohsiung county, Tianchi , elevation 2,171 m, on Alnus formosana, 5 Nov. 2007, Shen, Y.-M. TNM L00004745. Kaohsiung county, Tianchi, elevation 2,171 m, on Salix fulvopubescens var. fulvopubescens, 5 Nov. 2007, Shen, Y.-M. TNM L00004746. Nantou county, Meifeng, elevation 2,033 m, on Alnus formosana, 16 Nov. 2007. Shen, Y.-M. TNM L00004753. Nantou county, Meifeng, elevation 1,946 m, 16 Nov. 2007. Shen, Y.-M. TNM L00004755. Notes. Usnea orientalis is characterized by a glossy surface, an inflated, loose medulla, ceratina-type plectenchymatous cortex, apothecia with a green to pale yellow disc surface, and rarely branched circum-fibrils. It resembles U. sinensis in having the same disc surface color on the apothecia. Usnea sinensis differs in having an uninflated, dense to compact medulla and highly branched circum-fibrils. Salazinic acid was the major lichen substance produced by U. orientalis. The ITS sequence of U. orientalis matched best with that of U. pygmoidea (AB051657-58), with a 98% identity. Usnea orientalis and U. pygmoidea are close in ITS sequences. The two species share some morphological and chemical characteristics in common, but the former produces apothecia and the latter does not. Therefore they probably represent a species pair. To our knowledge, the sequence of U. orientalis is here reported for the first time. Our specimens fit the description of U. orientalis in Ohmura (2001) morphologically and chemically. Usnea orientalis is a

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commonly encountered holomorphic species of Usnea in Taiwan where it is mainly found at the altitudes of 1,940–2,960 m. The species has been reported from China, Japan, and southern Asia (Motyka, 1936–1938; Ohmura, 2001). Usnea pseudogatae Asahina, J. Jpn. Bot. 45: 131. 1970. ㎪䲐㑷㫍㘼哽Figs. 1D–G) Thalli corticolous, erect, branched anisotomic-dichotomously, cylindrical at base, up to 7 cm long, (1.0)–1.2–(1.5) mm diam at main branches; surface matt, foveolate at places, grayish-green, smooth to cracked and brown to paler than thalli at base; branches gradually tapering, terete to slightly ridged; lateral branches sparse, cylindrical at base; fibrils common; maculae common, patch-like; pseudocyphellae absent; papillae common, verrucose to cylindrical; cortex thin, merrillii-type plectenchymatous, without red pigments; medulla inflated, loose to dense, white; axis solid, pale orange; ratio of cortex, medulla, and axis (2.8)–6.7– (11.4) : (21.2)–31.4–(36.1) : (18.8)–23.8–(34.8). Soralia absent or sparsely formed at main branches, originating from papillae and scars, punctiform, narrower than radius of branches, discrete, raised, convex at top, lacking cortical margin; isidiomorphs commonly formed on soralia. Apothecia commonly produced, lateral or serial, frequently grouped together, occasionally proliferated on circum-fibrils, cup-shaped to everted, up to 7 mm diam, with a pale orange disc and a whitish rim; circum-fibrils sparse to absent, fallen easily, simple, broadened at base; amphithecial fibrils absent or rare, spinose; juvenile apothecia knob-shaped, without circumfibrils, pale orange at center; epihymenium 2.5– 12.5 m thick; hymenium 47.5–62.5 m thick; hypothecium 25–40 m thick. Asci clavate,

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eight-spored. Ascospores hyaline, broadly ellipsoidal, 1-celled, (6.3)–9.4–(14.0) × (5.0)–6.7– (8.8) m. Chemistry. salazinic acid (M), norstictic acid (M), protocetraric acid (M),unknown lichen substance 14 (LS14), usnic acid (m). K+ yellow then slowly turning orange, C–, P+ bright yellow. Specimens examined. Kaohsiung county, Tianchi, elevation 2,171 m, 5 Nov. 2007, Shen, Y.-M. TNM L00004743, accession number at GenBank: FJ494947. Kaohsiung county, Zhongzhiguan trail, elevation 1,995 m, 5 Nov. 2007, Shen, Y.-M. TNM L00004749, accession number at GenBank: FJ494948. Chiayi county, Alishan, elevation 2,410 m, on Prunus campanulata, 24 Mar. 2007, Shen, Y.-M. TNM L00004609. Nantou county, Cuifeng, elevation 2,314 m, 6 May 2007, Shen, Y.-M. TNM L00004624. Kaohsiung county, Tianchi, elevation 2,171 m, on Prunus campanulata, 5 Nov. 2007, Shen, Y.-M. TNM L00004744. Notes. Usnea pseudogatae is characterized by a matt surface, merrillii-type plectenchymatous cortex, commonly produced apothecia with a pale orange disc, and circum-fibrils lacking or, when produced, sparse. It is distinguished from other apothecium-producing Usnea species by the lack or sparsity of circum-fibrils. Other noticeable features include occasional formation of discrete, punctiform soralia with isidiomorphs and the proliferation of apothecia on circum-fibrils. Salazinic acid, norstictic acid, and protocetraric acid were the major lichen substances produced by U. pseudogatae. The closest ITS sequence at NCBI with that of U. pseudogatae we obtained was AB051640 of U. himalayana, with a 98% identity. Even though the sequences were quite similar, U. pseudoga-

tai and U. himalayana differ significantly in morphology of apothecia and soralia, and in texture of thallus surface. To our knowledge, the sequence of U. pseudogatai has not been reported previously. Usnea pseudogatai was first reported by Asahina (1970), based on a specimen collected at Alishan in Taiwan by Ogata (1935), with additional specimens collected by Yasue and Asahina in Taiwan during the period of 1933–1937. Our specimens fit well the descriptions of U. pseudogatae in Asahina (1970) and Ohmura (2001) except for the matt surface and the presence of maculae. The distribution of U. pseudogatae in Taiwan is at the altitudes of 1,990–2,410 m. It has only been reported from Taiwan. Usnea shimadae Asahina, J. Jpn. Bot. 45: 131. 1970. ⮴䏮㫍㘼哽 (Figs. 1H–J) Thalli corticolous, erect, branched anisotomic-dichotomously, slightly attenuate at base, up to 7 cm long, (1.5)–1.8–(2.1) mm diam at main branches; surface matt, occasionally decorticated at places, grayish-green, cracked and decorticated, paler than thalli at base; branches gradually tapering, terete to ridged; lateral branches common, cylindrical at base; fibrils common; maculae sparse; pseudocyphellae absent; papillae common, verrucose to cylindrical; cortex thin, ceratina-type plectenchymatous, without red pigments; medulla inflated, dense, white; axis solid, pale orange to orange; ratio of cortex, medulla, and axis (3.5)–7.6–(9.9) : (26.7)– 29.6–(32.6) : (23.3)–25.6–(27.8). Soralia absent. Apothecia commonly produced, lateral and occasionally serial, cup-shaped to flat, up to 17 mm diam, wrinkled at larger ones, with an orange disc and a whitish rim; circum-fibrils common, simple to branched, cylindrical to

Apothecium-producing Usnea in Taiwan broadened at base; amphithecial-fibrils absent to common, spinose; juvenile apothecia lipshaped, without circum-fibrils, orange all over the surface; epihymenium 7.5–20 m thick; hymenium 45–57.5 m thick; hypothecium 42.5–65 m thick. Asci clavate, eight-spored. Ascospores hyaline, broadly ellipsoidal, 1celled, (5.3)–8.6–(10.8) × (5.0)–6.4–(7.5) m. Chemistry. salazinic acid (M), galbinic acid (M), norstictic acid (M), usnic acid (M). K+ yellow, C–, P+ yellow. Specimen examined. Hsinchu county, Zhenxibao (Cinsbu tribe), elevation 1,576 m, on Albizia julibrissin, 11 Sep. 2007, Shen, Y.-M. TNM L00004691, accession number at GenBank: FJ494952. Notes. Usnea shimadae is characterized by a matt surface, terete to ridged branches, ceratina-type plectenchymatous cortex, large and wrinkled apothecia with an orange disc, and lipshaped juvenile apothecia. It resembles U. masudana in having an orange disc. However, U. masudana has a glossy surface, merrillii-type plectenchymatous cortex, and smaller apothecia. In addition, U. masudana has helicoknobshaped juvenile apothecia and slightly larger ascospores. Salazinic acid, galbinic acid and norstictic acid were the major lichen substances produced by U. shimadae. The ITS sequence of U. shimadae had the highest identity with that of U. rubrotincta (DQ232664) at NCBI currently, followed by that of U. merrillii (AB051649). To our knowledge, the sequence of U. shimadae is here reported for the first time. Usnea shimadae was first reported by Asahina (1970), based on a specimen collected from Shinchiku, Taiwan, by Shimada in 1928. The specimens that we collected fit well with the descriptions of U. shimadae in Asahina (1970) and Ohmura

39

(2001). The altitudinal distribution of U. shimadae in Taiwan is about 1580 m. The species was first reported from Taiwan by Asahina (1970) and later reported from South America by Clerc (2004). Usnea sinensis Motyka, Lich. Gen. Usnea Stud. Monogr. Pars Syst. 1: 248. 1936. ᳫ◉㘼哽 (Figs. 2G–J) Thalli corticolous, erect, branched anisotomic-dichotomously, cylindrical at base, up to 9 cm long, (1.1)–1.3–(1.5) mm diam at main branches; surface glossy, yellowish-green to grayish-green, cracked and brown to paler than thalli at base; branches gradually tapering, terete; lateral branches common, cylindrical at base; fibrils common; maculae sparse to common, irregular; pseudocyphellae absent; papillae common, verrucose to cylindrical; cortex thick, ceratina-type plectenchymatous, without red pigments; medulla uninflated, dense to compact, white; axis solid, pale orange; ratio of cortex, medulla, and axis (10.8)–12.7–(14.1) : (15.7)–19.5–(21.6) : (29.3)–35.5–(43.8). Soralia absent. Apothecia commonly produced, terminal to lateral, occasionally proliferated from amphithecial-fibrils, cup-shaped to flat, up to 8 mm diam, with a green to gray disc, and a yellowish-green to grayish-green rim; circumfibrils common, long and well-developed, highly branched, cylindrical to slightly broadened at base; amphithecial-fibrils common, spinose to aculeate, simple to branched; juvenile apothecia knob-shaped, with or without circum-fibrils, pale orange to pale yellow at center; epihymenium 7.5–15 m thick; hymenium 45–60 m thick; hypothecium 32.5–55 m thick. Asci clavate, eight-spored. Ascospores hyaline, broadly ellipsoidal, 1-celled, (7.0)–9.2–(10.8) × (4.5)–

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6.4–(8.0) m. Chemistry. usnic acid (M), norstictic acid (m). K–, C–, P+ medulla slowly turning yellow. Specimens examined. Nantou county, Meifeng, elevation 2,015 m, on Alnus formosana, 16 Nov. 2007, Shen, Y.-M. TNM L00004766, accession number at GenBank: FJ494953. Taichung county, Zhijiayangdashan, on Pinus taiwanensis, 2 Jan. 2008, Shih, H.-H. TNM L00004774. Notes. Usnea sinensis is characterized by a glossy surface, ceratina-type plectenchymatous cortex, an uninflated, dense to compact medulla, yellowish-green to grayish-green apothecia with highly branched circum-fibrils. It resembles U. orientalis in having the same disc surface color on the apothecia but differs in having an uninflated and dense to compact medulla. Only norstictic acid and usnic acid were detected on our collections of U. sinensis. The closest ITS sequence at NCBI so far was U. nipparensis (AB051652), with a 94% identity compared with the sequence that we obtained. To our knowledge, the sequence of U. sinensis has not been reported previously. The specimens that we collected in general fit the description of U. sinensis in Ohmura (2001) except for the surface texture and the absence of caperatic acid. The altitudinal distribution of U. sinensis in Taiwan is about 2,020 m. It has also been reported from China (Wei, 1991; Ohmura, 2001).

Discussion Characteristics and development of apothecia were included in the present study. Apothecia that bear fibrils are found in the five Usnea species. Two types of fibrils were noted in this study: circum-fibrils located at rims of apothe-

cia, and amphithecial-fibrils located at the backside of apothecia. Length of a spinose amphithecial-fibril was shorter than diameter of the apothecium; length of a aculeate amphithecial-fibril was longer than diameter of the apothecium. In addition, we observed proliferated apothecia in specimens of U. pseudogatae and U. orientalis. A proliferated apothecium is produced from a fibril of another apothecium (Fig. 1G). We distinguished three stages in the development of an apothecium, namely, juvenile stage, young stage, and mature stage. The juvenile stage is from developing a primary structure which determines to form an apothecium until cortex breaking, exposes part of the disc of an apothecium (Fig. 2D). The young stage is from cortex breaking until ascospores mature (Fig. 2E). The mature stage occurs after the ascospores mature (Fig. 2F). The juvenile apothecia vary among the Usnea species studied, knob-shaped in U. orientalis, U. pseudogatae, and U. sinensis but helocoknob-shaped, and lipshaped in U. masudana and U. shimadae, respectively. Chemical constitutions differ among Usnea species. Usnic acid, salazinic acid, and norstictic acid were confidently identified, but one lichen substance in U. pseudogatae with a retention time of 19.8 minutes has not been clearly recognized. The lichen substance denominated LS14 has maximum UV absorptions of 215, 244, and 320 nm (Fig. 3). LS14 was not found in the retention indices in Feige et al. (1993); the compound is absent in specimens of U. pseudogatae collected by Ogata as well (Ohmura, 2001). It is notable that we also found LS14 in a large amount in a chemical race of U. pectinata. Substrate and environmental affinities of the

Apothecium-producing Usnea in Taiwan

41

Fig. 3. HPLC chromatograms of acetone extracts from Usnea masudana, U. orientalis, U. pseudogatae, U. shimadae, and U. sinensis. UV spectra of usnic acid (usn), salazinic acid (sal), unknown lichen substance 14 (LS14), galbinic acid (gal), and norstictic acid (nor) are provided.

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apothecium-producing Usnea species are noted in the study. All the five species are attached to bark and branches of woody plants. In Taiwan, they were most frequently on Alnus formosana, followed by Prunus campanulata and Photinia niitakayamensis. The three plant species mainly grow in areas with sufficient sunlight. Although there is no obvious latitudinal preference for the studied Usnea species in Taiwan (Fig. 4), U. orientalis, U. pseudogatae, and U. sinensis tend to aggregate at the central mountainous areas. Therefore, their habitative elevations are higher than those of U. masudana and U. shimadae. Halonen (2000) has reported that Usnea species

have a relatively southern distribution in Fennoscandia and that apothecium-producing species are less frequently found in boreal areas than in temperate areas. We also notice that the richness of apothecium-producing Usnea species is higher in the tropics or subtropics than that in temperate zones. In a study by Ohmura (2001), eight species of the genus Usnea that commonly produce apothecia were recorded in Taiwan but only three species were found in Japan. Therefore it appears worth conducting further investigations at sites of higher altitudes located in lower latitudes for more Usnea species that produce apothecia.

Fig. 4. Distribution maps of apothecium-producing Usnea species in Taiwan. A, U. masudana; B, U. orientalis; C, U. pseudogatae; D, U. shimadae; E, U. sinensis. Maps are based on collections in this study (dots) and historical records in Ohmura (2001) (triangles).

Apothecium-producing Usnea in Taiwan

Acknowledgments We appreciate valuable advices and helps given from Mr. Chung-Kang Lin, Dr. Yu-Ming Ju, and Dr. Chi-Yu Chen. Thanks are extended to Dr. Yoshihito Ohmura for detailed discussion of Usnea species.

References Asahina, Y. 1970. Lichenologische Notizen 235. On some Formosan Usneae with terminal peltate apothecia. J. Jpn. Bot. 45: 129– 133. Brodo, I.M., S.D. Sharnoff, and S. Sharnoff. 2001. Lichens of North America. Yale University Press, New Haven, Connecticut. Clerc, P. 1998. Species concepts in the genus Usnea (lichenized ascomycetes). Lichenologist 30: 321–340. Clerc, P. 2004. Notes on the genus Usnea Adanson. II. Bibl. Lichenol. 88: 79–90. Feige, G.B., H.T. Lumbsch, S. Huneck, and J.A. Elix. 1993. Identification of lichen substances by a standardized high-performance liquid-chromatographic method. J. Chromatogr. 646: 417–427. Halonen, P. 2000. Studies on the lichen genus Usnea in east Fennoscandia and Pacific North America. Oulu University Library, Oulu. Herrera-Campos, M.A., P. Clerc, and T.H.

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Nash. 1998. Pendulous species of Usnea from the temperate forests in Mexico. Bryologist 101: 303–329. Lin, C.K. 2007. The lichen genus Usnea at Meifeng, central Taiwan. Coll. and Res. 20: 1–7. Motyka, J. 1936–1938. Lichenum generis Usnea studium monographicum. Pars Systematica, Leopoli. Kirk, P.M., P.F. Cannon, J.C. David, and J.A. Stalpers. 2001. Dictionary of the fungi, 9th edn. CABI International, New York. Kornerup, A. and J.H. Wanscher. 1978. Methuen handbook of colour. E. Methuen, London. Ohmura, Y. 2001. Taxonomic study of the genus Usnea (lichenized Ascomycetes) in Japan and Taiwan. J. Hattori Bot. Lab. 90: 1– 96. Stevens, G.N. 1999. A revision of the lichen family Usneaceae in Australia. J. Cramer, Berlin. Wei, J.C. 1991. An enumeration of lichens in China. International Academic Publishers, Beijing. Zahlbruckner, A. 1930. Catalogus lichenum universalis. Johnson Reprint, New York. Zahlbruckner, A. 1933. Flechten der Insel Formosa (Fortsetzung und Schluß). Feddes Repertorium specierum novarum regni vegetabilis 33: 22–68.

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傸㼡ᵒ䣬⊭䏠䏝⨎█䖢䕂㘼哽⬪◮圡 ؕনҕ1, 2! ᗂྶᏐ2! ဨषⶎ3! ࢹਕଖ2 1. 埴㓧昊彚㤕⥼⒉㚫冢ᷕ⋨彚㤕㓡列⟜ 2. ⚳䩳冢䀋⣏⬠㢵䈑䕭䎮冯⽖䓇䈑⬠䲣 3. 埴㓧昊彚㤕⥼⒉㚫㜿㤕娎槿㇨

ᄢ! ! ौ ⣏⣂㔠䓊䓇⫸♲䚌䘬㜦嗧⛐冢䀋⶚䴻⼰ᷭ㛒塓䘤䎦Ḯ炻ㆹᾹ慵㕘㈦⇘Ḽᾳ䧖栆炻啱䓙↮㜸⼊ンˣ⊾ ⬠ˣ↮⫸屯㕁炻↮⇍揹⭂䁢㜦䓘㮷㜦嗧 (Usnea masudana)ˣ㜙㕡㜦嗧 (U. orientalis)ˣ㒔䵺㕡㮷㜦嗧 (U. pseudogatae)ˣⲞ䓘㮷㜦嗧 (U. shimadae)ˣᷕ⚳㜦嗧 (U. sinensis)ˤ⊭⏓忁Ḽᾳ䈑䧖䘬⋩㡅 ITS ⸷↿⛐㬌 椾㫉塓⭂⸷↢Ἦˤ侴憅⮵⎬䈑䧖ᷳ↮ⶫ炻晾䃞⛐冢䀋䘬↮ⶫ㰺㚱䶗⹎ⶖ䔘炻ỮṆ䅙ⷞ⍲䅙ⷞ䘬檀㴟㉼ ⛘⋨ㅱ⏓㚱寸⭴䘬䓊䓇⫸♲䚌ᷳ㜦嗧ˤ 撚戳夜濣↮栆⬠ˣ䓇ン⬠ˣ⛘堋ˣ㜦嗧ˣ檀㓰㵚䚠刚Ⰼ↮㜸ˤ

Fung. Sci. 27(1): 45–50, 2012

傸㼡䏠Ῠ斃㓝㘭䖢劊榒⟔怳䯞ᴉ⃛㨣㉠壌 ஻ဎϛ! ֔छᝋ* 冢⊿ⶪ䩳㔁做⣏⬠⛘䎫䑘⠫㙐䓇䈑屯㸸⬠䲣炻10042 冢⊿ⶪッ⚳大嶗 1 嘇 (㍍⍿㖍㛇烉101 ⸜ 3 㚰 1 㖍)

ᄢ! ! ौ 㛔䞼䨞⇑䓐⚢ン➡梲➢炻忚埴㟠䚌却䥹䘬⃒晭㗇㜗䚌却 (Asterocalyx mirabilis) ↮㱴橼⢾愝䳈䘬䈡⿏↮ 㜸炻娎槿ἧ䓐 Hankin 冯 Anagnostakis ℑ㮷ᷳ㕡㱽炻㷔娎娚却㗗⏎℟㚱㝄先⍵⺷㴰⍣愞 (pectate transeliminase)ˣ㝄先⍣倂⎰愞 (pectin depolymerase) ␴㝄先愞 (pectinase)ˤ᷎忚ᶨ㬍㍊妶℞㽙䰱↮妋㳣⿏ (amylolytic activity)ˣ傪偒↮妋㳣⿏ (lipolytic activity)ˣ噳䘥㯜妋㳣⿏ (proteolytic activity)ˣ䢟愠愞㳣⿏ (phosphatase activity)ˣ⯧䳈愞㳣⿏ (urease activity) ␴⸦ᶩ岒愞㳣⿏ (chitinase activity)烊⎎ẍ Yeoh 㮷䫱 Ṣᷳ㕡㱽炻㷔娎娚却䧖䘬举䵕岒↮妋㳣⿏ (cellulolytic activity)烊᷎ẍ Pointing 㮷ᷳ㕡㱽㷔娎㛐倂態㳣⿏ (xylanase)ˤ㕤 23°C ⿮㹓➡梲 7 ⣑炻䳸㝄㛔却䧖㇨䓊䓇䘬傆⢾愝䳈炻⊭㊔:㝄先⍣倂⎰愞ˣ㝄先愞ˣ傪 偒愞ˣ举䵕䳈愞ˣ⯧䳈愞ˣ㽙䰱愞ˣ噳䘥岒愞␴㛐倂態愞ˤ 撚戳夜濣⃒晭㗇㜗䚌却ˣ橼⢾愝䳈ˤ

䛇却屯㸸⶚塓ṢᾹ⣏慷䞼䨞冯⇑䓐炻⤪曰 剅ˣ⅔垚⢷勱䫱㇨墥忈䘬喍⑩ˣᾅ‍梇⑩䫱 ⛯℟㚱旵埨䱾ˣ㈿儓䗌ˣ旵埨⡻䘬≇䓐 (䌳炻2010烊∱䫱Ṣ炻2009)炻侴䚌却䘬Ḵ㫉 ẋ 嫅 䈑 ḇ 㤝 ℟ 喍 䓐 攳 䘤 ₡ ῤ 炻 ⤪ Mollisia caesia Fuckel. ㇨↮㱴䘬 mollisin炻Monilinia fructicola Winter. ↮㱴䘬 scleroporin炻⎗䔞㈿ 䛇却㳣⿏ᷳ喍∹炻Lachnellula fuscosanguinea Rehm. ↮㱴䘬 lachnellulone炻Lachnum papyraceum Karst. ↮㱴䘬 lachnumon ⎗ 䁢㈿⽖ 䓇 䈑 喍 ∹ 炻 Mollisia ventosa Karst. ↮ 㱴 ᷳ KS-504a,b 冯 Trichopeziza mollissima Fuckel. ↮㱴ᷳ scyphostain 傥 䁢愝䳈㈹⇞∹ (Hosoya, 1998)ˤ㬌⢾姙⣂䛇却塓ㅱ䓐⛐ἄ䈑䕭 ⭛ㆾ䓇䈑旚㱣炻⤪⇑䓐 Phlebiopsis gigantea Jülich Ἦ 旚 㱣 㜦 㧡 㟡 悐 僸 㓿 䕭 炻 ᷎ ᶼ ⇑ 䓐 Beauveria bassiana Vuill. ˣ Metarizium an*

isopliae Sorokin. 忚埴⭬䓇囙䘬旚㱣 (Cook and Baker, 1983; Pratt et al., 2000; Kaaya and Hassan, 2000)ˤ㚨役㚦㚱⬠侭⟙⮶冢䀋䓊 8 䧖䚌却⮵ 5 䧖㢵䈑䕭⍇却忚埴 40 䳬⮵ⲁ䳬 ⎰䘬㊖㈿⮎槿炻䳸㝄栗䣢忁 8 㟒䚌却⮵Ⓒ卾 呚䀘溜却 (Botrytis gladiolorum Timmerm.)ˣ 刳䕭却 (Phytophthora colocasiae Racib.)ˣ厠 勱䕓暱却 (Phytophthora nicotianae Breda de Haan.) 㚱㊖㈿䎦尉炻℞ᷕ庇䚌却 (Mollisia tax. Sp.1) ⮵Ⓒ卾呚䀘溜却 (B. gladiolorum) 忼⇘ 81.67% 䘬㊖㈿≃炻℟㚱䓇䈑旚㱣䘬㼃 ≃ (檀␴⏛炻2011烊檀炻2009)ˤ 䓇䈑旚㱣䘬㨇⇞炻ᶨ凔悥㗗⇑䓐䛇却䘬Ḵ 㫉ẋ嫅䈑ㆾ䈡㬲愝䳈Ἦ㈹⇞䕭⍇却䓇攟ˤ㬌 ⢾䛇却䘬愝䳈ḇ⎗䓐⛐梇⑩ㆾㅱ䓐㕤℞Ṿⶍ 㤕炻⤪:㝄先愞⛐梇⑩㤕ᷕ炻⎗㷃⮹␾┉冯勞 䘬䘤愝㗪攻⍲⡆≈厫⍾梇䓐㱡䘬慷␴䆇梲䈑

忂妲ἄ侭炻e-mail: [email protected] or [email protected]

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岒 (Kashyap et al., 2001)炻ᶼ傪偒愞⎗⸓≑ḛ 愒䅇ㆸ炻⡆≈桐␛⍲ᾖ梦寮㻧䘬桐␛ (Seitz, 1974)ˤ⃒晭㗇㜗䚌却 (Asterocalyx mirabilis Höhn.) ⛐㗍⬋㗪炻℞㕷佌⎗⣏慷䓇攟㕤䤷Ⱉ 㢵䈑⚺ℏ唐栆䘬叱㝬ᶲ炻㍉普⼴㕤⮎槿⭌ẍ ╖⬊➡梲炻℞䓇攟忇䌯 (1 忙䲬攟 4 ℔↮) 㭼 ᶨ凔䚌却 (1 ᾳ㚰攟 4–5 ℔↮ⶎ⎛) ⾓忇 (檀炻2009)炻⚈㬌㛔䞼䨞㒔⮵㛔却䧖ᷳ⎬䧖 愝䳈炻⊭㊔⎗↮妋䳘傆⡩䘬䈑岒⍲℞ẋ嫅 䈑炻⤪:傪偒愞ㆾ㽙䰱愞䫱愝䳈ᷳ䈡⿏忚埴↮ 㜸炻ẍἄ䁢㛒Ἦ攳䘤㬌栆䛇却䘬屯㸸ᷳㅱ 䓐ˤ 㕤 2011 ⸜⮯㍉冒䤷Ⱉ㢵䈑⚺炻䓇攟⛐冢 䀋㠓㪷 (Cyathea spinulosa Wall.) 叱㝬ᶲ䘬⃒ 晭㗇㜗䚌却㇨↮暊⼿⇘䘬╖⬊炻⇑䓐 23°C PDA ⚢橼➡梲➢➡梲却㟒炻᷎⮯↮暊䘬却㟒 ⬀ 㕤 梇 ⑩ ⶍ 㤕 䞼 䨞 ㇨ 炻 却 䧖 䶐 嘇 BCRC 34832ˣ34833 ␴ 34834ˤ 忚ᶨ㬍⛐㷔娎㛔却䧖䘬 10 䧖愝䳈䈡⿏ᷳ ⮎槿㗪炻ἧ䓐ᶵ⎴惵㕡㷔娎ᶵ⎴愝䳈䘬↮妋 䈡⿏ˤ㝄先⍵⺷㴰⍣愞 (pectate transeliminase)ˣ㝄先⍣倂⎰愞 (pectin depolymerase)ˣ 㝄先愞 (pectinase)ˣ㽙䰱↮妋㳣⿏ (amylolytic activity) ˣ 傪 偒 ↮ 妋 㳣 ⿏ (lipolytic activity) ˣ 噳 䘥 㯜 妋 㳣 ⿏ (proteolytic activity) ˣ 䢟 愠 愞 㳣 ⿏ (phosphatase activity)ˣ⯧䳈愞㳣⿏ (urease activity)ˣ⸦ᶩ 岒愞㳣⿏ (chitinase activity) ㊱䄏 Hankin 冯 Anagnostakis ℑ㮷 (1975) ᷳ㕡㱽炻举䵕岒↮ 妋㳣⿏ (cellulolytic activity) ὅ Yeoh 㮷䫱Ṣ (1985) 䘬㕡㱽炻㛐倂態㳣⿏ (xylanase) ⇯ἧ 䓐 Pointing 㮷 (1999) ᷳ㕡㱽忚埴㷔⭂ˤ⍰⚈ ⇑䓐!PC 兄 (polycarbonate membrane) ⮵䛇却 !

橼 ⢾ 愝 䳈 ᷳ 㷔 娎 䳸 㝄 庫 㖻 奨 ⮇ (Chang et al.,1992*炻㛔䞼䨞⇑䓐!PC 兄 (0.2 m, 47 mm diam or 90 mm diam; Nuclepore Co.) 㓦⛐ 㭷ᶨᾳ㫚㷔娎䘬➡梲➢ᷳᶲ炻ℵ㍍䧖却䧖㕤 兄ᶲ炻䴻忶7⣑⼴炻⮯却䧖冯兄䦣昌䚜㍍奨 ⮇炻ㆾ≈ᶲ䈡⭂愝䳈㩊槿喍∹⼴奨⮇⍵ㅱ䳸 㝄炻㛔⮎槿⮯却㟒昼叿!PC 兄➡梲䘬⃒溆㗗 ⛐⼴临䳸㝄奨⮇㗪炻⎗性⃵却䴚⼙枧䳸㝄䘬 ⏰䎦ˤ ⮎槿䳸㝄栗䣢冒䤷Ⱉ㢵䈑⚺㇨↮暊ᷳ A. mirabilis 㖶栗℟㚱↮㱴举䵕岒愞ˣ㝄先⍣倂 ⎰愞ˣ㝄先愞ˣ㽙䰱愞ˣ噳䘥岒愞ˣ傪偒愞 ␴㛐倂態愞䘬傥≃炻ḇ℟㚱⯧䳈愞䘬㳣⿏Ữ 庫ᶵ㖶栗 (堐ᶨ⍲⚾ᶨA–T)ˤ举䵕岒愞䘬娎 槿栗䣢炻却䴚䓇攟䓊䓇愝䳈⮯➡梲➢ℏ䘬⣂ 態栆↮妋炻㓭䡀㵚≈ℍ⼴ᶵ⏰㡽湹刚炻侴↢ 䎦德㖶⚰ (⚾ᶨA炻⚈⃱㸸⍿␐⚵㶙㡽刚⼙ 枧炻侴⏰䎦湫刚)炻⮵䄏䳬⇯䃉㬌䎦尉 (⚾ᶨ B)ˤ㝄先⍣倂⎰愞␴㝄先愞娎槿栗䣢炻㚱䓊 䓇愝䳈⇯㝄先㚫塓↮妋炻堐朊䓊䓇䘥㽩⋨ (⚾ᶨC)炻侴⮵䄏䳬⇯䃉㬌䎦尉 (⚾ᶨD)ˤ㽙 䰱愞娎槿栗䣢㚱愝䳈䓊䓇炻㽙䰱㚫塓↮妋炻 㓭䡀㵚≈ℍ⼴ᶵ⏰㡽湹刚炻侴↢䎦德㖶⚰ (⚾ᶨE炻⚈⃱㸸⍿␐⚵㶙㡽刚⼙枧炻侴⏰䎦 湫刚)炻⮵䄏䳬⇯䃉㬌䎦尉 (⚾ᶨF)ˤ傪偒愞 娎槿栗䣢㚱愝䳈䓊䓇炻➡梲➢ℏ䘬傪偒塓↮ 妋侴↢䎦姙⣂䘥㽩㰱㽙䈑!(⚾ᶨG)炻⮵䄏䳬 ⇯䃉㬌䎦尉 (⚾ᶨH)ˤ㛐倂態㳣⿏娎槿栗䣢 㚱愝䳈䓊䓇炻⣂態栆塓↮妋㓭䡀㵚≈ℍ⼴炻 ᶵ⏰㡽湹刚侴↢䎦德㖶⚰ (⚾ᶨI炻⚈⃱㸸⍿ ␐⚵㶙㡽刚⼙枧炻侴⏰䎦湫刚)炻⮵䄏䳬⇯ 䃉㬌䎦尉 (⚾ᶨJ)ˤ! 噳䘥岒愞娎槿劍㚱㬌䧖愝䳈炻≈ℍ sulpho-!

圦Ჾ澱⃒晭㗇㜗䚌却⋩䧖愝䳈㳣⿏㷔娎* 㝄先⍵⺷ 㴰⍣愞 –

㝄先⍣倂 ⎰愞ˣ㝄 先愞 +

㽙䰱↮妋 㳣⿏

傪偒↮妋 㳣⿏

+

+

* +: 㚱㳣⿏⍵ㅱ烊!: ⽖㳣⿏⍵ㅱ烊–: 䃉㳣⿏⍵ㅱ

!

噳䘥㯜妋 㳣⿏!

䢟愠愞 㳣⿏

⯧䳈愞 㳣⿏

⸦ᶩ岒愞 㳣⿏!

+

–

!

–

!

举䵕岒↮ 妋㳣⿏! +

!

㛐倂態 㳣⿏! +

⃒晭㗇㜗䚌却橼⢾愝䳈

47

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ◔Ჾ澱⃒晭㗇㜗䚌却⋩䧖愝䳈娎槿ˤA. 举䵕岒愞⮎槿䳬炻栗䣢㛔䧖℟㚱愝䳈农ἧᷕ攻䓊䓇德㖶⚰ (⚈⃱㸸⍿␐⚵ ! 㶙㡽刚⼙枧炻ἧ㛔⚾⏰䎦湫刚)烊B. 举䵕岒愞⮵䄏䳬炻䃉德㖶⚰䓊䓇ˤC. 㝄先⍣倂⎰愞␴㝄先愞⮎槿䳬炻⚈愝䳈䓊 ! 䓇农ἧ堐朊䓊䓇䘥㽩⋨ (⤪䭕柕㇨䣢䭬⚵)烊D. 㝄先⍣倂⎰愞␴㝄先愞⮵䄏䳬䃉䘥㽩⋨䓊䓇ˤE. 㽙䰱愞⮎槿䳬炻⚈ ! 愝䳈农ἧ䓊䓇⼊ㆸᷕ攻德㖶⚰ (⚈⃱㸸⍿␐⚵㶙㡽刚⼙枧炻ἧ㛔⚾⏰䎦湫刚)烊F. 㽙䰱愞⮵䄏䳬䃉德㖶⋨䓊䓇ˤG. 傪偒愞⮎槿䳬炻⚈℟㬌䧖愝䳈侴䓊䓇姙⣂䘥㽩㰱㽙䈑烊H. 傪偒愞⮵䄏䳬䃉䘥㽩㰱㽙䈑䓊䓇ˤI. 㛐倂態愞⮎槿䳬炻 ! ⚈愝䳈农ἧ䓊䓇ᷕ攻德㖶⚰ (⚈⃱㸸⍿␐⚵㶙㡽刚⼙枧炻ἧ㛔⚾⏰䎦湫刚)烊J. 㛐倂態愞⮵䄏䳬⇯䃉德㖶⚰䘬⼊ ! ㆸˤK. 噳䘥岒愞⮎槿䳬炻⚈℟㬌䧖愝䳈㓭䓊䓇德㖶⚰ (⤪䭕柕㇨䣢䭬⚵炻⚈䄏䚠㗪ἧ䓐湹刚側㘗炻㇨ẍ德㖶⋨↢ ! 䎦湹刚)烊L .噳䘥岒愞⮵䄏䳬⇯ᶵ䓊䓇德㖶⚰ˤM. ⯧䳈愞⮎槿䳬炻⚈⽖慷愝䳈⍵ㅱ侴⏰䎦䵈刚烊N. ⯧䳈愞⮵䄏䳬 䃉愝䳈䓊↢㓭䵕㊩湫刚ˤO. ⸦ᶩ岒愞⮎槿䳬炻䃉愝䳈䓊䓇㓭䃉德㖶⚰⼊ㆸ烊P. ⸦ᶩ岒愞⮵䄏䳬Ṏᶵ⼊ㆸ德㖶⚰ˤ ! Q. 㝄先⍵⺷㴰⍣愞⮎槿炻䃉㬌䧖愝䳈䓊䓇㓭堐朊ᶵ⼊ㆸ䘥㽩⋨烊R. 㝄先⍵⺷㴰⍣愞⮎槿⮵䄏䳬Ṏᶵ䓊䓇䘥㽩⋨ˤ ! S. 䢟愠愞⮎槿䳬炻㛔却䧖䃉㬌愝䳈䓊䓇炻㓭➡梲➢㛒嬲ㆸ䰱䲭刚烊T. 䢟愠愞⮵䄏䳬Ṏ䃉嬲刚⍵ㅱˤ ! ! salicylic acid 㚫ἧ➡梲➢䓊䓇德㖶⚰炻䳸㝄 湫刚炻⚈㛔却䧖⏰刚⍵ㅱ䁢䵈刚炻侫ㄖ刚⼑ 栗䣢炻㛔䧖℟㚱㬌䧖愝䳈 [⮎槿䳬⚾ᶨK (⚈ ⬠ᷕ湫刚≈啵刚䁢䵈刚炻䓙㬌㍐婾㛔却䧖℟ 䄏䚠㗪ἧ䓐湹刚側㘗炻㇨ẍ德㖶⋨↢䎦湹 㚱⽖慷⯧䳈愞 (⚾ᶨM)炻⮵䄏䳬⇯䁢湫刚 刚) ␴⮵䄏䳬⚾ᶨL]ˤ⯧䳈愞娎槿劍㚱娚愝 (⚾ᶨN)ˤ⸦ᶩ岒愞娎槿劍㚱㬌䧖愝䳈⇯⮎ 䳈炻⮎槿䳬⮯↢䎦啵刚炻䃉愝䳈䓊䓇㗪⇯䁢 槿䳬䘬➡梲➢㚫↢䎦德㖶⚰炻Ữ㛔䞼䨞䳸

48

Fung. Sci. 27(1), 2012

㝄炻⮎槿䳬!(⚾ᶨO) ␴⮵䄏䳬 (⚾ᶨP) 䚠 ⎴炻悥⏰䎦⍇㷔娎➡梲➢㶟㽩䘬⼊ン炻㓭嫱 㖶䃉㬌䧖愝䳈ˤ㝄先⍵⺷㴰⍣愞娎槿劍㚱㬌 䧖愝䳈炻⇯⛐㷔娎➡梲➢堐朊ᶲ↢䎦䘥刚㶟 㽩⍵ㅱ䈑炻䓙⮎槿䳸㝄䘬⮎槿䳬!(⚾ᶨQ) ␴ ⮵䄏䳬 (⚾ᶨR) ⼿䞍㛔却ᶵ℟㬌䧖愝䳈ˤ䢟 愠愞娎槿栗䣢㛔却䧖䃉䢟愠↮妋炻⚈䁢≈ℍ 㯓㯏⊾扐⼴炻➡梲➢᷎㛒嬲ㆸ䰱䲭刚ㆾ䲭刚! )⚾ᶨS)炻⮵䄏䳬⇯ḇ㗗㬌䎦尉 (⚾ᶨT)ˤ ! ὅ㛔却䧖ᷕ昌℟㚱↮妋姙⣂⣂態䘬傥≃ᷳ ⢾᷎℟㚱傪偒愞␴噳䘥愞炻⎗䞍㛔却䧖晾ẍ ⣂態 䁢ᷣ天䡛䳈䆇梲Ἦ㸸炻Ữ䳸㝄ḇ姤㖶 忁䧖䚌却Ṏ⎗忚ᶨ㬍⇑䓐傪偒愞␴噳䘥愞ẋ 嫅炻ἄ䁢℞Ṿ傥慷Ἦ㸸ˤ䃞侴⬫䃉⸦ᶩ岒 愞炻⏓㤝⮹慷⯧䳈愞炻㬌Ṏ⌘嫱℞䁢ỽ傥 ⣈⣏慷䓇攟㕤唐栆叱㝬忁䧖➢岒䘬䓇攟䑘 ⠫炻⚈䁢冢䀋㠓㪷䘬叱⫸䳬ㆸㆸ↮炻⏓㚱 43% 䡛ˣ0.8% 㯖ˣ0.03% 䢟␴ 43% 㛐岒䳈 (楔䫱Ṣ炻2009)ˤ ⃒晭㗇㜗䚌却㇨↮㱴䘬愝䳈炻⏓㚱举䵕岒 愞ˣ㝄先愞ˣ㽙䰱愞⍲㛐倂態愞炻忁ṃ悥⎗ ↮妋⣏↮⫸態栆ㆸ庫⮷䘬↮⫸炻ḇ姙⎗ẍ䮑 怠↢檀䓊慷却㟒炻⇑䓐⛐忈䳁ⶍ㤕 (Beg et al., 2001) ㆾ 梇 ⑩ ⶍ 㤕 (Kashyap et al., 2001; Rode, 1999; De Souza et al., 2010) ᶲˤ 㚱䞼䨞㊯↢垚䓇䛇却℟㚱㝸ṃ噳䘥愞炻⛐ 屓䨧㖮垚橼堐忶䦳ᷕ㈖㺼朆ⷠ慵天奺刚 (䯉 䫱Ṣ炻2006)ˤᶨ凔⽖䓇䈑℟㚱䘬噳䘥岒愞 䓐徼⺋㲃炻忂ⷠㅱ䓐㕤㶭㻼∹ˣ墥喍ˣ墥 朑ˣ⺊㡬䈑嗽䎮ㆾ梇⑩␴梤㕁䘬㶣≈ (Godfrey and West, 1996)炻㚨役㚱䭯婾㔯䞼䨞㊯ ↢⇑䓐Pichia pastoris廱⼊却㟒炻㇨䓇䓊䛇却 却䧖 Candida rugosa 傪偒愞䫔ᶱ✳ (CRL3)炻 ⛐⌘⇟⟙䳁僓⡐㓰䌯䚠䔞㕤ἧ䓐⊾⬠僓⡐∹ ᷳ僓⡐㓰㝄炻⮵㕤ⶍ㤕ㅱ䓐ᶲ㤝℟䘤⯽㼃≃ (悕ṕ⏃炻2007)炻⎎⢾⚈㛔却䧖⛐ 10 䧖㷔娎 䘬愝䳈ᷕ炻⌛℟㚱 7 䧖愝䳈䘬↮㱴傥≃炻㛒 Ἦ⎗ 愝䳈⭂慷↮㜸炻ḇ姙㛔娎槿却㟒㛒Ἦ ⎗塓⺋㲃ㅱ䓐㕤ⶍˣ⓮㤕䓐徼ˤ

ᗂ! ! ᇬ デ嫅ᷕ冰⣏⬠㞗㔯晬㔁㌰岰復ᷳ PC 兄嬻 㛔䞼䨞枮⇑忚埴炻⍰▱佑⣏⬠唕㔯沛㔁㌰㍸ ὃ㔯䌣炻⮵㛔䞼䨞㚱卓⣏䘬≑䙲炻⛐㬌ᶨἝ 农嫅ˤ

୤ՃМᝦ 䌳尒. 2010. 曰剅冐⸲㕘䓐䯉ṳ. 㖶忂慓喍 406: 22–23. 楔⃫ᷡ炘㰇㳒炘ἁ㧡ℐ炘䩯㥖洔炘悕➡➡炘 䌳⼔. 2009. ᶵ⎴崟㸸㗪攻䘬㢵䈑叱ⅳ句䈑 ⛐ ᷕ Ṇ 䅙 ⷞ 䘬 ↮ 妋 䈡 ⿏ . 䓇 ン ⬠ ⟙ 26: 5327–5345. 檀䳡ヰ. 2009. 冢䀋℟㚱喍䓐㼃≃䘬䃉♲味䚌 却ᷳ䓇攟⍲℞㈿䓇⮎槿䞼䨞. 冢⊿ⶪ䩳㔁 做⣏⬠. 冒䃞䥹⬠䲣䡑⢓䎕⬠ỵ婾㔯. 檀䳡ヰ炘⏛伶渿. 2011. 冢䀋䓊拀冴却䚖却䧖 ⮵㢵䈑䕭⍇却㊖㈿ἄ䓐ᷳ⮎槿䞼䨞. 㴟ⲥ ℑⱠ䫔⋩⯮却䈑⬠㙐䫔ᶱ⯮梇喍䓐却⬠埻 䞼妶㚫婾㔯㐀天普: 288. 悕ṕ⏃. 2007. Pichia pastoris 䓇䓊 Candida rugosa 傪偒愞䫔ᶱ✳䘬䲼⊾冯⿏岒↮㜸⍲℞ ⛐⺊⟙䳁僓⡐ᶲᷳㅱ䓐. 冢䀋⣏⬠. ⽖䓇䈑 冯䓇⊾⬠䞼䨞㇨䡑⢓䎕⬠ỵ婾㔯. ∱ⅈ幵炘䌳廅炘堩剛. 2009. ⅔垚⢷勱喍䎮ἄ 䓐⍲冐⸲ㅱ䓐䵄徘. 湹漵㰇慓喍 22: 886– 88. 䯉ⶏ㱣炘唕㔯沛炘佩䥱暾. 2006. ⇑䓐 API ZYM 䲣䴙↮㜸䘥㭕却⍲冀噏廒㝅却ᷳ傆 ⢾愞↮㱴䧖栆⍲㗪䦳. 冢䀋㖮垚 26: 319– 328. Beg, Q.K., M. Kapoor, L. Mahajan, and G.S. Hoondal. 2001. Microbial xylanases and their industrial applications: a review. Appl. Microbiol. Biotechnol. 56: 326–338. Chang, T.T., X.Y. Yang, and W.H. Ko. 1992. A sensitive method for detecting production of

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Study on the extracellular enzyme of Asterocalyx mirabilis in Taiwan Yi-Chung Chang and Mei-Lee Wu Department of Earth and Life Science, Taipei Municipal University of Education, 1, Ai-Guo West Road, Taipei 10042, Taiwan.

ABSTRACT Solid media were used for the detection of enzyme production on different media from Asterocalyx mirabilis, Sclerotiniaceae. Hankin and Anagnostakis methods were used for the enzyme tests, including the enzymes of pectate transeliminase, pectin depolymerase, pectinase, amylolytic enzyme, lipolytic enzyme, proteolytic enzyme, phosphatase, urease, and chitinase. Cellulase were also detected by the method of Yeoh et al. Xylanase were further detected by the method of Pointing growing at 23°C for 7 days. There were pectin depolymerase, pectinase, lipolytic, cellulase, amylolytic enzyme, proteolytic enzyme and xylanase in the A. mirabilis but there were no pectate transeliminase, phosphatase, urease and chitinase. Keywords:!Asterocalyx mirabilis- extracellular enzyme.

Fung. Sci. 27(1): 51–54, 2012

ぴ傥䤄㐗㉆ ֔ᖐ๼ ⚳䩳冒䃞䥹⬠⌂䈑棐㢵䈑⬠䳬炻冢ᷕⶪ棐⇵嶗ᶨ嘇炻404

冏䧮㔁㌰㗗ᷕ⚳叿⎵卯却↮栆⬠⭞炻Ⱉ㜙 Ṣ炻1930 ⸜䓇㕤䄁⎘炻2011 ⸜ 11 㚰 10 㖍 㕤㖮㖶⍣ᶾ炻ṓ⸜ 81 㬚ˤ冏㔁㌰䇞奒㗗慹 圵⭞炻⸜⮹⛐ᶲ㴟⬠佺炻䔊㤕㕤喯ⶆ㜙⏛⣏ ⬠䓇䈑䲣ˤ㜙⏛⣏⬠㗗㔁㚫⬠㟉炻⟙⏲ẍ劙 㔯⮓ἄ炻⚈㬌冏㔁㌰恋ᶨẋ䘬ᷕ⚳㛔⛇䛇却 ⬠⭞ᷕ炻Ṿ䘬劙㔯㗗㚨⤥䘬ˤ⣏⬠䔊㤕⼴䔁 㟉㑼ả㔁借炻䳸嬀ᷕ⚳剼嗂⬠ᷳ䇞昛恎㜘㔁 ㌰䘬⬠䓇湶冰㰇⤛⢓炻᷎䳸⨂ˤ 湶㔁㌰㗗叿⎵剼嗂⬠侭炻⣏⬠䔊㤕⼴㕤ᷕ 䥹昊⊿Ṕ㢵䈑䞼䨞㇨ả借炻ᷳ⼴ℵ䌚倀㕤㖮

㖶㢵䈑䞼䨞㇨ả借军徨ẹˤ冏㔁㌰㕤 1973 ⸜崟Ṏ⍿倀㖮㖶㢵䈑䞼䨞㇨䚜军徨ẹˤ冏ˣ 湶ℑ㔁㌰ᶵデね㶙⍂炻ḇ㚱↮栆⬠ℙ⎴冰 嵋炻⮵㕤ᷕ⚳䛇却冯剼嗂⬠䞼䨞⋻㚱届䌣ˤ ㆹ冯冏㔁㌰⣓⨎⇅嬀㕤 1990 ⸜⇅⢷炻䔞 㗪ㆹ⛐剔嗕崓䇦彃➢⣏⬠ⓠ⌂⢓ˤ恋墉⎔攳 㜙Ṇ剼嗂⚳晃䞼妶㚫炻ᷕ⚳ᷣ天䘬剼嗂⬠侭 悥ἮḮ炻昌Ḯ攳㚫炻ḇ 䔁ᶨ㭝㗪攻䞼䨞崓 ⣏寸⭴㓞啷䘬剼嗂㧁㛔ˤ冏㔁㌰ᾳ⿏䇥㚿炻 䪹⎋ⷠ攳炻⭴㕤Ṣね␛ˤṾ㑭攟㚠䔓炻╄㬉 ⓙ㇚冯㬟⎚㓭ḳ炻㔯慯ṎἛˤ冏㔁㌰⣓Ṣ湶 㔁㌰ḇ㗗‍婯䅙ねˤᶨ㫉ㆹ⇘≽䈑䲣䘬⚾㚠 棐⸓ᾳ冢䀋⎴⬠⼙⌘㖮垚⬠䘬侩㔯䌣ˤ⛐⺊ 䳁➮ᷕ夳⇘ᶨỵ⣏映䔁⬠䓇⮓䘬娑ἄ炻℞ᷕ 㚱㍸⇘ℕ⚃䘬 デ㔯⎍炻娑㔯⮓⼿㤝⤥炻⯙ 㑧崟忁⸦䭯娑㔯炻⽆会⎵㈦⇘忁ᾳ⌂⢓䓇炻 ␴Ṿ俲Ḯ⸦⎍ˤ㚱㓧㱣㕡朊䘬栏ㄖ⏏炻Ṿ栗 ⼿㚱ṃᶵ冒⛐ˤㆹ㈲娑ἄⷞ⚆䴎冏㔁㌰䚳炻 Ṿ枿㚱デ妠炻㰱⏇䇯⇣⌛⮓⯙ᶨ椾䳽⎍デ゛ Ṍ䴎ㆹˤ㔯⿅㓷㌟⎎ㆹ⎫樂ˤ 䔞⸜ℓ㚰ㆹ䓙剔嗕⇘⽟⚳暟㟡㕗⟉ (Regensburgh) ⍫≈⚳晃䛇却⬠䞼妶㚫炻冏㔁㌰ ⣓⨎ḇ↢ⷕ忁枭㚫嬘ˤ忯⇘冢⊿慓⣏䘬喯ㄞ 厗㔁㌰炻倥⇘冏㔁㌰␴喯㔁㌰㍸崟ℙ⎴䘤堐 㔯䪈䘬ḳ炻⼴Ἦ゛崟⯙㗗㧇剅㕘䧖䘬㔯䪈ˤ 㚫⼴ㆹ␴冏㔁㌰⣓⨎㏕⽟⚳㜄屻㟡⣏⬠㫸⮞ 㹓㟤 (Oberwinker) 㔁㌰䘬ὧ干㊄姒㜄屻㟡ˤ ㆹ冯㫸⮞㹓㟤㔁㌰␴Ṿ䘬⬠䓇 Ewald Langer ⣓⨎ (䔞㗪㗗䓟⤛㚳⍳) 㗗⇵ᶨ⸜ṾᾹἮ冢㍉ 㧁㛔㗪娵嬀䘬ˤㆹᾹ啱叿⇘⽟⚳攳㚫ᷳὧ㊄ 姒ṾᾹˤ

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ㆹ 1990 ⸜⸽䔊㤕炻佴⸜⇅䓛婳⇘䥹⌂棐 ⶍἄˤ1992 ⸜ㆹ婳ᶨᾳ⣂㚰℔`⇘ᷕ⚳⣏映 ⸦ᾳ㢵䈑㧁㛔棐䞼䨞ㆹ䡑⢓昶㭝䞼䨞䘬咻剼 䥹㧁㛔ˤ⛐剔嗕㗪娵嬀⸦ỵ⣏映剼嗂⬠侭炻 ḇ゛⍣娵嬀⣏映䘬⬠埻䞼䨞ね㱩ˤ䫔ᶨ䪁⇘ ⊿Ṕ㢵䈑㇨炻∃⤥冏㔁㌰⣓⨎⇘⊿Ṕ攳㚫∃ 䳸㜇炻㈦ㆹ⍣俲⣑ˤ冏㔁㌰⇲ᶨ柮嗧咼䴎 ㆹ炻䓄傮⎗ẍ䓇⎫䘬炻墉朊㗗䲭倱炻Ṿ婒㗗 ⊿Ṕ䈡䓊⑩䧖炻὿䧙ˬ⽫墉伶˭炻⇘⊿Ṕᶨ ⭂天☸☸ˤ⊿Ṕᷳ⼴ㆹ⍣㾳春ˣᶲ㴟ˣ⺋ⶆ ␴㖮㖶䚳㧁㛔ˤ⛐㖮㖶㢵䈑㇨䚳䘬咻剼䥹㧁 㛔炻娵䁢㚱ℑᾳ䓊冒大啷⡐僓䘬㧁㛔⎗傥㗗 㕘䧖ˤ⣂⸜⼴湶冰㰇㔁㌰䘤堐㕘䧖炻婒ㆹ䔞 ⸜⛐㖮㖶㚱䞼䨞忶炻≈ㆹ䁢ℙ⎴ἄ侭ˤ㖮㖶 㚱ᶨ⣑炻䔞⛘梇䓐却䞼䨞㇨怨婳䔞⛘⸦ỵ卯 栆⮰⭞⹏婯炻ᷣ柴㗗㕘梇䓐却䘬攳䘤⓷柴炻 冏㔁㌰ḇ嬻ㆹᶨ崟⍣炻䔞㗪⚈ℑⱠ㛒⤪䎦⛐ 攳㓦炻Ṿ婒ㆹ㗗Ἦ冒⺋㜙⽖䓇䈑㇨ˤ⸦ỵ⮰ ⭞䘬䘤妨⣏橼䘮ⅈℽ➪䘯䓂军ㄟㄐ㽨㖪ˤ冏 㔁㌰晾㗗卯栆叿⎵⬠侭炻⎒㗗㺧共䪹⭡炻㊃ ␤俲⣑炻⹏婯ᷕ㰺Ṩ湤䘤妨ˤ⚆䦳ᷕṾ婒䔞 ⣑⸦ỵ䘬䘤妨㰺┍シ佑炻Ṿ悥ᶵ゛嫃娙ˤ䫔 Ḵ⣑ᶨ⣏㖑炻ㆹ䘤䎦䚠㨇⛐⇵ᶨ⣑䘬℔干⹏ ỵ⾀Ḯ㊧炻冏㔁㌰倥ㆹ婒Ḯ炻楔ᶲㇻ暣娙⇘ ℔干╖ỵ炻⛸干堅忶⍣㈲䚠㨇㊧⚆ἮˤㆹᾹ ⛸⛐℔干㚨⼴㌺炻㇨㚱Ḁ⭊⊭㊔⎠㨇悥㰺㲐 シ⇘忁ᾳ䚠㨇ˤ 冏㔁㌰䇥㚿⤥⭊炻劙㔯⤥炻⢾⚳Ṣ⇘ᷕ⚳  慶⢾ⶍἄ炻悥╄㬉婳Ṿ⬱㌺ˤ1995 ⸜ㆹ⍫ ≈⛐㖮㖶冱彎䘬䫔Ḵ⯮ℑⱠ䛇却⬠䞼䨞㚫炻 㚫⇵⍫≈䓙冏㔁㌰ⷞ柀炻㫸⮞㹓㟤㔁㌰␴ℑ ỵ㱽⚳⬠侭ḇ≈ℍ炻⇵⼨㹯大⊿渿㰇␴㹯⋿ 大暁䇰䲵䘬䛇却㍉普ˤ冏㔁㌰䘬⬠䓇㣲䤅列 ⛐㫸⮞㹓㟤㔁㌰恋墉ⓠ⌂⢓炻ḇᶨ⎴⇵⼨ˤ ᷳ⼴㚱⸦㫉⇵⼨暚⋿䘬慶⢾ⶍἄḇ䓙冏㔁㌰ ㆾ㣲䤅列⌂⢓ⷞ柀ˤ冏㔁㌰⣂㫉ⷞ柀㖍㛔⬠ 侭⛀昲⛐⣏映䘬慶⢾ⶍἄ炻ㆹ⛐ 1998 ⸜⍫ ≈ᶨ㫉ṾᾹ䘬㹯㜙⊿ᷳ埴炻冏㔁㌰⬱㌺䳘 ⽫炻屻ᷣ䚉㬉ˤ

冏㔁㌰⣓⨎ℑ㫉姒⓷冢䀋炻䫔ᶨ㫉㗗 1993 ⸜↢ⷕ䫔ᶨ⯮ℑⱠ䛇却⬠埻䞼妶㚫炻䔞㗪⣏ 映ẋ堐⛀ㇳ临崽彎ᶵ⍲炻䞼妶㚫⚈㬌⺞㛇炻 ⍵侴冏㔁㌰⣓⨎䓙㖍㛔忶Ἦ⤪㛇㉝冢炻∃⤥ ⍫≈䛇却⬠㚫⸜㚫ˤ恋嵇冏㔁㌰怬⛐䥹⌂棐  ⮰柴㺼嫃炻⍲嫃佺⣏✳䛇却揹⭂炻⼕慹様 ⍲昛⺢⎵⌂⢓㚱↢ⷕ嫃佺㚫ˤ冏㔁㌰⛐嫃佺 㚫ᷕṳ䳡卯却䧖栆㗪炻㇨⮓↢䘬㉱ᶩ⬠⎵忋 ⎴ἄ侭⎵ᶨἝ⮓↢炻㗗ㆹ军Ṳ⽆㛒夳䘬炻夳 嬀⇘冏㔁㌰ᷳ⌂倆⻟姀ˤ 1999 ⸜ㆹ䓙⚳䥹㚫䓛婳墄≑怨婳冏㔁㌰⇘ 䥹⌂棐䞼䨞䛇却㧁㛔ᶱᾳ㚰ˤ湶㔁㌰ᶨ⎴忶 Ἦ炻佑⊁⋼≑揹⭂䥹⌂棐䘬剼嗂㧁㛔ˤ㛇㺧 ⇵ㆹℵ䓛婳䥹⌂棐㔯㔁➢慹㚫墄≑ℑỵᶨᾳ 㚰⛐䥹⌂棐䞼䨞㧁㛔ˤ㛇攻␐棐攟婳ℑỵ⎫ 梗俲⣑ˤ㶉⣏⊾⬠䲣⏛▱渿㔁㌰Ἦ㊄姒⤥⍳ 冏㔁㌰⣓⨎炻ḇ婳冏㔁㌰䔓ᶨⷭ暚⋿䘬侩勞 㧡炻ἄ䁢⏛㔁㌰ᶨ㛔㕘㚠䘬⮩朊ˤ冰⣏嫅㔯 䐆㔁㌰⬱㌺ℑỵ⇘⋿悐崘ᶨ嵇炻ṾᾹ⮵ᷕ⚳ 㬟⎚㚱ℙ⎴娙柴ˤ㜙㴟⣏⬠㜿┬晬㔁㌰怨婳 冏㔁㌰⣓⨎ᶨ崟⇘⭞墉忶俾娽⣄ˤ冏㔁㌰⣓ ⨎䘬⤥⍳岜㖶㳚㔁㌰ᶨ㫉ⷞṾᾹ↢⍣炻⚆Ἦ ⼴冏㔁㌰㳍㳍㦪忻婒⍣⍫奨ᶨỵ㢵䈑ッ⤥侭 ⺢䩳䘬㢵䈑⚺ˤᷳ⼴ㆹṎ㚱㨇㚫㊄姒忁ỵ湫 ㄞ岊⃰䓇䘬㢵䈑⚺ˤ冏㔁㌰⣓⨎㉝冢䫔ᶨ㘂 ⃰ỷㆹ⭞炻㫉㖍ㆹ⬱㌺ṾᾹ䚳䦇㇧炻℞ᷕᶨ 攻㗗ㆹ⭞旬役䘬㺪ṖℑⰌ㲳㇧炻⯳ᷣ夳⇘㗗 ⣏映⬠侭炻⤥⽫婒怈㕡侴Ἦㅱ娚天䄏栏炻栀 シẍ㚰䦇Ḽ⋫⃫䘬䈡⇍₡↢䦇Ḵ㦻㔜Ⰼ炻旬 ℐ⣿⭞℟ˤ⎎ᶨ攻㗗䥹⌂棐旬役䘬⣿㇧炻庫 ⮷ᶼ栗⼿昛冲炻㚰䦇天ᶫ⋫⃫ˤㆹẍ䁢ṾᾹ 㚫怠㑯⇵侭炻晾䃞⛸℔干天⋲⮷㗪⇘䥹⌂ 棐炻忁⛐⢾⚳㗗⼰㬋ⷠ䘬炻㱩ᶼḇ⎗㏕ㆹ䘬 干ᶨ崟ᶲᶳ䎕ˤ冏㔁㌰⣓⨎⯭䃞怠Ḯ⼴侭炻 ṾᾹ婒㗗Ἦⶍἄ䘬炻ỷ䥹⌂棐㕩ㇵ㕡ὧˤ᷎ ᶼ䔞⣑⯙㏔埴㛶忶⍣炻ᶵ゛⣂湣䄑ㆹᾹˤ 冏㔁㌰㚱㔠⋩⸜䘬䱾⯧䕭⎚炻㘂⸜㚜♜ 慵炻⼙枧⇘䛤䜃␴埴≽ˤ2004 ⸜ℑⱠ䛇却⬠ 䞼妶㚫⛐㕘䔮䁷欗㛐滲冱埴炻ㆹ忂䞍冏㔁㌰

ㅞ冏䧮㔁㌰ ⣓⨎⍲岜㖶㳚⣓⨎⎴⍣⍫≈ˤ冏㔁㌰⣓⨎恋 嵇⃰枮忻⍣䚳㔎䃴䞛䩇炻Ḯ⌣⣂⸜⽫栀ˤ 2005 ⸜ᶲ㴟⎔攳䘬⚳晃梇䓐却䞼妶㚫冏㔁㌰ ḇ⍿怨㍸↢⮰柴⟙⏲炻恋㗪Ṿ埴≽⶚ᶵὧ炻 婒㗗⽆㕘䔮⚆Ἦ⼴幓橼䨩䃞ᶵ埴Ḯˤㆹ⍣Ṿ ỷ䘬㕭棐䚳Ṿ炻暊攳㗪冏㔁㌰晾埴崘ᶵὧ炻 ṵ➭㊩ᶨ嶗復ㆹ⇘㕭棐攨⎋ˤ 2007 ⸜ℑⱠ䛇却䞼妶㚫⛐攟㗍⎔攳炻ㆹℵ 忂䞍冏㔁㌰⣓⨎⍫≈炻㚫⼴ℙ忲攟䘥Ⱉ炻忁 ⸜冏㔁㌰䘬幓橼⍰⤥廱Ḯ炻崘嶗䃉⣏⚘暋ˤ ⣂⸜⇵冏㔁㌰㍸⇘Ṿ䇞ˣ⃬䘮㖑必炻Ṿ⎗傥 ḇᶵ攟⢥炻ᶼ怢⁛䘬斄Ὢᶵ㗗⚈叿䗴䕯⯙㗗 䱾⯧䕭ˤ冏㔁㌰ṓ⢥ 81 㬚炻ㅱ娚崭↢Ṿ㖑 ⸜゛⁷炻Ṿ㘂⸜梇䓐䢐䰱曰剅炻ㆾ姙㚱≑㕤 幓橼‍⹟ˤ冏㔁㌰㍸忶␴Ṿ⎴⸜䘬㚱⻝㧡⹕ 㔁㌰ˣ昛䐆曺㔁㌰␴⏪䎮䅲⌂⢓ˤ2009 ⸜⛐ 䥹⌂棐⎔攳Ṇ㳚䛇却⬠䞼妶㚫炻ㆹㇻ暣娙ⶴ 㛃冏㔁㌰⣓⨎傥↢ⷕ炻᷎㒔㸾⁁⮰Ṣ䄏栏炻

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Ṿ㚨䳪怬㗗⚈幓橼䉨㱩ᶵἛ炻ᶵ栀湣䄑⇍Ṣ 侴嫅䳽Ḯˤ 冏㔁㌰㤝倘㖶炻⌣㗗夾⎵⇑⤪㴖暚炻⮵䈑 岒㡅ẞ天㯪⼰ỶˤṾ⣂㔠㗪῁⤪⻴≺ἃ凔䪹 ␝␝炻⣏倚婒娙炻⣏倚㊃␤Ṣˤ䚜儠⫸侴䅙 ね㲳㹊炻㦪㕤≑Ṣ炻ḳ⽭奒〕ᶵ⊆䄑ṾṢ炻 㮓䃉㝞⫸ˤṾ婯⇘ᶨṃṢḳ䈑炻㽨≽䘬㗪῁ 䛤䜃ⷠ㗗⏓叿㯜⃱ˤ冏㔁㌰⛐ 1999 ⸜姽䥹 ⬠昊昊⢓᷎忚ℍ㚨⼴昶㭝炻䃞㰺㚱ㆸ≇炻Ṿ 䚳⼿⸛ⷠ炻婒ẍ⼴ᶵ怠Ḯˤㆹデ奢䛇却⬠䞼 䨞⮵冏㔁㌰侴妨ᶵ㗗枪Ḯᶵ崟䘬ḳˤṾ⮵㕤 丒䔓ˣ㚠㱽ˣ普悝ˣ䞛柕㓞啷ˣⓙ㇚ˣ㬟⎚ 䞼䨞ˣㇻ⣒㤝㊛ẍ⍲㚳⍳Ṍ⼨Ụ᷶怬㚜Ἦ ≩ˤ㭷⇘⸜䭨Ṿ⯙⮓ᶨ➮⌉䇯ˣ㚠ᾉ䴎⚳ ℏˣ⢾㚳⍳ˤㆹ奢⼿冏㔁㌰㚜⁷㗗喅埻⭞侴 ᶵ⁷䥹⬠⭞炻⤪㝄崘喅埻ㆾ㬟⎚䞼䨞⎗傥㖑 ⯙䔞ᶲḮ昊⢓ˤᶨ㫉ㆹ⓷冏㔁㌰⤥⍳炻叿⎵ 䘬䓇䈑䔓⭞㚦⬅㽪⃰䓇炻⮵㕤冏㔁㌰㚠䔓䘬

◔ᲾƱ冏䧮㔁㌰ 2006 ⸜㕤㖮㖶㢵䈑䞼䨞㇨ˤ

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Fung. Sci. 27(1), 2012

◔ᵊƱⶎ崟㣲䤅列⌂⢓ˣ昛䥨䍵⮷⥸ˣ冏䧮㔁㌰ˣ昛⽑䏜⌂⢓␴㛔㔯ἄ侭 2006 ⸜㕤㖮㖶㢵䈑䞼䨞㇨ˤ

姽婾ˤ㚦⃰䓇㚱ṃ⏓呬炻婒冏㔁㌰䘬⫿㭼庫 䨑⭂ˤ冏㔁㌰䘬䔓㚱ṃ㔯Ṣ䔓䘬␛忻炻䫮⡐ ᶵ⣏䷩墯炻㭼庫䯉╖䘬堐忼⼊尴嵋␛炻ᶵ⣒ 徥㯪㈨ⶏ⍲喅埻ㆸ⯙炻ℐ䃞䘬⾈㕤冒⶙ˤṾ 姀ㅞ㤝⻟炻⮵㕤㬟⎚Ṣ䈑⍲ḳẞ婒崟Ἦ㗗⤪ 㔠⭞䍵ˤ⣂⸜Ἦ冏㔁㌰⛐˪ᷕ⚳梇䓐却˫⮓ ˬ却䈑⬠⭞䥹㴟厵巒˭⮰㪬炻ṳ䳡⚳晃却⬠ ⭞䘬䓇⸛ḳ帇炻ᶨ⮓⯙㗗ℑ䘦⣂⇯炻⎗夳Ṿ ⮵㕤Ṣ䈑奨⮇ᷳ冰嵋ˣ⺋⌂冯㶙ℍˤ 冏㔁㌰⿍⿏⫸ᶼ≽ἄ⾓炻䔞㗪ἧ䓐䘬栗⽖ 掉㓰㝄ᶵἛ炻⣂⫼却᷎朆℞㑭攟炻≈ᶲ喅埻 ⭞⿏㟤炻䘤堐㧇剅㕘䧖㗪⛐ᶨṃ䈡⽝㍷徘ᶲ ↢Ḯ拗婌炻⺽崟ᷳ⼴㧇剅⬠⎵ἧ䓐䘬䇕嬘ˤ 䓙㕤Ṿ䘬㧇剅㕘䧖䘤堐䫎⎰㢵䈑␥⎵㱽夷䘬 夷⭂炻㧉⺷㧁㛔㗗㧇剅⫸⮎橼炻侴ᶼṾ䔞⇅ ㍷徘拗婌嗽⶚⼿⇘ᾖ㬋冯㼬㶭ˤ2010 ⸜ 11 㚰⛐⚳晃㢵䈑␥⎵㱽夷⥼⒉㚫䛇却⥼⒉㚫䘬 妶婾ᷕ炻㓗㊩㧇剅⬠⎵枰ẍṾ␴喯ㄞ厗㔁㌰

䘬䘤堐⬠⎵䁢ὅ㒂ˤ 2010 ⸜ 7 㚰ㆹ⇘㖮㖶旬役 慶⢾ⶍἄ炻ḇ ⍣㍊㛃冏㔁㌰⣓⨎炻㗗㚨⼴夳⇘冏㔁㌰ˤ䔞 㗪Ṿ埴崘ᶵὧ炻⸦᷶ᶵ↢攨Ḯ炻ᷣ天⛐⭞ⶍ ἄˤ晾侩䞋炻柕儎怬⼰㶭㤂曰㳣ˤ1990 ⸜⛐ 剔嗕㗪炻ᶨ㫉ᶳ⋰勞炻岜㖶㳚㔁㌰⭋ⶫ婳冏 㔁㌰⣓⨎䶐⮓˪ᷕ⚳⬊⫸㢵䈑录℠˫炻2011 ⸜⇅ㆹ㓞⇘冏㔁㌰⣓⨎⭬䴎ㆹℑ㛔⍂⮎ℏ⭡ 寸⭴∃↢䇰䘬㚠ˤṾᾹ⬴ㆸḮ 20 ⸜⇵䘬ⶍ ἄ㈧媦炻婳ㆹ廱Ṍᶨ㛔䴎岜㔁㌰⣓Ṣˤ岜㔁 ㌰⸦⸜⇵劙⸜㖑必炻⎗や㛒傥夳⇘㬌㚠↢ 䇰ˤ 㛒㚱ᶵ㔋䘬⭜ⷕ炰冏㔁㌰㺧儡䘬ㇵね冯㬟 ⎚㓭ḳ炻寸⭴䘬⬠ˣ⍳Ṍ㳩䳪㚱 㬊ᷳ㖍炻 晾检枛ṵ丆㦹ᶵ⶚ˤ冏㔁㌰䘬⬠嬀ˣ尒怩ˣ 㔯喅ˣ⮔⺋ˣ㦪㕤≑Ṣ冯㹓㘾≃慷炻ㆸ䁢㤝 ℟⼙枧冯デ㝻≃䘬Ṣ攻䴻℠ˤ