Volume 94, pp October-December 2005 ZONGQI LIANG" YANFENG HAN' Key Words-hyphomycete, taxonomy, morphology

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1 MYCOTAXON Volume 94, pp October-December 2005 Some entomogenous fungi from Wuyishan and Zhangjiajie Nature Reserves 2. Three new species of the genus Hirsutella* ZONGQI LIANG" YANFENG HAN' AIYING Lm' & JIANZHONG HUANG2 zqliang472( yahoo. com. cn' swallow ( yahoo.com. cn ayliu6( yahoo. com. cn 1 The Institute of Fungus Resources, Guizhou University Guiyang , China hjz( jjnu.edu.cn 2 The Key Laboratory of Microbiological Engineering Fujian Normal University, Fuzhou , China Abstract-Three new entomogenous fungi of the genus Hirsutella, H. zhangjiajiensis; H. hunanensis and H. erinita, collected from Wuyishan (Wuyi Mountains) and Zhangjiajie Nature Reserves, are described and ilustrated. Some problems of the identification of these fungi are briefly discussed. Key Words-hyphomycete, taxonomy, morphology Introduction Hirsutella Pat. is one of the most abundant and important entomogenous fungi. In 1990, we reviewed the development, taxonomic characteristics of the genus, and proposed a key to 62 taxa. A few doubtful species were discussed briefly (Liang 1990a, b). Seifert & Boulay (2004) suggested that Hirsutella consists of 65 species. From our study, we refer 90 species around the world to the genus of Hirsutella. The genus Hirsutella plays an important role in the natural control of pest insects (Evans 1974, 1982). H. gigantea Petch can infect many larvae and pupae of Lepidoptera in the Kuankuoshui Preserve in Guizhou, China (Liang 1991a). Besides this fungus, H. rhossiliensis Minter & B. 1. Brady, discovered in the early 1980's, has a stronger lethiferous effect on many plant parasitic nematodes, such as Ditylenchus dipsaci, Meloidogyne incognita, Aphelenchoides fragariae and Criconemella xenoplax (Jaffee et al 1982; Cayrol & Frankowski 1986; Cayrol et al 1986). It is known that many species in the genus Hirsutella are anamorphs of Cordyceps Fr. For example, an anamorph of the famous Chinese traditional medicine C. sinensis (Berk.) Sacc. is H. sinensis (Liu et al., 1989). Some valuable bioactive compounds have been recently discovered from members of Hirsutella. A protein toxic to insects, hirsutelln 'Corresponding author; Zongqi Liang

2 350 A (HtA), has been purified from the fungal mite pathogen, H. thompsonii F. E. Fisher (Mazet & Vey, 1995). HtA is considered to be one of many ribonuclease inactivator proteins (RIPs) (Maimala et al. 2002). Vongvanich et al.(2002) found hirsutellde A from a new species of Hirsutella, an interesting antimycobacterial cyclohexadepsipeptide. The synthesis of its key precursor has been studied (Xu et al. 2005). From 1989 to 2001, Chinese mycologists reported seven new species of Hirsutella (Table 1). The present paper describes three additional new species of Hirsutella and their taxonomic position within the genus. Table 1. Previously reported species of Hirsutella in China Fungal Name H. leizhouensis H.M. Fang & S.M. Tan H. changbeishanensis Z.Q. Liang H. polycolluta Z.Q. Liang H. yunnanensis Z.Q. Liang & A.Y. Liu H. yunnanensis var. tenuisynnemata Z.Q. Liang & A.Y. Liu H. sinensis X.T. Liu et ai. H. longissima C.R. Li et ai. H. huangshanensis C.R. Li et ai. References Fang & Tan 1992 Liu et ai., 1993 Liang et ai.,1997 Liu et ai., 1989 Li et ai., 1999 Li et al I i ~ i I i Materials and Methods All collected specimens were routinely oven -dried at 40C to prevent growth of contaminant fungi. For examination by light microscopy, slide preparations were made of a snippet of outer layer tissue from the delaminated synnemata by mounting in lactophenol and cotton blue. ~I J I ~~ Description of new species Fig. 1 Synneim Hirsutellazhangjiajiensis Z.Q. Liang & A.Y. Liu sp. novo Figs. 1-1,4 Stromatibus solitariis vel binariis, cylindraceis, simplicibus, 100 x 2 mm. Stipite et capitulo brunneo vel ochraceo. Phialides e ascosporis exorentibus, subulatiae graciles, x flm, vel basi inflata ellipsoida, 4.5 x 3f1m. Conidia lanceolata or leviter curvata, (-10) x flm, mucigeri, lemoniformes, 10 x 4 flm. Holotypus: GZDXIFR Sti:oma single or 2, cylindrical, 100 x 2 mm, not ramified, leathery. Stipe and fertile cycle part brown to snuff-colored. Conidiogenous structure deriving from the micro conidiation of ascospores from Cordyceps zhangjiajiensis. Phialides slender awl-shaped, x m or inflated ellpsoidal at basal portion, 4.5 x 3.m. Conidia lanceolate an orange segment, (10) x m, embedded in a mucous or the shape of sheath, limoniform, lox 4.m. Specimen studied: GZDXIFR was collected from Zhangjiajie Nature Reserve, Hunan Provjnce by LIANG Zongqi, LIU Aiying et ai. in VII structures and H. uinta. 1-3, Habitat: Pup: Teleomorph: Distribution Hirsutella 1 Syiinem. nigro-bri cilu11 0 x t Synnemata j dark brown,

3 351 iompsonii F. E. Fisher onuclease inactivator id hirsutellde A from iexadepsipeptide. The, species of Hirsutella cies of Hirsutella and J 21 ~ ~~ References Fang & Tan 1992 Liu et ai., 1993 Liang et ai., Liu et ai., 1989 Li et ai., 1999 Li et al l~ = ) prevent growth of ~parations were made nata by mounting in Figs. 1-1,4 ~. Stipite et capitulo ciles, x ter curvata, ~DXIFR iery. Stipe and fertile from the micro cycle :s slender awl-shaped, m. Conidia lanceolate ibedded in a mucous Ie Nature Reserve, 1= I Fig. 1 Synnemata and conidiogenous structures of three species of Hirsutella. 1-1, 4: Conidiogenous structures and synnemata of H. zhangjiajiensis. 1-2, 5: Conidiogenous structures and synnemata of H. crinta. 1-3,6: Conidiogenous structures and synnemata of H. hunanensis l~ I~ I~ I:: Bars1, 2 =lofi, Bar3 =5f1m Habitat: Pupa of Lepidoptera. Teleomorph: Cordyceps zhangjiajiensis Z.Q. Liang & A.Y. Liu (Liang et al. 2002). Distribution-Zhangjiajie Nature Reserve: Hunan Province, China. Hirsutella hunanensis Z.Q. Liang sp. novo Figs. 1-3,6 Synnemata erecta, filiformes, simplices, 5-55 mm longa, 1 mm crassa, flexiles velligneae, nigro-brunneae. Phialides x p.m, e basi inflate cylindrica vel pyriformibus in cilum p.m crassum angustatae, prolifcis. Conidia ellipsoidea velleviter curvata, 6 x p.m, mucigeri, ellipsoideis, 6 x 4.5 p.m. Holotypus: GZDXIFR Synnemata filiform, erect, unbranched, 5-55 mm long, Imm wide, flexible to ligneous, dark brown, arising from between head and thorax parts of host insect. Phialides solitary

4 352 or crowded along synnemata, mostly with cylindrical or pyriform inflated basal portion, x flm and with one or 2 slender, thin necks, x fl. Conidia long ellpsoidal to the shape of an orange segment, 6 x flm; embedded in a mucous sheath, ellpsoidal, 6 x 4.5 flm. Teleomorph not observed. Specimen studied: GZDXIFR was collected from Zhangjiajie Nature Reserve, Hunan Province by LIANG Zongqi, LIU Aiying et al. in VII Habitat: A larva of Lepidoptera. Distribution: Zhangjiajie Nature Reserve-Hunan Province, China. Hirsutella crinita Z.Q. Liang sp. novo Figs. 1-2, 5 Synnemata erecta, filiformes vel cylindrica, simplices, 170 mm longa, mm crassa, flexiles velligneae, nigro-brunneae, caespitosa. Phialides 4.5-6(-10) x ,m, e basi inflata globosa vel cylindrical in collum ,m crassum angustatae, prolifcis. Conidia rara, clavata angusta, 6-9 x 1-1.5,m, exmuca. Holotypus: GZDXIFR Synnemata slender cylindrical or filiform, unbranched, flexible to ligneous, dark brown, caespitose, arising from the head and thorax parts of host insect, very long (up to 170 mm), slightly swollen in the basal region, mm wide, in middle part to upside, mm wide. Phialides somewhat scattered, forming a loose hymenium, arising as lateral cells from the outer hyphae of the synnemata, hemispheric to globose or cylindrical, inflated at basal portion, 4.5-6( - 10) x flm, abruptly narrowing into a short thin neck, flm long and flm wide, always proliferating 2-4, sometimes forming inflated, sterile cylindrical hyphae, 15 x flm. Conidia infrequent, narrowly anisomerous obclavate, 6-9 x fl, absence of distinct mucus layer. Teleomorph not observed. Specimen studied: GZDXIFR was collected from Wuyishan, Hujian Province by LIANG Zongqi, LIU Aiying et al. in V Habitat: A larva of Lepidoptera x mm. Distribution: Wuyishan, Fujian Province, China. Discussion Evans & Samson (1982a,b, 1984) reported several species with phialides of two tyes (Table 2): A-phialides, which are lateral and compacted in a layer below the head, and B-phialides, which are terminal, compact, and awl-shaped. The new species, H. zhangjiajiensis, forms both A- and B-phialides during the ascosporic micro cycle conidiation (Fig. 1-1) and produces B- phialides that are solitary and compacted. Species in the genus Hirsutella that have awl-shaped phialides more than 40 flm long include H. aphidis, H. stilbelliformis var. stilbelliformis, H. stilbelliformis var. dolichoderi, H. sp'àrodochialis, H. darwinii, H. guignardii, H. sinensis, and H. zhangjiajiensis. Among them, species that ~ave both A- and B-phialides and some phialides greater than 40flm long are H. sporodochialis, H. stilbelliformis var. stilbelliformis, H. stilbelliformis var. dolichoderi and H. zhangjiajiensis (Table 2). Possession of rough-walled hyphae and echinate phialides separates the three former species from H. zhangjiajiensis. In the genus Hirsutella, some species that have proliferating phialides are H. besseyi, H. guyana, H. versicola, H. verticilioides, and H. yunnanensis var. tenuisynnemata. All Table 2. A cor H. tipliidis Pclch Species H. ljcssc)'i F.E. Fisher *H.uiiiiln H. iìi7riviliii H.C. Evans &: H. glligllirdii ( Samson et al. H.gli)'llIil '-linter & B.L. --H.1i1l111lt'sis H.liilllltSotcIiSi. S.Y. Chen et al H. lltcl1trix ivlinter et al. H. siiieisis lìu et al., H. sporodocliiali H.C. Evans & H. stilbelliformi: H.C. Ev:ins & H. stilbellifon doliclioderi H.C. Evans & H. versicoa Petch H. verticilloide; Charles H. yiiiilmien teii u is)''l1e11 Z.Q. Liang & *H. dimigjinjiei

5 I basal portion, Table 2. A comparison of three new Hirsutella with related species 8.. Conidia Phialides ed in a mucous Length Species Shape inflm Type Conidia (11m) Reference (max.) ire Reserve, H. aphidis Cymbiform Awl ;.40 B Petch 9x Petch 1942 H. besseyi Awl or ;.40 B Ellipsoid or Minter & Brady EE. Fisher cylindrical limoniform x *H. crinita Base inflated: ;.10 A Narrowly clavate, This work Figs. 1-2,5 sub globose 6-9 x mm crassaj H. darwinii Awl ;.40 B Fusiform Evans & Samson 5 p.m, e basi H.C. Evans & Samson x1.S a icis. Conidia H. guignardii (Maheu) Awl ;.40 B Ellipsoid or fusiform Samson et al. 1. Samson et al. 7-13x US, dark brown, H. guyana Base inflated: 0.0 A Ellpsoid or an Minter & Brady long (up to 170 Minter & B.L. Brady cylindric orange segment x3-7 part to upside, *H. hunanensis Base inflated: ;.10 A Ellipsoid or an This work 1enium, arising cylindric to orange segment c to globose or pyriform 6 x iarrowing into a H. minnesotensis Base inflated: ;.10 A Globose Chen et a! S.Y. Chen et al. subglobose 4-6 (in length),2-4, sometimes quent, narrowly H. necatrix Base inflated: ;.5 A& Ovoid or ellipsoid Minter et al releomorph not Minter et al. subglobose ;.10 B 3-4x2.5-3 & awl Globose, -3.5 l Province by H. sinensis Awl ;.40 B Reniform or Liu et ai., 1989 LiuetaJ. ellpsoid x3.2-S H. sporodochialis Base inflated: ;.80 Ato Fusiform Evans & Samson H.C. Evans & Samson flask-shaped B lo-27x or awl H. stilbelliformis Base inflated: ;.10 A& Clavate 7-9x1.S-2.5 Evans & Samson, H.C. Evans & Samson ellpsoid ;.100 B Ovoid 8-12x4-S 1982b echinate & ies of two tyes awl Delow the head, H. stilbelliformis var. Base inflated: ;.40 A& Cylindrical to ovoid Evans & Samson, dolichoderi ellpsoid ;.100 B x b 1e new species, H.C. Evans & Samson echinate & 10riC miclocycle awl npacted. Species H. versicola Base inflated: ;.40 A Fusiform Minter & Brady Petch ellpsoid 4-7x m long include H. 'verticilloides Base inflated: ;.40 A Ellipsoid or an Minter & Brady dolichoderi, H. Charles cylindrical orange segment x3-5 ajiensis. Among 'eater than 40iim H. yunnanensis var. Base inflated: ;.10 A Cylindrical or Liang et al.,1997 tt;nuisynnemata ellpsoid or obclavate tilbelliformis var. Z.Q. Liang & A.Y. Liu subglobose x Ued hyphae and *H. zhangjiajiensis Awl ;.40 A& Lanceolate or an This work 'iensis. & base B orange segment, inflated: x es are H. besseyi, ellipsoid uisynnemata. All

6 354 of these species are similar to the new species H. hunanensis in fusiform, ellipsoidal and orange conidia. H. hunanensis can be distinguished from the above-mentioned species by phialides less than 10flm length and smaller conidia, around 6 x flm. Minter et al. (1983) described a new species, H. necatrix, that also has phialides with a subglobose or globose swollen base portion. Possession of ovate or ellipsoidal separates this fungus from H. crinita, which has narrowly clavate conidia (Table 2). The new species H. crinita is also closely related to H. minnesotensis and H. yunnanensis var. tenuisynnemata with similar phialides containing a subglobose basal portion. Globose conidia tyically found in H. minnesotensis distinguish it from H. crinita. H. yunnanensis var. tenuisynnemata has fusiform or ellpsoidal conidia and can produce yellow caespitose synnemata. Acknowledgments This project was supported by the National Natural Science Foundation of China. We are grateful to Drs. Shengjun Chang and Jielin Liu for their comments on the manuscript. And we really appreciate editorial revisions. References Cayrol JC, Frankowski JP Influence of the number of parasitizing conidia of Hirsutella rhossiliensis on the mortality of Ditylenchus dipsaci. Revue Nématol. 9(4): Cayrol JC, Castet R, Samson RA Comparative activity of different Hirsutella species towards three plant parasitic nematodes. Revue Nématol. 9(4): Chen SY, Liu XZ, Chen FJ Hirsutella minnesotensis sp. nov., a new pathogen of the soybean cyst nematode. Mycologia 92(5): Evans HC, Samson RA. 1982a. Entomogenous fungi from the Galápagos Islands. Can. J. Bot. 60: Evans HC, Samson RA. 1982b. Cordyceps species and their anamorphs pathogenic on ants (Formicidae) in tropical forest ecosystems II. The Camponotus (Formicinae) complex. Trans. Br. Mycol. Soc. 82 (1): Evans HC, Samson RA Cordyceps species and their anamorphs pathogenic on ants (Formicidae) in tropical forest ecosystems 1. The Cephalotes (Myrmicinae) complex. Trans. Br. Mycol. Soc. 79(3): Evans He Natural control of arthropods with special reference to ants by fungi in the tropical high forest of Ghana. J. Appl. Ecology (11): Evans He Entomogenous fungi in tropical forest ecosystem: An appraisal. Ecological Entomology (7) Fang HM, Tan SM A new species of Hirsutella, H. leizhouensis Fang & Tan. Acta Mycologica Si~ica 11(1): 28-3L. Jafee BA, Zehr EL Parasitism pf the nematode Criconemella xenoplax by the fungus Hirsutella rhossiliensis. PhytopatliOlogy 72: L. Li CR, Huang B, Fan MZ, Li ZZ Cordyceps longissima and its Hirsutella anamorph. Proceedings of 4th Sino-Korean Joint Symposium on Mycology. J. Anhui Agri. Univ. 26(3): Li CR, Xia CR, Lin YR, Fan MZ & Li ZZ Hirsutella huangshanensis sp.nov., the anamorph of Cordyceps formosana and its infection on Tenebrio molitor Mycosystema 24(3): Liang ZQ. 1990a. taxonomy. Jou Liang ZQ. 1990b. ( Pat. Southwest Liang ZQ. 1991a. JournalofGui Liang ZQ b. Journal of Agr Liang ZQ, Liu AY Natural Resen Liang ZQ, Liu AY, in Guizhou II. Liu AY, Liang ZQ, area. Southwes Liu XI, Guo YL, Y Cordyceps sinei Maimala S, Tartar from HirsLllelh Mazet 1 & Vey A. Microbiology I Minter OW; Brady Br. Mycol. Soc. Petch T Noti Samson RA, Rom troglobiotic en Seifert KA & Bout 96(4): Vongvanich N, Ki TIiebtaranonth the entomopa~ Xu YI, Ouan XM, precursor of H

7 355 ellpsoidal and itioned species, flm. i has phialides ; or ellpsoidal l (Table 2). The f. yunnanensis basal portion. l H. crinita. H. d can produce l. We are grateful t. And we really idia of Hirsutella a species towards ~n of the soybean s. Can. J. Bot. 60: :hogenic on ants ) complex. Trans. hogenic on ants implex. Trans. Br. Liang ZQ. 1990a. Classification of the genus Hirsutella Pat. i. Advance and the characteristics of taxonomy. Journal of Guizhou Agricultural College 9 (1): Liang ZQ. 1990b. Classification of the genus Hirsutella Pat. II. Key to the taxa of the genus Hirsutella Pat. Southwest China Journal of Agricultural Sciences 3 (3): China. Liang ZQ. 1991a. Several entomopathogenic new species and records of Stilbelaceae in Journal of Guizhou Agricultural College 10 (1): Liang ZQ. 1991b. Two new entomopathogenic fungi of the genus Hirsutella. Southwest China Journal of Agricultural Sciences 4 (1): Liang ZQ, Liu AY & Huang JZ Some entomogenous fungi from Wuyishan and Zhangjiajie Natural Reserves i. Cordyceps spp. Mycosystema 21(2): Liang ZQ, Liu AY, Huang JZ & Jiao yc The genus and its alles from Kuankuoshui Preserve in Guizhou II. Mycosystema 16 (1): Liu AY, Liang ZQ, Cao L & Yang HM A few new and rare entomogenous fungi in Kunming area. Southwest China Journal of Agricultural Sciences 6 (1): 87-90, Liu XJ, Guo YL, Yu YX & Zeng W Isolation and identification of the anamorphic state of Cordyceps sinensis (Berk.) Sacco Acta Mycologica Sinica 8(1): Maimala S, Tartar A, Boucias D & Chandrapatyab A Detection of the toxi Hirsutellin A from Hirsutella thompsonii. Journal of Invertebrate Pathology 80: Mazet 1& Vey A Hirsutelln A, a toxic protein produced in vitro by Hirsutella thompsonii. Microbiology 141: Minter DW, Brady BL. & Hall RA Five hyphomycetes isolated from eriophyid mites. Trans. Br. Mycol. Soc. 81(3): Petch T Notes on entomogenous fungi. Trans. Br. Mycol. Soc. 25: Samson RA, Rombach MC & Seifert K Hirsutella guignardii and Stilbella kervilei, two troglobiotic entomogenous hyphomycetes. Persoonia 12(2): Seifert KA & Boulay H Hirsutella uncinata, a new hyphomycete from Australia. Mycologia 96(4).: Vongvanich N, Kittakoop P, Iaska M, Trakulnaleamsai S, Vimuttipong S, Tanticharoen M & Thebtaranonth y Hirsutellide A, a new antimycobacterial cyclohexadepsi peptide from the entomopathogenic fungus Hirsutella kobayasii. J. Nat. Prod. 65(9): XU YJ, Duan XM, Li ML, Jiang LQ, Zhao GL, Meng Y & Chen LG Synthesis of the key precursor of Hirsutellde A. Molecules 10: ngi in the tropical,raisal. Ecological. Acta Mycologica ~ fungus Hirsutella ;utella anamorph. Agri. Univ. 26(3): " the anamorph of 3):