Cryptogamie, Mycologie, 2006, 27 (1): 11-20 © 2006 Adac. Tous droits réservés Echinosphaeria macrospora sp. nov., teleomorph of Vermiculariopsiella endophytica sp. nov. GAWAS PUJA1#, B.D. SHENOY 2, K.D. HYDE 2 & D.J. BHAT 1* 1 Department of Botany, Goa University, Goa-403 206, India. 2 Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, The University of Hong Kong, Pokfulam Road, Hong Kong SAR Abstract – Echinosphaeria macrospora is a novel endophyte isolated from stems of Centella asiatica (Apiaceae/Umbelliferae) with its novel, hyphomycetous anamorph, Vermiculariopsiella endophytica. The fungus first produced the conidial state, followed by development of its teleomorph after 4 weeks of incubation. Echinosphaeria macrospora differs from the type of this monotypic genus, E. canescens, in having wider asci and larger ascospores. This is the first report of a sexual state amongst the species of Vermiculariopsiella and the third asexual stage of Echinosphaeria. Anamorph-teleomorph connection / perithecium / Helminthosphaeriaceae / sporodochia / Western Ghats INTRODUCTION Fungi are pleomorphic, i.e., they are capable of producing more than one form or type of spore in their life cycle (Sugiyama, 1987; Cai et al. 2005; Fernández & Huhndorf, 2004, 2005; Huhndorf & Fernández, 2005). The complete lifecycle of many fungi is poorly understood and therefore anamorph-teleomorph connections as and when established, attain significance. One such anamorphic genus with hitherto unknown teleomorph is Vermiculariopsiella Bender (Bender, 1932). During studies on biodiversity of microfungi of the Western Ghat forests in Goa, India (Pratibha et al., 2005; Shenoy et al., 2005), we isolated a novel species of hyphomycetous, endophytic fungus, Vermiculariopsiella endophytica, from living stems of Centella asiatica. The fungus in culture produced its sporodochial conidial state in 4 days. After one month of incubation at 23-25 °C, perithecia developed in small groups on tiny, inconspicuous, stromatic base amongst the sporodochia. The ascocarp, asci and ascospores of the teleomorph were similar to Echinosphaeria A.N. Mill. & Huhndorf (Miller & Huhndorf, 2004). The anamorph and teleomorph distinctly differ from hitherto known species in respective genera and therefore are described as novel taxa, in this paper. This is the first report of sexual state amongst the species of Vermiculariopsiella and the third asexual stage of Echinosphaeria. * Corresponding author: e-mail < bhatdj@rediffmail.com> # e-mail: < pujabg@yahoo.co.in> 12 Gawas Puja, B.D. Shenoy, K.D. Hyde & D.J. Bhat MATERIALS AND METHODS Isolation of the fungus from host tissue Fresh stem and leaves of Centella asiatica were processed for isolation of endophytic fungi following the procedure described by Petrini & Fisher (1986). The surface sterilized stem and leaf tissues were cut into pieces of 0.5 cm2, plated in 2% malt extract agar (MEA) medium and incubated for 7-14 days at 25°C. Fungal mycelium emerging out of cut ends of the tissue was aseptically transferred onto fresh MEA plates. The plates were incubated for over 2 months or until the fungus produced both it anamorphic and teleomorphic forms in the medium. Confirmation of anamorph-teleomorph connection The perithecium developed in culture was transferred onto a flamesterilized slide and carefully dissected in a drop of sterile distilled water to separate individual ascospores. The ascosporic suspension when spread on a 2% MEA plate, germinated readily. Germinated ascospores were individually transferred into slants and incubated at 25°C until sporulation effected. The anamorph developed in culture was in conformity with Vermiculariopsiella endophytica. TAXONOMY Echinosphaeria macrospora Puja, Bhat & K.D. Hyde sp. nov. (Figs 1-9) Ascocarpis peritheciis, pyriformis, gregariis, nigris, velvetis, aggregatis, cupulatis exaresco, 410-490 µm longis, 150-265 µm latit ad medius ora; oriundus brevis stromatic pessum. Ostiolis brevis, conicus, cum centralis apicalis. Peridiumii pseudoparenchymati, duo-layeri, cum angulari, leviter tenuibis cellulae. Extrenus layera atrum brunnea, cum 5-7 rows arto, pariter, profundus, angularis cellulae, 3-7-µm diametro. Penitus layera hyalinis vel subhyalinis, cum 4-6 rows arto, substrictus, parietibus tenuibus cellulae. Paraphyses absens. Asci oriundus penitus peridium pessum cellulae, octospori, clavati, unitunicati, pedicillati, 120-165 × 1417.5 µm; leviter substricti ad apice, iodo noncoerulescenti provisi, cum emineo apice orbis. Ascosporae 41-45 × 6-11 µm allantoideae vel vermiformae, hyalinae vel subhyalinae, eseptatae, guttulatae, laevia, biseriatae. Etymology: Larger size of the ascospores as compared to the type. Ascomata perithecial, pyriform, 410-490 µm high, 150-265 µm wide at the middle broadest region, gregarious, often growing in groups of 2-8 on a small stromatic base, black, velvety, cupulate when dry, with short, conical, centrally located apical ostiole. Peridium pseudoparenchymatous, 2-layered, composed of angular, slightly flattened cells. Outer layer dark brown, with 5-7 row of compactly laid, uniformly thickened, angular cells 3-7 µm diam. Inner layer hyaline to subhyaline, with 4-6 rows of closely packed, narrow, thin-walled cells. Paraphyses not observed. Asci 120-165 × 14-17.5 µm (mean = 150 × 16 µm), arising from the basal cells of inner peridium, 8-spored, clavate, unitunicate, pedicillate, slightly narrower at the tip, nonamyloid, with conspicuous apical ring. Ascospores 41-45 × 6-11 µm (mean = 43 × 8 µm) allantoid to vermiform, hyaline to subhyaline, aseptate, guttulate, smooth-walled, biseriately arranged in the asci. Echinosphaeria macrospora sp. nov. 13 Figs 1-8. Echinosphaeria macrospora 1. Ascocarp with attached anamorph (arrowed). 2. Asci. 3. Vertical section through ascoma. 4. Peridium. 5. Immature ascus with conspicuous apical ring (arrowed). 6. Ascus with biseriately arranged ascospores. 7, 8. Ascospores. 14 Gawas Puja, B.D. Shenoy, K.D. Hyde & D.J. Bhat Fig 9. Echinosphaeria macrospora. Ascocarp, asci and ascospores. Echinosphaeria macrospora sp. nov. 15 Anamorph – Vermiculariopsiella endophytica Puja, Bhat & K.D. Hyde sp. nov. Habitat – Centella asiatica. Known distribution – India Holotype: INDIA, Western Ghats, Goa, Colem, endophyte in stems of Centella asiatica, 24 January 2005, Puja Gawas, Dried culture mat, GUBH (Goa University Botany Herbarium) No. CaEnC-3. Vermiculariopsiella endophytica Puja, Bhat, K.D. Hyde sp. nov. (Figs 10-15) Coloniae in vitro aliquanta vel celer proventa, pervenio 5.5-6 cm diametro in 7 dies, platy, cum irregulari vel rhizoidali labrum, pallens-albo vel pallide brunnea, inverto palide brunnea. Sporodochia oriundus brevis stromatic pessum, sparsa, cream vel peach-coloris, setosae. Conidiophora laevia, septata, parum tremes, hyalinis vel palide-coloris, 75-85 × 6-9 µm. Setae 3-15, laevia, 2-6-septata, haud-tremes, erecta vel leviter curvata ad pessum, atrum brunnea, acuminata ad apicem, 180318 µm longis, 10-11 µm latit ad pessum, 6-7 µm latit ad medius; oriundus parietibus crassi, brunnea 5-7 µm diametro stromal cellulae. Cellulae conidiogenae monophialideae, integratae vel discretae, sine emineo collarettae, 22-25 × 10-11 µm. Conidia solitaria, cylindrica, teres ad duo extremitas, laevia, eseptata, hyalina, 32-42 × 10-11 µm, una peach-coloris. Etymology – Refers to endophytic nature. Colonies moderate to fast growing in culture, attaining diam of 5.5-6 cm in 7 days, flat, with irregular to rhizoidal margin, off-white to pale brown, reverse pale brown. Sporodochia develop on small stromatic base, scattered, cream to peachcoloured, setose, with smooth, septate, sparsely branched, hyaline to pale-coloured 75-85 × 6-9 µm conidiophores; setae 3-5, smooth, 2-6-septate, unbranched, straight to slightly curved at base, dark brown, pointed at the tip, 180-318 µm long, 10-11 µm wide (mean = 240 × 10 µm) at base, 6-7 µm wide at the center; arising from basal thick-walled, brown 5-7 µm diam stromal cells. Conidiogenous cells monophialidic, integrated to discrete, 22-25 × 10-11 µm, without a conspicuous collarette. Conidia solitary, cylindrical, rounded at both ends, smooth, aseptate, hyaline, 32-42 × 1011 µm (mean = 36 × 10.5 µm), in mass peach-coloured. Habitat – Centella asiatica. Known distribution – India Holotype: INDIA, Western Ghats, Goa, Colem, endophyte in stems of Centella asiatica, 24 January 2005, Puja Gawas, Dried culture mat, GUBH No. CaEnC-3. DISCUSSION The phylogenetic analyses of partial nuclear large subunit (LSU) rDNA sequences have shown the “Lasiosphaeria-complex” to be highly polyphyletic in that species segregated into seven monophyletic clades dispersed among several orders (Miller & Huhndorf, 2004). Consequently, the generic circumscription of Lasiosphaeria has been narrowed, with an addition of three novel genera, Echinosphaeria A.N. Mill. & Huhndorf, Hiberina A.N. Mill. & Huhndorf and Immersiella (Lasiosphaeriaceae) A.N. Mill. & Huhndorf. Echinosphaeria has phylogenetic affinities with the members of family Helminthosphaeriaceae (Miller & Huhndorf, 2004). The monotypic ascomycetous genus, Echinosphaeria is typified by E. canescens (Pers: Fr.) A.N. Mill. & Huhndorf. The type species is a basionym of Lasiosphaeria canescens (Pers.) Karst. Mycoth. fenn. (Helsinki) 2: 162, 1873 16 Gawas Puja, B.D. Shenoy, K.D. Hyde & D.J. Bhat Figs 10-14. Vermiculariopsiella endophytica. 10. Stereo-microscopic image with V. endophytica (black arrowed) and E. macrospora (white arrowed). 11, 12. Sporodochial conidiomata with setae and conidiophores. 13. Conidia 14. Phialidic conidiogenous cells Echinosphaeria macrospora sp. nov. 17 Fig 15. Vermiculariopsiella endophytica. Sporodochial conidiomata with setae, conidiophores, conidiogenous cells and conidia. Species Setae Conidiophores Conidiogenous cells 18 Table 1. Distinguishing features of Vermiculariopsiella species described so far. Conidia Ref. Aseptate, fusiform, 15-19.5 µm long 5 Cylindrical, curved near acuminate apex 4 Data not available 4 Branched Monophialidic, subcylindric to lageniform with recurved cylindric neck recurved with a flared collarette Monophialidic, no conspicuous collarette Aseptate, 20-27 µm long 3 Unbranched Rarely branched Monophialidic, cylindrical with distinct collarette 4 V. immersa (Desm.) Bender. Unbranched Rarely branched V. indica Keshavaprasad, D’souza & Bhat V. parva Keshavaprasad, D’souza & Bhat V. parvula Nawawi, Kuthub. & Sutton Unbranched Branched Monophialidic, subcylindric to lageniform with recurved cylindric neck recurved with a flared collarette Monophialidic, no conspicuous collarette 3-septate, guttulate, falcate with pointed and curved apex, truncate to rounded base 36-47 µm long Aseptate, guttulate, cylindrical with pointed and curved apex, base obtuse to rounded 13-23 µm long. Aseptate, cylindrical 12-15 µm long 3 Unbranched Branched Monophialidic, no conspicuous collarette Aseptate, cylindrical 22-30 µm long 3 Unbranched Branched Monophialidic, subcylindric to lageniform, flared collarette 4 V. ramosa (Sutton) Nawawi, Kuthub. & Sutton V. spiralis Crous, Wingf. & B. Kendr. V. endophytica Puja, Bhat & K.D. Hyde Once dichotomously branched Rarely branched Monophialidic, subcylindric to lageniform with recurved cylindric neck recurved with a flared collarette Monophialidic, subcylindric to lageniform, with recurved ends, collarette Monophialidic, inconspicuous collarette, sub cylindric Aseptate, guttulate, cylindrical with apex slightly curved and pointed, base rounded to obtuse 8-13 µm long Data not available Unbranched Branched Thrice dichotomously Unbranched branched Branched with primary and secondary branches Unbranched Unbranched spirally twisted Unbranched Rarely branched Rarely branched 1 4 Aseptate, cylindric apex curved and pointed, 2 base obtuse rounded 15-19 µm long Aseptate, cylindrical, 32-42 µm long and Present 10-13 µm wide study 1: Bender (1992), 2: Crous et al. (1995), 3: Keshavaprasad et al. (2003), 4: Nawawi & Kuthubutheen (1990), 5: Pasqualetti & Zucconi (1992). Gawas Puja, B.D. Shenoy, K.D. Hyde & D.J. Bhat Mono- to polyphialidic, lageniform with flared collarette Polyphialidic, obclavate to cylindrical V. arcicula Pasqual. & Zucconi V. cornuta (Rao & de Hoog) Nawawi, Kuthub. & Sutton V. cubensis (Castañeda) Nawawi, Kuthub. & Sutton V. elegans Keshavaprasad, D’souza & Bhat V. falcata Nawawi, Kuthub. & Sutton Echinosphaeria macrospora sp. nov. 19 Table 2. Distinguishing features of known species of Echinosphaeria Species Ascocarp Ascus Ascospore Refs. E. canescens (Pers: Fr.) Mill. & Huhndorf Sub-globose to ovoid Cylindric-clavate, 10-12 µm wide Uniseptate, 20-28 × 4-5 µm wide 1, 2 E. macrospora Puja, Bhat & K.D. Hyde Pyriform Clavate, 120-165 µm long Aseptate, 41-45 µm long and 6-11 µm wide Present study 1: Saccardo (1883); 2: Miller & Huhndorf (2004) (= Sphaeria canescens Pers., Syn. Meth. Fung.: 72, 1801). The genus is characterised by perithecial ascomata with 8-spored, unitunicate, nonamyloid asci containing allantoid, guttulate, hyaline, smooth-walled, biseriately arranged ascospores (Saccardo, 1883; Miller & Huhndorf, 2004). Echinosphaeria canescens was previously reported to have Endophragmiella anamorph and a Selenosporella-like synanamorph (Hughes, 1979; Sivanesan, 1983) and in this study E. macrospora was found to have a Vermiculariopsiella anamorph. Echinosphaeria macrospora is typical of the genus in having carbonaceous, shining, soft ascomata, unitunicate, nonamyloid, 8-spored asci and hyaline, allantoid ascospores. Echinosphaeria macrospora differs from E. canescens in having wider asci (14-17.5 µm vs. 10-12 µm) and greatly larger ascospores (41-45 × 6-11 µm vs. 20-28 × 4-5 µm) (Tab. 2). The length of asci was not indicated in the description of the type species [= Lasiosphaeria canescens (Pers.) Karst.] and hence could not be considered for comparison (Saccardo, 1883; Miller & Huhndorf, 2004). Vermiculariopsiella, typified by V. immersa (Desm.) Bender (Bender, 1932) is characterised by setose sporodochia, with hyaline, non-septate conidia produced in slimy mass on compact columns of cylindrical to obclavate phialidic conidiogenous cells. Recently, three new species have been added to the genus from India by Keshavaprasad et al. (2003), who also provided a key to the existing species. The taxa within the genus differ in organization of sporodochia, shape and size of setae, branching of conidiophores and phialides and, shape and size of conidia. An important, notable taxonomic rearrangement associated with Vermiculariopsiella is segregation of two species, V. microsperma Castañeda & Kendrick and V. ludoviciana Castañeda, Cano & Guarro (Pirozynski, 1962; Kirk & Sutton, 1985; Arambarri & Cabello, 1989; Castañeda & Kendrick, 1992; Pasqualetti & Zucconi, 1992; Arambarii et al., 1997; Castañeda et al. 1997; Index Fungorum 2005) from the genus. All recognized species of the genus are listed and compared in Table 1. Amongst the species described in the genus Vermiculariopsiella (Tab. 1), V. endophytica is close to V. falcata only in conidial dimension. The conidia are 36-47 µm long in V. falcata and 31-36 µm long in V. endophytica. However, conspicuous phialidic collarettes and 3-septate, falcate conidia of V. falcata are not present in V. endophytica. Though the shape and architecture of V. parva, V. elegans and V. indica are similar to V. endophytica, the conidia in the latter differ markedly in size. 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