Mycologia, 98(4), 2006, pp. 616–627. # 2006 by The Mycological Society of America, Lawrence, KS 66044-8897 Studies on Morinia: Recognition of Morinia longiappendiculata sp. nov. as a new endophytic fungus, and a new circumscription of Morinia pestalozzioides Javier Collado1 Gonzalo Platas Gerald F. Bills Ángela Basilio Francisca Vicente J. Rubén Tormo Pilar Hernández M. Teresa Dı́ez Fernando Peláez INTRODUCTION The monotypic genus Morinia Berl. & Bres. was based on Morinia pestalozzioides Berl. & Bres. and encompassed an anamorphic fungus forming appendagebearing and muriform conidia in acervular conidiomata. The fungus was found on dried stems of Artemisia camphorata Vill. (Berlese and Bresadola 1889). Passerini described the fungus as Pestalotia artemisiae Pass. from a later collection on the same host and assigned it to his new subgenus Pestalozziana Pass. (Passerini 1891). The synonymy was established in 1892 (Saccardo 1892, 1901; Guba 1961; Nag Raj 1993). Rinomia pestalozzioides (Berl. & Bres.) Nieuwl. subsequently was proposed as the valid name for the fungus by Nieuwland, who considered that the Linnaean genus Morina (Dipsacaceae, Phanerogam) rendered the Morinia Berl. & Bres. a homonym (Nieuwland 1916). Currently, Rinomia is considered as a homotypic synonym of Morinia (Kirk et al 2001). While studying the endophytic fungi in plants in southeastern and central Spain we isolated into culture six strains producing conidia that matched the characteristics of Morinia. Preliminary morphological analysis indicated that one of the isolates differed in micromorphology. High-performance liquid chromatography (HPLC) analysis and DNA sequencing of fragments of actin, chitin synthase and elongation factor 1 (EF1) genes along with the ITS region indicated that the strain’s distinct morphology was consistently separated from the other five, which were almost identical among themselves according to the results of the DNA sequence analyses. Systematic morphological comparison of the strain that showed different HPLC profile and DNA sequences and the only strain out of the remaining five isolates that retained capacity of producing conidia in culture confirmed the differences observed in the molecular study. In addition morphological comparison with an authentic specimen of M. pestalozzioides showed that the strain having different morphological and molecular characteristics was close to M. pestalozzioides. The results supported the recognition of a new species in the genus for the rest of isolates. In this work we describe the new endophytic species of Morinia. The type species M. pestalozzioides is redescribed based on morphology data that were absent in the original description as well as on molecular data. The taxonomic position of genus Morinia is assessed based on ITS sequence analyses. The production of Centro de Investigación Básica (CIBE). Merck Research Laboratories, Merck, Sharp and Dohme de España, Josefa Valcárcel 38 E-28027 Madrid, España Abstract: A new coelomycete, Morinia longiappendiculata sp. nov., isolated from living stems of four plant species in central Spain, is described. The distinctive morphological characteristics of this fungus are the production of conidia with long basal and apical appendages on filiform conidiogenous cells that contrasts with the short-appendaged conidia and cylindrical conidiogenic cells of the type species, M. pestalozzioides. Comparative sequence analysis of the ITS rDNA region and fragments of the translation elongation factor 1a, actin and chitin synthase 1 genes and the study of the HPLC profiles of the M. longiappendiculata and M. pestalozzioides isolates supported the recognition of the new species. Comparison of the ITS rDNA sequences of the Morinia isolates with GenBank sequences indicated that the genus belongs to the Amphisphaeriaceae with the highest similarity to Bartalinia and Truncatella. Bresadola’s original definition of M. pestalozzioides is updated by adding information on conidiogenesis and molecular data. A lectotype and epitype are designated for the species. A study of bioactive metabolites revealed that M. pestalozzioides cultures produced moriniafungin, a novel sordarin analog with potent antifungal activity. Key words: Amphisphaeriaceae, appendaged conidia, Coelomycetes, DNA-sequencing, HPLC, moriniafungin, typification Accepted for publication 21 May 2006. 1 Corresponding author. E-mail: javier_colladomartinez@merck. com 616 COLLADO ET AL: STUDIES ON MORINIA moriniafungin, an antifungal diterpene glycoside, by M. pestalozzioides also is discussed. MATERIALS AND METHODS Plant collections.—These plant species were sampled in three surveys carried out in central and southeastern Spain: Helichrysum stoechas (L.) Moench, Santolina rosmarinifolia L., Thymus mastichina L. (Torrelodones, Madrid, Jun 1995), Sedum sediforme (Jacq.) Pau (Sierra Alhamilla, Almerı́a, Sep 1998), and Calluna vulgaris (L.) Hull. (Riaza, Segovia, Jan 1999). Samples of fresh and apparently healthy stems and leaves were collected and taken to the laboratory where they were processed 24– 48 h after collection. Fungal isolation, strain identification and culture description.—Leaf and stem fragments about 1 cm long were surface-sterilized by successive dipping in ethanol 70%, hypochlorite 4%, ethanol 70% (Collado et al 1996). Isolation plates were prepared with YM (1 g yeast extract, 10 g malt extract, 5 mg streptomycin; 50 mg terramycin, 20 g agar, 1 L distilled water) and YMC (YM + 4 mg cyclosporin A). Plates were incubated at 22 C and 80% relative humidity 2–3 wk. Emerging fungal colonies were transferred individually to potato-dextrose agar (PDA, Difco) purification plates. The agar media used for morphological characterization and culture description were PDA, oatmeal agar (OTM, Difco) and cornmeal agar (CMA, Difco). Purification and characterization plates were incubated 21 d under the same conditions as isolation plates. In addition Morinia isolates were subcultured up to three times on PDA for 21 d to assess the variation in the morphology associated with colony transfer. For morphological analysis microscope slides were prepared from sporulated cultures on PDA and examined with a Leitz Diaplan microscope equipped with differential interference contrast optics. Morphology data were reported as the range of 20 measurements of each character. Morinia strains were compared morphologically with a herbarium specimen of M. pestalozzioides from Artemisia camphorata Vill., U.S. National Fungus Collections, Beltsville, Maryland (BPI 453814). Isolates were preserved in the CIBE Culture Collection as frozen agar plugs in glycerol 10% at 280 C unless noted otherwise. DNA extraction, PCR amplification, DNA sequencing and data analysis.—DNA extraction was performed by the methodology described in Peláez et al (1996). The amplification of both internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of these isolates was performed with primers ITS5 (White et al 1990) and ITS4a (Larena et al 1998). Amplifications of portions of the EF1, actin, and chitin synthase 1 genes were performed respectively with primers EF1-728F/EF1986R, ACT-512F/ACT.783R and CHS-79F/CHS-354R (Carbone and Kohn 1999). PCR reactions were performed according to standard procedures (5 min at 93 C followed by 40 cycles of 30 s at 617 93 C, 30 s at 53 C and 2 min at 72 C) with Taq DNA polymerase (Q-bioGene) following the recommendations of the manufacturer. The amplification products (0.1 mg/mL) were sequenced with the Bigdye Terminators version 1.1 (Applied Biosystems, Foster City, California). For all the amplification products each strand was sequenced with the same primers used for the initial amplification. Sequencing was performed in an ABI PRISM 3700 DNA Analyzer (Applied Biosystems). The partial sequences were assembled manually and a consensus sequence was generated. Sequences were aligned manually. Phylogenetic analysis was performed by maximum parsimony, using the branchand-bound algorithm of PAUP 4.0 (Swofford 1993). The robustness of the branches was assessed by bootstrap analysis (Felsenstein 1985), resampling the data with 1000 bootstrap replicates. To asses the taxonomic position of Morinia, ITS sequences of M. pestalozzioides and M. longiappendiculata isolates were compared to a set of ITS sequences available in GenBank TreeBASE submission numbers of the DNA sequence alignments and GenBank accession numbers of the sequences analyzed in this work are provided (FIGS. 7, 8). HPLC analyses.—Liquid inocula of M. longiappendiculata and M. pestalozzioides isolates were prepared from fresh slants (Peláez et al 1998). Two mL aliquots of inoculum cultures were used to seed 250 mL Erlenmeyer flasks containing 50 mL OP26-NLW production medium (125 g glycerol; 25 g glucose, 20 g pectin; 5 g ardamine PH, 4 g [NH4]2 SO4, 2 g glycine, 4 g KH2PO4, 0.1 g CoCl2?6H2O, 1 L distilled water). The flasks were incubated 21 d in the same conditions as inocula. Ten mL aliquots of this production culture were extracted with 14 mL methyl-ethyl-ketone (MEK) while stirring 1 h at room temperature. The liquid extract was dried under a stream of nitrogen. The residue was reconstituted in 1 mL HPLC-grade methanol to a final concentration of 73 and filtered through a 0.2 mm membrane. Metabolite analysis relied on HPLC reverse-phase gradient chromatography with a diode-array HPLC detector recording simultaneously at 210 and 280 nm (Julian et al 1998). Diode-array HPLC gradient characterizations were performed in a ZORBAX Rx-C8 4.6 3 250 mm column. A 10–100% gradient of acetonitrile in water with a flow-rate gradient of 0.9–1.2 mL/min was programmed in a 1100 HP Agilent ChemStation at 20 C during each 20-min analysis. Trifluoroacetic acid (TFA, 0.01%) was added for pH control (Tormo et al 2003). For analysis of the HPLC chromatograms, a relative 2% cut-off value of the total area was used as peak detection threshold. A neighbor joining dendrogram based on Dice’s similarity coefficients was generated with the BioNumericsTM commercial software (Tormo and Garcı́a 2005). RESULTS Morphological characterization.—Six endophytic Morinia strains were isolated from the stems of plant samples in three surveys (TABLE I). Preliminary 618 TABLE I. MYCOLOGIA Collection and isolation data of Morinia strains Strain identification M. M. M. M. M. M. Strain no. Isolation medium Host F-048003 F-048007 F-048041 F-048079 F-095552 F-090354 YMC YMC YMC YMC YMC YM Santolina rosmarinifolia Santolina rosmarinifolia Helichrysum stoechas Thymus mastichina Calluna vulgaris Sedum sediforme longiappendiculata longiappendiculata longiappendiculata longiappendiculata longiappendiculata pestalozzioides morphological analysis (data not shown) revealed that five of the strains (F-048003, F-048007, F048041, F-048079 and F-095552), isolated from plants in two locations in central Spain, had identical micromorphology and differed from the remaining strain (F-090354) isolated from a plant sample collected in southeastern Spain. Except for F-095552 and F-090354 the isolates lost the capacity of producing conidia in culture after first colony transfer, but when F-095552 was cultured under the same conditions as F-090354 for systematic comparison the differences in morphology were confirmed. Morphological comparison with a voucher specimen of M. pestalozzioides showed that isolate F-090354 coincided with most of the characteristics of M. pestalozzioides (TABLE III) (Basilio et al 2005). Isolate F-095552 and the closely related F-048003, F-048007, F048041 and F-048079 strains consequently were assigned to the new species M. longiappendiculata. The five M. longiappendiculata isolates exhibited a significant degree of variation in colony morphology when they were cultured on PDA. Culture of strains F-048003 and F-048007, both isolated from TABLE II. Strain no. Geographic origin Torrelodones, Madrid Torrelodones, Madrid Torrelodones, Madrid Torrelodones, Madrid Riaza, Segovia Sierra Alhamilla, Almerı́a Santolina rosmarinifolia, resembled each other more than the rest of strains, which were more heterogeneous (TABLE II, FIGS. 1–6). The resemblance of the two strains also was observed when they were cultured on OTM and CMA (data not shown). Furthermore variation in micromorphology was observed in F095552 after successive colony transfers on PDA. After the third transfer the strain produced larger conidia with apical and basal appendages considerably longer than those observed in the original culture (TABLE III). M. pestalozzioides (F-090354) did not exhibit such variation upon subculturing (data not shown). DNA sequences.—The lengths of the amplification products from the six fungal strains were within the range of 538–539 bp for ITS (including ITS1, ITS2 and 5.8S gene), 350–367 bp for EF1a, 248– 291 bp for actin, and 271–282 bp for chitin synthase 1. The nucleotide sequence similarity between M. longiappendiculata isolates (F-095552, F-048003, F-048007, F-048041 and F-048079) was 99–100% for ITS, 100% for EF1a, 100% for actin and 99–100% for chitin synthase 1. The sequence similarity between M. pestalozzioides (F-090354) and Colony characteristics of five M. longiappendiculata strains cultured on PDA after 21 d incubation Diameter (mm) Texture Topography F-095552 32 Glabrous Flat; radially sulcate F-048003 50 Floccose Flat; not sulcate F-048007 48 Floccose Flat; not sulcate F-048041 30 Slightly Flat; radially Floccose sulcate F-048079 40 Slightly Flat; radially Floccose sulcate Color Exudate Mostly in Yellowish brown colony (5E5, 5F5); edge center; yellowish white Hyaline (4A2) Greenish gray (30F2); At colony edge; center and edge hyaline gray (30B1) Greenish gray (30F2); At colony edge white edge; hyaline Grayish orange (6B3); Throughout center brown (6E6); the colony; hyaline edge white Grayish brown (5E3); Throughout center grayish yellow the colony; hyaline (4B3); edge white Diffusible pigment Reverse Edge None Dark blond Irregular (5D4) None Brownish orange (5C5) Brownish orange (5C5) Brown (6E8) None Yellowish brown (5D6) Yellowish brown (5D6) Brown (6E8) Semiregular Regular Irregular Irregular COLLADO ET AL: STUDIES ON MORINIA 619 FIGS. 1–6. Morinia isolates on PDA after 21 d at 22 C. 1. M. longiappendiculata F-048003. 2. M. longiappendiculata F048007. 3. M. longiappendiculata F-048041. 4. M. longiappendiculata F-048079. 5. M. longiappendiculata F-095552. 6. M. pestalozzioides F-090354. M. longiappendiculata isolates was 98% for ITS, 95% for EF1a, 98% for actin and 97% for chitin synthase. Maximum parsimony analysis of the sequences of the combined DNA markers (FIG. 7) indicated that M. pestalozzioides (F-090,354) was genotypically distinct from M. longiappendiculata strains, and the distinction was supported by a high bootstrap value. Two subclusters were observed for M. longiappendiculata, one well supported by bootstrapping, which included strains F-048079 and F-095552, isolated from different hosts and exhibiting different colony morphology, and another with less statistical support that included the macroscopically similar F-048003 and F-048007 strains isolated from the same host species. The comparison of the ITS sequences from Morinia isolates with fungal sequences deposited in GenBank indicated that Morinia strains were closer to species of Truncatella Steyaert and Bartalinia Tassi rather than to other species in the Amphisphaeriaceae, although this relationship is not clearly supported by bootstrapping (FIG. 8). Notably 95% similarity was observed with Bartalinia robillardoides Tassi and 94% with Truncatella angustata (Pers.) S. Hughes. HPLC analyses.—Metabolite profiles were compared among the six Morinia isolates (FIG. 9). M. 620 TABLE III. MYCOLOGIA Comparative morphological data on strains of M. pestalozzioides and M. longiappendiculata M. pestalozzioides (Bresadola’s M. pestalozzioides M. pestalozzioides (BPI 453814) (F-090354) description) Conidia Shape Color Size (mm) Transverse septa Vertical septa Oblique septa Median cells longitude (mm) No. of apical appendages Apical appendages longitude (mm) No. of basal appendages Basal appendages longitude (mm) Conidiogenous cells Shape Branching Size (mm) Septation Color a M. longiappendiculata M. longiappendiculata (F-095552) Subcul(F-095552) tureda Fusiform, ellipsoid, pyriform End cells hyaline, median cells brown 22–24 3 8–10 5 1–3 — — Fusiform, ellipsoid, pyriform End cells hyaline, median cells brown 16–24 3 7–8 5 1–4 0–(1) 14–16 Fusiform, ellipsoid, pyriform End cells hyaline, median cells brown 20–25 3 6–8 (5)–6–7 1–2 1–2 15–17 Fusiform, ellipsoid, pyriform End cells hyaline, median cells brown 25–31 3 9–11 5–6 (1)–2–(3) (1)–2 17–20 Fusiform, ellipsoid, pyriform End cells hyaline, median cells brown 25–42 3 7–10 (5)–6–7 (1)–2-(3) (1)–2 16–26 3 2–3 (2)–3 (2)–3 (2)–3 14–20 13–23 9–11 14–26 28–39 1 1 0–(1)–(2) 1 1 — 5 3 15–25 20–32 — — — — — Cylindrical Yes 8–18 3 2–3 Yes Hyaline Cylindrical Yes 10–19 3 2–3 Yes Hyaline Filiform Yes 23–42 3 2–3 Yes Hyaline Filiform Yes 20–43 3 2–3 Yes Hyaline Data recorded after 3rd colony transfer. pestalozzioides (F-090354) clustered apart from M. longiappendiculata strains (F-048007, F-048041, F048079, F-048003 and F-095552). M. longiappendiculata strains were metabolically heterogeneous. The highest similarity was observed between F048003 and F-048041 strains (88.9%) and F048079 and F-095552 strains (87.5%). F-048007 showed the most distinctive metabolite profile of the five M. longiappendiculata strains. TAXONOMY Morinia longiappendiculata J. Collado et G. Platas, sp. nov. FIGS. 10–12 Hyphae septatae, hyalinae vel galbinae, 5 mm diam. Acervula atra, globosa vel hemiglobosa, semiimmersa, sparsa, texturae angularis. Conidiophora vel simplicia vel in base ramosa. Cellae conidiiferae laeves, hyalinae, filiformes, multiseptatae, 23–42 3 2–3 mm. Conidia muriforma, fusiforma vel ellipsoidea, recta vel subcurvata, laeva, 25–31 3 9– 11 mm, cum quinque vel sex septis transversis et uno-tribus, plerumque duobus, septis longitudinalibus et obliquis, loculo medio brunneo, 17–20 mm longitudinem, cellis basilaribus et apicalibus hyalinis vel subhyalinis; cella basilare conica, cum una apendice simplice, centrale vel laterale, 15–25 mm longitudinem; cella apicale hemisphaerica, cum tribus, parce duobus, apendicibus cellularibus simplicibus, 14–26, rectis vel subcurvatis. Status sexualis absens. Mycelium branched, septate, with hyaline or greenish-yellow hyphae up to 5 mm wide. Conidiomata acervular, dark, globose to subglobose, partially immersed, sparse; wall textura angularis; conidiophores simple or branched at the base; conidiogenous cells smooth, hyaline, filiform, with several septa, 23–42 3 2–3 mm; conidia muriform, fusiform to ellipsoid, straight to slightly curved, smooth, 25–31 3 9–11 mm, with five or six transverse septa and 1–3 (mostly 2) vertical and oblique septa, versicolored, with basal and apical cells hyaline to subhyaline; median cells brown, 17–20 mm long; basal cell conical, with a single appendage, 15–25 mm long, attached either in central or lateral position; apical cell hemispherical, crowned with three (rarely two) COLLADO ET AL: STUDIES ON MORINIA 621 FIG. 7. Relationship of Morinia isolates inferred by maximum parsimony consensus of aligned sequences of a chimerical sequence of 1390 bases including: ITS1-5.8-ITS2 genes, intron-containing fragment of the actin gene, an intron containing fragment of the Elongation factor 1a, and one intron containing fragment of the chitin synthase gen. Statistical support (1000 bootstrap) values . 50% indicated at branch points. Tree parameters: total characters 5 1431, constant characters 5 1115, variable characters parsimony uninformative 5 307, variable characters parsimony informative 5 9, tree length 5 321, consistency index (CI) 5 0.997 and retention index (RI) 5 0.889. Outgroup taxon: Truncatella angustata (F110654). GenBank accession numbers: ITS1-5.8S-ITS2: F-048003, AY929323; F-048007, AY929320; F-048041, AY929321; F048079, AY929322; F-090354, AY929325; F-095552, AY929324; F-110,564, AY929326. EF1a: F-048003, AY929315; F-048007, AY929313; F-048041, AY929317; F-048079, AY929318; F-090354, AY929314; F-095552, AY929316; F-110,564, AY929319. ACTIN: F-048003, AY929328; F-048007, AY929329; F-048041, AY929330; F-048079, AY929331; F-090354, AY929327; F-095552, AY929332; F-110,564, AY929333. CHITIN SYNTHASE 1: F-048003, AY929308; F-048007, AY929309; F-048041, AY929310; F048079, AY929307; F-090354, AY929306; F-095552, AY929311; F-110,564, AY929312. TreeBASE submission no. SN2451-9342. single cellular appendages, straight or slightly curved, 14–26. Specimens examined. SPAIN. SEGOVIA: Riaza. In living stems of Calluna vulgaris (L.) Hull, Jan 1999, V. González. (HOLOTYPE: MA), MA-Fungi-61115, dried culture of CBS 117603 (5F-095552). (EXTYPE: CBS), living culture, CBS 117603. Etymology. Latin longus, appendix 5 conidia bearing long appendages. Colonies of M. longiappendiculata (F-095552) on PDA attaining 32 mm; flat, glabrous, with radial grooves, yellowish brown (5E5, 5F5) (Kornerup and Wanscher 1978), becoming yellowish white (4A2) at the edge, margin irregular; reverse dark blond (5D4); hyaline exudates produced predominantly at the center. Colonies on OTM 65 mm diam; flat, floccose; grayish brown (5C3) at the center, becoming grayish orange (5B3) toward the edge; margin regular; reverse centrally colored in golden yellow (5B7) and orange gray (5B2) in 622 MYCOLOGIA FIG. 8. Relationship among Morinia isolates and strains of Amphisphaeriaceae deposited in GenBank based on the analysis of ITS sequences. Statistical support (1000 bootstrap) values . 50% indicated at branch points. Tree parameters: total characters 5 528, constant characters 5 406, variable characters parsimony uninformative 5 23, variable characters parsimony informative 5 99, tree length 5 188, consistency index (CI) 5 0.729 and retention index (RI) 5 0.898. GenBank accession numbers: Amphisphaeria sp. 1, AF375998; Amphisphaeria sp. 2, AF346545; Bartalinia laurina (Mont.) Nag Raj, AF405302; B. robillardoides AF405301; Discostroma fuscellum (Berk. & Broome) Huhndorf, AF377284; D. tricellulare Okane, Nakagiri & Tad. Ito, AF377285; Pestalotiopsis maculans (Corda) Nag Raj, AF405296; P. neglecta (Thüm.) Steyaert, AY682935; Pestalotiopsis sp., AF405295; P. virginiana Oudem., AF409959; Seiridium ceratosporum (de Not.) Nag Raj, AY687314, Seiridium sp., AF377297; Truncatella angustata (authors’ collection, F-110654), AY929326; T. angustata, AF405306. For GenBank accession no. of Morinia ITS sequences see footer to FIG. 7. TreeBASE submission no. SN2451-9343. COLLADO ET AL: STUDIES ON MORINIA 623 FIG. 9. Dendrogram showing the relationship among six Morinia endophytic isolates, based on similarity among high performance liquid chromatography profiles (UPGMA, Dice . 50% Mean). outer parts; exudate hyaline. Colonies on CMA attaining 48 mm; flat; aerial mycelium scant, more abundantly produced toward the center; hyaline; margin somewhat irregular; reverse hyaline, with center brownish orange (6C5); exudate hyaline. Odors absent in all media. Diffusible pigment brownish orange (6C8), sometimes visible around the colony in PDA cultures. Morinia pestalozzioides Berl. & Bres., Ann. Soc. Alpinisti Tridentini 14:81.[1887-88]. 1889. FIGS. 13–15 ; Rinomia pestalozzioides (Berl. & Bres.) Nieuwl. [as ‘pestalozziodes’], Am. Midl. Nat. 4: 381. 1916. 5 Pestalotia (Pestalozziana) artemisiae Pass., Atti R. Accad. Lincei, Sci. fisiche. mat. nat., Ser. 4:7, fasc. 2, 51. 1891. Mycelium branched, septate, with hyaline and olive yellow hyphae 5 mm wide. Conidiomata acervular, globose; textura angularis; conidiophores lining the cavity of the conidioma, simple or branched at the base; conidiogenous cells smooth, hyaline, cylindrical, septate, 10–19 3 2–3 mm. Conidia muriform, ellipsoid, pyriform, straight to slightly curved, smooth, 20– 25 3 6–8 mm, with 6 or 7 transverse septa and 1 or 2 vertical and oblique septa; versicolored, with basal and apical cells hyaline to subhyaline; median cells brown, 15–17 mm long; basal cell conic, mostly lacking appendages but sometimes bearing one more or less central short cellular appendage (rarely two and lateral) 3 mm long; apical cell hemispherical, crowned with three single cellular appendages, straight or slightly curved, 9–11 mm long. Teleomorph not observed. Specimens examined. ITALY. dead stems of Artemisia camphorata Vill., Apr 1888, Bresadola. (LECTOTYPE: BPI), microscope slide, BPI 453814. SPAIN. 624 MYCOLOGIA COLLADO ET AL: STUDIES ON MORINIA ALMERÍA: Sierra Alhamilla. In living stems of Sedum sediforme (Jacq.) Pau, Sep 1998, J. Collado & F. Arenal (EPITYPE: MA), MA-Fungi-61116, dried culture of ATCC No. PTA-3862 (5 F-090354). (EXTYPE: ATCC), living culture, ATCC No. PTA3862. Colonies of M. pestalozzioides F-090354 on PDA attaining 30 mm; wrinkled, with radial grooves; center velvety, olive gray (3D2), with brown (7E8) exudates and surrounded by olive (3E8) mycelium; colony edge two-colored, with and external white ring that surrounds an olive (2E4) fringe; margin irregular; reverse light brown (6D4); diffusible light brown (7D7) pigment produced. Odors absent. Colonies on OTM 70 mm diam; flat, velvety to slightly floccose; with alternate pinkish white (7A2) and grayish red (7B3) fringes disposed concentrically; margin somewhat irregular; odors and exudates absent; reverse brownish yellow (5C8); soluble pigments absent. Colonies on CMA 65 mm diam; aerial mycelium poorly developed; flat, glabrous, hyaline, with a small area of white mycelium in the center; margin regular; reverse colorless; soluble pigments absent; odors and exudates absent. DISCUSSION Based on morphological data, isolate F-090354 was identified as Morinia pestalozzioides. It produced conidia somewhat narrower and with shorter apical appendages than Bresadola’s specimen but matched the morphology of M. pestalozziodes in most respects (TABLE III). The slight morphological differences observed might be an artifact. The voucher specimen consisted of a few conidia and conidiogenous cells fixed in a resin under a cover slip and mounted on a glass slide. The mounting resin and long-time storage of the voucher specimen (since 1888) might have caused some alteration in conidial morphology. In addition its geographical origin in Italy might be a factor that contributed to the differences. Further analyses of more M. pestalozzioides strains from different geographical origins would be needed to asses the degree of morphological variation within the species with respect to geographical origin, host or other factors. No holotype was designated for M. pestalozzioides (Berlese and Bresadola 1889). Guba (1961) reported to have examined a specimen from Bresadola deposited in the Swedish Museum of 625 Natural History, Stockholm, Sweden (S). Nag Raj (1993) indicated this specimen was missing and that there was no material available of M. pestalozziodes type in major Italian herbaria. We examined a herbarium specimen from Bresadola’s collection deposited in the US National Fungus Collections (BPI 453814), which was examined previously by Guba. According to data, this specimen might be compatible with the material described by Bresadola in Micromycetes Tridentini (1889), but we have not been able to find an explicit mention anywhere that BPI 453814 corresponds to either the holotype or lectotype for M. pestalozzioides or whether it has any relation to the specimen at S examined by Guba. Given that data on host, collector and date of this voucher specimen match those of the specimen described by Bresadola in his original description of M. pestalozzioides, we have designated it as lectotype for M. pestalozzioides and have considered our M. pestalozzioides isolate as an epitype. M. longiappendiculata F-095552 produces larger and more fusiform conidia than M. pestalozzioides (TABLE III). Both basal and apical appendages are much longer in M. longiappendiculata than in M. pestalozzioides conidia. Furthermore the conidia in M. longiappendiculata are generated on filiform conidiogenous cells, in contrast with the cylindrical shape of the conidiogenous cells in M. pestalozzioides (FIGS. 10–15). Despite the fact that only one out of the five M. longiappendiculata isolates could be compared morphologically with the M. pestalozziodes isolate due to lack of sporulation in the rest of M. longiappendiculata isolates, the contrasting morphological features, the extensive analysis of DNA sequences that indicated statistically well supported genetic differences (FIG. 7) and the differences in HPLC profiles (FIG. 9) led us to recognize M. longiappendiculata as a new species. The results of ITS sequence analysis placed Morinia within the Amphisphaeriaceae. Highest homologies of the ITS rDNA sequences of our Morinia isolates were with GenBank sequences of Bartalinia and Truncatella (anamorphic Amphisphaeriaceae) species. Previously reported analyses of 28S rDNA sequences indicated that Bartalinia and Truncatella shared a common ancestor and were associated with the Amphisphaeriaceae, which includes other morphologically related genera of coelomycetes e.g., Pestalotiopsis Steyaert, Seimatosporium Corda and Seiridium Nees (Jeewon et al 2002). As previously mentioned we noticed variations of r FIGS. 10–15. Conidiogenous cells and conidia of M. longiappendiculata and M. pestalozzioides. 10–12. M. longiappendiculata. 13–15. M. pestalozzioides. Bars 5 10 mm. 626 MYCOLOGIA conidial morphology in M. longiappendiculata strains after successive colony transfer (TABLE III). Worapong et al (2002) reported that conidia of Pestalotiopsis microspora (Speg.) Bat. & Peres can be converted, via UV irradiation, into conidia that bear a resemblance to those of taxa like Truncatella, Monochaetia (Sacc.) Allesch. or Seiridium spp. The authors concluded that the convertibility among conidial types of such closely related coelomycete genera potentially makes current taxonomic classification schemes artificial. That conclusion was supported by a study on the relationships among Pestalotiopsis spp. in which some diagnostic morphological characters currently used in Pestalotiopsis taxonomy were proved to be phylogenetically insignificant by comparison of morphology and rDNAbased phylogenies (Jeewon et al 2003). Except for the presence of vertical and oblique septa, Morinia conidia are almost morphologically identical to those of Pestalotiopsis. However as mentioned above the comparison of the ITS sequences of our Morinia isolates with other anamorphic Amphisphaeriaceae indicates that the genus is closer to Truncatella and Bartalinia than to Pestalotiopsis or Pestalotia de Not. The result supports the idea that taxonomic classifications of this group of coelomycetes based exclusively on morphological characters are unreliable. Colony morphology, genotype and HPLC profile were not well correlated among M. longiappendiculata strains (TABLE II, FIGS. 7 and 9). Isolates F-048003 and F-048007 produced similar colonies on PDA and clustered together in the alignment of the DNA sequences, although this was not clearly supported by bootstrapping. Nevertheless metabolite production was qualitatively different in the two strains. On the other hand a correlation between DNA sequence and HPLC profile was observed for isolates F-048079 and F-095552, which exhibited different colony morphology. Strains F-048003 and F-048007 were isolated from the same host, Santolina rosmarinifolia and as mentioned above presented similar gross morphology and DNA sequence. We did not find additional relationships among any of the morphological and molecular markers and the host. During screening of antifungal metabolites in our laboratory a compound with antifungal activity in MEK extracts of M. pestalozzioides (F-090354) cultures in OP26-NLW was observed. The compound was detected at RT 5 15.085 min in the HPLC chromatogram (data not shown) and corresponded to a novel sordarin analog named moriniafungin after the fungus. Moriniafungin is a new protein synthesis inhibitor targeting the eukaryotic elongation factor 2, which has wider antifungal spectrum and lower minimal inhibitory concentration value than sordarin (Basilio et al 2005). The production of moriniafungin in culture was not observed in M. longiappendiculata isolates or in strains of taxonomically related fungi such as Bartalinia, Truncatella and Pestalotiopsis spp. ACKNOWLEDGMENTS The authors thank Dr Amy Y. Rossman (director of U.S. National Fungus Collections, Beltsville, Maryland) for the loan of the M. pestalozzioides voucher specimen. 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