INTRODUCTION

Species of the genus Xylaria Hill ex Schrank usually grow on the rotten wood, but can be found in soil or on various substrates such as fallen leaves, petioles, herbaceous stems, dung, grasses, seeds or fruits and wood (Rogers 1986). The genus has been studied in various parts of the world such as America, Europe, Africa and Asia (Ellis & Everhart 1887a,b, Dennis 1956, 1957, 1958, 1961, 1964, Martin 1970, Ju & Tzean 1985, Rogers 1986, Rogers & Samuels 1986, Rogers et al. 1988, San Martin Gonzalez & Rogers 1989, Ju & Rogers 1999, San Martín et al. 2001, Rogers et al. 2008, Kshirsagar et al. 2009, Ma et al. 2011, 2012, 2013, Ju et al. 2012, Rogers & Ju 2012, Whalley et al. 2012), but there are only a few reports on Xylaria species from Iran (Soleimani 1976, Riedl & Ershad 1977, Daneshpazhuh 1980, Arefipour et al. 2004, Zare & Morid 2006, Zare & Asef 2008, Ershad 2009, Hashemi et al. 2013, 2014). More Xylaria species are reported from Guilan province of Iran in this paper.

MATERIALS AND METHODS

Isolates, media and morphological observation

Fungal materials were collected from Guilan Province, Iran. Cultures were obtained from ascospores as previously explained by Ju and Rogers (1996) and Ho and Ko (1997). Colony characteristics were recorded on fresh oatmeal agar (OA) [30 g rolled oats, 20 g agar and 1000 ml distilled water] (Stevens 1981) at 20 ± 0.5°C, 12/12 h light/dark regime. Growth rates were measured in duplicates after 10 days. Observations and measurements were made from specimens examined in distilled water for ascospores, in 10% Lactic acid for asci, and in Melzer's reagent for ascus apical rings. At least 30, 10 and 5 measurements were determined for ascospores, asci and ascus apical rings, respectively. A VANOX AHBS3 Olympus light-microscope was used to examine fungal structures and a M1000 Leica light-microscope equipped with a Canon EOS 600D digital camera was applied for photography. Fungal species were determined morphologically according to relevant literature (Dennis 1956, 1957, 1958, 1961, Rogers 1979, 1986, Rogers & Samuels 1986, Rogers et al. 1988, San Martin Gonzalez & Rogers 1989, Rogers & Ju 1998, Ju & Rogers 1999, Rogers et al. 2008, Hladki & Romero 2010, Rogers & Ju 2012). Specimens were deposited in the Fungal Reference Collection of the Ministry of Jihad-e Agriculture (IRAN) located at Iranian Research Institute of Plant Protection, Tehran and the fungal collection of the Department of Plant Protection, Faculty of Agricultural Sciences, University of Guilan, Iran (GUM).

Molecular studies

Total DNA was extracted from the scooped content of perithecia according to Chelex method (Walsh et al. 1991). Internal Transcribed Spacer (ITS) region was amplified using ITS5 [5′–gga agt aaa agt cgt aac aag g] and ITS4 [5′–tcc tcc gct tat tga tat gc] primers for the first PCR, and ITS1 [5′–tcc gta ggt gaa cct gcg g] and ITS4 for semi-nested PCR (Whiteet al. 1990). The PCR reaction mixture (25 μl) contained 1X PCR buffer, 2 mM MgCl₂, 5 pmol of each primer, 0.2 mM dNTPs (Bioflux biotech), 0.75 U Taq DNA polymerase (Bioflux biotech) and 5 µl of template DNA for the first PCR or 0.5 µl of the first PCR product for semi-nested PCR. The PCR amplification conditions were as follows: initial denaturation of 90 sec at 95°C followed by 35 cycles of 95°C/30 sec, 52°C/ 30 sec, 72°C/30 sec and a final extension of 6.5 min at 72°C. PCR amplification was carried out using a thermal cycler MyGenie® (Bioneer, South Korea). The PCR products were electrophoresed in 1% agarose gel containing 1 μl CinnaGen DNA safe stain (CinnaGen, Tehran)/30 ml agarose gel and visualized by a gel documentation tool (UVITEC^®, Cambridge). Amplified products were purified and sequenced by an ABI 3730XL capillary DNA sequencer (Sequetech, California).

Phylogenetic analysis

Sequence files were evaluated by Chromas 2.4 (Technelysium Pty Ltd., South Brisbane, Australia). Alignments were initially obtained using the Pairwise Alignment option in GeneDoc 2.7.000 (Nicholas et al. 1997). Phylogeny was inferred using Neighbor-Joining (p-distance) method with Pairwise Deletion option and the bootstrap test by 1000 replicates (Felsenstein 1985, Saitou & Nei 1987, Nei & Kumar 2000). Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). Relevant sequences representing nearly related taxa toour isolates were obtained from GenBank using a Blast search and incorporated to the analyses (Table 1). Hypoxylon fragiforme (JN979420) was used as outgroup.

RESULTS AND DISCUSSION

Three Xylaria species, X. longissima sp. nov., X. cf. striata and X. xylarioides are identified and illustrated. Xylaria cf. striata and X. xylarioides are reported as new records to Iranian mycobiota.

Xylaria longissimaHashemi, Khodaparast, Zare & Elahinia, sp. nov. —MycoBank MB 810915; Fig. 1

Etymology–Referring to long acute sterile apices of stromata.

Stromata caespitose, 55–71 mm long, fertile portion 6–8 × 1–1.5 mm diam., with very long (30–45 mm long) setiform and branched sterile apices; stipe pubescent, 17–20 × 1 mm diam.; stromatal surface

Table 1: Isolates and ITS sequences of Xylaria species used in this study. Underlined sequences are newly generated.

^†This sequence appears in GenBank as X. persicaria but it has been renamed as X. liquidambar (=X. liquidambaris in Index Fungorum and Mycobank) by Rogers et al. (2000).

roughened from evidently to slightly perithecial elevations, with light brown peeling outer layer splitting in longitudinal bands; ostioles slightly papillate. Asci cylindrical, 8-spored, 165–180 × 6–7 μm, the spore bearing part 90–100 μm, with apical ring bluing in Melzer’s iodine reagent, rectangular, (3–)3.5–4(–4.5) × 2.5 μm; ascospores (15–)16–18(–20) × 5–6 μm, inequilateral to navicular, with straight spore-length germ-slit.

Cultural characteristics — Colony growth on OA was 32 ± 6.9, 53.2 ± 10.1 and 76.8±9.1 mm after 10, 16 and 25 days at 20 ± 0.5°C and 12/12 h light/dark regime, respectively. Colony was at first white with gray center, and then turned to vinaceous buff with dark concentric rings which produced dark, branched and setiform stromata with white tip (Fig. 1H–I).

Fig. 1. Xylaria longissima holotype (IRAN 16581 F). a. Stromata on wood in natural conditions, b. Stromata with very long, setiform and branched sterile apices, c. Close-up of surface of stromata with brown peeling outer layer, d. Pubescent stipe, e. Ascospores and rectangular ascus apical ring bluing in Melzer's reagent, f. Close-up of ascospores with straight spore-length to nearly spore-length germ-slit, g. Colony on OA after 21 days at 20±0.5°C in 12/12 h D/L regime, h. Close-up of stromata produced on OA. Scale bar: E, F= 10 µm.

Specimens examined.IRAN, Guilan Province, Rasht, Saravan Forest Park, on the wood of unknown dicotyledonous plant, 30 Nov. 2012, S.A. Hashemi, holotype Herb. IRAN 16581F, culture ex-type IRAN 2268 C; Guilan Province, Shaft, Dorud Khan, on the wood of unknown dicotyledonous plant, 4 Oct. 2012, S.A. Hashemi, Herb. IRAN 16582F, culture ex-type IRAN 2269 C).

Notes —Phylogenetic analysis showed that X. longissima formed a distinct lineage (Fig. 4). Both morphology and rDNA sequences confirmed that our material represents a new species in Xylaria. Xylaria longissima is characterized by very long setiform sterile apices, more or less conspicuous perithecia, light brown peeling outer layer and 16–18 × 5–6 μm ascospores with straight spore-length germ-slit. Stromata were more robust (2 mm diam.) and longer (98 mm long), with longer (63 mm long) and unbranched sterile setiform apices in the second specimen collected from Shaft (IRAN 16582 F). However, it generally fits X. longissima. Comparison of its ITS sequence also shows 100% homology with the holotype of X. longissima (Fig. 4).

Morphologically, X. longissima is somewhat close to X. filiformis, X. theissenii var. macrospora, X. juruensis and X. arbuscula. Xylaria filiformis has individual naked perithecia dispread along the filiform stromata without peeling outer layer, wider ascospores 14–18 × 8–8.5 μm, and occurs on leaves and herbaceous debris (Ellis & Everhart 1887a, Rogers & Samuels 1986). Xylaria theissenii var. macrospora possesses very long (34–40 μm) ascospores (Rogers & Samuels 1986). Xylaria juruensis has naked individual perithecia dispread along the stromata, more or less pubescent stromata and 12–17 × 4–5 μm ascospores (Hennings 1904). X. arbuscula differs from X. longissima mainly based on its completely immersed perithecia, lack of very long acute sterile apices, inverted hat shape ascus apical ring and length of germ-slit (San Martin Gonzalez & Rogers 1989). Even though no sequences of the X. filiformis and X. theissenii are deposited at GenBank, X. longissima isolates formed a distinct lineage from our previously reported X. filiformis (Hashemi et al. 2014). Moreover, the ITS sequence of X. longissima differs from Xylaria juruensis and X. arbuscula sequences (Fig. 4).

Based on a BLAST search using ITS sequences, the closest taxa to X. longissima were X. bambusicola (KF381074, JX256820, JX256819 and JX256818) and X. grammica (HM752504) with 95% homology. Among them, X. bambusicola (KF381074) and X. grammica (HM752504) specimens have been reported as endophytes. Xylaria longissima differs from X. bambusicola mainly in its host plant, surface and apices of stromata and size of ascospore. Xylaria bambusicola which occurs on monocotyledonous material, has completely immersed perithecia, short acute sterile apices, with 9.5–11(–12.5) × 4–5 μm ascospore, and occasionally a tiny hyaline cellular appendage on one end (Ju & Rogers 1999). Xylaria longissima differs from X. grammica described by San Martin & Rogers (1989) mainly in ascospore size, diameter and tip of stromata and the color of peeling outer layer. Xylaria grammica has stromata with 5–8 mm diameter, rounded and fertile or acute and sterile apices, with whitish areas that appears as vertical strips, and (9.5–)10–12(–12.5) × (4.5–)5–5.5 μm ascospore.

Xylaria cf. striata Pat., Journ. Bot. (Morot) 1: 247. 1887. Fig. 2

Stromata gregarious on soil, several arising from common base, 44–120 mm long, fertile portion cylindrical, 9–18(–22) × (2–)2.5–3(–4) mm diam., with acute sterile apices; stipe root-like, 70–100 mm long, stromatal surface more or less roughened from whitish to pale peeling outer layer and minute wrinkles; perithecia almost immersed, 0.38–0.56 mm diam.; ostioles papillate to slightly papillate. Asci degenerated and not seen; apical ring bluing in Melzer’s iodine reagent, rectangular, 3–3.5(–4) × 2(–3) μm; ascospores (13–)15–19(–22.5) × 5–7(–9) μm, fusiform to ellipsoid, often apiculate at one end when produced on stromata under natural conditions (Fig. 2E) but not apiculate when produced on stromata under laboratory conditions (Fig. 2F), with straight about 1/3 of spore-length.

Cultural characteristics — Colony growth on fresh OA 12±3.2 mm after 10 days at 20±0.5 °C in 12 h dark/ 12 h fluorescent light regime. Colonies covering 9 cm diam. Petri plates in 6–7 wk, at first white then becoming blackish brown at center, margins regular, zonate, with blackish brown furrows with dark and unbranched mature stromata with buff tip. Reverse side with dark furrows and zones. Anamorph not produced (Fig. 2H–I).

Specimen examined. IRAN, Guilan Province, Lahijan, Koshal village, on soil, 7 Feb. 2014, S.A. Hashemi, Herb. GUM 1150.

Notes — Morphology of Iranian specimen fits X. striata except for the shape of ascospore. Ascospores of X. striata are reported 16–18(–19) × 4.5–5 μm (San Martínet al. 1999) or 15–20 × 5–8 μm (Saccardo 1891, Lloyd 1917). Dennis (1957) described ascospores of X. deserticola (a species that San Martín et al. (1999) believed to be synonymous with X. striata) as 16–18 × 5–7 μm. Ascospores of X. striata have always been described as lacking apiculate ends, while in this study spores of the Iranian specimen are often apiculate at one or rarely two ends when produced on stromata under natural conditions (Fig. 2E). They are however not apiculated when produced on stromata in culture (Fig. 2F). Colony of Iranian material differs from San Martin et al. (1999) description by slower growth rate, blackish brown furrows and dark furrows and zones at the reverse side (Fig. 2H–I). Phylogenetic analysis of ITS sequences showed that our isolate was close to X. striata (GU300089) with high bootstrap support value (Fig. 4). Although the deeply buried stipe in the soil

Fig. 2. Xylaria cf. striata. a. Gregarious stromata on soil, b. Stromata with long rooting base, c. Close-up of stromatal surface with whitish to buff color peeling outer layer, d. Stromata produced on OA with drops of ascospores logged from mature perithecia, e. Mostly papillate ascospores produced on stromata from natural conditions, f. Not apiculated ascospores produced on stromata in OA, g. Rectangular apical ring bluing in Melzer’s iodine reagent, h. and i. Front and reverse sides of colony on OA after 44 days at 20±0.5°C in 12/12 h D/L regime, respectively. Scale bars = 10 µm.