Two new records of Lopadostoma for mycobiota of Iran

INTRODUCTION

The Xylariales is a large order of perithecial ascomycetes that contains 209 genera and 2487 species (Kirk et al. 2008). Xylariaceae is the type and largest family of the Xylariales with 85 genera and a total of 1343 species (Kirk et al., 2008). The genus Lopadostoma (Nitschke) Traverso is a member of family Xylariaceae that was founded by Traverso (1906) and typified by L. turgidum. The genus is characterized by perithecial ascomata immersed in and erumpent from bark, standing on the wood, with only an ectostromatic discvisible or the disc surrounded by blackened bark surface; cylindrical asci with an flat ring-like part bluing in iodine reagent; ascospores which are oblong to narrowly ellipsoid, lack a dwarf cell, dark to blackish brown at maturity, with a straight germ slit across the entire spore length present on one side or circumferential side; and a Libertella-like anamorph (Jaklitsch et al. 2014).

Molecular delimitation of Lopadostoma and related genera, including Anthostoma, Anthostomella and Barrmaelia has been accomplished by sequencing three genes including the internal transcribed spacer (ITS) region, the nuclear large subunit rDNA (LSU) and the RNA polymerase II subunit B (rpb2) (Jaklitsch et al. 2014). According to revision of Jaklitsch et al. (2014), the genus consisted of twelve species: L. turgidum, L. fagi, L. juglandinum, L. quercicola, L. gastrinum, L. lechatii, L. dryophilum, L. linospermum, L. ailanthi, L. insulare, L. americanum and L. meridionale, which among them L. ailanthi and L. juglandinum are only known from morphology. L. pouzarii, L. polynesium and L. amoenum however are not included in Lopadostoma because molecular data do not support this conclusion (Jaklitsch et al. 2014). Vasilyeva and Stephenson (2014) also described a new species of Lopadostoma (L. cryptosphaeroides) on the bark of Quercus sp. in Virginia on the basis of morphological characters.

The aim of this study was to describe and illustrate two new records of Lopadostoma genus for Iranian mycobiota that have been collected during a taxonomic and phylogenetic study of Diatrypaceae in the northern Iran.

MATERIALS AND METHODS

Morphological studies

Branch samples were collected from Ardabil and East Azerbaijan provinces. Several unsuccessful attempts were made to isolate and culture the fungus from single ascospore on potato dextrose agar (PDA, Difco) and malt extract agar (MEA, Merck) at 25^°C in darkness. Morphological measurements and photomicrographs were made according to Mehrabi et al. (2015). Dry specimens were deposited in the herbarium of Iranian Research Institute of Plant Protection (IRAN).

DNA extraction and amplification

For extraction of genomic DNA, the content of several perithecia with a sterile needle transferred to an empty 1500 µl Eppendorf tube. Thirty microliters of TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA) was added to the tube, the tube was closed and transferred to liquid nitrogen. The mixture was grounded by a sterile peg. This was repeated several times. The tube was vortexed and then spun in a microfuge for 1 min at 12000 rpm. The supernatant liquid was collected and transferred to a clean Eppendorf tube. The supernatant contained genomic DNA and was used directly for PCR. PCR was performed in 25 µl reaction mixture containing 1 µl of each primer (10 pmol/µl, Takapouzist Inc.), 4 µl genomic DNA mentioned above (10 ng/µl), 2.5 µl 10× high yield PCR buffer (Jena Bioscience, Germany), 1.5 unit Taq polymerase (Jena Bioscience, Germany) ,1 mM MgCl₂ and 0.5 mM dNTP. Amplifications were performed by using primers ITS1 and ITS4 (White et al. 1990) in a PC-320 PCR System (ASTEC Co., Japan), which was programmed 4 min at 94˚C for denaturation , followed by 35 cycles at 94˚C/45 s, 58˚C/35 s and 72˚C/90 s, with a final elongation step at 72˚ C/10 min. The PCR products were sent out for sequencing in one direction (Macrogen company, South Korea).

Sequence analysis

The newly obtained nucleotide sequences were checked with FinchTV v. 1.4.0 (Geospiza Inc.). The sequences obtained were compared with those in the GenBank databases using the BLAST program. Based on the BLAST results, sequences were retrieved from GenBank for the comparative phylogenetic analysis. DNA sequences were aligned with Clustal W (Thompson et al. 1994), within the MEGA 6 (Tamura et al. 2013). Phylogenetic analyses of the aligned dataset were performed with Neighbor-Joining (NJ) method (Saitou and Nei 1987) with complete deletion option for gaps/missing data and the bootstrap test by 1000 replicates (Hillis and Bull 1993). Based on the Bayesian information criterion of MEGA 6, Kimura 2-parameter model with gamma distribution (K2 + G) was selected for the NJ analysis. Xylaria hypoxylon (AM993141) was used as the outgroup. The sequences of our isolates were deposited in GenBank.

RESULTS AND DISCUSSION

Molecular phylogeny

Two new ITS sequences were obtained in this study (GenBank accession numbers KR999997 and KR999998) and aligned with 13 sequences retrieved from GenBank (Table 1). Size of our sequences were 549 bp for Lopadostoma dryophilum and 516 bp for L. fagi. Based on both morphology and molecular sequence data, the occurrence of these two spices in Iran was confirmed with 100% bootstrap values.

Table 1: Isolates used in phylogenetic analysis, with data referring to the newly generated sequence marked in boldface.

Lopadostoma dryophilum (G.H. Otth) Jaklitsch, J. Fourn.&Voglmayr, Persoonia 32: 61. 2014. Fig. 2.

Basionym: Phaeosperma dryophilum G.H. Otth, Mitt. Naturf.Ges. Bern Nr. 654–683: 42. 1868.

Synonyms are given by Jaklitsch et al. (2014).

Stromata immersed in the bark of dead branches (1.5 cm diam.), pustulate, erumpent, 2–3.5 mm diam., often with slightly projecting black ostioles, delimited by a black zone in the host tissues, the latter 100–200 μm thick, with groups of 8–20 perithecia. Ostioles dark, opening separately in the disc. Perithecia dark, circinately arranged, globoid to subgloboid, monostichous, 300–800 μm diam., surrounded by brownish entostroma. Asci narrow cylindric, containing (6–)8 uniseriate ascospores, (74–) 90–110 × 7–8 μm, with stalks up to 30 μm long. Ascospores (–9)10–15(–16.5) × 3.4–4.7 μm, narrowly ellipsoid or narrowly fusiform, aseptate, dark brown to nearly black, with straight, circumferential germ slit and 2 large and sometimes several small guttules.

Specimens examined: IRAN, EAST AZERBAIJAN, Aghoyeh, on dead branch of Quercus sp., 6 August. 2014, M. Mehrabi, IRAN 16685F.

Note: This taxon has suggested by Jaklitsch et al. (2014) as a new combination. The studied material fits with L. dryophilum as described by Jaklitsch et al. (2014). The phylogenetic analyses of the ITS sequences confirmed the morphological identification with 100% bootstrap value (Fig. 1). Based on a megablast search and the phylogenetic tree inferred from ITS sequences (Fig. 1), the closest sequence to our fungus is L. dryophilum (GenBank KC774570 and KC774571; Identities = 547/550(99%), Gaps = 1/550(0%)). This is the first report of this species from Iran.

Fig. 1. Phylogenetic tree of Lopadostoma species inferred from ITS (ITS1–5.8S–ITS2) sequences using Neighbor Joining method in MEGA6 with 1000 bootstrap replications. The bootstrap values (>50%) are shown at the nodes. The new sequences obtained in this study are indicated in boldface.

Fig. 2. Lopadostoma dryophilum. a. Habit of ascostromata on bark; b. Ectostromatic discs; c. Transverse section through the ascoma; d. Longitudinal section through the stroma; e-f. Asci; g. Ascospores; h. Ascospore with straight spore-length germ slit. — Scale bars: a = 3mm; b-d = 1 mm: e-h= 10 μm.