Two new records of Lopadostoma for mycobiota of Iran

Document Type: Short Article

Authors

Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Iran

Abstract

Xylariaceous fungi are typically saprobes, but are also commonly isolated as endophytes and some species are pathogens. Two species of Lopadostoma (Xylariaceae, Xylariales) are reported for the first time from Iran. L. dryophilum was found from dead branches of Quercus sp. in East Azerbaiejan and L. fagi from dead branches of Fagus sp. in Ardabil province. Based on morphology and sequence data (ITS), the two species, L. dryophilum and L. fagi are confirmed as new records for mycobiota of Iran. A detailed description of the two species are provided. This is the first report of the genus in Iran.

Keywords

Main Subjects


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 AnthostomaAnthostomella 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. pouzariiL. 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.

 

Taxon name

 

Host

GenBank

accession no.

 

Origin

Lopadostoma americanum

Quercus sp

KC774568

USA

Lopadostoma dryophilum

Quercus petraea

KC774570

Austria

Lopadostoma dryophilum

Quercus petraea

KC774571

France

Lopadostoma dryophilum

Quercus sp.

KR999998

Iran

Lopadostoma fagi

Fagus sylvatica

KC774577

Austria

Lopadostoma fagi

Fagus sylvatica

KC774574

Austria

Lopadostoma fagi

Fagus sp.

KR999997

Iran

Lopadostoma gastrinum

Carpinus betulus

KC774579

Austria

Lopadostoma insulare

Quercus coccifera

KC774588

Greece

Lopadostoma lechatii

Carpinus betulus

KC774590

France

Lopadostoma linospermum

Pistacia lentiscus

KC774591

Italy

Lopadostoma meridionale

Quercus ilex

KC774598

Croatia

Lopadostoma quercicola

Quercus petraea

KC774604

Austria

Lopadostoma turgidum

Fagus sylvatica

KC774616

Austria

Xylaria hypoxylon

Sorbus aucuparia

AM993141

Sweden

 

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

BasionymPhaeosperma 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) 90110 × 78 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.

 

 

Lopadostoma fagi Jaklitsch, J. Fourn. & Voglmayr, Persoonia 32: 63. 2014. Fig. 3.

Stromata densely immersed in the bark of dead branches (1.5 cm diam.), pustulate, covered by the epidermis which is not discolored, 1–1.5 mm diam., slightly erumpent with tiny, black, rounded or slightly elliptical ectostomatic disc, with groups of 3–7 perithecia. Ostioles dark, converging toward the disc; tissue between the ostioles blackish, opening separately in the disc. Perithecia dark, circinately arranged, globoid to subgloboid, monostichous, 300–800 μm diam., tissue surrounding perithecia yellowish brown. Asci cylindric, containing 8 uniseriate ascospores, 6070 × 56 μm, with stalks up to 34 μm long. Ascospores 7–10.5(–11.3) × 3–4 μm, oblong or narrowly ellipsoid, aseptate, brown to nearly black, smooth, with straight, circumferential germ slit and 2 large guttules,.

Specimens examined: IRAN, ARDABIL, Khalkhal, on dead branch of Faqus sp., 5 August. 2014, M. Mehrabi, IRAN 16686F.

Note: This taxon has described by Jaklitsch et al. (2014) on the basis of material from Austria. The Iranian material was consistent with L. fagi as described by Jaklitsch et al. (2014). Based on both morphology and molecular sequence data, the occurrence of L. fagi in Iran was confirmed with 100% bootstrap values (Fig. 1). Based on a megablast search of NCBI GenBank nucleotide database, the closest sequence to our fungus is L. fagi (GenBank KC774577 and KC774574; Identities = 480/483(99%), Gaps = 0/483(0%))This is the first report of this species from Iran.

   

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.

  

Fig. 3. Lopadostoma fagi.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 showing germ slit. — Scale bars: a = 3mm; b-d = 1 mm: e-h= 10 μm.

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