INTRODUCTION

Dena is one of the protected areas in western Iran, and part of its range has been assigned as Dena Biosphere Reserve in 2010. The area is located in the central Zagros region, in Kohgiluyeh and Boyer-Ahmad province, and has the typical vegetation in Zagros, consisting of open Quercus woodlands and angiosperm shrubs, with sporadic gymnosperm stands at higher elevation. The main oak species in the area is Q. brantii (Sagheb Talebi et al. 2014, Mozaffarian 2005), which has been subjected to a severe decline in the past years (e.g. Attarod et al. 2016, Hosseini 2011, Mirabolfathi 2013, Ghobad-Nejhad 2016). The possible link between endophytic fungi and oak decline has not been studied in Zagros. Broadly, “endophytes are microorganisms that spend at least parts of their life cycle inside plants” (Hardoim et al. 2015), and they include fungi and bacteria. In contrast to some conventional definitions which restrict endophytes to a certain lifestyle within only living plant tissues (see Hardoim et al. 2015), this simple definition reflects multifaceted biology of endophytes substantiated by recent empirical studies (Peršoh 2015). It is corroborated that an endophytic species may change its mode of nutrition and function during its life cycle in a response to the host health conditions (Wrzosek et al. 2017, Szink et al. 2016), therefore the taxonomy of the fungus is not necessarily directly linked to its trophic habit. While the most recent count on magnitude of global fungal diversity (Hawksworth & Lücking 2017) estimates 2.2 to 3.8 million species of fungi to be present in the world, other studies approximate the number of endophytic fungi alone to be as high as 0.5–1.3 million species, reflecting their enormous diversity (Sieber 2007, Dreyfuss & Chapela 1994). Fungal endophytes of native oak stands in the Zagros region have not been adequately studied (Ghobad-Nejhad et al. 2018 provided a short review on published records of fungal endophytes isolated from Q. brantii). To know more about the fungal endophytes of damaged oak trees, some fungal endophytes were isolated from twigs of Q. brantii. We focused on tree stands which were visibly attacked by the basidiomycetous polypore fungus Inonotus krawtzewii (Pilát) Pilát (oak’s Inonotus, see http://www.mycolich.ir/species/3283, accessed on 30 October 2017). Then, samples were taken from the tree stands with as identical conditions as possible, as e.g. recommended by Torres et al. (2014) in endophyte studies. Moreover, we aimed to test if this polypore would be present endophytically inside oak twig tissue. The polypore has recently been reported from Iran, and has been shown to cause damage to Zagros oak stands (Ghobad-Nejhad 2016).

MATERIALS AND METHODS

Sampling and isolation

Twigs of Quercus brantii trees were collected in November 2016, in Dena National Park, Dashtak, in Kohgilouyeh and Boyer-Ahmad Province. Samples were taken from stands infected with visible basidiocarps of the polypore basidiomycete Inonotus krawtzewii (see Ghobad-Nejhad 2016 for a full description of this polypore). In the collecting area, five trees were randomly selected, and five twigs from each tree were sampled, and transported to the lab. Twigs were surface-sterilized following Unterseher (2011). From each twig, three segments were cut and put onto Petri dished with PDA medium kept at room temperature. Isolates were checked periodically for about one month and grown mycelia were moved to new dishes. Cultures are preserved at the Iranian Fungal Culture Collection (IRAN…C) at the Iranian Research Institute of Plant Protection, Tehran, Iran. Morphological examination of the cultured isolates and application of additional culture media for stimulating sporulation were done following Hajizadeh et al. (2015).

Molecular study

Total DNA was extracted using DNA Extraction Mini Kit (YT9030, Taiwan). The ITS region (covering ITS1, 5.8S, ITS2, partial SSU/LSU) was amplified with Super PCR Master Mix (YT1553-5, Taiwan) mainly using the primer pair ITS1F/ITS4 (White et al. 1990; Gardes & Bruns 1993). The parameters for PCR reactions follow Ghobad-Nejhad & Langer (2017). The PCR product was purified and sequenced by Macrogen (Korea, Macrogen Inc.). The obtained DNA sequences were assembled using DNA Baser Sequence Assembler v4, and were submitted to GenBank. Their identification is reported in Table 1, with the accession numbers reported in Table 2. The dataset for phylogenetic analyses was constructed using the newly obtained sequences together with additional relevant GenBank sequences retrieved from Blastn searches (Altschul et al. 1990), with 98–100 % identity and query coverage, and with particular notion to type strains or generic types, as much as possible. Doubtful and unpublished sequences and the sequences from environmental studies were avoided. To exclude the effect of flanking regions of 28S and 18S regions, the ITS region was partitioned into segments using ITSx (Bengtsson-Palme et al. 2013). Partitions were then blasted with massBlaster against the reference sequences at PlutoF (Abarenkov et al. 2010). The top-most matching sequences of Species Hypothesis (see Abarenkov et al. 2010) were added to the final ITS dataset. The sequences were aligned using MUSCLE 3.8 (Edgar 2004), and analyzed using MrBayes v. 3.2.6 (Ronquist & Huelsenbeck 2003), with the same parameters as used by Ghobad-Nejhad (2016). The outgroup species was Lentinus tigrinus AF516518.

RESULTS

In total, ca. 40 pure isolates were obtained in this study. After morphological studies, DNA was extracted from selected isolates and 16 ITS sequences were obtained (Table 1). Final dataset consisted of 56 taxa and 462 characters from which, 234 characters were informative, and 182 characters were constant. The Bayesian phylogram resulted from the phylogenetic analysis (using SYM+I+G model of nucleotide evolution as suggested by MrModeltest 2.3 (Nylander 2004)) is shown in Fig. 1. The identified isolates (except one) belonged to ascomycetes (Pezizomycotina), to the classes Dothide-omycetes, Sordariomycetes, and Pezizomycetes, the orders Diaporthales, Hypocreales, Pezizales, Pleosporales, Sordariales, and Xylariales, and the families Coniochaetaceae, Diatrypaceae, Didymosphaeriaceae, Lopadostomataceae, Ophiocordycipitaceae, Phaeosphaeriaceae, Pleosporaceae, Pyronemataceae, and Valsaceae. There was only one basidiomycete fungus Lentinus tigrinus (Polyporales, Polyporaceae), while I. krawtzewii was not isolated. Several isolates remained as sterile mycelia, with no reproductive organs (Table 1) and these were studied using phylogenetic analysis based on careful taxon sampling as described above. The isolates identified at species level were as following: Alternaria alternata, A. cf. arborescens, Kalmusia variispora, Lentinus tigrinus, Lopadostoma quercicola, and Purpureocillium lilacinum. A number of isolates could be identified at generic level, and they belonged to the genera Coniochaeta (Sacc.) Cooke, Cytospora Ehrenb., Diatrype Fr., Pyronema Carus, and Ulocladium Preuss. The rest of isolates were identified only at family (Phaeosphaeriaceae), and order (Pleosporales) levels (Table 1). Brief description and microscopic illustrations are given for isolates as much as possible.

Notes on some taxa

Alternaria cf. arborescens E.G. Simmons, Mycotaxon 70: 356 (1999). Fig. 2 c-g

The conidia with 1–3 longitudinal septa are much abundant in the isolate K3-4d1. Additional molecular examination would be required in the future to examine it at fine level. Although the examined isolates of Alternaria alternata and A. cf. arborescens are morphologically distinct (Simmons 2007, Ellis 1971), their ITS sequences are highly similar and collapsed in polytomy. (Fig. 1), corroborating the known idea that ITS is not the most appropriate marker for discriminating species of Alternaria. Future monographic studies on Alternaria-like taxa in Iran would apply that.

Table 1. Taxa identified in this study. All strains were obtained from twigs of Quercus brantii collected in November 2016 in Dena National Park, Dashtak, in Kohgilouyeh and Boyer-Ahmad Province. Strains in bold are sterile. Classification is based on MycoBank.

Coniochaeta sp. 1   Fig. 3

A phialosporic fungus. Small conidia are formed on denticles or branched conidiophores in heads. This anamorph resembles both Lecythophora Nannf. anamorph of Coniochaeta (Sacc.) Cooke and Collophora Damm & Crous. We compared the ITS sequence of this isolate with sequences of type species of Lecythophora (current name Coniochaeta, typified with C. ligniaria (Grev.) Cooke) and Collophora (typified with C. rubra Damm & Crous). As seen in Fig. 1, sequence of the isolate K3-5b is nested in a polytomy composed of Fimetariella rabenhorstii (Niessl) N. Lundq. and Coniochaeta sp./Coniochaeta taeniospora, rooted with C. ligniaria, while C. rubra found a distant position. Even though we tried to select the reliable sequences, which cannot excluded the possibility of misidentification in these GenBank sequences. For now, we name the isolate K3-5b as Coniochaeta sp. 1 (older generic name) until more details become available.

Coniochaetasp. 2

The studied isolate was sterile. Blast searches hit Fimetariella rabenhorstii and Coniochaeta sp. with high scores (99 % identity over 100 % query coverage). To infer its phylogenetic affiliation, authentic sequences of the generic type of Fimetariella N. Lundq. 1964 (i.e., F. rabenhorstii) and the type of Coniochaeta (i.e., C. ligniaria) were added to the dataset. As mentioned above, and shown in Fig. 1, F. rabenhorstii and Coniochaeta sp./Coniochaeta taeniospora do not appear as distinct clades, but their polytomy is outgrouped with C. ligniaria. For the time being, we regard the isolate K4-2c as an unidentified isolate of the genus Coniochaeta.

Kalmusia variispora(Verkley, Göker & Stielow) Ariyawansa & K.D. Hyde, Fungal Diversity 68: 85 (2014).                Fig. 4

Colonies dark fuscus to rusty brown, more or less paler at center and at margins, producing long synnemata-like bodies on PDA. Conidiophores light brown, smooth, highly branched, moderately thick-walled, 3–4 µm wide, with numerous septa, producing light brown, smooth, globose to subglobose aleuroconidia or chlamydospore-like bodies in mid-segments or terminally, ca. 5.5–9.8 × 4–9.2 µm.

The ITS sequence of this isolate matches very well with authentic Kalmusia variispora sequences in GenBank, including the ex-type sequence. Dendrothyrium variisporum Verkley, Göker & Stielow, a pycnidial fungus, has been synonymized under Kalmusia variispora (Ariyawansa et al. 2014, Verkley et al. 2014). However, our isolate produces long synnemata-like bodies on PDA, producing aleuroconidia or swellings on repeatedly branched, dendroid conidiophores (Fig. 4) that are absent in both Dendrothyrium Verkley, Göker & Stielow and Kalmusia Niessl. Therefore, sequencing an additional gene region like β-tubulin would be needed to decide if this isolate can be introduced as a novel anamorph for K. variispora. Abdollahi Aghdam & Fotouhifar (2017) recently reported D. variisporum as new to Iran, and their identification was based on ITS Blast (although no accession number was given in their study). The sterile isolate K1-a also seems to be conspecific with K3-3a, and their sequences match each other with 100 % identity over 98 % query coverage.