Mycologia Iranica
Volume:8 Issue: 2, Summer and Autumn 2021

Saprolegniales have been studying for nearly 150 years and have been recognized mainly as freshwater animal pathogens. Similar to the global trend, studies on Saprolegniales in Iran have mainly focused on their pathological aspects. Therefore, this review discusses the state of the art of Saprolegniales studies in Iran and pinpoints present deficiencies. More than 80% of Iranian studies have examined the impact of various plant extracts and other plant compounds on Saprolegnia parasitica, responsible for causing deadly diseases on fish species. On the other hand, recent taxonomic and ecological reports on Saprolegniales have addressed this topic in a more abstract manner. Finally, we give recommendations to how to more systematically study Saprolegniales in Iran. This review calls mycologists in Iran and elsewhere to study Saprolegniales more seriously and in a more coordinated manner.

Arbuscular Mycorrhizal Fungi (AMF) are one of the most essential beneficial soil microorganisms that can form a mutualistic symbiotic relationship with plants. AMF receive carbon from the host plant to complete their life cycle. In return, these fungi have copious roles for the host plants, including plant protection against pathogens, increasing abiotic stresses tolerance (drought and salinity), and enhancing water and mineral nutrient acquisition. Formerly, AMF was identified and classified merely based on morphological features of fungal spores such as mode of formation, wall structure, and subtending hyphal characteristics. Later on, molecular procedures were incorporated for AMF identification. PCR-based techniques led to the direct identification of AMF species that existed in plant roots or the rhizosphere. Several primers have been developed to increase the accuracy of AMF identification. Nowadays, classification systems of AMF are based on both morphological and molecular techniques. This paper aims to review the researches on the identification of AMF in Iran. Identification of AMF has received increasing interest over the past few decades. Authors have used a combination of morphological characteristics and DNA-based techniques to the identification of AMF. So far, more than 115 AMF species belonging to 22 genera have been identified and reported from different regions and plant communities such as croplands, forests, grasslands, etc. Understanding the community composition and diversity of AMF is vital for using them as biofertilizer in agriculture to reduce chemical inputs and increase sustainable crop production

Endophytic fungi are microorganisms with the ability to colonize plant tissues without any symptoms, in whole or part of their life cycle. These fungi have been found in every plant species examined to date. In this study, 417 isolates of endophytic fungi were obtained from healthy and symptomless fruits, leaves and branches of 70 analyzed wild (Malus orientalis) and Iranian endemic (Malus domestica) apple cultivars trees in the north of Iran. Among the identified fungi, species Coniochaeta endophytica and Curvularia hominis were new for the Funga of Iran based on morphological features and molecular data. Furthermore, these species are reported for the first time as endophytic fungi of apple trees in the world.

Molecular techniques are necessary for reliable identification of Trichoderma species. For this, sequencing of several genomic loci, such as ITS, TEF1 and RPB2, are advised, but this is costly when a lot of isolates are to be analyzed. RFLP of the ITS region, on the other hand, is a simple and cost-effective method for differentiation between large numbers of Trichoderma species. In the current work, 77 Trichoderma isolates were classified using the ITS-RFLP technique by combination of three restriction enzymes (MspI, NlaIII and NlaIV). Regarding the results, the isolates were divided into 8 groups. Out of these, 14 representative isolates were selected for sequencing of ITS, TEF1 and RPB2. In order to evaluate the enzymes used in this study, 227 ITS sequences of Trichoderma species from the RefSeq database of NCBI were analyzed. Based on these, about 65% of the tested Trichoderma sequences can be categorized into small groups including less than 10 species and thus allowing a low-cost pre-classification. Our results showed that the ITS-RFLP method and the enzymes used in this study can make easy the differentiation of a large numerous of Trichoderma species. We present the ITS-RFLP method using the combination of three restriction enzymes as an efficient and reliable method for the identification of Trichoderma isolates.

Since the publication of the latest monograph of powdery mildews by Braun and Cook in 2012, no comprehensive revision has been done to check the accuracy of the hitherto recorded species of the genus Erysiphe in Iran. In this study, we present a taxonomic revision of Erysiphe sect. Uncinula in Iran using morphological traits of voucher specimens and ITS-LSU rDNA sequence analysis. Thirty-eight voucher specimens were obtained from the University of Guilan Mycological herbarium (fungarium, GUM) and the Fungus Reference Collection of Herbarium Ministerii Iranici Agriculturae (IRAN), as well as newly collected specimens during 2019–2021, were examined morphologically. The ITS sequences were generated for 26 selected specimens, and the LSU sequences were generated for 16 specimens. Two ITS sequences of E. paradoxa (OM574846 and OM574845), as well as ITS and LSU sequences of E. celtidis (OM574855 and OM574834, respectively), were sequenced for the first time in this study. Precise morphological observations and molecular analyses of Iranian specimens changed the number of accepted species of Erysiphe sect. Uncinula from six taxa in 2009 to 10 taxa in 2021. All species identified from Iran are here described and illustrated. A key to species of Erysiphe sect. Uncinula in Iran is also provided.

Oomycete species occupy many different environments and many ecological niches. The family Saprolegniaceae from Oomycota includes widely distributed water molds which usually behave as saprophytes on plants and animal debris. Members of some species may also be pathogenic for plants and fish. Oomycete species identification based on DNA is well established, but DNA barcoding with cytochrome c oxidase subunit I (COXI) and II (COX II) are a relatively new approach. In this study, 57 isolates were obtained from water samples in mazandaran province, Iran. After morphological identification by morphological keys, ITS, COXI , and COX II gene regions of 10 representatives of the isolates were sequenced and 3 genera and 6 species (Newbya recurva, Achlya bisexualis, Dictyuchus monosporus, Saprolegnia ferax, S. bulbosa , and S. debaryana) were identified through BLASTn in NCBI gene bank. The results described in this paper were indicated that except for ITS, COXI and COXII sequencing could also be valuable resources to Saprolegniaceae identification. Except for S. ferax, other described species were new reports for oomycete biota of Iran.

Nuts are among Iran’s most important crops consumed by many people due to their nutritional and nutraceutical properties. Fungi from the genus Aspergillus contaminate them during pre- and post-harvest stages. Aspergillus species are responsible for various agricultural products' secondary spoilage, and they can produce mycotoxins harmful to humans and animals. The present study evaluated the fungal contamination of nuts marketed in local stores in Kerman. Samples of pistachio, walnut, and hazelnut were collected throughout Kerman province, Iran, to characterize Aspergillus species contaminating nuts marketed in retail shops. Aspergillus species were examined by morphological and molecular criteria to explore the diversity of this genus. The phylogenetic relationships of these species were determined using sequences from partial β-tubulin and calmodulin sequences. Aspergillus species were identified as A. flavus, A. parasiticus, A. arachidicola, A. tamarii, A. caelatus, A. nomius, A. leporis, A. quadrilineatus, A. unguis, A. spelunceus, A.ochraceus, A. auricomus, A. westerdijkiae, A. montevidensis, A. pseudoglaucus, A. subalbidus and A. taichungensis. Populations of Aspergillus species on nuts, how these populations vary among different types of nuts, and their mycotoxin production potential are discussed.

Leptosphaeria maculans is a fungus of the phylum Ascomycota that is a causal agent of blackleg disease on canola (Brassica napus L.). Due to the high diversity and worldwide distribution, L. maculans has been widely studied as a model phytopathogenic fungus. Simple sequence repeats (SSRs) are robust molecular markers widely used for population diversity research. This study utilized whole-genome sequencing data of four Iranian L. maculans isolates (Pk4, Ar3, Ar5, and Alam10). We compared them with the JN3 reference genome to identify and compare different types of SSRs, including perfect (SSRs), compound (cSSR), imperfect (iSSR) and variable tandem number repeats (VNTR) motifs. The average length of SSRs was estimated to be 155.692 kb, accounting for 0.36% of the total genome. An average of 7138 SSR motifs with a frequency of one SSR per 169.5 bp, including an average of 33.86% tri, 25.69% di, 14.48% mono, 10.87% tetra, 8.52% hexa, 6.58% penta-nucleotide repeats, were identified from assembled genomic sequences. Of the total SSRs identified in the Pk4 isolate, 459 motifs were identified in CDS regions. Approximately 13% of the identified SSRs were linked to cSSRs. The average cSSR loci density for four isolates was 487.32 bp/Mb, and C, AG and AC were the most frequent SSR motifs. The assessed isolates' cSSRs lengths ranged from 24 to 295 bp. The largest common cSSRs in four isolates were identified as a motif (GA)26-(CAGAGA)15 with a length of 142 bp. The tri-nucleotide (AAT) was the most common iSSRs motif, followed by di, tetra, mono, hexa, and penta-nucleotides. About 30% of iSSRs contained the AAT, AT, and AAG motifs. Among the 7 to 30 nucleotide motifs, 7, 8, 9, and 10 motifs showed the most occurrences. In addition, 11 motifs with more than 100 nucleotides were found in the studied isolates and the reference genome. The data demonstrate that these results can be used to characterize L. maculans isolates from diverse hosts and geographic locations and are transferable to other isolates of L. maculans.

 Bark beetles dig galleries in bark tissue and can transmit fungi in the forest. Their association with the members of the orders Microascales and Ophiostomatales are among the most exciting examples of symbioses in nature. In the present study, several synnematous fungal isolates were recovered from bark beetle galleries on declining woody hosts in Arasbaran and Toskestan forests in the northwestern and north zone of Iran. By the integration of morphological features with sequence data of ITS-rDNA region and TUB gene, the isolates were identified as Scedosporium minutisporum. A phylogeny inferred based on combined data set of  the ITS-rDNA and TUB sequences clustered our isolate together with reference stain of S. minutisporum in the family Microascaceae. To the best of our knowledge S. minutisporum is new to the mycobiota of Iran. The pathogenic relevance of this

The Didymellaceae is one of the most species-rich families in the Pleosporales, and contains numerous plant pathogenic, saprobic and endophytic specie, inhabiting a wide range of ecosystems. To identify the fungal species of the Didymellaceae in Iran, we investigated nine unidentified phoma-like strains obtained from the Iranian Fungal Culture Collection (IRAN) and six additional strains obtained from various diseased plants in Khuzestan Province, during 2019–2021. The strains under study were recognized by combining the results obtained from multi-gene phylogenetic analyses (ITS, LSU, rpb2 and tub2), and morphological comparisons. Accordingly, six species belonging to four genera of the Didymellaceae were identified and described as follow: Didymella aquatica, D. segeticola, Ectophoma multirostrata, Epicoccum dendrobii, E. tobaicum and Neomicrosphaeropsis Juglandis. All these species are new records for the fungi of Iran. Furthermore, several new hosts are reported for these taxa.

In this study, we re-examined a collection from IRAN herbarium that was identified as Erysiphe deutziae on Deutzia gracilis. Precise morphological studies supplemented with rDNA ITS sequencing disclosed that the powdery mildew on this collection is a member of the genus Arthrocladiella. Moreover, re-examination of host plant confirmed that host plant has been misidentified and it belongs to the genus Lycium. Arthrocladiella is a monotypic member of family Erysiphaceae (Ascomycota, Helotiales), which has only been reported from Lycium spp. (Solanaceae). According to our findings, Arthrocladiella mougeotii infects Lycium sp. in Iran and is reported as a new genus for Iranian Mycobiota.

Pyronema is a genus of fungi in the family Pyronemataceae (Pezizales, Ascomycota) (Yasin et al. 2016). It was described by German naturalist Carl Gustav Carus in 1835. Forty-six and forty-four of Pyronema species are recorded in the MycoBank (https://www.mycobank.org) and Index Fungorum (http://www.indexfungorum.org/names/names.asp) databases, respectively. About forty-six records of Pyronema species have been described by mycologists of various countries, out of them two are valid names under the genus. There are two currently accepted species of Pyronema: P. domesticum and P. omphalodes (= P. confluens). Pyromena species have been known to be rapid responders to fire and grow rapidly in situations where there are few competitors (Bruns et al. 2020). In April 2022, Pyronema ascocarps were observed on a sterile soil provided for tomato planting, in a greenhouse located at College of Agriculture, Razi University, Kermanshah, Iran. Ascocarps were measured, photographed and their macroscopic characters described. The voucher specimen was deposited in the Fungal Collection of the Iranian Research Institute of Plant Protection (IRAN18161F). Total genomic DNA was extracted using a fungi DNA isolation kit (CinnaGen, Iran) according to manufacturer’s instructions. Universal primers of internal transcribed spacers (ITS) of nuclear ribosomal DNA (ITS1: 5̍-TCCGTAGGT-GAACCTGCGG-3̍; ITS4: 5̍-TCCTCCGCTTATTGATA TGC-3̍) (White et al. 1990) were utilized for amplifying two specimens using T-Personal thermocycler (Biometra, Germany). The PCR was performed in a final volume of 25 μM reaction containing 20 ng genomic DNA, 1 μM of each primer, 100 μM of each dNTP, 0.5 U Taq DNA polymerase (CinnaGen, Iran), 1.5 mM of MgCl2, 2.5 μM of 10 × PCR buffer (CinnaGen, Iran), and 14.5 μM H2O. The PCR cycle included one cycle of initial denaturation at 94 for 3 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 60 s, and a final extension of 10 min at 72 °C after cycling. A portion (5 μM) of the amplified product was run on 1% TBE-agarose gel and the presence of a single band (ca. 600 bp) was considered as successful amplification. Apothecia 0.5-1 mm across, cup-shaped, round to lenticular, sessile, pink to red, and forms a mass of few square mm to few square cm and resides on a white surface mycelium. Hymenium usually convex, pink-red with an almost smooth margin. Paraphyses unbranched, straight, cylindrical, hyaline, septate, enlarged at the apex up to 8.78-10.75 μm, and 170 (160-200) µm. Asci cylindrical, non-amyloid, thin-walled, uniseriate, eight-spored, hyaline, and 125 (120-160) × 12 (11-14) µm. Ascospores broadly elliptical, smooth, hyaline, not guttulate, 12.05-17 ×  9.2-11.24 µm (x̄ = 14.31 × 10.24 µm, n = 100), and the ratio of length/width (Q) 1.39 (Fig 1. a-i).     Based on morphological criteria such as the shape and color of the apothecia, spore size and shape, and size of asci and ascospores, the studied specimens found in sterilized soils of greenhouse were identified as Pyronema sp. (Sowerby) Sacc (Moore & Korf 1963). BLAST analysis of ITS region (at least 600 bp in length) revealed 100% identity of our specimens to Pyronema domesticum from Austria (GenBank HQ115722) (Gorfer et al. 2011) and Germany (GenBank MG098284) (Bußkamp et al. 2020). This confirmed the identity of the specimens. Phylogenetic analyses based on ITS sequence of our isolates and 10 selected isolates of Pyronema showed that our isolates are closely related to P. domesticum (Fig. 2). The DNA sequences of examining two specimens RU-PyDo1 and RU-PyDo2 were deposited in GenBank under accession numbers ON368078 and ON368079, respectively. Pyronema domesticum has been associated with charred soil, damp whitewash, wet walls and damp paper, and reported from Germany, India, Indonesia, USA, China, Finland, Ukraine and other countries (Moore & Korf 1963; Dougoud 2011; Dzhagan et al. 2020). Pyronema domesticum and P. omphalodes are indistinguishable microscopically, but in nutrient-rich culture only P. domesticum forms sclerotia (Moore & Korf 1963). In this study, sclerotia formed on potato dextrose agar (PDA) after seven days in slant tube (Fig. 3 a-d). To our knowledge, this is the first report of P. domesticum from Iran.