نخستین گزارش از گونه Neocosmospora vasinfecta جداسازی شده از خاک‌های بکر در ایران

Abstract

Neocosmospora E.F. Smith is a filamentous ascomycete fungal genus belong to Hypocreales order and contains several species mainly pathogenic for plants (Cannon & Hawksworth 1982). Species of Neocosmospora are known to live in the soil of tropical or subtropical areas and often in association with plant roots. During summer 2017, for isolation of Fusarium species from uncultivated soil (foothill) in Qasr-e Shirin (Kermanshah province, Iran), we recovered one isolate of Neocosmospora. Soil samples were collected from 0–20 cm depth. The isolates were recovered using a soil dilution plate method directly from uncultivated soil. Isolation was performed from soil using komada and potato dextrose agar. Genomic DNA was extracted using CTAB method (Garde et al. 1991). The internal transcribed spacers (ITS) of nuclear ribosomal DNA and apart of the Ef1-α translation elongation factor (TEF1) gene were amplified by PCR using the primer pair ITS1 (5-TCCGTAGGT-GAACCTGCGG-3)/ITS4 (5-TCCTCCGCTTATTGATATGC-3) (White et al. 1990) and EF1F (5-ATGGGTAAGGAGGACAAGACTC-3)/EF1R (5-TGGAGATACCAGCCTCGAAC-3) (O’Donnell et al. 2008) 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. Amplifications were conducted in a T-Personal thermocycler (Biometra, Germany) for 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, with initial denaturation of 3 min at 94 °C before cycling 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. 540 bp) was a check for successful amplification. Phylogenetic analyses were conducted with maximum likelihood (ML) in the Molecular Evolutionary Genetic Analysis (MEGA) Ver. 5 program (Tamura et al. 2011). The best fit nucleotide substitution model (Jukes-Cantor) was based on the bayesian information criterion and was implemented in MEGA 5. Sequence produced in this study is deposited in GenBank under accession numbers MG811579 (ITS region), and MH976665 (Ef1-α). Mycelium white to creamish (Fig. 1A). Perithecia orange to pinkish red, globose to subpyriform with small neck, ostiolate, 240–350 µm tall and periphyses present (Fig. 1B). Asci cylindrical, 8-spored, thin-walled, not amyloid, with truncated apex with a short pedicel, and 80–100 × 10–12 μm (Fig. 1C). Ascospores were uniseriate, rugose ornamentation, globose to ellipsoidal, hyaline when immature and brown at maturity, sometimes with oil droplets, and 11.23(10–12.58) × 9.63(8.03–10.45) µm (Fig. 1D). The fungal isolate was identified as N. vasinfecta using morphological characteristics and sequence analysis of ITS region and apart of the Ef1-α translation elongation factor. Based on a BLASTn search of NCBI GenBank nucleotide database, the closest sequence to our fungus (GenBank Accession Nos: MG811579 for ITS, and MH976665 for Ef1-α translation elongation factor) was N. vasinfecta isolated from soil from India (GenBank KM231804; identities = 99%) and France (GenBank JX997934; identities = 100%). The phylogenetic analysis of the sequenced ITS fragment positioned the our isolate within the N. vasinfecta clade (94 %) (Fig. 2). Neocosmospora vasinfecta has been reported from soil in South Africa and India (Lombard et al. 2015), clinical materials in France and Senegal (Ben Hamida et al. 1993, Dromer et al. 1997, Kac et al. 1999, Gabriel et al. 2013), and animal dungs (Doveri 2011). This species have been also reported as phytopathogenic fungus from peanuts in Vietnam, Australia, South Africa and Taiwan (Dau et al. 2010, Fuhlbohm et al. 2007, Huang et al. 1992, Baard & van Wyk 1985), Arachis hypogaea plant in Guinea (Lombard et al. 2015), and other plants (Manikandan et al. 2011). A culture of the fungus is deposited at the Iranian Fungal Culture Collection, Iranian Research Institute of Plant Protection, Tehran, Iran. To our knowledge, this is the first report of N. vasinfecta from Iran. Specimen examined: Iran: Kermanshah province, Qasr-e Shirin, 45° 95΄ E, 33° 96΄ N, 26.06.2017, from uncultivated soil, Sh. Saeedi (IRAN 3320 C).