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Ekim 2017 2.Cdr Ekm(2017)8(2)137-142 Do :10.15318/Fungus.2 017.44 01.06.2017 25.09.2017 Research Artcle Molecular Identification and Phylogeny of Some Hypocreales Members Isolated from Agricultural Soils Rasime DEMİREL Department of Biology, Faculty of Science, Anadolu University, TR26470 Eskişehir, Turkey Abstract: Hypocreanlean fungi are include important plant pathogens around the world. Head blight and crown rot disease of cereals caused by these species are responsible for large economic losses due to reduction in seed quality and contamination of grain with their mycotoxins. Although morphological and biochemical tests are still fundamental there is an increasing more towards molecular diagnostics of these fungi. This paper reviews to PCR identification of Hypocreanlean fungi isolated from agricultural soil from Eskisehir City. Five Hypocreanlean fungi belong to 4 different genera as Bionectria, Fusarium, Gibberella and Nectria were isolated from 56 soil samples. DNA of these strains were isolated by glass beads and vortexing extraction method and used for PCR amplification with universal fungal specific primers. The internal transcribed spacer (ITS) regions of fungal ribosomal DNA (rDNA) were sequenced by CEQ 8000 Genetic Analysis System. The ITS-5.8S sequences obtained in this study were compared with those deposited in the GenBank Database. Phylogenetic position of investigated closely related Hypocreanlean fungi was determined. Key words: Hypocreanlean; PCR; ITS; Phylogeny; Eskisehir Tarımsal Topraklardan İzole Edlen Bazı Hypocreales Üyelernn Moleküler Teşhs ve Flogens Öz: Hypocreanlean mantarları Dünya'dak öneml btk patojenlerdr. Bu türlern sebep olduğu baş tahrbat ve tahıl hasar hastalığı, tohum kaltesnde azalma ve tahılın mkotoksnler le bulaşması nedenyle büyük ekonomk kayıplardan sorumludur. Morfolojk ve byokmyasal testler hala temel olsa da, bu mantarların moleküler teşhsne yönelm gderek artmaktadır. Bu makale, Eskşehr'dek tarım topraklarından zole edlmş olan Hypocreanlean mantarlarının PCR le dentfkasyonunu ncelemektedr. Bonectra, Fusarum, Gbberella ve Nectra olmak üzere 4 farklı cnse at 5 Hypocreanlean mantarı 56 farklı toprak numunesnden zole edlmştr. Bu suşların DNA'sı, cam boncuklar ve vorteks ekstraksyon yöntem le zole edlmş ve ünversal fungal spesfk prmerler le PCR amplfkasyonu çn kullanılmıştır. Fungal rbozomal DNA'nın (rDNA) ç transkrpsyonlu ayırıcı (ITS) bölgeler, CEQ 8000 Genetk Analz Sstem le dzlenmştr. Bu çalışmada elde edlen ITS-5.8S dzler, GenBank ver tabanında depolanan dzlerle karşılaştırılmıştır. İncelenen brbrne yakın akraba Hypocreanlean mantarlarının flogenetk konumu belrlenmştr. Anahtar kelmeler: Hypocreanlean; PCR; ITS; Flogen; Esksehr Introduction Gibberella, Nectria and Bionectria that are The best-known Hypocreanlean fungi mainly seen in agricultural, ecological, or are a broad order that are include members of biodiversity studies (Howard, 2002; Stone et Fusarium and Acremonium genera. They are al., 2004). anamorphs of teleomorph genera, such as Corresponding Author: [email protected] 137 Ekm(2017)8(2)137-142 These genera are widely distributed in soil al., 2005). All strains were stored in suitable and on organic substrates and have been conditions at the Culture Collections of KUKENS isolated from permafrost in the arctic and from (WDCM101), the Centre for Research and the sand of Sahara. They are amongst the fungi Application of Culture Collections of most frequently isolated by the plant pathologist. Microorganisms. Cultures were maintained at The predominant interest the genus has been 4°C on potato dextrose agar (PDA) for use in the and still is in their role as plant pathogens (Booth, present study. 1971; Ismail et al., 2015) as well as there are DNA Extraction, PCR Amplification species which are highly mycotoxigenic, and Sequencing producing a range of toxins affecting wildlife, Genomic DNA extraction were conducted livestock and humans (Antonissen et al., 2014). with strains grown on PDA for 7 days at 25ºC The current fungal taxonomic systems have using a modified method of Van Burik et al. been still identified by macroconidia and (1998). DNA concentration were estimated m i c r o c o n i d i a i n t h e a s e x u a l s t a g e , visually in 1% agarose gels containing 5 µg/mL morphological character of chlamydospore, host ethidium bromide by comparing band intensity range, and secondary metabolites. However, the with known quantities of DNA high range plasticity and intergradations of the phenotypic markers and the extracted DNA was stored at traits offered difficulty in identifying the −20ºC. To examine the phylogenetic relationship filamentous fungi (Ismail et al., 2015; Young et among the test strains of Fusarium, the nuclear al., 2000). In addition, because of their capacity ribosomal ITS1-5.8S-ITS2 region was amplified for rapid change, species identification presents w i t h p r i m e r s I T S 1 ( 5 ´ - certain problems (Booth, 1971; Hsuan et al., TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´- 2011). For these reasons, the molecular TCCTCCGCTTATTGATATGC-3´) (White et al., biological method has been recently introduced 1990). PCRs were performed using Techne in Hypocreanlean fungi systematic and the Thermal Cycler (Techgene, Techne, UK) in 25- molecular variation at the DNA level has been mL solution containing 1 µL of genomic DNA, 2.5 studied in many works (Young et al., 2000). In µL of 2.5-µM forward and reverse primers, 2.5 µL addition to DNA sequencing, phylogenetic of Taq buffer + KCl-MgCl2 (Fermentas), 2.5 µL of analyses have been supported strong 25-mM MgCl2 (Fermentas), 2 µL of 2.5-mM information about genetic relationship of closely dNTPmix, 0.25 µL of 5-U/µL Taq DNA related Hypocreanlean fungi (Hsuan et al., polymerase (Fermentas) and 11.75 µL of sterile 2011). This paper evaluates the use of ITS deionised water. The amplification conditions sequences for identification and phylogenetic consisted of initial denaturation at 95 ºC for 2 analysis of closely related Hypocreanlean fungi min, followed by 35 cycles of denaturation at 95 isolated from agricultural soils in Eskisehir ºC for 1 min, annealing at 55 ºC for 45 s, province. extension at 72 ºC for 1.30 s and final extension at 72 ºC for 5 min. To confirm the amplification, 5 Material and Method µl of the PCR product together with marker Fungal Strains (GeneRuler DNA Lader 50 bp Fermentas) was All of the strains used in this study were resolved by gel electrophoresis on 1% agarose obtained from agricultural soils in Eskisehir gel containing 5 µg/mL ethidium bromide in 1X province and identified using traditional methods TAE buffer. Gel were photographed by Gel according to the Booth (1971), Gerlach & Documentation system (Uvitec M02 4611) Nirenberg (1982) and Nelson et al. (1983). (Demirel, 2016). Additional information on these and related strains can also be found elsewhere (Demirel et 138 Ekm(2017)8(2)137-142 After agarose gel blocks containing DNA phylogenetic tree based on ITS region was fragment were cut out and purified with Promega structured at higher divergence levels. For Wizard® SV Gel and PCR Clean-Up System, investigated mainly closely related members, Cycle sequencing products were purified with identical positions and four sections for specific Dynabeads® Sequencing Clean-Up to remove clades such as Bionectria, Fusarium, Gibberella unincorporated dye-labeled nucleotides. Then, and Nectria genera were found. all the sequencing reactions were performed The genus Fusarium is the anamorph using CEQ™ DTCS Quick Start Kit (Beckman stage of Gibberella genus (Samuels et al., 2001). Coulter) by CEQ 8000 Genetic Analysis System. The members of these genera are known as Data Analysis main and wide plant pathogens (Howard, 2002; The ITS sequences were blasted with Stone et al., 2004; Dragich and Nelson, 2014; GenBank sequences (Altschul et al., 1990) to Chehri, 2016). These two genera have been verify their identity; the closest Blast results are distinguished with especially teleomorph reported for each taxon (Table 1). The structures of Gibberella genus. The complexity alignments were performed using the Muscle in about the their morphologic and microscopic MEGA 6.0 software package, together with the identification has been related with varies other sequences of morphologically and problems such as depending on the host, loosing phylogenetically related species that were of stock cultures, limitations associated with obtained from NCBI GenBank (Tamura et al., morphological characters (Summerell et al., 2013). The aligned data sets were investigated 2003; Hsuan et al., 2011; Antonissen et al., using ML analysis based on the Tamura–Nei 2014). The findings of this study demonstrated model (Tamura and Nei, 1993) as implemented the efficiency of ITS region and phylogenetic in the MEGA 6.0 with 1000 bootstrap analysis of belong to these two genera. Figure 2 replications. All the positions containing gaps shows that Fusarium and Gibberella genera and missing data were eliminated. Fusarium have considerably identical topology with the ITS oxysporum (KT794176) was used as the out locus. The genus Gibberella occurred in two group. The obtained sequence data have been main clades and two clear divergences, namely deposited in GenBank with accession numbers. Gibberella avenacea and Gibberella tricincta, were noted. Furthermore, investigated members Results and Discussion of Fusarium and Gibberella genera are The PCR products (570 bp) were obtained polyphyletic. from all of the species by using the universal The genus Nectria
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