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ŠUMARSKI LIST 3-4/2019 str. 11     <-- 11 -->        PDF

extension step at 72 °C for 5 min. The resulting PCR products were sequenced using primer ITS 4 at the DNA sequencing facility of Macrogen Europe (Amsterdam, Netherlands). After processing raw data using the BioEdit Sequence Alignment Editor v.7.2.5 software (Hall 1999), sequences were identified by comparison with reference sequences in NCBI GenBank using BLAST tool (Altschul et al. 1990). Sequences with 98 – 100% similarity were identified to the species level and with 94 – 97% of similarity to the genus level (Bakys et al. 2011).
Analysis of DNA from seeds – Analiza DNA iz sjemena
Twenty seeds from each of five locations were analyzed for fungal presence using a nested PCR method. After surface disinfection of samaras by immersing them in 35% H2O2 for three minutes, seeds were aseptically removed, cut into small pieces (1 – 2 mm long), placed in separate 2 ml centrifuge tubes and freeze-dried for 24 h (Cleary et al. 2013). Samples were homogenized in TissueLyser II (Qiagen, Hilden, Germany) at 30 Hz for two minutes. DNA was extracted following the protocol according to Minas et al. (2011). First PCR was conducted using the primers ITS1-F (Gardes and Bruns 1993) and ITS 4 (White et al. 1990) under the same cycling conditions and with same reagents concentrations as in the described PCR protocol used for DNA analysis of isolated mycelia. The PCR products were size separated by gel electrophoresis on 2% agarose gels stained with GelStar Nucleic Acid Gel Stain (Lonza, Rockland, USA) and visualised under UV light. All bands were aseptically excised from the gel, purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA) and re-amplified in a second PCR using the primers ITS 1 and ITS 4 (White et al. 1990) under the same cycling conditions and with same reagents concentrations as in the first one. The resulting PCR products were sequenced using primer ITS 4 at the DNA sequencing facility of Macrogen Europe (Amsterdam, Netherlands) and identified using NCBI GenBank database as already described in this paper.
Detection of Hymenoscyphus fraxineus in seeds – Utvrđivanje prisutnosti gljive Hymenoscyphus fraxineus u sjemenu
DNA extracted from seeds, as previously described, was additionally checked for the presence of Hymenoscyphus fraxineus in a PCR reaction with species specific primers: forward (5’AGCTGGGGAAACCTGACTG) and reverse (5’ACACCGCAAGGACCCTATC) (Johansson et al. 2010), and with same reagents concentrations as in previous analysis. The thermal cycling was carried out as follows: an initial denaturation step at 94 °C for 5 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 60 s, extension at 72 °C for 30 s and a final extension step at 72 °C for 7 min (Hayatgheibi 2013). DNA of confirmed Hymenoscyphus fraxineus isolate obtained from earlier research (isolated from Fraxinus angustifolia stem collar, Kranjec 2017) was used as a positive control in each PCR reaction. PCR products were run on 1% agarose gels stained with GelStar Nucleic Acid Gel Stain (Lonza, Rockland, USA) and visualised under UV light.
RESULTS
REZULTATI
Analysis of Fraxinus angustifolia seeds by mycelia isolation on MEA medium and nested PCR revealed fungal presence in 20 – 58% of screened seeds, depending on the method used and location they originated from (Table 2). Isolation of mycelia on MEA medium resulted in growth of 26 fungal isolates belonging to 15 different taxa, 10 of which were identified to the species level (Table 3). The nested PCR analysis resulted in identification of 19 different fungal taxa, 10 of which were identified to the species level (Table 4).
Most frequently detected taxa were Sphaerulina berberidis and Alternaria sp. with Alternaria alternata and A. tenuissima identified to the species level. Among the most frequently detected were also seven sequences obtained in nested PCR which corresponded to Fungal endophyte isolate 4480 according to NCBI GenBank and might be a species of genus Sphaerulina, which is next closest match in the given database. Species of Alternaria occurred in the seeds from all five locations included in this research and Sphaerulina berberidis occurred in seeds from four of those locations (not confirmed only in seeds from stand HR-FAN-SI-111/030 in Vukovar).
Neither of sequences obtained by first two described methods belonged to Hymenoscyphus fraxineus. Presence of this pathogenic fungus in seeds was not confirmed by using