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ŠUMARSKI LIST 3-4/2019 str. 64     <-- 64 -->        PDF

fragments did not allow adequate differentiation of Armillaria species. For identification of species of Armillaria group digestion of IGS region with AluI was used (Table 3). According to this results A. ostoyae and A. cepistipes were identified as wood rot fungi of Norway spruce.
Among 37 analysed samples, 28 resulted in successful fungal DNA isolation. Twelve DNA isolates were identified as species of Heterobasidion or Armillaria. Other causitive agents of wood decay, according to size of DNA fragments of ITS region, belonged to group of other fungi. Identification of species of this group is not done yet (Table 4).
DNA of wood decay fungi was isolated directly from infected wood. In most cases, enough quantity of DNA was obtained from decayed wood mass of 10-20 mg, but in several samples DNA wasn’t isolated because of small quantity of fungal tissue in the wood. Identification of Heterobasidion species was carried out using species specific primers. Specifity of primers MJ-F and MJ-R for identification of H. annosum (100 bp fragment) and primers KJ-F and KJ-R for identification of H. parviporum (350 bp fragment) was confirmed for use in Bosnia and Herzegovina. Previously, specificity of these primers was confirmed by Hantula and Vainio (2003) for use in the Scandinavian region.
Nine of 37 DNA samples isolated from decayed wood of Norway spruce (Table 3) were identified as H. parviporum, which was expected, since this species most commonly occurs on Norway spruce (Łakomy and Werner, 2003; Niemelä and Korhonen, 1998; Korhonen et al., 1997). According to Lockman et al. (2016) fungi of the genus Heterobasidion in mixed stands of conifers are causative agents of wood decay on about 9% of the trees, while in the stands of Norway spruce they cause rot on about 14% of the trees. Lehtijarvi et al. (2012) found in Abies nordmanniana ssp. bornmülleriana that 33% of the decay on the trees was caused by fungi of the genus Heterobasidion. H. annosum was not detected within the object of research, as it occurs most frequently on Scots pine (Łakomy and Werner, 2003; Niemelä and Korhonen, 1998; Korhonen et al., 1997). Specificity of the primers for this species was tested on DNA isolated from the decayed wood of Scots pine, collected from the site near Travnik (Table 2, Figure 1).
In case of Armillaria species, there are numerous methods of identifying these species using the techniques of PCR and AFLP or RFLP. ITS region is less used in studies of the genus Armillaria because it is more uniform and requires the application of a large number of enzymes for its digestion. More recently, for the identification of species of this genus IGS-1 region of DNA is used. PCR amplification of this region is achieved with primers LR12R and O-1 (Duchesne and Anderson, 1990). The best results in the digestion of IGS region are obtained with AluI. Treštić (2006) performed the reliable identification of fungi of the genus Armillaria by analysis of differences in the structure of the ITS and IGS regions of rDNA which was previously obtained from rhizomorphs. In the identification process, the following endonuclease were used: AluI, TaqI and HinfI. Three species were identified: A. cepistipes, A. gallica and A. ostoyae. In the research of Keča et al. (2006), in Serbia, A. cepistipes, A. ostoyae, A. mellea, A. gallica and A. tabescens were identified by analysing IGS region of their DNA.
For the purposes of this research, Armillaria fungi were determined, in the first step, according to the size of the ITS region obtained by using primers ITS1 and ITS4. DNA fragments were 820-860 bp. Reliable identification of A. ostoyae and A. cepistipes was carried out by digestion of the IGS region with endonuclease AluI (a second step).
Both species appear on conifers, but A. cepistipes is more characteristic for Norway spruce. The results obtained in this research are not sufficient for detailed studies of the structure of the populations of the present species of fungi of the genera Heterobasidion and Armillaria, but they provide basic data for future research in terms of optimizing the process of identifying fungi species of these two genera. PCR amplification of the ITS region of numerus isolates of DNA resulted in products of 630-650 bp. Determination of the species of fungi from this group would require significant material costs and therefore was not carried out. Out of 37 analysed samples 17 belong to this group (Table 4). According to Jasalavich et al. (2000), which conducted research using primers ITS-1F and ITS-4B, it was found that these primers can detect many fungi that cause white and brown rot. The extraction of the DNA was unsuccessful on 8 samples. It was not clear which group of microorganisms cause decay of wood on these samples. It is possible that they are decayed by bacteria or that the fungal DNA is significantly damaged and decomposed by their activity, which prevented its extraction.
According to the results of this research, the reliable identification of species of genus Heterobasidion can be made by