DIGITALNA ARHIVA ŠUMARSKOG LISTA
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ŠUMARSKI LIST 3-4/2019 str. 63     <-- 63 -->        PDF

for 30 seconds, annealing for 35 seconds at 67°C, and extension at 72°C for 1 minute.
The most frequently used amount of primers, water and extract DNA in micro tubes for PCR for Armillaria spp. (White et al., 1990; Treštić, 2006; Coetzee et al., 2005) were: a) ITS region - 1 µl of primer ITS-1, 1 µl of primer ITS-4, 8 µl of extract DNA and 15 µl of distilled water;  b) IGS region - 1 µl of primer LR12, 1 µl of primer O-1, 8 µl of extract DNA and 15 µl of distilled water. Amplification of ITS region of the species Armillaria were performed by PCR reactions consisting of an initial denaturation at 95°C for 2 minutes and 30 seconds, 30 cycles of amplification (each cycle consisted of denaturation at 95°C for 30 seconds, annealing for 30 seconds at 55°C, and extension at 72°C for 30 seconds), and a final extension at 72°C for 5 minutes. Amplification of IGS region of the species Armillaria were performed by PCR reactions consisting of an initial denaturation at 95°C for 1 minutes and 35 seconds, 30 cycles of amplification (each cycle consisted of denaturation at 95°C for 30 seconds, annealing for 40 seconds at 60°C, and extension at 72°C for 2 minutes), and a final extension at 72°C for 10 minutes. PCR products were separated by electrophoresis in 2% (wt/vol) agarose gels in 1X TBE (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) with ethidium bromide (EtBr) at 100 ng/ml in the gel and running buffer. DNA bands were visualized by the fluorescence of the intercalated EtBr under UV light and photographed.
PCR products of amplification of IGS region for Armillaria were digested by endonuclease AluI (Amersham Bioscience) in thermocycler. The mixture in tubes for this reaction was prepared by adding the following components: 0.5 µl of the corresponding enzyme-endonuclease, 2.0 µl of the supporting buffer to enzyme, 8.0 µl of PCR product, 9.5 µl of distilled water (dH2O) and then incubated for 6 h at 37°C. Restriction fragments were separated by electrophoresis in 2% (wt/vol) agarose gels in 1X TBE (89 mM Tris-borate, 89 mM boric acid, 2 mM EDTA) with ethidium bromide (EtBr) at 100 ng/ml in the gel and running buffer. DNA bands were visualized by the fluorescence of the intercalated EtBr under UV light and photographed.
More reliable interpretation of the profiles on the agarose gel was achieved by adding marker in the two outer lanes. In this study the marker 100 bp DNA Ladder (Carl Roth GmbH + Co. Kg) was used.
Rezultati
Results
In order to identify Heterobasidion by PCR amplification of ITS region using total DNA isolated from colonized Norway spruce wood we surveyed 37 samples. The size of segments of ITS region of fungus after amplification are shown in Table 2 and Figure 1. The lanes on the scheme are marked with the first letters of the scientific name (e.g. H. parviporum – H.p.), whereby the first and last lanes labelled “M” represent 100 bp DNA Ladder.
PCR amplification of ITS region of Armillaria spp. DNA resulted in 820-860 bp DNA fragments. These DNA