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ŠUMARSKI LIST 3-4/2019 str. 62     <-- 62 -->        PDF

is why we need a fast and accurate method for identification of pathogens. Techniques used to identify fungal organisms based on morphological approach are time consuming and require a lot of knowledge of the taxonomy of the pathogen. Other limitations include the difficulty of some species to be grown in vitro (Goud and Termorshuizen, 2003). All limitations and disadvantages of morphological techniques have led to the development of molecular methods for pathogen identification.
The advantage of molecular analysis is that wood decay can be detected in the early stages of development (Tomikawa et al., 1990; Mattheck and Breloer, 1993; Habermehl et al., 1999). Different molecular methods for the detection, identification and quantification of plant pathogenic fungi, are much faster, more accurate and more sensitive. This is one of the reasons why molecular methods are used in our research. Detection of fungal organisms by analysis of DNA isolated directly from the wood were used by other authors too (Nowakowska et al., 2013; Nicolotti et al., 2009; Hantula et al., 1999 and 2003; Guglielmo et al., 2007 and 2009; Škipars et al., 2011).
Decaying fungi reduce the vitality of infested trees and damage the best part of the tree that is used in the production of timber. Because of this multiple influence it is necessary to explore the infectious potential of these fungi in the forests of Bosnia and Herzegovina, in order to be able to prevent and reduce the damage they cause. Spruce in Bosnia and Herzegovina represent one of economically most important trees. Because of that, this research is focused on role and importance of spruce wood rot fungi.
Research Area and Methods – Područje i metode istraživanja
The research was carried out in the stands of Norway spruce in the area of ​​the mountain Zvijezda, the forest management area “Gornjebosansko”, Vareš municipality (Table 1). The object of research was department 65 of economic unit “Gornja Stavnja”, where secondary forest of fir and Norway spruce exists on predominantly deep distric cambisole, luvisols, pseudogleys and podsols on silicate and silicate-carbonate sedimentary substrates and acidic volcanic stones. A total area of department 65 is ​​39.38 ha. Determining the presence of decay was done on harvested trees of Norway spruce in a systematic network of 100 m x 100 m. Near the intersection points in the network, Norway spruce tree with specific symptoms of decay fungi (decayed wood, fruiting bodies, mycelium, and rhizomorphs) was selected.
The selected trees were harvested and then a sample of wood decay was taken. The samples were collected from three sites of the part of the stem that was affected by the process of decay (base, middle and peak zone of decay). At the sampling points the discs of wood thickness of 5 cm were cut. Then from each disc the three samples of wood decay were taken (a total of 9 samples per tree). The wood samples were packed in separate bags with data/information on: number of the tree, the part of the stem affected by the decay, as well as the number of the sample.
Probes for DNA isolation were prepared according to the methodology of Tel-Zur et al. (1999). The DNA probe weight was weighed by Sartorius BP 410 with accuracy of 0.01 grams. The DNA probe consisted of a mixture of wood mass of 10-20 mg. From the three samples of wood obtained from the same disk, a unique probe for DNA isolation was prepared. During the preparation of wood mass for isolation of DNA grinding of wood probes by grinder (TissueLyser, Qiagen) served as good solution for avoiding cross-contamination of samples. Besides the wood samples, fruiting bodies of species of the genus Heterobasidion were collected from locality Igman (Table 1). Isolation of DNA from fruiting bodies enables reliable testing of the specificity of the primers used for identification of species of the genus Heterobasidion. Presence of the species Heterobasidion annosum was confirmed on a sample of decayed wood from the Scots pine that was collected in the vicinity of Travnik because it is one of most important decaying fungi on this tree species (Table 1).
Table 1. Locations on which were collected samples of wood decay and fruiting bodies
Tablica 1. Lokacije na kojima je vršeno prikupljanje uzoraka truleži drveta i plodonosnih tijela
The analyses of samples were carried out in the laboratory of the Faculty of Forestry, University of Sarajevo. Amplification of the target segments of DNA was performed in the tubes with prepared reagents (ReadyToGo PCR beads, Amersham Bioscience). The tubes supplemented to 25 µl contained: 1.5 units of Taq DNA polymerase, 10 mM Tris-HCl (pH 9.0 at ambient temperature), 50 mM KCL, 1.5 mM MgCl2, 200 uM dNTPs and 200 uM stabilizer.
The most frequently used amounts of primers for the ITS region, water and extract DNA in micro tubes for PCR of Heterobasidion spp. were: 1 µl of primer MJ-F, 1 µl of primer MJ-R, 1 µl of primer KJ-F, 1 µl of primer KJ-R, 8 µl of extract DNA and 13 µl of distilled water. This protocol was used by Hantula and Vainio (2003). PCR reactions consisted of an initial denaturation at 95°C for 10 minutes, 40 cycles of amplification, and a final extension at 72°C for 7 minutes; each cycle of amplification consisted of denaturation at 95°C