DISTRIBUTION OF HETEROBASIDION GENETS ON A NORWAY SPRUCE SITE : CASE STUDY IN NATIONAL PARK “ KOPAONIK “

Heterobasidion annosum s.l. causes great economic loss in coniferous forests worldwide. Recent studies showed that three European Heterobasdion species are present in forest ecosystems in Serbia. Aim of this study was to find which Heterobasidion species are present in studied Norway spruce stand (National Park “Kopaonik“), to identify Heterobasidion genets, and present distribution of genets.


INTRODUCTION
Root and butt rot caused by Heterobasidion annosum is, in economic terms, the most impоrtant disease of conifers in the forests of northern temperate regions.Other fungal pathogens may be of greater local importance, but Heterobasidion occurs in most, if not all managed coniferous forests of the northern hemisphere, as far north as central Finland, and south as far as northern Africa and Central America (Wo o d w a r d et al. 1998).
During last 25 years our picture of the root-rot fungus H. annosum and the honey fungus Armillaria mellea has changed considerably because both of these fungi has been split into several species (K o r h o n e n , 2004).Because morphological differences were ГЛАСНИК ШУМАРСКОГ ФАКУЛТЕТА, БЕОГРАД, 2008БЕОГРАД, , бр. 98, стр. 117126 BIBLID: 03534537, (2008)) , 1990).Most recent studies showed that all three species are present in coniferous forests in Serbia (K e č a , 2008).
Studies about population structure, size of genets, spread and distribution of Heterobasidion haven't been performed earlier in Serbia.Fifty to 60% of the Heterobasidion genets identified in the final cutting Norway spruce stands had infected only one tree (P i r i et al., 1990).Most of available data are obtained from cultures and from Scandinavian countries.There are almost no data about structure and size of Heterobasidion genets from South-East Europe and also from Balkan region.
The aim of present case study was to obtain detailed information about the presence, size and spatial distribution of Heterobasidion genets in an selection Norway spruce forest.Heterobasidion genets are mapped and data about tree condition are collected.

Study site
This study was carried out in a Norway spruce stand in the National Park "Kopaonik".Compartment № 96 (Management Unit "Samokovska reka") is on slope (1115°), north aspect, altitude ranging 1,500-1,520 m a.s.l.Parent material -granite, compact structure without decomposition.The site was of the picetum excelsae oxalidetosum (Miš.et Pop.) on a brown podzolic soil, which is a typical spruce site for Serbia.According to the last forest inventory survey stand density is 493 trees•ha -1 , volume is estimated on 409,5 m 3 •ha -1 .Diameter of average tree is 29 cm on breast height and height is estimated on 20,5 m.

Field work
The study plot, 90×40 m in size, was established in May 2006.Trees showing chlorosis, declining processes, and all wind -and snow -snapped trees were mapped and the diameter of the stumps and trees (breast height) was recorded.For isolation of the decay fungus, wood samples were taken aseptically from the stem base and main roots of all mentioned categories of trees with an increment borer.Isolation was also performed from all carpophores found on site.In total, 23 living trees were examined, plus 6 windsnapped trees, 5 recently died trees and 2 carpophores.

Isolation and identification of Heterobasidion genotypes
Heterobasidion were isolated onto a modified selective medium (H u n t , C o b b , 1971) containing (per liter): 20 g malt extract (Merck, Darmstadt, Germany) and 15 g agar (Torlak, Belgrade, Serbia), 5 mg Benomyl WP 50 (Zorka, Šabac, Serbia), 4 mg dichloran (Merck).Following autoclaving at 105 kpa for 20 min, 100 mg•L -1 of streptomycin sulphate was added aseptically.Wood fragments, taken with increment borer, were surface sterilized in sodium hypochlorite (2 g•L -1 active chlorine) for 1 minute and small tissue fragments were removed aseptically before placing on selective medium.Cultures were incubated at room temperature for two weeks and emerging hyphae transferred into glass tubes containing 1.5% MeA and stored at 4°c.The genets were identified with somatic incompatibility test, by pairing the isolates in all possible combinations on MAE and recording the line of demarcation.The isolates were identified by pairing with 4 haploid tester isolates from the three european Heterobasidion species (isolates provided by K. Korhonen, Finish Forest Research Institute, Vantaa, Finland).

RESULTS
Eighteen Heterobasidion isolates and 12 isolates of rotting fungi were collected, each from a separate tree or other substrate (Table 1).All the Heterobasidion isolates found on the study plot belong to the species H. parviporum.There was no significant diference (data not shown) between isolation of H. parviporum from healthy (visually) -8, chlorotic -3, declining -3 and wind-snapped -4 trees.Sprophores, rather big, were found only on root of wind-snapped trees.During three year survey it is observed that, in this forest type, carpophores formation starts from end of August and lasts till end of October.
Eleven Heterobasidion genets were identified on the study plot (Figure 1).Five genets included only one tree, other five two trees and only one genet occupied larger area infecting three adjacent spruce trees.In the cases where two trees were occupied by the same Heterobasidion genet, excavation showed that, in four of five cases, root contact was present between spruce trees.Vegetative spread through root contact may be more important in selection forests than in even-aged stands and conifer plantations, because young trees are always in contact with roots of old potentially infected trees.
Three species causing rot of wood were found on declining and decaying trees sampled on studied site (Table 1).Fomitopsis pinicola (Swartz:Fr.)Karst. is one of the most frequently observed lignicolous fungi in the confer forests in Serbia.This species was observed on declining spruce causing intense brown rot.Rhizomorphs of Armillaria cepistipes Velen.were present as epiphyte on roots of declining spruce trees.One isolate of A. cepistipes was obtained from decaying wood sample.clymacocystis borealis (Fr.)Kotl.& Pouzar may cause root and butt rot in old forest at all altitudes.

DISCUSSION
Eleven Heterobasidion genets were present in the relatively small studied plot in spruce stand in National park "Kopaonik".Fifty to 60% of the Heterobasdion genets identified in stands at the end of rotation had infected only one tree (Va s i l i a u s k a s , S t e n l i d , 1998).In present study only one genet had spread on tree neighbouring trees and occupied area of about 120 m 2 .Only a few larger genets encompass 10 or more trees have been identified, but even on old spruce sites they seem to be rather uncommon (S t e n l i d , 1985, P i r i et al ., 1990).Observation of many small genets indicate that basidiospores play important role in establishing of new infections.Further development of genets is based on vegetative spread through root contact.In selection forests managers should be careful, because naturally regenerated advance-growth spruces are susceptible to infection (P i r i , K o r h o n e n , 2001) and the vegetative spread from old trees or stumps cannot be excluded because of root contact.
Management in selection forests should provide conditions which will favour stump decomposition.This study showed that even relatively small stumps (diameter ~30 cm) produced carpophores 27 years after tree was cut.In southern Finland it is estimated that Heterobasidion in not able to survive in spruce stumps for more than 50 years (p i r i , 1996).Studied carried out in Sweden have shown that the growth rate of Heterobasidion in the roots of Norway spruce increases almost three time after tree is cut (B e n d z -H e l l g r e n et al., 1999).young spruce trees growing around infected old stumps could be easily infected with Heterobasidion through root contact.P i r i and K o r h o n e n (2007) concluded that only stumps with greatest infection potential and with sufficient root contact are able to transfer the disease to the next stand generation.Some other authors also stress that spore infection is less important, for mortality in next generation, than infection established in roots before felling (G r e i g , B u r d e k i n , 1970).young trees may not necessarily decline and die, but infection could spread into the central part of trunk and cause rot.Artificial infections, carried out on spruce showed that central rot can spread up to 12 m in height (M a r i n k o v i ć et al ., 1990), which means that such tree can not be used a timber.Economic loss is highly infected stands could reach up to 25% of volume (Keča, ranković, unpublished).
Carpophores are produced during autumn, but fortunately only from August till first snow (M a r i n k o v i ć et al ., 1990).Size of fruit bodies found in present study was very big, and some of them occupied the whole root system of wind-snapped trees.Spore deposition is c. 30.000 spore per dm 2 •h -1 at a distance of 1 m (R e d f e r n , S t e n l i d , 1998).So, it is obvious that if fresh cut stumps or wounded trunks and roots of standing trees are present in stand infection will arise.Large roots of wind-snapped trees should be removed from stands as soon as possible, before they start to produce carpophores.
According to the results obtained in this case study could be concluded that if selection forests of Norway spruce are managed properly the impact of the Heterobasidion disease can be held under control.

CONCLUSIONS
In present study all Heterobasidion isolates are identified as Heterobasidion parviporum.Species is widely distributed in spruce forests type -picetum excelsae oxlalidetosum in N.p."Kopaonik".Fiftyeight percent of sampled trees were infected with Heterobasidion.Eleven Heterobasidion genets are present on studied site.Five genets had infected only one and five other two trees, while only one genet infected three neighboring trees.Carpophores were produced from middle of August till end of October.Management in the stands, endangered by Heterobasidion, should be directed to protection of freshly cut stamps and to providing of condition which favour decomposition of stumps.борова), H. abietinum (интерстерилна група Ф, са јеле) и H. parviporum (интерстерилна гру па С, са смрче).

Figure 1 .
Figure 1.Distribution of Heterobasidion parviporum genets in the study plot (compartment 96, Management Unit "Samokovska reka", N.P. Kopaonik -trees encircled by a dash line are infected by the same H. parviporum genet) Слика 1. Распоред Heterobasidion parviporum индивидуа на огледној површини (ГЈ "Само ковска река", одељење 96, Н.П."Копао ник" испрекидане линије пред стављају ста бла инфицирана истом индивидуом H. parviporum) Distribution of Heterobasidion genets on a norway spruce site: case study in national park "kopaonik".Bulletin of the Faculty of Forestry 98: 117126.alsoobserved in the fruit bodies N i e m e l ä & K o r h o n e n (1998) raised the three European intersterility groups (P, S, F) to the level of species: H. annosum (Fr.)Bref., H. parvipirum Niemelä & Korhonen, and H. abietinum Niemelä & Korhonen.Three Heterobasidion species are present in Europe and two intersterility groups are present in North America.History of study of Heterobasidion in Serbia goes back in 1963, when strong attack if Fomes annosus on Kopaonik was reported.At the same time, the disease was found in natural forests of spruce on Zlatar, Durmitor (Montenegro), Jahorina (Bosnia and Herzegovina) (M a r i n k o v i ć et al.

Table 1 .
List of isolates, including host condition, species identification and genets, collected in Norway spruce stand in National park "Kopaonik"

Table 1 .
List of isolates, including host condition, species identification and genets, collected in Norway spruce stand in National park "Kopaonik"