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Nenad Keča

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

Abstract: Heterobasidion annosum s.l. causes great economic loss in coniferous forests worldwide. Recent studies showed that three European Heterobasdion spe-cies 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.Key words: Norway spruce, Heterobasidion parviporum, genets, distribution

ПРИСУСТВО ИНДИВИДУА HETEROBASIDION ВРСТА НА СТАНИш­ТУ СМРчЕ У НАЦИОНАЛНОМ ПАРКУ „КОПАОНИК”

Извод: Heterobasidion annosum s.l. је врста која узрокује велике економске ште­те у четинарским шумама широм света. Новија истраживања показала су да су у шумским екосистемима у Србији присутне све три европске Heterobasidion врсте. Циљ ових истраживања био је да се установе које су Heterobasidion врсте присутне у истраживанoj састојини смрче у националном парку „Копаоник“, да се идентификују број индивидуа Heterobasidion­а и да се прикаже њихова ве личина и просторни распоред.Кључне речи: смрча, Heterobasidion parviporum, индивидуе, распоред

1. 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 fun-gal 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 Fin-land, and south as far as northern Africa and Central America (Woodward 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 (Korhonen , 2004). Because morphological differences were

ГЛАСНИК ШУМАРСКОГ ФАКУЛТЕТА, БЕОГРАД, 2008, бр. 98, стр. 117­126

BIBLID: 0353­4537, (2008), 98, p 117­126

UDK: 630*443:582.284.4 Heterobasidio n annosu m (497.11-751.2 Kopaonik)Оригинални научни рад

др Ненад Кеча, доцент, Универзитет у Београду - Шумарски факултет, Београд

Keča N. 2008. Distribution of Heterobasidion genets on a norway spruce site: case study in na-tional park “kopaonik“. Bulletin of the Faculty of Forestry 98: 117­126.

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also observed in the fruit bodies Niemelä & Korhonen (1998) raised the three Europe-an intersterility groups (P, S, F) to the level of species: H. annosum (Fr.) Bref., H. parvipi-rum 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 Herze-govina) (Mar inkov ić et al., 1990). Most recent studies showed that all three species are present in coniferous forests in Serbia (Keča , 2008).

Studies about population structure, size of genets, spread and distribution of Het-erobasidion 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 Scandina-vian 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 pres-ence, size and spatial distribution of Heterobasidion genets in an selection Norway spruce forest. Heterobasidion genets are mapped and data about tree condition are collected.

2. MATERIAL AND METHODS

2.1. Study site

This study was carried out in a Norway spruce stand in the National Park “Kopa-onik“. 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 struc-ture 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 m3∙ha–1. Diameter of average tree is 29 cm on breast height and height is estimated on 20,5 m.

2.2 Field work

The study plot, 90×40 m in size, was established in May 2006. Trees showing chlo-rosis, 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.

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2.3. Isolation and identification of Heterobasidion genotypes

Heterobasidion were isolated onto a modified selective medium (Hun t , Cobb , 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 sul-phate 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 Hetero-basidion species (isolates provided by K. Korhonen, Finish Forest Research Institute, Van-taa, Finland).

3. 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 impor-tant 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.

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

Табела 1. Листа изолата прикупљених у састојини смрче у националном парку „Копаоник“, укључујући стање домаћина, идентификоване врсте и индивидуе

№ Isolate lab.Озн. изол.

Host conditionСтање домаћ.

Fungal tissue isolatedИзол. из ткива гљиве

IdentificationИдентификација

Gen.Инд.

1 S21­2006 Chlorotic Heterokaryotic mycelium Heterobasidion parvipirumIX

2 S41­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum3 S42­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum VIII4 S23­2006 Declining Heterokaryotic mycelium Heterobasidion parvipirum VII5 S26­2006 Chlorotic Heterokaryotic mycelium Heterobasidion parvipirum IV6 S27­2006 Wind-snapped Heterokaryotic mycelium Heterobasidion parvipirum

III7 S83­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum8 S29­2006 Wind-snapped Heterokaryotic mycelium Heterobasidion parvipirum

V9 S29K­2006 carpophore homokaryotic Heterobasidion parvipirum10 S51­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum11 S43­2006 Wind-snapped Heterokaryotic mycelium Heterobasidion parvipirum

X12 S43K­2006 carpophore homokaryotic Heterobasidion parvipirum13 S45­2006 Declining Heterokaryotic mycelium Heterobasidion parvipirum14 S47­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum15 S52­2006 Wind-snapped Heterokaryotic mycelium Heterobasidion parvipirum

VI16 S53­2006 Chlorotic Heterokaryotic mycelium Heterobasidion parvipirum17 S84­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum XI18 S86­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum

II19 S87­2006 Declining Heterokaryotic mycelium Heterobasidion parvipirum20 S88­2006 Healthy Heterokaryotic mycelium Heterobasidion parvipirum I21 S22­2006 Wind-snapped Rhizomorphs Armillaria cepistipes22 S24­2006 Declining Mycelium Fomitopsis pinicola23 S25­2006 Wind-snapped Mycelium Fomitopsis pinicola24 S30­2006 Chlorotic Mycelium Armillaria cepistipes25 S44­2006 Declining Rhizomorphs Armillaria cepistipes

26 S46­2006 Declining Mycelium clymacocystis borea-lis (Fr.) Kotl. & Pouzar

27 S48­2006 Dieback, decayed Mycelium Fomitopsis pinicola28 S49­2006 Declining Mycelium Not identified29 S50­2006 Declining Mycelium Fomitopsis pinicola30 S89­2006 Healthy - Nothing isolated

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

Табела 1. Листа изолата прикупљених у састојини смрче у националном парку „Копаоник“, укључујући стање домаћина, идентификоване врсте и индивидуе

№ Isolate lab.Озн. изол.

Host conditionСтање домаћ.

Fungal tissue isolatedИзол. из ткива гљиве

IdentificationИдентификација

Gen.Инд.

31 S85­2006 Declining Mycelium Fomitopsis pinicola32 S90­2006 Recently dead - Nothing isolated33 S91­2006 Recently dead - Nothing isolated34 S92­2006 Recently dead - Nothing isolated35 S93­2006 Recently dead - Nothing isolated36 S94­2006 Recently dead - Nothing isolated

4. 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 iden-tified in stands at the end of rotation had infected only one tree (Vas i l i auskas , S t en l id , 1998). In present study only one genet had spread on tree neighbouring trees and occu-pied area of about 120 m2. 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 ten l id , 1985, Pi r i et al., 1990). Observation of many small genets indicate that basidiospores play im-portant 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 , Korhonen , 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 esti-mated 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 Heteroba-sidion in the roots of Norway spruce increases almost three time after tree is cut (Bendz-He l lg ren 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 Korhonen (2007) concluded that only stumps with greatest infection potential and with sufficient root con-tact 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 (Gre ig , Burdek in , 1970).

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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 (Mar inkov ić 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 (Mar inkov ić 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 dm2∙h–1 at a distance of 1 m (Redfe rn , S t en l id , 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 se-lection forests of Norway spruce are managed properly the impact of the Heterobasidion disease can be held under control.

5. CONCLUSIONS

In present study all Heterobasidion isolates are identified as Heterobasidion parvi-porum. Species is widely distributed in spruce forests type - picetum excelsae oxlalideto-sum in N.p. “Kopaonik”. Fifty­eight percent of sampled trees were infected with Heteroba-sidion. 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.Acknowledgements: Thank are due to K. Korhonen, METLA, Finland for the donation of the dif-

ferent haploid testers of the European Heterobasidion species.

LITERATURE

Bendz­He l lg ren M., Brand tbe rg p.O., Johansson M., Swed jemark g., S t en l id J. (1999): Growth rate of Heterobasidion annosum in picea abies established on for-est land and arable land, Scandinavian Journal of Forest Research 14 (402-407)

Gre ig B.J.W., Burdek in D.A. (1970): control and eradication of Fomes annosus in Great brit-ain, proceedings of the Third IUFr O Conference on Fomes annosus Denmard (ed. by Hodges C.S., rIshbeth K., yde­Andersen A.), USDA, Forest Service, washing-ton (21-32)

Hun t R.S., Cobb F.W. Jr. (1971): Selective medium for the isolation of wood-rotting Basidiomyce-tes, Canаdian Journal of Botany 49 (2064­2065)

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Keča N. (2008): Identification of Heterobasidion species in Serbia and possibility of its control, Plant Doctor 35 (44–50) (in Serbian with English summary)

Korhonen K. (2004): Fungi belonging to the genera Heterobasidion and Armillaria in eurasia, „Fungal Communities in forest ecosystems, Coordinating Investigation”, Vol. 2, Ed. by Storozhenko V.G., Krutov V.I., Moscow - Petrozavodsk (89-113)

Mar inkov ić p., Šmi t S., popov ić J. (1990): Disease of the root of spruce, Fomes annosus (Fr.) Cooke, the importance of this phenomenon in maintaining and restoring spruce for-ests on kopaonik, proceedings „Nature of Kopaonik ­ protection and use”, Institute for Tourism PMF, Belgrade (235-240) (in Serbian with English summary)

Niemelä T., Korhonen K. (1998): taxonomy of the Genus Heterobasidion, „Heterobasidion annosum ­ Biology, ecology, Impact and Control” (eds. S. woodward, J. Stenlid, r. Karjalainen, A. Hüttermann), CAB International, wallingford (27­33)

p i r i T. (1996): the spreading of the s type of Heterobasidion annosum from norway spruce stumps to the subsequent tree stand, european Journal of Forest pathology 26 (193–204)

Pi r i T., Korhonen K. (2001): infection of advance regeneration of norway spruce by Heterobasi-dion parviporum, Canаdian Journal of Forest research 31 (937­942)

Pi r i T., Korhonen K. (2007): spatial distribution and persistence of Heterobasidion parviporum genets on a norway spruce site, Forest Pathology37 (1-8)

P i r i T., Korhonen K., Sa i r anen A. (1990): occurrence of Heterobasidion annosum in pure and mixed spruce stands in southern Finland, Scandinavian Journal of Forest Resea-rch 5 (113-125)

Redfe rn B.D., S t en l id J. (1998): spore Dispersal and infection, „Heterobasidion annosum - Bi-ology, ecology, Impact and Control” (ed. by S. woodward, J. Stenlid, r. Karjalain-en, A. Hüttermann, CAB International, wallingford (105­124)

Sten l id J. (1985): population structure of Heterobasidion annosum as determined by somatic in-compatibility, sexual incompatibility, and isoenzyme patterns, Canаdian Journal of Botany 63 (2268­2273)

Vas i l i auskas R., S t en l id J. (1998): spread of s and p group isolates of Heterobasidion anno-sum within and among picea abies trees in central Lithuania, Canаdian Journal of Forest research 28 (961­966)

woodward S., S t en l id J., Kar j a l a inen r., Hü t t e rmann A. (1998): Heterobasidion anno-sum - Biology, Ecology, Impact and Control, CAB International, Wallingford (589)

Кеча Ненад

ПРИСУСТВО ИНДИВИДУА HETEROBASIDION ВРСТА НА СТАНИш ТУ СМРчЕ У НАЦИОНАЛНОМ ПАРКУ „КОПАОНИК”

РезимеHeterobasidion annosum s.l. je најзначајнији представник проузроковача трулежи ко­

рена и приданка и присутан је у готово свим четинарским шумама северне хемисфере. Ис­товремено представља један од најозбиљнијих проблема у газдовању четинарским шумама и културама. Новија истраживања показала су да су у четинарским састојинама у Србији присутна сва три европска представника овога рода H. annosum (интерстерилна група П, са

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борова), H. abietinum (интерстерилна група Ф, са јеле) и H. parviporum (интерстерилна гру­па С, са смрче).

У састојини смрче у НП „Копаоник“ издвојена је огледна површина 90×40 m на којој су узети узорци са стабала смрче која су показивала знаке хлорозе, одумирања или су била из ваљена. Састојина се налази у Г.Ј. „Самоковска река“ у одељењу 96. Стабла са којих су узе­ти узорци су обележена бројевима како је то приказано у табели 1. Узорци су узети помоћу свр дла, а затим је извршена изолација гљиве на хранљивој подлози ­ малц екстракт агар (1,5% малц w/v и 1,5% aгар w/v). После изолације извршена је идентификација Heterobasi-dion изолата, парењем са хаплоидним тестерима добијеним од К. Корхонена (MeTLA, Фин­ска). Идентификација индивидуа Heterobasidion извршена је међусобним укрштањем изолата и посматрањем појаве демаркационе линије на контакту двеју индивидуа.

Изолацијом је добијено 18 изолата Heterobasidion и 12 изолата трулежница (табела 1). Сви Heterobasidion изолати припадали су H. parviporum. Није било статистички значајних разлика у појави H. parviporum на здравим ­ 8, хлоротичним ­ 3, одумирућим ­ 3 и изваљеним ­ 4 стаблима (подаци нису приказани). Карпофоре су пронађене само на изваљеним стаблима и имале су велике димензије. Једна карпофора се распростирала на читавој површини кореновог система заузимајући површину од више десетина dm2. Утврђено је да се карпофоре образују од краја августа до краја октобра, тј. до првих снегова.

Из 18 изолата идентификовано је 11 индивидуа Heterobasidion­а (слика 1). Пет ин­дивидуа су биле присутне на само једном стаблу, других пет индивидуа било је присутно на по два суседна стабла, а само једна индивидуа се распростирала на три суседна стабла. За индивидуе које су се простирале на два и више стабала проверено је присуство контакта корења и установљено је да је у четири случаја био присутан конатакт између корења двају стабала. Вегетативно ширење кроз контакт корења може бити од значаја у пребирној састојини, због сталног присуства контакта између корења млађег и старијег дрвећа.

Добијени резултати указују да се газдовање мора усмерити на заштиту свеже посечених пањева у састојинама у којима H. parviporum није присутан, а у састојинама где је гљива већ присутна неопходно је обезбедити услове да што пре дође до декомпозиције пањева.

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