Zbornik gozdarstva in lesarstva, 52, 1997, s. 279 - 322 GDK 181.45: 181.351: 161.4: 160.201: 160.203: 174.7 Picea abies Karst: (497.12) Prispelo/ Arrived: 17.2.1997 Sprejeto / Accepted: 5. 3.1997 MIKOBIOINDIKACIJA ONESNAžENOSTI DVEH GOZDNIH RASTIŠČ Hajka KRAIGHER. Izvleček Na dveh različno onesnaženih, rastiščno primerljivih gozdnih raziskovalnih ploskvah v imisijski coni termoelektrarne Šoštanj smo v vegetacijski sezoni 1993 v volumsko enakih vzorcih zemlje raziskovali pojavljanje tipov ektomikorize. Na kratko smo opisali dvajset morfotipov ektomikorize, dva tipa ektomikorize, Lactarius lignyotus x Picea abies in Hydnum rufescens x Picea abies, smo natančno opisali z anatomskimi in molekularnimi metodami. Pojavljanje trosnjakov ni ustrezalo pojavljanju tipov ektomikorize v vzorcih tal. Tip ektomikorize Hydnum rufescens x Picea abies bi lahko uporabili za kazalca neonesnaženih rastišč, Paxillus involutus x Picea abies pa za kazalca onesnaženih gozdnih rastišč v Sloveniji. Ključne besede: tipi ektomikorize, Picea abies, anatomska karakterizacija, molekularne metode, mikobioindikacija MYCOBIOINDICATION OF POLLUTION OF TWO FOREST SITES Abstract In two differently polluted forest research plots in the emission area of the Coal Power Plant in Šoštanj, which were comparable regarding their site characteristics, the types of ectomycorrhizae were studied during the vegetation season of 1993, in soil cores of equal volumes. Twenty morphotypes of ectomycorrhizae were briefly described, while two types, Lactarius lignyotus x Picea abies and Hydnum rufescens x Picea abies, were comprehensively characterised by anatomical and molecular methods. The occurrence of fungal fruitbodies did not correspond to the occurrence of ectomycorrhizal types. It might be proposed that type Hydnum rufescens x Picea abies be used as a bioindicatior of unpolluted sites, while Paxillus involutus x Picea abies as bioindicator of polluted forest sites in Slovenia. Key words: types of ectomycorrhizae, Picea abies, anatomical characterisation, molecular methods, mycobioindication Doc. dr., dipl. biol., dipl. inž.gozd., Gozdarski inštitut Slovenije, Večna pot 2, 1000 Ljubljana, SLO 280 Zbornik gozdarstva in lesarstva, 52 CONTENTS 1 INTRODUCTION / UVOD .................................................................................. 281 1.1 MYCORRHIZAL SYMBIOSIS / M/KOR/ZNA SIMBIOZA ................................... 281 1. 2 CHANGES IN SPECI ES OIVERSITY OF ECTOMYCORRHIZAL FUNGI / SPREMEMBE V VRSTNI PESTROSTI EKTOMIKORIZNIH GLIV .................... 282 1.3 MYCOBIOINDICATION OF FOREST SITE POLLUTION / MIKOB/0/NDIKACiJA ONESNAŽENOSTI GOZDNIH RASTIŠČ ...................... 284 1.4 OBJECTIVES OF OUR STUOY / CILJI RAZISKAVE ........................................ 285 2 SITE PRESENTATION / PREDSTAVITEVOBJEKTA ..................................... 285 3 METHODS / METODE ..................................................................................... 286 4 RESULTS / REZULTATI ................................................................................... 287 4.1 TYPES OF ECTOMYCORRHIZAE OCCURING IN TWO FOREST RESEARCH PLOTS / TIPI EKTOMIKORIZE NA DVEH GOZDNIH RAZISKOVALNIH PLOSKVAH ......................................................................... 287 4.2 OCCURRENCE OF ECTOMYCORRHIZAE IN SOIL CORES / POJAVLJANJE EKTOMIKOR/ZE V TALNIH VZORCIH .................................... 295 4.3 COMPARISON OF THE OCCURENCE OF FRUITBOOIES ANO THE TYPES OF ECTOMYCORRHIZAE IN TWO FOREST RESEARCH PLOTS ! PRIMERJAVA POJAVLJANJA GLIV IN TIPOV EKTOMIKOR/ZE NA DVEH GOZDNIH RAZISKOVALNIH PLOSKVAH ............................................. 298 5 DISCUSSION / RAZPRAVA ............................................................................. 299 5.1 CHARACTERISATION ANO IOENTIFICATION OF ECTOMYCORRHIZAL TYPES IN TWO FOREST RESEARCH PLOTS / OPIS IN IDENTIFIKACIJA TIPOV TIPI EKTOMIKORIZE NA DVEH GOZDNIH RAZISKOVALNIH PLOSKVAH ......................................................................... 299 5.2 OCCURRENCE OF TYPES OF ECTOMYCORRHIZAE IN TWO FOREST RESEARCH PLOTS / POJAVLJANJE TIPOV TIPI EKTOMIKORIZE NA DVEH GOZDNIH RAZISKOVALNIH PLOSKVAH ............................................. 302 5.3 COMPARISON OF FRUITBOOY ABUNOANCE ANO ECTOMYCORRHIZAE / PRIMERJAVA POGOSTNOSTI GLIV IN EKTOMIKORIZE ............................................................................................... 306 5.4 APPLICATION OF ECTOMYCORRHIZAL STUOIES FOR BIOINOICATION / UPORABNOST EKTOMIKORIZNIH RAZISKAV ZA BIOINDIKACIJO ............................................................................. '. .................. 307 6 CONCLUSIONS I ZAKLJUČKI ......................................................................... 308 7 SUM MARY ........................................................................................................ 308 8 POVZETEK ....................................................................................................... 310 9 REFERENCES / VIRI ........................................................................................ 313 10 ACKNOWLEDGEMENTS I ZAHVALA ............................................................. 322 1 INTRODUCTION 281 Kraigher H.: Mikobioindikacija onesnaženosti ... Forest ecosystems are, functionally and structurally, highly organized systems of biotic and abiotic components, linked into a sensitive dynamic equilibrium. Air pollution can influence all and each of the components of these systems at different levels. Directly it can influence the physiology of forest trees by damaging their photosynthetic apparatus, leaves and needles at the cellular level (SCH0TT et alf./. 1984, REHFUESS 1988, BLANK/ ROBERTS / SKEFFINGTON 1988, FINK 1988, WOLFENDEN / MANSFIELD 1991 etc.). lndirectly it can induce changes in mycorrhizosphere by influencing root systems, by changes in the living component in forest soil and by influences on the principal linking component in the forest floor, the mycelium of fungi, which live in a symbiosis with roots of higher plants (SCHLECHTE 1985, MC COOL 1988, COWLING 1989 etc.). Different methods are in use for detection of forest site pollution: direct measurements of emissions are largely complemented by methods of bioindication, whereby bioindication organisms react to specific environmental conditions (or to a specific pollutant) by their specific physiological response. Biomonitoring of forest site pollution implies the analysis of damage and abundance of chosen "indicator" organisms in the ecosystem, the method which is known as passive biomonitoring; while active biomonitoring implies the introduction of "monitor" organisms into the site, for which a specific physiological reaction is known to occur under the influence of a specific pollutant (STEUBING / JAEGER 1981, ARNDT /NOBEL/ SCHWEIZER 1987, KREEB 1990, BATIČ 1994). 1.1 MYCORRHIZAL SYMBIOSIS Permanent associations of roots with hyphae of fungi were first named 'mycorrhiza' by FRANK in 1885. He distinguished two main forms of mycorrhiza, ectotrophic (now named ectomycorrhiza) and endotrophic mycorrhiza (now named endomycorrhiza). In symbiosis a bidirectional transport of nutrients occur: assimilates from the plant into fungus, and water and mineral nutrients from the 282 Zbornik gozdarstva in lesarstva, 52 fungus into the host plant (HARLEY / SMITH 1983). Each mycorrhizal form is associated with an ecosystem and soil environment with distinctive characteristics regarding the rate of decomposition, mineralization, availability of nutrients, and the dynamics of the ecosystem (READ 1991 ). A review on the role of ectomycorrhizae in forests has recently appeared (KRAIGHER 1996) in this journal, therefore only a short presentation of the role of ectomycorrhizae in forest ecosystems is shown below. Forest trees with ectomycorrhiza dominate acid soils with surface litter accumulation and seasonally released organic and inorganic nutrients (key factors being N, P and C/N ratio) (READ 1991 ). In late-stage forest successions ectomycorrhizal roots are known to develop mainly in the upper organic soil horizons (MIKOLA / LAIHO 1962). There, mycorrhizae can short-circuit nutrient cycles by directly reacquiring nutrients in organic form from plant and fungal litter, and they may reallocate resources between different plant individuals, preventing the loss of resources from the entire ecosystem (PANKOW / BOLLER / WIEMKEN 1991). Hyphal connections between different plants of the same or different species which transferred water, minerals and also assimilates between roots of different plants were demonstrated (FRANCIS / READ 1984, MILLER ALLEN 1992, SIMARD 1996). In symbiosis functional compatibility of different fungal species and strains in combination with forest trees differs regarding their physiological effectiveness (GIANINAZZI-PEARSON 1984), while any anthropogenic impacts can influence this highly interactive forest environment (AMARANTHUS / PERRY 1994). 1. 2 CHANGES IN SPECIES DIVERSITY OF ECTOMYCORRHIZAL FUNGI Since the early seventies an increasing number of reports have been concerned with a decrease in species diversity and the abundance of sporocarps of macromycetes in Europe (reviews and comments in papers by ARNOLDS 1991, JAENIKE 1991, PILTAVER 1990, GOGALA 1990, KRAIGHER / BATIČ / AGERER 1995, KRAIGHER 1996). 283 Kraigher H.: Mikobioindikacija onesnaženosti ... ARNOLDS (1988) has investigated the species diversity of different functional groups of macromycetes in the Netherlands by: i) comparing the lists of records of 15 forays made between 1912 and 1954 with those made between 1973 and 1982; ii) mapping of selected species in different periods; iii) repeated analysis of permanent plots with mycocoenological methods. AII methods showed similar changes in the fruiting of the macromycete flora of the Netherlands: i) the group of terrestrial saprophytes in grass- and heathlands have declined; ii) lignicolous fungi as a whole appeared to have increased; iii) the most dramatic change was shown by ectomycorrhizal fungi, among which 77% (100 sps) of 130 investigated species, showed a decrease in numbers. The greatest established decrease was for fungi predominantly associated with coniferous trees (66%), mainly for hydnaceous fungi (Cantharellaceae, Hydnaceae, Gomphidiaceae and the Corlinarius, Dermocybe, Tricholoma and Suil/us) genera. Similar observations were done by GUILLITE 1987, HOILAND 1986,, JAKUCS et alf./. 1986, P.J.JANSEN 1985, A.E.JANSEN and DE VRIES 1989, SCHAFFERS / TERMOSHUIZEN 1989, AGERER 1989, BLASCHKE 1986, GOBL 1986, 1989, HOLOPAINEN 1989, KOWALSKI 1987, MEJSTRIK / CUDLIN 1987, GULDEN et alf. 1992 and others. Direct fumigation studies are also available, showing that the vitality of ectomycorrhizal root tips depends on the species of ectomycorrhizae (WOLLMER / KOTTKE 1990, QIAN et alf. 1993), and that the occurence of fruitbodies does not necessarily correlate with the percentage of ectomycorrhizal root tips per type (SHAW et alf. 1992). Possible causes for the decline of ectomycorrhizal fungi were discussed by ARNOLDS (1991) with regard to natural successions, sporocarp removal, forest management, and air pollutants, which can act directly by influencing fungi, by forest soil acidification, nitrogen deposition, litter and herb layer removal or assimilate partitioning through influences on tree vitality. A widespread decline, predominantly in categories of macromycetes, associated with old broad-leaved and coniferous forests, especially in mycorrhiza-formers, has been found to account for more than half of total species listed (ING 1994 ). 284 Zbornik gozdarstva in lesarstva, 52 1.3 MYCOBIOINDICATION OF FOREST SITE POLLUTION FELLNER (1989) founded a myco-bioindication method far forest site pollution, based in part on evaluation of the ratio of mycorrhiza forming fungi to all macromycetes found at the locality studied, and in part on the degree of impoverishment of mycorrhizal mycocoenoses. He distinguished three phases of such impoverishment: 1) inhibition of sporocarp production; 2) loss of species diversity with the proportion of mycorrhizal species in the total count of macromycetes dropping to ca 35%; 3) partial to total destruction of the assemblages of ectomycorrhizal fungi which then contributed only 10-20% of the total number of macromycete species. ARNOLDS (1991) has tested Fellner's phases in respect of decline in macromycetes in the Netherlands and suggested an adaptation in respect to different sensitivities of different ectomycorrhizal species. Far instance, it was demonstrated that fruitbodies of hydnoid fungi ceased to be produced eventhough overall sporocarp productivity had not been significantly decreased. Therefore, he further elaborated Fellner's scheme far poor and acidic soils in the Netherlands, proposing the lists of most critical species - thise with a tendency to disappear - and of less critical species, which seem to replace the disappearing ones by increasing abundance in moderately polluted areas. Only in the last phase a total absence of ectomycorrhizal sporocarps can occur. The long-lasting tolerant species may vary in different forest communities and regions. FELLNER (1989), from observations in Krkonoše, suggested that the fungus Russu/a mustelina might be used as a bioindication mycorrhizal species far the estimation of forest soil pollution by acid depositions. However, the occurrence of fruitbodies depends on a range of climatic factors in different years, while ectomycorrhizal types are supposed to be present in the soils at any tirne of the year and in any climatical conditions. Furthermore, a range of ectomycorrhizal fungi, such as Fungi lmperfecti, do not produce any sporocarps. As a result, a more detailed study of ectomycorrhizal potential of forest sites by ectomycorrhizal types determination was proposed at the 3rd lnternational Mycological Congress in Regensburg in 1990, with a view to apply the results to bioindication: 1.4 OBJECTIVES OF OUR STUDY The objectives of our studies were: 285 Kraigher H.: Mikobioindikacija onesnaženosti ... • determination and characterisation of ectomycorrhizal types on Norway spruce from two different forest sites by anatomical and molecular tools; • possible application of these studies tor the development of a new bioindication method, which would be based on the determination of naturally occuring types of ectomycorrhizae on Norway spruce as "indicators" of forest site pollution. 2 SITE PRESENTATION Two forest research plots, which showed comparable site characteristics but were differently influenced by the emissions of the local Coal Power Plant (Šoštanj CPP) were chosen tor our studies. In the two forest research plots, the predominant rock types are acidophilous silicates, the corresponding soil types are distric cambisols of different depths (URBANČIČ 1990). The predominant phytocenological association belongs to Querco - Luzulo Fagetum (MARINČEK 1987). The predominant tree species is Norway spruce (/bid.). The torests have been surveyed tor damage classes (IGLG 1988, FERLIN 1990, KOLAR 1989), which result from the more or less direct influences of the Šoštanj CPP. The two forest research plots (at 800 - 900 m above sea level) were established tor ecological studies in the eighties (LOVREC 1989, LEŠNJAK et ali. 1989). The predominant rock types are acidophilous silicates, the corresponding soil types are distric cambisols of different depths (URBANČIČ 1990). The predominant phytocenological association belongs to Querco - Luzulo Fagetum (MARINČEK 1987). The predominant tree species is Norway spruce (/bid.). The torests have been surveyed tor damage classes (IGLG 1988, FERLIN 1990, KOLAR 1989). The main difference between the two plots is the influence of the emissions from the Šoštanj CPP. Regarding the relief, climatic, previous forest decline and lichenological studies, the plot in Zavodnje is more influenced by emissions from the Šoštanj CPP, which lies approximately 6 km to the east from the plot. The plot in Mislinjski jarek, which is situated approximately 20 km north- 286 Zbornik gozdarstva in lesarstva, 52 east of the CPP, represents a relatively unpolluted site. Air pollution in the two research plots was assessed by mapping of epiphytic lichens on Norway spruce (BATIČ 1991). lndex of air purity (IAP) was determined by mapping of the frequency and cover of lichen thalli types as described in BATIČ (1990). IAP values can be between O (polluted) and 54 (clean air). Zavodnje: IAP between 8 and 12 (representing very polluted air) Mislinja: IAP between 25 and 40 (representing clean air and protected exposition) The station far automated measurements of SO2, NO2 and 03 near the plot in Zavodnje showed maximum one-hour concentrations of SO2 in 1993 at 3612 µ g/m3; maximum one-hour concentrations of NO2 in 1993 at 129 µg/m3; maximum one-hour concentrations of 03 in 1993 at 204 µg/m3 (CIGLAR et alf. 1994). 3 METHODS Soil cores were taken far the determination and estimation of the abundance of ectomycorrhizae in the vegetation season 1993 (6 times, in 2 to 5 replicates per plot, with soil cores of upper diameter 4.8 cm and lower diameter 4.0 cm, 18 cm deep, which equalled 274 ml soil velurne). The samples were stored at 4 - 8° C in plastic bags. Far comprehensive characterisation and identification of ectomycorrhizae, soil cores from directly underneath fruitbodies of mycorrhizal fungi were cut with a sharp knife, wrapped in foil and stored at 4 - 8° C until processing. Far the identification of ectomycorrhizae the wrapped samples were soaked in water and mycelial connections were followed from fruitbodies to ectomycorrhizae under a dissecting microscope. Two types were comprehensively described after the method by AGERER (1987-1993; 1991a) and by molecular tools (after GARDES / BRUNS 1993) as presented in KRAIGHER (1994), KRAIGHER / AGERER / JAVORNIK (1995), AGERER / KRAIGHER/ JAVORNIK (1996) and KRAIGHER (1996)). Far the determination of ectomycorrhizal types in soil cores the morphotypes were separated and all ectomycorrhizal root tips from each morphotype from samplings in 1993 were counted (modified after GRAND / HARVEY 1984). lf 287 Kraigher H.: Mikobioindikacija onesnaženosti ... possible, the morphotypes were determined after their anatomical characteristics in respect to all adequate literature: AGERER (1987-1993, 1986, 1991a, 1991b, 1992 etc), GRONBACH (1988), WEISS (1988), TREU (1990), UHL (1988), BRANO (1991), INGLEBY et ali., (1990), HAUG / PRITSCH (1992), PILLUKAT / RAIDL / AGERER (1992), WALLER et ali. (1993) and others. The morphotypes, which could not be determined sufficiently for distinction after the above literature, were either comprehensively characterised, following the prescribed methodology, or a brief description was done and the distinctive morphotypes were given a reference number. The types which could not be described (they were either too old, too young or they were too rare to be analysed sufficciently for disctinction from other types), were categorized as 'unidentifiable'. Representative ectomycorrhizal systems were fixed in FAA, marked and stored at the lnstitute's Herbarium LJU FU2 SLO collection. In the cases when identification of ectomycorrhizae through connections to fruitbodies was achieved, these were stored as exicates in Herbarium LJU FU2 collection. Fungal species were determined at the plot and checked for their identity at the Institute by Andrej PILTAVER, according to: MOSER 1978 (Polyporales, Boletales, Agaricales, Russulales}, BON 1987, JOLICH 1984 (Aphyllophorales, Heterobasidiomycetes, Gastromycetes), BREITENBACH / KRANZLIN, 1981-1991. At the tirne of sampling the occurrence of fruitbodies was noted. Their abundance was estimated as single observation (+), a few (++), frequent (+++), massive occurrence (++++). 4 RESULTS 4.1 TYPES OF ECTOMYCORRHIZAE OCCURING IN TWO FOREST RESEARCH PLOTS Type 148 • white mycorrhiza with ochre tint, monopodial-pyramidal ramifications, straight to slightly bent unramified ends, length of ramified system up to 6.5 mm, of unramified ends 0.4 - 1.1 mm, diameter of unramified ends and axes 0.5 mm; mantle surface shiny, sometimes irregular patches of brown cortical cells 288 Zbornik gozdarstva in lesarstva, 52 visible, surrounded by a loose woolly cover of emanating hyphae, soil particles can remain on surface; • mantle in surface view plectenchymatous, type A, hyphal cells with thick walls at ramifications, globular hyphal ends, slightly tortuous, no clamps, hyphal ramifications at right angles; typical inflations throughout the mantle, intrahyphal hyphae, repair mechanism; • smooth, brownish rhizomorphs, in lactic acid reddish, 0.08 mm thick, undifferentiated; in rhizomorphs irregularly shaped emanating hyphae, with inflations, H-anastomoses, open or with septa, with thick walls, formed between two hyphae running alongside, simple septa, globule at the septum onesided; • colour reactions negative for most chemicals, with FeSO4 hyaline - decolored to bluish, guiaiacol on mantle bluish, on rhizomorphs red-brown, KOH on rhizomorphs ochre; • new type, the suggested name - a comprehensive description will follow - is Piceirhiza inf/ata; • Zavodnje SLO 00148 (07.10.1992). Type 149 • whitish mycorrhiza with olive-ochre patches, monopodial-pyramidal ramifications, up to 20 mm long systems, slightly bent; • with severa! white, smooth, thick rhizomorphs; • mantle structure plectenchymatous, type A, anastomoses rare, septa with doliporus-like structures, no clamps, diameter of hyphae 5 to 7 mm, cell walls hyaline, non-thickened; rhizomorphs 1 O to 80 mm thick, highly differentiated, with one single or a few central hyphae, type E or F, diameter of middle hyphae 8 to 16 mm; • type determined as belonging to Xerocomus badius group; • Zavodnje SLO 00149 (07.10.1994). Type 150 • pyramidal to coralloid ramification, soil particles difficult to remove; mantle surface smooth to grainy, greyish; • in surface view of mantle parallel agglutinated hyphae, plectenchymatous; no 289 Kraigher H.: Mikobioindikacija onesnaženosti ... clamps, occasionally found bottle-like cystidia; • could belong to Russu/a xerampelina or R. il/ota group; • Zavodnje SLO 00150 (15.10.1994). Type 156 • dark grey to black mycorrhiza, older parts light grey, monopodial-pyramidal ramification, mantle surface smooth; • mantle in surface view with very parallel oriented hyphae; septa thick, cross- walls can be seen together with longitudinal walls and depositions on with a different refraction index (bluish), the walls are collapsing, greyish-brown; • possibly similar to Piceirhiza para/leta (HAUG / PRITSCH 1992) • Zavodnje SLO 00156 (15.10.1992). Type 164 • long ochre-brown, scarcely ramified, monopodial-pyramidal ECM, mantle surface slightly grainy; • mantle type P, pseudoparenchymatous - epidermoid with a net of thin laticifers on top; cells irregularly shaped to roundish, irregularly indented, 1 O to 15 mm; laticifers with orange latex, which looks like irregularly shaped thick walled cells or incrustrations, but disappear in lactic acid; 5 to 7.5 mm in diameter, without septa; • belonging to Lactarius theioga/us group (laticifers without septa, but thinner than described by GRONBACH 1988); • Zavodnje, SLO 00164 (05.11.1992). Type 163 • monopodial-pyramidal system, totally covered with soil particles (Figure 2); • mantle surface with short emanating hyphae, appearance warty to wooly, colour violet-brownish; • mantle plectenchymatous, in deeper layers well pronounced stellar pattern, cells colourless, surface densely covered with soil particles, hyphae thickwalled and tightly glued together; rhizomorphs not observed, emanating hyphae colourless to yellowish, clampless, smooth, if rough because of adhering soil particles; repair mechanism: new hyphae grow out of broken 290 Zbornik gozdarstva in lesarstva, 52 ones; • foreign hyphae also present; rather thinwalled hyphae filled with contents on the very tip; • new type, the suggested name, after a comprehensive description will have been done, is Piceirhiza terraphila; • Zavodnje SLO 00163 (05.11.1992). Type 185 • black simple or monopodially ramified ectomycorrhizae with straight emanating hyphae, predominantly from its tips; • stellate patterned mantle; • belonging to group Cenococcum geophi/um; • Mislinja SLO 00214, Zavodnje SLO 00185. Type 186 • irregularly monopodial-pyramidal, greyish to olive-grey mycorrhizae, covered with soil particles; • mantle surface plectenchymatous; hyphae (1) 2 - 3 mm in diameter, 10 - 40 mm distance between septa, with clamps, Y-ramified or with simple anastomoses; • resembling Piceirhiza harenosa (BERG 1989); • Mislinja SLO 00186. Type 196 • irregularly pinnate ramification, white mycorrhiza covered with white, flattened rhizomorphs; • mantle surface plectenchymatous, septa with doliporus-like structures, bluish- white autofluorescence under UV-filter; emanating hyphae with clamps, 3 - 5 mm in diameter; rhizomorphs undifferentiated, anastomoses with contact- clamps; • resembling Cortinarius obtusus group; • Zavodnje SLO 00196, Mislinja SLO 00222. Type 204 291 Kraigher H.: Mikobioindikacija onesnaženosti ... • irregularly monopodial-pyramidal ramifications, brownish colour with silvery patches, wooly emanating hyphae, covered with soil particles; • mantle surface plectenchymatous, septa with central globular thickenings; inner mantle layers with hyaline gelatinous matrix inbetween hyphae; emanating hyphae 2 - 3.5 mm in diameter, without clamps, frequently ramified, forming anastomoses with septum; granulated, often with gelatinous substances on outer celi walls, with irregular crystalloid appositions; • resembling Elaphomyces granulatus (Piceirhiza g/utinosa) group; • Zavodnje SLO 00204. Type 207 • irregularly pinnate or unramified mycorrhizae, of bright orange colour, covered with emanating hyphae and flattened rhizomorphs; • mantle surface plectenchymatous; emanating hyphae with clamps, 3.5 - 5 mm in diameter, smooth; rhizomorphs undifferentiated, hyphae loosely connected; anastomoses open, no contact clamps, very thick walled; • resembling Piceirhiza aurea group; • Zavodnje SLO 00207. Type 213 • irregularly monopodial-pyramidal ramified mycorrhiza, brownish colour of root cortex cells visible, very tips whitish, rhizomorphs of no distinct colour, smooth; • mantle surface plectenchymatous, with a dense net of oily droplets containing hyphae, inner surface pseudoparenchymatous; hyphae without clamps, 2.5 - 5 mm in diameter; rhizomorphs rare, undifferentiated; • resembling Piceirhiza o/eiferans (WALLER et ali. 1993); • Mislinja SLO 00213. Type 241 • unramified or monopodially ramified mycorrhiza, of dark brown to black colour, emanating hyphae black; older parts carbonizing; • mantle surface plectenchymatous; outer hyphal net formed of dark hyphae in simple three-directional stellate pattern; inner layers of uncoloured hyphae, 292 Zbornik gozdarstva in lesarstva, 52 hyphae, forming the Hartig net, dark; emanating hyphae 2-3 mm in diameter, young hyphae hyaline, older brown to black coloured, warty; simple septa, ramifications rare, anastomoses simple; • identified as belonging to the Piceirhiza horti-atrata group; • Zavodnje SLO 00241. Type 244 • monopodial-pyramidally ramified mycorrhiza of olive-green colour with smooth to slightly warty surface with adhering soil particles; without rhizomorphs and very few emanating hyphae; • mantle surface pseudoparenchymatous, with epidermoid cells, occasionally dark coloured, interwoven by a loose net of thin laticifers of 4-5 mm in diameter; thick walled but rather thin emanating hyphae, without clamps; • Lactarius sp., possibly belonging to the Lactarius necator group (AGERER, pers.comm.); • Zavodnje SLO 00244. Type 247 • monopodial-pyramidal ectomycorrhiza with woolly emanating mycelium and yellowish rhizomorphs; • mantle plectenchymatous, ca 1 O mm thick, innermost layers showing palmetti- structures; emanating hyphae granulated, with clamps, 2.5 - 4 mm in diameter; rhizomorphs consisting of loosely interwoven hyphae, not differentiated, oval to roundish, 1 O - 80 mm in diameter, anastomoses resembling H, or clamp- connections; • similar to Amphinema byssoides; • Mislinja SLO 00247. Type 250 • monopodially-pinnate ramification, ochre-brown, surface smooth to slightly spiny, rhizomorphs ochre to reddish brown; • mantle surface plectenchymatous, emanating hyphae and cystidia thick walled, 3.5 - 4.5 mm in diameter, with clamps; rhizomorphs slightly differentiated, type C, up to 150 mm in diameter; 293 Kraigher H.: Mikobioindikacija onesnaženosti ... • identified as belonging to the Thelephora terrestris group; • Zavodnje SLO 00250, Mislinja SLO 00385. Type 291 • monopodial-pyramidal systems of yellowish to yellow-olive colour with brighter yellow dots , most frequent close to ramifications; • mantle structure pseudoparenchymatous with angular cells and with conical heaps of fiat cells; with yellowish autofluorescence under UV-filter; emanating hyphae 2-3 mm in diameter, without clamps; rhizomorphs rare; • identified as belonging to the Russula ochroleuca group; • Zavodnje SLO 00291, Mislinja SLO 00315. Type 310 • irregularly monopodial-pyramidal ramified mycorrhiza, rather thin, bright ochre, with silvery shiny patches, 2 - 15 mm long, nonramified ends 0.3-0.4 mm in diameter; • rhizomorphs mostly roundish, ochre, with sclerotia; rhizomorphs with centrally arranged hyphae of conspicuously thicker diameter; hyphae with clamps; sclerotia smooth, round, shiny, 0.2-0.4mm in diameter; • identified as belonging to the Paxillus involutus group; • Zavodnje SLO 00310. Type 316 • Monopodially ramified dark brown to black mycorrhiza, beaded, with occasionally emanating hyphae in bundles; • mantle surface pseudoparenchymatous, formed of angular cells with heaps of roundish cells on outer surface; emanating hyphae 2.5 - 5.5 mm in diameter, with clamps, brownish coloured; cystidia rare, spherical to conical; • identified as belonging to the Piceirhiza nigra group; • Mislinja SLO 00316. Type 349 • Monopodially-pyramidal ramifications, bright-brown to whitish coloured, young tips slightly violet tinted, woolly emanating hyphae; 294 Zbornik gozdarstva in lesarstva, 52 • mantle surface densely plectenchymatous, inner layers forming palmetti structures; emanating hyphae 2 - 3.5 mm in diameter, with clamps, simple anastomoses, rhizomorphs undifferentiated, type NB; • identified as belonging to the Laccaria bicolor group; • Zavodnje SLO 00349. Characterization of Lactarius lignyotus Fr. x Picea abies (L.) Karst. (characterised comprehensively in KRAIGHER/ AGERER / JAVORNIK 1995) • The Lactarius lignyotus x Picea abies type is characterised basically by its irregular-monopodial-pyramidal mycorrhizal system of white colour, plectenchymatous mantle, laticifers, (4)5-7 mm in diameter, and cystidia-like protruding hyphal ends. • Using molecular characterisation, this type could be distinguished from a closely related Lactarius picinus x Picea abies by the different restriction digest pattern of the amplified ITS region by the enzyme Hinf 1. The amplified ITS region far both fungi shows a distinct fragment at approximately 850 bp. Using Alu1 restriction enzyme two bands are observed far both fungi at a position of 530 bp and 250 bp. Using Hinf1 restriction enzyme, Lactarius picinus shows two bands at 360 bp and 250 bp region, while Lactarius lignyotus and its ectomycorrhiza only show one band at 360 bp region. Characterization of Hvdnum rufescens Fr. x Picea abies (L.}Karst. (characterised comprehensively in AGERER /KRAIGHER/ JAVORNIK 1996) • The Hydnum rufescens x Picea abies type is primarily characterised by its neat monopodial-pyramidal mycorrhizal systems of a peachy-white colour with frequent rhizomorphs, plectenchymatous mantle, emanating hyphae (1.5)2-4 mm in diameter, with clamps and trumpet-like inflations, rhizomorphs irregularly shaped, an intermediate type between A and D organization type (AGERER 1991 a), with highly complicated ramifications, several hyphae growing out of a single inflated hyphal end, most hyphae contain oily droplets, outer hyphae warty. • The amplified ITS region shows a distinct band at approximately 840 bp. This fragment can be digested into two bands using the A/u1 enzyme at 480 and 160 bp, or into three bands with the enzyme Hinf1 at 330, 290 and 130 bp position. 295 Kraigher H.: Mikobioindikacija onesnaženosti ... 4.2 OCCURRENCE OF ECTOMYCORRHIZAE IN SOIL CORES Number of root tips (nonmycorrhizal, unidentified ectomycorrhizae and ectomycorrhizal types) from six samplings with soil cores in 1993, each from 2 to 5 replicates per plot, are represented in Table 1 and in Figures 1,2,3, 4. Table 1: Occurence of nonmycorrhizal and different ectomycorrhizal root tips in soil cores. Preglednica 1: Pojavljanje nemikoriznih in različnih ektomikoriznih korenin v vzorcih zemlje. Plot/ M M M M M M M z z z z z z z Tvoe 08.04. 07.05. 15.06. 19.08. 16.09. 21.10. Tota! 08.04. 07.05. 15.06. 19.08. 16.09. 21.10. Tota! Roots 105 36 74 130 69 99 513 136 66 204 50 74 39 569 SNT 3465 914 2235 5009 1338 4777 17738 2455 1143 1819 2170 1984 1682 11253 148 - - - - - - - 89 64 272 654 28 1364 2471 149 30 16 - - - 5 51 76 - 673 350 - 1009 2108 150 - 50 - 275 15 161 501 - - - - - - - 156 - - - - - - - - - 159 105 - 949 1213 163 - - - - - - - - - 1629 1343 29 - 3001 164 - - - - - 9 9 47 - 6 - - - 53 185 382 40 1157 407 425 524 2935 - 13 98 35 105 79 330 186 1075 572 - - - - 1647 - - - - - - - 196 - 51 - 69 15 34 169 11 - - 227 - 73 311 204 - - - 38 - - 38 72 - - 119 - 172 363 207 - - 9 25 - - 34 138 - - - - - 138 213 841 263 - 20 - 1301 2425 - - - - - - - 241 - - - - - - - - 106 - - - - 106 244 - - - 364 - - 364 - 270 - 16 - - 286 247 127 214 - 894 272 2122 3629 - - - - - - - 250 - 33 - - 48 231 312 77 - 154 - 33 27 291 291 13 - 72 100 - 171 356 139 - 938 278 425 - 1780 310 862 - - 47 51 - 960 78 25 307 222 - 2605 3237 316 - - - 60 63 80 203 - - - - - 76 76 349 - - - 269 - - 269 5 - - 413 289 - 707 404 73 701 535 512 1357 2068 5246 - - - - - - - 428 - 33 148 39 473 185 878 - - - - - - - Other - - - 37 3 185 225 - 38 - 56 - 56 150 No.t. 8 10 5 16 10 15 24 10 6 9 12 6 10 17 No.s. 3 3 2 5 3 5 21 3 3 2 5 3 5 21 M=Mislinja, Z=Zavodnje, SNT=old unidentifiable types; 148 = Piceirhiza inflata, 149 = Xerocomus badius, 150 = Russula sp., 156 = Piceirhiza paralle/a, 163 = Piceirhiza terraphila, 164 = Lactarius theiogalus, 185 = Cenococcum geophilum, 186 = Piceirhiza harenosa, 196 = Cortinarius sp., 204 = Elaphomyces granulatus, 207 = Piceirhiza aurea, 213 = Piceirhiza oleiferans, 241 = Piceirhiza horti- atrata, 244 = Lactarius sp., 247 = Amphinema byssoides, 250 = Thelephora terrestris, 291 = Russula ochro/euca, 310 = Paxillus involutus, 316 = Piceirhiza nigra, 349 = Laccaria bicolor, 404 = Hydnum rufescens, 428 = Lactarius lignyotus, Other: other undescribed types (can include more Ihan one type); No.t.= number of types; No.s.=number of soil cores (274 ml volume each) analysed per sampling; ali numbers as total no. of mycorrhizal (or nonmycorrhizal = Roots) root tips per sampling (dates indicated below plot sign); Tota! = total numbers for 6 samplings in 2 to 5 replicates per plot. 296 Zbornik gozdarstva in lesarstva, 52 Throughout the year 1993 the most frequent types occurring in Zavodnje were Paxillus involutus, Piceirhiza inflata, Xerocomus badius and Russula ochroleuca. In Mislinja the most frequent types were Hydnum rufescens,which was also highly constant in all samplings, Russula sp., Amphinema byssoides, Cenococcum geophilum; a highly constant type in all samplings was also Lactarius /ignyotus. The occurrence of the types of ectomycorrhizae and roots are shown in Figures 1,2,3, 4. The percentages are calculated from total countings of 38502 tips from Mislinja and 28443 from Zavodnje. The percentages of types are calculated from 20251 vital and identifiable ectomycorrhizal types from Mislinja and 16621 from Zavodnje. 53% Figure 1: Slika 1: Figure 2: Slika 2: 1% 46% • Roots (i] SNT l!fi!ECM Percentage of nonmycorrhizal root tips (Roots), unidentifiable ectomycorrhizae (SNT) and viable ectomycorrhizal root tips (ECM) in soil cores from Mislinja. Deleži nemikoriznih korenin (Roots), nedoločljivih tipov (SNT) in vitalnih tipov ektomikorize (ECM) v vzorcih tal iz Mislinje. 2% 40% • Roots [ill]SNT ITIJECM Percentage of nonmycorrhizal root tips (Roots), unidentifiable ectomycorrhizae (SNT) and viable ectomycorrhizal root tips (ECM) in soil cores from Zavodnje. Deleži nemikoriznih korenin (Roots), nedoločljivih tipov (SNT) in vitalnih tipov ektomikorize (ECM) v vzorcih tal iz Zavodenj. Figure 3: Slika 3: Figure 4: Slika 4: 3% 18% 297 Kraigher H.: Mikobioindikacija onesnaženosti ... D Russula sp. lfll Ccnocm:cum geophilum • Pkeirhiza harenm.a D Pkeirhiza oleifora D Lactarius sp. D Amphinema hyssoidcs ~ Thelcphora tcrrestris II Russula odrrolcm:a IJ Paxillus involutus ~ Hydnum rufescens m Lactarius lignyotus Ectomycorrhizal types, which occurred in more than 1 % of all viable types in soil cores from Mislinja. Tipi ektomikorize, ki so predstavljali več kot 1 % vseh vitalnih tipov ektomikorize v vzorcih tal iz Mislinje. 4% 13% D Piceirhiza inflata ril Xerocomus hadius • Piceirhiza paralella D Piccirhiza terraphila ETI Cenococcum geophilum O Cortinarius sp. III Elaphomyces granulatus III Lactarius sp. B8l Thelephora terrestris ~ Russula uchroleuca [[] Pax.Hlus involutus ~ Laccaria bicolor Ectomycorrhizal types, which occurred in more than 1 % of all viable types in soil cores from Zavodnje. Tipi ektomikorize, ki so predstavljali več kot 1 % vseh vitalnih tipov ektomikorize v vzorcih tal iz Zavodenj. 298 Zbornik gozdarstva in lesarstva, 52 4.3 COMPARISON OF THE OCCURENCE OF FRUITBODIES AND THE TYPES OF ECTOMYCORRHIZAE IN TWO FOREST RESEARCH PLOTS The occurrence of fruitbodies was taken down and their abundance was estimated by ranking them into rare-single specimen (+), few (++), frequent {+++) and massive (++++) occurrence. In Table 2 a comparison of the occurrence of fruitbodies and the types of ectomycorrhizae in the two forest research plots on one sampling day is shown. Table 2: Occurrence of the types of ectomycorrhizae (Mycorrhiza) in soil samples (as% of all types, but only the most frequent types are shown) and fungal fruitbodies (Fruitbody by ranks: -/none, +/occasional, ++/frequent, +++/massive occurence; only the most frequent species are listed) in two forest research plots on 21/10/1993. The total number of roots in soil cores from the same day from Mislinja was 11.972 , from which 99 were nonmycorrhizal, 4.777 were unidentifiable, and in soil cores from Zavodnje 8.146, from which 39 were nonmycorrhizal ans 1.682 were unidentifiable types. Preglednica 2: Pojavljanje tipov ektomikorize (Mycorrhiza) v vzorcih zemlje (kot delež vseh tipov ektomikorize v %, navedeni samo pogostejši tipO in trosnjakov gliv (Fruitbody, rangi: -/ni, +/posamezni, ++/pogosti, +++/masovno pojavljanje; navedeni samo trosnjaki pogostejših gliv) na dveh raziskovalnih ploskvah ob vzorčenju dne 21.10.1993. Skupno število analiziranih korenin vzorčenj tega dne je bilo v vzorcih iz Mislinje 11972, od tega 99 nemikoriznih korenin in 4777 starih nevitalnih tipov ektomikorize in v vzorcih iz Zavodenj 8146, od tega 39 nemikoriznih korenin in 1682 starih nevitalnih tipov ektomikorize. Fungus Zavodnie Mislinia Fruitbodv Irank) Mvcorrhiza /%\ Fruitbodv irank\ Mvcorrhiza (%) Paxil/us involutus +++ 41 ++ o Amanita aemmata +++ o - o The/eohora terrestris +++ 1 - 1 Xerocomus badius ++ 16 ++ 1 Tricholoma sanonaceum ++ o - o Piceirhiza inflata - 21 - o Piceirhiza narallela - 15 - o Lactarius mitissimus ++ o +++ o Laccaria amethvstina ++ o +++ o Lactarius /ianvotus - o +++ 4 Russula ochroleuca - o +++ 1 Cortinarlus 73 snnf - 1 ++ 1 Hvdnum rufescens - o + 29 Amohinema bvssoides - o - 30 Piceirhiza oleiferans - o - 18 Cenococcum aeoohilum - 1 - 7 299 Kraigher H.: Mikobioindikacija onesnaženosti ... For each of the plots the same fungal species were observed in different sampling years. However, the most abundant fruitbody occurrence was in autumn 1993 (the occurrence from one sampling day are shown above). 5 DISCUSSION In our project the identification and characterisation of ectomycorrhizal types on Norway spruce from two different forest sites by anatomical and molecular tools were used for the studies of their abundance on the two differently polluted forest research plots throughout the vegetation period. A possible application of the basic studies for the development of a bioindication method, based on identification of naturally occurring types of ectomycorrhizae on Norway spruce as "indicators", was suggested. 5.1 CHARACTERISATION ANO IDENTIFICATION OF ECTOMYCORRHIZAL TYPES IN TWO FOREST RESEARCH PLOTS By 1993, 82 types of ectomycorrhizae on Norway spruce were characterised using the anatomical method (by AGERER 1991 a) and included into the Colour Atlas of Ectomycorrhizae (AGERER 1987 - 1993). The method is based on morphological and anatomical characteristics of ectomycorrhizae, whereby the range of features have to be recognized for each type and compared to ali other known ectomycorrhizae. Therefore the identifications can not only be based on the key (as described in the Atlas), but have to be confirmed by comparisons with original publications, containing comprehensive descriptions of each type as well. Furthermore, from ecological studies of fungal fruitbodies (TRAPPE 1962, AGERER 1985, DEACON / FLEMING 1992) 300 to 500 fungal species are expected to form ectomycorrhizal associations with Norway spruce. Some authors expect even 2100 or more species of fungi which may be mycorrhiza - formers with North American trees (SMITH 1971 ). The numbers can be especially high in pronaturally managed forests with high natura! diversity, as is the case in Slovenian forests. Therefore only 1/3 to 1/1 O of ali ectomycorrhizal 300 Zbornik gozdarstva in lesarstva, 52 types on spruce could be described so far, and in each project, concerning the types of ectomycorrhizae, new characterizations can be expected. The comprehensive morphological and anatomical characterization of each type of ectomycorrhizae is a project which might last far severa! growth seasons. A number of characteristics can only be described on fresh material. These comprise all morphological characteristics regarding the shape and dimensions of ectomycorrhizae, surface views, colour, occurrence of rhizomorphs and emanating hyphae, most chemical tests and autofluorescence. In addition, the observation of severa! anatomical features of plan views of mantle, such as laticifers, colour of hyphal contents and their surface, and the same for rhizomorphs and emanating hyphae have to be done on fresh material (AGERER 1987-1993}. Positive identifications of the fungal partner can also imply severa! years to trace mycelial connections from ectomycorrhizae to fruitbodies, which may not be produced every year. Furthermore, these connections are in severa! cases difficult to obtain, which especially holds far fungal species, which do not ar can only rarely form rhizomorphs (such as the Russu/a and Lactarius genera}. Therefore in ecological studies (HAUG / PRITSCH 1992, ZHAO 1989, SCHERFOSE 1990, QIAN et ali. 1993) brief descriptions of ectomycorrhizae are a prerequisite far further comparisons regarding site or pollution effects. These morphological and anatomical descriptions can lately be supplemented by molecular methods (GARDES et alf. 1991 ). In our approach we have joined brief descriptions of a fairly large number of ectomycorrhizal morphotypes to two comprehensive descriptions of new types of ectomycorrhizae, which have proved to be of interrest regarding their growth potential in unpolluted sites only. These two identifications of the fungal symbiont were confirmed by molecular method based on PCR - amplification, using basidiomycete specific primers, and the restriction digests of the amplified products (described in KRAIGHER/ AGERER / JAVORNIK 1995, as modified from GARDES / BRUNS 1993). Brief descriptions of morphotypes in soil cores from the two forest research plots 301 Kraigher H.: Mikobioindikacija onesnaženosti ... in the emission zone of the CPP Šoštanj, which could be differentiated from one another, were based on the descriptions of most distinguishable characteristics of ectomycorrhizae. Whenever possible these types were compared to an already described type or a 'group' type. The introduction of this term was necessary to emphasise the possibility that the type considered could show all the characteristics of one single fungal species, but due to the number of related fungal species the characteristics described might also belong to another fungus. For example Lactarius theiogalus x Picea abies, as described already by GRONBACH 1988, showed the same characteristics as the morphotype described in our studies, but there is stili a whole range of Lactarius spp. from the same Tabidi section (BON 1987), whose ectomycorrhizae have not been described on Norway spruce so far, therefore any of these fungi could also account for the relevant features from GRONBACH 1988. In two cases (regarding the brief descriptions of morphotypes) no similarities could be found with any characterised type of ectomycorrhizae. Considering them as new types, longer descriptions were needed and new names for them, regarding their most obvious characteristics, were suggested: Piceirrhiza inflata for the type with reference number SLO 00148 and Piceirhiza terraphila for the type with reference number SLO 00163. Piceirrhiza inflata is predominantly characterised by its white colour with ochre tint and brownish rhizomorphs, hyphae without clamps but with well distinguishable inflations, which occur throughout the mantle and in rhizomorphs. From the presence of the globular thickenings (here onesided) at the septum it is possible to conclude that the fungal partner belongs to Basidiomycetes. Piceirrhiza terraphila is characterized first by being totally covered with soil particles, adhering on a violet-brownish warty to wooly mantle, consisting of clampless hyphae which are tightly glued together and smooth emanating hyphae with a pronounced repair mechanism: a new hypha can grow out of a broken one. Any relationships could not be established so far. A few more types were dubious regarding their identity - those which might belong to severa! Russu/a or to severa! Lactarius sps. In one case (Amphinema 302 Zbornik gozdarstva in lesarstva, 52 byssoides) the hyphae of this fungus were found also to invade other (miner) types which were then (for ease) included among descriptions under the name of Amphinema byssoides. lf the hyphae of this fungus were not too numerous and the primary type was recognized, then the type would be included under the original type's name. Similar 'pirate' hyphae were also observed for Cenococcum geophilum and a few unidentified dark brown hyphae, which are also known to grow on the surface of other types (pers. comm. with colleagues from Munich, TObingen and Scotland). Fram the discussions on Lactarius lignyotus x Picea abies (KRAIGHER / AGERER / JAVORNIK 1995) and on Hydnum rufescens x Picea abies (AGERER /KRAIGHER/ JAVORNIK 1996) we would hereby only like to stress the easy and fast application of the molecular methods, and the necessity of the combination of the classical anatomical methods of characterisation and determination of types of ectomycorrhizae with molecular markers. Based on these studies a conclusion was made that molecular biology approaches can not replace classical taxonomical procedures but they may simplify them and add to the objectivity, due to the possibility to compare restriction patterns of r-DNA of fruitbodies and of mycorrhizae which grew within the area of a single fungal population. The molecular methods can be employed as helpful tool to implement the anatomical characterisation and identification of ectomycorrhizae; the latter could be adapted to a simplified version if complemented by molecular methods; thus temporal and to a certain extent also spatial studies of ectomycorrhizal communities can be made friendlier. 5.2 OCCURRENCE OF TYPES OF ECTOMYCORRHIZAE IN TWO FOREST RESEARCH PLOTS The two forest research plots were analysed far abundance of ectomycorrhizal types by regular sampling during the vegetation period 1993. Preparatory work on fungal types and methods of sampling was carried out in the years 1990 to 1992. As mycorrhizal fungi spread predominantly in upper soil horizons (MIKOLA / LAIHO 1962) and such distribution was also established in the Norway spruce forests on the Pohorje mountain range in north Slovenia (KRAIGHER 1991}, the 303 Kraigher H.: Mikobioindikacija onesnaženosti ... sampling was limited to organic soil horizons. To allow volumetric or percental comparisons of the occurrence of nonmycorrhizal and mycorrhizal root tips as well as the abundance of different ectomycorrhizal types, it was decided to compare identical volumes of soil at each sampling in the two plots. Due to high heterogeneity of soil biotic and abiotic structure, several replicates were needed per each sampling period. It was planned to analyse 5 replicates at different locations per plot per sampling in 1993 in order to minimize the variability in each sampling period. The actual number of replicates per sampling varied from 2 to 5 due to the occurrence of new types, which needed brief descriptions, and to additional samplings of soil cubes from underneath fruitbodies in the autumn months with a view to generate comprehensive descriptions of ectomycorrhizae. Between the percentages of nonmycorrhizal root tips, unidentifiable ectomycorrhizae and viable ectomycorrhizal root tips from Mislinja (Figures 1,3) and Zavodnje (Figures 2,4), only minor differences were observed. In total (Table 1 ), the same volumes of soil showed greater numbers of root and mycorrhizal root tips in samplings from Mislinja (a total of 38502) in comparison to Zavodnje (a total of 28443). Taking these different totals into account, no differences were established in the proportion of nonmycorrhizal or mycorrhizal root tips from both plots. Similar results were also reported by TERMORSHUIZEN / SCHAFFERS 1989a, 1989b, who found no significant differences in total counts of mycorrhizae under the influences of NH3 or SO2 pollution. However, organic soil horizons in Mislinja (see above) seem to be better overgrown with fine roots of Norway spruce in comparison to Zavodnje. This might be explained by previous work of WEISS / AGERER 1986 in which it was shown that either the lack of Ca and Mg fertilization or, separately, ozone treatment resulted in a reduction of fine root biomass. Similar data were reported by KOCOUREK / BYSTRIČAN 1989 for heavily polluted Krušne hory in comparison to unpolluted šumava mountains. Regarding the occurrence of different types of ectomycorrhiza (Table 1) the differences between the two plots were clear. In respect of the number of types, a greater number was found to occur in Mislinja (24) in comparison to Zavodnje 304 Zbornik gozdarstva in lesarstva, 52 (17). In the plot in Zavodnje the following types, as calculated from pooled samplings, were dominating throughout the year: Paxillus involutus (19.5%), Piceirhiza terraphila (18.1%), Piceirhiza inflata (14.9%), Xerocomus badius (12.7%), Russula ochroleuca (10.7%} and Piceirhiza para/Jela (7.3%). The three identified types from this list have been shown previously to occur on polluted sites: QIAN et ali. 1993 have shown that the viable mycorrhizae from the polluted plots in the TObingen area belonged predominantly to Xerocomus badius type; TURNAU / KOTTKE / OBERWINKLER 1993 have stressed their studies of heavy metal tolerance predominantly on Paxillus involutus, as this fungus was found previously to persist on heavily polluted plots, where it was accompanied also by Russu/a ochro/euca; this last fungus was found to grow well independently of pollution or liming also in HOglwald (TAYLOR / BRANO 1991). However, also opposite results were shown regarding Paxillus involutus by TERMORSHUIZEN / van der EERDEN / DUECK 1989 who have reported on decrease of mycorrhiza formation by this fungus under the influences of S02 treatment. SHAW et a/1. 1992, who have found high numbers of fruitbodies of the same fungus on fumigated plots, have discussed their results in connection to the above Dutch paper as an artefact, due to the presence of this fungus on the plots already before the fumigation started. In the view of our data, Paxillus invo/utus and the other above mentioned ectomycorrhizae do seem to share the ability to persist on heavily polluted sites, whereby a range of other fungi tend to disappear. Also Laccaria bicolor (occuring at 4.3% on this plot) was reported previously to persist on heavily polluted plots (TERMOSHUIZEN / van der EERDEN / DUECK 1989). However, as the period of ontogenetical development of this type is relatively short (WEISS 1988) it rnight be replaced by other fungi on a single mycorrhizal system, or in our samplings it might have been included among the unidentifiable types (as too old to be identified). In the plot in Mislinja the following types as calculated frorn pooled samplings were either dominating or occurring regularly throughout the year: Hydnum rufescens (25.9%), Russu/a sp. (25%), Amphinema byssoides (17.9%), Cenococcum geophilum (14.5%), Piceirhiza oleifera (12%), Piceirhiza harenosa (8.1%) and Lactarius /ignyotus (4.3%). Among these types we would like especially to stress on the constant and high frequency of Hydnum rufescens, 305 Kraigher H.: Mikobioindikacija onesnaženosti ... which is known to be among the first fungal species, disappearing under the influences of pollution (ARNOLDS 1991 ). From this point of view this ectomycorrhiza can be applied as a bioindicating type for unpolluted sites and the plot in Mislinja can be considered as an unpolluted plot. This fungus did not appear on the plot in Zavodnje at all. The other type, occuring constantly in all samplings from Mislinja, although in lower frequency, was Lactarius lignyotus. This fungus or its ectomycorrhiza was not studied previously, as it is relatively rare in central Europe (AGERER pers.comm.). We suggest that it might also be used as a bioindicator of unpolluted sites. The other ectomycorrhizae, occurring in the plot in Mislinja in relatively high frequencies, can not be considered as possible bioindication types from the following reasons: Russu/a sp. was not identified to species or group leve! and it could as well represent two different species. Amphinema byssoides was found to occur frequently in the plot in Mislinja, but its hyphae can also be found on other types. In our own studies we have found this ectomycorrhizae to occur as the predominant ectomycorrhizal type also in the lnstitute's nursery in Ljubljana, which might be considered as a relatively polluted site (unpublished). Cenococcum geophilum in our case seems to be more frequent in the plot in Mislinja, which might contradict the previous reports on this fungus as being well adapted to stress conditions (MEYER 1987). However, this ectomycorrhizae, probably formed by an asexual stadium of Deuteromycotina (Fungi lmperfecti), has been considered as a complex type, formed by severa! different fungal symbionts (AGERER pers.comm.). Therefore it might be possible that severa! of these fungi, showing similar characteristics in ectomycorrhizae, can be differently suited to different environmental conditions or can even show different competitiveness in natura! forest ecosystems. Paxillus involutus was also found in severa! samplings in Mislinja, representing 4.7% of all ectomycorrhizal types from this plot. This might suggest that this ectomycorrhiza has a broad range of distribution, but due to higher competitiveness of other fungal partners in nonpolluted sites, can not dominate in Norway spruce ectomycorrhizae in such plots where natura! diversity is high. It merely takes over the position of the disappearing types due to pollution in heavily polluted plots. 306 Zbornik gozdarstva in lesarstva, 52 5.3 COMPARISON OF FRUITBODY ABUNDANCE AND ECTOMYCORRHIZAE The autumn of 1993 was very productive regarding fungal fruitbody occurrence in the whole of Slovenia. Therefore also the comparison on fruitbody abundance and ectomycorrhizae could best be done on the last sampling date in October 1993. The predominance in fruitbodies did not linearily follow the abundance in ectomycorrhizal types, i.e. in mycelium which is present in the soils. However, as the fruitbodes were not counted, but their presence was estimated by four ranks, no statistical correlations could be done. In the plot in Zavodnje, the great number of fruitbodies of Paxillus involutus corresponded well to the percentage (41 %} of the ectomycorrhizal types sampled at this date. Xerocomus badius fruitbodies were also fairly frequent at the plot, as well as the ectomycorrhizae of this type (16%). However, Piceirhiza inflata and Piceirhiza paralle/a could not be connected with any of the fungal fruitbodies occurring on the plot at this samplig date. Therefore the presence or absence of fruitbodies could not be correlated with the occurrence of different types of ectomycorrhizae. Such results were also reported by other authors (i.e. SHAW et ali. 1992, MEHMANN et ali. 1995, ICOM 1996). Furthermore, the presence of severa! ectomycorrhizae could be established throughout the vegetation season, while fruitbodies only appeared for a few weeks in the autumn. The comparisons of fruitbody occurrence with ectomycorrhizae in the plot in Mislinja showed even fewer correlations. The predominant type of ectomycorrhiza was shown to be Hydnum rufescens, while its fruitbody was only noticed once on the plot. A few more have occurred in the vicinity of the plot. Neither Amphinema byssoides nor any other corticiaceous fungal species was noticed at all on the slopes of Mislinja and yet its 'pirate' hyphae and the type itself were rather frequent formers of ectomycorrhiza. A few fruitbodies of Paxillus involutus were found in the October sampling in Mislinja. However, at this sampling date, no ectomycorrhizae of this type were noticed. Therefore far this plot, it was even more apparent that no clear correlations were possible between fungal fruitbody occurrence and ectomycorrhizal types occurrence. 307 Kraigher H.: Mikobioindikacija onesnaženosti ... 5.4 APPLICATION OF ECTOMYCORRHIZAL STUDIES FOR BIOINDICATION Fram the occurrence of ectomycorrhizae in the two forest research plots and comparison with fungal fruitbody occurrence, we suggested the following (see also KRAIGHER 1996, KRAIGHER/ BATIČ/ AGERER 1996): 1. pollution can influence the distribution of mycorrhizal fungi, whereby several species can disappear while the others can profit from the new less competitive stage in the pollution-stressed forest ecosystem; 2. mycobioindication through mycocoenoses is a possible method to apply in bioindication of forest sites (as suggested by FELLNER 1989 and ARNOLDS 1988, 1991); 3. in Slovenia, mycobioindication through selective sensitive, in comparison to unsensitive, fungal species, i.e. the presence and abundance of the representatives of each of these two groups, seems to be the main choice far the application of this method, whereby the sensitive species in our case would be Hydnum rufescens and the nonsensitive one would be Paxillus involutus; 4. from the comparisons of fruitbody occurrence and types of ectomycorrhizae, the latter seems to be superior for the establishing of the presence of a certain fungus in a forest site, since: i) ectomycorrhizae can be present in forest soils throughout the year and in different vegetation seasons, while the presence of fruitbodies is restricted to a certain period in the year, and their presence can vary between different years due to general climatic conditions; ii) a great number of ectomycorrhizal fungi do not form sporocarps (Deuteromycotina), or belong to corticiaceous fungi, whose sporocarps are difficult to observe, or belong to hypogeus fungi, whose sporocarps need special sampling procedures, as they are nicely hidden bellow ground; iii) in several cases in our plots the identity of the fungal partner in ectomycorrhiza was not known, but ectomycorrhizae seemed to be restricted either to the polluted plot (such as Piceirhiza inflata and Piceirhiza terraphila) or to the unpolluted plot (such as Piceirhiza o/eifera and Piceirhiza harenosa), which means that these fungi might also be suspected as differentially sensitive to pollution. 308 Zbornik gozdarstva in lesarstva, 52 6 CONCLUSIONS In our studies a range of ectomycorrhizal types was briefly described tor the assessment of distribution of ectomycorrhizae in two research plots. Based thereupon, two comprehensive characterisations were done for Lactarius lignyotus x Picea abies and for Hydnum rufescens x Picea abies, which had not been characterised previously. The funga! partner was additionally characterized and identified by the electrophoretogram of the PCR amplified DNA fragment and the sizes of its restriction digest fragments. This characterization proved to be fast and objective. We suggest that PCR methods be used and further developed with every anatomical description of ectomycorrhizae in the future. Regarding the occurrence of ectomycorrhizal types a difference was shown in respect of their occurrence and abundance in the two forest research plots. It has been shown that pollution can influence the distribution of mycorrhizal fungi, therefore mycobioindication through mycocoenoses is a possible method to be applied in the bioindication of forest site pollution. In Slovenia mycobioindication through selectively sensitive fungal species - in cornparison to insensitive ones (two 'indicator' organisms) - seems to be the main choice for the application of this method, whereby the sensitive species in our case would be Hydnum rufescens and the nonsensitive one Paxilfus involutus. Frorn comparisons of fruitbody occurrence and types of ectomycorrhizae the latter seern to be superior for the establishing of the presence of a certain fungus in a forest site. With the application of molecular rnethods a large scale screening could also be possible in differently polluted forest sites. 7 SUMMARY In our studies severa! taxonornical and ecological aspects of ectornycorrhizae of Norway spruce were studied with respect to their potential for use in the bioindication of forest site pollution in Slovenia. Therefore two comparable forest research plots of the Forestry Institute of Slovenia were chosen in the ernission zone of the Coal Power Plant in Šoštanj, differing in the pollution impacts, the plot in Zavodnje representing the polluted site and the plot in Mislinja the relatively unpollutedone. 309 Kraigher H.: Mikobioindikacija onesnaženosti ... For taxonomical studies of ectomycorrhizae the classical method based on morphological and anatomical characteristics of the symbiotic organs was applied. It was supplemented with a molecular method based on the PCR amplification of the ITS region of ribosoma! repeats using basidiomycete-specific primers and two restriction enzymes for the analysis of the amplified product. A range of ectomycorrhizal morphotypes were briefly described for the assesment of ectomycorrhizal distribution in two forest research plots. Therefrom two comprehensive descriptions were done for Lactarius lignyotus x Picea abies and for Hydnum rufescens x Picea abies, which had not been characterised previously. The fungal partner was additionally characterized and identified by the electrophoretogram of the PCR amplified DNA fragment and the sizes of its restriction digest fragments. Besides these two types 20 other types were found to occur on the two plots in the vegetation season 1993. At least two of them will still need comprehensive descriptions in the future as they have not been described so far. In regular samplings with soil cores of ca 270 ml volume in 1993, a total of 21 soil cores were analysed from each plot. Total countings of all nonmycorrhizal and mycorrhizal root tips per plot showed 38502 tips in samples from Mislinja and 28443 tips in samples from Zavodnje, which showed a reduction in fine root biomass in the polluted plot. Regarding the occurrence of ectomycorrhizal types, a difference was clear as to the number of types, their occurrence and abundance in the two forest research plots. Based on our data it has been established that pollution can influence the distribution of mycorrhizal fungi, mycobioindication through mycocoenoses is a possible method to be applied in the bioindication of forest sites, mycobioindication through selectively sensitive - in comparison to insensitive - fungal species, i.e. the presence and abundance of the representatives of each of these two groups, seems to be the main application choice of this method in Slovenia; from comparisons of fruitbody occurrence and types of ectomycorrhizae it can be concluded that the latter seem to be superior for the establishing of the presence of a certain fungus in a forest site. 310 Zbornik gozdarstva in lesarstva, 52 8 POVZETEK Gozdni ekosistemi so funkcionalno in strukturno visoko organizirani sistemi biotskih in abiotskih komponent, povezanih v občutljivo dinamično ravnovesje. Onesnaževanje vpliva na vse in na posamezne komponente teh sistemov na različnih ravneh in se odraža v spremembah fiziologije gozdnih drevesnih vrst ter na spremembah vrstne sestave in abundanci vrst v gozdni flori in mikoflori. V prispevku s pomočjo taksonomskih in ekoloških raziskav tipov ektomikorize pri smreki prikazujemo enega od pristopov k študiju vplivov onesnaževanja na gozdne ekosisteme. Postopke in rezultate raziskav smo poskušali uporabiti za bioindikacijo onesnaženosti gozdnih rastišč. Terenski del raziskav smo izvajali na dveh različno onesnaženih, rastiščno primerljivih ploskvah na imisijskem območju termoelektrarne Šoštanj. Gozdna raziskovalna ploskev Gozdarskega inštituta Slovenije v Zavodnjah je predstavljala ploskev, ki je pod neposrednimi vplivi emisij, gozdna raziskovalna ploskev v Mislinji pa je predstavljala razmeroma neonesnaženo ploskev. Obe ploskvi ležita v pretežno smrekovih sestojih starosti 80 - 100 let, na nadmorski višini 850 m, talna podlaga je distrični kambisol, rastlinska združba je Querco-Luzu/o-Fagetum oziroma Luzulo-Fagetum il/yricum. Indeks čistosti zraka z analizami lišajev (IAP) je pokazal, da je ploskev v Zavodnjah zelo onesnažena, ploskev v Mislinji pa relativno neonesnažena (BATIČ 1991). Taksonomske raziskave tipov ektomikorize pri smreki smo izvajali po klasični anatomsko-morfološki metodi (AGERER 1991a in 1987-1993) s svetlobnim mikroskopom z dodatki za nomarski interferenčni kontrast, fazni kontrast in epifluorescenco in z lupo s standardiziranimi (predpisanimi) postopki za fotografijo in meritve. Dva popolna opisa tipov ektomikorize smo dopolnili z molekularno metodo analize prednostno amplificirane regije ITS ribosomalnih genov (po GARDES / BRUNS 1993) (ITS = notranji transkripcijski presledek) ter encimsko cepljenih fragmentov produkta amp!ifikacije. Z uporabo bazidiomicetno specifičnih komplementarnih oligonukleotidov (začetnikov, angl. 'primer') je bilo mogoče prednostno pomnožiti ITS regijo ribosomalnih genov glivnega partnerja v simbiozi (opisano v KRAIGHER / AGERER / JAVORNIK 1995, AGERER / KRAIGHER/ JAVORNIK 1996, metode v KRAIGHER 1996). 311 Kraigher H.: Mikobioindikacija onesnaženosti ... Na ploskvah smo vzorčili med leti 1990 in 1993. Prvotne študije smo omejili na spoznavanje tipov ektomikorize pri smreki na obeh ploskvah. Pri tem bi poudarili, da je bilo dotlej pri smreki opisanih 82 tipov ektomikorize (AGERER 1987-1993), glede na število gliv, ki se pojavljajo v smrekovih gozdovih severnega zmernega in borealnega pasu, pa pričakujejo, da bi bilo število tipov ektomikorize lahko od nekaj sto do tisoč (AGERER 1985, DEACON / FLEMING 1992). Zato je potrebno v vsaki ekološko usmerjeni študiji tipov ektomikorize le-te najprej na kratko ali popolno opisati in spoznati. V vegetacijski sezoni 1993 smo na obeh ploskvah izvedli 6 vzorčenj v 2 - 5 ponovitvah (zaradi velike biotske in abiotske heterogenosti zgornjih horizontov tal) s sondo volumna 270 ml (velikost vzorca je bila omejena z možnostjo časovne izvedbe analiz med vzorčenji). V vsakem vzorcu smo prešteli vse nemikorizne in mikorizne kratke korenine. Med mikoriznimi smo izločili stare, nedoločljive tipe in tiste, ki so bili premalo razviti ali jih je bilo premalo za opis. Vse določljive tipe smo na kratko opisali in prešteli. Dva tipa ektomikorize s ploskve v Mislinji smo popolno opisali in identificirali glivnega partnerja s sledenjem povezavi med ektomikorizo in trosnjakom glive in z molekularno metodo. Na ploskvi v Mislinji smo v skupno 21 sondah prešteli 38 502 kratkih korenin, na ploskvi v Zavodnjah pa 20251 kratkih mikoriznih in nemikoriznih korenin smreke (preglednica 1 ). Sklepali smo lahko na zmanjšanje biomase kratkih korenin na bolj onesnaženi ploskvi. O podobnih rezultatih so poročali WEISS / AGERER 1986 ter KOCOUREK / BYSTRIČAN 1989 za ploskve na Bavarskem in Češkem. Glede na število in pojavljanje tipov ektomikorize smo ugotovili razlike med obema ploskvama (preglednici 1, 2, slike 1, 2, 3, 4). Na ploskvi v Mislinji smo določili skupno 24 tipov ektomikorize, na ploskvi v Zavodnjah pa 17. Tam smo skozi vso vegetacijsko dobo popisovali naslednje tipe ektomikorize: Paxil/us involutus (20 %), Piceirhiza terraphila (18 %), Piceirhiza inflata (15 %), Xerocomus badius ( 13 % ), Russu/a ochroleuca ( 11 % ) in Piceirhiza para/leta (7 %). Nekatere od teh tipov so tudi drugi avtorji opisovali kot pogoste na onesnaženih ploskvah: QIAN in sod. 1993 so poročali, da je najpogostejši vitalni tip na onesnaženih ploskvah v okolici T0bingena Xerocomus badius, TURNAU in 312 Zbornik gozdarstva in lesarstva, 52 sod. 1993 pa so svoje raziskave omejevali na vrsti Paxillus involutus in Russu/a ochroleuca, ker sta uspevali na močno onesnaženih področjih v okolici Krakova. Na ploskvi v Mislinji smo skozi vse leto opisovali naslednje tipe ektomikorize: Hydnum rufescens (26 %), Russula sp. (25 %), Amphinema byssoides (18 %), Cenococcum geophilum (15 %), Piceirhiza oleifera (12 %), Piceirhiza harenosa (8 %) in Lactarius lignyotus (4 %). Med temi bi želeli poudariti predvsem pogosto pojavljanje in veliko številčnost tipa Hadnum rufescens. Te glive na ploskvi v Zavodnjah sploh nibilo. Za vse hidnoidne vrste je namreč značilno, da so med prvimi, ki izginejo z onesnaženih rastišč (ARNO LDS 1991 ). Zato bi lahko to glivo uporabili kot kazalec neonesnaženih rastišč in ploskev v Mislinji označili za neonesnaženo ploskev. Iz primerjave med popisi tipov ektomikorize in popisi trosnjakov gliv smo ugotovili, da ni mogoče neposredno primerjati številčnosti posameznih vrst gliv v obeh razvojnih oblikah. Trosnjaki gliv so se pojavljali predvsem v jesenskem obdobju, medtem ko so tipi ektomikorize prisotni na gozdnih rastiščih skozi vse leto. Trosnjaki posameznih vrst gliv, npr. Hydnum rufescens, so bili prisotni na ploskvi v Mislinji le posamič, medtem ko je med tipi ektomikorize ta tip prevladoval. Pri nekaterih tipih ektomikorize, ki so se značilno pojavljali le na onesnaženi ali le na neonesnaženi ploskvi, pa glivnega partnerja še nismo identificirali. Glede na rezultate naših raziskav smo sklepali naslednje: 1. onesnaževanje lahko vpliva na distribucijo mikoriznih gliv, med katerimi lahko nekatere izginejo, medtem ko druge lahko izkoristijo novonastali manj kompetitivni stadij v zaradi onesnaženja osiromašenem gozdnem ekosistemu; 2. mikobioindikacijo z analizo mikocenoz je mogoče uporabiti v ugotavljanju onesnaženosti gozdnih rastišč (kot sta predlagala FELLNER 1989 in ARNOLDS 1988 in 1991); 3. v Sloveniji bi predlagali metodo mikobioindikacije z analizo prisotnosti občutljivih v primerjavi z na onesnaženje neobčutljivimi vrstami gliv, to je z raziskavo prisotnosti in abundance predstavnikov vsake od teh skupin; v naših raziskovalnih razmerah se je pokazala kot občutljiva vrsta Hydnum 313 Kraigher H.: Mikobioindikacija onesnaženosti ... rufescens, kot neobčutljiva pa Paxil/us involutus; 4. iz primerjave med pojavljanjem trosnjakov in tipi ektomikorize sklepamo, da so slednji primernejši za ugotavljanje prisotnosti določene vrste gliv na gozdnem rastišču, iz naslednjih vzrokov: • ektomikoriza je v gozdnih tleh prisotna skozi vse leto in v različnih vegetacijskih sezonah, medtem ko je pojavljanje trosnjakov omejeno na določeno obdobje v letu, odvisno pa je od klimatskih dejavnikov v celem letu; • veliko število ektomikoriznih gliv ne tvori trosnjakov (Deuteromycotina) ali se uvršča med skorjaste glive, katerih trosnjake je težko opaziti, ali sodi med podzemne glive, katerih trosnjaki so skriti pod zemljo; • v nekaj primerih smo zabeležili povezanost določenih tipov ektomikorize bodisi z onesnaženo ali le z neonesnaženo ploskvijo, vendar pri njih glivnega partnerja še nismo identificirali; kljub temu bi te tipe lahko uvrstili med razlikovalno občutljive na onesnaženje. 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Molecular methods were applied by the author in the laboratory of the Center far Plant Breeding and Biotechnology, Ljubljana, as part of a joint project L4-6287-0404/94 (MST & MAF). The publication was prepared as part of the current projects L4-7402 (MST & MAF) and 1487/8 (MAF). Special thanks are due also to Mrs. Jana Janša far her help in sample preparation and countings.