FOLIA BIOLOGICA ET GEOLOGICA 65/1, 53–63, LJUBLJANA 2024 ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) IN SLOVENIA EKTOMIKORIZNI SIMBIONTI NAVADNE JELKE (ABIES ALBA MILL.) V SLOVENIJI Tina UNUK NAHBERGER1, Hojka KRAIGHER1 & Tine GREBENC1 http://dx.doi.org/10.3986/fbg0107 ABSTRACT Ectomycorrhizal symbionts of silver fir in Slovenia Ectomycorrhizal symbionts of silver fir have been in Slovenia rarely analyzed and identified, thereby little is known about their diversity and distribution across Slove- nia. The aim of this study was to identify ectomycorrhizal fungal symbionts of silver fir in silver fir natural distribu- tion area in Slovenia. Ectomycorrhizal fungi were identified based on morphological and anatomical descriptions and by ITS DNA barcoding. Altogether we identified 86 different ectomycorrhizal symbionts of silver fir, many of them were in symbiosis with silver fir, in our study identified for the first-time. The most abundant ECM fungal taxa were as- signed to Russula, Tomentella and Lactarius genus. Keywords: silver fir, ectomycorrhizal symbionts, ITS barcoding, Slovenia IZVLEČEK Ektomikorizni simbionti navadne jelke v Sloveniji Ektomikorizni simbionti navadne jelke v Sloveniji, so do sedaj bili le redko predmet raziskav, zato je poznavanje njihove pestrosti in razširjenosti v Sloveniji pomanjkljivo. Namen raziskave je bil identificirati glivne vrste, ki tvorijo ektomikorizno simbiozo z navadno jelko na naravnem ob- močju razširjenosti navadne jelke v Sloveniji. Ektomikori- zne simbionte smo identificirali s pomočjo morfološko-ana- tomskih značilnosti ter ITS DNK barkod. Skupaj smo iden- tificirali 86 različnih ektomikoriznih glivnih vrst, pri čemer smo veliko vrst v ektomikorizni simbiozi z navadno jelko v naši študiji identificirali prvič. Med številčnejše ektomikori- zne glive so se uvrstile glive iz rodov Russula, Tomentella in Lactarius. Ključne besede: navadna jelka, ektomikorizni simbionti, ITS barkoda, Slovenija 1 Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana, tina.unuk@gozdis.si; hojka.kraigher@gozdis.si, tine.grebenc@gozdis.si UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 54 FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Mountainous silver fir (Abies alba Mill.) has a central European distribution, with a continuous population in Alps and central Europe, where its distribution lim- its on mountainous regions of eastern, western, south- ern Europe (Wolf 2003). Silver fir distribution in Slo- venia represents a central area of the species total areal, where it similarly grows natively in the mountainous areas. It represents 14 % of the total forest cover in Slo- venia (Bončina et al. 2002). It rarely forms pure stands, as populations usually grow mixed with European beech (Fagus sylvatica L.) and/or Norway spruce (Picea abies (L.) H. Karst.). In central European forests, silver fir has been successful tree species as it has an ability to grow in low-light conditions, thereby silver fir is fa- vored by the selection thinning forest management system, widely applied in Slovenia. Combining this management system with a silver fir characteristics commonly results in silver fir dominated forests (Klopčič & Bončina 2011). Silver fir is an ectomycorrhizal (ECM) tree species, forming symbiosis with ECM fungi from Ascomycota and Basidiomycota. In the past, silver fir received only little attention as ECM host tree. In most studies ECM symbionts of silver fir have been identified only based on morphological and anatomical descriptions of ECM or based on fruit bodies occurrence (Agerer 1987; Berndt et al. 1990; Comandini et al. 1998; Pacioni et al. 2001; De Román & De Miguel 2005). This ap- proach is in most cases deficient, especially at a spe- cies-level. Only few studies were published in which authors identified ECM fungal symbionts using mo- lecular methods (Eberhardt et al. 2000; Schirkon- yer et al. 2013; Ważny 2014; Rudawska et al. 2016; Ważny & Kowalski 2017; Unuk et al. 2019; Mrak et al. 2020). ECM fungal symbionts of silver fir are also poorly represented in an online database DEEMY (http://www.deemy.de), where after latest update in 2019 only twelve were described for silver fir, among all 554 description available therein (Agerer & Rambold 2004). In Slovenia only recently some papers emerged publishing ECM fungal symbionts diversity with silver fir (Unuk et al. 2019) impacting plant growth and vigor by influencing the quality, direction, and flow of nutrients and water between plants and fungi. Link- ages of plant phenological characteristics with below- ground root-associated fungal communities have rare- ly been investigated, and thus our aim was to search for an interplay between contrasting phenology of host ectomycorrhizal trees from the same location and root-associated fungal communities (ectomycorrhizal, endophytic, saprotrophic and pathogenic root-associ- ated fungi while other common tree species, such as beech and spruce were frequently studied (Kraigher et al., 1995, Kraigher 1996; Agerer et al., 1996, Pučko et al. 2005; Kraigher et al. 2007; Grebenc & Kraigher 2007; De Groot et al. 2016). The aim of this study was to identify ectomy- corrhizal symbionts of silver fir in Slovenia. 1 INTRODUCTION 2 METHODS Samplings were performed in three silver fir (Abies alba Mill.) dominated stands at sites Jelovški boršt (45.05 N, 15,05 E), Lehen na Pohorju (46.33 N, 15.20 E) and Ljubelj (46.24 N, 14.15 E). At each site one silver fir dominating sampling plot (20 m × 20 m) was estab- lished. Inside individual site 5 adult silver fir trees, which showed no signs of illness or damages were se- lected for sampling. Samplings were performed once per month from March-October in year 2016 and 2017. One soil core sample (10 cm in diameter and 20 cm deep) per month was sampled for each selected silver fir tree at individual site (1 soil core x 5 silver fir trees x 3 sites), according to protocol described in Kraig- her (1996). Roots from samples were soaked in tap water, before being washed to remove all the soil par- ticles attached to the roots. Further, roots were sepa- rated into coarse and fine roots, according to Žele- znik et al. (2007, 2016). Fine roots were characterized according to the method of AGERER (Agerer 1987) until 250 root tips per individual soil core sample were characterized. Three to five root tips of each individu- al characterized morphotype were freeze dried and used for ITS DNA barcoding. Total DNA from each morphotype was extracted with a DNeasy Plant Mini kit (Qiagen, Hilden, Germa- ny) following manufacturer’s instructions. The ITS re- gion of nuclear ribosomal DNA was amplified from isolated DNA using the fungus specific primer pair ITS1F and ITS4 (Gardes & Bruns, 1993; White et al., 1990), following the modified procedure described in Sulzbacher et al. (Sulzbacher et al. 2016) Lawrence, KS 66044-8897. Restingomyces reticulatus gen. et sp. nov. is a recently discovered false truffle species from Atlantic \”restinga\” rainforest in northeastern Brazil. UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 55FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Molecular and morphological characters separate this new sequestrate species from other described taxa in the order Phallales (Phallomycetidae, Basidiomycota. PCR products were run on 1.5 % agarose gels in 0.5 x TBE buffer. Amplified DNA fragments were cut out of agarose gels and purified with innuPREP DOUBLE- pure Kit (Analytik Jena AG, Jena, Germany) following manufacturer’s instructions. After the DNA fragments’ purification, sequencing was performed at a commer- cial sequencing laboratory (Macrogen Inc., Seoul, South Korea). All morphotypes were sequenced in both direc- tions with the primers ITS1F and ITS4 (Gardes & Bruns, 1993; White et al., 1990). The obtained se- quences were processed in Geneious version 11.1.4 (https://www.geneious.com, (Kearse et al. 2012). BLASTN algorithm from NCBI website (National Cen- ter for Biotechnology Information; https://blast.ncbi. nlm.nih.gov/Blast.cgi) and from UNITE website (https://unite.ut.ee/, (Nilsson et al. 2019)) was used to assess the similarity of obtained ITS sequences to se- quences in GenBank and UNITE public sequence data- bases. Sequences that remain unclassified at a kingdom or family level were discarded. Final criteria for a data- base match were as followed: query cover ≥ 80 % and sequence similarity > 92 % (representing approximate cut-off value at genus level) or sequence similarity ≥ 97 % - 100 % (representing approximate cut-off at spe- cies level) (Porras-Alfaro et al. 2014; Raja et al. 2017). 3 RESULTS With the 16 sampling dates pooled together and 243 root samples, this study revealed the 86 ECM symbi- onts of silver fir in Slovenia, belonging to 32 different genera. Sixty-eight ECM fungal taxa were successfully amplified, sequenced, and identified using genetic tools, meanwhile 18 ECM fungal taxa were identified solely based on morphological-anatomical descrip- tions. Among 86 identified ECM fungal taxa, 77 (89 %) were identified to species level and 9 (11 %) to genus. In order of abundance, the most abundant ECM fungal taxa were assigned to Russula, Tomentella and Lactari- us, with average abundance higher than 10 % per sam- ple. More specifically, the most common ECM taxa with relative abundance higher than 1 % per sample and were in symbiosis with silver fir identified before were Amanita rubescens, Amphinema byssoides, Clavu- lina corraloides, Lactarius salmonicolor, Lactarius sub- dulcis, Lactifluus vellereus, Neoboletus erythropus, Rus- sula chloroides, Russula cyanoxantha, Russula ochrole- uca, Russula nigricans, Sebacina epigeae, Thelephora wakefilediae, Tomentella stuposa, Tylospora fibrillosa and Xerocomellus pruinatus. Among 86 identified ECM fungal taxa, 48 ECM taxa were identified in sym- biosis with silver fir for the first time. The most abun- dant (with relative abundance higher than 1 % per sample) were Elaphomyces granulatus, Inocybe assimi- lata, Lactarius tabidus, Russula delica, Russula hete- rophylla, Russula illota, Russula turci, Sebacina incru- stans, Terfezia sp. and Tricholoma virgatum (Figure 1, Table 1). UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 56 FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Figure 1: Most common ECM taxa in symbiosis with silver fir. A: Amanita rubescens Pers., B: Amphinema byssoides (Pers.) J. Erikss., C: Clavulina corraloides (L.) J. Schröt., D: Elaphomyces granulatus Fr., E: Inocybe assimilata (Britzelm.) Sacc., F: Lactarius salmonicolor R. Heim. & Leclair, G: Lactarius subdulcis (Pers. Ex. Fr.) Gray, H: Lactarius tabidus (Fr.) Kuntze, I: Neoboletus erythropus (Pers.), J: Russula badia Beeli, K: Russula chloroides (Krombh.) Bres., L: Russula cyanoxantha (Schaeff.) Fr., M: Russula delica Fr., N: Russula heterophylla (Fr.) Fr., O: Russula illota Romagn., P: Russula ochroleuca Pers., R: Russula nigricans Fr., S: Russula turci Bres., T: Sebacina epigeae (Berk & Broome) Neuhoff, U: Sebacina incrustans (Pers.) Tul. & C. Tul., V: Terfezia sp. (Tul. & C.Tul) Tul. & C. Tul., Z: Thelephora wakefilediae Zmitr., X: Tomentella stuposa (Link) Stalpers, Y: Tricholoma virgatum (Fr.) P. Kumm., W: Tylospora fibrillosa (Burt) Donk, Q: Xerocomellus pruinatus (Fr. & Hök) Šutara. UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 57FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Slika 1: Najpogostejši ektomikorizni taksoni, ki so se pojavljali v simbiozi z navadno jelko. A: Amanita rubescens Pers., B: Amphinema byssoides (Pers.) J. Erikss., C: Clavulina corraloides (L.) J. Schröt., D: Elaphomyces granulatus Fr., E: Inocybe assimilata (Britzelm.) Sacc., F: Lactarius salmonicolor R. Heim. & Leclair, G: Lactarius subdulcis (Pers. Ex. Fr.) Gray, H: Lactarius tabidus (Fr.) Kuntze, I: Neoboletus erythropus (Pers.), J: Russula badia Beeli, K: Russula chloroides (Krombh.) Bres., L: Russula cyanoxantha (Schaeff.) Fr., M: Russula delica Fr., N: Russula heterophylla (Fr.) Fr., O: Russula illota Romagn., P: Russula ochroleuca Pers., R: Russula nigricans Fr., S: Russula turci Bres., T: Sebacina epigeae (Berk & Broome) Neuhoff, U: Sebacina incrustans (Pers.) Tul. & C. Tul., V: Terfezia sp. (Tul. & C.Tul) Tul. & C. Tul., Z: Thelephora wakefilediae Zmitr., X: Tomentella stuposa (Link) Stalpers, Y: Tricholoma virgatum (Fr.) P. Kumm., W: Tylospora fibrillosa (Burt) Donk, Q: Xerocomel- lus pruinatus (Fr. & Hök) Šutara. UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 58 FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Figure 2: Mean root tips number of individual most common taxa in symbiosis with silver fir. Slika 2: Povprečno število koreninskih vršičkov posameznega pogostejšega taksona, ki je tvoril simbiozo z navadno jelko. EC M ta xa Russula cyanoxantha Inocybe assimilata Russula heterophylla Tomentella stuposa Tricholoma virgatum Lactarius salmonicolor Sebacina epigaea Clavulina coralloides Amanita rubescens Russula badia Sebacina incrustans Russula ochroleuca Theleophora wakefilediae Russula nigricans Russula turci Xerocomellus pruinatus Elaphohomyces granulatus Russula ilota Tylospora fibriliosa Terfezia sp Russula delica Neoboletus erythropus Lactarius subdulcis Russula chloroides Lactarius tabidus 0 20 40 60 80 100 120 Mean root tips no. per sample UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 59FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Table 1: Most common ECM taxa in symbiosis with silver fir, with morphotype description, accession number and closest GenBank match. Preglednica 1: Najpogostejši ektomikorizni taksoni v simbiozi z navadno jelko z morfološkimi opisi, pristopno številko in najbljižjim GenBank ujemanjem. ECM taxon Morphotype description Accession no. (representative sequence) Closest Genbank match Identities Similarity E-value Amanita rubescens (Pers.) brown-reddish grainy mantle with hyphae MN265475 Amanita rubescens MF954678.1 613/615 99.67 % 0.0 Amphinema byssoides (Pers.) J. Erikss. white to yellow woolly mantle with rhizomorphs MN265483 Amphinema byssoides MH248042.1 481/481 100 % 0.0 Clavulina corra- loides (L.) J. Schrõt. light brown grainy mantle with hyphae MN265498 Uncultured Clavulina cristata KT020818.1 653/654 99.85 % 0.0 Elaphomyces granu- latus Fr. brown smooth shiny mantle MN265528 Elaphomyces granulatus KX238835.1 644/644 100 % 0.0 Inocybe assmilata (Britzelm.) Sacc. beige grainy mantle with substrate MN265542 Inocybe assimilata MN047063.1 488/488 100 % 0.0 Lactarius salmoni- color R. Heim & Leclair orange smooth mantle with substrate MN265578 Lactarius salmonicolor MK028450.1 646/647 99.85 % 0.0 Lactarius subdulcis (Pers. Ex. Fr.) Gray brown-orange smooth to grainy mantle with hyphae MN265567 Lactarius subdulcis MN959786.1 638/638 100 % 0.0 Lactarius tabidus (Fr.) Kuntze brown mantle, with bended unramified ends MN265596 Uncultured Lactifluus clone MK820103 Lactarius tabidus KR364106.1 680/680 670/674 100 % 99.41 % 0.0 0.0 Lactifluus vellereus (Fr.) Fr. brown grainy mantle MN265592 Lactarius vellereus MH125241.1 654/659 99.24 % 0.0 Neoboletus erythro- pus (Pers.) dark brown grainy mantle with bended, light brown to orange unramified ends MN265815 Neoboletus erythropus MK492598.1 458/459 99.78 % 0.0 Russula badia Beeli brown shiny grainy mantle MN265655 Russula badia MG679813.1 638/639 99.84 % 0.0 Russula chloroides (Krombh.) Bres. ocher with substrate MN265643 Russula chloroides KX034108.1 660/663 99.55 % 0.0 Russula cyanoxan- tha (Schaeff.) Fr. white covered with substrate MN265612 Uncultured Russula clone JF519199.1 621/622 99.84 % 0.0 Russula delica Fr. dark brown mantle covered with substrate MN265678 Russula sp. KM576518.1 Russula delica AF418605.1 601/604 600/604 99.5 % 99.34 % 0.0 0.0 Russula heterophylla (Fr.) Fr. ocher grainy mantle with bended unramified ends MN265625 Russula heterophylla DQ422006.1 556/557 99.82 % 0.0 Russula illota Romagn. brown orangish grainy mantle with substrate MN265669 Russula illota MG687367.1 653/655 99.69 % 0.0 Russula ochroeluca Pers. dark ocher warty mantle with yellow dots MN265640 Russula ochroleuca MN959793.1 532/534 99.63 % 0.0 Russula nigricans Fr. brown grainy mantle covered with substrate MN265606 Russula nigricans voucher MK028892.1 479/480 99.79 % 0.0 Russula turci Bres. light brown to white mantle MN265615 Russula turci KF002747.1 654/665 98.35 % 0.0 UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 60 FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Using molecular markers, with the 16 sampling dates pooled together from three study sites in Slovenia, we were able to identify 86 different ECM fungal taxa be- longing to 32 different ECM fungal genera. Among most abundant ECM fungal taxa in symbiosis with sil- ver fir in Slovenia were Tomentella stuposa, Lactarius salmonicolor, Amanita rubescens, Russula chloroides, Thelephora wakefilediae, Xerocomellus pruinatus etc., for which ectomycorrhizal symbiosis formation with silver fir was already reported by several authors (Laganà et al. 2000, 2002; Cremer 2009; Ważny 2014; Rudawska et al. 2016; Ważny & Kowalski 2017). Identified ECM fungal taxa are also among the more common members of ECM fungal communities of temperate and boreal forest in Europe and many of them exhibit broad host ranges (Kennedy et al. 2003; Nara 2006). These taxa have been previously reported from ectomycorrhizal root tips studies from different locations in Slovenia (Kraigher 1996; Pučko et al. 2005; Grebenc & Kraigher 2007). Observed result confirm high potential for diversity of ECM fungi in silver fir stands, as was already reported in our previ- ous study (Unuk & Grebenc 2017). To compare, 48 morphotypes were recorded in five silver fir forests in Italy (Comandini et al. 2004), 25 morphotypes were recorded in two forest stands in Central Italy (Pacioni et al. 2001) as well as in five different stands on the Taunus Mountains in Germany (Schirkonyer et al. 2013). In Polish Pomerania Rudawska et al. (2016)na- tive to the mountainous regions of Europe but has been also widely introduced in the lowlands outside its na- tive range. Like most forest tree species, A. alba forms obligate mutualisms with ectomycorrhizal (ECM, re- corded 35 ECM taxa in four forest stands, meanwhile 63 and 53 ECM taxa, were recorded on silver fir seed- lings from three or six Poland forest stands, respective- ly (Ważny 2014; Ważny & Kowalski 2017)p = 0.0001. However, as many as 48 fungal ECM taxa, were in our study in symbiosis with silver fir identified for the first time, for example Elaphomyces granulatus, Inocy- be assimilata, Lactarius tabidus, Russula delica, Russu- la heterophylla, Russula illota, Russula turci, Sebacina incrustans, Terfezia sp. and Tricholoma virgatum, etc. Their occurrence in symbiosis with numerous host trees from family Pinaceae have been previously re- ported (Buée et al. 2011; Ding et al. 2011; Gao et al. 2013; Kernaghan and Patriquin 2015; Argüelles- -Moyao et al. 2017), however till date there were no Sebacina epigeae (Berk & Broome) Neuhoff light orange to white wooly mantle MN265696 Sebacina epigeae JQ665513.1 557/565 98.58 % 0.0 Sebacina incrustans (pers.) Tul. & C. Tul. light brown to white smooth mantle MN265710 Uncultured Sebacina HE687124.1 Sebacina incrustans MN947392.1 578/580 562/581 99.66 % 97 % 0.0 0.0 Terfezia sp. (Tul. & C. Tul.) Tul. & C. Tul. brown mantle with emanating hyphae MN265716 Pezizaceae sp. KM576471.1 593/593 100 % 0.0 Thelephora wakefil- ediae Zmitr. dark brown wooly mantle MN265765 Tomentella sublilacina KY693713.1 539/539 100 % 0.0 Tomentella stuposa (Link) Stalpers dark brown to black with emanating hyphae MN265719 Tomentella stuposa MK602778.1 628/630 99.68 % 0.0 Tricholoma virga- tum (Fr.) P. Kumm. brown wooly mantle, shiny with emanating hyphae and rhizomorphs MN265777 Uncultured ectomycor- rhizal fungus AB828043.1 Tircholoma virgatum DQ389735.1 649/651 645/657 99.69 % 98.17 % 0.0 0.0 Tylospora fibrillosa (Burt) Donk light brown with emanat- ing hyphae and cistidia MN265788 Uncultured Tylospora clone MK820144.1 Tylospora fibrillosa KP783485.1 565/565 563/565 100 % 99.65 % 0.0 0.0 Xerocomellus pruinatus (Fr. & Hök) Šutara ocher grainy mantle, shiny with bended un- ramified ends MN265793 Xerocomus pruinatus MN959798.1 715/717 99.72 % 0.0 4 DISCUSSION UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 61FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 reports about their ectomycorrhizal symbiosis forma- tion with silver fir. In this study we have identified numerous ECM fungal species associated with silver fir fine roots, where many of them were identified in symbiosis with silver fir for the first time. With this study the range of fungal species that can form ectomycorrhizal symbio- sis with silver fir expanded. A lot of species were iden- tified for the first time, which indicates poor current knowledge of silver fir ectomycorrhizal symbionts and indicates on high potential for diversity of ECM fungi in silver fir stands. POVZETEK Ektomikorizni simbionti navadne jelke so v Sloveniji bili le redko analizirani in identificirani. Namen študi- je je bil tako analizirati in identificirati ektomikorizne simbionte navadne jelke na treh z navadno jelko pre- vladujočih območjih v Sloveniji, in sicer na Jelovškem borštu, Ljubelju in Lehnu na Pohorju. Na vsaki izmed lokacij smo postavili eno ploskev, velikosti 20 m x 20 m. Znotraj posamezne ploskve smo izbrali po 5 zdravih in odraslih dreves navadne jelke, katere smo v razmaku enega meseca v času od marca do oktobra v letih 2016 in 2017 vzorčili po protokolu opisanem v Kraigher (1996). Drobne korenine smo analizirali in med seboj ločili na podlagi morfološko-anatomskih lastnostih opisanih v AGERER (1987-), do končnega števila 250 analiziranih koreninskih vršičkov na vzo- rec. Tri do pet vršičkov posameznega morfotipa smo vključili v nadaljnje analize določanja z uporabo ITS DNK barkod. Na skupno treh območjih in 16 združe- nih mesecih vzorčenja, smo tako določili kar 86 različ- nih ektomikoriznih simbiontov navadne jelke v Slove- niji, pripadajočih 32 različnim rodov. Od tega smo kar 68 ektomikoriznih taksonov navadne jelke uspešno pomnožili, sekvencirali in identificirali z uporabo ITS DNK barkodiranja, medtem ko smo 18 ektomikroi- znih taksonov identificirali samo na osnovi morfolo- ško-anatomskih značilnosti. Najpogostejši ektomiko- rizni taksoni navadne jelke prihajajo iz rodov Russula, Tomentella in Lactarius, pri čemer smo kar 48 takso- nov v simbiozi z navadno jelko v naši študiji identifici- rali prvič, na primer Elaphomyces granulatus, Inocybe assimilata, Lactarius tabidus, Russula delica, Russula heterophylla, Russula illota, Russula turci, Sebacina incrustans, Terfezia sp. and Tricholoma virgatum, itd. V študiji smo identificirali veliko število različnih ektomikoriznih simbiontov navadne jelke, pri čemer smo jih kar nekaj v ektomikorizni simbiozi z navadno jelko identificirali prvič, kar kaže slabo poznavanje ek- tomikoriznih simbiontov navadne jelke in kaže na velik potencial pestrosti ektomikoriznih gliv v sestojih navadne jelke. 5 REFERENCES - LITERATURA Agerer, R., Kraigher, H. & Javornik, B., 1996: Identification of ectomycorrhizae of Hydnum rufescens on Nor- way spruce and the variability of the ITS region of H. rufescens and H. repandum (Basidiomycetes). Nova Hedwigia, 63: 183-194 Agerer, R. : 1987: Colour Atlas of Ectomycorrhizae, 1st-12th edn. Einhorn-Verlag, Schwäbisch Gmünd, Germany Agerer, R. & Rambold, G., 2004–2018: DEEMY – An Information System for Characterization and Determina- tion of Ectomycorrhizae. In: München, Ger. Argüelles-Moyao, A., Garibay-Orijel, R., MÁrquez-Valdelamar, L.M. & Arellano-Torres, E., 2017: Cla- vulina-Membranomyces is the most important lineage within the highly diverse ectomycorrhizal fungal com- munity of Abies religiosa. Mycorrhiza 27:53–65. https://doi.org/10.1007/s00572-016-0724-1 Berndt, R., Kottke, I. & Oberwinkler, F., 1990: Ascomycete mycorrhizas from pot-grown silver-fir seedlings (Abies alba Mill.). New Phytol 115:471–482. https://doi.org/10.1111/j.1469-8137.1990.tb00473.x Bončina, A., Diaci, J. & Cenčič, L., 2002: Comparison of the two main types of selection forests in Slovenia: Distribution, site conditions, stand structure, regeneration and management. Forestry. https://doi.org/10.1093/ forestry/75.4.365 Buée, M., Maurice, J.P., Zeller, B., et al., 2011: Influence of tree species on richness and diversity of epigeous fungal communities in a French temperate forest stand. Fungal Ecol. https://doi.org/10.1016/j.fune- co.2010.07.003 UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 62 FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Comandini, O., Haug, I., Rinaldi, A.C. & Kuyper, T.W., 2004: Uniting Tricholoma sulphureum and T. bufonium. Mycol Res 108:1162–1171. https://doi.org/10.1017/S095375620400084X Comandini, O., Pacioni, G. & Rinaldi, A.C., 1998: Fungi in ectomycorrhizal associations of silver fir (Abies alba Miller) in Central Italy. Mycorrhiza 7:323–328. https://doi.org/10.1007/s005720050200 Cremer, E., 2009: Population genetics of silver fir (Abies alba Mill.) in the Northern Black Forest – preconditions for the recolonization of windthrow areas and associated ectomycorrhizal communities. Phillips-Universität Marbg 1:103. https://doi.org/10.1017/CBO9781107415324.004 De Groot, M., Eler, K. & Flajšman, K., et al., 2016: Differential short-term response of functional groups to a change in forest management in a temperate forest. For Ecol Manage 376:256–264. https://doi.org/10.1016/j. foreco.2016.06.025 De Román, M. & De Miguel, A.M., 2005: Post-fire, seasonal and annual dynamics of the ectomycorrhizal com- munity in a Quercus ilex L. forest over a 3-year period. Mycorrhiza 15:471–482. https://doi.org/10.1007/s00572- 005-0353-6 Ding, Q., Liang, Y., Legendre, P., et al., 2011: Diversity and composition of ectomycorrhizal community on seed- ling roots: The role of host preference and soil origin. Mycorrhiza. https://doi.org/10.1007/s00572-011-0374-2 Eberhardt, U., Oberwinkler, F., Verbeken, A., et al., 2000: Lactarius ectomycorrhizae on Abies alba: Morpho- logical description, molecular characterization, and taxonomic remarks. Mycologia 92:860–873. https://doi. org/10.2307/3761582 Gao, C., Shi, N.N., Liu, Y.X., et al., 2013: Host plant genus-level diversity is the best predictor of ectomycorrhizal fungal diversity in a Chinese subtropical forest. Mol Ecol. https://doi.org/10.1111/mec.12297 Gardes, M. & Bruns, T.D., 1993: ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118. https://doi.org/10.1111/j.1365-294X.1993. tb00005.x Grebenc, T. & Kraigher, H., 2007: Changes in the community of ectomycorrhizal fungi and increased fine root number under adult beech trees chronically fumigated with double ambient ozone concentration. Plant Biol 9:279–287. https://doi.org/10.1055/s-2006-924489 Kearse, M., Moir, R., Wilson, A., et al., 2012: Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. https://doi. org/10.1093/bioinformatics/bts199 Kennedy, P.G., Izzo, A.D. & Bruns, T.D., 2003: There is high potential for the formation of common mycorrhizal networks between understorey and canopy trees in a mixed evergreen forest. J Ecol 91:. https://doi.org/10.1046/ j.1365-2745.2003.00829.x Kernaghan, G. & Patriquin, G., 2015: Diversity and host preference of fungi co-inhabiting Cenococcum mycor- rhizae. Fungal Ecol. https://doi.org/10.1016/j.funeco.2015.05.001 KlopČIČ, M. & BonČIna, A., 2011: Stand dynamics of silver fir (Abies alba Mill.)-European beech (Fagus sylvati- ca L.) forests during the past century: A decline of silver fir? Forestry 84:259–271. https://doi.org/10.1093/for- estry/cpr011 Kraigher, H., Javornik, B. & Agerer, R., 1995: Ectomycorrhizae of Lactarius lignyotus on Norway spruce, char- acterized by anatomical and molecular tools. Mycorrhiza, 3: 175-180. Kraigher, H., 1996: Tipi ektomikorize - taksonomija, pomen in aplikacihe - Types of ectomycorrhizae - their taxonomy, role and applocation. Acta Silvae et Ligni 49:33–66 Kraigher, H., Al Sayegh Petkovšek, S., Grebenc, T. & Simončič, P., 2007: Types of ectomycorrhiza as pollu- tion stress indicators: Case studies in Slovenia. Environ Monit Assess 128:31–45. https://doi.org/10.1007/ s10661-006-9413-4 Laganà, A., Angiolini, C., Loppi, S., et al., 2002: Periodicity, fluctuations and successions of macrofungi in fir forests (Abies alba Miller) in Tuscany, Italy. For Ecol Manage 169:187–202. https://doi.org/10.1016/S0378- 1127(01)00672-7 Laganà, A., Salerni, E., Barluzzi, C., et al., 2000: Mycocoenology in Abies alba Miller woods of Central-South- ern tuscany (Italy). Acta Soc. Bot. Pol. 69:293–298 Mrak, T., Hukić, E., Štraus, I., et al., 2020: Ectomycorrhizal community composition of organic and mineral soil horizons in silver fir (Abies alba Mill.) stands. Mycorrhiza 30:. https://doi.org/10.1007/s00572-020-00970-y Nara, K., 2006: Ectomycorrhizal networks and seedling establishment during early primary succession. New Phytol 169:. https://doi.org/10.1111/j.1469-8137.2005.01545.x UNUK NAHBERGER, KRAIGHER & GREBENC : ECTOMYCORRHIZAL SYMBIONTS OF SILVER FIR (ABIES ALBA MILL.) 63FOLIA BIOLOGICA ET GEOLOGICA 65/1 – 2024 Nilsson, R.H., Larsson, K.H., Taylor, A.F.S., et al., 2019: The UNITE database for molecular identification of fungi: Handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res 47:D259–D264. https:// doi.org/10.1093/nar/gky1022 Pacioni, G., Comandini, O. & Rinaldi, A.C., 2001: An assessment of below-ground ectomycorrhizal diversity of Abies alba Miller in central Italy. Plant Biosyst - An Int J Deal with all Asp Plant Biol 135:337–350. https://doi. org/10.1080/11263500112331350960 Porras-Alfaro, A., Liu, K.L., Kuske, C.R. & Xiec, G., 2014: From genus to phylum: Large-subunit and internal transcribed spacer rRNA operon regions show similar classification accuracies influenced by database com- position. Appl Environ Microbiol 80:829–840. https://doi.org/10.1128/AEM.02894-13 Pučko, M., Grebenc, T., Božič, G., et al., 2005: Identification of types of ectomycorrhizae on seedlings in a beech provenance trial. Zb gozdarstva Lesar 75:87–104 Raja, H.A., Miller, A.N., Pearce, C.J. & Oberlies, N.H., 2017: Fungal identification using molecular tools: a primer for the natural products research community. J Nat Prod 80:756–770. https://doi.org/10.1021/acs. jnatprod.6b01085 Rudawska, M., Pietras, M., Smutek, I., et al., 2016: Ectomycorrhizal fungal assemblages of Abies alba Mill. out- side its native range in Poland. Mycorrhiza 26:57–65. https://doi.org/10.1007/s00572-015-0646-3 Schirkonyer, U., Bauer, C. & Rothe, G.M., 2013: Ectomycorrhizal diversity at five different tree species in for- ests of the Taunus Mountains in Central Germany. Open J Ecol 03:66–81. https://doi.org/10.4236/oje.2013.31009 Sulzbacher, M.A., Grebenc, T., Cabral, T.S., et al., 2016: Restingomyces , a new sequestrate genus from the Brazilian Atlantic rainforest that is phylogenetically related to early-diverging taxa in Trappeaceae (Phallales). Mycologia 108:954–966. https://doi.org/10.3852/15-265 Unuk, T. & Grebenc, T., 2017: Silver fir (Abies alba Mill.) ectomycorrhiza across its areal – a review approach / Ektomikorizni simbionti bele jelke (Abies alba Mill.) na naravnem območju razširjenosti – pregled. Folia Biol Geol 58:. https://doi.org/10.3986/fbg0025 Unuk, T., Martinović, T., Finžgar, D., et al., 2019: Root-associated fungal communities from two phenologi- cally contrasting silver fir (Abies alba Mill.) groups of trees. Front Plant Sci 10:1–11. https://doi.org/10.3389/ fpls.2019.00214 Ważny, R., 2014: Ectomycorrhizal communities associated with silver fir seedlings (Abies alba Mill.) differ large- ly in mature silver fir stands and in Scots pine forecrops. Ann For Sci 71:801–810. https://doi.org/10.1007/ s13595-014-0378-0 Ważny, R. & Kowalski, S., 2017: Ectomycorrhizal fungal communities of silver-fir seedlings regenerating in fir stands and larch forecrops. Trees - Struct Funct 31:929–939. https://doi.org/10.1007/s00468-016-1518-y White, T.J., Bruns, T., Lee, S., Taylor, J., 1990: Amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. In: PCR Protocols. Elsevier, pp 315–322 Wolf, H., 2003: EUFORGEN Technical Guidelines for genetic conservation and use for silver fir (Abies alba). Int Plant Genet Resour Institute, Rome, Italy. https://doi.org/10.1016/j.jaci.2010.08.025 Železnik, P., Hrenko, M., Then, C., et al., 2007: CASIROZ: Root parameters and types of ectomycorrhiza of young beech plants exposed to different ozone and light regimes. Plant Biol 9:298–308. https://doi. org/10.1055/s-2006-955916 Železnik, P., Vilhar, U., Starr, M., et al., 2016: Fine root dynamics in Slovenian beech forests in relation to soil temperature and water availability. Trees - Struct Funct 30:375–384. https://doi.org/10.1007/s00468-015-1218-z