1. EUFORINNO (RegPot No. 315982) MULTIPLE SYMBIOSES ON ALDER (ALNUS SPP.) Tine Grebenc, Melita Hrenko, Hojka Kraigher

2. EUFORINNO (RegPot No. 315982) ALDER Alder – Alnus spp. Order: Fagales Family: Betulaceae Genus: Alnus Key identification features for the genus: Mainly deciduous shrubs or wooded trees Flowers are catkins (male and female are on the same plant) Female catkins are woody (vs. Betula) Mainly wind pollinated

3. EUFORINNO (RegPot No. 315982) ALDER Global diversity About 35 species worldwide (several known crossings) Distributed throughout the north temperate zone with a few species extending into Central America and the northern Andes with the origin in East Asia 4 species in Europe (A. glutinosa, A. incana, A. viridis & A. cordata) 3 known in Slovenia and SE Europe (A. glutinosa, A. incana, A. viridis)

4. EUFORINNO (RegPot No. 315982) ALDER Alders in Europe in past Douda et al. 2014

5. EUFORINNO (RegPot No. 315982) ALDER The three species in SE Europe and Slovenia Alnus glutinosa Alnus incana Alnus viridis Figures sources: www.euforgen.org www.rogerstreesandshrubs. com www.rogerstreesandshrubs. com www.landforms.eu www.pfaf.org dryades.units.it www.forestryimages.org www.wikipedia.org

6. EUFORINNO (RegPot No. 315982) ALDER The global diversity of alders – three subgenera Subgenus Alnus: Alnus acuminata Kunth Alnus cordata (Loisel.) Duby Alnus cremastogyne Burkill Alnus firma Siebold & Zucc. Alnus glutinosa (L.) Gaertn. Alnus incana (L.) Moench Alnus hirsuta (Spach) Rupr. Alnus oblongifolia Torr. Alnus rugosa (Du Roi) Spreng. Alnus tenuifolia Nutt Alnus japonica (Thunb.) Steud. Alnus jorullensis Kunth Alnus mandshurica (Callier) Hand.-Mazz Alnus nepalensis D.Don Alnus orientalis Decne. Alnus pendula Matsum. Alnus rhombifolia Nutt. Alnus rubra Bong. Alnus serrulata (Aiton) Willd. Alnus sieboldiana Matsum. Alnus subcordata C.A.Mey. Alnus trabeculosa Hand.-Mazz. Subgenus Clethropsis: Alnus formosana (Burkill) Makino Alnus maritima (Marshall) Muhl. ex Nutt. Alnus nitida (Spach) Endl. Subgenus Alnobetula: Alnus alnobetula (Ehrh.) K.Koch (syn. Alnus viridis A. Gray)

7. EUFORINNO (RegPot No. 315982) ALDER The global diversity of Alnus ITS based phylogeny of Alnus spp. showing: a.) unadjusted phylogenetic tree b.) ultrametric tree (Põlme et al. 2013)

8. EUFORINNO (RegPot No. 315982) ALDER AND SYMBIOSES Types of symbioses of Alders: 1.Endomycorrhiza 2.Ectomycorrhiza 3.Actinorhiza (N – fixing actinomycetes) 4.Other rhizosphere interactions (other fungi and bacteria not forming specialised structures)

9. EUFORINNO (RegPot No. 315982) 1. SYMBIOSES – ENDOMYCORRHIZA Arbuscular mycorrhiza (AM) of Alnus: See presentation: Dr. Michail Orfanoudakis: Alnus glutinosa below ground diversity and root dynamics Alnus glutinosa AMF symbiosis in the field (Greece) Interactions of AMF and Frankia – how they influence the root growth Remember important biotechnology/environmental applications

10. EUFORINNO (RegPot No. 315982) 1. SYMBIOSES - ENDOMYCORRHIZA Endomycorrhiza - arbuscular mycorrhiza (AM) fungi: Photos: 1-2: Grebenc et al. (unpub.) 3-6: Peterson et al. 2006 Schüßler et al. 2001

11. EUFORINNO (RegPot No. 315982) 2. SYMBIOSES - ECTOMYCORRHIZA Ectomycorrhiza in Alnus spp. is mainly formed by basidiomycetes and ascomycetes „Ectomycorrhizal fungi associated with Alnus are not a rich community„ (Molina et al. 1992) Tomentella sublilacina X Alnus acuminata (Pritsch et al. 2010) Ectomycorrhizal communities in Alnus stands is very distinctive (Kennedy 2011) Gyrodon lividus (www.funghiitaliani.it)

12. EUFORINNO (RegPot No. 315982) 3. SYMBIOSES - ACTINORRHIZA Phylogeny of Frankia and relation to plant partners Clawson et al., 2003

13. EUFORINNO (RegPot No. 315982) 2. SYMBIOSES - ACTINORRHIZA Ecology of Frankia: Free living in soil In root nodules of symbiotic plants In laboratory conditions www.scinetificamerican.com http://www.morning-earth.org/

14. EUFORINNO (RegPot No. 315982) 4. SYMBIOSES – OTHER RHIZOSPHERE INTERACTIONS Not much known – a few examples Plant-rhizobacteria interaction in Alnus (Probanza et al. 1996) Several Bacillus sp. (B. pumilus & B. licheniformis) promoted growth Pseudomonas fluorescens had a significant negative influence on Alnus growth Rhizosphere community structure was seriously altered by adding rhizosphere promoting bacteria (B. pumilus) (Ramos et al. 2003) Smith and Read 2008

15. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 1. GLOBAL ANALYSIS Purpose: 1.To analyse the global diversity of Alnus-associated ectomycorrhizal fungi 2.To disentangle the relative effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhiza Methodology: See Polme et al. 2013

16. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 1. GLOBAL ANALYSIS Global diversity of Alnus-associated ectomycorrhizal fungi Outcomes: Cluster dendrogram of Alnus-associated ectomycorrhizal (ECM) fungal communities by biogeographic regions. rDNA sequence analysis revealed 146 taxa of ECM fungi from 22 Alnus species across 96 sites worldwide Molina: „Ectomycorrhizal fungi associated with Alnus are not a rich community„ Panamanian Land Bridge North Atlantic Land Bridge (Põlme et al. 2013)

17. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 1. GLOBAL ANALYSIS Effects of biological, geographical and edaphic variables on the distribution of Alnus-associated ectomycorrhiza Outcomes: 1.Relations among host plants and geographical links explained 43% and 10% of ECM fungal community composition 2.Soil calcium concentration positively influenced taxonomic richness (Põlme et al. 2013)

18. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 2. ALNUS FRG IN SLOVENIA Purpose: 1.To translate EUFORGEN technical guidelines upgrade/ adapt them for Slovenia

19. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 3. DIVERSITY OF FRANKIA Purpose: To analyze the diversity of Frankia strains from A. glutinosa and A. incana Methodology: 16S V7 hyper-variable region and nifDK gene were amplified Outcomes: No differences among Frankia DNA extracted from Alnus nodules Grebenc et al. 1999 Kraigher et al. 2010

20. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 4. SEEDLINGS INOCULATIONS Purpose: To establish in vitro inoculation procedures Methodology: Cultivated Frankia or homogenised nodules was introduced to germinating seedlings Outcomes: Inoculations from culture – failed Inoculation with homogenised nodules: Aglu Aglu - OK Aglu Ainc - OK Aglu->Avir - failed Ainc->Avir - failed (Grebenc 2000)

21. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 5. DIVERSITY OF ECTOMYCORRHIZA Purpose: To analyze the diversity of ectomycorrhizal fungi from A. viridis and A. glutinosa Methodology: Anatomorphology of ectomycorrhizal root tips (regional study) Outcomes: Alnus glutinosa 5 morphotypes Tomentella sp. Lactarius sp. 3x unidentified Alnus viridis 13 morphotypes L. alpinus. C. geophilum Cortinarius sp. 10x unidentified (Hrenko et al 2010)

22. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 6. MYCOREMEDIATION OF STERILE GROUND Purpose: To prove the suitability of A. glutinosa for revitalisation of sterile grounds Methodology: Non-inoculated seedligns were planted into an artificial revitalisation substrate as a „revitalisation package“. Applied for revitalisation of: 1.Depositions of ashes (TPP) 2.Road banks 3.Querries 4.Landslides (fitostabilisation) (Piškur B. et al. 2011)

23. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 5. MYCOREMEDIATION OF STERILE GROUND Purpose: To prove the suitability of A. glutinosa for revitalisation of sterile grounds. Outcomes: (Piškur B. et al. 2011) Alnus glutinosa genotype adapted to dry and polluted sites! ->High potential for further studies and applications

24. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 6. MYCOREMEDIATION OF MANURE Purpose: To test A. glutinosa as a potential plant for recycling of manure Methodology: Seedlings were planted in pots and regularly fertilised Number of Frankia root nodules was counted Outcomes: Kalan et al. 2004 – research report

25. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 7. HYPOGEOUS FUNGI Purpose: To analyse the diversity of hypogeous fungi from Alnus viridis stands Methodology: Dog-associated or manual digging for sporocarps of hypogeous fungi under on in the vicinity of Alnus viridis stands Outcomes: Grebenc et al. 2014 – The regional collection of hypogeous fungi at SFI 

26. EUFORINNO (RegPot No. 315982) SFI EXPERIENCES 7. HYPOGEOUS FUNGI Purpose: To analyse the diversity of hypogeous fungi from Alnus viridis stands Methodology: Dog-associated or manual digging for sporocarps of hypogeous fungi under on in the vicinity of Alnus viridis stands Outcomes: Grebenc et al. 2014 – Regional collection of hypogeous fungi at SFI Alpova diplophloeus Tuber dryophilum Hydnotria cerebrifor mis Tuber rufum Tuber excavatum TE1 TE2 TE3 P A B