1. Mycorrhiza, cytokinins and abscisic acid in the response of beech to 2xO 3 Tine Grebenc, Joanna Winwood, Adrienne Pate, Andrew O’Brien, David Hanke & Hojka Kraigher Institute of Forestry, Slovenija, and University of Cambridge, UK

2. Slovenian Forestry Institute ECTOMYCORRHIZAE affect nutrient cycling & biodiversity in forest ecosystems Rainfall, throughfall... Gaseous losses & gains Litter Leaves, buds, flowers, fruits, shoots, branches Inputs Retranslocation in time & space CWD Immobilization MineralisationWeathering of minerals Uptake & retranslocation of water & nutr. From: KRAIGHER 2003, as modif. after DIGHTON & BODDY 1998 Common mycelial networks

3. Slovenian Forestry Institute The mycelium of mycorrhizal fungi:  affects the field performance of forest trees through: capture & uptake of nutrients protection against pathogens & toxic elements extending feeder root longevity spatial & temporal linkages between sinks & sources of nutrients.  depends on: functional compatibility of species & strain of the fungus & the plant therefore identification of the fungal partner is important. Photos by H.Kraigher Mycorrhiza is considered a microbial key species in soils, which are defined as organisms that possess important functions in the soil ecosystem or are of human health concern (Nielsen & Winding: Microorganisms as indicators of soil health, 2002)

4. Slovenian Forestry Institute Standardized soil core for sampling (270 ml,  3,5 cm, 0-18 cm deep, 2 - 5 per sampling date, 5 - 30 per plot), cleaning, sorting of morphotypes & documenting. Counting (root tips above 1 mm length) and image analyses of vital types, old un-identifiable types and non-mycorrhizal roots. Anatomical and molecular identification or characterisation of the sorted morphotypes, comparisons to the reference material, PCR-ITS-RFLP library or GenBank. Presentation (lists, tables, graphs), biodiversity indices (Atlas, Bartha 1981), statistics (CANOCO, …). Search for physiological / ecological roles of the identified sp. Modelling with environmental parameters. Identification, quantification, qualification of ECM: Photos by H.Kraigher Right: CANOCO by T.Grebenc

5. Slovenian Forestry Institute Identification of types of ectomycorrhizae: Anatomical characteristics (AGERER 1987-2004), Molecular methods (PCR-ITS-RFLP & sequencing) (GARDES & BRUNS 1993, as descr. in KRAIGHER & al. 1995; sequencing.: as descr. in MARTĺN 2000) ©T.Bruns http:/ Photos by H.Kraigher Right: Extract from the PCR-ITS-RFLP database (containing ca 900 isolates of fungi & types of ECM) of the Slovenian Forestry Institute (Grebenc 2005)

6. Slovenian Forestry Institute Response of frequently occurring species to 2xO 3 fumigation The chronic ozone effect seems to change the succession stage of ECM in the stand causing a shift of some ectomycorrhizal species either to become more dominant (C. geophilum, R. fellea) or to decrease in abundance and finally disappear from the treated plots (R. cyanoxantha, Xerocomus chrysenteron & R. ochroleuca). Results & discussion Species with > 5% of all ECM root tips Response to two-fold ozone fumigation of tree canopies Cenococcum geophilum↑ Russula cyanoxantha↓ Xerocomus chrysenteron↓ Russula fellea↑ Russula illota↑ Russula ochroleuca↓ Russula sp. 2↑

7. Slovenian Forestry Institute Cenococcum geophilum was the most common and abundant type of ectomycorrhize with highly significant response to ozone treatment. Results & discussion C. geophilum, magnif. 45x Photo:P. Zeleznik C. geophilum, outer mantle layer photo T.Grebenc

8. Slovenian Forestry Institute Shannon-Weaver index showed a non-significantly higher diversity in 2xO 3 compared to 1xO 3 Species richness showed a trend of increase in 2xO3 compared to 1xO3 Total number of vital ectomycorrhizal and old&non- mycorrhizal root tips were significantly higher (indicating a higher fine root turnover) for trees under 2xO 3 Results & discussion Parameters describing ectomycorrhizae and short root dynamics Response to the fumigation of beech crowns with two- fold ozone Shannon-Weaver index↑ (n.s.) Species richness↑ Sum of vital ectomycorrhizal root tips↑ Old and non-mycorrhizal root tips↑

9. Slovenian Forestry Institute Chronic exposure of trees to increased ozone concentration caused changes in below-ground processes. The community structure of ectomycorrhizal fungi was changed, i.e. some species became more frequent, although only trends in species richness indices were observed. The increase in fine root turnover in increased ozone exposure was significant. Conclusions

11. Root levels

12. Xylem Levels

13. Isoprenoid Levels: 2003

14. Aromatic Levels: 2004

15. Z O-Glucoside Levels: 2004

16. Increased cytokinin content of roots and leaf xylem in 2xO 3 accords with prediction from root growth and mycorrhiza Decreased leaf content does not, and suggests a higher rate of destruction of cytokinin in leaves in 2xO 3

17. Changing ABA content of leaves: 2003 and 2004 Mean ABA content of beech leaves (nmol g-1 FW)

18. Changes in phloem ABA concentration ABA concentration (nmol mL -1 ) 2004 2003

19. ABA in sun and shade leaves plotted with AOT40 and COU in 2004 Mean ABA content of leaves(nmol/g FW)

20. Diurnal changes in leaf ABA content in July 2004 Mean ABA content of leaves(nmol/g FW) Ratio 2xO3/1xO3

21. Because the effect of 2xO 3 on leaf ABA content is positive, at the site of synthesis, and ABA is a stress signal inducing protective responses, O 3 -induced increases in leaf ABA are more likely to be part of the response to damage. By contrast, O 3 - induced decreases in leaf cytokinin content are more likely to be part of the damage and not the response to it

22. Acknowledgements: Thanks to Amanda Price and Maydelin Dorado Bermudez for help with hormone analyses

23. Correlations with other measured parameters Negative correlation with stomatal conductance in sun leaves in 2003 and 2004. Stronger correlation in control leaves than in 2 x O 3 leaves. High levels of ozone my reduce the capacity of ABA to close stomata.

25. Isoprenoid Levels: 2004

26. Aromatic Levels: 2003

27. Z O-Glucoside Levels: 2003