Fungal diversity in a transgenic poplar plantation and the role of ectomycorrhizal fungi for tree performance under field and controlled drought stress conditions

The worldw ide increasing energy demand comes along with diminishing fossil fuel resources. Thus, research on alternative bioenergy sources is urgently needed. Poplars with optimized properties for bioethanol production are available and have to be tested for sustainable usage in field experiments. It is an important issue to study possible environmental impacts of transgenic poplars on the biodiversity of associated organisms. Fungi play an important role in ecosystem functioning and information on their composition in the soil and on poplar roots of biomass plantations is rare. Poplars gain nutritional benefits from ectomycorrhizal (ECM) symbiosis and there is emerging evidence that ECM fungi could lead to enhanced water stress resistance in their host plants. The role of ECM symbiosis for poplar productivity and stress resistance is an important topic of research, especially in biomass plantations. In this w ork, fungal biodiversity in soil and roots of a poplar plantation w ere analyzed. In addition the role of ECM fungal diversity for poplar productivity and the potential role of ECM in amelioration of drought resistance in poplar w ere investigated. The follow ing research goals w ere pursued: (I) The fungal communities in a short rotation plantation with P. × canescens wildtype (WT) and tw o transgenic lines w ith suppressed cinnamyl alcohol dehydrogenase (CAD) activity were investigated to elucidate (1) if the fungal composition in the soil served as a large species-rich reservoir for the establishment of the fungal composition in roots of WT and the tw o transgenic lines and (2) if the fungal community in soil and roots was affected by the modification of the tw o transgenic lines in comparison to the WT. To investigate the soil/root fungal communities of WT and two transgenic CAD poplar lines, the pyrosequencing approach w as used and to detect temporal dynamics of ECM communities on roots pyrosequencing w as combined w ith the morphotyping/ Sanger-sequencing technique. Estimated species richness was highest in soil and decreased in the habitat order soil > root > root associated ECM. It w as also shown that the soil serves as a fungal-rich reservoir for fungal species colonizing the roots. Analysis of the life style of the fungi in soil revealed dominance of saprophytic fungi follow ed by ECM, pathogenic and endophytic fungi, while in roots ECM fungi w ere the dominant group. Temporal dynamics of ECM fungi colonizing the poplar roots show ed an increase in species richness after one year. Most species detected by morphotyping/ Sanger-sequencing in 2009 and 2010 w ere already detected by pyrosequencing in roots in 2009. The alteration of the CAD gene in poplars had no effect on the fungal community, neither in soil nor in roots. Summary VI (II) The biodiversity of ECM fungi in two short rotation plantations, one w ith commercial P. deltoides × P. nigra WT clones and the other with P. × canescens WT and seven transgenic lines w ith suppressed activities of CAD, caffeate/5-hydroxyferulate O-methyltransferase (COMT) or cinnamoyl-CoA reductase (CCR), w ere investigated to elucidate (1) if the ECM communities on the roots of poplars were affected by the gene modification of the transgenic lines compared to the WT and (2) if stem biomass and nutrient status in WT and transgenic lines w ere correlated w ith ECM colonization and community composition. To investigate the ECM community on the roots of poplar and to investigate if ECM fungi are linked w ith stem biomass production and nutrition, roots of three clones of P. deltoides × P. nigra (WT) in 2010 and roots of P. × canescens (seven transgenic lines and the WT) in 2009 and 2010 w ere analyzed by morphotyping/ Sanger-sequencing approach. Stem material of P. × canescens lines was used for analyzing the nutritional status of the poplars. Non metric multidimensional scaling (NMDS) revealed a similar fungal community structure of the different genotypes in 2009, while in 2010 a clustering of fungal communities w as detected. However, the variation w as in the range of fungal community structures obtained in the commercial poplar field. Comparison of the fungal community structure of the WT from 2009 and 2010 revealed a highly dynamic succession. Fungal community structures of the transgenic lines w ere not affected by gene modifications of poplars. Furthermore, these results demonstrate that multiple poplar genotypes increase the ECM community composition in poplar plantations. Differences in growth and nutrient element concentrations in w ood of transgenic poplars were found. A general mixed model revealed a link between the main factors for stem biomass prediction, ECM colonization and inverse w ood N concentration. (III) Drought stress responses of mycorrhizal and non-mycorrhizal P. × canescens plants were investigated in a controlled drought stress experiment to elucidate (1) if the ECM fungus Paxillus involutus improved the physiological responses of P. × canescens under water stress conditions and (2) if P. involutus enhanced the nutrition status of its host under drought stress conditions and (3) if the enhanced nutrition status w as related to the extent of mycorrhization. To elucidate the drought stress response of mycorrhizal and non-mycorrhizal drought stressed P. × canescens plants, the w ater supply w as slow ly decreased. The results show ed that the gravimetric soil w ater content under mild and medium w ater stress w as higher in mycorrhizal than non-mycorrhizal control and drought stressed plants. This effect w as also reflected in a slower decreasing relative w ater content of leaves in mycorrhizal compared to Summary VII non-mycorrhizal drought stressed plants. The efficiency of photosystem II (PSII) w as enhanced in mycorrhizal control and drought stressed plants and in case of drought treated plants the efficiency decreased only after severe water limitation. In contrast, the stomatal conductance w as mainly affected by drought even under mild drought stress, w hile the effect of mycorrhiza was only apparent in combination w ith drought and time. Most of the stress related genes investigated w ere up- or down-regulated in non-mycorrhizal and mycorrhizal drought stressed plants compared to non-mycorrhizal control plants. The nutrient status in leaves of mycorrhizal plants w as enhanced compared to non-mycorrhizal plants. ANCOVA results of leaves revealed a positive effect of mycorrhizal colonization on nutrient status in drought stressed plants. In conclusion, the present study show ed that soil serves as reservoir for ECM fungi establishing symbiotic interactions w ith poplar roots. Links between poplar productivity and nutrition status and ECM colonization w ere established and it w as demonstrated that ECM fungi ameliorate the stress responses and nutrition status of poplars under drought stress conditions. Thus, the results of this work provide information w hich underpins the significant role of the ectomycorrhizal symbiosis in relation to nutrient status of the poplar under drought stress conditions, and in relation to stem biomass production in a poplar plantation. These informations could be of crusial importance in the establishment phase of a poplar plantation as w ell as in relation to predicted increasing extreme climate events w hich could have negative impacts on biomass production