SPP 1090 Böden als Quelle und Senke von CO 2

1. Brown forest soil (Steigerwald) Frequences (number) Podzolic forest soil (Waldstein) I II III IV 200bp 1200bp 200bp DNA Cu1 PCR Cu4 PCR Cu2 Nested PCR Cu3 Nested PCR Cu2 PCR Regions encoding Cu- binding sites Degenerated primers tested Laccase genes Degenerated primers used for PCR amplifications Of-h 60.0% 52.0% 40.0% 50.0 % 76.9 % 60.8 % Ah 40.0% 46.1% 36.8% 46.6% 42.1 % 46.6% Bv 28.0% 45.4% 29.5% 35.7% 42.1% 45.4% Assessment of the primer pair specificity on fungal culture strains, 100bp DNA ladder (1), negative control (16) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 (Number of different laccases/number of clones sequenced) basidiomycetes ascomycetes % of laccase diversity 2:Pycnoporus cinnabarinus 3:Ganoderma lucidum 4:Pleurotus ostreatus 5:Trametes versicolor 6:Lentinulaedodes 7:Hypholoma sp. 8: Gymnopus fusipes 9:Neurospora crassa 10:Aspergillus nidulans 11:Cladosporium sp. 12:Cryphonectria parasitica 13:Podospora anserina 14:Morchella esculenta 15:Cenococcum geophilum % of Corg 19.0% 3.38% 1.19% Soil RNA extraction 1 2 3 4 5 6 The protocol developed gives high concentrations of soil total RNA. After purification, the remaining concentration allows to perform a RT-PCR 100pb DNA ladder (1 & 6), total RNA obtained from soil before (2 & 3) and after purification (4 & 5) PCR on cDNA obtained from soil RNA 1 2 3 4 5 6 7 8 9 10 Amplification products from cDNA gave fragments of the expected size (142pb) 100pb DNA ladder (1 & 10), PCR products obtained on cDNA which are respectively synthesized with 15 amplification cycles (2 & 3), 18 cycles (4 & 5), 21 cycles (6 & 7) and 24 cycles (8 & 9). Conclusions • The optimized primers allow to amplify approximately 200 bp fragments of laccase genes in a broad range of Basidiomycetes • Analyses on soils revealed a high soil and horizon specificity of the fungal laccase genes • Ectomycorrhizal fungi seems to have a wider vertical distribution compared to the others functional fungi groups • First RT-PCR on soil RNA followed by cDNA amplification was realized. Additional sample analyses and sequencing should confirm of the effectiveness of the method SPP 1090 Böden als Quelle und Senke von CO2 Diversity of basidiomycete laccase genes in soil samples Patricia Luis1,2, Grit Walther1, Francis Martin2 and François Buscot1 1 Friedrich Schiller University of Jena, Institute of Ecology, Department of Environmental Sciences 2 Centre INRA of Nancy, UMR INRA/UHP 1136 “Interactions Arbres/Micro-organismes” Summary Vertical diversity of laccase genes in 2 forest soils • Introduction and goal • Fungi are one of the major organism groups involved in formation and decomposition of soil organic matter (SOM) • By producing oxidative exo-enzymes without substrate specificity, they fully mineralize organic compounds or recombine organic radicals in stable polymers • Among the exo-enzymes, phenol oxidases (laccases) are produced by the broadest range of fungi. Therefore, they were chosen as model to develop a technique to monitor fungi with an oxidative potential in soils, without taking them in culture Comparison Podzolic\Brown forest soil Design of degenerated primers specific for basidiomycetes Brown forest soil replicates Higher diversity in O horizons  Stronger horizon specificity in the Podzol Small number of common laccases between soils  Replicate heterogeneity Diversity decreases with the depth The primers appear adequate to specifically amplify laccase genes from basidiomycetes For both soils, the diversity is stronger in O horizons (highest concentration of SOM) and generally decreases with the depth Comparing laccase-genes of soil & fruit-bodies First extraction of soil RNA & PCR on cDNA Laccase gene sequences are species specific Most fungi possess a family of laccase genes  Sequence and genera clades rarely overlap  Diversity of laccase genes is higher than the one of fungi Off 70 soil sequences, only 18 correspond to laccases of the collected fruit-bodies(12 saprophytic, 1 pathogen & 5 ectomycorrhizal fungi) Fruit bodies don’t reflect correctly the soil fungi community (seasonal fruiting)  Unknown groups of laccase genes (bold bars) were detected, especially in mycorrhizal fungi Ectomycorrhizal fungi seem to have a wide vertical distribution  Analysis of fungal laccase gene expression in soils seems feasible  The primers detect laccases in a broad range of Basidiomycota of all functional groups (saprophytes, pathogens & mycorrhiza) Contact: Prof. Dr. François Buscot, Institute of Ecology, Department of Environmental Sciences, Friedrich-Schiller-University of Jena, Dornburger Str. 159, D-07743 Jena / Mail: francois.buscot@uni-jena.de