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SPP 1090 Böden als Quelle und Senke von CO2 Diversity of basidiomycete laccase genes in soil samples Patricia 1,2 Luis , Grit 1 Walther , 2 Martin Francis and François 1 Buscot 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” 1 Summary Vertical diversity of laccase genes in 2 forest soils Introduction and goal Comparison Podzolic\Brown forest soil Fungi are one of the major organism groups involved in formation and decomposition of soil organic matter (SOM) 8 7 By producing oxidative exo-enzymes without substrate specificity, they fully mineralize organic compounds or recombine organic radicals in stable polymers Design of degenerated primers specific for basidiomycetes I 200bp II 1200bp III Cu1 PCR Cu2 Nested PCR 200bp IV 6 Of-h 5 Ah 4 Frequences (number) 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 Brown forest soil (Steigerwald) 9 DNA Cu4 PCR Cu3 Nested PCR Bv 3 2 1 0 lac1 lac2 lac3 lac4 lac5 lac6 lac7 lac8 lac9 lac10 lac11 lac12 lac13 lac14 lac15 lac16 lac17 lac18 lac19 lac20 lac21 lac22 lac23 Podzolic forest soil (Waldstein) 8 7 6 5 Of-h 4 Ahe 3 Bhs-sv 2 Cu2 PCR 1 0 lac1 Regions encoding Cu- binding sites Degenerated primers tested Degenerated primers used for PCR amplifications lac2 lac3 lac4 5 6 7 8 basidiomycetes lac7 lac8 lac9 lac10 lac11 lac12 lac13 lac14 lac15 lac16 lac17 lac18 lac19 lac20 lac21 Brown forest soil replicates Assessment of the primer pair specificity on 9 10 11 12 13 14 15 16 fungal culture strains, 100bp DNA ladder (1), negative control (16) ascomycetes % of laccase diversity 3 4 lac6 Laccase genes Of-h 1 2 lac5 2: Pycnoporus cinnabarinus 9: Neurospora crassa 10: Aspergillus nidulans 3: Ganoderma lucidum 11: Cladosporium sp. 4: Pleurotus ostreatus 12: Cryphonectria parasitica 5: Trametes versicolor 13: Podospora anserina 6: Lentinula edodes 14: Morchella esculenta 7: Hypholoma sp. 15: Cenococcum geophilum 8: Gymnopus fusipes The primers appear adequate to specifically amplify laccase genes from basidiomycetes 60.0% 40.0% 28.0% 52.0% 46.1% 45.4% 40.0% 36.8% 29.5% 50.0 % 46.6% 35.7% 76.9 % 42.1 % 42.1% 60.8 % 46.6% 45.4% % of Corg 19.0% 3.38% 1.19% are species specific 1 2 3 4 5 6 Soil RNA extraction The protocol developed gives high concentrations of soil total RNA. After purification, the remaining concentration allows to perform a RT-PCR Most fungi possess a family of laccase genes 100pb DNA ladder (1 & 6), total RNA obtained from soil before (2 & 3) and after purification (4 & 5) Sequence and genera clades rarely overlap Diversity of laccase genes is higher than the one of fungi Replicate heterogeneity Diversity decreases with the depth First extraction of soil RNA & PCR on cDNA Laccase gene sequences Bv 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 Ah Higher diversity in O horizons Stronger horizon specificity in the Podzol Small number of common laccases between soils 1 2 3 4 5 6 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). Off 70 soil sequences, only 18 correspond to laccases of the collected fruit-bodies (12 saprophytic, 1 pathogen & 5 ectomycorrhizal fungi) 7 8 9 10 PCR on cDNA obtained from soil RNA Analysis of fungal laccase gene expression in soils seems feasible 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 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 The primers detect laccases in a broad range of Basidiomycota realized. Additional sample analyses and sequencing should of all functional groups (saprophytes, pathogens & mycorrhiza) confirm of the effectiveness of the method Contact: Prof. Dr. François Buscot, Institute of Ecology, Department of Environmental Sciences, Friedrich-Schiller-University of Jena, Dornburger Str. 159, D-07743 Jena / Mail: [email protected]