Transcript Kein Folientitel - Chair of Soil Science

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]