Presentation on theme: "T-RFLP study of soil microbial community at elevated chloride concentration M. Gryndler Institute of Microbiology CAS, Prague In collaboration with: Miroslav."— Presentation transcript:
T-RFLP study of soil microbial community at elevated chloride concentration M. Gryndler Institute of Microbiology CAS, Prague In collaboration with: Miroslav Matucha and Jana Rohlenová, Institute of Experimental Botany CAS, Prague and Jan Kopecký, Institute of Microbiology CAS, Prague
Questions: Does elevated Cl - concentration affect established soil microbial community? Does elevated Cl - concentration affect de- -novo developing microbial community? Which organisms are affected? Is a change in composition of soil microbial community followed by changes in degradation of chlorinated SOM or chlorination of SOM?
Methodology: Nonsterile or irradiated/recolonized 128 g soil samples, 20% humidity Podzolic O H soil from spruce monoculture Two levels of total soil Cl: 20 or 500 mg/kg Counting microbial CFU Extraction of DNA, TRFLP analysis of prokaryotic and eukaryotic ribotypes 14 C-TCA degradation 36 Cl - incorporation into humic substances
Design of the experiment (n=3) Low chloride, intact microflora High chloride, intact microflora Low chloride, sterilized/recolonized High chloride, sterilized/recolonized
Restriction cleavage: Taq I. restriction endonuclease for eukaryotic DNA ALU I. restriction endonuclease for eubacterial DNA Forward primers HEX-labeled Analysis using capillary electrophoresis, LIF detector
Effect of soil sterilization/recolonization: 3 eubacterial and 1 fungal TRF disappeared 1 eukaryotic and 1 fungal TRF detected
Effect of increased Cl - 1 eubacterial and 1 fungal TRF disappeared 1 bacterial and 1 eukaryotic TRF detected
Eukaryotic TRF 135 bp Absent from any treatment but nonsterilized soil high in chloride In 3 replicates constitutes 33.5, 11.4 and 7.0 molar % of all TRF !
Identity? Forward primer 5´- ITS1 -............. TRF……....….......... T– 3´ 3´-................. 2-stranded.......... A GC– 5´ =TaqI Fragment lacks known reverse primer sequence Oligonucleotide adapter is necessary for PCR amplification
Identity? Forward primer 5´- ITS1 -.......……TRF………….......... T– 3´ 3´-................. 2-stranded.......... A GC– 5´ =TaqI Forward primer 5´- ITS1 -.............TRF.......................... T/CG AAT TCT CCG TCT CGC TCC G – 3´ 3´-................. 2-stranded.......... A GC/TTA AGA GGC AGA GCG AGG C – 5´ =TaqI reverse primer
Identity? 5´ ‑ TCCGTAGGTGAACCTGCGGAAGGATCATTGGAGAGAGAAAG GGGGAGAGAGTTGGAATGTGATGAGACGAGAGATTCAAACT ATATAGTGAATGATCATACAACTGCTGACAATGGATCTCTGGG CTCTTGCGTCGAATTCTCCGTCTCGCTCCG-3´ This sequence is 100% identical with the sequence registered in GenBank under accession number DQ309135 ("uncultured fungus isolate RFLP-145") and involves a part of 18S ribosomal RNA gene and a part of internal transcribed spacer 1. This sequence was amplified from DNA extracted directly from Ericaceous roots along a moorland-forest gradient in Scotland. At this stage, its identity is unknown (Prof. John W. G. Cairney, University of Western Sydney, Australia, personal communication).DQ309135
Identity? 5´ ‑ TCCGTAGGTGAACCTGCGGAAGGATCATTGGAGA GAGAAAGGGGGAGAGAGTTGGAATGTGATGAGAC GAGAGATTCAAACTATATAGTGAATGATCATACAA CTGCTGACAATGGATCTCTGGGCTCTTGCGTCG-3´ Sequence does not contain information sufficient to identify its bearer. However, it may be used to design specific primer to amplify larger rDNA fragments, perhaps suitable for identification by „BLASTing“
Primer CLF1 (forward) 5´-GAGTTGGAATGTGATGAGACG-3´ + Primer NL4 (reverse)
Efects of increased chloride on organic and inorganic Cl - ?
Conclusions Sodium Chloride at „realistic“ concentrations affects soil microflora TRFLP and downstream techniques can be used to detect and identify organisms responding to chloride Inorganic and organic Cl behaviour is affected by Cl - concentration Links between specific organisms and soil Cl fluxes are probable
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