t-rflp study of soil microbial community at elevated chloride concentration m. gryndler institute of...
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T-RFLP study of soil microbial community at elevated chloride
concentrationM. Gryndler
Institute of Microbiology CAS, Prague
In collaboration with:
Miroslav Matucha and Jana Rohlenová, Institute of Experimental BotanyCAS, 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 OH 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• 14C-TCA degradation • 36Cl- 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
DNA extraction(125 d) and
amplification:
Eubacteria
Eukaryota
Fungi
Lanes 1,2,3 – 20N
Lanes 4,5,6 – 500N
Lanes 4,8,9 – 20S
Lanes 10,11,12 – 500S
Eukaryotic ITS region,Eubacterial SSU rDNA
Primers:
• Eubacteria: 16Seu27f, 783r-a,b,c(Sakai et al. 2004)
• Eukaryota: ITS1, ITS4(White et al. 1990)
• Fungi: ITS1F, ITS4(Gardes and Bruns 1993)
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
Terminal restriction fragments:
0
200
400
600
800
1000
1200
1400
1600
1800
2000
60 110 160 210 260 310 360
bp
Numbers of TRFTreatment Sample Eubact. Eukaryota Fungi
20N 1 52 104 112 59 75 90
3 57 68 113
500N 1 50 73 572 43 126 17
3 225 159 100
20S 1 59 129 1422 32 56 92
3 29 100 16
500S 1 40 129 112 47 77 14
3 35 105 12
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
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
130 135 140 145
bp
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 primer5´- 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 primer5´- ITS1 - .......……TRF………….......... T– 3´3´- ................. 2-stranded .......... A GC– 5´
=TaqI
Forward primer5´- 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´‑TCCGTAGGTGAACCTGCGGAAGGATCATTGGAGAGAGAAAGGGGGAGAGAGTTGGAATGTGATGAGACGAGAGATTCAAACTATATAGTGAATGATCATACAACTGCTGACAATGGATCTCTGGGCTCTTGCGTCGAATTCTCCGTCTCGCTCCG-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).
Identity?
5´‑TCCGTAGGTGAACCTGCGGAAGGATCATTGGAGAGAGAAAGGGGGAGAGAGTTGGAATGTGATGAGACGAGAGATTCAAACTATATAGTGAATGATCATACAACTGCTGACAATGGATCTCTGGGCTCTTGCGTCG-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“
CLF1-ITS4/NL1-NL4
GAGTTGGAATGTGATGAGACGAGAGATTCAAACTATATAGTGAATGATCATACAACTGCTGACAATGGATCTCTGGGCTCTTGCGTCGATGAAGAACGCAGCAAAACGCGAAAAGTGTTATGATGTGCAGTCTTTGAGAATCATGAATGTTTGAACGCACCTTGCACCACCGAGCGATTGGGGGTATGCCTGTTTGAGCGGGGGATAAAATTGAGTGAACTGTGGTTTATTGTGGGGTACTAGGTAACACCTTGCCCTGAAAGACAGATCTCGTGTACTCTTGGGATAATCCATCAAGAAGCACATTTACAGTATACCACCTCAAATCAGGCAAGATGACCCGCTGAACTTAAGCATATCAGTAAGCGGAGGAAAAGAAACTAACCAGGATTCCCGCAGTAACGGCGAGTGAAGAGGGAACAGCTCACATTTGGAAACGATCATGAAAAAAGTGGTCGAGTTGTCAGTGATAGCATGGGAGCGGGATTTGGAAAGGGTGAGCGAGTCTGCTGGAAAGCAGCGCCAGAGAGGGTGACAGCCCCGTGGCTTGCCTTGCACAAGTTTACGGAGCCATGCGACGAGTCGGACTGTTTGGGAATGCAGTCCTAAATGGGTGATACGTGTCATCTAAAGCTAAATAGCGGCAAGAGACCGATAGCGAACAAGTACCGTGAGGGAAAGATGAAAAGAACTCTTGGCAGAGAGTGAAAAGTGCGTGAAATTGTCAGCAGGGAAGCGATGGTGGTGGGGAGGTGTGCCAAGAGAAGCAACTTCGTCAGAAGTTGCCAATTAAAAAGCACGAGCGAGGCGAGTGGATGAGGGGGAAGATAGGTGGGAAGAGAGAGTAATTCATTACAATTTCTTCAAACTAAACCCCCCACACACACCCACCTCGCAGCCCACCACGACCGACCCGTCTTGAAACACGGACC
Homology?
Glomus walkeri (AJ972467, E=1x10-36), Glomus drummondi (AJ970465, E=4x10‑36)
Orpinomyces sp. (AJ864475, E=2x10-36)
Entophlyctis sp. (DQ273782, E=6x10-36)
Cyllamyces aberensis (DQ273829, E=2x10-35) Neocallimastix sp. (DQ273822, E=2x10‑35)
Efects of increased chloride on organic and inorganic Cl-?
0
100
200
300
0 10 20 30 40 50 60 70 80
Time (h)
Rel
ease
d r
adio
acti
vity
(k
Bq
)
20N
500N
20S
500S
36Cl-TCA mineralization
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