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

Microbial CFU counts

0

5

10

15

20

25

30

35

40

20N 500N 20S 500S

CFU

x10

-5

Bacteria

Fungi

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)

rDNA cassette

18S rDNA

5.8S rDNA

28S rDNAITS1 ITS2 D1 D2

ITS1 ITS4

TRFLP

Terminal restriction fragment length polymorphism

Restriction cleavage

Restriction profile

A

BA+B

Restriction cleavage

Restriction profile

Restriction cleavage

Restriction profile

A

BA+B

A

BA+B

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“

Primer CLF1 (forward)

• 5´-GAGTTGGAATGTGATGAGACG-3´

+ Primer NL4 (reverse)

rDNA cassette

18S rDNA

5.8S rDNA

28S rDNAITS1 ITS2 D1 D2

CLF1 ITS4

ITS1F-ITS4

ITS1-ITS4

27f-783r

CLF1-ITS4

rDNA cassette

18S rDNA

5.8S rDNA

28S rDNAITS1 ITS2 D1 D2

CLF1 NL4

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-?

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

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