Diversity of Soil Microbes powerpoint.ppt

Diversity of Soil Microbes

1. Diversity of Soil Microbes

2. Approaches for Assessing Diversity
Microbial
community
Organism
isolation
Culture Nucleic acid extraction
Molecular
characterization
Phenotype

3. Nucleic acids as biomarkers

4. Sequence Conservation Level
nearly universal
intermediate
hypervariable
16S rRNA

5. Phylogenetic categories of organisms
Comparative analysis of 16S
ribosomal RNA genes
Ancient Taxa:
The Kingdoms

6. Remotely Related Taxa: The Classes and Divisions (Phyla)
Not shown are candidate divisions, organisms detected by PCR-16S analysis,
but no currently isolated and cultured representative

7. Moderately Related Bacterial Taxa:
The Major Intradivisional
Groupings
(Orders and Families)

8. Highly Related Taxa: The Genera and Species
Example: Phylogeny of
Ammonia-oxidizing bacteria
Organisms of the same species
are >97% identical in 16S rRNA
gene.

17. 16SrRNAanalysis=
changesinonegene
(DNAsequence)atone
siteinthegenome
REP=shortsequences
thatareoccurinmultiple
locationsthroughoutthe
bacterialgenome
REP-PCR assays
variationinsequenceat
multiplesitesthroughout
thegenome
Patternsdifferentiate
bacteriaatsubspecies
level
www.bacbarcodes.com
Genomic DNA
denatured to expose
repetitive sequences
Primers bind the
repetitive sequences
DNA sequences
between repetitive
sequences are
amplified
DNA fragments of
various lengths are
generated
Fragments are resolved by
gel electrophoresis yielding
complex fragment patterns
Similar strains show
similar patterns
Reptitive element (REP)-PCR Genomic fingerprinting

18. REP-PCR Analysis of fluorescent
Pseudomonas isolates from
different sites and continents
Fluorescent Pseudomonas:
Produce bright pigments in culture
Commoly isolated from soils and plant
rhizospheres
Metabolically diverse
Are strains globally mixed or endemic?
Examine REP-PCR patterns of bacteria isolated from
different continents, and different sites within
continents.

19. REP-PCR Analysis of
fluorescent Pseudomonas
isolates from different
continents
Genotypes of isolates from
different locations form
separate clusters
Indicates genotypes were
endemic

20. Summary: Geographic distribution
•Fluorescentpseudomonadsandothersareglobally-
distributed
•Developmentofendemicspeciesoccursatfinerscales
•Spatial(geographic)isolationaffectsbacterial
diversification

21. Effects of plant growth on microbial community
composition
How do plants alter the composition of soil microbial
communities?
Are certain bacteria preferentially selected for
colonization of the rhizosphere?
Do certain plants select for certain bacteria?
What has a greater impact on the composition of plant
rhizospheres? The plant type or the soil?

22. Rhizosphere: General effects
Density (abundance) of bacteria increases in rhizosphere
relative to bulk soil
Bacterial diversity in rhizosphere decreases relative to soil.
Increase in abundance of Proteobacteria relative
to other phyla

23. Comparison of rhizosphere vs. soil effects
Unique to Soil 1
Unique to Soil 2
Different populations of pseudomonads colonize the rhizosphere of
the same plants grown in different soils

24. Rhizosphere
Effects
Soil 1 PopulationSoil 2 Population
Selection imposed by
rhizosphere
varies with plant, and plants
(root) age
Growth and enrichment in
plant rhizosphere
Sub-populations selected
for growth from each soil

25. Rhizosphere Effects
Plant selects for organisms from a pool that has
developed and established in the soil.
Different parts of the pool may be selected by different
plants
Since plant changes with time, selection also varies with
time for a given plant

26. Rhizosphere variability over time
DGGE profiles of Pseudomonas rhizosphere communities at the early (A) and late (B)
flowering stage and at the senescent growth stage (C). Different lanes (1–3) represent
rhizosphere sample from different pots. GM, transgenic plants without herbicide; G,
transgenic plants with Basta application; MM, wild-type plant without herbicide application;
B, wild-type plants with Butisan S application. Bands 2a, 2b, 2c and 2d had the same
mobilities as bands 1a (3a), 3b, 1f and 1d.
FEMS Microbiol. Ecol.41:181-190

27. Perterbations: General effects on
soil microbial communities
Perterbations: Typically decrease diversity, select for a
proliferation of subpopulations
Examples:
Plants-rhizosphere
Animals -Earthworm casts
Geological -volcanic eruption
Climatic -Fire
Anthropogenic -Pollution, agricultural practices

28. Do bacterial community structure changes (decreased
diversity) induced by perturbations affect function?
Stability in transformation of A -->D is supported by diversity
in organisms and establishment of redundancy in the
activities they possess.
How is decreased diversity (less redundacy) reflected in
community activity?
A B C D
Transformations
Functions possesed by the indicated group
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6

29. Community Dilution Experiment
Hypothesis: Reduced diversity, reduces redundancy,
and reduces the capability of soils to respond to stress
Approach:
Sterile soil inoculated with serial dilution of soil
suspension
Incubated 9 months
Measured biomass, assessed community diversity,
and activities

30. Dilution experiment results: Biomass and diversity
The overall biodiversity in sterile soil inoculated with dilutions of a
soil suspension (A, 10 0; B, 10 2; C, 10 4; D, 10 6). The number of
species within the individual populations measured (i.e. soil
bacterial DNA bands, cultivable bacterial morphotypes, cultivable
fungal morphotypes and protozoan species) was normalized
relative to the maximum number observed, summed and divided
by four (i.e. the number of taxonomic groups) to give the
biodiversity index. The bar represents ±one standard error, n=3
No significant difference in biomass, but
Decreasing diversity as function of dilution

31. Dilution experiment results: Molecular analysis of
bacterial diversity
The DNA banding pattern of soil bacteria obtained by DGGE analysis of
eubacterial-primer based amplicons from sterile soil inoculated with dilutions
of a soil suspension (A, 10
0
; B, 10
2
; C, 10
4
; D, 10
6
). The three lanes of each
treatment represent individual replicates, n=3
Decreasing number of bands, decreasing diversity

32. Dilution experiment results: Activity as a function of
diversity
Effect of stresses, in the
form of Cu addition or heat
treatment, on the ability of
sterile soil inoculated with
dilutions of a soil suspension
(A, 10 0; B, 10 2; C, 10 4; D,
10 6) to decompose grass
residues at increasing time
intervals following the
application of the stress.
Bars represent one standard
error, n=3.

33. Community Dilution Experiment-Conclusion
Diversity could be experimentally manipulated
No detectable change in activity with the level of diversity
reduction achieved.
Minimum level of diversity (functional redundancy) to
support process stability in soil is unknown.