diversity of soil microbes. approaches for assessing diversity microbial community organism...
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Nucleic acids as biomarkersTRANSCRIPT
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Diversity of Soil Microbes
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Approaches for Assessing Diversity
Microbial community
Organism isolation
Culture Nucleic acid extraction
Molecular characterization
Phenotype
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Nucleic acids as biomarkers
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Sequence Conservation Level nearly universalintermediatehypervariable
16S rRNA
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Phylogenetic categories of organisms
Comparative analysis of 16S ribosomal RNA genes
Ancient Taxa:The Kingdoms
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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
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Moderately Related Bacterial Taxa:
The Major Intradivisional Groupings (Orders and Families)
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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.
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16S rRNA analysis = changes in one gene (DNA sequence) at one site in the genome
REP = short sequences that are occur in multiple locations throughout the bacterial genome
REP-PCR assays variation in sequence at multiple sites throughout the genome
Patterns differentiate bacteria at subspecies level
www.bacbarcodes.com
Genomic DNA denatured to exposerepetitive sequences
Primers bind therepetitive sequences
DNA sequencesbetween repetitivesequences areamplified
DNA fragments ofvarious lengths aregenerated
Fragments are resolved by gel electrophoresis yieldingcomplex fragment patterns
Similar strains show similar patterns
Reptitive element (REP)-PCR Genomic fingerprinting
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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.
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REP-PCR Analysis of fluorescent Pseudomonas
isolates from different continents
Genotypes of isolates from different locations form separate clusters
Indicates genotypes were endemic
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Summary: Geographic distribution
•Fluorescent pseudomonads and others are globally- distributed
•Development of endemic species occurs at finer scales
•Spatial (geographic) isolation affects bacterial diversification
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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?
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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
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Comparison of rhizosphere vs. soil effects
Unique to Soil 1Unique to Soil 2
Different populations of pseudomonads colonize the rhizosphere of the same plants grown in different soils
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Rhizosphere Effects
Soil 1 Population Soil 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
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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
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Rhizosphere variability over time
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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
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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
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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 1Group 2Group 3Group 4Group 5Group 6
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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
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Dilution experiment results: Biomass and diversity
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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, butDecreasing diversity as function of dilution
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Dilution experiment results: Molecular analysis of bacterial diversity
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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, 100; B, 102; C, 104; D, 106). The three lanes of each treatment represent individual replicates, n=3
Decreasing number of bands, decreasing diversity
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Dilution experiment results: Activity as a function of diversity
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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.
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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.