Diversity of Soil Microbes

Approaches for Assessing Diversity

Microbial community

Organism isolation

Culture Nucleic acid extraction

Molecular characterization

Phenotype

Nucleic acids as biomarkers

Sequence Conservation Level nearly universalintermediatehypervariable

16S rRNA

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

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

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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

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

Summary: Geographic distribution

•Fluorescent pseudomonads and others are globally- distributed

•Development of endemic species occurs at finer scales

•Spatial (geographic) isolation affects bacterial diversification

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?

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

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

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

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

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

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

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

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

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

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

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.

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.