Methods of Studying Rhizosphere Ecology

Understanding rhizosphere ecology and the interactions between plants and soil-borne organisms often requires determination of the identity and distribution of the vast diversity of organism(s) (bacteria, fungi, arthropods, etc.). However, it is this diversity that makes the rhizosphere one of the most difficult communities to study, often

Table 1 Summary of some methods for studying the rhizosphere General methodology Example techniques

Direct observation

Molecular markers

Functional measures dictating the use of a variety of methodologies dependent upon the research objectives and organisms of interest. Traditional methods based on the isolation and growth of live organisms in culture may significantly limit the population being evaluated. For example, it has been suggested that only 1% of a bacterial population can be cultured by common laboratory techniques. It is unknown if this limited sample is representative of the entire population and is unculturable due to a physiological state, or a highly selective sample that is phenotypically and/or genetically suited for laboratory growth on artificial media. To overcome these problems, a variety ofmethods have been developed including direct observation, fatty acid analysis, chemical, and molecular techniques (DNA and RNA), etc. A detailed discussion of each of these techniques is beyond the scope of this article and the reader should consult one of the many reviews that discuss the advantages and disadvantages in more detail. A brief introduction to some of the available techniques is shown in Table 1. As should be obvious from Table 1, the methodology chosen may significantly influence the type and diversity of organisms identified in rhizosphere ecology studies. In addition, the research must also consider a number of other factors in the design and analysis of rhizosphere ecology studies. For example, considerable temporal and spatial heterogeneity may be observed associated with factors such as plant species and distribution, microclimate, soil physical and chemical properties, and the life stage or physiological state of rhizosphere microorganisms.

Detection and/or isolation by any of the above methods may not lead to a definitive identification of an organism. For many taxonomic groups, there is no official definition of species. For example, the genetic plasticity of bacteria allowing DNA transfer through plasmids, bacter-iophages, and transposons complicates the concept of species. Fungal taxonomy has similar problems in identifying vegetative structures, as most taxonomy is based on

Target organism

Fungi, bacteria, arthropods Macroinvertebrates, roots

Arthropods Fungi, bacteria Fungi, bacteria

All - primer specific Bacteria Diazotrophs Bacteria, fungi, roots sexual structures. Species-level arthropod identification is time consuming and typically conducted by systematists, especially when examining immature (larval) specimen. Molecular techniques may alleviate some of these problems but are still limited due to incomplete databases, genetic polymorphyisms, multiple gene copies, and intraspecies variation. The utility of some molecular approaches for studying rhizosphere ecology may also be limited by their inability to separate organisms that are dormant and/or not participating in rhizosphere processes from those organisms that play key roles in the rhizosphere.

Larger-scale techniques such as microscopy and rhizo-tron-based observation have also been used to examine higher-order rhizosphere structure and organization. Fluorescent microscopy of bacteria, either naturally fluorescing (such as Pseudomonas spp.) or strains expressing a fluorescent protein, allows for detailed spatial organization of microbial communities in a relatively intact setting. Rhizotrons are essentially buried glass-walled containers that allow viewing of intact rhizosphere as it expands to the glass surface. Such devices are of limited use for microbial spatial dynamics, but can be valuable as tools for studying larger arthropods and root-root interactions.

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