SUMMARY

Current advances in biotechnology have allowed the construction of genetically engineered plants possessing novel and useful characteristics for applications such as crop protection, phytoremediation of polluted soils and production of specialty chemicals. Commercial development and field testing of engineered plants is far exceeding the knowledge of the potential ecological effects of their introduction into the environment (Krattiger, 1994). Concerns regarding the effects of transgenic plant introductions on soil and rhizosphere microorganisms have been raised by the scientific community (Seidler and Levin, 1994; Donegan et al, 1995). In addition, a more thorough understanding of rhizosphere microbial communities has been identified as a research need by a recent workshop on phytoremediation (U.S. DOE, 1994). A particular area of need is in the understanding of plant-microbe interactions. We have developed a novel approach to rhizosphere microbial community analysis using two of the most promising new techniques in microbial ecology, community metabolic profiles and community DNA fingerprinting. This study describes the application of this new methodology for evaluation and characterization of rhizosphere microbial communities of parental and transgenic alfalfa.

A new approach to the characterization of microbial communities based on communitylevel sole-carbon-source utilization patterns was reported by Garland and Mills (1991). The authors used Biolog Gram negative (GN) microplates (Biolog, Inc., Hayward, CA) inoculated with soil bacterial extracts of environmental samples to generate "metabolic fingerprints" of microbial communities. Substrate utilization patterns can then be used as indicators of the metabolic potential, genetic diversity and species diversity of microbial communities.

A recently developed molecular technique in microbial ecology relies on the amplification of enterobacterial repetitive intergeneric consensus (ERIC) DNA sequences by the polymerase chain reaction to fingerprint soil bacteria (De Bruijn, 1992). ERIC-PCR generates multiple distinct amplification products of sizes ranging from approximately 100 to 3,000 bp. Until this study ERIC-PCR has only been used to fingerprint pure cultures. We present its use in fingerprinting pure cultures, defined mixtures of microbes and rhizosphere microbial communities of environmental samples.

For this study isogenic strains of a parental and two transgenic alfalfa were used. The two transgenic strains express fungal genes for Mn dependent lignin peroxidase and bacterial genes for alpha amylase respectively (Austin et al, 1995). Community-level metabolic analysis detected subtle differences between the rhizosphere microbial communities of the parental (P), alpha amylase (AA) and lignin peroxidase (LP) alfalfa strains. Similarity analysis of the rhizosphere microbial community metabolic fingerprints demonstrated clustering based on genotype. Binarypattern comparison and multivariate analysis identified several significant substrates which can be used to differentiate the rhizosphere communities.

ERIC-PCR was used to fingerprint DNA extracted from the microbial consortia (elective cultures) present in the Biolog substrate wells. These DNA fingerprints were used to characterize and compare specific heterotrophic populations of the alfalfa rhizosphere communities. ERIC-PCR screening of bacterial colonies obtained from the Biolog elective cultures allowed the isolation and identification of several key species associated with the alfalfa strains. Use of this new methodology will enable us to better characterize plant-microbe relationships and to identify potential interactions between microbial populations in the rhizosphere.

REFERENCES

Austin, S., E.T. Bingham, D.E. Matthews, M.N. Shahan, J. Will and R.R. Burgess. 1995. Production and field performance of transgenic alfalfa (Medicago sativa L.) expressing alphaamylase and manganese-dependent lignin peroxidase. Euphytica 85:381-393.

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