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Rhizobium Meliloti Field Tests

Research Update - Plants And Microbes

Charles Hagedorn, Ph.D., Virginia Polytechnic Institute
"NBIAP News Report." U.S. Department of Agriculture (December 1994)

The results of recent field trials in Wisconsin with engineered Rhizobium meliloti strains on alfalfa have been reported (Bosworth et al. 1994. Appl. Environ. Microbiol. 60:3815-3832). While the construction of rhizobial strains that increase plant biomass under controlled conditions has been reported previously, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions found in the field.

Experiments in the new report tested the hypothesis that expression of additional copies of nifa (a regulatory nitrogen fixation gene) and dctABD (genes involved in energy transport) in strains of Rhizobium meliloti would increase alfalfa yield in the field. The hypothesis was based on the positive regulatory role that nifa plays in the expression of the nif regulon, and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule.

Seven recombinant strains of R. meliloti that contained an extra copy of nifa and/or dct genes inserted into the chromosome of two wild-type strains were used in the study to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass.

Recombinant strain RMBPC-2, which has an additional copy of both nifa and dctABD, increased alfalfa biomass by 12.9% compared with wild-type, and 17.9% over the uninoculated controls, at a site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. No significant increases or decreases in alfalfa biomass were observed with the six other recombinant strains at that site.

At three other sites where the experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. The authors concluded that RMBPC-2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient soil moisture.

Yield increases in the field as a result of legume inoculation with genetically engineered rhizobial strains are difficult to observe for a number of reasons. Most importantly, nitrogen is often not limiting under field conditions. If nitrogen is not limiting, increased nitrogen availability to the plant by biological fixation will have no effect on yield even though plant tissue may contain a higher concentration of nitrogen. The investigators attempted to select field sites where soil nitrogen levels were low and where irrigation could be used to be certain that moisture was adequate. Lack of moisture depresses yields and reduces the plants' demand for nitrogen.

Other factors that can limit the effects on enhanced nitrogen fixation include the presence of a large native rhizobial population, the selection of genes chosen to enhance the rhizobia, the management/harvest system used to grow the inoculated crop, and the selection of experimental design. The authors developed a strategy in their field trials to address all of these potential problems. Even though seed inoculation with the engineered strains was performed at high levels (seed coating with 2X to 3X the commercial rate), nodulation with the engineered strains was still very low at the one site where large populations of native rhizobia occurred. At the one site where a yield response occurred (low soil nitrogen, organic matter, and native rhizobial populations), the results indicated that additional copies of both the nifa and dctABD are needed, as strains that contained extra copies of either one of the sequences alone did not provide a yield response.

Lastly, this research demonstrates the need to address other issues such as nodulation and plant nitrogen demand, as well as biological nitrogen fixation by engineered strains. Enhancing any one aspect may not provide the most desirable results if there are limitations in one of the other parameters that reduce the plants' need for nitrogen or the ability of the engineered strains to form nodules.

While the authors have provided the first report of a yield increase in field trials as a result of inoculation with an engineered strain of R. meliloti, the potential usefulness of such a strain remains limited at this time since the yield increase only occurred at a location where both soil nitrogen content and native rhizobial populations were low. Such locations are difficult to find, especially in the Midwest.


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