Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression

Abstract

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nifregulon 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. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9%o compared with the yield with the wild-type strain RMBPC and 17.9%o over that in the uninoculated control plot at the 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. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC-2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

Dicarboxylate transport by rhizobia

“…Expression of the nifH structural gene of nitrogenase, which is under NifA control, also starts at the II-III interzone [83]. An observation that may be related to the ASA is that the presence of additional copies of nifA and dctABD increases symbiotic effectiveness in S. meliloti [86], an effect that is not observed when dctA is constitutively expressed [30]. The presence of a plasmid carried Klebsiella pneumoniae nifA and S. meliloti dctABD in S. inorhizobium fredii also improved symbiotic effectiveness of the strain [87].…”

A field study with genetically engineered alfalfa inoculated with recombinantSinorhizobium meliloti: effects on the soil ecosystem

“…The cells were kept frozen at ±70 C overnight, thawed and centrifuged to remove the glycerol, mixed with sterile water to a concentration of c. 10 7 cells ml ±1 , and used to inoculate the alfalfa plants. The inoculum was also plated on MRDM-N medium, a modi®ed Bergensen's medium (Bergersen 1961;Bosworth et al 1994) with and without antibiotics (500 mg ml ±1 spectinomycin, 200 mg ml ±1 streptomycin and 10 mg ml ±1 trimethoprim), to verify inoculum concentrations. Groups of 64 alfalfa plants were each placed in containers with 200 ml of sterile water (uninoculated treatment) or 200 ml inoculum of one of the three strains of S. meliloti for c. 30 min prior to planting in the ®eld plot.…”

Fate of genetically modified Rhizobium leguminosarum biovar viciae during long‐term storage of commercial inoculants

“…Until now, mainly wild-type strains obtained by conventional selection procedures have been tested (Wilson and Lindow 1993). However, the sensible application of recombinant DNA techniques has shown, during the last decade, the potential for genetically improved micro-organisms to be used as soil or seed inoculants in open environments (Bishop et al 1988;Jones et al 1988;Birkenhead et al 1990;Lindow 1991;Bosworth et al 1994). During the same period, the biosafety issues pertaining to the marketing of genetically engineered micro-organisms Correspondence to: Dr Marco P. Nuti Gradenlgo 6,35131 Padova,.…”