Field Efficacy of Inoculation of Rhizobium etli CE3 that Produces the Antibiotic Trifolitoxin

Abstract

Trifolitoxin (TFX) is a potent peptide antibiotic that inhibits (α-Proteobacteria, and confers competition for nodulation in Rhizobium (1, 2). We investigated field efficacy of TFX-producing inoculants at Arlington, Wisconsin. We inoculated seeds of Pinto beans (Phaseolus vulgaris) with mixtures of a TFX-sensitive strain with either a TFX non-producing or a TFX-producing strain at different ratios. Nodule occupancy of the TFX-producing strain was significantly higher than the non-producing strain. Inoculation with a mixture containing only 10 percent of the TFX-producing strain resulted in 45 percent of the nodules occupied by this strain. Nodule occupancy by the TFX non-producing strain was similar to its representation in the inoculum. Nodule occupancy by the TFX-producing strain did not adversely affect grain yield. Thus, TFX production increases nodule occupancy of the inoculant strains under field conditions. To determine whether the increase in nodulation competitiveness is dependent on the amount produced by a strain, we constructed a TFX overproducer plasmid and mated it into CE3. We cloned the tfx genes from pTFX24, a pBluescript derivative, into the HinDIII site of pUCD1002 (3), a cloning vector containing a rhizobium origin of replication that is maintained under no selection. The resulting construct, pTFXHCP, was mated into Rhizobium etli CE3 and conferred TFX production. Trifolitoxin overproduction and its effects on the range of inhibition and nodulation competitiveness are yet to be determined.