Inhibition of \u3cem\u3eRhizobium etli\u3c/em\u3e Polysaccharide Mutants by \u3cem\u3ePhaseolus vulgaris\u3c/em\u3e Root Compounds

Crude bean root extracts of Phaseolus vulgaris were tested for inhibition of the growth of several polysaccharide mutants of Rhizobium etli biovar phaseoli CE3. Mutants deficient only in exopolysaccharide and some mutants deficient only in the O-antigen of the lipopolysaccharide were no more sensitive than the wild-type strain to the extracts, whereas mutants defective in both lipopolysaccharide and exopolysaccharide were much more sensitive. The inhibitory activity was found at much higher levels in roots and nodules than in stems or leaves. Inoculation with either wild-type or polysaccharide-deficient R. etli did not appear to affect the level of activity. Sequential extractions of the crude root material with petroleum ether, ethyl acetate, methanol, and water partitioned inhibitory activity into each solvent except methanol. The major inhibitors in the petroleum ether and ethyl acetate extracts were purified by C18 high-performance liquid chromatography. These compounds all migrated very similarly in both liquid and thin-layer chromatography but were distinguished by their mass spectra. Absorbance spectra and fluorescence properties suggested that they were coumestans, one of which had the mass spectrum and nuclear magnetic resonances of coumestrol. These results are discussed with regard to the hypothesis that one role of rhizobial polysaccharides is to protect against plant toxins encountered during nodule development

Isolation of Phaseolus Vulgaris Compounds Inhibitory to Rhizobium Leguminosarum Biovar Phaseoli

The role that lipopolysaccharide plays in the infection process between rhizobia bacteria and bean has not been determined. One possible role is to protect the bacteria against compounds encountered during the infection process. As one test of this hypothesis, crude extracts derived from Phaseolus vulgaris bean roots have been tested for inhibition of the growth of Rhizobium leguminosarum biovar phaseoli wild type and mutant strains. Five day old bean roots have been extracted in hot 95% ethanol, filtered and concentrated. Mutant deficient in the 0-antigen of the lipopolysaccharide (Ops), while defective in nodulation, were no more sensitive than the wild type strain to compounds in the crude extract. Previous data had shown that a mutant defective in EPS (Exo) was also no more affected by the crude extract than the wild type (Jeanett Perez-Lesher, unpublished). However, a mutant defective in both LPS and EPS (Ops, Exo) was more strongly inbihited by the root extract than the wild type. Since there is some evidence that bacterial EPS is not synthesized during infection, it is suggested that a defect in the LPS of the bacteria could be shielded by the EPS ex planta but not within the infection thread. Sequential extractions of the crude root material with petroleum ether, ethyl acetate, methanol and water partitioned strong inhibitory activity into the petroleum ether, ethyl acetate and water fractions. The ethyl acetate fraction exhibited substantially more inhibitory activity toward the double mutant than to the wild type, and this activity was mainly confined to a single peak after high performance liquid chromatography (HPLC). Varying HPLC conditions allowed for purification of one of the constituents comprising this peak. The ultraviolet spectrum and fluorescence properties of the isolated compound are typical of a subclass of isoflavonoid compounds known as coumestans. Mass spectral, nuclear magnetic resonance data and relative chromatographic migration on TLC and HPLC suggest that the compound is coumestrol, a coumestan known to be present in legume roots