Defective infection and nodulation of clovers by exopolysaccharide mutants of Rhizobium leguminosarum bv trifolii

The biological and symbiotic properties of two types of exopolysaccharide deficient (Exo(-) phenotype) mutants of Rhizobium leguminosarum were investigated. One mutant, a pss1 derivative (ANU437) of R. I. by. trifolii, produces very low levels of acidic exopolysaccharide (EPS) and of the acidic oligosaccharide (AOS) repeating unit lacking its O-acetyl substituent. The second strain, originally a R. I. by. viciae derivative, but containing a clover pSym plasmid (Exo-l mutant ANU54(pBRIAN)), did not produce any EPS polymer or detectable oligosaccharide repeating unit. The Exo(-) mutants formed no capsules and their growth in laboratory media was comparable to their parent strains but was inhibited by the addition of the phytoalexins medicarpin and kievitone, but not by the phytoalexin pisatin. Both Exo(-) mutants were slow to induce clover root hair curling (Hac(+)), and formed growing infection threads (Inf(-)) only on white clovers. However, the extent of these early symbiotic defects depended on the plant growth conditions used. Both mutants induced small non-nitrogen-fixing (Fix(-)) noduies on white and subterranean clovers. Within the white clover nodules induced by mutant ANU437, bacteria were released into plant cells. On white I:lovers at elevated light and temperature conditions, the Exo-l mutant ANU54(pBRIAN) usually failed to initiate infection threads and thus formed empty nodules which do not contain any bacteria In addition, this mutant formed only empty nodules without infection threads on subterranean clovers under all growth conditions tested. The outermost layers of plant cells in these empty nodules had thickened cell walls and deposited materials. These experiments show that EPS synthesis is essential for the induction of rapid root hair curling, proper infection thread formation and the ability to sustain the growth of induced nodules on various clovers.