Calcium and acid stress interact to affect the growth of Rhizobium leguminosarum bv. trifolii

The effect of acidity on the growth and survival of six strains of Rhizobium leguminosarum bv. trifolii (WU95, NA3001, WSM409, TA1, NA3025 and NA3039) was studied. Acid conditions reduced the growth rate of all strains; mean generation times were 50–60% slower at pH 5.0 than at pH 7.0. The critical pH for growth on solid media was in the range of 4.3–4.6. This is consistent with growth of the strains at different pHs in liquid culture, with NA3001 being the only strain to exhibit a normal growth pattern at pH 4.5. The interaction of acidity and calcium on the growth and survival of three of the strains (WU95, WSM409 and TA1) was studied in the presence of high (300 μm) and low (20 μm) concentrations of phosphate. A region of “acid-stress” somewhere below pH 5.0 was observed where growth rate slowed rapidly over 0.2–0.3 of a pH unit. The presence of 300 μm phosphate did not affect the critical pH for growth or growth rate within the “acid-stress” zone, but did reduce the mean generation time of all strains studied at pH above the “acid-stress” zone. At pH 7.0, increasing calcium from 300 μm to 3000 μm had little effect on growth rate, but high calcium increased growth rate within the “acid-stress” zone and enabled growth at a lower pH than that observed with the low calcium concentrations. A four-zone model for the response of root nodule bacteria to acidity is proposed.