Effects of pH and aluminium on nodulation and growth of Trifolium subterraneum cultivars

Abstract

Effects of pH and aluminium on Trifolium subterraneum (subterranean clover), Rhizobium trifolii, and the symbiotic association between them were examined in solution culture experiments.The growth of 11 cultivars of subterranean clover was independent of pH over the range 4.0 to 6.5 when they were grown in flowing solution culture with adequate inorganic nitrogen. However, a solution pH of 3.5 was lethal to all cultivars.Acid tolerance of 14 strains of R. trifolii was evaluated using a defined liquid medium. All grew at ≥ pH 5.0, four grew at 4.5, and none grew at pH 4.0. Strains NA25-3 and UT13 were identified as the most acid-tolerant. Growth of R. trifolii in the rhizosphere of subterranean clover plants grown in non-sterile nutrient solutions was then examined using an antibiotic-resistant selection (RSP-24) of NA25-3. RSP-24 was more sensitive to acidity (pH 4.5) in the rhizosphere than when grown in the defined liquid medium. The lack of a readily available energy source in the rhizosphere was considered to be partially responsible for the enhanced acid sensitivity.When cultivars of subterranean clover were grown in flowing solution culture without nitrogen, they differed in their nodulation and growth responses to pH. All plants of the 14 cultivars tested were able to form nodules at pH 4.5 to 6.0. At pH 4.0, seven cultivars formed a few nodules, while the remainder formed none. In the cultivar Dwalganup, nodule number and dry weight and whole plant dry weight were independent of pH over the range 4.5 to 6.5, whereas in cultivar Clare, these attributes were reduced at pH ≤ 5.0. In the remaining cultivars, nodule numbers were reduced at pH 4.5, while nodule dry weights were reduced only in cultivars Bacchus Marsh, Daliak, Dinninup and Howard. However, the reduction in whole plant dry matter yield at pH 4.5, in all cultivars except Dwalganup, suggests that nodule function was impaired at this pH. A further flowing solution culture experiment showed that the 14 cultivars of subterranean clover were less tolerant of aluminium at concentrations ≥ 25 µM, when grown with adequate inorganic nitrogen, than had been previously reported. The higher ionic activities of monomeric aluminium species in the nutrient solutions of low ionic strength used in this experiment were considered to be mainly responsible for the lower tolerance of the cultivars to aluminium. The cultivars Trikkala, Larisa, Clare, Daliak and Dinninup were less tolerant to aluminium than nine other cultivars.Subsequent experiments showed that solution aluminium concentrations as low as 6.3 µM inhibited the growth of R. trifolii in defined nutrient media, plant culture solutions, and in the rhizosphere of subterranean clover plants.Static solution culture experiments with dilute, nitrogen-free nutrient solutions showed that nodulation of 13 cultivars of subterranean clover was even more sensitive than growth of host plants to aluminium. Aluminium at 6.3 and 12.5 µM delayed nodule appearance, reduced the proportion of plants which nodulated, and reduced nodule number and dry weight per nodulated plant, whereas root length and shoot and root dry weight of all cultivars were unaffected. The extent of the depressive effect of aluminium on nodulation varied among cultivars. At 12.5 µM aluminium, all plants of the cultivar Tallarook nodulated, whereas no plants of the cultivars Clare, Woogenellup, Dwalganup, Daliak and Seaton Park nodulated. Nodule dry weight per nodulated plant was only minimally reduced in cultivar Tallarook, Howard and Geraldton at 12.5 µM aluminium, whereas substantial decreases were observed in the other cultivars. Aluminium at low concentrations (6.3, 12.5 µM) reduced the density of root hairs and caused root hair deformities particularly in those cultivars in which nodulation was most severely inhibited.Solution aluminium concentrations similar to those observed in acid soils limited nodulation and growth of subterranean clover more severely than acidity at pH 4.5. The finding that cultivars differed in their tolerance to acidity only when inoculated with R. trifolii, together with the lack of a relationship between aluminium tolerance rankings of inoculated and nitrogen-fed plants indicates that plants supplied with adequate inorganic nitrogen cannot be used to evaluate the acid-aluminium tolerance of field-grown subterranean clover.