Effects of pH, Ca and Al on the exudation from clover seedlings of compounds that induce the expression of nodulation genes in Rhizobium trifolii

The expression of nodulation genes in R. trifolii is induced by flavone compounds present in clover root exudates. In the present experiments a bioassay with an indicator strain of R. trifolii, which contained the lacZ gene from Escherichia coli fused to the R. trifolii nodA gene, was used to measure the level of nod gene expression in R. trifolii. Compounds that stimulated nodA gene expression were shown to be present in exudates of white clover (Trifolium repens L.) and nine cultivars of subterranan clover (T. subterraneum L.) seedling sgrown at a range of pH between pH 3.0 and pH 8.0. The nod gene-induction activity of exudates was, however, reduced when seedlings of all clover species were grown at pH>7.0 and at pH<4.0 and pH<5.0 for white clover and subterranean clover respectively. No major differences were apparent in the activity of exudates from seedlings of the various cultivars of subterranean clover. Nod gene-induction activity of exudates was shown to increase markedly with seedling age. The presence of Ca at concentrations up to 10 m M in seedling culture solutions also resulted in marked increases in the nod gene-induction activity of seedling exudates. Increases in activity due to the presence of Ca were most apparent at low pH where between 5 and 10-fold increases were observed for white clover and subterranean clover respectively. Conversely, the presence of Al at concentrations up to 60 μM in seedling culture solutions had no effect on the nod gene-induction activity of seedling exudates. The observations that both low pH and Ca concentrations affected the nod gene-induction activity of seedling exudates suggested that the net presence of stimulatory flavones in root exudates was an important contributing factor to the 'acid-sensitive' step in nodule formation.