The effect of aromatic metabolism on the competitiveness and persistence of Rhizobium legumino sarum biovar trifolii WU95

Fiona Rynne

The work in this thesis investigates the effect of the ability to metabolise some aromatic compounds on the competitiveness and persistence of Rhizobium leguminosarum bv. trifolii WU95. A prerequisite for the study was therefore the isolation of mutants defective in the protocatechuate pathway, the sole route for aromatic catabolism in WU95. Five mutants (MNF9010-9014) unable to metabolise 4-hydroxybenzoate (4-HB') were isolated using transposon Tn5-233 mutagenesis. All of these mutants were similar to the wild-type in growth rates on glucose as the sole carbon source motility and symbiotic characteristics on subterranean clover {Trifolium subterraneum cv Seaton Park). However, the growth rates of three of these mutants (MNF9011, MNF9012 and MNF9014) were lower than the wild type on media containing glucose plus 4-hydroxybenzoate. The five 4-HB' mutants were each blocked at different sites in the protocatechuate pathway and could therefore be used to examine its regulation. The structural genes for the pathway were induced by protocatechuate (protocatechuate 1,2 dioxygenase) (pcaA), B-carboxy cis-cis-muconate (B-carboxy cis-cis muconate lactonising enzyme, B-carboxymuconate decarboxylase and enol-lactone hydroxylase (pcaBCD) and 3-oxoadipate (transferase) (pcaE), a novel regulation system not previously reported. In the course of this work spontaneous antibiotic-resistant mutants of WU95 were isolated which were identical to the wild-type in all major growth and symbiotic characteristics but their competitiveness was dramatically reduced compared to the wild-type. These data point to the importance of full characterisation of antibiotic-resistant mutant strains prior to their use in field trials. The survival characteristics of three 4-HB' strains (MNF9010, MNF9011 and MNF9013) were further examined in glasshouse and field trials. Mixed inocula containing a 4-HB' mutant plus WU95 were used to inoculate clover plants (in untreated yellow sand or Medina soil) or to inoculate native soil directly. The competitiveness and the persistence (over 6 months) of the 4-HB' mutants were found to be identical to that of the wild-type. Field trials were performed at two sites (Medina and Wooroloo) for three and two years respectively. Two types of experimental plots were used to investigate the competitiveness and persistence (saprophytic competence and cross-row plots) and also the spread (cross-row plots) of the wild-type and the 4-HB' mutants. The data from the field trials correlate quite well with the glasshouse results and the data are similar at the two sites. The results suggest that the 4-HB' mutants perform as well as the wild-type except for the cross-row experiments in the third year. The conclusion from both glasshouse and field trials is that the ability to metabolise aromatic compounds does not affect competitiveness and if it has any effect on persistence this is only observed when the rhizobial populations are low.

The effect of aromatic metabolism on the competitiveness and persistence of Rhizobium legumino sarum biovar trifolii WU95

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