Physical and biochemical characterization of a competition and nodulation defective mutant of Azorhizobium caulinodans ORS571

Abstract

The competitive ability of Azorhizobium caulinodans strain ORS57 1 was assessed using induced mutagenesis, which resulted in isolation of two mutants that were either defective in symbiotic properties or in competition for nodulation of Sesbania roscrata. One of the mutants was non-nodulating whereas the other one exhibited reduced competitiveness when inoculated along with the parent strain. Further characterization revealed that the non-nodulating mutant exhibited altered protein and lipopolysaccharide (LPS) profiles and the absence of flagella.One of the limitations in the practical realisation of legume-Rhizobium symbiotic nitrogen fixation technology is the Rhizobium competition problem -the failure of superior inoculant to occupy nodules in soils with a large population of indigenous Rhizobium. A number of strategies have been suggested to overcome the Rhizobium competition problem ranging from inoculation of plants with large numbers of Rhizobium (MEADE et al. 1985) over a long period to the stable transfer of genes involved in nodulation competitiveness to superior strains of Rhizobium (TRIPLET 1990).However, the basis of competitiveness among Rhizobium strain is largely unknown, and attempts to modify it by genetic manipulation have had varied results. Some Tn5 insertions in R. trijolii that reduce competitiveness are correlated with loss of motility or lack of trifoliotoxin production (TRIPLET and BATRA 1987). Chromosomal deletions or insertions in cryptic plasmids of R. meliloti have been shown to reduce strain competitiveness (HAHN and HENNECKE 1988). Similarly, competition defective mutants of R. fredii have been generated by Tn5 mutagenesis (MCLOUGHLIN et al. 1987). In R. loti, however, removal of a cryptic plasmid has been shown to increase competitiveness (PANKHURST et al. 1986). It has also been correlated with exopolysaccharide (EPS) production in R. fredii where induced mutants having reduced EPS production were competitively superior to the wild type of Bradyrhizobiurn (ZDOR and PUEPPKE 199 1).For the development of efficient inoculants, the most important issue is the selection and identification of a competitively superior inoculum strain. A number of tagging markers used for strain identification includes cultural characteristics, serological properties, intrinsic antibiotic resistance, phage typing, probes ("*P-labelled genomic DNA probes) of n8fi.x and nod genes; enzyme polymorphism, and SDS-PAGE protein and LPS patterns. All these techniques employed for determining nodule occupancy have one or the other practical limitation and the Rhizobium competition problem is still a major obstacle in the development of superior inoculants.Tn5 (carrying gusA (P-glucuronidase) as a reporter gene) has been used as a marker for Rhizobium in a wide range of studies (WILLSON et al. 1991). In the present study we describe the use of a Tn5 gusA marker system for assessing the competitive ability of Azorhizobium caulinoduns strain ORS57 1 with respect to other Rhizobium strains belonging to the genus Sesb...