Symbiotic characteristics and genetic diversity of rhizobia nodulating some native leguminous trees and shrubs

Abstract

The nodulation and nitrogen fixation potential of 157 strains of rhizobia isolated from 11 native legumes (six Acacia spp.), selected for revegetation of waste rock dumps of Kakadu National Park, Northern Territory, Australia, as studied in glasshouse eXPeriments. Growth properties, symbiotic effectiveness and host range of the various strains (designated as ARR, for Alligator Rivers Region) were determined. The genetic diversity of representative strains within the collection was analysed using phenotypic and genotypic approaches. Carbon substrate utilisation, intrinsic antibiotic resistance, pH change of growth media and tolerance to NaCI were examined. Multilocus enzyme electrophoresis (MLEE) was performed and partial analysis of 165 rRNA gene sequence of a 220-base pair 5' region corresponding to positions 29 to 249 of the Escherichia coli sequence, was determined from polymerase chain reaction (PCR) amplified DNA. These data were used to determine the phylogenetic relatedness of the rhizobia! strains nodulating the native legumes. The rhizobial isolates exhibited a wide range of symbiotic effectiveness ranging from being fully effective to totally ineffective as compared to either Nitrogen fed or uninoculated control plants, respectively. Growth rate category of the isolates had no apparent relationship to symbiotic effectiveness. Acacia holosericea Cunn. ex Benth., Chamaecrista mimosoides L. were effectively nodulated by the highest proportion of homologous isolates (i.e. strains originally isolated from that species) and A. holosericea was the most promiscuous host with non-homologous isolates. In contrast, Crotalaria medicaginea Lam. was the most selective in its rhizobial requirements, with the homologous strains ARR661 and ARR681 performing equal to that of the nitrogen fed plants. Tn general, all of the isolates obtained from Acacia spp. showed broader host range than the non-Acacia isolates. Based on the results from this study an appropriate host species by rhizobial strain combination was identified for each of the 11 leguminous host plants that exhibited potential for use in the revegetation of mine soils in KNP. Phenotypic characteristics carried out with 45 isolates selected on the basis of their symbiotic effectiveness on their homologous hosts were subjected to a principalcoordinate analysis which classified the strains into three distinct groups which was consistent with their growth rate categories of fast growing, slow growing and veryslow growing. MLEE analysis of the isolates at 13 enzyme loci, revealed a high level of genetic diversity (mean diversity per locus of 0.81) within the strains with 85% of strains differing at 9 or more of the 13 loci. Cluster analysis of the enzyme electrophoresis data confirmed the high level of diversity among isolates and suggested that the population consisted of a genetically diverse group, constituted by different species and genera. Phylogenetic relationships, as determined by partial analysis of 165 rRNA gene sequence from 14 representative strains, were determined by comparison of DNA sequence with previously published sequences of the family Rhizobiaceae. The sequence of a fast growing strain (ARR661) showed 98.6% homology to Rhizobium huakii and differed by up to 68 nucleotides (from a possible 220) from other ARR strains. Four strains had sequence homology ranging from 99.1% to 99.5% to Bradyrhizobium spp. and therefore clustered within the Bradyrhizobium-Rhodopseudomonas-Agronomas- Blastobacter rRNA complex. The remaining 9 ARR strains had sequence similarity of only 71% to 90% to Bradyrhizobium spp. and thus confirmed the very high level of diversity evident within this collection of rhizobia. However, these strains shared with Bradyrhizobium a set of unique 'signature' nucleotide sequences which were not present in sequences of strains in the Rhizobium, Azorhizobium or Sino rhizobium groups. On the basis of this results and other nucleotide comparisons, these strains are proposed to belong to a new genus tentatively assigned as the, Pseudo-bradyrhizobium. Moreover, within this group of rhizobia it is suggested on the basis of partial 165 rRNA sequence diversity that several species may be represented. However, further molecular and phenotypic studies are required before specific taxonomic designations are validly assigned to these species.