Genetic diversity and symbiotic effectiveness of rhizobium isolated from perennial trifolium species
Creators: name: Tesfaye, Mesfin
Publisher: name: University of British Columbia
Year: 2009

description: Soil bacteria that form functional nodules on the genus Trifolium (clover), a taxa of about 240 plant species, are included in a single species, Rhizobium leguminosarum bv trifolii. The taxonomic criterion for this bacterial group is their ability to interact symbiotically with a narrow range of plant species originating from temperate regions, although several effectiveness groups which differ in their cross-inoculation patterns have been described. T. semipilosum Fresen (Kenya white clover), is one of the perennial clover species of agricultural importance in tropical and sub-tropical farming systems. The objectives of this study were to use nodulation characteristics, substrate utilization patterns and DNA analyses to determine the phylogenetic relationships of Rhizobium isolated from temperate and tropical perennial Trifolium species. Nodulation and symbiotic effectiveness of compatible and incompatible bacterial strains were investigated using the T. semipilosum host. The symbiotic association of 22 R. I. bv trifolii strains appeared to be highly specific: no single R. I. bv trifolii strain was able to nodulate all six perennial Trifolium species included as hosts in cross-inoculation experiments. Bacterial strains that were effective on temperate perennial species including T. repens, T. pratense, T. hybridum and T. fragiferum produced NodVFix" phenotypes on T. semipilosum and vice versa. This anomalous nodulation was characterized by the formation of significantly greater numbers of nodules, which were white and variable in size, but generally smaller than wild-type nodules. Substrate utilization analysis using the Biolog™ system suggested that R. I. bv trifolii strains effective on T. semipilosum may have a broader metabolic profile than strains effective on other Trifolium species. Genetic relationships of R. I. bv trifolii strains were obtained by DNA analyses using four polymerase chain reaction (PCR) based techniques: Randomly Amplified Polymorphic DNA (RAPD-PCR), Enterobacterial Repetitive Intergeneric Consensus (ERIC-PCR), and PCR- based nucleotide sequence analysis of 16S and 23S rDNA regions. A considerable level of genetic diversity was found using RAPD- and ERIC-PCR. Rhizobium I. bv trifolii strains that are effective on the tropical perennial clover, T. semipilosum, formed a tight cluster, especially with ERIC-PCR, that was distinct from R. I. bv trifolii effective on temperate hosts. 16S rDNA nucleotide sequences were found to be highly conserved among R. I. bv trifolii. Comparative nucleotide sequence analysis of 23S rDNA regions clustered R. I. bv trifolii effective on T. semipilosum, T. repens, T. pratense, T. hybridum and T. fragiferum into two distinct groups, which were consistent with the pattern of symbiotic effectiveness observed in cross-inoculation experiments. The DNA sequences used as PCR primers for 23S rDNA analysis were found to be conserved among a wide range of rhizosphere bacterial species. Unique features identified by secondary structure analysis of the sequenced 23S rDNA region were used to design two 20-bp primers that provided group-specific differentiation and detection of Rhizobium by PCR. Detailed analyses using extracted DNA from many rhizosphere bacterial species confirmed the ultimate value of such groupspecific primers for phylogenetic and ecological analyses. In compatible interactions, both T. semipilosum and T. repens were infected via root hairs. Furthermore, the anatomy of nodules induced by effective strains on T. semipilosum was analogous to that reported for other indeterminate nodules including T. repens. Nodules induced by effective strains on T. semipilosum were localized near the upper region of the tap root where fewer root hairs are located; nodules of T. repens were distributed largely on lateral roots. A Rhizobium strain ANU843, effective on T. repens, caused root hair branching and twisting on T. semipilosum, although infection threads were not detected in any of the plants examined. Microsymbionts for T. semipilosum and T. repens were transformed with a constitutively expressed gusA gene to provide a visual assay of rhizobial infection and nodulation. Strain ANU843 was shown to enter the root system of T. semipilosum mainly at the epidermal sites of emerging lateral roots. However, only 33% of the nodules from this incompatible interaction showed a positive GUS reaction. Of these, 70% were localized at the junction between the tap and lateral roots.