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PCR assay for direct specific detection of Bradyrhizobium elite strain BR 3262 in root nodule extracts of soil-grown cowpea

Ophelia Osei, Jean L. Simões Araújo, Jerri E. Zilli, Robert M. Boddey, Benjamin D. K. Ahiabor, Robert C. Abaidoo, Luc F. M. Rouws
Plant and soil 2017 v.417 no.1-2 pp. 535-548
Bradyrhizobium, DNA, Rhizobium, aseptic conditions, bacteria, cowpeas, crops, genome, germ-free animals, nucleotide sequences, polymerase chain reaction, root nodules, soil
AIMS: Successful inoculation of legume crops with rhizobia depends on dominating nodule occupancy with highly efficient strains. The aim of this study was to develop a rapid and reliable conventional PCR methodology to specifically detect an elite Bradyrhizobium strain in root nodule extracts from soil-grown cowpea plants. METHODS: The draft genome sequence of Bradyrhizobium pachyrhizi BR 3262 was compared to the closely related strain PAC 48ᵀ. BR 3262-specific regions were selected to design specific primer pairs, which were tested with respect to PCR amplification specificity and efficiency on extracted DNA, bacterial cells and root nodules from cowpea plants grown under gnotobiotic conditions and in soil. RESULTS: Eleven designed primer pairs were specific for BR 3262 amplification and two of them (pairs 2645 and 2736) were highly sensitive and selected for further analyses. Experiments with gnotobiotic and soil-grown plants showed that both primer pairs were suitable to reliably determine nodule occupancy and confirmed the competitiveness of strain BR 3262 in natural soil. CONCLUSIONS: Primer pairs 2645 and 2736 are novel tools to accompany the fate of strain BR 3262 in inoculation experiments of cowpea in soil. This strategy should be applicable to other rhizobium/legume symbioses in the field.