Validation of species-specific PCR assays for the detection of Pantoea ananatis, P. agglomerans, P. allii and P. stewartii

Species of Pantoea represent a group of plant pathogenic bacteria that infect a variety of agro-economically important plant species. Among these, a complex of P. ananatis, P. allii, P. agglomerans, and P. stewartii subsp. indologenes cause Center rot in onion, resulting in significant economic losses. As species of Pantoea are phenotypically closely related, identification of Pantoea species relies on the sequencing and phylogenetic analysis of housekeeping genes. To aid in rapid identification of Pantoea species, efforts have been made in developing species-specific primers to be used in PCR assays. In the current study, two P. ananatis, one P. allii, one P. agglomerans and three P. stewartii published primers as well as newly developed P. agglomerans PagR primers were evaluated for their specificity against 79 Pantoea strains, belonging to 15 different species. To ensure that selected primers are evaluated against accurately identified species, sequencing and phylogenetic analysis of housekeeping gene infB was conducted thereafter PCR assays using selected species-specific primers were performed. The results showed that previously described P. ananatis-specific PANA_1008, P. allii-specific allii-leuS, and P. stewartii-specific PANST_rpoB, 3614galE, and DC283galE primers and one newly designed P. agglomerans-specific PagR primer pairs were highly specific for their target Pantoea species and accurately identified these strains into their species and, in some cases, sub-species level. The findings of the current study will facilitate rapid and reliable identification of P. ananatis, P. agglomerans, P. allii and P. stewartii.