A diagnostic multiplex PCR scheme for identification of plant-associated bacteria of the genus Pantoea

Abstract Background The genus Pantoea forms a complex of more than 25 species, among which several cause diseases of several crop plants, including rice. Notably, strains of Pantoea ananatis and Pantoea stewartii have been found to cause bacterial leaf blight of rice in Togo and Benin, while other authors have observed that Pantoea agglomerans can also cause bacterial leaf blight of rice. The contribution of these and perhaps other species of Pantoea to plant diseases and yield losses of crop plants is currently not well documented, partly due to the lack of efficient diagnostic tools. Result Using 34 whole genome sequences of the three-major plant-pathogenic Pantoea species, a set of PCR primers that specifically detect each of the three species, P. agglomerans , P. ananatis , and P. stewartii , was designed. A multiplex PCR protocol which can distinguish these three species and also detects members of other Pantoea species was further developed. Upon validation on a set of reference strains, 609 suspected Pantoea strains that were isolated from rice leaves or seeds originating from 11 African countries were screened. In total, 41 P. agglomerans strains from eight countries, 79 P. ananatis strains from nine countries, 269 P. stewartii strains from nine countries and 220 unsolved Pantoea strains from ten countries were identified. The PCR protocol allowed detecting Pantoea bacteria grown in vitro, in planta and in rice seeds. The detection threshold was estimated at 5 ng/mL of total genomic DNA and 1 × 10 5 CFU/mL of heated cells. Conclusion This new molecular diagnostic tool will help accurately diagnose major plant-pathogenic species of Pantoea . Due to its robustness, specificity, sensitivity, and cost efficiency it will be very useful for plant protection services and for the epidemiological surveillance of these important crop-threatening bacteria.