Genomics-informed multiplex PCR scheme for rapid identification of rice-associated bacteria of the genus Pantoea

The genus Pantoea forms a complex of more than 25 species, among which several cause diseases of various crop plants, including rice. Notably, strains of Pantoea ananatis and Pantoea stewartii have been repeatedly reported to cause bacterial leaf blight of rice, whereas 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. Using 32 whole genome sequences of the three major plant-pathogenic Pantoea species, a set of PCR primers that detect each of the three species, P. agglomerans, P. ananatis, and P. stewartii, was designed. A multiplex PCR scheme 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, 607 suspected Pantoea strains that were isolated from rice leaves or seeds originating from eleven 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 218 unresolved 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 0.5 ng/μl of total genomic DNA and 1×10^4 CFU/ml of heated cells. This new molecular diagnostic tool will help accurately diagnosing 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.