Genomics-Informed Multiplex PCR Scheme for Rapid Identification of Rice-Associated Bacteria of the Genus <i>Pantoea</i>

Abstract

The genus <i>Pantoea</i> forms a complex of more than 25 species, among which several cause diseases of various crop plants, including rice. Notably, strains of <i>Pantoea ananatis</i> and <i>P. stewartii</i> have been repeatedly reported to cause bacterial leaf blight of rice, whereas other authors have observed that <i>P. agglomerans</i> can also cause bacterial leaf blight of rice. The contribution of these and perhaps other species of <i>Pantoea</i> 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 <i>Pantoea</i> spp., a set of PCR primers that detect each of the three species <i>P. agglomerans</i>, <i>P. ananatis</i>, and <i>P. stewartii</i> was designed. A multiplex PCR scheme which can distinguish these three species and also detects members of other <i>Pantoea</i> spp. was further developed. Upon validation on a set of reference strains, 607 suspected <i>Pantoea</i> strains that were isolated from rice leaves or seed originating from 11 African countries were screened. In total, 41 <i>P. agglomerans</i> strains from 8 countries, 79 <i>P. ananatis</i> strains from 9 countries, 269 <i>P. stewartii</i> strains from 9 countries, and 218 unresolved <i>Pantoea</i> strains from 10 countries were identified. The PCR protocol allowed detection of <i>Pantoea</i> bacteria grown in vitro, in planta, and in rice seed. The detection threshold was estimated as total genomic DNA at 0.5 ng/µl and heated cells at 1 × 10⁴ CFU/ml. This new molecular diagnostic tool will help to accurately diagnose major plant-pathogenic species of <i>Pantoea</i>. 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.