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

Abstract

AbstractBackgroundThe genusPantoeaforms a complex of more than 25 species, among which several cause diseases of several crop plants, including rice. Notably, strains ofPantoea ananatisandPantoea stewartiihave been found to cause bacterial leaf blight of rice in Togo and Benin, while other authors have observed thatPantoea agglomeranscan also cause bacterial leaf blight of rice. The contribution of these and perhaps other species ofPantoeato plant diseases and yield losses of crop plants is currently not well documented, partly due to the lack of efficient diagnostic tools.ResultUsing 34 whole genome sequences of the three-major plant-pathogenicPantoeaspecies, a set of PCR primers that specifically detect each of the three species,P. agglomerans,P. ananatis, andP. stewartii, was designed. A multiplex PCR protocol which can distinguish these three species and also detects members of otherPantoeaspecies was further developed. Upon validation on a set of reference strains, 609 suspectedPantoeastrains that were isolated from rice leaves or seeds originating from 11 African countries were screened. In total, 41P. agglomeransstrains from eight countries, 79P. ananatisstrains from nine countries, 269P. stewartiistrains from nine countries and 220 unsolvedPantoeastrains from ten countries were identified. The PCR protocol allowed detectingPantoeabacteria grown in vitro, in planta and in rice seeds. The detection threshold was estimated at 5 ng/mL of total genomic DNA and 1 × 105CFU/mL of heated cells.ConclusionThis new molecular diagnostic tool will help accurately diagnose major plant-pathogenic species ofPantoea. 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.