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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.

12 13 Background: The genus Pantoea forms a complex of more than 25 species, among which 14 several cause diseases of several crop plants, including rice. Notably, strains of Pantoea 15 ananatis and Pantoea stewartii have been found to cause bacterial leaf blight of rice in Togo 16 and Benin, while other authors have observed that Pantoea agglomerans can also cause 17 bacterial leaf blight of rice. The contribution of these and perhaps other species of Pantoea to 18 plant diseases and yield losses of crop plants is currently not well documented, partly due to 19 the lack of efficient diagnostic tools. 20 21 Result: Using 34 whole genome sequences of the three-major plant-pathogenic Pantoea 22species, a set of PCR primers that specifically detect each of the three species, 23 P. agglomerans, P. ananatis, and P. stewartii, was designed. A multiplex PCR protocol which 24 can distinguish these three species and also detects members of other Pantoea species was 25 2 further developed. Upon validation on a set of reference strains, 609 suspected Pantoea 26 strains that were isolated from rice leaves or seeds originating from 11 African countries were 27 screened. In total, 41 P. agglomerans strains from eight countries, 79 P. ananatis strains from 28 nine countries, 269 P. stewartii strains from nine countries and 220 unsolved Pantoea strains 29 from ten countries were identified. The PCR protocol allowed detecting Pantoea bacteria 30 grown in vitro, in planta and in rice seeds. The detection threshold was estimated at 5 ng/mL 31 of total genomic DNA and 1×10 5 CFU/mL of heated cells. 32 33 Conclusion: This new molecular diagnostic tool will help accurately diagnose major plant-34 pathogenic species of Pantoea. Due to its robustness, specificity, sensitivity, and cost 35 efficiency it will be very useful for plant protection services and for the epidemiological 36 surveillance of these important crop-threatening bacteria. 37 38 39 41 3 Background 42 43 The genus Pantoea was first described in 1989 and was recently taxonomically classified as a 44 member of the Erwiniaceae family [1]. More than 25 species of this genus have been 45 described and reported worldwide [2,3]. Etymologically, the genus name Pantoea is derived 46 from the Greek word 'Pantoios', which means "of all sorts or sources" and reflects the diverse 47 geographical and ecological sources from which the bacteria have been isolated. Several 48 species of the genus are qualified as versatile and ubiquitous bacteria because they have been 49 isolated from many different ecological niches and hosts [2,4]. Remarkably, some species 50 have the ability to colonize and interact with members of both the plant and the animal 51 Kingdom [5]. Among the plant-interacting species, Pantoea ananatis, Pantoea agglomerans 52 and Pantoea stewartii are well known for their phytopathogenic characteristics. They are 53 recognized as the causal agent of several diseases, such as leaf blight, spot disease, dieback, 54 grain discoloration, seed stalk rot, center rot, stem necros...

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