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

International audienceThe 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 P. stewartii have been repeatedly reported to cause bacterial leaf blight of rice, whereas other authors have observed that P. 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 spp., 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 spp. was further developed. Upon validation on a set of reference strains, 607 suspected Pantoea strains that were isolated from rice leaves or seed originating from 11 African countries were screened. In total, 41 P. agglomerans strains from 8 countries, 79 P. ananatis strains from 9 countries, 269 P. stewartii strains from 9 countries, and 218 unresolved Pantoea strains from 10 countries were identified. The PCR protocol allowed detection of Pantoea 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 × 10 4 CFU/ml. This new molecular diagnostic tool will help to 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

Genome Resources of Three West African Strains of Pantoea ananatis Causing Bacterial Blight and Grain Discoloration of Rice

International audienceMembers of the genus Pantoea have been reported as pathogens for many economically important crops, including rice. Little is known about their host−pathogen interactions at the molecular level and the lack of comprehensive genome data impedes targeted breeding strategies toward resistant rice cultivars. Here, we describe the structural and functional annotation of the draft genome sequences of three rice-pathogenic Pantoea ananatis strains, ARC272, ARC310, and ARC311, which were isolated in Burkina Faso, Togo, and Benin, respectively. The genome sequences of these strains will help in developing molecular diagnostic tools and provide new insight into common traits that may enable P. ananatis to infect rice

Protocol for RYMV Inoculation and Resistance Evaluation in Rice Seedlings

International audienceRice yellow mottle virus (RYMV), a mechanically transmitted virus that causes serious damage to cultivated rice plants, is endemic to Africa. Varietal selection for resistance is considered to be the most effective and sustainable management strategy. Standardized resistance evaluation procedures are required for the identification and characterization of resistance sources. This paper describes a protocol for mechanical inoculation of rice seedlings with RYMV and two methods of resistance evaluation - one based on a symptom severity index and the other on virus detection through double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA)

Fine mapping of RYMV3: a new resistance gene to Rice yellow mottle virus from Oryza glaberrima

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Design of a new multiplex PCR assay for rice pathogenic bacteria detection and its application to infer disease incidence and detect co-infection in rice fields in Burkina Faso

International audienceCrop diseases are responsible for considerable yield losses worldwide and particularly in sub-Saharan Africa. To implement efficient disease control measures, detection of the pathogens and understanding pathogen spatio-temporal dynamics is crucial and requires the use of molecular detection tools, especially to distinguish different pathogens causing more or less similar symptoms. We report here the design a new molecular diagnostic tool able to simultaneously detect five bacterial taxa causing important diseases on rice in Africa: (1) Pseudomonas fuscovaginae, (2) Xanthomonas oryzae, (3) Burkholderia glumae and Burkholderia gladioli, (4) Sphingomonas and (5) Pantoea species. This new detection tool consists of a multiplex PCR, which is cost effective and easily applicable. Validation of the method is presented through its application on a global collection of bacterial strains. Moreover, sensitivity assessment for the detection of all five bacteria is reported to be at 0.5 ng DNA by μl. As a proof of concept, we applied the new molecular detection method to a set of 256 rice leaves collected from 16 fields in two irrigated areas in western Burkina Faso. Our results show high levels of Sphingomonas spp. (up to 100% of tested samples in one field), with significant variation in the incidence between the two sampled sites. Xanthomonas oryzae incidence levels were mostly congruent with bacterial leaf streak (BLS) and bacterial leaf blight (BLB) symptom observations in the field. Low levels of Pantoea spp. were found while none of the 256 analysed samples was positive for Burkholderia or Pseudomonas fuscovaginae. Finally, many samples (up to 37.5% in one studied field) were positive for more than one bacterium (co-infection). Documenting co-infection levels are important because of their drastic consequences on epidemiology, evolution of pathogen populations and yield losses. The newly designed multiplex PCR for multiple bacterial pathogens of rice is a significant improvement for disease monitoring in the field, thus contributing to efficient disease control and food safety

Structure of African Populations of Pyricularia oryzae from Rice

International audienceRice blast, caused by the filamentous ascomycete Pyricularia oryzae, is one of the most devastating diseases of rice. Four genetic clusters were previously identified, and three have a large geographic distribution. Asia is the center of diversity and the origin of most migrations to other continents, and sexual reproduction persisted only in the South China-Laos-North Thailand region, which was identified as the putative center of origin of all P. oryzae populations on rice. Despite the importance of rice blast disease, little is known about the diversity and the population structure of the pathogen in Africa (including Madagascar). The present study was intended to describe the structure of African populations of P. oryzae and identify the relationship between African and worldwide genetic clusters. A set of 2,057 strains (937 African and 1,120 Madagascan strains) were genotyped with 12 simple sequence repeat markers to assess the diversity and the population structure of P. oryzae. Four genetic clusters were identified in Africa and Madagascar. All four clusters previously identified are present in Africa. Populations from West Africa, East Africa, and Madagascar are highly differentiated. The geographic structure is consistent with limited dispersion and with some migration events between neighboring countries. The two mating types are present in Africa with a dominance of Mat1.2, but no female-fertile strain was detected, supporting the absence of sexual reproduction on this continent. This study showed an unsuspected high level of genetic diversity of P. oryzae in Africa and suggested several independent introductions