Diversity and plant growth promoting ability of rice root-associated bacteria in Burkina-Faso and cross-comparison with metabarcoding data.

Plant-associated bacteria are essential partners in plant health and development. In addition to taking advantage of the rapid advances recently achieved in high-throughput sequencing approaches, studies on plant-microbiome interactions require experiments with culturable bacteria. A study on the rice root microbiome was recently initiated in Burkina Faso. As a follow up, the aim of the present study was to develop a collection of corresponding rice root-associated bacteria covering maximum diversity, to assess the diversity of the obtained isolates based on the culture medium used, and to describe the taxonomy, phenotype and abundance of selected isolates in the rice microbiome. More than 3,000 isolates were obtained using five culture media (TSA, NGN, NFb, PCAT, Baz). The 16S rRNA fragment sequencing of 1,013 selected isolates showed that our working collection covered four bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes) and represented 33% of the previously described diversity of the rice root microbiome at the order level. Phenotypic in vitro analysis of the plant growth promoting capacity of the isolates revealed an overall ammonium production and auxin biosynthesis capacity, while siderophore production and phosphate solubilisation were enriched in Burkholderia, Ralstonia, Acinetobacter and Pseudomonas species. Of 45 representative isolates screened for growth promotion on seedlings of two rice cultivars, five showed an ability to improve the growth of both cultivars, while five others were effective on only one cultivar. The best results were obtained with Pseudomonas taiwanensis ABIP 2315 and Azorhizobium caulinodans ABIP 1219, which increased seedling growth by 158% and 47%, respectively. Among the 14 best performing isolates, eight appeared to be abundant in the rice root microbiome dataset from previous study. The findings of this research contribute to the in vitro and in planta PGP capacities description of rice root-associated bacteria and their potential importance for plants by providing, for the first time, insight into their prevalence in the rice root microbiome.

Dynamics of the rice yellow mottle disease in western Burkina Faso: Epidemic monitoring, spatio-temporal variation of viral diversity, and pathogenicity in a disease hotspot.

The rice yellow mottle virus (RYMV) is a model in plant virus molecular epidemiology, with the reconstruction of historical introduction routes at the scale of the African continent. However, information on patterns of viral prevalence and viral diversity over multiple years at a local scale remains scarce, in spite of potential implications for crop protection. Here, we describe a 5-year (2015-9) monitoring of RYMV prevalence in six sites from western Burkina Faso (geographic areas of Bama, Banzon, and Karfiguela). It confirmed one irrigated site as a disease hotspot and also found one rainfed lowland (RL) site with occasional high prevalence levels. Within the studied fields, a pattern of disease aggregation was evidenced at a 5-m distance, as expected for a mechanically transmitted virus. Next, we monitored RYMV genetic diversity in the irrigated disease hotspot site, revealing a high viral diversity, with the current coexistence of various distinct genetic groups at the site scale (ca. 520 ha) and also within various specific fields (25 m side). One genetic lineage, named S1bzn, is the most recently emerged group and increased in frequency over the studied period (from 20 per cent or less in 2015-6 to more than 65 per cent in 2019). Its genome results from a recombination between two other lineages (S1wa and S1ca). Finally, experimental work revealed that three rice varieties commonly cultivated in Burkina Faso were not different in terms of resistance level, and we also found no significant effect of RYMV genetic groups on symptom expression and viral load. We found, however, that infection outcome depended on the specific RYMV isolate, with two isolates from the lineage S1bzn accumulating at the highest level at early infections. Overall, this study documents a case of high viral prevalence, high viral diversity, and co-occurrence of divergent genetic lineages at a small geographic scale. A recently emerged lineage, which comprises viral isolates inducing severe symptoms and high accumulation under controlled conditions, could be recently rising through natural selection. Following up the monitoring of RYMV diversity is required to confirm this trend and further understand the factors driving the local maintenance of viral diversity.

Genetic diversity and structure of Bipolaris oryzae and Exserohilum rostratum populations causing brown spot of rice in Burkina Faso based on genotyping-by-sequencing.

In recent years, Brown spot disease of rice (BSR) has been observed on leaves and seeds of rice in all rice-growing areas of Burkina Faso. Bipolaris oryzae and Exserohilum rostratum are the main fungal species isolated from BSR infected tissues and they are frequently observed in the same field. However, we are lacking information on the genetic diversity and population structure of these fungi in Burkina Faso. The mode of reproduction is also unknown. The genetic diversity of isolates of B. oryzae (n=61) and E. rostratum (n=151), collected from major rice-growing areas of Burkina Faso, was estimated using genotyping-by-sequencing (GBS). The mean values for nucleotide diversity (π) were 1.9 x10-4 for B. oryzae and 4.8 x10-4 for E. rostratum. There is no genetic differentiation between the geographical populations of each species. The analysis of molecular variance revealed that 89% and 94% of the genetic variances were within the populations of B. oryzae and E. rostratum, respectively. For each species, four genetic clusters were identified by two clustering methods (DAPC and sNMF). The distribution of these genetic groups was independent of the geographical origin of the isolates. Evidence of recombination was detected in the populations of B. oryzae and E. rostratum. For B. oryzae balanced mating type ratios were supporting sexual reproduction. For E. rostratum overrepresentation of MAT1-2 isolates (79%) suggested a predominant asexual reproduction. This study provides important information on the biology and genetics of the two major fungi causing brown spot disease of rice in Burkina Faso.

The impact of the rice production system (irrigated vs lowland) on root-associated microbiome from farmer's fields in western Burkina Faso.

Due to their potential applications for food safety, there is a growing interest in rice root-associated microbial communities, but some systems remain understudied. Here, we compare the assemblage of root-associated microbiota in rice sampled in 19 small farmer's fields from irrigated and rainfed lowlands in Burkina Faso, using an amplicon metabarcoding approach of the 16S rRNA gene (prokaryotes, three plant samples per field) and ITS (fungi, one sample per field). In addition to the expected structure by root compartments (root vs rhizosphere) and geographical zones, we showed that the rice production system is a major driver of microbiome structure. In irrigated systems, we found a higher diversity of prokaryotic communities from the rhizosphere and more complex co-occurrence networks, compared to rainfed lowlands, while fungal communities exhibited an opposite pattern (higher richness in rainfed lowlands). Core taxa were different between the two systems, and indicator species were identified: mostly within Bacillaceae in rainfed lowlands, and within Burkholderiaceae and Moraxellaceae in irrigated areas. Finally, a higher abundance in rainfed lowlands was found for mycorrhizal fungi (both compartments) and rhizobia (rhizosphere only). Our results highlight deep microbiome differences induced by contrasted rice production systems that should consequently be considered for microbial engineering applications.

Spatiotemporal Survey of Multiple Rice Diseases in Irrigated Areas Compared to Rainfed Lowlands in the Western Burkina Faso.

Multiple constraints affect rice yields in West Africa. Among these constraints are viral, bacterial, and fungal pathogens. We aimed to describe the spatiotemporal patterns of occurrence and incidence of multiple rice diseases in farmers' fields in contrasting rice growing systems in the western Burkina Faso. For this purpose, we selected a set of three pairs of sites, each comprising an irrigated area and a neighboring rainfed lowland, and studied them over four consecutive years. We first performed interviews with the rice farmers to better characterize the management practices at the different sites. This study revealed that the transplanting of rice and the possibility of growing rice twice a year are restricted to irrigated areas, while other practices, such as the use of registered rice cultivars, fertilization, and pesticides, are not specific but differ between the two rice growing systems. Then, we performed symptom observations at these study sites to monitor the following four diseases: yellow mottle disease, Bacterial Leaf Streak (BLS), rice leaf blast, and brown spot. The infection rates were found to be higher in irrigated areas than in rainfed lowlands, both when analyzing all observed symptoms together (any of the four diseases) and when specifically considering each of the two diseases: BLS and rice leaf blast. Brown spot was particularly prevalent in all six study sites, while yellow mottle disease was particularly structured geographically. Various diseases were frequently found together in the same field (co-occurrence) or even on the same plant (coinfection), especially in irrigated areas.

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.

Crop 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.

Virus-Bacteria Rice Co-Infection in Africa: Field Estimation, Reciprocal Effects, Molecular Mechanisms, and Evolutionary Implications.

Simultaneous infection of a single plant by various pathogen species is increasingly recognized as an important modulator of host resistance and a driver of pathogen evolution. Because plants in agro-ecosystems are the target of a multitude of pathogenic microbes, co-infection could be frequent, and consequently important to consider. This is particularly true for rapidly intensifying crops, such as rice in Africa. This study investigated potential interactions between pathogens causing two of the major rice diseases in Africa: the Rice yellow mottle virus (RYMV) and the bacterium Xanthomonas oryzae pathovar oryzicola (Xoc) in order to: 1/ document virus-bacteria co-infection in rice in the field, 2/ explore experimentally their consequences in terms of symptom development and pathogen multiplication, 3/ test the hypothesis of underlying molecular mechanisms of interactions and 4/ explore potential evolutionary consequences. Field surveys in Burkina Faso revealed that a significant proportion of rice fields were simultaneously affected by the two diseases. Co-infection leads to an increase in bacterial specific symptoms, while a decrease in viral load is observed compared to the mono-infected mock. The lack of effect found when using a bacterial mutant for an effector specifically inducing expression of a small RNA regulatory protein, HEN1, as well as a viral genotype-specific effect, both suggest a role for gene silencing mechanisms mediating the within-plant interaction between RYMV and Xoc. Potential implications for pathogen evolution could not be inferred because genotype-specific effects were found only for pathogens originating from different countries, and consequently not meeting in the agrosystem. We argue that pathogen-pathogen-host interactions certainly deserve more attention, both from a theoretical and applied point of view.