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.

Quantification of diversity sampling bias resulting from rice root bacterial isolation on popular and nitrogen-free culture media using 16S amplicon barcoding.

Culturing bacteria from plant material is well known to be conducive to strong bias compared to the actual diversity in the original samples. This bias is related to the bacterial cultivability, chemical composition of the media and culture conditions. Recovery bias is often observed but has never been quantified on different media using an amplicon barcoding approach whereby plant microbiota DNA extractions are compared to DNA extracted from serial dilutions of the same plant tissues grown on bacterial culture media. In this study, we: i) quantified the bacterial culturing diversity bias using 16S amplicon barcode sequencing by comparing a culture-dependent approach (CDA) focused on rice roots on four commonly used bacterial media (10% and 50% TSA, plant-based medium with rice flour, nitrogen free medium NGN and NFb) versus a culture-independent approach (CIA) assessed with DNA extracted directly from root and rhizosphere samples; ii) assessed enriched and missing taxa detected on the different media; iii) used biostatistics functional predictions to highlight metabolic profiles that could potentially be enriched in the CDA and CIA. A comparative analysis of the two approaches revealed that among the 22 phyla present in microbiota of the studied rice root samples, only five were present in the CDA (Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Verrucomicrobia). The Proteobacteria phylum was the most abundant in all CDA samples, showing high gamma-Proteobacteria enrichment. The diversity of the combined culture media represented about a third of the total microbiota diversity, and its genus diversity and frequency was documented. The functional prediction tool (PICRUSt2) detected nitrogenase enzyme enrichment in bacterial taxa sampled from nitrogen-free media, thus validating its predictive capacity. Further functional predictions also showed that the CDA mostly missed anaerobic, methylotrophic, methanotrophic and photosynthetic bacteria compared to the CIA, thereby generating valuable insight that could enable the design of ad-hoc culture media and conditions to increase the rice-associated microbiota cultivability.

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.