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.

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.

Diallel Analysis of Early Leaf Spot (Cercospora arachidicola Hori) Disease Resistance in Groundnut.

Early leaf spot (ELS) is one of the major biotic constraints of groundnut production in West and Central Africa. A study using 6 × 6 F2 full diallel populations from six parents (NAMA, B188, PC79-79, QH243C, TS32-1, and CN94C) was conducted to assess the mode of inheritance of ELS resistance traits. The F2 and parents were grown in a randomized complete block design with three replications. Data was collected on ELS disease severity, and an area under disease progress curve (AUDPC) was estimated. The results revealed that additive and non-additive gene actions were involved in the inheritance of the ELS resistance traits, but additive gene action was predominant. Significant reciprocal cross effect was observed, suggesting cytoplasmic effect on ELS resistance. Graphical analysis also revealed the predominance of additive gene action for ELS resistance. The results suggest that early generation selection should be effective for ELS resistance. Looking at the distribution of array points along with the regression line, parental lines NAMA, PC79-79, and B188 would be suitable as good donors in an ELS disease resistance breeding program.