Study on bacteria associated with rice roots and their potential to promote rice growth

The increasing world population demands a considerable increase in stable crop yield to assure the food supply. When rice is considered, this means an increase in rice production up to 1 billion tonnes by 2025. To boost the crop yield, chemical fertilizers, and especially nitrogen (N), are applied in tremendous amounts. However, this common agricultural practice can have a significant adverse impact on the environment. Therefore, countless studies have investigated sustainable alternatives to ensure the cereal crop yield, while reducing the chemical fertilization, including the exploitation of plant growth-promoting rhizobacteria (PGPR) as biofertilizers. This PhD thesis was aimed to investigate the plant growth-promoting capacity of a model rice root-colonizing PGPR, Pseudomonas stutzeri A15. However, to be effective, an intimate interaction between strain A15 with the rice plant should be established. Also, the natural occurrence of P. stutzeri in rice farming systems need to be investigated, which enables elucidating the ecological functions in agricultural contexts. Furthermore, the diversity of the endogenous bacterial community associated with rice farming systems need to be explored because it might be useful for evaluating of their impact on strain A15’s ability to colonize rice roots and its competitiveness in the rhizosphere. In addition, it can provide a culturable collection of other PGPRs that might be exploited as bio-fertilizers. Therefore the main objectives of this PhD research was to: (i) explore the diversity of the root-associated bacterial community in wetland rice cultivars in Vietnam; (ii) unequivocally demonstrate the plant growth-promoting ability of strain A15; (iii) assess the contribution of its nitrogen-fixation activity to the pant growth-promoting effect; and (iv) identify P. stutzeri A15 genes that are important in colonization and invasion of rice roots.

To investigate the diversity of the root-colonizing bacterial community associated with wetland rice in Vietnam, we applied a cultivation-independent approach by 16S rRNA gene amplicon sequencing, as well as a cultivation-dependent approach by bacterial enrichment isolation. Our cultivation-independent approach showed that the root-associated bacterial community composition in the endosphere is less diverse than in the rhizosphere. Furthermore, compared to the rhizosphere, the root endosphere environment is enriched for members belonging to α- and γ-Proteobacteria, and Actinobacteria, and limits the association with Acidobacteria. These trends were also reported in other studies on root-associated bacteria of greenhouse- and field-grown rice plants as well as of other plant species, including Arabidopsis, poplar, and wheat. In contrast to few other studies examining rice root-associated bacterial community, our data revealed a similar relative abundance of Bacteroidetes and Firmicutes across the two studied root fractions. This might be specific for the structure of the bacterial community colonizing wetland rice in Vietnam. In addition, our 16S rRNA gene-based profiling revealed that Proteobacteria and Enterobacteriaceae were among the most abundant phyla and families, respectively, both in the rhizosphere and endosphere-enriched fractions. This is in agreement with other studies on rice root-colonizing bacteria that applied 16S rRNA-based finger-printing, 16S rRNA amplicon sequencing, or a metagenomics approach. Furthermore, this suggests that members of these taxonomic groups are indeed important for rice growth in agricultural contexts.

In our cultivation-dependent approach, the bacterial isolation from surface-sterilized root macerates revealed the isolation of several strains belonging to the genera Acinetobacter, Enterobacter, Klebsiella,Pseudomonas, and Pantoea. Interestingly, some members of these genera were previously characterized as rice endophytes or PGPR. It would therefore be of interest to further explore the plant growth-promoting ability of the isolates collected in this study. On the other hand, our isolation did not yield P. stutzeri, possibly owing to the low abundance or absence within the bacterial community colonizing wetland rice cultivars in Vietnam. Altogether, the results of our ecology study contribute to the knowledge of the bacterial community composition associated with rice root, and more specifically, of wetland rice cultivars in the Mekong Delta of Vietnam.

To elucidate the root colonization mechanisms of P. stutzeri A15, we exploited the in vivo expression technology (IVET) system developed previously to identify additional genes that are specifically upregulated during root colonization and invasion (cii genes). Along with the re-isolation of previously identified genes, new rice root-induced genes were identified. Overall, the cii genes identified are involved in motility and chemotaxis, carbohydrate or nucleic acid metabolisms, stress response and adaptation, biofilm formation, regulatory functions, or have an unknown function. In addition, we investigated the expression of three newly identified cii genes maeB, purM, and etfBA, encoding NADP+-dependent malic enzyme, phosphoribosylaminoimidazole synthetase, and subunits of the electron transferring flavoprotein, respectively. To this purpose, fusions of the 1-kb region upstream of each of these genes/operon with the promoterless gusA reporter gene were constructed. Histochemical staining confirmed that these transcriptional fusions as well as the original IVET clones were specifically expressed in rice seedling root, but not on minimal medium or medium supplemented with macerated rice root extract. However, further studies are required to elucidate the ecological functions of the identified cii genes, and in particular their role in rice root colonization.

To examine the plant growth-promoting ability of P. stutzeri A15, we measured shoot and root dry dryweight, as well as the shoot length of rice seedlings inoculated with the wild-type A15 in greenhouse trials. Our results demonstrate that P. stutzeri A15 could induce significant growth promotion when compared to non-inoculated rice seedlings under normal or even augmented N fertilization. This clearly points to the potential of this bacterium as biofertilizer to reduce chemical fertilization. Moreover, to the best of our knowledge, this is the first benchmark for the plant growth-promoting effects elaborated by P. stutzeri A15. To assess the contribution of nitrogen fixation to the plant growth-promoting effect, we also examined growth of rice seedlings inoculated with the nitrogen fixation-deficient mutant. Our results suggest that biological nitrogen fixation was (at best) only partially contributing to the plant growth-promoting effect, and that other traits in addition to BNF are involved in the plant growth-promoting activities of this endophytic bacterium. Furthermore, our results inspire further research including the optimization of N levels and inoculum concentrations, the comprehensive evaluation of biomass and yield over the entire rice growth cycle, and the characterization of other plant growth-promoting mechanisms of this endophytic PGPR. 

In conclusion, this research contributes to our understanding of the plant growth-promoting activities of the model PGPR P. stuteri A15 and the genes that are important to establish an intimate interaction bewteen strain A15 and the rice roots. Furthermore, this study provides a benchmark for the study of the bacterial community composition in real rice farming systems. The results obtained here can provide valuable information to improve the practical use of biofertilizers in rice crop production and our understanding of the plant-PGPR interaction mechanisms.