Isolation and selection of plant-associated microbes for the formulation of new inocula to be used in sustainable agriculture

The experimental plan included the employment of two different crops: Solanum lycopersicum var. Tarzan, and Oryza sativa var. Vialone nano. The culture-dependent approach has been focused on spore-forming bacteria (especially those belonging to the Bacillus genus) and arbuscular mycorrhizal fungi. We hypothesize that those microbes, recruited by plants under drought and after a recovery phase that has been set following the stress imposition, may improve drought stress tolerance in crops. After a period of drought stress, gradually imposed on plants, a recovery phase has been induced and the stressed plants received a full amount of water, as the control ones. Setting these two timepoints allowed us to analyse how the communities in the rhizosphere and in the root endosphere changed during the stress and upon rewatering. This project includes three main analytical approaches: (1) Metabarcoding analysis: to describe the changes in the microbial communities, next-generation sequencing techniques and subsequent bioinformatics analyses allowed the description of the bacterial and fungal diversity present in the soil before planting, in the rhizosphere and inin the endosphere under drought stress and control conditions; (2) Bacteria isolation and selection: bacteria were isolated and identified from rhizosphere and endosphere compartments. The plant growth-promoting abilities of isolated strains from the Bacillus genus have been evaluated through specific in vitro tests and the application of selected bacteria on plant roots has been carried out in two different greenhouse trials; (3) Arbuscular mycorrhizal fungi (AMF) isolation and selection: the mycorrhizal component from both rice and tomato pots has been isolated by trap culture technique and subsequent steps of single species cultures The isolated AMF morphotypes have been molecularly characterized and kept in pure culture. Chapter 1 will introduce the main concepts, state of the art and rationale related to our research. Chapter 2 constitutes the main part of the whole project. Metabarcoding analysis described how the microbial communities associated with plant roots are shaped by the imposition of drought stress and upon re-watering. Then, a culture-dependent approach was applied to isolate the bacterial (Bacillus strains) and the fungal (AMF) component from the main greenhouse drought experiment. The isolated bacteria have been selected through different plant growth promoting (pgp) tests and finally evaluated as inoculum in a greenhouse trial on lettuce under drought stress conditions. The AMF components have been propagated, isolated and identified through Sanger sequencing. In Appendix A are included additional experiments that have been performed, after the in vitro pgp tests, to select the bacterial strains that performed best. In Appendix B the work that has been performed on the AMF component is developed more in depth, including the trap culture methodology and the identification of the isolated AMF strains. Chapter 3 highlights the main findings of this PhD work, showing how drought stress and recovery shape plant-associated microbial communities. Overall, this study contributes to the understanding of plant–microbe interactions under drought and provides microbial candidates with potential use as biostimulants in sustainable agriculture. This project has been carried out thanks to the collaboration with Green Has Italia S.p.A., an Italian company, located in Canale d’Alba, that produces biostimulants and fertilizers for agriculture, which cofounded this project. During my Ph.D I spent a period abroad in Grasse, hosted by the French company Mycophyto which is active in the development and production of arbuscular mycorrhizal inocula for agriculture.