Investigation of the potential of new microorganisms and their derivatives as novel biostimulants

The rising global population and increasing food demand have compelled traditional agricultural practices to rely heavily on chemical fertilizers to boost yields. In response to concerns about depleting oil reserves, environmental consequences, declining soil fertility, and climate change, global organizations such as the Food and Agriculture Organization of the United Nations (FAO)[1] , the European Commission[2], alongside academic institutions, are championing the development and adoption of sustainable practices. This doctoral project aligns with these initiatives, focusing on plant biostimulants (PBS) as innovative tools to address current and future agricultural challenges. PBS, encompassing biological substances (BS) and beneficial microorganisms, provide sustainable approaches to enhance crop yield, productivity, quality, and tolerance to abiotic stresses[3]. This PhD project tackles a crucial challenge in the field of microbial biostimulants: the identification of effective and commercially viable candidates. The objective is to establish a systematic, stepwise workflow for selecting microbial candidates actively contributing to developing effective PBS. The research methodology involved a comprehensive process applied to a pool of seventeen bacterial cultures, including the following critical criteria: (i) initial identification of microbial candidates; (ii) evaluation of biosafety of the associated microbial species; (iii) assessment of regulatory compliance of biostimulants; (iv) examination of potential antagonistic activity of selected strains; (v) characterization of plant growth-promoting (PGP) traits through in vitro assays; and (vi) experimental validation. Specifically, in step (vi), strains exhibiting significant PGP traits were further tested on A. thaliana and pepper (C. annuum) as representative agronomic crops. This validation aimed to confirm the in vitro results on plant growth promotion and finalize the selection of the most promising microbial strains. Additionally, the compatibility between selected microbial strains and common BS used in biostimulant formulations was explored to exploit synergies between the two components. A critical aspect of the proposed workflow involves the adoption of MALDI-TOF MS as a powerful tool for fast and reliable microbial identification. This technology plays a pivotal role in streamlining the identification process. Furthermore, a parallel study explores the integration of proteomic analysis with statistical methods to predict and explore differences between closely related strains, demonstrating potential applications in typing and differentiating large microbial datasets. In conclusion, this research simplifies the biostimulant development process by integrating robust microbial identification, addressing biosafety and regulatory aspects, evaluating PGP traits, and exploring synergies to enhance microbial activity. Such a multifaceted approach not only streamlines development but also fosters the release of effective and sustainable solutions in the field of PBS.