Isolation and evaluation of beneficial microorganisms for their potential use in agriculture as microbial biostimulants

Abstract

[eng] Conventional agricultural practices often rely on the extensive application of fertilizers to ensure maximum crop yields, leading to significant economic costs and negative environmental impacts. Nitrogen (N) and phosphorus (P) are critical nutrients for plant growth; however, excessive nitrogen use contributes to ecosystem pollution and disrupts the nitrogen cycle, while phosphorus, typically derived from limited mining sources, necessitates constant applications to maintain nutrient levels and high yields. In this context, microbial biostimulants emerged as a promising alternative to conventional chemical fertilizers, being defined as materials containing one or more microorganisms that, when applied to plants or the rhizosphere, stimulate natural processes to enhance nutrient use efficiency, seed quality, plant growth promotion, and accessibility on confined nutrients in soil. Promising microorganisms for biostimulant development include Bacillus subtilis, Pseudomonas fluorescens, Azospirillum spp., and Azotobacter spp., for their abilities to solubilize phosphorus, fix nitrogen, promote plant growth, and improve soil health. This thesis is part of an industrial PhD program, a scholarship from Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, Resolution EMC/460/2020), Biocontrol Technologies, S.L. and Universitat de Barcelona. The general objective of the research was to isolate, characterize, and explore the potential application of these genera to plants, with the aim, to develop a microbial biostimulant that complies with Spanish regulations (Royal Decree 999/2017) or European regulations (Regulation (EU) 2019/1009) by combining in vitro characterization experiments, plant trials, and soil studies. The isolation and characterization identified several strains of interest, including 16 Bacillus subtilis, 16 Pseudomonas fluorescens, 4 Azotobacter spp., and 9 Azospirillum spp. with some P. fluorescens excelling for Indole-3-Acetic acid production (IAA), B. subtilis for IAA, siderophores production and phosphorus solubilizing capacities while Azotobacter spp. demonstrated higher mineral phosphorus solubilization and Azospirillum spp. nitrogen fixing capabilities. Plant trials revealed Bacillus subtilis B7 and B17 as promising candidates for the development of biofertilizers according to Spanish regulations when applied at the substrate at a concentration of 107 CFU mL-1 which significantly enhanced seed germination by 10% and 13%, respectively, and promoted plant growth in cucumber, lettuce, and maize compared to non-inoculated plants. Furthermore, with the inoculation of B7 and B17 applied at the plant rhizosphere at a concentration of 107 CFU mL-1 the maize phosphorus use efficiency was improved under conditions of soluble phosphorus limitation, where B7 increased maize biomass by 34% and B17 enhanced plant phosphorus accumulation by 59% compared to non-inoculated plants. Moreover, Azospirillum brasilense 21F221 and Azospirillum aestuarii 21F226 were selected as candidates for the development of microbial biostimulants under European regulations due to their significant increase in nitrogen use efficiency when applied to maize and rice seeds at 108 CFU g seed-1 cultivated with different N fertilization regimes. Strain 21F221 notably

enhanced plant growth (16% and 4%) and plant nitrogen accumulation (33% and 34%) and 21F226 boosted plant biomass (20% and 11%) and yield (148% and 37%) compared to non- inoculated plants in maize and rice respectively, suggesting the possibility to significantly reduce fertilizer. The study of these Azospirillum spp. strains' effects on soil dynamic revealed no nitrogen fixing in the absence of plants and efficient nutrient mining from organic matter, as well as significant impacts on native microbial populations, underscoring the need for tailored application strategies. The studies conducted in the framework of this PhD thesis successfully bridges academic research with industrial application, supporting the initial steps for the development of a microbial biostimulant that meets stringent Spanish and European regulatory standards. Further studies would be necessary focused on development of growth, sporulation and formulation methods of selected strains, optimization of application strategies and field trials to validate these strains under diverse agricultural conditions.