Maria Clara Santana Aguiar¹, Moacir Rossi Forim¹

Essential oils (EOs) are volatile organic compounds extracted from plants, known for various biological activities and considered potential plant biostimulants. However, their molecular-level effects are not yet well understood, and their volatile, easily degradable nature restricts practical application. To address these limitations, we investigated the potential of essential oils from Rosmarinus officinalis, Lippia alba, and Citrus sinensis as plant biostimulants. We used tomato cultivars Solanum pimpinellifolium (an introduced plant) and S. lycopersicum (a commercial plant) as models.

To enhance EO stability, we developed biopolymeric microcapsules using modified cassava starch and adjuvants like banana, pequi, and lignin residues. These formulations were soil-applied to the tomato cultivars. We then evaluated plant responses through growth parameters (shoot and root), foliar metabolomic analyses (UHPLC-qTOF-MS/MS and GC-MS), and rhizosphere metagenomic analyses to assess the bacterial community. Robust analytical methods for extraction and characterization were developed for these analyses.

Our formulations successfully microencapsulated EOs with over 85% recovery, significantly increasing EO retention in the soil (two-fold compared to emulsions) with controlled, diffusion-dependent release. Root growth stimulation was observed in both cultivars treated with EOs; however, the EO mixture did not exhibit a synergistic effect. Interestingly, in S. pimpinellifolium, the mixture reduced shoot growth but increased leaf area, while the biopolymer alone reduced stem diameter. Foliar metabolomic analyses revealed complex and cultivar-specific modulations. For S. pimpinellifolium, EOs induced changes in secondary metabolites that differentiated treated plants from controls: R. officinalis notably induced purine nucleosides and carbohydrate-derived compounds, while L. alba, C. sinensis, and the mixture increased the production of carbohydrate-derived compounds and trans-hex-2-enal.

Notably, all EOs decreased alpha-tomatine and fatty acids, suggesting defense remodeling. In S. lycopersicum, L. alba particularly induced quinic acid and other metabolites linked to primary and energy metabolism. Our rhizospheric soil metagenomics provided an unexpected insight: bacterial communities were influenced more by the cultivar than by EO treatments. However, applying the biopolymer with L. alba essential oil did increase Nitrospirae abundance in S. pimpinellifolium. Since these microorganisms are less affected by nutrient changes, this suggests that EOs can influence the nitrogen cycle and nutrient availability, beyond their direct plant effects, raising questions about the complexity of soil-plant interactions mediated by these biostimulants.

The developed formulations demonstrate the potential of EOs as biostimulants to promote root growth and modulate foliar metabolism in a cultivar-dependent manner, independent of mixture synergy. Crucially, the discovery of increased Nitrospirae with Lippia alba EO in S. pimpinellifolium adds a significant layer of complexity, indicating that biostimulant effects can involve intricate interactions with the soil microbiome. While this work represents an initial step, future studies are essential to fully understand these complex effects and their relevance under various condition.

Agradecimentos: The authors thank the MCTI/Financiadora de Estudos e Projetos (MCTI/FINEP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the support of the projects.