Integration and benefits of root inoculation with endophytic entomopathogenic fungus <i>Metarhizium brunneum</i> in the propagation of olive tree seedlings in nurseries

Despite the recognized benefits of endophytic entomopathogenic fungi (EEPF) as biological control agents, their potential role in promoting plant growth and inducing systemic resistance (ISR) in olive nurseries remains poorly understood. Many fungi live in close association with plants, and a deeper understanding of the interactions among beneficial fungi is required to unravel the complex relationships among olive cultivars and phytopathogens, which can be fundamental for improving crop production and protection. EEPF are important soil-inhabiting microorganisms that can provide several benefits to olive plants beyond insect pest control. This study investigates the role of <i>Metarhizium brunneum</i> in the olive rhizosphere during the nursery stage, focusing on its potential to promote plant growth and induce systemic resistance in Picual and Manzanilla cultivars. Conidia and microsclerotia of the <i>M. brunneum</i> EAMa 01/58-Su strain, together with an arbuscular mycorrhizal fungus (AMF), were applied to olive plants. Fungal colonization, growth promotion, and induction of systemic resistance were evaluated. Microbiological, qPCR, and droplet digital PCR analyses showed that <i>M. brunneum</i> colonized roots via the apoplastic pathway when applied as conidia or microsclerotia. Mycorrhization rates were higher in Manzanilla, but elevated mycorrhization inhibited <i>M. brunneum</i> colonization. Growth-promoting effects were more pronounced in Picual. Application of the EAMa 01/58-Su strain induced specific subsets of systemic resistance (SR) genes associated with the ISR pathway. Additionally, Picual plants treated with both AMF and microsclerotia exhibited increased leaf nitrogen and potassium levels. Overall, this strain demonstrates promising potential as a plant growth promoter during the early nursery stage of olive cultivation.IMPORTANCEOlive cultivation is an economically and culturally significant crop; however, early-stage plant growth in nurseries can be limited by nutrient uptake, abiotic stress, and pathogen pressure. Endophytic entomopathogenic fungi (EEPF), such as <i>Metarhizium brunneum</i>, are traditionally used for biological control of insect pests, but their potential to promote plant growth and induce systemic resistance has been overlooked and is currently of interest to olive nursery companies. This study demonstrates that <i>M. brunneum</i> EAMa 01/58-Su can effectively colonize olive roots, improve certain vegetative growth parameters, and activate systemic resistance pathways, influenced by both cultivar and the application with arbuscular mycorrhizal fungi prior to field transplantation. These findings highlight the dual role of the <i>M. brunneum</i> EAMa 01/58-Su strain in olive nurseries, providing both pest protection and growth-promoting benefits. Integrating EEPF into nursery management could enhance plant health, nutrient status, and stress resilience, offering a sustainable strategy for early-stage cultivation and contributing to more productive and resilient olive orchards.