IDENTIFICATION, PURIFICATION AND BIOLOGICAL EVALUATION OF COMPOUNDS OR PLANT EXTRACTS FOR PLANT PROTECTION WITH LOW ENVIRONMENTAL IMPACT

In recent years, interest in natural alternatives to synthetic products has grown significantly, driven by multiple environmental, health, and economic factors. The spread of resistance among agricultural pathogens has reduced the effectiveness of many conventional pesticides, requiring more diverse and sustainable control strategies. At the same time, growing awareness of the ecological impact of pesticides and their persistence in soil and water has raised concerns about biodiversity and non-target organisms, including pollinators and beneficial soil microorganisms. Added to these concerns is the need to ensure greater food safety for human consumption and to respond to European policies promoting the reduction of synthetic pesticides with the adoption of integrated pest management and biological control practices. A further incentive comes from the abundant availability of plant biomass and agro-industrial by-products, which represent an economical and renewable resource for the extraction of bioactive metabolites with potential antimicrobial or insecticidal activity. In this context, plant secondary metabolites represent a promising alternative to synthetic pesticides, thanks to their wide structural diversity, natural bioavailability, and ability to exert targeted biological activities, opening new perspectives for sustainable crop protection and for the valorisation of natural resources. In this context, the present study is part of the phytochemical research applied to integrated crop protection, with particular attention to the biological potential of plants belonging to the genus Citrus L. (Rutaceae) and Salvia L. (Lamiaceae). The genus Citrus is widely recognized for the high content of bioactive compounds present not only in the fruits, but also in the by-products of industrial processing. In this context, a study was conducted to evaluate the antibacterial and antifungal activity of essential oils (EOs) extracted from the peels of two Citrus sinensis (L.) varieties. In particular, Navel and Tarocco varieties were selected for their relevance in the Mediterranean area, leading to a large supply of waste material derived from industrial processing. The Citrus peels, rich in volatile compounds with bioactive potential, represent a matrix of particular interest for valorisation strategies in the context of the circular economy. After a preliminary screening phase on several phytopathogenic fungi and bacteria, attention was focused on pathogens of primary importance in horticultural crops: Fusarium oxysporum f. sp. radicis-lycopersici, Botrytis cinerea, and Pseudomonas syringae pv. tomato. For F. oxysporum f. sp. radicis-lycopersici, a vascular pathogen known to colonize the root system preferentially, the activity of the oils was evaluated on tomato rootlets germinated under controlled conditions, to simulate the direct interaction between the root system and the fungal inoculum. The aim was to observe the curative effect of the oils on the progression of the infection in an early phase of plant development. For B. cinerea, antifungal activity was tested on ripe tomato fruits, a tissue typically affected by this necrotrophic pathogen, responsible for post-harvest gray mold. In both cases, EOs showed significant inhibitory activity, reducing infection and disease progression. In addition to in vivo tests on plant tissues, a specific test was performed to evaluate the antibacterial activity of EOs under simulated sanitization conditions of inert surfaces. The agronomic instruments were treated with EO-containing solutions after being experimentally contaminated with P. syringae pv. tomato. The findings demonstrated a considerable decrease in bacterial viability and an efficient sanitizing effect. This test validates EOs' adaptability for use on plants and fruits as well as in preventative sanitization interventions, which may be useful for the combined management of post-harvest safety and phytosanitary measures. The results obtained confirm the potential of citrus derivatives as natural antibacterial and antifungal agents, supporting their use as a resource that can be valorised within circular and environmentally low-impact agricultural supply chains. Attention was subsequently turned to the Salvia genus, another group of aromatic plants of great phytochemical interest, known for its high production of secondary metabolites. As previously done for the Citrus genus, the objective was to evaluate the biological potential of natural extracts and isolated compounds for controlling phytopathogens of agronomic interest. Therefore, this work examines several species of Salvia, including Salvia rosmarinus Spenn., Salvia somalensis Vatke, Salvia chamaedryoides Cav., and Salvia interrupta Schousb., evaluating their bioactive constituents and their antifungal and insecticidal activities. The chemical and biological characterisation of S. rosmarinus's volatile components was the main goal of the first investigation. Tuta absoluta, a leaf-mining moth that is one of the most destructive to tomato crops worldwide, was used to investigate the insecticidal properties of rosemary EOs. Additionally, an enzymatic experiment that targeted acetylcholinesterase activity was carried out to support the interpretation of its mechanism of action. Finally, phytotoxicity tests have demonstrated the tomato plants' excellent tolerance to treatments, supporting the possible use of rosemary EOs in the agronomic sector. Salvia somalensis has proven to be a promising species due to its ability to produce carnosic acid, an abietane diterpenoid known for its biological properties. In a first study, attention was paid to the chemical characterization of the dichloromethane extract obtained from the fresh aerial parts of plants grown in Liguria and to evaluate its antifungal activity against important fungal pathogens. Using callus cultures and micropropagated plants, a second study examined S. somalensis's potential as a sustainable supply of agrochemicals. To test their biological activity against fungal infections and assess their capacity to manufacture carnosic acid, in vitro plants were developed. In a study to determine the biological potential of various extractive fractions of the plant's aerial parts, the antifungal activity of S. chamaedryoides was assessed. These fractions were prepared using four solvents of increasing polarity to determine which class of metabolites was primarily responsible for the observed activity. The initial phase of the study involved in vitro.screening of a broad spectrum of phytopathogenic fungi, including several species of interest to horticultural and industrial crops. Among the strains tested, Phoma betae, the pathogen responsible for sugar beet infections, proved particularly sensitive to the extracts. Given these findings, antifungal activity was analyzed in vivo on sugar beet seeds to determine its activity, performed under two different applications: (1) the spraying of the seed surface and (2) the cultivation of sugar beet seeds on poisoned substrates containing the extract. Results from the two strategies were not the same, highlighting that the method of administration also affects efficacy. Overall, the observation of these effects suggests that S. chamaedryoides represents a rich source of specialized metabolites of interest in the development of alternative treatments against emerging pathogens, particularly in economically important crops such as sugar beet. S. interrupta, which had not previously been explored from either a chemical or microbiological perspective, was the last species examined. Here, it was investigated to evaluate its antifungal potential against B. cinerea, a pathogen known for its increasing tolerance to conventional fungicides. To account for its genetic variability and its marked resistance, the antifungal activity was tested in vitro against different strains of B. cinerea, selected to represent different levels of aggressiveness. In parallel, the efficacy of the S. interrupta extract was also evaluated in vivo, on artificially infected tomato fruits. In both experimental systems, the extract demonstrated a significant ability to reduce pathogen development and limit damage, suggesting a direct effect on the fungus’s mycelial and conidial structures. A phytotoxicity study was also conducted to demonstrate that S. interruptata extracts do not have phytotoxic effects on tomato plants. Considering the lack of previous studies on this species and the promising results obtained, S. interrupta is proposed as a new plant resource of interest for organic and integrated agriculture, to be further investigated, including its characterization as a source of active constituents. The data collected highlight the central role of the Salvia genus in the development of multifunctional biopesticides of plant origin, compatible with environmentally friendly agronomic practices.