AGRONOMIC AND ECO-PHYSIOLOGICAL RESPONSES OF BRASSICACEAE SUBJECTED TO BIOSTIMULANT TREATMENT AND ABIOTIC STRESS

The European Union is committed to achieving climate neutrality by 2050 through its Green Deal, focusing on sustainable agriculture to secure an eco-friendly food supply. The strategy involves "producing more while consuming less," emphasising efficient resource utilisation and fair compensation in the supply chain by 2030. In this context, biostimulants can be represent an environmentally friendly alternatives, optimising fertiliser and pesticide use while enhancing crop productivity and supporting the Green Deal's objectives. The aim of this thesis is the agronomic and eco-physiological evaluation of potential biostimulant products applied to horticultural crops. Biostimulants are substances which can promote the nutrient uptake and assimilation, thus maximise the action of fertilisers, resulting in higher yields with fewer inputs. Additionally, biostimulants have demonstrated positive effects in resisting abiotic stress, such as drought, salinity, heat, and cold. Developing new biostimulant products involves a complex process involving several steps, including screening, investigating the mode of action, and efficacy validation. Determining the appropriate dosage and timing for applying biostimulants ensures their efficacy. Since biostimulants can stimulate plants differently, studies on physiological, biochemical, and molecular effects are pivotal to deepening the knowledge of their mode of action. Fast and economical methods to evaluate physiological responses in vivo, such as the evaluation of photosynthetic capacity and gas changes, are essential tools for preliminary trials. After an initial screening, more sophisticated and specific trials can be designed to investigate plant changes at the molecular level. It is possible to deepen the view of the mode of action by analysing the differentially regulated genes by adopting several bioinformatic tools, and understand which biological processes are the most interesting. The data obtained can be helpful to determine in which situation a specific product can be used. For example, if genes for heat stress are activated in non-stressed conditions, in a sort of priming mechanism, it could be interesting to try that product in a stressful situation for the plants. A step-by-step approach, which corresponds to the thesis's four chapters, is proposed to evaluate the potential application of biostimulants: - Characterisation of novel biostimulant from raw materials: Screening for dosage optimisation and characterisation of the transcriptomic response. - Efficacy validation of a biostimulant prototype in a model plant to contrast heat stress, testing application before and after the temperature increase. - Identify the best timing for applying a biostimulant product before a heat stress period. - Validation of the efficacy of a biostimulant as a curative application to counteract heat stress events. In the first chapter, three different biostimulant products were tested on wild rocket (Diplotaxis tenuifolia L.): two of them derived from brewery and bakery yeast, while the latter is a botanical hydrolysate obtained from corn. Two different trials were conducted: the first investigated plants' physiological response to different doses of the three treatments, while the second looked into both physiological and molecular responses of the most promising dose of each treatment. All the products resulted in attractive biostimulant candidates. The stimulation of transcriptome regulation is explicit, with interesting effects on photosynthesis and secondary metabolism, partially confirmed by physiological analysis. In the following three chapters, several biostimulant products at different development stages (one prototype and two commercial products) were tested in different times to verify their efficacy against heat stress. In all trials, plants were exposed to cycles of supra-optimal temperatures, matching the maximum (37°C ± 1 °C) corresponding to the photoperiod peaks. The heat treatment was applied for up to 4 days for 4 hours to induce a stressful condition. The second chapter aimed to understand better the molecular and physiological responses of a model plant, Arabidopsis thaliana (L.) Heynh., subjected to heat stress and to the application of a biostimulant prototype, obtained from bacterial fermentation of sugarcane. Two distinct trials were designed to investigate the efficacy of counteracting the adverse effects of heat stress: in the first trial, the aim was to establish the possibility of a priming effect by applying a biostimulant before the stress period. In the second experiment, the biostimulant was applied after high-temperature exposure to determine its possible curative effects. The product was found to be an effective biostimulant in both cases; however, its preventive application showed more promise in mitigating the effects of heat stress. We found that the preventive application of the biostimulant helped maintain a higher leaf functionality and nitrate metabolism and led to the accumulation of heat shock proteins (HSPs). The third chapter aimed to evaluate the effect of the different application times of a commercial biostimulant product, based on Ascophyllum nodosum extracts, applied to wild rocket plants prior to a heat stress period. After identifying the most promising application time as 24 hours before heat stress, the trial investigated the different molecular responses to underline the product's mode of action. The biostimulant maintains the photosynthetic activity in stressed and biostimulated plants, thanks to the protective effects of the product on photosystems efficiency and the photosynthetic machinery in general. The positive and protective effects are probably due to the overexpression of HSP genes, which directly regulate heat shock proteins responsible for ensuring proper protein folding and preventing cellular damage. In the last chapter, a physiological and transcriptomic evaluation was carried out to assess the impact of an amino acid-based biostimulant product applied to wild rocket plants subjected to heat stress. The experimental objective was to investigate the different physiological and molecular responses and highlight the product's mode of action as a curative application when applied following stress. The analysis confirmed the positive effect of biostimulants on plant recovery: the application of the biostimulant reduced the energy dissipated during the photosynthetic process, contributed to maintaining the nitrate metabolism in stressed plants and promoted the transcription of heat shock regulatory elements. In the presented approach, certain aspects, such as utilising varying crop types and conditions different from the protected environment, have yet to be fully explored. Nonetheless, the methodologies employed in this research can be adapted and applied to other products grown in diverse settings and exposed to varied forms of stress. In conclusion, biostimulant products hold great promise for agriculture and have the potential to provide solutions to the challenges of sustainable farming. The development and application of new biostimulant products must be based on a targeted approach involving a thorough understanding of the product's mode of action and the optimum dosage and timing of application. The proposed protocol outlined in this thesis constitutes a flexible framework that can be tailored to suit specific applications.