MECHANISMS OF ACTION OF BIOSTIMULANTS IN CROPS

Abiotic stresses can affect plant growth and lead to great losses in yield and quality. Among them water availability is a current issue and intensive agriculture has now reached a critical point in the negative effects on natural resources. Hence, the main challenge is to achieve an “ecological intensification” of agriculture minimizing negative externalities. To cope with this situation, in recent years, a notable diffusion of biostimulant products has been observed. Biostimulants are complex mixtures of compounds and substances able to promote plants growth, improving stress tolerance, quality, and yield. However, this variability in composition creates difficulties in the comprehension of the mode of action and the effectiveness of these products. The purpose of this Ph.D. research project is to study the effect of different biostimulants prototypes on leafy vegetables subjected to abiotic stresses. The attention is focused on physiological and molecular changes induced by the stress and on the potential biostimulant effect of treatments. Qualitative and physiological parameters such as photosynthetic pigments, polyphenols, chlorophyll a fluorescence, transcription factors, and genes involved in plant stress responses have been analysed. The first activity regarded the preparation and the evaluation of aqueous extracts obtained from leaves and flowers of Borago officinalis L. grown in a greenhouse. The extracts were prepared with different times of maceration and were applied on rocket and lettuce crops as foliar spray two times during the growing cycles. Results showed that the maceration time affected the efficacy of the products and different trends emerged from their applications on the two species. Moreover, about twenty transcription factors (TFs) from the NAC, MYB, bZIP and other families have been chosen as potential stress markers and primers for qRT-PCR analysis have been designed. A borage extract was tested to evaluate the transcriptional changes induced in rocket grown under salt stress. The physiological responses linked to the primary and secondary metabolism of the leaves subjected to high salinity were monitored by measuring the changes in chlorophyll content, carotenoids, anthocyanins, lipid peroxidation, and in chlorophyll a fluorescence-related indices. At molecular level, the stress responses were studied by measuring the changes in the expression of the selected TFs within 24 hours after the beginning of the stress application. Results obtained showed that the treatment affects the gene expression in different ways. DtRd29a, a stress-responsive gene, was generally more expressed in stressed plants treated with borage extract. In general, salt stress induced the expression of all the TFs examined. Results obtained have allowed to point out the complex plant response to a sudden exposition to high level of salinity, to the treatment with borage extract and to the interaction between these two factors. Moreover, it has been possible to get information on different gene expression patterns during time. Some of these transcription factors were involved in the regulation of several pathways including sugars metabolism (DtbZIP63), cuticular wax biosynthesis (DtMYB30), brassinosteroids signaling (DtMYB30, DtbHLH122, DtBEE2, DtHBI1, DtIBH1, DtWRKY54, DtNAC72) and intercellular transport (DtRABC2B). At the same time a collaboration with a private company was carried out with the aim to evaluate the effectiveness of a biostimulant prototype against water and salt stress conditions. In order to better understand the mode of action, the experimental plan included the study of the plant responses to the application of the prototype alone, in combination with proline and glutamic acid solutions. Treatments were applied on lettuce plants (Lactuca sativa var. acephala ‘Chiara’) subjected to a period of water deprivation of 30%. Results obtained showed that the addition of proline to the formulation did not affect the efficacy of the product in a significant way. Some interesting results were obtained after the application of glutamic acid. An increase of chlorophyll and carotenoids content and a higher water use efficiency was observed in plants grown with a lower water availability. Based on the above-mentioned results, the prototype formulation was slightly changed and the new product was tested on romaine lettuce (Lactuca sativa var. longifolia) grown under water stress and salt stress. Physiological and biochemical traits of plant responses to stressful environments and biostimulant treatment were investigated. Moreover, the study of the plant response at molecular level, focusing in particular on the genes involved in oxidative stress and antioxidant defence was performed. The expression of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR), and glutathione reductase (GR) was examined. Generally, the results obtained showed that stress conditions had a more significant effect than treatments and the expression levels of selected genes significantly decreased in response to stressful conditions. The last part of the research project was carried out at Cardiff University aiming to evaluate the effect of a treatment with glutamic acid on the aroma of rocket salad subjected to a period of salt stress. Volatile organic compound (VOC) profiles have been assessed using a thermal desorption gas chromatography time-of-flight mass spectrometry (TD-GC–TOF-MS). Data were processed using MSD ChemStation software deconvoluted and integrated with AMDIS (NIST14) using a retention-indexed mass spectral library. The identification of each peak has been performed comparing the mass spectrum against a rocket library and a NIST database. PerMANOVA analysis indicated that the aroma of rocket was significantly affected by salt stress. About 190 compounds were identified from all the rocket salad samples. 3-Hexen-1-ol, acetate, (E)-, 3-Hexen-1-ol, (Z)-, 1,2-Benzenedicarboxylic acid, butyl 2-ethylhexyl ester, Butanoic acid, 3-hexenyl ester, (Z)-, "1-Penten-3-ol, Dimethyl sulfone, Sulfur dioxide, Acetic acid, hexyl ester, 3-Pentanone and an undefined branched alkane were the most abundant compounds found in control plants.