Savarese, Claudia (2022) Humic biostimulants from green compost and synergies with mycorrhizal fungi. [Tesi di dottorato]

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Currently, agricultural intensification is considered as the main strategy to ensure the growing need for food production. However, conventional agriculture already has major global environmental and human impacts, such as soil fertility and biodiversity reduction, about one-quarter of global greenhouse gas (GHG) emission, and environmental pollution by the extensive use of chemical products (i.e. fertilizer and pesticides). Therefore, the present thesis work aimed to evaluate various approaches to provide a truly effective solution for maintaining crop productivity and food security, thus supporting the development of sustainable agriculture to replace conventional agronomic practices. Conventional agronomic practices can drastically reduce the quantity and quality of soil organic matter (SOM), as well as the storage of soil organic carbon (SOC). SOM plays a fundamental role in the agro-ecosystems, as it regulates the global carbon and nitrogen cycle, the development of plants and microorganisms, the fertility and stabilization of soil structure. Despite its importance for the development of a sustainable agriculture, the molecular dynamics of soil organic matter under different cropping systems is still poorly understood. Hence, in the first two studies of this research the molecular dynamics of organic matter in soils subjected to long-term field experiments (20 years) under conventional maize monoculture or maize-leguminous crop rotation, were evaluated by both traditional analytical techniques and an innovative chemical sequential fractionation named Humeomics. The application of off-line pyrolysis TMAH-GC-MS (thermochemolysis) and solid state 13C NMR spectroscopy for the direct molecular characterization of OM components of both the bulk soils and their humic extracts revealed that the long-term cultivation under conventional tillage destabilizes SOM molecular conformation, though to a different extent, as a function of the cropping system. In particular, 20 consecutive years of maize mono-cultivation led to a decrease in alkyl and aliphatic compounds, and an increase in hydrophilic labile components, while the crop-rotated soils 5 showed a partial preservation of the pristine SOM composition by maintaining the content of hydrophobic and lipid constituents. The application of Humeomics technique, coupled to characterization of separated fractions by GC-MS and high-resolution Orbitrap LC-MS, on the same soils confirmed these results. In particular, Humeomics showed that the ratio of organosoluble to hydrosoluble components significantly decreased passing from untreated to long-term cultivated soils, thus revealing that the loss of humic hydrophobic compounds, such as long-chain esters and fatty acids, rendered more physically and chemically fragile the protection of organic matter in soil. Moreover, most of N-containing compounds in cropped soils were found to be bound to iron, thus implying that different forms of nitrogen entering soil are progressively sequestered into recalcitrant organic pools. These findings indicate that a detailed knowledge on the molecular dynamics of soil Humeome can be archive by Humeomics fractionation, which could be an innovative tool to identify new environmentally sustainable technologies in agriculture. Another objective of this thesis was to estimate the effectiveness of mixed plant biostimulants as an eco-friendly strategy to increase plant growth, while concomitantly ensuring high levels of agricultural productivity and environmentalsecurity. To this purpose, the bioactivity of two different humic materials, a potassium humate from leonardite (KH) and compost tea (CT) from a green compost made of coffee husks, and their combination (1:1), was evaluated toward basil seeds germination and maize early growth. After their thorough chemical and molecular characterization, a relation between structure and bioactivity was also investigated. The results of this experiment showed that the high polar CT stimulated both the epicotyl and root development of basil seeds, while the mostly hydrophobic KH exerted a significant bioactivity on maize early growth. On the other hand, the application of a mixed solution of both humic materials to hydroponically grown maize plantlets resulted in a biostimulant effect similar to KH but greater than the individual CT treatment. The molecular characterization ofthe humic materials allowed to explain these results by a cage effect of the polar bioavailable CT compounds, such as oxidized lignin fragments, saccharides, and peptides within the hydrophobic domains of the mainly apolar KH. These findings thus suggest that 6 a calibrated mixture of humic materials of selected molecular composition may represent an innovative and ecologically viable method to build up sustainable products with diverse mechanisms of plant biostimulation. Moreover, in a second experiment the bioactivity of the same mixed humic biostimulants was investigated in synergy with microbial bioeffectors (Micosat TABPLUS, M+) on lettuce productivity, nutritional status, and metabolism. The synergistic interaction between KH, CT and M+ significantly increased lettuce biomass production, and uptake of both macro- and micronutrients as compared to their individual application. Furthermore, the humic-microbial combination (MIX_M+) positively affect the overall plants metabolism. In particular, the GC-MS analysis of leaves primary metabolites revealed an improve in the biosynthesis of essential amino acids and saccharides following the MIX_M+ treatment, thus indicating a potential role of humic substances in supporting the survival of beneficial microorganisms in the soil environment, aswell as in promoting their colonization capacity. Similarly, the UHPLC-MS-IT-TOF analysis of lettuce polyphenols metabolism showed an accumulation of important antioxidant compounds in plants treated with mixed humic materials, thereby supporting the cage effect of bioavailable CT compounds in the hydrophobic components of KH that may enhance the conformational stability of the humic assembly essential for the release of bioactive molecules and the effectiveness of their biological activity. Hence, the findings of this second experiment indicate that a calibrate mixture of humic extracts, containing different type of bioactive molecules, in combination with microbial consortia is a potential tool to improve plants both productivity and nutritional status, as well as to modulate plants metabolome for the development of novel functional crops. Finally, an additional goal of this doctoral research was to investigate the possible applications of innovative hydrogels based on humic matter from green compost to improve plant growth. The potential use of a hydrogel containing sodium alginate and humic extracts from green compost cross linked by calcium nitrate and able of maintaining shape after 3D bio printing, as a substrate for soilless cultivation was explored. The preliminary results of this experimentation showed that the addition of humic substances improved the printability of the alginate gel, ensuring the right mechanical 7 proprieties essential for 3D printing, and thus resulting in well-assembled and easly printable structures. Furthermore, the biostimulant activity of humic extracts toward plants development allowed an increased growth of both basil and lettuce seedlings in the hydrogel bio-composite compared to the sodium alginate alone. These findidings may indicate that humic-hydrogels could be innovative biomaterials for several agricultural applications.

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