Molecular characteristics of humic substances from different origins and their effects on growth and metabolism of Pinus laricio callus

Humic susbtances (HS) are increasingly applied as biostimulants in agriculture, though their mechanism of action is not yet completely understood due to their complex and heterogeneous composition. Here, we isolated thirteen different humic and fulvic acids from different sources, such as soils, composts, peat, leonardite and lignite, and characterized the distribution of their carbon components by 13 C-CPMAS–NMR spectroscopy. Callus of Pinus Laricio , as a model plant species, was treated with different humic extracts and its growth and content of carbohydrates, phenols, and enzyme related to the nitrogen metabolism (invertase, glutamine synthetase, glutamate synthase, phosphoenoly pyruvate carboxylase, malate dehydrogenase) and stress resistance (catalase) were monitored. While a multivariate statistical analysis of NMR results well-separated the HS characteristics based on their origin, humic materials generally increased callus growth, as expected, with largest effects being exerted by the mostly polar humic acids from composts. However, the rest of measured parameters were not linearly related to hydrophobicity and aromaticity of humic isolates as well as their origin, but their conformational dynamics had to be advocated to explain their effects on callus cellular components. This work confirms that HS of various origin can act as sustainable biostimulants of plant growth, though the comprehension of their effects on plants biochemical activities requires further research and additional understanding of their molecular composition and conformational behaviour. Graphical Abstract