Biostimulation of humic acids on Lepidium sativum L. regulated by their content of stable phenolic O⋅ radicals

Background: Humic acid affects plant growth. Its source and structure may play a central role to its functionality. The relationship between humic acid and plant bioactivity is still unclear. This study investigated the biostimulation effects of two natural humic acids derived from soil (SHA) and lignite (LHA) on Lepidium sativum in comparison to a synthetic humic acid model (HALP) with known structure. Results: All humic acids positively affected cress seed germination and root elongation. Greater root hairs density and dry matter, compared to control, were observed using concentration of 5 mg L−1 for HALP, 50 mg L−1 for LHA, and 100 mg L−1 for SHA. The germination index was the largest (698% more effective than control) with 50 mg L−1 of SHA, while it was 528% for LHA, and 493% for HALP at 5 mg L−1. SHA contained the lowest aromatic and phenolic C content, the largest pK2 value of 9.0 (7.7 for LHA and 7.6 for HALP), the least ratio between the aromaticity index and lignin ratio (ARM/LigR) of 0.15 (0.66 for LHA and 129.92 for HALP), and at pH 6.3 the lowest amount of free radicals with a value of 0.567 × 1017 spin g−1 (1.670 × 1017 and 1.780 × 1017 spin g−1 for LHA and HALP, respectively), with the greatest g value of 2.0039 (2.0035 for LHA and 2.0037 for HALP). Conclusions: The overall chemical structure of humic acids exerted a biostimulation of cress plantlets. The level of the intrinsic stable free radicals identified by EPR in the humic acids resulted well correlated to the ARM/LigR ratio calculated by NMR. Our results suggested that HA biostimulation effect is related to its applied concentration, which is limited by its free radical content. The modulation of the humic supramolecular structure by ROS and organic acids in root exudates can determine the release of bioactive humic molecules. When the content of the intrinsic humic free radicals is high, possible molecular coupling of the bioactive humic molecules may hinder their biostimulation activity. In such cases, a low humic acid concentration appears to be required to achieve the optimum biostimulation effects.