Effect of Liquid Humalite on Symbiotic Nitrogen Fixation in Red Clover (Trifolium pratense L.)

Symbiotic nitrogen fixation (SNF) in forage legumes is a key biological process that provides nitrogen inputs and enhances soil fertility in agroecosystems. Enhancing SNF efficiency helps reduce dependence on synthetic nitrogen fertilizers and improve nitrogen use efficiency. Humic substances (HS) have been reported to stimulate root development and nutrient acquisition in different crop species. However, their effect on root nodulation and SNF in forage legumes remains poorly understood. This study investigated the effects of a humic acid-based soil amendment (liquid Humalite) on root growth, nodulation, SNF, and plant nitrogen acquisition in red clover. Red clover seedlings were grown in a modified Leonard jar system placed under controlled environmental conditions and treated with 0.1, 0.2, 0.4, and 0.8% (v/v) liquid Humalite. Plant biomass, root morphological traits, nodulation parameters, SNF, and shoot nitrogen accumulation were assessed after 6 weeks. SNF was quantified using the 15N-isotope dilution method. Application of liquid Humalite significantly affected plant growth and SNF responses in red clover. The 0.2% liquid Humalite treatment significantly increased root and total plant biomass by 35% compared to the untreated control. Total root length, surface area, and volume increased by 20–25% at 0.2% liquid Humalite. Nodule number, nodule dry weight, shoot nitrogen concentration, and shoot C:N ratio did not differ among treatments. However, the percent nitrogen derived from the atmosphere (%Ndfa) was significantly higher under the 0.2% liquid Humalite treatment (47%) as compared to the untreated control (28%). Under the 0.2% liquid Humalite, total fixed and accumulated shoot nitrogen increased by 134% and 37%, respectively, compared to the control. Overall, these findings indicate that humic substances may enhance SNF and nitrogen accumulation in red clover, highlighting their potential as a management strategy to improve forage productivity.