Determination of Optimal Gamma Irradiation Dose of Humic Acid for Improving Some Agronomic Traits and Yield of Bell Pepper Using Fourier Transform Infrared Spectroscopy (FTIR)

Background and Objectives
Nowadays, the adoption of environmentally friendly and sustainable practices in horticultural crop production has become a critical necessity. The use of soil amendments and biostimulants, particularly humic substances, has gained considerable attention as an effective strategy to enhance plant growth, nutrient efficiency, and overall crop productivity. Humic acid (HA), as a major component of soil organic matter, is increasingly recognized for its ability to improve soil physicochemical properties, increase nutrient bioavailability, stimulate root development, enhance chlorophyll concentration, and boost photosynthetic efficiency. These effects collectively lead to stronger vegetative growth and higher yields. However, the biological efficacy of humic acid is highly dependent on its molecular structure, molecular weight, functional group composition, concentration, and application method. Recent evidence suggests that physical or chemical modification of humic substances can significantly alter their bioactivity. Among various modification techniques, gamma irradiation has emerged as a promising, clean, and residue-free method for inducing molecular fragmentation, oxidation, and structural changes in organic compounds. Gamma irradiation can break chemical bonds in hydrocarbon and aliphatic chains, reduce molecular weight, increase the abundance of functional groups such as hydroxyl and carboxyl, and improve solubility. These structural modifications potentially enhance the interaction of humic acid with plant roots and its capacity for cellular signaling. Despite the recognized benefits of humic acid in bell pepper (Capsicum annuum L.) cultivation–an important greenhouse vegetable with a global market value of approximately $7.5 billion in 2024–no previous study has investigated the combined effect of gamma-irradiated humic acid and its application rate on bell pepper growth and yield. Therefore, this research was conducted to determine the optimal gamma irradiation dose for modifying the molecular structure of humic acid and to evaluate its interaction with different application levels on key agronomic traits and yield of bell pepper under greenhouse conditions.
Materials and Methods
This experiment was carried out in a research greenhouse at the University of Zanjan, Iran, under controlled environmental conditions (average temperature 17.5–34.3 ^°C, relative humidity 24–33%, and natural light intensity ~10,000 lux). A commercial liquid humic acid (containing 10% humic acid, 3% fulvic acid, 3% potassium, and 5% nitrogen) was subjected to gamma irradiation at doses of 0, 500, 1000, 2000, and 4000 Gy using a calibrated gamma radiation source at the Nuclear Agricultural Research Institute in Karaj, Iran. The irradiation process was performed prior to soil application. The study was done as a factorial experiment based on a completely randomized design and three replications. Treatments consisted of combinations of five gamma doses and five humic acid application rates (0, 1.5, 3.0, 4.5, and 6.0 g HA per kg of soil). Humic acid was applied to the soil in three splits during the plant growth period. Bell pepper seeds (cv. Nirvin) were sown in plastic pots filled with 5 kg sandy loam soil. To evaluate structural changes in humic acid induced by gamma irradiation, Fourier Transform Infrared Spectroscopy (FTIR) analysis was performed. Approximately 2 mg of each HA sample (non-irradiated and irradiated at different doses) was mixed with 200 mg of spectroscopic-grade KBr and pressed into transparent pellets. FTIR spectra were recorded in the wavenumber range of 400–4000 cm⁻¹ with a resolution of 4 cm⁻¹ and 32 scans per sample. Major functional groups including hydroxyl (O–H), carbonyl (C=O), carboxyl (COOH), aromatic (C=C), and aliphatic (C–H) were compared between irradiated and non-irradiated samples. Vegetative and yield parameters of plants were measured 92 days after cultivation. Growth parameters included plant height, stem diameter, leaf number, leaf area, root dry weight, shoot dry weight, and total dry weight. Yield parameters included fruit number per plant, average fruit fresh weight, average fruit dry weight, and total marketable yield per plant. Data were analyzed using SPSS (Ver. 19) and means were compared using Duncan's multiple range test at p<0.01.
Results
Analysis of variance revealed that the simple effects of gamma irradiation dose and humic acid application rate were highly significant (p<0.01) for all measured traits, including fruit number, fruit fresh weight, fruit dry weight, root dry weight, total dry weight, plant height, leaf number, leaf area, and shoot dry weight. The interaction effect (gamma dose × HA rate) was also significant (p< 0.01) for fruit number, fruit fresh weight, fruit dry weight, Root DW, Total DW, and leaf area, but non-significant for plant height, leaf number, and shoot dry weight. FTIR analysis revealed that gamma irradiation at 2000 Gy induced the most favorable structural modifications, including increased hydroxyl and carboxyl functional groups, reduced aliphatic C–H peaks (indicating fragmentation of hydrocarbon chains), decreased aromatic C=C content (suggesting breakdown of large aromatic rings), and overall reduction in molecular weight, leading to improved solubility. In contrast, the 4000 Gy dose caused extensive degradation, with a sharp reduction in all peak intensities, near-complete loss of carboxyl structure, dehydroxylation, and formation of new peaks corresponding to adsorbed CO₂, indicating severe molecular destruction. Among all treatments, 3 g HA irradiated at 2000 Gy/kg soil exhibited the best performance. This treatment increased fruit fresh weight by 15.6%, fruit dry weight by 19.6%, leaf area by 39%, total dry weight by 54.5%, and fruit number by 62% as compared to 1.5 g non-irradiated HA. The 4000 Gy dose significantly reduced all measured traits compared to the 2000 Gy dose, confirming the detrimental effects of excessive irradiation. For instance, in 1.5 g HA at 4000 Gy, leaf area dropped to 214.4 cm²/plant and total dry weight decreased to 7.61 g/plant, which were even lower than those of the non-irradiated control treatments.
Conclusion
Based on the comprehensive evaluation of structural modifications and plant responses, gamma irradiation at 2000 Gy was identified as the optimal dose for improving the molecular structure of humic acid. At this dose, desirable structural changes–including molecular weight reduction, increased hydroxyl and carboxyl functional groups, and enhanced solubility–were achieved, while lower doses (500 and 1000 Gy) produced insufficient modifications and the higher dose (4000 Gy) caused extensive degradation. The application rate of 3 g irradiated humic acid per kg of soil was determined as the optimal concentration, as higher rates did not provide additional benefits and, in some cases, led to relative yield reduction, indicating a non-linear dose-response relationship. The treatment of 3 g HA irradiated at 2000 Gy/kg soil was identified as the best treatment, with increases of 15.6% in fruit fresh weight, 19.6% in total dry weight, and 39% in leaf area compared to the control. FTIR analysis clearly demonstrated that gamma irradiation at 2000 Gy effectively fragmented humic acid molecular chains, increased active functional groups, and improved solubility, which in turn enhanced HA uptake and bio-efficacy by the plant. The 4000 Gy dose, due to extensive molecular destruction, caused a significant decline in yield compared to the 2000 Gy treatment, emphasizing the necessity of precise dose optimization before commercial application. Overall, gamma irradiation technology can serve as a clean, cost-effective, efficient, and residue-free method to enhance the bioactivity of humic acid as a biostimulant in greenhouse bell pepper cultivation. Furthermore, it is recommended that future research investigate the effects of this optimized treatment on fruit quality attributes (vitamin C, soluble solids, anthocyanins, carotenoids, and nitrate/nitrite reduction), nutrients uptake, soil biological and chemical properties, and field-scale validation under diverse climatic conditions.