Enhancement of Bioactive Compounds Concentration in Opuntia Cactus Using Humic Acid, Seaweed Extract, and Mycorrhizal Fungi

Background and Objectives
Climate change and global warming have severely impacted agricultural productivity and crop quality worldwide, especially in arid and semi-arid regions where water scarcity, soil degradation, and increasing environmental stresses challenge sustainable farming. Opuntia species are recognized as strategic multipurpose crops due to their drought tolerance, nutritional and medicinal values, and ecological functions such as soil stabilization and desertification control. Among their notable traits, Opuntia species produce significant amounts of bioactive compounds- especially antioxidants like flavonoids, phenolics, carotenoids, and vitamin C- which are crucial for human health and plant stress resilience. Enhancement of these bioactive compounds can add substantial value to cactus products in pharmaceutical and functional food industries. Amidst efforts to improve the growth and phytochemical content of Opuntia, the integrated use of soil amendments including humic acid, seaweed extracts, and arbuscular mycorrhizal fungi has demonstrated promising results in various crops by improving nutrients uptake, physiological traits, and stress tolerance. However, there remain gaps in the comprehensive understanding of how these biostimulants affect the bioactive profile of Opuntia under controlled conditions. This study aimed to investigate the individual and combined effects of humic acid, seaweed extracts, and mycorrhizal fungi on the antioxidant capacity, flavonoid, phenol, carotenoid, and vitamin C contents of Opuntia plants, while also assessing related morphological and physiological parameters, to provide insights for sustainable enhancement of cactus quality and productivity.
Materials and Methods
The experiment was conducted under controlled pot culture conditions, applying a factorial design with eight treatments: 1- control (no amendment), 2- mycorrhiza inoculation (My), 3- seaweed extract application (SW), 4- humic acid use (HA), 5- HA + SW, 6- My + SW, 7- My + HA, and 8- My + HA + SW. Each treatment was applied according to standardized protocols for inoculation or soil amendment. Morphological traits including plant height, new cladode (pad) number, cladode thickness, and leaf surface area were measured. Physiological analyses encompassed chlorophyll (a, b, and total), carotenoid content, and relative water content. Bioactive compounds were quantified by spectrophotometric methods: total phenolics, total flavonoids, vitamin C content, and antioxidant capacity using DPPH radical scavenging assay. Data were statistically analyzed by SPSS software.
Results
The application of mycorrhiza, seaweed extract, and humic acid showed significant positive effects on both growth and biochemical parameters in Opuntia plants, with notable differences depending on individual or combined treatments. The treatment with SW induced the highest increase in plant height, up to 30% greater than the control (19.7 cm vs. 15.2 cm), and significantly increased the number of new cladodes to about 6 pads per plant, more than doubling the control number (~2.3 pads). The combied treatment of My + SW also showed synergistic benefits, especially in enhancing leaf surface area by over 80% compared to the control, indicating improved photosynthetic capacity and plant vigor. Cladode thickness was maximized under combined HA + SW, reaching a 54% increase over the control, suggesting firmer and better-developed plant structures. The chlorophyll content responded robustly to HA and SW treatments, with HA + SW combination achieving a remarkable 123% increase in total chlorophyll relative to the control. Carotenoids content, essential for photoprotection, was most elevated in mycorrhiza treated plants (84% increase), highlighting the role of symbiotic fungi in stimulating antioxidant pigment biosynthesis. The relative water content was also improved in HA + SW treatment, reflective of better water retention and stress tolerance. The total phenolic content exhibited substantial elevation in mycorrhiza inoculated plants, with nearly 49% increase compared to the control, indicating enhanced secondary metabolite production linked to plant defense and health-promoting attributes. The accumulation of flavonoids was highest in seaweed extract treated plants, with 42% higher levels than control, supporting the stimulatory role of natural biostimulants on phenolic biosynthesis. The content of vitamin C was dramatically increased (by 85%) in the My + HA treatment, suggesting synergistic enhancement of antioxidant defenses and nutrient quality when both mycorrhiza and humic acid were applied. Antioxidant capacity reflected these biochemical enhancements. Humic acid treatment exhibited the highest DPPH radical scavenging activity, exceeding the control by 5%. Combinations containing My and HA maintained elevated antioxidant activity levels, confirming the role of integrated biostimulant treatments in improving the overall defensive capacity of the plant against oxidative stress.
Conclusion
This comprehensive study demonstrates that application of humic acid, seaweed extract, and mycorrhizal fungi- either individually or combined- significantly improved the morphological development, physiological health, and bioactive compound content of Opuntia cactus plants. While seaweed extract promoted vegetative growth and flavonoid synthesis, humic acid primarily enhanced photosynthetic pigments and antioxidant capacity. Mycorrhizal inoculation notably increased carotenoids, total phenolics, and vitamin C content. The combined use of mycorrhiza with humic acid yielded the most pronounced increase in contents of vitamin C and phenolic compounds, indicating a synergistic effect on plant biochemical quality. These findings underscore the potential of integrated biostimulant applications as sustainable agronomic strategies to improve crop quality and stress resilience in arid and semi-arid regions where Opuntia spp. are cultivated. Enhancing antioxidant and bioactive compounds content not only benefits plant health but also adds value to cactus products for pharmaceutical and functional food applications. Future field studies under diverse environmental conditions are encouraged to validate these results and optimize amendment protocols for large-scale adoption.
Author Contributions
Conceptualization, M.S. and A.H.; methodology, M.S. and S.A.; software, M.N.H; validation, M.N.H.; formal analysis, M.S. and S.A.; investigation, M.S., S.A., M.N.H., and A.H.; resources, M.N.H. and A.H.; data curation, M.S. and S.A.; writing—original draft preparation, M.N.H. and A.H.; writing–review and editing, M.N.H. and A.H.; visualization, M.N.H.; supervision, A.H.; project administration, A.H.; funding acquisition, A.H. All authors have read and agreed to the published version of the manuscript.
Data Availability Statement
Data is available on reasonable request from the authors.
Acknowledgements
This paper is published as a part of a Master's thesis supported by the Vice Chancellor for Research and Technology of the University of Zanjan, Zanjan, Iran. The authors are thankful to the University of Zanjan for financial supports.
Conflict of interest
The authors declare no conflict of interest.
Ethical considerations
The authors avoided data fabrication, falsification, plagiarism, and misconduct.