Introduction: Ajwain (Carum copticum L.) is a medicinal herb from the Apiaceae family that is widely cultivated in Europe, Iran, India, Egypt, and Pakistan. Pharmacological and clinical trials indicate that C. copticum is effective in treating pharyngitis, showcasing benefits like antioxidant, antiparasitic, expectorant, and antilithiatic effects. Although this herb can withstand damage from water stress, regular and sufficient watering will increase its yield. Inadequate water supply, particularly in terms of quantity and allocation throughout growth and developmental stages, leads to reduced achievement of the crop's intrinsic potential, eventually decreasing the farmer's production and livelihood. Water deprivation is a major limiting factor in semi-arid and arid regions, affecting all aspects of plant morphology, physiology, growth, development, and yield. Chitosan facilitates the formation of secondary compounds like polyphenolics, flavonoids, and lignin, which are beneficial for various herb species, and for the activation of herb defense enzymes. Humic acid can especially influence the growth of medicinal herbs by affecting cell membrane permeability, metabolic pathways, cellular respiration, photosynthetic efficiency, enzyme activation, and cell growth. Melatonin improves plant tolerance against various environmental stresses, including extreme temperatures, cold, salinity, chemical compounds, heavy metals, and pathogens. The use of selenium improve photosynthesis, antioxidant activity, carbohydrate accumulation, and synthesis of secondary metabolites under drought stress. Therefore, this study aims to assess the effects of stress modifiers; namely chitosan, humic acid, melatonin, and selenium on the physiological and yield traits of ajwain when exposed under water stress.
Materials and Methods: A two-year field trial was performed in the 2022 and 2023 years of cultivation at the University of Mohaghegh Ardabili in Ardabil, Iran. The experimental design was factorial, using a complete randomized block design with three replications. The factors were the stress modulator treatment (MEN: melatonin, CHN: chitosan, HUC: humic acid, and SEM: selenium and CON: control) and irrigation treatments (watering at 100%, 75%, and 50% field capacity (FC) as normal,  moderate deficit irrigation and severe deficit irrigation, respectively). All plots consisted of 8 rows of herbs spaced 20 cm from each other, with plants spaced 40 cm apart within each row. Seeds of ajwain (Carum copticum L.) were sown on March 20, 2022 and 2023. During the vegetative phase of ajwain, foliar spraying was applied on the leaves in the three stages at ten-day intervals, coinciding with periods of moisture stress. Weeds were carefully controlled when needed.  The soil was collected from a depth of 0-30 cm, analyzed, and found to be silty clay (36% Silt, 33% Clay, 31% Sand) with an electrical conductivity of 0.37 dS /m, pH of 7.8, 1.04% of organic matter, 0.11% total N, 10.04, and 344 mg kg⁻¹ available P and K, respectively. A 2-year investigation analysis of variance (ANOVA) was conducted operating the general linear model (GLM) (SAS 9.1.3) combining the findings from 2022 and 2023. The LSD trial was evaluated for comparing means at p < 0.05.
Results and Discussion: The results showed that under normal irrigation conditions, ajwain plants sprayed with melatonin had the seed yield (733.35 kg/ha), and essential oil yield (22.11 kg/ha). The maximum absorption of nitrogen, phosphorus and potassium (3.09%, 0.40%, and 1.65%, respectively) was obtained under normal irrigation conditions and foliar spraying with melatonin. While the highest amount of total phenol and flavonoid (29.55 and 3.33 mg/g dw, respectively), DPPH radical inhibition percentage, nitric acid radical inhibition percentage, and superoxide radical collection capacity were 78.85, 67.88, and 138.47% respectively, which were observed in the conditions of severe stress and foliar spraying with melatonin. Also, melatonin foliar application improved the amount of essential oil of ajwain (3.30%) under medium stress conditions. Water stress hinders plant growth and development by impacting many physiologic processes, including nutrient absorption, stomata function, transpiration rate, sugar synthesis, hormone production, assimilation, and photosynthesis. Stressful environments were shown to accelerate the synthesis of EOs, as evidenced by the increased EO content under stress. This is likely due to the role of secondary metabolites in helping plants withstand ecological stresses. The application of MEN, CHA, HUM, and SEM directly enhanced photosynthetic pigments, improved photosynthetic efficiency, increased the concentration and activity of RuBisCo, and promoted ATP and NADPH production to enhance plant productivity. Drought stress triggers the production of secondary metabolites and essential oils, which act as protective molecules against environmental pressures. The biostimulant composition, which includes macro and microelements, vitamins, amino acids, and growth hormones, plays a crucial role in promoting plant growth, as well as the percentage and yield of essential oil. When soil moisture availability is restricted, the uptake of water, the solubility of minerals in the soil, and their absorption by roots decline, leading to adverse effects on photosynthesis and transpiration rates. In these circumstances, as previously demonstrated, the foliar application of MEN, CHA, HUM, and SEM positively influences stomatal opening adjustment and chlorophyll abundance, facilitating the growth, division, and elongation of herb root cells. As a result, foliar spraying of MEN, CHA, HUM, and SEM likely maximized this influence by reactivating various biosynthetic and biological pathways and genes that regulate secondary metabolite production, while also enhancing the glands that manufacture essential oils in the flowers and leaves of plants under water restriction. Since hydrocarbons are required as precursors for the production of phenols, an increase in their rate and concentration influences the enhancement of phenolic combinations. Applying MEN, and CHA has been shown to be beneficial in reducing the impact of free radicals and increasing the production of phenolic compounds. This effect may be related to the role of MEN, and CHA in improving antioxidant activity against ROS.
Conclusion: Overall, among all the tested stress modulators, MEN and CHN emerged as valuable tools for improving growth, physiological characteristics, essential oil, and fixed production in ajwain herb. They can help reduce the detrimental impacts of water stress and establish a sustainable framework for optimizing ajwain performance even under moisture-deficit conditions in semi-arid and arid regions of Iran.