Role of NO3−/NH4+ ratio and humic substances in modulating photosynthetic pigments and improving essential oil quantity and quality of Melissa officinalis L

Melissa officinalis L. (lemon balm) has become a valuable medicinal plant due to its diverse bioactive compounds, highlighting the need for strategies to optimize/increase their production. This study aimed to investigate the effect of different sources of nitrogen (25/75, 50/50, and 75/25 Nitrate/Ammonium: NO 3 - /NH 4 + ratios) and humic substances: HS (0, 10, 20, 40, and 60 mg L -1 ) on some morphophysiological properties and essential oil (EO) compositions of M. officinalis . The results showed that different ratios of NO 3 - /NH 4 + and different concentrations of HS and their interaction had a significant effect on morphological characteristics and EO compositions. The greatest aerial part length (47.7 cm) was recorded in 50/50 NO 3 - /NH 4 + ratio and HS 40 mg L -1 (50/50-40), while the highest root length (38.3 cm) and root dry weight (5.3 g) occurred in treatment 25/75-0. In addition, the highest chlorophyll a, b, total, and total carotenoid content were observed under treatment 50/50-60. The results indicated that the NO 3 - /NH 4 + ratio and humic concentration significantly affected the percentage and yield of EO of M. officinalis , showing an increasing trend particularly at intermediate levels, with the highest percentage (1 %) under treatment 75/50-60 and the greatest yield (9.3 ml m -2 ) under treatment 50/50-60. Based on the results of GC-MS analysis, 26 compounds were identified, 3 of which included citronellal, neral, and geranial as the main compounds. Their concentrations increased with higher NO 3 - /NH 4 + ratio and humic levels, reaching maximum values of 7.7%, 33.2%, and 47.8%, respectively, under treatment 75/25-60. Multivariate analysis of treatments placed the chemical compounds into 5 separate clusters, with the three main compounds, along with 6-methyl-5-hepten-2-one, linalool, nerol, and β-Caryophyllene, in 5 cluster, with the highest levels of these compounds observed in the 75/25-60 treatment. In general, high NO 3 - /NH 4 + ratio and the presence of HS increase key M. officinalis EO compounds. • Greatest aerial part length (47.67 cm) recorded in 50/50 NO 3 - /NH 4 + with 40 mg L -1 humic substances. • Humic substances improved chlorophyll and carotenoid contents in Melissa officinalis. • Highest EO percentage (1.04%) achieved with 75/25 NO 3 - /NH 4 + and 60 mg L -1 humic substances. • Maximum EO yield (9.30 mL/m 2 ) observed under 50/50 NO 3 - /NH 4 + and 60 mg L -1 humic substances. • Citronellal, neral, and geranial peaked under 75/25 NO 3 - /NH 4 + + 60 mg L -1 humic substances.