Ziziphora clinopodioides L. is a valuable medicinal and aromatic plant; however, its special requirements for proper growth and enhanced secondary metabolite composition have limited its production and availability. The lack of appropriate fertilizer dosage recommendations is one of these limiting factors for ex situ conservation and large-scale cultivation. This study investigated the effects of nitrogen (N) fertilization on Z. clinopodioides in both open field and unheated greenhouse conditions. The determined research factor was N dosage (0, 40, 80, and 120 kg N ha⁻¹ in the field) and (0, 200, 400, and 600 mg N pot⁻¹ in the greenhouse). It was found that Z. clinopodioides plants could grow successfully outside their natural habitat with sufficient N fertilization yielding a satisfactory amount of metabolites per plantation area. Specifically, among the tested dosages, the 120 kg N ha⁻¹ (which showed no significant difference from 80 kg N ha⁻¹) and 400 mg N pot⁻¹ fertilizers increased the aboveground dry and fresh biomass, essential oil (EO) content, and yield. Nitrogen fertilization showed a direct correlation with menthone, neo-menthol, menthol, pulegone, E-ocimenone, and spathulenol, but a negative correlation with α-pinene, β-pinene, limonene, 1,8-cineol, trans-pulegol, and iso-menthone. This study is the first to evaluate the effects of N fertilization on the genus Ziziphora. The results indicate that utilizing N fertilizer at a moderate rate (80 kg N ha⁻¹ in the field and 400 mg N pot⁻¹ in the greenhouse) can improve the agronomic traits and EO of Z. clinopodioides.

Nitrogen (N) availability often limits primary productivity in terrestrial ecosystems, and arbuscular mycorrhizal fungi (AMF) can enhance plant N acquisition. This study investigated the interactive effects of N fertilization and AMF inoculation on N uptake, plant performance and phenolic acid content in Hyssopus officinalis L., with the aim of promoting sustainable N management in H. officinalis cultivation. A factorial randomized complete block design was employed to evaluate four AMF inoculation strategies (no inoculation, root inoculation, soil inoculation and combined root–soil inoculation) across three N application rates (0, 0.5 and 1,1 g N pot⁻¹ (7 L)) in a controlled greenhouse environment. Combined root and soil AMF inoculation alongside moderate N fertilization (0.5 mg N pot⁻¹) optimized N use efficiency, maximizing plant biomass and bioactive compound production. Compared to non-inoculated controls, this treatment combination increased N uptake by 30%, phosphorus uptake by 24% and potassium uptake by 22%. AMF colonization increased chlorophyll content and total phenolic compounds under moderate N supply. However, excessive N application (1 g N pot⁻¹) reduced AMF effectiveness and secondary metabolite accumulation. Notably, AMF inoculation without N fertilization yielded the highest levels of anthocyanin and salicylic acid, indicating differential N-dependent regulation of specific biosynthetic pathways. The interaction between AMF and N demonstrated that moderate N fertilization (0.5 g N pot⁻¹) combined with dual inoculation strategies can reduce total N input requirements by 50%, while maintaining optimal plant performance. These findings provide practical insights for developing N-efficient cultivation protocols in medicinal plant production systems, contributing to sustainable agricultural practices that minimize environmental N losses.