Salvia miltiorrhiza (S. miltiorrhiza) Bunge is one of the most economically important medicinal crops in China. In traditional Chinese medicine, its root is used as an important ingredient in formulas for treatment of atherosclerosis-related disorders. The continuous cropping of S. miltiorrhiza increases the proportion of dried seedlings and decreases the biomass of the shoots and roots and the contents of active components. In this study, three field experiments were conducted to investigate the effects of Glomus mosseae (G. mosseae) inoculation on the growth and contents of active ingredients and nutrients in continuously cropped S. miltiorrhiza. The results showed that inoculation with G. mosseae increased the shoot biomass of S. miltiorrhiza by 48.1% and the root biomass by 39.2%, and decreased the dried seedling rate by nearly 75%. Inoculation with G. mosseae also increased the salvianolic acid B concentration by 21.9% in the shoots and 9.2% in the roots of S. miltiorrhiza, and also significantly increased Mn concentration in the roots and shoots (by 65.1% and 93.4%, respectively) and Fe concentration in the roots (by 75%). The accumulation of salvianolic acid B, Mn, and Fe in G. mosseae inoculated S. miltiorrhiza may be a mechanism that imparts tolerance to continuous cropping. Inoculation of S. miltiorrhiza with G. mosseae can serve as an effective approach of biocontrol to improve the performance of continuously cropped S. miltiorrhiza.

Nitrogen (N) strongly affects plant growth and metabolism. Although ammonium toxicity has been reported, the effects of nitrogen on shikonin biosynthesis remain obscure. In this study, we tested four different concentrations of NH₄⁺ on Arnebia euchroma hairy roots (AEHR) to clarify the influence of NH₄⁺ on the growth of AEHR and on shikonin accumulation in them and the possible mechanisms. The results showed that compared with the 0% NH₄⁺ treatment (only nitrate as a nitrogen source), the 10% NH₄⁺ treatment increased the fresh weight and the dry weight of AEHR and promoted the synthesis of shikonins. In contrast, the 20% NH₄⁺ treatment started to show inhibition effects on the growth of and shikonin accumulation in AEHR, and the 30% NH₄⁺ treatment exhibited the strongest inhibition effects. With an increased percentage of NH₄⁺, the AEHR became shorter and thicker, with more branches. To further elucidate the mechanisms, we analyzed the time course of nitrogen assimilation, the gene expression level of key enzymes involved in shikonin biosynthesis pathway, and the content of various endogenous hormones in the presence of toxic NH₄⁺ concentrations. The results indicated that auxin and cytokinin might regulate the growth and architecture of AEHR under NH₄⁺ toxicity and revealed that the jasmonate level was reduced along with the inhibition of shikonin biosynthesis. This first comprehensive investigation of the effects of the ammonium/nitrate ratio on shikonin biosynthesis not only provides valuable data for optimizing the in vitro culture of A. euchroma and its shikonin production, but also suggests potential fertilizing strategies for its cultivation.