Negative effects of artemisinin on phosphorus solubilizing bacteria in vitro

The anti-malarial drug artemisinin is extracted from the leaves of Artemisia annua L. Due to toxicity to some microorganisms, the release of artemisinin from this medicinal plant in commercial cultivation might produce a potential risk for phosphorus (P) solubilizing bacteria (PSB). Therefore, the growth, P mobilization, and proton and organic acid efflux by two PSB isolates, Bacillus subtilis and Pseudomonas fluorescens, obtained from the soil without growing A. annua L. in history in the region for growing A. annua L., Chongqing, China, were studied through soil and solution incubations with different nominal concentrations of artemisinin (0, 2.5, 5.0, and 10.0 mg/kg or mg/L). Addition of artemisinin into soil and culture solutions decreased significantly the number of PSB except P. fluorescens at a low artemisinin concentration (2.5 mg/L) in culture solution which remained unchanged in comparison with the control (without artemisinin). This suggests high artemisinin inhibited the cell division or led to the death of PSB, and the different species responded differently to artemisinin. Compared with original soil, PSB inoculation significantly increased Olsen P, whilst the addition of artemisinin decreased this P form in soil. There was a positive correlation between the number of PSB and Olsen P content in soils (r² = 0.824, n = 8), indicating the involvement of PSB in P mobilization of insoluble minerals. Oxalate and acetate were commonly found in the bacterial culture solutions, which accounted for 73.6-84.4% of all organic acids in the culture medium without artemisinin. Malate was detected in the culture solution of B. subtilis, and citrate and succinate in P. fluorescens. The percentage of tricalcium phosphate solubilization (PTPS) positively correlated to the concentrations of protons and all organic acids (r²_proton＝0.901, n＝8, P＜0.01; r²_{organic acids}＝0.923, n＝8, P＜0.01). The concentrations of protons, organic acids and soluble inorganic P in culture solutions, and PTPS were decreased simultaneously as nominal artemisinin concentrations increased. For these decreases it implies the metabolic inhibition and the death of PSB caused by artemisinin could be the main reasons for the less efflux of protons and organic acids, presumably resulting in the decreased ability of PSB to mobilize inorganic P. Therefore, artemisinin released from A. annua L. in commercial and continual cultivation could adversely affect the community structure and inorganic P mobilization of PSB in soils.