| Nowadays, approximately 40% of the population in the world are now under the threat of malaria infection. So far artemisinin is the best choice, recommended by the World Health Organization (WHO), to cure this disease. It can only be extracted from Artemisia annua L., a herb medicine, which has been used in China for over 2000 years.This medicinal plant is now intensively grown on a large scale for commercial use in China, for its high artemisinin concentrations in the leaves. About 90% of A. annua L. and artemisinin in the world was grown and produced in China. Three Gorges area of Chongqing is the place for growing A.annua L. with high-quality and the yield of artemisinin and planting area of A. annua L. accounted for 70% and 80% respectively in China. Many studies from abroad show that A. annua L. can release various kinds of allelochemicals into soils either via dead plant decomposition, rain leaching, or root exudation in the growth period, inhibiting the growth and development of succeeding and adjacent crops. Soil microorganism is an important component of soil, motivating biochemistry reaction of the soil, such as soil organic matter mineralization, poison degradation and nutrient transformation and supply. Therefore, it is necessary to understand allelopathic effect of A.annua L.cultivation on soil microorganism. It is crucial to estimate scientifically the ecological risks for the cultivation of A. annua L. as to reduce the harmfulness for the succeeding and adjacent crops, as well as maintain the fertility and sustainability of soil for the cultivation of A. annua L.In the current research, based on field investigation and field experiments, we primarily chose the wild and cultivated A. annua L.’s soil as the experimental objects to investigate the content and distribution of artemisinin derivatives, and the propenties of soil physical chemistry and microorganism in soil by routine analysis, GC-MS and Illumina MiSeq pyrosequencing. Pot experiments were carried out subsequently to study the effect of artemisinin and litter decomposition from A. annua L. on soil microbial community structure and diversity by routine analysis of phosphorus lipid fatty acids (PLFAs), and 454 pyrosequencing. Then, we had further studied the allelopathic effect and mechanism of artemisinin on some functional soil microbes, mainly including rhizobium, azotobacters and ectomycorrhizal fungi. The main results are as follows:1 Artemisinin and flavonoids in root region soils of wild A.annua L. and their influence on microbesThe highest contents of artemisin in leaf was found at the squaring period. The biomass of leaves decreased significantly from this developmental stage, and reached to 55.13% of the maximum at full-bloom period. The allelochemical contents changed in following sequence:squaring period>first flowering period>full-bloom period> Vegetative growth period (artemsinin in leaves and root region soil) and stem>leaf> root>flower (flavonoids in plants). The mean content of deoxyartemsinin was highest and followed by artemisic acid and artemisinin in soil and the sum of artemisinin derivatives was 516.93 ug·kg-1 dry soil. All of the three compounds were much higher in both root surface soil and rhizosphere soil than non-rhizosphere, which showed highest in squaring period compared to other growth periods. Soil flavonoids changed in the sequence:root surface>rhizosphere>non-rhizosphere, which increased continually as the growth period of A. annua L. prolonged and reached highest at full-bloom stage (434.77 μg·kg-1 dry soil). Flavonoids could thus be released into soils through root exudation. The number of bacteria and actinomycetes showed significant negative correlations with artemisinin (r=-0.508* and r=-0.478*, n=24). There was also a negative correlation between deoxyartemsinin contents and actinomycete numbers (r=-0.528**, n=24). But soil flavonoids and artemisic acid contents were not significantly correlated with bacteria, fungi and actinomycetes numbers. In summary, artemisinin derivatives released from A.annua L. might inhibit microbial growth and reproduction whereby to influence biochemical reactions in soils.2 The influence of A.annua L. Cultivation on enzyme activities and microbial communities in soilAfter continuous cropping of A.annua L., indexes like soil microbial biomass carbon and nitrogen, pH, activity of dehydrogenase and urease, and the number of 16S rRNA sequence were obviously decreased. New cropping had an evident increased in the number of ITS sequence and dominant index in contrast to fungal and bacteria phylotypes, richness index (Ace and Chao) and diversity index of of fungal community, and especially the relative abundance of Ascomycota was up to 95.28%. Cropping succession A. annua L. (3-5 years) declined the sequences number of soil fungi, and two kinds of pathogen of the artemisia were detected, namely Erysiphe artemisiae and Puccinia tanaceti. Principal component analysis (PCA) and cluster analysis showed there were significant differences among microorganism communities in soils with A. annua L.’different planting years. The results obtained show that continuous cropping of A.annua L. would deteriorate soil environment, reduce the number of soil microorganisms and community, and could cause an increase in the population of fungi and soil microbial community simplification, and increase the incidence of crop disease.3 The influence of artemisinin or litter from A. annua L. on soil microorganism community structure and diversityThe litter decomposition from A. annua L. and artemisinin inhibited selectively the growth of soil microorganism. The number of fungus increased but actinomycetes, azotobacter, nitrobacteria and nitrite bacteria decreased significantly. The litter decomposition from A. annua L. and artemisinin significantly inhibited soil respiration, decreased microbial quotient but increased metabolic quotient. In addition, type and total content of phosphorus lipid fatty acids(PLFAs), the signature of microbes, actinomycete and protozoa decreased as artemisinin added into soil. The decrease in diversity and evenness indexes of microbial community in soil added with the litter or artemisinin. By 454 high-throughput sequencing techniques, the number of 16S rRNA sequence in soils changed in following sequence:control treatment (soil without litter and artemisinin) (9135)>with litter(7343)≈with artemisinin (7512), manifesting that A. annua L. and artemisinin inhibited the growth and reproduction of bacteria in soil. In addition, there were obvious differences among bacterial communities of three treatments, and artemisinin caused notable reduction of bacterial species(OTUs), diversity and evenness index of bacterial community. Only 13 out of 20 predominant bacteria were the same as the control, some function microorganism including Planctomycetaceae, Rhizobium, Actinomycetales, Acidimicrobiaceae had not been detected in soil added with litter or artemisinin, and richness of Cyanobacteria reduced by 97.5%. All of these indicated soil ecosystem deterioration and reduction of microbial groups and densities in soil. Therefore, allelopathic chemicals released form A annua L. could change the microbial community structure and result in serious soil problems by continual cropping of this medicinal plant.4 Studies on allelopathic effect of artemisinin on rhizobiumA significant inhibition of the reproduction and growth of rhizobium by artemisinin. After about 8 hours by adding 40 mg·L-1 artemisinin into the culture medium, the number of rhizobia was less than half of those in normal culture. The utilization of sucrose and glucose, the activities of extracellular protease and acid phosphatase released from rhizobia decreased significantly as the concentration of artemisinin increased in the culture medium. y=e-ax+b reflected the relationships bwtween nitrogenase activities (y) and concentrations of artemisinin (x). In the culture medium with 48 mg·L-1 of artemisinin, nitrogenase activities were about zero, resulting in the inactivation of nitrogenase in nodules formed. In general, artemisinin refluxed from A. annua L. could thus inhibit the reprouction and growth of rhizobia, formation of root nodules and interfere with energy supply and reception between bacteroid and host cells, leading to less nitrogen supply, poor growth and development, and low yields of beans.5 Allelopathic effects of artemisinin on azotobacterIn the liquid medium, artemisinin significantly inhibited the growth of azotobacters. Adding artemisinin into the culture medium, which increased the rate of proton efflux by azotobacters, In the solution with 80 mg·L-1 of artemisinin, the rate of proton efflux by azotobacters were 1.61 times (Ab 09) and 1.03 times (Ab 10) compared with control (without artemisinin) respectively. However, artemisinin inhibited the activities of extracellular protease, nitrogenase and the contents of citric acid released from azotobacters, but malic acid was quite opposite. In the solution with 48 mg-L"1 of artemisinin, the nitrogenase activities were about zero, which could interfered with the metabolism of azotobacters. In general, artemisin in A. annua L. grown soils may inhibit the growth of azotobacters, organic acids secretion capacity, activities of extracellular protease and nitrogen biofixation, which could affect the microbial population and the ability of nitrogen fixation, phosphorus and potassium-dissolving.6 Allelopathic effects of artemisinin on ectomycorrhizal fungiIn solution culture, artemisinin inhibited significantly the growth of four studied fungi. With 25 mg artemisinin·L-1 added, fungal biomass was decreased by 78.62%(S. luteus1),96.75%(S.luteus13),77.78%(S.luteus11) and S6.81%(S.subluteus12) compared with the control (without artemisinin). The amount of proton and oxalic acid efflux by the fungal isolates also decreased as nominal artemisinin concentrations increased, indicating the limited ability of ectomycorrhizal fungi to mobilize nutrients from soil minerals. However, nominal artemisinin significantly increased the rate of fungal oxalate efflux, suggesting membrane damage and the abnormal opening of anion channels on hyphae cell membranes. Nominal artemisinin also decreased the uptake of nitrogen, phosphorus and potassium by the fungal isolates, and there was no nutrient absorption by S. luteus 13,77.8% S. luteus 11 and 86.8% S. subluteus 12 under 25 mg artemisinin·L-1, showing the inhibition of nutrient uptake by ectomycorrhizal fungal under artemisinin treatment. Our results suggest that artemisin in forest soils from A. annua L. will alter the growth and nutrient uptake of ectomycorrhizal fungi, and the inhibition effect varies with different fungal strains. |
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