Analysis of artemisinin in Artemisia annua L. by LC-MS with selected ion monitoring

A liquid chromatography--mass spectrometry (LC-MS) method with selected ion monitoring (SIM) was developed and validated for the analysis and standardization of artemisinin in Artemisia annua L. This method is simple and accurate and requires only an 11 min per sample running time for the direct detection and quantification of artemisinin, a sesquiterpene lactone with antimalarial activity. To accurately analyze artemisinin, SIM was used to record the abundance of the [M - 18 + H]+ ion peak at m/z 265.3, with a scan range between m/z 250 and 270. Quantification was based on the LC-MS peak area of artemisinin, and the standard curve was used for calculation. This method was then validated and applied to several populations of A. annua to assess the population means and the diversity within a selected population. The distribution of artemisinin was found to vary by plant origin with population means ranging from 0.03 to 0.71% artemisinin dry leaf weight. Individual plants within the selected population that exhibited the highest artemisinin mean were then sampled, and the contents ranged from trace amounts to 1.5% artemisinin dry leaf weight basis, making these single plants accumulating artemisinin in concentrations >1.0% promising candidates for plant breeding and varietal development for high-yielding artemisinin.     

栽培密度和施肥水平对黄花蒿生长特性和青蒿素的影响

采用大田试验,研究不同密度和施肥水平对黄花蒿生长、生物量分配和青蒿素含量的影响。试验设3个密度水平: 高密度（111111株/hm2）、中密度（55555株/hm2）和低密度（27778株/hm2）。 各密度设3个施肥水平(复合肥,N-P2O5-K2O为15-15-15): 不施肥、低肥（60 kg/hm2）和高肥（120 kg/hm2）。结果表明,黄花蒿对密度和养分条件变化的适应性较强,其中密度是植株大小、生物量分配和产量有关参数的主要决定因子,而青蒿素含量由施肥水平决定。相同施肥水平下,黄花蒿的基径与分枝数均随密度的降低而显著增大,其单株生物量也随密度的降低而显著增大; 中、高密度黄花蒿的支持结构生物量分数均显著大于低密度,低密度黄花蒿的根生物量分数和根/冠比显著大于中、高密度。相同密度下,施肥水平对黄花蒿的单株生物量影响不显著,但显著影响其生物量分配。低密度下,黄花蒿根生物量分数和根/冠比随施肥水平的增高而显著降低; 高密度下,黄花蒿叶生物量分数随施肥水平的增高而显著增大。所有处理中,低密度低施肥水平黄花蒿的青蒿素含量最高,中密度低施肥水平的叶产量和青蒿素产量最大。本试验条件下,黄花蒿栽培以密度55555株/hm2、 施肥60 kg/hm2为宜。     

Nutrient deficiency in the production of artemisinin, dihydroartemisinic acid, and artemisinic acid in Artemisia annua L

Artemisia annua became a valuable agricultural crop after the World Health Organization recommended artemisinin as a component of ACT (artemisinin-combination based therapies) for malaria in 2001. A cloned, greenhouse-grown, A. annua (Artemis) subjected to an acidic soil and macronutrient deficit was evaluated for artemisinin production. Lack of lime (L) and macronutrients (N, P, and K) reduced leaf biomass accumulation. When L was provided (pH 5.1), the highest average leaf biomass was achieved with the "complete" (+N, +P, +K, and +L) treatment (70.3 g/plant), and the least biomass was achieved with the untreated (-N, -P, -K, and -L) treatment (6.18 g/plant). The nutrient least required for biomass accumulation per plant (g) was K (49.0 g), followed by P (36.5 g) and N (14.3 g). The artemisinin concentration (g/100 g) was significantly higher (75.5%) in -K plants when compared to plants under the complete treatment (1.62 vs 0.93%). Although the artemisinin total yield (g/plant) was 21% higher in -K plants, it was not significantly different from plants under the complete treatment (0.80 vs 0.66 g/plant). There were no marked treatment effects for concentration (g/100 g) or yield (g/plant) of both dihydroartemisinic acid and artemisinic acid, although higher levels were achieved in plants under the complete or -K treatments. There was a positive and significant correlation between artemisinin and both artemisinic acid and dihydroartemisin acid, in g/100 g and g/plant. This is the first report where potassium deficiency significantly increases the concentration (g/100 g) of artemisinin. Thus, under a mild potassium deficiency, A. annua farmers could achieve similar gains in artemisinin/ha, while saving on potassium fertilization, increasing the profitability of artemisinin production.     

Utilization of Sparingly Soluble Phosphate by Red Clover in Association with Glomus mosseae and Bacillus megaterium

A pot experiment was conducted to investigate the mobilization of sparingly soluble inorganic and organic sources of phosphorus (P) by red clover (Trifolium pratense L.) whose roots were colonized by the arbuscular mycorrhizal (AM) fungus Glomus mosseae and in association with the phosphate-solubilizing (PS) bacterium Bacillus megaterium ACCC10010. Phosphate-solubilizing bacteria and rock phosphate had a synergistic effect on the colonization of plant roots by the AM fungus. There was a positive interaction between the PS bacterium and the AM fungus in mobilization of rock phosphate, leading to improved plant P nutrition. In dual inoculation with the AM fungus and the PS bacterium, the main contribution to plant P nutrition was made by the AM fungus. Application of P to the low P soil increased phosphatase activity in the rhizosphere. Alkaline phosphatase activity was significantly promoted by inoculation with either the PS bacterium or the AM fungus.     

Effect of Superphosphate and Arbuscular Mycorrhizal Fungus Glomus mosseae on Phosphorus and Arsenic Uptake in Lentil (Lens culinaris L.)

Arsenic (As)-contaminated irrigation water is responsible for high As levels in soils and crops in many parts of the world, particularly in the Bengal Delta, Bangladesh and West Bengal, India. While arbuscular mycorrhizal (AM) fungi markedly improve phosphorus (P) uptake, they can also alleviate metal toxicity. In this study, the effects of superphosphate and inoculation with the AM fungus Glomus mosseae on P and As uptake of lentil were investigated. Plant height, shoot dry weight, shoot/root P concentration, and shoot P content increased due to mycorrhizal inoculation. However, As concentration in roots/shoots and root As content were reduced, plant height, shoot dry weight, shoot/root P concentration/content, and root As concentration and content increased due to superphosphate application. Root P concentration decreased with increasing As concentration. It was apparent that As concentration and content in shoots/roots increased with increasing As concentration in irrigation water. Superphosphate interaction with G. mosseae reduced the role of mycorrhizal infection in terms of enhancing P nutrition and reducing uptake of potentially toxic As into plant parts. The role and relationship of mycorrhizal in respect of P nutrition and As remediation efficiency in plant parts was established. In conclusion, it was worth alluding to that lentil with AM fungal inoculation can reduce As uptake and improve P nutrition. However, in retrospect superphosphate increased P and As uptake and decreased the role of the mycorrhizal association. This resulted in stimulating increased P uptake while decreasing As uptake in lentil.     

Effects of inoculation with Glomus mosseae, Azorhizobium caulinodans and rock phosphate on the growth of and nitrogen and phosphorus accumulation in Sesbania rostrata

A study was carried out to evaluate the influence of single or dual inoculation with Glomus mosseae and Azorhizobium caulinodans with and without rock phosphate on the growth of and N, P and K accumulation in Sesbania rostrata grown in sterilized soil. Height of plants inoculated with G. mosseae in combination with A. caulinadans and/or rock phosphate was significantly higher than that of uninoculated plants or plants inoculated with A. caulinodans alone before but not after 28 days. However, the oven-dry weight of shoots and roots from all treatments did not vary significantly. Generally, concentration and uptake of N and P but not K in shoots and roots of plants were improved by dual inoculation with G. mosseae and A. caulinodans plus or minus P. Mycorrhizal infection of roots was enhanced significantly by A. caulinodans. Received: 18 March 1996     

Synergistic effect of arbuscular mycorrhizal fungi and Bacillus subtilis on the biomass and essential oil yield of rose-scented geranium (Pelargonium graveolens)

Four different arbuscular mycorrhizal (AM) fungi, Glomus aggregatum, Glomus fasciculatum, Glomus intraradices and Glomus mosseae, were used alone and in combinations with Bacillus subtilis to evaluate their capability to increase the productivity of geranium. Mycorrhizal treatment increased the growth and total biomass invariably over non-mycorrhizal control plants. In AM alone treatment, the best result was obtained for G. mosseae treatment, where 380.9 and 335.3 g fresh herb yield per pot was recorded in 2005–2006 and 2006–2007, respectively, an increase of 75.3 and 85.9% over controls. Plants inoculated with B. subtilis alone yielded 287.8 and 252.3 g fresh herb, an increase of 32.4 and 39.9% over uninoculated controls. However, B. subtilis in combination with G. mosseae produced the highest herb yield, i.e. 410.8 and 347.8 g herbs pot^?1, an increase of 89.4 and 92.9% over untreated controls. The field experimental data validated the results of the pot experiment. Treatment with G. mosseae alone increased herb yield by 49.4%, whereas in combination with B. subtilis, it increased herb yield by 59.5%. Treatment with AM fungi and B. subtilis did not affect the essential oil content of the plant, but total oil yield was significantly increased because of the increase in biomass production.     

枯草芽孢杆菌和绿色木霉协同促进芹菜生长的研究

为了芹菜高产栽培的需要,利用枯草芽孢杆菌(BLG010)和绿色木霉(H06)对芹菜进行促生长试验,试验设置了蒸馏水处理、BLG010处理、H06处理、BLG010+H06处理。结果表明,BLG010和 H06具有促进芹菜生长协同增效的现象。因此,通过测定芹菜的防御酶活性和代谢物的变化来探明 BLG010和 H06对芹菜生长呈现协同增效效果的原因。酶活性测定结果表明,相对于蒸馏水处理,BLG010和 H06均能提高芹菜的 CAT和 POD活性,并且 BLG010+H06处理的 CAT和 POD活性均比蒸馏水处理、BLG010处理、H06处理高。利用 GC-MS技术分析不同处理代谢物的结果表明,苹果酸、甘露醇和蔗糖这 3种物质的含量在不同处理间呈现规律性差异。     

Impact of soil nitrogen concentration on Glomus spp.-Sinorhizobium interactions as affecting growth, nitrate reductase activity and protein content of Medicago sativa

Our objective was to evaluate how increasing levels of N in the medium (0, 4, 8 and 16 mmol N added kg^-1 soil) affect the interaction between Sinorhizobium and arbuscular mycorrhiza (AM) fungi in the tripartite symbiosis with Medicago sativa. Growth response, nutrient acquisition, protein content, and nitrate reductase (NR) activity were measured both in plant shoots and roots. Results showed that N levels in soil did not affect mycorrhizal colonization but they strongly influenced nodulation, particularly of mycorrhizal plants. Mycorrhizal colonization was required for a proper nodulation when no N was applied to soil. In contrast, the addition of 4 mmol N kg^-1 soil reduced nodulation only in mycorrhizal plants and 8 mmol N added kg^-1 soil allowed nodule formation only in non-mycorrhizal plants. Nodulation was totally inhibited in all treatments with the addition of 16 mmol N added kg^-1 soil. N addition enhanced NR activity in all the treatments, while AM colonization increased the proportion of NR allocated to roots. This effect was more pronounced under the lowest N levels in the medium. The two AM fungal species showed different distribution pattern of enzymatic activities in plant tissues indicating specific physiological traits. Protein content as well as the relative proportion of protein in roots were greatly increased after mycorrhizal colonization. Glomus intraradices-colonized plants had the highest protein content in shoot and root. Mycorrhizal effects on growth, N acquisition and biochemical variables cannot be interpreted as an indirect P-mediated effect since P content was lower in mycorrhizal plants than in those which were P fertilized. Mycorrhizal colonization increased the N content in plants irrespective of the N level, but the effectiveness of AM fungi on plant N acquisition depended on the AM fungus involved, G. intraradices being the most effective, particularly at the highest N rate. N₂ fixation, enhanced by AM colonization, contributed to N acquisition when a moderate N quantity was available in the soil. Nevertheless, under a high N amount the nodulating process and/or fixing capacity by Sinorhizobium was reduced in AM plants. In contrast, the AM fungal mycelium from a particular mycorrhizal fungus may continue to contribute efficiently to the N uptake from the soil even at high N levels. These results demonstrate the particular sensitivity of AM fungal species in terms of their growth and/or function to increasing N amounts in the medium. A selection of AM fungi used to address specific environmental conditions, such as N fertilization regimes comparable to those used in agronomic practices, is required for a better use of N applied to soil.     

氮肥对菊芋生物量、 热值和灰分含量的影响

探讨氮肥对菊芋生物量、热值和灰分含量的影响,为菊芋氮肥管理提供理论参考。于2010年在内蒙古锡林河流域利用弃耕地进行菊芋种植,设置0、2.5、5、7.5、10 g/m2 5个施氮水平,对不同施氮水平条件下菊芋的生物量、热值和灰分进行测定。结果表明:氮肥施入能够提高菊芋生物量、能量积累量和热值,最佳施氮量5～7.5 g/m2;各器官热值顺序为根系＞茎秆＞块茎＞叶片;能量积累量顺序为块茎＞茎秆＞叶片＞根系;灰分含量顺序为叶片＞茎秆＞根系＞块茎。     

Estimation of synergistic effect of humic fertilizer and Bacillus subtilis on lettuce plants by reflectance measurements

Previous research showed that the use of plant growth promoting rhizobacteria helped to increase nutrient use efficiency. The individual and combined effects of combined action of humic fertilizer and rhizobacteria Bacillus subtilis No. 2 on the lettuce yield, chlorophyll, total nitrogen and nitrate-(N) contents in lettuce leaves was studied. Traditional biochemical analysis and crop reflectance method were compared. Vegetation Indices and key spectrum characteristics - a median of frequency spectrum and bandwidth of frequency spectrum were used to estimate chlorophyll content in plant leaves.

The synergistic effect of bacteria and humic fertilizer was evidenced by increase in N and chlorophyll contents and in decreased nitrates content in lettuce leaves. Humic fertilizer resulted in decreased nitrates concentration in plants, whereas bacillus (B). subtilis No. 2 increased total N and chlorophyll contents. Results indicated that the application rate of humic fertilizer may be reduced when B. subtilis No. 2 is also applied.     

The effect of different nitrogen sources on the symbiotic interaction between Sorghum bicolor and Glomus intraradices: Expression of plant and fungal genes involved in nitrogen assimilation

In the arbuscular mycorrhizal (AM) symbiosis, plants take up part of the nitrogen (N) through a mycorrhizal pathway. In this study, we assessed the effect of different N sources on the expression of genes coding for enzymes and transporters of the mycorrhizal N uptake pathway, using Sorghum bicolor and Glomus intraradices as a model. Some of the genes investigated were differentially regulated in the intraradical and in the extraradical mycelium depending on the N source. In AM roots, some fungal and plant genes were co-regulated, suggesting an interdependence of both partners in the mycorrhizal N uptake pathway. Mycorrhizal N transfer may have a preference for glycine (plant growth and N uptake stimulation).     

施氮量对白萝卜硝酸盐含量和土壤硝态氮淋溶的影响

在保护地栽培条件下,通过6个施氮水平的田间小区试验,结合土层原位渗滤装置,研究了施用氮肥对白萝卜（Raphanus sativus L.）产量和硝酸盐含量及土壤硝态氮淋溶的影响。结果表明,施氮处理白萝卜产量比不施氮处理仅增加6.04%～10.92%,当尿素氮施用量大于N 100 kg/hm2时,增产幅度开始下降。不同施氮处理白萝卜产量没有显著差异,说明在土壤基础肥力较高的情况下,增施氮肥不能明显提高白萝卜的产量;单施有机肥白萝卜体内硝酸盐含量为 196.86 mg/kg,比不施氮处理降低 5.08%。在此基础上加施尿素后,硝酸盐含量随氮肥施用量的增加显著升高（p0.05）;0—100cm土壤剖面硝态氮累积量随氮肥施用量的增加而增加,且与氮肥施用量显著正相关（r=0.993, r0.01=0.917）;白萝卜生长期间收集到的土壤淋溶液中硝态氮浓度较高,平均为32.88 mg/L,硝态氮的淋失量为 4.42～6.14 kg/hm2,不同施氮量处理之间没有显著差异。     

Role of soil water content in the carbon and nitrogen dynamics of Lumbricus terrestris L. burrow soil

We evaluated the role of soil water content in controlling C and N dynamics within the drilosphere created by the anecic earthworm Lumbricus terrestris (L.). Mesocosms (volume = 3.1 l) were each amended with corn litter and three earthworms. Control treatments received no earthworms and no other earthworm species were present in the soil. WET and DRY treatments received a total of 9.25 cm and 3.25 cm of water, respectively. Water was added on weeks 1, 3, 7, and 10 at a rate of 2.0 cm per mesocosm for WET treatments and 0.5 cm per mesocosm for DRY treatments. Mesocosms were sampled destructively after incubation at 18–20 °C for 0, 3, 7, and 13 weeks. The water content of WET burrow soil ranged from 0.12 g g⁻¹ to 0.18 g g⁻¹ and was significantly higher than in the DRY treatment throughout the incubation period. The live weight of earthworms was significantly higher in the WET treatment only on week 13, whereas litter consumption was significantly lower in the DRY treatment for week 13. Carbon mineralization, measured as CO₂ evolved after a 24-h incubation, was consistently higher in WET than in DRY burrow soil. Effects of differences in soil water content were also apparent for biomass C and metabolic quotient. Soil water content did no affect the total C concentration of burrow soil. DRY burrow soil had consistently lower levels of nitrate than WET soil throughout the experiment. Lower levels of ammonium and inorganic N were observed for WET burrow soil on weeks 3 and 7. Water content did not have a significant effect on burrow soil total N. We concluded that the water content of the drilosphere affects both C and N dynamics and can affect the speciation of inorganic N; yet, the effects of soil water content do not appear to result from differences in the feeding activities of anecic earthworms.     

低温处理对青蒿叶片脯氨酸、SOD和CAT的含量及蛋白质的影响

青蒿是一种重要的抗痢疾药物的原材料,低温是影响青蒿的青蒿素含量的重要因素。本试验以2个不同产地的青蒿为材料,研究低温处理对青蒿叶片脯氨酸、SOD和CAT的含量及蛋白质的影响。结果表明,低温处理条件下,青蒿叶片脯氨酸、超氧化物歧化酶活性(SOD)和过氧化氢酶活性(CAT)与室温相比均增加,且差异显著。SOD含量的增加,会增强青蒿植株抵抗低温的能力;CAT含量的增加,有利于缓解过量的过氧化物对青蒿叶片细胞的伤害;脯氨酸含量的增加,有利于调节细胞渗透压,促使细胞正常生长。进一步研究表明,在低温和室温条件下,青蒿叶片蛋白质电泳图谱存在强弱差异条带,说明低温处理对青蒿叶片中蛋白质产生影响。本文结果将为今后青蒿的栽培生产提供有价值的参考。     

枯草芽孢杆菌B11基因文库的构建及与拮抗物质的生物合成相关基因的克隆

枯草芽孢杆菌菌株B 11对广泛的植物病原真菌和细菌都具有拮抗作用。以柯斯质粒pW EB∷TNC为载体构建了枯草芽孢杆菌菌株B 11的基因文库,文库含9 000个克隆。文库克隆中插入的DNA片段平均为42.1 kb,该文库含有菌株B 11基因组中任一基因的概率为99.99%。采用平板活性检测法筛选文库,筛选到1个对茄青枯假单胞菌菌株P 13具有拮抗活性的文库克隆GXN 9527,该克隆的重组质粒pGXN 9527含有50 kb的菌株B 11的DNA。文库克隆对革兰氏阴性植物病原细菌如水稻黄单胞菌水稻变种也具有拮抗活性,而对革兰氏阳性细菌如地衣芽孢杆菌和植物病原真菌如尖孢镰刀菌西瓜专化型、立枯丝核菌、水稻稻灰梨孢菌则没有拮抗活性。分别含有pGXN 9527的18、12、9、8 kb B amHⅠ片段的亚克隆对P 13均没有拮抗活性,说明编码该拮抗物质的生物合成基因很可能成簇存在。     

施氮对小白菜产量和品质的效应

田间试验研究了施氮对两种肥力土壤小白菜产量、硝酸盐含量和营养品质的影响。结果表明,施氮量与小白菜产量呈显著(低肥力)和极显著(中肥力)的二次回归关系,中、低肥力土壤施氮小白菜分别增产141.2%~309.4%和149.5%~617.6%;低肥力土壤小白菜产量仅为中肥力土壤的28.6%~49.5%。施氮75~300kg.hm-2提高小白菜硝酸盐含量9.9%~33.3%(中肥力,75 kg.hm-2处理除外)和240.3%~363.6%(低肥力),硝酸盐含量以中肥力土壤>低肥力土壤(N 150 kg.hm-2处理除外)。施氮降低中肥力土壤小白菜Vc含量、提高氨基酸含量,对可溶性糖影响不大;低肥力土壤施氮降低小白菜Vc、氨基酸、可溶性糖含量。     

施氮量和花后土壤含水量对小麦氮代谢特性和子粒蛋白质含量的影响

在池栽条件下,研究了施氮量和花后土壤相对含水量对小麦氮代谢特性和籽粒蛋白质含量的影响。结果表明,在同一施氮量下,旗叶和子粒硝酸还原酶（NR）和谷氨酰胺合成酶（GS）活性表现为花后土壤相对含水量（Soil relative water content , SRWC）以60%～70%最高,过低（40%～50%）或过高(80%～90%)均降低NR和GS活性。旗叶蛋白酶活性随土壤相对含水量增加而降低;花后土壤相对含水量过低不利于叶片游离氨基酸含量的提高,过高则前期氨基酸合成少,后期向子粒转运不彻底。子粒游离氨基酸和蛋白质含量也随土壤相对含水量增加而降低;子粒蛋白质积累量以花后土壤相对含水量为60%～70% 时最高,过高和过低均不利于子粒蛋白质积累。在同一土壤含水量下,旗叶和子粒NR和GS活性表现为随着施氮量的增加而升高,蛋白酶活性随着施氮量增加而降低;旗叶和子粒游离氨基酸含量、子粒蛋白质含量和积累量随施氮量增加而提高,但施氮量过多,蛋白质积累量增加幅度减小。试验表明,小麦生产中可以通过施用氮肥和控制花后土壤水分含量技术,调节植株氮代谢,提高子粒蛋白质含量。     

多粘类芽孢杆菌BRF-1和枯草芽孢杆菌BRF-2对黄瓜和番茄枯萎病的防治效果

采用室内培养方法,对生防细菌多粘类芽孢杆菌BRF-1和枯草芽孢杆菌BRF-2不同稀释倍数的代谢产物抑制黄瓜尖孢镰刀菌、番茄枯萎病菌分生孢子萌发和菌丝生长的试验结果表明,当两菌株代谢产物稀释5倍时,对分生孢子萌发的抑制率达到80％以上;两菌株代谢产物在稀释2倍和5倍时,对2种病原真菌菌丝生长的抑制率在40％-80％之间,与对照差异极显著（P〈0．01）。盆栽试验表明,BRF-1和BRF-2生防细菌菌悬液及其无菌代谢物对黄瓜和番茄枯萎病不仅具有较好的防治效果,而且具有明显的促进生长作用。