Piriformospora indica alter root-associated microbiome structure to enhance Artemisia annua L. tolerance to arsenic.

Microorganisms in the rhizosphere are crucial allies for plant stress tolerance. Recent research suggests that by interacting with the rhizosphere microbiome, microorganisms can aid in the revegetation of soils contaminated with heavy metal(loid)s (HMs). However, it is unknown that how Piriformospora indica influences the rhizosphere microbiome to mitigate arsenic-toxicity in arsenic-enriched environments. Artemisia annua plants were grown in the presence or absence of P. indica and spiked with low (50) and high (150 µmol/L) concentrations of arsenic (As). After inoculation with P. indica, fresh weight increased by 37.7% and 10% in control and high concentration treated plants, respectively. Transmission electron microscopy showed that cellular organelles were severely damaged by As and even disappeared under high concentration. Furthermore, As was mostly accumulated by 5.9 and 18.1 mg/kg dry weight in the roots of inoculated plants treated with low and high concentrations of As, respectively. Additionally, 16 S and ITS rRNA gene sequencing were applied to analyze the rhizosphere microbial community structure of A. annua under different treatments. A significant difference was observed in microbial community structure under different treatments as revealed by non-metric multidimensional scaling ordination. The bacterial and fungal richness and diversity in the rhizosphere of inoculated plants were actively balanced and regulated by P. indica co-cultivation. Lysobacter and Steroidobacter were found to be the As-resistant bacterial genera. We conclude that P. indica inoculation could alter rhizosphere microecology, thereby mitigating As-toxicity without harming the environment.

A metabolomic approach to target antimalarial metabolites in the Artemisia annua fungal endophytes.

Fungal endophytes are a major source of anti-infective agents and other medically relevant compounds. However, their classical blinded-chemical investigation is a challenging process due to their highly complex chemical makeup. Thus, utilizing cheminformatics tools such as metabolomics and computer-aided modelling is of great help deal with such complexity and select the most probable bioactive candidates. In the present study, we have explored the fungal endophytes associated with the well-known antimalarial medicinal plant Artemisia annua for their production of further antimalarial agents. Based on the preliminary antimalarial screening of these endophytes and using LC-HRMS-based metabolomics and multivariate analyses, we suggested different potentially active metabolites (compounds 1-8). Further in silico investigation using the neural-network-based prediction software PASS led to the selection of a group of quinone derivatives (compounds 1-5) as the most possible active hits. Subsequent in vitro validation revealed emodin (1) and physcion (2) to be potent antimalarial candidates with IC50 values of 0.9 and 1.9 µM, respectively. Our approach in the present investigation therefore can be applied as a preliminary evaluation step in the natural products drug discovery, which in turn can facilitate the isolation of selected metabolites notably the biologically active ones.

The ameliorative effects of exogenous inoculation of Piriformospora indica on molecular, biochemical and physiological parameters of Artemisia annua L. under arsenic stress condition.

Aim of the current study was to investigate the effect of exogenously inoculated root endophytic fungus, Piriformospora indica, on molecular, biochemical, morphological and physiological parameters of Artemisia annua L. treated with different concentrations (0, 50, 100 and 150 µmol/L) of arsenic (As) stress. As was significantly accumulated in the roots than shoots of P. indica-inoculated plants. As accumulation and immobilization in the roots is directly associated with the successful fungal colonization that restricts most of As as compared to the aerial parts. A total of 4.1, 11.2 and 25.6 mg/kg dry weight of As was accumulated in the roots of inoculated plants supplemented with 50, 100 and 150 µmol/L of As, respectively as shown by atomic absorption spectroscopy. P. indica showed significant tolerance in vitro to As toxicity even at high concentration. Furthermore, flavonoids, artemisinin and overall biomass were significantly increased in inoculated-stressed plants. Superoxide dismutase and peroxidase activities were increased 1.6 and 1.2 fold, respectively under 150 µmol/L stress in P. indica-colonized plants. Similar trend was followed by ascorbate peroxidase, catalase and glutathione reductase. Like that, phenolic acid and phenolic compounds showed a significant increase in colonized plants as compared to their respective control/un-colonize stressed plants. The real-time PCR revealed that transcriptional levels of artemisinin biosynthesis genes, isoprenoids, terpenes, flavonoids biosynthetic pathway genes and signal molecules were prominently enhanced in inoculated stressed plants than un-inoculated stressed plants.

Flavobacterium artemisiae sp. nov., isolated from the rhizosphere of Artemisia annua L. and emended descriptions of Flavobacterium compostarboris and Flavobacterium procerum.

A Gram-stain-negative, strictly aerobic, yellow-coloured, motile by gliding and elongated rod-shaped bacterial strain, designated SYP-B1015T, was isolated from the rhizosphere of Artemisia annua L. Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain SYP-B1015T belonged to the genus Flavobacterium and had highest 16S rRNA gene sequence similarity to Flavobacterium compostarboris JCM 16527T (98.1 %) and Flavobacterium procerum JCM 30113T (97.2 %). The predominant respiratory quinone for the strain was MK-6, and the major cellular fatty acids were iso-C15 : 0, iso-C15 : 0 3-OH and iso-C17 : 0 3-OH. The polar lipid profile contained phosphatidylethanolamine as a major compound. The DNA G+C content of strain SYP-B1015T was 33.5 mol%. The DNA-DNA relatedness values between strain SYP-B1015T and F. compostarboris JCM 16527T and F. procerum JCM 30113T were 56.5±0.4 and 48.9±1.2 %, respectively. Combining the data from morphological, physiological, biochemical and chemotaxonomic characterizations presented in this study, strain SYP-B1015T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium artemisiae sp. nov. is proposed. The type strain is SYP-B1015T (=CGMCC 1.16115T=KCTC 62025T).

Cumulative role of bioinoculants on growth, antioxidant potential and artemisinin content in Artemisia annua L. under organic field conditions.

Artemisia annua L. is mostly known for a bioactive metabolite, artemisinin, an effective sesquiterpene lactone used against malaria without any reputed cases of resistance. In this experiment, bioinoculants viz., Streptomyces sp. MTN14, Bacillus megaterium MTN2RP and Trichoderma harzianum Thu were applied as growth promoting substances to exploit full genetic potential of crops in terms of growth, yield, nutrient uptake and particularly artemisinin content. Further, multi-use of the bioinoculants singly and in combinations for the enhancement of antioxidant potential and therapeutic value was also undertaken which to our knowledge has never been investigated in context with microbial application. The results demonstrated that a significant (P < 0.05) increase in growth, nutrient uptake, total phenolic, flavonoid, free radical scavenging activity, ferric reducing antioxidant power, reducing power and total antioxidant capacity were observed in the A. annua treated with a combination of bioinoculants in comparison to control. Most importantly, an increase in artemisinin content and yield by 34 and 72 % respectively in the treatment having all the three microbes was observed. These results were further authenticated by the PCA analysis which showed positive correlation between plant macronutrients and antioxidant content with plant growth and artemisinin yield of A. annua. The present study thus highlights a possible new application of compatible bioinoculants for enhancing the growth along with antioxidant and therapeutic value of A. annua.

Overexpression of a Novel NAC Domain-Containing Transcription Factor Gene (AaNAC1) Enhances the Content of Artemisinin and Increases Tolerance to Drought and Botrytis cinerea in Artemisia annua.

The NAC (NAM, ATAF and CUC) superfamily is one of the largest plant-specific transcription factor families. NAC transcription factors always play important roles in response to various abiotic stresses. A NAC transcription factor gene AaNAC1 containing a complete open reading frame (ORF) of 864 bp was cloned from Artemisia annua. The expression of AaNAC1 could be induced by dehydration, cold, salicylic acid (SA) and methyl jasmonate (MJ), suggesting that it might be a key regulator of stress signaling pathways in A. annua. AaNAC1 was shown to be localized to the nuclei by transforming tobacco leaf epidermal cells. When AaNAC1 was overexpressed in A. annua, the content of artemisinin and dihydroartemisinic acid was increased by 79% and 150%, respectively. The expression levels of artemisinin biosynthetic pathway genes, i.e. amorpha-4,11-diene synthase (ADS), artemisinic aldehyde Δ11(13) reductase (DBR2) and aldehyde dehydrogenase 1 (ALDH1), were increased. Dual luciferase (dual-LUC) assays showed that AaNAC1 could activate the transcription of ADS in vivo. The transgenic A. annua exhibited increased tolerance to drought and resistance to Botrytis cinerea. When AaNAC1 was overexpressed in Arabidopsis, the transgenic Arabidopsis were markedly more tolerant to drought. The transgenic Arabidopsis showed increased resistance to B. cinerea. These results indicate that AaNAC1 can potentially be used in transgenic breeding for improving the content of artemisinin and drought tolerance in A. annua.

Dual symbiosis between Piriformospora indica and Azotobacter chroococcum enhances the artemisinin content in Artemisia annua L.

At present, Artemisia annua L. is the major source of artemisinin production. To control the outbreaks of malaria, artemisinin combination therapies (ACTs) are recommended, and hence an ample amount of artemisinin is required for ACTs manufacture to save millions of lives. The low yield of this antimalarial drug in A. annua L. plants (0.01-1.1%) ensues its short supply and high cost, thus making it a topic of scrutiny worldwide. In this study, the effects of root endophyte, Piriformospora indica strain DSM 11827 and nitrogen fixing bacterium, Azotobacter chroococcum strain W-5, either singly and/or in combination for artemisinin production in A. annua L. plants have been studied under poly house conditions. The plant growth was monitored by measuring parameters like height of plant, total dry weight and leaf yield with an increase of 63.51, 52.61 and 79.70% respectively, for treatment with dual biological consortium, as compared to that of control plants. This significant improvement in biomass was associated with higher total chlorophyll content (59.29%) and enhanced nutrition (especially nitrogen and phosphorus, 55.75 and 86.21% respectively). The concentration of artemisinin along with expression patterns of artemisinin biosynthesis genes were appreciably higher in dual treatment, which showed positive correlation. The study suggested the potential use of the consortium P. indica strain DSM 11827 and A. chroococcum strain W-5 in A. annua L. plants for increased overall productivity and sustainable agriculture.

Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels.

It is becoming increasingly evident that the formation of arbuscular mycorrhiza (AM) enhances secondary metabolite production in shoots. Despite mounting evidence, relatively little is known about the underlying mechanisms. This study suggests that increase in artemisinin concentration in Artemisia annua colonized by Rhizophagus intraradices is due to altered trichome density as well as transcriptional patterns that are mediated via enhanced jasmonic acid (JA) levels. Mycorrhizal (M) plants had higher JA levels in leaf tissue that may be due to induction of an allene oxidase synthase gene (AOS), encoding one of the key enzymes for JA production. Non-mycorrhizal (NM) plants were exogenously supplied with a range of methyl jasmonic acid concentrations. When leaves of NM and M plants with similar levels of endogenous JA were compared, these matched closely in terms of shoot trichome density, artemisinin concentration, and transcript profile of artemisinin biosynthesis genes. Mycorrhization increased artemisinin levels by increasing glandular trichome density and transcriptional activation of artemisinin biosynthesis genes. Transcriptional analysis of some rate-limiting enzymes of mevalonate and methyl erythritol phosphate (MEP) pathways revealed that AM increases isoprenoids by induction of the MEP pathway. A decline in artemisinin concentration in shoots of NM and M plants treated with ibuprofen (an inhibitor of JA biosynthesis) further confirmed the implication of JA in the mechanism of artemisinin production.

[A new indole derivative from endophyte Myrothecium roridum IFB-E091 in Artemisia annua].

Three compounds were isolated from solid culture of endophyte Myrothecium roridum IFB-E091 in Artemisia annua. Their structures were determined as (S)-(-)-N-[2-(3-hydroxy-2-oxo-2,3-dihydro-1H-indol-3-yl)-ethyl]-acetamide (1), N-(4-hydroxyphenethyl)acetamide (2) and asperfumoid (3), in which compound 1 was a new indole derivative. In cytotoxicity assay, the compound 1 had no obvious inhibition activity in human hepatoma cell line SMMC-7721 and human cervical carcinoma cell line HeLa.

AaORA, a trichome-specific AP2/ERF transcription factor of Artemisia annua, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea.

· Six transcription factors of APETALA2/ethylene-response factor (AP2/ERF) family were cloned and analyzed in Artemisia annua. Real-time quantitative polymerase chain reaction (RT-Q-PCR) showed that AaORA exhibited similar expression patterns to those of amorpha-4,11-diene synthase gene (ADS), cytochrome P450-dependent hydroxylase gene (CYP71AV1) and double bond reductase 2 gene (DBR2) in different tissues of A. annua. · AaORA is a trichome-specific transcription factor, which is expressed in both glandular secretory trichomes (GSTs) and nonglandular T-shaped trichomes (TSTs) of A. annua. The result of subcellular localization shows that AaORA is targeted to the nuclei and the cytoplasm. · Overexpression and RNA interference (RNAi) of AaORA in A. annua regulated, positively and significantly, the expression levels of ADS, CYP71AV1, DBR2 and AaERF1. The up-regulated or down-regulated expression levels of these genes resulted in a significant increase or decrease in artemisinin and dihydroartemisinic acid. The results demonstrate that AaORA is a positive regulator in the biosynthesis of artemisinin. · Overexpression of AaORA in Arabidopsis thaliana increased greatly the transcript levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2), HEVEIN-LIKE PROTEIN (HEL) and BASIC CHITINASE (B-CHI). After inoculation with Botrytis cinerea, the phenotypes of AaORA overexpression in A. thaliana and AaORA RNAi in A. annua demonstrate that AaORA is a positive regulator of disease resistance to B. cinerea.

AaERF1 positively regulates the resistance to Botrytis cinerea in Artemisia annua.

Plants are sessile organisms, and they can not move away under abiotic or biotic stresses. Thus plants have evolved a set of genes that response to adverse environment to modulate gene expression. In this study, we characterized and functionally studied an ERF transcription factor from Artemisia annua, AaERF1, which plays an important role in biotic stress responses. The AaERF1 promoter had been cloned and GUS staining results of AaERF1 promoter-GUS transgenic A. annua showed that AaERF1 is expressed ubiquitiously in all organs. Several putative cis-acting elements such as W-box, TGA-box and Py-rich element, which are involved in defense responsiveness, are present in the promoter. The expression of AaERF1 can be induced vigorously by methyl jasmonate as well as by ethephon and wounding, implying that AaERF1 may activate some of the defense genes via the jasmonic acid and ethylene signaling pathways of A. annua. The results of electrophoretic mobility shift assay (EMSA) and yeast one-hybrid experiments showed that AaERF1 was able to bind to the GCC box cis-acting element in vitro and in yeast. Ectopic expression of AaERF1 could enhance the expression levels of the defense marker genes PLANT DEFENSIN1.2 (PDF1.2) and BASIC CHITINASE (ChiB), and increase the resistance to Botrytis cinerea in the 35S::AaERF1 transgenic Arabidopsis. The down-regulated expression level of AaERF1 evidently reduced the resistance to B. cinerea in A. annua. The overall results showed that AaERF1 positively regulated the resistance to B. cinerea in A. annua.

Indole-3-acetic acid production by endophytic Streptomyces sp. En-1 isolated from medicinal plants.

Plant-associated actinobacteria are rich sources of bioactive compounds including indole-derived molecules such as phytohormone indole-3-acetic acid (IAA). In view of few investigations concerning the biosynthesis of IAA by endophytic actinobacteria, this study evaluated the potential of IAA production in endophytic streptomycete isolates sourced from medicinal plant species Taxus chinensis and Artemisia annua. By HPLC analysis of IAA combined with molecular screening approach of iaaM, a genetic determinant of streptomycete IAA synthesis via indole-3-acetamide (IAM), our data showed the putative operation of IAM-mediated IAA biosynthesis in Streptomyces sp. En-1 endophytic to Taxus chinensis. Furthermore, using the co-cultivation system of model plant Arabidopsis thaliana and streptomycete, En-1 was found to be colonized intercellularly in the tissues of Arabidopsis, an alternative host, and the effects of endophytic En-1 inoculation on the model plant were also assayed. The phytostimulatory effects of En-1 inoculation suggest that IAA-producing Streptomyces sp. En-1 of endophytic origin could be a promising candidate for utilization in growth improvement of plants of economic and agricultural value.

Streptomyces endophyticus sp. nov., an endophytic actinomycete isolated from Artemisia annua L.

Three filamentous actinomycetes, strains YIM 65594(T), YIM 65638 and YIM 65642, were isolated from the surface-sterilized roots of Artemisia annua L. collected from Yunnan province, south-west China. These strains were found to have morphological and chemotaxonomic characteristics typical of the genus Streptomyces. The organisms formed an extensively branched substrate mycelium, with abundant aerial hyphae that differentiated into spores. The cell wall of the isolates contained ll-diaminopimelic acid and the menaquinones were MK-9(H(8)) and MK-9(H(6)). The major fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0) and iso-C(16 : 0). Phylogenetic analysis of the 16S rRNA gene sequences of these strains revealed that the strains clustered together and were most closely related to Streptomyces kunmingensis NBRC 14463(T), with 98.5-98.6 % 16S rRNA gene sequence similarity. The results of DNA-DNA hybridization and physiological tests allowed the genotypic and phenotypic differentiation of strains YIM 65594(T), YIM 65638 and YIM 65642 from related species. However, the high level of DNA-DNA relatedness between them showed that these three strains belong to the same species. Strain YIM 65594(T) (= DSM 41984(T) = CCTCC AA 209036(T)) was selected as the type strain to represent this novel species, for which the name Streptomyces endophyticus sp. nov. is proposed.

Artemisinin production and precursor ratio in full grown Artemisia annua L. plants subjected to external stress.

The concentration of the lifesaving antimalarial compound artemisinin (AN) in cultivated Artemisia annua (A. annua) plants is relatively low, and thus research in improving the content is important. In the present study, external stress was applied to adult plants of A. annua and the effect was examined on the concentrations of AN and its immediate precursors in leaves, and these concentrations were related to densities and sizes of the glandular trichomes (GT). Plants were stress treated weekly five times by sandblasting or spraying with salicylic acid, chitosan oligosaccharide, H2O2, and NaCl solutions. Contents of AN-related compounds (AN-c) were analysed in leaf samples from an upper and a lower position of the plants, and GT were quantified and measured. In lower leaves, several stress treatments had significant negative effects on concentrations of AN-c, whereas the ratios between compounds showed an increased conversion to AN. In the upper leaves, no changes were observed compared to controls. Linear relations were found between the concentrations of metabolites and the density of GT in both upper and lower leaves, and size of GT in lower leaves. Results suggested that older and younger leaves may respond differently to applied stress. A part of the plants were infected by powdery mildew, and this caused significantly different compositions of the AN-c, compared to uninfected plants. In conclusion, changes in concentrations of AN-c seemed largely to be related to changes in GT densities and sizes.

An endophytic Pseudonocardia species induces the production of artemisinin in Artemisia annua.

Endophytic actinobacteria colonize internal tissues of their host plants and are considered as a rich and reliable source of diverse species and functional microorganisms. In this study, endophytic actinobacterial strain YIM 63111 was isolated from surface-sterilized tissue of the medicinal plant Artemisia annua. We identified strain YIM 63111 as a member of the genus Pseudonocardia. A. annua seedlings grown under both sterile and greenhouse conditions were inoculated with strain YIM 63111. The growth of A. annua seedlings was strongly reduced when YIM 63111 was inoculated at higher concentrations under sterile conditions. However, no growth inhibition was observed when A. annua was grown under greenhouse conditions. Using an enhanced green fluorescent protein (EGFP) expressing YIM 63111 strain, we also observed the endophytic colonization of A. annua seedling using confocal laser-scanning microscopy. The transcription levels of the key genes involved in artemisinin biosynthesis were investigated using real time RT-PCR, revealing that cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 oxidoreductase (CPR) expression were up-regulated in A. annua upon inoculation with strain YIM 63111 under certain conditions. The up-regulation of these genes was associated with the increased accumulation of artemisinin. These results suggest that endophytic actinobacteria effectively stimulate certain plant defense responses. Our data also demonstrate the use of Pseudonocardia sp. strain YIM 63111 as a promising means to enhance artemisinin production in plants.

[Plasmid pCQ4 and its phage phiCQ4 of endophytic Streptomyces sp. from Artemisia annua L].

OBJECTIVE: Large plasmid pCQ4 was detected in Streptomyces sp. W75 from Artemisia annua L. We clonined, sequenced, analyzed and characterized pCQ4. METHODS: Southern hybridization was used to determine restriction map of pCQ4. To clone the full-length of pCQ4, conjugation and recombinational cloning in a BAC vector were used. RESULTS: The complete nucleotide sequence of pCQ4 consisted of 84833-bp, encoding 129 ORFs which 40 ORFs resembled these of bacterial phages. W75 culture could infect W75 cured of pCQ4 and formed plaques on plate. Phage particle (phiCQ4) was observed by transmission electron microscopy. LinearphiCQ4 DNA was detected on pulsed-field gel electrophoresis. Comparison to Streptomyces plasmid-phage pZL12, genes encoding major phage structural proteins resembled that of pCQ4. CONCLUSION: Streptomyces plasmid pCQ4 could be transformed into lytic phagephiCQ4, and the phage segment on pCQ4 might be a mobile unit.

Pseudonocardia antimicrobica sp. nov., a novel endophytic actinomycete associated with Artemisia annua L. (sweet wormwood).

A Gram-reaction-positive, non-motile, endophytic actinomycete, designated strain YIM 63235(T), was isolated from the surface-sterilized stems of Artemisia annua L., and characterized to determine its taxonomic position. The strain YIM 63235(T) formed well-differentiated aerial and substrate mycelia on media tested. The phylogenetic tree based on 16S rRNA gene sequences showed that the new isolate formed a distinct lineage within the genus Pseudonocardia, and the strain YIM 63235(T) was closely related to Pseudonocardia parietis 04-St-002(T) (99.1%). However, DNA-DNA relatedness demonstrated that strain YIM 63235(T) was distinct from the closest phylogenetic neighbor. The chemotaxonomic properties of strain YIM 63235(T) were consistent with those of the genus Pseudonocardia: the diagnostic diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid and MK-8(H(4)) was the predominant menaquinone. The major fatty acids were iso-C(16:0) and iso-C(16:1) H. The DNA G+C content of strain YIM 63235(T) was 71.0 mol%. On the basis of the phenotypic and phylogenetic distinctiveness, the novel isolate was identified as representing a novel species of the genus Pseudonocardia, for which the name Pseudonocardia antimicrobica sp. nov. (type strain YIM 63235(T) =CCTCC AA 208080(T)=DSM 45303(T)) is proposed.

Control de calidad de drogas vegetales: lavado y desinfección de Artemisia annua L y Tagetes lucida Cav / Quality control of vegetable drugs: washing and disinfection of Artemisia annua L and Tagetes lucida Cav

Introducción: utilizar el lavado y desinfección química como método que permita disminuir la contaminación microbiana en el material vegetal cosechado de Artemisia annua L y Tagetes lucida Cav. Objetivos: garantizar la calidad microbiológica de las drogas vegetales obtenidas de estas 2 especies medicinales. Métodos: se utilizó material vegetal procedente de las parcelas establecidas en la Estación Experimental de Plantas Medicinales Dr Juan Tomás Roig, en suelo ferralítico rojo hidratado (ferralsols). Se emplearon 2 testigos, uno sin tratar y otro tratado con agua potable; para las pruebas a nivel de laboratorio las muestras de 300 g en A. annua y de 250 g de T lucida fueron lavadas con agua potable y después sometidas a un proceso de desinfección en una solución de hipoclorito de sodio 0,5 por ciento y 1,0 por ciento, sumergidas durante 5 y 10 min en ambos casos; para las pruebas de escalado se seleccionó el tratamiento químico que a nivel de laboratorio resultó más efectivo, se emplearon 3 réplicas de la droga cruda de 10 kg en el caso de A annua y de 6 kg en T lucida, con posterioridad las muestras se escurren sobre bastidores y después pasan a un proceso de secado en estufa de 35 a 40 ºC durante 2 d Luego de secadas se realizan los correspondientes análisis microbiológico y físico-químico. En el primer caso se determinó el conteo total de bacterias, conteo total de hongos y otras enterobacterias, además de microorganismos aislados. En el control físico- químico, los porcentajes de materia orgánica extraña; materia inorgánica extraña y hojas ennegrecidas; los índices numéricos como humedad, cenizas totales, sustancias solubles en agua, sustancias solubles en etanol 30 por ciento, sustancias solubles en etanol 70 por ciento, así como mediante tamizaje fitoquímico, la presencia de metabolitos secundarios...

Introduction: the use of washing and chemical disinfection as a method allows reducing microbial pollution in the harvested vegetal material of Artemisia annua L. and Tagetes lucida Cav Objectives: to assure the microbiological quality of vegetal drugs from these two medicinal species. Methods: there was used some vegetal material from lots on red hydrated ferralitic soils (ferrasols) located in Dr Juan Tomás Roig Experimental Center of Medicinal Plants. Two controls, one treated with drinking water and the other untreated, were used. For lab tests, the 300g A. annua and 250g T lucida samples were washed with drinking water and then were subjected to disinfection process using 0,5 and 1,0 percent sodium hypochloride in which both samples were submerged for 5 and 10 minutes. The most effective chemical treatment at lab was selected for the scaling test of three replicas of the crude drug from 10 kg of A annua and from 6 kg T lucida respectively. The samples were placed upon frames to be dried later at 35 to 40 ºC for 2 hours. After drying, the corresponding microbiological and physical-chemical analyses were performed. The microbiological analysis determined the total count of bacteria, of fungi and other enterobacteria in addition to isolated microorganisms. The physical and chemical analysis estimated the percentages of foreign organic matter, foreign inorganic matter and blackened leaves; and the indexes of humidity, total ashes, soluble substances in water, soluble substances in 30 percent etanol and soluble substances in 70 percent etanol...

Control de calidad de drogas vegetales: lavado y desinfección de Artemisia annua L y Tagetes lucida Cav / Quality control of vegetable drugs: washing and disinfection of Artemisia annua L and Tagetes lucida Cav

Introducción: utilizar el lavado y desinfección química como método que permita disminuir la contaminación microbiana en el material vegetal cosechado de Artemisia annua L y Tagetes lucida Cav. Objetivos: garantizar la calidad microbiológica de las drogas vegetales obtenidas de estas 2 especies medicinales. Métodos: se utilizó material vegetal procedente de las parcelas establecidas en la Estación Experimental de Plantas Medicinales Dr Juan Tomás Roig, en suelo ferralítico rojo hidratado (ferralsols). Se emplearon 2 testigos, uno sin tratar y otro tratado con agua potable; para las pruebas a nivel de laboratorio las muestras de 300 g en A. annua y de 250 g de T lucida fueron lavadas con agua potable y después sometidas a un proceso de desinfección en una solución de hipoclorito de sodio 0,5 por ciento y 1,0 por ciento, sumergidas durante 5 y 10 min en ambos casos; para las pruebas de escalado se seleccionó el tratamiento químico que a nivel de laboratorio resultó más efectivo, se emplearon 3 réplicas de la droga cruda de 10 kg en el caso de A annua y de 6 kg en T lucida, con posterioridad las muestras se escurren sobre bastidores y después pasan a un proceso de secado en estufa de 35 a 40 ºC durante 2 d Luego de secadas se realizan los correspondientes análisis microbiológico y físico-químico. En el primer caso se determinó el conteo total de bacterias, conteo total de hongos y otras enterobacterias, además de microorganismos aislados. En el control físico- químico, los porcentajes de materia orgánica extraña; materia inorgánica extraña y hojas ennegrecidas; los índices numéricos como humedad, cenizas totales, sustancias solubles en agua, sustancias solubles en etanol 30 por ciento, sustancias solubles en etanol 70 por ciento, así como mediante tamizaje fitoquímico, la presencia de metabolitos secundarios...(AU)

Introduction: the use of washing and chemical disinfection as a method allows reducing microbial pollution in the harvested vegetal material of Artemisia annua L. and Tagetes lucida Cav Objectives: to assure the microbiological quality of vegetal drugs from these two medicinal species. Methods: there was used some vegetal material from lots on red hydrated ferralitic soils (ferrasols) located in Dr Juan Tomás Roig Experimental Center of Medicinal Plants. Two controls, one treated with drinking water and the other untreated, were used. For lab tests, the 300g A. annua and 250g T lucida samples were washed with drinking water and then were subjected to disinfection process using 0,5 and 1,0 percent sodium hypochloride in which both samples were submerged for 5 and 10 minutes. The most effective chemical treatment at lab was selected for the scaling test of three replicas of the crude drug from 10 kg of A annua and from 6 kg T lucida respectively. The samples were placed upon frames to be dried later at 35 to 40 ºC for 2 hours. After drying, the corresponding microbiological and physical-chemical analyses were performed. The microbiological analysis determined the total count of bacteria, of fungi and other enterobacteria in addition to isolated microorganisms. The physical and chemical analysis estimated the percentages of foreign organic matter, foreign inorganic matter and blackened leaves; and the indexes of humidity, total ashes, soluble substances in water, soluble substances in 30 percent etanol and soluble substances in 70 percent etanol...(AU)

Rhodococcus artemisiae sp. nov., an endophytic actinobacterium isolated from the pharmaceutical plant Artemisia annua L.

A Gram-positive, non-motile actinobacterium, designated YIM 65754T, was isolated from the stem of Artemisia annua L., collected from Yunnan province, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM 65754T comprised an evolutionary lineage within the genus Rhodococcus. The isolate clustered with Rhodococcus pyridinivorans PDB9T, Rhodococcus gordoniae W 4937T and Rhodococcus rhodochrous DSM 43241T, with which it shared 98.4, 97.9 and 97.8 % 16S rRNA gene sequence similarities, respectively. However, DNA-DNA relatedness demonstrated that strain YIM 65754T was distinct from its closest phylogenetic neighbours. The cell-wall peptidoglycan contained meso-diaminopimelic acid, arabinose, galactose, mannose and glucose (cell-wall chemotype IV). The major menaquinone was MK-8(H2) and the predominant fatty acids were C16:0 (27.83 %), iso-C15:0 2-OH and/or C16:1ω7c (20.21 %) and 10-methyl C18:0 (17.50 %). The DNA G+C content was 66.2 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic evidence, the isolate represents a novel species of the genus Rhodococcus, for which the name Rhodococcus artemisiae sp. nov. is proposed; the type strain is YIM 65754T (=CCTCC AA 209042T=DSM 45380T).