Volatile constituents of four Inula species of Bulgarian origin

In this study the volatile components of four Inula species (Inula germanica L., I. bifrons (L.) L., I. ensifolia L., and I. salicina L.) were analysed by GC-FID/MS. A total of 141 chemical compounds were identified. A distinct volatile chemical profiles with high variation in the type of compounds was observed. Inula germanica was rich in oxygenated monoterpenes (54.7%), with cis-carvyl acetate (20.7%) and 1,8-cineole (14.6%) as main components. Sesquiterpenoids dominated in I. bifrons (60.3%), while the relatively high percentage of fatty acids characterized the other two species I. ensifolia and I. salicina (44.1 and 39.8%, respectively). Principal component analysis (PCA) and cluster analysis (CA) were used to investigate the variations in the volatiles of different Inula species.     

Antichemotactic and Antifungal Action of the Essential Oils from Cryptocarya aschersoniana,Schinus terebinthifolia,and Cinnamomum amoenum

The purpose of this work was to determine the chemical composition and evaluate the antichemotactic, antioxidant, and antifungal activities of the essential oil obtained from the species Cryptocarya aschersoniana Mez , Cinnamomum amoenum (Ness & Mart .) Kosterm. , and Schinus terebinthifolia Raddi , as well as the combination of C. aschersoniana essential oil and terbinafine against isolates of dermatophytes. Allo‐aromadendrene, bicyclogermacrene, and germacrene B were identified as major compounds in essential oils. The essential oil of C. aschersoniana shown 100 % inhibitory effect on leukocyte migration at the concentration of 10 μg/mL while S. terebinthifolia oil presented 80.1 % inhibitory effect at the same concentration. Only S. terebinthifolia oil possessed free‐radical‐scavenging activity which indicates its antioxidant capacity. The essential oils were also tested against fungal isolates of dermatophyte species (Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum), resulting in MIC ranging from 125 μg/mL to over 500 μg/mL. C. aschersoniana oil combined with terbinafine resulted in an additive interaction effect. In this case, the essential oil may act as a complement to conventional therapy for the topical treatment of superficial fungal infections, mainly because it is associated with an anti‐inflammatory effect.     

The antagonistic/synergistic effects of some medicinal plant essential oils,extracts and powders combined with Diatomaceous earth on red flour beetle,Tribolium castaneum Herbst (Coleoptera: Tenebrionidae)

Abstract

Red flour beetle, Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) is a prominent pest of stored products particularly cereal flour. Since resistance of this pest to common chemical insecticides is well documented, we were examined the synergistic/antagonistic interaction between Satureja hortensis L., Trachyspermum ammi L., Ziziphora tenuior L., Cuminum cyminum L. and Foeniculum vulgare Miller essential oils, ethanolic extracts and powders with Diatomaceous earth (DE) against T. castaneum adults under laboratory conditions at 27 ± 1 °C, 65 ± 5% RH and continuous darkness. We assayed repellency of ethanolic extracts and essential oils of mentioned plants on the pest. Results showed that DE had high toxicity to the pest. Plant essential oils and ethanolic extracts (except ziziphora) synergized the performance of DE. Nevertheless, plant powders elicited antagonistic effects (except ziziphora that exhibited synergistic effect). The most repellent EO and extract was cumin which exhibited mean repellency value on adult insect equivalent to 92.58 and 51.47%, respectively.     

Optimization of culture media for large‐scale lutein production by heterotrophic Chlorella vulgaris

Lutein is a carotenoid with a purported role in protecting eyes from oxidative stress, particularly the high‐energy photons of blue light. Statistical optimization was performed to growth media that supports a higher production of lutein by heterotrophically cultivated Chlorella vulgaris. The effect of media composition of C. vulgaris on lutein was examined using fractional factorial design (FFD) and central composite design (CCD). The results indicated that the presence of magnesium sulfate, EDTA‐2Na, and trace metal solution significantly affected lutein production. The optimum concentrations for lutein production were found to be 0.34 g/L, 0.06 g/L, and 0.4 mL/L for MgSO₄·7H₂O, EDTA‐2Na, and trace metal solution, respectively. These values were validated using a 5‐L jar fermenter. Lutein concentration was increased by almost 80% (139.64 ± 12.88 mg/L to 252.75 ± 12.92 mg/L) after 4 days. Moreover, the lutein concentration was not reduced as the cultivation was scaled up to 25,000 L (260.55 ± 3.23 mg/L) and 240,000 L (263.13 ± 2.72 mg/L). These observations suggest C. vulgaris as a potential lutein source. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:736–743, 2014     

A novel biotransformation process of 4′-demethylepipodophyllotoxin to 4′-demethylepipodophyllic acid by Bacillus fusiformis CICC 20463, Part II: process optimization

This work optimized the novel biotransformation process of 4′-demethylepipodophyllotoxin (DMEP) into 4′-demethylepipodophyllic acid (DMEPA) by Bacillus fusiformis CICC 20463. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/L of yeast extract and 10 g/L of peptone were optimal for DMEPA production (i.e., 2.81 ± 0.21 mg/L), while not beneficial for the cell growth of B. fusiformis. This indicated that the biosynthesis of DMEPA was not corresponded well to the cell growth of B. fusiformis. 40 g/L of sucrose was optimal for DMEPA production (i.e., 2.94 ± 0.17 mg/L), and 3 g/L of NaCl was the best for DMEPA production (i.e., 4.10 ± 0.18 mg/L). Secondly, the production of DMEPA was significantly enhanced by the control of substrate concentration and culture pH. 100 mg/L of substrate was optimal for DMEPA production (i.e., 6.47 ± 0.35 mg/L), and DMEPA concentration was enhanced to 38.78 mg/L by controlling culture pH at 9.0 in the stirred-tank bioreactors. The fundamental information obtained in this study provides a simple and efficient way to produce DMEPA by biotransformation.     

Enhanced production of the pharmaceutically important polyphenolic compounds in Vitex agnus castus L. shoot cultures by precursor feeding strategy

Agitated Vitex agnus castus L. shoot cultures were established to analyse the content of selected pharmaceutically important flavonoids and phenolic acids. Two variants (selected from nine ones) of MS medium were prepared: A (BAP 1 mg/L; NAA 0.5 mg/L; GA₃ 0.25 mg/L) and B (BAP 2 mg/L; NAA 0.5 mg/L). The biomass was harvested after 1, 2, 3,4, 5 and 6 weeks. Four‐week cultures (variant A) were selected to perform the precursor feeding experiment. The L‐phenylalanine dose of 1.6 g/L appears to be the most advantageous. Compared to the control cultures, the content of the individual compounds increased in a range from 1.4 to 17.3‐fold (e.g. p‐coumaric acid – 17.3 fold; casticin – 4.8‐fold). The biomass from in vitro cultures is richer in neochlorogenic acid (16‐fold), p‐coumaric acid (5.3‐fold), rutin (2.8‐fold), caffeic acid (1.5‐fold) and cinaroside (1.5‐fold) than the leaves of its parent greenhouse‐cultivated plants. Extracts contained 30 mg/100 g DW of casticin, but after the hydrolysis its amount increased up to 200 mg/100 g DW and twice exceeded the content in the greenhouse leaves. The results indicate that V. agnus castus agitated shoot cultures might be considered as a potential biotechnological source of some pharmaceutically important compounds, especially casticin, rutin, neochlorogenic and p‐coumaric acids.     

Preparative separation of chlorogenic acid by centrifugal partition chromatography from highbush blueberry leaves (Vaccinium corymbosum L.)

Introduction – Blueberries (genus Vaccinium) have gained worldwide focus because of the high anthocyanin content of their fruits. In contrast, the leaves of blueberry have not attracted any attention, even though they contain large quantities of chlorogenic acid, a strong antioxidant compound. Objective – The aim of this investigation was the quantification and preparative isolation of chlorogenic acid (5‐caffeoylquinic acid, 5‐CQA) from blueberry leaves using a new separation scheme, centrifugal partition chromatography (CPC). Methodology – A water fraction containing a high concentration of 5‐CQA (14.5% of dry weight extract) was obtained by defatting a crude methanol extract from blueberry leaves. CPC was applied to isolate 5‐CQA from this water fraction using a two‐phase solvent system of ethyl acetate–ethanol–water at a volume ratio 4:1:5 (v/v/v). The flow‐rate of mobile phase was 2 mL/min with the ascending mode while rotating at 1200 rpm. The eluate was monitored at 330 nm. The structure of chlorogenic acid in the CPC fraction was confirmed with HPLC, UV, ESI/MS and NMR spectra. Results – The HPLC chromatogram showed that the fractions collected by CPC contained chlorogenic acid with 96% purity based on peak area percentage. The total amount of chlorogenic acid isolated from 0.5 g of a water fraction was 52.9 mg, corresponding to 10.6% of the water fraction. The isolated compound was identified successively as 5‐CQA with MS (parent ion at m/z 355.1 [M + H]⁺) and ¹H NMR spectra [caffeoyl moiety in the down field (δ 6.0–8.0 ppm) and quinic acid moiety in the up field (δ 2.0–5.5 ppm)]. Conclusion – 5‐CQA was successfully isolated from blueberry leaves by the CPC method in a one‐step procedure, indicating a further potential use for blueberry leaves. Copyright © 2010 John Wiley & Sons, Ltd.     

Phytochemical Profile and Biological Activities of Helianthemum canum l. baumg. from Turkey

In the current study, antioxidant, antibacterial activities, and the phenolic compositions of extracts from Helianthemum canum L. Baumg . (Apiaceae) aerial parts were investigated for the first time. The H . canum was extracted with 70% methanol (HCM eOH ) and water (HCW ). Both extracts were determined by total phenolic contents (3 mg/ml), flavonoids (1.5 mg/ml), flavonols (1.5 mg/ml), qualitative–quantitative compositions, iron (II ) chelation activities (0.1 – 5 mg/ml), free radical scavenging activities (DPPH ^?: 0.01 – 0.6 mg/ml and ABTS ^+?: 0.125 – 0.5 mg/ml) and the effect upon inhibition of β ‐carotene/linoleic acid co‐oxidation (1 mg/ml). The peroxidation level was also determined using the thiobarbituric acid method (0.01 – 1.5 mg/ml). The results of the activity tests given as IC ₅₀ values were estimated from non‐linear algorithm and compared with standards. Antibacterial activities of extracts and standards were evaluated against Gram‐ negative and ‐positive ten standard strains using disc diffusion and broth microdilution methods. The MIC results (312.5 – 2500 μg/ml) against tested microorganisms varied from 625 to 2500 μg/ml. In HPLC analysis, 3,5‐dihydroxybenzoic acid was found as the main substance in both extracts. These results showed that HCM eOH was richer in phenolic compounds (284.13 ± 0.30 mg GAE /g extract) from HCW (244.55 ± 0.35 mg GAE /g extract)_. In conclusion, H . canum extracts showed in vitro antibacterial and antioxidant activities.     

Labeling of Polyethylenimine with Fluorescent Dye to Image Nucleus,Nucleolus, and Chromosomes in Digitonin-Permeabilized HeLa Cells

The effect of a koji (Aspergillus awamori mut.) extract on the caffeoylquinic acid derivatives purified from sweetpotato (Ipomoea batatas L.) leaves was examined to develop the mass production of caffeic acid. A koji extract hydrolyzed the caffeoylquinic acid derivatives, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4,5-tri-O-caffeoylquinic acid, to caffeic acid. Furthermore, the koji extract also converted the major polyphenolic components from sweetpotato, burdock (Arctium lappa L.), and mugwort (Artemisia indica var. maximowiczii) leaves to caffeic acid. These results suggest that the production of caffeic acid from plant resources containing caffeoylquinic acid derivatives is possible.     

Site-directed mutagenesis improves the practical application of L-glutamic acid decarboxylase in Escherichia coli

γ-Aminobutyric acid (GABA) is a kind of non-proteinogenic amino acid which is highly soluble in water and widely used in the food and pharmaceutical industries. Enzymatic conversion is an efficient method to produce GABA, whereby glutamic acid decarboxylase (GAD) is the key enzyme that catalyzes the process. The activity of wild-type GAD is usually limited by temperature, pH or biotin concentration, and hence directional modification is applied to improve its catalytic properties and practical application. GABA was produced using whole cell transformation of the recombinant strains Escherichia coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively. The results showed that Gad B-T62S and Gad B-Q309A are two effective mutation sites. These findings may contribute to ideas for constructing potent recombinant strains for GABA production. Practical Application : Enzymatic properties of the GAD from Escherichia coli and GAD site-specific mutants were examined by analyzing their conserved sequences, substrate contacts, contact between GAD amino acid residues and mutation energy (ΔΔG) of the GAD mutants. The enzyme activity and stability of Gad B-T62S and Gad B-Q309A mutants were improved compared to Gad B. The kinetic parameters K_m and V_max of Gad B, Gad B-T62S, and Gad B-Q309A mutants were 11.3 ± 2.1 mM and 32.1 ± 2.4 U/mg, 7.3 ± 2.5 mM and 76.1 ± 3.1 U/mg, and 7.2 ± 3.8 mM and 87.3 ± 1.1 U/mg, respectively. GABA was produced using whole cell transformation of the recombinant strains E. coli BL21(DE3)-Gad B, E. coli BL21(DE3)-Gad B-T62S, and E. coli BL21(DE3)-Gad B-Q309A. The corresponding GABA concentrations in the fermentation broth were 219.09, 238.42, and 276.66 g/L, and the transformation rates were 78.02%, 85.04%, and 98.58%, respectively.     

Quantification of Phytochemicals and Metal Ions as well as the Determination of Volatile Compounds,Antioxidant, Antimicrobial and Antacid Activities of the Mimosa pudica L. Leaf: Exploration of Neglected and Under-Utilized Part

Mimosa pudica L. (MP) is well-known plant in traditional medicinal system, especially in India. Unfortunately, leaves of MP are less explored. To determine the food and nutritional value of the neglected part of Mimosa pudica L. (MP), that is MP leaves, phytochemicals and metal ions of MP were quantified by newly developed HPLC and ICPOES-based methods. The content of phytochemicals observed using HPLC analysis for chlorogenic acid, catechin, and epicatechin was 141.823 (±8.171), 666.621 (±11.432), and 293.175 (±12.743) μg/g, respectively. Using GC/MS/MS analysis, fatty acid like oleic acid were identified. In ICP-OES analysis, a significant content of Na, K, Ca, Cu, Fe, Mg, Mn, and Zn was observed. The observed TPC and TFC for MP leaf extracts was 44.327 (±1.041) mg GAE/ g of wt. and 214.217 (±4.372) mg QCE/ g of wt., respectively. The DPPH assay depicted a strong antioxidant activity of MP leaf extracts with IC₅₀ values of 0.796 (±0.081) mg/mL and a TEAC value of 0.0356 (±0.0003). A significant antacid activity (666 mg MP+400 mg CaCO₃ >400 mg CaCO₃ ≫666 mg Gelusil) of MP leaves was noticed. The methanolic extract of MP leaves demonstrated anti-microbial activity against Staphylococcus aureus (15±2mm), Pseudomonas aeruginosa (12±2mm) and Escherichia coli (10±2mm). In silico studies confirmed the in vitro results obtained for antioxidant, antiacid, and anti-microbial activities. In addition, in silico studies revealed the anti-cancerous and anti-inflammatory potential of the MP leaves. In summary, this study demonstrated the medicinal significance of MP leaves and the conversion of agro-waste or the under-utilized part of MP into pharmaceutical potent materials. Consequently, the present study highlighted that MP leaves alone have medicinal importance with good nutritional utility and possess large promise in the pharma industry along with improving bio-valorization and the environment.     

Effects of feeding rate on the growth performance of gynogenetic albino sterlet,Acipenser ruthenus (Linnaeus, 1758) larvae

The feeding rate effects were studied on the growth performance of gynogenetic diploid larvae of sterlet Acipenser ruthenus during the first 4 weeks of exogenous feeding. The experimental rearing was conducted from 7 to 38 days post‐hatch (dph) in a circulation system. This was set up in four groups with three replicates (440 individuals/replicate), viz: AC‐control larvae fed Artemia sp., CFC‐control larvae fed compound feed, AG‐gynogenetic larvae fed Artemia sp., and CFG‐gynogenetic larvae fed compound feed. The larvae were reared in glass tanks (44 L volume, 10 individuals/L) with the temperature maintained at 18 ± 0.5°C, photoperiod of 12L:12D and water flow regime of 1‐L/min and fed 50%, 25%, 25%, and 9% of their total biomass/day during feeding. Highest TL and WBW of gynogenetic diploid larvae (AG) were observed with 50.6 ± 1.2 mm and 607.3 ± 36.1 mg (n = 30) at 38 dph. Highest TL and WBW of control larvae (AC) were recorded with 49.5 ± 3.8 mm and 600.8 ± 88.0 mg (n = 30), respectively, with 73.1% ± 11.4% survival; the lowest survival rate was at 46.4% ± 7.1% (n = 30) for the CFG group. The results indicate that the gynogenetic and normal larvae of sterlet fed with live food (Artemia nauplii) from 7 dph can achieve higher growth and survivability compared to the larvae fed with formulated test feed. Results of this study suggest that the effective rearing of sterlet larvae from 7 to 38 dph strongly depends upon the types of feed rather than the genome manipulation performed.     

Secolignans with antiangiogenic activities from Peperomia dindygulensis

Two new secolignans, peperomins G and H ( 1 and 2 , resp.), were isolated from the whole plant of Peperomia dindygulensis, together with five known secolignans, peperomin A ( 3 ), peperomin E ( 4 ), peperomin B ( 5 ), 2,3‐trans‐2‐methyl‐3‐{(3‐hydroxy‐4,5‐dimethoxyphenyl)[5‐methoxy‐3,4‐(methylenedioxy)phenyl]methyl}butyrolactone ( 6 ), 2,3‐cis‐2‐(hydroxymethyl)‐3‐{bis[5‐methoxy‐3,4‐(methylenedioxy)phenyl]methyl}butyrolactone ( 7 ). Their structures and configurations were elucidated by spectroscopic methods including 2D‐NMR techniques. Antiangiogenic effects of all compounds were evaluated using human umbilical vein endothelial cells (HUVEC) proliferation and tube‐formation tests, with compounds 4 and 5 being active in the bioassay. Compounds 4 and 5 induced obvious cell toxicity to HUVEC with IC₅₀ values of 1.64±0.19 and 8.44±0.4 μM , respectively. Compounds 4 and 5 also exhibited significant HUVEC tube formation‐inhibiting activity with IC₅₀ values of 3.13±0.09 and 6.24±0.12 μM , respectively.     

CipA-mediating enzyme self-assembly to enhance the biosynthesis of pyrogallol in Escherichia coli

Pyrogallol is a valuable phenolic compound and displays various physiological and pharmaceutical functions. Chemical synthesis of pyrogallol suffered from many issues, including environmental pollution, high cost, and low yield. Here, to address the above drawbacks, an artificial pathway for de novo pyrogallol production was established and this pathway only needed two exogenous enzymes (Y385F/T294A PobA and 3,4-dihydroxybenzoic acid decarboxylase (PDC)). Y385F/T294A PobA is a mutant of PobA which is a hydroxylase from Pseudomonas aeruginosa, while PDC is a decarboxylase from Klebsiella pneumoniae subsp. pneumoniae. First, the conversion efficiency of PDC was tested and 1800 ± 100 mg/L pyrogallol was generated from 4 g/L gallic acid (GA). Subsequently, assembly of the whole pathway enabled 33 ± 6 mg/L pyrogallol production from simple carbon sources. After that, based on the assembling property of CipA (a hydrophobic protein) and to enhance the hydroxylation of 3,4-dihydroxybenzoic acid, CipA was employed to organize its fusion (Y385F/T294A PobA) into protein crystalline inclusions (PCIs). Remarkably, the formation of CipA-Y385F/T294A PobA PCIs increased the pyrogallol production to 60 ± 6 mg/L, a 1.8 ± 0.4-fold higher value as compared to the strain without enzyme self-assembly. Additionally, the titer of pyrogallol was enhanced to 80 ± 1 mg/L through yeast extract concentration optimization. This work not only realizes the biosynthesis of pyrogallol from renewable carbon sources but also demonstrates that using CipA-mediating enzyme self-assembly could reinforce the hydroxylation efficiency of Y385F/T294A PobA, resulting in the enhancement of pyrogallol production.

     

Untersuchungen zur physiologischen Spezialisierung von Erysiphe graminis DC

Zusammenfassung In den vorliegenden Untersuchungen wurde der Wirtspflanzenbereich einer Mehltaupopulation von Dactylis glomerata L., Sorte Motterwitzer, überprüft. Von 460 infizierten Grasarten blieben 420 befallsfrei, 40 erwiesen sich als anfällig. Am stärksten befallen wurden die drei Arten Dactylis aschersoniana Graebner, Dactylis glomerata L. und Dactylis polygama Horvat sowie die Subspecies D. glomerata L. spp. aschersoniana (Graebn.) Thell., die aber insgesamt in ihrem Verhalten sehr unausgeglichen waren. Eine unerwartet geringe Anfälligkeit von Dactylis woronowii Ovcz. müßte nochmals überprüft werden. Als sehr anfällig erwiesen sich die drei Grasarten Bouteloua hirsuta Lag., Danthonia provinzialis DC. und Sesleria latifolia (Adam) Degen. Geringer Befall konnte auch an einigen Wildgräsern der Gattungen Agropyron, Bromus, Festuca, Mibora, Poa und Sesleria nachgewiesen werden. Vom Getreide wird nur die Gerste vom Knaulgras-Mehltau befallen. Da sowohl bei der Wildform als auch bei der Zuchtsorte Motterwitzer von Dactylis glomerata L. über 10% befallsfreie Pflanzen gefunden wurden, erscheint die Züchtung mehltauresistenter Sorten von Dactylis glomerata L. nicht ohne Aussicht auf Erfolg.

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Studies on the physiological specialization of Erysiphe graminis DCIV. The host plants of cocksfoot mildew

Summary In the described experiments the interaction of 460 grass species with a mildew population obtained from Dactylis glomerata L. Motterwitzer was examined. 420 species remained free of symptoms, while 40 species were susceptible. The most susceptible ones were Dactylis aschersoniana Graebner, Dactylis glomerata L., Dactylis glomerata spp. aschersoniana (Graebn.) Thell., and Dactylis polygama Horvat, but all showed differences in their behaviour. The unexpectedly low susceptibility of Dactylis woronowii Ovcz. should be examined again. Three grass species Bouteloua hirsuta Lag., Danthonia provinzialis DC., and Sesleria latifolia (Adam) Degen were also highly susceptible to cocksfoot mildew, while on wild grasses of the species Agropyron, Bromus, Festuca, Mibora, Poa, and Sesleria only few symptoms could be seen. Among cereals only barley could be infected by cocksfoot mildew. Not only in the wild form of Dactylis glomerata L. but also in the cultivated variety Motterwitzer, above 10% were found to be free from infection. Therefore the breeding of resistant varieties of Dactylis glomerata L. seems to be feasible.

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Für die freundliche Unterstützung der Arbeiten möchten wir den Botanischen Gärten der Deutschen Demokratischen Republik sowie der Deutschen Bundesrepublik unseren herzlichen Dank aussprechen.

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Angenommen durch H. Stubbe     

Arbuscular mycorrhizal fungi alter thymol derivative contents of Inula ensifolia L.

Individuals of Inula ensifolia L. (Asteraceae), a valuable xerothermic plant species with potential therapeutic value, were inoculated under laboratory conditions with different strains of arbuscular mycorrhizal fungi (AMF): (1) Glomus intraradices UNIJAG PL-Bot, (2) G. intraradices UNIJAG PL-Kap, (3) Glomus clarum UNIJAG PL13-2, and (4) AMF crude inoculum from natural stands of I. ensifolia. We found AMF species specificity in the stimulation of thymol derivative production in the roots of I. ensifolia. There was an increase in thymol derivative contents in roots after G. clarum inoculation and at the same time the decreased production of these metabolites in the G. intraradices treatments. Moreover, no correlation between the extent of AMF colonization and the effects of the fungal symbionts on the plant was observed. A multilevel analysis of chlorophyll a fluorescence transients (JIP test) permitted an evaluation of plant vitality, expressed in photosynthetic performance index, influenced by the applied AMF strains, which was found to be in good agreement with the results concerning thymol derivative production. The mechanisms by which AMF trigger changes in phytochemical concentration in plant tissues and their consequences for practice are discussed.     

THE BIOLOGICAL ACT'IVITIES OF YELLOW AZALEA,RHODODENDRON MOLLE G. DON AGAINST THE VEGETABLE LEAFMINER,LIRIOMYZA SATIVAE (DIPTERA: AGROMYZIDAE) *

Abstract The results of laboratory and greenhouse bioassays indicated that Rhodojaponin‐ III (Abbr. R‐ 1) and extracts of flowers from Rhododendron molle G. Don possessed signficant feeding inhibition and insectcidal properties against the larvae and adults of Liromyzia sativae. Treated with 500 mg/L R‐ III 1 000 mg/L molosul‐tap, and 10 000 mg/L methanol(MeOH) ethyl acetate (EtOAc), CH₂Cl₂, methanol‐water (MeOH‐H₂O) extracts the rates of feeding inhibition were 77. 34 % 74.30 % 82.15 % 77.50 % 67. 33 % 62.85 % against the 2nd instar larvae, and were 67.66% 55.21 % 49.72% 54.26% 46.81 % 38.53% against the 3rd instar larvae, respectively;LC₅₀ values against the 2nd instar larvae were 208.65, 166.05, 2.74 ± 10³,766.72, 5.95 ± 10³, 1.85 ± 10³mg/L, and against 3rd larvae were 300.62, 256.00, 4.33 ±10³, 1.03 ± 10³,9.79 ± 10³, and 2.62± 10³mg/L, respectively. Against the adults, LC₅₀ values of R‐III EtOAc extract and molosultap were 159.07.723.87 and 134.55mgL respectively after treatment for 24 h.     

Enhancing epi‐cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi‐cedrol synthase

Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi‐cedrol synthase (AaECS). The stop codon at the N‐terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS‐ECS produced sesquiterpene 8‐epi‐cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K_m values for FPPS‐ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi‐cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi‐cedrol in fermentation medium. The strain having fusion plasmid (pET32a‐FPPS‐ECS) produced 1.084 ± 0.09 mg/L epi‐cedrol, while the strain harbouring mixed plasmid (pRSETB‐FPPS and pET28a‐ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I‐TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N‐ C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.