Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae)

In this study, interactions between Cry1Ac, a toxic crystal protein produced by Bacillus thuringiensis (Berliner), and Beauveria bassiana on the mortality and survival of Ostrinia furnacalis was evaluated in the laboratory. The results showed that Cry1Ac is toxic to O. furnacalis. Not only were larval growth and development delayed, but pupation, pupal weight and adult emergency also decreased when larvae were fed on artificial diet containing purified Cry1Ac toxin. When third instars O. furnacalis were exposed to combination of B. bassiana (1.8 × 10⁵, 1.8 × 10⁶ or 1.8 × 10⁷ conidia ml⁻¹) and Cry1Ac, (0.2 or 0.8 μg g⁻¹), the effect on mortality was additive, however, the combinations of sublethal concentrations showed antagonism between Cry1Ac (3.2 or 13 μg g⁻¹) and B. bassiana (1.8 × 10⁵ or 1.8 × 10⁶ conidia ml⁻¹). When neonates were reared on sublethal concentrations of Cry1AC until the third instar, and survivors exposed B. bassiana conidial suspension, such treatments showed additive effect on mortality of O. furnacalis except for the combination of Cry1Ac (0.2 μg g⁻¹) and B. bassiana (1.8 × 10⁶ conidia ml⁻¹) that showed antagonism.     

Effects of Bacillus thuringiensis toxin Cry1Ac and cytoplasmic polyhedrosis virus of Helicoverpa armigera (Hübner) (HaCPV) on cotton bollworm (Lepidoptera: Noctuidae)

In this study, interactions on the mortality and debilitating effects between Cry1Ac, a toxic protein produced by Bacillus thuringiensis (Berliner) and HaCPV (Chinese strain) on first and third instars larvae of Helicoverpa armigera were evaluated in laboratory. When first instar was exposed to combination of Bt cotton leaf discs containing HaCPV (6 × 10⁶, 1 × 10⁷, and 3 × 10⁷ PIB ml⁻¹) the effect on mortality was additive, when such instar larvae exposed to combination of Cry1Ac (0.9, 2.7, or 8.1 μg g⁻¹) and the same concentrations of HaCPV the effect on mortality was additive except for the combination of Cry1Ac (0.3 μg g⁻¹) and HaCPV concentrations that showed synergism. When third instars of H. armigera were infected using a suspension containing both HaCPV and Cry1Ac, most combinations of them showed additive effect except for the combination of Cry1Ac (0.3 μg g⁻¹) and HaCPV (3 × 10⁷ PIB ml⁻¹) that showed synergism. However, when they exposed to Bt cotton leaf discs and HaCPV the effect on mortality was synergism except combination of Bt cotton leaf discs and HaCPV (6 × 10⁶ PIB ml⁻¹) that showed additive. Most of the combinations are showed additive effect in the toxicity and in combinations of Cry1Ac at lowest and HaCPV at highest concentrations synergism is observed. Not only were larval growth and development delayed, but pupation and pupal weight also decreased when larvae were fed on artificial diet containing Cry1Ac and HaCPV or transgenic Bt cotton leaf discs specially in first instar.     

Characterization of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase multigene family of Malus domestica Borkh

Two 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes have been cloned from RNA isolated from leaf tissue of apple (Malus domestica cv. Royal Gala). The genes, designated MD-ACO2 (with an ORF of 990 bp) and MD-ACO3 (966 bp) have been compared with a previously cloned gene of apple, MD-ACO1 (with an ORF of 942 bp). MD-ACO1 and MD-ACO2 share a close nucleotide sequence identity of 93.9% in the ORF but diverge in the 3′ untranslated regions (3′-UTR) (69.5%). In contrast, MD-ACO3 shares a lower sequence identity with both MD-ACO1 (78.5%) and MD-ACO2 (77.8%) in the ORF, and 68.4% (MD-ACO1) and 71% (MD-ACO2) in the 3′-UTR. Southern analysis confirmed that MD-ACO3 is encoded by a distinct gene, but the distinction between MD-ACO1 and MD-ACO2 is not as definitive. Gene expression analysis has shown that MD-ACO1 is restricted to fruit tissues, with optimal expression in ripening fruit, MD-ACO2 expression occurs more predominantly in younger fruit tissue, with some expression in young leaf tissue, while MD-ACO3 is expressed predominantly in young and mature leaf tissue, with less expression in young fruit tissue and least expression in ripening fruit. Protein accumulation studies using western analysis with specific antibodies raised to recombinant MD-ACO1 and MD-ACO3 produced in E. coli confirmed the accumulation of MD-ACO1 in mature fruit, and an absence of accumulation in leaf tissue. In contrast, MD-ACO3 accumulation occurred in younger leaf tissue, and in younger fruit tissue. Further, the expression of MD-ACO3 and accumulation of MD-ACO3 in leaf tissue is linked to fruit longevity. Analysis of the kinetic properties of the three apple ACOs using recombinant enzymes produced in E. coli revealed apparent Michaelis constants (K_m) of 89.39 μM (MD-ACO1), 401.03 μM (MD-ACO2) and 244.5 μM (MD-ACO3) for the substrate ACC, catalytic constants (K_cat) of 6.6 × 10⁻² (MD-ACO1), 3.44 × 10⁻² (Md-ACO2) and 9.14 × 10⁻² (MD-ACO3) and K_cat/K_m (μM s⁻¹) values of 7.38 × 10⁻⁴ μM s⁻¹ (MD-ACO1), 0.86 × 10⁻⁴ M s⁻¹ (MD-ACO2) and 3.8 × 10⁻⁴ μM s⁻¹ (MD-ACO3). These results show that MD-ACO1, MD-ACO2 and MD-ACO3 are differentially expressed in apple fruit and leaf tissue, an expression pattern that is supported by some variation in kinetic properties.     

Light-dependent phosphate uptake of a submersed macrophyte Myriophyllum spicatum L.

The uptake kinetics of phosphate (Pi) by Myriophyllum spicatum was determined from adsorption and absorption under light and dark conditions. Pi uptake was light dependent and showed saturation following the Michaelis-Menten relation (in light: V = 16.91 × [Pi](1.335 + [Pi]), R² = 0.90, p < 0.001; in the dark: V = 5.13 × [Pi](0.351 + [Pi]), R² = 0.77, p < 0.001). Around 77% of the loss of Pi in the water column was absorbed into the tissue of M. spicatum, and only 23% was adsorbed on the surface of the plant shoots. Our study shows that M. spicatum shoots have a much higher affinity (in light: 3.9 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 3.7 μmol g⁻¹ dw h⁻¹ μM⁻¹) and V_max (maximum uptake rate, shoot light) for Pi uptake than many other aquatic macrophytes (in light: 0.002-0.23 μmol g⁻¹ dw h⁻¹ μM⁻¹; in the dark: 0.002-0.19 μmol g⁻¹ dw h⁻¹ μM⁻¹), which may provide a competitive advantage over other macrophytes across a wide range of Pi concentrations.     

The human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 (NAT1) is irreversibly inhibited by inorganic (Hg) and organic mercury (CH3Hg): Mechanism and kinetics

Human arylamine N-acetyltransferase 1 (NAT1) is a xenobiotic-metabolizing enzyme that biotransforms aromatic amine chemicals. We show here that biologically-relevant concentrations of inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury inhibit the biotransformation functions of NAT1. Both compounds react irreversibly with the active-site cysteine of NAT1 (half-maximal inhibitory concentration (IC₅₀) = 250 nM and k_inact = 1.4 × 10⁴ M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 1.4 μM and k_inact = 2 × 10² M⁻¹ s⁻¹ for CH₃Hg⁺). Exposure of lung epithelial cells led to the inhibition of cellular NAT1 (IC₅₀ = 3 and 20 μM for Hg²⁺ and CH₃Hg⁺, respectively). Our data suggest that exposure to mercury may affect the biotransformation of aromatic amines by NAT1.     

Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores

Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80 °C, and for the decolorization of indigo carmine at pH 8.0 and 60 °C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2 h at 37 °C. The apparent K_m of the enzyme displayed on spores was 443 ± 124 μM for ABTS with a V_max of 150 ± 16 U/mg spores. Notably, 1 mg of spores displaying B. subtilis laccase (3.4 × 10² U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2 h. The spore reactor (0.5 g of spores corresponding to 1.7 × 10⁵ U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60 °C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.     

Hydrolysis of the soluble fluorescent molecule carboxyumbelliferyl-beta-D-glucuronide by E. coli beta-glucuronidase as applied in a rugged, in situ optical sensor

Techniques utilizing β-glucuronidase (GUS) activity as an indicator of Escherichia coli (E. coli) presence use labeled glucuronides to produce optical signals. Carboxyumbelliferyl-β-d-glucuronide (CUGlcU) is a fluorescent labeled glucuronide that is soluble and highly fluorescent at natural water pHs and temperatures and, therefore, may be an ideal reagent for use in an in situ optical sensor. This paper reports for the first time the Michaelis-Menten kinetic parameters for the binding of E. coli GUS with CUGlcU as K_m = 910 μM, V_max = 41.0 μM min⁻¹, V_max/K_m 45.0 μmol L⁻¹ min⁻¹, the optimal pH as 6.5 ± 1.0, optimal temperature as 38 °C, and the Gibb's free energy of activation as 61.40 kJ mol⁻¹. Additionally, it was found CUGlcU hydrolysis is not significantly affected by heavy solvents suggesting proton transfer and solvent addition that occur during hydrolysis are not limiting steps. Comparison studies were made with the more common fluorescent molecule methylumbelliferyl-β-d-glucuronide (MUGlcU). Experiments showed GUS preferentially binds to MUGlcU in comparison to CUGlcU. CUGlcU was also demonstrated in a prototype optical sensor for the detection of E. coli. Initial bench testing of the sensor produced detection of low concentrations of E. coli (1.00 × 10³ CFU/100 mL) in 230 ± 15.1 min and high concentrations (1.05 × 10⁵ CFU/100 mL) in 8.00 ± 1.01 min.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.     

A simple bacterial transformation method using magnesium- and calcium-aminoclays

An efficient and user-friendly bacterial transformation method by simple spreading cells with aminoclays was demonstrated. Compared to the reported transformation approaches using DNA adsorption or wrapping onto (in)organic fibers, the spontaneously generated clay-coated DNA suprastructures by mixing DNA with aminoclay resulted in transformants in both Gram-negative (Escherichia coli) and Gram-positive cells (Streptococcus mutans). Notably, the wild type S. mutans showed comparable transformation efficiency to that of the E. coli host for recombinant DNA cloning. This is a potentially promising result because other trials such as heat-shock, electroporation, and treatment with sepiolite for introducing DNA into the wild type S. mutans failed. Under defined conditions, the transformation efficiency of E. coli XL1-Blue and S. mutans exhibited ~ 2 × 10⁵ and ~ 6 × 10³ CFU/μg of plasmid DNA using magnesium-aminoclay. In contrast, transformation efficiency was higher in S. mutans than that in E. coli XL1-Blue for calcium-aminoclay. It was also confirmed that each plasmid transformed into E. coli and S. mutans was stably maintained and that they expressed the inserted gene encoding the green fluorescent protein during prolonged growth of up to 80 generations.     

Accumulation of lead by free and immobilized cyanobacteria with special reference to accumulation factor and recovery

Lead accumulation by free and immobilized cyanobacteria, Lyngbya majuscula and Spirulina subsalsa was studied. Exponentially growing biomass was exposed to 1-20 mg L⁻¹ of Pb(II) solution at pH 6, 7 and 8 for time periods ranging from 10 min to 48 h. L. majuscula accumulated 10 times more Pb (13.5 mg g⁻¹) than S. subsalsa (1.32 mg g⁻¹) at pH 6 within 3 h of exposure to 20 mg L⁻¹ Pb(II) solution and 76% of the Pb could be recovered using 0.1 M EDTA. This chelator (2 μM) did not influence Pb accumulation whereas 100 μM citrate increased that of S. subsalsa 6- to 8-fold. L. majuscula filaments enmeshed in a glass wool packed in a column removed 95.8% of the Pb from a 5 mg L⁻¹ Pb solution compared to free and dead biomass which removed 64 and 33.6% Pb respectively. A 92.5% recovery of accumulated Pb from the immobilized biomass suggests that repeated absorption-desorption is possible.     

Antifeedant activity of ethanolic extract from Flourensia oolepis and isolation of pinocembrin as its active principle compound

The ethanolic extract from Flourensia oolepis aerial parts showed strong antifeedant activity against the pest larvae, Epilachna paenulata, with an antifeedant index (AI%) of 99.1% at 100 μg/cm². Based on chromatographic fractionation of the extract, guided by bioassays on E. paenulata, the flavanone pinocembrin (1) was isolated as the most active principle. In a choice assay, compound 1 showed strong antifeedant activity against E. paenulata, Xanthogaleruca luteola and Spodoptera frugiperda with an AI% of 90, 94 and 91% (p < 0.01) respectively, at 50 μg/cm². The dosages necessary for 50% feeding inhibition of the insects (ED₅₀) were 7.98, 6.13 and 8.86 μg/cm², respectively. The feeding inhibitory activity of 1 against E. paenulata was compared with the activity of other structurally related flavonoids like naringenin, which was inactive up to 100 μg/cm², catechin which was nearly 6 times less active than 1, and quercetin which was equally active as 1. The effect of these on the feeding behavior of E. paenulata was also studied.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.     

Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light

Microbial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10⁷ spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3 × 10⁴ spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3 × 10⁵ spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

Quantification of carnosine-related peptides by microchip electrophoresis with chemiluminescence detection

A microchip electrophoresis (MCE) method with chemiluminescence (CL) detection was developed for the determination of carnosine-related peptides, including carnosine, homocarnosine, and anserine, in biological samples. A simple integrated MCE-CL system was built to perform the assays. The highly sensitive CL detection was achieved by means of the CL reaction between hydrogen peroxide and N-(4-aminobutyl)-N-ethylisoluminol-tagged peptides in the presence of adenine as a CL enhancer and Co²⁺ as a catalyst. Experimental conditions for analyte labeling, MCE separation, and CL detection were studied. MCE separation of the above-mentioned three peptides took less than 120 s. Detection limits (signal/noise ratio [S/N] = 3) of 3.0 × 10⁻⁸, 2.8 × 10⁻⁸, and 3.4 × 10⁻⁸ M were obtained for carnosine, anserine, and homocarnosine, respectively. The current MCE-CL method was applied for the determination of carnosine, anserine, and homocarnosine in human cerebrospinal fluid (CSF) and canine plasma. Homocarnosine was detected at the micromolar (μM) level in the CSF samples analyzed, whereas the levels of carnosine and anserine in these samples were below the detection limit of the assay. Interestingly, both carnosine and anserine were detected in the canine plasma samples, whereas homocarnosine was not.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Efficient regeneration for enhanced steviol glycosides production in Stevia rebaudiana (Bertoni)

An efficient method of regeneration for antidiabetic plant (Stevia rebaudiana) has been established for healthy biomass and main steviol glycosides (SGs) production, using different PGRs and agar concentrations. Higher callus induction (93.3%) was recorded when leaf explants were placed on an MS medium supplemented with 3.5 gL⁻¹ agar and 2.0 mgL⁻¹ 2,4-D. The addition of 7.0 gL⁻¹ agar and BA (1.0, 2.0 and 4.0 mgL⁻¹) significantly (P < 0.01) influences shooting response (100%). A maximum mean shoot length (13.03 cm) and 28 shoots per explant were observed on a medium containing 1.0 mgL⁻¹ BA. However, the maximum number of leaves (132.67) was encouraged by the addition of BA (1.0 mgL⁻¹) and Kin (1.0 mgL⁻¹). Lower agar (3.5 gL⁻¹), IAA (2.0 mgL⁻¹), and NAA (2.0 mgL⁻¹) concentrations significantly influence the rooting percent (100%), the mean root length (2.9 cm), and the number of roots per plantlet (26.3). These plantlets were successfully acclimatized in the soil. The BA (3.0 mgL⁻¹) in combination with Kin (3.0 mgL⁻¹) and 3.5 gL⁻¹ agar increases dulcoside-A content (Dul-A; 71.8 μg/g-DW) in shoots compared to control (50.81 μg/g-DW). Similar PGRs with 7.0 gL⁻¹ significantly increases the production of steviosides (Stev. 82.48 μg/g-DW). A higher rebaudioside-A content (Reb-A; 12.35 μg/g-DW) was observed in shoots that underwent the addition of BA (1.0 mgL⁻¹) and 7.0 gL⁻¹ agar than in control (07.39 μg/g-DW). Hereby, we developed an efficient and cost-effective method for regeneration and major SGs production, which could be helpful for future studies on this species.     

Pathogenecity of a baculovirus isolated from Arctornis submarginata (Walker) (Lepidoptera:Lymantriidae), a potential pest of tea growing in the Darjeeling foothills of India

A granulosis virus (GV) was isolated from the diseased caterpillars of Arctornis submarginata (Walker) (Lymantriidae), a defoliating pest of tea from Darjeeling foothill region. The phase contrast and transmission electron microscopic studies identified the virus as granulosis virus. SDS-PAGE analysis of major protein of the occlusion bodies was found to be 31 kDa, characteristic for granulin. The total genomic DNA was isolated. The major band found was of molecular weight 16 kDa. Bioassay conducted with the occlusion bodies (OBs) of the virus showed LC₅₀ value of 4.46 × 10⁴ OBs/ml for the second instar caterpillars. Median lethal time (LT₅₀) were 6.6 days for 1 × 10⁴ OBs/ml, 5.09 days for 1 × 10⁵ OBs/ml, 4.45 days for 1 × 10⁶ OBs/ml and 3.87 days for 1 × 10⁷OBs/ml concentrations. The results indicated the potential of the virus for its future application as microbial pesticide against A. submarginata in future.     

Inter-population variability of leaf morpho-anatomical and terpenoid patterns of Pistacia atlantica Desf. ssp. atlantica growing along an aridity gradient in Algeria

Three Algerian populations of female Pistacia atlantica shrubs were investigated in order to check whether their terpenoid contents and morpho-anatomical parameters may characterize the infraspecific variability. The populations were sampled along a gradient of increasing aridity from the Atlas mountains into the northwestern Central Sahara.As evidenced by Scanning Electron Microscopy, tufted hairs could be found only on seedling leaves from the low aridity site as a population-specific trait preserved also in culture. Under common garden cultivation seedlings of the high aridity site showed a three times higher density of glandular trichomes compared to the low aridity site. Increased aridity resulted also in reduction of leaf sizes while their thickness increased. Palisade parenchyma thickness also increases with aridity, being the best variable that discriminates the three populations of P. atlantica.Analysis of terpenoids from the leaves carried out by GC-MS reveals the presence of 65 compounds. The major compounds identified were spathulenol (23 μg g⁻¹ dw), α-pinene (10 μg g⁻¹ dw), verbenone (7 μg g⁻¹ dw) and β-pinene (6 μg g⁻¹ dw) in leaves from the low aridity site; spathulenol (73 μg g⁻¹ dw), α-pinene (25 μg g⁻¹ dw), β-pinene (18 μg g⁻¹ dw) and γ-amorphene (16 μg g⁻¹ dw) in those from medium aridity and spathulenol (114 μg g⁻¹ dw), α-pinene (49 μg g⁻¹ dw), germacrene D (29 μg g⁻¹ dw) and camphene (23 μg g⁻¹ dw) in leaves from the high aridity site. Terpene concentrations increased with the degree of aridity: the highest mean concentration of monoterpenes (136 μg g⁻¹ dw), sesquiterpenes (290 μg g⁻¹ dw) and total terpenes (427 μg g⁻¹ dw) were observed in the highest arid site and are, respectively, 3-, 5- and 4-fold higher compared to the lower arid site. Spathulenol and α-pinene can be taken as chemical markers of aridity. Drought discriminating compounds in low, but detectable concentrations are δ-cadinene and β-copaene. The functional roles of the terpenoids found in P. atlantica leaves and principles of their biosynthesis are discussed with emphasis on the mechanisms of plant resistance to drought conditions.