Evaluation of Chilo partellus and Busseola fusca susceptibility to δ-endotoxins in Bt maize

Susceptibility of Chilo partellus (Lepidoptera, Crambidae) and Busseola fusca (Lepidoptera, Noctuidae) populations to Cry proteins from the bacterium, Bacillus thuringiensis (Bt), the δ-endotoxins Cry1Ab and Cry1Ba in Bt-maize, were evaluated under biosafety greenhouse conditions. Larval feeding on Bt-maize was adjusted to deliver sub-lethal doses of δ-endotoxins from the two events; survivors were reared on artificial diet to obtain successive generations. Eight generations of three C. partellus populations and five generations of a B. fusca population were screened for susceptibility on each event. Mean proportion of surviving larvae from Bt-maize plants, and the corresponding pupal weights of survivors for each population, were lower for individuals exposed to δ-endotoxins. Both Bt Cry proteins expressed in maize leaves controlled C. partellus and showed stability in control, with no indication of a change in susceptibility among generations. Neither toxin, however, provided complete control of B. fusca, but no changes in susceptibility were observed after five generations of selection. Implications for development of future transgenic Bt maize events, and research for East Africa are discussed.     

Identification and characterization by PCR–RFLP analysis of the genetic variation for the Glu-A1x and Glu-B1x genes in rivet wheat (Triticum turgidum L. ssp. turgidum)

Many studies have suggested that the true variation of high-molecular-weight (HMW) glutenin subunits in wheat could be under-estimated due to the anomalous mobility of these proteins in one-dimensional electrophoresis (SDS-PAGE). An alternative technique used is the specific PCR amplification of these genes, however the scarce variation in size among the different alleles could also under-estimate the true variability. In this study, the variability of HMW glutenin subunits found in one Spanish collection of rivet wheat (Triticum turgidum L. ssp. turgidum) was evaluated by a combination of two techniques (PCR amplification and digestion with endonucleases). The data allowed detection of allelic variations that were not clearly detected by SDS-PAGE or PCR analysis alone. The approach used in the current study could allow identification of alleles contributing to achieve desirable quality in modern wheat.     

Are insensitivities of Venturia inaequalis to myclobutanil and fenbuconazole correlated?

In the UK, two demethylation inhibitor (DMI) fungicides, myclobutanil and fenbuconazole, are recommended for controlling apple scab. Experiments were conducted to determine whether the sensitivity of V. inaequalis to myclobutanil was correlated with that to fenbuconazole. There was a marked reduction in the sensitivity of V. inaequalis to myclobutanil; averaged ED₅₀ values increased from 0.338 mg L⁻¹ in the baseline population to 2.945 mg L⁻¹ for isolates from a commercial orchard. Overall, the average ED₅₀ value for fenbuconazole was only about 20% of that for myclobutanil for the baseline population. There was an overall significant positive correlation in the fungal sensitivities to myclobutanil and fenbuconazole, but such correlation only accounted for 40% of the total observed variability. In addition, the magnitude of this cross-resistance varied among orchards. The finding is discussed in the context of fungicide deposition in relation to disease control.     

Pasting characteristics and in vitro digestibility of γ-irradiated RS4 waxy maize starches

The effect of γ-irradiation on the physicochemical properties of cross-linked waxy maize resistant starches was examined. The cross-linked waxy maize starches contained resistant starch (RS) of 56.1 and 63.5%, respectively for 5 and 10% sodium trimetaphosphate (STMP)/sodium tripolyphosphate (STPP) cross-linking, and the RS contents slightly decreased as the irradiation dose increased whereas the RS content in unmodified waxy maize starch increased with an increase in irradiation dose. For both native and cross-linked starches, the rapidly digestible starch (RDS) content increased and the slowly digestible starch (SDS) content decreased by the irradiation. The solubility of the native and cross-linked starches increased as the irradiation dose increased. The cross-linked starches did not swell in boiling water without showing pasting viscosity. However, the starches became swellable, forming pastes by irradiation, and the pasting viscosity gradually increased with an increase in irradiation dose. The crystallinity as determined by an X-ray diffraction analysis remained unchanged upon cross-linking and γ-irradiation. However, the gelatinization enthalpy of the cross-linked starches decreased in proportion with irradiation dose. The melting temperatures of cross-linked starches gradually decreased and the temperature range for melting increased with an increase in irradiation dose.     

Structural and Functional Characterization of a Novel α-Conotoxin Mr1.7 from Conus marmoreus Targeting Neuronal nAChR α3β2, α9α10 and α6/α3β2β3 Subtypes

In the present study, we synthesized and, structurally and functionally characterized a novel α4/7-conotoxin Mr1.7 (PECCTHPACHVSHPELC-NH₂), which was previously identified by cDNA libraries from Conus marmoreus in our lab. The NMR solution structure showed that Mr1.7 contained a 3₁₀-helix from residues Pro⁷ to His¹⁰ and a type I β-turn from residues Pro¹⁴ to Cys¹⁷. Electrophysiological results showed that Mr1.7 selectively inhibited the α3β2, α9α10 and α6/α3β2β3 neuronal nicotinic acetylcholine receptors (nAChRs) with an IC₅₀ of 53.1 nM, 185.7 nM and 284.2 nM, respectively, but showed no inhibitory activity on other nAChR subtypes. Further structure-activity studies of Mr1.7 demonstrated that the PE residues at the N-terminal sequence of Mr1.7 were important for modulating its selectivity, and the replacement of Glu² by Ala resulted in a significant increase in potency and selectivity to the α3β2 nAChR. Furthermore, the substitution of Ser¹² with Asn in the loop2 significantly increased the binding of Mr1.7 to α3β2, α3β4, α2β4 and α7 nAChR subtypes. Taken together, this work expanded our knowledge of selectivity and provided a new way to improve the potency and selectivity of inhibitors for nAChR subtypes.     

Some Characteristics of β-D-Xylopyranosidases, α-L-Arabinofuranosidases and an Arabinoxylan α-L-arabinofuranohydrolase from Wheat Bran and Germinated Wheat

Enzymes from wheat bran and germinated wheat involved in the degradation of arabinoxylan and arabinoxylooligosaccharides were investigated. Fourp-nitrophenyl-α-l-arabinofuranoside hydrolysing activities (ArafI-IV) and threep-nitrophenyl-β-d-xylopyranoside hydrolysing activities (XylpI-III) were identified in wheat kernels and germinating wheat. Two of these activities, ArafI and XylpII, were purified about 10 000-fold from wheat bran. Both enzymes were inactive towards polymeric arabinoxylan. ArafI produced no arabinose but some xylose from arabinoxylooligosaccharides, while XylpII gave only xylose upon incubation with this substrate. An arabinoxylan arabinofuranohydrolase (AXH) was found in wheat bran and germinated wheat. This enzyme was active towards the polymeric substrate, but was unable to hydrolysep-nitrophenyl-α-l-arabinofuranoside. TheM_rs of these enzymes were determined by size exclusion chromatography and were in the range of 40–50 000, except for ArafIII, for which aM_rof 104 000 was determined. During germination, the levels of these enzymes increased markedly between the third and fifth day, after which some of them decreased again by the seventh day. ArafI-IV were inhibited strongly by arabinonic acid-γ-lactone, while xylonic acid-γ-lactone was a good inhibitor of XylpI-III. The latter lactone also inhibited ArafI. Neither of these lactones inhibited AXH. Endoxylanase activity was demonstrated but not quantified.     

Does intercropping winter wheat (Triticum aestivum) with red fescue (Festuca rubra) as a cover crop improve agronomic and environmental performance? A modeling approach

The introduction of a living cover crop during a cash crop growth cycle (relay intercropping) and its maintenance after the cash crop harvest may help to preserve biodiversity, increase soil organic matter content and carbon sequestration and provide other ecosystem services, such as natural pest regulation or nutrient recycling, by increasing useful biotic interactions within the agroecosystem. We studied the impact of various approaches to manage a red fescue cover crop in a winter wheat crop in terms of light, water and nitrogen competition, using the STICS crop model adapted for intercropping. The STICS model for wheat/fescue intercropping was first evaluated on two years of experimental data obtained in the field. It gave satisfactory statistical results for the prediction of dry matter, leaf area index (LAI) and nitrogen accumulation in the two species, and for nitrogen and water dynamics in the soil. By simulating unmeasured variables, such as transpiration, the model improves our understanding of the performance of the intercrop in the field. For example, we showed that the intercropping system was more efficient that wheat grown as a sole crop, in terms of nitrogen accumulation and decreasing soil nitrogen levels before the leaching period. However, it also resulted in lower wheat yields. We then used the STICS model to compare four intercropping management scenarios differing in terms of the date of red fescue emergence, over 35 climatic years. We found that, in most climatic scenarios, the emergence of the fescue crop during the late tillering phase of the wheat crop gave the best compromise between wheat yield overall nitrogen accumulation and radiation interception.     

Inhibitory Activity of Cinnamon Bark Species and their Combination Effect with Acarbose against Intestinal α-glucosidase and Pancreatic α-amylase

Inhibition of α-glucosidase and pancreatic α-amylase is one of the therapeutic approaches for delaying carbohydrate digestion, resulting in reduced postprandial glucose. The aim of this study was to evaluate the phytochemical analysis and the inhibitory effect of various cinnamon bark species against intestinal α-glucosidase and pancreatic α-amylase. The results showed that the content of total phenolic, flavonoid, and condensed tannin ranged from 0.17 to 0.21 g gallic acid equivalent/g extract, from 48.85 to 65.52 mg quercetin equivalent/g extract, and from 0.12 to 0.15 g catechin equivalent/g extract, respectively. The HPLC fingerprints of each cinnamon species were established. Among cinnamon species, Thai cinnamon extract was the most potent inhibitor against the intestinal maltase with the IC₅₀ values of 0.58 ± 0.01 mg/ml. The findings also showed that Ceylon cinnamon was the most effective intestinal sucrase and pancreatic α-amylase inhibitor with the IC₅₀ values of 0.42 ± 0.02 and 1.23 ± 0.02 mg/ml, respectively. In addition, cinnamon extracts produced additive inhibition against intestinal α-glucosidase and pancreatic α-amylase when combined with acarbose. These results suggest that cinnamon bark extracts may be potentially useful for the control of postprandial glucose in diabetic patients through inhibition of intestinal α-glucosidase and pancreatic α-amylase.     

Toxicity of several δ-endotoxins of Bacillus thuringiensis against the cotton pest Earias insulana (Lepidoptera: Noctuidae)

The toxicity of nine Bacillus thuringiensis Cry proteins against neonate Earias insulana larvae was tested using a mixture of crystals and spores. The mean lethal concentration (LC₅₀) of Cry1Ac was 1.99 μg/ml. Cry1Fa, Cry1Ca, Cry1Ja and Cry2Aa were more active than Cry1Ac, with LC₅₀ values of 0.22, 0.24, 0.29, 0.43 μg/ml, respectively. Cry1Da and Cry1Aa were considerably less active than Cry1Ac. The remaining proteins, Cry1Ba and Cry1Ab, displayed no activity. Relative potencies were also calculated. Cry1Ja and Cry1Fa were significantly more active (7.72 and 5.71 times, respectively) than Cry1Ac, while Cry1Ca was significantly (1.95 times) more active than Cry2Aa.     

Investigating the impact of α-amylase, α-glucosidase and glucoamylase action on yeast-mediated bread dough fermentation and bread sugar levels

Wheat flour is generally supplemented with α-amylases to increase maltose levels in bread dough and increase loaf volume. While the preference of yeast for glucose and fructose over maltose as substrate for fermentation is well documented, the impact of maltose versus glucose producing enzymes on bread dough fermentation kinetics and bread sugar levels is ill documented. Hence the impact of α-amylase, α-glucosidase and glucoamylase action on both aspects was investigated. Glucoamylase and α-amylase increase the total fermentable sugar content of dough, while α-glucosidase only affects the glucose/maltose ratio. Due to their effect on total fermentable sugar levels, addition of α-amylase or glucoamylase prolongs the total productive fermentation time, while this is not the case for α-glucosidase. In contrast to α-amylase, both glucoamylase and α-glucosidase supplementation leads to higher CO₂ production rates during the initial stages of fermentation. In the final bread product, different sugar levels are observed depending on the dosage and type of starch-degrading enzyme. The results of this study imply that long and short fermentation processes benefit from α-amylase and α-glucosidase addition, respectively, while glucoamylase supplementation is suitable for both long and short fermentation times.     

How many Orius laevigatus are needed for effective western flower thrips, Frankliniella occidentalis, management in sweet pepper?

Frankliniella occidentalis (Pergande) is a primary pest of greenhouse crops worldwide, in organic and integrated pest management control practices, Orius spp. are frequently released for thrips control. However, Orius spp. are relatively expensive to produce. More cost-efficient rearing systems and reduced release rate might reduce the expense. In these trials, we released Orius laevigatus (Fieber) at different rates with or without simultaneous release the predatory mite Amblyseius swirskii Athias-Henriot, another known thrips predator, which is less expensive to rear. There was no significant difference in the number of O. laevigatus recovered in which either 2 or 6 individuals were released per square meter, and there was no difference in thrips control among any of the release strategies using O. laevigatus, suggesting that a reduced release rate can maintain effective thrips control. There was no significant difference in the quality or quantity of the pepper yield between treatments in which either 2 or 6 Orius/m² or Orius plus A. swirskii were released.     

Characterisation of a ω-gliadin gene in Triticum tauschii

A ω-gliadin gene at the Gli-D^t1 locus of Triticum tauschii accession AUS18913 was isolated using PCR primers, designed from published sequences of ω-gliadin genes of bread wheat cv Cheyenne, and deduced sequences of the N-terminal amino acids of ω-gliadin proteins. Further, the derived protein was isolated from A-PAGE and was sequenced. The protein sequence contained a signal peptide of 19 amino acids followed by a short N-terminal sequence of 11 amino acids, a central repetitive domain that covers approximately 90% of the sequence and a short C-terminal domain of 12 amino acids. The sequence comparison with other ω-gliadins showed a high level of similarities between them. Further analysis of the ω-gliadins using A-PAGE revealed that there are three ω-gliadin proteins in AUS18913 accession. Comparison of N-terminal sequences of these proteins revealed that two of these proteins have very high homologies with ω-gliadins of Cheyenne while the third one was significantly different.     

Towards an optimal process for gelatinisation and hydrolysis of highly concentrated starch–water mixtures with alpha-amylase from B. licheniformis

The enzymatic hydrolysis of starch is usually carried out with 30–35 w/w% starch in water. Higher substrate concentrations (50–70 w/w%) were reached by using a twin-screw extruder for gelatinisation and for mixing enzyme with gelatinised starch prior to enzymatic hydrolysis in a batch reactor. The aim of this study was to determine which parameters are important for gelatinisation of wheat starch and to investigate the effects of different extrusion conditions on the enzymatic hydrolysis. After extrusion, the degree of gelatinisation was measured. During hydrolysis, the carbohydrate composition, the dextrose equivalent (DE) and the alpha-amylase activity were measured. Gelatinisation measurements showed that mechanical forces lowered the temperature required for complete gelatinisation. During hydrolysis experiments, high DEs were observed even if starch was not completely gelatinised during extrusion. Due to high substrate concentrations, the residual alpha-amylase activity remained high throughout enzymatic hydrolysis, although high temperatures were used. Increased substrate concentrations did not affect the carbohydrate composition of the product. Furthermore, the time required for the batch hydrolysis step could be varied by choosing a different enzyme-to-substrate ratio. This article provides a basis for detailed optimisation of this process to develop an industrial-scale process at high substrate concentrations.     

Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli

α-Conotoxin LvIA is derived from Conus lividus, native to Hainan, and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. In this study, an efficient approach for the production of recombinant α-Conotoxin LvIA is described. Tandem repeats of a LvIA gene fragment were constructed and fused with a KSI gene and a His₆ tag in a Escherichia coli (E. coli) expression vector pET-31b(+). The recombinant plasmids were transformed into E. coli and were found to express well. The KSI-(LvIA)_n-His₆ fusion protein was purified by metal affinity chromatography and then cleaved with CNBr to release recombinant LvIA (rLvIA). High yields of fusion protein ranging from 100 to 500 mg/L culture were obtained. The pharmacological profile of rLvIA was determined by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing rat nAChR subtypes. The rLvIA antagonized the α3β2 nAChR subtype selectively with a nano-molar IC₅₀. The rLvIA was analgesic in a mouse hot-plate test model of pain. Overall, this study provides an effective method to synthesize α-conotoxin LvIA in an E. coli recombinant expression system, and this approach could be useful to obtain active conopeptides in large quantity and at low cost.     

Two fraction extraction of α-zein from DDGS and its characterization

Zein was recovered from corn distiller's dried grains with solubles (DDGS) by a modified method using 70% (w/w) aqueous 2-propanol (70-IPA) or 70% (v/v) aqueous ethanol (70-EtOH) solvents, and a commercial method using 88% (w/w) aqueous 2-propanol (88-IPA). Yield, purity, and film properties of the isolated zein were determined. The modified procedure extracted two fractions of zeins: a mostly α-zein fraction, and a mostly γ-zein fraction. The modified method increased α-zein yield from 4% to 14%. Enzyme cellulase pretreatment did not improve zein yield, but grinding did. The α-zein fraction showed electrophoretic bands at 40, 22, 19, and 10 kDa, corresponding to α-zein dimer, α₁-zein, α₂-zein, and δ-zein, respectively. The α-zein of DDGS retained its film forming capability. The α-zein film of unmodified DDGS was cloudy and rough, unlike the clear and smooth films of α-zeins isolated from corn gluten meal and enzyme-treated DDGS.     

Reversed phase HPLC analysis of α- and β-carotene from selected raw and cooked vegetables

Traditional AOAC colorimetric procedures for carotenoid analysis are known to lack specificity and accuracy. Newer HPLC methods provide the investigator with a more precise tool for carotenoid quantification in foods and tissues. In the present studies, reverse phase HPLC was utilized to evaluate the - and -carotene content in raw and cooked leaves of lettuce, spinach and winged bean as well as in the carrot root. The vegetables were boiled or steamed and the true retention of - and -carotene in the cooked products was determined. Boiling for 30 minutes resulted in a 53 and 40% loss of -carotene from lettuce and carrots, respectively. Full retention or even an increase in -carotene content in boiled winged bean leaves and spinach was noted. Steaming resulted in very good retention of - and - carotene in all vegetables (83–139% retention). Thus, although cooking procedures (especially boiling) may result in oxidative loss of carotenoids in some vegetables, heat treatment increases the chemical extractability of - and -carotene in others. The presence of carotenoproteins in some vegetables may effect the heat stability of extractability of - and - carotene.     

Quantitative analyses of individual γ-Oryzanol (Steryl Ferulates) in conventional and organic brown rice (Oryza sativa L.)

γ-Oryzanol (steryl ferulates; SF) has been shown to be a major bioactive compound in rice. To determine the content of individual γ-oryzanols in brown rice by high performance liquid chromatography (HPLC), purification of individual SF for use as an external standard is required. Four main SF were isolated from a commercial γ-oryzanol mixture and identified as cycloartenyl ferulate (1), 24-methylenecycloartanyl ferulate (2), campesteryl ferulate (3), and sitosteryl ferulate (4) based on mass spectrometry and nuclear magnetic resonance spectroscopic data. The SF contents between conventional and organic brown rice were qualitatively determined by HPLC using SF isolated from a commercial γ-oryzanol mixture as the external standard. The total γ-oryzanol content (mg/100 g) in organic brown rice (65.6 ± 2.7) was slightly higher (P < 0.05) than that found in conventional brown rice (60.2 ± 1.8). The content (mg/100 g) of 1 (21.2 ± 0.9) and 4 (9.8 ± 0.4) in organic brown rice was higher (P < 0.05) than that observed in conventional brown rice (1, 18.2 ± 1.1; 4, 8.5 ± 0.3). However, the content of 2 and 3 in the conventional and organic brown rice samples did not differ significantly. These results indicate that the cultivation methods do significantly alter the γ-oryzanol content for conventional and organic brown rice.     

A mutation of the cellulose-synthase-like (CslF6) gene in barley (Hordeum vulgare L.) partially affects the β-glucan content in grains

The chemical induced barley mutant m351 was first selected for its low level of mixed-linkage (1–3,1–4) beta-D-glucan (MLG) in an experimental effort to search for barley lines with varied grain MLG contents. The MLG decrease in m351 was associated with increased levels of fructans and crude fiber, but maintained the same plant characteristics under field conditions. The mutation was mapped to a genetic locus flanked by two SSR markers, Bmag369 and Bmag564, on chromosome 7H. Molecular cloning of the CslF6 gene from the m351 line revealed the presence of a point mutation, causing a substitution of an alanine for threonine at position 849 in the amino acid sequence of the corresponding protein. The resultant protein retains some functionality and affects other components in the m351 grain. Those metabolic changes associated with MLG reduction in m351 is the first case reported of a partially functional CslF6 gene in cereal grains. The results contribute to better understanding of the functional effects of the CslF6 gene and the mutant has potential implications in grain end-use quality improvement.