Characterization of depolymerized residues from extremely low acid hydrolysis (ELA) of sugarcane bagasse cellulose: Effects of degree of polymerization, crystallinity and crystallite size on thermal decomposition

Sugarcane bagasse cellulose was subjected to the extremely low acid (ELA) hydrolysis in 0.07% H₂SO₄ at 190, 210 and 225 °C for various times. The cellulose residues from this process were characterized by TGA, XRD, GPC, FTIR and SEM. A kinetic study of thermal decomposition of the residues was also carried out, using the ASTM and Kissinger methods. The thermal studies revealed that residues of cellulose hydrolyzed at 190, 210 and 225 °C for 80, 40 and 8 min have initial decomposition temperature and activation energy for the main decomposition step similar to those of Avicel PH-101. XRD studies confirmed this finding by showing that these cellulose residues are similar to Avicel in crystallinity index and crystallite size in relation to the 110 and 200 planes. FTIR spectra revealed no significant changes in the cellulose chemical structure and analysis of SEM micrographs demonstrated that the particle size of the cellulose residues hydrolyzed at 190 and 210 °C were similar to that of Avicel.     

Optimization of ethanol production by Saccharomyces cerevisiae UFPEDA 1238 in simultaneous saccharification and fermentation of delignified sugarcane bagasse

Ethanol production by Saccharomyces cerevisiae UFPEDA1238 was performed in simultaneous saccharification and fermentation of delignified sugarcane bagasse. Temperature (32 °C, 37 °C), agitation (80; 100 rpm), enzymatic load (20 FPU/g cellulose and 10%, v/v β-glucosidase or 10 FPU/g cellulose and 5% β-glucosidase) and composition of culture medium were evaluated. Ethanol concentration, enzymatic convertibility of cellulose and volumetric productivity were higher than 25 g/L, 72% and 0.70 g/L h, respectively, after 30 h, when the culture medium 1 and 20 FPU/g cellulose/10%, v/v β-glucosidase or the culture medium 2 and 10 FPU/g cellulose/5% β-glucosidase were used in SSF at 37 °C and 80 rpm. In the SSF with culture medium 2 (supplemented with ammonium, phosphate, potassium and magnesium), 150 L ethanol/t bagasse was achieved, with minimum enzyme loading (10 FPU/g cellulose and 5%, v/v β-glucosidase) for 8%, w/v of solids, which is often an important requirement to provide cost-efficient second generation ethanol processes.     

Utilization of pretreated bagasse for the sustainable bioproduction of cellulase by Aspergillus nidulans MTCC344 using response surface methodology

Response surface methodology (RSM) was used to optimize the conditions for the production of endo β-1,4 glucanase, a component of cellulase by Aspergillus nidulans MTCC344 under solid state fermentation, using bagasse as the chief substrate. A four-factor-five-level central composite design was employed for experimental design and analysis of the results. Maximum cellulase activity (CMCase was 28.96 U g⁻¹) can be attained at the optimum conditions, 16.8 mm bagasse bed height, 60% moisture content, pH 4.25 and temperature 40 °C in the solid state fermenter. These data were rather close to the experimental results obtained (CMCase was 28.84 U g⁻¹). A. nidulans MTCC344 was able to hydrolyze pretreated bagasse completely after 8 days of incubation with significant endo β-1,4 glucanase activities. The results of Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) of bagasse showed structural changes through pretreatment, in favor of enzymatic hydrolysis. Bagasse with alkali pretreatment using sodium hydroxide is a source of lignocelluloses able to improve the yield of endo β-1,4 glucanase by the strain of A. nidulans. The endo β-1,4 glucanase produced during the bioconversion of cellulose to glucose by A. nidulans MTCC344 is strongly dependent on the pretreatment given before hydrolysis.     

Release of doxycycline through cellulose acetate symmetric and asymmetric membranes produced from recycled agroindustrial residue: Sugarcane bagasse

Cellulose acetate is one of the components employed in drug controlled-release systems in the form of membranes. The aim of this study was to examine the controlled-release of doxycycline employing cellulose acetate symmetric and asymmetric membranes as matrices. The cellulose triacetate was produced from sugarcane bagasse through a homogeneous acetylation reaction, using acetic acid as the solvent, acetic anhydride as the acetylating agent and sulfuric acid as the catalyst. The viscosity average molecular weight of the cellulose acetate produced was 39,000 g mol⁻¹. The symmetric membranes were produced using a system solvent of dichloromethane/ethanol (9:1, v/v) and the asymmetric membranes were produced from the same solvent system and 10% of water. For the formulation of both, 5% of doxycycline was used. The membranes were characterized by thermal analysis (DSC and TGA) and scanning electron microscopy SEM. The release of doxycycline through cellulose triacetate matrices was examined using spectrophotometric analysis in the ultraviolet-visible region, at 275 nm. The results revealed that asymmetric membranes release 80% of the drug in 100 min, while symmetric membranes release 14% of the drug during the same time interval.     

Bioethanol production from sweet sorghum bagasse by Mucor hiemalis

The present work deals with production of ethanol from sweet sorghum bagasse by a zygomycetes fungus Mucor hiemalis. The bagasse was treated with phosphoric acid and sodium hydroxide, with or without ultrasonication, prior to enzymatic hydrolysis by commercial cellulase and β-glucosidase enzymes. The phosphoric acid pretreatment was performed at 50 °C for 30 min, while the alkali treatment performed with 12% NaOH at 0 °C for 3 h. The pretreatments resulted in improving the subsequent enzymatic hydrolysis to 79-92% of the theoretical yield. The best hydrolysis performance was obtained after pretreatment by NaOH assisted with ultrasonication. The fungus showed promising results in fermentation of the hydrolyzates. In the best case, the hydrolyzate of NaOH-ultrasound pretreated bagasse followed by 24 h fermentation resulted in about 81% of the corresponding theoretical ethanol yield. Furthermore, the highest volumetric ethanol productivity was observed in the hydrolyzates of NaOH pretreated bagasse, especially after ultrasonication in pretreatment stage.     

Composite materials of thermoplastic starch and fibers from the ethanol-water fractionation of bagasse

The aim of this study was to evaluate the potential of the fibrous material obtained from ethanol-water fractionation of bagasse as reinforcement of thermoplastic starches in order to improve their mechanical properties. The composites were elaborated using matrices of corn and cassava starches plasticized with 30 wt% glycerin. The mixtures (0, 5, 10 and 15 wt% bagasse fiber) were elaborated in a rheometer at 150 °C. The mixtures obtained were pressed on a hot plate press at 155 °C. The test specimens were obtained according to ASTM D638. Tensile tests, moisture absorption tests for 24 days (20-23 °C and 53% RH, ASTM E104), and dynamic-mechanical analyses (DMA) in tensile mode were carried out. Images by scanning electron microscopy (SEM) and X-ray diffraction were obtained. Fibers (10 wt% bagasse fiber) increased tensile strength by 44% and 47% compared to corn and cassava starches, respectively. The reinforcement (15 wt% bagasse fiber) increased more than fourfold the elastic modulus on starch matrices. The storage modulus at 30 °C (E_30 °C′) increased as the bagasse fiber content increased, following the trend of tensile elastic modulus. The results indicate that these fibers have potential applications in the development of biodegradable composite materials.     

Steam explosion pretreatment reproduction and alkaline delignification reactions performed on a pilot scale with sugarcane bagasse for bioethanol production

This work was focused on the steam explosion pretreatment reproduction and alkaline delignification reactions on a pilot scale for the ethanol production, through different varieties of natural sugarcane bagasse, pretreated bagasse and delignified pretreated bagasse (cellulosic pulp). The possible chemical composition differences of the various types of bagasse, as well as the chemical composition variations of the materials in the 20 processes of pretreatment and delignification on the pilot scale were verified. The analytical results of the 20 samples of most diverse varieties and origins of natural sugarcane bagasse considering planting soils, planting periods and weather; show no significant chemical differences. It is evident that only with the chemical composition it is not possible to verify the differences between the varieties of sugarcane bagasses. The research results may offer some evidences of these varieties, but it is not a reliable parameter. The pilot process of steam explosion pretreatment and the alkaline delignification process of pretreated material showed through analytical results a good capacity of reproduction, as the standard differences were below 2.7. The average allowed in the pretreatment and alkaline delignification processes were 66.1 ± 0.8 and 51.5 ± 2.6 respectively, ensuring an excellent reproduction capacity of the processes obtained through chemical characterizations.     

Effects of washing, milling and loading enzymes on the enzymatic hydrolysis of a steam pretreated sugarcane bagasse

Enzymatic hydrolysis of steam pretreated sugarcane bagasse was performed to investigate the production of glucose and xylose. A blend of industrial enzymes (Novozymes) was used. The enzymes were used either without having been washed or having been washed with water or 1% aqueous NaOH solution. The influence of the size of sugarcane bagasse and the proportion of the enzymatic cocktail, which was composed of endoglucanases/cellobiohydrolases (Celluclast 1.5L) and β-glucosidase (Novozym 188) cellulases mixtures, was investigated. The tests were performed at a temperature of 50 °C and at a pH of 4.8 during a period of 72 h. The assays that were conducted with a pretreated sugarcane bagasse that was washed with a 1% aqueous NaOH solution without milling led to the largest amounts of glucose after 72 h, which was independent of the proportions of enzymes used in experiments with the smallest amounts of enzymes. The largest amount of xylose was obtained with a pretreated sugarcane bagasse that was not washed.     

Carboxymethyl lignin as stabilizing agent in aqueous ceramic suspensions

Identifying new uses for residues of industries that process large quantities of biomass, as in bioethanol production, is essential for a sustainable development with reduced impact on the environment, which is the reason why many efforts have been devoted to find noble uses for lignins. In this study, a lignin obtained from sugarcane bagasse in a bioethanol producing plant was carboxymethylated to yield the water-soluble carboxymethyl lignin (CML), which was then used as stabilizing agent in aqueous alumina (Al₂O₃) suspensions. CML had a degree of substitution 0.46 ± 0.01, in relation to the C9 unit of lignin, and behaved as a polyelectrolyte in a large pH range owing to the dissociation of carboxylic groups. The action of CML as stabilizing agent of alumina aqueous suspensions was investigated using viscometry, zeta potential, and photon correlation spectroscopy (PCS) measurements, mainly as a function of pH and time. Overall, the results showed that CML had a good performance as a deflocculating agent, because it led to dispersions with low viscosity and small change in particle size as a function of time. The positive effect from the addition of CML was confirmed in the morphological features of the material obtained from the alumina suspensions after elimination of water, as indicated by scanning electron microscopy. The stabilization of alumina suspensions afforded by CML opens the way for similar applications of modified lignins, whose electrical and structural properties may be tuned for specific uses in various industries, including the ceramic industry.     

Comprehensive utilization of the hydrolyzed productions from rice hull

The study based on pretreatment, hydrolyzation and separation processes with the raw material rice hull, provides a comprehensive utilization of the hydrolyzed productions, such as glucose (C₆H₁₂O₆) from cellulose, silica (SiO₂), and byproduct crystalline sodium sulfate (Na₂SO₄·10H₂O). The optimum hydrolysis conditions are as follows: the concentration of H₂SO₄ is 72% (wt.%), the temperature is 50 °C, the ratio of H₂SO₄ solution volume (mL) to the rice hull mass (g) is 10:1 and the time is 5 min, the glucose yield rate reaches 45.6% (wt.%). The concentration of glucose solution could be 0.1 g/mL after neutralization measured by ultraviolet spectrophotometer (UV-VIS). Silica powder was below 50 nm characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The main byproduct crystalline sodium sulfate was featured by XRD and photographs.     

Production of activated carbon from organic by-products from the alcoholic beverage industry: Surface area and hardness optimization by using the response surface methodology

The ability of activated carbon to remove pollutants from water in packed column systems is dependent on granular material with mechanical strength sufficient to avoid attrition caused by stream flow. Therefore, an appropriate balance between surface area and hardness is essential when using activated carbon in real systems. The purpose of this research is to determine the optimal production conditions that generate activated carbon with adequate physical properties to be used in packed systems from agave bagasse, a waste product from the mezcal industries in Mexico. Activated carbons were produced by chemical activation (ZnCl₂ or H₃PO₄). Response surface methodology (RSM) was used to evaluate the effect of the activation temperature (250-550 °C), activation time (0-50 min), and the concentration of activating agent (0.2-1.4; g activating agent/g bagasse) on both surface area and hardness. The production conditions that generated optimal characteristics in the activated carbon were 392 °C, 1.02 g activating agent/g bagasse and 23.8 min for H₃PO₄ activated samples and 456 °C, 1.08 g activating agent/g bagasse and 23.8 min for ZnCl₂ activated samples. The surface area and hardness of the activated carbon produced from bagasse under these conditions were similar to activated commercial carbons (surface area > 800 m²/g and hardness > 85%).     

Biological pretreatment of sugar cane bagasse for the production of cellulases and xylanases by Penicillium echinulatum

In this study, sugar cane bagasse was pretreated with the white rot fungus Pleurotus sajor-caju PS 2001, and this biomass was subsequently used in the production of cellulases and xylanases by the fungus Penicillium echinulatum. Despite the environmental advantages offered by this type of pretreatment, the enzymatic activity obtained with biologically pretreated sugar cane bagasse (PSCB) was lower than that of the control treatments, which were carried out with untreated sugar cane bagasse (SCB) and cellulose. For medium supplemented with PSCB, the average peak activities obtained were 0.13, 1.0, 0.18, and 0.33 U ml^?1 for FPA, endoglucanase, β-glucosidases, and xylanases, respectively. For the cellulose, control values of 0.52, 1.20, 0.20, and 1.46 U ml^?1, and SCB values of 0.95, 1.60, 0.21, and 1.49 U ml^?1 were obtained, respectively. Although the enzymatic activities of the culture with biologically pretreated sugar cane bagasse were lower than the cultures carried out with untreated sugar cane bagasse, it should be noted that production of enzymes of the cellulase and hemicellulase complex after production of the mushrooms is another way to add value to this agricultural residue.     

High-intensity ultrasound irradiated modification of sugarcane bagasse cellulose in an ionic liquid

This current work is concerned with the glutarylation of sugarcane bagasse cellulose in ionic liquid 1-butyl-3-methylimidazolium chloride by ultrasound irradiation without catalyst. The degree of substitution ranging from 0.22 to 1.20 was obtained in one-step homogeneous modification, which increased with ultrasound irradiation time, temperature, and the molar ratio of glutaric anhydride/anhydroglucose unit in cellulose. The structural characterization of the glutarylated cellulose was carried out by using FT-IR and CP/MAS ¹³C NMR and the results showed that the glutarylation reaction occurred. The thermal stability of the glutarylated cellulose decreased upon chemical modification.     

Pest-damage relationships for Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on soybean (Glycine max) and dry bean (Phaseolus vulgaris) during pod-fill

The response of soybean (Glycine max) and dry bean (Phaseolus vulgaris) to feeding by Helicoverpa armigera during the pod-fill stage was studied in irrigated field cages over three seasons to determine the relationship between larval density and yield loss, and to develop economic injury levels. H. armigera intensity was calculated in Helicoverpa injury equivalent (HIE) units, where 1 HIE was the consumption of one larva from the start of the infestation period to pupation. In the dry bean experiment, yield loss occurred at a rate 6.00 ± 1.29 g/HIE while the rates of loss in the three soybean experiments were 4.39 ± 0.96 g/HIE, 3.70 ± 1.21 g/HIE and 2.12 ± 0.71 g/HIE. These three slopes were not statistically different (P > 0.05) and the pooled estimate of the rate of yield loss was 3.21 ± 0.55 g/HIE. The first soybean experiment also showed a split-line form of damage curve with a rate of yield loss of 26.27 ± 2.92 g/HIE beyond 8.0 HIE and a rapid decline to zero yield. In dry bean, H. armigera feeding reduced total and undamaged pod numbers by 4.10 ± 1.18 pods/HIE and 12.88 ± 1.57 pods/HIE respectively, while undamaged seed numbers were reduced by 35.64 ± 7.25 seeds/HIE. In soybean, total pod numbers were not affected by H. armigera infestation (out to 8.23 HIE in Experiment 1) but seed numbers (in Experiments 1 and 2) and the number of seeds/pod (in all experiments) were adversely affected. Seed size increased with increases in H. armigera density in two of the three soybean experiments, indicating plant compensatory responses to H. armigera feeding. Analysis of canopy pod profiles indicated that loss of pods occurred from the top of the plant downwards, but with an increase in pod numbers close to the ground at higher pest densities as the plant attempted to compensate for damage. Based on these results, the economic injury levels for H. armigera on dry bean and soybean are approximately 0.74 HIE and 2.31 HIE/m², respectively (0.67 and 2.1 HIE/row-m for 91 cm rows).     

Relationship between P and N concentrations in maize and wheat leaves

Simple plant-based diagnostic tools can be used to determine crop P status. Our objectives were to establish the relationships between P and N concentrations of the uppermost collared leaf (P_L and N_L) of spring wheat (Triticum aestivum L.) and maize (Zea mays L.) during the growing season and, in particular, to determine the critical leaf P concentrations required to diagnose P deficiencies. Various N applications were evaluated over six site-years for wheat and eight site-years for maize (2004-2006) with adequate soil P for growth. Phosphorus and N concentrations of the uppermost collared leaf were determined weekly and the relationships between leaf N and P concentrations were established using only the sampling dates from the stem elongation stage for wheat and from the V8 stage of development for maize. Leaf P concentration generally decreased with decreasing N fertilization. Relationships between P_L and N_L concentrations (mg g⁻¹ DM) using all site-years and sampling dates were described by significant linear-plateau functions in both maize (P_L = 0.82 + 0.089 N_L if N_L ≤ 32.1 and P_L = 3.7 if N_L > 32.1; R² = 0.41; P < 0.001) and wheat (P_L = 0.02 + 0.106 N_L if N_L ≤ 33.2 and P_L = 3.5 if N_L > 33.2; R² = 0.42; P < 0.001). Variation among sampling dates in the relationships were noted. By restricting the sampling dates [413-496 growing degree days (5 °C basis) in wheat (i.e., stem elongation) and 1494-1579 crop heat units in maize (i.e., silking), relationships for wheat (P_L = 0.29 + 0.073 N_L, R² = 0.66; P < 0.001) and maize (P_L = 1.04 + 0.084 N_L, R² = 0.66; P < 0.001) were improved. In maize, expressing P and N concentrations on a leaf area basis (P_LA and N_LA) at silking further improved the relationship (P_LA = 0.002 + 0.101 N_LA, R² = 0.80; P < 0.001). Predictive models of critical P concentration as a function of N concentration in the uppermost collared leaf of wheat and maize were established which could be used for diagnostic purposes.     

Repellent effects of five monoterpenoid odours against Tribolium castaneum (Herbst) and Rhyzopertha dominica (F.) in Calabar, Nigeria

Secondary plant compounds are recognised as important components of plant defence system against herbivores and pathogens. Five monoterpenoids, (R)-linalool, 1,8-cineole, (S)-2-heptyl acetate, (S)-2-heptanol and citral, which are natural components of the essential oils of Aframomum melegueta (K. Schum) and Zingiber officinale (Roscoe), were tested at the ratios in which they occur naturally for repellent activity against Tribolium castaneum (Herbst.) and Rhyzopertha dominica (F.) in a 4-way olfactometer. The results showed the repellent properties of the compounds as both beetles spent less time in the olfactometer arm containing the test stimuli. (R)-linalool and (S)-2-heptanol were stronger repellent compounds than the others. Linalool showed good repellent activity against T. castaneum (P = 0.001) as the insect spent 1.22 min in the test arm compared to the control arms (2.78 min), and R. dominica (P = 0.001) with 0.89 min in the test arm compared to 2.87 min in the control arms. With (S)-2-heptanol, T. castaneum spent 1.23 min in the test arm compared to 2.83 min in the control arms. R. dominica spent 1.61 min in the test arm and 2.69 min in the control arms. For the number of entries or visits made, while both insects were significantly repelled (P < 0.05) by the linalool-treated arm than the control, only R. dominica was repelled by the (S)-2-heptanol-treated arm (P = 0.038) compared to the control arms. The results indicate that A. melegueta and Z. officinale essential oils and their components could be suitable as safer repellents or fumigants against T. castaneum and R. dominica.     

Processing of Lespedeza stalks by pretreatment with low severity steam and post-treatment with alkaline peroxide

The steam pre-treatment with low severity preserves valuable biomass components, and further delignification with alkaline peroxide could improve hydrolysis. A combination of low severity steam pretreatment and alkaline peroxide post-treatment of Lespedeza stalks was investigated. The post-treatment of steam-pretreated Lespedeza stalks with alkaline peroxide significantly increased the cellulose content and changed the structure of the cellulose-rich fractions. A glucose yield of 503.5 mg g⁻¹ raw material from enzyme hydrolysis was obtained when the steam-pretreated material (184 °C for 4 min) was post-treated with 2% hydrogen peroxide at 60 °C for 24 h with a substrate concentration of 3.3%. Its hydrolysis yield is 88.8%, which is higher than that of samples processed by steam pretreatment alone (63.7%). The samples obtained by post-treatment with alkaline peroxide were found to have a smoother surface and looser structure in scanning electron microscopy images. The isolated lignin preparations had a yield range from 10.9 to 14.7 (% dry matter). The lignin was characterized by thermogravimetric analysis/differential thermal analysis, Fourier transform infrared spectroscopy, and gel permeation chromatography. Alkaline peroxide treatment increased the thermal stability of lignin, and decreased the amounts of all functional groups. Depolymerization and repolymerization occurred during the alkaline peroxide treatment.     

Temporal dynamics of light and nitrogen vertical distributions in canopies of sunflower, kenaf and cynara

To enhance eco-physiological and modelling studies, we quantified vertical distributions of light and nitrogen in canopies of three Mediterranean bio-energy crops: sunflower (Helianthus annuus), kenaf (Hibiscus cannabinus) and cynara (Cynara cardunculus). Field crops were grown with and without water stress in 2008 and 2009. Canopy vertical distributions of leaf area index (LAI), photosynthetically active radiation (PAR), specific leaf area (SLA), nitrogen concentration (N_conc) and specific leaf nitrogen (SLN) were assessed over time for each crop × year × water input combination. Light and nitrogen distributions were quantified by the Beer's law (exponential model) and extinction coefficients for light (K_L) and nitrogen (K_N) were calculated. Within a year, K_L did not change significantly over the studied period in all irrigated crops, but differences in K_L were significant between years (sunflower: 0.74 vs. 0.89; kenaf: 0.62 vs. 0.71; cynara: 0.77). K_L estimates were always lower (−48 to −65%) in water-stressed sunflower and kenaf crops because of the reduction in leaf angle. These results should be taken into account, when simulating water-limited biomass production. Vertical SLN distributions were found in canopies when LAI was >1.5 (40 from 51 cases). These distributions were significantly correlated with the cumulative LAI from the top (r² = 0.75-0.81; P < 0.05), providing parameters to upscale photosynthesis from leaf to canopy levels. Vertical SLN distributions followed species-specific patterns over the crop cycle and varied less compared to PAR distributions between years. Lastly, we observed strong associations between SLN and PAR distributions in irrigated sunflower and kenaf canopies (r² > 0.66; P < 0.001). However, observed SLN distributions were less steep than the distributions that would maximize canopy photosynthesis.     

Adventitious root cultures of Castilleja tenuiflora Benth. as a source of phenylethanoid glycosides

Castilleja tenuiflora is a highly valued medicinal plant that grows in pine-oak woods in Mexico. In this study, we identified for the first time verbascoside and isoverbascoside as the major phenylethanoid glycosides (PhGs) in C. tenuiflora. These compounds have proven biological activities, including anti-inflammatory, antioxidant, and cytotoxic activities, which may be related to the traditional uses of this plant. We developed a reverse-phase high-performance liquid chromatography (RP-HPLC) procedure to analyze PhGs, and determined their concentrations in various different tissues of wild plants. Verbascoside accumulated mainly in roots and inflorescences (9.23 and 7.88 mg g⁻¹ dry biomass, respectively), while isoverbascoside accumulated mainly in the roots (7.13 mg g⁻¹ dry biomass). To provide an alternative source of material for production of bioactive compounds, we established in vitro adventitious root cultures in which roots were grown in B5 medium containing either 10 μM indole 3-acetic acid (IAA) or 10 μM α-naphthaleneacetic acid (NAA). The greatest dry biomass yield (30 g L⁻¹) was achieved at 30 days after transfer of roots into IAA-containing medium. The highest specific yields of PhGs were also obtained using this auxin; the maximum level of verbascoside was 14.62 mg g⁻¹ dry root biomass (438.6 mg L⁻¹) at 30 days after root transfer, and the maximum yield of isoverbascoside was 37.32 mg g⁻¹ dry root biomass (522.48 mg L⁻¹) at 23 days after root transfer. Adventitious root cultures of C. tenuiflora are a promising system for further studies on scale-up and phenylethanoid glycosides biosynthesis.     

Pest-damage relationships for Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) on vegetative soybean

The response of vegetative soybean (Glycine max) to Helicoverpa armigera feeding was studied in irrigated field cages over three years in eastern Australia to determine the relationship between larval density and yield loss, and to develop economic injury levels. Rather than using artificial defoliation techniques, plants were infested with either eggs or larvae of H. armigera, and larvae allowed to feed until death or pupation. Larvae were counted and sized regularly and infestation intensity was calculated in Helicoverpa injury equivalent (HIE) units, where 1 HIE was the consumption of one larva from the start of the infestation period to pupation. In the two experiments where yield loss occurred, the upper threshold for zero yield loss was 7.51 ± 0.21 HIEs and 6.43 ± 1.08 HIEs respectively. In the third experiment, infestation intensity was lower and no loss of seed yield was detected up to 7.0 HIEs. The rate of yield loss/HIE beyond the zero yield loss threshold varied between Experiments 1 and 2 (−9.44 ± 0.80 g and −23.17 ± 3.18 g, respectively). H. armigera infestation also affected plant height and various yield components (including pod and seed numbers and seeds/pod) but did not affect seed size in any experiment. Leaf area loss of plants averaged 841 and 1025 cm²/larva in the two experiments compared to 214 and 302 cm²/larva for cohort larvae feeding on detached leaves at the same time, making clear that artificial defoliation techniques are unsuitable for determining H. armigera economic injury levels on vegetative soybean. Analysis of canopy leaf area and pod profiles indicated that leaf and pod loss occurred from the top of the plant downwards. However, there was an increase in pod numbers closer to the ground at higher pest densities as the plant attempted to compensate for damage. Defoliation at the damage threshold was 18.6 and 28.0% in Experiments 1 and 2, indicating that yield loss from H. armigera feeding occurred at much lower levels of defoliation than previously indicated by artificial defoliation studies. Based on these results, the economic injury level for H. armigera on vegetative soybean is approximately 7.3 HIEs/row-metre in 91 cm rows or 8.0 HIEs/m².