Methylene blue for malaria in Africa: results from a dose-finding study in combination with chloroquine

Background

Atovaquone is part of the antimalarial drug combination atovaquone-proguanil (Malarone^®) and inhibits the cytochrome bc₁ complex of the electron transport chain in Plasmodium spp. Molecular modelling showed that amino acid mutations are clustered around a putative atovaquone-binding site resulting in a reduced binding affinity of atovaquone for plasmodial cytochrome b, thus resulting in drug resistance.

Methods

The prevalence of cytochrome b point mutations possibly conferring atovaquone resistance in Plasmodium falciparum isolates in atovaquone treatment-naïve patient cohorts from Lambaréné, Gabon and from South Western Ethiopia was assessed.

Results

Four/40 (10%) mutant types (four different single polymorphisms, one leading to an amino acid change from M to I in a single case) in Gabonese isolates, but all 141/141 isolates were wild type in Ethiopia were found.

Conclusion

In the absence of drug pressure, spontaneous and possibly resistance-conferring mutations are rare.     

Streptococcus danieliae sp. nov., a novel bacterium isolated from the caecum of a mouse

We report the characterization of one novel bacterium, strain ERD01G^T, isolated from the cecum of a TNF^deltaARE mouse. The strain was found to belong to the genus Streptococcus based on phylogenetic analysis of partial 16S rRNA gene sequences. The bacterial species with standing name in nomenclature that was most closely related to our isolate was Streptococcus alactolyticus (97 %). The two bacteria were characterized by a DNA–DNA hybridization similarity value of 35 %, demonstrating that they belong to different species. The new isolate was negative for acetoin production, esculin hydrolysis, urease, α-galactosidase and β-glucosidase, was able to produce acid from starch and trehalose, grew as beta-hemolytic coccobacilli on blood agar, did not grow at >40 °C, did not survive heat treatment at 60 °C for 20 min and showed negative agglutination in Lancefield tests. On the basis of these characteristics, strain ERD01G^T differed from the most closely related species S. alactolyticus, Streptococcus gordonii, Streptococcus intermedius and Streptococcus sanguinis. Thus, based on genotypic and phenotypic evidence, we propose that the isolate belongs to a novel bacterial taxon within the genus Streptococcus, for which the name Streptococcus danieliae is proposed. The type strain is ERD01G^T (= DSM 22233^T = CCUG 57647^T).     

Simultaneous separation of atovaquone, proguanil and its metabolites on a mixed mode high-performance liquid chromatographic column

An isocratic high-performance liquid chromatographic (HPLC) method for simultaneous separation of the components in the antimalarial combination drug Malarone^® with UV detection is described. An HPLC system using a mixed mode column composed of 50% C₁₈ phase and 50% strong cation-exchanger has been optimised for the simultaneous separation of atovaquone, proguanil and its two main metabolites. The mobile phase was optimised for factors such as pH, counter ion concentration and acetonitrile. Elimination of interferences from other antimalarial drugs was achieved by adding sodium perchlorate to the mobile phase. With a mobile phase of acetonitrile-phosphate buffer (60:40, v/v) pH 6.8, 50.7 mmol l⁻¹ K⁺ and 10 mmol l⁻¹ Na·ClO₄, separation was achieved within a run time shorter than 17 min.     

Identifying patients with undetected renal tract cancer in primary care: An independent and external validation of QCancer® (Renal) prediction model

Introduction: To evaluate the performance of QCancer^® (Renal) for predicting the absolute risk of renal tract cancer in a large independent UK cohort of patients from general practice records. Materials and methods: Open cohort study to validate QCancer^® (Renal) prediction model. Record from 365 practices from United Kingdom contributing to The Health Improvement Network (THIN) database. 2.1 million patients registered with a general practice surgery between 01 January 2000 and 30 June 2008, aged 30–84 years (3.7 million person years) with 2283 renal tract cancer cases. Renal tract cancer was defined as incident diagnosis of renal tract cancer during the 2 years after study entry. Model discrimination was measured using the receiver operating characteristics derived area under the curve. Calibration plots examined the relationship between predicted and observed probabilities of undetected renal tract cancer. Results: The results from this independent and external validation of QCancer^® (Renal) demonstrated good performance data on a large cohort of general practice patients. QCancer^® (Renal) had very good discrimination with areas under the ROC curve of 0.92 and 0.95 for women and men respectively. QCancer^® (Renal) was well calibrated across all tenths of risk and over all age ranges with predicted risks closely matching observed risks. QCancer^® (Renal) explained 74.4% and 74.2% of the variation in men and women respectively. A limitation of our study is the recording of symptoms might be less complete, as patients with mild symptoms may not visit their general practitioner or not report mild symptoms. Conclusions: QCancer^® (Renal) are useful tools to help in identifying undetected cases of undiagnosed renal tract cancer in primary care in the UK.     

Abscisic acid,gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima

The seaweed extract Kelpak^® made from the kelp Ecklonia maxima is registered as a biostimulant for use in agriculture. It elicits many beneficial responses including improved root and shoot growth, higher yields and greater resistance to abiotic and biotic stresses. Previously, cytokinins, auxins and polyamines were identified in Kelpak^®. The aim of the present study was to quantify other groups of plant growth regulators (PGRs)—abscisic acid (ABA), gibberellins (GAs) and brassinosteroids—that may be present in E. maxima and Kelpak^®. Kelpak^® samples harvested between 2008 and 2010 and stored for up to 26 months were analysed using ultra performance liquid chromatography tandem mass spectrometry. ABA levels were below the limits of detection in E. maxima but were detected in low concentrations in Kelpak^®, ranging from 0.31 to 20.70 pg mL^?1 Kelpak^®. Eighteen GAs were found in E. maxima and Kelpak^® with concentrations from 187.54 to 565.96 pg mL^?1 Kelpak^®. The biologically active GAs (GA₁, GA₃, GA₄, GA₅, GA₆ and GA₇) comprised less than 3 % in Kelpak^®. Although GA₁₃ (a final product in the metabolic pathway) was present in low concentrations in E. maxima, very high concentrations were present in Kelpak^®. The brassinosteroids brassinolide (BL) and castasterone (CS) were identified in E. maxima and Kelpak^®. Concentrations varied with harvest and storage time, ranging from 384.72 to 793.23 pg BL mL^?1 Kelpak^® and 62.84 to 567.51 pg CS mL^?1 Kelpak^®. It is likely that this cocktail of natural PGRs present in Kelpak^® may act individually or in concert and thus contribute to the numerous favourable physiological responses elicited by Kelpak^® application to plants.     

Can Kelp Extract (KELPAK®) be Useful in Seaweed Mariculture?

The addition of low concentrations of commercial kelp extract (Ecklonia maxima: Kelpak^®) in addition to fertiliser has proven to be beneficial in agriculture. It triggers rooting in field crops, increases yields and has other useful effects, such as parasite reduction. Its efficacy has been attributed to the fact that Kelpak^® is produced by a cold process, and is a high auxin/low cytokinin product. The aim of this study was to investigate if seaweeds (which do not have a root system) grown in culture systems, would benefit from the addition of Kelpak^® or a combination of Kelpak^® and fertilizer. A preliminary laboratory experiment was carried out by growing excised 15 mm tips of the red alga Gracilaria gracilis in culture dishes containing Provasoli Enriched Seawater medium to which various concentrations of Kelpak^® were added. Gracilaria tips in some of the Kelpak^® treatments (1:2500; 1:1000; 1:500) grew significantly better than the control. Further experiments were carried out on a pilot commercial scale at Jacobsbaai Sea Products Ltd. on the South African west coast. Ulva lactuca was grown in effluent from fish (turbot) culture, with additions of 1:5000, 1:2500 and 1:500 concentrations of Kelpak^® once a week. The intermediate Kelpak^® concentration (1:2500) produced the highest growth of Ulva in the turbot water, while the highest Kelpak^® concentration (1:500) inhibited Ulva growth. In another Ulva experiment, various combinations of aquaculture effluent water, commercial fertiliser and Kelpak^® at 1:2500 were used. Best growth of Ulva was obtained in turbot water containing both fertiliser and Kelpak^®. The results suggest that Kelpak^® could be useful in commercial seaweed mariculture operations.     

Self-Nanoemulsifying Drug Delivery System of Nifedipine: Impact of Hydrophilic–Lipophilic Balance and Molecular Structure of Mixed Surfactants

A simple but novel mixed surfactant system was designed to fabricate a self-nanoemulsifying drug delivery system (SNEDDS) based on hydrophilic–lipophilic balance (HLB) value. The impacts of HLB and molecular structure of surfactants on the formation of SNEDDS were investigated. After screening various oils and surfactants, nifedipine (NDP)-loaded liquid SNEDDS was formulated with Imwitor^® 742 as oil and Tween^®/Span^® or Cremophor^®/Span^® as mixed surfactant. Droplet size of the emulsions obtained after dispersing SNEDDS containing Tween^®/Span^® in aqueous medium was independent of the HLB of a mixed surfactant. The use of the Cremophor^®/Span^® blend gave nanosized emulsion at higher HLB. The structure of the surfactant was found to influence the emulsion droplet size. Solid SNEDDS was then prepared by adsorbing NDP-loaded liquid SNEDDS comprising Cremophor^® RH40/Span^® 80 onto Aerosil^® 200 or Aerosil^® R972 as inert solid carrier. Solid SNEDDS formulations using higher amounts (30–50% w/w) of Aerosil^® 200 exhibited good flow properties with smooth surface and preserved the self-emulsifying properties of liquid SNEDDS. Differential scanning calorimetry and X-ray diffraction studies of solid SNEDDS revealed the transformation of the crystalline structure of NDP due to its molecular dispersion state. In vitro dissolution study demonstrated higher dissolution of NDP from solid SNEDDS compared with NDP powder.     

Characterisation of Isaria fumosorosea isolates and their virulence toward the Diamondback Moth,Plutella xylostella

Some morphological and physiological characteristics of an Isaria fumosorosea isolate with diminished virulence, IFCF01-D, and its parent isolate, IFCF01, were evaluated and laboratory bioassays were performed to assess their virulence against Plutella xylostella. The relationship among these traits and virulence against P. xylostella is discussed. There were no significant differences in conidial viability, spore production and the time required for 50% germination (GT₅₀). Spore viability after incubation for 24 h at 25°C was greater than 98% for both isolates tested. Spore production on potato dextrose agar after 14 days incubation at 25°C was 4.68 × 10⁸ and 4.59 × 10⁸ conidia/mL for IFCF01 and IFCF01-D, respectively. When exposed to high temperatures (40, 45, 50 or 55°C) through a water bath for 10 min, conidial germination ranged from 0.83% to 84.0% for IFCF01 and 0% to 86% for IFCF01-D. Germination rate showed a negative relationship with the exposure temperature for both isolates. The per cent germination of isolate IFCF01 24 h after ultraviolet (UV) radiation (18 W, 240–260 nm) varied from 0% to 92% and 0% to 81% for IFCF01-D. Germination rate and the exposure time exhibited a negative correlation for both isolates tested. Conidial surface hydrophobicity of IFCF01 (60%) was significantly higher than that of isolate IFCF01-D (53%). Subsequently, using the cicada exuviae as the substrate for enzymatic analysis, Pr1 and chitinase activity demonstrated the contrasting virulence traits: higher specific activities for the more virulent IFCF01 and lower enzymatic levels for isolate IFCF01-D.     

Response of adult parasitoids ofBemisia tabaci (Hom.: Aleyrodidae) to leaf residues of selected cotton insecticides

The contact toxicity of eight insecticides to adults of four parasitoids of the sweetpotato whiteflyBemisia tabaci was evaluated in the laboratory. Two common Texas species,Eretmocerus sp. andEncarsia pergandiella Howard, and two exotic species,Eretmocerus mundus Mercet from Spain andEncarsia formosa Gahan from Greece were tested. Insecticides, applied as sprays to greenhouse-grown cotton plants at recommended rates were amitraz (Ovasyn^®), azinphosmethyl (Guthion^®), bifenthrin (Capture^®), buprofezin (Applaud^®), cypermethrin (Ammo^®), methyl parathion and thiodicarb (Larvin^®), with a water control. Parasitoid adults were confined on discs cut from leaves (1) sprayed the same day and (2) sprayed two days previously. Survival in both treatments was measured two and four days following exposure. Significant differences in toxicity were detected among the insecticides. Buprofezin was not toxic to any of the four parasitoids. When caged on leaves sprayed two days previously, only amitraz of the remaining compounds allowed significant general parasitoid survival after two days.E. mundus exhibited the greatest overall tolerance to insecticides, with 40% or more surviving 48 hr after confinement on leaves sprayed with amitraz, thiodicarb and cypermethrin. Survival was generally much reduced after 96 hr. In a separate test, fresh residues of endosulfan (Thiodan^®) were highly toxic at the two rates tested, but two day old residues at the lower rate allowed 76.7% survival ofE. mundus and 35% survival ofE. pergandiella after 48 hrs.     

EPs® 7630, an extract from Pelargonium sidoides roots inhibits adherence of Helicobacter pylori to gastric epithelial cells

Helicobacter pylori specifically adheres to gastric host cells, mainly based on carbohydrate-mediated cell–cell interaction. The extract of Pelargonium sidoides roots (EPs^® 7630), a South African herbal remedy, is currently used to treat acute bronchitis. EPs^® 7630 prevents bacteria from attaching to cell membranes. Therefore, the ability of EPs^® 7630 to interfere with H. pylori growth and adhesion to gastric epithelial cells (AGS cells) was tested in vitro. EPs^® 7630 inhibited H. pylori growth and with higher potency adhesion to gastric AGS cells. EPs^® 7630 (50 and 100 μg/ml) reduced bacterial count attached to AGS cells by 77% and 91%, respectively. The results suggest that the mode of action of EPs^® 7630 is mainly related to its anti-adhesive activity.     

Influence of an Acrylic Polymer Blend on the Physical Stability of Film-Coated Theophylline Pellets

The purpose of this study was to investigate the physical stability of a coating system consisting of a blend of two sustained release acrylic polymers and its influence on the drug release rate of theophylline from coated pellets. The properties of both free films and theophylline pellets coated with the polymer blend were investigated, and the miscibility was determined via differential scanning calorimetry. Eudragit^® RS 30 D was plasticized by the addition of Eudragit^® NE 30 D, and the predicted glass transition temperature (T _g) of the blend was similar to the experimental values. Sprayed films composed of a blend of Eudragit^® NE 30 D/Eudragit^® RS 30 D (1:1) showed a water vapor permeability six times greater than films containing only Eudragit^® NE 30 D. The presence of quaternary ammonium functional groups from the RS 30 D polymer increased the swellability of the films. The films prepared from the blend exhibited stable permeability values when stored for 1 month at both 25°C and 40°C, while the films which were composed of only Eudragit^® NE 30 D showed a statistically significant decrease in this parameter when stored under the same conditions. Eudragit^® NE 30 D/Eudragit^® RS 30 D (1:1)-sprayed films decreased in elongation from 180% to 40% after storage at 40°C for 1 month, while those stored at 25°C showed no change in elongation. In coated pellets, the addition of Eudragit^® RS 30 D to the Eudragit^® NE 30 D increased the theophylline release rate, and the pellets were stable when stored at 25°C for a period of up to 3 months due to maintenance of the physico-mechanical properties of the film. Pellets stored at 40°C exhibited a decrease in drug release rate over time as a result of changes in film physico-mechanical properties which were attributed to further coalescence and densification of the polymer. When the storage temperature was above the T _g of the composite, instabilities in both drug release rate and physical properties were evident. Stabilization in drug release rate from coated pellets could be correlated with the physico-mechanical stability of the film formulation when stored at temperatures below the T _g of the polymer.     

The effect of phosphorus binding clay (Phoslock®) in mitigating cyanobacterial nuisance: a laboratory study on the effects on water quality variables and plankton

This laboratory study examined the lanthanum modified clay Phoslock^® for its effectiveness to bind soluble reactive phosphorus (SRP), release of nutrients from this modified clay, its influence on water quality variables (pH, oxygen saturation %, conductivity and turbidity), effects on phytoplankton growth (green alga Scenedesmus obliquus, cyanobacteria Microcystis aeruginosa and Anabaena sp.), and, lastly, its effect on the population growth of the rotifer Brachionus calyciflorus. A clear dose–response for SRP binding by the modified clay was observed. A small amount of ammonium is released from Phoslock^®. We found no effect of Phoslock^® on pH or oxygen saturation. Conductivity increased with the increasing concentration of Phoslock^®. An application of Phoslock^® caused a transient increase of turbidity up to 211 NTU. However, due to rapid settlement, turbidity fell below 13 NTU (~1 m Secchi depth), after 6 h. Phoslock^® addition caused a reduction in growth of all phytoplankton species tested that we attribute to the combined effects of light limitation, flocculation with the bentonite and binding of SRP to Phoslock^®. We estimated the EC₅₀ of Phoslock^® on the population growth of rotifer B. calyciflorus to be 0.15 g Phoslock^® l^?1. Overall, the results of our study indicate Phoslock^® seems to be suitable for field applications.     

Selectivity of different biological products to the egg parasitoid Telenomus remus (Hymenoptera: Platygastridae)

The selectivity of five entomopathogens and a chemical insecticide (positive control) to pupae and adults of the egg parasitoid Telenomus remus (Nixon) was evaluated in the laboratory under controlled environmental conditions according to protocols established by the International Organization for Biological Control. Baculovirus anticarsia (Baculovirus AEE^®), Bacillus thuringiensis var. kurstaki (Thuricide^®), Bacillus thuringiensis var. aizawai (Agree^®), Beauveria bassiana (Boveril^®), Metarhizium anisopliae (Metarril^®) and Trichoderma harzianum (Trichodermil^®) are harmless to T. remus pupae, and adults. Thus, our results suggest that the insect control strategies applied here are compatible since entomopathogens were classified as harmless to T. remus in most examined cases and therefore facilitate a joint application to control different pests. Bacillus thuringiensis var. kurstaki (Dipel^®), despite being classified as slightly harmful in some of the evaluations, can still be considered compatible for use together with T. remus, especially when compared with chemical insecticides such as chlorpyrifos that might be considered harmful to the parasitoid survival.     

Risk assessment of Cry toxins of Bacillus thuringiensis on the predatory mites Euseius concordis and Neoseiulus californicus (Acari: Phytoseiidae)

Genetically modified plants carrying Cry toxins of Bacillus thuringiensis (Bt) are widely used for pest control. Possible adverse effects as a result of the use of this control technique to non-target organisms is still a concern; however, few studies have addressed the effects of Bt crops on phytoseiid predatory mites. Phytoseiids are important for the natural control of phytophagous mites, but they can also feed on pollen, plant exudates, etc. Thus, phytoseiids may ingest Bt toxins through several pathways. In this paper, we evaluate the direct effect of Bt-toxins by feeding the predators on Bt cell suspensions, on solution of a Bt toxin and the tri-trophic effect by Bt expressed in transgenic plants. We present a method of conducting toxicological tests with Phytoseiidae which can be useful in studies of risk analysis of toxins to be expressed by genetically engineered plants. This method was used to evaluate the potential effect of ingestion of suspensions of Bt (1.25 × 10⁸ spores/ml) and of purified protein Cry1Ia12 (0.006 mg/ml and 0.018 mg/ml) on Euseius concordis, a predatory mite that develops and reproduces best on pollen. The effects of genetically modified Bollgard^® cotton, which carries the Cry1Ac protein, on Neoseiulus californicus, a selective predator that feeds more on spider mites than on pollen or insects, was determined by feeding them with Tetranychus urticae reared in Bollgard^® cotton and on the non-transgenic isoline. When E. concordis was fed with suspension of Bt isolate derived from product Dipel^® PM, no significant effects were detected. Similarly, Cry1Ia12 Bt toxin, at a concentration of 0.006 mg/ml, did not affect E. concordis. At a concentration of 0.018 mg/ml, however, the intake of this protein reduced the reproduction of E. concordis. There were no effects of Bollgard^® cotton on the biological traits and on the predatory capacity of N. californicus. Results indicate that the Cry toxins of B. thuringiensis studied, at the concentrations used in the field or expressed in transgenic plants, should not affect the predatory mites E. concordis and N. californicus.     

Effects of adjuvant and conidial concentration on the efficacy of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> for the control of the two spotted spider mite, <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Greenhouse experiments were conducted on various crops (cucumber, tomato, eggplant, green bean) to ascertain the effects of Break-thru^® (polyether-polymethylsiloxane-copolymer, a silicone surfactant) and an oil emulsion, on Beauveria bassiana (Balsamo) Vuillemin (Bb) applications for the control of the two spotted spider mite, Tetranychus urticae Koch. The objectives were to compare a) the efficacy of Bb control when applied in aqueous Break-thru^® or an oil emulsion; b) the effects of various concentrations of Bb conidia, as affected by each surfactant; and c) the effects of Break-thru^® on the activity of the fungus. Conidia were suspended either in an aqueous Break-thru^® or an emulsifiable formulation at different conidial concentrations (1.05 × 10⁶, 2.1 × 10⁶ and 4.2 × 10⁶ conidia ml^?1) and sprayed onto leaves 2 weeks after artificial pest inoculation. Two sprays were performed, with an interval of one week from one spray to another, and T. urticae population counts (both motile and egg stages) were made on plant leaves 7 days after each spray. Bb conidia in Break-thru^® were more efficacious than conidia in emulsifiable formulation. With the highest rate of conidia (4.2 × 10⁶ conidia ml^?1), mortality of adult mites ranged from 60 ± 4.2 (mean ± SE) to 85.7 ± 4.3% in the Break-thru^® suspension and 39.4 ± 7 to 61.3 ± 6% in the oil emulsion. Leaf damage index was also substantially reduced from 70% in the unsprayed control to 40% by the application of Bb conidia at the highest rate with Break-thru^®. Break-thru^® can be combined with Bb in the integrated management of T. urticae and Isolate R444 is a promising candidate for the control of the pest.     

Importance of Molecular Methods to Determine Whether a Probiotic is the Source of <Emphasis Type="Italic">Lactobacillus</Emphasis> Bacteremia

There has been an increasing interest in the use of probiotic products for the prevention of Clostridium difficile infection (CDI). Bio-K+^® is a commercial probiotic product comprising three strains of lactobacilli—Lactobacillus acidophilus CL1285^®, Lact. casei LBC80R^® and Lact. rhamnosus CLR2^®—that have been applied to prevent CDI. Generally considered as safe, lactobacilli have potential to cause bacteremia, endocarditis and other infections. The source of Lactobacillus bacteremia can be normal human flora or lactobacilli-containing probiotic. The aim of this study was to assess whether probiotic lactobacilli caused bacteremia and to show the value of molecular identification and typing techniques to determine probiotic and patient strain relatedness. We report an episode of Lactobacillus bacteremia in a 69-year-old man admitted to a hospital with severe congestive heart failure. During his hospitalization, he required long-term antibiotic therapy. Additionally, the patient received Bio-K+^® probiotic as part of a quality improvement project to prevent CDI. Subsequently, Lactobacillus bacteremia occurred. Two independent blinded laboratory evaluations, using pulse field gel electrophoresis, 16S rRNA gene sequencing and DNA fingerprint analysis (rep-PCR), were performed to determine whether the recovered Lact. acidophilus originated from the probiotic product. Ultimately, the patient strain was identified as Lact. casei and both laboratories found no genetic relation between the patient’s strain and any of the probiotic lactobacilli. This clinical case of lactobacillus bacteremia in the setting of probiotic exposure demonstrates the value of using discriminatory molecular methods to clearly determine whether there were a link between the patient’s isolate and the probiotic strains.     

Seaweed-Derived Biostimulant (Kelpak<Superscript>®</Superscript>) Influences Endogenous Cytokinins and Bioactive Compounds in Hydroponically Grown <Emphasis Type="Italic">Eucomis autumnalis</Emphasis>

The effect of a seaweed-derived biostimulant (Kelpak^® at 1, 2.5 and 5 % dilution; v/v) on the growth, endogenous cytokinin (CK) and phytochemical content in Eucomis autumnalis (Mill.) Chitt. under hydroponic conditions was evaluated. After 4 months, the stimulatory effect of Kelpak^® treatments was more noticeable in the underground organs than in the aerial organs. Total endogenous CK was also higher in plants treated with Kelpak^® (c.a. 1000–1200 pmol g^?1 DW) compared to control plants (860 pmol g^?1 DW). Isoprenoid CKs (which mainly accumulated in the aerial organs) were more dominant than aromatic-type CKs across all the treatments. A total of 11 bioactive chemicals (8 phenolic acids and 3 flavonoids) and eucomic acid known for their diverse biological activities were quantified in the samples. The most abundant compound was p-coumaric acid (6.5 µg g^?1 DW) and it was approximately sevenfold higher in 2.5 % Kelpak^®-treated plants than in the control. It was also noteworthy that syringic acid only occurred in the underground organs of 5 % Kelpak^®-treated plants. Eucomic acid which is a major bioactive compound in E. autumnalis was significantly enhanced in Kelpak^® treatments, and the leaves accounted for more than 70 % of the overall content. Thus, Kelpak^® elicited a significant influence on the growth, endogenous CK and phytochemical content in E. autumnalis. These findings provide additional evidence of the enormous potential of Kelpak^® as a useful biostimulant with practical applications in various agricultural endeavours.     

Supplementation of 2nd break mushroom compost with isoleucine, leucine, valine, phenylalanine, Fermenten® and SoyPlus®

Three mushroom (Agaricus bisporus) crops (Crops 1, 2, 3) were grown to evaluate the effects of re-supplementing “spent” mushroom compost (MC) with the crystalline amino acids isoleucine (ile), leucine (leu), valine (val) and phenylalanine (phe) singly or in combination with Fermenten^® or SoyPlus^® on mushroom yield. Fermenten^® is a rumen fermentation enhancer while SoyPlus^® is a commercial delayed release mushroom nutrient. The most important single amino acid found for stimulating mushroom yield from 2nd break MC was ile. Crystalline ile added to 2nd break MC at 3.6% (dry wt) increased mushroom yields by 28.3% and 68.7% (average 48.5%) in Crops 1 and 2, respectively, compared to the non-supplemented control. In Crop 3, the addition of 5% or 10% ile to Fermenten^® and SoyPlus^® (3.6% total combined dry wt) did not significantly improve mushroom yield over treatments containing Fermenten^® or SoyPlus^® (3.6% total dry wt) alone. However, mixtures of equal quantities of Fermenten^®, ile and val significantly increased yield over Fermenten^® alone. Use of ile and val as supplements to stimulate mushroom yield from 2nd break MC is not economically viable because these amino acids are not commercially available at feed grade prices.