DypB peroxidase for aflatoxin removal: New insights into the toxin degradation process.

Aflatoxin B1 (AFB1) is one of the most potent carcinogens and a widespread food and feed contaminant. As for other toxins, many efforts are devoted to find efficient and environmentally-friendly methods to degrade AFB1, such as enzymatic treatments, thus improving the safety of food and feed products. In this regard, the dye decolorizing peroxidase of type B (DypB) can efficiently degrade AFB1. The molecular mechanism, which is required to drive protein optimization in view of the usage of DypB as a mycotoxin reduction agent in large scale application, is unknown. Here, we focused on the role of four DypB residues in the degradation of AFB1 by alanine-scanning (residues 156, 215, 239 and 246), which were identified from biochemical assays to be kinetically relevant for the degradation. As a result of DypB degradation, AFB1 is converted into four products. Interestingly, the relative abundancy of these products depends on the replaced residues. Molecular dynamics simulations were used to investigate the role of these residues in the binding step between protein and manganese, a metal ion which is expected to be involved in the degradation process. We found that the size of the haem pocket as well as conformational changes in the protein structure could play a role in determining the kinetics of AFB1 removal and, consequently, guide the process towards specific degradation products.

Penicillium diversity in Canadian bat caves, including a new species, P. speluncae.

Penicillium species were commonly isolated during a fungal survey of bat hibernacula in New Brunswick and Quebec, Canada. Strains were isolated from arthropods, bats, rodents (i.e. the deer mouse Peromyscus maniculatus), their dung, and cave walls. Hundreds of fungal strains were recovered, of which Penicillium represented a major component of the community. Penicillium strains were grouped by colony characters on Blakeslee's malt extract agar. DNA sequencing of the secondary identification marker, beta-tubulin, was done for representative strains from each group. In some cases, ITS and calmodulin were sequenced to confirm identifications. In total, 13 species were identified, while eight strains consistently resolved into a unique clade with P. discolor, P. echinulatum and P. solitum as its closest relatives. Penicillium speluncae is described using macroand micromorphological characters, multigene phylogenies (including ITS, beta-tubulin, calmodulin and RNA polymerase II second largest subunit) and extrolite profiles. Major extrolites produced by the new species include cyclopenins, viridicatins, chaetoglobosins, and a microheterogenous series of cyclic and linear tetrapeptides.

A plant biostimulant made from the marine brown algae Ascophyllum nodosum and chitosan reduce Fusarium head blight and mycotoxin contamination in wheat.

Fusarium head blight (FHB) caused by Fusarium graminearum is a disease that results in yield loss and mycotoxin contamination in wheat globally. This study assessed the effect of a plant biostimulant prepared from a brown macroalga Ascophyllum nodosum (Liquid Seaweed Extract; LSE) alone and in combination with chitosan in controlling Fusarium. Wheat seedlings drenched with LSE and chitosan in combination showed reduced severity of F. graminearum infection on leaves as evidenced by a significant reduction in necrotic area and fewer number of conidia produced in the necrotic area. Gene expression studies showed that the combination of LSE and chitosan amplified the response of pathogenesis-related genes (TaPR1.1, TaPR2, TaPR3, TaGlu2) in wheat seedlings infected with Fusarium spores above that observed for the individual treatments. The combination treatments were more effective in enhancing the activity of various defense related enzymes such as peroxidase and polyphenol oxidase. FHB studies on adult plants showed a reduction of bleached spikes in wheat heads treated with the combination of LSE and chitosan. Mycotoxin content appeared to be correlated with FHB severity. Combination treatments of LSE and chitosan reduced the levels of mycotoxins deoxynivalenol and sambucinol in wheat grains. Systemic disease resistance appears to be induced by LSE and chitosan in response to F. graminearum in wheat by inducing defense genes and enzymes.

Plant growth regulator-mediated anti-herbivore responses of cabbage (Brassica oleracea) against cabbage looper Trichoplusia ni Hübner (Lepidoptera: Noctuidae).

Plant elicitors can be biological or chemical-derived stimulators of jasmonic acid (JA) or salicylic acid (SA) pathways shown to prime the defenses in many crops. Examples of chemical elicitors of the JA and SA pathways include methyl-jasmonate and 1,2,3-benzothiadiazole-7-carbothioate (BTH or the commercial plant activator Actigard 50WG, respectively). The use of specific elicitors has been observed to affect the normal interaction between JA and SA pathways causing one to be upregulated and the other to be suppressed, often, but not always, at the expense of the plant's herbivore or pathogen defenses. The objective of this study was to determine whether insects feeding on Brassica crops might be negatively affected by SA inducible defenses combined with an inhibitor of detoxification and anti-oxidant enzymes that regulate the insect response to the plant's defenses. The relative growth rate of cabbage looper Trichoplusia ni Hübner (Lepidoptera: Noctuidae) fed induced cabbage Brassica oleraceae leaves with the inhibitor, quercetin, was significantly less than those fed control cabbage with and without the inhibitor. The reduced growth was related to the reduction of glutathione S-transferases (GSTs) by the combination of quercetin and increased levels of indole glucosinolates in the cabbage treated with BTH at 2.6× the recommended application rate. These findings may offer a novel combination of elicitor and synergist that can provide protection from plant disease and herbivores in cabbage and other Brassica crops.

Post-acquisition filtering of salt cluster artefacts for LC-MS based human metabolomic studies.

Liquid chromatography-high resolution mass spectrometry (LC-MS) has emerged as one of the most widely used platforms for untargeted metabolomics due to its unparalleled sensitivity and metabolite coverage. Despite its prevalence of use, the proportion of true metabolites identified in a given experiment compared to background contaminants and ionization-generated artefacts remains poorly understood. Salt clusters are well documented artefacts of electrospray ionization MS, recognized by their characteristically high mass defects (for this work simply generalized as the decimal numbers after the nominal mass). Exploiting this property, we developed a method to identify and remove salt clusters from LC-MS-based human metabolomics data using mass defect filtering. By comparing the complete set of endogenous metabolites in the human metabolome database to actual plasma, urine and stool samples, we demonstrate that up to 28.5 % of detected features are likely salt clusters. These clusters occur irrespective of ionization mode, column type, sweep gas and sample type, but can be easily removed post-acquisition using a set of R functions presented here. Our mass defect filter removes unwanted noise from LC-MS metabolomics datasets, while retaining true metabolites, and requires only a list of m/z and retention time values. Reducing the number of features prior to statistical analyses will result in more accurate multivariate modeling and differential feature selection, as well as decreased reporting of unknowns that often constitute the largest proportion of human metabolomics data.

Fifteen new species of Penicillium.

We introduce 15 new species of Penicillium isolated from a diverse range of locations, including Canada, Costa Rica, Germany, Italy, New Zealand, Tanzania, USA and the Dry Valleys of Antarctica, from a variety of habitats, including leaf surfaces in tropical rain forests, soil eaten by chimpanzees, infrabuccal pockets of carpenter ants, intestinal contents of caterpillars and soil. The new species are classified in sections Aspergilloides (1), Canescentia (2), Charlesia (1), Exilicaulis (3), Lanata-Divaricata (7) and Stolkia (1). Each is characterised and described using classical morphology, LC-MS based extrolite analyses and multigene phylogenies based on ITS, BenA and CaM. Significant extrolites detected include andrastin, pulvilloric acid, penitrem A and citrinin amongst many others.

Repellent and Attractive Effects of α-, ß-, and Dihydro-ß- Ionone to Generalist and Specialist Herbivores.

In plants, the oxidative cleavage of carotenoid substrates produces volatile apocarotenoids, including α-ionone, ß-ionone, and dihydro-ß-ionone, compounds that are important in herbivore-plant communication. For example, ß-ionone is part of an induced defense in canola, Brassica napus, and is released following wounding by herbivores. The objectives of the research were to evaluate whether these volatile compounds would: 1) be released in higher quantities from plants through the over-expression of the carotenoid cleavage dioxygenase1 (CCD1) gene and 2) cause herbivores to be repelled or attracted to over-expressing plants relative to the wild-type. In vivo dynamic headspace collection of volatiles coupled with gas chromatography-mass spectrometry was used to determine volatile organic compounds (VOC) in the headspace of the Arabidopsis thaliana ecotype Columbia-0 (L.) over-expressing the AtCCD1 gene. The analytical method allowed the detection of ß-ionone in the Arabidopsis headspace where emission rates ranged between 2 and 5-fold higher compared to the wild type, thus corroborating the in vivo enhancement of gene expression. A two chamber choice test between wild type and AtCCD1 plants revealed that crucifer flea beetle Phyllotreta cruciferae (Goeze) adults were repelled by the AtCCD1 plants with the highest transcription and ß-ionone levels. α-Ionone and dihydro-ß-ionone were not found in the headspace analysis, but solutions of the three compounds were tested in the concentration range of ß-ionone found in the Arabidopsis headspace (0.05 to 0.5 ng/µl) in order to assess their biological activity with crucifer flea beetle, two spotted spider mite Tetranychus urticae (Koch), and silverleaf whiteflies Bemisia tabaci (Gennadius). Choice bioassays demonstrated that ß-ionone has a strong repellent effect toward both the flea beetle and the spider mite, and significant oviposition deterrence to whiteflies. In contrast, dihydro-ß-ionone had attractant properties, especially to the crucifer flea beetle, while α-ionone did not show any significant activity. These findings demonstrate how regulating genes of the carotenoid pathway can increase herbivore deterrent volatiles, a novel tool for insect pest management.