Mycotoxins in Flanders’ Fields : occurrence and correlations with fusarium species in whole-plant harvested maize

Mycotoxins are well-known contaminants of several food- and feedstuffs, including silage maize for dairy cattle. Climate change and year-to-year variations in climatic conditions may cause a shift in the fungal populations infecting maize, and therefore alter the mycotoxin load. In this research, 257 maize samples were taken from fields across Flanders, Belgium, over the course of three years (2016-2018) and analyzed for 22 different mycotoxins using a multi-mycotoxin liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. DNA of Fusarium graminearum, F. culmorum and F. verticillioides was quantified using the quantitative polymerase chain reaction (qPCR). Multi-mycotoxin contamination occurred frequently, with 47% of samples containing five or more mycotoxins. Nivalenol (NIV) was the most prevalent mycotoxin, being present in 99% of the samples, followed by deoxynivalenol (DON) in 86% and zearalenone (ZEN) in 50% of the samples. Fumonisins (FUMs) were found in only 2% of the samples in the wet, cold year of 2016, but in 61% in the extremely hot and dry year of 2018. Positive correlations were found between DON and NIV and between F. graminearum and F. culmorum, among others. FUM concentrations were not correlated with any other mycotoxin, nor with any Fusarium sp., except F. verticillioides. These results show that changing weather conditions can influence fungal populations and the corresponding mycotoxin contamination of maize significantly, and that multi-mycotoxin contamination increases the risk of mycotoxicosis in dairy cattle

Multi-mycotoxin contamination of maize silages in Flanders, Belgium : monitoring mycotoxin levels from seed to feed

Maize silage, which in Europe is the main feed for dairy cattle in winter, can be contaminated by mycotoxins. Mycotoxigenic Fusarium spp. originating from field infections may survive in badly sealed silages or re-infect at the cutting edge during feed-out. In this way, mycotoxins produced in the field may persist during the silage process. In addition, typical silage fungi such as Penicillium spp. and Aspergillus spp. survive in silage conditions and produce mycotoxins. In this research, 56 maize silages in Flanders were sampled over the course of three years (2016-2018). The concentration of 22 different mycotoxins was investigated using a multi-mycotoxin liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and the presence of DNA of three Fusarium spp. (F. graminearum, F. culmorum and F. verticillioides) was analyzed in a selection of these samples using quantitative polymerase chain reaction (qPCR). Every maize silage contained at least two different mycotoxins. Nivalenol (NIV) and deoxynivalenol (DON) were the most prevalent (both in 97.7% of maize silages), followed by ENN B (88.7%). Concentrations often exceeded the EU recommendations for DON and zearalenone (ZEN), especially in 2017 (21.3% and 27.7% of the maize silages, respectively). No correlations were found between fungal DNA and mycotoxin concentrations. Furthermore, by ensiling maize with a known mycotoxin load in a net bag, the mycotoxin contamination could be monitored from seed to feed. Analysis of these net bag samples revealed that the average concentration of all detected mycotoxins decreased after fermentation. We hypothesize that mycotoxins are eluted, degraded, or adsorbed during fermentation, but certain badly preserved silages are prone to additional mycotoxin production during the stable phase due to oxygen ingression, leading to extremely high toxin levels

Genome editing in macroalgae: advances and challenges

This minireview examines the current state and challenges of genome editing in macroalgae. Despite the ecological and economic significance of this group of organisms, genome editing has seen limited applications. While CRISPR functionality has been established in two brown (Ectocarpus species 7 and Saccharina japonica) and one green seaweed (Ulva prolifera), these studies are limited to proof-of-concept demonstrations. All studies also (co)-targeted ADENINE PHOSPHORIBOSYL TRANSFERASE to enrich for mutants, due to the relatively low editing efficiencies. To advance the field, there should be a focus on advancing auxiliary technologies, particularly stable transformation, so that novel editing reagents can be screened for their efficiency. More work is also needed on understanding DNA repair in these organisms, as this is tightly linked with the editing outcomes. Developing efficient genome editing tools for macroalgae will unlock the ability to characterize their genes, which is largely uncharted terrain. Moreover, given their economic importance, genome editing will also impact breeding campaigns to develop strains that have better yields, produce more commercially valuable compounds, and show improved resilience to the impacts of global change

Reappraisal of the toxicity test method using the green alga Ulva pertusa Kjellman (Chlorophyta),

This study was aimed to develop an objective way of quantifying the reproductive status of the green macroalga, Ulva pertusa using a vital stain and programmed automated analysis (by Image J program). The EC50 values (with 95% CI), the concentrations of toxicants inducing a reduction of 50% in sporulation after 96 h exposure, from the newly developed method were similar to those obtained by the conventional method: 0.651 (0.598-0.705) mg l(-1) for Cd, 0.144 (0.110-0.162) mg l(-1) for Cu, 0.180 (0.165-0.195) mg l(-1) for atrazine, 0.076 (0.049-0.094) mg l(-1) for diuron and 30.6 (26.5-34.4) ml l(-1) for DMSO, respectively. When the EC50 values from this study were compared to that those from literatures, the sensitivity for some toxicants was similar or higher than that of U. fasciata (1.930 mg l(-1) for germination for Cd), U. armoricana (0.250 mg l(-1) for Fv/Fm for Cu), U. reticulata (0.126-1.585 mg l(-1) for growth for Cu), and U. intestinalis (0.650 mg l(-1) for Fv/Fm for atrazine). The subjective views of the experimental performers can be eliminated using the newly developed method. The Ulva method gave consistent responses to Cu and Cd of internationally allowable ranges for effluents, implying that the method is a useful tool for monitoring industrial wastewaters containing these metals

Defining Major Surgery: A Delphi Consensus Among European Surgical Association (ESA) Members

Background: Major surgery is a term frequently used but poorly defined. The aim of the present study was to reach a consensus in the definition of major surgery within a panel of expert surgeons from the European Surgical Association (ESA). Methods: A 3-round Delphi process was performed. All ESA members were invited to participate in the expert panel. In round 1, experts were inquired by open- and closed-ended questions on potential criteria to define major surgery. Results were analyzed and presented back anonymously to the panel within next rounds. Closed-ended questions in round 2 and 3 were either binary or statements to be rated on a Likert scale ranging from 1 (strong disagreement) to 5 (strong agreement). Participants were sent 3 reminders at 2-week intervals for each round. 70% of agreement was considered to indicate consensus. Results: Out of 305 ESA members, 67 (22%) answered all the 3 rounds. Significant comorbidities were the only preoperative factor retained to define major surgery (78%). Vascular clampage or organ ischemia (92%), high intraoperative blood loss (90%), high noradrenalin requirements (77%), long operative time (73%) and perioperative blood transfusion (70%) were procedure-related factors that reached consensus. Regarding postoperative factors, systemic inflammatory response (76%) and the need for intensive or intermediate care (88%) reached consensus. Consequences of major surgery were high morbidity (>30% overall) and mortality (>2%). Conclusion: ESA experts defined major surgery according to extent and complexity of the procedure, its pathophysiological consequences and consecutive clinical outcomes

Simple and efficient modification of Golden Gate design standards and parts using oligo stitching

The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble de novo sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitching's versatility and high efficiency, we expect that it will be a useful addition to the molecular biologist's toolbox

Application of a programmed semi-automated Ulva pertusa bioassay for testing single toxicants and stream water quality

A toxicity test based on inhibition of reproduction in the green macroalga Ulva pertusa involves quantifying the change in thallus color as reproduction progresses. However, interpretation of this color change is reliant on the skill level of the examiner. This study aimed to validate a new toxicity test based on inhibition of reproduction in the green macroalga U. pertusa using a vital stain and programmed semi-automated analysis (using Image J) of the change in thallus color. The toxicity rank by inverse EC50 values was: irgarol (0.048 mg L-1) > Ag (0.132 mg L-1) > As (0.172 mg L-1) > simazine (0.378 mg L-1) > formaldehyde (0.442 mg L-1) > DCOIT (0.783 mg L-1) > ZnPT (3.556 mg L-1) > medetomidine (11.600 mg L-1) > phenol (29.316 mg L-1) > methanol (2,736 mg L-1) > ethanol (3,306 mg L-1). The sensitivity of the U. pertusa test to stream waters was similar to or lower than those of the commonly-used Lemna minor and Daphnia magna bioassays. The U. pertusa bioassay is sensitive to, and suitable for, testing various toxicants including metals, volatile organic compounds, herbicide, antifouling agents and phenol and can also be applied to testing freshwater quality after salinity adjustment

IMPLANT: a new technique for transgene copy number estimation in plants using a single end-point PCR reaction

Background: Copy number determination is one of the first steps in the characterization of transgenic plant lines. The classical approach to this, Southern blotting, is time-consuming, expensive and requires massive amounts of high-quality genomic DNA. Other PCR-based techniques are either inaccurate, laborious, or expensive. Results: Here, we propose a new technique, IMPLANT (Insertion of competitive PCR calibrator for copy number estimation), a competitive PCR-based technique in which the competitor (based on an endogenous gene) is also incorporated in the T-DNA, which then gets integrated in the genome together with the gene of interest. As the number of integrated competitor molecules directly corresponds to the number of transgene copies, the transgene copy number can be determined by a single PCR reaction. We demonstrate that the results of this technique closely correspond with those obtained by segregation analysis in Arabidopsis and digital PCR In rice, indicating that it is a powerful alternative for other techniques for copy number determination. Conclusions: We show that this technique is not only reliable, but is also faster, easier, and cheaper as compared with other techniques. Accurate results are obtained in both Arabidopsis and rice, but this technique can be easily extended to other organisms and as such can be widely adopted in the field of biotechnology