Abstracts of Presentations to the Working Session on Improving Predictive Modeling of Mycotoxin Risk for Africa Held at the 3rd ASM2022 on 7 September 2022, in Stellenbosch, South Africa.

In 2008, the African Postharvest Losses Information Systems project (APHLIS, accessed on 6 September 2022) developed an algorithm for estimating the scale of cereal postharvest losses (PHLs). The relevant scientific literature and contextual information was used to build profiles of the PHLs occurring along the value chains of nine cereal crops by country and province for 37 sub-Saharan African countries. The APHLIS provides estimates of PHL figures where direct measurements are not available. A pilot project was subsequently initiated to explore the possibility of supplementing these loss estimates with information on the aflatoxin risk. Using satellite data on drought and rainfall, a time series of agro-climatic aflatoxin risk warning maps for maize was developed covering the countries and provinces of sub-Saharan Africa. The agro-climatic risk warning maps for specific countries were shared with mycotoxin experts from those countries for review and comparison with their aflatoxin incidence datasets. The present Work Session was a unique opportunity for African food safety mycotoxins experts, as well as other international experts, to meet and deepen the discussion about prospects for using their experience and their data to validate and improve agro-climatic risk modeling approaches.

An observational study of wounds treated with hydro-responsive wound dressings.

OBJECTIVE: Acute and hard-to-heal wounds are a significant burden to both a patient's quality of life and resources in healthcare systems. Here, we evaluate the outcomes of a non-comparative case series study in which Ringer's solution-preactivated polyacrylate dressings were used to treat acute and hard-to-heal wounds (the presence of Ringer's solution provides a wound dressing that allows, upon application, the immediate hydration of the underlying wound tissue). METHOD: Patients with acute and hard-to-heal wounds were enrolled into an open-labelled, non-comparative observational study. Patients were treated with Ringer's solution-preactivated polyacrylate dressings to enable wound debridement and wound cleansing for up to 12 weeks. RESULTS: A total of 303 patients were enrolled in the study and 278 were included in the analysis. Wound size decreased, from a median of 3.6cm2 (interquartile range (IQR): 1.2-9.3] at baseline to a median of 2.6cm2 (IQR: 1.1-7.8] at 84 days. Relative wound area reduction (WAR) was 43.1% at 84 days and estimated probability of achievement of a WAR of ≥40% and ≥60% was 68.7% and 53.4%, respectively. Median time to achieve a WAR of ≥40% and ≥60% was 54 days and 75 days, respectively. The median percentage of wound area covered by fibrin had decreased from 50.0% to 10% and granulation tissue had increased from 25% to 50% after 84 days. In addition, periwound skin condition, local signs of infection and pain all showed improvement. The majority of the wounds were assessed as 'healed' or 'better' at the conclusion of the evaluation period. CONCLUSION: Based on the findings of this study, the use of Ringer's solution-preactivated polyacrylate dressings in daily practice has the potential to improve clinical outcomes, including healing, in patients with acute and hard-to-heal wounds.

Zearalenone and ß-Zearalenol But Not Their Glucosides Inhibit Heat Shock Protein 90 ATPase Activity.

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant-pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant-pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, AtHSP90.1 and wheat TaHSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants.

A barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium Head Blight resistance in transgenic wheat.

Fusarium Head Blight is a disease of cereal crops that causes severe yield losses and mycotoxin contamination of grain. The main causal pathogen, Fusarium graminearum, produces the trichothecene toxins deoxynivalenol or nivalenol as virulence factors. Nivalenol-producing isolates are most prevalent in Asia but co-exist with deoxynivalenol producers in lower frequency in North America and Europe. Previous studies identified a barley UDP-glucosyltransferase, HvUGT13248, that efficiently detoxifies deoxynivalenol, and when expressed in transgenic wheat results in high levels of type II resistance against deoxynivalenol-producing F. graminearum. Here we show that HvUGT13248 is also capable of converting nivalenol into the non-toxic nivalenol-3-O-ß-d-glucoside. We describe the enzymatic preparation of a nivalenol-glucoside standard and its use in development of an analytical method to detect the nivalenol-glucoside conjugate. Recombinant Escherichia coli expressing HvUGT13248 glycosylates nivalenol more efficiently than deoxynivalenol. Overexpression in yeast, Arabidopsis thaliana, and wheat leads to increased nivalenol resistance. Increased ability to convert nivalenol to nivalenol-glucoside was observed in transgenic wheat, which also exhibits type II resistance to a nivalenol-producing F. graminearum strain. Our results demonstrate the HvUGT13248 can act to detoxify deoxynivalenol and nivalenol and provide resistance to deoxynivalenol- and nivalenol-producing Fusarium.

Time-course expression QTL-atlas of the global transcriptional response of wheat to Fusarium graminearum.

Fusarium head blight is a devastating disease of small grain cereals such as bread wheat (Triticum aestivum). The pathogen switches from a biotrophic to a nectrotrophic lifestyle in course of disease development forcing its host to adapt its defence strategies. Using a genetical genomics approach, we illustrate genome-wide reconfigurations of genetic control over transcript abundances between two decisive time points after inoculation with the causative pathogen Fusarium graminearum. Whole transcriptome measurements have been recorded for 163 lines of a wheat doubled haploid population segregating for several resistance genes yielding 15 552 at 30 h and 15 888 eQTL at 50 h after inoculation. The genetic map saturated with transcript abundance-derived markers identified of a novel QTL on chromosome 6A, besides the previously reported QTL Fhb1 and Qfhs.ifa-5A. We find a highly different distribution of eQTL between time points with about 40% of eQTL being unique for the respective assessed time points. But also for more than 20% of genes governed by eQTL at either time point, genetic control changes in time. These changes are reflected in the dynamic compositions of three major regulatory hotspots on chromosomes 2B, 4A and 5A. In particular, control of defence-related biological mechanisms concentrated in the hotspot at 4A shift to hotspot 2B as the disease progresses. Hotspots do not colocalize with phenotypic QTL, and within their intervals no higher than expected number of eQTL was detected. Thus, resistance conferred by either QTL is mediated by few or single genes.

Joint Transcriptomic and Metabolomic Analyses Reveal Changes in the Primary Metabolism and Imbalances in the Subgenome Orchestration in the Bread Wheat Molecular Response to Fusarium graminearum.

Fusarium head blight is a prevalent disease of bread wheat (Triticum aestivum L.), which leads to considerable losses in yield and quality. Quantitative resistance to the causative fungus Fusarium graminearum is poorly understood. We integrated transcriptomics and metabolomics data to dissect the molecular response to the fungus and its main virulence factor, the toxin deoxynivalenol in near-isogenic lines segregating for two resistance quantitative trait loci, Fhb1 and Qfhs.ifa-5A. The data sets portrait rearrangements in the primary metabolism and the translational machinery to counter the fungus and the effects of the toxin and highlight distinct changes in the metabolism of glutamate in lines carrying Qfhs.ifa-5A. These observations are possibly due to the activity of two amino acid permeases located in the quantitative trait locus confidence interval, which may contribute to increased pathogen endurance. Mapping to the highly resolved region of Fhb1 reduced the list of candidates to few genes that are specifically expressed in presence of the quantitative trait loci and in response to the pathogen, which include a receptor-like protein kinase, a protein kinase, and an E3 ubiquitin-protein ligase. On a genome-scale level, the individual subgenomes of hexaploid wheat contribute differentially to defense. In particular, the D subgenome exhibited a pronounced response to the pathogen and contributed significantly to the overall defense response.

chromoWIZ: a web tool to query and visualize chromosome-anchored genes from cereal and model genomes.

BACKGROUND: Over the last years reference genome sequences of several economically and scientifically important cereals and model plants became available. Despite the agricultural significance of these crops only a small number of tools exist that allow users to inspect and visualize the genomic position of genes of interest in an interactive manner. DESCRIPTION: We present chromoWIZ, a web tool that allows visualizing the genomic positions of relevant genes and comparing these data between different plant genomes. Genes can be queried using gene identifiers, functional annotations, or sequence homology in four grass species (Triticum aestivum, Hordeum vulgare, Brachypodium distachyon, Oryza sativa). The distribution of the anchored genes is visualized along the chromosomes by using heat maps. Custom gene expression measurements, differential expression information, and gene-to-group mappings can be uploaded and can be used for further filtering. CONCLUSIONS: This tool is mainly designed for breeders and plant researchers, who are interested in the location and the distribution of candidate genes as well as in the syntenic relationships between different grass species. chromoWIZ is freely available and online accessible at http://mips.helmholtz-muenchen.de/plant/chromoWIZ/index.jsp.

Zearalenone-16-O-glucoside: a new masked mycotoxin.

This paper reports the identification of a barley UDP-glucosyltransferase, HvUGT14077, which is able to convert the estrogenic Fusarium mycotoxin zearalenone into a near-equimolar mixture of the known masked mycotoxin zearalenone-14-O-ß-glucoside and a new glucose conjugate, zearalenone-16-O-ß-glucoside. Biocatalytical production using engineered yeast expressing the HvUGT14077 gene allowed structural elucidation of this compound. The purified zearalenone-16-O-ß-glucoside was used as an analytical calibrant in zearalenone metabolization experiments. This study confirmed the formation of this new masked mycotoxin in barley seedlings as well as in wheat and Brachypodium distachyon cell suspension cultures. In barley roots, up to 18-fold higher levels of zearalenone-16-O-ß-glucoside compared to the known zearalenone-14-O-ß-glucoside were found. Incubation of zearalenone-16-O-ß-glucoside with human fecal slurry showed that this conjugate can also be hydrolyzed rapidly by intestinal bacteria, converting the glucoside back to the parental mycotoxin. Consequently, it should be considered as an additional masked form of zearalenone with potential relevance for food safety.

Quantitative trait loci-dependent analysis of a gene co-expression network associated with Fusarium head blight resistance in bread wheat (Triticum aestivum L.).

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium graminearum Schwabe is one of the most prevalent diseases of wheat (Triticum aestivum L.) and other small grain cereals. Resistance against the fungus is quantitative and more than 100 quantitative trait loci (QTL) have been described. Two well-validated and highly reproducible QTL, Fhb1 and Qfhs.ifa-5A have been widely investigated, but to date the underlying genes have not been identified. RESULTS: We have investigated a gene co-expression network activated in response to F. graminearum using RNA-seq data from near-isogenic lines, harboring either the resistant or the susceptible allele for Fhb1 and Qfhs.ifa-5A. The network identified pathogen-responsive modules, which were enriched for differentially expressed genes between genotypes or different time points after inoculation with the pathogen. Central gene analysis identified transcripts associated with either QTL within the network. Moreover, we present a detailed gene expression analysis of four gene families (glucanases, NBS-LRR, WRKY transcription factors and UDP-glycosyltransferases), which take prominent roles in the pathogen response. CONCLUSIONS: A combination of a network-driven approach and differential gene expression analysis identified genes and pathways associated with Fhb1 and Qfhs.ifa-5A. We find G-protein coupled receptor kinases and biosynthesis genes for jasmonate and ethylene earlier induced for Fhb1. Similarly, we find genes involved in the biosynthesis and metabolism of riboflavin more abundant for Qfhs.ifa-5A.

Transcriptomic characterization of two major Fusarium resistance quantitative trait loci (QTLs), Fhb1 and Qfhs.ifa-5A, identifies novel candidate genes.

Fusarium head blight, caused by Fusarium graminearum, is a devastating disease of wheat. We developed near-isogenic lines (NILs) differing in the two strongest known F. graminearum resistance quantitative trait loci (QTLs), Qfhs.ndsu-3BS (also known as resistance gene Fhb1) and Qfhs.ifa-5A, which are located on the short arm of chromosome 3B and on chromosome 5A, respectively. These NILs showing different levels of resistance were used to identify transcripts that are changed significantly in a QTL-specific manner in response to the pathogen and between mock-inoculated samples. After inoculation with F. graminearum spores, 16 transcripts showed a significantly different response for Fhb1 and 352 for Qfhs.ifa-5A. Notably, we identified a lipid transfer protein which is constitutively at least 50-fold more abundant in plants carrying the resistant allele of Qfhs.ifa-5A. In addition to this candidate gene associated with Qfhs.ifa-5A, we identified a uridine diphosphate (UDP)-glycosyltransferase gene, designated TaUGT12887, exhibiting a positive difference in response to the pathogen in lines harbouring both QTLs relative to lines carrying only the Qfhs.ifa-5A resistance allele, suggesting Fhb1 dependence of this transcript. Yet, this dependence was observed only in the NIL with already higher basal resistance. The complete cDNA of TaUGT12887 was reconstituted from available wheat genomic sequences, and a synthetic recoded gene was expressed in a toxin-sensitive strain of Saccharomyces cerevisiae. This gene conferred deoxynivalenol resistance, albeit much weaker than that observed with the previously characterized barley HvUGT13248.

Functional characterization of two clusters of Brachypodium distachyon UDP-glycosyltransferases encoding putative deoxynivalenol detoxification genes.

Plant small-molecule UDP-glycosyltransferases (UGT) glycosylate a vast number of endogenous substances but also act in detoxification of metabolites produced by plant-pathogenic microorganisms. The ability to inactivate the Fusarium graminearum mycotoxin deoxynivalenol (DON) into DON-3-O-glucoside is crucial for resistance of cereals. We analyzed the UGT gene family of the monocot model species Brachypodium distachyon and functionally characterized two gene clusters containing putative orthologs of previously identified DON-detoxification genes from Arabidopsis thaliana and barley. Analysis of transcription showed that UGT encoded in both clusters are highly inducible by DON and expressed at much higher levels upon infection with a wild-type DON-producing F. graminearum strain compared with infection with a mutant deficient in DON production. Expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae revealed that only two B. distachyon UGT encoded by members of a cluster of six genes homologous to the DON-inactivating barley HvUGT13248 were able to convert DON into DON-3-O-glucoside. Also, a single copy gene from Sorghum bicolor orthologous to this cluster and one of three putative orthologs of rice exhibit this ability. Seemingly, the UGT genes undergo rapid evolution and changes in copy number, making it difficult to identify orthologs with conserved substrate specificity.

Validation of a candidate deoxynivalenol-inactivating UDP-glucosyltransferase from barley by heterologous expression in yeast.

Resistance to the virulence factor deoxynivalenol (DON) due to formation of DON-3-O-glucoside (D3G) is considered to be an important component of resistance against Fusarium spp. which produce this toxin. Multiple candidate UDP-glycosyltransferase (UGT) genes from different crop plants that are either induced by Fusarium spp. or differentially expressed in cultivars varying in Fusarium disease resistance have been described. However, UGT are encoded by a very large gene family in plants. The study of candidate plant UGT is highly warranted because of the potential relevance for developing Fusarium-spp.-resistant crops. We tested Arabidopsis thaliana genes closely related to a previously identified DON-glucosyltransferase gene by heterologous expression in yeast and showed that gene products with very high sequence similarity can have pronounced differences in detoxification capabilities. We also tested four candidate barley glucosyltransferases, which are highly DON inducible. Upon heterologous expression of full-length cDNAs, only one gene, HvUGT13248, conferred DON resistance. The conjugate D3G accumulated in the supernatant of DON-treated yeast transformants. We also present evidence that the product of the TaUGT3 gene recently proposed to encode a DON-detoxification enzyme of wheat does not protect yeast against DON.