The mycotoxin deoxynivalenol (DON) poses severe threats to human and animal well-being globally. Enzymatic degradation is the most effective way to eliminate DON toxicity, yet no catalytic process for complete degradation of DON has been uncovered. Here, we show that a metabolic pathway initiated by C3-epimerization and C8-reduction is responsible for complete degradation of DON in the DON-metabolizing bacterium Nocardioides sp. S5-5. Two horizontally transferred aldo-keto reductase genes, DONepi and DONrd, have evolved to orchestrate C3-epimerization and C8-reduction respectively. Notably, the octameric-structured DONepi alone catalyzes C3-epimerization of DON by steering the rigid-body rotation of the transient 3-keto intermediate for stereoinverting reduction. Moreover, DONrd can catalyze the C8-reduction of DON and its C3-epimerized product 3-epi-DON simultaneously to form C8-hydroxyl products, which facilitates the further degradation by a potential oxidase and other putative enzymes. DONepi expression in transgenic plants confers resistance to DON, representing potential for controlling mycotoxin contamination pre- and postharvest.

Biocontrol microorganisms and their derived metabolites with antagonistic activity represent promising alternatives to chemical fungicides in managing plant pathogens. The lipopeptides (LPs) iturin and fengycin derived from Bacillus amyloliquefaciens S76-3 exhibit highly inhibitory effects against pathogenic fungi, especially Fusarium graminearum (Fg), the primary pathogen causing Fusarium head blight (FHB) in cereals. However, the specific target of iturin and fengycin in Fg and the underlying mechanism of antagonistic activity remain unclear. Here, global transcriptome sequencing, combined with both genetic and chemical approaches, demonstrates that the LPs exhibit antagonism toward Fg by binding to multiple components in the cell membrane of Fg cells, including ergosterol, phospholipids, glycosylphosphatidylinositol, and ankyrin. Lipopeptides result in cell swelling by inducing cell wall remodeling and osmotic substance glycerol synthesis mediated by cell wall integrity and high-osmolarity glycerol signaling pathways. Furthermore, we found that LPs can activate the induced systemic resistance in wheat against FHB and deoxynivalenol accumulation. Additionally, LPs were able to promote wheat growth by regulating auxin, cytokinin, and gibberellin signaling pathways while also delaying seed germination through the stimulation of abscisic acid and ethylene signaling pathways. These findings advance knowledge on the underlying mechanism of iturin and fengycin antagonistic activity and provide a new avenue for developing agricultural and clinical broad-spectrum antifungal agents and identifying plant growth regulators in the future.

Aspergillus flavus and the produced aflatoxins are common hazardous contaminants in grains and food, which generated great damage to food security and human health. Acetyl-CoA carboxylase (ACC), catalyzing acetyl-CoA to malonyl-CoA, is the first and speed-determining step in fatty acid biosynthesis (cell membrane composition) and aflatoxins biosynthesis process in A. flavus. Here, we demonstrated that dimethyl trisulfide (DMTS), emitted from bacteria, could interact with ACC, decrease the enzymatic activity from 377.62 to 191.42 kat/g in vitro, down-regulate the expression of ACC and other important genes in fatty acid and aflatoxin biosynthesis pathway, reduce the content of hexadecanoic acid, octadecanoic acid and eicosanoic acid, and finally prevent A. flavus growth and aflatoxins production. Two binding sites containing 19 amino acids were detected between DMTS and ACC through molecular docking analysis. These amino acids were highly conserved in different filamentous fungi, which accounted for the broad antifungal spectrum of DMTS. Therefore, we concluded that ACC is the molecular target of DMTS on A. flavus growth and aflatoxins production. These results elucidate the inhibitory mechanism of DMTS on fungal pathogens, and provide basic theories for the development of novel antifungal agents.

Deoxynivalenol (DON) is the most widespread mycotoxin contaminant hazardous to human and animal health globally. It acts as a crucial virulence factor to stimulate the spread of pathogenic Fusarium within wheat plants. Control of DON and Fusarium disease contributes enormously to food safety, which relies on chemical fungicides. Here, we report the biodegradation of DON using a novel soil bacterium, Devosia insulae FS10-7, and its biocontrol effect against Fusarium crown rot. We demonstrated that strain FS10-7 degraded DON to 3-epi-DON by forming a 3-keto-DON intermediate. Such degradation activity can be maintained at a wide range of pH (4 to 10) and temperature (16 to 42°C) values under aerobic conditions. Notably, strain FS10-7 exhibited practical inhibitory effects on Fusarium crown rot disease caused by F. graminearum and F. pseudograminearum in the in vitro Petri dish test under laboratory conditions and the pot experiment under greenhouse conditions. The mechanisms underlying the biocontrol ability of strain FS10-7 were preliminarily investigated to be associated with its high DON-degrading activity rather than direct antagonism. These results establish the foundation to develop further bioagents capable of biodegrading mycotoxins in cereals and derived products and, accordingly, biocontrol plant diseases caused by DON-producing pathogens.

Fusarium head blight (FHB) and the presence of mycotoxin deoxynivalenol (DON) pose serious threats to wheat production and food safety worldwide. DON, as a virulence factor, is crucial for the spread of FHB pathogens on plants. However, germplasm resources that are naturally resistant to DON and DON-producing FHB pathogens are inadequate in plants. Here, detoxifying bacteria genes responsible for DON epimerization were used to enhance the resistance of wheat to mycotoxin DON and FHB pathogens. We characterized the complete pathway and molecular basis leading to the thorough detoxification of DON via epimerization through two sequential reactions in the detoxifying bacterium Devosia sp. D6-9. Epimerization efficiently eliminates the phytotoxicity of DON and neutralizes the effects of DON as a virulence factor. Notably, co-expressing of the genes encoding quinoprotein dehydrogenase (QDDH) for DON oxidation in the first reaction step, and aldo-keto reductase AKR13B2 for 3-keto-DON reduction in the second reaction step significantly reduced the accumulation of DON as virulence factor in wheat after the infection of pathogenic Fusarium, and accordingly conferred increased disease resistance to FHB by restricting the spread of pathogenic Fusarium in the transgenic plants. Stable and improved resistance was observed in greenhouse and field conditions over multiple generations. This successful approach presents a promising avenue for enhancing FHB resistance in crops and reducing mycotoxin contents in grains through detoxification of the virulence factor DON by exogenous resistance genes from microbes.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic messenger RNAs. m6A was discovered in wheat about 40 years ago; however, its potential roles in wheat remain unknown. In this study, we profiled m6As in spikelets transcriptome at the flowering stage of hexaploid wheat and found that m6As are evenly distributed across the A, B, and D subgenomes but their extents and locations vary across homeologous genes. m6As are enriched in homeologous genes with close expression levels and the m6A methylated genes are more conserved. The extent of m6A methylation is negatively correlated with mRNA expression levels and its presence on mRNAs has profound impacts on mRNA translation in a location-dependent manner. Specifically, m6As within coding sequences and 3′UTRs repress the translation of mRNAs while the m6As within 5′UTRs and start codons could promote it. The m6A-containing mRNAs are significantly enriched in processes and pathways of “translation” and “RNA transport,” suggesting the potential role of m6As in regulating the translation of genes involved in translation regulation. Our data also show a stronger translation inhibition by small RNAs (miRNA and phasiRNA) than by m6A methylation, and no synergistical effect between the two was observed. We propose a secondary amplification machinery of translation regulation triggered by the changes in m6A methylation status. Taken together, our results suggest translation regulation as a key role played by m6As in hexaploid wheat.

Aspergillus flavus and the produced aflatoxins cause great hazards to food security and human health across all countries. The control of A. flavus and aflatoxins in grains during storage is of great significance to humans. In the current study, bacteria strain YM6 isolated from sea sediment was demonstrated effective in controlling A. flavus by the production of anti-fungal volatiles. According to morphological characteristics and phylogenetic analysis, strain YM6 was identified as Pseudomonas stutzeri. YM6 can produce abundant volatile compounds which could inhibit mycelial growth and conidial germination of A. flavus. Moreover, it greatly prevented fungal infection and aflatoxin production on maize and peanuts during storage. The inhibition rate was 100%. Scanning electron microscopy further supported that the volatiles could destroy the cell structure of A. flavus and prevent conidia germination on the grain surface. Gas chromatography/mass spectrometry revealed that dimethyl trisulfide (DMTS) with a relative abundance of 13% is the most abundant fraction in the volatiles from strain YM6. The minimal inhibitory concentration of DMTS to A. flavus conidia is 200 µL/L (compound volume/airspace volume). Thus, we concluded that Pseudomonas stutzeri YM6 and the produced DMTS showed great inhibition to A. flavus, which could be considered as effective biocontrol agents in further application.

Controlling the devastating fungal pathogen Fusarium graminearum (Fg) is a challenge due to inadequate resistance in nature. Here, we report on the identification of RNAi molecules and their applications for controlling Fg in wheat through silencing chitin synthase 7 (Chs7), glucan synthase (Gls) and protein kinase C (Pkc). From transgenic Fg strains four RNAi constructs from Chs7 (Chs7RNAi−1, −2, −3, and −4), three RNAi constructs from Gls (GlsRNAi−2, −3, and −6), and one RNAi construct from Pkc (PkcRNAi−5) were identified that displayed effective silencing effects on mycelium growth in medium and pathogenicity in wheat spikes. Transcript levels of Chs7, Gls and Pkc were markedly reduced in those strains. Double-strand RNAs (dsRNAs) of three selected RNAi constructs (Chs7RNAi-4, GlsRNAi-6 and PkcRNA-5) strongly inhibited mycelium growth in vitro. Spray of those dsRNAs on detached wheat leaves significantly reduced lesion sizes; the independent dsRNAs showed comparable effects on lesions with combination of two or three dsRNAs. Expression of three targets Chs7, Gls, and Pkc was substantially down-regulated in Fg-infected wheat leaves. Further application of dsRNAs on wheat spikes in greenhouse significantly reduced infected spikelets. The identified RNAi constructs may be directly used for spray-induced gene silencing and stable expression in plants to control Fusarium pathogens in agriculture.

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium–wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, 67HXXXG71, and exhibits DON affinity with a dissociation constant (KD) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC50 values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium–wheat interactions.

The major causal agents Fusarium graminearum (F. graminearum) and Fusarium asiaticum could produce multiple mycotoxins in infected wheat, which threatens the health of humans and animals. Specifically, deoxynivalenol (DON) and its derivatives 3- and 15-acetyldeoxynivalenol (3-ADON and 15-ADON) are commonly detected mycotoxins in cereal grains. However, the good chromatographic separation of 3-ADON and 15-ADON remains challenging. Here, an LC-MS/MS method for the chemotype determination of Fusarium strains was developed and validated. 3- and 15-ADON could be separated chromatographically in this study with sufficiently low limits of detection (LODs; 4 μg/kg) and limits of quantification (LOQs; 8 μg/kg). The satisfying intraday and interday reproducibility (both %RSDr and %RSDR were <20%) of this method indicated good stability. The recoveries of all analytes were in the range of 80–120%. In addition, three F. graminearum complex (FGC) strains, i.e., PH-1 (chemotype 15-ADON), F-1 (chemotype 3-ADON) and 5035 (chemotype 15-ADON), were selected to verify the accuracy of the method in differentiating phenotypes. The validation results showed that this LC-MS/MS method based on sample pretreatment is effective and suitable for the chromatographic separation of 3-ADON and 15-ADON in wheat.

转基因技术是现代生物技术的核心,也是当今最易引起公众争论的社会议题之一.为了全面了解高校学生对转基因生物(genetically modified organisms,GMO)的认知现状,本研究对我国31个省/市174所高校的1085名学生进行了问卷调查与分析.发现目前高校学生了解转基因的主要渠道依次为电脑(或手机)网络>电视>报刊杂志>日常交流>课程或讲座.高校学生支持/反对转基因的比例(3.22/1),是普通公众支持/反对转基因之比(0.29/1)的11倍;支持转基因与学历、学科、专业知识高度相关.在购物时,近55％的高校学生不会关注是否为转基因食品,约36.6％愿意或按需购买转基因食品,50.6％会综合考虑"性价比"等因素后再做决定,12.8％拒绝或抵制购买.同时,近1/4非常看好我国转基因产业化发展前景,1/10不看好.基于这些调查分析,本研究对目前高校的转基因科普宣传及相关通识课程设置与改革提出了一些思考和建议,还概述了我国转基因作物研发和产业发展的新态势,并提出一些对策,有助于有序推进我国转基因产业化的健康发展.

The Fusarium mycotoxin deoxynivalenol (DON) is typically controlled by fungicides. Here, we report DON detoxification using enzymes from the highly active Devosia strain D6-9 which degraded DON M 2.5 mu g/min/10(8) cells. Strain D6-9 catabolized DON to 3-keto-DON and 3-epi-DON, completely removing DON in wheat. Genome analysis of three Devosia strains (D6-9, D17, and D13584), with strain D6-9 transcriptomes, identified three genes responsible for DON epimerization. One gene encodes a quinone-dependent DON dehydrogenase QDDH which oxidized DON into 3-keto-DON. Two genes encode the NADPH-dependent aldo/keto reductases AKR13B2 and AKR6D1 that convert 3-keto-DON into 3-epi-DON. Recombinant proteins expressed in Escherichia coll. efficiently degraded DON in wheat grains. Molecular docking and site-directed mutagenesis revealed that residues S497, E499, and E535 function in QDDH's DON-oxidizing activity. These results advance potential microbial and enzymatic elimination of DON in agricultural samples and lend insight into the underlying mechanisms and molecular evolution of DON detoxification.

Trichothecenes are the most common mycotoxins contaminating small grain cereals worldwide. The C12,13 epoxide group in the trichothecenes was identified as a toxic group posing harm to humans, farm animals, and plants. Aerobic biological de-epoxidation is considered the ideal method of controlling these types of mycotoxins. In this study, we isolated a novel trichothecene mycotoxin-de-epoxidating bacterium, Desulfitobacterium sp. PGC-3-9, from a consortium obtained from the soil of a wheat field known for the occurrence of frequent Fusarium head blight epidemics under aerobic conditions. Along with MMYPF media, a combination of two antibiotics (sulfadiazine and trimethoprim) substantially increased the relative abundance of Desulfitobacterium species from 1.55% (aerobic) to 29.11% (aerobic) and 28.63% (anaerobic). A single colony purified strain, PGC-3-9, was isolated and a 16S rRNA sequencing analysis determined that it was Desulfitobacterium. The PGC-3-9 strain completely de-epoxidated HT-2, deoxynivalenol (DON), nivalenol and 15-acetyl deoxynivalenol, and efficiently eliminated DON in wheat grains under aerobic and anaerobic conditions. The strain PGC-3-9 exhibited high DON de-epoxidation activity at a wide range of pH (6-10) and temperature (15-50 °C) values under both conditions. This strain may be used for the development of detoxification agents in the agriculture and feed industries and the isolation of de-epoxidation enzymes.

Graphene oxide (GO), which has been emerging as a novel antimicrobial agent against plant pathogens, has aroused great research interest and is considered as highly promising in agricultural science.

The contamination of food with Aspergillus flavus and subsequent aflatoxins is considered as one of the most severe safety problems in the world. The application of microorganisms and the produced bio-active compounds is considered as the most promising method for controlling foodborne pathogens and mycotoxins contamination both in pre- and post-harvest. Vt-7, identified as Enterobacter asburiae, was able to completely inhibit the growth of Aspergillus flavus (AF) and other seven important fungal pathogens by the production of volatiles. Additionally, it can also significantly inhibited AF infection on peanuts in storage, down-regulated the gene expression of aflatoxin biosynthesis and eventually prevented aflatoxins production. Scanning electron microscope further proved that Vt-7 volatiles prevented conidia germination of AF on peanut surface, and severely destroyed the conidia structure. Gas chromatography - tandem mass spectrometry revealed that two abundant compounds (1-Pentanol and Phenylethyl Alcohol) were involved in the volatile profiles. They showed great antagonistic activity against AF with minimal inhibitory concentration at 200 mu L/L. Therefore, E. asburiae Vt-7, and volatiles 1-Pentanol and Phenylethyl Alcohol were effective agents in controlling AF and aflatoxins in peanut during storage. They will provide novel strategies for the application of microbe and bio-active compounds against fungal pathogens and mycotoxins in food and grains during storage.

Controlling aflatoxigenic Aspergillus flavus and aflatoxins (AFs) in grains and food during storage is a great challenge to humans worldwide. Alcaligenes faecalis N1-4 isolated from tea rhizosphere soil can produce abundant antifungal volatiles, and greatly inhibited the growth of A. flavus in un-contacted face-to-face dual culture testing. Gas chromatography tandem mass spectrometry revealed that dimethyl disulfide (DMDS) and methyl isovalerate (MI) were two abundant compounds in the volatile profiles of N1-4. DMDS was found to have the highest relative abundance (69.90%, to the total peak area) in N1-4, which prevented the conidia germination and mycelial growth of A. flavus at 50 and 100 μL/L, respectively. The effective concentration for MI against A. flavus is 200 μL/L. Additionally, Real-time quantitative PCR analysis proved that the expression of 12 important genes in aflatoxin biosynthesis pathway was reduced by these volatiles, and eight genes were down regulated by 4.39 to 32.25-folds compared to control treatment with significant differences. And the A. flavus infection and AFs contamination in groundnut, maize, rice and soybean of high water activity were completely inhibited by volatiles from N1-4 in storage. Scanning electron microscope further proved that A. flavus conidia inoculated on peanuts surface were severely damaged by volatiles from N1-4. Furthermore, strain N1-4 showed broad and antifungal activity to other six important plant pathogens including Fusarium graminearum, F. equiseti, Alternaria alternata, Botrytis cinerea, Aspergillus niger, and Colletotrichum graminicola. Thus, A. faecalis N1-4 and volatile DMDS and MI may have potential to be used as biocontrol agents to control A. flavus and AFs during storage.

SummaryMicroRNA‐like RNAs (milRNAs) post‐transcriptionally down‐regulate target genes. We investigated Fusarium graminearum (Fg) milRNA expression during fungal vegetative growth and infection of wheat. Small RNA sequencing identified 36 milRNAs from Fg, one of which, Fgmil‐2, had >100 transcripts per million in conidia, mycelia and infected wheat, with the highest expression in conidia and the lowest expression in colonized wheat tissue. Fgmil‐2 displays perfect homology to the 3ʹ‐untranslated region (3ʹ‐UTR) of an FgbioH1 messenger RNA that is involved in biotin biosynthesis. Poly(A) polymerase‐mediated rapid amplification of cDNA ends combined with sequencing analysis demonstrated that cleavage at a specific site by FgDicer2 in the 3ʹ‐UTR of FgbioH1 transcripts generated the Fgmil‐2 precursor with a typical hairpin structure. Deletion of FgbioH1 or FgDicer2 genes abolished Fgmil‐2 biogenesis. FgbioH1 had an inversely correlated pattern of expression to that of Fgmil‐2 and FgDicer2. Deletion of FgbioH1 also showed that it is required for mycelial growth, virulence, mycotoxin biosynthesis and expression of biotin‐dependent carboxylase genes. This study reveals in Fg a novel mode of inversely correlated post‐transcriptional regulation in which Fgmil‐2 originates from its own target transcript, FgbioH, to govern biotin biosynthesis.

Fusarium head blight (FHB) mainly resulting from Fusarium graminearum (Fg) Schwabe is a notorious wheat disease causing huge losses in wheat production globally. Fg also produces mycotoxins, which are harmful to human and domestic animals. In our previous study, we obtained two Fg mutants, TPS1- and TPS2-, respectively, with a single deletion of trehalose 6-phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPS2) compared with the wild type (WT). Both mutants were unable to synthesize trehalose and produced fewer mycotoxins. To understand the other biochemical changes induced by TPS gene deletion in Fg, we comprehensively analyzed the metabolomic differences between TPS- mutants and the WT using NMR together with gas chromatography-flame ionization detection/mass spectrometry. The expression of some relevant genes was also quantified. The results showed that TPS1- and TPS2- mutants shared some common metabolic feature such as decreased levels for trehalose, Val, Thr, Lys, Asp, His, Trp, malonate, citrate, uridine, guanosine, inosine, AMP, C10:0, and C16:1 compared with the WT. Both mutants also shared some common expressional patterns for most of the relevant genes. This suggests that apart from the reduced trehalose biosynthesis, both TPS1 and TPS2 have roles in inhibiting glycolysis and the tricarboxylic acid cycle but promoting the phosphopentose pathway and nucleotide synthesis; the depletion of either TPS gene reduces the acetyl-CoA-mediated mycotoxin biosynthesis. TPS2- mutants produced more fatty acids than TPS1- mutants, suggesting different roles for TPS1 and TPS2, with TPS2- mutants having impaired trehalose biosynthesis and trehalose 6-phosphate accumulation. This may offer opportunities for developing new fungicides targeting trehalose biosynthesis in Fg for FHB control and mycotoxin reduction in the FHB-affected cereals.

脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)是由镰刀菌在侵染小麦等禾谷类作物过程中产生的一种有毒次级代谢产物,是目前小麦及其制品中污染最为普遍的一种真菌毒素.DON能够对真核细胞产生多种毒性作用,严重危害人畜健康.DON又是一种毒力因子,促进镰刀菌扩展蔓延,加重赤霉病发病程度.利用脱毒菌、脱毒酶对DON毒素进行生物脱毒是最好的脱毒方式之一,其可将DON转化成低毒或无毒代谢产物,减少毒素对人畜健康的危害.脱毒基因还可作为新型抗源用于小麦赤霉病抗性改良,加速抗性品种的选育,从源头防止DON毒素的污染.本文概述了DON毒素生物脱毒的类型、代谢产物的毒性、脱毒基因的鉴定以及脱毒材料的应用等方面的研究进展,以期为DON毒素的生物防控和小麦赤霉病抗性改良提供参考.

Fusarium head blight disease resulting from Fusarium graminearum (FG) infection causes huge losses in global production of cereals and development of FG-resistant plants is urgently needed. To understand biochemistry mechanisms for FG resistance, here, we have systematically investigated the plant metabolomic phenotypes associated with FG resistance for transgenic Arabidopsis thaliana expressing a class-I chitinase (Chi), a Fusarium-specific recombinant antibody gene (CWP2) and fused Chi-CWP2. Plant disease indices, mycotoxin levels, metabonomic characteristics, and expression levels of several key genes were measured together with their correlations. We found that A. thaliana expressing Chi-CWP2 showed higher FG resistance with much lower disease indices and mycotoxin levels than the wild-type and the plants expressing Chi or CWP2 alone. The combined metabonomic and quantitative RT-PCR analyses revealed that such FG-resistance was closely associated with the promoted biosynthesis of secondary metabolites (phenylpropanoids, alkanoids) and organic osmolytes (proline, betaine, glucose, myo-inositol) together with enhanced TCA cycle and GABA shunt. These suggest that the concurrently enhanced biosyntheses of the shikimate-mediated secondary metabolites and organic osmolytes be an important strategy for A. thaliana to develop and improve FG resistance. These findings provide essential biochemical information related to FG resistance which is important for developing FG-resistant cereals.