Detoxifying bacterial genes for deoxynivalenol epimerization confer durable resistance to Fusarium head blight in wheat.

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.

A prospective study of total urinary deoxynivalenol in adolescents in Shanghai, China.

There is growing interest in deoxynivalenol (DON) exposure during puberty because experimental evidence shows that DON-exposed to adolescents are more sensitive to DON and have limited detoxification ability. Nevertheless, there have been few surveys of DON exposure for adolescents in China. Furthermore, little is known about the effects of collection times on risk exposure. In the study, we estimated the risk of DON exposure for adolescents in Shanghai and explore the effects of collection time, areas, sex and BMI on intake estimates. A total of 315 adolescents aged 14-16 years, including 161 boys and 154 girls, were recruited. Urine samples were collected for three consecutive days and digested with ß-glucuronidase and then purified using a DON-immunoaffinity column (IAC). Total deoxynivalenol levels were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with a stable isotope dilution assay (SIDA). DON was detected in 945 morning urine samples taken from 315 individuals for three consecutive days. The mean estimated dietary intake of DON did not exceed the tolerable daily intake (TDI) of l µg/kg·bw/day, showing that exposure of adolescents in Shanghai is not of concern. However, 10-20% of probable daily intake (PDI) values exceed the TDI, indicating potential adverse effects. In addition, the DON concentration at the population level did not differ for urine samples collected at different times except for those of overweight adolescents. Therefore, assessment of exposure to DON by monitoring the morning urine of a healthy adolescent, except for overweight people, provides an appropriate estimate of exposure and related risk at the population level, but intake estimates for individuals are uncertain; these could be used to assess exposure of adolescents to DON rapidly and effectively for epidemiological investigations.

A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins.

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.

Novel Soil Bacterium Strain Desulfitobacterium sp. PGC-3-9 Detoxifies Trichothecene Mycotoxins in Wheat via De-Epoxidation under Aerobic and Anaerobic Conditions.

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.

A quinone-dependent dehydrogenase and two NADPH-dependent aldo/keto reductases detoxify deoxynivalenol in wheat via epimerization in a Devosia strain.

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 at 2.5 µg/min/108 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 coli 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.

Variation in the Microbiome, Trichothecenes, and Aflatoxins in Stored Wheat Grains in Wuhan, China.

Contamination by fungal and bacterial species and their metabolites can affect grain quality and health of wheat consumers. In this study, sequence analyses of conserved DNA regions of fungi and bacteria combined with determination of trichothecenes and aflatoxins revealed the microbiome and mycotoxins of wheat from different silo positions (top, middle, and bottom) and storage times (3, 6, 9, and 12 months). The fungal community in wheat on the first day of storage (T0) included 105 classified species (81 genera) and 41 unclassified species. Four species had over 10% of the relative abundance: Alternaria alternata (12%), Filobasidium floriforme (27%), Fusarium graminearum (12%), and Wallemia sebi (12%). Fungal diversity and relative abundance of Fusarium in wheat from top silo positions were significantly lower than at other silo positions during storage. Nivalenol and deoxynivalenol in wheat were 13⁻34% higher in all positions at 3 months compared to T0, and mycotoxins in wheat from middle and bottom positions at 6 to 12 months were 24⁻57% higher than at T0. The relative abundance of toxigenic Aspergillus and aflatoxins were low at T0 and during storage. This study provides information on implementation and design of fungus and mycotoxin management strategies as well as prediction models.

An aldo-keto reductase is responsible for Fusarium toxin-degrading activity in a soil Sphingomonas strain.

Degradation of toxins by microorganisms is a promising approach for detoxification of agricultural products. Here, a bacterial strain, Sphingomonas S3-4, that has the ability to degrade the mycotoxin deoxynivalenol (DON) was isolated from wheat fields. Incubation of Fusarium-infected wheat grains with S3-4 completely eliminated DON. In S3-4 DON is catabolized into compounds with no detectable phytotoxicity, 3-oxo-DON and 3-epi-DON, via two sequential reactions. Comparative analysis of genome sequences from two DON-degrading strains, S3-4 and Devosia D17, and one non-DON-degrading strain, Sphingobium S26, combined with functional screening of a S3-4 genomic BAC library led to the discovery that a novel aldo/keto reductase superfamily member, AKR18A1, is responsible for oxidation of DON into 3-oxo-DON. DON-degrading activity is completely abolished in a mutant S3-4 strain where the AKR18A1 gene is disrupted. Recombinant AKR18A1 protein expressed in Escherichia coli catalyzed the reversible oxidation/reduction of DON at a wide range of pH values (7.5 to 11) and temperatures (10 to 50 °C). The S3-4 strain and recombinant AKR18A1 also catabolized zearalenone and the aldehydes glyoxal and methyglyoxal. The S3-4 strain and the AKR18A1 gene are promising agents for the control of Fusarium pathogens and detoxification of mycotoxins in plants and in food/feed products.

Aerobic De-Epoxydation of Trichothecene Mycotoxins by a Soil Bacterial Consortium Isolated Using In Situ Soil Enrichment.

Globally, the trichothecene mycotoxins deoxynivalenol (DON) and nivalenol (NIV) are among the most widely distributed mycotoxins that contaminate small grain cereals. In this study, a bacterial consortium, PGC-3, with de-epoxydation activity was isolated from soil by an in situ soil enrichment method. Screening of 14 soil samples that were sprayed with DON revealed that 4 samples were able to biotransform DON into de-epoxydized DON (dE-DON). Among these, the PGC-3 consortium showed the highest and most stable activity to biotransform DON into dE-DON and NIV into dE-NIV. PGC-3 exhibited de-epoxydation activity at a wide range of pH (5-10) and temperatures (20-37 °C) values under aerobic conditions. Sequential subculturing with a continued exposure to DON substantially reduced the microbial population diversity of this consortium. Analyses of the 16S rDNA sequences indicated that PGC-3 comprised 10 bacterial genera. Among these, one species, Desulfitobacterium, showed a steady increase in relative abundance, from 0.03% to 1.55% (a 52-fold increase), as higher concentrations of DON were used in the subculture media, from 0 to 500 µg/mL. This study establishes the foundation to further develop bioactive agents that can detoxify trichothecene mycotoxins in cereals and enables for the characterization of detoxifying genes and their regulation.

The Shewanella algae strain YM8 produces volatiles with strong inhibition activity against Aspergillus pathogens and aflatoxins.

Aflatoxigenic Aspergillus fungi and associated aflatoxins are ubiquitous in the production and storage of food/feed commodities. Controlling these microbes is a challenge. In this study, the Shewanella algae strain YM8 was found to produce volatiles that have strong antifungal activity against Aspergillus pathogens. Gas chromatography-mass spectrometry profiling revealed 15 volatile organic compounds (VOCs) emitted from YM8, of which dimethyl trisulfide was the most abundant. We obtained authentic reference standards for six of the VOCs; these all significantly reduced mycelial growth and conidial germination in Aspergillus; dimethyl trisulfide and 2,4-bis(1,1-dimethylethyl)-phenol showed the strongest inhibitory activity. YM8 completely inhibited Aspergillus growth and aflatoxin biosynthesis in maize and peanut samples stored at different water activity levels, and scanning electron microscopy revealed severely damaged conidia and a complete lack of mycelium development and conidiogenesis. YM8 also completely inhibited the growth of eight other agronomically important species of phytopathogenic fungi: A. parasiticus, A. niger, Alternaria alternate, Botrytis cinerea, Fusarium graminearum, Fusarium oxysporum, Monilinia fructicola, and Sclerotinia sclerotiorum. This study demonstrates the susceptibility of Aspergillus and other fungi to VOCs from marine bacteria and indicates a new strategy for effectively controlling these pathogens and the associated mycotoxin production during storage and possibly in the field.

A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains.

Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv-AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10(-2) µg mL(-1), superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10(-3) mg g(-1) of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food.

Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of Fusarium graminearum.

Based on the intergenic sequences of Tri5-Tri6 genes involved in the mycotoxin pathways of Fusarium species, a generic PCR assay was developed to detect a 300 bp fragment of deoxynivalenol (DON)-chemotypes and a 360 bp sequence of nivalenol (NIV)- chemotypes of Fusarium graminearum. Mycotoxin chemotypes identified by the PCR assays were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates from 12 provinces of China showed that 310 were DON-chemotypes and 54 were NIV-chemotypes. Sequence analyses revealed that DON-chemotypes display more variations than NIV-chemotypes. This PCR assay could be used to detect mycotoxin-producing Fusarium-species and may thus help to develop strategies to avoid or reduce mycotoxin contamination of cereals. Also this assay may provide useful alternatives to antibody-based mycotoxin tests.