Bacterial Epimerization as a Route for Deoxynivalenol Detoxification: the Influence of Growth and Environmental Conditions.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by several Fusarium species that infest wheat and corn. Food and feed contaminated with DON pose a health risk to both humans and livestock and form a major barrier for international trade. Microbial detoxification represents an alternative approach to the physical and chemical detoxification methods of DON-contaminated grains. The present study details the characterization of a novel bacterium, Devosia mutans 17-2-E-8, that is capable of transforming DON to a non-toxic stereoisomer, 3-epi-deoxynivalenol under aerobic conditions, mild temperature (25-30°C), and neutral pH. The biotransformation takes place in the presence of rich sources of organic nitrogen and carbon without the need of DON to be the sole carbon source. The process is enzymatic in nature and endures a high detoxification capacity (3 µg DON/h/10(8) cells). The above conditions collectively suggest the possibility of utilizing the isolated bacterium as a feed treatment to address DON contamination under empirical field conditions.

Toxicology of 3-epi-deoxynivalenol, a deoxynivalenol-transformation product by Devosia mutans 17-2-E-8.

Microbial detoxification of deoxynivalenol (DON) represents a new approach to treating DON-contaminated grains. A bacterium Devosia mutans 17-2-E-8 was capable of completely transforming DON into a major product 3-epi-DON and a minor product 3-keto-DON. Evaluation of toxicities of these DON-transformation products is an important part of hazard characterization prior to commercialization of the biotransformation application. Cytotoxicities of the products were demonstrated by two assays: a MTT bioassay assessing cell viability and a BrdU assay assessing DNA synthesis. Compared with DON, the IC50 values of 3-epi-DON and 3-keto-DON were respectively 357 and 3.03 times higher in the MTT bioassay, and were respectively 1181 and 4.54 times higher in the BrdU bioassay. Toxicological effects of 14-day oral exposure of the B6C3F1 mouse to DON and 3-epi-DON were also investigated. Overall, there were no differences between the control (free of toxin) and the 25 mg/kg bw/day or 100 mg/kg bw/day 3-epi-DON treatments in body and organ weights, hematology and organ histopathology. However, in mice exposed to DON (2 mg/kg bw/day), white blood cell numbers and serum immunoglobulin levels were altered relative to controls, and lesions were observed in adrenals, thymus, stomach, spleen and colon. Taken together, in vitro and in vivo studies indicate that 3-epi-DON is substantially less toxic than DON.

An epimer of deoxynivalenol: purification and structure identification of 3-epi-deoxynivalenol.

In an investigation of deoxynivalenol (DON)-transformation products by Devosia mutans 17-2-E-8, the major product was identified as 3-epi-DON. This DON-transformation product was analysed by liquid chromatography and identified by congruent retention time and UV/Vis spectrum, as well as mass spectrometric data. Nuclear magnetic resonance (NMR) experiments including correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC) and nuclear overhauser effect (NOE) were conducted for structural characterisation of 3-epi-DON. High-speed counter-current chromatography (HSCCC) was applied to scale up the separation of 3-epi-DON from DON in a D. mutans 17-2-E-8 culture. From the culture where 100 mg DON was applied, 56 mg of 3-epi-DON (purity of 96.8%) was obtained from the HSCCC. The purified 3-epi-DON will be used for toxicological characterisation studies of this chemical.

Purification of deoxynivalenol from Fusarium graminearum rice culture and mouldy corn by high-speed counter-current chromatography.

Deoxynivalenol (DON) is a mycotoxin produced by several Fusarium species and is toxic to a wide range of organisms, including human beings and livestock. To produce large amounts of pure DON for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Rice cultured with Fusarium graminearum and field mouldy corn infected by F. graminearum were extracted with methanol and found to contain 1.16 and 1.30 mg DON/g, respectively. The extracts were concentrated and then separated using a biphasic solvent system consisting of ethyl acetate-water (1:1, v/v). Collected fractions were analyzed by high-performance liquid chromatography (HPLC) and identified by congruent retention time and UV/vis spectrum and mass spectrometric data. Fractions containing DON were combined and freeze-dried. This method produced 116 mg and 65 mg DON with a purity of greater than 94.9% from 200 g of the rice culture and the mouldy corn, respectively. The HSCCC method had a recovery rate of DON at 88% from the crude extracts of both samples. This one-step purification method provided a simple and effective tool for obtaining a large amount of DON, an essential material for studies related to toxicology and detoxification of this mycotoxin.

Lateral Clipping of Canopy Influences the Microclimate and Development of Apothecia of Sclerotinia sclerotiorum in Carrots.

Four canopy management treatments were evaluated in carrot (Daucus carota) production in Bradford Marsh, Ontario, in 2001 and 2002: (i) unclipped control; (ii) unclipped canopy with manual removal of collapsed senescing leaves at 2-week intervals following the first appearance on the soil; (iii) lateral clipping of the canopy at the initial emergence of apothecia, leaving the debris in the furrow, and (iv) lateral clipping of the canopy with manual removal of the debris from the furrow. Clipping reduced the canopy width by ca. 20% on both sides of the carrot bed by cutting off overlapping leaves above the furrow and senescing foliage on the soil surface. Maximum air and soil temperatures were up to 9.2 and 3.1°C lower, respectively, and relative humidity was up to 30% higher in unclipped canopies than in clipped canopies. The total number of apothecia in clipped plots was reduced by 74 and 76% compared with unclipped plots in 2001 and 2002, respectively. Canopy clipping reduced the quantity of apothecia in the crop by creating an unfavorable microclimate for the development of S. sclerotiorum without affecting the fresh foliar and root weights of carrot at harvest. The presence of clipped foliar debris in the furrow affected the number of apothecia in 2001; however, apothecia under the debris are unlikely to contribute to the overall inoculum in the crop. Lateral clipping also appeared to control Sclerotinia rot of carrot where it occurred (in 2001). This is the first report that documents and quantifies the effects of canopy architecture on the microclimate, development of apothecia of S. sclerotiorum, and Sclerotinia rot in carrot crops.

Evaluation of Composts for Suppression of Dollar Spot (Sclerotinia homoeocarpa) of Turfgrass.

The use of composts in turfgrass disease management allows for a reduction in pesticide use in chemical control practices. Disease suppressive properties of composts rely on a number of factors including microbial activity, microbial population dynamics, nutrient concentrations, and other associated chemical and physical factors. Five composts were evaluated for suppression of dollar spot caused by Sclerotinia homoeocarpa. The dollar spot disease suppressive properties of selected compost formulations prepared in different years was evaluated. A third objective was to examine the effects of storage of compost (1 year) on the suppression of dollar spot. Field experiments were conducted in 1998 with compost prepared in 1997 to 1998. Applications of compost every 3 weeks throughout the season suppressed dollar spot of turf to levels not significantly different than applications of chlorothalonil fungicide applied at the manufacturer's lowest recommended preventative rate of 38.4 ml a.i./100 m2 every 2 weeks (P = 0.05). Single applications of composts at the start of the 1998 season were not effective in reducing disease. Field experiments in 1999 evaluated batches of two selected compost formulations, one batch produced in 1998 to 1999, another stored since production in 1997 to 1998. Composts were effective in suppressing disease to levels not significantly different than the fungicide controls, which showed up to 33% disease in 1998 and up to 31% disease in 1999 (P = 0.05). Storage of composts for up to 1 year did not affect their ability to reduce dollar spot severity (P = 0.05). The use of composts as plant disease suppressants is not likely to replace the use of commercial fungicides in dollar spot management. However, multiple applications of compost may reduce incidence and severity of dollar spot to levels at which chemical control may be reduced or eliminated for a significant portion of the season.