Development and validation of dry reagent time-resolved fluoroimmuno assays for zeranol and alpha-zearalenol to assist in distinguishing zeranol abuse from Fusarium spp. toxin contamination in bovine urine

Zeranol, an oestrogenic growth promoter in food animals, is banned within the European Union (EU). However, commercially available immunoassay kits for zeranol cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the validation of a specificity enhanced, rapid dry reagent time-resolved fluoroimmunoassay (TR-FIA) for zeranol (recovery 99%, limit of detection 1.3 ng ml(-1)) demonstrating that up to 150 ng ml(-1) of Fusarium spp. toxins in urine do not lead to false-positive results. This assay will assist EU Member States to implement Council Directive 961 23\EC, which requires states to monitor for potential abuses of zeranol. A similar TR-FIA for the Fusarium spp. toxin a-zearalenol, using the same sample extract, is also described (recovery 68%, limit of detection 5.6 ng ml(-1)). Only the addition of diluted sample extract is required to perform these dry-reagent TR-FIAs, the results being available within 1 h of extract application. The EU-funded project 'Natural Zeranol' (FAIR5-CT97-3443) will use these fluoroimmunoassays to screen bovine urine in four Member States to gather data on the seasonality of Fusarium spp. toxin contamination of urine and the incidence of zeranol screening test positives.

A pilot survey for Fusarium mycotoxin biomarkers in women from Golestan, northern Iran

Fusarium mycotoxins are frequent contaminants of cereals in many world regions, and are suggested risk factors for various acute and chronic human diseases. To date a lack of exposure tools has restricted epidemiological studies of the potential health effects. Recently established exposure biomarkers for deoxynivalenol (DON) and fumonisins are now available and here a pilot biomarker survey of 110 women (aged 39 to 72 years) from Golestan, northern Iran was conducted on samples collected at one time point during August-September 2007. Urinary DON and DON-glucuronide combined were detected frequently (79/110, 72%), mean 1.3 ng DON/ml urine, range not detected (nd)-6.5 ng/ml; mean creatinine adjusted levels were 1.5 ng DON/mg creatinine, range nd-7.1 ng/mg). Neither urinary de-epoxy DON (DOM-1) and DOM-1 glucuronide combined, nor urinary fumonisin B-1 were detected. This study is the first reported biomarker based exposure assessment of DON and fumonisins in this region. Overall DON exposure at this time point appears modest compared to other world regions where data are available.

Anther Extrusion and Fusarium Head Blight Resistance in European Wheat

Anther extrusion has been widely discussed as a factor influencing fusarium head blight (FHB) resistance in wheat. This is despite a paucity of quantitative information on its importance, between cultivars, in contrast to that for heading date and plant height. We describe a method applicable to a plant breeding
situation at 10 days postanthesis, for assessing the distinct characteristics of anther retention (anthers held within the spikelet) and trapped anthers (partially
extruded and trapped between the lemma and palea of the wheat spikelet). FHB resistance was tested in field experiments in 2004 and 2005. In these experiments designed to resemble applications to a plant breeding selection scheme anther retention was significantly correlated with FHB in 2004 (r = 0.26; P < 0.05) and 2005 (r = 0.26; P < 0.05). A higher proportion of anthers retained relating, albeit weakly, with increased FHB susceptibility in European wheat.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells.

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.

Distribución de cepas de fusarium moniliforme productoras de fumonisina en B1 en maíz, cultivado en el estado de Nuevo León

Tesis (Maestría en Ciencias con Especialidad en microbiología) UANL

Micorrización de Pinus greggii Engelm. como una medida de prevención del establecimiento de tres cepas de Fusarium oxysporum Schlecht. causantes de damping-off

Tesis (Maestría en Ciencias Forestales) U.A.N.L.

Supresión de Fusarium moniliforme por Bacillus thuringienses

Tesis (Doctorado en Ciencias con Especialidad en Microbiología) UANL

Determinación del efecto inhibitorio del aceite esencial y diferentes extractos de orégano (lippia berlandieri Schauer) sobre el crecimiento de fusarium oxysporum de tanto in vitro como en plántula de tomate.

Tesis (Doctor en Ciencias Biológicas con Acentuación en Alimentos) UANL, 2010.

Antifungal activity of Bacillus subtilis filtrate to control Fusarium oxysporum f.sp. lentis, the causal organism of lentil vascular wilt

In dual cultures, the supernatant filtrate of the biological control agent Bacillus subtilis was evaluated against (Fusarium oxysporum f.sp. lentis) the causal organism of lentil vascular wilt. The antagonistic activity was evaluated as percent reduction of fungal growth (certainly due, in part, to the antifungal metabolites produced by the antagonistic bacterium). The in-vitro experiments showed that B. subtilis filtrate, whether solid or liquid media, had a strong inhibiting activity on the spore germination and mycelial growth of F. oxysporum f. sp. lentis. In a glasshouse experiment, soil was drenched with B. subtilis filtrate at 30 ml/kg (vol/wt) around seedlings of a susceptible lentil line (ILL 4605). In this treatment there was only 31% mortality compared with 100% kill of plants in the control treatment (P≤0.05).

Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of lentil vascular wilt caused by Fusarium oxysporum f. sp lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log(10) CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log(10) CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc-based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.

Antagonistic potential of bacterial isolates associated with entomopathogenic nematodes against tomato wilt caused by Fusarium oxysporum f.sp., Lycopersici under greenhouse conditions

Three concentrations of Xenorhabdus nematophila and Xenorhabdus spp., (4x10(5,) 4x10(6,) 4x10(7) cells/ml) were evaluated in the laboratory and in pot experiments to test their antagonistic effects on Fusarium oxysporum f.sp., lycopersici. All concentrations effectively inhibited its growth on agar plates. In soil under greenhouse conditions treatments with each bacterium at 4x10(7) cells/ml reduced the disease incidence of tomato by up to 40.38 and 47.54% respectively and there were significant increases of plant biomass by 198 and 211% respectively. The rhizosphere population of Fusarium oxysporum f.sp., lycopersici was reduced by 97%. The Xenorhabdus spp., was comparatively more effective than X. nematophila.