Niels Henrik Spliid

Real-time PCR for quantification of eleven individual Fusarium species in cereals

Contamination of cereals with Fusarium species is one of the major sources of mycotoxins in food and feed. Quantification of biomass of Fusarium species is essential to understand the interactions of individual species in disease development. In this study quantitative real-time PCR assays based on the elongation factor 1 alpha (EF1alpha) gene for the 11 Fusarium species F. graminearum, F. culmorum, F. poae, F. langsethiae, F. sporotrichioides, F. equiseti, F. tricinctum, F. avenaceum, F. verticillioides, F. subglutinans and F. proliferatum were developed and tested on 24 wheat and 24 maize field samples. The assays were found to be specific and sensitive. Generally, the results from the quantitative real-time PCR assays corresponded well with mycotoxin data of the field samples.

Evaluation of chloride and pesticide transport in a fractured clayey till using large undisturbed columns and numerical modeling

Saturated groundwater flow and tracer experiments using fluorescent dye, chloride, and the herbicides mecoprop and simazine were carried out in the laboratory using three large-diameter (0.5 m) undisturbed columns of fractured clayey till. Hydraulic conductivity of the columns ranged from 10−5 m/s in the shallowest column (1 m dept)) to 10−7 m/s in the deepest column (4 m depth) and were similar to field-measured values for these deposits. Results of the tracer experiments are consistent with a conceptual model of advective transport along the fractures combined with diffusion into the fine-grained matrix between the fractures. Arrival of the chloride tracer in the effluent was highly retarded relative to fracture flow velocities calculated on the basis of the cubic law and measured values of fracture spacing and hydraulic conductivity. The herbicides were more strongly retarded than the chloride at low flow rates, but at higher flow rates the herbicides arrived with the chloride, indicating the influence of nonequilibrium sorption of the herbicides to fracture walls and the matrix solids. The columns were dismantled following the tracer experiments and mapping under UV light showed that nearly all of the visible, weathered fractures (and the few root holes in the case of the shallowest sample) were active transport pathways, with the dye appearing mainly on the fracture surfaces and as a “rim” in the adjacent matrix. Concentration profiles measured perpendicular to the fracture surfaces showed that the herbicides had also moved into the matrix, apparently by diffusion. Simulations of solute transport with a discrete fracture flow/matrix diffusion model showed that the simulations could be “fit” to the data if all of the visible fractures were hydraulically active, but could not be fit if all or most of the flow was channelled through just the primary fractures (defined by prominent oxidation stains). Simulations with an equivalent porous media (EPM) model could not fit the data using the measured total porosity as the effective porosity. The simulations could likely be fit with a smaller value of effective porosity, but this would limit applicability to field situations because fitted effective porosity is expected to change with physical scale and residence time of the solute in the soil.

Fusarium Head Blight of Cereals in Denmark: Species Complex and Related Mycotoxins

Quantitative real-time polymerase chain reaction differentiating 10 Fusarium spp. and Microdochium nivale or M. majus was applied to a total of 396 grain samples of wheat, barley, triticale, oat, and rye sampled across Denmark from 2003 to 2007, along with selected samples of wheat and barley from 1957 to 2000, to determine incidence and abundance of individual Fusarium spp. The mycotoxins deoxynivalenol (DON), nivalenol, zearalenone, T-2, and HT-2 were quantified using liquid chromatography-double mass spectrometry. Major differences in the Fusarium species complex among the five cereals as well as great yearly variation were seen. Fusarium graminearum, F. culmorum, and F. avenaceum were dominant in wheat, with DON as the dominant mycotoxin. F. langsethiae, F. culmorum, and F. avenaceum were dominant in barley and oat, leading to relatively high levels of the mycotoxins T-2 and HT-2. F. graminearum, F. culmorum, and F. avenaceum dominated in triticale and rye. The nontoxigenic M. nivale/majus were present in significant amounts in all cereal species. Wheat and barley samples from 1957 to 1996 exhibited no or very low amounts of F. graminearum, indicating a recent increase of this pathogen. Biomass and mycotoxin data exhibited good correlations between Fusarium spp. and their corresponding mycotoxins under field conditions.

Fate and availability of glyphosate and AMPA in agricultural soil

The fate of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) was studied in soil. Labeled glyphosate was used to be able to distinguish the measured quantities of glyphosate and AMPA from the background values since the soil was sampled in a field where glyphosate had been used formerly. After addition of labeled glyphosate, the disappearance of glyphosate and the formation and disappearance of AMPA were monitored. The resulting curves were fitted according to a new EU guideline. The best fit of the glyphosate degradation data was obtained using a first-order multi compartment (FOMC) model. DT50 values of 9 days (glyphosate) and 32 days (AMPA) indicated relatively rapid degradation. After an aging period of 6 months, the leaching risk of each residue was determined by treating the soil with pure water or a phosphate solution (pH 6), to simulate rain over a non-fertilized or fertilized field, respectively. Significantly larger (p < 0.05) amounts of aged glyphosate and AMPA were extracted from the soil when phosphate solution was used as an extraction agent, compared with pure water. This indicates that the risk of leaching of aged glyphosate and AMPA residues from soil is greater in fertilized soil. The blank soil, to which 252 g glyphosate/ha was applied 21 months before this study, contained 0.81 ng glyphosate/g dry soil and 10.46 ng AMPA/g dry soil at the start of the study. Blank soil samples were used as controls without glyphosate addition. After incubation of the blank soil samples for 6 months, a significantly larger amount of AMPA was extracted from the soil treated with phosphate solution than from that treated with pure water. To determine the degree of uptake of aged glyphosate residues by crops growing in the soil, 14C-labeled glyphosate was applied to soil 6.5 months prior to sowing rape and barley seeds. After 41 days, 0.006 ± 0.002% and 0.005 ± 0.001% of the applied radioactivity was measured in rape and barley, respectively.

Investigation of the effect of nitrogen on severity of Fusarium Head Blight in barley

The effect of nitrogen on Fusarium Head Blight (FHB) in a susceptible barley cultivar was investigated using gel-based proteomics. Barley grown with either 15 or 100kgha(-1)N fertilizer was inoculated with Fusarium graminearum (Fg). The storage protein fraction did not change significantly in response either to N level or Fg, whereas eighty protein spots in the water-soluble albumin fraction increased and 108 spots decreased more than two-fold in intensity in response to Fg. Spots with greater intensity in infected plants contained fungal proteins (9 spots) and proteolytic fragments of plant proteins (65 spots). Identified fungal proteins included two superoxide dismutases, L-xylulose reductase in two spots, peptidyl prolyl cis-trans isomerase and triosephosphate isomerase, and proteins of unknown function. Spots decreasing in intensity in response to Fg contained plant proteins possibly degraded by fungal proteases. Greater spot volume changes occurred in response to Fg in plants grown with low nitrogen, although proteomes of uninfected plants were similar for both treatments. Correlation of proteome changes with measurement of Fusarium-damaged kernels, fungal biomass and mycotoxin levels indicated that increased Fusarium infection occurred in barley with low N and suggests control of N fertilization as a possible way to minimise FHB in barley.

Particle-facilitated pesticide leaching from differently structured soil monoliths.

The leaching of soil particles and surface applied 14C-labeled glyphosate and pendimethalin from intact soil columns (height: 50 cm; diameter: 30 cm) were investigated, and the relative significance of particle-facilitated pesticide transport was quantified. Investigations were performed with a recently plowed (four columns) and an untilled (five columns) sandy loam soil. Leaching was driven by three irrigation events (15 mm h(-1); 2 h each). Samples of the leachate were filtered immediately (within 1.5 minutes) using 20 nm filters, and the 14C-pesticide content was determined for filtered and unfiltered samples. Pesticide leaching was driven by preferential water flow in macropores. For the plowed structure, 68+/-10% of the leached glyphosate (average of 6 events+/-std.) was bound to particles whereas significantly less glyphosate was bound to particles in leachate from minimally disturbed columns (17+/-12%). Thus, the results suggest that soil structure affected the mode of transport of glyphosate. It is likely that glyphosate sorbed strongly when applied on recently plowed soil (Kd=503 L kg(-1) for the soil), and that it could be mobilized and transported independently of soil particles more easily when applied on the minimally disturbed soil covered in part with crop residues (Kd<1 L kg(-1) for straw). Significantly less amounts of soil particles were leached from minimally disturbed (119-247 mg) than from recently plowed (441-731 mg) columns. The significance of particle-facilitated pendimethalin leaching could not be accurately quantified due to disagreement between control measurements based on both 14C-activity and chemical analyses.

Leaching of pesticides through normal-tillage and low-tillage soil--a lysimeter study. II. Glyphosate.

Glyphosate is a widely used non‐selective herbicide. Leaching of glyphosate (N‐(phosphonomethyl)glycine) and/or its metabolite AMPA (aminomethylphosphonic acid) was studied in four lysimters, two of them being replicates from a low‐tillage field (lysimeter 3 and 4), the other two being replicates from a normal tillage field (lysimeter 5 and 6). In both cases the soil was a sandy loam soil with 13–14% clay. The lysimeters had a surface area of 0.5 m2 and a depth of 110 cm. Lysimeter 3 and 4 were sprayed with a mixture of 14C‐labelled glyphosate and unlabelled glyphosate, while lysimeter 5 and 6 were sprayed with unlabelled glyphosate. The spraying took place September 18, 1997. The total amount of glyphosate sprayed onto each lysimeter was 40 mg, corresponding to 0.8 kg active ingredient per ha. The lysimeters were installed in an outdoor system in Research Centre Flakkebjerg and were thus exposed to normal climatic conditions of the area. A mean of 260 l drainage water were collected from lysimeter 3 and 4 and a mean of 375 litres from lysimeter 5 and 6. The mean yearly concentration of leached glyphosate and/or AMPA was significantly below 0.1 μg/l from both sets of lysimeters, and thus no significant difference between the two lysimeter sets was shown. However, in both sets of lysimeters several single findings at concentrations above 0.1 μg/l was seen, which might be due to the leaching of particle‐bound compounds. A significant difference between the soil residual concencentrations of AMPA was seen, the higher concentration was found in the set of lysimeterw where low‐tillage had been practiced and where Round Up had been used several times in the years before sampling of the lysimeter soil.

Fate in Soil of Flavonoids Released from White Clover (Trifolium repens L.)

White clover is frequently used as a leguminous cover crop, serving as green manure, and is also included with grasses in cattle feed mixtures. Numerous biological effects reported for clover cultivation have been attributed to the production of bioactive secondary metabolites. Thus far the presence in soil of bioactive secondary metabolites from clover has received limited attention. In this paper we examine for the first time the release of flavonoids both from field-grown white clover and from soil-incorporated white clover plants of flavonoids, as analyzed by LC-MS/MS. The dominant flavonoid aglycones were formononetin, medicarpin, and kaempferol. Soil-incorporated white clover plants generated high concentrations of the glycosides kaempferol-Rha-Xyl-Gal and quercetin-Xyl-Gal. Substantial amounts of kaempferol persisted in the soil for days while the other compounds were degraded faster. These compounds should be considered in future studies of soil fatigue, allelopathic activity, and possible environmental risks from extended clover cultivation.

Influence of surfactants on the leaching of bentazone in a sandy loam soil

BACKGROUND Surfactants are very often used for more efficient pesticide spraying, but knowledge about their influence on the leaching potential for pesticides is very limited. In the present study, the leaching of the herbicide bentazone [3-isopropyl-1H-2, 1,3-benzothiadiazin-4(3H)-one 2,2-dioxide] was measured in columns with sandy loam soil with or without the addition of a non-ionic surfactant, octylphenol ethylene oxide condensate (Triton X-100, Triton), and an anionic surfactant, sodium dodecylbenzenesulfonate (SDBS), and in the presence of both surfactants (SDBS + Triton). RESULTS The mobility of bentazone (B) increased in the following order: B + Triton (slowest) < B + SDBS + Triton < B < B + SDBS (fastest). When Triton X-100 was applied to the soil together with bentazone, the leaching of bentazone in the soil decreased significantly compared with leaching of bentazone without the addition of surfactant. SDBS and Triton X-100 neutralised their influence on the leaching speed of bentazone in the soil columns when both surfactants were applied with bentazone. CONCLUSION From the study it can be concluded that, depending on their properties, surfactants can enhance or reduce the mobility of bentazone. By choosing a non-ionic surfactant, bentazone mobility can be reduced, giving time for degradation and thereby reducing the risk of groundwater pollution.

Determination of volatilization (dissipation) and secondary deposition of pesticides in a field study using passive dosimeters

The volatilization and secondary deposition of the herbicides prosulfocarb and pendimethalin were measured using passive dosimeters with Chinese cabbage and soil as collective material. Passive dosimeters were placed in the field before spraying and at three distances downwind after spraying. The volatilization of prosulfocarb and pendimethalin determined as dissipation was 80 and 60% of the applied amount from the cabbage surface, while no significant loss was measured from soil surfaces after 48 h. The secondary deposition of prosulfocarb and pendimethalin 25 m from the spray zone was 2 and 4% on the leaf surface as well as on the soil surface. At 5 m distance, 6 and 10% of the field dose of prosulfocarb and pendimethalin could be determined on dosimeters with leaves, and 4 and 8% of the field dose could be determined on soil surfaces.