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.

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.

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. DT(50) 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, (14)C-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.

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels.

Identification of Fusarium species by traditional methods requires specific skill and experience and there is an increased interest for new molecular methods for identification and quantification of Fusarium from food and feed samples. Real-time PCR with probe technology (Taqman) can be used for the identification and quantification of several species of Fusarium from cereal grain samples. There are several critical steps that need to be considered when establishing a real-time PCR-based method for DNA quantification, including extraction of DNA from the samples. In this study, several DNA extraction methods were evaluated, including the DNeasy Plant Mini Spin Columns (Qiagen), the Bio robot EZ1 (Qiagen) with the DNeasy Blood and Tissue Kit (Qiagen), and the Fast-DNA Spin Kit for Soil (Qbiogene). Parameters such as DNA quality and stability, PCR inhibitors, and PCR efficiency were investigated. Our results showed that all methods gave good PCR efficiency (above 90%) and DNA stability whereas the DNeasy Plant Mini Spin Columns in combination with sonication gave the best results with respect to Fusarium DNA yield. The modified DNeasy Plant Mini Spin protocol was used to analyse 31 wheat samples for the presence of F. graminearum and F. culmorum. The DNA level of F. graminearum could be correlated to the level of DON (r(2) = 0.9) and ZEN (r(2) = 0.6) whereas no correlation was found between F. culmorum and DON/ZEA. This shows that F. graminearum and not F. culmorum, was the main producer of DON in Swedish wheat during 2006.

Leaching and degradation of 21 pesticides in a full-scale model biobed.

Filling and cleaning of pesticide sprayers presents a potential risk of pollution of soil and water. Three different solutions for handling sprayers have been suggested: Filling and cleaning in the field, filling and cleaning on hard surfaces with collection of the waste water, and filling and cleaning on a biobed, which is an excavation lined with clay and filled with a mixture of chopped straw, sphagnum and soil with turf on top, and with increased sorption capacity and microbial activity for degradation of the pesticides. In the present study the degradation and leaching of 21 pesticides (5 g of each) was followed in an established full-scale model biobed. Percolate was collected and analysed for pesticide residues, and the biobed material was sampled at three different depths and analysed by liquid chromatography double mass spectrometry (LC-MSMS). During the total study period of 563 days, no traces of 10 out of 21 applied pesticides were detected in the percolate (detection limits between 0.02 and 0.9 microg l(-1)) and three pesticides were only detected once and at concentrations below 2 microg l(-1). During the first 198 days before second application, 14% of the applied herbicide bentazone was detected in the leachate with maximum and mean concentrations of 445 and 172 microg l(-1), respectively. About 2% of the initial mecoprop and fluazifop dose was detected in the percolate, with mean concentrations of 23 microg l(-1), while MCPA and dimethoate had mean concentrations of 3.5 and 4.7 microg l(-1), respectively. Leachate concentrations for the remaining pesticides were generally below the detection limit (0.02-0.9 microg l(-1), below 1% of applied). Sorption studies of five pesticides showed that compounds with a low K(d) value appeared in the leachate. After 169 days, all pesticides in the biobed profile were degraded to a level below 50% of the calculated initial dose. Pesticides with K(oc) values above 100 were primarily found in the uppermost 10 cm and degraded slowest due to the low bioavailability. The 11 most degradable pesticides were all degraded such that less than 3% remained in the biobed after 169 days. Following second pesticide application of the biobed, leachate was sampled 215 and 365 days after the treatment. This showed the same pesticides to be leached out and at concentrations comparable to those of the first treatment. The same pesticides as after the first treatment were retained in the biobed.

Species-specific sensitivity of aquatic macrophytes towards two herbicides.

The s-triazine herbicide terbutylazine, an inhibitor of photosystem II, is often found in surface waters in concentrations < 1 microg L(-1), but concentrations up to 13 microg L(-1) have been measured. To study the effect on the aquatic flora, we tested the sensitivity of 10 aquatic macrophyte species and a natural epiphyte community in a 2-week laboratory multispecies test at constant terbutylazine concentrations and two irradiance regimes. The data were described by a log-logistic concentration-response model and species sensitivity distributions (SSDs) were created from the EC50 and EC10 values. The 5% hazard concentration (HC5) of the EC10-based SSD for terbutylazine was 1 and 3 microg L(-1); hence the low chronic terbutylazine concentrations measured in the environment are not likely to affect the macrophyte community. To compare the species sensitivity between different groups of herbicides, SSDs were constructed from a published study on the sulfonylurea metsulfuron-methyl, an inhibitor of acetolactate synthase. There was no correlation between species-specific sensitivity to the two herbicides; hence, the combined exposure of different herbicides might affect the macrophyte community more broadly rather than seriously affecting a few susceptible species. Evaluating the standard procedure of leaving at least a factor of 100 between the EC50 of standard tests on Lemna sp. and the predicted environmental concentration seems to be protective for at least 95% of the macrophyte species for both terbutylazine and metsulfuron-methyl.

Sensitivity of aquatic plants to the herbicide metsulfuron-methyl.

The sensitivity of 12 aquatic plant species to the herbicide metsulfuron-methyl was tested in microcosm experiments under two growth conditions. As reference species, barley (Hordeum vulgare) and oil-seed rape (Brassica napus) were grown with their roots submerged in the microcosms. Two response variables were chosen: relative growth rate and specific leaf area (SLA). SLA was the most sensitive response variable, with 11 of the 12 aquatic species responding to the herbicide. EC(50) values varied 56-fold between species, with the commonly used aquatic test species Lemna minor being one of the most sensitive. Fast-growing species with a small exposed leaf area proved to be more sensitive to the herbicide than slow growing species with a large exposed leaf area, which was believed to be primarily due to variations in growth rates rather than to variations in exposed leaf area. The aquatic plants displayed high tolerance in growth to metsulfuron compared with the sensitive crop oil-seed rape. Hence, possible spray-drift events and leaching of the herbicide applied at agricultural rates are not considered to have a large impact on the growth of the aquatic flora tested.

Capacity of model biobeds to retain and degrade mecoprop and isoproturon.

Biobeds are used to increase the adsorption and degradation of pesticide spillage on sites used for mixing and loading and for cleaning of sprayers. The adsorption and the rate of degradation of 14C-labelled isoproturon and mecoprop (MCPP) at concentrations from 0.0005 to 25 000 mgkg(-1) were determined in biobed soil. Further leaching of the two herbicides was determined in a model biobed with a surface area of 2 m2. The biobed material showed enhanced ability to adsorb the two herbicides. Kd was 5.2 litre kg(-1) for isoproturon and 1.6 litre kg(-1) for MCPP in biobed material, which is higher than in natural soil. In different experiments with natural soil, Kd ranges from 0.07 to 0.6 litrekg(-1) for MCPP and from 1.5 to 4.6 litre kg(-1) for isoproturon in soils with varying organic carbon content. Degradation of MCPP was rapid at concentrations from 0.0005 to 500 mg kg(-1), delayed at 5000 mg kg(-1), and very slow at 25 000 mg kg(-1). For isoproturon, the relative degradation was most rapid at the lowest concentration and decreasing with increasing concentrations. After 120 days, between 55% and 8% 14C was evolved as 14CO2 at concentrations between 0.0005 and 25 000 mg kg(-1). The rate of evolution of 14CO2 indicated that degradation rates at low concentrations were of first-order and at higher concentrations of zero-order. Leaching of MCPP and isoproturon was determined in a newly established model biobed during a 2-year period. About 13% of applied MCPP and 1.4% of applied isoproturon leached out during the winter following the first autumn application (worst-case scenario). Leaching was completely prevented when the biobed had a well-developed grass cover and was covered during the winter.

Leaching of pesticides through normal-tillage and low-tillage soil--a lysimeter study. I. Isoproturon.

Isoproturon is a herbicide, which was used in Denmark against grass weeds and broad-leaved weeds until 1998. Isoproturon has frequently been detected in ground water monitoring studies. Leaching of isoproturon (N,N-dimethyl-N'-(4-(1-methylethyl)-phenyl)urea) and its metabolites, N'-(4-isopropylphenyl)-N-methylurea and N'-(4-isopropylphenyl)urea was studied in four lysimetres, 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 lysimetres had a surface area of 0.5 m2 and a depth of 110 cm. Lysimeter 3 and 4 were sprayed with unlabelled isoproturon while lysimeter 5 and 6 was sprayed with a mixture of 14C-labelled and unlabelled isoproturon. The total amount of isoproturon sprayed onto each lysimeter was 63 mg, corresponding to 1.25 kg active ingredient per ha. The lysimeters were sprayed with isoproturon on October 26, 1997. The lysimetres were installed in an outdoor system in Research Centre Flakkebjerg and were thus exposed to normal climatic conditions of the area. A mean of 360 l drainage water were collected from lysimeter 3 and 4 and a mean of 375 litres from lysimeter 5 and 6. Only negligible amounts of isoproturon and its primary metabolites were found in the drainage water samples, and thus no significant difference between the two lysimeter sets was shown. In a total of 82 drainage water samples, evenly distributed between the four lysimetres isoproturon was found in detectable amounts in two samples and N'-(4-isopropylphenyl)urea was found in detectable amounts in two other samples. The detection limit for all the compounds was 0.02 microg/l. 48% and 54% of the added radioactivity were recovered from the upper 10 cm soil layer in lysimeter 5 and 6, respectively, and 17 and 14% from 10-20 cm's depth. By extraction first with an aquatic CaCl2 solution 0.49% of the added radioactivity was extracted from the upper 10 cm layer in lysimeter 5. In the subsequent extraction with acetonitril, 1.19% of the added radioactivity was extracted. In lysimeter 6, upper 10 cm, 0.2% were extracted with water and 0.56% were extracted with acetonitril. Below 10 cm's depth no measurable amounts could be extracted.

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 lysimeters, 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 microg/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 microg/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 lysimeter where low-tillage had been practiced and where Round Up had been used several times in the years before sampling of the lysimeter soil.