Taqman qPCR Quantification and Fusarium Community Analysis to Evaluate Toxigenic Fungi in Cereals.

Fusarium head blight (FHB) is an economically important plant disease. Some Fusarium species produce mycotoxins that cause food safety concerns for both humans and animals. One especially important mycotoxin-producing fungus causing FHB is Fusarium graminearum. However, Fusarium species form a disease complex where different Fusarium species co-occur in the infected cereals. Effective management strategies for FHB are needed. Development of the management tools requires information about the diversity and abundance of the whole Fusarium community. Molecular quantification assays for detecting individual Fusarium species and subgroups exist, but a method for the detection and quantification of the whole Fusarium group is still lacking. In this study, a new TaqMan-based qPCR method (FusE) targeting the Fusarium-specific elongation factor region (EF1α) was developed for the detection and quantification of Fusarium spp. The FusE method was proven as a sensitive method with a detection limit of 1 pg of Fusarium DNA. Fusarium abundance results from oat samples correlated significantly with deoxynivalenol (DON) toxin content. In addition, the whole Fusarium community in Finnish oat samples was characterized with a new metabarcoding method. A shift from F. culmorum to F. graminearum in FHB-infected oats has been detected in Europe, and the results of this study confirm that. These new molecular methods can be applied in the assessment of the Fusarium community and mycotoxin risk in cereals. Knowledge gained from the Fusarium community analyses can be applied in developing and selecting effective management strategies for FHB.

Updated survey of Fusarium species and toxins in Finnish cereal grains.

The aim of the project was to produce updated information during 2005-14 on the Fusarium species found in Finnish cereal grains, and the toxins produced by them, as the last comprehensive survey study of Fusarium species and their toxins in Finland was carried out at the turn of the 1960s and the 1970s. Another aim was to use the latest molecular and chemical methods to investigate the occurrence and correlation of Fusarium species and their mycotoxins in Finland. The most common Fusarium species found in Finland in the FinMyco project 2005 and 2006 were F. avenaceum, F. culmorum, F. graminearum, F. poae, F. sporotrichioides and F. langsethiae. F. avenaceum was the most dominant species in barley, spring wheat and oat samples. The occurrence of F. culmorum and F. graminearum was high in oats and barley. Infection by Fusarium fungi was the lowest in winter cereal grains. The incidence of Fusarium species in 2005 was much higher than in 2006 due to weather conditions. F. langsethiae has become much more common in Finland since 2001. F. graminearum has also risen in the order of importance. A highly significant correlation was found between Fusarium graminearum DNA and deoxynivalenol (DON) levels in Finnish oats, barley and wheat. When comparing the FinMyco data in 2005-06 with the results of the Finnish safety monitoring programme for 2005-14, spring cereals were noted as being more susceptible to infection by Fusarium fungi and the formation of toxins. The contents of T-2 and HT-2 toxins and the frequency of exceptionally high DON concentrations all increased in Finland during 2005-14. Beauvericin (BEA), enniatins (ENNs) and moniliformin (MON) were also very common contaminants of Finnish grains in 2005-06. Climate change is leading to warmer weather, and this may indicate more changes in Finnish Fusarium mycobiota and toxin contents and profiles in the near future.

Analysis of neonicotinoids from plant material by desorption atmospheric pressure photoionization-mass spectrometry.

RATIONALE: Neonicotinoids are widely used insecticides which have been shown to affect the memory and learning abilities of honey bees, and are suspected to play a part in the unexplainable, large-scale loss of honey bee colonies. Fast methods, such as ambient mass spectrometry (MS), for their analysis from a variety of matrices are necessary to control the use of forbidden products and study the spreading of insecticides in nature. METHODS: The feasibilities of two ambient MS methods, desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI), for the analysis of five most used neonicotinoid compounds, thiacloprid, acetamiprid, clothianidin, imidacloprid and thiamethoxam, were tested. In addition, DAPPI was used to analyze fresh rose leaves treated with commercially available thiacloprid insecticide and dried and powdered turnip rape flowers, which had been collected from a field treated with thiacloprid-containing insecticide. RESULTS: DAPPI was found to be more sensitive than DESI, with 2-11 times better signal-to-noise ratios, and limits of detection at 0.4-5.0 fmol for the standard compounds. DAPPI was able to detect thiacloprid from the rose leaves even 2.5 months after the treatment and from the turnip rape flower samples collected from a field. The analysis of plant material by DAPPI did not require extraction or other sample preparation. CONCLUSIONS: DAPPI was found to be suitable for the fast and direct qualitative analysis of thiacloprid neonicotinoid from plant samples. It shows promise as a fast tool for screening of forbidden insecticides, or studying the distribution of insecticides in nature.

Birch (Betula spp.) wood biochar is a potential soil amendment to reduce glyphosate leaching in agricultural soils.

Glyphosate (N-(phosphonomethyl) glycine), a commonly used herbicide in agriculture can leach to deeper soil layers and settle in surface- and ground waters. To mitigate the leaching of pesticides and nutrients, biochar has been suggested as a potential soil amendment due to its ability to sorb both organic and inorganic substances. However, the efficiency of biochar in retaining agro-chemicals in the soil is likely to vary with feedstock material and pyrolysis conditions. A greenhouse pot experiment, mimicking a crop rotation cycle of three plant genera, was established to study the effects of pyrolysis temperature on the ability of birch (Betula sp.) wood originated biochar to reduce the leaching of (i) glyphosate, (ii) its primary degradation product AMPA and (iii) phosphorus from the soil. The biochar types used were produced at three different temperatures: 300 °C (BC300), 375 °C (BC375) and 475 °C (BC475). Compared to the control treatment without biochar, the leaching of glyphosate was reduced by 81%, 74% and 58% in BC300, BC375 and BC475 treated soils, respectively. The respective values for AMPA were 46%, 39% and 23%. Biochar had no significant effect on the retention of water-soluble phosphorus in the soil. Our results corroborate earlier findings on pesticides, suggesting that biochar amendment to the soil is a promising way to reduce also the leaching of glyphosate. Importantly, the ability of biochar to adsorb agro-chemicals depends on the temperature at which feedstock is pyrolysed.

Molecular Quantification and Genetic Diversity of Toxigenic Fusarium Species in Northern Europe as Compared to Those in Southern Europe.

Fusarium species produce important mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV) and T-2/HT-2-toxins in cereals. The highest DON and T-2/HT-2 toxin levels in northern Europe have been found in oats. About 12%-24% of Finnish oat samples in 2012 contained >1.75 mg·kg-1 of DON, which belongs to type B trichothecenes. Fusarium graminearum is the most important DON producer in northern Europe and Asia and it has been displacing the closely related F. culmorum in northern Europe. The 3ADON chemotype of F. graminearum is dominant in most northern areas, while the 15ADON chemotype of F. graminearum is predominating in Central and southern Europe. We suggest that the northern population of F. graminearum may be more specialized to oats than the southern population. Only low levels of F. culmorum DNA were found in a few oat samples and no correlation was found between F. culmorum DNA and DON levels. DNA levels of F. graminearum were in all cases in agreement with DON levels in 2011 and 2012, when DON was measured by gas chromatography-mass spectrometry (GC-MS). When the RIDA®QUICK SCAN kit results (DON) were compared to DNA levels of F. graminearum, the variation was much higher. The homogenization of the oats flour by grinding oats with 1 mm sieve seems to be connected to this variation. There was a significant correlation between the combined T-2 and HT-2 and the combined DNA levels of F. langsethiae and F. sporotrichioides in Finland in 2010-2012.

Effects of soil phosphorus status on environmental risk assessment of glyphosate and glufosinate-ammonium.

The increased use of herbicides poses a risk to the aquatic environment. Easy and economical methods are needed to identify the fields where specific environment protection measures are needed. Phosphorus (P) and organophosphorus herbicides compete for the same adsorption sites in soil. In this study the relationship between P obtained in routine Finnish agronomic tests (acid ammonium acetate [P(AC)]) and adsorption of glyphosate and glufosinate-ammonium was investigated to determine whether P(AC) values could be used in the risk assessment. The adsorption of glyphosate ((N-(phosphonomethyl)glycine) and glufosinate-ammonium (2-amino-4-(hydroxymethylphosphinyl)butanoic acid) was studied in a clay and a sandy loam soil enriched with increasing amounts of P added as potassium dihydrogen phosphate. Desorption was also determined for some P-enriched soil samples. The adsorption of both herbicides diminished with increasing P(AC) value. The correlations between Freundlich adsorption coefficients obtained in the adsorption tests and P(AC) were nonlinear but significant (r > 0.98) in both soils. The exponential models of the relationship between soil P(AC) values and glyphosate adsorption were found to fit well to an independent Finnish soil data set (P < 0.1 for glyphosate and P < 0.01 for glufosinate-ammonium). The desorption results showed that glufosinate-ammonium sorption is not inversely related to soil P status, and the high correlation coefficients obtained in the test of the model were thus artifacts caused by an abnormal concentration of exchangeable potassium in soil. The solved equations are a useful tool in assessing the leaching risks of glyphosate, but their use for glufosinate-ammonium is questionable.

Fate of the herbicides glyphosate, glufosinate-ammonium, phenmedipham, ethofumesate and metamitron in two Finnish arable soils.

The fate of five herbicides (glyphosate, glufosinate-ammonium, phenmedipham, ethofumesate and metamitron) was studied in two Finnish sugar beet fields for 26 months. Soil types were sandy loam and clay. Two different herbicide-tolerant sugar beet cultivars and three different herbicide application schedules were used. Meteorological data were collected throughout the study and soil properties were thoroughly analysed. An extensive data set of herbicide residue concentrations in soil was collected. Five different soil depths were sampled. The study was carried out using common Finnish agricultural practices and represents typical sugar beet cultivation conditions in Finland. The overall observed order of persistence was ethofumesate > glyphosate > phenmedipham > metamitron > glufosinate-ammonium. Only ethofumesate and glyphosate persisted until the subsequent spring. Seasonal variation in herbicide dissipation was very high and dissipation ceased almost completely during winter. During the 2 year experiment no indication of potential groundwater pollution risk was obtained, but herbicides may cause surface water pollution.

Adsorption of sugar beet herbicides to Finnish soils.

Three sugar beet herbicides, ethofumesate, phenmedipham and metamitron, are currently used on conventional sugar beet cultivation, while new varieties of herbicide resistant (HR) sugar beet, tolerant of glyphosate or glufosinate-ammonium, are under field testing in Finland. Little knowledge has so far been available on the adsorption of these herbicides to Finnish soils. The adsorption of these five herbicides was studied using the batch equilibrium method in 21 soil samples collected from different depths. Soil properties like organic carbon content, texture, pH and partly the phosphorus and oxide content of the soils were tested against the adsorption coefficients of the herbicides. In general, the herbicides studied could be arranged according to their adsorption coefficients as follows: glyphosate>phenmedipham>ethofumesate approximately glufosinate-ammonium>metamitron, metamitron meaning the highest risk of leaching. None of the measured soil parameters could alone explain the adsorption mechanism of these five herbicides. The results can be used in model assessments of risk for leaching to ground water resulting from weed control of sugar beet in Finland.