Two distinct Fusarium graminearum populations colonized European wheat in the past two decades.

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB) disease in wheat in Europe. To reveal population structure and to pinpoint genetic targets of selection we studied genomes of 96 strains of F. graminearum using population genomics. Bayesian and phylogenomic analyses indicated that the F. graminearum emergence in Europe could be linked to two independently evolving populations termed here as East European (EE) and West European (WE) population. The EE strains are primarily prevalent in Eastern Europe, but to a lesser extent also in western and southern areas. In contrast, the WE population appears to be endemic to Western Europe. Both populations evolved in response to population-specific selection forces, resulting in distinct localized adaptations that allowed them to migrate into their environmental niche. The detection of positive selection in genes with protein/zinc ion binding domains, transcription factors and in genes encoding proteins involved in transmembrane transport highlights their important role in driving evolutionary novelty that allow F. graminearum to increase adaptation to the host and/or environment. F. graminearum also maintained distinct sets of accessory genes showing population-specific conservation. Among them, genes involved in host invasion and virulence such as those encoding proteins with high homology to tannase/feruloyl esterase and genes encoding proteins with functions related to oxidation-reduction were mostly found in the WE population. Our findings shed light on genetic features related to microevolutionary divergence of F. graminearum and reveal relevant genes for further functional research aiming at better control of this pathogen.

Whole-genome single nucleotide polymorphism analysis for typing the pandemic pathogen Fusarium graminearum sensu stricto.

Recent improvements in microbiology and molecular epidemiology were largely stimulated by whole- genome sequencing (WGS), which provides an unprecedented resolution in discriminating highly related genetic backgrounds. WGS is becoming the method of choice in epidemiology of fungal diseases, but its application is still in a pioneer stage, mainly due to the limited number of available genomes. Fungal pathogens often belong to complexes composed of numerous cryptic species. Detecting cryptic diversity is fundamental to understand the dynamics and the evolutionary relationships underlying disease outbreaks. In this study, we explore the value of whole-genome SNP analyses in identification of the pandemic pathogen Fusarium graminearum sensu stricto (F.g.). This species is responsible for cereal diseases and negatively impacts grain production worldwide. The fungus belongs to the monophyletic fungal complex referred to as F. graminearum species complex including at least sixteen cryptic species, a few among them may be involved in cereal diseases in certain agricultural areas. We analyzed WGS data from a collection of 99 F.g. strains and 33 strains representing all known cryptic species belonging to the FGSC complex. As a first step, we performed a phylogenomic analysis to reveal species-specific clustering. A RAxML maximum likelihood tree grouped all analyzed strains of F.g. into a single clade, supporting the clustering-based identification approach. Although, phylogenetic reconstructions are essential in detecting cryptic species, a phylogenomic tree does not fulfill the criteria for rapid and cost-effective approach for identification of fungi, due to the time-consuming nature of the analysis. As an alternative, analysis of WGS information by mapping sequence data from individual strains against reference genomes may provide useful markers for the rapid identification of fungi. We provide a robust framework for typing F.g. through the web-based PhaME workflow available at EDGE bioinformatics. The method was validated through multiple comparisons of assembly genomes to F.g. reference strain PH-1. We showed that the difference between intra- and interspecies variability was at least two times higher than intraspecific variation facilitating successful typing of F.g. This is the first study which employs WGS data for typing plant pathogenic fusaria.

Diversity of Mobile Genetic Elements in the Mitogenomes of Closely Related Fusarium culmorum and F. graminearum sensu stricto Strains and Its Implication for Diagnostic Purposes.

Much of the mitogenome variation observed in fungal lineages seems driven by mobile genetic elements (MGEs), which have invaded their genomes throughout evolution. The variation in the distribution and nucleotide diversity of these elements appears to be the main distinction between different fungal taxa, making them promising candidates for diagnostic purposes. Fungi of the genus Fusarium display a high variation in MGE content, from MGE-poor (Fusarium oxysporum and Fusarium fujikuroi species complex) to MGE-rich mitogenomes found in the important cereal pathogens F. culmorum and F. graminearum sensu stricto. In this study, we investigated the MGE variation in these latter two species by mitogenome analysis of geographically diverse strains. In addition, a smaller set of F. cerealis and F. pseudograminearum strains was included for comparison. Forty-seven introns harboring from 0 to 3 endonucleases (HEGs) were identified in the standard set of mitochondrial protein-coding genes. Most of them belonged to the group I intron family and harbored either LAGLIDADG or GIY-YIG HEGs. Among a total of 53 HEGs, 27 were shared by all fungal strains. Most of the optional HEGs were irregularly distributed among fungal strains/species indicating ancestral mosaicism in MGEs. However, among optional MGEs, one exhibited species-specific conservation in F. culmorum. While in F. graminearum s.s. MGE patterns in cox3 and in the intergenic spacer between cox2 and nad4L may facilitate the identification of this species. Thus, our results demonstrate distinctive traits of mitogenomes for diagnostic purposes of Fusaria.

BotRisk: simulating the annual bunch rot risk on grapevines (Vitis vinifera L. cv. Riesling) based on meteorological data.

The aim of the present investigations was to simulate the annual risk of bunch rot (Botrytis cinerea) on Vitis vinifera L. cv. Riesling grapes based on three long-term (n = 3 × 7 = 21 cases) assessment data sets originating from three Central European grape-growing regions. Periods when meteorological parameters were significantly (p < 0.01) correlated with the cumulative degree day (CDD7;18;24) reaching 5% disease severity were determined by Window Pane analysis. Analyses revealed five critical weather constellations ("events") influencing annual epidemics: relatively low temperatures after bud break, dry conditions during flowering, high temperatures after flowering, and low temperatures and high precipitation sums during/after veraison were all associated with thermal-temporal early epidemics. Meteorological data in each of the five events served as input for the bunch rot risk model "BotRisk." The multiple linear regression model resulted in an adjusted coefficient of determination (R2adj.) of 0.63. BotRisk enables (i) the simulation of the thermal-temporal position of the annual epidemic and, based on this, (ii) the classification of the annual bunch rot risk into three classes: low, medium, or high risk. According to leave-one-out cross-validation, 11 of 21 case studies were correctly classified. No systematic bias caused by location was observed, indicating that the transfer of the model into other locations with comparable climatic conditions could be possible. BotRisk (i) represents a novel viticultural decision support tool for crop cultural and chemical measures against bunch rot and (ii) enables an estimation of the bunch rot risk under changing environmental conditions.

Searching molecular determinants of sensitivity differences towards four demethylase inhibitors in Fusarium graminearum field strains.

Demethylase inhibitors (DMIs) also referred to as azoles or triazoles are currently the main fungicides used for controlling Fusarium diseases and associated toxins in cereals. DMIs also represent an important class of fungicides used in the medical domain. The level of sensitivity of a set of F. graminearum strains (n = 23), collected over the period 1994-2010 in Luxembourg, Germany, Canada, USA, Italy and Belgium against three DMIs (cyproconazole, propiconazole, tebuconazole) used in agriculture and one DMI used in medicine (tioconazole) was assessed using a microplate test. Median molar EC50 values varied 113-fold among DMIs and on average 11-fold within DMIs with cyproconazole and tebuconazole being the least and the most effective ones, respectively. The EC50 values of the two DMIs registered for use against Fusarium species on cereals (propiconazole and tebuconazole) were significantly correlated (r = 0.597**), while no evidence for cross-resistance was obtained for other fungicide combinations. Haplotypes for CYP51A and CYP51C were defined based on snps determining amino acid variations in the two genes. EC50 values of strains with the CYP51A haplotype A0 and the CYP51C haplotype D1 varied greatly for the agricultural DMIs tebuconazole, propiconazole and cyproconazole, but not for the medical DMI tioconazole. None of the mutations and snps that were previously reported to be associated with resistance towards propiconazole was unambiguously related with resistance to tioconazole, because the mutations and snps were found in strains with low as well as with high EC50 values. Our results show that (1) DMI sensitivity of F. graminearum mycelium has been largely stable between 1994 and 2010, (2) effects of snps on sensitivity towards one DMI detected in one set of strains cannot be extrapolated to other DMIs and sets of strains and (3) F. graminearum strains responded differently to DMIs used in agriculture and to a representative of a medical DMI with no evidence for cross-resistance.

An improved life cycle impact assessment principle for assessing the impact of land use on ecosystem services.

In order to consider the effects of land use, and the land cover changes it causes, on ecosystem services in life cycle assessment (LCA), a new methodology is proposed and applied to calculate midpoint and endpoint characterization factors. To do this, a cause-effect chain was established in line with conceptual models of ecosystem services to describe the impacts of land use and related land cover changes. A high-resolution, spatially explicit and temporally dynamic modeling framework that integrates land use and ecosystem services models was developed and used as an impact characterization model to simulate that cause-effect chain. Characterization factors (CFs) were calculated and regionalized at the scales of Luxembourg and its municipalities, taken as a case to show the advantages of the modeling approach. More specifically, the calculated CFs enable the impact assessment of six land cover types on six ecosystem functions and two final ecosystem services. A mapping and comparison exercise of these CFs allowed us to identify spatial trade-offs and synergies between ecosystem services due to possible land cover changes. Ultimately, the proposed methodology can offer a solution to overcome a number of methodological limitations that still exist in the characterization of impacts on ecosystem services in LCA, implying a rethinking of the modeling of land use in life cycle inventory.

Pesticide residue profiles in bee bread and pollen samples and the survival of honeybee colonies-a case study from Luxembourg.

Pesticide residues (112 compounds) were quantified by GC-MS/MS or LC-MS/MS in 85 bee bread samples and 154 pollen samples obtained from five apiaries each with three or four colonies (genotype Buckfast) in Luxembourg over the period 2011-2013. Thiacloprid, chlorfenvinphos, tebuconazole, and methiocarb were found most frequently in bee bread while thiacloprid, permethrin-cis, and permethrin-trans were detected most frequently in the pollen samples. Three neonicotinoid insecticides (clothianidin, imidacloprid, and thiamethoxam) that were restricted by an EU regulation in 2013 after our sampling campaign was finished were each found in less than 8% of the pollen or bee bread samples. The maximum concentrations of thiacloprid, metazachlor, and methiocarb measured in the pollen collected by a group of honeybee colonies (n = 5) without survivors within the 3-year period of observation were 86.20 ± 10.74 ng/g, 2.80 ± 1.26 ng/g, and below the limit of quantification, respectively. The maximum concentrations of the same compounds measured in the pollen collected by a group of honeybee colonies with significantly (P = 0.02) more survivors (7 out of 9) than expected, if the survivors had been distributed randomly among the groups of colonies, were 11.98 ± 2.28 ng/g, 0.44 ± 0.29 ng/g, and 8.49 ± 4.13 ng/g, respectively. No honeybee colony that gathered pollen containing more than 23 ng/g thiacloprid survived the 3-year project period. There was no statistically significant association between pesticide residues in the bee bread and the survival of the colonies. Actions already taken or planned and potential further actions to protect bees from exposure to pesticides are discussed.

Development of a Highly Sensitive FcMito qPCR Assay for the Quantification of the Toxigenic Fungal Plant Pathogen Fusarium culmorum.

Fusarium culmorum is a ubiquitous, soil-borne fungus (ascomycete) causing foot and root rot and Fusarium head blight on cereals. It is responsible for yield and quality losses as well as grain contamination with mycotoxins, which are a potential health hazard. An extremely sensitive mitochondrial-based qPCR assay (FcMito qPCR) for quantification of F. culmorum was developed in this study. To provide specificity, the FcMito assay was successfully validated against 85 F. culmorum strains and 53 isolates of 30 other fungal species. The assay efficiency and sensitivity were evaluated against different F. culmorum strains with various amounts of pure fungal DNA and in the presence of background wheat DNA. The results demonstrated the high efficiency of the assay (97.2⁻106.0%, R²-values > 0.99). It was also shown that, in the presence of background DNA, 0.01 pg of fungal template could be reliably quantified. The FcMito assay was used to quantify F. culmorum DNA using 108 grain samples with different trichothecene levels. A significant positive correlation was found between fungal DNA quantity and the total trichothecene content. The obtained results showed that the sensitivity of the FcMito assay was much higher than the nuclear-based qPCR assay for F. culmorum.

Winter honey bee colony losses, Varroa destructor control strategies, and the role of weather conditions: Results from a survey among beekeepers.

Sets of treatments that were applied against varroa mites in the Luxembourgish beekeeper community were surveyed annually with a questionnaire between the winters 2010/11 and 2014/15. The average temperature and the precipitation sum of the month, when the respective varroa control method was applied were considered as co-variables when evaluating the efficacy of varroa control regimes. Success or failure of control regimes was evaluated based on the percentage of colonies lost per apiary in the winter following the treatment(s). Neither a positive nor a negative effect of formic acid (concentration 60%, w/v) on the colony losses could be found, irrespective of the weather conditions around the time of application. The higher concentration of 85% formic acid was linked with reduced colony losses when applications were done in August. Colony losses were reduced when Thymovar was applied in July or August, but applications in September were associated with increased losses compared with apiaries not treated with Thymovar during the same period. Apilife application in July as well as Apivar applications between July and September were associated with reduced colony losses. The removal of the drone brood and trickled oxalic acid application had beneficial effects when being done in April and December, respectively. Relatively warm (3.0±1.3°C) and wet (507.0±38.6mm/2months) conditions during the winter months December and January and relatively cool (17.2±1.4°C average monthly temperature) and wet (110.8±55.5mm/month) conditions in July were associated with elevated honey bee colony losses.

Antifungal Activity of Saponins from the Fruit Pericarp of Sapindus mukorossi against Venturia inaequalis and Botrytis cinerea.

The antifungal activity of an aqueous extract (AE) and the solid fraction of a chloroform-methanol fruit pericarp extract (CME) of Sapindus mukorossi resolved in water was tested for the first time against Venturia inaequalis and Botrytis cinerea-two important fungal pathogens worldwide. In the greenhouse, a CME (1% vol/vol) spray significantly reduced V. inaequalis symptoms and sporulation (99%) on apple seedling leaves (P ≤ 0.05). In field trials, applications of AE (1% vol/vol) reduced the disease severity of B. cinerea on grape, on average, by 63%. Extracts were fractionated by high-performance liquid chromatography and the bioefficacy of the fractions was tested in vitro. Some components of the most fungicidal fraction were identified by liquid chromatography-high resolution mass spectrometry as saponins: sapindoside B (accounting for ≥98% of the total constituents), hederagenin-pentosylhexoside, and oleanolic acid-hexosyl-deoxyhexosyl-hexoside. This fraction inhibited the mycelial growth of V. inaequalis and B. cinerea by 45 and 43%, respectively.

The Luxembourg database of trichothecene type B F. graminearum and F. culmorum producers.

Data specific to 486 strains belonging to Fusarium graminearum and Fusarium culmorum were manually collected from Luxembourg field monitoring campaigns between the year 2007 ad 2013. It is of interest to store such data in a web-enabled advanced database to help in epidemiological studies. Hence, we describe the design and development of a Fusarium database added to the Luxembourg Microbial Culture Collection (LuxMCC™) web interface at the Luxembourg Institute of Science and Technology (LIST). The database has three main features: (1) filter search, (2) detailed viewer of isolate information, and (3) excel export function of the dataset. Information on fungal strains includes genetic chemotypes, data on selected agronomic factors and crop management issues with geographic localization. The database constitutes a rich source of data for addressing epidemiological issues related to these two species. It will be regularly updated with improved features for advancement and utility.

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes.

Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000-2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum, 479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.

Development of an FgMito assay: A highly sensitive mitochondrial based qPCR assay for quantification of Fusarium graminearum sensu stricto.

An ascomycete fungus, Fusarium graminearum sensu stricto (s.s.), is the major cause of Fusarium head blight (FHB), a devastating disease of cereals worldwide. The fungus contaminates crops with mycotoxins, which pose a serious threat to food and feed safety. In this study, we developed a highly sensitive mitochondrial based qPCR assay (FgMito qPCR) for quantification of F. graminearum s.s. To ensure high sensitivity of the assay, primers and a Minor-groove binding (MGB) probe were designed based on multi-copy mitochondrial DNA. The FgMito assay was successfully validated against a range of geographically diverse F. graminearum s.s. strains to ensure uniformity of the assay at an intraspecific level, as well as with other fungal species to ensure specificity. The assay was further evaluated in terms of efficiency and sensitivity against a test panel of different F. graminearum s.s. strains with various levels of pure fungal DNA and in the presence of wheat background DNA. The results showed a high efficiency of the assay developed, ranging from 93% to 101% with r(2)-values of >0.99. We further showed that three low concentrations of fungal template 2 pg, 0.6 pg and 0.2 pg could be reliably quantified in the presence of wheat background DNA. The FgMito assay was used to quantify F. graminearum s.s. DNA on 65 field samples from a range of hosts with defined levels of trichothecenes. We revealed a significant positive correlation between fungal DNA quantity and the sum of trichothecenes. Lastly, we showed a higher sensitivity of the FgMito assay than the nuclear based qPCR assay for F. graminearum s.s. by comparing Ct-values from both assays.

Correlations between land covers and honey bee colony losses in a country with industrialized and rural regions.

High levels of honey bee colony losses were recently reported from Canada, China, Europe, Israel, Turkey and the United States, raising concerns of a global pollinator decline and questioning current land use practices, in particular intense agricultural cropping systems. Sixty-seven crops (data from the years 2010-2012) and 66 mid-term stable land cover classes (data from 2007) were analysed for statistical relationships with the honey bee colony losses experienced over the winters 2010/11-2012/13 in Luxembourg (Western Europe). The area covered by each land cover class, the shortest distance between each land cover class and the respective apiary, the number of plots covered by each land use class and the size of the biggest plot of each land cover class within radii of 2 km and 5 km around 166 apiaries (2010), 184 apiaries (2011) and 188 apiaries (2012) were tested for correlations with honey bee colony losses (% per apiary) experienced in the winter following the season when the crops were grown. Artificial water bodies, open urban areas, large industrial facilities including heavy industry, railways and associated installations, buildings and installations with socio-cultural purpose, camping-, sports-, playgrounds, golf courts, oilseed crops other than oilseed rape like sunflower or linseed, some spring cereals and former forest clearcuts or windthrows were the land cover classes most frequently associated with high honey bee colony losses. Grain maize, mixed forest and mixed coniferous forest were the land cover classes most frequently associated with low honey bee colony losses. The present data suggest that land covers related to transport, industry and leisure may have made a more substantial contribution to winter honey bee colony losses in developed countries than anticipated so far. Recommendations for the positioning of apiaries are discussed.

Evidence for a reversible drought induced shift in the species composition of mycotoxin producing Fusarium head blight pathogens isolated from symptomatic wheat heads.

Fusarium species are fungal plant pathogens producing toxic secondary metabolites such as deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15AcDON) and nivalenol (NIV). In Luxembourg, the Fusarium species composition isolated from symptomatic winter wheat heads was dominated by Fusarium graminearum sensu stricto strains (genetic 15AcDON chemotype) between the years 2009 and 2012, except for 2011, when Fusarium culmorum strains (genetic NIV chemotype) dominated the pathogen complex. Previous reports indicated that F. graminearum sensu stricto (genetic 15AcDON chemotype) was also most frequently isolated from randomly sampled winter wheat kernels including symptomatic as well as asymptomatic kernels in 2007 and 2008. The annual precipitation (average of 10 weather stations scattered across the country) decreased continuously from 924.31mm in 2007 over 917.15mm in 2008, to 843.38mm in 2009, 736.24mm in 2010, and 575.09mm in 2011. In 2012, the annual precipitation increased again to 854.70mm. Hardly any precipitation was recorded around the time of wheat anthesis in the years 2010 and 2011, whereas precipitation levels >50mm within the week preceding anthesis plus the week post anthesis were observed in the other years. The shift to genetic NIV chemotype F. culmorum strains in 2011 was accompanied by a very minor elevation of average NIV contents (2.9ngg(-1)) in the grain. Our data suggest that high NIV levels in Luxembourgish winter wheat are at present rather unlikely, because the indigenous F. culmorum strains with the genetic NIV chemotype seem to be outcompeted under humid in vivo conditions by F. graminearum DON producing strains on the one hand and seem to be inhibited - even though to a lower extent than DON producing strains - under dry in vivo conditions on the other hand.

Differences between the succinate dehydrogenase sequences of isopyrazam sensitive Zymoseptoria tritici and insensitive Fusarium graminearum strains.

Forty-one Zymoseptoria tritici strains isolated in Luxembourg between 2009 and 2010 were highly sensitive towards the new succinate dehydrogenase inhibitor (SDHI) isopyrazam, with concentrations inhibiting fungal growth by 50% (EC50) ranging from 0.0281 to 4.53µM, whereas 41 Fusarium graminearum strains isolated in Europe and Northern America between 1969 and 2009 were insensitive with the average rate of inhibition converging towards 28% with increasing isopyrazam concentration. Seven isolates of both species covering the range of isopyrazam sensitivities observed in the present study were selected for the sequencing of the subunits B, C and D of the succinate dehydrogenase (sdh) gene. Predicted sdh amino acid sequences of subunits B, C and D were identical among F. graminearum strains. By comparing with fungal strains where resistance towards SDHIs was previously reported, three variations were unique to F. graminearum; B-D130N located in the iron-sulfur cluster [2Fe-2S], B-A275T located in the [3Fe-4S] cluster and an additional S at amino acid position 83-84 of sdhC, probably modifying structurally the ubiquinone binding site and therefore the biological activity of the fungicide. No variation was found among the Z. tritici strains in subunits B and D. Two variations were observed within the subunit C sequences of Z. tritici strains: C-N33T and C-N34T. The difference in EC50 values between Z. tritici strains with the NN and TT configuration was non-significant at P=0.289. Two outliers in the Z. tritici group with significantly higher EC50 values that were not related to mutations in the sdhB, sdhC, or sdhD were detected. The role of isopyrazam for the control of F. graminearum and Z. tritici in Luxembourg is discussed.

FcStuA from Fusarium culmorum controls wheat foot and root rot in a toxin dispensable manner.

Fusarium culmorum is one of the most harmful pathogens of durum wheat and is the causal agent of foot and root rot (FRR) disease. F. culmorum produces the mycotoxin deoxynivalenol (DON) that is involved in the pathogenic process. The role of the gene FcStuA, a StuA ortholog protein with an APSES domain sharing 98.5% homology to the FgStuA protein (FGSG10129), was determined by functional characterisation of deletion mutants obtained from two F. culmorum wild-type strains, FcUk99 (a highly pathogenic DON producer) and Fc233B (unable to produce toxin and with a mild pathogenic behavior). The ΔFcStuA mutants originating from both strains showed common phenotypic characters including stunted vegetative growth, loss of hydrophobicity of the mycelium, altered pigmentation, decreased activity of polygalacturonic enzymes and catalases, altered and reduced conidiation, delayed conidial germination patterns and complete loss of pathogenicity towards wheat stem base/root tissue. Glycolytic process efficiency [measured as growth on glucose as sole carbon (C) source] was strongly impaired and growth was partially restored on glutamic acid. Growth on pectin-like sources ranked in between glucose and glutamic acid with the following order (the lowest to the highest growth): beechwood xylan, sugarbeet arabinan, polygalacturonic acid, citrus pectin, apple pectin, potato azogalactan. DON production in the mutants originating from FcUK99 strain was significantly decreased (-95%) in vitro. Moreover, both sets of mutants were unable to colonise non-cereal plant tissues, i.e. apple and tomato fruits and potato tubers. No differences between mutants, ectopic and wild-type strains were observed concerning the level of resistance towards four fungicides belonging to three classes, the demethylase inhibitors epoxiconazole and tebuconzole, the succinate dehydrogenase inhibitor isopyrazam and the cytochrome bc1 inhibitor trifloxystrobin. StuA, given its multiple functions in cell regulation and pathogenicity control, is proposed as a potential target for novel disease management strategies.

Estimating deoxynivalenol contents of wheat samples containing different levels of Fusarium-damaged kernels by diffuse reflectance spectrometry and partial least square regression.

Fusarium head blight is a fungal disease causing yield losses and mycotoxin contamination in wheat and other cereals. Wheat kernels (cultivar Ritmo) were sampled in 2001, 2002, 2003, and 2006 and Fusarium-damaged kernels were separated from sound grain based on visual assessment. Subsequently, grain lots containing 0, 20, 40, 60, 80, and 100% of damaged kernels were compiled. Each lot was split and the spectrometric reflectance (wavelengths 350-2500nm) was measured using subgroup one, while the concentration of the mycotoxin deoxynivalenol (DON) was determined by high-performance liquid chromatography in subgroup two. DON concentrations in batches classified as sound were not significantly different from 0. Estimating DON contents from the percentage of Fusarium-damaged kernels was impeded by vast variability, resulting in a coefficient of determination of 0.49. Using spectrometric data subjected to partial least square regression allowed estimating DON contents with higher accuracy, in particular at elevated percentages of damaged kernels. The coefficient of determination was 0.84 for the relationship between DON contents estimated based on spectrometric data and the DON contents measured. The intercept of a regression line fitted through a plot of estimated versus measured DON contents was 0.89+/-3.61mg/kg. Since intercept+standard error was larger than the actual legal limit (1.25mg DON per kg dry grain in the European Union), the spectrometric procedure was still not precise enough to allow a reliable separation of grain samples with DON contents below 1.25mg/kg from samples with DON contents above the limit. However, spectrometric data also allowed estimating the DON content of the average damaged kernel within a given lot composed of sound and damaged kernels, which is probably the reason for the reduction of the fraction of unexplained variance by 35% compared to the visual approach and illustrates that spectrometric approaches can make a contribution to reducing DON contents of wheat grain.

Estimating mycotoxin contents of Fusarium-damaged winter wheat kernels.

Winter wheat (Triticum aestivum L., cultivars Ritmo and Dekan) grain was sampled in Northern Germany between 2001 and 2006. Kernels damaged by fungi of the genus Fusarium were separated from sound grain by visual assessment. Samples containing 0%, 20%, 40%, 60%, 80% and 100% of Fusarium-damaged kernels were compiled and analyzed for the Fusarium type B trichothecenes deoxynivalenol (DON, 2001-2006), nivalenol (NIV, 2006), 3-acetyl-deoxynivalenol (3AcDON, 2006) and 15-acetyl-deoxynivalenol (15AcDON, 2006). The relationship between mycotoxin contents and the percentage of Fusarium-damaged kernels was calculated for each lot of grain. Apart from one exception, relationships between the percentage of Fusarium-damaged kernels and NIV, 3AcDON or 15AcDON were non-significant. In contrast, close relationships between the percentage of Fusarium-damaged kernels and the DON content were observed (r(2)=0.93-0.99). The y-axis intercepts were not significantly different from zero, but the DON content of the damaged kernels varied by a factor of 11.59 between years and by a factor of 1.87 between cultivars. Fusarium-damaged kernels contained between 0.21 and 2.39 microg DON kernel(-1). The overall average DON content of a Fusarium-damaged wheat kernel was 1.29 +/- 0.11 microg. The DON content of diseased kernels was affected by environment and wheat genotype but not by genotype x environment interaction. On average, Fusarium-damaged kernels contained 9.7-fold more DON than 15AcDON, 19.5-fold more DON than NIV, and 26.9-fold more DON than 3AcDON. 3AcDON and 15AcDON contents per wheat kernel were not significantly different between cultivars. On average, 4.27% of Fusarium-damaged kernels were sufficient to reach the 1.25 mg DON kg(-1) grain limit for unprocessed cereals in the EU. Given the low percentages of Fusarium-damaged kernels that are equivalent to current legal DON limits, grading accuracies >96% would be needed when using automatic grading systems for separating sound from damaged kernels.

Fate of deoxynivalenol in contaminated wheat grain during preparation of Egyptian 'balila'.

Soaking and boiling whole wheat kernels in water are the key steps in the preparation of an Egyptian dish called 'balila'. The effects of washing, soaking and boiling wheat kernels in tap water or in 0.1 M Na2CO3 solution on the deoxynivalenol (DON) content of the wheat kernels were studied. Boiling contaminated wheat kernels in water reduced the DON content of the grain by 70%. The mechanism of decontamination due to boiling is probably a leaching of DON out of the grain into the boiling medium. A combined treatment of soaking in 0.1 M Na2CO3 solution (pH 11) with subsequent boiling reduced the DON content of the grain by 93%. Data suggest that apart from leaching DON out of the kernels into the boiling medium, a degradation of DON occurred in alkaline medium. Modifying the traditional process of 'balila' preparation by using Na2CO3 solution may be useful to reduce the risk of mycotoxin exposure via 'balila'.