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Contamination of Fusarium moulds and subsequent production of mycotoxins is a common problem in cereals grown in Northern Europe. Oats is the third most important cereal crop in Sweden and is commonly contaminated by Fusarium. To date there are no published reports on the distribution of trichothecene-producing Fusaria in Swedish oats and correlation to T-2 and HT-2, the two most dominant toxins. Identification of Fusarium species by traditional methods requires specific skill and experience and quantification of specific species is not possible. Using modern molecular methods such as real-time PCR both identification and quantification of specific Fusarium species are possible directly from the food or feed samples. In this study, specific methods for Fusarium langsethiae, Fusarium sporotrichioides, Fusarium graminearum, Fusarium culmorum and Fusarium poae as well as for the total amount of DNA from trichothecene-producing species were evaluated with respect to precision, robustness and efficiency and then used in the analysis of oat samples. Sixty-two oat samples were taken from field trial harvests from 2006-2008 in the central and southern parts of Sweden and analysed for the presence of Fusarium species by using species-specific real-time PCR-based methods with TaqMan or SYBR Green biochemistry. The T-2 and HT-2 toxin content was analysed by gas chromatography with electron capture detection of the toxins' pentafluoropropyl derivatives. The results showed that F. langsethiae and F. poae were the most dominant Fusarium species among those tested in Swedish oats during this period. The DNA level of F. langsethiae correlated well with the level of T-2 and HT-2 toxin (r=0.7), indicating that this species was the most important producer of T-2 and HT-2 toxins in Swedish oats during the sampling period (2006 to 2008). It can also be concluded that real-time PCR is a powerful tool when studying Fusarium species distribution in oats.

A survey of moulds and mycotoxins was performed on 99 rice samples taken from the Swedish retail market. The main objective was to study the mould and mycotoxin content in basmati rice and rice with a high content of fibre. Samples of jasmine rice as well as long-grain rice were also included. The samples were analysed for their content of ochratoxin A (high-performance liquid chromatography (HPLC)), aflatoxin B(1), B(2), G(1), and G(2) (HPLC, RIDA(R)QUICK), and mould (traditional cultivation methods in combination with morphological analysis). The majority of samples were sampled according to European Commission Regulation 401/2006. Subsamples were pooled and mixed before milling and both mould and mycotoxin analyses were performed on milled rice. The results showed that the majority of basmati rice (71%) and many jasmine rice samples (20%) contained detectable levels of aflatoxin B(1) (level of quantification = 0.1 microg aflatoxin kg(-1) rice). Two samples of jasmine rice and ten basmati rice samples contained levels over the regulated European maximum limits of 2 microg kg(-1) for aflatoxin B(1) or 4 microg kg(-1) for total aflatoxins. Aspergillus was the most common mould genus isolated, but also Penicillium, Eurotium, Wallemia, Cladosporium, Epicoccum, Alternaria, and Trichotecium were found. The presence of Aspergillus flavus in 21% of the samples indicates that incorrect management of rice during production and storage implies a risk of mould growth and subsequent production of aflatoxin. Rough estimates showed that high rice consumers may have an intake of 2-3 ng aflatoxin kg(-1) bodyweight and day(-1) from rice alone. This survey shows that aflatoxin is a common contaminant in rice imported to Europe.

A diploid and a haploid strain of Pichia anomala were tested for their biocontrol ability against the spoilage mould Penicillium roqueforti in glass tubes filled with grain at two water activities (aw). At aw 0.98, the two yeast strains grew and inhibited mould growth equally well and showed similar patterns of ethyl acetate production, reaching maximum values of 10–14 μg ml−1 headspace. At aw 0.95, both growth and biocontrol performance of the haploid strain were reduced. Ethyl acetate formation was also substantially reduced, with maximum headspace concentrations of 4 μg ml−1. We conclude that ethyl acetate is a major component of the anti-mould activity. The inhibitory effect of ethyl acetate was confirmed in a bioassay where the pure compound reduced biomass production of P. roqueforti.

The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (A), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (A), but continued to be produced in (B). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (B). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (B) than in (A), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.

The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3°C and 37°C, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.

The sigmaB transcription factor of the bacterium Bacillus subtilis is activated by growth-limiting energy or environmental challenge to direct the synthesis of more than 100 general stress proteins. Although the signal transduction pathway that conveys these stress signals to sigmaB is becoming increasingly well understood, how environmental or energy stress signals enter this pathway remains unknown. We show here that two PP2C serine phosphatases - RsbP, which is required for response to energy stress, and RsbU, which is required for response to environmental stress - each converge on the RsbV regulator of sigmaB. According to the current understanding of sigmaB regulation, in unstressed cells the phosphorylated RsbV anti-anti-sigma is unable to complex the RsbW anti-sigma, which is then free to bind and inactivate sigmaB. We can now advance the model that either PP2C phosphatase, when triggered by its particular class of stress, can remove the phosphate from RsbV and thereby activate sigmaB. The action of the previously described RsbU is known to be controlled by dedicated upstream signalling components that are activated by environmental stress. The action of the RsbP phosphatase described here requires an energy stress, which we suggest is sensed, at least in part, by the PAS domain in the amino-terminal region of the RsbP phosphatase. In other bacterial signalling proteins, similar PAS domains and their associated chromophores directly sense changes in intracellular redox potential to control the activity of a linked output domain.

Fusarium moulds frequently contaminate oats and other cereals world-wide, including those grown in Northern Europe. To investigate the presence of toxigenic Fusarium species and their toxins in oats, samples were taken during 2010 and 2011 in three geographical regions of Sweden (east, west, south). The samples were analysed by real-time PCR for the specific infection level of seven Fusarium species associated with oats and other cereals (Fusarium poae, Fusarium graminearum, Fusarium langsethiae, Fusarium culmorum, Fusarium tricinctum, Fusarium sporotrichioides and Fusarium avenaceum) and with a multi-mycotoxin method based on liquid chromatography/electrospray ionisation-tandem mass spectrometry (HPLC/ESI-MS/MS) for the detection of many fungal metabolites, including deoxynivalenol (DON), zearalenone (ZEA), nivalenol (NIV), T-2 toxin, HT-2 toxins, moniliformin (MON), beauvericin (BEA) and enniatins (ENNs). Most samples contained at least four of the seven Fusarium species analysed and F. poae, F. langsethiae and F. avenaceum were present in approximately 90-100% of all samples. The most common toxins detected were DON, NIV, BEA and ENNs, which were present in more than 90% of samples. Most Fusarium species and their toxins occurred in higher concentrations in 2010 than in 2011, with the exception of DON and its main producer F. graminearum. Significant regional differences were detected for some moulds and mycotoxins, with higher levels of F. graminearum, DON and ZEA in western Sweden than in the east (P

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 (r2 = 0.9) and ZEN (r2 = 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.

We amplified, sequenced and studied the transcriptional regulation of genes of the alcoholic fermentation pathway in the biocontrol and non-Saccharomyces wine yeast, Pichia anomala. Two ADH isogenes, PaADH1 and PaADH2, and one PDC gene, PaPDC1, were amplified from genomic P. anomala DNA by a two-step PCR approach, using degenerated primers against conserved regions of the respective genes for cloning core regions, and PCR-based gene walking for cloning the respective 5′ and 3′-ends. According to sequence analysis, ADH1 and PDC1 are most likely cytoplasmatic proteins, while ADH2 is most probably localized in the mitochondria. PaADH1 was expressed during aerobic growth on glucose, ethanol and succinate, but was nine-fold upregulated in response to oxygen limitation when grown on glucose. The gene seems to be involved in both production and consumption of ethanol. Only low expression of PaADH2 was detected during growth on glucose and ethanol, but it was highly expressed during growth on the non-fermentable carbon source succinate and repressed by the addition of glucose. PaPDC1 was expressed during aerobic growth on glucose and was upregulated four-fold in response to oxygen limitation. PaPDC1 expression was lower in cells grown on ethanol and succinate than on glucose and was up- regulated two- and four-fold, respectively, after glucose addition. Our results demonstrate that transcription of genes of the fermentative pathway is regulated by hypoxia and carbon source but posttranscriptional regulation may play a major role in regulating the metabolic flux. Copyright © 2006 John Wiley & Sons, Ltd.

The ascomycetous yeast Pichia anomala is frequently associated with food and feed products, either as a production organism or as a spoilage yeast. It belongs to the nonSaccharomyces wine yeasts and contributes to the wine aroma by the production of volatile compounds. The ability to grow in preserved food and feed environments is due to its capacity to grow under low pH, high osmotic pressure and low oxygen tension. A new application of P. anomala is its use as a biocontrol agent, which is based on the potential to inhibit a variety of moulds in different environments. Although classified as a biosafety class-1 organism, cases of P. anomala infections have been reported in immunocompromised patients. On the other hand, P. anomala killer toxins have a potential as antimicrobial agents. The yeast can use a broad range of nitrogen and phosphor sources, which makes it a potential agent to decrease environmental pollution by organic residues from agriculture. However, present knowledge of the physiological basis of its performance is limited. Recently, the first studies have been published dealing with the global regulation of the metabolism of P. anomala under different conditions of oxygenation.

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non- Saccharomyces wine yeast Pichia anomala . In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose −1 ) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.

A diploid and a haploid strain of Pichia anomala were tested for their biocontrol ability against the spoilage mould Penicillium roqueforti in glass tubes filled with grain at two water activities (aw). At aw 0.98, the two yeast strains grew and inhibited mould growth equally well and showed similar patterns of ethyl acetate production, reaching maximum values of 10-14 microg ml(-1) headspace. At aw 0.95, both growth and biocontrol performance of the haploid strain were reduced. Ethyl acetate formation was also substantially reduced, with maximum headspace concentrations of 4 microg ml(-1). We conclude that ethyl acetate is a major component of the anti-mould activity. The inhibitory effect of ethyl acetate was confirmed in a bioassay where the pure compound reduced biomass production of P. roqueforti.