Whole-genome sequence of an Aspergillus parasiticus strain isolated from Kenyan soil.

Aspergillus parasiticus is an important aflatoxigenic fungus, frequently found in soil samples. Here, we report the sequencing of A. parasiticus strain MRI410 using Illumina MiSeq and Oxford Nanopore platforms. This strain was isolated from soil of a Kenyan maize field.

Whole-Genome Sequences of Two Kenyan Aspergillus minisclerotigenes Strains.

Here, we report the sequencing of the whole genome, including the mitochondrial DNA, of the two highly aflatoxigenic Aspergillus minisclerotigenes strains MRI390 and MRI400 using the MiSeq and PacBio platforms and the generated assemblies. The strains were isolated from Kenyan maize kernels.

Formation of B- and M-group aflatoxins and precursors by Aspergillus flavus on maize and its implication for food safety.

Aflatoxins count to the most toxic known mycotoxins and are a threat to food safety especially in regions with a warm and humid climate. Contaminated food reaches consumers globally due to international trade, leading to stringent regulatory limits of aflatoxins in food. While the formation of aflatoxin (AF) B1 by the filamentous fungus Aspergillus flavus is well investigated, less is known about the formation kinetics of its precursors and further aflatoxins. In this study, autoclaved maize kernels were inoculated with A. flavus and incubated at 25 °C for up to 10 days. Aflatoxins and precursors were analyzed by a validated UHPLC-MS method. Additional to AFB1 and AFB2, AFM1 and AFM2 were detected, confirming the ability of the formation of M-group aflatoxins on cereals by A. flavus. The measured relative levels of AFB2, AFM1, and AFM2 on maize compared to the level of AFB1 (mean of days 5, 7, and 10 of incubation) were 3.3%, 1.5%, and 0.2%, respectively. The occurrence and kinetics of the measured aflatoxins and their precursors sterigmatocystin, O-methylsterigmatocystin, 11-hydroxy-O-methylsterigmatocystin, aspertoxin, and 11-hydroxyaspertoxin (group 1) as well as of dihydrosterigmatocystin and dihydro-O-methylsterigmatocystin (group 2) supported the so far postulated biosynthetic pathway. Remarkable high levels of O-methylsterigmatocystin and aspertoxin (17.4% and 4.9% compared to AFB1) were found, raising the question about the toxicological relevance of these intermediates. In conclusion, based on the study results, the monitoring of O-methylsterigmatocystin and aspertoxin as well as M-group aflatoxins in food is recommended.

Draft Genome Sequence of an Aflatoxin-Producing Aspergillus flavus Strain Isolated from Food.

Aspergillus flavus is the main producer of carcinogenic aflatoxins and thus is one of the most important fungal food contaminants. Here, we report that the genome of A. flavus strain MRI19 was sequenced using MiSeq and PacBio platforms and that a hybrid assembly was generated.

Analysis of the competitiveness between a non-aflatoxigenic and an aflatoxigenic Aspergillus flavus strain on maize kernels by droplet digital PCR.

Non-aflatoxigenic Aspergillus flavus strains are used as a biocontrol system on maize fields to decrease the aflatoxin biosynthesis of aflatoxigenic A. flavus strains. A. flavus strain AF36 was the first commercially available biocontrol strain and is authorized for use on maize fields by the US Environmental Protection Agency, e.g., in Texas and Arizona. A droplet digital PCR (ddPCR) assay was developed to analyze the mechanisms of competition and interaction of aflatoxigenic and non-aflatoxigenic A. flavus strains. This assay enables the parallel identification and quantification of the biocontrol strain A. flavus AF36 and the aflatoxigenic A. flavus strain MRI19. To test the assay, spores of both strains were mixed in varying ratios and were incubated on maize-based agar or maize kernels for up to 20 days. Genomic equivalent ratios (genome copy numbers) of both strains were determined by ddPCR at certain times after incubation and were compared to the spore ratios used for inoculation. The aflatoxin biosynthesis was also measured. In general, A. flavus MRI19 had higher competitiveness in the tested habitats compared to the non-aflatoxigenic strain, as indicated by higher final genomic equivalent ratios of this strain compared to the spore ratios used for inoculation. Nevertheless, A. flavus AF36 effectively controlled aflatoxin biosynthesis of A. flavus MRI19, as a clear aflatoxin inhibition was already seen by the inoculation of 10% spores of the biocontrol strain mixed with 90% spores of the aflatoxigenic strain compared to samples inoculated with only spores of the aflatoxigenic A. flavus MRI19.

Influence of Light and Water Activity on Growth and Mycotoxin Formation of Selected Isolates of Aspergillus flavus and Aspergillus parasiticus.

Aspergillus flavus and A. parasiticus are the main causes of aflatoxin contamination in various foods, particularly grains, as they can thrive in environments with lower water activity and higher temperatures. The growth of Aspergillus and the formation of the mycotoxins aflatoxin and cyclopiazonic acid are strongly influenced by environmental stimuli and can be reduced by modulating parameters such as water activity, pH, temperature and light during the storage. This study has two objectives-on the one hand, to assess how global warming and an increase in exposure to sunlight affect growth and mycotoxin formation, and on the other hand, how the findings from these experiments can be used to reduce fungal growth and mycotoxin formation in stored foods. Using growth substrates with two different water activities (aw 0.95, aw 0.98), together with a light incubation device consisting of different chambers equipped with diodes emitting visible light of five different wavelengths (455 nm, 470 nm, 530 nm, 590 nm, 627 nm) plus white light, we analyzed the growth and mycotoxin formation of selected Aspergillus flavus and A. parasiticus isolates. It was shown that light with a wavelength of 455/470 nm alone, but especially in combination with a lower water activity of aw 0.95, leads to a significant reduction in growth and mycotoxin formation, which was accompanied by reduced transcriptional activity of the responsible mycotoxin biosynthetic genes. Therefore, these results can be used to significantly reduce the growth and the mycotoxin formation of the analyzed fungi during storage and to estimate the trend of fungal infestation by Aspergillus flavus and A. parasiticus in water activity- and light exposure-equivalent climate change scenarios. Mycotoxin-producing aspergilli can be effective and sustainably inhibited using a combination of short-wave light and lowered water activity in the substrate. A higher annual mean temperature accompanying climate change may lead to an increased spread of aflatoxin-producing fungi in areas that were previously too cold for them. On the other hand, there will be regions in the world where contamination with aflatoxin-producing fungi will be reduced due to increased drought and sun exposure.

Whole-Genome Sequencing of the Fungus Penicillium citrinum Reveals the Biosynthesis Gene Cluster for the Mycotoxin Citrinin.

Penicillium citrinum is a food-contaminating ascomycete that consistently produces large amounts of the mycotoxin citrinin. Citrinin exhibits, besides its toxicity, antibiotic effects and thus potentially forces antibiotic resistance. Within the genome sequence, we identified the biosynthesis gene cluster for citrinin, which appears to be highly conserved within the genus Penicillium.

Sequencing and Analysis of the Genome of the Filamentous Fungus Penicillium verrucosum BFE808, Which Produces the Mycotoxins Citrinin and Ochratoxin.

Penicillium verrucosum is a filamentous ascomycete that occurs worldwide. Various cereals and the products thereof are the main habitats of this fungal species, where it produces the mycotoxins ochratoxin and citrinin. Here, we report the first draft genome sequence of P. verrucosum strain BFE808, isolated from wheat kernels.

Citrinin as an accessory establishment factor of P. expansum for the colonization of apples.

Penicillium expansum is the causal agent of blue mold decay of apples. This fungal species can produce the two important mycotoxins patulin and citrinin. It was previously shown that patulin represents a colonization factor for the infection of apples. No definitive information about the importance of citrinin for the colonization of apples is currently available. The pksCT gene of the citrinin cluster codes for the citrinin polyketide synthase. Mutants of P. expansum in which the pksCT was inactivated showed a drastic decrease in the citrinin production. In addition, the pksCT mutants were also reduced in the ability to colonize apples. Externally added citrinin restored the capacity of the mutants to colonize apples roughly to that of the wild type. A kinetic analysis of the expression of the two respective pks genes of patulin (patK) and citrinin (pksCT) revealed that both genes are highly expressed in the first phase during the colonization process. The production of patulin in the apple matrix coincides with the expression of the patK gene. Almost no citrinin could be identified analytically during the first phase but only at a later stage of the colonization. It could be demonstrated that citrinin is degraded in apples and can tightly be bound to pectin. Overall the results suggest that citrinin may have an accessory function for the establishment of the colonization guided by other factors.

Influence of Environmental Factors on the Production of Penitrems A-F by Penicillium crustosum.

Filamentous fungi produce a multitude of secondary metabolites, some of them known as mycotoxins, which are toxic to vertebrates and other animal groups in low concentrations. Among them, penitrems, which belong to the group of indole-diterpene mycotoxins, are synthesized by Penicillium and Aspergillus genera and exhibit potent tremorgenic effects. This is the first complex study of the penitrems A-F production under the influence of different abiotic factors, e.g., media, incubation time, temperature, pH, light, water activity, and carbon and nitrogen source as well as oxidative and salt stress. For this purpose, penitrems A-F were isolated from Penicillium crustosum cultures and used as analytical standards. Among the carbon sources, glucose supplemented to the media at the concentration of 50 g/L, showed the strongest inducing effect on the biosynthesis of penitrems. Among nitrogen sources, glutamate was found to be the most favorable supplement, significantly increasing production of these secondary metabolites. CuSO4-promoted oxidative stress was also shown to remarkably stimulate biosynthesis of all penitrems. In contrast, the salt stress, caused by the elevated concentrations of NaCl, showed an inhibitory effect on the penitrem biosynthesis. Finally, cheese model medium elicited exceptionally high production of all members of the penitrems family. Obtained results give insides into the biosynthesis of toxicologically relevant penitrems A-F under different environmental factors and can be utilized to prevent food contamination.

Sulfoglucosides as Novel Modified Forms of the Mycotoxins Alternariol and Alternariol Monomethyl Ether.

The mycotoxins alternariol and alternariol-9-O-methyl ether have recently been reported to be extensively conjugated with glucose and malonyl glucose in tobacco suspension cells. However, only trace amounts of glucosylated conjugates were detected in tomatoes inoculated with Alternaria alternata in the present study. Instead, mostly sulfate conjugates were observed. In studies using cultures of A. alternata and incubations of alternariol and alternariol-9-O-methyl ether with tomato tissue in the absence of the fungus, it was clarified that sulfate conjugates were produced by the fungus, whereas tomato tissues converted alternariol and alternariol-9-O-methyl ether to glucosylated metabolites. Alternariol-3-sulfate, alternariol-9-sulfate, and alternariol-9-O-methyl ether-3-sulfate were unambiguously identified as fungal metabolites using MS and 1H and 13C NMR spectroscopy. When these sulfate conjugates were incubated with tobacco suspension cells or ex planta tomato tissues, three sulfoglucosides of alternariol and one sulfoglucoside of alternariol-9-O-methyl ether were formed. Using NMR spectroscopy, the chemical structures of alternariol-3-sulfate-9-glucoside, alternariol-9-sulfate-3-glucoside, and alternariol-9-O-methyl ether-3-sulfate-7-glucoside were established. These conjugates were also detected in the A. alternata-inoculated tomato. This is the first report on a mixed sulfate/glucoside diconjugate of a mycotoxin. Diconjugates of this novel type may be formed by all mycotoxins and their phase I metabolites with two or more hydroxyl groups and should be taken into account in the future analysis of modified mycotoxins.

Arginine acts as an inhibitor of the biosynthesis of several mycotoxins.

It is well known that the type and the availability of nitrogen have a great influence on the biosynthesis of certain mycotoxins. Here it is shown that some amino acids have no influence, some others strongly support and a third group inhibits the biosynthesis of ochratoxin (OTA) by Penicillium nordicum even in a complex medium, such as PDA. Arginine (Arg) is one of the strong OTA inhibiting amino acids. It was shown that Arg not only inhibits OTA in Penicillium but also citrinin (CIT) biosynthesis in Penicillium verrucosum, Penicillium expansum and Penicillium citrinum and alternariol (AOH), alternariol monomethylether (AME) and tenuazonic acid (TeA) biosynthesis in Alternaria alternata. The minimal inhibitory concentration of Arg differs depending on the mycotoxin and the species analysed. However, the OTA biosynthesis by P. verrucosum and P. nordicum was most sensitive. Growth, on the other hand, was much less affected by Arg. Urea, a metabolite of Arg catabolism, shows a similar inhibitory activity. In wheat medium containing 50mM Arg almost no OTA was produced by Penicillium, in contrast to plain wheat medium.

Influence of Different Nanomaterials on Growth and Mycotoxin Production of Penicillium verrucosum.

Nanoparticles are ubiquitous in the environment. They originate from anthropogenic or natural sources or they are intentionally produced for different purposes. There exist manifold applications of nanoparticles in modern life leading unavoidably to a confrontation and interaction between nanomaterial and living organisms. Based on their wide distribution tending to increase steadily, the influence of particles based on silica and silver, exhibiting nominal sizes between 0.65 nm and 200 nm, on the physiology of the mycotoxigenic filamentous fungus Penicillium verrucosum was analyzed. The applied concentration and time-point, the size and the chemical composition of the particles was shown to have a strong influence on growth and mycotoxin biosynthesis. On microscopic scale it could be shown that silver nanoparticles attach to the mycelial surface. Moreover, silver nanoparticles with 0.65 nm and 5 nm in size were shown to internalize within the cell, form agglomerates in the cytoplasm and associate to cell organelles.

Impacts of environmental stress on growth, secondary metabolite biosynthetic gene clusters and metabolite production of xerotolerant/xerophilic fungi.

This paper examines the impact that single and interacting environmental stress factors have on tolerance mechanisms, molecular ecology and the relationship with secondary metabolite production by a group of mycotoxigenic species of economic importance. Growth of these fungi (Aspergillus flavus, A.ochraceus, A.carbonarius, Penicillium nordicum and P. verrucosum) is influenced by water and temperature interactions and type of solute used to induce water stress. Such abiotic stresses are overcome by the synthesis of increased amounts of low molecular weight sugar alcohols, especially glycerol and erythritol, to enable them to remain active under abiotic stress. This is accompanied by increased expression of sugar transporter genes, e.g., in A. flavus, which provides the nutritional means of tolerating such stress. The optimum conditions of water activity (a w) × temperature stress for growth are often different from those for secondary metabolite production. The genes for toxin production are clustered together and their relative expression is influenced by abiotic interacting stress factors. For example., A. flavus synthesises aflatoxins under water stress in non-ionic solutes. In contrast, P. nordicum specifically occupies a high salt (0.87 a w = 22% NaCl) niche such as cured meats, and produces ochratoxin A (OTA). There is differential and temporal expression of the genes in the secondary metabolite clusters in response to a w × temperature stress. We have used a microarray and integrated data on growth, relative expression of key genes in the biosynthetic pathways for secondary metabolite production and toxin production using a mixed growth model. This was used to correlate these factors and predict the toxin levels produced under different abiotic stress conditions. This system approach to integrate these different data sets and model the relationships could be a powerful tool for predicting the relative toxin production under extreme stress conditions, including climate change scenarios. This approach will facilitate a better functional understanding of the influence that environmental stress has on these mycotoxigenic fungi and enable better prevention strategies to be developed based on this system-based approach.

Oxidative stress induces the biosynthesis of citrinin by Penicillium verrucosum at the expense of ochratoxin.

Penicillium verrucosum is a fungus that can produce ochratoxin A and citrinin, two structurally related nephrotoxic mycotoxins. P. verrucosum usually occurs on wheat but can occasionally also be found in NaCl rich habitats such as salted cheeses or olives, indicating that this fungus can adapt to different environments. The ratio of ochratoxin A to citrinin produced by P. verrucosum is shifted to one of either mycotoxin at the expense of the other dependent on the environmental conditions. High NaCl concentrations shift secondary metabolite biosynthesis towards ochratoxin A production. P. verrucosum copes with NaCl stress by increased ochratoxin A biosynthesis, ensuring chloride homeostasis. Ochratoxin A carries chlorine in its molecule and can excrete chlorine from the cell. It was further shown that the regulation of ochratoxin A by high NaCl conditions is mediated by the HOG MAP kinase signal transduction pathway. Here it is shown that high oxidative stress conditions, evoked for example by increasing concentrations of Cu(2+) cations in the growth medium, shift secondary metabolite biosynthesis of P. verrucosum from ochratoxin A to citrinin. The production of citrinin normalizes the oxidative status of the fungal cell under oxidative stress conditions leading to an adaptation to these environmental conditions and protects against increased oxidative stress caused by increased Cu(2+) concentrations. Moreover citrinin also protects against light of short wavelength, which may also increase the oxidative status of the environment. The biosynthesis of citrinin is apparently regulated by a cAMP/PKA signaling pathway, because increasing amounts of external cAMP reduce citrinin biosynthesis in a concentration dependent manner. These conditions lead to the cross-regulation of the ochratoxin A/citrinin secondary metabolite pair and support the adaptation of P. verrucosum to different environments.

Comparative proteome analysis of Penicillium verrucosum grown under light of short wavelength shows an induction of stress-related proteins associated with modified mycotoxin biosynthesis.

In this study the differentially expressed protein population of Penicillium verrucosum grown either in the dark or under light with a wavelength of 450nm has been analyzed. Light of short wavelength led to oxidative stress in the fungal cell; under this condition the mycotoxin biosynthesis revealed a mutual shift from ochratoxin A to citrinin. Using a proteomic approach combining an optimized protein extraction method with 2-dimensional SDS-PAGE followed by HPLC-ESI-TOF-MS/MS mass spectrometric analysis, initially 56 significantly differential proteins (light vs. dark) were detected comprising proteins of a broad range of isoelectric points and molecular masses. In total, 46 proteins could be identified further by database query, most of these proteins are assumed to be involved in response to stress (e.g. antioxidative proteins, heat shock proteins) and general metabolic processes (e.g. glycolysis, ATP supply). Proteome analyses are necessary to unravel the regulation of secondary metabolite biosynthesis at a translational level. This may enable identification of proteins which are involved in mycotoxin biosynthesis, adaption processes or even stress compensation mechanisms. This study depicts the first proteome analysis of P. verrucosum.

Differences in the regulation of ochratoxin A by the HOG pathway in Penicillium and Aspergillus in response to high osmolar environments.

Penicillium verrucosum, P. nordicum and Aspergillus carbonarius are three important ochratoxin A producing species. P. verrucosum is in addition able to produce citrinin. It has been shown earlier that P. nordicum is adapted to NaCl rich environments like salt rich dry cured foods or even salines. In this organism, the biosynthesis of ochratoxin A plays an adaptive role in this habitat. P. verrucosum generally can be found on cereals, but occasionally also on salt rich dry cured foods. In contrast A. carbonarius usually cannot be found in NaCl rich environments, but it occurs in another environment with high concentration of solutes, e.g., in sugar rich substrates like grapes and grape juices. Usually osmotic challenging conditions activate the HOG MAP kinase signal cascade, which in turn activates various osmo-regulated genes. In the current analysis, it could be demonstrated that in case of P. nordicum and P. verrucosum the NaCl induced production of ochratoxin A is correlated to the phosphorylation status of the HOG MAP kinase. Just the opposite was true for A. carbonarius. In this case, also higher amounts of NaCl in the medium lead to an increased phosphorylation status of HOG, but no increase in ochratoxin biosynthesis was observed. In contrast to the Penicillia, higher NaCl concentrations lead to a rapid cessation of growth by A. carbonarius. High glucose concentrations have much less impact on growth and the phosphorylation of HOG.

Intraspecific variability of HOG1 phosphorylation in Penicillium verrucosum reflects different adaptation levels to salt rich habitats.

Mycotoxin biosynthesis in Penicillium verrucosum is modulated by different molecular regulation mechanisms. One important mechanism is the HOG1 (high osmolarity glycerol) MAP kinase signaling pathway. In a comparative analysis three different P. verrucosum strains were selected from six strains with different ability to produce the mycotoxins ochratoxin and citrinin. The fungal strains were grown on laboratory medium supplemented with different concentrations of NaCl. It could be shown that there exists an interrelationship between the growth rate, the level of HOG phosphorylation and the mycotoxin biosynthesis under the respective growth condition. The weak to non ochratoxin producing P. verrucosum strain, BFE875, showed only a poor growth rate but the strongest HOG1 phosphorylation; the strong ochratoxin and citrinin producing strain BFE575 showed a reasonable HOG1 phosphorylation with an average growth rate; and the strong ochratoxin and weak citrinin producing strain BFE495 showed only a poor phosphorylation but the highest growth rate in 7days of incubation at 25°C. The magnitude of phosphorylation of the HOG1 protein seems to be inversely correlated with the degree of adaption of the fungus to hyperosmotic growth conditions.

Integrating toxin gene expression, growth and fumonisin B1 and B2 production by a strain of Fusarium verticillioides under different environmental factors.

The objective of this study was to integrate data on the effect of water activity (a(w); 0.995-0.93) and temperature (20-35 °C) on activation of the biosynthetic FUM genes, growth and the mycotoxins fumonisin (FB1, FB2) by Fusarium verticillioides in vitro. The relative expression of nine biosynthetic cluster genes (FUM1, FUM7, FUM10, FUM11, FUM12, FUM13, FUM14, FUM16 and FUM19) in relation to the environmental factors was determined using a microarray analysis. The expression was related to growth and phenotypic FB1 and FB2 production. These data were used to develop a mixed-growth-associated product formation model and link this to a linear combination of the expression data for the nine genes. The model was then validated by examining datasets outside the model fitting conditions used (35 °C). The relationship between the key gene (FUM1) and other genes in the cluster (FUM11, FUM13, FUM9, FUM14) were examined in relation to aw, temperature, FB1 and FB2 production by developing ternary diagrams of relative expression. This model is important in developing an integrated systems approach to develop prevention strategies to control fumonisin biosynthesis in staple food commodities and could also be used to predict the potential impact that climate change factors may have on toxin production.

Wavelength-dependent degradation of ochratoxin and citrinin by light in vitro and in vivo and its implications on Penicillium.

It has previously been shown that the biosynthesis of the mycotoxins ochratoxin A and B and of citrinin by Penicillium is regulated by light. However, not only the biosynthesis of these mycotoxins, but also the molecules themselves are strongly affected by light of certain wavelengths. The white light and blue light of 470 and 455 nm are especially able to degrade ochratoxin A, ochratoxin B and citrinin after exposure for a certain time. After the same treatment of the secondary metabolites with red (627 nm), yellow (590 nm) or green (530 nm) light or in the dark, almost no degradation occurred during that time indicating the blue light as the responsible part of the spectrum. The two derivatives of ochratoxin (A and B) are degraded to certain definitive degradation products which were characterized by HPLC-FLD-FTMS. The degradation products of ochratoxin A and B did no longer contain phenylalanine however were still chlorinated in the case of ochratoxin A. Citrinin is completely degraded by blue light. A fluorescent band was no longer visible after detection by TLC suggesting a higher sensitivity and apparently greater absorbance of energy by citrinin. The fact that especially blue light degrades the three secondary metabolites is apparently attributed to the absorption spectra of the metabolites which all have an optimum in the short wave length range. The absorption range of citrinin is, in particular, broader and includes the wave length of blue light. In wheat, which was contaminated with an ochratoxin A producing culture of Penicillium verrucosum and treated with blue light after a pre-incubation by the fungus, the concentration of the preformed ochratoxin A reduced by roughly 50% compared to the control and differed by > 90% compared to the sample incubated further in the dark. This indicates that the light degrading effect is also exerted in vivo, e.g., on food surfaces. The biological consequences of the light instability of the toxins are discussed.