Alternariol as virulence and colonization factor of Alternaria alternata during plant infection.

The filamentous fungus Alternaria alternata is a potent producer of many toxic secondary metabolites, which contaminate food and feed. The most prominent one is the polyketide-derived alternariol (AOH) and its derivative alternariol monomethyl ether (AME). Here, we identified the gene cluster for the biosynthesis of AOH and AME by CRISPR/Cas9-mediated gene inactivation of several biosynthesis genes in A. alternata and heterologous expression of the gene cluster in Aspergillus oryzae. The 15 kb-spanning gene cluster consists of a polyketide synthase gene, pksI, an O-methyltransferase, omtI, a FAD-dependent monooxygenase, moxI, a short chain dehydrogenase, sdrI, a putative extradiol dioxygenase, doxI and a transcription factor gene, aohR. Heterologous expression of PksI in A. oryzae was sufficient for AOH biosynthesis. Co-expression of PksI with different tailoring enzymes resulted in AME, 4-hydroxy-alternariol monomethyl ether (4-OH-AME), altenusin (ALN) and altenuene (ALT). Hence, the AOH cluster is responsible for the production of at least five different compounds. Deletion of the transcription factor gene aohR led to reduced expression of pksI and delayed AOH production, while overexpression led to increased expression of pksI and production of AOH. The pksI-deletion strain displayed reduced virulence on tomato, citrus and apple suggesting AOH and the derivatives as virulence and colonization factors.

The life and times of yeasts in traditional food fermentations.

Yeasts are eukaryotic microorganisms which have a long history in the biotechnology of food production, as they have been used since centuries in bread-making or in the production of alcoholic beverages such as wines or beers. Relative to this importance, a lot of research has been devoted to the study of yeasts involved in making these important products. The role of yeasts in other fermentations in association with other microorganisms - mainly lactic acid bacteria - has been relatively less studied, and often it is not clear if yeasts occurring in such fermentations are contaminants with no role in the fermentation, spoilage microorganisms or whether they actually serve a technological or functional purpose. Some knowledge is available for yeasts used as starter cultures in fermented raw sausages or in the production of acid curd cheeses. This review aimed to summarize the current knowledge on the taxonomy, the presence and potential functional or technological roles of yeasts in traditional fermented plant, dairy, fish and meat fermentations.

Comprehensive proteomic analysis of Penicillium verrucosum.

Mass spectrometric identification of proteins in species lacking validated sequence information is a major problem in veterinary science. In the present study, we used ochratoxin A producing Penicillium verrucosum to identify and quantitatively analyze proteins of an organism with yet no protein information available. The work presented here aimed to provide a comprehensive protein identification of P. verrucosum using shotgun proteomics. We were able to identify 3631 proteins in an "ab initio" translated database from DNA sequences of P. verrucosum. Additionally, a sequential window acquisition of all theoretical fragment-ion spectra analysis was done to find differentially regulated proteins at two different time points of the growth curve. We compared the proteins at the beginning (day 3) and at the end of the log phase (day 12).

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.

Expansion of the multi-locus gene alignment approach to improve identification of the fungal species Alternaria alternata.

Alternaria alternata is part of a genus comprised of over 600 different species that occur all over the world and cause damage to humans, plants and thereby to the economy. Yet, even though some species are causing tremendous issues, the past years have shown that assigning newly found isolates to known species was rather inconsistent. Most identifications are usually done on the basis of spore morphology, chemotype and molecular markers. In this work we used strains isolated from the wild as well as commercial strains of the DSMZ (German collection of microorganisms and cell cultures) as a reference, to show, that the variation within the Alternaria alternata species is comparable to the variation between different species of the genus Alternaria in regards to spore morphology and chemotype. We compared the different methods of identification and discerned the concatenation of multiple molecular markers as the deciding factor for better identification. Up until this point, usually a concatenation of two or three traditional molecular markers was used. Some of those markers being stronger some weaker. We show that the concatenation of five molecular markers improves the likeliness of a correct assignment, thus a better distinction between the different Alternaria species.

Comparative analysis of the genomes and aflatoxin production patterns of three species within the Aspergillus section Flavi reveals an undescribed chemotype and habitat-specific genetic traits.

Aflatoxins are the most dangerous mycotoxins for food safety. They are mainly produced by Aspergillus flavus, A. parasiticus, and A. minisclerotigenes. The latter, an understudied species, was the main culprit for outbreaks of fatal aflatoxicosis in Kenya in the past. To determine specific genetic characteristics of these Aspergillus species, their genomes are comparatively analyzed. Differences reflecting the typical habitat are reported, such as an increased number of carbohydrate-active enzymes, including enzymes for lignin degradation, in the genomes of A. minisclerotigenes and A. parasiticus. Further, variations within the aflatoxin gene clusters are described, which are related to different chemotypes of aflatoxin biosynthesis. These include a substitution within the aflL gene of the A. parasiticus isolate, which leads to the translation of a stop codon, thereby switching off the production of the group 1 aflatoxins B1 and G1. In addition, we demonstrate that the inability of the A. minisclerotigenes isolates to produce group G aflatoxins is associated with a 2.2 kb deletion within the aflF and aflU genes. These findings reveal a relatively high genetic homology among the three Aspergillus species investigated. However, they also demonstrate consequential genetic differences that have an important impact on risk-assessment and food safety.

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.

The biosynthesis of ochratoxin A by Penicillium as one mechanism for adaptation to NaCl rich foods.

Penicillium.nordicum is an ochratoxin A producing filamentous fungus, which is adapted to sodium chloride and protein rich food environments like certain cheeses or dry cured meats. Penicillium.verrucosum usually occurs on cereals but can also be isolated from brined olives. It could be shown that sodium chloride has a profound influence on the regulation of ochratoxin A biosynthesis in both Penicillium species. High amounts of ochratoxin A are produced by P. nordicum over a wide concentration range of NaCl (5-100 g/l) with a weak optimum at about 20 g/l after growth on YES medium. P. verrucosum shifts secondary metabolite biosynthesis after growth on YES medium from citrinin at low to ochratoxin at elevated NaCl concentrations. The ochratoxin A biosynthesis of P. nordicum is accompanied by an induction of the otapksPN gene, the gene of the ochratoxin A polyketide synthase. A mutant strain unable to produce ochratoxin showed a drastic growth reduction under high NaCl conditions. Determination of the dry weight and the chloride content in the mycelium of the P. nordicum wild type strain and a non-ochratoxin A producing mutant strain showed a much higher increase of both parameters in the mutant compared to the wild type. These results suggest, that the constant biosynthesis and excretion of ochratoxin A, which itself contains a chloride atom, ensures a partial chloride homeostasis in the fungal cell. This mechanism may support the adaptation of ochratoxin A producing Penicillia to NaCl rich foods.

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.

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.

Sequencing and Analysis of the Entire Genome of the Mycoparasitic Fungus Trichoderma afroharzianum.

Trichoderma sp. is a globally occurring fungal ascomycete. The genus Trichoderma belongs to the order of Hypocreales in the class of Sordariomycetes. Due to its importance as a mycoparasite and biocontrol fungus that antagonizes phytopathogenic and mycotoxin-producing fungi, the genome of the Trichoderma afroharzianum strain BFE349 from the fungal strain collection of the Max Rubner-Institut was sequenced and analyzed.

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.

Stress induction of mycotoxin biosynthesis genes by abiotic factors.

Systematic expression analysis of mycotoxin biosynthesis genes by real-time PCR and microarray was carried out to examine the relationship between growth and general expression patterns in relation to single environmental factors such as temperature, water activity (a(w)) and pH and water activity x temperature interactions. For single parameters, one major peak of expression occurred close to optimum growth conditions. However, a second minor peak was observed under suboptimal growth conditions, when intermediate environmental stress was imposed on Aspergillus parasiticus (afl genes), Penicillium verrucosum (ota genes) and Fusarium culmorum (tri genes). This expression profile pattern was more pronounced in relation to changes in temperature and a(w) than to pH. In a two-factorial experimental design with temperature xa(w) regimes, again two peaks of expression were observed for cluster genes after microarray analysis, one close to those giving optimal growth and one under imposed stress conditions. Interestingly, when the activity of single genes of the microarray data were plotted in relation to the two parameters, again a two-peak expression profile became obvious independently for both parameters. Expression of the mycotoxin biosynthesis genes was followed exactly by phenotypic mycotoxin production. This expression profile appears to be generic across the mycotoxigenic fungi examined.

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.

A microarray for monitoring the production of mycotoxins in food.

A microarray, which covers most of the known relevant mycotoxin biosynthesis genes, has been developed. The microarray carries oligonucleotides of the fumonisin, the aflatoxin, the ochratoxin, the trichothecene (type A and B) and the patulin biosynthesis pathways. For trichothecene producing Fusaria the biosynthesis cluster of trichothecene producing Fusarium sporotrichioides (type A) and of Gibberrella zeae (type B, teleomorph of F. graminearum) have been spotted. The aflatoxin cluster carries oligonucleotides specific for Aspergillus flavus. The ochratoxin pattern is specific for ochratoxin A producing Penicillia, the fumonisin cluster is specific for G. moniliformis (teleomorph of F. verticillioides) and the patulin genes have been obtained from Penicillium expansum. The microarray is designed in a way that newly identified pathway genes can be added easily at any time. The microarray was used to detect the activation of all gene clusters under conditions conducive for mycotoxin biosynthesis. According to the results the obtained signals were specific under the hybridization conditions used and only insignificant cross-hybridizations occurred. The microarray was used to demonstrate differences in mycotoxin pathway gene expressions after growth on various media for trichothecene and ochratoxin A biosynthesis. It was used further to study and compare the expression kinetics of the trichothecene biosynthesis genes of Fusarium on different trichothecene supporting media. An expression pattern indicative for trichothecene biosynthesis could be identified.

Physiological relationship between food preservatives, environmental factors, ochratoxin and otapksPV gene expression by Penicillium verrucosum.

There is significant interest in trying to understand the relationship between environmental factors, preservative concentration and expression of genes involved in mycotoxin production. However, little information is available on the link between physiological stress factors and expression of genes responsible for mycotoxin production. This study has examined the effect of interactions between ionic and non-ionic water availability modified with glycerol or NaCl (a(w), 0.98, 0.95 and 0.93) and sub-optimal concentrations of calcium propionate and potassium sorbate (150, 300 ppm) at 25 degrees C on growth, ochratoxin A (OTA) and otapksPV gene expression by the mycotoxigenic species Penicillium verrucosum. Growth was inhibited between 25-35% by the preservatives at each a(w) level. However, OTA production was stimulated by 150 and 300 ppm of both preservatives, especially at 0.95 and 0.93 a(w). If water activity as a single stress factor was changed, a typical OTA production and otapksPV expression profile occurred, indicating that OTA biosynthesis is activated under two conditions, i.e., under optimal growth conditions and under weak stress conditions. Temporal analysis of otapksPV expression showed that there was an optimum after 8-9 days incubation. Statistical analyses indicated good correlation between sub-optimal concentrations of preservatives, intermediate a(w) levels and genotypic and phenotypic gene and toxin production. This is the first time that genotypic information has been linked to phenotypic mycotoxin production in relation to such interacting stress factors.