A sensitive and inexpensive yeast bioassay for the mycotoxin zearalenone and other compounds with estrogenic activity

Zearalenone (ZON) is a nonsteroidal estrogenic mycotoxin produced by plant-pathogenic species of Fusarium. As a consequence of infection with Fusarium culmorum and Fusarium graminearum, ZON can be found in cereals and derived food products. Since ZON is suspected to be a cause of human disease, including premature puberty syndrome, as well as hyperestrogenism in farm animals, several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We developed a low-cost method for monitoring ZON contamination in grain based on a sensitive yeast bioassay. The indicator Saccharomyces cerevisiae strain YZRM7 is unable to grow unless an engineered pyrimidine biosynthetic gene is activated by the expressed human estrogen receptor in the presence of exogenous estrogenic substances. Deletion of the genes encoding ATP-binding cassette (ABC) transporters Pdr5p and Snq2p increases net ZON uptake synergistically. Less than 1 microg of ZON per liter of medium is sufficient to allow growth of the indicator strain. To prevent interference with pyrimidines potentially present in biological samples, we also disrupted the genes FUR1 and URK1, blocking the pyrimidine salvage pathway. The bioassay strain YZRM7 allows qualitative detection and quantification of total estrogenic activity in cereal extracts without requiring further cleanup steps. Its high sensitivity makes this assay suitable for low-cost monitoring of contamination of maize and small grain cereals with estrogenic Fusarium mycotxins.     

Development and applications of a yeast-based bioassay for the mycotoxin zearalenone

Zearalenone (ZON) is a non-steroidal estrogenic mycotoxin produced by plant pathogenic species ofFusarium. As a consequence of infection withF. culmorum andF. graminearum, ZON can be found in cereals and derived food products. Several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We have developed a sensitive yeast bioassay allowing detection of the estrogenic activity of ZON in cereal extracts without requiring further clean up steps. The high sensitivity makes this assay suitable for low cost monitoring of contamination of small grain cereals with estrogenicFusarium mycotoxins, but also attractive as a tool for basic research. We have successfully used yeast indicator strains to screen for mutants ofF. graminearum which no longer produce detectable amounts of ZON, and have identified a plant cDNA encoding a ZON detoxification enzyme. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Fusarium Species from Nepalese Rice and Production of Mycotoxins and Gibberellic Acid by Selected Species

Infection of cereal grains with Fusarium species can cause contamination with mycotoxins that affect human and animal health. To determine the potential for mycotoxin contamination, we isolated Fusarium species from samples of rice seeds that were collected in 1997 on farms in the foothills of the Nepal Himalaya. The predominant Fusarium species in surface-disinfested seeds with husks were species of the Gibberella fujikuroi complex, including G. fujikuroi mating population A (anamorph, Fusarium verticillioides), G. fujikuroi mating population C (anamorph, Fusarium fujikuroi), and G. fujikuroi mating population D (anamorph, Fusarium proliferatum). The widespread occurrence of mating population D suggests that its role in the complex symptoms of bakanae disease of rice may be significant. Other common species were Gibberella zeae (anamorph, Fusarium graminearum) and Fusarium semitectum, with Fusarium acuminatum, Fusarium anguioides, Fusarium avenaceum, Fusarium chlamydosporum, Fusarium equiseti, and Fusarium oxysporum occasionally present. Strains of mating population C produced beauvericin, moniliformin, and gibberellic acid, but little or no fumonisin, whereas strains of mating population D produced beauvericin, fumonisin, and, usually, moniliformin, but no gibberellic acid. Some strains of G. zeae produced the 8-ketotrichothecene nivalenol, whereas others produced deoxynivalenol. Despite the occurrence of fumonisin-producing strains of mating population D, and of 8-ketotrichothecene-producing strains of G. zeae, Nepalese rice showed no detectable contamination with these mycotoxins. Effective traditional practices for grain drying and storage may prevent contamination of Nepalese rice with Fusarium mycotoxins.     

Monitoring of fusarium toxins in cereals and feed-stuffs from thuringia (1998 - 2001)

From 1998 to 2001 a total of about 1172 conventionally and organically produced samples of wheat, rye, barley and triticale were examined for the presence of deoxynivalenol (DON) and zearalenone (ZON). Furthermore, feedstuffs for pigs were included in the monitoring of Fusarium toxins. DON and ZON analyses were performed using ELISA or HPLC. The incidences and levels of toxins varied from year to year. Overall contamination levels were highest in wheat and triticale, followed by rye and barley. The highest DON contaminations were found in 1998. The probes of the years 1999-2001 showed lower incidences of Fusarium toxins. The second examined mycotoxin ZON was detected at lower levels in cereals. Similar results were observed in the monitoring of feedstuffs.     

Analysis of theFusarium toxins Deoxynivalenol and Zearalenone in Austrian feeds of the crop years 2002 and 2003

164 feed samples were collected in Lower Austria, Upper Austria, Styria and Carynthia in the crop years 2002 and 2003 and were analysed by means of the RIDASCREEN^® ELISA-assay for theFusarium toxins Deoxynivalenol (DON) and Zearalenone (ZON). The collected samples consisted of swine feeds, cereals and silages. DON concentrations were found up to the range of mg/kg (ppm), in contrast ZON results remained significantly lower.     

Characterization of Two Polyketide Synthase Genes Involved in Zearalenone Biosynthesis in Gibberella zeae

Zearalenone, a mycotoxin produced by several Fusarium spp., is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is a polyketide derived from the sequential condensation of multiple acetate units by a polyketide synthase (PKS), but the genetics of its biosynthesis are not understood. We cloned two genes, designated ZEA1 and ZEA2, which encode polyketide synthases that participate in the biosynthesis of zearalenone by Gibberella zeae (anamorph Fusarium graminearum). Disruption of either gene resulted in the loss of zearalenone production under inducing conditions. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Quantitative PCR analysis of both PKS genes and six flanking genes supports the view that the two polyketide synthases make up the core biosynthetic unit for zearalenone biosynthesis. An appreciation of the genetics of zearalenone biosynthesis is needed to understand how zearalenone is synthesized under field conditions that result in the contamination of grain.     

Induction of a zearalenone degrading enzyme caused by the substrate and its derivatives

The Fusarium toxin zearalenone (ZON) is very harmful to animal and man due to its estrogenic effect, immunotoxicity and genotoxicity. Therefore, it is of high importance to establish a system for the detoxification of ZON. In large screening programmes, only the mycoparasiteGliocladium roseum (DSM 62726) was found to be capable of detoxifying ZON, by not yet characterized enzyme(s). It is the only known microorganism hydrolyzing the lactonic bond within the macrocyclic ring system of ZON. The resulting products are less toxic because they loose their estrogenic capacity. The extent of toxin degradation is enhanced when enzyme production inG roseum is induced by the substrate ZON itself and its derivatives. ZON and its derivatives differ in the ability to induce enzyme production. This was investigated underin vitro conditions. Differences were found in the required amount of the inducing substances and time optimum of induction in order to get maximal degradation of ZON. The regulation and biochemical properties of the enzyme are to be characterized as a prerequisite to develop applications aimed at the detoxification of ZON in food and feeding-stuff. Our aim is to isolate the unknown enzyme which is capable of the detoxification of this mycotoxin.     

Comparison of the efficacy of chitosan with that of a fluorescent pseudomonad for the control of Fusarium head blight disease of cereals and associated mycotoxin contamination of grain

Fusarium culmorum can cause Fusarium head blight (FHB) disease of cereals, resulting in yield loss and contamination of grain with the trichothecene mycotoxin, deoxynivalenol (DON). In this study, we compared the efficacy of a biological control agent (Pseudomonas fluorescens strain MKB 158) with the biochemical chitosan (the deacetylated derivative of chitin) in controlling FHB disease of wheat and barley. Both agents were equally effective in reducing DON contamination of grain caused by F. culmorum. Under both glasshouse and field conditions, treatment with commercially available crabshell-derived chitosan reduced the severity of FHB symptom development on wheat and barley by ?74% (P ? 0.050). While treatment with P. fluorescens reduced the severity of FHB symptom development on these cereals by ?48% (P ? 0.050). Chitosan and P. fluorescens respectively prevented ?58 and ?35% of the FHB-associated reductions in 1000-grain weight in wheat and barley (P ? 0.050). Both agents significantly reduced the DON content of wheat and barley under both glasshouse and field conditions (P ? 0.050) and were equally efficacious in doing so (?74 and ?79% reductions due to chitosan and P. fluorescens, respectively). Crude chitin extracts from crabshells and crude chitosan-based formulations prepared from crabshells and eggshells were also tested under field conditions, but were not as effective as the commercial crabshell-derived preparation in controlling FHB disease. This is the first report on the use of chitosan for the control of Fusarium head blight disease and DON contamination of grain.     

A novel actinomycete derived from wheat heads degrades deoxynivalenol in the grain of wheat and barley affected by Fusarium head blight

Deoxynivalenol (DON) is a hazardous and globally prevalent mycotoxin in cereals. It commonly accumulates in the grain of wheat, barley and other small grain cereals affected by Fusarium head blight (caused by several Fusarium species). The concept of reducing DON in naturally contaminated grain of wheat or barley using a DON-degrading bacterium is promising but has not been accomplished. In this study, we isolated a novel DON-utilising actinomycete, Marmoricola sp. strain MIM116, from wheat heads through a novel isolation procedure including an in situ plant enrichment step. Strain MIM116 had background degradation activity, and the activity was enhanced twofold by the consumption of DON. Among Tween 20, Triton X-100 and Tween 80, we selected Tween 80 as a spreading agent of strain MIM116 because it promoted DON degradation and the growth of strain MIM116 in the presence of DON. The inoculation of MIM116 cell suspension plus 0.01% Tween 80 into 1,000 harvested kernels of wheat and barley resulted in a DON decrease from approximately 3 mg kg^?1 to less than 1 mg kg^?1 of dry kernels, even when cells had only basal levels of DON-degrading activity. To the best of our knowledge, this is the first report that describes (1) the isolation of a DON-degrading bacterium from wheat heads, (2) the effects of surfactants on the biodegradation of DON and (3) the decrease of DON levels in naturally contaminated wheat and barley grain using a DON-degrading bacterium.     

Fusarium cerealis Associated with Barley Seeds in Argentina

Fusarium head blight is a fungal disease caused by a complex of Fusarium species on cereals, such as barley and wheat. It has economic impacts due to yield reductions and mycotoxin contamination. As barley production has increased considerably in the last 5 years in Argentina, a survey was conducted for identifying Fusarium species associated with barley grains. Fusarium cerealis was isolated and identified based on morphological and molecular analysis. The potential production of nivalenol and zearalenone was assessed using specific PCR assays. Koch′s postulates were carried out to confirm the pathogenicity of the fungus.     

Metabolism and Resistance of Fusarium spp. to the Manzamine Alkaloids via a Putative Retro Pictet-Spengler Reaction and Utility of the Rational Design of Antimalarial and Antifungal Agents

As a part of our continuing investigation of the manzamine alkaloids we studied the in vitro activity of the β-carboline containing manzamine alkaloids against Fusarium solani, Fusarium oxysporium, and Fusarium proliferatum by employing several bioassay techniques including one-dimensional direct bioautography, dilution, and plate susceptibility, and microtiter broth assays. In addition, we also studied the metabolism of the manzamine alkaloids by Fusarium spp. in order to facilitate the redesign of the compounds to prevent resistance of Fusarium spp. through metabolism. The present research reveals that the manzamine alkaloids are inactive against Fusarium spp. and the fungi transform manzamines via hydrolysis, reduction, and a retro Pictet-Spengler reaction. This is the first report to demonstrate an enzymatically retro Pictet-Spengler reaction. The results of this study reveal the utility of the rational design of metabolically stable antifungal agents from this class and the development of manzamine alkaloids as antimalarial drugs through the utilization of Fusarium’s metabolic products to reconstruct the molecule.     

Toxinogenicity of<Emphasis Type="Italic">Microdochium nivale (Fusarium nivale)</Emphasis> isolates from cereals in Poland

Microdochium nivale (Fusarium nivale) was found to be frequently occuring in Poland pathogen of small grain cereals heads, causing symptoms similar to those observed after infection ofFusarium species. In consecutive years since 1985 till 1989 the following percentage of wheat and rye ears infected withM. Nivale and withFusarium head blight symptoms was found: 34%, 21%, 42%, 9%, 46% (wheat) and 57%, 43%, 65%, 4%, 47% (rye) heads.However, in naturally infected rye and wheat samples (kernels and chaff), we did not detect toxins usually present in samples infected with fungi of genusFusarium — such as deoxynivalenol and derivatives. TypicalFusarium trichothecene metabolites were also not present in cultures of 11M. nivale strains, growing 3–5 weeks on rice (45% water content) at 20°C. Cultures of two typical isolates on wheat grain (strain KF 1124) and on rice (KF 245) were found to be non toxic to broiler chickens when present in amount 20–40% in their diet. It can be concluded thatM. nivale (F. nivale) representatives in Poland did not produce toxic metabolites neither under laboratory condition nor after cereal ears infection under field conditions.     

Effects of<Emphasis Type="Italic">Fusarium-</Emphasis>toxincontaminated wheat in ruminant nutrition

An experiment was carried out to examine the effects of aFusarium-contaminated wheat grain as a component of the concentrate portion (10 mg deoxynivalenol and 0.76 mg zearalenone, ZON, per kg dry matter) on performance of growing bulls, and on carry over of ZON into tissues and body fluids. In a second study, rumen physiological parameters were investigated in wethers equipped with rumen fistulae. Moreover, the influences of a detoxifying agent (Mycofix®, MP, Biomin GmbH, Herzogenburg, Austria) were considered as an additional experimental factor beside the contamination of the wheat (uncontaminated control wheat,Fusarium-toxincontaminated wheat).The fattening experiment with bulls (n=14 per treatment) covered the live weight range between 244 kg and 460 kg. Daily dry matter intake and live weight gain (kg per animal and day) were 7.40, 7.52, 7.51 and 7.49 and 1.367, 1.296, 1.380 and 1.307 for bulls fed the unsupplemented control wheat, the supplemented control wheat, the unsupplemented andFusarium toxin contaminated wheat and the supplementedFusarium toxin contaminated wheat, respectively. Concentration of ZON and its metabolites in edible tissues were lower than the detection limits of the applied HPLC-method.The results of the rumen physiological investigations revealed that the molar ratios of short chained volatile fatty acids and ammonia concentration in rumen fluid remained unchanged in response to dietary treatments whereas the addition of MP to the diets buffered the postprandial decrease in rumen pH.     

Effects of Fusarium Toxin Contaminated Wheat and of a Detoxifying Agent on Performance of Growing Bulls,on Nutrient Digestibility in Wethers and on the Carry Over of Zearalenone

Experiments were carried out to examine the effects of a Fusarium contaminated wheat (10mg deoxynivalenol and 0.76mg zearalenone, ZON, perkg dry matter) and of a detoxifying agent (Mycofix ®Plus, Biomin GmbH, Herzogenburg, Austria) on the growing performance of bulls, carry-over of ZON and its metabolites into body fluids and tissues, and on nutrient digestibility in wethers. The experiments were designed according to a complete two by two factorial approach which meant that both the uncontaminated control wheat and the Fusarium toxin contaminated wheat were tested both in the absence and presence of Mycofix ®Plus. The growing experiment with bulls (n = 14 per treatment) covered the live weight range between 244kg and 460kg. The respective wheat batches were included in the concentrate portion at 65%. Concentrates were fed according to plan whereas maize silage was offered for ad libitum consumption. Daily dry matter intake and live weight gain [kg per animal and day] were 7.40, 7.52, 7.51 and 7.49 and 1.367, 1.296, 1.380 and 1.307 for bulls fed the unsupplemented control wheat, the supplemented control wheat, the unsupplemented and Fusarium toxin contaminated wheat and the supplemented Fusarium toxin contaminated wheat, respectively. ZON and its metabolites were not detected in edible tissues. The most striking effects of feeding the Fusarium toxin contaminated wheat on carcass characteristics were a reduced dressing percentage, an increased weight of the emptied gastro-intestinal tract and a reduced weight of the testicles. No effect of the detoxifying agent was seen for these parameters whereas heart weight increased independently of Fusarium toxin contamination of the concentrates. Nutrient digestibility of the two wheat batches, unsupplemented or supplemented with Mycofix ®Plus was evaluated according to the difference method using wethers. Presence of Fusarium toxins in wheat did not influence its feeding value. The effects of the addition of the detoxifying agent were mycotoxin unspecific and resulted in an increase in apparent digestibility of crude protein and a decrease in crude fiber digestibility. It is concluded that feeding of Fusarium toxin contaminated wheat did not adversely affect performance of growing bulls (approximately 2.2mg DON and 0.1mg ZON perkg complete ration at a reference dry matter content of 88%) or nutrient digestibility in wethers. The effects of the detoxifying agent Mycofix ®Plus on growing performance and on nutrient digestibility were rather Fusarium toxin unspecific. The slightly negative effects on growing performance needs to be examined further.     

Cleavage of Zearalenone by Trichosporon mycotoxinivorans to a Novel Nonestrogenic Metabolite

Zearalenone (ZON) is a potent estrogenic mycotoxin produced by several Fusarium species most frequently on maize and therefore can be found in food and animal feed. Since animal production performance is negatively affected by the presence of ZON, its detoxification in contaminated plant material or by-products of bioethanol production would be advantageous. Microbial biotransformation into nontoxic metabolites is one promising approach. In this study the main transformation product of ZON formed by the yeast Trichosporon mycotoxinivorans was identified and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and LC-diode array detector (DAD) analysis. The metabolite, named ZOM-1, was purified, and its molecular formula, C₁₈H₂₄O₇, was established by time of flight MS (TOF MS) from the ions observed at m/z 351.1445 [M-H]⁻ and at m/z 375.1416 [M+Na]⁺. Employing nuclear magnetic resonance (NMR) spectroscopy, the novel ZON metabolite was finally identified as (5S)-5-({2,4-dihydroxy-6-[(1E)-5-hydroxypent-1-en-1-yl]benzoyl}oxy)hexanoic acid. The structure of ZOM-1 is characterized by an opening of the macrocyclic ring of ZON at the ketone group at C6′. ZOM-1 did not show estrogenic activity in a sensitive yeast bioassay, even at a concentration 1,000-fold higher than that of ZON and did not interact with the human estrogen receptor in an in vitro competitive binding assay.Zearalenone (ZON) is the main member of a growing family of biologically important “resorcylic acid lactones” (RALs), which have been found in nature. ZON is produced by several Fusarium species, which colonize maize, barley, oat, wheat, and sorghum and tend to develop ZON during prolonged cool, wet growing and harvest seasons (38). Maize is the most frequently contaminated crop plant, and therefore, ZON can be found frequently in animal feeding stuff. Occurrence, toxicity, and metabolism data of ZON were summarized by the European Food Safety Authority (EFSA) (5) and in recent reviews (12, 38).The potent xenohormone ZON leads to hyperestrogenism symptoms and in extreme cases to infertility problems, especially in pigs (15). Ovarian changes in pigs have been noted with toxin levels as low as of 50 μg/kg in the diet (1). Ruminants are more tolerant to ZON ingestion; however, hyperestrogenic syndrome, including restlessness, diarrhea, infertility, decreased milk yields, and abortion, have been well documented with cattle and sheep (4, 29).Because widespread ZON contamination in feed can occur in problematic years, efficient ways to detoxify are desirable. The transformation of mycotoxins to nontoxic metabolites by pure cultures of microorganisms or by cell-free enzyme preparations (3) is an attractive possibility. Microbial metabolization of ZON to alpha-ZOL and beta-ZOL cannot be regarded as detoxification, because both ZOL products are still estrogenic (14). Also, formation of ZON-glucosides and -diglucosides (8, 17) and ZON-sulfate (7) cannot be considered true detoxification but rather formation of masked mycotoxins, because the conjugates may be hydrolyzed during digestion (11, 23), releasing ZON again (2).As the estrogenic activity of ZON and its derivates can be explained by its chemical structure, which resembles natural estrogens (20), it can be expected that cleavage of the lactone undecyl ring system of ZON results in permanent detoxification.El-Sharkawy and Abul-Hajj (9) were the first to report inactivation of ZON after opening of the lactone ring by Gliocladium roseum. This filamentous fungus was capable of metabolizing ZON in yields of 80 to 90%. Also Takahashi-Ando et al. (31) described the degradation reaction of ZON with Clonostachys rosea (synonym of G. roseum). A hydrolase (encoded by a gene designated ZHD101) cleaves the lactone ring, and as recently proved (37; unpublished data) by subsequent decarboxylation of the intermediate acid, the compound 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1′E-undecene-6′-one is formed. In contrast to ZON and 17β-estradiol, which showed potent estrogenic activity, this cleavage product did not show any estrogenic activity in the human breast cancer MCF-7 cell proliferation assay (16). Further details, e.g., on the conditions of the maximum activity of ZHD101 and its exploitation in genetically modified grains, can be found in later published work of this research group (32, 33).Only a few authors reported the loss of estrogenicity in microbial metabolites of ZON, which are based on reactions other than cleavage of the lactone undecyl ring system. El-Sharkawy and Abul-Hajj demonstrated (10) that binding to rat uterine estrogen receptors requires a free 4-OH phenolic group (devoid of methylation or glycosylation). Loss of estrogenicity was, for instance, observed with 2,4-dimethoxy-ZON, one of the metabolites produced by Cunninghamella bainieri ATCC 9244B. Nevertheless, this rule cannot be generalized, as 8′-hydroxyzearalenone formed by Streptomyces rimosus NRRL 2234, despite having a free 4-phenolic hydroxyl group, did not bind to the estrogen receptor. Also, other authors reported that 8′-hydroxyzearalenone and 8′-epi-hydroxyzearalenone are nonestrogenic (13). However, so far, no practical application in feed or food detoxification has been found for the microorganisms producing these compounds.It has been shown previously that the yeast Trichosporon mycotoxinivorans has a very high capability to degrade both ochratoxin A (OTA) and ZON (22, 26, 27). When T. mycotoxinivorans is used as a feed additive preparation, microbial degradation of the mycotoxins is assumed to take place in the gastrointestinal tract of the animal after consumption of contaminated feed. The protective effect of T. mycotoxinivorans against OTA toxicity has already been shown with broiler chicken (24).In the present study we report the isolation, analytical characterization, and structure elucidation, as well as the evaluation, of the estrogenic activity of the main degradation product of ZON produced by T. mycotoxinivorans.     

The Fusarium Mycotoxin Deoxynivalenol Can Inhibit Plant Apoptosis-Like Programmed Cell Death

The Fusarium genus of fungi is responsible for commercially devastating crop diseases and the contamination of cereals with harmful mycotoxins. Fusarium mycotoxins aid infection, establishment, and spread of the fungus within the host plant. We investigated the effects of the Fusarium mycotoxin deoxynivalenol (DON) on the viability of Arabidopsis cells. Although it is known to trigger apoptosis in animal cells, DON treatment at low concentrations surprisingly did not kill these cells. On the contrary, we found that DON inhibited apoptosis-like programmed cell death (PCD) in Arabidopsis cells subjected to abiotic stress treatment in a manner independent of mitochondrial cytochrome c release. This suggested that Fusarium may utilise mycotoxins to suppress plant apoptosis-like PCD. To test this, we infected Arabidopsis cells with a wild type and a DON-minus mutant strain of F. graminearum and found that only the DON producing strain could inhibit death induced by heat treatment. These results indicate that mycotoxins may be capable of disarming plant apoptosis-like PCD and thereby suggest a novel way that some fungi can influence plant cell fate.     

Purification and characterization of a zearalenone degrading enzyme produced by<Emphasis Type="Italic">Gliocladium roseum</Emphasis>

Enzymatic inactivation of fungal toxins is an attractive strategy for the decontamination of food and feeding stuff. A constitutively expressed enzyme opening the lactone linkage within the macrocyclic ring system of zearalenone (ZON) was isolated fromGliocladium roseum. The enzyme has been shown to catalyze the transformation of the mycotoxin ZON and therefore has been named ZON degrading enzyme. The resulting products of the enzymatic reaction are less toxic because they have lost their estrogenic capacity. In this study, we used scanning electron microscopy to evaluate the possible mycoparasitism betweenFusarium graminearum andG. roseum. The ZON-degrading enzyme could be isolated fromG. roseum cultures and biochemically characterized. It has been found to be similar to superoxide-dismutases at its N-teminus.     

Heterologous Expression of Arabidopsis UDP-Glucosyltransferases in Saccharomyces cerevisiae for Production of Zearalenone-4-O-Glucoside

Zearalenone, a secondary metabolite produced by several plant-pathogenic fungi of the genus Fusarium, has high estrogenic activity in vertebrates. We developed a Saccharomyces cerevisiae bioassay strain that we used to identify plant genes encoding UDP-glucosyltransferases that can convert zearalenone into zearalenone-4-O-glucoside (ZON-4-O-Glc). Attachment of the glucose moiety to zearalenone prevented the interaction of the mycotoxin with the human estrogen receptor. We found that two of six clustered, similar UGT73C genes of Arabidopsis thaliana encode glucosyltransferases that can inactivate zearalenone in the yeast bioassay. The formation of glucose conjugates seems to be an important plant mechanism for coping with zearalenone but may result in significant amounts of “masked” zearalenone in Fusarium-infected plant products. Due to the unavailability of an analytical standard, the ZON-4-O-Glc is not measured in routine analytical procedures, even though it can be converted back to active zearalenone in the digestive tracts of animals. Zearalenone added to yeast transformed with UGT73C6 was converted rapidly and efficiently to ZON-4-O-Glc, suggesting that the cloned UDP-glucosyltransferase could be used to produce reference glucosides of zearalenone and its derivatives.     

A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides

Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler^® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize.