Two-dimensional environmental profiles of growth,deoxynivalenol and nivalenol production by Fusarium culmorum on a wheat-based substrate

AIMS: To determine the effect of interacting conditions of water activity (aw, 0.99-0.85), temperature (15, 25 degrees C) and time (40 days) on growth and production of the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) by Fusarium culmorum on a wheat-based agar medium. METHODS AND RESULTS: Fusarium culmorum grew optimally at 0.995aw and minimally at 0.90 at both 15 and 25 degrees C. No growth was observed at <0.90aw. Overall, temperature, aw and their interaction had a statistically significant effect on the growth rate of F. culmorum. Production of both DON and NIV were over a much narrower range (0.995-0.95aw) than that for growth. The highest concentrations of DON and NIV levels were produced at 0.995aw and 0.981aw at 25 degrees C, respectively, after 40 days of incubation. Statistically, aw, temperature and incubation time, and aw x temperature and temperature x incubation time had a statistically significant effect on DON/NIV production. CONCLUSIONS: This is the first detailed report on the two-dimensional environmental profiles for DON/NIV production by F. culmorum in the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of a hazard analysis critical control point (HACCP) approach, this type of information is critical in monitoring critical control points for prevention of DON/NIV entering the wheat production chain.     

Occurrence and Distribution of Microdochium and Fusarium Species Isolated from Durum Wheat in Northern Tunisia and Detection of Mycotoxins in Naturally Infested Grain

An outbreak of Fusarium Head Blight of durum wheat occurred in 2004 being localized in sub-humid and higher semi-arid region of Northern Tunisia. A mycological survey carried out throughout these regions, revealed that 78% of the prospected fields were infested. Results of the morphological and molecular identification, showed that the most common species isolated from diseased wheat spikes was Microdochium nivale var. nivale (63.5%), followed by Fusarium culmorum (26%), F. pseudograminearum (9%) and F. avenaceum (1.5%). To evaluate mycotoxin content of naturally infected grain, the amounts of trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from 45 fields were quantified by RIDASCREEN DON Enzyme Immunoassay Kit (ELISA) . This study showed that the infection levels in freshly harvested grain were very low and the maximum deoxynivalenol (DON) level of the positive samples was 53 ppb. This is the first report on the natural occurrence of DON in naturally infected wheat grain sampled from Northern Tunisia.     

Influence of the interactions among ecological variables in the characterization of zearalenone producing isolates of Fusarium spp

To carry out the physiological characterization of Fusarium graminearum and F. culmorum isolates with regard to its zearalenone producing ability, an in-depth experiment with a full factorial design was conducted. The effects and mutual interactions of temperature, moisture, substrate and isolate on the production of the toxin were studied. The study was done with twelve isolates of Fusarium (7 of F. graminearum and 5 of F. culmorum). The analysis of variance shows that there is a complex interaction of all of these factors, which can influence the relative concentrations of the mycotoxin produced, and hence, the correct physiological characterization of the strain. All the tested cultures were susceptible to invasion by Fusarium. The moisture content of grains (water activity values 0.960, 0.970 and 0.980) did not constitute a limiting factor for fungal growth or ZEA production, but incubation temperature (15 degrees C, 20 degrees C, 28 degrees C, and 32 degrees C) affected the rate of zearalenone synthesis. Very low or undetectable ZEA production was observed at 32 degrees C. All tested isolates showed a characteristic behavior concerning the optimum temperature for ZEA production, which was usually 20 degrees C maintained during the whole incubation period. This finding, which does not agree with other reports obtained with strains from different origins, suggests that there are genetic differences that would explain the particular physiological behavior of each isolate related to the optimal production conditions for ZEA. The existence of significant differences regarding the susceptibility of the assayed cereal grains (wheat, corn and rice) used for ZEA production by the different Fusarium species (F. graminearum and F. culmorum) is described for the first time in this paper.     

Water, temperature and gas composition interactions affect growth and ochratoxin A production by isolates of Penicillium verrucosum on wheat grain

AIMS: To examine the effect of interactions between water, temperature and gas composition on growth and ochratoxin A (OTA) production by isolates of Penicillium verrucosum in vitro and in situ on grain-based media and wheat grain. METHODS AND RESULTS: Three isolates of P. verrucosum were examined in relation to radial growth rate and OTA production, and to interacting conditions of water activity (a(w)), temperature and gas composition on a milled wheat medium. Subsequently, detailed temporal studies were carried out on gamma irradiated wheat grain over the range 0.75-0.995 a(w), 10-25 degrees C and air, 25 or 50% CO(2). This showed that optimum growth of P. verrucosum was at 0.98 a(w) in vitro at 25 degrees C, but at 0.95 a(w) and 25 degrees C on wheat grain. The a(w) minimum for growth was about 0.80 a(w), although no OTA was produced under this condition even after 56 days. Significant inhibition of growth and OTA production occurred with 50% CO(2), and 0.90-0.995 a(w) at 25 degrees C. CONCLUSIONS: The optimum and marginal conditions for growth and OTA production on wheat grain have been identified. At least 50% CO(2) is needed to inhibit growth and OTA production by >75% in moist grain (0.90-0.995 a(w)). SIGNIFICANCE AND IMPACT OF THE STUDY: First detailed identification of optimal and marginal interacting conditions of water/temperature and gas composition on growth and OTA production by P. verrucosum on wheat grain. This is a critical component of the postharvest management strategy for minimizing contamination by this important mycotoxin and predicting risk, based on environmental conditions, during drying and storage.     

The influence of gamma radiation and substrate on mycotoxin production by Fusarium culmorum IMI 309344

Mycotoxin production (deoxynivalenol (DON), acetyl deoxynivalenol (A DON) and zearalenone) by Fusarium culmorum inoculated on to maize (heat sterilized, irradiation sterilized and non-sterile) and irradiated to 1 kGy or 3 kGy, or unirradiated, was investigated over a period of time. Lowest mycotoxin production was observed on non-sterile maize which may be due to the presence of a competitive microflora on non-sterile maize. In general, mycotoxin production was higher on heat-sterilized grain as compared to irradiation-sterilized maize. It was suggested that this pattern of mycotoxin production was possibly caused by changes in the grain brought about by autoclaving, which favoured mycotoxin production and possibly induced changes in irradiation-sterilized maize which inhibited mycotoxin production. On sterile maize, there was no significant difference in DON production by unirradiated, 1 kGy and 3 kGy irradiated cultures up to 56 d of incubation; between days 56 and 77 of incubation, DON production increased rapidly with largest increases occurring in irradiated (1 kGy and 3 kGy) cultures. On non-sterile grain, neither DON nor A DON were detected in unirradiated cultures of F. culmorum but were detected in cultures irradiated to 1 kGy and 3 kGy. In practice grain should be stored under conditions of temperature and moisture content which prevent fungal growth. However, in this study, the grain was stored under conditions that were approaching ideal for growth of the test organism. The results highlight that irradiation disinfestation of grain must be combined with good grain handling practices so that excessive mycotoxin production can be prevented during storage.     

Toxin production by Fusarium culmorum IMI 309344 and F. graminearum NRRL 5883 on grain substrates

The production of deoxynivalenol, acetyl deoxynivalenol and zearalenone by Fusarium culmorum and F. graminearum on autoclave-sterilized grain (maize, rice, wheat and barley) was investigated. Fusarium culmorum produced significantly greater levels of toxins than F. graminearum. The four substrates examined differed in their ability to support toxin production. Toxin production on maize and rice was significantly greater than toxin production on barley or wheat.     

Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue

The Ascomycete pathogen Fusarium graminearum can infect all cereal species and lower grain yield, quality and safety. The fungus can also cause disease on Arabidopsis thaliana. In this study, the disease-causing ability of two F. graminearum mutants was analysed to further explore the parallels between the wheat (Triticum aestivum) and Arabidopsis floral pathosystems. Wild-type F. graminearum (strain PH-1) and two isogenic transformants lacking either the mitogen-activated protein kinase MAP1 gene or the trichodiene synthase TRI5 gene were individually spray- or point-inoculated onto Arabidopsis and wheat floral tissue. Disease development was quantitatively assessed both macroscopically and microscopically and deoxynivalenol (DON) mycotoxin concentrations determined by enzyme-linked immunosorbent assay (ELISA). Wild-type strain inoculations caused high levels of disease in both plant species and significant DON production. The map1 mutant caused minimal disease and DON accumulation in both hosts. The tri5 mutant, which is unable to produce DON, exhibited reduced pathogenicity on wheat ears, causing only discrete eye-shaped lesions on spikelets which failed to infect the rachis. By contrast, the tri5 mutant retained full pathogenicity on Arabidopsis floral tissue. This study reveals that DON mycotoxin production is not required for F. graminearum to colonize Arabidopsis floral tissue.     

长江流域禾谷镰孢菌群部分菌株系统发育学、产毒素化学型及致病力研究

从采集自长江流域引起小麦赤霉病的禾谷镰孢菌群(Fusarium graminearum clade)菌株中选取了31株,扩增并测定了这些菌株的EF-1α(translation elongation factor)、PHO(phosphate permease)基因序列,利用相关软件进行了系统发育分析。对这些菌株的产毒素化学型进行了分子检测。同时,用两个小麦品种(扬麦158和安农8455)测定了菌株的致病力。系统发育分析表明绝大多数菌株与F.asiaticum聚为一枝,只有一个菌株11027与F.graminearum聚类。30株F.asiaticum中有24株产脱氧雪腐镰孢菌烯醇(deoxynivalenol,DON)和3-乙酰脱氧雪腐镰孢菌烯醇(3-acetyldeoxynivalenol,3-AcDON),另外6株产雪腐镰孢菌烯醇(Nivalenol,NIV)。一株F.graminearum菌株11027产脱氧雪腐镰孢菌烯醇(DON)和15-乙酰脱氧雪腐镰孢菌烯醇(15-acetyldeoxynivalenol,15-AcDON)。在扬麦158上,菌株间的致病力分化较为明显,产NIV毒素的菌株致病力普遍较弱,强致病力的菌株都产3-AcDON毒素。结果表明在我国长江流域,产3-AcDON毒素的F.asiaticum是引起小麦赤霉病的优势种群,中抗赤霉病的小麦品种扬麦158可以有效评价菌株的致病力强弱。     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

Method evaluation of Fusarium DNA extraction from mycelia and wheat for down-stream real-time PCR quantification and correlation to mycotoxin levels

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 (r(2) = 0.9) and ZEN (r(2) = 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.     

RNA silencing of mycotoxin production in Aspergillus and Fusarium species

Mycotoxins are natural fungal products that are defined by their harmful effects on humans and animals. Aflatoxin contamination of maize by Aspergillus species and trichothecene contamination of small grains by Fusarium species are two of the most severe mycotoxin problems in the United States. We are investigating RNA silencing in an effort to identify novel ways to control mycotoxin contamination of crops. Transformation of two Aspergilli (A. flavus and A. parasiticus) and a Fusarium (F. graminearum) with inverted repeat transgenes (IRT) containing sequences of mycotoxin-specific regulatory genes suppressed mycotoxin production in all three plant-pathogenic fungi. This atoxigenic phenotype was stable during infection on corn and wheat, and importantly, F. graminearum IRT strains were less virulent on wheat than were wild type. The IRT did not alter physiological characteristics of the fungi, such as spore production and growth rate on solid media. These results indicate that RNA silencing exists in Aspergillus and Fusarium plant pathogens and suggest that RNA silencing technology may be a useful tool for eliminating mycotoxin contamination of agricultural products.     

Trichothecene Content of Rye and Wheat Genotypes Inoculated with a Deoxynivalenol- and a Nivalenol-producing Isolate of Fusarium culmorum

Head blight caused by Fusarium culmorum may lead to yield reduction and the contamination of cereal grain with the mycotoxins deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-ADON), nivalenol (NIV), fusarenone-X (FUS), and others. In this study, the covariation between DON and NIV accumulation of 12 rye and eight wheat genotypes that differed in resistance were analysed by inoculating them with a DON-and a NIV-producing isolate, respectively, in three locations. The resistance traits head blight rating and plot yield relative to the uninoculated plots of the same genotype were assessed and the contents of DON, 3-ADON, NIV, and FUS in the grain were analysed by gas chromatography with mass spectrometry. The NIV-producing isolate was significantly (P=0.05) less aggressive and led to a considerably lower mean NIV content in the grain compared with the aggressiveness and mean DON content of the DON-producing isolate (19.5 mg NIV/kg grain versus 48.4 mg DON/kg). Wheat and rye genotypes significantly differed in their DON and NIV accumulation. All genotypes reacted in a similar manner to both chemotypes of F. culmorum for the resistance traits and the respective mycotoxin contents with the exception of one wheat variety, that caused a change in rank order for mycotoxin content. In conclusion, resistance to head blight and tolerance to mycotoxin accumulation seems to be most likely the same for DON- and NIV-producing isolates of F. culmorum .     

Mycotoxin-producing Fusarium Species Occurring in Winter Wheat in Belgium (Flanders) During 2002–2005

Deoxynivalenol (DON) content and Fusarium spp. ( Fusarium graminearum , Fusarium culmorum , Fusarium avenaceum , Microdochium nivale and Fusarium poae ) of mycotoxin-producing Fusarium species in winter wheat in Belgium (Flanders) were determined. Field trials were set up in the varietal testing network of Flanders Agricultural Centre for Small Grains (Roeselare – Beitem, Belgium) and followed up during growing seasons 2001–2002, 2002–2003, 2003–2004 and 2004–2005. Fusarium infection and DON contamination were mainly influenced by location and environmental parameters. Mean DON levels ranged from 0 to 15 mg/kg. Over the period of four growing seasons cvs Deben, Ordeal and Napier had the highest DON contamination. Seasonal and local weather conditions before and during flowering together with local crop husbandry measures (crop rotation, soil preparation) seemed to be of great importance in explaining the variation in results obtained. At Bottelare a positive correlation between disease index and DON content was found for the growing seasons 2001–2002 and 2002–2003, but not the season 2003–2004. Fusarium graminearum and F. culmorum were in general the most frequently occurring Fusarium spp. in Flanders over the 4 years but the composition of the Fusarium population varied strongly from location to location and from year to year. Fusarium graminearum predominated in areas especially where maize was cultivated, whereas in areas with more small cereals in the crop rotation more F. culmorum was present. Also temperature played a role in the composition of Fusarium spp.     

Differences in susceptibility of winter wheat varieties for Fusarium species under Belgian growing conditions

Fusarium head blight is an important disease of cereal crops caused by Fusarium species. It causes not only a reduction in yield, but most Fusarium species (F. graminearum. F. culmorum, F. avenaceum. F. poae) produce also a range of toxic metabolites such as deoxynivalenol (DON) and zearalenone (ZEA). The evaluation of Fusarium species was followed up under natural infection conditions during the growing seasons 2001--2002 and 2002--2003 in two varietal winter wheat experiments on the experimental farm of the Hogeschool Gent at Bottelare. Disease pressure, DON and ZEA content, different Fusarium species as well as growth and yield parameters were determined. In both years there were significant differences between the varieties concerning the susceptibility to Fusarium and the DON content. ZEA was not found in the kernels. The mean deoxynivalenol (DON) content was in 2002 (1,126 mg/kg) higher than in 2003 (0.879 mg/kg) although the mean disease severity was bigger in 2003 than in 2002 what means that the DON content was not always correlated with the disease severity. The Fusarium species most frequently identified in our two field trials (Bottelare) were F. graminearum and F. culmorum Varietal differences in susceptibility to Fusarium species and DON contamination could be detected.     

Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat

We developed a PCR-based assay to quantify trichothecene-producing Fusarium based on primers derived from the trichodiene synthase gene (Tri5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing Fusarium species. Amounts of the trichothecene-producing Fusarium and the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing Fusarium and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producing Fusarium compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing Fusarium in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing Fusarium within the FHB disease complex and not by altering the rate of DON production. The Tri5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed.     

A simple method with liquid chromatography/tandem mass spectrometry for the determination of the six trichothecene mycotoxins in rice medium

A selective and speedy LC-MS/MS method was developed to determine six trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin) in rice medium where Fusarium graminearum were cultivated for in vitro tests. The analytes were extracted from the rice medium with acetonitrile/water (85/15, v/v), and diluted with acetonitrile/water (5/95, v/v) in order to minimize the effects of matrices. Diluted solutions were analyzed by LC-MS/MS with electrospray ionization (ESI) interface in negative or positive ion mode and the multiple reaction monitoring mode. Recovery rates were 76-106% with a spiked level at 1-6 microg/kg of mycotoxins that corresponded to the limit of quantitation. The method was applied to study the time courses of trichothecene production and the biomass of fungi by three Fusarium graminearum strains. Three strains have different mycotoxin biosynthesis pathways, wFg14 and 03E-1 were DON producer, and 03N-1 was NIV producer.     

Fitness of three Fusarium pathogens of wheat

Crown rot and head blight of wheat are caused by the same Fusarium species. To better understand their biology, this study has compared 30 isolates of the three dominant species using 13 pathogenic and saprophytic fitness measures including aggressiveness for the two diseases, saprophytic growth and fecundity and deoxynivalenol (DON) production from saprophytic colonization of grain and straw. Pathogenic fitness was generally linked to DON production in infected tissue. The superior crown rot fitness of Fusarium pseudograminearum was linked to high DON production in the stem base tissue, while Fusarium culmorum and Fusarium graminearum had superior head blight fitness with high DON production in grains. Within each species, some isolates had similar aggressiveness for both diseases but differed in DON production in infected tissue to indicate that more than one mechanism controlled aggressiveness. All three species produced more DON when infecting living host tissue compared with saprophytic colonization of grain or straw, but there were significant links between these saprophytic fitness components and aggressiveness. As necrotrophic pathogens spend a part of their life cycle on dead organic matter, saprophytic fitness is an important component of their overall fitness. Any management strategy must target weaknesses in both pathogenic fitness and saprophytic fitness.     

Effects of different carbon sources on trichothecene production and Tri gene expression by Fusarium graminearum in liquid culture

Fusarium head blight caused by Fusarium graminearum is a disease of cereal crops that not only reduces crop yield and quality but also results in contamination with trichothecenes such as nivalenol and deoxynivalenol (DON). To analyze the trichothecene induction mechanism, effects of 12 carbon sources on the production of DON and 3-acetyldexynivalenol (3ADON) were examined in liquid cultures incubated with nine strains of 3ADON-producing F. graminearum. Significantly high levels of trichothecene (DON and 3ADON) production by sucrose, 1-kestose and nystose were commonly observed among all of the strains tested. On the other hand, the levels of trichothecene biosynthesis induced by the other carbon sources were strain-specific. Tri4 and Tri5 expressions were up-regulated in the sucrose-containing medium but not in glucose. Trichothecene accumulation in the sucrose-containing medium was not repressed by the addition of glucose, indicating that trichothecene production was not regulated by carbon catabolite repression. These findings suggest that F. graminearum recognizes sucrose molecules, activates Tri gene expression and induces trichothecene biosynthesis.     

Bioprospecting for trichothecene 3-O-acetyltransferases in the fungal genus Fusarium yields functional enzymes with different abilities to modify the mycotoxin deoxynivalenol

The trichothecene mycotoxin deoxynivalenol (DON) is a common contaminant of small grains, such as wheat and barley, in the United States. New strategies to mitigate the threat of DON need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that are able to modify DON and reduce its toxicity. Recent work has highlighted differences in the activities of TRI101 from two different species of Fusarium (F. graminearum and F. sporotrichioides), but little is known about the relative activities of TRI101/TRI201 enzymes produced by other species of Fusarium. We cloned TRI101 or TRI201 genes from seven different species of Fusarium and found genetic identity between sequences ranging from 66% to 98%. In vitro feeding studies using transformed yeast showed that all of the TRI101/TRI201 enzymes tested were able to acetylate DON; conversion of DON to 3-acetyl-deoxynivalenol (3ADON) ranged from 50.5% to 100.0%, depending on the Fusarium species from which the gene originated. A time course assay showed that the rate of acetylation varied from species to species, with the gene from F. sporotrichioides having the lowest rate. Steady-state kinetic assays using seven purified enzymes produced catalytic efficiencies for DON acetylation ranging from 6.8 × 10(4) M(-1)·s(-1) to 4.7 × 10(6) M(-1)·s(-1). Thermostability measurements for the seven orthologs ranged from 37.1°C to 43.2°C. Extended sequence analysis of portions of TRI101/TRI201 from 31 species of Fusarium (including known trichothecene producers and nonproducers) suggested that other members of the genus may contain functional TRI101/TRI201 genes, some with the potential to outperform those evaluated in the present study.