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 .     

Trichothecene production by Fusarium species isolated from grain and pasture throughout New Zealand

Liquid cultures of 200 Fusarium isolates selected to represent the most common species found in autumn pasture (70 isolates) and in grain (130 isolates) grown in New Zealand were analysed for trichothecenes and related compounds. Production of butenolide, cyclonerodiol derivatives and culmorins was also measured. The principal trichothecenes produced were derivatives of either nivalenol (NIV), deoxynivalenol (DON) or scirpentriol (Sctol), in order of frequency. The principal trichothecene producing species were F. crookwellense, F. culmorum and F. graminearum. Isolates of the first two species were predominantly NIV-chemotypes with one or two isolates respectively as Sctol-chemotypes. F. graminearum showed equal quantities of NIV- and DON-chemotypes, with the DON-chemotypes producing primarily 15-acetyldeoxynivalenol (15-ADON).     

Real-time PCR detection and quantification of Fusarium poae,F. graminearum,F. sporotrichioides and F. langsethiae in cereal grains in Finland and Russia

Abstract

TaqMan real-time quantitative PCR assays were developed for the accurate detection and quantification of DNA from Fusarium poae and F. graminearum species, which are able to produce trichothecenes. These and other PCR assays were used for the quantification of trichothecene-producing Fusarium fungi in cereal grains. A correlation was found between the levels of F. poae DNA and nivalenol and enniatins in barley and between the levels of F. graminearum DNA and deoxynivalenol in oats. The correlations between F. poae DNA and nivalenol and F. graminearum DNA and deoxynivalenol levels were higher than those between these mycotoxins and morphologically determined F. poae and F. graminearum/F. culmorum contamination levels. The use of F. poae specific primers and probe together with F. sporotrichioides/F. langsethiae specific primers and probe in a multiplex qPCR assay yielded results in accordance with those obtained using these primers and probes separately.     

Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F.?poae isolates. A total of 125 F.?poae, four F.?cerealis, two F.?culmorum, one F.?langsethiae, one F.?sporotrichioides and seven F.?graminearum, plus F.?austroamericanum, F.?meridionale, F.?graminearum sensu stricto and F.?cortaderiae from the NRRL collection were analysed, and only F.?poae isolates gave a positive result for the presence of a 296-bp partial tri7 DNA fragment. Moreover, the primer set was tested from cereal seed samples where F.?poae and other Fusarium species with a negative result for the specific reaction ( F.?graminearum, F.?oxysporum, F.?chlamydosporum, F.?sporotrichioides, F.?equiseti and F.?acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F.?poae isolates.     

Comparison of Canadian Fusarium graminearum isolates for aggressiveness,vegetative compatibility,and production of ergosterol and mycotoxins

Fusarium graminearum is the predominant pathogen causing fusarium head blight of cereals in North America. Fifteen Canadian isolates of Fusarium graminearum were highly diverse in terms of vegetative compatibility grouping (VCG) and varied for production of ergosterol and mycotoxin production in rice culture. Aggressiveness was assessed by scoring the disease severity incited in wheat spikes by each isolate. Two inoculation methods, single-floret injection and spray of entire spikes, were used to screen 4 wheat varieties for reaction to the F. graminearum isolates. All isolates were of broadly similar aggressiveness, with disease severity ranging from 17.2 to 39.1 for single floret injection, and 39.1 to 69.0 for spray inoculation. Disease severity, ergosterol production, and mycotoxin development were not correlated. Using nitrate non-utilizing mutants the 15 isolates were grouped into 14 VCGs. Deoxynivalenol (DON) was produced by all isolates in rice culture, at levels between 0.2 and 249 ppm. 15-acetyldeoxynivalenol was produced by 14 of the 15 isolates at levels between 0.4 and 44.6 ppm. These results reveal a high level of diversity for several characteristics among F. graminearum isolates from Canada. This revised version was published online in June 2006 with corrections to the Cover Date.     

The occurrence of HT-2 toxin and other trichothecenes in Norwegian cereals

A total of 449 grain samples, 102 barley, 169 wheat and 178 oat samples were collected from different regions of Norway from 1996–1998 crops, mainly from grain loads and silos. The samples were analysed for type A and B trichothecenes, the largest groups of mycotoxins produced by the Fusarium species, by gas chromatography with mass spectrometric detection (GC-MS). Factors affecting the presence of the different trichothecenes are discussed. Deoxynivalenol (DON) and HT-2 toxin were the trichothecenes most frequently detected, followed by T-2 toxin, nivalenol, and scirpentriol, scirpentriol being detected only in seven samples (>20 g/kg).Oats were the grain species most heavily contaminated with an incidence(% >20 g/kg) and mean concentration of positive samples of 70%(115 g/kg) for HT-2 toxin, 30% (60 g/kg) for T-2 toxin, 57%(104 g/kg) for DON, and 10% (56 g/kg) for nivalenol. The corresponding values for barley were 22% (73 g/kg), 5% (85 g/kg),17% (155 g/kg) and 6% (30 g/kg), and for wheat 1.2% (20 g/kg),0.6% (20 g/kg), 14% (53 g/kg) and 0% for HT-2, T-2, DON and nivalenol, respectively. Norwegian oats were found to contain HT-2 and T-2 toxin in concentrations that might be at threat to human health for high consumers of oats. The amount of DON was significantly lower than in the crop from previous years.This revised version was published online in October 2005 with corrections to the Cover Date.     

Short review: Metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants

Plants have a high capacity to transform and thereby detoxify deleterious or poisonous compounds, like mycotoxins. The formation of glucose conjugates has a central role in this process. Mammals, however, are able to (partly) release the precursor substances during digestion, reactivating the mycotoxins. This short review provides a brief summary about the metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants. Two examples are discussed in greater detail. First, the formation of deoxynivalenol-3-glucoside in wheat is linked to a quantitative trait locus that is often used forFusarium head blight resistance breeding. Secondly, the metabolism of zearalenone inArabidopsis thaliana results in at least 17 different metabolites, all of which are potentially hazardous for humans and animals. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006 Financial support: Christian Doppler Society, the Austrian Genome Research Initiative GEN-AU, the Lower Austrian government, the Austrian Science Fund FWF     

Fusarium crookwellense,a newly isolated fungus from wheat in Japan: Its mycotoxin production and pathogenicity to wheat and barley

Fusarium crookwellense was isolated for the first time in Japan from scabby wheat harvested in Hokkaido in 1991. Four isolates were obtained and examined for their mycological features on culture media, mycotoxin production, and pathogenicity to wheat and barley. The texture of fungal colonies and the morphology of macroconidia on a potato dextrose agar medium were similar to those ofFusarium graminearum. AllF. crookwellense isolates examined produced nivalenol, 4-acetylnivalenol, and zearalenone on rice media at levels ranging from 0.9 to 22.5 µg/g, 0.5 to 25.0 µg/g, and 1.4 to 162.5 µg/g, respectively. All were pathogenic toward the wheat and barley strains tested, and scab symptoms were found on the heads and leaves of plants. This is the first report on domestic isolates ofF. crookwellense from the crop field in Japan.     

Auxins of non-flowering plants. I. Occurrence of 3-indoleacetic acid and phenylacetic acid in vegetative and fertile fronds of the ostrich fern (Matteucia struthiopteris)

Gas chromatography-mass spectrometry evidence is presented for the presence of both phenylacetic acid (PAA) and 3-indoleacetic acid (IAA) in vegetative and fertile tissues of the sporophyte of the ostrich fern [ Matteucia struthiopteris (L.) Todaro]. 3-Indolepropionic acid, tryptamine and 3-indoleacetonitrile were not found in tissue extracts, although small amounts of 3-indolebutyric acid and tryptophol may have been present. PAA was present in amounts higher than those found in flowering plants, while LAA levels fall within the angiosperm range. The levels of both auxins were higher in the younger vegetative tissues than in mature vegetative or fertile pinnae. Recent evidence for the occurrence of angiosperm growth hormones in ferns is discussed.     

Testicular shape and its relationship to sperm production in mature Holstein bulls

This study was conducted to determine the relationship between testicular shape, scrotal circumference (SC) and sperm production. Twenty-seven mature Holstein bulls were evaluated subjectively and objectively for testicular shape as indicated by testicular length and width, then placed in 1 of 3 groups. Group 1 contained 17 bulls with a normal ovoid testicular shape and a length to width ratio of 1.61:1 +/- 0.01 (SEM). Group 2 was composed of 4 bulls with a long, slender testicular shape and a length to width ratio of 1.95:1 +/- 0.06 (SEM). Group 3 was comprised of 6 bulls with spheroid-shaped testicles and a length to width ratio of 1.3:1 +/- 0.03 (SEM). All the groups were statistically different for length to width ratios (P < 0.05). Length measurements from cranial to caudal pole of the testis proper were also different between groups (P < 0.05). Width or testicular diameter was different between Group 2 and Group 3 at P < 0.05; however, there was no difference between Group 1 and Group 2 or between Group 1 and Group 3. Predicted volumes and weights of testicles were not significantly different between groups. Scrotal circumference measurements were significantly different between groups (P < 0.05). Group 1 had an average SC of 43.07 +/- 0.36 cm (SEM), Group 2 of 39.33 +/- 1.18 cm (SEM) and Group 3 of 46.22 +/- 0.69 cm (SEM). Sperm production for a twice daily, 2-day-per-week collection schedule revealed a statistically significant difference for sperm output. A total of 2742 ejaculates was evaluated. A total of 1818 ejaculates was evaluated in Group 1, 440 ejaculates in Group 2 and 484 ejaculates in Group 3. The mean spermatozoal harvest per day for Group 1 bulls was 13.62 +/- 0.09 x 10(9) (SEM). Group 2 bulls with the longer-shaped testicles produced 14.82 +/- 0.18 x 10(9) (SEM) spermatozoa per day, and Group 3 bulls, with the more rounded testicle shape and the significantly larger SC produced 11.72 +/- 0.64 x 10(9)(SEM) sperm cells per day. All 3 groups were statistically different at the P = 0.05 level. The results suggest that prediction of sperm production may be dependent on factors other than SC, testicular volume, or weight. Testicular shape may influence sperm output in mature Holstein bulls.     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Genetic and mutational tools for investigating the genetics and molecular biology of trichothecene production inGibberella pulicaris (Fusarium sambucinum)

Gibberella pulicaris (Fusarium sambucinum) is a promising organism for studying the genetics and regulation of trichothecene biosynthesis; conditions for obtaining fertile crosses have been defined (Desjardins & Beremand, 1987) and crosses between natural variants have provided some information about the number, location, arrangement, and role of genes which determine trichothecene production (Desjardins & Beremand, 1987; Beremand & Desjardins, 1988). The development of some additional experimental tools and methodologies required for the further genetic analysis of trichothecene production inG. pulicaris are described in the present study. A highly fertile, isogenic line was constructed forG. pulicaris strain R-6380. The ability to readily generate mutants in this strain was also demonstrated. Both biochemical and morphological mutants were obtained following UV-mutagenesis. The inheritance of some of these mutations through meiosis indicated that they will be useful genetic markers for crosses and mapping studies. Since strain R-6380 is also transformable (Salch & Beremand, 1988), it is an excellent choice for transmission and molecular genetic studies involving trichothecene production.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Effects of infection time and moisture on development of ear blight and deoxynivalenol production by Fusarium spp. in wheat

Wheat ears were inoculated with conidia of Fusarium spp. at different growth stages between ear emergence and harvest and moist conditions were maintained for up to 7 days subsequently by mist irrigation. Of the fungi tested (Fusarium culmorum, F. avenaceum, F. tricinctum, F. sporotrichioides and Microdochium nivale), only F. culmorum produced ear blight symptoms and grain samples were found subsequently to contain deoxynivalenol. Most ear infection and deoxynivalenol formation occurred following inoculation at about mid-anthesis. Small amounts of deoxynivalenol were formed and some F. culmorum was isolated even in the absence of ear blight symptoms. An overnight wet period was sufficient to initiate infection and deoxynivalenol formation but both were increased by extending the wet period up to at least 3 days. Recovery of Fusarium spp. from harvested grain was usually possible whether or not symptoms developed. F. culmorum usually persisted and often increased to moderately high levels after storage for 7 wk in a range of moisture conditions.     

On the relationship between sample plot size and beta diversity in boreal coniferous forests

Nested sample plots of three sizes (16, 1, and 1/16 sq. m) from three different studies of Norwegian coniferous forests have been subjected to DCA ordination using the same choice of options. At each sample plot size, species quantities are recorded as frequency in 16 subplots. Beta diversity, measured as length of the first DCA axis, invariably increased upon lowering of sample plot size. The same applied to the eigenvalues of the axes. This is explained as a consequence of the weakening of structure in the data matrices when the fine-grained patterns of the vegetation are emphasized.     

Need to determine the relative developmental risks of Fusarium mycotoxin dexoynivalenol (DON) and Benomyl (BEN) in wheat

The wheat supply is known to have levels of deoxynivalenol (DON) from fungal infections. The fungal infections can be suppressed with benomyl (BEN). Under certain exposure laboratory conditions DON and BEN are developmental toxins. Since use of BEN may result in concurrent dietary exposure to both compounds, evaluation of the relative developmental risks is needed. The dietary NOAELs for pup decreased body weight for DON and BEN are 0.375 mg/kg/day in mice and 5 mg/kg/day in rat, respectively. Human exposure to DON can be estimated from surveys of wheat grain and finished products which gave a mean DON concentration of 0.86 ppm. The tolerance level for BEN in grain is 0.2 ppm. Presence of DON and BEN in wheat at these concentrations would not impact wheat consumption. A modified margin of exposure (modMOE) was developed using “a”; kg (food) /kg (body weight) as the constant for wheat consumption. The modMOE for DON is 28/a and that for BEN is 1667/a. Comparative relative risks can be expressed as the ratio of BEN risks to DON risks (60). Treatment with BEN at the most efficacious time decreased DON levels to 34% of controls, and decreased the comparative risk ratio from 60 to 18. The potential for fungicides to decrease mycotoxin health risks is important in the safety assessment for foodstuffs. This approach improves on current practices for food safety assessment by including the expected reductions in risks from naturally occurring mycotoxins in the regulatory analysis.     

吸烟对肺组织D4-GDI表达的影响及其与慢性阻塞性肺疾病相关性的蛋白质组学研究

慢性阻塞性肺疾病(COPD)是环境因素与遗传因素共同作用的结果,而吸烟是导致COPD发生的最主要危险因素,然而,吸烟导致COPD的机制及COPD的遗传易感机制目前尚未完全阐明．蛋白质组学研究方法具有高效和信息含量丰富的特点,为COPD研究提供了有力的帮助,被认为在COPD的研究领域具有广阔的前景．运用二维凝胶电泳和基质辅助激光解吸电离飞行时间质谱蛋白质组学研究方法结合生物信息学技术,对不吸烟者、非COPD吸烟者和吸烟COPD患者的肺组织进行蛋白质组比较,共鉴定出24种表达差异蛋白．研究发现,非COPD吸烟者肺组织ρ-GDP解离抑制因子2(D4-GDI)表达水平为不吸烟者的1.7倍,吸烟COPD患者肺组织D4-GDI表达水平接近非COPD吸烟者的2倍．通过免疫组织化学染色和Western-blotting检测对肺组织D4-GDI表达水平进行验证,获得了与蛋白质组学研究相一致的结果．结果首次说明：吸烟能够导致肺组织D4-GDI表达水平升高,D4-GDI参与了COPD的发病机制而且可能与COPD易感性相关．     

Differential proteomic analysis during the vegetative phase change and the floral transition in Malus domestica

In order to identify the proteomic changes of apple (Malus domestica Borkh.) during the vegetative phase change and the floral transition, leaf protein of juvenile, adult vegetative and reproductive phase in a seedling ('Jonathan' × 'Golden Delicious') was extracted and analyzed by 2-D electrophoresis and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Seventy two gel spots with significant expression differences between ontogenetic phases were obtained. Five protein spots were only detected in leaves of juvenile phase and 11 were not; 17 spots were found exclusively in adult vegetative leaves; and only one spot solely appeared in reproductive leaves while 12 did not. Twenty six of the differentially expressed proteins identified were involved in photosynthesis. Seven enzymes were related to respiration and carbohydrate metabolism. Fifteen other proteins also presented qualitative or quantitative differences among developmental phases. The spatial distribution of one differentially expressed protein, serine hydroxymethyltransferase, was confirmed by enzyme linked immunosorbent assay and immunohistochemistry. These results strongly support the idea that the vegetative phase change and the floral transition are regulated independently during developmental process.     

Cytotoxicity of Fusarium species mycotoxins and culture filtrates of Fusarium species isolated from the medicinal plant Tribulus terrestris to mammalian cells

Ayurvedic medicine, which uses decoctions made of medicinal plants, is used to cure diseases in many Asian countries including Sri Lanka. Although proper storage facilities for medicinal plants are unavailable in Sri Lanka, neither the potential for growth of toxigenic fungi nor their ability to produce mycotoxins in stored medicinal plants has been investigated. We isolated three Fusarium species, F. culmorum, F. acuminatum and F. graminearum from the medicinal plant Tribulus terrestris. Culture extracts of the 3 Fusarium spp. were cytotoxic to mammalian cell lines BHK-21 and HEP-2. Three toxic metabolites produced by Fusarium spp; T-2 toxin, zearalenone, and diacetoxyscirpenol were also cytotoxic to the same mammalian cell lines. The 3 Fusarium spp. grown on rice media produced zearalenone. Plant material destined for medicinal use should be stored under suitable conditions to prevent growth of naturally occurring toxigenic fungi prior to its use.