Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab.

During the past decade, the plant disease called scab or Fusarium head blight of wheat and barley has reached epidemic proportions in North America and elsewhere in the world. Scab is an economically devastating plant disease, not only because it causes significant reduction in seed yields and quality, but also because infested seeds are often contaminated with trichothecene and estrogenic mycotoxins that pose a serious threat to animal health and food safety. To test whether the primary etiological agent of scab, the fungus Fusarium graminearum, is panmictic throughout its range, allelic genealogies were constructed from six single-copy nuclear genes from strains selected to represent the global genetic diversity of this pathogen. Excluding one hybrid strain, all six genealogies recovered the same seven biogeographically structured lineages, suggesting that they represent phylogenetically distinct species among which gene flow has been very limited during their evolutionary history. Parsimony analysis of the combined data set comprising 7,120 aligned nucleotide characters resolved most relationships among the seven lineages of the F. graminearum clade and related fusaria included in the study. Phylogenetic evidence is also presented for introgressive hybridization and intragenic recombination among lineages of the F. graminearum clade in nature.

Fusarium Species Pathogenic to Barley and Their Associated Mycotoxins.

Epidemics of Fusarium head blight (FHB) occurred on barley in Minnesota, North Dakota, and South Dakota from 1993 to 1998. The Red River Valley region was most severely impacted by the disease based on assessments of FHB severity in grain samples harvested from commercial fields. Fusarium graminearum was the primary pathogen causing these FHB epidemics. It comprised from 62 to 64% of all Fusarium species isolated from infected kernels from 1994 to 1996. Fusarium poae (range of isolation 13 to 20%),F. sporotrichioides (10 to 17%), and F. avenaceum (6 to 10%) also were isolated from barley kernels and were likely involved in causing some FHB infection, but to a very limited extent. All four Fusarium species were pathogenic on barley in inoculation tests conducted in both the greenhouse and the field. Mycotoxin screens were performed on barley spikes inoculated with the respective species in the greenhouse. Spikes infected with F. graminearum contained deoxynivalenol and 15-acetyldeoxyni-valenol; those infected with F. sporotrichioides contained T-2 toxin, HT-2 toxin, and T-2 tetraol; and those infected with F. poae contained nivalenol. Some isolates of F. poae also produced 15-acetoxyscirpenol and scirpentriol. Although F. graminearum and DON are recognized as the primary FHB pathogen and mycotoxin, respectively, in barley, the possible presence of other Fusarium species and mycotoxins should not be overlooked.

The effect of grain storage conditions on the viability of Fusarium and deoxynivalenol production in infested malting barley.

A continuing outbreak of Fusarium head blight occurred on barley in the upper Midwest from 1993 to 1995. This resulted in barley with levels of the mycotoxin deoxynivalenol (DON) reaching levels of concern for maltsters and brewers. This study evaluated the effect of 7 months of storage under different conditions (ambient outdoor temperature from October to April), -20 degrees C, 4 degrees C, 24 degrees C with quiescent air, and 24 degrees C with forced air) on the viability of Fusarium and Alternaria infesting stored grain. Additionally, the ability of Fusarium to produce DON after storage and during malting was evaluated. Initial levels of infestation of barley by Fusarium and Alternaria were 85 and 75%, respectively. All storage condition reduced the viability of both molds slightly and significantly for Fusarium. Forced air ventilation at 24 degrees C was the type of storage most effective in reducing the viability of Fusarium, dropping the percentage of infected kernels to 66%. DON levels did not change after 7 months with respect to storage conditions. However, DON levels were lower in malt produced from barley stored at 24 degrees C with or without aeration. On-farm storage of infected barley at elevated temperatures may provide a means to reduce the level of DON in finished malts.

Molecular phylogenetic, morphological, and mycotoxin data support reidentification of the Quorn mycoprotein fungus as Fusarium venenatum.

Molecular phylogenetic, morphological, and mycotoxin data were obtained in order to investigate the relationships and identity of the Quorn mycoprotein fungus within Fusarium and to examine Quorn strains and commercial Quorn food products for trichothecene mycotoxins. Phylogenetic analyses of aligned DNA sequences obtained via the polymerase chain reaction from the nuclear 28S ribosomal DNA, nuclear ribosomal internal transcribed spacer region, and beta-tubulin gene exons and introns indicate that the Quorn fungus is Fusarium venenatum, rather than F. graminearum as previously reported. All of the Quorn strains examined were morphologically degenerate aconidial colonial mutants except for NRRL 25139, which produced chlamydospores in recurved terminal chains together with mostly 5-septate sporodochial conidia on doliform monophialides diagnostic of F. venenatum. Bootstrap and decay analyses provide strong support for a monophyletic lineage containing F. venenatum and several other type A trichothecene-producing species, while reference strains of F. graminearum were nested in a separate clade of species that produce type B trichothecenes and/or zearalenone. Analysis of mycotoxins from rice cultures inoculated with Quorn strain NRRL 25416 revealed that four type A trichothecenes are produced, but at low levels relative to strain NRRL 22198 of F. venenatum. No trichothecene mycotoxins, however, were detected from the analysis of three commercial Quorn products marketed for human consumption in England.

Individual and combined effects of fumonisin B1 present in Fusarium moniliforme culture material and T-2 toxin or deoxynivalenol in broiler chicks.

The individual and combined effects of feeding diets containing 300 mg fumonisin B1 (FB1), and 5 mg T-2 toxin (T-2)/kg of diet, or 15 mg/kg deoxynivalenol (DON, vomitoxin) from naturally contaminated wheat were evaluated in two studies in male broiler chicks from day of hatch to 19 or 21 d of age in Experiments 1 and 2, respectively. When compared with controls, body weight gains were reduced 18 to 20% by FB1, 18% by T-2, 2% by DON, 32% by the FB1 and T-2 combination, and 19% by the FB1 and DON combination. The efficiency of feed utilization was adversely affected by FB1 with or without T-2 or DON. Mortality ranged from none for the controls to 15% for the FB1 and T-2 combination. Relative weights of the liver and kidney were significantly increased by FB1 with or without T-2 or DON. Serum concentrations of cholesterol were increased in chicks fed FB1 with or without T-2 or DON. Activities of aspartate aminotransferase, lactate dehydrogenase, and gamma glutamyltransferase were increased in chicks fed FB1 at 300 mg/kg alone and in combination with T-2 or DON, indicating possible tissue damage and leakage of the enzymes into the blood. Results indicate additive toxicity when chicks were fed diets containing 300 mg FB1 and 5 mg T-2/kg of diet and less than additive toxicity when chicks were fed 300 mg FB1 and 15 mg DON/kg of diet. Of importance to the poultry industry is the fact that toxic synergy was not observed for either of these toxin combinations and the likelihood of encountering FB1 at this concentration in finished feed is small. However, under field conditions with additional stress factors, the toxicity of these mycotoxins could be altered to adversely affect the health and performance of poultry.

Moniliformin from Fusarium fujikuroi culture material and deoxynivalenol from naturally contaminated wheat incorporated into diets of broiler chicks.

The effects of feeding diets containing 100 mg moniliformin (M)/kg of feed from culture material and 16 mg deoxynivalenol (DON)/kg of feed from naturally contaminated wheat were evaluated in growing broiler chicks from 1 day to 21 days of age. Body weight (BW), body-weight gain, and feed consumption were decreased by feeding M and M plus DON diets. Relative heart weight was increased by the M diet, whereas relative weights of proventriculus, gizzard, and heart were increased by the M plus DON diet. The M diet increased alanine transferase and aspartate transaminase activities and creatinine concentration and decreased mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration (MCHC). The M and DON diet decreased glucose, hemoglobin, and MCHC. Histopathological lesions from the M diet were limited to the kidney and consisted of extensive renal tubular epithelial degeneration plus luminal mineralization. A moderation of the severity of lesions was seen in the tissues of the M plus DON-fed chicks, consisting of generally mild tubular epithelial degeneration. None of the parameters measured were affected by the DON diet. Results indicate additive or less-than-additive toxicity for most parameters when chicks were fed diets containing 100 mg M plus 16 mg DON/kg of feed. Although the concentration of M in this study was high compared with that reported for feedstuffs, additional information on the occurrence and toxicity of M will need to be collected in order to assess the importance of M to the poultry industry.

Effects of dietary fumonisin B1-containing culture material, deoxynivalenol-contaminated wheat, or their combination on growing barrows.

OBJECTIVE: To examine the toxic effects of fumonisin B1 (FB1)-containing culture material and deoxynivalenol (DON)-contaminated wheat diets on barrows. ANIMALS: 24, 7-week-old crossbred barrows allotted to 4 equal groups of 3 replicates of 2 barrows/replicate. PROCEDURE: Barrows were fed diets for 28 days that were formulated as follows: no additional FCM or DON/kg of feed (control); 100 mg FB1/kg of feed; 5 mg DON/kg of feed; or 100 mg FB1 plus 5 mg DON/kg of feed. Body weight and feed consumption were monitored weekly. On day 28, blood samples were obtained for serum biochemical, hematologic, and immunologic measurements. On day 29, barrows were euthanatized and necropsies were performed. RESULTS: Analyzed mycotoxin content of diets were: none detected (control); 47 mg of FB1/kg of feed (FB1 diet); 4.5 mg of DON/kg of feed (DON diet); and 56 mg of FB1 and 3.7 mg of DON/kg of feed (FB1 plus DON diet). Differences were detected among groups of barrows for clinical performance, serum biochemical analytes, immunologic response, and histopathologic lesions. CONCLUSIONS: Combining FB1-containing material and DON-contaminated wheat in the diets of growing barrows induces a more toxic response than that induced by either toxin singly. For many variables, the response could be described as additive; however, for some variables, responses were interactive in a greater-than-additive manner. CLINICAL RELEVANCE: Caution should be exercised when formulating swine diets that could contain FB1 and DON, because the condition induced by their combination is more severe than that predicted for each mycotoxin's toxicity.

Determination of deoxynivalenol in wheat, barley, and malt by column cleanup and gas chromatography with electron capture detection.

A rapid and reliable method to determine deoxynivalenol (DON) in wheat, barley, and malt is described. Samples are extracted with acetonitrile-water (84 + 16). Extracts are eluted through a C18-alumina (1 + 3) column, evaporated to dryness, and derivatized with trimethylsilylimidazole-trimethylchlorosilane (100 + 1). DON is identified and quantitated by capillary gas chromatography with electron capture detection. This method can quantitate DON levels from 0.2 to 40 ppm. Multiple analyses of wheat spiked at 2 ppm and of naturally contaminated wheat, malt, and barley resulted in coefficients of variation of 5.1, 5.1, 6.0, and 9.8%, respectively. Recoveries of DON spikes at 3 levels were 94-100% for wheat, 100-105% for barley, and 100-105% for malt. Results for wheat sample analyzed with this procedure (1.9 +/- 0.1 ppm DON) compared well with results for the same sample analyzed by enzyme-linked immunosorbent assay (1.9 ppm DON) and by liquid chromatography (1.7 ppm DON).

Use of epidemiologic principles to identify risk factors associated with the development of diarrhea in calves in five beef herds.

The objective of the study reported here was to identify and measure risk factors associated with the incidence of diarrhea in calves in 5 beef herds in North Dakota. Risk factors associated with calves developing diarrhea during the 1992 calving season were used to develop the following intervention strategies that were implemented during 1993: heifers calved in a separate location, diarrheal calves and their dams were placed in a quarantine area, and pregnant cattle were fed a diet supplemented with protein and copper. The risk of calves developing diarrhea was 5 times higher during the first year, compared with the second year, suggesting that the management changes made during the second year were contributing factors in reducing the percentage of calves with diarrhea. The risk of diarrhea developing in calves born to heifers was 3.9 times greater than that in calves born to cows. Analysis of our data indicated that the use of an epidemiologic approach can identify risk factors, which can lead to the development of intervention procedures that may decrease the incidence of diarrhea in calves in beef herds.

Two new modified trichothecenes from Fusarium sporotrichioides.

Two new modified trichothecenes, 2-deoxy-11-epi-3 alpha-hydroxysambucoin [1] and 2-deoxy-11-epi-12-acetyl-3 alpha-hydroxysambucoin [2], were isolated from Fusarium sporotrichioides culture. This is the first report of modified trichothecenes where the two six-membered rings are cis-fused. Structures were elucidated using gc-ms, nmr, X-ray crystallography, and other spectroscopic techniques. Compounds 1 and 2 were screened for relative cytotoxicity in cultured baby hamster kidney (BHK-21) cells and found to be non-toxic.

Properties of a 4-ene-3-ketosteroid-5 alpha-reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144.

When Eubacterium sp. 144 was grown in the presence of progesterone, extracts of these cells contained a 4-ene-3-ketosteroid-5 alpha-reductase (5 alpha-reductase). No evidence for the presence of a 5 beta-steroid-reductase or a 5 alpha to 5 beta-steroid-isomerase was found. 5 alpha-Reductase activity was dependent on reduced methyl viologen as the electron donor and this could be generated biologically by adding pyruvate or H2 to cell extracts or chemically by adding sodium dithionite. NADH or NADPH with or without flavin nucleotides were not electron donors for 5 alpha-reductase. Most of the 5 alpha-reductase activity (60-65%) of crude extracts was located in the membrane fraction and the enzyme was solubilized by treatment with 1% Triton X-100. Optimum 5 alpha-reductase activity occurred at pH 7.0-7.5 in potassium phosphate buffer but was stimulated by Tris-HCl buffer (pH 8.0-9.0). 5 alpha-Reductase activity was highest at 10% (v/v) methanol and was progressively inhibited by higher methanol concentrations. Sulfhydryl reagents strongly inhibited 5 alpha-reductase but the enzyme was not affected by other metabolic inhibitors. Extracts prepared from cells induced with 16-dehydroprogesterone and grown without hemin contained 5 alpha-reductase and 16-dehydroprogesterone-reductase activities equivalent to those found in extracts of induced cells grown with hemin. This indicates that hemin is not required for the synthesis of active steroid double bond-reductases in strain 144.

Two new trichothecenes from Fusarium sporotrichioides.

Two new trichothecenes, 8-n-pentanoylneosolaniol and 8-n-hexanoylneosolaniol, were isolated from corn grits cultured with Fusarium sporotrichioides. The structures of these compounds were elucidated using gc-ms, nmr, X-ray crystallography, and other spectroscopic techniques. Seven known trichothecenes were also isolated, and the relative cytotoxicity of these nine trichothecenes in cultured baby hamster kidney (BHK-21) cells was determined.

Novel occurrence of cyclopiazonic acid in sunflower seeds.

Cyclopiazonic acid (CPA), a secondary metabolite produced by several species of Aspergillus and Penicillium, was detected in moldy sunflower seed screenings fed to sows who experienced conception problems and feed refusal. Tandem mass spectrometry confirmed the CPA which was estimated by thin-layer chromatography (TLC) to be present at 10 ppm. A Penicillium species was isolated from the moldy seeds, but in culture it did not produce CPA.

Evaluation of vitamin K3 feed additive for prevention of sweet clover disease.

Sweet clover poisoning in cattle is caused by an anticoagulant (dicumarol) that is formed in moldy sweet clover hay. Previous experiments with vitamin K3 and vitamin K1 in therapy trials indicated that vitamin K1 was effective in reducing prothrombin times but vitamin K3 was not. As a possible alternative in the use of toxic sweet clover hays, vitamin K3 was evaluated to see if it would prevent hemorrhagic crises when fed to cattle consuming toxic sweet clover hay. Vitamin K3 levels of 0, 0.45, 4.5, 11, and 45 mg/kg body weight/day were fed to 173-235-kg steers consuming toxic (40-50 ppm dicumarol) sweet clover. The 45-mg K3/kg/day supplement was not palatable and had to be discontinued. The 0.45, 4.5, and 11-mg K3/kg/day supplements did not significantly reduce the prothrombin times as compared to the 0-mg K3/kg/day group.

Tissue fluoroacetate residues in prairie dogs dosed with low-level sodium monofluoroacetate.

A total of 83 black-tailed prairie dogs (Cynomys ludovicianus) from South Dakota were subjected to low-level treatment with sodium monofluoroacetate (Compound 1080) in the laboratory (0.01-0.30 mg 1080/kg). The acute oral median lethal dose (LD50) of 1080 administered by oral gavage was established at 0.173 mg/kg. To assay fluoroacetate residues, 8 kinds of tissue from each of 10 prairie dogs dead of low-level 1080 poisoning were analyzed by capillary gas chromatography-mass spectrometry. Of the total of 79 tissues analyzed, 73 contained less than 100 ppb fluoroacetate, and 67 contained less than 50 ppb fluoroacetate. To test the effect of secondary poisoning on non-target species, 8 European ferrets (Mustela furo) were fed ground whole carcasses of prairie dogs dead of low-level 1080 poisoning, with no observable ill effects on the ferrets.

Fluoroacetate residues in ground squirrel and coyote tissues due to primary or secondary 1080 poisoning.

Fluoroacetate residues in various tissues of 1080-poisoned ground squirrels and coyotes are listed. The tissues (excluding the stomach) of squirrels poisoned with an average of 0.8 mg 1080/kg (low dose) contained from 182 to 1309 ppb fluoroacetate. In squirrels poisoned with an average of 4.8 mg 1080/kg (high dose), the tissue residues ranged from 535 to 9754 ppb fluoroacetate. Tissues from coyotes which died after consuming 1080-poisoned ground squirrels were also analyzed for fluoroacetate residues. Residues in these coyote kidneys and livers ranged from less than 10 ppb to 95 ppb fluoroacetate. The residue findings in this research indicate that a diagnostic assay for 1080 in tissues must be reliable at 10 ppb (or less) fluoroacetate.

Vitamin K treatment of sweet clover poisoning in calves.

Dicumarol poisoning was reproduced by feeding naturally spoiled, sweet clover hay, which contained a minimum of 90 ppm dicumarol. Vitamin K1 administered IM was effective in treating the disease at dosages of 1.1, 2.2, and 3.3 mg/kg of body weight. Vitamin K3 treatment by various routes and dosages was ineffective.

Capillary gas chromatographic-mass spectrometric determination of fluoroacetate residues in animal tissues.

A method for the quantitative determination of fluoroacetate (FAC) residues in animal tissues is described. The procedure involves tungstic acid extraction, partitioning into ethyl acetate, evaporation of ethyl acetate, derivatization with pentafluorobenzyl bromide (PFB), and analysis of the resulting derivative (PFB-FAC) by capillary gas chromatography-mass spectrometry (CGC-MS) with specific ion monitoring (SIM). The tungstic acid system extracted 96.8 +/- 4.2% of the endogenous 14C-1080 residues in rat tissues. Recovery of FAC during the extraction, purification, and derivatization procedures is established by use of a 14C-FAC spike. 1,2-Dibromobenzene is used as an internal standard for the CGC-MS analysis. PFB-FAC is identified on the basis of comparative retention times and the relative intensities of m/z 257.9 and 181.0. PFB-FAC is quantitated by comparing the response at m/z 257.9 to a PFB-FAC standard curve. Routine sensitivity of the method allows determination of 10 ppb fluoroacetate in tissue.