Developmental toxicity of fumonisin in Syrian hamsters

The effects of fumonisin on development of Syrian hamster fetuses were studied using fumonisin B₁ and B₂ extracted fromFusarium moniliforme corn-culture and purified fumonisin B₁. A significant increase in litters with fetal deaths occurred with the high doses of purified (18 mg FB₁/kg) and culture-extracted (18 mg FB₁ plus 4.5 mg FB₂) fumonisin. It is concluded that prenatal exposure to fumonisin on days 8 and 9 of gestation is detrimental to fetal hamster survivability but does not induce clinical maternal intoxication at these doses. Equivalent doses of fumonisin B₁, whether from culture-extract or pure solution produced similar results.     

Mycoflora and fumonisin-producing strains ofFusarium moniliforme in mixed poultry feeds and component raw material

In a survey of the mycoflora and mycotoxins in foods and feeds, 66 samples of mixed poultry feeds and some component raw materials were investigated. Fungal counts ranged from < 10² to 1.3 × 10⁶ CFU/g.Fusarium spp. counts ranged from 10² to 1.0 × 10⁶ CFU/g. TheFusarium spp. strains isolated were screened for their potential to produce fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in maize cultures. Samples and maize cultures were analysed for FB₁ and FB₂ using TLC and fluorescamine-derivative HPLC. No fumonisins were detected in the samples (<6 ppm).Fusarium moniliforme was isolated in 59.1% of samples, and 97.4% of the strains produced FB₁ and 79.4% of strains produced FB₂ in maize cultures. Some isolates produced higher FB₁ and FB₂ levels than the reference strainF. moniliforme MRC 826.     

Lymphocyte cytotoxicity and erythrocytic abnormalities induced in broiler chicks by fumonisins B1 and B2 and moniliformin fromFusarium proliferatum

Peripheral blood lymphocytes were isolated from broiler chicks that had ingested feed amended with autoclavedFusarium proliferatum culture material containing fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and moniliformin. Lymphocyte viability was determined for birds that were placed on amended rations at day 1 or day 7 of age at three different levels of mycotoxins, ranging from 61–546 ppm FB₁, 14–94 ppm FB₂ and 66–367 ppm moniliformin. Reduction of the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], to yield MTT formazan, based on mitochondrial metabolic activity, was used to assess cell viability. Lymphocyte cytotoxic effects were observed in all treatment groups on day 21; chicks that started on amended feed at day 1 of age were affected more than those that started at day 7. Abnormal erythrocytes resembling early stages of erythroblasts were observed in peripheral blood from test chicks. Abnormally shaped red cells (poikilocytes) having a spindle-shape with one or both ends pointed were present. Some red cells appeared to be undergoing mitosis. Both reduced lymphocyte viability and abnormal erythrogenesis occurred in chicks given feed amended withF. proliferatum culture material containing FB₁, FB₂ and moniliformin.     

Fumonisin and Beauvericin Induce Apoptosis in Turkey Peripheral Blood Lymphocytes

Fumonisins, a family of mycotoxins produced by Fusarium verticillioides (synonym Fusarium moniliforme Sheldon) and F. proliferatum, have been associated with various deleterious effects in different animal species. Serological, hematological and pathological effects and mortality have previously been observed in broiler chicks fed F. proliferatum culture material containing known concentrations of fumonisin, moniliformin and beauvericin. Turkey peripheral blood lymphocytes were exposed in vitro for 72 hours to fumonisin B₁(FB₁), fumonisin B₂(FB₂), hydrolyzed fumonisin B₁ (HFB₁), moniliformin and tricarballylic acid (TCA) (0.01-25 g/ml). A decrease in cell proliferation, as determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] bioassay, occurred in the order: FB₂ > FB₁ > HFB₁, with IC₅₀ = 0.6 M, 1 M and 10 M, respectively. Internucleosomal DNA fragmentation and morphological features characteristic of apoptosis were observed following exposure to fumonisin B₁ and beauvericin; cytoplasmic condensation and membrane blebbing were seen by light microscopy. Tricarballylic acid and moniliformin did not interfere with cell proliferation. Results suggested that fumonisin B₁ and beauvericin may affect immune functions by suppressing proliferation and inducing apoptosis of lymphocytes.     

Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids growth in Argentina

Fifty commercial corn hybrids with different endosperm characteristics, vegetative cycle length and cross class grown in the same geographical area (Cordoba Province, Argentina) were analysed for fumonisin accumulation. All hybrids analysed showed fumonisin B₁ and B₂ contamination ranging from 185 to 27,050 ng/g for FB₁ and from 40 to 9950 ng/g for FB₂. Although most of the hybrids analysed had flint-type endosperm, two hybrids with dent-type endosperm (e.g. Prozea 10 and AX 746) showed the highest level of fumonisin (37,000 ng/g) and more FB₂ than FB₁ (FB₂/FB₁ ratio 2.42), respectively. There was no correlation between fumonisin concentration and length of the vegetative cycle. Among 18 hybrids examined for Fusarium species contamination there was also no correlation between fumonisin contamination and the level of infection with Fusarium species (Section Liseola). Eighteen hybrids showed fumonisin levels lower than 1000 ng/g. This result suggests that there is some possibility of selecting hybrids resistant or less susceptible to fumonisin and Fusarium contamination.     

Production of a phage-displayed mouse ScFv antibody against fumonisin B1 and molecular docking analysis of their interactions

Fumonisins produced by Fusarium pathogens are mycotoxins present in maize and other grains in the field as well as during storage worldwide and pose a serious threat to humans and domestic animals. Fumonisin B consists of different chemotypes, and fumonisin B₁ (FB₁) is the most predominant fumonisin found in food/feed commodities. Recombinant antibody can be deployed to analyze the fumonisin toxicological mechanism and develop a simple and cost-effective method for the detection of fumonisins, which is vitally important for monitoring and preventing fumonisins from entering food/feed chains. In this study, FB₁ conjugated to keyhole limpet hemocyanin was used to immunize mice, from which RNA was isolated to construct a recombinant antibody library. Successive panning of the library by phage display was used to select monoclonal phage clones reactive to FB₁ conjugated to bovine serum albumin. Subsequent phage ELISA and sequencing analyses revealed four different reactive scFv antibodies specific to FB₁. Soluble expression and ELISA analysis showed that one scFv antibody, FBMA1, had the highest reactivity and could be purified from bacterial cells in large quantities. Surface plasmon resonance measurements further revealed that the FBMA1 scFv antibody had a binding kinetics of K _D = 1.89 × 10^–7 M. Molecular modeling and docking analyses suggested that the FBMA1 antibody shaped a proper cavity to embed the whole FB₁ molecule and that a steady-state complex was formed relying on intermolecular forces, including hydrogen bonding, electrostatic force and hydrophobic interactions. Thus, the scFv antibody can be applied for mechanistic studies of intermolecular interactions and fumonisin toxicity, and for the development of an immunoassay for fumonisin-contaminated food/feed samples.     

Production of fumonisins byFusarium species of Taiwan

Twenty-nineFusarium species isolated from various sources in different districts of Taiwan were tested for their ability to produce fumonisins in corn cultures. OnlyFusarium moniliforme produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). The finding that the other 28Fusarium species produced neither FB₁ nor FB₂ is preliminary because only one strain per species was studied. The detection of FB₁ and FB₂ in cultures ofF. moniliforme was demonstrated by TLC and HPLC, and FB₁ was further confirmed by mass spectrometry. In a separate experiment, in which 38 strains ofF. moniliforme were tested for fumonisins, approximately 66% (25/38) produced FB₁ and/or FB₂. Of the 25 strains, 14 produced only FB₁ and 11 produced both FB₁ and FB₂, and the amounts of FB₁ and FB₂ produced by different strains varied greatly. This is the first report that fumonisins are found in corn cultures experimentally infected withF. moniliforme strains from Taiwan. It is safe to assume that fumonisin producing strains ofF. moniliforme are widely distributed among the economic crops such as corn, rice, sugarcane, and sorghum throughout the Island.Abbreviations FB₁ Fumonisin B₁ - FB₂ Fumonisin B₂ - OPA o-phthalidialdehyde     

Co-occurrence of five Fusarium toxins in corn-Dried Distiller’s Grains with Solubles in Thailand and comparison of ELISA and LC-MS/MS for fumonisin analysis

Dried Distiller’s Grains with Solubles (DDGS), a by-product of bio-ethanol production from maize and other cereals, is increasingly used as a feed additive. In this study, five Fusarium toxins, including fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), deoxynivalenol (DON), zearalenone (ZEN) and beauvericin (BEA) were quantified by LC-MS/MS in 59 corn-DDGS samples. In addition, the fumonisin level in 30 randomly selected-samples was compared using an ELISA detection technique. No sample was free from mycotoxin contamination, and 50.8 % of the samples were co-contaminated with all five mycotoxins. Moreover, toxin levels were generally high, with mean levels of 9 mg kg^?1 FB₁, 6 mg kg^?1 FB₂, 1.2 mg kg^?1 DON, 0.9 mg kg^?1 ZEN, and 0.35 mg kg^?1 BEA. Maximum levels for FB₁ (143 mg kg^?1) and FB₂ (125 mg kg^?1) are of acute toxicological relevance. The ELISA method had a tendency to underestimate the fumonisin content when compared with LC-MS/MS. Finally, this is the first reported beauvericin contamination in corn-DDGS.     

Fum3p, a 2-Ketoglutarate-Dependent Dioxygenase Required for C-5 Hydroxylation of Fumonisins in Fusarium verticillioides

Fumonisins are polyketide-derived mycotoxins produced by several agriculturally important Fusarium species. The B series fumonisins, FB₁, FB₂, FB₃, and FB₄, are fumonisins produced by wild-type Fusarium verticillioides strains, differing in the number and location of hydroxyl groups attached to the carbon backbone. We characterized the protein encoded by FUM3, a gene in the fumonisin biosynthetic gene cluster. The 33-kDa FUM3 protein (Fum3p) was heterologously expressed and purified from Saccharomyces cerevisiae. Yeast cells expressing the Fum3p converted FB₃ to FB₁, indicating that Fum3p catalyzes the C-5 hydroxylation of fumonisins. This result was verified by assaying the activity of Fum3p purified from yeast cells. The C-5 hydroxylase activity of purified Fum3p required 2-ketoglutarate, Fe²⁺, ascorbic acid, and catalase, all of which are required for 2-ketoglutarate-dependent dioxygenases. The protein also contains two His motifs that are highly conserved in this family of dioxygenases. Thus, Fum3p is a 2-ketoglutarate-dependent dioxygenase required for the addition of the C-5 hydroxyl group of fumonisins.     

AAL Toxins,funionisms (biology and chemistry) and host-specificity concepts

The AAL toxins and the fumonisins (FB₁ and FB₂) are structurally related and produced respectively by Alternaria alternata f.sp. lycopersici and Fusarium moniliforme. AAL toxin is characterized as a hostspecific toxin, toxic to tomato, whereas fumonisin B₁ causes equine leukoencephalomalacia. FB₁ and FB₂ were biologically active in susceptible tomato tissue (Earlypak-7) and animal tissue culture (rat hepatoma H4TG and dog kidney MDCK). Conversely, AAL toxin was also active in the rat and dog tissue culture cells. Both fungi produce toxin/s in culture that cause death in rats; these toxins are other than AAL and fumonisin. The peracetylated derivatives of AAL and FB₁ are biologically inactive in both the tomato bioassay and the animal tissue culture systems. Acetylation of the amine renders AAL inactive. The hydrolysis product of AAL (pentolamine) is toxic to the susceptible tomato line whereas the pentolamine of fumonisin is not.AAL and FB₁ can be analyzed by Continuous Flow Fast Atom Bombardment (CFFAB) and Ionspray Mass Spectrometry (ISM), both sensitive to the picomole range. The N-acetyl of the TFA hydrolysis product of AAL and FB₁ is determined by comparing the fragment ions at m/z 86 and 140 for FB₁ and 72 and 126 for AAL.Published as paper No. 19,598 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under Project 22–34H, supported by HATCH funds.     

Mutagenicity of potentially carcinogenic mycotoxins produced byFusarium moniliforme

The mutagenic behaviour of two potentially carcinogenic mycotoxins produced byFusarium moniliforme was investigated in theSalmonella mutagenicity test using tester strains TA97a, TA98, TA100, and TA102. The mutagenic response obtained with fusarin C (1, 5, and 10μg/plate) against tester strains TA98 and TA100 in the presence of microsomal activation confirmed previous observations on the mutagenic behaviour of this mutagen while that obtained against TA97a is reported for the first time. No dose-response relationship could be detected for the concentration levels (0.2, 0.5, 1, 5, 10 mg/plate) tested for FB₁, FB₂, and FB₃ against any of the tester strains used in either the plate incorporation and / or the pre-incubation tests. A cytotoxic effect was obtained at concentration levels of 5 and 10mg/plate in the absence of the microsomal activation mixture. From the studies it became evident thatF moniliforme produces two compounds, a mutagenic compound, fusarin C which has been shown to lack carcinogenic activity in rats and the non-mutagenic fumonisin B mycotoxins of which FB₁ is known to be responsible for the hepatocarcinogenicity of the fungus in rats.     

Variability and characterization of mycotoxin-producing Fusarium spp isolates by PCR-RFLP analysis of the IGS-rDNA region

In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB₁, FB₂, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be␣divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA␣and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.     

Ear-rot fungi and mycotoxins in South African corn of the 1989 crop exported to Taiwan

A shipment of South African corn (1989) exported to Taiwan, was analyzed for various ear-rot fungi andFusarium mycotoxins. Two sets of samples, one from the points of origin in South Africa prior to shipment, and the other from the end-point distributors in Taiwan, were studied. Surface-sterilized kernels were plated onto two different agar media and the fungal colonies identified. High Performance Liquid Chromatography was used to analyze mycotoxin levels. The predominant ear-rot fungi, in decreasing order of isolation frequency, wereFusarium subglutinans, F. moniliforme, Diplodia maydis andF. graminearum. Aspergillus flavus andA. parasiticus were not isolated from samples prior to export, but a small number ofA. flavus isolates were found after shipment. The predominant mycotoxins were fumonisins B₁ (0–865 ng/g) and B₂ (0–250 ng/g). Low levels of moniliformin (390 ng/g) were detected in some samples before shipment. Zearalenone (25 ng/g), and nivalenol (120 ng/g) were detected in two out of 32 samples taken in Taiwan. The samples contained no detectable levels of either aflatoxins (>0.5 ng/g) or deoxynivalenol (>100 ng/g) before or after shipment.Abbreviations RSA South Africa(n) - FB₁ fumonisin B₁ - FB₂ fumonisin B₂ - ETVL eastern Transvaal - WTVL western Transvaal     

Fumonisin mycotoxins in human hair

This study shows for the first time the accumulation of fumonisin mycotoxins in human hair of population clusters exposed to contaminated maize, and thus the feasibility of human hair analysis for the assessment of past fumonisin exposure. Composite hair samples were obtained from the Bizana, Butterworth and Centane districts within the Transkei region of the Eastern Cape Province of South Africa. Following methanol extraction and strong anion exchange clean up, the fumonisins FB₁, FB₂ and FB₃ were detected using high performance liquid chromatography coupled to electrospray ionization-mass spectrometry (HPLC-ESI-MS). Hair from Centane and Butterworth showed mean levels of FB₁ of 26.7 and 23.5 μg kg^?1 hair, respectively. FB₂ was only detected in hair from Centane and in one sampling point in Butterworth, with mean levels of 6.5 and 5.7 μg kg^?1 hair, respectively. Hair samples from Bizana, on the other hand, were found to contain higher levels of FB 1 (mean 33.0 μg kg^?1 hair) and FB 2 (mean 11.1 μg kg^?1 hair). No samples contained more than trace levels of FB 3 . Recoveries from spiked hair samples using this method ranged from 81% to 101%, demonstrating the applicability of hair analysis in assessing human exposure to fumonisin mycotoxins.     

Occurrence of fumonisin B1 and B2 in corn-based foodstuffs in Taiwan market

Corn-based human foodstuffs purchased in Taiwan were analyzed for fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) using high-performance liquid chromatography. Fifty-two (33.9%) and 32 (20.9%) of 153 samples were found to contain FB₁ (73–2395 ng/g) and FB2 (120–715 ng/g), respectively. The highest frequency of detection and also the highest FB1 concentrations were found in sweetcorn (50%, 1089 ng/g) and cornflour (50%, 608 ng/g), followed by corn snacks (33.3%, 2395 ng/g), miscellaneous corn products (33.3%, 73 ng/g), popcorn (31.8%, 1003 ng/g) and cornflakes (23.5%, 1281 ng/g). 16 corn snacks (= approximately 20.5% of the samples) had an average FB₁ and FB₂ content of 456 and 145 ng/g, respectively, while six sweetcorn (= 25%) samples were contaminated with an average of 400 ng/g of FB₁ and 65 ng/g of FB₂. Of the 22 pop-corn samples examined, 7 had an average of 347 ng/g and 116 ng/g of FB₁ and FB₂, respectively. During an analysis of the distribution pattern for the combined fumonisin levels of FB₁ and FB₂, it became apparent that more than 69% of tested samples had fumonisin concentrations below 100 ng/g, while 11.1% (or 17 samples) contained in excess of 600 ng toxins per g. These results clearly illustrated that commercially available corn-based foodstuffs for human consumption in Taiwan are frequently contaminated with FB₁ and FB₂.This revised version was published online in October 2005 with corrections to the Cover Date.     

In vivo effects of fumonisin B1-producing and fumonisin B1-nonproducing Fusarium moniliforme isolates are similar: Fumonisins B2 and B3 cause hepato- and nephrotoxicity in rats

Fumonisins are mycotoxins produced by Fusarium moniliforme, F. proliferatum, and related Fusarium species found on corn. They occur naturally in corn-based feeds and foods and are suspected human esophageal carcinogens. Fumonisin B₁ (FB₁), the most common homologue, causes the animal diseases associated with F. moniliforme. Hepato- and nephrotoxicities, disrupted sphingolipid metabolism, and liver cancer have been found in rats fed FB₁. To determine the in vivo effects of diets containing fumonisins B₂ (FB₂) or B₃ (FB₃), male rats were fed culture materials (CM) of FB₁ non-producing F. moniliforme isolates to provide low (4.6–6.7 ppm), mid (32–49 ppm) or high (219–295 ppm) dietary levels of either FB₂ (FB₂CM) or FB₃ (FB₃CM). Other groups were fed culture material of an FB₁ producing isolate (FB₁CM) providing 6.9, 53 or 303~ppm total fumonisins (FB₁ : FB₂ : FB₃ = 1.0 : 0.38 : 0.15) and a tenth group was fed a control diet having no detectable fumonisins. One-half (n = 5/group) the animals were killed after three weeks, at which time the toxicological and histopathological effects of the three culture materials were similar, mimicked the effects of FB₁, and included decreased body weight gains, serum chemical indicators of hepatotoxicity, decreased kidney weights, and apoptosis of hepatocytes and kidney tubular epithelium. FB₁CM, FB₂CM, and FB₃CM affected sphingolipids, causing increased sphinganine to sphingosine ratios (Sa/So) in both liver and kidneys. The remaining animals (n = 5/group) were fed a control diet for three additional weeks. All body weight and tissue specific effects, including increased Sa/So, induced by the FB₂CM, FB₃CM and low level FB₁CM diets were absent following the recovery period. Except for mild biliary lesions found in the high dose FB₁CM group and a few apoptotic hepatocytes present in one mid- and two high-dose FB₁CM rats, no evidence of toxicity remained in these groups following the recovery period.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mortality in broiler chicks on feed amended withFusarium proliferatum culture material or with purified fumonisin B1 and moniliformin

Two hundred twenty-eight male chicks (Columbia × New Hampshire) were given feed amended with autoclaved culture material (CM) ofFusarium proliferatum Containing fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and moniliformin in 3 separate feeding trials. Purified FB₁ and moniliformin were given separately and in combination in a fourth feeding trial. Birds were given amended rations at day 1 (Trial 1 and 4), day 7 (Trial 2), and day 21 (Trial 3) and their respective ration was given for 28 days (Trial 1), 21 days (Trial 2), 7 days (Trial 3), and 14 days (Trial 4). FB₁ concentrations were 546, 193, and 61 ppm; FB₂ were 98, 38 and 14 ppm; and moniliformin were 367, 193, and 66 ppm in the first 3 feeding trial regimens. Chicks in Trial 4 were given dietary concentrations of purified FB₁ at 274 and 125 ppm, and moniliformin at 154 and 27 ppm. FB₁ and moniliformin, both alone and in combination, produced dose-responsive clinical signs, reduced weight gains and mortality in chicks. Age of birds given amended feeds had little difference in the clinical response; however, those given the rations from days 7 or 21 were slightly less susceptible than those given rations beginning at 1 day of age. Additive effects were noted when the toxins were given in combination. When toxins were given separately, adverse effects took longer to occur. A system to monitor pattern and rate of defecation (RD) was developed for assessing the chicks' approach to feed, water and heat source as illness progressed. Our results indicate that chicks fed corn heavily infected withF. proliferatum under field conditions could suffer acute death similar to that described for spiking mortality syndrome during the first 3 weeks of age.     

Screening toxicity study in young carp (Cyprinus carpio L.) on feed amended with fumonisin B1

Fumonisin B₁ (FB₁) is one of several mycotoxins produced by Fusarium moniliforme, a major fungal pathogen of corn and widely spread throughout the world. FB₁ produces a wide range of biological effects, some of which are specific for particular organs or species and some are common to all investigated animals. In this study we have evaluated subchronic toxicosis features in young carp (Cyprinus carpio L.) exposed to 0.5 and 5.0 mg FB₁ kg^–1 body weight for 42 days through nutritionally balanced diet. During the trial we observed loss of body weight in both treated groups, together with higher incidence of infective bacterial dermatological lesions erythrodermatitis cyprini (Aeromonas salmonicida subsp. nova) in the group treated with the higher FB₁ dose. Several hematological parameters (erythrocyte count, platelet count) and serum chemical concentrations (creatinin, total bilirubin) and activities (aspartate aminotransferase, AST and alanine aminotransferase, ALT) were greater in the fumonisin treated groups than in the control group. Our results indicate that long-term dietary exposure to 0.5 and 5.0 mg FB₁ kg^–1 body weight is not lethal to young carp, but can produce adverse physiological effects. These findings also suggest that primary target organs of FB₁ in the carp are kidney and liver, as it has already been observed in other animal species tested. Specifically changed red blood cell-parameters reveal that FB₁ probably causes erythrocyte membrane defect or interferes with carp's respiratory process.This revised version was published online in October 2005 with corrections to the Cover Date.     

Reducing production of fumonisin mycotoxins in <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> by RNA interference

The fungus Fusarium verticillioides is a maize pathogen that can produce fumonisin mycotoxins in ears under certain environmental conditions. Because fumonisins pose health risks to humans and livestock, control strategies with minimal risk to the environment are needed to reduce fumonisin contamination. Host-induced gene silencing is a promising technique in which double-stranded RNA expressed in the plant host is absorbed by an invading fungus and down-regulates genes critical for pathogenicity or mycotoxin production in the fungus. A key preliminary step of this technique is identification of DNA segments within the targeted fungal gene that can effectively silence the gene. Here, we used segments of the fumonisin biosynthetic gene FUM1 to generate double-stranded RNA in F. verticillioides. Several of the resulting transformants exhibited reduced FUM1 gene expression and fumonisin production (24- to 3675-fold reduction in fumonisin FB₁). Similar reductions in fumonisin production resulted from double-stranded RNA constructs with segments of FUM8, another fumonisin biosynthetic gene (3.5- to 2240-fold reduction in fumonisin FB₁). FUM1 or FUM8 silencing constructs were transformed into three isolates of F. verticillioides. Whole genome sequence analysis of seven transformants revealed that reductions in fumonisin production were not due to mutation of the fumonisin biosynthetic gene cluster and revealed a complex pattern of plasmid integration. These results suggest the cloned FUM1 or FUM8 gene segments could be expressed in maize for host-induced gene silencing of fumonisin production.     

Practical aspects of fumonisin production under laboratory conditions

Studies were performed to develop an efficient method for fumonisin toxin production in sufficient quantities for animal toxicological experiments, on the basis of three earlier published fumonisin toxin production methods. Three absolutely necessary factors were taken into account and tested in a serial experiment. TheFusarium verticillioides strain MRC 826 was directly inoculated onto soaked, autoclaved, whole maize kernels (50 g/1.71 jar). The inoculation was performed by standard spore suspension (l×l0⁶/ml), a 5/2 surface/volume culture was prepared and incubated at 25 °C for 5 weeks. To maintain the optimal a_w of approximately 1.00, the evaporated water was re-filled weekly. A final concentration of 4454±1060.9 ppm fumonisin B₁ was reached, with good repeatability. In the laboratory practice, consistent production of constant amounts of FB₁ can be obtained by applying the above settings.