Production of fumonisins by Fusarium moniliforme and Fusarium proliferatum isolates associated with equine leukoencephalomalacia and a pulmonary edema syndrome in swine.

Fumonisin B1 (FB1) and FB2 were isolated from corn cultures of both Fusarium moniliforme and Fusarium proliferatum. Respective concentrations in culture materials of FB1 and FB2 ranged from 960 to 2,350 and 120 to 320 micrograms/g for F. moniliforme and from 1,670 to 2,790 and 150 to 320 micrograms/g for F. proliferatum. Thin-layer chromatography, gas chromatography-mass spectroscopy, high-performance liquid chromatography, and liquid secondary ion mass spectroscopy were used for detection. Fumonisins from F. proliferatum have not previously been reported.     

Survey of fumonisin production by Fusarium species

Fumonisins B1 (FB1) and B2 (FB2), two structurally related mycotoxins with cancer-promoting activity, were recently isolated from corn cultures of Fusarium moniliforme MRC 826. These toxins have been reported to be produced also by isolates of F. proliferatum. Contamination of foods and feeds by F. moniliforme has been associated with human esophageal cancer risk, and FB1 has been shown to be the causative agent of the neurotoxic disease leukoencephalomalacia in horses. Because of the toxicological importance of the fumonisins, the potential to produce FB1 and FB2 was determined in a study of 40 toxic Fusarium isolates representing 27 taxa in 9 of the 12 sections of Fusarium, as well as two recently described species not yet classified into sections. With the exception of one isolate of F. nygamai, fumonisin production was restricted to isolates of F. moniliforme and F. proliferatum, in the section Liseola. The F. nygamai isolate produced 605 micrograms of FB1 g-1 and 530 micrograms of FB2 g-1, and the identity of the toxins was confirmed by capillary gas chromatography-mass spectrometry. This is the first report of the production of the fumonisins by F. nygamai.     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Mycotoxin Production by Fusarium Species Isolated from Bananas

The ability of Fusarium species isolated from bananas to produce mycotoxins was studied with 66 isolates of the following species: F. semitectum var. majus (8 isolates), F. camptoceras (3 isolates), a Fusarium sp. (3 isolates), F. moniliforme (16 isolates), F. proliferatum (9 isolates), F. subglutinans (3 isolates), F. solani (3 isolates), F. oxysporum (5 isolates), F. graminearum (7 isolates), F. dimerum (3 isolates), F. acuminatum (3 isolates), and F. equiseti (3 isolates). All isolates were cultured on autoclaved corn grains. Their toxicity to Artemia salina L. larvae was examined. Some of the toxic effects observed arose from the production of known mycotoxins that were determined by thin-layer chromatography, gas chromatography, or high-performance liquid chromatography. All F. camptoceras and Fusarium sp. isolates proved toxic to A. salina larvae; however, no specific toxic metabolites could be identified. This was also the case with eight isolates of F. moniliforme and three of F. proliferatum. The following mycotoxins were encountered in the corn culture extracts: fumonisin B(inf1) (40 to 2,900 (mu)g/g), fumonisin B(inf2) (150 to 320 (mu)g/g), moniliformin (10 to 1,670 (mu)g/g), zearalenone (5 to 470 (mu)g/g), (alpha)-zearalenol (5 to 10 (mu)g/g), deoxynivalenol (8 to 35 (mu)g/g), 3-acetyldeoxynivalenol (5 to 10 (mu)g/g), neosolaniol (50 to 180 (mu)g/g), and T-2 tetraol (5 to 15 (mu)g/g). Based on the results, additional compounds produced by the fungal isolates may play prominent roles in the toxic effects on larvae observed. This is the first reported study on the mycotoxin-producing abilities of Fusarium species that contaminate bananas.     

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities ( a _w, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B₁ (FB₁) and B₂ (FB₂) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing a _w and best at 30°C. All three isolates produced more FB 1 than FB₂ regardless of a _w or temperature. Very little FB₁ and FB₂ were produced at 0.925 a _w, with maximum produced at 0.956 and 0.968 a _w at both temperatures tested. Most FB₁ and FB₂ were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all a _w levels and both temperatures there was an increase in FB₁ and FB₂ concentration with time. Statistical analyses of a _w, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB₁ and FB₂ production.     

Swine refusal factors elaborated by Fusarium strains and identified as trichothecenes.

Fusarium poae (Peck) Wollenw. NRRL 3287, F. nivale (Fr.) Ces. NRRL 3289, and F. moniliforme Sheldon NRRL 3197, each grown on cracked corn (13 days at 28 degrees C), produced refusal factors in pig bioassays. Substantial quantities of trichothecenes were detected in the refused corn: T-2 toxin (30 micrograms/g) was detected in corn fermented with the F. poae strain; the level of vomitoxin (1 microgram/g) in corn cultured with F. nivale did not account for the 48% refusal response in the pigs tested. The F. moniliforme concomitantly produced T-2 toxin (33 micrograms/g) and vomitoxin (1.5 micrograms/g). This strain's taxonomic position was reexamined, and it is shown to be a cultural variant of the species F. tricinctum (Cda.) Sacc.     

Thermostability of Fumonisin B(1), a Mycotoxin from Fusarium moniliforme, in Corn

Fumonisin B(1) (FB(1)) is a mycotoxin from Fusarium moniliforme that is frequently associated with corn. Thermal treatments are used in many processes concerning this cereal and its derivatives. The thermostability of this toxin in dry contaminated corn, resulting from F. moniliforme culture, was studied in different time-temperature combinations. FB(1) was quantified by instrumentalized thin-layer chromatography after a two-step sequential development and postchromatographic derivatization by p-anisaldehyde. The identity of FB(1) in extracts, before and after heat treatments, was confirmed by high-pressure liquid chromatography. For each temperature, the natural logarithm of the ratio of resulting FB(1) on initial content (In C/C(0)) is linearly correlated to exposure time. The calculated half-lives (L(50)), corresponding to the 50% value, were 10 min, 38 min, 175 min, and 8 h at 150, 125, 100, and 75 degrees C, respectively. There is a linear relationship between calculated L(50)s on a logarithmic scale and temperature. Therefore FB(1) is not significantly destroyed by the main drying processes of corn or thermal treatments used for its derivatives. Other associated means are required for detoxification.     

临床常见镰刀菌的鉴别

目的从分子生物学角度寻找一种快速准确鉴定临床常见镰刀菌的方法。方法将受试镰刀菌接种于PDA培养基,观察其菌落及镜下形态,在此基础上PCR扩增受试镰刀菌的rDNA ITS并测其序列,在GenBank核酸序列数据库进行同源序列搜索及分析。选择限制性内切酶Dra Ⅱ和Cfr13 Ⅰ进行RFLP。设计了茄病镰刀菌的种特异性引物Sol1、Sol2,初步验证其特异性。结果形态学鉴定结果显示,茄病镰刀菌所占比例最高,除2株串珠镰刀菌外,其余镰刀菌ITS序列分析的结果与形态学鉴定结果一致。茄病、层生和串珠镰刀菌的Dra Ⅱ、Cfr13 I酶切带形互不相同。用Sol1、Sol2扩增受试菌的rDNA ITS,只有茄病镰刀菌为阳性。结论rDNA ITS序列测定及其PCR-RFLP可用于初步鉴别几种临床常见镰刀菌,合适的种特异性引物可以初步快速鉴定茄病镰刀菌。     

Fusarin C production by North American isolates of Fusarium moniliforme

A liquid culture medium was developed to screen North American isolates of Fusarium moniliforme Sheldon and Fusarium subglutinans (Wollenw. and Reink.) Nelson, Toussoun, and Marasas for their ability to produce fusarin C. Parameters which were important for the optimal biosynthesis of fusarin C included pH (3.0 to 4.0), aeration, and sugar concentration (30 to 40%). Of seven sugars tested, sucrose and glucose were the best carbohydrate sources for mycotoxin production, resulting in levels of fusarin C of greater than 60 ppm (greater than 60 micrograms/g) in liquid culture (28 degrees C; 7 days). A time-course study of fusarin C production was done over a 21-day period, during which time pH values, glucose concentrations, nitrogen levels, and fungal biomass were determined. Of the two Fusarium spp. studied, 13 of 16 isolates of F. moniliforme produced fusarin C in liquid medium (14 of 16 in corn), while none of the 15 isolates of F. subglutinans studied was found to produce the compound. Levels of fusarin C produced by Fusarium sp. isolates growing on corn ranged from 18.7 to 332.0 micrograms/g.     

FUM cluster divergence in fumonisins-producing Fusarium species

Fumonisins are polyketide-derived mycotoxins, produced by several Fusarium species, and its biosynthetic pathway is controlled by the FUM cluster--a group of genes exhibiting a common expression pattern during fumonisin biosynthesis. The most common are the B analogues with fumonisin B(1) (FB(1)) being the most prevalent. At least a part of the inter- and intraspecific variation in FBs synthesis level can be explained by the sequence differences inside FUM cluster. The aim of our study was to evaluate the toxin production and sequence variability in FUM genes and intergenic regions among thirty isolates of seven species reported as potential fumonisins producers: Fusarium anthophilum, Fusarium fujikuroi, Fusarium nygamai, Fusarium oxysporum, Fusarium proliferatum, Fusarium subglutinans and Fusarium verticillioides, particularly with respect to FBs synthesis. Fumonisins were produced in high amounts (over 1mg g(-1)) by one isolate of F. subglutinans, three of F. verticillioides and all F. proliferatum isolates except one, regardless of the host organism. The remaining isolates produced low amounts of FBs and two F. verticillioides isolates didn't produce it at all. The lowest variation in amount of toxin produced was found among F. proliferatum isolates. Based on the translation elongation factor 1α (tef-1α) sequence of F. fujikuroi, a species-specific marker was developed. The intergenic region presents similar opportunity for F. nygamai identification. The phylogenetic reconstruction based on FUM1 gene generally reflects the scenario presented by tef-1α sequences. Although the sequence similarities for intergenic regions were lower than in coding regions, there are clearly conserved patterns enabling separation of different subsets of species, including the non-producer species.     

Fusarin C production by North American isolates of Fusarium moniliforme.

A liquid culture medium was developed to screen North American isolates of Fusarium moniliforme Sheldon and Fusarium subglutinans (Wollenw. and Reink.) Nelson, Toussoun, and Marasas for their ability to produce fusarin C. Parameters which were important for the optimal biosynthesis of fusarin C included pH (3.0 to 4.0), aeration, and sugar concentration (30 to 40%). Of seven sugars tested, sucrose and glucose were the best carbohydrate sources for mycotoxin production, resulting in levels of fusarin C of greater than 60 ppm (greater than 60 micrograms/g) in liquid culture (28 degrees C; 7 days). A time-course study of fusarin C production was done over a 21-day period, during which time pH values, glucose concentrations, nitrogen levels, and fungal biomass were determined. Of the two Fusarium spp. studied, 13 of 16 isolates of F. moniliforme produced fusarin C in liquid medium (14 of 16 in corn), while none of the 15 isolates of F. subglutinans studied was found to produce the compound. Levels of fusarin C produced by Fusarium sp. isolates growing on corn ranged from 18.7 to 332.0 micrograms/g.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme.

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

Contamination of pine nuts by fumonisin produced by strains of Fusarium proliferatum isolated from Pinus pinea

AIMS: To test the ability of Fusarium proliferatum strains isolated from Pinus pinea to synthesize fumonisin B(1) (FB(1)) in pine nuts. METHOD AND RESULTS: Eleven strains were inoculated in moist sterile shelled pine nuts and whole pine nuts, and incubated for 28 days. Moist sterile maize was inoculated in parallel as an optimum substrate for FB(1) production by Fusarium species. CONCLUSIONS: Six of the strains produced FB(1) in shelled pine nuts, and two of them did so in whole pine nuts. IMPACT AND SIGNIFICANCE OF THE STUDY: F. proliferatum is able to grow and produce FB(1) in the husk of pine nuts and the mycotoxin can be found in the nut once shelled. Consequently, FB(1) may be an important hazard in pine nuts.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture.

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

Extracellular enzymatic activity of Fusarium section Liseola isolates

Extracellular enzymatic activity of Fusarium species from section Liseola was determined by the Api-Zym^R test, a semiquantitative method for assaying different cellular enzymes. This enzyme testing system was evaluated for possible use as a species identification aid. Twenty-two strains of Fusarium section Liseola were used: 8 of F. moniliforme, 6 of F. proliferatum, 6 of F. subglutinans and 2 of F. anthophilum. The optimal spore concentration was also determined for maximal activity in Api-Zym^R assay. According to our findings, the species showed little difference in Api-Zym^R enzymatic profiles, being not suitable for identification of these species. Only F. subglutinans and F. anthophilum showed a different enzymatic pattern. This revised version was published online in August 2006 with corrections to the Cover Date.     

Moniliformin production and toxicity of different Fusarium species from Southern Africa.

Four new moniliformin-producing species of Fusarium were found, viz., F. acuminatum, F. concolor, F. equiseti, and F. semitectum. Isolates of F. acuminatum and F. concolor produced large amounts of moniliformin (3.4 and 9.5 g/kg, respectively), whereas isolates of the other three species yielded less than 30 mg/kg. The production of moniliformin by isolates of F. oxysporum and F. avenaceum from southern Africa is described. All 14 toxic isolates of F. oxysporum produced moniliformin. Most isolates of F. fusarioides and all six isolates of Fusarium moniliforme va. subglutinans tested produced moniliformin, as did 28 of 36 toxic isolates of F. moniliforme. A number of F. moniliforme isolates produced greater than 10 g/kg, and one isolate yielded 33.7 g/kg in corn after incubation for 5 weeks at 25 degrees C. Moniliformin production in the field in corn ears was shown by inoculating plants with known moniliformin-producing isolates of three Fusarium species. Yields of up to 645 mg/kg were recorded. Isolates of F. acuminatum, F. equiseti, F. fusarioides, and F. moniliforme were found that were highly toxic to ducklings but which did not produce moniliformin.     

Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.