Prevalence of Fusarium species of the Liseola section on selected Colombian animal feedstuffs and their ability to produce fumonisins

A total of 57 samples of feedstuffs commonly used for animal nutrition in Colombia (corn, soybean, sorghum, cottonseed meal, sunflower seed meal, wheat middlings and rice) were analyzed for Fusarium contamination. Fusarium fungi were identified at species level by means of conventional methods and the ability to produce fumonisins of the most prevailing species was determined. A total of 41 of the feedstuffs analyzed (71.9%) were found to contain Fusarium spp. Most contaminated substrates were corn (100%), cottonseed meal (100%), sorghum (80%), and soybean (80%). Wheat middlings and rice showed lower levels of contamination (40% and 20%, respectively), while no Fusarium spp. could be isolated from sunflower seed meal. The most prevalent species of Fusarium isolated were F. verticilliodes (70.8%), F.␣proliferatum (25.0%), and F. subglutinans (4.2%). All of them correspond to section Liseola.Production of fumonisins on corn by the isolated Fusarium was screened through liquid chromatography. Almost all strains of F. verticilliodes (97.1%) produced FB1 (5.6–25,846.4 mg/kg) and FB2 (3.4–7507.5 mg/kg). Similarly, almost all strains of F.␣proliferatum (91.7%) produced fumonisins but at lower levels than F.␣verticilliodes (FB1 from 6.9 to 3885.0 mg/kg, and FB2 from 34.3 to 373.8 mg/kg), while F. subglutinans did not produce these toxins. This is the first study in Colombia describing toxigenic Fusarium isolates from␣animal feedstuffs.     

Diversity of Fusarium species and mycotoxins contaminating pineapple

Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B₁–B₃, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB₁ content was 250 μg g^?1 in pineapple skin and 20 μg ml^?1 in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.     

Occurrence and toxicity ofFusarium subglutinans from Peruvian maize

Twenty-five samples of maize kernels collected at harvest time from geographically different corn fields in Peru, were examined for the occurrence of toxigenicFusarium species. The most frequently recovered species wereF. subglutinans (48%),F. moniliforme (46%), andF. equiseti (5%). OtherFusarium species isolated (up to 1%) includedF. graminearum, F. acuminatum, F. solani, F. oxysporum, andF. culmorum. Assays ofFusarium culture extracts usingArtemia salina larvae, showedF. subglutinans as one of the most toxigenic species, and its toxicity was mostly correlated to the capability to produce beauvericin (BEA). All eight tested isolates ofF. subglutinans grown on autoclaved corn kernels produced BEA (from 50 to 250 mg/Kg) as well as moniliformin (M) (from 70 to 270 mg/Kg). This is the first report on BEA and M production by maize isolates ofF. subglutinans from South America.     

Effects of beauvericin to mammalian tissue and its production by Austrian isolates ofFusarium proliferatum and Fusarium subglutinans

Austrian isolates ofFusarium subglutinans andFusarium proliferatum were studied for their ability to produce beauvericin, moniliformin and fumonisin B₁ and B₂ under laboratory conditions. Analytical methodology for beauvericin was specially adapted for this task. Our analyses showed that the strains produced beauvericin up to 687 mg /kg maize and moniliformin up to 70 mg/kg. The culture ofF. proliferatum in addition produced fumonisin B₁ and B₂ at levels of 106 and 61 mg/kg,respectively. The preliminary toxicity experiments performed in this study clearly indicated a toxic effect of beauvericin on the contractility of mammalian smooth muscle and thus on mammalian cells.     

Production of fumonisins B1, B2 and B3 byFusarium proliferatum Isolated from rye grains

Using the seed- plate technique, we have isolated a strain ofF. proliferatum from rye grains that produces 3 fumonisins, fumonisin B₁ (FB₁), FB₂ and FB₃ on inoculated rice and corn. Inoculated corn and rice were extracted with an aqueous methanol solution and fumonisin concentrations estimated using high performance liquid chromatography. Production of all 3 fumonisins (FB₁, FB₂ and FB₃) was much higher on rice than corn; ranging from 3816, 1068 and 985 ppm to 1643, 350 and 162 ppm respectively. We conclude that all natural substrates whereF. proliferatum is present as a component of the mycoflora should be monitored for the presence of fumonisins.     

Gas chromatography-mass spectrometry characterisation of the anti-Listeria components of Garcinia kola seeds

Adsorption chromatography was used to separate the bioactive constituents of the crude n-hexane extract of Garcinia kola seeds. The silica gel 60 column fractions were eluted using the solvent combination of benzene:ethanol:ammonium hydroxide (BEA) in the ratio combination of 36:4:0.4 v/v. The fractions were tested for anti-Listeria activities by determining their MIC₅₀, MIC₉₀ or MIC against 4 Listeria isolates. The fractions were labelled BEA1 to BEA5 and 3 out of the 5 fractions eluted were active against the test Listeria species with MIC’s ranging from MIC 0.157 mg/mL to MIC₅₀ 0.625 mg/mL. The most active fractions, BEA2 and BEA3, were subjected to gas chromatography coupled to mass spectrometry (GC-MS) to identify their composition. Fraction BEA2 constituted of 18 compounds mostly sterols and the BEA3 fraction contained 27 compounds with the most abundant compounds being fatty acids derivatives. The BEA2 fraction’s interactions with antibiotics proved to be 100% synergistic with ciprofloxacin and ampicillin whilst it exhibited 50% additivity and 50% synergism with penicillin G. However, all the interactions of the BEA2 fraction with each of the conventional antibiotics used were synergistic against the human listeriosis causative bacteria Listeria monocytogenes.     

珍珠岩液体培养基制备镰刀菌素C的研究

串珠镰刀菌可利用羟基脯氨酸、蔗糖、甘油和珍珠岩(P)等组成的P液体培养基合成镰刀菌素c(Fc),其最高量为93 6mg/kg有机物,比在玉米渣培养基中形成的Fc量较高。用P液体培养基制备Fc,受蔗糖浓度、胺类和培养温度及培养时间等的影响。实验证明,由1g百姓遭基因氨酸、40g蔗糖和珍珠岩组或的P液体培养基．在28℃培养二周是形成Fc的理想条件。液体培养基中加八珍珠岩,Fc的形成量增加500多倍。     

An Endophytic Sanguinarine-Producing Fungus from Macleaya cordata,Fusarium proliferatum BLH51

Fermentation processes using sanguinarine-producing fungi other than Macleaya cordata may be an alternative way to produce sanguinarine (SA), which is a quaternary benzo[c]phenanthridine alkaloid possessing antibacterial, anthelmintic, and anti-inflammatory properties. In this study, a SA-producing endophytic fungus strain BLH51 was isolated from the leaves of M. cordata grown in the Dabie Mountain, China. Strain BLH51 produced SA when grown in potato dextrose liquid medium. The amount of SA produced by this endophytic fungus was quantified to be 178 μg/L by HPLC, substantially lower than that produced by the host tissue. The fungal SA—which was analyzed by thin layer chromatography and high-performance liquid chromatography—was shown to be identical to authentic SA. Strain BLH51 was identified as Fusarium proliferatum based on the morphological characteristics and nuclear ribosomal DNA ITS sequence analysis. To the best of our knowledge, this is the first report concerning the isolation and identification of endophytic SA-producing fungi from the host plant, which further proved that endophytic fungi are valuable reservoirs of bioactive compounds.     

Occurrence of fumonisins in asparagus (asparagus officinalis L.) and garlic (allium sativum L.) from Germany

Fusarium proliferatum is able to produce fumonisins and is considered a pathogen of many economically important plants (e.g. corn, rice, asparagus) [1]. The occurrence of fumonisin FB₁ inF. proliferatum infected asparagus spears from Germany was investigated using a liquid chromatography/electrospray ionization-mass spectrometry (LC-ESI-MS) method with isotopically labeled fumonisin FB₁-d₆ as internal standard. Asparagus samples were harvested in July 2000 and screened forFusarium species. AltogetherF. oxysporum, F. proliferatum and F. sambucinum were isolated from the spears. The samples infected with F.proliferatum were subsequently analyzed for fumonisins. FB₁ was detected in 9 of the 10 samples in amounts ranging from 36.4 ng/g to 4513.7 ng/g (based on dry weight). Fumonisins FB₂ and FB₃ were found in six samples in lower concentrations. In asparagus spears of June 2002 we could findF. proliferatum in 6% of the samples, however no fumonisins were detectable. Furthermore the capability of producing FB₁ by the fungus in garlic bulbs was investigated. Therefore garlic was cultured inF. proliferatum contaminated soil and the bulbs were screened for infection with F.proliferatum and for the occurrence of fumonisins by LC-MS. F.proliferatum was detectable in the garlic tissue and all samples contained FB₁ (26.0 ng/g to 94.6 ng/g). This is the first report of the natural occurrence of FB₁ in German asparagus spears and furthermore our findings suggest a potential for natural contamination of garlic bulbs with fumonisins. For detailed results and methods see Ref. [2].     

Moniliformin production byFusarium species isolated from Argentinian corn

Forty-one isolates ofFusarium obtained from the main Argentinian corn production area were tested for their ability to produce moniliformin. One of 22 isolates ofF. moniliforme, 2/10 of F.proliferatum and 3/9 ofF. subglutinans, produced moniliformin in a range between 0,3 to 2,7 mg/g. These data represent the first report of the production of moniliformin byFusarium species from section Liseola in Argentina.     

Potential of kenaf (Hibiscus cannabinus L.) and corn (Zea mays L.) for phytoremediation of dredging sludge contaminated by trace metals

The potential of kenaf (Hibiscus cannabinus L.) and corn (Zea mays L.) for accumulation of cadmium and zinc was investigated. Plants have been grown in lysimetres containing dredging sludge, a substratum naturally rich in trace metals. Biomass production was determined. Sludge and water percolating from lysimeters were analyzed by atomic absorption spectrometry. No visible symptoms of toxicity were observed during the three- month culture. Kenaf and corn tolerate trace metals content in sludge. Results showed that Zn and Cd were found in corn and kenaf shoots at different levels, 2.49 mg/kg of Cd and 82.5 mg/kg of Zn in kenaf shoots and 2.1 mg/kg of Cd and 10.19 mg/kg in corn shoots. Quantities of extracted trace metals showed that decontamination of Zn and Cd polluted substrates is possible by corn and kenaf crops. Tolerance and bioaccumulation factors indicated that both species could be used in phytoremediation.     

Occurrence of Fusaproliferin and Beauvericin in Fusarium-Contaminated Livestock Feed in Iowa

Fusarium fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. Fusarium moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B₁ was produced by all nine F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.     

Optimization of a liquid medium for beauvericin production in <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial culture

Beauvericin (BEA) is a proven and potent antibiotic compound useful for bio-control and a potential antifungal and anticancer agent for human. This study was to evaluate and optimize the nutrient medium for BEA production in mycelial liquid culture of a high BEA-producing fungus Fusarium redolens Dzf2 isolated from a medicinal plant. Among various organic and inorganic carbon and nitrogen sources, glucose and peptone were found the most favorable for the F. redolens Dzf2 mycelial growth and BEA production. Through a Plackett-Burman screening test on a basal medium, glucose, peptone, and medium pH were identified as the significant factors for mycelial growth and BEA production. These factors were optimized through central composite design of experiments and response surface methodology, as 49.0 g/L glucose, 13.0 g/L peptone and pH 6.6, yielding 198 mg/L BEA (versus 156 mg/L in the basal medium). The BEA yield was further increased to 234 mg/L by feeding 10 g/L glucose to the culture during exponential phase. The results show that F. redolens Dzf2 mycelial fermentation is a feasible and promising process for production of BEA.     

Enhanced beauvericin production with in situ adsorption in mycelial liquid culture of Fusarium redolens Dzf2

Fusarium redolens Dzf2, an endophytic fungal species, is a high producer of the antibiotic compound beauvericin (BEA). However, the BEA produced by the F. redolens Dzf2 fungus was retained mainly as an intracellular product. This study was to evaluate an integrated fermentation-in situ product recovery process for enhancement of BEA production in F. redolens Dzf2 myelical culture. A macroporous polystyrene resin (X-5) was selected as the sorbent and added to the mycelial culture flasks (enclosed in a nylon bag). With 2 g resin added to 40 ml medium in each flask in the early stationary growth phase (day 5), the volumetric BEA yield (on day 7) was increased from 194 to 265 mg l^?1, with 65% being adsorbed to the resin phase. With resin renewal plus glucose feeding (on day 7), the BEA production was increased even more dramatically to 400 mg l^?1 (on day 9), double of the yield in the batch control culture. The results show that in situ adsorption was an effective strategy for enhancing the BEA production and also facilitating its recovery in the mycelial liquid culture.     

Mycotoxin Production by Fusarium proliferatum isolates from rice with Fusarium sheath rot disease

Twenty samples of unpolished (rough) rice collected in Arkansas and Texas during the 1995 harvesting season from fields exhibiting Fusarium sheath rot disease or panicle blight were previously shown to include 8 samples positive for fumonisin B₁(FB₁) in the range 2.2–5.2 ppm, and moniliformin (MON), but no beauvericin (BEA), deoxynivalenol, its derivatives or zearalenone were detected. Fifteen cultures of F. proliferatum were established from the 20 rough rice samples. Single spore isolates of each culture were grown on rice and tested for the production of fumonisins (FB₁, FB₂, FB₃, etc.), MON and BEA. All 15 isolates produced FB₁, FB₂, MON and BEA in culture on rice. No deoxynivalenol, its derivatives orzearalenone were detected. Seven cultures produced FB₁ at >50ppm (range 80–230 ppm), with therest producing FB₁ in the range 14–43 ppm.FB₂ was produced in the range 5–47 ppm, and those cultures which produced the most FB₁ also produced the most FB₂. Of the 15 cultures producing MON, 11 produced it at >100 ppm in the range 188–6018 ppm, with the rest producing in the range 7–64 ppm. BEA was produced in the range 109–1350 ppm. Other derivatives of fumonisins, including FA₁, FA₂ and partially hydrolyzed FB₁, as well asseveral unknown metabolites including a compound with MW 414, were identified in culture extracts by continuous flow fast atom bombardment with ion spraymass spectrometry (CF/FAB/MS). Further study is needed to identify the factors that control production of FB₁, MON and BEA by F.proliferatu in culture and in field samples. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production of allantoin, rabdosiin and rosmarinic acid in callus cultures of the seacoastal plant Mertensia maritima (Boraginaceae)

A callus culture of the extreme halophyte seacoastal plant Mertensia maritima (Boraginaceae) was established from apical shoots of the plant using a modified Murashige and Skoog medium supplemented with 6-benzyladenine (0.5?mg/L) and ??-naphthaleneacetic acid (2.0?mg/L). Three main compounds, (?)-R-allantoin, (+)-rabdosiin and rosmarinic acid, were isolated from extracts of M. maritima calli by liquid chromatography and identified by ¹H and ¹³C NMR, UV, ECD and HPLC?CMS. Quantitative HPLC analysis showed that the calli produce (+)-rabdosiin (0.14% dry wt), rosmarinic acid (0.74% dry wt) and (?)-R-allantoin (3.7% dry wt). Allantoin was detected in plant cell cultures for the first time. All of these metabolites were also present in lower quantities in different parts of the plant. The presence of rabdosiin and rosmarinic acid, in combination with the skin-conditioning agent (?)-R-allantoin, represents a potentially useful novel composition for skin protection.     

Occurrence of Fumonisin B<Subscript>1</Subscript> in Corn from the Main Corn-Producing Areas of China

Fumonisin B₁ (FB₁) is the most abundant of the fumonisin mycotoxins, mainly produced in maize by F. verticillioides and F. proliferatum. A total of 282 corn samples harvested in 2005 from six provinces, the main corn-producing areas of China, were analyzed for FB₁ using high-performance liquid chromatography. All samples except one were (99.6%) positive for FB₁ at levels varying from 3 to 71,121 ng/g with mean and median levels for all samples of 6,662 and 1,569 ng/g, respectively. During an analysis of the distribution pattern for FB₁, it became apparent that 43.6% of tested samples had FB₁ concentrations below 1,000 ng/g, while 25.2% contained in excess of 5,000 ng/g. The average exposure to FB₁ (1.1 μg/kg body weight/day) is within the provisional maximum tolerable daily intake of 2 μg/kg body weight/day set by the Joint FAO/WHO Expert Committee on Food Additives.     

Diacetoxyscirpenol production by Fusarium sambucinum strain KF 735 on solid and liquid media

The yield of diacetoxyscirpenol (DAS) production by F. sambucinum strain No KF 735, isolated from potato tuber with dry rot symptoms, cultured on solid media and on liquid medium, has been examined.The amount of DAS produced within 28 days at 25°C in the cultures grown on solid media (wheat, rye, rice, oats, corn, barley, triticale and malt) reached 238mg/kg±9 to 789±16mg/kg (mean ± standard error; n=3), on potato cubes ?55±3mg/kg and on the potato extract ?147±5mg/dcm³.The best substracts for crystalline compound production were malt and barley grain.     

Sequential Isolation of Superoxide Dismutase and Ajmaline from Tissue Cultures of Rauwolfia serpentinaBenth

Two biologically active compounds, the enzyme superoxide dismutase (EC 1.15.1.1) and the anti-arhythmic indole alkaloid ajmaline, were isolated from a callus culture of Rauwolfia serpentinaBenth. Sequential isolation of biologically active compounds by metal–chelate affinity chromatography followed by azoadsorbent affinity chromatography allowed us to obtain highly purified products. The yields of superoxide dismutase and ajmaline were 180 mg/kg biomass and 16.5 g/kg dry weight, respectively.     

Microbial production of squalene by a nicotinic acid-resistant mutant derived from Fusarium sp. no. 5-128B

A nicotinic acid-resistant mutant, designated NA201, was obtained from Fusarium sp. no. 5-128B by treatment with ultraviolet light. This mutant strain could grow in the presence of up to 500 mM nicotinic acid in the culture medium, although the parent strain could not grow at concentrations of nicotinic acid above 200 mM. The NA201 strain exhibited morphological mutations, neither forming aerial hyphae nor secreting a red-brown pigment. However, it retained the resistance to kabicidin at 25 mg l^−t of the parent strain. The mutant NA201 cells contained high levels of squalene and low levels of ergosterol, about 53 times higher and five to six times lower, respectively, than those of the parent strain under standard culture conditions. The volumetric oxygen transfer coefficient (Kd) affected the level of squalene in the mutant cells. The Kd for the maximum production of squalene by the mutant was 24 mmol O₂I⁻¹h⁻¹atm⁻¹ and the level of squalene in the mutant cells was 26 mg (g cell)⁻¹ on a dry weight basis. The greatest accumulation of squalene by the NA201 strain, corresponding to 323 mg per liter of culture medium and 35 mg (g cell)⁻¹ on a dry weight basis, was achieved in a culture in which the Kd was changed from a high to a low value on the third day, with the simultaneous addition of 3% glucose (w/v).