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.     

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.     

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.     

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.     

Immunohistochemistry of fumonisin in poultry using avidin-biotin-peroxidase system

Using monoclonal anti-fumonisin B1 antibody (anti-FB1) and avidin-biotin-peroxidase system, liver and kidneys of broiler chicks were evaluated for the detection and distribution of fumonisins (FBs). One hundred and fifty micrograms of FB1 or culture extract of Fusarium moniliforme str. 113F containing 150 microg of FB1 and 4 microg of FB2 were administered into the vitelline sac of 1-day old, specific pathogen-free chicks. The animals were killed 24 h after injection, and renal and hepatic tissues submitted for immunohistochemical analysis. FBs were detected in the epithelial cells of convoluted distal and proximal tubules of the kidneys, as well as in the cytoplasm of hepatocytes. This novel immunohistochemical method developed is expected to be an efficient way for monitoring the target of the FB toxins in tissues.     

Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and E mating populations of Gibberella fujikuroi (Fusarium moniliforme).

Two biological species of Gibberella fujikuroi (A and F mating populations) share the Fusarium moniliforme anamorph. Twenty strains of each of these biological species were tested for the ability to produce fumonisins B1, B2, and B3 and moniliformin and for toxicity to 1-day-old ducklings. Most of the members of the A mating population (19 of 20 strains) produced more than 60 micrograms of total fumonisins per g, whereas only 3 of 20 members of the F mating population produced more than trace levels of these toxins and none produced more than 40 micrograms of total fumonisins per g. In addition, only 3 of 20 members of the A mating population produced more than 1 microgram of moniliformin per g (and none produced more than 175 micrograms/g), while all 20 strains of the F mating population produced more than 85 micrograms of this toxin per g and 1 strain produced 10,345 micrograms/g. The duckling toxicity profiles of the strains of the two mating populations were similar, however, and the level of either toxin by itself was not strongly correlated with duckling toxicity. On the basis of our data we think that it is likely that the members of both of these mating populations produce additional toxins that have yet to be chemically identified. These toxins may act singly or synergistically with other compounds to induce the observed duckling toxicity.     

Fumonisin production by field strains of Fusarium nygamai (Gibberella nygamai) and ascospore progeny of laboratory crosses

Field strains of Fusarium nygamai (Gibberella nygamai) are important producers of the fumonisin mycotoxins. Such strains were mated on carrot agar to obtain ascospore progeny. Field strains and ascospore progeny of F. nygamai produced differential levels of fumonisin B1 (FB1) and B2 (FB2) suitable for genetic analyses. Most of the strains produced higher levels of FB1 than FB2. Ascospore progeny from crosses segregated in 1:1 ratios for loci controlling FB1 production and mating type. These findings can be used as the basis to elucidate the genetics of fumonisin production by F. nygamai.     

Fumonisin B1 production by Fusarium species other than F. moniliforme in section Liseola and by some related species.

Strains of Fusarium proliferatum, F. subglutinans, F. anthophilum, F. annulatum, F. succisae, F. beomiforme, F. dlamini, F. napiforme, and F. nygamai from a variety of substrates and geographic areas were tested for the production of fumonisin B1 in culture. None of the cultures of F. subglutinans (0 of 23), F. annulatum (0 of 1), F. succisae (0 of 2), or F. beomiforme (0 of 15) produced fumonisin B1 in culture. Strains of F. proliferatum (19 of 31; 61%) produced fumonisin B1 in amounts ranging from 155 to 2,936 ppm, strains of F. anthophilum (3 of 17; 18%) produced fumonisin B1 in amounts ranging from 58 to 613 ppm, strains of F. dlamini (5 of 9; 56%) produced fumonisin B1 in amounts ranging from 42 to 82 ppm, strains of F. napiforme (5 of 33; 15%) produced fumonisin B1 in amounts ranging from 16 to 479 ppm, and strains of F. nygamai (10 of 27; 37%) produced fumonisin B1 in amounts ranging from 17 to 7,162 ppm. Of the species tested, F. proliferatum is the most important producer of fumonisin B1 because of its association with corn and animal mycotoxicoses such as porcine pulmonary edema. F. napiforme and F. nygamai also may be important because of their association with the food grains millet and sorghum. At present, F. anthophilum and F. dlamini are of minor importance because they are not associated with corn or other major food grains and have only a limited geographic range. This is the first report of the production of fumonisins by F. anthophilum, F. dlamini, and F. napiforme.     

Fumonisin B1 production by Fusarium species other than F. moniliforme in section Liseola and by some related species.

Strains of Fusarium proliferatum, F. subglutinans, F. anthophilum, F. annulatum, F. succisae, F. beomiforme, F. dlamini, F. napiforme, and F. nygamai from a variety of substrates and geographic areas were tested for the production of fumonisin B1 in culture. None of the cultures of F. subglutinans (0 of 23), F. annulatum (0 of 1), F. succisae (0 of 2), or F. beomiforme (0 of 15) produced fumonisin B1 in culture. Strains of F. proliferatum (19 of 31; 61%) produced fumonisin B1 in amounts ranging from 155 to 2,936 ppm, strains of F. anthophilum (3 of 17; 18%) produced fumonisin B1 in amounts ranging from 58 to 613 ppm, strains of F. dlamini (5 of 9; 56%) produced fumonisin B1 in amounts ranging from 42 to 82 ppm, strains of F. napiforme (5 of 33; 15%) produced fumonisin B1 in amounts ranging from 16 to 479 ppm, and strains of F. nygamai (10 of 27; 37%) produced fumonisin B1 in amounts ranging from 17 to 7,162 ppm. Of the species tested, F. proliferatum is the most important producer of fumonisin B1 because of its association with corn and animal mycotoxicoses such as porcine pulmonary edema. F. napiforme and F. nygamai also may be important because of their association with the food grains millet and sorghum. At present, F. anthophilum and F. dlamini are of minor importance because they are not associated with corn or other major food grains and have only a limited geographic range. This is the first report of the production of fumonisins by F. anthophilum, F. dlamini, and F. napiforme.     

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.     

Identity of Fusarium nygamai isolates with long and short microconidial chains from millet,sorghum and soil in Africa

Several Fusarium species have been found associated with millet and sorghum in Nigeria, Lesotho and Zimbabwe. Amongst these, some isolates were originally identified as short- and long-chained types of F. nygamai. However, there was some question as to the correct identification of the long chained types. This study reclassified some of the isolates with long microconidial chains as F. moniliforme. Morphologically, these strains do not produce chlamydospores like F. nygamai, but produce swollen hyphal cells or resistant hyphae. The isolates in this study were crossed with the mating-type tester strains of Gibberella fujikuroi (F. moniliforme and G. nygamai (F. nygamai). Of the isolates with long chains of microconidia and other characteristics of F. moniliforme, 36% crossed with mating population 'A' of G. fujikuroi. Of the isolates with characteristics of F. nygamai, 65% crossed with the testers used to produce the teleomorph of F. nygamai. Mating tests support the separation of the sample population into F. moniliforme and F. nygamai. The results of this study show that genetics can be an aid in resolving some problems in fungal taxonomy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Production of fumonisins by Fusarium moniliforme strains isolated from corn grain

Fusarium moniliforme is the predominant fusarium species in the grain mycoflora of corn grown in Northern Caucasus, accounting for 95% of fusarium isolates. Eighty-five Fusarium moniliforme strains were grown on grain substrate and checked for the presence of fumonisins (B1 + B2 + B3) by indirect solid-phase enzyme immunoassay (EIA). All strains were capable of producing fumonisins (0.95 to 32,000 mg/kg). Strains sampled in the Krasnodar krai produced the highest fumonisin levels (averaging 5490 mg/kg).     

A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis for B versus C fumonisin production

Most species of Fusarium that produce fumonisin mycotoxins produce predominantly B fumonisins (FBs). However, Fusarium oxysporum strain O-1890 produces predominantly C fumonisins (FCs). In this study, the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890 was determined. The order and orientation of FUM genes were the same as in the previously described clusters in Fusarium verticillioides and Fusarium proliferatum. Coding regions of F. oxysporum and F. verticillioides FUM genes were 88-92% identical, but regions flanking the clusters did not share significant identity. The FUM cluster gene FUM8 encodes an alpha-oxoamine synthase, and fum8 mutants of F. verticillioides do not produce fumonisins. Complementation of a fum8 mutant with the F. verticillioidesFUM8 restored FB production. Complementation with F. oxysporumFUM8 also restored production, but the fumonisins produced were predominantly FCs. These data indicate that different orthologues of FUM8 determine whether Fusarium produces predominantly FBs or FCs.     

Differentiation of Fusarium subglutinans f. sp. pini by histone gene sequence data.

Fusarium subglutinans f. sp. pini (= F. circinatum) is a pathogen of pine and is one of eight mating populations (i.e., biological species) in the Gibberella fujikuroi species complex. This species complex includes F. thapsinum, F. moniliforme (= F. verticillioides), F. nygamai, and F. proliferatum, as well as F. subglutinans associated with sugarcane, maize, mango, and pineapple. Differentiating these forms of F. subglutinans usually requires pathogenicity tests, which are often time-consuming and inconclusive. Our objective was to develop a technique to differentiate isolates of F. subglutinans f. sp. pini from other isolates identified as F. subglutinans. We sequenced the histone H3 gene from a representative set of Fusarium isolates. The H3 gene sequence was conserved and contained two introns in all the isolates studied. From both the intron and the exon sequence data, we developed a PCR-restriction fragment length polymorphism technique that reliably distinguishes F. subglutinans f. sp. pini from the other biological species in the G. fujikuroi species complex.     

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.     

Production of deoxynivalenol by Fusarium isolates from samples of wheat associated with a human mycotoxicosis outbreak and from sorghum cultivars.

Fusarium isolates from specific diseased sorghum plants and rain-soaked wheat and wheat flour associated with human mycotoxicosis in India have been screened for their toxigenic potential. Of the 322 isolates screened, 11 isolates were found to produce deoxynivalenol in concentrations ranging from 0.01 to 186 micrograms g-1. The occurrence of deoxynivalenol-producing fusaria in a nontemperate region and deoxynivalenol production in low concentrations by Fusarium moniliforme are reported for the first time.     

Production of deoxynivalenol by Fusarium isolates from samples of wheat associated with a human mycotoxicosis outbreak and from sorghum cultivars

Fusarium isolates from specific diseased sorghum plants and rain-soaked wheat and wheat flour associated with human mycotoxicosis in India have been screened for their toxigenic potential. Of the 322 isolates screened, 11 isolates were found to produce deoxynivalenol in concentrations ranging from 0.01 to 186 micrograms g-1. The occurrence of deoxynivalenol-producing fusaria in a nontemperate region and deoxynivalenol production in low concentrations by Fusarium moniliforme are reported for the first time.     

Production of gibberellic acids by an orchid-associated Fusarium proliferatum strain

The rice pathogen Fusarium fujikuroi is well known for its ability to produce the plant hormones gibberellins (GAs). However, the majority of closely related Fusarium species is unable to produce GAs although the GA gene cluster is present in their genomes. In this study, we analyzed five orchid-associated Fusarium isolates for their capacity to produce GAs. Four of them did not produce any GAs and were shown not to contain any GA biosynthetic genes. However, the fifth isolate, which has been identified as F. proliferatum based on five molecular markers, produced significant amounts of GAs in contrast to previously characterized F. proliferatum strains. We focused on the molecular characterization of two GA-specific genes, ggs2 and cps/ks, both inactive in F. proliferatum strain D-02945. Complementation of a F. fujikuroi Deltaggs2 mutant with the ET1 ggs2 gene fully restored GA biosynthesis, confirming that the orchid-associated isolate contains an active gene copy. A possible correlation between GA production and their role in plant-fungal interactions is discussed.     

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.