Control of Aspergillus growth and aflatoxin production using antioxidants at different conditions of water activity and pH

AIMS: The effect of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trihydroxybutyrophenone (THB) and propyl paraben (PP) (at concentrations of 1, 10 and 20 mmol l(-1)) on germination, growth and aflatoxin B1 production by Aspergillus section Flavi was evaluated. METHODS AND RESULTS: Studies on the percentage of spore germination, elongation rate, growth rate and aflatoxin B1 production were carried out in vitro in relation to water activity (aw) at 0.982, 0.937, 0.809 and 0.747 values. At 0.809 and 0.747aw values none of the isolates was able to germinate. Overall, PP and BHA were the antioxidants most effective at inhibiting germination of both species. In the presence of the lowest concentration of BHA and PP (1 mmol l(-1)) the conidial germination percentage ranged from 2 to 19% after 15 h of incubation at the highest water activity tested. BHA and PP at 10-20 mmol l(-1) completely inhibited conidial germination. The antioxidants more efficient in controlling Aspergillus elongation rate were PP, BHT and BHA. All strains were much more sensitive to all antioxidants tested on the percentage of spore germination and growth rate at 0.937aw. The antioxidants PP and BHA completely inhibited aflatoxin B1 production by all strains when added at 1 mmol l(-1). Decreased aflatoxin B1 levels in comparison with the control, were observed with BHT at 1, 10 and 20 mmol(-1) with the strain T20 at 0.982aw. In contrast, stimulation was observed with the antioxidant THB at 10 and 20 mmol l(-1) at 0.937aw with the strains T20 and T23. The effect of BHA and PP at 1 mmol l(-1) on lag phase and growth rate was maintained in the pH range between 6 and 8. At all pH values the inhibitory effect of BHA was higher than PP. No aflatoxin B1 was detected at all pH values. CONCLUSIONS: The data show that BHA and PP could be considered as effective fungitoxicants for A. flavus and A. parasiticus. SIGNIFICANCE AND IMPACT OF THE STUDY: The information obtained show promise for controlling growth and aflatoxin B1 in stored maize. Futher studies should be carried out to examine the potential for antioxidants, such as BHA and PP to effectively control both growth and aflatoxin production.     

In vitro effect of phenolic antioxidants on germination, growth and aflatoxin B accumulation by peanut Aspergillus section Flavi

AIMS: The effectiveness of the food-grade antioxidants butylated hydroxytoluene (BHT), trihydroxybutyrophenone (THB), propyl paraben (PP) and butylated hydroxyanisole (BHA) at 1, 10 and 20 mmol l(-1) concentrations on germination, growth, and aflatoxin B(1) (AFB(1)) production by Aspergillus section Flavi strains was determined. METHODS AND RESULTS: Assays on the lag phase of germination, germination percentage, germ tube elongation rate, lag phase, growth rate and AFB(1) production by three strains of Aspergillus flavus and three of Aspergillus parasiticus were carried out in vitro on peanut extract meal agar conditioned at different water activities (a(w): 0.982, 0.971, 0.955, 0.937). The antioxidants PP and BHA efficiently inhibited the germination of the two species tested at the doses 10 and 20 mmol(-1). The antioxidants PP and BHA at 1 mmol l(-1) and THB at 20 mmol l(-1) reduced the germ tube elongation rate most effectively, regardless of a(w) levels. An increase in the lag time and a reduction in the growth rate of 100% of the strains was observed, this was due to the action of BHT at the doses 10 and 20 mmol(-1) at 0.982, 0.971 and 0.955 a(w), although these treatments stimulated the AFB(1) accumulation in most of the fungi tested. The more effective antioxidants were PP and BHA, which increased the lag phase, reduced the growth rate and AFB(1) production in all of the strains at the four a(w) assayed. At concentrations 10 and 20 mmol l(-1), these antioxidants totally inhibited fungal development. CONCLUSIONS: The study shows that the antioxidants BHA and PP are effective fungal inhibitors to peanut Aspergillus section Flavi in wide range of water activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that phenolic antioxidants, BHA and PP, can be effective fungitoxicants on aflatoxigenic strains in peanut at industrial level.     

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.     

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.     

Aflatoxin B1 and fumosin B1 in mixed cultures of Aspergillus flavus and Fusarium proliferatum on maize

Production of aflatoxin B1 and fumonisin B1 in pure and mixed cultures of Aspergillus flavus and Fusarium proliferatum were determined on irradiated maize seeds inoculated with different spore concentrations at 0.97 water activity (a(w)) and a temperature of 25 degrees C. The highest levels of aflatoxin B1 were produced by A. flavus at the lowest levels of inoculum (10(3) spore ml(-1)). There was no spore concentration influence on fumonisin B1 production after 10, 20 and 35 days of incubation. When A. flavus was co-inoculated with F. proliferatum, aflatoxin B1 production was inhibited. The higher the inocula levels of Fusarium produced, the higher the inhibition and this inhibition increased during the incubation period. Total inhibition was reached at 35 days of incubation. There was no interaction influence on fumonisin B1 production at all inoculum levels assayed. These results suggest that under optimal environmental conditions of substrate, water activity and temperature, the interaction between A. flavus and F proliferatum could produce inhibition of aflatoxin B1 and stimulation of fumonisin B1.     

Two-dimensional environmental profiles of growth,deoxynivalenol and nivalenol production by Fusarium culmorum on a wheat-based substrate

AIMS: To determine the effect of interacting conditions of water activity (aw, 0.99-0.85), temperature (15, 25 degrees C) and time (40 days) on growth and production of the mycotoxins deoxynivalenol (DON) and nivalenol (NIV) by Fusarium culmorum on a wheat-based agar medium. METHODS AND RESULTS: Fusarium culmorum grew optimally at 0.995aw and minimally at 0.90 at both 15 and 25 degrees C. No growth was observed at <0.90aw. Overall, temperature, aw and their interaction had a statistically significant effect on the growth rate of F. culmorum. Production of both DON and NIV were over a much narrower range (0.995-0.95aw) than that for growth. The highest concentrations of DON and NIV levels were produced at 0.995aw and 0.981aw at 25 degrees C, respectively, after 40 days of incubation. Statistically, aw, temperature and incubation time, and aw x temperature and temperature x incubation time had a statistically significant effect on DON/NIV production. CONCLUSIONS: This is the first detailed report on the two-dimensional environmental profiles for DON/NIV production by F. culmorum in the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: As part of a hazard analysis critical control point (HACCP) approach, this type of information is critical in monitoring critical control points for prevention of DON/NIV entering the wheat production chain.     

In vitro influence of bacterial mixtures on Fusarium verticillioides growth and fumonisin B1 production: effect of seeds treatment on maize root colonization

AIMS: Enterobacter cloacae, Microbacterium oleovorans, Pseudomonas solanacearum and Bacillus subtilis were investigated in order to evaluate: (i) the inoculum size of two bacterial mixtures on Fusarium verticillioides growth and fumonisin B1 production in vitro at different water activities and (ii) the efficacy of a seed treatment with the best bacterial mixture on F. verticillioides root colonization in greenhouse studies. METHODS AND RESULTS: The influence of bacterial mixtures (1 = E. cloacae and M. oleovorans and 2 = P. solanacearum and B. subtilis) to antagonize 13 F. verticillioides strains at different inoculum concentrations (10(8), 10(9) and 10(10) cells ml(-1)) and water activities (0.937, 0.955 and 0.982 aW) were examined. Antibiosis, growth rate and fumonisin B1 production were determined. Bacterial mixture 1 proved to exert the most effective control. Seed treatment with mixture 1 at 10(8) cells ml(-1) had the best inhibitory effect on F. verticillioides root colonization. CONCLUSIONS: These results suggest that the combination E. cloacae and M. oleovorans has the potential for the biological control of F. verticillioides as a maize seed inoculant. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of this knowledge contributes to prevent the vertical transmission of F. verticillioides.     

The FvMK1 mitogen-activated protein kinase gene regulates conidiation, pathogenesis, and fumonisin production in Fusarium verticillioides

Fusarium verticillioides is one of the most important fungal pathogens to cause destructive diseases of maize worldwide. Fumonisins produced by the fungus are harmful to human and animal health. To date, our understanding of the molecular mechanisms associated with pathogenicity and fumonisin biosynthesis in F. verticillioides is limited. Because MAP kinase pathways have been implicated in regulating diverse processes important for plant infection in phytopathogenic fungi, in this study we identified and functionally characterized the FvMK1 gene in F. verticillioides. FvMK1 is orthologous to FMK1 in F. oxysporum and GPMK1 in F. graminearum. The Fvmk1 deletion mutant was reduced in vegetative growth and production of microconidia. However, it was normal in sexual reproduction and increased in the production of macroconidia. In infection assays with developing corn kernels, the Fvmk1 mutant was non-pathogenic and failed to colonize through wounding sites. It also failed to cause stalk rot symptoms beyond the inoculation sites on corn stalks, indicating that FvMK1 is essential for plant infection. Furthermore, the Fvmk1 mutant was significantly reduced in fumonisin production and expression levels of FUM1 and FUM8, two genes involved in fumonisin biosynthesis. The defects of the Fvmk1 mutant were fully complemented by re-introducing the wild type FvMK1 allele. These results demonstrate that FvMK1 plays critical roles in the regulation of vegetative growth, asexual reproduction, fumonisin biosynthesis, and pathogenicity.     

Impact of essential oils on growth rate, zearalenone and deoxynivalenol production by Fusarium graminearum under different temperature and water activity conditions in maize grain

AIMS: The effect of five essential oils (oregano, cinnamon, lemongrass, clove and palmarose) on growth rate, zearalenone (ZEA) and deoxynivalenol (DON) production by Fusarium graminearum strains was assessed. METHODS AND RESULTS: The influence of the essential oils was tested on irradiated maize at two concentrations (500 and 1000 mg kg-1), at different water activity (aw) (0.95 and 0.995) and temperature (20 and 30 degrees C) levels. At 0.995 aw all essential oils tested had an inhibitory effect on growth rate of F. graminearum at both temperatures studied. At this aw level, DON production in general was inhibited by all essential oils at 30 degrees C and, although palmarose and clove were the only essential oils with statistically significant inhibitory effect on ZEA production, an inhibitory trend was observed when cinnamon and oregano oils were added to maize grain. CONCLUSIONS: Antifungal and antimycotoxigenic activity of the essential oils assayed was shown to depend on environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: It is apparent that essential oils should be considered as alternative preharvest natural fungicides. Further investigation on natural maize grain might be useful to study the effectiveness of these essential oils in the presence of natural mycoflora of maize grain.     

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.     

Competitive interactions between Fusarium sambucinum Fuckel and Phoma glomerata (Corda) Wollenweber & Hochapfel under in vitro conditions

The effects of temperature (15 and 25 degrees C), water activity (0.85, 0.90, 0.95, 0.98 y 0.995) and the interaction between Fusarium sambucinum and Phoma glomerata in rice extract agar on fungus growth were investigated. Fungi interactions were given a numerical score to obtain an Index of Dominance (ID) and to observe possible variations under different conditions of temperature and water activity (aw) changed. F. sambucinum and P. glomerata grew most rapidly -both individually and paired- at 0.995 aw and 25 degrees C. On the other hand, F. sambucinum presented higher growth rates than P. glomerata and it was dominant over P. glomerata under all the tested conditions. Water activity and temperature showed a significant effect on fungus growth.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Production of Fumonisins by Fusarium Verticillioides Strains on Solid and in a Defined Liquid Medium — Effects of L-Methionine and Inoculum

In order to evaluate the toxicological and carcinogenic effects of fumonisins, large amounts of fumonisins need to be purified, which requires optimal conditions for production in culture. Five strains of F. verticillioides were compared for their ability to produce fumonisins in solid and liquid media with and without the addition of methionine, a fumonisin precursor. Inoculations were made either with lyophilized cultures or a concentrated inoculum. Growth in liquid medium, measured by biomass, was directly correlated to total fumonisin production when a lyophilized inoculum was used. Fumonisin production was stimulated by the addition of 0.2% L-methionine to solid media for most strains. Levels ranged from 1500-3900 mg/kg in rice, and 2900-12500 mg/kg in maize cultures inoculated with lyophilized cultures; 200-4800 mg/kg in rice, and 1500-4200 mg/kg in maize cultures inoculated with concentrated inoculum. Strains that produced relatively high levels of fumonisins in solid media did not necessarily do so in liquid medium and vice versa. Total fumonisin levels in liquid medium ranged from 40-590 mg/l inoculated with lyophilized cultures and < 1-110 mg/l inoculated with concentrated inoculum, without adding the precursor. F. verticillioides strains therefore varied in their ability to produce fumonisins, and conditions for production need to be optimized individually for each strain.     

Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings

The filamentous ascomycete Fusarium verticillioides is a pathogen of maize and produces the fumonisin mycotoxins. However, a distinct population of F. verticillioides is pathogenic on banana and does not produce fumonisins. Fumonisin-producing strains from maize cause leaf lesions, developmental abnormalities, stunting, and sometimes death of maize seedlings, whereas fumonisin-nonproducing banana strains do not. A Southern analysis of banana strains did not detect genes in the fumonisin biosynthetic gene (FUM) cluster but did detect genes flanking the cluster. Nucleotide sequence analysis of the genomic region carrying the flanking genes revealed that the FUM cluster was absent in banana strains except for portions of FUM21 and FUM19, which are the terminal genes at each end of the cluster. Polymerase chain reaction analysis confirmed the absence of the cluster in all banana strains examined. Cotransformation of a banana strain with two overlapping cosmids, which together contain the entire FUM cluster, yielded fumonisin-producing transformants that were pathogenic on maize seedlings. Conversely, maize strains that possess the FUM cluster but do not produce fumonisins because of mutations in FUM1, a polyketide synthase gene, were not pathogenic on maize seedlings. Together, the data indicate that fumonisin production may have been lost by deletion of the FUM cluster in the banana population of F. verticillioides but that fumonisin production could be restored by molecular genetic complementation. The results also indicate that fumonisin production by F. verticillioides is required for development of foliar disease symptoms on maize seedlings.     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.