Wheat kernel black point and fumonisin contamination by Fusarium proliferatum

Fusarium proliferatum is a major cause of maize ear rot and fumonisin contamination and also can cause wheat kernel black point disease. The primary objective of this study was to determine whether nine F. proliferatum strains from wheat from Nepal can cause black point and fumonisin contamination in wheat kernels. For comparison, the study included three Fusarium strains from US maize. In test 1, all the strains but one produced significant symptoms of kernel black point; two strains decreased kernel yield; and four strains contaminated kernels with fumonisins B₁, B₂ and B₃ as determined by liquid chromatography-mass spectroscopy. Strain Ggm202 from Nepal, which produced the highest levels of fumonisins (mean = 49 µg g^-1) on five wheat cultivars in test 1, was confirmed to produce fumonisins (mean = 38 µg g^-1) on two cultivars in test 2. The data indicate a potential for fumonisin contamination of wheat infected with F. proliferatum.     

Control of growth and fumonisin B1 production by Fusarium verticillioides and Fusarium proliferatum isolates in moist maize with propionate preservatives.

The effect of propionic acid, its sodium salt or a commercial formulation of propionates (0.03, 0.05 and 0.07%), on growth and fumonisin B1 production by Fusarium verticillioides and F. proliferatum isolated was evaluated on irradiated maize at different water activities (aw, 0.93, 0.95, 0.98) and temperatures (15, 25 degrees C). The four isolates grew at all aw x temperature treatments in the absence of propionates. At the highest propionate concentration tested (0.07%), however, growth was restricted to 0.98 aw, for F. proliferatum isolates but not for those of F. verticillioides. Inhibition of growth was maximum when propionates were added in the acid form. In the presence of low propionate concentrations (0.03%), growth was sometimes enhanced probably due to assimilation of these compounds by the fungus. Water activity, temperature, concentration and source of propionate, as well as most two-, three-, four-, and five-way interactions had a significant influence on growth of Fusarium isolates. None of the assayed treatments had any effect on fumonisin B1 production by F. verticillioides isolates. For F. proliferatum, higher fumonisin B1 production occurred in the absence of propionates, and in general concentration decreased with increasing doses of preservatives. Single factors (aw, propionate concentrations and temperature) and temperature x aw and propionate concentration x temperature interactions had a significant effect on fumonisin production (p < 0.01). Moreover, propionate concentration was the single most important factor, besides temperature, which affected fumonisin B1 production.     

Potential use of antioxidants for control of growth and fumonisin production by Fusarium verticillioides and Fusarium proliferatum on whole maize grain

The effect of interactions between two food grade antioxidants butylated hydroxyanisole (BHA) and propyl paraben (PP, 100, 200, 500 microg g(-1)) and water activity (a(w), 0.995, 0.98, 0.95) of irradiated maize on lag phase prior to growth, growth rate and fumonisin production by Fusarium verticillioides and Fusarium proliferatum was evaluated at 25 degrees C. Both antioxidants had an effect on growth characteristics, and fumonisin production. However, this was dependent on the dose used and the a(w) treatment. At 500 microg g(-1) BHA and PP increased the lag phase prior to growth, and reduced the growth rate of both Fusarium species significantly, especially at 0.95 a(w). Both antioxidants significantly reduced the production of fumonisin by both Fusarium species, especially at 0.98 and 0.95 a(w). These results suggest that these antioxidants have potential for treatment of maize grain for controlling growth of these mycotoxigenic species and prevent fumonisin accumulation.     

Inhibitory effect of cinnamon, clove, lemongrass, oregano and palmarose essential oils on growth and fumonisin B1 production by Fusarium proliferatum in maize grain

The effect of cinnamon, clove, oregano, palmarose and lemongrass oils on growth and FB1 production by three different isolates of F. proliferatum in irradiated maize grain at 0.995 and 0.950 aw and at 20 and 30 degrees C was evaluated. The five essential oils inhibited growth of F. proliferatum isolates at 0.995 aw at both temperatures, while at 0.950 aw only cinnamon, clove and oregano oils were effective in inhibiting growth of F. proliferatum at 20 degrees C and none of them at 30 degrees C. Cinnamon, oregano and palmarose oils had significant inhibitory effect on FB1 production by the three strains of F. proliferatum at 0.995 aw and both temperatures, while clove and lemongrass oils had only significant inhibitory effect at 30 degrees C. No differences were found using 500 or 1000 microg essential oil g(-1). At 0.950 aw, none of the essential oils had any significant effect on FB1 production. The results suggest that mainly cinnamon and oregano oils could be effective in controlling growth and FB1 production by F. proliferatum in maize under preharvest conditions.     

Effect of water activity and temperature on growth and the relationship between fumonisin production and the radial growth of Fusarium verticillioides and Fusarium proliferatum on corn

The two major fumonisin-producing Fusarium species are Fusarium verticillioides and Fusarium proliferatum. The growth and fumonisin production of these two isolates on corn was studied at water activities (a(w)) between 0.860 and 0.975 and at temperatures between 15 and 30 degrees C. Growth rates (g, mm/day) were obtained by linear regression during the linear phase of growth. In general, growth rates for both isolates increased significantly (P < 0.05) with increases in a(w) and temperature. Both fumonisin production and radial growth (mycelial development) for both isolates increased with a(w) at all temperatures investigated, but the effect of temperature on this relationship was not obvious. The effect of temperature on fumonisin production at high a(w) values optimal for growth was only marginal, whereas at lower a(w) values the effect of temperature was more pronounced, with more fumonisin production occurring at temperatures not optimal for growth. The optimum temperature for fumonisin production was between 15 and 25 degrees C. For F. proliferatum, the optimum temperature for growth at all a(w) values, 30 degrees C, resulted in the poorest fumonisin production. For both isolates, the slowest initial rate of fumonisin production was at 15 degrees C, the temperature at which the slowest growth rates were obtained.     

Comparison of fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides

Fusarium verticillioides produces a group of mycotoxins known as fumonisins in maize kernels. Fumonisins are associated with a variety of mycotoxicoses in humans and animals; thus, their presence in food is a considerable safety issue. This study addressed fumonisin B1 (FB1) production in two components of the maize kernel, namely the germ tissues and the degermed kernel. Growth of F. verticillioides was similar in colonized germ tissue and degermed kernels, but FB1 production was at least five times higher in degermed maize kernels than in germ tissue. Expression of the fumonisin polyketide synthase gene, FUM1, as measured by beta-glucuronidase (GUS) and Northern blot analysis, followed the same pattern as FB1 production. Also correlated to FB1 was a concomitant drop in pH of the colonized degermed kernels. A time course experiment showed that degermed kernels inoculated with F. verticillioides became acidified over time (from pH 6.4 to 4.7 after 10 days of incubation), whereas colonized germ tissue became alkaline over the same period (from pH 6.5 to 8.5). Because conditions of acidic pH are conducive to FB1 production and alkaline pH is repressive, the observed correlation between the acidification of degermed kernels and the increase in FB1 provides one explanation for the observed differences in FB1 levels.     

Experimental infection of Fusarium proliferatum in Oryza sativa plants; fumonisin B1 production and survival rate in grains

Fusarium proliferatum is a plant pathogenic fungus associated with crops such as asparagus and corn, and it possesses the ability to produce a range of mycotoxins, including fumonisins. In Asia, rice (Oryza sativa) is a staple cereal and is occasionally colonized by this fungus without obvious physiological changes. F. proliferatum is closely related to Gibberella fujikuroi (anamorph F. fujikuroi) responsible for Bakanae disease in rice; however there are few reports of F. proliferatum as a rice pathogen. In this study, we examined the pathogenic potential of F. proliferatum in rice plants with respect to browning, fumonisin production, and survival rates in rice grains. Fungal inoculation was conducted by spraying a conidial suspension of F. proliferatum onto rice plants during the flowering period. Browning was found on the stalk, leaf, and ear of rice. Fumonisin B(1) was detected at levels from trace to 21 ng/g grains, using tandem mass spectrometry. Fungal recovery after 6 months indicated that F. proliferatum had high affinity to rice plants being still viable in grains. From this study, it can be concluded that F. proliferatum is a possible pathogen of rice and possesses a potential to produce fumonisin B(1) in rice grains in the field.     

Fumonisin B1 production by Fusarium moniliforme and Fusarium proliferatum as affected by cycling temperatures

The effects of temperatures cycling between 5 and 20 degrees C, 10 and 25 degrees C, and 15 and 30 degrees C on the production of fumonisin B1 (FB1) and ergosterol by Fusarium moniliforme and Fusarium proliferatum on rice was studied. Temperatures were cycled at 12-h intervals by manually moving cultures from one temperature to another. Constant temperature incubation at 25 degrees C and a low temperature stress were compared with the cycling temperature incubations. Low temperature stress was achieved by incubating rice cultures at 25 degrees C for 2 weeks followed by 15 degrees C for 4 weeks. The maximum yields of FB1 were found to be 247 microg/g by F. moniliforme at temperatures that cycled between 10 and 25 degrees C after 2 weeks and 284 microg/g by F. proliferatum when the temperatures cycled between 5 and 20 degrees C after 6 weeks. Ergosterol content of the rice cultures was also monitored. Overall, the two Fusarium species showed differences in production of FB1 and ergosterol under the various temperature treatments. The most notable differences were that the temperature treatments that stimulated greatest FB1 production were different for each species: cycling temperatures between 10 and 25 degrees C for F. moniliforme and cycling temperatures between 5 and 25 degrees C for F. proliferatum. At most temperatures, F. proliferatum produced more ergosterol than F. moniliforme. Maximum production of ergosterol by F. proliferatum occurred at 6 weeks, with temperatures that cycled between 10 and 25 degrees C, whereas F. moniliforme produced maximum amounts of ergosterol at 6 weeks, with temperatures that cycled between 15 and 30 degrees C.     

Two-dimensional profiles of fumonisin B1 production by Fusarium moniliforme and Fusarium proliferatum in relation to environmental factors and potential for modelling toxin formation in maize grain

This study has examined in detail the effect of temperature (7-37 degrees C) and water availability (water activity, a(w), 0.89-0.97) on fumonisin B1 (FB1) production by an isolate of Fusarium moniliforme and F. proliferatum on irradiated maize grain after incubation for 28 days. The optimum conditions for F. moniliforme and F. proliferatum were 30 degrees C at 0.97 a(w) and 15 degrees C at 0.97 a(w), respectively. The maximum concentrations were 2861 mg kg(-1) and 17,628 mg kg(-1) dry wt. maize grain, respectively. At marginal a(w)/temperature conditions for growth (e.g. 0.89-0.91 a(w)) no FB1 was detected (<0.1 mg kg(-1)). A high variability was found between replicates for F. moniliforme, but not for F. proliferatum. These data were used to construct two-dimensional diagrams of all the a(w) x temperature conditions favourable for FB1 production for the first time. The data were also subjected to a polynomical regression, which demonstrated that there was a very good fit for the 15-30 degrees C range of temperature and at 0.97 a(w). However, at marginal environmental conditions this was not possible. This suggests that it may be possible to predict within a limited environmental range the potential for significant FB1 production.     

The effect of fungal competition on colonization of maize grain by Fusarium moniliforme, F. proliferatum and F. graminearum and on fumonisin B1 and zearalenone formation

The effect of water activity (0.98, 0.95, 0.93) and temperature (15, 25 degrees C) on fungal growth and toxin production from interactions between isolates of Fusarium moniliforme and F. proliferatum producing fumonisin, and an isolate of F. graminearum producing zearalenone, incubated at the same time on irradiated maize grains were determined in vitro. Populations (CFUs) of F. moniliforme and F. proliferatum were reduced to a greater or lesser extent by the presence of F. graminearum under all conditions tested, while that the presence of F. moniliforme or F. proliferatum had a minor inhibitory effect on fungal populations of F. graminearum. Fumonisin B, production by F. proliferatum was inhibited under all conditions tested, while fumonisin B1 production by F. moniliforme was inhibited at 15 degrees C and enhanced at 25 degrees C in the presence of F. graminearum. The level of zearalenone was not significantly modified in the presence of F. moniliforme and F. proliferatum under the conditions tested.     

Trichoderma viride suppresses fumonisin B1 production by Fusarium moniliforme

Biocontrol activity against Fusarium moniliforme was analyzed for a Trichoderma viride strain isolated from root segments of corn plants grown in Piedmont Georgia. The isolate suppressed radial extension of F. moniliforme colonies during cocultivation on potato dextrose agar and fumonisin B1 (FB1) production during incubation of both fungi on corn kernels. T. viride decreased radial extension of F. moniliforme by 46% after 6 days and by 90% after 14 days. Furthermore, the colony diameter of F. moniliforme was less at 14 days than at 5 days, suggesting that F. moniliforme mycelia were undergoing lysis. FB1 production by F. moniliforme on corn kernels decreased by 85% when both organisms were inoculated the same day onto corn kernels and by 72% when inoculation of T. viride was delayed by 7 days after F. moniliforme inoculation. These results are the first to demonstrate that T. viride can suppress FB1 production by F. moniliforme, thereby functioning to control mycotoxin production. Thus, this isolate may be useful in biological control to inhibit F. moniliforme growth as a preharvest agent to prevent disease during plant development and/or as a postharvest agent during seed storage to suppress FB1 accumulation when kernels are dried inadequately.     

Evaluating three commonly used growth media for assessing fumonisin analogues FB1, FB2 and FB3 production by nine Fusarium verticillioides isolates

ABSTRACT

Maize is most often infected by the fumonisin-producing Fusarium verticillioides. Total fumonisins of natural infected grain is made up of FB₁, FB₂ and FB₃ with FB₁ occurring naturally at higher levels. A maize plant can be infected with more than one F. verticillioides isolate, and finding a reliable method to elucidate the toxigenic potential of these isolates is important to extrapolate the possible fumonisin risk to consumers of grain. It is not clear whether F. verticillioides produces similar fumonisin levels, as well as fumonisin analogue ratios, across media. In this study, nine F. verticillioides isolates were subjected to three methods of fumonisin testing using liquid media, maize patties and a field trial (silk inoculation of grain) in Potchefstroom, South Africa. Spore concentrations of 1 × 10⁶ conidia ml^–1 of each isolate were used to inoculate the different media and levels fumonisin analogues were measured using HPLC. Fumonisin production per isolate was highly variable and was influenced by the two-way interaction of F. verticillioides isolate × growth media. Total fumonisins produced in the liquid medium ranged from 0 to 21.3 ppm, on maize patties fumonisins they ranged from 0 to 21.5 ppm, and in the silk inoculation technique they ranged from 0 to 15.5 ppm. The fumonisin analogue FB₁ occurred at higher levels followed by FB₃ in both in vitro studies. In the silk inoculation technique, fumonisin analogue FB₂ was the second highest occurring analogue after FB₁. Isolate GCI 282 produced higher FB₂ and FB₃ levels than FB₁ in the patties and grain, respectively. In order not to miscalculate the fumonisin and analogue ratio levels per F. verticillioides isolate, the growth medium will have to be optimised for each isolate and more than one growth medium used.     

Acidic pH as a determinant of TRI gene expression and trichothecene B biosynthesis in Fusarium graminearum

Reducing production of type B trichothecenes by Fusarium graminearum on cereals is necessary to control contamination, prevent yield reduction and protect human and animal health. Thus, an understanding of how trichothecene biosynthesis is induced is essential. The effect of ambient pH on fungal growth, toxin biosynthesis and expression of TRI genes was studied during in vitro liquid culture of F. graminearum on minimal medium. Fungal development stopped at day 3 after a sharp pH drop in the medium. At the same time, induction of TRI gene expression was observed and toxin began accumulating 1 day later. Acidification seems a determinant of induction, as neither the toxin nor the TRI genes were detected when the pH was maintained neutral. Shifting from neutral to acidic pH by mycelium transfer induced TRI gene expression and toxin accumulation. The regulation of toxin production by ambient pH appears to be specific to some TRI genes since TRI5, located in the core FgTRI5 cluster, showed an immediate induction while TRI101, located elsewhere in the genome, showed a more progressive response. The regulation of trichothecene biosynthesis by the ambient pH appears to be a general mechanism, independent of strain or chemotype, as all tested strains, including F. graminearum and F. culmorum species, showed a regulation of toxin production in response to the ambient pH. We conclude that, in vitro, external acidification is required for induction of TRI gene expression.     

The influence of modified atmospheres and their interaction with water activity on the radial growth and fumonisin B(1) production of Fusarium verticillioides and F. proliferatum on corn. Part II: The effect of initial headspace oxygen concentration

This paper is the second in a series of two that describe the effect of modified atmospheres on the growth and mycotoxin production of Fusarium verticillioides and Fusarium proliferatum on corn. In this part, the effect of initial headspace (IH) oxygen concentration and its interaction with water activity (a(w)) on growth and fumonisin B(1) production was investigated. In addition, the impact of vacuum packaging and in-cooperation of O(2) scrubbing sachets was also studied. It was observed that at all a(w) values studied, reduction of IH O(2) concentration from 20 to 2% had no significant effect on the colony growth rate (g, mm d(-1)) and lag phase duration (lambda, d). However, g and lambda were positively and negatively correlated to a(w). The IH O(2) concentration was determined to have a a(w) dependent effect on the oxygen consumption rate. Although the maximum colony diameter (D(max), mm) decreased with the reduction of the IH O(2) level, the greatest mycelial density occurred at 10% IH O(2) for both isolates. This observation was accompanied by a trend of a decrease in the value of the IH O(2) level at which the most fumonisin B(1) was produced from 15 to 5% when the a(w) was decreased from 0.976 to 0.930 for F. verticillioides. For F. proliferatum the optimum conditions for fumonisin B(1) production shifted from 20% at a(w) 0.976 to 10% at both 0.951 and 0.930. Vacuum packaging and the in-cooperation of O(2) absorbing sachets completely inhibited the growth of both isolates. These results together with those reported in Part I of the study indicate that O(2) should preferably be completely excluded from modified atmospheres that are employed to protect stored corn from fungal growth and mycotoxin production.     

Survey of maize from south-western Nigeria for zearalenone, alpha- and beta-zearalenols, fumonisin B1 and enniatins produced by Fusarium species.

A survey for the natural occurrence of Fusarium mycotoxins in maize for human consumption in four south-western states of Nigeria using High Performance Liquid Chromatography coupled with Mass Spectroscopy (HPLC/MS) showed that 93.4% of the samples were contaminated with zearalenone (ZON), alpha- and beta-zearalenols (alpha- and beta-ZOL), fumonisin B(1) (FB(1)) or enniatins (ENNs). The fractions of contaminated samples were 73% for FB(1) (mean:117 microg kg(-1), range:10-760 microg kg(-1)); 57% for ZON (mean:49 microg kg(-1), range:115-779 microg kg(-1)) and 13% for alpha-ZOL (mean: 63.6 microg kg(-1), range:32-181 microg kg(-1)), while ENNs A1, B and B(1) were present in 3, 7 and 3% of the samples respectively. There was no beta-ZOL present above the quantification limits of 50 microg kg(-1). Only the FB(1) content was significantly different at the 95% confidence level among the four states. The Fusarium species most frequently isolated from maize seeds were F. verticillioides (70%), followed by F. sporotrichioides (42%), F. graminearum (30%), F. pallidoroseum (15%), F. compactum (12%), F. proliferatum (12%), F. equiseti (9%), F. acuminatum (8%) and F. subglutinans (4%). This is the first report of the occurrence of alpha-zearalenol and enniatins in Nigerian maize.     

The influence of modified atmospheres and their interaction with water activity on the radial growth and fumonisin B(1) production of Fusarium verticillioides and F. proliferatum on corn. Part I: the effect of initial headspace carbon dioxide concentration

The effect of modified atmospheres on the growth and fumonisin B(1) production of Fusarium verticillioides and Fusarium proliferatum on corn is presented in a series of two papers. In this, the first part, the effect of initial headspace (IH) carbon dioxide concentration and its interaction with water activity (a(w)) on growth and fumonisin B(1) production was evaluated. It was observed that at all a(w) values studied, increase in the IH CO(2) concentration generally resulted in a decrease in the colony growth rate (g, mm day(-1)) and maximum colony diameter (D(max), mm) and an increase in the lag phase duration (lambda, day). Although both a(w) and IH CO(2) concentration had significant and synergistic effects on g, a(w) had the largest effect. As little as 10% IH CO(2) completely inhibited the production of fumonisin B(1) by F. verticillioides. F. proliferatum was more resistant and required 40, 30 and 10% IH CO(2) at a(w) 0.984, 0.951 and 0.930, respectively, to completely inhibit fumonisin B(1) production. These results demonstrate that modified atmospheres containing high CO(2) levels could potentially be employed for the protection of corn from fungal spoilage and mycotoxin contamination during the post-harvest period.     

Kinetics of fumonisin B1 formation in maize ears inoculated with Fusarium verticillioides

The kinetics of fumonisin B₁ (FB₁) biosynthesis have been examined in ears of four botanical varieties Zea mays var. indentata, Zea mays var. indurata, Zea mays var. saccharata and Zea mays var. everta inoculated with F. verticillioides isolates at silking stage. The level of mycotoxin accumulated in kernels was correlated with sample harvest time (r?=?0.73) and ergosterol content (r?=?0.70). FB₁ biosynthesis was influenced by amylose, starch and moisture contents, which undergo dynamic changes during grain formation. A comparative analysis of ear infection rates and efficiency of FB₁ biosynthesis showed that these are separate characters and their expression could be independent.     

Associations of planting date,drought stress,and insects with Fusarium ear rot and fumonisin B1 contamination in California maize

Fusarium ear rot, caused by Fusarium verticillioides, is one of the most common diseases of maize, causing yield and quality reductions and contamination of grain by fumonisins and other mycotoxins. Drought stress and various insects have been implicated as factors affecting disease severity. Field studies were conducted to evaluate the interactions and relative influences of drought stress, insect infestation, and planting date upon Fusarium ear rot severity and fumonisin B₁ contamination. Three hybrids varying in partial resistance to Fusarium ear rot were sown on three planting dates and subjected to four irrigation regimes to induce differing levels of drought stress. A foliar-spray insecticide treatment was imposed to induce differing levels of insect injury. Populations of thrips (Frankliniella spp.), damage by corn earworm (Helicoverpa zeae), Fusarium ear rot symptoms, and fumonisin B₁ levels were assessed. There were significant effects of hybrid, planting date, insecticide treatment, and drought stress on Fusarium ear rot symptoms and fumonisin B₁ contamination, and these factors also had significant interacting effects. The most influential factors were hybrid and insecticide treatment, but their effects were influenced by planting date and drought stress. The more resistant hybrids and the insecticide-treated plots consistently had lower Fusarium ear rot severity and fumonisin B₁ contamination. Later planting dates typically had higher thrips populations, more Fusarium ear rot, and higher levels of fumonisin B₁. Insect activity was significantly correlated with disease severity and fumonisin contamination, and the correlations were strongest for thrips. The results of this study confirm the influence of thrips on Fusarium ear rot severity in California, USA, and also establish a strong association between thrips and fumonisin B₁ levels.     

Incidence of Fusarium verticillioides and levels of fumonisin B1 and B2 in corn in Turkey

A total of 100 corn samples conforming collected from local farmers and markets from districts of Samsun, Turkey, were analyzed for Fusarium verticillioides, fumonisin B₁ and B₂ contamination. Ninety-three corn samples were found to contain F. verticillioides, 52 samples fumonisin B₁, and 25 samples fumonisin B₂. Fumonisin B₁ contamination ranged from 0.05 to 25.72 mg/kg and B₂ from 0.05 to 5.7 mg/kg, respectively. This figure indicated widespread contamination of fumonisin B₁ and B₂ in maize grown in different areas of Samsun, Turkey.