Trans-2-hexen-1-ol as a tool for the control of Fusarium verticillioides in stored maize grains

Fusarium verticillioides infects stored maize grains. In the present study, we first evaluated the effects of ten volatile organic compounds on F. verticillioides growth in culture medium. Subsequently, based on the antifungal activity obtained in the present study and the insecticidal effect against Sitophilus zeamais reported previously, trans-2- hexen-1- ol was selected to analyzed its effect on the fungus in stored maize. All the compounds tested showed inhibitory activity on fungal development, being the most active compounds geraniol, nerol, isovaleraldehyde and linalool, followed by trans-2-hexen-1-ol, cis-3-hexen-1-ol, cis-2-hexen-1-ol and trans-3-hexen-1-ol. Besides its antifungal activity against F. verticillioides, trans-2-hexen-1-ol inhibited FB₁ production. In this study it was also demonstrated that S. zeamais favors F. verticillioides dispersion, confirming the importance of these insects as mechanical vectors of fungal spores. These results reveal the ability of trans-2-hexen-1-ol as a potential tool to control F. verticillioides in a natural approach as an alternative to synthetic pesticides.     

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.     

Characterization of Fusarium verticillioides strains isolated from maize in Italy: fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B₁, B₂, and B₃ were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg⁻¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.     

Effect of alsD deletion and overexpression of nox and alsS on diacetyl and acetoin production by Lacticaseibacillus casei during milk fermentation

Diacetyl and acetoin are key aroma components of fermented milk but are produced in low concentrations by starter cultures. In this study, we expressed NADH oxidase, acetolactate synthase, and inactivated acetolactate decarboxylase in Lacticaseibacillus casei TCS to generate recombinant L. casei strains, and investigated the effects of the genes encoding these enzymes on diacetyl and acetoin production during milk fermentation. In the single-gene recombinant strains tested, diacetyl concentrations were highest in milk fermented by L. casei TCSI-nox (nox gene overexpressed, 3.68 mg/kg), whereas acetoin concentrations were highest in milk fermented by L. casei TCS-ΔalsD (alsD gene deleted, 32.94 mg/kg). Moreover, diacetyl and acetoin concentrations were higher in the inducible strains than in the corresponding constitutive strains (e.g., TCSI-nox vs. TCSC-nox, and TCSI-ΔalsD-nox vs. TCSC-ΔalsD-nox). This phenomenon was also reflected in the protein expression levels and enzyme activities. In the double-gene recombinant strains tested, the highest concentrations of diacetyl and acetoin were produced by L. casei TCSI-ΔalsD-nox (nox overexpressed and alsD deleted, 4.66 mg/kg, 69.62 mg/kg, respectively). The triple-gene recombinant L. casei TCS-ΔalsD-nox-alsS produced the highest concentrations of diacetyl and acetoin, which were 2.38 and 11.19 times, respectively, the concentrations produced by the original strain. These results show that the nox, alsS, and alsD genes make key contributions to the biosynthesis of diacetyl and acetoin by L. casei. The modification of multiple genes had a synergistic effect, leading to greatly increased synthesis of diacetyl and acetoin by L. casei during its fermentation of milk.     

Analysis of Volatile Compounds Produced by 2 Strains of Lactococcus lactis Isolated from Leben (Tunisian Fermented Milk) Using Solid-Phase Microextraction-Gas Chromatography

ABSTRACT:  The volatile compounds that characterize Leben during fermentation with 2 Lactococcus lactis strains (SLT6 and SLT10) in flasks, in a 100-L fermentor, and during storage at 4 °C, were investigated and compared to those from commercial Leben. Volatile compounds from Leben were concentrated by a Carboxen–PDMS fiber and analyzed by GC-MS. These compounds include acids, alcohols, aldehydes, ketones, sulfur compounds, and hydrocarbons. Commercial Leben presented a poor volatile profile compared to the laboratory-made Leben. The mixed culture of 2 Lactococcus lactis strains resulted in higher volatile compound formation than the single strain culture. The GC volatile profiles of Leben produced in flask and in the 100-L fermentor were similar. Changes in volatile compounds were observed during storage at 4 °C. The effect of culture conditions on production of volatiles by SLT6 strain was studied. Aeration (0.1 mL/min) and agitation enhanced the production of diacetyl, acetoin, 3-methylbutanal, and 3-methylbutanol. Fermentation at pH 5 had no effect on volatile production.     

Citrate metabolism in Lactococcus lactis subsp. lactis var. diacetylactis strains

The formation of diacetyl, acetoin, 2,3-butylene glycol, acetaldehyde, ethanol and lactic acid during 24 h of cultivation in milk with 0.19 and 0.5 % of citrate has been studied. Depending on the strain, bacteria produced 1.5 - 1.9 mg of diacetyl, 212 - 311 mg of acetoin and 137 - 156 mg of butylene glycol in 1 1 milk. An increase of the citrate concentration in milk to 0.5 % resulted in an increase in the production of diacetyl from 58 to 74 % and of acetoin by 2.8 - 3.7 times. The strains of distinct activity of acetoin reductase produced in these conditions 2.3 - 2.7 times as much as 2,3-butylene glycol. The recovery of citrate in the from of C₄-compounds ranged from 76 to 98 %, yet barely 0.18 - 0.44 % in the from of diacetyl. Increased concentration of citrate in milk stimulated the production of diacetyl and acetaldehyde to the similar extent, thereby it did not result in the deterioration of organoleptic qualities of starters and milk products. Within the doses used citrate did not significantly affect growth and acidifying activity of the bacteria.     

Effect of alpha-acetolactate decarboxylase inactivation on alpha-acetolactate and diacetyl production by Lactococcus lactis subsp. lactis biovar diacetylactis

Strains of Lactococcus lactis subsp. lactis biovar diacetylactis deficient in alpha-acetolactate decarboxylase produce alpha-acetolactate. This unstable compound is a precursor of acetoin and an aromatic compound, diacetyl. Following random mutagenesis of strain CNRZ 483, alpha-acetolactate decarboxylase-negative mutant 483 M1 was selected. When grown in milk, its growth and acidification characteristics were similar to those of the parental strain. In anaerobic conditions, the parental strain produced 2.10 mM acetoin and less than 0.05 mM diacetyl. The mutant accumulated up to 2.11 mM alpha-acetolactate, which spontaneously degraded to acetoin and diacetyl. After 24 h of culture, the alpha-acetolactate concentration was only 0.49 mM and the acetoin and diacetyl concentrations reached 1.50 mM and 0.26 mM, respectively. Diacetyl production by both strains increased in aerobic conditions, as well as when citrate was added. In contrast to cultures of the parental strain, however, diacetyl and acetoin concentrations in mutant cultures continued to increase without reaching a plateau. The results also showed that diacetyl production by wild type L. lactis subsp. lactis biovar diacetylactis strains cannot be explained uniquely by the spontaneous decarboxylation of the alpha-acetolactate produced in the culture medium.     

Effect of fungal volatile organic compounds on a fungus and an insect that damage stored maize

The silo is an environment where a large number of biological interactions take place such as: insect-microorganism-grain interactions, which can generate great economic losses due to the deterioration in quality of the grain and the presence of mycotoxins. In recent years, particular interest has been focused on the search for environmentally friendly insecticides that will provide pest control in stored grains. The volatile organic compounds (VOCs), of a fungal origin, were evaluated for the control of maize grain pests: the insect Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae), the fungus Fusarium verticillioides and its mycotoxin, fumonisin B₁ (FB₁). The most active fumigant compound tested was 1-octen-3-ol (LD₅₀ = 27.7 μL/L air), followed by 3-octanol and 3-octanone (LD₅₀ = 43.2 and 219.7 μL/L air, respectively). The fungal VOCs also showed repellent activity against S. zeamais, with antifugal activity against F. verticillioides growth being inhibited at concentrations greater than 0.53 mM, while its mycotoxin production capacity was inhibited depending on the compound concentration. At the repellent concentration, the fungal VOCs showed low phytotoxicity activities. The results presented in this paper demonstrate the potential of fungal VOCs as biopesticides, because they may control granivorous insects, fungal growth and FB₁ production, which consequently is of economic importance and might improve food safety of stored grains.     

Volatile organic compounds from the interaction between Fusarium verticillioides and maize kernels as a natural repellents of Sitophilus zeamais

Maize kernels are exposed to Sitophilus zeamais attack and Fusarium verticillioides infestation during storage, which can result in product deterioration and economic losses. The objective of this study was to evaluate the involvement of the Volatile Organic Compounds (VOCs) emitted by the fungi-corn system in grain-insect interactions. Volatiles emitted by healthy maize kernels were different from those emitted by fungal infected kernels, with the latter being enriched by alcohols, ketones and sesquiterpenes, which were considered early indicators of fungal contamination. The results demonstrated that the kernels exposed to the fungal VOCs and their pure compounds (1-octen-3-ol and 3-octanol) were less attractive and less damaged by S. zeamais than controls. In addition to compound adsorption, other processes may have caused the protective effect of exposed kernels against insect damage. This is the first contribution of the role of the fungal volatiles on the behavior of S. zeamais, and could provide an important contribution to the conservation of stored grains and pest management and an early indicator of fungal contamination.     

Effect of essential oils of cinnamon,clove, lemon grass,oregano and palmarosa on growth of and fumonisin B1 production by Fusarium verticillioides in maize

The ability of cinnamon, clove, lemon grass, oregano and palmarosa essential oils to prevent growth of and fumonisin B₁ (FB₁) production by Fusarium verticillioides at different water activity (0.95 and 0.995 a_w) and temperature (20 and 30 °C) levels in irradiated maize grain was evaluated. All the essential oils inhibited growth of F verticillioides isolates under all conditions tested, but FB₁ production was only inhibited at 30 °C and 0.995 a_w. Moreover, stimulation of toxin production was found under certain environmental conditions. None of the essential oils showed a significantly greater ability to inhibit FB₁ production when compared with the others. At 1000 mg essential oil kg^?1 maize the essential oils showed a greater inhibitory effect on growth of F verticillioides than at 500 mg kg^?1, but there was no difference in FB₁ production between the two levels of essential oil. Copyright © 2004 Society of Chemical Industry     

Recent research on fumonisins: a review

Fumonisins are well known mycotoxins produced by Fusarium verticillioides, F. proliferatum and other Fusarium species. Many new fumonisins and fumonisin-like compounds have been detected by mass spectrometry in cultures of F. verticillioides. Recently, fumonisins B₂ and B₄ were produced by Aspergillus niger isolated from coffee and fumonisin B₂ in A. niger from grapes. Fumonisin B₂ was itself detected in coffee beans, wine and beer, adding to the list of foodstuffs and feedstuffs other than corn (maize) and sorghum in which fumonisins have been found in recent years. Fumonisin B₁ (FB₁) can bind to proteins (PB FB₁) and to other matrix components during food processing involving heat. The occurrence of bound fumonisins in processed corn foods is common. Another type of binding (or association) relates to observed instability of fumonisins in rice flour, corn starch and corn meal at room temperature; this can affect the immunoaffinity column clean-up procedure in analysis of naturally contaminated starch-containing corn foods for fumonisins. The occurrence of N-fatty acylated fumonisin derivatives in retail fried corn foods has also been demonstrated. Bioaccessibility of free FB₁ and total bound FB₁ (TB FB₁) present in corn flakes has been estimated by in vitro digestion experiments. Intentional binding of fumonisins to cholestyramine has been demonstrated in vivo and is a potential means of detoxification of animal feed.     

Fatty acid esters of fumonisins: first evidence of their presence in maize

Fumonisin derivatives obtained by esterification of fumonisin B₁ (FB₁) with palmitic, oleic and linoleic fatty acids have been recently described, but never reported in raw maize so far. In this study, the presence of oleoyl-EFB₁ (EFB₁OA) and linoleoyl-EFB₁ (EFB₁LA) in raw maize is reported by means of a suitable LC-ESI-MS/MS method. In addition, the production of EFB₁ derivatives by three Fusarium verticillioides strains is described on malt extract–based media and on corn meal–based growth media. EFB₁OA and EFB₁LA were produced by all considered strains in corn meal medium, with EFB₁LA > EFB₁OA. On the contrary, EFB₁OA and EFB₁LA were never observed in Fusarium cultures grown on a malt extract medium, suggesting that the esterification of FB₁ can occur only in a complex matrix such as maize.     

Influence of acetaldehyde on growth and acetoin production by Leuconostoc citrovorum

Addition of acetaldehyde (100 μg/ml) to glucose-citrate broth stimulated growth and approximately doubled the production of acetoin plus diacetyl by Leuconostoc citrovorum. Radioactive acetaldehyde was converted to ethanol; the acetoin, diacetyl and acetic acid produced were not radioactive. A discussion of the mechanisms by which species of Leuconostoc utilize citrate and carbohydrate suggests that the added acetaldehyde enhanced production of acetoin and diacetyl by increasing the availability of hydroxyethylthiamine pyrophosphate and acetyl-coenzyme A, and that it stimulated growth by permitting greater conversion of acetyl-coenzyme A and acetyl-phosphate to acetate and adenosine 5′-triphosphate.     

Short communication: Enzymatic perspective of galactosidases reveals variations in lactose metabolism among Lactococcus lactis strains

To date, most studies of lactose utilization have focused on the genetic diversity of lactic acid bacteria or its influence on product quality, but phenotypic evaluation has rarely been based on metabolic characteristics. In the present study, we investigated the growth, acid production, β-galactosidase, and 6-phospho-β-galactosidase activities of 16 Lactococcus lactis strains obtained from various habitats with lactose as the sole carbon source. The 15 L. lactis strains obtained from various habitats exhibited significant differences in growth and acid production characteristics in the de Man, Rogosa, and Sharpe-lactose broth, and 4 strains consumed more lactose when cultured in skim milk than the type strain ATCC 19435. Among these strains, DQHXNQ38–12 mainly produced acetoin and diacetyl when cultured in skim milk, whereas the strains 15M2 and 5G2 produced high levels of acid and formed curd rapidly. We concluded that the use of lactose is necessary for strain adaptation to the dairy niche. Comprehensive studies of lactose use and the fermentation characteristics of L. lactis are of significant importance.     

Solvent Desorption Dynamic Headspace Method for Diacetyl and Acetoin in Buttermilk

Major buttermilk volatiles recovered included diacetyl, acetic acid, and acetoin. Detection limits were diacetyl 0.2 and acetoin 2.0 μg/g. Mean percent recoveries were 112% for diacetyl and 8.08% for acetoin. Normalized detector responses were linear over the range of concentrations tested (R² > 0.999) for diacetyl and acetoin. Percent relative standard deviations from quadruplicate analysis of 7-day-old buttermilk were <8% for diacetyl, acetoin, and acetic acid. The method enabled quantitative estimation of diacetyl and acetoin in <30 min, including sample preparation time.     

Occurrence of the C-series fumonisins in maize from the former Transkei region of South Africa

Fumonisins are a group of structurally related mycotoxins produced mainly in maize by Fusarium verticillioides and F. proliferatum. The most abundant naturally occurring analogue is fumonisin B₁ (FB₁), with lesser amounts of fumonisin B₂ (FB₂) and fumonisin B₃ (FB₃) occurring. The C-series fumonisins (FCs) are structurally analogous to the B-series but lack the C-1 methyl group. Good and mouldy subsistence-grown maize samples were collected from the Centane and Bizana districts in the former Transkei region of South Africa. After extraction with methanol/water and clean-up on strong anion exchange solid phase extraction cartridges, FB₁, FB₂, FB₃, FC₁, FC₃ and FC₄ were determined by reversed-phase LC–MS/MS using positive ion electrospray ionisation. FB₁ levels in both good and mouldy maize from Centane (means (±SD) 2.75?±?2.24 and 23.4?±?12.5?mg?kg^?1, respectively) were higher than the corresponding levels in maize samples from Bizana (means 0.056?±?0.157 and 3.71?±?5.01?mg?kg^?1, respectively). Similarly, FC₁ levels in both good and mouldy maize from Centane (means 0.107?±?0.099 and 0.814?±?0.391?mg?kg^?1, respectively) were higher than in Bizana, where FC₁ was detected in only one (0.018?mg?kg^?1) of 19 good maize samples and occurred in mouldy maize with a mean of 0.102?±?0.135?mg?kg^?1. A significant correlation (r?=?0.982, p?<?0.01) was observed between FB₁ and FC₁ levels in all samples, with FC₁ levels at 3.3% of the corresponding FB₁ levels. FC₄ levels were similar to FC₁, whereas only low amounts of FC₃ were detected.     

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1α partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B₁ and B₂ production levels. Among the isolated strains, 96 were identified as Fusarium verticillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1α sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verticillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r = 0.95 and r = 0.79 (correlation of FUM1 with FB₁ and FB₂, respectively, P < 0.0001); r = 0.93 and r = 0.78 (correlation of FUM19 with FB₁ and FB₂, respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity.     

Redirection of Pyruvate Pathway of Lactic Acid Bacteria to Improve Cheese Quality

Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh ^d) and diacetyl reductase (dar ^d) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh ^d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.     

Identification,mycotoxin risk and pathogenicity of Fusarium species associated with fig endosepsis in Apulia,Italy

In a survey carried out on 87 rotted fig fruits samples collected in the Apulia region of Italy, the authors isolated 126 Fusarium strains identified as F. ramigenum (69 strains), F. solani (49), F. proliferatum (five) and three not identified. Investigation on the fertility of the strains belonging to F. proliferatum and F. ramigenum revealed that only strains of F. proliferatum were fertile. The identity of F. ramigenum strains was confirmed by sequencing a portion of the translation elongation factor-1α gene. When Fusarium species were analysed for their toxigenicity, 37/69 strains of F. ramigenum produced fusaric acid (FA) up to 525 mg kg^?1; 30 strains produced beauvericin (BEA) up to 190 mg kg^?1; 60 strains produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) up to 1575 mg kg^?1 of total FBs; and two strains produced fusaproliferin (FUP) up to 345 mg kg^?1; all five strains of F. proliferatum produced FA at low levels; two strains produced BEA up to 205 mg kg^?1; one strain produced FB₁ and FB₂, 1100 and 470 mg kg^?1, respectively; and one strain produced FUP, 820 mg kg^?1; F. solani (30 strains) produced FA, 13 strains up to 215 mg kg^?1. Few fungal extracts showed high toxicity toward brine shrimp larvae and in some cases in relation to BEA and FA content. A pathogenic assay on fig fruits showed that all three species were pathogenic, with higher virulence of F. ramigenum. These data report for the first time the production of BEA and FB₁/FB₂ by F. ramigenum and show that it is a main agent of fig endosepsis in Apulia and can contribute to fumonisin contamination of fresh and dried figs.