Evaluation of chemically characterised essential oils of Coleus aromaticus,Hyptis suaveolens and Ageratum conyzoides against storage fungi and aflatoxin contamination of food commodities

The result of the present investigation explores the efficacy of chemically characterised essential oils (EOs) of Coleus aromaticus, Hyptis suaveolens and Ageratum conyzoides as antifungal and antiaflatoxigenic agent against some storage fungi and the toxigenic strain of Aspergillus flavus (Saktiman 3NSt). Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of these EOs were also determined against the toxigenic strain of A. flavus (Saktiman 3NSt). The EO from C. aromaticus was found to be most effective exhibiting MIC and MFC at 0.1μL mL^?1. The EOs also completely checked aflatoxin B₁ synthesis in concentration‐dependent manner. In addition, fumigation of stored wheat samples with EOs exhibited remarkable protection (>80%) from fungal infestation showing their efficacy during in vivo storage conditions. Based on the results of the present investigation, the EOs of C. aromaticus, H. suaveolens and A. conyzoides may be recommended as novel plant‐based antifungal and aflatoxin B₁ suppressor over the synthetic preservatives.     

Relative populations and toxin production by Aspergillus flavus and Fusarium verticillioides in artificially inoculated corn at various stages of development under field conditions

The presence, development and production of mycotoxins by Aspergillus flavus and Fusarium verticillioides were studied in corn ears under field conditions after artificial contamination of corn silks. The planted area was divided into five treatments: T1, inoculated with A. flavus solution containing 1 × 10⁸ spores, ears covered; T2, inoculated with F. verticillioides solution containing 1 × 10⁸ spores, ears covered; T3, inoculated with F. verticillioides plus A. flavus solution containing 1 × 10⁸ spores of each, ears covered; T4, sprayed with sterile phosphate‐buffered saline, ears covered; T5, non‐sprayed silks, uncovered ears. Soil and air samples were also collected and analysed for the occurrence of fungi. Water activity, relative air humidity, rainfall and temperature were determined to assess the correlation between abiotic factors and the presence of fungi in the samples. Contamination with the inoculated fungus predominated in T1 and T2. In the other treatments, F. verticillioides was the most frequently isolated contaminant irrespective of treatment. Considering the production of mycotoxins, a positive relation between the production of fumonisins B₁ and B₂ and the frequency of F. verticillioides was statistically verified in all treatments. Copyright © 2007 Society of Chemical Industry     

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.     

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.     

Characterization of Fusarium verticillioides strains by PCR‐RFLP analysis of the intergenic spacer region of the rDNA

Thirty‐three Fusarium verticillioides strains from diverse origins and hosts have been analysed for fumonisin production and characterized in order (i) to detect the variability present in this species and (ii) to discriminate among isolates. The method used was a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) generated by restriction endonucleases applied to the IGS region (intergenic spacer of rDNA). All the F. verticillioides strains associated with crops produced fumonisins B₁ and B₂ except those isolated from banana. Analysis of the IGS region by PCR‐RFLP proved to be useful to detect variability within F. verticillioides and allowed discrimination of two related groups of isolates belonging to distinct lineages differing in fumonisin production and host preferences: the fumonisin‐producing group associated with cereals and the fumonisin non‐producing group associated with banana. The method used facilitates early detection and characterization of F. verticillioides strains required to control both types of pathogens and to evaluate plant exposure to the toxin, quality of the raw material to be processed and the potential fumonisin contamination in order to prevent fumonisins entering the food chain. Copyright © 2005 Society of Chemical Industry     

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.     

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     

Assessment of Pelargonium graveolens oil as plant‐based antimicrobial and aflatoxin suppressor in food preservation

BACKGROUND: Contamination of stored food commodities by moulds and mycotoxins results in qualitative as well as quantitative losses. Most of the synthetic antimicrobials used for preservation of stored food items produce side effects in the form of residual and mammalian toxicity. Recently some higher plant products have been recommended as safe alternatives of such synthetic antimicrobials. In the present investigation antifungal efficacy of some essential oils was evaluated against two toxigenic strains of Aspergillus flavus with special reference to the oil of Pelargonium graveolens to investigate its potential to inhibit aflatoxin B₁ secretion. RESULTS: Essential oil of P. graveolens exhibited absolute fungitoxicity against both the toxigenic strains of A. flavus. The minimum inhibitory concentration of the oil was found to be 0.75 g L^?1 and exhibited a fungistatic nature. It was found superior over the synthetic fungicides tested and exhibited a broad fungitoxic spectrum. The oil showed excellent anti‐aflatoxigenic efficacy as it completely inhibited aflatoxin B₁ production even at 0.50 g L^?1. CONCLUSION: This is the first report on the aflatoxin B₁ inhibitory nature of P. graveolens oil. It may be recommended as a novel plant‐based antimicrobial as well as aflatoxin B₁ suppressor over synthetic preservatives in food protection. Copyright © 2008 Society of Chemical Industry     

Comparative evaluation of antioxidant and anti food-borne bacterial activities of essential oils from some spices commonly consumed in Iran

The antioxidant potency, anti food borne bacterial activity, and total phenolic contents of essential oils (EOs) from avishane shirazi (Zataria multiflora), clove (Syzgium aromaticum), cinnamon (Cinnamomum zeylanicum), cumin (Cuminum cyminum), black cumin (Bunium persicum), spearmint (Mentha spicata), horsemint (Mentha longifolia), coriander (Coriandrum sativum), sage (Salvia officinalis), and ginger (Zingiber officinale) were evaluated. In 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, free radical scavenging activities of clove and avishane shirazi EOs were 90.69% and 88.63%, respectively. In reducing power assay, the EO of clove showed the highest reducing capacity. The highest concentrations of total phenolics (66.01 mg and 44.81 mg GAE/gram sample) were also detected for the EOs of clove and avishane shirazi, respectively. The results of disc diffusion assay showed that the EOs of avishane shirazi, cinnamon, and clove strongly inhibited growth of the tested bacteria. The EO of cinnamon had the lowest minimal inhibitory concentration (MIC) (0.312 mg/mL).     

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.     

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.     

Control of fumonisin B 1 accumulation in naturally contaminated maize inoculated with Fusarium verticillioides and Fusarium proliferatum, by cinnamon, clove, lemongrass, oregano and palmarosa essential oils

The effect of cinnamon, clove, oregano, palmarosa and lemongrass oils on fumonisin B₁ (FB₁) accumulation by one isolate each of Fusarium verticillioides and Fusarium proliferatum in non-sterilised naturally contaminated maize grain at 0.995 and 0.950 a_w and at 20 and 30 °C was evaluated. The concentration used was 500 mg kg^-1 maize. Under these conditions it was shown that antimycotoxigenic ability only took place at the higher water availabilities, and mostly at 20 °C. Only cinnamon, lemongrass and palmarosa oils were somewhat effective. Moreover, it was suggested that competing mycoflora plays an important role in FB₁ accumulation. It was concluded that the efficacy of essential oils in real substrates, such as cereals, may be much lower than in synthetic media; different essential oils may be found to be useful and at different concentrations. Their effectiveness is highly dependent on both abiotic and biotic factors involved.     

Impact of environment and interactions of Fusarium verticillioides and Fusarium proliferatum with Aspergillus parasiticus on fumonisin B1 and aflatoxins on maize grain

Fusarium verticillioides and F proliferatum isolates were inoculated in mixed cultures with Aspergillus parasiticus on irradiated maize grain at two different inoculum concentrations (2 × 10⁵ and 2 × 10² conidia g^?1 dry maize). The treatments were 0.93–0.98 water activity (a_w) and 15 and 25 °C for 28 days. A complex relationship was found between a_w, temperature, inoculum concentration and the interactions which took place between fumonisin and aflatoxin producers. In general, A parasiticus reduced F verticillioides and F proliferatum populations (by 6–36%) but did not affect fumonisin B₁ production by these species. In contrast, while the Fusarium species were not able to decrease A parasiticus populations, they significantly reduced aflatoxin B₁ accumulation (by 30–93%). © 2001 Society of Chemical Industry     

Analysis of fumonisin contamination and the presence of Fusarium in wheat with kernel black point disease in the United States

The ability of the fungus Fusarium proliferatum to cause kernel black point disease in wheat was previously established, but natural contamination of black point wheat with both F. proliferatum and fumonisin mycotoxins has not been studied in the United States. Low levels of fumonisins were detected in nine of 43 wheat samples with kernel black point disease that were obtained from across the United States. A subset of samples was contaminated with F. proliferatum as well as with F. fujikuroi, F. nygamai, F. thapsinum and F. verticillioides, species closely related to F. proliferatum and morphologically similar to it in that they produce chains of asexual spores, or conidia. Nevertheless, of conidial chain-forming fusaria isolated from symptomatic wheat, F. proliferatum dominated. In greenhouse tests, isolates of F. proliferatum and the other species recovered from wheat samples were able to cause symptoms of kernel black point and, in some cases, low levels of fumonisin contamination of wheat. These data add to the understanding of the risk of fumonisin contamination of wheat and the potential for Fusarium species to cause kernel black point disease and fumonisin contamination of wheat. Further, the results of this study indicate that while US-grown wheat can sporadically be contaminated by fumonisins, the natural contamination levels seem to be low. The observations made provide evidence that fumonisins are not likely to be a factor contributing to the ability of Fusarium to cause kernel black point disease.     

Occurrence of fumonisins in Catalonia (Spain) and an exposure assessment of specific population groups

Fumonisin B₁ (FB₁) and B₂ (FB₂) are mycotoxins produced by Fusarium verticillioides and F. proliferatum and common contaminants of cereal crops. The objectives of this study were to (1) study the occurrence of fumonisins in Catalonia (north-eastern region of Spain) and (2) assess the exposure of the Catalonian population to these mycotoxins. Contamination data was provided by a wide survey where 928 individual samples were pooled to analyse 370 composite samples. Fumonisins were extracted and purified using immunoaffinity columns and determined by HPLC with fluorescence detection. The raw consumption data came from a nutritional study specifically designed to assess the dietary intake of the main foodstuffs related to fumonisin contamination for all population age groups. In addition, two specific groups were selected with respect to maize consumption: immigrants and celiac sufferers. Contamination and consumption data were combined by simulation using an essentially parametric–parametric (P-P) method. The P-P method draws sampling values from distribution functions fitted to consumption and contamination datasets. Moreover, to quantify the accuracy and reliability of the statistical estimates, we built related confidence intervals using a Pseudo-Parametric bootstrap method. The results of this study show that fumonisins are commonly found in some commodities on the Catalonian market, such as beer, corn snacks and ethnic foods; however, the values were well below the permitted maximum EU levels. The most exposed group were infants followed by immigrants but, in all cases, they were below the TDI of 2?µg/kg bw/day.     

Impact of volatile compounds generated by essential oils on Aspergillus section Flavi growth parameters and aflatoxin accumulation

BACKGROUND: The advantage of essential oils is their bioactivity in the vapour phase, a characteristic that makes them attractive as possible fumigants for stored grain protection. In this study the antifungal and antiaflatoxigenic effects of the volatile fractions of five essential oils (EOs) were evaluated by vapour contact on Aspergillus section Flavi isolates. RESULTS: In maize meal extract agar the volatile fractions of Pimpinella anisum L. (anise), Pëumus boldus Mol. (boldus), Hedeoma multiflora Benth. (mountain thyme), Lippia turbinata var. integrifolia (Griseb.) (poleo) and Syzygium aromaticum L. (clove) were able to decrease the growth rate and lag phase of aflatoxigenic isolates. Boldus EO showed the best antifungal effect on Aspergillus section Flavi growth rate. In sterilised maize grains, boldus and poleo EOs showed antifungal effects on growth rate and aflatoxin accumulation. The volatile fraction of boldus EO completely inhibited the growth of isolates at water activity (a_w) levels of 0.955, 0.930 and 0.900, while poleo EO showed this effect only at the lower a_w levels (0.930 and 0.900). All aflatoxigenic isolates showed reduced total aflatoxin accumulation in the presence of boldus EO under all a_w conditions. CONCLUSION: These findings clearly indicate that the volatile fraction of boldus EO could be used to control aflatoxigenic fungi in stored maize. Copyright © 2009 Society of Chemical Industry     

Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis of Fusarium verticillioides and maize kernels

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize, even if frequently asymptomatic, producing a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins result as signals able to modulate the secondary metabolism in fungi. In keeping with this, a novel, quantitative LC-MS/MS method was designed to quantify up to 17 different oxylipins produced by F. verticillioides and maize kernels. By applying this method, we were able to quantify oxylipin production in vitro – F. verticillioides grown into Czapek–Dox/yeast extract medium amended with 0.2% w/v of cracked maize – and in vivo, i.e. during its growth on detached mature maize ears. This study pinpoints the role of oxylipins in a plant pathogen such as F. verticillioides and sets up a novel tool aimed at understanding the role oxylipins play in mycotoxigenic pathogens during their interactions with respective hosts.     

Mycoflora and fumonisin contamination in Brazilian sorghum from sowing to harvest

BACKGROUND: The aim of this study was to characterise the mycoflora and the presence of fumonisin in sorghum grains, correlating the results with the environment and abiotic factors. RESULTS: Fifty samples (five collections of ten samples each) of sorghum were analysed. All samples were found to be contaminated with fungi, with higher frequencies of Cladosporium spp. (61.8%) and Helminthosporium spp. (33.4%). Fusarium verticillioides was isolated from 15.1% of the samples, with 38% of them being contaminated with fumonisin B₁ (FB₁) at levels ranging from 50 to 368.78 ng g^?1. Regarding abiotic factors, temperature, water activity and rainfall showed a positive correlation with the frequency of F. verticillioides and FB₁ production. There was a significant positive correlation between relative air humidity and FB₁ production. The results obtained from sexual crosses between standard F mating tester strains and the isolated strains confirmed that the strains isolated were F. verticillioides. CONCLUSION: It can be concluded that the decrease in F. verticillioides and fumonisin contamination occurred owing to atypical climatic factors during the period of sorghum cultivation, when there was any occurrence of rain and the level of water activity of grains did not reach 0.58. Copyright © 2010 Society of Chemical Industry     

Application of essential oils in maize grain: impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation

The antifungal activity of Pimpinella anisum L. (anise), Pëumus boldus Mol (boldus), Hedeoma multiflora Benth (mountain thyme), Syzygium aromaticum L. (clove), and Lippia turbinate var. integrifolia (griseb) (poleo) essential oils (EOs) against Aspergillus section Flavi was evaluated in sterile maize grain under different water activity (a_w) condition (0.982, 0.955, and 0.90). The effect of EOs added to maize grains on growth rate, lag phase, and aflatoxin B₁ (AFB₁) accumulation of Aspergillus section Flavi were evaluated at different water activity conditions. The five EOs analyzed have been shown to influence lag phase and growth rate. Their efficacy depended mainly on the essential oil concentrations and substrate water activity conditions. All EOs showed significant impact on AFB₁ accumulation. This effect was closely dependent on the water activity, concentration, and incubation periods. Important reduction of AFB₁ accumulation was observed in the majority of EO treatments at 11 days of incubation. Boldus, poleo, and mountain thyme EO completely inhibited AFB₁ at 2000 and 3000 μg g⁻¹. Inhibition of AFB₁ accumulation was also observed when aflatoxigenic isolates grew with different concentration of EOs during 35 days.