Effect of Equisetum arvense and Stevia rebaudiana extracts on growth and mycotoxin production by Aspergillus flavus and Fusarium verticillioides in maize seeds as affected by water activity

Cereals are a very important part of the human and animal diets. However, agricultural products can be contaminated by moulds and their mycotoxins. Plant extracts, particularly those of Equisetum arvense and Stevia rebaudiana have been reported previously to contain antioxidant compounds which may have antifungal properties. In this study, E. arvense and S. rebaudiana extracts were tested for their control of mycotoxigenic fungi in maize. The extracts were tested separately and as a mixture for their effect on growth of Aspergillus flavus and Fusarium verticillioides. Extracts were added to unsterilised inoculated maize at different water activity (a(w)) levels (0.85-0.95). Moulds were inoculated and incubated for 30 days. Results confirmed that the extract of E. arvense and a mixture 1:1 of Equisetum-Stevia may be effective for the inhibition of both growth of A. flavus and aflatoxin production at high water activity levels (pre-harvest conditions). In general, growth of the F. verticillioides was reduced by the use of plant extracts, especially at 0.95 a(w). However, fumonisin presence was not significantly affected. E. arvense and S. rebaudiana extracts could be developed as an alternative treatment to control aflatoxigenic mycobiota in moist maize.     

Efficacy of bioactive compounds isolated from Albizia amara and Albizia saman as source of antifungal and antiaflatoxigenic agents

The antifungal and antiaflatoxigenic activities of budmunchiamine A (BUA) isolated from Albizia amara and pithecolobine (PI) isolated from Albizia saman were assessed. The present study reports the broad-spectrum and concentration-dependent antifungal activities of BUA and PI with zone of inhibitions and minimum inhibitory concentrations ranging from 6.8 to 19.6 mm and 0.015–0.5 mg/mL, respectively. Aspergillus flavus growth and its aflatoxin B₁ production were completely inhibited in vitro by BUA and PI at concentration of 1 mg/mL. BUA severely inhibited the growth of wide range of seed-borne fungi of maize including aflatoxigenic A. flavus with an increased seedling vigour index in vivo at a lower concentration (1.0 g/kg) than PI (2.0 g/kg). Enhanced seedling vigour was observed in BUA and PI treatments with no adverse effect on seed germination. The present findings indicate the possible use of BUA and PI as antifungal agents against post harvest fungal infestation of food commodities and mycotoxin contamination.     

Natural Control of Corn Postharvest Fungi Aspergillus flavus and Penicillium sp. Using Essential Oils from Plants Grown in Argentina

The objective in this study was to evaluate the antifungal activity of essential oils from native and commercial aromatic plants grown in Argentina against corn postharvest fungi and to link the essential oil bioactivity with lipid oxidation and morphological changes in fungus cell membrane. Essential oil (EO) of oregano variety Mendocino (OMen), Cordobes (OCor), and Compacto (OCom), mint variety Inglesa (Mi), and Pehaujo (Mp), Suico (Sui); rosemary (Ro), and Aguaribay (Ag) were tested in vitro against 4 corn fungi: A. flavus (CCC116–83 and BXC01), P. oxalicum (083296), and P. minioluteum (BXC03). The minimum fungicidal concentration (MFC) and the minimum inhibitory concentration (MIC) were determined. The chemical profiles of the EOs were analyzed by GC‐MS. Lipid oxidation in cell membrane of fungi was determined by hydroperoxides and related with essential oil antifungal activity. The major compounds were Thymol in OCor (18.66%), Omen (12.18%), and OCom (9.44%); menthol in Mi and Mp; verbenone in Sui; dehydroxy‐isocalamendiol in Ag; and eucaliptol in Ro. OCor, Omen, and OCom showed the best antifungal activity. No antifungal activity was observed in Ag and Ro EO. The hydroperoxide value depended on the fungi (P < 0.001) and the antimicrobial agent (P < 0.001).Membrane lipids were oxidized by Sui EO in A. flavus BXC01 and A. flavus CCC116–83 (0.021 and 0.027 meqO₂/kg, respectively). The results suggest that the EOs of OCor, OMen, OCom, Mi, Mp, and Sui grown in Argentina can be used as natural alternatives to control fungi that produce mycotoxin in maize.     

In vitro antifungal activity of terpinen-4-ol,eugenol, carvone, 1,8-cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens

The aim of this study was to examine the effect of five naturally occurring compounds from essential oils on 10 different species of mycotoxigenic fungi involved in several plant diseases. The antifungal activities of terpinen-4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol were observed in vitro on Fusarium subglutinans, Fusarium cerealis, Fusarium verticillioides, Fusarium proliferatum, Fusarium oxysporum, Fusarium sporotrichioides, Aspergillus tubingensis, Aspergillus carbonarius, Alternaria alternata and Penicillium sp. The naturally occurring compounds tested showed toxic effects on in vitro mycelium growth of all fungal species but with different level of potency. The results are encouraging for further investigations of in planta antifungal activities of these essential oils components.     

Promising antifungal effect of some Euro-Asiatic plants against dangerous pathogenic and toxinogenic fungi

BACKGROUND: Increasing evidence of fungicide‐resistant toxinogenic and pathogenic fungal species is obvious. Looking for new possibilities of antifungal treatment or sources of antifungal substances is a major problem. Some medicinal plants exert strong antifungal properties and could be conveniently used as a promising alternative source for presently problematic antifungal treatment in many areas with respect to their natural origin. Methanol extracts of 46 medicinal plants from the Eurasian area were used in a screening assay for antifungal activity in this study. The growth inhibitory effect was tested against six significant pathogenic and toxinogenic fungal species: Fusarium oxysporum, F. verticillioides, Penicillium expansum, P. brevicompactum, Aspergillus flavus and A. fumigatus. RESULTS: For 14 plant species, the possibility of using them as natural fungicides was indicated. The extract from Grindelia camporum showed significant activity against all target fungal species. The most sensitive target fungus was the toxinogenic and human pathogenic species A. fumigatus. CONCLUSION: This study has identified 14 extracts of medicinal plants with a potential use as an antifungal treatment in various areas. One of them showed promising efficiency against all selected significant pathogenic and toxinogenic fungal species. Copyright © 2010 Society of Chemical Industry     

Efficacy of Some Essential Oils Against Aspergillus flavus with Special Reference to Lippia alba Oil an Inhibitor of Fungal Proliferation and Aflatoxin B1 Production in Green Gram Seeds during Storage

During mycofloral analysis of green gram (Vigna radiata (L.) R. Wilczek) seed samples taken from different grocery stores by agar and standard blotter paper methods, 5 fungal species were identified, of which Aspergillus flavus exhibited higher relative frequency (75.20% to 80.60%) and was found to produce aflatoxin B₁. On screening of 11 plant essential oils against this mycotoxigenic fungi, Lippia alba essential oil was found to be most effective and showed absolute inhibition of mycelia growth at 0.28 μL/mL. The oil of L. alba was fungistatic and fungicidal at 0.14 and 0.28 μL/mL, respectively. Oil had broad range of fungitoxicity at its MIC value and was absolutely inhibited the AFB1 production level at 2.0 μL/mL. Chemical analysis of this oil revealed geranial (36.9%) and neral (29.3%) as major components followed by myrcene (18.6%). Application of a dose of 80 μL/0.25 L air of Lippia oil in the storage system significantly inhibited the fungal proliferation and aflatoxin production without affecting the seed germination rate. By the virtue of fungicidal, antiaflatoxigenic nature and potent efficacy in storage food system, L. alba oil can be commercialized as botanical fungicide for the protection of green gram seeds during storage.     

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.     

The interaction between fusaria and their mycotoxins in grass silage

Perennial ryegrass was inoculated with a mycotoxigenic strain of Fusarium roseum or F. tricinctum and ensiled in laboratory silos to study the ability of the fungi to grow and produce toxin. Neither strain persisted in the silage or produced toxin. The addition of preformed toxin to the grass produced no detectable detrimental effect on the overall fermentation process.     

Effects of Tropical Citrus Essential Oils on Growth, Aflatoxin Production, and Ultrastructure Alterations of Aspergillus flavus and Aspergillus parasiticus

Ethyl acetate extracts and hydrodistillated essential oils from five cultivars of tropical citrus epicarps were evaluated for their inhibitory activities against Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, and Penicillium sp. using disk diffusion and broth microdilution assays. Essential oils prepared from kaffir lime (Citrus hystrix DC) and acid lime (Citrus aurantifolia Swingle) epicarps exhibited stronger antifungal activity to all fungi than their ethyl acetate extracts with minimum inhibitory concentration and minimum fungicidal concentration values of 0.56 and 1.13 mg/ml (dry matter), respectively, against aflatoxin-producing A. flavus and A. parasiticus. The dominant components of the essential oil from kaffir lime were limonene, citronellol, linalool, o-cymene, and camphene, whereas limonene and p-cymene were major components of acid lime essential oil. Pure limonene, citronellal, and citronellol were five to six times less fungicidal than the natural essential oils, indicating the synergistic activity of many active compounds present in the oils. Kaffir and acid lime essential oils significantly reduced aflatoxin production of A. flavus and A. parasiticus, particularly lime essential oil, which completely inhibited growth and aflatoxin production of A. flavus at the concentration of 2.25 mg/ml. Target cell damage caused by acid lime essential oil was investigated under transmission electron microscopy. Destructive alterations of plasma and nucleus membrane, loss of cytoplasm, vacuole fusion, and detachment of fibrillar layer were clearly exhibited in essential-oil-treated cells.     

Original article: Antifungal activities of cinnamon oil against Rhizopus nigricans,Aspergillus flavus and Penicillium expansum in vitro and in vivo fruit test

The postharvest pathogens such as R. nigricans, A. flavas and P. expansum are the causal agents of jujube or orange fruit, therefore, in vitro and in vivo antifungal activities of cinnamon oil to inactivate these fungi were investigated. Cinnamaldehyde is the main constituent of cinnamon oil. The minimum inhibitory concentrations of cinnamon oil against Rhizopus nigricans, Aspergillus flavus and Penicillium expansum were 0.64% (v/v), 0.16% (v/v) and 0.16% (v/v), respectively. The antifungal activity of cinnamon oil against A. flavus and P. expansum was stronger than that against R. nigricans and the activity was improved with increasing its concentration. In an in vivo study, cinnamon oil with concentrations of 2.0% (v/v) and 3.0% (v/v) showed complete control the growth of fungi in wound‐inoculated Lingwu Long Jujube and Sand Sugar Orange fruits. These results revealed that cinnamon oil has a good potential to be as a natural antifungal agent for fruit applications.     

Phenolic extracts of cachaça aged in different woods and quantifying antioxidant activity and antifungal properties

The antioxidant and antifungal activities of the phenolic compounds in different alcoholic extracts of aged cachaça were evaluated. The physico‐chemical analyses were performed in the Laboratório de Qualidade de Aguardente of the Universidade Federal de Lavras according to the methods of the Ministério Agricultura Pecuária e abastecimento. Total phenol content was determined by the Folin–Ciocalteu method, quantification of these compounds was performed by HPLC and antioxidant activities were determined by methods involving inhibition of the DPPH (1,1‐diphenyl‐2 picrylhydrazyl) radical, the β‐carotene/linoleic acid system, ABTS (2,2 azinobis‐[3‐ethyl‐6‐benzothiazolinesulfonic acid]) radical, reducing power and thiobarbituric acid. Determination of antifungal activity was accomplished through the technique of dissemination in discs using Aspergillus carbonarius, Aspergillus niger, Aspergillus flavus, Penicillium commune and Penicillium cladosporoides fungi at the Laboratório de Micologia de Alimentos. The values obtained for the phenolic compounds ranged from 0.41 to 9.69 mg L^?1; syringaldehyde, vanillic acid and gallic acid were predominant. A satisfactory antioxidant activity was observed in all of the tests with the alcoholic extracts. A moderate activity against P. commune and P. cladosporoides, but no inhibition of the growth of A. carbonarius, A. niger or A. flavus was observed. Copyright © 2016 The Institute of Brewing & Distilling     

Recent advances in plant-based compounds for mitigation of mycotoxin contamination in food products: current status,challenges and perspectives

There are numerous strategies to control the growth of mycotoxigenic fungi and reduce mycotoxins in food, including physical, chemical and biological treatments. However, consumers prefer organic food and natural inhibitors because they are biodegradable and safe for human and animal health. This review summarises the current advances in plant-based compounds to mitigate contamination of food products by mycotoxigenic fungi and their toxins. In addition, a clear understanding of the roles of plant-based extracts in food products, their mechanisms of action and challenges and perspectives in mycotoxin degradation are presented. Essential oils and plant-based extracts are complex mixtures of major and minor chemical compounds with antimicrobial and antioxidant properties. In general, the mechanisms of antifungal activity of plant-based compounds are attributed to the reduction of the ergosterol content, disruption of cell membrane integrity, enhancing of membrane ion leakage and permeability, disruption to the organisation of mitochondrial structure, interference in enzymatic reactions of cell wall synthesis, disturbance of oxidative balance, inhibition of carbohydrate metabolism, suppression of mycotoxin biosynthetic genes and alterations in the molecular structure of mycotoxins.     

In vitro evaluation of antifungal activity of soybean (Glycine max) seed coat proteins

Proteins in the soybean seed coat have previously been characterized; however, the function of these proteins is unknown. We show that a soybean seed coat protein fraction was able to inhibit the growth of Fusarium lateritium and Fusarium oxysporum phytopathogenic fungi. The antifungal fraction isolated by DEAE-Sepharose chromatography revealed the presence of peroxidase, vicilin and a 24 kDa protein homologous to acid phosphatases. Germination experiments revealed that both acid phosphatase and peroxidase were exuded during seed imbibition. We suggest that the set of seed coat antifungal proteins may help protect seeds from colonization by phytopathogenic fungi.     

Evaluation of Cinnamomum tamala Oil and Its Phenylpropanoid Eugenol for Their Antifungal and Antiaflatoxigenic Activity

During screening of 20 plant extracts against toxigenic strain of Aspergillus flavus (SK 3NSt), the extract of Cinnamomum tamala was found to exhibit absolute fungitoxic activity (100% growth inhibition). Hence, essential oil of C. tamala was extracted and selected for further investigations. The selected oil was subsequently standardised through physico-chemical and fungitoxic properties. Minimum inhibitory concentration (MIC) of the oil for absolute inhibition of growth of the toxigenic strain of A. flavus (SK 3NSt) was found to be 150 μl/l and oil showed fungicidal nature at its respective MIC. The oil had a broad fungitoxic spectrum. It was found to be absolutely inhibitory to almost all the 11 fungi tested when its fungitoxic spectrum was assayed. The antifungal potency of oil was found to be greater in comparison to some prevalent synthetic fungicides. The oils had the potency to withstand high inoculum density. The oil remained active up to 2 years and was thermostable. In addition, the oil showed significant efficacy in arresting aflatoxin B₁ secretion by the toxigenic strain (SK 3NSt) of A. flavus at 750 μl/l. GC-MS analysis of the oil led to the identification of main components of oil viz. eugenol (45.58%), β-pinene (10.03%), β-myrcene (9.73%), β-ocimene (4.51%), β-costol (3.88%) and thujyl alcohol isomer (2.51%). The efficacy of C. tamala oil as aflatoxin B₁ suppressor is being reported for the first time. Eugenol, the major component of oil showed absolute antiaflatoxigenic efficacy even at 250 μl/l. The high LD₅₀ value (16.94 ml/kg body weight) of oil recorded on mice indicates its non-mammalian toxicity and suggests the recommendation of the oil as a novel and safe post-harvest biological preservative of food commodities for their preservation from contaminating fungi.     

Phytopathogenic organisms and mycotoxigenic fungi: Why do we control one and neglect the other? A biological control perspective in Malaysia

In this review, we present the current information on development and applications of biological control against phytopathogenic organisms as well as mycotoxigenic fungi in Malaysia as part of the integrated pest management (IPM) programs in a collective effort to achieve food security. Although the biological control of phytopathogenic organisms of economically important crops is well established and widely practiced in Malaysia with considerable success, the same cannot be said for mycotoxigenic fungi. This is surprising because the year round hot and humid Malaysian tropical climate is very conducive for the colonization of mycotoxigenic fungi and the potential contamination with mycotoxins. This suggests that less focus has been made on the control of mycotoxigenic species in the genera Aspergillus, Fusarium, and Penicillium in Malaysia, despite the food security and health implications of exposure to the mycotoxins produced by these species. At present, there is limited research in Malaysia related to biological control of the key mycotoxins, especially aflatoxins, Fusarium‐related mycotoxins, and ochratoxin A, in key food and feed chains. The expected threats of climate change, its impacts on both plant physiology and the proliferation of mycotoxigenic fungi, and the contamination of food and feed commodities with mycotoxins, including the discovery of masked mycotoxins, will pose significant new global challenges that will impact on mycotoxin management strategies in food and feed crops worldwide. Future research, especially in Malaysia, should urgently focus on these challenges to develop IPM strategies that include biological control for minimizing mycotoxins in economically important food and feed chains for the benefit of ensuring food safety and food security under climate change scenarios.     

Antifungal Effect of Allium tuberosum,Cinnamomum cassia,and Pogostemon cablin Essential Oils and Their Components Against Population of Aspergillus Species

Antifungal activity of Allium tuberosum (AT), Cinnamomum cassia (CC), and Pogostemon cablin (Patchouli, P) essential oils against Aspergillus flavus strains 3.2758 and 3.4408 and Aspergillus oryzae was tested at 2 water activity levels (a_w: 0.95 and 0.98). Main components of tested essential oils were: allyl trisulfide 40.05% (AT), cinnamaldehyde 87.23% (CC), and patchouli alcohol 44.52% (P). The minimal inhibitory concentration of the plant essential oils against A. flavus strains 3.2758 and 3.4408 and A. oryzae was 250 ppm (A. tuberosum and C. cassia), whereas Patchouli essential oil inhibited fungi at concentration > 1500 ppm. The essential oils exhibited suppression effect on colony growth at all concentrations (100, 175, and 250 ppm for A. tuberosum; 25, 50, and 75 for C. cassia; 100, 250, and 500 for P. cablin essential oil). Results of the study represent a solution for possible application of essential oil of C. cassia in different food systems due to its strong inhibitory effect against tested Aspergillus species. In real food system (table grapes), C. cassia essential oil exhibited stronger antifungal activity compared to cinnamaldehyde.     

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     

Lactic Acid Bacteria as Antifungal and Anti‐Mycotoxigenic Agents: A Comprehensive Review

Fungal contamination of food and animal feed, especially by mycotoxigenic fungi, is not only a global food quality concern for food manufacturers, but it also poses serious health concerns because of the production of a variety of mycotoxins, some of which present considerable food safety challenges. In today's mega‐scale food and feed productions, which involve a number of processing steps and the use of a variety of ingredients, fungal contamination is regarded as unavoidable, even good manufacturing practices are followed. Chemical preservatives, to some extent, are successful in retarding microbial growth and achieving considerably longer shelf‐life. However, the increasing demand for clean label products requires manufacturers to find natural alternatives to replace chemically derived ingredients to guarantee the clean label. Lactic acid bacteria (LAB), with the status generally recognized as safe (GRAS), are apprehended as an apt choice to be used as natural preservatives in food and animal feed to control fungal growth and subsequent mycotoxin production. LAB species produce a vast spectrum of antifungal metabolites to inhibit fungal growth; and also have the capacity to adsorb, degrade, or detoxify fungal mycotoxins including ochratoxins, aflatoxins, and Fusarium toxins. The potential of many LAB species to circumvent spoilage associated with fungi has been exploited in a variety of human food and animal feed stuff. This review provides the most recent updates on the ability of LAB to serve as antifungal and anti‐mycotoxigenic agents. In addition, some recent trends of the use of LAB as biopreservative agents against fungal growth and mycotoxin production are highlighted.     

Aspergillus flavus infection and aflatoxin production in mixtures of high-moisture and dry maize

High-moisture (26·6–27·9% m.c.) and dry (9·8% m.c.) fractions of white and yellow maize were examined for fungal development and aflatoxin production during an 8-week incubation at 25°C. Treatment procedures included blending of either high-moisture white with dry yellow or high-moisture yellow with dry white maize fractions (average moisture in blend, 14%) and inoculation of some test maizes with A. flavus spores. At sampling time white and yellow components of maize blends were manually separated and all of the maize samples were analyzed for levels of moisture, fungal infection and aflatoxin. Moisture levels in maize blends equilibrated rapidly during the initial 2–4 days of incubation; neither dry yellow nor dry white exceeded 13% moisture during the trial period. Only a limited incidence of A. flavus was observed on uninoculated maize. but in samples treated with A. flavus spores a high infection rate developed; from 58 to 98% of the kernels in dry fractions of inoculated blends were infected with A. flavus during the trial. Aflatoxin was detected in high-moisture maize and in both high-moisture and dry fractions of inoculated maize blends. Up to 500 μg aflatoxin B₁/kg of corn was found after the 8-week incubation in a dry fraction of inoculated maize blends.