Volatilome of the maize phytopathogenic fungus Fusarium verticillioides: potential applications in diagnosis and biocontrol.

BACKGROUND: Fusarium verticillioides is a maize fungal phytopathogen and a producer of volatile organic compounds (VOCs) and fumonisin B1 (FB1). Our aim was to study the volatilome, conidial production, ergosterol and FB1 biosynthesis in maize cultures over a 30-day incubation period (5, 10, 15, 20, 25, 30 days post inoculation [DPI]). The effect of pure VOCs on the same parameters was then evaluated to study their potential role as biocontrol agents. RESULTS: In total, 91 VOCs were detected, with volatile profiles being more similar between 5 and 10 DPI compared with 15, 20, 25 and 30 DPI. Ergosterol content increased steadily with incubation time, and three growth stages were identified: a lag phase (0 to 15 DPI), an exponential phase (15 to 20 DPI) and a stationary phase (20 to 30 DPI). The maximum concentration of FB1 was detected at 25 (0.030 µg FB1/µg ergosterol) and 30 DPI (0.037 µg FB1/µg ergosterol), whereas conidial production showed a maximum value at 15 DPI (4.3 ± 0.2 × 105 conidia/µg ergosterol). Regarding pure VOCs, minimal inhibitory concentration values ranged from 0.3 mm for 4-hexen-3-one to 7.4 mm for 2-undecanone. Pure VOCs reduced radial growth, conidial production and ergosterol and FB1 biosynthesis. CONCLUSIONS: The marked resemblance between VOC profiles at 5 and 10 DPI suggests that they could act as early indicators of fungal contamination, particularly 4-ethylguaiacol, 4-ethyl-2-methoxyanisole, heptanol and heptyl acetate. On the other hand, their role as inhibitors of fungal growth and FB1 biosynthesis prove their great potential as safer alternatives to control phytopathogenic fungi. © 2024 Society of Chemical Industry.

Sulcatone as a Plant-Derived Volatile Organic Compound for the Control of the Maize Weevil and Its Associated Phytopathogenic Fungi in Stored Maize.

Stored maize is frequently attacked by different pests, such as insects and microorganisms. The aim of the present study was to evaluate the bioactivities of sulcatone (6-methyl-5-hepten-2-one) against the maize weevil Sitophilus zeamais and the phytopathogenic fungi Fusarium verticillioides, Aspergillus flavus, and A. parasiticus. Sulcatone showed a strong repellent effect with a maximum value of -92.1 ± 3.2% at 40 µM in two-choice olfactometer bioassays and an LC95 value of 17.2 µL/L air (95% 16.5-18.1) in a fumigant toxicity experiment. The antifungal effect of sulcatone was evaluated through the fumigant method, reporting MIC values of 3.5, 3.8, and 3.9 mM for F. verticillioides, A. parasiticus, and A. flavus, respectively. Additionally, a silo-bag experiment containing all pests was conducted to evaluate the potential use of sulcatone in a real storage system. Sulcatone caused 71.69 ± 1.57% weevil mortality in silo-bags and proved to be effective as a fungicidal and antimycotoxigenic agent since both ergosterol and fumonisin B1 content were significantly reduced by 60% in silo-bags containing sulcatone. This study demonstrated that sulcatone has the potential to be used for the control of both insects and fungi of stored maize, without affecting the germination of grains.

Exploring Contact Toxicity of Essential Oils against Sitophilus zeamais through a Meta-Analysis Approach.

Sitophilus zeamais is a primary pest of maize. Our aim was to perform a qualitative review and meta-analyses with 56 scientific articles published from 1 January 2000 to 1 October 2022 dealing with direct (topical application) and indirect (impregnation of essential oils, EOs, onto filter paper or maize grains) contact toxicity of EOs against S. zeamais. Three independent meta-analyses of single means of LD50 (direct contact) and LC50 (indirect contact) were conducted using a random effect model. Essential oils more frequently evaluated were those belonging to Asteraceae, Apiaceae, Lamiaceae, Myrtaceae, Piperaceae, and Rutaceae. The LC50 global mean values were 33.19 µg/insect (CI95 29.81-36.95) for topical application; 0.40 µL/cm2 (CI95 0.25-0.65) for filter paper indirect contact; and 0.50 µL/g maize (CI95 0.27-0.90) for maize grains indirect contact. The species Carum carvi, Salvia umbratica, Ilicium difengpi, Periploca sepium, Cephalotaxus sinensis, Murraya exotica, Rhododendron anthopogonoides, Ruta graveolens, Eucalyptus viminalis, Ocotea odorifera, Eucalyptus globulus, Eucalyptus dunnii, Anethum graveolens, Ilicium verum, Cryptocarya alba, Azadirachta indica, Chenopodium ambrosioides, Cupressus semperivens, Schinus molle, Piper hispidinervum, Mentha longifolia, and Croton pulegiodorus showed LC50 or LD50 values lower than the global means, indicating good insecticidal properties. Our results showed that EOs have great potential to be used as bioinsecticides against S. zeamais.

Effect of Carbon Sources on the Production of Volatile Organic Compounds by Fusarium verticillioides.

The aim of the present study was to evaluate the effect of different carbon sources on the hydrocarbon-like volatile organic compounds (VOCs) of Fusarium verticillioides strain 7600 through a Principal Component Analysis approach, and to explore their diesel potential by using data from the literature. The fungus was cultivated in GYAM culture medium, and five carbon sources were evaluated: glucose, sucrose, xylose, lactose, and fructose. The VOCs were collected using a close-loop apparatus and identified through GC-MS. The same profile of 81 VOCs was detected with all treatments, but with different relative percentages among carbon sources. The production of branched-chain alkanes (30 compounds) ranged from 25.80% to 38.64%, straight-chain alkanes (12 compounds) from 22.04% to 24.18%, benzene derivatives (12 compounds) from 7.48% to 35.58%, and the biosynthesis of branched-chain alcohols (11 compounds) was from 6.82% to 16.71%, with lower values for the remaining groups of VOCs. Our results show that F. verticillioides has the metabolic potential to synthesize diesel-like VOCs. Further research should include the optimization of culture conditions other than carbon sources to increase the production of certain groups of VOCs.

Fungal diversity and mycotoxins detected in maize stored in silo-bags: a review.

Silo-bags are hermetic storage systems that inhibit fungal growth because of their atmosphere with low humidity, as well as low pH and O2 concentrations, and a high CO2 concentration. If a silo-bag with stored maize loses its hermetic nature, it favors the development of fungi and the production of mycotoxins. To the best of our knowledge, this is the first review on the diversity of fungal species and mycotoxins that were reported in maize stored under the environmental conditions provided by silo-bags. The genera Penicillium, Aspergillus and Fusarium were found more frequently, whereas Acremonium spp., Alternaria sp., Candida sp., Cladosporium sp., Debaryomyces spp., Epiconum sp., Eupenicillium spp., Eurotium sp., Eurotium amstelodami, Hyphopichia spp., Hyphopichia burtonii, Moniliella sp., Wallemia sp. and genera within the orden Mucorales were reported less recurrently. Despite finding a great fungal diversity, all of the studies focused their investigations on a small group of toxins: fumonisins (FBs), aflatoxins (AFs), deoxynivalenol (DON), zearalenone (ZEA), patulin (PAT), toxin T2 (T2) and ochratoxin (OT). Of the FBs, fumonisin B1 and fumonisin B2 presented higher incidence percentages, followed by fumonisin B3 . Of the AFs, the only one reported was aflatoxin B1. The mycotoxins DON, ZEA and OT were found with lower incidences, whereas PAT and T2 were not detected. Good management practices of the silo-bags are necessary to achieve a hermetically sealed environment, without exchange of gases and water with the external environment during the storage period. © 2022 Society of Chemical Industry.

Chemical composition and antifungal properties of commercial essential oils against the maize phytopathogenic fungus Fusarium verticillioides / Composición química y propiedades antifúngicas de aceites esenciales comerciales contra el hongo fitopatógeno de maíz Fusarium verticillioides

ABSTRACT The aim of the present study was to analyze the chemical composition ofCurcuma longa,Pimenta dioica,Rosmarinus officinalis, andSyzygiumaromaticumessential oils (EOs) and their antifungal and anti-conidiogenic activity againstFusarium verticillioides. The chemical profile of the EOs was determined by GC/MS. The antifungal and anti-conidiogenic activities were evaluated by the agar dilution method. The tested concentrations were 1000ppm, 500ppm, 250ppm and 125ppm.S. aromaticumEO exhibited the highest antifungal effect, followed byP. dioicaand to a lesser extentC. longa. The major compounds of these EOs were eugenol (88.70% inS. aromaticumand 16.70% inP. dioica), methyl eugenol (53.09% inP. dioica), and α-turmerone (44.70%), β-turmerone (20.67%), and Ar-turmerone (17.27%) inC. longa.Rosmarinus officinalispoorly inhibited fungal growth; however, it was the only EO that inhibited conidial production, with its major components being 1,8-cineole (53.48%), α-pinene (15.65%), and (−)-camphor (9.57%). Our results showed that some compounds are capable of decreasing mycelial growth without affecting sporulation, and vice versa. However, not all the compounds of an EO are responsible for its bioactivity. In the present work, we were able to identify different major compounds or mixtures of major compounds that were responsible for antifungal and anti-conidiogenic effects. Further experiments combining these pure components are necessary in order to achieve a highly bioactive natural formulation against the phytopathogenic fungusF. verticillioides.

RESUMEN El objetivo del presente estudio fue analizar la composición química de los aceites esenciales (AE) deCurcuma longa,Pimenta dioica,RosmarinusofficinalisySyzygiumaromaticum, y su actividad antifúngica y anti-esporuladora contraFusarium verticillioides. La composición de los AE se analizó por CG-EM. La actividad antifúngica y anti-esporuladora se evaluó a través del método de dilución en agar usando las siguientes concentraciones: 1.000, 500, 250 y 125ppm. El AE deS. aromaticumdemostró el mayor efecto antifúngico, seguido del deP. dioicay, en menor medida, delC. longa. Los compuestos principales de estos AE fueron eugenol (88,70% enS. aromaticumy 16,70% enP. dioica), metileugenol (53,09% enP. dioica) y α-turmerona (44,70%), β-turmerona (20,67%) y Ar-turmerona (17,27%) enC. longa. El AE deR. officinalisfue el que menor efecto inhibitorio presentó sobre el crecimiento fúngico, pero fue el único que inhibió la producción de conidias; sus principales componentes fueron 1,8-cineol (53,48%), α-pineno (15,65%) y (−)-alcanfor (9,57%). Nuestros resultados demostraron que algunos compuestos son capaces de disminuir el crecimiento del micelio deF. verticillioidessin afectar la esporulación, y vice versa. Sin embargo, no todos los compuestos de un AE son responsables de su bioactividad. En el presente trabajo, pudimos identificar diferentes compuestos o mezclas de compuestos que fueron responsables de los efectos antifúngicos y anti-esporuladores. Se necesitan nuevos experimentos que evalúen la combinación de estos compuestos puros para lograr una formulación bioactiva y de origen natural para el control deF. verticillioides.

Carbon sources to enhance the biosynthesis of useful secondary metabolites in Fusarium verticillioides submerged cultures.

Fusarium verticillioides is a prolific producer of useful secondary metabolites such as naphthoquinone pigments, monoterpenes, and sesquiterpenes, as well as the harmful mycotoxins fumonisins. A strategy to increase their production includes creating a proper nutritional environment that enables the fungus to produce the compounds of interest. The aim of the present work was to study the effect of different carbon sources (glucose, fructose, xylose, sucrose, and lactose) on secondary metabolites biosynthesis in F. verticillioides submerged cultures. The production of volatile terpenes was evaluated through gas chromatography coupled to mass spectrometry. The quantification and identification of pigments was conducted using a UV/VIS spectrophotometer and NMR spectrometer, respectively. The quantification of fumonisin B1 and fumonisin B2 was performed by high-performance liquid chromatography. Our results showed that the biosynthesis of naphthoquinone pigments, monoterpenes, and sesquiterpenes was highest in cultures with fructose (13.00 ± 0.71 mmol/g), lactose [564.52 × 10-11 ± 11.50 × 10-11 µg/g dry weight (DW)], and xylose (54.41 × 10-11 ± 1.55 × 10-11 µg/g DW), respectively, with fumonisin being absent or present in trace amounts in the presence of these carbon sources. The highest biosynthesis of fumonisins occurred in sucrose-containing medium (fumonisin B1: 7.85 × 103 ± 0.25 × 103 µg/g DW and fumonisin B2: 0.38 × 103 ± 0.03 × 103 µg/g DW). These results are encouraging since we were able to enhance the production of useful fungal metabolites without co-production with harmful mycotoxins by controlling the carbon source provided in the culture medium.

Chemical composition and antifungal properties of commercial essential oils against the maize phytopathogenic fungus Fusarium verticillioides.

The aim of the present study was to analyze the chemical composition of Curcuma longa, Pimenta dioica, Rosmarinus officinalis, and Syzygium aromaticum essential oils (EOs) and their antifungal and anti-conidiogenic activity against Fusarium verticillioides. The chemical profile of the EOs was determined by GC/MS. The antifungal and anti-conidiogenic activities were evaluated by the agar dilution method. The tested concentrations were 1000ppm, 500ppm, 250ppm and 125ppm. S. aromaticum EO exhibited the highest antifungal effect, followed by P. dioica and to a lesser extent C. longa. The major compounds of these EOs were eugenol (88.70% in S. aromaticum and 16.70% in P. dioica), methyl eugenol (53.09% in P. dioica), and α-turmerone (44.70%), ß-turmerone (20.67%), and Ar-turmerone (17.27%) in C. longa. Rosmarinus officinalis poorly inhibited fungal growth; however, it was the only EO that inhibited conidial production, with its major components being 1,8-cineole (53.48%), α-pinene (15.65%), and (-)-camphor (9.57%). Our results showed that some compounds are capable of decreasing mycelial growth without affecting sporulation, and vice versa. However, not all the compounds of an EO are responsible for its bioactivity. In the present work, we were able to identify different major compounds or mixtures of major compounds that were responsible for antifungal and anti-conidiogenic effects. Further experiments combining these pure components are necessary in order to achieve a highly bioactive natural formulation against the phytopathogenic fungus F. verticillioides.

Effect of Selected Volatiles on Two Stored Pests: The Fungus Fusarium verticillioides and the Maize Weevil Sithophilus zeamais.

New agronomic practices and technology enabled Argentina a larger production of cereal grains, reaching a harvest yield of 26.5 million metric tons of maize, of which, about 40% was exported. However, much of the maize production is lost annually by the attack of fungi and insects (2.6 million tons). In this study, the antifungal effect of selected volatiles on Fusarium verticillioides, its mycotoxin production, and the repellent and insecticidal activities against the weevill Sithophilus zeamais, an insect vector of F. verticillioides, were evaluated. The compounds tested were (2E)-2-hexenal, (2E)-2-nonenal, (2E,6Z)-2,6-nonadienal, 1-pentanol, 1-hexanol, 1-butanol, 3-methyl-1-butanol, pentanal, 2-decanone, and 3-decanone, which occur in the blend of volatile compounds emitted by various cereal grains. The most active antifungals were the aldehydes (2E)-2-nonenal, (2E)-2-hexenal, and (2E,6Z)-2,6-nonadienal (minimum inhibitory concentration values of <0.03, 0.06, and 0.06 mM, respectively). The occurrence of fumonisin B1 also was prevented because these compounds completely inhibited fungal growth. The best insecticidal fumigant activities against the maize weevil were shown by 2-decanone and 3-decanone (lethal concentration ≤ 54.6 µL/L (<0.28 mM)). Although, all tested compounds showed repellent activity against S. zeamais at a concentration of 4 µL/L, (2E,6Z)-2,6-nonadienal was the most active repellent compound. These results demonstrate the potential of (2E,6Z)-2,6-nonadienal to be used as a natural alternative to synthetic pesticides on F. verticillioides and S. zeamais.

Bioactivities of Ketones Terpenes: Antifungal Effect on F. verticillioides and Repellents to Control Insect Fungal Vector, S. zeamais.

Maize is one the most important staple foods in the world. However, numerous pests, such as fungal pathogens, e.g., Fusarium verticillioides, and insects, such as Sitophlilus zeamais, attack maize grains during storage. Many F. verticillioides strains produce fumonisins, one of the most important mycotoxin that causes toxic effects on human and animal health. This situation is aggravated by the insect fungal vector, Sitophlilus zeamais, which contributes to the dispersal of fungal spores, and through feeding damage, provide entry points for fungal infections. The aim of this study was to evaluate in vitro bioassays, the antifungal activity on F. verticillioides M3125 and repellent effects against S. zeamais of ketone terpenes. In addition, we performed Quantitative structure-activity relationship (Q-SAR) studies between physico-chemical properties of ketone terpenes and the antifungal effect. Thymoquinone was the most active compound against F. verticillioides (Minimum Inhibitory Concentration, MIC: 0.87) affecting the lag phase and the growth rate showing a total inhibition of growth at concentration higher than 2 mM (p < 0.05). The Q-SAR model revealed that the antifungal activity of ketone compounds is related to the electronic descriptor, Pi energy. Thymoquinone showed a strong repellent effect (-77.8 ± 8.5, p < 0.001) against S. zeamais. These findings make an important contribution to the search for new compounds to control two stored pests of maize.

Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B1 adsorption ability for use in poultry feedstuffs.

In this study the aflatoxin B1 (AFB1) removal capacity, the tolerance to salivary and gastrointestinal conditions, autoaggregation and coaggregation with pathogenic bacteria of Saccharomyces cerevisiae strains isolated from broiler feces, were evaluated. Only four of twelve isolated strains were identified as Saccharomyces cerevisiae using molecular techniques. The results obtained in AFB1 binding studies indicated that the amount of AFB1 removed was both strain and mycotoxin-concentration dependent. Therefore, a theoretical model was applied in order to select the most efficient strain to remove AFB1 in a wide range of mycotoxin concentration. The results indicated that S. cerevisiae 08 and S. cerevisiae 01 strains were the most efficient microorganisms in the mycotoxin removal. Viability on simulated salivary and gastrointestinal conditions was investigated and S. cerevisiae 08 strain showed the best results, achieving 98% of total survival whereas S. cerevisiae 01 reached only 75%. Autoaggregation and coaggregation assays showed S. cerevisiae 08 as the most appropriate strain, mainly because it was the unique strain able to coaggregate with the four bacterial pathogens assayed. Consequently, S. cerevisiae 08 is the best candidate for future in vivo studies useful to prevent aflatoxicosis. Further quantitative in vitro and in vivo studies are required to evaluate the real impact of yeast-binding activity on the bioavailability of AFB1 in poultry. However, this study could be useful in selecting efficient strains in terms of AFB1 binding and provide an important contribution to research into microorganisms with potential probiotic effects on the host.

Analysis of fumonisin B1 removal by microorganisms in co-occurrence with aflatoxin B1 and the nature of the binding process.

The objectives of this investigation were to evaluate the ability of Saccharomyces cerevisiae CECT 1891 and Lactobacillus acidophilus 24 to remove fumonisin B(1) (FB(1)) from liquid medium; to determine the nature of the mechanism involved in FB(1)-microorganism interaction and to analyze whether the presence of aflatoxin B(1) (AFB(1)) interferes with the removal of FB(1) and vice versa. The results obtained indicated that: (i) both microorganisms were able to remove FB(1) from liquid medium; (ii) the removal was a fast and reversible process; (iii) cell viability was not necessary; (iv) the amount of FB(1) removed was both toxin- and microorganism concentration-dependent; (v) the process did not involve chemical modification of FB(1) molecules; and (vi) cell wall structural integrity of the microorganisms was required for FB(1) removal. Consequently, we propose that the mechanism involved in the removal of FB(1) is a physical adsorption (physisorption) of the toxin molecule to cell wall components of the microorganisms. It is highly probable that FB(1) and AFB(1) co-occur in contaminated foods, since the fungal genera Aspergillus and Fusarium frequently occur simultaneously. Therefore, we analyzed whether the presence of AFB(1) interferes with the removal of FB(1) by the microorganisms previously evaluated, and vice versa. Studies of co-occurrence of both mycotoxins clearly showed that they did not compete for binding sites on the microorganism cell wall and the presence of one toxin did not modify the efficiency of the organism in the removal of the other mycotoxin. These findings may be useful for optimization of mycotoxin binding and provide an important contribution to research on microorganisms with ability to remove these secondary metabolites.

Physical adsorption of aflatoxin B1 by lactic acid bacteria and Saccharomyces cerevisiae: a theoretical model.

The ability of lactic acid bacteria (LAB) and Saccharomyces cerevisiae to remove aflatoxin B1 (AFB1) from liquid medium was tested. The experimental results indicated that (i) AFB1 binding to microorganisms was a rapid process (no more than 1 min); (ii) this binding involved the formation of a reversible complex between the toxin and microorganism surface, without chemical modification of the toxin; (iii) the amount of AFB1 removed was both toxin- and bacteria concentration-dependent; and (iv) quantitatively similar results were obtained with viable and nonviable (heat-treated) bacteria. According to these details, a physical adsorption model is proposed for the binding of AFB1 to LAB and S. cerevisiae, considering that the binding (adsorption) and release (desorption) of AFB1 to and from the site on the surface of the microorganism took place (AFB1 + S <--> S - AFB1). The model permits the estimation of two parameters: the number of binding sites per microorganism (M) and the reaction equilibrium constant (K(eq)) involved, both of which are useful for estimating the adsorption efficiency (M x K(eq)) of a particular microorganism. Application of the model to experimental data suggests that different microorganisms have similar K(eq) values and that the differences in toxin removal efficiency are mainly due to differences in M values. The most important application of the proposed model is the capacity to select the most efficient microorganism to remove AFB1. Furthermore, it allows us to know if a modification of the adsorption efficiency obtained by physical, chemical, or genetic treatments on the microorganism is a consequence of changes in M, K(eq), or both.