Encapsulation of Ammoides pusila Essential Oil into Mesoporous Silica Particles for the Enhancement of Their Activity against Fusarium avenaceum and Its Enniatins Production.

Essential oils (EOs) that have antifungal activity and mycotoxin reduction ability are candidates to develop bioactive alternatives and environmentally friendly treatment against Fusarium species in cereals. However, their practical use is facing limitations such as high volatility, UV sensitivity, and fast oxidation. Encapsulation techniques are supposed to provide protection to the EOs and control their release into the environment. Ammoides pusilla essential oil (AP-EO) proved to be an efficient inhibitor of Fusarium avenaceum growth and its enniatins (ENNs) production. In the present work, AP-EO was encapsulated, using the impregnation method, into mesoporous silica particles (MSPs) with narrow slit pores (average diameter = 3.1 nm) and coated with chitosan. In contact assays using an agar medium, the antifungal activity of AP-EO at 0.1 µL mL-1 improved by three times when encapsulated into MSPs without chitosan and the ENNs production was significantly inhibited both in coated and non-coated MSPs. Controls of MSPs also inhibited the ENNs production without affecting the mycelial growth. In fumigation experiments assessing the activity of the EO volatile compounds, encapsulation into MSPs improved significantly both the antifungal activity and ENNs inhibition. Moreover, coating with chitosan stopped the release of EO. Thus, encapsulation of an EO into MSPs improving its antifungal and antimycotoxin properties is a promising tool for the formulation of a natural fungicide that could be used in the agriculture or food industry to protect plant or food products from the contamination by toxigenic fungi such as Fusarium sp. and their potential mycotoxins.

Ammoides pusilla Essential Oil: A Potent Inhibitor of the Growth of Fusarium avenaceum and Its Enniatin Production.

Enniatins are mycotoxins produced by Fusarium species contaminating cereals and various agricultural commodities. The co-occurrence of these mycotoxins in large quantities with other mycotoxins such as trichothecenes and the possible synergies in toxicity could lead to serious food safety problems. Using the agar dilution method, Ammoides pusilla was selected among eight Tunisian plants for the antifungal potential of its essential oil (EO) on Fusarium avenaceum mycelial growth and its production of enniatins. Two EO batches were produced and analyzed by GC/MS-MS. Their activities were measured using both contact assays and fumigant tests (estimated IC50 were 0.1 µL·mL-1 and 7.6 µL·L-1, respectively). The A. pusilla EOs and their volatiles inhibited the germination of spores and the mycelial growth, showing a fungistatic but not fungicidal activity. The accumulation of enniatins was also significantly reduced (estimated IC50 were 0.05 µL·mL-1 for the contact assays and 4.2 µL·L-1 for the fumigation assays). The most active batch of EO was richer in thymol, the main volatile compound found. Thymol used as fumigant showed a potent fungistatic activity but not a significant antimycotoxigenic activity. Overall, our data demonstrated the bioactivity of A. pusilla EO and its high potential to control F. avenaceum and its enniatins production in agricultural commodities.

Characterization of Fusarium acuminatum: A Potential Enniatins Producer in Tunisian Wheat.

Fusarium Head Blight (FHB), caused by multiple species of Fusarium in small grain cereals, is a significant and long-standing problem anywhere in the world. Knowing regional Fusarium spp. present on non-symptomatic grains and their potential for mycotoxin production is of concern for identifying novel actions for FHB and mycotoxin management, such as treatments with essential oils. Analyzing the mycotoxin content of grains from non-symptomatic ears of different wheat varieties cultivated in Tunisia, we isolated Fusaria specimens identified as F. culmorum and F. acuminatum using analysis of the partial DNA sequence of the ß-tubulin gene and ITS region. Two isolates of the latter species, uncommon in cereal grains in this region until now, were shown to be effective producers of enniatins in vitro, with 1390 and 3089 µg g-1 mycelial biomass (dry) in 11-day-old cultures. The susceptibility of an isolate of F. acuminatum to the fungistatic and antimycotoxin effects of eight essential oils was measured. Essential oils from Ammoides pusilla and Thymus capitatus used at 0.1 µL mL-1 in an agar culture medium, affected the mycelial growth by 55% and 79%, respectively and reduced the accumulation of enniatins per unit of mycelial colony by 26% and 52%, respectively. Finally, F. acuminatum was shown to be a contaminant of wheat grains in Tunisia and it may contribute to the contamination in enniatins. Two essential oils of Tunisian plants could be used for developing a biofungicide limiting both its mycelial growth and its accumulation of mycotoxins in grains.

Induction of Fusarium lytic Enzymes by Extracts from Resistant and Susceptible Cultivars of Pea (Pisum sativum L.).

Being pathogenic fungi, Fusarium produce various extracellular cell wall-degrading enzymes (CWDEs) that degrade the polysaccharides in the plant cell wall. They also produce mycotoxins that contaminate grains, thereby posing a serious threat to animals and human beings. Exposure to mycotoxins occurs through ingestion of contaminated grains, inhalation and through skin absorption, thereby causing mycotoxicoses. The toxins weaken the host plant, allowing the pathogen to invade successfully, with the efficiency varying from strain to strain and depending on the plant infected. Fusariumoxysporum predominantly produces moniliformin and cyclodepsipeptides, whereas F. proliferatum produces fumonisins. The aim of the study was to understand the role of various substrates and pea plant extracts in inducing the production of CWDEs and mycotoxins. Additionally, to monitor the differences in their levels when susceptible and resistant pea plant extracts were supplemented. The cultures of F. proliferatum and F. oxysporum strains were supplemented with various potential inducers of CWDEs. During the initial days after the addition of substrates, the fungus cocultivated with pea extracts and other carbon substrates showed increased activities of ß-glucosidase, xylanase, exo-1,4-glucanase and lipase. The highest inhibition of mycelium growth (57%) was found in the cultures of F. proliferatum strain PEA1 upon the addition of cv. Sokolik extract. The lowest fumonisin content was exhibited by the cultures with the pea extracts and oat bran added, and this can be related to the secondary metabolites and antioxidants present in these substrates.

Multimycotoxin Determination in Tunisian Farm Animal Feed.

Mycotoxins presence was evaluated in animal feed marketed in Tunisia for the first time ever. A QuEChERS method was performed to analyze the natural copresence of 22 mycotoxins (enniatins, beauvericin, ochratoxin A, aflatoxins, alternariol monomethyl ether, alternariol, tentoxin, zearalenone, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, neosolaniol, diacetoxyscirpenol, T-2 toxin, and HT-2 toxin) in 122 Tunisian marketed feed samples, intended for poultry (n = 43), cattle (n = 35), rabbit (n = 12), sheep (n = 16), and horse (n = 16). Analytes detection and quantification were done using both liquid chromatography and gas chromatography coupled to tandem mass spectrometry. The analytical method showed good linearity (R > 0.996) and sensitivity, the limits of quantification ranged from 0.1 ng/g (enniatin A1) to 225 ng/g (3-acetyldeoxynivalenol). Eighty-five percent of the analyzed samples were positive. Poultry (n = 43) and rabbit (n = 12) feed samples were the most contaminated. Enniatin B was the most prevalent mycotoxin with values ranged between 0.5 ng/g for horse feed and 40 ng/g for poultry feed, followed by deoxynivalenol detected from 16 ng/g in cattle feed to 250 ng/g in poultry feed. None exceeded the limits set by EU recommendations for animal feed. Mycotoxins co-occurrence was observed at most by five different mycotoxins (26%) and up to eight mycotoxins was recorded in 5% of samples. Furthermore, a relatively high copresence rate of different fusariotoxins was registered. Even if no toxicological concern was clearly revealed, the contamination is a real fact and will probably present influence on meat production and on food safety.

Universal direct PCR amplification system: a time- and cost-effective tool for high-throughput applications.

Taking into account the limits of current genotyping methodologies, we have established a versatile direct PCR method on intact microtissue samples without prior DNA isolation. A simple and standard protocol was developed and validated on a wide range of living organisms including bacterial and fungal strains, plant species and human samples. This allows reliable amplification of target genomic DNA fragment directly from source material using minimal amount of tissue which makes DNA purification irrelevant for a number of biological applications. The direct PCR technique established here represents an excellent alternative to traditional amplification methods used for real-time detection. Since this approach was efficiently and universally applied for high-throughput molecular screening, its implementation will offer new insights for several investigations in human health, biomedical diagnosis, plant biotechnology, as well as in applied environmental and food microbiology.

Changes in the Fusarium Head Blight Complex of Malting Barley in a Three-Year Field Experiment in Italy.

In this study, conducted for three years on eleven malting barley varieties cultivated in central Italy, the incidence of different mycotoxigenic fungal genera, the identification of the Fusarium species associated with the Fusarium Head Blight (FHB) complex, and kernels contamination with deoxynivalenol (DON) and T-2 mycotoxins were determined. The influence of climatic conditions on Fusarium infections and FHB complex composition was also investigated. Fusarium species were always present in the three years and the high average and maximum temperatures during anthesis mainly favored their occurrence. The FHB complex was subject to changes during the three years and the main causal agents were F. poae, F. avenaceum, F. tricinctum and F. graminearum, which, even if constantly present, never represented the principal FHB agent. The relative incidence of Fusarium species changed because of climatic conditions occurring during the seasons. The FHB complex was composed of many different Fusarium species and some of them were associated with a specific variety and/or with specific weather parameters, indicating that the interaction between a certain plant genotype and climatic conditions may influence the presence of Fusarium spp. causing infections. With regard to mycotoxin contamination, T-2 toxin, in some cases, was found in kernels at levels that exceeded EU recommended values.

Evaluation of Alternaria mycotoxins in strawberries: quantification and storage condition.

Alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) are Alternaria mycotoxins produced by the most common post-harvest pathogens of fruits. The production of these metabolites depends on several environmental factors, mainly temperature, water activity, pH and the technological treatments that have been applied to the product. In this study, the occurrence of AOH, AME and TEN was evaluated in strawberries samples stored at different temperatures ranges (at 22 ± 2 or 6 ± 2°C) and different periods (up to 1 month) simulating the current practice of consumer's storage conditions. Sample extraction was performed using a liquid-liquid extraction method prior to LC-MS/MS analysis. AOH was the most prevalent mycotoxins with a 42% at strawberries stored at (22 ± 2)°C and 37% stored at (6 ± 2)°C. The highest AOH levels were found in samples conserved at (22 ± 2)°C ranging between 26 and 752 ng g(-1). AME levels ranged between 11 and 137 ng g(-)(1), which were found mainly in stored samples at (6 ± 2)°C for more than 28 days. None sample presented levels of TEN in either of the studied conditions.

Presence of mycotoxins in sorghum and intake estimation in Tunisia.

Sorghum samples (n = 60) from Tunisian markets were analysed for the occurrence of 22 of both traditional and emerging mycotoxins. Samples were extracted with a QuEChERS-like method and mycotoxins were detected by LC-MS/MS. This method was validated and adequate analytical parameters were obtained. All samples had contamination with mycotoxins and several samples had higher contamination levels than European Union legislative limits (MLs). The most frequently found mycotoxins were ENB (100%), OTA (98%), ENA1 (63%), ENB1 (56%), BEA (48%), AFB1 (38%) and STG (33%). Mean contaminations were 30.7, 1.93, 33.2, 51.0, 15.4, 1.49 and 20.5 µg kg(-1), respectively. While two samples were contaminated with FB2 and FB3 at mean values of 16.2 and 45.9 µg kg(-1), respectively, one sample was contaminated with AFB2 and ZEA at levels of 0.82 and 45.0 µg kg(-1), respectively. The results were used to estimate the daily intake of mycotoxins through sorghum consumption with regard to normal consumers (low-risk population) and high consumers such as babies (high-risk consumers) who are facing an alarming situation.

Multi-mycotoxin determination in cereals and derived products marketed in Tunisia using ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry.

The aim of the study was the use of a fast and simple method using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the simultaneous determination of aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), fumonisins (FB1 and FB2), deoxynivalenol (DON), T2 and HT2 toxins in 58 samples of raw wheat (n = 34), barley (n = 5), sorghum (n = 3), processed wheat (n = 13) and breakfast cereals (n = 3) from Tunisian markets. The frequency of contamination of total samples with the analyzed mycotoxins was 50%. AFG2 was the most frequently detected in 11 samples (4 wheat, 4 barley and 3 sorghum) and it was detected at 5.2-52.4 µg/kg. HT2 toxin contaminated seven samples (4 wheat and 3 barley) and it was detected at 5.0-11.1 µg/kg. FB2 was detected in one wheat, sorghum, semolina and breakfast cereal samples at 5.0-61.5 µg/kg. FB1 was detected in three samples (2 sorghum and one barley) at 6.4-120 µg/kg. AFB1 was only found in two sorghum samples at 14 and 79.9 µg/kg. OTA was detected in one sorghum sample at concentrations below limit of quantification (5 µg/kg). The analytical results also showed that all the analyzed samples were not contaminated with DON, AFG1, AFB2 and T2 toxin.

Water activity and temperature effects on fungal growth and ochratoxin A production by ochratoxigenic Aspergillus carbonarius isolated from Tunisian grapes.

This study investigated the effects of temperature (15 to 37 degrees C) and water activity (0.90 to 0.99) on the growth and production of ochratoxin A (OTA) by Aspergillus carbonarius cultured on synthetic nutrient medium (SNM) after 5 and 10 d of incubation. Total of 8 ochratoxigenic A. carbonarius, isolated from vineyards located in different regions of Tunisia, were used. Growth data were modeled by the flexible model of Baranyi and growth rates at each set of conditions were obtained. For both growth and OTA production, optimal water activity was 0.99; however, optimal temperature varied. The optimal temperature for growth was 30 degrees C. At 37 degrees C, the growth rate decreased significantly (P < 0.05). Maximum toxin production occurred at temperatures in the range of 15 to 25 degrees C with the optimum one depending on the isolate tested. Significant amounts of OTA were produced after only 5 d of incubation. Our results showed that A. carbonarius isolated from Tunisian grapes behave as those from European and Australian grapes, as reported in the literature, although some differences in trends for growth and OTA production were observed.

Alternating temperatures and photoperiod effects on fungal growth and Ochratoxin A production by Aspergillus carbonarius isolated from Tunisian grapes.

The effect of three alternating temperatures cycles (20/30, 20/37 and 25/42 degrees C) and photoperiod on growth and Ochratoxin A (OTA) production of six isolates of Aspergillus carbonarius on synthetic nutrient medium were investigated. The different temperature regimes affected significantly both the mycelial growth and the OTA production. The best growth and OTA production were recorded at 20/30 degrees C. The isolates from the region of Baddar produced the highest OTA yields. A 24 h light cycle generally enhanced the growth of A. carbonarius. Growth rates cycles of 11 h/13 h light/darkness and 24 h darkness were often similar for individual isolates, such conditions enhanced OTA production in two of the six isolates tested.