Fumonisins, carcinogenic mycotoxins produced by various Fusarium species, occur naturally in maize and maize-based food products. They are hazards for animal and human health as they cause cancer in rodents and have been associated with oesophageal cancer and neural tube defects in humans. The most abundant naturally occurring fumonisins analogues in maize are fumonisin B1, B2 and B3 (FB1, FB2 and FB3). For analytical determination, they mostly require suitable extraction, clean-up and pre or post-column derivatization together with reversed-phase HPLC separation. o- Phthaldialdehyde (OPA) had been adopted as the most widely used derivatization reagent for fumonisins as they lack useful chromophores or fluorophores. Alternative derivatization reagents, naphthalene-2,3- dicarboxaldehyde (NDA) and dansyl chloride (DnS-Cl), were investigated in this study

Scopo di questa tesi di dottorato è stato quello di monitorare la presenza di micotossine lungo la filiera produttiva del mais. Inizialmente è stata valutata la contaminazione da micotossine della granella di mais, proveniente da due regioni italiane. Le micotossine, la cui presenza era più estesa e rilevante, erano le fumonisine. Sono state indagate le condizioni di umidità e temperatura più favorevoli alla produzione di AFB1 da parte di un ceppo di Aspergillus flavus. L'ibrido DKC5353 ha presentato le minori contaminazioni, in tutte le località in cui è stato coltivato, per quanto riguarda la contaminazione da fumonisine. Tra i vari trattamenti insetticidi e fungicidi testati per l'efficacia nella riduzione della contaminazione da fumonisine, solo uno ha dato riscontro positivo. È stata valutata la ripartizione delle fumonisine nelle frazioni di molitura e nei sottoprodotti della lavorazione dell'olio di mais. Entrambi i processi si sono dimostrati efficaci metodi di decontaminazione. Dalle analisi sulle farine di mais per polenta emerge la necessità di intensificare i controlli e sorvegliare i punti critici del processo di molitura. / The aim of this study was to monitor the occurrence of mycotoxins over productive cycle of maize. Samples of maize kernels, coming from two Italian regions, were analysed to quantify mycotoxins contamination: the mycotoxins, that showed the most widespread and significant occurrence, were fumonisins. The conditions of humidity and temperature, in which an Aspergillus flavus strain produced the greatest quantity of AFB1, were investigated. Considering fumonisins content, hybrid DKC5353 showed the least contaminations, in all places it was grown. Only one of the various insecticide and fungicide treatments tested was effective. The distribution of fumonisins in several milling fractions and by-products of corn oil manufacture: both processes proved successful methods of decontamination. The necessity of increase controls and monitor the critical points of milling process emerged from the analyses on polenta maize flour.

Fumonisins, carcinogenic mycotoxins produced by various Fusarium species, occur naturally in maize and maize-based food products. They are hazards for animal and human health as they cause cancer in rodents and have been associated with oesophageal cancer and neural tube defects in humans. The most abundant naturally occurring fumonisins analogues in maize are fumonisin B1, B2 and B3 (FB1, FB2 and FB3). For analytical determination, they mostly require suitable extraction, clean-up and pre or post-column derivatization together with reversed-phase HPLC separation. o- Phthaldialdehyde (OPA) had been adopted as the most widely used derivatization reagent for fumonisins as they lack useful chromophores or fluorophores. Alternative derivatization reagents, naphthalene-2,3- dicarboxaldehyde (NDA) and dansyl chloride (DnS-Cl), were investigated in this study

Potential vegetative and reproductive increases in yield, as well as the biological efficacy against Fusarium verticillioides and F. proliferatum causing ear and stem rot in maize crops of commercially-formulated micro-organism formulation T-Gro (Trichoderma harzianum isolate DB103 WP) combined with Spartacus (Beauveria bassiana isolate DB 105 WP), T-Gro combined with Armenius (Bacillus subtilis isolate DB 109 WP), T-Gro combined with Maximus (Bacillus subtilis isolate DB 108 WP), T-Gro combined with Shelter (Bacillus subtilis isolate DB 101), T-Gro combined with Bismarck (Microbacterium maritypicum isolate DB 107 WP), as well as individual treatments of T-Gro, Armenius, Bismarck, Maximus and Shelter, were investigated when applied to maize seed and soil under field conditions. All the micro-organism treatments were compared with Thiram 750WP (750g/kg thiram WP) and an untreated control. The micro-organism treatments showed an increase in vegetative as well as reproductive yields when compared to the reference product Thiram 750 WP and the untreated control. There were no observations of adverse effects on the germination of maize seed in all the treatments that were applied. The three isolates B. subtilis, T. harzianum, and M. maritypicum, showed a significant reduction in vascular tissue discolouration of the main and ear stems, indicating a potential to be used in the reduction and control of diseases caused by Fusarium spp. Results also showed poor to very good increases of stem and foliage biomass as well as cob yield per plant produced by the micro-organism treatments when compared to the untreated control. The highest cob yield per plant that differed significantly from the untreated control was produced by T-Gro and Shelter. No phytotoxicity of any kind was observed with the application of the micro-organism formulations and they could therefore be deemed suitable to be used for the treatment of maize seed. The micro-organism formulations containing fungal and bacterial biological control agents have the potential to be used in commercial maize production to increase vegetative and reproductive yields and reduce the severity of ear and stem rot in maize. / Agriculture Animal Health and Human Ecology / M.Sc. (Agriculture)

O objetivo foi avaliar a expressão dos genes FUM como indicativo da contaminação por Fusarium verticillioides e por fumonisinas nos grãos de milho transgênico durante todo o amadurecimento dos grãos. Constatou-se contaminação fúngica em 100% das amostras. As frequências de Fusarium foram de 3,85% a 100%, sendo 97% F. verticillioides. Foi detectada FB1 em 100% das amostras e FB2 em 18,41%. Os níveis de FB1 variaram de 0,02 mg/g a 5,12 mg/g e FB2 de níveis não detectáveis a 1,74 mg/g. Todos os genes FUM foram amplificados em 33,8% das amostras, e as expressões foram em média entre 3,88 e 7,65 (valores obtidos considerando a amostra calibradora). Não houve correlação positiva entre a porcentagem de F. verticillioides, FB1 e FB2 e os fatores abióticos (p>0,05), o mesmo ocorreu entre a expressão relativa dos genes FUM e os percentuais de isolamento de F. verticillioides e os níveis de FB1 e FB2 (p>0,05), e constatou-se correlação positiva entre a probabilidade de amplificação dos genes FUM e a fase de amadurecimento do grão (p<0,05). / This study evaluated the FUM gene expression in corn grains from sowing to harvest as evidence of Fusarium verticillioides contamination and fumonisins production. All samples (100%) were contaminated with fungi. The isolation frequencies of Fusarium genre ranged from 3,85 % to 100 % and 97 % were F. verticillioides. FB1 was detected in 100% of the samples and FB2 in 18,41 %. The levels of FB1 ranged from 0,02 mg/g to 5,12 mg/g, while FB2 ranged from undetectable levels to 1,74 mg/g. All FUM genes were amplified in 33,8 % of the samples, with average ranging from 3.88 to 7,65, when compared to the calibrator sample. Positive correlation among the F. verticillioides isolation percentage, FB1 e FB2 production and the abiotic factors was not detected (p>0,05), positive correlation was also not detected among the FUM gene cluster relative expressions, F. verticillioides isolation percentage and FB1 and FB2 production (p>0,05). However, a positive correlation was detected between the probability of FUM genes expression and the kernel maturity (p<0,05).

Masters of Science / Fumonisins, carcinogenic mycotoxins produced by various Fusarium species, occur naturally in maize and maize-based food products. They are hazards for animal and human health as they cause cancer in rodents and have been associated with oesophageal cancer and neural tube defects in humans. The most abundant naturally occurring fumonisins analogues in maize are fumonisin B1, B2 and B3 (FB1, FB2 and FB3). For analytical determination, they mostly require suitable extraction, clean-up and pre or post-column derivatization together with reversed-phase HPLC separation. o- Phthaldialdehyde (OPA) had been adopted as the most widely used derivatization reagent for fumonisins as they lack useful chromophores or fluorophores. Alternative derivatization reagents, naphthalene-2,3- dicarboxaldehyde (NDA) and dansyl chloride (DnS-Cl), were investigated in this study. / South Africa

Les fumonisines (FBs) sont les principales mycotoxines produites par Fusarium verticillioides et Fusarium proliferatum, qui se retrouvent partout dans le monde dans le maïs et ses produits dérivés. Les doses toxiques et les signes cliniques de toxicité provoqués par les FBs varient dune espèce à lautre. La toxicité des FBs est généralement liée à leur capacité à bloquer le métabolisme des sphingolipides chez les espèces animales, y compris chez les espèces aviaires. De précédentes études ont démontré que les canards présentent une plus grande sensibilité à la toxicité des FBs que les dindes, alors que laccumulation de sphinganine (Sa) dans les tissues est plus importante chez les dindes que chez les canards. Lobjectif de nos travaux était de comprendre les différences de toxicité entre les dindes et les canards los dune exposition aux FBs. Les trois hypothèses suivantes ont été explorées : i) La toxicocinétique de la fumonisine B2 chez les dindes et les canards. ii) La capacité des cellules aviaires à se protéger de limportante accumulation de sphingolipides libres en augmentant leur catabolisme (phosphorylation). iii) Des mécanismes de toxicité des FBs autre que leur altération via le métabolisme des sphingolipides (stress oxydatif et les réponses inflammatoires). Lanalyse des paramètres de toxicocinétique de la fumonisine B2 na pas mis en évidence de différence significative entre les dindes et les canards. Les mesures de la toxicité simultanée de plusieurs FBs chez les dindes et les canards ont confirmé la forte sensibilité des canards. Laccumulation de shingasine-1-phosphate (Sa1P) dans le foie a également été corrélée avec la quantité de Sa mais pas avec les paramètres hépatiques de toxicité. De plus cette étude a mis en évidence que la quantité de Sa dans le foie était fortement dépendante de la teneur en FBs. Cependant les FBs nont eu aucun effet sur les paramètres de stress oxydatif pour les deux espèces. De manière intéressante, les FBs ont eu une légère réponse inflammatoire chez les canards mais pas chez les dindes. Des investigations plus poussées sur les effets des FBs sur le métabolisme des céramides et sur les processus inflammatoires seraient nécessaires pour comprendre les différences de toxicité entre les dindes et les canards exposés aux FBs. / Fumonisins (FBs) are the major mycotoxins produced by Fusarium verticillioides and Fusarium proliferatum, which are found worldwide in maize and maize products. FBs toxic dose and clinical signs of toxicity vary from one species to another. FBs toxicity is commonly linked to their ability on blocking sphingolipids metabolism in all animal species, including avian species. Previous studies have demonstrated that ducks exhibit higher sensitivity to FBs toxicity than turkeys, whereas, the accumulation of sphinganine (Sa) in tissues is more pronounced in turkeys than in ducks. The objectives of our works were to investigate the causes which lead to different toxicity between ducks and turkeys to FBs exposure. The following three hypotheses were investigated: i) Toxicokinetics of fumonisin B2 in ducks and turkeys. ii) Ability of bird cells to protect themselves against high accumulation of free sphingolipids by increasing their catabolism (phosphorylation). iii) Other toxicity mechanisms of FBs rather than their alteration of sphingolipids metabolism (oxidative stress damage and inflammatory responses). The analysis of toxicokinetic parameters of fumonisin B2 did not provide a significant difference between ducks and turkeys. The measurement of simultaneous toxicity of FBs in ducks and turkeys confirmed higher sensibility of ducks. Also the accumulation of Sphingasine-1-Phosphate (Sa1P) in the liver correlated with the amount of Sa but not parameters of hepatic toxicity. Moreover, this study revealed that the amount of Sa in the liver was strongly dependent on the amount of FBs. On the other hand, FBs had no effect on oxidative damages parameters in both species. Interestingly, FBs had mild inflammatory response effect in ducks but not in turkeys. Further investigation on the effects of FBs on ceramide metabolism and inflammatory processes would be necessary to understand the different toxicity between ducks and turkeys to FBs exposure.

<p>Ispitana&nbsp; je&nbsp; mogućnost ekstrakcije fumonizina iz kukuruza&nbsp;sa vodom i fosfatnim puferom, umesto sa sme&scaron;om&nbsp;acetonitril&ndash;metanol&ndash;voda.&nbsp; Ekstrakcione metode&nbsp;neorganskim&nbsp; rastvaraĉima&nbsp; su&nbsp; uspe&scaron;no&nbsp; primenjene za&nbsp;odreĊivanje&nbsp; fumonizina&nbsp; B₁, B_2&nbsp;i&nbsp; B_3&nbsp;metodom&nbsp; tečne&nbsp;hromatografije sa fluorescentnom detekcijom, kao i&nbsp;ukupnih fumonizina pomoću&nbsp; imunohemijske metode.&nbsp;Analiziran&nbsp; je&nbsp; veći broj&nbsp; uzoraka na&nbsp; prisustvo i sadržaj&nbsp;fumonizina.&nbsp; Ispitani su uzorci&nbsp; kukuruza (235), sakupljeni&nbsp;tokom vi&scaron;egodi&scaron;njeg perioda (2005.&nbsp; i 2009&minus;2013.&nbsp; godine)&nbsp; i&nbsp;p&scaron;enice (83) roda 2010. i 2012. godine, sa područja&nbsp; severne&nbsp;Srbije.&nbsp; Ispitana je kontaminiranost kukuruza sa područja&nbsp;<br />severne Srbije fumonizinima, drugim mikotoksinima i&nbsp;plesnima, kao i mogući uticaj klimatskih faktora na stepen&nbsp;kontaminacije.&nbsp; Rezultati kontaminiranosti useva&nbsp;fumonizinima&nbsp; za svaku godinu&nbsp; pojedinačno&nbsp; su povezivani&nbsp;sa vremenskim prilikama koje su&nbsp; pratile istu. Različita&nbsp;hrana na bazi žitarica je analizirana ELISA metodama.&nbsp;Razvijena je ELISA i metoda&nbsp; tečne hromatografije sa&nbsp;fluorescentnom detekcijom&nbsp; za određivanje fumonizina u tri&nbsp;vrste lekovitog bilja sa područja Srbije.&nbsp; Ispitana je&nbsp;mogućnost&nbsp; primene infracrvene spektroskopije sa&nbsp;Furijeovim transformom&nbsp; za &nbsp;odredivanje&nbsp; fumonizina.&nbsp;Razvijen je&nbsp; ekspertni sistem za re&scaron;avanje problema izbora&nbsp;<br />optimalnog postupka određivanja fumonizina u kukuruzu.</p> / <p>Possibility of fumonisin extraction from maize using water and phosphate buffer instead of acetonitrile-methanol-water mixture was examined. The methods of extraction without organic solvents have successfully been applied for the determination of fumonisins&nbsp; B1, B2 and&nbsp; B3 using&nbsp; liquid chromatography-fluorescence detection&nbsp; method,&nbsp; as well as total fumonisins using enzyme-linked immunosorbent&nbsp; assay&nbsp; (ELISA).&nbsp; Large number of samples was analyzed for the presence and content of fumonisins. Maize samples (total 235) collected throughout several-year period (2005 and 2009-2013) and wheat samples (total 83) from 2010 and 2012 harvest originating from the territory of northern Serbia were analyzed. Contamination of maize originating from the territory of northern Serbia with&nbsp;fumonisins and other mycotoxins and moulds was examined, as well as the potential impact of climatic factors on contamination level.&nbsp; The results on the fumonisin-contamination of cereals obtained for each individual year are related with the climatic&nbsp; conditions characteristic for the relevant year. A variety of cereal-based food was analyzed using ELISA methods. Improved&nbsp; liquid chromatography-fluorescence&nbsp; detection method and ELISA for the determination of fumonisins in three &nbsp;medicinal plant&nbsp; species from the territory of Serbia were developed.&nbsp; Possible application of&nbsp; Fourier transform&nbsp; infrared spectroscopy&nbsp; (FTIR) for quantification of fumonisin was investigated.&nbsp; An expert system&nbsp; to solve the problem of selecting an optimal&nbsp;method for&nbsp; determination of fumonisins in&nbsp; maize has been developed.</p>

>Magister Scientiae - MSc / Subsistence farmers may contribute significantly to food production, food security, and employment in South Africa. However poor storage practices and contamination with mycotoxins, particularly fumonisins and aflatoxins impacts adversely on production, food safety and food security. Mycotoxins are toxic natural food-borne compounds which frequently contaminate agricultural produce worldwide. They are hazardous to humans and animals and result in significant production losses for farmers. This study focused on former Bantustans in Northern South Africa, namely Vhembe District Municipality (Limpopo) and Gert Sibande District Municipality (Mpumalanga). The aim was to assess mycological and mycotoxin contamination of crops grown by subsistence farmers. A semi-structured questionnaire was administered to randomly thirty-nine households. Data on demographics, storage practices and production during period of 2011 and 2012 cropping seasons were collected. One hundred and fifteen (115) crop samples (maize, beans and peanuts) were collected for analysis. Standard mycological methods and validated mycotoxin analysis methods (HPLC and LC- MS/MS) were used. It was found that maize was the staple food in both provinces, with a significant difference (p = 0.0184) in its production between the two districts; Vhembe produced 0.6 tonnes compared to 2.4 tonnes in Gert Sibande. The majority of the farmers for storage used traditional open wooden cribs (15/20) and steel tanks (5/20) while VDM farmers used sealed store houses 5/19 and 15/19 used polystyrene sacks. Aflatoxin occurrence was low with <1% of GSDM samples contaminated compared to 11% of VDM samples. No significant difference (p > 0.05) was observed in the aflatoxin contamination in VDM samples between the year 2011 and 2012. Samples from VDM households had higher Aspergillus fungal infection (maximum incidence 69%) compared to GSDM (27%) over both seasons. The most frequently isolated Fusarium species in VDM samples was F. verticillioides (92%; 93%), and F. subglutinans (97%; 80%) in GSDM samples over seasons 2011 and 2012, respectively. Highest levels of fumonisins (FB1+ FB2) ranged between 1010 μg/kg and 12168 μg/kg with less than 30% extremely contaminated above the regulated limit in 91% of samples from Limpopo over both seasons (2011 and 2012). Fumonisin levels between the two seasons in VDM showed no significant difference (p>0.05). Only three (less than 5%) from 68% GSDM contaminated maize samples were above the FB1 and FB2 limit. In 2011, there were two highly contaminated maize samples (1762 μg/kg and 4598 μg/kg) with the other samples less than 600 μg/kg, whereas in season two (2012) all samples were below 200 μg/kg, except one highly contaminated sample (26115 μg/kg). None of the beans and peanuts from Mpumalanga was contaminated with mycotoxins above the recommended limit, but from Limpopo 1/5 peanuts was found contaminated with aflatoxin G1 (41 μg/kg). Natural occurrence and contamination of both fumonisin and aflatoxin in stored home-grown maize from VDM was significantly (p < 0.0001) higher than GSDM over both seasons. In general, Limpopo farmers’ experience lower harvests and greater mycotoxin contamination of agricultural produce. This may be attributed in part to poor storage practices and environmental and climatic conditions in that agro-ecological zone.

Maize is the principal staple food/feed crop exposed to mycotoxins, and the co-occurrence of multiple mycotoxins and their metabolites has been well documented. Dietary (co)-exposure to mycotoxins is associated with human and animal health concerns as well as economic losses. The present thesis aims to apply a holistic approach for the risk assessment of mycotoxin mixtures in food and feed, i.e. from fungal production and occurrence to harmonised risk characterisation. This was done in three folds. Firstly, available environmental, ecological, and agronomic factors that may affect the relative abundance of co-occurring mycotoxins in the contaminated crops were collected from peer-reviewed literature, with focus on maize (Chapter I). Secondly, (co-)occurrence data on mycotoxins in core cereals was extracted from available articles in the scientific literature and analysed to estimate potential pattern of co-exposure in humans and animals (Chapter II). Finally, Chapter III investigates the applicability of the EFSA guidance to multiple mycotoxins through a scenario of possible co-exposure in humans and animals, using maize as a case study. In particular, a human and animal risk assessment to mycotoxin mixture in maize was conducted using a modelled component-based approach for selected mixture of mycotoxins, that, according to our data, co-occur in maize based feed and food products. / Maize is the principal staple food/feed crop exposed to mycotoxins, and the co-occurrence of multiple mycotoxins and their metabolites has been well documented. Dietary (co)-exposure to mycotoxins is associated with human and animal health concerns as well as economic losses. The present thesis aims to apply a holistic approach for the risk assessment of mycotoxin mixtures in food and feed, i.e. from fungal production and occurrence to harmonised risk characterisation. This was done in three folds. Firstly, available environmental, ecological, and agronomic factors that may affect the relative abundance of co-occurring mycotoxins in the contaminated crops were collected from peer-reviewed literature, with focus on maize (Chapter I). Secondly, (co-)occurrence data on mycotoxins in core cereals was extracted from available articles in the scientific literature and analysed to estimate potential pattern of co-exposure in humans and animals (Chapter II). Finally, Chapter III investigates the applicability of the EFSA guidance to multiple mycotoxins through a scenario of possible co-exposure in humans and animals, using maize as a case study. In particular, a human and animal risk assessment to mycotoxin mixture in maize was conducted using a modelled component-based approach for selected mixture of mycotoxins, that, according to our data, co-occur in maize based feed and food products.