Le micotossine sono metaboliti tossici prodotti da funghi in grado di svilupparsi sulle derrate alimentari. Diverse strategie sono state considerate per risolvere questo problema studiando la crescita di funghi/produzione di micotossine. Questa tesi è focalizzata sullo sviluppo/validazione di modelli matematici per prevedere la contaminazione di micotossine (deossinivalenolo, fumonisine e aflatossine) in cereali (mais/grano) sulla base di dati meteorologici. Il primo capitolo fornisce un’introduzione sulla teoria dei modelli e sui pat-sistemi modelizzati. Il secondo si concentra sulla presenza di tricoteceni e zearalenone nel frumento coltivato in Italia. Nel cap.3 sono stati confrontate le differenze predittive di modelli empirici/meccanicistici per la contaminazione di deossinivalenolo nel grano. Nel cap.4 è stata descritta la contaminazione da fumonisine e aflatossine in mais coltivato in Italia. I capitoli 5 e 6 analizzano il pato-sistema mais-Aspergillus flavus, il primo si concentra sulla sporulazione di A. flavus , il secondo sullo sviluppo di un modello per prevedere la contaminazione da aflatossina. Un altro modello meccanicistico per prevedere la presenza di fumonisina nel mais è descritto nel cap.7. L'ultimo capitolo riassume l'attività svolta nel progetto europeo MYCORED in cui sono stati coinvolti diversi paesi in tutto il mondo che hanno fornito i dati necessari per la validazione dei modelli. / Mycotoxin are toxic secondary metabolite produced by fungi able to colonize crops and thus posing a potential menace to human/animal health. Several strategies have been considered to mitigate the problem studying the variables related to mould growth and mycotoxin production. This thesis focuses on the development and validation of mechanistic models to predict mycotoxins (deoxinivalenol, fumonisins and aflatoxins) contamination in cereals (maize/ wheat) based on meteorological data. The first chapter introduce modelling theory, and patho-systems analysed. Chapter 2 focuses on trichothecenes and zearalenone occurrence in wheat produced in Italy. Predictive performance of empirical and mechanistic models for deoxnivalenol contamination in wheat were discussed in chapter 3. Chapter 4 described fumonisins and aflatoxins occurrence in maize grown in Italy. Chapters 5 and 6 analised the patho-system maize-Aspergillus flavus; the former focuses on the dynamics of A. flavus sporulation the lalatter on the development of a mechanistic model to predict aflatoxin produced by A. falvus. Another mechanistic model for Fusarium ear rot and fumosin production in maize (chapter 7). The last chapter summarised the activity done in the European project MYCORED in which several countries worldwide were involved and wheat and/or maize samples collected with data necessary for model validation.

A survey involving 71 farmers from rural communities in Mpumalanga, South Africa was conducted to gather information regarding the importance and utilisation of cowpea. Cowpea was rated third most important in contributing to household security, preceded by maize and vegetable production. Cowpea was mainly produced for own consumption, as a source of income and as fodder for livestock to a lesser extent. The crop was used by 8.5% of the farmers for medicinal purposes. Results indicated that 20% of the farmers encountered problems with mouldiness during storage, with insect infestation to a lesser degree. Cowpea seed samples from South Africa and Benin, West Africa were analysed for seed mycoflora and various fungal genera, particularly Aspergillus, Phoma and Lasiodiplodia were recorded. The results indicated an array of Fusarium spp. including F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. scirpi, F. semitectum and F. subglutinans. The seed samples and the F. proliferatum isolates, cultured on maize patty media, were analysed for fumonisin production. Samples were extracted with methanol/water (70:30) and cleaned-up on strong anion exchange solid phase extraction cartridges. High-performance liquid chromatography with pre-column derivatisation using o-phthaldialdehyde was used for the detection and quantification of FB1, FB2 and FB3. The cowpea cultivars from South Africa had levels of FB1 ranging between 0.12 – 0.61 µg/g. All the F. proliferatum isolates produced FB1, FB2 and FB3 with total fumonisin concentration levels between 0.80 - 25.30 µg/g. The highest level of FB1 detected was 16.86 µg/g. Surface-disinfected seeds were imbibed in sterile distilled water amended with FB1 to yield final concentrations of 10, 25, 50 and 100 ppm. Percentage germination was determined in paper towels according to the International Seed Testing Association (ISTA) rules. Root and shoot length was measured after 9 days. All the toxin concentrations significantly decreased seed germination whilst root and shoot elongation was inhibited by the 50 and 100 ppm concentrations. Embryonic seed tissue treated with FB1 indicated compaction of the protoplasm and separation of the plasma membrane from the cell wall. Lipid bodies accumulated and seemed to line the cell wall. Acetone and ethanol extracts of the leaves of two cowpea cultivars exhibited significant inhibition of the growth of fungal plant pathogens at 5.0 mg/ml, with the exception of Fusarium equiseti. The growth of some fungi, in particular Alternaria alternate, was also reduced by lower concentrations of certain extracts. Acetone extracts of the Bechwana White cultivar inhibited growth of Staphylococcus aureus and Enterococcus faecalis at 2.5 mg/ml and Bacillus cereus, B. subtilis and Enterobacter cloacae at 5.0 mg/ml. Ethanol extracts of the same cultivar showed antibacterial activity against E. faecalis and E. cloacae at 5.0 mg/ml. This study represents the first report on the natural occurrence of fumonisins on cowpea seed and the potential of F. proliferatum isolates from cowpea seed to produce fumonisins. The phytotoxic effects of FB1 on cowpea seeds as well as the antimicrobial potential of cowpea leaf extracts were demonstrated for the first time. / Thesis (PhD)--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

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Propomos avaliar a influência da rotação de culturas na infecção de milho transgênico (Bt) e cobertura morta por F. verticillioides e na presença de fumonisinas em amostras provenientes de Palmital e Capão Bonito. O isolamento fúngico da cobertura morta e do milho foi realizado em DG18 e DRBC, respectivamente. Os fungos isolados pertencentes ao gênero Fusarium foram identificados até espécie. Utilizamos, para análise de fumonisinas, Cromatografia Líquida de Alta Eficiência. Micobiota da cobertura morta revelou maior frequência de Cladosporium spp. Nas amostras de milho, F. verticillioides foi o fungo mais isolado. Dentro do gênero Fusarium, a espécie F. verticillioides foi a mais frequente. Análise micotoxicológica do milho revelou a presença de fumonisinas em 88,9% das amostras provenientes de Palmital e em 86,1% de Capão Bonito. Dos isolados de F. verticillioides 84,6% foram produtores de FB1 + FB2. A presença de F. verticillioides e fumonisinas nos grãos de milho estabelece um problema econômico e de saúde pública. / The objective of this study was to evaluate the influence of crop rotation systems on the infection of maize grains and mulch by F. verticillioides and on the presence of fumonisins in maize samples collected in Palmital and Capão Bonito. Surface seeding (DG18) and direct seeding (DRBC) were employed for fungal isolations from mulch and maize, respectively. Identified isolates belonging to the genus Fusarium were further classified at the species level. Analysis of the mulch mycobiota revealed a higher frequency of Cladosporium spp., whereas in maize samples, F. verticillioides was the most frequently isolated fungus. High-performance liquid chromatography (HPLC) was used for mycotoxicological analysis of the grain samples. Maize from Palmital and Capão Bonito presented fumonisins in 88.9% and 86.1% of the samples, respectively. From the F. verticillioides isolates, 84.6% were producing FB1 + FB2. The high incidence of F. verticillioides in maize grains represents a serious problem, due to its potential in causing diseases in humans and animals.

Thesis (D.Tech.: Clinical Technology)-Durban Institute of Technology, 2005 xxiv, 235 leaves ; ill. ; 30 cm / Fumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They ex¬ert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancer-promoting metabo¬lites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunomodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL –1, IL-6, IL-8, IL-10 and TNF-) were determined using ELISA kits.

Thesis (D.Tech.: Clinical Technology)-Durban Institute of Technology, 2005 xxiv, 235 leaves ; ill. ; 30 cm / Fumonisin B1 (FB1) and ochratoxin A (OTA) represent examples of mycotoxins of greatest public health and agro-economic significance. They ex¬ert adverse effects on humans, animals and crops that result in illnesses and economic losses. Fumonisin B1 are cancer-promoting metabo¬lites of Fusarium proliferatum and F verticillioides, (formerly moniliforme), and are implicated in oesophageal cancer. Ochratoxins are metabolites of both Aspergillus and Penicillium species. These compounds are known for their nephrotoxic effects in all animal species and may promote tumours in humans. In man OTA exhibits unusual toxicokinetics, with a half-life in blood of 840 h (35 days) after oral ingestion. Although much is known regarding the toxicology of these toxins, little is known of the effects of these toxins on the immune system. The aim of this study was to determine and compare the immunomodulating effects of FB1 and OTA in human carcinoma. Initial experiments involved isolating lymphocytes and neutrophils from healthy volunteers. The isolated cells were exposed to either FB1 or OTA on a dose and time dependent level and LD50 of the toxins was determined. Thereafter, challenge tests were performed, whereby lymphocytes and neutrophils isolated from volunteers, oesophageal cancer patients and breast cancer patients were exposed to the LD50 dose of either FB1 or OTA for the appropriate time. The effect of the toxins was demonstrated by viability studies, light microscopy and electron microscopy. Cytokine receptors (CK, TNF and CSF) were evaluated by immuno-cytochemical methods and the levels of circulating cytokines (IL –1, IL-6, IL-8, IL-10 and TNF-) were determined using ELISA kits.

Propomos avaliar a influência da rotação de culturas na infecção de milho transgênico (Bt) e cobertura morta por F. verticillioides e na presença de fumonisinas em amostras provenientes de Palmital e Capão Bonito. O isolamento fúngico da cobertura morta e do milho foi realizado em DG18 e DRBC, respectivamente. Os fungos isolados pertencentes ao gênero Fusarium foram identificados até espécie. Utilizamos, para análise de fumonisinas, Cromatografia Líquida de Alta Eficiência. Micobiota da cobertura morta revelou maior frequência de Cladosporium spp. Nas amostras de milho, F. verticillioides foi o fungo mais isolado. Dentro do gênero Fusarium, a espécie F. verticillioides foi a mais frequente. Análise micotoxicológica do milho revelou a presença de fumonisinas em 88,9% das amostras provenientes de Palmital e em 86,1% de Capão Bonito. Dos isolados de F. verticillioides 84,6% foram produtores de FB1 + FB2. A presença de F. verticillioides e fumonisinas nos grãos de milho estabelece um problema econômico e de saúde pública. / The objective of this study was to evaluate the influence of crop rotation systems on the infection of maize grains and mulch by F. verticillioides and on the presence of fumonisins in maize samples collected in Palmital and Capão Bonito. Surface seeding (DG18) and direct seeding (DRBC) were employed for fungal isolations from mulch and maize, respectively. Identified isolates belonging to the genus Fusarium were further classified at the species level. Analysis of the mulch mycobiota revealed a higher frequency of Cladosporium spp., whereas in maize samples, F. verticillioides was the most frequently isolated fungus. High-performance liquid chromatography (HPLC) was used for mycotoxicological analysis of the grain samples. Maize from Palmital and Capão Bonito presented fumonisins in 88.9% and 86.1% of the samples, respectively. From the F. verticillioides isolates, 84.6% were producing FB1 + FB2. The high incidence of F. verticillioides in maize grains represents a serious problem, due to its potential in causing diseases in humans and animals.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this research was to evaluate variability and performance of two maize sampling plans for mycotoxins quantification. Eleven maize lots were sampled using two distinct sampling methods: manual, using a sampling spear, and automatic, using a continuous flow sampling. Total variability and variability from each step of the analysis procedure (sampling, sample preparation, and analysis) for results of aflatoxins, fumonisin, and zearalenone were determined in 8, 11, and 5 lots respectively for each sampling plan. Those variances were compared, for each mycotoxin, between the two sampling plans using a multifactor analysis of variance. Distribution of the quantification results for aflatoxins and zearalenone were compared among four theoretical distribution models. The acceptance/rejection probabilities of a lot with determined aflatoxins or zearalenone concentration were determined using calculated variances and the information on the selected distribution to plot an operation characteristic curve (OC). Sampling and total variances were lower on the automatic sampling plan for aflatoxins, fumonisins, and zearalenone quantification. The OC curve from automatic sampling plan for aflatoxins and zearalenone quantification reduces the consumer end seller risks when compared with the OC curve from the manual sampling plan. The automatic sampling plan is more efficient than the manual one for mycotoxins quantification in maize, reducing lot classification error, and providing more safety in commercial transactions. / O objetivo deste trabalho foi avaliar a variabilidade e o desempenho de dois planos de amostragem de milho para quantificação de micotoxinas. Amostraram-se 11 lotes de milho, através de dois planos de amostragem: o manual, utilizando-se o calador graneleiro e o automático, utilizando-se a amostragem em fluxo contínuo. De cada plano amostral determinaram-se a variabilidade total e as variabilidades das etapas de amostragem, preparação e análise dos resultados de aflatoxinas, fumonisinas e zearalenona em 8, 11 e 5 lotes, respectivamente. Essas variâncias foram comparadas, para cada micotoxina, entre os dois planos de amostragem utilizando a análise de variância multifatorial. As distribuições dos resultados de quantificação das aflatoxinas e zearalenona foram comparadas entre quatro modelos de distribuições teóricas. Determinaram-se as probabilidades de aceitação e rejeição de um lote com determinadas concentrações de aflatoxinas ou zearalenona, utilizando-se a variância e as informações da distribuição selecionada para montar as curvas características de operação (CO). A variância da amostragem e a variância total foram menores no plano de amostragem automático para quantificação de aflatoxinas, fumonisinas e zearalenona. A curva CO do plano de amostragem automático, para quantificação de aflatoxinas e zearalenona reduz os riscos do consumidor e do produtor em relação à curva CO do plano de amostragem manual. O plano de amostragem automático é mais eficiente que o plano de amostragem manual para quantificação de micotoxinas em milho diminuindo, portanto, o erro na classificação de lotes significando, pois, maior segurança nas relações comerciais.

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 & 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).