This project focused on the development and validation of an ultra-performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS) method for the determination of Patulin in apple-based products. Patulin is one of the many mycotoxins that are secondary metabolites from about 60 filamentous fungi. The mold often appears as black or blue on fruit, vegetables or crops. To determine the concentration of Patulin in consumer products is important since it may affect consumer health. The symptoms are often flu-like and can lead to kidney-failure and neurotic damage. The Swedish Food Agency is tasked to analyze consumer products to determine if they are safe to ingest. The European Commission has set maximum residue limits for several toxins that can potentially appear in groceries on the market. Using an UPLC-MS/MS allows for the accurate qualification and quantification of Patulin in apple juice and purees. The method was validated by analyzing several lots of apple juices and a proficiency test from Fapas®. The recovery rate ranged between 70.5-103.8% and were accepted because they met the recovery criteria in Regulation (EC) No. 401/2006 for Patulin.

Industry demand for local sources of grain for animal feed has increased sorghum production in the mid-Atlantic region of the U.S. Sorghum anthracnose (causal agent Colletotrichum sublineola) and the grain mold complex, which includes mycotoxin-producing Fusarium spp., limit the yield and quality of grain sorghum in humid climates worldwide. A majority of U.S. grain sorghum production is in arid regions, and management strategies have not been developed for the mid-Atlantic U.S. where warm, wet conditions favor disease. The specific objectives of this research were to: (1) determine the effectiveness of fungicides and their application timing for the management of sorghum foliar anthracnose, (2) compare five grain sorghum hybrids for their susceptibility to foliar anthracnose, grain mold and mycotoxin contamination under field conditions, (3) integrate host resistance and fungicide application to manage anthracnose and grain mold, and (4) identify Fusarium spp. associated with grain mold and mycotoxin contamination of sorghum in the mid-Atlantic U.S. For Objective 1, it was determined that a single application of pyraclostrobin-containing fungicide no later than flowering reduced anthrancose, protected yield and maximized farm income. Objective 2 focused on sorghum hybrid selection as a disease management tactic, and it was determined that hybrids with high yield potential and moderate disease resistance should be selected for mid-Atlantic sorghum production in order to maximize grain yield and quality while minimizing the need for fungicide inputs. Objective 3 focused on integrated management and demonstrated that under moderate disease pressure, a high-yielding susceptible hybrid required a single application of pyraclostrobin-based fungicide to minimize fungal diseases and maintain acceptable yields, whereas under high disease pressure it was necessary to integrate hybrid resistance and judicous applications of fungicides. The aim of Objective 4 was to characterize potential causal agents of mycotoxin contamination in mid-Atlantic sorghum, and thirteen phylogenetically distinct Fusarium species (F. lacertarum, F. graminearum. F. armeniacum, F. proliferatum, F. fujikuroi, F. verticillioides, F. thapsinum and several in Fusarium incarnatum-equiseti species complex) were found to be associated with grain mold and fumonisin and/or deoxynivalenol contamination of sorghum grain. This work has provided insights into the impacts of fungal diseases on grain sorghum yield and quality in the mid-Atlantic and has aided in development of best management practices for the region. / Doctor of Philosophy / Sorghum is grown in tropics, sub-tropics and semi-arid region worldwide for food, feed, forage and fuel. Sorghum acreage in the mid-Atlantic is increasing due the demand for locally grown grain by poultry and swine industries. During the growing season, warm and humid conditions are common in the southeastern and mid-Atlantic states favoring fungal diseases development that reduce the grain yield and quality. Anthracnose and grain mold, which includes toxic mycotoxin-producing Fusarium species, are the two major constraints in sorghum production in the region. However, management alternatives have not been developed. The main goal of this research was to develop management strategies to protect yield and maximize farm profitability by controlling anthracnose and grain mold of sorghum using chemicals and/or host resistance. The specific objectives were to: (1) determine the effectiveness of fungicides and their application timing for the management of sorghum foliar anthracnose, (2) compare grain sorghum hybrids for their susceptibility to foliar anthracnose, grain mold and mycotoxin contamination under field conditions, (3) assess the value of integrating host resistance and judicious use of fungicides to manage sorghum anthracnose and grain mold, and (4) identify Fusarium spp. associated with grain mold and mycotoxin contamination of sorghum in the mid-Atlantic U.S. Results from this research indicate that a single application of pyraclostrobin-containing fungicides no later than flowering reduces anthrancose, protects yield, and increases farm income. Sorghum hybrids varied in susceptibility to anthrancnose and grain mold, and planting a moderately resistant hybrid and applying a fungicide under high disease risk conditions provided the greatest return on investment. Both fumonisin and deoxynivalenol were frequently detected from sorghum grain, and mycotoxin contamination was associated with 13 different Fusarium species from three distinct species complexes. Based on the results of this work, best management practices for minimizing sorghum disease losses were developed for the mid-Atlantic region.

The objective of this research was to investigate the occurrence of mycoflora in rice plants and rice seeds in South Africa and their negative impact. A total of six species of Fusarium were isolated from diseased rice plants and rice seeds and identified as F. anthophilum, F. chlamydosporum, F. compactum, F. equiseti, F. fujikuroi and F. semitectum. In the translation elongation factor data set, Fusarium equiseti isolates grouped together within the F. incarnatum - equiseti Species Complex (FIESC). The isolates from rice clustered together in a single clade with the F. equiseti and F. incarnatum isolates forming two separate sub-clades.The isolates of F. equiseti present a new phylogenetically distinct species in FIESC. In the pathogenicity tests, isolates of both F. anthophilum and F. fujikuroi caused bakanae disease to rice plants. Fifty four rice cultivars and lines were tested by the standardized test tube inoculation method for resistance and susceptibility against bakanae isolate of F. anthophilum and the bakanae isolate of F. fujikuroi. None of the rice cultivars and lines was found to be resistant to bakanae isolates of Fusarium spp. The fungicide, benomyl was found to be most effective as a seed treatment for controlling bakanae disease of rice due to isolates of both F. anthophilum and F. fujikuroi. Thiram was found to be the least effective fungicide for controlling bakanae disease of rice caused by isolates of both the Fusarium spp. Apart from Fusarium species, other fungi that were also isolated from diseased rice plants and rice seeds were identified as Alternaria alternata, Alternaria longipes, Cochliobolus miyabeanus, Nigrospora sphaerica, Phoma eupyrena, Phoma jolyana, Phoma sorghina and Pithomyces sp. In mycotoxin tests, the isolates of both F. anthophilum and F. fujikuroi produced moniliformin. None of the isolates of F. anthophilum and F. fujikuroi produced fumonisins. This research is important as it identifies many fungal species in rice plants and seeds in South Africa for the first time. Currently, there is very little literature that makes reference to such findings under South African conditions. In addition, this investigation unravels previously unknown information on the resistance of rice to bakanese disease. Finally, information is provided on the effectiveness of commonly used fungicides (benomyl and thiram) to control rice diseases. This knowledge is crucial information that is useful to plant pathologists, the farming community and the scientists that are involved in strategies of fighting or reducing rice diseases so as to help contribute to food security. / Environmental Sciences / D. Phil. (Environmental Science)

The objective of this research was to investigate the occurrence of mycoflora in rice plants and rice seeds in South Africa and their negative impact. A total of six species of Fusarium were isolated from diseased rice plants and rice seeds and identified as F. anthophilum, F. chlamydosporum, F. compactum, F. equiseti, F. fujikuroi and F. semitectum. In the translation elongation factor data set, Fusarium equiseti isolates grouped together within the F. incarnatum - equiseti Species Complex (FIESC). The isolates from rice clustered together in a single clade with the F. equiseti and F. incarnatum isolates forming two separate sub-clades.The isolates of F. equiseti present a new phylogenetically distinct species in FIESC. In the pathogenicity tests, isolates of both F. anthophilum and F. fujikuroi caused bakanae disease to rice plants. Fifty four rice cultivars and lines were tested by the standardized test tube inoculation method for resistance and susceptibility against bakanae isolate of F. anthophilum and the bakanae isolate of F. fujikuroi. None of the rice cultivars and lines was found to be resistant to bakanae isolates of Fusarium spp. The fungicide, benomyl was found to be most effective as a seed treatment for controlling bakanae disease of rice due to isolates of both F. anthophilum and F. fujikuroi. Thiram was found to be the least effective fungicide for controlling bakanae disease of rice caused by isolates of both the Fusarium spp. Apart from Fusarium species, other fungi that were also isolated from diseased rice plants and rice seeds were identified as Alternaria alternata, Alternaria longipes, Cochliobolus miyabeanus, Nigrospora sphaerica, Phoma eupyrena, Phoma jolyana, Phoma sorghina and Pithomyces sp. In mycotoxin tests, the isolates of both F. anthophilum and F. fujikuroi produced moniliformin. None of the isolates of F. anthophilum and F. fujikuroi produced fumonisins. This research is important as it identifies many fungal species in rice plants and seeds in South Africa for the first time. Currently, there is very little literature that makes reference to such findings under South African conditions. In addition, this investigation unravels previously unknown information on the resistance of rice to bakanese disease. Finally, information is provided on the effectiveness of commonly used fungicides (benomyl and thiram) to control rice diseases. This knowledge is crucial information that is useful to plant pathologists, the farming community and the scientists that are involved in strategies of fighting or reducing rice diseases so as to help contribute to food security. / Environmental Sciences / D. Phil. (Environmental Science)

Thesis (M.Tech.: Biotechnology)- Dept. of Biotechnology & Food Technology, Durban University of Technology, 2007 xv, 102 leaves / For decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.

Tout au long de la chaîne alimentaire, des moisissures peuvent se développer et produire des mycotoxines. Ce sont des composés toxiques naturels issus du métabolisme secondaire des moisissures, susceptibles de contaminer l'alimentation animale et humaine, provoquant de nombreuses pathologies (hépatotoxicité, néphrotoxicité, neurotoxicité, mutagénicité, tératogénicité, cancérogénicité,…). La première étape de ce travail était d'évaluer la présence simultanée de l'ochratoxine A (OTA), de la citrinine (CIT), des aflatoxines (AFs), de la zéaralénone (ZEA), de la fumonisine (FB) et des trichothécènes dans des aliments destinés aux humaines (céréales, lait, café, jambon) et aux animaux (croquettes de chat et chien, foins). En général plusieurs mycotoxines coexistaient. Certains échantillons pour les humains dépassaient les limites autorisées en mycotoxines dans l'Union Européenne. Suite à l'étude de simulation d'apport en mycotoxines dans une ration quotidienne, nous avons constaté que les doses journalières admissibles (DJA) peuvent être dépassées. La deuxième phase consistait à étudier l'impact des mycotoxines seules ou en combinaison sur la viabilité cellulaire et la génotoxicité sur des modèles cellulaires (cellules rénales d'opossum (OK), cellules rénales humaines (HK2), cellules humaines de glandes mammaires (MCF7)) et chez des animaux (porc, rat). Nous avons montré que la CIT, la FB1 et la ZEA agissent en synergie sur la génotoxicité de l'OTA. Chez les animaux, nous avons montré qu'à des doses (5 ng d'OTA/kg poids corporel/ jour et de 200ng FB1/kg pc/j) correspondantes aux DJA, il y avait des effets génotoxiques (formation d'adduits à l'ADN). Nous avons mis en évidence l'implication des mycotoxines dans l'alimentation animale sur la baisse de fertilité et la tératogénicité chez les chats, ainsi que sur la mort des chevaux. Au cours de la troisième partie de cette étude, nous avons testé sur des cultures cellulaires (HK2 et MCF7) et in vivo (poulet) l'effet protecteur du glutathion (GSH) et de la sélénométhionine (SeMet) contre l'OTA responsable de cancers de voie urinaire et la ZEA responsable de baisse de fertilité. Le GSH est un puisant antioxydant et le sélénium est un oligoélément indispensable qui intervient comme co-facteur de nombreuses enzymes ayant des propriétés antioxydantes, comme les glutathion peroxydases. D'une manière générale, au niveau des cellules rénales, le GSH seul et la levure correspondante ont un effet bénéfique vis-à-vis de la génotoxicité de l'OTA ; par contre la sélénométhionine et la levure séléniée augmentent la génotoxicité de l'OTA et de la ZEA. Dans les cellules des glandes mammaires, il y a une nette amélioration vis-à-vis de la génotoxicité des deux mycotoxines lorsque les cellules sont exposées à une seule mycotoxine simultanément au GSH, à la sélénométhionine et aux levures enrichies. Chez les poulets, la diminution de la génotoxicité n'est pas exclusivement corrélée à la capacité des parois de levure ou des levures à adsorber l'OTA. Ces dérivés de levure ont gardé la propriété de partiellement métaboliser l'OTA dans l'intestin. Les parois de levures et les levures enrichies en GSH ont un meilleur pouvoir protecteur que celles enrichies en SeMet / Throughout the food chain, mold can grow and produce mycotoxins. These are toxic compounds "natural" from the secondary metabolism of molds that may contaminate the feed and food, causing many diseases (hepatotoxicity, nephrotoxicity, neurotoxicity, mutagenicity, teratogenicity, carcinogenicity, ...). The first stage of this work was to assess the level of multi-contamination by mycotoxins (OTA, CIT, Afs, ZEA, FB, DON) in food (cereals, milk, coffee, ham) and feed (pet food). Some samples analyzed exceeded the limits of mycotoxins in the European Union. Through the simulation study of mycotoxin intake in a daily diet, we found that the acceptable daily intake (ADI) may be exceeded. The second phase was to study the impact of mycotoxins alone or in combination on cell proliferation, genotoxicity in cellular models (OK, HK2, and MCF7) and animal (pig, rat). We have demonstrated genotoxic effects (formation of DNA adducts) at doses (5 ng OTA / kg bw / day and 200 ng FB1/kg bw / day) considered safe (ADI). We have shown that the CIT, FB1 and ZEA act synergistically on the genotoxicity of OTA. We pointed to the involvement of mycotoxins in animal feed on declining fertility and teratogenicity in cats, as well as the death of horses. In the third part of this study, we tested in cell cultures (HK2 and MCF7) and in vivo (chicken) the protective effect of glutathione (GSH) and selenomethionine (SeMet) against OTA responsible for urinary tract cancers and ZEA reducing fertility. GSH is considered as a potent antioxidant and selenium is a trace essential element that acts as a cofactor of enzymes such glutathione peroxidase. In summary, in kidney cells, GSH and GSH enriched yeast decrease OTA genotoxicity whereas SeMet and SeMet enriched yeast increase genotoxicity of OTA and ZEA. In mammary cells, whatever the compounds gentoxicty of OTA and ZEA significantly decrease. Decrease of OTA genotoxicity in chicken kidney cannot be exclusively explained by adsorption of OTA on yeast by products. The yeast products retain their ability to metabolize the OTA. GSH enriched yeast and yeast cell wells are more efficient than SeMet enriched yeast

Das Ziel der Untersuchungen war die Ermittlung der Grenzbedingungen von Lagerungsdichte und äußerer Luftabschlußgüte (Gasdurchlässigkeit des Zudeckmaterials), unter denen Schimmelpilzwachstum während der Silagelagerung eingeschränkt wird. Den Schwerpunkt bildeten 10 Praxis- und Laborsilierversuche mit extensiv erzeugtem Grünfutter, das aufgrund relativ hoher Rohfasergehalte allgemein schwer verdichtbar ist. In den Silagen wurde die Pilzkeimzahl sowie der Gehalt an Ergosterin und Roquefortin C bestimmt. Für die Analyse des Ergosteringehaltes wurde eine neue Methode entwickelt. Die Gasdurchlässigkeit von 1 - 8 Folienlagen der verwendeten Silierstretchfolie wurde radiometrisch gemessen. Im Versuchszeitraum (1995 - 1997) wurde Grünfutter von jahreszeitlich verschiedenen Aufwüchsen unter landwirtschaftlichen Praxisbedingungen einsiliert. Mit Hilfe drei verschiedener Ballenpressen wurden insgesamt 165 Silageballen mit unterschiedlichen Lagerungsdichten erzeugt. Die Folienlagenzahl und die Lagerdauer bei den Silageballen wurde gestaffelt. Hierdurch sollte der Einfluß des Luftabschlusses auf Gärqualität, Pilzbefall und Mykotoxingehalt in den Silagen untersucht werden. Im Labor wurden Einflußfaktoren wie der Trockenmassegehalt (T-Gehalt) des Siliergutes geprüft. In den Praxisversuchen trat bei einem T-Gehalt < 400 g/kg unabhängig von Lagerungsdichte und äußerem Luftabschluß eine relativ starke Buttersäurebildung in den Silagen auf. Dagegen wurden bei einem T-Gehalt > 450 g/kg bei ausreichendem Luftabschluß buttersäurefreie (£ 0,3 % T) Silagen erzielt. Hier lag offenbar ein ausreichender T-Gehalt zur Sicherung einer guten Gärqualität vor. In allen Versuchen stellte die Erhöhung der Lagerungsdichte die primäre Grundlage zur Erzeugung eines ausreichenden Luftabschlusses in den Silagen dar. Die äußere Luftabschlußgüte besaß im Vergleich dazu sekundären Charakter. Als notwendige Grenzbedingungen des Luftabschlusses zur Erzeugung einer guten Gärqualität sowie zur Einschränkung von Pilzbefall in den Silagen erwiesen sich: (1) eine Lagerungsdichte von mindestens 200-210 kg T/m³ und (2) eine maximale Gasdurchlässigkeit des Zudeckmaterials von 1,7 l/m² in 24 Stunden (6 Folienlagen der verwendeten Silierstretchfolie). Ein ausreichender Luftabschluß war die Voraussetzung für die Wirksamkeit von Silierzusätzen hinsichtlich der Einschränkung von Pilzbefall und der Verbesserung der Gärqualität in den Silagen. Die Mykotoxinbildung in den Silagen, die am Beispiel des Vorkommens von Roquefortin C (ROF) untersucht worden ist, wurde ungeachtet von Lagerungsdichte und äußerem Luftabschluß vor allem durch den T-Gehalt des Siliergutes beeinflußt. Bei einem T-Gehalt < 450 g/kg enthielten etwa 88 % der in diesem T-Bereich vorliegenden Silagen Roquefortin C. Bei einem T-Gehalt zwischen 450 und 550 g/kg enthielten noch etwa 10 % der hier vorliegenden Silagen Roquefortin C, überwiegend aber im Bereich der Nachweisgrenze von ³ 0,05 mg ROF/kg T. Bei einem T-Gehalt > 550 g/kg wurde in den Silagen Roquefortin C nicht nachgewiesen. Die insgesamt in den Silagen gemessenen Gehalte an Roquefortin C waren mit < 1,0 mg ROF/kg T relativ niedrig. Sie sind bei Verfütterung der Silagen an Wiederkäuer nach dem gegenwärtigen Erkenntnisstand toxikologisch als nicht kritisch einschätzbar. / The goal of the investigation was to determine the boundary conditions of compactness and hermetic level of covering material (permeability of the covering material) to inhibit mould growth during silage storage. The emphasis was based on 10 practical and lab ensiling experiments with green forage having a relatively high content of raw fiber, which in general is difficult to compress. The silages were investigated for their mould count, as well as for their content of ergosterol and roquefortine C. A new method was developed to analyse the ergosterol content. The permeability of 1 - 8 numbers of wraps of the used ensiling stretch film was measured by radiometric methods. During the experimental time (1995 - 1997), green forage from seasonally different bites were ensiled under practical agricultural conditions. Using three different balers, a total of 165 bales were wrapped at various compactness levels. The numbers of wraps and the storage period of the bales were staggered. Hereby the influence of air exclusion on fermentation quality, mould growth and mycotoxin content in the silage should be tested. In the lab, factors such as the dry matter content (d-content) of the green forage were tested. In the practical experiments, the results showed that at a d-content of < 400 g/kg, a relatively high amount of butyric acid formed in the silages, independent of the compactness and hermetic level of the covering material. Whereas, at a d-content of > 450 g/kg, no butyric acid (£ 0,3 % dry matter) was found in the silages with sufficient air exclusion. Here, the d-content to ensure a good fermentation quality was sufficient. In all experiments, the primary way to generate sufficient air exclusion in the silages was to increase the compactness. Compared with this, the hermetic level of covering material had secondary character. To get a sufficient fermentation quality, as well as an inhibition of mould growth in the silages, necessary boundary conditions of air exclusion were: (1) a compactness of at least 200-210 kg T/m³ and (2) a maximum permeability of the covering material of 1,7 l /m² in 24 hours (6 numbers of wraps of the used ensiling stretch film). Sufficient air exclusion was necessary for the effectiveness of the silage additives in inhibiting mould growth and improving the fermentation quality in the silages. The mycotoxin formation in the silages, investigated by measuring the occurrence of roquefortine C (ROF), was influenced mainly by the dry matter content of the ensiled material, regardless of the compactness and hermetic level of covering material. At a dry matter content of < 450 g/kg, about 88 % of the silages contained roquefortine C. Between 450 to 550 g/kg dry matter about 10 % of the silages containing roquefortine C, however, at low levels in the range of the detectable content of ³ 0,05 mg ROF/kg dry matter. At a dry matter content of > 550 g/kg, no roquefortine C was found in the silages. Summarised, the measured amounts of < 1,0 mg ROF/kg roquefortine C in the silages is considered to be relatively low. Within the actual state of knowledge of toxicology, it is not considered dangerous to feed ruminant animals with silages containing these low amounts of roquefortine C.

A cultura de sorgo granífero no país é amplamente utilizada na alimentação animal. Através deste estudo, decidimos avaliar a micobiota e a ocorrência de ácido tenuazônico em grãos de sorgo cultivados em duas safras. Além disso, através de abordagem polifásica, caracterizamos cepas de E. sorghinum, bem como avaliamos os aspectos ecofisiológicos desta espécie. E. sorghinum foi o fungo mais prevalente nos de grãos de sorgo. O ácido tenuazônico foi detectado na totalidade das amostras avaliadas, com maiores níveis nos grãos cultivados na safra verão. Foi possível verificar considerável variabilidade genética nas cepas de E. sorghinum, apresentando-se como complexo de espécies filogenéticas. Avaliando os fatores abióticos ocorridos no campo, bem como acessando a ecofisiologia de E. sorghinum, foi possível verificar que condições quentes e úmidas são favoráveis a produção de ácido tenuazônico. Estes resultados alertam para presença de compostos tóxicos em grãos de sorgo cultivados no país, bem como contribuem com informações aplicáveis para o manejo desta cultura. / The culture of sorghum in Brazil is widely used in animal feed. The aim of this study was evaluate the mycobiota and occurrence of tenuazonic acid in sorghum grains cultivated in two sowing periods. In addition, through a polyphasic approach, we characterized strains of Epicoccum sorghinum, as well as evaluated the ecophysiological of this species. E. sorghinum was the most prevalent fungus in sorghum grains. Tenuazonic acid was detected in all the samples, with higher levels in the grains grown in the summer crop. It was possible to verify considerable genetic variability in the strains of E. sorghinum, presenting as a complex of phylogenetic species. By evaluating the abiotic factors occurring in the field, as well as the access to an ecophysiology of E. sorghinum, it was possible to verify that hot and wet conditions are favorable for tenuazonic acid production. These results indicate the presence of toxic compounds in sorghum grains cultivated in Brazil, as well as contribute with information for crop management.

A cultura do milho é responsável por 40% dos grãos produzidos no país, sendo considerada um dos segmentos mais importantes para a economia do agronegócio brasileiro. Devido ao seu alto valor nutritivo, o milho possui grande suscetibilidade ao crescimento de fungos, com destaque para Fusarium verticillioides, que além de causar danos nas raízes, colmos e espigas, é responsável por produzir fumonisinas, um grupo de micotoxinas termoestáveis e possivelmente carcinogênico, ao homem e animais. É sabido que apesar da importância não existem muitas informações associando a influência de diferentes fatores sobre a sua produção, principalmente nas condições tropicais brasileiras. Desta forma, estudos foram realizados com o objetivo geral de avaliar a influência de fatores ambientais, genótipos e métodos de inoculação de F. verticillioides na severidade da podridão de espigas, qualidade sanitária das sementes, produtividade do milho, na produção de fumonisinas e histopatologia em frangos de corte. As investigações aqui relatadas foram divididas em quatro capítulos. No primeiro capítulo, avaliou-se o efeito de fatores ambientais, genótipos e métodos de inoculação de F. verticillioides na produção de fumonisinas em grãos de milho. No segundo capítulo, comparou- se em locais diferentes métodos de inoculação artificial (spray, injetável) e natural de F. verticillioides em dez genótipos de milho pela quantificação da severidade da doença e produtividade. No terceiro capítulo, realizou-se um levantamento da população de F. verticillioides através de análise sanitária e transmissão do fungo à campo das plantas para as sementes e das sementes para as plântulas inoculados com diferentes métodos de inoculação. Por fim, o quarto capítulo, avaliou-se na safra de 2015/2016, procedentes de lavouras experimentais contendo cultura de milho, híbridos comerciais inoculados com F. verticillioides e determinou-se os parâmetros zootécnicos e histopatológicos em frangos de corte. O delineamento experimental para o primeiro e segundo capítulos, desenvolvidos em campo, foi igualmente realizado nos quatros locais estudados, sendo o de blocos ao acaso em fatorial de 10x3, com três repetições, sendo dez genótipos de milho e três métodos de inoculação. No terceiro capítulo, desenvolvido em laboratório, os experimentos tiveram o delineamento inteiramente casualizado, em fatorial de 10x3 com quatro repetições, sendo dez genótipos de milho e três métodos de inoculação. Utilizou-se as sementes provenientes dos híbridos que foram inoculados pelos três métodos de inoculação. Cada unidade experimental consistiu de um Gerbox contendo 25 sementes. No quarto capítulo, o experimento foi conduzido no aviário experimental da Escola de Medicina Veterinária e Zootecnia da Universidade Federal do Tocantins, no campus de Araguaína-TO. Foram utilizados 160 pintos de um dia, frangos de corte da linhagem Cobb 500, distribuídos em delineamento experimental inteiramente casualizado, com dois tratamentos (ração basal de milho com ausência de fumonisina e a ração de grãos de milho com fumonisina), com 10 repetições contendo 8 aves cada. Todas as análises da concentração fumonisinas foram realizadas no Laboratório de Micotoxologia (LAMIC), da Universidade Federal de Santa Maria (UFSM), e utilizou-se a metodologia de quantificação denominada LC-MS/MS (Liquid Chromatography Mass Spectrometry) POP 45. Com relação aos resultados obtidos, para o primeiro capítulo, concluiu-se que não foi possível assegurar que grãos sintomáticos e assintomáticos quanto à infecção de Fusarium verticillioides, estejam livres de contaminação por fumonisinas e os métodos de inoculação por aspersão e o inoculado no centro da espiga não influenciaram em diferentes concentrações de fumonisinas. A produção de fumonisinas nos grãos de milho é potencializada por condições ambientais de temperatura elevada e pela susceptibilidade do genótipo e alta severidade de F. verticillioides. Nestas condições, houve tendência de produzir maiores teores de fumonisinas para a maioria dos genótipos avaliados. A produtividade de grãos foi reduzida pela incidência de F. verticillioides. Para o segundo capítulo, verificou-se que o método de inoculação artificial através de aspersão no estilo-estigma e o injetável no centro da espiga, proporcionaram maior intensidade da podridão da espiga, possibilitando diferenciar do método natural. Em Planaltina, verificou-se que o método de inoculação por aspersão resultou em maior severidade da doença, causada por Fusarium verticillioides do que o método de inoculação injetável no centro da espiga. Os híbridos de milho P4285, DKB390PRO2 e DKB310PRO2 foram os mais resistentes à podridão da espiga, nos quatro ambientes, enquanto que o híbrido 32R48YH apresentou a maior severidade da doença. O método de inoculação artificial foi o mais eficiente em ambientes de climas amenos e esta informação é importante na padronização do método para seleção e caracterização de genótipos de milho em programas de melhoramento. Para o terceiro capítulo, concluiu-se que as sementes oriundas de espigas inoculadas com spray apresentaram maior grau de incidência do patógeno, sendo este, o método mais eficiente de inoculação artificial de F. verticillioides na fase R1 para a avaliação de emergência de sementes. Os híbridos 32R48YH e o DKB240PRO2 tiveram o maior nível de infecção por F. verticillioides pois obtiveram a maior incidência do fungo. Enquanto que os híbridos P3630H e P4285H demostraram ser os mais resistentes, pois obtiveram as menores incidência do patógeno. Os híbridos P3250, 32R48YH e 30K75Y apresentaram menor percentagem de emergência quando a inoculação foi realizada pelo método injetável enquanto que os híbridos P4285H, 30K75Y e DOW30A37PW apresentaram menor percentagem de emergência quando a inoculação foi realizada pelo método spray. A transmissão da semente para a planta foi confirmada pela análise molecular. Verificou- se que, apesar das plantas não terem apresentado sintomas visíveis, pôde-se confirmar que houve infecção latente. A técnica de PCR foi capaz de revelar a presença do patógeno nas sementes de milho em menor nível de incidência avaliado (1%). No quarto capítulo, foi concluído que a concentração de 2,78μg/g de fumonisina determinada na ração não foi suficiente para provocar alterações nos parâmetros zootécnicos (peso das aves e no consumo de ração) e histopatológico (tecidos do intestino, fígado e coração) de aves da linhagem COBB 500, alimentadas até o 21° dia. As análises dos parâmetros zootécnicos e histopatológicos permitiram revelar concentrações mínimas e seguras de fumonisina que não provocaram danos toxicológicos em aves da linhagem COBB 500. Este é um importante parâmetro que pode contribuir nas etapas de produção de carnes e de produtos avícolas. / The corn crop is responsible for 40% of the grains produced in the country, being considered one of the most important segments for the Brazilian agribusiness economy. Due to its high nutritional value, maize is highly susceptible to fungus growth, especially Fusarium verticillioides, which besides causing damage to the roots, stalks and ears, is responsible for producing fumonisins, a group of thermostable mycotoxins and possibly carcinogenic, to man and animals. It is known that despite the importance, there is not much information associating the influence of different factors on its production, mainly in Brazilian tropical conditions. Studies were carried out with the general objective of evaluating the influence of environmental factors, genotypes and methods of inoculation of F. verticillioides on the severity of ear rot, seed quality, maize productivity, fumonisin production and histopathology in broilers. The investigations reported here were divided into four chapters. In the first chapter we evaluated the effect of environmental factors, genotypes and methods of inoculation of F. verticillioides on the production of fumonisins in corn grains. In the second chapter, was compared in different locations, methods of artificial (spray and injectable) and natural inoculation of F. verticillioides in ten maize genotypes by quantification of disease severity and productivity. In the third chapter a population of F. verticillioides was carried out through sanitary analysis and transmission of the fungus from plants to the seeds and from seeds to the seedlings inoculated with different methods of inoculation. Finally, the fourth chapter evaluated the harvest of 2015/2016 coming from crops containing commercial hybrids corn inoculated with F. verticillioides and the zootechnical and histopathological parameters were determined in broilers. The experimental design for the first and second chapters, conducted in the field, was also performed in the four localities studied, being a randomized block design in a 10x3 factorial scheme, with three replications, being ten maize genotypes and three inoculation methods. In the third chapter the experiments had a completely randomized design, in a 10x3 factorial scheme with four replications, being ten maize genotypes and three inoculation methods. Were used seeds from the hybrids that were inoculated by the three methods of inoculation. Each replicate consisted of a Gerbox containing 25 seeds. In the fourth chapter, the experiment was conducted in the experimental aviary of the School of Veterinary Medicine and Animal Science of the Federal University of Tocantins, on the campus of Araguaína-TO. A total of 160 broilers of Cobb 500 lineage (of one day old), were used in a completely randomized experimental design with two treatments (basal corn meal with no fumonisin and corn grain feed with fumonisin), with 10 replicates containing 8 broiler chicks each. All fumonisin concentration analyzes were performed at the Laboratory of Mycotoxology (LAMIC), Federal University of Santa Maria (UFSM), and the quantitative methodology called LC-MS / MS (Liquid Chromatography Mass Spectrometry) POP 45 was used. Regarding the results obtained, for the first chapter, it was concluded that it was not possible to assure that symptomatic and asymptomatic grains for Fusarium verticillioides infection are free from fumonisin contamination and the methods of inoculation by spraying and inoculation in the center of the ear did not influence different concentrations of fumonisins. Fumonisin production in maize grains is potentiated by high temperature environmental conditions and the susceptibility of the genotype and high severity of F. verticillioides. Under these conditions, there was a tendency to produce higher levels of fumonisins for most genotypes evaluated. The grain yield was reduced by the incidence of F. verticillioides. For the second chapter, it was verified that the method of artificial inoculation by sprinkling in the style-stigma and the injectable in the center of the spike, provided a greater intensity of the rot of the ear, allowing to differentiate of the natural method. In Planaltina, it was verified that the spray inoculation method resulted in a greater severity of the disease, caused by F. verticillioides than the injectable inoculation method in the center of the spike. Hybrids of maize P4285, DKB390PRO2 and DKB310PRO2 were the most resistant to ear rot in the four environments, while the hybrid 32R48YH showed the highest severity of the disease. The artificial inoculation method was the most efficient in mild climate environments and this information may be important in the standardization of the method for selection and characterization of maize genotypes in breeding programs. For the third chapter, it was concluded that the seeds from ears inoculated with spray presented a higher level of incidence of the pathogen, being this the most efficient method of artificial inoculation of F. verticillioides in the R1 phase for seed emergence evaluation. The hybrids 32R48YH and DKB240PRO2 had the highest level of infection by F. verticillioides because they had the highest incidence of the fungus. While the hybrids P3630H and P4285H proved to be the most resistant because they obtained the lowest incidence of the pathogen. Hybrids P3250, 32R48YH and 30K75Y presented a lower percentage of emergence when the inoculation was performed by the injectable method, while the hybrids P4285H, 30K75Y and DOW30A37PW showed a lower percentage of emergence when the inoculation was performed by the spray method. The transmission of the seed to the plant was confirmed by molecular analysis. It was found that, although the plants showed no visible symptoms, it was possible to confirm that there was a latent infection. The PCR technique was able to reveal the presence of the pathogen in maize seeds at a lower incidence level (1%). In the fourth chapter, it was concluded that the concentration of 2.78 μg/g of fumonisin determined in the diet was not sufficient to cause alterations in the zootechnical parameters (broilers weight and feed consumption) and histopathological parameters (intestinal, liver and heart tissues) of broilers of the COBB 500 lineage, fed until the 21st day. Analyzes of the zootechnical and histopathological parameters allowed to reveal minimum and safe concentrations of fumonisin that did not cause toxicological damages in broilers of Cobb 500 lineage. This is an important parameter that can contribute in the stages of production of meat and broiler chicks products.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-20T18:20:05Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TAIS FERNANDA ANDRZEJEWSKI KAMINSKI.pdf: 736237 bytes, checksum: 7739abe4dcd9ebf1aa4595b41498419a (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-20T18:20:34Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TAIS FERNANDA ANDRZEJEWSKI KAMINSKI.pdf: 736237 bytes, checksum: 7739abe4dcd9ebf1aa4595b41498419a (MD5) / Made available in DSpace on 2017-06-20T18:20:34Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TAIS FERNANDA ANDRZEJEWSKI KAMINSKI.pdf: 736237 bytes, checksum: 7739abe4dcd9ebf1aa4595b41498419a (MD5) Previous issue date: 2017-02-21 / Os fungos produzem vários metabólitos secundários, onde muitos destes têm sido associados com a indução de efeitos tóxicos em animais e seres humanos. O efeito toxicológico, incluindo o carcinogênico, tem sido observado em mais de 300 micotoxinas estruturalmente conhecidas. As Fumonisinas são micotoxinas produzidas principalmente por Fusarium verticillioides e Fusarium proliferatum, as quais frequentemente contaminam vários alimentos, especialmente milho e seus derivados, induzindo ao aparecimento de quadros patológicos em humanos. A Fumonisina B1 (FB1) é a mais prevalente, respondendo por aproximadamente 70% do total das micotoxicoses. Esta micotoxina está associada com leucoencefalomalácia (LEM) em cavalos, edema pulmonar em suínos e hepatocarcinoma em ratos, além de estar relacionada à inibição da biossíntese de esfingolípideos e ao aumento do risco de cancro esofágico em seres humanos. O presente estudo teve como objetivo investigar os efeitos citotóxico e genotóxico da fumonisina B1 através do teste de viabilidade celular e do teste de proliferação celular em cultura de leucócitos humanos. As concentrações testadas foram de 200; 100; 50; 5; 0,5; 0,05; 0,005 μg/mL e 300; 30; 3; 1; 0,1; 0,01 fg/mL. Todos os ensaios foram realizados em triplicatas sendo que, como controle positivo foi utilizado H2O2 4mM e, como controle negativo, tampão PBS 7,4. Com exceção das concentrações de 3fg/mL, 0,1fg/mL e 0,01fg/mL, todas as concentrações testadas demonstraram ser citotóxicas (p<0,05). Em relação ao teste de genotoxicidade, exceto as concentrações de 0,1fg/mL e 0,01fg/mL, demonstraram interferir significativamente na proliferação celular. Podemos concluir, de forma inédita, que somente em concentrações extremamente baixas, na ordem de fentogramas por mililitro, a Fumonisina B1 diminuiu os danos induzidos em leucócitos humanos. / Fungi produce several secondary metabolites, where many of these have been associated with the induction of toxic effects in animals and humans. The toxicological effect, including the carcinogenic, has been observed in more than 300 structurally known mycotoxins. Fumonisins are mycotoxins produced mainly by Fusarium verticillioides and Fusarium proliferatum, which often contaminate various foods, especially corn and its derivatives, leading to the appearance of pathological conditions in humans. Fumonisin B1 (FB1) is the most prevalent, accounting for approximately 70% of the total mycotoxicosis. This mycotoxin is associated with leukoencephalomalacia (LEM) in horses, pulmonary edema in pigs and hepatocarcinoma in rats, besides being related to the inhibition of sphingolipid biosynthesis and to the increasing risk of esophageal cancer in humans.The present study aimed to investigate the cytotoxic and genotoxic effects of fumonisin B1 through the cell viability and the cell proliferation test in human leukocyte culture. The tested concentrations tested were 200; 100; 50; 5; 0,5; 0,05; 0,005 μg/mL e 300; 30; 3; 1; 0,1; 0,01 fg/mL. All the tests were performed in triplicates,and it was used H2O2 4mM as a positive control, and PBS 7.4 bufferas a negative control. All concentrations tested were cytotoxic (p <0.05), except the 3fg/mL, 0,1fg/mL and 0,01fg/mL concentrations. Regarding to the genotoxicity test, except the 0.1fg/mL and 0.01fg/mL concentrations of, they demonstrated to significantly interfere in the cell proliferation. In an unprecedented way , it can be concludedthat only in extremely low concentrations, in the order of phentograms per milliliter, the Fumonisin B1 decreased the induced damage in human leukocytes.