O objetivo deste estudo foi avaliar a presença de fungos e micotoxinas [aflatoxinas e ácido ciclopiazônico (ACP)] em amostras de castanha-do-Brasil coletadas em diferentes estados da região Amazônia brasileira: Acre, Amazonas, Amapá e Pará. Um total de 200 amostras de casca e 200 amostras de amêndoa foram semeadas em ágar Aspergillus flavus-parasiticus para pesquisa da micobiota. As micotoxinas foram analisadas por cromatografia líquida de alta eficiência. A morfológica clássica e a análise molecular revelou a presença dos seguintes fungos, em ordem decrescente de frequência: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. e Aspergillus spp. A presença de aflatoxinas e ACP foi detectada em amostras de amêndoa, na frequência de 11% e 22%, respectivamente. Aspergillus nomius e Aspergillus flavus foram as espécies mais isoladas de Aspergillus. A presença de fungos não implica necessariamente na contaminação por micotoxinas, porém a amêndoa da castanha-do-Brasil parece ser um bom substrato para o crescimento de fungos. / The aim of this study was to evaluate the presence of fungi and mycotoxins [aflatoxin and cyclopiazonic acid (ACP)] in Brazil nut samples collected in different States of the Brazilian Amazon: Acre , Amazonas , Amapá and Pará. A total of 200 almond and 200 husk samples were plated on agar Aspergillus flavus - parasiticus for mycobiota isolation. Mycotoxins were analyzed by high performance liquid chromatography. The classical morphological and molecular analysis revealed the presence of the following fungi , in decreasing order of frequency: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. and Aspergillus spp. The presence of aflatoxins and ACP was detected in almonds samples at a frequency of 11% and 22% , respectively. Aspergillus nomius and Aspergillus flavus species were the most isolated from Aspergillus. The presence of fungi does not necessarily imply the mycotoxin contamination, but the almond samples of Brazil nut seem to be a good substrate for the growth of fungi.

A maioria dos patógenos responsáveis pelas principais doenças do milho (Zea mays L.) é transmitida pelas sementes, trazendo consequências como a podridão de sementes e o tombamento de plântulas, redução da germinação e do vigor, diminuindo assim a qualidade das sementes de milho. Este trabalho teve por objetivo avaliar a patogenicidade, transmissão e incidência de Fusarium verticilliodes em sementes de milho oriundas de espigas inoculadas com diferentes métodos. No presente trabalho foram realizados testes de detecção de F. verticillioides para confirmação da presença do patógeno nas plantas de milho inoculadas nas formas de spray, injetável e natural. Foi avaliada a porcentagem de emergência, qualidade fisiológica e sanitária de sementes de milho produzidas com diferentes graus de infecção por F. verticillioides, relacionando-se com a produtividade. Foi possível identificar a sensibilidade das técnicas de PCR, que amplificaram as concentrações de DNA testadas. A técnica de PCR utilizada foi capaz de detectar até 1% de infecção. Foi observado que as inoculações feitas nas espigas das plantas com spray resultaram em maior incidência do patógeno nas sementes quando comparada aos demais tratamentos. Os dez híbridos inoculados apresentaram incidência de F. verticillioides nas sementes que foram submetidas às três formas de inoculação. Quanto à emergência das plântulas verificou-se porcentagem superior à 90% em todos os híbridos estudados. / Most of the pathogens responsible for the main diseases of maize (Zea mays L.) are transmitted by seeds, with consequences such as seed rot and seedling tipping, germination and vigor reduction, thus reducing the quality of maize seeds. The objective of this work was to evaluate the pathogenicity, transmission and incidence of Fusarium verticilliodes in corn seeds from spikes inoculated with different methods. In the present work, Fusarium verticillioides detection tests were performed to confirm the presence of the pathogen in maize plants inoculated in spray, injectable and natural forms. The percentage of emergence, physiological and sanitary quality of maize seeds produced with different degrees of infection by F. verticillioides was evaluated, being related to productivity. The PCR technique used was able to detect up to 1% of infection. It was observed that the inoculations on the spikes of the spray plants resulted in a higher incidence of the pathogen in the seeds when compared to the other treatments. The ten inoculated hybrids showed incidence of F. verticillioides in the seeds that were submitted to all forms of inoculation. To verify the transmissibility of the seed pathogen to the plants, after extractions of leaf and stem tissues, DNA extraction analyzes with specific primer of F. verticillioides were made. Seedling emergence showed a percentage higher than 90% in all hybrids studied.

O objetivo deste estudo foi avaliar a presença de fungos e micotoxinas [aflatoxinas e ácido ciclopiazônico (ACP)] em amostras de castanha-do-Brasil coletadas em diferentes estados da região Amazônia brasileira: Acre, Amazonas, Amapá e Pará. Um total de 200 amostras de casca e 200 amostras de amêndoa foram semeadas em ágar Aspergillus flavus-parasiticus para pesquisa da micobiota. As micotoxinas foram analisadas por cromatografia líquida de alta eficiência. A morfológica clássica e a análise molecular revelou a presença dos seguintes fungos, em ordem decrescente de frequência: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. e Aspergillus spp. A presença de aflatoxinas e ACP foi detectada em amostras de amêndoa, na frequência de 11% e 22%, respectivamente. Aspergillus nomius e Aspergillus flavus foram as espécies mais isoladas de Aspergillus. A presença de fungos não implica necessariamente na contaminação por micotoxinas, porém a amêndoa da castanha-do-Brasil parece ser um bom substrato para o crescimento de fungos. / The aim of this study was to evaluate the presence of fungi and mycotoxins [aflatoxin and cyclopiazonic acid (ACP)] in Brazil nut samples collected in different States of the Brazilian Amazon: Acre , Amazonas , Amapá and Pará. A total of 200 almond and 200 husk samples were plated on agar Aspergillus flavus - parasiticus for mycobiota isolation. Mycotoxins were analyzed by high performance liquid chromatography. The classical morphological and molecular analysis revealed the presence of the following fungi , in decreasing order of frequency: Phialemonium spp., Penicillium spp., Fusarium spp., Phaeoacremonium spp. and Aspergillus spp. The presence of aflatoxins and ACP was detected in almonds samples at a frequency of 11% and 22% , respectively. Aspergillus nomius and Aspergillus flavus species were the most isolated from Aspergillus. The presence of fungi does not necessarily imply the mycotoxin contamination, but the almond samples of Brazil nut seem to be a good substrate for the growth of fungi.

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

O trabalho teve como objetivo avaliar a presença de fungos e aflatoxinas e ácido ciclopiazônico (ACP) em castanhas-do-brasil a campo e armazenadas, no solo e ar a fim de estabelecer vias de contaminação fúngica. A pesquisa de fungos foi feita pela técnica de semeadura em superfície em meio Ágar batata dextrose e Ágar Aspergillus flavus parasiticus e a determinação de micotoxinas, por cromatografia líquida de alta eficiência. As amostras a campo foram analisadas em: Dia 0; amostras na árvore, Dias 5, 10 e 15; em contato com solo 5, 10 e 15 dias, respectivamente. As armazenadas foram analisadas mensalmente por 11 meses. A campo, os fungos mais prevalentes foram: A. flavus em ouriços e amêndoas; Fusarium spp. em cascas. No solo foram isolados Penicillium spp. e Aspergillus flavus e no ar, Fusarium spp. e Penicillium spp. No armazenamento, em amêndoas, foi constatado A. flavus, Fusarium spp. e A. nomius; e em cascas, Fusarium spp. e A. flavus. Aflatoxinas e ACP não foram detectados. O aumento do tempo de contato das castanhas-do-brasil com o solo foi acompanhado de maior isolamento de A. flavus; sugerindo contaminação da castanha durante etapa a campo. A. nomius e A. parasiticus foram isolados apenas no armazenamento, indicando contaminação no estoque das amêndoas. / The objective of this present study was analyse, in the field and in the storage, mycobiota and the contamination by (aflatoxins and cyclopiazonic acid from Brazil nuts, soil and air samples to determine route of fungi contamination. The Brazil nuts mycobiota was determine by diluition plating method in Potate Dextrose Agar and Aspergillus flavus parasiticus agar and, micotoxins was done by high performance liquid chromatography. Field samples were collected in: day 0, samples still on the tree; days 5, 10 and 15, samples in contact with soil for 5, 10 and 15 days, respectively. The most prevalent fungi were Aspergillus flavus in fruit pods and nuts and Fusarium spp. in shells. Penicillium spp. and A. flavus were isolated from soil, and Fusarium spp. and Penicillium spp. from air. The storage samples were analysed montly during 11 months; and showed predominance A. flavus, Fusarium spp. e A. nomius in nuts, and Fusarium spp. and A. flavus in shells. Aflatoxins and cyclopiazonic acid were not detected. These findings indicate soil as the main source of fungal contamination of Brazil nuts. A. nomius e A. parasiticus were isolated only in storage, suggesting Brazil nuts contamination occurs in this period.

O trabalho teve como objetivo avaliar a presença de fungos e aflatoxinas e ácido ciclopiazônico (ACP) em castanhas-do-brasil a campo e armazenadas, no solo e ar a fim de estabelecer vias de contaminação fúngica. A pesquisa de fungos foi feita pela técnica de semeadura em superfície em meio Ágar batata dextrose e Ágar Aspergillus flavus parasiticus e a determinação de micotoxinas, por cromatografia líquida de alta eficiência. As amostras a campo foram analisadas em: Dia 0; amostras na árvore, Dias 5, 10 e 15; em contato com solo 5, 10 e 15 dias, respectivamente. As armazenadas foram analisadas mensalmente por 11 meses. A campo, os fungos mais prevalentes foram: A. flavus em ouriços e amêndoas; Fusarium spp. em cascas. No solo foram isolados Penicillium spp. e Aspergillus flavus e no ar, Fusarium spp. e Penicillium spp. No armazenamento, em amêndoas, foi constatado A. flavus, Fusarium spp. e A. nomius; e em cascas, Fusarium spp. e A. flavus. Aflatoxinas e ACP não foram detectados. O aumento do tempo de contato das castanhas-do-brasil com o solo foi acompanhado de maior isolamento de A. flavus; sugerindo contaminação da castanha durante etapa a campo. A. nomius e A. parasiticus foram isolados apenas no armazenamento, indicando contaminação no estoque das amêndoas. / The objective of this present study was analyse, in the field and in the storage, mycobiota and the contamination by (aflatoxins and cyclopiazonic acid from Brazil nuts, soil and air samples to determine route of fungi contamination. The Brazil nuts mycobiota was determine by diluition plating method in Potate Dextrose Agar and Aspergillus flavus parasiticus agar and, micotoxins was done by high performance liquid chromatography. Field samples were collected in: day 0, samples still on the tree; days 5, 10 and 15, samples in contact with soil for 5, 10 and 15 days, respectively. The most prevalent fungi were Aspergillus flavus in fruit pods and nuts and Fusarium spp. in shells. Penicillium spp. and A. flavus were isolated from soil, and Fusarium spp. and Penicillium spp. from air. The storage samples were analysed montly during 11 months; and showed predominance A. flavus, Fusarium spp. e A. nomius in nuts, and Fusarium spp. and A. flavus in shells. Aflatoxins and cyclopiazonic acid were not detected. These findings indicate soil as the main source of fungal contamination of Brazil nuts. A. nomius e A. parasiticus were isolated only in storage, suggesting Brazil nuts contamination occurs in this period.

O presente estudo objetivou avaliar a microbiota fúngica e a ocorrência de micotoxinas por Cromatografia Líquida de Alta Eficiência acoplada à Espectrometria de Massas em amostras de grãos de trigo recém-colhidas e armazenadas. Os resultados revelaram a predominância do gênero Alternaria nas amostras de todas as coletas, seguido de Epicoccum (12,6%), Fusarium (8,3%) e mais 16 outros gêneros de fungos filamentosos. As espécies de Fusarium e Alternaria foram identificadas utilizando métodos moleculares, sendo F. graminearum e A. alternata as espécies mais frequentes. De 70 amostras, 69 (98,6%) estavam contaminadas com a toxina DON (210-2910 ppb) e 3 (4,3%) amostras contaminadas com a toxina ZEA (20-30,1 ppb). O nível de contaminação por DON é inferior aos limites estabelecidos recentemente pela legislação brasileira (3000 ppb), porém superam os limites da Comunidade Européia (1750 ppb). É prudente que se realize um monitoramento contínuo de grãos de trigo a fim de avaliar a exposição dos consumidores às contaminações e estabelecer diretrizes de segurança alimentar. / The present study aimed to evaluated the mycoflora and occurrence of mycotoxins by high performance liquid chromatography coupled to tandem mass in freshly harvested and stored wheat samples. The results showed the predominance of genus Alternaria in all samples, followed by Epicoccum (12.6%), Fusarium (8.3%) and over 16 other genera of filamentous fungi. The species of Fusarium and Alternaria were identified using molecular methods, and F. graminearum and A. alternata were the most frequent species. Of 70 samples, 69 (98.6%) were contaminated with toxin DON (210-2910 ppb), and 3 (4.3%) samples contaminated with the toxin ZEA (20-30.1 ppb). The level of DON contamination is below of the limits recently established by Brazilian legislation (3000 ppb), but exceed the limits of the European Community (1750 ppb). It is prudent to perform a continuous monitoring of wheat to assess consumer exposure to contaminants and establish guidelines for food safety.

O trabalho avaliou micobiota, contaminação por aflatoxinas (AFB), ácido ciclopiazônico (ACP) e fitoalexina (trans-resveratrol) nas variedades de amendoim Runner IAC 886 e IAC-Caiapó, no campo e armazenamento. Nas amostras de campo, na IAC 886 predominou Fusarium spp. nos grãos e cascas, contaminação por AFB em 30% dos grãos, ACP em 70% e trans-resveratrol em 5%. Na IAC-Caiapó prevaleceu Fusarium spp., em todas as amostras, AFB em 25% dos grãos, ACP em 55% e trans-resveratrol em 15%. Nas folhas, trans-reveratrol estava presente em 70%, nas variedades. No armazenamento, observou-se maior freqüência de A. flavus, A. parasiticus. Na IAC 886 AFB foi detectada em 20% nos grãos e 23% nas cascas e ACP em 80% dos grãos. Na IAC-Caiapó, AFB foi encontrada em 13% tanto nas amostras de grãos, como nas cascas e ACP em 70% dos grãos. Os resultados do potencial toxigênico de A. flavus revelaram 81% produtores de aflatoxinas, 95% de ACP e 75% de ambas. Todos A. flavus produtores de esclerócios pertenciam ao grupo L. / This study had assessed mycobiota, contamination by aflatoxins (AFB), cyclopiazonic acid (CPA) and phytoalexin (trans-resveratrol) in the peanuts varieties Runner IAC 886 and IAC-Caiapó, in the field and storage. In the Field samples, in IAC 886 it was predominated Fusarium spp. in grains and shells, contamination by AFB in 30% of the grains, CPA in 70% and trans-resveratrol in 5%. In IAC-Caiapó, it was prevailed Fusarium spp. in all samples, AFB in 25% of the grains, CPA in 55% and trans-resveratrol in 15%. In the leaves, trans-resveratrol was present in 70% in the varieties. In the storage, it was observed a higher frequency of A. flavus, A. parasiticus. In IAC 886, it was detected AFB in 20% of the grains and 23% in the shells, and CPA in 80% of the grains. In IAC-Caiapó, it was found AFB in 13% both in grains as in the shells samples, and CPA in 70% of the grains. The toxigenic potential results of A. flavus revealed 81% aflatoxins producers, 95% CPA producers and 75% both producers. All strains of A. flavus sclerotia producers belonged to the L group.

O presente estudo objetivou avaliar a microbiota fúngica e a ocorrência de micotoxinas por Cromatografia Líquida de Alta Eficiência acoplada à Espectrometria de Massas em amostras de grãos de trigo recém-colhidas e armazenadas. Os resultados revelaram a predominância do gênero Alternaria nas amostras de todas as coletas, seguido de Epicoccum (12,6%), Fusarium (8,3%) e mais 16 outros gêneros de fungos filamentosos. As espécies de Fusarium e Alternaria foram identificadas utilizando métodos moleculares, sendo F. graminearum e A. alternata as espécies mais frequentes. De 70 amostras, 69 (98,6%) estavam contaminadas com a toxina DON (210-2910 ppb) e 3 (4,3%) amostras contaminadas com a toxina ZEA (20-30,1 ppb). O nível de contaminação por DON é inferior aos limites estabelecidos recentemente pela legislação brasileira (3000 ppb), porém superam os limites da Comunidade Européia (1750 ppb). É prudente que se realize um monitoramento contínuo de grãos de trigo a fim de avaliar a exposição dos consumidores às contaminações e estabelecer diretrizes de segurança alimentar. / The present study aimed to evaluated the mycoflora and occurrence of mycotoxins by high performance liquid chromatography coupled to tandem mass in freshly harvested and stored wheat samples. The results showed the predominance of genus Alternaria in all samples, followed by Epicoccum (12.6%), Fusarium (8.3%) and over 16 other genera of filamentous fungi. The species of Fusarium and Alternaria were identified using molecular methods, and F. graminearum and A. alternata were the most frequent species. Of 70 samples, 69 (98.6%) were contaminated with toxin DON (210-2910 ppb), and 3 (4.3%) samples contaminated with the toxin ZEA (20-30.1 ppb). The level of DON contamination is below of the limits recently established by Brazilian legislation (3000 ppb), but exceed the limits of the European Community (1750 ppb). It is prudent to perform a continuous monitoring of wheat to assess consumer exposure to contaminants and establish guidelines for food safety.

O trabalho avaliou micobiota, contaminação por aflatoxinas (AFB), ácido ciclopiazônico (ACP) e fitoalexina (trans-resveratrol) nas variedades de amendoim Runner IAC 886 e IAC-Caiapó, no campo e armazenamento. Nas amostras de campo, na IAC 886 predominou Fusarium spp. nos grãos e cascas, contaminação por AFB em 30% dos grãos, ACP em 70% e trans-resveratrol em 5%. Na IAC-Caiapó prevaleceu Fusarium spp., em todas as amostras, AFB em 25% dos grãos, ACP em 55% e trans-resveratrol em 15%. Nas folhas, trans-reveratrol estava presente em 70%, nas variedades. No armazenamento, observou-se maior freqüência de A. flavus, A. parasiticus. Na IAC 886 AFB foi detectada em 20% nos grãos e 23% nas cascas e ACP em 80% dos grãos. Na IAC-Caiapó, AFB foi encontrada em 13% tanto nas amostras de grãos, como nas cascas e ACP em 70% dos grãos. Os resultados do potencial toxigênico de A. flavus revelaram 81% produtores de aflatoxinas, 95% de ACP e 75% de ambas. Todos A. flavus produtores de esclerócios pertenciam ao grupo L. / This study had assessed mycobiota, contamination by aflatoxins (AFB), cyclopiazonic acid (CPA) and phytoalexin (trans-resveratrol) in the peanuts varieties Runner IAC 886 and IAC-Caiapó, in the field and storage. In the Field samples, in IAC 886 it was predominated Fusarium spp. in grains and shells, contamination by AFB in 30% of the grains, CPA in 70% and trans-resveratrol in 5%. In IAC-Caiapó, it was prevailed Fusarium spp. in all samples, AFB in 25% of the grains, CPA in 55% and trans-resveratrol in 15%. In the leaves, trans-resveratrol was present in 70% in the varieties. In the storage, it was observed a higher frequency of A. flavus, A. parasiticus. In IAC 886, it was detected AFB in 20% of the grains and 23% in the shells, and CPA in 80% of the grains. In IAC-Caiapó, it was found AFB in 13% both in grains as in the shells samples, and CPA in 70% of the grains. The toxigenic potential results of A. flavus revealed 81% aflatoxins producers, 95% CPA producers and 75% both producers. All strains of A. flavus sclerotia producers belonged to the L group.