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Orientador: Glaucia Maria Pastore / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T11:37:31Z (GMT). No. of bitstreams: 1 Barros_FranciscoFabioCavalcante_M.pdf: 816271 bytes, checksum: 1ac93e87eef201f2b03cad5fc4da0cac (MD5) Previous issue date: 2007 / Resumo: A bactéria Bacillus subtilis tem a capacidade de produzir biossurfactantes do grupo dos lipopeptídeos, dentre os quais, o que mais se destaca é a surfactina, um dos que possui maior atividade superficial. Esse composto é capaz de reduzir a tensão superficial da água a 20°C de 72 para 27 mN/m em concentrações menores que 20 _M. A aplicação de resíduos industriais como substrato para produção de biossurfactante de Bacillus subtilis tem sido estudada como forma de reduzir custos associados à produção destes. A manipueira, que é o resíduo líquido da produção de farinha e fécula, tem sido apontada como potencial meio de cultura para processos biotecnológicos, incluindo produção de biossurfactantes. Esse uso tem significativa relevância quando se consideram os resultados de redução de tensão superficial e de produtividade obtidos. Este trabalho estudou o processo produtivo, as propriedades e a estabilidade de biossurfactante produzido pela linhagem LB5a de Bacillus subtilis em escala piloto utilizando manipueira como substrato. O composto produzido foi capaz de reduzir a tensão superficial da água de 72 para 27 mN/m além de apresentar concentração micelar crítica de 12 mg/l. Manteve estabilidade frente à temperatura de 100°C por 140 minutos e 121°C por 60 minutos. Também foi estável na faixa de pH de 6 a 10 e suportou concentrações salinas testadas (de até 20%). A eficiência da extração primária realizada através da coleta de espuma mostrou bons resultados, sendo perfeitamente aplicável ao processo. Além disso, os parâmetros envolvidos no preparo da manipueira foram otimizados visando um melhor aproveitamento do substrato. Os resultados apontam temperatura de aquecimento ótima de 95°C (máxima temperatura testada), tempo de aquecimento de 1 minuto, a aceleração centrífuga de 17,85 G x 103 e o tempo de centrifugação de 14,86 minutos. Os resultados apresentados são bastante animadores em relação à possibilidade de aplicações do biossurfactante produzido em diversos setores. Além de permitir um melhor aproveitamento da manipueira / Abstract: The bacteria Bacillus subtilis is well known by their capacity of production surfactants lipopeptides. Among these, the most studied is surfactin, a powerfull surfactant that reduces the superficial tension of the water from 72 to 27 mN/m in concentrations less than 20_M. The application of industrial wastewaters as substrate for production of biosurfactant by Bacillus has been studied in order to reduce manufacturing costs. Manipueira is the residue from cassava industrialization process to the production of flour and starch. It has been pointed as potential culture medium for biotechnological processes, including production of biosurfactants. This work studied the productive process, the properties and the stability of biosurfactant produced by Bacillus subtilis strain LB5a in pilot scale using manipueira as substrate. The produced compounds were capable of reducing the superficial tension of the water from 72 to 27 mN/m beyond presenting critical micelar concentration of 12 mg/l. It remaing stable on temperature of 100°C during 140 minutes and 121°C during 60 minutes. It was also stabile in the range of pH from 6 to 10 and in saline concentrations (until 20%). The efficiency of the primary extration by foam collection showed good results, being perfectly applicable to the process. Moreover, the involved parameters in the preparation of the manipueira has been optimized with the objective of better using of the substrate. The results presented the optimal points of heating temperature was 95°C (maximum tested temperature), warm up time of 1 minute, the acceleration centrifugal of 17,85 G x 103 and the centrifugalization time of 14,86 minutes. The results showed that the biosurfactant produced have potential applications in several industrial sectors, beyond allowing one better exploitation of the manipueira / Mestrado / Mestre em Ciência de Alimentos

Submitted in fulfilment of the requirements for the Degree of Doctor of Technology: Biotechnology, Durban University of Technology, 2010. / Cassava, (Manihot esculenta Crantz), is used for the production of a variety of West African foods and ranks fourth in the list of major crops in developing countries after rice, wheat and maize. Gari is one of the most popular foods produced from cassava. Cassava may contain high levels of linamarin, a cyanogenic glucoside, which in its natural state is toxic to man. Therefore, some processing methods that can enhance the detoxification of cassava and lead to the improvement of the quality and hygienic safety of the food are vitally important for less toxic products to be obtained. Quality, safety and acceptability of traditional fermented foods may be improved through the use of starter cultures. There has been a trend recently to isolate wild-type strains from traditional products for use as starter cultures in food fermentation. A total of 74 bacterial strains and 21 yeast strains were isolated from a cassava mash fermentation process in a rural village in Benin, West Africa. These strains were assessed, together with 26 strains isolated at the CSIR from cassava samples sent from Benin previously, for phenotypic and technological properties. Twenty four presumptive lactic acid bacteria (LAB) were selected for further phenotypic, genotypic and technological characterization during a research visit to the BFE (now Max Rubner Institute of Nutrition and Food). After assessment, the strains VE 20, VE 36, VE 65b, VE 77 and VE 82 were chosen for further study as starter cultures. These L. plantarum strains were chosen on the basis of predominance and possession of suitable technological properties. The investigation of this study was complemented by further, similar studies on further Gari isolates in Germany by the BFE. That study was done independently from this study, but both studies served to select potential starter cultures for cassava fermentation for the production of Gari, as this was the common goal of the project. Thus, a wider final selection of potential starter cultures was decided on at the project level and this selection was further tested in fermentation experiments. A total of 17 strains were grown in optimized media in 2 L fermenters. These strains were freeze-dried and thereafter tested in lab-scale cassava mash fermentation trials. xiii The strains performed well in the small scale bucket fermentations. There was a rapid acidification evidenced by the increase in titratable acidity, ranging from 1.1 to 1.3 % at 24 hours, and 1.3 to 1.6 % at 48 hours. The effect of the starter was obvious in that it lowered the pH much faster and to lower levels than the control. It appeared that both the processing and starter culture addition played a role in the removal of cyanide during processing of the cassava into Gari. This was evident from the lower cyanide values obtained for fermentations that included starter cultures. The study also showed that especially the L. plantarum group strains could be produced as starter cultures at lower costs than compared to L. fermentum, W. paramesenteroides or L. mesenteroides strains. Overall the results of this study were crucial for the project in showing that a starter culture which is easy and economical to produce and which has the desired attributes is a feasible possibility for application in the field.

A mandioca (Manihot esculenta Crantz) é uma das principais culturas do mundo, havendo grande variabilidade genética. As variedades são classificadas com base na palatabilidade e toxicidade das raízes, em mansas ou doces e bravas ou amargas. Apesar da importância, o potencial da mandioca é pouco explorado, não sendo conhecidos, em nível molecular, os elementos determinantes para as suas características. Assim, pretendeu-se identificar, empregando a 2D-PAGE, proteínas que possam estar associadas com as diferenças físico-químicas das raízes tuberosas de variedades de mesa (IAC 576-70 e IAC 06-01), indústria (Cigana Preta, IAC 12 e IAC 90) e de uso misto (Vassourinha Paulista). Após extração de proteínas e separação por 2D-PAGE, as imagens dos géis foram analisadas no programa Delta2D (DECODON), sendo realizada análise estatística utilizando-se ANOVA (p<0,01), Heat Map e Análises de Componentes Principais (ACP) e de Agrupamentos. Os 146 spots de interesse foram removidos dos géis e suas proteínas digeridas e sequenciadas por espectrometria de massas. Algumas proteínas refletiram as características fenotípicas das variedades em estudo, especialmente entre as de mesa e indústria. Pela ACP, foram explicados 54,54% da variabilidade entre as amostras. A primeira componente separou as variedades exclusivamente de mesa de todas as demais, enquanto a segunda separou a IAC 90 de todas as outras, sendo esta caracterizada por um perfil proteico diferente das demais amostras de uso industrial. A IAC 576-70 e a IAC 12 apresentaram alta correlação positiva, assim como, a Vassourinha e a Cigana. A Análise de Agrupamentos corroborou as informações da ACP, revelando que o proteoma das raízes tuberosas refletiu diferenças fenotípicas entre as variedades. / Cassava (Manihot esculenta Crantz) is a main crop with large genetic variability. The varieties are classified according palatability and toxicity of the roots as sweet or bitter cassavas. Despite its importance, little is known about the molecular basis of phenotypic characteristics. Therefore, this study aimed to identify proteins associated to the differences between the sweet (\'IAC 576-70\' e \'IAC 06-01\'), bitter (\'Cigana Preta\', \'IAC 12\' e \'IAC 90\') and the mixed-use (\'Vassourinha Paulista\') varieties by 2D-PAGE. After the protein extraction and separation by 2D-PAGE, the gel images were analyzed through the software Delta 2D (DECODON), and the statistical analysis were performed with ANOVA (p<0,01), Heat Map, Principal Component Analysis (PCA) and Cluster Analysis. The 146 significant spots were removed from the gels, digested and sequenced by mass spectrometry. Some proteins were related to the physico-chemical characteristics of the varieties, especially between the sweet and the bitter. Variability of the samples was explained at the level of 54,54% by the PCA. The first component separated the sweet varieties from all others while the second one separated the \'IAC 90\' from all others. This variety was characterized by a different protein profile among the bitter cassavas. The \'IAC 576-70\' and the \'IAC 12\' were positively correlated, as well as, \'Vassourinha\' and the \'Cigana\'. Cluster Analysis agreed the PCA information, revealing that the proteomes of the tuberous roots reflected phenotypic differences among the varieties.

Pubagem é o processo de fermentação natural de raízes de mandioca para produção de puba, um alimento tradicional nas regiões Norte e Nordeste do Brasil. Além da fermentação lática, uma ação combinada da pectina-metil-esterase endógena e enzimas microbianas despolimerizantes causa o amolecimento das raízes, que é importante para sua completa desintegração. No entanto, o reconhecimento do ponto final de fermentação e as condições que propiciam puba de boa qualidade não são completamente esclarecidos. O objetivo do trabalho foi estudar a influência da temperatura de fermentação e da adição de enzima como auxiliar de pubagem na textura das raízes e rendimento de puba, procurando associar estes parâmetros para detectar o ponto final desta etapa do processo. Cerca de 1Kg de raízes descascadas e 2 litros de água foram colocados em recipientes plásticos. Diferentes concentrações (0,1 e 2mL) de pectinase comercial de Aspergillus aculeatus/Kg de raiz foram adicionadas em tratamentos separados. Amostras foram incubadas a 25, 30 e 35ºC, por períodos de 48, 72 e 96 horas. Foram efetuadas análises de acompanhamento do processo fermentativo (pH, textura e rendimento) e da composição da farinha de puba obtida. O maior rendimento, de 77% após 72 horas, foi observado no tratamento a 25ºC com 2mL de enzima por Kg de raiz, quando a textura que era de 18,62lbf/g na matériaprima passou para 3,92lbf/g na raiz pubada. Outros rendimentos mais próximos desse valor, 75,7 e 74,7%, foram obtidos respectivamente na fermentação natural a 35ºC e na amostra tratada com 2mL de enzima incubada a 30ºC/48 horas. Os valores de textura foram de 2,50lbf/g para o primeiro tratamento e 2,89lbf/g para o segundo. A relação entre textura e rendimento foi significativa para a temperatura de 30ºC e todos os períodos de 72 horas. A associação entre os dados de textura e rendimento permite concluir que quando a textura alcança valores da ordem de 4lbf/g os rendimentos de puba são maiores, indicando o final do processo. Os rendimentos mais baixos ocorreram para os valores de textura próximos a 2,30lbf/g de amostra, geralmente obtidos em fermentações mais longas e com maior concentração de enzima. / "Retting" is a natural fermentation of cassava roots for the production of puba, a traditional food in the North and Northeast of Brazil. Besides the lactic fermentation, the combined action of the endogenous pectin-methyl-esterase and depolymeryzyng enzymes, causes a softening of the roots which is important for its complete disintegration. However, the recognition of the end point of fermentation and the conditions that lead to good quality puba are not completely understood. The objective of this work was to study the influence of the temperature and enzyme concentration on the yield and texture, aiming at associating these parameters to detect the end point of fermentation. The physicochemical properties of the puba flour were also determined. About 1Kg of peeled roots and 2 liters of water were placed in plastic containers. One or two milliliters of commercial pectinase from Aspergillus aculeatus were added / Kg root, in separate treatments. In the control treatment no enzyme was added. Samples were incubated at 25, 30 and 35ºC and portions removed after 48, 72 and 96 hours for the determination of pH, texture, yield and dry matter. Fibers, starch, total soluble sugars and amylose were determined in the flour of the dried puba. The highest yield of 77% was obtained after 72 hours for the treatment at 25ºC with 2mL of enzyme/kg of root, when the texture values decreased from 18.62lbf/g (raw material) to 3.92lbf/g (retted root). A high yield of 75.7% was also observed in same period, for the natural fermentation at 35ºC, when texture values of 2.50lbf/g were reached. The relation between texture and yield was significant for a temperature of 30ºC and a period of 72 hours. The association between the texture and yield data suggests that when the texture reaches values of the order of 4lbf/g, the yields are higher, indicating the end of the process. The yields were lower when texture values were close to 2.30lbf/g, usually achieved for long fermentations and higher enzyme concentrations.

Atualmente o consumo de xarope de maltose vem crescendo devido ao seu uso em cervejarias e está substituindo progressivamente os adjuntos amiláceos. O xarope de maltose é, tradicionalmente, produzido por meio da hidrólise ácida e/ou enzimática de amido ou flocos de milho. Este trabalho teve como objetivo analisar a possibilidade de obtenção de maltose a partir de outras matérias-primas amiláceas como a mandioca e a puba (produto derivado da fermentação da mandioca) pela ação da a-amilase bacteriana e da a-amilase fúngica sem que fosse necessária a extração do amido. Amostras de mandioca e puba com 10, 20 e 30% de sólidos foram incubadas com a-amilase bacteriana termoestável durante 10, 20 e 30 minutos a 80 0 C, adicionando-se em seguida, µ-amilase fúngica e incubando-se as amostras durante 48 horas a 55 0 C. O grau de sacarificação, expresso em dextrose equivalente (DE), foi determinado pelo método DNS em vários intervalos de tempo. Glicose e maltose foram determinadas por HPLC após 48 horas de sacarificação. Os resultados mostraram que o tempo de ação da a-amilase bacteriana não causou diferenças significativas no grau de hidrólise entre as amostras, mas a concentração de sólidos influiu significativamente no grau da liquefação das amostras. O comportamento das curvas do grau de sacarificação foi semelhante para todos os tratamentos tanto da mandioca quanto da puba. O conteúdo de maltose nas amostras variou entre 30-60% e a glicose entre 0-10% caracterizando um xarope com alto teor de maltose. A eficiência de hidrólise ficou abaixo do esperado. Entretanto, esse fato pode ser explicado pela utilização da mandioca sem extração prévia do amido e pelas dificuldades na extração dos sólidos por centrifugação. Pode-se afirmar que tanto a mandioca quanto à puba podem ser utilizadas como matéria prima em substituição ao milho na obtenção de xarope de maltose através da hidrólise enzimática. A puba, porém, é de mais fácil manuseio sendo que o tratamento com 20% de sólidos, exposto durante 10 minutos a a-amilase bacteriana proporcionou maior rendimento, atingindo 4,2 kg de maltose e 0,3 kg de glicose por 100 kg de mandioca fresca além de proporcionar menor quantidade de resíduo sólidos de 13,7 kg. / Nowadays the consumption of maltose syrups is increasing due to its utilization in breweries where it replaces starch adjuncts. Traditionally maltose syrup has been produced from cornstarch or pellets using acid and/or enzymatic hydrolysis. The objective of this work was to evaluate the possibility of obtaining maltose from starch sources other than maize, such as cassava roots or puba, a fermented cassava product, without extraction of the starch, using bacterial a-amylase and fungal a-amylase for starch hydrolysis. Cassava and puba samples with 10, 20 and 30% dry matter were incubated with termostable bacterial a-amylase for 10, 20 and 30 minutes at 80 0 C, followed by the addition of fungal a-amylase. The samples were incubated for 48 hours at 55 0 C. The degree of saccharification, expressed as dextrose equivalent (DE), was determined by the DNS method. The glucose and maltose contents of the hydrolysate were determined after 48 hours by HPLC. The results showed that the time of action of a-amylase did not influence the degree of saccharification of the samples but the solids concentration significantly affected the hydrolysis degree. The saccharification degree curves were similar for both cassava and puba. The maltose content of the samples varied between 30-60% and the glucose content between 0-10%, which characterized them as "High maltose syrups". The hydrolysis efficiency was lower than expected. However, this fact could be explained by the use of cassava without extraction of the starch and by the difficulty of extracting the solids by centrifugation. It was concluded that for replacement of corn starch, both cassava roots and puba could be used as raw materials for maltose syrup production by enzymatic hydrolysis. However the puba was easier to handle than cassava. The puba treatment consisting of 20% of solids and 10 minutes exposure to a-amylase gave the highest yield reaching 4.2 kg of maltose and 0.3 kg of glucose per 100 kg of raw cassava, in addition to a lower solids residue of 13.7 kg.

Cassava Manihot esculenta Crantz (Euphorbiaceae), is a source of food for more than 700 million people in developing countries and is cultivated in estimated global area of 18.6 million hectares with total annual production of 238 million tonnes. Diseases however, take a substantial toll of yield, with CMD being the most important disease and major constraint for cassava production in Tanzania and Africa. The disease causes an estimated loss of over US$ 14 million per annum. A study was undertaken in 2006/2007 to investigate the influence of satellite DNA molecules on severity of cassava begomoviruses and the breakdown of resistance to cassava mosaic disease (CMD) in Tanzania. The goal was to appraise the nature of resistance to CMD in indigenous and improved cassava cultivars in the presence of resistance-breaking satellites. Three specific aims were earmarked: to identify and characterize cassava mosaic virus isolates and satellite DNA molecules in major cassava growing areas of Tanzania; to screen cassava cultivars for resistance to begomoviruses in presence and absence of the satellite DNA molecules; and to determine the nature of interaction between begomovirus DNAs and Satellite DNA molecules in Nicotiana benthamiana. To achieve these aims, a survey was done in the major cassava growing areas of Tanzania to investigate occurrence of cassava mosaic begomoviruses and associated satellites namely, SatDNA-II and SatDNA-III. Stems from plants showing CMD symptoms were collected from field. The stems were re-planted in screenhouse to study more about the symptoms. Symptomatic leaves from sprouting cuttings were collected for DNA extraction to be used in two downstream assays - amplification of EACMV, ACMV, SatDNA-II and SatDNA-III by polymerase chain reaction (PCR) and sequencing. In another experiment to evaluate cassava cultivars for resistance to CMD in presence of satellites, stem cuttings of the classical CMD-resistant cultivars were planted in greenhouse. Infectious clones of EACMV-TZ and EACMV-UG2 comprising both DNA-A and DNA-B components of bipartite begomoviruses (EACMV-TZ and EACMV-UG2) as well as infectious clones of SatDNA-II and SatDNA-III were bombarded onto the greenhouse cassava plants using a gene gun. Emerging disease symptoms on inoculated plants were scored using standard procedure. Total nucleic acid extraction from the inoculated plants was done and PCR was performed to amplify AC1 and βC1 genes as well as full length SatDNA-II and SatDNA-III. Southern blot analysis was performed to determine the presence of AC1, βC1, SatDNA-II and SatDNA-III on the DNA. In order to study interaction between cassava mosaic begomovirus (EACMV-TZ) and satellites, infectious clones of EACMV-TZ (DNA-A and DNA-B) and that of SatDNA-II and SatDNA-III were used. The clones (DNAs) were used to infect Nicotiana benthamiana by abrasion. Inoculated plants were covered with a plastic dome and placed in insect-free growth chamber for symptom development, which were scored on a standard scale of 1 to 5. Total DNA was extracted from the N. benthamiana leaves and used for Southern blot analysis. Results from the field survey showed that disease incidences varied from 60 to 90% in the Lake Victoria Zone and from 10 to 90% in the Eastern Zone. Cultivar Lyongo had the highest disease symptom severity in the Lake Victoria Zone while in the Eastern zone plants with high severity levels were from cvs Maiza and Tabora. In the screenhouse, some sprouted cuttings remained healthy up to 16 days after planting (DAP) and others recovered from the disease. Reversion was also observed in some cultivars. Using PCR, East African cassava mosaic Tanzania virus (EACMV-TZ) was amplified from 72.8% of tested samples while African cassava mosaic virus (ACMV) was amplified from 4.3%. Five percent of plants had dual infection of the two viruses. While ACMV was detected in samples collected from Lake Victoria, EACMV-TZ was mostly found on samples from the Eastern zone. Sequencing showed the presence of two new virus isolates: EACMV-TZ [TZ113] and EACMV-TZ [TZ108]. Seventy five percent of plants, which showed reversion of symptoms, contained SatDNA-II. It was found that full length SatDNA-II occurred in both zones, while SatDNA-III was exclusive to the Lake Zone. Multiple DNA bands were noted in PCR agarose gels, more so in SatDNA-II than SatDNA-III. For SatDNA-II, the multiple bands were more evident for samples collected from Eastern zone than for those from the Lake Zone. Using primers based on expressed sequence tags (EST-primers) for SatDNA-II (895 bp) and SatDNA-III (306 bp), genome integrated forms of the satellites were amplified from 68% and 71.17% of samples, respectively. Thirty percent of the samples showed co-infection of the satellites. While EST-primers for detection of the integrated forms of SatDNA-III produced single bands on gels, those of SatDNA-II still produced additional bands, most noteworthy being the closely spaced „double bands‟. Upon sequencing, the satellite DNA isolates showed similarity with sequences deposited in the genebank and bearing accession numbers AY836366 and AY836367 for SatDNA-II and SatDNA-III isolates, respectively. Alignment reports (Clustal W) revealed presence of GC-rich regions, TATA protein binding motifs (TATAAAT) and CAAT boxes as well as poly (A) signal. GC-rich regions in SatDNA-II were mostly trinucleotides (CGC) and hexanucleotides (CCGCCG) while in SatDNA-III the regions were trinucleotides (CGC) and pentanucleotides (CCGCC). Following biolistic inoculation, five-week scoring for the symptoms showed that plants from cvs AR37-92, CR27-24 and AR16-3 remained symptomless while plants from cv T200 were symptomatic. PCR amplification of βC1 gene five weeks post inoculation (wpi) gave PCR products in 19.6% of the samples while AC1 was amplified from only two plants. Full-length SatDNA-II was amplified from 70% of DNA samples, mostly from plants in which a begomovirus was co-inoculated with SatDNA-II. Amplification of full-length SatDNA-III from bombarded plants was unsuccessful. Amplification of integrated fragments of SatDNA-II from bombarded plants using EST-primers gave a PCR product in 93.7% of the samples. PCR amplification of the fragments from DNAs extracted from plants of cvs AR17-5 and CR27-24 previously inoculated with EACMV-TZ + SatDNA-II and EACMV-UG2 + SatDNA-III, respectively, gave closely spaced bands on 13% of the DNA samples. Amplification of integrated forms of SatDNA-III gave bands in 52.4% of samples. Probing for full-length SatDNA-II, SatDNA-III and AC1 from DNAs extracted from plants pre-inoculated with these DNAs using DIG- labeled probes gave hybridization signals in 60%, 83% and 68% of the samples, respectively. Further analysis of the signals in the context of screening suggested that cvs AR37-92 and AR37-96 were highly resistant to CMD while cv AR40-10 was susceptible. In the interaction experiment, Nicotiana benthamiana plants inoculated with an infectious clone of EACMV-TZ developed moderate CMD symptoms 7 days post inoculation (dpi) with symptoms consisting of leaf distortion and moderate stunting of plants. There were also plants which recovered from the symptoms by 35 dpi. Plants inoculated with EACMV-TZ + SatDNA-II produced similar symptoms with N. benthamiana plants developing symptoms 7 dpi that became severe by 14 dpi and without recovery even after 35 dpi. Very severe symptoms were also observed when N. benthamiana plants were inoculated with EACMV-TZ + SatDNA-II + SatDNA-III. Plants inoculated with SatDNA-II or SatDNA-III alone remained asymptomatic even after 35 dpi. Southern blot analysis showed clear increase in DNA accumulation when EACMV-TZ was inoculated together with both SatDNA-II and SatDNA-III as compared to when EACMV-TZ was inoculated alone or with SatDNA-II only and probed with EACMV-TZ. In conclusion, symptom recovery and reversion of symptoms in screenhouse plants is associated with virus resistance. There is a wide occurrence of satellites (SatDNA-II and SatDNA-III) across the sampled regions consistent with distribution of their helper cassava begomoviruses. The satellites are of a wider occurrence and diversity in Eastern zone than elsewhere in the country. The occurrence of SatDNA-III was not confined to the Lake zone as previously thought. There is evidence for satellite sequence integration into host plant genome, a further indication that the satellites are wider spread in cassava germplasm than earlier conceptualized. In few instances, both SatDNA-II and SatDNA-III isolates co-existed in the same plant though its effect on symptom enhancement could not be immediately established. The observed recovery in screening studies is thought to result from resistance introduced in the plant materials involved. Since labeled probes for satellites that were used in hybridization had been prepared from satellite sequences considered to be integrated, the hybridization signals did not depend on whether the leaf samples were picked from symptomatic or asymptomatic plants. From the study, three observations clearly suggest that SatDNA-II and SatDNA-III are biologically functional and that their effects on host plants are distinctly different. The study has demonstrated enhanced cassava begomovirus symptoms in N. benthamiana in the presence of satellite DNA molecules. This is the first detailed study undertaken to highlight the occurrence and role played by satellite DNA molecules in breaking the resistance to CMD of cassava cultivars grown in Tanzania. Keywords: Cassava mosaic disease, Cassava mosaic begomoviruses, Satellite DNA molecules, Tanzania.

Phosphorus uptake cassava (Manihot esculanta Crantz) were tested using on thirty-six plants per plot under dryland conditions at four different sites selected Bioresource group 1 (BRG 1) of northern KwaZulu-Natal of South Africa, which is described as Moist, Coastal Forest, Thorn and Palm veld, exhibiting sub-tropical characteristics. Soils in this region are very low in Phosphorus (P) due to high fixation by iron and aluminium oxides. With its high root yields coupled with efficient nutrients miner, cassava removes large quantities of N, P, K and Mg. With the ever-increasing prices of P fertilizer, which impact on the socio-economic livelihood of smallscale farmers, there is the need to look into improving the P uptake by the crop by alternative means apart from using mineral fertilizers. The objective of this study was to investigate the use of Arbuscular Mycorrhizal Fungi (AMF) to improve P uptake by cassava in agricultural soils. Laboratory studies were conducted at Soil Fertility and Analytical Services in Cedara (Pietermaritzburg). A Latin Square design (LSD) was used. Four treatments used were Untreated (Control), P-fertilizer, AMF, and P + AMF. Correlation and path-coefficient (probabilities) were computed. The P + AMF were significantly (p<0.05) taller than those in P-fertilizer treatment plots but were significantly (p<0.05) similar to those in Control and AMF-treated plots. Percent leaf P was statistically similar at the four sites with grand mean of 0.4%. Adding AMF and P+AMF to the soil substantially increased leaf P concentration to 0.5%. Tubers collected from P+AMF-treated plots were significantly (p<0.05) the longest, while those from Control plots were the shortest. P, and AMF-treated plots increased tuber length relative to the Control. All soil treatments significantly (p<0.05) increased tuber yields over the Control-treatment plots. However, AMF and P+AMF treated plots were significantly higher than P and control plots. This study suggests that using AMF or P+AMF can improve cassava yield as compared P alone or control – untreated cassava plants. Cassava producers in northern KwaZulu- Natal should consider using AMF or P+AMF to optimize tuber yield. A further study into the economic implications of the use of AMF is recommended. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2014.

Submitted by Bruno Rossitto de Marchi (bruno_dmarchi@hotmail.com) on 2018-08-08T18:14:08Z No. of bitstreams: 1 Bruno De Marchi Tese Definitivo.pdf: 5408910 bytes, checksum: 6c7b13538e592fdcfc85ae1e3eb12212 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-08-08T18:50:48Z (GMT) No. of bitstreams: 1 de marchi_ br_dr_botfca.pdf: 5386545 bytes, checksum: 8a0580cd031695c504bc6270ee8317c9 (MD5) / Made available in DSpace on 2018-08-08T18:50:48Z (GMT). No. of bitstreams: 1 de marchi_ br_dr_botfca.pdf: 5386545 bytes, checksum: 8a0580cd031695c504bc6270ee8317c9 (MD5) Previous issue date: 2018-07-31 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A mosca-branca, Bemisia tabaci (Gennadius) é uma praga de distribuição global que afeta centenas de diferentes plantas hospedeiras incluindo grandes culturas, olerícolas e ornamentais. B. tabaci causa danos principalmente através da transmissão de vírus de plantas como os begomovirus, crinivirus, ipomovirus, torradovirus e carlavirus. Atualmente, B. tabaci é considerada um complexo de pelo menos 40 espécies crípticas que apresentam diversidade genética, biológica e diferentes composições de bactérias endossimbiontes facultativas. No Brasil, tanto espécies nativas quanto exóticas de mosca-branca são encontradas e ainda há uma escassez de dados genômicos destas populações e das bactérias endossimbiontes. Na África Oriental, altas populações de moscas-brancas estão disseminando diferentes vírus de plantas que causam epidemias na cultura da mandioca (Manihot esculenta) e com perdas na produtividade. A principal forma de manejo desses vírus na África é através da utilização de variedades tolerantes. Portanto, é essencial a identificação de novos genes de resistência para o desenvolvimento de variedades e um manejo eficiente das doenças. O sequenciamento de transcriptomas é uma ferramenta que possibilita uma análise genômica da mosca-branca, dos vírus transmitidos por ela, das bactérias endossimbiontes e das plantas hospedeiras dessa praga. Portanto, os dados genômicos obtidos dão suporte para o desenvolvimento de novas técnicas que podem se tornar futuras alternativas de controle de mosca-branca e dos vírus associados. No Capítulo 1, dados de transcriptomas foram obtidos das diferentes espécies de B. tabaci encontradas no Brasil, tendo sido possível a obtenção de genomas mitocondriais completos de espécies exóticas e nativas de mosca-branca, além de genomas parciais do endossimbionte facultativo Hamiltonella. A análise filogenética revelou que as diferenças genéticas presentes no gene mtCOI entre as espécies nativas e as espécies exóticas se estendem ao genoma mitocondrial e ao endossimbionte facultativo Hamiltonella. Além disso, foi possível verificar uma deleção de aminoácidos somente no gene GroEL de Hamiltonella que está presente em populações de moscas-brancas nativas. Esse gene é conhecido por estar associado a transmissão de vírus de plantas. No Capítulo 2, foram sequenciados transcriptomas de plantas de mandioca naturalmente infectadas com vírus coletadas no campo em diferentes países da África. A partir desses dados, foi avaliada a diversidade de genes do cloroplasto e a relação com diferentes espécies de vírus. Há uma baixa diversidade dentre as cultivares de mandioca atualmente plantadas na África e não foi possível verificar nenhuma relação entre os genes de cloroplastos avaliados e as espécies de vírus detectadas ocorrendo naturalmente no campo. Os dados obtidos reforçam a necessidade da introdução de novos materiais genéticos para aumentar a diversidade genética das cultivares de mandioca plantadas nesses países, a fim de melhorar as alternativas de manejo das epidemias de vírus. / The whitefly, Bemisia tabaci (Gennadius) is a global pest that affects hundreds of different plant hosts including vegetable, fiber and ornamental crops. B. tabaci causes damage mainly by the transmission of plant viruses such as begomoviruses, criniviruses, ipomoviruses, torradoviruses, and carlaviruses. Currently, B. tabaci is known as a complex of at least 40 putative cryptic species that shows genetic and biological diversity and a different composition of bacterial facultative endosymbionts. In Brazil, both exotic and indigenous species of whiteflies are found and there is still a lack of genomic data available among these populations and also their bacterial endosymbionts. In East Africa, high populations of whiteflies are transmitting different plant viruses that are causing epidemics in cassava crops (Manihot esculenta) and leading to great yield losses. Currently, the management of these viral diseases in Africa is carried out mainly by growing cassava tolerant varieties. Therefore, is essential to identify new target genes for the development of new varieties for an efficient management of viral diseases. Transcriptome sequencing is a tool that allows a genomic analysis of the whitefly, whitefly-transmitted viruses, bacterial endosymbionts and plant hosts. Therefore, the genomic data obtained gives support for the development of new technics that might aid for future management alternatives of whiteflies and their associated viruses. In Chapter 1, transcriptome data were obtained from different B. tabaci species found in Brazil which allowed to obtain complete mitochondrial genomes from different whitefly species and draft genomes of the facultative endosymbiont Hamiltonella. The phylogenetic analysis revealed that the genetic differences among exotic and native populations present in the mtCOI gene extends to the mitochondrial genome and to the facultative endosymbiont Hamiltonella. In addition, it was verified an amino acid deletion in the GroEL gene from Hamiltonella present only in native populations of whiteflies. This gene is known to be associated with the transmission of plant viruses. In Chapter 2, transcriptome data were obtained from virus-infected cassava plants collected in the field in different African countries. The data allowed to evaluate the diversity of chloroplast genes and their relationship with different virus species. The data revealed a low genetic diversity among cassava currently grow in East Africa. In addition, there was no direct relationship between the evaluated chloroplast genes and the virus species detected. The obtained data reinforce the need of introduction of new genetic accession to increase the genetic diversity of the currently grown cassava in Africa in order to improve the alternatives of management of viral diseases. / CNPq 131324/2015-5 / CNPq 200826/2015-8

Made available in DSpace on 2014-06-11T19:24:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-25Bitstream added on 2014-06-13T20:52:18Z : No. of bitstreams: 1 cassoni_v_me_botfca.pdf: 551302 bytes, checksum: ad5f4e495789c704c091c046c8b51b44 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Tucupi é um molho ácido preparado e usado na culinária dos estados do Norte do país. Há poucas informações sobre seu preparo. A pesquisa teve por objetivo verificar a viabilidade de fermentação acética da manipueira, água residual do processamento de farinha. A manipueira foi obtida em laboratório a partir da Fécula Branca, cultivar de polpa branca, indicada tanto para fim industrial como alimentício, cultivada em Campo Grande-MS, onde foi realizado o experimento. A manipueira foi caracterizada para cianeto livre, Brix, pH, açúcar redutor, açúcar redutor total e minerais. Para fins de comparação e compreensão do processo de acetificação da manipueira, foram estabelecidos ensaios simultâneos com suco de laranja, considerando que as frutas são tidas como substrato ideal para produção de vinagre. O suco de laranja também foi caracterizado como a manipueira e para ajustar a concentração de açúcares fermentescíveis adicionou-se sacarose comercial a manipueira com o intuito de se igualar o Brix de ambos os substratos. Os ensaios constaram de fermentação alcoólica seguida de acetificação. A fermentação alcoólica da manipueira e do suco de laranja foram realizadas com inoculação com 10 g L-1 de fermento seco da marca Fleischmann. Os fermentados alcoólicos obtidos foram caracterizados para °GL, AR, ART, acidez, pH, dosagem de nitrogênio, fósforo, potássio, cálcio, manganês, enxofre, ferro, zinco, cobre e ácidos orgânicos. Para iniciar o processo de acetificação, a fase inicial constou do isolamento de bactérias acéticas a partir de frutas fermentadas naturalmente em temperatura ambiente. Em seguida, os microrganismos foram purificados, mantidos e multiplicados por isolamento em meio de cultura seletivo adaptado do meio de Frateur e a partir destes foi preparado o inóculo. As bactérias isoladas em meio de Frateur... / The cassava waste water from Brazilian flour processing is used as base for the tucupi manufacture. Tucupi is a sauce prepared and culinary used in the north region of the country and are few information about it in the scientific literature. The research had as objective the evaluation of the cassava wastewater acetification in the tucupi processing. The cassava wastewater was obtained in laboratory from the Fécula Branca with white pulp cultivar, indicated for both industrial and culinary uses, cultivated in Campo Grande-MS, where the experiment was performed. The cassava waste water was characterized for free cyanide, Brix, pH, acidity, reducing sugar, total reducing sugar and minerals.In order to compare and understanding the acetification process of the cassava waste water there were established simultaneous assays using orange juice, considering that fruits are known as ideal subtract for vinegar production. The orange juice was characterized as the cassava wastewater and to adjust the fermentable sugar to the same concentration it was used commercial sugar cane in cassava wastewater. The essays were alcoholic fermentation following by acetification. The alcoholic fermentation of both cassava wastewater and orange juice were performed by the inoculation dried Fleischmann yeast 10 g l-1. The alcoholic fermented obtained were characterized for ºGL, reducing sugar, total reducing sugar, acidity, pH, nitrogen, phosphorus, potassium, calcium, manganese, sulfur, iron, zinc, copper and organic acids To beginning the acetic fermentation it was isolated the acetic bacteria from natural fermented fruits in room conditions. The microorganisms were purified, maintained and multiplied by isolation in a selective medium adapted from Frateur's medium, and from those was prepared the inoculums. The isolated bacteria in the Frateur's medium presented morphologic and biochemical characteristics...(Complete abstract, click electronic access below)