Dissertations / Theses on the topic 'Fungal sporulation'

Apesar da espécie Vigna unguiculata (L.) Walp. ser bastante rústica e estar adaptada às condições adversas de clima e solo brasileiros, seu rendimento é muito baixo. Diversas causas têm sido levantadas para explicar esse comportamento; entre elas destacam-se as doenças fúngicas, sobretudo aquelas cujos patógenos são transmitidos por sementes, em especial a podridão cinzenta do caule, causada por Macrophomina phaseolina (Tassi) Goid. A abordagem analítica desse patossistema revelou alguns problemas emergentes. Entre eles, destacam-se: a) desconhecimento da qualidade sanitária das sementes de caupi, utilizadas para semeadura; b) desunifomidade na metodologia usada para detectar os patógenos presentes nas semente; c) dificuldade na esporulação do patógeno, máxime de alguns isolados reticentes em esporular em meios artificiais de cultivo, cujo comportamento dificulta os trabalhos de seleção de genótipos resistentes; d) carência de medidas de controle do patógeno, que empreguem práticas naturais, como uso de sementes sadias, de indutores de resistência e de cultivares resistentes, de fácil uso e passível de adoção por parte dos produtores. Na estruturação da matriz lógica do presente estudo, referidos problemas foram transformados em objetivos. Os trabalhos foram conduzidos no Departamento de Entomologia, Fitopatologia e Zoologia Agrícola da ESALQ/USP, em Piracicaba-SP. Os resultados indicaram o teste de sanidade de sementes de caupi empregando o método do papel de filtro com restrição hídrica utilizando NaCl a –0,8Mpa, como o mais adequado para detecção dos fungos presentes nas sementes de caupi, especialmente M. phaseolina. A análise sanitária das amostras de sementes originadas de vários estados brasileiros revelou que, em 62% das amostras analisadas, o fungo M. phaseolina estava presente, sendo as amostras originadas do estado da Paraíba, Piauí, Pará e Bahia as que apresentaram maiores níveis de incidência do patógeno. Os melhores níveis de esporulação do patógeno foram conseguidos com a combinação de sobreposição de discos de folhas de trigo ao meio BDA, com temperatura de 25oC. Quanto à identificação de indutores de resistência, capazes de controlar M. phaseolina, os resultados revelaram que o acibenzolar-S-metil (ASM) foi o mais eficiente, quando comparado com quitosana e com um produto silicatado derivado de rocha micronizada (PSiM), apresentando um controle residual por mais de 40 dias após a semeadura. A maior eficiência verificada pelo ASM ocorreu devido a sua capacidade de ativar mecanismos bioquímicos de defesa, configurando-se em efetivo ativador da resistência induzida nas plantas de caupi, por atuar na cinética de importantes enzimas relacionadas à defesa, como a fenilalanina amônia-liase, peroxidase e quitinase. Quanto à reação de cultivares de caupi à doença, foi possível verificar razoável nível de resistência de algumas cultivares, tendo sido consideradas resistentes Mulato, Guariba e Maratauã.
Notwithstanding the specie Vigna unguiculata (L.) Walp is sufficiently rustic and adapted to the adverse conditions of the Brazilian soil and climate, its improvement is very low. Many causes have been raised in order to explain such behavior; among them the fungal diseases stand out, over all those whose pathogens are transmitted by the seeds especially the charcoal rot disease caused by Macrophomina phaseolina (Tassi) Goid. The analytical approach of such pathosystem has revealed some emerging problems. Among them, it stands out: a) the ignorance of the sanitary quality of the cowpea seeds used for sowing; b) the non-uniformity in the used methodology in order to detect the pathogens, which are present in the seed; c) the difficult in pathogen sporulation, principally of some isolated reticent in forming spores in cultivation artificial environments whose behavior hampers the selection works of the resistant genotypes; d) lack of pathogen control measures, which utilize natural practices, such as the use of healthy seeds, resistance inducers and resistant cultivars of easy utilization and liable to adoption by the producers. In structuring the logical matrix of this study, such problems were transformed into objectives. The works were conducted at the Entomology, Phytopathology and Agricultural Zoology Departments of ESALQ/USP, in Piracicaba-SP. The results have pointed out the sanity test of the cowpea seeds through the method of filter paper with hydric restriction using NaCI – 0,8Mpa, as the most suitable for detecting the current fungus in cowpea seeds, especially M. phaseolina. The sanitary analysis of the seeds samples originated from several Brazilian states has revealed that in 63% of the analyzed samples, the fungus M. phaseolina was present, and the samples originated from the states of Paraíba, Piauí, Pará and Bahia were those that have presented higher incident levels of pathogen. The best levels of sporulation were obtained with the combination of the superposition of wheat leaves disks in the middle of BDA in 25ºC. As to the identification of the resistance inducers, capable of controlling the M. phaseolina, the results have revealed that the acinbezolar-S-methyl (ASM) was more efficient when compared to chitosan and with a silicate product originated from micronized rock (PsiM), presenting a residual control for more than 40 days after the sowing. The greatest efficiency ascertained by ASM has occurred due to its capacity of activate the defense biochemistries mechanisms, forming itself in an activator effect of the induced resistance in cowpea plants because it acts in the kinetic of important enzymes related to the defense, such as the phenylalanine ammonia-lyase, peroxidase and chitinase. As to the cowpea cultivars reaction to the disease, it was possible to ascertain a reasonable resistance level of some cultivars, and BR 14 Mulato, Guariba e Maratauã were considered as resistant.

Stagonospora nodorum is a necrotrophic fungal pathogen that is the causal agent of leaf and glume blotch on wheat. Very little is currently known about the molecular mechanisms required for pathogenicity of S. nodorum, despite its major impact on Australian agriculture. S. nodorum is a polycyclic pathogen. Rain-splashed pycnidiospores attach to and colonise wheat tissue and subsequently sporulate within 2-3 weeks. Several cycles of infection are needed to build up inoculum for the damaging infection of flag leaves and heads, sporulation is therefore a critical component of the infection cycle of S. nodorum; our aim is to determine the genetic and biochemical requirements for sporulation for development of control of the pathogen. Disease progression of S. nodorum on wheat cv. Amery was monitored by light microscopy to determine the time point when pycnidia development began. Early pycnidia development was evident 12 days post-infection. This information was used to guide a genomics and a metabolomics based approach to determine the requirements for sporulation in S. nodorum. The genomics approach utilised two cDNA libraries created from sporulating and non-sporulating cultures. EST frequency was used to determine highly expressed genes under the two developmental states. Gene expression from the most highly represented genes during sporulation were confirmed using quantitative PCR. A gene encoding an arabitol 4-dehydrogenase (Abd1), was mutagenised, in its absence sporulation was reduced by approximately 20%. The metabolomics approach isolated metabolites from both in planta infection and in vitro growth. Rapid changes in the abundance of metabolites were detected during the onset of sporulation. Key fungal metabolites identified include mannitol and trehalose. The concentration of both mannitol and trehalose increased dramatically in concert with pycnidia formation. Both mannitol and trehalose have also been linked to pathogenicity in filamentous fungi. Creation of deletion mutants of the gene encoding trehalose 6-phosphate synthase showed the synthesis of trehalose is required for full sporulation of S. nodorum in planta and in vitro.

The fluffy gene of Neurospora crassa is required for asexual sporulation. It encodes an 88 kDa polypeptide containing a typical fungal Zn2Cys6 DNA binding motif. To identify the target genes on which FL may act, I sought to identify target sequences to which the FL protein binds. Several strategies were attempted to obtain purified FL protein. Purification was achieved by expressing the DNA binding domain of FL in Escherichia coli as a fusion with glutathione S-transferase followed by affinity purification using glutathione sepharose chromatography. DNA binding sites were selected by in vitro binding assays. Comparison of the sequences of selected clones suggested that FL binds to the motif 5??-CGG(N)9CCG-3??. A potential binding site was found in the promoter region of the eas (ccg-2) gene, which encodes a fungal hydrophobin. In vitro competitive binding assays revealed a preferred binding site for FL in the eas promoter, 5??-CGGAAGTTTCCTCCG-3??, which is located 1498 bp upstream of the eas translation initiation codon. In vivo experiments using a foreign DNA sequence tag confirmed that this sequence is a target site for FL regulation. Using Saccharomyces cerevisiae as an experimental system, I demonstrated that the C-terminal portion of FL functions in transcriptional activation. Microarray analysis was performed to study the role of fl in gene regulation on a large scale. mRNA levels in a fl mutant were compared to those in a strain overexpressing the fl gene. Experiments with cDNA microarray containing 13% of the total number of predicted N. crassa genes revealed 122 genes differentially expressed in response to overexpression of fl. Among these, eas displayed the greatest level of response. The cDNA microarray approach also revealed a number of genes that may be indirectly regulated by fl but may be involved in development. This information provides a foundation for further analysis of the role of fl in conidial development.

Resumo: Objetivou-se o ajuste de metodologia e seleção de genótipos de guandu para resistência a Macrophomina phaseolina a partir de material obtido pela Embrapa Pecuária Sudeste, e verificar o desenvolvimento micelial e esporulação do fungo em meios de cultura. O trabalho foi conduzido em casa de vegetação na UNESP/Jaboticabal no período de agosto de 2004 a dezembro de 2005. Para o ajuste de metodologia e seleção de genótipos resistentes ao fungo as sementes foram submetidas a escarificação com lixa d'água e inoculação artificial através do método de exposição das mesmas ao patógeno por diferentes períodos, que variaram de O a 72 horas. Foram avaliadas porcentagem de plantas sobreviventes e massa fresca. Já para o crescimento micelial e esporulação do fungo foi utilizado o método de sobreposição de discos de diferentes hospedeiros no meio de cultura. A escarificação das sementes contribuiu para a penetração do fungo nas mesmas o período de 24h de exposição das sementes ao fungo são suficientes para detectar diferenças no grau de resistência dos genótipos. Os genótipos mais resistentes são g167-97, g124-95, g27-94, g40-95, g154-95, g127-97 e g9m-97, e os mais suscetíveis são g48-95, g123-95, g8-95, g168-99 e g1m-95. A sobreposição de discos foliares de guandu em meio BDA e folha de papel de filtro em meio sojinha proporcionam um incremento na esporulação de M. phaseolina.
Abstract: This work had the objective of determining the best schedule for artificial inoculation and select pigeon pea genotypes resistant to Macrophomina phaseolina in material obtained by Embrapa Pecuária Sudeste, and verify the mycelial growth and sporulation of the fungi in middle of culture. The work were carried in greenhouse at the UNESP/Jaboticabal, from August 2004 to December 2005. For the methodology and selection adjustment of resistant genotypes to the fungi the seeds were submitted scarified with water sandpaper and artificial inoculation the seeds were the contact method to fungi for different periods, which varied from O to 72 hours. They were evaluated percentage of surviving plants and fresh mass. For the mycelial growth and sporulation of the fungi was used the superposition of disks method of different hosts in the middle of culture. The scarified of the seeds contributed for penetration of the fungi at the seeds; the period of 24h of contact of the seeds to the fungi enough to detect differences in the resistance degree ofthe genotypes. The genotypes g167-97, g124-95, 927-94, g40-95, g154-95, g127-97 and g9m-97 were found to be the most resistant and most susceptible were g48-95, g123-95, g8-95, g168-99 and g1m-95. The treatment with superposition of the leaf disks of pigeon pea in BDA and disks of filter paper in middle of soybean extract were the treatments that provided better sporulation levei in the conditions of that experiment were half.
Orientador: Rita de Cássia Panizzi
Coorientador: Rodolfo Godoy
Banca: Antonio de Goes
Banca: Patrícia Menezes Santos
Mestre

Arbuscular mycorrhizal (AM) fungi are key soil symbiotic microorganisms, intensively studied for their roles in improving plant fitness and their ubiquity in terrestrial ecosystems. Research on AM fungi is difficult because their obligate biotrophic nature makes it impossible to culture them in the absence of a host. Over the last three decades, Ri T-DNA transformed roots have been the gold standard to study AM fungi under in vitro conditions. However, only two host plant species (Daucus carota and Cichorium intybus) have been routinely used to in vitro propagate less than 5% of the known AM fungal species. There is much evidence that host identity can significantly affect AM symbioses, therefore, we investigated any potential host-specific effects of two novel Ri T-DNA transformed root species, Medicago truncatula and Nicotiana benthamiana, by associating them with seven AM fungal species selected based on their contrasting behaviors when grown with Ri T-DNA transformed D. carota roots. To evaluate the performance of new Ri T-DNA transformed roots to host and propagate AM fungal species, a factorial set-up was used to generate nine unique pairs of hosts (M. truncatula, N. benthamiana, D. carota) and AM fungi (Rhizophagus irregularis, R. clarus, Glomus sp.). Using statistical modeling, all pairs of hosts and AM fungi were compared by their symbiosis development (SD) and sporulation patterns in the hyphal compartments (HCs) of two-compartment Petri dishes. Our results show that 1) most of the variation between host and AM fungus pairs relating to SD or HC sporulation was explained by an interaction between host and AM fungal identity, i.e., host identity alone was not sufficient to explain AM fungal behaviour, 2) AM symbioses involving different combinations of symbiont identities trigger heterogenous fungal behaviours. This work provides a robust framework to develop and evaluate new Ri T-DNA roots for the in vitro propagation of AM fungi, an important asset for germplasm collections and biodiversity preservation.

The ex situ fungal cultures collections represent important biological heritage and is useful for mycologists and plant pathologists, supporting several scientific works. They provide viable pathogens anytime and assisting at identification, morpho-physiological aspects, life cycle, epidemiology, resistance to fungicides and breeding programs in resistance of diseases. However, there is not a universal preservation method that is efficient and suitable for the different groups of fungi. The most appropriate is the one that maintain, even after long periods, the original characteristics of culture: viability, sporulation and pathogenicity, excluding mutations and undesirable contamination. The choice will depend of the laboratory infrastructure, micro-organism, objectives, preferences and knowledge of the researcher. For necrotrophic fungi, after passing their life cycle stage as saprophytes they can be isolated in growing medium, using different preservation methods, especially: periodic transfer, dried host tissues, sterile water (Castellani), mineral oil, sterile soil, freezing, silica gel, lyophilization and cryopreservation. The study aimed to describe the efficiency of sterile soil (68 isolates), resistant structures (Sclerotinia sclerotiorum) in 4°C (10 strains), gelatin (17 strains), mineral oil (31 strains) and silica gel (14 strains) on the maintenance of viability, sporulation and colonization-pathogenicity of phytopathogenic necrotrophic fungi preserved in different dates, in Laboratório de Micologia e Proteção de Plantas (LAMIP), Uberlândia (MG), and in Laboratório de Microbiologia e Fitopatologia (LAMIF), Monte Carmelo (MG). The gelatine method has never been tested for fungi. The viability remained in 38 strains of sterile soil; three of mineral oil, 10 of gelatin. Sclerotia s maximum time of preservation was four years, and all fungal strains were viable on silica gel.
As coleções ex situ de culturas fúngicas são um importante patrimônio biológico, úteis à micologia e fitopatologia como suporte em trabalhos científicos. Disponibilizam patógenos a qualquer momento para: identificação, estudos morfofisiológicos, ciclo de vida, epidemiologia, resistência aos fungicidas e programas de melhoramento, visando resistência a doenças. Não há um método de preservação que seja eficiente e recomendado para os diferentes grupos de fungos, sendo mais adequado aquele que mantiver, mesmo após longos períodos, as características originais da cultura viabilidade, esporulação e patogenicidade , evitando mutações e contaminações indesejadas. A escolha irá depender da infraestrutura do laboratório, do microrganismo em estudo, dos objetivos do trabalho e de preferências e conhecimentos do pesquisador. Para os fungos necrotróficos, que passam alguma fase de seu ciclo de vida como saprófitas, podendo ser isolados em meio de cultivo, utilizam-se: repicagens periódicas, tecidos secos do hospedeiro, Castellani, óleo mineral, terriço, congelamento, sílica-gel, liofilização e criopreservação. O trabalho objetivou avaliar a eficiência de métodos de preservação e o tempo máximo de manutenção da viabilidade, esporulação, colonização/patogenicidade de fungos fitopatogênicos necrotróficos utilizados no Laboratório de Micologia e Proteção de Plantas (LAMIP) em Uberlândia (MG) e no Laboratório de Microbiologia e Fitopatologia (LAMIF) em Monte Carmelo (MG), preservados pelos métodos: terriço (68 isolados), escleródios (Sclerotinia sclerotiorum) em 4 °C (10 isolados), gelatina (17 isolados), óleo mineral (31 isolados) e sílica-gel (14 isolados), em diferentes datas. Mantiveram-se viáveis 38 isolados em terriço, três isolados em óleo mineral e 10 isolados em gelatina. O tempo máximo de preservação de escleródios foi de quatro anos, sendo que todos os isolados em sílica-gel permaneceram viáveis.
Mestre em Agronomia

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
Objetivou-se o ajuste de metodologia e seleção de genótipos de guandu para resistência a Macrophomina phaseolina a partir de material obtido pela Embrapa Pecuária Sudeste, e verificar o desenvolvimento micelial e esporulação do fungo em meios de cultura. O trabalho foi conduzido em casa de vegetação na UNESP/Jaboticabal no período de agosto de 2004 a dezembro de 2005. Para o ajuste de metodologia e seleção de genótipos resistentes ao fungo as sementes foram submetidas a escarificação com lixa d'água e inoculação artificial através do método de exposição das mesmas ao patógeno por diferentes períodos, que variaram de O a 72 horas. Foram avaliadas porcentagem de plantas sobreviventes e massa fresca. Já para o crescimento micelial e esporulação do fungo foi utilizado o método de sobreposição de discos de diferentes hospedeiros no meio de cultura. A escarificação das sementes contribuiu para a penetração do fungo nas mesmas o período de 24h de exposição das sementes ao fungo são suficientes para detectar diferenças no grau de resistência dos genótipos. Os genótipos mais resistentes são g167-97, g124-95, g27-94, g40-95, g154-95, g127-97 e g9m-97, e os mais suscetíveis são g48-95, g123-95, g8-95, g168-99 e g1m-95. A sobreposição de discos foliares de guandu em meio BDA e folha de papel de filtro em meio sojinha proporcionam um incremento na esporulação de M. phaseolina.
This work had the objective of determining the best schedule for artificial inoculation and select pigeon pea genotypes resistant to Macrophomina phaseolina in material obtained by Embrapa Pecuária Sudeste, and verify the mycelial growth and sporulation of the fungi in middle of culture. The work were carried in greenhouse at the UNESP/Jaboticabal, from August 2004 to December 2005. For the methodology and selection adjustment of resistant genotypes to the fungi the seeds were submitted scarified with water sandpaper and artificial inoculation the seeds were the contact method to fungi for different periods, which varied from O to 72 hours. They were evaluated percentage of surviving plants and fresh mass. For the mycelial growth and sporulation of the fungi was used the superposition of disks method of different hosts in the middle of culture. The scarified of the seeds contributed for penetration of the fungi at the seeds; the period of 24h of contact of the seeds to the fungi enough to detect differences in the resistance degree ofthe genotypes. The genotypes g167-97, g124-95, 927-94, g40-95, g154-95, g127-97 and g9m-97 were found to be the most resistant and most susceptible were g48-95, g123-95, g8-95, g168-99 and g1m-95. The treatment with superposition of the leaf disks of pigeon pea in BDA and disks of filter paper in middle of soybean extract were the treatments that provided better sporulation levei in the conditions of that experiment were half.

A incidência e severidade da mancha de cercospora, causada por Cercospora zeae-maydis Tehon & Daniels, aumentou significativamente em território brasileiro a partir do ano 2000, sendo hoje considerada uma das principais doenças foliares da cultura do milho. Mesmo assim, poucos estudos com este patossistema foram realizados no Brasil. Este trabalho teve por objetivo determinar meio de cultura e regime luminoso para adequada esporulação de C. zeae-maydis, estudar a reação de um grupo de 118 linhagens endogâmicas de milho quanto a resistência ao patógeno em dois ambientes distintos (Indianópolis-MG e Jardinópolis-SP), observar aspectos microscópicos da esporulação, germinação e penetração em hospedeira suscetível e avaliar diferenças morfológicas, genéticas e de agressividade entre isolados coletados na região centro-sul do país. Os resultados indicaram que a melhor esporulação do fungo foi obtida em meio V8 e suco de tomate temperado quando submetidos a fotoperíodo 12/12h (luz/escuro). Quanto a reação das linhagens à doença, foi possível verificar interação diferencial significativa entre genótipo de milho e os dois ambientes, indicando que fatores ambientais ou patogênicos, distintos entre os locais, podem ter contribuído para os discrepantes comportamentos de alguns genótipos. Também foi possível verificar elevado nível de resistência em 12 linhagens em ambos locais, demonstrando a existência de genótipos mais estáveis para resistência com possibilidade de uso em programas de melhoramento da cultura. Através da análise do padrão de restrição gerado pela digestão da região ITS-5.8S do rDNA, de 104 locos AFLP e de mensurações morfométricas dos conídios, foi possível verificar a existência de dois grupos geneticamente distintos de C. zeae-maydis em território brasileiro. Estes são relatados na literatura como grupos I e II ou espécies afins (siblings species). Estes grupos foram detectados em todos os locais de coleta do território brasileiro, com exceção de Goiás, onde o grupo I não foi observado. Quanto aos aspectos microscópicos deste patógeno, foi possível verificar que sob condições ambientais adequadas a germinação dos esporos ocorre 13 horas após o contato do esporo com a hospedeira, e a penetração, via estômato, tem início 16 horas após a inoculação. Também foi observado o fenômeno da conidiação microcíclica nos isolados brasileiros. Vinte e seis por cento daqueles pertencentes ao grupo I produziram microconídios, enquanto nenhum do grupo II apresentou esta característica. Deste modo, este é o primeiro relato da existência deste fenômeno no grupo I e ausência no grupo II. Estes estudos demonstram que a população brasileira de C. zeae-maydis se assemelha àquelas existentes nos Estados Unidos e na África, com a prevalência dos dois grupos genéticos.
The incidence and severity of cercospora leaf spot, caused by Cercospora zeae-maydis Tehon & Daniels, increased significantly in Brazil in 2000, being considered today one of the major leaf disease of the crop. Despite this, few researches about the pathosystem come being carried in Brazil. The aims of this work were to identify the suitable culture media and light conditions for sporulation of C. zeae-maydis; to study the reaction of 118 mayze genotypes to pathogen in two different locations (Indianópolis – Minas gerais State and Jardinópolis – São Paulo State); to observe some microscopical aspects of esporulation, germination and penetration in a susceptible maize genotype; and finally to assess morphological and genetic differences among a group of isolates collected in center-south Brazil. The results showed that the better culture media for esporulation was the V8 media and tomato juice, under 12-hours photoperiod. Concerning to genotype reaction to disease, it was possible to verify significant interaction between genotypes and environment, indicanting that environmental or pathogenic factors, distinct between locations, may have influenced the reactions of some genotypes. It was possible to identify highly level of resistance in 12 lines in both places, evidencing the existence of stable genotypes that can be used in breeding programs. Analysis of restriction fragments from ITS-5.8S of rDNA, 104 AFLP loci, and conidial measurements, showed the existence of two genetically divergent groups of C. zeae-maydis in Brazil. These groups are similar to the ones reported previously reported as I and II groups or siblings species. Both groups were detected in all sampled regions, except Goiás State where no isolates from group I were detected. Concerning to microscopic traits, it was possible to verify that the brazilian isolates of this pathogen have the ability for production of microconidia. Twenty six percent of the isolates of the group I produced microconidia, while none of the group II showed this trait. Thus, this is the first report with presence of MC in the group I but absence in the group II. The results showed that Brazilian isolates are very similar to isolates from USA and Africa, occurring both genetic groups.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior-CAPES
The aim of this study was to observe the germination, production of glomalin and monitor development of species of mycorrhizal fungi (AMF) of the germplasm bank of Embrapa in root organ culture (ROC) of basil and bryophytes in vitro, analyzing their interaction with the hosts and the influence of the culture medium enriched with humic acids on fungal growth and bryophyte Lunularia cruciata. For this, some AMF species were selected and had their glomerospores extracted and subjected to surface disinfection process, placed in water-agar medium and temperature-controlled chamber to germinate. A germination test was conducted for 15 days, and the results were analyzed by ANOVA and Tukey test applied to 5% probability. Species with germinated glomerospores (Gigaspora margarita, Glomus manihots, Scutellospora heterogama and Glomus proliferum) were placed in ROC of purple basil where they had their growth observed until 100 days after inoculation. Also as part of the characterization of AMF species it was quantified the level of glomalin in the samples of multiplication and the results were subjected to analysis of variance and Scott-Knott test at 5% probability. In the second chapter it was investigated the effect of mycorrhizal association in ROC of purple basil, and in the third chapter the influence of different concentrations of humic acid and association with growth of Lunularia cruciata (area and length). The results were submitted to ANOVA and Tukey test at 5% probability. Scutellospora heterogama was the species with higher germination rates of glomerosporos, followed by Gigaspora margarita. The species of Glomus sporulated after formation of symbiosis. The amount of glomalin produced by different AMF was distinct, especially in total glomalin fraction. Different AMF species did not show difference in efficiency to promote development of Ocimum basilicum transformed roots. The growth of basil transformed roots in the MSR was extended from 15 days after inoculation with mycorrhizal fungi. The usage of humic acids in the culture medium in concentrations of 20 and 80 mg CL-1 enhanced growth of bryophyte L. cruciata, and its association with mycorrhizal fungi, as well as promoted the highest number of spores of Gl. proliferum. The association L. cruciata and AMF was characterized as mutualistic, since both had advantages in growth and sporulation. Gigaspora margarita and Glomus proliferum increased growth of Lunularia cruciata.
O objetivo do trabalho foi observar a germina??o e produ??o de glomalina e acompanhar desenvolvimento de esp?cies de fungos micorr?zicos arbusculares (FMA) do banco de germoplasma da Embrapa em ra?zes geneticamente modificadas de manjeric?o e em bri?fitas in vitro. Ainda, avaliar sua intera??o com os hospedeiros e a influ?ncia de meio de cultura enriquecido com ?cidos h?micos no crescimento do fungo e da bri?fita Lunularia cruciata. Para isso algumas esp?cies de FMAs foram selecionadas e tiveram seus glomerosporos extra?dos e submetidos ao processo de desinfesta??o superficial, colocados em meio Agar?gua e c?mara com temperatura controlada para germinar. Realizou-se teste de germina??o por 15 dias e os resultados foram submetidos a an?lise de vari?ncia e aplicado teste de Tukey ? 5% de probabilidade. Esp?cies com glomerosporos germinados (Gigaspora margarita, Glomus manihots, Scutellospora heterogama e Glomus proliferum) foram colocadas em ra?zes modificadas de manjeric?o roxo onde tiveram seu crescimento observado at? 100 dias ap?s a inocula??o. Ainda como parte da caracteriza??o de esp?cies de FMAs foi realizado a quantifica??o dos teores de glomalina nas amostras de multiplica??o sendo os resultados submetidos a an?lise de vari?ncia e aplicado teste de Scott-Knott ? 5% de probabilidade. No segundo cap?tulo foi verificado o efeito da associa??o FMAs em ra?zes modificadas de manjeric?o roxo e no terceiro cap?tulo a influ?ncia da associa??o ?cido h?mico em diferentes concentra??es, bri?fita Lunularia cruciata (?rea e comprimento) e FMAs. Os resultados foram submetidos ? an?lise de vari?ncia e teste de Tukey a 5% de probabilidade. Scutellospora heterogama foi a esp?cie com maiores taxas de germina??o de glomerosporos, seguida da Gigaspora margarita. As esp?cies de Glomus esporularam logo ap?s a forma??o da simbiose. A quantidade de glomalina produzida pelos diferentes FMAs foi distinta, em especial na fra??o glomalina total. As diferentes esp?cies de FMAs n?o apresentaram distin??o na efici?ncia de promover o desenvolvimento das ra?zes transformadas de Ocimum basilicum. O crescimento de ra?zes transformadas de manjeric?o em meio MSR foi ampliado a partir dos 15 dias ap?s a inocula??o de fungos micorr?zicos. O uso de ?cidos h?micos no meio de cultura em concentra??es de 20 e 80 mg C.L-1 incrementou o crescimento da bri?fita Lunularia cruciata e sua associa??o com fungos micorr?zicos arbusculares, assim como promoveram a maior esporula??o de Gl. proliferum. A associa??o Lunularia cruciata e FMAs foi caracterizada como mutualista j? que ambos apresentaram benef?cios em crescimento e esporula??o. Gigaspora margarita e Glomus proliferum promoveram maior crescimento de Lunularia cruciata.

Neste trabalho realizamos a análise das variações na expressão gênica global durante a fase de esporulação do fungo aquático Blastocladiella emersonii utilizando a tecnologia dos microarranjos de cDNA em lâminas contendo 3.773 genes distintos. Ao todo 615 genes foram classificados como induzidos enquanto 645 foram classificados como reprimidos ao longo da esporulação. As categorias funcionais mais representadas entre os genes induzidos foram: microtúbulo e citoesqueleto, transmissão de sinal, atividade de ligação ao íon Ca2+, proteólise (apenas no início da esporulação) e biogênese e organização do cromossomo (apenas no final da esporulação). Dentre os genes reprimidos, as categorias funcionais mais representadas foram: biossíntese de proteína, transporte de carboidratos e metabolismo energético. A comparação dos dados de expressão gênica da esporulação com aqueles obtidos recentemente em nosso laboratório para a germinação mostrou um grande número de genes regulados inversamente ao longo das duas fases de diferenciação do ciclo de vida de B. emersonii. Muitos genes induzidos na esporulação são reprimidos na germinação e vice versa. Analisamos também o efeito de glicose e triptofano sobre a expressão gênica durante a formação dos zoósporos, tendo em vista que tais nutrientes são capazes de inibir a esporulação de B. emersonii. Nossos resultados mostraram que na presença de glicose (1%) genes envolvidos na composição e atividade do citoesqueleto foram superexpressos, enquanto na presença do aminoácido triptofano houve um aumento na expressão de genes envolvidos no processo de enovelamento de proteínas e proteólise, e na resposta ao estresse oxidativo. Além disso, genes envolvidos no processo de esporulação propriamente dito foram reprimidos durante o tratamento com triptofano. Investigamos também a via de sinalização por GMP cíclico (cGMP), cujos níveis aumentam consideravelmente durante a esporulação de B. emersonii. Iniciamos o estudo com uma busca no banco de ESTs de B. emersonii (http://blasto.iq.usp.br) por seqüências que codificassem enzimas envolvidas na síntese e degradação de cGMP. Foram encontradas três ESTs que codificam domínios catalíticos que parecem pertencer a três diferentes guanilato ciclases e uma EST codificando uma fosfodiesterase com alta similaridade com fosfodiesterases que possuem alta afinidade por cGMP. Experimentos de microarranjos de cDNA validados por RT-PCR quantitativo em tempo real mostraram que os quatro transcritos são expressos durante esporulação, com picos de indução durante a fase tardia da esporulação, momento em que ocorre a biogênese dos zoósporos. Além disso, dados obtidos a partir de experimentos in vivo e in vitro utilizando inibidores das enzimas guanilato ciclase e óxido nítrico sintase, sugeriram a participação do íon Ca2+ e do radical livre óxido nítrico (•NO) na atividade de guanilato ciclase, em uma via do tipo Ca2+-•NO-cGMP.
In the present work, we analyzed global gene expression changes during the sporulation phase of the aquatic fungus Blastocladiella emersonii using cDNA microarray technology with chips containing 3773 distinct genes. A total of 615 genes were upregulated and 645 were down-regulated along the sporulation of the fungus. The overrepresented functional categories among the induced genes were: microtubule and cytoskeleton, signal transduction, Ca2+ binding activity, proteolysis (only at the beginning of sporulation), and chromosome biogenesis and organization (only at the end of sporulation). Among the down-regulated genes, the over-represented functional categories were: protein biosynthesis, carbohydrate transport, and energetic metabolism. Sporulation gene expression data were compared with those obtained recently in our laboratory for the germination phase, showing that a great number of genes are inversely regulated along the two differentiation stages of B. emersonii life cycle. We also analyzed the effects of glucose and tryptophan on gene expression during biogenesis of the zoospores, as such nutrients are able to inhibit B. emersonii sporulation. Our results showed that in the presence of glucose (1%) genes related to activity and composition of cytoskeleton were over-expressed, while in the presence of tryptophan genes involved in protein folding, proteolysis and oxidative stress were induced. In addition, genes involved in the sporulation process per se were downregulated by tryptophan treatment. We also investigated the cyclic GMP signaling pathway, as the levels of this cyclic nucleotide increase considerably during B. emersonii sporulation. Firstly, we searched for sequences encoding enzymes involved in cGMP synthesis and degradation using the B. emersonii EST databank (http://blasto.iq.usp.br). Three sequences were found encoding distinct guanylate cyclase catalytic domains, and one showed high similarity with phosphodiesterases that exhibit high affinity for cGMP. Microarray experiments, validated by real time quantitative RT-PCR, showed that the four transcripts are induced during sporulation, reaching maximum levels at the late stages of sporulation, when zoospore biogenesis occurs. In addition, data obtained from in vivo and in vitro experiments using inhibitors for the enzymes guanylate cyclase and nitric oxide synthase indicated the involvement of the ion Ca2+ and the free radical nitric oxide (•NO) in guanylate cyclase activity, suggesting the existence of a Ca2+-• NO-cGMP signaling pathway.

Com o intuito de gerar subsídios para melhoria do processo de produção de Metarhizium anisopliae, o presente estudo teve como principais objetivos, determinar os efeitos de inibidores de proteinases de soja no crescimento vegetativo, esporulação e virulência do fungo e comparar a produção, viabilidade e virulência dos conídios produzidos em diferentes tipos de arroz e aditivos. A adição de 5 g.L-1 de inibidores de proteinases semi-purificados de soja ou 0,5 g.L-1 de inibidor purificado do tipo Kunitz no meio de cultura ME resultou em grande aumento na esporulação (de duas a 75 vezes) sem afetar a viabilidade dos conídios de quatro isolados (ESALQ-1037, IBCB348, E9, F20) de M. anisopliae. A presença destes inibidores de proteinases também alterou a morfologia dos conídios produzidos em ME. Os mecanismos responsáveis por estas alterações fisiológicas não foram determinados, mas provavelmente estejam associados à ação anti-nutricional, diminuindo a absorção protéica e estimulando a esporulação. Os conídios produzidos no meio com adição de 0,5 g.L-1 de inibidor de proteinase do tipo Kunitz ou com 2,5 g.L-1 de albumina de soro bovino e no meio BDA apresentaram virulência superior aos conídios produzido no meio ME sem inibidores. Quando o inibidor de proteinase do tipo Kunitz foi adicionado à suspensão de conídios do fungo antes da pulverização de lagartas de Diatraea saccharalis, a eficiência de controle foi 35,1%, inferior ao apresentado pelos demais tratamentos sem a presença deste inibidor. Nos estudos para determinação dos melhores tipos de arroz para produção de M. anisopliae, tentou-se correlacionar à produção de conídios com características destes substratos como valor nutricional, teor de resíduos de agrotóxicos e densidade de microorganismos. O arroz parboilizado foi responsável pela maior produção de conídios (4,38 x 109 conídios.g-1). Este tipo de arroz apresentou teor de proteína bruta menor do que a maioria dos arrozes e o maior teor de umidade (41,3% após a autoclavagem). Além disto, os grãos após autoclavagem ficaram menos gelatinosos e mais soltos, o que facilita o processo de produção do fungo. Enquanto que os arrozes dos tipos brancos polidos, canjicão e integral ficaram pegajosos e formaram grumos, o que provavelmente deve acarretar em menor superfície de desenvolvimento para o fungo. O segundo melhor arroz, o canjicão, com produção de 3,42 x 109 conídios.g-1, teve a maior quantidade de fungos contaminantes nos grãos crus. Quantidades intermediárias de conídios foram produzidas pelos arrozes branco polido irrigado, de terras altas e orgânico. O integral foi o que resultou na menor quantidade de conídios (1,53 x 109 conídios.g-1), sendo o mais rico em minerais, proteína bruta e extrato etéreo. Nenhum dos aditivos (farelo de soja, grãos de soja partidos, extrato de soja, peptona de soja, inibidores de proteinases de soja semipurificados e purificado do tipo Kunitz, polpa cítrica e levedura) resultou em aumento na produção de conídios em comparação com o arroz parboilizado sem aditivos. Os conídios produzidos em todos os arrozes e aditivos apresentam viabilidade superior a 99%. As vantagens da utilização do arroz parboilizado levando-se em consideração custo, facilidade de manuseio e produtividade são discutidas.
In order to optimize the production process of Metarhizium anisopliae, the present study aimed to determine the effects of soybean proteinase inhibitors on growth, sporulation and virulence of the fungus and to compare yield, viability and virulence of M. anisopliae conidia produced in different types of rice and additives. The addition of 5 g.L-1 of semi-purified soybean proteinase inhibitor or 0.5 g.L-1 of Kunitz-type inhibitor purified on the culture medium ME resulted in large increases in sporulation (two to 75 times) without affecting the viability of conidia of four M. anisopliae isolates (ESALQ-1037, IBCB348, E9, F20). The presence of proteinase inhibitors altered morphology of conidia produced in ME. Mechanisms responsible for these physiological changes in the fungus have not been determined, but it is probably associated to an anti-nutritional action, reducing the absorption of protein and stimulating sporulation. Spores produced in the medium with the addition of 0.5 g.L-1 Kunitz-type inhibitor purified or 2.5 g.L-1 of bovine serum albumin (BSA) and PDA medium showed higher virulence of conidia produced in ME without inhibitors. When the Kunitz-type inhibitor was added to conidial suspension of the fungus before spraying larvae of Diatraea saccharalis, control efficiency was 35.1% lower than that presented in other inhibitor-free treatments. In the studies aiming to determine the best types of rice for production of M. anisopliae, we tried to correlate production of conidia with characteristics of these substrates such as nutritional content, pesticide residues and density of microorganisms. The parboiled rice was responsible for greater production of conidia (4.38 x 109 conidia.g-1). This type of rice showed crude protein content lower than most rice and the highest moisture content (41.3% after autoclaving). Besides that, grains became less gelatinous and loose after autoclaving, and these feature favored fungus production. While types of polished white, brown rice and course (broken) rice grain were sticky and formed clumps, providing a smaller area for fungus development. The second best rice, course rice grain, with production of 3.42 x 109 conidia.g-1, had the highest amount of fungal contaminants in raw grains. Intermediate amounts of conidia were produced by white irrigated polished rice, upland rice and organic rice. The brown rice was the kind that resulted in fewer conidia (1.53 x 109 conidia.g-1), being the richest in minerals, protein and lipids. None of the additives (soybean meal, soybean parties, soy extract, soy peptone, semi-purified soybean proteinase inhibitor, Kunitz-type inhibitor purified, citrus pulp and yeast) resulted in increased production of conidia compared to parboiled rice without additives. Conidia produced in all types of rice and additives presented viability greater than 99%. The advantages of the use of parboiled rice taking into consideration the cost, easy handling and productivity are discussed.

La croissance, la sporulation, la germination ainsi que la production de metabolites de champignons filamenteux et de levures sont tres sensibles aux variations energetiques de l'eau du milieu (activite de l'eau, pression osmotique). Des modeles mathematiques permettent de predire l'influence de l'eau sur les microorganismes.

Stagonospora nodorum is the causal agent of the wheat disease Stagonospora nodorum blotch (SNB). It is of major economic importance within Australia where, in recent growing seasons, it has caused upwards of A$100 million in losses. The severity of the disease is dependent upon several factors, of which asexual sporulation is a major contributor. The aim of this study was to identify proteins that are differentially abundant between a series of S. nodorum strains across sporulation, and using this knowledge, investigate the role they facilitate within asexual development. A quantitative multiplexed 2D LC-MALDI-TOF/TOF MS analysis of S. nodorum, and three mutants previously identified as perturbed at various stages in the development of asexual sporulation, sch1, stuA, and mpd1, was performed using the iTRAQ 8-plex labelling system. This analysis identified a selection of proteins with possible roles in asexual development. The identified proteins were evaluated against a range of criteria, including postulated roles, gene expression, and across which interactions they were altered. These comparisons resulted in the selection of four proteins for further study. These proteins were an inducible near-UV protein (Uvi1), a formate dehydrogenase (Fod1), a predicted HSCARG dehydrogenase (Hsc1), and lastly, a protein of unknown function, SNOG_08052. The gene encoding for each protein of interest was identified, and through homologous recombination knockout mutants were constructed. Characterisation of the four mutants was performed to determine the role of each of these proteins during asexual sporulation. The first three mutants presented with phenotypes not significantly different from the wildtype strain. The SNOG_08052KO strains showed a reduction in sporulation when grown on MM agar supplemented with sucrose as the sole carbon source. This phenotype was complemented by the addition of mannitol to the media. In addition the SNOG_08052KO strains showed significantly increased enzymatic activities for pathways related to mannitol metabolism and reduced pathogenicity in planta. To investigate the biochemical aspects of this strain further, a metabolite analysis was performed. This identified a number of key differences between the wildtype and knockout strains of S. nodorum. An increase in abundance of the metabolite glucose was accompanied by decreases in the abundances of a range of polyols. This suggests that disruption of SNOG_08052 causes an inability of the mutants to accumulate or process simple sugars. The metabolites mannitol and trehalose, which have previously been linked to sporulation, were differentially abundant within the mutants further confirming their involvement in sporulation. This study has utilised a shotgun proteomics workflow and identified a selection of proteins linked to sporulation, and whilst some of these proteins have previous links to sporulation, many do not. Through the use of reverse genetics this study successfully disrupted the genes encoding for four of those proteins, of which one, SNOG_08052, has been found to play a role in development of asexual sporulation. Metabolic assessment of the non-sporulating SNOG_08052KO mutants revealed the ability to accumulate and utilise specific sugars is central to sporulation.

碩士
輔仁大學
生物學研究所
83
In this study,we used tomato and Fusarium oxysporum to study the interactions between plants and pathogens. The protein extracts from tomato cultivar 299 and CF4 can induce sporulation of Fusarium oxysporum f. sp. lycopersici FO26, but the protein extract from tomato cultivar 571 does not have this effect. We further purified the protein which can induce sporulation of FO26 from tomato cultivar CF4 by using DEAE chromatography.It can only be found in tomato cultivars CF4 and 299 which were susceptible to FO26 and can't be found in tomato cultivar 571 which was resistant to FO26.This protein was heat- stable and it's molecular weight was about 33 KDa.The induction of sporulation of FO26 by the partial purified plant protein was influenced by the treatment of divalent cations, various pH value and light. 0.1mM Mn+2 can inhibit the sporulation of FO26. The optimal pH for sporulation was 6.5 . Conidial production increased 4-fold in continueous light than in darkness. This protein has differential effects of inducing sporulation in different Fusarium oxysporum races. Based on these results, we hypothesized that the protein inducing sporulation was involved in tomato susceptibility toward pathogen.

Spore formation is an essential step in the fungal life cycle that contributes to the dispersal of the organism and also to survival under harsh environmental conditions. The morphology of spores shows an astonishing diversity in the fungal kingdom and varies from very simple round and small spores to very complex multi-armed or sigmoid structures. With exception of the regulation of ascospore formation in Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are well-characterized model organisms for spore development in fungi, little is currently known about the regulation of more complex spore morphologies. In this study, the filamentous ascomycete Ashbya gossypii is used as a model system for the investigation of a complex and composite spore morphology. A. gossypii produces linear, needle-shaped spores possessing a length of 30 µm, which can be divided into three major segments: a rigid tip segment, a more fragile membrane compartment and a stable tail-cap. Furthermore, the different compartments were shown to correlate with distinct materials. While the tip segment and the tail-cap of the spores consist of stabilizing materials like chitin and chitosan, these materials are absent from the compartment in the middle. The actin cytoskeleton plays an essential role in several steps of spore formation in A. gossypii. Different regions of actin accumulation were identified that directly correlate with the developing spores. Especially the developing tip segment is characterized by heavy-bundled linear actin structures. Furthermore, proteins of the formin family, a class of actin organizing proteins, were identified to be directly involved in spore formation in A. gossypii. The formin AgBnr2 fulfills an actin-related key function during spore development by linking actin to the spindle pole body during sporulation. Downregulation of AgBNR2 leads to severe sporulation defects, indicating a central function in spore development. Moreover, AgBni1, another representative of the formin family, also has a regulatory function in size determination of the typical needle-shaped spores of A. gossypii. Using a modified yeast two-hybrid approach, four potential activators of the formin AgBni1 were identified: the Rho-type GTPases AgRho1a, AgRho1b, AgRho3 and AgRho4. The interaction of AgBni1 with the two Rho1 GTPases plays an important role during spore development. In this study, the Rho binding domain of AgBni1 was further examined to identify amino acids that are essential for the interaction with the Rho-type GTPases. Using random mutagenesis combined with a two-hybrid screen, the point mutation S250P in the Rho binding domain of AgBni1 was identified to reduce the interaction of the formin with the Rho1 GTPases. Integration of AgBni1 S250P causes an increase in spore length, suggesting a direct effect of this signaling pathway in spore length determination. An actin-regulating protein network that includes the formin AgBni1, the Rho-type GTPases AgRho1a and AgRho1b and the paxillin-like protein AgPxl1 was identified to be mainly involved in the regulation of the spore length. Thereby, this network seems to be involved in the arrangement of the different spore compartments via the actin cytoskeleton.