Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 101-112). Also available on the World Wide Web.

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

Most plant oxylipins, a large class of diverse oxygenated polyunsaturated fatty acids and their derivatives, are produced through the lipoxygenase (LOX) pathway. Recent progress in dicots has highlighted the biological roles of oxylipins in plant defense responses to pathogens and pests. In contrast, the physiological function of LOXs and their metabolites in monocots is poorly understood. We cloned and characterized three maize LOXs ZmLOX10 ZmLOX11 and ZmLOX12. Both ZmLOX10 and ZmLOX11 apeared to be 13-LOX, whereas ZmLOX12 is a unique 9-LOX. Whereas leaf was the preferential site of ZmLOX10 expression, ZmLOX11 was strongly expressed in silks. Induction of these ZmLOX10 and ZmLOX12 by wounding and defense-related compounds suggested their role in plant resistance mechanisms against pests and pathogens. Abscisic acid, however, was the only inducer of ZmLOX11 in leaves. Higher increase in ZmLOX10 transcripts in maize infected by fungus Cochliobolus carbonum implicated this gene in resistance responses to necrotrophic pathogens. In addition, ZmLOX10 was shown to be the first reported LOX to be regulated by a circadian clock. It was found that ZmLOX10 was also inducible by low temperatures. Phenotypical studies of wild type and mutant near isogenic lines showed that expression of ZmLOX12, specific to underground organs, was required for pathogenesis of F. verticillioides on maize mesocotyls.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

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No. of bitstreams: 1 Dissertação_Completa_Henia_VF-2.pdf: 4622430 bytes, checksum: d09b1f29bbf344aeaacc5347c72ee181 (MD5) / CNPq e Fapesb / “ESTUDO QUÍMICO E AVALIAÇÃO DAS ATIVIDADES ANTIMICROBIANA, ANTICOLINESTERÁSICA E LARVICIDA DO FUNGO Curvularia lunata E LEVANTAMENTO QUIMIOTAXONÔMICO DO COMPLEXO Curvularia- Bipolaris-Cochliobolus” A revisão bibliográfica sobre os metabólitos secundários isolados de espécies fúngicas dos gêneros Curvularia, Bipolaris e Cochliobolus foi realizada com o objetivo de comparar o perfil metabólico deste três gêneros visando contribuir para a classificação taxonômica deste complexo. Como resultados, foram encontrados metabólitos secundários pertencentes às classes dos policetídeos (incluindo antraquinonas, lactonas macrociclicas e compostos fenólicos), terpenoides (sesterpenoies e sesquiterpenoides), esteróides e alcalóides. Os policetídeos como as lactonas macrociclicas curvularinas foram descritas somente para fungos do gênero Curvularia e alguns Cochliobolus, enquanto que os sesterpenoides foram encontrados nos gêneros Bipolaris e Cochliobolus, permitindo sugerir que as lactonas sejam marcadores quimiotaxonômicos para o gênero Curvularia e as espécies de Cochliobolus relacionadas (teleomorficas), e que os sesterpenoides são marcadores do gênero Bipolaris e os teleomorfos do gênero Cochliobolus. A análise dos extratos oriundos do cultivo de Curvularia lunata em diferentes meios de cultura foi realizada para verificação da influencia da fonte de carbono sobre a produção de metabólitos secundários. Os métodos utilizando a cromatografia em camada delgada comparativa, cromatografia líquida de alta eficiência e ressonância magnética nuclear de hidrogênio permitiram estimar a diversidade química conforme a fonte de carbono usada. Os meios de cultura Czapeck, batata e dextrose e extrato de malte apresentaram mais sinais nos cromatogramas e nos espectros de RMN de 1H com relação aos demais, por isto estes meios foram considerados os meio de culturas ideais para o estudo do metabolismo secundário. Além disso, foi possível inferir a presença da 11-α-metoxicurvularina nos extratos do fungo cultivado em batata e dextrose e farinha de arroz. Os extratos obtidos foram submetidos a ensaios biológicos (atividade antimicrobiana, anticolinesterásica e larvicida). Os extratos obtidos do cultivo do fungo em batata e dextrose apresentaram atividade contra a levedura Candida albicans, enquanto os extratos oriundos do meio contendo extrato de levedura XV mostraram atividade antibacteriana contra Micrococcus luteus, Staphylococcus aureus e Bacillus subtilis. Com relação à atividade anticolinesterasica, todos os extratos brutos oriundos do micélio continham um sinal com fator de retenção igual 0.7 que mostrou inibição da atividade da enzima acetilcolinesterase, destacando os extratos fúngicos resultantes do cultivo em meio contendo extrato de malte e meio Czapeck enriquecido com 2% de extrato de levedura por apresentarem maior halo de inibição. Nenhum dos extratos apresentou atividade contra larvas do mosquito Aedes aegypti. Finalmente o fungo Curvularia lunata foi cultivado em larga escala em meio contendo extrato de malte selecionado pela sua atividade anticolinesterasica. A investigação química inicial resultou no isolamento de três derivados perilenoquinônicos: dois já descritos na literatura como dihidroalterperilenol e alterperilenol, mas obtidos pela primeira vez no gênero Curvularia e o perileno de estrutura inédita, identificado como 12-metil- stemfitriol. / “CHEMICAL STUDY AND BIOLOGICAL ASSAYS (ANTIMOCROBIAL, ANTICHOLINESTERASIC AND INSECTICIDAL) FROM THE FUNGUS Curvularia lunata AND QUIMIOTAXONOMIC SEARCH FROM THE FUNGAL COMPLEX Curvularia-Bipolaris-Cochliobolus” A literature revision of secondary metabolites isolated from fungal species of Curvularia, Bipolaris and Cochliobolus genera was performed with the aim of get their metabolic profile in order to contribute to the taxonomic classification on this complex. As result were found secondary metabolites belonging to the polyketides (including anthraquinones, macrocyclic lactones and phenolic), terpenoids (mainly sesterpenoids), steroids and alkaloids. Polyketides like the macrocyclic lactone curvularins were described only for fungi from the genus Curvularia and the related teleomorphic Cochliobolus species, while sesterpenoids such as ophiobolins was described for Bipolaris species as well as their related Cochliobolus, suggesting these lactones and sesterpenoids as quimiotaxonomic marker for Curvularia and Bipolaris genera, respectively. An analysis of extracts derived from cultivation of Curvularia lunata on several media was performed to investigate the effect of carbon source on secondary metabolism of the fungus. As analytical strategy based on TLC (thin layer chromatography), high performance liquid chromatography with diode array detection and nuclear magnetic resonance of hydrogen allowed to get an estimative about chemical diversity under different carbon sources. Malt extract, Czapeck plus 2% of yeast extract and potato and dextrose extracts were the media that exhibited the most signals in chromatograms and 1H NMR spectra, indicating them as the ideal culture media for secondary metabolism studies. Furthermore, we can suggest that the compounds 11-α-methoxycurvularin were found in fungal extracts derived from the cultivation of Curvularia in potato and dextrose and rice flour media. In addition, those extracts were also evaluated for their biological activities (antimicrobial, anticolinesterásico and insecticidal). Extracts obtained from fungal growth in potato and dextrose media exhibited activity against the yeast Candida albicans, while extracts derived from the yeast extract cultivation showed antibacterial activity against M. luteus, S. aureus and B. subtillis. With respect the anticholinesterasic activity, all mycelial extract had a signal (Rf 0.7) that inhibited the enzyme AChE activity, being the fungal extracts from malt extracts and czapeck plus 2% yeast extract that had a marked inhibition halo. None of these XVII extracts had an activity against larval stage of the mosquito Aedes aegypti. Finally, the fungus Curvularia lunata was cultivated in large scale on malt extract medium, selected for its anticholinesterasic activity. Initial chemical investigation had resulted in the isolation of three perylene derivates: known dihydroalterperylenol and alterperylenol but being described for the first time in Curvularia genera and the novel perylene having a metabolic group, which was named as 12-methyl-stemphytriol.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-06T16:54:49Z No. of bitstreams: 1 texto completo.pdf: 650913 bytes, checksum: b9f17c4a57a8b906d67c928beb6ed056 (MD5) / Made available in DSpace on 2017-04-06T16:54:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 650913 bytes, checksum: b9f17c4a57a8b906d67c928beb6ed056 (MD5) Previous issue date: 2015-07-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os fungos endofíticos constituem um grupo de micro-organismos produtores de metabólitos secundários bioativos. Dentro desta perspectiva, foi estudado o potencial antimicrobiano do endofítico Cochliobolus sativus, isolado do feijoeiro comum Phaseolus vulgaris. Foi investigada a capacidade deste fungo em produzir metabólitos secundários com atividade antifúngica e antibacteriana em diferentes condições de crescimento, levando em consideração o meio de cultura, o fotoperíodo e o tempo de incubação. Os extratos gerados nas diferentes condições foram testados contra quatro fitopatógenos de importância agrícola e bactérias Gram-positivas e Gram-negativas de interesse médico e industrial, utilizando o método de diluição em ágar e difusão em ágar, respectivamente. O extrato que apresentou os melhores resultados de inibição determinou a melhor condição de crescimento para a síntese desses compostos. Foi observado que a atividade do extrato E4 nos micro- organismos testados foi superior a atividade dos demais extratos, apresentando inibição significativa de 52%, 29,69% e 8,16% do crescimento micelial dos fungos Sclerotinia sclerotiorum, Rhizoctonia solani e Fusarium oxysporum. A inibição do fitopatógeno Colletotrichum lindemuntianum não foi significativa. Quanto as bactérias, a atividade dos extratos brutos só foi observadas nas Gram-positivas Staphylococcus aureus, Bacillus subtilis e Enterococcus faecalis, apresentando os melhores halos de inibição, pelo extrato bruto 4, de 12 mm, 10 mm e 6,8 mm, respectivamente. A menor MIC (concentração mínima inibitória) do extrato bruto foi contra S. aureus (MIC = 32 μg mL -1 ), enquanto em B. subtillis e E. faecalis foi de 68 μg mL -1 . Sendo assim, foi selecionada a condição de crescimento correspondente ao extrato bruto E4 para otimização da síntese de metabólitos secundários em larga escala. Ensaios biológicos foram realizados, para averiguação da influência do pH do filtrado fúngico na extração com acetato de etila. Os resultados indicaram que não há necessidade de acidificação ou alcalinização do filtrado para se obter os compostos bioativos. Posteriormente, os extratos brutos foram submetidos a análises de cromatografia à gás, apontando inicialmente, para dois compostos majoritários. Procedimentos futuros serão empregados para o isolamento e caracterização completa desses compostos e será testada a capacidade do fungo para inibir o crescimento de fitopatógenos in vivo. / From this perspective, we investigated the potential antimicrobial activity of Cochliobolus sativus endophyte, isolated from the common bean Phaseolus vulgaris. We investigated the ability of this fungus to produce antifungal and antibacterial secondary metabolites in different growth conditions, taking into account the culture medium, photoperiod and incubation time. Extracts of the culture media generated under the various growth conditions were tested against four phytopathogenic fungi of agricultural importance and against gram- positive and gram-negative bacteria of medical and industrial interest, using the agar dilution method and agar diffusion, respectively. The extract that showed the best inhibition results determined the best growth condition for the synthesis of these compounds. Extract E4 showed the higher antifungal and antibacterial activities inhibiting 52%, 29.69%, and 8.16% of the mycelial growth of the fungus Sclerotinia sclerotiorum, Rhizoctonia solani and Fusarium oxysporum, respectively. Also, this extract presented significant activity against gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis,producing inhibition zones of 12 mm, 10 mm, and 6.8 mm in diameter, respectively. MIC (minimum inhibitory concentration) values were determined as 32 ug mL -1 for S. aureus and 68 ug mL -1 for B. subtilis and E. faecalis . Therefore, the growth conditions that lead to extract E4 were selected for large scale fermentation and isolation of bioactive metabolites. Biological assays were performed to investigate the influence of the pH of the culture filtrates on the of extraction of the metabolites. The results indicated that there is no need for acidification or alkalinization of the filtrate to improve the extraction of the bioactive compounds. Subsequently, the crude extracts were subjected to analysis by Gas-Chromatography-Mass Spectrometry, pointing initially to two major compounds. Future procedures will be applied to the complete isolation and characterization of these compounds, which will be tested for its ability to inhibit in vivo growth of pathogens.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21158号 / 農博第2284号 / 新制||農||1060(附属図書館) / 学位論文||H30||N5132(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Made available in DSpace on 2015-03-26T13:51:50Z (GMT). No. of bitstreams: 1 texto completo.pdf: 474764 bytes, checksum: dc1db5661d6bff9e0de8af890d4b1836 (MD5) Previous issue date: 2009-07-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Gypsy/Ty3 group retrotransposons are the main transposable elements found in the genome of pathogenic fungi. In this work, two retrotransposons termed MpSaci and Sophie were analyzed in Moniliophthora perniciosa and Cochliobolus heterostrophus, respectively. MpSaci was used to assess molecular markers based on transposable elements IRAP and REMAP. When 70 isolates of M. perniciosa were analyzed, a total of 43 loci were amplified by generating 46.51% polymorphism. Significant differences were found between populations of M. perniciosa divided in relation to biotope and the geographical origin, showing that populations are structured on the geographical origin and the host (biotype). The cluster analysis of different geographical regions of biotype C were observed two large groups that show two main entrances of the pathogen in the state of Bahia. Searches done in the database of the Genome Project of C. heterostrophus (JGI- Genome) showed the presence of DNA sequences with similarity to reverse transcriptase of Class I elements belonging to the group Gypsy/Ty3. Based on these sequences could be found seven different copies of an intact transposable element named Sophie. The analysis of 37 sequences of the reverse transcriptase gene showed a possible silencing mechanism similar to RIP. The seven copies of the element Sophie had 7426 bp 7512 bp. The retrotransposon sequences Sophie has two open reading frames (ORFs) that encode the Gag protein and the Pol polyprotein. The presence of different target sites suggests that Sophie is an element with recent activity in the genome of C. heterostrophus can have a great impact on the evolution of the genome of its host. / Retrotransposons do grupo Gypsy/Ty3 são os principais elementos transponíveis encontrados no genoma de fungos fitopatogênicos. Neste trabalho, dois retrotransposons denominados de MpSaci e Sophie foram analisados em Moniliophthora perniciosa e Cochliobolus heterostrophus, respectivamente. MpSaci foi utilizado para avaliar os marcadores moleculares baseados em elementos transponíveis IRAP e REMAP. Quando 70 isolados de M. perniciosa foram analisados, o total de 43 locos foram amplificados gerando 46,51% de bandas polimórficas. Diferenças significativas foram encontradas entre as populações de M. perniciosas divididas em relação ao biótipo e à origem geográfica, demonstrando que as populações encontram-se estruturadas quanto à origem geográfica e ao hospedeiro (biótipo). Pela análise de agrupamento de diferentes regiões geográficas do biótipo C foram observados dois grandes grupos que evidenciam duas principais entradas do patógeno no estado da Bahia. Buscas feitas no banco de dados do Projeto Genoma de C. heterostrophus (JGI - Genome) revelaram a presença de seqüências de DNA com similaridade a transcriptase reversa de elementos da Classe I pertencentes ao grupo Gypsy/Ty3. Com base nessas seqüências foi possível encontrar sete cópias diferentes e intactas de um elemento transponível que foi denominado Sophie. A análise de 37 seqüências do gene da transcriptase reversa demonstrou possível mecanismo de silenciamento semelhante a RIP. As sete cópias do elemento Sophie apresentaram 7.426 pb a 7.512 pb. O retrotransposon Sophie possui duas seqüências de leitura abertas (ORFs) que codificam a proteína Gag e a poliproteína Pol. A presença de diferentes sítios-alvo sugere que Sophie é um elemento com atividade recente no genoma de C. heterostrophus podendo ter grande impacto na evolução do genoma do seu hospedeiro.

Victoria blight, caused by fungus Cochliobolus victoriae, is a disease originally described on oats and recapitulated on Arabidopsis. Victoria blight is used as a model plant disease that conforms to an inverse gene-for-gene interaction. C. victoriae virulence is dependent upon its production of victorin, a host-specific toxin that induces programmed cell death in sensitive plants. In oats, victorin sensitivity and disease susceptibility is conferred by the Vb gene, which is genetically inseparable from the Pc-2 crown rust resistance gene. In Arabidopsis, victorin sensitivity and disease susceptibility is conferred by the LOCUS ORCHESTRATING VICTORIN EFFECTS 1 (LOV1) gene which encodes a NB-LRR protein, a type of protein commonly associated with disease resistance. LOV1-mediated cell death occurs when victorin binds Thioredoxin-h5 (TRX-h5) and LOV1 appears to "guards" TRX-h5. Together, these results suggest C. victoriae causes disease by inducing a resistance response. The work presented here aimed to determine if the response mediated by LOV1 is functionally related to a resistance response. We genetically characterized the response mediated by LOV1 with virus-induced gene silencing. We determined SUPPRESSOR OF THE G2 ALLELE OF SKP1 (SGT1), a gene required for the function of many resistance genes, is required for victorin sensitivity and involved in LOV1 protein accumulation. We screened a normalized library and identified six genes that suppressed victorin-mediated cell death and cell death induced by expression of the RESISTANCE TO PERONOSPORA PARASITICA PROTEIN 8 (RPP8) resistance gene: a mitochondrial phosphate transporter, glycolate oxidase, glutamine synthetase, glyceraldehyde 3-phosphate dehydrogenase and the P- and T-protein of the glycine decarboxylase complex. Silencing the latter four also inhibited cell death induced by the expression of an autoactive form of the resistance gene PTO, and reduced PTO-mediated resistance to Pseudomonas syringae pv. tabaci. These results provide evidence that victorin-mediated cell death is functionally similar to a resistance response, further supporting the hypothesis that a resistance response is exploited by C. victoriae for pathogenesis in Victoria blight. Resistance function of LOV1 was evaluated by observing Pseudomonas syringae pv. tomato virulence upon LOV1 activation. The LOV1 response pathway in Arabidopsis was adapted to activate upon infection with Pseudomonas syringae pv. tomato expressing the type III-dependent effector protein AvrRpt2, a well-characterized protease. We developed a construct to express a beta-glucuronidase (GUS) and TRX-h5 fusion protein separated by an AvrRpt2 proteolytic cleavage site, in which GUS sterically inhibits TRX-h5 function in LOV1-mediated cell death. The fusion is cleaved upon infection by P. syringae pv. tomato expressing avrRpt2, thereby leading to TRX-h5-mediated activation of LOV1 in the presence of victorin. However, when this strain was inoculated with victorin into transgenic LOV1 trx-h5 plants expressing the GUS/TRX-h5 fusion protein, no decrease in pathogen virulence was observed. Technical shortcomings likely prevented observable LOV1 resistance function. ��� / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Oct. 9, 2012 - Oct. 9, 2013

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-17T13:55:35Z No. of bitstreams: 1 Isadora Fernandes de Franca.pdf: 1420687 bytes, checksum: 6cec52a0fb3778ca5533af6c71262265 (MD5) / Made available in DSpace on 2017-03-17T13:55:36Z (GMT). No. of bitstreams: 1 Isadora Fernandes de Franca.pdf: 1420687 bytes, checksum: 6cec52a0fb3778ca5533af6c71262265 (MD5) Previous issue date: 2011-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The yam (Dioscorea spp.) has great social importance for the Northeast region of Brazil, due to the excellent nutritional quality and energy of their tubers and very useful for human alimentation. Leaf-spots on white yam, caused by the fungus Curvularia eragrostidis, is responsible for damage to yam in northeast Brazil, mainly in the states of Paraíba and Pernambuco. The aim of this study was to analyze the population structure of isolates of C. eragrostidis pathogenic to yam, D. cayennensis, in the states of Alagoas, Pernambuco and Paraiba, and to study the phylogenetic relationships of these with other species in the genus Cochliobolus. In the first study, 151 isolates were obtained from leaves of yam, which were analyzed the sequences of part of the ITS region and -tubulin gene and gpd and the profiles of DNA with ISSR. It was observed that the DNA sequences had no variation, and ISSR data showed the presence of six haplotypes. The pathogen population is characterized by low genotypic diversity, predominance of one or two genotypes evenly distributed, the predominance of asexual reproduction, and the presence of genotypic flow between the regions of cultivation. In the second study, it was conducted phylogenetic analysis sequences of 81 isolates of species of the family Pleosporaceae using the methods of maximum likelihood and Bayesian inference. The 16 isolates of C. eragrostidis from yam analyzed are related to the genus Cochliobolus species which has smaller conidia and they are phylogenetically closer to the species C. clavata, C. brachyspora, C. oryzae and C. intermedia. / A cultura do inhame (Dioscorea spp.) apresenta grande importância socioeconômica para a região Nordeste do Brasil, devido à excelente qualidade nutritiva e energética de suas túberas e a utilidade para a alimentação humana. A queima das folhas, conhecida também como pinta preta, causada pelo fungo Curvularia eragrostidis, é responsável por grandes prejuízos à cultura do inhame no Nordeste brasileiro, principalmente nos Estados da Paraíba e Pernambuco, que são os maiores produtores. Este trabalho teve como objetivos analisar a estrutura populacional de isolados de C. eragrostidis patogênicos ao inhame, D. cayennensis, nos estados de Alagoas, Pernambuco e Paraíba, bem como estudar as relações filogenéticas destes com as demais espécies do gênero Cochliobolus. No primeiro estudo, foram obtidos 151 isolados, dos quais foram analisados as sequências de parte da região ITS, e dos genes -tubulina e gpd e os perfis de DNA com marcadores ISSR. Observou-se que as sequências de DNA não apresentaram variabilidade, e os dados de ISSR mostraram a presença de seis haplótipos. A população do patógeno é caracterizada por baixa diversidade genotípica, predominância de um ou dois genótipos uniformemente distribuídos, predominância de reprodução assexual, presença de fluxo genotípico entre as regiões de cultivo. No segundo estudo, foram realizadas analises filogenéticas de sequências de 81 isolados de espécies da família Pleosporaceae, através dos métodos de máxima verossimilhança e inferência bayesiana. Os 16 isolados de C. eragrostidis de inhame analisados estão relacionados com espécies do gênero Cochliobolus que apresentam conídios menores e mais próximos filogeneticamente de C. clavata, C. brachyspora, C. oryzae e C. intermedia.