Os fungos endofíticos constituem uma fonte promissora para descoberta de novos compostos ativos de interesse biotecnológico. Nas últimas décadas, os fungos endofíticos têm recebido atenção no meio científico devido aos compostos que produzem, com estruturas únicas e atividades biológicas de relevância em diversas áreas, inclusive agronômica. Um dos maiores problemas nesta área são as doenças causadas por fungos fitopatogênicos que acometem as culturas. Dentre elas, a antracnose é uma das principais doenças que atinge diversas culturas no Brasil, gerando grandes perdas agrícolas, principalmente as culturas do morango, guaraná e citrus, causadas por fungos do gênero Colletotrichum. Esse trabalho teve como objetivo investigar extratos de linhagens dos fungos endofíticos Fusarium e Penicillium, isolados de folhas do guaranazeiro, com atividade à fitopatógenos do gênero Colletotrichum isolados das culturas de guaraná, morango e citrus. Para tanto foi realizado uma triagem inicial de duas linhagens de fungos, dentre nove, por ensaios biológicos in vitro de cultivo pareado na atividade antifúngica aos três fitopatógenos. As duas linhagens selecionadas, apresentaram os maiores índices de antagonismo contra os três fitopatógenos e foram identificadas como Fusarium sp e Penicillum pinophilum. Os extratos e as frações semipurificadas se mostraram com grande potencial na inibição do crescimento do fitopatógeno do gênero Colletotrichum, causador da antracnose, que provoca grandes perdas nas culturas de importância econômica do Brasil. / Endophitic fungi are a promising source for discovery of new active compounds of biotechnological interest. In recent decades, endophytic fungi have received attention in the scientific community because of compounds they produce, with unique structures and biological activities of relevance in several areas, including agronomy. One of the greatest problems in this is diseases caused by phytopathogenic fungi that affect the crops. Among them, anthracnose is a major disease that affects several crops in Brazil, generating large agricultural losses, especially strawberry crops, guarana and citrus caused by fungi of the genus Colletotrichum. This study investigated endophytic fungi strains of Fusarium and Penicillium extracts isolated from guarana leaves, with activity to phytopathogens of the genus Colletotrichum isolated from guarana crops, strawberry and citrus. For that, it was conducted an initial screening of two strains of fungi, among nine, using in vitro biological assays of cultivation paired in antifungal activity to the three pathogens. The two strains selected had the highest rates of antagonism against the three phytopathogens and were identified as Fusarium sp and Penicillium pinophilum. Extracts and semi-purified fractions were shown with great potential to inhibit pathogen growth of the genus Colletotrichum, which causes anthracnose and, thus, great losses to crops of economic importance in Brazil.

O guaraná, Paullinia cupana var. sorbilis, espécie nativa do Brasil é uma cultura de relevância para o país, principalmente nos estados da Bahia e Amazonas. O fruto contém substâncias estimulantes, como a cafeína, que conferem ao guaraná um grande potencial exploratório, visto que esta é uma das substâncias estimulantes mais consumidas no mundo. Apesar de sua importância, a cultura do guaraná é pouco estudada, portanto pouco se sabe sobre sua genética e as interações microbianas existentes, tanto em relação aos microrganismos endofíticos e epifíticos, quanto em relação aos patógenos. De tal forma que a cultura do guaraná na região Amazônica vem sendo afetada por condições fitossanitárias desfavoráveis, como a presença do fungo do gênero Colletotrichum, causador da antracnose, doença considerada uma ameaça à produção comercial do guaraná. Assim, visando compreender a dinâmica envolvida na interação planta - microrganismos e o possível controle da antracnose, o presente trabalho acessou a comunidade bacteriana associada às folhas com e sem sintomas de antracnose, de forma independente e dependente de cultivo; bem como, investigou o potencial biotecnológico e de biocontrole dos isolados bacterianos. A comunidade bacteriana acessada de forma dependente e independente de cultivo apresentaram similaridades, como maior valor de riqueza e menor diversidade bacteriana em folhas assintomáticas, quando comparadas com folhas sintomáticas, e com relação ao filo Proteobacteria, mais abundantemente acessado nas duas comunidades. Comparações realizadas com isolados bacterianos acessados epi e endofiticamente de folhas com e sem sintoma da antracnose apontaram que o aparecimento da doença antracnose na folha pareceu ser o fator que mais influenciou na estrutura da comunidade bacteriana. Com relação às diferenças entre tecidos sintomáticos e assintomáticos, foi possível identificar alguns gêneros abundantes mais frequentemente acessados em plantas sintomáticas, sendo que os gêneros Pseudomonas, Stenotrophomonas e Pantoea foram acessados em grande frequência tanto de forma dependente como independente de cultivo. Porém, de forma independente de cultivo foram acessados outros táxons abundantes que diferiram significativamente entre as plantas, sendo que folhas sintomáticas apresentaram Acinetobacter, Acidobacter_GP1 e Sphingobacteria, e em folhas assintomáticas foram acessados os táxons Methylobacterium, Beijerinckia, Bacilli e um grupo de Rhizobiales não classificados. Além disto, os microrganismos endofíticos e epifíticos cultivados foram testados com o fitopatógeno Colletotrichum sp. em ensaios de antagonismos, sendo que isolados do gênero Bacillus, Stenotrophomonas, Pantoea, Erwinia, Entereobacter, Pseudomonas, entre outros, apresentaram inibição do crescimento do fungo fitopatogênico. As bactérias cultivadas ainda foram testadas quanto ao potencial biotecnológico de produção de enzimas hidrolíticas e sideróforo. Muitos isolados apresentaram produção de protease e sideróforo. Foi possível também correlacionar a produção das enzimas amilase, lipase e poligalacturanase com os isolados provenientes de tecidos sintomáticos. / Guarana, Paullinia cupana var. sorbilis, is a native culture of Brazil that has relevance to the country, mainly in the states of Bahia and Amazonas. The fruit contains stimulants substances, such as caffeine, that gives a high exploration potential to the culture, since this is one of the most consumed stimulant substance in the world. Despite its importance, the culture of guarana hasnt gotten much of attention, and little is known about its genetics and microbial interactions with both epiphytic and endophytic microorganisms, and in relation to pathogens. So, the guarana culture in the Amazon region has been affected by bad phytosanitary conditions, such as the presence of the fungi Colletotrichum, which causes anthracnose disease thats considered a threat to guarana commercial production. Thus, to understand the dynamics involved in the interaction plant-microorganism and the possible control of anthracnose, this study accessed the bacterial community associated to leaves with and without anthracnose symptoms, combining culture-dependent and -independent methodologies and also investigated the biotechnology potential and biocontrol of culture-dependent bacteria. The bacterial community accessed by culture-dependent and -independent manner presented similarities, both presented higher bacterial richness but lower bacterial diversity in asymptomatic leaves when compared with symptomatic leaves, and both communities presented the Proteobacteria phylum as the most abundant one. Comparisons between the endophytic and epiphytic bacterial isolates associated with leaves with and without anthracnose symptoms showed that the onset of anthracnose disease on leaf seemed to be the most important factor that modified the bacterial community structure. Regarding the differences between symptomatic and asymptomatic tissue it was possible to identify some genera most frequently accessed in symptomatic plants such as Pseudomonas, Stenotrophomonas and Pantoea accessed in both culture-dependent and independent methodologies. However, the cultureindependent approach of bacterial accessed others abundants taxa that differed significantly between plants. Symptomatic leaves showed Acinetobacter, Acidobacter_GP1 and Sphingobacteria and asymptomatic leaves presented taxa Methylobacterium, Beijerinckia, group Bacilli and a group of unclassified Rhizobiales. In addition, endophytic and epiphytic isolates microorganisms were tested with the pathogen Colletotrichum sp. in antagonism assays, and isolates from the genus Bacillus, Stenotrophomonas, Pantoea, Erwinia, Entereobacter, Pseudomonas, and others, showed inhibition of growth of the fungus. The bacterial isolates were also tested for production of hydrolytic enzymes and siderophore. Many isolates showed protease and siderophore production. It was possible to correlate the production of amylase, lipase and poligalacturanase with isolates from symptomatic tissue.

Orientador: Antonio de Góes / Coorientador: Janete Apparecida Desidério / Banca: Ronai Ferreira Ramos / Banca: Viviani Vieira Marques / Banca: Luciana Rossini Pinto / Banca: Margarete Camargo / Resumo: A produção média de fruta por ano no mundo é aproximadamente de 800 milhões de toneladas. O Brasil é o terceiro colocado no ranking das principais nações produtoras, estando atrás apenas da China e da Índia, respectivamente. Desta forma, verifica-se que o Brasil possui um grande potencial a ser explorado no contexto das exportações. Para isso, no entanto, há necessidade de investimentos visando minimizar os danos pré e pós-colheita, que representam um fator limitante, tanto na produção, como nas exportações. Entre os danos pós-colheita incluem-se os de natureza fúngica, com destaque ao complexo antracnose, causada por fungos do gênero Colletotrichum. O presente trabalho teve como objetivo identificar taxonomicamente as espécies de Colletotrichum associadas a frutos de abacate, banana, goiaba e manga, bem como estabelecer relações filogenéticas entre as espécies identificadas. Duzentos isolados de Colletotrichum spp. de frutos de abacateiro, bananeira, goiabeira e mangueiras com sintomas de antracnose foram submetidos a análises morfocultural, molecular e teste de patogenicidade cruzada. Embora mediante análises morfoculturais tenham identificado diversidade entre os isolados de Colletotrichum avaliados, essas foram insuficientes para distinguir espécies do gênero. Os isolados de Colletotrichum spp. encontrados em lesões de antracnose em frutos de abacate, banana, goiaba e manga não apresentaram especificidade fisiológica. Colletotrichum musae e C. siamense estão associadas aos sintomas de antracnose em abacate no Brasil. O agente causal da antracnose em banana não é constituído por um único grupo monofilético, C. musae, contando-se com espécies adiconais de patógenos. Além de C. acutatum e C. gloeosporioides, C. siamense e C. musae são também patógenos responsáveis por sintomas de antracnose em frutos de goiaba. A antracnose em frutas de mangueira, no... / Abstract: The average production of fruit per year worldwide is about 800 million tons. The Brazil is third in ranking of the major producing nations. It is behind only to China and India, respectively. It acertain that Brazil has great potential to be explored in the context of exports. Among the post-harvest damage includes up the fungal nature, highlighting the complex anthracnose, caused by fungi of the genus Colletotrichum and present in all the producing countries of fruit. Among the postharvest damage includes up the fungal nature, highlighting the complex anthracnose, caused by fungi of the genus Colletotrichum and present in all the producing countries of fruit. This study aimed to identify the taxonomic species of Colletotrichum associated with avocado fruit, banana, guava and mango, as well as to establish phylogenetic relationships among the species identified. Two hundred isolates of Colletotrichum spp. fruit avocado, banana, guava and mango trees, underwent Morphocultural analysis, molecular and pathogenicity. According to the results, cultural and morphological characterization still enabling the detection of phenotypic diversity among isolates, do not provide reliable results for identification of species of Colletotrichum evaluated. The Colletotrichum spp. found in avocado fruit, banana, guava and mango showed no physiological specificity. C. musae and C. siamense are associated with anthracnose symptoms on avocado in Brazil. The causal agent of anthracnose in banana is not composed of a single monophyletic group of C. musae, suggesting the existence of more than one species as a pathogen of this crop. In addition to C. acutatum and C. gloeosporioides, C. siamanese and C. musae are also pathogen responsible for anthracnose symptoms in fruits of guava. The anthracnose in hose fruits in the state of São Paulo, is associated with the species C. asianum and C. dianesii / Doutor

Fungos do gênero Colletotrichum são considerados um dos principais fitopatógenos do mundo, comprometendo diversas culturas e causando danos econômicos e sociais para diversos países, inclusive o Brasil. Apesar de medidas de controle já serem empregadas, estas nem sempre são eficazes, motivo pelo qual se faz necessária a busca por novas opções de controle que possam atuar no manejo integrado. Dentre estas, encontram-se os metabólitos secundários produzidos por fungos endofíticos, os quais estão envolvidos na produção de compostos de interesse biotecnológico, com aplicações em diversas áreas, inclusive a agronômica. Neste contexto, o presente trabalho de doutorado teve por objetivo explorar o potencial biológico e químico de metabólitos secundários produzidos por fungos endofíticos isolados de guaranazeiros da Amazônia, avaliando sua atividade antifúngica \"in vitro\" a Colletotrichum gloeosporioides, isolado da cultura do guaranazeiro, a C. acutatum e C. gloeosporioides, isolados da cultura do pimentão. Para tanto, 16 linhagens de fungos endofíticos foram avaliadas em ensaio biológico por cultivo pareado e pelo método de difusão em disco contra Colletotrichum spp. Observou-se que quatro linhagens se mostraram mais promissoras, as quais foram selecionadas para o estudo de bioprospecção. A partir do extrato bruto do endófito Aspergillus flavus (272) foi isolado o composto ativo asperfuran, que apresentou atividade inibitória aos três fitopatógenos, com CI50 < 100 &mu;g disco-1, semelhante ao fungicida comercial difenoconazol. A partir do extrato bruto de Xylaria sp. (214) foi isolado o composto ativo citocalasina D, que apresentou atividade inibitória a C. gloeosporioides isolado do guaranazeiro, com CI50 > 500 &mu;g disco-1. A partir do extrato bruto de Xylaria sp. (249) foi isolado o composto ativo ácido pilifórmico, que apresentou atividade inibitória a C. gloeosporioides isolado do guaranazeiro, com CI50 de 500 &mu;g disco-1 e à C. acutatum isolado o pimentão, com CI50 de 300 &mu;g disco-1. E, a partir do extrato bruto de Talaromyces aculeatus (507) foram isolados três compostos, da classe dos ésteres ftalicos, sendo um ativo. A fração antecessora destes três compostos apresentou atividade inibitória a C. gloeosporioides isolado do guaranazeiro, com CI50 > 500 &mu;g disco-1. A partir do extrato bruto de T. aculeatus também foi isolado um composto ativo parcialmente identificado e um composto ativo semi-purificado. A fração antecessora destes dois compostos apresentou atividade inibitória aos dois C. gloeosporioides, com CI50 > 300 &mu;g disco-1 e a C. acutatum, com CI50 em torno de 150 &mu;g disco-1. A atividade antifúngica destes compostos a fungos do gênero Colletotrichum está sendo relatada pela primeira vez neste estudo. / Colletotrichum fungi are considered one of the main pathogens in the world, affecting different cultures and causing social and economic damage to several countries, including Brazil. Despite control measures already being employed, these are not always effective, which is why the search for new control options that can act in the integrated management is necessary. Among these, the secondary metabolites produced by endophytes are involved in the production of compounds of biotechnological interest, with applications in several areas, including the agronomic. In this context, this study aimed to explore the biological and chemical potential of secondary metabolites produced by endophytic fungi isolated from the Amazonian guarana, evaluating their antifungal activity \"in vitro\" to Colletotrichum gloeosporioides, isolated from the culture of guarana and C. acutatum and C. gloeosporioides, isolated from the red pepper crop. For this purpose, 16 strains of endophytic fungi were evaluated in biological assay of dual culture and disk diffusion method against Colletotrichum spp. Four strains of these strains presented promising results which were selected for bioprospecting. From the crude extract of the endophyte Aspergillus flavus (272) was isolated the active compound asperfuran, which showed inhibitory activity to three pathogens, with IC50 <100 &mu;g disk-1, similar to the commercial fungicide difenoconazole. From the crude extract of Xylaria sp. (214) was isolated the active compound cytochalasin D, which showed inhibitory activity against C. gloeosporioides isolated from guarana (IC50> 500 &mu;g disk-1). From the crude extract of Xylaria sp. (249) was isolated the active compound piliformic acid, which showed inhibitory activity against C. gloeosporioides isolated from guarana (IC50 of 500 &mu;g disk-1) and C. acutatum isolated from red pepper (IC50 of 300 &mu;g disk-1). And from the crude extract of Talaromyces aculeatus (507) were isolated three compounds, of phthalate esters class, one of them with activity. The predecessor fraction of these three compounds showed inhibitory activity against C. gloeosporioides isolated from guarana (IC50> 500 &mu;g disk-1). From the crude extract of T. aculeatus it was also isolated a partially identified active compound and a semi-purified active compound. The predecessor fraction of these two compounds showed inhibitory activity to both C. gloeosporioides (IC50> 300 &mu;g disk-1) and to C. acutatum (IC50 around 150 &mu;g disk-1). The antifungal activity of the compounds isolated in this study is being reported for the first time against fungus of the genus Colletotrichum.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-03-16T14:18:00Z No. of bitstreams: 1 Ariele Carneiro de Andrade.pdf: 1066650 bytes, checksum: 26d95d8f20aec5260de9adb89d2195cd (MD5) / Made available in DSpace on 2017-03-16T14:18:00Z (GMT). No. of bitstreams: 1 Ariele Carneiro de Andrade.pdf: 1066650 bytes, checksum: 26d95d8f20aec5260de9adb89d2195cd (MD5) Previous issue date: 2015-07-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The bell pepper (Capsicum annum L.) crop is one of the most important vegetables grown in Brazil, and has been showing significant growth. However, this culture can be affected by a number of diseases that cause economic damage and anthracnose, still represent a serious obstacle to production, both in the field and at home-de-vegetation. Anthracnose in Capsicum annum can be caused by several species of fungi of the genus Colletotrichum, but in Brazil, the majority of reports identify Colletotrichum gloeosporioides as the main causative agent of the disease. Infections characterized in lesions aerial parts of the plants such fruits, stems and leaves. The control method of the infections is used in fungicidal applications, however currently search for more sustainable alternatives. Thus the use of resistance inducers has been outstanding and can be applied preventively, by acting on the physiological and biochemical processes of plants. Given the above, the objective of this study was to investigate the efficacy of acibenzolar-S-methyl inducer for the control of anthracnose in pepper plants as well as the activation of enzymes related to pathogenicity and they peroxidase, polifeniloxidase, catalase, ascorbate peroxidase and β-1,3-glucanase.. The study was conducted in a greenhouse in Garanhuns-PE municipality, and the treatments were arranged in subdivided portion, in which the portion consisted of collection times and the subplot of different inducer of doses, with four replications. The plants were treated with the treatments as follows: 0.15g L-1; 0.30g L-1; 0.45g L-1, 0.60g L-1 acibenzolar-S-methyl (Syngenta®) and control (distilled water only). The results were analyzed by the software program SAEG 5.0 to 5% probability. The peroxidase activity was not significant for analysis of variance at 5% probability, however the dose 0.45g L-1 promoted greater activity. For peroxidase although not significant ascorbate activity to analysis of variance, the dose 0.15g L-1 showed increased activity of this enzyme is superior to the control. The increase in catalase activity was significant when the plants were treated with the highest dose of 0.60g L-1 inducer. On the eighth day after inoculation was higher by polyphenol oxidase activity, standing out from the others collection times. The dose 0.60g L -1 promoted greater activity of β-1,3-glucanase enzyme. When the plants medium subjected to the application of acibenzolar-S-methyl there was a reduction in the severity as the doses increased, thereby showing that prior treatment with resistance inducer consists of an efficient method for control of anthracnose disease. / A cultura do pimentão (Capsicum annum L.) é uma das mais importantes hortaliças cultivadas no Brasil. Entretanto essa cultura pode ser acometida por uma série de doenças que acarretam danos econômicos e a antracnose, ainda representa um sério entrave à produção, tanto em campo quanto em casa-de-vegetação. A antracnose nesta olerícola pode ser causada por várias espécies de fungos do gênero Colletotrichum, mas no Brasil, a maioria dos relatos identificam Colletotrichum gloeosporioides como o principal agente causal da doença. As infecções se caracterizam por lesões nas partes aéreas das plantas como nos frutos, caules e folhas. O método de controle das infecções mais utilizado consiste em aplicações de fungicidas, no entanto atualmente se busca por alternativas mais sustentáveis. Desse modo o uso de indutores de resistência vem se destacando podendo ser aplicado de forma preventiva, por atuar nos processos fisiológicos e bioquímicos das plantas. Diante do exposto, o objetivo deste estudo foi verificar a eficácia do indutor acibenzolar-S-metil para o controle da antracnose nas plantas de pimentão bem como a ativação de enzimas relacionadas à patogenicidade sendo elas peroxidase, polifeniloxidase, catalase, ascorbato peroxidase e β-glucanase. O estudo foi conduzido em casa de vegetação no município de Garanhuns-PE. Os tratamentos foram dispostos em parcela subdivida, na qual a parcela consistiu das épocas de coleta e a subparcela das diferentes doses do indutor, com quatros repetições. As plantas receberam os tratamentos da seguinte forma: 0,15g L-1, 0,30g L-1, 0,45g L-1, 0,60g L-1 do acibenzolar-S-metil (Syngenta®) e Controle (somente água destilada). Os resultados obtidos foram analisados pelo emprego do programa software SAEG 5.0 a 5% de probabilidade. A atividade peroxidase não foi significativa para análise de variância a 5% de probabilidade, no entanto a dose 0,45g L-1 promoveu maior atividade. Para a ascorbato peroxidase embora não significativo para análise de variância, a dose 0,15g L-1 apresentou maior atividade desta enzima sendo superior ao controle. O aumento na atividade catalase foi significativo quando as plantas foram tratadas com a maior dose do indutor 0,60g L-1. No oitavo dia após a inoculação houve maior atividade da enzima polifenoloxidase, destacando-se das demais épocas de coletas. A dose 0,60g L-1 promoveu maior atividade da β-1,3-glucanase. Quando as plantas foram submetidas à aplicação do acibenzolar-S-metil verificou-se uma redução da severidade à medida que as doses aumentavam, mostrando com isso que o tratamento prévio com indutor de resistência consiste em um método eficiente para o controle da doença antracnose.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-21T14:38:30Z No. of bitstreams: 1 Luiz Henrique Rocha Lopes.pdf: 2118185 bytes, checksum: 93ff9ca19a00eb532e526f9d4c773b3a (MD5) / Made available in DSpace on 2017-03-21T14:38:30Z (GMT). No. of bitstreams: 1 Luiz Henrique Rocha Lopes.pdf: 2118185 bytes, checksum: 93ff9ca19a00eb532e526f9d4c773b3a (MD5) Previous issue date: 2015-07-27 / One of the main onion crop diseases in Brazil is anthracnose, caused by especies of genus Colletotrichum. In this work it was used several Colletotrichum isolates obtained from plants do Allium genus with symptoms of anthracnose. The isolates were obtained from different regions of Brazil and characterized with the aid of morphological methods, molecular and cultural features. The Bayesian Analysis Method of inference was used for an initial analysis involving the genomic region β-tubulin (tub2). A representative group of isolates, selected in the first analysis, was sequenced for the partial region of actin (act) and ApMat. Two groups of haplotypes were generated and analyzed separately, one by concatenating tub2 and act and Other tub2, act and ApMat. The analysis revealed along with morphological analyzes that onion anthracnose is probably caused by several Colletotrichum species belonging to complexes acutatum and gloesporioides. All the representative isolates used in this study were pathogenic on onion, being able to cause of induce typical symptoms of anthracnose, especially on bulbs with variable aggressiveness range. All the results obtained in this work lead us to believe that onion anthracnose is caused by several species different of what has been reported so far. / Uma das principais doenças no cultivo de cebola no Brasil é a antracnose, causada por espécies do gênero Colletotrichum. Neste trabalho foram utilizados 42 isolados obtidos de plantas do gênero Allium com sintomas de antracnose de todas as regiões geográficas do Brasil e caracterizados com o auxílio de métodos morfológicos, moleculares e de características culturais. O método de análise de inferência Bayesiana foi utilizado para uma análise inicial envolvendo o gene β-tubulina (tub2). Um outro grupo, oriundo desta análise, foi seqüenciado para os genes parciais actina (act) e ApMat. Dois grupos de haplótipos foram gerados e analisados separadamente, um concatenando tub2 e act e outro tub2, act e ApMat. A análise revelou juntamente com análises morfológicas que a antracnose em cebola é causada, por várias espécies inseridas dentro dos complexos acutatum e gloesporioides. Todos os isolados representativos usados no estudo foram patogênicos sendo capazes de induzir sintomas típicos de antracnose, sobretudo em bulbos de cebola com variação de agressividade. Todas as informações deste trabalho levam a crer que, a antracnose em cebola é causada por um conjunto de espécies, diferente do que se tem relatado até o momento.

L'anthracnose constitue pour plusieurs productions végétales une maladie importante qui engendre des pertes post récoltes considérables. Colletotrichum musae est le responsable de cette maladie chez la banane dessert. L'utilisation d'antimicrobiens naturels comme le système lactoperoxydase (LPS) représente une voie naturelle de lutte intéressante contre l'anthracnose. Dans cette étude, nous avons amélioré le LPS en ajoutant de l'iode dans le système existant ou en substituant le thiocyanate par de l'iode. La substitution du thiocyanate à l'iode a permis d'avoir un effet fongicide du LPS. De plus, d'autres substances comme la lactoferrine, le Bioxeda® et l'huile de Neem ont été étudiées pour leur effet antifongique. Les deux dernières substances ont donné une inhibition supérieure respectivement à 90% et à 40%., tandis qu'il n'y a eu aucun effet de la lactoferrine

Colletotrichum graminicola is a hemibiotrophic pathogen of maize that causes anthracnose leaf and stalk rot diseases. The pathogen penetrates the host and initially establishes an intracellular biotrophic infection, in which the hyphae are separated from the living host cell by a membrane that is elaborated by the host, apparently in response to pathogen signals. A nonpathogenic mutant (MT) of C. graminicola was generated that germinates and penetrates the host normally, but is incapable of establishing a normal biotrophic infection. The mutated gene is Cpr1, conserved in eukaryotes and predicted to encode a component of the signal peptidase complex. How can we explain why the MT is normal in culture and during early stages of pathogenicity, but is deficient specifically in the ability to establish biotrophy? To address this, first I characterized the insertion in the 3’ UTR of the MT strain in detail, something that had not been done before. The wild-type (WT) transcript did not differ from predictions, but the MT produced several aberrant transcript species, including truncated and non-spliced transcripts, and the normal one. Aberrant splicing of MT cpr1 was observed both in RNAseq transcriptome data and reverse-transcription polymerase chain reaction (RT-PCR), under different growth conditions and in planta. I also conducted a bioinformatic analysis of other conserved components of the secretory pathway in the MT and WT in planta. One explanation for nonpathogenicity of the MT is that it cannot cope with an increase in secretory activity during infection, and fails to produce necessary pathogenicity factors. With the transcriptome data, I was able to identify effector proteins that were expressed in the WT but not in the MT. Another possible explanation for the MT phenotype is that the MT can’t adapt to stress imposed by the plant. I developed a growth assay to characterize the effect of chemical stressors in vitro. The MT was more sensitive to most stressors, when compared to the WT. The transcriptome data indicates that the genes involved in different stress pathways are expressed in planta in both WT and MT, although very few genes are differentially expressed across the different growth stages.

Soil application of a known activator of Systemic Acquired Resistance (SAR), benzo(1,2,3)thiadiazole-7-carbothioic acid-S-methyl ester (BTH), and Harmonizer, a polychlorinated copper (II) phthalocyanine pigment, reduced severity of Colletotrichum orbiculare in Nicotiana benthamiana by 99% and 38%, respectively. BTH induced expression of nine SAR/progammed cell death-related genes and primed expression of two Induced Systemic Resistance (ISR)-related genes, while Harmonizer induced expression of only one SAR-related gene. Soil application of Harmonizer also reduced severity of Sclerotinia homoeocarpa in Agrostis stolonifera up to 39%, whereas BTH was ineffective. Next generation sequencing identified over 1000 genes in A. stolonifera with two-fold or higher increased expression following Harmonizer treatment relative to a water control, and induced expression of three defense-related genes was confirmed by relative RT-PCR. These results demonstrate that Harmonizer can activate systemic resistance in a dicot and a monocot, but changes in expression of genes indicated that it differed from BTH-activated SAR. / Petro-Canada, Natural Sciences and Engineering Research Council of Canada, Ontario Turfgrass Research Foundation

Colleotrichum graminicola is the causal agent of anthracnose stalk rot, and is one of the most common and aggressive pathogens of maize. The goal of my Ph.D. project was to contribute to a better understanding of the biology of the interaction between C. graminicola and its host. C. graminicola produces two type of asexual spores: one is produced on the surface of infected tissues and is thought to be involved in the spread of the disease in the field. The second type of spore, oval in shape, is produced inside the infected plant tissues, and was believed to be involved in the movement of the pathogen inside the plant tissues via the vascular system. I tested this hypothesis with both cytological and molecular approaches. I used strains of C. graminicola expressing green fluorescent proteins (GFP) to inoculate wounded plants, and followed the development of the pathogen over time. This study revealed that C. graminicola is not a vascular pathogen. C. graminicola primarily moved through the rind and vascular fibers. Oval spores were produced in colonized parenchyma cells and remained dormant, and did not appear to be involved in the movement of the pathogen, at least during the early stages of the disease development. I also studied pathogen ingress in the absence of a wound. I inoculated unwounded plants with the GFP expressing strains. C. graminicola efficiently colonized the epidermis and, given enough time, penetrated and colonized the deeper parenchyma tissues, after first moving through the fibers. To further test the role of sporulation in colonization of maize tissues, I used targeted mutagenesis to disrupt a major gene known to regulate sporulation and vegetative growth in several other fungi. The gene Cgg1, orthologue of the A. nidulans fadA, was disrupted using the split marker method. The Cgg1 mutants were less pathogenic than the wildtype to wounded plants. This was associated with an apparent increase in production of spores and primary infection hyphae. This suggests that Cgg1 signaling pathway plays a role in maximizing colonization of host tissues, and that this involves negative regulation of sporulation and primary hyphae production in planta.