Le biocontrôle est défini comme un ensemble de méthodes de protection des végétaux par l’utilisation de mécanismes naturels. Son principe repose sur la gestion des équilibres des populations d’agresseurs plutôt que sur leur éradication. La protection des cultures de la pomme de terre Solanum tuberosum contre les bactéries pectinolytiques (Dickeya et Pectobacterium) a été précédemment proposée comme une application du biocontrôle. Il s’agit ici de perturber (quencher) la communication quorum-sensing (QS) utilisée par l’agent pathogène pour coordonner son attaque et sa virulence. Afin d’optimiser cette méthode de lutte par quorum-quenching (QQ) et d’en contrôler l’efficacité, nous avons étudié la voiecatabolique des -lactones d’un agent de biocontrôle, la bactérie Rhodococcus erythropolis. Cette voie est impliquée dans la dégradation des signaux N-acyl-homoserine lactones du pathogène. Nous avons d’abord étudié le rôle du répresseur QsdR ainsi que la régulation transcriptionnelle de l’opéron qsd impliqué dans la dégradation des signaux. La compréhension de cette régulation a permis de générer des biosenseurs capables de monitorer les activités QS du pathogène et QQ du protecteur. Sous microscopie confocale à balayage laser, ces outils ont apporté des preuves visuelles du rôle et du lien entre ces deux activités dans les tissus du tubercule. Enfin, la faible spécificité du répresseur QsdR pour ses ligands, apermis de proposer la -caprolactone, un analogue structural des signaux de QS, comme inducteur de l’opéron qsd. Dans l’ensemble, ces travaux permettent d’approfondir nos connaissances sur le rôle et le fonctionnement du QQ chez R. erythropolis. Ils permettent aussi d’envisager le contrôle de la maladie via un agent dont l’activité de QQ pourra être biostimulée par des lactones peu coûteuses lors de la formulation puis de l’épandage aux champs. / Biocontrol is defined as a set of plant protection methods through the use of natural mechanisms. Its principle involves the control of populations of aggressors rather than their eradication. The protection of the potato Solanum tuberosum against soft-rot bacteria (Dickeya and Pectobacterium) has been previously proposed as an application of biocontrol. This involves disturbing the quorum-sensing (QS) communication used by the pathogen to coordinate its attack and virulence. In order to optimize this quorum-quenching (QQ) biocontrol method and to control its effectiveness, we have studied the catabolic pathway of -lactones of a biocontrol agent, the Rhodococcus erythropolis bacterium. This pathway is involved in the degradation of the pathogen N-acyl-homoserine lactones signals. We firststudied the role of the QsdR repressor as well as the transcriptional regulation of the qsd operon involved in signal degradation. The understanding of this regulation has made it possible to generate biosensors capable of monitoring the QS of the pathogen and QQ of the protector. Under confocal laser scanning microscopy, these tools provided visual evidence of the role and link between these two activities in the tuber tissues. Finally, the low specificity of the QsdR repressor for its ligands made it possible to propose the -caprolactone, a structural analog of QS signals, as an inducer of the qsd operon. Overall, this work provides insight into the role and function of QQ in R. erythropolis. It also allows to envisage the control of the disease using a biocontrol agent whose QQ activity can be biostimulated by inexpensive lactones during formulation then spreading in the field.

My diploma thesis deals with genetic variability of entomopathogenic fungi of the Isaria genus encountered in the Czech Republic. Individual representative of the genus can be found in soil where they attack all developmental stages of insects, giving preference to larvae and pupae. The Isaria fungi find application first and foremost where plants have to be provided biological protection. In case of mitosporic fungi is the precise identification very difficult, taxonomy is often unclear in many genera, including the genus Paecilomyces/Isaria to demonstrate their polyphyletic nature. The fungi are classified primarily with reliance on morphological studies. The most common markers used to identify fungi are the shapes and sizes of their conidia and the biological properties (germination of spores, tests of biological efficiency). Identification made in consideration of the morphological markers is inaccurate and very variable. To overcome those accuracies, there are very useful molecular DNA markers, which can be relevant in ecology, biology and in fungi genetics. This paper relies on applying the ITS region (Internal Transcribed Spacer) as a molecular marker. ITS regions are partial constituent rDNA carrying no code - that is why the regions are likely to accumulate evolutionary changes in the DNA sequence, which makes them suitable for extensive use in taxonomic analyses of many organisms. The study results in a phylogenetic trees constructed by comparing different sequences of ITS regions obtained from the samples of entomopathogenic fungi of the Isaria genus gathered in the Czech Republic during the monitoring stage 2013 to 2014. Thereunder detection of Isaria sp. occurring in the Czech Republic.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The genus Pinus is highlight in the forestry sector, especially in southern Brazil, by production of wood and cellulose. However, their seeds are vulnerable to attack by fungi causing diseases in nurseries. The present study aims to evaluate the physiological and sanitary quality of Pinus spp. seeds. from different origins; morphological and molecular identification of fungal species isolated from seeds of Pinus sp.; evaluate the pathogenic potential these fungi; and testing the efficacy of biocontrol agents for the treatment of seeds. For initial characterization of seed were evaluated germination and sanity tests. Four lots were utilized and samples were composed of 400 seeds for each test. The morphological characterization of the fungal isolates was accomplished based on specific keys to the genera Fusarium and Lasiodiplodia. For the molecular identification, ITS region and elongation factor, were sequenced. The pathogenicity test consists of the inoculation of the pine seeds by contact with a fungal culture of the pathogen, the substrate was sterilized sand, the test was kept in a growth room (25 ± 3 °C and photoperiod of 12 h) during 45 days. For biocontrol tests were used commercial products based on Trichoderma and Bacillus; and an isolated Bacillus themselves obtained from pine seeds. The in vitro control was conducted by direct confrontation betweem antagonist and pathogen. The in vivo test was carried out in conditions of vegetation for 60 days, the variables evaluated were emergence, stem diameter, fresh weight and dry seedling. The germination percentage of lots was higher 70%. The fungal genera associated with seeds were: Fusarium, Lasiodiplodia, Aspergillus, Penicillium and Trichoderma. Based on morphological and molecular characteristics identified the species F. oxysporum, F. verticillioides and Lasiodiplodia theobromae as pathogenic species to Pinus spp., causing pre and post-emergence damping-off. The antagonists show control potential of F. oxysporum, F. verticillioides and Lasiodiplodia theobromae in vitro. In vivo tests does not interfere with the development of the seedlings, until the 60 days of test. / O gênero Pinus destaca-se no setor florestal, especialmente na região Sul do Brasil, pela produção de madeira e celulose. Entretanto, suas sementes apresentam vulnerabilidade sanitária e doenças causadas por fungos são ocorrências decisivas na fase de produção de mudas nos viveiros. Diante disso, o presente estudo tem como objetivo avaliar a qualidade fisiológica e sanitária de sementes de Pinus spp. oriundas de diferentes procedências; identificar morfologicamente e molecularmente os isolados fúngicos associados as sementes; avaliar o potencial patogênico desses fungos; e testar a eficiência de agentes biocontroladores no tratamento das sementes. Para a caracterização inicial das sementes foram utilizados os testes de germinação e sanidade, avaliando-se quatro lotes e as amostras foram compostas por 400 sementes, para cada teste. A caracterização morfológica dos isolados fúngicos foi realizada com base em chaves específicas para os gêneros Fusarium e Lasiodiplodia, já para a identificação molecular, foram sequenciadas as regiões ITS e fator de elongação. O teste de patogenicidade consistiu na inoculação das sementes de Pinus via contato com a cultura fúngica do patógeno, sendo o substrato utilizado areia esterilizada com o teste mantido em sala de germinação (25 ± 3 °C e fotoperíodo de 12 h) durante 45 dias. Para os testes de biocontrole foram utilizados produtos comerciais à base de Trichoderma e Bacillus e um isolado de Bacillus obtido das próprias sementes de Pinus. O controle in vitro foi realizado pelo método de confronto pareado de culturas (antagonista x patógeno) e o teste in vivo foi desenvolvido em condições de casa de vegetação, durante 60 dias; as variáveis avaliadas foram emergência, diâmetro do colo, peso fresco e seco de mudas. O percentual de germinação dos lotes foi superior a 70%. Os gêneros fúngicos identificados nas sementes foram: Fusarium, Lasiodiplodia, Aspergillus, Penicillium e Trichoderma. Com base nas características morfológicas e moleculares identificaram-se as espécies Fusarium oxysporum, F. verticillioides e Lasiodiplodia theobromae como patogênicas a espécie em estudo, causando damping off de pré e pós-emergência. Os agentes antagonistas mostraram potencial de controle in vitro sobre F. oxysporum, F. verticillioides e L. theobromae e quando confrontados in vivo com L. theobromae não interferiram no desenvolvimento das mudas, até os 60 dias de condução do teste.

Orientador: Samia Maria Tauk-Tornisielo / Banca: Sandra Regina Ceccato Antonini / Banca: Regina Teresa Rosim Monteiro / Banca: Antonio Ismael Bassinelo / Banca: Carlos Renato Corso / Resumo: Nas usinas, no início da safra, a obtenção de matéria-prima de boa qualidade é maximizada com a aplicação de fitorreguladores, os quais aumentam o teor de sacarose da cana-de-açúcar. Em áreas onde eles são aplicados, tem se observado melhor desenvolvimento e perfilhamento das plantas. Avaliou-se aqui o efeito da aplicação dos fitorreguladores Ethrel e Moddus sobre o crescimento da cana-de-açúcar, de forma direta e indiretamente, através da modificação da microbiota rizosférica. Além disso, objetivou-se também avaliar o uso de rizobactérias, isoladas dos experimentos com fitorreguladores, para o biocontrole de doenças e seus possíveis mecanismos de ação. Os efeitos dos fitorreguladores sobre os microrganismos do solo foram avaliados em meios de cultura acrescidos de Ethrel e Moddus em concentrações de 0 a 1000 ppm. Estes fitorreguladores foram aplicados via foliar e via solo para análise do desenvolvimento da cana-de-açúcar (variedades RB72454, RB835486 e RB855156) em casa-de-vegetação, utilizando-se solo sem tratamento e tratado com brometo de metila. Após dez meses, foram avaliadas a brotação, altura e matéria seca da parte aérea e das raízes das plantas cultivadas. Rizobactérias foram isoladas dos solos contidos nos vasos e avaliadas in vitro quanto à capacidade de controle de fungos fitopatogênicos (Thielaviopsis paradoxa, Fusarium spp. e Hendersonina sacchari), e in vivo, quanto à capacidade de promoção de crescimento de plântulas de cana-de-açúcar. Alguns mecanismos de ação das rizobactérias foram também estudados, como produção de ácido indol acético, ácido cianídrico, sideróforos e solubilização de fosfato inorgânico. Constatou-se que as populações de fungos foram mais sensíveis à adição dos fitorreguladores do que outros grupos de microrganismos, com redução...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: For sugar and alcohol industries, at the start of harvesting, to obtain good quality raw material is potentially possible with the application of plant regulators, which have a role in natural sugar cane maturation, increasing sucrose content. In areas where they have been applied, better plant development and shooting have been observed. The aim here was to evaluate the application of plant regulators Ethrel and Moddus on sugar cane growth, not only in a direct way, but also indirectly, through the modification of rhizosphere microorganisms. Besides, this work also aimed the evaluation of rhizobacteria isolated from the experiments using plant regulators upon the disease biocontrol and their action mechanisms in this respect. The effects of plant regulators upon the soil microorganisms were verified in culture media where Ethrel and Moddus were added in concentrations ranging from 0 to 1000 ppm, while the effects of these substances (applied in leaves and in soil) upon the sugar cane development (varieties RB72454, RB835486 and RB855156) were surveyed in greenhouse, using soil without treatment and treated with methyl bromide. After a ten-month period, the experiments were finished, and sprouting, height and aerial part and root dry matter were analyzed. Soil samples were taken from the pots for rhizobacteria isolation, which were evaluated initially in vitro regarding their ability to control plant pathogenic fungi (Thielaviopsis paradoxa, Fusarium spp. and Hendersonina sacchari), and in vivo, regarding their ability to promote sugar cane growth. Some action mechanisms were also studied, as indol acetic acid, cyanide acid and siderophore production and inorganic phosphate solubilization. It was verified that the fungi populations were more sensitive to the addition of plant regulators than other microorganisms, reducing their colony-forming unit (CFU)...(Complete abstract, click electronic access below) / Doutor

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Xanthomonas campestris pv. campestris is a phytopathogenic bacterium, the causative agent of black rot in crucifers. For the control of plant pathogens diseases, there is the use of bacteria with activity antagonistic to the pathogen. Recent studies show that Bacillus species have on X. campestris a strong biological control. One of the mechanisms of this control is the production of secondary metabolites by these species. The objective of this work was to select bacteria X. campestris and antagonists to evaluate the antimicrobial activity of extracellular filtered bacteria (FEB) antagonist activity. To this, 257 bacteria isolated from a suppressive soil. They were evaluated in vitro antagonist activity by the technique of double layer. Ninety-two isolates (44.6%) were able to inhibit growth of the target pathogen (X.campestris). Of the 92 isolates selected on double layer of the test, 51 (55.43%) showed inhibition of growth of X. campestris on the inhibition assays with FEB in liquid medium. Thirteen of 50% or more inhibited the growth of the target pathogen, and the FEB-8, FEB-31-FEB 68, FEB 74-FEB-87 and were able to inhibit 100% growth of X. campestris. The FEB isolated TC-DT08, belonging to the genus Paenibacillus, it was used for in vivo tests in plant farming kale. The artificial inoculation kale with X. campestris pretreated with FEB-08 showed that the bacterium loses the ability to colonize and cause the cabbage black rot, indicating the potential use of this isolate to protect kale butter infection by X. campestris. / Xanthomonas Campestris. pv campestris é uma bactéria fitopatogênica, agente causal da podridão negra em crucíferas. Dentre os mecanismos para o controle de doenças de fitopatógenos, destaca-se o uso de bactérias com atividade antagonista ao patógeno. Estudos recentes mostram que espécies de Bacillus exercem sobre X. Campestris um forte controle biológico. Um dos mecanismos deste controle é a produção de metabólitos secundários por essas espécies. O objetivo deste trabalho foi selecionar bactérias antagonistas a X. campestris e avaliar a atividade antimicrobiana dos filtrados extracelulares das bactérias (FEB) com atividade antagonista. Para isso, 257 bactérias isoladas de solos supressivos foram avaliadas quanto a atividade antagonista in vitro pela técnica da dupla camada. Noventa e dois isolados (44,6%) foram capazes de inibir o crescimento do fitopatógeno alvo (X.campestris). Dentre os 92 isolados selecionados no teste da dupla-camada, 51 (55,43%) apresentaram inibição do crescimento da X. campestris nos ensaios de inibição com os FEB em meio líquido. Treze destes inibiram 50% ou mais do crescimento do fitopatógeno-alvo, sendo que os FEB-08, FEB-31, FEB-68, FEB-74 e FEB-87 foram capazes de inibir 100% do crescimento de Xanthomonas campestris. O FEB do isolado TC-DT08, pertencente ao gênero Paenibacillus, foi utilizado para testes in vivo em plantas de couve-manteiga, em condições de casa de vegetação. A inoculação artificial de couve-manteiga com X. campestris pré-tratada com o FEB-8 demonstrou que a bactéria perde a habilidade de colonizar a couve e causar a podridão negra, o que indica o potencial do uso deste isolado para proteger a couve-manteiga da infecção por X. campestris.

Made available in DSpace on 2014-07-29T15:16:30Z (GMT). No. of bitstreams: 1 Tese Mestrado FINAL.pdf: 1488636 bytes, checksum: c1d993759439dd885efc25277ad9b4d3 (MD5) Previous issue date: 2011-01-28 / The hydrolytic activity of enzymes produced by Trichoderma asperellum, immobilized biodegradable films, as growth inhibitor of microorganisms was tested. The inhibitory activity was demonstrated on Aspergillus niger, Penicillium sp. and Sclerotinia sclerotiorum, microorganisms usually related to the attack and/or food contamination at the field or packaged. We used two polymer blends with different compositions, cassava starch and poly-butylene adipate-co-terephthalate (Ecoflex®, BASF Chemical Company) and other composed for polyvinyl alcohol (PVA) and polysaccharide cashew gum (PEJU). T. asperellum was induced to produce enzymes involved in the attack mycoparasite (N-acetylglucosaminidases, β-1,3-glucanases, chitinases and proteases) by the addition of crude chitin in the growth medium. The enzymes produced in major quantity were N-acetylglucosaminidase and chitinase. The pool of enzymes produced in the experiments was then used for immobilization tests. The immobilization process was performed in films by two methods: covalent and ionic bonding. In both methods, the presence of immobilized hydrolytic enzymes resulted in reduced growth of microorganisms, but the covalent immobilization of the results were more expressive. S.sclerotiorum was the microorganism most sensitive, followed by A. niger and Penicillium sp. To confirm the action of hydrolytic enzymes produced by T. asperellum and evaluate the effects they produce cell wall of microorganisms and other structures, the films with enzyme immobilized by covalent bonding were subjected to scanning electron microscopy. The structures most affected were hyphae and spores. Overall, the synergistic action of all enzymes produced by T. asperellum, reduced the growth of microorganisms when immobilized on the surface of the films Starch-Ecoflex® and PVA-PEJU. Moreover, the polymer blends tested exhibited desirable characteristics for future use in food packaging and most importantly, also provide efficient systems for the immobilization of enzymes. / O trabalho foi conduzido com o objetivo de testar a ação de enzimas hidrolíticas produzidas por Trichoderma asperellum, imobilizadas em filmes biodegradáveis, como agente inibidor do crescimento de microrganismos. A atividade inibitória foi testada sobre Aspergillus niger, Penicillium sp. e Sclerotinia sclerotiorum, microrganismos geralmente relacionados com o ataque e, ou, contaminação de alimentos, seja no campo ou embalados. Foram utilizadas duas blendas poliméricas de diferentes composições, uma a base de amido de mandioca e poli-butileno-adipato-co-tereftalato (Ecoflex®; BASF Chemical Company) e outra a base de álcool polivinílico (PVA) e polissacarídeo de caju (PEJU). T. asperellum foi induzido a produzir as enzimas envolvidas no processo de ataque micoparasita (N-acetilglicosaminidases, β-1,3-glicanases, quitinases e proteases) pela adição de quitina bruta no meio de crescimento. As enzimas produzidas em maior quantidade foram N-acetilglicosaminidases e quitinases. O pool de enzimas produzidas foi então utilizado nos experimentos de imobilização. O processo de imobilização foi realizado nos filmes por dois métodos: ligação covalente ou ligação iônica. Em ambos os métodos, a presença de enzimas hidrolíticas imobilizadas resultou em redução do crescimento dos microorganismos, porém na imobilização por ligação covalente os resultados foram mais expressivos. S.sclerotiorum foi o microrganismo mais sensível, seguido por A. niger e Penicillium sp. Para confirmar a ação hidrolítica das enzimas produzidas por T. asperellum e avaliar os efeitos por elas produzidos na parede celular e demais estruturas dos microrganismos, os filmes com enzima imobilizada por ligação covalente foram submetidos à microscopia eletrônica de varredura. As estruturas mais afetadas foram hifas e esporos. De forma geral, a ação sinérgica de todas as enzimas produzidas por T. asperellum reduziu visivelmente o crescimento dos microrganismos quando imobilizadas na superfície dos filmes Amido-Ecoflex® e PVA-PEJU. Além disso, as blendas poliméricas testadas apresentaram características desejáveis para futuro uso em embalagens de alimentos e principalmente, também constituem sistemas eficientes para imobilização de enzimas.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-06-02T18:09:18Z No. of bitstreams: 2 Dissertação - Rafaela Araújo Guimarães - 2014.pdf: 1551849 bytes, checksum: b39011c17618c122fad17d142b0288ec (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-06-03T12:41:12Z (GMT) No. of bitstreams: 2 Dissertação - Rafaela Araújo Guimarães - 2014.pdf: 1551849 bytes, checksum: b39011c17618c122fad17d142b0288ec (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-06-03T12:41:12Z (GMT). No. of bitstreams: 2 Dissertação - Rafaela Araújo Guimarães - 2014.pdf: 1551849 bytes, checksum: b39011c17618c122fad17d142b0288ec (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-08-21 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Sarocladium oryzae, the causal agent of rice sheath rot disease is described as antagonistic to rice pathogens. Rice blast is a major rice disease and is responsible for losses up to 100% in productivity. The disease control is done by integrated management, where the main practices are use of resistant cultivars and chemical control. The biocontrol agents or their metabolites may to be more practical to be including them as components in the management. The in objectives of this study a consist to evaluate of S. oryzae isolates for the morphological variability, genetics, biochemistry and antagonistic activity to rice pathogens; evaluate effect of S. oryzae filtrade on conidial germination and appressorium formation of M. oryzae; evaluate potential of conidia and filtrate S. oryzae in the suppression of leaf blast severity and quantify activity of enzymes involved in interaction M. oryzae x rice plant x S. oryzae. Isolates were characterized for color, texture, colony diameter, conidia size and hyphae thickness. In genetic studies, we used RAPD marker primers, and cerulenin production was quantified by HPLC. Antagonism in vitro was assessed by dual culture method. The effect of S. oryzae on conidial germination and appressorium formation of M. oryzae was evaluated using hydrophobic surface. Rice cultivar BRS Primavera, M. oryzae isolate (Py 10.900) and S. oryzae, isolate So 03, were utilized to study plant-pathogen-antagonist relationship. Plants were sprayed, with conidial suspension (CS), 3x105 conídios.mL-1 and culture filtrate (CF) 100% of S. oryzae, two days before inoculation with M. oryzae. S. oryzae isolates showed morphological variability, polymorphism in DNA. A majority of S. oryzae isolates (60%) ware able to produce cerulenin and over 55% were antagonistic to C. miyabeans, M. oryzae, M. albescens and T. cucumeris. The isolate So 29 showed largest inhibition zone. Filtrate of isolates So 03 and So 29 delayed conidia germination by 89.5% and inhibited appressoria formation of M. oryzae by 85%. CS reduced of leaf blast severity in 68.8% and CF in 75.5%. The enzymes β-1,3-glucanase and peroxidase exhibited maximum activity in plants sprayed with CF, when as the activity was high for SA and lipoxygenase in relation to CS treatment, compared to their respective controles, in the absence of M. oryzae. After inoculation with M. oryzae, the lipoxygenase and phenylalanine ammonia-lyase activity in both treatments CF and CS, showed differences compared to controls (plants inoculated with M. oryzae and water only). S. oryzae presented variability to characteristics evaluated and showed potential antagonism against to rice pathogens. Changes in enzyme activity indicate their role in induction resistance in plants in M. oryzae x rice x S. oryzae interaction. / Sarocladium oryzae, agente causal da podridão da bainha do arroz, é descrito como antagonista a patógenos de arroz. A brusone, principal doença do arroz é responsável por perdas de até 100% na produtividade. O controle desta doença é feito pelo uso do manejo integrado, onde as principais práticas são o uso de cultivares resistentes e o controle químico. O uso de agentes de biocontrole ou seus metabólitos pode ser mais uma prática a ser inserida ao manejo. Os objetivos deste estudo foram: avaliar isolados de S. oryzae quanto à variabilidade morfológica, genética, bioquímica e quanto à atividade antagônica aos patógenos de arroz; avaliar o efeito do filtrado de S. oryzae na germinação de conídios e na formação de apressórios de M. oryzae; avaliar o potencial da suspensão de conídios e do filtrado de S. oryzae na supressão da brusone foliar e quantificar a atividade das enzimas relacionadas à patogênese e do fitohormônio ácido salicílico (AS) envolvidos na interação M. oryzae x arroz x S. oryzae. Os isolados foram caracterizados quanto à cor, textura, diâmetro da colônia, tamanho dos conídios e espessura das hifas. No estudo genético, utilizou-se marcador RAPD (Random Amplified Polymorphic DNA) e a produção de cerulenina foi quantificada em HPLC. O antagonismo in vitro foi avaliado pelo método da cultura pareada. A ação de S. oryzae sobre a germinação de conídios e a formação de apressórios de M. oryzae foi avaliada em superfície hidrofóbica. Plantas de arroz da cultivar BRS Primavera, um isolado virulento de M. oryzae (Py 10.900) e o isolado So 03 de S. oryzae, foram avaliados no estabelecimento das relações planta-patógeno-antagonista. As plantas foram pulverizadas com S. oryzae, na forma de suspensão de conídios (SC) - 3x105 conídios.mL-1 e filtrado (FI) 100% concentrado, dois dias antes da inoculação com M. oryzae. Os isolados de S. oryzae apresentaram variabilidade morfológica e polimorfismo no DNA. A maioria dos isolados de S. oryzae (60%) foi capaz de produzir cerulenina e mais de 55% foram antagônicos a C. miyabeans, M. oryzae, M. albescens e T. cucumeris. O isolado So 29 apresentou o maior halo de inibição no pareamento. Os filtrados dos isolados So 03 e So 29 retardaram a germinação dos conídios em 89,5% e inibiram a formação dos apressórios de M. oryzae em 85%. A SC reduziu a severidade da brusone foliar em 68,8% e o FI em 75,5%. Os maiores valores de atividade enzimática específica em relação ao controle antes da presença de M. oryzae foram para β-1,3-glucanase e peroxidase no tratamento com FI, enquanto que, na SC foram lipoxigenase e AS. Depois da presença de M. oryzae a lipoxigenase e a fenilalanina-amônia liase apresentaram atividade tanto com FI quanto na SC, diferindo dos controles (plantas inoculadas com água e com M. oryzae, somente). S. oryzae apresentou variabilidade para as características avaliadas e potencial antagônico aos patógenos do arroz. As alterações na atividade enzimática indicam a indução de resistência em plantas na interação M. oryzae x arroz x S. oryzae.

Avec la prise de conscience de l'impact environnemental et sanitaire des pesticides et l'apparition de phénomènes de résistance face à ces produits, les exigences règlementaires européennes (UE 1107/2009) encadrant leur mise sur le marché sont de plus en plus drastiques. Pour répondre à ces exigences, de nouveaux types de produits de protection des cultures font leur apparition : les bio-pesticides, ou produits de biocontrôle. Il s'agit de produits naturels d'origine végétale, animale ou microbienne, à priori moins rémanent et impactant pour l'environnement que les pesticides conventionnels. C'est dans ce contexte que mes travaux de thèse ont eu pour but le développement de biopesticides d'origine végétale destinés à lutter contre les champignons pathogènes des plantes, pour lesquels il existe encore peu ou pas de moyens de lutte naturels. Au cours de cette thèse, j'ai pu sélectionner à travers différents criblages, écologique, économique et biologique deux extraits végétaux efficaces sur des cibles biologiques d'intérêt et présentant une toxicité sur organismes non cibles bien plus faible que des fongicides conventionnels. Les molécules responsables de l'activité ont été identifiées par fractionnement bio-guidé et analyses spectrales. L'activité des extraits a pu être validée en champs notamment sur mildiou de la vigne (Plasmopara viticola) et moniliose de la nectarine (Monilinia fructigena) après une optimisation de leur formulation. / With the awareness of the environmental and sanitary impact of pesticides and emergence of resistance phenomena, the European regulation requirements (EU 1107/2009) supervising their marketing authorization are more and more drastic. To meet these requirements, new phytoprotection products are emerging: biopesticides also called biocontrol products. They are natural product of plant, animal or microbial origins, likely to be less persistent and more environmentally friendly than conventional pesticides.It is against this background that my thesis work aimed to develop some biopesticides from plant origin, for protection against phytopathogenic fungi for which there are no or few natural product.In this work, I selected, by ecological, economical and biological screening, two plant extracts, effective against biological targets and far less toxic against non-target organism than tested conventional pesticides.Active molecules were identified by bioguided fractionation and spectral analysis. Extracts activities have been evaluated in field test against vine downy mildew (Plasmopara viticola) and Moniliose infection (Monilinia fructigena)

Thesis (PhD (Chemical Engineering))--Cape Peninsula University of Technology, 2019 / The agro-processing industry is currently facing losses due to microbial spoilage of agricultural produce and associated value-added products such as beverages. Decay and undesired fermentation of fruit and beverages by fungal, yeast and bacterial spoilage organisms are among the major contributors of product losses in the food industry. When looking at the different level of food spoilage, it is common to find different spoilage organisms occurring in the same food item; which usually requires food producers to utilise a mixture of synthetic preservatives for spoilage organism control. Some of the synthetic chemical compounds with growth inhibition properties that have been used in food preservation are sulphur dioxide, benzoic, lactic, sorbic and acetic acid. These compounds act against a variety of spoilage microorganisms. In post-harvest control of fungi, triazoles, hydroanilide fenhexamid, dicarboximides and succinate dehydrogenase are also being used. Some spoilage organisms have been found to be resistant to the use of synthetic chemical preservatives which usually favour the use of higher dosage of preservatives in food. The use of synthetic chemicals as preservative and as postharvest control agents has been found to present serious health risks such as cardiovascular diseases, muscles and stomach pains, eyesight and skin damages and impairment of brain functions. The problem posed by the current use of synthetic chemicals in food put pressure on food producers and exporters to seek alternatives that will allow for the eradication of the use of synthetic chemicals as preservative in beverages and as postharvest control agents on fruits. Yeasts have been found to have the ability to grow at a faster rate on cheap media and to colonise dried surfaces rapidly. It has also been found that yeasts produce extracellular compounds of proteinaceous and volatile organic nature with growth inhibition properties against spoilage organisms. The current findings lack some engineering concept that could assist in the design of a production system for high scale production of biopreservation compounds from yeasts. The availability of a cost effective production media, the growth and production kinetics data using a cheaply available nutrient sources as well as the biological thermodynamic data are some of the gaps in biopreservation bioprospecting. Although several yeasts have already been studied to have great inhibition properties against fruit fungal pathogens, it was still unclear what was the minimum inoculum dose to be able to have a fungistatic and fungicidal effect on the growth of fruit spoilage organisms. The concept of combination of biopreservatives and the interaction effect of their biopreservation activity against consortia of spoilage organisms has also been lacking. As an attempt to seek alternatives to the use of synthetic chemicals as preservatives or postharvest control agents, Candida pyralidae Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 strains were assessed for antimicrobial activity against spoilage yeasts (Dekkera bruxellensis, Dekkera anomala, Zygosaccharomyces bailii) and spoilage fungi (Botrytis cinerea, Colletotrichum acutatum and Rhizopus stolonifer). As alternative to refined media, a cost effective approach was explored whereby the use of agro-waste, i.e. grape pomace extracts (GPE), as production medium for biopreservation compounds, was studied. Production kinetics using modified existing models, subsequent to optimization using response surface methodology (RSM) for biopreservation compounds production was studied for the three biocontrol yeasts using GPE broth as the fermentation medium. The evaluation of the interaction study between mixtures of crude biopreservatives against consortia of common spoilage organisms present in beverages was also conducted by producing the crude biopreservation compounds separately from yeasts and then formulating growth inhibition combinations (GICs); GIC 1 (Candida pyralidae Y1117 and Pichia kluyveri Y1125); GIC 2 (C. pyralidae Y1117 and P. kluyveri Y1164), GIC 3 (P. kluyveri Y1125 and Pichia kluyveri Y1164); GIC 4 (C. pyralidae, P. kluyveri Y1125 and P. kluyveri Y1164). The spoilage organism consortia combinations, i.e. SC1, D. anomala and D. bruxellensis; SC2 (D. anomala and Z. bailii); SC3 (D. bruxellensis and Z. bailii) and SC4 (D. anomala, D. bruxellensis and Z. bailii) were also prepared. This study also investigated the effect of varying inoculum dose (ID) of Candida pyralidae strain Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 on the biocontrol of Botrytis cinerea by contaminating the headspace of the growth medium with a fungal plug subsequent to biotreatment with different initial inoculum dose of the respective biocontrol yeasts. Finally, grape pomace extracts was used as fermentation medium to study the biological thermodynamics of biopreservation compound production from the three biocontrol yeasts. The results obtained demonstrated some interesting results. The antagonistic properties of C. pyralidae and P. kluyveri were observed on cheap solidified medium (grape pomace extracts) as well as on fruits (grapes and apples). These yeasts produced extracellular volatile organic compounds (VOCs) that could be responsible for yeast and fungal growth inhibition. Twenty-five VOCs in the category of alcohols, organic acids and esters were identified by GC-MS. The results of the kinetic study showed that the highest volumetric zone of inhibition (VZI) was 1.24 L contaminated solidified media (CSM) per mL biopreservation compounds used (BCU) when Candida pyralidae Y1117 was inoculated in a pH 3-diluted GPE broth (150 g L−1) incubated at 25 °C for 24 h. Similar conditions were applied for Pichia kluyveri Y1125 and P. kluyveri Y1164, albeit under slightly elongated fermentation periods (up to 28 h), prior to the attainment of a maximum VZI of only 0.72 and 0.76 L CSM mL−1 ACU, respectively. The potential biopreservation compounds produced were identified to be isoamyl acetate, isoamyl alcohol, 2-phenyl ethylacetate and 2-phenyl ethanol. The growth inhibition interaction study showed a variation in growth inhibition proficiency depending on the spoilage organisms or the consortia of spoilage organisms being deactivated. It was then suggested that, a food environment contaminated with a consortium of spoilage organisms can be controlled by employing either the crude biopreservation compounds from individual yeast or those of the following yeast combinations, GIC1-4, which showed a better growth inhibition proficiency against SC1-3. The fungistatic and fungicidal effects on the fungal pathogen were dose dependent. The fungistatic characteristics against Botrytis cinerea were displayed after 7 days when 102-105 cells mL-1 of Candida pyralidae Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 were independently used in-vitro and in-vivo. However, 106-108 cells mL-1 inoculum doses displayed fungicidal characteristics. Additionally, the fungicidal property of yeasts studied was also confirmed on table grape (in vivo studies) using closed jar method. The biological thermodynamic study showed that, dried biomass molecular weight of 28.9 g/C-mol, 29.163 g/C-mol, and 27.176 g/C-mol were obtained for Candida pyralidae strain Y1117, Pichia kluyveri Y1125 and Pichia kluyveri Y1164 respectively. The results obtained successfully established useful biological thermodynamic data applicable to the design of adequate biopreservatives production system from yeasts using cheaply available nutrients source.

Les pertes alimentaires mondiales se chiffrent à environ un tiers des aliments destinés à la consommation humaine, soit environ 1,3 milliards de tonnes par an (FAO). Une large fraction de ces pertes est due aux altérations microbiologiques des denrées alimentaires. L’utilisation de produits phytosanitaires reste aujourd’hui la solution la plus largement utilisée en agriculture pour limiter ces pertes. Cependant, avec le plan EcoPhyto 2, le gouvernement français a pour objectif de réduire de 50% l’usage des pesticides chimiques d’ici 2025, en particulier en promouvant l’émergence du biocontrôle. Pour développer cette approche, il est cependant nécessaire de comprendre, pour mieux les maitriser, les mécanismes sous-jacents. Les différents modes d’action de biocontrôle par les microorganismes décrits sont la stimulation des défenses naturelles des plantes, la production de substances antimicrobienne et la compétition nutritionnelle. L'originalité de ce projet est d'intégrer le mode de vie en biofilm dans les mécanismes de bioprotection (compétition spatiale et nutritionnelle, libération de principes antimicrobiens). Dans la filière Française des champignons de couche (Agaricus bisporus), l’agent de biocontrôle utilisé depuis 2008 par plus de 80 % de la filière, est Bacillus subtilis QST713. Ce biofongicide montre une nette efficacité contre Trichoderma aggressivum, la principale moisissure à l’origine de pertes économiques lors de la culture d’A. bisporus. Afin d’accompagner la filière dans cette voie biologique, nous avons entrepris de séquencer et étudier le génome de cette souche, afin de déterminer son potentiel de biocontrôle et sa capacité à former des biofilms. Nous avons également évalué l’impact de ce biofongicide sur la dynamique des communautés microbiennes du compost de culture d’A. bisporus exposé ou non à T. aggressivum. Enfin, l'étude de la reprogrammation cellulaire de cet agent de biocontrôle lors de sa culture en micromodèles axéniques, nous a permis une meilleure compréhension des phénomènes de colonisation des substrats et d'inhibition des flores indésirables. Ce projet a permis d’enrichir les connaissances vis-à-vis des mécanismes de biocontrôle dans la filière des champignons et pourra permettre une possible application à d’autres filières agricoles. / Worldwide, food losses amount for about one-third of food for human consumption, 1.3 billion tons per year (FAO). A large fraction of these losses are due to microbiological alterations. The use of phytosanitary products remains today the most widely used solution in agriculture to limit these losses. However, with the EcoPhyto 2 plan, the French government aims to reduce the use of chemical pesticides by 50% by 2025, in particular by promoting the emergence of biocontrol. To develop this approach, it is necessary to understand the underlying mechanisms. The different modes of action of biocontrol by the microorganisms described are the stimulation of the natural defenses of the plants, the production of antimicrobial substances and the nutritional competition. The originality of this project is to integrate the biofilm mode of life into bioprotection mechanisms (spatial and nutritional competition, release of antimicrobial principles). In the French sector of the button mushrooms (Agaricus bisporus) culture, the biocontrol agent used since 2008 by more than 80% of the sector, is Bacillus subtilis QST713. This biofungicide shows a clear efficacy against Trichoderma aggressivum, the main mold causing economic losses during the cultivation of A. bisporus. To accompany the sector in this biological pathway, we have sequenced and studied the genome of this strain, in order to determine its biocontrol potential and its ability to form biofilms. We also evaluated the impact of this biofungicide on the dynamics of microbial communities in A. bisporus culture compost exposed or not to T. aggressivum. Finally, the study of the cellular reprogramming of this biocontrol agent during the culture in axenic micromodels allowed us a better understanding of the substrates colonization phenomenon and the inhibition of undesirable flora. This project will enrich the knowledge of the biocontrol mechanisms used in the mushroom industry and may allow a possible application to other agricultural sectors.