Este estudo descreve a diversidade de actinobactérias isoladas da esponja marinha Aplysina fulva e o potencial destes microorganismos como produtores de metabólitos bioativos com propriedades fungicidas e herbicidas. Actinobactérias são prolíficas produtoras de compostos farmacologicamente importantes, pois cerca de 70% dos antibióticos naturalmente derivados que estão atualmente em uso clínico são produzidos por estes microorganismos. Entretanto este valor é ainda inexpressivo na indústria agrícola. Agroquímicos sintéticos ainda são dominantes no mercado apesar de estarem menos efetivos contra plantas daninhas e patógenos cada vez mais resistentes. Neste trabalho, um total de 21 actinobactérias foram isoladas com a utilização de meios seletivos. Análises filogenéticas baseadas no sequenciamento parcial do gene que codifica para o rRNA 16S mostrou que estes microorganismos pertencem a oito gêneros do filo Actinobacteria: Kocuria; Citricoccus; Terrabacter; Gordonia; Agrococcus; Tsukamurella; Brevibacterium e Streptomyces. Os extratos de todos os isolados foram testados para verificar a produção de metabólitos secundários com propriedades fungicidas contra os fungos fitopagênicos de importância agrícola: Pythium aphanidermatum; Phytophthora capsici e Magnaporthe grisea. O extrato bruto de 43% dos isolados mostrou atividade fungicida para ao menos um dos patógenos. O perfil químico do extrato dos isolados com bioatividade positiva foram similares mesmo entre gêneros diferentes. Os metabólitos do Streptomyces ASPSP 103 foram mais eficientes devido à forte inibição contra todos os patógenos testados. Portanto este isolado foi selecionado e testado para atividade herbicida por meio de screening que teve início com testes de atividade algicida contra a microalga Selenastrum capricornutum. Acreditamos que actinobactérias associadas a esponjas marinhas desempenham um papel de defesa química contra microalgas que possam obstruir os porócitos asfixiando o animal, e que estes compostos algicidas possivelmente tenham ação herbicida. Foi verificada atividade do extrato bruto do Streptomyces ASPSP 103 contra S. capricornutum, e a atividade herbicida pré-emergência com um efeito fraco em Lactuca sativa (dicotiledônea) e uma forte inibição em Agrostis stolonifera (monocotiledônea). A purificação do extrato bruto para isolamento do composto bioativo foi guiado por bioensaio contra Pythium aphanidermatum, um oomiceto de rápido crescimento e sensível aos metabólitos de ASPSP 103 previamente testados. Foi identificado o composto da classe butenolida com atividade herbicida préemergência contra Agrostis stolonifera (IC50 33.43 μg/mL). Este é o primeiro relato da atividade de butenolida para atividade herbicida. Estudos aprofundados em taxonomia mostraram que as características filogenéticas, morfológicas e químicas do isolado ASPSP 103 são consistentes com o gênero Streptomyces. Portanto devido algumas diferenças em parâmetros taxonômicos, ASPSP 103T foi proposto como linhagem tipo para uma nova espécie de Streptomyces, para qual o nome Streptomyces atlanticus sp. nov. foi sugerido. Estes resultados enfatizam o potencial de Streptomyces marinhos para produzir compostos bioativos com potencial de aplicação em agrobiotecnologia. / Actinobacteria are producers of important pharmacological compounds. About 70% of natural antibiotics are derived from these microorganisms. However, the use of natural compounds are still limited in the agricultural industry, even considering that synthetic pesticides are less effective against pathogens and weed plants. This study describes the diversity of actinobacteria associated with the marine sponge Aplysina fulva and their potential as producers of bioactive compounds with fungicidal and herbicidal properties. In this study, a total of 21 actinomycetes were isolated with the use of selective media. Phylogenetic analyzes based on partial sequencing of the gene encoding for 16S rRNA showed that these microorganisms belong to eight Actinobacteria genera, including Kocuria, Citricoccus, Terrabacter, Gordonia, Agrococcus, Tsukamurella, Brevibacterium and Streptomyces. The extracts of all isolates were tested for the production of secondary metabolites with fungicidal properties against the following phytopathogenic fungi: of Pythium aphanidermatum, Phytophthora capsici and Magnaporthe grisea. The crude extract of 43% of the isolates showed fungicidal activity for at least one of the pathogens. The chemical profiles of the actinobacteria extracts with positive bioactivity were similar even among different genus. The metabolites of Streptomyces ASPSP 103 were more efficient because of the strong inhibition against all tested pathogens. So, the isolate ASPSP 103 was selected and tested for herbicide activity through screening for algaecide activity towards microalgae Selenastrum capricornutum. We believe that actinobacteria associated with marine sponges play a role in chemical defense against algae that can obstruct the pores, choking the animal. These algaecides compounds possibly have herbicide action. Activity of the Streptomyces ASPSP 103 crude extract against S. capricornutum was observed. In addition, it was observed a weak pre-emergence herbicide activity on Lactuca sativa (dicot) and a strong inhibition in Agrostis stolonifera (monocot). The purification of the crude extract to isolate the bioactive compound was guided by bioassay against Pythium aphanidermatum, a fast growing oomycete and sensitive to metabolites from ASPSP 103 previously tested. The butenolide compound was identified with pre-emergence herbicidal activity against Agrostis stolonifera (IC50 33.43 μg/mL). This is the first report of butenolide activity with herbicide activity. Taxonomy studies showed that the phylogenetic, morphological and chemical characteristics of the isolated ASPSP 103 are consistent with the Streptomyces genus. Then, considering some differences in taxonomic parameters, ASPSP 103T was proposed as line type for a new species of Streptomyces, for which the name Streptomyces atlanticus sp. nov. was suggested. These results emphasize the potential of marine Streptomyces to produce bioactive compounds with potential biotechnological application in agricultural industry.

Dermatophytes (order Onygenales, Ascomycota) are microscopic filamentous keratinophilic fungi that can cause skin infections known as dermatophytosis. The most diverse but not very studied genus Arthroderma has been revised recently (Míková 2018) which was essential for further research. This genus comprises mostly species with a supposed reservoir in soil. Lack of information about their ecology and frequent isolation of some species from the hair of free- living mammals (mainly rodents) may testify a strong host association. Rodents could thus represent the hidden reservoir of this species. For this thesis, I have chosen three ecologically distinct rodent species: Mus musculus, Apodemus flavicollis, and Clethrionomys glareolus. I obtained the material by brushing the hair of asymptomatic individuals and used this material for cultivation on selective medium. I identified the isolates of dermatophytes (n = 30) using molecular methods. I used sequences of three highly variable loci (ITS, tubb a tef1α) to incorporate these isolates in the phylogenetic analysis based on the monography of the genus Arthroderma (Míková 2018). I characterized the phenotype of selected strains based on morphological and physiological data including the ability to utilize keratin and the production of siderophores. The...

Une recherche de souches bactériennes lactiques productrices de bactériocine a été entreprise à partir daliments traditionnels dorigine sénégalaise et les souches les plus actives ont été mises en uvre comme barrière en supplément du sel (NaCl) contre la croissance de la flore contaminante de différents poissons maigre [sompat (Pomadasys jubelini)], moyennement gras [capitaine (Polydactylus quadrifilis)], et gras [mâchoiron (Arius heudeloti)] de production artisanale au Sénégal. La prévalence des bactéries lactiques a été déterminée à 109 UFC/(g ou ml) dans les produits fermentés traditionnels à base de céréale et de lait, et 103 UFC/g dans les produits de la pêche fermentés. Douze souches bactériennes à activité inhibitrice de type bactériocine ont été détectées au sein de 220 souches de bactéries lactiques isolées de 32 échantillons de ces différents types de produits. Les 11 souches ont été caractérisées (API 50 CH, 16S rDNA) comme étant des souches de Lactococcus lactis subsp. lactis qui portent le gène codant pour la nisine, la souche restante est une souche dEnterococcus faecium et possède le gène codant pour lEntérocine B. Les deux souches, Lactococcus lactis subsp. lactis CWBI-B1410, isolée de farine de mil fermentée et productrice de nisine, et la souche Lactobacillus curvatus CWBI-B28 provenant de la collection du CWBI de la FUSAGx, produisant trois bactériocines de type curvalicine 28a, b et c, ont été mises en uvre en combinaison avec du sel comme barrière contre la croissance de la flore contaminante dans les poissons. La flore totale dans les poissons de production artisanale atteingnait 5,70 log UFC/g. La transformation des poissons par fermentation naturelle à 30°C (sans traitement préalable : procédé traditionnel), entraîne une prolifération de bactéries Gram-négatives comme Proteus sp, Shewanella putrefaciens et des souches de Bacillus sp qui atteignent 109-10 UFC/g en moins de 24 h dans les produits. Lutilisation de CWBI-B1410 (107 UFC/g) comme ferment avec ajout de glucose (1% m/m) dans les filets, entraîne une production in situ de substances antagonistes (acides organiques et bactériocine). Il en résulte une réduction du niveau de la flore contaminante entérique de 2 (P. quadrifilis) et de 4 (P. jubelini) log UFC/g par rapport aux poissons préparés traditionnellement. Lajout de sel dans des produits ainsi fermentés réduit la flore entérique. Laddition des surnageants de culture neutralisés (pH 6) bactéricides issus de CWBI-B1410 ou de CWBI-B28 en combinaison avec du chlorure de sodium (0,14 g/ml) sur les filets crus (100 ml/100 g) incubés à 10°C réduit le niveau de la contamination bactérienne de 1,5 log UFC/g et retarde laugmentation de la flore qui reste à un niveau < 106 UFC/g pendant 13 à 18 jours dincubation à 10°C selon le type de poisson, alors que pour les filets contrôles, traités avec des surnageants de culture neutralisés de souches non productrices de bactériocines (Lactobacillus curvatus LMG 21688 et Lactococcus lactis LMG 6890) salés (NaCl 0,14 g/ml), la flore contaminante dépasse 106 UFC/g au bout de 3 à 7 jours. Les effets antibactériens des surnageants de cultures neutralisés bactéricides et salés (NaCl 0,14 g/ml), sont par ailleurs, plus importants que ceux dune solution salée (NaCl 0,14 g/ml) supplémentée de sels de benzoate et de sorbate en concentration de 0,5 mg/ml chacun sur les poissons riches en lipides. Ces résultats suggèrent que, la combinaison des effets antibactériens des bactéries lactiques productrices de bactériocines et du sel, plus particulièrement lutilisation de SCN salés, comme préservatifs, peut constituer un moyen approprié damélioration de la conservation et de la qualité microbiologique des produits de la pêche artisanale au Sénégal. Le traitement des poissons selon lune ou lautre des stratégies décrites précédemment, combiné à un système de séchage devrait permettre de produire des produits halieutiques traditionnels de meilleures qualités microbiologiques et de durée de conservation plus longue, justifiant limportance de continuer cette étude sur lamélioration de la productivité de bactériocine par CWBI-B1410, et les impacts des nouveaux traitements sur les qualités organoleptiques et nutritionelles des produits. A screening of bacteriocin-producing lactic acid bacteria from Senegalese traditional food products was undertaken and the most active strains were tested in combination with sodium chloride as barrier to control spoilage bacteria growth on different artisanal fish products [lean sumpat grunt (Pomadasys jubelini), moderarely fat giant African threadfin (Polydactylus quadrifilis) and fat smoothmouth sea catfish (Arius heudeloti)] in Senegal. The prevalence of lactic acid bacteria was determined at 109 CFU/(g or ml) in traditional fermented cereals and fermented milk products, and 103 CFU/g in fermented seafood products. Twelve bacteriocin-producing strains were detected of 220 lactic acid bacteria strains randomly selected from such products. The eleven were characterized (API 50CH and 16S rDNA) as Lactococcus lactis subsp lactis strains and contain gene encoding nisin, and the remaining one an Enterococcus faecium strain which contains gene encoding Enterocin B. Two strains, Lactococcus lactis subsp. lactis CWBI-B1410, a nisin producer isolated from fermented millet flour from Senegal, and Lactobacillus curvatus CWBI B28 which produces three bacteriocins (Curvalicin 28a, b et c) from CWBI collection, were tested in combination with sodium chloride as barrier to control spoilage bacteria growth on fish from artisanal production. The total bacterial counts of fishes reached 5.7 log CFU/g. The processing of such products by natural fermentation at 30°C (without any preliminary treatment as in traditional process) result in proliferation of Gram-negative bacteria such as Proteus sp and Shewanella putrefaciens, and some Bacillus sp strains, which reached 109-10 CFU/g in products within less than 24 h of incubation. When using CWBI-B1410 living culture (107 CFU/g) with glucose (1% wt/wt) supplementation as barrier against bacterial growth in fishes, a depression of the pH as well as a bacteriocin-like activity were noted, resulting in a decrease of the Gram negative strains counts of 4 log CFU/g (P. jubelini) and 2 log CFU/g (P. quadrifilis) in comparison to fishes prepared in using traditional process. The addition of salt in such fermented products reinforced the antimicrobial effect by CWBI-B1410 strain. The addition of neutralized (pH 6) culture supernatants of CWBI-B1410 or CWBI-B28 strains in combination with sodium chloride (0.14 g/ml) as preservatives on fishes (100 ml/100 g) incubated at 10°C, declined the level of total bacterial counts of 1.5 log CFU/g within 48 h, and delayed the increase of bacteria number in fishes: bacterial counts remained under 106 CFU/g for 13 to 18 days of storage at 10°C following the fish, whereas they were over 106 CFU/g after 3 to 7 days fish storage at 10°C in controls, treated with salted (NaCl 0.14 g/ml) neutralized (pH 6) culture supernatants of non-bacteriocin producers (Lactococcus lactis LMG 6890 and Lactobacillus curvatus LMG 216888). The antimicrobial effects by salted neutralized culture supernatants of the bacteriocin producers were higher to those of salted solution (NaCl 0.14 g/ml) supplemented with benzoate and sorbate acid salts each at concentration of 0.5 mg/ml on the moderate and fat fishes. These data suggest that the use of bacteriocin-producing lactic acid bacteria in combination with sodium chloride as described above, particularly the use of salted bactericidal culture supernatant, as preservatives, can be a suitable strategy to enhance the storage and the bacterial quality of traditional fish products in Senegal. The treatment of fishes in using one of the strategies as described above combined with a drying process could permit production of seafood commodities with higher bacterial quality and time of storage, justifying to continue this investigation on the enhancement of the bacteriocin-productivity by CWBI-B1410, and the evaluation of the effects of the new treatments on the organaoleptic and nutritional quality of products.

The genus Arthroderma contains predominantly geophilic dermatophytes (naturally occuring in soil). Some species, especially those from Trichophyton terrestre complex, cause human and animal dermatomycosis. In the past, the species boundaries were determined mainly on the basis of biological species concept using in vitro mating experiments. But these nearly 70-years-old findings have not been tested by means of modern taxonomic methods. In total 194 species of the genus Arthroderma (including all available ex-type strains) originating predominantly in USA, Canada and Europe were studied in this thesis. They were mostly isolated from soil (n = 77), animals (n = 50), human clinical material (n = 41) and cave sediment (n = 9). The main goal of the thesis was to elucidate the species boundaries between species A. insingulare, A. lenticulare and A. quadrifidum, that were classified into the T. terrestre complex because of their seemingly identical asexual stage. Further, this work aimed to resolve the relationship between Arthroderma species using the multigene phylogeny and clarify which species are clinically relevant. A multigene phylogeny of the genus Arthroderma was based on the sequences of the ITS rDNA region, β-tubulin (TUB2) and translation elongation factor 1α (TEF1α) genes. The genus...