Dissertationen zum Thema „Microbial antagonists“

The cost of losses and control measures attributed to late blight of potatoes caused by Phytophthora infestans, are estimated to exceed $5 billion annually. Breeding for resistance is difficult owing to the tetraploid genotype of potato and current strains of the pathogen have developed resistance to chemical control. Consequently the search for biological control has assumed greater importance. In this investigation an in vivo bioassay was used to select soils antagonistic to late blight of potatoes, caused by Phytophthora infestans. Four out of eight samples demonstrated reproducible antagonism as determined by a reduction in the volume of tuber tissue rotted. A total of 292 bacterial and yeast samples and 20 fungal samples were recovered from suppressive soils using a variety of non-selective and selective media. When these organisms were tested individually against P. infestans in the assay, 15 isolates suppressed tuber rotting by >85% in at least three out of four assays. The antagonists were characterised as Pseudomonas spp. (3 strains), Enterobacter spp. (4 strains), Bacillus spp. (1 strain), Pantoea spp. (2 strains), Citrobacter spp. (1 strain), Buttiauxella spp. (1 strain), Trichosporon spp. (2 strains) and Geotrichum spp. (1 strain) by routine bacteriological tests, fatty acid profiling and partial sequencing of the gene encoding 16S or 18S (where appropriate) ribosomal RNA. Subsequently the possible mechanisms by which the potential biocontrol agents inhibited the disease were examined. Nine isolates showed some evidence of antibiotic production with a Pantoea spp. producing a compound that caused the hyphae of P. infestans to kink and permanently cease growth. Three isolates colonised hyphae of the pathogen and eleven produced siderophores in liquid culture. Hydrogen cyanide, proteolytic, cellulolytic and beta-1,3-glucanase activity was also evident in some species. Significant promotion of axenically grown tomato seedlings, as determined by increased stem and main root elongation, was achieved by ten of the isolates. Three population levels of the isolates were retested for disease inhibition at the end of the investigation. Isolates 3, 7 and 14 exhibited the highest levels of consistent inhibition at the lowest population levels and were therefore tested in combination. This achieved disease suppression that, at an antagonist concentration of 25 cfu/nL, was more consistent than isolate 3 alone and was over 30% greater than either isolate 7 or 14.

As formigas-cortadeiras (Hymenoptera: Formicidae), gêneros Atta e Acromyrmex, são as pragas mais importantes da silvicultura, encontradas exclusivamente nas Américas. Sua importância econômica se deve ao seu hábito alimentar de cortar material vegetal fresco – sendo algumas importantes espécies cultivadas - que serve de substrato para o fungo simbionte mutualista Leucoagaricus gongylophorus (Basidiomycota: Agaricales) o qual é cultivado em câmaras subterrâneas. As fêmeas fecundadas são responsáveis pela fundação de uma nova colônia e sofrem influência direta de microrganismos presentes no solo, assim existem evidências que fêmeas de Atta spp. recém-fecundadas evitam solos férteis, com alta carga microbiana e mesmo as áreas de matas para fundação dos seus ninhos. Os fungos entomopatogênicos e fungos antagonistas de L. gongylophorus, presentes naturalmente no solo, podem ser usados no controle biológico de formigas-cortadeiras. Os objetivos deste trabalho foram avaliar em laboratório o desenvolvendo de colônias jovens de Atta sexdens em contato com solos de área de mata e de área desnuda, realizar a bioprospecção de isolados de fungos antagonistas de L. gongylophorus (Trichoderma spp.), em solos da região de Gurupi - TO, e testar o efeito antagônico desses fungos contra L. gongylophorus. Após 106 dias da revoada, as operárias de A. sexdens das 36 colônias, logo que iniciado o experimento, removeram o solo em todos os tratamentos, e esse comportamento favoreceu a sobrevivência das colônias, apresentando uma taxa de sobrevivência de 91,67% durante as 13 semanas. Verificou-se que, após as colônias terem passado pelo período crítico de fundação, o tipo de solo parece exercer pouca influência na sobrevivência das operárias e das colônias. O solo de área de mata apresenta maior quantidade e diversidade de fungos filamentosos que o solo de área desnuda; A utilização de Tenebrio molitor como isca-viva é eficiente para bioprospecção por fungos entomopatogênicos. Todos os isolados de Trichoderma foram capazes de inibir o crescimento in vitro de L. gongylophorus; Trichoderma spirale isolado 2 apresentou crescimento micelial lento em placa de Petri e inibição contra L. gongylophorus, podendo este ser um isolado promissor em testes em campo.
The leaf-cutting ants (Hymenoptera: Formicidae), Atta and Acromyrmex genera, are the most important forest pests. They are found exclusively in Americas. Its economic importance is due to behavior to cut fresh plant material that serves as a substrate for the mutualistic symbiotic fungus Leucoagaricus gongylophorus (Basidiomycota: Agaricales) that are grown in underground chambers. Fertilized females are responsible for the founding of a new colony and they suffer direct influence of soil microorganisms. Therefore, newly fertilized females of Atta spp. should avoid fertile soils with high microbial load and even forested areas to found their nests. Entomopathogenic fungi and antagonists of L. gongylophorus are naturally present in the soil and they could be used in biological control of leaf-cutting ants. The objectives of this study were to evaluate and to compare the developing of young colonies of Atta sexdens in contact with soil from forested area and in contact with bare soils. Also, this and others procedures served to bioprospect antagonistic fungi of L. gongylophorus (Trichoderma spp.) from Gurupi region soil, and test the antagonistic effect of Trichoderma spp. against L. gongylophorus. In the first hours of start of experiments, Atta workers removed soil from colony recipients. This behavior could have favored colony survival, with a survival rate of 91,67 % during the 13 weeks. It was found that after the colonies have passed the critical period of foundation, the soil appears to exert little influence on the survival of workers and colonies. The soil from forested area had greater quantity and diversity of filamentous fungi than that from bare soil. The use of Tenebrio molitor as bait alive is efficient for bioprospecting by entomopathogenic fungi. All isolates of Trichoderma were able to inhibit in vitro growth of L. gongylophorus. Trichoderma spirale isolated 2 showed slow mycelial growth in Petri dish and strong level of inhibition against L. gongylophorus in vitro. So, this isolate is promising and should be tested in field.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
As formigas da tribo Attini são conhecidas pela complexa simbiose que mantêm com fungos, os quais cultivam como alimento. É sabido que além desse fungo, outros microrganismos podem ser encontrados nos ninhos desses insetos e estudos prévios apontaram que alguns microfungos (i.e. leveduras e fungos filamentosos) podem ser importantes nessa simbiose. O objetivo do presente trabalho foi avaliar o papel desses microfungos associados aos jardins dessas formigas. Analisando várias espécies do gênero Acromyrmex do sul do Brasil, demonstrou-se que as formigas importam uma comunidade diversa de microfungos para seus ninhos, provavelmente provenientes do solo e do substrato vegetal que as formigas utilizam para cultivar seu fungo. Num segundo estudo, avaliando formigas Attini da América do Norte (Atta texana, Trachymyrmex septentrionalis e Cyphomyrmex wheeleri) observou-se que a estrutura das comunidades de microfungos nos jardins desses insetos não se correlaciona com a variação sazonal, sugerindo que não existam relações espécie-específicas entre as formigas e os microfungos. Apesar de tais microrganismos não serem especialistas dos jardins desses insetos, é sugerido que os microfungos atuem como antagonistas do fungo simbionte. Ainda, descobriu-se que o parasita especializado Escovopsis spp. parece ser menos freqüente nas populações de formigas da América do Sul em relação as Attini da América Central, porém estudos adicionais são necessários para estabelecer a epidemiologia desse parasita nos ninhos das Attini. Num terceiro estudo, demonstrou-se que leveduras presentes nos jardins de fungos da formiga cortadeira A. texana inibem o crescimento de Escovopsis spp., sugerindo que esses insetos utilizam outros microrganismos, além das bactérias presentes em suas cutículas (Pseudonocardia spp.), para inibir esse parasita. Esse achado traz importantes implicações para essa...
Ants in the tribe Attini are well-known social insects that maintain a symbiotic relationship with fungi which they cultivate as food. Besides of the cultivated fungi, fungus gardens contain several other microorganisms considered to be potential players in this symbiosis. The aim of the present study was to evaluate the possible roles of microfungi (i.e. yeasts and filamentous fungi) in attine gardens. Our microbial profiling of gardens from several species in the genus Acromyrmex from South Brazil revealed that ants can harbor a diverse community of microfungi that probably originated from the surrounding soil or from the substrate used to manure the cultivated fungus. In this sense, additional studies of North American attine species (Atta texana, Trachymyrmex septentrionalis and Cyphomyrmex wheeleri) demonstrated that the structure of microfungal communities in gardens of these ants did not correlate with seasonal changes over a one year period, again suggesting there are no species-specific relationships among ants and microfungi species. Although, the microfungi are not specialized parasites of the attine ant-fungus symbiosis we suggest they can be considered antagonists to the cultivated fungus. Moreover, we demonstrated that the specialized parasite Escovopsis spp. is probably less frequent in South America than in Central America and we reinforce that additional studies are necessary to unravel the epidemiology of this parasite in attine gardens. In another study, we showed that yeasts isolated from gardens of the leafcutter ant A. texana can significantly inhibit the growth of Escovopsis sp. This interesting finding suggests that attine ants may use additional microbes to protect their gardens against Escovopsis spp. and not only actinomycete bacteria (Pseudonocardia spp.) found in their cuticles. Finally, we studied microfungi relationships with female alates (gynes) in two... (Complete abstract click electronic access below)

Little is known about how the leaf bacterial community is affected by the seed microbiota at different stages of plant development. The bacterial populations of spinach seed and leaves after germination were compared using DGGE, to assess bacterial community richness, and real-time PCR to compare the abundance of select phyla (total bacteria, Actinobacteria, Bacteroidetes, Firmicutes, α-Proteobacteria and β- Proteobacteria). To determine the effect of environment, the plants were grown in the field and growth chambers. Vertical transmission of bacterial community members was evident; the developmental stage of the plant affected the richness and abundance of select bacterial phyla. The bacterial richness of plants grown in the two environments was not affected. However, overall numbers of bacteria increased in field grown samples in comparison to those produced in growth chambers during development. A statistically significant interaction was seen between growth stage and environment with each of the selected phyla. Populations on cotyledons were smaller than mature leaves, but were not significantly different than the 3-4 leaf stage plants. The culturable populations of bacteria on seeds (~5 log CFU/g) were significantly smaller than determined using real time PCR (~7 log copies). Of these bacteria cultured from spinach seeds, isolates belonging to the genera Pantoea were found to inhibit growth of E. coli O157:H7 in vitro. This study highlights the importance of vertical transmission on the bacterial community of plants and suggests the importance of developing strategies to influence these communities on seed to control human and plant pathogens on the leaf surface.

Master of Science in Life Sciences

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O controle biológico tem recebido atenção dos pesquisadores, principalmente devido aos movimentos, de preservação do ambiente. Têm sido amplamente aplicado em espécies consideradas importantes pragas agrícolas, porém não indicado para as formigas cortadeiras. Tais formigas apresentam um conjunto de estratégias físicas, químicas e comportamentais que as operárias realizam a fim de evitar a própria contaminação e da colônia. Entretanto alguns microorganismos conseguem causar mortalidade de alguns membros da colônia, como os fungos entomopatogênicos. Pretende-se fornecer conhecimentos básicos para desenvolvimento de novas estratégias de controle microbiano das formigas cortadeiras em um futuro próximo. Para tanto estudamos quatro fungos com grande potencial patogênico, Metarhizium anisopliae, Beauveria bassiana, Aspergillus flavus e Trichoderma asperellus. Primeiramente foram estudados a patogenicidade dos fungos (entomopatogeno e oportunista) em operarias isoladas da colônia. Estudou-se a susceptibilidade de larvas e adultos de formigas cortadeiras Atta sexdens rubropilosa a conídios de B. bassiana, por meio de técnicas histológicas e de microscopia. Finalmente encapsulados com conídios foram veiculados para o jardim de fungo simbionte visando o controle de mini colônias em laboratório. Propomos a elaboração de novas alternativas aos manejos existentes.

Leafy greens such as spinach have been the object of several recent food-borne pathogen outbreaks. The purpose of this study was to isolate bacteria spinach epiphytic bacteria that inhibit growth of E. coli O157:H7, which we describe as antagonism. The mechanism of antagonism was investigated and we attempted to improve the antagonistic potential in vitro and on spinach leaves when cellobiose, a carbon source utilized by the antagonists but not E. coli O157:H7, was added. There were larger culturable populations of bacteria on the leaves of savoyed cultivars compared to flat. From the isolated colonies, 47 displayed antagonism towards E.coli O157:H7, and were identified as members of 11 different genera and sixteen species. A representative isolate from each species was evaluated for three possible mechanisms of antagonism: acid production, secretion of an inhibitory compounds or secreted protease. The majority (14/16) produced at least a moderate level of acid. Two of these strains, Paenibacillus polymyxa and Pseudomonas espejiana, were found to secrete a non- protease antagonistic compound. These antagonists varied in their reduction of E.coli O157:H7 numbers in vitro, but all significantly reduced numbers in 48 hours of co-culturing in nutrient rich media. Five antagonists resulted in a significant reduction in E.coli O157:H7 populations when co-cultured on spinach leaves. Application of cellobiose did not improve the amount of antagonism in vitro or on the leaf surface after 24 hours.
Master of Science

LIMA, José Vinícius Leite. Populações microbianas e antagonismo de actinobactérias sobre rizóbios em solos do semiárido. 2017. 81 f. Tese (Doutorado em Ecologia e Recursos Naturais)-Centro de Ciências, Universidade Federal do Ceará, Fortaleza, 2017.
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The structure of the communities living in the soil is driven by various mechanisms and the ecological interactions established between the living organisms. In the same way, the components of the microbiota present harmonic or non-harmonic relationships, for example, between actinobacteria and rhizobia. Interspecific competition among microorganisms occurs when many species require the same resources, and the negative effect on the availability of common resources adversely affects the others. In this context, the objective of this study was to quantify the microbial population in the soil and leaf litter in the vegetation of the caatinga and carrasco in the Brazilian semiarid region (Chapter I) and to characterize and evaluate the antagonistic effect of actinobacteria on rhizobia (Chapter II). The soil samples were collected at the Ecological Station of Aiuaba (Aiuaba, Ceará), characterized by the vegetation of caatinga and carrasco. In chapter I, the population density of the microbial community was evaluated by sampling of soil and litter in the vegetation of caatinga and carrasco at the Aiuaba Ecological Station. The microorganisms were isolated from the soil samples in flasks containing 0.8% saline solution and cultured, after consecutive dilutions, in culturing media specific to cultivable populations of total bacteria, actinobacteria, cellulolytic bacteria, phosphate solubilizing bacteria and fungi. Population estimation was then performed by standard counting on plates and the values were expressed in CFU.g-1. The microbial populations from the soil and leaf litter differed each other quantitatively and in the two vegetations, but in general were greater in the leaf litter. Thus, the knowledge of the population structure of the microbial community can be extended to the semiarid soils. In Chapter II, the strains of actinobacteria isolated in the first study were characterized and tested for “in vitro” inhibitory effect on strains of rhizobia also isolated from semiarid soils. The actinobacteria were purified in their respective culture medium (CDA medium). The strains were characterized for their color and morphology of the colonies, tolerance to pH levels, production of melanin and use of carbon sources. The “in vitro” antagonism of actinobacteria on rhizobia was evaluated in Petri dish containing yeast mannitol agar (YMA) medium by the formation of inhibition zone. The actinobacteria and rhizobia that had greater antagonistic effect or did not presented inhibition zones were molecularly identified. Sixty strains were identified in seven genera of actinobacteria in which had tolerance to variations in pH, low melanin production and generalist use of carbon sources. It was also observed “in vitro” antagonism of actinobacteria species, standing out the genus Streptomyces, on strains of Rhizobium tropici, Bradyrhizobium yuanmingense and two other rhizobia strains not identified. This is the first work that addresses this ecological interaction between microorganisms of the Brazilian semiarid region and reveals the occurrence of new species related to this negative interaction. Thus, the presence of antagonism among these organisms may lead to “in vivo” studies, contributing to future agricultural and/or ecological uses.
A estrutura das comunidades que vivem no solo é direcionada por vários mecanismos e as interações ecológicas estabelecidas entre os organismos. Da mesma forma, os componentes da microbiota apresentam relações harmônicas ou não entre si, como por exemplo, entre actinobactérias e rizóbios. A competição interespecífica entre os micro-organismos acontece quando muitas espécies procuram pelos mesmos recursos, e o efeito depressor que cada uma tem na disponibilidade dos recursos comuns afeta adversamente os outros. Neste contexto, teve-se como objetivo neste estudo quantificar em região semiárida a população microbiana no solo e serrapilheira nas fisionomias vegetais de caatinga e carrasco (Capítulo I) e caracterizar e avaliar o efeito antagônico de actinobactérias sobre rizóbios do semiárido (Capítulo II). O estudo foi realizado na Estação Ecológica de Aiuaba (Aiuaba, Ceará) caracterizada pelas fisionomias de caatinga e carrasco. No capítulo I, avaliou-se a densidade populacional da comunidade microbiana por meio de coleta de amostras do solo e serrapilheira nas fisionomias vegetais de caatinga e carrasco na Estação Ecológica de Aiuaba. Os micro-organismos foram isolados das amostras em solução salina 0,8% e cultivados, após diluições seriadas, em meios de cultura específicos para populações cultiváveis de bactérias totais, actinobactérias, bactérias celulolíticas, bactérias solubilizadoras de fosfato e fungos. Em seguida foi realizada a contagem das populações pela contagem padrão em placas usando a técnica de espalhamento em superfície, e os valores foram expressos em UFC.g-1. As populações microbianas oriundas do solo e serrapilheira diferiram quantitativamente entre elas e nas duas fisionomias vegetais, mas no geral foram maiores na serrapilheira. Dessa forma, pode-se ampliar o conhecimento da estrutura populacional da comunidade microbiana em solos de clima semiárido. No capítulo II, caracterizaram-se as cepas de actinobactérias isoladas das populações obtidas do primeiro estudo e testou-as para efeito inibidor in vitro sobre estirpes de rizóbios também oriundas de solos semiáridos. As actinobactérias foram purificadas no seu respectivo meio de cultura (meio CDA). As cepas foram caracterizadas quanto a atributos cromogênicos e morfológicos, para tolerância a níveis de pH, na produção de melanina e uso de fontes de carbono. O antagonismo das actinobactérias in vitro sobre rizóbios foi avaliado em meio de cultura ágar manitol levedura (YMA) pela formação de halo de inibição em placa de Petri. Em seguida, as actinobactérias e rizóbios que tiveram maior efeito antagônico ou que não formaram halo inibidor foram caracterizadas molecularmente. Foram obtidas 60 cepas identificados em sete gêneros de actinobactérias que tiveram tolerância a variações de pH, baixa produção de melanina e uso generalista de fontes de carbono. Foi observado antagonismo in vitro com formação de halo inibidor de espécies de actinobactérias, com destaque para o gênero Streptomyces, sobre os rizóbios Rhizobium tropici, Bradyrhizobium yuanmingense e outras duas estirpes não identificadas. Este é o primeiro trabalho que aborda essa relação ecológica em micro-organismos do semiárido e revela a ocorrência de novas espécies relacionados com essa interação negativa. Com isso, confirma-se a presença de antagonismo entre esses organismos que pode direcionar estudos de certificação in vivo, contribuindo para futuros usos agrícolas e/ou ecológicos.

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FAPESB
A Baía de Todos os Santos (BTS), segunda maior baía do Brasil, abriga grande diversidade de ambiente, flora e fauna, porém pouco se sabe sobre sua diversidade microbiológica e seu potencial de bioprospecção. Este trabalho teve por objetivo a busca de novas substâncias antimicrobianas, capazes de inibir o crescimento de bactérias patogênicashumanas e de animais marinhos, isolados do solo de cinco praias da BTS. No total foram isoladas 107 bactérias das quais dez apresentaram produção de substâncias antimicrobianas. Após o sequenciamento verificou-se que dos dez isolados, somente oito foram de fato identificadas molecularmente, sendo sete destes membros da família Bacillaceae, grupo ao qual pertencem diversas produtoras de substâncias antimicrobianas. Entre os isolado que apresentaram produção de substâncias inibidoras do crescimento constatou-se que os isolados 21, 50 e 54 tinham maior poder de inibição. O extrato destes três isolados foi confeccionado através da acidificação do sobrenadante livre de células, isolado após o período de incubação. Os extratos do isolado 21 e do isolado 50 foram capazes de inibir a bactéria Klebsiella rizophyla, e o extrato 21 conseguiu ainda inibir a Vibrio vulnificus. Após verificação molecular através de primers específicos de sete substâncias antimicrobianas produzidas por Bacillus, constatou-se que os isolados 21 e 50 são produtores da substância ericina.
The All Saints´s Bay (BTS), the second largest bay in Brazil, where can be founded great diversity of flora and fauna including diversity of landscapes, but unknown about their microbial diversity and potential to bioprospecting. This study aimed to search for new antimicrobial substances that can be able to inhibit the growth of human pathogenic bacterias and marine animals, isolated from sediment from five BTS beaches. Was isolated a total of 107 bacterias where 10 showed antimicrobial substances production. After 16S sequencing, was found that from the ten isolates, only eight were identified by amplification PCR and seuqencing, seven members of the Bacillaceae family, where antimicrobial substances belonging this group are known. It was found that the isolates numbers 21, 50 and 54 had a greater power of inhibition. The extract of these three strains was made by acidification of the cell-free supernatant, isolated after the incubation period. The extracts of the isolate 21 and 50 were able to inhibit the bacteria Kocuria rhizophyla , and the same extract could also inhibit Vibrio vulnificus. In advance, was realized a molecular bioprospecting using specific molecular primers from conserved regions belongs to seven antimicrobial substances produced by Bacillus. It was found that the isolates 21 and 50 are Erycina producing.

Fusarium verticillioides est une moisissure phytopathogène et mycotoxinogène dont l’occurrence est fréquente dans le sol et dans de nombreux céréales et végétaux et plus particulièrement dans le maïs et le blé. La contamination des cultures par F. verticillioides conduit à des maladies variées et est souvent associée avec une accumulation de mycotoxines. Les fumonisines B1 (FB1) and B2 (FB2) sont les toxines les plus dangereuses produites par F. verticillioides. Des conséquences létales de la consommation de produits alimentaires contaminés par les fumonisines ont été reportées chez les animaux et l’homme. Différentes démarches ont été utilisées pour lutter contre cette moisissure et ses toxines mais souvent liées à l’utilisation de fongicides chimiques par ailleurs reconnus pour leurs effets néfastes pour l’homme, les animaux et l’environnement. Des pratiques alternatives ont été développées pour préserver les récoltes en respectant les écosystèmes et la santé humaine et animale. L’utilisation d’amendements organiques est apparue comme une stratégie intéressante de contrôle des maladies des plantes en assurant un apport de PGPM (Plant Growth Promoting Microorganisms) et agents de biocontrôle. Notre étude a donc eu pour but de caractériser les communautés microbiennes, et plus particulièrement celles d’intérêt pour le biocontrôle, d’amendements organiques et sols amendés ainsi que de tester leur potentiel antifongique envers F. verticillioides. Les mécanismes de biocontrôle ont été étudiés en utilisant des approches métabolomiques et transcriptomiques par séquençage haut débit. L’étude des communautés microbiennes dans les amendements et les sols amendés a été réalisée par pyroséquençage de marqueurs qui sont des portions du gène ribosomal 16S et de la séquence ITS2. Le microbiote des amendements est essentiellement constitué par les familles des Pseudonocardiaceae, Bacillaceae et Trichocomaceae qui incluent différents PGPM et agents antifongiques. Par ailleurs les amendements semblent favoriser l’installation des familles d’intérêt dans les sols tout en limitant celle des pathogènes tels les Nectriaceae qui comprennent des Fusarium pathogènes. Les tests d’antagonisme ont montré que les sols amendés réduisaient la croissance de F. verticillioides de façon plus importante que le sol de référence en inhibant la production de microconidies. La production de fumonisines a également été fortement réduite en présence des métabolites produits par le microbiote des sols amendés (plus de 68 % et 92 % pour FB1 and FB2 respectivement). Des souches d’actinomycètes isolées des amendements, et identifiées comme appartenant au genre Streptomyces, ont montré une activité antifongique envers F. verticillioides. Parmi celles-ci, Streptomyces AV05 a été sélectionnée pour les tests supplémentaires en raison de son fort potentiel d’inhibition vis-à-vis de F. verticillioides. Des comparaisons des endométabolomes et transcriptomes des 2 souches cultivées seules ou en confrontation ont été réalisées et de nombreuses différences ont été remarquées pour chacune entre les 2 modalités. Ainsi, 29 métabolites impliqués dans les modifications du métabolome de F. verticillioides en co-culture avec Streptomyces AV05 ont été identifiés et différentes voies métaboliques semblent avoir été affectées. L’étude du transcriptome des 2 souches a donné des résultats allant dans le même sens que celle du métabolome. En effet des changements significatifs de niveau d’expression ont été observés au niveau transcriptomique pour 800 gènes de F. verticillioides et 115 gènes de Streptomyces AV05 en situation de confrontation. L’identification de ces gènes est en cours et plusieurs d’entre eux semblent impliqués dans les modifications des profils métaboliques observées chez les 2 souches. L’intégration des données métabolomiques et transcriptomiques devrait permettre d’améliorer a compréhension des mécanismes mis en jeu au cours de la confrontation
Fusarium verticillioides is a phytopathogenic and mycotoxigenic filamentous fungus that can be found with high occurrence in soils and in a wide range of cereals and vegetables, particularly in corn and wheat. The F. verticillioides contamination in crops leads to various diseases and is usually associated with an accumulation of mycotoxins. Fumonisin B1 (FB1) and fumonisin B2 (FB2) are the most dangerous mycotoxins produced by Fusarium verticillioides. Lethal consequences caused by fumonisins have been reported in animals and in human due to the consumption of contaminated food products. To deal with this pathogenic fungus and its mycotoxins, several approaches have been applied but they are usually based on the use of chemical agents that are reported to be unsafe for humans, animals and ecosystems. Alternative practices that maintain the quality and the abundance of crops while preserving the ecosystems and human and animal health have been developed. The application of organic amendments has been reported as an interesting strategy for controlling diseases by providing an abundant source of PGPM (Plant Growth Promoting Microorganisms) and biocontrol agents. The aim of our study was to characterize the microbial communities of organic amendments and amended soils focusing on the microbial families of interest for biocontrol and to assess the antifungal potential of amended soils and their microbiota towards F. verticillioides. Mechanisms of biocontrol were studied using metabolomics and transcriptomics approaches. Evaluation of microbial communities in amendments and amended soils using pyrosequencing of the 16S rDNA and the ITS genes showed that the amendments contained mainly the families of Pseudonocardiaceae, Bacillaceae and Trichocomaceae that were believed to include various PGPM and antifungal agents. Furthermore, the amendments were expected to promote the families of interest in soil and also to limit those of pathogens such as Nectriaceae that might contain many pathogenic Fusarium. Antifungal assays showed that the amended soils reduced the F. verticillioides growth better than the reference soil by inhibiting the microconidia production. The fumonisin production was strongly reduced by the metabolites produced by the amended soils’ microbiota (up to 68 % and 92 % for FB1 and FB2 respectively). Some actinomycete isolates from these amendments were identified as Streptomyces and they demonstrated antifungal activity against F. verticillioides. Among the Streptomyces strains, Streptomyces AV05 was selected for further studies because of its strong inhibition towards F. verticillioides. The interaction between the Streptomyces strain AV05 as antagonist agent and F. verticillioides was investigated. The study of the endometabolome and the transcriptome of the two microorganisms was carried out in two different conditions: strains cultivated alone or in confrontation. Many modifications have been noticed into the endometabolome of the 2 microorganisms during the direct confrontation and 29 metabolites involved in the endometabolome alteration of F. verticillioides in co-culture with Streptomyces AV05 were identified. Many fungal metabolic pathways were suggested to have been affected. The results of the study of the mRNA of the 2 strains appeared to be in accordance with the metabolomics results. A change in the transcriptomic level was observed: 800 genes of F. verticillioides and 115 genes of Streptomyces AV05 were found to be expressed differently when the strains were cultivated alone or in confrontation. The identification of these genes is in progress. Many of them are expected to be responsible for the change in the metabolic profiles observed in the bacterial-fungal interaction. The integration of the metabolomic and transcriptomic informations could improve the understanding of the mechanisms of biocontrol during direct confrontation

Foods are complex ecosystems, due to the influence of either intrinsic and extrinsic factors such as the chemical composition, microbial communities and the environment that can have an effect on the final product. In cheese microbes are the most influencing factor because they can effect both shelf-life and quality flavor/taste of the food. In addition microbiome establishes complex interactions within the bacterial community itself and between the food environment, such that nowadays these dynamics have been only partially highlighted with traditional culture-dependent technique. Thus, culture-independent methods such as Next Generation Sequencing (NGS) approach have become a powerful tool to study in depth microbial communities contained in a variety of ecological niches, including food. In fact they should be increasingly considered as a complementary technique to culture-dependent methods in microbiology. In this comprehensive study we have evaluated and applied a 16S Next Generation Sequencing approach to the study of Ricotta cheese shelf life, its raw materials and to food technology studies in soft or ripened cheeses using adjunct cultures. In all these studies NGS method have always been supported with culture-dependent approaches, other molecular methods and biochemical analysis. The different studies are separated in the subsequent chapters of this dissertation. First of all using a mock community we evaluated the impact of 16S NGS pipeline on the final community structure and identification, showing that bioinformatic analysis, and above all the OTU-picking strategy, can affect the alpha-diversity (within the sample) of the community but not its beta-diversity (between samples). To this extent both strategies can be used in order to compare community structures. Moreover the protocol was showed as robust and repeatable. Secondly we evaluated the raw materials (wheys and cream) used for ricotta production showing that the microbiological quality of these materials must be improved. As they showed a very biodiverse micro flora (mainly Streptococcaceae and Pseudomonadaceae) and a high concentration of aerobic spores (10 to 104 CFU/ml, not present in cream) their usage for Ricotta-making could damage the final product. Moreover NGS combined with Gas Cromatography approach on organoleptic non satisfactory frozen wheys showed that under-hygienic storage of the raw materials drove to the production of fermentation or lipid oxidation compounds influenced by particular bacteria such as Acinetobacter, Lactococcus and Pseudomonas. In the third chapter we evaluated Ricotta shelf life during winter and summer season, showing that Ricotta microbiome was mainly composed of spore-forming bacteria, Bacillus, Paenibacillus and Clostridium (more than 80% of relative abundance). Moreover we found a summer ricotta with a pink discoloration and, applying a molecular Multi-locus Sequence Typing approach, we demonstrated that the bacterium causing the defect belonged to the Bacillus cereus group, in particular it was a B. mycoides/wehienstephanensis strain. This work has been published in Food Microbiology journal. In the last chapter we applied NGS analysis to the technological use of adjunct cultures on soft or ripened cheeses in order to evaluate their anti-spoilers effect. We evaluated the anti-gammaproteobacteria effect of some strains such as L. rhamnosus, L. sakei and Carnobacterium maltoaromaticum in fresh industrial cheeses prone to package swelling and premature spoiling. From a NGS point of view we found that C. maltoaromaticum managed to eliminate almost all contaminants at different storage temperatures. In conclusion with NGS approach we could improve food microbiological quality assessment and support food technology research. In fact the results obtained in the present studies demonstrate how the application of NGS technologies, describing in-depth microbial communities, could in the next future become a scalable, cost-effective and suitable tool in the food industry for improving the quality and the safety of products.
Gli alimenti sono ecosistemi complessi in quanto vengono influenzati sia da fattori intrinseci che da fattori estrinseci, come la composizione chimica, il profilo microbiologico e l'ambiente, che possono avere forte impatto sul prodotto finale. In particolare nei formaggi i microbi sono il fattore più influente perché possono modificare sia la vita commerciale che il sapore ed il gusto del prodotto. Inoltre il microbiota stabilisce delle complesse interazioni internamente alla comunità batterica e con l'ambiente, a tal punto che al giorno d'oggi queste dinamiche sono state solo in parte evidenziate con metodi tradizionali coltura-dipendenti. Di conseguenza i metodi coltura-indipendenti, come il Sequenziamento di Nuova Generazione (NGS), stanno diventando un potente strumento per studiare in profondità le comunità microbiche presenti in una varietà di nicchie ecologiche, compresi gli alimenti. È probabile che ormai tali tecniche debbano essere considerate complementari ai metodi di microbiologia coltura-dipendenti. In questo ampio progetto abbiamo valutato e applicato un approccio NGS sul gene 16S rRNA allo studio della vita commerciale della Ricotta, delle sue materie prime e ad alcuni studi di tecnologia degli alimenti su formaggi molli o stagionati addizionati con colture batteriche antagoniste. In tutti questi studi i metodi NGS sono sempre stati supportati da approcci cultura-dipendenti, altri metodi molecolari e analisi biochimiche. I vari studi sono suddivisi tra i successivi capitoli di questa tesi. Per prima cosa utilizzando una comunità batterica artificiale abbiamo valutato l'impatto del workflow di processo del protocollo 16S-NGS sulla struttura e sull'identificazione della comunità reale. Con questo approccio abbiamo dimostrato che l'analisi bioinformatica, e principalmente la strategia di raggruppamento delle sequenze in OTU (Operational Taxonomic Unit), può influenzare la diversità della comunità di tipo alfa (intra-campione), ma non la sua beta-diversità (inter-campioni). In questo senso entrambe le strategie possono essere utilizzate al fine di confrontare le strutture delle comunità microbiche. Inoltre il protocollo si è dimostrato robusto e ripetibile. In secondo luogo abbiamo valutato le materie prime (siero e panna) utilizzate per la produzione di ricotta, dimostrando che la qualità microbiologica di queste materie deve essere migliorata. Poiché queste hanno mostrato una forte biodiversità (principalmente di Streptococcaceae e Pseudomonadaceae) e un'alta concentrazione di spore aerobie (da 10 a 104 UFC/ml, non presenti nella panna), il loro utilizzo per la produzione della ricotta potrebbe danneggiare il prodotto finale. Non di meno l'approccio combinato NGS con Gas Cromatografia su sieri congelati con difetti organolettici ha mostrato che lo stoccaggio non igienico delle materie prime conduce alla produzione di composti di fermentazione o di ossidazione lipidica, guidati anche da particolari batteri come Acinetobacter, Lactococcus e Pseudomonas. Nel terzo capitolo abbiamo valutato la vita commerciale della Ricotta durante l'inverno e l'estate, dimostrando che il microbioma della Ricotta era composto principalmente da batteri sporigeni, Bacillus, Paenibacillus e Clostridium (oltre l'80% di abbondanza relativa). Inoltre abbiamo trovato una ricotta estiva con colorazione rosa e, applicando l'approccio molecolare Multi-locus Sequence Typing, abbiamo dimostrato che il batterio che causava il difetto apparteneva al gruppo del Bacillus cereus, in particolare si trattava di un ceppo di B. mycoides/wehienstephanensis. Tale lavoro è stato pubblicato nella rivista Food Microbiology. Nell'ultimo capitolo abbiamo applicato l'analisi NGS a studi di culture ausiliari aggiunte a formaggi a pasta molle o stagionati, al fine di evidenziare il loro effetto antibatterico. Abbiamo così valutato l'effetto anti-Gammaproteobacteria di alcuni ceppi come L. rhamnosus, L. sakei e Carnobacterium maltoaromaticum in formaggi freschi industriali con problematiche di rigonfiamento della confezione e deterioramento prematuro. Dal punto di vista NGS abbiamo evidenziato che C. maltoaromaticum riusciva ad eliminare quasi tutti i contaminanti a temperature di conservazione differenti. In conclusione, con l'approccio NGS, è stato possibile migliorare la valutazione della qualità microbiologica alimentare e sostenere la ricerca tecnologica alimentare. Infatti i risultati ottenuti in questo studio dimostrano come l'applicazione di tecnologie NGS, descrivendo approfonditamente le comunità microbiche, potrebbe diventare nel prossimo futuro uno strumento scalabile ed efficace, adatto per migliorare la qualità e la sicurezza alimentare, nonché favorire la produzione di nuovi alimenti.

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As bebidas lácteas simbióticas possuem micro-organismos probióticos e compostos prebióticos, e estão inseridas na classe dos alimentos funcionais. Estes produtos apresentam representatividade no mercado consumidor devido às características sensoriais agradáveis e também pelos benefícios à saúde. O objetivo desta pesquisa foi observar o potencial da atividade antagonista de Lactobacillus acidophilus e Bifidobacterium lactis contra micro-organismos patogênicos em soro lácteo fermentado, bem como avaliar a composição de fibras e componentes com atividade antioxidante em graviolas, visando justificar o desenvolvimento de uma bebida simbiótica utilizando estes alimentos. Os probióticos foram avaliados quanto a atividade antagonista contra micro-organismos patogênicos (Bacillus cereus, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus) e quanto a resistência ao ácido clorídrico (pH 4 e pH 2) e a bile (0,3%). Graviolas das variedades Crioula, Lisa e Morada, em estádio de maturação fisiológica (MF) e madura (M), foram comparadas quanto a composição centesimal, fibra alimentar total, solúvel e insolúvel, compostos fenólicos totais e atividade antioxidante. As bebidas simbióticas produzidas com soro lácteo residual da produção de queijo de coalho e polpa de graviola (n=10) foram avaliadas quanto as características sensoriais e vida de prateleira realizada após 0, 7, 14, 21 e 28 dias de produção. Os resultados mostram que Lactobacillus acidophilus e Bifidobacterium lactis apresentam atividade probiótica demonstrada pela resistência à bile e baixa sensibilidade ao ácido clorídrico, tanto em caldo Man Rogosa Sharp (MRS) quanto no soro lácteo. Além disso, apresentam alta e intermediária atividade antagônica comprovada frente micro-organismos patogênicos. Quando foi utilizado o soro lácteo como substrato para fermentação, observou-se maior atividade inibitória diante de E. faecalis, L. monocytogenes e S. aureus. Ficou evidenciado que as graviolas da variedade Morada apresentam teores de cinzas, superiores às demais, porém a composição em proteína, lipídeo, carboidrato e teor de umidade não diferenciam. A variedade Lisa-MF apresentou menor teor de fibra alimentar total e fibras insolúveis. Todas as variedades mostraram-se semelhantes quanto à composição de fibras solúveis. Os valores de compostos fenólicos totais entre as variedades de graviola não diferem estatística (p<0,05) entre os estádios de maturação, contudo a graviola Lisa madura apresentou maior teor de fenólicos totais, diferindo-se apenas da Crioula-MF. A variedade Crioula apresentou maior capacidade de sequestro do radical DPPH•, diferindo estatisticamente das graviolas da variedade Morada, e por isso possuem maior atividade antioxidante. As análises das bebidas simbióticas mostram que a formulação com 71,5% de soro apresenta boa aceitabilidade sensorial, ressaltando que 25% da polpa de graviola favorece a aceitação quanto ao sabor e aroma. A bebida proposta manteve as características durante a vida de prateleira, principalmente as bactérias probióticas em níveis recomendados pela legislação vigente. Pode ser concluído que a bebida láctea de soro fermentado por micro-organismos probióticos e saborizada com graviola apresenta viabilidade de produção. No processo final há agregação de valor econômico, nutricional e funcional, além de minimização no impacto ambiental.

Resumo: As formigas da tribo Attini são conhecidas pela complexa simbiose que mantêm com fungos, os quais cultivam como alimento. É sabido que além desse fungo, outros microrganismos podem ser encontrados nos ninhos desses insetos e estudos prévios apontaram que alguns microfungos (i.e. leveduras e fungos filamentosos) podem ser importantes nessa simbiose. O objetivo do presente trabalho foi avaliar o papel desses microfungos associados aos jardins dessas formigas. Analisando várias espécies do gênero Acromyrmex do sul do Brasil, demonstrou-se que as formigas importam uma comunidade diversa de microfungos para seus ninhos, provavelmente provenientes do solo e do substrato vegetal que as formigas utilizam para cultivar seu fungo. Num segundo estudo, avaliando formigas Attini da América do Norte (Atta texana, Trachymyrmex septentrionalis e Cyphomyrmex wheeleri) observou-se que a estrutura das comunidades de microfungos nos jardins desses insetos não se correlaciona com a variação sazonal, sugerindo que não existam relações espécie-específicas entre as formigas e os microfungos. Apesar de tais microrganismos não serem especialistas dos jardins desses insetos, é sugerido que os microfungos atuem como antagonistas do fungo simbionte. Ainda, descobriu-se que o parasita especializado Escovopsis spp. parece ser menos freqüente nas populações de formigas da América do Sul em relação as Attini da América Central, porém estudos adicionais são necessários para estabelecer a epidemiologia desse parasita nos ninhos das Attini. Num terceiro estudo, demonstrou-se que leveduras presentes nos jardins de fungos da formiga cortadeira A. texana inibem o crescimento de Escovopsis spp., sugerindo que esses insetos utilizam outros microrganismos, além das bactérias presentes em suas cutículas (Pseudonocardia spp.), para inibir esse parasita. Esse achado traz importantes implicações para essa... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Ants in the tribe Attini are well-known social insects that maintain a symbiotic relationship with fungi which they cultivate as food. Besides of the cultivated fungi, fungus gardens contain several other microorganisms considered to be potential players in this symbiosis. The aim of the present study was to evaluate the possible roles of microfungi (i.e. yeasts and filamentous fungi) in attine gardens. Our microbial profiling of gardens from several species in the genus Acromyrmex from South Brazil revealed that ants can harbor a diverse community of microfungi that probably originated from the surrounding soil or from the substrate used to manure the cultivated fungus. In this sense, additional studies of North American attine species (Atta texana, Trachymyrmex septentrionalis and Cyphomyrmex wheeleri) demonstrated that the structure of microfungal communities in gardens of these ants did not correlate with seasonal changes over a one year period, again suggesting there are no species-specific relationships among ants and microfungi species. Although, the microfungi are not specialized parasites of the attine ant-fungus symbiosis we suggest they can be considered antagonists to the cultivated fungus. Moreover, we demonstrated that the specialized parasite Escovopsis spp. is probably less frequent in South America than in Central America and we reinforce that additional studies are necessary to unravel the epidemiology of this parasite in attine gardens. In another study, we showed that yeasts isolated from gardens of the leafcutter ant A. texana can significantly inhibit the growth of Escovopsis sp. This interesting finding suggests that attine ants may use additional microbes to protect their gardens against Escovopsis spp. and not only actinomycete bacteria (Pseudonocardia spp.) found in their cuticles. Finally, we studied microfungi relationships with female alates (gynes) in two... (Complete abstract click electronic access below)
Orientador: Fernando Carlos Pagnocca
Coorientador: Mauricio Bacci Junior
Banca: Carlos Augusto Rosa
Banca: Lara Durães Sette
Banca: Luiz Carlos Forti
Banca: Maria José Aparecida Hebling
Doutor

Rarely are natural conditions constant, but generally biologists study microbes in artificially constant environments in the laboratory. I relaxed these assumptions of constant environments through time and space as I investigated how microbial populations evolve. First, I examined how bacteriophage evolved in the presence of permissive and nonpermissive hosts. I found that bacteriophage evolved discrimina- tion in mixed environments as well as in one of two environments with homogeneous, permissive hosts. This showed the asymmetry of host-shifting in viruses as well as the possibility of large, and somewhat unpredictable, pleiotropic effects. Secondly, I reconstructed ancestral environmental conditions for soil bacteria groups using phy- logenetics and environmental variables of extant species’ habitats. These generaliza- tions suggested characteristic phenotypes for several phylogenetic groups, including uncultured Acidobacteria. Lastly, I collected genetic sequences and global collection information for 65 bacteria genera across the domain. In examining the relation- ship between genetic distance, environmental conditions, and geography, I observed positive relationships specifically between genetic distance and geography or genetic distance and environmental conditions for bacteria from land sites but not from wa- ter sites. Phylogenic classifications or phenotypes of the genera could not predict these correlations. In all of these projects, variations in the environment created evolutionary signals that hinted at past environments of microbial populations.
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Greenhouse and laboratory experiments in 2011 determined that Clonostachys rosea can effectively prevent Botrytis cinerea infection in Vaccinium angustifolium blossoms. In vitro testing demonstrated that C. rosea germination was not significantly affected by the presence of Switch®, but was by either Pristine® or Maestro®. Field experiments completed during the summer of 2010 and 2011 indicated that the dispenser designs tested had no significant effects on Bombus impatiens foraging behaviours, aside from hive-activity. There was also no difference in the quantity of C. rosea applied by each to bees, the distribution of product in the field, or for blossoms exposed to bees from each dispenser to resist infection by B. cinerea. However, B. cinerea prevalence in blossoms from both treatments was significantly different from the control, with infection reduced by 10-20%. Technical issues with dispensers currently appear to be the limiting factor for application within commercial wild blueberry production.

Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Phytophthora cinnamomi é um fitopatogéneo do solo altamente difundido que está associado à doença da tinta dos castanheiros. Apesar dos esforços para controlo desta doença através da aplicação de fungicidas específicos, estes não se têm revelado eficazes. Assim, os agentes de controlo biológico (BCA) têm-se tornado uma alternativa de crescente interesse aos fungicidas. Este trabalho teve como objectivo o isolamento e screening de isolados bacterianos com actividade antagonista para com P. cinnamomi. A actividade antagonista foi avaliada in vitro tendo sido estudados um total de 339 isolados. Apenas 12% destes isolados demonstraram uma boa actividade antagonista (≥50%) nos testes em cocultura. Todos os isolados foram analisados quanto à produção de enzimas hidrolíticas, sideróforos e solubilização do fosfato. A diversidade genética foi estudada através da análise dos perfis de M13, PCR-RFLP e a análise filogenética através da sequenciação da região 16S rDNA. Os géneros Bacillus, Paenibacillus, Pseudomonas e Streptomyces foram os maioritariamente obtidos nos testes de antagonismos. Os isolados antagonistas foram subsequentemente seleccionados para inoculação em bioensaios de sorgo e milho de modo a verificar a interacção e influência destes microrganismos no crescimento das plantas. Devido aos seus resultados, os isolados BC7, B106, B167A, B20-B, BC8-B, BC133 e BC27 foram considerados potenciais promotores do crescimento das plantas e promissores BCAs para controlo da doença da tinta dos castanheiros.
Phytophothora cinnamomi is one of the most widespread soil-borne pathogen and is associated with the chestnut ink disease. Although efforts are being made to control the disease using specific fungicides, they are not been effective. Biological control agents (BCA) are becoming an increasingly interesting alternative to chemical fungicides. The aims of this study were isolate and screen the bacterial strains showing highest antifungal activity against P. cinnamomi. A total of 339 bacterial strains were isolated and tested for the in vitro antagonism against P.cinnamomi. Among these isolates, only 12 bacterial strains exhibited a maximum antagonistic activity in dual culture assay (≥50%). The isolates where also tested for the production of hydrolytic enzymes, siderophores and phosphorous solubilization. The genetic diversity of the selected isolates was studied by M13 analysis, PCR-RFLP and phylogenetic analysis of 16S rDNA sequences. Based on the 16S rDNA sequencing, Bacillus, Paenibacillus, Pseudomonas and Streptomyces were the most in vitro antagonist isolates identified. Isolates were further assayed in greenhouse bioassays with maize and sorghum plants for investigation of their interaction and influence on plant´s growth. Among these isolates the BC7, B106, B167A, B20-B, BC8-B, BC133 and BC27 are considered potential plant growth promoters and also promissory BCAs for chestnut ink disease.