Made available in DSpace on 2015-04-11T13:38:51Z (GMT). No. of bitstreams: 1 Laryssa da Silva Melo.pdf: 834446 bytes, checksum: 1dbffe1130e351818bfc66f05b6077ae (MD5) Previous issue date: 2009-10-14 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Trichoderma are anamorphic fungi belonging to the class of Hifomicetos, also called imperfect fungi, or asexual conidial and whose gender Hypocrea teleomorph. They are free-living fungi, distributed throughout the world and found in different soil temperatures, especially those containing organic matter. Usually are not considered important human pathogens, but there are some reports indicating occasional pathogenicity of some species. Its easy handling and in vitro, stability and viability of colonies preserved this kind are a big target for biotechnology research. Because of these characteristics were isolated Trichoderma from plant Victoria amazonica, Rollinia sp. Murraya paniculata and Strychnos cogens; wood Scleronema micranthum, known as cardeiro; jatoba (Hymenaea courbaril), land of cubiu culture (Solanum sessiliflorum) and Indian black earth, in order to identify the molecular biology, the species level, such isolates and to assess their ability to produce cellulolytic enzymes. Of the 30 lines obtained were cultured spore, which were preserved in mineral oil, Castellani and method in 10% glycerol. From the suspension in glycerol, each sample was inoculated in 20ml of 10μL BD and cultivated 26oC, 100 rpm for 40:00 h. Next was extracted genomic DNA, performed PCR of specific regions of the ITS-1 and ITS-2 ribosomal DNA sequencing and subsequently. For the production of enzymes, the isolates were first grown in induction medium. Were inoculated 10μL of spore solution (glycerol 10%) in 50 mL of solution Manachini where the substratum was used to carboxymethylcellulose. The fungi were incubated at 27 ° C, 120 rpm for 120 hours. The dosage of CMCase was performed using the method of acid Dinitrosalicílico. For the determination of β-glucosidase was used p-nitrophenyl-β-D-glucopyranoside (PNPG) as substrate for the enzyme. The total protein concentration was determined by the Bradford method, using the reagent concentrate commercial Bio-RadTM and bovine serum albumin (ASB) as standard. The result of molecular identification of the first 13 samples revealed the species: T. harzianum, T. koningii, T. asperellum, T. viride, T. ovaslisporum, T. hamatum, T. piluliferum and T. koningiopsis, with a percentage between 96 and 99% identity and 100% reliability. The results indicated CMCase enzyme to low values, less than 0.100 U / mL with the exception of T. koningii (MPCE 10 3.2), T. harzianum (MPCE 2 2.2a), both isolates of M. paniculata, and isolate 1437 identified as Trichoderma sp., from Indian black earth, which showed slightly higher levels of 0.112 and 0.103 and 0.105 U / mL, respectively. For β-glucosidase, the results showed high activity in the vast majority of isolates with emphasis on T. harzianum MPCE 3 3.1 (10.45 U / mL) and T. piluliferum Vrc 2 3.2 (9.71 U / mL). All isolates produced protein in culture medium containing carboxymethylcellulose as substrate inducer / Trichoderma são fungos anamórficos pertencentes à classe dos hifomicetos, também chamados fungos imperfeitos, assexuais ou conidiais e que têm como teleomorfo o gênero Hypocrea. São fungos de vida livre, distribuídos em todo o mundo e encontrados em solos de diversas temperaturas, especialmente naqueles que contém matéria orgânica. Geralmente não são considerados importantes patógenos humanos, mas existem alguns relatos indicando patogenicidade ocasional em algumas espécies. Sua fácil manipulação e cultivo in vitro, estabilidade e viabilidade das colônias preservadas, fazem desse gênero um grande alvo para as pesquisas biotecnológicas. Por tais características, foram isolados Trichoderma das plantas Victoria amazonica, Rollinia sp., Murraya paniculata e Strychnos cogens; da madeira Scleronema micranthum, conhecida como cardeiro; de jatobá (Hymenaea courbaril), do solo de cultura de cubiu (Solanum sessiliflorum) e de terra preta de índio, com o objetivo de identificar, pela biologia molecular, em nível de espécie, tais isolados, bem como avaliar sua capacidade de produção de enzimas celulolíticas. Das 30 linhagens obtidas foram realizadas culturas monospóricas, as quais foram preservadas em óleo mineral, método Castellani e em glicerol 10%. A partir da suspensão em glicerol, de cada amostra foi inoculado 10μL em 20mL de BD e cultivado a 26oC, a 100 rpm por 40:00h. Em seguida foi extraído o DNA genômico, deste realizada a PCR específica para as regiões ITS-1 e ITS-2 do DNA ribossômico e posteriormente o sequenciamento. Para a produção de enzimas, os isolados foram previamente cultivados em meio indutor. Foram inoculados 10μL da solução de esporos (Glicerol 10%) em 50mL de solução de Manachini onde o substrato indutor utilizado foi a carboximetilcelulose. Os fungos foram incubados a 27°C, 120 rpm durante 120 horas. A dosagem de CMCase foi efetuada com base no método do Ácido Dinitrosalicílico. Para a dosagem de β-glucosidase foi utilizado o pnitrofenil- β-D-Glucopiranosídeo (pNPG) como substrato para a enzima. A concentração de proteínas totais foi determinada pelo método de Bradford, utilizando-se o reagente concentrado comercial da Bio-RadTM e albumina de soro bovino (ASB), como padrão. O resultado da identificação molecular das primeiras 13 amostras nos revelaram as espécies: T. harzianum, T. koningii, T. asperellum, T. viride, T. ovaslisporum, T. hamatum, T. piluliferum e T. koningiopsis, com um percentual entre 96 e 99% de identidade e 100% de confiabilidade. Os resultados enzimáticos para CMCase indicaram valores baixos, inferiores a 0,100 U/mL com exceção de T. koningii (MPCe 10 3.2), T. harzianum (MPCe 2 2.2a), ambos isolados de M. paniculata, e o isolado 1437 identificado como Trichoderma sp., proveniente de terra preta de índio, que apresentaram valores um pouco mais altos de 0,112 e 0,103 e 0,105 U/mL, respectivamente. Para β-glucosidase, os resultados apresentados mostraram alta atividade na grande maioria dos isolados com destaque para T. harzianum MPCe 3 3.1(10,45U/mL) e T. piluliferum Vrc 2 3.2 (9,71 U/mL). Todos os isolados produziram proteínas em meio de cultura contendo carboximetilcelulose como substrato indutor

Für die Identifizierung bzw. Differenzierung von Candida- Spezies und -Stämmen sowie für die Bestimmung der genetischen und epidemiologischen Verwandtschaft von Stämmen der gleichen Spezies wurde eine PCR-Fingerprint-Technik und eine RFLP-Analyse der amplifizierten ITS-Region angewandt. Das PCR-Fingerprinting amplifiziert anonyme Sequenzen in der chromosomalen DNA, die über das gesamte Genom verteilt sind. Die ITS-Region ist Bestandteil des ribosomalen Operons, welches in ca. 50-100 Kopien/Zelle vorhanden ist. 1.a. Beide molekularbiologischen Verfahren wurden zur Unterscheidung von routinemäßig schwer differenzierbaren klinischen Candida famata und Candida guilliermondii-Isolaten genutzt. Von insgesamt 37 fraglichen Stämmen konnten 31 als C. guilliermondii und 3 als C. famata identifiziert werden, die drei verbliebenen Stämme waren mit diesen Techniken nicht identifizierbar. Mit der Biochemotypie gelang nur die Zuordnung eines der 3 C. famata-Isolate sowie von 23 der 31 C. guilliermondii-Isolate. 14 Isolate wurden mit den konventionellen Methoden gar nicht oder falsch identifiziert. 1b. Mit dem PCR-Fingerprinting wurde auch die Spezieszugehörigkeit phänotypisch veränderter Candida albicans-Isolate überprüft. Alle atypischen Stämme, bei denen solche für C.albicans charakteristischen Merkmale wie die Bildung von Chlamydosporen, die Verwertung der Aminozucker Glukosamin und N-Acetylglukosamin sowie die Assimilation von 2-Ketogluconat und Xylose nicht ausgeprägt waren, wiesen die für C. albicans typischen Fingerprintmuster auf. Unsere Studie zeigte, daß die biochemische Typisierung an Grenzen stößt, wenn typische Stoffwechselreaktionen nicht nachgewiesen werden können. 2. Bei 6 verschiedenen C. albicans-Populationen aus Angola, Madagaskar, Deutschland und Portugal wurde die Variabilität phänotypischer und genotypischer Merkmale untersucht, wobei in diese Analyse auch atypische Stämme miteinbezogen wurden. Während die phänotypischen Eigenschaften, bis auf die der atypischen Stämme, kaum variierten, wurden für die insgesamt 212 C. albicans-Isolate 87 unterschiedliche PCR-Fingerprint-Genotypen nachgewiesen. Eine Analyse der Beziehungen zwischen den Fingerprint-Genotypen wurde mit der UPGMA-Distanz-Methode durchgeführt. 3. Weiterhin wurden Candida-Vaginalisolate von Patientinnen mit rezidivierenden Episoden von Candida-Vaginitis mit Stämmen verglichen, die aus anderen Körperregionen stammten bzw. bei ihren Partnern isoliert wurden. Es konnte gezeigt werden, daß Stammaustausche zwischen den Partnern vorkommen, ein Stamm ohne oder mit geringfügigen genotypischen Veränderungen trotz Therapie persistieren kann und daß eine Reinfektion auch durch einen neuen Stamm möglich ist. Das Problem der Erregeridentifizierung in der mykologischen Labordiagnostik ist sowohl klinisch als auch epidemiologisch relevant. Molekularbiologische Methoden sollen gut funktionierende konventionelle Methoden zur Erregeridentifizierung nicht ersetzen, können aber bei Problemfällen eine wertvolle Ergänzung für die mykologische Diagnostik vorzugsweise in fachständigen Referenzlaboratorien darstellen. / A PCR fingerprinting approach and a RFLP analysis of the amplified ITS region were used to differentiate Candida species and strains as well as to assess genetic and epidemiological relationships of strains belonging to the same species. The PCR fingerprinting amplifies anonymous DNA sequences sampled throughout the whole genome. The ITS region is part of the ribosomal operon which occurs in tandem arrays of nearly 50-100 copies in one cell. 1.a. Both methods were used to identify clinical isolates of C. famata and C. guilliermondii which were difficult to differentiate with routine methods. Out of 37 ambiguous isolates 31 could be identified as C. guilliermondii, 3 as C. famata and the other 3 were not identifiable. Biochemical typing (Api 32 C V.1) identified only 23 out of 31 C. guilliermondii and 1 out of 3 C. famata whereas 14 isolates were misidentified or not identified at all. 1.b. By using the PCR fingerprinting technique strains of C. albicans with altered phenotypes could be identified at species level. Atypical isolates which did not express those characteristics which are thought to be typical for C.albicans like the formation of clamydospores, the ability to metabolise the amino sugars glucosamine and N-acetylglucosamine as the sole carbon source or to assimilate 2-ketogluconate and xylose showed the PCR patterns typical for C.albicans. Our study revealed that biochemical and morphological methods of species identification are limited if some of the key reactions fail. 2. We investigated the variability of phenotypic and genotypic properties of 6 different C. albicans populations from different countries (Angola, Madagascar, Portugal and Germany) including atypical strains. Except for the atypical strains only very little phenotypical variation was observed. However, 87 different genotypes were found among the 212 strains. The relatedness of the fingerprint-genotypes were analysed by measuring genetic distances with the UPGMA method. 3. Vaginal isolates of Candida spp. obtained from patients with recurrent episodes of vaginitis were compared with isolates from different body locations of the women and from their male partners. It has been shown that strain exchanges between the partners occur, that the original strain with or without minor genotypic changes can persist despite of the therapy, but also that reinfection by a new strain is possible. The identification of an ethiological agent by the mycological diagnostic laboratory is of clinical and epidemiological importance. Molecular biological methods should not replace well established conventional methods but they can supplement the identification of fungal pathogens in specialised reference laboratories if diagnosis cannot be achieved easily by conventional diagnostic procedures.

ABSTRACT COMPARATIVE SEQUENCE ANALYSIS OF THE INTERNAL TRANSCRIBED SPACER 2 REGION OF TURKISH RED PINE (Pinus brutia TEN.) AND NATURAL ALEPPO PINE (Pinus halepensis MILL.) POPULATIONS FROM TURKEY Tozkar, &Ouml / zge M.S., Department of Biology Supervisor: Prof. Dr. Zeki Kaya April, 2007, 107 pages Turkish red pine (Pinus brutia) is wide-spread and an important forest tree species in Turkey, occurring mainly in southern, western and north-western Turkey and as small isolated populations in the Black Sea region. Aleppo pine (Pinus halepensis) has naturally found only in Adana and Mugla provinces as small population in mixture with Turkish red pine. Although Turkish red pine and Aleppo pine are morphologically different, Turkish red pine has been regarded as subspecies of Aleppo pine by some taxonomists due to occurrence of natural hybridization between these two species. However, the phylogenic relationship between these species needs to be explored further. In the present study, by sampling overlapped populations of both species from Mugla and Adana provinces (4 populations of Turkish red pine and 3 populations of Aleppo pine), internal transcribed spacer (ITS) region of ribosomal DNA were comparatively studied with sequence analysis. Although ITS1, 5.8s and ITS2 regions of ribosomal DNA were studied with ITS primers, only ITS2 region was successfully amplified with polymerase chain reaction (PCR). The complete data set for this region was analysed using MEGA3.1 and Arlequin softwares. Analysis of molecular variance (AMOVA) demonstrated the highest genetic differentiation between Turkish red pine and Aleppo pine in Mugla with 100 percentage of variation. AMOVA analysis also indicated the possibility of low-level migration of genes between Turkish red pine and Aleppo pine populations in Adana with 50.65 percent of molecular variance. Haplotype comparison revealed that two major haplotypes were represented Based on the results of ITS2 region sequence analysis, Turkish populations of Aleppo pine and Turkish red pine populations could not be fully differentiated. In Mugla province Turkish red pine and Aleppo pine revealed more differentiation due to reproductive isolation. But in Adana province, two species shared more common genetic background due to possible hybridization. Since ITS2 region of nuclear ribosomal DNA revealed a few variable and parsimony informative sites for both species, thus, only ITS2 region of ribosomal DNA does not appear to be sufficient for fully resolving genetic relationships between Turkish red pine and Aleppo pine populations. Further studies including ITS1 and 5.8s regions of ribosomal DNA and populations included from major Aleppo pine distribution areas will be useful to understand the evolutionary relationship between Aleppo pine and Turkish red pine populations in Turkey.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The genus Scleroderma Pers.: Fr. belongs to Order Boletales, Phylum Basidiomycota, and involves a group of fungi whose spore production occurs in an enclosed hymenium with passive basidiospores release. The main morphological characteristic of this genus are the size and ornamentation of the spores and mycorrhizal association with various tree species. The aim of this study is to identify Scleroderma species of Pampa biome region by using techniques of morphological analysis and molecular biology following specific methodologies. For an accurate analysis of the basidiospore ornamentation, scanning electron microscopy (SEM) was used. The specimens collected were deposited in the Herbarium SMDB of the Department of Biology, Federal University of Santa Maria - UFSM. For the analysis of phylogenetic relationships of selected species of Scleroderma was sequenced the rDNA (ITS1-5.8S-ITS2) region of some specimens collected or obtained from other herbaria for comparison with sequences deposited in GenBank. Two species were identified: S. albidum; S. citrinum and a specimen to genus level (Scleroderma sp.). We have generate 20 new ITS sequences, clustering most of them, in S. albidum clade, contrasting, however, with basidiome morphological variation initially observed; they represent the first records of this species for the ITS region in GenBank database. Sequences in clade S. albidum showed high similarity with some other nominated as S. bovista, S. aurantium and Sceroderma sp. from GenBank so, the latter sequences are considered as conspecific in the clade. S. citrinum clustered together with homonymous sequences from China, United States and Germany being conspedifics, and its basidiomes were the most identifable in study area by their morphological characteristics. The specimen ICN:154625, from Rio Grande do Sul, State nominated as S. verrucosum, showed high similarity with sequences of S. areolatum and were identified under the latter name. The phylogenetic analysis confirms the literature trend on formation of two major infrageneric clades combining, respectively, echinulate spores plus simple-septate hyphae and reticulate spores plus fibulae. Scleroderma sp. appeared phylogenetically isolated from these two main clusters suggesting a probable formation of a new clade combining echinulate spores and fibulae. / O gênero Scleroderma Pers.: Fr. pertence à Ordem Boletales, Filo Basidiomycota, e envolve um grupo de fungos cuja produção dos esporos ocorre em um himênio fechado, com liberação passiva dos basidiósporos. As principais características morfológicas deste gênero são o tamanho e ornamentação dos esporos e a associação micorrízica com várias espécies arbóreas. Este trabalho tem por objetivo buscar a identificação das espécies do gênero Scleroderma de ocorrência no bioma Pampa, utilizando, para esta finalidade, técnicas morfológicas e de moleculares, segundo metodologias específicas para o estudo do grupo. Para a análise inequívoca da ornamentação dos basidiósporos, foi utilizada a Microscopia Eletrônica de Varredura MEV. Os espécimes coletados foram depositados no Herbário SMDB do Departamento de Biologia da Universidade Federal de Santa Maria - UFSM. Para a análise das relações filogenéticas das espécies selecionadas de Scleroderma, foram sequenciadas as regiões ITS1-5.8S-ITS2 do rDNA de alguns espécimes coletados ou obtidos de outros herbários para comparação com sequências depositadas no GenBank. Duas espécies foram identificadas: S. albidum; S. citrinum e um espécime a nível somente de gênero (Scleroderma sp.). Foram obtidas 20 novas sequencias de ITS, a maioria delas agrupando-se no clado para S. albidum contrapondo a variação morfológica inicialmente verificada nos basidiomas e representando os primeiros registros desta espécie para a região ITS no banco de dados do GenBank; as sequências deste clado também apresentaram alta similaridade com outras do GenBank nominadas como S. bovista, S. aurantium e Scleroderma sp. (da Estônia e Montenegro), permitindo, assim, a retificação nas suas identidades para S. albidum, S. citrinum alinhou com sequências homônimas da China, Estados Unidos e Alemanha mostrando-se conspecíficas, e seus basidiomas podem ser considerados os de mais fácil identificação da área de estudo pelas suas características morfológicas. O espécime ICN:154625, nominado como S. verrucosum, anteriormente citado para o Rio Grande do Sul, mostrou alta similaridade com sequências de S. areolatum do GenBank, formando um clado S. areolatum. A análise filogenética confirma a tendência na literatura para a formação de dois grandes clados infragenéricos combinando, respectivamente, esporos reticulados e hifas fíbuladas ou esporos equinulados e septos simples. Scleroderma sp. isolou-se filogeneticamente dos dois grupos anteriores sugerindo a provável formação de um novo clado combinando com esporos equinulados e hifas fíbuladas.

Made available in DSpace on 2015-03-26T13:51:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1135397 bytes, checksum: ae470991496aa9e9f575bf2c2ec32c0b (MD5) Previous issue date: 2010-07-27 / Soybean is one of the most important crops used in human and animals food. Fungal endophytes are microorganisms that live inside the plant tissue at some stage of their life cycle without causing apparent disease to the host. Many studies have shown the use of fungal endophytes as agents of biological control of pests and plants diseases, and to improve the plant growth and induce systemic resistance of plants. The goal of this work was to isolate and identify the endophytic fungi from leaves of soybean cultivars Monsoy and Conquista using two method of isolation (tissue fragmentation and extinction cultivation). Furthermore, was determining the species richness and the diversity of fungi and compared between cultivars. 188 morphologically distinct isolates were obtained representing 52 taxa identified by sequencing the ITS region of rDNA. The phylum Ascomycota was the dominant represented by 99 and 96 % of the isolates to Monsoy and Conquista cultivars respectively. Species richness of cultivars Monsoy (31 species) and Conquista (37 species) was greater when both isolation method were used. Diversity of endophytic fungi was similar for both cultivars using the same isolation methods. The nucleotide sequences of the ITS region was used for phylogenetic analysis and allowed the grouping of fungal isolates according to their respective Order (when present), Class and Phylum. This is one of the first studies using the extinction culturing method for endophytic fungal isolation in soybean. / A soja é uma das mais importantes culturas agrícolas mundiais, sendo utilizada principalmente na alimentação humana e de animais. Os fungos endofíticos são micro-organismos que habitam o interior do tecido vegetal durante alguma fase do seu ciclo de vida, mas sem causar doença aparente ao hospedeiro. Muitos trabalhos têm mostrado o potencial do uso de fungos endofíticos como agentes no controle biológico de doenças e pragas em plantas, na indução de resistência sistêmica e na promoção de crescimento vegetal. O objetivo deste estudo foi isolar e identificar os fungos endofíticos de folhas de soja das cultivares Conquista e Monsoy, utilizando duas técnicas de isolamento, a fragmentação do tecido e a técnica de cultivo por extinção, para determinar a riqueza de espécies e comparar a diversidade de fungos encontrados entre as cultivares. Um total de 188 morfotipos foram obtidos representando 52 taxa identificados pela região ITS do rDNA. O filo Ascomycota foi o dominante, representado por 99 e 96 % dos isolados para as cultivares Monsoy e Conquista, respectivamente, enquanto o filo Basidiomycota foi representado por 1 e 4 % dos isolados, para as mesmas cultivares. A riqueza de espécies para a cultivar Monsoy (31 espécies) e para a cultivar Conquista (37 espécies) foi maior quando ambas as técnicas de isolamento foram utilizadas. A diversidade de fungos endofíticos foi semelhante para ambas as cultivares, utilizando a mesma técnica de isolamento. A utilização das sequências da região ITS para a análise filogenética permitiu o agrupamento dos isolados fúngicos de acordo com a sua respectiva Ordem (quando definida), Classe e Filo. Esse é o primeiro trabalho que utiliza a técnica de cultivo por extinção para o isolamento de fungos endofíticos da soja.

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.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The chemical profile of a fermented product depends upon the raw basic material and the fermentative microbiota. The yeast microbiota is responsible for fermentation and contributes to fermented product aroma by several mechanisms. The study of this microbiota is relevant because the microorganisms utilize the constituents of the raw basic material and transform them into aroma or flavour impacting components. Autochthonous yeasts belonging to the blueberry microbiota of two different cultivars, Florida M and Climax, were investigated by analyzing the fermentative capacity, the hydrogen sulfide (H2S) production, the film-forming ability, killer feature, sensitivity and neutrality to killer factor. Three methods employed to distinguish autochthonous yeast species were used: MALDI-TOF MS, PCR and PCR-RFLP of ITS region of rRNA gene and, if necessary, genetic sequencing of the D1/D2 26S rRNA region. The species Hanseniaspora uvarum, Hanseniaspora opuntiae, Candida sorboxylosa, Metschnikovia kunwiensis, Candida bentonensis, Candida oleophila/Candida railenesis and Candida quercitrusa were found on the surface of Florida M. Hanseniaspora uvarum, Issatchenkia terricola, Candida sorboxylosa, Candida asiatica, Candida oleophila/railenensis, Issatchenkia hanoiensis and Kazachstania intestinalis were of berries of Climax variety. Only three species were found in both varieties: H. uvarum, C. sorboxylosa and C. oleophila/railenensis. The species H. uvarum was the predominant in both cultivars, representing 70.4% and 78% of yeasts isolated from the surface of the Florida M and Climax, respectively. The killer feature was not detected in any strain isolated from Florida M. From strains isolated from Climax, only one strain C. asiatica 133 MCMCF/14 was killer against the 26B. Moreover, the K. intestinalis 91 MCMCF/14 was the only sensitive when tested against both the patterns killer S. cerevisiae K1 (Lallemand), EMBRAPA 1B, EMBRAPA 91B and the killer yeast C. asiatica 133 MCMCF/14. All strains showed low fermentative capacity and all of them were non-Saccharomyces yeasts. All yeasts isolated from Florida M produced H2S. Only four strains from Climax did not produce H2S. The highest evolution of H2S was observed with the genus Issatchenkia and with the majority strains of C. sorboxylosa. The species C. oleophila/C. railenensis showed low production of this parameter. The fermentation process of fermented products from blueberries of both Florida and Climax can be severely affected by this kind of autochthonous yeasts. / O perfil químico de um produto fermentado depende da matéria prima de base e da microbiota que participam do processo fermentativo. No presente estudo, leveduras pertencentes à microbiota do mirtilo foram investigadas, isolando-se estes micro-organismos da superfície de duas diferentes cultivares de mirtilo, Florida M e Climax. Foram avaliadas a capacidade fermentativa, produção de sulfeto de hidrogênio (H2S), formação de filme, característica killer, sensibilidade e neutralidade a este fator. Três metodologias para identificação foram empregadas: espectrometria de massas MALDI-TOF, análise da região ITS do gene rRNA por PCR-RFLP e, quando necessário, sequenciamento genético da região D1/D2 do 26S do gene rRNA. A cultivar Florida M apresentou em sua superfície as espécies Hanseniaspora uvarum, Hanseniaspora opuntiae, Candida sorboxylosa, Metschnikovia kunwiensis, Candida bentonensis, Candida oleophila/Candida railenensis e Candida quercitrusa. Na cultivar Climax, foram encontradas as espécies Hanseniaspora uvarum, Issatchenkia terricola, Candida sorboxylosa, Candida asiatica, Candida oleophila/Candida railenesis, Issatchenkia hanoiensis e Kazachstania intestinalis. Apenas três espécies foram comuns as duas cultivares, sendo elas H. uvarum, C. sorboxylosa, Candida oleophila/Candida railenesis. H. uvarum foi a espécie predominante em ambas cultivares, representando 70,4% e 78% do total de leveduras isoladas da cv. Florida M e Climax, respectivamente. A característica killer não foi detectada em nenhuma linhagem isolada da cv. Florida M. Na cv. Climax, apenas a linhagem C. asiatica 133 MCMCF/14 se comportou como killer. A linhagem K. intestinalis 91 MCMCF/14 foi a única que demonstrou sensibilidade em relação a todas as linhagens killer padrão K1 (Lallemand), EMBRAPA 1B, EMBRAPA 91B. Esta mesma linhagem se mostrou sensível também à linhagem isolada da cultivar Climax C. asiatica 133 MCMCF/14. Todas as linhagens isoladas de ambas cultivares apresentaram baixa capacidade fermentativa e todas elas foram identificadas como sendo não-Saccharomyces. Houve de moderada a alta produção de H2S em 21 e 34% nas linhagens isoladas das cultivares Florida M e Climax, respectivamente. A produção máxima de H2S se destacou principalmente em linhagens das espécies I. terricola e C. sorboxylosa. A maioria das linhagens de leveduras da espécie C. oleophila/C. railenensis apresentaram baixa produção deste gás. O processo de produção de fermentados de mirtilo tanto da cultivar Florida M como da cultivar Climax pode ser severamente afetado por este tipo de leveduras autóctones.

New Jersey, Georgia, and the Eastern Shore of Virginia (ESV) are important snap bean (Phaseolus vulgaris L.) growing regions, but profitability is threatened by Pythium blight. Causal agents of Pythium blight on snap bean were identified using morphological characterization and sequence analysis of the rDNA-internal transcribed spacer (ITS) regions of 100 isolates. Most isolates were Pythium aphanidermatum (Edson) Fitzp. (53%), and also included Pythium deliense Meurs (31%; all from Georgia), Pythium ultimum Trow (12%), Pythium myriotylum Drechsler (2%), Pythium catenulatum Matthews (1%), and unknown Pythium sp. (1%). To our knowledge, this is the first report of P. deliense in Georgia and on common bean and squash (Cucurbita pepo L.); as well as the first report of P. catenulatum on lima bean (Phaseolus lunatus L.) and in New Jersey. Fungicide labeling and cultivar selection for Pythium blight management is hindered by difficulties associated with conducting successful trials, because the disease occurs sporadically and clustered in the field. Three P. aphanidermatum-infested inoculum substrates were evaluated at three concentrations. The vermiculite/V8 juice (5:3 weight to volume) inoculum (10,000 ppg/0.3 m) consistently caused at least 50% disease in 3 field trials. Sensitivity of the Pythium blight pathogens was determined in vitro against five fungicides. Twenty-two Pythium isolates representing P. aphanidermatum, P. deliense, P. ultimum, and P. myriotylum were inoculated to media amended with each active ingredient at 0, 100μg/ml, the concentration equivalent to the field labeled rate if applied on succulent beans at 187 L/ha, and the equivalent if applied at 374 L/ha. All isolates were completely sensitive (100% growth reduction, or GR) to all active ingredients at the labeled rates, except azoxystrobin. At 100μg/ml azoxystrobin, one P. deliense isolate had 8.9% GR. All isolates had 100% GR to copper hydroxide at 100μg/ml, and the lowest GR on mefenoxam-amended medium was 91.9%. At 100μg/ml cyazofamid, all P. deliense isolates were completely sensitive and variation was observed in P. aphanidermatum isolates. At 100μg/ml potassium phosphite, significant GR similarities were recorded within isolates of the same species, and less than 50% GR was observed in all P. deliense isolates. / Ph. D.

Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados / Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)

Cryptococcus neoformans (C. neoformans), um fungo que se encontra disseminado em várias partes do mundo, inclusive no Brasil, é o responsável pela criptococose infecção oportunista mais comum em pacientes com a síndrome da imunodeficiência adquirida (SIDA). O caráter sistêmico da criptococose pode levar esses pacientes a óbito. A finalidade de se obter um diagnóstico laboratorial rápido e acurado de C. neoformans, principalmente para o seguimento dos pacientes HIV nos levou a investigar \"seqüências iniciadoras\" (Si) A, B e C das regiões 18SrDNA, 5,8SrDNA e ITS do Cryptococcus spp. Pela Nested PCR com estas seqüências, sugerimos a melhor delas para o desenvolvimento de um diagnóstico molecular em relação aos métodos usuais. Para tal, foram avaliadas amostras de soro e líquor de 39 pacientes, que já haviam recebido tratamento clínico. Todos os casos foram selecionados em grupos, como segue:7 com criptococose (GIII), 14 HIV positivos (GIV), 18 HIV positivos associados com a criptococose (GV) em relação a 10 controles - indivíduos sadios (GI) e amostras de culturas referência (GII). Os resultados obtidos pela Nested PCR com as \"Sis\" A, B e C foram comparados àqueles obtidos pelos métodos de diagnóstico convencionais. As análises desse estudo mostraram que as \"Sis\" A, B e C detectam C. neoformans com especificidade variada, tanto no soro (SiA 91,66%, SiB-100%, SiC 75%), como no líquor (SiA 83,33%, SiB 100% e SiC 75%) mas não apresentaram falso positivo, quando esses resultados foram comparados aos obtidos das culturas heterólogas (GVI). A SiB, em líquor, apresentou sensibilidade, acurácia, valores preditivo positivo e negativo, e especificidade de 100% para a detecção de C. neoformans da mesma forma que no soro, porém neste o valor preditivo negativo foi 89%, acurácia 94% e a sensibilidade 88%. Em amostras de soro e líquor, os testes Tinta da China e Látex, mostraram resultados falso positivos para o GIV e falso negativos nos grupos GIII e GV. As análises comparativas entre as técnicas mostraram que a ordem de eficiência da sensibilidade para detecção de C. neoformans no soro foi SiB>SiA=Látex>SiC e no líquor foi SiB> SiA>tinta da China>Látex=SiC. No soro, a especificidade entre as técnicas foi SiB>SiA=Látex>SiC e no líquor foi SiB=tinta da China>Látex>SiA>SiC. De acordo com nossos dados, concluímos que, independente da doença associada (HIV) ou se o paciente for tratado, a SiB foi a melhor seqüência para a detecção de C. neoformans, tanto em amostras diretamente de soro, como de líquor para todos os grupos estudados. Tendo em vista os fatos, acreditamos que, a aplicação da técnica da Nested PCR com a \"SiB\", em amostras de líquor e soro, é um método viável e acurado para realizar o diagnóstico molecular do C. neoformans em pacientes HIV. O uso de amostras de soro, para o segmento dos pacientes com SIDA, durante o tratamento, pode ser a forma menos invasiva em relação ao líquor para a detecção do C. neoformans, com vantagens sobre os métodos utilizados / Cryptococcus neoformans (C. neoformans), a fungus that is widespread in many parts of the world, including Brazil, is responsible for cryptococcosis the most common opportunistic infection in patients with acquired immunodeficiency syndrome (AIDS). The systemic character of cryptococcosis may be fatal. In order to obtain a rapid and accurate laboratory diagnosis to follow - up of HIV-cryptococcosis patients led us to investigate the sensibility and especificity of three primers (A, B and C) of 18SrDNA, 5,8SrDNA and ITS regions of Cryptococcus spp. Using Nested PCR with those primers we suggest the best among them to be used as a method of molecular diagnosis in relation to the usual techniques. For this purpose, the serum and cerebrospinal fluid (CSF) of 39 patients, who had received medical treatment, were evaluated. All cases were separated in groups, as follows: 7 with cryptococcosis (group III), 14 HIV positive (group IV), 18 HIV positive associated with cryptococcosis (group V) were compared to, 10 healthy subjects (group I) controls, as well as to reference cultures (group II) samples. The results obtained by nested PCR with primers A and B and C were compared to those obtained by conventional diagnostic methods. The analyses of primers A, B and C detected C. neoformans both in serum (SiA 91,6%, SiB-100%, SiC 75%), and in CSF (SiA 83,3%, SiB 100% e SiC 75%). Besides, they were specific for the identification of C. neoformans and showed that there were no false positives when compared with heterologous cultures (group VI) samples. The primer B in CSF showed 100% sensitivity, 100% accuracy and 100% predictive values (positive and negative), and 100% specificity for the detection of C. neoformans, the same that occurs in serum, but in this case, with 88% in sensitivity, 89% predictive value negative and 94% accuracy. In serum and CSF samples the China Ink and the Latex tests showed false positive results for group IV and false negative for III and V groups. The comparative analysis among the techniques (Nested PCR, China Ink, Latex) indicated that the efficiency order of sensitivity for the detection of C. neoformans were PrB> PrA = Latex> PrC in serum and PrB > PrA > China Ink> Latex = PrC in CSF. For the serum and CSF specificities the same techniques were used with the following results: PrB>PrA=Latex>PrC and PrB=China Ink>Latex>PrA>PrC. According to our data we conclude that, whether the patients had been under treatment or not, the Nested PCR by PrB was the best way to detect C. neoformans both in serum and in CSF for all groups. The following up of AIDS patients, throughout the course of therapy was found to be feasible, accurate and less invasive to detect C. neoformans by using serum samples (directly)