Culturable endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane and its non-transgenic isolines

The diversity of endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane plants and its isoline was evaluated by cultivation followed by amplified rDNA restriction analysis (ARDRA) of randomly selected strains. Transgenic and non-transgenic cultivars and their crop management (herbicide application or manual weed control) were used to assess the possible non-target effects of genetically modified sugarcane on the fungal endophytic community. A total of 14 ARDRA haplotypes were identified in the endophytic community of sugarcane. Internal transcribed spacer (ITS) sequencing revealed a rich community represented by 12 different families from the Ascomycota phylum. Some isolates had a high sequence similarity with genera that are common endophytes in tropical climates, such as Cladosporium, Epicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of molecular variance indicated that fluctuations in fungal population were related to both transgenic plants and herbicide application. While herbicide applications quickly induced transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. These results represent the first draft on composition of endophytic filamentous fungi associated with sugarcane plants. They are an important step in understanding the possible effects of transgenic plants and their crop management on the fungal endophytic community.

Process Optimization for Mass Production of Marine Yeast Candida sp. S 27 and its Nutritional Characterization

The main source of protein for human and animal consumption is from the agricultural sector, where the production is vulnerable to diseases, fluctuations in climatic conditions and deteriorating hydrological conditions due to water pollution. Therefore Single Cell Protein (SCP) production has evolved as an excellent alternative. Among all sources of microbial protein, yeast has attained global acceptability and has been preferred for SCP production. The screening and evaluation of nutritional and other culture variables of microorganisms are very important in the development of a bioprocess for SCP production. The application of statistical experimental design in bioprocess development can result in improved product yields, reduced process variability, closer confirmation of the output response to target requirements and reduced development time and overall cost.The present work was undertaken to develop a bioprocess technology for the mass production of a marine yeast, Candida sp.S27. Yeasts isolated from the offshore waters of the South west coast of India and maintained in the Microbiology Laboratory were subjected to various tests for the selection of a potent strain for biomass production. The selected marine yeast was identified based on ITS sequencing. Biochemical/nutritional characterization of Candida sp.S27 was carried out. Using Response Surface Methodology (RSM) the process parameters (pH, temperature and salinity) were optimized. For mass production of yeast biomass, a chemically defined medium (Barnett and Ingram, 1955) and a crude medium (Molasses-Yeast extract) were optimized using RSM. Scale up of biomass production was done in a Bench top Fermenter using these two optimized media. Comparative efficacy of the defined and crude media were estimated besides nutritional evaluation of the biomass developed using these two optimized media.

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract Background Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes. Results ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups. Conclusion These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.

Evaluation of inflammatory response induced by different types of daily contact lenses

Dissertação de mestrado em Genética Molecular

Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation.

The present study was performed to assess the interlaboratory reproducibility of the molecular detection and identification of species of Zygomycetes from formalin-fixed paraffin-embedded kidney and brain tissues obtained from experimentally infected mice. Animals were infected with one of five species (Rhizopus oryzae, Rhizopus microsporus, Lichtheimia corymbifera, Rhizomucor pusillus, and Mucor circinelloides). Samples with 1, 10, or 30 slide cuts of the tissues were prepared from each paraffin block, the sample identities were blinded for analysis, and the samples were mailed to each of seven laboratories for the assessment of sensitivity. A protocol describing the extraction method and the PCR amplification procedure was provided. The internal transcribed spacer 1 (ITS1) region was amplified by PCR with the fungal universal primers ITS1 and ITS2 and sequenced. As negative results were obtained for 93% of the tissue specimens infected by M. circinelloides, the data for this species were excluded from the analysis. Positive PCR results were obtained for 93% (52/56), 89% (50/56), and 27% (15/56) of the samples with 30, 10, and 1 slide cuts, respectively. There were minor differences, depending on the organ tissue, fungal species, and laboratory. Correct species identification was possible for 100% (30 cuts), 98% (10 cuts), and 93% (1 cut) of the cases. With the protocol used in the present study, the interlaboratory reproducibility of ITS sequencing for the identification of major Zygomycetes species from formalin-fixed paraffin-embedded tissues can reach 100%, when enough material is available.

Molecular characterization and pathogenicity of isolates of Beauveria spp. to fall armyworm

The objective of this work was to evaluate the pathogenicity of 24 Beauveria isolates to Spodoptera frugiperda larvae, and characterize them molecularly through rDNA-ITS sequencing and RAPD markers. Sequencing of rDNA-ITS fragments of 570 bp allowed the identification of isolates as B. bassiana or B. brongniarti by sequence comparison to GenBank. Sixty seven polymorphic RAPD fragments were capable to differentiate 20 among 24 Beauveria isolates, grouping them according to the derived host insect and to pathogenicity against maize fall armyworm larvae. Three RAPD markers were highly associated to the pathogenicity against S. frugiperda, explaining up to 67% of the phenotypic variation. Besides identification and molecular characterization of Beauveria isolates, ITS sequence and RAPD markers proved to be very useful in selecting the isolates potentially effective against S. frugiperda larvae and in monitoring field release of these microorganisms in biocontrol programs.

Black yeasts from the slope sediments of Bay of Bengal: phylogenetic and functional characterization

Occurrence of black yeasts in the slope sediments of Bay of Bengal was investigated during FORV Sagar Sampada cruises 236 and 245. The black yeast population was found to be very scanty in the area and the isolates could be obtained from 200m to 1000m depth regions in the slope sediments. The isolates were identified as Hortaea werneckii by Internal Transcribed Spacer (ITS) sequencing. The biodegradation potential of these strains was found to be very high with all the strains exhibiting protease, lipase and amylase production. The optimum growth conditions were pH 8, salinity 30 ppt and temperature 30oC. The pigment melanin, in these organisms was identified to be of dihydroxynaphthalene type by NMR. The melanin was found to exhibit inhibitory activity against different human and fish pathogens. Melanin degrading enzyme could also be extracted from these organisms

Fungal communities in the garden chamber soils of leaf-cutting ants

Leaf-cutting ants modify the properties of the soil adjacent to their nests. Here, we examined whether such an ant-altered environment impacts the belowground fungal communities. Fungal diversity and community structure of soil from the fungus garden chambers of Atta sexdens rubropilosa and Atta bisphaerica, two widespread leaf-cutting ants in Brazil, were determined and compared with non-nest soils. Culture-dependent methods revealed similar species richness but different community compositions between both types of soils. Penicillium janthinellum and Trichoderma spirale were the prevalent isolates in fungus chamber soils and non-nest soils, respectively. In contrast to cultivation methods, analyses of clone libraries based on the internal transcribed spacer (ITS) region indicated that richness of operational taxonomic units significantly differed between soils of the fungus chamber and non-nest soils. FastUnifrac analyses based on ITS sequences further revealed a clear distinction in the community structure between both types of soils. Plectania milleri and an uncultured Clavariaceae fungus were prevalent in fungus chamber soils and non-nest soils, respectively. FastUnifrac analyses also revealed that fungal community structures of soil from the garden chambers markedly differed among ant species. Our findings suggest that leaf-cutting ants affect fungal communities in the soil from the fungus chamber in comparison to non-nest soils. © 2013 WILEY-VCH Verlag GmbH & Co.

Lack of host specificity of Colletotrichum spp. isolates associated with anthracnose symptoms on mango in Brazil

The aim of the present study was to analyse the genetic and pathogenic variability of Colletotrichum spp. isolates from various organs and cultivars of mango with anthracnose symptoms, collected from different municipalities of São Paulo State, Brazil. Colletotrichum gloeosporioides isolates from symptomless citrus leaves and C. acutatum isolates from citrus flowers with post-bloom fruit drop symptoms were included as controls. Sequencing of the ITS region allowed the identification of 183 C. gloeosporioides isolates from mango; only one isolate was identified as C. acutatum. amova analysis of ITS sequences showed larger genetic variability among isolates from the same municipality than among those from different populations. fAFLP markers indicated high levels of genetic variability among the C. gloeosporioides isolates from mango and no correlation between genetic variability and isolate source. Only one C. gloeosporioides mango isolate had the same genotype as the C. gloeosporioides isolates from citrus leaves, as determined by ITS sequencing and fAFLP analysis. Pathogenicity tests revealed that C. gloeosporioides and C. acutatum isolates from either mango or citrus can cause anthracnose symptoms on leaves of mango cvs Palmer and Tommy Atkins and blossom blight symptoms in citrus flowers. These outcomes indicate a lack of host specificity of the Colletotrichum species and suggest the possibility of host migration. © 2012 British Society for Plant Pathology.

Objetos de aprendizagem para universitários sobre prevenção de acidentes de trânsito

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização epidemiológica da infecção pelo vírus respiratório sincicial bovino (BRSV) em rebanhos bovinos leiteiros do estado de São Paulo

Pós-graduação em Medicina Veterinária - FCAV

Bases moleculares da resistência ao mercúrio em bactérias Gram-negativas da Amazônia brasileira

Mercúrio é um dos elementos mais tóxicos tanto para seres humanos como os demais animais. Em ambientes naturais seus compostos podem ter origem em fontes naturais ou em decorrência da ação do homem. A atividade microbiana tem papel crítico na biorremediação. A resistência ao mercúrio nas bactérias está associada a um conjunto de genes organizados no operon mer. Considerando este cenário realizamos um estudo com bactérias Gram-negativas isoladas de sedimento de rios de duas áreas com distintos graus de atividade antropogênica, Barreiras e Caxiuanã. A resistência ao mercúrio foi avaliada em ensaios de crescimento in vitro da bactéria em meio contendo Hg. A presença do operon mer foi determinada por PCR para os genes RTPCABD, componentes do operon. O ensaio in vitro, determinou que apenas 2 isolados dos 107 avaliados, Acinetobacter baumannii de Caxiuanã e Pseudomonas stutzeri de Barreiras, apresentavam resistência ao Hg. Um operon mer foi identificado e caracterizado apenas do isolado Acinetobacter baumannii. Este apresenta os genes RTPCAD e sua organização e seqüência nucleotídica possui identidade total com o operon mer de um isolado de Acinetobacter calcoaceticus da Rússia.

Narrow Fungal Mycorrhizal Diversity in a Population of the Orchid Coppensia doniana

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Deep-sequencing identification of the genomic targets of the cytidine deaminase AID and its cofactor RPA in B lymphocytes

The cytidine deaminase AID hypermutates immunoglobulin genes but can also target oncogenes, leading to tumorigenesis. The extent of AID's promiscuity and its predilection for immunoglobulin genes are unknown. We report here that AID interacted broadly with promoter-proximal sequences associated with stalled polymerases and chromatin-activating marks. In contrast, genomic occupancy of replication protein A (RPA), an AID cofactor, was restricted to immunoglobulin genes. The recruitment of RPA to the immunoglobulin loci was facilitated by phosphorylation of AID at Ser38 and Thr140. We propose that stalled polymerases recruit AID, thereby resulting in low frequencies of hypermutation across the B cell genome. Efficient hypermutation and switch recombination required AID phosphorylation and correlated with recruitment of RPA. Our findings provide a rationale for the oncogenic role of AID in B cell malignancy.

PCR-Internal Transcribed Spacer (ITS) genes sequencing and phylogenetic analysis of clinical and environmental Aspergillus species associated with HIV-TB co infected patients in a hospital in Abeokuta, southwestern Nigeria.

Background: Aspergillosis has been identified as one of the hospital acquired infections but the contribution of water and inhouse air as possible sources of Aspergillus infection in immunocompromised individuals like HIV-TB patients have not been studied in any hospital setting in Nigeria. Objective: To identify and investigate genetic relationship between clinical and environmental Aspergillus species associated with HIV-TB co infected patients. Methods: DNA extraction, purification, amplification and sequencing of Internal Transcribed Spacer (ITS) genes were performed using standard protocols. Similarity search using BLAST on NCBI was used for species identification and MEGA 5.0 was used for phylogenetic analysis. Results: Analyses of sequenced ITS genes of selected fourteen (14) Aspergillus isolates identified in the GenBank database revealed Aspergillus niger (28.57%), Aspergillus tubingensis (7.14%), Aspergillus flavus (7.14%) and Aspergillus fumigatus (57.14%). Aspergillus in sputum of HIV patients were Aspergillus niger, A. fumigatus, A. tubingensis and A. flavus. Also, A. niger and A. fumigatus were identified from water and open-air. Phylogenetic analysis of sequences yielded genetic relatedness between clinical and environmental isolates. Conclusion: Water and air in health care settings in Nigeria are important sources of Aspergillus sp. for HIV-TB patients.