A search for markers of sugarcane evolution

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

Genetic diversity and antifungal susceptibility testing of Trichosporon asahii isolated of Intensive Care Units patients

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evidence that the Ceratobasidium-like white-thread blight and black rot fungal pathogens from persimmon and tea crops in the Brazilian Atlantic Forest agroecosystem are two distinct phylospecies

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

Cryptic hammerhead shark lineage occurrence in the western South Atlantic revealed by DNA analysis

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

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Intraspecific variation and emendation of Hannaella kunmingensis

In order to investigate the intraspecific variability in Hannaella kunmingensis, 11 isolates, including the type strain, were analyzed for their morphological and biochemical traits. The combined internal transcribed spacer region (ITS), D1/D2 domains of the large subunit rDNA (LSU), and cytochrome b gene were examined using phylogenetic and parsimony network analyses. Our investigations revealed differences in colony morphology as well as differences in 31 out of 64 phenotypic characteristics examined, including growth in lactose, vitamin free medium, xylitol, L-arabinitol, and nitrite. Growth in the presence of 0. 1 % cycloheximide was also highlighted in H. kunmingensis. All the 11 strains were conspecific in the LSU; however, variations of about 2. 5 % were found in the ITS while isolate CBS 8356 exhibited a 27. 3 % divergence from the other strains in the cytochrome b gene. Parsimony network analysis revealed the existence of three haplotypes among the H. kunmingensis strains studied but excluded CBS 8356 from the network connecting these haplotypes. This study contributes to the knowledge of the intraspecific diversity of H. kunmingensis. To accommodate such intraspecific variations, an emendation of the species diagnosis is proposed. © 2012 German Mycological Society and Springer.

Diversity of Mycosphaerella spp. and Teratosphaeria spp. in Eucalyptus globulus plantations in Brazil

Among the most important diseases affecting Eucalyptus is Mycosphaerella Leaf Disease (MLD) caused by Mycosphaerella spp. and Teratosphaeria spp. MLD has led to significant losses in eucalypt plantations in the South and Southeast Region of Brazil, as well as in several countries such as Portugal, Spain, South Africa and Australia. Symptoms of MLD include localized necrotic spots, early defoliation in juvenile plants, stem cankers, early death of branches, and in some cases, atrophy and death. In the present study, single spore isolations from leaves of E. globulus from five locations in Brazil allowed the differentiation of species of Mycosphaerella and Teratosphaeria based on ascospore germination and growth in culture. These isolates were also subjected to sequence analysis of the ribosomal RNA internal transcribed spacer regions, which allowed their identification to species level. The results of this study showed that six species of Mycosphaerella and four species of Teratosphaeria were associated with leaves showing symptoms of MLD in E. globulus plantations in various locations of Brazil. © 2013 KNPV.

Caracterização molecular de isolados de Paracoccidioides brasiliensis obtidos de Dasypus novemcinctus (tatu) e sua correlação com virulência e origem geográfica

Pós-graduação em Patologia - FMB

Análises isoenzimáticas, moleculares e ultraestruturais em camarões dulcícolas do gênero Macrobrachium

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

New light on the systematics of fungi associated with attine ant gardens and the description of escovopsis kreiselii sp nov

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

Detecção do Paracoccidioides spp. em amostras ambientais e diferenciação do complexo P. brasiliensis da espécie P. lutzii por Nested PCR e hibridização in situ

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

3-Hydroxypropionic Acid as an Antibacterial Agent from Endophytic Fungi Diaporthe phaseolorum

Endophytic fungi are considered a rich source of active compounds resulting from their secondary metabolism. Fungi from marine environment grow in a habitat with unique conditions that can contribute to the activation of metabolic pathways of synthesis of different unknown molecules. The production of these compounds may support the adaptation and survival of the fungi in the marine ecosystem. Mangroves are ecosystems situated between land and sea. They are frequently found in tropical and subtropical areas and enclose approximately 18.1 million hectares of the planet. The great biodiversity found in these ecosystems shows the importance of researching them, including studies regarding new compounds derived from the endophytic fungi that inhabit these ecosystems. 3-hydroxypropionic acid (3-HPA) has been isolated from the mangrove endophytic fungus Diaporthe phaseolorum, which was obtained from branches of Laguncularia racemosa. The structure of this compound was elucidated by spectroscopic methods, mainly 1D and 2D NMR. In bioassays, 3-HPA showed antimicrobial activities against both Staphylococcus aureus and Salmonella typhi. The structure of this antibiotic was modified by the chemical reaction of Fischer-Speier esterification to evaluate the biologic activity of its chemical analog. The esterified product, 3-hydroxypropanoic ethyl ester, did not exhibit antibiotic activity, suggesting that the free carboxylic acid group is important to the pharmacological activity. The antibiotic-producing strain was identified with internal transcribed spacer sequence data. To the best of our knowledge, this is the first report of antibacterial activity by 3-HPA against the growth of medically important pathogens.

Systematics of the Oswaldoi Complex (Anopheles, Nyssorhynchus) in South America

Abstract Background Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex. Methods DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies. Results Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate. Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Species identification, distributioin and reproductive interactions of common smoot-hound and blackspotted smooth-hound shark (Genus mustelus) in the Adriatic Sea

Longstanding taxonomic ambiguity and uncertainty exist in the identification of the common (M. mustelus) and blackspotted (M. punctulatus) smooth-hound in the Adriatic Sea. The lack of a clear and accurate method of morphological identification, leading to frequent misidentification, prevents the collation of species-specific landings and survey data for these fishes and hampers the delineation of the distribution ranges and stock boundaries of the species. In this context, adequate species-specific conservation and management strategies can not be applied without risks of population declining and local extinction. In this thesis work I investigated the molecular ecology of the two smooth-hound sharks which are abundant in the demersal trawl surveys carried out in the NC Adriatic Sea to monitor and assess the fishery resources. Ecological and evolutionary relationships were assessed by two molecular tests: a DNA barcoding analysis to improve species identification (and consequently the knowledge of their spatial ecology and taxonomy) and a hybridization assay based on the nuclear codominant marker ITS2 to evaluate reproductive interactions (hybridization or gene introgression). The smooth-hound sharks (N=208) were collected during the MEDITS 2008 and 2010 campaigns along the Italian and Croatian coasts of the Adriatic Sea, in the Sicilian Channel and in the Algerian fisheries. Since the identification based on morphological characters is not strongly reliable, I performed a molecular identification of the specimens producing for each one the cytochrome oxidase subunit 1 (COI) gene sequence (ca. 640 bp long) and compared them with reference sequences from different databases (GenBank and BOLD). From these molecular ID data I inferred the distribution of the two target species in the NC Adriatic Sea. In almost the totality of the MEDITS hauls I found no evidence of species sympatry. The data collected during the MEDITS survey showed an almost different distribution of M. mustelus (confined along the Italian coasts) and M. punctulatus (confined along the Croatian coasts); just one sample (Gulf of Venice, where probably the ranges of the species overlap) was found to have catches of both the species. Despite these data results suggested no interaction occurred between my two target species at least during the summertime (the period in which MEDITS survey is carried out), I still wanted to know if there were inter-species reproductive interactions so I developed a simple molecular genetic method to detect hybridization. This method is based on DNA sequence polymorphism among species in the nuclear ribosomal Internal Transcribed Spacer 2 locus (ITS2). Its application to the 208 specimens collected raised important questions regarding the ecology of this two species in the Adriatic Sea. In fact results showed signs of hybridization and/or gene introgression in two sharks collected during the trawl survey of 2008 and one collected during the 2010 one along the Italian and Croatian coasts. In the case that it will be confirmed the hybrid nature of these individuals, a spatiotemporal overlapping of the mating behaviour and ecology must occur. At the spatial level, the northern part of the Adriatic Sea (an area where the two species occur with high frequency of immature individuals) could likely play the role of a common nursery area for both species.

Systematics, phylogeny and biogeography of Juncaginaceae

I investigated the systematics, phylogeny and biogeographical history of Juncaginaceae, a small family of the early-diverging monocot order Alismatales which comprises about 30 species of annual and perennial herbs. A wide range of methods from classical taxonomy to molecular systematic and biogeographic approaches was used. rnrnIn Chapter 1, a phylogenetic analysis of the family and members of Alismatales was conducted to clarify the circumscription of Juncaginaceae and intrafamilial relationships. For the first time, all accepted genera and those associated with the family in the past were analysed together. Phylogenetic analysis of three molecular markers (rbcL, matK, and atpA) showed that Juncaginaceae are not monophyletic. As a consequence the family is re-circumscribed to exclude Maundia which is pro-posed to belong to a separate family Maundiaceae, reducing Juncaginaceae to include Tetroncium, Cycnogeton and Triglochin. Tetroncium is weakly supported as sister to the rest of the family. The reinstated Cycnogeton (formerly included in Triglochin) is highly supported as sister to Triglochin s.str. Lilaea is nested within Triglochin s. str. and highly supported as sister to the T. bulbosa complex. The results of the molecular analysis are discussed in combination with morphological characters, a key to the genera of the family is given, and several new combinations are made.rnrnIn Chapter 2, phylogenetic relationships in Triglochin were investigated. A species-level phylogeny was constructed based on molecular data obtained from nuclear (ITS, internal transcribed spacer) and chloroplast sequence data (psbA-trnH, matK). Based on the phylogeny of the group, divergence times were estimated and ancestral distribution areas reconstructed. The monophyly of Triglochin is confirmed and relationships between the major lineages of the genus were resolved. A clade comprising the Mediterranean/African T. bulbosa complex and the American T. scilloides (= Lilaea s.) is sister to the rest of the genus which contains two main clades. In the first, the widespread T. striata is sister to a clade comprising annual Triglochin species from Australia. The second clade comprises T. palustris as sister to the T. maritima complex, of which the latter is further divided into a Eurasian and an American subclade. Diversification in Triglochin began in the Miocene or Oligocene, and most disjunctions in Triglochin were dated to the Miocene. Taxonomic diversity in some clades is strongly linked to habitat shifts and can not be observed in old but ecologically invariable lineages such as the non-monophyletic T. maritima.rnrnChapter 3 is a collaborative revision of the Triglochin bulbosa complex, a monophyletic group from the Mediterranean region and Africa. One new species, Triglochin buchenaui, and two new subspecies, T. bulbosa subsp. calcicola and subsp. quarcicola, from South Africa were described. Furthermore, two taxa were elevated to species rank and two reinstated. Altogether, seven species and four subspecies are recognised. An identification key, detailed descriptions and accounts of the ecology and distribution of the taxa are provided. An IUCN conservation status is proposed for each taxon.rnrnChapter 4 deals with the monotypic Tetroncium from southern South America. Tetroncium magellanicum is the only dioecious species in the family. The taxonomic history of the species is described, type material is traced, and a lectotype for the name is designated. Based on an extensive study of herbarium specimens and literature, a detailed description of the species and notes on its ecology and conservation status are provided. A detailed map showing the known distribution area of T. magellanicum is presented. rnrnIn Chapter 5, the flower structure of the rare Australian endemic Maundia triglochinoides (Maundiaceae, see Chapter 1) was studied in a collaborative project. As the morphology of Maundia is poorly known and some characters were described differently in the literature, inflorescences, flowers and fruits were studied using serial mictrotome sections and scanning electron microscopy. The phylogenetic placement, affinities to other taxa, and the evolution of certain characters are discussed. As Maundia exhibits a mosaic of characters of other families of tepaloid core Alismatales, its segregation as a separate family seems plausible.