High-resolution melt and morphological analyses of mealybugs (Hemiptera: Pseudococcidae) from cacao: tools for the control of Cacao Swollen Shoot Virus spread

BACKGROUND: Mealybugs (Hemiptera: Coccoidea: Pseudococcidae) are key vectors of badnaviruses, including Cacao Swollen Shoot Virus (CSSV) the most damaging virus affecting cacao (Theobroma cacao L.). The effectiveness of mealybugs as virus vectors is species dependent and it is therefore vital that CSSV resistance breeding programmes in cacao incorporate accurate mealybug identification. In this work the efficacy of a CO1-based DNA barcoding approach to species identification was evaluated by screening a range of mealybugs collected from cacao in seven countries. RESULTS: Morphologically similar adult females were characterised by scanning electron microscopy and then, following DNA extraction, were screened with CO1 barcoding markers. A high degree of CO1 sequence homology was observed for all 11 individual haplotypes including those accessions from distinct geographical regions. This has allowed for the design of a High Resolution Melt (HRM) assay capable of rapid identification of the commonly encountered mealybug pests of cacao. CONCLUSIONS: HRM Analysis (HRMA) readily differentiated between mealybug pests of cacao that can not necessarily be identified by conventional morphological analysis. This new approach, therefore, has potential to facilitate breeding for resistance to CSSV and other mealybug transmitted diseases.

Comparing species and measures of landscape structure as indicators of conservation importance

P>1. The use of indicators to identify areas of conservation importance has been challenged on several grounds, but nonetheless retains appeal as no more parsimonious approach exists. Among the many variants, two indicator strategies stand out: the use of indicator species and the use of metrics of landscape structure. While the first has been thoroughly studied, the same cannot be said about the latter. We aimed to contrast the relative efficacy of species-based and landscape-based indicators by: (i) comparing their ability to reflect changes in community integrity at regional and landscape spatial scales, (ii) assessing their sensitivity to changes in data resolution, and (iii) quantifying the degree to which indicators that are generated in one landscape or at one spatial scale can be transferred to additional landscapes or scales. 2. We used data from more than 7000 bird captures in 65 sites from six 10 000-ha landscapes with different proportions of forest cover in the Atlantic Forest of Brazil. Indicator species and landscape-based indicators were tested in terms of how effective they were in reflecting changes in community integrity, defined as deviations in bird community composition from control areas. 3. At the regional scale, indicator species provided more robust depictions of community integrity than landscape-based indicators. At the landscape scale, however, landscape-based indicators performed more effectively, more consistently and were also more transferable among landscapes. The effectiveness of high resolution landscape-based indicators was reduced by just 12% when these were used to explain patterns of community integrity in independent data sets. By contrast, the effectiveness of species-based indicators was reduced by 33%. 4. Synthesis and applications. The use of indicator species proved to be effective; however their results were variable and sensitive to changes in scale and resolution, and their application requires extensive and time-consuming field work. Landscape-based indicators were not only effective but were also much less context-dependent. The use of landscape-based indicators may allow the rapid identification of priority areas for conservation and restoration, and indicate which restoration strategies should be pursued, using remotely sensed imagery. We suggest that landscape-based indicators might often be a better, simpler, and cheaper strategy for informing decisions in conservation.

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies.

Molecular characterization and fumonisin production by Fusarium verticillioides isolated from corn grains of different geographic origins in Brazil

Gibberella moniliformis is most commonly associated with maize worldwide and produces high levels of fumonisins, some of the most agriculturally important mycotoxins. Studies demonstrate that molecular methods can be helpful for a rapid identification of Fusarium species and their levels of toxin production. The purpose of this research was to apply molecular methods (AFLP, TEF-1 alpha partial gene sequencing and PCR based on MAT alleles) for the identification of Fusarium species isolated from Brazilian corn and to verify if real time RT-PCR technique based on FUM1 and FUM19 genes is appropriated to estimate fumonisins B(1) and B(2) production levels. Among the isolated strains, 96 were identified as Fusarium verricillioides, and four as other Fusarium species. Concordant phylogenies were obtained by AFLP and TEF-1 alpha sequencing, permitting the classification of the different species into distinct clades. Concerning MAT alleles, 70% of the F. verricillioides isolates carried the MAT-1 and 30% MAT-2. A significant correlation was observed between the expression of the genes and toxin production r=0.95 and r=0.79 (correlation of FUM1 with FB(1) and FB(2), respectively, P < 0.0001): r=0.93 and r =0.78 (correlation of FUM19 with FB(1) and FB(2). respectively, P < 0.0001). Molecular methods used in this study were found to be useful for the rapid identification of Fusarium species. The high and significant correlation between FUM1 and FUM19 expression and fumonisins production suggests that real time RT-PCR is suitable for studies considering the influence of abiotic and biotic factors on expression of these genes. This is the first report concerning the expression of fumonisin biosynthetic genes in Fusarium strains isolated from Brazilian agricultural commodity. (c) 2010 Elsevier B.V. All rights reserved.

Caracterização de rizóbios indicados para produção de inoculantes por meio de sequenciamento parcial do 16S rRNA

O objetivo deste trabalho foi confrontar as sequências parciais do gene 16S rRNA de estirpes padrão de rizóbios com as de estirpes recomendadas para a produção de inoculantes no Brasil, com vistas à verificação da confiabilidade do sequenciamento parcial desse gene para a identificação rápida de estirpes. Foram realizados sequenciamentos através de reação em cadeia da polimerase (PCR) com iniciadores relativos à região codificadora do gene 16S rRNA entre as bactérias estudadas. Os resultados foram analisados pela consulta de similaridade de nucleotídeos aos do Basic Local Alignment Search Tool (Blastn) e por meio da interpretação de árvores filogenéticas geradas usando ferramentas de bioinformática. A classificação taxonômica das estirpes Semia recomendadas para inoculação de leguminosas com base em propriedades morfológicas e especificidade hospedeira não foi confirmada em todas as estirpes. A maioria das estirpes estudadas, consultadas no Blastn, é consistente com a classificação proposta pela construção de árvores filogenéticas das sequências destas estirpes, com base na similaridade pelo sequenciamento parcial do gene considerado.

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.

HPLC-ESIMSn Profiling, Isolation, Structural Elucidation, and Evaluation of the Antioxidant Potential of Phenolics from Paepalanthus geniculatus

The methanol extract of the flowers of Paepalanthus geniculatus Kunth. showed radical-scavenging activity in the TEAC assay. An analytical approach based on HPLC:ESIMSn was applied to obtain the metabolite profile of this extract and led to the rapid identification of 19 polyphenolic compounds comprising flavonoids and naphthopyranones. The new naphthopyranone (10, 16), quercetagetin (1, 5, 7, 13), and galetine derivatives (9, 11, 17, 19), and a flavonol glucoside cyclodimer in the truxillate form (12), were identified. Compounds 2, 6, and 7 showed the highest antioxidant capacity and ability to affect the levels of intracellular ROS in human prostate cancer cells (PC3).

Molecular fingerprinting methods for the discrimination between C-albicans and C-dubliniensis

Opportunistic fungal pathogens are becoming increasingly important causes of both community-acquired and nosocomial infections. The most important fungal pathogens are yeast species belonging to the genus Candida. These species show differences in levels of resistance to antifungal agents and mortality. Consequently, it is important to correctly identify the causative organism to the species level. Identification of Candida dubliniensis in particular remains problematic because of the high degree of phenotypic similarity between this species and Candida albicans. However, as the differences between both are most pronounced at the genetic level, several studies have been conducted in order to provide a specific and rapid identification fingerprinting molecular test. In most candidal infectious, no single DNA fingerprinting technique has evolved as a dominant method, and each method has its advantages, disadvantages and limitations. Moreover, the current challenge of these techniques is to compile standardized patterns in a database for interlaboratory use and future reference. This review provides an overview of most common molecular fingerprinting techniques currently available for discrimination of C. albicans and C. dubliniensis.

Characterization of flavonoids and naphthopyranones in methanol extracts of Paepalanthus chiquitensis herzog by HPLC-ESI-IT-MSn and their mutagenic activity

A HPLC-ESI-IT-MSn method, based on high-performance liquid chromatography coupled to electrospray negative ionization multistage ion trap mass spectrometry, was developed for rapid identification of 24 flavonoid and naphthopyranone compounds. The methanol extracts of the capitulae and scapes of P. chiquitensis exhibited mutagenic activity in the Salmonella/microsome assay, against strain TA97a. © 2013 by the authors.

Characterization of flavonoids and phenolic acids in Myrcia bella cambess. Using FIA-ESI-IT-MSn and HPLC-PAD-ESI-IT-MS combined with NMR

The leaves of Myrcia DC. ex Guill species are used in traditional medicine and are also exploited commercially as herbal drugs for the treatment of diabetes mellitus. The present work aimed to assess the qualitative and quantitative profiles of M. bella hydroalcoholic extract, due to these uses, since the existing legislation in Brazil determines that a standard method must be developed in order to be used for quality control of raw plant materials. The current study identified eleven known flavonoid-O-glycosides and six acylated flavonoid derivatives of myricetin and quercetin, together with two kaempferol glycosides and phenolic acids such as caffeic acid, ethil galate, gallic acid and quinic acid. In total, 24 constituents were characterized, by means of extensive preparative chromatographic analyses, along with MS and NMR techniques. An HPLC-PAD-ESI-ITMS and FIA-ESI-IT-MSn method were developed for rapid identification of acylated flavonoids, flavonoid-O- glycosides derivatives of myricetin and quercetin and phenolic acids in the hydroalcoholic M. bella leaves extract. The FIA-ESI-IT-MS techinique is a powerful tool for direct and rapid identification of the constituents after isolation and NMR characterization. Thus, it could be used as an initial method for identification of authentic samples concerning quality control of Myrcia spp extracts. © 2013 by the authors.

Microespectroscopia infravermelha por transformada de fourier: identificação e discriminação de cepas clínicas de Candida albicans e Candida glabrata

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Gonadal development in the freshwater crab Sylviocarcinus pictus (H. Milne Edwards, 1853) (Brachyura: Trichodactylidae) from the Guamá river, state of Pará, Brazil

Os estádios de desenvolvimento das gônadas de machos e fêmeas de caranguejos Sylviocarcinus pictus (H. Milne Eduards, 1853) foram descritos por meio de observações macroscópicas e microscópicas (técnica histológica). A descrição histológica foi baseada em 40 espécimes (20 de cada sexo). Foram identificados quatro estádios de desenvolvimento para as fêmeas: imaturo, em maturação, maturo e em reabsorção. As seguintes células foram encontradas: ovogônias, ovócitos em vitelogênese inicial, ovócitos em vitelogênese avançada, células foliculares e folículos pós-ovulatórios. Três estádios de desenvolvimento foram encontrados para os machos: imaturo, em maturação e maturo, com indicação de: espermatogônias, espermatócitos, espermátides, espermatozóides e espermatóforos. Tais dados sugerem o padrão descrito na literatura. O tamanho da maturidade sexual foi de 32,3 mm de largura da carapaça para machos e 31,5 mm para fêmeas. Os estádios gonadais observados macroscopicamente por meio do volume e da coloração das gônadas foram validados pela análise histológica, sendo um critério útil e ágil para a identificação da maturidade sexual para a espécie. O presente estudo oferece informações inéditas sobre a biologia reprodutiva de Sylviocarcinus pictus.

Compostos fenólicos de Paepalanthus geniculatus (Eriocaulaceae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Isolation and characterisation of aerobic endospore forming Bacilli from sugarcane rhizosphere for the selection of strains with agriculture potentialities

Eighteen aerobic endospore forming strains were isolated from sugarcane rhizosphere in N-free medium. A phenotypic description and analysis of the 5' end hypervariable region sequences of 16S rRNA revealed a high diversity of Bacillus and related genera. Isolates were identified, and four genera were obtained: seven strains belonged to Bacillus (Bacillaceae family), four belonged to Paenibacillus, six belonged to Brevibacillus and one strain was identified as Cohnella (Paenibacillaceae family). Four Brevibacillus strains showed in vitro inhibitory activity against plant pathogens fungi Curvularia and Fusarium. Seventy-four percent of the isolated bacteria grew on pectin as the only carbon source, showing polygalacturonase activity. Pectate lyase activity was detected for the first time in a Brevibacillus genus strain. All isolates showed endoglucanase activity. Calcium phosphate solubilisation was positive in 83.3% of the isolates, with higher values than those reported for Bacillus inorganic phosphate solubilising strains. High ethylene plant hormone secretion in the culture medium was detected in 22% of the bacteria. This is the first report of ethylene secretion in Paenibacillaceae isolates. Indole-3-acetic acid production was found in a Brevibacillus genus isolate. It was reported for the first time the presence of Cohnella genus strain on sugarcane rhizosphere bearing plant growth promoting traits. The sugarcane isolate Brevibacillus B65 was identified as a plant growth inoculant because it showed wider spectra of plant stimulation capabilities, including an antifungal effect, extracellular hydrolases secretion, inorganic phosphate solubilisation and plant hormone liberation. In this work, sugarcane was shown to be a suitable niche for finding aerobic endospore forming 'Bacilli' with agriculture biotechnological purposes.

Advances in methods to detect, isolate and quantify foodborne pathogens

Foodborne diseases impact human health and economies worldwide in terms of health care and productivity loss. Prevention is necessary and methods to detect, isolate and quantify foodborne pathogens play a fundamental role, changing continuously to face microorganisms and food production evolution. Official methods are mainly based on microorganisms growth in different media and their isolation on selective agars followed by confirmation of presumptive colonies through biochemical and serological test. A complete identification requires form 7 to 10 days. Over the last decades, new molecular techniques based on antibodies and nucleic acids allow a more accurate typing and a faster detection and quantification. The present thesis aims to apply molecular techniques to improve official methods performances regarding two pathogens: Shiga-like Toxin-producing Escherichia coli (STEC) and Listeria monocytogenes. In 2011, a new strain of STEC belonging to the serogroup O104 provoked a large outbreak. Therefore, the development of a method to detect and isolate STEC O104 is demanded. The first objective of this work is the detection, isolation and identification of STEC O104 in sprouts artificially contaminated. Multiplex PCR assays and antibodies anti-O104 incorporated in reagents for immunomagnetic separation and latex agglutination were employed. Contamination levels of less than 1 CFU/g were detected. Multiplex PCR assays permitted a rapid screening of enriched food samples and identification of isolated colonies. Immunomagnetic separation and latex agglutination allowed a high sensitivity and rapid identification of O104 antigen, respectively. The development of a rapid method to detect and quantify Listeria monocytogenes, a high-risk pathogen, is the second objective. Detection of 1 CFU/ml and quantification of 10–1,000 CFU/ml in raw milk were achieved by a sample pretreatment step and quantitative PCR in about 3h. L. monocytogenes growth in raw milk was also evaluated.