Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.

Application of molecular techniques to evaluate the methanogenic archaea and anaerobic bacteria in the presence of oxygen with different COD : sulfate ratios in a UASB reactor

In this paper, the microbial characteristics of the granular sludge in the presence of oxygen (3.0 +/- 0.7 mg O-2 1(-1)) were analyzed using molecular biology techniques. The granules were provided by an upflow anaerobic sludge blanket (UASB) operated over 469 days and fed with synthetic substrate. Ethanol and sulfate were added to obtain different COD/SO42- ratios (3.0, 2.0, and 1.6). The results of fluorescent in situ hybridization (FISH) analyses showed that archaeal cells, detected by the ARC915 probe, accounted for 77%, 84%, and 75% in the COD/SO42- ratios (3.0, 2.0, and 1.6, respectively). Methanosaeta sp. was the predominant acetoclastic archaea observed by optical microscopy and FISH analyses, and confirmed by sequencing of the excised bands of the DGGE gel with a similarity of 96%. The sulfate-reducing bacterium Desulfovibrio vulgaris subsp. vulgaris (similarity of 99%) was verified by sequencing of the DGGE band. Others identified microorganism were similar to Shewanella sp. and Desulfitobacterium hafniense, with similarities of 95% and 99%, respectively. These results confirmed that the presence of oxygen did not severely affect the metabolism of microorganisms that are commonly considered strictly anaerobic. We obtained mean efficiencies of organic matter conversion and sulfate reducing higher than 74%. (C) 2008 Elsevier Ltd. All rights reserved.

DESIGN OF CONCRETE PLATFORMS OFFSHORE USING THE THREE-LAYER SHELL THEORY

The concrete offshore platforms, which are subjected a several loading combinations and, thus, requires an analysis more generic possible, can be designed using the concepts adopted to shell elements, but the resistance must be verify in particular cross-sections to shear forces. This work about design of shell elements will be make using the three-layer shell theory. The elements are subject to combined loading of membrane and plate, totalizing eight components of internal forces, which are three membrane forces, three moments (two out-of-plane bending moments and one in-plane, or torsion, moment) and two shear forces. The design method adopted, utilizing the iterative process proposed by Lourenco & Figueiras (1993) obtained from equations of equilibrium developed by Gupta (1896) , will be compared to results of experimentally tested shell elements found in the literature using the program DIANA.

Role of price and enforcement in water allocation: Insights from Game Theory

As many countries are moving toward water sector reforms, practical issues of how water management institutions can better effect allocation, regulation, and enforcement of water rights have emerged. The problem of nonavailability of water to tailenders on an irrigation system in developing countries, due to unlicensed upstream diversions is well documented. The reliability of access or equivalently the uncertainty associated with water availability at their diversion point becomes a parameter that is likely to influence the application by users for water licenses, as well as their willingness to pay for licensed use. The ability of a water agency to reduce this uncertainty through effective water rights enforcement is related to the fiscal ability of the agency to monitor and enforce licensed use. In this paper, this interplay across the users and the agency is explored, considering the hydraulic structure or sequence of water use and parameters that define the users and the agency`s economics. The potential for free rider behavior by the users, as well as their proposals for licensed use are derived conditional on this setting. The analyses presented are developed in the framework of the theory of ""Law and Economics,`` with user interactions modeled as a game theoretic enterprise. The state of Ceara, Brazil, is used loosely as an example setting, with parameter values for the experiments indexed to be approximately those relevant for current decisions. The potential for using the ideas in participatory decision making is discussed. This paper is an initial attempt to develop a conceptual framework for analyzing such situations but with a focus on the reservoir-canal system water rights enforcement.

Galerkin finite element method for incompressible thermofluid flows framed within the bond graph theory

In this paper a bond graph methodology is used to model incompressible fluid flows with viscous and thermal effects. The distinctive characteristic of these flows is the role of pressure, which does not behave as a state variable but as a function that must act in such a way that the resulting velocity field has divergence zero. Velocity and entropy per unit volume are used as independent variables for a single-phase, single-component flow. Time-dependent nodal values and interpolation functions are introduced to represent the flow field, from which nodal vectors of velocity and entropy are defined as state variables. The system for momentum and continuity equations is coincident with the one obtained by using the Galerkin method for the weak formulation of the problem in finite elements. The integral incompressibility constraint is derived based on the integral conservation of mechanical energy. The weak formulation for thermal energy equation is modeled with true bond graph elements in terms of nodal vectors of temperature and entropy rates, resulting a Petrov-Galerkin method. The resulting bond graph shows the coupling between mechanical and thermal energy domains through the viscous dissipation term. All kind of boundary conditions are handled consistently and can be represented as generalized effort or flow sources. A procedure for causality assignment is derived for the resulting graph, satisfying the Second principle of Thermodynamics. (C) 2007 Elsevier B.V. All rights reserved.

Fast Transforms for Acoustic Imaging-Part I: Theory

The classical approach for acoustic imaging consists of beamforming, and produces the source distribution of interest convolved with the array point spread function. This convolution smears the image of interest, significantly reducing its effective resolution. Deconvolution methods have been proposed to enhance acoustic images and have produced significant improvements. Other proposals involve covariance fitting techniques, which avoid deconvolution altogether. However, in their traditional presentation, these enhanced reconstruction methods have very high computational costs, mostly because they have no means of efficiently transforming back and forth between a hypothetical image and the measured data. In this paper, we propose the Kronecker Array Transform ( KAT), a fast separable transform for array imaging applications. Under the assumption of a separable array, it enables the acceleration of imaging techniques by several orders of magnitude with respect to the fastest previously available methods, and enables the use of state-of-the-art regularized least-squares solvers. Using the KAT, one can reconstruct images with higher resolutions than was previously possible and use more accurate reconstruction techniques, opening new and exciting possibilities for acoustic imaging.

Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants

We progressively reduced the complexity of humic matter by a mild sequential removal of unbound or free components, weakly, and strongly bound molecules. The auxin-like response of residues from each step was tested using tomato (cv. Micro-Tom) seedlings expressing DR5 auxin synthetic promoter fused to the beta-glucuronidase (GUS) reporter gene and the low auxin-sensitivity diageotropica (dgt) mutant. Both exogenous auxin and humic matter promoted lateral root emergence in the control, but failed to induce lateral roots in the dgt mutant. When strongly bound components were removed from humic matter by breaking the ester and ether bonds, the humic residues lost their ability to induce the DR5

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis x Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Molecular detection and differentiation of Brazilian and African isolates of the entomopathogen Neozygites tanajoae (Entomophthorales: Neozygitaceae) with PCR using specific primers

Neozygites tanajoae is an entomopathogenic fungus which has been used for biocontrol of the cassava green mite (Mononychellus tanajoa, CGM) in Africa. Establishment and dispersal of Brazilian isolates which have been introduced into some African countries in recent years to improve CGM control was followed with specific PCR assays. Two primer pairs, NEOSSU_F/NEOSSU_R and 8DDC_F/8DDC_R, were used to differentiate isolates collected from several locations in Brazil and from three countries in Africa, Benin, Ghana and Tanzania. The first primer pair enabled the species-specific detection of Neozygites tanajoae, while the second differentiated the Brazilian isolates from those of other geographical origin. PCR assays were designed for detection of fungal DNA in the matrix of dead infested mites since N. tanajoae is difficult to isolate and culture on selective artificial media. Our results show that all isolates (Brazilian and African) that sporulated on mummified mites were amplified with the first primer pair confirming their Neozygites tanajoae identity. The second pair amplified DNA from all the Brazilian isolates, but did not amplify any DNA samples from the African isolates. None of the two primers showed amplification neither from any of the non-sporulating mite extracts nor from the dead uninfected mites used as negative controls. We confirmed that the two primer pairs tested are suitable for the detection and differential identification of N. tanajoae isolates from Brazil and Africa and that they are useful to monitor the establishment and spread of the Brazilian isolates of N. tanajoae introduced into Benin or into other African countries for improvement of CGM biocontrol.

Molecular Identification and Phylogenetic Analysis of a Group 16SrI-B Phytoplasma Associated with Sugarcane Yellow Leaf Syndrome in Brazil

Yellow leaf syndrome was a serious problem in the beginning of the 1990s in Brazil, when yield losses were estimated to be around 50%. The disease is currently endemic, but it is considered potentially important. Previous studies have revealed only the presence of a luteovirus associated with the disease in Brazil. We report that a phytoplasma of 16SrI-B is also associated with this disease. This is the first demonstration of the presence of a group 16SrI-B phytoplasma in association with sugarcane yellow leaf in Brazil.

Molecular and morphological markers for rapid distinction between 2 Colletotrichum species

Endophytic microorganisms reside asymptomatically within plants and are a source of new bioactive products for use in medicine, agriculture, and industry. Colletotrichum (teleomorph Glomerella) is a fungus widely cited in the literature as a producer of antimicrobial substances. Identification at the species level, however, has been a problem in this type of study. Several authors have reported the presence of endophytic fungi from the medicinal plant Maytenus ilicifolia (espinheira-santa) in Brazil that has antimicrobial activity against various pathogens. Therefore, Colletotrichum strains were isolated from M. ilicifolia and identified based on morphology, RAPD markers, sequence data of the internal transcribed spacer regions (ITS-1 and ITS-2), the 5.8S gene, and species-specific PCR. The analyses suggested the presence of 2 species, Colletotrichum gloeosporioides and Colletotrichum boninense. Two morphological markers were characterized to allow C. gloeosporioides and C. boninense to be distinguished quickly and accurately. The molecular diagnosis of C. boninense was confirmed by using Coll and ITS4 primers. This species of Colletotrichum is reported for the first time in M. ilicifolia.

Molecular evidence of phytoplasma associated with witches` broom on passion fruit in five Brazilian States

Phytoplasmas are cell wall-less prokaryotes and phloem-inhabitants associated with diseases that affect several crops. Passion fruit is a tropical plant species cultivated in various Brazilian regions. Diseases are among the factors that may cause damage to this crop, and witches` broom is a very important one. This disease, associated with a phytoplasma, has been reported only in Brazil, where it was described in Rio de Janeiro and Pernambuco States at the beginning of the 1980`s. In 2005-2006, symptomatic plants Suspected of phytoplasma infection were sampled in areas located in Sao Paulo, Parana, Rio de Janeiro, Sergipe and Bahia. Amplification of DNA fragments of 1.2kb from nested PCR primed by the pairs 16mF2/mR1 and 16F2n/R2 revealed the presence of phytoplasma in the tissue of plants from all sampled regions. Molecular detection of the agent allowed confirmation of the diagnosis based on the symptomatology; demonstrated the strong association between diseased plants and a phytoplasma, confirming previous investigations based on electron microscopy;, and revealed the present occurrence of the agent in the States of Bahia, Parana, Rio de Janeiro, Sergipe, and Sao Paulo.

Localization of Pantoea ananatis inside lesions of maize White Spot Disease using transmission electron microscopy and molecular techniques

The etiological agent of maize white spot (MWS) disease has been a subject of controversy and discussion. Initially the disease was described as Phaeosphaeria leaf spot caused by Phaeosphaeria maydis. Other authors have Suggested the existence of different fungal species causing similar symptoms. Recently, a bacterium, Pantoea ananatis, was described as the causal agent of this disease. The purpose of this Study was to offer additional information on the correct etiology of this disease by providing visual evidence of the presence of the bacterium in the interior of the MWS lesions by using transmission electron microscopy (TEM) and molecular techniques. The TEM allowed Visualization of a large amount of bacteria in the intercellular spaces of lesions collected from both artificially and naturally infected plants. Fungal structures were not visualized in young lesions. Bacterial primers for the 16S rRNA and rpoB genes were used in PCR reactions to amplify DNA extracted from water-soaked (young) and necrotic lesions. The universal fungal oligonucleotide ITS4 was also included to identity the possible presence of fungal structures inside lesions. Positive PCR products from water-soaked lesions, both from naturally and artificially inoculated plants, were produced with bacterial primers, whereas no amplification was observed when ITS4 oligonucleotide was used. On the other hand, DNA amplification with ITS4 primer was observed when DNA was isolated from necrotic (old) lesions. These results reinforced previous report of P. ananatis as the primary pathogen and the hypothesis that fungal species may colonize lesions pre-established by P. ananatis.

Histological, physiological and molecular investigations of Fagus sylvatica seedlings infected with Phytophthora citricola

P>The aim of the work was to shed light into histological, physiological and molecular changes of Fagus sylvatica seedlings infected with the root pathogen Phytophthora citricola with the final goal to distinguish between local and systemic responses. Real-time quantitative PCR analysis proved that P. citricola was able to grow from infected roots into hypocotyl and epicotyl tissue of F. sylvatica seedlings. Light microscopy showed many collapsed parenchyma cells of the cortex without being penetrated by the pathogen. Hyphae were mainly growing intracellular in parenchyma and xylem tissue. Transmission electron microscopy displayed disintegration of xylem vessels and of parenchyma cells. Inhibition of water uptake of infected beech seedlings was positively correlated with the concentration of zoospores used in the experiment. In addition, a split root experiment indicated that invertases were possibly involved locally and systemically in the conversion of sucrose of P. citricola infected roots. During the growth of the pathogen in roots, a transient expression of the 1-aminocyclopropane-1-carboxylic acid (ACC)-oxidase gene was quantified in leaves which was detected in parallel with the first peak of a biphasic ethylene outburst. Additionally a systemic upregulation of aquaporin transcripts was mainly detected in leaves of beech seedlings infected with P. citricola.

Cloning, expression, molecular modelling and docking analysis of glutathione transferase from Saccharum officinarum

Sugarcane yield and quality are affected by a number of biotic and abiotic stresses. In response to such stresses, plants may increase the activities of some enzymes such as glutathione transferase (GST), which are involved in the detoxification of xenobiotics. Thus, a sugarcane GST was modelled and molecular docked using the program LIGIN to investigate the contributions of the active site residues towards the binding of reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). As a result, W13 and I119 were identified as key residues for the specificity of sugarcane GSTF1 (SoGSTF1) towards CDNB. To obtain a better understanding of the catalytic specificity of sugarcane GST (SoGSTF1), two mutants were designed, W13L and I119F. Tertiary structure models and the same docking procedure were performed to explain the interactions between sugarcane GSTs with GSH and CDNB. An electron-sharing network for GSH interaction was also proposed. The SoGSTF1 and the mutated gene constructions were cloned and expressed in Escherichia coli and the expressed protein purified. Kinetic analyses revealed different Km values not only for CDNB, but also for GSH. The Km values were 0.2, 1.3 and 0.3 mM for GSH, and 0.9, 1.2 and 0.5 mM for CDNB, for the wild type, W13L mutant and I119F mutant, respectively. The V(max) values were 297.6, 224.5 and 171.8 mu mol min(-1) mg(-1) protein for GSH, and 372.3, 170.6 and 160.4 mu mol min(-1) mg(-1) protein for CDNB.