Genome reconstruction and comparative genomics of pneumocystis jirovecii

Pneumocystis jirovecii is a fungus belonging to a basal lineage of the Ascomycotina, the Taphrinomycotina subphylum. It is a parasite specific to humans that dwells primarily in the lung and can cause severe pneumonia in individuals with debilitated immune system. Despite its clinical importance, many aspects of its biology remain poorly understood, at least in part because of the lack of a continuous in vitro cultivation system. The present thesis consists in the genome reconstruction and comparative genomics of P. jirovecii. It is made of three parts: (i) the de novo sequencing of P. jirovecii genome starting from a single broncho- alveolar lavage fluid of a single patient (ii) the de novo sequencing of the genome of the plant pathogen Taphrina deformans, a fungus closely related to P. jirovecii, and (iii) the genome scale comparison of P. jirovecii to other Taphrinomycotina members. Enrichment in P. jirovecii cells by immuno-precipitation, whole DNA random amplification, two complementary high throughput DNA sequencing methods, and in silico sorting and assembly of sequences were used for the de novo reconstruction of P. jirovecii genome from the microbiota of a single clinical specimen. An iterative ad hoc pipeline as well as numerical simulations was used to recover P. jirovecii sequences while purging out contaminants and assembly or amplification chimeras. This strategy produced a 8.1 Mb assembly, which encodes 3,898 genes. Homology searches, mapping on biochemical pathways atlases, and manual validations revealed that this genome lacks (i) most of the enzymes dedicated to the amino acids biosyntheses, and (ii) most virulence factors observed in other fungi, e.g. the glyoxylate shunt pathway and specific peptidases involved in the degradation of the host cell membrane. The same analyses applied to the available genomic sequences from Pneumocystis carinii the species infecting rats and Pneumocystis murina the species infecting mice revealed the same deficiencies. The genome sequencing of T. deformans yielded a 13 Mb assembly, which encodes 5,735 genes. T. deformans possesses enzymes involved plant cell wall degradation, secondary metabolism, the glyoxylate cycle, detoxification, sterol biosynthesis, as well as the biosyntheses of plant hormones such as abscisic acid or indole-3-acetic acid. T. deformans also harbors gene subsets that have counterparts in plant saprophytes or pathogens, which is consistent with its alternate saprophytic and pathogenic lifestyles. Mating genes were also identified. The homothallism of this fungus suggests a mating-type switching mechanism. Comparative analyses indicated that 81% of P. jirovecii genes are shared with eight other Taphrinomycotina members, including T. deformans, P. carinii and P. murina. These genes are mostly involved in housekeeping activities. The genes specific to the Pneumocystis genus represent 8%, and are involved in RNA metabolism and signaling. The signaling is known to be crucial for interaction of Pneumocystis spp with their environment. Eleven percent are unique to P. jirovecii and encode mostly proteins of unknown function. These genes in conjunction with other ones (e.g. the major surface glycoproteins) might govern the interaction of P. jirovecii with its human host cells, and potentially be responsible of the host specificity. P. jirovecii exhibits a reduced genome in size with a low GC content, and most probably scavenges vital compounds such as amino acids and cholesterol from human lungs. Consistently, its genome encodes a large set of transporters (ca. 22% of its genes), which may play a pivotal role in the acquisition of these compounds. All these features are generally observed in obligate parasite of various kingdoms (bacteria, protozoa, fungi). Moreover, epidemiological studies failed to evidence a free-living form of the fungus and Pneumocystis spp were shown to co-evolved with their hosts. Given also the lack of virulence factors, our observations strongly suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, and which causes disease only in individuals with compromised immune system. The same conclusion is most likely true for all other Pneumocystis spp in their respective mammalian host. - Pneumocystis jirovecii est un champignon appartenant à ine branche basale des Ascomycotina, le sous-embranchement des Taphrinomycotina. C'est un parasite spécifique aux humains qui réside principalement dans les poumons, et qui peut causer des pneumonies sévères chez des individus ayant un système immunitaire déficient. En dépit de son importance clinique, de nombreux aspects de sa biologie demeurent,largement méconnus, au moins en partie à cause de l'absence d'un système de culture in vitro continu. Cette thèse traite de la reconstruction du génome et de la génomique comparative de P. jirovecii. Elle comporte trois parties: (i) le séquençage de novo du génome de P. jirovecii à partir d'un lavage broncho-alvéolaire provenant d'un seul patient, (ii) le séquençage de novo du génome d'un champignon pathogène de plante Taphrina deformans qui est phylogénétiquement proche de P. jirovecii, et (iii) la comparaison du génome de P. jirovecii à celui d'autres membres du sous-embranchement des Taphrinomycotina. Un enrichissement en cellules de P. jirovecii par immuno-précipitation, une amplification aléatoire des molécules d'ADN, deux méthodes complémentaires de séquençage à haut débit, un tri in silico et un assemblage des séquences ont été utilisés pour reconstruire de novo le génome de P. jirovecii à partir du microbiote d'un seul échantillon clinique. Un pipeline spécifique ainsi que des simulations numériques ont été utilisés pour récupérer les séquences de P. jirovecii tout en éliminant les séquences contaminants et les chimères d'amplification ou d'assemblage. Cette stratégie a produit un assemblage de 8.1 Mb, qui contient 3898 gènes. Les recherches d'homologies, de cartographie des voies métaboliques et des validations manuelles ont révélé que ce génome est dépourvu (i) de la plupart des enzymes dédiées à la biosynthèse des acides aminés, et (ii) de la plupart des facteurs de virulence observés chez d'autres champignons, par exemple, le cycle du glyoxylate ainsi que des peptidases spécifiques impliquées dans la dégradation de la membrane de la cellule hôte. Les analyses appliquées aux données génomiques disponibles de Pneumocystis carinii, l'espèce infectant les rats, et de Pneumocystis murina, l'espèce infectant les souris, ont révélé les mêmes déficiences. Le séquençage du génome de T. deformans a généré un assemblage de 13.3 Mb qui contient 5735 gènes. T. deformans possède les gènes codant pour les enzymes impliquées dans la dégradation des parois cellulaires des plantes, le métabolisme secondaire, le cycle du glyoxylate, la détoxification, la biosynthèse des stérols ainsi que la biosynthèse d'hormones de plantes telles que l'acide abscissique ou l'acide indole 3-acétique. T. deformans possède également des sous-ensembles de gènes présents exclusivement chez des saprophytes ou des pathogènes de plantes, ce qui est consistent avec son mode de vie alternatif saprophyte et pathogène. Des gènes impliqués dans la conjugaison ont été identifiés. L'homothallisme de ce champignon suggère mécanisme de permutation du type conjuguant. Les analyses comparatives ont démontré que 81% des gènes de P. jirovecii sont présent chez les autres membres du sous-embranchement des Taphrinomycotina. Ces gènes sont essentiellement impliqués dans le métabolisme basai. Les gènes spécifiques au genre Pneumocystis représentent 8%, et sont impliqués dans le métabolisme de l'ARN et la signalisation. La signalisation est connue pour être cruciale pour l'interaction des espèces de Pneumocystis avec leur environnement. Les gènes propres à P. jirovecii représentent 11% et codent en majorité pour des protéines dont la fonction est inconnue. Ces gènes en conjonction avec d'autres (par exemple, les glycoprotéines de surface), pourraient être déterminants dans l'interaction de P. jirovecii avec les cellules de l'hôte humain, et être potentiellement responsable de la spécificité d'hôte. P. jirovecii possède un génome de taille réduite à faible pourcentage en GC et récupère très probablement des composés vitaux comme les acides aminés et le cholestérol à partir des poumons humains. De manière consistante, son génome code pour de nombreux transporteurs (22% de ses gènes), qui pourraient jouer un rôle essentiel dans l'acquisition de ces composés. Ces caractéristiques sont généralement observées chez les parasites obligatoires de plusieurs règnes (bactéries, protozoaires, champignons). De plus, les études épidémiologiques n'ont pas réussi à prouver l'existence d'ime forme vivant librement du champignon. Etant donné également l'absence de facteurs de virulence, nos observations suggèrent que P. jirovecii est un parasite obligatoire spécialisé dans la colonisation des poumons humains, ne causant une maladie que chez des individus ayant un système immunitaire compromis. La même conclusion est très probablement applicable à toutes les autres espèces de Pneumocystis dans leur hôte mammifère respectif.

The differentiation of fibre- and drug type Cannabis seedlings by gas chromatography/mass spectrometry and chemometric tools

Cannabis cultivation in order to produce drugs is forbidden in Switzerland. Thus, law enforcement authorities regularly ask forensic laboratories to determinate cannabis plant's chemotype from seized material in order to ascertain that the plantation is legal or not. As required by the EU official analysis protocol the THC rate of cannabis is measured from the flowers at maturity. When laboratories are confronted to seedlings, they have to lead the plant to maturity, meaning a time consuming and costly procedure. This study investigated the discrimination of fibre type from drug type Cannabis seedlings by analysing the compounds found in their leaves and using chemometrics tools. 11 legal varieties allowed by the Swiss Federal Office for Agriculture and 13 illegal ones were greenhouse grown and analysed using a gas chromatograph interfaced with a mass spectrometer. Compounds that show high discrimination capabilities in the seedlings have been identified and a support vector machines (SVMs) analysis was used to classify the cannabis samples. The overall set of samples shows a classification rate above 99% with false positive rates less than 2%. This model allows then discrimination between fibre and drug type Cannabis at an early stage of growth. Therefore it is not necessary to wait plants' maturity to quantify their amount of THC in order to determine their chemotype. This procedure could be used for the control of legal (fibre type) and illegal (drug type) Cannabis production.

Toward improved immunocompetence of adoptively transferred CD8+ T cells.

Adoptive transfer of autologous or allogenic T cells to patients is being used with increased frequency as a therapy for infectious diseases and cancer. However, many questions remain with regard to defining optimized procedures for preparation and selection of T cell populations for transfer. In a new study in this issue of the JCI, Gattinoni and colleagues used a TCR transgenic mouse model to examine in vitro-generated tumor antigen-specific CD8+ T cells at various stages of differentiation for their efficacy in adoptive immunotherapy against transplantable melanoma. The results confirm that CD8+ T cells progressively lose immunocompetence with prolonged in vitro cultivation and suggest that effector CD8+ T cells alone may be considerably less potent at protecting hosts with advanced tumors than are less differentiated T cells.

Comparação dos sistemas de cultivo nativo e adensado de erva mate, Ilex paraguariensis St. Hil., quanto à ocorrência e flutuação populacional de insetos

This research was carried out in order to compare the occurrence of insects in two maté cultivation systems, native and high tree density. It was performed from August/2000 to September/2001, in a private property in São Mateus do Sul county, in Paraná State, Brazil. Visual inspections of trees and light traps were used to evaluate insect populations in both areas. For Hedypathes betulinus (Klug) (Coleoptera, Cerambycidae), only six adults were observed in the dense area. Based on presence of sawdust at the basis of the trunk, it was obtained that the number of attacked trees did not surpass 11% in either area. For Gyropsylla spegazziniana (Lizer y Trelles) (Hemiptera, Psyllidae), the number of galls per tree was counted and it was observed that the population peak occurred from November to January. For Hylesia spp. (Lepidoptera, Saturniidae) and Thelosia camina Schaus (Lepidoptera, Eupterotidae), the presence of caterpillars on the trees was noticed from September to February, with the population peak in November and December. Adults of Hylesia spp. were more numerous in February and March. Two species that were not previously recorded for Brazil on maté were identified: Hylesia paulex Dognin (83%) and Hylesia remex Dyer (17%), collected with light traps. The maté caterpillar, T. camina was not collected with these traps. Nymphs and adults of Ceroplastes grandis Hempel (Hemiptera, Coccidae) were observed along the year on the branches, with population peak between April and June for the nymphs and from September to November for the adults. It should be considered that despite higher insect incidence in the dense area compared to the native area, the first presents higher yield, and that with a good pest management program the insect problems can be minimized.

The In Vitro Mass-Produced Model Mycorrhizal Fungus, Rhizophagus irregularis, Significantly Increases Yields of the Globally Important Food Security Crop Cassava.

The arbuscular mycorrhizal symbiosis is formed between arbuscular mycorrhizal fungi (AMF) and plant roots. The fungi provide the plant with inorganic phosphate (P). The symbiosis can result in increased plant growth. Although most global food crops naturally form this symbiosis, very few studies have shown that their practical application can lead to large-scale increases in food production. Application of AMF to crops in the tropics is potentially effective for improving yields. However, a main problem of using AMF on a large-scale is producing cheap inoculum in a clean sterile carrier and sufficiently concentrated to cheaply transport. Recently, mass-produced in vitro inoculum of the model mycorrhizal fungus Rhizophagus irregularis became available, potentially making its use viable in tropical agriculture. One of the most globally important food plants in the tropics is cassava. We evaluated the effect of in vitro mass-produced R. irregularis inoculum on the yield of cassava crops at two locations in Colombia. A significant effect of R. irregularis inoculation on yield occurred at both sites. At one site, yield increases were observed irrespective of P fertilization. At the other site, inoculation with AMF and 50% of the normally applied P gave the highest yield. Despite that AMF inoculation resulted in greater food production, economic analyses revealed that AMF inoculation did not give greater return on investment than with conventional cultivation. However, the amount of AMF inoculum used was double the recommended dose and was calculated with European, not Colombian, inoculum prices. R. irregularis can also be manipulated genetically in vitro, leading to improved plant growth. We conclude that application of in vitro R. irregularis is currently a way of increasing cassava yields, that there is a strong potential for it to be economically profitable and that there is enormous potential to improve this efficiency further in the future.

Percepções das Comunidades Rurais sobre os Diques de Contenção de Sedimentos e Água na Bacia Hidrográfica de Águas Belas

Na sequência do projecto “Ordenamento e Valorização da Bacia Hidrográfica de Águas Belas”, da ilha de Santiago, que visou a construção de estruturas de conservação do solo e água para combater a erosão hídrica e a desertificação, disponibilizar recursos hídricos para consumo doméstico e agricultura e proteger o ambiente, estudou-se o modo como as comunidades rurais percepcionam as funções, gestão e conservação dessas estruturas, bem como o aproveitamento de recursos a elas associados. No estudo utilizou-se a informação contida no original do projecto referido, realizou-se o levantamento de dados de campo, aplicou-se um inquérito às populações beneficiadas pelas infra-estruturas construídas e utilizaram-se programas relativos a sistemas de informação geográfica. Constatou-se que as comunidades beneficiadas percepcionam os diques como infra-estruturas que disponibilizam mais meios de produção, como água para rega e abastecimento doméstico, solo e área de cultivo e a retenção de solo e água, ou seja, meios que têm uma estreita ligação com a actividade agrícola e as necessidades de sobrevivência. Observou-se igualmente que os mais jovens e as mulheres valorizam mais a segurança e, estas últimas, a maior disponibilidade de água para rega e uso doméstico. Para ajustar os usos que as comunidades fazem destas estruturas são necessárias acções de sensibilização e informação junto das comunidades sobre as funções e requisitos de manutenção das estruturas de conservação do solo e água.

Thrips species (Thysanoptera, Terebrantia) inhabiting irrigated rice and surrounding habitats in Cachoeirinha, state of Rio Grande do Sul, Brazil

In Brazil, almost nothing is known about the thrips fauna associated with rice crops. This is especially true for Rio Grande do Sul State (RS), the major rice producer in Brazil, where no systematic surveys have been conducted. Thus, this work records preliminarily the species of suborder Terebrantia inhabitanting irrigated rice (RC) and three nearby habitats: the area selected for rice, before cultivation (BR), grassland (GR) and forest border (FB) in Cachoeirinha, RS (50º58'21"W; 29º55'30"S). Eight field trips were carried out between October/2004 and February/2005 to Estação Experimental do Arroz in Cachoeirinha. Thrips specimens were collected with sweeping net and transported to laboratory for identification. A total of 623 adults terebrantians were recorded, belonging to two families and 18 species. Frankliniella rodeos (55.5%), Neohydatothrips cf. paraensis (30.3%) and Frankliniella schultzei (5.6%), were the most abundant, comprising more than 90% of the individuals. FB showed the highest species richness (S=15), followed by GR (S=10), RC (S=9) and BR (S=6). FB presented a low number of individuals (14.0%), contrasting with GR (44.8%) and BR (36.7%). In general, thrips abundance in RC was low at EEA (4.5%), and few species seems to effectively use rice as a host-plant. Two species previously considered of economic importance were found, F. rodeos and F. schultzei, the latter a new record for rice in Brazil. Five species are new records to RS.

Percepções das Comunidades Rurais sobre os Diques de Contenção de Sedimentos e Água na Bacia Hidrográfica de Águas Belas

Na sequência do projecto “Ordenamento e Valorização da Bacia Hidrográfica de Águas Belas”, da ilha de Santiago, que visou a construção de estruturas de conservação do solo e água para combater a erosão hídrica e a desertificação, disponibilizar recursos hídricos para consumo doméstico e agricultura e proteger o ambiente, estudou-se o modo como as comunidades rurais percepcionam as funções, gestão e conservação dessas estruturas, bem como o aproveitamento de recursos a elas associados. No estudo utilizou-se a informação contida no original do projecto referido, realizou-se o levantamento de dados de campo, aplicou-se um inquérito às populações beneficiadas pelas infra-estruturas construídas e utilizaram-se programas relativos a sistemas de informação geográfica. Constatou-se que as comunidades beneficiadas percepcionam os diques como infra-estruturas que disponibilizam mais meios de produção, como água para rega e abastecimento doméstico, solo e área de cultivo e a retenção de solo e água, ou seja, meios que têm uma estreita ligação com a actividade agrícola e as necessidades de sobrevivência. Observou-se igualmente que os mais jovens e as mulheres valorizam mais a segurança e, estas últimas, a maior disponibilidade de água para rega e uso doméstico. Para ajustar os usos que as comunidades fazem destas estruturas são necessárias acções de sensibilização e informação junto das comunidades sobre as funções e requisitos de manutenção das estruturas de conservação do solo e água.

Pyrroloquinoline quinone biosynthesis gene pqqC, a novel molecular marker for studying the phylogeny and diversity of phosphate-solubilizing pseudomonads.

Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity, we identified one phylogenetic group with high solubilization activity. In summary, we demonstrate that the gene pqqC is a novel molecular marker that can be used complementary to housekeeping genes for studying the diversity and evolution of plant-beneficial pseudomonads.

Control de la derivació de l'aigua de reg de Masjoan

RESUM Masjoan és una masia del segle XII on resideix la família Masferrer des del 1710. Actualment a Masjoan l’activitat principal és el cultiu i venta de plantes ornamentals, i l’explotació forestal per obtenir fusta. Pel que fa a l’aigua, tant per a la realització de l’activitat professional com per al subministrament de la casa, aquesta prové de mines d’aigua natural situades en la finca. L’objectiu d’aquest TFC ha estat dissenyar un sistema autònom per automatitzar el procés de derivació de l’aigua procedent d’una mina, i d’aquesta manera aprofitar millor els recursos naturals dels que és disposa. El desenvolupament d’aquest sistema, comprèn la selecció i configuració de sensors i actuadors, el disseny del circuit amb la realització de la placa, el disseny del sistema d’alimentació autònom, el software que controla el sistema, i dimensionar la resta d’elements de la instal•lació. Tot el sistema està controlat per un microcontrolador PIC16F876 i alimentat per un mòdul solar de 4W. En el disseny s’ha procurat, sobredimencionar les diferents etapes per possibles ampliacions o modificacions del sistema i que el circuit procures optimitzar el consum d’energia. Com a conclusions cal dir que s’han assolit els objectius proposats amb èxit. S’ha aconseguit un disseny funcional i estable que actualment es troba en funcionament.

Does wheat genetically modified for disease resistance affect root-colonizing pseudomonads and arbuscular mycorrhizal fungi?

This study aimed to evaluate the impact of genetically modified (GM) wheat with introduced pm3b mildew resistance transgene, on two types of root-colonizing microorganisms, namely pseudomonads and arbuscular mycorrhizal fungi (AMF). Our investigations were carried out in field trials over three field seasons and at two locations. Serial dilution in selective King's B medium and microscopy were used to assess the abundance of cultivable pseudomonads and AMF, respectively. We developed a denaturing gradient gel electrophoresis (DGGE) method to characterize the diversity of the pqqC gene, which is involved in Pseudomonas phosphate solubilization. A major result was that in the first field season Pseudomonas abundances and diversity on roots of GM pm3b lines, but also on non-GM sister lines were different from those of the parental lines and conventional wheat cultivars. This indicates a strong effect of the procedures by which these plants were created, as GM and sister lines were generated via tissue cultures and propagated in the greenhouse. Moreover, Pseudomonas population sizes and DGGE profiles varied considerably between individual GM lines with different genomic locations of the pm3b transgene. At individual time points, differences in Pseudomonas and AMF accumulation between GM and control lines were detected, but they were not consistent and much less pronounced than differences detected between young and old plants, different conventional wheat cultivars or at different locations and field seasons. Thus, we conclude that impacts of GM wheat on plant-beneficial root-colonizing microorganisms are minor and not of ecological importance. The cultivation-independent pqqC-DGGE approach proved to be a useful tool for monitoring the dynamics of Pseudomonas populations in a wheat field and even sensitive enough for detecting population responses to altered plant physiology.

Emergence of secretion-defective sublines of Pseudomonas aeruginosa PAO1 resulting from spontaneous mutations in the vfr global regulatory gene.

Pseudomonas aeruginosa undergoes spontaneous mutation that impairs secretion of several extracellular enzymes during extended cultivation in vitro in rich media, as well as during long-term colonization of the cystic fibrosis lung. A frequent type of strong secretion deficiency is caused by inactivation of the quorum-sensing regulatory gene lasR. Here we analyzed a spontaneously emerging subline of strain PAO1 that exhibited moderate secretion deficiency and partial loss of quorum-sensing control. Using generalized transduction, we mapped the secretion defect to the vfr gene, which is known to control positively the expression of the lasR gene and type II secretion of several proteases. We confirmed this secretion defect by sequencing and complementation of the vfr mutation. In a reconstruction experiment conducted with a 1:1 mixture of wild-type strain PAO1 and a vfr mutant of PAO1, we observed that the vfr mutant had a selective advantage over the wild type after growth in static culture for 4 days. Under these conditions, spontaneous vfr emerged in a strain PAO1 population after four growth cycles, and these mutants accounted for more than 40% of the population after seven cycles. These results suggest that partial or complete loss of quorum sensing and secretion can be beneficial to P. aeruginosa under certain environmental conditions.

Nitrogen application timing and soil inorganic nitrogen dynamics under no-till oat/maize sequential cropping

The timing of N application to maize is a key factor to be considered in no-till oat/maize sequential cropping. This study aimed to evaluate the influence of pre-planting, planting and sidedress N application on oat residue decomposition, on soil N immobilisation and remineralisation and on N uptake by maize plants in no-till oat/maize sequential cropping. Undisturbed soil cores of 10 and 20 cm diameter were collected from the 0-15 cm layer of a no-till Red Latossol, when the oat cover crop was in the milk-grain stage. Two greenhouse experiments were conducted simultaneously. Experiment A, established in the 10 cm diameter cores and without plant cultivation, was used to asses N dynamics in soil and oat residues. Experiment B, established in the 20 cm diameter cores and with maize cultivation, was used to assess plant growth and N uptake. An amount of 6.0 Mg ha-1 dry matter of oat residues was spread on the surface of the cores. A rate of 90 kg N ha-1 applied as ammonium sulphate in both experiments was split in pre-planting, planting and sidedress applications as follows: (a) 00-00-00 (control), (b) 90-00-00 (pre-planting application, 20 days before planting), (c) 00-90-00 (planting application), (d) 00-30-60 (split in a planting and a sidedress application 31 days after emergence), (e) 00-00-00* (control, without oat residue) and (f) 90-00-00* (pre-planting application, without oat residue). The N concentration and N content in oat residues were not affected during decomposition by N fertilisation. Most of the fertiliser NH4+-N was converted into NO3--N within 20 days after application. A significant decrease in NO3--N contents in the 0-4 cm layer was observed in all treatments between 40 and 60 days after the oat residue placement on the soil surface, suggesting the occurrence of N immobilisation in this period. Considering that most of the inorganic N was converted into NO3- and that no immobilisation of the pre planting fertiliser N occurred at the time of its application, it was possible to conclude that pre-planting applied N was prone to losses by leaching. On the other hand, with split N applications, maize plants showed N deficiency symptoms before sidedress application. Two indications for fertiliser-N management in no-till oat/maize sequential cropping could be suggested: (a) in case of split application, the sidedress should be earlier than 30 days after emergence, and (b) if integral application is preferred to save field operations, this should be done at planting.

Hepatitis E virus: a zoonosis adapting to humans.

Hepatitis E virus (HEV) infection is gaining global attention, not only because of the increasing burden of the disease in low endemicity countries, in terms of morbidity and mortality rates, but also due to recent advances in the molecular virology and epidemiology of this emerging pathogen. HEV infection spread can be described as the evolution of a zoonosis towards an established human infection. As known from other viruses, such as the human immunodeficiency virus or the influenza viruses, crossing the species barriers from animals to humans is a recurrent phenomenon. Albeit slow at the beginning, once the virus has adapted to humans, the person-to-person spread can proceed very quickly. Although an optimal cell culture system for HEV is not yet available, outstanding progress has been made with the in vitro expression of HEV-like particles. These new tools have fostered new research to understand the molecular, structural and immunological aspects of human HEV infection. Although some promising data from Phase II vaccine trials are available, recent discoveries will certainly open new avenues for HEV-specific prophylaxis and therapy.

Protective effect of divalent cations against aluminum toxicity in soybean

A large proportion of soybean fields in Brazil are currently cultivated in the Cerrado region, where the area planted with this crop is growing considerably every year. Soybean cultivation in acid soils is also increasing worldwide. Since the levels of toxic aluminum (Al) in these acid soils is usually high it is important to understand how cations can reduce Al rhizotoxicity in soybean. In the present study we evaluated the ameliorative effect of nine divalent cations (Ca, Mg, Mn, Sr, Sn, Cu, Zn, Co and Ba) in solution culture on Al rhizotoxicity in soybean. The growth benefit of Ca and Mg to plants in an acid Inceptisol was also evaluated. In this experiment soil exchangeable Ca:Mg ratios were adjusted to reach 10 and 60 % base saturation, controlled by different amounts of CaCl2 or MgCl2 (at proportions from 100:0 up to 0:100), without altering the soil pH level. The low (10 %) and adequate (60 %) base saturation were used to examine how plant roots respond to Al at distinct (Ca + Mg)/Al ratios, as if they were growing in soils with distinct acidity levels. Negative and positive control treatments consisted of absence (under native soil or undisturbed conditions) or presence of lime (CaCO3) to reach 10 and 60 % base saturation, respectively. It was observed that in the absence of Aluminum, Cu, Zn, Co and Sn were toxic even at a low concentration (25 µmol L-1), while the effect of Mn, Ba, Sr and Mg was positive or absent on soybean root elongation when used in concentrations up to 100 µmol L-1. At a level of 10 µmol L-1 Al, root growth was only reverted to the level of control plants by the Mg treatment. Higher Tin doses led to a small alleviation of Al rhizotoxicity, while the other cations reduced root growth or had no effect. This is an indication that the Mg effect is ion-specific and not associated to an electrostatic protection mechanism only, since all ions were divalent and used at low concentrations. An increased exchangeable Ca:Mg ratio (at constant soil pH) in the acid soil almost doubled the soybean shoot and root dry matter even though treatments did not modify soil pH and exchangeable Al3+. This indicates a more efficient alleviation of Al toxicity by Mg2+ than by Ca2+. The reason for the positive response to Mg2+ was not the supply of a deficient nutrient because CaCO3 increased soybean growth by increasing soil pH without inducing Mg2+ deficiency. Both in hydroponics and acid soil, the reduction in Al toxicity was accompanied by a lower Al accumulation in plant tissue, suggesting a competitive cation absorption and/or exclusion of Al from plant tissue stimulated by an Mg-induced physiological mechanism.