In vitro EVALUATION OF EUCALYPTUS ECTOMYCORRHIZAE ON SUBSTRATE WITH PHOSPHORUS DOSES FOR FUNGAL PRE-SELECTION

The benefit promoted by ectomycorrhizal depends on the interaction between symbionts and phosphorus (P) contents. Phosphorus effect on ectomycorrhizal formation and the effectiveness of these in promoting plant growth for fungal pre-selection were assessed under in vitro conditions. For P effect evaluation, Eucalyptus urophylla seedlings inoculated with four Pisolithus sp. isolates and others non-inoculated were grown on substrate containing 0.87, 1.16 and 1.72 mg P per plant. For evaluation of effectiveness and fungal pre-selection, other 30 isolates of Pisolithus sp., Pisolithus microcarpus ITA06 isolate, Amanita muscaria AM16 isolate, Scleroderma areolatum SC129 isolate were studied. D26 isolate promoted the highest plant heights for the three P doses, D51 at the lower dose and D72 at the intermediate dose. P doses did not influenced shoot fresh weight and fungal colonization. In the pre-selection of fungi, 14 isolates of Pisolithus sp., P. microcarpus ITA06 isolate and S. areolatum SC129isolate increased plant height and fresh weight. D82 isolate of Pisolithus sp. had effect singly on plant height while D17 and D58 on fresh weight. Of these, only D15, D17, D58 and ITA06 had typical ectomycorrhizae. The cultivation in vitro has shown adequate for pre-selection of ectomycorrhizal fungi. Colonization and benefits depend on species and isolate. D15, D17 and D58 of Pisolithus sp. and P. microcarpus isolate ITA06 are the most promising for nursery studies.

PRODUCTION OF AUSTRALIAN CEDAR SEEDLINGS INOCULATED WITH ARBUSCULAR MYCORRHIZAL FUNGI IN DIFFERENT TYPES OF CONTAINERS

ABSTRACT The present study aimed to evaluate the growth and the levels of N, P, K, Ca and Mg in Australian cedar seedlings which had been inoculated with arbuscular mycorrhizal fungi (AMF) in different types of containers. The experiment was carried out in a greenhouse and the experimental design was that of randomized complete blocks (RCB), with a 4 x 4 factorial design consisting of four inoculation treatments with AMF (Rhizophagus clarum, Gigaspora margarita, a mixed inoculation (R. clarum + G. margarita) and the control (with no AMF inoculation); four types of containers (plastic bags measuring 250 cm3, tubes of 55 and 130 cm3 and pressed blocks 440 cm3. plant-1), with four repetitions. The height, the diameter of the stem base, the aerial part dry weight (APDW), the dry weight of the root (DWR) and the total plant dry weight (DW) were measured, along with the Dickson quality index, the percentage of mycorrhizal colonization and the levels of N, P, K, Ca and Mg in the aerial part dry weight. One hundred and thirty eight days (138) days after sowing, the greatest growth and/or the highest levels of P, K and Ca could be observed in the aerial part dry weight of the Australian cedar seedlings which had been planted in the pressed block container and inoculated with a mixture of the two AMF species (G. margarita + R. clarum) or with just R. clarum. Thus it can be seen that AMF can make a significant contribution to the production of Australian cedar seedlings.

The impact of the law 10,267/2001 in the brazilian rural registration

The publication of the Law 10,267 of 08/28/2001 changed the paradigm of rural registration in Brazil, because this law known as the "Law of Georeferencing" has created the National Registration of Rural Property, that unifies in a common basis different registrations present in several government agencies, such as the National Institute for Colonization and Agrarian Reform (INCRA), the Secretariat of Federal Revenue, the Brazilian Institute of Environment and Natural Resources, and the National Indian Foundation. Also, this new registration system has a graphical component which has not existed until such date, where the boundaries of rural property are georeferenced to the Brazilian Geodetic System. This new paradigm has resulted in a standardization of the survey and its representation of rural properties according to the Technical Standard for Georeferencing of Rural Properties, published by INCRA in compliance with the new legislation. Due to the georeferencing, the creation of a public GIS of free access on the Internet was possible. Among the difficulties found it may be observed the great Brazilian territory, the need for specialized professionals, and especially the certification process that INCRA has to perform for each georeferenced property. It is hoped that this last difficulty is solved with the implementation of the Land Management System that will allow automated and online certification, making the process more transparent, agile and fast.

Administration of Bifidobacterium animalis subsp. lactis BB-12 and xylitol with a novel pacifier in early childhood

Probiotic bifidobacteria are used in the prevention and treatment of childhood diseases. On the other hand, these bacteria are also connected to dental caries. The purpose of the present work was to test a food supplement containing Bifidobacterium animalis subsp. lactis BB-12 (B. lactis BB-12) and xylitol, and to investigate its health effects, properties and safety when used in a novel pacifier in early childhood. In a double-blind, placebo-controlled trial, newborn infants (n=163) were assigned randomly to receive B. lactis BB-12, xylitol, or sorbitol from the age of 1– 2 monthsto 2 years with a pacifier or a spoon. Children were followed up to four years of age. A part of the parents participating in the clinical trial evaluated the feasibility of the novel administration method. The pattern of tablet release from the pouch of the pacifier was tested in adults. The food supplement tablet containing B. lactis BB-12 and xylitol could be delivered in a safe and controlled way with the novel pacifier. The early administration of B. lactis BB-12 did not result in permanent oral colonization of this probiotic or affect the colonization of mutans streptococci in early childhood. Moreover, B. lactis BB-12 did not increase the occurrence of caries. Controlled administration of B. lactis BB-12 significantly reduced the incidence of respiratory infections during the first eight months of life in a Finnish population with breastfed infants. To conclude, administration of B. lactis BB-12 in early childhood is safe with regard to the future dental health of the child. In addition, B. lactis BB-12 may add to the protection against respiratory infections provided by human breast milk in infancy.

Immunohistochemical detection of Tritrichomonas foetus in experimentally infected mice

The need to intensify knowledge of the pathogenesis of bovine genital trichomoniasis (BGT) led to the use of alternative animal models such as the mouse. Nevertheless, it is necessary to elucidate the dynamics of the infection in this animal species, evaluating different stages of the colonization and evolution of the pathological alterations. The immunohistochemistry (IHC) offers advantages over the routine histopathological staining techniques for the detection of the protozoan in tissues, cellular detritus and inside the macrophages. The goal of the present study was to demonstrate the presence of Tritrichomonas foetus in the reproductive tract of infected mice using an IHC technique. Female BALB/c mice were infected with a suspension of T. foetus by intravaginal route, in the estrum phase, detected by exfoliative vaginal cytology. After 10 weeks, the animals were sacrificed; uterus and vagina were fixed and histologically processed. Some slides were stained with HE. The rest of the slides were processed for IHC. An immunoadsorbed polyclonal serum against T. foetus was used. The avidine-biotine technique (HistoMouse, Zymed™) was employed. The histopathological studies showed a dilation of the uterine glands, presence of macrophages in the lumen of the organ and inner part of the endometrial glands. No T. foetus was identified using this method. The IHQ allowed additionally the identification of the protozoan in the endometrium, endometrial glands, uterine lumen and inside neutrophils and macrophages. The cytological studies stained with IHC showed either isolated T. foetus adhered to epithelial cells or inside macrophages. This technique proves to be a useful tool for the study of the pathogenesis of bovine genital trichomoniasis (BGT) in an experimental model.

Two-Photon Excited Fluorescence Detection Technology in Screening for Methicillin-Resistant Staphylococcus aureus

Control of the world-wide spread of methicillin-resistant Staphylococcus aureus (MRSA) has been unsuccessful in most developed countries. A few countries have been able to maintain a low MRSA prevalence, plausibly due to their strict MRSA control policies. Such policies require wide-scale screening of patients with suspected MRSA colonization, in order to nurse the MRSA-positive patients in contact isolation. The aim of this study was to develop and introduce a 2-photon excited fluorescence detection (TPX) technique for screening of MRSA directly from clinical samples. The assay principle involves specific online immunometric monitoring of S. aureus growth under selective antibiotic pressure. After the novel TPX approach had been set up, its applicability for the detection of MRSA was evaluated using a large MRSA collection including practically all epidemic MRSA strains identified in Finland between 1991 and 2009. The TPX assay was found both sensitive (97.9%) and specific (94.1%) in this epidemiological setting, illustrating that the method is tolerant to wide biological variation as well as to environments with rapidly emerging MRSA strains. When MRSA was screened directly from colonization samples, all patients positive for MRSA by conventional methods were positive also by the TPX assay. The assay capacity was 48 samples per a test run, and the median time required for confirmation of a true-positive screening test result was 3 h 26 min. Collectively, the findings presented in this thesis suggest that the TPX MRSA screening assay could be applicable for direct screening of MRSA colonization samples without any prior steps of isolation. This can potentially mean that contact isolation of suspected carriers testing negative could be discontinued earlier, thereby reducing the costs and burden associated with the containment of MRSA. In case of infection, a positive test result would ensure an early onset of effective therapy.

Biodiversity and ecosystem functioning in angiosperm communities in the Baltic Sea

The biological variation in nature is called biodiversity. Anthropogenic pressures have led to a loss of biodiversity, alarming scientists as to what consequences declining diversity has for ecosystem functioning. The general consensus is that diversity (e.g. species richness or identity) affects functioning and provides services from which humans benefit. The aim of this thesis was to investigate how aquatic plant species richness and identity affect ecosystem functioning in terms of processes such as primary production, nutrient availability, epifaunal colonization and properties e.g. stability of Zostera marina subjected to shading. The main work was carried out in the field and ranged temporally from weeklong to 3.5 months-long experiments. The experimental plants used frequently co-occur in submerged meadows in the northern Baltic Sea and consist of eelgrass (Z. marina), perfoliate pondweed (Potamogeton perfoliatus), sago pondweed (P. pectinatus), slender-leaved pondweed (P. filiformis) and horned pondweed (Zannichellia palustris). The results showed that plant richness affected epifaunal community variables weakly, but had a strong positive effect on infaunal species number and functional diversity, while plant identity had strong effects on amphipods (Gammarus spp.), of which abundances were higher in plant assemblages consisting of P. perfoliatus. Depending on the starting standardizing unit, plant richness showed varying effects on primary production. In shoot density-standardized plots, plant richness increased the shoot densities of three out of four species and enhanced the plant biomass production. Both positive complementarity and selection effects were found to underpin the positive biodiversity effects. In shoot biomass-standardized plots, richness effects only affected biomass production of one species. Negative selection was prevalent, counteracting positive complementarity, which resulted in no significant biodiversity effect. The stability of Z. marina was affected by plant richness in such that Z. marina growing in polycultures lost proportionally less biomass than Z. marina in monocultures and thus had a higher resistance to shading. Monoculture plants in turn gained biomass faster, and thereby had a faster recovery than Z. marina growing in polycultures. These results indicate that positive interspecific interactions occurred during shading, while the faster recovery of monocultures suggests that the change from shading stress to recovery resulted in a shift from positive interactions to resource competition between species. The results derived from this thesis show that plant diversity affects ecosystem functioning and contribute to the growing knowledge of plant diversity being an important component of aquatic ecosystems. Diverse plant communities sustain higher primary productivity than comparable monocultures, affect faunal communities positively and enhance stability. Richness and identity effects vary, and identity has generally stronger effects on more variables than richness. However, species-rich communities are likely to contain several species with differing effects on functions, which renders species richness important for functioning. Mixed meadows add to coastal ecosystem functioning in the northern Baltic Sea and may provide with services essential for human well-being.

A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model

The ability of thymidine kinase (tk)-deleted recombinant bovine herpesvirus 5 (BoHV-5tkΔ) to establish and reactivate latent infection was investigated in lambs. During acute infection, the recombinant virus replicated moderately in the nasal mucosa, yet to lower titers than the parental strain. At day 40 post-infection (pi), latent viral DNA was detected in trigeminal ganglia (TG) of all lambs in both groups. However, the amount of recombinant viral DNA in TGs was lower (9.7-fold less) than that of the parental virus as determined by quantitative real time PCR. Thus, tk deletion had no apparent effect on the frequency of latent infection but reduced colonization of TG. Upon dexamethasone (Dx) administration at day 40 pi, lambs inoculated with parental virus shed infectious virus in nasal secretions, contrasting with lack of infectivity in secretions of lambs inoculated with the recombinant virus. Nevertheless, some nasal swabs from the recombinant virus group were positive for viral DNA by PCR, indicating low levels of reactivation. Thus, BoHV-5 TK activity is not required for establishment of latency, but seems critical for efficient virus reactivation upon Dx treatment.

Development of porous glass-fiber reinforced composite for bone implants. Evaluation of antimicrobial effect and implant fixation

Cranial bone reconstructions are necessary for correcting large skull bone defects due to trauma, tumors, infections and craniotomies. Traditional synthetic implant materials include solid or mesh titanium, various plastics and ceramics. Recently, biostable glass-fiber reinforced composites (FRC), which are based on bifunctional methacrylate resin, were introduced as novel implant solution. FRCs were originally developed and clinically used in dental applications. As a result of further in vitro and in vivo testing, these composites were also approved for clinical use in cranial surgery. To date, reconstructions of large bone defects were performed in 35 patients. This thesis is dedicated to the development of a novel FRC-based implant for cranial reconstructions. The proposed multi-component implant consists of three main parts: (i) porous FRC structure; (ii) bioactive glass granules embedded between FRC layers and (iii) a silver-polysaccharide nanocomposite coating. The porosity of the FRC structure should allow bone ingrowth. Bioactive glass as an osteopromotive material is expected to stimulate the formation of new bone. The polysaccharide coating is expected to prevent bacterial colonization of the implant. The FRC implants developed in this study are based on the porous network of randomly-oriented E-glass fibers bound together by non-resorbable photopolymerizable methacrylate resin. These structures had a total porosity of 10–70 volume %, of which > 70% were open pores. The pore sizes > 100 μm were in the biologically-relevant range (50-400 μm), which is essential for vascularization and bone ingrowth. Bone ingrowth into these structures was simulated by imbedding of porous FRC specimens in gypsum. Results of push-out tests indicated the increase in the shear strength and fracture toughness of the interface with the increase in the total porosity of FRC specimens. The osteopromotive effect of bioactive glass is based on its dissolution in the physiological environment. Here, calcium and phosphate ions, released from the glass, precipitated on the glass surface and its proximity (the FRC) and formed bone-like apatite. The biomineralization of the FRC structure, due to the bioactive glass reactions, was studied in Simulated Body Fluid (SBF) in static and dynamic conditions. An antimicrobial, non-cytotoxic polysaccharide coating, containing silver nanoparticles, was obtained through strong electrostatic interactions with the surface of FRC. In in vitro conditions the lactose-modified chitosan (chitlac) coating showed no signs of degradation within seven days of exposure to lysozyme or one day to hydrogen peroxide (H2O2). The antimicrobial efficacy of the coating was tested against Staphylococcus aureus and Pseudomonas aeruginosa. The contact-active coating had an excellent short time antimicrobial effect. The coating neither affected the initial adhesion of microorganisms to the implant surface nor the biofilm formation after 24 h and 72 h of incubation. Silver ions released to the aqueous environment led to a reduction of bacterial growth in the culture medium.

Effects of Corynespora cassiicola on Lantana camara

The present study combines the examination of toxins produced by C. cassiicola and the effects of the fungus colonization on L. camara. C. cassiicola was cultivated on solid media and the crude extracts CAE and CE were produced. Both extracts were submitted to a seed germination and growth assay utilizing Physalis ixocarpa, Trifolium alexandrinum, Lolium multiflorum and Amaranthus hypochodriacus. The effect of the extracts on the ATP-synthesis in isolated spinach chloroplasts was also tested. Bioassay guided chromatographic fractionation identified the most active extract (CAE). From this extract ergosta-4,6,8(14),22-tetraen-3-one (C1) and fatty acids were isolated. The C1 compound reduce ATP synthesis in isolated spinach chloroplasts. The interference of fatty acids with ATP synthesis and also with weed growth provides one explanation of the phytogrowth-inhibitory properties of such fungal extracts. Histological observations involving fungus-plant interaction were made on L. camara plants inoculated with C. cassiicola conidia suspension. After inoculations, fragments of the leaf blades were prepared for observation by light and scanning electron microscopy. Fungal colonization of Lantana camara was typical of a necrotroph and penetration initiated a hypersensitive response. L. camara reacted to the pathogen penetration through thickening of the epidermis walls, cytoplasm granulation and a cicatrisation tissue.

Aquatic plant community in porto primavera reservoir

Aiming to identify the populations of aquatic plants present in the Porto Primavera reservoir and evaluate the behavior of Hydrilla verticillata colonization of this water body a survey was carried out in 2007. The data was based on presence or absence, only were assessed the presence or absence of the species and the data were subjected to cluster analysis to establish differences in distribution and occurrence of populations. The community of aquatic plants showed 24 species distributed in 16 botanical families. Cyperaceae and Pontederiaceae were the most representative in terms of species richness. The submerged macrophyte Hydrilla verticillata showed the highest frequency of occurrence in the water body, showing a different behavior from the other populations of the water body. Species like Eichhornia crassipes, Eichhornia azurea, Typha dominguensis and Oxycaryum cubense also showed different behavior in relation to other populations within the community, forming large populations in lagoons and backwater areas.

Glyphosate drift affects arbuscular mycorrhizal association in coffee

Mycorrhizal association promotes better survival and nutrition of colonized seedling on field, and consequently, increasing of productivity. However, the weed management can interfere on this association, due to incorrect use of glyphosate. This work has assessed the effects of glyphosate drift on the growth and nutrition of arabica coffee plants (Catuaí Vermelho - IAC 99) colonized with arbuscular mycorrhizal fungi (AMF). The experiment was conducted in 2 x 5 factorial scheme, and included inoculated and non-inoculated plants, and five glyphosate subdoses (0.0, 57.6, 115.2, 230.4, and 460.8 g ha-1 of glyphosate), in randomized blocks with five replication. The inoculation was carried during the greenhouse phase of seedlings production with a mixture of Rhizophagus clarus and Gigaspora margarita, and after to transplanting, when the plants had seven pairs of leaves, glyphosate subdoses were applied. The product caused intoxication in up to 60% of non-inoculated and 45% on inoculated plants, when the highest dose of 460.8 g a.e. ha-1 was applied. A negative effect was noted on the growth and phosphorus content of coffee plants, this effect increased depending on glyphosate subdose, but regardless of inoculation. Glyphosate drift reduces the growth and nutrition of plants colonized by species of AMF and native fungi, negatively affecting root colonization of plants treated.

Soil microorganisms and their role in the interactions between weeds and crops

The competition between weeds and crops is a topic of great interest, since this interaction can cause heavy losses in agriculture. Despite the existence of some studies on this subject, little is known about the importance of soil microorganisms in the modulation of weed-crop interactions. Plants compete for water and nutrients in the soil and the ability of a given species to use the available resources may be directly affected by the presence of some microbial groups commonly found in the soil. Arbuscular mycorrhizal fungi (AMF) are able to associate with plant roots and affect the ability of different species to absorb water and nutrients from the soil, promoting changes in plant growth. Other groups may promote positive or negative changes in plant growth, depending on the identity of the microbial and plant partners involved in the different interactions, changing the competitive ability of a given species. Recent studies have shown that weeds are able to associate with mycorrhizal fungi in agricultural environments, and root colonization by these fungi is affected by the presence of other weeds or crops species. In addition, weeds tend to have positive interactions with soil microorganisms while cultures may have neutral or negative interactions. Competition between weeds and crops promotes changes in the soil microbial community, which becomes different from that observed in monocultures, thus affecting the competitive ability of plants. When grown in competition, weeds and crops have different behaviors related to soil microorganisms, and the weeds seem to show greater dependence on associations with members of the soil microbiota to increase growth. These data demonstrate the importance of soil microorganisms in the modulation of the interactions between weeds and crops in agricultural environments. New perspectives and hypotheses are presented to guide future research in this area.

Anatomy and physiology of Cattail as related to different population densities

The objective of this work was to evaluate the effects of the population density of Typha angustifolia plants in the anatomical and physiological characteristics. Plants were collected from populations of high density (over 50% of colonization capacity) and low density (less than 50% of colonization capacity) and cultivated under controlled greenhouse conditions. Plants from both populations were grown in plastic trays containing 4 L of nutritive solution for 60 days. At the end of this period, the relative growth rate, leaf area ratio, net assimilatory rate, root/shoot ratio, leaf anatomy, root anatomy, and catalase and ascorbate peroxidase activities were evaluated. Plants from high density populations showed increased growth rate and root/shoot ratio. Low density populations showed higher values of stomatal index and density in leaves, as well as increased palisade parenchyma thickness. Root epidermis and exodermis thickness as well as the aerenchyma proportion of high density populations were reduced, these plants also showed increased vascular cylinder proportion. Only catalase activity was modified between the high and low density populations, showing increased values in low density populations. Therefore, different Typha angustifolia plants show differences in its anatomy and physiology related to its origins on high and low density conditions. High density population plants shows increased growth capacity related to lower apoplastic barriers in root and this may be related to increased nutrient uptake capacity.

Mycorrhizal Association and Microbial Activity of Soil Cultivated with Cassava After Application of Mesotrione and Fluazifop-p-Butyl

Soil is a very heterogeneous environment that allows the establishment of wide range of microorganisms populations, whose balance is affected by biotic and abiotic factors. This study has aimed to assess the effect of doses of mesotrione and fluazifop-p-butyl herbicides and two assessment periods on microbial activity and biomass of soil cultivated with cassava Cacau-UFV cultivar, besides the root colonization by arbuscular mycorrhizal fungi. Two trials were conducted in a protected environment where was realized post-emergence application of mesotrione in the doses of 72, 108, 144 and 216 g ha-1 and fluazifop-p-butyl in the doses of 100, 150, 200 and 300 g ha-1, besides a control without application. Soil samples were collected for determination of soil respiratory rate (RR), microbial biomass carbon (MBC), metabolic quotient (qCO2), and colonization of roots by arbuscular mycorrhizal fungi at the 30 and 60 days after applications (DAA) of the herbicides. Fluazifop-p-butyl increased the RR, MBC and the percentage of cassava roots colonized by mycorrhizal fungi in the assessment performed at 60 DAA. The larger effects of mesotrione on soil microbial indicators were up to 30 DAA, being the changes minimized at 60 DAA. It is concluded that the herbicides alter the soil microbial indicators, with effects dependent of the product, of dose applied and also of the period of assessment.