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.

Production of a bioherbicide agent in liquid and solid medium and in a biphasic cultivation system

The use of fungi in weeds control programs depends upon the conidia production in large scale. Therefore, this study aimed to evaluate liquid and solid culture media and the cultivation by biphasic system for the conidia production of Bipolaris euphorbiae Muchovej & Carvalho a specific pathogen of Euphorbia heterophylla. The liquid media were obtained from agro-industrial waste or by-products, and the solid media were prepared with mixtures of grains and grain derivatives. The liquid medium made with sugar cane molasses stood out from the others because it provided great sporulation (23 x 10(4) conidia mL-1 of medium), conidial viability (99.7%), and formation of mycelial fungal biomass (1.26 g 100 mL-1 of medium). On solid media conidial production was markedly higher than in liquid media, especially the medium composed by a blend of sorghum grain (40%) and soybean hulls (60%) where the fungus produced 2.3 x 10(7) conidia g-1 of medium. The cultivation of B. euphorbiae in biphasic system not promoted a significant increase in the production of conidia. The solid media were more effective for the mass production of fungus and mixtures of grains and derivatives were effective for increasing conidia production.

Light and storage on the germination of spores of Dicksonia sellowiana (Presl.) Hook., Dicksoniaceae

Spores of Dicksonia sellowiana are positively photoblastic and reach the maximum percentage of germination at 23 ± 2°C in white light after seven days of imbibition. The pre-induction phase for spores induced by white or red light for 24 hours was 72 hours. Gametophytes grown in white light were plane and bidimensional, while those grown under red light were filamentous. The higher the number of hours of light applied per day during 10 days, the higher the percentage of germination. Germination was higher for long white light treatments applied on a daily basis. The effect of different light intensities on germination was also investigated here. The lower percentages of germination were observed for spores kept under 43% and 2% of full sunlight, while those kept under 26, 19 and 4% presented higher percentages. Spores presented circa 82% of germination after 731 days of storage under refrigeration at aproximately 10°C.

Anatomical and ultrastructural aspects of root and mycorrhiza of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) Altschul (Leguminosae-Mimosoideae)

A. peregrina var. falcata form mutualistic symbiosis with arbuscular mycorrhizal fungus. An anatomical and ultrastructural study was carried out to analyze some aspects of this simbiotic association as well as some root features. The results evidenced the presence of fibers with non-lignified thicked secondary walls in the stele and sparse papillae on root surface. A. peregrina var. falcata mycorrhizas presented features of Arum-type (intercellular hyphae) and Paris-type (extensive coils) arbuscular mycorrhiza. Their general appearance with extraradical hyphae, intracellular coils, intercellular hyphae and arbuscules, is in agreement with arbuscular mycorrhizas of several plants. The ultrastructural observations showed that in intercellular hyphae and arbuscules vacuoles were dominant and that in rough endoplasmatic reticulum and small vesicles seems to be associated with arbuscule senescence process.

Purification and characterization of a phytoalexin elicitor from spores of the saprobe Mucor ramosissimus

Plants accumulate antimicrobial compounds (phytoalexins) in response to a wide variety of microorganisms. Mucor ramosissimus Samutsevitsch is a saprobe capable of inducing phytoalexin production in soybean cotyledons and in the leaves of tropical Rubiaceae on whose surface it has been found. In the present study, the elicitor from M. ramosissimus was partially purified and the activity compared to that of a glucan elicitor isolated from Phytophthora sojae. Optimal isolation of the elicitor (based on fungal growth, yield of spores and elicitor activity) was achieved by autoclaving spores obtained from nine day-old cultures of the fungus. The elicitor was precipitated with ethanol and purified by chromatography on an anion exchange column, which retained the elicitor, and a Concanavalin A-affinity matrix, to which the elicitor did not bind. The purification resulted in a considerable increase (six-fold) in the specific activity of the elicitor. Neutral sugar composition, analyzed by HPLC, revealed the predominance of mannose, followed by glucose and galactose, whereas colorimetric quantification showed the presence of uronic acids. GC-MS analysis of the elicitor revealed the predominance of glucuronic acid and mannose. These results suggest that fragments of mucoran-type polysaccharides are the phytoalexin elicitors present in the spores of the saprobe M. ramosissimus. Our results also indicate for the first time that soybean cotyledon tissues can recognize fragments of glucuronic-acid heteropolymers as phytoalexin elicitors.

First occurrence of Leightoniomyces phillipsii (Berk. & Leight.) D. Hawksw. & B. Sutton for South America

Leightoniomyces phillipsii (Berk. & Leight.) D. Hawksw. & B. Sutton, an anamorphic fungus, is described and illustrated for the first time for South America. This synnematous fungus with typical coarsely verrucose conidia was previously known to be associated only with lichens, but can be associated with plant roots. This discovery extends its habitat, geographical distribution, and ecosystem roles.

Differential in vitro pathogenicity of predatory fungi of the genus Monacrosporium for phytonematodes, free-living nematodes and parasitic nematodes of cattle

In vitro tests were carried out on the pathogenicity of nine isolates of the predatory fungi of the genus Monacrosporium (5 M. sinense isolates, 3 M. appendiculatum and 1 M. thaumasium isolate) for a phytonematode (second stage juveniles from Meloidogyne incognita, race 3), a free-living nematode (Panagrellus spp), and two gastrointestinal parasitic nematodes of cattle (infective larvae of Cooperia punctata and Haemonchus placei). A suspension containing 2,000 nematodes from each species was added to Petri dishes containing fungi and grown on 2% water-agar medium at 25oC in the dark for up to 7 days. The dishes were examined every other day for 7 days and predation-free nematodes were counted. The results showed that the free-living nematodes, Panagrellus spp, were the most susceptible (P<0.05), followed by the phytonematode M. incognita, while the controls were ³98.5% viable. However, a variable susceptibility of the nematodes to different fungi was observed. This indicates that the use of predatory fungi for the environmental control of nematodes will be limited by the multiplicity of nematodes in the environment and their differential susceptibility to fungal isolates of the same genus.

Expression of extracellular matrix components and their receptors in the central nervous system during experimental Toxoplasma gondii and Trypanosoma cruzi infection

Alterations in extracellular matrix (ECM) expression in the central nervous system (CNS) usually associated with inflammatory lesions have been described in several pathological situations including neuroblastoma and demyelinating diseases. The participation of fibronectin (FN) and its receptor, the VLA-4 molecule, in the migration of inflammatory cells into the CNS has been proposed. In Trypanosoma cruzi infection encephalitis occurs during the acute phase, whereas in Toxoplasma infection encephalitis is a chronic persisting process. In immunocompromised individuals such as AIDS patients, T. cruzi or T. gondii infection can lead to severe CNS damage. At the moment, there are no data available regarding the molecules involved in the entrance of inflammatory cells into the CNS during parasitic encephalitis. Herein, we characterized the expression of the ECM components FN and laminin (LN) and their receptors in the CNS of T. gondii- and T. cruzi-infected mice. An increased expression of FN and LN was detected in the meninges, leptomeninges, choroid plexus and basal lamina of blood vessels. A fine FN network was observed involving T. gondii-free and T. gondii-containing inflammatory infiltrates. Moreover, perivascular spaces presenting a FN-containing filamentous network filled with a4+ and a5+ cells were observed. Although an increased expression of LN was detected in the basal lamina of blood vessels, the CNS inflammatory cells were a6-negative. Taken together, our results suggest that FN and its receptors VLA-4 and VLA-5 might be involved in the entrance, migration and retention of inflammatory cells into the CNS during parasitic infections.

Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

Searching for the role of protein phosphatases in eukaryotic microorganisms

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.

Extended hydrogen photoproduction by nitrogen-fixing cyanobacteria

Cyanobacteria are the only prokaryotic organisms performing oxygenic photosynthesis. They comprise a diverse and versatile group of organisms in aquatic and terrestrial environments. Increasing genomic and proteomic data launches wide possibilities for their employment in various biotechnical applications. For example, cyanobacteria can use solar energy to produce H2. There are three different enzymes that are directly involved in cyanobacterial H2 metabolism: nitrogenase (nif) which produces hydrogen as a byproduct in nitrogen fixation; bidirectional hydrogenase (hox) which functions both in uptake and in production of H2; and uptake hydrogenase (hup) which recycles the H2 produced by nitrogenase back for the utilization of the cell. Cyanobacterial strains from University of Helsinki Cyanobacteria Collection (UHCC), isolated from the Baltic Sea and Finnish lakes were screened for efficient H2 producers. Screening about 400 strains revealed several promising candidates producing similar amounts of H2 (during light) as the ΔhupL mutant of Anabaena PCC 7120, which is specifically engineered to produce higher amounts of H2 by the interruption of uptake hydrogenase. The optimal environmental conditions for H2 photoproduction were significantly different between various cyanobacterial strains. All suitable strains revealed during screening were N2-fixing, filamentous and heterocystous. The top ten H2 producers were characterized for the presence and activity of the enzymes involved in H2 metabolism. They all possess the genes encoding the conventional nitrogenase (nifHDK1). However, the high H2 photoproduction rates of these strains were shown not to be directly associated with the maximum capacities of highly active nitrogenase or bidirectional hydrogenase. Most of the good producers possessed a highly active uptake hydrogenase, which has been considered as an obstacle for efficient H2 production. Among the newly revealed best H2 producing strains, Calothrix 336/3 was chosen for further, detailed characterization. Comparative analysis of the structure of the nif and hup operons encoding the nitrogenase and uptake hydrogenase enzymes respectively showed minor differences between Calothrix 336/3 and other N2-fixing model cyanobacteria. Calothrix 336/3 is a filamentous, N2-fixing cyanobacterium with ellipsoidal, terminal heterocysts. A common feature of Calothrix 336/3 is that the cells readily adhere to substrates. To make use of this feature, and to additionally improve H2 photoproduction capacity of the Calothrix 336/3 strain, an immobilization technique was applied. The effects of immobilization within thin alginate films were evaluated by examining the photoproduction of H2 of immobilized Calothrix 336/3 in comparison to model strains, the Anabaena PCC 7120 and its ΔhupL mutant. In order to achieve optimal H2 photoproduction, cells were kept under nitrogen starved conditions (Ar atmosphere) to ensure the selective function of nitrogenase in reducing protons to H2. For extended H2 photoproduction, cells require CO2 for maintenance of photosynthetic activity and recovery cycles to fix N2. Application of regular H2 production and recovery cycles, Ar or air atmospheres respectively, resulted in prolongation of H2 photoproduction in both Calothrix 336/3 and the ΔhupL mutant of Anabaena PCC 7120. However, recovery cycles, consisting of air supplemented with CO2, induced a strong C/N unbalance in the ΔhupL mutant leading to a decrease in photosynthetic activity, although total H2 yield was still higher compared to the wild-type strain. My findings provide information about the diversity of cyanobacterial H2 capacities and mechanisms and provide knowledge of the possibilities of further enhancing cyanobacterial H2 production.

Endothelial cells, tissue factor and infectious diseases

Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

Oxygen Photoreduction in Cyanobacteria

Cyanobacteria are well-known for their role in the global production of O2 via photosynthetic water oxidation. However, with the use of light energy, cyanobacteria can also reduce O2. In my thesis work, I have investigated the impact of O2 photoreduction on protection of the photosynthetic apparatus as well as the N2-fixing machinery. Photosynthetic light reactions produce intermediate radicals and reduced electron carriers, which can easily react with O2 to generate various reactive oxygen species. To avoid prolonged reduction of photosynthetic components, cyanobacteria use “electron valves” that dissipate excess electrons from the photosynthetic electron transfer chain in a harmless way. In Synechocystis sp. PCC 6803, flavodiiron proteins Flv1 and Flv3 comprise a powerful electron sink redirecting electrons from the acceptor side of Photosystem I to O2 and reducing it directly to water. In this work, I demonstrate that upon Ci-depletion Flv1/3 can dissipate up to 60% of the electrons delivered from Photosystem II. O2 photoreduction by Flv1/3 was shown to be vital for cyanobacteria in natural aquatic environments and deletion of Flv1/3 was lethal for both Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 under fluctuating light conditions. The lethal phenotype observed in the absence of Flv1/3 results from oxidative damage to Photosystem I, which appeared to be a primary target of reactive oxygen species produced upon sudden increases in light intensity. Importantly, cyanobacteria also possess other O2 photoreduction pathways which can protect the photosynthetic apparatus. This study demonstrates that respiratory terminal oxidases are also capable of initiating O2 photoreduction in mutant cells lacking the Flv1/3 proteins and grown under fluctuating light. Photoreduction of O2 by Rubisco was also shown in Ci-depleted cells of the mutants lacking Flv1/3, and thus provided the first evidence for active photorespiratory gas-exchange in cyanobacteria. Nevertheless, and despite the existence of other O2 photoreduction pathways, the Flv1/3 route appears to be the most robust and rapid system of photoprotection. Several groups of cyanobacteria are capable of N2 fixation. Filamentous heterocystous N2- fixing species, such as Anabaena sp. PCC 7120, are able to differentiate specialised cells called heterocysts for this purpose. In contrast to vegetative cells which perform oxygenic photosynthesis, heterocysts maintain a microoxic environment for the proper function of the nitrogenase enzyme, which is extremely sensitive to O2. The genome of Anabaena sp. PCC 7120 harbors two copies of genes encoding Flv1 and Flv3 proteins, designated as “A” and “B” forms. In this thesis work, I demonstrate that Flv1A and Flv3A are expressed only in the vegetative cells of filaments, whilst Flv1B and Flv3B are localized exclusively in heterocysts. I further revealed that the Flv3B protein is most responsible for the photoreduction of O2 in heterocysts, and that this reaction plays an important role in protection of the N2-fixing machinery and thus, the provision of filaments with fixed nitrogen. The function of the Flv1B protein remains to be elucidated; however the involvement of this protein in electron transfer reactions is feasible. Evidence provided in this thesis indicates the presence of a great diversity of O2 photoreduction reactions in cyanobacterial cells. These reactions appear to be crucial for the photoprotection of both photosynthesis and N2 fixation processes in an oxygenic environment.

Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae)

Leaf-cutting ants of the genera Atta and Acromyrmex (tribe Attini) are symbiotic with basidiomycete fungi of the genus Leucoagaricus (tribe Leucocoprineae), which they cultivate on vegetable matter inside their nests. We determined the variation of the 28S, 18S, and 5.8S ribosomal DNA (rDNA) gene loci and the rapidly evolving internal transcribed spacers 1 and 2 (ITS1 and ITS2) of 15 sympatric and allopatric fungi associated with colonies of 11 species of leafcutter ants living up to 2,600 km apart in Brazil. We found that the fungal rDNA and ITS sequences from different species of ants were identical (or nearly identical) to each other, whereas 10 GenBank Leucoagaricus species showed higher ITS variation. Our findings suggest that Atta and Acromyrmex leafcutters living in geographic sites that are very distant from each other cultivate a single fungal species made up of closely related lineages of Leucoagaricus gongylophorus. We discuss the strikingly high similarity in the ITS1 and ITS2 regions of the Atta and Acromyrmex symbiotic L. gongylophorus studied by us, in contrast to the lower similarity displayed by their non-symbiotic counterparts. We suggest that the similarity of our L. gongylophorus isolates is an indication of the recent association of the fungus with these ants, and propose that both the intense lateral transmission of fungal material within leafcutter nests and the selection of more adapted fungal strains are involved in the homogenization of the symbiotic fungal stock.