Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga,Fucus

Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immunoglobulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues by indirect immunofluorescence. Each antibody labelled tissues with a distinctive distribution pattern in cell walls and extracellular matrix regions, demonstrating that each antibody was specific for a different extracellular epitope (i.e., antigenic determinant). Most antibodies also labelled intracellularly on at least one cell type. Punctate, fibrous or clumped labelling was characteristic of individual antibodies and provided information related to carbohydrate structure and solubility. These antibodies are molecular probes for small regions on cell wall polymers and should be valuable in studies of cell wall synthesis, secretion, assembly and modification as well as carbohydrate fine structure and function.Abbreviations EDTA ethylenediaminetetraacetic acid - EIA enzyme immunoassay - Ig immunoglobulin (IgG, IgM and IgA are immunoglobulin types)     

Revision and reclassification of three Plasmopara species based on morphological and molecular phylogenetic data

Based on the results of morphological and DNA sequence (partial D1–D3/D7–D8 nuLSU and partial nuSSU-ITS1-5.8S rDNA) data, three species of Plasmopara are revised and reclassified. A species of Plasmopara parasitic on Scorzonera, invalidly published several times, is assigned to a new genus and species under Novotelnova scorzonerae. Plasmopara euphrasiae sp. nov. is segregated from P. densa, and P. centaureae-mollis is revised and relegated to synonymy of Bremia centaureae. All taxa are described and illustrated.     

Paracoccidioides brasiliensis: chemical and molecular tools for research on cell walls, antifungals, diagnosis, taxonomy

Paracoccidioides brasiliensis is a dimorphic fungus, a causative agent of paracoccidioidomycosis, one of the most frequent systemic mycoses that affect the rural population in Latin America, only geographical region in which this fungus is to be found. In this work, we discuss matters related to (a) cell wall studies based on the cloning and analysis of genes involved in the synthesis of cell wall components, and their possible roles in virulence and dimorphism in P. brasiliensis, (b) molecular taxonomy and the molecular classification of P. brasiliensis as an Ascomycete belonging in the Order Onygenales, (c) phylogeny of P. brasiliensis and the possible existence of cryptic species within the genus Paracoccidioides, and (d) new experimental antifungal drugs such as azasterols or sterol hydrazones, compounds that affect the activity of Δ²⁴⁽²⁸⁾ sterol methyl reductase (SMR) and/or Δ⁽²⁴⁾-sterol methyl transferase (SMT), and (e) specific primers for the molecular detection of P. brasiliensis in vitro and in clinical samples.     

An investigation of the fine structure, cell surface carbohydrates, and appeal of the diatom Extubocellulus sp. as prey for small flagellates

Summary. The fine structure and surface exopolymers of a coastal planktonic nanodiatom of the sparsely reported genus Extubocellulus were studied respectively by scanning electron microscopy and confocal microscopy in conjunction with fluorescent lectins. Monitoring the suitability of the species as prey food for other protists was also investigated by video microscopy coupled with digital film. Cells are rectangular in girdle view, with a pervalvar axis longer than the apical axis. Valves are almost circular with a diameter of 2.8 to 3.6 μm. The valve face bears randomly distributed areolae (ca. 50 in 10 μm), which may be either open or occluded. Two small raised ocelluli occur at the apices, with a rim devoid of perforations and about 6–7 porelli. Glucose and N-acetyl-glucosamine moieties present on the surface of the live diatom were labelled with fluorescent lectins, and a differential pattern of distribution for both carbohydrates was observed. The potential role of fluorescent lectins as cellular probes of taxonomic value in small diatoms is compared with that of nucleotide and antibody probes. We provide the first illustrative evidence of the presence of Extubocellulus sp. in the cytoplasm of the nanoflagellate Goniomonas amphinema and of the egestion of diatom frustules. Results obtained are discussed in the light of the present knowledge of the role of carbohydrate–protein interactions in phagocytosis of prey by free-living protozoa. Correspondence (present address): M. Martin-Cereceda, Department of Ecology and Evolutionary Biology, Haworth Hall, 1200 Sunnyside Avenue, University of Kansas, Lawrence, KS 66045, U.S.A.     

Production of Bacteriocin ST33LD, Produced by Leuconostoc mesenteroides subsp. mesenteroides, as Recorded in the Presence of Different Medium Components

Summary Bacteriocin ST33LD, produced by Leuconostoc mesenteroides subsp. mesenteroides, is approximately 2.7 kDa in size and inhibits Enterococcus faecalis, Escherichia coli, Lactobacillus casei and Pseudomonas aeruginosa. Good growth was recorded in the presence of 10% (w/v) soy milk or 10% (w/v) molasses, but there was no bacteriocin production. Growth in MRS broth adjusted to pH 4.5 yielded low bacteriocin levels (800 AU/ml). However, the same medium adjusted to pH 5.0, 5.5 and 6.5, respectively, yielded 3200 AU/ml. Tween 80 decreased bacteriocin production by more than 50%. Growth in the presence of tryptone yielded maximal activity (12,800 AU/ml), whereas different combinations of tryptone, meat extract and yeast extract produced activity levels of 1600 AU/ml and less. Growth in the presence of 2.0% (w/v) sucrose, or maltose, yielded much higher levels of bacteriocin activity (12,800 AU/ml) compared to growth in the presence of 2.0% (w/v) glucose or lactose (6400 AU/ml). Lower yields were also recorded in the presence of fructose and mannose. KH₂PO₄ at 10.0% (w/v) stimulated bacteriocin production. Glycerol concentrations of 0.5% (w/v) and higher (up to 5.0%, w/v) repressed bacteriocin production by 50%. The addition of cyanocobalamin, thiamine and L-ascorbic acid to MRS broth (1.0 ppm) yielded 12,800 AU/ml bacteriocin, whereas the addition of DL-6,8-thioctic acid yielded only 6 400 AU/ml.     

A molecular protocol using quenched FRET probes for the quarantine surveillance of Tilletia indica, the causal agent of Karnal bunt of wheat

The current surveillance protocol for Karnal bunt of wheat in most countries, including the USA, European Union (EU), and Australia, involves the tentative identification of the spores based on morphology followed by a molecular analysis. Germination of spores is required for confirmation which incurs a delay of about two weeks, which is highly unsatisfactory in a quarantine situation. A two-step PCR protocol using FRET probes for the direct detection and identification of Tilletia indica from a very few number of spores (≤10) is presented. The protocol involves amplification of the ITS1 DNA segment in the highly repeated rDNA unit from any Tilletia species, followed by FRET analysis to detect and unequivocably distinguish T. indica and the closely related T. walkeri. This rapid, highly sensitive, fluorescent molecular tool is species-specific, and could supersede the conventional microscopic diagnosis used in a quarantine surveillance protocol for Karnal bunt which is often confounded by overlapping morphological characters of closely related species.     

Induction of apoptosis by the mistletoe lectins: A review on the mechanisms of cytotoxicity mediated by Viscum album L.

This review focuses on the cytotoxic properties of Viscum album L. (VAL). Apart from well-established results of protein synthesis inhibition by the mistletoe lectins (MLs), namely their catalytic A chain, there is now convincing evidence that the VAL-mediated cytotoxicity is mainly due to an induction of apoptosis. Among the more than 1,000 proteins detected in VAL, the MLs and the viscotoxins (VTs) are the predominant toxic proteins. Using purified components, such as the D-galactose-specific ML I, the N-acetyl-D-galactosamine-specific ML II and ML III, crude VTs and oligosaccharides, only the MLs induced apoptosis. The in vitro studies suggest that interaction of lectin B chains with appropriate receptors on the cell surface activates distinct signalling pathways that ultimately leads to apoptosis in a large fraction of cells, while others survive, however, with a conservation of their DNA. Inhibition of protein synthesis by the A chain of the hololectin probably accelerates the B chain-induced course of events.     

Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans

The multilayered cell wall of the cyanobacterium Anacystis nidulans was studied by the freezeetching technique. A characteristic fracture face in the outer cell wall was demonstrated which is densely packed with particles of a diameter of 60–75 Å. This particle layer is comparable with layers which have been described in many cell walls of Gram-negative prokaryotes.The outer membrane of the cell wall was solubilised by extraction with phenol/water or sodium dodecyl sulfate (SDS). In the SDS-extract 31 bands were separated by polyacrylamide gel electrophoresis, among them 3–5 major proteins with molecular weights of approximately 60, 40, and 10 kdaltons, respectively. Several polypeptides of the Anacystis cell wall were comparable in their mobility with polypeptides extracted from cell walls of different Gramnegative bacteria. The analysis of the SDS-unsoluble electron dense layer (sacculi) revealed the typical components of peptidoglycan diaminopimelic acid, muramic acid, glutamic acid, glucosamine and alamine in the molar ratio of 1.0:0.9:1.1:1.5:1.9. In addition, other amino acids (molar ratio from 0.05–0.36), mannosamine (molar ratio 0.54), and lipopolysaccharide components were detected in low concentration.Abbreviations SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetate     

Snowdrop and wheatgerm lectins and avidin as antimetabolites for the control of sugarcane whitegrubs

Snowdrop and wheatgerm lectins were found to be insecticidal and growth inhibiting dietary proteins for larvae of the sugarcane whitegrub Antitrogus parvulus. At concentrations as low as 0.5 mg of snowdrop lectin per gram of semi-artificial diet, growth was inhibited by 21 days of feeding and significant mortality was apparent by 28 days. Wheatgerm lectin was active at similar concentrations, although expression of the effects was slower. Avidin was found to be a growth inhibiting dietary protein for larvae of Antitrogus consanguineus. At levels as low as 0.01 mg g^-1 of diet, growth was inhibited by 28 days of feeding. Avidin caused no significant mortality after 35 days of feeding. Snowdrop and wheatgerm lectins and avidin are insect growth-inhibiting proteins whose genes potentially could be manipulated into sugarcane and improve host-plant resistance to whitegrubs.     

Studies on the cell cycle of Myxobacter AL-1

Myxobacter AL-1 was synchronized by size fractionation of asynchronous cultures on a 5–20% equivolumetric sucrose gradient in a Ti—15 zonal rotor. The degree of synchronization obtained by this method was determined 1. by a comparison of the size distribution of an asynchronous culture to the distribution of cell numbers in the fractions of the zonal run, 2. by studying the size distributions in the different fractions obtained with the zonal rotor and 3. by following the growth and size distribution of a fraction from a zonal run during one division cycle. The results indicate that this method is suitable for the study of the cell cycle of Myxobacter AL-1.     

Morphological differences and molecular phylogeny of freshwater blooming species, Peridiniopsis spp. (Dinophyceae) from China

In 2008–2010, several freshwater dinoflagellate blooms caused by Peridiniopsis spp. were observed in China. P. penardii and P. niei sampled from various geographical localities were examined by means of light and scanning electron microscopy. After comparing morphological and molecular differences, the new freshwater variety Peridiniopsis penardii var. robusta var. nov. (Peridiniales, Dinophyceae) found in Manwan Reservoir, Yunnan Province was described. The new variety differed from P. penardii since it possessed numerous robust antapical spines and a conspicuous apical spine. Molecular phylogenetic analyses based on SSU, LSU and ITS indicated P. niei, P. penardii and P. penardii var. robusta were closely related with P. kevei, and clustered into a monophyletic clade. The new variety possessed an endosymbiotic diatom which was similar to P. penardii and P. kevei, whereas the endosymbiont was not present in cells of P. niei. The endosymbiont SSU and ITS phylogenies showed that the endosymbionts of these three dinoflagellates were closely related to members of Thalassiosirales. Furthermore it was concluded that the endosymbionts might originate from Discostella-like species.     

The optical brightener Calcofluor White disorders thecal architecture and inhibits cell division ofCryothecomonas longipes Schnepf et Kühn, a marine nanoflagellate incertae sedis

Summary Calcofluor White inhibits the division of the marine nanoflagellateCryothecomonas longipes and causes the development of monstrously lobed cells. It disturbs the assembly of a putative noncellulosic fibrillar polysaccharide in the theca. The compact layer of the theca becomes thicker and wider, less densely packed and less rigid. The deposition of thecal components becomes uncoordinated. In consequence the initially unaffected coat of the theca and loose portions of the compact layer protrude from the cell surface. These are then convoluted, with the coat on their convex faces. The spacing of the coat ridges is increased here. The tension between the different layers obviously stabilizes the theca and contributes to its rigidity and elasticity.Abbreviations CW Calcofluor White - DCB 2-chloro-6-dichlo-robenzonitrile     

Intercalibration of classical and molecular techniques for identification of Alexandrium fundyense (Dinophyceae) and estimation of cell densities

A workshop with the aim to compare classical and molecular techniques for phytoplankton enumeration took place at Kristineberg Marine Research Station, Sweden, in August 2005. Seventeen different techniques – nine classical microscopic-based and eight molecular methods – were compared. Alexandrium fundyense was the target organism in four experiments. Experiment 1 was designed to determine the range of cell densities over which the methods were applicable. Experiment 2 tested the species specificity of the methods by adding Alexandrium ostenfeldii, to samples containing A. fundyense. Experiments 3 and 4 tested the ability of the methods to detect the target organism within a natural phytoplankton community. Most of the methods could detect cells at the lowest concentration tested, 100 cells L⁻¹, but the variance was high for methods using small volumes, such as counting chambers and slides. In general, the precision and reproducibility of the investigated methods increased with increased target cell concentration. Particularly molecular methods were exceptions in that their relative standard deviation did not vary with target cell concentration. Only two of the microscopic methods and three of the molecular methods had a significant linear relationship between their cell count estimates and the A. fundyense concentration in experiment 2, where the objective was to discriminate that species from a morphologically similar and genetically closely related species. None of the investigated methods were affected by the addition of a natural plankton community background matrix in experiment 3. The results of this study are discussed in the context of previous intercomparisons and the difficulties in defining the absolute, true target cell concentration.     

Fine root architecture, morphology, and biomass of different branch orders of two Chinese temperate tree species

We have limited understanding of architecture and morphology of fine root systems in large woody trees. This study investigated architecture, morphology, and biomass of different fine root branch orders of two temperate tree species from Northeastern China—Larix gmelinii Rupr and Fraxinus mandshurica Rupr —by sampling up to five fine root branch orders three times during the 2003 growing season from two soil depths (i.e., 0–10 and.10–20 cm). Branching ratio (R _b) differed with the level of branching: R _b values from the fifth to the second order of branching were approximately three in both species, but markedly higher for the first two orders of branching, reaching a value of 10.4 for L. gmelinii and 18.6 for F. mandshurica. Fine root diameter, length, SRL and root length density not only had systematic changes with root order, but also varied significantly with season and soil depth. Total biomass per order did not change systematically with branch order. Compared to the second, third and/or fourth order, the first order roots exhibited higher biomass throughout the growing season and soil depths, a pattern related to consistently higher R _b values for the first two orders of branching than the other levels of branching. Moreover, the differences in architecture and morphology across order, season, and soil depth between the two species were consistent with the morphological disparity between gymnosperms and angiosperms reported previously. The results of this study suggest that root architecture and morphology, especially those of the first order roots, should be important for understanding the complexity and multi-functionality of tree fine roots with respect to root nutrient and water uptake, and fine root dynamics in forest ecosystems.     

Nest architecture of a bagworm species: Rhythmic pattern in the arrangement of sticks

Nest architecture of a bagworm species,Clania crameri was examined. Fortytwo bags (nests) were collected from the host plant,Clerodendron indicum and number of sticks used in each bag was counted. Furthermore, length of each stick in each nest was measured (in mm) clockwise one after another serially beginning with the longest stick. The data obtained were subjected to frequency analysis and power spectrum analysis. Results clearly reveal that the larvae of bagworms do not glue together sticks of different size randomly but with a definite pattern.     

Detection of hemicelluloses specific to the cell wall of tracheary elements and phloem cells by fluorescein-conjugated lectins

Summary Binding of fluorescein-conjugated wheat-germ agglutinin (F-WGA) and some other lectins to tissues from various plants were examined by epifluorescence microscopy. F-WGA bound specifically to the walls of tracheary elements (TEs) and phloem cells of pea roots. The binding sites in TEs were localized only in the secondary thickening and became evident at very early stages of differentiation. Fluorescein-conjugated derivatives ofSolanum tuberosum lectin,Lycopersicon esculentum lectin, andDatura stramonium lectin, which bind N-acetylglucosamine residues as WGA, also bound to the secondary thickening of TEs of pea roots. The binding sites for F-WGA were not removed by extraction with hot EDTA and proteinase K, but removed by extraction with an alkali solution. The alkali-extracted binding sites from the roots were precipitated together with hemicelluloses by 80% ethanol. These results indicate that the binding sites are not present on pectins, proteins, or cellulose, but hemicelluloses. Localized distribution of the binding sites for F-WGA in TEs was found also in a variety of angiosperm plants.Abbreviations BSL-II Bandeiraea simplicifolia lectin II - DSL Datura stramonium lectin - F fluorescein-conjugated - LEL Lycopersicon esculentum lectin - MT microtubule - STL Solanum tuberosum lectin - TE tracheary element - WGA wheat-germ agglutinin     

Observations on the teleomorph of the white root rot fungus,Rosellinia necatrix, and a related fungus,Rosellinia aquila

The process of teleomorph development in the white root rot fungusRosellinia necatrix is described on diseased roots of Japanese pear. Stromata were also found on dead plants in nonagricultural lands such as yards and forests. The stroma ofR. aquila is also described. Research based on the program for Promotion of Basic Research Activities for Innovative Biosciences.     

Studies on the molecular ecology of Blastomyces dermatitidis

The microecology of Blastomyces dermatitidis, the dimorphic etiologic agentof the potentially fatal systemic fungal infection, blastomycosis, is not well defined.Blastomyces dermatitidis may occur periodically at natural sites, perhaps aided by rotting organic material, animal droppings and physical changes. Semi-quantitative growth studies of B. dermatitidis on 2% agar plates determined the ability toutilize or tolerate a variety of substrates including simple and complex molecules as carbon source, and organic and inorganic nitrogen sources. Allantoin, creatinine, quanidoacetic acid, guanidine and cysteine may be used as sole nitrogen source. Allantoin in combination with dextrose, glycerol, lichenen, celloboise and other wood by-products support growth of B. dermatitidis at room temperature. The nutritional conversion of the fungus to the yeast form at room temperature, well demonstrated on allantoin/glycerol/yeast extract media, appears to be affected by certain inorganic compounds. The organism tolerates low to moderate levels of alpha-pinene, tannic acid, shikimic acid, veratryl alcohol, vanillic acid, and polyethyleneglycol-200. There are significant differences among isolates regarding growth on various substances at 20° and 37° centigrade. It appears that a variety of wood by-products and animal waste substrates, in combination, support the growth of B. dermatitidis. Their role in the ecological niche of B. dermatitidis, and the importance of nutritional dimorphism in the natural environment warrants further investigation. This revised version was published online in June 2006 with corrections to the Cover Date.