Xylem structure and connectivity in grapevine (Vitis vinifera) shoots provides a passive mechanism for the spread of bacteria in grape plants

BACKGROUND AND AIMS: Bacterial leaf scorch occurring in a number of economically important plants is caused by the xylem-limited bacterium Xylella fastidiosa (Xf). In grapevine, Xf systemic infection causes Pierce's disease and is lethal. Traditional dogma is that Xf movement between vessels requires the digestion of inter-vessel pit membranes. However, Yersinia enterocolitica (Ye) (a bacterium found in animals) and fluorescent beads moved rapidly within grapevine xylem from stem into leaf lamina, suggesting open conduits consisting of long, branched xylem vessels for passive movement. This study builds on and expands previous observations on the nature of these conduits and how they affect Xf movement. METHODS: Air, latex paint and green fluorescence protein (GFP)-Xf were loaded into leaves and followed to confirm and identify these conduits. Leaf xylem anatomy was studied to determine the basis for the free and sometimes restricted movement of Ye, beads, air, paint and GFP-Xf into the lamina. KEY RESULTS: Reverse loading experiments demonstrated that long, branched xylem vessels occurred exclusively in primary xylem. They were observed in the stem for three internodes before diverging into mature leaves. However, this stem-leaf connection was an age-dependent character and was absent for the first 10-12 leaves basal to the apical meristem. Free movement in leaf blade xylem was cell-type specific with vessels facilitating movement in the body of the blade and tracheids near the leaf margin. Air, latex paint and GFP-Xf all moved about 50-60% of the leaf length. GFP-Xf was never observed close to the leaf margin. CONCLUSIONS: The open vessels of the primary xylem offered unimpeded long distance pathways bridging stem to leaves, possibly facilitating the spread of bacterial pathogens in planta. GFP-Xf never reached the leaf margins where scorching appeared, suggesting a signal targeting specific cells or a toxic build-up at hydathodes.     

Susceptibility of the ant-cultivated fungus Leucoagaricus gongylophorus (Agaricales: Basidiomycota) towards microfungi

The aim of this study was to select virulent strains of microfungi against Leucoagaricus gongylophorus, a symbiotic fungus cultivated by leaf-cutting ants. The results from in vitro assays showed that microfungal strains had a variable and significant impact on the colony development of L. gongylophorus. Specifically, Trichoderma harzianum, Escovopsis weberi CBS 810.71 and E. weberi A088 were more effective, inhibiting the L. gongylophorus colonies by 75, 68 and 67%, respectively (P < 0.05) after 15 days. Strain E. weberi A086 and Acremonium kiliense were less effective: 43 and 26%, respectively (P < 0.05). In spite of the current negative perspective of a microbiological control approach for these ants, the present work discusses the possibility of using mycopathogenic fungi for the control of these insects, and points out the importance of encouraging more studies in this area.     

Rapid and efficient extraction of soluble proteins from Gram-negative microorganisms without disruption of cell walls

The ability of buffer solutions containing low concentrations of nonionic detergents (Triton X-100, Tween 20, Brij 58, and Lubrol PX) and the anionic detergent sodium deoxycholate, as well as mixtures of these detergents with chaeotropes (urea and guanidine hydrochloride), to extract intracellular proteins of Gram-negative microorganisms (Escherichia coli and Pseudomonas aeruginosa) was studied. It was established that the solutions containing Triton X-100 and sodium deoxycholate and the mixtures of these detergents with urea are the most effective. It was shown that the extraction of proteins from bacterial cells under the studied conditions is not accompanied by a release of DNA into solution but is associated with extraction of low-molecular RNAs. The level of protein extraction reaches 80%. No disruption of the bacterial cell wall occurs during the extraction, and proteins probably penetrate through meshes of the murein network. The efficiencies of our buffer mixtures are close to or higher than that of the commercial reagent CelLytic B (Sigma, United States). The practical uses of the chaeotropic mixtures developed are discussed.     

Plant pathogens drive density-dependent seedling mortality in a tropical tree

One explanation for the extraordinary diversity of tropical forest trees is that density-dependent mortality from herbivores or pathogens puts locally rare species at an advantage. Density-dependent mortality of seeds and small seedlings is particularly intense in tropical forests, but its causes remain uncertain. Here, we show experimentally that pathogens from the Oomycota are associated with intense mortality in seedlings of a neotropical tree, Sebastiana longicuspis . Seedlings in untreated plots experienced eight times higher mortality compared with seedlings in plots treated with fungicide. Mortality was strongly density dependent: in fungicide-treated plots survival was unaffected by density, but survival in unsprayed plots was over three times higher at low density. Density-dependent mortality observed in a simultaneous, non-manipulative study was highly transient, suggesting that short-term observational studies may underestimate the intensity and form of pathogen-induced mortality. If such effects are widespread, plant pathogens may play a key role in maintaining and structuring tropical diversity.     

植物病原物的群体遗传学

品种单一化、生产密集型和一年多茬的现代农业特点导致病原物呈现出进化速度加快、致病力增强及流行风险增大趋势。深入研究病原物群体遗传学对认识病害的流行、有效选育和使用抗性品种乃至控制病害具有重要意义。文章阐述了植物病原物群体遗传学的研究目标和内容、突变、基因迁移、基因重组、随机遗传漂变和自然选择5大遗传机制在植物病原物进化过程中的作用,以及目前植物病原物群体遗传学研究的现状。     

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.     

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell^{1, 2}. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli^3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence^6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods⁹. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann¹⁰, is ideal for LPS extraction.     

Complete genome sequence of the thermophilic sulfate-reducing ocean bacterium Thermodesulfatator indicus type strain (CIR29812(T))

Thermodesulfatator indicus Moussard et al. 2004 is a member of the Thermodesulfobacteriaceae, a family in the phylum Thermodesulfobacteria that is currently poorly characterized at the genome level. Members of this phylum are of interest because they represent a distinct, deep-branching, Gram-negative lineage. T. indicus is an anaerobic, thermophilic, chemolithoautotrophic sulfate reducer isolated from a deep-sea hydrothermal vent. Here we describe the features of this organism, together with the complete genome sequence, and annotation. The 2,322,224 bp long chromosome with its 2,233 protein-coding and 58 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1(T)), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaeta

Spirochaeta coccoides Dröge et al. 2006 is a member of the genus Spirochaeta Ehrenberg 1835, one of the oldest named genera within the Bacteria. S. coccoides is an obligately anaerobic, Gram-negative, non-motile, spherical bacterium that was isolated from the hindgut contents of the termite Neotermes castaneus. The species is of interest because it may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut. Here we provide a taxonomic re-evaluation for strain SPN1^T, and based on physiological and genomic characteristics, we propose its reclassification as a novel species in the genus Sphaerochaeta, a recently published sister group of the Spirochaeta. The 2,227,296 bp long genome of strain SPN1^T with its 1,866 protein-coding and 58 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.