Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Structure of hydroxylated galactocerebrosides from myelin at the air-water interface.

Hydroxy-galactocerebrosides (mixed chain length, constituent of myelin membranes) from bovine brain are investigated as monolayers at the air-water interface with isotherms, fluorescence microscopy, x-ray reflectivity and grazing incidence diffraction. With grazing incidence diffraction a monoclinic tilted chain lattice is found in the condensed phase. According to x-ray reflectivity, the longest chains protrude above the chain lattice and roughen the lipid/air interface. On compressing the chain lattice, the correlation length increases by approximately 65%; obviously, the sugar headgroups are flexible enough to allow for lattice deformation. With fluorescence experiments, small coexisting fluid and ordered domains are observed, and there is lipid dissolution into the subphase as well. The dissolved hydroxy-galactocerebroside molecules reenter on monolayer expansion. The electron density profiles derived from x-ray reflectometry (coherent superposition) show that the chain-ordering transition causes the molecules to grow into the subphase.     

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305-316, 1997.     

High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9

The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence‐specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T‐DNA, biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10‐fold enhancements in GT frequencies have been achieved using DNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV‐based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.     

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.     

Ethyl acetate extraction procedure and isocratic high-performance liquid chromatographic assay for testosterone metabolites in cell microsomes

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for separation of testosterone and its main metabolites over the nominal range 20 to 40 μg/ml and 280 to 4600 ng/ml, respectively. Mobile phase composition (phosphate buffer–methanol–acetonitrile, 50:38.5:11.5) was optimised by studying the influence of numerous chromatographic parameters. The most critical one was the ratio CH₃CN/CH₃OH. Good recoveries (around 90% for all compounds) and an improved specificity were assessed by a double ethyl acetate extraction of biological samples. According to the performance criteria tested, the method could be applied to enzymatic inhibition and induction in vitro studies.