A PCR diagnostic for cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum

A molecular diagnostic was used to examine the conservation of cyclodiene resistance associated mutations between different strains of Tribolium castaneum (Herbst.). An improved insecticide bioassay for discrimination between resistant genotypes was developed and seven resistant strains were established from five different continents. In order to develop a molecular diagnostic a partial cDNA of the cyclodiene insecticide resistance gene Rdl, a γ-aminobutyric-acid-gated chloride-ion channel, was cloned and sequenced. This cDNA spans exon 7, the region containing the resistance-associated mutation, and part of exon 8. An ‘allele-specific’ oligonucleotide primer, carrying the resistance-associated mutation at its 3′ end, was used in combination with a flanking ‘allele-independent’ primer in the polymerase chain reaction to selectively amplify a single resistance-associated mutation from all seven strains collected worldwide. The implications of these findings for the population genetics of insecticide resistance and its management in pest insects via quarantine are discussed.     

Interactions Between Strains of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere of Wheat

ABSTRACT Strains of fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG) are among the most effective rhizobacteria controlling diseases caused by soilborne pathogens. The genotypic diversity that exists among 2,4-DAPG producers can be exploited to improve rhizosphere competence and biocontrol activity. Knowing that D-genotype 2,4-DAPG-producing strains are enriched in some take-all decline soils and that P. fluorescens Q8r1-96, a representative D-genotype strain, as defined by whole-cell repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer, is a superior colonizer of wheat roots, we analyzed whether the exceptional rhizosphere competence of strain Q8r1-96 on wheat is characteristic of other D-genotype isolates. The rhizosphere population densities of four D-genotype strains and a K-genotype strain introduced individually into the soil were significantly greater than the densities of four strains belonging to other genotypes (A, B, and L) and remained above log 6.8 CFU/g of root over a 30-week cycling experiment in which wheat was grown for 10 successive cycles of 3 weeks each. We also explored the competitive interactions between strains of different genotypes inhabiting the same soil or rhizosphere when coinoculated into the soil. Strain Q8r1-96 became dominant in the rhizosphere and in nonrhizosphere soil during a 15-week cycling experiment when mixed in a 1:1 ratio with either strain Pf-5 (A genotype), Q2-87 (B genotype), or 1M1-96 (L genotype). Furthermore, the use of the de Wit replacement series demonstrated a competitive disadvantage for strain Q2-87 or strong antagonism by strain Q8r1-96 against Q2-87 in the wheat rhizosphere. Amplified rDNA restriction analysis and sequence analysis of 16S rDNA showed that species of Arthrobacter, Chryseobacterium, Flavobacterium, Massilia, Microbacterium, and Ralstonia also were enriched in culturable populations from the rhizosphere of wheat at the end of a 30-week cycling experiment in the presence of 2,4-DAPG producers. Identifying the interactions among 2,4-DAPG producers and with other indigenous bacteria in the wheat rhizosphere will help to elucidate the variability in biocontrol efficacy of introduced 2,4-DAPG producers and fluctuations in the robustness of take-all suppressive soils.     

Pesticide resistance via transposition-mediated adaptive gene truncation in Drosophila

To study adaptation, it is essential to identify multiple adaptive mutations and to characterize their molecular, phenotypic, selective, and ecological consequences. Here we describe a genomic screen for adaptive insertions of transposable elements in Drosophila. Using a pilot application of this screen, we have identified an adaptive transposable element insertion, which truncates a gene and apparently generates a functional protein in the process. The insertion of this transposable element confers increased resistance to an organophosphate pesticide and has spread in D. melanogaster recently.     

Coherent two-dimensional nanoscopy

We introduce a spectroscopic method that determines nonlinear quantum mechanical response functions beyond the optical diffraction limit and allows direct imaging of nanoscale coherence. In established coherent two-dimensional (2D) spectroscopy, four-wave-mixing responses are measured using three ingoing waves and one outgoing wave; thus, the method is diffraction-limited in spatial resolution. In coherent 2D nanoscopy, we use four ingoing waves and detect the final state via photoemission electron microscopy, which has 50-nanometer spatial resolution. We recorded local nanospectra from a corrugated silver surface and observed subwavelength 2D line shape variations. Plasmonic phase coherence of localized excitations persisted for about 100 femtoseconds and exhibited coherent beats. The observations are best explained by a model in which coupled oscillators lead to Fano-like resonances in the hybridized dark- and bright-mode response.     

Biological control of take-all by fluorescent Pseudomonas spp. from Chinese wheat fields

Take-all disease of wheat caused by the soilborne fungus Gaeumannomyces graminis var. tritici is one of the most important root diseases of wheat worldwide. Bacteria were isolated from winter wheat from irrigated and rainfed fields in Hebei and Jiangsu provinces in China, respectively. Samples from rhizosphere soil, roots, stems, and leaves were plated onto King's medium B agar and 553 isolates were selected. On the basis of in vitro tests, 105 isolates (19% of the total) inhibited G. graminis var. tritici and all were identified as Pseudomonas spp. by amplified ribosomal DNA restriction analysis. Based on biocontrol assays, 13 strains were selected for further analysis. All of them aggressively colonized the rhizosphere of wheat and suppressed take-all. Of the 13 strains, 3 (HC9-07, HC13-07, and JC14-07, all stem endophytes) had genes for the biosynthesis of phenazine-1-carboxylic acid (PCA) but none had genes for the production of 2,4-diacetylphloroglucinol, pyoluteorin, or pyrrolnitrin. High-pressure liquid chromatography (HPLC) analysis of 2-day-old cultures confirmed that HC9-07, HC13-07, and JC14-07 produced PCA but no other phenazines were detected. HPLC quantitative time-of-flight 2 mass-spectrometry analysis of extracts from roots of spring wheat colonized by HC9-07, HC13-07, or Pseudomonas fluorescens 2-79 demonstrated that all three strains produced PCA in the rhizosphere. Loss of PCA production by strain HC9-07 resulted in a loss of biocontrol activity. Analysis of DNA sequences within the key phenazine biosynthesis gene phzF and of 16S rDNA indicated that strains HC9-07, HC13-07, and JC14-07 were similar to the well-described PCA producer P. fluorescens 2-79. This is the first report of 2-79-like bacteria being isolated from Asia.     

Biological control of Rhizoctonia root rot on bean by phenazine- and cyclic lipopeptide-producing Pseudomonas CMR12a

Pseudomonas CMR12a was previously selected as an efficient biocontrol strain producing phenazines and cyclic lipopeptides (CLPs). In this study, biocontrol capacity of Pseudomonas CMR12a against Rhizoctonia root rot of bean and the involvement of phenazines and CLPs in this ability were tested. Two different anastomosis groups (AGs) of Rhizoctonia solani, the intermediately aggressive AG 2-2 and the highly aggressive AG 4 HGI, were included in growth-chamber experiments with bean plants. The wild-type strain CMR12a dramatically reduced disease severity caused by both R. solani AGs. A CLP-deficient and a phenazine-deficient mutant of CMR12a still protected bean plants, albeit to a lesser extent compared with the wild type. Two mutants deficient in both phenazine and CLP production completely lost their biocontrol activity. Disease-suppressive capacity of CMR12a decreased after washing bacteria before application to soil and thereby removing metabolites produced during growth on plate. In addition, microscopic observations revealed pronounced branching of hyphal tips of both R. solani AGs in the presence of CMR12a. More branched and denser mycelium was also observed for the phenazine-deficient mutant; however, neither the CLP-deficient mutant nor the mutants deficient in both CLPs and phenazines influenced hyphal growth. Together, results demonstrate the involvement of phenazines and CLPs during Pseudomonas CMR12a-mediated biocontrol of Rhizoctonia root rot of bean.     

Construction of a recombinant strain of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> producing both phenazine-1-carboxylic acid and cyclic lipopeptide for the biocontrol of take-all disease of wheat

Four Neofabraea species are responsible for bull’s eye rot, which is an important postharvest disease of apples and pears. The species diversity of its causal agents in Europe has not been thoroughly explored using molecular genetic methods. Eighty-one Neofabraea isolates were obtained mostly from apples with bull’s eye rot symptoms in the Czech Republic over a two year period. The isolates were identified using PCR fingerprinting and DNA sequencing of the ITS rDNA region, the mitochondrial SSU rDNA and the β-tubulin and EF1α genes. The most common species was N. alba (89 %), followed by N. perennans (5 %) and N. kienholzii (5 %). This is the third published record of N. kienholzii in Europe. The species identity of the isolate CPPF507, which was placed close to N. kienholzii, remains unclear. EF1α was shown to be a suitable marker for the identification of species of the genus Neofabraea and was comparable to the previously used β-tubulin gene. Furthermore, the aggressiveness of individual species was compared and species distribution across Europe was summarized. N. perennans and isolate CPPF507 proved to be the most aggressive, whereas the least aggressive was N. kienholzii. Two N. alba isolates isolated from symptomless apple fruits and leaves were pathogenic to apples in the infection tests.     

Vibrational circular-dichroism spectroscopy of homologous cyclic peptides designed to fold into β helices of opposite chirality

Cyclic β-helical peptides have been developed as model structured biomolecules for examining peptide adsorption and conformation on surfaces. As a key prerequisite to circular-dichroism (CD) analysis of these model peptides on surfaces, their conformations and the corresponding vibrational spectra in the 1400-1800 cm?1 range were analyzed by vibrational circular-dichroism (VCD) spectroscopy in solution. The two model peptides ("β Leu and β Val") were examined in chloroform, where they each fold into a homogeneous well-defined antiparallel double-stranded β-helical species, as determined previously by NMR and electronic CD spectroscopy. Because the β-helical conformations of β Leu and β Val are well characterized, the VCD spectra of these peptides can be unambiguously correlated with their structures. In addition, these two β-helical peptides differ from one another in two key respects that make them uniquely advantageous for CD analysis--first, while their backbone conformations are topologically similar, β Leu and β Val form helices of opposite chiralities; second, the two peptides differ in their sequences, i.e., composition of the side chains attached to the backbone. The observed VCD spectra for β Leu and β Val are roughly mirror images of each other, indicating that the VCD features are dominated by the chirality and conformation of the peptide backbone rather than by the peptide sequence. Accordingly, spectra similarly characteristic of peptide secondary structure can be expected for peptides designed to be structural analogs of β Leu and β Val while incorporating a variety of side chains for studies of surface adsorption from organic and aqueous solvents.     

High-throughput identification of catalytic redox-active cysteine residues

Cysteine (Cys) residues often play critical roles in proteins; however, identification of their specific functions has been limited to case-by-case experimental approaches. We developed a procedure for high-throughput identification of catalytic redox-active Cys in proteins by searching for sporadic selenocysteine-Cys pairs in sequence databases. This method is independent of protein family, structure, and taxon. We used it to selectively detect the majority of known proteins with redox-active Cys and to make additional predictions, one of which was verified. Rapid accumulation of sequence information from genomic and metagenomic projects should allow detection of many additional oxidoreductase families as well as identification of redox-active Cys in these proteins.