PCR-based sensitive and specific detection of Pectobacterium atrosepticum using primers based on Rhs family gene sequences

A sensitive and specific assay was developed to detect potato blackleg caused by Pectobacterium atrosepticum in potato. Primers PEAF and PEAR from the Rhs family gene homologous to RhsA (cell envelope biogenesis – outer membrane) were used to amplify a 904 bp DNA fragment. PCR was used to detect the pathogen in artificially inoculated potato seed tubers. The PCR product was only produced from 12 isolates of P. atrosepticum from various countries among 36 isolates of other species of Pectobacterium , Pseudomonas , Xanthomonas , as well as Escherichia coli and the soilborne fungus Fusarium oxysporum f.sp. dianthi .     

Distribution of pathotypes with regard to host cultivars in French wheat leaf rust populations

ABSTRACT Isolates of wheat leaf rust collected from durum and bread wheat cultivars in France during 1999-2002 were analyzed for virulence on 18 Thatcher lines with single genes for leaf rust resistance (Lr genes). Sampling focused on the five most widely grown bread wheat cultivars (two susceptible and three resistant) to allow statistical comparison of diversity indexes between the cultivars. Leaf rust populations from durum and bread wheats were different. The diversity of the bread wheat leaf rust pathotypes, as measured by the Shannon index, ranged from 2.43 to 2.76 over the 4 years. Diversity for wheat leaf rust resistance was limited in the host since we postulated only seven seedling resistance genes in the 35 cultivars most widely grown during 1999-2002. Leaf rust populations were strongly differentiated for virulence within bread wheat cultivars, and diversity was higher on those that were resistant, mainly due to a more even distribution of virulence phenotypes than on susceptible cultivars. The pathogen population on the susceptible cv. Soissons was largely dominated by a single pathotype (073100), whereas all other pathotypes virulent on cv. Soissons either decreased in frequency or remained at a low frequency during the period studied. Several pathotypes including the most complex one were found only on resistant cultivars, even though most of them were virulent on the susceptible cv. Soissons. Specific interactions were necessary, but not always sufficient, to account for pathotype distribution and frequencies on the cultivars, suggesting that selection for virulence to host resistance genes is balanced by other selective forces including selection for aggressiveness.     

Fumigant activity of plant essential oils and components from horseradish (Armoracia rusticana), anise (Pimpinella anisum) and garlic (Allium sativum) oils against Lycoriella ingenua (Diptera: Sciaridae)

Plant essential oils from 40 plant species were tested for their insecticidal activities against larvae of Lycoriella ingénue (Dufour) using a fumigation bioassay. Good insecticidal activity against larvae of L. ingenua was achieved with essential oils of Chenopodium ambrosioides L., Eucalyptus globulus Labill, Eucalyptus smithii RT Baker, horseradish, anise and garlic at 10 and 5 microL L(-1) air. Horseradish, anise and garlic oils showed the most potent insecticidal activities among the plant essential oils. At 1.25 microL L(-1), horseradish, anise and garlic oils caused 100, 93.3 and 13.3% mortality, but at 0.625 microL L(-1) air this decreased to 3.3, 0 and 0% respectively. Analysis by gas chromatography-mass spectrometry led to the identification of one major compound from horseradish, and three each from anise and garlic oils. These seven compounds and m-anisaldehyde and o-anisaldehyde, two positional isomers of p-anisaldehyde, were tested individually for their insecticidal activities against larvae of L. ingenua. Allyl isothiocyanate was the most toxic, followed by trans-anethole, diallyl disulfide and p-anisaldehyde with LC(50) values of 0.15, 0.20, 0.87 and 1.47 microL L(-1) respectively.     

The vagus nerve is one route of transneural invasion for intranasally inoculated influenza a virus in mice

Intranasally inoculated neurotropic influenza viruses in mice infect not only the respiratory tract but also the central nervous system (CNS), mainly the brain stem. Previous studies suggested that the route of invasion of virus into the CNS was via the peripheral nervous system, especially the vagus nerve. To evaluate the transvagal transmission of the virus, we intranasally inoculated unilaterally vagectomized mice with a virulent influenza virus (strain 24a5b) and examined the distribution of the viral protein and genome by immunohistochemistry and in situ hybridization over time. An asymmetric distribution of viral antigens was observed between vagal (nodose) ganglia: viral antigen was detected in the vagal ganglion of the vagectomized side 2 days later than in the vagal ganglion of the intact side. The virus was apparently transported from the respiratory mucosa to the CNS directly and decussately via the vagus nerve and centrifugally to the vagal ganglion of the vagectomized side. The results of this study, thus, demonstrate that neurotropic influenza virus travels to the CNS mainly via the vagus nerve.     

Comparison of phenotypic and genotypic methods for the species identification of coagulase-negative staphylococcal isolates from bovine intramammary infections

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system.     

Enhancement of resistance to soft rot (Pectobacterium carotovorum subsp. carotovorum) in transgenic Brassica rapa

Brassica rapa (chinese cabbage) is one of the main vegetable crops grown in Asian countries. Pectobacterium carotovorum subsp. carotovorum causes severe economic loss in this crop as well as in other Brassica crops through soft rot disease. Cysteine proteases like bromelain, papain or ficin show toxic effects to herbivorous insects and pathogenic bacteria. They have been known to be critical factors in plant defence mechanisms. The current study investigated the effect of bromelain gene (BL1) of pineapple (Ananas comosus L. Merrill) on enhancing resistance to soft rot in transgenic Brassica rapa ‘Seoulbaechu’. Three homozygous T₂ lines were inoculated with Pectobacterium carotovorum subsp. carotovorum and BL8-2 line showed the lowest rate of infected leaves (RIL) in both wound inoculation and non-wound inoculation, when the non-infected line showed 100 % RIL in both cases. The highest expression of BL1 gene was also observed in BL8-2 homozygous line. Thus, the over-expressed BL1 gene conferred enhanced resistance to soft rot in Brassica rapa.     

Photoprotection for deltamethrin using chitosan-coated beeswax solid lipid nanoparticles

BACKGROUND: In the natural environment, photodegradation is one of the most common degradative processes of pesticides. In order to reduce the photodegradation of pesticides, and so increase their killing activity against target pests, chitosan‐coated beeswax solid lipid nanoparticles (CH‐BSLNs) were prepared by a combination of hot homogenization and sonication, with deltamethrin as an active ingredient. RESULTS: Under optimal conditions, the highest encapsulation efficiency (95%) and a high payload of deltamethrin (approximately 12.5%) were achieved. In direct photolysis, in the case of CH‐BSLNs after UV irradiation for 24 h, 37.3% of deltamethrin remained, as opposed to only 14.6% of the free‐form deltamethrin. In addition, in indirect photolysis, in the case of CH‐BSLNs after UV irradiation for 2 h in 2% acetone solution, approximately 74.5% of deltamethrin remained, as opposed to only 37.6% of the free‐form deltamethrin. CONCLUSION: CH‐BSLNs showed good protection for deltamethrin against photodegradation. This novel nanocarrier may be useful in crop protection as an economical strategy to enhance the effect of pesticides in the field and protect the environment as well. Copyright © 2012 Society of Chemical Industry     

Identification of <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis> using real-time PCR and high resolution melting (HRM) analysis

To develop a molecular method to identify Pseudoperonospora cubensis in cucumber leaves with signs of downy mildew, we compared the nucleotide sequences of ribosomal DNA-internal transcribed spacer, cytochrome oxidase II, and β-tubulin genes of P. cubensis and P. humuli isolates. Seven single nucleotide polymorphisms (SNPs) distinguished P. cubensis and P. humuli based on variations in β-tubulin sequences, and one specific primer set was designed for further analysis. Real-time PCR and high resolution melting analysis showed that the primer set can be used to specifically identify P. cubensis in cucumber leaves with downy mildew.     

Allometric equations for estimating the aboveground volume of five common urban street tree species in Daegu,Korea

Quantifying urban tree biomass and carbon (C) storage by using allometric equations is required for various studies such as assessing the inventory, modelling, and measuring ecosystem services of urban trees. However, the lack of urban-specific allometric equations leads to uncertainty when estimating urban tree biomass and C storage. Therefore, we followed a nondestructive approach and developed allometric equations specifically for Acer buergerianum Miq., Ginkgo biloba L., Platanus orientalis L., Prunus yedoensis Matsum., and Zelkova serrata (Thunb.) Makino in Daegu, Korea. Diameter at breast height (DBH)-based and DBH-and-height-based allometric equations were highly accurate at estimating the aboveground volume (R² > 0.92), while the allometric equations for P. orientalis and Z. serrata developed for traditional forests overestimated volume by 68% and 427%, respectively. The addition of a height variable into the DBH-based allometric equations did not increase the reliability of the allometric equations at a local level. The mean aboveground C storage of urban street trees was 24.9 Mg C/ha except for P. orientalis with a mean of 69.7 Mg C/ha, and the total aboveground C storage of urban street trees in Daegu was 10.6 Gg C. Alternatively, a generalized allometric equation which compiled species-specific equations can be applied for large-scale estimation. The generalized equations developed in this study and those found in the literature may suggest a constant value (～2.3–2.4) for the scaling exponent in the generalized equations. Allometric equations developed from natural or artificial stands may overestimate the volume of urban street trees; therefore, estimating urban tree biomass and C storage requires urban-specific allometric equations.