Over-expression of a scopoletin glucosyltransferase in Nicotiana tabacum leads to precocious lesion formation during the hypersensitive response to tobacco mosaic virus but does not affect virus resistance

Nicotiana tabacum Togt encodes a scopoletin glucosyltransferase (UDPglucose:scopoletin O -beta-D-glucosyltrans- ferase, EC 2.4.1.128) known to act in vitro on many different substrates including the 6-methoxy-7-hydroxy- coumarin scopoletin. This phenolic compound accumulates in vast amounts, essentially in its glucosylated form scopolin, in tobacco during the hypersensitive response (HR) to tobacco mosaic virus (TMV). To identify the physiological role of this pathogen-inducible UDP-Glc glucosyltransferase (UGT), we generated TOGT over-expressing transgenic plants. Although no endogenous scopoletin or scopolin could be detected before infection, the accumulation of both the aglycone and the glucoside was found to be 2-fold higher in transgenic plants after inoculation with TMV than in wild-type plants. Scopoletin UGT activity in plants over-expressing Togt was significantly higher during the HR than in control plants. This up-regulated activity was associated with a strong increase of the bright blue fluorescence surrounding the HR-necrotic lesions under UV light, which is known to correlate with scopoletin and scopolin abundance. Necrosis appeared sooner in transgenic plants and lesions developed faster, suggesting an accelerated HR. Unexpectedly, the viral content in each lesion was not significantly different in transgenic and in wild-type plants. These results are discussed in relation to the role of TOGT as the major UDP-Glc: scopoletin glucosyltransferase and to the importance of scopoletin accumulation during the HR.     

Prey stage preference and functional response of Euseius hibisci to Tetranychus urticae (Acari: Phytoseiidae, Tetranychidae)

The aims of this study were: (a) determine the prey stage preference of female Euseius hibisci (Chant) (Phytoseiidae) at constant densities of different stages of Tetranychus urticae Koch (Tetranychidae), (b) assess the functional response of the predator females to the varying densities of eggs, larvae, or protonymphs of T. urticae, and (c) estimate the functional response of E. hibisci when pollen of Ligustrum ovalifolium was present as well. We conducted experiments on excised pieces of strawberry leaf arenas (Fragaria ananassa) under laboratory conditions of 25 ± 2 °C, 60 ± 5% RH and 12 h photophase. Our results indicated that the predator consumed significantly more prey eggs than other prey stages. Consumption of prey deutonymphs and adults was so low that they were excluded from the non-choice functional response experiments. The functional response on all food items was of type II. The two parameters of the functional response were estimated for each prey type by means of the adjusted non-linear regression model. The highest estimated value a (instantaneous rate of discovery) and the lowest value of T_h (handling time, including digestion) were found for the predator feeding on prey eggs, and a was lowest and T_h highest when fed protonymphs. Using the jack-knife method, the values for the functional response parameters were estimated. The values of a and T_h produced by the model were similar among all prey types except for the eggs, which were different. Using pollen simultaneously with prey larvae decreased the consumption of the latter over the full range of prey densities The suitability of this predator for biological control of T. urticae on strawberry is discussed.     

Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.     

Adaptive response and split-dose effect of radiation on the survival of mice

Although the importance of radiation-induced adaptive response has been recognized in human health, risk assessment and clinical application, the phenomenon has not been understood well in terms of survival of animals. To examine this aspect Swiss albino mice were irradiated with different doses (2–10 Gy) at 0015 Gy/s dose rate and observed on a regular basis for 30 days. Since almost 50% lethality was seen with 8 Gy, it was selected as the challenging dose for further studies. Irradiation of mice with conditioning doses (0.25 or 0.5 Gy) and subsequent exposure to 8 Gy caused significant increase in the survival of mice compared to irradiated control. The splitting of challenging dose did not influence the efficiency of conditioning doses (0.25 Gy and 0.5 Gy) to induce an adaptive response. However conditioning doses given in fractions (0.25 Gy + 0.25 Gy) or (0.5 Gy + 0.5 Gy) were able to modulate the response of challenging dose of 8 Gy. These results clearly showed the occurrence of adaptive response in terms of survival of animals. The conditioning dose given in small fractions seemed to be more effective. The findings have been discussed from a mechanistic point of view. The possible biological implications, potential medical benefits, uncertainties and controversies related to adaptive response have also been addressed     

Design, synthesis and anti-microbial activity of 1H-pyrazole carboxylates

In a SAR study, we have synthesized a few 1H-pyrazole carboxylate related microbicides using Vilsmeier reagent. The anti-microbial screening results of 1H-pyrazole-3-carboxylate are reported here for the first time. The effect of 1H-pyrazole carboxylates on the mycelial growth of plant pathogenic fungi is revealed. The first X-ray structure in the family of microbicidal 1H-pyrazole-4-carboxylates is presented.     

Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli

AIMS: To investigate the influence of the source of tryptone in the growth medium on the resistance of Escherichia coli to various types of oxidative stress. METHODS AND RESULTS: Cultures of Escherichia coli MG1655 were grown in Luria-Bertani (LB) medium at 37 degrees C to stationary phase, harvested, and subsequently subjected to various types of oxidative stress. A marked difference in oxidative stress sensitivity was observed depending on the origin of the tryptone in the LB medium used to grow the cultures. Cells harvested from LB containing tryptone from source x (LBx) were more sensitive to inactivation by the superoxide generating compound plumbagin and by t-butyl peroxide, and to growth inhibition by the lactoperoxidase enzyme system, than cells harvested from LB containing tryptone from source y (LBy). By monitoring expression of a panel of stress gene promotors linked to the gfp (green fluorescent protein) gene, and using Delta2-22 alkaline phosphatase as a probe for disulphide bridge formation from protein sulphydryl groups, it was demonstrated that a greater cytoplasmic oxidative stress existed in cells during growth in LBy than in LBx. CONCLUSIONS: Depending on the source of tryptone, bacteria may experience different levels of oxidative stress in tryptone-containing nonselective growth media. Although these levels of oxidative stress are subinhibitory, they may trigger a stress response that makes the bacteria more resistant to a subsequent exposure to a lethal or inhibitory level of oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the importance of controlling very subtle differences in composition of nonselective growth media in studies on bacterial physiology.     

The relationship between concentration of a dual marker strain of Salmonella Typhimurium in bovine faeces and its probability of detection by immunomagnetic separation and culture

AIMS: To modify a strain of Salmonella serotype Typhimurium to express unique marker traits and then define how the concentration of the marker in bovine faeces affects the probability of its detection by culture preceded by immunomagnetic separation (IMS). METHODS AND RESULTS: DNA encoding for the production of green fluorescent protein (gfp) and resistance to kanamycin was inserted into the bacterial chromosome of Salm. Typhimurium. Transposon insertion was demonstrated by Southern blot hybridization. Varying amounts of one electroporant (gfpSal-1) were inoculated into suspensions of bovine faeces and attempts made to isolate gfpSal-1 using a protocol based on pre-enrichment incubation, IMS and enrichment in selective media. Isolates of gfpSal-1 were differentiated from wild strains of Salmonella using fluorescence under u.v. light and expression of kanamycin resistance. A logistic and Gompertz function each derived from the dose-response data partially explained the observations with the fit of the Gompertz function judged to be superior. The 10, 50 and 90% limits of detection from the Gompertz function were estimated to be 1.92, 2.03 and 2.27 CFU g(-1) respectively. CONCLUSIONS: Reliance on the traditional concept of 'limit of detection' could introduce unacceptable errors in the interpretation of test findings when the concentration of Salm. Typhimurium in bovine faeces (pooled or individual) is below ca 3 CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The dose-response curve can be used to aid the design of protocols for detecting Salmonella in individual and pooled faecal specimens. The experiments demonstrate that both reporter genes in tandem are useful for studying the performance of culture-based methods for detecting pathogens in faeces.     

Production and purification of recombinant DP1B silk-like protein in plants

Spider dragline silk of Nephila clavipes consists of two highly repetitive proteins, spidroin 1 and spidroin 2. To develop a plant platform for production of recombinant silk-like protein, two plant-optimized DP1B genes were synthesized to mimic spidroin 1. DP1B-8P encodes for a 64 kD DP1B silk-like protein and DP1B-16P for a 127 kD DP1B silk-like protein. Both genes have been introduced into Arabidopsis for leaf-based production driven by the 35S promoter and for seed-specific production driven by the -conglycinin subunit promoter, respectively. They have also been expressed in somatic soybean embryos under the control of the -conglycinin subunit promoter. The results demonstrate the synthesis and accumulation of DP1B silk-like protein in leaves and seeds of Arabidopsis, as well as in somatic soybean embryos. They suggest that seeds are the more preferred tissue for DP1B production since they offer higher production yield and quality. In addition, a simplified protocol for purifying DP1B from plant tissue is discussed.     

A practical approach to assess the native status of a rare plant species: the controverse of Buxus sempervirens L. in northern France revisited

Whether a species is native or introduced in a given geographic area is of major interest within the framewok of biological conservation. A practical approach combining phytosociological, ecological, phytogeographical and historical data is proposed to explore this status for rare plant species, and applied to Boxtree in northern France. Buxus sempervirens L. is an evergreen sub-mediterranean species whose wild populations in northern France are very rare and threatened. Its status – native or introduced – has long been be controversial. Three types of Box-woodland were found in the study area: 1) a Taxus baccata – Buxus sempervirens community which is strongly linked to post-Renaissance castles, 2) a Fraxinus excelsior–Mercurialis perennis community which may be related both to steep chalk slopes where Box was expected to be native and to Medieval castles, 3) a Quercus pubescens–Buxus sempervirens community which may be considered as an immature anthropogenic woodland. In all cases Buxus sempervirens was observed close to archaeological sites and together with exotic and/or nitrophilous plant species. Consequently Box is probably originally an introduced species in northern France and should be considered as both an archaeophyte and a feudal plant. This method offers an interesting alternative to determine the indigenity status of a rare plant species in its localities that would provide sufficiently accurate criteria in most of the cases.