Implementation of IPM programs on European greenhouse tomato production areas: Tools and constraints

Whiteflies and whitefly-transmitted viruses are some of the major constraints on European tomato production. The main objectives of this study were to: identify where and why whiteflies are a major limitation on tomato crops; collect information about whiteflies and associated viruses; determine the available management tools; and identify key knowledge gaps and research priorities. This study was conducted within the framework of ENDURE (European Network for Durable Exploitation of Crop Protection Strategies). Two whitefly species are the main pests of tomato in Europe: Bemisia tabaci and Trialeurodes vaporariorum. Trialeurodes vaporariorum is widespread to all areas where greenhouse industry is present, and B. tabaci has invaded, since the early 1990’s, all the subtropical and tropical areas. Biotypes B and Q of B. tabaci are widespread and especially problematic. Other key tomato pests are Aculops lycopersici, Helicoverpa armigera, Frankliniella occidentalis, and leaf miners. Tomato crops are particularly susceptible to viruses causingTomato yellow leaf curl disease (TYLCD). High incidences of this disease are associated to high pressure of its vector, B. tabaci. The ranked importance of B. tabaci established in this study correlates with the levels of insecticide use, showing B. tabaci as one of the principal drivers behind chemical control. Confirmed cases of resistance to almost all insecticides have been reported. Integrated Pest Management based on biological control (IPM-BC) is applied in all the surveyed regions and identified as the strategy using fewer insecticides. Other IPM components include greenhouse netting and TYLCD-tolerant tomato cultivars. Sampling techniques differ between regions, where decisions are generally based upon whitefly densities and do not relate to control strategies or growing cycles. For population monitoring and control, whitefly species are always identified. In Europe IPM-BC is the recommended strategy for a sustainable tomato production. The IPM-BC approach is mainly based on inoculative releases of the parasitoids Eretmocerus mundus and Encarsia formosa and/or the polyphagous predators Macrolophus caliginosus and Nesidiocoris tenuis. However, some limitations for a wider implementation have been identified: lack of biological solutions for some pests, costs of beneficials, low farmer confidence, costs of technical advice, and low pest injury thresholds. Research priorities to promote and improve IPM-BC are proposed on the following domains: (i) emergence and invasion of new whitefly-transmitted viruses; (ii) relevance of B. tabaci biotypes regarding insecticide resistance; (iii) biochemistry and genetics of plant resistance; (iv) economic thresholds and sampling techniques of whiteflies for decision making; and (v) conservation and management of native whitefly natural enemies and improvement of biological control of other tomato pests.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConA looked very similar. However, docking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, revealed conformational changes in side chains of the amino acid residues involved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConA requires conformational chances of its monosaccharide-binding site.

Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia.

Methylmalonic and propionic acidemia (MMA/PA) are inborn errors of metabolism characterized by accumulation of propionic acid and/or methylmalonic acid due to deficiency of methylmalonyl-CoA mutase (MUT) or propionyl-CoA carboxylase (PCC). MMA has an estimated incidence of ~ 1: 50,000 and PA of ~ 1:100'000 -150,000. Patients present either shortly after birth with acute deterioration, metabolic acidosis and hyperammonemia or later at any age with a more heterogeneous clinical picture, leading to early death or to severe neurological handicap in many survivors. Mental outcome tends to be worse in PA and late complications include chronic kidney disease almost exclusively in MMA and cardiomyopathy mainly in PA. Except for vitamin B12 responsive forms of MMA the outcome remains poor despite the existence of apparently effective therapy with a low protein diet and carnitine. This may be related to under recognition and delayed diagnosis due to nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity.

Diethyldithiocarbamic acid inhibits the octadecanoid signaling pathway for the wound induction of proteinase-inhibitors in tomato leaves

The induction of proteinase inhibitor I synthesis in tomato (Lycopersicon esculentum) leaves in response to wounding is strongly inhibited by diethyldithiocarbamic acid (DIECA). DIECA also inhibits the induction of inhibitor I synthesis by the 18-amino acid polypeptide systemin, polygalacturonic acid (PCA), and linolenic acid, but not by jasmonic acid, suggesting that DIECA interferes with the octadecanoid signaling pathway. DIECA only weakly inhibited tomato lipoxygenase activity, indicating that DIECA action occurred at a step after the conversion of linolenic acid to 13(S)-hydroperoxylinolenic acid (HPOTrE). DIECA was shown to efficiently reduce HPOTrE to 13-hydroxylinolenic acid (HOTrE), which is not a signaling intermediate. Therefore, in vivo, DIECA is likely inhibiting the signaling pathway by shunting HPOTrE to HOTrE, thereby severely reducing the precursor pool leading to cyclization and eventual synthesis of jasmonic acid. Phenidone, an inhibitor of lipoxygenase, inhibited proteinase inhibitor I accumulation in response to wounding, further supporting a role for its substrate, linolenic acid, and its product, HPOTrE, as components of the signal-transduction pathway that induces proteinase inhibitor synthesis in response to wounding, systemin, and PCA.

Regulation of gastric and pancreatic lipase secretion by CCK and cholinergic mechanisms in humans.

Gastric lipase (HGL) contributes significantly to fat digestion. However, little is known about its neurohormonal regulation in humans. We studied the role of CCK and cholinergic mechanisms in the postprandial regulation of HGL and pancreatic lipase (HPL) secretion in six healthy subjects. Gastric emptying of a mixed meal and outputs of HGL, pepsin, acid, and HPL were determined with a double-indicator technique. Three experiments were performed in random order: intravenous infusion of 1) placebo, 2) low-dose atropine (5 micrograms.kg-.h-1), and 3) the CCK-A receptor antagonist loxiglumide (22 mumol.kg-.h-1). Atropine decreased postprandial outputs of HGL, pepsin, gastric acid, and HPL (P < 0.03) while slowing gastric emptying (P < 0.05). Loxiglumide markedly increased the secretion of HGL, pepsin, and acid while distinctly reducing HPL outputs and accelerating gastric emptying (P < 0.03). Plasma CCK and gastrin levels increased during loxiglumide infusion (P < 0.03). Atropine enhanced gastrin but not CCK release. Postprandial HGL, pepsin, and acid secretion are under positive cholinergic but negative CCK control, whereas HPL is stimulated by cholinergic and CCK mechanisms. We conclude that CCK and cholinergic mechanisms have an important role in the coordination of HGL and HPL secretion to optimize digestion of dietary lipids in humans.

Coffee consumption and digestive tract cancers.

The relationship between coffee drinking and the risk of digestive tract neoplasms was analyzed in a case-control study of 50 cases of cancer of the mouth or pharynx, 209 of the esophagus, 397 of the stomach, 455 of the colon, 295 of the rectum, 151 of the liver, 214 of the pancreas, and 1944 control subjects admitted for acute, non-digestive tract disorders. There was no significant or consistent association between coffee and cancers of the mouth or pharynx, esophagus, stomach, liver, or pancreas. In particular, for pancreatic cancer, the multivariate relative risks for the intermediate and upper tertiles were 1.05 and 1.01, respectively. There were significant inverse trends in risk with measures of coffee consumption for colon and rectal cancers, the multivariate relative risks according to tertiles of coffee consumption being 0.86 and 0.64 for colon and 0.97 and 0.66 for rectum. This apparent protection is in agreement with some (but not all) previous epidemiological evidence and finds a possible biological interpretation in terms of interference on bile secretion, causing reduced bile acid and neutral sterol concentrations in the bowel. In conclusion, the results of this study, the major interest of which lies in the opportunity of drawing up an overall pattern of risk for various digestive neoplasms, offer further reassurance as regards the effects of coffee on digestive tract carcinogenesis.

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

Carbon isotopic ratio analysis by gas chromatography/combustion/isotope ratio mass spectrometry for the detection of gamma-hydroxybutyric acid (GHB) administration to humans.

Since GHB (gamma-hydroxybutyric acid) is naturally produced in the human body, clinical and forensic toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. To suggest an alternative to the use of interpretative concentration cut-offs, the detection of exogenous GHB in urine specimens was investigated by means of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GHB was isolated from urinary matrix by successive purification on Oasis MCX and Bond Elute SAX solid-phase extraction (SPE) cartridges prior to high-performance liquid chromatography (HPLC) fractioning using an Atlantis dC18 column eluted with a mixture of formic acid and methanol. Subsequent intramolecular esterification of GHB leading to the formation of gamma-butyrolactone (GBL) was carried out to avoid introduction of additional carbon atoms for carbon isotopic ratio analysis. A precision of 0.3 per thousand was determined using this IRMS method for samples at GHB concentrations of 10 mg/L. The (13)C/(12)C ratios of GHB in samples of subjects exposed to the drug ranged from -32.1 to -42.1 per thousand, whereas the results obtained for samples containing GHB of endogenous origin at concentration levels less than 10 mg/L were in the range -23.5 to -27.0 per thousand. Therefore, these preliminary results show that a possible discrimination between endogenous and exogenous GHB can be made using carbon isotopic ratio analyses.

Inducible malondialdehyde pools in zones of cell proliferation and developing tissues in Arabidopsis.

Malondialdehyde (MDA) is a natural and widespread genotoxin. Given its potentially deleterious effects, it is of interest to establish the identities of the cell types containing this aldehyde. We used in situ chemical trapping with 2-thiobarbituric acid and mass spectrometry with a deuterated standard to characterize MDA pools in the vegetative phase in Arabidopsis thaliana. In leaves, MDA occurred predominantly in the intracellular compartment of mesophyll cells and was enriched in chloroplasts where it was derived primarily from triunsaturated fatty acids (TFAs). High levels of MDA (most of which was unbound) were found within dividing cells in the root tip cell proliferation zone. The bulk of this MDA did not originate from TFAs. We confirmed the localization of MDA in transversal root sections. In addition to MDA in proliferating cells near the root tip we found evidence for the presence of MDA in pericyle cells. Remodeling of non-TFA-derived MDA pools occurred when seedlings were infected with the fungus Botrytis cinerea. Treatment of uninfected seedlings with mediators of plant stress responses (jasmonic acid or salicylic acid) increased seedling MDA levels over 20-fold. In summary, major pools of MDA are associated with cell division foci containing stem cells. The aldehyde is pathogen-inducible in these regions and its levels are increased by cellular mediators that impact defense and growth.

Toxicokinetics of captan and folpet biomarkers in dermally exposed volunteers

To better assess biomonitoring data in workers exposed to captan and folpet, the kinetics of ring metabolites [tetrahydrophthalimide (THPI), phthalimide (PI) and phthalic acid] were determined in urine and plasma of dermally exposed volunteers. A 10  mg kg(-1) dose of each fungicide was applied on 80  cm(2) of the forearm and left without occlusion or washing for 24  h. Blood samples were withdrawn at fixed time periods over the 72  h following application and complete urine voids were collected over 96  h post-dosing, for metabolite analysis. In the hours following treatment, a progressive increase in plasma levels of THPI and PI was observed, with peak levels being reached at 24  h for THPI and 10  h for PI. The ensuing elimination phase appeared monophasic with a mean elimination half-life (t(½) ) of 24.7 and 29.7 h for THPI and PI, respectively. In urine, time courses PI and phthalic acid excretion rate rapidly evolved in parallel, and a mean elimination t(½) of 28.8 and 29.6  h, respectively, was calculated from these curves. THPI was eliminated slightly faster, with a mean t(½) of 18.7  h. Over the 96  h period post-application, metabolites were almost completely excreted, and on average 0.02% of captan dose was recovered in urine as THPI while 1.8% of the folpet dose was excreted as phthalic acid and 0.002% as PI, suggesting a low dermal absorption fraction for both fungicides. This study showed the potential use of THPI, PI and phthalic acid as key biomarkers of exposure to captan and folpet.

Dietary proteins and atherosclerosis.

More than one hundred years ago the "protein hypothesis" of the pathogenesis of atherosclerosis and its association with cardiovascular disease was put forward on the basis of animal experiments; however, it has so far never been verified in humans. This theory was soon replaced by the "lipid hypothesis", which was confirmed in humans as of 1994. Epidemiological ecological studies in the 1960 s showed significant associations between dietary animal protein and mortality from cardiovascular disease. However, animal protein intake was also significantly correlated with saturated fatty acid and cholesterol intake. In the last decades two prospective cohort studies demonstrated a decreased cardiovascular risk in women during high- versus low-protein intake when adjusting for other dietary factors (e. g., saturated fats) and other cardiovascular risk factors. A direct cholesterol lowering effect of proteins has not been shown. Despite earlier research indicating that soy protein has cardioprotective effects as compared to other proteins, these observations have not been confirmed by randomized placebo-controlled trials. However, most experts recommend the consumption of foods rich in plant proteins as alternatives to meat and dairy products rich in saturated fat and containing cholesterol. There are no scientific arguments to increase the daily protein intake to more than 20 % of total energy intake as recommended by the guidelines, in order to improve cardiovascular health.

Are light traps baited with kairomones effective in the capture of Lutzomyia longipalpis and Lutzomyia intermedia? An evaluation of synthetic human odor as an attractant for phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae)

Phlebotomine sand flies are often captured with human bait and/or light traps, either with or without an animal bait. More recently, synthetic attractants have been used as bait in traps to improve the capture of phlebotomine sand flies as well as other insects of medical and veterinary importance. The aim of the present study was to evaluate the effects of the kairomone 1-octen-3-ol (octenol) and the synthetic human odor BG-Mesh LureTM (BGML - lactic acid, caproic acid and ammonia) baited in modified CDC light traps on the capture of phlebotomine sand flies. The experiments followed the 5x5 Latin square design. Among the species caught, Lutzomyia intermedia apparently presented a dose-dependent response to octenol. The response obtained with the BGML, alone or in combination with octenol (5 mg/h), indicated some degree of attractiveness of these baits to different phlebotomine sand fly species. Octenol seems to be more attractive to L. intermedia than to Lutzomyia longipalpis, while the BGML presented a higher success in capturing L. longipalpis. When the components of the BGML were used separately, there was no increase in catching the female of L. intermedia. Apparently, there was no synergistic effect between the octenol and the BGML. In conclusion, the octenol and the BGML were demonstrated to be possible baits to attract some phlebotomine sand fly species.

Divinyl ether fatty acid synthesis in late blight-diseased potato leaves.

We conducted a study of the patterns and dynamics of oxidized fatty acid derivatives (oxylipins) in potato leaves infected with the late-blight pathogen Phytophthora infestans. Two 18-carbon divinyl ether fatty acids, colneleic acid and colnelenic acid, accumulated during disease development. To date, there are no reports that such compounds have been detected in higher plants. The divinyl ether fatty acids accumulate more rapidly in potato cultivar Matilda (a cultivar with increased resistance to late blight) than in cultivar Bintje, a susceptible cultivar. Colnelenic acid reached levels of up to approximately 24 nmol (7 microgram) per g fresh weight of tissue in infected leaves. By contrast, levels of members of the jasmonic acid family did not change significantly during pathogenesis. The divinyl ethers also accumulated during the incompatible interaction of tobacco with tobacco mosaic virus. Colneleic and colnelenic acids were found to be inhibitory to P. infestans, suggesting a function in plant defense for divinyl ethers, which are unstable compounds rarely encountered in biological systems.

Construction and Quantitative Evaluation of a Dual Specific Promoter System for Monitoring the Expression Status of Stra8 and c-kit Genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

Detection of live and antibiotic-killed bacteria by quantitative real-time PCR of specific fragments of ribosomal RNA

RÉSUMÉ Le but d'un traitement antimicrobien est d'éradiquer une infection bactérienne. Cependant, il est souvent difficile d'en évaluer rapidement l'efficacité en utilisant les techniques standard. L'estimation de la viabilité bactérienne par marqueurs moléculaires permettrait d'accélérer le processus. Ce travail étudie donc la possibilité d'utiliser le RNA ribosomal (rRNA) à cet effet. Des cultures de Streptococcus gordonii sensibles (parent Wt) et tolérants (mutant Tol 1) à l'action bactéricide de la pénicilline ont été exposées à différents antibiotiques. La survie bactérienne au cours du temps a été déterminée en comparant deux méthodes. La méthode de référence par compte viable a été comparée à une méthode moléculaire consistant à amplifier par PCR quantitative en temps réel une partie du génome bactérien. La cible choisie devait refléter la viabilité cellulaire et par conséquent être synthétisée de manière constitutive lors de la vie de la bactérie et être détruite rapidement lors de la mort cellulaire. Le choix s'est porté sur un fragment du gène 16S-rRNA. Ce travail a permis de valider ce choix en corrélant ce marqueur moléculaire à la viabilité bactérienne au cours d'un traitement antibiotique bactéricide. De manière attendue, les S. gordonii sensibles à la pénicilline ont perdu ≥ 4 log10 CFU/ml après 48 heures de traitement par pénicilline alors que le mutant tolérant Tol1 en a perdu ≥ 1 log10 CFU/ml. De manière intéressant, la quantité de marqueur a augmenté proportionnellement au compte viable durant la phase de croissance bactérienne. Après administration du traitement antibiotique, l'évolution du marqueur dépendait de la capacité de la bactérie à survivre à l'action de l'antibiotique. Stable lors du traitement des souches tolérantes, la quantité de marqueur détectée diminuait de manière proportionnelle au compte viable lors du traitement des souches sensibles. Cette corrélation s'est confirmée lors de l'utilisation d'autres antibiotiques bactéricides. En conclusion, l'amplification par PCR du RNA ribosomal 16S permet d'évaluer rapidement la viabilité bactérienne au cours d'un traitement antibiotique en évitant le recours à la mise en culture dont les résultats ne sont obtenus qu'après plus de 24 heures. Cette méthode offre donc au clinicien une évaluation rapide de l'efficacité du traitement, particulièrement dans les situations, comme le choc septique, où l'initiation sans délai d'un traitement efficace est une des conditions essentielles du succès thérapeutique. ABSTRACT Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether ribosomal RNA (rRNA) could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts, and compared to quantitative real-time PCR amplification of either the 16S-rRNA genes (rDNA) or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost ≥ 4 log10 CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Toll mutant lost ≤ 1 log10 CFU/ml. Amplification of a 427-base fragment of 16S rDNA yielded amplicons that increased proportionally to viable counts during bacterial growth, but did not decrease during drug-induced killing. In contrast, the same 427-base fragment amplified from 16S rDNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Toll mutant (≥4 log10 CFU/ml and ≤1 lo10 CFU/ml, respectively), and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments the experiments were repeated by amplifying a 119-base region internal to the origina1 427-base fragment. The amount of 119-base amplicons increased proportionally to viability during growth, but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical to differentiate between live and dead bacteria.