Biological control by Clonostachys rosea as a key component in the integrated management of strawberry gray mold

Gray mold, caused by Botrytis cinerea, is an important strawberry disease. As gray mold control is difficult, there is a need to evaluate integrated methods to successfully manage the disease. The efficiency of integrating Clonostachys rosea sprays, fungicide sprays, and crop debris removal to manage gray mold was evaluated in field experiments conducted in 2006 and 2007. Leaf colonization by C. rosea (LAC), average number of B. cinerea conidiophores (ANC), gray mold incidence in both flowers (Iflower) and fruits (Ifruit), and yield were evaluated weekly. In both years, LAC was higher in the treatments with no fungicide. When compared to the check, ANC, Iflower and Ifruit were most reduced in treatments that included C. rosea sprays. Maximal reductions were achieved with the combination of C. rosea sprays, fungicide sprays and debris removal (96.62%, 86.54% and 65.33% reductions of ANC, Iflower and Ifruit, respectively). Otherwise, maximal yield (103.14% increase as compared to the check) was achieved with the combination of the three treatments. With just C. rosea sprays, ANC, Iflower and Ifruit were reduced by 92.01%, 68.48% and 65.33%, respectively, whereas yield was increased by 75.15%. Considering the individual effects, application of C. rosea was the most efficient treatment. Chemical control was effective only in plots without debris removal. Elimination of crop debris was the least effective method in reducing gray mold incidence in both flowers and fruits. The integrated control approach enhanced the efficacy of the individual methods of gray mold control and provided high strawberry yield. An important component of this integrated approach it the biological control with C. rosea.     

Dengue Virus 2 American-Asian Genotype Identified during the 2006/2007 Outbreak in Piauí, Brazil Reveals a Caribbean Route of Introduction and Dissemination of Dengue Virus in Brazil

Dengue virus (DENV) is the most widespread arthropod-borne virus, and the number and severity of outbreaks has increased worldwide in recent decades. Dengue is caused by DENV-1, DENV- 2, DENV-3 and DENV-4 which are genetically distant. The species has been subdivided into genotypes based on phylogenetic studies. DENV-2, which was isolated from dengue fever patients during an outbreak in Piaui, Brazil in 2006/2007 was analyzed by sequencing the envelope (E) gene. The results indicated a high similarity among the isolated viruses, as well as to other DENV-2 from Brazil, Central America and South America. A phylogenetic and phylogeographic analysis based on DENV-2E gene sequences revealed that these viruses are grouped together with viruses of the American-Asian genotype in two distinct lineages. Our results demonstrate the co-circulation of two American-Asian genotype lineages in northeast Brazil. Moreover, we reveal that DENV-2 lineage 2 was detected in Piauí before it disseminated to other Brazilian states and South American countries, indicating the existence of a new dissemination route that has not been previously described.     

Endocannabinoids measurement in human saliva as potential biomarker of obesity

Background

The discovery of the endocannabinoid system and of its role in the regulation of energy balance has significantly advanced our understanding of the physiopathological mechanisms leading to obesity and type 2 diabetes. New knowledge on the role of this system in humans has been acquired by measuring blood endocannabinoids. Here we explored endocannabinoids and related N-acylethanolamines in saliva and verified their changes in relation to body weight status and in response to a meal or to body weight loss.

Methodology/Principal Findings

Fasting plasma and salivary endocannabinoids and N-acylethanolamines were measured through liquid mass spectrometry in 12 normal weight and 12 obese, insulin-resistant subjects. Salivary endocannabinoids and N-acylethanolamines were evaluated in the same cohort before and after the consumption of a meal. Changes in salivary endocannabinoids and N-acylethanolamines after body weight loss were investigated in a second group of 12 obese subjects following a 12-weeks lifestyle intervention program. The levels of mRNAs coding for enzymes regulating the metabolism of endocannabinoids, N-acylethanolamines and of cannabinoid type 1 (CB₁) receptor, alongside endocannabinoids and N-acylethanolamines content, were assessed in human salivary glands.The endocannabinoids 2-arachidonoylglycerol (2-AG), N-arachidonoylethanolamide (anandamide, AEA), and the N-acylethanolamines (oleoylethanolamide, OEA and palmitoylethanolamide, PEA) were quantifiable in saliva and their levels were significantly higher in obese than in normal weight subjects. Fasting salivary AEA and OEA directly correlated with BMI, waist circumference and fasting insulin. Salivary endocannabinoids and N-acylethanolamines did not change in response to a meal. CB₁ receptors, ligands and enzymes were expressed in the salivary glands. Finally, a body weight loss of 5.3% obtained after a 12-weeks lifestyle program significantly decreased salivary AEA levels.

Conclusions/Significance

Endocannabinoids and N-acylethanolamines are quantifiable in saliva and their levels correlate with obesity but not with feeding status. Body weight loss significantly decreases salivary AEA, which might represent a useful biomarker in obesity.     

STM2209-STM2208 (opvAB): a phase variation locus of Salmonella enterica involved in control of O-antigen chain length

STM2209 and STM2208 are contiguous loci annotated as putative protein-coding genes in the chromosome of Salmonella enterica. Lack of homologs in related Enterobacteria and low G+C content suggest that S. enterica may have acquired STM2209-STM2208 by horizontal transfer. STM2209 and STM2208 are co-transcribed from a promoter upstream STM2209, and their products are inner (cytoplasmic) membrane proteins. Analysis with the bacterial adenylate cyclase two-hybrid system suggests that STM2209 and STM2208 may interact. Expression of STM2209-STM2208 is subjected to phase variation in wild type Salmonella enterica serovar Typhimurium. Switching frequencies in LB medium are 6.1×10(-5) (OFF→ON) and 3.7×10(-2) (ON→OFF) per cell and generation. Lack of DNA adenine methylation locks STM2209-STM2208 in the ON state, and lack of the LysR-type factor OxyR locks STM2209-STM2208 in the OFF state. OxyR-dependent activation of STM2209-STM2208 expression is independent of the oxidation state of OxyR. Salmonella cultures locked in the ON state show alteration of O-antigen length in the lipopolysaccharide, reduced absorption of bacteriophage P22, impaired resistance to serum, and reduced proliferation in macrophages. Phenotypic heterogeneity generated by STM2209-STM2208 phase variation may thus provide defense against phages. In turn, formation of a subpopulation unable to proliferate in macrophages may restrain Salmonella spread in animal organs, potentially contributing to successful infection.     

Antifungal activity of altenusin isolated from the endophytic fungus Alternaria sp. against the pathogenic fungus Paracoccidioides brasiliensis

BackgroundAltenusin is a biphenyl derivative isolated from different species of fungi, which presents several biological activities.AimsWe report the antifungal activity of the altenusin isolated from the endophytic fungus Alternaria sp., against clinical isolates of Paracoccidioides brasiliensis, and its action on cell walls of P. brasiliensis and the nonpathogenic yeast Schizosaccharomyces pombe.MethodsIn vitro antifungal activity of altenusin was evaluated using the broth microdilution method against 11 strains of P. brasiliensis and one strain of S. pombe. The effects of the altenusin on the cell wall were estimated using the sorbitol protection assay.ResultsThe altenusin presented strong activity against P. brasiliensis with MIC values ranging between 1.9 and 31.2 μg/ml, and 62.5 μg/ml for S. pombe. Our results demonstrated that the MIC values for altenusin were increased for P. brasiliensis Pb18 and for S. pombe when the medium was supplemented with sorbitol. Additionally, S. pombe cells treated with altenusin were more rounded in shape than untreated cells.ConclusionsAltenusin showed activity against clinical strains of P. brasiliensis at the concentration tested, and this compound probably affects fungal cell walls. These findings suggest that altenusin could act through the inhibition of cell wall synthesis or assembly in P. brasiliensis and S. pombe, and could be considered as a lead compound for the design of new antifungals.     

Antifungal and other Biological Activities from Oudemansiella Canarii(Basidiomycota)

Summary The ethyl acetate extract from the fungus Oudemansiella canarii grown in malt extract medium was evaluated against (a) the recombinant enzyme trypanothione reductase from Trypanosoma cruzi, (b) lymphocyte proliferation in human peripheral blood mononuclear cells (PBMCs) stimulated with phytohaemaglutinin, (c) the human tumour cell lineages MCF-7, TK-10 and UACC-62, and (d) the phytopathogenic fungus Cladosporium sphaerospermum. At 10 μg/ml, the crude extract was inactive against PBMC but inhibited the growth of UACC-62 cells by 47% and the enzyme trypanothione reductase (TryR). It also presented strong inhibition in the bioautographic assay with C. sphaerospermum. Chromatographic fractionation guided by this assay allowed the isolation of oudemansin A (1), a known fungitoxic compound that showed a minimum inhibitory concentration (MIC) of 1.25 μg/spot in the bioautographic assay. As oudemansin A was not active in the other assays, other components in the extract may be responsible for the observed activities by the crude extract against the UACC-62 cells or the TryR enzyme.     

Food Intake‐independent Effects of CB1 Antagonism on Glucose and Lipid Metabolism

Overactivity of the endocannabinoid system (ECS) has been linked to abdominal obesity and other risk factors for cardiovascular disease and type 2 diabetes. Conversely, administration of cannabinoid receptor type 1 (CB1) antagonists reduces adiposity in obese animals and humans. This effect is only in part secondary to the anorectic action of CB1 agonists. In order to assess the actions of CB1 antagonism on glucose homeostasis, diet‐induced obese (DIO) rats received the CB1 antagonist rimonabant (10 mg/kg, intraperitoneally (IP)) or its vehicle for 4 weeks, or were pair‐fed to the rimonabant‐treated group for the same length of time. Rimonabant treatment transiently reduced food intake, while inducing body weight loss throughout the study. Rats receiving rimonabant had significantly less body fat and circulating leptin compared to both vehicle and pair‐fed groups. Rimonabant, but not pair‐feeding, also significantly decreased circulating nonesterified fatty acid (NEFA) and triacylglycerol (TG) levels, and reduced TG content in oxidative skeletal muscle. Although no effects were observed during a glucose tolerance test (GTT), rimonabant restored insulin sensitivity to that of chow‐fed, lean controls during an insulin tolerance test (ITT). Conversely, a single dose of rimonabant to DIO rats had no acute effect on insulin sensitivity. These findings suggest that in diet‐induced obesity, chronic CB1 antagonism causes weight loss and improves insulin sensitivity by diverting lipids from storage toward utilization. These effects are independent of the anorectic action of the drug.     

Leishmanicidal Metabolites from Cochliobolus sp., an Endophytic Fungus Isolated from Piptadenia adiantoides (Fabaceae)

Protozoan parasites belonging to genera Leishmania and Trypanosoma are the etiological agents of severe neglected tropical diseases (NTDs) that cause enormous social and economic impact in many countries of tropical and sub-tropical areas of the world. In our screening program for new drug leads from natural sources, we found that the crude extract of the endophytic fungus Cochliobolus sp. (UFMGCB-555) could kill 90% of the amastigote-like forms of Leishmania amazonensis and inhibit by 100% Ellman''s reagent reduction in the trypanothione reductase (TryR) assay, when tested at 20 µg mL⁻¹. UFMGCB-555 was isolated from the plant Piptadenia adiantoides J.F. Macbr (Fabaceae) and identified based on the sequence of the internally transcribed spacer (ITS) regions of its ribosomal DNA. The chromatographic fractionation of the extract was guided by the TryR assay and resulted in the isolation of cochlioquinone A and isocochlioquinone A. Both compounds were active in the assay with L. amazonensis, disclosing EC₅₀ values (effective concentrations required to kill 50% of the parasite) of 1.7 µM (95% confidence interval = 1.6 to 1.9 µM) and 4.1 µM (95% confidence interval = 3.6 to 4.7 µM), respectively. These compounds were not active against three human cancer cell lines (MCF-7, TK-10, and UACC-62), indicating some degree of selectivity towards the parasites. These results suggest that cochlioquinones are attractive lead compounds that deserve further investigation aiming at developing new drugs to treat leishmaniasis. The findings also reinforce the role of endophytic fungi as an important source of compounds with potential to enter the pipeline for drug development against NTDs.