Sporulation rate in culture and mycoparasitic activity, but not mycohost specificity, are the key factors for selecting Ampelomyces strains for biocontrol of grapevine powdery mildew (Erysiphe necator)

To develop a new biofungicide product against grapevine powdery mildew, caused by Erysiphe necator, cultural characteristics and mycoparasitic activities of pre-selected strains of Ampelomyces spp. were compared in laboratory tests to the commercial strain AQ10. Then, a 2-year experiment was performed in five vineyards with a selected strain, RS1-a, and the AQ10 strain. This consisted of autumn sprays in vineyards as the goal was to reduce the number of chasmothecia of E. necator, and, thus, the amount of overwintering inocula, instead of targeting the conidial stage of the pathogen during spring and summer. This is a yet little explored strategy to manage E. necator in vineyards. Laboratory tests compared the growth and sporulation of colonies of a total of 33 strains in culture; among these, eight strains with superior characteristics were compared to the commercial product AQ10 Biofungicide® in terms of their intrahyphal spread, pycnidial production, and reduction of both asexual and sexual reproduction in E. necator colonies. Mycoparasitic activities of the eight strains isolated from six different powdery mildew species, including E. necator, did not depend on their mycohost species of origin. Strain RS1-a, isolated from rose powdery mildew, showed, togetherwith three strains from E. necator, the highest rate of parasitism of E. necator chasmothecia. In field experiments, each strain, AQ10 and RS1-a, applied twice in autumn, significantly delayed and reduced early-season development of grapevine powdery mildew in the next year. Therefore, instead of mycohost specificity of Ampelomyces presumed in some works, but not confirmed by this study, the high sporulation rate in culture and the mycoparasitic patterns became the key factors for proposing strain RS1-a for further development as a biocontrol agent of E. necator

Reproducibility of adipogenic responses to metabolism disrupting chemicals in the 3T3-L1 pre-adipocyte model system: An interlaboratory study

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812)

Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol

Use of systems analysis to develop plant disease models based on literature data: grape black-rot as a case-study

The available knowledge on black-rot of grape was retrieved from literature, analyzed, and synthesized to develop a mechanistic model of the life cycle of the pathogen (Guignardia bidwelii) based on the systems analysis. An analysis of published, quantitative information was conducted to develop a mechanistic model driven by weather and vine phenology. The model was then evaluated for its ability to represent the real system and its usefulness for understanding black-rot epidemics by using three typical epidemics. Finally, weaknesses in our knowledge are discussed

Effect of temperature and wetness duration on infection by Plasmopara viticola and on post-inoculation efficacy of copper

A weather-driven model was developed to predict P. viticola population dynamics on grape leaf surfaces during a discrete wet period. To develop this model, the relationships between temperature, wetness duration, zoospore release, and infection were investigated with environmentally controlled experiments, and equations were developed that fitted the experimental data

Sporulation rate in culture and mycoparasitic activity, but not mycohost specificity, are the key factors for selecting Ampelomyces strains for biocontrol of grapevine powdery mildew (Erysiphe necator)

To develop a new biofungicide product against grapevine powdery mildew, caused by Erysiphe necator, cultural characteristics and mycoparasitic activities of pre-selected strains of Ampelomyces spp. were compared in laboratory tests to the commercial strain AQ10