Associazione molecolare tra Trichoderma virens e pianta in diversi sistemi

La ricerca di metodi alternativi alla lotta chimica ai patogeni delle piante mediante l’utilizzo di microrganismi ha subito, in particolar modo negli ultimi anni, una notevole espansione. Gli agenti di biocontrollo sono molto vantaggiosi in quanto totalmente privi di rischi per la salute umana e ad impatto ambientale praticamente trascurabile. Nel presente lavoro di tesi è stato preso in esame il ceppo I10 di Trichoderma virens, specie fungina che riveste notevole importanza in ambito biotecnologico in quanto promotore della crescita vegetale, agente di biocontrollo dei fitopatogeni ed induttore di resistenza. Trichoderma riveste un ruolo importante nel controllo biologico delle colture grazie alle sue caratteristiche fisiologiche ed ecologiche: questo microorganismo instaura un’associazione di tipo simbiotico, opportunistico ed avirulento, con la pianta colonizzandone le radici e dovendo perciò penetrare all’interno delle cellule vegetali. Il primo ostacolo alla penetrazione è costituito dai primi strati della parete, ricchi in pectina: tra gli enzimi che degradano la parete cellulare (CWDE: Cell Wall Degrading Enzymes), i pectinolitici sono tra i primi ad essere prodotti. Le endopoligalatturonasi (endoPG), i più efficaci tra gli enzimi peptici, possono avere un ruolo pre-elicitante per lo sviluppo della resistenza, mediante la liberazione di oligogalatturonidi (elicitori) dai costituenti della parete vegetale. Sulla base di queste premesse e dei risultati ottenuti in lavori pregressi, lo scopo di questo lavoro di tesi è stato quello di analizzare l’interazione che il fungo instaura con le radici di Phaseulus vulgaris, Arabidopsis thaliana e Solanum lycopersicum cv. Micro-Tom dal punto di vista molecolare. Per la realizzazione delle analisi sono stati utilizzati due diversi sistemi sperimentali: • Interazione pianta-fungo su perlite in Vasi Microbox da colture in vitro (per Phaseulus vulgaris e Arabidopsis thaliana) • Interazione pianta-fungo su dischi di chellopane in piastre Petri (per Arabidopsis thaliana e Solanum lycopersicum cv. Micro-Tom) Il primo sistema sperimentale consente l’accrescimento di T. virens direttamente sulle radici delle diverse plantule; nel secondo le piantine vengono fatte interagire con il fungo adagiando su di esse un disco di chellopane contenente il micelio fungino, precedentemente cresciuto in incubatore. Nel secondo sistema i due organismi interagiscono pur rimanendo separati dai dischi, così da permettere la raccolta di campioni molto più puliti rispetto al sistema in perlite. Per quanto riguarda pomodoro si sono confrontati i risultati ottenuti in piastra con quelli scaturiti da un precedente lavoro di tesi, realizzato in un sistema sperimentale analogo a quello in perlite utilizzato in questo lavoro. Per Arabidopsis invece si sono realizzate prove in entrambe i sistemi sperimentali considerati. L’associazione molecolare tra T. virens e Fagiolo è stata studiata solamente in perlite. E’ stata indagata l’interazione molecolare che si stabilisce fra T. virens e le radici vegetali delle diverse specie di piante, con particolare attenzione alla degradazione della parete cellulare vegetale da parte del fungo ed alla eventuale reazione inibitoria da parte della pianta che può rappresentare una risposta di resistenza. Allo scopo è stata valutata l’espressione di 2 geni (Tvpg1 ed Tvpg2) codificanti per endopoligalatturonasi in T. virens e l’induzione di: • un gene (corrispondente a Lepgip1, già identificato in pomodoro) codificante per una proteina inibitrice delle endopoligalatturonasi in S. lycopersicum. • due geni (corrispondenti a bpgip1 e bpgip2, precedentemente identificati in fagiolo) codificanti per due proteine inibitrici delle endopoligalatturonasi in P. vulgaris. • due geni (corrispondenti a pgip1 e pgip2, precedentemente identificati in Arabidopsis) codificanti per due proteine inibitrici delle endopoligalatturonasi in A. thaliana. Uno dei due geni del fungo, Tvpg2, è risultato costitutivo in quanto espresso in tutte le condizioni saggiate, inclusi i controlli, in tutti e due i sistemi sperimentali adottati. Relativamente al gene Tvpg1, in una precedente tesi si era osservata, nelle analisi relative all’interazione tra T. virens e S. lycopersicumin in perlite, un’evidente induzione dell’espressione genica fra le 48 e le 72 ore dall’inoculazione del fungo sull’apparato radicale della pianta; allo stesso intervallo di tempo era stata registrata l’espressione del gene Lepgip1 di S. lycopersicum nelle radici inoculate con T. virens. In questo lavoro non si è potuta confermare l’induzione del suddetto gene fungino nell’interazione con pomodoro, saggiata questa volta in un sistema diverso (piastra Petri), in quanto esso non è risultato espresso a nessun tempo nei campioni indotti testati e neppure nei controlli. Non si è riscontrata l’espressione del gene Tvpg1 neppure nei campioni indotti relativi alle altre piante saggiate: i dati ottenuti in questo lavoro suggeriscono la mancata induzione del gene da parte delle radici di pomodoro, nel nuovo sistema testato, e delle altre piante utilizzate, in entrambe i sistemi sperimentali adottati. Per quanto riguarda i geni di pianta codificanti per inibitori delle endopoligalatturonasi, ad esclusione di pomodoro per cui non è mai stata osservata l’espressione del gene Lepgip1, sono stati ottenuti i seguenti risultati: • P. vulgaris → il gene bpgip1 sembra essere costitutivo in quanto si è rilevata la presenza del trascritto ad ogni tempo di campionamento considerato e nel controllo della pianta; il gene bpgip2 presenta lo stesso pattern di espressione risultando anch’esso di tipo costitutivo. • A. thaliana → il gene pgip1 sembra inducibile siccome si è rilevata la presenza del trascritto a 24 ore dall’inoculazione, nel sistema sperimentale in perlite, e a 48 ore nel sistema in piastra Petri, mentre la banda attesa risulta invece assente a tutti gli altri tempi saggiati e nel controllo della pianta; il gene pgip2 è risultato costitutivo in tutti e due i sistemi sperimentali adottati: il trascritto è stato rilevato nei campioni indotti, a tutti e quattro i tempi considerati, e nel controllo della pianta. Le differenze di timing riscontrate nell’espressione del gene pgip1 di Arabidopsis nei due sistemi sono probabilmente dovute alle caratteristiche intrinseche dei due metodi: in perlite, dove il contatto tra i due organismi è diretto, l’espressione del gene in questione si verifica all’incirca 24 ore prima rispetto al sistema in piastra Petri. Ulteriori indagini sono necessarie per avvalorare questi risultati e confermare l’esistenza di una comunicazione molecolare, tra il fungo e le piante prese in considerazione, che coinvolge le funzioni geniche analizzate

Design Method for Contra-Rotating Propellers for High-Speed Crafts: Revising the Original Lerbs Theory in a Modern Perspective

The main theoretical and numerical aspects of a design method for optimum contrar-rotating (CR) propellers for fast marine crafts are presented. We propose a reformulated version of a well-known design theory for contra-rotating propellers, by taking advantage of a new fully numerical algorithm for the calculation of the mutually induced velocities and introducing new features such as numerical lifting surface corrections, use of an integrated modern cavitation/strength criteria, a modified method to consider different numbers of blades among the two propellers, and to allow for an unloading function in the search for the optimal circulation distribution. The paper first introduces the main theoretical principles of the new methods and then discusses the influence of the main design parameters on an emblematic example of application in the case of counter rotating propellers for a pod propulsor designed for fast planing crafts (35 knots and above)

SPH simulation of periodic wave breaking in the surf zone. A detailed fluid dynamic validation

The estimation of wave breaking and run-up on sloped beaches is a relevant issue in different coastal engineering applications. The present study stresses on the capabilities of a Smoothed Particle Hydrodynamics (SPH) solver, with optimal numerical and physical parameters, to accurately simulate the complex flow field in the surf zone and run-up region. Numerical results are compared with high quality experimental measurements of the local flow field in terms of instantaneous and phase averaged values. The selected test case regards the propagation and breaking of regular non-linear waves on a smooth impermeable plane slope. The comparison is based on a complete set of 128 consecutive non-linear regular waves. The level of accuracy of the numerical results and the ability of the model to reproduce the periodic flow in the surf-zone is provided. Current limitations and uncertainty sources are identified and discussed to guide future developments

Short-Term CO2 Treatment of Harvested Grapes (Vitis vinifera L., cv. Trebbiano) before Partial Dehydration Affects Berry Secondary Metabolism and the Aromatic Profile of the Resulting Wine

High CO2 concentrations applied to harvested horticultural products can modify primary and secondary metabolism. This work reports the metabolic responses to short-term CO2 treatments of white-skinned grapes (cv Trebbiano) undergoing postharvest partial dehydration. The influence of CO2 treatments on the aroma profile of the derived sweet wine was also assessed. Harvested grapes were treated with gaseous CO2 (30%) or air (control) for 24 h and then dehydrated (about 45% of weight loss) before vinification. Lipophilic and phenolic compounds of grape skin and the wine aroma profile were analyzed. In CO2-treated berries, the lipophilic and phenolic compounds decreased at a reduced and faster rate, respectively, during dehydration. Aroma profile of wine from CO2-treated grapes showed a slight but significantly higher content of glycosylated C13 and terpene compounds, and a decrease/absence of free acids, vanillin derivates and other phenol volatiles. The higher content of volatile alcohols in wine from treated berries suggests that the alcoholic fermentation was triggered. CO2 application before the withering process of Trebbiano grapes affects the aroma profile of the resulting wine by altering the free:glycosylated volatiles ratio. This study provides information on the possible use of CO2 as metabolic elicitor to modulate the aroma profile of the resulting wines obtained after grape dehydratio

Transition from shear-dominated to Rayleigh-Taylor turbulence

Turbulent mixing layers in nature are often characterized by the presence of a mean shear and an unstable buoyancy gradient between two streams of different velocity. Depending on the relative strength of shear versus buoyancy, either the former or the latter may dominate the turbulence and mixing between the two streams. In this paper, we present a phenomenological theory that leads to the identification of two distinct turbulent regimes: an early regime, dominated by the mean shear, and a later regime dominated by the buoyancy. The main theoretical result consists of the identification of a cross-over time-scale that discerns between the shear- and the buoyancy-dominated turbulence. This cross-over time depends on three large-scale constants of the flow, namely the buoyancy difference, the velocity difference between the two streams, and the gravitational acceleration. We validate our theory against direct numerical simulations (DNSs) of a temporal turbulent mixing layer compounded with an unstable stratification. We observe that the cross-over time correctly predicts the transition from shear to buoyancy driven turbulence, in terms of turbulent kinetic energy production, energy spectra scaling and mixing layer thickness

Universal alignment in turbulent pair dispersion

Countless processes in nature and industry, from rain droplet nucleation to plankton interaction in the ocean, are intimately related to turbulent fluctuations of local concentrations of advected matter. These fluctuations can be described by considering the change of the separation between particle pairs, known as pair dispersion, which is believed to obey a cubic in time growth according to Richardson's theory. Our work reveals a universal, scale-invariant alignment between the relative velocity and position vectors of dispersing particles at a mean angle that we show to be a universal constant of turbulence. We connect the value of this mean angle to Richardson's traditional theory and find agreement with data from a numerical simulation and a laboratory experiment. While the Richardson's cubic regime has been observed for small initial particle separations only, the constancy of the mean angle manifests throughout the entire inertial range of turbulence. Thus, our work reveals the universal nature of turbulent pair dispersion through a geometrical paradigm whose validity goes beyond the classical theory, and provides a novel framework for understanding and modeling transport and mixing processes.Comment: 20 pages, three figure

Fiber Tracking Velocimetry for Two-Point Statistics of Turbulence

We propose and validate a novel experimental technique to measure two-point statistics of turbulent flows. It consists of spreading rigid fibers in the flow and tracking their position and orientation in time and is therefore named “fiber tracking velocimetry.” By choosing different fiber lengths, i.e., within the inertial or dissipative range of scales, the statistics of turbulence fluctuations at the selected length scale can be probed accurately by simply measuring the fiber velocity at its two ends and projecting it along the transverse-to-fiber direction. By means of fully resolved direct numerical simulations and experiments, we show that these fiber-based transverse velocity increments are statistically equivalent to the (unperturbed) flow transverse velocity increments. Moreover, we show that the turbulent energy-dissipation rate can be accurately measured exploiting sufficiently short fibers. The technique is tested against standard particle tracking velocimetry (PTV) of flow tracers with excellent agreement. Our technique overcomes the well-known problem of PTV to probe two-point statistics reliably because of the fast relative diffusion in turbulence that prevents the mutual distance between particles to remain constant at the length scale of interest. This problem, making it difficult to obtain converged statistics for a fixed separation distance, is even more dramatic for natural flows in open domains. A prominent example is oceanic currents, where drifters (i.e., the tracer-particle counterpart used in field measurements) disperse quickly, but at the same time their number has to be limited to save costs. Inspired by our laboratory experiments, we propose pairs of connected drifters as a viable option to solve the issue

CTLA4-Ig interacts with cultured synovial macrophages from rheumatoid arthritis patients and downregulates cytokine production

Introduction: Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.Methods: All macrophages were co-cultured for 24 hours with activated T cells, without or with CTLA4-Ig (10, 100, 500 \u3bcg/ml for 1 hour, 3 hours and overnight, respectively). Immunofluorescence (IF) staining for B7.2, and an analysis of inflammatory cytokine expression (interleukin (IL) -6, tumor necrosis factor (TNF) \u3b1, IL-1\u3b2, transforming growth factor (TGF) \u3b2) by immunocytochemistry (ICC), western blot (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed.Results: Macrophages showed intense B7.2 expression. CTLA4-Ig/B7.2 masking was evident for all macrophages, even after only 1 hour of cell culture (range from 10 to 100 \u3bcg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNF\u3b1, IL-1\u3b2 and TGF\u3b2). Data were confirmed by WB and RT-PCR analysis.Conclusions: Optimal concentrations of CTLA4-Ig for the CTLA4-Ig/B7.2 masking on activated macrophages were identified and were found to induce significant downregulation in the cell production of IL-6, TNF\u3b1, IL1-\u3b2 and TGF\u3b2. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA

Correction: Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages

[This corrects the article DOI: 10.1371/journal.pone.0166433.]