RICERCA ITALIANA     

Research program

Methodological innovations for the nursery production of high quality artichoke plantlets

University Co-ordinator

Università degli Studi di BARI - BIOLOGIA E PATOLOGIA VEGETALE - BARI(BA)

Research Unit Leader

Franco CICCARESE

Description

Activities of the Operating Unity "UO5" will regard the evaluation of reaction towards Verticillium dahliae of mycorrizal artichoke seedlings obtained by micropropagation and directly by adult plants grown in healthy soil as offshoot.
Tests will be carried out on mycorrhizal seedlings available at the "UO4" Operating Unity and on new material obtained by the same Unity and by "UO1" Operating Unity during the project. At the same time on mycorrhizal artichoke plants the analysis for establishing the variations of some biochemical constituent of tissues of plant in consequence of colonization of roots by mycorrhizal fungi, will be carried out. Tests on reaction towards Verticillium wilt of mycorrhizal fungi artichoke will be realized in open field on artificial inoculated soil at the experimental field of Faculty. Isolate Vd-20 of V. dahliae, obtained from artichoke plant naturally infected and with high characteristic of virulence will be used.
The inoculum will be prepared growing isolate Vd-20 of V. dahliae on vermiculite added with nutritive solution. The obtained product will be included in the soil. The effect of mycorrhizal fungi will be recorded at three inoculum concentrations. The same tests will be carried out on healthy soil and artichoke plants will be inoculated with isolate Vd-20, before transplanting, by dipping the roots in the fungal suspension at 1x107, 1x105 and 1x103 CFU (Colony forming units) concentrations. The disease progress will be evaluated by periodic records of symptoms using an empiric scale of assessment formed by 6 classes (from 0 to 5) where 0 = healthy plant and 5 = plant with severe symptoms or death plant.
The final record, at the end of growing, will regard the evaluation of vascular browning caused by Verticillium dahliae infection and the isolation of pathogen by plant tissues. In the same experimental conditions and with the same techniques of soil inoculation and of disease symptoms evaluation, the effect of mycorrhizal fungi on artichoke Verticillium wilt will be compared with the antagonistic activity of Aphanocladium album, a biocontrol agent with mycoparasitic and necrotrophic activity for production of hydrolytic enzymes, and with the efficacy of Acibenzolar-S-methyl, analogous of salicylic acid, inducing SAR. The biocontrol agent, A. album, will be grown on vermiculite added with nutritive solution and in this formulation will be included in soil artificially inoculated with V. dahliae.
The foliar treatments with Acibenzolar-S-methyl will include three semestral cycles and, for each cycle, three treatments will be carried out.
The analysis on the variations of some biochemical constituents of plants colonized by mycorrhizal fungi, infected and non infected by V. dahliae, will regard the hydrolytic β-1,3-Glucanase and chitinase, catalase, peroxidase and ascorbic-glutathion cicle components known for their involvement in the defence mechanisms of plants towards the pathogens. In the analysis of β-1,3-glucanase, the crude proteic extract obtained by vegetal material will be centrifugated and an aliquot of supernatant will be used for the assessment of protein contents by the method of Bradford (1976). A soluble fraction of purified β-glucans will be used as substrate for β-1,3-Glucanase activity. The proteic extract will be in contact with the soluble fraction of β-glucans and after electrophoresis and staining, under long wave UV light, the β-1,3-Glucanase activity will be evaluated. The chitinase activity will be analyzed by native electrophoresis in order to gain information on the presence and/or activation of enzyme isoforms. Enzymatic native electrophoresis will be made on polyacrylamide gel. The gels will be incubated in suitable substrate and by staining will be possible to visualize the enzymatic bands.
It will be determined the ascorbic acid dehydroascorbic and reduced and oxidised glutathione content with colorimetric reaction, after having deproteinized the material that will be analized with meta-phosphoric acid (5%).