Extraction, purification and characterization of two antiphytoviral substance (s) produced by zucchini yellow mosaic virus

The ability of Pseudomonas viridilivida strain Zagazig University, Faculty of Science (ZUFS) to inhibit Zucchini Yellow Mosaic Virus (ZYMV) in cucumber plants was studied in vitro by mixing it with sap containing virus and in vivo by seed and soil treatments. The production, extraction, purification and characterization of the antiviral substance(s) from strain ZUFS was done. P. viriddivida stimulated the growth of cucumber (Cucumis sativus L.) and inhibit the symptoms of zucchini yellow mosaic virus (ZYMV) by 90%, when mixed with virus suspension in equal volumes (v/v). Treatment of cucumber seeds with bacterial pellets increase the plant growth and showed 100% viral inhibition. The virus concentration was detected by DAS-ELISA technique in leaves of cucumber plants, whose seeds were treated with P. viridilivida. The ELISA value was 0.805 near to the negative control of ZYMV (0.697), while the positive virus control was 1.884. The P. viridili vida detected by electron microscopy inside the root tissue of seed treated cucumber plants, (root-colonizing). The treatment of natural and sterile soil by liquid bacterial culture gave 70 and 89.3% inhibition, the ELISA value of cucumber leaves grown on treated sterile soil was 1.231 compared to (1.884) for positive ZYMV control. ZYMV-infection induced severe modification in number and ultra structure of chloroplasts, where lower number and irregular structure of chloroplasts were observed in ZYMV infected leaf in comparison with control and treated plants. Scrolls, pinwheels, bundles and laminated aggregates were induced in the cytoplasm of infected cells. Physical properties of ZYMV showed that the virus was sap transmissible, its thermal inactivation point is 65 degree C, dilution end point is longevity in vitro is 24 h at room temperature. The antiviral substance (s) produced by culture of P. viridilivida were extracted by using different solvents followed by thin laye r chromatography (TLC) purification. Two spots blue and green detected under UV lamp had Rf of 0.80 and 0.93, respectively. The IR, UV, NMR, mass spectrum and elemental analysis of blue and green compounds showed that the molecular formula of these compounds suggested to be C19H35O4N and C17H34O4N and molecular weight 341 and 316, respectively. Purified substance (s), were tested against ZYMV in cucumber plants giving inhibition 100%. The ELISA values were 0.975, 0.928 for virus concentration in cucumber leaves extract treated with blue and green compounds, respectively in comparison with 1.884 for mechanically inoculated ZYMV.