Studies on Phytoplasma Seed Transmission in Different Biological Systems

The transmission of phytoplasmas by seed in Sesamum indicum, Brassica napus, Solanum lycopersicum and Zea mays, was studied. The seeds, derived from infected mother-plants, were sown in sterile substrates and their germination percentage was evaluated. The seedlings were analyzed for the presence of phytoplasmas in different stages of growth by "nested"-PCR/RFLP analysis. The four species resulted positive for phytoplasmas belonging to different ribosomal groups and the number of positive plants decreased in the later stages of growth. For S. lycopersicum samples the presence of phytoplasmas was also analyzed in second generation plants: 7 seedlings out of the 60 tested resulted positive for phytoplasmas. Phytoplasma isolation in artificial medium CB was performed for all samples resulted positive, to verify the viability of the phytoplasmas. From Z. mays samples, colonies positive to phytoplasmas belonging to ribosomal groups 16SrI and 16SrXII were obtained. These colonies positive to 16SrI were reproducible for at least three subsequent passages liquid/solid media carried out every 5 days. Some of these samples produced colonies also from broth maintained for seven months at 25°C after isolation. These preliminary results indicate the viability of 16SrI phytoplasmas isolated from corn seedlings and confirm seed transmission of viable phytoplasmas. A quantitative PCR assay with SYBR Green chemistry, with generic "primers" to detect phytoplasmas belonging to different ribosomal groups was successfully applied both to seedlings and symptomatic field infected plants. Contrasting results were obtained from phytoplasma in liquid media and colonies. This technique demonstrated high sensitivity for phytoplasma at low concentrations and high specificity for the Mollicutes that can be differentiated from non- Mollicutes by the analysis of melting temperatures. In carrot samples from Gran Canaria Island (Spain), symptoms of shoot and root malformation were observed. ‘Candidatus Liberibacter solanacearum’, haplotype D, and phytoplasmas belonging to the ribosomal group 16SrI were detected.