Phytoplasma cultivation: proofs, problems and possible solutions

Phytoplasmas are pretty well characterized from the genetic perspective after about twenty years of research on this matter, while their biology is still largely unknown also due to the restricted ability of their growth in chemically defined media. However a deep and sound knowledge of phytoplasma biology is needed to allow the definition of feasible and practical solutions to reduce their disease impact in the worldwide agriculture. The cultivation of phytoplasmas without concurrent growth of other plant phloem colonizing prokaryotes is still slowly in progress. New chemically defined media were tested in order to improve their selectivity for phytoplasma growth, and diverse purification procedures allowed to obtain phytoplasma containing colonies from different infected plant species. Moreover treatments with plasma activated water (PAW) of micropropagated shoots before isolation in liquid media were also carried out to verify their effectiveness in improving cultivation procedures. Proofs of phytoplasma isolation with colony formation from plants harboring agronomic relevant diseases such as “flavescence dorée”, “bois noir”, “stolbur”, aster yellows and apple proliferation were achieved. In a number of cases the same phytoplasma associated with the disease by molecular identification was detected in the colonies, however also other phytoplasmas were often isolated from the same symptomatic plants. By insect acquisition trials by feeding them on colonies the presence of phytoplasma nucleic acid in a large number of insects after 4 to 15 days from the feeding was confirmed. PAW treatments on phytoplasma infected micropropagated periwinkle shoots allow to improve their molecular detection and reduced the shoot symptomatology, meanwhile the lack of colony formation in agar media from these treated shoots induce to speculate an interaction between PAW and phytoplasma viability. On the other hand preliminary results are confirming the viability of phytoplasmas isolated from seedlings obtained from infected mother plants providing the first biological proof of seed phytoplasma transmission.