Search

Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1–3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26 for phytoplasma diagnostic methods).

Phytoplasma identification has proved difficult due to the pathogen’s lack of morphological traits and their inability to be maintained in vitro. DNA bar-coding is an identification method based on comparison of a short DNA sequence with known sequences from a database. A DNA bar-coding tool was developed for phytoplasma identification. While other sequence-based methods may be well adapted to identification of particular strains of phytoplasmas, they not always can be used for identification of phytoplasmas from different groups simultaneously. This phytoplasma DNA bar-coding protocol based on Tuf and 16Sr genes allows identification of the following phytoplasma groups at group and in several cases subgroup levels: 16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrVI, 16SrVII, 16SrIX, 16SrX, 16SrXI, 16SrXII, 16SrXII, 16SrXIV, 16SrXV, 16SrXVIII, 16SrXX, 16SrXXI.