Sensitive detection of a phytoplasma in Catharanthus roseus L. (G.) Don

Catharanthus roseus L. (G.). Don., a high-valued medicinal plant, is severely affected by phytoplasmas present in the sieve tubes of the phloem. The infected plant shows malformation of leaves and flowers as well as virescence of flowers. As a first step to regenerate phytoplasma-free C. roseus plants through in vitro culture, a nested-PCR based sensitive system was standardized to detect phytoplasma from various parts of naturally as well as in vitro propagated phytoplasma-infected plants. Universal primers, from the 16S rDNA and the 16S-23S rDNA spacer region sequences of phytoplasma, were applied in direct/nested-PCR. Total DNA extracts from different parts of naturally infected as well as in vitro propagated phytoplasma-infected plants were subjected to direct PCR. The direct PCR products were subsequently used as templates in nested PCR. It yielded a DNA fragment of 1.2 kb representing the 16S rDNA of phytoplasma associated with C. roseus. Further, it was sensitive enough to amplify direct PCR products obtained from crude DNA template (symptomatic leaf tissues) diluted to 10(-3). The authenticity of the PCR-amplified DNA fragment was confirmed after nucleotide sequence determination and restriction fragment length polymorphism pattern. The developed nested PCR based system should facilitate indexing of the phytoplasma in different stages of in vitro regenerated C. roseus plants following phytoplasma-elimination procedures.