Deep amplicon sequencing for detection of mixed phytoplasma infections in plants

Phytoplasmas within single plants can be considered as populations of individuals and as phytoplasmas are obligate parasites of plants, single clones cannot be obtained. For identification of phytoplasmas, PCR followed by RFLP, sequencing PCR products directly or sequencing cloned PCR products are standard procedures. These procedures will, however, not show the diversity of populations within single plants, as only the most frequent genotypes will be detected and identified. A number of grapevine samples, in which mixed phytoplasma infections were detected by a nested PCR, were used for deep amplicon sequencing on the Roche Genome Sequencer FLX system. 50,926 phytoplasma sequences from the 5’end of the 16Sr DNA gene were obtained from 16 phytoplasma-infected grapevine samples. After clustering and alignment to phytoplasma reference sequences it was shown that the phytoplasmas in the grapevine plants belonged to diverse 16Sr groups, mainly the same groups that were detected using nested/PCR/RFLP. Furthermore, a high number of single nucleotide polymorphisms were present in individual samples.