Universal and Specific 16S-23Sr RNA PCR Primers forIdentification of Phytoplasma associated with sesame in Egypt

Abstract

Sesame (Sesamum indicumL.), familyPedaliaceaeis one of the most ancient cultivated oilseed cropsamong the edible annualgroup in Egypt and all over the world. Phytoplasmas are pathogens of many plant species throughout the world including sesamecausing a large variety of symptoms ranged from mild yellowing to death of infected plants.Symptomaticsamples includinggreen leaf-like floral organs,virescence, phyllody and proliferationwere collected from infected sesame field in GizaGovernorate.This research was undertaken to develop a diagnostic tool to identify phytoplasmas recently discovered infectingsesame in Egypt, and to classify them according to their phylogenetic group. Direct and nested PCR primers of 16S-23SrRNAgene, paired with phytoplasma universal primer followed by strain-specific PCR primers were used in this study. Spacer Region(SR) primers were employed for identification of Phytoplasma group associated with sesame symptoms. Results showed thepresences of mixed infections of phytoplasma in the tested sesame samples which collected from the field with different typeofPhytoplasma like symptoms. Results also indicated thatpolymerase chain reaction with primers from sequencing of 16S-32SrRNA and from SR opened new paths for research on phytoplasma identification and classification. Nested PCR has been appliedto overcome problems related to sensitivity of phytoplasma detection, although this approach is more time consuming and subjectto template. Unfortunately, nested-PCR also meets some difficulties: unspecific bands, false positives or negatives caused byDNA and contaminationof single or nested PCR. Therefore, confirmation of PCR results by using different primer paircombinations (generic and group-specific) seems to be the way for correct phytoplasma identification in the examined sesamesamples