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

Abstract

Sesame (Sesamum indicum L.), family Pedaliaceae is one of the most ancient cultivated oilseed crops among the edible annual group in Egypt and all over the world. Phytoplasmas are pathogens of many plant species throughout the world including sesame causing a large variety of symptoms ranged from mild yellowing to death of infected plants. Symptomatic samples including green leaf-like floral organs, virescence, phyllody and proliferation were collected from infected sesame field in Giza Governorate. This research was undertaken to develop a diagnostic tool to identify phytoplasmas recently discovered infecting sesame in Egypt, and to classify them according to their phylogenetic group. Direct and nested PCR primers of 16S-23SrRNA gene, 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 the presences of mixed infections of phytoplasma in the tested sesame samples which collected from the field with different type of Phytoplasma like symptoms. Results also indicated that polymerase chain reaction with primers from sequencing of 16S-32S rRNA and from SR opened new paths for research on phytoplasma identification and classification. Nested PCR has been applied to overcome problems related to sensitivity of phytoplasma detection, although this approach is more time consuming and subject to template. Unfortunately, nested-PCR also meets some difficulties: unspecific bands, false positives or negatives caused by DNA and contamination of single or nested PCR. Therefore, confirmation of PCR results by using different primer pair combinations (generic and group-specific) seems to be the way for correct phytoplasma identification in the examined sesame samples.

Effect of Phytoplasma Associated with Sesame Phyllody on Ultrastructural Modification, Physio-Biochemical Traits, Productivity and Oil Quality

“…Light microscopy and transmission electron microscopy have been carried out to detect phytoplasmas [18,26,27]. Over the last ten years, molecular DNA-based techniques such as polymerase chain reaction (PCR) and sequence analysis have been used to characterize and differentiate phytoplasma strains by amplifying the highly conserved gene coding in the dispersed regions of the 16S rRNA [28]. The nested-PCR assay is designed to overcome the low concentration of phytoplasma in infected samples through increasing both sensitivity and specificity [29].…”