Molecular studies on Spiroplasma citri for the development of sensitive and specific genotyping assay.

Abstract

Stubborn disease of citrus is one of the main causes of quality deterioration of citrus fruits in Egypt. The early detection and the molecular characterization of the causal agent (Spiroplasma citri) are vital for revealing its real distribution and for management. Citrus included sweet orange (C. sinensis) and mandarin (C.reticulate Planco) showing typical symptoms of stubborn disease were detected from different fields located at Qualubia, Ismailia and Kafr El-Sheikh governorates in Egypt. The detected S. citri from diseased samples were cultured in the artificial C-3G liquid medium and the color gradually changed from red to yellow, an indication of the presence of Spiroplasma in the cultured samples. The Spiroplasma citri, when growing on low-agar medium, forms a fried-egg, fuzzy colonies with occasional surrounding satellite colonies due to the ability of the Spiroplasma cells to move through the agar matrix. DNA extracted from symptomatic samples were used as a template for amplification of products of 675 bp using primer pair Spiralin- f / Spiralin- r by PCR. Furthermore the spiralin gene was cloned, sequenced and the obtained isolate was characterized molecularly by sequence analysis showing close relationship with Egyptian isolates, Iran isolate and France isolate. Keywords: Spiroplasma citri, Stubborn Disease, C-3G Media, PCR, Spiralin Gene, Sequence Analysis.