Improvement of Detection Methods and Further Characterization of Spiroplasma citri, the Causal Agent of Citrus Stubborn Disease in Egypt

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 are vital for revealing its real distribution and for management. In 2011, several samples were collected at different times of the year from stubborn suspected symptomatic trees within the main citrus growing area in Egypt, the Nile-delta region. After culturing the causal agent on artificial LD8 media from the field fresh samples, two new and improved methods of biological indexing were set up and compared with the traditional method in order to increase the detection efficiency by increasing the greenhouse transmission rate; which reached 85% with the new inverse inoculation method. Different PCR primer pairs were evaluated for their detection efficiency of the Egyptian Isolates of Spiroplasma citri and the most specific primer pair for these local isolates was determined. Improving the efficiency of biological indexing, along with determining the most specific and efficient PCR primer pair for the detection, will enhance and facilitate the citrus certification programs in Egypt, making them better tools for the early detection of stubborn disease. Furthermore obtained Egyptian isolates were characterized molecularly by the analysis of the obtained sequences showing close relationship with the Moroccan strain (GII3).