Detection of Citrus Viroids by CF-11 Nucleic Acid Extraction Method

Cachexia (Citrus cachexia viroid, CCaVd) and exocortis (Citrus exocortis viroid, CEVd) are the most important citrus viroid diseases in the world. Detection of the viroids through the common serological methods is not feasible and the current methods are time consuming and costly with some limitation. Purification and accurate detection of the viroids was studied by column chromatography with CF-11 cellulose powder in presence of ethanol (common method for viruses’ dsRNA detection) in comparison to the conventional methods. During 2010-2012, sampling was done from twigs of 22 Mandarin and 15 orange trees have symptoms of cachexia and exocortis, respectively, in East Mazandaran. Leaf samples were ground and mixed with extraction buffer. Purification carried out in CF-11 column and viroid molecules extracted with ethanol and precipitated by adding sodium acetate. The nucleic acid extracts were analyzed by 1% agarose gel electrophoresis and specific bands of the two viroids with molecular sizes of 7000-7800bp (circular form) and 300-400bp (linear form) were detected in warm and all months, respectively. The dsRNA nature of the bands was confirmed by nuclease treatment (DNaseI and RNaseA) and 2M LiCl extraction methods.Viroid entity of the each nucleic acid sample was recognized by RT-PCR method using specific primers of cachexia and exocortis viroids. 500 orange and 300 mandarin seedlings were tested in healthy citrus seedling production program.As regards of advantages of this method (repeatability, reliability, saving time and price) and confirmation by RT-PCR, the method is recommended for viroid detection in healthy citrus seedling production programs.