Detection and Identification of Tomato Yellow Leaf Curl Virus-EG Using Molecular Technique

摘要

TOMATO Yellow Leaf Curl vims (TYLCV-Eg) was isolated from whiteflies-infected tomato (Lycopersicon esculentum cv. Castle rock) plants growing in Nubaria and El-Behera Governorate. The infected plants exhibited systemic viral symptoms in the form of severe leaf curling, leaf crinkle with marginal yellowing, stem upright, twisted and stunted. TYLCV-Eg reacted positively with polyclonal antibodies specific to TYLCV using DAS-ELISA. It was transmitted by both syringe injection and whiteflies with transmission efficiency of about 80% and 100%, respectively. TYLCV-Eg isolate was transmitted to different species belonging to families Cucurbitaceae, Fabaceae, Solanaceae and Chenopodiaceae. TYLCV had TIP (Thermal Inactivation Point) of 70°C, DEP (Dilution End Point) of 10~(-7) and LIV (Longevity) of about 6 days. Electron micrograph of the partially purified TYLCV revealed the presence of monomer and dimmer gemini particles with dimensions of 22 nm and 20 x 30 nm to 24 x 30 nm, respectively when negativelystained with uranyl acetate. DNA from infected plants was extracted and amplified successfully by polymerase chain reaction (PCR) using degenerate oligonucleotide primers V324(+) and C889 (-) producing approx 500 bp fragment from infected tomato plants.The viral genome was detected by specific DNA probe using dot blot hybridization technique. Comparative nucleotide sequence analysis showed a similarity of 98% between TYLCV-EG and other isolates.