Molecular and Serological Studies for Detection and Identification of Cotton Leaf Curl Virus in Cotton Plant (G. barbadense L.) in Egypt
Azza Makhlouf *
Genetics Department, Faculty of Agriculture, Alexandria University, Egypt
D. G. Asse
Arid Lands Research Institute (ALRI), A City for Scientific Research and Technology Applications, Alexandria, Egypt.
E. E. Hafez
Arid Lands Research Institute (ALRI), A City for Scientific Research and Technology Applications, Alexandria, Egypt.
M. A. EL Seehy
Genetics Department, Faculty of Agriculture, Alexandria University, Egypt.
*Author to whom correspondence should be addressed.
Abstract
Aims: Cotton production contributes in economic security in Egypt. Cotton leaf curl virus (CLCuV) belongs to the Begomovirus genus and Geminiviridae family that is transmitted by the whitefly Bemisia tabaci. It is the greatest current threat where cotton is grown. In Egypt, a distinct Begomovirus causing leaf curl has been identified in okra, tomato, hollyhock and other malvaceous species suggesting that it could possibly infect Egyptian cotton. The aim of this study was the detection and isolation of this Virus from Egyptian cotton fields.
Methodology: Diseased leaves of cotton, okra and tomato plants were collected from different localities during 2009; samples were subjected to DAS-ELISA for viral detection. Coat protein (CP) gene of the virus was amplified by PCR, sequenced and aligned with the others Begomovirus available in the GenBank. Cloning and sub cloning of CP gene for cotton virus were performed and its recombinant protein was separated on SDS-PAGE. DBIA was also used to identify protein of the virus.
Results: The amplified fragment of the CP gene was about 280bp in all samples. The CP gene of CLCUV in our study shared 97% identity with CLCuV isolate from Pakistan while, CP gene amplicon of okra leaf curl virus (OLCUV) showed identity of 84% with three isolates of tomato leaf curl virus (TYLCV) from Chian. The recombinant protein of CLCUV was about 24kDa and positive relatedness was shown between this recombinant protein and TYLCuV antiserum.
Conclusion: our study showed the existence of such virus in malvaceous species as okra plant may be causes a serious problem for cotton production in Egypt. Also, the CP gene is a good tool for examination the existence of the virus in the infected fields. The purified protein of CLCUV can use as an antigen to produce its antibodies.
Keywords: CP gene, CLCUV, OLCUV, TLCUV, DAS- ELISA, DBIA