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Original Article

JVS. 2019; 5(1): 97-113
doi: 27011


Molecular Characterization of Alfalfa Mosaic Virus and Its Effect on Basil (Ocimum basilicum) Tissues in Egypt

Ahmed K. El-Attar,Samah A. Mokbel,Om-Hashem M. EL-Banna.

Abstract
Background: Various isolates of Alfalfa Mosaic Virus (AMV) found throughout the world affect a wide variety of aromatic and medical plants. In March 2016, several symptoms of leaf necrosis, bright yellow mosaic and malformation of leaves suggested viral infection of AMV on basil plants grown in Beni Suef governorate.
Objectives: The present study aimed to characterize the virus at the molecular level and described the ultrastructural changes or other histopathological alterations in basil cells following infection by AMV.
Methods: Studies were conducted to elucidate the etiology of the disease. The diagnostic tools used were the transmission electron microscope for rapid diagnosis, host reactions, serological double-antibody sandwich (DAS)-ELISA, reverse transcription (RT)-PCR and sequence determination. Ultra-structural responses of basil leaf cells infected with a morphologically distinct RNA virus, AMV, were studied. Molecular characterization and phylogenetic analysis were performed for the AMV coat protein (CP) gene. An amplicon of the predicted size (∼666 bp) derived from O. basilicum isolate was purified and cloned in pCR4-TOPO vector before proceeding to DNA sequencing and the alignment of sequences.
Results: Electron microscope examination of negatively stained preparations from symptomatic basil leaves revealed viral particles have a bacilliform structure with particles size of 112.5 nm in length and 57.5 nm in width. Initial microscopic analysis suggested that the described symptoms are caused by AMV. On the basis of mechanical transmission, symptoms induced were similar to those caused by AMV. Presence of AMV in basil plant was further confirmed by the results obtained from the laboratory-based techniques such as (DAS)-ELISA using the commercially prepared polyclonal antiserum to AMV, and RT-PCR using a pair of primers specific to the AMV-CP gene. Phylogenetic analysis results indicated that AMV-Egypt (accession no. MH625710) is most closely related to the AMV-Spain strain (96.4%, HE591387) isolated from Hibiscus plants. The major effects on cells of yellow parts infected with AMV included disappearance of nucleolus, disruption of nuclear membranes, vacuolated cytoplasm, plasmodesmal dilation, abnormality of chloroplast shape, disorganization of the palisade mesophyll cells and necrosis in the zone of vascular cells.
Conclusion: Pathological investigation may provide insights into the alterations of the cell after viral infection and understanding of the data concerning the behavior of the virus. Phylogenetic analysis revealed highest identity with the Hibiscus isolate of the AMV that help in the molecular epidemiology of the virus. Particular attention should be given to the possibility of continuous monitoring of isolates of AMV at the molecular level to implement appropriate control measures.

Key words: Basil; Alfalfa mosaic virus; RT-PCR; Cloning; Nucleotide Sequence Analysis; Histopathology; Cell Pathology.



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