Molecular Characterization of Alfalfa Mosaic Virus and Its Effect on Basil (Ocimum basilicum) Tissues in Egypt
Molecular Characterization of Alfalfa Mosaic Virus and Its Effect on Basil (Ocimum basilicum) Tissues in Egypt
Ahmed K. El-Attar1, Samah A. Mokbel1 and Om-Hashem M. EL-Banna2
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 nucleotide
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 E.Coli
into pCR®4-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. The major effects on cells 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. 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, and RT-PCR using a pair of primers specific to the AMV-CP gene.
Phylogenetic analysis results indicated that AMV-Egypt that isolated from Basil is most closely
related (96.4%) to the AMV-Spain strain isolated from Hibiscus plants.
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.
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January 2019
Vol. 5, Pages 1-141
