Molecular characterization of Cucumber mosaic virus infecting Gladiolus, revealing its phylogeny distinct from the Indian isolate and alike the Fny strain of CMV

Abstract

The majority of Gladiolus plants growing in the botanical garden at NBRI, Lucknow, India and adjoining areas exhibited symptoms of mosaic, color breaking, stunting of spikes and reduction in flower size. The occurrence of Cucumber mosaic virus (CMV) was suspected in symptomatic Gladiolus plants. Cucumber mosaic virus, the type species of the genus Cucumovirus of the family Bromoviridae, is an important plant virus worldwide, which infects many plants and causes quantity and quality losses. For virus characterization, total RNA was isolated from leaves of infected plants and used in reverse transcriptase polymerase chain reaction with a primer set designed in the Cucumber mosaic virus coat protein region. Viral amplicons of the expected 657 bp size were obtained from infected plants. No viral amplicon was obtained from healthy control plants. Viral amplicons were cloned and sequenced (DQ295914). Molecular characterization was performed and phylogenetic relationship determined by the comparison of coat protein gene nucleotide and amino acid sequences with other Cucumber mosaic virus isolates reported from India and worldwide. The nucleotide and amino acid percentage comparison and phylogenetic tree results revealed that Cucumber mosaic virus infecting Gladiolus show resemblance with the Fny strain, which is not common in the Asian continent.

Biological and Molecular Characterization of Cucumber mosaic virus Subgroup II Isolate Causing Severe Mosaic in Cucumber

“…Broad host range of CMV from subgroup I and II have been identified in India, which includes: Chrysanthemum [45]; Ocimum sanctum and Zinnia elegans [34]; Amaranthus tricolor, Datura innoxia and Hyoscyamus muticus [44]; Gerbera [53]; Vanilla [28]; Pelargonium [52]; Piper longum and Piper betel [19]; Rouvolfia serpentine [35]; Jatropha curcas [36]; Catharanthus roseus [40]; Daucas carrota [1]; Banana [24,29]; Lycopersicum esculentum [15,25,47]; Gladiolus [13]. Although many CMV strains were isolated from India, but incomplete information is available at the genome level about these isolates.…”

Expression of coat protein gene ofCucumber mosaic virus(CMV-subgroup IA)Gladiolusisolate inNicotiana tabacum

“…The mechanism of resistance against virus could be mediated either by the expressed viral protein or by the transcript produced from the transgene, also known as post-transcriptional gene silencing (PTGS), or both (Varma et al 2002). In this report, we demonstrate virus resistance in transgenic N. tabacum model plants expressing the CP gene of CMV isolated from Gladiolus, (CMV-G) in India and characterized as a member of the subgroup IA (Dubey et al 2010). Though there are several reports from different parts of the world demonstrating transgenic resistance utilizing the CP gene from CMV subgroup IB or II, a report from a subgroup IA strain is still awaited from Asian continent as this strain is not common here.…”

“…DQ295914) was introduced in between the CaMV 35S promoter and the NOS terminator of the binary vector (pBI 121), which also has the NPTII marker gene for kanamycin resistance (Dubey et al 2010). The construct was mobilized into Agrobacterium tumefaciens LBA 4404 through electroporation.…”

“…The transformed leaf-discs were regenerated on selective Murashige Skoog's (MS) medium containing Gamborg's B5 vitamins, antibiotics (500 mg/l cefotaxim and 100 mg/l kanamycin) and hormones (1 mg/l 6-benzyl aminopurine [BAP] and 0.1 mg/l α-naphthalene acetic acid [NAA]) and cultured kept in a growth chamber. The callus/shoots regenerating from the explants were subsequently transferred to MS medium containing 1.0 mg/l each of BAP and α-indole acetic acid (IAA) for shoot elongation and finally to a medium containing antibiotics and IAA 0.1 mg/l for rooting (Dubey et al 2010). The putative transgenic plants were finally planted in pots and grown further in a glasshouse.…”

“…To verify the presence of the gene in T 0 plants, polymerase chain reaction (PCR) was carried out using the total DNA isolated from each plant by using the CP-specific primers of CMV-Gladiolus (Dubey et al 2010). The PCR products were electrophoresed with standard DNA in 1% agarose gel.…”