Raja Ram

Coat protein gene diversity among Chrysanthemum virus B isolates from India

Summary.The complete coat protein (CP) sequences from 29 Indian isolates of Chrysanthemum virus B (CVB) were determined and analysed in relation to other previously characterized carlaviruses. The CP genes of the Indian CVB isolates were highly heterogeneous, sharing nucleotide sequence identities of 74–98%. Based on phylogenetic analyses, the isolates formed three groups potentially representing either two or three major CVB strain groupings. Recombination analysis revealed at least one definite recombination event involving the exchange of sequences between members of different groups. To our knowledge this is the first reported evidence of homologous recombination in carlaviruses.

Coat protein sequence shows that Cucumber mosaic virus isolate from geraniums (Pelargonium spp.) belongs to subgroup II

A viral disease was identified on geraniums (Pelargonium spp.) grown in a greenhouse at the Institute of Himalayan Bioresource Technology (IHBT), Palampur, exhibiting mild mottling and stunting. The causal virus (Cucumber mosaic virus, CMV) was identified and characterized on the b0asis of host range, aphid transmission, enzyme linked immunosorbent assay (ELISA), DNA-RNA hybridization and reverse transcription polymerase chain reaction (RT-PCR). A complete coat protein (CP) gene was amplified using degenerate primers and sequenced. The CP gene showed nucleotide and amino acid homology up to 97%–98% and 96%–99%, respectively with the sequences of CMV subgroup II. The CP gene also showed homologies of 75%–97% in nucleotide and 77%–96% in amino acid with the CMV Indian isolates infecting various crops. On the basis of sequence homology, it was concluded that CMV-infecting geraniums in India belong to subgroup II

Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein.

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91–100% and 70–98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.

Genomic sequence analysis of four new chrysanthemum virus B isolates: evidence of RNA recombination

Chrysanthemums worldwide suffer from a high incidence of infection with chrysanthemum virus B (CVB), a member of the genus Carlavirus, family Betaflexiviridae. Three major lineages or strains of this virus have been found in India, but none have been characterized beyond the genetic variation they display in their coat protein genes. Here, we describe the analysis of four near-complete genome sequences (from the three lineages) representing the genetic diversity of these strains. Ranging in size from 8815 to 8855 nucleotides (excluding the polyA tail), these four isolates have a genome organization very similar to that of the recently reported Japanese isolate of CVB, with which they share between 70 and 73% genome-wide sequence identity. We present further evidence that recombination may feature quite prominently in the evolution of CVB.

Determination of Major Viral and Sub Viral Pathogens Incidence in Apple Orchards in Himachal Pradesh

Apple is the major commercial horticulture crop in Himachal Pradesh and other hill states of Jammu & Kashmir, Uttarakhand and some parts of Northeastern states of India. In order to gather data on health status and incidence of virus and virus-like pathogens in apple orchards, survey was conducted in the month of June and September, 2010 in Hatkoti, Rohru, Kuthara, Jubbal and Khadapathar areas of major apple producing Shimla district of Himachal Pradesh. A total of 250 samples were collected and analyzed by DAS-ELISA, NASH and RT-PCR. NASH results indicated that a total of 117 samples were infected with Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV) and Apple scar skin viroid (ASSVd). Results showed that ASSVd is predominant in these areas with highest infection rate of 27.6% followed by ASPV (17.2%), ACLSV (16.8%), ApMV (15.2%) and ASGV (12%). Mixed infection of these viruses and viroid was frequently detected in apple trees in Himachal Pradesh. The trees, which were positive for viruses and viroids, showed a variety of fruit deformation and rusting symptoms besides leaf deformation, mosaic and chlorosis.

Cucumber mosaic virus (CMV) infecting Alstroemeria hybrids in India

Infection of Alstroemeria hybrids in India with a subgroup 1 strain ofCucumber mosaic virus (CMV) is reported for the first time. The virus was identified on the basis of host range, transmission byAphis gossypii andMyzus persicae in the non-persistent manner, ELISA, electron microscopy and RT-PCR using CMV-specific primers. CMV was detected in nine hybrids and 61% of plants by dot-blot hybridisation.

Screening of chrysanthemum cultivars for Chrysanthemum stunt viroid in an Indian scenario

Chrysanthemum stunt viroid (CSVd) belonging to Pospiviroidae was found prevailing in some of the chrysanthemum plant samples collected from various geographical regions of India. In this study 70% cultivars of chrysanthemum were tested positive for CSVd by reverse transcription polymerase chain reaction (RT-PCR) and DNA-RNA hybridisation. The viroid was found to be mechanically transmissible to in vitro grown healthy chrysanthemum cv. Regol Time. The RT-PCR amplified product of CSVd was sequenced (348 bp length) and was directly used as a probe for hybridisation. Isolates from Palampur, Mandi, Karnataka, Bangalore and Sikkim showed positive results for CSVd. This analysis shows that CSVd is prevailing in these regions of India.

Molecular characterization and variability analysis of Apple scar skin viroid in India

Apple scar skin viroid (ASSVd) infection is a major limitation to apple fruit quality and causes huge economic losses. In surveys of apple orchards in the northern Indian state of Himachal Pradesh, fruits with dappling symptoms were noticed. ASSVd was detected from these fruits and molecularly characterized. Ten clones from three isolates were sequenced, of which seven were new sequence variants of ASSVd. The clones had significant sequence variability (94–100%) with each other. Variability was more common in the pathogenic domain of the viroid genome. Four of the clones were 330 nucleotides (nt) long, and the other six had an additional nucleotide. Phylogenetic analysis showed close affinity of the present isolates with some Chinese and Korean isolates. The study reports seven new variants of ASSVd and also provides the first molecular evidence of viroid infection (ASSVd) in apple in India.

Molecular studies on Tomato aspermy virus isolates infecting chrysanthemums

Abstract Tomato aspermy virus (TAV), an important viral pathogen of chrysanthemums, was detected by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) from chrysanthemums exhibiting mottling and deformed inflorescence grown in various states of India. Out of 15 cultivars (cvs.) tested, 11 (73.3%) were found to be positive for TAV. On sap inoculation the virus produced local lesions on Chenopodium spp. and Cucumis sativus. Nicotiana clevelandii, N. glutinosa, N. megalosiphon and N. tabacum reacted systemically to the virus producing severe mosaic, leaf deformation and characteristic leaf enations. Tomato plants produced malformed fruits (2-3/plant) with a few seeds. Myzus persicae and Aphis gossypii transmitted the virus non-persistently. Electron microscopy of partially purified viral preparations revealed polyhedral virions (ca. 29 nm dia). Cytopathology of infected leaves of N. clevelandii showed crystalline inclusions containing virions in the central vacuole of infected cells. The virions showed very good clumping with antiserum to TAV in liquid phase immuno-electron microscopy. Slot blot hybridization was performed to detect the virus in various chrysanthemum cultivars. The virus positivity was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing of coat protein gene (CP). The amplified 657 bp fragment was about 97% identical with respect to CP sequences of other TAV isolates available in the database. In terms of derived amino acid sequence similarity, the homology value was 99%. In order to amplify RNA 1, RNA 2 and RNA 3, multiplex RT-PCR was performed. High genetic similarities of CP gene of TAV Indian isolates with that of other isolates of TAV indicate their probable common ancestry.

Simultaneous detection and identification of four cherry viruses by two step multiplex RT-PCR with an internal control of plant nad5 mRNA.

A multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed and standardized for the simultaneous detection of four cherry viruses: Cherry virus A (CVA, Genus; Capillovirus), Cherry necrotic rusty mottle virus (CNRMV, unassigned species of the Betaflexiviridae), Little cherry virus 1 (LChV-1, Genus; Closterovirus) and Prunus necrotic ringspot virus (PNRSV, Genus; Ilarvirus) with nad5 as plant internal control. A reliable and quick method for total plant RNA extraction from pome and stone fruit trees was also developed. To minimize primer dimer formation, a single antisense primer for CVA and CNRMV was used. A mixture of random hexamer and oligo (dT) primer was used for cDNA synthesis, which was highly suited and economic for multiplexing. All four viruses were detected successfully by mRT-PCR in artificially created viral RNA mixture and field samples of sweet cherry. The identity of the viruses was confirmed by sequencing. The assay could detect above viruses in diluted cDNA (10(-4)) and RNA (10(-3), except PNRSV which was detected only till ten times lesser dilution). The developed mRT-PCR will not only be useful for the detection of viruses from single or multiple infections of sweet cherry plants but also for other stone and pome fruits. The developed method will be therefore quite helpful for virus indexing, plant quarantine and certification programs. This is the first report for the simultaneous detection of four cherry viruses by mRT-PCR.