Sequence analysis of six full-length bean yellow mosaic virus genomes reveals phylogenetic diversity in India strains, suggesting subdivision of phylogenetic group-IV.

We report the complete genome sequence of five bean yellow mosaic virus (BYMV) isolates (CK-GL1, CK-GL3, CK-GL4, CK-GL5 and Vfaba2) that share 74.6-98.9% (nucleotide) and 81.5-99.1% (amino acid) identity with globally available BYMV sequences. Phylogenetic analysis clustered them specifically in BYMV phylogenetic group-IV within the existing nine groups. The CK-GL1, CK-GL2, CK-GL4 and CK-GL5 isolates formed a discrete cluster within group-IV. The present study suggests subdivision of group-IV into subgroup-IVa and IVb. Moreover, infectivity assays using in vitro RNA transcripts from subgroup-IVa (CK-GL3 isolate) and IVb (CK-GL1 isolate) showed distinct biological differences between the isolates supporting subdivision.

Paenibacillus lentimorbus Inoculation Enhances Tobacco Growth and Extenuates the Virulence of Cucumber mosaic virus.

Previous studies with Paenibacillus lentimorbus B-30488" (hereafter referred as B-30488), a plant growth promoting rhizobacteria (PGPR) isolated from cow's milk, revealed its capabilities to improve plant quality under normal and stress conditions. Present study investigates its potential as a biocontrol agent against an economically important virus, Cucumber mosaic virus (CMV), in Nicotiana tabacum cv. White Burley plants and delineates the physical, biophysical, biochemical and molecular perturbations due to the trilateral interactions of PGPR-host-CMV. Soil inoculation of B-30488 enhanced the plant vigor while significantly decreased the virulence and virus RNA accumulation by ~12 fold (91%) in systemic leaves of CMV infected tobacco plants as compared to the control ones. Histology of these leaves revealed the improved tissue's health and least aging signs in B-30488 inoculated tobacco plants, with or without CMV infection, and showed lesser intercellular spaces between collenchyma cells, reduced amount of xyloglucans and pectins in connecting primary cells, and higher polyphenol accumulation in hypodermis layer extending to collenchyma cells. B-30488 inoculation has favorably maneuvered the essential biophysical (ion leakage and photosynthetic efficiency) and biochemical (sugar, proline, chlorophyll, malondialdehyde, acid phosphatase and alkaline phosphatase) attributes of tobacco plants to positively regulate and release the virus stress. Moreover, activities of defense related enzymes (ascorbate peroxidase, guaiacol peroxidase, superoxide dismutase and catalase) induced due to CMV-infection were ameliorated with inoculation of B-30488, suggesting systemic induced resistance mediated protection against CMV in tobacco. The quantitative RT-PCR analyses of the genes related to normal plant development, stress and pathogenesis also corroborate well with the biochemical data and revealed the regulation (either up or down) of these genes in favor of plant to combat the CMV mediated stress. These improvements led tobacco plant to produce more flowers and seeds with no negative impact on plant health. The present study may advocate the applicability of B-30488 for crop yield improvement in virus infested areas.

Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India.

Severe yellow mosaic disease was observed in three ornamental species of Jatropha: J. integerrima, J. podagrica and J. multifida grown in gardens at Lucknow, India, during a survey in 2013. The causal pathogen was successfully transmitted from diseased to healthy plants of these species by whitefly (Bemisia tabaci). The infection of begomovirus was initially detected in naturally infected plant samples by PCR using begomovirus universal primers. The begomovirus was characterized having a monopartite genome based on sequence analyses of the cloned ∼2.9kb DNA-A genome amplified by rolling circle amplification using Phi-29 DNA polymerase. The genome contained 2844 nucleotides that could be translated into seven potential open reading frames. The nucleotide sequences of DNA-A genome of the begomovirus isolates: JI (KC513823), JP (KF652078) and JM (KF652077) shared 94-95% identities together and 93-95% identities with an uncharacterized begomovirus isolated from J. curcas (the only sequences available in GenBank database as GU451249 and EU798996 under the name jatropha leaf curl virus). These shared highest identity of 61% and highly distant phylogenetic relationships with other begomoviruses reported worldwide. Based on 61% sequence identities (much less than 89%, the species demarcation criteria for a new begomovirus) the isolates under study were identified as members of a new Begomovirus species for which the name was proposed as "Jatropha mosaic Lucknow virus (JMLV)". The recombination analysis also suggested that JMLV was not a recombinant species, hence considered as unidentified Begomovirus species. Koch's postulates were also established by agroinfiltration assay of agroinfectious clone of JMLV. Characterization of JMLV associated with yellow mosaic disease of J. integerrima, J. podagrica and J. multifida is being reported for the first time.

Molecular Characterization of a Begomovirus and Betasatellite Causing Yellow Vein Net Disease of Ageratum houstonianum.

Ageratum houstonianum was introduced in India as an annual ornamental plant and is grown in beds for blue head flowers. Yellow vein net disease was observed on A. houstonianum plants with about 9.0% disease incidence during a survey in February 2012 at gardens of NBRI, Lucknow, India. Association of a begomovirus and betasatellite with the disease was characterized based on sequence analyses of their cloned full length genome isolated from diseased A. houstonianum. Sequence analysis of the begomovirus showed presence of the six open reading frames in its genome, similar to the arrangement of Old World begomoviruses. The begomoviral genome shared 95 to 97% sequence identities with various strains of Ageratum enation virus (AEV); however, it showed distinct phylogenetic relationships with them, and hence was identified as a variant of AEV based on more than 94% sequence homology, the criteria defined by ICTV. The sequence analysis of associated betasatellite revealed highest 93% sequence identity and close phylogenetic relationships with Ageratum leaf curl betasatellite (ALCB) molecules; therefore, it was identified as an isolate of ALCB (based on 93% sequence homology). Agroinfiltration of partial dimers of the AEV variant and ALCB induced similar systemic yellow vein net and leaf curl symptoms on A. houstonianum when infiltrated in combination, fulfilling Koch's postulates. Characterization of AEV and ALCB causing yellow vein net disease of A. houstonianum is being reported for the first time.

Molecular identification of Cucumber mosaic virus isolates of subgroup IB associated with mosaic disease of eggplant in India.

Association of Cucumber mosaic virus (CMV) with severe mosaic disease of eggplant (Solanum melongena L.) collected from Lucknow and Kanpur, India was initially detected by host reaction and serological assay and confirmed by RT-PCR employing coat protein gene specific primers. Further, molecular identification of the virus isolates was done by cloning and sequence analysis of the complete RNA3 genome. Based on 97-99 % identities and phylogenetic relationships, the virus isolates infecting eggplant were identified as members of CMV subgroup IB.

Molecular characterization of an Indian isolate of Banana bunchy top virus based on six genomic DNA components.

Banana bunchy top virus (BBTV) is a single-stranded circular DNA virus of the genus Babuvirus, belonging to family Nanoviridae. The six genomic DNA components of Indian (Lucknow) isolate of BBTV were amplified by polymerase chain reaction (PCR) with specific primers using total DNA extracted from banana tissues showing typical symptoms of banana bunchy top disease (BBTD). The resulting ~1.1 Kb amplicons were cloned and sequenced. Analysis of sequence data revealed the presence of six full-length components of BBTV: DNA-R (1111 bp), DNA-U3 (1060 bp), DNA-S (1075 bp), DNA-M (1048 bp), DNA-C (1018 bp), and DNA-N (1096 bp). Comparisons of sequence data of the six DNA components of the BBTV Lucknow isolate revealed highest identities with sequences of other BBTV isolates from the South Pacific group: [DNA-R (98%), DNA-U3 (93%), DNA-S (100%), DNA-M (98%), DNA-C (97%), and DNA-N (99%)]. A phylogenetic analysis revealed a close relationship of the Lucknow isolate with BBTV isolates of South Pacific group rather than those of the Asian group. Based on these analyses the virus has been classified as BBTV Lucknow, a new member of South Pacific group.

An umbraviral protein, involved in long-distance RNA movement, binds viral RNA and forms unique, protective ribonucleoprotein complexes.

Umbraviruses are different from most other viruses in that they do not encode a conventional capsid protein (CP); therefore, no recognizable virus particles are formed in infected plants. Their lack of a CP is compensated for by the ORF3 protein, which fulfils functions that are provided by the CPs of other viruses, such as protection and long-distance movement of viral RNA. When the Groundnut rosette virus (GRV) ORF3 protein was expressed from Tobacco mosaic virus (TMV) in place of the TMV CP [TMV(ORF3)], in infected cells it interacted with the TMV RNA to form filamentous ribonucleoprotein (RNP) particles that had elements of helical structure but were not as uniform as classical virions. These RNP particles were observed in amorphous inclusions in the cytoplasm, where they were embedded within an electron-dense matrix material. The inclusions were detected in all types of cells and were abundant in phloem-associated cells, in particular companion cells and immature sieve elements. RNP-containing complexes similar in appearance to the inclusions were isolated from plants infected with TMV(ORF3) or with GRV itself. In vitro, the ORF3 protein formed oligomers and bound RNA in a manner consistent with its role in the formation of RNP complexes. It is suggested that the cytoplasmic RNP complexes formed by the ORF3 protein serve to protect viral RNA and may be the form in which it moves through the phloem. Thus, the RNP particles detected here represent a novel structure which may be used by umbraviruses as an alternative to classical virions.