C. N. Lakshminarayana Reddy

Molecular characterization of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India

Yellow vein mosaic disease of okra is a whitefly transmitted begomovirus causing heavy economic loss in different parts of India. The okra isolate (OY131) of this virus from a bhendi plant [(Abelmoschus esculentus L.) Moench] showing yellow vein mosaic, vein twisting, reduced leaves, and a bushy appearance in the Palem region, New Delhi, India, was characterized in the present study. The complete DNA-A and DNA-B sequences have been determined and are comprised of 2,746 and 2,703 nucleotides, respectively. The betasatellite (DNA-β) component was absent in the sample. The genome organization was typically of biparite begomoviruses, which were characterized earlier. Comparison of DNA-A component with other known begomoviruses suggest that this virus, being only distantly related (<85.9% similarity with its nearest relative, BYVMV) to other known begomoviruses, is a new species. We have tentatively assigned the genome to a novel geminivirus species Bhendi yellow vein mosaic Delhi virus [BYVDV-IN (India: Delhi: okra)]. DNA-B showed highest sequence identity (87.8% identical) to that of a ToLCNDV (AY158080). The phylogenetic analysis of the present isolate is distinct from all other viruses; however clusters with ToLCNDV group infect different crops. The recombination analysis revealed that this isolate has sequences originated from ToLCNDV. This is the first known bhendi yellow vein mosaic disease associated bipartite begomovirus from India.

Diversity and phylogeography of begomovirus-associated beta satellites of okra in India

BackgroundOkra (Abelmoschus esculentus; family Malvaceae) is grown in temperate as well as subtropical regions of the world, both for human consumption as a vegetable and for industrial uses. Okra yields are affected by the diseases caused by phyopathogenic viruses. India is the largest producer of okra and in this region a major biotic constraint to production are viruses of the genus Begomovirus. Begomoviruses affecting okra across the Old World are associated with specific, symptom modulating satellites (beta satellites). We describe a comprehensive analysis of the diversity of beta satellites associated with okra in India.ResultsThe full-length sequences of 36 beta satellites, isolated from okra exhibiting typical begomovirus symptoms (leaf curl and yellow vein), were determined. The sequences segregated in to four groups. Two groups correspond to the beta satellites Okra leaf curl beta satellite (OLCuB) and Bhendi yellow vein beta satellite (BYVB) that have previously been identified in okra from the sub-continent. One sequence was distinct from all other, previously isolated beta satellites and represents a new species for which we propose the name Bhendi yellow vein India beta satellite (BYVIB). This new beta satellite was nevertheless closely related to BYVB and OLCuB. Most surprising was the identification of Croton yellow vein mosaic beta satellite (CroYVMB) in okra; a beta satellite not previously identified in a malvaceous plant species. The okra beta satellites were shown to have distinct geographic host ranges with BYVB occurring across India whereas OLCuB was only identified in northwestern India. Okra infections with CroYVMB were only identified across the northern and eastern central regions of India. A more detailed analysis of the sequences showed that OLCuB, BYVB and BYVIB share highest identity with respect βC1 gene. βC1 is the only gene encoded by beta satellites, the product of which is the major pathogenicity determinant of begomovirus-beta satellite complexes and is involved in overcoming host defenses based on RNAi.ConclusionThe diversity of beta satellites in okra across the sub-continent is higher than previously realized and is higher than for any other malvaceous plant species so far analyzed. The beta satellites identified in okra show geographic segregation, which has implications for the development and introduction of resistant okra varieties. However, the finding that the βC1 gene of the major okra beta satellites (OLCuB, BYVB and BYVIB) share high sequence identity and provides a possible avenue to achieve a broad spectrum resistance.

Association of Tomato leaf curl Joydebpur virus and a betasatellite with leaf curl disease of eggplant

Leaf samples (five) from brinjal/eggplant fields showing upward leaf curling symptoms were collected from Varanasi, Uttar Pradesh state, India. The full length genome of begomovirus and associated betasatellite were amplified by PCR, cloned and sequenced. Sequences of homologous DNA-A and its betasatellite in all samples were the same. The samples failed to amplify DNA-B, suggesting that the begomovirus associated with leaf curl disease of eggplant was monopartite. The complete genome (homologous of DNA-A) consists of 2758 nts, whereas the betasatellite has 1352 nts and the genome organization is typical of Old World begomoviruses. The sequence analysis showed high levels of nucleotide sequence identity (79.8–91.7%) of virus with Tomato leaf curl Joydebpur virus (ToLCJoV) infecting chilli in India, suggesting it as a strain of ToLCJoV based on the current ICTV taxonomic criteria for begomovirus strain demarcation. However, the betasatellite associated was identified as a variant of Tomato leaf curl Bangladesh betasatellite (ToLCBDB), with which it shared highest sequence identity of 84.7–94.8%. Phylogenetic analyses of the genome further supported the above results. The recombination analyses of both genome and betasatellite showed that a major part of genome sequences are derived from begomoviruses (ToLCJoV, ChiLCuV, AEV) infecting chilli, tomato, ageratum and betasatellite from PaLCuB as the foremost parents in evolution, suggesting this as a new recombinant virus strain. This is the first report of a monopartite begomovirus and a betasatellite molecule associated with the leaf curl disease of eggplant.

‘Candidatus Phytoplasma’ belonging to the 16SrVI phytoplasma group, is associated with witches broom disease of Azadirachta indica in India

Five samples from neem trees exhibiting witches broom (NeWB) symptoms were collected from the Raichur district, Karnataka State, India. The identity of the phytoplasma associated with all five neem samples was confirmed through PCR using phytoplasma 16Sr RNA gene specific universal primers. The amplified products were cloned, sequenced and nucleotide (nt) sequence comparisons were made with published phytoplasmas 16S rRNA gene nt sequences available at NCBI database. The 16Sr RNA gene nt sequence of NeWB phytoplasma had 99 to 99.8% identity with ‘Candidatus Phytoplasma’ group (16SrVI) isolates reported from different parts of the world. This was supported by the close clustering of NeWB phytoplasma in the current study with members of clover proliferation group-16SrVI in the phylogenetic analysis. The virtual RFLP pattern generated for the phytoplasma from neem was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group VI and subgroup D (Brinjal little leaf-16VI-D, NCBI Ac.No.: X83431). The analysis further confirmed that the phytoplasma associated with NeWB disease of neem belongs to 16Sr group VI and subgroup 16Sr IV-D. This is the first report of ‘Candidatus Phytoplasma’ belonging to the 16SrVI phytoplasma group associated with witches broom disease of Azadirachta indica from India.