V. Venkataravanappa

Nucleotide sequencing and an improved diagnostic for screening okra (Abelmoschus esculentus L.) genotypes for resistance to a newly described begomovirus in India

ABSTRACT Ten okra (Abelmoschus esculentus L.) plants showing distinct yellow vein mosaic disease (YVMD) symptoms were collected from different fields in Karnataka State, India. The genomic DNA of the isolated viruses was amplified, cloned, and sequenced. Sequence analysis revealed that the DNA-A-like sequences of all ten isolates were identical. Sequence analysis of a representative virus isolate (OYSK2) with other begomovirus sequences available in GenBank showed ≥90% sequence identity with Bhendi yellow vein Maharashtra virus (BYVMaV; EU482411) and ≤89% homology with full-length Bhendi yellow vein mosaic virus (BYVMV) infecting okra on the Indian subcontinent. These results suggested that a new strain of BYVMaV was present in all ten samples collected from the field. A source of resistance to BYVMaV and naturally present virus isolates causing YVMD was identified by screening okra genotypes under artificial and natural inoculation conditions, respectively. None of the genotypes tested showed complete immunity to BYVMaV. However, the okra genotypes ‘Tulasi’ and ‘Trisha’ were only moderately susceptible under glasshouse and field conditions. The new begomovirus strain could be detected by dot-blot hybridisation using a non-radioactive DNA probe in the virus samples collected from both symptomless and symptomatic okra plants.

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.

Detection, Characterization and In-Silico Analysis of Candidatus Phytoplasma australasiae Associated with Big Bud Disease of Tomato in India

Tomato plants showing witches broom symptoms were collected from different states of India. The presence of phytoplasma infection was confirmed by PCR using phytoplasma-specific primer of 16S rRNA and SecY gene. The sequence analysis of 16S rRNA and SecY gene of eight tomato big bud phytoplasmas showed maximum nucleotide (nt) identity of 95–100% with Peanut WB group (16SrII). Further in-silico RFLP analysis of 16S rRNA gene of TBB-Pun1, TBB-Ban, TBB-mal, TBB-Guj and TBB-Vns showed similarity coefficient of 0.68–0.95. Since threshold similarity coefficient for classifying the phytoplasma into new subgroup is set at 0.97, the strain under study significantly distinct from the representative strains in the subgroups of pea nut witches broom. Further, the phylogenetic analysis of tomato big bud phytoplasmas revealed that, they are closely clustered with peanut witches’-broom strains (16Sr II), specifically within the 16Sr II-D and 16Sr II-A subgroups. A comprehensive recombination analysis showed the evidence of both intra and inter-species recombination in seven tomato big bud isolates with most part of their 16Sr RNA F2nR2 fragments descending from Ca.P.brasiliense (16Sr XV) as major parent, except isolate TBB-Vns which had an intra species recombination with Cactus witches-broom-16Sr II-L as major parent. Similarly, in case of SecY gene, all the seven isolates have intra-species recombination with major portion descending from Vinca virescence-[16Sr VI-A] and Potato purple top wilt-[16Sr XVIII-B]. The genetic similarities and the potential threat of this new phytoplasma belonging to 16Sr II group of Peanut witches’ broom’ group infecting tomato in India are discussed.

Molecular detection and characterization of phytoplasma in association with begomovirus in eggplant

The samples from eggplants showing mixed symptoms of little leaf and mosaic were collected from two districts (Mirzapur and Varanasi) of Uttar Pradesh, India. The total nucleic acid extracted from these samples was amplified by PCR using universal 16S rRNA primers specific to phytoplasma and primers specific to DNA-A-like sequence of begomovirus. A total of eighteen eggplant samples showing the symptoms of little leaf and mosaic tested positive for the presence of both begomovirus and phytoplasma. The phytoplasma associated with the mixed symptoms of mosaic and little leaf in the eggplant samples was identified as a member belonging to Clover proliferation group (16SrVI) (nucleotide sequence identity of 97.5-97.8%). The characterized begomovirus from the eggplant samples was identified as a strain of previously described bipartite begomovirus tomato leaf curl Palampur virus (ToLCPalV) (92.5-94.1% nucleotide sequence identity), which is known to infect cucurbits and solanaceous crops in India and Ireland. Further, putative recombination events were detected within the 16S rRNA gene F2n/R2 fragment of phytoplasma and DNA-A of strain of ToLCPalV. Most of the sequence variations observed within the phytoplasma were due to intra and interspecific recombination events between eggplant little leaf-16SrVI-D, Ca. P. asteris-16SrI and Ca. P. pruni-16SrIII. Similarly, most of the DNA fragments of newly characterized strain of ToLCPalV appear to have been derived from tomato leaf curl New Delhi virus (ToLCNDV), squash leaf curl China virus (SLCCNV) and ToLCPalV like ancestors. This perhaps is the first evidence of mixed infection of both phytoplasma-begomovirus in eggplant in India.