Bitter gourd plants showing symptoms of little leaf disease are prevalent in farmers' fields in the Bangalore rural district, Karnataka state, India. Twenty leaf samples from different locations were collected to determine the etiology of the disease. Using PCR and phytoplasma 16S rRNA gene-specific universal primers, we observed positive amplification for the phytoplasma specific primers in five out of twenty samples. The amplified products were cloned, sequenced and nucleotide (NT) sequence comparisons were made with the available phytoplasmas' 16S rRNA gene NT sequences in the NCBI database. The 16S rRNA gene NT sequence of bitter gourd phytoplasma shared highest identity of 81.7-96.0% with 'Candidatus Phytoplasma asteris' (Ca. P. asteris) 16Sr I group isolates from different parts of the world. This was supported by close clustering of phytoplasma of the current study with the Ca. P. asteris 16Sr I subgroup by phylogenetic analysis. The virtual restriction fragment length polymorphism (RFLP) pattern generated for the Phytoplasma from bitter gourd was in congruence with the in vitro RFLP pattern for the six enzymes. This was typical to Ca. P. asteris from the 16Sr I group. Further, virtual RFLP analysis with 11 more enzymes used for RFLP pattern prediction revealed differences only in the Mse I RFLP pattern, with a similarity coefficient of 0.91, which is less than the threshold similarity coefficient for a new subgroup. We propose that the phytoplasma detected in the present study that infects bitter gourd and causes littleleaf disease should be considered as a new subgroup of group 16Sr I (Ca. P. asteris). This is the first report of phytoplasma associated with littleleaf disease of bitter gourd from India.
Sixteen ivy gourd (Coccinia grandis (L.) Voigt) plant samples showing severe mosaic symptoms were collected from New Delhi and Varanasi (Uttar Pradesh) in India. Begomovirus infection was confirmed by PCR using begomovirusspecific primers. Amplified PCR products (1.2 kb fragments) were cloned and the sequence was characterized. Based on sequence analysis, begomovirus associated with the majority of ivy gourd samples (16) was found to be a member of a bipartite begomovirus species, which is closely related to tomato leaf curl New Delhi virus (ToLCNDV). Therefore, two samples of ivy gourd, IVG1-ND and IVG2-Var, were selected for full-length genome (DNA-A and DNA-B-like sequence) amplification by the rolling circle DNA amplification (RCA) method. Sequence analysis performed using the Species Demarcation Tool (SDT) program revealed that they share 89.5-91.3% (IVG1-ND) and 93.4-96.8% (IVG2-Var) nucleotide (nt) identity with the DNA-A-like sequence of ToLCNDV isolated from cucurbits and chilli, respectively. The IVG1-ND and IVG2-Var isolates shared 90% nt identity among themselves, indicating that they are two different strains of ToLCNDV. Similarly, SDT analysis of the DNA-B-like sequence of IVG1-ND and IVG2-Var exhibited showed 82.7-93.3% nt identity with the DNA-B-like sequences of ToLCNDV infecting cucurbits. The recombination analysis of DNA-A and DNB-B-like sequences showed that the greater part of their genome most likely originated from previously reported begomoviruses that are known to infect chilli and cucurbits through recombination.
Papaya (Carica papaya L.) is one of the most important fruit crops grown in tropical and subtropical regions of the world. Papaya leaf curl disease is one of the greatest concerns next to Papaya ring spot disease for India and the world. A survey was conducted during the year 2019 to 2021 for assessing the leaf curl disease incidence in five major papaya-growing districts of Karnataka State, India. The incidence ranged from 10 to 21 percent, with plants expressing typical begomovirus symptoms. Thirty-two virus-infected papaya samples (PLC-1 to PLC-32), collected from different farmer’s fields, gave positive amplification for begomovirus detection. Based on the partial genome analysis, 13 representative papaya leaf curl isolates were selected for complete genome amplification by rolling circle DNA amplification (RCA). The RCA products were cloned, sequenced and analyzed. Based on the analysis and strain classification criteria for begomoviruses, five isolates (PLC-2, 3, 9, 11 and 18) were considered variants of Chilli leaf curl virus (ChiLCV). Isolate PLC-22 is considered a strain of ChiLCV, with 93.5% nt identity sharing. Similarly, isolate PLC-28 is considered a strain of Croton yellow vine mosaic virus (CYVMV), and isolates PLC-25 and PLC-31 were considered as strains of Papaya leaf curl virus (PaLCuV). Among the remaining four isolates, three (PLC-1, PLC-4 and PLC-7) share more than 91% nt identity among them and less than 91% nt identity with all other reported begomovirus isolates. Hence, they are considered to be isolates of the novel begomovirus, and the name Papaya leaf curl Bagalkote virus [India:Karnataka:Bagalkote:Papaya:2021] is proposed. One isolate (PLC-32) is also found to be distinct from all other begomovirus isolates, including the isolates in the current study also considered to be novel begomovirus, for which we propose the name Papaya leaf curl Haveri virus [India:Karnataka:Haveri:Papaya:2021]. The putative recombination analysis of all 13 papaya isolates showed that a major part of the viral genome was likely descended from the begomoviruses reported previously. This is the first report on the diversity and a distribution of the begomoviruses infecting papaya in Karnataka, India. The current investigation results revealed five major papaya-infecting begomoviruses (PaLCuBKV, ChiLCV, PaLCuV, CYVMV and PaLCuHV) in the sampled regions.
The incidence and severity of begomovirus diseases have been increasing around the world recently, and the ridge gourd [Luffa acutangula (Roxb.) L.] is the latest example of a crop that has become highly susceptible to the outbreak of the tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in India. Accurate diagnosis of causal agents is important in designing disease management strategies. In this study the coat protein (CP) gene from a ToLCNDV-Rg ridge gourd isolate was used to produce polyclonal antibodies (ToLCNDV-Rg-CP-PAb) in a rabbit. The antibodies successfully detected a 30.5 kDa ToLCNDV-Rg-CP in extracts of symptomatic ridge gourd leaf samples by several assays, such as Western Blotting (WB), Dot Immuno Binding Assay (DIBA), Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA), Immuno Capture Polymerase Chain Reaction (IC-PCR), and Immuno Capture Loop-Mediated Isothermal Amplification (IC-LAMP) assays. However, none of the negative samples tested positive in either of the detection methods. Among all the methods tested, the immunocapture assay, IC-LAMP, was the most sensitive in detecting ToLCNDV-Rg. Furthermore, antibodies generated in this study also detected other commonly occurring begomoviruses in South India, such as tomato leaf curl Palampur virus and squash leaf curl China virus in cucurbits. Together, ToLCNDV-Rg-CP-PAb can be used for detecting at least three species of begomoviruses infecting cucurbits. The obtained antibodies will contribute to monitoring disease outbreaks in multiple crops.
Papaya ringspot virus (PRSV) is a significant threat to global papaya cultivation, causing ringspot disease, and it belongs to the species Papaya ringspot virus, genus Potyvirus, and family Potyviridae. This study aimed to assess the occurrence and severity of papaya ringspot disease (PRSD) in major papaya-growing districts of Karnataka, India, from 2019 to 2021. The incidence of disease in the surveyed districts ranged from 50.5 to 100.0 percent, exhibiting typical PRSV symptoms. 74 PRSV infected samples were tested using specific primers in RT-PCR, confirming the presence of the virus. The complete genome sequence of a representative isolate (PRSV-BGK: OL677454) was determined, showing the highest nucleotide identity (nt) (95.8%) with the PRSV-HYD (KP743981) isolate from Telangana, India. It also shared an amino acid (aa) identity (96.5%) with the PRSV-Pune VC (MF405299) isolate from Maharashtra, India. Based on phylogenetic and species demarcation criteria, the PRSV-BGK isolate was considered a variant of the reported species and designated as PRSV-[IN:Kar:Bgk:Pap:21]. Furthermore, recombination analysis revealed four unique recombination breakpoint events in the genomic region, except for the region from HC-Pro to VPg, which is highly conserved. Interestingly, more recombination events were detected within the first 1710 nt, suggesting that the 5’ UTR and P1 regions play an essential role in shaping the PRSV genome. To manage PRSD, a field experiment was conducted over two seasons, testing various treatments, including insecticides, biorationals, and a seaweed extract with micronutrients, alone or in combination. The best treatment involved eight sprays of insecticides and micronutrients at 30-day intervals, resulting in no PRSD incidence up to 180 days after transplanting (DAT). This treatment also exhibited superior growth, yield, and yield parameters, with the highest cost–benefit ratio (1:3.54) and net return. Furthermore, a module comprising 12 sprays of insecticides and micronutrients at 20-day intervals proved to be the most effective in reducing disease incidence and enhancing plant growth, flowering, and fruiting attributes, resulting in a maximized yield of 192.56 t/ha.
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