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General Begomovirus A genomic DNA component map showing the relative annealing positions of the degenerate primers (Av514 and Ac1048, core coat protein primers; PALIv1978 and PARIc715, top half primers; PALIc1960 and PARIv722, bottom half primers) and corresponding amplicons and amplicon sizes
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Whitefly-transmissible geminiviruses (family Geminiviridae, genus Begomovirus) were detected by the polymerase chain reaction (PCR) in total nucleic acid preparations of tomato and squash leaf samples from different areas in the country. Begomovirus DNA fragments were detected by PCR using 3 sets of degenerate primers that amplify different regions...
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Begomoviruses (family Geminiviridae, genus Begomovirus) are responsible for considerable losses in several tropical crops, more commonly in bean and tomato. Begomoviruses comprise the most numerous and economically destructive group within Geminiviridae. Characteristic symptoms of begomovirus infection in Capsicum chinense (chili pepper, Solanaceae...
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... One pair of generic primer of tomato-infecting begomovirus designed in this study yielded expected size of amplification with all the four cloned DNA-A genome of ToLCNDV, ToLCPalV, ToLCBV and ToLCJoyV. Although, previous studies reported the degenerate primers for geminiviruses [11,28], which were widely used for the detection of many different geminiviruses in the host and insect-vector [1,3,15,19]. Many times, these primers failed to detect tomato-infecting begomoviruses [21]. ...
Multiple begomovirus species are known to cause leaf curl disease in tomato in India. In order to develop specific and generic PCR based diagnostics for the tomato-infecting begomoviruses, in this study, we attempted to design primers initially based on the multiple alignment of the complete genome sequence of DNA-A component. However, the specific nucleotide stretches adequate for preparing specific primers could not be obtained. Alternatively, the online Primer-BLAST tool that offers designing of target-specific PCR primers was attempted to prepare specific primers targeting three clones (DNA-A) of tomato-infecting begomovirus species (Tomato leaf curl New Delhi virus, Tomato leaf curl Palampur virus and Tomato leaf curl Joydebpur virus) selected based on their sequence identity and phylogenetic relatedness. The primers derived from Primer-BLAST tool showed high level of cross-reaction among these begomovirus species and therefore were not able to differentiate these target begomovirus species. In order to understand the reason of cross-reactivity further sequence analysis revealed the high occurrence of single nucleotide variations (SNVs) compared to the multi-nucleotide stretches. There was no SNV hot-spot in the genome, rather the SNVs were randomly distributed throughout the genome of these begomovirus species. This pattern of nucleotide diversities among these tomato-infecting begomoviruses seriously implicated on developing specific PCR diagnostics. On the contrary, sequence analysis showed high sequence conservancy, which enabled to develop a generic PCR diagnostic for these begomoviruses. Our study, thus showed that the genome sequence diversity pattern among the tomato-infecting begomoviruses in India poses challenges in developing PCR based specific diagnostics.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13337-022-00785-9.
... 4368814). The cDNA synthesis reaction was performed in a final volume of 20 µl PCR reaction mixture and the synthesized cDNA was used as a template for RNA viruses, while the genomic DNA was used for detecting DNA viruses, the total genomic DNA was isolated from the plants of virus-infected leaf (Sambrook and Russell 2001 Dennis and Narceo (2007) denaturation at 94 °C for 4 min followed by 35 cycles with 45 s of denaturation at 94 °C, 45 s of annealing at different temperature (Table 1), extension for 90 s at 72 °C followed by a final extension for 10 min at 72 °C, the PCR products were subjected to electrophoresis in 2% agarose gel. ...
The molecular identification of different viruses was done, because identification of viral disease based on phenotypic symptoms is difficult and complicated. The molecular detection results revealed that the incidence of Chilli Veinal Mottle Virus during kharif crop was predominant, whereas the chilli virus samples collected from late kharif (August-September), rabi (October-November) and summer (March-April) crop resulted in predominance incidence of Chilli Leaf Curl Virus (ChiLCV) among the samples. Whereas the Chilli Leaf Curl Virus (ChiLCV) and Chilli Veinal Mottle Virus in early kharif (May) crop favours the virus complex in chilli. Furthermore, by phenotyping of genotypes for virus disease in the field and genotyping with molecular markers linked to chilli virus resistance, virus-resistant genotypes were identified namely DCA-295, DCA-107, DCA-154, DCA-222, DCA-224 and DCA-232. They were recorded to have least viral disease incidence, which reveals their resistance against studied chilli viruses, and therefore these genotypes were considered as field resistant to the chilli virus disease.
... 4368814). The cDNA synthesis reaction was performed in a final volume of 20 µl PCR reaction mixture and the synthesized cDNA was used as a template for RNA viruses, while the genomic DNA was used for detecting DNA viruses, the total genomic DNA was isolated from the plants of virus-infected leaf (Sambrook and Russell 2001 Dennis and Narceo (2007) denaturation at 94 °C for 4 min followed by 35 cycles with 45 s of denaturation at 94 °C, 45 s of annealing at different temperature (Table 1), extension for 90 s at 72 °C followed by a final extension for 10 min at 72 °C, the PCR products were subjected to electrophoresis in 2% agarose gel. ...
The molecular identification of different viruses was done, because identification of viral disease based on phenotypic symptoms is difficult and complicated. The molecular detection results revealed that the incidence of Chilli Veinal Mottle Virus during kharif crop was predominant, whereas the chilli virus samples collected from late kharif (August–September), rabi (October–November) and summer (March–April) crop resulted in predominance incidence of Chilli Leaf Curl Virus (ChiLCV) among the samples. Whereas the Chilli Leaf Curl Virus (ChiLCV) and Chilli Veinal Mottle Virus in early kharif (May) crop favours the virus complex in chilli. Furthermore, by phenotyping of genotypes for virus disease in the field and genotyping with molecular markers linked to chilli virus resistance, virus-resistant genotypes were identified namely DCA-295, DCA-107, DCA-154, DCA-222, DCA-224 and DCA-232. They were recorded to have least viral disease incidence, which reveals their resistance against studied chilli viruses, and therefore these genotypes were considered as field resistant to the chilli virus disease.
... The positive correlation of whitefly and leaf curl incidence was observed in linear regression with R 2 = 0.095. Y=3.7036x R 2 =0.095 by several mono and bipartite begomoviruses and it was confirmed by specific primer pairs (Anfoka et al., 2005;Dennis and Narceo, 2007;Thakuria et al., 2012 andMuniyappa et al., 2000). Comprehensive observations of leaf curl in tomato plants were undertaken to find critical factors involved in the epidemiology of leaf curl virus. ...
... To investigate the type of virus infection, total DNA was extracted from leaves of twenty infected plants using the cetyl trimethyl ammonium bromide (CTAB) method (Manen et al., 2005) and subjected to PCR using a pair of degenerate primers specific to the coat protein region of begomovirus. The forward primer sequence was GGRTTDGARGCATGHGTACATG (AC 1048) and the reverse primer sequence was GCCYATR TAYAGRAAGCCMAG (AV 494) (Bela-ong and Bajet, 2007). The primers have been used previously in screening of variety of Begomoviruses such as Tomato leaf curl virus (JN009664), Croton yellow vein mosaic Hissar virus (JN000701), Radish leaf curl virus (JN998450), Chilli leaf curl virus (JN000700), Cotton leaf curl virus (JQ693143), etc. Positive PCR reaction as an expected amplification of~750 bps was obtained from highly conserved coat protein confirms the presence of Begomovirus infection. ...
Leaves with vein yellowing were observed on Spinach (Spinacia oleracea) in Rajasthan province of India. The
plants were severely stunted in growth and exhibited symptoms typical of Begomovirus infection. Here we report a comprehensive study of complexity and recombination of novel begomovirus associated with spinach yellow vein disease. It is probably a perpetuation of Papaya leaf curl virus in absence of its main host and thus raises concern about its spread to other crops. Analysis of samples collected in the survey indicates that SYVD infected plants are associated with a begomoviruses (novel species showed < 91% nucleotide sequence similarity) and its associated betasatellites. Interestingly, most of the begomoviruses were found to be inter-species recombinants and the betasatellites possess high nucleotide variability, due to recombination. This suggested the existence of a complex recombination pattern to form “gene pool” of the crop-infecting begomovirus–betasatellites complexes. The Nucleotide diversity (π) and nucleotide substitution rates were determined for the DNA-A of begomoviruses SYVSV is 0.10382 and 2.436 × 10− 3substitutions site− 1 year− 1 respectively. Thus mutation and recombination are driving forces for the emergence and evolution of new begomoviruses. This strengthens the hypothesis for increase of host range of begomovirus and indicates that the high evolutionary rate of the begomoviruses is pri- marily due to frequent recombination and mutation.
... Gen-793 primer was used to detect SLCPV in Benincasa hipsida [27]. Av494 primer and Ac1048 primer were used to amplify Begomovirus coat protein [28]. However, further observations related to the kinds of isolates of Begomovirus infecting melon Hikapel used in this research should be carried out, because only four samples from ten samples showed the 770 bp DNA band. ...
... ToLCNDV-potato is widely spreading in India at fast rate due to polyphagous nature, high multiplication rate and broader host range and quick adaptability of its whitefly vectors. Effective management of the viral diseases entails early detection and breeding for resistance (Bela-ong and Bajet, 2007). As of now, breeding for ToLCNDV-potato resistance in potato is in its infancy, therefore early and quick diagnosis, and elimination of the virus is one of the best options for the management of viruses. ...
Squash and tissue-print PCR protocols were designed to detect Tomato leaf curl New Delhi virus-potato in Bemisia tabaci and Trialeurodes vaporariorum. Both whiteflies species were given an acquisition feeding of 24 hours separately on pure culture of this virus. Then, the individual whitefly tissues were directly printed on nitrocellulose membrane in tissue print method, and whiteflies were crushed in PBST buffer and spotted on membrane in squash print method. The uniplex PCR assay of eluted DNA revealed a sharp amplicon of 491 bp region of coat protein gene of ToLCNDV-potato. In simultaneous detection with mt COI gene as internal control, a sharp amplicon of 161, 300 and 491 bp for T. vaporariorum, B. tabaci and ToLCNDV-potato respectively, were observed in a single reaction. Gene sequencing results showed 99-100% similarity with reported sequences of ToLCNDV-potato. The protocols were validated for their sensitivity and reproducibility using ethanol preserved whiteflies collected from potato fields of Punjab, Uttar Pradesh, Madhya Pradesh and Maharashtra, and concurrent results were observed on repetition. This is the first report to detect ToLCNDV-potato in single whitefly along with internal control which will not only strengthen the virus diagnostics but also help to screen the field collected whiteflies for their viruliferous nature and to understand the epidemiology of virus vector which would guide us in taking timely vector management decisions.
... To detect any DNA-B component in the diseased trees, the primer pair PCRc1 and PBLlv2040 was used (Rojas et al. 1993;Bela-ong and Bajet 2007) as the pair displays the same PCR conditions and reaction as that in begomovirus DNA-A. To test whether a satellite molecule was associated with the isolate GD, a universal primer pair specific to alphasatellite and betasatellite was also used to amplify the putative DNA (Briddon et al. 2002;Bull et al. 2003). ...
The begomovirus infection in plants has been widely reported throughout the world. The chief carrier of this virus is the whitefly. All of the reports,however, concern plants that grow at a stumpy height from the ground; moreover, the whitefly transmits the begomovirus infection to plants at this low height only by residing under their leaves. To date, there has been no record of the begomovirus infection in trees as the prevalence of the whitefly at tree level is unlikely. For this reason, this study focuses on and presents the first report of airborne begomovirus infection in an ornamental tree—the Melia azedarach (or Pride of India) found on the Indian subcontinent
... To detect any DNA-B component in the diseased trees, the primer pair PCRc1 and PBLlv2040 was used (Rojas et al. 1993;Bela-ong and Bajet 2007) as the pair displays the same PCR conditions and reaction as that in begomovirus DNA-A. To test whether a satellite molecule was associated with the isolate GD, a universal primer pair specific to alphasatellite and betasatellite was also used to amplify the putative DNA (Briddon et al. 2002;Bull et al. 2003). ...
The begomovirus infection in plants has been widely reported throughout the world. The chief carrier of this virus is the whitefly. All of the reports, however, concern plants that grow at a stumpy height from the ground; moreover, the whitefly transmits the begomovirus infection to plants at this low height only by residing under their leaves. To date, there has been no record of the begomovirus infection in trees as the prevalence of the whitefly at tree level is unlikely. For this reason, this study focuses on and presents the first report of airborne begomovirus infection in an ornamental tree—the Melia azedarach (or Pride of India) found on the Indian subcontinent.
... For the detection of any DNA-B component in diseased plant, primer pair PCRc1 and PBLlv2040 was used (Rojas et al. 1993;Bela-ong et al. 2007), having the same PCR condition and reaction as used in the case of begomovirus DNA-A. To test whether a DNA satellite molecule was associated with begomovirus, a universal primer pair specific for alpha-satellite and beta-satellite (Briddon et al. 2002;Bull et al. 2003) was also used to amplify the putative DNA. ...
Infected leaf samples of an ornamental plant Chrysanthemum indicum showing yellowing of leaf veins were collected from gardens of New Delhi (India). An expected PCR product of size ~500 bp was amplified from total DNA extracts of symptomatic leaf samples with universal primers on the gene of coat protein region of begomovirus DNA-A component. The presence of begomoviruses was also confirmed by Southern blot analysis using control cloned DNA-A probe of Cotton leaf curl virus. Sequence analysis of the virus infecting Chrysanthemum indicum showed 99% nucleotide sequence identity with Clerodendron yellow mosaic virus(EF408037).
