Millettia pinnata (Pongame oiltree, Sukh chain; family Fabaceae) is an important leguminous tree widely grown and utilised as a herb in Southeast Asia and the Indian subcontinent. In October 2020, symptoms of stunted growth, leaf curling (upward and downward) and vein thickening (Figures 1 and 2), which are associated with infection by Begomovirus species in other hosts, were observed on M. pinnata, growing near cotton fields (30.3°N, 73.0°E), in Faisalabad, Punjab Province, Pakistan. DNA was isolated from ten leaves (S1 to S10), one each from ten diseased plants, and one leaf from two asymptomatic plants (AS11 and AS12) using a cetyl trimethyl ammonium bromide protocol (Doyle & Doyle, 1990). Rolling circle amplification in combination with PCR using begomovirus universal primers (BegomoF/BegomoR) (Briddon & Markham, 1994) amplified a c. 2.8 kb product from all the diseased samples. PCR assays for the Begomovirus alphasatellite (primer pair DNA101F/DNA101R) (Bull et al., 2003) and betasatellite (primer pair Beta01F/Beta02F) (Briddon et al., 2002) each amplified a c. 1.4 kb product from all the diseased samples. Neither begomovirus genome nor satellite DNA was detected in the asymptomatic samples. Amplicons from the begomovirus genome (AV-2 and AV-3 from S1 and S2; MZ268122-MZ268123), alphasatellite (AV-36 from S1; MZ057253) and betasatellite (AV-8 and AV-10 from S1, AV-11 and AV-12 from S2, and AV-13 from S3; MZ057231- MZ057235), were cloned in a general purpose PTZ57R/T cloning vector (Thermo Fisher Scientific, USA), Sanger sequenced, and the sequences deposited in GenBank. The genome sequences shared >99% nucleotide identity with Cotton leaf curl Multan virus (CLCuMuV) and were most closely related to CLCuMuV strain NAS-14 (MK357257), which was found in cotton in the same region of Faisalabad (Ahmed et al., 2021). They also had 94% and 87% nt similarity with the CLCuMuV-Raj (AF363011) and CLCuMuV-Dar strains (EU365613) respectively. Moreover, the alphasatellite and betasatellite sequences shared 98% similarity with Cotton leaf curl Multan alphasatellite (CLCuMuA; MK357286.1) and betasatellite (CLCuMuB; MT920327.1), respectively. The five CLCuMuB betasatellite sequences (MZ057231-MZ057235) shared 99% identity and the two Begomovirus genomic sequences (MZ268122-MZ268123) from the M. pinnata samples had 99% identity with each other. Southern hybridization (Zubair et al., 2017) confirmed the detection of Begomovirus genomic DNA and quantitative real-time PCR (Shafiq et al., 2017) assays confirmed the detection of CLCuMuV in three samples, S1, S2 and S3, selected for further analysis. To the best of our knowledge, this is the first report of CLCuMuV in Millettia pinnata which may serve as an alternative host of the virus in the off season when cotton is not grown. Routine monitoring and detection of CLCuMuV is essential to prevent the devastating leaf curl disease it causes from spreading worldwide. The authors acknowledge support in Sanger sequencing from Dr. Brian Scheffler and his group from USDA-ARS.

Two field-collected tomato plants, with severe and mild tomato leaf curl disease symptoms, were shown to be infected with tomato leaf curl New Delhi virus. The plant with severe symptoms was shown to additionally contain tobacco leaf curl betasatellite (TobLCuB). Inoculation of Nicotiana benthamiana plants with the cloned components showed the severe symptoms to be due to the presence of TobLCuB. A shorter latent period was associated with the presence of TobLCuB except in the presence of the DNA-A and DNA-B components of the severe type. The DNA-B component from the mild type also reduced the latent period, more so than the DNA-B from the severe type, except in the interaction with the DNA-A from the mild type. These differences in the effects of the virus components from the two isolates may possibly be due to mutations in the DNA-B from the severe type. The results show that betasatellites can enhance the virulence of bipartite begomoviruses, even for isolates that induce quite mild symptoms.

Whitefly-transmitted viruses of the genus Begomovirus (the family Geminiviridae) have become a limiting factor for agricultural productivity in many warmer parts of the world. The economies of Pakistan and India have, since the early 1990s, suffered losses due to cotton leaf curl disease (CLCuD). The disease is caused by begomoviruses, the most important of which at this time is cotton leaf curl Kokhran virus strain Burewala (CLCuKoV-Bu), and a disease-specific betasatellite, cotton leaf curl Multan betasatellite (CLCuMuB). Efforts to minimize losses due to CLCuD rely mainly on the use of insecticides to kill the whitefly vector; no resistant cotton varieties are currently commercially available. The study described here has investigated RNA interference technology for its potential to yield resistance against CLCuKoV-Bu and three other begomoviruses; CLCuKoV, tomato leaf curl New Delhi virus (ToLCNDV) and Pedilanthus leaf curl virus (PeLCV). Three fragments of the virion-sense V2 gene of CLCuKoV-Bu were transformed into Nicotiana benthamiana in antisense orientation and transgenic lines expressing virus-specific short RNAs were assessed for their ability to yield resistance. Only CLCuKoV-Bu with the V2 sequence closest to the promoter was resistant. Inoculation of CLCuKoV-Bu with CLCuMuB into transgenic plants did not significantly affect the outcome, although viral DNA was detected in number of plants, suggesting that the betasatellite may impair RNAi resistance. Overall the results indicate that targeting the 5' end of V2 gene using antisense-RNA has the potential to deliver resistance against begomoviruses and that RNAi-based resistance imparts some degree of resistance to heterologous viruses. Keywords: geminivirus; begomovirus; RNAi; resistance; CLCuKoV-Burewala; CLCuMuB.

Gut-expressed aphid genes, which may be more easily inhibited by RNA interference (RNAi) constructs, are attractive targets for pest control efforts involving transgenic plants. Here we show that expression of cathepsin L, a cysteine protease that functions in aphid guts, can be reduced by expression of an RNAi construct in transgenic tobacco. The effectiveness of this approach is demonstrated by up to 80% adult mortality, reduced fecundity, and delayed nymph production of Myzus persicae (green peach aphids) when cathepsin L expression was reduced by plant-mediated RNAi. Consistent with the function of cathepsin L as a gut protease, M. persicae fed on the RNAi plants had a lower protein content in their bodies and excreted more protein in their honeydew. Larvae of Coccinella septempunctata (seven-spotted ladybugs) grew more slowly on aphids having reduced cathepsin L expression, suggesting that prey insect nutritive value, and not just direct negative effects of the RNAi construct, needs to be considered when producing transgenic plants for RNAi-mediated pest control. Highlights Silencing expression of cathepsin L by RNA interference reduces protein content of Myzus persicae (green peach aphid) bodies. Honeydew of aphids with cathepsin L silenced contains elevated protein. Cathepsin L is required for efficient protein uptake from phloem sap. Aphids with cathepsin L expression silenced have increased mortality and fewer offspring. Coccinella septempunctata (seven-spotted ladybugs) grow more slowly on aphids with expression of cathepsin L silenced.

Alphasatellites, formerly known as DNA 1, are a satellite-like components associated with begomoviruses (the family Geminiviridae) that require betasatellite for symptom induction but depend on DNA-A for systemic movement. We have converted alphasatellite into gene-silencing vector (modified alphasatellite (∆DNA 1)) by deleting its A-rich region that does not affect the replication nor the movement of the helper virus. Insertion of a transgene green florescence protein (GFP) into ∆DNA 1 resulted in the silencing g of the cognate gene in Nicotiana benthamiana. The silencing persisted for more than one and half month and was associated with the decreased level of mRNA of the target gene. This satellite-like DNA vector induced gene silencing (VIGS) promises to be applicable to other begomovirus/alphasatellite systems, thereby providing the powerful approach to gene discovery and the analysis of gene functions in malvaceous crops. Keywords: cotton; begomovirus; alphasatellite; RNAi.

HomePlant DiseaseVol. 101, No. 5First Report of Pedilanthus leaf curl virus, Tobacco leaf curl betasatellite, and Guar leaf curl alphasatellite Infecting Radish (Raphanus sativus) in Pakistan PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Pedilanthus leaf curl virus, Tobacco leaf curl betasatellite, and Guar leaf curl alphasatellite Infecting Radish (Raphanus sativus) in PakistanH. Ismail, I. Hassan, M. Zubair, M. Z. Khan, S. Serfraz, N. Jamil, S. Mansoor, S. Asad, and I. AminH. IsmailSearch for more papers by this author, I. HassanSearch for more papers by this author, M. ZubairSearch for more papers by this author, M. Z. KhanSearch for more papers by this author, S. SerfrazSearch for more papers by this author, N. JamilSearch for more papers by this author, S. MansoorSearch for more papers by this author, S. AsadSearch for more papers by this author, and I. AminSearch for more papers by this authorAffiliationsAuthors and Affiliations H. Ismail I. Hassan M. Zubair M. Z. Khan S. Serfraz N. Jamil S. Mansoor S. Asad I. Amin , National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan. Published Online:23 Feb 2017https://doi.org/10.1094/PDIS-09-16-1265-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Begomoviruses are whitefly (Bemisia tabaci)-transmitted single-stranded DNA viruses of the family Geminiviridae that cause extensive crop losses across the warmer parts of the world. During a survey in October 2015 to monitor begomovirus infection in radish (Raphanus sativus), plants were noted with symptoms typical of begomovirus infection, consisting of upward leaf curling, stunted growth, and vein yellowing in the Chiniot region of Punjab province. Symptoms were observed in three fields and infection ranged from 10 to 20%. Leaf samples were collected from seven symptomatic and five non-symptomatic radish plants and total genomic DNA was extracted using the CTAB method (Doyle and Doyle 1990). PCR with a primer pair designed to amplify all begomoviruses (BegomoF/BegomoR; Shahid et al. 2007) resulted in amplification of product of the expected size (∼2.8 kb) from one out of the seven symptomatic samples while no amplification was observed in healthy tissues. The amplified product was cloned in pTZ57R/T (Thermo Scientific, U.S.A.), sequenced and analyzed through nBLAST (www.ncbi.nlm.nih.gov/blast). The results showed the sequence of the clone to have the highest nucleotide sequence identity (94%) with an isolate of Pedilanthus leaf curl virus (PeLCV) from spinach originating from Pakistan (accession no. HF568781). The sequence is available in the nucleotide sequence database under the accession number LT600729. To further confirm the presence of a begomovirus, total DNA from five symptomatic (including the plant from which the clone was obtained) and one non-symptomatic plant were resolved on agarose gel, transferred to nylon membrane, and hybridized with DIG-labeled PeLCV probe produced by PCR with primer pair CLCV1/CLCV2 (Hussain et al. 2003). Hybridization was detected for two symptomatic samples (including the one positive by PCR) but not for the sample lacking symptoms. The radish samples were also assessed for the presence of alpha- and betasatellites. PCR with universal primer pair for amplification of alphasatellites (DNA101/DNA102; Bull et al. 2003) yielded a product of the expected ∼1.4 kb from one symptomatic sample (from which the virus was cloned). The fragment was cloned and sequenced. The sequence was 1,358 bp in length and an nBLAST analysis showed 98% sequence identity with an isolate of Guar leaf curl alphasatellite (GuLCA: HGA17075). Similarly, PCR with a primer pair designed to amplify all betasatellites (beta01/beta02; Briddon et al. 2002) resulted in the expected ∼1.4 kb product for one sample (from which the alphasatellite and virus were cloned). This sequence was 1,363 bp in length and showed 97% identity with an isolate of Tobacco leaf curl Pakistan betasatellite (TbLCB: AM922485) upon nBLAST. The sequences of alpha- and betasatellites are available in the nucleotide sequence databases under accession numbers LT600724 and LT600728, respectively. Southern blot analyses were conducted using the cloned satellites as probes, which showed the presence of both satellites in the two samples in which PeLCV was detected. To the best of our knowledge, this is the first report of PeLCV with TbLCB and GuLCA infecting radish in Pakistan.References:Briddon, R. W., et al. 2002. Mol. Biotechnol. 20:315. https://doi.org/10.1385/MB:20:3:315 Crossref, ISI, Google ScholarBull, S. et al. 2003. Mol. Biotechnol. 23:83. Google ScholarDoyle, J. J., and Doyle, J. L. 1990. Focus 12:13. Google ScholarHussain, M. et al. 2003. Plant Pathol. 52:809. Google ScholarShahid, M. et al. 2007. Arch Virol. 152:2131. Google ScholarDetailsFiguresLiterature CitedRelated Vol. 101, No. 5 May 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 14 Apr 2017Published: 23 Feb 2017First Look: 9 Jan 2017Accepted: 23 Dec 2016 Pages: 845-845 Information© 2017 The American Phytopathological SocietyCited byAnalysis of the effects of the mutation of selected genes of pedilanthus leaf curl virus on infectivity, symptoms and the maintenance of tobacco leaf curl betasatellite25 May 2022 | Canadian Journal of Plant Pathology, Vol. 44, No. 5Tobacco leaf curl betasatelliteCABI Compendium, Vol. CABI CompendiumPedilanthus leaf curl virusCABI Compendium, Vol. CABI CompendiumAssociation of an isolate of papaya leaf curl virus and papaya leaf curl betasatellite with leaf curl disease of radish in India26 January 2021 | Journal of Plant Pathology, Vol. 103, No. 2Interaction of eukaryotic proliferating cell nuclear antigen (PCNA) with the replication-associated protein (Rep) of cotton leaf curl Multan virus and pedilanthus leaf curl virus2 January 2021 | 3 Biotech, Vol. 11, No. 1Codiaeum variegatum in Pakistan harbours pedilanthus leaf curl virus and papaya leaf curl virus as well as a newly identified betasatellite23 May 2020 | Archives of Virology, Vol. 165, No. 8Identification of two further agriculturally important begomoviruses and their associated satellites infecting the weed Digera arvensis in Pakistan14 June 2019 | European Journal of Plant Pathology, Vol. 155, No. 2Raphanus sativus (Radish)6 June 2020Characterization, phylogeny and recombination analysis of Pedilanthus leaf curl virus-Petunia isolate and its associated betasatellite31 August 2018 | Virology Journal, Vol. 15, No. 1Identification of pedilanthus leaf curl virus and associated betasatellite infecting turnip in Pakistan24 May 2018 | Journal of Plant Pathology, Vol. 100, No. 2

HomePlant DiseaseVol. 101, No. 6First Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in Pakistan Previous DISEASE NOTES OPENOpen Access licenseFirst Report of Tomato leaf curl New Delhi virus on Calotropis procera, a Weed as Potential Reservoir Begomovirus Host in PakistanS. S. Zaidi, S. Shakir, H. J. Malik, M. Farooq, I. Amin, and S. MansoorS. S. Zaidihttp://orcid.org/0000-0002-2976-4624Search for more papers by this author, S. ShakirSearch for more papers by this author, H. J. MalikSearch for more papers by this author, M. FarooqSearch for more papers by this author, I. AminSearch for more papers by this author, and S. MansoorSearch for more papers by this authorAffiliationsAuthors and Affiliations S. S. Zaidi , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan S. Shakir , Virology Laboratory, Center for Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan, and Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan H. J. Malik M. Farooq I. Amin S. Mansoor , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. Published Online:19 Apr 2017https://doi.org/10.1094/PDIS-10-16-1539-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In 2015–16, during field surveys of Sindh and Punjab provinces of Pakistan to observe virus symptoms on cultivated crops and nearby weeds, symptoms of leaf yellowing and yellow mosaic were observed on leaves of Calotropis procera growing around the cultivated fields. C. procera, also known as rubber bush or aak, is a common weed in Pakistan. To detect the possible presence of begomoviruses, five symptomatic plants and one asymptomatic plant were collected from Punjab and Sindh, and total DNA was extracted. Southern blot hybridization was performed using a DIG labeled probe to the Tomato leaf curl New Delhi virus (ToLCNDV) C1 gene. A strong hybridization signal was observed in symptomatic plants, while there was no signal in asymptomatic plants collected from the same fields. To further confirm the association of ToLCNDV, rolling circle amplification (RCA) was performed to enrich for the circular DNA molecules of the begomovirus genome, using phi29 DNA polymerase (Thermo Fisher Scientific, Waltham, MA). The RCA product was purified and sequenced by the Illumina MiSeq sequencing system. The Illumina NeoPrep automation system (Illumina, San Diego, CA) was used with the Illumina TruSeq Nano DNA Library Prep Kit (NP-101-1001). After sequencing, adapters were trimmed by the MiSeq Reporter Software and sequencing data were analyzed by the CLC Genomics Workbench 7.5 (https://www.qiagenbioinformatics.com). De novo assemblies were prepared following the standard quality parameters (quality score: 0.001 and Phred score: 30). The NCBI BLASTn results of assembled de novo reads confirmed the presence of ToLCNDV DNA-A and ToLCNDV DNA-B (sequence available in the database with the accession number KX710158 and KX710159, respectively). The species demarcation of ToLCNDV was also confirmed using the Sequence Demarcation Tool (SDT) program (http://web.cbio.uct.ac.za/∼brejnev/), following the revised taxonomic criteria of begomoviruses (Brown et al. 2015). The nBLAST analysis of ToLCNDV DNA-A reported in this study indicated that it was 98% identical to the ToLCNDV DNA-A reported previously in chili from Khanewal, Pakistan (DQ116880); while the nBLAST analysis of ToLCNDV DNA-B reported in this study indicated that it was 95% identical to the ToLCNDV DNA-B reported previously in Cucurbita pepo from Rawalpindi, Pakistan (KT948073). No evidence for the presence of alphasatellites or betasatellite either in the Illumina data or by PCR using universal primers for amplification of alphasatellites or betasatellites was found. Whitefly transmitted ToLCNDV (Gilbertson et al. 2015) infects several important crops like tomato, potato, cucurbits (Zaidi et al. 2016a), and cotton (Zaidi et al. 2016b). The occurrence of ToLCNDV on C. procera indicates the increasing host range of ToLCNDV, which is serious threat for other economically important crops. C. procera has been reported as a potential host for Papaya leaf curl virus (JQ407224; formerly known as Croton yellow vein mosaic virus) and croton yellow vein mosaic betasatellite (HQ631430) in India (Prajapat et al. 2012). C. procera is a common weed and may therefore serve as an alternative virus reservoir, with an important role in the epidemiology of the disease. To our knowledge, this is the first report of ToLCNDV infecting C. procera in Pakistan.References:Brown, J. K., et al. 2015. Arch. Virol. 160:1593. Crossref, ISI, Google ScholarGilbertson, R. L., et al. 2015. Annu Rev Virol. 2:67. https://doi.org/10.1146/annurev-virology-031413-085410 Crossref, ISI, Google ScholarPrajapat, R., et al. 2012. Arch Phytopathol. Plant Prot. 45:1980. Crossref, Google ScholarZaidi, S. S., et al. 2016a. Mol. Plant Pathol. (in press). https://doi.org/10.1111/mpp.12481 Google ScholarZaidi, S. S., et al. 2016b. PLoS One 11:e0155520. https://doi.org/10.1371/journal.pone.0155520 Crossref, ISI, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 101, No. 6 June 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 17 May 2017Published: 19 Apr 2017First Look: 7 Feb 2017Accepted: 31 Jan 2017 Page: 1071 Information© 2017 The American Phytopathological SocietyCited byTomato yellow leaf curl virus (leaf curl)CABI Compendium, Vol. CABI CompendiumTomato leaf curl New Delhi virus (Tomato New Delhi virus)CABI Compendium, Vol. CABI CompendiumA Major QTL Located in Chromosome 8 of Cucurbita moschata Is Responsible for Resistance to Tomato Leaf Curl New Delhi Virus20 March 2020 | Frontiers in Plant Science, Vol. 11Engineering Molecular Immunity Against Plant Viruses

HomePlant DiseaseVol. 101, No. 6First Report of Tomato leaf curl New Delhi virus and a Tomato yellow leaf curl Thailand betasatellite Causing Severe Leaf Curl Disease of Potato in Pakistan PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Tomato leaf curl New Delhi virus and a Tomato yellow leaf curl Thailand betasatellite Causing Severe Leaf Curl Disease of Potato in PakistanA. Hameed, M. N. Tahir, I. Amin, and S. MansoorA. Hameed, M. N. Tahir, I. Amin, and S. MansoorAffiliationsAuthors and Affiliations A. Hameed , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan; and Department of Bioinformatics and Biotechnology, Government College University, Faisalabad, Pakistan M. N. Tahir I. Amin S. Mansoor , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan. Published Online:14 Mar 2017https://doi.org/10.1094/PDIS-09-16-1335-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In April 2011, during a field survey, a severe leaf curl disease was observed on potato (Solanum tuberosum) in potato-growing areas of Punjab, Pakistan (approximate coordinates 30.3551°N, 72.5321°E). Severe upward and downward leaf curling, vein swelling, and leaf thickening, resulting in conspicuous bushy appearance suggested that the disease might be caused by begomoviruses. The disease symptoms were widespread throughout the potato growing districts with an incidence of 5 to 10%. To confirm the association of begomoviruses with the disease, total DNA was extracted from 10 symptomatic samples using CTAB protocol. Samples without any symptoms were also collected as negative controls. Universal primers designed on conserved sequences of begomoviruses DNA A were used in PCR (Shahid et al. 2007). A PCR product of expected size (∼2.8 kb) was obtained in three out of 10 symptomatic samples and confirmed the association of begomoviruses with the disease. The amplified products were cloned and sequenced. Complete nucleotide sequence of two clones were submitted in GenBank (accession numbers LN908935 and LN908936). NCBI BLASTn analysis showed maximum nucleotide identity with the isolates of Tomato leaf curl New Delhi virus (ToLCNDV), a highly prevalent bipartite begomovirus in the Indian subcontinent reported from several crops such as cotton, chilli, potato, etc. (Zaidi et al. 2016). Sequence demarcation tool (SDT) analysis reveals that the ToLCNDV isolates from Pakistan have >99% sequence identity with the ToLCNDV isolates identified from potato plants in India (KC874503 and KC874505). Previously, the association of ToLCNDV with leaf curl disease of potato has been reported from India (Usharani et al. 2004). For infectivity analysis, all efforts to mechanically transmit the ToLCNDV isolates in potato were unsuccessful under our conditions. In order to confirm the presence of begomoviruses, Southern hybridization was performed with ToLCNDV AV2 probe (a DIG labeled probe, target specificity to AV2 gene of ToLCNDV). All PCR positive plants showed hybridization with ToLCNDV AV2 probe in Southern blot analysis while in healthy plants, no hybridization signals were observed. The symptom phenotype on potato suggested that DNA satellites might be associated with the severe disease in some of symptomatic samples. In order to check the presence of DNA satellites, universal primers for alphasatellites and betasatellites were used in PCR (Briddon et al. 2002). No PCR product was amplified by alphasatellite primers, but use of betasatellite primers in PCR resulted in amplification of a product of desirable size (∼1.4 kb) in two out of three symptomatic DNA A positive samples. The amplified product of one sample was cloned, sequenced, and submitted in GenBank (LK933548). BLASTn showed that the sequence shared maximum sequence identity (>96%) with Tomato yellow leaf curl Thailand betasatellite (TYLCTHB) isolates FR819710, EF068245, and HG531760. To induce systemic infection, DNA A of ToLCNDV does not necessarily require DNA B as the association of DNA A of ToLCNDV as a helper virus with betasatellite has been proven (Saeed et al. 2007). To our knowledge, this is the first report of a bipartite begomovirus, ToLCNDV, with leaf curl disease of potato in Pakistan. In addition, this is the first report of the association of ToLCNDV and TYLCTHB infecting potato in Pakistan. This is an indication of the increased incidence of and expansion in the host range of begomovirus disease complexes, which can pose serious future threat to agriculture in Pakistan.References:Briddon, R. W., et al. 2002. Mol. Biotechnol. 20:315. https://doi.org/10.1385/MB:20:3:315 Crossref, ISI, Google ScholarSaeed, M., et al. 2007. J. Gen. Virol. 88:2881. https://doi.org/10.1099/vir.0.83049-0 Crossref, ISI, Google ScholarShahid, M. S., et al. 2007. Arch. Virol. 152:2131. https://doi.org/10.1007/s00705-007-1043-9 Crossref, ISI, Google ScholarUsharani, K. S., et al. 2004. Plant Pathol. 53:235. https://doi.org/10.1111/j.0032-0862.2004.00959.x Crossref, ISI, Google ScholarZaidi, S. S., et al. 2016. Mol. Plant Pathol. Online. doi:10.1111/mpp.12481 Google ScholarDetailsFiguresLiterature CitedRelated Vol. 101, No. 6 June 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 17 May 2017Published: 14 Mar 2017First Look: 23 Jan 2017Accepted: 13 Jan 2017 Pages: 1065-1065 Information© 2017 The American Phytopathological SocietyCited byCotton leaf curl Multan betasatellite impaired ToLCNDV ability to maintain cotton leaf curl Multan alphasatellite1 January 2024 | Brazilian Journal of Biology, Vol. 84Tomato yellow leaf curl Thailand betasatelliteCABI Compendium, Vol. CABI CompendiumTomato leaf curl New Delhi virus (Tomato New Delhi virus)CABI Compendium, Vol. CABI CompendiumPotato apical leaf curl disease: current status and perspectives on a disease caused by tomato leaf curl New Delhi virus13 April 2021 | Journal of Plant Diseases and Protection, Vol. 128, No. 4Interaction of a tomato leaf curl New Delhi virus with a betasatellite enhances symptom severity in field-infected tomato plants19 January 2021 | Tropical Plant Pathology, Vol. 46, No. 2Molecular characterization of tomato leaf curl Joydebpur virus and tomato leaf curl New Delhi virus associated with severe leaf curl symptoms of papaya in Bangladesh6 August 2020 | European Journal of Plant Pathology, Vol. 158, No. 2Pest categorisation of non‐EU viruses and viroids of potatoEFSA Journal, Vol. 18, No. 1Evaluation of Solanum species for resistance to Tomato leaf curl New Delhi virus using chip grafting assayScientia Horticulturae, Vol. 256Evolutionary Factors in the Geminivirus Emergence13 July 2019Replication of DNA Satellites and Their Role in Viral Pathogenesis13 July 2019Solanum tuberosum (Potato)6 June 2020Tomato Leaf Curl New Delhi Virus: An Emerging Virus Complex Threatening Vegetable and Fiber Crops21 September 2017 | Viruses, Vol. 9, No. 10Engineering Molecular Immunity Against Plant Viruses

HomePlant DiseaseVol. 101, No. 6First Report of a Novel Strain of Tomato yellow leaf curl virus Causing Yellow Leaf Curl Disease on Cluster Bean in Pakistan PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a Novel Strain of Tomato yellow leaf curl virus Causing Yellow Leaf Curl Disease on Cluster Bean in PakistanS. S. Zaidi, S. Shakir, M. Farooq, I. Amin, and S. MansoorS. S. Zaidihttp://orcid.org/0000-0002-2976-4624Search for more papers by this author, S. ShakirSearch for more papers by this author, M. FarooqSearch for more papers by this author, I. AminSearch for more papers by this author, and S. MansoorSearch for more papers by this authorAffiliationsAuthors and Affiliations S. S. Zaidi , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan S. Shakir , Virology Laboratory, Center for Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan; and Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan M. Farooq I. Amin S. Mansoor , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. Published Online:7 Apr 2017https://doi.org/10.1094/PDIS-12-16-1859-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In 2015–16, during field surveys of Sindh and Punjab provinces of Pakistan, characteristic Begomovirus-like symptoms of leaf yellowing and curling were observed on Cyamopsis tetragonoloba. C. tetragonoloba, also known as cluster bean or guar, is a leguminous crop commonly cultivated in India and Pakistan. To test for possible begomoviruses, one symptomatic plant and one asymptomatic plant was collected from Sindh and two symptomatic and one asymptomatic plant was collected from Punjab. Total DNA was extracted from leaf tissue of all plants using a CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle 1987). Southern blot hybridization was performed using a DIG labeled probe to the Tomato yellow leaf curl virus (TYLCV) C1 gene. A strong hybridization signal was observed in symptomatic plants only. To confirm the association of TYLCV, DNA from one positive sample was used in rolling circle amplification (RCA) for viral DNA enrichment using phi29 DNA polymerase (Thermo Fisher Scientific, Waltham, MA, U.S.A.). The RCA product was purified and sequenced by the Illumina MiSeq system. The Illumina NeoPrep automation system was used with the Illumina TruSeq Nano DNA Library Prep Kit (NP-101-1001). Adapters were trimmed using MiSeq Reporter Software and sequencing data were analyzed using CLC Genomics Workbench 7.5 (https://www.qiagenbioinformatics.com). The total number of raw reads obtained was 264,578. De novo assemblies were prepared following the standard quality parameters (quality score: 0.001 and Phred score: 30). Sequence analysis resulted in one contig corresponding to the full-length genome of TYLCV (∼2800 nucleotides) with a coverage of 107,574 reads. An NCBI BLASTn of the contig confirmed the presence of TYLCV (deposited in GenBank as KX710157). A BLASTn analysis of the contig sequence showed that TYLCV from Pakistan was 92% identical to TYLCV reported in 2005 on tomato from Al-Batinah, Oman. TYLCV PCR primers were designed from the contig sequence as follows: TYLCV-PK-F 5′-CGACCAGTCTGAGGCTGTAAT-3′ and TYLCV-PK-R 5′-CCCTTTAATTTGAATGGG-3′. DNA from each symptomatic sample tested positive for TYLCV by PCR and the amplification products (511 bp) were cloned into a T/A cloning vector (pTZ57R/T; ThermoFisher Scientific). Clones were Sanger sequenced. Sequences were assembled using Lasergene software (DNA Star Inc.) and a consensus sequence was deposited in GenBank as KY769274. A BLASTn analysis of the consensus sequence showed 97% identify to TYLCV-Iran isolate from tomato in Iran. The species and strain demarcation of TYLCV was confirmed using the Sequence Demarcation Tool (SDT) program (http://web.cbio.uct.ac.za/∼brejnev/), following the taxonomic criteria of begomoviruses (Brown et al. 2015). A MUSCLE alignment was performed using default settings of SDT, using all 347 available sequences of TYLCV from the ICTV (http://www.ictvonline.org/virustaxonomy.asp). SDT-MUSCLE analysis indicated that the TYLCV, found on cluster bean, is a novel strain, to which we purpose the name TYLCV-Pakistan (TYLCV-PK), following the standard nomenclature for begomoviruses (Brown et al. 2015). TYLCV has been detected in several countries and is a serious threat to economically important crops such as tomato and cucurbits (Mabvakure et al. 2016). The occurrence of TYLCV-PK on cluster bean indicates the increasing geographical and host range of TYLCV. This is the first report of TYLCV-PK, a novel strain of TYLCV; this is also the first report of TYLCV infecting cluster bean or any other crop in Pakistan.References:Brown, J. K., et al. 2015. Arch. Virol. 160:1593. https://doi.org/10.1007/s00705-015-2398-y Crossref, ISI, Google ScholarDoyle, J. J., and Doyle, J. L. 1987. Phytochem. Bull. 19:11. Google ScholarMabvakure, B., et al. 2016. Virology 498:257. https://doi.org/10.1016/j.virol.2016.08.033 Crossref, ISI, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 101, No. 6 June 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 17 May 2017Published: 7 Apr 2017First Look: 27 Feb 2017Accepted: 18 Feb 2017 Pages: 1071-1071 Information© 2017 The American Phytopathological SocietyCited byMultiple begomoviruses infecting soybean; a case study in Faisalabad, Pakistan14 December 2022 | Biologia, Vol. 78, No. 2Engineering Molecular Immunity Against Plant Viruses

Cotton leaf curl disease (CLCuD) is a serious threat to cotton production across the Indian subcontinent. In the Punjab province of Pakistan the disease is associated with a distinct strain, the Burewala strain, of cotton leaf curl Kokhran virus (CLCuKoV-Bu) and a distinct strain of the symptom-determining betasatellite, cotton leaf curl Multan betasatellite-Burewala (CLCuMuBBur). At this time all commercial varieties of Gossypium hirsutum are susceptible to CLCuD. Two exotic G. hirsutum wild accessions, AS0039 and AS0099 from Caribbean, were found to be naturally resistant to CLCuD. However, the nature of the resistance has remained undefined. Graft-inoculation and whitefly transmission assays were conducted as a preliminary analysis of the resistance of these cultivars to CLCuKoV-Bu/CLCuMuBBur complex. By whitefly transmission under conditions where all plants of the susceptible cultivar CIM-496 became symptomatic AS0039 and AS0099 plants remained non-symptomatic and no virus was detected. Graft-inoculation of AS0039 and AS0099 with severely infected scions from G. hirsutum cv. CIM-496 showed that these accessions could support the replication and long-distance spread of the virus/betasatellite-complex. Several leaves developing at the time of grafting close to and distal to the grafts in these plants showed mild symptoms with low amounts of viral/betasatellite DNA; significantly less than the levels seen in susceptible CIM-496 plants. Although symptoms spread to leaves away from the initial point of infection, the youngest leaves at the apex of both AS0039 and AS0099 plants remained symptomless with very low virus/betasatellite- complex. The results showed that the resistance of AS0039 and AS0099 plants is either due to reduced virus/ satellite replication or perhaps interference in the short distance (cell-to-cell) spread of the virus/betasatellite-complex.

HomePlant DiseaseVol. 101, No. 1First Report of Alternanthera yellow vein virus From Eclipta prostrata in Pakistan Previous DISEASE NOTES OPENOpen Access licenseFirst Report of Alternanthera yellow vein virus From Eclipta prostrata in PakistanS. S. Zaidi, S. Shakir, M. Farooq, I. Amin, and S. MansoorS. S. ZaidiSearch for more papers by this author, S. ShakirSearch for more papers by this author, M. FarooqSearch for more papers by this author, I. AminSearch for more papers by this author, and S. MansoorSearch for more papers by this authorAffiliationsAuthors and Affiliations S. S. Zaidi , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan S. Shakir , Virology Laboratory, Center for Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad, Pakistan, and Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan M. Farooq I. Amin S. Mansoor , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. Published Online:10 Nov 2016https://doi.org/10.1094/PDIS-08-16-1164-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Alternanthera yellow vein virus (AlYVV) is a Begomovirus species that is transmitted by the silverleaf whitefly Bemisia tabaci. In July 2015, during a field survey of the Tando Mohammad Khan area in Sindh province and the Faisalabad area in Punjab province, Pakistan, symptoms of vein yellowing and swelling were observed on leaves of Eclipta prostrata (family Asteraceae). E. prostrata, also known as false daisy or bhangra,is a common weed in Pakistan and was recently reported as a host for AlYVV in China (He et al. 2008). To detect the possible presence of AlYVV, two symptomatic plants and one asymptomatic plant were collected and total DNA was extracted. Southern blot hybridization was performed using a DIG labeled probe to the AlYVVC1 gene. A strong hybridization signal was observed in symptomatic plants, while there was no signal in asymptomatic plants collected from the same fields. To further confirm the association of AlYVV, rolling circle amplification (RCA) was performed to enrich the circular DNA molecules of begomovirus genome, using phi29 DNA polymerase (Thermo Fisher Scientific, Waltham, MA). The RCA product was purified and sequenced by the Illumina MiSeq sequencing system. The Illumina NeoPrep automation system (Illumina, San Diego, CA) was used with the Illumina TruSeq Nano DNA Library Prep Kit (NP-101-1001). After sequencing, adapters were trimmed by the MiSeq Reporter Software and sequencing data were analyzed by CLC Genomics Workbench 7.5 (https://www.qiagenbioinformatics.com). De novo and reference-based assemblies were prepared following the standard quality parameters (quality score: 0.001 and Phred score: 30). The NCBI-nBLAST results of 2,917 assembled reads confirmed the presence of AlYVV, available in the database with the accession number KX710155. This AlYVV sequence showed 96% nucleotide identity with AlYVV (FN432361) reported on Sonchus arvensis from Faisalabad, Pakistan; another weed species belonging to the family Asteraceae. The species demarcation of AlYVV was also confirmed using the sequence demarcation tool (SDT) program (http://web.cbio.uct.ac.za/∼brejnev/), following the revised taxonomic criteria of begomoviruses (Brown et al. 2015). Neither in the Illumina data nor by PCR using universal primers for amplification of DNA-B (Zaidi et al. 2016), alphasatellites (Bull et al. 2003), or betasatellites (Briddon et al. 2002), was evidence found for the presence of DNA-B, alphasatellites, or betasatellites. E. prostrata has been reported as a host for multiple begomoviruses like AlYVV (He et al. 2008), and could therefore serve as an alternative virus reservoir, with an important role in the epidemiology of the disease. To our knowledge, this is the first report of AlYVV on E. prostrata in Pakistan.References:Briddon, R. W., et al. 2002. Mol. Biotechnol. 20:315. https://doi.org/10.1385/MB:20:3:315 Crossref, ISI, Google ScholarBrown, J. K., et al. 2015. Arch. Virol. 160:1593. https://doi.org/10.1007/s00705-015-2398-y Crossref, ISI, Google ScholarBull, S. E., et al. 2003. Mol. Biotechnol. 23:83. https://doi.org/10.1385/MB:23:1:83 Crossref, ISI, Google ScholarHe, Z. F., et al. 2008. J. Phytopathol. 156:496. https://doi.org/10.1111/j.1439-0434.2007.01381.x Crossref, ISI, Google ScholarZaidi, S. S., et al. 2016. PLoS One 11:e0155520. https://doi.org/10.1371/journal.pone.0155520 Crossref, ISI, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 101, No. 1 January 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 22 Dec 2016Published: 10 Nov 2016First Look: 17 Oct 2016Accepted: 23 Sep 2016 Pages: 266-266 Information© 2017 The American Phytopathological SocietyCited byNigrospora sphaerica causing leaf spot in a new host, Eclipta prostrata (False Daisy), in China20 January 2022 | Journal of Phytopathology, Vol. 170, No. 4Alternanthera yellow vein virusCABI Compendium, Vol. CABI CompendiumEvolutionary Factors in the Geminivirus Emergence13 July 2019Eclipta prostrata (False daisy)6 June 2020Bemisia tabaci-mediated facilitation in diversity of begomoviruses: Evidence from recent molecular studiesMicrobial Pathogenesis, Vol. 123Engineering Molecular Immunity Against Plant Viruses

Co-infection of Carrot red leaf virus (CtRLV), Carrot mottle virus (CMoV) and Carrot red leaf virus associated RNA (CtRLVaRNA) causes Carrot motley dwarf (CMD) disease. This study examined the capacity of the aphid Myzus persicae at transmitting viruses associated with CMD. M. persicae exposed to CMD-infected chervil plants transmitted CtRLV-, CMoV- and CtRLVaRNA to disease-free chervil, fennel, celery, carrot, cilantro, and parsley, as shown by RT-PCR using specific primers. Recipient plants developed typical CMD symptoms. Sequence analysis of the amplified virus genes showed high sequence diversity with corresponding sequences available in GenBank. This study expands on Cavariella aegopodii, the only previously recognized aphid vector of CMD-causing viruses.

HomePlant DiseaseVol. 99, No. 11First Report of Tomato leaf curl Gujarat virus, a Bipartite Begomovirus on Cotton Showing Leaf Curl Symptoms in Pakistan PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Tomato leaf curl Gujarat virus, a Bipartite Begomovirus on Cotton Showing Leaf Curl Symptoms in PakistanS. S. A. Zaidi, Z. Iqbal, I. Amin, and S. MansoorS. S. A. Zaidi, Z. Iqbal, I. Amin, and S. MansoorAffiliationsAuthors and Affiliations S. S. A. Zaidi , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, and Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad, Pakistan Z. Iqbal I. Amin S. Mansoor , Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan. Published Online:8 Sep 2015https://doi.org/10.1094/PDIS-02-15-0195-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Cotton leaf curl is one of the most important constraints on cotton (Gossypium hirsutum) in Pakistan and northwestern India. The disease is associated with several monopartite begomoviruses, which essentially require a specific DNA satellite called Cotton leaf curl Multan betasatellite. The disease complex is evolving rapidly by component capture and recombination, which supposedly helps the complex to overcome resistance (Sattar et al. 2013). During a routine survey for monitoring cotton leaf curl disease in October 2013, several leaf samples of infected cotton plants showing typical symptoms of disease such as leaf curling and vein thickening were collected from the Gojra region of Punjab, and were checked for the presence of begomoviruses and associated satellites. Total DNA extracted from infected plants was resolved on agarose gel, transferred to nylon membrane, and probed with Cotton leaf curl Burewala virus probe. The probe detected begomovirus DNA forms found in two out of six infected plants and confirmed the association of begomovirus with the disease. Universal primers for begomoviruses (Akhtar et al. 2009) were used in PCR for confirmation of begomoviruses, and PCR products of expected size (∼2.8 kb) were obtained. For the detection of begomoviruses, the amplified products were cloned in a TA cloning vector and were sequenced. The complete nucleotide sequences of two clones showed 99% sequence identity with Tomato leaf curl Gujarat virus (ToLCGuV), available in database under accession numbers LN794214 and LN794215, a bipartite begomovirus reported from India (Chakraborty et al. 2003). ToLCGuV was previously found on a weed species in Pakistan but effort to find cognate DNA B was not successful (Mubin et al. 2012). In order to confirm the bipartite nature of the virus, specific primers were designed on DNA B (forward primer 5′-GGTACCCGTAACGATCTTGAACTATGTCCC-3′ and reverse primer 5′-GGTACCCTATCTGGCTATAGGTCCGAACG-3′). The use of these primers in PCR resulted in amplification of a full length ∼2.7 kb product, confirming the bipartite nature of virus. PCR analysis of 40 plant samples was done, among which 14 showed amplification of expected size. The amplified product was cloned and fully sequenced. The sequence analysis confirmed the identity as DNA B of Tomato leaf curl New Delhi virus (ToLCNDV), available in database under accession number LN713269, which is 92% identical to the ToLCNDV DNA B isolated from Sonchus arvensis, a weed found in Pakistan (Mubin et al. 2010). To our knowledge, this is the first report of a bipartite begomovirus on cotton showing leaf curl disease symptoms; however, further work is needed to confirm Koch's postulate on cotton. The association of a bipartite virus may help the virus complex to overcome host disease resistance or expansion of the host range.References:Akhter, A., et al. 2009. Plant Dis. 93:962. https://doi.org/10.1094/PDIS-93-9-0962B Link, ISI, Google ScholarChakraborty, S., et al. 2003. Phytopathology 93:1485. https://doi.org/10.1094/PHYTO.2003.93.12.1485 Link, ISI, Google ScholarMubin, M., et al. 2010. Virus Genes 40:452. https://doi.org/10.1007/s11262-010-0470-y Crossref, ISI, Google ScholarMubin, M., et al. 2012. Virus Genes 44:112. https://doi.org/10.1007/s11262-011-0662-0 Crossref, ISI, Google ScholarSattar, M. N., et al. 2013. J. Gen. Virol. 94:695. https://doi.org/10.1099/vir.0.049627-0 Crossref, ISI, Google ScholarDetailsFiguresLiterature CitedRelated Vol. 99, No. 11 November 2015SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 4 Nov 2015Published: 8 Sep 2015First Look: 4 May 2015Accepted: 29 Apr 2015 Page: 1655 Information© 2015 The American Phytopathological SocietyCited byEmergence of Asian endemic begomoviruses as a pandemic threat28 September 2022 | Frontiers in Plant Science, Vol. 13Cotton leaf curl disease complex (leaf curl disease of cotton)CABI Compendium, Vol. CABI CompendiumTomato leaf curl Gujarat virusCABI Compendium, Vol. CABI CompendiumMolecular insight into cotton leaf curl geminivirus disease resistance in cultivated cotton ( Gossypium hirsutum )30 September 2019 | Plant Biotechnology Journal, Vol. 18, No. 3Cotton Diseases and Their Management6 March 2020Non-cultivated Cotton Species (Gossypium spp.) Act as a Reservoir for Cotton Leaf Curl Begomoviruses and Associated Satellites14 May 2019 | Plants, Vol. 8, No. 5Evolutionary Factors in the Geminivirus Emergence13 July 2019Gossypium spp. (Cotton)6 June 2020Bemisia tabaci-mediated facilitation in diversity of begomoviruses: Evidence from recent molecular studiesMicrobial Pathogenesis, Vol. 123Association of tomato leaf curl Gujarat virus and tomato leaf curl Bangladesh betasatellite on papaya showing typical leaf curl symptoms in North India8 May 2018 | 3 Biotech, Vol. 8, No. 5Identification of a new begomovirus infecting Duranta repens in Pakistan9 December 2017 | Archives of Virology, Vol. 163, No. 3Multiple begomoviruses found associated with cotton leaf curl disease in Pakistan in early 1990 are back in cultivated cotton6 April 2017 | Scientific Reports, Vol. 7, No. 1An Insight into Cotton Leaf Curl Multan Betasatellite, the Most Important Component of Cotton Leaf Curl Disease Complex29 September 2017 | Viruses, Vol. 9, No. 10Tomato leaf curl New Delhi virus : a widespread bipartite begomovirus in the territory of monopartite begomoviruses17 October 2016 | Molecular Plant Pathology, Vol. 18, No. 7The Prediction of a New CLCuD Epidemic in the Old World19 April 2017 | Frontiers in Microbiology, Vol. 8Status and Diversity of Begomoviruses in Pakistan15 October 2017Engineering Molecular Immunity Against Plant VirusesFrequent Occurrence of Tomato Leaf Curl New Delhi Virus in Cotton Leaf Curl Disease Affected Cotton in Pakistan23 May 2016 | PLOS ONE, Vol. 11, No. 5

China rose (Hibiscus rosa-sinensis) is a perennial ornamental plant grown throughout the tropics and subtropics. China rose plants with severe vein thickening/greening, leaf curling, and enations on the lower leaf surface were found near cotton fields at the Nuclear Institute for Agriculture and Biology (NIAB) in Faisalabad (Pakistan). These symptoms were very much similar to those of cotton leaf curl disease (CLCuD). DNA was extracted from 10 naturally infected symptomatic China rose plants and subjected to PCR using begomovirus- and betasatellite-specific primers. The expected products of 2.8 kb and 1.4 kb for begomoviruses and betasatellites, respectively, were amplified. The begomovirus and its cognate betasatellite isolated from China rose were sequenced and submitted to GenBank with accession Nos. HG003876 and HG003877. Owing to a 99% nucleotide sequence identity, the virus under study was identified as an isolate of Cotton leaf curl Burewala virus (CLCuBuV). The sequenced betasatellite showed 96% nucleotide sequence identity with Cotton leaf curl Multan betasatellite (CLCuMuB). Induction of CLCuD symptom upon indexing in LRA-5166, a CLCuBuV susceptible cotton genotype that resists Cotton leaf curl Multan virus (CLCuMuV), further confirmed the presence of CLCuBuV in China rose. To our knowledge, this is the first report of CLCuBuV and its cognate betasatellite in China rose.

The diploid cotton species belonging to A, D and tetraploids with AD genome were screened for the presence/absence of components of cotton leaf curl complex. These cotton species are being maintained in a living herbarium for more than fourdecades under natural conditions. As expected, the two diploid species (Gossypium arboreum and G. herbaceum) of A genome, were found to be free of virus when screened by PCR and Φ29 DNA polymerase. The two cotton species of D genome (G. thurberii and G. aridum) showed the presence of begomovirus. The species belonging to AD genome were found to be susceptible to CLCuD due to the presence of both begomovirus and betasatellite. However, the interesting exception among D genome species was G. gossypioides. The results from present study suggest that G. gossypioides is a valuable resource fo r mapping of resistance by developing segregating populations and for developing synthetic tetraploids.

RNA silencing technology has become the tool of choice for inducing resistance against viruses in plants. A significant discovery of this technology is that double-stranded RNA (dsRNA), which is diced into small interfering RNAs (siRNAs), is a potent trigger for RNA silencing. By exploiting this phenomenon in transgenic plants, it is possible to confer high level of virus resistance by specific targeting of cognate viral RNA. In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have constructed a chimeric expression vector containing three partial gene sequences derived from the ORF2 gene of Potato virus X, Helper Component Protease gene of Potato virus Y and Coat protein gene of Potato leaf roll virus. Solanum tuberosum cv. Desiree and Kuroda were transformed with this chimeric gene cassette via Agrobacterium tumefaciens-mediated transformation and transgenic status was confirmed by PCR, Southern and double antibody sandwich ELISA detection. Due to simultaneous RNA silencing, as demonstrated by accumulation of specific siRNAs, the expression of partial triple-gene sequence cassette depicted 20% of the transgenic plants are immune against all three viruses. Thus, expression of a single transgene construct can effectively confer resistance to multiple viruses in transgenic plants.