Identification of a New, Monopartite Begomovirus Associated with Leaf Curl Disease of Cotton in Gezira, Sudan

Cotton leaf curl disease (CLCuD) was first reported in Sudan in 1931. Disease symptoms in cotton were characterized by vein thickening and leaf curling, and the suspect causal agent was shown to be transmitted by the whitefly Bemisia tabaci (Genn.) among cotton, okra, and several weed species (2). Although begomovirus etiology was suspected based on symptomatology and vector transmission, no evidence was available that confirmed or disputed this hypothesis. During 1994 to 1996, four cotton samples exhibiting typical CLCuD symptoms were collected from different fields in the Gezira region in Central Sudan and examined for presence of begomovirus DNA. Total nucleic acids were isolated from cotton plants and subjected to polymerase chain reaction (PCR) using degenerate primers (pAV 2644 and pAC 1154) to amplify begomovirus coat protein (Cp) gene and its flanking sequences (1). An amplicon of the expected size (1,300 bp) was obtained by PCR from each sample, and their nucleotide (nt) sequences were determined. Virus-specific primers designed around the Cp sequence were used to amplify an apparent full-length DNA component. Amplicons were cloned and their sequences were determined, yielding a begomoviral component of approximately 2,761 nt (AF260241). Despite exhaustive attempts to amplify a putative viral B-component using degenerate primers based on the intergenic region sequence of the putative “A-component,” or sequences that are highly conserved for other begomoviruses, no B component was detected. The four cotton isolates shared 99.9 to 100% nt sequence identity, and the number and arrangement of predicted open reading frames were similar to those known for other monopartite begomoviruses. Phylogenetic analysis of the putative CLCuV genome with other begomoviruses indicated that its closest relative was Althea rosea enation virus (AREV) from Egypt (AF014881) with which it shares 79% sequence identity, indicating that CLCuV is a unique begomovirus species with a probable origin in the Eastern Hemisphere. CLCuV shared 66% identity with its second closest relative, Cotton leaf curl virus-Pakistan (CLCuV-PK) (AJ002448). These data provide the first direct evidence for the association of a monopartite begomovirus with the leaf curl disease of cotton in Gezira, Sudan, that is distinct from all other begomoviral species described to date. Herein, we provisionally designate this unique begomoviral species as Cotton leaf curl virus from Sudan (CLCuV-SD). References: (1) A. M. Idris and J. K. Brown. Phytopathology 88:648, 1998. (2) A. M. Nour and J. J. Nour. Emp. Cott. Gr. Rev. 41:27, 1964.

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Evidence for the Association of a Bipartite Geminivirus with Tomato Leaf Curl Disease in Pakistan

Plant Disease ◽

10.1094/pdis.1997.81.8.958c ◽

1997 ◽

Vol 81 (8) ◽

pp. 958-958 ◽

Cited By ~ 11

Author(s):

S. Mansoor ◽

S. H. Khan ◽

M. Saeed ◽

A. Bashir ◽

Y. Zafar ◽

...

Keyword(s):

Tomato Leaf ◽

Cotton Leaf ◽

Degenerate Primers ◽

Tomato Plants ◽

Tomato Leaf Curl Disease ◽

Eastern Hemisphere ◽

Tomato Leaf Curl ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Tomato leaf curl disease is the most important constraint on tomato production in Pakistan, where it is found throughout the country. The disease, which occurs in high incidence in Punjab and Sindh provinces, causes 30 to 40% yield losses in the spring crop and uneconomically high losses when grown as an autumn crop. The symptoms of the disease include upward or downward leaf curling, vein thickening, and stunting of the plant. The disease is transmitted by Bemisia tabaci whiteflies (non-B, biotype K) and is suspected to be caused by a geminivirus. For the detection of geminivirus, total DNA was extracted from infected plants, fractionated in an agarose gel, transferred to a nylon membrane, and Southern blotted. A full-length clone of DNA-A of cotton leaf curl virus from Pakistan (S. Mansoor, I. Bedford, M. S. Pinner, A. Bashir, R. Briddon, J. Stanley, Y. Zafar, K. A. Malik, and P. G. Markham, unpublished) was labeled with [32P]dCTP by the oligo-labeling method and hybridized at medium stringency. Geminivirus DNA forms that are normally found in infected plants were detected in plants with tomato leaf curl disease but not in healthy plants. To further confirm the presence of a whiteflytransmitted geminivirus, universal primers for dicot-infecting geminiviruses (1) were used in polymerase chain reaction (PCR) and a product of expected size (approximately 2.7 kb) was detected. The 2.7-kb PCR-amplified DNA from diseased tomato plants was labeled with [32P]dCTP and used as probe in Southern hybridization. This probe also detected geminivirus DNA forms at medium stringency. Both monopartite and bipartite geminiviruses transmitted by whiteflies have been reported to cause leaf curl symptoms on tomato from the Eastern hemisphere. Degenerate primers (PBLv2040 and PCRc1), which amplify B component DNA, were used to determine if tomato leaf curl was monopartite or bipartite (2). A product of expected size (0.65 kb) was amplified, suggesting this virus to be bipartite. DNA-B PCR product obtained from diseased tomato plants was hybridized as described above and detected geminivirus DNA forms at medium stringency. Samples of diseased tomato plants were collected from tomato fields throughout Punjab. DNA-A was detected in all 20 samples whereas DNA B was detected in 17 samples when hybridized by dot blot method at medium stringency. Our data show that tomato leaf curl virus from Pakistan is a bipartite geminivirus. This is the first evidence for a bipartite geminivirus in tomato plants from Pakistan. References: (1) R. W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1993. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

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Widespread Occurrence of Cotton leaf curl virus on Radish in Pakistan

Plant Disease ◽

10.1094/pdis.2000.84.7.809b ◽

2000 ◽

Vol 84 (7) ◽

pp. 809-809 ◽

Cited By ~ 7

Author(s):

S. Mansoor ◽

S. Mukhtar ◽

M. Hussain ◽

I. Amin ◽

Y. Zafar ◽

...

Keyword(s):

Host Range ◽

Full Length ◽

Cotton Leaf ◽

Degenerate Primers ◽

Nylon Membrane ◽

Punjab Province ◽

Cotton Leaf Curl Virus ◽

Kitchen Gardens ◽

Leaf Curl Virus ◽

Leaf Curl

The current epidemic of cotton leaf curl disease (CLCuD) in Pakistan started in 1988 with the natural host range limited to a few plant species in the family Malvaceae. However, we have observed expansion in the host range of the virus, and several non-Malvaceous plants were found to be infected with the virus. Characteristic symptoms of CLCuD such as leaf curl and enations have been observed on radish plants, primarily in kitchen gardens. However, in 1999, levels of infection of 10 to 90% were observed both in commercial fields and kitchen gardens in the Punjab province of Pakistan. Both symptomatic and nonsymptomatic samples were collected from five different locations. Total DNA was isolated, dot-blotted on nylon membrane, and a full-length clone corresponding to DNA A of cotton leaf curl virus was labeled with 32P dCTP and used as a probe for the detection of a begomovirus. Strong signals were observed in symptomatic plants while no signals were observed in nonsymptomatic plants. Infection with a begomovirus was further confirmed by polymerase chain reaction (PCR) using degenerate primers for DNA A (1). Primers specific for the two distinct begomoviruses associated with CLCuD were also used in PCR reactions (2), and products of the expected size were obtained from all symptomatic samples, confirming infection with begomoviruses similar to those associated with CLCuD. A full-length probe of a nanovirus-like molecule associated with cotton leaf disease (3), called DNA 1 was labeled with 32P dCTP and detected the virus only in symptomatic plants. Similarly, primers specific for DNA 1 (3) amplified a product of expected size when used in PCR. On the basis of symptomatology and the detection of specific viral components associated with the disease, we confirmed that radish plants are infected with Cotton leaf curl virus (CLCuV). Since radish is a short duration crop, infection of CLCuV in radish may not serve as a direct source of infection for the next cotton crop. However, it is a potential threat to tomato crops which overlap with radish in the Punjab province. The detection of CLCuD in radish is another example of the mobilization of begomoviruses to previously unknown hosts. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) S. Mansoor et al. Pak. J. Bot. 31:115, 1999. (3) Mansoor et al. Virology 259:190, 1999.

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Cotton leaf curl virus (leaf curl disease of cotton).

10.1079/cpc.16813.20210102471 ◽

2021 ◽

Author(s):

Judith K Brown

Keyword(s):

South Asia ◽

Ornamental Plants ◽

Seed Transmission ◽

Growing Season ◽

The Philippines ◽

Cotton Leaf ◽

Cotton Leaf Curl Virus ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Abstract Leaf curl disease of cotton caused by the CLCuD-complex of begomoviruses is endemic to Pakistan and India and perhaps other nearby locales in south Asia. It has been introduced from there to China and the Philippines on ornamental plants, from where it has spread to infect cotton and okra in China. Losses are difficult to assess, but early estimates (pre-1990) range up to 20% when infection occurs early in the growing season and/or with highly susceptible cultivars. Viruliferous whiteflies on infested/infected plants harbouring CLCuD-begomoviruses imported to other cotton-growing countries, in particular, are of concern in preventing introduction under optimal circumstances. No seed transmission is known to occur.

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First Report of Papaya Leaf Curl Disease in Pakistan

Plant Disease ◽

10.1094/pdis.1997.81.11.1333b ◽

1997 ◽

Vol 81 (11) ◽

pp. 1333-1333 ◽

Cited By ~ 10

Author(s):

A. Nadeem ◽

T. Mehmood ◽

M. Tahir ◽

S. Khalid ◽

Z. Xiong

Keyword(s):

Nucleic Acids ◽

Virus Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Cotton Leaf ◽

First Report ◽

Cotton Leaf Curl Virus ◽

Dna Fragment ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Papaya plants with virus-disease-like symptoms were observed in back yards and commercial groves in Multan, Pakistan. Leaves of the diseased plants displayed downward curling and thickened, dark green veins. Leaf-like enations grew from the base of the diseased leaves. These symptoms are similar to those of cotton leaf curl disease. In addition, diseased papayas were stunted and distorted. Leaf extracts from 3 diseased and 2 healthy papayas were tested in enzyme-linked immunosorbent assay against antibodies to geminiviruses. SCRI-52 and SCRI-60, two monoclonal antibodies to Indian cassava mosaic virus (2), reacted positively (more than 7× healthy background) with the diseased samples but not with the healthy ones. Total nucleic acids from the papaya samples were used as templates in polymerase chain reaction with primers F500 and R1800 (1), which are capable of amplifying a region of DNA A component of the whitefly-transmitted geminiviruses. A DNA fragment of approximately 1.4 kb was amplified from the nucleic acids of the diseased but not the healthy papayas. Under high stringency conditions (1), cloned DNA A fragments of both cotton leaf curl virus and cotton leaf crumple virus cross-hybridized with the amplified DNA fragment, but the hybridization signals were much weaker than those of the homologous hybridization. This is the first report of the papaya leaf curl disease in Pakistan. These data demonstrated that a geminivirus may be the causative agent of this papaya disease. We are currently determining the relationship between the geminivirus infecting papaya and cotton leaf curl virus. References: (1) A. Nadeem et al. Mol. Plant Pathol. (On-line: /1997/0612nadeem). (2) M. M. Swanson et al. Ann. Appl. Biol. 211:285, 1992.

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Association of a Begomovirus and Nanovirus-like Molecule with Ageratum Yellow Vein Disease in Pakistan

Plant Disease ◽

10.1094/pdis.2000.84.1.101a ◽

2000 ◽

Vol 84 (1) ◽

pp. 101-101 ◽

Cited By ~ 9

Author(s):

S. Mansoor ◽

S. H. Khan ◽

M. Hussain ◽

Y. Zafar ◽

M. S. Pinner ◽

...

Keyword(s):

Full Length ◽

Yellow Vein ◽

Cotton Leaf ◽

Degenerate Primers ◽

Total Dna ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Vein Disease ◽

Full Length Clone ◽

Leaf Curl

Whitefly-transmitted geminiviruses (begomoviruses) cause heavy losses to many food and fiber crops in Pakistan. Many weeds also show symptoms typical of begomoviruses. Ageratum (Ageratum conyzoides) is a common perennial weed in Pakistan, growing along irrigation canals, that often shows symptoms, such as yellow vein and mosaic, suggesting infection by a begomovirus. To confirm this, symptomatic and asymptomatic ageratum plants were collected from three locations in the Punjab Province of Pakistan, and total DNA was isolated, subjected to agarose gel electrophoresis, transferred to a nylon membrane, and Southern blotted. Total DNA isolated from cotton infected with Cotton leaf curl virus (CLCuV), tomato infected with Tomato leaf curl virus from Pakistan (TLCV-Pak), tobacco infected with African cassava mosaic virus (ACMV) from Nigeria, and healthy tobacco were included as controls. A full-length clone of CLCuV DNA A was labeled with [32P]dCTP by oligo-labeling and hybridized at medium stringency. The probe detected characteristic geminivirus DNA forms in symptomatic ageratum and plants infected with CLCuV, TLCV-Pak, and ACMV, while no signal was detected in asymptomatic ageratum from the field or healthy tobacco. To confirm infection by a begomovirus, degenerate primers WTGF (5′-GATTGTACGCGTCCDCCTTTAATTT GAAYBGG-3′), designed in the rep gene of begomoviruses, and WTGR (5′-TANACGCGTGGC TTCKRTACATGGCCTDT-3′), designed in the coat protein gene of DNA A of begomoviruses, were used in polymerase chain reaction (PCR). Degenerate primers (PBLv2040 and PCRc1) also were used in PCR (2). A product of expected size (≈1.4 kb) was obtained with DNA A primers from symptomatic ageratum, while no product was obtained with DNA B primers in the same sample. Previously we were unable to detect a DNA component equivalent to begomovirus DNA B in cotton showing symptoms of cotton leaf curl disease (1). We recently reported a novel circular DNA molecule that was approximately half as long as the full-length DNA A (CLCuV DNA-1) associated with CLCuV that share homology to plant nanoviruses (1). The supercoiled replicative form of viral DNA isolated from infected ageratum plants indicated the presence of smaller molecules, as was found in cotton leaf curl disease, suggesting that a nanovirus-like molecule might be associated with ageratum yellow vein disease. A duplicate blot of samples used in Southern hybridization with the DNA A probe was prepared, and a probe of the full-length clone of the nanovirus-like molecule (CLCuV DNA-1) was prepared as described for DNA A. The probe detected characteristic nanovirus DNA forms in ageratum with yellow vein symptoms and cotton infected with CLCuV, while no signal was detected in plants infected with TLCV-Pak or ACMV, healthy tobacco, or asymptomatic ageratum. Abutting primers PB2-F and PB2R (1), designed based on the CLCuV DNA-1 sequence, were unable to amplify a PCR product from ageratum with yellow vein symptoms, suggesting the nanovirus-like molecule associated with ageratum yellow vein disease is distinct from CLCuV DNA-1. Our results show that yellow vein disease of ageratum in Pakistan is associated with a begomovirus infection and single-stranded circular DNA molecule with similarity to CLCuV DNA-1. References: (1) S. Mansoor et al. Virology 259:190, 1999. (2) M. R. Rojas et al., Plant Dis. 77:340, 1993.

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A New Begomovirus Species Causing Leaf Curl Disease of Radish in India

Plant Disease ◽

10.1094/pdis-91-8-1053b ◽

2007 ◽

Vol 91 (8) ◽

pp. 1053-1053 ◽

Cited By ~ 5

Author(s):

A. K. Singh ◽

B. Chattopadhyay ◽

P. K. Pandey ◽

A. K. Singh ◽

S. Chakraborty

Keyword(s):

Sequence Analysis ◽

Electron Microscopic Observation ◽

Begomovirus Species ◽

Infected Leaf ◽

Electron Microscopic ◽

Degenerate Primers ◽

Sequence Identity ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Leaf curl disease of radish (RLCD) was observed for the first time in India in commercial fields and kitchen gardens of the Varanasi District and adjoining areas of eastern Uttar Pradesh during November 2003. Infected plants exhibited typical upward and downward leaf curling, leaf distortion, reduction of leaf area, and conspicuous enations on the underside of the leaves. Incidence of RLCD was estimated to be between 10 and 40% depending on the cultivars used. Electron microscopic observation revealed typical geminate particles in infected leaf samples. The causal virus could be transmitted to radish cv. Minu Early by whiteflies (Bemisia tabaci) and grafting. Inoculated plants developed symptoms similar to those observed in naturally infected radish plants. Viral DNA was isolated from artificially inoculated symptomatic radish plants (4) followed by concentration of super-coiled DNA by alkaline denaturation (1). The presence of a geminivirus was confirmed by PCR using DNA-A degenerate primers (3), and a 1.5-kb amplified product was obtained from six artificially and three naturally infected plants. Amplification of the full-length DNA-A was achieved using a primer combination derived from sequences obtained from a 1.5-kb amplicon. Amplification of 1.3-kb DNA-β sequences was achieved using specific primers (2) in three infected plants. Sequence analysis revealed that DNA-A (GenBank Accession No. EF 175733) contained 2,756 nt and DNA-β contained 1,358 nt (GenBank Accession No. EF 175734). DNA-A of the causal virus shares 87.7% identity with Tomato leaf curl Bangladesh virus (GenBank Accession No. AF 188481) and 62% identity with Mungbean yellow mosaic India virus (GenBank Accession No. AF126406). The begomovirus DNA-A sequence associated with RLCD contained seven open reading frames (AV1, AV2, AC1, AC2, AC3, AC4, and AC5). The DNA-β associated with RLCD shared the highest nucleotide sequence identity (84.9%) with DNA-β of Tobacco leaf curl virus isolate NIB 12-1 (GenBank Accession No. AJ316033) reported from Pakistan. Despite exhaustive attempts to amplify a putative viral B-component using degenerate primers based on the intergenic region sequence of the DNA-A or sequences that are highly conserved for other begomoviruses, no DNA-B component was detected. On the basis of DNA-A sequence analysis, the ICTV species demarcation criteria of 89% sequence identity, and genome organization, the virus causing RLCD should be considered a new Begomovirus species, for which the name Radish leaf curl virus (RLCV) is proposed. To our knowledge, this is the first report of the association of a Begomovirus with a disease of radishes in India. References: (1) H. C. Birnboim and J. Doly. Nucleic Acids Res. 7:1513, 1979. (2) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995.

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Multiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan

Microbiology ◽

10.1099/0022-1317-81-7-1839 ◽

2000 ◽

Vol 81 (7) ◽

pp. 1839-1849 ◽

Cited By ~ 81

Author(s):

Ana I. Sanz ◽

Aurora Fraile ◽

Fernando García-Arenal ◽

Xueping Zhou ◽

David J. Robinson ◽

...

Keyword(s):

Indian Subcontinent ◽

Multiple Infection ◽

Pcr Analysis ◽

Cotton Leaf ◽

Evolutionary Divergence ◽

Cotton Leaf Curl Virus ◽

Geographical Regions ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Begomoviruses occur in many plant species in Pakistan and are associated with an epidemic of cotton leaf curl disease that has developed since 1985. PCR analysis with primer pairs specific for each of four already sequenced types of DNA-A of cotton leaf curl virus (CLCuV-PK types a, 26, 72b and 804a), or for okra yellow vein mosaic virus (OYVMV), indicated that many individual naturally infected plants of cotton and other malvaceous species contained two or three begomovirus sequences. Similarly, sequence differences among overlapping fragments of begomovirus DNA-A, amplified from individual naturally infected plants, indicated much multiple infection in malvaceous and non-malvaceous species. Some cotton plants contained DNA-A sequences typical of begomoviruses from non-malvaceous species, and some non-malvaceous plants contained sequences typical of CLCuV-PK. Some DNA-A sequences were chimaeric; they each included elements typical of different types of CLCuV-PK, or of different malvaceous and/or non-malvaceous begomoviruses. Often an apparent recombination site occurred at the origin of replication. No complete CLCuV-PK DNA-A sequence was found in malvaceous or non-malvaceous species collected in Pakistan outside the area of the cotton leaf curl epidemic but chimaeric sequences, including a part that was typical of CLCuV-PK DNA-A, did occur there. We suggest that recombination among such pre-existing sequences was crucial for the emergence of CLCuV-PK. Recombination, following multiple infection, could also explain the network of relationships among many of the begomoviruses found in the Indian subcontinent, and their evolutionary divergence, as a group, from begomoviruses causing similar diseases in other geographical regions.

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