First Report of Okra enation leaf curl virus and Associated Cotton leaf curl Multan betasatellite and Cotton leaf curl Multan alphasatellite Infecting Cotton in Pakistan: A New Member of the Cotton Leaf Curl Disease Complex

Cotton (Gossypium hirsutum L.) is an important and widely cultivated crop in Pakistan, upon which many rely for economic security. Cotton leaf curl disease (CLCuD) is caused by a complex comprising of more than eight species in the genus Begomovirus (family Geminiviridae) with associated betasatellite and alphasatellites. During 2011, characteristic symptoms of leaf curl disease were widespread (>40%), and the whitefly Bemisia tabaci (Genn.) vector of the leaf curl complex was abundant in commercial cotton fields in Burewala, Pakistan. Symptoms included vein thickening, upward or downward leaf curling, and foliar enations. To test for the presence of a begomovirus(es), total DNA was extracted from 100 mg of symptomatic leaf tissues from five different plants (isolates CLCuDBur1 to 5) using the CTAB method (1). Total DNA extracts were used for rolling circle amplification (RCA) using TempliPhi DNA Amplification Kit (GE Healthcare). Of the five field isolates, the RCA product for only one, CLCuDBur3, digested with HindIII, produced an apparently full-length ~2.7 kb fragment, suggesting that CLCuD-Bur3 represented a distinct isolate. The 2.7-kb fragment was cloned into the plasmid vector pGEM-3Zf+ (Promega, Madison, WI). To test for the presence of associated alphasatellites and betasatellites, the PCR primers, AlphaF/R and BetaF/R (2), were used to amplify the putative 1.4-kbp molecules. The resultant 1.4-kb PCR products were ligated into the pGEMT-Easy vector and cloned. Cloned inserts for each were subjected to DNA sequencing, bidirectionally. The cloned monopartite, helper begomovirus genome (HF567945), one betasatellite (HF567946), and one alphasatellite (HF567947) sequences were determined and found to be 2,742, 1,358, and 1,376 bases long, respectively. Pairwise sequence comparisons were carried out for each using the 10 most closely related species or strains (identified in GenBank using BLASTn) using MEGA5 software. The CLCuDBur3 genome sequence shared its highest identity (99.6%) with Okra enation leaf curl virus (OELCuV) (KC019308), so CLCuDBur3 is a variant of OELCuV, a begomovirus reported previously from Abelmoschus esculentus (L.) (okra) plants in India. The betasatellite and alphasatellite shared their highest nt identity at 96 and 98.7% with Cotton leaf curl Multan betasatellite (CLCuMB) (AM774311) and Cotton leaf curl Multan alphasatellite (CLCuMA), respectively (misnamed as CLCuBuA in GenBank) (FN658728). Additionally, the HindIII-digested RCA products were analyzed by Southern blot hybridization using a DIG-labeled DNA probe specific for the intergenic region of either Cotton leaf curl Burewala virus (CLCuBuV) or OELCuV. The OELCuV, but not the CLCuBuV, probe hybridized with HindIII digested RCA products (CLCuDBur3 genome), confirming the presence of OELCuV and the absence of CLCuBuV, the latter being the most prevalent begomovirus species infecting cotton in Pakistan. This is the first report of OELCuV infecting cotton plants in Pakistan, underscoring the discovery of yet another begomovirus member of the CLCuD complex. Further, the possible co-infection of cotton by OELCuV and other recognized species of the CLCuD complex could facilitate further diversification (potentially, through recombination) and lead to the emergence of new variants with the potential to cause damage to the cotton crop in Pakistan. References: (1) J. J. Doyle and J. L. Doyle. Focus. 12:13, 1990. (2) M. Zia-Ur-Rehman et al. Plant Dis. 97:1122, 2013.

Download Full-text

First Detection of Cotton leaf curl Burewala virus and Cognate Cotton leaf curl Multan betasatellite and Gossypium darwinii symptomless alphasatellite in Symptomatic Luffa cylindrica in Pakistan

Plant Disease ◽

10.1094/pdis-12-12-1159-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1122-1122 ◽

Cited By ~ 9

Author(s):

M. Zia-Ur-Rehman ◽

H.-W. Herrmann ◽

U. Hameed ◽

M. S. Haider ◽

J. K. Brown

Keyword(s):

Plasmid Vector ◽

Cotton Leaf ◽

Cotton Production ◽

Alpha Satellite ◽

Luffa Cylindrica ◽

First Report ◽

Cotton Leaf Curl Disease ◽

Total Dna ◽

Leaf Curl Disease ◽

Leaf Curl

Cotton leaf curl disease (CLCuD) is the major plant viral constraint to cotton production on the Indian subcontinent (2). CLCuD is primarily caused by begomovirus, Cotton leaf curl Burewala virus (CLCuBuV), and Cotton leaf curl Multan betasatellite (CLCuMB). During 2011 in Burewala, Pakistan, plants in a production field of Luffa cylindrica (Ghia tori) were infested with the whitefly Bemisia tabaci (Genn.), and ~60% of the plants exhibited leaf curling and stunting symptoms, reminiscent of those caused by begomoviruses (Geminiviridae). Total DNA was extracted from five different symptomatic leaf samples using the CTAB method (1), and extracts were analyzed by Southern blot hybridization. As a probe, we used a 1.1-kbp fragment of CLCuBuV and a positive signal was obtained from all five samples. Total DNA was used as template for rolling circle amplification (RCA) using the TempliPhi DNA Amplification Kit (GE Healthcare, Little Chalfont, United Kingdom). The amplified RCA products were digested with EcoRI, and the resulting ~2.7-kbp fragments from each isolate were directionally cloned into the EcoRI digested, pGEM-3Zf+ (Promega, Madison, WI) plasmid vector. PCR was used to amplify the prospective, associated betasatellite and alphasatellite molecules using the primers BetaF5′-GGTACCGCCGGAGCTTAGCWCKCC-3′ and BetaR5′-GGTACCGTAGCTAAGGCTGCTGCG-3′, and AlphaF5′-AAGCTTAGAGGAAACTAGGGTTTC-3′ and AlphaR5′-AAGCTTTTCATACARTARTCNCRDG-3′, respectively. The putative satellite amplicons, at ~1.4 kbp each were cloned in the plasmid vector pGEMT-Easy (Promega, Madison, WI) and sequenced. BLASTn comparisons of the apparently full-length begomoviral genomes, at 2,753 nt, against the NCBI database revealed that all five isolates were most closely related to CLCuBuV (FR750321). In addition, one each of beta- and alpha-satellite were amplified from all five samples at 1,393 and 1,378 bases, respectively. The beta- and alpha-satellites were most closely related to CLCuMB (HE985228) and the Gossypium darwinii symptomless alphasatellite (GDaSA) (FR877533), respectively. Pairwise sequence comparisons of the top 10 BLASTn hits using MEGA5 indicated that the helper begomovirus shared 99.9% identity with CLCuBuV (FR750321), the most prevalent helper virus currently associated with the leaf curl complex in Pakistan. Based on the ICTV demarcation for begomoviral species at <89%, it is considered a variant of CLCuBuV. The resultant beta- and alpha-satellite sequences were 98.1% and 97.8% identical to CLCuMB (HE985228) and GDaSA (FR877533), respectively, and are the most prevalent satellites associated with the CLCuD complex in Pakistan and India (2). To our knowledge, this is first report of the CLCuBuV-CLCuMB-GDaSA complex infecting a cucurbitaceous species, and the first report of L. cylindrica as a host of the CLCuD complex. This discovery of CLCuBuV and associated satellites in a cucurbitaceous host that is widely grown in Pakistan and India where this complex infects cotton indicates that the host range of CLCuBuV is broader than expected. This new information will aid in better understanding of cotton leaf curl disease epidemiology in the current epidemic areas. References: (1) J. J. Doyle and J. L. Doyle. Focus 12:13, 1990. (2) S. Mansoor et al. Trends Plant Sci. 11:209, 2006.

Download Full-text

Identification of Chili leaf curl virus Causing Leaf Curl Disease of Petunia in Oman

Plant Disease ◽

10.1094/pdis-06-13-0678-pdn ◽

2014 ◽

Vol 98 (4) ◽

pp. 572-572 ◽

Cited By ~ 5

Author(s):

A. A. Al-Shihi ◽

S. Akhtar ◽

A. J. Khan

Keyword(s):

Indian Subcontinent ◽

Rolling Circle Amplification ◽

Rapid Evolution ◽

Post Inoculation ◽

Multiple Sequence ◽

Rolling Circle ◽

Begomovirus Genome ◽

Leaf Curl Virus ◽

Leaf Curl Disease ◽

Leaf Curl

Petunias (Petunia × hybrida) are the most important ornamental plants in Oman. In 2012, petunias were observed in public parks and airport landscape in Dhofar region with symptoms of upward leaf curling, yellowing and vein clearing, and size reduction in leaves. Almost all plants in the surveyed landscape showed high infestation of Bemisia tabaci and symptoms that suggested infection with a begomovirus. Six symptomatic samples were collected from three different sites. All symptomatic samples were found PCR-positive with diagnostic primers for begomovirus (3) when DNA extracted from infected leaves was used as template. Nucleic acids extracted from the symptomatic leaves were used to amplify circular DNA molecules by rolling circle amplification method. The amplified concatameric products were digested with restriction enzyme PstI, which yielded a product ∼2.8 kb in size. The putative begomovirus fragment was cloned and sequenced in both orientations. Partial sequences of six clones were 99 to 100% similar and thus only two clones, PT-2 and PT-3, were fully sequenced. The whole genomes of both clones were 2,761 bp, and both were deposited in GenBank under accession numbers HF968755 and HF968756 for the isolates PT-2 and PT-3, respectively. Both sequences had six open reading frames; Rep, TrAP, REn, and C4 genes in complementary sense; and CP and V2 genes in virion-sense, typical of the begomovirus genome organization. Upon alignment, the two sequences showed 99.4% nucleotide identity with each other, thus representing isolates of a single begomovirus species. BlastN comparison showed PT-2 and PT-3 from petunia were 94 to 95% identical to the sequences of ChCLV from Oman (JN604490 to JN604500), which were obtained from other hosts. ClustalV multiple sequence alignment showed that isolates PT-2 and PT-3 shared maximum sequence identity of 93.3 and 92.8%, respectively, with an isolate of ChLCV-OM (JN604495). According to ICTV rules for begomoviruses, PT-3 should be considered to be a new strain of ChLCV-OM and PT-2 a variant of the already existing ChLCV-OM strain. We propose the name for this new strain as the “Petunia strain” of Chili leaf curl virus (ChLCV-Pet). Two infectious clones were constructed from the PT-2 and PT-3 sequences, clones as 1.75-genome sequences in a binary vector, suitable for agroinfection to confirm their infectivity. Both clones, PT-2 and PT-3, produced typical leaf curl disease symptoms upon inoculation on petunia 18 days post inoculation. The presence of the same virus in symptomatic field infected and inoculated petunia was confirmed by Southern blot using 650 bp DIG labeled probe prepared from CP region of PT-3 isolate. ChLCV-OM, a monopartite begomovirus, is widely associated with leaf curl disease of tomato and pepper in Oman, with its origin traced to the Indian subcontinent (2). Identification of a new strain of ChLCV from petunia provides evidence of an ongoing rapid evolution of begomoviruses in this region. Although petunia has been tested as an experimental host for some begomoviruses (1,4), this is the first report of petunia as natural host for ChLCV, a begomovirus previously reported in tomato and pepper in Oman. References: (1) Cui et al. J. Virol. 78:13966, 2004. (2) Khan et al. Virus Res. 177:87, 2013. (3) Khan et al. Plant Dis. 97:1396, 2013. (4) Urbino et al. Arch. Virol. 149:417, 2003.

Download Full-text

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.

Download Full-text

First report of cotton leaf curl disease affecting chili peppers

Plant Pathology ◽

10.1111/j.1365-3059.2003.00931.x ◽

2003 ◽

Vol 52 (6) ◽

pp. 809-809 ◽

Cited By ~ 8

Author(s):

M. Hussain ◽

S. Mansoor ◽

I. Amin ◽

S. Iram ◽

Y. Zafar ◽

...

Keyword(s):

Cotton Leaf ◽

First Report ◽

Cotton Leaf Curl Disease ◽

Chili Peppers ◽

Leaf Curl Disease ◽

Leaf Curl

Download Full-text

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.

Download Full-text

First Report of Cotton Leaf Curl Disease in Central and Southern Sindh Province in Pakistan

Plant Disease ◽

10.1094/pd-90-0826a ◽

2006 ◽

Vol 90 (6) ◽

pp. 826-826 ◽

Cited By ~ 9

Author(s):

S. Mansoor ◽

L. Amrao ◽

I. Amin ◽

R. W. Briddon ◽

K. A. Malik ◽

...

Keyword(s):

Southern Hybridization ◽

Universal Primers ◽

Cotton Leaf ◽

Pcr Product ◽

Sindh Province ◽

Cotton Leaf Curl Disease ◽

Total Dna ◽

Low Incidence ◽

Leaf Curl Disease ◽

Leaf Curl

Cotton leaf curl is a devastating disease of cotton that has resulted in severe losses (estimated at more than US$87 million per annum) in Pakistan. The epidemic is centered in Punjab, the province that contributes approximately 80% of Pakistan's cotton. Previously, the disease had been observed sporadically on single plants in the northern Sindh Province but did not cause economically significant damage. During the years 2004 and 2005, a high incidence (approximately 20%) of the disease was observed in Shahdadpur and parts of District Sanghar, located in central Sindh Province. The disease was also observed at low incidence (<1%) in southern Sindh. To confirm the identity of the causal agent of the disease, 18 samples from three districts in central southern Sindh (Sanghar, Hala, and Hyderabad) were collected, and total DNA was extracted using cetyltrimethylammoniumbromide (2). Universal primers for begomoviruses based on conserved sequences as follows were used in polymerase chain reaction (PCR): BegomoF (5′-CCGTGCTGCTGCCCCCATTGTCCGCGTCAC-3′) and BegomoR (5′-CTGCCACAACCATGGATTCACGCACAGGG-3′). Universal primers for amplification of DNA β with PCR were also used (1). A full-length clone of Cotton leaf curl Multan virus (CLCuMV) was labeled with alpha-32PdCTP by the oligo-labeling method and used as a probe in Southern hybridization for the detection of geminivirus DNA forms (2). Similarly, cotton leaf curl disease associated DNA β was also labeled and used as a probe in Southern hybridization. The use of universal primers for begomoviruses resulted in amplification of viral DNA of the expected size from all samples while no PCR product was obtained from healthy plants. PCR results confirmed that all plants were infected with begomoviruses. Southern hybridization with CLCuMV and DNA β probes detected begomovirus DNA forms associated with virus replication when washed at medium stringency, further confirming that the plants were infected with the cotton leaf curl geminivirus complex (2). Our results indicate that cotton leaf curl complex has become established in central and southern districts of Sindh Province and it poses a major threat to cotton grown in the region. References: (1) R. W. Briddon et al. Mol. Biotechnol. 20:315, 2002. (2). S. Mansoor et al. Arch. Virol. 148:1969, 2003.

Download Full-text