Engineering broad-spectrum resistance against RNA viruses in potato.

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.

Mobilisation into cotton and spread of a recombinant cotton leaf curl disease satellite.

Analysis of a DNA beta satellite associated with a recently identified cotton leaf curl disease (CLCuD) strain indicated it to be recombinant, with most of the molecule originating from CLCuD DNA beta but with some sequence from a satellite isolated from tomato. Analysis of both archival (pre 2001) and recent cotton samples, shows the recombinant satellite is confined to a small area but was not present in cotton prior to 2001. This indicates that the recombinant DNA beta was recently mobilized into cotton, likely from tomato, and that recombination plays a role in the evolution of these satellites.

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

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 ß 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 ß 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 ß 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.

Transgenic tobacco expressing geminiviral RNAs are resistant to the serious viral pathogen causing cotton leaf curl disease.

Cotton, the major cash crop in Pakistan, suffers 30% losses to cotton leaf curl disease, caused by the geminivirus, cotton leaf curl virus DNA A, plus a satellite component, DNA beta responsible for symptom development with plants failing to produce cotton bolls. We constructed transgenic tobacco expressing sense and antisense RNAs representing: [i] the 5' half of the viral DNA replication gene, AC1, [ii] the 3' half of AC1, [iii] two overlapping genes, AC2, a transcription activator, and AC3, a replication enhancer. In contrast to controls, 25% of 72 transgenic tobacco lines tested showed heritable resistance [T(1) - T(3) generations]: symptom-free and no replication of DNA A or DNA beta even after 120 days of continuous exposure to viruliferous whiteflies. As geminiviral and transgene RNAs are not detected in resistant lines following infection, and selected uninfected resistant tobacco sense lines reveal double-stranded and small interfering RNAs, the most likely mechanism is via post-transcriptional gene silencing.

Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA beta.

For bipartite begomoviruses (family Geminiviridae) trans-replication of the DNA B component by the DNA A-encoded replication-associated protein (Rep) is achieved by virtue of a shared sequence, the "common region", which contains repeated motifs (iterons) which are sequence-specific Rep binding sites and form part of the origin of replication. Recently cotton leaf curl disease (CLCuD), a major constraint to cotton production on the Indian subcontinent, has been shown to be caused by a monopartite begomovirus ( Cotton leaf curl Multan virus [CLCuMV]) and a novel single-stranded DNA satellite molecule termed CLCuD DNA beta. The satellite molecule is trans-replicated by CLCuMV but does not possess the iteron sequences of this virus. We have investigated the ability of CLCuD DNA beta to interact with three further clones of monopartite begomoviruses, isolated from cotton, that have distinct Rep binding specificities. All three cloned viruses were capable of trans-replicating the satellite molecule and inducing CLCuD symptoms in cotton, indicating that the interaction between begomovirus and DNA beta is relaxed in comparison to the interaction between DNA A and DNA B components. Field surveys across all the cotton growing regions of Pakistan indicate that dual and multiple infections are the norm for CLCuD with no evidence of synergism. Despite the diversity of begomoviruses associated with CLCuD, only a single class of DNA beta has been detected, suggesting that this satellite has the capacity to be recruited by unrelated begomoviruses.

Association of a Monopartite Begomovirus Producing Subgenomic DNA and a Distinct DNA Beta on Croton bonplandianus Showing Yellow Vein Symptoms in Pakistan.

The recent discovery that monopartite begomoviruses on ageratum and cotton essentially require a DNA satellite called DNA ß (2,4) is leading to identification of several other hosts that have similar disease complexes. A weed species (Croton bonplandianus) belonging to the family Euphorbiaceae is one such example. C. bonplandianus is widely distributed on wastelands throughout the Punjab Province in Pakistan. It very often shows yellow vein symptoms indicating infection by a begomovirus. To detect a begomovirus, both symptomatic and asymptomatic plants were collected from several widely separated locations in the Punjab Province. Total DNA was isolated from these samples by the cetyltrimethylammoniumbromide (CTAB) method, resolved in an agarose gel, and blotted on a nylon membrane (2). A full-length clone of DNA A of Cotton leaf curl virus (CLCuV) labeled with 32PdCTP was used as a probe in Southern hybridization (2). The probe detected hybridizing bands only in symptomatic plants, confirming the presence of a begomovirus. In addition to hybridizing bands of the expected sizes, smaller bands were also detected, suggesting the presence of subgenomic molecules derived from DNA A. Universal polymerase chain reaction (PCR) primers for dicot-infecting geminiviruses (1) were used in PCR for amplification of DNA A of the begomovirus associated with the disease. The use of these primers in PCR was expected to result in amplification of full-length DNA A. In addition to a product of the expected size (2.7 to 2.8 kb), another product of approximately 1.4 kb was amplified. The presence of subgenomic DNAs that are derived from DNA A is an indicator of the monopartite nature of begomoviruses, because in bipartite begomoviruses subgenomic DNAs are derived solely from DNA B. The presence of a DNA ß, a DNA satellite associated with certain monopartite begomoviruses, was suspected because of symptoms and the possible monopartite nature of the virus. Universal primers for amplification of DNA ß (3) were used in PCR for amplification of a putative DNA ß. The PCR reaction yielded a product of expected size (≈1.4 kb). A probe from the amplified product was made by the oligolabeling method. The probe detected hybridizing bands in all symptomatic samples collected from three locations, confirming the association of a DNA ß with the disease. A duplicate blot when hybridized with a DNA ß associated with ageratum yellow vein disease did not hybridize to these samples. These results confirm that yellow vein disease on this weed is associated with a monopartite begomovirus and a distinct DNA ß. References: (1) R. W. Briddon et al. Mol. Biotechnol. 1:202, 1994. (2) R. W. Briddon et al. Virology 285:234, 2001. (3) R. W. Briddon et al. Mol. Biotechnol. In press. (4) K. Saunders et al. Proc. Natl. Acad. Sci. U S A 97:6890, 2000.

Identification of dna components required for induction of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is a major constraint to cotton production in Pakistan. Infectious clones of the monopartite begomovirus cotton leaf curl virus (CLCuV), associated with diseased cotton, are unable to induce typical symptoms in host plants. We have identified and isolated a single-stranded DNA molecule approximately 1350 nucleotides in length which, when coinoculated with the begomovirus to cotton, induces symptoms typical of CLCuD, including vein swelling, vein darkening, leaf curling, and enations. This molecule (termed DNA beta) requires the begomovirus for replication and encapsidation. The CLCuV/DNA 1/DNA beta complex, together with a similar complex previously identified in Ageratum conyzoides, represent members of an entirely new type of infectious, disease-causing agents. The implications of this finding to our understanding of the evolution of new disease-causing agents are discussed.

Association of a Disease Complex Involving a Begomovirus, DNA 1 and a Distinct DNA Beta with Leaf Curl Disease of Okra in Pakistan.

Okra leaf curl disease (OLCD), characterized by either upward or downward leaf curl and stunted plant growth, is one of the major diseases of okra (Hibiscus esculentis L.) in Pakistan. OLCD is transmitted by the whitefly Bemisia tabaci and is suspected of being associated with a whitefly-transmitted geminivirus (Genus Begomovirus). Total DNAs isolated from both symptomatic and healthy okra plants collected from several locations in Pakistan were resolved on agarose gels and blotted to nylon membranes. A full-length DNA A clone of Cotton leaf curl virus (CLCuV) from Pakistan (2) was labeled with 32PdCTP and used as a probe at medium stringency. The probe detected the presence of characteristic geminivirus DNA forms in infected plants, while no hybridization was observed to healthy plant extracts, confirming the association of a begomovirus with OLCD. Degenerate oligonucleotide primers based on conserved sequences of DNA B components of begomoviruses were used in PCR for the detection of a potential DNA B (3). No amplification was observed with these primers from okra plants, while amplification of a product of expected size was obtained from plants infected with African cassava mosaic virus, suggesting the lack of a genomic component equivalent to DNA B. We have reported previously that monopartite begomoviruses on cotton and Ageratum conyzoides in Pakistan are associated with a disease complex involving a DNA component termed DNA 1, which shows homology to components of nanoviruses that encode the replication-associated protein (2). Recently, another molecule, DNA beta, has been identified, associated with Ageratum yellow vein disease from Singapore (4) and with cotton leaf curl disease (CLCuD) from Pakistan (1). These molecules are DNAs satellite and are essential for the development of typical disease symptoms in their respective hosts. Duplicate blots were probed for the presence of DNAs homologous to DNA 1 and DNA beta (using full-length clones of these molecules isolated from CLCuD originating from Pakistan [1,2]) and washed at medium stringency. The probes detected bands hybridizing to DNA 1 in extracts from infected okra plants but not DNA beta. No hybridizing bands were detected for either probe in extracts from healthy okra. A pair of primers, designed to conserved sequences in DNA beta molecules (4), were used in PCR for the amplification of DNA beta from symptomatic plants. The use of these primers amplified a product of the expected size (approximately 1.35 kb) from extracts of infected okra plants. The amplified DNA was cloned in TA cloning vector and labeled with 32PdCTP. The use of this as a probe detected the presence of a hybridizing band in infected okra plants, while no signal was observed in extracts from cotton plants showing symptoms of CLCuD. These results show that OLCD in Pakistan is associated with a DNA beta molecule that is distinct from that reported on cotton and Ageratum. In particular, the DNA beta of CLCuD and OLCD originating from Pakistan are sufficiently diverse not to cross-hybridize under the conditions used here, and are most likely different disease complexes. To our knowledge this is the first report of the association of a whitefly-transmitted begomovirus/DNA 1/DNA beta complex with okra leaf curl disease. References: (1) R. W. Briddon et al. Virology, 2001 (In press). (2) S. Mansoor et al. Virology 259:190, 1999. (3) M R. Rojas et al. Plant Dis. 77: 340, 1993. (4) K. Saunders et al. PNAS 97:6890, 2000.

Widespread Occurrence of Cotton leaf curl virus on Radish in Pakistan.

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.

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

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.

Evidence that Watermelon Leaf Curl Disease in Pakistan Is Associated with Tomato Leaf Curl Virus-India, a Bipartite Begomovirus.

Whitefly-transmitted geminiviruses (begomoviruses) have emerged as major constraints on food and fiber crops worldwide, and there are several examples of begomovirus mobilization in previously unknown host plants. Here we report on evidence that leaf curl disease of watermelon in Pakistan is caused by Tomato leaf curl virus-India (TLCV-India). Leaf curl disease of watermelon, characterized by leaf curling and mottling and stunted plant growth, was observed at several locations in the Punjab Province of Pakistan. Symptomatic and asymptomatic leaf samples were collected from three locations, and total DNA was isolated by the cetyltrimethylammoniumbromide method and resolved in agarose gel. A full-length clone of Cotton leaf curl virus DNA A was labeled with [32P]dCTP and used as a general probe in Southern hybridization. The probe detected characteristic geminivirus DNA forms in infected watermelon plants, whereas no signal was detected in asymptomatic plants. The association of a begomovirus was confirmed further by polymerase chain reaction (PCR) amplification with degenerate primers PAL1V and pAR1c (2). Samples were screened for infection by TLCV-India, because of symptom similarity. A full-length clone of DNA B of TLCV-India (1) was labeled with [32P]dCTP by random priming and was used as a specific probe in Southern hybridization. The probe detected geminivirus DNA forms, showing that the disease is associated with TLCV-India. Primers TLCV1 (GAGGTACCAAAACTTGTCGTTTTGATTCGG), in the virion-sense, and TLCV2 (GCCCATGGTTCTTTGCTCGGAGAACAAGAA), in the complementary-sense, were designed based on the sequence of DNA A of TLCV-India. These primers were used in PCR and amplified a product of the expected size from infected plants. Similarly, primers TLCVBC1 (GCGGATCCTTATTCCGTAATTATATCTGCA), in the virion-sense, and TLCV BC2 (CACCATGGCAATAGGAAATGATGGTATGGG), in the complementary-sense, were designed based on the sequence of DNA B of TLCV-India (1). These primers amplified a product of expected size when used in PCR. The results show that watermelon leaf curl disease in Pakistan is associated with TLCV-India. This the first report of detection of a begomovirus in watermelon in Pakistan and the first report of detection of TLCV-India on a plant other than tomato from Southeast Asia. References: (1) M. Padidam et al. J. Gen. Virol. 76:25, 1995. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993.

Identification of a novel circular single-stranded DNA associated with cotton leaf curl disease in Pakistan.

Recent reports have suggested that cotton leaf curl virus (CLCuV), a geminivirus of the genus Begomovirus, may be responsible for cotton leaf curl disease in Pakistan. However, the causal agent of the disease remains unclear as CLCuV genomic components resembling begomovirus DNA A are unable to induce typical disease symptoms when reintroduced into plants. All attempts to isolate a genomic component equivalent to begomovirus DNA B have been unsuccessful. Here, we describe the isolation and characterisation of a novel circular single-stranded (ss) DNA associated with naturally infected cotton plants. In addition to a component resembling DNA A, purified geminate particles contain a smaller unrelated ssDNA that we refer to as DNA 1. DNA 1 was cloned from double-stranded replicative form of the viral DNA isolated from infected cotton plants. Blot hybridisation using probes specific for either CLCuV DNA or DNA 1 was used to demonstrate that both DNAs co-infect naturally infected cotton plants from different geographical locations. DNA 1 was detected in viruliferous Bemisia tabaci and in tobacco plants infected under laboratory conditions using B. tabaci, indicating that it is transmitted by whiteflies. Sequence analysis showed that DNA 1 is approximately half the size of CLCuV DNA but shares no homology, indicating that it is not a defective geminivirus component. DNA 1 has some homology to a genomic component of members of Nanoviridae, a family of DNA viruses that are normally transmitted by aphids or planthoppers. DNA 1 encodes a homologue of the nanovirus replication-associated protein (Rep) and has the capacity to autonomously replicate in tobacco. The data suggest that a nanovirus-like DNA has become whitefly-transmissible as a result of its association with a geminivirus and that cotton leaf curl disease may result from a mutually dependent relationship that has developed between members of two distinct DNA virus families that share a similar replication strategy.

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

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.

Genetic diversity evaluation of some elite cotton varieties by RAPD analysis.

Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the genetic diversity of elite commercial cotton varieties. Twenty two varieties belonging to Gossypium hirsutum L. and one to G. arboreum L. were analyzed with 50 random decamer primers using the polymerase chain reaction (PCR). Forty nine primers detected polymorphism in all 23 cotton varieties, while one produced monomorphic amplification profiles. A total of 349 bands were amplified, 89.1% of which were polymorphic. Cluster analysis by the unweighted pair group method of arithmetic means (UPGMA) showed that 17 varieties can be placed in two groups with a similarity ranging from 81.51% to 93.41%. G. hirsutum L. varieties S-12, V3 and MNH-93 showed a similarity of 78.12, 74.46 and 69.56% respectively with rest of the varieties. One variety, CIM-1100, showed 57.02% similarity and was quite distinct. The diploid cotton G. arboreum L. var. Ravi was also very distinct from rest of its tetraploid counterparts and showed only 55.7% similarity. The analysis revealed that the intervarietal genetic relationships of several varieties is related to their center of origin. As expected, most of the varieties have a narrow genetic base. The results obtained can be used for the selection of possible parents to generate a mapping population. The results also reveal the genetic relationship of elite commercial cotton varieties with some standard "Coker" varieties and the diploid G. arboreum L. var. Ravi (old world cotton).