Use of the cotton leaf curl Multan alphasatellite as a silencing or expression vector.

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.

The antisense 5' end of the V2 gene confers enhanced resistance against the monopartite begomovirus cotton leaf curl Kokhran virus-Burewala strain.

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.

Molecular characterization of a distinct monopartite begomovirus associated with betasatellites and alphasatellites infecting Pisum sativum in Nepal.

Pea (Pisum sativum) plants exhibiting leaf distortion, yellowing, stunted growth and reduction in leaf size from Rampur, Nepal were shown to be infected by a begomovirus in association with betasatellites and alphasatellites. The begomovirus associated with the disease showed only low levels of nucleotide sequence identity (<91%) to previously characterized begomoviruses. This finding indicates that the pea samples were infected with an as yet undescribed begomovirus for which the name Pea leaf distortion virus (PLDV) is proposed. Two species of betasatellite were identified in association with PLDV. One group of sequences had high (>78%) nucleotide sequence identity to isolates of Ludwigia leaf distortion betasatellite (LuLDB), and the second group had less than 78% to all other betasatellite sequences. This showed PLDV to be associated with either LuLDB or a previously undescribed betasatellite for which the name Pea leaf distortion betasatellite is proposed. Two types of alphasatellites were identified in the PLDV-infected pea plants. The first type showed high levels of sequence identity to Ageratum yellow vein alphasatellite, and the second type showed high levels of identity to isolates of Sida yellow vein China alphasatellite. These are the first begomovirus, betasatellites and alphasatellites isolated from pea.

A Distinct Strain of Chickpea chlorotic dwarf virus Infecting Pepper in Oman.

During a field survey in 2011, pepper (Capsicum annum) plants showing symptoms suggestive of geminivirus infection were observed in three fields in the Al-Sharqiya region of Oman. Symptoms observed included upward leaf curling leading to cupping and stunting with 15 to 25% disease incidence in surveyed fields. Total DNA was extracted from the leaves of seven symptomatic plants and subjected to rolling circle amplification (RCA). The RCA product was digested with several restriction endonucleases to obtain unit length of ~2.6 to 2.8, typical of geminivirus. Out of seven samples, only four yielded a product of ~2.6 kb in size by KpnI digestion. The fragments were cloned in pUC19 and sequenced. The partial sequences of four isolates were >95% identical to each other at the nucleotide (nt) level and thus only one isolate (P-25) was fully sequenced, determined to be 2,572 nt in length, and its sequence deposited in GenBank (KF111683). The P-25 sequence showed a genome organization typical of a mastrevirus, with four open reading frames (ORFs), two in virion-sense and two in complementary-sense. The virion and complementary-sense ORFs were separated by a long intergenic region, containing a predicted hairpin structure with the nonanucleotide sequence (TAATATTAC) in the loop, and a short intergenic region. An initial comparison to all sequences in the NCBI database using BlastN showed the isolate to have the highest level of sequence identity with isolates of the dicot-infecting mastrevirus Chickpea chlorotic dwarf virus (CpCDV). Subsequent alignments of all available CpCDV isolates using the species demarcation tool (2) showed the isolate P-25 to share between 83.6 and 90.3% identity to isolates of CpCDV available in databases, with the highest (90.3%) to CpCDV strain A originating from Syria (FR687959) (3). Amino acid sequence comparison showed that the predicted proteins encoded by the four ORFs of P-25 (coat protein [CP], movement protein [MP], replication associated protein A [RepA], and RepB) share 91.5, 88.2, 89.1, and 90.8% amino acid sequence identity, respectively, with the homologous proteins of the CpCDV isolate from Syria. Based on the recently revised mastreviruses species and strain demarcation criteria (78 and 94% whole genome nt identity, respectively) proposed by Muhire et al. (2), the results indicate that isolate P-25 represents a newly identified strain (strain F) of CpCDV. The presence of CpCDV in symptomatic pepper plants was further confirmed by Southern blot hybridization technique using digoxygenin (DIG) labeled probe prepared from CpCDV isolate P-25. The genus Mastrevirus consists of geminiviruses with single component genomes that are transmitted by leafhoppers. Mastreviruses have so far only been identified in the Old World and infect either monocotyledonous or dicotyledonous plants (1). To our knowledge, this is the first report of a mastrevirus on the Arabian Peninsula and the first record of pepper as host of CpCDV. Recently, several begomoviruses of diverse geographic origins have been found infecting vegetable crops in Oman. The propensity of geminiviruses to evolve through recombination may lead to evolution of recombinant CpCDV with new host adaptability. Due to extensive agricultural/travel links of Oman with rest of the world, there exists high probability for the spread of this virus. References: (1) M. I. Boulton. Physiol. Mol. Plant Pathol. 60:243, 2002. (2) B. Muhire et al. Arch. Virol. 158:1411, 2013 (3) H. Mumtaz et al. Virus Genes 42:422, 2011.

Complete nucleotide sequence of a monopartite Begomovirus and associated satellites infecting Carica papaya in Nepal.

Carica papaya (papaya) is a fruit crop that is cultivated mostly in kitchen gardens throughout Nepal. Leaf samples of C. papaya plants with leaf curling, vein darkening, vein thickening, and a reduction in leaf size were collected from a garden in Darai village, Rampur, Nepal in 2010. Full-length clones of a monopartite Begomovirus, a betasatellite and an alphasatellite were isolated. The complete nucleotide sequence of the Begomovirus showed the arrangement of genes typical of Old World begomoviruses with the highest nucleotide sequence identity (>99 %) to an isolate of Ageratum yellow vein virus (AYVV), confirming it as an isolate of AYVV. The complete nucleotide sequence of betasatellite showed greater than 89 % nucleotide sequence identity to an isolate of Tomato leaf curl Java betasatellite originating from Indonesian. The sequence of the alphasatellite displayed 92 % nucleotide sequence identity to Sida yellow vein China alphasatellite. This is the first identification of these components in Nepal and the first time they have been identified in papaya.

A Distinct Strain of Tomato leaf curl Sudan virus Causes Tomato Leaf Curl Disease in Oman.

Tomato leaf curl disease (ToLCD) is a significant constraint for tomato production in the Sultanate of Oman. The disease in the north of the country has previously been shown to be caused by the monopartite begomoviruses (family Geminiviridae) Tomato yellow leaf curl virus and Tomato leaf curl Oman virus. Many tomato plants infected with these two viruses were also found to harbor a symptom enhancing betasatellite. Here an analysis of a virus isolated from tomato exhibiting ToLCD symptoms originating from south and central Oman is reported. Three clones of a monopartite begomovirus were obtained. One of the clones was shown to be infectious to tomato and Nicotiana benthamiana and to induce symptoms typical of ToLCD. Analysis of the cloned sequences show them to correspond to isolates of Tomato leaf curl Sudan virus (ToLCSDV), a virus that occurs in Sudan and Yemen. However, the sequences showed less than 93% nucleotide sequence identity to previously characterized ToLCSDV isolates, indicating that the viruses represent a distinct strain of the species, for which we propose the name "Oman" strain (ToLCSDV-OM). Closer analysis of the sequences showed them to differ from their closest relative, the "Tobacco" strain of ToLCSDV originating from Yemen, in three regions of the genome. This suggests that the divergence of the "Oman" and "Tobacco" strains has occurred due to recombination. Surprisingly, ToLCSDV-OM was not found to be associated with a betasatellite, even though the isolates of the other ToLCSDV strains have been shown to be. The significance of these findings and the possible reasons for the distinct geographic distributions of the tomato-infecting begomoviruses within Oman are discussed.

Xanthium strumarium: a weed host of components of begomovirus-betasatellite complexes affecting crops.

Xanthium strumarium is a common weed that often shows symptoms typical of begomovirus infection, such as leaf curling and vein thickening. The virus complex isolated from the weed consisted of two begomoviruses along with a betasatellite and an alphasatellite. The first begomovirus was shown to be an isolate of Cotton leaf curl Burewala virus, a new recombinant begomovirus species that is associated with resistance breaking in previously resistant cotton varieties in Pakistan, whereas the second was shown to be an isolate of Tomato leaf curl Gujarat virus (ToLCGV), a begomovirus previously reported to be bipartite. However, there was no evidence for the presence of the second genomic component, DNA B, of ToLCGV in X. strumarium. The betasatellite was shown to be an isolate of Tomato yellow leaf curl Thailand betasatellite, the first time this satellite has been identified in Pakistan. The alphasatellite associated with infection of X. strumarium was shown to be a species recently identified in potato and various weeds; Potato leaf curl alphasatellite. Although each component has been identified previously, this is the first time they have been identified in a single host. These findings reinforce the hypothesis that weeds are reservoirs of crop-infecting begomoviruses that may contribute to virus diversity by virtue of harboring multiple viruses and virus associated components, which may lead to interspecific recombination and component exchange.

Identification of Cotton leaf curl Gezira virus in Papaya in Oman.

Papaya is an important fruit crop in Oman covering some 130 ha with an annual production of 20 tonnes. In 2011, during surveys of farms in the Quriyat region of Oman, papaya plants were found severely affected by leaf curl disease. Leaves with severe curling, vein darkening, and vein thickening were collected for study. Disease incidence ranged from 30 to 50%, particularly in fields with young papaya. A begomovirus (family Geminiviridae) was suspected as the causal agent based on symptoms (1) and the presence of whiteflies in the field. Samples (four to five) were collected from three farms. Total nucleic acids extracted from symptomatic leaves using the CTAB method were used as templates to amplify circular DNAs using Φ29 DNA polymerase and products were digested with restriction enzymes to identify fragments of 2.6 to 2.8 kb typical of geminiviruses. PstI yielded a fragment of ~1.8 kb when the digested product was analyzed by electrophoresis on a 1% agarose gel. The fragment was cloned and sequenced using primer walking strategy in both directions. The sequencing confirmed the exact size (1,764 bp) and the sequence was deposited in GenBank (HE800524). The viral sequence from Oman (isolate Pap-2) showed four open reading frames (ORFs) in the complementary sense (replication associated protein [Rep] gene, the C2 gene, the replication enhancer protein [REn] gene, and the C4 gene) and the virion-sense ORFs (V1 and V2) were missing in the sequence. An initial comparison to NCBI database sequences using BLAST showed the clone from Oman had the highest level of sequence identity to Cotton leaf curl Gezira virus (CLCuGeV) (FJ868828) cloned from okra in Sudan. Subsequent pair wise sequence comparison was done using ClustalV algorithm. Full length sequences of CLCuGeV from database were trimmed according to the size and genomic coordinates of Pap-2 isolate. The Pap-2 isolate sequence was found to have 83.3 to 95.1% sequence identity to CLCuGeV sequences with maximum value to the Sudan isolate. Amino acid sequence comparison showed that the four predicted proteins (Rep, C2, REn, and C4) encoded by the Pap-2 isolate shared 95.3%, 97.8%, 97.7%, and 87.6% sequence identity, respectively, with the homologous proteins of CLCuGeV-SD (FJ868828). The absence of virion-sense protein sequences indicated it to be a subgenomic molecule of CLCuGeV. According to the recommendations of International Committee on Taxonomy of Viruses, these results indicate that the virus identified in association with papaya leaf curl disease in Oman is a variant of CLCuGeV. CLCuGeV is a begomovirus of African origin which is distinct from the begomoviruses of the Middle East and Asia. To our knowledge, this is the first report of CLCuGeV, or any other cotton infecting begomovirus, from papaya in Oman. The presence of a recombinant fragment of CLCuGeV in a Tomato yellow leaf curl virus isolate from Iran (2), and the association of CLCuGeV with cotton in Pakistan (3) and hollyhock in Jordan (GU945265) suggests this virus has moved into the Middle East and Asia from Africa. The identification of CLCuGeV in Oman shows the widespread occurrence of this virus species. This discovery is important since Oman, and other countries in the area, are a hub of international trade and travel, particularly by air and sea, meaning that the virus could spread further. References: (1) R. W. Briddon and P. G. Markham. Virus Res. 71:151, 2000. (2) P. Lefeuvre et al. PLoS Pathog. 6:e1001164, 2010. (3) M. N. Tahir et al. PLoS ONE 6:e20366, 2011.

Cotton leaf curl Multan betasatellite impaired ToLCNDV ability to maintain cotton leaf curl Multan alphasatellite.

Alphasatellites (family Alphasatellitidae) are circular, single-stranded (ss) DNA molecules of ~1350 nucleotide in size that have been characterized in both the Old and New Worlds. Alphasatellites have inherent ability to self-replicate, which is accomplished by a single protein, replication-associated protein (Rep). Although the precise function of alphasatellite is yet unknown, and these consider dispensable for infectivity, however, their Rep protein functions as a suppressor of host defence. While alphasatellites are most frequently associated with begomoviruses, particularly with monopartite than bipartite begomoviruses, they have recently been found associated with mastreviruses. The in planta maintenance of alphasatellites by helper geminivirus is still an enigma, with no available study on the topic. This study aimed to investigate whether a widely distributed bipartite begomovirus, tomato leaf curl New Delhi virus (ToLCNDV), can maintain cotton leaf curl Multan alphasatellite (CLCuMuA) in the presence or absence of cotton leaf curl Multan betasatellite (CLCuMuB). The findings of this study demonstrated that ToLCNDV or its DNA A could maintain CLCuMuA in Nicotiana benthamiana plants. However, the presence of CLCuMuB interferes with the maintenance of CLCuMuA, and mutations in the CP of ToLCNDV further reduces it. Our study highlighted that the maintenance of alphasatellites is impaired in the presence of a betasatellite by ToLCNDV. Further investigation is needed to unravel all the interactions between a helper virus and an alphasatellites.

Characterization of begomovirus components from a weed suggests that begomoviruses may associate with multiple distinct DNA satellites.

A begomovirus disease complex associated with Sonchus arvensis, a common weed in Pakistan was studied using cloning, nucleic acid sequencing and phylogenetic analysis. The complex associated with this weed consists of a monopartite begomovirus and several distinct betasatellites and alphasatellites. The monopartite begomovirus associated with yellow vein disease of Sonchus arvensis showed 95-99% nucleotide sequence identity with Alternanthera yellow vein virus (AlYVV) reported from China, Vietnam and India. Two betasatellites were isolated from S. arvensis: one sharing between 91.4 and 95.3% nucleotide sequence identity with isolates of Ageratum yellow leaf curl betasatellite (AYLCB), and the other sharing between 78.2 and 99.9% identity with isolates of Cotton leaf curl Multan betasatellite (CLCuMB). Two alphasatellites were identified: one was homologous to Potato leaf curl alphasatellite (PotLCuA), while the other was closely related to Hibiscus leaf curl alphasatellite (HLCuA). Thus, AlYVV in S. arvensis is associated with satellites shown previously to be associated with other begomoviruses in Pakistan. Our results suggest that monopartite begomoviruses may associate with distinct satellites that are prevalent in the region.

Diverse and recombinant DNA betasatellites are associated with a begomovirus disease complex of Digera arvensis, a weed host.

Weeds are considered as a source of new viruses and reservoirs of economically important viruses but are often neglected during diversity studies. Here, we report the complete nucleotide sequences and phylogenetic analyses of the components of a begomovirus disease complex associated with yellow vein disease of Digera arvensis, a common weed. The begomovirus associated with the disease showed 98% nucleotide sequence identity with Cotton leaf curl Rajasthan virus. Two species of betasatellite were identified. The first betasatellite species was an isolate of Ageratum yellow leaf curl betasatellite. The second was a recombinant consisting for the most part of sequence derived from a Tobacco leaf curl betasatellite but with the satellite conserved region (SCR) and some sequence between the SCR and adenine-rich (A-rich) region derived from a Cotton leaf curl Multan betasatellite. The alphasatellite isolated from this weed was near identical to an isolate recently characterized from potato. The presence of multiple and recombinant betasatellites in D. arvensis indicates that weeds can be important sources of multiple begomovirus components that affect crop plants. Furthermore, the presence of a recombinant betasatellite suggested that weeds are likely vessels for recombination and evolution of components of begomovirus complexes.

Cotton leaf curl Multan betasatellite impaired ToLCNDV ability to maintain cotton leaf curl Multan alphasatellite / O enrolamento da folha de algodão Multan betassatélite prejudica a capacidade de ToLCNDV de manter o enrolamento da folha de algodão alfassatélite Multan

Alphasatellites (family Alphasatellitidae) are circular, single-stranded (ss) DNA molecules of ~1350 nucleotide in size that have been characterized in both the Old and New Worlds. Alphasatellites have inherent ability to self-replicate, which is accomplished by a single protein, replication-associated protein (Rep). Although the precise function of alphasatellite is yet unknown, and these consider dispensable for infectivity, however, their Rep protein functions as a suppressor of host defence. While alphasatellites are most frequently associated with begomoviruses, particularly with monopartite than bipartite begomoviruses, they have recently been found associated with mastreviruses. The in planta maintenance of alphasatellites by helper geminivirus is still an enigma, with no available study on the topic. This study aimed to investigate whether a widely distributed bipartite begomovirus, tomato leaf curl New Delhi virus (ToLCNDV), can maintain cotton leaf curl Multan alphasatellite (CLCuMuA) in the presence or absence of cotton leaf curl Multan betasatellite (CLCuMuB). The findings of this study demonstrated that ToLCNDV or its DNA A could maintain CLCuMuA in Nicotiana benthamiana plants. However, the presence of CLCuMuB interferes with the maintenance of CLCuMuA, and mutations in the CP of ToLCNDV further reduces it. Our study highlighted that the maintenance of alphasatellites is impaired in the presence of a betasatellite by ToLCNDV. Further investigation is needed to unravel all the interactions between a helper virus and an alphasatellites.

Alfassatélites (família Alphasatellitidae) são moléculas de DNA circulares de fita simples (ss) de ~1350 nucleotídeos de tamanho, que foram caracterizadas tanto no Velho como no Novo Mundo. Os alfassatélites têm capacidade inerente de autorreplicação, o que é realizado por uma única proteína, a proteína associada à replicação (Rep). Embora a função precisa dos alfassatélites ainda seja desconhecida, e estes sejam considerados dispensáveis ​​para infectividade, entretanto, sua proteína Rep funciona como supressora da defesa do hospedeiro. Embora os alfassatélites sejam mais frequentemente associados a begomovírus, particularmente com begomovírus monopartidos do que bipartidos, eles foram encontrados recentemente associados a mastrevírus. A manutenção in planta de alfassatélites por helper geminivirus ainda é um enigma, sem estudos disponíveis sobre o tema. Este estudo teve como objetivo investigar se um begomovírus bipartido amplamente distribuído, o tomate leaf curl New Delhi virus (ToLCNDV), pode manter o alfassatélite Multan do enrolamento das folhas de algodão (CLCuMuA) na presença ou ausência do betassatélite Multan do enrolamento das folhas de algodão (CLCuMuB). Os achados deste estudo demonstraram que ToLCNDV ou seu DNA A poderia manter CL CuMuA em plantas de Nicotiana benthamiana. No entanto, a presença de CLCuMuB interfere na manutenção de CLCuMuA, e mutações no CP de ToLCNDV a reduzem ainda mais. Nosso estudo destacou que a manutenção de alfassatélites é prejudicada na presença de um betassatélite por ToLCNDV. Mais investigações são necessárias para desvendar todas as interações entre um vírus auxiliar e um alfassatélite.

Frequent occurrence of Mungbean yellow mosaic India virus in tomato leaf curl disease affected tomato in Oman.

Next generation sequencing (NGS) of DNAs amplified by rolling circle amplification from 6 tomato (Solanum lycopersicum) plants with leaf curl symptoms identified a number of monopartite begomoviruses, including Tomato yellow leaf curl virus (TYLCV), and a betasatellite (Tomato leaf curl betasatellite [ToLCB]). Both TYLCV and ToLCB have previously been identified infecting tomato in Oman. Surprisingly the NGS results also suggested the presence of the bipartite, legume-adapted begomovirus Mungbean yellow mosaic Indian virus (MYMIV). The presence of MYMIV was confirmed by cloning and Sanger sequencing from four of the six plants. A wider analysis by PCR showed MYMIV infection of tomato in Oman to be widespread. Inoculation of plants with full-length clones showed the host range of MYMIV not to extend to Nicotiana benthamiana or tomato. Inoculation to N. benthamiana showed TYLCV to be capable of maintaining MYMIV in both the presence and absence of the betasatellite. In tomato MYMIV was only maintained by TYLCV in the presence of the betasatellite and then only at low titre and efficiency. This is the first identification of TYLCV with ToLCB and the legume adapted bipartite begomovirus MYMIV co-infecting tomato. This finding has far reaching implications. TYLCV has spread around the World from its origins in the Mediterranean/Middle East, in some instances, in live tomato planting material. The results here may suggest that begomoviruses which do not commonly infect tomato, such as MYMIV, could be spread as a passenger of TYLCV in tomato.

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.

Cotton leaf curl virus disease.

Cotton is one of the most important crops of Pakistan, accounting for over 60% of foreign exchange earnings. The present epidemic of cotton leaf curl disease (CLCuD) originated in the Punjab region near the city of Multan and was first reported in 1985, although it was noted in this region as early as 1967. By the early 1990s, CLCuD had become the major limitation to cotton production in Pakistan and it has now spread into India and, more recently, south and west into other provinces of Pakistan. The very characteristic symptoms include leaf curling, darkened veins, vein swelling and enations that frequently develop into cup-shaped, leaf-like structures on the undersides of leaves. Identification of the vector of CLCuD as the whitefly Bemisia tabaci (Genn.) quickly led to the suggestion that the causative agent of the disease is a geminivirus. Researchers soon confirmed the presence of such a virus that is currently ascribed to the genus Begomovirus of the family Geminiviridae, However, in 1999, the aetiology of the disease was shown to be more complex than was originally assumed. Despite the identification of both a begomovirus and a so-called nanovirus-like component, the precise causal agent of CLCuD remains uncertain.

Rapid production of full-length, infectious geminivirus clones by abutting primer PCR (AbP-PCR).

The application of the polymerase chain reaction (PCR) method of DNA amplification for the isolation of full-length, infectious clones of geminiviruses is described. Non-overlapping, abutting 20-mer oligonucleotide primers were used to produce a linear product from the circular geminivirus genomic template. Clones of African cassava mosaic virus (ACMV) DNA A, obtained by this method, were infectious following mechanical inoculation (in the presence of ACMV DNA B) onto Nicotiana benthamiana. Normal ACMV symptoms resulted and typical geminate viral particles were detected by electron microscopy. The use of PCR for the detection and production of full-length, infectious geminivirus clones is discussed.

Universal primers for the PCR-mediated amplification of DNA 1: a satellite-like molecule associated with begomovirus-DNA beta complexes.

DNA 1 is a single-stranded DNA molecule of approximately 1370 nucleotides. It is associated with monopartite geminiviruses of the genus Begomovirus, which require a DNA beta component for symptomatic infection. The DNA 1 molecule requires the helper begomovirus for movement in plants, but is capable of self-replication. We designed two abutting primer pairs (DNA101/DNA102 and UN101/UN102) to conserved sequences of DNA 1. This allowed polymerase chain reaction-mediated amplification of the full-length molecule from total nucleic acid extracts produced from various host plants from geographically distinct, worldwide locations. These primers are useful both as diagnostic probes and for producing full-length infectious clones for in planta studies.

Universal primers for the PCR-mediated amplification of DNA beta: a molecule associated with some monopartite begomoviruses.

DNA beta is an approx 1350 nucleotide, single-stranded DNA molecule which has been shown to be associated with some monopartite geminiviruses of the genus Begomovirus. This component requires the helper begomovirus for replication in the cells of host plants and for insect transmission, possibly by trans-encapsidation. Sequence comparisons of the two available DNA beta sequences has identified a highly conserved region upstream of a predicted hairpin structure. Abutting primers designed to this conserved region allows PCR-mediated amplification of the full-length DNA beta component from total nucleic acid extracts isolated from infected plants originating from a variety of geographically distinct sources and host plants.

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.