Diversity of begomovirus DNA beta satellites of non-malvaceous plants in east and south east Asia.

Two previous analyses of the diversity of begomovirus-associated DNA beta satellites focused predominantly on molecules originating from the Indian sub-continent and southern China. They showed the satellites to group according to the hosts from which they were isolated, either malvaceous or non-malvaceous plants, and then to form sub-groups based upon geographic origin and host. In this study we analysed the diversity of DNA beta satellites in east and south east Asia. Here the satellites group by geographic location and are considerably more diverse than previously indicated. This probably reflects the limited movement of begomovirus/DNA beta complexes in this region and their subsequent diversification from a common ancestor to a variety of hosts.

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.

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 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.

Complementation of bipartite begomovirus movement functions by topocuviruses and curtoviruses.

The DNA A components of bipartite species of the genus Begomovirus (family Geminiviridae) encode all viral functions required for replication and encapsidation but require gene products encoded on DNA B for cell-to-cell movement in plants. We demonstrate that geminiviruses of the genera Curtovirus and Topocuvirus are able to trans-complement efficient movement of DNA A for two bipartite begomoviruses in the absence of their corresponding DNA B. It previously has been shown that DNA A can, at low efficiency and without inducing symptoms, spread in the absence of DNA B. The spread of DNA A independent of DNA B, following Agrobacterium-mediated inoculation, is shown to require coat protein whereas trans-complemented spread of DNA A can occur independent of the coat protein encoded on DNA A. The significance of these findings to our understanding of the geminiviral infection cycle is discussed.

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.

Exchange of three amino acids in the coat protein results in efficient whitefly transmission of a nontransmissible Abutilon mosaic virus isolate.

Geminiviruses are transmitted in a circulative manner by whiteflies, leafhoppers, or treehoppers. The whitefly species Bemisia tabaci (Genn.) is the vector for members of the genus Begomovirus. The closely related bipartite Central American begomoviruses Abutilon mosaic virus (AbMV), Sida golden mosaic virus originating from Costa Rica (SiGMV-CR), and Sida golden mosaic virus originating from Honduras (SiGMV-Hoyv) were used to study transmission by their insect vector. The AbMV isolate is defective in transmission, whereas the two Sida-infecting viruses are readily transmitted by B. tabaci. These three viruses are able to form pseudorecombinant viruses by exchange of genomic components. The pseudorecombinant virus SiGMV-Hoyv A/AbMV B was transmissible, whereas the reciprocal pseudorecombinant virus AbMV A/SiGMV-Hoyv B was not transmitted, indicating that DNA B is not involved in the transmission defect. However, the uptake of the pseudorecombinant virus AbMV A/SiGMV-Hoyv B was much better than AbMV itself, indicating that DNA B or DNA B gene products enhance uptake of viral DNA. Exchange of AbMV coat protein with that of SiGMV-CR resulted in a transmissible chimeric AbMV. Mutagenesis of the AbMV coat protein showed that the exchange of two amino acids, at positions 124 and 149, was sufficient to obtain a whitefly-transmissible AbMV mutant. However, when amino acid 174 was altered in addition to amino acids 124 and 149 AbMV was readily transmitted by B. tabaci. From this we conclude that it is not a concise motif, such as the amino acid triplet, aspartate-alanine-glycine (DAG), involved in aphid transmission of potyviruses, that determines transmissibility of begomoviruses by B. tabaci. Instead it is the composition of the coat protein domain from amino acid 123 to 149, as a minimal transmission domain, with the contribution of amino acids 149 to 174 for efficient transmission.

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.

Host range and symptom variation of pseudorecombinant virus produced by two distinct bipartite geminiviruses.

Within the whitefly group only the species Bemisia tabaci (Gennadius) is the vector. Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, DNAs A and B. Although they are closely related to each other, the production of viable pseudorecombinants between bipartite geminiviruses by reassortment of infectious cloned components is generally limited to strains of a particular virus. Following exchange of cloned genomic components of Sida golden mosaic virus (SiGMV/Hoyv) and Abutilon mosaic virus (AbMV), the pseudorecombinant viruses were infectious in various host plants. The symptom type of pseudorecombinant virus was in most cases determined by DNA B. However, in some host plants also DNA A of the pseudorecombinant virus was involved in the symptom phenotype.

Genetic analysis of Bemisia (Hemiptera: Aleyrodidae) populations by isoelectric focusing electrophoresis.

Twenty-one whitefly populations in the genus Bemisia were evaluated for genetic variation at 3 allozyme loci. Nine of the 22 populations that exhibited polymorphic loci were subjected to allozyme analysis using a minimum of 10 enzymes, representing 10 to 14 distinct loci. Among those nine variants examined, calculated genetic distances ranged between 0.03 and 0.52, with three main groups emerging from the analysis. One group comprised two closely related Western Hemisphere variants of B. tabaci: type A from California, United States and a geographically proximal population from Culiacan, Mexico. A second cluster contained five collections previously identified as B. tabaci type B and Bemisia argentifolii, while a third group contained a single population from Benin, Africa. The latter two groups were grouped separately from New World populations and are thought to have a recent origin in the Eastern Hemisphere.

A unique virus complex causes Ageratum yellow vein disease.

Ageratum conyzoides L., a weed species widely distributed throughout southeast Asia, frequently exhibits striking yellow vein symptoms associated with infection by Ageratum yellow vein virus (AYVV), a member of the Geminiviridae (genus Begomovirus). Most begomoviruses have bipartite genomes (DNAs A and B), but only a DNA A has been identified for AYVV. We demonstrate that yellow vein disease of A. conyzoides results from co-infection by AYVV DNA A (2,741 nt) and a circular DNA that is approximately half its size (1,347 nt) that we designate DNA beta. Apart from the sequence TAATATTAC, common to all geminiviruses and containing the initiation site of rolling circle replication, DNA beta shows negligible sequence homology either to AYVV DNA A or to DNA B associated with bipartite begomoviruses. DNA beta depends on DNA A for replication and is encapsidated by DNA A-encoded coat protein and so has characteristics of a DNA satellite. However, systemic infection of A. conyzoides by DNA A alone is sporadic and asymptomatic, and DNA A accumulation is reduced to 5% or less of its accumulation in the presence of DNA beta. Therefore, DNA A and DNA beta together form a previously unrecognized disease-inducing complex. Our data also demonstrate that the nanovirus-like DNA 1 component associated with infected A. conyzoides plays no essential role in the disease and represents a satellite-like DNA. Furthermore, the satellite DNA previously found associated with tomato leaf curl virus is probably a defective DNA beta homologue.

Clones of cotton leaf curl geminivirus induce symptoms atypical of cotton leaf curl disease.

The causative agent of cotton leaf curl disease has previously been shown to be transmissible by the whitefly Bemisia tabaci (Gennadius) and a begomovirus (Geminiviridae) was shown to be associated with the disease. This virus was provisionally called cotton leaf curl virus (CLCuV) although no causal relationship between virus and disease was shown. In the present study full-length clones of CLCuV, equivalent to the DNA A component of bipartite begomoviruses, were obtained. The clones of CLCuV were systemically infectious to both Nicotiana benthamiana and cotton. Infected plants did not exhibit symptoms characteristic of cotton leaf curl disease, producing mild leaf curling, yellowing and some stunting. Efforts to identify a second genomic component were not successful. These findings suggest that the begomovirus, CLCuV, is not or not the sole cause of cotton leaf curl disease. The transmission of cotton leaf curl disease by B. tabaci, however, may indicate that the begomovirus plays a part in the transmission of the disease. The implications of these findings are discussed.

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.

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.

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.

Identification of genes directly and indirectly involved in the insect transmission of African cassava mosaic geminivirus by Bemisia tabaci.

The inability to transmit progeny virus resulting from the cloned components of an isolate of African cassava mosaic virus originating from Kenya (ACMV-K) has been shown to be due to defects in both genomic components. This was achieved by the production of infectious pseudorecombinants between ACMV-K and the cloned components of a whitefly-transmissible ACMV isolate originating from Nigeria (ACMV-NOg). The exchange of gene fragments between ACMV-K and ACMV-NOg has been used to demonstrate that the defects responsible for lack of transmissibility reside on the coat protein and DNA B C1 gene of ACMV-K. The significance of these finding with respect to the present understanding of the function of these gene products are discussed.

A Novel Geminivirus of Ipomoea indica (Convolvulacae) from Southern Spain.

A cutting of Ipomoea indica displaying yellow vein symptoms was collected from Nerja in southern Spain in 1995, rooted, and maintained by vegetative propagation under glasshouse conditions at the John Innes Centre, Norwich. Although this member of the Convolvulaceae is native to the New World, it has escaped from cultivation as an ornamental and has now been naturalized in many tropical and warm temperate regions of the world, such as southern Spain. The same plant was found to host a population of whiteflies that were also brought back to containment facilities, and maintained in colony. Total plant DNA was extracted from the I. indica plant and universal primers for begomovirus A component (1) were used to amplify an approximately 2.8-kb fragment that was cloned and sequenced. The sequence is available in the DDJB, EMBL, and GenBank nucleotide sequence data bases under accession number AJ132548. A GENEMBL search with the complete sequence of the clone showed 70.8% identity to the AC1 gene of Ageratum yellow vein virus (AYVV). A search with the coat protein gene sequence showed highest homology to tomato leaf curl virus from southern India, another monopartite virus. Typical geminivirus vein yellowing symptoms, nucleotide sequence similarity, and EM detection of geminate virus particles strongly suggest that a geminivirus is present in this plant. The low level of homology to other sequenced geminiviruses suggests that it is an uncharacterized Begomovirus sp. With degenerate DNA-B primers (2), no B component has so far been detected. This virus is provisionally named Ipomoea yellow vein virus (IYVV). With techniques already established for identifying Bemisia spp. (3), the whiteflies collected with this Ipomoea plant were confirmed as Bemisia tabaci. Transmission studies to healthy I. indica showed that this whitefly population (named biotype S), the Q biotype from Spain, and the B biotype from Israel were all unable to transmit IYVV to healthy I. indica, tobacco, tomato, or nightshade. This may be due to many years of vegetative propagation of the host plant as an ornamental, resulting in loss of virus transmissibility by insects, which has occurred with Abutilon mosaic virus (AbMV) and honeysuckle yellow vein mosaic virus (HYVMV). This is the first report of a novel geminivirus on I. indica. It highlights the importance of weeds as hosts and potential reservoirs of both viruses and pests. We acknowledge support from the British Council, The Royal Society, BBSRC, and MAFF. References: (1) R.W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1994. (2) M. R. Rojas et al. Plant Dis. 77:340, 1993. (3) R. C. Rosell et al. Ann. Entomol. Soc. Am. 90:575, 1997.

Infectivity of African cassava mosaic virus clones to cassava by biolistic inoculation.

Clones of an African cassava mosaic virus isolate originating from Nigeria (ACMV-NOg) were shown to be infectious to cassava by biolistic inoculation. The production of pseudorecombinants between ACMV-NOg and clones of an ACMV isolate originating from Kenya (ACMV-K) indicated that the lack of infectivity of ACMV-K to cassava was due to defect(s) in the DNA B genomic component; this component encodes two proteins involved in cell-to-cell movement. This is the first demonstration of infectivity of a cloned geminivirus to cassava and conclusively proves that ACMV is the causative agent of cassava mosaic disease. The potential uses of infectious ACMV clones and the means by which to introduce them into cassava are discussed.