Temporal changes in the levels of virus and betasatellite DNA in B. tabaci feeding on CLCuD affected cotton during the growing season.

Cotton, a key source of income for Pakistan, has suffered significantly by cotton leaf curl disease (CLCuD) since 1990. This disease is caused by a complex of phylogenetically-related begomovirus (genus Begomovirus, family Geminiviridae) species and a specific betasatellite (genus Betasatellite, family Tolecusatellitidae), cotton leaf curl Multan betasatellite. Additionally, another DNA satellite called alphasatellite (family Alphasatellitidae), is also frequently associated. All these virus components are vectored by a single species of whitefly (Bemisia tabaci). While many factors affect cotton productivity, including cotton variety, sowing time, and environmental cues such as temperature, humidity, and rainfall, CLCuD is a major biotic constraint. Although the understanding of begomoviruses transmission by whiteflies has advanced significantly over the past three decades, however, the in-field seasonal dynamics of the viruses in the insect vector remained an enigma. This study aimed to assess the levels of virus and betasatellite in whiteflies collected from cotton plants throughout the cotton growing season from 2014 to 2016. Notably, begomovirus levels showed no consistent pattern, with minimal variations, ranging from 0.0017 to 0.0074 ng.µg-1 of the genomic DNA in 2014, 0.0356 to 0.113 ng.µg-1 of the genomic DNA in 2015, and 0.0517 to 0.0791 ng.µg-1 of the genomic DNA in 2016. However, betasatellite levels exhibited a distinct pattern. During 2014 and 2015, it steadily increased throughout the sampling period (May to September). While 2016 showed a similar trend from the start of sampling (July) to September but a decline in October (end of sampling). Such a study has not been conducted previously, and could potentially provide valuable insights about the epidemiology of the virus complex causing CLCuD and possible means of controlling losses due to it.

qPCR Assay as a Tool for Examining Cotton Resistance to the Virus Complex Causing CLCuD: Yield Loss Inversely Correlates with Betasatellite, Not Virus, DNA Titer.

Cotton leaf curl disease (CLCuD) is a significant constraint to the economies of Pakistan and India. The disease is caused by different begomoviruses (genus Begomovirus, family Geminiviridae) in association with a disease-specific betasatellite. However, another satellite-like molecule, alphasatellite, is occasionally found associated with this disease complex. A quantitative real-time PCR assay for the virus/satellite components causing CLCuD was used to investigate the performance of selected cotton varieties in the 2014-2015 National Coordinated Varietal Trials (NCVT) in Pakistan. The DNA levels of virus and satellites in cotton plants were determined for five cotton varieties across three geographic locations and compared with seed cotton yield (SCY) as a measure of the plant performance. The highest virus titer was detected in B-10 (0.972 ng·µg-1) from Vehari and the lowest in B-3 (0.006 ng·µg-1) from Faisalabad. Likewise, the highest alphasatellite titer was found in B-1 (0.055 ng·µg-1) from Vehari and the lowest in B-1 and B-2 (0.001 ng·µg-1) from Faisalabad. The highest betasatellite titer was found in B-23 (1.156 ng·µg-1) from Faisalabad and the lowest in B-12 (0.072 ng·µg-1) from Multan. Virus/satellite DNA levels, symptoms, and SCY were found to be highly variable between the varieties and between the locations. Nevertheless, statistical analysis of the results suggested that betasatellite DNA levels, rather than virus or alphasatellite DNA levels, were the important variable in plant performance, having an inverse relationship with SCY (-0.447). This quantitative assay will be useful in breeding programs for development of virus resistant plants and varietal trials, such as the NCVT, to select suitable varieties of cotton with mild (preferably no) symptoms and low (preferably no) virus/satellite. At present, no such molecular techniques are used in resistance breeding programs or varietal trials in Pakistan.

Complete genome sequence of hollyhock vein yellowing virus, a novel monopartite begomovirus infecting hollyhock in Pakistan.

Hollyhock (Alcea rosea, family Malvaceae) is an ornamental plant grown widely in gardens across South Asia. In a bed of ornamental plants near the village of Chakri (Punjab Province, Pakistan) in 2014, hollyhock plants showing two distinct symptom types were identified: yellow vein mosaic and leaf crumple. PCR amplification with universal primers amplified a begomovirus from separate nucleic acid extracts of single plants of each type but amplified a betasatellite only from the plant with the yellow vein mosaic symptoms. No potential begomovirus DNA B component or alphasatellite could be identified in either sample. After cloning, the genome sequences of two viruses, one from a plant of each symptom type, were determined and shown to share 99.9% nucleotide sequence identity with each other but less than 91% nucleotide sequence identity with all previously characterized begomoviruses, with the highest identity (90%) to an isolate of pedilanthus leaf curl virus (PeLCV). This indicates that the two hollyhock plants were infected with a newly identified begomovirus for which the name "hollyhock vein yellowing virus" (HoVYV) is proposed. HoVYV likely has a recombinant origin. The betasatellite showed the highest nucleotide sequence identity to an isolate of cotton leaf curl Multan betasatellite (CLCuMuB), a betasatellite associated with cotton leaf curl disease across Pakistan and northwestern India. These findings add to the diversity of known begomoviruses in South Asia and again highlight the role of hollyhock as a reservoir of the cotton leaf curl begomovirus betasatellite complex. The results also suggest that the yellow vein mosaic symptoms in hollyhock are due to the betasatellite rather than the virus.

Codiaeum variegatum in Pakistan harbours pedilanthus leaf curl virus and papaya leaf curl virus as well as a newly identified betasatellite.

Codiaeum variegatum (common name, garden croton) is an ornamental plant grown for its bright yellow variegated leaf morphology. Two C. variegatum plants with upward leaf curling and vein swelling symptoms were collected in Faisalabad, Pakistan. Sequencing of clones obtained by PCR amplification with specific primers showed one plant infected with the monopartite begomoviruses pedilanthus leaf curl virus (PeLCV) and papaya leaf curl virus (PaLCuV) and the other to be infected with only PeLCV. Both plants also harboured a betasatellite that was distinct from all previously identified betasatellites, for which the name "codiaeum leaf curl betasatellite" (CoLCuB) is proposed. This is the first identification of a begomovirus and an associated betasatellite infecting C. variegatum in Pakistan. Both PeLCV and PaLCuV cause problems in a number of crop plants, and C. variegatum may act as a reservoir for these agriculturally important viruses. The precise impact and geographical distribution of the newly identified CoLCuB will be investigated.

Molecular characterization of Chickpea chlorotic dwarf virus strain D in Chickpea (Cicer arietinum) from District Dera Ismail Khan Khyber Pakhtunkhwa, Pakistan.

Chickpea chlorotic dwarf virus (CpCDV), a member of genus Mastrevirus (family Geminiviridae) is an important viral pathogen of chickpea and other legume crops in Middle East, North Africa, India and Pakistan. Among sixteen known strains of CpCDV three are known to infect legume crops in Punjab province of Pakistan. In this study diversity of CpCDV was explored in Khyber Pakhtunkhwa (KP) province of Pakistan. In year 2016, during a survey in the chickpea growing areas of district Dera Ismail Khan, CpCDV infected plants were identified. Leaf samples were collected, and a diagnostic PCR confirmed mastreivrus infection in 4 out of 100 samples. From these samples full-length genome of CpCDV was amplified using specific back-to-back primers. Virus molecules were sequenced to their entirety and sequence analysis of a molecule KRF4 (GenBank accession # KY952837) showed the highest pair wise sequence identity of 97% with a CpCDV molecule (KM229787) isolated from chickpea plant. An SDT analysis revealed it to be the D strain of CpCDV and a recombination detection program (RDP) showed it to be a recombinant between C (KM229780) and L (KT634301) strains of CpCDV. Thus, further supporting the intra-species recombination for CpCDV and presence of the same strain in chickpea growing areas of Pakistan other than Punjab province. This is the first identification of CpCDV (genus Mastrevirus family Geminiviridae) from chickpea (Cicer arietinum) plants in District Dera Ismail Khan, KP province, Pakistan.

Molecular and biological characterization of Chilli leaf curl virus and associated Tomato leaf curl betasatellite infecting tobacco in Oman.

BACKGROUND: In Oman tobacco (Nicotiana tabacum; family Solanaceae) is a minor crop, which is produced only for local consumption. In 2015, tobacco plants exhibiting severe downward leaf curling, leaf thickening, vein swelling, yellowing and stunting were identified in fields of tobacco in Suhar Al-Batina region, Oman. These symptoms are suggestive of begomovirus (genus Begomovirus, family Geminiviridae) infection. METHODS: Circular DNA molecules were amplified from total DNA extracted from tobacco plants by rolling circle amplification (RCA). Viral genomes were cloned from RCA products by restriction digestion and betasatellites were cloned by PCR amplification from RCA product, using universal primers. The sequences of full-length clones were obtained by Sanger sequencing and primer walking. Constructs for the infectivity of virus and betasatellite were produced and introduced into plants by Agrobacterium-mediated inoculation. RESULTS: The full-length sequences of 3 begomovirus and 3 betasatellite clones, isolated from 3 plants, were obtained. Analysis of the full-length sequences determined showed the virus to be a variant of Chilli leaf curl virus (ChiLCV) and the betasatellite to be a variant of Tomato leaf curl betasatellite (ToLCB). Both the virus and the betasatellite isolated from tobacco show the greatest levels of sequence identity to isolates of ChiLCV and ToLCB identified in other hosts in Oman. Additionally clones of ChiLCV and ToLCB were shown, by Agrobacterium-mediated inoculation, to be infectious to 3 Nicotiana species, including N. tabacum. In N. benthamiana the betasatellite was shown to change the upward leaf rolling symptoms to a severe downward leaf curl, as is typical for many monopartite begomoviruses with betasatellites. CONCLUSIONS: The leaf curl disease of tobacco in Oman was shown to be caused by ChiLCV and ToLCB. This is the first identification of ChiLCV with ToLCB infecting tobacco. The study shows that, despite the low diversity of begomoviruses and betasatellites in Oman, the extant viruses/betasatellites are able to fill the niches that present themselves.

The Rep proteins encoded by alphasatellites restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

Alphasatellites are non-essential satellite-like components associated with geminiviruses. The precise selective advantage to a geminivirus infection of an alphasatellite remains unclear. The ability of the cotton leaf curl Multan alphasatellite (CLCuMuA)-encoded replication-associated protein (Rep) to suppress TGS was investigated by using Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant Potato virus X (PVX) vector carrying CLCuMuA Rep resulted in restoration of GFP expression. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. Inoculation with PVX vectors harbouring a further six Rep proteins, encoded by genetically distinct alphasatellites, were similarly shown to result in 16-TGS plants with restored GFP expression. These results indicate that the alphasatellite-encoded Rep can restore the expression of a transcriptionally silenced GFP transgene in N. benthamiana, indicating that alphasatellites are involved in overcoming host defence.

The V2 protein encoded by a monopartite begomovirus is a suppressor of both post-transcriptional and transcriptional gene silencing activity.

Papaya leaf curl virus (PaLCuV) is a begomovirus (genus Begomovirus; family Geminiviridae) with a monopartite genome that is usually associated with beta- and alphasatellites in plants. Geminiviruses are DNA viruses with small circular genomes that occur as minichromosomes in the nucleus and are susceptible to post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). Transient expression of the PaLCuV V2 (PV2) protein together with the green fluorescent protein (GFP) in Nicotiana benthamiana resulted in enhanced levels of GFP fluorescence and GFP mRNA, indicative of suppression of PTGS. Expression of PV2 from a Potato virus X vector restored GFP expression in N. benthamiana plants harbouring a transcriptionally silenced GFP transgene, indicative of suppression of TGS. The results show that the PV2 protein encoded by PaLCuV has both suppressor of PTGS and TGS activity and is an important factor in overcoming host RNA-silencing based defenses.

Transmission of cotton leaf curl disease: answer to a long-standing question.

Cotton leaf curl disease (CLCuD) has been a major constraint to cotton production across Pakistan and northwestern India since the early 1990s. The disease is caused by a number of begomoviruses, including Cotton leaf curl Multan virus (CLCuMuV), which associate with a specific host range and symptom determining betasatellite known as Cotton leaf curl Multan betasatellite (CLCuMuB). Bemisia tabaci is a complex of cryptic species that consists of numerous (> 44) morphologically indistinguishable and, at least partially, reproductively isolated species. CLCuD has recently been introduced into parts of China but has not, at least so far, become a problem in the major cotton regions. The disease in China has been shown to be caused by CLCuMuV with CLCuMuB, which is believed to have been introduced from South Asia in ornamental plants. To understand the basis for this lack of spread of CLCuD into the cotton-growing areas of China, Pan et al. (Phytopathology 108:1172-1183, 2018) investigated the transmission of CLCuMuV/CLCuMuB by B. tabaci. The study showed that, of the four cryptic B. tabaci species investigated, only the cryptic species Asia II 1 was able to efficiently transmit CLCuMuV/CLCuMuB. Significantly, Asia II 1 is not present in the major cotton-growing regions of China. The results of Pan et al. (Phytopathology 108:1172-1183, 2018) are discussed with particular emphasis on the situation of CLCuD in Pakistan and northwestern India, which differs significantly from the situation in China.

Identification of a dicot infecting mastrevirus along with alpha- and betasatellite associated with leaf curl disease of spinach (Spinacia oleracea) in Pakistan.

Spinach is a common vegetable crop and very little data is available about its virus infection. Symptomatic leaves of spinach were collected during field survey. Circular DNA molecules were amplified from symptomatic samples using rolling circle amplification (RCA). After restriction analysis, presumed bands of virus and satellites were cloned, sequenced and analyzed. Analysis of sequenced RCA product revealed the presence of chickpea chlorotic dwarf virus (CpCDV; Mastrevirus). Further analyses of the cloned virus showed that strain "C" of CpCDV was present in symptomatic samples of spinach collected from field associated with vein darkening, curling and enations on leaves. Amplification of alpha- and betasatellites with universal primers was performed. CpCDV showed association with cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellites (CLCuMA). Infectivity analysis of CpCDV and CpCDV/CLCuMB were done in N. benthamiana using particle bombardment method and the results showed that CpCDV was able to transreplicates CLCuMB in this host. To our knowledge, this is the first report of a dicot infecting mastrevirus (CpCDV) along with CLCuMB and CLCuMA associated with leaf curl disease of spinach in Pakistan. The significance of the results is discussed.

World Management of Geminiviruses.

Management of geminiviruses is a worldwide challenge because of the widespread distribution of economically important diseases caused by these viruses. Regardless of the type of agriculture, management is most effective with an integrated pest management (IPM) approach that involves measures before, during, and after the growing season. This includes starting with resistant cultivars and virus- and vector-free transplants and propagative plants. For high value vegetables, protected culture (e.g., greenhouses and screenhouses) allows for effective management but is limited owing to high cost. Protection of young plants in open fields is provided by row covers, but other measures are typically required. Measures that are used for crops in open fields include roguing infected plants and insect vector management. Application of insecticide to manage vectors (whiteflies and leafhoppers) is the most widely used measure but can cause undesirable environmental and human health issues. For annual crops, these measures can be more effective when combined with host-free periods of two to three months. Finally, given the great diversity of the viruses, their insect vectors, and the crops affected, IPM approaches need to be based on the biology and ecology of the virus and vector and the crop production system. Here, we present the general measures that can be used in an IPM program for geminivirus diseases, specific case studies, and future challenges.

Alphasatellitidae: a new family with two subfamilies for the classification of geminivirus- and nanovirus-associated alphasatellites.

Nanoviruses and geminiviruses are circular, single stranded DNA viruses that infect many plant species around the world. Nanoviruses and certain geminiviruses that belong to the Begomovirus and Mastrevirus genera are associated with additional circular, single stranded DNA molecules (~ 1-1.4 kb) that encode a replication-associated protein (Rep). These Rep-encoding satellite molecules are commonly referred to as alphasatellites and here we communicate the establishment of the family Alphasatellitidae to which these have been assigned. Within the Alphasatellitidae family two subfamilies, Geminialphasatellitinae and Nanoalphasatellitinae, have been established to respectively accommodate the geminivirus- and nanovirus-associated alphasatellites. Whereas the pairwise nucleotide sequence identity distribution of all the known geminialphasatellites (n = 628) displayed a troughs at ~ 70% and 88% pairwise identity, that of the known nanoalphasatellites (n = 54) had a troughs at ~ 67% and ~ 80% pairwise identity. We use these pairwise identity values as thresholds together with phylogenetic analyses to establish four genera and 43 species of geminialphasatellites and seven genera and 19 species of nanoalphasatellites. Furthermore, a divergent alphasatellite associated with coconut foliar decay disease is assigned to a species but not a subfamily as it likely represents a new alphasatellite subfamily that could be established once other closely related molecules are discovered.

Evaluation of carbon nanotube based copper nanoparticle composite for the efficient detection of agroviruses.

We report a biosensor that combines the construction of a three-dimensional nanocomposite with electrochemical methods for the detection of viruses in plants. This is the first report, where carbon nanotubes are used as a conductive frame to anchor highly electrolytic agglomerates of copper nanoparticles to detect agroviruses. Morphological analysis of nanocomposite revealed the presence of carbon nanotubes having a diameter of 50-100nm with copper nanoparticles of 20-100nm, attached in the form of bunches. This material was applied to assess the infection caused by geminiviruses which are a major threat to the cotton plants in Asian and African countries. The hybridization events were studied by monitoring differential pulse voltammetry signals using methylene blue as a redox indicator. In the presence of target DNA, sensor signals decreased from 7×10-4 to 1×10-4Ampere. The probe exhibited 97.14% selectivity and the detection limit was found to be 0.01ngµL-1. The developed biosensor is stable for at least four weeks, losing only 4.3% of the initial signal value. This sensor was able to detect the presence of viruses in sap extracted from cotton leaves, thus providing a promising platform to detect a range of other crops-infecting viruses.

Diversity and Distribution of Cryptic Species of the Bemisia tabaci (Hemiptera: Aleyrodidae) complex in Pakistan.

Bemisia tabaci (Gennadius; Hempitera: Aleyrodidae) is considered to be a cryptic (sibling) species complex, the members of which exhibit morphological invariability while being genetically and behaviorally distinct. Members of the complex are agricultural pests that cause direct damage by feeding on plants, and indirectly by transmitting viruses that cause diseases leading to reduced crop yield and quality. In Pakistan, cotton leaf curl disease, caused by multiple begomovirus species, is the most economically important viral disease of cotton. In the study outlined here, the diversity and geographic distribution of B. tabaci cryptic species was investigated by analyzing a taxonomically informative fragment of the mitochondrial cytochrome c oxidase 1 gene (mtCOI-3'). The mtCOI-3' sequence was determined for 285 adult whiteflies and found to represent six cryptic species, the most numerous being Asia II-1 and Middle East Asia Minor 1 (MEAM-1), the later also referred to as the B-biotype, which was previously thought to be confined to Sindh province but herein, was also found to be present in the Punjab province. The endemic Asia I was restricted to Sindh province, while an individual in the Asia II-8 was identified in Pakistan for the first time. Also for the first time, samples were collected from northwestern Pakistan and Asia II-1 was identified. Results indicate that in Pakistan the overall diversity of B. tabaci cryptic species is high and, based on comparisons with findings from previous studies, the distribution is dynamic.

Further changes in the cotton leaf curl disease complex: an indication of things to come?

Cotton leaf curl disease (CLCuD) has been a problem for cotton production in Pakistan and India since the early 1990s. The disease is caused by begomoviruses associated with a specific satellite, the cotton leaf curl Multan betasatellite (CLCuMB). In 2001, resistance introduced into cotton was broken by a recombinant begomovirus, Cotton leaf curl Kokhran virus strain Burewala (CLCuKoV-Bur). Unusually, in resistant cotton, this virus lacked an intact transcriptional activator protein (TrAP) gene, with the capacity to encode only 35 of the usual ~134 amino acids. Recently, isolates of CLCuKoV-Bur with a longer, but still truncated, TrAP gene have been identified in cotton breeding lines lacking the earlier resistance. This suggests that more pathogenic viruses with a full TrAP could return to cotton if the earlier resistance is not maintained in ongoing breeding efforts to produce CLCuD-resistant cotton varieties. This conclusion is supported by recent studies showing the reappearance of pre-resistance-breaking begomoviruses, with full-length TrAP genes, in cotton.

Real-time quantitative PCR assay for the quantification of virus and satellites causing leaf curl disease in cotton in Pakistan.

Cotton leaf curl disease (CLCuD) is the major biotic constraint to cotton production in Pakistan and northwestern India. The disease is caused by monopartite begomoviruses in association with a specific DNA satellite, Cotton leaf curl Multan betasatellite. The virus-betasatellite complex is also frequently associated with another DNA satellite-like molecule; an alphasatellite. A quantitative real-time PCR (qPCR) assay to detect all three components of the monopartite begomovirus/betasatellite/alphasatellite complex which causes CLCuD was established. This was used to investigate the relationship between symptoms and virus/satellite titre. Not surprisingly the analysis showed that, overall, there was a reasonable correlation between symptom severity and virus/satellite titre - more severe symptoms usually being associated with more virus/satellite. However, cotton plants were identified with no or very mild symptoms with relatively high virus/satellite titres and plants with severe symptoms but relatively low virus/satellite titres. This may be attributed to the resistance/susceptibility of the cotton variety - tolerant plants being able to sustain a relatively high virus/satellite titre whilst exhibiting mild symptoms. The usefulness of this qPCR procedure in the screening for resistance in cotton against CLCuD is discussed.

ICTV Virus Taxonomy Profile: Geminiviridae.

The geminiviruses are a family of small, non-enveloped viruses with single-stranded, circular DNA genomes of 2500-5200 bases. Geminiviruses are transmitted by various types of insect (whiteflies, leafhoppers, treehoppers and aphids). Members of the genus Begomovirus are transmitted by whiteflies, those in the genera Becurtovirus, Curtovirus, Grablovirus, Mastrevirus and Turncurtovirus are transmitted by specific leafhoppers, the single member of the genus Topocuvirus is transmitted by a treehopper and one member of the genus Capulavirus is transmitted by an aphid. Geminiviruses are plant pathogens causing economically important diseases in most tropical and subtropical regions of the world. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Geminiviridae which is available at www.ictv.global/report/geminiviridae.

Capulavirus and Grablovirus: two new genera in the family Geminiviridae.

Geminiviruses are plant-infecting single-stranded DNA viruses that occur in most parts of the world. Currently, there are seven genera within the family Geminiviridae (Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus). The rate of discovery of new geminiviruses has increased significantly over the last decade as a result of new molecular tools and approaches (rolling-circle amplification and deep sequencing) that allow for high-throughput workflows. Here, we report the establishment of two new genera: Capulavirus, with four new species (Alfalfa leaf curl virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus and Plantago lanceolata latent virus), and Grablovirus, with one new species (Grapevine red blotch virus). The aphid species Aphis craccivora has been shown to be a vector for Alfalfa leaf curl virus, and the treehopper species Spissistilus festinus is the likely vector of Grapevine red blotch virus. In addition, two highly divergent groups of viruses found infecting citrus and mulberry plants have been assigned to the new species Citrus chlorotic dwarf associated virus and Mulberry mosaic dwarf associated virus, respectively. These species have been left unassigned to a genus by the ICTV because their particle morphology and insect vectors are unknown.

Maintenance of Cotton Leaf Curl Multan Betasatellite by Tomato Leaf Curl New Delhi Virus-Analysis by Mutation.

Viruses of the genus Begomovirus (family Geminiviridae) are economically important phytopathogens that are transmitted plant-to-plant by the whitefly Bemisia tabaci. Most Old World (OW) begomoviruses are monopartite and many of these interact with symptoms and host range determining betasatellites. Tomato leaf curl New Delhi virus (ToLCNDV) is one of only a few OW begomoviruses with a bipartite genome (components known as DNA A and DNA B). Four genes [AV2, coat protein (CP), transcriptional-activator protein (TrAP), and AC4] of ToLCNDV were mutated and the effects of the mutations on infectivity, symptoms and the ability to maintain Cotton leaf curl Multan betasatellite (CLCuMuB) were investigated. Infectivity and virus/betasatellite DNA titer were assessed by Southern blot hybridization, PCR, and quantitative PCR. The results showed TrAP of ToLCNDV to be essential for maintenance of CLCuMuB and AV2 to be important only in the presence of the DNA B. AC4 was found to be important for the maintenance of CLCuMuB in the presence of, but indispensable in the absence of, the DNA B. Rather than being required for maintenance, the CP was shown to possibly interfere with maintenance of the betasatellite. The findings show that the interaction between a bipartite begomovirus and a betasatellite is more complex than just trans-replication. Clearly, multiple levels of interactions are present and such associations can cause additional significant losses to crops although the interaction may not be stable.

Engineering Dual Begomovirus-Bemisia tabaci Resistance in Plants.

The whitefly Bemisia tabaci is an important pest of many economically important crops and the vector of begomoviruses (family Geminiviridae). Recently, the expression of insecticidal proteins and/or toxins or double-stranded (ds)RNA homologous to B. tabaci genes has been demonstrated to provide the plant with protection against B. tabaci and the viruses that it transmits.