Detection and relationships of cotton leaf curl virus and allied whitefly-transmitted geminiviruses occurring in Pakistan

A stock culture of cotton leaf curl virus from Pakistan (CLCuV-PK), was transmitted by whiteflies (Bemisia tabaci) to seven plant species, including French bean, okra, tobacco and tomato, and caused vein thickening and leaf curl symptoms. It was readily detected in triple antibody sandwich ELISA (TAS-ELIS A) by 11 out of 31 monoclonal antibodies raised against the particles of three other geminiviruses: African cassava mosaic, Indian cassava mosaic and okra leaf curl viruses. Reaction strength was enhanced when the tissue extraction fluid contained sodium sulphite. Minor variations in epitope profile were found among virus isolates from cotton (Gossypium hirsutum) collected from different districts in Pakistan over a 5-year period. These epitope profiles were distinguishable from that of cotton leaf curl virus from G. barbadense in southern India but indistinguishable from the profiles of viruses causing yellow vein disease of okra in India or Pakistan, or leaf curl of okra {Abelmoschus esculentus), Hibiscus tiliaceus, radish or sunflower in Pakistan, suggesting that these plants are putative natural hosts of CLCuV-PK. The viruses in cotton, and in okra with leaf curl or yellow vein symptoms, were also detected by PCR with three pairs of CLCuV-PK-specific primers. Five additional whitefly-transmitted geminiviruses were found among isolates from 11 other naturally-infected species in Pakistan, and were distinguished by their epitope profiles. These viruses were associated, respectively, with tobacco leaf curl, squash yellow blotch, tomato yellow leaf curl, watermelon leaf crinkle and soybean yellow mosaic diseases. The first four of these viruses were detected readily by PCR with geminivirus general primers but only weakly, if at all, with two pairs of CLCuV-PK-specific primers. Pakistani crops are infected with a range of distinguishable but relatively closely related whitefly-transmitted geminiviruses, some of which resemble those found in India.     

Breeding for resistance to whitefly-transmitted geminiviruses

Geminiviruses comprise a large and diverse family of viruses that infect a wide range of important monocotyledonous and dicotyledonous crop species and cause significant yield losses. The family Geminiviridae is divided into three genera, one of which is Begomovirus. Species of this genus are transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner and infect dicotyledonous plants. Severe population outbreaks of B. tabaci are usually accompanied by a high incidence of begomoviruses. During the last two decades, there has been a worldwide spread of the B biotype of B. tabaci, accompanied by the emergence of whitefly‐transmitted geminiviruses. Control measures in infected regions are based mainly on limitation of vector populations, using chemicals or physical barriers. However, under conditions of severe whitefly attack, none of these control measures has sufficed to prevent virus spread. Thus, the best way to reduce geminivirus damage is by breeding crops resistant or tolerant to the virus, either by classical breeding or by genetic engineering. A number of begomoviruses have been the subject of much investigation, due to their severe economic impact. This review considers the most severe viral diseases of four major crops (tomato, bean, cassava and cotton). The approaches taken to breed for resistance to these viral diseases should provide a perspective of the issues involved in breeding for begomovirus resistance in crop plants.     

Occurrence of whitefly-transmitted geminiviruses in crops in Burkina Faso, and their serological detection and differentiation

Whitefly-transmitted geminiviruses were found to be associated with four diseases of crop plants in Burkina Faso: cassava mosaic, okra leaf curl, tobacco leaf curl and tomato yellow leaf curl. Tomato yellow leaf curl is an economically serious disease, reaching a high incidence in March, following a peak population of the vector whitefly, Bemisia tabaci, in December. Okra leaf curl is also a problem in the small area of okra grown in the dry season but is not important in the main period of okra production in the rainy season. The geminiviruses causing these four diseases, African cassava mosaic (ACMV), okra leaf curl (OLCV), tobacco leaf curl (TobLCV) and tomato yellow leaf curl (TYLCV) viruses, were each detected in field-collected samples by triple antibody sand-wich-ELISA with cross-reacting monoclonal antibodies (MAbs) to ACMV. Epitope profiles obtained by testing each virus isolate with panels of MAbs to ACMV, OLCV and Indian cassava mosaic virus enabled four viruses to be distinguished. ACMV and OLCV had similar but distinguishable profiles. The epitope profile of TobLCV was the same as that of one form of TYLCV (which may be the same virus) and was close to the profile of TYLCV from Sardinia. The other form of TYLCV reacted with several additional MAbs and had an epitope profile close to that of TYLCV from Senegal. Only minor variations within each of these four types of epitope profile were found among geminivirus isolates from Burkina Faso. Sida acuta is a wild host of OLCV.     

Recombination Among Begomoviruses on Malvaceous Plants Leads to the Evolution of Okra Enation Leaf Curl Virus in Pakistan

Whitefly transmitted begomoviruses (family Geminiviridae) are the major reason for significant yield losses of dicotyledonous crops in tropics and subtropics. Okra (Abelmoschus esculentus) is one of the important vegetable crops, and leaf curl disease caused by geminiviruses is the most important limiting factor for its production in Pakistan. Here, we report a new species of okra‐infecting begomovirus in south‐eastern region of Pakistan and the name Okra enation leaf curl virus (OELCuV) complex is proposed. This okra enation leaf curl disease complex (OELCuD) in Pakistan is found to be associated with Ageratum conyzoides symptomless alphasatellite (AConSLA). All efforts to clone the betasatellite were unsuccessful. Comprehensive sequence analyses suggest that intermalvaceous recombination between okra and cotton‐infecting begomoviruses resulted in the evolution of the new species. Surprisingly, Bhendi yellow vein mosaic virus (BYVMV) which has not been reported previously from Pakistan is the major parent while Cotton leaf curl Multan virus (CLCuMV) acts as a distant parent of the virus. Comparative recombination analysis also reveals that okra‐infecting begomoviruses from south and north‐western India is causing OELCuD in the Pakistan by recombining with CLCuMV at the Rep (1964–1513 nts). Recombination is common among geminiviruses and recombining of BYVMV and CLCuMV resulted in a new species: OELCuV. To the best of our knowledge, this evolution of a new species of okra‐infecting begomovirus is the first report of intermalvaceous recombination where Rep acts as the target region.     

Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus Invade South-east Coast of China

An epidemic outbreak of severe yellow leaf curl disease was reported in field grown tomato within Zhejiang Province of China in the autumn–winter cropping season of 2006. A molecular diagnostic survey was carried out based on comparisons of partial and complete viral DNA sequences. Comparison of partial DNA‐A sequences amplified with degenerate primers specific for begomoviruses confirmed the presence of two types of begomoviruses. The complete DNA sequences of five isolates, corresponding to the two types, were determined. Sequence comparisons and phylogenetic analysis revealed that they correspond to two previously identified begomoviruses, Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus. The satellite DNAβ molecule was not detected in these samples by either PCR or Southern blot hybridization analysis. There has been no previous report of geminivirus disease incidence in Zhejiang Province, indicating that the introduction of these two tomato infecting geminiviruses into the agro‐ecological zone of South‐eastern China is a fairly recent event. The implications for disease control are discussed.     

A mixture of begomoviruses in leaf curl-affected tobacco in Karnataka, South India

Tobacco leaf curl is widespread in several states in India including Andhra Pradesh, Gujarat, Karnataka, Bihar and West Bengal. Tobacco leaf curl virus (TbLCV) isolates collected from five different parts of India induced four distinct symptom phenotypes (group I, II, III & IV) on tobacco cultivars Samsun and Anand 119 (Valand & Muniyappa, 1992). PCR was performed on DNA extracted from group I and IV leaf curl‐affected tobacco from Karnataka, India using degenerate begomovirus‐specific primers. Subsequent cloning and sequencing of PCR products revealed preliminary evidence for the presence of at least three begomoviruses in the affected material following alignment of a 333 bp region of the coat protein gene (CP). The complete CP and common region (CR) of two putative begomoviruses, Tobacco leaf curl virus‐Karnataka1 (TbLCV‐Kar1) and Tobacco leaf curl virus‐Karnataka2 (TbLCV‐Kar2), were sequenced using PCR clones obtained with designed sequence‐specific primers. Phylogenetic analysis of the CP and CR of TbLCV‐Kar1 and TbLCV‐Kar2 placed them in the Asian Old World begomovirus cluster. The two viruses differed from each other significantly in both the CP gene and the CR (< 90% nucleotide sequence identity). This difference, in conjunction with distinct iterative sequences strongly suggests that these begomoviruses are distinct from one another. Group I and IV tobacco were also found to harbour a possible third begomovirus following the 333 bp CP alignment. Comparison of TbLCV‐Kar1 and TbLCV‐Kar2 with other geminiviruses, showed that both sequences shared high nucleotide sequence identity (> 90%) with other begomoviruses in either the CP or CR, thereby suggesting these viruses to be possible strains of other reported begomoviruses. Combined comparison of the CP and CR sequences however, suggests that the two viruses are not strains of other reported begomoviruses, but may be distinct begomoviruses that could have arisen through recombination events during mixed infections. Phylogenetic comparison demonstrated no significant homology between the Indian tobacco begomoviruses and a tobacco‐infecting begomovirus from Zimbabwe, again showing that as with other geminiviruses, there is a geographic basis for phylogenetic relationships rather than an affiliation with tobacco as a host.     

Detection of three whitefly-transmitted geminiviruses occurring in Europe by tests with heterologous monoclonal antibodies

Selected monoclonal antibodies (MAbs), prepared to particles of African cassava mosaic or Indian cassava mosaic geminiviruses, detected three geminiviruses that occur in Europe: abutilon mosaic virus in Abutilon pictum ‘Thompsonii’, tobacco leaf curl virus in Lonicera japonica var. aureo-reticulata and tomato yellow leaf curl virus in Lycopersicon esculentum. All three viruses were detected in indirect ELISA by MAbs SCR 17 and SCR 20 but they were differentiated by their reactions with SCR 18 and SCR 23. Tobacco leaf curl virus was detected only when reducing agents were included in the leaf extraction medium. Inclusion of sodium sulphite slightly improved detection of tomato yellow leaf curl virus but reducing agents were not needed for detection of abutilon mosaic virus.     

Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan β-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and βC1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and βC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.     

Particle purification, properties and epitope variability of Indian tomato leaf curl geminivirus

A whitefly-transmissible stock isolate of Indian tomato leaf curl geminivirus (ITmLCV) was cultured in graft-inoculated tomato plants and its particles purified from chloroform-clarified extracts in citrate buffer by precipitation with 70 g/litre polyethylene glycol, ultracentrifugation and sucrose density gradient centrifugation. Contaminating helical filaments were eliminated by banding in caesium sulphate gradients. ITmLCV particles had the shape typical for geminiviruses, measured c. 30 × 20 nm and contained a single major protein of estimated mol. wt c. 32 000. They reacted in immunosorbent electron microscopy with antisera to four other whitefly-transmitted geminiviruses. ITmLCV reacted with one out of 17 monoclonal antibodies specific for different epitopes in the particle protein of African cassava mosaic geminivirus and five or six out of 10 monoclonal antibodies to the particle protein of Indian cassava mosaic geminivirus. Virus isolates from tomato at nine locations in Karnataka State showed only slight differences in epitope profile, and isolates from four weed species in tomato fields were similar or identical to those from tomato.     

Recognition and differentiation of seven whitefly-transmitted geminiviruses from India, and their relationships to African cassava mosaic and Thailand mung bean yellow mosaic viruses

Particles resembling those of geminiviruses were found by immunosorbent electron microscopy in extracts of plants infected in India with bhendi yellow vein mosaic, croton yellow vein mosaic, dolichos yellow mosaic, horsegram yellow mosaic, Indian cassava mosaic and tomato leaf curl viruses. All these viruses were transmitted by Bemisia tabaci whiteflies, all reacted with at least one out of ten monoclonal antibodies to African cassava mosaic virus (ACMV), and all reacted with a probe for ACMV DNA-1, but scarcely or not at all with a full-length probe for ACMV DNA-2. Most of the viruses were distinguished by their host ranges when transmitted by whiteflies, and the rest could be distinguished by their pattern of reactions with the panel of monoclonal antibodies. Horsegram yellow mosaic virus was distinguished from Thailand mung bean yellow mosaic virus by its lack of sap transmissibility, ability to infect Arachis hypogaea, failure to react strongly with the probe for ACMV DNA-2 and its pattern of reactions with the monoclonal antibodies. Structures resembling a ‘string of pearls’, but not geminate particles, were found in leaf extracts containing malvastrum yellow vein mosaic virus. Such extracts reacted with two of the monoclonal antibodies, suggesting that this whitefly-transmitted virus too is a geminivirus. All seven viruses from India can therefore be considered whitefly-transmitted geminiviruses.