Detection and Molecular Characterization of Squash leaf curl virus (SLCV) in Jordan

Squash leaf curl virus (SLCV) was detected for the first time in Jordan using degenerated oligonucleotide primers. Two isolates of the virus, SLCV‐E and SLCV‐R, were detected using specific oligonucleotide primers in symptomatic Cucurbita pepo. SLCV was also found to occur naturally in Malva parviflora, which showed severe leaf curling, yellowing and stunting of the whole plants. The full‐length genomes of Squash leaf curl virus‐Malva (SLCV‐Malva) isolate were amplified using the bacteriophage Φ DNA polymerase enzyme. Nucleotide sequence analysis showed that SLCV‐Malva shared high nucleotide identity (98% and 97%) with SLCV‐EG and SLCV‐E from Egypt and USA, respectively. A survey using dot‐blot hybridization indicated that squash leaf curl disease occurred in all surveyed areas. The highest disease incidence (95%) was recorded in Dir Alla area, whereas disease incidence did not exceed 69% in squash samples collected from North Ghor.     

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.     

Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India

Background

Emerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India.

Results

We demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.

Conclusion

This is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.     

Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF βC1 in satellite DNA β which are predicted to function as silencing suppressors. In the present study suppressor function of ORF βC1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB–[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing βC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with βC1 gene from ToLCBB. However, in the case of 35S-βC1 CLCuMB and 35S-βC1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which βC1 interfere in RNA-silencing pathway is different.     

Tomato yellow leaf curl viruses: ménage à trois between the virus complex,the plant and the whitefly vector

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci. Taxonomy: The tomato yellow leaf curl virus‐like viruses (TYLCVs) are a complex of begomoviruses (family Geminiviridae, genus Begomovirus) including 10 accepted species: Tomato yellow leaf curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLVKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl Vietnam virus (TYLCVNV) and Tomato yellow leaf curl virus(TYLCV). We follow the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV), the most important of which is an 89% nucleotide identity threshold between full‐length DNA‐A component nucleotide sequences for begomovirus species. Strains of a species are defined by a 93% nucleotide identity threshold. Host range: The primary host of TYLCVs is tomato (Solanum lycopersicum), but they can also naturally infect other crops [common bean (Phaseolus vulgaris), sweet pepper (Capsicum annuum), chilli pepper (C. chinense) and tobacco (Nicotiana tabacum)], a number of ornamentals [petunia (Petunia×hybrida) and lisianthus (Eustoma grandiflora)], as well as common weeds (Solanum nigrum and Datura stramonium). TYLCVs also infect the experimental host Nicotiana benthamiana. Disease symptoms: Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.     

Genome affinities and epitope profiles of whitefly-transmitted geminiviruses from the Americas

The relationships among fifteen isolates of whitefly-transmitted geminiviruses (WTGs) from North, Central and South America and six from other continents were assessed (a) in nucleic acid hybridisation tests with sulphonated DNA probes for eight of the viruses, and/or (b) in triple-antibody-sandwich ELISA with panels of monoclonal antibodies (MAbs) to particles of African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV). Probes specific for DNA-A of four American viruses, abutilon mosaic (AbMV), bean golden mosaic (BGMV), squash leaf curl (SLCV) and tomato golden mosaic (TGMV), detected virtually all the American viruses but reacted weakly if at all with ICMV, ACMV or tomato yellow leaf curl virus from Thailand (TYLCV-T). Conversely, the probe for ACMV DNA-A did not detect any of the American viruses, and that for TYLCV-T DNA-A reacted weakly with SLCV and TGMV0020but did not detect the others. In contrast, probes specific for DNA-B of the four American viruses or ACMV detected only the homologous virus, except for slight reactions between the AbMV DNA-B probe and both chino del tomate virus (CdTV)-DNA and SLCV-DNA. However, a probe for DNA-B of bean calico mosaic virus (BCMoV) reacted weakly with BGMV-PR DNA, and a probe for DNA-B of CdTV from Mexico detected several American viruses. Six out of 17 MAbs specific for ACMV and six out of 10 MAbs specific for ICMV reacted with one or other of the 14 American virus isolates tested. Two and-ACMV MAbs reacted with all, and one anti-ACMV MAb and two anti-ICMV MAbs reacted with nearly all the American viruses, one anti-ACMV MAb reacted with about half the American viruses and six other MAbs reacted with only one or two of them. Of the American viruses, CdTV and AbMV were the least closely related to the others. The epitope profiles of BCMoV, BGMV, cotton leaf crumple virus, serrano golden mosaic virus and SLCV were virtually indistinguishable. TGMV, potato yellow mosaic virus (PYMV) and an euphorbia virus had profiles intermediate between those of the BGMV cluster and AbMV-CdTV. In general, the epitope profiles and the results of hybridisation tests with DNA-A probes show that the similarities among the American viruses are greater than those between the American viruses and the viruses from other continents; the hybridisation tests with DNA-B probes show that substantial differences exist between individual American viruses. In America, geminivirus evolution seems to have proceeded convergently from different progenitor viruses, or divergently from one ancestral form, with DNA-B diverging to a greater extent than DNA-A and its particle-protein gene.     

The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci— insights from studies with Tomato yellow leaf curl virus

Our current knowledge concerning the transmission of begomoviruses by the whitefly vector Bemisia tabaci is based mainly on research performed on the Tomato yellow leaf curl virus (TYLCV) complex and on a number of viruses originating from the Old World, such as Tomato leaf curl virus, and from the New World, including Abutilon mosaic virus, Tomato mottle virus, and Squash leaf curl virus. In this review we discuss the characteristics of acquisition, transmission and retention of begomoviruses by the whitefly vector, concentrating on the TYLCV complex, based on both published and recent unpublished data. We describe the cells and organs encountered by begomoviruses in B. tabaci. We show immunolocalisation of TYLCV to the B. tabaci stylet food canal and to the proximal part of the descending midgut, and TYLCV‐specific labelling was also associated with food in the lumen. The microvilli and electron‐dense material in the epithelial cells of the gut wall were also labelled by the anti TYLCV serum, pointing to a possible virus translocation route through the gut wall and to a putative site of long‐term virus storage. We describe the path of begomoviruses in their vector B. tabaci and in the non‐vector whitefly Trialeurodes vaporariorum, and we follow the rate of virus translocation in these insects. We discuss TYLCV transmission between B. tabaci during mating, probably by exchange of haemolymph. We show that following a short acquisition access to infected tomato plants, TYLCV remains associated with the B. tabaci vector for weeks, while the virus is undetectable after a few hours in the non‐vector T. vaporariorum. The implications of the long‐term association of TYLCV with B. tabaci in the light of interactions of the begomovirus with insect receptors are discussed.     

广东番茄曲叶病毒G2分离物基因组DNA-A的分子特征

从采集于广东的番茄曲叶病病株上分离到病毒分离物G2 ,序列分析结果表明 ,其DNA_A为单链环状 ,全长2 74 4nt,共有 6个ORF ,其中病毒链上编码AV1(CP)、AV2 ,互补链上编码AC1、AC2、AC3和AC4。BLAST结果显示 ,与G2基因组有同源关系的病毒均属双生病毒科菜豆金色花叶病毒属。序列比较结果显示 ,G2与菜豆金色花叶病毒属病毒的DNA_A序列同源率均不超过 83% ,其中同源率最高的是PaLCuCNV_[G10 ](82 8% )。进一步比较发现 ,它们的基因间隔区 (IR)变异最大 (同源率为 30 9%～ 81 8% ) ;CP氨基酸序列的同源率较高 (77 6 %～ 99 2 % ) ,AC4蛋白氨基酸序列的同源率较低 (4 3 5 %～ 78 8% )。系统进化关系分析结果也显示 ,G2与已报道的菜豆金色花叶病毒属病毒的亲缘关系均较远。因此 ,G2可能是双生病毒科菜豆金色花叶病毒属中一个未报道的新种 ,命名为广东番茄曲叶病毒 (TomatoleafcurlGuangdongVirus ,ToLCGDV)     

A rapid PCR method to discriminate between Tomato yellow leaf curl virus isolates

Tomato yellow leaf curl virus (TYLCV) was recently divided into two different species: Tomato yellow leaf curl virus‐Israel (TYLCV‐Is) and Tomato yellow leaf curl virus‐Sardinia (TYLCV‐Sar). There are no rapid methods by which TYLCV viruses may be assigned to either TYLCV‐Is or TYLCV‐Sar species. In the present work, using an extensive alignment of begomovirus sequences, TYLCV‐specific primers were designed and tested which allow the specific amplification of DNA fragments from any isolate of TYLCV. Also, a primer was designed and tested which allows the specific amplification of TYLCV‐Sar. Furthermore, a combination of these primers was selected to develop a duplex PCR method, which has the potential to detect either TYLCV‐Is or TYLCV‐Sar. The PCR methods were also highly effective with minimal sample preparation and allowed direct amplification of TYLCV from infected leaf extracts. This approach may be used in the laboratory as a tool for rapid, large‐scale diagnostics of TYLCV‐infected samples.     

Molecular Evidence for the Association of Tomato leaf curl Gujarat virus with a Leaf Curl Disease of Phaseolus vulgaris L.

A leaf curl disease with symptoms typical of begomoviruses was observed in bean (Phaseolus vulgaris) at the Main Research Farm of the Indian Institute of Pulses Research, Kanpur, India. Infected plants had severe distortion of leaves and the plants were unproductive. PCR indicated the involvement of French bean leaf curl virus (JQ866297), a recently described Begomovirus, and Tomato leaf curl Gujarat virus (ToLCGV). The full‐length genome of ToLCGV associated with leaf curl disease of bean was 2757 nucleotides long and had maximum identity (97–98%) with seven isolates of ToLCGV (AY234383, AF449999, EU573714, GQ994098, AY190290, FR819708, AF413671) and is designated as Tomato leaf curl Gujarat virus‐(IN:Knp:Bean:2013) (KF440686). To the best of our knowledge, this is the first record of ToLCGV infecting a leguminous host, P. vulgaris.     

Three variants of cotton leaf curl begomoviruses with their satellite molecules are associated with cotton leaf curl disease aggravation in New Delhi

Cotton leaf curl disease (CLCuD), caused by monopartite begomoviruses and its satellite molecules, is one of the serious constrains in cultivation of cotton in India. In the present study, five CLCuD-begomovirus and its associated satellite molecules were characterized based on rolling circle amplification and sequencing of complete genome. Sequence analysis showed 82–99 % nucleotide identity among them. The phylogenetic analysis and nt identity matrix determined that of the five CLCuD-begomovirus isolates, three IARI-34, IARI-42 and IARI-50 were members of Cotton leaf curl Multan virus (CLCuMuV)-Rajasthan isolates, designated as CLCuMuV-Rajasthan-34 and two, IARI-30 and IARI-45 of Cotton leaf curl Kokhran virus (CLCuKoV)-Burewala isolates, designated as CLCuKoV-Burewala-45. The present CLCuMuV-Rajasthan-34 is recombinant isolate showing recombination events in IR, C1 and C4 regions of its genome with high probality (P = 9.9 × 10^?10–3.2 × 10^?6). Same species of betasatellite (1371 nt) molecules obtained from both the present isolates was related with cotton leaf curl Multan betasatellite by 89–97 % nt identity. Three alphasatellites (1366–1396 nt) related to Cotton leaf curl Burewala alphasatellite and Gossypium darwinii symptomless alphasatellite by 86 % nt identity were also obtained. This is the first report of appearance of CLCuKoV-Burewala isolate and CLCuD associated alphasatellites in New Delhi. The present study demonstrated that CLCuD in New Delhi is caused by three kinds of variants, two are strains of CLCuMuV and one of CLCuKoV, either by single or mixed infection along with beta- and alpha-satellite molecues.     

Ipomoea crinkle leaf curl caused by a whitefly-transmitted gemini-like virus

A whitefly-transmitted infectious agent, associated with geminate particles, induced distinct symptoms on several Ipomoea species, but not on I. batatas cv. Georgia Jet. The virus was transmitted by Bemisia argentifolii in a persistent manner and by grafting, but not mechanically. No transmission to species outside Ipomoea was obtained. Extracts from infected Ipomoea plants hybridised with a bean yellow mosaic virus riboprobe and a tomato yellow leaf curl virus riboprobe, although not so strongly as hybridisation of these riboprobes with extracts from plants infected with the homologous viruses. Based on host range, we consider this virus to be distinct from sweet potato leaf curl virus reported from the Far East, and propose it be named “Ipomoea crinkle leaf curl virus” (ICLCV).     

Distribution and diversity of begomoviruses in tomato and sweet pepper plants in Costa Rica

Begomoviruses (genus Begomovirus, family Geminiviridae) have emerged as important plant pathogens in tropical and subtropical regions worldwide. Although these viruses were reported during the 1970s in Costa Rica, they are still poorly known. Therefore, the objective of this study was to analyse the diversity and distribution of begomoviruses in commercial tomato and sweet pepper fields from different agricultural production systems of the major growing regions of Costa Rica. A total of 651 plants were randomly sampled from greenhouses and open field crops during 2011 and 2012 in three different geographical locations. The bipartite begomoviruses Tomato yellow mottle virus, Tomato leaf curl Sinaloa virus and Pepper golden mosaic virus, and the monopartite begomovirus Tomato yellow leaf curl virus were detected in the collected samples. The complete genome of isolates from each species was cloned and sequenced. The frequency of detection of these four begomoviruses in the analysed samples ranged from 0 to 9%, the presence, and the prevalent virus varied largely according to the geographical location, the host (tomato and pepper), and the production system (greenhouses or open fields). An association between geographical region and begomovirus species was observed suggesting that in Costa Rica the heterogeneity on climate, topography and agricultural system might influence the distribution of begomovirus species in the country. A broader survey needs to be conducted to confirm it, although these preliminary results may contribute to the management of begomoviruses in Costa Rica.     

Distinct evolutionary histories of the DNA-A and DNA-B components of bipartite begomoviruses

Background  

Viruses of the genus Begomovirus (family Geminiviridae) have genomes consisting of either one or two genomic components. The component of bipartite begomoviruses known as DNA-A is homologous to the genomes of all geminiviruses and encodes proteins required for replication, control of gene expression, overcoming host defenses, encapsidation and insect transmission. The second component, referred to as DNA-B, encodes two proteins with functions in intra- and intercellular movement in host plants. The origin of the DNA-B component remains unclear. The study described here was initiated to investigate the relationship between the DNA-A and DNA-B components of bipartite begomoviruses with a view to unraveling their evolutionary histories and providing information on the possible origin of the DNA-B component.     

Inoculation of cucumber,melon and zucchini varieties with Tomato leaf curl New Delhi virus and evaluation of infection using different detection methods

The disease caused by Tomato leaf curl New Delhi virus (ToLCNDV), which is naturally transmitted by the whitefly Bemisia tabaci, causes important economic losses in cucurbit crops. The availability of simple and efficient inoculation protocols and detection methods is necessary for screening varieties and germplasm collections as well as for breeding populations. We evaluated the infectivity of ToLCNDV inocula prepared using three different buffers for mechanical sap inoculation in a susceptible variety of zucchini. We found that inoculum prepared with buffer III, which contains polyvinylpyrrolidone, is highly efficient for mechanical inoculation, with 100% of plants displaying severe symptoms 21 days post‐inoculation. Using this buffer, we mechanically inoculated 19 commercial varieties of cucurbit crops (six of cucumber, six of melon and seven of zucchini), evaluated the evolution of symptoms and diagnosed infection using nine different ToLCNDV detection methods (four based on serology, four based on molecular hybridization and one based on PCR detection). The results revealed that all varieties are susceptible, although cucumber varieties display less severe symptoms than those of melon or zucchini. All detection methods were highly efficient (more than 85% of plants testing positive) in melon and zucchini, but in cucumber, the percentage of positive plants detected with serology and molecular hybridization methods ranged from 20.4% with Squash leaf curl virus (SLCV) antiserum, to 78.5% with DNA extract hybridization. Overall, the best detection results were obtained with PCR, with 92.6%, 92.4% and 98.4% cucumber, melon and zucchini plants, respectively, testing positive. When considering the overall results in the three crops, the best serology and molecular hybridization methods were those using Watermelon chlorotic stunt virus (WmCSV) antiserum and DNA extract, respectively. The inoculation methodology developed and the information on detection methods are of great relevance for the selection and breeding of varieties of cucurbit crops that are tolerant or resistant to ToLCNDV.     

Occurrence of Six Begomoviruses Infecting Tomato Fields in Venezuela and Genetic Characterization of Potato Yellow Mosaic Virus Isolates

Begomoviruses are one of the major pathogens in tomato crops worldwide. In Venezuela, six begomovirus species have been described infecting tomato: Potato yellow mosaic virus (PYMV), Euphorbia mosaic Venezuela virus (EuMVV), Merremia mosaic virus (MeMV), Tomato chlorotic leaf distortion virus (ToCLDV), Tomato yellow margin leaf curl virus (TYMLCV) and Tomato yellow leaf curl virus (TYLCV). In this study, the occurrence of these viruses was analysed by PCR in 338 tomato plants exhibiting virus‐like symptoms. Sixty‐three per cent of the plants were positive at least to one of the begomoviruses tested. PYMV and TYLCV were the most frequent viruses showing 39.6 and 23.7% occurrence, respectively. Phylogenetic analyses revealed two groups of PYMV isolates from several Caribbean Basin countries. The first group clustered isolates from several countries, including Venezuela, and the second group clustered only Colombian isolates. Due to the high prevalence of PYMV and TYLCV in Venezuela, it is suggested that the surveillance and control strategies currently applied in the country should be focused on these two begomoviruses.     

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.     

Genetic diversity of Bemisia tabaci cryptic species in Nigeria and their relationships with endosymbionts and acquired begomoviruses

Bemisia tabaci is a species complex of at least 44 cryptic species with a worldwide distribution. It is a serious pest of many crop plants as well as a successful vector of at least 100 begomoviruses. Using B. tabaci collected from cassava and tomato fields in the southwestern and north central regions of Nigeria, we determined nucleotide sequences from the mitochondrial cytochrome c oxidase subunit I (COI) of 23 B. tabaci samples, the 16S and 23S ribosomal DNA of endosymbionts, and the coat protein gene of geminiviruses ingested by the whiteflies. The COI analysis identified three different genetic groups including the indigenous Sub-Saharan Africa 1 subgroup 1 (SSA1-SG1) and 5 (SSA1-SG5, which was most prevalent), and an invasive cryptic species (Mediterranean). SSA1 was infected by five known secondary endosymbionts, Arsenophonus, Cardinium, Hamiltonella, Rickettsia, and Wolbachia, and co-infections with two or three endosymbionts were common. Five begomoviruses, okra enation leaf curl virus, squash leaf curl China virus, tobacco curly shoot virus, tomato leaf curl New Delhi virus, and tomato yellow leaf curl virus, were detected from 43.5% of the B. tabaci samples. However, cassava mosaic disease that causes devastating cassava yield losses was not detected in this study. This study improves the current understanding of the genetic diversity of B. tabaci cryptic species, and it reveals their relationships with endosymbionts and geminiviruses in the cassava and tomato fields of Nigeria.     

Development of a multiplex RT-RPA assay for simultaneous detection of three viruses in cucurbits

Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.