Downregulation of dystrophin expression in pupae of the whitefly Bemisia tabaci inhibits the emergence of adults.

The whitefly Bemisia tabaci is a major pest to agriculture. Adults are able to fly for long distances and to colonize staple crops, herbs and ornamentals, and to vector viruses belonging to several important taxonomic groups. During their early development, whiteflies mature from eggs through several nymphal stages (instars I to IV) until adults emerge from pupae. We aim at reducing whitefly populations by inhibiting the emergence of adults from nymphs. Here we targeted dystrophin, a conserved protein essential for the development of the muscle system in humans, other animals and insects. We have exploited the fact that whitefly nymphs developing on tomato leaves feed from the plant phloem via their stylets. Thus, we delivered dystrophin-silencing double-stranded RNA to nymphs developing on leaves of tomato plantlets with their roots bathing in the silencing solution. Downregulation of dystrophin expression occurred mainly in pupae. Dystrophin silencing induced also the downregulation of the dystrophin-associated protein genes actin and tropomyosin, and disrupted F-actin. Most significantly, the treatment inhibited the emergence of adults from pupae, suggesting that targeting dystrophin may help to restrain whitefly populations. This study demonstrates for the first time the important role of dystrophin in the development of a major insect pest to agriculture.

First Report of Recombinant Potato virus Y Strains Infecting Potato in Jordan.

Potato (Solanum tuberosum L.) is an important vegetable crop in Jordan, occupying second position after olives. In 2012, potatoes were planted on about 6,000 ha with a production of about 141,000 t (2). Potato virus Y (PVY) is a serious problem for potato production worldwide. Recombinant strains of the virus were reported to cause tuber necrotic ringspot disease (PTNRD) in many potato-growing regions of the world. In the last few years, a new recombinant PVYNTN-NW that belongs to PVYZ (3) has been reported in the neighboring Syria. It included three recombination patterns, SYR-I, SYR-II, and SYR-III, and caused severe PTNRD (1). Since PVY is easily transmitted from one region to another by aphid vectors and infected potato seeds, this study was initiated to investigate the possible occurrence of PVY strains in Jordan. In October 2013, 33 leaf samples were collected from symptomatic potato plants cv. Spunta from Wadi Rum, Jordan (GPS coordinates 29°31'37.76″ N, 35°42'48.75″ E), the largest potato-producing area in Jordan. Sampled plants displayed leaf mottling and yellowing, symptoms similar to those caused by PVY. All samples were tested for PVY by DAS-ELISA using the ELISA kit (monoclonal cocktail) developed by BIOREBA (Reinach, Switzerland) to detect all PVY isolates. Twenty-nine samples were found positive for PVY by ELISA. To confirm virus infection, total RNA was extracted from all ELISA-positive samples and used as template in uniplex RT-PCR using strain-specific primers (1). The band pattern of PCR amplicons showed that 12 samples were infected with PVYNTN-NW genotype SYR-III and produced bands of 1,085, 441, and 278 bp. One sample was infected with PVYNTN (A) and produced bands of 1,307, 633, and 441 bp, and one other sample was infected with PVYNTN-NW genotype SYR-II and produced bands of 1,085 and 441 bp. Mixed infection with PVYNTN-NW genotype SYR-III and PVYNTN (B) was also detected in one sample producing bands of 278, 441, 1,085, and 1,307 bp. To confirm infection with the recombinant strains, PCR fragments of 278 bp amplified from three samples and 1,085 bp obtained from another three samples were directly sequenced and sequences were deposited in GenBank under accession numbers KJ159968, KJ159969, and KJ159970 for the 278-bp fragment and KJ159974, KJ159975, and KJ159976 for the 1,085-bp fragment. Sequence comparison with other PVY strains available in the NCBI database showed that the 278-bp fragment had the highest nucleotide sequence identity (100%) with PVY isolates SYR-III-A26 (AB461467) and SYR-III-2-4 (AB461457) from Syria. BLAST searches also showed that the 1,085-bp fragment shared 99% nucleotide identities with PVY isolates SYR-II-L3 (AB461482) and SYR-II-Be4 (AB461474) from Aleppo, Syria. To our knowledge, this is the first report of PVY recombinants in Jordan, and the first report of PVYNTN-NW recombinants infecting potato crop outside Syria. Since Europe is the main supplier of potato seeds for farmers in Jordan and Syria, the introduction of PVYNTN-NW to the region could have happened through infected potato seeds. Results of this study create new challenges for potato growers in Jordan as well as other countries in the region. References: (1) M. Chikh Ali et al. J. Virol. Methods 165:15, 2010. (2) FAO. http://faostat.fao.org/ (3) A. V. Karasev and S. M. Gray. Ann. Rev. Phytopathol. 51:571, 2013.

Sequencing and assembly of a full-length infectious clone of grapevine virus B and its infectivity on herbaceous plants.

Grapevine virus B (GVB) has been found associated with corky bark-diseased vines. Although the sequence of a 7.6-kb cDNA clone from a GVB isolate from Italy has been described, striking differences in sequences between GVB isolates prompted us to construct an additional full-length GVB clone from the isolate 94/971 and to determine its complete sequence. The cDNA of GVB 94/971 shared a nucleotide sequence identity of only 77% with the GVB isolate from Italy. The cDNA of GVB 94/971 was infectious on Nicotiana plants as demonstrated by symptoms and by means of Northern blot, Western blot and electron microscopic analyses.

Making a friend from a foe: expressing a GroEL gene from the whitefly Bemisia tabaci in the phloem of tomato plants confers resistance to tomato yellow leaf curl virus.

Some (perhaps all) plant viruses transmitted in a circulative manner by their insect vectors avoid destruction in the haemolymph by interacting with GroEL homologues, ensuring transmission. We have previously shown that the phloem-limited begomovirus tomato yellow leaf curl virus (TYLCV) interacts in vivo and in vitro with GroEL produced by the whitefly vector Bemisia tabaci. In this study, we have exploited this phenomenon to generate transgenic tomato plants expressing the whitefly GroEL in their phloem. We postulated that following inoculation, TYLCV particles will be trapped by GroEL in the plant phloem, thereby inhibiting virus replication and movement, thereby rendering the plants resistant. A whitefly GroEL gene was cloned in an Agrobacterium vector under the control of an Arabidopsis phloem-specific promoter, which was used to transform two tomato genotypes. During three consecutive generations, plants expressing GroEL exhibited mild or no disease symptoms upon whitefly-mediated inoculation of TYLCV. In vitro assays indicated that the sap of resistant plants contained GroEL-TYLCV complexes. Infected resistant plants served as virus source for whitefly-mediated transmission as effectively as infected non-transgenic tomato. Non-transgenic susceptible tomato plants grafted on resistant GroEL-transgenic scions remained susceptible, although GroEL translocated into the grafted plant and GroEL-TYLCV complexes were detected in the grafted tissues.

Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry.

The nuclear DNA content of the whitefly Bemisia tabaci (Gennnadius) was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. (Brassicaceae) as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex.

Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly Bemisia tabaci.

To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) - a whitefly-transmitted geminivirus - was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll-associated viruses (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.

Whiteflies (Bemisia tabaci) issued from eggs bombarded with infectious DNA clones of Tomato yellow leaf curl virus from Israel (TYLCV) are able to infect tomato plants.

We have reported previously that Tomato yellow leaf curl virus from Israel (TYLCV) penetrates the reproductive system of its vector, the whitefly Bemisia tabaci biotype B, and may be transmitted to progeny. In order to mimic this phenomenon and to understand how TYLCV accompanies the development of the insect, we have bombarded B. tabaci eggs with an infectious DNA clone of TYLCV. After a linear full-length genomic copy of TYLCV DNA was delivered to eggs, the DpnI-sensitive DNA became circular and DpnI resistant. When a dimeric copy of TYLCV DNA was delivered to eggs, the viral DNA was detected in all the whitefly developmental stages. Adult insects that developed from the treated eggs were able to infect tomato test plants with variable frequency. Viral DNA was detected in the progeny of whiteflies that developed from eggs bombarded with TYLCV. Similarly, when insect eggs were bombarded with a dimeric copy of an infectious clone of the genome of Tomato yellow leaf curl virus from Sardinia, Italy (TYLCSV), adults that eclosed from the treated eggs were able to infect tomato test plants.

Digestive, salivary, and reproductive organs of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) B type.

A microscopic analysis of the morphology and ultrastructure of the digestive, salivary, and reproductive systems of adult Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) B type was conducted using light, scanning, and transmission electron microscopy. The internal anatomy of B. tabaci was found to be similar to that reported for Trialeurodes vaporariorum. In a microscopic analysis of the salivary glands, we have shown that each primary salivary gland is composed of at least 13 cells varying in morphology and staining differentially, while the accessory salivary glands are composed of four morphologically similar cells. We analyzed the course of the alimentary canal in B. tabaci, demonstrated the internal morphology of the organs, and clarified the location of the filter chamber relative to other organs in the whitefly. Our observations confirm that the pair of structures extending from the connecting chamber are caeca that may aid in fluid movement through the midgut and are not Malpighian tubules, as previously suggested. We confirm an earlier finding that the whitefly lacks Malpighian tubules, having instead specialized Malpighian-like cells within the filter chamber at the juncture with the internal ileum. Finally, we provide the first scanning electron microscopic analysis showing the reproductive organs of B. tabaci. Our investigation provides clarified terminology for several components of the digestive and excretory system. We also provide drawings and micrographs that will aid future researchers in localizing the internal organs of B. tabaci. We expect our analysis to provide a valuable tool for studying B. tabaci / plant virus interactions and physiological and biological aspects of this insect.

Rate of Tomato yellow leaf curl virus Translocation in the Circulative Transmission Pathway of its Vector, the Whitefly Bemisia tabaci.

ABSTRACT Whiteflies (Bemisia tabaci, biotype B) were able to transmit Tomato yellow leaf curl virus (TYLCV) 8 h after they were caged with infected tomato plants. The spread of TYLCV during this latent period was followed in organs thought to be involved in the translocation of the virus in B. tabaci. After increasing acquisition access periods (AAPs) on infected tomato plants, the stylets, the head, the midgut, a hemolymph sample, and the salivary glands dissected from individual insects were subjected to polymerase chain reaction (PCR) without any treatment; the presence of TYLCV was assessed with virus-specific primers. TYLCV DNA was first detected in the head of B. tabaci after a 10-min AAP. The virus was present in the midgut after 40 min and was first detected in the hemolymph after 90 min. TYLCV was found in the salivary glands 5.5 h after it was first detected in the hemolymph. Subjecting the insect organs to immunocapture-PCR showed that the virus capsid protein was in the insect organs at the same time as the virus genome, suggesting that at least some TYLCV translocates as virions. Although females are more efficient as vectors than males, TYLCV was detected in the salivary glands of males and of females after approximately the same AAP.

The GroEL protein of the whitefly Bemisia tabaci interacts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system.

We have previously suggested that a GroEL homolog produced by the whitefly Bemisia tabaci endosymbiotic bacteria interacts in the insect hemolymph with particles of Tomato yellow leaf curl virus from Israel (TYLCV-Is), ensuring the safe circulative transmission of the virus. We have now addressed the question of whether the nontransmissibility of Abutilon mosaic virus from Israel (AbMV-Is) is related to a lack of association between GroEL and the virus coat protein (CP). Translocation analysis has shown that, whereas TYLCV-Is DNA is conspicuous in the digestive tract, hemolymph, and salivary glands of B. tabaci 8 h after acquisition feeding started, AbMV-Is DNA was detected only in the insect digestive tract, even after 96 h. To determine whether AbMV-Is particles were rapidly degraded in the hemolymph as a result of their inability to interact with GroEL, we have isolated a GroEL gene from B. tabaci and used a yeast two-hybrid assay to compare binding of the CP of TYLCV-Is and AbMV-Is to the insect GroEL. The yeast assay showed that the CPs of the two viruses are able to bind efficiently to GroEL. We therefore suggest that, although GroEL-CP interaction in the hemolymph is a necessary condition for circulative transmission, the nontransmissibility of AbMV-Is is not the result of lack of binding to GroEL in the B. tabaci hemolymph, but most likely results from an inability to cross the gut/hemolymph barrier.

Tomato leaf curl virus from Bangalore (ToLCV-Ban4): sequence comparison with Indian ToLCV isolates, detection in plants and insects, and vector relationships.

Tomato leaf curl virus (ToLCV) is a whitefly (Bemisia tabaci) transmitted geminivirus (family Geminiviridae, genus Begomovirus) causing a destructive disease of tomato in many regions of India, East Asia and Australia. While ToLCV isolates from Australia and Taiwan have a single genomic component (designated DNA-A), those from Northern India have two components (DNA-A and DNA-B). The ToLCV isolates from Southern India (Bangalore) previously cloned seem to have a DNA-A-like monopartite genome. We have used degenerate DNA-A-specific PCR primers to clone the genome of a ToLCV isolate (named ToLCV-Ban4) from field-infected tomato plants growing in Bangalore, India, in 1997. Degenerate DNA-B-specific PCR primers have not allowed to amplify a putative DNA-B from infected tomato, at the time when DNA-B fragments were amplified from plants infected by known bipartite begomoviruses. The full-length 2759 nucleotide-long DNA-A-like viral genome was sequenced. Similarly to other monopartite ToLCV and TYLCV isolates, ToLCV-Ban4 contains six open reading frames, two on the virion strand and four on the complementary strand. Sequence comparisons indicated that ToLCV-Ban4 is similar to the other three isolates from Bangalore previously sequenced, and is closely related to ToLCV-Ban2 (approximately 91% nucleotide sequence identity). Phylogenetic analysis showed that the ToLCV isolates from Bangalore constitute a group of viruses separated from those of Northern India. ToLCV-Ban4 was detected in tomato and in its whitefly vector Bemisia tabaci by one or by a combination of ELISA, Southern blot hybridization and PCR. Parameters of virus acquisition, retention and transmission by the whitefly vector were investigated in the laboratory. Single whiteflies were able to acquire ToLCV-Ban4 from infected tomato and to transmit the virus to tomato test plants, but five insects were necessary to achieve 100% transmission. Minimum acquisition access and inoculation access periods were 10 min and 20 min, respectively. A latent period of 6 h was required for B. tabaci to efficiently infect tomato test plants. Following a 24 h acquisition access period the insect retained its ability to infect tomato test plants for 12 days, but not for its entire life. In one insect/one plant inoculation tests, female whiteflies were more efficient (approximately 95%) than males (approximately 25%) in transmitting the virus.

Tomato yellow leaf curl geminivirus (TYLCV-Is) is transmitted among whiteflies (Bemisia tabaci) in a sex-related manner.

Tomato yellow leaf curl virus (TYLCV) is the name given to a complex of geminiviruses infecting tomato cultures worldwide. TYLCV is transmitted by a single insect species, the whitefly Bemisia tabaci. Herein we show that a TYLCV isolate from Israel (TYLCV-Is) can be transmitted among whiteflies in a sex-dependent manner, in the absence of any other source of virus. TYLCV was transmitted from viruliferous males to females and from viruliferous females to males but not among insects of the same sex. Transmission took place when insects were caged in groups or in couples, in a feeding chamber or on cotton plants, a TYLCV nonhost. The recipient insects were able to efficiently inoculate tomato test plants. Insect-to-insect virus transmission was instrumental in increasing the number of whiteflies capable of infecting tomato test plants in a whitefly population. TYLCV was present in the hemolymph of whiteflies caged with viruliferous insects of the other sex; therefore, the virus follows, at least in part, the circulative pathway associated with acquisition from infected plants. Taken as a whole, these results imply that a plant virus can be sexually transmitted from insect to insect.

A cDNA from tobacco codes for an inhibitor of virus replication (IVR)-like protein.

We have shown previously that localization of tobacco mosaic virus (TMV) in tobacco is associated with a ca. 23 kDa protein that inhibits replication of several plant viruses. This protein, named 'inhibitor of virus replication' (IVR), was purified from the medium of TMV-inoculated protoplasts derived from Nicotiana tabacum cv. Samsun NN. IVR was shown to be present also in induced-resistant leaf tissue of N. tabacum cv. Samsun NN. We prepared an expression cDNA library from such induced-resistant tissue and screened it with a polyclonal antibody raised against the IVR protein. A 1016 bp clone (named NC330) containing a 597 bp open reading frame, coding for a 21.6 kDa polypeptide, was isolated. The NC330 clone hybridized with RNA from induced-resistant tissue from N. tabacum cv. Samsun NN but not with RNA from non-induced tissue. Likewise, it did not hybridize with RNA from infected or uninfected tissue of N. tabacum cv. Samsun nn. Similarly, the NC330 cloned probe hybridized with the RT-PCR products from RNA of the induced-resistant tissue only. In Southern blot hybridization the NC330 DNA probe detected several genomic DNA fragments in both N. tabacum cv. Samsun NN and Samsun nn. The size of the DNA fragments differed in Samsun NN and Samsun nn. We suggest that DNA encoding the IVR-like protein is present in resistant and susceptible N. tabacum genotypes, but is expressed only in NN. We have inserted the NC330 into the expression vector pET22b and a 21.6 kDa protein was produced in Escherichia coli that reacted in immunoblots with the IVR antibody. This protein greatly reduced replication of TMV in N. tabacum cv. Samsun nn leaf disk assays.

A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus.

Evidence for the involvement of a Bemisia tabaci GroEL homologue in the transmission of tomato yellow leaf curl geminivirus (TYLCV) is presented. A approximately 63-kDa protein was identified in B. tabaci whole-body extracts using an antiserum raised against aphid Buchnera GroEL. The GroEL homologue was immunolocalized to a coccoid-shaped whitefly endosymbiont. The 30 N-terminal amino acids of the whitefly GroEL homologue showed 80% homology with that from different aphid species and GroEL from Escherichia coli. Purified GroEL from B. tabaci exhibited ultrastructural similarities to that of the endosymbiont from aphids and E. coli. In vitro ligand assays showed that tomato yellow leaf curl virus (TYLCV) particles displayed a specific affinity for the B. tabaci 63-kDa GroEL homologue. Feeding whiteflies anti-Buchnera GroEL antiserum before the acquisition of virions reduced TYLCV transmission to tomato test plants by >80%. In the haemolymph of these whiteflies, TYLCV DNA was reduced to amounts below the threshold of detection by Southern blot hybridization. Active antibodies were recovered from the insect haemolymph suggesting that by complexing the GoEL homologue, the antibody disturbed interaction with TYLCV, leading to degradation of the virus. We propose that GroEL of B. tabaci protects the virus from destruction during its passage through the haemolymph.

Nuclear import of the capsid protein of tomato yellow leaf curl virus (TYLCV) in plant and insect cells.

The tomato yellow leaf curl virus (TYLCV) found in Israel is a whitefly-transmitted monopartite geminivirus. Although geminiviruses have been found in the nuclei of phloem-associated cells, the mechanism of viral invasion is poorly understood. The possible role of the TYLCV capsid protein (CP), the only known component of the viral coat, in virus transport into the host cell nucleus was investigated by monitoring its specific nuclear accumulation in plant and insect cells. CP was fused to the beta-glucuronidase (GUS) reporter enzyme to assay nuclear import in petunia protoplasts, and micro-injection of purified fluorescently labeled CP was used to examine its nuclear uptake in Drosophila embryos. Both assays demonstrated that TYLCV CP is transported into plant- and insect-cell nuclei by an active process of nuclear import via a nuclear localization signal (NLS)-specific pathway. Using the GUS assay and deletion analysis, the TYLCV CP NLS sequence was identified in the amino-terminus of the protein.

Evidence for transovarial transmission of tomato yellow leaf curl virus by its vector, the whitefly Bemisia tabaci.

The whitefly Bemisia tabaci is the only vector of the tomato yellow leaf curl geminivirus (TYLCV). The insect transmits the virus in a persistent-circulative manner. TYLCV DNA was detected by polymerase chain reaction and by Southern blot hybridization in progeny (eggs, first and second instars, adults) of single viruliferous whiteflies that developed on eggplant or on cotton (two TYLCV nonhost plants). Furthermore, TYLCV DNA was present in the progeny of insects that had acquired the virus through the egg. The adult progeny of the viruliferous insects and their own progeny were able to infect tomato test plants, producing typical disease symptoms. Ovaries and maturing eggs of viruliferous insects contained viral DNA, as did eggs laid by viruliferous insects maintained on an artificial diet Eggs laid by nonviruliferous whiteflies on cotton plants previously caged with viruliferous insects did not acquire viral DNA from the plant. Hence, TYLCV can be transmitted through the egg for at least two generations. In the absence of an available plant host, the whitefly may serve as a reservoir of the virus between growing seasons.

Tomato Breeding Lines Resistant and Tolerant to Tomato Yellow Leaf Curl Virus Issued from Lycopersicon hirsutum.

ABSTRACT Two tomato yellow leaf curl virus (TYLCV)-resistant plants from accessions LA1777 and LA386 of the wild tomato species Lycopersicon hirsutum have been crossed. The resulting resistant F1 plants were crossed with the domesticated tomato L. esculentum, and a series of selfing was performed. At each generation, individuals were selected for resistance (no symptoms and undetectable viral DNA) and tolerance (no symptoms but with detectable viral DNA) following controlled massive and repeated inoculations with viruliferous whiteflies. A stable BC1F4 line (denominated 902) that does not segregate for resistance was obtained. This line does not support virus accumulation, even upon extensive whitefly-mediated inoculation of young seedlings, and does not need protection with nets or insecticides. Another stable BC1F4 line (denominated 908) was tolerant to the virus. Both lines have good horticultural characteristics and bear 80- to 120-g red fruits. Analysis of segregation of susceptibility, tolerance, and resistance during the BC1F1 to BC1F4 crosses indicated that tolerance is controlled by a dominant major gene and resistance by two to three additive recessive genes. The resistant and tolerant lines do not need to be protected by insecticides or nets.

A worldwide survey of tomato yellow leaf curl viruses.

The name tomato yellow leaf curl virus (TYLCV) has been given to several whitefly-transmitted geminiviruses affecting tomato cultures in many tropical and subtropical regions. Hybridization tests with two DNA probes derived from a cloned isolate of TYLCV from Israel (TYLCV-ISR) were used to assess the affinities of viruses in naturally infected tomato plants with yellow leaf curl or leaf curl symptoms from 25 countries. Probe A which included most of the intergenic region was expected to detect only isolates closely related to TYLCV-ISR, especially after high stringency washes. In contrast probe B, which included the full-length genome, was expected to detect a wide range of whitefly-transmitted geminiviruses. Tomato samples from six countries in the Middle East, from Cuba or the Dominican Republic proved to be closely related to TYLCV-ISR and probably were infected by strains of the same virus. Samples from Senegal and Cape Verde Islands were also related to the Middle Eastern virus. Samples from nine other countries in the western Mediterranean area, Africa, or South-East Asia were more distantly related and probably represent one or more additional geminivirus species. Samples from five countries in Africa, Central or South America gave hybridization signals with the full-length viral genome, only after low stringency wash, indicating that these samples were infected by remote viruses. These results were supported by DNA and protein sequence comparison, which indicate that tomato geminiviruses fall into three main clusters representing viruses from 1) the Mediterranean/Middle East/African region, 2) India, the Far East and Australia, and 3) the Americans. Within the first cluster, two sub-clusters of viruses from the western Mediterranean or from the Middle East/Caribbean Islands were distinguished. The incidence of tomato yellow leaf curl diseases has increased considerably between 1990 and 1996.

Long-term association of tomato yellow leaf curl virus with its whitefly vector Bemisia tabaci: effect on the insect transmission capacity, longevity and fecundity.

The association between tomato yellow leaf curl geminivirus (TYLCV, Israeli isolate) and its insect vector, the whitefly Bemisia tabaci, was investigated. Insects that emerged during a 24 h period were caged with TYLCV-infected plants for a 48 h acquisition access period, then with egg-plants--a TYLCV non-host--for the rest of their lives. While TYLCV DNA was associated with the whiteflies during their entire adult life, the amount of capsid protein rapidly decreased and was not detectable in the insect after approximately 12 days of age. The ability of the infected whiteflies to transmit TYLCV to tomato test plants steadily decreased with age but did not disappear completely. Transmission by viruliferous insects decreased from 100% to 10-20% during their adult lifetime, compared with a decrease from 100% to 50% for non-viruliferous insects. The association of TYLCV with adult B. tabaci led to a reduction of 17-23% in their life expectancy compared with insects that had not acquired the virus, and to a 40-50% decrease in the mean number of eggs laid. These results suggest that TYLCV has some features reminiscent of an insect pathogen.