Muhammad Zeidan

Tomato yellow leaf curl virus: A whitefly-transmitted geminivirus with a single genomic component

The genome of the tomato yellow leaf curl virus (TYLCV), a Bemisia tabaci-transmitted geminivirus, was cloned. All clones obtained were of one genomic molecule, analogous to DNA A of African cassava mosaic virus. Nucleotide sequence analysis of the TYLCV genome showed that it comprises 2787 nucleotides, encoding six open reading frames, two on the virion strand and four on the complementary strand. All of them have counterparts in other geminiviruses. Dimeric copies of the cloned viral genome were introduced into tomato plants by agroinoculation. Severe yellow leaf curl disease symptoms developed in all of them. Effective whitefly-mediated transmission of the virus from agroinoculated plants to test plants demonstrated that the cloned molecule carries all the information needed for virus replication, systemic infection, and transfer by whiteflies. Restriction and hybridization analyses of viral DNA forms in infected plants and viruliferous whiteflies did not support the presupposed existence of a second genomic component. This is the first report of a whitefly-transmitted geminivirus that possesses a single genomic molecule.

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

Summary. 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 (∼95%) than males (∼25%) in transmitting the virus.

Acquisition of tomato yellow leaf curl virus by the whitefly Bemisia tabaci

Tomato yellow leaf curl virus (TYLCV) genomic DNA can be detected by Southern blot analysis in nucleic acid extracted from a single whitefly. Acquisition of TYLCV by individual whiteflies in relation to the length of the access period, the virus concentration in, and the developmental stage of plant tissues was studied. The frequency of TYLCV detection increased with the length of the access-period; DNA was detected in 15% of whiteflies tested after a period of access to infected tissue of 30 min, regardless of whether it had a high or a low virus content (5 ng or 0.05 ng TYLCV DNA/micrograms plant chromosomal DNA), and in all insects tested after an 8 h period of access to all the plants. Those insects which had access to the youngest leaves of source plants, which have a high virus content, acquired detectable TYLCV DNA within 2 h. Insects which had access to a tissue for the same period acquired variable amounts of TYLCV DNA; insects feeding on plants with a low virus concentration acquired amounts of viral DNA comparable to those acquired by insects feeding on plants containing a 100-fold greater concentration of virus. Viruliferous insects retained TYLCV DNA for at least 13 days when placed on uninfected tomato plants. In these tests, whitefly could not acquire more than 600 million virus genomes (1 ng viral DNA), suggesting the existence of factors controlling the number of virions present in an insect.

Replication of Tomato yellow leaf curl virus in its whitefly vector Bemisia tabaci

ABSTRACT Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted exclusively by the whitefly Bemisia tabaci in a persistent, circulative manner. Replication of TYLCV in its vector remains controversial, and thus far, the virus has been considered to be nonpropagative. Following 8 h of acquisition on TYLCV-infected tomato plants or purified virions and then transfer to non-TYLCV-host cotton plants, the amounts of virus inside whitefly adults significantly increased (>2-fold) during the first few days and then continuously decreased, as measured by the amounts of genes on both virus DNA strands. Reported alterations in insect immune and defense responses upon virus retention led us to hypothesize a role for the immune response in suppressing virus replication. After virus acquisition, stress conditions were imposed on whiteflies, and the levels of three viral gene sequences were measured over time. When whiteflies were exposed to TYLCV and treatment with two different pesticides, the virus levels continuously increased. Upon exposure to heat stress, the virus levels gradually decreased, without any initial accumulation. Switching of whiteflies between pesticide, heat stress, and control treatments caused fluctuating increases and decreases in virus levels. Fluorescence in situ hybridization analysis confirmed these results and showed virus signals inside midgut epithelial cell nuclei. Combining the pesticide and heat treatments with virus acquisition had significant effects on fecundity. Altogether, our results demonstrate for the first time that a single-stranded DNA plant virus can replicate in its hemipteran vector. IMPORTANCE Plant viruses in agricultural crops are of great concern worldwide. Many of them are transmitted from infected to healthy plants by insects. Persistently transmitted viruses often have a complex association with their vectors; however, most are believed not to replicate within these vectors. Such replication is important, as it contributes to the viruss spread and can impact vector biology. Tomato yellow leaf curl virus (TYLCV) is a devastating begomovirus that infects tomatoes. It is persistently transmitted by the whitefly Bemisia tabaci but is believed not to replicate in the insect. To demonstrate that TYLCV is, in fact, propagative (i.e., it replicates in its insect host), we hypothesized that insect defenses play a role in suppressing virus replication. We thus exposed whitefly to pesticide and heat stress conditions to manipulate its physiology, and we showed that under such conditions, the virus is able to replicate and significantly influence the insects fecundity.

Diagnosis and Control of Cereal Viruses in the Middle East

Middle Eastern countries are major consumers of small grain cereals. Egypt is the biggest bread wheat producer with 7.4 million tons (MT) in 2007, but at the same time, it had to import 5.9 MT. Jordan and Israel import almost all the grains they consume. Viruses are the major pathogens that impair grain production in the Middle East, infecting in some years more than 80% of the crop. They are transmitted in nonpersistent, semipersistent, and persistent manners by insects (aphids, leafhoppers, and mites), and through soil and seeds. Hence, cereal viruses have to be controlled, not only in the field but also through the collaborative efforts of the plant quarantine services inland and at the borders, involving all the Middle Eastern countries. Diagnosis of cereal viruses may include symptom observation, immunological technologies such as ELISA using polyclonal and monoclonal antibodies raised against virus coat protein expressed in bacteria, and molecular techniques such as PCR, microarrays, and deep sequencing. In this chapter, we explore the different diagnoses, typing, and detection techniques of cereal viruses available to the Middle Eastern countries. We highlight the plant quarantine service and the prevention methods. Finally, we review the breeding efforts for virus resistance, based on conventional selection and genetic engineering.