Tomato Yellow Leaf Curl Virus DNA Research Articles

Pcr-amplification of tomato yellow leaf curl virus (TYLCV) DNA from squashes of plants and whitefly vectors: Application to the study of TYLCV acquisition and transmission

DNA of tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci, was amplified from squashes of infected tomato plants and of viruliferous vectors using the polymerase chain reaction (PCR). Samples of infected tissues as small as 1 mm2 were squashed onto a nylon membrane. A 1 × 2 mm strip containing the squash was introduced into a 25 µl PCR reaction mix. The reaction products were subjected to gel electrophoresis, blotted and hybridized with a radiolabeled virus-specific DNA probe. TYLCV DNA was amplified from squashes of leaves, roots, and stem of infected tomato and from individual viruliferous whiteflies. The same squash could be used several times to amplify different virus DNA fragments with various sets of primers. Thus plant and insect squashes can be used as templates for the amplification of geminiviral DNA with no need to prepare tissue extracts or purify nucleic acids. The squash-PCR procedure was applied to study whitefly transmission of TYLCV. Tomato plants were inoculated by placing a single viruliferous insect in the center of a young leaflet. In some plants TYLCV DNA was detected at the site of inoculation as early as 5 min after the beginning of the access feeding and in all plants after 30 min. The squash-PCR procedure also was applied to the study of TYLCV acquisition by the insect vector. TYLCV DNA was detected in the head of whiteflies as early as 5 min after the beginning of the access feeding on infected tomato plants. Viral DNA was detected in the thorax after 10 min and in the abdomen after 25 min.

Tomato yellow leaf curl virus DNA in callus cultures derived from infected tomato leaves

Callus cultures were induced from leaves of a tomato plant infected with tomato yellow leaf curl virus (TYLCV) and analyzed for viral DNA presence during successive subcultures. No TYLCV DNA was detected in calli sampled after eight months of culture. Considerable differences in the presence of TYLCV DNA were found within sectors of a callus culture and between different callus cultures, throughout the entire eight months period. Infected calli which were cultured at sub-optimal temperature (15°C) retained the viral DNA longer than at 25 °C. The results suggested that TYLCV disappearance during callus culture was due to a disruption of some of the cell-to-cell connections, resulting in islands of infected cells in the midst of uninfected tissue and/or to the competition between the rate of cell division and that of viral DNA replication.

Accumulation and Translocation of Tomato Yellow Leaf Curl Virus (TYLCV) in aLycopersicon esculentumBreeding Line Containing theL. chilenseTYLCV Tolerance GeneTy-1

The major tomato yellow leaf curl virus (TYLCV) tolerance gene, Ty-1, of the wild tomato species Lycopersicon chilense was mapped to chromosome 6 by using restriction fragment length polymorphism (RFLP) markers and introgressed into the domesticated tomato L. esculentum. Two nearly isogenic breeding lines were obtained. In infected fields, plants from line 32, which contained the Ty-1 allele, remained symptomless (tolerant line); plants from line 30, which did not contain the Ty-1 allele, were all symptomatic (susceptible line). The effect of the Ty-1 gene on TYLCV DNA accumulation and translocation was investigated by hybridizing plant DNA extracts with a viral DNA probe

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes.

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid of Agrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2-5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation, L. chilense LA1969 and L. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.

Use of the Polymerase Chain Reaction to Amplify Tomato Yellow Leaf Curl Virus DNA from Infected Plants and Viruliferous Whiteflies

The genomic DNA molecule of an Israeli isolate of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected plants (Lycopersicon esculentum 'M82') by the use of the polymerase chain reaction (PCR). Synthetic oligonucleotides complementary to different regions of the viral genome were used in different combinations to amplify the full-length as well as subgenomic fragments of the TYLCV DNA molecule (...)

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.