Tomato yellow leaf curl virus (TYLCV) is an economically important virus with tomato (Solanum lycopersicum L.) as its main host. The virus is widely distributed in subtropical areas and is transmitted by the tobacco whitefly (Bemisia tabaci) in a persistent manner. TYLCV has a quarantine status (IIAII) in the European Union (EU directive 2000/29/EC). It was not previously recorded in the Netherlands. In September 2007, symptoms were observed in tomato crops in a few greenhouses located in close proximity from each other in the western part of the Netherlands. Infected plants showed TYLCV-like symptoms, i.e., stunting, leaf curl, and marginal and interveinal chlorosis. Similar symptoms were evoked after grafting symptomatic tips onto healthy tomato seedlings, whereas no viruses were transmitted by mechanical inoculation to herbaceous test plants. Extracted DNA from symptomatic leaves was used in PCR with two sets of primers for universal detection of begomoviruses (1,2). Analysis of the overlapping amplified products revealed the highest identity to isolate TYLCV-Alm (GenBank Accession No. AJ489258) from Almeria, Spain. To amplify the remaining 60% of the virus genome, three additional primer sets were designed: TYLCV965F 5'-GGCAGCCAAGTACGAGAACC-3' and TYLCV1736R 5'-CCACTATCTTCCTCTGCAATCC-3'; TYLCV1598F 5'-TACTTGCGAACAGTGGCTCG-3' and TYLCV2282R 5'-TCCAAATCGATGGCAGATCAG-3'; TYLCV2229F 5'-ATGCGTCGTTGGCAGATTG-3' and TYLCV68R 5'-CAGTGACGTCTGTGGAACCCT-3'. Analysis of the five overlapping PCR products of one isolate revealed a total virus genome of 2,781 nucleotides. The complete sequence of the Netherlands Isolate (GenBank Accession No. FJ439569) showed 99.3% nucleotide identity to isolate TYLCV-Alm (AJ489258), and therefore, the virus was identified as TYLCV-Alm. After the initial identification, a survey was conducted in all tomato crops in a surrounding area of approximately 40 km2. TYLCV was found in 19 of 27 cultivations. The identity of one isolate per cultivation was confirmed by sequence analysis of the products obtained with the Wyatt and Brown primers (2) occasionally in combination with the Deng primers (with 99.1 to 100% and 99.2 to 100% nucleotide identity to the Netherlands isolate [FJ439569], respectively) (1). As many as 25 symptomatic plants were recorded per greenhouse. A subsequent survey of 34 randomly selected tomato growers in other areas of the country revealed no further infections. Results of the sequence analyses and surveys suggested that the outbreak resulted from a single introduction of the virus, whereas the insect vector B. tabaci accounted for local spread. Measures taken to eliminate the virus included the removal and subsequent destruction of infected tomato plants as well as eradication of B. tabaci. No TYLCV infections were found during surveys in 2008, and therefore, it is believed that the virus was eradicated effectively.
- polymerase chain-reaction
- degenerate primers