First Report of Tomato yellow leaf curl virus Infecting Tomatoes in Trinidad.

Tomato is one of the most important agricultural crops in Trinidad and Tobago, with an annual fruit production of ∼1,500 tonnes (http://faostat.fao.org). Over the past two years (2014 to 2016), several farmers’ fields planted with tomato in Trinidad were showing noticeable symptoms, such as severe stunting, upward curling of leaves, and reduction of leaf size. The surveys revealed up to 85% incidence with the above disease symptoms in several farmers’ fields. Symptoms were typical of those caused by begomoviruses (Akbar Behjatnia et al. 1998). The leaf samples from 100 symptomatic plants were collected from different farmers’ fields located at Maloney, Valencia, Tabaquite, Tortuga, and Gasparillo in Trinidad. DNA was isolated from all the samples using a commercial kit (Promega) and PCR was carried out with begomovirus specific primers such as PAL1v1978/PAR1c496 and PTYCPv369/PTYCPc1023 targeting a partial region of DNA-A and PBL1v2040/PCRc1 targeting a partial region of DNA-B (Navot et al. 1991; Rojas et al. 1993). Strong amplification was observed in 85% of the samples (85 of 100 samples) using a Tomato yellow leaf curl virus (TYLCV) specific PCR with PTYCPv369/PTYCPc1023 primers targeting a partial coat protein (∼654 bp) region. Ten TYLCV-specific PCR amplicons were cloned in pGEM-T vector and sequenced by Sanger sequencing method (Macrogen). BLAST analysis of all the nucleotide sequences shared 99.0 to 99.2% identity with the partial V1 gene of a TYLCV-Israel isolate (FM163455). Further, full length viral genomes of Trinidad isolates from 10 positive samples were amplified with MA13/MA26 primers targeting 1,292 bp and MA17/MA27 primers targeting 1,835 bp of circular DNA-A of TYLCV (Boukhatem et al. 2008). All the amplicons were sequenced and the complete genome (∼2,752 bp) of the Trinidad isolates was obtained. All the sequences of Trinidad isolates were submitted to GenBank under accession nos. KU981040 to 49 and K224405 to 10. The phylogenetic relationship of Trinidad isolates were compared with 47 complete genome isolates reported from 22 countries and a neighbor-joining tree was constructed with 1,000 bootstrap replications using MEGA 6.06 software. Trinidad isolates showed 98.6 to 98.7% nucleotide identity with isolate Grenada:Paradise (FR851298) from Grenada. These results suggest that TYLCV may have been introduced into Trinidad through imported seed and by the movement of whiteflies and that the virus may have originated from Grenada since both countries are in close proximity. The lowest nucleotide identity of 78.8 to 78.9% was with isolate Jeddah-Chor-1 (KT355023) from Saudi Arabia. TYLCV has been reported to be spread through whiteflies (Bemisia tabaci) (Gorovits et al. 2013). To confirm the spread in Trinidad, virus free whiteflies were transferred to TYLCV infected tomato plants in a greenhouse for 48 h for an acquisition access period. Ten replicate healthy tomato seedlings each had five viruliferous whiteflies transferred onto them. The plants were left for a 48 h inoculation access period in a netted greenhouse. After 14 days, the initiation of upward leaf curling was noticed in seven of the 10 seedlings. DNA was extracted from young leaf samples of all the seedlings and PCR was performed with the TYLCV coat protein specific primers (PTYCPv369/PTYCPc1023). TYLCV PCR amplicons were obtained from the seven symptomatic seedlings. TYLCV has not been reported previously in Trinidad and this study (under ACP-EU project), confirms presence of TYLCV and suggests the spread of this virus in Trinidad tomatoes via imported seed and by whiteflies. © 2016, American Phytopathological Society. All rights reserved.