Real-time qPCR assay for the TYLCV titer in relation to symptoms-based disease severity scales

Abstract

During the last few decades, Tomato yellow leaf curl disease (TYLCD) has resulted in heavy yield losses and increased incidences in tropical and subtropical regions. In Oman, six different species of begomoviruses were found to be associated with TYLCD. However, Tomato yellow leaf curl virus- Oman (TYLCV-OM) was found to be the most widely distributed species in the whole country. A real-time quantitative PCR (qPCR) assay with an internal control [tomato elongation factor 1(EF1)] was developed for TYLCV-OM, based on SYBRGreen chemistry for the rapid detection and quantification of the virus. This assay was carried out on field samples showing symptoms according to The World Vegetable Center (AVRDC) disease severity scales. The test was used to establish a relationship between virus load and phenotypic symptoms. Viral copies of 2.88 × 109 were detected in field infected tomato plants showing symptom severity according to AVRDC scale '3' (i.e. severe leaf curling and yellowing of leaves). The copy number of virus decreased with the relative decrease in symptom severity scale. Tomato plants exhibiting scale '1' and '2' have 7.7 × 104 and 7.47 × 106 viral copies, respectively. Very low copy number of the virus (564) was detected in tomato plants showing symptoms at a severity scale of '0', which were apparently symptomless. However, tomato plants developed by tissue culture in sterilized conditions, which were used as a negative control, did not show the presence of the virus. The developed qPCR assay could detect as low as 18 fg (femto gram) of virus from total nucleic acid equivalent to approximately 30 genomic units. This quantitative assay can be used to determine virus titer, in breeding programs for development of virus resistant plants and for epidemiological surveys to monitor viral populations and their intensities.