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 × 10 9 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 × 10 4 and 7.47 × 10 6 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.

RNA silencing of South African cassava mosaic virus in transgenic cassava expressing AC1/AC4 hp- RNA induces tolerance

“…Interestingly, at 65 DPI while both wild-type TME3 plants and O13-8 displayed signs of recovery, the viral load in line O13-8 was lower than CMD-tolerant TME3. Although decreased symptom severity correlated with a decrease in viral load in transgenic lines O12-2, O13-5, and O13-8, as shown in other studies [80], there are several reports where no correlation between viral load and symptom severity was found [81]. The most promising SACMV-transgenic cv.60444 line O13-8 warrants further investigation to unravel contributing factors to its tolerance to SACMV.…”

Spatial Virome Analysis of Zanthoxylum armatum Trees Affected With the Flower Yellowing Disease

“…In fact, observation of GSPNeV and satGSPNeV with high read proportions of the total reads sequenced from RNAs of symptomatic samples suggested their intense replication in the host. It is not difficult to imagine that they will compete with hosts for nutrient resources and may interfere with host normal physiological activity and cause visible symptoms ( Ammara et al, 2017 ). Higher viral titers may also help the biological vectors perform more efficient viral acquisition and transmission ( Gray et al, 1991 ), but not necessarily ( Linak et al, 2020 ).…”

Identifications and Validations of Reference Genes for Gene Expression Data Normalization of Chenopodium album

“…EF1α gene has also been validated as a stable internal control in Brachypodium grown under various stressed conditions or treated with different growth hormones (Hong et al, 2010).Recently, EF1 α4 hasbeen found to be a stable internal control gene for the expression of the abioticstressed in G. macrophylla (He et al, 2016). Additionally EF1 α has also been used as an internal control for the rapid detection and quantification of the virusintomato yellow leaf curl disease-OM (TYLCD-OM) (Ammara et al, 2017).…”