Dataset Information

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

ABSTRACT: Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

SUBMITTER: Botermans M

PROVIDER: S-EPMC7514017 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Botermans Marleen M Roenhorst Johanna W JW Hooftman Marinus M Verhoeven Jacobus Th J JTJ Metz Eveline E van Veen Esther J EJ Geraats Bart P J BPJ Kemper Mark M Beugelsdijk Debora C M DCM Koenraadt Harrie H Jodlowska Agata A Westenberg Marcel M

PloS one 20200924 9

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicu ...[more]

PMID: 32970706

Similar Datasets

Project description:BackgroundThe elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR.ResultsIn this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (CAC), SGN-U321250 (TIP41), SGN-U346908 ("Expressed") and SGN-U316474 (SAND) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process.ConclusionThis work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability.

Project description:Imports of seeds for sowing are a major pathway for the introduction of contaminant seeds, and many agricultural weeds globally naturalised originally have entered through this pathway. Effective management of this pathway is a significant means of reducing future plant introductions and helps minimise agricultural losses. Using a national border inspection database, we examined the frequency, origin and identity of contaminant seeds within seed for sowing shipments entering New Zealand between 2014-2018. Our analysis looked at 41,610 seed lots across 1,420 crop seed species from over 90 countries. Overall, contamination was rare, occurring in 1.9% of all seed lots. Among the different crop types, the arable category had the lowest percentage of seed lots contaminated (0.5%) and the forage category had the highest (12.6%). Crop seeds Capsicum, Phaseolus and Solanum had the lowest contamination rates (0.0%). Forage crops Medicago (27.3%) and Trifolium (19.8%) had the highest contamination rates. Out of 191 genera recorded as contaminants, Chenopodium was the most common. Regulated quarantine weeds were the rarest contaminant type, only occurring in 0.06% of seed lots. Sorghum halepense was the most common quarantine species and was only found in vegetable seed lots. Vegetable crop seed lots accounted for approximately half of all quarantine species detections, Raphanus sativus being the most contaminated vegetable crop. Larger seed lots were significantly more contaminated and more likely to contain a quarantine species than smaller seed lots. These findings support International Seed Testing Association rules on maximum seed lot weights. Low contamination rates suggest industry practices are effective in minimising contaminant seeds. Considering New Zealand inspects every imported seed lot, utilises a working sample size 5 times larger than International Seed Testing Association rules require, trades crop seed with approximately half of the world's countries and imports thousands of crop seed species, our study provides a unique overview of contaminant seeds that move throughout the seed for sowing system.

Dataset Information

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Publications

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets