Development of a Multiplexed Bead-Based Suspension Array for the Detection and Discrimination of Pospiviroid Plant Pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies

Reproducibility of the Luminex MagPlex-TAG pospiviroid array over ten independent tests.

<p>One sample of <i>Potato spindle tuber viroid</i> (isolate #3077695) was tested in ten independent reactions over several days. Mean natural log (ln) median fluorescence intensity (MFI) values are plotted; error bars show plus or minus (±) one standard deviation. The horizontal bars plotted on each of the bars shows the detection threshold obtained from our kernel density estimation method, for each assay of the array. The small standard deviation of the ln(MFI) values for positively reacting assays in the array (PSTVd, PospUni and PlantIC) demonstrate the high level of precision of the multiplexed bead-based array.</p

Predictions of the number of isolates of each pospiviroid species that will be detected using universal and species-specific assays of the Luminex MagPlex-TAG pospiviroid array.

<p>Predictions of the number of isolates of each pospiviroid species that will be detected using universal and species-specific assays of the Luminex MagPlex-TAG pospiviroid array.</p

Specificity of the Luminex MagPlex-TAG pospiviroid array when screened against sequence-characterized pospiviroid isolates.

<p>Performance of the 11-plex bead-based array when screened against a large panel of single and mixed infections of sequence-characterized pospiviroid isolates, obtained from natural infections of a variety of host plants (with mixed infections simulated). Data for healthy tomato (uninfected control) and blank (no template control) samples are included for reference. Asterisks denote mean natural log (ln) median fluorescence intensity (MFI) values that exceed the threshold for that assay within the multiplexed bead-based array.</p

Details of pospiviroid isolates used for validation of the Luminex MagPlex-TAG pospiviroid array.

<p>Details of pospiviroid isolates used for validation of the Luminex MagPlex-TAG pospiviroid array.</p

Percentages of false positive and false negative results for Luminex MagPlex-TAG pospiviroid array data when analyzed using three different methods for setting thresholds for positivity.

<p>Percentages of false positive and false negative results for Luminex MagPlex-TAG pospiviroid array data when analyzed using three different methods for setting thresholds for positivity.</p

Results of the blind testing of single and mixed infections of pospiviroids in plant samples, showing a comparison of three separate methods for setting thresholds for positivity.

<p>Results of the blind testing of single and mixed infections of pospiviroids in plant samples, showing a comparison of three separate methods for setting thresholds for positivity.</p

Characteristics of the oligonucleotide primers used in this study.

<p>Characteristics of the oligonucleotide primers used in this study.</p

Comparative evolutionary analyses of eight whitefly Bemisia tabaci sensu lato genomes: cryptic species, agricultural pests and plant-virus vectors

The genomes, transcriptomes, and predicted protein-coding sequences are available from Ensembl Metazoa (http://metazoa.ensembl.org) and are included within the references. Raw RNA-Seq datasets generated and/or analyzed during the current study are available from the European Nucleotide Archive database repository (https://www.ebi.ac.uk/ena) under the parent project accessions: PRJEB28507, PRJEB36965, PRJEB35304, PRJEB39408. All data generated during the analyses of these datasets are included in this published article, supplementary information files, and figshare repository (https://doi.org/10.6084/m9.figshare.23666799; https://doi.org/10.6084/m9.figshare.23666832.v4; https://doi.org/10.6084/m9.figshare.23666844).International audienceBackground: The group of > 40 cryptic whitefly species called Bemisia tabaci sensu lato are amongst the world's worst agricultural pests and plant-virus vectors. Outbreaks of B. tabaci s.l. and the associated plant-virus diseases continue to contribute to global food insecurity and social instability, particularly in sub-Saharan Africa and Asia. Published B. tabaci s.l. genomes have limited use for studying African cassava B. tabaci SSA1 species, due to the high genetic divergences between them. Genomic annotations presented here were performed using the 'Ensembl gene annotation system' , to ensure that comparative analyses and conclusions reflect biological differences, as opposed to arising from different methodologies underpinning transcript model identification. Results: We present here six new B. tabaci s.l. genomes from Africa and Asia, and two re-annotated previously published genomes, to provide evolutionary insights into these globally distributed pests. Genome sizes ranged between 616-658 Mb and exhibited some of the highest coverage of transposable elements reported within Arthropoda. Many fewer total protein coding genes (PCG) were recovered compared to the previously published B. tabaci s.l. genomes and structural annotations generated via the uniform methodology strongly supported a repertoire of between 12.8-13.2 × 10 3 PCG. An integrative systematics approach incorporating phylogenomic analysis of nuclear and mitochondrial markers supported a monophyletic Aleyrodidae and the basal positioning of B. tabaci Uganda-1 to the sub-Saharan group of species. Reciprocal cross-mating data and the co-cladogenesis pattern of the primary obligate endosymbiont 'Candidatus Portiera aleyrodidarum' from 11 Bemisia genomes further supported the phylogenetic reconstruction to show that African cassava B. tabaci populations consist of just three biological species. We include comparative analyses of gene families related to detoxification, sugar metabolism, vector competency and evaluate the presence and function of horizontally transferred genes, essential for understanding the evolution and unique biology of constituent B. tabaci. s.l species.Conclusions: These genomic resources have provided new and critical insights into the genetics underlying B. tabaci s.l. biology. They also provide a rich foundation for post-genomic research, including the selection of candidate gene-targets for innovative whitefly and virus-control strategies

Development of a multiplexed bead-based suspension array for the detection and discrimination of Pospiviroid plant pathogens

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies