Real-Time PCR Detection of Elsinoë spp. on Citrus

Elsinoë species are the causal agents of scab and spot diseases on many economically important plant species. Diagnosis based on symptomology is often problematic, and traditional methods are not appropriate as Elsinoë spp. are notoriously difficult to isolate and slow growing. Three Elsinoë species that infect Citrus are regulated as quarantine organisms within the European Union. Reliable and fast detection of Elsinoë species on citrus fruit is essential for effective phytosanitary control. In this study, a multiplex real-time PCR was designed for rapid and sensitive detection of Elsinoë species, and validation was focused on citrus fruit. The test was specific to the Elsinoë genus when tested against a range of nontarget pathogens and had the ability to detect all three regulated Elsinoë species on Citrus. The test also proved highly sensitive, with a limit of detection of 12 fg of DNA per reaction. This new test can be used as a tool in the diagnostic process for the rapid and sensitive detection of Elsinoë pathogens on symptomatic plant material. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Development and Validation of a High-Throughput Sequencing Test for Mitogenome and rDNA Assembly and Annotation, and Its Use in Support of Nematode Identification of Regulatory Concern

Nematoda is a diverse phylum, and representatives are found in most habitats, including in and on animals and plants. Nematodes are regarded as the most abundant group in terms of individuals in marine and terrestrial sediments. Plant-parasitic nematodes are globally responsible for an annual yield loss of $125 billion. Reliable species identification is essential to take appropriate phytosanitary measures. The introduction of validated Sanger sequencing of 18S, 28S, and cox1 barcode loci represented a powerful tool in support of nematode identification. However, technical challenges associated with PCR and Sanger sequencing and the need for additional loci for identification hamper the efficient use of sequence data. To overcome these challenges, we developed an automated bioinformatic pipeline for the assembly and annotation of mitochondrial genomes and ribosomal DNAs, and we defined and validated a standardized test protocol including controls for routine diagnostics (i.e., high-throughput sequencing [HTS] test). The HTS test can be performed on single nematode specimens and outperforms the Sanger-based sequencing by producing less ambiguous consensus sequences and by yielding additional sequence data offering additional diagnostic resolution when needed. Compared with Sanger sequencing, the HTS test represents a reduction in hands-on time. The HTS test is regarded as fit for the purpose of the molecular identification of single nematode specimens in support of nematode diagnostics of regulatory concern. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license