SODplex, a Series of Hierarchical Multiplexed Real-Time PCR Assays for the Detection and Lineage Identification of Phytophthora ramorum, the Causal Agent of Sudden Oak Death and Sudden Larch Death

Since its emergence in the 1990s, the invasive pathogen Phytophthora ramorum has spread in Europe and the west coast of North America, causing sudden oak death in the United States and sudden larch death in the United Kingdom, resulting in the mortality or destruction of millions of trees. Due to its invasive nature, its damage potential, its wide host range, and its ability to disseminate via the plant trade, P. ramorum has been placed on quarantine lists worldwide. Rapid and reliable detection of the pathogen and identification of its lineages are crucial to limit spread and inform mitigation and eradication efforts. SODplex, a suite of new multiplex real-time PCR tools, was developed to streamline the detection and identification of P. ramorum. It offers four multiplexed assays covering different use cases. SODplex-base combines primers and probes for the sensitive and accurate detection of Phytophthora spp. and P. ramorum. SODplex-ITS and SODplex-mito offer a single-step identification of P. ramorum and the EU1, NA1, and NA2 lineages present in the United States and Canada. SODplex-lin targets each of the four P. ramorum lineages present in Europe and North America in a single reaction. The assays have high levels of accuracy and are robust to the use of different instruments, different operators, and different temperatures. The redundancy within the assays reduces the likelihood of false negatives and false positives. The SODplex assays presented here improve the toolbox available for the detection of P. ramorum and its lineages. [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

Genome-Enhanced Detection and Identification (GEDI) of plant pathogens

Plant diseases caused by fungi and Oomycetes represent worldwide threats to crops and forest ecosystems. Effective prevention and appropriate management of emerging diseases rely on rapid detection and identification of the causal pathogens. The increase in genomic resources makes it possible to generate novel genome-enhanced DNA detection assays that can exploit whole genomes to discover candidate genes for pathogen detection. A pipeline was developed to identify genome regions that discriminate taxa or groups of taxa and can be converted into PCR assays. The modular pipeline is comprised of four components: (1) selection and genome sequencing of phylogenetically related taxa, (2) identification of clusters of orthologous genes, (3) elimination of false positives by filtering, and (4) assay design. This pipeline was applied to some of the most important plant pathogens across three broad taxonomic groups: Phytophthoras (Stramenopiles, Oomycota), Dothideomycetes (Fungi, Ascomycota) and Pucciniales (Fungi, Basidiomycota). Comparison of 73 fungal and Oomycete genomes led the discovery of 5,939 gene clusters that were unique to the targeted taxa and an additional 535 that were common at higher taxonomic levels. Approximately 28% of the 299 tested were converted into qPCR assays that met our set of specificity criteria. This work demonstrates that a genome-wide approach can efficiently identify multiple taxon-specific genome regions that can be converted into highly specific PCR assays. The possibility to easily obtain multiple alternative regions to design highly specific qPCR assays should be of great help in tackling challenging cases for which higher taxon-resolution is needed

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulosedegrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and fromprokaryotes. Acquisition andmaintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

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Genome-Enhanced Detection and Identification (GEDI) of plant pathogens

Abstract Plant diseases caused by fungi and Oomycetes represent worldwide threats to crops and forest ecosystems. Effective prevention and appropriate management of emerging diseases rely on rapid detection and identification of the causal pathogens. The increase in genomic resources makes it possible to generate novel genome-enhanced DNA detection assays that can exploit whole genomes to discover candidate genes for pathogen detection. A pipeline was developed to identify genome regions that discriminate taxa or groups of taxa and can be converted into PCR assays. The modular pipeline is comprised of four components: (1) selection and genome sequencing of phylogenetically related taxa, (2) identification of clusters of orthologous genes, (3) elimination of false positives by filtering, and (4) assay design. This pipeline was applied to some of the most important plant pathogens across three broad taxonomic groups: Phytophthoras (Stramenopiles, Oomycota), Dothideomycetes (Fungi, Ascomycota) and Pucciniales (Fungi, Basidiomycota). Comparison of 73 fungal and Oomycete genomes led the discovery of 5,939 gene clusters that were unique to the targeted taxa and an additional 535 that were common at higher taxonomic levels. Approximately 28% of the 299 tested were converted into qPCR assays that met our set of specificity criteria. This work demonstrates that a genome-wide approach can efficiently identify multiple taxon-specific genome regions that can be converted into highly specific PCR assays. The possibility to easily obtain multiple alternative regions to design highly specific qPCR assays should be of great help in tackling challenging cases for which higher taxon-resolution is needed