Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs.

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control.

An Efficient Triplex TaqMan Quantitative PCR to Detect a Blackleg-Causing Lineage of in Potato Based on a Pangenome Analysis.

is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it.

Unravelling hybridization in Phytophthora using phylogenomics and genome size estimation.

The genus Phytophthora comprises many economically and ecologically important plant pathogens. Hybrid species have previously been identified in at least six of the 12 phylogenetic clades. These hybrids can potentially infect a wider host range and display enhanced vigour compared to their progenitors.

The Pectobacterium pangenome, with a focus on Pectobacterium brasiliense, shows a robust core and extensive exchange of genes from a shared gene pool.

Background: Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus.

Application of Real-Time PCR for the Detection and Quantification of Oomycetes in Ornamental Nursery Stock.

Numerous and disease outbreaks have occurred in Europe following inadvertent introduction of contaminated ornamental plants. Detection and identification of pathogens are crucial to reduce risks and improve plant biosecurity in Europe and globally. Oomycete diversity present in roots and compost was determined in 99 hardy woody plants bought from nurseries, retailers and internet sellers, using both isolations and molecular analyses.

Comparative genomics of chytrid fungi reveal insights into the obligate biotrophic and pathogenic lifestyle of Synchytrium endobioticum.

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported.

Development and Evaluation of a Triplex TaqMan Assay and Next-Generation Sequence Analysis for Improved Detection of Xylella in Plant Material.

Xylella fastidiosa is a heterogenous gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. Since 2013, in Europe, the presence of the pathogen is increasing in a part of the Mediterranean area, but it causes in particular severe disease problems in olive orchards in the Southern part of Italy. Various subspecies of the pathogen were also diagnosed in natural outbreaks and intercepted ornamental plants in Europe, among them Olea europaea, Coffea arabica, and Nerium oleander.

Genetic relationships in an international collection of Puccinia horiana isolates based on newly identified molecular markers and demonstration of recombination.

The obligate biotrophic pathogen Puccinia horiana is the causal agent of chrysanthemum white rust. Although P. horiana is a quarantine organism, it has been able to spread to most chrysanthemum-producing regions in the world since the 1960s; however, the transfer routes are largely obscure.

Pyrosequencing as a tool for the detection of Phytophthora species: error rate and risk of false Molecular Operational Taxonomic Units.

Aims: To evaluate the accuracy of pyrosequencing for the description of Phytophthora communities in terms of taxa identification and risk of assignment for false Molecular Operational Taxonomic Units (MOTUs).

Methods And Results: Pyrosequencing of Internal Transcribed Spacer 1 (ITS1) amplicons was used to describe the structure of a DNA mixture comprising eight Phytophthora spp. and Pythium vexans.

A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes.

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp.

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer.

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak.

Clonal expansion of the Belgian Phytophthora ramorum populations based on new microsatellite markers.

Co-existence of both mating types A1 and A2 within the EU1 lineage of Phytophthora ramorum has only been observed in Belgium, which begs the question whether sexual reproduction is occurring. A collection of 411 Belgian P. ramorum isolates was established during a 7-year survey.

Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum.

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P.

Robust detection and identification of multiple oomycetes and fungi in environmental samples by using a novel cleavable padlock probe-based ligation detection assay.

Simultaneous detection and identification of multiple pathogenic microorganisms in complex environmental samples are required in numerous diagnostic fields. Here, we describe the development of a novel, background-free ligation detection (LD) system using a single compound detector probe per target. The detector probes used, referred to as padlock probes (PLPs), are long oligonucleotides containing asymmetric target complementary regions at both their 5' and 3' ends which confer extremely specific target detection.

Natural Hybrids of Phytophthora nicotianae and Phytophthora cactorum Demonstrated by Isozyme Analysis and Random Amplified Polymorphic DNA.

ABSTRACT Three similar isolates of Phytophthora (Phytophthora sp-h) were obtained from diseased Spathiphyllum and Primula plants. Cultural characteristics did not fit any known description of Phytophthora species. The Phytophthora sp-h isolates are papillate, are homothallic, possess 80 to 86% amphigynous antheridia, and have a maximum temperature for growth of 36.

Gene Genealogies and AFLP Analyses in the Fusarium oxysporum Complex Identify Monophyletic and Nonmonophyletic Formae Speciales Causing Wilt and Rot Disease.

ABSTRACT The monophyletic origin of host-specific taxa in the plant-pathogenic Fusarium oxysporum complex was tested by constructing nuclear and mitochondrial gene genealogies and amplified fragment length polymorphism (AFLP)-based phylogenies for 89 strains representing the known genetic and pathogenic diversity in 8 formae speciales associated with wilt diseases and root and bulb rot. We included strains from clonal lineages of F. oxysporum f.

Molecular Characterization of Natural Hybrids of Phytophthora nicotianae and P. cactorum.

ABSTRACT Hybrid isolates of Phytophthora nicotianae x P. cactorum from five different hosts (Cyclamen, Lavandula, Lewisia, Primula, and Spathiphyllum spp.) were identified by their atypical morphology and their well-defined heterozygous isozyme patterns.

Diagnostic Values and Utility of Immunological, Morphological, and Molecular Methods for In Planta Detection of Phytophthora ramorum.

ABSTRACT In this study, six methods for the detection of Phytophthora ramorum in planta were compared using naturally infested rhododendron plant material. The methods included two immunological methods, one an enzyme-linked immunosorbent assay (ELISA) and the other using a lateral flow format (LFD). Three molecular tests based on the polymerase chain reaction (PCR) using TaqMan chemistry also were assessed, including two assays designed for specific detection of P.

A Rapid Diagnostic Test to Distinguish Between American and European Populations of Phytophthora ramorum.

ABSTRACT A new devastating disease in the United States, commonly known as Sudden Oak Death, is caused by Phytophthora ramorum. This pathogen, which previously was described attacking species of Rhododendron and Viburnum in Germany and the Netherlands, has established itself in forests on the central coast of California and is killing scores of native oak trees (Lithocarpus densiflora, Quercus agrifolia, Q. kelloggii, and Q.

Nonpathogenic Isolates of the Citrus Black Spot Fungus, Guignardia citricarpa, Identified as a Cosmopolitan Endophyte of Woody Plants, G. mangiferae (Phyllosticta capitalensis).

ABSTRACT The population structure of Guignardia citricarpa sensu lato (anamorph: Phyllosticta citricarpa), a fungus of which strains pathogenic to citrus are subject to phytosanitary legislation in the European Union and the United States, was investigated. Internal transcribed spacer sequences revealed two phylogenetically distinct groups in G. citricarpa.

Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays.

Background: Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens.

Use of hybridization melting kinetics for detecting Phytophthora species using three-dimensional microarrays: demonstration of a novel concept for the differentiation of detection targets.

Microarray-based detection is limited by variable and inconsistent hybridization intensities across the diversity of probes used in each array. In this paper, we introduce a novel concept for the differentiation of detection targets using duplex melting kinetics. A microarray assay was developed on a PamChip microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes.

Microsatellite markers identify three lineages of Phytophthora ramorum in US nurseries, yet single lineages in US forest and European nursery populations.

Analysis of 12 polymorphic simple sequence repeats identified in the genome sequence of Phytophthora ramorum, causal agent of 'sudden oak death', revealed genotypic diversity to be significantly higher in nurseries (91% of total) than in forests (18% of total). Our analysis identified only two closely related genotypes in US forests, while the genetic structure of populations from European nurseries was of intermediate complexity, including multiple, closely related genotypes. Multilocus analysis determined populations in US forests reproduce clonally and are likely descendants of a single introduced individual.

Diagnostic application of padlock probes--multiplex detection of plant pathogens using universal microarrays.

Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection.