Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites

Summary Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on variable number of tandem repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools, and VNTR markers were developed and validated. A Multiple-Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region

Erwinia amylovora Novel Plasmid pEI70: Complete Sequence, Biogeography, and Role in Aggressiveness in the Fire Blight Phytopathogen

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5–92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora

Minimum performance parameters to select tests for validation and selection of laboratories for TPS

The aim of deliverable 1.1. is to prepare criteria to select tests for validation and to select laboratories for TPS (test performance study). Criteria for selection of tests for the TPS for each pest have been set (see Tables 7-12). These criteria have been divided in five groups: 1) validation data, 2) applicability, 3) protocols, 4) chemicals and 5) equipment. For selection of participants for the TPS selection criteria have also been set (see Table 13). Amongst the most important criteria for selection for participants of TPS are technical expertise for the pest group and the method, authorization to work with the specific pest and that the participating laboratory has quality assurance in place. These criteria enable evaluation of whether participants are proficient to perform the tests, have the necessary equipment and a permit to work with viable regulated organism. The scope of the testing for specific pests was set and common rules for each selection process was defined

Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites

Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on variable number of tandem repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools, and VNTR markers were developed and validated. A Multiple-Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region

List of tests for validation

We prepared a list of methods and tests for validation in test performance study (TPS) Round 1, both for laboratory and on-site use, for 6 selected pests: Erwinia amylovora, Pantoea stewartiisubsp. stewartii, citrus tristeza virus, plum pox virus, Fusarium circinatum and Bursaphelenchus xylophilus. The listed tests were first validated in preliminary studies by TPS organizers in order to select the final tests for TPS, based on the scope and criteria prepared in D1.1

Ex post evaluation of the activities of the joint research centre under Horizon 2020 and Euratom 2014-2020

The report is the result of the external Panel ex post evaluation of the JRC activities under H2020 and Euratom 2014-2020. It provides the independent assessment requested in the Council Decisions concerning the specific programmes to be carried out by means of direct actions by the Joint Research Centre implementing the Horizon 2020 Framework Programme (2014-2020) of the European Commission and of the European Atomic Energy Community (Euratom). The evaluation has been conducted by a panel of independent external experts under the chairmanship of Dr Rolf-Dieter Heuer. In this report the Panel concludes positively on the effectiveness of the JRC as the Commission’s science service in support of Euratom and EU policies. Besides a number of recommendations for incremental improvement of the JRC, the Panel has flagged that the JRC is in a unique position as a provider of independent scientific evidence inside the European Commission, but, because of this, the JRC and its research work are less visible to the outside world than they merit. The Panel also flags that the JRC and its stakeholders, internal and external to the Commission, would also benefit from more communication and interactions. The Panel has particularly appreciated the meetings with the stakeholders that gave much insight into the cooperation between the JRC and the other parts of the Commission, supporting our positive assessment and our suggestions for improvement

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35GA, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by 1.2- fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and 1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%– 8% and 5%–10%, respectively). CONCLUSIONS: This work validates dPCR as an SItraceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine