Next generation biosecurity: Towards genome based identification to prevent spread of agronomic pests and pathogens using nanopore sequencing.

The unintentional movement of agronomic pests and pathogens is steadily increasing due to the intensification of global trade. Being able to identify accurately and rapidly early stages of an invasion is critical for developing successful eradication or management strategies. For most invasive organisms, molecular diagnostics is today the method of choice for species identification. However, the currently implemented tools are often developed for certain taxa and need to be adapted for new species, making them ill-suited to cope with the current constant increase in new invasive species. To alleviate this impediment, we developed a fast and accurate sequencing tool allowing to modularly obtain genetic information at different taxonomical levels. Using whole genome amplification (WGA) followed by Oxford nanopore MinION sequencing, our workflow does not require any a priori knowledge on the investigated species and its classification. While mainly focusing on harmful plant pathogenic insects, we also demonstrate the suitability of our workflow for the molecular identification of bacteria (Erwinia amylovora and Escherichia coli), fungi (Cladosporium herbarum, Colletotrichum salicis, Neofabraea alba) and nematodes (Globodera rostochiensis). On average, the pairwise identity between the generated consensus sequences and best GenBank BLAST matches was 99.6 ± 0.6%. Additionally, assessing the generated insect genomic dataset, the potential power of the workflow to detect pesticide resistance genes, as well as arthropod-infecting viruses and endosymbiotic bacteria is demonstrated.

Dispersal of harmful fruit fly pests by international trade and a loop-mediated isothermal amplification assay to prevent their introduction.

Global trade of plant products represents one of the major driving forces for the spread of invasive insect pests. This visualization illustrates the problem of unintended dispersal of economically harmful fruit fly pests using geospatial maps based on interception data from the Swiss import control process. Furthermore, it reports the development of a molecular diagnostic assay for rapid identification of these pests at points of entry such as sea- and airports as a prevention measure. The assay reliably differentiates between target and non-target species within one hour and has been successfully evaluated for on-site use at a Swiss point of entry.

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

Developing diagnostic SNP panels for the identification of true fruit flies (Diptera: Tephritidae) within the limits of COI-based species delimitation.

BACKGROUND: Rapid and reliable identification of quarantine pests is essential for plant inspection services to prevent introduction of invasive species. For insects, this may be a serious problem when dealing with morphologically similar cryptic species complexes and early developmental stages that lack distinctive characters useful for taxonomic identification. DNA based barcoding could solve many of these problems. The standard barcode fragment, an approx. 650 base pairs long sequence of the 5'end of the mitochondrial cytochrome oxidase I (COI), enables differentiation of a very wide range of arthropods. However, problems remain in some taxa, such as Tephritidae, where recent genetic differentiation among some of the described species hinders accurate molecular discrimination. RESULTS: In order to explore the full species discrimination potential of COI, we sequenced the barcoding region of the COI gene of a range of economically important Tephritid species and complemented these data with all GenBank and BOLD entries for the systematic group available as of January 2012. We explored the limits of species delimitation of this barcode fragment among 193 putative Tephritid species and established operational taxonomic units (OTUs), between which discrimination is reliably possible. Furthermore, to enable future development of rapid diagnostic assays based on this sequence information, we characterized all single nucleotide polymorphisms (SNPs) and established "near-minimal" sets of SNPs that differentiate among all included OTUs with at least three and four SNPs, respectively. CONCLUSIONS: We found that although several species cannot be differentiated based on the genetic diversity observed in COI and hence form composite OTUs, 85% of all OTUs correspond to described species. Because our SNP panels are developed based on all currently available sequence information and rely on a minimal pairwise difference of three SNPs, they are highly reliable and hence represent an important resource for developing taxon-specific diagnostic assays. For selected cases, possible explanations that may cause composite OTUs are discussed.

Genome sequence of an Erwinia amylovora strain with pathogenicity restricted to Rubus plants.

Here, we present the genome of a strain of Erwinia amylovora, the fire blight pathogen, with pathogenicity restricted to Rubus spp. Comparative genomics of ATCC BAA-2158 with E. amylovora strains from non-Rubus hosts identified significant genetic differences but support the inclusion of this strain within the species E. amylovora.

Quantitative assessment of heteroplasmy levels in Senecio vulgaris chloroplast DNA.

Heteroplasmy in coding chloroplast DNA was only recently shown to occur and was so far not quantitatively assessed. We present a quantitative analysis of cpDNA heteroplasmy levels at a triazine-resistance determining site within and between individual Senecio vulgaris plants. Detectable levels of heteroplasmic haplotypes were observed in all tested plants. As expected, the levels of heteroplasmy vary greatly between plants. However, even within individual plants, the fraction of one haplotype may cover a range from below 1% to well over 90%. Our results suggest that heteroplasmy may be a common phenomenon in S. vulgaris. Possible consequences for molecular diagnostics of chloroplast encoded traits as well as evolutionary consequences of chloroplast heteroplasmy are discussed.

Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format.

A system for microarrays was developed to detect and differentiate Cucumber mosaic virus (CMV) serogroups and subgroups. The coat protein genes of 14 different isolates were amplified using cy3-labelled generic but species-specific primers. These amplicons were hybridized against a set of five different serotype and subgroup specific 24-mer oligonucleotides bound to an aldehyde-coated glass slide via an aminolinker. The results of the hybridization revealed that the method allowed a clear differentiation of the 14 different CMV isolates into the serogroupes 1 and 2, and in addition was able to assign 9 out of 10 different serogroup 1 isolates correctly into subgroups 1a and 1b. This differentiation was not possible by RFLP analysis with the restriction enzyme MspI. The use of amplicons larger than 700 base pairs and their successful differentiation by hybridization to specific oligonucleotides opens avenues to highly parallel, yet sensitive assays for plant viruses.

Structure and bonding of the vanadium(III) hexa-aqua cation. 1. Experimental characterization and ligand-field analysis.

Spectroscopic and crystallographic data are presented for salts containing the [V(OH(2))(6)](3+) cation, providing a rigorous test of the ability of the angular overlap model (AOM) to inter-relate the electronic and molecular structure of integer-spin complexes. High-field multifrequency EPR provides a very precise definition of the ground-state spin-Hamiltonian parameters, while single-crystal absorption measurements enable the energies of excited ligand-field states to be identified. The EPR study of vanadium(III) as an impurity in guanidinium gallium sulfate is particularly instructive, with fine-structure observed attributable to crystallographically distinct [V(OH(2))(6)](3+) cations, hyperfine coupling, and ferroelectric domains. The electronic structure of the complex depends strongly on the mode of coordination of the water molecules to the vanadium(III) cation, as revealed by single-crystal neutron and X-ray diffraction measurements, and is also sensitive to the isotopic abundance. It is shown that the AOM gives a very good account of the change in the electronic structure, as a function of geometric coordinates of the [V(OH(2))(6)](3+) cation. However, the ligand-field analysis is inconsistent with the profiles of electronic transitions between ligand-field terms.

Origin of intra-individual variation in PCR-amplified mitochondrial cytochrome oxidase I of Thrips tabaci (Thysanoptera: Thripidae): mitochondrial heteroplasmy or nuclear integration?

The mitochondrial genome is increasingly being used as a species diagnostic marker in insects. Typically, genomic DNA is PCR amplified and then analysed by restriction analyses or sequencing. This analysis system may cause some serious problems for molecular diagnosis. Besides the errors introduced by the PCR process, mtDNA sequence variation of amplified fragments may originate from mtDNA heteroplasmy or from nuclear integrations of mtDNA fragments, both of which have been shown to occur in insects. Here we document abundant variation in PCR-amplified sequences of the mitochondrial cytochrome oxidase I gene of Thrips tabaci. We confirm that the most common haplotype is of mitochondrial origin. Some of the observed mutations were introduced by the amplification process. However, the occurrence of some haplotypes at elevated frequencies indicates that within-individual variation of the respective fragment exists at low levels in T. tabaci. The frequencies of these sequences are too low to negatively affect mtDNA-based molecular diagnosis of T. tabaci. The possible origin of these variant haplotypes is discussed.