Technology development for the early detection of plant pests: a framework for assessing Technology Readiness Levels (TRLs) in environmental science.

Unlabelled: Innovation in environmental fields such as plant health is complex because of unbounded challenges and lack of certainty of commercial uptake. In this paper we present a Technology Readiness Level (TRL) framework, specifically to assist with assessment of technologies to support detection of tree pests and pathogens, but also for wider potential adaptation. Biosecurity can be enhanced by improved early detection of pests and pathogens, but development and deployment of new technologies requires robust scrutiny.

Genome sequences of 12 isolates of the EU1 lineage of , a fungus-like pathogen that causes extensive damage and mortality to a wide range of trees and other plants.

Here we present genome sequences for twelve isolates of the invasive pathogen EU1. The assembled genome sequences and raw sequence data are available BioProject accession number PRJNA177509. These data will be useful in developing molecular tools for specific detection and identification of this pathogen.

Transcriptome sequencing identifies novel persistent viruses in herbicide resistant wild-grasses.

Herbicide resistance in wild grasses is widespread in the UK, with non-target site resistance (NTSR) to multiple chemistries being particularly problematic in weed control. As a complex trait, NTSR is driven by complex evolutionary pressures and the growing awareness of the role of the phytobiome in plant abiotic stress tolerance, led us to sequence the transcriptomes of herbicide resistant and susceptible populations of black-grass and annual rye-grass for the presence of endophytes. Black-grass (Alopecurus myosuroides; Am) populations, displaying no overt disease symptoms, contained three previously undescribed viruses belonging to the Partititiviridae (AMPV1 and AMPV2) and Rhabdoviridae (AMVV1) families.

The development of monoclonal antibodies to the secA protein of Cape St. Paul wilt disease phytoplasma and their evaluation as a diagnostic tool.

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins.

Methods in virus diagnostics: from ELISA to next generation sequencing.

Despite the seemingly continuous development of newer and ever more elaborate methods for detecting and identifying viruses, very few of these new methods get adopted for routine use in testing laboratories, often despite the many and varied claimed advantages they possess. To understand why the rate of uptake of new technologies is so low, requires a strong understanding of what makes a good routine diagnostic tool to begin. This can be done by looking at the two most successfully established plant virus detection methods: enzyme-linked immunosorbant assay (ELISA) and more recently introduced real-time polymerase chain reaction (PCR).

Real-time quantitative PCR based sensitive detection and genotype discrimination of Pepino mosaic virus.

Over the last decade a new virus disease caused by Pepino mosaic virus (PepMV) has been threatening the tomato industry worldwide. Reliable detection is vitally important to aid disease control. Methods must be both sensitive and capable of detecting the range of distinct genotypes that have been identified.

Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology.

A novel, unbiased approach to plant viral disease diagnosis has been developed which requires no a priori knowledge of the host or pathogen. Next-generation sequencing coupled with metagenomic analysis was used to produce large quantities of cDNA sequence in a model system of tomato infected with Pepino mosaic virus. The method was then applied to a sample of Gomphrena globosa infected with an unknown pathogen originally isolated from the flowering plant Liatris spicata.

Direct detection of plant viruses in potato tubers using real-time PCR.

Virus indexing of seed potatoes can be carried out by growing eye plugs to produce small plants and then testing them by ELISA, but this method is time consuming. Direct testing of the eye plugs by ELISA is not reliable, and so a method has been developed for the routine testing of seed potatoes for virus by PCR.

Panel of 23S rRNA gene-based real-time PCR assays for improved universal and group-specific detection of phytoplasmas.

Primers and probes based on the 23S rRNA gene have been utilized to design a range of real-time PCR assays for routine phytoplasma diagnostics. These assays have been authenticated as phytoplasma specific and shown to be at least as sensitive as nested PCR. A universal assay to detect all phytoplasmas has been developed, along with a multiplex assay to discriminate 16SrI group phytoplasmas from members of all of the other 16Sr groups.

Phytoplasma phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of 'Candidatus Phytoplasma'.

Phytoplasma phylogenetics has focused primarily on sequences of the non-coding 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (16-23S ISR), and primers that enable amplification of these regions from all phytoplasmas by PCR are well established. In this study, primers based on the secA gene have been developed into a semi-nested PCR assay that results in a sequence of the expected size (about 480 bp) from all 34 phytoplasmas examined, including strains representative of 12 16Sr groups. Phylogenetic analysis of secA gene sequences showed similar clustering of phytoplasmas when compared with clusters resolved by similar sequence analyses of a 16-23S ISR-23S rRNA gene contig or of the 16S rRNA gene alone.

Microarrays for rapid identification of plant viruses.

Many factors affect the development and application of diagnostic techniques. Plant viruses are an inherently diverse group that, unlike cellular pathogens, possess no nucleotide sequence type (e.g.

Advances in molecular phytodiagnostics - new solutions for old problems.

In the last decade, developments in molecular (nucleic acid-based) diagnostic methods have made significant improvements in the detection of plant pathogens. By using methods such as the polymerase chain reaction (PCR), the range of targets that can now be reliably diagnosed has grown to the extent that there are now extremely few, known pathogens that cannot be identified accurately by using laboratory-based diagnostics. However, while the detection of pathogens in individual, infected samples is becoming simpler, there are still many scenarios that present a major challenge to diagnosticians and plant pathologists.

The reliable detection of Barley yellow and mild mosaic viruses using real-time PCR (TaqMan).

Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) are economically-important, soil-borne pathogens of winter barley. Until recently, laboratory diagnosis of these pathogens has relied upon ELISA using polyclonal antiserum. However, due to the unstable nature of these viruses, combined with the inability to sap transmit BaYMV, high quality antiserum has been difficult to obtain and as a result the performance of ELISA is often unsatisfactory.