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High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, fungi and fungus-like protists, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health.

DNA collections are a valuable type of Natural Science collection, enabling the validation of past research, serving as a source for new genomic studies and supporting ex situ conservation. The DiSSCo Flanders DNA collection working group, aiming to advance and "unlock" their DNA collections, identified the need for: 1) actively sharing best practices regarding the management of DNA collections; and 2) providing guidance on how to bring theory into practice. By combining best practice examples from within the working group with available literature and brainstorming ideas, the working group co-created two outputs, referred to as: the "Challenges" and the "Key". The Challenges are a list of obstacles to DNA collection management, which shape the structure of the linked Key and can also be used to spark discussion amongst stakeholders. The Key is a tool that guides users through the maturation process of their DNA collection in a standardised way. It stimulates holistic growth, breaks down the needed work into manageable steps and helps to decide priorities during the process. Furthermore, the Key facilitates communication with both internal stakeholders and external DNA collection managers. The Key distinguishes itself from other self-assessment tools in several ways: it includes (re)investigation of the collection's purpose and context; it is specialised for DNA collections; it delivers concrete goals linked to relevant information and shared experience; and it is inclusive, targeting all Natural Science DNA collections, regardless of their context or size. [ABSTRACT FROM AUTHOR]

Background Xanthomonas fragariae (Xf) is a bacterial strawberry pathogen and an A2 quarantine organism on strawberry planting stock in the EU. It is taxonomically and metabolically distinct within the genus Xanthomonas, and known for its host specificity. As part of a broader pathogenicity study, the genome of a Belgian, virulent Xf strain (LMG 25863) was assembled to draft status and examined for its pathogenicity related gene content. Results The Xf draft genome (4.2 Mb) was considerably smaller than most known Xanthomonas genomes (~5 Mb). Only half of the genes coding for TonB-dependent transporters and cellwall degrading enzymes that are typically present in other Xanthomonas genomes, were found in Xf. Other missing genes/regions with a possible impact on its plant-host interaction were: i) the three loci for xylan degradation and metabolism, ii) a locus coding for a ßketoadipate phenolics catabolism pathway, iii) xcs, one of two Type II Secretion System coding regions in Xanthomonas, and iv) the genes coding for the glyoxylate shunt pathway. Conversely, the Xf genome revealed a high content of externally derived DNA and several uncommon, possibly virulence-related features: a Type VI Secretion System, a second Type IV Secretion System and a distinct Type III Secretion System effector repertoire comprised of multiple rare effectors and several putative new ones. Conclusions The draft genome sequence of LMG 25863 confirms the distinct phylogenetic position of Xf within the genus Xanthomonas and reveals a patchwork of both lost and newly acquired genomic features. These features may help explain the specific, mostly endophytic association of Xf with the strawberry plant. [ABSTRACT FROM AUTHOR]

Eruca vesicaria subsp. sativa (Mill.) Thell. (arugula or rocket) is a leafy vegetable originating from the Mediterranean region primarily being sold in bagged salads. From 2014 to 2017, plants (cv. Montana) exhibiting blackened leaf veins and irregular V-shaped chlorotic to necroic lesions at the leaf margins were observed in commercial greenhouses in Flanders, Belgium (Figure S1A). Symptoms started after harvest of the first cut, indicating that leaf injury favours disease development. By the last cut, infections had spread uniformly across the plots, with symptoms advanced to the point where harvesting was no longer profitable. Excised surface-sterilized necrotic leaf tissue and seeds were homogenized in phosphate buffer (PB), followed by dilution plating on Pseudomonas Agar F containing sucrose. After four days at 28°C, bright yellow round, mucoid, convex Xanthomonas-like colonies were obtained, both from leaves and seeds. For confirmation, DNA was extracted from pure cultures after which a partial fragment of gyrB was amplified and sequenced (Holtappels et al. 2022). Amplicons were trimmed to 530 nucleotides (Genbank ON815895-ON815900) according to Parkinson et al. (2007) and compared with the NCBI database. Strain GBBC 3139 shares 100% sequence identity with Xanthomonas campestris pv. campestris (Xcc) type strain LMG 568 and with RKFB 1361-1364, isolated from arugula in Serbia (Prokić et al. 2022). The other isolates from Belgian rocket - GBBC 3036, 3058, 3077, 3217 and 3236 - all have a gyrB sequence 100% identical to that of Xcc strain ICMP 4013, among others. To determine the genetic relatedness to other pathogenic Xc strains, the genomes of GBBC 3077, 3217, 3236 and 3139 were sequenced using a MinION (Nanopore) and non-clonal sequences were submitted to NCBI (BioProject PRJNA967242). Genomes were compared by calculating Average Nucleotide Identity (ANI). This revealed that the Belgian strains cluster together with Xc isolates originating from Brassica crops and separate from strains identified as Xc pv. barbareae, pv. incanae and pv. raphani (Figure S2A). Their designation as pv. campestris is supported by maximum likelihood clustering of concatenated gyrB-avrBs2 sequences (EPPO, 2021; Figure S2B,C). Finally, pathogenicity was verified on five-week-old rocket 'Pronto' plants grown in a commercial potting mix by cutting the leaves along the midrib with scissors dipped into a suspension of 108 cfu/ml of each strain or PB as control (4 plants/strain). Plants were kept in closed polypropylene boxes for 48 hr to support high humidity and facilitate infection. They were then maintained at 25 ± 2 °C. Lesions like those observed on commercial plants developed on the inoculated leaves within one week (Figure S1B). Bacterial colonies reisolated from symptomatic tissue were identified based on gyrB as the strains used for inoculation, thereby fulfilling Koch's postulates. To the best of our knowledge, this is the first report of black rot disease in arugula caused by Xcc in Belgium. Previously, Xcc on arugula has been reported in Argentina, California and Serbia as well (Romero et al. 2008; Rosenthal et al. 2017; Prokić et al. 2022). Arugula being a minor crop in Belgium, challenged by Xcc infections and strong import competition, many growers have abandoned the sector in recent years. Therefore, this study makes a strong case for early detection of disease symptoms and timely application of relevant management strategies in vulnerable crop settings.