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Slide of Euphresco project 2021-A-378 'Inventory and validation of quality control procedures for the extraction of nucleic acids for real-time PCR used for the diagnosis of pests (IVENAD)', Project funded through the Euphresco network

Nematodes of the genus Xiphinema (dagger nematodes) are migratory root ectoparasites with a broad host range. There are more than 260 species, of which approximately 60 belong to the Xiphinema americanum group (X. americanum sensu lato). Some of the species can specifically transmit certain nepoviruses of phytosanitary concern. Several subgroups of nepoviruses exist, each with their own specific vector species. Based on morphological and morphometrical data, the identification up to Xiphinema species level is quite difficult. Most of the time, it is impossible due to lack of specimens or appropriate nematode stadia. Currently, there is also a lack of molecular information of all Xiphinema species to develop reliable diagnostic tools such as PCR or barcoding. Direct damage caused by Xiphinema species is usually limited, the main issue is that some specimens are potential carriers of viruses. Whereas the whole X. americanum group had a quarantine status before the new Plant Health Legislation (December 2019), only 7 species retained this status and two species became regulated non-quarantine organisms. Still, their identification up to species level is very difficult. Focusing on a direct detection of these viruses in the nematodes could be an alternative to the difficult task of correctly identifying the Xiphinema species. This way, only specimens of the X. americanum group that are actually carrying viruses can be considered as a Plant Health risk, and consignments carrying Xiphinema spp. free of viruses could be allowed to enter trade. The aim of the project was to optimise a generic classic nepovirus detection test for nematodes of the genus Xiphinema and ultimately to validate it through the organisation of an interlaboratory test. Based on literature, methods were selected to develop a diagnostic procedure for nepovirus detection in Xiphinema nematodes. Nematodes (X. diversicaudatum, X. index, and X. americanum s.s.) and nepoviruses (Arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV) and tomato ringspot virus (ToRSV)) were procured, virus transmission assays were carried out and the selected methods were compared and validated in the laboratory. This resulted in a recommended diagnostic protocol consisting of the Automated Zonal Centrifuge (AZC) for nematode extraction from soil, a method of choice for physical disruption of the isolated nematodes (slicing, bead beating and bead beating with collagenase pre-treatment) and the KingFisher MagMax Kit for RNA extraction. Due to inadequate sensitivity of generic nepovirus subgroup detection tests, nepovirus detection still relies on specific single nepovirus real-time PCR tests. The MinION nanopore technology was evaluated as an alternative for the future. MinION nanopore sequencing failed to detect GFLV and ToRSV in single nematode specimens. However, its potential was demonstrated by successful ArMV detection in six X. diversicaudatum individuals.

The increasing introduction of new crops in Europe are an opportunity for growers to produce for niche markets. These newly introduced exotic crops, as well as “forgotten” crops, are mainly grown and marketed outside the general, large-scale commercial agriculture. This local production, and the associated short food supply chain, obtain their planting materials (seed, tubers, cuttings) from different sources, e.g. multipliers in the South of Europe or directly from overseas areas, internet shops, etc. The phytosanitary status of this material is very rarely checked. Entry and spread of possible plant pathogens and pests could pose a threat to traditional crops, besides hampering the cultivation of the new crops. Especially when planting material is obtained from non-European countries, phytosanitary risks can be high. The project focussed on gathering information on a selection of vegetatively propagated tuber producing crops that are gaining a lot of interest, but for which only limited information on their phytosanitary status is available. A survey was performed on yacon (Smallanthus sonchifolius), ulluco (Ullucus tuberosus), sweet potato (Ipomoea batatas), crosne (Stachys affinis), mashua (Tropaeolum tuberosum), oca (Oxalis tuberosa) and Jerusalem artichoke (Helianthus tuberosus). The survey was complemented with a desktop study to map the distribution, pathways and extent of these niche crops. The focus of risk identification was put on viruses and nematodes due to their high potential for introduction without being noticed due to lack of typical symptoms. In addition, specifically for viruses, screening through HTS (high throughput sequencing) was performed to detect targeted and untargeted viruses. A limited number of biological assessments was performed for selected viruses and nematodes (e.g. host status of other crops) to gather information in support of pest risk analyses. Literature studies were performed to identify control options for selected pests., Report of the Euphresco project 2018-A-293 'Phytosanitary risks of newly introduced crops (PRONC)'

Success story of the Euphresco project 2018-A-293 'Phytosanitary risks of newly introduced crops (PRONC)'

Plant pest diagnosis is performed by official laboratories upon request of National Plant Protection Organizations (NPPOs), growers or traders, in samples that inspectors have collected in situ (a consignment, a place of production, an outbreak area, a buffer zone, etc.). Resources allocated to official laboratories have decreased over time, while trade in plants and plant products, and consequently the material to be tested, have increased steadily. As indicated in the Euphresco Strategic Research Agenda (priority R-6), on-site detection and identification tests, that are both high throughput and scalable at contained costs should be developed and validated to accelerate diagnosis (especially in the case of perishable goods) and to relieve pressure on laboratories. In particular, the application of on-site detection and surveillance methods for plant viruses and viroids is needed for the quick assessment of the health status of plant material. New protocols and technologies based on sequencing are under development (such as Oxford nanopore direct RNA sequencing - cDNA sequencing), but need to be tested, optimised and validated in order to be applicable in routine on-site testing. Furthermore, current barriers for the use of novel on-site technologies need to be addressed. The VIRFAST project explored the possibility of on-site virus diagnosis with the MinION technology (Oxford Nanopore sequencing). Early results indicated that the traditional RNA extraction protocol could be bypassed resulting in a quicker and low-tech solution for practical diagnostics on-site. However, skipping the extraction needs additional development for diagnostic purposes, as the sequencing threshold and, consequently, the sensitivity of the test is greatly reduced. However, it should be noted that validation was performed using dehydrated samples and moreover some participating laboratories used the Oxford nanopore sequencer on crude extract material for the first time. Better analytical sensitivity is expected when working with fresh tissue and if the users are familiar with the use of crude extract material. Two sample preparation kits were also compared starting from purified RNA extracts. In summary, better results for virus/viroid detection when using cDNA-PCR library preparation approach, compared to direct RNA sequencing approach, due to the higher relative amount of sequencing reads of viral origin. The increased analytical sensitivity when using the cDNA-PCR library preparation opens the possibility of using this method to monitor an outbreak in the field, in a middle-tech laboratory or in remote locations with limited access to plant diagnostics facilities. A survey of 26 NPPO laboratories showed that while only 15% were using high-throughput sequencing (HTS) in their diagnostic workflows in 2019, almost all wished to adopt it and that improved staff training and reduced costs would be essential for this uptake. The results of a survey on on-site testing allowed also to observe interesting trends. Twelve laboratories were already using on-site testing with an estimated volume ranging from 25 to 2,500 tests per year. The main context for using on-site testing kits was field inspection (63%), followed by screening test in the laboratory (19%). Time saving was the most important advantage for use. The laboratories also highlighted the ease of use. The three main limitations for a wider use of HTS were the lack of validation data, the limited sensitivity and the cost., Report of the Euphresco project 2017-A-243 'Faster, cheaper identification of emerging virus problems (VIRFAST)'

Tomato brown rugose fruit virus (ToBRFV) is a relatively recently described tobamovirus (Salem et al., 2016) causing problems in tomato and pepper cultivation worldwide (Luria et al., 2017, Salem et al., 2019). ToBRFV has been reported to cause yellow spots, green spots, necrotic lesions and occasional rugose symptoms on tomato fruits, chlorosis and mosaic symptoms on leaves and occasional leaf narrowing (Cambrón-Crisantos et al., 2018; Luria et al., 2017; Menzel et al., 2019). For pepper, stunting of young plants, puckering and yellow mottling of leaves, and misshapen fruits have been reported (Salem et al., 2019). These symptoms render affected fruits non-marketable. Tobamoviruses are very stable and will remain infectious for long periods of time when present in crop debris, soil and on surfaces. Infectivity on seeds is preserved for up to several years (Dombrovsky and Smith, 2017). ToBRFV is easily transmitted mechanically (via contaminated tools, hands, clothing, direct plant-to-plant contact, and bumble bees) and seed-to-seedling transmission is expected to play a role in the spread of the virus (Levitzky et al., 2019, Salem et al., 2021). As of November 1, 2019, emergency measures (EU) 2019/1615 are in place in the EU to prevent introduction and further spread of ToBRFV. The initial measures have been replaced by Commission Implementing Regulation (EU) 2021/1809. These measures include an obligatory annual survey, and specific requirements for movement and introduction of plants for planting, including seeds, of Solanum lycopersicum and its hybrids and Capsicum spp. In addition, there is a requirement for testing of at least 20% of the consignments of seeds and plants for planting of Solanum lycopersicum and Capsicum spp. upon entry into the EU. For consignments from Israel, the required testing rate is even 50% and for seeds from China 100%. Therefore, reliable and harmonised protocols for the detection of ToBRFV in tomato and pepper plants and seeds are needed. In the framework of the EU H2020 research project VALITEST (www.valitest.eu) a test performance study (TPS) has been conducted on leaf and fruit material of tomato and pepper. In addition, however, there is a need for reliable and harmonised test protocols for the detection of ToBRFV in tomato and pepper seeds. A TPS was organised in the framework of this Euphresco project to compare the suitability of different available methods and tests for the detection of ToBRFV in tomato and pepper seeds. Since the number of tests that can be included in a TPS is limited, comparative experiments were conducted in order to select the most suitable tests for the TPS. The main results of these comparative experiments were that DAS-ELISA was not sensitive enough to reliably detect ToBRFV in tomato seeds with a medium level of the virus. In addition, for molecular tests using guanidine hydrochloride (GH+) buffer for grinding and RNA extraction resulted in a more sensitive detection of ToBRFV in tomato compared to phosphate buffer. Based on the results of the comparative experiments, the TPS included two real-time RT-PCR tests (ISHI-Veg, 2019; Menzel & Winter, 2021), two end-point RT-PCR tests (Loewe kit, adapted from Rodriguez-Mendoza et al., 2019; Alkowni et al., 2019) and two isothermal amplification tests (Sarkes et al., 2020; Agdia AmplifyRP® kit). GH+ buffer was chosen as the extraction buffer to be used. Twenty-six laboratories worldwide, but mostly from Europe and the Mediterranean region, participated in this study. The project results showed that the real-time RT-PCR tests (ISHI-Veg, 2019 and Menzel & Winter, 2021) allowed to diagnose ToBRFV in all samples with only a few percent false negative and false positive results. In contrast, end-point RT-PCR and isothermal amplification tests appeared unsuitable for the reliable detection of ToBRFV in tomato and pepper seeds, because they were not sensitive enough. The real-time RT-PCR tests (ISHI-Veg, 2019 and Menzel & Winter, 2021) have been recommended for the detection of ToBRFV in seeds by the European and Mediterranean Plant Protection Organisation (EPPO, 2021) and are currently required for testing of tomato and pepper seeds under EU emergency measures. The TPS results confirm the suitability of the real-time RT-PCR tests for the detection of ToBRFV in tomato and pepper seeds., Revised report of the Euphresco project 2019-A-327 'Validation of molecular tests for the detection of tomato brown rugose fruit virus (ToBRFV) in seed of tomato and pepper'. This revised report includes additional data to improve transparency and detailing of the conclusions of the test performance study.

High-throughput sequencing (HTS, formerly also known as next-generation sequencing or pyrosequecing) technologies have seen a tremendous evolution in terms of technical developments. These technologies are more and more used for plant virus discovery, metagenomic and ecological studies but are also increasingly being used in plant virus diagnostic settings, post-entry quarantine investigations or pre-export diagnostics. Conventional virus detection methods based on serology (i.e., enzyme-linked immunosorbent assays) or nucleic acids (polymerase chain reaction (PCR), real-time PCR), requires in depth knowledge for the target pathogen to develop antisera or primers limiting these methods to the detection of mostly known viruses. The use of HTS for virus detection is not dependent on a priori knowledge about the viruses to be detected which is a major advance in diagnostic testing. This project aimed to harmonise sample enrichment strategies and HTS workflows for plant virus and viroid detection within a diagnostic framework. Existing bioinformatics approaches were investigated during a proficiency test and a test performance study which demonstrated the power of HTS for diagnostics but also the limitations if diagnosticians are not experienced with the analyses of HTS data. HTS and newly developed sequencing technologies such as Oxford Nanopore Sequencing have a great potential for diagnostics but further work is required to set up guidelines for validation of HTS workflows., Report of the Euphresco project 2015-F-172 'The application of Next-Generation Sequencing technology for the detection and diagnosis of non-culturable organisms: viruses and viroids (NGSdetect)'

The main goal of the project was to generate more information on insect vectors of bacteria and phytoplasmas of phytosanitary concern, more specifically: a) on phloem and xylem feeding insects that should be tested as possible vectors for bacteria and phytoplasmas b) on the fruit and field crop range that the vectored bacteria and phytoplasmas cause damage to c) on the capability of the insects to vector specific bacteria and phytoplasmas of phytosanitary concern d) on the specific association between vector and plant pathogen. The main objectives of the project were to gain insight into the following questions: -Should yet-unknown phloem and xylem feeding insects (Auchenorryncha) be regarded as vectors of the pathogens in selected major crops in Europe? -What monitoring and trapping techniques are optimal to efficiently survey the phloem and xylem feeding insect vectors? -Are there reliable and validated diagnostic tools available to detect/monitor the prokaryotic plant pathogens inside the vectors? -Can we generate extra information on the transmission pathways by monitoring phloem and xylem feeding insect vectors in and around selected infested fields? -Can we generate extra information on the host range of the prokaryotic pathogens by performing vector-transmission trials between different test crops? -Can we reduce the number of treatments of pesticides due the better understanding of relation vector/pathogen/environment and thereby enable a better integrated management of vectors and diseases in the crops., Report of the Euphresco project 2015-D-168 'Tracking vectors of bacteria and phytoplasmas threatening Europe's major crops (VECTRACROP)'