Mediterranean Plant Protection Organization Research Articles

Viroids of the Mediterranean Basin.

There has been substantial progress in the Mediterranean countries regarding research on viroids. Twenty-nine viroid species, all belonging to Pospiviroidae and Avsunviroidae genera, have been detected in the Mediterranean Basin. Not only have detection methods, such as reverse transcription-quantitative polymerase chain reaction and next-generation sequencing, been used for viroid detection, along with molecular hybridization techniques allowing for rapid detection, identification, and characterization of known and novel viroids in these countries, but eradication measures have also been taken that allowed for the efficient elimination of certain viroids in a number of Mediterranean countries. The eradication measures were followed as recommended by the European and Mediterranean Plant Protection Organization, which is known by its abbreviation, EPPO. The Mediterranean Region has been a niche for viroids since ancient times due to the warm climate and the socio-cultural conditions that facilitate viroid transmission among different host plant species.

Field-Deployed Spectroscopy from 350 to 2500 nm: A Promising Technique for Early Identification of Powdery Mildew Disease (Erysiphe necator) in Vineyards

This study explores spectroscopy in the 350 to 2500 nm range for detecting powdery mildew (Erysiphe necator) in grapevine leaves, crucial for precision agriculture and sustainable vineyard management. In a controlled experimental vineyard setting, the spectral reflectance on leaves with varying infestation levels was measured using a FieldSpec 4 spectroradiometer during July and September. A detailed assessment was conducted following the guidelines recommended by the European and Mediterranean Plant Protection Organization (EPPO) to quantify the level of infestation; categorising leaves into five distinct grades based on the percentage of leaf surface area affected. Subsequently, spectral data were collected using a contact probe with a tungsten halogen bulb connected to the spectroradiometer, taking three measurements across different areas of each leaf. Partial Least Squares Regression (PLSR) analysis yielded coefficients of determination R2 = 0.74 and 0.71, and Root Mean Square Errors (RMSEs) of 12.1% and 12.9% for calibration and validation datasets, indicating high accuracy for early disease detection. Significant spectral differences were noted between healthy and infected leaves, especially around 450 nm and 700 nm for visible light, and 1050 nm, 1425 nm, 1650 nm, and 2250 nm for the near-infrared spectrum, likely due to tissue damage, chlorophyll degradation and water loss. Finally, the Powdery Mildew Vegetation Index (PMVI) was introduced, calculated as PMVI = (R755 − R675)/(R755 + R675), where R755 and R675 are the reflectances at 755 nm (NIR) and 675 nm (red), effectively estimating disease severity (R2 = 0.7). The study demonstrates that spectroscopy, combined with PMVI, provides a reliable, non-invasive method for managing powdery mildew and promoting healthier vineyards through precision agriculture practices.

Naturalised Hakea. What species are we actually talking about in Europe?

ABSTRACT Hakea sericea Schrad. & J.C.Wendl. (Proteaceae) is a shrub or small tree native to south-eastern Australia. The species has been introduced to other countries; it has naturalised and become invasive in France, New Zealand, Portugal, and South Africa. During the review process of the Pest Risk Analysis for the European and Mediterranean Plant Protection Organization (EPPO) on Hakea sericea in 2017, the actual identity of the plant causing major negative biodiversity impact in Portugal was questioned. Flowering material from Portugal and additional collections from South Africa and New Zealand were collected in order to clarify the taxonomical status. To make the survey on the proper identity of the Hakea plants invasive in Portugal more concise, all Hakea species encountered in Portugal, France, South Africa and New Zealand were included. For morphological distinction of the species the length of the pistil and fruit characters were used. For molecular analysis Illumina sequencing in combination with de novo assembly and extraction of loci was used. Results show that invasive populations of Hakea in Portugal belong to H. decurrens R.Br a species that, although very similar and sometimes considered synonymous with H. sericea, can be distinguished both morphologically and on the basis of cpDNA. Both species occur in France and in New Zealand, while only H. sericea occurs in South Africa. Both species are invasive and seem to have similar impacts on biodiversity in invaded ranges. The morphologically similar species H. gibbosa (J.White) Cav. occurs in both New Zealand and South Africa. By adopting the H. sericea sensu lato concept for inclusion of the species on the List of Union Concern pursuant of Regulation (EU) No 1143/2014, a potential legislative controversy can be prevented.

Testing plant growth promoting microorganisms in the field - a proposal for standards.

In the European Union and worldwide there are a burgeoning markets for plant growth promoting microorganisms (PGPM) and other biological agents as soil improvers, bio-fertilizers, plant bio-stimulants, and biological control agents or bio-pesticides. Microbial agents have a major share in this development. The use of such products is often advertised with the promise of contributing to sustainable agricultural practices by increasing crop growth and yield and offering an alternative or substitute to decrease the dependency of agriculture on hazardeous agrochemicals. In contrast to registered microbial plant protection products, PGPM that are marketed in the EU as soil improvers or plant biostimulants, are not strictly required to have proven minimum efficacy levels under field conditions. Manufacturers only have to ensure that these products do not pose unacceptable risks to human, animal or plant health, safety or the environment. Uniform guidelines comparable to the EPPO - standards (European and Mediterranean Plant Protection Organisation) to test the efficacy in field trials are not available. This paper attempts to fill the gap. It proposes guidelines for PGPM field trial design and implementation, as well as recommendations for the type and scope of data collection and evaluation. Selected research papers from literature were evaluated to analyze, whether and to what extent the requirements are already met. The majority of the papers had a clear experimental design followed by proper data evaluation. Frequent deficiencies were the low number of tested environments and crop species, insufficient site and agronomic management description and missing data on soil humidity and temperature. Using the suggested standards is assumed to increase the expressive power of tested microbial products.

Pan-Genomic Analysis Identifies the Chinese Strain as a New Subspecies of Xanthomonas fragariae.

Xanthomonas fragariae is classified as a quarantine pathogen by the European and Mediterranean Plant Protection Organization. It commonly induces typical angular leaf spot (ALS) symptoms in strawberry leaves. X. fragariae strains from China (YL19, SHAQP01, and YLX21) exhibit ALS symptoms in leaves and more severe symptoms of dry cavity rot in strawberry crowns. Conversely, strains from other countries do not cause severe dry cavity rot symptoms in strawberries. After employing multilocus sequence analysis (MLSA), average nucleotide identity (ANI), and amino acid identity (AAI), we determined that Chinese strains of X. fragariae are genetically distinct from other strains and can be considered a new subspecies. Subsequent analysis of 63 X. fragariae genomes published at NCBI using IPGA and EDGAR3.0 revealed the pan-genomic profile, with 1,680 shared genes present in all 63 strains, including 71 virulence-related genes. Additionally, we identified 123 genes exclusive to all the Chinese strains, encompassing 12 virulence-related genes. The qRT-PCR analysis demonstrated that the expression of XopD, XopG1, CE8, GT2, and GH121 out of 12 virulence-related genes of Chinese strains (YL19) exhibited a constant increase in the early stages (6, 24, 54, and 96 hours postinoculation [hpi]) of strawberry leaf infected by YL19. So, the presence of XopD, XopG1, CE8, GT2, and GH121 in Chinese strains may play important roles in the early infection process of Chinese strains. These findings offer novel insights into comprehending the population structure and variation in the pathogenic capacity of X. fragariae.

Testing a modified version of the EPPO decision-support scheme for release of classical biological control agents of plant pests using Ganaspis cf. brasiliensis and Cleruchoides noackae as case studies

The 6/04 standard of the European and Mediterranean Plant Protection Organisation (EPPO) on the safe use of biological control is a decision-support scheme (DSS) for the import and release of biological control agents in Europe. It was recently developed by the Joint EPPO/International Organisation of Biological Control (IOBC) Panel on Biological Control Agents. The DSS can be used to assess the potential environmental impacts of biological control agents. It is valid for different types of biological control: classical and augmentative biological control of invertebrates, pathogens and weeds. However, the DSS is not yet widely implemented in Europe and, during preliminary trials, it was found that its broad range of usages could lead to some confusion or misunderstandings, as well as requiring unnecessary information in some cases. Thus, the scheme was modified to specifically assess classical biological control against plant pests, i.e. the introduction of exotic natural enemies of plant pests for establishment and long-term control. The new version of the scheme was then tested on two parasitoids that are presently being released in Europe, the figitid Ganaspis cf. brasiliensis against the spotted wing drosophila Drosophila suzukii and the mymarid Cleruchoides noackae against the Eucalyptus bronze bug Thaumastocoris peregrinus. Both parasitoids were successfully assessed with the new version of the DSS. No major issues were encountered during the assessments and most questions were answered with low levels of uncertainty. Both assessments concluded that the parasitoids were safe to release in the impact assessment areas, with positive impacts exceeding negative impacts. Suggestions for potential improvements are provided.

First Report of Root-Knot Nematode Meloidogyne enterolobii on Antirrhinum majus in China.

Antirrhinum majus L. is a medicinal and ornamental herb commonly grown in China. In October 2022, A. majus plants were observed stunted in growth with yellowish leaves and containing a large number of galls on roots in a field in Nanning, Guangxi, China (N22°47'23.35″, E108°23'4.26). Ten samples were collected randomly from rhizosphere soil and roots of A. majus. Second-stage juveniles (J2) were isolated from fresh soil with a Baermann funnel, and a mean of 36 ± 2.9 per 500 cm3 of soil was recorded. Gall roots were dissected using a microscope, where 2+ 0.42 males per sample were recovered. The species was determined to be Meloidogyne enterolobii based on morphological characteristics, including the female perineal pattern, and DNA studies. Female perineal patterns and morphometric data were similar to the original description of M. enterolobii Yang and Eisenback 1983 from Enterolobium contortisilquum (Vell.) Morong in China (Yang and Eisenback 1983). The measurements of males (n = 10) included body length, 1600.7 ± 55.32 (1421.3 to 1924.3) µm; body diameter = 41.3 ± 0.80 (37.8 to 45.4) µm, stylt length = 20.5 ± 0.40 (19.1 to 22.2) µm, spicules length = 30.0 ± 0.47 (28.2 to 32.0) µm and DGO = 4.5 ± 0.3 (3.8 to 5.2) µm. Measurements of J2 (n = 20) included body length, 441.9 ± 5.42 (403.2 to 493.3) µm; body diameter = 16.6 ± 0.30 (14.4 to 8.7) µm, a = 26.8 ± 0.54 (21.9 to 31.2), c = 8.7 ± 0.27 (6.4 to 10.8), stylet length = 12.6 ± 0.17 (11.2 to 14.3) µm, DGO = 3.8 ± 0.10 (2.9 to 4.8) µm, tail length = 51.6 ± 1.27 (42.3 to 63.1) µm and hyaline tail terminus length = 11.7 ± 0.15 (10.2 to 13.1) µm. These morphological characteristics are similar to the original description of M. enterolobii (Yang and Eisenback 1983). Pathogenicity tests were conducted on A. majus 'Taxiti' plants directly germinated from seeds in a 10.5-cm-diameter pot filled with 600 ml of sterilized peat moss/sand (1:1, v/v) soil in the glasshouse. After 1 week, fifteen plants were inoculated with 500 J2/pot (nematode culture collected from the original field) and five uninoculated plants served as a control. After 45 days, aboveground parts of all inoculated plants showed symptoms similar to those observed in the field. No symptoms were observed on control plants. The RF value of the inoculated plants was determined by the method of Belair and Benoit (1996) 60 days after inoculation, and the average was 14.65. J2 were used in this test and sequenced on 28S rRNA-D2/D3, ITS, COII -16SrRNA 3 region and confirmed to be M. enterolobii. Species identification was confirmed by using polymerase chain reaction primers D2A/D3B (De Ley et al. 1999), F194/5368r (Ferris et al. 1993), C2F3/1108 (Powers and Harris, 1993). The sequences obtained GenBank accession numbers OP897743 (COII), OP876758 (rRNA) and OP876759 (ITS) were 100% similar to other M. enterolobii populations from China (MN269947), (MN648519) and (MT406251). M. enterolobii is a highly pathogenic species and has been reported in vegetables, ornamental plants, guava (Psidium guajava L.), and weeds in China, Africa and America (Brito et al. 2004; Xu et al. 2004; Yang and Eisenback 1983). The medicinal plant Gardenia jasminoides J. Ellis was also infected by M. enterolobii in China (Lu et al. 2019). Of concern is its ability to develop on crop genotypes carrying RKN resistance genes in tobacco (Nicotiana tabacum L.), tomato (Solanum lycopersicum L.), soybean (Glycine max (L.) Merr.), potato (Solanum tuberosum L.), cowpea (Vigna unguiculata (L.) Walp.), sweetpotato (Ipomoea batatas (L.) Lam.), and cotton (Gossypium hirsutum L.). Consequently, this species was added to the European and Mediterranean Plant Protection Organization A2 Alert List in 2010. This is the first natural infection report of M. enterolobii in Guangxi, China on the medicinal and ornamental herb A. majus. Acknowledgments This research was funded by the National Natural Science Foundation of China (31860492), Natural Science Foundation of Guangxi (2020GXNSFAA297076), and Guangxi Academy of Agricultural Sciences Fund, China (2021YT062, 2021JM14, 2021ZX24).

International Cooperation to Support the Diagnosis of Forestry Pests: The Role of EPPO and Euphresco

Plant pests are a threat to biodiversity, food security, and the economy. The challenges posed by the introduction of plant pests have triggered the establishment of cooperative mechanisms such as the International Plant Protection Convention (IPPC), an international treaty to protect plant health, but also of Regional Plant Protection Organizations (RPPOs), such as the European and Mediterranean plant Protection Organizations (EPPO). RPPOs participate in activities in their regions to promote and achieve the objectives of the IPPC. Because accurate and reliable detection and identification of pests are essential in order to be able to take appropriate measures against a pest and thus avoid or reduce the economic, social, and environmental costs that it can cause, the IPPC and EPPO have established a specific program on diagnostics. The main aim of this article is to present the diagnostic activities conducted by the European and Mediterranean Plant Protection Organization and Euphresco. Some information is also presented on other initiatives in diagnostics.

Development and validation of real‐time PCR tests for specific identification of Xanthomonas species causing bacterial spot disease on tomato (Solanum lycopersicum) and pepper (Capsicum annuum)

AbstractBacterial spot is a significant disease of tomato and pepper crops caused by members of four Xanthomonas pathogens from three species, namely Xanthomonas euvesicatoria pv. euvesicatoria, Xanthomonas euvesicatoria pv. perforans, Xanthomonas vesicatoria and Xanthomonas hortorum pv. gardneri. These Gram‐negative bacterial plant pathogens are recommended for regulation as quarantine pests by the European and Mediterranean Plant Protection Organization and are currently regulated on tomato and pepper seeds in many regions of the world. Tomato and pepper seeds are a transmission pathway for all three species. Real‐time PCR TaqMan tests were developed and validated for specific identification of isolates from tomato and pepper seeds of these pathogens. These tests are designed to be used in seed health tests after isolation on semiselective media to reduce the number of isolates that need testing in follow‐up pathogenicity tests. A second set of real‐time TaqMan PCR tests was subsequently developed targeting two markers for each taxonomic group of strains. The analytical specificity of all developed tests was evaluated with in vitro and in silico analyses. Diagnostic specificity and sensitivity were also validated on collections of isolates in several laboratories.

Effectiveness of insecticides against the adult stage of western corn beetle (Diabrotica virgifera virgifera LeConte, 1868)

Goal. Assessment of chemical insecticides for WCR (Diabrotica virgifera virgifera LeConte, 1868) adults control for using in integrated corn pest management systems.
 Methods. Fields method. Trials conducted in 2021—2022 in farms in Bilyi Rukav Vinnytsia region and Ivanivka Kyiv region where corn was sown for 4 years. Techniques approved by the EPPO (Mediterranean Plant Protection Organization) are counting using yellow sticky traps, direct counting on plants, counting dead beetles, others.
 Results. 1—3 DAAT, the most effective was Avaunt EC (indoxacarb 150 g/l) at the rates of 0.17 l/ ha and 0.25 l/ ha with % reduction in population compared to the control 65.69 and 62.84, respectively, Karate Zeon SC (lambda-cyhalotryn 50 g/l) 0.3 l/ ha with 54.26% and Engio SC (thiamethoxam 141 g/l, lambda-cyhalotryn 50 g/l) 0.18 l/ ha — 48.54%. 12—14 DAAT, Avaunt EC insecticide at the rate of 0.17 l/ ha with 57.14% and Engio SC with 47.62% showed the highest percentage of population reduction, respectively. The records conducted during 2021—2022 were not fully statistically reliable. In 2021, the indicators of the calculations carried out using the technique of «direct counting on plants» were statistically reliable, in 2022 — counting using yellow sticky trap. When comparing the number of pests caught in yellow and blue sticky traps, the number in yellow traps was many times greater than in blue.
 Conclusions. Avaunt EC and Engio SC insecticides showed the most effective efficiency — 65.69% and 54.26%, the lower efficacy — Coragen SC with 11.38%. The highest efficiency can be observed 1—3 days after application, which is due to the direct contact of pests with CPP. 12—14 days after application, most of CPP showed a slight decrease in the number of pests, except Avaunt EC and Engio SC with 57.14 and 47.62%, respectively.

EPPO ontology: a semantic-driven approach for plant and pest codes representation.

The agricultural industry and regulatory organizations define strategies and build tools and products for plant protection against pests. To identify different plants and their related pests and avoid inconsistencies between such organizations, an agreed and shared classification is necessary. In this regard, the European and Mediterranean Plant Protection Organization (EPPO) has been working on defining and maintaining a harmonized coding system (EPPO codes). EPPO codes are an easy way of referring to a specific organism by means of short 5 or 6 letter codes instead of long scientific names or ambiguous common names. EPPO codes are freely available in different formats through the EPPO Global Database platform and are implemented as a worldwide standard and used among scientists and experts in both industry and regulatory organizations. One of the large companies that adopted such codes is BASF, which uses them mainly in research and development to build their crop protection and seeds products. However, extracting the information is limited by fixed API calls or files that require additional processing steps. Facing these issues makes it difficult to use the available information flexibly, infer new data connections, or enrich it with external data sources. To overcome such limitations, BASF has developed an internal EPPO ontology to represent the list of codes provided by the EPPO Global Database as well as the regulatory categorization and relationship among them. This paper presents the development process of this ontology along with its enrichment process, which allows the reuse of relevant information available in an external knowledge source such as the NCBI Taxon. In addition, this paper describes the use and adoption of the EPPO ontology within the BASF's Agricultural Solutions division and the lessons learned during this work.

Real-time on-site detection of the three 'Candidatus Liberibacter' species associated with HLB disease: a rapid and validated method.

Huanglongbing (HLB) is a devastating disease that affects all commercial citrus species worldwide. The disease is associated with bacteria of three species of the genus 'Candidatus Liberibacter' transmitted by psyllid vectors. To date, HLB has no cure, so preventing its introduction into HLB-free areas is the best strategy to control its spread. For that, the use of accurate, sensitive, specific, and reliable detection methods is critical for good integrated management of this serious disease. This study presents a new real-time recombinase polymerase amplification (RPA) protocol able to detect the three 'Ca. Liberibacter' species associated with HLB in both plant and insect samples, validated according to European and Mediterranean Plant Protection Organization (EPPO) guidelines and tested on 365 samples from nine different geographic origins. This new protocol does not require nucleic acid purification or specialized equipment, making it ideal to be used under field conditions. It is based on specific primers and probe targeting a region of fusA gene, which shows a specificity of 94%-100%, both in silico and in vitro, for the 'Ca. Liberibacter' species associated with HLB. The analytical sensitivity of the new protocol is excellent, with a reliable detection limit in the order of 101 copies per microliter in HLB-infected plant and insect material. The repeatability and reproducibility of the new methods showed consistent results. Diagnostic parameters of the new RPA protocol were calculated and compared with the gold standard technique, a quantitative real-time PCR, in both crude extracts of citrus plants and insect vectors. The agreement between the two techniques was almost perfect according to the estimated Cohen's kappa index, with a diagnostic sensitivity and specificity of 83.89% and 100%, respectively, and a relative accuracy of 91.59%. Moreover, the results are obtained in less than 35min. All these results indicate the potential of this new RPA protocol to be implemented as a reliable on-site detection kit for HLB due to its simplicity, speed, and portability.

What Did We Achieve Through VALITEST, an EU Project on Validation in Plant Pest Diagnostics?

Ensuring the reliability of diagnostic activities is an essential cornerstone of plant health strategies to reduce the risk of entry and spread of plant pests in a region and ultimately their impacts. Diagnostic tests should be validated to ensure that they are fit for purpose. Validation is usually done by diagnostic laboratories, although companies commercializing diagnostic kits also produce validation data for their products. Due to the high number of pest, matrix, and method combinations and given the significant resources required to validate tests, it is essential that validation data are shared with the entire diagnostic community and produced in a harmonized way to facilitate their use by different stakeholders. Indeed, the selection of tests to be used in specific contexts is not the sole responsibility of diagnostic laboratories but also involves national plant protection organizations. The VALITEST EU project (2018 to 2021) was established to tackle all these issues. New validation data for tests targeting important pests for the European and Mediterranean Plant Protection Organization region were produced. Guidelines to improve and harmonize the validation framework were developed. Sharing of validation data and experience was ensured through the development of new or existing databases, the organization of training courses, and the dissemination of the project outputs in scientific publications and standards. Finally, the involvement of researchers, diagnosticians, policy makers, inspectors, and industries and the establishment of the European Plant Diagnostic Industry Association were important actions to strengthen the interactions between plant health stakeholders.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Citizen science at school increases awareness of biological invasions and contributes to the detection of exotic ambrosia beetles

The serious and growing threat posed by biological invasions to biodiversity and livelihoods means that public engagement in dealing with problems of invasive alien species is ever more urgent and necessary hence a citizen science experiment was carried out in north-eastern Italy. The study aimed i) to raise awareness of invasive alien species threatening trees and forests, and ii) to perform monitoring activities of a group of wood borers as an example, involving teachers and students (aged 11 to 18) of high schools. Students and teachers were given background knowledge and trained about the protocol for collecting data in schoolyards/grounds. Native (Anisandrus dispar (Fabricius, 1792), Xyleborinus saxesenii (Ratzeburg, 1837)) and exotic ambrosia beetles (Anisandrus maiche Kurentsov, Cnestus mutilatus (Blandford), Xylosandrus crassiusculus (Motschulsky, 1866), Xylosandrus germanus (Blandford, 1894)) were used as the target species, as they could be easily detected through accessible and low-cost traps requiring limited effort. The traps were exposed for 24 hours weekly between March and June 2021. The experiment also aimed to strengthen public involvement, connecting environmental education and experiential outdoor learning. The mutual collaboration resulted in a wider knowledge of the potential impact of exotic species. It also led to new geographical citizen-science records of two alien ambrosia beetles considered to be quarantine pests by the European Union: C. mutilatus, new for the European part of the EPPO (European and Mediterranean Plant Protection Organization) region, and A. maiche, which was previously found only in Eastern European EPPO member countries.

Molecular Diagnostics in Tomato: Chip Digital PCR Assays Targeted to Identify and Quantify Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum in planta

Clavibacter michiganensis subsp. michiganensis (Cmm) and Ralstonia solanacearum (Rs) are important bacterial pathogens of tomatoes (Solanum lycopersicum), are included in A2 list in the EPPO (European and Mediterranean Plant Protection Organization) region and are recommended for regulation as quarantine pests. The control of quarantine pathogens requires accurate and rapid detection tools. In this study, a method based on chip digital PCR (cdPCR) was developed to identify and quantify Cmm and Rs. The assays were tested on pure bacteria samples and on tomato samples naturally contaminated or spiked with bacteria DNA. For a better estimation of infection level in host plants, duplex assays that are able to simultaneously amplify plant and bacteria DNA were developed. The two cdPCR assays proposed can be used for the rapid and timely detection of this group of high-risk quarantine bacteria to prevent the spread of pathogens and the occurrence of disease in other areas.

A Quantitative Real-Time PCR Assay for Early Detection and Quantification of Ramularia mali, an Emerging Pathogen of Apple Causing Dry Lenticel Rot.

Ramularia mali is an emerging pathogen of apple (Malus domestica) causing dry lenticel rot. No preventive measures have been adopted due to the lack of knowledge about the life cycle and epidemiology of this pathogen. In a preliminary survey to identify the agents of dry lenticel rot of apple, R. mali was constantly associated with the disease. Using isolates from this survey, a SYBR Green quantitative PCR (qPCR) assay was developed, using calmodulin as target gene, for the detection and quantification of R. mali in apple fruit. The qPCR assay was validated in terms of specificity, sensitivity, repeatability, and reproducibility following the international European and Mediterranean Plant Protection Organization standard PM 7/98. The primers amplified a region of 237 bp specific to R. mali. The specificity was validated with 20 fungal species commonly found on apple, and 36 strains of R. mali and closely related species of the R. eucalypti species complex. Positive amplifications were obtained only with DNA of R. mali and no cross-reaction was detected with the other fungal species. Sensitivity was assessed with serial dilutions of target DNA and the limit of detection was 100 fg. No influence of host DNA was observed when target DNA was diluted on the DNA of Ambrosia and Golden Delicious apple. The assay permitted to detect and quantify R. mali in symptomatic and asymptomatic fruit. The presence of R. mali on asymptomatic Ambrosia and Golden Delicious apple fruit was demonstrated, and the presence of the pathogen was reported for the first time on Jeromine, Gala, Opal, and Story Inored fruit. This assay could be useful in clarifying the life cycle of this pathogen in order to build up an effective disease management strategy. Furthermore, the early detection of the pathogen on asymptomatic apple fruit could be used to forecast the development of dry lenticel rot, supporting the packinghouse operators in deciding the storage length of apple lots.

First report of Tomato leaf curl New Delhi virus in Türkiye

A leaf curl disease has been observed frequently in greenhouses where cucumber (Cucumis sativus), melon (Cucumis melo) and squash (Cucurbita pepo) are produced in Antalya since 2019. The infected plants showed symptoms including apical leaf curl, vein swelling, severely stunted, wrinkled leaves, fruit shape and texture disorder and a conspicuous mosaic pattern (Figure 1-5). These symptoms are similar to those caused by Tomato leaf curl New Delhi virus (ToLCNDV), an important member of the genus Begomovirus, family Geminiviridae. ToLCNDV is transmitted by Bemisia tabaci in a persistent circulative manner, causing serious damage to a wide variety of plant species, especially in the Solanaceae and Cucurbitaceae families (Briddon, 2015). ToLCNDV was first identified on solanaceous crops in India (Padidam et al., 1995) but the virus is recognised as an emerging threat worldwide due to the severity of the disease and its increasing prevalence. Outbreaks have recently occurred in countries in Africa, Asia and Europe (European and Mediterranean Plant Protection Organization, 2023). Samples were collected from eight adjoined greenhouses in Antalya, four growing cucumber, two melon and two squash. Three leaf samples and one fruit sample were collected individually from a diseased plant in each of the greenhouses, that is 32 samples in total. Total nucleic acid was extracted from the leaf and fruit samples and analysed by PCR with specific ToLCNDV primers (ToA1-F and ToA1-R) which amplify a 504 bp fragment of viral DNA-A (Saez et al., 2016). The following conditions were used for PCR: 95°C for 5 min; 35 cycles of 30 s at 95°C, 45 s at 55°C and 45 s at 72°C, followed by a final 72°C extension step for 10 min. Sequences were obtained from samples from each of the eight plants and were found to be identical (GenBank Accession No. MZ458535). BLAST analysis showed that there was 99% identity with a ToLCNDV isolate (MH475397). Antalya is a major area of greenhouse and seed production in Türkiye and the Mediterranean basin. ToLCNDV and other plant viruses are serious threats to solanaceous and cucurbit crops grown in the region since these crops may be infected through mechanical contact, seeds and insects (Kil et al., 2020). Quarantine remains the most effective way to prevent and control such viral infections. Diseased plants in the affected greenhouses were destroyed and the houses sanitised using solarization and disinfectant. Further surveys were done in 2020 and 2021 in the affected and neighbouring greenhouses and potentially virus-infected plants (~ 84 cucurbit samples) were tested by PCR as described above. No ToLCDV infection was found indicating that the virus had been eradicated. To our knowledge, this is the first report of ToLCNDV in Türkiye.

Introduction to PM 7 Standards on Diagnostics

EPPO BulletinVolume 53, Issue 1 p. 40-41 EUROPEAN AND MEDITERRANEAN PLANT PROTECTION ORGANIZATION Introduction to PM 7 Standards on Diagnostics The following revised Standards are included in this issue: PM 7/31 (2), PM 7/44(2). First published: 29 March 2023 https://doi.org/10.1111/epp.12921Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume53, Issue1April 2023Pages 40-41 RelatedInformation

The Need and a Vision for a Diagnostic Assay Validation Network

Plant biosecurity depends heavily on early detection of biological pest and disease threats and accurate diagnosis of causal agents. The information from these activities is disseminated and communicated with decision makers to promote effective mitigation. In the United States, most land-grant university, state, and private diagnostic and expert pest identifier labs are successfully networked via the U.S. Department of Agriculture Animal Plant Health Inspection Service, Plant Protection and Quarantine (USDA APHIS PPQ; https://www.aphis.usda.gov/ ), the National Plant Diagnostic Network (NPDN; https://www.npdn.org ), and the National Clean Plant Network (NCPN; https://www.nationalcleanplantnetwork.org/ ). These organizations provide access to training, shared protocols, and standardized communications. For example, the NPDN has secure communications tools that network leadership built into its IT infrastructure and deploys a formal communications process and protocol for regulatory and high-risk samples. Additionally, the diagnostic networks adopt protocols from regulatory organizations such as the USDA-APHIS, European and Mediterranean Plant Protection Organization (EPPO), North American Plant Protection Organization (NAPPO), and other international and national plant protection organizations (NPPOs). However, plant health diagnosticians also need access to validated protocols for endemic and nonregulated organisms. Although there is strong networking of diagnostic laboratories, plant disease diagnostic assay development and validation are siloed, representing a critical gap in our biosecurity infrastructure, especially for new or emerging pathogens. Most assays are developed in independent research labs and then chosen independently by the end-use laboratory. Assay developers, those who provide reference materials, labs that can provide ring testing for validation, and assay end users could be connected and integrated for more streamlined operations. Terminology and validation protocols need to be standardized within the United States and harmonized with our trade partners to ensure understanding. The ideal would be availability of standard, taxon-specific validation protocols, essential reference materials, and appropriate control materials for use to quickly develop and deploy assays in emergencies, as well as for day-to-day testing. This perspective article provides a summary of principles of assay validation, fitness-for-purpose concepts, and the need for continuous evaluation of assay performances. We describe the existing capacity and resources needed to efficiently develop, validate, and use diagnostic tests, as well as the inefficiencies and resource shortfalls currently faced. We propose development of a Diagnostic Assay Validation Network (DAVN) system to coordinate resources at the national level and harmonize with our partners at the international level. Here, we outline a project to set up the DAVN with the objectives of standardizing terminology and statistics and facilitating location of reference materials used in diagnostic assay development and validation via a portal of tools designed by and for the plant disease research and extension community. Although many of the concepts are also relevant to insects and other pests, the focus of this article is primarily plant pathogens.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Development of New Preventive Strategies for Pine Pitch Canker Caused by Fusarium circinatum in Irrigation Water and Evaluation in a Real Nursery Context

Fusarium circinatum is one of the many threats to forests and Pinus nurseries all over the world, being classified as a quarantine organism by several organizations and governing bodies, such as the European and Mediterranean Plant Protection Organization (EPPO) and the European Union (EU), with associated phytosanitary measures in place to prevent its spread through the various means of dispersal. One such means of dispersal is the water used for irrigation in nurseries, which can contain fungal propagules. Three different treatments, namely, Desogerme, Intra Hydrocare and sodium hypochlorite (NaClO), were tested for their efficacy in eliminating F. circinatum spores in water at several concentrations. Those that showed 100% disinfection rates were selected for further assays regarding seed germination and water quality impact. From these studies, Desogerme 1% and Intra Hydrocare 4% were then selected for large-scale seed germination and plant certification assays in nurseries, where they showed promising results in regard to the prevention of infections in nurseries, and in this way, contribute to the efforts of mitigating this disease.