European Plant Protection Organization Research Articles

Анализ распространения фитоплазм почернения древесины и золотистого пожелтения на виноградниках Краснодарского края

The purpose of research is to study the spread of phytoplasma infection in the vineyards of the Krasnodar Region using the PCR method. The object of the study is grape plants of various varieties with external symptoms of phytoplasma infection. The material for total DNA extraction was symptomatic leaves (their veins and petioles) collected from plantings of Shiraz, Chardonnay, Cabernet Sauvignon, Saperavi, Mizar, Moldova, Augustine, Pinot Blanc, Riesling, and Aligote varieties. DNA extraction was carried out using a buffer based on the cationic detergent CTAB (cetyltrimethylammonium bromide), according to the protocol recommended by EPPO (European plant protection organization) for the isolation of phytoplasmas from grape plant material. Sampling was carried out in industrial vineyards of the Krasnodar Region in 9 different geographical locations of the Novorossiysk, Temryuk and Anapa Regions. DNA was isolated from symptomatic leaves according to the protocol recommended by EPPO (European plant protection organization). Determination of the presence of phytoplasma infection was carried out by real-time PCR on a Quant Studio 5 amplifier using a commercial kit designed to detect phytoplasmas – Candidatus Phytoplasma solani and Candidatus Phytoplasma vitis (Synthol) and a test system recommended by EPPO. Using a kit produced by Synthol, 239 samples were analyzed, including 12 rechecked with markers recommended by EPPO. As a result of the analysis, it was found that DNA of Candidatus Phytoplasma solani was present in 113 samples studied. DNA from the phytoplasma Candidatus Phytoplasma vitis, which causes golden yellowing of leaves, was not detected in any of the analyzed samples.

First report of Cryptostroma corticale causing sooty bark disease in California and first worldwide report of silver maple as a host.

In November 2022 and February 2023, CAL FIRE tree health experts examined four maples (Acer spp.) planted decades earlier in a residential setting in Elk Grove, Sacramento Co., California (USA). Three of the trees were silver maples (Acer saccharinum ) and one was a Norway maple (A. platanoides); both species are exotic to California. The trees were in an irreversible state of decline, with the canopy substantially thinned and browning. Extensive bark cankers elongating longitudinally along the stem were visible on all trees (Fig. S1). Cankers were filled by fungal stromata protruding through the bark and producing masses of elliptical dark brown conidia (Fig. S2), approximately 5.5 x 3.7 um in size, giving the cankers a sooty appearance. The cankered bark could be peeled off easily, revealing dark and discrete lesions in the phloem and xylem. Samples from the three trees were shipped to the U.C. Berkeley Forest Pathology and Mycology Laboratory and to the CDFA PPDC in Sacramento, CA. In the laboratories, small wood chips were taken from the margins of the lesions, surface sterilized by dipping them for 30 seconds in 70% Ethanol, rinsed for 30 seconds in sterile water and plated onto 2.5% Malt Extract Agar amended with 0.3g/L Streptomycin or onto one-half strength acidified potato dextrose agar (APDA). Two morphologically identical cultures were obtained, one (T2) from a silver maple and one (T6) from the Norway maple. Cultures were then grown in liquid 2.5% malt extract broth and, after one week, DNA was extracted using the Qiagen Plant DNeasy DNA extraction kit. The ITS sequences are diagnostic for this fungus (Li et al. 2021) and those of the two cultures (GB OR064033 and OR933565) were 100% homologous to GenBank sequences of Cryptostroma corticale ( e.g. GB OP474010-11). The RPB2 sequence of T2 ( GB OR992132) was 100% homologous to that of C. corticale (GB HG934116.1). The isolate obtained from silver maple was inoculated in four potted silver maples by removing a bark disk 50 mm in diameter with a cork borer in three spots staggered at different heights and sides on the stem, placing a colonized agar plug of C. corticale in contact with the phloem, replacing the bark disk and wrapping with parafilm. Two control trees were mock inoculated using sterile agar plugs. Trees were in 57 L pots, had an average stem caliper of 2.7 cm, an average height of 3.5 m and were kept in a lath house at average high temperatures of 18-24 degrees C. After ten weeks, average lesion length was 15.4 cm (SE= 4.6) and 4.3 cm (SE=2.3) in the fungus-inoculated and control trees, respectively. An ANOVA test, nesting lesions sizes within tree, determined lesions lengths were different between inoculated and mock trees (P= 0.04). The fungus was reisolated from all points in all inoculated trees but never from control trees. C. corticale was first described in the UK from sycamore maple (Acer pseudoplatanus) (Gregory et al. 1949) and is an emerging problem in Europe (Muller et al. 2023). In North America, it has been reported from A. negundo, A. campestre, A. macrophyllum and Cornus nuttallii (Worral 2020), and it appears to be present in the Pacific Northwest (Brooks et al. 2023, Goree 1969). Norway maple is included in the European Plant Protection Organization list of hosts for C. corticale (EPPO 2023), however our finding of C. corticale on silver maple is a first report of this host worldwide and of this pathogen in California. This report is noteworthy, given that C. corticale is also a human pathogen infecting the respiratory system (Braun et al. 2021).

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.

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.

First Report of the Root-Knot Nematode Meloidogyne luci on Tomato in Serbia.

Root-knot nematode (RKN) Meloidogyne luci Carneiro, Correa, Almeida, Gomes, Deimi, Castagnone-Sereno, and Karssen, 2014 was described from Brazil, Chile and Iran, parasitizing in various crops (Carneiro et al. 2014). It was later also described from Slovenia, Italy, Greece, Portugal, Turkey and Guatemala (review in Gerič Stare et al. 2017). It is considered an extremely damaging pest as it has a wide host range and infects numerous higher plants, including monocotyledons and dicotyledons as well as herbaceous and woody plants. This species was included in the European Plant Protection Organisation Alert List of harmful organisms. In Europe, M. luci has been detected in both greenhouse and field agricultural production (review in Gerič Stare et al. 2017). Furthermore, M. luci has been shown to survive winter in the field under continental and sub-Mediterranean climatic conditions (Strajnar et al. 2011). In August 2021, an official survey for quarantine RKN in Serbia (Province Vojvodina) revealed in a greenhouse in the village of Lugovo (43043'32,562; 19008'55,168), near Sombor, yellowing, stunning and extensive root galls on tomato (Solanum lycopersicum L.) cultivar Diva F1 caused by an unknown Meloidogyne sp. (Fig. 1). As correct identification is essential for effective pest management program, the next step was to identify the nematode species. Morphological characterization performed on freshly isolated females revealed perineal patterns similar to M. incognita (Kofoid and White, 1919) Chitwood, 1949. The shape was oval to squarish with the dorsal arch rounded to moderately high and without shoulders. The dorsal striae were wavy and continuous. The ventral striae were smooth and the lateral lines were weakly demarcated. The perivulval region was without striae (Fig. 2). The female stylet was robust with well-developed knobs and the stylet cone slightly curved dorsally. Although morphological characters was very variable, the nematode was suspected as M. luci based on comparison with originally described M. luci and M. luci populations from Slovenia, Greece and Turkey. Identification was achieved with subsequent species-specific PCR and sequence analysis. The nematode was determined to belong to the tropical RKN group and the M. ethiopica group using two PCR reactions as described by Gerič Stare et al. (2019) (Figs. 3 and 4). Identification was confirmed by species-specific PCR of M. luci as described by Maleita et al. (2021), and a band of approximately 770 bp was obtained (Fig. 5). In addition, the identification was confirmed by sequence analyses. The region of mtDNA was amplified with primers C2F3 and 1108 (Powers and Harris 1993), cloned, sequenced (acc. no. OQ211107), and compared to other Meloidogyne spp. sequences from the Genbank. The determined sequence is 100% identical to an unidentified Meloidogyne sp. from Serbia, while the next highest scores are sequences of M. luci from Slovenia, Greece and Iran, all of which have 99.94% sequence identity. In phylogenetic tree, all M. luci sequences including the sequence from Serbia belong to a single clade. Egg masses isolated from infected tomato roots were used to establish a nematode culture in greenhouse and they caused typical root galls on cultivar Maraton of tomato. The galling index assessed 110 days-post-inoculation was in the range 4-5 according to the scoring scheme (1-10) for field evaluation of RKN infestations (Zeck 1971). To our knowledge, this is the first report of M. luci in Serbia. The authors hypothesize that climate change and higher temperatures could lead to much greater spread and damage to various agricultural crops in the field by M. luci in the future. National surveillance program for RKN in Serbia continued in 2022 and 2023. A management program to control the spread and damage from M. luci will be implemented in Serbia in 2023. Acknowledgments: This work was financially supported by the Serbian Plant Protection Directorate of MAFWM in the frame of Program of Measures in Plant Health in 2021, the Slovenian Research Agency in the frame of Research Programme Agrobiodiversity (P4-0072) and the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia in the frame of Expert work in the field of plant protection (C2337).

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.

Transcriptome analysis reveals the high ribosomal inhibitory action of 1,4-naphthoquinone on Meloidogyne luci infective second-stage juveniles.

The root-knot nematode (RKN) Meloidogyne luci presents a threat to the production of several important crops. This nematode species was added to the European Plant Protection Organization Alert list in 2017. The scarce availability of efficient nematicides to control RKN and the phasing out of nematicides from the market have intensified the search for alternatives, such as phytochemicals with bionematicidal properties. The nematicidal activity of 1,4-naphthoquinone (1,4-NTQ) against M. luci has been demonstrated; however, knowledge of the potential mode(s) of action of this compound is still scarce. In this study, the transcriptome profile of M. luci second-stage juveniles (J2), the infective stage, in response to 1,4-NTQ exposure was determined by RNA-seq to identify genes and pathways that might be involved in 1,4-NTQ's mode(s) of action. Control treatments, consisting of nematodes exposed to Tween® 80 (1,4-NTQ solvent) and to water, were included in the analysis. A large set of differentially expressed genes (DEGs) was found among the three tested conditions, and a high number of downregulated genes were found between 1,4-NTQ treatment and water control, reflecting the inhibitory effect of this compound on M. luci, with a great impact on processes related to translation (ribosome pathway). Several other nematode gene networks and metabolic pathways affected by 1,4-NTQ were also identified, clarifying the possible mode of action of this promising bionematicide.

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 .

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

A new and accurate qPCR protocol to detect plant pathogenic bacteria of the genus ‘Candidatus Liberibacter’ in plants and insects

Four pathogenic bacterial species of the genus ‘Candidatus Liberibacter’, transmitted by psyllid vectors, have been associated with serious diseases affecting economically important crops of Rutaceae, Apiaceae and Solanaceae families. The most severe disease of citrus plants, huanglongbing (HLB), is associated with ‘Ca. Liberibacter asiaticus’ (CaLas), ‘Ca. Liberibacter americanus’ (CaLam) and ‘Ca. Liberibacter africanus’ (CaLaf), while ‘Ca. Liberibacter solanacearum’ (CaLsol) is associated with zebra chip disease in potatoes and vegetative disorders in apiaceous plants. Since these bacteria remain non-culturable and their symptoms are non-specific, their detection and identification are done by molecular methods, mainly based on PCR protocols. In this study, a new quantitative real-time PCR protocol based on TaqMan probe, which can also be performed in a conventional PCR version, has been developed to detect the four known phytopathogenic species of the genus Liberibacter. The new protocol has been validated according to European Plant Protection Organization (EPPO) guidelines and is able to detect CaLas, CaLam, CaLaf and CaLsol in both plants and vectors, not only using purified DNA but also using crude extracts of potato and citrus or psyllids. A comparative analysis with other previously described qPCR protocols revealed that this new one developed in this study is more specific and equally or more sensitive. Thus, other genus-specific qPCR protocols have important drawbacks regarding the lack of specificity, while with the new protocol there was no cross-reactions in 250 samples from 24 different plant and insect species from eight different geographical origins. Therefore, it can be used as a rapid and time-saving screening test, as it allows simultaneous detection of all plant pathogenic species of ‘Ca. Liberibacter’ in a one-step assay.

CHARACTERISTICS OF THE FIRE BLIGHT PATHOGEN ERWINIA AMYLOVORA ISOLATED FROM FRUIT CULTURES IN SOUTHEASTERN KAZAKHSTAN

One of the most dangerous and quarantine diseases of fruit crops of the Rosaceae family is fire blight which affected on plants. Its causative agent Erwinia amylovora causes necrosis of all organs of host plants. In the territory of Republic of Kazakhstan, the disease belongs to a limited number of quarantine species. A special place is given to laboratory diagnostics of E.amylovora, which is necessary for reliable detection of the pathogen. Bacteriological analyses of diseased samples of fruit crops were carried out under laboratory conditions to isolate pathogens into pure cultures. Isolating of the quarantine and causative agent of fire blight into a pure culture was carried out on King B nutrient media, Levan and Miller Schroth selective media. They were tested for pathogenicity on indicator plants of indoor geranium by hypersensitivity reaction by the infectious-infiltration method of Clement. The pathogenicity of isolated bacteria was also tested by testing on unripe pear fruits, on which the damaging bacteria caused the release of milky-white exudate. After the selected isolates of the fire blight pathogen were identified by two pairs of specific primers PEANT F/PEANT2 R and FER F/rgER2-R approved by the European and Mediterranean Plant Protection Organization. For molecular differentiation, the nucleotide sequences of 16S rRNA genes were compared

Prioritization of absent quarantine pests in Brazil through the Analytical Hierarchy Process.

Introducing an Absent Quarantine Pest (AQP) can cause severe economic, social and environmental impacts, generating food insecurity. The Analytical Hierarchy Process (AHP) method is an excellent tool for prioritizing APQs, allowing countries to better prepare against these threats. This study aimed to determine which AQPs should be prioritized in Brazil. For this, 20 AQPs were selected from the Brazilian official list. The selection was based on pests intercepted by Brazil between 2015 and 2018 and by countries of the European and Mediterranean Plant Protection Organization, in the international movement of plants. It can be concluded that out of the 20 AQPs studied, 17 are the priority and that the AHP method is effective for this purpose. Other countries from different continents can use this methodology to prioritize PQAs and thus create strategic plans to prevent entry into their territories and economic, social, and environmental impacts.

Susceptibility of crop plants to the root-knot nematode Meloidogyne luci, a threat to agricultural productivity

The root-knot nematode (RKN) Meloidogyne luci is included in the Alert List of the European Plant Protection Organization, because it has potential negative impacts on economically important crops. Identification of plant species/cultivars resistant to M. luci is important for its management. Susceptibility of 35 commercial plant species/cultivars, from nine families to a M. luci isolate from Portugal was evaluated in pot assays, assessing root gall index (GI) and reproduction factor (Rf) 60 d after inoculation, with tomato ‘Coração-de-Boi’ used as the positive susceptible experimental control. Presence/absence of RKN resistance genes was also determined in the tomato and pepper cultivars. One cultivar of cabbage, three of lettuce, ten of pepper, one of sugar beet, and all the cultivars of Cucurbitaceae (five), Fabaceae (two) and Poaceae (one) were susceptible to M. luci (GI = 4-5; Rf = 2.1-152.3). One cultivar each of carrot, passion fruit, lettuce ‘Cocktail’, cabbage ‘Bacalan’, ‘Coração’ and ‘Lombarda’, and spinach ‘Tayto’ were resistant/hypersensitive (Rf < 1; GI > 2). The tomato ‘Actimino’, ‘Briomino’, ‘Veinal’ and ‘Vimeiro’, which carried at least one copy of the Mi-1.2 gene, were resistant to the nematode (GI = 1-2; 0.0 < Rf < 0.1). These results indicate that the tomato cultivars have potential to contribute to reduction of M. luci populations in agro-ecosystems and improve the crop yields.

Introduction to Diagnostics Standards

EPPO BulletinVolume 52, Issue 1 p. 65-66 EUROPEAN AND MEDITERRANEAN PLANT PROTECTION ORGANIZATION Introduction to Diagnostics Standards The following revised Standards are included in this issue: PM 7/99 (2), PM 7/30 (3) First published: 15 April 2022 https://doi.org/10.1111/epp.12842Read 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 onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume52, Issue1April 2022Pages 65-66 RelatedInformation

Evaluation of Droplet Digital PCR for the Detection of Black Canker Disease in Tomato.

Clavibacter michiganensis subsp. michiganensis, the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is widely distributed in the European and Mediterranean Plant Protection Organization region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the C. michiganensis subsp. michiganensis pathogen from other closely related subspecies of C. michiganensis, especially the nonpathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital PCR (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of C. michiganensis subsp. michiganensis. This new primers/probe set has been evaluated by real-time PCR (qPCR) and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all five C. michiganensis subsp. michiganensis strains, and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/ml for both pure C. michiganensis subsp. michiganensis cell suspensions and infected tomato seed, which was 100-fold more sensitive than qPCR performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect C. michiganensis subsp. michiganensis even with low bacterial load, which could facilitate both C. michiganensis subsp. michiganensis inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.