European And Mediterranean Plant Protection Organization Research Articles

Identification, detection and eradication ofHymenoscyphus fraxineusfrom ash (Fraxinus excelsior) seeds

AbstractThe European and Mediterranean Plant Protection Organization (EPPO) real-time PCR protocol for the detection of ash dieback (Hymenoscyphus fraxineus (T. Kowalski) Barak, Queloz, Hosoya) was optimized for diagnostics on ash (Fraxinus excelsior L.) fruits. Ash fruits were collected from a range of sites in Britain, from trees with various levels of infection. To assess the potential pathway and the presence of H. fraxineus, tissue components of ash fruits as well as ash flowers were tested for the presence of H. fraxineus DNA and RNA. The fungus was detected on ash fruits from trees on all five sampled sites including symptomless, lightly and highly infected sites. DNA of H. fraxineus was detected on the pericarps of all fruit lots and on seeds from fruit lots that included damaged and empty fruits, but not on embryos or flowers. RNA of H. fraxineus was never detected on any of the samples or sample types, indicating that H. fraxineus was only detected in an inactive form (i.e. dormant or dead). The absence of RNA from the pathogen suggests that only spores of H. fraxineus are present on ash fruits. A double hot-water treatment was evaluated as a control measure for the eradication of H. fraxineus on ash fruits; these were treated before and after warm stratification. Large proportions of ash fruits survived the double hot-water treatments, with a 60.4 ± 28.4 per cent germination success rate depending on crop year. Double hot-water treatment of ash fruits proved to be an efficient, low cost and low-tech approach for the eradication of H. fraxineus on ash fruits.

Hylotelephium spectabile, a New Host for Carnation Tortrix Moth (Cacoecimorpha pronubana) and Molecular Characterization in Greece.

Cacoecimorpha pronubana (Hübner) (Lepidoptera, Tortricidae) is a highly polyphagous pest of a wide range of crop and ornamental plants. It is of Mediterranean origin and widespread in European and Mediterranean Plant Protection Organization (EPPO) region. For the first time, infestations of Hylotelephium spectabile (Boreau) Ohba (syn.: Sedum spectabile Boreau) (Saxifragales, Crassulaceae) ornamental plants by C. pronubana larvae, in private gardens in urban area of Drama, Greece, were found. Species identification was conducted based on morphology of female genitalia. In addition, due to reports on occurrence of cryptic C. pronubana species within Europe, DNA barcoding was carried out to determine the molecular status of the pest. This communication reports a new host of C. pronubana and places the Greek pest population along with European species clade.

Solanum linnaeanum and Solanum sisymbriifolium as a sustainable strategy for the management of Meloidogyne chitwoodi

Root-knot nematodes (RKN), Meloidogyne spp., are important crop pests that cause severe losses in crop production worldwide, reducing both productivity and crop quality. Meloidogyne chitwoodi Golden, O'Bannon, Santo & Finley, 1980 is considered a quarantine organism by the European and Mediterranean Plant Protection Organization (EPPO) causing damage in tomato and potato crops. The development of nonchemical and sustainable management strategies to reduce nematode damage is crucial. The resistance of Solanum linnaeanum Hepper & P.-M.L. Jaeger and S. sisymbriifolium Lamarck cv. Sis 6001 to M. chitwoodi was evaluated based on gall index (GI), the Bridge & Page (1980) rating chart and reproduction factor (RF). Both plant species were resistant to M. chitwoodi. Solanum linnaeanum had an average of 519 small root swellings/plant, with 45% adult nematodes inside the roots, all males. Solanum sisymbriifolium had GI ≤ 2 and RF ≤ 1 with a high percentage (69%) of nematodes inside the roots that did not develop beyond the sexually undifferentiated second-stage. The use of S. linnaeanum as a new source of resistance is a good alternative for the control of RKN in the quest to develop nonchemical and sustainable management strategies to protect crops.

Genomics informed design of a suite of real-time PCR assays for the specific detection of each Xylella fastidiosa subspecies.

Existing methods for the identification of the subspecies of Xylella fastidiosa are time-consuming which can lead to delays in diagnosis and the associated plant health response to outbreaks and interceptions. Diagnostic markers were identified using a comparative genomics approach to allow fine differentiation of the very closely related subspecies. Five qPCR assays were designed to allow specific detection of X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, X. fastidiosa subsp. pauca, X. fastidiosa subsp. morus and X. fastidiosa subsp. sandyi. All assays were validated according to the European and Mediterranean Plant Protection Organisation (EPPO) standard PM7/98(2). All of the assays were shown to be specific to the target subspecies and all the assays could be used to detect femtogram quantities of X. fastidiosa DNA. At present, diagnosing the subspecies of X. fastidiosa requires multiple conventional PCR assays (although only available for three of the five subspecies) or multi-locus sequence typing which takes several days. By comparison, the new assays provide a substantial reduction in the turnaround time for direct identification to the subspecies level in as little as 75min.

Weed invasive

Biological invasions are increasingly attracting the attention of ecologists, so it seems that this phenomenon is relatively new. However, the beginning of the study of biological invasions is publication of the book "The Ecology of Invasions by Animals and Plants" (1958), which is classified as classic works of invasions. Research of biological invasion is focused on the study of crucial biological and ecological characteristics which enable species invasiveness, as well as the characteristics of the recipient community. Despite the intensive study of this phenomenon, there are still differing views whether only introduced alien species or even native species can be considered as invasive species. While some researchers consider that native species cannot belong to the invasive category, others thought that a species that is native, when expend to adjacent habitats, may also be considered as invasive. Understanding biological invasions as if there no difference between native and introduced allochthonous species eliminates the dilemma that arises in the case of cryptogenic species (species that cannot be clearly classified into one of these two groups), whether it is native or introduced species, the success of the spreading depends on ecological adaptations. In addition, there is also disagreement about the stages of biological invasions. One point of view is, invasion processes take place through three phases (introduction, colonization and naturalization), while to other, the introduction is an elementary requirement for invasion, colonization is an integral part of the concept of naturalization, and naturalization which is referred in to previous point of view, is the phase that corresponds to the term "invasiveness". The time and route of introduction, as well as the mode and pathways of spreading for a large number of adventitious weed species have not been precisely established. Depending on whether weed species are introduce accidentally or intentionally, they are divided into boyletophytes (intentionally introduce) and aboyletophytes (accidentally introduce), while according to period of introduction they are divided into archeophytes (introduced in the Paleozoic to Neolithic period), paleophytes (introduced during the Old and Middle Ages until the discovery of America), neophytes (introduced after the discovery of America until to World War II), and neotophytes (introduced from the World War II until nowadays). Invasive processes are very complex, and it is not easy to predict and evaluate the behavior of introduced species in a new environment. Depending on the shape, structure and size of the reproductive organs, invasive weeds can be spread in several ways: autochthonous (selfpropagating), anemohoric (spreading through the wind), hydrohorn (spreading through water), zohornous (spreading by animals) and anthroporous (spreading by human). The success of invasive processes depends on the ecological characteristics of species, environmental resources and degree of ecosystem damage into which species has reached. According to success of survival in the new environment, allochthonous species can have status of the ephemeral (species which temporarily retained in the new environment and do not form stable populations), naturalized (species that are maintained in the new habitat but do not have an expansive character) and invasive (species that are adapted to the new habitat, spreading, forming indigenous part of flora and leave fertile offspring). The ecological-genetic potential of these species is the result of genetic diversity and their capacity for ecological adaptation; mutations, recombination, hybridization and introgression are the main processes responsible for genetic diversity. In the area of introduction, allochthonous species manifest numerous negative impacts on agriculture, ecosystems, human and domestic animal health, socioeconomic relationships and can jeopardize the survival of native species. Invasions are generally classified as negative occurrences, although in some circumstances they may also have a positive effect on the environment. For example, introduction of allochthonous species can enrich biodiversity, in case when number of introduced species far exceeds the number of extinct species. Despite the great scientific and technological progress, the problem of biological invasions cannot be completely resolved, but it is possible to limit these processes and reduce their negative consequences. The European and Mediterranean Plant Protection Organization (EPPO) dedicate special attention to the risks of invasive plant species and recommends measures to prevent their introduction and spread, with special reference to organisms that cause damage in agriculture. Control of these species implies application of all available preventive and direct measures, integrated into a system of integral measures.

Invasive Agricultural Pest Drosophila suzukii (Diptera, Drosophilidae) Appeared in the Russian Caucasus.

Simple SummarySpotted-wing drosophila, Drosophila suzukii, is one of the most important invasive pests of fruit and wine production worldwide. This species feeds on cherry, raspberry, blackberry, blueberry, strawberry, peach, plums, grape, and other soft fruits. It causes significant damage because, unlike most other Drosophila species, it oviposits and feeds on healthy fruits. Drosophila suzukii is native to East Asia and has been rapidly spreading through Europe, where it is considered a quarantine pest, since 2008. Herein, we report the first records of spotted wing drosophila in European Russia. In 2017 and 2020, we placed baited traps in different districts of the resort city of Sochi (Black sea Coast of the Caucasus, Krasnodar Territory, Russia) and collected 49 adults of this species. They were identified by the typical female ovipositor, spotted wings of the males, and other characters. Krasnodar Territory is one of the main fruit-producing regions of Russia. Therefore, populations of this pest should be monitored and measures to minimize economic damage should be taken.Spotted-wing drosophila Drosophila suzukii (Matsumura) is one of the most important invasive pests of fruit and wine production worldwide. This species feeds on Prunus spp., Rubus spp., Fragaria spp. (Rosaceae), Vaccinium spp. (Ericaceae), Vitis spp. (Vitaceae), and other soft fruits. It causes significant damage because, unlike most other Drosophila species, it oviposits and feeds on healthy fruits. Drosophila suzukii is a quarantine pest that is included on the European and Mediterranean Plant Protection Organization (EPPO) A2 List. This species is native to East Asia and has been rapidly spreading through Europe since 2008. Herein, we report the first records of D. suzukii in European Russia. In 2017 and 2020, we placed baited traps in different districts of the resort city of Sochi (Black sea Coast of the Caucasus, Krasnodar Territory, Russia). Three specimens of D. suzukii were collected in June 2017, two specimens in September 2017, and 44 specimens in September 2020. Specimens were identified by the typical female ovipositor, spotted wings of the males, and other morphological characters. Krasnodar Territory is one of the main fruit-producing regions of Russia. Therefore, populations of this pest should be monitored and measures to minimize economic damage should be taken.

Strawberry vein bordering virus (SVBV) is a dangerous strawberry pathogen

Strawberry vein bordering virus (SVBV) is a harmful strawberry pathogen and is spread in a number of European countries that supply the planting material of this crop to the Russian Federation. The virus spreads effectively with infected planting material and several aphid vectors. The climatic and agroecological conditions of many regions of the Russian Federation are favorable for the acclimatization and wide spread of this pathogen. SVBV is a quarantine object of European and Mediterranean Plant Protection Organization (EPPO) and several countries of the world. However, this dangerous pathogen is not regulated by the quarantine lists of the Eurasian Economic Union (EAEU) and the Russian Federation. The most effective measure to prevent the spread of SVBV is the use of virus-free planting material obtained in accordance with an internationally recognized certification scheme. The most important measures to prevent the spread of SVBV are the control of aphid vectors and the reduction of the duration of the operation of the strawberry plantations. In existing plantations, due to the lack of registered antiviral drugs, the fight against viruses is difficult and consists in the removal and destruction of infected plants.

A simple PCR-based approach for rapid detection of Ips typographus and Ips duplicatus in the presence of (associated) symbionts and parasites

In plant pest diagnosis, Sanger sequencing of marker genes (DNA-barcoding) is the most applied and appropriate method for the identification of insects. Standard PM7/129 of the European and Mediterranean Plant Protection Organization (EPPO) includes a number of primers and PCR protocols for diagnosing insect pests. LCO1490 and HCO2198 primers recommended herein were shown to be excellent tools for amplifying a fragment of the COI gene from a vast range of arthropods. The COI barcoding region is available for thousands of arthropod taxa in public databases and ready-to-use for evolutionary studies. However, we found that LCO1490 and HCO2198 primers are not working for bark beetles of genus Ips. The attempt to amplify this gene fragment from an individual organism using the barcoding primers led to DNA amplification of associated wasps and nematodes, which were apparently vectored by the beetle. Thus, new primers for Ips that bind specifically to another (non-barcoding) region of the COI gene were developed in the past years. These primers were successfully applied in phylogenetic analyses of this genus, resulting in the adverse effect that COI-based Ips phylogenies cannot be expanded to higher systematic categories without sequencing the outgroups (as they are not available in databases yet). Here we provide new primers for Ips that differ significantly from DNA sequences of Ips-associated wasps and nematodes and bind to a COI fragment that largely overlaps with the barcoding region proposed in the EPPO standard. Furthermore, using these primers we developed a quick PCR-based test for detecting Ips duplicatus, a quarantine pest currently emerging in many European countries.

Оccurrence of bacterial spot caused by Xanthomonas arboricola pv. pruni on peach and apricot in Serbia.

Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of leaves and fruits, defoliation, fruit dropping and twigs, branches or trunk cankers in most cultivated and ornamental Prunus species. The bacterium is listed as an EPPO (European and Mediterranean Plant Protection Organisation) A2 quarantine pathogen. Xap was first detected in 2019 on peach [Prunus persica L., unknown cultivar (cv.)] leaves in a 13-year-old orchard located in Irig (GPS: 45°6'10.538'' N, 19°54'8.04'' E), with a disease incidence of 10-20%. Thereafter, Xap was detected in 2020 on apricot (Prunus armeniaca L., cvs. NS4, NS Rodna and Roxana) leaves and fruits in a 5-year-old orchard located in Bešenovo (GPS: 45°04'59.0'' N, 19°41'23.0'' E), with disease incidence of 30-50%. Symptoms on leaves appeared along leaf midribs or margins in form of brown to black spots, with a pale green to yellow halo, evident on both leaf surfaces. The diseased area on leaves dropped out giving a shot-hole appearance, leaves turn yellow and drop prematurely. Bacterial spots of apricot fruits appeared in form of water-soaked or dark brown sunken lesions. Primarly detection of Xap in collected samples was obtained using polymerase chain reaction (PCR) with Xap - specific primers XapY17-F/XapY17-R, after amplifying target fragments of 943-bp (1). Xap reference strain NCPPB 3156 served as positive control. Isolations performed on yeast extract-dextrose-calcium carbonate agar (YDC) resulted in pale yellow, circular, raised and mucoid colonies after 3 days at 26 °C. A total of 20 representative isolates were aerobic, gram negative, catalase positive, oxidase and arginine dihydrolase negative; they induced hypersensitivity reaction, reduced nitrates, hydrolyzed aesculin and gelatine, but not starch, produced H2S, not produced indole (2). NCBI BLASTn search of the ftsX (ABC transporter ATP-binding protein) sequences (1) indicated 100% identity of Serbian isolates with Xap strains XAP HU2 (P. persica, Acc. no. MG049921) and XAP HU1 (P. armeniaca, Acc. no. KY039173) from Hungary. The nucleotide sequences of one isolate from peach (Xp219) and one from apricot (Xp320) were deposited in GenBank under Acc. nos. MT890969 and MT890970, respectively. Pathogenicity was performed on detached peach and apricot leaves for all 20 isolates and on leaves and shoots of potted 1-year-old plants of peach (cv. Vineyard peach) and apricot (cv. NS Rodna) for three isolates from each host (3, 4). Sterile distilled water and reference strain NCPPB 3156 were used as negative and positive controls. On detached leaves all isolates caused typical water-soaked spots 3 days after inoculation (DAI), while 10 DAI spots became brown and necrotic. Same symptoms were appeared on the leaves of potted plants. On peach and apricot shoots water-soaked, slightly reddish lesions emerged on inoculation sites 7-10 DAI, while 20 DAI lesions become dark, circular to elliptical 3.5-4 and 2.5-3 cm in size for peach and apricot, respectively. Negative controls were symptomless. Reisolated bacteria were confirmed to be the same as the original using PCR (1), fulfilling Koch's postulates. This is the first report of Xap in Serbia, which has occurred with a limited distribution in the Fruška Gora region (Vojvodina). Only two orchards in Serbia have been deteched with Xap so far. In the diseased peach orchard Xap was eradicated by uprooting trees. The apricot orchard is still under official control to limit disease spread. Appropriate cultivation practices, national inspection and surveillance is in place to prevent further pathogen spread and establishment to new hosts and regions in Serbia.

Antibacterial Activity of Essential Oils and Trametes versicolor Extract against Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum for Seed Treatment and Development of a Rapid In Vivo Assay.

Clavibacter michiganensis subsp. michiganensis (Smith) Davis et al. (Cmm) and Ralstonia solanacearum Yabuuchi et al. (Smith) (Rs) are important seed-borne bacterial pathogens of tomato (Solanum lycopersicum) listed as A2 pests in the EPPO (European and Mediterranean Plant Protection Organization) region. At present, there are few strategies to control these pathogens, and seed control with eco-compatible approaches is widely encouraged. In this work, the essential oils (EOs) of oregano (Origanum vulgare), garlic (Allium sativum), basil (Ocimum basilicum), cinnamon (Cinnamomum zeylanicum), clove buds (Syzygium aromaticum), thyme (Thymus vulgaris), and Trametes versicolor extract (Tve) were tested in vitro for their antimicrobial activity against Cmm and Rs (broth microdilution method). The tested EOs and the Tve extract caused a significant inhibition of bacterial growth, with very promising MBC (minimum bactericidal concentration) and MIC90 (minimum inhibitory concentration causing a 90% growth inhibition) values. Moreover, an in vivo germination test showed no major reduction in seed germination when the substances were applied as seed treatment. A rapid molecular screening method has been developed, through real-time PCR, for the specific quantification of Cmm in the presence of a vegetable matrix to test in vivo the antimicrobial efficacy of oregano and cinnamon oil on seed treatment without resorting to whole plant essays, which are time- and space-consuming.

Specific Real-Time PCR for the Detection and Absolute Quantitation of Grapevine Roditis Leaf Discoloration-Associated Virus, an EPPO Alert Pathogen.

Grapevine Roditis leaf discoloration-associated virus (GRLDaV) is an emerging grapevine pathogen included in the European and Mediterranean Plant Protection Organization (EPPO) alert list due to its ability to damage grapevine crops and cause production losses. This work aimed to develop a specific and reliable diagnostic tool that would contribute to preventing the spread of this pathogen. Therefore, a TaqMan real-time quantitative PCR was developed. The method was validated according to EPPO guidelines showing a high degree of analytical sensitivity, analytical specificity, selectivity, and repeatability and reproducibility. The sensitivity of this method is much higher than the sensitivity reached by previously reported methods even when tested in crude extracts, which could allow rapid testing by avoiding nucleic acid extraction steps. The method was also able to detect GRLDaV isolates from all the geographic origins reported so far, despite their high degree of genetic diversity. In addition, this new technique has been successfully applied for the quantitative detection of GRLDaV in plant material and two mealybug species, Planococcus citri and Pseudococcus viburni. In conclusion, the methodology developed herein represents a significant contribution to the diagnosis and control of this emerging pathogen in grapevine.

Analysis of Trade Flows of Ornamental Citrus Fruits and Other Rutaceae in the Mediterranean Basin and Potential for Xantomonas citri Introduction

In this study, we built a basic scenario for risk assessment of the introduction of Xantomonas citri (X. citri), an agent of bacterial citrus canker, through international trade activities. According to the international phytosanitary authority European Food Safety Agency (EFSA), X. citri is currently included in the European Union A1 list (quarantine pests not present in the area) of the European and Mediterranean Plant Protection Organization (EPPO). Therefore, at the moment, to counter the spread of X. citri, some pest-specific phytosanitary requirements are foreseen in the case of citrus fruit commercial activities. One possible introduction route is through some ornamental Rutaceae, which are widely cultivated in Mediterranean countries, where they are economically important and have a social impact on the employees involved and the related industries. To assess the risk of introducing X. citri, we distinguished the import and export territories and the type of import material, and formulated a basic hypothesis linked to the positive correlation between commercial dependence on citrus imports from countries of the Mediterranean Basin and potential risk of invasion.

The Risk of Bark and Ambrosia Beetles Associated with Imported Non-Coniferous Wood and Potential Horizontal Phytosanitary Measures

Many bark and ambrosia beetle species (Coleoptera: Scolytinae and Platypodinae) are known to have spread worldwide in relation to international trade. Concerns have been expressed within the European and Mediterranean Plant Protection Organization (EPPO) about recent introductions of non-indigenous species of these groups. Regulation of the non-coniferous wood trade into many EPPO member countries is currently not sufficient to cover such risks. In 2018–2019, an EPPO study on the risk of bark and ambrosia beetles associated with imported non-coniferous wood was carried out, and the key characteristics contributing to the pest risk from introduced species were determined using expert consensus. This paper summarizes the key findings of the study, which are available in full detail on the EPPO website. The study identified biological and other risk factors and illustrated them with examples from 26 beetle species or groups of species known to be invasive or posing a threat to plant health. These representative species were classified into three categories based on known damage and level of uncertainty. In the present article, factorial discriminant analyses were used to identify features of bark and ambrosia beetle biology associated with damage caused and invasiveness. Based on the information assembled and consideration of the risk factors, it was recommended that in order to prevent the introduction of new bark and ambrosia beetles via non-coniferous wood commodities, horizontal phytosanitary measures should be adopted, irrespective of the host plant species and the origin (i.e., for all genera of non-coniferous woody plants and from all origins). Phytosanitary measures are presented here for various wood commodities.

Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species within the Genus Clavibacter, Including Nonpathogenic Tomato-Associated Strains.

Members of the genus Clavibacter are economically important bacterial plant pathogens infecting a set of diverse agricultural crops (e.g., alfalfa, corn, potato, tomato, and wheat). Tomato-associated Clavibacter sp. strains account for a great portion of the genetic diversity of the genus, and C. michiganensissensu stricto (formerly C. michiganensis subsp. michiganensis), causing bacterial canker disease, is considered one of the most destructive seed-borne agents for the crop worldwide. However, current taxonomic descriptions of the genus do not reflect the existing diversity of the strains, resulting in unsatisfactory results in quarantine surveys for the pathogens. In this study, we used all the available genome sequences of Clavibacter sp. strains, including the type strains of newly described subspecies, to provide precise insight into the diversity of tomato-associated members of the genus and further clarify the taxonomic status of the strains using genotypic and phenotypic features. The results of phylogenetic analyses revealed the existence of nine hypothetical new species among the investigated strains. None of the three new subspecies (i.e., C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis, and C. michiganensis subsp. phaseoli) is included within the tomato-pathogenic C. michiganensissensu stricto lineage. Although comparative genomics revealed the lack of chp and tomA pathogenicity determinant gene clusters in the nonpathogenic strains, a number of pathogenicity-related genes were noted to be present in all the strains regardless of their pathogenicity characteristics. Altogether, our results indicate a need for a formal taxonomic reconsideration of tomato-associated Clavibacter sp. strains to facilitate differentiation of the lineages in quarantine inspections.IMPORTANCEClavibacter spp. are economically important bacterial plant pathogens infecting a set of diverse agricultural crops, such as alfalfa, corn, pepper, potato, tomato, and wheat. A number of plant-pathogenic members of the genus (e.g., C. michiganensissensu stricto and C. sepedonicus, infecting tomato and potato plants, respectively) are included in the A2 (high-risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Although tomato-associated members of Clavibacter spp. account for a significant portion of the genetic diversity in the genus, only the strains belonging to C. michiganensissensu stricto (formerly C. michiganensis subsp. michiganensis) cause bacterial canker disease of tomato and are subjected to the quarantine inspections. Hence, discrimination between the pathogenic and nonpathogenic Clavibacter sp. strains associated with tomato seeds and transplants plays a pivotal role in the accurate detection and cost-efficient management of the disease. On the other hand, detailed information on the genetic contents of different lineages of the genus would lead to the development of genome-informed specific detection techniques. In this study, we have provided an overview of the phylogenetic and genomic differences between the pathogenic and nonpathogenic tomato-associated Clavibacter sp. strains. We also noted that the taxonomic status of newly introduced subspecies of C. michiganensis (i.e., C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis, and C. michiganensis subsp. phaseoli) should be reconsidered.

Scaphoideus titanus Ball, 1932 (Hemiptera: Cicadellidae) - a new invasive pest of grapes on the territory of the Donbass

In August 2019, in the course of reconnaissance monitoring of insect pest complex of grapevine in the territory of the Donbass, the invasive North American grapevine leafhopper Scaphoideus titanus Ball, 1932 (Hemiptera: Cicadellidae) was first recorded. A pest outbreak has been registered in the territory of the Novoazovsk region. A targeted search for the species in other areas ended up with a negative result. Scaphoideus titanus is a specialized pest of grapevine and a pathogenic agent of Grapevine flavescence doree - phytoplasma 'Candidatus Phytoplasma vitis' (Phytoplasma vitis), included in the list of pests of the European and Mediterranean Plant Protection Organization (EPPO) and the Common List of Quarantine Pests of the Eurasian Economic Union. The paper presents information on the history of invasion and distribution of this leafhopper throughout Europe, host plants, biology, life cycle and pathways of phytoplasma ‘ Candidatus Phytoplasma vitis’. The range expansion of S. titanus threatens with penetration of the causative agent of grapevine flavescence doree into the Donbass and requires purposeful studies on the features of biology, distribution and population condition of grapevine leafhopper in the region.

Aleurothrixus trachoides (Back) can transmit begomovirus from Duranta to potato, tomato and bell pepper

Solanum whitefly, Aleurothrixus trachoides (Back). (Hemiptera: Aleyrodidae) was considered as a non-virus vector by European and Mediterranean Plant Protection Organization (EPPO) reports. However, in the present study it was found to transmit Duranta leaf curl virus (DLCV) to tomato, bell pepper and potato. A. trachoides infested field samples of Duranta sp (100%) and tomato (20%) tested positive for begomovirus by PCR using begomovirus degenerate primers and primers specific to Tomato leaf curl New Delhi virus showing amplicon of 520 bp and 2.7 Kb respectively. The DNA samples of A. trachoides collected from virus positive duranta and tomato plants also tested positive for the virus. Virulent whiteflies from duranta could successfully transmit DLCV to bell pepper (26%) and tomato (13 %) plants as confirmed by Rolling Circle Amplification. The rate of virus transmission by A. trachoides from DLCV inoculated tomato to bell pepper and tomato to potato was 100% and tomato to tomato was 80%. The results suggest whitefly A. trachoides as the vector for DLCVand to the best of our knowledge, this is the first report for A. trachoides as vector of begomovirus. These findings suggest need for reconsideration of A. trachoides as a virus-vector. This will have great impact on solanaceous vegetable cultivation in India and other parts of the world.

Multiple Introductions of Tomato Pathogen Clavibacter michiganensis subsp. michiganensis into Iran as Revealed by a Global-Scale Phylogeographic Analysis.

Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is one of the most important seed-borne tomato diseases around the globe. The disease was initially reported in 1993 in Iran, and it became a rising threat for the multibillion dollar tomato industry of the country during the last decade. In this study, using phylogeographic analyses, we determined genetic diversity and geographic distribution of C. michiganensis subsp. michiganensis in Iran. Our field surveys showed that the pathogen is expanding into the southern and eastern areas of the country. Furthermore, multilocus sequence analysis and typing (MLSA/MLST) using the sequences of five housekeeping genes (atpD, gyrB, ppk, recA, and rpoB) revealed that 37 C. michiganensis subsp. michiganensis strains isolated in Iran had high genetic diversity and placed in 15 sequence types (STs), while all the available 184 worldwide C. michiganensis subsp. michiganensis sequences were placed in 43 STs. MLSA divided the worldwide C. michiganensis subsp. michiganensis strains into two phylogroups (I and II). Among the 37 strains isolated in Iran, 30 strains clustered in phylogroup I, while 7 strains clustered in phylogroup II. Phylogeographic data inferred from the allelic profile of the five housekeeping genes suggested multiple introductions of C. michiganensis subsp. michiganensis inoculum into Iran, while the geographic origin of the Iranian C. michiganensis subsp. michiganensis strains remains undetermined. Further analyses using higher numbers of strains are warranted to decipher the evolutionary history of C. michiganensis subsp. michiganensis in Iran. Additionally, stricter seed/transplant inspections are recommended to reduce the risk of pathogen expansion to areas with no history of the disease.IMPORTANCEClavibacter michiganensis subsp. michiganensis, the causal agent of tomato bacterial canker disease, is one of the economically important pathogens of solanaceous crops (e.g., eggplant, pepper, and tomato) around the world. The disease occurs in many countries, with a particular importance in regions characterized by high precipitation and humid environmental conditions. As a seed-borne pathogen, C. michiganensis subsp. michiganensis is included in the A2 (high risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Bacterial canker disease was reported for the first time in 1993 in Iran, while the geographic distribution, genetic diversity, and phylogenetic position of the causal agent remain undetermined. In this study, using the multilocus sequence analysis and typing (MLSA/MLST) approach, we provided a phylogeographic scheme for the C. michiganensis subsp. michiganensis strains isolated in Iran. Furthermore, global-scale phylogenetic analyses led to determination of phylogenetic position of Iranian C. michiganensis subsp. michiganensis strains among worldwide population of the pathogen. Based on diversity parameters and population structure, we suggest relatively higher genetic diversity of the bacterial canker pathogen in Iran than has so far been observed in the other areas of the world. Results obtained in this study provide a novel insight into the genetic diversity and population structure of the bacterial canker pathogen on a global scale.

Molecular Typing Reveals High Genetic Diversity of Xanthomonas translucens Strains Infecting Small-Grain Cereals in Iran.

This study provides a phylogeographic insight into the population diversity of Xanthomonas translucens strains causing bacterial leaf streak disease of small-grain cereals in Iran. Among the 65 bacterial strains isolated from wheat, barley, and gramineous weeds in eight Iranian provinces, multilocus sequence analysis and typing (MLSA and MLST) of four housekeeping genes (dnaK, fyuA, gyrB, and rpoD), identified 57 strains as X. translucens pv. undulosa, while eight strains were identified as X. translucens pv. translucens. Although the pathogenicity patterns on oat and ryegrass weed species varied among the strains, all X. translucens pv. undulosa strains were pathogenic on barley, Harding's grass, rye (except for XtKm35) and wheat, and all X. translucens pv. translucens strains were pathogenic on barley and Harding's grass, while none of the latter group was pathogenic on rye or wheat (except for XtKm18). MLST using the 65 strains isolated in Iran, as well as the sequences of the four genes from 112 strains of worldwide origin retrieved from the GenBank database, revealed higher genetic diversity (i.e., haplotype frequency, haplotype diversity, and percentage of polymorphic sites) among the Iranian population of X. translucens than among the North American strains of the pathogen. High genetic diversity of the BLS pathogen in Iran was in congruence with the fact that the Iranian Plateau is considered the center of origin of cultivated wheat. However, further studies using larger collections of strains are warranted to precisely elucidate the global population diversity and center of origin of the pathogen.IMPORTANCE Bacterial leaf streak (BLS) of small-grain cereals (i.e., wheat and barley) is one of the economically important diseases of gramineous crops worldwide. The disease occurs in many countries across the globe, with particular importance in regions characterized by high levels of precipitation. Two genetically distinct xanthomonads-namely, Xanthomonas translucens pv. undulosa and X. translucens pv. translucens-have been reported to cause BLS disease on small-grain cereals. As seed-borne pathogens, the causal agents are included in the A2 list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Despite its global distribution and high economic importance, the population structure, genetic diversity, and phylogeography of X. translucens remain undetermined. This study, using MLSA and MLST, provides a global-scale phylogeography of X. translucens strains infecting small-grain cereals. Based on the diversity parameters, neutrality indices, and population structure, we observe higher genetic diversity of the BLS pathogen in Iran, which is geographically close to the center of origin of common wheat, than has so far been observed in other areas of the world, including North America. The results obtained in this study provide a novel insight into the genetic diversity and population structure of the BLS pathogen of small-grain cereals on a global scale.

Identification of larval salivary gland polytene chromosomes of the peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae)

Peach fruit fly, Bactrocera zonata (Saunders) (Diptera: Tephritidae) is native to South and South-East Asia. It is a polyphagous species and has a high reproductive potential (approx. 600 eggs in a lifetime), high biotic potential, i.e., several generations of progeny per year. The species has the rapid dispersal ability and can be active throughout the year. Its establishment may have a serious impact on the environment following the initiation of chemical and/or biological control programmes. B. zonata is of quarantine significance to EPPO (European and Mediterranean Plant Protection Organization) countries and it is classified on the A1 list of pests recommended for regulation as quarantine pests by Centre for Agriculture and Biosciences International (CABI). The present study initiates an attempt to identify the larval (3rd instar) salivary gland polytene chromosome arms of B. zonata using Olympus phase contrast microscope CX 41. A photographic representation of the polytene chromosomes of this species associates with identifying tips as well as well defined characteristics of each polytene chromosome arms are described. Five long chromosomes with their identifying tips are recognized in each polytene nucleus. Polytene chromosomes are of special interest to construct a Genetic Sexing Strain (GSS) of a pest species and it can play an important role for the successful application of Sterile Insect Technique (SIT) in field application as part of the biological control programme under Integrated Pest Management (IPM) scheme.

Eastern chestnuts nuts — dangerous chestnut pest

Goal. Determine the main ways of entry into the country of the dangerous quarantine type of eastern chestnut walnut-fruit (Asian chestnut gall Trach).
 Methods. Analytical informational messages of the European and Mediterranean Plant Protection Organization (EPPO), as well as data of EU phytosanitary services, literature and Internet resources.
 Results. Information is given on the new economically important species of Dryocosmus kuriphilus Yas., which is rapidly spreading in the countries of Europe. In Ukraine, the eastern chestnut nut walnut has a quarantine status. The threat of entering the species Dryocosmus kuriphilus with planting material (seedlings) of certain types of chestnuts from European countries in Ukraine exists. The absence of natural enemies contributes to the active resettlement of the pest. The focus should be on the study of the entomophages of the walnutworm. It is known that in Japan it is found in 26 species of parasitoids that control reproduction, in China — 11, in Korea and Italy — 15 species. The rapid spread of the pest in the EU is of great concern to scientists. The lack of natural enemies, as well as effective drugs approved for use, threaten the disappearance of chestnut plantations. The experience of some European countries shows that it effectively regulates the number of its specialized Torymus sinensis parasitoids, which was introduced into Europe from China and Japan.
 Conclusions There is a danger of entering into Ukraine the species Dryocosmus kuriphilus, which has quarantine status and is a threat to chestnut plantations.