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Synchytrium endobioticum (Schilb.) Percival. is a pathogen of potato wart disease and has a restricted distribution on the territory of the Russian Federation. Its main pathways are infected potato tubers and different planting material containing soil particles infected with spores of the fungus. One of the main problems is the use of toxic chemicals during detecting the disease in laboratory methods of direct soil testing to identify resting spores. This paper presents the assessment of molecular methods of soil diagnosis for detection of S. endobioticum by direct extraction of fungal DNA from soil samples using the MetaGen reagent kit. Identification was performed using the Fitoskrin. Synchytrium endobioticum-RT kit. The kit was pre-tested using DNA isolated from potato warts by various commercial kits. It was found that the optimal method of DNA isolation from the warts was using the FitoSorb-Avtomat 48 kit at the Tecan robotic station. Studies have shown that the sensitivity of the direct DNA extraction method from soil samples with various infection levels is the same as that of flotation method using carbon tetrachloride. Moreover, this method makes it possible to work with soil samples of different types, including peaty soils.

The family Drosophilidae includes over 3750 species worldwide and over 2000 of these are species of Drosophila . Spotted wing drosophila (SWD), Drosophila suzukii is one of the most dangerous species in this family. The insects live on undamaged ripening fruits, using its peculiar serrated ovipositor to break the skin of fresh ripening fruits and lay eggs in it. Drosophila species are very difficult and practically impossible to detect at larval stages. The present investigation was conducted at the All-Russian Plant Quarantine Center and Agrarian and Technological Institute of RUDN University, Moscow, Russia in 2018—2020. The aim of this study was to investigate the method of accurate and rapid identification of D. suzukii , and to design specific primer pairs for pest identification by Real-Time PCR method. The real-time quantitative PCR is a fast, sensitive, repeatable and accurate method for quantifying gene transcript levels. In this study, we designed specific primers (4.Dsuz.FRP) for Real-Time PCR to identify D . suzukii from other relative species. Although D. suzukii is absent in the Russian Federation and has not been reported so far, the project could be a precautionary measure.

In plant pathology, the correct naming of a species is essential for determining the causal agents of disease. Species names not only serve the general purpose of concise communication, but also are critical for effective plant quarantine, preventing the introduction of new pathogens into a territory. Many phytopathogenic genera have multiple species and, in several genera, disagreements between the multiple prevailing species concept definitions result in numerous cryptic species. Some of these species were previously called by various names; forma speciales (specialised forms), subspecies, or pathotypes. However, based on new molecular evidence they are being assigned into new species. The frequent name changes and lack of consistent criteria to delineate cryptic species, species, subspecies, forms, and races create increasing confusion, often making communication among biologists arduous. Furthermore, such ambiguous information can convey misleading evolutionary concepts and species boundaries. The aim of this paper is to review these concepts, clarify their use, and evaluate them by referring to existing examples. We specifically address the question, “Do plant pathogens require a different ranking system?” We conclude that it is necessary to identify phytopathogens to species level based on data from multiple approaches. Furthermore, this identification must go beyond species level to clearly classify hitherto known subspecies, forms and races. In addition, when naming phytopathogenic genera, plant pathologists should provide more information about geographic locations and host ranges as well as host specificities for individual species, cryptic species, forms or races. When describing a new phytopathogen, we suggest that authors provide at least three representative strains together with pathogenicity test results. If Koch’s postulates cannot be fulfilled, it is necessary to provide complementary data such as associated disease severity on the host plant. Moreover, more sequenced collections of species causing diseases should be published in order to stabilise the boundaries of cryptic species, species, subspecies, forms, and races.

Citrus farming is a major source of revenue for large and small-scale farmers in Kenya. Citrus production is confronted with threats from pests and diseases. Surveys of citrus farms in Kilifi, Kwale, Machakos and Makueni counties, Kenya in July/August (dry season) and in November/December (wet season), 2019 were conducted to identify scale insect pests (Hemiptera: Coccomorpha) attacking the trees (Sapindales: Rutaceae), and their related biota. A total of 22 scale insect species belonging to four families, namely Diaspididae (armoured scales), Coccidae (soft scales), Pseudococcidae (mealybugs), and Monophlebidae (giant mealybugs) were found infesting citrus trees in the two regions surveyed. Among the scale insects reported, four species were newly introduced in Kenya; three armoured scales Parlatoria ziziphi (Lucas), Parlatoria pergandii (Comstock), Aonidiella comperei (McKenzie), and a soft scale, Pulvinaria polygonata (Cockerell). The scale insects were closely associated with predators (coccinellids and lacewings) and attendant ants. This information will be helpful in the development of efficient management strategies against the scale insect pests, thus improving citrus production in Kenya. The scale insect pests identified in this study will be useful to plant quarantine facilities in Kenya to help to prevent and detect accidental introductions of exotic scale insect species.

International exchange of plants/planting material carries an unintended risk of introduction of exotic pests or their new virulent races/strains into new areas. History has evidenced several examples of dangerous pests introduced along with plants/planting material/plant products that have led to serious socio-economic consequences. Plant quarantine is a statutory requirement to regulate the entry of seed/planting material, plant products, living organisms and soil etc. so as to prevent unintentional entry of pests and pathogens. International exchange of plant genetic resources (PGR) is important to broaden the genetic base of crops in order to develop improved crop varieties. The Government of India has legislated the Plant Quarantine (Regulation of Import into India) Order in 2003 to regulate the import of plant material. Under this Order, ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR) has been delegated powers to issue Import Permit and to carry out quarantine processing of imported PGR including transgenics and for issue of Phytosanitary Certificate for PGR meant for export. The Division of Plant Quarantine at ICAR-NBPGR, New Delhi has developed an efficient and step-by-step protocol for quarantine processing for pest diagnostics, salvaging and containment to uphold biosecurity during exchange of PGR and to be transparent during exchange internationally. Meticulous quarantine examination of 5,45,945 samples of PGR during 2015–2020 has resulted in interception of several insect pests, fungi, bacteria, viruses, nematodes and weeds. Some of these are yet not reported from India, if reported, have a restricted distribution; have an extensive host range and/or cause huge economic losses or have more infectious/several races/strains etc. All infested/infected/contaminated samples are either disinfected/disinfested prior to release or incinerated depending on the type of pest intercepted. During the past five years from 2015 to 2020, a total of 36 pests of quarantine significance including insects/mites (7), fungi (8), viruses (13) and weeds (28) were intercepted at ICAR-NBPGR, New Delhi. Had any of these pests not been intercepted and had escaped, they could have entered and established in the country and subsequently caused devastation to our agriculture. Presently, exchange of PGR has become more difficult under the Convention on Biological Diversity, hence, all attempts were made to salvage the germplasm and 7393 samples were salvaged and 2813 samples were rejected. Although, ICAR-NBPGR has contemporary facilities to properly take up this task, more wide-ranging efforts are needed in the national plant quarantine system to develop adapt and adopt latest detection and eco-friendly disinfestation/disinfection techniques to minimalize the risk of pest escape in quarantine. Additionally, the legislation on quarantine needs to be upgraded to the Agricultural Biosecurity Bill as drafted by the Ministry of Agriculture and Farmers Welfare, Government of India to have an all-inclusive approach towards biosecurity in India.

Xylella fastidiosa Wells et al. causes grapevine bacteriosis and is among most hazardous malicious bacterial phytopathogens affecting a wide variety of important crops and ornamental plants. The agent colonises plant xylem and transmits with insects feeding on xylem sap. The insect vectors of X. fastidiosa belong to the order Hemiptera, suborder Auchenorrhyncha, families Cicadellidae, Cercopidae, Aphrophoridae and Cicadidae. A phytosanitary control survey conducted by the All-Russian Research Institute for Plant Quarantine in 2014 identified a high risk of X. fastidiosa introduction and adaptation in the Russian Federation. The Crimean Peninsula is a potential introduction area of the Pierce’s disease agent due to suitable climatic conditions, the ample availability of major host plants, as well as insect vectors. During a research monitoring in 2018-2020, the Institute assessed the phytosanitary status of the Crimean territory. The survey sampled vegetative parts of grapevines, stone fruits (peach, cherry, plum, merry, almond), selected essential-oil and ornamental shrubs and trees. Diagnostic procedures were performed in accordance with the international standards. A three-year phytosanitary survey of the Crimean plantations revealed no presence of X. fastidiosa. Two protocols of sample preparation and DNA extraction from various substrates have been tested. A real-time PCR-based protocol was proved highly specific for zero false positive and nonspecific rates.

A new pathway of travels by invasive pest and disease pathogen through railways, as sneakers, were noticed for the first time. The Indian railways passing through the soybean fields infested with Spodoptera litura, after evening attracted the moth of Spodoptera by the illuminating lights in the railways compartment and the moth enters into the compartment through open compartment windows. These moths remain in the railway compartment until the dawn and get out of the compartment as the early morning sun light enters into the compartments and are thus the sneakers crop pest in railways due to their unnoticed travels/transport. Such sneakers crop pest travels up to a distance of 600 km during the period of night from one ecological region to another ecological region having the same crop to infest or the alternative host crop of the pest to cause the fresh infestation. This phenomenon of pest travel was observed in the region of Lalitpur in Uttar Pradesh, India, having soybean infestation. This is a quick travel by the pest into different areas, unseen and unreported earlier and may occur in any part of the world where the railways pass through the infested crop areas and attract the positive phototaxis pest and transport them, as sneakers, in to another ecological region. In another instance, the Sigatoka disease pathogen of banana was also noticed to travels from one ecological region to another ecological region through the railways, as sneakers without notice. This phenomenon of disease pathogen’s travels was noticed in Jalgaon region from where the banana produce is transported to different parts of India. In the trading and transport of banana through railways, the loaders use the banana leaves infected with the sigatoka pathogen as a packaging material for banana bunches while loading the banana produce in the railway wagons for their transportation. Thus, the sigatoka pathogen travels up to a distance of 1200 km/day along with the banana produce through the railways. The sigatoka infected leaves, with the unloading of banana produce also sneaks into the new ecological region and spread to infect the banana crop available in the region. Thus, the positive phototaxis insect pest are the sneakers in the railways for their travels while diseases pathogens with healthy crop produce travels and embarks to sneaks into a new ecological region and thus the railways transport system unintentionally transport the crop pest and disease pathogen from one ecological region to another. The knowledge of this new travel pathway will be useful in finalizing the strategies of plant quarantine and management of invasive pest and disease pathogens.

Coccids are a group of pest insects with pronounced sexual dimorphism. Winged males don’t havefeed organs. Wingless females lead an immobile life under a wax structure (scale cover) and feed on plantjuices piercing them with a long proboscis. At early stages of life cycle, the larvae of scale insects are able to move, and the larvae of soft scales are mobile throughout the entire life-time. They are able to move with air flows or creep along the feed substrate, forming extensive colonies. These large colonies deplete and deform the host plants, making them vulnerable to diseases and even can cause plant death. Considering the geographical location, it should be noted that the majority of coccid species live in areas with tropical and subtropical climate. The pests can passively migrate with seedlings, fruits or vegetables.Coccids that live on seedlings or potted plants are the most dangerous. Those living on fruits do less harm.The study of the species composition of coccids on fruits and roots imported to Syktyvkar has not been carried out yet. Here, we present the results of laboratory studies of fruits and root crops for the presence of coccids for the period 2017–2019. For each species, we indicate the economically valuable host plants. The identification of coccids was made by the microstructure features of terminal segments of the abdomen in females (pygidium).According to the results conducted in the laboratory of the «Russian Plant Quarantine Center» in the KomiRepublic, 13 species of scale insects (Diaspididae) and one species of coccids (Coccoidae) were identified.Four species have the status of a quarantine object on the territory of the Eurasian Economic Union (EAEU), among them three are quarantine for the Russian Federation.

D. melanogaster is one of the most harmful citrus fruit flies having a large number of host plants. The molecular diagnostic method has been created for identification the D. melanogaster from another non-quarantine species Drosophila spp. The proposed method for differentiation is to use the mitochondrial DNA cytochrome oxidase I gene region 709-bp. We amplified samples of DNA with primers Droso-S391 and Droso-A381 by D. melanogaster, D. suzukii, and D. Simulans collections in the laboratory samples from many countries and contrasted with sequences of other GenBank Drosophila taxa. The findings of a polymerase chain reaction (PCR) based on DNA sequence polymorphisms showed that these primers accurately identify the area of the gene as well as the unique primers of Drosophila melanogaster.

A new species of Coccidohystrix Lindinger, intercepted by plant quarantine in Japan from imported Madagascar plant species, Pachypodium inopinatum Lavranos (Apocynaceae), and Pachypodium sp., is herein described and illustrated based on adult female. The new species is quite distinct in the genus in having anterior and posterior ostioles and trilocular pores on marginal cerarii and on elevated dorsal cerarii. The species is similar to Coccidohystrix primigenia Gavrilov-Zimin from Madagascar in having some morphological features mentioned above. However, the new species differs from C. primigenia in lacking multilocular pores on the dorsum, having fewer circuli, having numerous translucent pores on the back side surface of the hind tibiae, having 7–9 conical setae on anal lobe marginal cerarii, having only 2–3 conical setae on penultimate cerarii and having only 1–5 (mostly 1–2) conical setae on submarginal dorsal cerarii. An identification key for the genus, including the new species, is also provided. www.zoobank.org/urn:lsid:zoobank.org:pub:9CEDFC41-D0C9-4569-9E7D-317B02AE424F

Rice blast caused by Pyricularia oryzae is considered as one of the most dangerous diseases in Egypt and worldwide. The fungus can infect rice plants at any growth stage. P. oryzae can be transmitted from seeds to seedlings and increase the possibilities of spreading the disease and could cause epidemics, so that blast was referred to seed-borne disease. Current investigation aimed to develop an advanced technique or procedure that would allow reliable, reasonably fast and accurate detection of dangerous seed-borne fungal diseases such as P. oryzae in rice. The most common seed health tests including washing test (WT), blotter method (BM), washing after incubation, modified (WAI), deep freezing method (DF) and potato dextrose agar method (PDA) were applied for detecting seed-borne fungi from rice seeds. Among other fungi, the blast fungus P. oryzae was recorded only for three times from rice seed samples with very low frequency values of 1.0 and 1.5 in 2015 season using WT and WAI, respectively, and 2.0 in 2016 season using WAI. PCR has many beneficial characteristics that make it highly applicable for detecting P. oryzae in seeds. So that, side by side, PCR reaction was used for detecting and identifing P. oryzae to overcome the time consuming and inacuracy of the common seed heath tests. Metallic nanoparticles, silver and iron oxide nanoparticles were also involved to enhance DNA extraction which considered an important step in the PCR technology. Silver and Iron oxide nanoparticles were able to enhance the quantity and purity of DNA template for successful PCR assay. Preferably, seed health methods should be sensitive, specific, rapid, robust, inexpensive and simple to be applicable and fit for plant quarantine purposes. Many detection methods occur for different seed-borne pathogens, but, few of the minimum requirements for adequate seed tests were satisfied. In conclusion Nano based PCR method could be applied as an appropriate test for rapid detection of rice pathogenic fungus P. oryzae.

The Russian Federation imports large quantities of planting and grafting material of grapes, including from countries where dangerous harmful organisms are spread to vineyards. Plant protection specialists in this industry, as well as grape producers, must understand the possible risk associated with the danger of the entry of quarantine disease pathogens into the Russian Federation and the potential damage if infected plant material is imported. Studies have been carried out by the staff of All-Russian Plant Quarantine Center to assess the phytosanitary risk of pests associated with the import of seedlings, stocks and grape cuttings. Based on these studies, the list of pests for the vine that have quarantine status and potentially quarantine status for the Russian Federation, in particular bacterioses, phytoplasmas and viral diseases has been established. This article describes the main diseases that affect grapes and are included in the Unified List of Quarantine Pests of the Eurasian Economic Union, as well as potentially dangerous viral diseases that are not currently included, but planned for inclusion in the Unified List, which pose a serious threat if they penetrate to the territory of the Russian Federation. From quarantine pathogens, grapes are affected by grape bacteriosis (Pierce disease of grapevine) (Xylella fastidiosa Wells et al.), bacterial blight of grapevine (Xylophilus ampelinus (Panagopoulos) Willems et al.), Flavescence dorée of grapevine (Candidatus Phytoplasma vitis), from the planned inclusion grapes are affected by red spotting of grape leaves (Grapevine red blotch-associated virus), marbling of grape leaves (Grapevine vein clearing virus) and discoloration of the leaves of Roditis grape (Grapevine Roditis leaf discoloration-associated virus). The article considers the biological peculiarities of pathogens, ways of their possible penetration and spread on the territory of the cultivation of grape in the Russian Federation, host plants of quarantine and dangerous pests, the main symptoms of diseases on plants, possible vectors, damage caused by quarantine pests in the countries of spreading, diagnostic methods, as well as areas of possible harmfulness for the Russian Federation.

Monilia fruit rot is one of the most common diseases of various fruit and subtropical crops on the Black Sea coast of the Caucasus. Moniliosis of fruit crops is caused by three fungi species of the genus Monilinia — Monilinia fructigena (Pers.) Honey, M. laxa (Aderh. & Ruhland) Honey and M. fructicola (G. Winter) Honey. According to the literature data, among the species of the genus Monilinia , the most common and harmful phytopathogenic species on the Black Sea coast of the Caucasus is M. fructigena . About the presence of the species M. fructicola in the Krasnodar Territory, in particular, and in Russia as a whole, there was no information before the present studies. This pathogen is included both in the EPPO Alert List and in the Unified List of Quarantine Objects of the Eurasian Economic Union. Traditionally, the identification of species of the genus Monilinia occurs by visual method according to cultural-and-morphological characteristics. However, this method does not provide accurate data on the Monilinia species presented in the region. The subject of the experiment was samples of pome fruits and stone fruits (fruits, shoots, buds) with obvious symptoms of moniliosis. Samples were collected at different points in the humid subtropics of the Krasnodar Territory (Russia) and the Republic of Abkhazia. Species identification was carried out using speciesspecific markers. In the process of PCR-analysis for the species-specificity of fungi of the genus Monilinia , a quarantine species M. fructicola was revealed, which was not observed earlier in the territory of the European Economic Union and in the Republic of Abkhazia. M. fructicola was found on peach collected in Sochi and in Abkhazia (Gulripshsky district). To confirm the correctness of the species determination of the discovered fungi of the genus Monilinia , sequencing was performed. The molecular diagnostic method has proved to be promising for the accurate identification of fungi of the genus Monilinia , the causative agent of fruit rot of fruit and subtropical crops.

Background In July 2015, the carcasses of 11 cockatiels were submitted for disease diagnosis to the Avian Disease Division of the Animal and Plant Quarantine Agency of Korea. The cockatiels, which appeared dehydrated and underweight, had exhibited severe diarrhea and 22 % mortality over 2 weeks. Traditional diagnosis did not reveal the causes of these symptoms. Methods We conducted metagenomics analysis on intestines and livers from the dead cockatiels using Illumina high-throughput sequencing. To obtain more accurate and longer contigs, which are required for further genetic characterization, we compared the results of three de novo assembly tools (metaSPAdes, MEGAHIT, and IDBA-UD). Results Sequence reads of Campylobacter jejuni (C. jejuni) and Chlamydia psittaci (C. psittaci) were present in most of the cockatiel samples. Either of these bacteria could cause the reported symptoms in psittaciformes. metaSPAdes (ver.3.14.1) identified the 1152 bp flaA gene of C. jejuni and the 1096 bp ompA gene of C. psittaci. Genetic analysis revealed that flaA of C. jejuni was recombinant between C. jejuni and Campylobacter coli, and that ompA of C. psittaci isolated from cockatiel was closely related to strains isolated from humans. Conclusions C. jejuni and C. psittaci were detected in cockatiels in the Republic of Korea using metagenomic analysis. This approach is useful for understanding pathogens of pet birds. Three de novo assemblers were compared to obtain accurate contigs from large quantities of reads, and sequences of C. jejuni and C. psittaci generated by metaSPAdes were analyzed.

Burkholderia glumae, before mid-2018, is categorized as plant quarantine pest A2 Group 1 that its existence has been detected in Indonesia. B. glumae has been known to spread in the central production of rice in Java, Sumatra, Borneo dan Sulawesi. This review aimed to explain the strategies for B. glumae detection through its characteristics and to prevent the divergence of this bacterium in Indonesia. The previous studies reported that the bacteria could reduce yield up to 75% and caused the decrease of weight-grain or the increase of empty grain. The disease intensity is affected by environmental and physiological factors such as warm temperature at nighttime and high rainfall intensity. The optimum temperature for the development of the disease is 30–35°C. Moreover, the pathogen could survive at a temperature of 41°C. The tropical area of 32-36°C are suitable for B. glumae. Recently, the effective control of the disease in the field has not been found yet. Meanwhile, early detection of the disease is not yet determined, even though it is necessary to prevent its spread in rice cultivation in Indonesia. Detection of the disease by Agricultural Quarantine Agency as a frontline is needed to check the entry of the disease carried by the import activities of the seed. Detection in the suspected field by protection institutes through frequent surveillance in central production areas of rice should be considered as an important task.. The effective techniques to prevent B. glumae are the use of resistant varieties, the practice of seed treatments (using antibacterial, bactericide, heat treatment or plant extract), and the application of oxolinic acid to the crops.

The recommended EPPO diagnostic protocol for detection and identification of Fusarium oxysporum f.sp. albedinis (FOA), the causal agent of Bayoud disease of date palm (Phoenix dactylifera) was validated under Egyptian laboratory conditions. The validation process was carried out in The Plant Quarantine Pathogens Laboratory (PQPL), Plant Pathology Research Institute, Agricultural Research Center (ARC). The performance criteria were assessed for the conventional PCR methods and recommend by EPPO as PM 7/16(1) (2002, 2009). The sensitivity, specificity, repeatability, and reproducibility of the method were tested for different concentrations of pure culture of FOA. The method was proved to be unambiguously specific to detect FOA. The limit of detection (LOD) of the method was 1ng µl-1 and 100% reproducibility and repeatability were also achieved. Moreover, the validated method was then implemented for testing date palm samples presenting lots of imported planting materials (43 samples) from different origins in addition to different samples collected from domestic plantations (14 samples) presenting different locations in Egypt. Although F. oxysporum was isolated from most of the tested samples but the presence of FOA in all samples was nil.

Ascochyta blight of chickpea is caused by Ascochyta rabiei (Pass.) Labr. which is primarily seedborne. For rapid detection and precise identification of A. rabiei, a sequence-characterized amplified region (SCAR) marker was developed for detection of genomic DNA and infected plant DNA. An SSR primer amplified monomorphic band was cloned in pGEM®-T easy vector and sequenced. The best primer pair was selected and validated on A. rabiei. The specificity and sensitivity of the SCAR-based marker designated as MBAR was evaluated using conventional PCR and real-time PCR. The marker produced consistently an amplicon size of 196 bp in all A. rabiei isolates tested. The sensitivity of the marker was 0.1 ng of genomic fungal DNA and 0.5 ng of plant DNA by conventional PCR and 0.5 pg of A. rabiei DNA and 1.0 pg of plant DNA by real-time PCR. This is the first SCAR marker having high specificity and sensitivity towards A. rabiei. The marker may be useful in detecting the pathogen before the disease appearance and in plant quarantine program to detect the pathogen in seed lots.

The Indian citrus ringspot virus (ICRSV) that causes ringspot disease, especially to ‘Kinnow mandarin’ hampers the sustainability of crop production. Presently, the disease is not amenable for control through host resistance or the introduction of chemicals, hence raising virus-free plants is one of the most effective approaches to manage the disease. Consequently, it is necessary to develop rapid, sensitive, specific, and early diagnostic methods for disease control. In the present study, newly designed primers targeting a 164 bp region of the ICRSV coat protein gene were used to develop and optimize a SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay, for the detection of ICRSV. The RT-qPCR assay was evaluated and confirmed using viral RNA extracted from ICRSV infected plants maintained in screen house as well as field samples. The standard curves displayed a dynamic linear range across eight log units of ICRSV-cRNA copy number ranging from 9.48.1 fmol (5.709 × 10(9)) to 0.000948 amol (5.709 × 10(2)), with detection limit of 5.709 × 10(2) copies per reaction using serial tenfold diluted in vitro transcribed viral cRNA. The developed RT-qPCR is very specific to ICRSV does not react to other citrus pathogens, and approximately 100-fold more sensitive than conventional RT-PCR. Thus, this assay will be useful in laboratories, KVKs, and nurseries for the citrus budwood certification program as well as in plant quarantine stations. To our knowledge, this is the first study of the successful detection of ICRSV by RT-qPCR.

A survey on 13 regions of Kazakhstan was carried out to identify foci of fire blight, the extent of its spread, the range of affected crops, and the identification of infection reserves. The disease was mainly concentrated in the main fruit growing zone.at the south and southeast of Kazakhstan. Symptoms of fire blight were characteristic of two bacterial diseases: namely, necrosis, by Pseudomonas syringae Van. Hall. and fire blight by Erwinia amylovora (Burrill) Winslow et al. The authors performed bacteriological analyses to isolate and identify the causative agent of fire blight from various fruit and wild cultures of the Rosaceae family, using classical bacteriological and molecular genetic methods. Two types of bacteria were isolated from the samples affected by the disease, namely E. amylovora, the causative agent of fire blight, and P. syringae, the causative agent of bacterial necrosis. The results of the studies on the identification of bacterial species E. amylovora and P. syringae were confirmed by Swiss scientists from the Agroscope research center based on an immuno-chromatographic test and by Russian scientists at the All-Russian Research Institute of Plant Quarantine using enzyme-linked immunosorbent assay, FLASH polymerase chain reaction, and polymerase chain reaction in real-time.

Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.

Global trade has increased the invasion risk of exotic organisms and damaged agricultural and natural ecosystems. The Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ) handles quarantine services of animal- and plant-associated pests and diseases in Taiwan. The predatory mite family Phytoseiidae (Acari: Mesostigmata) is a well-known group due to the potential use of certain species as biocontrol agents for small phytophagous pests. Some species are available in commercial markets and frequently used in biological control in many agricultural systems, especially in greenhouse crops. However, exotic biological control agents may interfere with natural or naturalised populations of predatory mites and they may threaten indigenous populations via intraguild predation. The present study aims to provide a checklist of phytoseiid mite species found in plant quarantine from 2006–2013. Twenty-five species belonging to two subfamilies and eight genera were found in samples imported to Taiwan from twelve countries, including one new species Typhlodromus (Anthoseius) ueckermanni sp. nov. from South Africa. The checklist provides distribution, remarks, and also an identification key for all species.

Plant virus diseases are one of the major constraints to the global agriculture industry. Early and accurate detection and diagnosis of plant viruses, both in plants and carrier vectors, play a critical role in reducing disease spread and developing effective management strategies. Conventional diagnostic techniques based on biological and physical properties related to interaction of the virus with its host and/or vector and to its size and shape (e.g., symptoms, electron microscopy) are time-consuming, cumbersome, and not feasible for large-scale testing. For the last four decades, detection tests based on virus protein (e.g., enzyme-linked immunosorbent assays [ELISAs]) have been widely used for routine and large-scale testing of plant viruses. Nucleic acid-based methods (e.g., nucleic acid hybridization, polymerase chain reaction [PCR]) provide greater flexibility, and increased sensitivity and specificity than the serological methods for rapid and reliable diagnosis of plant virus diseases. In this chapter, recent developments in the area of plant virus detection and diagnosis, which include the techniques that can be performed in the field (e.g., lateral flow immunoassay), methods to detect many viruses in a single test (e.g., multiplex-PCR), and cutting edge technologies suited for the discovery of newly emerging plant viruses (e.g., next-generation sequencing [NGS]), are discussed. These diagnostic methods are useful in plant virus disease management strategies, characterization studies, disease surveillance surveys, epidemiological studies, plant quarantine, germplasm screening, seed certification, and breeding programs, thereby playing a key role in global crop protection and maintaining sustainable agriculture.

Heterobasidion species are amongst the most intensively studied polypores because several species are aggressive white rot pathogens of managed coniferous forests mainly in Europe and North America. In the present study, both morphological and multilocus phylogenetic analyses were carried out on Heterobasidion samples from Asia, Oceania, Europe and North America. Three new taxa were found, i.e., H. armandii, H. subinsulare, and H. subparviporum are from Asia and are described as new species. H. ecrustosum is treated as a synonym of H. insulare. So far, six taxa in the H. annosum species complex are recognized. Heterobasidion abietinum, H. annosum, and H. parviporum occur in Europe, H. irregulare, and H. occidentale in North America, and H. subparviporum in East Asia. The North American H. irregulare was introduced to Italy during the Second World War. Species in the H. annosum complex are pathogens of coniferous trees, except H. subparviporum that seems to be a saprotroph. Ten species are found in the H. insulare species complex, all of them are saprotrophs. The pathogenic species are distributed in Europe and North America; the Asian countries should consider the European and North American species as entry plant quarantine fungi. Parallelly, European countries should consider the American H. occidentale and H. irregulare as entry plant quarantine fungi although the latter species is already in Italy, while North America should treat H. abietinum, H. annosum s.s., and H. parviporum as entry plant quarantine fungi. Eight Heterobasidion species found in the Himalayas suggest that the ancestral Heterobasidion species may have occurred in Asia.

Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120‍ ‍min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens.

Global trade has increased the invasion risk of exotic organisms and damaged agricultural and natural ecosystems. The Bureau of Animal and Plant Health Inspection and Quarantine (BAPHIQ) handles quarantine services of animal- and plant-associated pests and diseases in Taiwan. The predatory mite family Phytoseiidae (Acari: Mesostigmata) is a well-known group due to the potential use of certain species as biocontrol agents for small phytophagous pests. Some species are available in commercial markets and frequently used in biological control in many agricultural systems especially in greenhouse crops. However, exotic biological control agents may interfere with native or naturalized populations of predatory mites and they may threaten indigenous populations via intraguild predation. The present study aims to provide the phytoseiid mite species found in plant quarantine from 2006–2013. Twenty-five species belonging two subfamilies and eight genera were found in samples imported to Tawan from twelve countries, including one new speciesTyphlodromus(Anthoseius)ueckermannisp. nov.from South Africa. The checklist provides distribution, remarks, and also an identification key for all species.

Wheat streak mosaic virus (WSMV), a member of the genus Tritimovirus in the family Potyviridae, has been designated as a plant quarantine pathogen in South Korea. Several diagnostic methods can be applied to diagnose viral infections in plants, but polymerase chain reaction and enzyme-linked immunosorbent assay, which can identify viral species with speed and accuracy, are mainly used in Korean plant quarantine. Many variants of different viral species with highly divergent genomic sequences are constantly being discovered by high-throughput sequencing technology. This means that previously established primers may no longer be suitable for diagnostic use. In this study, we developed a reverse transcription polymerase chain reaction assay for detecting WSMV isolates/strains using all of the WSMV sequences available in NCBI GenBank. All 13 primer sets were able to produce amplicons of the expected sizes from WSMV-infected samples. To check whether nonspecific reactions occur, some closely related viruses (one tritimovirus and five potyviruses) and target imported plants (wheat, maize, oat, and proso millet) were tested. Consequently, four primer sets, which did not produce nonspecific bands, were finally selected among the 13 primer sets. Concentration-dependent amplification tests showed that the four primer sets are adequate for use in the diagnosis of WSMV in Korean plant quarantine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13337-020-00646-3) contains supplementary material, which is available to authorized users.

A stray female cat of unknown age, presenting bright red watery diarrhea, was submitted to the Animal and Plant Quarantine Agency for diagnosis. In the small intestines extracted from the necropsied cat, numerous white oval-shaped organisms were firmly embedded in the mucosa and there was thickening of intestinal wall. Histopathological analysis revealed severe necrotizing enteritis, together with atrophied intestinal villi, exfoliated enterocytes, and parasitic worms. Recovered worms were identified as Pharyngostomum cordatum by morphological observation and genetic analysis. Although P. cordatum is known to occur widely in Korea, this is the first clinical description of an infection by P. cordatum causing severe feline enteritis.

The highly invasive Madeira mealybug Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae) is a polyphagous pest which seriously harms crops of staple foods, fruits, vegetables, and ornamental plants. A method for rapid and accurate identification is needed to slow further spread of this pernicious pest. DNA barcoding and taxonomic keys were used to identify specimens of P. madeirensis collected in Rwanda and China. We designed a species-specific PCR (SS-PCR) primer pair for P. madeirensis based on variable sequences of the mitochondrial cytochrome c oxidase subunit I gene among P. madeirensis and 29 related species. This SS-PCR assay allows for rapid and precise identification. With this assay, no cross-reaction was detected from the 21 closely related mealybug species. All specimens from all ontogenic stages, adult debris, and individuals from 11 populations of P. madeirensis from different geographical areas and/or host plants were detected. The detection limit of this method was 192.58 ± 16.48 pg μL−1 of P. madeirensis DNA. These demonstrated the high sensitivity and effectiveness of this method. Using this method, we found the first described occurrence of P. madeirensis in Southern Province, Rwanda, indicating ongoing spread, and an entirely new invasion in Fujian, China. Invaded regions of China were delineated by a nationwide field study. The SS-PCR assay developed in this study is a rapid, straightforward, and reliable method for the identification of P. madeirensis, and will prove practical in plant quarantine, thus slowing or preventing further invasion and spread, and allowing for sustainable and effective management of this cosmopolitan invader.

Grain export is an important branch of the food business in the Russian Federation. The countries of Europe, Asia, Africa, and South America are importers of Russian grain. Each importing country has its own requirements for the phytosanitary condition of imported products. One important requirement for importers is the absence of pathogens that can cause bacterial diseases of grain crops, such as Pectobacterium rhapontici, Rathayibacter tritici, Pseudomonas fuscovaginae, Pseudomonas syringae pvs., Acidovorax avenae subsp. avenae, Xanthomonas translucens pvs., Rathayibacter rathayi, and Pseudomonas cichorii. Reliable information on the distribution of these bacterial strains in the Russian Federation is limited. Methods for the isolation and identification of these bacterial pathogens have not been developed to date, which increases the risk of the spread of phytopathogens that could cause significant economic harm to agriculture. The purpose of this study was to isolate and identify the causative agents of bacterial diseases of wheat and barley. In order to do this, we collected samples of plant material of wheat and barley in the Rodionovo-Nesvetaysky, Myasnikovsky, Zernogradsky, Azovsky, and Martynovsky districts of the Rostov Oblast. Various bacterial strains were isolated from the obtained samples using the appropriate cultural media. The strains were tested by polymerase chain reaction (PCR) using primers designed for the 16S ribosomal RNA (PSF/PSR and 8UA/519B) and SyD1/SyD2 primers selected for the Pseudomonas syringae genome (GenBank CP047267.1) with subsequent sequencing according to the Sanger method. As a result, the following bacterial strains were isolated and identified from wheat and barley samples: Curtobacterium sp., Paenibacillus sp., Enterobacteriaceae, Pseudomonas azotoformans, P. poae, P. azotoformans, P. hibiscicola, P. fluorescens, Stenotrophomonas sp., P. syringae pv. syringae, P. syringae pv. atrofaciens, Bacillus sp., Erwinia sp., Pantoea sp., and Pantoea agglomerans.

Most species attack the seed bearing organs of plants, including soft fruits and flowers. The taxonomy of the Tephritidae, or the true fruit flies, has applications in pest management and plant quarantine, weed biocontrol, and evolutionary biology. In the Oriental fruit fly species complex, about 60 sibling species are recognized so far, most of which are of limited distribution. The frequency of fruit fly symposia and the attendance they attract indicate that many hundreds of entomologists worldwide must be involved with the group of flies. A proportion of these entomologists work in areas with a high diversity of fruit flies or in areas with specific quarantine threats from areas with a diversity of species. Clearly there is a need for non-taxonomists to do a proportion of their own identification work, and this need has been addressed through training and the compilation of a general guide to pest fruit flies.

The distribution of the plant quarantine species – Ambrosia artemisifolia L. ( Asteraceae ) in Lutsk city has been investigated, which grows in territories with different ecological conditions was investigated: on railroad, near rivers, landfills, cultivated fields, residential buildings. Primary distribution centers were marked near railway stations and roadside lanes of the road. The first massive appearance of A. artemisifolia on the territory of the city are observed at the end of April – the beginning of May, the flowering begins in mid-August and lasts until October. The basic localities were indentified and the map of A . artemisifolia distribution on the territory was compiled. There are five main areas of distribution of A. artemisifolia within the city: three areas of industrial units (northern, southern and eastern), railway area and area of building. The largest area of distribution of A. artemisifolia in the city is the area of the building, and the main centers of growth with the highest density fall into the railway area and the area of the southern industrial place. It was found that the distribution of A . artemisifolia deep into the city takes place from the railway and industrial areas as well, as from the entry roads – the streets Kovelska, Volodymyrska and Lvivska. It was hypothesis established that the successful area capture by this species within the city is connected with the environmental preferences of A . artemisifolia : in places of distribution there is a high level of illumination of the territory, an increased amount of exchangeable Са +, К + , Nа + in the soil, it neutral or light-grained soil pH reaction, violation of vegetation density, due to which the competitiveness of the investigated species and its further maintenance in phytocoenoses increases. Usage of only mechanical (periodic abatement) methods of combating with this species do not give positive results, so it is recommended that chemical and biological control measures be used in accessible places, as well as the introduction of legal aspects and raising public awareness of the harmfulness of this species.

Pathogen attacks often occur on oil palm plants, particularly on the central nursery. Factors that affect the attack are plant and environmental factors. This study aims to determine the location of planting (temperature and humidity) and the administration of KCL fertilizer on the invasion of fungal pathogens on oil palm plants (Elaeis guineensis Jacq.) In nurseries (Main - nursery). This research was carried out on Jalan Jendral Sudirman Km. 9, Pasir Putih Village, Baamang District, East Kotawaringin Regency, Sampit. The testing of palm oil seedling samples was carried out at the Plant Quarantine Laboratory, Palangka Raya Class II Agricultural Quarantine Center. The design of this study was Split Plots Design. The planting location factors were considered to be more influential, so they are placed as main plots with three levels, namely the location of the greenhouse, open space and using 80% paranet and KCL fertilizer as a subplot with two levels. Each plot was compiled with RAL with four replications. The results of the study obtained both planting systems, environmental factors in the form of temperature and humidity, as well as giving KCl fertilizer as a whole have a significant effect on the growth of fungal pathogens on oil palm plants. In cultivating oil palm plants, it is recommended to use the planting system in open land by providing KCl fertilizer.

Aim. To evaluate potato breeding material for resistance to pathotypes of Synchytrium endobioticum (Schilbersky) Percival (1909) known to be present in Ukraine (pathotypes 1(D 1 ), 11, 13, 18 and 22); to identify resistant registered and potential varieties for the usage in the national wart disease eradication programs and to recommend these se- lected (potential and registered) potato varieties for the breeding program targeted on the development of multiple resistance against pathotypes of S. endobioticum present in Ukraine. Methods. Evaluation of the potato breeding material and registered potato varieties for the resistance against common pathotype 1 (D 1 ) and four aggressive pa- thotypes of S. endobioticum (pathotypes 11, 13, 18 and 22) in climatic chamber and greenhouse tests of Ukrainian Scientifi c Research Plant Quarantine Station of Institute of Plant Protection NAAS (Boyany, Ukraine) following the Spieckermann and Glynne-Lemmerzahl methods (EPPO Standard PM7/28(2)). Field trials on naturally infected soils were conducted according to standard methods adapted to national requirements in the area of Chernivtsi, Zakarpattia and Ivano-Frankivsk regions. Results. 3,736 samples of potato breeding material from six breeding institutions of Ukraine were tested for resistance against S. endobioticum during 2011–2017 in the western region of the country. Among all samples tested, 3,389 were identifi ed as resistant to the widely spread pathotype 1 in the preliminary climatic chamber and greenhouse tests, and 130 of them proved to be resistant under fi eld conditions. Five out of 41 Ukrainian registered potato varieties (Bazys, Hlazurna, Solokha, Bozhedar and Santarka) were found to be resistant to all 5 pathotypes tested (1 (D 1 ), 11, 13, 18 and 22). Conclusions. The 130 samples of potato breed- ing material (which were found to be resistant against the common pathotype 1 of S. endobioticum in the laboratory, greenhouse as well as in the fi eld trials) were recommended for the state variety registration and further usage in an eradication program to localize potato wart outbreaks of the western part of Ukraine. The screening tests revealed that the national breeding program targeted on resistance against S. endobioticum pathotype 11 was the most effec- tive (49 % of samples tested proved to be resistant against this pathotype), whereas it was the least effective against pathotype 18, namely only 30 % of samples resistant. It was speculated that such a dissimilarity may be related to the differences in the genetic material used in the breeding process at various institutions, and which may be the subject of further analysis in order to improve the results of breeding programs. The already registered potato variet- ies Bazys, Hlazurna, Solokha, Bozhedar and Santarka which were found to have a multiple resistance to common pathotype 1 and four local aggressive pathotypes of S. endobioticum (11, 13, 18 and 22) were recommended for use in the breeding process as sources of resistance and also for the eradication programs in the western region of Ukraine, where S. endobioticum is mostly distributed (2409 hectares or 98 %).

Acanthiophilus helianthi (Rossi) (safflower fly) is known to host a wide range of plant species belonging to the tribe Cardueae (Asteraceae). It is also known as a serious pest of safflower in the dry regions and marginal areas of the world where safflower is an important oilseed crop. Due to its possible introduction to Korean peninsula, it is listed as a managed quarantine pest by the Korean Animal and Plant Quarantine Agency. We here report a natural occurrence of A. helianthi for the first time in Korea, based on a single female specimen collected from northern part of South Korea. Since the discovery of A. helianthi is of agricultural and quarantine importance in this part of world, we here provide its detailed morphological redescription, diagnosis, and color photographs including genitalic structures for accurate identification. We also provide a brief account of its biology and pest status summarized from previous literature. In order to make sure the correct identification of the newly discovered Korean specimen of A. helianthi as well as to investigate systematic position of the genus Acanthiophilus, we conducted a DNA barcoding analysis. Finally, we here suggest that A. helianthi should be removed from the Korean quarantine pest list, since its natural occurrence in Korea is confirmed by the present study.

The Jack Beardsley mealybug, Pseudococcus jackbeardsleyi Gimpel & Miller (Hemiptera: Pseudococcidae), a globally devastating, invasive polyphagous insect, causes great damage to many fruits, ornamentals, vegetables, and food crops worldwide. It is of Neotropical origin and has invaded throughout America and in parts of Asia, Africa and Oceania, and is still expanding its invasion ranges. Therefore, a method for quick and correct identification of this invasive species is crucial for quarantine and spreading interruption of it. In present study, we report the first record of P. jackbeardsleyi on the Chinese mainland, which would cause great damage to many crops. The identification of P. jackbeardsleyi was verified via morphological characters and DNA barcoding. One pair of species-specific polymerase chain reaction (SS-PCR) primers was designed based on variations in the sequences of the mitochondrial cytochrome c oxidase subunit I gene among P. jackbeardsleyi and 28 other mealybug species. No cross-reaction was detected among 21 closely related species using this SS-PCR assay, demonstrating the specificity of this marker. Furthermore, this method was successfully applied to detect individuals from different developmental stages and adult debris across four geographic populations of P. jackbeardsleyi, showing the high stability of the assay. Additionally, the detection limit of the marker was 55.94 ± 5.05 pg/μl of P. jackbeardsleyi DNA, illustrating the high sensitivity of the assay. The SS-PCR assay developed in this study provides a rapid, simple and reliable method for the identification of P. jackbeardsleyi, which should be crucial in the plant quarantine, early detection and sustainable management of this globally invasive pest.

Phytoplasmas from the apple proliferation (AP) group collected and isolated from the territory of the Russian Federation and some European countries have been studied, including their detection and identification. Analysis of 67 samples of fruit crops by nested PCR using universal primers for 16–23S ribosomal RNA regions has shown the presence of the AP group phytoplasmas in some samples. Candidatus Phytoplasma mali, a causal agent of the apple proliferation disease, has been revealed in samples from Spain; Ca. Phytoplasma pyri (pear decline disease) was found in samples from Italy, Spain, and the Republic of Dagestan; and Ca. Phytoplasma prunorum (European stone fruit yellows disease) was found in samples from the Czech Republic. Data on the DNA sequencing of the identified pathogens have been deposited into the database of the National Center for Biotechnology Information (NCBI).

Coconut case caterpillar or Bagworm, Mahasena corbetti Tams (Lepidoptera: Psychidae), is a destructive polyphagus pest in Southeast Asia and some islands of the Pacific. Considering the rapid expansion of the distribution range of M. corbetti, this species is included in the list of dangerous pests in plant quarantine act of Malaysia. M. corbetti is also considered as a pest of quarantine importance in many countries. During 2015–2016, severe incidence of M. corbetti was observed for the first time on arecanut (Areca catechu L.) plants in Sohbar and adjoining villages of East Khasi Hills district of Meghalaya state (India). This is the first report on occurrence of M. corbetti in India. We are reporting the infestation, natural field parasitism and description of life stages of M. corbetti infesting arecanut plants in India. Considering the taxonomic complexities in identification and destructive nature of bagworms, we have also developed DNA barcode for this species by sequencing standard barcoding region of mitochondrial cytochrome oxidase I (COI) gene. This report would serve as a preliminary foundation for reliable diagnosis of M. corbetti and also for creating awareness among the arecanut growers and other related functionaries in India and nearby countries.

Armoured scales (Hemiptera: Diaspididae) belong to the largest scale insect family and are among the most invasive insects in the world. Accurate identification of armoured scales is essential for systematic and phylogenetic studies; biogeography; trade and plant quarantine; and pest management, particularly biological control. Several species are serious pests of citrus. Records of past field surveys conducted in Viet Nam between 1967 and 2010 indicated the presence of 28 species on citrus there. Discrepancies in these records, and the retention of specimens of only five species in collections, led us to undertake surveys throughout the citrus-growing regions of the country in 2013 and 2014 to verify previous records and conserve voucher specimens. The presence of 21 diaspidid species was confirmed based on morphological and molecular data. The species observed were common but rarely abundant. Populations in commercial orchards may have been influenced by use of pesticides, but most species were recorded also in gardens and orchards where pesticide use was uncommon. Natural enemies were abundant, but were not thoroughly documented for all the diaspidids we observed. An identification key to the species collected is provided. Differences between our findings and previous records from Viet Nam, from Yunnan and Guangxi in China, and from Indochinese countries neighbouring Viet Nam, indicate the need for extensive surveys to fully document the diaspidid fauna on citrus in the region.

The production of wine grapes is gaining widespread popularity and being carried out on approximately 2,200 hectares of land in Japan. Scions grafted onto rootstocks generally have been imported from the EU, USA, New Zealand, and Australia into Japan. Unfortunately, viruses have spread in Japanese vineyards by slipping through the net of plant quarantine. Grapevine rupestris vein feathering virus (GRVFV), which was detected in a Greek grapevine accessions, is a member of genus Marafivirus in family Tymoviridae (El Beaino et al. 2001). GRVFV has been detected in many countries such as USA, Canada, Australia, New Zealand, Italy, Spain, Switzerland, Czech Republic, Uruguay, and Pakistan (Jo et al. 2015; Eichmeier et al. 2016; Xiao and Meng 2016; Blouin and MacDiarmid 2017; Reynard et al. 2017; Cho et al. 2018; Mahmood et al. 2019; Wu et al. 2020). Herein we report GRVFV infection in Vitis vinifera L. grapevines from Japan. In February 2021, dormant canes from 18 V. vinifera cv. Cabernet Sauvignon with leafroll-like disease symptoms, growing in a vineyard located in Kanagawa Prefecture, were collected. No typical vein banding symptom by GRVFV were observed in the grapevines during the growing season. Total RNA was isolated from the canes using an RNeasy Plant Mini Kit and QIAshredder (Qiagen, Valencia, CA), and subjected to cDNA synthesis using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara Bio, Shiga, Japan). RT-PCR was performed with GRVFV_6156F and GRVFV_6600R primers for GRVFV detection (Reynard et al. 2017). The expected 445 nucleotides (nt) amplification product was obtained from four of 18 grapevines. Sequence analysis of the products revealed 91% identities to corresponding sequences of GRVFV isolates CHASS (KY513702) and Mauzac (KY513701) from Switzerland. Genome walking to determine the whole-genome sequence of the GRVFV isolates from the four grapevines was performed. Briefly, the upstream and downstream of the 445 nt amplification product were amplified from first-strand cDNA using gene-specific primers designed from the product and CHASS-specific primers. Each amplified fragment was Sanger sequenced. Next, gene-specific primers were designed to obtain the complete genome of GRVFV as 13 overlapping DNA fragments from each of the four grapevine samples. An identical complete genome of 6,704 bp was assembled from the overlapping DNA fragments using MEGA 10 software and named as NA1 isolate (DDBJ accession no. LC619667). Phylogenetic analysis of the NA1 genome and corresponding sequences of GRVFV from other countries showed that NA1 formed a cluster with isolate NZ ChTK0004 from New Zealand (MF000326; Supplementary Figure 1). In pairwise comparisons, the complete NA1 genome was most identical at 88% and 87%, respectively to isolates NZ ChTK0004 and Mauzac. The predicted amino acid sequences of NA1 polyprotein shared high homologies (96%) to the corresponding polyprotein sequences of NZ ChTK0004 and Mauzac, suggesting that NA1 is genetically similar to GRVFV isolates from New Zealand and Switzerland. The NA1-infected Cabernet Sauvignon was co-infected with Grapevine leafroll-associated virus 3, Grapevine virus A, and Grapevine rupestris stem pitting-associated virus according to RT-PCR assay for grapevine virus detection (Nakaune and Nakano 2006). The results underscore the importance of intensifying quarantine measures to prevent introduction of exotic viruses via contaminated wine grape vegetative cuttings.

ICRISAT safely exported 182,605 seed samples to 127 countries during 1998–2019. The maximum number of exported samples were of sorghum (51,309) followed by chickpea (48,513) and pearl millet (39,771). A total of 2749 seed samples (chickpea 1301, groundnut 300, minor millets 50, pearl millet 338, pigeonpea 395 and sorghum 365) were rejected due to presence of seed-borne pathogens of quarantine significance. Thirty-one pathogenic fungal species were detected in the blotter test and seven pathogens detected in seed washing and sedimentation test. Maximum fungal/oomycetes species were detected in sorghum seed samples (18) followed by chickpea (16), pigeonpea (15), groundnut (10), pearl millet (9) and minor millets (6). Acremonium sp., Botrytis sp., Cercospora sp., Colletotrichum sp., Exserohilum turcicum, Fusarium sp., Phoma medicaginis, Phomopsis sp., Pyricularia grisea, Sclerotium rolfsii, Peronosclerospora sp., Sporisorium sorghi, Puccinia purpurea, Moesziomyces penicillariae, Claviceps fusiformis and Sphacelotheca sp. were detected in the seed samples.

Liposcelis entomophila (Psocodea: Liposcelididae) is one of the most economically important booklice in the world. This species damages stored grain and represents an allergenic health risk to humans. Because of the small size of booklice, they are difficult to identify using morphological characteristics. Traditional molecular identification, such as DNA barcoding and qRT-PCR, requires specialized equipment and takes a long time, which is not appropriate for practical usage by plant health inspectors at ports. Therefore, for storage psocid identification, we suggested a simple, rapid and relatively new technique known as loop-mediated isothermal amplification (LAMP). In our study, a new rapid molecular identification method using the LAMP technique based on the mitochondrial gene cytochrome c oxidase I (COI) was developed for L. entomophila with high specificity and efficiency. Evident amplification results could be presented if the DNA concentration was 0.001 ng/μL. Visual detection was also possible with our LAMP primers using hydroxy naphthol blue (HNB), relying on the clear change in color from violet to sky blue after amplification. This method successfully determined unknown species that were newly collected from grain stores and may facilitate L. entomophila identification in plant quarantine and stored product protection.

In 2015, a total of 31 samples belonging to superfamily Curculionoidea (Coleoptera) were intercepted from the cut flowers imported from South Africa at the Korean quarantine border. These samples were identified based on the available literature, and they were confirmed as 11 species belonging to four families. However, only three species were identified at the species level. The others were only classified into seven genera and one tribe level. Until now, there have been very few studies about weevils distributed in Africa. Consequently, there is a lack of available information for species identification of intercepted weevils at the quarantine border. This study aims to raise public awareness regarding the introduction of unwanted insect pests from the international trade of plants and/or plant products. It also suggests the need for international cooperation and taxonomic networking for the identification of intercepted pests. Keywords: Curculionoidea, cut flower, interception, plant quarantine, taxonomic networking system

Stored-product psocids (Psocoptera: Liposcelididae) are cosmopolitan storage pests that can damage stored products and cause serious economic loss. However, because of the body size (~1 mm) of eggs, nymphs, and adults, morphological identification of most stored-product psocids is difficult and hampers effective identification. In this study, 10 economically important stored-product Liposcelis spp. psocids (Liposcelis brunnea, L. entomophila, L. decolor, L. pearmani, L. rufa, L.mendax, L. bostrychophila, L. corrodens, L. paeta, and L. tricolor) were collected from 25 geographic locations in 3 countries (China, Czech Republic, and the United States). Ten species-specific probes for identifying these 10 psocid species were designed based on ITS2 sequences. The microarray method and reaction system were optimized. Specificity of each of the ten probes was tested, and all probes were found suitable for use in identification of the respective10 Liposcelis spp. psocids at 66 °C. This method was also used to identify an unknown psocid species collected in Taian, China. This work has contributed to the development of a molecular identification method for stored-product psocids, and can provide technical support not only to facilitate identification of intercepted samples in relation to plant quarantine, but also for use in insect pest monitoring.

Plum pox virus (PPV), the causal agent of sharka/plum pox disease, is considered the most harmful virus that threatens safe and successful cultivation of stone fruits (Prunus spp.; almond, apricot, cherry, nectarine, peach, and plum). Cambra et al. (2006) estimated that financial losses associated with sharka disease globally were approximately 3.6 billion (Bn) Euros for apricot for the preceding 30 years, 5.4 Bn Euros for plum for the preceding 30 years, and 0.58 Bn Euros for peach for the preceding 20 years. A considerable amount of research has been carried out on characterizing PPV, hence its identification as one of the top 10 plant viruses in molecular plant pathology (Scholthof et al., 2011). In spite of, and maybe because of, these research activities, the virus has been detected in almost every country where any significant commercial stone-fruit cultivation occurs, with the exception perhaps of Australia, New Zealand and South Africa. Are very strict plant quarantine programs or remoteness the keys to preventing the spread of PPV? Strategies for effectively controlling the spread of PPV must include simple, accurate and effective diagnostics (with PPV-appropriate sampling) that should be part of any quarantine program, survey activity, or phytosanitary certification program. Accurate information about the spread of PPV, better understanding of the various pathways for movement of the virus and taking the necessary steps to obstruct or remove these pathways are essential for preventing further spread of this very harmful virus.