Bursaphelenchus Species Research Articles

Bursaphelenchus glaucae n. sp. isolated from larval faeces of a longhorn beetle, Aeolesthes chrysothrix chrysothrix (Bates), in its pupal chamber constructed in the dead wood of Quercus glauca Thunb. from Japan

Summary A Bursaphelenchus species belonging to the hofmanni group (corneolus subgroup) was isolated from a small piece of larval faeces of the longhorn beetle Aeolesthes chrysothrix chrysothrix in its pupal chamber. The chamber was constructed in a dead wood section of Quercus glauca obtained from a live tree. The material was obtained during a management procedure of a garden tree to remove the dead part from live tree. The species is typologically characterised by its three-lined lateral field, the position of its secretory-excretory pore overlapping with the metacorpus anterior half, a very small spicule with distinctive broad and rounded condyles, a flattened trapezoid-shaped rostrum and small flattened cucullus, the presence of seven (P1, P2 and P3 papilliform and P5 gland) male genital papillae, a female vulva with a side flap and female tail forming an elongate conoid with a strongly ventrally curved posterior half and narrowly rounded or digitate tip. The species is typologically close to B. corneolus, sharing a small cucullus and female tail shape, but can be distinguished from the other species by the position of the secretory-excretory pore and the spicule rostrum shape. Phylogenetically, the new species forms a well-supported clade with B. corneolus, but can be distinguished by its clearly separable phylogenetic status inferred from SSU and D2-D3 LSU sequences and the difference in the ITS sequence. The new species is described as B. glaucae n. sp. Although the nematode was isolated from the larval faeces of A. c. chrysothrix, it has not been directly isolated from the insect body to date, thus, the carrier insect of the nematode has not been determined.

Molecular Detection of Phytophthora cinnamomi by RPA-CRISPR/Cas12a-Mediated Isothermal Amplification

Background: Phytophthora cinnamomi is one of the soil-borne pathogens that causes root rot and stem rot in many plants globally. P. cinnamomi has serious economic, social, and environmental impacts, threatening natural ecosystems and biodiversity. Methods: In this study, a molecular detection method based on Recombinant polymorphic amplification (RPA) combined using the CRISPR/Cas12a system was developed for P. cinnamomi. The method was found to be highly specific for P. cinnamomi. Results: The results showed that 10 P. cinnamomi isolates were positive; however, 21 Phytophthora species, 4 Phytopythium species, 18 fungal species, and 2 Bursaphelenchus species were negative. In total, 10 pg·µL−1 of P. cinnamomi genomic DNA can be detected. The detection process is performed within 20 min at 37 °C, which makes it fast and convenient for use. Discussion: In conclusion, the RPA-CRISPR/Cas12a system in this study is a promising tool for the rapid and sensitive detection of P. cinnamomi in plant samples.

Bursaphelenchus zealandicus sp. n. (Tylenchina: Aphelenchoididae), a new species of the hofmanni-group from Pinus radiata in New Zealand

Summary Bursaphelenchus zealandicus sp. n. was extracted from a dead pine tree (Pinus radiata) in Kinleith Forest, Taupo, New Zealand (NZ). This is the fourth Bursaphelenchus species from NZ. Bursaphelenchus zealandicus sp. n. is characterised by a body length of 562 (466-730) μm and 580 (470-706) μm of males and females, respectively, slim body (a = 34.7 and 33.1, respectively), stylet 11.4 (9.7-13.0) μm long for males and 10.7 (9.4-12.1) μm for females, lateral field with three lines, the mean value of the vulva position 71.8%, lacking a vulval flap in lateral view, length of the post-uterine branch ca 41.6% of the vulva-anus distance, the female tail long and conical (c = 19.3), tapering to a finely rounded terminus, ventrally curved like a hook, the male with spicules 12.3 μm long in chord, having an obvious developed rostrum and condylus, the rostrum sharply pointed, the male tail terminus with a distinct bursa seen in dorso-ventral position, a single pre-anal papilla, two pairs of ventro-sublateral papillae (one pair pre-anal (P2), one pair post-anal (P3) just in front of the bursa) and one pair of ventral mid-line glandular papillae (P4) present. The combination of three lateral lines and the position of caudal papillae reveal a relationship of the new species to other three-lined groups (hofmanni-group, eggersi-group, eremus-group, leoni-group); however, the shape of the spicules is different and unique among the known species of Bursaphelenchus. Its taxonomic status has been confirmed by analyses of the near full length small subunit (SSU), the internal transcribed spacer region (ITS1 and 2), D2-D3 expansion segments of the large subunit (LSU) of the ribosomal rDNA and the mitochondrial cytochrome c oxidase subunit I (COI) gene.

Utilizing a new target gene, Ppinihp1, identified from genome sequences to detect Phytophthora pini

Phytophthora pini is a globally distributed plant pathogen of the Phytophthora genus. This fungus-like organism was recently associated with foliage blight and shoot dieback disease of Rhododendron pulchrum Sweet, which threaten the health of natural ecosystems and horticultural crops in China. Early and accurate detection of P. pini is critical for effective disease prevention and management. In this study, a new target gene Ppinihp1, which is a hypothetical protein, identified from the whole-genome sequence of P. pini was used to establish a conventional polymerase chain reaction (PCR) assay for the detection of this pathogen. PCR results were proved to be positive for P. pini, but negative for other Phytophthora, Pythium, fungal isolates and Bursaphelenchus species. The test was sensitive enough to detect 100 pg of P. pini purified genomic DNA in a 50 μL reaction mixture. P. pini was also detected in artificially inoculated R. pulchrum samples. Finally, this novel Ppinihp1-PCR-assay was able to detect P. pini in 15 of 40 soil samples collected from R. pulchrum fields in Jiangsu, China. This method established the specificity, sensitivity, accuracy and convenience for P. pini detection, offering a dependable tool for the rapid screening of Phytophthora root and stem rot in R. pulchrum.

RPA-CRISPR/Cas12a mediated isothermal amplification for visual detection of Phytophthora sojae.

Phytophthora sojae is among the most devastating pathogens of soybean (Glycine max) and severely impacts soybean production in several countries. The resulting disease can be difficult to diagnose and other Phytophthora species can also infect soybean. Accurate diagnosis is important for management of the disease caused by P. sojae. In this study, recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system were used for detection of P. sojae. The assay was highly specific to P. sojae. The test results were positive for 29 isolates of P. sojae, but negative for 64 isolates of 29 Phytophthora species, 7 Phytopythium and Pythium species, 32 fungal species, and 2 Bursaphelenchus species. The method was highly sensitive, detecting as little as 10 pg.µL-1 of P. sojae genomic DNA at 37°C in 20 min. The test results were visible under UV light and readout coming from fluorophores. In addition, P. sojae was detected from natural inoculated hypocotyls of soybean seedlings using this novel assay. The rapidity and accuracy of the method were verified using 30 soybean rhizosphere samples. In conclusion, the RPA-CRISPR/Cas12a detection assay developed here is sensitive, efficient, and convenient, and has potential for further development as a kit for monitoring root rot of soybean in the field.

Bursaphelenchus ogawanus n. sp. isolated from bark material of Fagus japonica Maxim. from Japan

Summary A Bursaphelenchus species belonging to the abietinus group was isolated from a bark sample of Fagus japonica infested by an unidentified Dryocoetes bark beetle. The species is typologically characterised by its two-lined lateral field, mitten-shaped spicule possessing a well-developed and dorsally truncate condylus, narrow triangular rostrum and a distinct cucullus, the presence of seven (P1, P2, P3 papilliform and P5 gland-opening-like) male genital papillae, female vulva with side flap and the long, smoothly tapering and ventrally recurved female tail with strongly ventrally recurved distal part. The species is typologically close to B. irokophilus and B. rainulfi but can be distinguished from these two species by the shape of spicule rostrum, which is narrower in the new species. Phylogenetically, the new species forms a well-supported clade with B. rainulfi but can be distinguished from them by the differences in the SSU and D2-D3 LSU sequences. The new species is described as Bursaphelenchus ogawanus n. sp.

Two decades of epidemiological surveillance of the pine wood nematode in France reveal its absence despite suitable conditions for its establishment

Key messageThis study takes stock of the first 20 years (2000–2019) of monitoring the pine wood nematode (PWN) in metropolitan France. While PWN was never found in the wild during this period, it was reported in some wood-based commodities entering or circulating on French territory. This stresses the importance of remaining extremely vigilant, as the conditions found in France, especially weather conditions, could be particularly suitable for the pest’s establishment.ContextThe pine wood nematode (PWN) Bursaphelenchus xylophilus, responsible for pine wilt disease (PWD), is one of the most important forest tree pests worldwide. It is thus the focus of many monitoring programmes. In the European Union, for example, it is categorised as a priority quarantine pest, so each member state is obliged to monitor the PWN on its territory.AimsThe first objective of this paper was to describe PWN monitoring in metropolitan France, namely how it is organised and whether it has led to the nematode’s detection. Secondly, we wished to investigate what the levels of PWD expression for host pines infected by B. xylophilus would be in France. Thirdly, we wanted to find out whether other Bursaphelenchus species had been found on French territory during these two decades of PWN monitoring.MethodsWe analysed data from samples collected in the framework of the monitoring programme between 2000 and 2019 to track the PWN in its host pines, its insect vector (Monochamus spp.) and in wood-based commodities imported into or circulating in metropolitan France. We also generated risk maps of PWD expression based on an evapo-transpiration model using climate data for the period 2000–2019.ResultsThis monitoring, which was regularly reinforced from 2000 to 2019, consisted of sampling and analysing around 18,000 wood samples and 66,000 insects over this period. Although the PWN was not detected in pine stands or within its insect vector, some wood-based commodities were found to be contaminated. Risk maps of PWD expression show that in the most recent years (2015–2019), the weather conditions in a large fraction of metropolitan France were suited to PWD expression, mostly with a delay (i.e., latency) between infection and observable wilt symptoms. PWN monitoring has also revealed the presence of other Bursaphelenchus species, most of which were discovered for the first time in metropolitan France and are described herein.ConclusionWhile metropolitan France is still free of the PWN, this study emphasises the need to remain cautious as the French territory appears particularly suitable for this pest’s establishment. Furthermore, our research has led us to propose some ideas on how to improve PWN monitoring.

Rapid detection of the pine wood nematode Bursaphelenchus xylophilus using recombinase polymerase amplification combined with CRISPR/Cas12a

The pine wilt disease (PWD) caused by pine wood nematode (PWN), Bursaphelenchus xylophilus, is a destructive forest disease and causes global economic losses. Effective and affordable detection of PWN is critical for the management of this pest. In this study, we developed a rapid diagnostic assay using recombinase polymerase amplification (RPA) combined with the CRISPR/Cas12a system to detect PWN. The assay was validated as being fast, accurate, and reliable based on detection of PWN from pine wood samples. Test results for 33 PWN were positive, but negative for 11 isolates Bursaphelenchus species, 10 other nematodes, 20 isolates of 15 fungi species, and 6 isolates of Phytophthora species. This method allow us to have the results naked-eye directly. Results from this study indicated that this sensitive, specific, and rapid RPA-CRISPR/Cas12a assay has potentially significant applications to detect PWN, especially under time- and resource limited conditions.

Bursaphelenchus leoni Baujard, 1980 (Tylenchina: Aphelenchoididae)'nin Türkiye'nin Çam Ormanlarından Ilk Kaydı

Several surveys have been performed in Turkey in order to determine a possible presence of Bursaphelenchus xylophilus (Steiner and Buhrer, 1934), Nickle, 1970. As a result, several Bursaphelenchus species have been reported. During a study aimed to identify potential insect vectors of these Bursaphelenchus species using the trap tree method in the western region of Turkey, one Bursaphelenchus species was isolated from a wood chip sample of a Pinus brutia trap log located in Bergama town of the city of İzmir. According to morphological and molecular studies, the species was determined as Bursaphelenchus leoni Baujard, 1980. Morphological characteristics of the species matched well with the original description made by Baujard in 1980. 18S and 28S rRNA genes of B. leoni were sequenced and a phylogenetic tree was constructed that showed B. leoni was clustered with B. leoni in the NCBI database. This is the first report of B. leoni from Turkey. To determine presence of other known Bursaphelenchus species in Turkey, new studies should be performed.

Rapid detection of Phytophthora cinnamomi based on a new target gene Pcinn13739.

Phytophthora cinnamomi causes crown and root wilting in more than 5,000 plant species and represents a significant threat to the health of natural ecosystems and horticultural crops. The early and accurate detection of P. cinnamomi is a fundamental step in disease prevention and appropriate management. In this study, based on public genomic sequence data and bioinformatic analysis of several Phytophthora, Phytopythium, and Pythium species, we have identified a new target gene, Pcinn13739; this allowed us to establish a recombinase polymerase amplification–lateral flow dipstick (RPA-LFD) assay for the detection of P. cinnamomi. Pcinn13739-RPA-LFD assay was highly specific to P. cinnamomi. Test results for 12 isolates of P. cinnamomi were positive, but negative for 50 isolates of 25 kinds of Phytophthora species, 13 isolates of 10 kinds of Phytopythium and Pythium species, 32 isolates of 26 kinds of fungi species, and 11 isolates of two kinds of Bursaphelenchus species. By detecting as little as 10 pg.µl−1 of genomic DNA from P. cinnamomi in a 50-µl reaction, the RPA-LFD assay was 100 times more sensitive than conventional PCR assays. By using RPA-LFD assay, P. cinnamomi was also detected on artificially inoculated fruit from Malus pumila, the leaves of Rhododendron pulchrum, the roots of sterile Lupinus polyphyllus, and the artificially inoculated soil. Results in this study indicated that this sensitive, specific, and rapid RPA-LFD assay has potentially significant applications to diagnosing P. cinnamomi, especially under time- and resource-limited conditions.

Nematodes in the Pine Forests of Northern and Central Greece.

Simple SummaryPine wood nematode Bursaphelenchus xylophilus is the agent of pine wilt disease and one of the most important forest tree pathogens worldwide, transmitted through beetles of the Monochamus genus. As an invasive species, it has spread beyond its natural range by human activity mainly wood trade. The devastating impact it has on pine forests has led to severe environmental and economic damages in its introduced countries. The wide distribution of Monochamus spp. beetles in many parts of the world along with favourable climatic conditions, which are both important factors for the establishment of pine wilt disease, have raised awareness over its continuous expansion. Therefore, in an attempt to control and even inhibit its further spread and consequently its severely adverse impacts, appropriate measures have already been taken and implemented from countries across the globe.In the context of plants or plant products protection by harmful organisms, measures have been taken by EU countries in order to prevent their introduction and establishment into the EU, and also limit their expansion in case they do enter. Such a case is Bursaphelenchus xylophilus (Parasitaphelenchidae, Nematoda), already recorded in Portugal and Spain. So, Member States should take all the appropriate steps in order to monitor and confine if necessary susceptible plants and/or plant products. Such measures include annual surveys even in countries where pine wilt disease does not occur yet. Therefore, national survey programs are widely established, sampling and examining samples from pine trees showing suspicious symptoms that could potentially be attributed to B. xylophilus. In this direction, such a network has also been established in Greece collecting and examining wood samples nationwide. In total, 123 wood samples were collected from conifer trees of Northern and Central Greece. Though B. xylophilus was absent from all samples examined, four other Bursaphelenchus species were identified. In addition, other nematode taxa were also recorded, including several phytophagous, microbivorous as well as predatory nematode species. This highlights the fact that besides preventing the introduction of B. xylophilus in Greece, national survey programs can significantly contribute to and enhance our knowledge of the indigenous nematode species.

Effect of Bursaphelenchus spp. inoculation on carbohydrate concentrations of differentpine species in forest stands of Düzce Forest Directorate

The pinewood nematode (Bursaphelenchus xylophilus) is one of the most important pathogens of conifer forests worldwide that causes the pine wilt disease. This problem has increased the scientific interest in Bursaphelenchus species both in the world and in Turkey. Previously, six Bursaphelenchus species were isolated from wilted pine trees in Turkey. The main goal of this study was to investigate the effects of nematode inoculation on the total carbohydrate concentrations of tree species. Native six Bursaphelenchus species were inoculated on three pine species (Pinus pinaster, P. nigra, and P. sylvestris) under natural stand conditions. Two different doses of nematodes (2000 and 20,000) were used for inoculation. For each treatment, three trees of each species were inoculated. The total carbohydrate concentrations were differed significantly among tree species, nematode species and between nematode doses. The total carbohydrate concentration values were higher in P. pinaster than both P. nigra and P. sylvestris. These results may suggest that pine species responded to nematode inoculation by changing the amount of TCC.

First record of Bursaphelenchus hildegardae Braasch et al., 2006 (Nematoda) in New Zealand with updated information on morphology, sequencing and a key to species of the eggersi-group.

Bursaphelenchus hildegardae Braasch et al., 2006 was collected from pine wood (Pinus radiata) growing in Kaingaroa Timberlands, and a bark beetle, Hylastes ater Paykull, 1800 in New Zealand. This is a new record for B. hildegardae, occuring in New Zealand, and the second report from the southern hemisphere in addition to Australia. In general, the New Zealand isolate of B. hildegardae corresponds well with the description of B. hildegardae given by Braasch et al. (2006) from Germany. The New Zealand isolate is characterized by having an adult body length of 8071190 m, medium a ratios (47.558.5 for female and 44.660.1 for male), b ratios of 9.814.5 (female) and 10.212.7 (male), c ratios of 18.825.2 (female) and 21.632.4 (male), c ratios of 4.04.4 (female) and 2.12.7 (male), and is characterised by having three incisures in the lateral fields, thorn-shaped spicules with a distinctly dorsally-bent thin hook-like condylus, and a dorso-ventally visible terminal bursa. In addition, molecular phylogeny using near full length small subunit (SSU), D2/D3 expansion segments of the large subunit (LSU) and the internal transcribed spacer region (ITS1 and 2) of the ribosomal rDNA supports the identification. A key to Bursaphelenchus species in the eggersi-group is given.

Assessment of the PCR technique for identification of Bursaphelenchus mucronatus Mamiya & Enda 1979, B. xylophilus Steiner & Buhrer 1934 (Nickle 1970) and B. fraudulentus Rühm 1956 (Nematoda, Aphelenchoididae) in crude extract of nematodes isolated from wood

The pine wood nematode, Bursaphelenchus xylophilus Steiner & Buhrer 1934 (Nickle 1970) is the major causative agent of the pine wilt disease which has become devastating to Asian and European coniferous forests. These regions are also naturally occupied by two other native but nonpathogenic species, i.e. B. mucronatus Mamiya & Enda 1979 and B. fraudulentus Rühm 1956 which are closely related to the invasive B. xylophilus. Moreover, all these three species can colonize pine trees, and potentially be extracted from the same wood samples. Due to the cosmopolitan character and wide genetic variation within- and between existing populations the taxonomic distinction of these species based exclusively on their morphology is difficult or, almost impossible. The present quarantine regulations related to B. xylophilus require the most credible and simple methods which could allow for a possibly earliest detection and precise identification of this species in wood shipments and conifer forests stands. The main objectives of the presently reported research were to simplify the presently available procedures for possibly fast and precise detection and identification of B. xylophilus examined in the background of the remaining Bursaphelenchus species of the xylophilus group and other bacterio- and mycetophagous nematodes naturally present in the pine wood samples. The developed method is based on a direct examination of the crude nematode extract from wood samples and subsequent use of PCR technique with earlier designed specific reaction starters amplifying ITS1–28S rDNA regions.

BURSAPHELENCHUS SPECIES (NEMATODA: APHELENCHIDA) ASSOCIATED WITH AN OLIVE TREE IN CENTRAL ITALY

Olive cultivation is of great economic, ecological, and cultural interest in Italy, as well as in the rest of the Mediterranean basin. Among the pests of olive trees, several groups of insects, mites, and nematodes have been reported. Phytoparasitic nematodes especially of the genera Meloidogyne, Pratylenchus, Helicotylenchus, Xiphinema, Tylenchulus, Rotylenchulus, and Heterodera have usually been extracted from roots and soil around trees. On the other hand, no information is available concerning nematodes directly associated with the wood. At the end of September 2018, in a high-density cultivated olive grove in Tuscany (central Italy), several olive trees with decline symptoms were observed. Three Bursaphelenchus species, B. fungivorus, B. minutus, and B. sexdentati were extracted from the wood of one dead tree. Even though these species had already been reported in Italy, these findings were the first ones recorded in olive wood. Moreover, another undescribed Bursaphelenchusspecies was found associated with the bark beetle Hylesinus fraxini collected from olive trunks and branches. Further research is needed to investigate the role of insects and Bursaphelenchus spp. in the decline processes of olive trees.

Preliminary survey of nematodes associated with broadleaved trees in İzmit Forest Management Directorate, Turkey

AbstractA survey for Bursaphelenchus species was carried out in broadleaf forest sites of İzmit Forest Management Directorate. Nematodes were extracted from 128 wood samples, and 22 of them were cultured. Nematodes were identified using morphological characteristics and molecular analysis by amplifying D2‐D3 LSU with primers. Only one Bursaphelenchus species, Bursaphelenchus mucronatus kolymensis, was isolated from Quercus petraea representing the first report from broadleaved tree species in Turkey. Paraphelenchus acontioides and Aphelenchoides heidelbergi were identified and presented herein as the first reports of these two species from Turkey. In addition, five Aphelenchoides species were also found and reported as Aphelenchoides sp.

New observations on occurrence and distribution of Bursaphelenchus spp. (Nematoda: Aphelenchoididae) in conifers in Romania

AbstractSix species of the genus Bursaphelenchus were recovered as the result of a survey to assess the potential introduction of Bursaphelenchus xylophilus into the conifer forests of Romania. Bursaphelenchus xylophilus was not detected in the sampling regions. Bursaphelenchus dietrichi was recovered from one point, representing the first report of this species in the country. The other detected species, B. abietinus, B. andrassyi, B. hofmanni, B. fraudulentus and B. willibaldi, were characterized by their morphometric data and molecular analyses. The morphometrics of Romanian populations of B. hofmanni and B. willibaldi are presented for the first time. The phylogenetic relationships of the recovered species (except for B. abietinus, with no successful sequencing) were reconstructed using the sequences of the D2‐D3 expansion segments of the large subunit of ribosomal DNA (D2‐D3LSU rDNA). Some of the species were also characterized using the restriction fragment length polymorphism (RFLP) pattern of the internal transcribed spacer (ITS) fragment. The distribution, related host trees and phylogenetic relationships of the identified species are discussed.

The genus Berntsenus Massey, 1974 is a junior synonym of Bursaphelenchus Fuchs, 1937

Summary Stylet (feeding) dimorphism of Bursaphelenchus sinensis was experimentally confirmed during a recent study and its predatory form was considered similar in morphology to species of Berntsenus. Therefore, available type and voucher materials for members of Berntsenus were examined for their morphology and compared with Bursaphelenchus sinensis and B. aberrans, another related species showing feeding dimorphism. The typological characters of their co-habiting aphelenchoidid species were also examined. The type materials of Berntsenus spp. have characteristic squared lips, a thick stylet, large median bulb and short and stout pharyngeal glands; males, when present, have a P1 papilla and a tape-like bursa, suggesting that they are the predatory form of improperly classified Bursaphelenchus species. In addition, Aphelenchoides pityokteini and Ektaphelenchus tenuidens (= Aphelenchoides tenuidens), co-occurring with their respective host bark beetle isolates of Berntsenus, were typologically confirmed to belong to the aberrans-group of Bursaphelenchus and considered to be conspecific to each co-habiting Berntsenus species. Herein, Berntsenus is synonymised with Bursaphelenchus, and Bursaphelenchus brachycephalus n. comb. (= Berntsenus brachycephalus and E. tenuidens) and Bursaphelenchus pityokteini n. comb. (= A. pityokteini and Berntsenus labiosus) are proposed.

First detection of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on Monochamus sutor (Coleoptera: Cerambycidae) in Romania

AbstractAs a result of the detection of the pinewood nematode Bursaphelenchus xylophilus in Portugal, and its subsequent spread to Spain, intense surveys were conducted to screen for the presence of Bursaphelenchus species in Romania. Herein, we report recent surveys of insects potentially vectoring Bursaphelenchus species collected using trap trees or pheromone‐baited traps placed in the forest. Trap felled spruce trees (Picea abies) and pheromone‐baited traps were installed in six different counties in Romania (Brașov, Sibiu, Suceava, Hunedoara, Timiș and Dâmbovița). Ten different species of insects distributed among Curculionidae and Cerambycidae were obtained. Nematodes were extracted from insects and observed to validate the presence of Bursaphelenchus specimens. One female identified as Monochamus sutor was the only specimen carrying nematodes in the genus Bursaphelenchus. Nematodes were identified as B. mucronatus based on morphological and molecular features. This is the first detection and report of natural spread of B. mucronatus in Romania. The absence of B. xylophilus was confirmed in the areas of Romania surveyed in this work.

Report of Bursaphelenchus crenati Rühm, 1956 (Nematoda: Parasitaphelenchinae) from Belarus and Russia with a diagnostic key and phylogeny of the Sexdentati group

AbstractThe endoparasitic nematode, Bursaphelenchus crenati, in beetle tunnels of Hylesinus crenatus from the ash Fraxinus excelsior L. with signs of ash dieback, is recorded for the first time in Central Russia and Belarus. Third‐stage dauer juveniles were extracted from the bark beetle, H. crenatus, and cultured on Botrytis cinerea. Morphological, morphometric and molecular analyses of these populations are presented here. Some morphological differences of dauers and adults were found between Belarusian and Russian populations, which may be attributed to seasonal variations. For description of new Bursaphelenchus species, it is recommended to indicate not only the number of lateral incisures but also the number of bands. Recommendations are made to classify the male caudal papillae of Bursaphelenchus into two groups: papilliform and gland papillae with separate numerals. Emended diagnosis, tabular diagnostic polytomous key for the species of Sexdentati group with lists of their vectors and plants, is given. Phylogenetic relationships between some species of the Sexdentati group are also presented based on the analysis of partial 18S, ITS and 28S rRNA gene sequences.