Xylophilus Group Research Articles

How did Bursaphelenchus nematodes acquire a specific relationship with their beetle vectors, Monochamus?

For insect-borne pathogens, phoretic ability is important not only to spread more widely and efficiently but also to evolve virulence. Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is transmitted by the cerambycid beetle Monochamus alternatus, which is associated with pine tree host. Their specific phoretic ability to appropriate vectors depending on their life cycle is critical for efficient transfer to the correct host and is expected to enhance virulence. We evaluated how B. xylophilus acquired a specific relationship with M. alternatus with a focus on Bursaphelenchus okinawaensis, a close relative of B. xylophilus that has evolved a relationship with a cerambycid beetle vector. Bursaphelenchus okinawaensis has a single dispersal stage (dauer) larva (third-stage dispersal [DIII] larva), whereas B. xylophilus has two distinct dispersal stages (DIII and fourth-stage dispersal [DIV] larva). Also, the dauer formation in B. okinawaensis is not completely dependent on its beetle vector, whereas DIV larvae of B. xylophilus are induced by volatile from the beetle vector. We investigated the induction conditions of dauer larvae in B. okinawaensis and compared to with B. xylophilus. The dauer percentages of B. okinawaensis significantly increased when the nematode population on the plate increased or when we propagated the nematodes with a crude extract of cultured nematodes, which likely contained dauer-inducing pheromones. In addition, dauer formation tended to be enhanced by the crude extract at high temperatures. Furthermore, when we propagated the nematodes with M. alternatus pupae until the beetles eclosed, B. okinawaensis significantly developed into dauer larvae. However, only 1.3% of dauer larvae were successfully transferred to M. alternatus, the rate lower than that of B. xylophilus. DIII and DIV of B. xylophilus were induced by increasing the nematode population and the presence of the beetle vector, respectively. These results suggest that B. okinawaensis has acquired specificity for the cerambycid beetle through dauer formation, which is efficiently induced in the presence of the beetle, and the DIV larval stage, exclusive to the xylophilus group, may be crucial for high transfer ability to the beetle vector.

Female's war: a story of the invasion and competitive displacement between two xylophilus group nematode species

Female's war: a story of the invasion and competitive displacement between two xylophilus group nematode species

Transfer of four isolates of Bursaphelenchus doui into Monochamus alternatus and potential vector switching of the nematode

Summary To reinforce vector-switching potential of Bursaphelenchus doui in its ecological and evolutionary contexts, we tested our previous hypothesis that “B. doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad-leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and Bursaphelenchus xylophilus”. The affinity of four B. doui isolates and one B. xylophilus isolate for Monochamus alternatus was tested using our simple nematode-loading method to the beetle, and the affinity was assessed based on the nematode loads on the beetles. Phoretic stages of two B. doui isolates obtained from Monochamus saltuarius and Pinus densiflora showed loading levels similar to that of B. xylophilus, which were significantly higher than those of the other two B. doui isolates obtained from A. fraudatrix and M. subfasciatus. This result indicates that the first two isolates of B. doui derived from a conifer-using beetle and a coniferous tree adapt to M. alternatus better than the last two isolates associated with beetles using broad-leaved trees. The data reinforce that vector switching of B. doui could have occurred during the evolutionary history of the B. xylophilus group.

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.

Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes (Nematoda: Aphelenchoididae).

To show the importance of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we tested a hypothesis that “Bursaphelenchus doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad‐leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and B. xylophilus.” We used a simple nematode‐loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. Phoretic stages of B. doui appeared in all beetle species examined, namely Acalolepta luxuriosa, Psacothea hilaris, A. fraudatrix, and Monochamus alternatus, although the affinity of the nematode for M. alternatus was weak. This finding indicates that B. doui could switch vectors to conifer‐using Monochamus beetles after transfer by A. fraudatrix from broad‐leaved trees to conifers. We conclude that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group.

A fast and sensitive multiplex real-time PCR assay for simultaneous identification of Bursaphelenchus xylophilus, B. mucronatus and B. fraudulentus – three closely related species from the xylophilus group

The quarantine nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease in Asia and Europe. Differentiation between B. xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. In this paper we described the results of the optimized multiplex real-time PCR protocol that utilizes two universal primers and three specific TaqMan probes to identify and differentiate the three Bursaphelenchus species, simultaneously. Species-specific real-time PCR reactions gave expected products for B. xylophilus, B. mucronatus and B. fraudulentus templates. The optimized primer combination together with designed species-specific probes has produced reliable results in multiplex PCR assays with eleven geographically distant isolates of B. xylophilus, eleven of B. mucronatus, seven of B. fraudulentus, and no cross-reactions were observed between tested samples and with other species (i.e., B. piniperdae, B. pinophilus, B. populi and Parasitaphelenchus papillatus). All species-specific real-time PCR reactions performed on DNA extracted from a single nematode individual consistently gave specific amplicons, confirming the reproducibility of the method. Moreover, the conducted study revealed that 30 fg was the lowest DNA input still detected in multiplex real-time PCR analysis. The described approach is simple, time-saving, and reliable in comparison to conventional PCR, and it can be used for simultaneous identification of the above three species within the xylophilus group.

Multiplex polymerase chain reaction for simultaneous detection and identification of Bursaphelenchus xylophilus, B. mucronatus and B. fraudulentus – three closely related species within the xylophilus group

Differentiation between Bursaphelenchus xylophilus and other related, non-pathogenic species can be ambiguous when based exclusively on morphological characters. The morphology of B. mucronatus and B. fraudulentus most closely resembles that of B. xylophilus. Moreover, all of these nematodes are found in both Asia and Europe and can colonise various species of pine. Therefore, for phytosanitary purposes it is necessary to identify the three species precisely and rapidly. We report the results of a multiplex PCR that utilises four primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex PCR yielded DNA fragments of 767, 305 and 132 bp, for B. xylophilus, B. mucronatus and B. fraudulentus, respectively. This primer combination has produced reliable results in multiplex PCR assays with a number of different populations of the listed species, and no cross-reactions were observed with other Bursaphelenchus species. The described approach is simple, reliable and cheaper than other molecular methods presently used for simultaneous identification of the above three species within the xylophilus group.

Improved morphological key to the species of the xylophilus group of the genus BursaphelenchusFuchs, 1937

Correct identification of the pine wood nematode Bursaphelenchus xylophilus becomes more difficult, as more related or similar species are described. Spread of the nematode is being monitored worldwide, and in view of the fact that several new Bursaphelenchus species have been described, identification keys should be adjusted accordingly. Based on the identification key of the xylophilus group published by Braasch (2008) and the revised intrageneric grouping of Bursaphelenchus by Braasch et al. (2009), an improved dichotome morphological key including newly described species (B. paraluxuriosae, B. populi, B. macromucronatus, B. firmae, B. koreanus and B. gillanii) is presented. The recognition of the quarantine pest B. xylophilus using morphological characteristics in laboratories of the National Plant Protection Services is facilitated by a simplified key.

Bursaphelenchus gillanii sp. n. (Nematoda: Aphelenchoididae) – a new species of the xylophilus group in packaging wood imported from China

A new Bursaphelenchus species of the xylophilus group was detected in coniferous packaging wood imported with goods from China in 2011. The new species is described herein and compared with other species of the xylophilus group. Bursaphelenchus gillanii sp. n. has a slim body (a = 31 (28-34) and 33 (29-36) in females and males, respectively), c′ = 3.8 (3.2-4.5) and 2.1 (1.7-2.5) in females and males, respectively, a large vulval flap, a 5-7 μm long digitate mucro as a continuation of the female tail, excretory pore at or closely posterior to the median bulb, strongly arcuate spicules, 34 (31-37) μm long as measured along the median line, with prominent pointed rostrum and small cucullus. The ITS-RFLP pattern of the new species obtained by digestion of the PCR product with RsaI, HaeIII, MspI, HinfI and AluI is different from other known Bursaphelenchus species. Results of sequencing the ITS1/2 region demonstrate the close relationship of the new species to B. mucronatus and B. xylophilus.

Bursaphelenchus tiliae sp. n. (Nematoda: Parasitaphelenchidae), a nematode associate of the bark beetle Ernoporus tiliae (Panz.) (Coleoptera: Curculionidae, Scolytinae), in small-leaved lime, Tilia cordata Mill.

Bursaphelenchus tiliae sp. n. is described from the bark of dead branches of the small-leaved lime, Tilia cordata, infested with the lime bark beetle, Ernoporus tiliae. The nematode adults and propagative juveniles were present in larval galleries, while dispersal dauer juveniles colonised the Malpighian tubules of adult beetles. Bursaphelenchus tiliae sp. n. is characterised by the body length of 664 (568-817) μm in female and 622 (515-754) μm in male, moderately slender body (a = 41.0 (34.7-47.9) and 43.1 (37.8-51.9) in female and male, respectively), and spicules 20.0 (18.1-21.8) μm long. The extended anterior vulval lip in female, lateral fields with four incisures, long post-uterine sac, and number (7) and arrangement of male caudal papillae, may indicate that B. tiliae sp. n. is closely related to the xylophilus group. It differs from this group by smaller, arcuate spicules with relatively short rostrum, narrow capitulum (5.2-6.9 μm), and indistinct condylus in an almost continuous line with dorsal lamina. Distal third of spicule dorsal contour is usually almost straight. The spicules have a distinct, disk-like cucullus at the terminus. The status of the new species is confirmed by the unique molecular profile of the ITS region (ITS-RFLP) and its close relation to the xylophilus group by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The presence of B. tiliae sp. n. dauer juveniles in Malpighian tubules of their vector bark beetle, E. tiliae, caused limited pathogenicity expressed as a local expansion of the tubule basement membrane and partial degradation of the cellular epithelium.

Revision of Beardius Reiss et Sublette, 1985 (Diptera: Chironomidae), with the description of twenty new species.

The genus Beardius Reiss et Sublette, 1985 is revised and twenty new species, Beardius abbadi sp. n., B. arawak sp. n., B. bizzoi sp. n., B. bucephalus sp. n., B. curticaudatus sp. n., B. dioi sp. n., B. hirtidorsum sp. n., B. krenak sp. n., B. kumadueni sp. n., B. longicaudatus sp. n., B. mileneae sp. n., B. nebularius sp. n., B. neusae sp. n., B. novoairensis sp. n., B. phoenix sp. n., B. sapiranga sp. n., B. tupinamba sp. n., B. vanessae sp. n., and B. yperoig sp. n. from Brazil and Beardius chapala sp. n. from Mexico are described and figured. Further, the female, pupa and larva of B. urupeatan Pinho, Mendes et Andersen and the female and larva of B. cristhinae Trivinho-Strixino et Siqueira are described, and new records of Brazilian species are provided. Emended diagnoses for all stages and sexes are given. The phylogeny of the genus is outlined and seven tentative species groups, the parcus group, the truncatus group, the tupinamba group, the triangulatus group, the bucephalus group, the xylophilus group, and the roquei group, are proposed. Keys to males, females, pupae and larvae are provided.

Validation of morphological keys for identification of Bursaphelenchus xylophilus (Nematoda, Parasitaphelenchidae) to group and species level

Official analyses undertaken in the framework of an official survey, or import controls need reliable results. This can be achieved by using validated methods. For morphological tests, this validation process is rarely illustrated despite guidance provided in EPPO PM 7/98 (1) Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. This paper presents validation for the morphological identification of Bursaphelenchus xylophilus, with the evaluation results of published identification keys and of internally designed identification keys at B. xylophilus group and species levels. For published identification keys some criteria were shown not to be reliable for routine use: excretory pore position and number of caudal papillae. The key designed in the laboratory for xylophilus group identification was shown to be sensitive and specific when one male and one female were observed. The key designed for B. xylophilus species identification is sensitive, specific and reproducible if only one female of B. xylophilus is observed. The tools designed were validated as simple and reliable for routine analysis. The advantages and limitations of the validation process for morphological tools are discussed for process improvement.

An Entomoparasitic Adult Form inBursaphelenchus doui(Nematoda: Tylenchomorpha) Associated withAcalolepta fraudatrix

The nematode family Aphelenchoididae (Rhabditida: Tylenchomorpha) includes species with various feeding habitats. Bursaphelenchus, a member of the family, has for a long time been considered as a home for plant parasitic or mycophagous species (or both). However, recent intensive biological studies on the family revealed that the genus contains several insect parasitic species. Dauer juveniles of Bursaphelenchus doui were isolated from Acalolepta fraudatrix during a field study of longhorn beetle-Bursaphelenchus nematode associations. Two different insect-associated forms, an "entomoparasitic adult form" and a regular dauer juvenile, were isolated from a single individual beetle in a subsequent laboratory investigation of the B. doui-A. fraudatrix relationship. Thus these 2 distinct, insect-associated forms were confirmed to occur simultaneously. The entomoparasitic form is morphologically similar to that of Bursaphelenchus luxuriosae, with a dome-shaped head and vacuole-like spots assumed to be an internal structure of sensory organ, a stylet, a metacorpus (median bulb), and a moderately-developed and seemingly fully functional reproductive system. It is distinguishable from B. luxuriosae based on male spicule morphology and female tail morphology. A degenerate ingestive-digestive system distinguishes the entomoparasitic form from the propagative form and, unlike dauer juveniles, it has a moderately-developed reproductive system. The presence of this characteristic parasitic adult form is known only in these 2 Bursaphelenchus species. However, these 2 species did not form a clear monophyletic clade within the Bursaphelenchus xylophilus group and, thus, this characteristic parasitic form may occur independently in each species.

Bursaphelenchus masseyi sp. n. (Nematoda: Parasitaphelenchinae) – a nematode associate of the bark beetle, Trypophloeus populi Hopkins (Coleoptera: Curculionidae: Scolytinae), in aspen, Populus tremuloides Michx. affected by sudden aspen decline in Colorado

Bursaphelenchus masseyi sp. n. is described from trunks of unhealthy trembling aspen, Populus tremuloides, affected by sudden aspen decline (SAD) in Colorado, USA. All propagative stages of the nematode were present in larval galleries of a bark beetle, Trypophloeus populi. The dauer juveniles occupy the haemocoel of older larvae, pupae and adult beetles. The characteristic morphology of the male spicules with small but distinct cucullus, extended anterior vulval lip in female, lateral fields with four incisures, and number and arrangement of male caudal papillae, indicate that B. masseyi sp. n. is closely related to the xylophilus group. This relation has been confirmed by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The new species is characterised by the body length of 958 (765-1203) μm in female and 874 (691-1122) μm in male, moderately slender body (a = 38.2 (33.5-41.1) and 38.0 (33.1-44.1) in female and male, respectively), and spicules 29.2 (26.2-34.7) μm long with a small cucullus (1.3-1.5 μm in diam.) at their tips. Bursaphelenchus masseyi sp. n. can be separated from other species in the xylophilus group by the morphology of spicules which have a short capitulum and unique rostrum that is pointed somewhat anteriorly, relatively thick vulval flap, which is straight, parallel to the body long axis and bent towards the body wall at its distal end, and other morphological and morphometric characters. The new species most closely resembles B. trypophloei, but differs by the morphology of spicules (short but distinct condylus vs condylus in a continuous line with dorsal lamina, and smaller cucullus). The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). Diallelic cross-breeding in vitro revealed also that B. masseyi sp. n. and B. trypophloei are reproductively incompatible. The new species showed poor ability to develop and reproduce on Botrytis cinerea cultures, although it grew vigorously on laboratory cultures of Polish and Colorado isolates of Cytospora chrysosperma, the fungus naturally associated with galleries of the nematode vector, T. populi.

Bursaphelenchus koreanus sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from South Korea

Bursaphelenchus koreanus sp. n., isolated in Ningbo, P.R. China, from packaging wood made from Pinus sp. imported from South Korea, is described. It is characterised by a slim body (a = 28-35), lateral field with four lines, excretory pore located at level of, or slightly posterior to, median bulb, vulva at 75% of total body length, presence of a distinct vulval flap in lateral view, post-uterine branch extending for ca two-thirds of vulva-anus distance, female tail conical and ventrally bent with slightly pointed, irregular or roughened terminus (c′ = 4.1), spicules large and arcuate (27-34 μm) with pointed rostrum, cucullus visible but sometimes reduced to a small blunt extension. The new species belongs to the xylophilus group and is most similar to B. luxuriosae and B. paraluxuriosae. It is distinguished from other Bursaphelenchus species by morphology, ITS-RFLP patterns and sequencing results.

Wykorzystanie techniki real-time PCR do wykrywania kwarantannowego gatunku nicienia Bursaphelenchus xylophilus

The real-time PCR (polymerase chain reaction) based on TaqMan chemistry was used to detect and distinguish Bursaphelenchus xylophilus from other wood-inhabiting nematode species of the xylophilus group. In the study reported here DNA (deoxyribonucleic acid) samples of B. xylophilus and two other most common in Europe and both morphologically and genetically similar nematode species, i.e. B. fraudulentus and B. mucronatus, were examined in detail. The study confirmed a high efficiency of the reaction primers and TaqMan probe. These components allowed detection of B. xylophilus in such small target DNA samples as 3 pg. In contrast, in none of the examined concentrations of DNA samples derived from B. mucronatus and B. fraudulentus any signal was produced. These results showed high sensitivity and specificity of the real-time PCR assay and its potential for rapid and accurate molecular identification of B. xylophilus, even in samples contaminated with DNA of other, most closely related nematode species. Therefore, we conclude that this technique could be of a great value to plant quarantine inspection. Reakcję real-time PCR (polymerase chain reaction) (reakcja łańcuchowa polimerazy) przeprowadzano z użyciem specyficznych starterów BSatF i BSatRV zaprojektowanych dla kwarantannowego nicienia Bursaphelenchus xylophilus oraz sondy TaqMan. Ocenę wiarygodności tej metody przeprowadzono poddając analizie próby DNA (deoxyribonucleic acid) (kwas deoksyrybonukleinowy) B. xylophilus oraz dwóch powszechnych w Europie i najbardziej zbliżonych do niego morfologicznie i molekularnie gatunków B. mucronatus i B. fraudulentus. W przeprowadzonych badaniach potwierdzono wysoką skuteczność zaprojektowanych starterów wraz z sondą TaqMan. Przy ich zastosowaniu wykrywano B. xylophilus na podstawie już minimalnej ilości DNA, dochodzącej do 3 pg. W żadnym stężeniu DNA nie wykrywano zaś pokrewnych jemu gatunków B. mucronatus oraz B. fraudulentus. Dzięki badaniu przyrostu ilości produktu reakcji PCR w czasie rzeczywistym analiza DNA jest znacznie mniej pracochłonna i szybsza niż pozostałe, wykorzystywane do tego celu metody. Włączenie real-time PCR do praktyki, jako metody identyfikacji kwarantannowego gatunku B. xylophilus umożliwia łatwą eliminację wszystkich pozostałych, zbliżonych morfologicznie nicieni mogących występować w próbach drewna i tym samym może okazać się bardzo wartościowym rozwiązaniem dla służb inspekcji granicznej.

Bursaphelenchus paraluxuriosae sp. n. (Nematoda: Parasitaphelenchidae) in packaging wood from Indonesia

Bursaphelenchus paraluxuriosae sp. n., isolated in Ningbo, China, from packaging wood made from Alphitonia sp. (Rhamnaceae) imported from Indonesia is described. It is characterised by a slim body (a = 29-39), lateral field with four lines, excretory pore located at level of median bulb or slightly posterior, vulva at 75% of total body length, presence of a distinct vulval flap in lateral view, post-uterine branch ca two-thirds of the vulva-anus distance, female tail conical and slightly ventrally bent with irregular or roughened terminus (c′ = 4.4), spicules large and arcuate, with pointed rostrum and a cucullus, which is typical for the xylophilus group, but is reduced to a small blunt extension, and three pairs of ventro-sublateral papillae (one pair precloacal, two pairs postcloacal just anterior to the bursal flap and adjacent to each other) and a small single precloacal papilla present. The new species belongs to the xylophilus group and is most similar to B. luxuriosae. It is distinguished from other Bursaphelenchus species by morphology, ITS-RFLP patterns and sequencing results.

Bursaphelenchus firmae n. sp. (Nematoda: Aphelenchoididae), isolated from Monochamus grandis Waterhouse that emerged from dead firs, Abies firma Sieb. et Zucc.

Bursaphelenchus firmae n. sp. is described. This new species was isolated during a field survey of longhorn beetle-associated nematodes. The fourth-stage dispersal (dauer) juveniles of the new species were recovered from dissected bodies (tracheal system) of Monochamus grandis, which emerged from dead logs of Japanese fir, Abies firma, collected from Hachioji, Tokyo, Japan. The new species is mid-sized for the genus, with females 603-828 μm and males 530-698 μm long. Four lateral lines occur on the body surface and seven genital papillae are found in males (P1 ventral single papilla and P2-P4 pairs). A long and arcuate spicule forms a trapezium in lateral view and a rather large, sub-squared, bursal flap and a vulva with conspicuous vulval flap are present. The female tail is smoothly tapered and possesses a conspicuous and blunt mucro. Based upon its diagnostic morphological characters, the new species belongs to the B. xylophilus group of the genus, and is closely related to B. fraudulentus, B. mucronatus, B. doui, B. macromucronatus and B. populi. It is distinguished from these five species by the morphology of the male bursal flap and the female mucro and several morphometric values. Molecular phylogenetic analyses inferred from D2/D3 LSU suggest that the new species is close to B. mucronatus and B. xylophilus, i.e., these three species form a well supported monophyletic clade within the genus. Although the new species has a weak pathogenicity to pine trees, it does not seem to be a severe risk to native pine forests.

A multiplex one‐step PCR method for the simultaneous identification of Bursaphelenchus xylophilus, B. mucronatus and B. doui– three species within the xylophilus group

SummaryThe pine wood nematode, Bursaphelenchus xylophilus, causes severe damage to pines in Eastern Asia. Bursaphelenchus mucronatus and B. doui resemble closely B. xylophilus morphologically, moreover they were found frequently in this area recently. It is necessary to identify the three species precisely and rapidly. In this study, we report the results of a multiplex one‐step polymerase chain reaction (PCR) utilizing five primers to identify and discriminate the three Bursaphelenchus species simultaneously. The multiplex one‐step PCR yielded one fragment of about 1000 bp for all Bursaphelenchus populations tested. Futhermore, B. xylophilus, B. mucronatus and B. doui produced another fragment of about 100, 350 and 600 bp respectively. This approach is simple and reliable to simultaneously identify the above three species within the xylophilus group usually encountered together in a nematode assay.

Bursaphelenchus trypophloei sp. n. (Nematoda: Parasitaphelenchinae) – an associate of the bark beetle, Trypophloeus asperatus (Gyll.) (Coleoptera: Curculionidae, Scolytinae), in aspen, Populus tremula L.

Abstract Bursaphelenchus trypophloei sp. n. is described from weakened and dead twigs of living or recently fallen aspen trees, Populus tremula, in Poland. All propagative stages of the nematode were present in larval galleries of a bark beetle, Trypophloeus asperatus, and its dauer juveniles could be found in the haemocoel of older larvae, pupae and adult beetles. Characteristic morphology of the male spicules with distinct cucullus, extended anterior vulval lip, lateral fields with four incisures, and number and arrangement of male caudal papillae indicate that B. trypophloei sp. n. is closely related to the xylophilus group. This relation has been further confirmed by DNA sequencing and phylogenetic analysis of the 28S and ITS-1 rDNA region. Bursaphelenchus trypophloei sp. n. can be separated from other species in the xylophilus group by the morphology of spicules which have a shorter capitulum and unique rostrum which is pointed somewhat anteriorly, relatively thick vulval flap, which is straight, parallel to the body long axis or bent towards the body wall at its distal end, and other morphological and morphometric characters, i.e., shape of female tail, position of excretory pore at or posterior to nerve ring, spicule length of 25.7 (23.1-28.0) μm (as measured along arc) and shape, moderate length, i.e., L = 702 (603-946) μm in females and L = 679 (543-828) μm in males, and slender body (a = 39.2 (33.0-45.2) and 40.9 (32.0-46.4) in female and male, respectively). The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). Full reproductive incompatibility of B. trypophloei sp. n. with B. xylophilus, B. mucronatus, B. fraudulentus, B. populi and B. doui has been demonstrated in in vitro diallelic cross-breeding. The newly described nematode revealed limited ability to develop and reproduce on Botrytis cinerea cultures although it produced large populations on laboratory cultures of Cytospora chrysosperma – a fungus naturally associated with galleries of the nematode vector, T. asperatus.