Wolfgang Burgermeister

Identification of Bursaphelenchus spp. isolated from Chinese packaging wood imported to Austria.

Bursaphelenchus spp. found in packaging wood (pallets, crates, dunnage) imported with other goods from China to Austria in 1999 were identified, and their possible vector species of wood- and bark-breeding insects were recorded. Of the inspected consignments of coniferous and deciduous wood, 43 (78%) were either contaminated with insects or nematodes or showed grubholes or galleries of wood- and bark-breeding insects. Of the 33 samples of coniferous packaging wood with symptoms of insect attack, 48.5% had been attacked by longhorn beetles (Cerambycidae), with 11 samples harbouring living stages of Monochamus, the vector of Bursaphelenchus xylophilus and B. mucronatus. Living stages of non-European Scolytidae, possibly vectors of other Bursaphelenchus spp., were found in seven samples. Nematodes were found in 24 consignments, of which nine (=27% of the 33 coniferous wood samples) were contaminated with Bursaphelenchus spp. Seven samples contained B. mucronatus (East Asian genotype), whereas B. thailandae and B. aberrans were each found in two samples which were also contaminated with B. mucronatus. Morphological features and measurements for the three species found and ITS-RFLP patterns for B. thailandae and B. mucronatus are presented. Bursaphelenchus thailandae and B. aberrans were found for the first time in wood transported over long distances. Bursaphelenchus thailandae is recorded for the first time from China and its ITS-RFLP profile, enabling differentiation of the species from 17 other Bursaphelenchus spp., is presented.

Description of Bursaphelenchus paraparvispicularis n. sp. (Nematoda: Aphelenchoididae) found in packaging wood from Hongkong, China

Bursaphelenchus paraparvispicularis n. sp. is described and figured from pine packaging wood originating in Hongkong, China, and inspected in Ningbo harbour, China. The new species clearly belongs to the hofmanni group. It is characterised by a relatively stout body (a = 26.7 and 26.5 for males and females, respectively), three lines in the lateral field, seven caudal papillae, spicules relatively small (12.6-15.3 μm), mitten-shaped, with lamina dorsal line smoothly arcuate but calomus relatively straight, condylus squared or round, well developed, rostrum well developed with round terminus, cucullus absent, the shape of the female tail, which is short and ventrally bent with a bluntly pointed terminus, and vulval lips not forming a vulval flap. The new species is morphologically closest to B. parvispicularis and can be distinguished by smaller and stouter body, lower female ratio c′ (average 2.8 vs 4.4) and longer spicule condylus. The separate species status is supported by ITS-RFLP patterns and molecular phylogenetic analysis based on ITS1/2 and partial LSU sequences, which revealed that B. paraparvispicularis n. sp. is closest to B. parvispicularis.

Multiple displacement amplification of DNA for ITS-RFLP analysis of individual juveniles of Bursaphelenchus

Differentiation of the plant-pathogenic pinewood nematode, Bursaphelenchus xylophilus, from non-pathogenic Bursaphelenchus species is difficult because of high morphological similarities among closely related species. In recent years, ITS-RFLP analysis has become a useful tool for Bursaphelenchus species identification. Analysis of individual nematodes is hampered by the fact that sufficient template DNA for ITS-PCR cannot be extracted reliably. We have employed a whole genome amplification method, termed multiple displacement amplification (MDA), to 26 DNA extracts from individual juveniles to increase the amount of template DNA. Preamplification of the whole genomic DNA by MDA provided sufficient amounts of PCR product for ITS-RFLP analysis with 12 out of 20 B. xylophilus, two B. mucronatus, two B. fraudulentus and two B. eggersi samples tested. The introduction of MDA to ITS-RFLP analysis of nematodes may improve the reliability of diagnostic testing for limited samples and permit verification of analytical results.

Analysis of Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) Provenances Using ISSR and RAPD Fingerprints

The pinewood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease. In order to trace the origin of its recently introduced Portuguese population, two PCR-based techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR), were used to determine genetic relationships among 30 B. xylophilus isolates from the USA, Canada, Japan, China, South Korea and Portugal. Fingerprints obtained with both methods detected a reduced genetic variation of introduced isolates as compared to native North American isolates. Cluster analyses of genetic distances between isolates were carried out and bootstrap dendrograms were constructed. The results indicated that founders of the Portuguese isolates most likely were translocated one or two times to Portugal from their colonized sites in East Asia, but not from their native habitats in North America.

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 spp. in Wood Packaging Intercepted in China

Solid wood packaging material (including dunnage) made of unprocessed wood is known to be an important pathway for the introduction and spread of pests, but we may not know how serious this problem is. Another question is: Can a quarantine treatment certificate or the “HT” (“MB”) mark on the wood really assure that the wooden packaging is free of pests? In February of 2006, many living pinewood nematodes (PWN) were detected in wooden packaging exported from Portugal to China. The black mark “PT 048 HT” could clearly be seen on the wood (PT representing Portugal, 048 representing the number of the heat treatment site). Was the wood cut from the PWN affected zone in Portugal and not properly treated? At Ningbo Entry-exit Inspection and Quarantine Bureau, China, almost all wooden packaging imported through this harbour have been sampled and inspected since 1997. In recent years, the quarantine pest B. xylophilus was detected many times in large numbers (sometimes thousands of specimens) in wood samples from different countries, and a considerable number of other Bursaphelenchus species, among them several undescribed species, were also found. The morphological and molecular diagnostic work was stimulated and supported by a training course for identification of Bursaphelenchus species given by Helen Braasch and Wolfgang Burgermeister in Shanghai, China, in October 2002. From January, 2003 to June, 2006, wooden packaging material of 8720 batches was nematologically inspected in the Ningbo Entry-exit Inspection and Quarantine Bureau. Living nematodes were detected in 1772 batches, accounting for 20.3%. Most of them were identified as Rhabditida, Tylenchida and Aphelenchida (previously Aphelenchoides spp. and Bursaphelenchus spp., but also Aphelenchus spp., Paraphelenchus sp., Cryptaphelenchus spp. and Ruehmaphelenchus sp.). Bursaphelenchus spp. was detected in 343 batches from 26 different countries (Table 1). The following species were identified on the basis of their morphology and their ITSRFLP patterns: B. xylophilus, B. fungivorus, B. rainulfi, B. hylobianum, B. thailandae, B mucronatus, B. aberrans, B. lini, B. singaporensis, B. doui, B. conicaudatus,

Variation in ITS and 28S rDNA of Bursaphelenchus Species (Nematoda: Parasitaphelenchidae)

Internal transcribed spacers and 28S D2/D3 domain of rDNA were used to infer the phylogenetic relationships among Bursaphelenchus species, with special emphasis on members of the xylophilus and fungivorus groups. Sequence alignments and phylogenetic analysis using neighbour-joining and maximum parsimony algorithms resulted in trees with similar topologies. The 17 Bursaphelenchus species examined can be separated into two main branches: the first includes members of the xylophilus group and the second includes the species of the fungivorus group, separated from the remaining species B. eremus, B. hofmanni, B. rainulfi and B. yongensis. As far as known, the species of the xylophilus group are phoretically associated with longhorn beetles. The phylogenetic analysis revealed the digger bee associated B. abruptus as the basal taxon of the species investigated or at least of the xylophilus group. The remainder of the species are, as far as known, associated with bark beetles and a soil-dwelling bee in case of B. seani. The significantly supported groups are largely

Description of a new subspecies of Bursaphelenchus africanus (Nematoda: Aphelenchoididae) found in packaging wood from Russia

Bursaphelenchus africanus rossicus subsp. n. was detected from Russian packaging wood (Pinus sp.) arriving in China in August, 2009. The spicule shape and size are almost the same as in the B. africanus found in wood from South Africa, but it differs slightly from the South African isolate by longer (mean L=945 vs 691 μm and 1062 vs 766 μm, for males and females, respectively) and slimmer body (a=39.8 vs 35.0 and 40.0 vs 35.1, for males and females, respectively), higher male ratio c (mean c=37.0 vs 28.7) and higher female ratio c′ (mean c′=4.7 vs 3.4), longer female tail (58 vs 42 μm), and also by female tail shape (slightly ventrally bent vs straight). Their ITS-RFLP patterns are also slightly different. Based on the absence of clear morphological differences and relatively small ITS1/2 and D2/D3 LSU sequence divergences, the new isolate is considered as Bursaphelenchus africanus rossicus subsp. n.

Molecular Characterization of Isolates of the Bursaphelenchus sexdentati Group Using Ribosomal DNA Sequences and ITS-RFLP

Species-specific ITS-RFLP patterns have been established for more than 30 Bursaphelenchus species including 5 species of the ‘sexdentati’ group (B. sexdentati, B. vallesianus, B. pinophilus, B. poligraphi and B. borealis). Morphological species differentiation in the ‘sexdentati’ group is based on the shape of female tail and the spicules. However, observations on different isolates of B. sexdentati have revealed considerable variability of these features suggesting the existence of intraspecific genetic types which could not be differentiated by ITS-RFLP analysis using five enzymes. To improve intraspecific differentiation, we have determined ITS1/2 sequences of 17 isolates belonging to five species of the ‘sexdentati’ group. In some isolates, sequence heterogeneity at a few sites of ITS2 was detected. In the sequence-based phylogenetic tree, branching of clusters confirmed the affiliation of isolates to their respective species. In addition, isolates of B. sexdentati were separated in two groups suggesting the existence of a Central European and a South European type of this species or two separate species. This was supported by the differences in shape of female tails and spicules between the two types. The information obtained from sequencing was used to select three additional enzymes for extending the scope of ITS-RFLP analysis. In this way, improved distinction of species and differentiation of the two types of B. sexdentati was achieved.