Computerized key to the genus Bursaphelenchus Fuchs, analysis of species clusters based on morphology, using information of insect vectors and associated plants, with a revision of the genus.

The genus Bursaphelenchus includes B. xylophilus (Steiner et Buhrer, 1934) Nickle, 1981, which is of world economic and quarantine importance. Distinction among several species of the pinewood nematodes species complex (PWNSC) is often difficult. Besides standard morphology, morphometrics and molecular biology, new tools are welcome to better understand this group. The computerized (or e-) key of this genus, presented in this communication, includes 74 species (complete list of valid species of the world fauna) and 35 characters, that were used by the taxonomic experts of this group, in the original descriptions. Morphology of sex organs (male spicules and female vulval region) was digitized and classified to distinguish alternative types. Several qualitative characters with overlapping character states (expressions) were transformed into the morphometric indices with the discontinuous ranges (characters of ratios of the spicule dimensions). Characters and their states (expressions) were illustrated in detail and supplied by brief user-friendly comments. E-key was created in the BIKEY identification system (Dianov & Lobanov, 1996-2004). The system has built-algorithm ranging characters depending on their diagnostic values at each step of identification. Matrix of species and the character states (structural part of the e-key database) may be easily transformed using statistical packages into the dendrograms of general phenetic similarities (UPGMA, standard distance: mean character difference). It may be useful in the detailed analysis of taxonomy and evolution of the genus and in its splitting to the species groups based on morphology. The verification of the dendrogram using the information on the species links with insect vectors and their associated plants, provided an opportunity to recognize the five clusters (xylophilus, hunti, eremus sensu stricto, tusciae and piniperdae sensu stricto), which seem to be the natural species groups. The hypothesis about the origin and the first stages of the genus evolution is proposed. A general review of the genus Bursaphelenchus is presented.

The Pine Wood Nematode: a personal view

The first report of the disease (“pine wilt disease”) associated with the pinewood nematode, goes back to 1905, when Yano reported an unusual decline of pines from Nagasaki. For a long time thereafter, the cause of he disease was sought, but without success. Because of the large number of insect species that were usually seen around and on infected trees, it had always been assumed that the causal agent would prove to be one of these. However, in 1971, Kiyohara and Tokushike found a nematode of the genus Bursaphelenchus in infected trees. The nematode found was multiplied on fungal culture, inoculated into healthy trees and then re-isolated from the resulting wilted trees. The subsequent published reports were impressive: this Bursaphelenchus species could kill fully-grown trees within a few months in the warmer areas of Japan, and could destroy complete forests of susceptible pine species within a few years. Pinus densiflora, P. thunbergii und P. luchuensis were particularly affected. In 1972, Mamiya and Kiyohara described the new species of nematode extracted from the wood of diseased pines; it was a named Bursaphelenchus lignicolus. Since 1975, the species has spread to the north of Japan, with the exception of the most northerly prefectures. In 1977, the loss of wood in the west of the country reached 80%. Probably as a result of unusually high summer temperatures and reduced rainfall in the years 1978 and 1979, the losses were more than 2 million m3 per year. From the beginning, B. lignicolus was always considered by Japanese scientists to be an exotic pest. But where did it come from? That this nematode could also cause damage in the USA became clear in 1979 when B. lignicolus was isolated in great numbers from wood of a 39 year-old pine tree (Pinus nigra) in Missouri which had suddenly died after the colour of its needles changed to a reddish-brown colour (Dropkin und Foudin, 2 1979). In 1981, B. lignicolus was synonymised by Nickle et al. with B. xylophilus which had been found for the first time in the USA as far back as 1929, and reported by Steiner and Buhrer in 1934. It had originally been named Aphelenchoides xylophilus, the wood-inhabiting Aphelenchoides but was recognised by Nickle, in 1970,to belong in the genus Bursaphelenchus. Its common name in the USA was the "pine wood nematode" (PWN. After its detection in Missouri, it became known that B. xylophilus was widespread throughout the USA and Canada. It occurred there on native species of conifers where, as a rule, it did not show the symptoms of pine wilt disease unless susceptible species were stressed eg., by high temperature. This fact was an illuminating piece of evidence that North America could be the homeland of PWN. Dwinell (1993) later reported the presence of B. xylophilus in Mexico. The main vector of the PWN in Japan was shown to be the long-horned beetle Monochamus alternatus, belonging to the family Cerambycidae. This beetle lays its eggs in dead or dying trees where the developing larvae then feed in the cambium layer. It was already known in Japan in the 19th century but in the 1930s, it was said to be present in most areas of Japan, but was generally uncommon. However, with the spread of the pine wilt disease, and the resulting increase of weakened trees that could act as breeding sites for beetles, the populations of Monochamus spp. increased significantly In North America, other Monochamus species transmit PWN, and the main vector is M. carolinensis. In Japan, there are also other, less efficient vectors in the genus Monochamus. Possibly, all Monochamus species that breed in conifers can transmit the PWN. The occasional transmission by less efficient species of Monochamus or by some of the many other beetle genera in the bark or wood is of little significance. In Europe, M. galloprovincialis and M. sutor transmits the closely related species B. mucronatus. Some speculate that these two insect species are “standing by” and waiting for the arrival of B. xylophilus. In 1982, the nematode was detected and China. It was first found in dead pines near the Zhongshan Monument of Nanjing (CHENG et. al. 1983); 265 trees were then killed by pine wilt disease. Despite great efforts at eradication in China, the nematode spread further and pine wilt disease has been 3 reported from parts of the provinces of Jiangsu, Anhui, Guangdong, Shandong, Zhejiang and Hubei (YANG, 2003). In 1986, the spread of the PWN to Taiwan was discovered and in 1989, the nematode was reported to be present in the Republic of Korea where it had first been detected in Pinus thunbergii and P. densiflora. It was though to have been introduced with packing material from Japan. PWN was advancing. In 1984, B. xylophilus was found in wood chips imported into Finland from the USA and Canada, and this was the impetus to establish phytosanitary measures to prevent any possible spread into Europe. Finland prohibited the import of coniferous wood chips from these sources, and the other Nordic countries soon followed suit. EPPO (the European and Mediterranean Plant Protection Organization) made a recommendation to its member countries in 1986 to refuse wood imports from infested countries. With its Directive of 1989 (77/93 EEC), the European Community (later called the European Union or EU) recognised the potential danger of B. xylophilus for European forests and imposed restrictions on imports into the Europe. PWN was placed on the quarantine list of the EU and also of other European countries. Later, in 1991, a dispensation was allowed by the Commission of the EU(92/13 EEC) for coniferous wood from North America provided that certain specified requirements were fulfilled that would prevent introduction.

Bursaphelenchus pinophilus Brzeski & Baujard, 1997 (Nematoda: Parasitaphelenchinae) associated with nematangia on Pityogenes bidentatus (Herbst, 1783) (Coleoptera: Scolytinae), from the Czech Republic

The occurrence of Bursaphelenchus species in the Czech Republic is poorly known, the first report of the genus being made by Kubátová et al. (2000) who reported the association of B. eremus with the hyphomycetous microfungus, Esteya vermicola, and the bark beetle, Scolytus intricatus, collected from Quercus robur, in central Bohemia. To date, four other species have been reported from the country, namely B. fungivorus (Braasch et al., 2002), B. hofmanni (see Braasch, 2001), B. mucronatus (see Braasch, 2001) and B. vallesianus (Gaar et al., 2006). More recently, a survey for Bursaphelenchus species associated with bark- and wood-boring insects in the Czech Republic identified B. pinophilus Brzeski & Baujard, 1997 from the Moravia region. Although this represents a new country record, it was also associated with nematangia on the hind wings of a new insect vector. A total of 404 bark- and wood-boring insects were collected from declining or symptomatic trees and screened for the presence of Bursaphelenchus. Bark and longhorn beetles were captured manually after debarking parts of the trunk displaying symptoms of insect attacks. Longhorn beetle larvae were also collected together with logs cut from the trunk. Logs were kept at room temperature in the laboratory until insect emergence. Each adult insect was individually dissected in water and examined for nematodes. All nematodes resembling dauer juveniles of Bursaphelenchus were collected and identified by molecular characterisation using a region of ribosomal DNA (rDNA) containing the internal transcribed spacer regions ITS1 and ITS2. ITS-RFLP analyses using five restriction enzymes (AluI, HaeIII, HinfI, MspI, RsaI) were performed to generate the species-specific profile according to Burgermeister et al. (2009). Species identification was also confirmed by morphological data after culture of the dauers on Botrytis cinerea Pers. ex Ft., growing in 5% malt extract agar. During this survey, only species belonging to the Curculionidae, subfamily Scolytinae, revealed the presence of nematodes belonging to Bursaphelenchus. Dauers of this genus were found aggregated under the elytra in nematangia formed at the root of the hind wings (Fig. 1). The dauers were identified from 12 individuals of Pityogenes bidentatus (Herbst, 1783) (Coleoptera: Scolytinae) collected under the bark of Pinus sylvestris trunks. Each insect carried ca 10-100 dauers. The ITS-RFLP patterns of the dauers so obtained confirmed the identification of B. pinophilus associated with this insect species. Bursaphelenchus pinophilus has been found mainly in Europe and has been reported from various countries such as Poland (Brzeski & Baujard, 1997), Germany (Braasch, 2001), and Portugal (Penas et al., 2007). The recent detection of this species associated with dead P. koraiensis in Korea (Han et al., 2009) expands its geographical distribution and potential importance. It has been found associated only with Pinus species, but very little is known about the insect vector. The bark beetle, Hylurgus ligniperda, was initially suggested as the insect vector by Pe-nas et al. (2006), although the nematode associated with this insect was later reclassified as B. sexdentati by morphological and molecular analysis (Penas et al., 2007). According to the literature, P. bidentatus has been cited as a vector of Ektaphelenchus sp. (Kakuliya, 1966) in Georgia, and an unidentified nematode species in Spain (Roberston et al., 2008). Interestingly, B. pinophilus was found in the nematangia formed at the root of the hind wings of P. bidentatus. Although this phenomenon is not so common in other Bursaphelenchus species, B. rufipennis has been found recently in such a structure on the hind wings of the insect Dendroctonus rufipennis (Kanzaki et al., 2008). Although other nematode species (e.g., Ektaphelenchus spp.) are frequently found associated within the same nematangia (see Kanzaki et al., 2008), in this particular case, only dauers of B. pinophilus were identified. The association between B. pinophilus and P. bidentatus represents the first report of this biological association and the association with the Scolytinae strengthens the tight and specific links between this group of Bursaphelenchus species and members of the Scolytinae (Ryss et al., 2005).