Systematic Identification of the Xylophilus Group in the Genus Bursaphelenchus

The pine wood nematode (PWN) Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) Nickle, 1970 is the agent responsible for pine wilt disease (PWD). This nematode has been killing native pine trees (Pinus densiflora, P. thunbergii, P. luchuensis) in Japan since the early twentieth century. It is the number one forest pest in Japan and has been spread to China, Korea, Portugal, and Spain. The nematode is native to North America (Canada, USA, Mexico) and is thought to have been carried to Japan at the beginning of the twentieth century on timber exports. Up to now, the genus Bursaphelenchus Fuchs, 1937 comprises nearly 120 species (14 groups). Around 14 species very similar to B. xylophilus are put together and named the xylophilus group. This chapter presents the grouping history, subspecies or genetic types in species of the xylophilus group, and an identification key for 14 species of the xylophilus group, ITS-RFLP identification, and other molecular identification methods are also discussed.


Introduction
Pine wilt disease (PWD), which is caused by pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer [6]) Nickle [1], has been devastating Japanese pine forests since the beginning of the twentieth century. For many years, the mass mortality of pine trees was supposed by attacks of beetles. Until 1971, Bursaphelenchus sp. was demonstrated as the causal agent of PWD by inoculation tests on Pinus spp. [2], and subsequently the nematode was described as Bursaphelenchus lignicolus [3]. After that, the PWN was first reported in the United States in 1979 [4]. Extensive surveys revealed the widespread distribution of the nematode throughout the country [5], but no epidemic was found, and the disease occurred only on a few exotic pine species. The PWN was later proven to be the same species as the one described in Florida in 1934 [6], the name was then changed from B. lignicolus to B. xylophilus [7], and it has been indigenous to North America [8].
Later, the disease has spread into China in 1982, Korea in 1988, Mexico in 1993, Portugal in 1999, and Spain in 2011 [9], and it is now still a potential threat to pine forests worldwide.
In nature, B. xylophilus is spread from tree to tree through the activity of adult stages of woodinhabiting longhorn beetles of the genus Monochamus (Coleoptera: Cerambycidae) for short distance. This transmits the nematode either to the shoots of living trees during maturation feeding either by sex or by oviposition of females. But human activity is responsible for the long-distance spread. It is widely accepted that national and international trade of pine logs and related packaging wood is the causal of PWN spreading, so national and international regulations (e.g., ISPM 15: FAO, 2003, revised 2009) were accompanied by intensive sampling and laboratory investigations for the presence of PWD in imported wood worldwide in order to significantly reduce the risk of the pest's spread. So, it is important to identify B. xylophilus to manage its further spreading and conduct early eradication plan.
Before 2000, there were only other two closely related species: B. fraudulentus Rühm [10] and B. mucronatus Mamiya and Enda [11] (B. kolymensis Korenchenko [12] was later considered as being synonymous with B. mucronatus). For a long time, in diagnostic protocol of B. xylophilus, it was morphologically compared with only B. mucronatus and B. fraudulentus, many PCRbased methods also used only these three species samples.
Since 2000, with further study of packaging wood and phoretic insects, more Bursaphelenchus species were discovered. Now, there are 110-120 known species in this genus [9] Giblin and Kaya [24] first separated five groups within Bursaphelenchus mainly according to spicule morphology; the xylophilus group contains three species, namely, B. xylophilus, B. mucronatus, and B. fraudulentus, all have large, paired, arcuate spicules with a sharply pointed rostrum, and a disk-like expansion, cucullus, and females of this group have a vulval flap ( Table 1). Braasch [25] studied the morphological relationship between European Bursaphelenchus species in order to provide key characters for their taxonomic identification. She considered the number of incisures in the lateral field as a basic grouping feature, together with other features like spicule shape, number and position of caudal papillae, presence and size of a vulval flap, and the shape of female tail. Among the 28  [27]. B. crenati has a different position of the caudal papillae (the double pair in front of the bursa is missing), the presence of a vulval flap is questionable, and the spicules do not show a cucullus. Additionally, it is transmitted by a bark beetle, a scenario not typical for the xylophilus group. B. eroshenkii has five incisures in the lateral field, only five caudal papillae (seven in the xylophilus group) and no vulval flap [28]. The spicule shape of B. abruptus is not typical.

Grouping history
Braasch [29] stated that the xylophilus group of the genus Bursaphelenchus can be clearly distinguished from other species of the genus by the presence of four lateral lines, the presence of a vulval flap in females, a characteristic shape of the male spicules, and the arrangement of the seven caudal papillae. An identification key of the nine species of the xylophilus group was presented, and B. kolymensis was considered to be the European type of B. mucronatus.
Later, with the development of the molecular methods, especially sequencing technique, more Bursaphelenchus sequences are available in the GenBank. Based on morphological characters and phylogenetic analysis [27], the genus is divided into eight groups with four incisures in the lateral field (xylophilus, okinawaensis, africanus, fungivorus, cocophilus, kevini, tokyoensis and sexdentati groups), four groups with three incisures (eggersi, eremus, hofmanni, and leoni groups), and two groups with two incisures (abietinus and sinensis groups). Most of the groups are well separated by both morphological and molecular studies.

Subspecies or genetic types in species of the xylophilus group
Bursaphelenchus mucronatus Mamiya and Enda [11] was first found from pine trees in Japan.
Braasch [30] reported for the first time B. mucronatus in timber imports from Siberia and in forest trees in Germany. These populations (later on named "European genotype" or "European type") had shown morphological and morphometric deviations from Japanese B. mucronatus isolate. Separate species status for Japanese and European B. mucronatus was postulated on the basis of sequence differences of an amplified fragment of the heat shock 70A gene [31]. differences in the shape of female tail, length of mucro, position of excretory pore, and also small differences in spicule shape. They can be distinguished by their ITS-RFLP patterns based on restriction fragments obtained with enzymes Rsa I and Hae III. Based on sequence analysis of ribosomal ITS1/ITS2, LSU D2/D3, and mitochondrial COI regions, a clear subdivision of the two isolate groups (subspecies) has been confirmed.
Since the report of a mucronate ("M") form of B. xylophilus detected from balsam fir (Abies balsamea) in Minnesota and Wisconsin, USA [34], uncertainty in morphological distinction of B. xylophilus from related species became evident. For a long time, it is morphological and molecular characters are not clear. Gu et al. [35] made a morphological and molecular study based on five isolates of "M" form of Bursaphelenchus xylophilus, together with the round-tailed ("R") form of B. xylophilus and B. mucronatus (both subspecies), and founded that the spicules of these species (types or forms) are similar. The "M" form of B. xylophilus is distinguished from the "R" form of B. xylophilus by a distinct mucro at the female tail end. It differs from the B. mucronatus kolymensis by slightly shorter female tail mucro and position of excretory pore. It is distinguished from B. mucronatus mucronatus by female tail shape and shorter female tail mucro. The conventional five restriction endonucleases (Rsa I, Hae III, Msp I, Hinf I, and Alu I) used for obtaining ITS-RFLP patterns of Bursaphelenchus species cannot distinguish the "M" and "R" form of B. xylophilus, but the two forms can be differentiated by the use of two additional restriction endonucleases (Hpy188 I and Hha I). The molecular phylogenetic analysis based on the sequences of D2D3 LSU rDNA, ITS1/2 region, and mtCOI revealed that the "M" form of B. xylophilus is genetically closest to the "R" form of B. xylophilus, and that their sequence divergence is small.

Morphological characters of the xylophilus group
According to Braasch et al. [27], the xylophilus group is characterized by four lateral lines; seven caudal papillae; conspicuous P4, P3, and P4 papillae adjacent to each other (double pair) just anterior to bursa; spicules long, slender, and semicircular with angular lamina in posterior third; capitulum fattened with small condylus and distinct rostrum; cucullus present (for B. fraudulentus and B. paraluxuriosae, spicule cucullus is not clearly visible); and large vulval flap.
But lateral lines and caudal papillae are not easy to be seen sometimes, so typical male spicule shape and female vulval flap should be the main grouping characters [35].

Morphological identification of B. xylophilus with a key
Usually, R form of B. xylophilus is distinguished from other species by cylindrical female tail with bluntly rounded terminus, without mucro, or in some cases, some females will show a mucro, which is less than 2 μm.
But the mucro character of the R form of B. xylophilus is not always stable; it depends on different hosts and environmental situations. Braasch [36] reported that when an R form B. xylophilus isolate (US15) was re-extracted from trees 3 months after inoculation experiment, 35% of females were round-tailed, 8% had conical tails, and 17% had a distinct mucro (up to 4-5 μm), whereas 40% had a very small mucro of 1 μm length. Zheng et al. [37] reported that an R form B. xylophilus was detected from a pine tree in Ningbo, China; all females had a distinct mucro, ranging from 0.5 to 2.9 μm (mean 1.7 μm), but the mucro disappeared after culturing on B. fuckeliana. Gu et al. [35] also reported an R form B. xylophilus isolate (4049); about half of the females detected from packaging wood had a round tail, and the other half showed a  very small mucro about 0.5-1 μm long. But after culturing on B. fuckeliana for 1 month, more than half of females showed a mucro of about specimens, a mucro of less than 0.5 μm long, or no mucro. However, after being cultured on Pestalotiopsis sp., apart from some round-tailed females, most females had a bluntly pointed tail terminus (Figure 1). The following dichotomous key of species of the xylophilus group is based on the female tail shape (conical or cylindrical, with or without mucro, and mucro length), vulval flap shape (straight or bent), and spicule size and shape (with or without cucullus).

Identification of the xylophilus group species with ITS-RFLP method
Application of ITS-RFLP analysis to Bursaphelenchus species identification was first described in 1998 [38,39]. In this technique, a region of ribosomal DNA (rDNA), containing the internal transcribed spacer regions ITS1 and ITS2, is amplified by PCR method with forward primer F194 5'-CGTAACAAGGTAGCTGTAG-3′ (Ferris et al.) and reverse primer 5368 5'-TTTCACTCGCCGTTACTAAGG-3′ (Vrain) [40,41], and, subsequently, the PCR products were digested with five restriction endonucleases Alu I, Hae III, Hinf I, Msp I, and Rsa I to get the restriction fragment length polymorphisms. Using the same set of five restriction enzymes, species-specific ITS-RFLP reference patterns were compiled for 11 Bursaphelenchus species in 1999 [42] and extended to 26 species in 2005 [43]. The technique has proven to be a valuable tool in identification of nematodes isolated from imported wood in quarantine control or forest surveys [44][45][46][47].  [49], additional bands in the patterns of certain isolates or individual nematodes were observed which may be explained by ITS sequence microheterogeneity, i.e., the presence of ITS sequence variants within the number of rDNA tandem repeats, but they did not seriously impair identification of species based on the overall reference patterns. ITS-RFLP analysis has proven valuable not only for differentiation of the pathogenic pine wood nematode, B. xylophilus, from related species but also useful in other Bursaphelenchus identifications. In many recent descriptions of new Bursaphelenchus species, ITS-RFLP profiles have been used as additional species identification criteria.
The abovementioned traditional ITS-RFLP method cannot separate M and R form of B. xylophilus, but according to Gu et al. [33], the two forms can be differentiated by the use of two additional restriction endonucleases (Hpy188 I and Hha I).

Other molecular identification methods
Besides RFLP method, many species-specific PCR and real-time PCR methods were developed for B. xylophilus identification [50][51][52][53][54][55][56][57][58]. By real-time PCR [59] or loop-mediated isothermal amplification (LAMP) methods [60], B. xylophilus can be detected directly from wood. But we should notice that those methods were developed years ago, now more species in the xylophilus group are known, and the results may be questionable. And when molecular tests are used for quarantine purposes to detect B. xylophilus in wood products, it is essential to recognize that both live and dead nematodes can be detected by these tests.
More recently, Ye et al. [60] developed a real-time PCR assay for PWN identification [61].
Based on DNA sequence analysis on the ribosomal DNA small subunit, large subunit D2/D3, internal transcribed spacer (ITS), and mitochondrial DNA cytochrome oxidase subunit one on the aphelenchid species, they developed a rapid and accurate PWN identification method targeting the ITS-1. A total of 97 nematode populations were used to evaluate the specificity and sensitivity of this assay, including 45 populations of B. xylophilus; 36 populations of 21 other species of Bursaphelenchus which belong to the abietinus, cocophilus, eggersi, fungivorus, hofmanni, kevini, leoni, sexdentati, and xylophilus groups and one unassigned group from a total of 13 groups in the genus Bursaphelenchus; 15 populations of Aphelenchoides besseyi, A. fragariae, Aphelenchoides species, and Aphelenchus avenae; and one population of mixed nematode species from a soil sample. This assay proved to be specific to B. xylophilus only and was sensitive to a single nematode specimen regardless of the life stages present. This approach provides rapid species identification necessary to comply with the zero-tolerance export regulations.
Nucleic acid sequencing methods have undergone tremendous advances over the past decade. Now, many 18S, ITS, and 28S gene sequences have been determined for Bursaphelenchus species, and they are deposited in the GenBank database (http://www.ncbi.nlm.nih.gov/). In general, the comparison of those genes with reference data using sequence and phylogenetic analysis allows classification of nematode samples and establishing identification. Determinations of clades to which samples belong and the level of the interspecific variation are two approaches used together for molecular identification.
DNA sequencing method has been used widely in the last decade. But this method is not standard: different target genes and different primers are used, and sequences are analyzed with different methods in different labs.
DNA barcoding is a generic diagnostic method that uses a short standardized genetic marker in an organism's DNA to aid species identification. An organism is identified by finding the closest matching reference record in a database containing large amounts of barcode sequence data. The first genetic marker to be described as a "barcode" was the mitochondrial cytochrome c oxidase I (COI) gene which is used for species identification in the animal kingdom [62]. According to Quarantine Barcoding Of Life (QBOL) project financed by the Seventh Framework Program of the European Union (www.q-bank.eu), first, a 1600 bp fragment of the small subunit (SSU) 18S rDNA gene can be PCR amplified and sequenced using primers 988F, 1912R, 1813F, and 2646R [63]. The obtained sequence data is used for identification to the genus and sometimes to species level. However, in some cases the SSU does not contain sufficient variation for identification to the species level, and additional sequences of the LSU (28S) rDNA or COI gene may be required to confirm the identification.
He and Gu [64]  When sequencing is more easy, quick, and cheap, and more sequences are available in the database, DNA barcoding will be the best way for species identification for genus Bursaphelenchus, even for other genera in the future.

Conclusion
After devastating a vast area of pine forests in Asian countries, the pine wilt disease was spread into European forests in 1999 and was causing a worldwide concern. To date, about 120 species of the genus Bursaphelenchus have been described, and 14 groups is suggested. About 14 species very similar to B. xylophilus are put together and named the xylophilus group.
The xylophilus group is characterized by four lateral lines; seven caudal papillae; conspicuous P4, P3, and P4 papillae adjacent to each other (double pair) just anterior to bursa; spicules long, slender, and semicircular with angular lamina in posterior third; capitulum fattened with small condylus and distinct rostrum; cucullus present or not clearly visible; and large vulval flap. Subspecies (B. mucronatus kolymensis and B. mucronatus mucronatus) and two genetic types ("M" form and "R" form of B. xylophilus) exist in the group, and the mucro character of B. xylophilus is not always stable, which depends on different hosts and environmental situations, making identification complicated. Usually, R form of B. xylophilus is distinguished from other species by cylindrical female tail with bluntly rounded terminus, without mucro, or in some cases, some females will show a mucro, which is less than 2 μm. Due to a certain variation in characters between populations and different hosts and environmental situations, it is essential to perform molecular test in case of doubt. ITS-RFLP identification and other molecular identification methods are also discussed; DNA barcoding by using the 28S and ITS loci will be a reliable and convenient method in the future.