**6. 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 [44–47]. Wolfgang et al. (2009) produced ITS-RFLP reference profiles of 44 *Bursaphelenchus* species [48], including two intraspecific types in each of *B. mucronatus* and *B. leoni*. Though in the case of *B. corneolus*, *B. lini* (later identified as *Devibursaphelenchus lini*), *B. singaporensis*, *B. sexdentati*, and *B. doui* [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.

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

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He and Gu [64] evaluated the applicability of 28S, 18S, and ITS loci as candidate DNA barcode markers for the *xylophilus* group of the genus *Bursaphelenchus*; they demonstrated that the average intraspecific divergences of 28S (not distinguishing two subspecies of *B. mucronatus*), 28S (distinguishing two subspecies of *B. mucronatus*), 18S, and ITS were 0.0071, 0.0030, 0.0007, and 0.0043, respectively, and, for interspecific divergences, were 0.0476, 0.0454, 0.0052, and 0.1556, respectively. The genetic distances between intraspecific and interspecific divergences of 28S and 18S loci showed some overlapping, but ITS loci had some degree of barcoding gap. The NJ trees from 28S and ITS loci with reliable bootstrap value could effectively separate 14 species of the *B. xylophilus* group into an independent branch. Furthermore, 28S locus could identify two subspecies of *B. mucronatus* well. The NJ tree of 18S locus demonstrated that *B. gillanii*, *B. firmae*, and *B. mucronatus* were mixed and difficult to be separated each other. In conclusion, 28S and ITS loci were suggested as candidate barcode genes for the *B. xylophilus*

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*,

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,

(28S) rDNA or COI gene may be required to confirm the identification.

group due to their larger barcoding gap and higher species resolution.

even for other genera in the future.

**8. Conclusion**

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).

## **7. Other molecular identification methods**

Besides RFLP method, many species-specific PCR and real-time PCR methods were developed for *B. xylophilus* identification [50–58]. 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] evaluated the applicability of 28S, 18S, and ITS loci as candidate DNA barcode markers for the *xylophilus* group of the genus *Bursaphelenchus*; they demonstrated that the average intraspecific divergences of 28S (not distinguishing two subspecies of *B. mucronatus*), 28S (distinguishing two subspecies of *B. mucronatus*), 18S, and ITS were 0.0071, 0.0030, 0.0007, and 0.0043, respectively, and, for interspecific divergences, were 0.0476, 0.0454, 0.0052, and 0.1556, respectively. The genetic distances between intraspecific and interspecific divergences of 28S and 18S loci showed some overlapping, but ITS loci had some degree of barcoding gap. The NJ trees from 28S and ITS loci with reliable bootstrap value could effectively separate 14 species of the *B. xylophilus* group into an independent branch. Furthermore, 28S locus could identify two subspecies of *B. mucronatus* well. The NJ tree of 18S locus demonstrated that *B. gillanii*, *B. firmae*, and *B. mucronatus* were mixed and difficult to be separated each other. In conclusion, 28S and ITS loci were suggested as candidate barcode genes for the *B. xylophilus* group due to their larger barcoding gap and higher species resolution.

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.

## **8. Conclusion**

other *Bursaphelenchus* identifications. In many recent descriptions of new *Bursaphelenchus* spe-

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

Besides RFLP method, many species-specific PCR and real-time PCR methods were developed for *B. xylophilus* identification [50–58]. 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

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

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

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

are two approaches used together for molecular identification.

with different methods in different labs.

recognize that both live and dead nematodes can be detected by these tests.

cies, ITS-RFLP profiles have been used as additional species identification criteria.

additional restriction endonucleases (*Hpy188* I and *Hha* I).

**7. Other molecular identification methods**

58 Advances in Plant Pathology

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.

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## **Acknowledgements**

The research was supported by the State Key Research and Development Plan (2016YF-C1202104), Ningbo Science and Technology Innovation Team (2015C110018), and General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (AQSIQ) Science Program (2016IK168).
