Main morphological characters of the
Abstract
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.
Keywords
- morphology
- molecular
- ITS-RFLP
- DNA barcoding
1. Introduction
Pine wilt disease (PWD), which is caused by pine wood nematode (PWN),
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,
Before 2000, there were only other two closely related species:
Since 2000, with further study of packaging wood and phoretic insects, more
2. Grouping history
Giblin and Kaya [24] first separated five groups within
Ryss [26] considered that those characters like lateral lines, number and position of caudal papillae, and vulval flap are available for only some of the nominal species; thereby, their utility is limited. So, he studied 75 valid species of the genus
Braasch [29] stated that the
Later, with the development of the molecular methods, especially sequencing technique, more
3. Subspecies or genetic types in species of the xylophilus group
Braasch et al. [33] proposed the two
Since the report of a mucronate (“M”) form of
4. Morphological characters of the xylophilus group
According to Braasch et al. [27], the
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]. In all known
5. Morphological identification of B. xylophilus with a key
Usually, R form of
But the mucro character of the R form of
Typical R form of
The following dichotomous key of species of the
1. | (a) Posterior to the vulva | |
(b) Vulval flap bent and to the vulva not clear | 2 | |
2. | (a) Spicule cucullus not clearly expanded | 3 |
(b) Spicule cucullus expanded | 4 | |
3. | (a) Female tail cylindrical, c’ = 2.7–3.4, mucro present | |
(b) Female tail conical, c’ = 4–5, without mucro | ||
4. | (a) Average c’ > 4, female tail conical | 5 |
(b) Average c’ < 4, female tail cylindrical, subcylindrical, or conical | 9 | |
5. | (a) Female tail without mucro | 6 |
(b) Female tail with mucro | 8 | |
6. | (a) Spicule length along the curved median line 27–30 μm | |
(b) Spicule length along the curved median line more than 35 μm | 7 | |
7. | (a) Spicule length along the curved median line 35–44 μm, condylus set off from dorsal spicule line | |
(b) Spicule length along the curved median line 41–48 μm, condylus continuous with the dorsal spicule line | ||
8. | (a) Stylet with small knob, excretory pore ranging from median bulb to hemizonid, c’ = 3.6–5 | |
(b) Stylet without small knob, excretory pore at the position of median bulb, c’ = 3–4 | ||
9. | (a) Spicule length in chord 34~44 μm, the middle part nearly straight | |
(b) Spicule length in chord <34 μm, the middle part slightly ventrally curved | 10 | |
10. | (a) Female tail cylindrical, terminus broadly rounded, without mucro (some females may possess a short process at the tail terminus, usually less than 2 μm | |
(b) Female tail cylindrical, subcylindrical, or conical, terminus with mucro, more than 2 μm | 11 | |
11. | (a) Mucro usually continuous with tail | 12 |
(b) Mucro usually offset from tail | 15 | |
12. | (a) Spicule condylus dorsally not offset, body slim (a > 40) | |
(b) Spicule condylus dorsally offset, body stout (a < 40) | 13 | |
13. | (a) Female mucro terminus pointed | 14 |
(b) Female mucro terminus bluntly pointed | ||
14. | (a) Female tail straight | |
(b) Female tail slightly bent, dorsally stronger bent than ventrally | ||
15. | (a) Mucro mean length more than 4 μm | |
(b) Mucro mean length less than 3 μm |
6. Identification of the xylophilus group species with ITS-RFLP method
Application of ITS-RFLP analysis to
The abovementioned traditional ITS-RFLP method cannot separate M and R form of
7. Other molecular identification methods
Besides RFLP method, many species-specific PCR and real-time PCR methods were developed for
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
Nucleic acid sequencing methods have undergone tremendous advances over the past decade. Now, many 18S, ITS, and 28S gene sequences have been determined for
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
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
8. 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
Acknowledgments
The research was supported by the State Key Research and Development Plan (2016YFC1202104), 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).
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