Pine wilt disease caused by Bursaphelenchus xylophilus has caused serious loss to pine forestry around the world. The nematodes are transmitted by beetles. Monochamus alternatus is the most important vector of B.xylophilus (Ryss et al., 2005). It is the key point to diagnose the disease as early as possible for controlling it effectively, so the forecast of disease development trends has been studied based on the quantity of M. alternatus in the forest. A lot of research work about the relationships between the nematodes and the beetles has been done. Only adult beetles carry larvae nematodes. (Mamiya et al., 1979; Giblin-Davis et al., 1983; Kanzaki et al., 2001; Penas et al., 2006). Because nematodes are normally identified by their morphological characteristics of adult nematodes, it is difficult to identify the young nematodes carried by the beetles. A lot of molecular methods have been studied in order to identify the nematodes based on the condition that the nemtodes should be isolated from their host initially(Iwahori et al., 2000;Hoyer et al., 1998; Wu et al., 2005; Liao et al., 2001; Kang et al., 2004; Castagnone et al., 2005). As beetles usually carry several species of nematodes, it is helpful to develop the molecular method for identifying the nematodes(Wang et al., 2008). But until recently, there has been no report about the identification of nematodes from the tissue of M.alternatus based on PCR amplification.

In our study, we firstly find the proper DNA extraction method for following PCR amplification, then find one pair of B. xylophilus-specific primers for amplifying nematodes directly from the tissue of M.alternatus. Finally, a PCR-based method has been established for detecting B.xylophilus from M.alternatus without a separate DNA extraction step.

1 Materials and methods 1.1 Nematodes populations

13 isolates of B. xylophilus, B.mucronatus, B.aberrans, B.corneolus, B.leoni, B.hunanensis, B.teratospicularis, Aphelenchoides resinosi, Seinura steineri, Ditylenchus parvus, Odorhabdiplogaster xiphocaudatus, Rhabditida sp. and Parasitorhabditis sp. were isolated from chips of infected pine wood of Pinus massoniana in Guangdong Province in China by the Baermann method(Xie, 2000). The nematodes species were identified under the microscope. Isolates of B.xylophilus and B.mucronatus were reared on a lawn of Pestalotiopsis sp. cultured on PDA plates, at 25 ℃ in the dark. Collected nematode species were stored in water at 4 ℃.

1.2 Genomic DNA extraction

Genomic DNA of nematodes and beetle tissue were extracted, according to Zhang et al. (2001), with some modifications. 50 μL nematodes of mixed developmental stages of one species, were collected in 1.5 mL Eppendorf tube after being washed in sterilized, distilled water for 3 times, and then centrifuged at 500 g for 5 min with as much water removed as possible. Purified nematode individuals were frozen in liquid nitrogen and ground with a mortar and pestle. DNA was extracted according to a phenol/chloroform procedure. Following ethanol precipitation, DNA was re-suspended in 0.01 mol·L^-1 Tris, pH 8, and 0.01 mol·L^-1 EDTA and stored at -80 ℃.

DNA from single nematode of B. xylophilus, B.mucronatus, B.aberrans, B.corneolus, B.leoni, B.hunanensis, B.teratospicularis, A. resinosi, S. steineri, D. parvus, O.xiphocaudatus, Rhabditida sp., Parasitorhabditis sp., was isolated respectively based on the method of Zhang et al. (2001) by a simplified procedure: This involved a single adult or juvenile which was hand-picked and placed in 10 μL WLB(2.5 mmol·L^-1 DTT, 1.125% Tween 20, 0.025% Gelatin, 125 mmol·L^-1 KCl, 25 mmol·L^-1 Tris-HCl(pH 8.0), 3.75 mmol·L^-1 MgCl₂), and placed at -70 ℃ for 30 min. Each tube was incubated at 95 ℃ for 15 min, 65 ℃ for 5 min, then 1 μL 20 mg·mL^-1 proteinase K was added, with continued incubation at 65 ℃ for 1 h, and 95 ℃ for 15 min. At this stage, either the PCR reaction was performed directly in the same tube or the lysate was frozen at -20 ℃ for subsequent steps of the procedure. This procedure was modified for DNA isolation from a mixture of a single nematode + 1 mg nematode-free beetle tissues or beetle sample with B.xylophilus.

1.3 Development of species-specific PCR primer pairs

To design nucleotide sequences of species-specific PCR primer pair for B. xylophilus, DNA sequences in the ribosome region of B. xylophilus and M. alternatus had been searched from the NCBI(http://www.ncbi.nlm.nih.gov). But there is no available result for M. alternatus. Anyway, one pair of B.xylophilus-specific primers whose forward primer located in the 155^th bp of ITS1 region and designated P155(sequence:5′-CTACGTGCTGTTGTTGAGTTGGC-3′) with one reverse primer located in the 538^th bp of the ITS2 region and designated as P538 (sequence: 5′-TGGTGCC TAACATTGCGCGA-3′) was choosen (Fig. 1). PCR amplicon of B. xylophilus is 403 bp in the region of ribosome RNA (Hu, 2006). The primers are synthesized by Beijing SAIBAISHENG Biotic Company (Beijing, China).

1.4 PCR amplification

PCR amplification was performed in a total volume of 25 μL using a thermal cycle (TaKaRa). Each reaction mix contained 1.0 μL (20 ng) DNA template, 2.5 μL PCR buffer(10×), 2.5 μL MgCl₂(25 mmol·L^-1), 2 μL dNTP(2.5 mmol·L^-1), 1.0 μL primer P155 (10 mmol·L^-1)、1.0 μL primer P538 (10 mmol·L^-1), 0.1 μL rTaq polymerse 0.5 U (TaKaRa Biotech, Japan) and 14.9 μL ddH₂O.The cycling experimental conditions were as follows: 94 ℃ for 1 min, followed by 35 cycles at 94 ℃ for 30 s→62 ℃ for 30 s→72 ℃ for 30 s, ending with 1 cycle at 72 ℃ for 10 min and storage at 4 ℃. Amplification products were separated on a 1.5% agarose gel in 1×TAE buffer (54 mmol·L^-1 Tris, 27.5 mmol·L^-1 boric acid, and 10 mmol·L^-1 EDTA, pH 8.0) and stained with 1 μg of ethidium bromide per mL. The band pattern was photographed under UV light. Molecular size was evaluated according to a commercial marker (TaKARA, Bio Inc., Shiga, Japan).

For amplifying DNA of a single nematode, little modification was done based on the above conditions. The cycling experimental conditions were as follows:94 ℃ for 3 min, followed by 40 cycles at 94 ℃ for 45 s→50 ℃ for 30 s→72 ℃ for 1min, and ending with 1 cycle at 72 ℃ for 10 min and storage at 4 ℃.

1.5 Assessment of the species-specificity of the primers for B. xylophilus

Each of the 13 isolates of B. xylophilus, B.mucronatus, B.aberrans, B.corneolus, B.leoni, B.hunanensis, B.teratospicularis, A. resinosi, S. steineri, D. parvus, O. xiphocaudatus, R. sp., P. sp., and genomic DNA for M. alternatus was amplified with the (designated) primers respectively: Forward primer was P155 and reverse primer was P538. All amplification was performed from a single nematode. At least three replicates were performed for each treatment.

1.6 Detection of B. xylophilus from the breast gastube tissue of M. alternatus

Two sets of experiments had been done: firstly, PCR amplification was performed for one adult of B.xylophilus mixed with 2 mg nematode-free beetle tissue of M.alternatus. Six replicates were done for the treatment. Then B.xylophilus carried by the beetle tissue of M.alternatus was detected by the PCR method. Beetle breast gas tube tissue carries different numbers of nematodes (nematode number was countedbeforehand.The nematode number was from 97 to 10 860)(12 replicates). The DNA extraction procedure and PCR amplification condition was according to above-mentioned method.

2 Results 2.1 Genomic DNA extraction

Genomic DNA was extracted from B.xylophilus and M.alternatus (A1/A2=1.8~1.9), which is qualified for PCR amplification (Fig. 2, 4, 5, 6). DNA from one single nematode of B.xylophilus alone (Fig. 4), or a single nematode of B.xylophilus + nematode-free beetle tissue of M.alternatus (Fig. 5), or nematodes carried by beetle tissue, of M.alternatus, carrying nematodes were also isolated for successful PCR amplification (Fig. 6).

2.2 PCR amplification

Using the forward primer of P155 and the reverse primer of P538, it was found that the expected electrophoresis band 403 bp was successfully obtained for B.xylophilus from genomic DNA (Fig. 3), and from the mixture DNA of one single nematode and nematode-free beetle tissue of M.alternatus (Fig. 4), and from the beetle tissue of M.alternatus carrying nematodes B.xylophilus (Fig. 6).

2.3 Assessment of the species-specificity of the primers for B.xylophilus

Each of the 13 isolates of B. xylophilus, Rhabditida sp., A. resinosi, S. steineri, D. parvus, O. xiphocaudatus, P. sp., and genomic DNA for M. alternatus were PCR amplified with the designated primers. The electrophoresis band 403 bp was only successfully obtained for B.xylophilus(Fig. 4).

2.4 Detection of B. xylophilus from the breast gastube tissue of M. alternatus

The expected amplicon of 403 bp was successfully obtained for B.xylophilus from both the mixture of one single nematode + 2 mg breast gas tube tissue of M. alternatus (Fig. 5), and beetle breast gas tube tissue with different numbers of nematodes(12 replicates)(Fig. 6).

3 Discussion

Our results show that both Genomic DNA from B.xylophilus or M.alternatus, and DNA from a single nematode of B.xylophilus alone, or single nematode of B.xylophilus + nematode-free beetle tissue of M.alternatus were isolated for successful PCR amplification. For nematodes identification, the nematode was usually isolated from the host firstly, and molecular identification was then performed (Iwahori et al., 2000; Matsunaga et al., 2004). Only Takeuchi et al. (2005) have successfully extracted nematodes DNA in infected wood by the CTAB method using phenol-chloroform procedure. In our study, we simply isolated nematodes DNA in bettle tissue by freezing and thawing them in WLB solution, which will be more convenient and healthy for identifying nematode by PCR amplification for practical use. It is the first trial in which the nematodes DNA in infected beetle tissue is isolated in this way.

A pair of B. xylophilus-specific PCR primers were choosen. The forward primer located in the 155^th bp of ITS1 region was designated as P155(sequence:5′-CTACGTGCTGTTGTTGAGTTGGC-3′) with one reverse primer located in 518 th bp of ITS2 region and designated as P538 (sequence: 5′-TGGTGCCTAACATTGCGCGA-3′) (Fig. 1). PCR specific-amplicon was expected to be 403 bp (DQ855275) for B. xylophilus(Fig. 3). No amplification band was obtained for any of the other 12 nematode species related to pine wilt disease. 13 nematode species related to M.alternatus are B.xylopilus, R. sp., A. resinosi, S. steineri, D. parvus, O. xiphocaudatus, P. sp., respectively. (Fig. 4). It is the first finding that one pair of primers is effective to differentiate B. xylophilus from the 12 other nematode species related to M.alternatus according to the PCR amplification result '+/-'. It is important to continue to test the primers P155 and P538 for other nematode species related to pine wilt disease or other beetle species.

Specific PCR amplicon of 403 bp is successfully obtained by PCR amplification from the beetle tissue with B.xylophilus (Fig. 5, 6), which is the first trial to identify nematodes in beetle tissue by PCR amplification. So, it may be used in following situations: 1) Differentiating B.xylophilus from the 12 other species of nematodes as B.mucronatus, B.aberrans, B.corneolus, B.leoni, B.hunanensis, B.teratospicularis, A. resinosi, S. steineri, D. parvus, O. xiphocaudatus, R. sp. and P. sp.; 2) Detection of B.xylophilus in beetle tissue of M.alternatus, even when nematodes have already died as the result of heat, desiccation or other factors. To improve the overall usefulness of this method, further studies with more nematode species are needed.

Acknowledgements

The authors are very grateful to Dr. Esmenjaud D(INRA, France) and Dr.Eric Burt(one Canadian professor teaching English language in South China Agricultural University) for his helpful comments and linguistic corrections.