tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for Bursaphelenchus xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2,304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions or bands via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two non-identical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

The pine sawyer beetle, Monochamus alternatus, transmits the pinewood nematode (PWN, Bursaphelenchus xylophilus), causing the pine wilt disease (PWD), which gives rise to enormously economic as well as forest damage. However, PWN has been identified as a pathogen of PWD, it is very difficult to discriminate B. xylophilus from B. mucronatus in a short time, which are genetically and morphologically very similar. Therefore, it has been necessary to detect and eliminate PWN-infected trees in the forest area for the prevention of PWD transmission. Up to date, there is no report on biomarkers such as DNA and protein for the diagnosis of B. xylophilus. In this study, we produced a B. xylophilus monoclonal antiserum (D9-F10) from BalbC mice and screened its specificity with various proteins extracts. Western blot analysis revealed that the D9-F10 is only reactive with B. xylophilus protein extract among other tested protein extracts, indicating that the D9-F10 is specific for a B. xylophilus protein. Furthermore, two-dimensional electrophoresis showed the D9-F10 detects a very highly acidic protein, pI≒3.5. These results suggest that the D9-F10 monoclonal antibody is useful for the development of a diagnostic kit for the pine wilt disease.

The pinewood nematode (PWN, Bursaphelenchus xylophilus) causes the pine wilt disease, transmitted to pinewoods by the pine sawyer beetle, Monochamus alternatus. It is very difficult to discriminate B. xylophilus from B. mucronatus. Therefore, it has been necessary to detect PWN-infected trees for the prevention of pine wilt disease transmission in a short time. The development of biomarkers such as DNA and protein is important for diagnosis of B. xylophilus. However, there have been no reports regarding biomarker identifications of B. xylophilus. In this study, polyclonal antisera were raised against whole proteins of B. xylophilus in BalbC mice and were primarily screened with ELISA. Twenty five among over 500 cell lines releasing polyclonal antisera revealed B. xylophilus-specific immuno-reactivity, which was at least three times higher than that of B. mucronatus. Three cell lines among them were secreting monoclonal antibody through further screening. These cell lines only detect about a 33-kDa protein in B. xylophilus in the western blot. These results suggest that these monoclonal antibodies will be useful for the development of diagnostic kit for the pine wilt disease.

Total RNAs were isolated from cultured roots of Scopolia parviflora, poly(A)+ RNA was obtained through the mRNA purification, cDNA library of Hnh6h was constructed. Recombinant baculoviruses in Spodoptera frugiperda (Sf) cells were constructed by use of the transfer vector pBacPAK, which has the AcNPV sequence under the polyhedrin promoter. The expression vector carrying Hnh6h gene was transferred to S. parviflora and obtained transgenic hairy root lines. Our results confirmed the over expression of the H6H protein was used by anti-pBacPAK about cDNAs of S. parviflora. This study will served for production of tropane alkaloids by metabolic engineering.