Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.

There is emerging evidence showing that lncRNAs can be involved in various critical biological processes. Zebrafish is a fully developed model system being used in a variety of basic research and biomedical studies. Hence, it is an ideal model organism to study the functions and mechanisms of lncRNAs. Here, we constructed ZFLNC—a comprehensive database of zebrafish lncRNA that is dedicated to providing a zebrafish-based platform for deep exploration of zebrafish lncRNAs and their mammalian counterparts to the relevant academic communities. The main data resources of lncRNAs in this database come from the NCBI, Ensembl, NONCODE, zflncRNApedia and literature. We also obtained lncRNAs as a supplement by analysing RNA-Seq datasets from SRA database. With these IncRNAs, we further carried out expression profiling, co-expression network prediction, Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG)/Online Mendelian Inheritance in Man (OMIM) annotation and conservation analysis. As far as we know, ZFLNC is the most comprehensive and well-annotated database for zebrafish lncRNA.

A species-specific real-time PCR assay targeting the DNA topoisomerase I gene has been developed to detect Bursaphelenchus xylophilus. The specificity of the assay was confirmed by the lack of amplification of genomic DNA from other Bursaphelenchus or Seinura species. The sensitivity test showed that the limit of the reaction was 0.01 ng of genomic DNA or one individual nematode, as small as an egg. The validity of the real-time PCR assay was evaluated by analyzing mixed nematode samples extracted from diseased pine trees in which B. xylophilus was associated with several closely related species, B. mucronatus, B. hofmanni, Aphelenchoides macronucleatus, S. lii and S. wuae. These results demonstrate the potential of the assay to provide rapid, specific and sensitive molecular identification of B. xylophilus for use in pest risk assessment and quarantine regulations.

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