Pine wilt disease (PWD), a destructive disease for pine trees, is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus and additional bacteria. In this study, extracts of Zostera marina showed a high nematicidal activity against PWN and some of the bacteria that it carries. Light yellow crystals were obtained from extracts of Z. marina through solvent extraction, followed by chromatography on AB-8 resin and crystallization. The NMR and HPLC analysis showed that the isolated compound was rosmarinic acid (RosA). RosA showed effective nematicidal activity, of which the LC50 (50% lethal concentration) to PWN at 24 h, 48 h and 72 h was 1.18 mg/g, 1.05 mg/g and 0.95 mg/g, respectively. To get a high yield rate of RosA from Z. marina, single factor experiments and an L9 (34) orthogonal experiment were performed. This extraction process involved 70% ethanol for 3 h at 40 °C. The extraction dosage was 1:50 (w/v). The highest yield of RosA from Zostera was 3.13 mg/g DW (dried weight). The crude extracts of Zostera marina (10 mg/mL) and RosA (1 mg/mL) also showed inhibitory effects to some bacterial strains carried by PWN: Klebsiella sp., Stenotrophomonas maltophilia, Streptomyces sp. and Pantoea agglomerans. The results of these studies provide clues for preparing pesticide to control PWD from Z. marina.

Bacterial volatiles represent an important source for new natural nematicidal compounds that may be developed as novel nematicidal agents to control plant-parasitic nematodes. In this study, two marine bacteria,Pseudoalteromonas marinastrain H-42 andVibrio atlanticusstrain S-16, were isolated from seawater and the bay scallop (Argopecten irradians), respectively, collected from the subtidal beds at Qingdao, China.In vitrobioassays indicated that culture supernatants of the two bacteria displayed strong nematicidal activities with corrected mortalities of more than 93% after 24 h exposure against the pine wood nematode (PWN),Bursaphelenchus xylophilus. Maximal nematicidal activity was exhibited by culture supernatants of the two marine bacteria incubated at 15-20°C for 3 days in Zobell 2216E medium prepared with 100% seawater and initial pH 6.0-7.0, inoculating 19-h-old cultures. Through analysis using solid-phase microextraction-gas chromatography/mass spectrometry, the volatiles produced by the two bacteria were shown to contain mainly dimethyl disulphide, benzaldehyde, dimethyl trisulphide,tert-butylamine, acetone and dimethylamine, N-(diisopropylphosphino)methyl-. Results from nematicidal assay using pure commercial compounds instead of the candidate volatiles demonstrated that the four volatile compounds, dimethyl trisulphide (LC90 = 0.060 mmol l−1), benzaldehyde (LC90 = 0.309 mmol l−1), dimethyl disulphide (LC90 = 0.162 mmol l−1) andtert-butylamine (LC90 = 0.366 mmol l−1), showed strong nematicidal activities against PWN. This is the first report on the nematicidal activity of volatile organic compounds (VOC) from marine microorganisms. This study indicates that the nematicidal VOC produced by marine bacteria are potential substitutes for current chemical control options of pine wilt disease caused by PWN which greatly threatens global forest resources.

The ethanol extract of the Ficus carica L. leaves was tested to show strong nematicidal activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, causing 90.93% corrected mortality within 72 h at 1.0 mg/mL. From the ethyl acetate soluble fraction of the F. carica L. leaves extract, the main nematicidal constituents were obtained by bioassay-guided isolation and identified as linear furocoumarins bergapten (1) and psoralen (2) by mass and NMR spectral data analysis. Bergapten and psoralen had significant nematicidal activity against PWN with the LC50 values of 97.08 aKSnd 115.03  μ g/mL within 72 h, respectively. The two furocoumarins could inhibit the activities of amylase, cellulase and acetylcholinesterase (AchE) from PWN. The morphologies of PWNs changed much after they were treated by bergapten and psoralen. The physiological effects of bergapten and psoralen on PWN might provide helpful clues to elucidate their nematicidal mechanisms.

Crude extracts of the surface coat (SC) proteins of the pine wood nematode (PWN), Bursaphelenchus xylophilus, and purified flagellin from Pseudomonas fluorescens (GcM5-lA) isolate were bioassayed using symbiontic GcM5-lA and axenic PWNs, respectively. The results showed that the flagellin significantly increased the reproduction of axenic PWN and the SC protein extracts of the PWN promoted GcM5-l A muhiplication. The finding is a new step to further elucidate the symbiotic mechanism between the PWN and its associated bacteria and the SC protein function in pine wilt disease.

Peroxiredoxins (Prxs) are enzymatic antioxidants widely distributed in biological kingdoms, which constitute a family of heme-free peroxidases that reduce alkyl hydroperoxides and hydrogen peroxide. In this paper, an open reading frame (ORF) of 639 bp, which encoded a protein of 213 amino acid residues, was cloned from Pseudomonas fluorescens GcM5-1A carried by pine wood nematode. Amino acid sequence alignment showed that the encoded protein shared 99, 97, and 97 % identity with the thiol-specific antioxidant protein LsfA of P. fluorescens Q2-87, the peroxiredoxin of Pseudomonas sp. GM17 and 1-Cys peroxiredoxin of P. fluorescens Pf 0-1, respectively. The ORF was cloned into expressing vector pET-15b and introduced into Escherichia coli BL21 (DE3). Overexpression of a 27-kDa protein was achieved by IPTG induction. The recombinant protein was purified by affinity chromatography on a Ni(2+) matrix column. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that part of the recombinant appeared in dimer form. Bioassay results showed that purified recombinant protein had both peroxidase and thioredoxin activity. Furthermore, E. coli expressing the ORF showed tolerance to hydrogen peroxide stress, which indicated that the gene might help P. fluorescens GcM5-1A resist hydrogen peroxide generated by host pines after pine wood nematode associated with this bacterium infected pine trees.