Volatiles Produced By Bacillus Thuringiensis BRC-XQ12 Against Bursaphelenchus Xylophilus

The pine wood nematode (Bursaphelenchus xylophilus) is dangerous with fast spreading speed and difficult to be controlled, and might cause destructive diseases. In recent years, it has been found that microbial volatile metabolites may play an important role in the control of these plant parasitic nematodes. In this study, volatiles produced by Bacillus thuringiensis (Bt) strains and the active components of the volatiles against Bursaphelenchus xylophilus were identified. The finding here would be valurable for further exploitation and application of Bt volatile metabolites to control Bursaphelenchus xylophilus. The main research results of the thesis were as follows:1. Bursaphelenchus xylophilus cultured in petri dishes with the media containing Alternaria alternate, was applied as a target nematode to isolate Bt strains producing nematicidal volatiles.The nematodes were cultured at 22-27℃ (27/22℃, day/night) for 10 days until the nematode density was greater than 100,000 individuals per dish. A volatile-producing Bt strain BRC-XQ12 was obtained with a 3-partment-dish assay. When Bursaphelenchus xylophilus was exposed to the volatiles produced by Bt BRC-XQ12, they gradually lost their motility within 12 h and completely stopped movement after 24 h, indicating that the volatiles contained nematicidal compounds.2. Optimization of cultural conditions for Bt BRC-XQ12 volatile production through fermentation design.To determine the best nutritional sources for Bt BRC-XQ12 for optimum nematicidal activity,10 kinds of carbons (dextrose, citric acid, maltose, sucrose, lactose, corn meal, rice meal, soluble starch, inositol and mannitol) and 10 kinds of nitrogens (ammonium sulfate, ammonium nitrate, ureophil, casein, L-cystine, L-lysine, yeast extract, soya peptone, fish meal and beef extract) were tested in liquid cultural conditions:(1) The effects of nutrition on volatile production were determined by single factor analysis method. The results suggested that lactose and yeast extract were the best carbon and nitrogen sources, respectively, for nematicidal activity. (2) Plackett-Burman design was used to determine the most important factors for production of the nematicial volatiles of Bt BRC-XQ12. These factors were Na2HPO4, yeast extract, and the pH values of the media. (3) Response surface methodology (RSM) was applied to further optimize the nutritional components and fermentation conditions. The resulted data was treated with appropriate statistical methods and a quadratic regression model equation was obtained. The optimal culture medium were determined based on the model, which were lactose 6.00 g/L, yeast extract 6.99 g/L, MgSO4·7H2O 1.20 g/L, Na2HPO40.47 g/L, KH2PO40.60 g/L, NaCl 1.20 g/L, pH 7.97, and the best culture conditions were incubation at 37℃ with shaking at 160 r/min for 24 h. In this optimal condition, the theoretical nematicidal activity of Bt BRC-XQ12 was 99.25% in our model. The actual activity observed reached a maximum of 100% within 24 h, indicating the model equation precisely represented the actual cultural conditions.3. Identification of volatile organic compounds (VOCs) produced by Bt BRC-XQ12.In order to identify the nematicidal VOCs of Bt BRC-XQ12, the VOCs were collected with Solid-phase micro-extraction (SPME) method and then separated and measured by GC-MS.19 main compounds were detected. They were 3-methyl-2-pentanone, phenylacetaldehyde, ethylbenzene, o-xylene, 1-(2-furanyl)-ethanone, n-propylbenzene, isotridecanol,1,2,3-trimethylbenzene,1-decanol, undecane, tetradecane, phenylethylalcohol, azulene, benzothiazole, hexadecane,2-hexyl-1-octanol, nonadecane,2,4-bis(1,1-dimethylethyl)-phenol and n-dotriacontane. Commercially available pure chemicals of the 19 candidates were investigated for their anti-nematode activities. The results indicated that 3-methyl-2-pentanone, phenylacetaldehyde, 1-(2-furanyl)-ethanone,1-decanol, phenylethylalcohol, benzothiazole,2-hexyl-l-octanol and 2,4-bis(1,1-dimethylethyl)-phenol had high nematicidal activities and killed 100% of the tested nematodes with a dosage of less than 0.0200 g within 8 h. The minimum lethal concentration for these compounds was followed:34.31±1.64×10-5 g/cm3,30.43±1.88×10-5 g/cm3,53.13±2.06×105 g/cm3,17.69±0.55×10-5g/cm3,4.94±0.30×10-5 g/cm3,8.55±0.51×10-5 g/cm3, 7.08±0.49×10-5 g/cm3 and 23.06±0.78×10-5 g/cm3. In addition,1,2,3- trimethylbenzene 0.0600 g also showed 100% nematicidal activity within 8 h and the minimum lethal concentration for this compound was about 106.16±1.77×10-5 g/cm3.