Analysis Of Marine Bacteria Isolated Kill Pine Wood Nematode, Identification And Active Substances

Pine wood nematode (PWN), Bursaphelenchus xylophilus, is an important pathogen to conifer trees. Looking for microbes with nematicidal activity and analysing their active substances are an important way to develop a novel nematicide of microbial origin with the features of high activity to parasitic nematodes, low toxicity to mammals and other non-target organisms, and better environmental compatibility. In order to screen high nematicidal marine bacteria, in this study, forty-nine marine bacterial strains were obtained from the samples collected from the subtidal beds at Qingdao and were assayed in vitro for their nematicidal activities against PWNs using immersion test. Five of these strains exhibited higher nematicidal activities to the tested nematodes and the revised mortalities of PWNs treated with the culture supernatants of the five strains for24h were more than80%. On the basis of morphology, biochemical-physiological characters and16S rDNA sequence analysis, these five stains were identified as Pseudoalteromonas marina, P. nigrifaciens, Vibrio atlanticus, Bacillus thuringiensis, B. indicus, respectively. The results from the single-factor experiments demonstrated that the optimal cultivation conditions of strain H-42, S-16and G-23for the production of nematicidal substances were as follows:inoculating19h-old culture into Zobell2216E medium prepared with100%seawater, with initial pH7.0(H-42),6.0(S-16) and8.0(G-23) and cultivating at15℃(H-42),20℃(S-16) and25℃(G-23) for3days. The determination of active ingredient constituents indicated that the crude volatile extracts from the culture supernatants of H-42and S-16could kill100%nematodes in24h. Through analysis by using SPME-GC/MS (solid-phase microextraction-gas chromatography/mass spectrum), the volatiles were proved to contain mainly dimethyl trisulfide (LC9o=0.060mmol/L), benzaldehyde (LC90=0.309mmol/L), dimethyl disulfide (LC90=0.162mmol/L), tert-butylamine (LC90=0.366mmol/L). In the experiment of stability, the results indicated that the nematicidal substances produced in the liquid medium by G-23contained both highly water soluble and low polarity substances which were heat stable, relatively stable under weakly alkaline (pH8.0) conditions. This study provides an important theoretical basis for the utilization of marine microorganism resources and the development of new nematicides of microbial origin.