Cultivation Characteristics And Pilot Study Of The Insecticidal Substances From Symbiotic Bacterium Xenorhabdus Bovieniibe Of Entomopathogenic Nematode Steinernera Feltiae

The combination of Entomopathogenic nematodes and their symbiotic bacteria show high security to animals and human,while high toxicity to many insects.Because the nematodes has the ability of searching host,they have the potential to become a new biocontrol substances.In this study,the symbiotic bacterium Xenorhabdus bovienii BE strain was extracted from the Belgian population of entomopathogenic nematode Steinernera feltiae.In order to provide basic information for developing a new biological control resource,the growing and the optimal fermentation conditions were tested,which can provide the precondition for the advanced research.Furthermore,the toxicities of different ingredients extracted from bacterium's fermentation solution were tested,from which the high toxic substance was found.The character of the toxic substances was investigated in order to provide a new resource for biocontrol.This research includes three parts:â…°) optimal fermentation and growth condition of Xenorhabdus bovienii BE have been determined;â…±) toxicities of different ingredients extracted from bacterium's fermentation solution have been testified andâ…²) the high toxic substances have been found.The relativity of different indexes was analyzed and the oral and hemocoel cavities 50%lethal dose to second-instar and fifth-instar larvae were tested. The results are as follows:1 The lag phase,logarithmic phase,stationary phase,and dead phase of the symbiotic bacterium Xenorhabdus bovienii BE were 0～6 h,6～12 h,12～22 h and over 22 h respectively.In NA fermention liquid,the optimal temperature is 27.5℃with incubation speed 180 r/min,pH is 7.0～7.5.The inoculation quantity is 6%and the liquid volume is 35ml in conical flask with the volume of 250ml,which indicated that the optimal quality of the fermention is 14%of the container.2 The symbiotic bacterium Xenorhabdus bovienii BE strain was extracted from the Belgian population of entomopathogenic nematode Steinernera feltiae.High toxic activity to Galleria mellonella fifth-instar larvae was shown when the insect hemocoel cavities were injected with the fermentation solutions of the bacteria.Studies on the insecticidal activity of substances from fermentation solution indicated that the toxic substances are proteins existed in both extracellular and intracellular,and the toxicity of intracellular proteins is higher than those from extracellular.SDS-PAGE analysis demonstrated that there are five bands from intracellular proteins and no band from extracellular proteins when running the gel with the molecular weight marker ranged from14.3 to 97.2kDa.The 50%lethal dose(LD₅₀) of intracellular crude proteins were 4.7316g/L and 1.5715g/L against insect larvae in 24h and 48h respectively,and its 50% lethal dose(LD₅₀) was 253.7579μg/g against insect larvae oral cavity in 5d.The crude proteins showed four absorption peaks after sephadex gel filtration and only two peaks showed obvious bands in SDS-PAGE analysis.The active substances of one band showed very high toxic against insect instar larvae.SDS-PAGE showed that the substances combined two proteins with the molecular weights ranged from 20.1 to 29.0kDa.The 50%lethal dose(LD₅₀) of the substances against G.mellonella larvae in 24 h and 48 h were 0.3772g/L and 0.1238g/L respectively.The 50%lethal dose(LD₅₀) was 93.0021μg/g against insect larvae oral cavity in 5d.3 The characters of intracellular crude proteins are:the heat-stable temperature is 70℃and the optimize saturation of aminium sulphate is 80%.the concentrations of the intracellular proteins and extracellular proteins are 316.67μg/ml and 580.89μg/ml, respectively which were obtained from the standard equation by method of Coomassie brilliant blue G-250.