Identification Of Entomopathogenic Nematodes Symbiotic Bacteria 09FLYB1 And Purification Of It's Insecticidal Proteins

There are three entomopathogenic nematodes (EPNs) symbiont bacteria genera, Xenorhabdus, Photorhabdus and Serratia, which are important members of the family Enterobacteriaceae. These bacteria are Gram-negative and symbotic with the EPNs of Steinernematidae, Heterorhabditidae and the genus of Heterorhabditidoides of Rhabditidae, respectively.The new EPNS strain 09FLY1 was obtained from a soil sample collected from Yixing, Jiangsu province using the insect baiting method. A high virulent symbiotic bacterium strain,09FLYB1, was isolated from the intestine of the entomopathogenic nematode 09FLY1. The bacterium strain is gram-negative, with flagellum, no spores, short rod-shaped.The growth curve of strain 09FLYB1 was in accordance with that of the most other bacteria. The bacterium begins to exit extracellular insecticidal material in the early logarithmic growth and the largest insecticidal activity phase was found in the late logarithmic growth. The mortality of 100% of G. mellonella larvae was obtained with culture filtrate of strain 09FLYB1 cultured after 28 hours, which indicated that the optimal fermentation time for insecticidal activity was 28 hours.The phylogenetic tree constructed based on 16S rDNA sequences data showed that the strain 09FLYB1 belongs to the genus Serratia. The 16S rDNA sequences similarities between 09FLYB1 and Serratia nematodiphila DZ0503SBS11, Serratia marcescens subsp Sakuensis KREDT are 99.855% and 99.855%, respectively. The physiological and biochemical properties between strains 09FLYB1 and Serratia nematodiphila DZ0503SBS1T is the closest, only has 3 traits different; numerical classification showed that strain Serratia nematodiphila DZ0503SBS1T and 09FLYB1 also has the largest coefficient of similarity (0.86). These results indicated that the strain 09FLYB1 is belong to Serratia nematodiphila.The insecticiadal activity test of 09FLYB1 culture filrate showed that the culture filrate has insecticiadal activity whether oral or injection. The oral toxin activity test indicated that 1g food with 100μg culture filtrate killed over 32.6% of insects and the growth inhabiting rates to G. mellonella larvae was 48.2% within 168 h. The insecticidal activity of culture filrate was sensitive to protease K, showed that protein play a most part in insecticidal activity. And the half lethal dose of the crude insecticidal protein was 1.7μg·μL-1.Two protein peaks were isolated from the crude extracts of insecticidal protein by Sephadex G-75 gel filtration, named 09FLYB1G-I and 09FLYB1G-Ⅱ, respectively. Injected 10μL 09FLYB1G-Ⅰand 09FLYB1G-Ⅱ(concentration is 2μg·μL-1) to the haemocoel of the G. mellonella larvae, the mortality are 94.7% and 25%, respectively. The oral activity test indicted the 1 g food with 100μg of the peak 09FLYB1G-Ⅰkilled 52.8% of insects and the growth inhabiting rates to G. mellonella larvae was 75.3%. The mortality and the growth inhabiting rates of 09FLYB1G-Ⅱwere 11.5% and 25.6%, respectively.Only one protein peaks were isolated and purified from the 09FLYB1G-Ⅰby DEAE Sepharose FF, named 09FLYB1D-Ⅰ. The oral activity test indicated that mixting 1g food with 100μg of the two protein peaks,09FLYB1D-Ⅰkilled 53.6% of the insects and the growth inhabiting rates to G. mellonella larvae was 78.2%. Injected 10μL 09FLYB1D-I to G. mellonella larvae with concentration 2μg·μL-1, the mortality was 100%.Electrophoresis showed that the purified insecticidal proteins migrated as a single band on nature and reducing on SDS-PAGE with estimated molecular weight about 53.24 kDa, named as 09FLYB1P53. The LC50 of blood cavity toxicity of the insecticidal protein on larvae of G. mellonella was 0.4μg·μL-1. The LC50 of oral toxicity of the insecticidal protein on larvae of G. mellonella was 96.94μg per g artificial diet.