Identification Of A Pathogenic Bacteria Strain BP-1of Galleria Mellonella,Purification And Toxicity Of Its Insecticidal Proteins

Galleria mellonella is widely distributed, and belong to Lepidoptera. Galleria mellonella cause serious harm to sino-bee with3-5instar larvae feeding bee comb, so that bees can not be sealed, resulting in the formation of bald chrysalis within the colony. Bald chrysalis can not eclosion, even if reluctantly eclosion, the bees would be trapped in the nest room by the silk secreted by Galleria mellonella,. which has brought great loss to the world beekeeping industry. In nature, the main food of Galleria mellonella is bee honeycomb, biological control is an ideal choice to control G. mellonella preventing bees from harm during the control utilization. Researchers had brought lots of useful methods to control G.. mellonella, especially finding the most widely useful biocontrol microorganism in genera Bacillus and some fungi, such as Bacillus thuringiensis and Metarhizium. Among biocontrol microorganism, Bacillus thuringiensis is the widespread used microorganism to control G. mellonella. However, the long-term dependence on a single insecticidal bacteria will produce a considerably greater G. mellonella.resistance, leading to the biological effects of pesticides declined. Therefore, it is necessary to expand the range of pathogens in order to develop new pathogens for useful control Galleria mellonella.In this study, one pathogen strain BP-1, isolated from naturelly dead Galleria mellonella, was identificated. The toxin proteins of the strain were purified and conducted toxity test. The main results were as following.Galleria mellonella were repeatedly infected by BP-1, then appeared the similar symptom as before and the same pathogen was isolated from dead Galleria mellonella. infected. Its pathogenicity was testified by law of KOCH. The optimum growth pH range of the strain BP-1was7-8, and the optimum salt concentration was1%. The growth curve of BP-1showed that the logarithmic phase was2-7h and the stationary phase was8-24hours after cultured. BP-1was gram-positive bacteria, VP-positive, nitrate reduction positive. It can be hydrolyzed starch, make gelatin liquefaction. It does not produce hydrogen sulfide, can use propionate, citric acid salt, and can be able to use glucose, sucrose, and lactose. The16S rDNA sequences analysis combined the phenotyped characteristics indicated that the strain BP-1was identified as Bacillus amyloliquefaciens.The bioassay showed that the corrected mortality rate of G. mellonella was91.23%±3.04%with injection of20μL BP-1fermentation supernatant to its hemolymph within24h. The corrected mortality rate of G. mellonella were43.25%±4.365%and the weight inhibition rate was59.16%±2.250%when feeding G. mellonella larvae with100μL pathogen BP-1fermentation supernatant per gram artificial diet within148h. The best fermentation time of the pathogen strain BP-1is16h. Insecticidal activity test showed that the corrected mortality rate of fermentation supernatant, treated with proteinase K, to G.. mellonella was8.77%±3.04%, which indicated that the material that has the insecticidal activity is the insecticidal protein of the fermentation supernatant of the pathogen strain BP-1.Insecticidal protein crude extraction was obtained by ammonium sulfate precipitation saturated fermentation supernatant of BP-1. Insecticidal protein crude extraction was purified by Sephadex G-75gel filtration, and two peaks appeared. Stomach toxin activity and blood toxicity tests showed that cavity peak1was active peak. After DEAE-Sephacryl FF purification, just one peaks appeared. The corrected mortality rate was63.86%±4.25%and the weight inhibition rate was68.64%±0.4473%with1g artificial diet plus100Hg of freeze-dried powder of the peak feeding newly hatched larvae of G. mellonella within168h. The blood cavity toxicity testing showed that the corrected mortality rate was93.58%±5.268%within24h when njecting16μg freeze-dried powder of active peak to the hemolymph of G. mellonella larvae.SDS-PAGE electrophoresis showed that insecticidal protein was a single-chain protein with comparative molecular weight37kDa, named as BA1P37.The LC50of blood cavity toxicity of the insecticidal protein active peak on larvae of G.. mellonella was0.457μg.μL-1. The LC50of oral toxicity of the insecticidal protein active peak on larvae of G. mellonella was56.973μg per g artificial diet within168h.