Isolation And Insecticidal Activities Study Of A Bacillus Subtillis Strain

Lepidoptera is a worldwide insect that cause billions of US dollars lose every year. Detailed inspecting the bacterial resources and screening the broad spectrum strain have an significant meaning on the construction of engineering bacterial and the development of environmental friendly pesticide.In this research, a Bacillus subtillis strain has been separated from soil, which is named HX08,by using the methods of bacterial shape and microscope observation, using the optical microscope,the laser scanning confocal microscope and16srRNA genetic homology analysis. Then16S rRNA gene sequence has been submitted to the Genbank database, gained the accession number KF774302.We do some behavior pilot study on this strain, including growth curve, optimal pH value, optimal temperature, pH change during culturing and antibiotic resistance etc.At the same time, we studied on the physiological-biochemical characteristic on this strain. Including Gram staining, Citrate utilization, gelatin liquefaction, indole test, methyl red test, V.P test, lecithinase test, starch hydrolysis test, Cellulose hydrolysis experiments, L-Arab sugar hydrolysis experiments, D-glucose hydrolysis experiments, D-xylose hydrolysis experiments, etc.Studied on the insecticide activity of this strain to Helicoverpa armigera under different fermentation period and sample concentration. All the results show that the fermentation broth at60h of this strain HX08also has significant insecticide activity towards some kinds among staphylococcus aureus, salmonella typhimurium, escherichia coli, pscudomonasaemginosa, enteroaerogen etc.Some elementary research has been implemented to the protein from the fermentation broth, such as cut the target protein stripe from SDS-PAGE gel and enzymolysis, LTQ-MS analysis, as a result of, the strain HX08has insecticidal activity because of the oxalate decarboxylase.As the same time, has built the HPLC separation system,The HPLC using a molecular sieve Surperdex200, H2O as mobile phase, elution flow with0.5mL/min, detection wavelength at215nm,254nm and280nm. the coarse separation substance which from the2nd peak at31min retention time has the activity.According to the results we discussed the reason of this high insecticide toward Helicoverpa armigera.This studying built a foundation for the enrichment of strain resource, the active material discovery and further investigation the ntibacterial active component of this strain.