Isolation And Identification Of Bacillus Pumilus CH-1 Strain From Insect And Optimization Of Fermentation Conditions

A total of four strains of suspected pathogenic bacterium were isolated from Apocheima cinerarius larvae,and a strain CH-1 with insecticidal activity was finally screened according to the Koch’s law.Using the Birger’s bacterial identification manual,physiological and biochemical indicators and 16S rRNA gene molecular identification method to determine that the pathogen is Bacillus pumilus.In order to detect the insecticidal activity of the strain on lepidoptera larvae,the larvae of Plutella xylostella,Spodoptera exigua and Spodoptera frugiperda were measured respectively.It is determined that the medium composition and fermentation conditions suitable for the growth of the bacteria will be optimized,so as to provide a scientific basis for subsequent research.The main findings are as follows:(1)The pathogenic strain CH-1 was isolated from the larvae of the Apocheima cinerarius,and it was identified as Bacillus pumilus CH-1 strain by morphology,physiology,biochemistry and molecular determination.(2)Bacillus pumilus CH-1 fermentation solution and upper serum have an active and lethal effect on Lepidoptera larvae such as Plutella xylostella,and the effect of fermentation solution is more obvious.After 4 days of treatment,the mortality rates of Plutella xylostella,Spodoptera exigua and Spodoptera frugiperda treated with fermentation solution are 96.67%,100.00%and 100.00%respectively,which is 40.00%,70.00%and 40.00%higher than that under the control of clear water,respectively,compared with the treatment of the upper serum.The mortality rate is 0%,13.33%and 60.00%respectively.(3)Through the combination of single-factor test and orthogonal test,shaker fermentation is used to optimize the medium composition and fermentation conditions of the Bacillus pumilus CH-1 strain.Finally,the best medium composition and fermentation conditions are obtained:the best carbon source of the medium is glucose,the added amount is 1.5%,and the best nitrogen source is raw soybean flour,the added amount is 2%,the inorganic salt ion is 1%NaCl,the optimal temperature is 33℃,the optimal initial pH value is 7,the optimal rotation speed is 180rmp,the optimal liquid is 40mL/250mL,and the spore concentration in the final fermentation solution is 1.792×1010 cfu/mL.