Analysis Of Synergism Between Extracellular Polysaccharide And Insecticidal Proteins From Bacillus Thuringensis

Bacterial exopolysaccharides have been widely used in food,cosmetics,agriculture and pharmaceutical industries due to their unique and complex chemical structures.Bacillus thuringiensis(Bt)is the most productive and widely used microbial insecticide,which has brought enormous economic,social and ecological benefits to human beings.However,studies on the function of Bt exopolysaccharide were lacking.It was first reported by our laboratory that Bt strain 4D19 exopolysaccharide could improve the insecticidal activity of Cry1Ac protoxin against Plutella xylostella,and monosaccharide composition of 4D19 exopolysaccharide was analyzed.Based on the established extracellular polysaccharide purification system and previous research results,this study analyzed the proportion of synergistic effect of extracellular polysaccharide in representative strains of Bt standard strain library,and analyzed the universality of synergistic effect of extracellular polysaccharide from the perspective of target pests and insecticidal proteins.The main results are as follows:1.24 representative Bt strains,including 4D19,were selected from the standard Bt strain library from the perspective of strain affinity and serotype,12 strains of crude extracellular polysaccharide extracts could significantly improve the insecticidal activity of Cry1Ac protein against Spodoptera frugiperda,accounting for 50%of the total.The results showed that the synergistic effect of Bt extracellular polysaccharide was universal.2.4D19 exopolysaccharide could enhance the insecticidal activity of CrylAc protein against different target pests.The results showed that added 2 mg/g of 4D19 crude extracellular polysaccharide reduced the LC50 of Cry1Ac to from 66.24μg/g to 28.36 μg/g,and reduced the LC50 of H.armigera from 10.57 μg/g to 5.12 μg/g.After 50 μg/g CrylAc was added with 0.5 mg/g purified 4D19 extracellular polysaccharide,the corrected mortality rate of S.frugiperda increased by 20.8%.The corrected mortality of H.armigera was increased by 15.2%with the addition of 0.5 mg/g purified 4D19 extracellular polysaccharide at 12 μg/g CrylAc.These results indicated that the synergistic effect of 4D19 exopolysaccharide was universal to target pests.3.4D19 extracellular polysaccharide could enhance the activity of Vip3Aa11 protein against S.frugiperda and H.armigera.The bioassay results showed that the LC50 of Vip3Aa11 to S.frugiperda reduced from 1.03 μg/g to 0.49 μg/g,and the LC50 of H.armigera reduced from 11.93μg/g to 5.06μg/g by adding 2 mg/g crude extracellular polysaccharide 4D19.After adding 0.5 mg/g purified 4D19 extracellular polysaccharide to 0.8μg/g Vip3Aa11,the corrected mortality of S.frugiperda increased by 18%.When 16 μg/g Vip3Aa11 was added with 0.5 mg/g purified 4D19 extracellular polysaccharide,the corrected mortality of H.armigera increased by 15.3%.4.The crude extracellular polysaccharide extract of 4D19 could improve the activity of CrylAb and Cry1Ah proteins against Plutella xylostella.The bioassay results showed that the corrected mortality rate of Plutella xylostella was significantly increased by 25.5%after 20 μg/mL CrylAb was added with 5 mg/mL extracellular polysaccharide crude extract of strain 4D19.After adding 5 mg/mL extracellular polysaccharide crude extract from strain 4D19 to 0.8μg/mL Cry1Ah,the corrected mortality of Plutella xylostella was significantly increased by 42.9%.5.ELISA results showed that 4D19 extracellular polysaccharide could bind to Vip3Aa11 protein,and the dissociation constant Kd=6.43±1.31 nmol/L.4D19 extracellular polysaccharide made CrylAc protoxin and Vip3Aa11 protein more stable in the midgut fluids of S.frugiperda.and H.armigera.In this study,it was found that most of the extracellular polysaccharides of Bt strains had synergistic activity,and this synergistic activity was applied to Lepidopteran pests of different families and insecticidal proteins with different structures,indicating that the synergistic effect of extracellular polysaccharides of Bacillus thuringiensis was universal.The results laid a foundation for improving the virulence of Bt products and the application of extracellular polysaccharide.