Screening Of Bt Strains And Identification Of Insecticidal Genes With High Activity Against Spodoptera Frugiperda

The fall armyworm（Spodoptera frugiperda）is a major international migratory agricultural pest,causing serious damage to corn and other food and economic crops.So far,it has spread in 126 countries and regions around the world.Fall armyworm invaded my country in January 2019 and quickly spread to 27 provinces（municipalities,autonomous regions）.Bacillus thuringiensis（Bt）has the characteristics of high efficiency and specificity against pests,environmental friendliness,and safety for humans and animals,and is widely used in countries all over the world.Due to the sudden invasion of Spodoptera.frugiperda,there is still a lack of sustainable green control microbial pesticides against Spodoptera.frugiperda in China.It is urgent to screen and obtain more new strains of Bt with higher insecticidal activity against Spodoptera.frugiperda and new gene.（1）In this study,14 Bt strains with high activity against Spodoptera frugiperda were screened from 880 strains in the strain storage by high-efficiency insecticidal activity assay of Bt strains.Through the insecticidal activity assay on neonate of Spodoptera frugiperda,it was found that the lethal concentration(LC₅₀)of SP25 strain was 0.289μg/g,and the LC₅₀of BJ53 strain was 0.368μg/g.The insecticidal activity of the above two strains against Spodoptera frugiperda is 4.6 times and 3.4 times that of the commercially applied Bt product"Jinweijun"G033A,respectively,and the insecticidal activities of the other three strains BJ1,TY58,and J157 are comparable to G033A.（2）By analyzing the insecticidal genes of 589 Bt genomes downloaded from the National Center for Biotechnology Information（NCBI）and 1368 Bt genomes in our laboratory database,it was confirmed that there is a common linkage relationship among Bt insecticidal genes.That is,multiple insecticidal genes active on the same target often co-exist in the same strain.Such as vip3A insecticidal gene and cry1A,cry1I,cry2A genes co-exist in multiple strains.According to the linkage relationship of insecticidal genes,the14 strains with high activity against Spodoptera frugiperda obtained in the previous part can be screened only by PCR screening with vip3A primers,which proves that this screening method is fast and effective.By this method,16 strains containing the vip3A gene were screened from 1186 Bt strains isolated from soil samples collected in Shanxi,Anhui and other places.Insecticidal activity bioassay showed that the LC₅₀of the S1-3strain against newly hatched larvae of Spodoptera frugiperda was 0.754μg/g,and the insecticidal activity was 1.5 times that of G033A.The insecticidal activity of the other two strains S5-11 and S1-17 was comparable to that of G033A.（3）Subsequently,the insecticidal genes in Bt strains were identified by genome high-throughput sequencing,and 575 insecticidal genes were found from 83 Bt strains.Among them,129 genes were active against S.frugiperda,including 10 new genes that were active against S.frugiperda.Including one cry1Ca,cry1Da,cry1Ea,cry1Ia,vip3Aa gene,two cry1Ab and cry2Ab genes.There is also a second-level new gene cry56-like with51%similarity to cry56Aa,which may have insecticidal activity against Spodoptera frugiperda.In this study,three Bt strains with high activity against Spodoptera frugiperda were finally obtained based on insecticidal activity and linkage analysis of insecticidal genes.The insecticidal genes in the strains were identified using high-throughput sequencing,and10 new genes with insecticidal potential against Spodoptera frugiperda were obtained.This study enriches the resource bank of Bt strains in my country;provides insecticidal gene reserves for the research and development of GM insect-resistant crops,and provides an important guarantee for the green control of major agricultural pests Spodoptera frugiperda.