Screening And Analysis Of Attractive Bacteria To Plutella Xylostella

Plutella xylostella(diamondback moth,DBM),Lepidoptera,Plutellidae,which mainly harms cruciferous plants.In agricultural production,it mainly relies on chemical pesticides for control,but DBM is resistant to most pesticides used in the field,causing a series of problems such as pesticide residues in environmental pollution and food safety.Using volatile odor to regulate insect behavior is a green control method for pest damage.In the earlier stage,our laboratory isolated and screened the surface bacteria of cruciferous vegetables and DBM eggs,and found that the volatiles of DBM egg surface bacteria showed different degrees of attraction to the 3rd instar larvae of DBM.We speculated whether diamondback moth egg surface bacteria could be transmitted vertically from the mother’s ovary.Will there be more strains in the ovaries that attract diamondback moth larvae? In order to further explore the resources of bacterial strains with attractive effects,we started with isolating bacterial strains from the ovaries of DBM(Plutella xylostella),and carried out the isolation and screening of bacterial strains with attractive effects on DBM larvae,the determination of bacterial genome by using three-generation Nanopore sequencing technology,and the analysis and identification of volatile components,etc.The main results are as follows:1.A total of 15 strains of bacteria were isolated from adult DBM(Plutella xylostella)ovary tissue by traditional plate culture method.The volatiles of 10 strains were found to attract the 3rd instar larvae of DBM.The LB-LC1 strain had the strongest attraction effect on the 3rd instar larvae,with the attraction index of 0.73(t = 20.878,P < 0.0001).After 16 S r DNA sequence analysis and comparison,LB-LC1 strain was classified as Pseudomonas and Gram-negative bacteria.A preliminary study on the ecological function of the strain showed that the strain had no effect on the behavior and oviposition of the adults,and probably only played an aggregation function on the larvae.2.The whole genome sequencing of LB-LC1 strain showed that the genome contained a complete annular chromosome with a size of 4,863,178 bp and a complete annular plasmid with a size of 1,804 bp,with GC content of61.66% and 59.7%,respectively.A total of 4475 genes and 4332 coding sequences(CDS)were predicted.The complete genomes of 20 Pseudomonas strains were collected from NCBI Gen Bank database,and the phylogenetic tree was constructed by using CVtree and PHYLIP.The results showed that LB-LC1 strain was Pseudomonas fulva.3.HS-SPME-GC-MS method was used to collect and determine the headspace volatiles of LB-LC1 strain growing in LB medium.A total of 6 compounds were detected,mainly alkenes,especially rich 1-undecene.In different growth medium and growth conditions,the volatiles produced by bacteria will change.After inoculating LB-LC1 strain into LB,NB and TSB media,it was found that the attraction activity of LB-LC1 strain decreased significantly in NB and TSB media.The volatiles of LB-LC1 strain growing in these three media were compared.It was found that in NB medium,besides alkenes and alkanes,there were also various cyclic aromatic hydrocarbons.In TSB medium,only two alkenes were detected.The behavioral effects of the volatiles from LB-LC1 strain were determined.It was found that 1-nonene and 1-undecene exhibited behavioral effects on larvae,and these two volatiles were active volatiles for the strain to attract larvae.In conclusion,in this study,a number of strains with attraction effect on the 3rd instar larvae were successfully isolated from ovarian tissue,among which P.fulva had the strongest attraction effect on the 3rd instar larvae,and volatiles of this strain were determined and screened.This study is expected to provide a method for screening strains that are attractive to lepidopteran insects in the future and provide a new idea for pest control technology in the future.