Isolation And Identification Of Bacteria With The Function Of Attracting Plutella Xylostella

The diamondback moth(DBM),Plutella xylostella(L.)(Lepidoptera: Plutellidae),mainly harm cruciferous vegetables,resulting in serious reduction of vegetables,newly hatched larvae first eat eggshell,and then harm the leaves of vegetables,therefore,the contents of this study to research bacteria on the surface of eggshell and Phyllospheric,explore the two place whether include the volatile bacteria which affected Plutella xylostella behavior.This study isolated bacteria from leaf surface and eggshell surface by traditional culture method,extracting genomic,PCR and 16 S rDNA sequencing and identification of bacteria,comparing with the NCBI database,analyzing the bacteria on the genus level;In addition,the V4 hypervariable region of the 16 S rDNA gene was selected for amplicon sequencing of the phyllosphere microbial community using of non-cultured method of Illumina HISeq sequencing technology,based on the common method of extracting leaf surface total bacterial DNA,the study using new method of directly bacterial pellets to analyze phyllosphere;the traditional method isolated 46 strains of leaf surface bacteria and 4 strains of eggshell surface bacteria,leaf surface bacteria assigned to 18 genera,eggshell surface bacteria assigned to 2 genera;compared with the common method,the new method in high-throughput sequencing had less chloroplast pollution,higher bacterial community diversity,in the top 10 dominant genera,Pseudomonas also exists in the cultivable bacteria.A screening device was designed to choose the bacteria that were attractive to the Plutella xylostella,the screening device includes three parts of trap A and B,selection hole and odor selection area,trap A and B are respectively used for placing odor samples,and traps can capture test insects and realize unattended screening;selection hole link with odor selection area and trap,the smaller selection hole improves the selection accuracy,high distance with trap bottom,avoid insects return odor selection area,located in the central region to avoid error caused by lateral light.The odor selection area has smaller height that is conducive to diffusion of odor molecules in space,larger area providing sufficient activity space,giving test insects the freedom of choice,and improving the selection accuracy.In order to verify the reliability of the screening device,using vegetable cabbage leaves to experiments,the results show that the device is reliable and can be used for screening the bacteria.The bacteria that attracted the diamondback moth were screened by way of a device,with three replicates per strain,The preliminary screening results showed that 4 strains of Phyllosphere bacteria and 1 strains of Eggshell bacteria were more attractive to the 3 instar larvae of Plutella xylostella,After screening,except for Eggshell bacteria,the other 4 bacteria showed a downward trend.In order to verify the accuracy of the test,a video acquisition instrument is used to screen.The results showed that the egg surface bacteria had obvious attraction to the 3 instar larvae of Plutella xylostella,the bacteria was identified Pseudomonas by 16 Sr DNA,the antibiotic sensitivity test showed that it is resistant to ampicillin,chloramphenicol and etracycline,but no resistance to kanamycin and rifampicin;using five different methods for the trapping of Plutella xylostella to further verify the function of bacteria in cabbage field experiment,the four methods were centrifugation mycelium,12 h fermentation liquid,24 hours fermentation liquid and plate lineatio.The positive control was the lure,and the negative control was sterile water,comparison of four different methods,24 hours of fermentation liquid has a certain attraction,but compared with the positive control,the number of Plutella xylostella were rare.The genome sequencing analysis of the bacteria showed that the genome size was 6 499 289,consisting of one plasmid and one chromosome,6132 genes were obtained by gene annotation analysis,the KEGG library functions as a functional annotation of chromosome genes,obtaining metabolic pathways of metabolism terpenoids and polyketides may be related to bacterial volatiles.