| The Asian citrus psyllid(ACP),Diaphorina citri Kuwayama,belongs to Homopetra Chermidae,and it is one of the important insect pests because it transmits primarily citurs huanglongbing(HLB)causing economic losses in citrus production worldwide.At present,the chemical methods are frequently used to prevent and control ACP,but the irrational use of chemical insecticides leads to the resistance development and has negative effects on the environment.Therefore,a new biopesticide for the control of ACP is need to be explored.The bacteria KH-001 was isolated from an infection nymph of ACP,which caused high mortality of ACP and called as Serratia marcescens,and its cell-free fermentation supernatant also could cause high mortality of ACP.To explore the insecticidal mechanism of the KH-001,the colonization ability of KH-001 in the ACP was detected by real–time PCR,and the effects of symbiotic strains of ACP were detected with high-throughput sequencing.Chitinase activity of KH-001 strain was determined by DNS method,and then the Chitinase chia gene was cloned.Finally,the crude extract of prodigiosin was extracted from fermentation of strain KH-001,and it was made into an emulsion preparation,which could effectively control ACP.this study provided a new method and basis for the field application.The main results were summarized as follows:(1)As the time incresed the activity of ACP decreased and died eventually.ACP became redder and redder after being infected by the strain KH-001 with the time increasing,and they shed red fungus pus when dead insects were touched.(2)The 16 S rDNA of the strain KH-001 was amplified and sequenced through its DNA.A phylogenetic tree was constructed,showing the strain KH-001 belonged to Serratia marcescens.The physiological–biochemical test showed the strains KH-001 could utilize mannitol,sucrose,citric acid,raffinose and glucose to produce acid,but it can't use malonic acid,and the V-P test was positive,while indole and methyl red determination were negative.The strain KH-001 grew quickly in LB fluid media at 37 ? with rotation speed at 180 r/min.After 6 h,it entered the logarithmic phase,and entered the stable phase after12 h.After reaching the maximum growth at20 h,it begain to decline.(3)The colonization ability of KH-001 strain on ACP nymphs was detected by real-time PCR.The results showed that,the number of KH-001 strain increased with time,and the relative content of KH-001 after 24 h treatment was significantly higher than that of the 1-min and 6-h treatment groups,but there was no significant difference with the 12-h treatment group.(4)1331 OUTs were found in ACP nymphs infected by strains KH-001 with high-throughput sequencing,and the similarity of 10 microbial groups with the largest proportion were distinguished by UPGMA,among which four groups Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes were more than 90%.The bacterial community analysis found that ACP nymphs treated with four methods had 34 same OTUs,and the relative abundance of Wolbachia,Anaplasmataceae,Alphaproteobacteria,Gammaproteobacteria,Rickettsiales and Carsonella in PBS(C)group were significantly higher than that in clear liquid of the fermentation(A),DH5 alpha(E)and s.marcescens group(S).The abundance of Serratia in group S was higher than other groups(P<0.01),and Wolbachia in group C was significantly higher than that in group S(P<0.01),whilie Profftella and Carsonella in group S was significantly lower than that ingroups C,A,and E(P<0.01).(5)The activity of chitinase was 44.55 U/mL after fermentation 4days at 37 ?,however,it decreased to 29.95 U/mL at 28 ?.1863 bp fragment was cloned from strain KH-001,which contained a chitinase gene chia with a length of 1692 bp.(6)The red crude extract was extracted from KH-001 fermentation.The emulsion is made up of 10% of the crude red extract,2.5% of the emulsifier for milk 500 # and T80,and 0.45% of fast penetrating agent T,lastly using sec-butyl acetate to make up to 100%.it was found that the emulsion had good self-dispersibility,good emulsification and qualified emulsion stability The corrected mortality rate of citrus psyllid treated with 200 times of dilution was 88.89 % with no significant differences from 85.19 % with 500 times of dilution,which was significantly higher than that 40.74 % with1000 times of dilution... |
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