Mechanism Of Bt-resistant In Plutella Xylostella And Its IPM In Hunan Province

The diamondback moth, Plutella xylostella (L.), is one of the world wide agricultural pest of cruciferous vegetables, and has developed resistance to a wide array of insecticides. The environmentally friendly insecticide crystal proteins produced by Bacillus thuringiensis (Bt) are of great scientific interest because of their potency and specificity to a wide range of insect pests. The resistance mechanism of Bt-resistance strain of P. xylostella was studied by constructing the life table to compare the biological characteristics between the resistance and susceptible Bt strains, cloning the aminopeptidase N (APN4) gene of P. xylostella, and using quantitative real-time PCR to studied gene expression of potential Bt receptor of P. xylostella. On the other hand, the Integrated Pest Management (IPM) of P. xylostella in Hunan province was proposed on the basis of the occurrence rules and resistance monitoring of P. xylostella. The main contents and conclusions are as follows:1. Exploring valid reference genes in P. xylostellaIn this study, a total of eight candidate reference genes were evaluated under various experimental conditions throughout the entire qRT-PCR workflow. Based on the comprehensive analysis, a suite of internal references are recommended to accurately normalize and quantify gene expression in P. xylostella. The results showed that under the test conditions should be selected different reference gene, compared, EF1and RPL32in most experimental conditions showed a good stability. This study not only provides a standardized procedure for quantification of gene expression in the diamondback moth, but also lays a solid foundation for the genomics and functional genomics research in this emerging insect model.2. Cloning and sequencing of Aminopeptidase N (APN4) gene from P.xylostella susceptible and resistant to CrylAc toxincDNA of aminopeptidase N (APN4) from CrylAc toxin susceptible and resistant strains of P.xylostella was cloned and sequenced. It contained a2835bp open reading frame that encoded944amino acids. The sequences had a N-terminal siganal peptide with16amino acid. GPI-Anchor sites showed that the first496amino acid glutamine (Gln) is the potential C-terminal GPI modification sites. ScanProsit showed that APN4has13N-terminal acylated sites,9tyrosine kinase2phosphorylation sites,4N-glycosylation sites,9protein kinase C phosphorylation sites and2tyrosine kinase phosphorylation sites. There was5amino acids mutation in APN4from susceptible strain of P. xylostella relative to that of resistant strain. This study provides a certain foundation for studing the system structure and function of APN4in P.xylostella.3. The gene expression analysis of Bt potential receptors of P. xylostellaThe gene expression of Bt potential receptors (cadherin (CAD), aminopeptidase N (APN) and alkaline phosphatase (ALP)) in P. xylostella was studied. The results showed that the cadherin gene expression of susceptible strain of P. xylostella were higher than resistant strain except for the1st larvae. The gene expression of APN1and APN2of susceptible strain were higher than resistant strain in the most developmental stage larvae. The gene expression of APN3of susceptible strain were lower than resistant strain except for the3rd and4th larvae, while expression of susceptible strain were higher than resistant strain in the midgut.There were no significant difference between the gene expression of APN4of susceptible and resistant strain. There were no significant difference between the gene expression of ALP of susceptible and resistant strain in different development stages, while the gene expression of susceptible strain were higher than resistan strain in the midgut. This study contributes to the determination of the Bt receptor of P. xylostella and its molecular mechanism of resistance.4. The life characteristic of resistant P. xylostella populationObjective to compare biological characteristics and population dynamics of Bt-resistant and susceptible strain of P. xylostella in the laboratory, the two sex life table was established. The results showed that the intrinsic rate of increase (r), the net reproductive rate (R0) and the the gross reproduction rateis (GRR) of susceptible strain is higher than resistant strain, while the mean length (T) of susceptible strain is shorter then Bt-resistant strain. The fitness cost of restant strain is0.78compared to the susceptible strain.This study found that Bt-resistant strain has a higher resistance to CrylAb (59.68-fold) and CrylAc (67.68-fold), lower resistance to CrylCa (3.32-fold), and no complete resistance to CrylAh (1.64-fold), respectively.This study helps to define the biological characteristics of Bt-resistant P. xylostella and the resistance of resistant strain to different Bt toxin protein.5. The occurrence rules and resistance monitoring of P. xylostella in Hunan provinceThe occurrence rules of P. xylostella in Changsha and Huaihua was ascertained with systemic investigation and field census. The results showed that the maximum occurrence of the adult of was diamondback moth in the late mid-April and the early November in Changsha and in the early May and the mid October in Huaihua, respectively.The relative susceptible toxicity baselines of P. xylostella to major insecticides in the laboratory condition were established with leaf dipping method. The Changsh and Huaihua population showed a low-level resistance to Bt, Chlorantraniliprole and Diafenthiuron, a middle-level resistance to Spinosad, Chlorfluazuron and Chlorfenapyr, and a high-level resistance to Abamectin, Cypermethrin and Indoxacarb, respectively.Studies to detect incripient resistant in the filed are very important to design effective strategies to avoid resistance development.6. The IPM of P.xylostella in Hunan provinceBased on the experiment between the yield loss and the the population density of P. xylostella, it is suggested that the economic threshold is53.65in seedling,90.84in rosette stage,67.21in early heading stage and607.51in the late period of the heading stage imported cabbage larvae on one hundred cabbages.The toxicities of emamectin benzoate, chlorfluazuron and their mixture on3rd instar larvae of P.xylostella were tested in laboratory. It was found that the synergism was the most significant when the proportion of emamectin benzoate and chlorfluazuron was1:10and the co-toxicity coefficient was154.58.Six different control systems for P. xylostella on autumn wild cabbage were established, and the population dynamics of P. xylostella, quantity of natural enemy, quality of wild cabbage and economic benefit in each control system were studied. The results showed that the effects of pest control, natural enemy protection, crop yield and crop quality in integrated control area were greater than that in other control areas, and the rate of return on investment was the best (1:17.39).The study evaluated the single system control techniques and integrated control system for the effective control of P. xylostella in Hunan Province which provide a model for IPM of P. xylostella in Hunan.