Analysis Of Controlling Effects Of Predatory Natural Enemies On Soybean Aphid By Fluorescent Quantitative PCR Technique

Soybean aphid (Aphis glycines Matsumura) is one of the more serious soybean seedling pests in recent years, a large number of chemicals used not only to soybean aphid resistance, but also destroyed the controlling action of natural enemies, so that biological control is gradually promotion and application. To conduct biological control by soybean aphid predators, first of all, to clear the dominant populations of soybean aphid enemies, but also to evaluate ability of predation and control of enemies to soybean aphid, and then making out the feasible control measures.In this study, on the basis of analysing of soybean aphid cytochrome oxidaseⅡ(COⅡ) gene sequences, the specific primers were designed. RNA of wingless soybean aphid adults used as template, DNA fragment of COⅡgene was obtain by RT-PCR, which was loaded into the plasmid pMD18-T vector by T-A cloning method. The standard was obtain through transformation and plasmid extraction, which was used to template, carried out Real-time PCR after gradienting dilution. Standard curve of detecting soybean aphid COⅡgene copies was established. The pests components in the body of predators were detected and analysed. At last, control ability of Harmonia axyridis larvae, Chrysopa septempunctata larvae, Propylaea japonica larvae and Orius similes larvae to soytean aphid was comprehensive evaluated by comprehensiving the number of collected natural enemies, the positive rate of natural enemies, the content of target pests in natural enemies and the decay rate of objective pest in intra-abdomina of the natural enemies, in order to provide theoretical evidence for biological control of soybean aphid.The results of this experiment are as follows:1) Design and synthesis of soybean aphid-specific primers and verify that the primers has no amplification to other aphid species occurred, including green peach aphid and corn aphid, Rhopalosiphum padi, Sitobion avenae and electrophoresis results showed that the specific primers could be used for quantitative PCR reaction.2) Initial detection of predation between predator and soybean aphid using PCR technology, different types of predators were collected in the field, including of Harmonia axyridis, and adults and larvae of P. japonica, adults and larvae of pallens, adults and larvae of flowers stink bug, the results show that soybean aphid gene fragments can not be fully detected in different instars, detection rate as a standard, in the same natural enemy the larvae predation has higher detection rate than the adult, in four kinds of predatory natural enemies and different instars, the predatory effect detected rate in the larvae of Propylaea japonica, Harmonia axyridis larva , Chrysopa septempunctata larvae and Orius similes adults and larvae of flowers stink bugr were 80%, 66.7%, 73.3%, 56%, 60%.3)The standard curve of soybean aphid cytochrome oxidaseⅡ(COⅡ)gene expression was successfully constructed using SYBR GreenⅠfluorescence quantitative PCR, there is good linear regression between the gene copy number and Ct value through analysis the standard curve, amplification curve and melting curve, there has no primer dimmer. So this method can be used for quantitative study.4) Detect the copy number of COⅡgene in positive individual predators tusing quantitative PCR reaction system and procedures. The results showed that Harmonia axyridis larvae's abdomen containing the largest amount of; Chrysopa septempunctata larvae; Propylaea japonica larvae; Orius similes larvae, the larvae of has the smallest amount of COⅡgene copies.5) The gene in natural enemies decay show that with delay the time of digestion, detected fragments's positive individuals gradually decreased, and the copy number of fragments in predator were decreased with the delay the time of digestion. Calculate the relevant formula between digestion time after the natural enemies feed soybean aphids and copy number, The relevant formula of Harmonia axyridis larvae, is A= -0.6517B+3.3024 (R~2=0.983); The relevant formula of Propylaea japonica larvae feeding on soybean aphid is C= -0.4824D+3.2952 (R~2 =0.965); The relevant formula of Chrysopa septempunctata larvae feeding on soybean aphid is E= -0.4715F+3.4319(R~2=0.975); The relevant formula of Orius similes larvae feeding on soybean aphids is G= -0.4464H+3.2439(R~2=0.939).6) Comprehensive consider four factors from the number of collected natural enemies, the positive rate of natural enemies, the content of target pests in natural enemies and the decay rate of objective pest in intra-abdomina of the natural enemies. Evaluate the ability of controlling pests of Harmonia axyridis larvae, P. japonica larvae, pallens flowers stink bug larvae, from the advantage natural enemies, the controlling ability from strong to weak as follows: Harmonia axyridis larvae (R=5.2290×10~6), Chrysopa septempunctata larvae (R=1.9628×10~6), Propylaea japonica larvae (R=5.7623×10~5), Orius similes larvae (R=5.4086×10~5).