Preliminary Study On The Insecticidal Mechanism Of Serratia Marcescens PS-1 Strains Against Spodoptera Exigua Larvae

Beet armyworms, Spodoptera exigua Hübner, belonging to the spodoptera littoralis genus, noctuidae, Lepidoptera, are polyphagous agricultural pests spreading all over the world. They mainly harm the vegetable and have a strong drug resistance, found to be resistant to many pesticides（such as organophosphorus, pyrethroid pesticides, carbamate insecticides and insect growth regulators） and Bt preparations. Therefore, it is an urgent need to find a novel biological agent to prevent and control beet armyworms.Serratia marcescens PS-1 strains（hereinafter referred to as PS-1 strains） are new pathogenic bacteria obtained from larvae of Phyllotreta striolata through separation, and have a significant inhibition effect on the growth, development and survival of the larvae of beet armyworms; and the supernatants of the bacterial culture have a strong insecticidal activity for the beet armyworms. To clarify the insecticidal mechanism of the PS-1 strains, this paper conducted the physiological and biochemical test on the PS-1 strains, observed the symptoms of the larvae of the beet armyworms after being infected with PS-1, and studied the pathological changes of the midgut tissues of infected larvae of the beet armyworms; and measured the influence of the PS-1 strains on the intestinal symbiotic bacteria of the beet armyworms and on the activity of digestive enzyme, so as to further illustrate the insecticidal mechanism of the PS-1 strains, provide a new method for the biological control on beet armyworms and provide basis for the research and development and field application of the PS-1 strains. The main results are summarized as follows:1. Physiological-biochemical characteristics of PS-1 strainsThe PS-1 strains growth quickly in the LB fluid media with constant temperature at 28℃and rotation speed at 160r/min; they enter the logarithmic phase about 4 h later, then enter the stable phase in the period from 20 h to 32 h and reach the maximum growth value, and finally enter the decline phase 32 h later. The physiological-biochemical test indicates that PS-1 strains are gram-negative bacteria which can utilize glucose, mannitol and inositolbut cannot utilizesorbitol, rhamnose and arabinose, and the utilization effect: ismannitol>glucose> inositol.The PS-1 strains do not generate hemolysis, the M.R. test is negative, the H₂O₂ test generates gases, and both the lipolytic activity test and the indole testare positive, the antibiotic susceptibility test indicates that the PS-1 strains are sensitive tochloramphenicol, spectinomycin and tetracycline, and from big to small, the sequence of the diameter of the inhibition zone is spectinomycin> chloramphenicol> tetracycline; and the PS-1 strains are insensitive to ampicillin.2. Damage of PS-1 strains to intestinal cell and organelle structure of larvae of beet armywormsThe influence of the PS-1 strains on the structure of intestinal cells in the midgut tract of larvae of the beet armyworms was observed by using the microtomy and themicrotechnique. The result indicated that the PS-1 strains can damage the intestinal wall cells of the midgut of larvae of the beet armyworms, causing the intestinal wall cells to deform or even fall in the gut cavity, and the damage effect was strengthened along with the extension of the treatment time. Furthermore, the influence on the intestinal tract organelle of the larvae of the beet armyworms already subject to the PS-1 effect was observed with the transmission electron microscopy method. The result showed that the cellular microvilli of the infected larvae of the beet armyworms were disordered in arrangement and some even fell,the mitochondria and the nucleoli had lesion, partial organelle disappeared, and there were cavities in the cells.3. Influence of PS-1 strains on composition of intestinal symbiotic bacteria of beet armywormsAfter the larvae of the beet armyworms were fed with feeds containing PS-1 bacterial suspension（2.0×108cfu/g） and the intestinal cultivable symbiotic bacteria in the larvae were subjected to isolated culture, three kinds of symbiotic bacteriawere identified, namely Klebsiella aerogenes, bacillus subtilis and staphylococcus aureus; the types and composition of the intestinal symbiotic bacteria in the beet armyworms were identified through a high-throughput sequencing method, with 314 OUT copolymerized and 7 phyla, 11 classes,38 ordered, 50 families and 71 genera annotated, among which thedominant bacterial community mainly included the bacteria of the firmicutes and the proteobacteria, accounting for respectively 90.05% and 9.65%. In terms of the genusclassification, providencia is an advantageous genus, accounting for 30.85%. The top fifteen symbiotic bacteria with high abundance include providencia, acinetobacter, escherichia, veillonella, wolbachia,sphingomonas, pseudomonas, stenotrophomonas, neisseria, enterococcus, staphylococcus,lactobacillus, croynebacterium, mycoplasma and streptococcus in sequence. Compared with the control group, the abundance of thesymbiotic bacteria in the midguts of the larvae ofthe beet armyworms treated with the PS-1 bacterium suspension changed greatly,with providencia increased significantly, the stenotrophomonas reduced significantly and the acinetobacter also increased. The analysis result showed that the PS-1 strains remarkably influenced the composition of the intestinal symbiotic bacteria of larvae of the beet armyworms.4. Inhibition effect of PS-1 strains on activity of digestive enzymes in beet armyworm intestinal tractsThe influence of the PS-1 strains on the activity of amylase and protein enzymes in the midgets of larvae of the beet armyworms was determined by combining the metabolicenzymology method. The result showed that the PS-1 strains had an obvious inhibition effect on the activity of amylase in the midgets of larvae of the beet armyworms, with no significant difference even when being subjected to different concentration treatment. The PS-1 strains also had the inhibition effect on the activity of protein enzymes, which was obviously enhanced along with the increase of the treating concentration. The inhibition effect on theactivity of the protein enzymes in the midgets of larvae of the beet armyworms was the best, with the inhibition ratio reaching 44.42%, when the PS-1 strains with 2.0×108cfu/g concentration were used.