Serratia marcescens with efficient insecticidal activity on lepidoptera larvae and application of serratia marcescens
By screening and applying Serratia syrhizalella Sm001 strain, the preparation of bio-drug agents solved the problem of control of Lepidoptera pests, achieved efficient, low-toxic and low-residue pest control effects, and provided environmentally friendly biological control methods.
Patent Information
- Application Number
- CN202510834619.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-20
- Publication Date
- 2025-08-29
AI Technical Summary
The existing chemical control methods have drug resistance, residue and environmental pollution problems in the prevention and control of Lepidopteran pests such as Beet Fertilizer, Cotton Bollworm, Twill Fertilizer, etc., and it is urgent to develop low-toxic, low-residue and environmentally friendly biological control methods.
A Serratia marcescens Sm001 strain was screened and preserved. Pest control was carried out by preparing bio-drug agents, including seed culture and fermentation culture. The preparation process used LB liquid culture medium with a concentration of 1×108~2×109CFU/mL, and was used in lepidopteran pests such as lepidopteran, such as lepidopteran, serratia, twill, etc.
Serratia marzipan Sm001 showed efficient insecticidal activity against lepidoptera pests. The corrected mortality rate of beet serrata within 3 days reached more than 96%, and the mortality rate of beet serrata within 3 days reached more than 80%, providing an environmentally friendly biological control strategy.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of agricultural microorganisms, and more specifically relates to a strain of Serratia marcescens having high insecticidal activity against lepidopteran larvae and its application. Background Art
[0002] Lepidopteran pests such as the beet armyworm, cotton bollworm, armyworm, and armyworm are known for their high reproductive capacity, wide-ranging diet, and adaptability to their environments. They severely damage a variety of important vegetable varieties, including cabbage, kale, and radish. Currently, chemical control is dominant. However, due to the long-term and extensive use of pesticides, the "3R" problem—resistance, resurgence, and residue—is now unavoidable.
[0003] With increasing public concern about environmental safety, food safety, and pesticide resistance, the development of new, low-toxic, low-residue, and environmentally friendly insecticides has become a pressing issue. Microbial biocontrol agents are gaining increasing attention in this field due to their environmentally friendly nature, as they are less likely to produce chemical residues or pollute the environment. The inventors of this case discovered that a strain of Serratia marcescens, belonging to the genus Serratia in the family Enterobacteriaceae, exhibited highly effective insecticidal properties, achieving a corrected mortality rate of over 96% against beet armyworms within three days. Therefore, this strain has the potential to be developed into a novel biocontrol agent for use in biological control strategies against Lepidoptera pests such as beet armyworm. Summary of the Invention
[0004] The present invention addresses the defects and shortcomings of the prior art by screening a superior strain of Serratia marcescens Sm001 that has highly effective insecticidal activity against Lepidoptera larvae, thereby enhancing pest control. This invention is thus completed.
[0005] The present invention aims to provide a Serratia marcescens strain Sm001 with highly effective insecticidal activity against Lepidoptera larvae. The strain was deposited with the China General Microbiology Center (CGMCC) on May 19, 2025, under the accession number CGMCC No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain is classified as Serratia marcescens.
[0006] The second object of the present invention is to provide the application of the Serratia marcescens Sm001 strain.
[0007] The second aspect of the present invention provides the use of the Serratia marcescens in controlling lepidopteran pests.
[0008] The third aspect of the present invention provides a biocontrol agent comprising the Serratia marcescens Sm001.
[0009] A fourth aspect of the present invention provides a method for preparing a biocontrol agent, comprising: activating Serratia marcescens Sm001; inoculating the activated Serratia marcescens Sm001 into a seed culture medium for seed culture to obtain a seed culture solution; inoculating the seed culture solution into a fermentation culture medium for fermentation culture to obtain a fermentation solution, which is the biocontrol agent.
[0010] Furthermore, the seed culture medium and fermentation medium are LB liquid culture medium; Preferably, the formula of LB liquid medium is: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, distilled water to 1 L, and pH adjusted to 7.0; Preferably, the seed culture solution is inoculated into the fermentation medium at an inoculation rate of 1% (v / v).
[0011] Furthermore, the seed culture conditions are: 37°C, 200 rpm / min, continuous culture for 8 to 10 hours; The fermentation culture conditions are: 37° C., 200 rpm / min, and continuous culture for 48 to 52 hours.
[0012] Furthermore, the concentration of the Serratia marcescens Sm001 seed culture solution is 1×10 8 ~2×10 9 CFU / mL; The concentration of the Serratia marcescens Sm001 fermentation broth was 3.36×10 9 ~3.53×10 9 CFU / mL.
[0013] A fifth aspect of the present invention provides use of the biocontrol agent, the bioinsecticide, or the biocontrol agent prepared by the preparation method in controlling lepidopteran pests such as beet armyworm and cotton bollworm.
[0014] Furthermore, when the biocontrol agent is applied, the concentration of Serratia marcescens in the biocontrol agent is 1×10 8 ~1×10 9 CFU / mL.
[0015] The present invention provides a Serratia marcescens Sm001 strain having highly effective insecticidal activity against lepidopteran larvae. Studies of the present invention have shown that the Sm001 strain has good control effects on lepidopteran pests including beet armyworm, cotton bollworm, Spodoptera litura, and armyworm. Among them, at the same dosage, the Sm001 strain has the best insecticidal effect on Spodoptera exigua, causing healthy Spodoptera exigua to turn red and die, with a corrected mortality rate of over 96% within three days. For cotton bollworm, Spodoptera litura, and armyworm, the corrected mortality rate within three days reaches over 80%, indicating that the Sm001 strain is suitable for the control of lepidopteran pests. The Serratia marcescens Sm001 strain involved in the present invention can be used as an environmentally friendly green biological control agent to replace chemical pesticides and other control agents that pose potential hazards to the environment, thereby achieving effective prevention and control of lepidopteran pests such as beet armyworm and cotton bollworm, providing a new microbial strain and control strategy for the field of biological control of agricultural pests. The strain has significant environmentally friendly characteristics, has no adverse effects on human health, and exhibits excellent biological control effects against target pests.
[0016] Biomaterial deposit information: The deposit date of the Serratia marcescens Sm001 strain is May 19, 2025. The depository unit is: China General Microbiology Culture Collection Center (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), the deposit number is CGMCC No. 34594, and the classification name is: Serratia marcescens. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a colony photo of Serratia marcescens Sm001; Figure 2 This is the 16S rDNA phylogenetic analysis of Serratia marcescens Sm001 (Note: Each branch is marked with: GenBank sequence number + strain name); Figure 3 This is a photo of a blood plate colony of Serratia marcescens Sm001; Figure 4 This is the corrected survival curve of Serratia marcescens Sm001 against beet armyworm, cotton bollworm, Spodoptera litura, and armyworm; Figure 5 The adjusted mortality rate of Serratia marcescens Sm001 suspension concentrate against beet armyworm, cotton bollworm, Spodoptera litura and armyworm. DETAILED DESCRIPTION
[0018] The present invention is described in detail below in conjunction with specific examples. The following examples help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. The experimental methods used in the following examples are conventional methods unless otherwise specified.
[0019] Example 1 The isolation and purification method for Serratia marcescens Sm001 requires all steps to be performed in a sterile environment. The specific steps are as follows: 1) Disinfect the surface of infected beet armyworm larvae with 75% ethanol; 2) Rinse three times with sterile water to remove any residual ethanol from the insect's surface; 3) Place the surface-disinfected insect body into a 1.5mL sterile centrifuge tube, add 1mL of sterile water, and grind with a grinding rod until the insect tissue is completely broken down; 4) Dilute the grinding solution 100-1000 times with sterile water, apply 100μL of the solution to a LB solid medium plate, spread evenly, and incubate at 30°C for 12 hours; 5) Pick a single colony for purification and culture to obtain a single colony. LB solid medium consists of 10g tryptone, 5g yeast extract, 10g sodium chloride, 14g agar, and distilled water to 1L, with the pH adjusted to 7.0. Blood agar plates were purchased from Qingdao Haibo Biotechnology Co., Ltd.
[0020] The morphology of the Sm001 strain is as follows Figure 1 As shown, the colony morphology is round, red, smooth, convex in the center, and opaque. The morphology of blood agar plate culture is as follows Figure 3 As shown, the colony morphology of the Sm001 strain on the blood agar plate is round and red, with a smooth and raised surface and no hemolytic ring around it, indicating that no hemolysis occurs.
[0021] The single colony was identified as Serratia marcescens using universal primers for bacterial 16S rDNA. The sequencing results were analyzed in the NCBI database, ultimately confirming the strain as Serratia marcescens, which was named Sm001. The sequence was blasted against the rRNA / ITS database in NCBI. After selecting and downloading the closely related sequences of the strain, multiple sequence alignment analysis was performed using ClustalW software. A phylogenetic tree was then constructed using the neighbor-joining method using Mega X software. The bootstrap value was adjusted and the reliability of the phylogenetic tree was verified. The results are shown in Figure 2. Figure 2 As shown, the strain had the highest similarity to Serratia marcescens E10 (AP028495), and was assigned to Serratia marcescens and named Sm001.
[0022] Example 2 The preparation method for the Serratia marcescens Sm001 bacterial suspension requires all steps to be performed in a sterile environment. The specific steps are as follows: 1) Add 30 mL of LB liquid medium to a sterile test tube; 2) Use a sterile inoculating loop to collect a small amount of seed liquid and add it to the test tube medium; 3) Incubate at 37°C, 200 rpm / min, for 10 hours, using sterile medium as a control; 4) Adjust the bacterial suspension to an OD600 of 1, or a concentration of 1.02 × 109 CFU / mL. LB solid medium consists of 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and distilled water to a volume of 1 L. The pH is adjusted to 7.0.
[0023] The insecticidal activity assays of Examples 3 and 4 were performed using the Serratia marcescens Sm001 bacterial solution prepared in Example 2 and a sterile culture medium.
[0024] Example 3 Method 1 for determining the insecticidal activity of Serratia marcescens Sm001: 1) Weigh 160 g of rabbit chow, 150 g of wheat germ flakes, 10 g of yeast powder, and 30 g of agar, mix them in a feed bucket, add 800 ml of distilled water, and stir evenly. Ferment in an incubator (37°C) for 15-20 min, then transfer to a high-pressure steam sterilizer (121°C, 20 min). Cool to approximately 45°C, add 1 g of sorbic acid, 3.8 g of paraben, and 8 g of vitamin C, stir evenly, and transfer to a feed box. After returning to room temperature, store in a cool cabinet at 4°C until use.
[0025] Completely immerse 2g of feed in freshly prepared bacterial solution for 1 minute, discard the bacterial solution, and dry aseptically at room temperature for 5 minutes; 2) Place the infected feed in an insect rearing box; 3) Place one third-instar larva of the test insect (beet armyworm, cotton bollworm, Spodoptera litura, or armyworm) in each insect rearing box as a treatment group, with a sample size of 30; 5) Using sterile culture medium as a control, treat the artificial feed in the same manner and place one third-instar larva of the test insect (beet armyworm, cotton bollworm, Spodoptera litura, or armyworm) in the insect rearing box as a control, with a sample size of 30; 6) Observe and record the growth status of the test insects for 72 hours, repeat three times, and calculate the mortality rate of different test insects in the control and treatment groups. The results are shown in Table 1. After 72 hours of feeding, the mortality rates of the control group were 2.22%, 1.11%, 0%, and 2.22%, respectively, and the mortality rates of the treatment groups were 96.67%, 84.44%, 88.89%, and 82.22%, respectively. This indicates that Serratia marcescens Sm001 has strong insecticidal activity against beet armyworm, cotton bollworm, Spodoptera litura, and armyworm in a short period of time, and the lethal time is short.
[0026] Table 1 Mortality (%) of beet armyworm, cotton bollworm, Spodoptera litura, and armyworm larvae after feeding on artificial diet containing Serratia marcescens Sm001 solution for 72 hours
[0027] Example 4 Method 2 for determining the insecticidal activity of Serratia marcescens Sm001 is as follows: The preparation methods for the artificial diet containing bacteria and the artificial diet for the sterile control group are as described in Example 3. Fifteen third-instar test insects (beet armyworm, cotton bollworm, Spodoptera litura, and armyworm) are placed in each insect rearing box. This is repeated three times. The mortality rate of the test insects in the control and treatment groups is calculated to calculate the adjusted mortality rate (adjusted mortality rate = (treatment group mortality rate - control group mortality rate) / (1 - control group mortality rate) × 100). The results are shown in Figure 2. Figure 4 As shown, the mortality rate of beet armyworm larvae after feeding on artificial diet containing Serratia marcescens Sm001 solution for 72 hours was over 90%.
[0028] Example 5
[0029] Preparation of biocontrol suspension: Activate frozen Serratia marcescens Sm001. Inoculate the activated Serratia marcescens Sm001 into 10 mL of LB liquid medium and incubate at 37°C, 200 rpm / min, for 12 hours to obtain a seed culture. Inoculate the seed culture into LB liquid medium at a 1% (v / v) inoculum and incubate at 37°C, 200 rpm / min, for 48 hours to obtain a fermentation broth. The OD value of the fermentation broth is 0.04. 600 Adjust to 1-1.5, the fermentation liquid is the biocontrol agent, and Sm001 suspension concentrate is prepared according to the formula in Table 2. When the biocontrol suspension concentrate is used to control beet armyworm, cotton bollworm, Spodoptera litura, and armyworm, the concentration of Serratia marcescens in the biocontrol suspension concentrate is 1×10 9 CFU / mL. The insecticidal activity of the biocontrol suspension was determined using the protocol of Example 3 and Example 4. The results are as follows: Figure 5 After the larvae of beet armyworm, cotton bollworm, Spodoptera litura, and armyworm fed on artificial diet soaked with biocontrol suspension for 72 hours, the corrected mortality rates were 94.44%, 83.33%, 88.88%, and 80.00%, respectively, indicating that the biocontrol suspension of Serratia marcescens Sm001 has a good insecticidal effect on the above pests.
[0030] Table 2, Serratia marcescens suspension formulation
[0031] The above embodiments are only intended to describe preferred embodiments of the present invention and are not intended to limit the scope of the present invention.
Claims
1. A Serratia marcescens, characterized in that Its deposit number is CGMCC No.34594 and the deposit date is May 19, 2025.
2. A biocontrol agent, characterized in that The biocontrol agent uses Serratia marcescens as an active component.
3. The biocontrol agent according to claim 2, characterized in that The formulation of the biocontrol agent is an oil suspension.
4. The biocontrol agent according to claim 3, characterized in that The weight percentage of the active component in the oil suspension is 5-20%.
5. The biocontrol agent according to claim 3, characterized in that The active ingredient in the oil suspension is calculated as 1×10 6 ~1×10 8 CFU / mL.
6. The use of Serratia marcescens according to claim 1, characterized in that Used to prepare microbial agents.
7. The use according to claim 5, characterized in that The dosage form of the microbial agent is an oil suspension.
8. The use of the microbial agent according to claim 6 or 7, characterized in that: Used to control lepidopteran pests.
9. The use of the microbial agent according to claim 8, characterized in that: The lepidopteran pests are selected from the group consisting of beet armyworm, cotton bollworm, Spodoptera litura or armyworm.
10. The use according to claim 9, characterized in that When the microbial agent is applied, the concentration of Serratia marcescens in the microbial agent is 1×10 8 ~1×10 9 CFU / mL.