Separation and culture method for spawn surface bacteria of spodoptera frugiperda and spawning avoiding application

Abstract

The invention relates to the technical field of spodoptera frugiperda repellent strains, in particular to a spodoptera frugiperda egg surface bacterium separation and culture method and spawning repellent application, and is characterized by comprising the following steps: S1, feeding to-be-tested insects; s2, disinfecting spodoptera frugiperda pupae; s3, culturing and collecting an egg surface microorganism fermentation solution without environmental bacteria; s4, preliminary purification and separation identification of ovum surface microorganisms; s4-1, screening and purifying the strain; s4-2, separation and identification of spodoptera frugiperda egg surface microorganisms; s5, detecting the oviposition avoiding activity of the lactobacillus; s5-1, planting the plants; s5-2, determining the avoiding activity by a spawning cage method; and S5-3, performing field simulation determination on the spawning avoiding activity. According to the invention, the condition that the lactobacillus on the spawning surface of the spodoptera frugiperda can generate obvious spawning avoiding activity on the spodoptera frugiperda under indoor and field simulation conditions is clear, the positive effect on biological control of the spodoptera frugiperda is achieved, the drug resistance of the spodoptera frugiperda to drugs can be reduced, a strategy is provided for pest control, and a certain theoretical basis is provided for biological control.

Description

Technical Field

[0001] The invention relates to the technical field of fall armyworm repellent strains, and specifically to a fall armyworm egg-surface bacteria separation and cultivation method and an egg-laying repellent application. Background Art

[0002] The fall armyworm (Spodopterafrugiperda, JSmith) belongs to the Noctuidae family of the Lepidoptera order. It is a major cross-border migratory pest that my country has paid increasing attention to in recent years, and it has seriously affected the safety of my country's agricultural production. The adults of the fall armyworm migrate and spread quickly, and the larvae have a short growth cycle and a large appetite, which makes its prevention and control difficult and requires a long prevention and control cycle. Currently, there is a bottleneck in chemical control, and the fall armyworm has developed resistance to some pesticides.

[0003] The relationship between insects and microorganisms is very delicate. Symbiotic microorganisms are closely linked to the life activities of insects, which is extremely important for the growth and development of insects and population reproduction. To ensure the subsequent growth and development of larvae, female insects rely on their sense of smell to receive volatiles released by the host to locate at a distance to check whether the host meets the conditions for laying eggs. Chemical substances released by microorganisms, plants and insects themselves play an important regulatory role in insect egg-laying behavior. Considering the diversity of bacterial niches, the study of bacterial volatiles and insect behavior will be an emerging research field with great potential.

[0004] Insect symbiotic microorganisms release a series of chemical information substances during their growth, such as microbial metabolites - volatile organic compounds, which is a way for insects to "indirectly" contact with microorganisms. Lepidoptera egg surface microorganisms play an important role in their life cycle. For example, Botrytis cinerea or Actobacter aceti can regulate the egg-laying behavior of Lobesia botrana and inhibit female insects from laying eggs.

[0005] By measuring the effect of different doses of egg surface bacteria fermentation liquid on the oviposition behavior of fall armyworm, we determined whether the egg surface bacteria of fall armyworm have an oviposition repellent effect. The repellent strain isolated from the egg surface is a sustainable resource, which can reduce the damage to natural resources and alleviate the difficulties in the development of repellent drugs caused by the scarcity of natural resources.

[0006] In view of this, it is of great significance to explore the isolation and cultivation of an active strain for the biological control of fall armyworm. Summary of the invention

[0007] The technical problem to be solved by the present invention is to solve the shortcomings of the above-mentioned existing control technology system, preliminarily separate and culture the microorganisms on the surface of the fall armyworm eggs, and isolate a total of 60 culturable strains. After 16SrDNA sequence determination and blast comparison analysis in NCBI, the strains were identified as Lactobacillus ( Lactococcuslactis ), Leuconostoc pseudomesentericum ( Leuconostocpseudomesenteroides ), Enterococcus peroxidis ( Enterococcushaemoperoxidus ). Among them, Lactobacillus ( Lactococcuslactis ) exhibits significant oviposition deterrent activity against fall armyworm at the tested concentration, which can effectively solve the problems raised in the above-mentioned background technology.

[0008] In order to solve the above problems, the technical solution adopted by the present invention is: a method for isolating and culturing bacteria on the surface of fall armyworm eggs and an application for egg-laying repellent, characterized in that it comprises the following steps: S1: raising test insects; S2: Disinfection of fall armyworm pupae; S3: Culture and collection of egg surface microbial fermentation broth without environmental bacteria; Collect the eggs laid by the fall armyworm in a sterile oviposition bottle, take 400-600 fresh eggs from different egg masses within 16-24 hours, put the eggs into 200-300 mL BHI liquid culture medium, shake and culture them in a shaker overnight, set the shaker speed to 160-200 r / min and the temperature to 25-35°C, measure the OD600 values ​​to 0.5 and 1.0 respectively by an enzyme marker, and store them in a refrigerator at 3-5°C; S4: Preliminary purification, isolation and identification of egg surface microorganisms; S4-1: strain screening and purification; Take 800-1200 fresh eggs within 16h-24h of spawning, add them to 1-3mL sterile water and shake and wash them for 1-3min. The washing solution after shaking is diluted 10 times with sterile water to 10 -3 times, draw 100 μL and spread it evenly on the BHI solid culture medium plate, and spread 100 μL of sterile water as a blank control, seal the plate and culture it in a constant temperature incubator at 25-35℃ for 36-60h; S4-2: Isolation and identification of microorganisms on the surface of fall armyworm eggs; Under sterile conditions, a single colony on the culture medium was selected, and multiple separation and purification were performed. The purified strain was inoculated into 1-3 mL of BHI liquid culture medium, and cultured at 25-35°C and 180 r / min for 20-28 hours. After culture until turbidity, bacterial-specific primers were used for strain PCR and sequencing. After 16S rDNA sequence determination and blast comparison analysis in NCBI, the culturable strains on the plate were identified as Lactobacillus, Pseudomonas pseudomesentericus, and Enterococcus peroxidase. S5: detection of egg-laying deterrent activity of lactobacilli; S5-1: Plant cultivation; S5-2: Deterrent activity assay by the oviposition cage method; S5-3: Field simulation assay of oviposition deterrent activity.

[0009] Preferably, in S1, the mated pairs of adults are placed in an egg-laying cage made of white paper and a culture dish, the paper pieces with egg masses are cut and transferred into a feeding box, which is then placed in a light incubator for hatching and feeding, during which fresh corn shoots are placed, and the larvae are fed with fresh corn shoots until they are 3 years old and are placed in a six-well cell plate for single feeding.

[0010] Preferably, in S2, the fall armyworm pupae are soaked in 75% ethanol for 30-60 seconds, rinsed with sterile water for 2-5 times, each rinse for 20-40 seconds, the last rinse solution is added to the antibiotic-free BHI solid culture medium, evenly spread, and then inverted in an incubator at 25-35°C for culture, and after 36-60 hours, check whether there are bacterial plaques to determine that the surface disinfection of the pupae is complete, and after disinfection, place them in a sterile environment for eclosion, and after mating, transfer them to a sterile oviposition bottle for egg laying, during which the adults are fed with filtered and sterilized honey water.

[0011] Preferably, in S3, the eggs laid by the fall armyworm are collected in a sterile oviposition bottle, 500 fresh eggs from 5 different egg masses within 16-24 hours are taken, the eggs are placed in 250 mL of BHI liquid culture medium, and cultured overnight in a shaker with the shaker set at a speed of 180 r / min and a temperature of 30°C. The OD600 values ​​are measured by an ELISA instrument to be 0.5 and 1.0, respectively, and the eggs are stored in a 4°C refrigerator.

[0012] Preferably, in S4-1, 1000 fresh eggs are added to 1 mL of sterile water and shaken for 1 min, and the plate is sealed and cultured in a 30° C. constant temperature incubator for 48 h; In the S4-2, the purified strain was inoculated into 1 mL of BHI liquid culture medium and cultured at 30° C. and 180 rpm for 24 h.

[0013] Preferably, in S5-1, the plant to be planted is corn. Corn seeds are taken, soaked in tap water for 6-10 hours, fished out, cleaned and planted in seedling pots covered with paper towels, 80-100 plants are planted in each pot, and all seedling pots are placed in a light culture room for culture. When the corn seedlings grow to 3 leaves and are 25-30 cm in height, seedling pots with healthy plant growth and no lodging are selected for biological testing.

[0014] Preferably, in S5-2, the egg-laying deterrent activity of the fermentation broth of lactobacillus is determined by the left-right selection method, and three doses are set in the experiment, namely 1 mL, 0.25 mL and 0.1 mL of the fermentation broth when OD600 is 1.0.

[0015] Preferably, in S5-3, 400-600 fresh eggs from different egg masses of fall armyworm within 16-24 hours of egg laying are collected in a sterile oviposition bottle, the eggs are placed in 200-300 mL BHI liquid culture medium, and shake-cultured overnight in a shaker; the shaker speed is set to 160-200 r / min and the temperature is set to 25-35°C; the OD600 values ​​are measured by an ELISA instrument to be 0.5 and 1.0, respectively, and the eggs are stored in a refrigerator at 2-6°C; In the bioassay, a total of four doses of bacterial fermentation liquid with an OD600 of 1.0 were set, namely 1 mL, 5 mL, 10 mL, and 20 mL, and the volume was uniformly fixed to 20-30 mL with sterile water. The control was sterile water or liquid culture medium BHI. Both the control and the treatment contained one thousandth volume of Tween 80. The liquid was evenly sprayed on the corn seedlings, air-dried, and placed in a bioassay cage. Two pots of corn seedlings in the treatment group and two pots of corn seedlings in the control group were placed on the left and right of the bioassay cage. 3-8 pairs of mature and first mating fall armyworm adults were placed in each bioassay. The experimental duration was 24h, 48h, and 72h, respectively. The number of egg masses and egg grains on the corn seedlings and pots were recorded. Six replicates were set for each bioassay. The directions of the treatment and control positions were swapped for each replicate. The bioassay temperature was 26℃-27℃, and the humidity was maintained at 50-70%. The egg-laying deterrent rate was used to evaluate the egg-laying deterrent effect of the fermentation liquid: ODR=(C−T) / (C+T)×100%, where C and T represent the number of eggs on untreated leaves and treated leaves, respectively. The paired sample T-test method was used for statistical analysis of experimental data.

[0016] Preferably, according to the aforementioned use of the bacteria on the egg surface of fall armyworm to repel the oviposition of fall armyworm, it is characterized in that the fermentation liquid of lactobacillus is sprayed on the plant seedlings.

[0017] Compared with the prior art, the present invention provides a method for isolating and culturing bacteria on the surface of fall armyworm eggs and an application for egg-laying prevention, which has the following beneficial effects: The present invention proposes a method for isolating and culturing bacteria on the egg surface of fall armyworm, and at the same time clarifies that a strain of lactic acid bacteria on the egg surface of fall armyworm can produce significant oviposition repellent activity against fall armyworm under conditions simulated indoors and in the field, has a positive effect on the biological control of fall armyworm, can reduce its resistance to pesticides, provides a strategy for green and efficient pest control, and provides a certain theoretical basis for technical innovation of biological control. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 a is the result of applying the rinsing liquid on the surface of fall armyworm pupae without disinfection; Figure 1 b is the result of applying the first rinse solution of sterile water to the surface of fall armyworm pupae after alcohol disinfection; Figure 1 c is the result of applying the third rinse solution of sterile water to the surface of fall armyworm pupae after alcohol disinfection; Figure 2 The egg-laying cage selection result of the egg-surface microbial fermentation liquid of the present invention; Figure 3 The oviposition selection results of female fall armyworms in the fermentation liquid of the present invention (OD600=1.0) at different concentrations and at different time points compared with the control group; Figure 4 The present invention shows the effect of the culture medium BHI on the egg-laying behavior of female fall armyworm. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0020] As a specific embodiment of the present invention: 1. Test insects and breeding methods Female fall armyworms can reach sexual maturity and begin mating two days after emergence. The peak egg-laying period is within 48 hours after mating, and artificial egg-laying is used during the egg-laying period. The specific method is: put the mated pairs of adults into an egg-laying cage made of white paper of size 10.5cm*14.85cm and a culture dish with a diameter of 10cm. When the number of egg masses is appropriate, cut the paper with egg masses and transfer them to the breeding box, put them in the light incubator for incubation and breeding, and place fresh corn shoots during the period. The conditions of the light incubator are 27℃±1℃ and the humidity is 70%±5%. The larvae are fed with fresh corn shoots until they are 3 years old and are placed in a six-well cell plate for single feeding. Corn shoots were purchased from Jinan Chengyi E-commerce Co., Ltd. During the experiment, female and male insects that mated for the first time were selected for biological testing within 72 hours.

[0021] 2. Disinfection of Fall Armyworm Pupae In order to ensure that the isolated bacteria were insect-borne bacteria, a surface disinfection experiment of fall armyworm pupae was conducted.

[0022] The whole process was performed in a clean bench. The fall armyworm pupae were soaked in 75% ethanol for 45 seconds, rinsed three times with sterile water, each rinse for 30 seconds, and the last rinse solution was added to the antibiotic-free BHI solid culture medium, evenly spread on it, and then inverted in a 30°C incubator for culture. After 48 hours, check whether there are bacterial plaques to determine that the surface disinfection of the pupae is complete. After disinfection, they are placed in a sterile environment for eclosion. After mating, they are transferred to a sterile oviposition bottle for laying eggs. During this period, the adults are fed with 10% honey water that has been filtered and sterilized.

[0023] 3. Cultivation and collection of egg surface microbial fermentation liquid without environmental bacteria 500 fresh eggs of fall armyworm within 16-24 hours of egg-laying were collected from 5 different egg masses in a sterile oviposition bottle, the eggs were placed in 250 mL of BHI liquid culture medium, and cultured overnight in a shaker with the shaker speed set at 180 r / min and the temperature at 30°C. The OD600 values ​​were measured by an enzyme marker to be 0.5 and 1.0, respectively. The eggs were stored in a 4°C refrigerator, and the fermentation broth was used for subsequent bioassays.

[0024] 4. Preliminary purification, separation and identification of egg surface microorganisms (1) Strain screening and purification Take 1000 fresh eggs within 16h-24h of spawning, add them to 1mL of sterile water and shake them for 1min. Dilute the washing solution 10 times with sterile water to 10 -3 100 μL was evenly spread on the BHI solid medium plate, and 100 μL of sterile water was spread as a blank control, with 3 replicates each. The plate was sealed and cultured in a 30°C constant temperature incubator for 48 h.

[0025] (2) Isolation and identification of microorganisms on the surface of Spodoptera frugiperda eggs Under sterile conditions, a representative single colony on the culture medium was selected and separated and purified multiple times. The purified strain was inoculated into 1 mL of BHI liquid culture medium and cultured at 30°C and 180 r / min for 24 hours. After culturing until turbidity, bacterial-specific primers were used for strain PCR and sequencing. After 16S rDNA sequence determination and blast comparison analysis in NCBI, the culturable strains on the plate were identified as Lactobacillus ( Lactococcuslactis ), Leuconostoc pseudomesentericum ( Leuconostocpseudomesenteroides ), Enterococcus peroxidis ( Enterococcushaemoperoxidus ).

[0026] 5. Detection of egg-laying deterrent activity of Lactobacillus (1) Corn planting The black glutinous corn seeds of 168 used in the biological test were purchased from Shouguang Shancheng Agricultural Technology Co., Ltd. After soaking in tap water for 8 hours, the seeds were taken out, cleaned and planted in seedling pots covered with paper towels (pot height 7 cm, pot length 32.5 cm, pot width 26 cm), with 90 plants planted in each pot. All seedling pots were placed in a light culture room with a photoperiod of L:D=16:8. When the corn seedlings grew to 3 leaves and a height of about 25-30 cm, the seedling trays with healthy plant growth and no lodging were selected for biological testing.

[0027] (2) Deterrent activity determination using the egg-laying cage method In this experiment, the left-right selection method was used to determine the oviposition deterrent activity of the fermented liquid of lactobacillus. Figure 2 shown. Figure 2 The control group CK was blank, and the treatment group CT was fermentation broth with different doses; ns means the result is not significant, and * indicates a significant difference at the probability level of P < 0.05.

[0028] The experiment set three doses, namely OD 600 The fermentation liquid was 1mL, 0.25mL, and 0.1mL when the concentration was 1.0. According to the chi-square analysis, at the 1mL bacterial liquid dosage, the treatment and the control formed a significant difference (p=0.017<0.05, df=1, c 2 =5.730); when the dosage was reduced to 0.25 mL, the number of eggs laid by the control group was significantly higher than that of the treatment group, and the female moths still showed egg-laying avoidance behavior (p=0.013<0.05, df=1, c 2 =5.730); at the lowest dose of 0.1 mL of fermentation liquid, most of the fall armyworms still laid eggs on the filter paper of the control group, and the number of eggs laid in the treatment group was significantly higher than that in the control group (p=0.017<0.05, df=1, c 2 =6.119). Under the action of three doses of bacterial fermentation liquid, female moths all showed oviposition avoidance behavior. The results showed that the strain contained metabolites that had oviposition-repellent activity on female moths, and the substances could affect the oviposition site selection of female moths.

[0029] (3) Field simulation test of egg-laying deterrent activity 500 fresh eggs of fall armyworm within 16-24 hours of egg laying were collected in a sterile oviposition bottle from 5 different egg masses, and the eggs were placed in 250 mL BHI liquid culture medium and cultured overnight in a shaker. The shaker was set at a speed of 180 r / min and a temperature of 30°C. The OD values ​​were measured by an ELISA instrument. 600 The values ​​are 0.5 and 1.0 and stored in a 4°C refrigerator.

[0030] The treatment group in the bioassay is the bacterial fermentation broth (OD600 =1.0) and set four doses, 1mL, 5mL, 10mL, and 20mL, respectively. The volume was uniformly fixed to 30mL with sterile water. The control was sterile water or liquid culture medium BHI. The control and treatment contained one thousandth volume of Tween 80. The liquid was evenly sprayed on the corn seedlings, air-dried, and placed in a test cage (100cm*50cm*55cm). Two pots of corn seedlings in the treatment group and the control group were placed on the left and right of the test cage. Five pairs of mature and first mating fall armyworm adults were placed in each test. The experimental duration was 24h, 48h, and 72h, respectively. The number of egg masses and egg grains on the corn seedlings and pots were recorded. Six replicates were set for each test. The directions of the treatment and control positions were replaced each time. The test temperature was 26℃-27℃, and the humidity was kept at about 60%.

[0031] The oviposition deterrent rate (ODR) was used to evaluate the oviposition deterrent effect of the fermentation liquid: ODR = (C-T) / (C+T) × 100%, where C and T represent the number of eggs on untreated leaves and treated leaves, respectively. The paired sample T-test method was used for statistical analysis of experimental data.

[0032] Figure 3 In the table, 24 h, 48 h, and 72 h are the number of egg-laying masses or egg grains within the egg-laying time point of Spodoptera frugiperda; ns means the result is not significant, and *, **, and *** indicate significant differences at the probability levels of P < 0.05, P < 0.001, and P < 0.0001, respectively.

[0033] At 48h and 72h, the fermentation liquid showed significant oviposition deterrent activity for female fall armyworms at the dosages of 5 mL, 10 mL, and 20 mL ( Figure 3 af).

[0034] The number of eggs was counted. Figure 3 a, b), at a dosage of 20 mL of fermentation liquid, the egg-laying avoidance rates of female moths at 24 h, 48 h, and 72 h were 85.82%, 84.92%, and 80.32%, respectively; ( Figure 3 c, d), at a dosage of 10 mL of fermentation liquid, the avoidance rates of female moths at three time points were 73.57%, 72.25%, and 73.94%, respectively; ( Figure 3 e, f), at a dosage of 5 ml of fermentation liquid, the avoidance rates at the corresponding time were 57.87%, 62.22%, and 62.65%, respectively; ( Figure 3g, h), at a dose of 1 mL, the number of egg masses in the treatment group was significantly less than that in the control group only at 72 h, and the egg-laying avoidance rates at the three time points were 22.34%, 11.81%, and 13.36%, respectively. There was no significant difference in the number of eggs between the treatment group and the control group at the three time points.

[0035] Depend on Figure 3 It can be seen that the microbial fermentation liquid on the egg surface of the fall armyworm has an oviposition-repellent effect, the minimum dose that produces significant activity is 5 mL, and the sensitivity of the fall armyworm to the oviposition-repellent activity decreases as the dose of the fermentation liquid decreases.

[0036] In 5 mL of clear fermentation broth (OD 600 =1.0) was the lowest dose of the fermentation liquid to produce egg-laying deterrent activity, and we further analyzed whether the egg-laying deterrent behavior of the fall armyworm was produced by the liquid culture medium BHI itself. 600 =1) vs. liquid medium BHI and liquid medium BHI vs. water for egg laying selection experiments, the results are shown in Figure 4 . At the same time, 5 mL of fermentation broth (OD 600 = 0.5) vs. water for spawning selection experiments. The results are shown in Figure 4 .

[0037] To verify whether the liquid medium BHI would affect the egg-laying deterrent effect of the fermentation liquid, we performed 5 mL of fermentation liquid (OD 600 =1) vs BHI medium and BHI medium vs water. The results showed that the fermentation broth (OD 600 =1.0) compared with BHI medium, it has significant egg-laying deterrent activity, and the egg-laying deterrent egg rates at 24h, 48h, and 72h are 81.93%, 72.83%, and 69.94% ( Figure 4 a, b). There was no significant difference in the number of egg masses and egg laying amount of female moths on water and culture medium. The egg laying selection rates at the three time points were 46.27%, 29.24%, and 24.42% ( Figure 4 c, d). It can be seen that liquid BHI medium has no effect on the oviposition behavior of Spodoptera frugiperda, and the components in the fermentation liquid that play an oviposition-repellent role are mainly bacterial metabolites.

[0038] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for isolating and culturing bacteria on the surface of fall armyworm eggs, It is characterized in that The steps include: S1: rearing of test insects; S2: Disinfection of fall armyworm pupae; S3: Culture and collection of egg surface microbial fermentation broth without environmental bacteria; Collect the eggs laid by the fall armyworm in a sterile oviposition bottle, take 400-600 fresh eggs from different egg masses within 16-24 hours, put the eggs into 200-300 mL BHI liquid culture medium, shake and culture them in a shaker overnight, set the shaker speed to 160-200 r / min and the temperature to 25-35°C, measure the OD600 values ​​to 0.5 and 1.0 respectively by an enzyme marker, and store them in a refrigerator at 3-5°C; S4: Preliminary purification, isolation and identification of egg surface microorganisms; S4-1: strain screening and purification; Take 800-1200 fresh eggs within 16h-24h of spawning, add them to 1-3mL sterile water and shake and wash them for 1-3min. The washing solution after shaking is diluted 10 times with sterile water to 10 -3 times, draw 100 μL and spread it evenly on the BHI solid culture medium plate, and spread 100 μL of sterile water as a blank control, seal the plate and culture it in a constant temperature incubator at 25-35℃ for 36-60h; S4-2: Isolation and identification of microorganisms on the surface of fall armyworm eggs; Under sterile conditions, a single colony on the culture medium was selected, and multiple separation and purification were performed. The purified strain was inoculated into 1-3 mL of BHI liquid culture medium, and cultured at 25-35°C and 180 r / min for 20-28 hours. After culture until turbidity, bacterial-specific primers were used for strain PCR and sequencing. After 16S rDNA sequence determination and blast comparison analysis in NCBI, the culturable strains on the plate were identified as Lactobacillus, Pseudomonas pseudomesentericus, and Enterococcus peroxidase. S5: detection of egg-laying deterrent activity of lactobacilli; S5-1: Plant cultivation; S5-2: Deterrent activity assay by the oviposition cage method; S5-3: Field simulation assay of oviposition deterrent activity.

2. The method for isolating and culturing bacteria on the surface of fall armyworm eggs according to claim 1, It is characterized in that In the S1, the mated pairs of adults are placed in an egg-laying cage made of white paper and a culture dish, the paper pieces with egg masses are cut off and transferred into a feeding box, which is then placed in a light incubator for hatching and feeding, during which fresh corn shoots are placed, and the larvae are fed with fresh corn shoots until they are 3 years old and are placed in a six-well cell plate for single feeding.

3. The method for isolating and culturing bacteria on the surface of fall armyworm eggs according to claim 1, It is characterized in that In the S2, the fall armyworm pupae are soaked in 75% ethanol for 30-60 seconds, rinsed with sterile water for 2-5 times, each rinse for 20-40 seconds, and the last rinse liquid is added to the antibiotic-free BHI solid culture medium, evenly coated and then inverted in a 25-35°C incubator for culture. After 36-60 hours, check whether there are bacterial plaques to determine that the surface disinfection of the pupae is complete. After disinfection, place them in a sterile environment for eclosion, and after mating, transfer them to a sterile oviposition bottle for egg laying. During this period, the adults are fed with filtered and sterilized honey water.

4. The method for isolating and culturing bacteria on the surface of fall armyworm eggs according to claim 1, It is characterized in that In the S3, the eggs laid by the fall armyworm are collected in a sterile oviposition bottle, and 500 fresh eggs from 5 different egg masses within 16-24 hours are taken, and the eggs are placed in 250 mL of BHI liquid culture medium, and cultured overnight in a shaker with a setting speed of 180 r / min and a temperature of 30°C. The OD600 values ​​are measured by an ELISA instrument to be 0.5 and 1.0, respectively, and the eggs are stored in a 4°C refrigerator.

5. The method for isolating and culturing bacteria on the surface of fall armyworm eggs according to claim 1, It is characterized in that In the S4-1, 1000 fresh eggs were added to 1 mL of sterile water and shaken for 1 min. The plate was sealed and cultured in a 30° C. constant temperature incubator for 48 h. In the S4-2, the purified strain was inoculated into 1 mL of BHI liquid culture medium and cultured at 30° C. and 180 rpm for 24 h.

6. The method for isolating and culturing bacteria on the surface of fall armyworm eggs according to claim 1, It is characterized in that In the S5-1, the plant to be planted is corn. Corn seeds are taken, soaked in tap water for 6-10 hours, fished out, cleaned and planted in seedling pots covered with paper towels, 80-100 plants are planted in each pot, and all seedling pots are placed in a light culture room for cultivation. When the corn seedlings grow to 3 leaves and are 25-30 cm in height, seedling pots with healthy plant growth and no lodging are selected for use in biological testing.

7. The method for isolating and culturing bacteria on the surface of fall armyworm eggs according to claim 6, It is characterized in that In S5-2, the egg-laying deterrent activity of the fermentation liquid of lactobacillus was determined by the left-right selection method, and three doses were set in the experiment, namely 1 mL, 0.25 mL and 0.1 mL of the fermentation liquid when OD600 was 1.

0.

8. The method for isolating and culturing bacteria on the surface of fall armyworm eggs according to claim 7, It is characterized in that In the S5-3, 400-600 fresh eggs from different egg masses of fall armyworm within 16-24 hours of egg laying are collected in a sterile oviposition bottle, the eggs are placed in 200-300 mL BHI liquid culture medium, and shake-cultured overnight in a shaker; the shaker speed is set to 160-200 r / min and the temperature is set to 25-35° C.; the OD600 values ​​are measured by an ELISA instrument to be 0.5 and 1.0, respectively, and the eggs are stored in a refrigerator at 2-6° C.; In the bioassay, a total of four doses of bacterial fermentation liquid with an OD600 of 1.0 were set, namely 1 mL, 5 mL, 10 mL, and 20 mL, and the volume was uniformly fixed to 20-30 mL with sterile water. The control was sterile water or liquid culture medium BHI. Both the control and the treatment contained one thousandth volume of Tween 80. The liquid was evenly sprayed on the corn seedlings, air-dried, and placed in a bioassay cage. Two pots of corn seedlings in the treatment group and two pots of corn seedlings in the control group were placed on the left and right of the bioassay cage. 3-8 pairs of mature and first mating fall armyworm adults were placed in each bioassay. The experimental duration was 24h, 48h, and 72h, respectively. The number of egg masses and egg grains on the corn seedlings and pots were recorded. Six replicates were set for each bioassay. The directions of the treatment and control positions were swapped for each replicate. The bioassay temperature was 26℃-27℃, and the humidity was maintained at 50-70%. The egg-laying deterrent rate was used to evaluate the egg-laying deterrent effect of the fermentation liquid: ODR=(C−T) / (C+T)×100%, where C and T represent the number of eggs on untreated leaves and treated leaves, respectively. The paired sample T-test method was used for statistical analysis of experimental data.

9. Use of a fall armyworm egg-surface bacterium according to any one of claims 1 to 8 to deter fall armyworm egg-laying, It is characterized in that The fermented liquid of lactobacillus is sprayed on the plant seedlings.

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