CN113832076B

CN113832076B - Bacterial strain with lepidoptera pest insecticidal activity and culture method and application thereof

Info

Publication number: CN113832076B
Application number: CN202111283970.3A
Authority: CN
Inventors: 金琳; 沈晨辉; 李志伟; 刘璐; 李国清
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2023-04-21
Anticipated expiration: 2041-11-01
Also published as: CN113832076A

Abstract

The invention discloses a bacterial strain with lepidoptera pest insecticidal activity, a culture method and application thereof, wherein the bacterial strain is enterobacter bacteria Enterobacter cancerogenus and is separated from dead larvae of lepidoptera pest cotton bollworms. The strain has high-efficiency, specific and stable insecticidal activity on lepidoptera pests, effectively kills lepidoptera larvae through stomach poisoning activity, has the advantages of low preparation cost, low environmental pollution and the like, has the potential of being developed into a biological control agent for controlling lepidoptera pests, and meets the important requirements of China on green sustainable comprehensive control of pests.

Description

Bacterial strain with lepidoptera pest insecticidal activity and culture method and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a strain with lepidoptera pest insecticidal activity, and a culture method and application thereof.

Background

Lepidoptera (Lepidoptera) is the second largest class of the class of insects, which includes a variety of agricultural pests that are extremely damaging to crop production, such as cotton bollworms, asparagus caterpillar, spodoptera frugiperda, prodenia litura, plutella xylostella, and the like. Lepidopteran pests have characteristics such as strong reproductive capacity, feeding capacity, easy diffusion, easy resistance to chemical pesticides, and the like, so that control thereof is difficult. For a long time, chemical pesticides are taken as an important means for pest control, so that various lepidoptera pests are effectively controlled, and the key effect of protecting the agricultural industry is achieved. However, chemical pesticide applications face a number of problems: for example, pest resistance caused by improper use leads to a significant reduction in the useful life of the agent; the innovative medicament for attacking the new target has long research and development period and extremely high cost, and has serious hysteresis when introduced from abroad; can pollute the environment, poison higher animals and non-target organisms and destroy ecological safety. The above drawbacks have delayed the development and field application of chemical pesticides, and thus the development of new alternatives is urgent.

Compared with the synthetic chemical pesticide, the bacterial pesticide or the transgenic insect-resistant crop developed by using natural pathogenic bacteria and mycotoxin has the characteristics of strong insecticidal specificity, environmental safety, difficult evolution resistance of the insect, low research and development cost and the like, is an important mode in the field of green prevention and control of the insect, and has great application potential. For example, the entomopathogenic bacteria bacillus thuringiensis Bt can kill various pests, and the expressed Cry1Ac insecticidal protein genes are constructed to form transgenic Bt cotton to control cotton bollworms, so that large-area planting in China for more than 20 years can create huge economic and ecological benefits for peasants. Bacterial insecticidal preparations developed based on Bt bacteria and Equilibrium brevifolium respectively occupy most of the market share of microbial insecticide in China and are recommended to be main selection agents for green prevention and control of pests.

However, since Bt cotton expressing a single insecticidal protein is used in the north China cotton area for a long period of time, cotton bollworm field populations have evolved resistance to Bt cotton, and the service life of Bt cotton is severely compromised. Meanwhile, bacterial pesticide preparations which can be used for emergency and long term in the pesticide market in China are only two types, namely Bt bacterial pesticide preparations and Equilibrium brevis pesticides, and cannot meet the requirements of disaster prevention and reduction of pests and sustainable green prevention and control. Therefore, development and utilization of pathogenic bacterial strains and strains with characteristics of high host specificity, high-efficiency insecticidal toxicity, easiness in-vitro culture and propagation and the like are one of effective measures for solving the problem of Bt resistance evolution of cotton bollworms, and the research short plates of China in the aspect of green high-efficiency bacterial insecticide can be supplemented, so that the important requirements of China on green sustainable comprehensive prevention and control of pests are met.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides the bacterial strain Haec1 with the insecticidal activity of lepidoptera pests, which is a high-efficiency, specific and stable entomopathogenic bacterial strain and can be used for carrying out green sustainable comprehensive prevention and control on lepidoptera pests such as cotton bollworms, spodoptera exigua, spodoptera frugiperda, prodenia litura, plutella xylostella and the like.

The invention also provides a culture method of the strain Haec1.

The invention also provides application of the strain Haec1 in preventing and controlling lepidoptera pests.

The invention also provides a biological control agent with the strain Haec1.

According to one aspect of the invention, a bacterial strain Haec1 with lepidopteran pest insecticidal activity is provided, wherein the preservation number of the bacterial strain Haec1 is CGMCC No.23485. The preservation unit of the strain Haec1 is China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CGMCC), and the preservation time is 2021, 9 and 26 days.

In some embodiments of the invention, the strain HaEc1 has at least one of the following features (i) - (iii): (i) is enterobacter bacteria Enterobacter cancerogenus; (ii) a cell rod shape; (iii) the cells are about 0.7 microns in diameter and about 1.9 microns in length.

In the invention, the strain is enterobacter bacteria Enterobacter cancerogenus by utilizing the phylogenetic tree analysis of the serial 16s rDNA sequence of the housekeeping gene atpD, guaA, mutM, ppsA and recA of the strain.

In the present invention, the enterobacteria Enterobacter cancerogenus are gram-negative long-rod-shaped bacteria, which are a class of bacteria widely existing in natural environments. At present, the bacteria have no pathogenicity to insects and have no report of bacteria which are separated from lepidoptera pests and have killing activity to pest larvae such as cotton bollworms, beet armyworms, spodoptera frugiperda, prodenia litura, plutella xylostella and the like.

In some embodiments of the invention, the strain HaEc1 is isolated from a lepidopteran pest, cotton bollworm, dead larva.

In some preferred embodiments of the invention, the strain HaEc1 is isolated from a three-instar larva dying from a cotton bollworm infection. In the invention, the whole larva corpse is blackened, softened and has no peculiar smell.

In some embodiments of the invention, the lepidopteran pest includes at least one of a noctuidae pest and a plutella xylostella pest.

In some preferred embodiments of the present invention, the noctuidae pest includes at least one of cotton bollworm, asparagus caterpillar, spodoptera frugiperda and prodenia litura; or the plutellaceae pest is plutella xylostella.

In some more preferred embodiments of the invention, the strain HaEc1 has a high level of stomach poisoning activity on the lepidopteran pests.

According to still another aspect of the present invention, there is provided a method for culturing the strain HaEc1 as described above, comprising the steps of: the culture was performed at a temperature of about 28℃using LB medium.

In some embodiments of the invention, the LB medium contains ampicillin.

In some preferred embodiments of the present invention, the steps specifically include: the Haec1 is diluted by sterile water and evenly coated on the surface of an LB solid culture medium, ampicillin in the culture medium avoids contamination by mixed bacteria, a flat plate is placed in a 28 ℃ incubator, and dark culture is carried out for 24 hours until a yellow-white colony with a smooth surface and a slightly protruding center is grown. In the present invention, the method is a solid culture method.

In some preferred embodiments of the present invention, the steps specifically include: adding the Haec1 bacterial suspension or colony into LB liquid culture medium, wherein the culture medium contains ampicillin to avoid contamination by mixed bacteria, and culturing the bacterial liquid in a shaking table at 28 ℃ at 200rpm under shaking for more than 12 hours until the bacterial liquid is turbid. In the present invention, the method is a liquid culture method.

According to a further aspect of the invention, the use of the strain HaEc1 as described above for controlling lepidopteran pests is proposed.

According to still another aspect of the present invention, there is provided a biocontrol agent comprising the above strain HaEc1 capable of controlling lepidopteran pests.

In some embodiments of the invention, the biocontrol agent is a bacterial suspension of strain HaEc1.

In some preferred embodiments of the invention, the concentration of the bacterial suspension ranges from 10 ⁷ -10 ⁹ cfu/mL。

In some preferred embodiments of the invention, the biocontrol agent is prepared and used in particular by diluting the bacterial suspension formed by the liquid culture method of the strain HaEc1 with sterile water, spraying on the larva feed surface, flattening the feed surface, and having a surface area of 2cm ² The corresponding bacterial suspension volume was 200. Mu.L.

In the present invention, the strain Haec1 is formed into 10 by a liquid culture method ⁹ The corrected mortality rate of the bacterial suspension with the concentration of cfu/mL, which is orally treated for 5 days on cotton bollworms, beet armyworms and spodoptera frugiperda 2-year larvae, reaches about 90 percent.

The invention has the beneficial effects that: 1) Host dependence is weak: the strain is separated from digestive tracts and haemolymph of the morbid larvae of cotton bollworms, is not found in normal larvae, can be cultured in vitro in large quantity on an artificial culture medium, and is a typical facultative pathogen and has weak dependence on hosts; 2) The stability is strong: the strain bacterial suspension can continuously cause diseases to pest larvae under the condition of no embedding protection, is not influenced by feed antibiotic environment and insect immunity environment, and has strong relative stability; 3) Stomach toxic activity: the strain causes larval septicemia and further death in an oral ingestion mode, and has good stomach toxicity activity; 4) The in vitro culture is quick and has low cost: the strain can be greatly and rapidly proliferated by using a common LB culture medium, achieves the dosage and state required by pest control, has low requirements on culture conditions such as temperature, humidity, nutrient components and the like, is easy to achieve, and has relatively low cost; 5) The insecticidal specificity is strong: the strain has good insecticidal effect on lepidoptera pests, in particular cotton bollworms, spodoptera exigua and spodoptera exigua larvae which are pests of the nocturnal moth family, and has general insecticidal effect on coleoptera pests and diptera pests and strong insecticidal specificity.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 shows the cell morphology of the strain Haec1 of example 1 under an electron microscope, wherein A is the cell morphology under a transmission electron microscope, and B is the cell morphology under a scanning electron microscope;

FIG. 2 is a phylogenetic tree of the Haec1 strain constructed by using the housekeeping gene atpD, guaA, mutM, ppsA and recA tandem 16S rRNA sequence in example 1 of the present invention;

FIG. 3 shows the LC of the strain Haec1 of example 2 of the present invention for the oral killing of 2-year larvae of cotton bollworms, asparagus caterpillar, spodoptera frugiperda, prodenia litura and plutella xylostella ₅₀ A figure;

FIG. 4 is a graph showing the oral corrected mortality rate of the biocontrol insecticidal formulation of strain Haec1 of example 4 of the present invention against 2-year larvae of Heliothis armigera, spodoptera exigua and Spodoptera frugiperda.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1: identification and culture method of strain Haec1

This example shows that a strain Haec1 of Enterobacter Enterobacter cancerogenus having stomach poisoning activity against lepidopteran pests is obtained. The strain Haec1 is preserved in China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CGMCC) with the preservation number of CGMCC No.23485 and the preservation time of 2021, 9 months and 26 days.

1. Strain acquisition pathway: the strain is obtained by separating and purifying the dead third-instar larva of cotton bollworm, and the whole larva body is blackened, softened and odorless, accords with the characteristics of bacterial infection, and is specifically separated from the digestive tract and haemolymph of the sick larva of cotton bollworm.

2. Morphological characteristics of the strain: the strain forms a single colony which is yellow and white, has smooth surface and slightly raised center on LB fixed culture medium, has a rod shape of bacterial cells, has a diameter of about 0.7 microns and a length of about 1.9 microns, grows flagellum, and has the morphological characteristics shown in figure 1, wherein A is the bacterial morphology of the strain Haec1 under a transmission electron microscope, and B is the bacterial morphology of the strain Haec1 under a scanning electron microscope.

3. Identification of the genus of the strain: the strain has housekeeping genes atpD (SEQ ID No. 1), guaA (SEQ ID No. 2), mutM (SEQ ID No. 3), ppsA (SEQ ID No. 4) and recA (SEQ ID No. 5) connected in series with 16s rDNA (SEQ ID No. 6) and other bacteria for phylogenetic tree analysis, which shows that the strain is enterobacter bacteria Enterobacter cancerogenus, named HaEc1 and the phylogenetic tree analysis is shown in figure 2.

The enterobacter Enterobacter cancerogenus strain HaEc1 in this example was cultured using the following method and procedure:

1. solid culture method: the Haec1 is diluted by using sterile water, the dilution gradient is 10 times, bacterial solutions with various concentrations are uniformly coated on the surface of an LB solid culture medium, ampicillin in the culture medium avoids other bacterial pollution, a flat plate is placed in a 28 ℃ incubator, and the culture is carried out in darkness for 24 hours, so that a yellow-white colony with a smooth surface and a slightly protruding center can be grown. Final concentration composition of LB solid medium: 10g/L of sodium chloride, 5g/L of yeast extract, 0g/L of tryptone L, 15g/L of agarose, pH7.2 and distilled water as a solvent.

2. Liquid culture method: the Haec1 bacterial suspension or colony is added into LB liquid culture medium, the culture medium contains ampicillin to avoid other bacterial pollution, and the bacterial liquid is placed in a shaking table for shaking culture for more than 12 hours until turbidity, the temperature is 28 ℃, and the rotating speed is 200rpm. LB liquid medium final concentration composition: 10g/L of sodium chloride, 5g/L of yeast extract, 0g/L of tryptone, pH7.2 and distilled water as solvent.

Example 2: insecticidal Activity of the Strain Haec1

1. The toxin coating method is adopted in the embodiment to determine the insecticidal toxicity level of the strain: the liquid culture method in the example 1 is used for obtaining a high-concentration mother solution of the bacterial suspension of Haec1, and sterile water is used for dilution, and the dilution gradient is 3 times; spraying the above materials on the surface of larva fodder, with the fodder surface being flat and the surface area being 2cm ² The volume of the bacterial suspension with corresponding concentration is 200 mu L; naturally airing and feeding lepidoptera pests such as cotton bollworms, asparagus caterpillar, spodoptera frugiperda, prodenia litura, plutella xylostella and the like into the second-instar larvae, detecting the death rate of the larvae after 5 days, and calculating to obtain the concentration of bacterial liquid which kills 50% of the larvae, namely LC ₅₀ . According to the method, LC of the strain against cotton bollworms, beet armyworms, spodoptera frugiperda, prodenia litura and plutella xylostella is determined ₅₀ Respectively 5.0 x 10 ⁷ cfu/mL、3.0×10 ⁷ cfu/mL、1.5×10 ⁸ cfu/mL、1.3×10 ⁸ cfu/mL and 5.6X10 ⁸ cfu/mL, the results are shown in Table 1 below:

TABLE 1 study of insecticidal Activity of the strain Haec1 against different species of lepidopteran pests

The results prove that the poisoning mode of the strain Haec1 on insect larvae is that the larvae die after oral ingestion to exert stomach toxicity, and the larvae die after oral ingestion to cause septicemia, so that the strain has good stomach toxicity activity. The strain Haec1 has strong insecticidal activity on lepidoptera pests such as cotton bollworms, asparagus caterpillar, spodoptera frugiperda, prodenia litura and plutella xylostella.

2. This example prepared a biocontrol insecticidal formulation based on strain HaEc1 for control of cotton bollworms, asparagus caterpillar and spodoptera frugiperda, the biocontrol insecticidal formulation obtained by the liquid culture method of example 1 at a concentration of 10 ⁹ cfu/mL of Haec1 bacterial suspension. The toxin coating method is adopted to determine the control effect of the preparation on cotton bollworms, beet armyworms and spodoptera frugiperda larvae, and the specific experimental process is as follows: spraying the bacterial suspension on the surface of larva fodder, and fodderThe surface is flat, the surface area is 2cm ² The volume of the bacterial suspension with corresponding concentration is 200 mu L; after naturally airing, feeding cotton bollworms, asparagus caterpillar and spodoptera frugiperda second-instar larvae, detecting the death rate of the larvae after 5 days, and calculating to obtain the corrected death rate of the preparation on the larvae. The corrected mortality rates of the formulations for cotton bollworms, spodoptera exigua and spodoptera exigua larvae were determined to be 97.7%, 95.6% and 89.6%, respectively, according to this method, and the results are shown in fig. 3.

The Haec1 strain single strain provided by the invention can be rapidly propagated in a large quantity in an LB culture medium, and the bacterial suspension can be applied to control of lepidoptera pests such as cotton bollworms, asparagus caterpillar, spodoptera frugiperda, prodenia litura, plutella xylostella and the like. Biological assay results of these lepidopteran pests by using a toxin coating method show that the strain can be orally taken by LC for the second-instar larvae of cotton bollworms ₅₀ Is 5.0X10 ⁷ cfu/mL; oral administration of LC to two-instar larvae of spodoptera exigua, spodoptera frugiperda, spodoptera litura and plutella xylostella ₅₀ 3.0X10 respectively ⁷ cfu/mL、1.5×10 ⁸ cfu/mL、1.3×10 ⁸ cfu/mL and 5.6X10 ⁸ cfu/mL; 1.1X10 obtained by propagation for 12 hours using LB medium ⁹ The oral corrected mortality of cfu/mL bacterial suspension on cotton bollworms, asparagus caterpillar and spodoptera frugiperda 2-year larvae reached 97.7%, 95.6% and 89.6%, respectively. The above results indicate that HaEc1 has a high level of stomach poisoning activity against at least 5 lepidopteran pests, with significant potential for development as a bacterial pesticide against lepidopteran pests.

Example 3: verification of insecticidal Activity of Strain Haec1 against different species of cotton bollworms

The application method provided by the embodiment is used for detecting the sensitivity of the cotton bollworm field population to the strain Haec1, and the strain Haec1 is proved to have good insecticidal activity to the cotton bollworm field population and important potential for developing into bacterial insecticide.

The specific implementation steps of this embodiment are as follows:

1. activation of strain HaEc 1: taking out the strain Haec1 frozen in an ultralow temperature refrigerator at the temperature of minus 80 ℃, activating by a liquid culture method, inoculating the Haec1 in an LB liquid culture medium, and placing the LB liquid culture medium in a shaking table for shake culture for 8 hours at the temperature of 28 ℃ and the rotating speed of 200rpm; LB liquid medium comprises tryptone 10g, sodium chloride 10g, yeast extract 5g, distilled water 1L, pH7.2, and sterilizing at 121deg.C for 30min.

2. Expansion culture of strain HaEc 1: and (3) taking activated Haec1 bacterial suspension, performing expansion culture by a liquid culture method, adding the suspension into LB liquid culture medium according to the ratio of 1:500, and placing the culture medium into a shaking table for shake culture for 12 hours at the temperature of 28 ℃ and the rotating speed of 200rpm.

3. Measuring the concentration of the bacterial suspension obtained after the expansion culture: colony Forming Unit (CFU) number was determined by plate colony counting, and the bacterial suspension was diluted to 3 ^-9 、3 ^-11 、3 ^-13 And uniformly coating 200 mu L of the total concentration on an LB solid culture medium, repeating each concentration for 3 times, placing the culture medium in a constant temperature incubator at 28 ℃, counting the number of colonies after the colonies grow out, and calculating the concentration of the bacterial liquid according to the formula of 'concentration of bacterial liquid = number of colonies multiplied by dilution multiplied by 5'. The LB solid medium comprises 10g of tryptone, 10g of sodium chloride, 5g of yeast extract, 15g of agarose, 1 liter of distilled water, pH value of 7.2 and sterilization at 121 ℃ for 30min.

4. Preparation of 24-well plates for bioassays: surface area per well of 2cm ² Ultraviolet sterilizing the 24-hole culture plate for 30min; weighing wheat germ powder and soybean powder which are main components of the artificial feed, mixing, fully crushing in a wall breaking machine, and preparing according to an artificial feed preparation method; before the feed is cooled to about 60 ℃ and is not solidified, 1mL of feed is added into each hole of a 24-hole culture plate by a liquid transfer device, the surface of each hole of feed is flattened by beating, and the prepared culture plate is stored at 4 ℃ for standby after being cooled at room temperature.

5. Preparing a bacterial strain Haec1 bacterial solution concentration gradient solution: diluting the Haec1 bacteria suspension mother liquid with sterilized 0.01M PBS solution or sterile water, respectively diluting 3 times as dilution gradient ^-1 、3 ^-2 、3 ^-3 、3 ^-4 、3 ^-5 And 3 ^-6 Multiple 6 concentrations; each concentration was sprayed onto 2 24-well plates, 200 μl per well, with plates sprayed with PBS solution or sterile water as controls; naturally airing at room temperature, and taking care to avoid separation of the feed and the hole wall of the culture plate.

6. Starvation treatment for test insects in advance: picking cotton bollworm sensitive strain with consistent body size and cotton bollworm field population 2-year initial larvae collected in different areas of cotton areas of China north China in the same year by using tweezers, starving the cotton bollworm field population 2-year initial larvae in an empty box for 4 hours, and covering black cloth to prevent escape.

7. Feeding and treating the test insects: the hunger-treated healthy larvae are quickly pulled into 24-hole culture plates by a writing brush, 1 head of larvae is added into each hole, black cloth and a cover are added to prevent the larvae from escaping, the plates are covered by rubber bands, labels are written, and the healthy larvae are put into a non-light incubator at 26 ℃ for 5 days.

8. And (3) detecting mortality rate of test insects: larval death was detected, and either complete larval death or weight less than 5mg (growth severely inhibited) was considered dead, and the number of larval deaths at each concentration treatment was recorded and mortality calculated.

9. LC of strain Haec1 on different populations of Heliothis armigera was calculated using PoLoPlus software ₅₀ Numerical values, 95% confidence limits, and virulence regression curve slopes.

The results of the above experiments were: the bioassay results show that the sensitivity of the cotton bollworm field population to the strain HaEc1 of the present invention is shown in table 2:

TABLE 2 sensitivity studies of different groups of cotton bollworms on the strain Haec1

The strain Haec1 has high-level insecticidal activity on different field populations of cotton bollworms, and the concentration of bacterial liquid capable of causing 50% mortality of larvae of the field populations is lower than 1 multiplied by 10 ⁹ cfu/mL. The sensitivity of the Hebei gallery, shandong Xiajin and Anhui Wangjiang field population to the Haec1 strain is not significantly different from that of the indoor sensitive strain SCD. The sensitivity of Henan Kaifeng and Jiangsu salt city field population to Haec1 is significantly lower than that of the sensitive strain, but the concentration is 10 ⁹ The cfu/mL Haec1 bacterial liquid can effectively kill almost all larvae of the two populations, and shows high insecticidal toxicity. Therefore, the strain Haec1 provided by the invention has good insecticidal activity on different field populations of cotton bollworms, and has the characteristics of high development ratePotential of potent bacterial pesticides.

Example 4: insecticidal activity verification of strain Haec1 on different lepidopteran pests

The biological control insecticidal preparation based on the strain Haec1 and the application method provided by the invention are used for detecting the sensitivity of different lepidoptera pests to the strain Haec1, so that the strain Haec1 has good insecticidal activity to lepidoptera pests and has important potential for developing bacterial insecticide for preventing and controlling lepidoptera pests.

The specific implementation steps of this embodiment are as follows:

1. activation of strain HaEc 1: taking out the strain Haec1 frozen in the ultralow temperature refrigerator at-80 ℃, activating by a liquid culture method, inoculating the Haec1 in an LB liquid culture medium, and placing the LB liquid culture medium in a shaking table for shake culture for 8 hours at the temperature of 28 ℃ and the rotating speed of 200rpm.

2. Expansion culture of strain HaEc 1: and (3) taking activated Haec1 bacterial suspension, performing expansion culture by a liquid culture method, adding the suspension into LB liquid culture medium according to the ratio of 1:500, and performing shaking culture for 12 hours by a shaking table to form bacterial suspension, wherein the temperature is 28 ℃, and the rotating speed is 200rpm.

3. Preparation of 24-well plates for bioassays: surface area per well of 2cm ² Ultraviolet sterilizing the 24-hole culture plate for 30min; respectively weighing the main components of the corresponding artificial feed of cotton bollworms, beet armyworms, spodoptera frugiperda, prodenia litura and plutella xylostella, fully crushing in a wall breaking machine, and preparing according to an artificial feed preparation method; before the feed is not solidified, 1mL of feed is added into each hole of a 24-hole culture plate by a liquid transfer device, the surface of each hole of feed is flattened by beating, and the prepared culture plate is cooled at room temperature and then stored at 4 ℃ for standby.

4. Spraying an insecticidal preparation: spraying the suspension of the Haec1 bacteria onto 2 culture plates with 24 holes, wherein 200 mu L of each hole is used as a control, and the culture plates sprayed with PBS solution or sterile water are arranged; naturally airing at room temperature, and taking care to avoid separation of the feed and the hole wall of the culture plate.

5. Starvation treatment for test insects in advance: picking initial larvae of test insects with consistent body sizes from tweezers, starving the initial larvae into an empty box for 4 hours, and covering black cloths to prevent escape.

6. Feeding and treating the test insects: the hunger-treated healthy larvae are quickly pulled into 24-hole culture plates by a writing brush, 1 head of larvae is added into each hole, black cloth and a cover are added to prevent the larvae from escaping, the plates are covered by rubber bands, labels are written, and the healthy larvae are put into a non-light incubator at 26 ℃ for 5 days.

7. And (3) detecting mortality rate of test insects: larval death was detected, and either complete larval death or weight less than 5mg (growth severely inhibited) was considered dead, and the number of larval deaths at each concentration treatment was recorded and corrected mortality was calculated.

The bioassay results show that the corrected mortality of the biocontrol insecticidal formulation based on the strain HaEc1 of this example on spodoptera exigua, spodoptera frugiperda, spodoptera litura and plutella xylostella sensitive strain larvae is shown in fig. 4. The Haec1 biocontrol agent used in this example had a concentration of 8.9X10 ⁸ cfu/mL, the preparation has higher insecticidal activity on various lepidopteran pest larvae. The activity of the larva against spodoptera exigua and spodoptera exigua is highest, and the larva correction mortality is 92% and 90% respectively; the corrected mortality rates of the prodenia litura and the plutella xylostella larvae are 75% and 50% respectively, which shows that the preparation can be used for developing bacterial insecticidal preparations aiming at lepidoptera nocturnal moth pests.

The results of the above examples show that the strain of the invention has important potential for developing efficient, specific and low-cost bacterial pesticides, is helpful for solving the problem of Bt resistance evolution of lepidoptera pests, and supplements the research short plates of China in the aspects of green and efficient biological control agents and bacterial pesticides, thereby meeting the important requirements of China on green sustainable comprehensive control of pests.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims

1. Bacterial strain with lepidoptera pest insecticidal activityHaec1, characterized in that said strain Haec1 is enterobacter oncogenic @Enterobacter cancerogenus) The preservation number is CGMCC No.23485.

2. A method for culturing the strain Haec1 as defined in claim 1, comprising the steps of: the culture was performed at 28℃using LB medium.

3. Use of the strain HaEc1 according to claim 1 for controlling lepidopteran pests.

4. A biocontrol agent against lepidopteran pests, characterized in that it contains the strain HaEc1 according to claim 1.

5. The biocontrol agent of claim 4, wherein said biocontrol agent is a bacterial suspension of strain HaEc1.

6. The biocontrol agent of claim 5, wherein said bacterial suspension is at a concentration in the range of 10 ⁷ -10 ⁹ cfu/mL。