CN111549004A

CN111549004A - Composite biocontrol bacterium agent of masson pine caterpillar cytoplasmic polyhedrosis virus DpCPV and beauveria bassiana and application thereof

Info

Publication number: CN111549004A
Application number: CN202010492015.XA
Authority: CN
Inventors: 靳亮; 王金昌; 关丽梅; 占智高; 况文东; 黄朝; 梁小文
Original assignee: INSTITUTE OF MICROBIOLOGY JIANGXI ACADEMY OF SCIENCES; Jiangnan University
Current assignee: INSTITUTE OF MICROBIOLOGY JIANGXI ACADEMY OF SCIENCES
Priority date: 2020-06-03
Filing date: 2020-06-03
Publication date: 2020-08-18
Anticipated expiration: 2040-06-03
Also published as: CN111549004B

Abstract

The invention discloses a composite biocontrol microbial inoculum of a dendrolimus pinus massoniana cytoplasmic polyhedrosis virus DpCPV and beauveria bassiana and application thereof, and discloses a composite biocontrol microbial inoculum of a nangchang virus Nanchang strain DnCPV-2 and beauveria bassiana, which is characterized by comprising effective active ingredients of a virus suspension of the Nanchang strain DnCPV-2 of the dendrolimus pinus cytoplasmic polyhedrosis virus, a virus suspension of a CCTCC NO (V201607) of the dendrolimus pinus cytoplasmic polyhedrosis virus and a Beauveria bassiana CCTCC NO. 7998. The beauveria bassiana CCTCC NO.7998 can further enhance the toxicity of the nanchang strain DnCPV-2 of the nereisenia similis virus, has good killing effect on various insects, and particularly has good effect on the moths of the moths and the corn borers.

Description

Composite biocontrol bacterium agent of masson pine caterpillar cytoplasmic polyhedrosis virus DpCPV and beauveria bassiana and application thereof

Technical Field

The invention relates to a composite biological control microbial preparation of Dendrolimus massoniana cytoplasmic polyhedrosis virus DpCPV, oleander hawkmoth virus Nanchang strain DnCPV-2 and beauveria bassiana, belonging to the technical field of microorganisms.

Background

The insect virus pesticide has strong specificity, and has the common advantages of high efficiency, strong selectivity, greenness, safety, low chemical residue, high decomposition speed and other biological pesticides in the pest control process. At present, the more studied insect viruses of the family Gelidae are the nuclear polyhedrosis viruses of the corn borer.

In 10 months of 2015, we found dead bodies of Nerium oleander larvae in Nerium oleander forest in the wetland park of Exi lake, academy of sciences of Jiangxi, speculated that the virus is a new CPV type which is not reported at present and is tentatively named as Nerii polyhedrosis virus Nanchang (Daphnis Cypovirus-NanChang, DnCPV-NC for short). The prior application of the applicant has proposed an oleander moth virus, which is preserved in China center for type culture Collection with the preservation number: v201730, namely virus DnCPV-NC. In practical use, the strain is found to have a good killing effect only on nerium oleander hornworm, has a poor effect on corn borer, and is basically not found to have good effect on other insects.

The Dendrolimus Punctatus Cytoplasmic Polyhedrosis Virus (DPCPV) is a pathogeny of pine caterpillar, a major forest pest in China, and has a good control effect on pine caterpillar. In 1989, Belloncik S researches find that Cytoplasmic Polyhedrosis Virus (CPV) infects insect midgut epithelial cells, and the method has the outstanding characteristics of no toxicity and harm to the environment, good continuous infection effect and easy pest epidemic disease. Research by Zhao Tonghai et al (2004) finds that DpCPV has a wide host range and can infect and attack 36 lepidopteran insects in 10 families. The prevention and control principle is as follows: pine moth cytoplasmic polyhedrosis virus is released in forests, and large-area epidemic disease of pine moth is caused by feeding of pine moth, which finally leads to mass death. Generally, the infection rate of the current generation is more than 70 percent, thereby greatly reducing the population density and promoting the virtuous cycle of ecology. In addition, the virus also has the effects of horizontal and vertical transmission, does not pollute the environment, does not kill natural enemies, and can play a role in continuous prevention and control, thereby not only greatly reducing the prevention and control cost, but also increasing the benefit of wood, simultaneously reducing the using amount of chemical pesticides and improving the ecological environment of pine forest.

Beaueria bassiana (Balsampo) Vuillemin) is an entomopathogenic fungus, is used as a living biological pesticide, and is propagated in a large amount in a host insect body to change the function of body fluid and cause metabolic disturbance to death. Beauveria bassiana is nontoxic, tasteless and pollution-free, and is widely used for preventing and controlling pests in agriculture and forestry.

The invention aims to provide a composite biocontrol microbial preparation of an oleander hawkmoth virus Nanchang strain DnCPV-2, a Dendrolimus Punctatus Cypovirus (DPCPV) and beauveria bassiana, wherein three microorganisms have a synergistic effect and have a good killing effect on various insects.

Disclosure of Invention

The invention aims to provide a composite biocontrol microbial preparation of an oleander hawkmoth virus Nanchang strain DnCPV-2, Dendrolimus Punctatus Cytoplasmic Polyhedrosis Virus (DPCPV) and beauveria bassiana, wherein three microorganisms have a synergistic effect and have a good killing effect on various insects.

The nandromous virus DnCPV-2 has a preservation number: CCTCC NO: V201927, which has broader spectrum of insecticidal activity, and the date of harvest (storage date): 2019.4.29.

the pine moth cytoplasmic polyhedrosis virus strain is preserved in China center for type culture Collection at 10 months and 15 days 2014, and the preservation number is as follows: CCTCC NO (V201607); beauveria bassiana is preserved in China center for type culture Collection in 2013, 8 months and 22 days, and the preservation numbers are as follows: CCTCC NO. 7998; . Both strains have been disclosed in the patent documents of the prior application.

Based on the oleander hornworm virus V201730 disclosed in the prior application, a large number of chemical mutagenesis experiments are carried out, and finally a novel oleander hornworm virus with broad-spectrum insecticidal activity and strong toxicity is screened, wherein the culture name (classified name) is as follows: the nandromous virus DnCPV-2 with the preservation number: CCTCC NO: V201927, date of receipt (date of preservation): 2019.4.29, which has a broader spectrum of pesticidal activity.

We utilized the concentration: 1*10⁶The DnCPV-2 of the PIB/ML infects 3-4 d-age neriidae larvae, the symptoms of the 3-4 d-age neriidae larvae are similar to those of the neriidae virus V201730, no obvious external characteristics are shown in the initial stage, the larvae have symptoms after 3-4 days, the appetite of the larvae is reduced, the larvae grow slowly, the body color of the larvae gradually changes into grey brown after the onset of diseases, the actions are abnormal, the appetite is gradually lost, and finally the body surface is softened, but the body of the dead larvae is not liquefied.

The DnCPV-2 virus is subjected to electron microscope negative staining observation and electron microscope section observation, the shape of the DnCPV-2 virus is similar to that of the original virus namely oleander hawkmoth virus V201730, the virus is hexagonal or pentagonal mostly in polyhedron, and the diameter is about 1.8-3.2 mu m; hexagonal and nearly circular virus particles (including whole virus particles and empty-shell virus particles) can be observed from the polygonal ultra-thin slices, the diameter is between 65 and 83nm, and the structural size of the polygonal ultra-thin slices is similar to the size of the known masson pine moth cytoplasmic polyhedrosis virus and is similar to the oleander moth virus V201730.

The camphor tree is a predominant tree in Jiangxi, namely the camphor nest borer Orthaga achatina, and is an insect in the lepidoptera, namely the Bombycidae and Convallaria nubilalis. Is an important pest on camphor trees, and takes place for 2-3 generations in 1 year. Distributed in the east China. The host comprises Cinnamomum camphora and fructus Piperis. Taking camphor tree leaves as larvae. Feeding leaves for 1-2 instar larvae, spinning and conjugating twigs and leaves for 3-5 instar larvae to form a bird nest-like insect nest. Some whole leaves are almost eaten, and the growth of the camphor trees is seriously influenced.

The corn borer, also called corn borer, belongs to lepidoptera, snout moth's moth family, and the corn borer generated in China has two kinds of corn borers in Asia and European, mainly harms corn, sorghum, millet and the like, can also be harmed crops such as cotton, sugarcane, hemp, sunflower, rice, beet, sugarcane beans and the like, and belongs to worldwide pests.

The invention discovers that the nanchang strain DnCPV-2 of the nereistraria piniperi virus, the cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) of the pine moth and the beauveria bassiana CCTCC NO.7998 have synergistic effect, have good killing effect on various insects, and particularly have good effect on the antrodia camphorata borer and the corn borer.

The technical problem to be solved by the invention can be realized by the following technical scheme.

A composite biological antimicrobial preparation of an oleander hawkmoth virus Nanchang strain DnCPV-2, a pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) and beauveria bassiana is characterized by comprising effective active ingredients of an oleander hawkmoth virus Nanchang strain DnCPV-2 virus suspension, a pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) virus suspension and a beauveria bassiana CCTCC NO. 7998.

The nandromous virus DnCPV-2 has the special preservation number: CCTCC NO: V201927.

The DnCPV-2 has stronger toxicity to nerium oleander hornworm, and in addition, has toxicity to camphora nest borer, can kill various harmful insects once, and has greater progress in effect compared with the DnCPV-1. Beauveria bassiana CCTCC NO.7998, and pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) can further enhance the toxicity of the nanchang strain DnCPV-2 of the nereistra oleander virus.

The preservation unit of the invention is as follows: china center for type culture Collection;

the address of the preservation unit of the invention is as follows: wuhan university school of eight-channel 299 # in Wuchang area of Wuhan city, Hubei province.

The invention has the advantages that:

the high-value strain DnCPV-2 is obtained by mutagenesis and screening, has stronger toxicity to neriidae indocalamus, has toxicity to antrodia camphorata moth, can kill various harmful insects at one time, and has great improvement in effect compared with DnCPV-1. The beauveria bassiana CCTCC NO.7998 and the pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) can further enhance the toxicity of the nanchang strain DnCPV-2 of the nereisria oleander virus, have good killing effect on various insects, and particularly have good effect on the moths and the corn borers.

Detailed Description

The following examples of the present invention are described in detail, and are only for the purpose of illustrating the present invention and are not to be construed as limiting the present invention.

Specific examples of the present invention are described below.

Drawings

The figure is a comparison of the bodies of the infected virus and normal worms, the normal worms are listed above, and the bodies infected with the virus DnCPV-2 are listed below.

Example 1

Based on the nerium oleander sky moth virus V201730, a large number of chemical mutagenesis experiments are carried out, and finally a novel nerium oleander sky moth virus with broad-spectrum insecticidal activity and strong toxicity is screened, and the culture name (classified name) is as follows: the nandromous virus DnCPV-2 with the preservation number: CCTCC NO: V201927, which has broader insecticidal activity.

The obtained novel oleander hornworm virus obtained by mutagenesis has the following culture names (classified names): the nandromous virus DnCPV-2 with the preservation number: CCTCC NO: V201927, date of receipt (date of preservation): 2019.4.29.

Example 2

(1) Toxicity determination of oleander hawkmoth virus Nanchang strain DnCPV-2 on camphor nest borer

The titer was 1 × 10¹¹The stock solution of the Nanchang strain DnCPV-2 of the oleander hawkmoth virus of PIBs/mL is diluted to the concentration of 1 × 10³PIBs/mL、1×10⁴PIBs/mL、1×10⁷PIBs/mL、1×10⁸PIBs/mL are ready for use.

Negative control worms were fed with normal LB broth medium, and the artificial infection experiments were performed on healthy antrodia camphorata nestling, respectively, and the experiments were repeated 3 times. The mothball nest moths of the same batch are taken and cultured in a laboratory in groups, and after three days, the mothballs are still healthy and serve as tested worms. The camphor leaves are respectively immersed into the polyhedron suspension with each concentration for 1 minute, and the camphor leaves are put into the polyhedron suspension after air drying. The test insects were allowed to feed naturally (equivalent to inoculation of infection via the digestive tract). The morbidity or mortality is regularly observed and recorded every day after inoculation, the infected morbidity and mortality show the morbidity characteristics of the naturally diseased (dead) antrodia camphorata moth, the original infectious bacteria can be separated and recovered from dead insect bodies to serve as the judgment index of the pathogenic effect, and meanwhile, an experimental control only inoculated with a sterile common LB broth culture medium is established. And (4) checking for 7-10 times according to survival conditions of different batches of test insects, and recording the number of dead insects, wherein the results are shown in the following table.

The death rate (%) < dead insect number/test insect number x 100; the toxicity of DnCPV-2 to the Antrodia camphorata Walker and the infection test results of the Antrodia camphorata Walker are shown in the following table 1:

TABLE 1

Therefore, the high-concentration nandina peach hornworm virus Nanchang strain DnCPV-2 also has obvious toxicity to the camphor nest borer, and the killing effect is poor at low concentration.

(2) Determination of toxicity of beauveria bassiana CCTCC NO.7998 to mothball

Inoculating Beauveria bassiana CCTCC NO.7998 to 5mL of common LB broth culture medium, culturing at 37 ℃ for 12h with a shaker (180rpm) to activate strains, taking 1mL of activated strain suspension, inoculating to 250mL of LB broth culture medium, culturing at 28 ℃ with a shaker (180rpm) for 16h, and determining the concentration of the strain to be 1 × 10 by a colony counting method⁷cfu/ml and 1 × 10⁸cfu/ml for use.

Infection test of Antrodia camphorata: negative control worms were fed with normal LB broth medium, and the artificial infection experiments were performed on healthy antrodia camphorata and the experiments were repeated 3 times, respectively. The same batch of the moths is taken and cultured in a laboratory in groups, and after three days, the moths are still healthy and used as tested worms. Immersing camphor leaves into the bacterial suspensions with different concentrations for 1 minute; the test insects were allowed to feed naturally (equivalent to inoculation of infection via the digestive tract). The morbidity or mortality is regularly observed and recorded every day after inoculation, the infected morbidity and mortality show the morbidity characteristics of the naturally diseased (dead) antrodia camphorata moth, the original infectious bacteria can be separated and recovered from dead insect bodies to serve as the judgment index of the pathogenic effect, and meanwhile, an experimental control only inoculated with a sterile common LB broth culture medium is established. And (4) checking for 7-10 times according to survival conditions of different batches of test insects, and recording the number of dead insects, wherein the results are shown in the following table.

The death rate (%) < dead insect number/test insect number x 100; the toxicity of beauveria bassiana CCTCC NO.7998 to the Antrodia camphorata nest borer and the infection test result of the Antrodia camphorata nest borer are shown in the following table 2:

TABLE 2

It can be seen that beauveria bassiana CCTCC NO.7998 has low toxicity to the antrodia camphorata.

(3) Toxicity determination of pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) on camphor moth borer

The titer was 1 × 10¹¹PIBs/mL stock solution of Dendrolimus Punctatus cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) with dilution concentration of 1 × 10³PIBs/mL、1×10⁴PIBs/mL are ready for use.

The death rate (%) < dead insect number/test insect number x 100; the toxicity of the pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) to the moths, and the infection test results of the moths are shown in the following table 3:

TABLE 3

It can be seen that the pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) has low toxicity to the Antrodia camphorata.

(4) Toxicity determination of composite biocontrol agent on mothball

The titer was 1 × 10¹¹The stock solution of the Nanchang strain DnCPV-2 of the oleander hawkmoth virus of PIBs/mL is diluted to the concentration of 1 × 10³PIBs/mL、1×10⁴PIBs/mL are ready for use.

Low concentration group 1 × 10³PIBs/mL Nerium indicum virus Nanchang strain DnCPV-2 virus suspension and 1 × 10⁷cfu/ml beauveria bassiana CCTCC NO.7998 suspension, 1 × 10³PIBs/mL pine moth cytoplasmic polyhedrosis virus strain CCTCCNO (V201607) virus suspension is mixed.

High concentration group 1 × 10⁴PIBs/mL Nerium indicum virus Nanchang strain DnCPV-2 virus suspension and 1 × 10⁸cfu/ml beauveria bassiana CCTCC NO.7998 suspension, 1 × 10⁴PIBs/mL pine moth cytoplasmic polyhedrosis virus strain CCTCCNO (V201607) virus suspension is mixed.

Infection test of Antrodia camphorata: negative control worms were fed with normal LB broth medium, and the artificial infection experiments were performed on healthy antrodia camphorata nestling, respectively, and the experiments were repeated 3 times. The camphor nest borers of the same batch are taken and cultured in a laboratory in groups, and after three days, the camphor nest borers are still healthy and serve as tested insects. The camphor leaves are respectively immersed into the mixed suspension with each concentration for 1 minute, so that the test insects feed naturally (equivalent to inoculation infection through the digestive tract). The morbidity or mortality is regularly observed and recorded every day after inoculation, the infected morbidity and mortality show the morbidity characteristics of the naturally diseased (dead) antrodia camphorata moth, the original infectious bacteria can be separated and recovered from dead insect bodies to serve as a judgment index of the pathogenic effect, and meanwhile, an experimental control only inoculated with a sterile common LB broth culture medium is established. And (4) checking for 7-10 times according to the survival conditions of the test insects in different batches, and recording the number of dead insects, wherein the results are shown in the table.

The death rate (%) < dead insect number/test insect number x 100; the toxicity of the oleander hawkmoth virus V201730 on the moths, and the infection test results of the moths are shown in the following table 4:

therefore, after the compound, the toxicity of the DnCPV-2 virus suspension to the mothball at low concentration can be obviously increased by the Beauveria bassiana CCTCC NO. 7998.

Example 3:

(1) toxicity determination of oleander hawkmoth virus Nanchang strain DnCPV-2 on corn borer

The titer was 1 × 10¹¹The stock solution of the Nanchang strain DnCPV-2 of the oleander hawkmoth virus of PIBs/mL is diluted to the concentration of 1 × 10⁷PIBs/mL、1×10⁸PIBs/mL are ready for use.

The same batch of healthy 5d old corn borers are taken and cultured in a laboratory in groups, and after three days, the corn borers are still healthy and serve as test insect bodies. Starvation treatment is carried out for 12 hours before experiments, negative control corn borers are fed by sterile water treatment leaves, 10 corn borers are added in each group, and each treatment is repeated by 5 groups.

Picking the three-instar corn borers into a feeding box, respectively soaking the fresh corn kernels into the polyhedron suspension with each concentration for 1 minute, and putting the corn borers after air drying. 5 treated corn kernels were placed in each box, and the negative control was leaf treated with sterile water. Feeding in insect-culturing room at 27 + -1 deg.C, relative humidity of 70% -80%, and illumination time of 16L-8D. After 24h, all the leaves were replaced with fresh virus-free leaves, cleaned and observed once a day, and the number of live insects, typical diseased deaths and other factor deaths were recorded. The experiment was carried out for 8 days, and the results are shown in table 5 below:

TABLE 5 toxicity test results of different concentrations of the Nerium indicum Hemsl Virus Nanchang strain DnCPV-2 on Zea mays

As can be seen from Table 1: the nandromous virus DnCPV-2 has no obvious toxicity to corn borer.

(2) Determination of toxicity of beauveria bassiana CCTCC NO.7998 to corn borer

Inoculating Beauveria bassiana CCTCCNO.7998 in 5mL of common LB broth culture medium, culturing at 37 deg.C with shaker (180rpm) for 12h to activate strain, taking 1mL from activated strain suspension, inoculating in 250mL of LB broth culture medium, culturing at 28 deg.C with shaker (180rpm) for 16h, and determining the concentration of strain to be 1 × 10 by colony counting method⁷cfu/ml and 1 × 10⁸cfu/ml for use.

The same batch of healthy three-year old corn borers are taken and cultured in a laboratory in groups, and after three days, the corn borers are still healthy and serve as test insect bodies. Starvation treatment is carried out for 12 hours before experiments, negative control corn borers are fed by sterile water treatment leaves, 10 corn borers are added in each group, and each treatment is repeated by 5 groups.

Picking the three-instar corn borers into a feeding box, respectively immersing fresh corn kernels into the bacterial suspensions with various concentrations for 1 minute, and putting the corn kernels after air drying. 5 treated corn kernels were placed in each box, and the negative control was leaf treated with sterile water. Feeding in insect-raising room at 27 + -1 deg.C, relative humidity of 70% -80%, and illumination time of 16L-8D. After 24h, all leaves were replaced with fresh virus-free leaves, cleaned and observed once a day, and the number of live insects, typical diseased deaths, and other factor deaths were recorded. The experiment was carried out for 8 days, and the results are shown in Table 6 below.

TABLE 6 determination of toxicity of Beauveria bassiana CCTCC NO.7998 on Zea mays Guenee at different concentrations

As can be seen, different concentrations of beauveria bassiana CCTCC NO.7998 have obvious toxicity to the corn borer.

(3) Toxicity determination of pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) on corn borer

The titer was 1 × 10¹¹PIBs/mL pine moth cytoplasmic polyhedraStock solution of the virus strain CCTCC NO (V201607) with the dilution concentration of 1 × 10⁷PIBs/mL、1×10⁸PIBs/mL are ready for use.

TABLE 7 toxicity test results of different concentrations of Dendrolimus punctatus cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) to Zea mays

As can be seen from Table 1: the nandromous virus Nanchang strain DnCPV-2 has slight toxicity to corn borer.

(4) Toxicity determination of composite biocontrol agent on mothball

Picking the three-instar corn borers into a feeding box, respectively immersing the fresh corn kernels into the mixed suspension with each concentration for 1 minute, and putting the corn kernels after air drying. 5 treated corn kernels were placed in each box, and the negative control was leaf treated with sterile water. Feeding in insect-raising room at 27 + -1 deg.C, relative humidity of 70% -80%, and illumination time of 16L-8D. After 24h, all leaves were replaced with fresh virus-free leaves, cleaned and observed once a day, and the number of live insects, typical diseased deaths, and other factor deaths were recorded. The experiment was carried out for 8 days, and the results are shown in Table 8 below.

The death rate (%) < dead insect number/test insect number x 100;

TABLE 8

Therefore, after compounding, the toxicity of the beauveria bassiana CCTCC NO.7998 to the corn borer can be obviously increased by the DnCPV-2 and the pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607). The nandromous disease virus DnCPV-2 has synergistic effect with Beauveria bassiana CCTCC NO.7998 and pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607), has good killing effect on various insects, and especially has good effect on the moths and the corn borers. The concept is considered that bacteria and viruses have a synergistic interaction in terms of bactericidal virulence, which may be associated with efficient colonization and infection of insects, and may also be caused by secretory changes such as certain cytokines during the pathogenic process, and more likely to act together.

It should be understood, however, that the foregoing description is only a preferred embodiment of the invention,

variations that do not depart from the gist of the invention are intended to be within the scope of the invention.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A complex microbial inoculant, which comprises:

the effective active ingredients comprise suspension of Nanchang strain DnCPV-2 virus of the Nerium indicum Hemsl moth virus, suspension of pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) virus, and Beauveria bassiana CCTCC NO. 7998.

2. The method for preparing the complex microbial inoculum of claim 1, which is characterized in that:

the titer was 1 × 10¹¹The stock solution of the Nanchang strain DnCPV-2 of the oleander hawkmoth virus of PIBs/mL is diluted to the concentration of 1 × 10³PIBs/mL、1×10⁴PIBs/mL is reserved;

inoculating Beauveria bassiana CCTCC NO.7998 to 5mL of common LB broth culture medium, culturing for 12h at 37 ℃ in a shaking table (180rpm) for strain activation, taking 1mL from the activated strain suspension, inoculating to 250mL of LB liquid culture medium, culturing for 16h at 28 ℃ in a shaking table (180rpm), and determining the concentration of the strain to be 1 × 10 by adopting a colony counting method⁷cfu/ml and 1 × 10⁸The cfu/ml concentration is reserved;

the titer was 1 × 10¹¹PIBs/mL stock solution of Dendrolimus Punctatus cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) with dilution concentration of 1 × 10³PIBs/mL、1×10⁴PIBs/mL is reserved;

low concentration bacterial preparation 1 × 10³PIBs/mL Nerium indicum virus Nanchang strain DnCPV-2 virus suspension and 1 × 10⁷cfu/ml beauveria bassiana CCTCC NO.7998 suspension, 1 × 10³Mixing PIBs/mL pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) virus suspensions;

high-concentration bacterial preparation prepared from 1 × 10⁴PIBs/mL Nerium indicum virus Nanchang strain DnCPV-2 virus suspension and 1 × 10⁸cfu/ml beauveria bassiana CCTCC NO.7998 suspension, 1 × 10⁴PIBs/mL pine moth cytoplasmic polyhedrosis virus strain CCTCC NO (V201607) virus suspension is mixed.

3. The use of the complex microbial agent of claim 1 as a pesticide.

4. The complex microbial inoculant of claim 1, which is used for killing corn borers.

5. The complex microbial inoculant according to claim 1, which is used for killing antrodia camphorata.