CN111607570A

CN111607570A - Novel oleander hornworm cytoplasmic polyhedrosis virus and application thereof

Info

Publication number: CN111607570A
Application number: CN202010509406.8A
Authority: CN
Inventors: 靳亮; 占智高; 关丽梅; 王金昌; 况文东; 刘卓荣; 黄朝
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: 2020-09-01
Anticipated expiration: 2040-06-03
Also published as: CN111607570B

Abstract

The invention discloses a novel nerium oleander hornworm virus strain, which is characterized in that on the basis of nerium oleander hornworm virus V201730, a large number of chemical mutagenesis experiments are carried out to screen a novel nerium oleander hornworm virus with broad-spectrum insecticidal activity and strong toxicity, and the culture names (classified names) are 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.

Description

Novel oleander hornworm cytoplasmic polyhedrosis virus and application thereof

Technical Field

The invention relates to a novel nerium oleander hornworm cytoplasmic polyhedrosis virus and application thereof, belonging to the technical field of microorganisms.

Background

The oleander hornworm (Daphnis nerii (Linnaeus)), also named as Pinna farina, hawk moth, oleander hornworm, belongs to Lepidoptera (Lepidotera), and Hypogaeae (Sphingideae), is a worldwide pest and is domestically distributed in Guangdong, Guangxi, Taiwan, Fujian, Jiangxi, Yunnan, Hunan, Sichuan and other provinces. The nerium oleander is mainly harmful to nerium oleander, and also harmful to rauvolfia vomitoria (Rauvdfiasp.) of medicinal plants of rauvolfia (Rauvdfiasp.) and other tree species in the family, and plants of apocynaceae such as allamanda cathartica, nerium oleander and catharanthus roseus.

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 bean moth nucleopolyhedrosis viruses.

In 2015, 10 months, we found dead bodies of oleander hawkmoth larvae naturally dead in oleander forests in an Exi lake wetland park near the academy of sciences in Jiangxi. And grinding the dead bodies, centrifuging and purifying, and infecting the neriidae again to obtain more dead bodies with virus-like death symptoms. After sample purification and electron microscope section observation again, the pathogen is judged to be a polyhedrosis virus in the first step. Extracting a genome from the separated and purified sample, and identifying the separated and purified sample through electrophoresis, wherein the electrophoresis pattern of the sample is in accordance with the characteristics of the cytoplasmic polyhedrosis, but the genome band of the sample is different from 22 kinds of electrophoretic polyhedrosis viruses found at present; the genomic cDNA of CPV was cloned by using FLAC (full Length Amplification of cDNAs) technique, and the sequencing work of the genomic S2 fragment and S10 fragment was completed. Sequencing results show that the CPV genome S2 fragment encodes RNA polymerase, and the S10 fragment encodes polyhedrin. The two terminal conserved sequences are the same, and the terminal conserved sequence 5 'AGUCAAA. AGC 3' exists at the 5 'end and the 3' end of the plus strand respectively. Evolutionary analysis based on the amino acid sequences of RNA polymerase and polyhedrin showed that this polyhedrosis virus has a close relationship with type 19 and type 5 polyhedrosis viruses, but there is a clear difference in electrophoretic patterns. Therefore, the virus is presumed to be a new type of CPV which is not reported at present and is tentatively named as the Nerium oleander polyhedrosis virus NanChang strain (Daphnis nerii 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 the 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 bean hornworm, and is basically not found to have application with a good effect on other insects.

Disclosure of Invention

The invention aims to provide a novel nerium oleander hornworm virus, and the name of a culture (classified name) is as follows: the nandromous virus DnCPV-2 with the preservation number: CCTCC NO of V201927, which has broader spectrum and stronger insecticidal activity.

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, date of receipt (date of preservation): 2019.4.29, which has a broader spectrum, more potent insecticidal 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 disease occurs, the larvae have abnormal actions, the appetite is gradually lost, the body surface is softened finally, but the larvae dieThe worm bodies do not liquefy.

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

A novel oleander hornworm virus, culture name (taxonomic nomenclature): the nandromous virus DnCPV-2 with the 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.

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.

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

FIG. 1 is a comparison of the bodies of infected virions and normal worms, with normal worms listed above and those infected with the virus DnCPV-2 listed below.

FIG. 2 is a linear plot of the virulence assay of example 2.

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.

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 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 bean hawkmoth

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 × 10²PIBs/mL、1×10³PIBs/mL、1×10⁴PIBs/mL、1×10⁵PIBs/mL、 1×10⁶PIBs/mL、1×10⁷PIBs/mL、1×10⁸PIBs/mL are ready for use.

The same batch of healthy three-year-old bean hawkmoths are taken and cultured in a laboratory in groups, and after three days, the three-year-old bean hawkmoths are still healthy and serve as test insect bodies. Starvation was carried out for 12 hours before the experiment, and the negative control bean hawkmoths were fed with sterile water-treated leaves, 10 bean hawkmoths per group, and 5 treatment groups were repeated.

Picking up the three-instar bean hawkmoth into a breeding box, respectively soaking fresh bean leaves into the polyhedron suspension with each concentration for 1 minute, and putting the bean leaves after air drying. 5 treated bean leaves were placed in each box, and the negative control was the 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 factors were recorded. The experiment was carried out for 8 days, and the results are shown in table 1 below:

TABLE 1 toxicity test results of different concentrations of Nerium indicum Homopoliae virus Nanchang strain DnCPV-2 on Dolichee

As can be seen from Table 1: the nandromous virus DnCPV-2 has obvious toxicity to the bean hawkmoth, when the concentration of the DnCPV-2 is 10³The mortality rate reaches 54 percent in 5 days and the corrected mortality rate is 51.06 percent at the concentration of 10 when PIB/mL is adopted⁸The 7-day mortality reached 100% at PIB/mL, with a corrected mortality of 100%.

The test method comprises the following steps: mortality and corrected mortality were calculated, a regression equation for virulence was developed and LC50 values were calculated. If the control mortality is greater than 10%, the test is considered invalid. The calculation formula is as follows: the mortality rate (%) — the number of live insects before treatment-the number of live insects after treatment/the number of live insects before treatment × 100; corrected mortality (%) - (treatment-control mortality)/(100-control mortality) × 100; solving a toxicity regression equation: y ═ b + aX, R values, and LC50 values.

From the line graph (FIG. 2), we know that the virulence equation of this virus strain DnCPV-2 is: Y0.123X +0.1317, R²0.9553, LC50 value of 10³

(2) Toxicity determination of oleander hawkmoth virus V201730 on bean hawkmoth

The titer was 1 × 10¹¹Stock solution of PIBs/mL Nerium indicum Lee Virus Nanchang strain V201730 with dilution concentration of 1 × 10²PIBs/mL、1×10³PIBs/mL、1×10⁴PIBs/mL、1×10⁵PIBs/mL、 1×10⁶PIBs/mL、1×10⁷PIBs/mL、1×10⁸PIBs/mL are ready for use.

Picking up the three-instar bean hawkmoth into a breeding box, respectively soaking fresh bean leaves into the polyhedron suspension with each concentration for 1 minute, and putting the bean leaves after air drying. 5 treated bean leaves were placed in each box, and the negative control was the 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 factors were recorded. The experiment was carried out for 8 days, and the results are shown in Table 2 below.

TABLE 2 toxicity test results of different concentrations of the Adenophora Nebrodensis virus Nanchang strain V201730 on Bean hawkmoth

It is known that the nandromous virus Nanchang strain V201730 has low toxicity to the bean hawkmoths.

Example 3

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

The titer was 1 × 10¹¹PIBs/mL Nerium indicumThe stock solution of virus Nanchang strain DnCPV-2 is diluted to the concentration of 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 3:

TABLE 3

Therefore, the high-concentration nandrolimus disease virus Nanchang strain DnCPV-2 also has obvious toxicity to the camphor nest borer.

(2) Toxicity determination of oleander hawkmoth virus V201730 on moths

The titer was 1 × 10¹¹Stock solution of PIBs/mL oleander hornworm virus V201730 with the dilution concentration of 1 × 10⁷PIBs/mL、1×10⁸PIBs/mL are ready for use.

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:

TABLE 4

Therefore, the toxicity of the high-concentration nandromous disease virus Nanchang strain V201730 on the camphor nest borer is not high and is obviously lower than that of the nandromous disease virus Nanchang strain DnCPV-2.

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. It is speculated that it may be that the receptor binding protein has been mutated such that the spectrum of disease is increased. In addition, virulence production may also be altered, and thus it is more virulent.

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. An oleander hornworm virus, which is characterized in that: named as the nanchang strain DnCPV-2 of the nereisenia similis virus with the preservation number: CCTCC NO: V201927.

2. Use of the oleander hornworm virus of claim 1 as an insecticide.

3. A pesticide comprising the Nanchang strain DnCPV-2 of Nerium indicum Tenebrio Virus of claim.

4. A pesticide according to claim 3, characterized in that it further comprises an adjuvant.

5. The insecticide of claim 3 or 4, which is used to kill bean hawkmoth.

6. The insecticide of claim 3 or 4, which is used to kill Antrodia camphorata.