Beauveria bassiana with strong pathogenicity to elm leaf beetles, fungicide and application of beauveria bassiana

Abstract

The invention belongs to the technical field of biocontrol preparations, and particularly relates to beauveria bassiana with high pathogenicity to elm leaf beetles, a fungicide and application of the beauveria bassiana. The beauveria bassiana is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2024, and the preservation number is CGMCC No.41634. The beauveria bassiana is named as Beauveria bassiana. The determination of the lethal concentration and the required time of the beauveria bassiana on the schizophrassica juncea and the schizophrassica juncea in a room shows that the strain has strong pathogenicity on the schizophrassica juncea and the schizophrassica juncea, and can be used for preparing a biocontrol preparation for preventing and treating insect pests caused by the schizophrassica juncea; the invention provides a new resource for biological prevention and control of the elm leaf beetles, and provides a theoretical basis for development and utilization of high-toxicity strains.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control preparations, and particularly relates to a Beauveria bassiana with strong pathogenicity to elm leaf beetles, a bacterial agent and application thereof. Background Art

[0002] Elm is one of the important native tree species in northern China. It is highly adaptable, easy to plant, and resistant to pruning. It is often used in garden landscape greening. Pyrrhalta aenescens (Fairmaire), also known as elm blue leaf beetle, elm hairy breast leaf beetle, elm green hairy leaf beetle, and elm green golden flower beetle, belongs to the Chrysomelidae family of the Coleoptera order. It mainly harms elm plants such as white elm Ulmus pumila Linnaeus, weeping elm Ulmus pumila Linnaeus cv. Tenue, and golden leaf elm Ulmus pumilacv. jinye. It is one of the main pests in urban gardens. Both adults and larvae can feed on leaves. In severe cases, the crown of the tree turns yellow and the entire plant leaves are not intact. When mature larvae gather on the main trunk to pupate, they often secrete liquid, which causes fungal parasitism and causes the branches and trunks to turn black, seriously damaging the urban greening landscape. In addition, during the adult period in July, they often fly into residents' homes in large numbers, affecting the lives of urban residents.

[0003] Pyrrhalta maculicollis (Motschulsky), also known as the elm spotted neck firefly leaf beetle, elm yellow leaf beetle, and black-horned hairy breast firefly leaf beetle, belongs to the family Chrysomelidae in the order Coleoptera and is also a major harm to elm trees. The newly hatched larvae gather around the egg shell to feed on the mesophyll, and then disperse to cause damage, chewing the elm leaves to leave only the veins. In severe cases, the entire tree crown turns yellow.

[0004] At present, the research related to the control of elm leaf beetles mainly includes: (1) using natural enemies for control. The population of Oenopia conglobata (Linnaeus), Aiolocaria mirabills (Motschulsky), Arma chinesis (Fallou) and Tetrastichus gallerucae (Fonscolombe) are controlled. In recent years, although there has been great progress in the research on the control of elm leaf beetles with natural enemies, there is still a lack of systematic research and it has not been widely promoted in practical applications. (2) Using microsporidia for control. In 1987, Wen Jinzeng et al. discovered a microsporidia in the adult of elm green hairy leaf beetles. The infection rate of overwintering adults can reach 96%. After indoor test, it was found that it has great control potential, but it has not been applied yet. (3) The use of chemical pesticides such as cypermethrin, chlorfenapyr, emamectin benzoate, chlorpyrifos, phoxim, and furadan to control elm leaf beetles is still the first choice. The drugs used to control elm leaf beetles are mainly nicotinoid insecticides, and the dosage and concentration of the drugs are relatively high. Therefore, it is increasingly urgent to timely understand the sensitivity level of elm leaf beetle populations to insecticides, slow down the development of their resistance, and find new effective alternatives. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Beauveria bassiana with strong pathogenicity to elm leaf beetles, a fungal agent and application thereof, so as to solve the above technical problems.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In the first aspect, the present invention provides a strain of Beauveria bassiana that is highly pathogenic to elm leaf beetles. The Beauveria bassiana was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 20, 2024, with a deposit number of CGMCC No.41634.

[0008] The present invention determines the indoor toxicity of the Beauveria bassiana to populations of green-haired elm leaf beetles and yellow-haired elm leaf beetles and the forest control effect. The results show that the Beauveria bassiana of the present invention has obvious treatment effects on all ages of the two types of elm leaf beetles, and has a good forest control effect, which can provide a reference for the biological control of elm leaf beetles in garden production.

[0009] In a second aspect, the present invention provides a use of the Beauveria bassiana in the preparation of a biocontrol agent, wherein the biocontrol agent controls insect pests caused by elm leaf beetles.

[0010] Furthermore, the elm leaf beetles include green elm leaf beetles and yellow elm leaf beetles.

[0011] In a third aspect, the present invention provides a bacterial agent containing the Beauveria bassiana.

[0012] Furthermore, the bacterial agent is the fermentation culture solution or freeze-dried powder of the Beauveria bassiana.

[0013] In a fourth aspect, the present invention provides the use of the bacterial agent in the preparation of a biocontrol agent, wherein the biocontrol agent controls insect pests caused by elm leaf beetles, which include elm green-haired firefly leaf beetles and elm yellow-haired firefly leaf beetles.

[0014] In a fifth aspect, the present invention provides a biocontrol agent, comprising the Beauveria bassiana described above, or the bacterial agent described above.

[0015] Furthermore, the biocontrol agent also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the auxiliary material includes a surfactant.

[0017] Furthermore, the surfactant includes Tween-80.

[0018] The present invention has the following beneficial effects:

[0019] The Beauveria bassiana of the present invention can effectively reduce the damage of the green-haired elm leaf beetle and the yellow-haired elm leaf beetle to elm trees. Indoor toxicity determination found that the pupal stage is the most difficult insect stage to control. They come down from the tree to pupate and their bodies are covered with hard pupal shells. The LD50 of the pupal stage is 1.08×10 7 / mL and 1.99×10 7 The lower the larval age, the worse the drug resistance and the easier it is to be infected by Beauveria bassiana. The LD50 of 1st to 3rd instar larvae is 1.27×10 6 / mL, 1.30×10 6 / mL, 1.37×10 6 / mL and 1.55×10 6 / mL, 1.64×10 6 / mL, 1.98×10 6 / mL. The LD50 of the egg stage is lower than that of the first-instar larvae, which are 1.19×10 6 / mL and 1.39×10 6 / mL, and the LD50 of the adult stage was equivalent to that of the third-instar larvae, which were 1.35×10 6 / mL and 1.29×10 6 Pieces / mL.

[0020] The control effect in the field is visible: about 3 days after the application of Beauveria bassiana to the elm green hairy firefly leaf beetle, the insect population began to decrease significantly, and the control effects from low concentration to high concentration were 0.3446, 0.6976 and 0.9303, respectively. The control effects after 5 days of treatment were 0.8156, 0.9176 and 0.9303, respectively. About 3 days after the application of Beauveria bassiana to the elm yellow hairy firefly leaf beetle, the insect population began to decrease significantly, and the control effects from low concentration to high concentration were 0.3420, 0.6534 and 0.8151, respectively. The control effects after 5 days of treatment were 0.8514, 0.9134 and 0.9306, respectively.

[0021] The invention provides a new resource for biological control of elm leaf beetles and a theoretical basis for the development and utilization of highly toxic strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the growth of mycelium after Beauveria bassiana was used to treat the elm green-haired firefly leaf beetle.

[0023] Figure 2 These are the lethal effects of Beauveria bassiana on different insect stages of the green firefly leaf beetle, among which A is the effect of Beauveria bassiana on the egg stage of the green firefly leaf beetle, B is the effect of Beauveria bassiana on the 1st instar larvae of the green firefly leaf beetle, C is the effect of Beauveria bassiana on the 2nd instar larvae of the green firefly leaf beetle, D is the effect of Beauveria bassiana on the 3rd instar larvae of the green firefly leaf beetle, E is the effect of Beauveria bassiana on the pupal stage of the green firefly leaf beetle, and F is the effect of Beauveria bassiana on the adults of the green firefly leaf beetle.

[0024] Figure 3 These are the lethal effects of Beauveria bassiana on different insect stages of the elm yellow hairy leaf beetle, among which A is the effect of Beauveria bassiana on the egg stage of the elm yellow hairy leaf beetle, B is the effect of Beauveria bassiana on the first instar larvae of the elm yellow hairy leaf beetle, C is the effect of Beauveria bassiana on the second instar larvae of the elm yellow hairy leaf beetle, D is the effect of Beauveria bassiana on the third instar larvae of the elm yellow hairy leaf beetle, E is the effect of Beauveria bassiana on the pupal stage of the elm yellow hairy leaf beetle, and F is the effect of Beauveria bassiana on the adults of the elm yellow hairy leaf beetle.

[0025] Figure 4 is the effect of Beauveria bassiana on the tropism of adults of the green-haired elm leaf beetle, where A is 1.0×10 6 The concentration of spore suspension was 1.0×10 / mL, and B was 1.0×10 7 The concentration of spore suspension was 1.0×10 / mL, and C was 1.0×10 8 The concentration of spores / mL was used for treatment.

[0026] Figure 5 is the effect of Beauveria bassiana on the tropism of adults of the yellow-haired leaf beetle, A is 1.0×10 6 The concentration of spore suspension was 1.0×10 / mL, and B was 1.0×107 The concentration of spore suspension was 1.0×10 / mL, and C was 1.0×10 8 The concentration of spores / mL was used for treatment. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0028] Example 1: Isolation and identification of Beauveria bassiana

[0029] The tested Beauveria bassiana strain was isolated from the larvae of the infected elm green-haired firefly leaf beetle, from Shenhe District, Shenyang City, Liaoning Province. The plate streak separation method was used to isolate single colonies using PDA medium. Select the colonies with fast-growing and dense mycelium, and then use the plate dilution method to spread to obtain a single colony, then streak, and repeatedly isolate and purify the strain three times, and transfer it to a plate for culture at 25-28°C for use. After one week of culture, the strain was examined under a microscope, and after systematic evolutionary analysis and comparison of the microscopic morphology of hyphae, conidiophores and conidia, it was identified as Beauveria bassiana. The strain is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.41634 and the deposit date of November 20, 2024.

[0030] Example 2: Indoor toxicity assay

[0031] 1. Experimental Materials

[0032] Test medium: PDA medium was used to separate, purify and culture the strains. PDA medium contained 200 g potato, 20 g glucose, 20 g agar and 1000 mL water.

[0033] Test insects: The tested elm leaf beetles and elm leaf beetles were collected from the Botanical Garden of Shenyang Agricultural University in Shenyang, Liaoning Province. The 2nd and 3rd instar larvae were collected from the forest stands where the elm leaf beetles were seriously infested; the collected larvae were placed in insect cages and fed with fresh elm leaves every day; larvae with basically the same size and strong activity were selected for bioassay.

[0034] 2. Experimental methods

[0035] The toxicity of the strains to elm leaf beetles was determined by the insect immersion method. Different Beauveria bassiana strains were cultured on PDA plates for 15 days, and the spores were washed off with sterile water containing 0.1% Tween-80. The concentration of the spore suspension was adjusted to 1.0×10 6 / mL, 1.0×107 / mL and 1.0×10 8 / mL; immerse the larvae in the spore suspension for about 5s, take them out, use sterile filter paper to absorb the moisture on the surface of the insect body, put them in a plastic insect box (10.8cm×10.8cm×7.5cm) covered with moist sterile filter paper, put fresh young leaves in the box, and cover the lid; put the insect box in a constant temperature incubator at 25℃ and relative humidity of 90.

[0036] Each treatment was repeated three times, with 15 beetles in each repeat. The treatment immersed in sterile water was used as the control (CK). The number of larval deaths and dead beetles of elm leaf beetles was observed and recorded every day, and the corrected mortality of larvae, dead beetle rate on the 7th day and LD50 were calculated.

[0037] Mortality rate (%) = number of dead larvae / total number of larvae × 100%.

[0038] Corrected mortality rate (%) = (mortality rate of each treatment - mortality rate of the control) / (1 - mortality rate of the control) × 100%.

[0039] Dead insect rate (%) = number of dead insects / total number of larvae × 100%.

[0040] 3. Experimental results

[0041] Beauveria bassiana has a significant effect on all ages of elm leaf beetles. White hyphae can be seen on the surface of the insects around 7 days after treatment. Figure 1 ).

[0042] Green-haired leaf beetle Figure 2 ): Dead insects began to appear on the 4th day after the egg treatment; on the 7th day after the treatment, the insect population reduction rates from low concentration to high concentration were 46.67%, 53.33%, and 73.33%, respectively, and no death occurred in the control. The LD50 of the egg stage was 1.19×10 6 / mL. On the second day of treatment of 1st instar larvae, 1.0×10 8 The dead insects began to appear in the group with 10 / mL. On the 7th day of treatment, the insect population reduction rates from low concentration to high concentration were 46.67%, 53.33%, and 73.33%, respectively. The LD50 of the first-instar larvae was 1.27×10 6 / mL. On the 4th day after treatment with the 2nd instar larvae, dead insects began to appear in the treatment group; on the 7th day after treatment, the insect population reduction rates from low concentration to high concentration were 73.33%, 93.33%, and 93.33%, respectively. The LD50 of the 2nd instar larvae was 1.30×10 6 / mL. On the 5th day after treatment, dead insects began to appear in the 3rd instar larvae. On the 7th day after treatment, the insect population reduction rates from low concentration to high concentration were 47.78%, 93.33%, and 87.78%, respectively. The LD50 of the 3rd instar larvae was 1.37×106 / mL. On the 7th day of pupae treatment, the concentration of the suspension was 1.0×10 8 / mL, dead insects began to appear; on the 7th day of treatment, the insect population reduction rates from low concentration to high concentration were 0.00%, 6.67%, and 40.00%, respectively, and the LD50 of the pupal stage was 1.08×10 7 / mL. On the 4th day of treatment with adults, the concentration of the suspension was 1.0×10 8 / mL, dead insects began to appear; on the 7th day of treatment, the insect population reduction rate from low concentration to high concentration was 100.00%, and the LD50 of adults was 1.35×10 6 Pieces / mL.

[0043] The yellow-haired leaf beetle Figure 3 ): On the 4th day after egg treatment, dead insects began to appear in the treatment group; on the 7th day after treatment, the insect population reduction rates from low concentration to high concentration were 26.67%, 40.00%, and 53.33%, respectively, and no dead insects appeared in the control group. The LD50 of the egg stage was 1.39×10 6 / mL. On the second day of treatment of 1st instar larvae, 1.0×10 8 The dead insects began to appear in the group with 100 / mL. On the 7th day of treatment, the insect population reduction rates from low concentration to high concentration were 80.00%, 66.67%, and 100.00%, respectively. The LD50 of the first-instar larvae was 1.55×10 6 / mL. On the third day after treatment with the second-instar larvae, dead insects began to appear in the treatment group; on the seventh day after treatment, the insect population reduction rates from low concentration to high concentration were 73.33%, 93.33%, and 93.33%, respectively, and the LD50 of the second-instar larvae was 1.64×10 6 / mL. On the 5th day after treatment, dead insects began to appear in the 3rd instar larvae. On the 7th day after treatment, the insect population reduction rates from low concentration to high concentration were 46.67%, 93.33%, and 86.67%, respectively. The LD50 of the 3rd instar larvae was 1.98×10 6 / mL. On the 7th day of pupae treatment, the concentration of the suspension was 1.0×10 7 / mL and 1.0×10 8 / mL, dead insects began to appear; on the 7th day of treatment, the insect population reduction rates from low concentration to high concentration were 0.00%, 6.67%, and 33.33%, respectively, and the LD50 of the pupal stage was 1.99×10 7 / mL. On the 4th day of treatment with adults, the concentration of the suspension was 1.0×10 7 / mL and 1.0×10 8 / mL, dead insects began to appear; on the 7th day of treatment, the insect population reduction rate from low concentration to high concentration was 100.00%, and the LD50 of adults was 1.29×10 6Pieces / mL.

[0044] Example 3: Feeding and spawning tropism

[0045] 1. Experimental methods

[0046] Transplant three-year-old elm seedlings into plastic pots for cultivation. Select seedlings with uniform growth and robust growth for the experiment. One pot of seedlings was treated with Beauveria bassiana, and the other was treated with water as a control. Two pots of elm seedlings were placed in the same container, and 10 to 15 elm leaf beetle adults were placed artificially to observe their feeding and egg-laying tendencies.

[0047] 2. Experimental results

[0048] Three-year-old elm seedlings with uniform growth and robust growth were placed in insect cages. One seedling was treated with Beauveria bassiana, and the other plant was treated with clean water to observe the feeding and egg-laying tendencies of the adults. During the treatment process, some adults did not choose any plant, but rested on the inner wall of the insect cage. However, most adults showed obvious avoidance effects. Both elm leaf beetles showed obvious avoidance to the treatment group ( Figure 4 and Figure 5 ), and the higher the concentration, the more obvious the avoidance effect.

[0049] Example 4: Evaluation of forest control effectiveness

[0050] 1. Experimental methods

[0051] The forest control effect test was conducted in the Botanical Garden of Shenyang Agricultural University. The elm species was "Jiayu", and the elm leaf beetle was more seriously infested. The cultivation and management conditions of the elms used in the test were consistent, and no other pesticides were used within the experimental range before the test. A suspension of Beauveria bassiana with a certain spore concentration was used, and a control of spraying water was set up at the same time. For each treatment, the same trees with a large number of 2-3 instar larvae of elm leaf beetles were selected as replicates in the same area. The pesticide was sprayed repeatedly until a small amount of liquid dripped. The number of elm leaf beetle larvae was investigated before and 1, 3, and 5 days after the application of the pesticide, and the insect population reduction rate and corrected control effect were calculated based on the survey results.

[0052] Insect population reduction rate = (insect population density before application - insect population density after application) / insect population density before application.

[0053] Control effect = (insect population reduction rate in the treatment area - insect population reduction rate in the blank control area) / (1 - insect population reduction rate in the blank control area).

[0054] Data analysis: After the data were sorted using Excel, SPSS20.2 was used to calculate the toxicity regression equation, correlation coefficient, LD50, and 95% confidence interval. The Duncan new multiple range method was used to analyze the significance of the differences between groups for the corrected efficacy.

[0055] 2. Experimental results

[0056] The number of elm leaf beetle larvae was investigated before application and 1d, 3d, and 5d after application, and the insect population reduction rate and corrected control effect were calculated based on the survey results. After 1d of treatment with elm green hairy firefly leaf beetle (Table 1), the population of the control group did not change; each treatment group had a decrease, and the control effect from low concentration to high concentration was 0.0588, 0.1842, and 0.2889, respectively. After 3d of treatment, the population of the control group decreased by 2; the control effect from low concentration to high concentration of the treatment group was 0.3446, 0.6976, and 0.9303, respectively. After 5d of treatment, the population of the control group remained unchanged compared with the 3d; the control effect from low concentration to high concentration of the treatment group was 0.8156, 0.9176, and 0.9303, respectively.

[0057] Table 1 Field efficacy of Beauveria bassiana against the green-haired elm leaf beetle

[0058]

[0059] After 1 day of treatment with the yellow-haired leaf beetle (Table 2), the population of the control group did not change; each treatment group had a decrease, and the control effects from low concentration to high concentration were 0.0204, 0.1458 and 0.2889, respectively. After 3 days of treatment, the population of the control group decreased by 2; the control effects from low concentration to high concentration of the treatment groups were 0.3420, 0.6534 and 0.8151, respectively. After 5 days of treatment, the population of the control group remained unchanged compared with the 3rd day; the control effects from low concentration to high concentration of the treatment groups were 0.8514, 0.9134 and 0.9306, respectively.

[0060] Table 2 Field control effect of Beauveria bassiana on the control of the yellow-haired leaf beetle

[0061]

[0062]

[0063] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0064] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0065] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A strain of Beauveria bassiana which is highly pathogenic to elm leaf beetles, characterized in that: The Beauveria bassiana was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on November 20, 2024, with the deposit number CGMCC No.41634.

2. The use of Beauveria bassiana according to claim 1 in the preparation of a biocontrol agent, characterized in that: The insect pests controlled by the biological control agent are caused by elm leaf beetles.

3. The use of Beauveria bassiana in the preparation of a biocontrol agent according to claim 2, characterized in that: The elm leaf beetles include the green elm leaf beetle and the yellow elm leaf beetle.

4. A bacterial agent containing the Beauveria bassiana according to claim 1.

5. The bacterial agent according to claim 4, characterized in that The bacterial agent is the fermentation culture solution or freeze-dried powder of the Beauveria bassiana.

6. Use of the bacterial agent according to any one of claims 4 to 5 in the preparation of a biocontrol agent, characterized in that: The insect pests controlled by the biological control agent are caused by elm leaf beetles, which include elm green firefly leaf beetles and elm yellow firefly leaf beetles.

7. A biocontrol agent, characterized in that: It comprises the Beauveria bassiana according to claim 1, or the bacterial agent according to any one of claims 4 to 5.

8. The biocontrol agent according to claim 7, characterized in that: The biocontrol preparation further comprises pharmaceutically acceptable adjuvants.

9. The biocontrol agent according to claim 8, characterized in that The auxiliary materials include surfactants.

10. The biocontrol agent according to claim 9, characterized in that The surfactant includes Tween-80.

Images