Paecilomyces lilacinus with strong pathogenicity to fall webworm, fungicide and application of paecilomyces lilacinus and fungicide

Abstract

The invention belongs to the technical field of biocontrol preparations, and particularly relates to paecilomyces lilacinus with high pathogenicity to fall webworms, a fungicide and application of the paecilomyces lilacinus and the fungicide. The paecilomyces lilacinus is preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2024, and the preservation number is CGMCC No.41635. The paecilomyces lilacinus is named as paecilomyces lilacinus. The determination of the lethal concentration and the required time of the paecilomyces lilacinus on the fall webworms in a room shows that the strain has strong pathogenicity on the fall webworms and can be used for preparing a biocontrol preparation for preventing and treating insect pests caused by the fall webworms, a new resource is provided for biological prevention and treatment of the fall webworms, and the application has wide application prospects. And a theoretical basis is provided for development and utilization of high-toxicity strains.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control preparations, and specifically relates to a strain of Paecilomyces lilacinus with strong pathogenicity to the American white moth, a bacterial agent and application thereof. Background Art

[0002] The American white moth Hyphantria cunea (Drury), also known as the American lantern moth, fall curtain caterpillar, and fall curtain moth, is native to North America and is a worldwide quarantine pest. Because of its omnivorous diet, large reproduction volume, strong adaptability, and wide transmission routes, it causes serious damage and is an invasive pest that is the focus of prevention and control. Paecilomyces lilacinus (Thorn.) Samson is a filamentous fungus with a complex life history of saprophytic-endophytic-parasitic. Studies have found that it has advantages in pest and disease control. At present, chemical pesticides are still the first choice for the prevention and control of American white moths, but long-term use will lead to problems such as rampant pests, large-scale occurrence of secondary pests, pesticide residues and environmental pollution. It is urgent to find a new way to prevent and control American white moths.

[0003] In the areas invaded by the gypsy moth, various parasitic natural enemies, predatory natural enemies and pathogenic microorganisms are widely present. Biological regulatory factors such as nuclear polyhedrosis virus, pathogenic bacteria (such as Bacillus thuringiensis), parasitic wasps (such as Chouioia cunea Yang, Pediobius elasmi (Ashmead), Tetrastichus septentrionalis Yang), parasitic flies (Exorista japonica (Townsend), Exorista fasciata (Fallén)), and ground beetles have been discovered and used. There are relatively few reports on the use of pathogenic fungi to control the gypsy moth, and the pathogenic fungi of the gypsy moth have not yet been fully developed and utilized. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Paecilomyces lilacinus with strong pathogenicity to the American white moth, a fungal agent and its application, so as to solve the above technical problems.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] In the first aspect, the present invention provides a strain of Paecilomyces lilacinus that is highly pathogenic to the American white moth. The Paecilomyces lilacinus was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on November 20, 2024, with a deposit number of CGMCC No.41635.

[0007] As a widely distributed pathogenic fungus, Paecilomyces lilacinus has a high potential for biological control in the prevention and control of leaf-feeding pests. Its previous studies focused on the prevention and control and utilization of larval and surface fungi, and the control effect of Paecilomyces lilacinus on various insect stages of the gypsy moth, especially the egg stage, is unknown. The present invention obtains a strain of Paecilomyces lilacinus that has strong pathogenicity to the gypsy moth. By measuring the indoor toxicity and forest control effect of the Paecilomyces lilacinus on the gypsy moth population, the results show that the Paecilomyces lilacinus of the present invention has a good control effect on gypsy moth pupae, which can provide a theoretical basis for further scientific and rational use of Paecilomyces lilacinus to control the gypsy moth.

[0008] In a second aspect, the present invention provides a use of the Paecilomyces lilacinus in the preparation of a biocontrol agent, wherein the biocontrol agent controls insect pests caused by the gypsy moth.

[0009] In a third aspect, the present invention provides a bacterial agent containing the Paecilomyces lilacinus.

[0010] Furthermore, the bacterial agent is the fermentation culture solution or freeze-dried powder of the Paecilomyces lilacinus.

[0011] In a fourth aspect, the present invention provides an application of the bacterial agent in the preparation of a biocontrol agent, wherein the biocontrol agent controls pests caused by the gypsy moth.

[0012] In a fifth aspect, the present invention provides a biocontrol agent, comprising the above-mentioned Paecilomyces lilacinus, or the above-mentioned bacterial agent.

[0013] Furthermore, the biocontrol agent also includes pharmaceutically acceptable excipients.

[0014] Furthermore, the auxiliary material includes a surfactant.

[0015] Furthermore, the surfactant includes Tween-80.

[0016] The present invention has the following beneficial effects:

[0017] The present invention studies the results of the toxicity determination of Paecilomyces lilacinus to the American white moth by measuring the lethal concentration and required time of Paecilomyces lilacinus to the American white moth indoors. The results show that the American white moth larvae have a certain repelling effect on the Paecilomyces lilacinus of the present invention when feeding and adults lay eggs. In the process of the hyphae of Paecilomyces lilacinus invading the egg, chitinase acts on the egg shell and digests the contents of the egg at the same time; the larvae in the net curtain stage have the ability to build a net to protect themselves; after entering the dispersion stage, with the increase of the age, the lethality speed is accelerated. It is speculated that the older the larvae are, the greater the food intake is, and the more pathogenic fungi enter the midgut through the mouth, which accelerates the lethal process; the pupal stage has stronger stress resistance than the larval stage, and it is not easy to observe the infection situation in a short period of time. The lethal effect can be greatly improved by applying bacterial liquid while tying grass bundles to lure pupae.

[0018] The invention provides a new resource for biological control of the gypsy moth and a theoretical basis for the development and utilization of highly toxic strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the effect of Pseudomonas lilacinus on the egg masses of the American moth. A is before treatment, B is 3 days after treatment, and C is 7 days after treatment.

[0020] Figure 2 Toxicity assay of Paecilomyces lilacinus against egg masses of Hyphanthus cunea.

[0021] Figure 3 These are indoor toxicity tests of Paecilomyces lilacinus on larvae and pupae, among which A is the indoor toxicity test of Paecilomyces lilacinus on screen larvae, B is the indoor toxicity test of Paecilomyces lilacinus on dispersed larvae, C is the indoor toxicity test of Paecilomyces lilacinus on mature larvae, and D is the indoor toxicity test of Paecilomyces lilacinus on pupae.

[0022] Figure 4 The effect of Paecilomyces lilacinus on the feeding tendency of nymphalid moth larvae, where A is a representative picture of nymphalid moth larvae showing obvious attraction and avoidance to leaves treated with Paecilomyces lilacinus liquid, and B is 1.0×10 6 The effect of Paecilomyces purpurogenum on the feeding tendency of larvae of Hyphanthus cunea, C is 1.0×10 7 The effect of Paecilomyces purpurogenum on the feeding tendency of larvae of Hyphanthus cunea, D is 1.0×10 8 Effect of Paecilomyces purpurogenus on the feeding preferences of larvae of the American moth. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1: Isolation and identification of Paecilomyces lilacinus

[0025] The tested Paecilomyces lilacinus strain was isolated from the infected American white moth pupae, 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, it 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 Paecilomyces lilacinus. The strain was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.41635 and the deposit date of November 20, 2024.

[0026] Example 2: Indoor toxicity assay

[0027] 1. Experimental Materials

[0028] 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.

[0029] Test insects: Egg masses were collected from untreated roadside trees in Shenyang, Liaoning Province, and a large number of insect sources with consistent growth indicators were obtained through indoor breeding.

[0030] 2. Experimental methods

[0031] The toxicity of the strain to the American white moth was determined by the insect immersion method. The spores of the Paecilomyces lilacinus strain cultured on the PDA plate for 15 days were washed off with 0.1% Tween-80 sterile water and the concentration of the spore suspension was adjusted to 1.0×10 6 / mL, 1.0×10 7 / mL, 1.0×10 8 / mL; immerse eggs, larvae and pupae in the spore suspension for about 5 seconds in turn, 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.

[0032] Each treatment was repeated three times, with 15 larvae in each repeat. The treatment immersed in sterile water was used as the control (CK). The number of larval deaths and dead insects of Paecilomyces lilacinus was observed and recorded every day, and the corrected mortality of larvae, dead insect rate on the 7th day and LT50 were calculated.

[0033] Mortality rate (%) = number of dead larvae / total number of larvae × 100%.

[0034] Corrected mortality rate (%) = (mortality rate of each treatment - mortality rate of the control) / (1 - mortality rate of the control) × 100%.

[0035] Dead insect rate (%) = number of dead insects / total number of larvae × 100%.

[0036] 3. Experimental results

[0037] Fresh egg masses of the American white moth were selected and disinfected with formaldehyde solution indoors before use in the experiment. Each group treated 15 eggs and the treatment effect was continuously observed. Sterile water was used as a control. After treatment for 3 days, 1.0×10 8 One egg mass in the treatment group showed infection symptoms; on the 5th day of treatment, each treatment group showed obvious infection; on the 7th day of treatment, all egg masses in the treatment groups were infected, and some egg masses were dissolved ( Figure 1 ). When Paecilomyces lilacinus was used to treat the egg masses of the American moth, the LT50 from low concentration to high concentration was 5.15d, 4.70d, and 4.25d, respectively. Figure 2 ). It is speculated that during the invasion of the eggs by mycelium, chitinase acted on the eggshell and digested the contents of the egg, confirming that Paecilomyces can promote the decomposition of many polymers.

[0038] Put the net curtain larvae soaked in bacterial liquid into the insect box, and the larvae in the net curtain will quickly find a dry place to re-weave the web. 6 / mL and 1.0×10 7 The number of larvae in the treatment group did not decrease significantly, only 1.0×10 8 The number of / mL treatment group decreased significantly, and the LT50 was 6.30d ( Figure 3 A). It was verified that the larval net curtain can provide a good growth environment for the larvae, and before they become dispersed larvae, they have the ability to weave a net to protect themselves.

[0039] The number of dispersed larvae soaked in bacterial solution decreased significantly day by day in the treatment group, while the number of control group remained basically unchanged. The number of survivors on the 3rd day of the control group and the treatment group was 14.33±0.4714, 10.33±0.4714, 9.67±2.5048 and 7.67±0.4714, respectively; the number of survivors on the 5th day was 14.33±0.4714, 7.00±0.8165, 5.67±0.9428 and 4.33±0.9428, respectively. The LT50 of the dispersed larvae of the American moth treated with Paecilomyces lilacinus from low concentration to high concentration was 5.40d, 4.90d and 4.40d, respectively. The number of successful pupations was 13.67±1.2472, 2.33±0.9428, 1.67±0.4714 and 0( Figure 3 B).

[0040] The number of mature larvae soaked in bacterial solution decreased significantly day by day in the treatment group, but no spores grew on the insect body, and the dead insect body dissolved after a period of time. It is speculated that strong light affects the production of spores, which is consistent with the result that "Paecilomyces lilacinus is most conducive to growth and spore production under full darkness, followed by half light, and the worst under full light." The number of control groups remained basically unchanged. The LT50 of Paecilomyces lilacinus treated mature larvae of the American white moth from low concentration to high concentration was 4.80d, 4.60d, 4.00d ( Figure 3 C).

[0041] The pupal stage has stronger resistance than the larval stage, and it is difficult to observe the infection situation in a short period of time. After 7 days of treatment, the mortality rates from low concentration to high concentration were 0.00%, 13.33%, 20.00% ( Figure 3 D)

[0042] Example 3: Feeding and spawning tropism

[0043] 1. Experimental methods

[0044] Three-year-old mulberry seedlings that had grown tender leaves after pruning were transplanted into plastic pots for cultivation. Seedlings with uniform growth and robust growth were selected for the experiment. One pot of seedlings was treated with Paecilomyces lilacinus, and the other plant was treated with water as a control. Two pots of mulberry seedlings were placed in the same container, and 10 to 15 larvae and adults of the American white moth were artificially placed to observe their feeding and egg-laying tendencies.

[0045] 2. Experimental results

[0046] The larvae of the American moth showed a significant avoidance effect on leaves treated with the liquid of Paecilomyces lilacinus ( Figure 4 A). 1.0×10 6 The leaves of the treatment group with 100 / mL were completely eaten in 12 hours, while the leaves of the control group began to be eaten in 12 hours, indicating that the low concentration of Paecilomyces lilacinus solution had an attractive effect on the feeding of the larvae of the American moth ( Figure 4 B). 1.0×10 7 The leaves of the treatment group with 100 / mL had 80.00% remaining after 12 hours, and the leaves of the control group had 60.00% remaining; the leaves of the treatment group with 24 hours had 75.00% remaining, and the leaves of the control group had 35.00% remaining; this indicates that under this concentration treatment, the larvae of the American white moth are more inclined to feed on the leaves of the control group ( Figure 4 C). 1.0×10 8 The leaves of the 100 / mL treatment group were not eaten during the observation period, while the leaves of the control group were eaten completely within 12 hours, indicating that even in the absence of food, the nymphs still did not choose the leaves treated with high concentrations of bacterial solution ( Figure 4 D).

[0047] The adults of the American white moth generally tended to lay eggs on the plants in the control group, with an average of 5.00, 4.67 and 4.33 eggs laid from low to high concentrations; some adults laid eggs on the insect cages, with an average of 4.00, 3.33 and 2.33 eggs laid from low to high concentrations; almost no adults chose to lay eggs on the plants in the treatment group (Table 1). The total number of eggs laid in the insect cages from low to high concentrations was 9.67, 8.33 and 6.67, respectively, and it is speculated that the high concentration of Paecilomyces lilacinus reduced the number of eggs laid by adults.

[0048] Table 1 Oviposition tendency of adult Hyphantria cunea

[0049] Concentration (pcs / mL) Control group (block) Processing group (block) Insect cage (block) Total(block) <![CDATA[1×10 6 ]]> 5.00±0.8165 0.67±0.9428 4.00±0.0000 9.67±0.4714 <![CDATA[1×10 7 ]]> 4.67±0.9428 0.33±0.4714 3.33±0.9428 8.33±1.2472 <![CDATA[1×10 8 ]]> 4.33±0.4714 0.00±0.0000 2.33±0.4714 6.67±0.4714

[0050] Example 4: Evaluation of forest control effectiveness

[0051] 1. Experimental methods

[0052] The forest control test was conducted on untreated street trees in Dongling South Street, Hunnan District, Shenyang City. The tree species was "Acer truncatum", and the gypsy moth was seriously damaged. A suspension of Paecilomyces lilacinus with a certain spore concentration was used, and water was sprayed as a control. The same trees in the same area were repeated. Spray the grass bundles tied at the base of the tree trunks, and spray the agent repeatedly until the grass bundles were soaked. The number of healthy gypsy moth pupae was investigated before and 5, 10, and 15 days after the application of the pesticide, and the insect population reduction rate and corrected control effect were calculated based on the survey results.

[0053] Insect population reduction rate = (insect population density before application - insect population density after application) / insect population density before application.

[0054] 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).

[0055] 2. Experimental results

[0056] After 5 days of treatment, the number of pupae trapped in the grass bundles was counted. There were 27 pupae in the blank control group and 1.0×10 6 23 heads / mL group, 1.0×10 7 pcs / mL group: 21 heads, 1.0×10 8The number of pupae in the 10-day treatment group was 13. Combined with the results of larval feeding tendency, it was inferred that the high concentration of bacterial solution affected the pupation of the gypsy moth. After 10 days of treatment, the number of pupae trapped in the grass bundles was counted, and the insect population reduction rate and corrected control effect compared with 5 days were calculated. It was found that 4 new pupae were added to the control group; the control effect of each treatment group from low concentration to high concentration was 0.0911, 0.5438 and 0.7990, respectively. After 15 days of treatment, the number of pupae trapped in the grass bundles was counted, and the insect population reduction rate and corrected control effect compared with 10 days were calculated. It was found that the population of the control group remained unchanged compared with the 10th day; the control effect of the treatment group from low concentration to high concentration was 0.6250, 0.7273 and 1.0000, respectively (Table 2). It was proved that Paecilomyces lilacinus had a good control effect on the pupae of the gypsy moth.

[0057] Table 2 Field efficacy of Paecilomyces lilacinus against gypsy moth

[0058]

[0059] 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.

[0060] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other 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.

[0061] 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 Paecilomyces lilacinus having strong pathogenicity to the gypsy moth, characterized in that: The described Paecilomyces lilacinus was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on November 20, 2024, with the deposit number CGMCC No.41635.

2. The use of Paecilomyces lilacinus 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 the gypsy moth.

3. A bacterial agent containing the Paecilomyces lilacinus according to claim 1.

4. The bacterial agent according to claim 3, characterized in that The bacterial agent is the fermentation culture solution or freeze-dried powder of the Paecilomyces lilacinus.

5. Use of the bacterial agent according to any one of claims 3 to 4 in the preparation of a biocontrol agent, characterized in that: The insect pests controlled by the biological control agent are caused by the gypsy moth.

6. A biocontrol agent, characterized in that: It comprises the Paecilomyces lilacinus described in claim 1, or the bacterial agent described in any one of claims 3 to 4.

7. The biocontrol agent according to claim 6, characterized in that The biocontrol preparation further comprises pharmaceutically acceptable adjuvants.

8. The biocontrol agent according to claim 7, characterized in that The auxiliary materials include surfactants.

9. The biocontrol agent according to claim 8, characterized in that The surfactant includes Tween-80.

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