A strain of Paecilomyces lilacinus with strong pathogenicity to the gypsy moth, a fungal agent and its application

Abstract

The present invention belongs to the technical field of biocontrol agents, and specifically relates to a strain of Paecilomyces lilacinus that has strong pathogenicity to the gypsy moth, a bacterial agent, and its application. 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. The present invention determines the lethal concentration and required time of Paecilomyces lilacinus to the gypsy moth indoors, indicating that the strain has strong pathogenicity to the gypsy moth and can be used to prepare biocontrol agents for preventing and controlling pests caused by the gypsy moth. The present invention provides a new resource for the biological control of the gypsy moth and provides a theoretical basis for the development and utilization of highly toxic strains.

Description

Technical Field

[0001] The invention belongs to the technical field of biocontrol preparations, and particularly relates to a strain of Paecilomyces lilacinus that is highly pathogenic to the gypsy moth, a bacterial agent and applications thereof. Background Art

[0002] The American gypsy 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. Due to its diverse diet, high reproductive output, strong adaptability, and wide range of transmission routes, it poses serious risks and is an invasive pest that requires key control. Paecilomyces lilacinus (Thorn.) Samson is a filamentous fungus with a complex saprophytic-endophytic-parasitic life cycle, and studies have shown that it has advantages in pest and disease control. Currently, chemical pesticides remain the primary choice for controlling the American gypsy moth, but long-term use can lead to rampant pest resurgence, widespread outbreaks of secondary pests, pesticide residues, and environmental pollution. There is an urgent need to find a new method for controlling the American gypsy moth.

[0003] In areas where the gypsy moth invades, a wide variety of parasitic and predatory natural enemies and pathogenic microorganisms are widespread. Biological control factors such as nuclear polyhedrosis viruses, pathogenic bacteria (such as Bacillus thuringiensis), parasitic wasps (such as Chouioia cunea Yang, Pediobius elasmi (Ashmead), and Tetrastichus septentrionalis Yang), parasitic flies (Exorista japonica (Townsend) and Exorista fasciata (Fallén)), and carabid beetles have been discovered and utilized. However, relatively few reports have been published on the use of pathogenic fungi for the control of the gypsy moth, and these pathogenic fungi have yet to be fully explored and utilized. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Paecilomyces lilacinus that is highly pathogenic to the gypsy 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 solutions:

[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 Culture Collection Administration on November 20, 2024, with the deposit number CGMCC No.41635.

[0007] Paecilomyces lilacinus, a widely distributed pathogenic fungus, has high biocontrol potential for leaf-feeding pests. Previous research focused on the control and utilization of larval and surface fungi, but its effectiveness against the various stages of the cuneiform moth, particularly the egg stage, was unknown. The present invention has obtained a strain of Paecilomyces lilacinus with strong pathogenicity against the cuneiform moth. The results of testing the indoor toxicity of the strain against cuneiform moth populations and its control efficacy in forests showed that the strain has a good control effect against cuneiform moth pupae, providing a theoretical basis for further scientific and rational use of Paecilomyces lilacinus for cuneiform moth control.

[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 pests caused by the nymphal 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 liquid or freeze-dried powder of the Paecilomyces lilacinus.

[0011] In a fourth aspect, the present invention provides a use 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 aforementioned Paecilomyces lilacinus or the aforementioned bacterial agent.

[0013] Furthermore, the biocontrol preparation 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 toxicity of Paecilomyces lilacinus to the cuneiform moth by measuring the lethal concentration and time required for Paecilomyces lilacinus to the cuneiform moth indoors. The results show that the Paecilomyces lilacinus of the present invention has a certain repellent effect on cuneiform moths both when feeding and when adults lay eggs. During the process of Paecilomyces lilacinus hyphae invading eggs, chitinase acts on the egg shell and digests the egg contents. Larvae in the net-curtain stage continue to be able to build webs for protection. After entering the dispersal stage, the lethality rate accelerates with age. It is speculated that as larvae age, they consume more food, and more pathogenic fungi enter the midgut through the mouth, accelerating the lethality process. The pupal stage is more resistant to stress than the larval stage, making infection difficult to observe in the short term. Tying straw bundles to lure pupae while applying the bacterial solution can greatly enhance the lethality.

[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 Paecilomyces lilacinus on the egg masses of the gypsy moth. A is before treatment, B is 3 days after treatment, and C is 7 days after treatment.

[0020] Figure 2 To determine the toxicity of Paecilomyces lilacinus to treat egg masses of Hyphantria cunea.

[0021] Figure 3 The figures are indoor toxicity tests of Paecilomyces lilacinus on larvae and pupae, among which A is the indoor toxicity test of Paecilomyces lilacinus on net-curtain 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, among which A is a representative picture of nymphalid moth larvae showing obvious attraction and avoidance to leaves treated with Paecilomyces lilacinus solution, B is 1.0×10 6 The effect of Paecilomyces purpurogenum on the feeding tendency of hymenopteran moth larvae is 1.0×10 7 The effect of Paecilomyces purpurogenum on the feeding tendency of hymenopteran moth larvae was 1.0×10 8 Effects of Paecilomyces purpurogenus on the feeding preferences of hymenopteran moth larvae. DETAILED DESCRIPTION

[0023] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they 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. Colonies with fast-growing and dense mycelium were picked up, and then the plate dilution method was used to spread them to obtain single colonies, which were then streaked. The purified strain was separated and transferred to a plate at 25-28°C for later use. After one week of cultivation, the strain was examined under a microscope. After phylogenetic 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 of the China General Microbiology Culture Collection (CGMCC) 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 isolate, purify and culture the strain. PDA medium contained 200 g of potato, 20 g of glucose, 20 g of agar and 1000 mL of 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 gypsy moth was determined by the insect immersion method. The spores of the Paecilomyces lilacinus strain cultured on a PDA plate for 15 days were washed off with sterile water containing 0.1% Tween-80, 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, dry the surface moisture of the insects with sterile filter paper, place them in a plastic insect box (10.8 cm × 10.8 cm × 7.5 cm) covered with moistened sterile filter paper, put fresh young leaves in the box, and cover it; place the insect box in a constant temperature incubator at 25°C and relative humidity of 90%.

[0032] Each treatment was replicated three times, with 15 larvae per replicate. The treatment immersed in sterile water served as the control (CK). The number of Paecilomyces lilacinus larvae that died and died was observed and recorded daily. The corrected mortality rate, the death rate on the seventh day, and the LT50 were calculated.

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

[0034] Corrected mortality (%) = (mortality of each treatment - mortality of the control) / (1 - mortality 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 sterilized 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 3 days of treatment, 1.0×10 8 One egg mass in the 1000 / mL 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 gypsy moth, the LT50 from low to high concentrations were 5.15d, 4.70d, and 4.25d, respectively. Figure 2 It is speculated that during the invasion of the eggs by mycelium, chitinase acts on the eggshell and digests the contents of the egg, confirming that Paecilomyces can promote the decomposition of many polymers.

[0038] Place the net curtain larvae soaked in bacterial solution 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, but only 1.0×10 8 The number of cells / mL treatment group decreased significantly, and the LT50 was 6.30d ( Figure 3 A) The results show that the larval net curtain can provide a good growth environment for the larvae, and that they have the ability to weave nets to protect themselves before they become dispersed larvae.

[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 surviving larvae on the 3rd day of the control and treatment groups was 14.33±0.4714, 10.33±0.4714, 9.67±2.5048 and 7.67±0.4714, respectively; the number of surviving larvae 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 to high concentrations 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 the bacterial solution decreased significantly day by day, but no spores grew on the insect bodies, and the dead insect bodies 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 grows and produces spores most favorably 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 to high concentrations was 4.80d, 4.60d, 4.00d ( Figure 3 C).

[0041] The pupal stage has stronger resistance to stress 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 to high concentrations are 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, pruned to produce young leaves, were transplanted into plastic pots for cultivation. Seedlings with consistent, robust growth were selected for the experiment. One pot of seedlings was treated with Paecilomyces lilacinus, while the other was treated with plain water as a control. The two pots of mulberry seedlings were placed in the same container and artificially inoculated with 10 to 15 larvae and adults of the gypsy moth to observe their feeding and oviposition tendencies.

[0045] 2. Experimental results

[0046] The larvae of the American white 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 treated group (1000mg / 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 100mg / mL had 80.00% of the residue left after 12 hours, while the leaves of the control group had 60.00% of the residue left. The leaves of the treatment group with 100mg / mL had 75.00% of the residue left after 24 hours, while the leaves of the control group had 35.00% of the residue left. 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 nymphalids still did not choose the leaves treated with high concentration of bacterial solution ( Figure 4 D).

[0047] Adults of the cuneiform moth generally preferred to lay eggs on plants in the control group, with an average of 5.00, 4.67, and 4.33 eggs laid, respectively, from low to high concentrations. Some adults also laid eggs in the insect cages, with an average of 4.00, 3.33, and 2.33 eggs laid, respectively, from low to high concentrations. Almost no adults chose to lay eggs on plants in the treated groups (Table 1). The total number of eggs laid in the insect cages in the low to high concentration treatments was 9.67, 8.33, and 6.67, respectively. It is speculated that the high concentration of Paecilomyces lilacinus reduced the number of eggs laid by adults.

[0048] Table 1 Ovipositional tendencies of adult gypsy moths

[0049] Concentration (pieces / 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 efficacy

[0051] 1. Experimental methods

[0052] A forest control efficacy trial was conducted on untreated roadside trees on Dongling South Street in Hunnan District, Shenyang. The trees were Acer rubrum, a compound leaf tree species, and were severely infested by the gypsy moth. A suspension of Paecilomyces lilacinus with a specific spore concentration was used, along with a water spray control. Identical trees in the same area served as replicates. The pesticide was applied to straw bundles tied to the base of the tree trunks, repeatedly spraying until the bundles were thoroughly soaked. The number of healthy gypsy moth pupae was measured before and 5, 10, and 15 days after application. The population reduction rate and corrected control efficacy 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 treated 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. The blank control group had 27 pupae, 1.0×10 6 23 heads / mL group, 1.0×10 7 21 heads / mL group, 1.0×10 8The control group had 13 individuals per mL of the bacterial solution. Combined with the results of larval feeding trends, it is speculated that the high bacterial concentration affected the nymph's ability to pupate from the tree. Ten days after treatment, the number of pupae trapped in the grass bundles was counted, and the population reduction and corrected efficacy compared to the 5-day period were calculated. Four new pupae were found in the control group. The control efficacy of each treatment group, from low to high concentration, was 0.0911, 0.5438, and 0.7990, respectively. Fifteen days after treatment, the number of pupae trapped in the grass bundles was counted, and the population reduction and corrected efficacy compared to the 10-day period were calculated. The control population in the control group remained unchanged compared to the 10-day period. The control efficacy of each treatment group, from low to high concentration, was 0.6250, 0.7273, and 1.0000, respectively (Table 2). This demonstrates that Paecilomyces lilacinus has a good control effect on nymphalid moth pupae.

[0057] Table 2 Field control effect 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 preferred embodiments.

[0060] 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.

[0061] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A strain of Paecilomyces lilacinus that is highly pathogenic to the American moth Paecilomyces lilacinus ) in the preparation of a biocontrol agent, characterized in that: The Paecilomyces lilacinus was deposited in the General Microbiology Center of the China Culture Collection Administration on November 20, 2024, with the deposit number CGMCC No. 41635; the insect pest controlled by the biocontrol agent is caused by the gypsy moth.

2. The use according to claim 1, characterized in that The biocontrol preparation further comprises pharmaceutically acceptable excipients.

3. The use according to claim 2, characterized in that The auxiliary materials include surfactants.

4. The use according to claim 3, characterized in that The surfactant includes Tween-80.

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