Cordyceps sobolifera strain 022017-9 as well as biocontrol agent and application thereof

Abstract

The invention provides a cordyceps sobolifera strain 022017-9 and a biocontrol agent and application thereof, and belongs to the technical field of functional microorganisms. According to the invention, one strain is separated from aphid bodies which are dead due to diseases, the number of the strain is 022017-9, and the strain is determined as Cordyceps cicadae through morphological characteristic observation and a molecular biological identification technology. The strain 022017-9 has a good prevention and control effect on the adult stage and / or the nymph stage of aphids. The strain 022017-9 can obviously reduce the survival rate of aphids and the egg laying amount, and meanwhile, the net reproduction rate Ro, the average generation duration T, the intrinsic growth rate rm, the weeklimit growth rate lambda and other parameters of an inoculated aphid population are all obviously changed. Therefore, the strain 022017-9 has relatively strong pathogenicity to the aphids and has a good application prospect in the aspect of biological control of the aphids.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial technology, specifically relating to a strain of Cordyceps militaris 022017-9, its biocontrol agents, and their applications. Background Technology

[0002] Cordyceps cicadae is a fungus that is not widely used. Research on Cordyceps cicadae has largely focused on its control of pests such as Lepidoptera and Coleoptera, or on its medicinal value. However, research on its application to aphids and other homopteran pests is relatively limited in the field of biological pest control. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a cicada flower strain 022017-9 that affects the pathogenicity, survival rate, lifespan and reproduction rate of aphids, and has a control effect.

[0004] This invention provides a *Cicada Flower* strain 022017-9, with the preservation number CGMCC No. 42076.

[0005] The present invention provides a biocontrol agent, the active ingredients of which include the *Cicada nymph* strain 022017-9 and / or metabolites of the *Cicada nymph* strain 022017-9.

[0006] Preferably, the cicada flower strain 022017-9 comprises spores and / or hyphae.

[0007] Preferably, the spore concentration of the *Cicada nymph* strain 022017-9 is 1×10⁻⁶. 5 cells / mL or 1×10 5 More than one per gram.

[0008] Preferably, the spore concentration of the *Cicada nymph* strain 022017-9 is 1×10⁻⁶. 6 ~1×10 7 per mL.

[0009] This invention provides the application of the cicada flower strain 022017-9 or the biocontrol agent in aphid control.

[0010] Preferably, the aphid control includes the control of adult aphids and / or the control of nymphal aphids.

[0011] Preferably, the aphid control includes reducing at least one of the following indicators: aphid lifespan, aphid reproduction capacity, and aphid growth and development.

[0012] Preferably, the aphid control targets include at least one of the following: corn, wheat, and tea trees.

[0013] The present invention provides a Cordyceps cicadae strain 022017-9, with accession number CGMCC No. 42076. This strain 022017-9 was isolated from infected and dead aphids. Morphological observation and molecular biological techniques identified strain 022017-9 as belonging to Cordyceps cicadae, located on the same branch of the phylogenetic tree. The pathogenicity of strain 022017-9 against adult aphids gradually increases with increasing spore concentration, with a bacterial suspension concentration of 1×10⁻⁶. 7 At 1 / mL, LT 50 The inoculation period was 2.54 days, and the corrected mortality rate of adult aphids reached 60 (±9.13)% 72 hours after inoculation; the concentration was 1×10⁻⁶. 6 At 1 / mL, LT 50 The inoculation period was 3.02 days. 72 hours after inoculation, the corrected mortality rate of adult aphids reached 35% (±4.08)%; the concentration was 1×10⁻⁶. 5 At 1 / mL, LT 50 The corrected mortality rate of adult aphids was only 17.5% (±1.44)% after 72 hours of treatment and inoculation, with a treatment duration of 3.88 days. Furthermore, regardless of whether the aphids were inoculated at the adult or nymph stage, strain 022017-9 significantly reduced the survival rate (lx) and egg production (mx) of aphids. Simultaneously, parameters such as net reproductive rate Ro, mean generation duration T, intrinsic growth rate rm, and cyclical growth rate λ of the inoculated aphid population all showed significant changes. In conclusion, strain 022017-9 exhibits strong pathogenicity against aphids and shows promising application potential in aphid biological control. Attached Figure Description

[0014] Figure 1 This is a diagram showing the growth morphology of *Cicada Flower* on PDA solid medium.

[0015] Figure 2 The image shows the morphological characteristics of strain 022017-9; where A is a conidiophore and B is a conidium.

[0016] Figure 3 Phylogenetic tree of strain 022017-9 constructed based on rDNA ITS sequence;

[0017] Figure 4 The images show the symptoms of aphids infected by strain 022017-9. A and B show the growth of white mycelium on the joints of the insect's legs and dorsal plate after 24 hours (A) and 72 hours (B) of infection. C shows the growth of mycelium on the brown and shriveled insect body after 120 hours of infection. D shows the production of a large number of mycelium on the surface of the insect carcass 144 hours after inoculation.

[0018] Figure 5 The results show the corrected mortality rate of aphids induced by spore suspension of strain 022017-9;

[0019] Figure 6 The results show the effects of infection with cicada flowers at different ages on the survival rate (lx) and reproductive capacity (mx) of aphids.

[0020] Certificate of Preservation of Biological Materials

[0021] The strain *Cordyceps cicadae* is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 6, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The biological accession number is CGMCC No. 42076, and the strain number is 022017-9. Detailed Implementation

[0022] This invention provides a *Cicada Flower* strain 022017-9, with the preservation number CGMCC No. 42076.

[0023] In this invention, the rDNA-ITS sequence of strain 022017-9 has 100% similarity to that of *Cicada nymph*, and is located on the same branch of the phylogenetic tree. The morphological characteristics of strain 022017-9 are as follows: Macroscopically, colonies growing on PDA medium are off-white to light yellow, with a velvety appearance and concentric rings; during sporulation, the colony surface is powdery; under a microscope, the mycelial conidiophores of strain 022017-9 are erect, growing in clusters or singly, with their bases connected. The conidiophores are commonly cylindrical with a pointed apex, and some may repeatedly branch, forming a cockscomb-like head structure. This head produces numerous flesh-colored or straw-yellow conidia, which are septate tubular structures. The branching of the conidiophores is typical, usually with 2-5 phialides clustered on short branches, which are arranged in whorls on the conidiophores. Conidia are columnar single cells with smooth, colorless, and transparent walls. They are mostly symmetrical, with a few slightly curved. Based on morphological characteristics and molecular identification, strain 022017-9 is identified as Cordyceps militaris.

[0024] In this invention, the *Cicada Flower* strain 022017-9 exhibits strong toxicity to aphids, and the toxicity (pathogenicity) increases with the concentration of the spore suspension. Infection for 5-7 days essentially leads to the death of all adult aphids, significantly reducing their survival rate. It is even more pathogenic to adult aphids, with a spore concentration of 1×10⁻⁶. 7 At a concentration of cells / mL, the lethal time LT 50The shortest survival time was 2.54 days. However, for nymphal aphids (2nd to 4th instar), the survival rate of aphids treated with the *Cicada nymph* strain 022017-9 did not show a significant decrease during the nymphal stage. Instead, a significant and uniform decrease in survival rate only began within 3-4 days of development into adulthood, indicating a delayed survival time for nymphal aphids compared to adults. Besides affecting aphid survival rate, *Cicada nymph* strain 022017-9 also impacted reproductive capacity. Inoculation with *Cicada nymph* strain 022017-9 in both nymphal and adult stages resulted in a significantly lower aphid reproductive rate (mx) compared to the control. Furthermore, *Cicada nymph* strain 022017-9 significantly altered parameters such as net reproductive rate Ro, average generation duration T, intrinsic growth rate rm, and cyclical growth rate λ of the aphid population. In conclusion, strain 022017-9 exhibits strong pathogenicity against aphids and shows promising application prospects in aphid biological control.

[0025] The present invention provides a biocontrol agent, the active ingredients of which include the *Cicada nymph* strain 022017-9 and / or metabolites of the *Cicada nymph* strain 022017-9.

[0026] In this invention, the biocontrol agent preferably comprises any life cycle of the *Cicada nymph* strain 022017-9, such as spores and / or hyphae. The biocontrol agent preferably also comprises metabolites of the *Cicada nymph* strain 022017-9, such as culture supernatant or fermentation supernatant.

[0027] In this invention, the biocontrol agent comprises an aqueous solution and / or a powder. The spore concentration of the *Cicadaeidae* strain 022017-9 is preferably 1 × 10⁻⁶. 5 cells / mL or 1×10 5 For cells / g or higher, it can be 1×10 6 ~1×10 7 cells / mL or 1×10 6 ~1×10 7 per g. This invention does not impose any particular limitations on the preparation methods of the aqueous and powder formulations; any preparation methods well-known in the art can be used.

[0028] This invention provides the application of the cicada flower strain 022017-9 or the biocontrol agent in aphid control.

[0029] In this invention, the *Cicada Flower* strain 022017-9 exhibits good control effects against all species within the genus *Aphid*. The aphids preferably include those from the family Aphididae. The family Aphididae preferably includes those from the genus *Aphidius*. In this embodiment, toxicity experiments were conducted using *Aphidius gracilis* as an example.

[0030] In this invention, aphid control preferably includes the control of adult aphids and / or nymphal aphids. The nymphal stage of the aphids includes 2 to 4 instars, and can be 3 instars. Experiments show that the younger the nymphal stage, the less sensitive they are to the application of the cicada flower strain 022017-9. Compared with nymphal insects, adult aphids are more sensitive to the application of the cicada flower strain 022017-9, and die rapidly after inoculation. This indicates that the pathogenicity of strain 022017-9 to aphid nymphs is slightly weaker than that to adults, but its control effect is still sustained. With the extension of treatment time, most nymphs die successively within 2 days after emergence.

[0031] In this invention, aphid control preferably includes reducing at least one of the following indicators: aphid lifespan, aphid reproduction capacity, and aphid growth and development. Pathogenicity is a key indicator for evaluating the effectiveness of entomopathogenic microorganisms in biological control. This invention explored the infectivity of the cicada nymph strain 022017-9 against aphids, clarifying that strain 022017-9 has strong pathogenicity against adult aphids. The results show that the toxicity of the spore suspension of 022017-9 against adult aphids exhibits a significant dose-response relationship with its concentration, and that LT increases with increasing spore concentration. 50 The incubation period is shortened accordingly. The results of the embodiments of the present invention show that strain 022017-9 has a significant impact on the survival rate, lifespan, and litter size of aphids. The intrinsic growth rate, net reproductive rate, and cyclical growth rate of the aphid population treated with strain 022017-9 were all significantly lower than those of the control, indicating that strain 022017-9 has a significant inhibitory effect on the growth, development, reproduction, and even population growth of aphids during the incubation period.

[0032] In this invention, the targets of aphid control preferably include at least one of the following: corn, wheat, and tea trees.

[0033] The following detailed description, in conjunction with embodiments, illustrates a *Cicadaeidae* strain 022017-9, its biocontrol agents, and their applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Isolation and Identification Method of a Cordyceps Strain 022017-9

[0036] 1. Isolation of strains

[0037] Strain 022017-9 was isolated from the infected *Aphidius gracilis* by the Insectivorous Fungi Research Laboratory of the Zhejiang Subtropical Crops Research Institute. The strain preserved on slant agar was transferred to potato dextrose agar (PDA) plates and incubated statically at 27±1℃ for 3 days in a BMJ-400 incubator (manufactured by Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory). Subsequently, colonies with good mycelial growth were selected, and a portion of the mycelium was streaked onto fresh PDA medium for subculture to isolate single colonies. After incubation at 27℃ for 10 days, spores were collected using a sieve in a sterilized laminar flow hood.

[0038] The spore powder collected from the above strains was dissolved in deionized water and 0.05% Tween-80 to prepare a concentration of 1×10⁻⁶. 7 A spore powder suspension of 1 spore / mL was prepared. Larvae of the cereal aphid were inoculated with the suspension, and the mummified larvae were collected. Tissue was extracted, and the *Cicadae* strain was re-isolated. Strains in good growth condition were selected as purified strains, and subsequent morphological and molecular identifications were performed on the purified strains.

[0039] 2. Strain identification

[0040] 2.1 Observation of strain morphology

[0041] Aseptic techniques were employed. A 5mm diameter mycelial disc was selected from a single colony of the purified strain and inoculated into the center of a PDA plate, with a sterile coverslip placed horizontally on top. The plate was then incubated at 28°C for 7 days. Colony growth was observed daily during the incubation period, and color and morphological changes were recorded. When the hyphae covered half the area of ​​the coverslip, the coverslip was carefully removed and placed on the stage of an optical microscope. The focus, aperture, and other parameters were adjusted to observe the morphology and structure of the hyphae and spores. Morphological identification was performed according to the *Handbook of Fungal Identification* to determine the fungal species (Wei Jingchao, 1979; Bi Keke et al., 2023).

[0042] 2.2 Molecular Identification

[0043] DNA was extracted from the strain according to the Ezup column-based fungal genomic DNA extraction kit (B518255). The rDNA ITS sequence was amplified and determined using universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO:1) and ITS4-R (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO:2). A 25 μL PCR reaction mixture contained 12.5 dμL of 10X PCR Buffer dNTPs (10 mM each), Taq Plus DNA Polymerase (5 U / μL), and 50 mM MgSO4, 1 μL each of forward and reverse primers, and 1 μL of DNA template. ddH2O was added to bring the total volume to 25 μL. PCR amplification conditions were: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, and 72℃ extension for 90 s; 30 cycles, with a final extension at 72℃ for 10 min. The amplified products were analyzed for DNA fragmentation using 1.5% agarose gel electrophoresis. PCR products were sent to Sangon Biotech Co., Ltd. (Shanghai) for sequencing. The sequences were aligned using NCBIBLAST, and multiple sequence alignment was performed using the Clustal X method in Mega 11.0.13 software. A molecular phylogenetic tree was constructed using the Maximum Likelihood Method (ML), with bootstrap values ​​estimated with 1000 replicates to determine the taxonomic position of the strains.

[0044] 3. Identification results of strain 022017-9

[0045] 3.1 Morphological characteristics of strain 022017-9

[0046] Strains 022017-9 were cultured on PDA medium at 28°C for 15 days, resulting in colonies with diameters between 69 and 75 mm. During growth, the colonies appeared off-white to pale yellow on the upper surface, with a velvety appearance and concentric rings. Sporulation began approximately 7 days after culture, and abundant sporulation occurred by day 14. At the time of sporulation, the colony surface was powdery. Figure 1 ).

[0047] Under a microscope, the hyphae of *C. cicada* are erect, growing in clusters or singly, connected at the base. The hyphae are commonly cylindrical with a pointed apex, sometimes repeatedly branching to form a cockscomb-like head structure. This head produces numerous flesh-colored or straw-yellow conidiophores, which are septate tubular structures. The branching of the conidiophores is typical, usually with 2-5 phialides clustered on short branches, which are arranged in whorls on the conidiophores. The conidia are cylindrical, unicellular, with smooth walls, colorless and transparent, mostly symmetrical, with a few slightly curved. Figure 2 ).

[0048] 3.2 Phylogenetic analysis of strain 022017-9

[0049] Using ITS1 and ITS4 as primers, PCR amplification was performed on the genomic DNA of strain 022017-9 as a template to obtain a target fragment of 562 bp (SEQ ID NO: 3). Comparison of the sequencing results with NCBI BLAST revealed that its rDNA ITS sequence was 100% similar to that of *Cordyceps cicadae*. Phylogenetic analysis showed that the rDNA ITS sequence of strain 022017-9 clustered on the same branch as *Cordyceps cicadae* / *Cordyceps sinclade* / *Cordyceps sinclade* (sequence numbers MF460361.2, MH937747.1, MZ373176.1, FJ765283.1, and OL653011.1, etc.). Figure 3 ).

[0050] Example 2

[0051] Virulence assay of Cicadae periostracum strain 022017-9

[0052] 1. Materials and Methods

[0053] 1.1. Test materials

[0054] Test strain: Cordyceps militaris strain 022017-9.

[0055] Test insects: Healthy *Aphidius gracilis*, required for toxicity testing, were collected from maize plants at the Zhejiang Subtropical Crops Research Institute and propagated single-aphids on maize leaves in a climate chamber.

[0056] Test culture medium: Potato dextrose agar (PDA solid culture), the main components of which are 200g peeled potato, 20g glucose, 15g agar, diluted with water to 1L, autoclaved at 121℃ for 20min, and ready for use.

[0057] Potato dextrose broth (PDB): 200g peeled potatoes, 20g glucose, diluted with water to 1L, autoclaved at 121℃ for 20min.

[0058] Bran culture medium (BCM): 800g oats, 200g buckwheat, add sterile water to submerge the wheat mixture, and autoclave at 121℃ for 20min.

[0059] Main reagents: Ezup column-based fungal genomic DNA extraction kit and SanPrep column-based DNA gel extraction kit were purchased from Sangon Biotech Co., Ltd.; Taq Plus DNA polymerase, agarose B, and 4S Red Plus nucleic acid staining agent (10,000X aqueous solution) were purchased from BBI; GeneRuler DNA LadderMix was purchased from Thermo Scientific. Primers used were synthesized by Sangon Biotech Co., Ltd. (Shanghai).

[0060] 1.2. Activation and culture of the tested strains

[0061] The test strain 022017-9, preserved on a slant, was transferred to potato dextrose agar (PDA) plates and incubated statically at 25±1℃ for 3 days in a BMJ-400 incubator (manufactured by Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory). Subsequently, colonies with good mycelial growth were selected, and a portion of the mycelium was streaked onto fresh PDA medium for subculture to isolate single colonies. After incubation at 25℃ for 10 days, spores were collected using a sieve in a sterilized laminar flow hood. The spores were used for subsequent bioassays of the cereal aphid.

[0062] The spore powder collected from the above strains was dissolved in deionized water and 0.05% Tween-80 to prepare a concentration of 1×10⁻⁶. 7 A spore suspension of 1 / mL was prepared. Larvae of the rice strangler aphid were inoculated with the suspension, and the mummified larvae were collected. Tissue was extracted, and the *Cicada nyctalor* strain was re-isolated. Strains in good growth condition were selected as purified strains, and subsequent morphological and molecular identifications were performed. The isolated *Cicada nyctalor* strains were inoculated into PDB medium, mixed, and transferred to a shaker for 3 days at 25°C and 140 rpm. The bacterial suspension was then inoculated into BCM medium, mixed, and cultured in the dark at 25°C for 7 days, followed by 14 days of light culture. After the culture period, the fruiting bodies were harvested, and the spores of each strain were collected.

[0063] 1.3 Determination of the toxicity of cicada flower to the cereal constrictor aphid

[0064] The tested strain was inoculated onto PDA plates and cultured. After the plates were confluent with conidia, spore powder was collected and washed with sterile water containing 0.02% Tween-80. The spores were counted under a biological microscope using a hemocytometer to determine the initial concentration. The spores were then diluted with sterile water containing 0.05% Tween-80 to a final concentration of 1×10⁻⁶. 5 1×10 5.5 1×10 6 1×10 6.5 1×10 7 Cells / mL, ready for use. Healthy adult *Aphidius gracilis* on the first day of emergence were selected, and different concentrations of *Cicadaeidae* spore suspension were sprayed onto the surface of the adult aphids, with 0.05% Tween 80 as the control group. Each group contained 20 nymphs; four replicates were set up. The aphids were reared at a temperature of 25±1℃ and a relative humidity of 65%±5%. The nymph survival rate was observed and recorded every 24 hours, and the corrected mortality rate was calculated according to Formula I.

[0065] Corrected mortality rate = (treatment group mortality rate - control group mortality rate) / (1 - control group mortality rate) × 100%

[0066] Formula I.

[0067] 1.4 Effects of Cordyceps militaris on the growth and development of the cereal aphid

[0068] Collect nymphs of the rice aphid at different instars, including 2nd, 3rd, and 4th instars and adults, at a ratio of 1×10⁻⁶. 7 spores·mL -1 After spraying the aphids with different concentrations, they were reared at a temperature of 25±1℃ and a relative humidity of 65%±5%. The number of surviving aphids and the number of offspring during the adult stage were observed and recorded every 24 hours until all adults died. The survival rate lx and the number of offspring mx were calculated.

[0069] 1.5 Data Processing and Analysis

[0070] The data obtained from the toxicity tests were analyzed using SPSS 25.0 software via Probit regression to obtain the regression equation and determine the median lethal concentration (LC50). 50 ) and lethal time (LT) 50 ).

[0071] The calculation methods for net proliferation rate and weekly growth rate of intrinsic population growth rate in the generation cycle of life parameters are based on the calculation methods of Liu (2019, 2021):

[0072] Net growth rate R o =∑lx×mx Formula II;

[0073] Generation cycle T = ∑x × lx × mx / ∑lx × mx (Formula III);

[0074] Intrinsic growth rate r m =LnR0 / T Formula IV;

[0075] Weekly growth rate λ = e rm Formula V;

[0076] In the formula, x represents time (d), lx represents the survival rate of aphids at time x, and mx represents the number of offspring produced per female aphid during the time interval from x-1 to x. e represents the natural constant (Euler number), which is an irrational number, approximately 2.71828.

[0077] One-way ANOVA was used to assess the significance of differences between treatment groups. When multiple comparisons were involved in the analysis, Tukey's test, built into IBM SPSS Statistics 27.0 software, was used.

[0078] 2. Results and Analysis

[0079] 2.1 Toxicity of Cicadae Periwinkle to Cephalotaxus fortunei

[0080] The pathogenicity of different concentrations of spore suspensions of strain 022017-9 against adult *Aphidius gracilis* is as follows: Figure 4 As shown, pathogenicity gradually increases with increasing concentration. 48 hours after inoculation, significant differences in the corrected mortality rate of adult *Aphidius gracilis* treated with spore suspensions of different concentrations began to appear. The optimal spore concentration was 1×10⁻⁶. 7 The corrected mortality rate of adult *Aphidius gracilis* in the *[number] / mL* treatment group was 25% (±)%, significantly higher than that in the 1×10[number] / mL* treatment group. 5.5 cells / mL (5±%) and 1×10 5 Cells / mL (2.5±%), 72 h after inoculation, 1×10 7The corrected mortality rate of adult aphids in the treatment group with a density of 1 × 10⁶ / mL reached 60 (±9.13)%, significantly higher than that in the group with a density of 1 × 10⁶ / mL. 5 Treatment with 17.5 ± 1.44% per mL. 96 h after inoculation, 1 × 10⁻⁶ cells / mL were administered. 7 cells / mL and 1×10 6 The corrected mortality rate of adult *Aphidius gracilis* treated with *[number] individuals / mL* reached over 80%, significantly higher than that of adults treated with 1.0 × 10⁻⁶ individuals / mL. 5 Treatment with spores / mL concentration (40.97±4.31)% Figure 5 After 5-7 days of infection with spore suspensions of various concentrations, almost all adult *Aphidius gracilis* insects died. The results indicate that *Cicada nymph* has high pathogenicity to *Aphidius gracilis*, capable of infecting adult insects and significantly reducing their survival rate. In most *Aphidius gracilis* insects, white mycelium grows on the joints of the legs and dorsal plates 24-72 hours after infection. Between 72-120 hours after infection, the insects turn brown, die, and shrivel, with mycelium continuing to grow. After 120 hours, the mycelium begins to multiply rapidly, gradually covering the entire body.

[0081] The lethal time (LT) of strain 022017-9 against adult *Aphidius gracilis* was determined. 50 The results showed that the lethal time gradually decreased with increasing conidial concentration. When the conidial concentration was 1×10⁻⁶, the lethal time decreased. 7 At a concentration of cells / mL, the lethal time LT 50 The shortest is 2.54 days. The next shortest is 1×10⁻⁶ days. 6.5 1×10 6 1×10 5.5 and 1×10 5， LT 50 The lethal concentrations were 2.84, 3.02, 3.52, and 3.88 days, respectively (Table 1). 50 The results showed that with the extension of inoculation time, the median lethal concentration (LC50) of strain 022017-9 against adult *Aphidius gracilis* decreased. 50 Decreasing; lethal median concentration (LC50) 72 hours after inoculation 50 3.52×10 6 The results indicate that strain 022017-9 exhibits high toxicity and rapid pathogenicity against adult *Aphidius gracilis*.

[0082] Table 1. LT (Low Temperature) of strain 022017-9 against adult *Aphidius gracilis* 50 and regression equation

[0083] Spore concentration (spores / mL) Probit regression equation <![CDATA[Lethal time and 95% confidence interval LT 50 (d)]]> <![CDATA[1×10 7 ]]> y = 10.891x - 4.401 2.54(2.17～2.87) <![CDATA[1×10 6.5 ]]> y = 10.370x - 4.696 2.84(2.44～3.20) <![CDATA[1×10 6 ]]> y = 11.086x - 5.315 3.02(1.05～4.49) <![CDATA[1×10 5.5 ]]> y = 13.183x - 7.203 3.52(3.13～3.89) <![CDATA[1×10 5 ]]> y = 16.382x - 9.6541 3.88(3.52～4.23)

[0084] Table 2. LC50 of strain 022017-9 against adult *Aphidius gracilis* 50 and regression equation

[0085] Vaccination time Probit regression equation <![CDATA[Lethal concentration and 95% confidence interval (×10 6 individuals / mL)]]> 72 y = 1.060x - 6.938 3.52(1.38～27.69) 96 y = 1.263x - 6.438 0.12(0.01～0.31)

[0086] 2.2 Effects of Cicadae periostracum infection on the survival rate and reproductive capacity of the cereal aphid

[0087] Inoculation with cicada flower was performed on *Aphidius gracilis* at different instars. The survival rate (lx) of adult aphids after inoculation generally decreased significantly from the 3rd day post-inoculation. Notably, when 2nd, 3rd, and 4th instar *Aphidius gracilis* were inoculated separately, the survival rate of these treatments did not show a significant decrease during the nymphal stage; instead, the survival rate began to decline sharply within 3-4 days of reaching adulthood. Furthermore, *Aphidius gracilis* in the late nymphal (4th instar) and adult stages all died within 5-6 days of entering adulthood after inoculation with cicada flower. In contrast, the survival time of early nymphs (2nd-3rd instar) was slightly delayed, typically dying within 7-8 days of entering adulthood. However, regardless of the inoculation stage, the survival rate of the inoculated groups was significantly lower than that of the control group. In the comparison of reproductive rate (mx), the reproductive rate (mx) of aphids inoculated at the 2nd, 3rd, and 4th instars and the adult stage was significantly lower than that of the control group. In contrast, the number of offspring produced by the control group remained relatively stable with increasing age, showing a slow downward trend. Figure 6 ).

[0088] 2.3 Effects of cicada flower infection on population parameters of the cereal constrictor aphid

[0089] The effects of infection with *Cicada spp.* at different instars on life parameters of *Aphis gracilis*, including net reproductive rate Ro, mean generation duration T, intrinsic growth rate rm, and periodic growth rate λ, are detailed in Table 3. *Cicada spp.* was used to inoculate *Aphis gracilis* at the 2nd, 3rd, and 4th instars, as well as in the adult stage.

[0090] The results showed that, compared with the control group, the net proliferation rate (R0) of *Aphidius gracilis* at all instars decreased significantly, with the most significant decreases observed in 2nd instar and adult inoculation, at 9.76 and 7.14, respectively. Regarding the mean generation duration (T), the T values ​​for all inoculation treatments were significantly lower than the control (9.19 days). Further analysis revealed that the mean generation duration of *Aphidius gracilis* inoculated at the 2nd instar (6.72 days) was significantly longer than that of individuals inoculated at the adult stage (5.79 days). However, in terms of intrinsic growth rate (rm) and periodic growth rate (λ), the 3rd instar (r... m＝ 0.45, λ=1.56) and 4-year-old (r m＝ The number of *Aphidius gracilis* inoculated with a growth rate of 0.43 (λ = 1.53) was significantly higher than that of the 2nd instar (r...). m＝ 0.34, λ=1.41) and adult stage (r m＝Individuals inoculated with 0.34, λ=1.40) (Table 3).

[0091] Table 3. Effects of infection with *Cicada nymph* at different ages on life parameters of *Aphidius gracilis*.

[0092] vaccination age <![CDATA[Net reproductive rate R0]]> Generation cycle T(d) <![CDATA[intrinsic growth rate r m > Weekly growth rate λ Comparison 36.59±1.73a 9.19±0.10a 0.39±0.00ab 1.48±0.00ab 2 years old 9.76±1.00c 6.72±0.33b 0.34±0.03b 1.41±0.04b 3 years old 16.59±0.97b 6.28±0.18bc 0.45±0.01a 1.56±0.02a 4 years old 15.30±1.17b 6.38±0.11bc 0.43±0.20a 1.53±0.03a adult 7.14±0.37c 5.79±0.08c 0.34±0.01b 1.40±0.01b

[0093] Note: Different lowercase letters in the same column indicate significant differences according to Tukey's test (P<0.05).

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Cordyceps cicadae, 022017-9, characterized in that, The accession number is CGMCCNo.42076.

2. A biocontrol agent, characterized in that, The active ingredients include the Cordyceps militaris strain 022017-9 of claim 1 and / or the metabolites of the Cordyceps militaris strain 022017-9.

3. The biocontrol agent according to claim 2, characterized in that, The *Cicada Flower* strain 022017-9 comprises spores and / or hyphae.

4. The biocontrol agent according to claim 2 or 3, characterized in that, The spore concentration of the *Cicada nymph* strain 022017-9 was 1×10⁻⁶. 5 cells / mL or 1×10 5 More than one per gram.

5. The biocontrol agent according to claim 4, characterized in that, The spore concentration of the *Cicada nymph* strain 022017-9 was 1×10⁻⁶. 6 ~1×10 7 per mL.

6. The application of the cicada nymph strain 022017-9 according to claim 1 or the biocontrol agent according to any one of claims 2 to 5 in aphid control.

7. The application according to claim 6, characterized in that, The aphid control measures include the control of adult aphids and / or the control of aphid nymphs.

8. The application according to claim 6, characterized in that, The aphid control measures include reducing at least one of the following indicators: aphid lifespan, aphid reproduction capacity, and aphid growth and development.

9. The application according to any one of claims 6 to 8, characterized in that, The targets of aphid control include at least one of the following: corn, wheat, and tea trees.

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