Lecanicillium attenuatum NBCC-L2 and application thereof

Abstract

The invention relates to Lecanicillium attenuatum NBCC-L2 and application of the Lecanicillium attenuatum NBCC-L2 in prevention and treatment of aphids and Coptotermes formosanus. The lecanicillium attenuatum NBCC-L2 is preserved in the China Center for Type Microbiological Culture Collection on October 23, 2024, and the preservation number of the lecanicillium attenuatum NBCC-L2 is CCTCC (China Center for Type Culture Collection) NO: M20242227. The invention also provides application of the strain in prevention and control of aphids and Coptotermes formosanus. The application comprises the following steps: culturing the strain to prepare a spore liquid or a spore-containing solid fungicide. The preparation method of the spore liquid comprises the following steps: eluting conidia by using sterile water containing Tween-80, carrying out magnetic stirring to obtain hypha-spore mixed liquid, and then filtering through double-layer lens wiping paper to finally obtain suspension liquid with the concentration of 106-108 spores / mL. The strain provided by the invention has a good biological control effect, can effectively control aphids and termites, and is helpful for reducing the use of pesticides and promoting the development of green agriculture as an environment-friendly and pollution-free substitute.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and specifically, it relates to a Lecanicillium attenuatum NBCC-L2 and its application. Background Art

[0002] Chemical pesticides have played an important role in the prevention and control of plant diseases and insect pests, but the environmental and health problems they bring are becoming increasingly severe. According to the latest research, the effective utilization rate of pesticides globally is still less than 30%. In China, the annual application area of chemical pesticides exceeds 280 million hectares, and the application amount reaches 500,000 - 600,000 tons. Among them, about 80% of the pesticides directly enter the environment, causing pollution to the soil, water bodies, and ecological systems (Wang Ming et al., 2022). The long-term and excessive use of chemical pesticides not only leads to a significant increase in the pest resistance, but also causes problems such as excessive pesticide residues in agricultural products, ecological imbalance, and pest resurgence, seriously threatening the sustainable development of agriculture and human health. To address these challenges, in recent years, green plant protection technologies have gradually become a research hotspot. For example, biological control technologies use natural regulation means such as natural enemy insects and microbial agents (such as entomogenous fungi and bacteria), showing the advantages of high efficiency and environmental protection (Zhang Wei et al., 2023). In addition, comprehensive measures such as physical control and agricultural ecological regulation have also achieved remarkable results in the control of plant diseases and insect pests. Therefore, in the future, it is necessary to accelerate the promotion of a diversified integrated management mode centered on biological control, combined with chemical control, physical control, and agricultural control, to build a green and sustainable plant protection system, providing strong support for the realization of high-quality agricultural development and ecological environment protection.

[0003] The green peach aphid (Myzus persicae) and the Formosan subterranean termite (Coptotermes formosanus) are important pests in the Aphididae family of Hemiptera and the Rhinotermitidae family of Isoptera, respectively, causing severe damage to the agricultural and construction sectors. The green peach aphid is tiny in size, highly fecund, capable of parthenogenesis, with severe generation overlap, and can form large populations in a short time, posing great challenges to control efforts. Currently, the control of the green peach aphid mainly relies on chemical pesticides. However, due to long-term irrational use, it has developed resistance to multiple pesticides, while also causing problems such as a reduction in natural enemy populations, excessive pesticide residues, and environmental pollution, seriously threatening the quality safety of agricultural products and ecological balance. In recent years, with global climate change and the intensive development of agriculture, the spread and damage of the green peach aphid have been further exacerbated, and it has become an important pest of various crops. The Formosan subterranean termite is known for its large colonies, clear division of labor, and strong reproductive ability, mainly damaging wooden structures and buildings. Its concealment makes control difficult. Traditional control methods rely on chemical agents, but long-term use not only leads to increased resistance of termites but also poses potential threats to the environment and human health. In addition, global trade and climate warming have also promoted the continuous expansion of the distribution range of the Formosan subterranean termite, making it an important global pest. Faced with these challenges, it is imperative to explore environmentally friendly control strategies. Biological control, as a sustainable management method, shows broad prospects in the control of the green peach aphid and the Formosan subterranean termite. For example, using entomopathogenic fungi to control the population of the green peach aphid and inhibit the Formosan subterranean termite can effectively reduce the use of chemical agents and reduce the harm to the environment and human health. Using Metarhizium fungi to control pests provides important support for green agriculture and integrated pest management, not only helping to ensure the safety of agricultural products and ecological balance but also providing new ideas and methods for dealing with the pest spread problems brought about by global climate change and the intensive development of agriculture, laying a solid foundation for achieving the goals of sustainable agriculture and environmental protection.

[0004] Akanthomyces attenuatum is an important entomopathogenic fungus belonging to the Ascomycota, Sordariomycetes, Hypocreales, and Cordycipitaceae. The research history of Lecanicillium can be traced back to 1861 when it was first discovered on Coccus viridis; in 1898, Zimmerman named it Cephalosporium lecanii Zimmerman; in 1939, Viegas classified it into the genus Verticillium based on its morphological characteristics and renamed it Verticillium lecanii. However, with the development of molecular biology techniques, Zare et al. reclassified Verticillium lecanii through morphological characteristics and rDNA-ITS sequence analysis and established the genus Lecanicillium, which mainly includes species such as Lecanicillium attenuatum, L. lecanii, and L. psalliotae. But in recent years, based on multi-gene phylogenetic analysis (such as ITS, LSU, etc. sequences), scholars have found that it has a closer genetic relationship with the genus Akanthomyces. In 2017, Kepler et al. reclassified some species originally belonging to Lecanicillium to Akanthomyces by integrating morphological and molecular data, thus establishing the current scientific name Akanthomyces attenuatum. Akanthomyces attenuatum is widely distributed in temperate and tropical regions of the world and mainly parasitizes various agricultural pests such as scale insects, aphids, and whiteflies. It attaches to the insect body surface through conidia and invades the body, secretes enzymes to decompose insect tissues, and ultimately causes the death of the host. Due to its high biological control potential, Akanthomyces attenuatum is widely used in organic agriculture and green agriculture. As an environmentally friendly biopesticide, it has the advantages of being safe for non-target organisms and not easily causing pest resistance; being able to isolate Akanthomyces attenuatum with excellent biocontrol effects can be used as an alternative for controlling some insects resistant to insecticides, thus having the effect of no pollution and no residue on the environment.

[0005] In summary, although Akanthomyces attenuatum has been widely studied, in practical applications, the control of aphids and Coptotermes formosanus is not yet mature. Existing biological control methods mostly rely on other entomopathogenic fungi, and the control effect and application scope are limited. Especially when dealing with aphids and Coptotermes formosanus with strong drug resistance, the effect is not obvious. Although Akanthomyces attenuatum has certain biological control potential against various pests under laboratory conditions, there is currently no mature application method for aphids and Coptotermes formosanus. Therefore, there is an urgent need to develop an efficient and environmentally friendly biocontrol strain to solve this problem, replace chemical pesticides, reduce environmental pollution and pest drug resistance, and promote the sustainable development of agriculture. Summary of the Invention

[0006] The first technical problem to be solved by the present invention is to provide an Akanthomyces attenuatum NBCC-L2 to solve the problems of drug resistance, environmental pollution, and excessive pesticide residues caused by the use of traditional chemical pesticides to control aphids and Coptotermes formosanus in the prior art.

[0007] To overcome the defects of the above prior art, the present invention provides an Akanthomyces attenuatum NBCC-L2, which was deposited at the China Center for Type Culture Collection on October 23, 2024. Its Latin name is Akanthomyces attenuatus NBCC-L2, and the deposit number is CCTCC NO: M20242227.

[0008] Compared with the prior art, the Akanthomyces attenuatum NBCC-L2 provided by the present invention is a natural biological control product that does not produce drug resistance problems. Using this strain to control pests can avoid environmental pollution and pesticide residue problems, and has less impact on non-target organisms, ensuring the stability of the ecosystem. The Akanthomyces attenuatum NBCC-L2 of the present invention can significantly increase the lethality rate of aphids and Coptotermes formosanus in a short time, ensuring its application effect in actual agricultural production. The principle includes: the Akanthomyces attenuatum NBCC-L2 in the present invention attaches to the surface of pests through its conidia and invades the body, releasing enzyme substances to decompose the tissues in the pests' bodies, thereby causing the pests to die quickly. This process effectively controls the pest population through the biological control effect of the strain and does not rely on the use of chemical pesticides. Therefore, it can overcome common problems such as drug resistance, pesticide residues, and environmental pollution in traditional technologies. In conventional technical means, the problems of difficult control of aphids and Coptotermes formosanus and enhanced drug resistance have been effectively solved. Through the Akanthomyces attenuatum NBCC-L2 of the present invention, the dependence on traditional pesticides can be significantly reduced, avoiding negative impacts such as pesticide resistance and environmental pollution, and providing an environmentally friendly alternative for modern agriculture.

[0009] In a possible implementation, the morphological characteristics of Lecanicillium attenuatum NBCC-L2 are described as follows: On PDA medium, the colony is velvety, white on the surface, orange-yellow on the back, with a small number of wrinkles; its conidia are cylindrical or oval, with a length of 3.7 ± 0.2 μm and a width of 1.33 ± 0.1 μm.

[0010] Compared with the prior art, by adopting the above technical solution, through optimizing the morphological characteristics and precisely controlling the growth conditions of the strain, the efficiency and stability of the strain can be ensured. Without relying on chemical pesticides, the strain of the present invention provides an efficient, safe and environmentally friendly biological control means, effectively solving the problems of enhanced pest resistance to pesticides, environmental pollution and pesticide residues, and providing a new solution for the sustainable development of agriculture.

[0011] Another technical problem to be solved by the present invention is to provide an application of Lecanicillium attenuatum NBCC-L2 to solve the problems of excessive dependence on chemical pesticides for the control of aphids and termites in the prior art, resulting in enhanced pest resistance to pesticides, environmental pollution and pesticide residues.

[0012] To overcome the defects of the above prior art, the present invention also provides an application of Lecanicillium attenuatum NBCC-L2, and the application includes the application of Lecanicillium attenuatum NBCC-L2 in the control of aphids and termites.

[0013] Compared with the prior art, the application of Lecanicillium attenuatum NBCC-L2 in the present application has the following advantages: In the application of the present invention, the use of chemical pesticides in the prior art is changed to a new strategy based on biological control. Utilizing the biological characteristics of Lecanicillium attenuatum, through natural control of the pest population, a more sustainable and environmentally friendly pest control is achieved. Lecanicillium attenuatum NBCC-L2 attaches to and invades the pest body through conidia, secretes enzymes to decompose the pest tissue, directly causing its death, making it highly pathogenic to the target pests and having a low environmental risk.

[0014] In a possible implementation, the aphid is Myzus persicae and the termite is Coptotermes formosanus.

[0015] Compared with the prior art, by adopting the above technical solution, two important pests that are significantly harmful in agricultural and urban environments are targeted for control, enhancing the targeting and practicality of the control. The Lecanicillium attenuatum NBCC-L2 of the present invention can adhere to the body surfaces of Myzus persicae and Coptotermes formosanus through its conidia, germinate and penetrate the cuticle, reproduce in the insect body and release various enzymes and toxins, destroying the tissue structure and causing the insects to die quickly.

[0016] In a possible implementation, the application includes culturing the Lecanicillium attenuatum NBCC-L2 strain and then making it into a spore liquid or a solid fungicide containing spores.

[0017] Compared with the prior art, adopting the above technical solution can significantly improve the application convenience of the strain and the flexibility of pesticide application, making it more suitable for various control scenarios such as field spraying, soil treatment, or bait placement. Through formulation treatment in liquid or solid form, the spore activity can be maintained and its stability and biological efficacy during storage, transportation, and application can be enhanced, ensuring its rapid attachment, germination, and pathogenesis after contacting the pest body surface.

[0018] In a possible implementation manner, the preparation method of the spore liquid includes the following steps: S1: Use sterile water containing Tween-80 to wash and elute the conidia of the strain of Lecanicillium attenuatum NBCC-L2, and then perform magnetic stirring to obtain a mycelium-spore mixture; S2: After filtering the mycelium-spore mixture treated in step S1, obtain the mother spore liquid, and finally prepare a suspension of 10 4 -10 8 spores / mL by gradient dilution.

[0019] Compared with the prior art, adopting the above technical solution effectively improves the spore activity, stability, and application effect of the prepared spore liquid, and provides a simple and effective production process, ensuring the long-term stability of the spore activity and control effect of the strain, providing a reliable solution for practical applications. By adjusting the spore concentration through the gradient dilution method, it ensures that the spore liquid has the best activity during application and can conveniently adjust the use concentration to adapt to different control requirements. And through filtration treatment, impurities and incompletely developed spores can be removed to ensure the final high-quality spore liquid.

[0020] In a possible implementation manner, in step S1, in the sterile water, the addition amount of Tween-80 is 0.05±0.02 wt%, and the conditions for the stirring are: magnetic stirring at an oscillation speed of 180 rpm for 25±5 min.

[0021] Compared with the prior art, adopting the above technical solution can effectively improve the separation efficiency and purity of the spores of Lecanicillium attenuatum NBCC-L2: As a non-ionic surfactant, Tween-80 can reduce the liquid surface tension and enhance the compatibility of conidia with water, making the spores more easily separated from the mycelium. And the setting of the Tween-80 concentration of 0.05±0.02 wt% can maximize the promotion of spore separation and avoid potential damage to the spores caused by excessive Tween-80, thereby improving the survival rate and activity of the spores.

[0022] In a possible implementation manner, in step S2, the filtering method is filtering through double-layer lens paper, and in the suspension, the spore concentration is 10 6 -108 spores / mL.

[0023] Compared with the prior art, adopting the above technical solution, the double-layer lens cleaning paper can provide multiple screening effects during the filtering process. The first-layer lens cleaning paper will intercept larger impurity particles, and the second-layer lens cleaning paper will further filter smaller impurities. In this way, it can ensure that the spores still maintain high activity and purity after passing through two layers of filtration, and the optimized control of the spore suspension concentration (10 6 -10 8 spores / mL) enables the spores to be evenly distributed during application, achieving the best control effect. Description of the Drawings

[0024] Figure 1 Figure showing a dead individual of Coptotermes formosanus covered by white hyphae; Figure 2 Figure showing the colony morphology of NBCC-L2 (the medium is PDA medium); Figure 3 Figure showing the spore morphology of NBCC-L2; Figure 4 Figure showing the electrophoresis pattern of the PCR amplification product in the molecular identification of NBCC-L2; Figure 5 Figure showing the schematic diagram of the molecular phylogenetic tree constructed for NBCC-L2. Detailed Embodiments

[0025] First of all, those skilled in the art should understand that these embodiments are only used to explain the technical principles of the embodiments of the present application and are not intended to limit the protection scope of the embodiments of the present application. Those skilled in the art can make adjustments according to needs to adapt to specific application scenarios.

[0026] The present invention provides an Akanthomyces attenuatus NBCC-L2, which was deposited at the China Center for Type Culture Collection on October 23, 2024. Its Latin name is Akanthomyces attenuatus NBCC-L2, and the deposit number is CCTCC NO: M20242227.

[0027] As a preferred solution, the morphological characteristics of the Akanthomyces attenuatus NBCC-L2 are described as follows: The colony on the PDA medium is velvety, white on the surface, orange-yellow on the back, with a small number of wrinkles; its conidia are cylindrical or elliptical, with a length of 3.7 ± 0.2 μm and a width of 1.33 ± 0.1 μm.

[0028] The present invention also provides the application of the Akanthomyces attenuatus NBCC-L2, and the application includes the application of the Akanthomyces attenuatus NBCC-L2 in controlling aphids and termites.

[0029] As a preferred embodiment, the aphids are Myzus persicae and the termites are Coptotermes formosanus.

[0030] As a preferred embodiment, the application includes culturing the strain Akanthomyces attenuatum NBCC-L2 and then preparing a spore liquid or a solid fungicide containing spores.

[0031] As a preferred embodiment, the method for preparing the spore liquid includes the following steps: S1: Using sterile water containing Tween-80 to wash the conidia of the strain Akanthomyces attenuatum NBCC-L2, and then performing magnetic stirring to obtain a mycelium-spore mixture; S2: After filtering the mycelium-spore mixture processed in step S1, obtaining a mother spore liquid, and finally preparing a suspension of 10 4 -10 8 spores / mL by gradient dilution.

[0032] As a preferred embodiment, in step S1, the addition amount of Tween-80 in the sterile water is 0.05±0.02 wt%, and the conditions for stirring are: magnetic stirring at an oscillation speed of 180 rpm for 25±5 min.

[0033] As a preferred embodiment, in step S2, the filtering method is filtering through double-layer lens paper, and in the suspension, the concentration of spores is 10 6 -10 8 spores / mL.

[0034] Since there are significant differences in the lethal effects of different strains of Akanthomyces attenuatum on aphids and termites, it is necessary to screen for highly effective strains. Biological experiments have shown that the Akanthomyces attenuatum NBCC-L2 strain of the present invention has excellent control effects on Myzus persicae and Coptotermes formosanus. The experimental results show that when the concentration is 10 4 -10 8 spores / mL, the cumulative mortality rate of Myzus persicae and the cumulative mortality rate of Coptotermes formosanus in Taiwan are higher than those of conventional technical means. When the concentration is 1×10 8 spores / mL, the cumulative mortality rate of Myzus persicae on the 6th day of NBCC-L2 is 100%, and the cumulative mortality rate of Coptotermes formosanus in Taiwan on the 5th day of NBCC-L2 is 99%. Therefore, the Akanthomyces attenuatum NBCC-L2 strain of the present invention can effectively control the populations of Myzus persicae and Coptotermes formosanus and has broad application prospects in the field of biological control. This strain can be used as an environmentally friendly alternative for controlling drug-resistant insects and has environmentally friendly characteristics such as pollution-free and residue-free.

[0035] The following are specific embodiments that specifically combine data and experimental verification to further supplement and explain the above-mentioned scope of the technical solution of the present invention: Example: This example provides an Akanthomyces attenuatus NBCC-L2, its specific acquisition method, and its application. The Akanthomyces attenuatus NBCC-L2 was deposited at the China Center for Type Culture Collection on October 23, 2024. Its Latin name is Akanthomyces attenuatus NBCC-L2, and the deposit number is CCTCC NO: M20242227. This strain was isolated and screened from the Coptotermes formosanus with mycelium covering its body in Ninghai District, Ningbo City, Zhejiang Province, China. On a PDA plate, its colony is velvety, white on the surface, orange-yellow on the back, with a small number of wrinkles, and the spores are cylindrical and elliptical, with a spore length of 3.7 μm and a width of 1.33 μm.

[0036] Specifically, the collection method of this strain is as follows: Termite nests were collected in the wild in Ninghai City, Ningbo City, Zhejiang Province, China, sealed in plastic bags and brought back to the laboratory for breeding. Dead Coptotermes formosanus with mycelium covering its body were found among the bred Coptotermes formosanus, and then moisturizing culture was carried out (as Figure 1 shown). After hyphae or conidia appeared on the surface of the insect body, a small amount of conidia was picked with an inoculation needle in a laminar flow hood; the conidia were inoculated on Potato Dextrose Agar medium, and the strain was obtained after culturing at a constant temperature of 25 °C for 3 - 7 days. Then, a single colony was cut with an inoculation loop and inoculated on a PDA plate for continued culture to obtain a strain that was preliminarily Akanthomyces from its morphology, named NBCC-L2. A mycelium block was cut and transplanted onto a PDA slant for continued culture and stored in a refrigerator at 4 °C.

[0037] Morphological identification was carried out on the strain collected above, including: The strain was inoculated onto a PDA plate and cultured at 25 °C for 5 days to observe the colony morphology: showing typical filamentous fungal characteristics. The colony was initially white or light yellow, with a texture of villous or cottony, and as the culture time extended, the color might gradually deepen to light brown. The edge of the colony was relatively neat at the beginning. The hyphae were slender and richly branched, transparent or light-colored, and obvious septate structures could be seen under a microscope. The conidia were elliptical or cylindrical, transparent or light-colored, the conidiophores were upright, and spore chains might be formed at the top. Based on morphological observation and judgment, it was preliminarily determined that the strains collected were all Akanthomyces attenuatus. Specifically as Figures 2-3 shown, where Figure 2 is the colony morphology diagram of NBCC-L2 (the medium is PDA medium), Figure 3 is the spore morphology diagram of NBCC-L2.

[0038] The strains of the above embodiments of the present invention were further identified by molecular biology, including: Molecular biology identification method: The molecular biology identification of the strains was carried out as follows: (1) Use a 1 mL sterile pipette tip to scrape the well-grown mycelium of the strain NBCC-L2, place it in a sterile mortar, quickly grind it into powder with liquid nitrogen, transfer it into a 1.5 mL centrifuge tube, and use a fungal genomic DNA extraction kit (Sangon Biotech, Shanghai) to extract the total fungal genomic DNA; (2) The primers used for PCR of fungal DNA were universal primers: The fungal universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATAGC-3’) were used to amplify its rDNA-ITS sequence. The PCR reaction system was 25 μL, including 1 μL of ITS1 and 1 μL of ITS4, 12.5 μL of PCR MasterMix, 1 μL of template DNA, and 9.5 μL of ultrapure water;

[0039] (3) PCR reaction conditions: Pre-denaturation at 95 °C for 3 min; denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 1.5 min, for 35 cycles; extension at 72 °C for 10 min; The PCR products were detected by 1% agarose gel electrophoresis, and the amplification results were observed under a gel imaging system. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The bases with poor quality at both ends of the obtained sequence were removed, and the optimized sequence was submitted to the NCBI website for BLAST alignment. Sequences with high homology to the target sequence were downloaded, and the Mega X software was used to construct the phylogenetic tree of the strain by the neighbour-joining method (NJ) with 1000 bootstrap validations.

[0040] The identification results were as Figure 4 shown. The electrophoretic bands in each lane were relatively clear, and the gene fragment of the strain could be accurately judged to be 591 bp according to the migration distance of the PCR product.

[0041] The ITS sequences of the obtained NBCC-L2 strain genotype (NCBI accession number: PQ357183) and the sequences related to Akanthomyces attenuatus downloaded from GenBank were combined to construct a molecular phylogenetic tree. The results were as Figure 5 shown, Figure 5 which was a schematic diagram of the molecular phylogenetic tree constructed for NBCC-L2, and it could be identified that the NBCC-L2 strain belonged to Akanthomyces attenuatus.

[0042] Biological assays were performed on the strains obtained in the above embodiments, including: Test strain: strain NBCC-L2 obtained in the embodiment; Test insects: The aphid population used in the test was collected from Taoshan, Fenghua District, Ningbo City and identified as Myzus persicae. The collected aphids were continuously reared on indoor potted radish seedlings; the Coptotermes formosanus used in the test was reared in this laboratory under the conditions of temperature 26 °C, relative humidity 65%, and photoperiod L:D = 12:12.

[0043] Biological assay test method: In this embodiment, spore liquid was used as the applied strain to control aphids and termites, including: (1) Preparation of spore suspension S1: After culturing on a PDA plate at 28 ± 1 °C for 7 days, the conidia of entomogenous fungi with sufficient sporulation were washed with 0.05% Tween-80 sterile water, stirred with a magnetic stirrer, and then placed on a shaker and shaken at 180 rpm and 25 °C for 25 min; S2: Filter the product of S1 with double-layer lens paper, then count with a hemocytometer, measure the concentration of the mother liquor, and prepare a spore suspension with a spore concentration of 1×10 4 -1×10 8 spores / mL.

[0044] (2) Virulence determination of entomogenous fungi against Myzus persicae The Potter Spray Tower method was used for determination: Specific determination steps: Place the water agar plate with aphids on the stage of the Potter Spray Tower (diameter 11 cm, area 95 cm 2 ), set the working pressure of the Potter Spray Tower to 7.5 lbs / in 2 , then start spraying and evenly spray 3.0 mL of spore suspension with different concentrations from the nozzle onto the water agar plate on the stage from top to bottom; Use the Tween water containing 0.05% Tween-80 as the control treatment, with 4 replicates for each treatment concentration. After treatment, cover with plastic wrap with pinholes and place in a constant temperature incubator at (25 ± 0.5) °C, L:D = 16:8 for rearing; Observe the test insects regularly every day, record the number of dead insects, and pick the dead insects to the edge of the plate to observe whether they are infected. If new nymphs appear, pick them out immediately and observe continuously for 7 days.

[0045] (3) Virulence determination of entomogenous fungi against Coptotermes formosanus The pathogenicity of the strain was determined by the drop method: First, filter paper was laid on the bottom of each 9-cm petri dish and moistened with water, and then 20 mature termite workers with good vitality were placed in the petri dish; using sterile water as a control, the spore suspension was dropped on the pronotum of the termite with a 0.1-μL micropipette (the dose dropped on each termite = 0.1 μL × the number of drops of the titration solution × the concentration of the bacterial solution). The concentration of the spore suspension used was 1×10 8 spores / mL. The mother liquor was diluted 4 times with sterile water at a 10-fold gradient, and the final spore solution concentrations obtained were 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 and 1×10 4 spores / mL. Each concentration of the drug in the experiment and the control were independently repeated 5 times. After inoculation, observations were made regularly every day until the mortality rate of the control reached 20%, and the number of dead larvae was recorded. The dead larvae were cultured under moisturized conditions for observation. When white hyphae grew on the surface of the dead insect body, it was considered to be infected and killed by Isaria farinosa.

[0046] (3) Data processing The experimental data were analyzed using SPSS 19.0 software. One-way analysis of variance was used to analyze the results, and Tukey's test was used to analyze the significance of differences.

[0047] Experimental results: 1. Results of the virulence determination of the strain against Myzus persicae The pathogenicity of the strain in the above-mentioned examples of the present invention against Myzus persicae showed that at high concentrations (>10 6 spores / ml), the death of nymphs began to occur on the 3rd day after inoculation; on the 4th day after inoculation, the death of nymphs occurred at each concentration gradient (10 4 - 10 8 spores / ml); at high concentration treatments (>10 6 spores / ml), the death peak of most strains was reached on the 5th and 6th days; when the observation ended on the 7th day, the corrected mortality rate of nymphs in the highest concentration treatment (10 8 spores / mL) of each strain measured reached over 95%; the corrected mortality rate of nymphs of the NBCC-L2 strain reached 83.7% on the 5th day when treated with 10 8 spores / mL; the cumulative mortality rate of Myzus persicae on the 6th day was 100%. The above results indicate that the strain showed high pathogenicity against Myzus persicae.

[0048] By calculating the median lethal concentration (LC 50 ) of the strain against Myzus persicae nymphs at different time periods, and the median lethal time (LC 50The calculation results (Tables 2 and 3) show that the median lethal concentration of the strain on the 6th day is 2.84×10 5 spores / mL, and the median lethal time at a concentration of 10 8 spores / mL is 4.11 days. The specific test results are shown in Tables 1 - 3:

[0049] Table 1 Lethality rate of strain NBCC-L2 against nymphs of Myzus persicae

[0050] Table 2 Median lethal concentration of strain NBCC-L2 against nymphs of Myzus persicae

[0051] Table 3 Median lethal time of strain NBCC-L2 against nymphs of Myzus persicae

[0052] Note: After Tukey test, different lowercase letters in the same column indicate significant differences in pathogenicity among different strains (P<0.05) 2. Virulence determination results of the strain against Coptotermes formosanus The virulence effect of NBCC-L2 against Coptotermes formosanus was determined by titration method: Through bioassay experiments, it was found that when treated with 1×10 8 spores / mL, the mortality rate of Coptotermes formosanus reached 99% first; at 4 days of treatment, in the treatment group with 1×10 8 spores / mL, the highest mortality rate reached 82.7%, and there were no significant differences among other treatment groups (P<0.05); at 5 days and 6 days of treatment, in the treatment group with 1×10 8 spores / mL, the highest mortality rate reached over 99%, higher than other treatment groups. At 7 days of treatment, the mortality rate of the treatment group with 1×10 8 spores / mL reached 100%.

[0053] The calculation results of the median lethal concentration (LC 50 ), and the median lethal time at different concentrations (LC 50 ), of the strain against Coptotermes formosanus over time show that the median lethal concentration of the strain on the 5th day is 1.22×10 6 spores / mL, and the median lethal time at a concentration of 10 8 spores / mL is 3.08 days, indicating that the strain has high pathogenicity to Coptotermes formosanus. The specific test results are shown in Tables 4 - 6: Table 4: Lethality rate of strain NBCC-L2 against Coptotermes formosanus

[0054] Table 5: Median lethal concentration of strain NBCC-L2 against Coptotermes formosanus

[0055] Table 6: Median lethal time of strain NBCC-L2 against Coptotermes formosanus Note: After Tukey test, different lowercase letters in the same column indicate significant differences in pathogenicity among different strains (P < 0.05). Verified by the above embodiments of the present invention, the Lecanicillium attenuatum NBCC-L2 (deposit number CCTCC NO: M20242227) provided by the present invention solves the key problems of the existing entomogenous fungi, such as a single host range, low lethality efficiency, and insufficient field adaptability. Traditional strains usually only target pests at the order level (such as Lepidoptera), while NBCC-L2 realizes efficient cross-order control of the hemipteran Myzus persicae and the isopteran Coptotermes formosanus through unique biological characteristics (the colony morphology is white and flocculent, and the spores are cylindrical / elliptical, 3.7 μm in length and 1.33 μm in width) and molecular markers. The above experimental data of the present invention show that at a concentration of 1×10 8 spores / mL, the corrected mortality rate of M. persicae reached 100% on the 6th day (Table 1), the mortality rate of C. formosanus reached 99.1% on the 5th day (Table 4), and the median lethal time (LT 50 ) was shortened to 4.11 days (Table 3) and 3.08 days (Table 6) respectively. Moreover, suspensions in the range of 1×10 4 -1×10 8 spores / mL also achieved good technical effects. In addition, the present invention also confirmed its taxonomic status through molecular phylogenetic tree analysis and realized rapid infection through direct spraying of the spore suspension, solving the technical bottlenecks of traditional strains, such as slow action, strong host specificity, and poor environmental tolerance, and providing an efficient and stable microbial resource for the green control of agricultural pests.

[0056] In the description of the present application, the descriptions referring to terms such as "one embodiment", "some embodiments", "in this embodiment", "specific examples", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without conflict, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0057] As described above, it is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present application should be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. An Isaria attenuata NBCC-L2, characterized in that, The Akanthomyces attenuatus NBCC-L2 was deposited at the China Center for Type Culture Collection on October 23, 2024. Its Latin name is Akanthomyces attenuatus NBCC-L2, and the deposit number is CCTCC NO: M20242227.

2. The Lecanicillium attenuatum NBCC-L2 according to claim 1, characterized in that, The morphological characteristics of the Akanthomyces attenuatus NBCC-L2 are described as follows: The colony on the PDA medium is velvety, white on the surface, orange-yellow on the back, with a small number of wrinkles; its conidia are cylindrical or elliptical, with a length of 3.7 ± 0.2 μm and a width of 1.33 ± 0.1 μm.

3. Use of Lecanicillium attenuatum NBCC-L2 according to any one of claims 1-2, characterized in that, The application includes the application of the Akanthomyces attenuatus NBCC-L2 in controlling aphids and termites.

4. Use of Lecanicillium attenuatum NBCC-L2 according to claim 3, characterized in that, The aphids are Myzus persicae, and the termites are Coptotermes formosanus.

5. Use of Lecanicillium attenuatum NBCC-L2 according to claim 3, characterized in that, The application includes culturing the Akanthomyces attenuatus NBCC-L2 strain and then making it into a spore liquid or a solid fungicide containing spores.

6. Use of Lecanicillium attenuatum NBCC-L2 according to claim 5, characterized in that, The preparation method of the spore liquid includes the following steps: S1: Use sterile water containing Tween-80 to wash the conidia of the Akanthomyces attenuatus NBCC-L2 strain, and then perform magnetic stirring to obtain a mycelium-spore mixture; S2: After filtering the mycelium-spore mixed solution processed in step S1, a spore mother liquor is obtained, and finally a suspension of 10 4 -10 8 spores / mL is prepared by gradient dilution.

7. Use of Lecanicillium attenuatum NBCC-L2 according to claim 6, characterized in that, In the step S1, in the sterile water, the addition amount of Tween-80 is 0.05 ± 0.02 wt%, and the stirring conditions are: magnetic stirring at an oscillation speed of 180 rpm for 25 ± 5 min.

8. Use of Lecanicillium attenuatum NBCC-L2 according to claim 6, characterized in that, In the step S2, the filtering method is filtering through a double-layer lens wiping paper. In the suspension, the concentration of spores is 10 6 -10 8 spores / mL.

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