Method for evaluating the susceptibility of spodoptera frugiperda to metarhizium rileyi and application thereof

Abstract

This invention discloses a method and application for evaluating the susceptibility of the fall armyworm to *Metarhizium anisopliae*. The method involves extracting epidermal compounds from fifth-instar larvae of the fall armyworm with methanol, then detecting the germination rate of these epidermal compounds against *Metarhizium anisopliae*, and evaluating the fall armyworm's susceptibility to *Metarhizium anisopliae* based on the germination rate results. This invention establishes for the first time the correlation between the compositional characteristics of insect epidermal compounds and their impact on *Metarhizium anisopliae* germination and the susceptibility to entomopathogenic fungi. It can evaluate the fungal susceptibility of pest populations in a relatively short time (more than 70% shorter than traditional bioassay methods), with high accuracy. This provides technical support for the rational selection, precise application, and effect prediction of biological pesticides in the field, and has broad application value.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biological control technology, specifically involving a method for rapidly evaluating the germination rate of Metarhizium anisopliae using the chemical composition of insect epidermis. This method can be used for predicting the effectiveness of biological pesticides before field application and for formulating pest population control strategies. Background Technology

[0002] The fall armyworm (Spodoptera frugiperda) is a major invasive pest native to the Americas. Since its invasion of my country in 2019, it has spread to most corn and rice-growing areas, posing a serious threat to food security. Metarhizium rileyi, a highly effective biological pesticide for controlling fall armyworm and other lepidopteran pests, has advantages such as being environmentally friendly and preventing pests from developing resistance, making it an important means of green control for fall armyworm.

[0003] However, existing research and production practices show that the control efficacy of *Metarhizium anisopliae* against fall armyworm populations fluctuates significantly: for the same strain and application concentration, the mortality rate of fall armyworm populations can vary between 20% and 100%, and the median survival time (MST) can fluctuate between 3.0 and 15.0 days. This instability in control efficacy poses difficulties for the widespread application of biopesticides in the field and is one of the key bottlenecks restricting the large-scale application of insect pathogenic fungal biopesticides.

[0004] Existing methods for assessing insect susceptibility to biopesticides mainly include:

[0005] (1) Indoor surface immersion or injection bioassays require feeding a large number of test insects and continuously observing and recording for more than 5-10 days, which is a long cycle and labor-intensive, and cannot meet the needs of rapid decision-making in the field.

[0006] (2) Transcriptome sequencing can reveal differences in immune response, but the inventors’ previous research found that the expression levels of epidermal and intestinal immune genes (Toll, Imd signaling pathway-related genes, etc.) of fall armyworm are negatively correlated with their actual resistance to Metarhizium anisopliae. That is, populations with high expression levels of immune genes are more sensitive to fungi. Therefore, transcriptome data cannot be used as a reliable indicator for sensitivity assessment. Moreover, this method is costly and time-consuming, making it difficult to promote its application.

[0007] (3) Existing technologies have not yet established a method to directly predict the susceptibility of insect pathogenic fungi by utilizing the chemical components of insect epidermis, and lack technical solutions to associate the types of epidermal compounds and their antibacterial / germination-promoting activities with the overall drug resistance of pests. Summary of the Invention

[0008] To address the problems of long bioassay cycles, high transcriptome detection costs, and unreliable indicators in existing technologies, this invention aims to provide a method for rapidly evaluating the susceptibility of lepidopteran pests such as the fall armyworm to entomopathogenic fungi such as Metarhizium anisopliae based on the characteristics of epidermal compounds. This method assesses the fungal susceptibility of pest populations in a shorter time (more than 70% shorter than traditional bioassay methods) by detecting the effect of insect epidermal extracts on fungal spore germination, providing technical support for the rational selection, precise application, and effect prediction of biopesticides in the field.

[0009] Therefore, one objective of this invention is to provide a method for evaluating the susceptibility of fall armyworm to Metarhizium anisopliae, comprising the following steps:

[0010] 1) Take a fifth-instar larva of the fall armyworm, freeze it on ice until stunned, place it in a 5 mL centrifuge tube, add 2 mL of methanol, shake well, and extract it at room temperature in the dark for 2 h to obtain a methanol extract.

[0011] 2) Transfer the extract to a sample vial, concentrate and dry the sample under a nitrogen stream, then add 1 mL of methanol to resuspend the sample to obtain the epidermal extract;

[0012] 3) Place 5 μL of epidermal extract in the center of a solid water agar. After the solvent evaporates, add 5 μL of 1×10⁻⁶ agar. 6 A suspension of Metarhizium anisopliae per mL was poured onto the center of the solid water agar and then incubated at 27°C and 100% humidity for 24 h. After incubation, five fields of view were selected under a microscope, and the number of germinations of 200 conidia in each field of view was counted and the spore germination rate was calculated.

[0013] 3) Based on the germination rate, give the results of the sensitivity evaluation of fall armyworm to Metarhizium anisopliae.

[0014] Of course, the above steps can simultaneously test the susceptibility of multiple fifth-instar larvae of fall armyworm, so as to better understand the fall armyworm population under a certain condition / environment, and make better evaluations and control measures.

[0015] Furthermore, the fall armyworm sensitivity evaluation results in step 3) include three levels: high sensitivity, medium sensitivity, and low sensitivity. The germination rate of the high sensitivity level is not less than 65% (greater than or equal to 65%), the germination rate of the medium sensitivity level is between 45% and 65% (greater than or equal to 45% and less than 65%), and the germination rate of the low sensitivity level is less than 45% (less than 45%).

[0016] Furthermore, the low-sensitivity epidermal extract contains compounds with strong antibacterial activity; the medium-sensitivity epidermal extract contains compounds with weak antibacterial activity; and the high-sensitivity epidermal extract does not contain compounds with significant antibacterial activity.

[0017] Furthermore, the strong antibacterial active compound includes hydroxybenzoic acid; the weak antibacterial active compound includes α-linolenic acid.

[0018] The second objective of this invention is to provide the application of the above-mentioned method in guiding the control of fall webworm by Metarhizium anisopliae in the field.

[0019] Preferably, for the highly sensitive level, the method for controlling fall webworm in the field is to directly apply the normal concentration of Metarhizium anisopliae; for the moderately sensitive and low sensitive levels, the method for controlling fall webworm in the field is to increase the application concentration and / or frequency of Metarhizium anisopliae, or to apply it together with other preparations that can produce a synergistic effect with Metarhizium anisopliae. Other preparations include methyl benzoate emamectin (Dongguan Ruifang National Defense Biotechnology Co., Ltd.) or Bt preparations (32000 IU / mg Bacillus thuringiensis wettable powder, Wuhan Kenuo Biotechnology Co., Ltd.).

[0020] The third objective of this invention is to provide a method for improving the control effect of Metarhizium anisopliae on fall armyworm, characterized by a step of reducing the content of antibacterial active compounds in the epidermis of fall armyworm, wherein the antibacterial active compounds include one or more of p-hydroxybenzoic acid, lactic acid, oleic acid and α-linolenic acid.

[0021] The fourth objective of this invention is to provide the application of hydroxybenzoic acid in screening highly virulent strains of Metarhizium anisopliae.

[0022] In their research, the inventors unexpectedly discovered that the methanol extract of the epidermis of fall armyworms that feed on artificial feed, corn leaves, and rice leaves showed significant differences in its ability to promote the germination of Metarhizium anisopliae spores. This discovery established for the first time the correlation between the compositional characteristics of insect epidermal compounds and their influence on the germination of Metarhizium anisopliae and the susceptibility of insect pathogenic fungi, providing a technical basis for this invention and thus obtaining a method for evaluating the susceptibility of fall armyworms to Metarhizium anisopliae.

[0023] Beneficial effects:

[0024] (1) Short detection cycle: Compared with the traditional body surface immersion method or injection method bioassay which requires an observation cycle of more than 5-10 days, the detection cycle of this invention only requires 24-72 hours, which improves efficiency by more than 70%.

[0025] (2) Reliable indicators: The germination rate and antibacterial activity indicators of the epidermal compounds used in this invention are highly consistent with the actual bioassay results (consistent with the mortality rate and MST change trends), overcoming the defects of the transcriptome immune gene expression indicators being negatively correlated with actual resistance and unreliable;

[0026] (3) Simple operation and low cost: The required equipment is conventional laboratory instruments (water agar plate, microscope, filter paper diffusion device), and there is no need to raise test insects on a large scale. The detection cost is significantly lower than that of bioassay and transcriptome sequencing.

[0027] (4) Clear application value: It can provide theoretical basis and technical support for the rational selection of biological pesticides such as Metarhizium anisopliae in the field, precise adjustment of pesticide concentration and prediction of control effect, which is of great significance for improving the scientificity and effectiveness of biological control;

[0028] (5) High applicability: The principle on which this method is based (epidermal compounds affect fungal spore germination) is universal and can be extended to evaluate the susceptibility of other lepidopteran pests (such as beet armyworm, cotton bollworm, etc.) to Metarhizium anisopliae and other insect pathogenic fungi of the same genus. Attached Figure Description

[0029] Figure 1 The germination of Metarhizium anisopliae on epidermal compounds;

[0030] Figure 2 The content of single compounds in the cuticle of the fall armyworm in response to different food sources;

[0031] Figure 3 The experiment on the antibacterial zone of epidermal compounds of fall armyworm in the artificial feed group included: A: p-hydroxybenzoic acid compound; B: lactic acid compound; C: oleic acid compound; D: palmitoleic acid compound; E: stearic acid compound.

[0032] Figure 4 The experiment on the antibacterial zone of epidermal compounds of fall armyworm in the maize group included: A: α-linolenic acid compound; B: salicylic acid compound; C: eicosadienoic acid compound; D: linoleic acid compound.

[0033] Figure 5 The experiment on the inhibition zone of epidermal compounds of fall armyworm in rice group. Among them, A: carnitine compound; B: acetylcarnitine compound; the middle is the negative control; the far right is the positive control; and the other three are compounds of different concentrations.

[0034] Figure 6 This study analyzed the survival rate of fall armyworms infected with Metarhizium anisopliae by immersion method and fed on different foods. In the figure, A: survival rate, B: median survival time.

[0035] Figure 7 This study analyzed the survival rate of 5th instar fall armyworms infected with Metarhizium anisopliae by immersion method and fed on different foods. In the figure, A: survival rate, B: median survival time.

[0036] Figure 8 This study analyzed the survival rate of fall armyworms infected with Metarhizium anisopliae via injection and fed on different foods. Detailed Implementation

[0037] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are all conventional commercially available products or can be obtained by known methods.

[0038] Test strain and spore suspension preparation: The test strain was *Metarhizium rileyi* GZUMr46, preservation number: CCTCC M 2023161, disclosed in patent CN116333892A. The strain was inoculated onto SMAY agar plates using an inoculation loop and incubated upside down in a constant temperature incubator at 28°C with a 14:10 (L:D) h photoperiod until colony formation. In a clean bench, the strain was scraped from SMAY agar into a 15 mL sterile centrifuge tube, and an appropriate amount of sterile 0.05% Tween-80 solution was added. The tube was vortexed for 20 min to allow for complete spore release. The bacterial suspension was filtered through sterile filter paper folded into a funnel shape into a new 15 mL sterile centrifuge tube. 5 μL of the spore suspension was aspirated onto a hemocytometer and observed and counted under an optical microscope to determine the spore concentration. Finally, the spores were diluted to 1×10⁻⁵ using sterile water containing 0.05% Tween-80. 8 cells / mL and 1×10 6 Prepare two concentrations of Metarhizium anisopliae spore suspensions per mL for later use.

[0039] Test insects: The inventors' laboratory collected *Spodoptera frugiperda*, a fall armyworm, from Tianba, Qiannan Prefecture, Guizhou Province. Larvae emerged and multiplied in the rearing chamber. These larvae were then fed corn leaves, rice leaves, and artificial feed, respectively, for more than 10 generations to establish stable feeding patterns for the experiment. The larvae were divided into three groups: a corn group, a rice group, and an artificial feed group.

[0040] Artificial feed formula for fall armyworm: 120g soybean flour, 120g wheat bran flour, 48g yeast powder, 24g casein, 24g agar powder, 2.4g sorbic acid, 0.24g cholesterol, 0.24g inositol, 9.6g L-ascorbic acid, 1.2g choline chloride, 2g p-hydroxybenzoic acid, and 1.2L water.

[0041] Experimental solution and culture medium formulations:

[0042] SMAY medium: 10 g / L yeast extract, 40 g / L sucrose, 10 g / L peptone, 20 g / L agar, 1 L ultrapure water, mixed thoroughly, and autoclaved at 121 °C for 20 min.

[0043] Solid water agar: 20 g / L agar, 1 L ultrapure water, mix well, and autoclave at 121 ℃ for 20 min.

[0044] 0.05% Tween-80 solution: Prepare a 0.05% Tween-80 solution by mixing Tween-80 solution and sterile water according to the specified ratio.

[0045] Data statistics and analysis:

[0046] Experimental data were compiled using Excel, and cumulative mortality and cumulative corrected mortality were calculated. GraphPad Prism 9 software was used to calculate the median survival time (MST) for insects that killed 50% of the target population in the indoor bioassay using Kaplan-Meier survival analysis. SPSS 26.0 software was used for statistical analysis. Independent samples t-tests or one-way ANOVA were selected based on data characteristics. Post-hoc multiple comparisons in ANOVA used the LSD method or Duncan's method. A p-value < 0.05 was used as the criterion for statistical significance. The relevant calculation formulas are shown below:

[0047] Cumulative mortality rate = (Total number of dead insects treated / Total number of insects treated) × 100%

[0048] Cumulative corrected mortality rate = (Cumulative mortality rate in the treatment group - Cumulative mortality rate in the control group) / (100 - Cumulative mortality rate in the control group) × 100%

[0049] Survival rate = 1 - cumulative corrected mortality rate.

[0050] Example 1: Statistical analysis of antifungal germination rates of epidermal compounds extracted from fall armyworms that feed on different foods.

[0051] Three different organic solvents—hexane, methanol, and chloroform—were used to extract epidermal compounds from fall armyworm larvae in three groups: corn, rice, and artificial feed. The procedure was as follows: Three 5th instar fall armyworm larvae of uniform size, feeding on different foods, were selected from each of the three groups. After being stunned on ice, each larva was placed in a 5 mL centrifuge tube (one larva per tube). Then, 2 mL of hexane, methanol, or chloroform was added to each tube (each organic solvent was added to the corresponding tube for the three groups of larvae feeding on different foods). After mixing, extraction was carried out at room temperature (25°C) in the dark for 2 hours. The extract was carefully transferred to sample vials using a pipette. The samples were concentrated and dried under nitrogen flow, then resuspended in 1 mL of the initial solvent (hexane, methanol, or chloroform) to obtain fall armyworm epidermal extracts (three types of hexane epidermal extracts, three types of methanol epidermal extracts, and three types of chloroform epidermal extracts). Take 5 μL of each of the nine samples (hexane, chloroform, methanol, and epidermal extract) and drop them onto the center of each solid water agar plate. After the solvents evaporate, add another 5 μL of 1×10⁻⁶ solution to each sample. 6 A suspension of *Metarhizium anisopliae* at a concentration of 1 / mL was also inoculated onto the center of a solid water agar plate. The plates were incubated at 27 °C and 100% humidity for 24 h. Each sample was taken in triplicate, i.e., three replicates. Five fields of view were selected under a microscope for each plate, and the number of germinating conidia (200 spores per field) was counted. The spore germination rate was calculated as follows: Total germination rate of a plate = (Number of germinations at position 1 + Number of germinations at position 2 + Number of germinations at position 3 + Number of germinations at position 4 + Number of germinations at position 5) / (Number of 200 spores × 5) × 100%; Final germination rate of each sample = (Rate of plate 1 + Rate of plate 2 + Rate of plate 3) / 3.

[0052] The above experiment used a single 5th instar larva as a biological replicate, with a total of 3 biological replicates.

[0053] Germination rate results as follows Figure 1 As shown, the germination rate of the chloroform extract did not differ significantly among fall armyworms feeding on different diets; the germination rates for the artificial feed group, corn group, and rice group were 42%, 43%, and 39%, respectively. In the hexane extract, the germination rates of the artificial feed group and corn group were lower than those of the rice group, at 28%, 26%, and 39%, respectively. In the methanol extract, the germination rate of the artificial feed group was significantly lower than that of the corn and rice groups, at 40%, 63%, and 66%, respectively. These significant differences among the three extraction solvents indicate that the components or contents of epidermal compounds extracted by different organic solvents are not identical.

[0054] Studies have found that *Metarhizium anisopliae* spores lack nutrients and require external nutrient sources to support germination. In this study, after 24 hours of treatment, the average germination rate of *Metarhizium anisopliae* treated with sterile water was only 8%. The epidermal compounds extracted with methanol, chloroform, and n-hexane all showed significantly higher germination rates for *Metarhizium anisopliae* than the control group. This indicates that fungi can utilize organic compounds on the cuticle surface as nutrients for germination and other pre-osmotic growth stages. However, the different compositions of these epidermal compounds lead to varying germination rates in *Metarhizium anisopliae*, which in turn result in varying mortality rates in *Pseudomonas repens*.

[0055] Example 2: Determination of the antifungal activity of epidermal compounds in fall armyworms fed on different foods

[0056] The methanol epidermal extracts from fall armyworms that fed on different foods, obtained in Example 1, were sent to Baiqu Biotechnology Co., Ltd. for processing and analysis to determine the composition of epidermal compounds. The compound content analysis of the methanol epidermal extracts is as follows: Figure 2 As shown, the artificial feed group contained the highest levels of p-hydroxybenzoic acid, palmitoleic acid, oleic acid, stearic acid, etc., the corn group contained linoleic acid, α-linolenic acid, salicylic acid, etc., and the rice group contained carnitine, acetylcarnitine, pyruvic acid, etc.

[0057] Uniform filter paper was selected and punched into circular discs of the same diameter. After sterilization and drying, 500 μl of Metarhizium anisopliae bacterial suspension was injected into agar plates cooled to approximately 50°C. The mixture was thoroughly mixed and poured into agar plates (approximately 15 mL / plate). The plates were allowed to solidify horizontally for later use. To detect the antifungal activity of the epidermal compounds, the five single compounds with the highest content in fall armyworm in the artificial feed group, the four single compounds with the highest content in the corn group, and the two single compounds with the highest content in the rice group were selected. 5 μL solutions of different concentrations (100 mg / ml, 50 mg / ml, 10 mg / ml) of each single compound were inoculated onto filter paper discs of the culture medium. Amphotericin B was used as a positive control, and the solvent was used as a negative control. Each treatment was repeated three times, and the inhibitory effect was measured after 3 days of incubation.

[0058] 2.1 Experiment on the antibacterial zone of epidermal compounds in the artificial feed group of fall armyworm

[0059] Through heatmap analysis of the content of single compounds in fall armyworms feeding on different diets, p-hydroxybenzoic acid, oleic acid, lactic acid, palmitoleic acid, and stearic acid, which were present in high amounts in the artificial feed group, were selected for inhibition zone experiments. Figure 3 As shown, p-hydroxybenzoic acid had the most significant inhibitory effect on the growth of *Metarhizium anisopliae*, while lactic acid and oleic acid had a slight inhibitory effect on the growth of *Metarhizium anisopliae*. The crystallization of palmitoleic acid and stearic acid on the culture medium did not indicate whether they inhibited the growth of *Metarhizium anisopliae*.

[0060] 2.2 Experiment on the antibacterial zone of epidermal compounds of fall armyworm in the maize group

[0061] Linoleic acid, α-linolenic acid, salicylic acid, and eicosapentaenoic acid, which are abundant in the corn group, were selected for the inhibition zone experiment. Figure 4 As shown, only α-linolenic acid has an inhibitory effect on the growth of Metarhizium anisopliae.

[0062] 2.3 Experiment on the inhibition zone of epidermal compounds of fall armyworm in rice group

[0063] Carnitine and acetylcarnitine, which are present in high concentrations in the rice group, were selected for inhibition zone experiments. For example... Figure 5 As shown, it has no antibacterial effect.

[0064] Example 3: Determination of the pathogenicity of *Metarhizium anisopliae* to fall armyworm larvae fed on different foods using the surface immersion method.

[0065] Second-instar fall armyworm larvae of similar size were selected from the corn, rice, and artificial feed groups, and 1 × 10⁻⁶ larvae were used. 8 Fall armyworms were soaked in a suspension of Metarhizium anisopliae spores per mL, with a control (CK) treated with 0.05% Tween-80 solution. After 15 seconds, the larvae were removed and placed on clean filter paper, then aliquoted into small plastic boxes. Each treatment contained 15 larvae, with three technical replicates. The treated larvae were then fed in an artificial climate incubator (temperature 28 ℃, relative humidity 70 ± 2%, photoperiod L:D = 16:8). Survival rates were recorded twice daily starting 0.5 days after treatment and continued until pupation.

[0066] The results of pathogenicity testing of fall armyworms that have fed on different foods using the immersion method are as follows: Figure 6 As shown, *Metarhizium anisopliae* exhibits significant pathogenicity against second-instar larvae of the fall armyworm that feed on different foods. Specifically, by day 8 post-infection, the mortality rate reached 100% in the rice group, 90% in the maize group, and 68% in the artificial feed group. Furthermore, the mortality progression differed among the three groups: the survival rate in the rice group decreased to 50% by day 4 post-infection, while in the maize group it decreased to 50% by day 6, and in the artificial feed group it decreased to 50% by day 8. This indicates that rice-feeding larvae are more susceptible to *Metarhizium anisopliae*. Figure 6 A). The median survival time (MST) of fall armyworm in the artificial feed group differed significantly from that in the maize and rice groups, and there was also a difference in median survival time between the maize and rice groups. The median survival times in the artificial feed group, maize group, and rice group were 6.95 ± 0.04 d, 4.56 ± 0.04 d, and 3.41 ± 0.1 d, respectively. Figure 6 B, Table 1).

[0067] Table 1. Median survival time (MST) of second-instar larvae of the fall armyworm fed with different diets by Metarhizium anisopliae.

[0068]

[0069] The pathogenicity of fifth-instar fall armyworm larvae in the corn, rice, and artificial feed groups was also determined using the same surface immersion method. The results were as follows: Figure 7 As shown in Table 2, similarly, rice-feeding larvae were most susceptible to Metarhizium anisopliae, followed by the maize group, and lastly the artificial feed group, consistent with the trend observed in the second instar fall armyworm larvae.

[0070] Table 2. Median survival time (MST) of 5th instar larvae of fall armyworm fed with different diets by Metarhizium anisopliae.

[0071]

[0072] Example 3: Determination of the pathogenicity of *Metarhizium anisopliae* to fall armyworm larvae fed on different diets using the injection method.

[0073] The fourth instar larvae were infected by injection. 5 μL of the solution was injected into the abdomen of the fall armyworm larvae using a microsyringe. The solution was prepared with 1×10⁻⁵ Tween-80 solution. 6 A spore suspension of 10⁶ spores / mL was used as a control (CK) treated with 0.05% Tween-80 solution. Healthy, uniformly sized 4th instar larvae of the fall armyworm were selected, and 1×10⁶ spores were injected into each larva. 6 4th instar larvae of the fall armyworm were infected by injection of a spore suspension at a concentration of 15 larvae per mL. Three technical replicates were set up for each treatment. The treated larvae were then fed a diet in an artificial climate incubator (temperature 28 ℃, relative humidity 70 ± 2%, photoperiod L:D = 16:8). Survival rates were recorded twice daily starting 0.5 days after treatment and continued until all larvae died.

[0074] The pathogenicity of fall armyworms fed on different foods was determined using an injection method. The results are as follows: Figure 8 As shown, the mortality rate reached 100% within 5 days of inoculation with Metarhizium anisopliae. Figure 8 The median survival time (MST) of the artificial feed group, corn group, and rice group were 3.25 ± 0.07 d, 3.05 ± 0.08 d, and 2.8 ± 0.19 d, respectively (Table 3), with little difference. This indicates that the fall armyworm's resistance to fungal infection mainly relies on its epidermis (as shown in Example 1) rather than its innate immunity.

[0075] Table 3. Median survival time (MST) of 4th instar larvae of the fall armyworm fed with different diets by Metarhizium anisopliae.

[0076]

[0077] Based on the above experimental results, the germination rate of methanol epidermal extract of fall armyworm fed on artificial feed was 40% 24 hours after inoculation with Metarhizium anisopliae spores, which is below the 45% threshold and is classified as low sensitivity. Mass spectrometry analysis revealed that it contains the potent antibacterial compound p-hydroxybenzoic acid. The germination rate of methanol epidermal extract of fall armyworm fed on corn leaves was 63% 24 hours after inoculation with Metarhizium anisopliae spores, which is between 45% and 65% and is classified as moderate sensitivity. Mass spectrometry analysis revealed that it contains the weak antibacterial compound α-linolenic acid. The germination rate of methanol epidermal extract of fall armyworm fed on rice leaves was 66% 24 hours after inoculation with Metarhizium anisopliae spores, which is above the 65% threshold and is classified as high sensitivity. Mass spectrometry analysis revealed that it does not contain any antibacterial components. The germination rate results of methanol epidermal extracts from fall armyworms feeding on different foods showed a consistent trend with the results of bioassays using the surface immersion method (mortality rate, survival rate, and MST). The germination rate results of epidermal extracts from other organic matter showed poor consistency with the bioassay results, indicating that methanol extraction is highly accurate. Based on sensitivity levels, fall armyworm populations at medium and low sensitivity levels cannot be controlled solely with the conventional application methods of *Metarhizium anisopliae*. It is necessary to increase the application concentration and / or frequency of *Metarhizium anisopliae*, or to apply other formulations that can produce synergistic effects with *Metarhizium anisopliae*, such as emamectin benzoate or Bt formulations. After obtaining the evaluation results using the sensitivity evaluation method of this invention, field managers can understand the fall armyworm population situation, gain a clear understanding, and provide technical support for rational selection, precise application, and effect prediction, thereby taking more appropriate control measures.

[0078] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for evaluating the susceptibility of fall armyworm to Metarhizium anisopliae, characterized in that, Includes the following steps: 1) Take a fifth-instar larva of the fall armyworm, freeze it on ice until stunned, place it in a 5 mL centrifuge tube, add 2 mL of methanol, shake well, and extract it at room temperature in the dark for 2 h to obtain a methanol extract. 2) Transfer the extract to a sample vial, concentrate and dry the sample under a nitrogen stream, then add 1 mL of methanol to resuspend the sample to obtain the epidermal extract; 3) Place 5 μL of epidermal extract in the center of a solid water agar. After the solvent evaporates, add 5 μL of 1×10⁻⁶ agar. 6 A suspension of *Metarhizium anisopliae* cells / mL was poured onto the center of the solid water agar, and then incubated at 27 °C and 100% humidity for 24 h. After incubation, five fields of view were selected under a microscope, the number of germinations of 200 conidia in each field of view was counted, and the spore germination rate was calculated. 3) Based on the germination rate, give the results of the sensitivity evaluation of fall armyworm to Metarhizium anisopliae.

2. The method as described in claim 1, characterized in that, Step 3) The fall armyworm sensitivity evaluation results include three levels: high sensitivity, medium sensitivity and low sensitivity. The germination rate of the high sensitivity level is not less than 65%, the germination rate of the medium sensitivity level is between 45% and 65%, and the germination rate of the low sensitivity level is less than 45%.

3. The method as described in claim 2, characterized in that, The low-sensitivity epidermal extract contains compounds with strong antibacterial activity; the medium-sensitivity epidermal extract contains compounds with weak antibacterial activity; and the high-sensitivity epidermal extract does not contain compounds with significant antibacterial activity.

4. The method as described in claim 3, characterized in that, The strong antibacterial active compound includes hydroxybenzoic acid; the weak antibacterial active compound includes α-linolenic acid.

5. The method described in any one of claims 1-4 is used to guide the control of fall armyworm by Metarhizium anisopliae in the field.

6. The application as described in claim 5, characterized in that, For the highly sensitive level, the method for controlling fall webworm in the field is to directly apply normal concentrations of Metarhizium anisopliae; for the moderately sensitive and lowly sensitive levels, the method for controlling fall webworm in the field is to increase the application concentration and / or frequency of Metarhizium anisopliae, or to apply it together with other preparations that can produce a synergistic effect with Metarhizium anisopliae.

7. A method for improving the control effect of Metarhizium anisopliae on fall armyworm, characterized in that, The method includes the step of reducing the content of antibacterial active compounds in the epidermis of the fall armyworm, said antibacterial active compounds including one or more of p-hydroxybenzoic acid, lactic acid, oleic acid and α-linolenic acid.

8. Application of hydroxybenzoic acid in screening highly virulent strains of Metarhizium anisopliae.

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