Composition for enhancing toxicity of entomopathogenic fungi to pests as well as preparation method and application of composition

Abstract

The invention discloses a composition for enhancing toxicity of entomopathogenic fungi to pests as well as a preparation method and application of the composition, and belongs to the technical field of biological control. The specific implementation way is as follows: suspending the entomopathogenic fungi in a mannan solution to obtain a spore suspension, wherein a surfactant can also be added into the spore suspension; the spore suspension is then applied to the pests. The invention also provides a synergistic composition containing mannan, a surfactant can be added into the composition, the composition provided by the invention can be used for remarkably enhancing the fungal toxicity, accelerating the lethal speed of pests and reducing the use dosage, and a new way is provided for developing efficient, specific and environment-friendly biological insecticides.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to a composition that enhances the toxicity of entomopathogenic fungi to pests, its preparation method, and its application. Background Technology

[0002] In modern agriculture, forestry, and public health management, effective pest control is crucial for ensuring crop yields, maintaining ecosystem health, and preventing disease transmission. For a long time, chemical pesticides have been the primary means of pest control. However, the excessive and inappropriate use of chemical pesticides has led to a series of serious problems, including environmental pollution, toxicity to non-target organisms such as pollinating insects, pesticide residues in food, and the increasingly serious problem of pesticide resistance in pests. These challenges have prompted the scientific and industrial communities to actively seek safer, more environmentally friendly, and more sustainable alternatives.

[0003] Against this backdrop, biopesticides, represented by entomopathogenic fungi, have demonstrated enormous application potential. Fungi such as *Metarhizium spp.* and *Beauveria bassiana* can naturally infect and kill a variety of pests, exhibiting significant advantages such as high host specificity, low susceptibility to resistance development, and environmental friendliness. They attach to the insect's body surface via conidia, which germinate under suitable conditions, producing an infective structure that penetrates the insect's cuticle and enters the hemocoel, ultimately causing the host's death through nutrient depletion and toxin secretion.

[0004] Despite its promising prospects, the commercial application of entomopathogenic fungi still faces a core bottleneck: their relatively slow rate of pathogenicity. Compared to fast-acting chemical pesticides, fungal insecticides typically require several days to kill pests, which may not be sufficient to contain losses in situations of high pest density and significant damage. This limitation in pathogenicity stems from the fact that host insects have evolved a complex and highly efficient immune defense system capable of recognizing pathogens and initiating a strong immune response in the early stages of fungal infection, thereby delaying or halting the infection process.

[0005] Therefore, this invention proposes a composition, preparation method, and application that enhances the toxicity of entomopathogenic fungi to pests, significantly enhancing fungal toxicity while accelerating pest mortality, providing a new approach for developing highly efficient, specific, and environmentally friendly biological insecticides. Summary of the Invention

[0006] This invention provides a series of innovative methods, compositions and their applications that can significantly enhance the toxicity of fungi to target pests and shorten their lethal time.

[0007] In view of this, one of the objects of the present invention is to provide a composition that enhances the toxicity of entomopathogenic fungi to pests, comprising entomopathogenic fungi and mannan.

[0008] Furthermore, surfactants are also added.

[0009] Furthermore, the surfactant is Tween-80.

[0010] Furthermore, the final concentration of the Tween-80 is 0.1% or 0.05%.

[0011] Furthermore, the entomopathogenic fungi include any one or more of Metarhizium anisopliae, Metarhizium septemlobus, and Beauveria bassiana.

[0012] Preferably, the *Metarhizium anisopliae* strain is *Metarhizium anisopliae* CQMa102, the *Metarhizium septemlobum* strain is *Metarhizium septemlobum* CQMa421, and the *Beauveria bassiana* strain is *Beauveria bassiana* ZJU435.

[0013] Furthermore, the final concentration of the entomopathogenic fungus is 4 × 10⁻⁶. 5 cells / mL, 2×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 2.5×10 8 per mL.

[0014] Preferably, the final concentration of the entomopathogenic fungus is 1×10⁻⁶. 7 per mL.

[0015] Furthermore, the mannan is yeast mannan.

[0016] Furthermore, the final concentration of the yeast mannan is 0.02% (w / v), 0.2% (w / v), or 2% (w / v).

[0017] Preferably, the final concentration of the yeast mannan is 0.2% (w / v).

[0018] A second objective of this invention is to provide a method for preparing any of the above-described compositions, comprising the step of suspending entomopathogenic fungi in a mannan solution to obtain a spore suspension.

[0019] A third objective of this invention is to provide the application of any of the above-described compositions in the control of pests.

[0020] Furthermore, the pests include any one or more of the following: the migratory locust, the brown planthopper, the yellow-striped flea beetle, and the fall armyworm.

[0021] Furthermore, the spore suspension is applied to pests for pest control.

[0022] Furthermore, the method of application includes dripping onto the dorsal plate of the pest or spraying onto the body of the pest.

[0023] This invention has a wide range of applications. By combining entomopathogenic fungi and mannan and applying them to pests, it can control pests including those in the orders Orthoptera, Lepidoptera, Coleoptera, and Homoptera. It aims to provide a new generation of efficient, specific, and environmentally friendly biological pest control solutions. Attached Figure Description

[0024] Figure 1 The results show the screening results for the optimal synergistic concentration of yeast mannan in this invention;

[0025] Figure 2 The results of the synergistic experiment of this invention against the East Asian migratory locust, the yellow-striped flea beetle, and the fall armyworm;

[0026] Figure 3 This is the result of a semi-field trial to verify the invention. Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of this invention, the following detailed description is provided in conjunction with embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. This invention is not limited to the following embodiments or examples; any modifications and variations made without departing from the spirit of this invention should be included within its scope. Unless otherwise specified, all reagents or materials mentioned in the embodiments are commercially available.

[0028] Example: Mannan as an adjuvant to enhance the virulence of Metarhizium anisopliae and Beauveria bassiana.

[0029] This embodiment aims to verify the broad-spectrum synergistic effect of mannan as an adjuvant on the infection of different species of pests by different entomopathogenic fungi.

[0030] 1. Insect rearing and strain culture

[0031] The migratory locust (Locusta migratoria), brown planthopper (Nilaparvata lugens), striped flea beetle (Phyllotreta striolata), and fall armyworm (Spodoptera frugiperda) used in this study were all raised and provided by the Genetic Engineering Research Center of Chongqing University, China. The migratory locusts were reared at 30±3 ℃, relative humidity 75%, with a photoperiod of 14 h / 10 h. The brown planthoppers were reared on rice seedlings at 27±1 ℃, relative humidity 80%, with a photoperiod of 14 h / 10 h. The striped flea beetle was reared on Chinese cabbage at 28±2 ℃, with a photoperiod of 14 h / 10 h. Fall armyworm eggs were collected from cornfields, and the larvae were reared individually in round plastic boxes with fresh corn leaves at 26±2 ℃, relative humidity 65%, with a photoperiod of 14 h / 10 h. The strains *Metarhizium acridum* CQMa102, *Metarhizium anisopliae* CQMa421, and *Beauveria bassiana* ZJU435 were provided by the Genetic Engineering Research Center of Chongqing University. Fungal spores were inoculated onto 1 / 4 SDAY medium and cultured at 28 ℃ for 15 days. Conidia were then collected for experimental use.

[0032] 2. Screening of the optimal synergistic concentration of yeast mannan

[0033] To determine the optimal application concentration of yeast mannan (Xintai Food Technology Co., Ltd.) as an adjuvant, a series of concentration gradient experiments were conducted. Conidia of *Metarhizium anisopliae* CQMa102 were suspended in solutions containing 0.02%, 0.2%, and 2% (w / v) yeast mannan (all containing 0.1% Tween-80 as a surfactant, Chengdu Kelong Chemical Co., Ltd.) to prepare a final spore concentration of 1×10⁻⁶. 7 The spore suspension was prepared at a concentration of 1 spore / mL. To rule out the potential toxicity of yeast mannan itself, a control group 1 was established, which treated *Metarhizium anisopliae* locusts with only a yeast mannan solution at the highest tested concentration of 2% (w / v) (containing 0.1% Tween-80, without fungal spores). Control group 2 consisted of *Metarhizium anisopliae* CQMa102 conidia suspended in a solution containing only 0.1% Tween-80, with a final concentration of 1×10⁻⁶. 7 Species / mL. Using the local titration method, 5 µL of spore suspension was dropped onto the dorsal plate of fifth-instar nymphs of the Oriental migratory locust. Each group consisted of 30 nymphs, and the assay was repeated three times. Mortality was recorded every 12 hours, and the median lethal time (LT) was calculated. 50 ).

[0034] The results showed that no deaths or adverse reactions occurred in the Oriental migratory locusts in control group 1 throughout the observation period, proving that the yeast mannan used was safe and non-toxic to the Oriental migratory locusts themselves. Compared with control group 2 without added yeast mannan, all three concentrations of yeast mannan showed an enhancing effect on fungal toxicity. The experimental groups with added 0.02%, 0.2%, and 2% yeast mannan had lower LT values. 50 The time to recovery was shortened by 0.29 days, 0.64 days, and 0.13 days respectively compared to the control group. Figure 1 The results indicate that yeast mannan has a synergistic effect within the tested concentration range, with the most significant effect observed at a concentration of 0.2% (w / v). Therefore, 0.2% was selected as the optimal application concentration of yeast mannan in subsequent adjuvant synergistic experiments of this invention.

[0035] 3. Synergistic effect experiment against the East Asian migratory locust (Metarhizium anisopliae)

[0036] Conidia of the beetle *Metarhizium anisopliae* strain CQMa421 were suspended in an aqueous solution containing 0.1% Tween-80 and 0.2% (w / v) yeast mannan to prepare a series of final concentration gradients (4 × 10⁻⁶). 5 , 2×10 6 1×10 7 5×10 7 2.5×10 8 Spore suspensions (conidia / mL) were prepared. Equal volumes of conidia were suspended in aqueous solutions containing only 0.1% Tween-80 to create spore suspensions with equal concentration gradients, serving as control groups. Healthy fifth-instar nymphs of the Oriental migratory locust were selected, with 30 nymphs per group. For both experimental and control groups, the local titration method was used; 5 µL of the corresponding concentration of spore suspension was carefully pipetted onto the dorsal plate of each Oriental migratory locust. The treated Oriental migratory locusts were continued to be reared under standard conditions, and the number of deaths was observed and recorded every 12 hours. The experiment was repeated three times. Compared with the control group, the addition of 0.2% yeast mannan significantly enhanced toxicity. On day 3, the median lethal dose (LD50) was significantly higher. 50 ) decreased by 55.96% ( Figure 2 A). More importantly, at 1×10 7 At a concentration of [number] cells / mL, the median lethal time (LT) 50 It shortened by 0.41 days. Figure 2 B).

[0037] 4. Synergistic effect experiment against the East Asian migratory locust (Beauveria bassiana)

[0038] Spores of *Beauveria bassiana* were suspended in a solution containing 0.1% Tween-80 and 0.2% (w / v) yeast mannan to prepare a spore suspension with the same concentration gradient as in step 3. The control group consisted of *Beauveria bassiana* conidia suspended in a solution containing only 0.1% Tween-80, prepared with the same concentration gradient as in step 3. *Ophiopogon japonicus* was treated using the same method as in step 3. Compared to the control group, the yeast mannan adjuvant reduced the LT (transmission rate) of *Beauveria bassiana*. 50 It shortened the treatment time by 0.63 days, a significant improvement. Figure 2 C)

[0039] 5. Synergistic effect experiment on flea beetle (Metarhizium anisopliae)

[0040] Spores of *Metarhizium anisopliae* CQMa421 were suspended in a solution containing 0.1% Tween-80 and 0.2% (w / v) yeast mannan to prepare 1×10⁻⁶ spores. 7 The control group consisted of spore suspensions at a concentration of spores / mL. The control group consisted of spores of *Metarhizium anisopliae* CQMa421 suspended in a solution containing only 0.1% Tween-80, with the concentration gradient the same as in step 3. Adult flea beetles were sprayed with this solution (200 μL per group), with the remaining treatments the same as in step 3. Compared to the control group, the addition of yeast mannan reduced the median lethal dose (LD50). 50 It decreased by 88.24% (day 3). Figure 2 D)

[0041] 6. Synergistic effect experiment against fall armyworm (Metarhizium anisopliae)

[0042] Spores of *Metarhizium anisopliae* CQMa421 were suspended in a solution containing 0.1% Tween-80 and 0.2% (w / v) yeast mannan to prepare spore suspensions with the same concentration gradient as in step 3. The control group consisted of *Metarhizium anisopliae* CQMa421 conidia suspended in a solution containing only 0.1% Tween-80, with the same concentration gradient as in step 3. Second-instar larvae of *P. fall armyworm* were sprayed with this solution (200 μL per group), with the remaining treatments the same as in step 3. Compared to the control group, the addition of yeast mannan resulted in a lower LD50. 50 A significant reduction of 73.48% (on day 3). Figure 2 E)

[0043] 7. Synergistic effect experiment against brown planthopper (Metarhizium anisopliae)

[0044] Spores of *Metarhizium anisopliae* CQMa421 were suspended in a solution containing 0.1% Tween-80 and 0.2% (w / v) yeast mannan to prepare spore suspensions with the same concentration gradient as in step 3. The control group consisted of *Metarhizium anisopliae* CQMa421 conidia suspended in a solution containing only 0.1% Tween-80, with the same concentration gradient as in step 3. Adult brown planthoppers were sprayed with this solution (200 μL per group), with the remaining treatments the same as in step 3. Compared to the control group, the addition of yeast mannan significantly accelerated the lethal rate. Compared to the control group, the addition of yeast mannan significantly reduced the LD50. 50 A significant reduction of 83.03% (day 3). Figure 2 F)

[0045] 8. Semi-field trial verification

[0046] To evaluate the practical feasibility of this strategy under real-world environmental conditions, we conducted a semi-field trial. The trial was conducted in an experimental field in the Western China (Chongqing) Science City (29.590587°N, 106.288897°E). Maize seedlings were pre-planted in the experimental area, which was divided into 11 plots. Each plot (1 m²) was covered with a nylon net (1 m × 1 m × approximately 0.4 m high). The net was supported by arched plastic poles, with its edges buried in the soil to prevent the escape of the Oriental migratory locust. Figure 3 A). Twenty-four hours prior to treatment, 40 fifth-instar Oriental migratory locusts were introduced into each net enclosure for acclimatization. The Metarhizium anisopliae preparation used in the treatment was prepared from a commercially available oil suspension of Metarhizium anisopliae CQMa421 provided by Chongqing Julixin Biotechnology Co., Ltd. The recommended standard effective dosage of this commercially available oil suspension of Metarhizium anisopliae CQMa421 is 2 × 10⁻⁶. 7 1 spore / ml.

[0047] The plots were divided into three groups, where n represents the number of plots in each group: (1) Control group (n=3): administered an aqueous solution containing 0.05% Tween-80; (2) Standard full-dose CQMa421 group (n=4): the commercial oil suspension of Metarhizium anisopliae CQMa421 was prepared to a final concentration of 2×10⁻⁶ containing 0.05% Tween-80. 7 (3) Half-dose + mannan group (n=4): The commercial oil suspension of Metarhizium anisopliae CQMa421 was prepared into a final concentration of 1×10⁻⁶ cells / mL containing 0.2% (w / v) yeast mannan and 0.05% Tween-80. 7The application rate was 1 / mL of Metarhizium anisopliae formulation for scarab beetles. For each treatment, a total volume of 25 mL of the solution was evenly sprayed onto corn leaves and Oriental migratory locusts in each 1 m² plot using a handheld electric sprayer to ensure consistent application. The mortality rate of Oriental migratory locusts was recorded daily for seven consecutive days. Throughout the experiment, the ambient temperature and relative humidity inside the netting were monitored and recorded using a temperature and humidity recorder (model COS-03, Shandong Jinan Renke Measurement and Control Technology Co., Ltd.). The experimental results strongly confirm that our research results can be successfully translated from laboratory to field application. Notably, the insecticidal rate and final mortality rate achieved by using half-dose (50%) of Metarhizium anisopliae formulation containing 0.2% (w / v) yeast mannan were significantly higher than those of the standard full-dose Metarhizium anisopliae CQMa421 group. Figure 3 B). Data analysis showed that, compared with the standard full-dose Metarhizium anisopliae CQMa421 group, the half-dose + mannan group had a significantly shorter median lethal time (LT). 50 It significantly shortened the time by 0.57 days. Figure 3 C). This result demonstrates that our proposed simple additive method can significantly reduce the effective dosage of commercial biopesticides in a field-like environment, highlighting its direct potential for industrial application.

[0048] In summary, mannan, as a highly effective adjuvant, exhibits a broad-spectrum synergistic effect. This invention, by co-application of yeast mannan with fungal spores, not only enhances the virulence of various entomopathogenic fungi (Metarhizium anisopliae, Beauveria bassiana), but also effectively targets pests of various orders, including Lepidoptera, Coleoptera, Orthoptera, and Homoptera, accelerating their mortality. It is a highly promising biopesticide synergist, providing strong support for pest control and the development of biological insecticides.

[0049] Operations not specifically described in the embodiments can be performed using conventional methods in the art. The above embodiments and / or experimental examples describe in detail the preferred embodiments of the present invention. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and all such simple modifications fall within the protection scope of the present invention.

Claims

1. A composition for enhancing the toxicity of entomopathogenic fungi to insect pests, characterized in that, It contains entomopathogenic fungi and mannan.

2. The composition according to claim 1, characterized in that, Surfactants are also added.

3. The composition according to claim 2, characterized in that, The surfactant is Tween-80.

4. The composition according to claim 1, characterized in that, The entomopathogenic fungi include any one or more of Metarhizium anisopliae, Metarhizium septemlobus, and Beauveria bassiana.

5. A method for preparing the composition according to any one of claims 1-4, characterized in that, This includes the step of suspending entomopathogenic fungi in a mannan solution to obtain a spore suspension.

6. The use of the composition according to any one of claims 1-4 in the control of pests.

7. The application as described in claim 6, characterized in that, The pests mentioned include any one or more of the following: Oriental migratory locust, brown planthopper, yellow-striped flea beetle, and fall armyworm.

8. The application as described in claim 6, characterized in that, The spore suspension is applied to pests for pest control.

9. The application as described in claim 8, characterized in that, The application method includes dripping onto the back plate of the pest or spraying it onto the body of the pest.

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