Application of butylated hydroxytoluene in enhancing biocontrol activity of entomopathogenic nematodes

Abstract

The invention discloses an application of butylated hydroxytoluene in enhancing the biocontrol activity of entomopathogenic nematodes. The entomopathogenic nematodes are pretreated by using the volatile butylated hydroxytoluene specifically released by the larvae of the red weevil, so that the activity of the entomopathogenic nematodes can be remarkably enhanced, and the quantitative enhancement of the behavior of the nematodes can be achieved; therefore, the parasitic lethal ability of entomopathogenic nematodes to hosts (larvae such as red palm weevils, greater wax moths and tomato leaf miners) is improved, and the control effect on various pests is effectively improved. Butylated hydroxytoluene has no toxic or side effect on entomopathogenic nematodes under an effective concentration, so that the survival and activity stability of the entomopathogenic nematodes are ensured. The invention provides a safe, efficient and wide-application-range strategy for improving biological prevention and control means based on entomopathogenic nematodes, and has good popularization and application prospects.

Description

Technical Field

[0001] This invention relates to the field of biological control and agricultural pest control, specifically to the application of butylated hydroxytoluene in enhancing the biocontrol activity of entomopathogenic nematodes. Background Technology

[0002] Insectivorous nematodes, such as *Strombus squarrosa* (a type of nematode), are pathogenic nematodes. Steinernema carpocapsae Due to their active host-seeking, highly efficient infection mechanisms, and safety for humans, livestock, and the environment, entomopathogenic nematodes are widely recognized as effective biological control agents for a variety of insect pests. They are extensively used in agricultural and forestry pest control, showing great potential, especially in controlling underground pests and concealed herbivorous insects. However, despite these advantages, entomopathogenic nematodes still have limitations in locating and infecting concealed insect hosts living in plant tissues or hidden environments. These concealed environments reduce the chemical signals available to nematodes, complicating host detection and thus lowering infection rates.

[0003] Recent studies have shown that host-derived volatile organic compounds (VOCs) in this nematode can profoundly influence its behavior. Specific VOCs released by the host larvae can act as attractants and behavioral initiators, enhancing the nematode's localization and infection efficiency.

[0004] Therefore, searching for specific chemical signals from these hosts holds promise for overcoming the natural limitations of detecting elusive pest hosts, thereby improving the practical effectiveness of nematode-based biological control. Summary of the Invention

[0005] The purpose of this invention is to provide the application of butylated hydroxytoluene (BHT) in enhancing the biocontrol activity of entomopathogenic nematodes. This is achieved by using the host-derived volatile compound BHT to enhance the motility and host lethality of entomopathogenic nematodes.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of butylated hydroxytoluene in the preparation of attractants for worm pathogenic nematodes.

[0007] Furthermore, the entomopathogenic nematode is *Strombus schoenleinii*.

[0008] Furthermore, the concentration of the butylated hydroxytoluene used is 2 ng / μL to 2000 ng / μL.

[0009] Secondly, the present invention provides the application of butylated hydroxytoluene in the preparation of products that enhance the biocontrol activity of entomopathogenic nematodes.

[0010] Furthermore, the biocontrol activity refers to the activity of the entomopathogenic nematodes and their lethality to pests, wherein the pests are the red palm weevil, the large wax moth, and the tomato leafminer.

[0011] Furthermore, the entomopathogenic nematode is *Strombus schoenleinii*.

[0012] Furthermore, the concentration of the butylated hydroxytoluene used is 2 ng / μL to 2000 ng / μL.

[0013] Thirdly, the present invention provides a product for improving the biocontrol activity of entomopathogenic nematodes, wherein the biocontrol product contains butylated hydroxytoluene, and the entomopathogenic nematode is *Strombus schoenleinii*.

[0014] Fourthly, the present invention provides a method for improving the biocontrol activity of entomopathogenic nematodes, specifically by placing the entomopathogenic nematodes in an aqueous solution of 2 ng / μL to 2000 ng / μL butylhydroxytoluene for 24 to 72 hours to improve the activity of the entomopathogenic nematodes and their lethality to pests. The entomopathogenic nematode is *Steinella stearothermite*, and the pests are red palm weevil, large wax moth, and tomato leafminer.

[0015] Compared with the prior art, the present invention has the following significant advantages: (1) Enhance pest mortality: Nematodes pretreated with BHT or used in conjunction with BHT have enhanced motility and pathogenicity, which can significantly increase the mortality rate of target pests.

[0016] (2) No toxic side effects on nematodes: At concentrations up to 2000 ng / μL, BHT had no adverse effects on the survival and physiological health of entomopathogenic nematodes, and no increase in nematode mortality was observed even during an exposure period of up to 16 days. This ensures the safety of the biocontrol agent and avoids potential harm to the environment and non-target organisms.

[0017] (3) Wide applicability: Although BHT is a volatile substance specifically released by the red palm weevil, applying BHT exogenously to other pests (such as the large wax moth and the tomato leafminer) can also effectively enhance the attraction and lethality of nematodes. This means that this method can be extended to a variety of hidden or non-hidden pests that do not naturally produce BHT, thus expanding the spectrum of biological control of entomopathogenic nematodes.

[0018] (4) Easy integration into existing biocontrol agents: Given the specificity, safety, and behavioral activation effects of BHT, this invention provides a feasible approach for developing improved entomopathogenic nematode agents or pretreatment programs. This invention combines chemical ecology with applied biocontrol technology, and this integration can significantly enhance the actual efficacy of nematode biopesticides in the field, especially when facing difficult-to-control, elusive pests. Attached Figure Description

[0019] Figure 1 The headspace volatiles of the red brown weevil larvae and the large wax moth larvae were analyzed using headspace solid-phase microextraction (SPME-GC-MS). Among them, 1 was Naphthalene; 2 was Heptacosane; 3 was Butylated hydroxytoluene (BHT); 4 was Propanoic acid; and Cont. was a contamination peak.

[0020] Figure 2 Analysis of headspace volatiles from larvae of the large wax moth, fall armyworm, and tomato leafminer.

[0021] Figure 3 Effects of BHT on movement behavior-related indicators of the small leaflet moth Stellaria scoparia.

[0022] Figure 4 Survival rate of St. John's nematode under different concentrations of BHT treatment over 16 days.

[0023] Figure 5 Effects of BHT on the ability of *Steinus stessori* to infect larvae of *Heliotropium indicum*.

[0024] Figure 6 Effects of BHT on the ability of St. John's leafminer larvae to infect tomatoes. Detailed Implementation

[0025] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0026] Experimental methods not specifically described in the following examples are generally performed using methods known in the art.

[0027] Butylated hydroxytoluene, chemical formula C 15 H 24 O.

[0028] Example 1 Host-specific recognition of butylated hydroxytoluene This embodiment first uses headspace solid-phase microextraction-gas chromatography-mass spectrometry to analyze 70 g of four-year-old reddish-brown weevils (… Rhynchophorus ferrugineus Analysis was conducted on the volatiles of the red-brown weevil larvae and the large wax moth larvae. Butylated hydroxytoluene (BHT) was identified as a volatile compound specifically released by the red-brown weevil larvae. Figure 1 In other pests tested, such as the large wax moth (…), Galleria mellonella Tomato leafminer ( Tuta absoluta ) and fall armyworm ( Spodoptera frugiperdaBHT was completely undetectable in the volatiles of ( ). Figure 2 This finding indicates that BHT is not a universal characteristic of all hosts, but rather a host-specific chemical signal of the red-brown weevil.

[0029] Example 2: Effect of butylated hydroxytoluene on the motility of entomopathogenic nematodes This embodiment uses the entomopathogenic nematode *Strombi skeletoni* (a type of nematode). Steinernema carpocapsae The infective stage (IJs) of these nematodes can be used as research and application subjects. These nematodes can be studied in live insect hosts (such as the larvae of the large wax moth). Galleria mellonella IJs were cultured in [a specific environment] and collected using the white trap method (see [link to documentation] for details). White, G. A method for obtaining infective nematode larvae from cultures. Science 66, 302-303 (1927) Nematodes of the small leafroller *Strombus skeletalus* were suspended in aqueous solutions containing BHT (at concentrations of 2 ng / μL, 20 ng / μL, 200 ng / μL, and 2000 ng / μL) at a density of 1000 nematodes / mL. Nematodes suspended in a BHT-free aqueous solution served as the control group (CK). After treatment at 25°C for 3 h, 6 h, 12 h, 24 h, and 36 h, a random sample of nematodes was tested for motility. Motility parameters (automatically generated by the software) were quantitatively analyzed using WormLab and other motion tracking software, including center point velocity (μm / s), absolute peristaltic velocity (μm / s), forward movement distance (μm), reverse movement distance (μm), cumulative forward movement time (s), and cumulative reverse movement time (s) to monitor the enhancement of their activity. The results, shown in Figure 3, revealed that BHT treatment increased the center point velocity and absolute peristaltic velocity of the nematodes, exhibiting a significant increase in motility after 24 h.

[0030] Example 3: Effect of butylated hydroxytoluene on the survival rate of entomopathogenic nematodes Nematodes (IJs) of the small leafroller moth *Strombus skeletalus* were suspended in aqueous solutions of BHT at concentrations ranging from 2 to 2000 ng / μL for 16 days at a density of 1000 IJs / mL. The number of surviving nematodes was recorded at 1, 2, 4, 8, 12, and 16 days. A control group (CK) of nematodes suspended in a BHT-free aqueous solution was also included to investigate the toxicity of BHT to nematodes. Results are as follows: Figure 4As shown, at the highest concentration tested (2000 ng / μL), BHT had no negative impact on the survival rate of *Strombus schoenleinii*. During the 16-day exposure period, the nematode survival rate remained statistically indistinguishable from the untreated control group. This indicates that BHT is a non-toxic behavior enhancer with no adverse effects on the survival and physiological health of entomopathogenic nematodes, ensuring the safety of biocontrol agents and avoiding potential harm to the environment and non-target organisms.

[0031] Example 4: Effect of butylated hydroxytoluene on the lethality of entomopathogenic nematodes 500 *Skesneferi* nematodes treated with 2 ng / μL and 2000 ng / μL BHT aqueous solutions were inoculated into soil or sand containers containing the target pests (great wax moth larvae and tomato leafminer larvae). *Skesneferi* nematodes suspended in BHT-free aqueous solution served as the control group (CK). Mortality rates of the target pests were observed and recorded periodically within 48–72 hours post-inoculation. Results are as follows: Figure 5 and Figure 6 As shown, the mortality rate of *Skesneferi* nematodes treated with BHT against *Heliotropium indicum* larvae was significantly increased by approximately 12% compared to the control group. *Tomato leafminer* larvae, a cryptic pest, exhibited a higher mortality rate from BHT-treated nematodes, significantly higher than the control group by approximately 15%, and showed good lethality even at lower BHT concentrations. Although BHT is a volatile substance specifically released by the red palm weevil, exogenous application of BHT to other pests (such as *Heliotropium indicum* and *Tomato leafminer*) can also effectively enhance nematode attraction and lethality.

[0032] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention shall be covered by the present invention.

Claims

1. Application of butylated hydroxytoluene in the preparation of attractants for worm pathogenic nematodes.

2. The application according to claim 1, characterized in that: The entomopathogenic nematode is *Strombus schoenleinii*.

3. The application according to claim 1, characterized in that: The concentration of butylated hydroxytoluene used is 2 ng / μL to 2000 ng / μL.

4. Application of butylated hydroxytoluene in the preparation of products that enhance the biocontrol activity of entomopathogenic nematodes.

5. The application according to claim 4, characterized in that: The biocontrol activity refers to the motility of entomopathogenic nematodes and their lethality to pests.

6. The application according to claim 4, characterized in that: The entomopathogenic nematode is *Strombus schoenleinii*.

7. The application according to claim 4, characterized in that: The concentration of butylated hydroxytoluene used is 2 ng / μL to 2000 ng / μL.

8. The application according to claim 5, characterized in that: The pests mentioned are the red palm weevil, the large wax moth, and the tomato leafminer.

9. A product for enhancing the biocontrol activity of entomopathogenic nematodes, characterized in that: The biocontrol active product contains butylated hydroxytoluene, and the entomopathogenic nematode is *Strombus schoenleinii*.

10. A method for improving the biocontrol activity of entomopathogenic nematodes, characterized in that: The entomopathogenic nematodes were placed in an aqueous solution of 2 ng / μL to 2000 ng / μL butylhydroxytoluene for 24 to 72 hours to enhance their activity and mortality rate against pests. The entomopathogenic nematodes were *Steinella stearothermiae*, and the pests were red palm weevil, large wax moth, and tomato leafminer.

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