A compound prevention and control method for anaphothrips and drug resistance of goodyera repens in an extremely high temperature climate

Abstract

The application discloses a compound prevention and control method for anaphelides and drug resistance of Anoectochilus roxburghii under extreme high temperature and application thereof. The compound prevention and control method is that the seedlings of the Anoectochilus roxburghii are disinfected, transplanted and planted in April to May, enter a mature period in June to July, high-temperature biological prevention and control and high-temperature physical prevention and control are carried out before the mature Anoectochilus roxburghii enters a high-temperature environment of 35 DEG C, high-temperature drug resistance prevention and control is carried out by spraying high-temperature special trehalose-nano emulsion diluent every mu under the continuous high-temperature environment of 35 DEG C, and harvesting is carried out in October to November. The 5% Trichoderma harzianum preparation, Neoseiulus californicus and the high-temperature special trehalose-nano emulsion have good compatibility, the growth, activity and prevention and control effect of the preparation on the biological preparation have no significant inhibition, and the preparation can be applied in the extreme climate prevention and control system of the Anoectochilus roxburghii in cooperation.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biological control technology, specifically relating to a combined control method for preventing and controlling mites and insects, as well as pesticide resistance, in *Anoectochilus roxburghii* under extreme high-temperature climates. Background Technology

[0002] Anoectochilus roxburghii, a rare edible and medicinal plant in my country, suffers from inhibited synthesis of its active ingredient, anoectochiloside, under extreme climate conditions. Related monitoring data shows that in areas with a yield of 155 kg per mu under normal climate conditions, the yield decreased by 37% during prolonged periods of high temperatures. The main reason for this is mite infestation. Current conventional pesticides have the following problems: 1) Poor adaptability: Commercially available emulsifiable concentrates break down at temperatures above 35℃, resulting in a 42% loss of active ingredients; 2) Lagging resistance monitoring: Traditional field observation methods cannot detect resistance mutations early when mite density is below the detection threshold at low temperatures; 3) Lack of integrated pest management: Single pesticides have an effective period of less than 3 days under extreme climate conditions and are not combined with agronomic measures to form a synergistic control system.

[0003] The applicant's research found that the reproductive cycle of the root mite is shortened to 7 days in environments above 35℃. Therefore, based on the applicant's research findings, this invention develops control measures for extreme high temperatures (continuous high temperature (≥35℃) environments), constructing a three-in-one control system of "pesticides-agronomy-biology" to effectively improve the survival rate of *Anoectochilus roxburghii* under extreme high temperatures. Summary of the Invention

[0004] The purpose of this invention is to propose a combined control method for the resistance to mites and insects and the resistance to pesticides in Anoectochilus roxburghii under extreme high-temperature climates, and to construct a three-in-one control system of "pesticides-agronomy-biology" to improve the survival rate of Anoectochilus roxburghii under extreme high temperatures.

[0005] The technical solution of this invention is a combined control method for mite and insect resistance and pesticide resistance in *Anoectochilus roxburghii* under extreme high-temperature climates. The combined control method involves disinfecting, transplanting, and planting *Anoectochilus roxburghii* seedlings in April, allowing them to reach maturity in July. Mature *Anoectochilus roxburghii* seedlings are then sprayed with a high-temperature-specific trehalose-nanoemulsion diluted solution under continuous high-temperature conditions ≥35℃ to control high-temperature pesticide resistance. The combined prevention and control method shall be carried out according to the following specific steps: (1) Disinfection treatment before planting: In April, before transplanting the seedlings of Anoectochilus roxburghii, disinfect them by drenching with 50% carbendazim at a dilution of 400-600 times, with a drenching volume of 20-30L per square meter, and adjust the soil EC value to 1.0-1.5mS / cm. Then, use 0.1% citric acid solution or wood ash to adjust the soil pH value to 5.0-7.0, and obtain disinfected Anoectochilus roxburghii seedlings. (2) Transplanting and planting: Use humus:perlite:coconut coir = 2.5-3.5:0.9-1.1:0.9-1.1 as the mixed substrate for transplanting Anoectochilus roxburghii seedlings. Disinfect the mixed substrate with 50% carbendazim at a dilution of 400-600 times. The dosage of 50% carbendazim at a dilution of 400-600 times is 20-30 L / m². 2 Inoculate with 5% Trichoderma harzianum 2-4 days after disinfection. The dosage of 5% Trichoderma harzianum 8-12 g / m³ is as follows. 2 Transplant the seedlings of *Anoectochilus roxburghii* 6-8 days later, with a spacing of 10cm x 15cm and a planting depth of 2-3cm. Plant the seedlings so that the rootstock is level with the substrate surface, with 60-70 seedlings per square meter. Immediately after transplanting, water thoroughly with deionized water (≤10μS / cm) at a rate of 20-30L per square meter, drip-irrigating until the substrate moisture reaches 65-75%. (3) High-temperature composite control: After the golden thread lotus enters the maturity period in July, when the temperature reaches ≥35℃, a 40-60% shade net is erected on the top and axial flow fan is used for forced ventilation to control the high temperature. The wind speed is 0.6-1.5m / s, and ventilation is carried out 2-4 times a day, each time for 1.8-2.2 hours. After ventilation, the highest temperature in the greenhouse should be ≤32℃, the day-night temperature difference should be ≤8℃, the relative humidity should be controlled at 65-75%, and the wind speed difference between the four corners and the central area of ​​the greenhouse should be ≤0.3m / s. After ≥3 consecutive days of high temperature of 35℃, a high-temperature special trehalose-nano emulsion is added. The trehalose-nanoemulsion is diluted with water to obtain a high-temperature-specific trehalose-nanoemulsion solution. This solution is sprayed every 4-6 days to control high-temperature resistance. Each application uses 45-55 L of the high-temperature-specific trehalose-nanoemulsion solution per acre. Spraying should be done before 9:00 AM or between 2:00 PM and 4:00 PM. Before spraying, the solution should be preheated to 22-28°C. After spraying, ventilation should be closed for 1.8-2.2 hours. Simultaneously, after three consecutive days of temperatures ≥35°C, 900-1100 Neoseios californicus mites per acre are released for high-temperature biological control. The aforementioned method for preparing the high-temperature trehalose-nanoemulsion diluent is to take 1L of the high-temperature trehalose-nanoemulsion and add it to 900-1100L of water before use, dilute and mix evenly to obtain the high-temperature trehalose-nanoemulsion diluent.

[0006] In the aforementioned step (1), disinfection treatment before planting: In April, before transplanting the seedlings of Anoectochilus roxburghii, disinfect them by drenching with 50% carbendazim at a dilution of 500 times, with a drenching volume of 25L per square meter, adjusting the soil EC value to 1.2mS / cm, and then using 0.1% citric acid solution or wood ash to regulate the soil pH value, controlling the pH value at 5.5-6.5, thus obtaining disinfected Anoectochilus roxburghii seedlings.

[0007] In step (1) above, the EC value adjustment method is as follows: when the EC value is <1.0mS / cm, spray a nitrogen, phosphorus and potassium water fertilizer with a concentration of 100mg / L, the mass ratio of nitrogen, phosphorus and potassium is 3:1:2, the amount of each spray is 20-30L per square meter, and the EC value is retested 24 hours after each adjustment until it is adjusted to 1.0-1.5mS / cm.

[0008] In step (2) above, transplanting and planting: humus:perlite:coconut coir = 3:1:1 was used as the mixed substrate for transplanting Anoectochilus roxburghii seedlings. The mixed substrate was disinfected with a 50% carbendazim solution diluted 500 times, and the dosage of the 50% carbendazim solution was 25 L / m². 2 Three days after disinfection, inoculate with 5% Trichoderma harzianum at a dosage of 10 g / m³. 2 Seven days later, transplant the seedlings of Anoectochilus roxburghii. The planting spacing is 10cm×15cm, and the planting depth is 2-3cm. The rhizome should be level with the substrate surface. Plant 75 seedlings per square meter. Immediately after transplanting, water the seedlings with deionized water with an electrical conductivity ≤10μS / cm. Use 25L per square meter and drip irrigate until the substrate moisture reaches 65-75%.

[0009] In step (3) above, high temperature compound control: After the golden thread lotus enters the maturity period in July, when the temperature reaches ≥35℃, a 50% shade net is erected on the top in conjunction with axial flow fans for forced ventilation to control the high temperature. The wind speed is 0.6-1.5m / s, and ventilation is carried out 3 times a day for 2 hours each time. After ventilation, the highest temperature in the greenhouse should be ≤32℃, the day-night temperature difference should be ≤8℃, the relative humidity should be controlled at 65-75%, and the wind speed difference between the four corners and the central area of ​​the greenhouse should be ≤0.3m / s. After ≥3 consecutive days of high temperature at 35℃, high temperature special trehalose is applied. - Dilute the nanoemulsion with water to obtain a trehalose-nanoemulsion solution. Spray the high-temperature-specific trehalose-nanoemulsion solution to control high-temperature resistance. Spray once every 5 days, with each application using 50L of the high-temperature-specific trehalose-nanoemulsion solution per acre. Spray before 9:00 AM or between 2:00 PM and 4:00 PM. Before spraying, preheat the high-temperature-specific trehalose-nanoemulsion solution to 25°C. After spraying, close the ventilation for 2 hours. Simultaneously, after 3 consecutive days of high temperatures ≥35°C, release 1000 Neoseios californica mites per acre for high-temperature biological control.

[0010] In step (3) above, the preparation method of high-temperature trehalose-nanoemulsion is carried out according to the following steps: Preparation of S1 oil phase: Weigh 210-230 kg of terpinene-4-ol, 4-6 kg of tea polyphenols, and 38-42 kg of diatomaceous earth, mix them, and stir at a constant temperature of 63-67℃ for 8-12 min until completely dissolved. The stirring speed is 400-600 rpm, and the particle size is controlled at CV≤7% to obtain the oil phase for later use. The purity of the terpinene-4-ol is ≥95%, the purity of the tea polyphenols is ≥98%, and the particle size of the diatomaceous earth is ≤5 μm. Preparation of S2 aqueous phase: Weigh 38-52 kg of Tween-80 and add it to 650-670 L of deionized water. Mix and heat to 63-67 °C to obtain the aqueous phase for later use. Preparation of S3 High-Temperature Special Trehalose-Nanoemulsion: The aqueous phase, controlled at 63-67℃, is slowly added dropwise to the oil phase, which is at 63-67℃, at a rate of 8-12 mL / min, while stirring at a stirring rate of 450-550 rpm. After the addition is complete, stirring is continued for 8-12 min. Then, 28-32 kg of trehalose and 2.5-3.5 kg of azadirachtin are added and sonicated for 8-12 min for secondary emulsification. The secondary emulsification is maintained at a temperature of 59-61℃ and an ultrasonic power of 190-210 W to obtain the high-temperature special trehalose-nanoemulsion, which is stored at 3-5℃ away from light for later use.

[0011] Specifically, in step (3) above, the preparation method of high-temperature trehalose-nanoemulsion is carried out according to the following steps: Preparation of S1 oil phase: Weigh 220 kg of terpinene-4-ol, 5 kg of tea polyphenols and 40 kg of diatomaceous earth and mix them. Stir at 65℃ for 10 min until completely dissolved. The stirring speed is 500 rpm and the particle size is controlled CV≤7% to obtain the oil phase for later use. The purity of terpinene-4-ol is ≥95%, the purity of tea polyphenols is ≥98%, and the particle size of diatomaceous earth is ≤5 μm. Preparation of S2 aqueous phase: Weigh 40 kg of Tween-80 and add it to 662 L of deionized water. Mix and heat to 63-67 °C to obtain the aqueous phase for later use. Preparation of S3 high-temperature trehalose-nanoemulsion: The aqueous phase, controlled at 65℃, is slowly added dropwise to the oil phase at 65℃ at a rate of 10mL / min, while stirring at a stirring rate of 500rpm. After the addition is complete, stirring is continued for 10min. Then, 30kg of trehalose and 3kg of azadirachtin are added and sonicated for 10min for secondary emulsification. The secondary emulsification is carried out at a temperature of 60℃ and a sonication power of 200w to obtain the high-temperature trehalose-nanoemulsion, which is stored at 4℃ away from light for later use.

[0012] In step (3) above, if the continuous high temperature environment of ≥35℃ exceeds 13 days, an additional spraying is required. The dosage of the high temperature special trehalose-nano emulsion dilution is halved, and 500 California neoseido mites per acre are released again simultaneously.

[0013] In step S4 above, the preparation of the trehalose-nanoemulsion dilution is as follows: when the mite density in 100g of soil is >5, take 1L of trehalose-nanoemulsion and add it to 999L of water before use, and dilute and mix evenly; when the air humidity is >85%, take 1L of trehalose-nanoemulsion and add it to 999L of water before use, and dilute and mix evenly.

[0014] In step (3) above, when the high temperature of ≥35℃ ends, a periodic consolidation spray of high temperature special trehalose-nano emulsion diluted solution is carried out once, with a dosage of 25L per mu.

[0015] Compared with the prior art, the present invention has the following advantages: 1. This invention overcomes the challenge of pesticide stability under extreme high temperatures, achieving emulsion-free operation at high temperatures. It establishes a special early warning mechanism for pesticide resistance under extreme high temperatures, advancing the detection time of resistance mutations by 7-10 days. It constructs a three-in-one control system of "pesticide-agronomy-biology," increasing the survival rate of *Anoectochilus roxburghii* (a type of orchid) under extreme high temperatures to over 90%. The high-temperature emulsion contains trehalose and azadirachtin. Trehalose forms a hydrogen bond network with tea polyphenols, increasing thermal stability by 40%. Azadirachtin inhibits the activity of mite detoxification enzymes at high temperatures, synergistically enhancing resistance regulation efficiency by 28% in conjunction with terpinen-4-ol.

[0016] 2. Improved efficiency in controlling extreme high temperatures: The incidence of mite infestation in high-temperature scenarios decreased from 32% to 3.8%, an improvement of 62% compared to the original technology (data from 200 mu of high-temperature experimental area in Boluo in 2025, n=20, t test P<0.01).

[0017] 3. Breakthrough in drug resistance management: The mutation frequency of G119S during the high-temperature period was controlled at 4.1% (12 months of monitoring, standard deviation ±0.3%), the resistance period was extended to 28 months, the drug resistance index of mites to the agent was reduced by 35% compared with the traditional method, and the inhibition rate of other metabolic resistance genes such as the CYP6 family of esterase genes reached 29%.

[0018] 4. Environmental and economic benefits: Reduces pesticide use by 2.3 tons per 100 acres during the high-temperature season and reduces carbon emissions by 28 tons (calculated according to ISO 14064-1). Attached Figure Description

[0019] Figure 1 Flowchart of the extreme high temperature prevention and control system; Figure 2 Schematic diagram of a high-temperature-specific nanoemulsion; Figure 3 Comparison chart of drug resistance monitoring data over 12 months. Detailed Implementation

[0020] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0021] Example 1:

[0022] 1. Preparation of high-temperature trehalose-nanoemulsion S1. Preparation of the oil phase: Weigh 220 kg of terpinene-4-ol (purity ≥95%), 5 kg of tea polyphenols (purity ≥98%) and 40 kg of diatomaceous earth (particle size ≤5 μm), mix them, and stir at a constant temperature of 63-67℃ until completely dissolved. The stirring speed is 300 rpm, and the particle size CV is controlled to be ≤7% to obtain the oil phase for later use. S2. Preparation of aqueous phase: Weigh 40 kg of Tween-80 and add it to 662 L of deionized water. Mix and heat to 63-67 °C to obtain the aqueous phase for later use. S3. Preparation of high-temperature trehalose-nanoemulsion: The aqueous phase, controlled at 65℃, is slowly added dropwise to the oil phase at 65℃ at a rate of 8-12 mL / min, while stirring at a stirring rate of 500 rpm. After the addition is complete, stirring is continued for 10 min. Then, 30 kg of trehalose and 3 kg of azadirachtin are added and sonicated for 10 min for secondary emulsification. The secondary emulsification is carried out at a temperature of 60℃ and a sonication power of 200 W to obtain the high-temperature trehalose-nanoemulsion, which is stored at 4℃ away from light for later use.

[0023] 2. Comprehensive control measures for disinfection, transplanting, and planting of *Anoectochilus roxburghii* seedlings, along with high-temperature management. (1) Disinfection treatment before planting: In April-May, before transplanting the seedlings of Anoectochilus roxburghii, disinfect them by drenching with 50% carbendazim at a dilution of 500 times. The drenching volume is 20-30L per square meter. The EC value is adjusted to 1.0-1.5mS / cm. Then, the pH value is adjusted by 0.1% citric acid solution or wood ash. The pH value is controlled at 5.5-6.5 to obtain disinfected Anoectochilus roxburghii seedlings. (2) Transplanting and planting: Use humus:perlite:coconut coir = 3:1:1 as the mixed substrate for transplanting Anoectochilus roxburghii seedlings. Disinfect the mixed substrate with 50% carbendazim solution diluted 500 times. The dosage of 50% carbendazim solution diluted 500 times is 25L / m². 2 On the third day after disinfection, inoculate with 5% Trichoderma harzianum at a dosage of 10 g / m³. 2 Seven days later, transplant the seedlings of Anoectochilus roxburghii. The planting spacing is 10cm×15cm, and the planting depth is 2-3cm. The rhizome should be level with the substrate surface. Plant 66 seedlings per square meter. Immediately after transplanting, water the seedlings with deionized water with an electrical conductivity ≤10μS / cm. Use 25L per square meter and drip irrigate until the substrate moisture reaches 65-75%.

[0024] (3) High-temperature combined control: After the golden thread lotus enters the maturity period in July, when the temperature reaches ≥35℃, a 50% shade net is erected on the top in conjunction with axial flow fans for forced ventilation to control the high temperature. The wind speed is 0.6-1.5m / s, and ventilation is carried out 3 times a day for 2 hours each time. After ventilation, the highest temperature in the greenhouse should be ≤32℃, the day-night temperature difference should be ≤8℃, the relative humidity should be controlled at 65-75%, and the wind speed difference between the four corners and the central area of ​​the greenhouse should be ≤0.3m / s. After ≥3 consecutive days of high temperature of 35℃, the high-temperature special Trehalose-nanoemulsion was diluted with water to obtain a trehalose-nanoemulsion solution. This solution was then sprayed to control high-temperature resistance. The dosage of the high-temperature trehalose-nanoemulsion solution was 50L per acre. Spraying was done before 9:00 AM or after 4:00 PM. Before spraying, the solution was preheated to 25°C. After spraying, ventilation was closed for 2 hours. Simultaneously, after three consecutive days of temperatures ≥35°C, 1000 Neoseios californicus mites were released per acre for high-temperature biological control. Preparation method of high-temperature special trehalose-nano emulsion dilution: When the mite density in 100g of soil is >5, add 1L of trehalose-nano emulsion to 999L of water before use, and dilute and mix evenly; when the air humidity is >85%, add 1L of trehalose-nano emulsion to 999L of water before use, and dilute and mix evenly.

[0025] (4) When the high temperature of ≥35℃ ends, apply a concentrated spray of high temperature special trehalose-nano emulsion diluted solution once, with a dosage of 25L per mu.

[0026] 3. Prevention and control effectiveness 1) Density of root mite: After high-temperature compound control with Anoectochilus roxburghii, the density of root mite ≤2 mites / 100g soil; 2) Drug resistance: The peak mutation frequency of G119S after high-temperature combined control of Anoectochilus roxburghii was only 4.1%; 3) Growth of Anoectochilus roxburghii: After high temperature and compound control, the yield of Anoectochilus roxburghii reached 118 kg per mu, and the content of Anoectochilus roxburghii glycoside was 1.68 mg / g.

[0027] Example 2:

[0028] 1. Preparation of high-temperature trehalose-nanoemulsion Preparation of S1 oil phase: Weigh 230 kg of terpinene-4-ol (purity ≥95%), 6 kg of tea polyphenols (purity ≥98%), and 38 kg of diatomaceous earth (particle size ≤5 μm), mix them, and stir at 67℃ for 8 min until completely dissolved. The stirring speed is 600 rpm, and the particle size CV is controlled to ≤7%. The oil phase is obtained and set aside. Preparation of S2 aqueous phase: Weigh 52 kg of Tween-80 and add it to 650 L of deionized water. Mix and heat at 67℃ to obtain the aqueous phase. Preparation of S3 high-temperature trehalose-nanoemulsion: The aqueous phase, controlled at 67℃, is slowly added dropwise to the oil phase at 67℃ at a rate of 8mL / min, while stirring at a stirring rate of 550rpm. After the addition is complete, stirring is continued for 8min. Then, 32kg of trehalose and 2.5kg of azadirachtin are added and sonicated for 10min for secondary emulsification. The secondary emulsification temperature is maintained at 61℃ and the ultrasonic power is 210w to obtain the high-temperature trehalose-nanoemulsion, which is stored at 3℃ away from light for later use.

[0029] 2. Comprehensive control measures for disinfection, transplanting, and planting of *Anoectochilus roxburghii* seedlings, along with high-temperature management. (1) Disinfection treatment before planting: In April, before transplanting the seedlings of Anoectochilus roxburghii, disinfect them by drenching with 50% carbendazim at a dilution of 600 times, with a drenching volume of 30L per square meter, and adjust the soil EC value to 1.0mS / cm. Then, use 0.1% citric acid solution or wood ash to adjust the soil pH value, and control the pH value at 6.0-7.0 to obtain disinfected Anoectochilus roxburghii seedlings; (2) Transplanting and planting: A mixture of humus, perlite, and coconut coir in a ratio of 2.5:0.9:1.1 was used as the substrate for transplanting Anoectochilus roxburghii seedlings. The substrate was disinfected with a 600-fold dilution of 50% carbendazim at a concentration of 30 L / m². 2 Two days after disinfection, inoculate with 5% Trichoderma harzianum at a dosage of 8 g / m³. 2 Eight days later, transplant the seedlings of Anoectochilus roxburghii. The planting spacing is 10cm×15cm, and the planting depth is 2-3cm. The rhizome should be level with the substrate surface. Plant 70 seedlings per square meter. Water immediately after transplanting. Use deionized water with an electrical conductivity ≤10μS / cm. Use 30L per square meter and drip irrigate until the substrate moisture reaches 75%. (3) High-temperature composite control: After the golden thread lotus enters the maturity period in July, when the temperature reaches ≥35℃, a 40% shade net is erected on the top in conjunction with axial flow fans for forced ventilation to control the high temperature. The wind speed is 1.5m / s, and ventilation is carried out twice a day for 1.8 hours each time. After ventilation, the highest temperature inside the greenhouse should be ≤32℃, the day-night temperature difference should be ≤8℃, the relative humidity should be controlled at 75%, and the wind speed difference between the four corners and the central area of ​​the greenhouse should be ≤0.3m / s. After ≥3 consecutive days of high temperature at 35℃, a high-temperature special trehalose-nano emulsion is added The trehalose-nanoemulsion was diluted with water to obtain a high-temperature-specific trehalose-nanoemulsion solution. This solution was sprayed every 4 days to control high-temperature resistance. The dosage of the high-temperature-specific trehalose-nanoemulsion solution was 45L per acre each time. Spraying was done before 9:00 AM or between 2:00 PM and 4:00 PM. Before spraying, the solution was preheated to 22°C. Ventilation was closed for 1.8 hours after spraying. Simultaneously, after three consecutive days of temperatures ≥35°C, 1100 Neoseios californicus mites were released per acre for high-temperature biological control. Preparation method of high-temperature special trehalose-nano emulsion dilution: When the mite density in 100g of soil is >5, add 1L of trehalose-nano emulsion to 1100L of water before use, and dilute and mix evenly; when the air humidity is >85%, add 1L of trehalose-nano emulsion to 1100L of water before use, and dilute and mix evenly.

[0030] (4) When the high temperature of ≥35℃ ends, apply a concentrated spray of high temperature special trehalose-nano emulsion diluted solution once, with a dosage of 25L per mu.

[0031] Example 3:

[0032] 1. Preparation of high-temperature trehalose-nanoemulsion Preparation of S1 oil phase: Weigh 210 kg of terpinene-4-ol (purity ≥95%), 4 kg of tea polyphenols (purity ≥98%), and 42 kg of diatomaceous earth (particle size ≤5 μm), mix them, and stir at 63℃ for 12 min until completely dissolved. The stirring speed is 400 rpm, and the particle size CV is controlled to ≤7%. The oil phase is obtained and set aside. Preparation of S2 aqueous phase: Weigh 38 kg of Tween-80 and add it to 670 L of deionized water. Mix and heat to 63℃ to obtain the aqueous phase. Preparation of S3 high-temperature trehalose-nanoemulsion: The aqueous phase, controlled at 63℃, is slowly added dropwise to the oil phase at 63℃ at a rate of 12mL / min, while stirring at a stirring rate of 450rpm. After the addition is complete, stirring is continued for 12min. Then, 28kg of trehalose and 2.5kg of azadirachtin are added and sonicated for 10min for secondary emulsification. The secondary emulsification temperature is maintained at 59℃ and the ultrasonic power is 190w to obtain the high-temperature trehalose-nanoemulsion, which is stored at 5℃ away from light for later use.

[0033] 2. Comprehensive control measures for disinfection, transplanting, and planting of *Anoectochilus roxburghii* seedlings, along with high-temperature management. (1) Disinfection treatment before planting: In April, before transplanting the seedlings of Anoectochilus roxburghii, disinfect them by drenching with 50% carbendazim at a dilution of 400 times, with a drenching volume of 20L per square meter, and adjust the soil EC value to 1.5mS / cm. Then, use 0.1% citric acid solution or wood ash to adjust the soil pH value, and control the pH value at 5.0-6.0 to obtain disinfected Anoectochilus roxburghii seedlings; (2) Transplanting and planting: A mixture of humus, perlite, and coconut coir in a ratio of 3.5:1.1:0.9 was used as the substrate for transplanting Anoectochilus roxburghii seedlings. The substrate was disinfected with a 400-fold dilution of 50% carbendazim at a concentration of 20 L / m². 2 Four days after disinfection, inoculate with 5% Trichoderma harzianum at a dosage of 12 g / m³. 2Six days later, transplant the seedlings of Anoectochilus roxburghii. The planting spacing is 10cm×15cm, and the planting depth is 2-3cm. The rhizome should be level with the substrate surface. Plant 60 seedlings per square meter. Water immediately after transplanting. Use deionized water with an electrical conductivity ≤10μS / cm. Use 20L per square meter and drip irrigate until the substrate moisture reaches 65%. (3) High-temperature composite control: After the golden thread lotus enters the maturity period in July, when the temperature reaches ≥35℃, a 60% shade net is erected on the top in conjunction with axial flow fans for forced ventilation to control the high temperature. The wind speed is 0.6m / s, and ventilation is carried out 4 times a day for 2.2 hours each time. After ventilation, the highest temperature in the greenhouse should be ≤32℃, the day-night temperature difference should be ≤8℃, the relative humidity should be controlled at 65%, and the wind speed difference between the four corners and the central area of ​​the greenhouse should be ≤0.3m / s. After ≥3 consecutive days of high temperature of 35℃, a high-temperature special trehalose-nano emulsion is added The trehalose-nanoemulsion was diluted with water to obtain a high-temperature-specific trehalose-nanoemulsion solution. This solution was sprayed every 6 days to control high-temperature resistance. The dosage was 55L per acre each time. Spraying was done before 9:00 AM or between 2:00 PM and 4:00 PM. The solution was preheated to 28°C before spraying, and ventilation was closed for 2.2 hours after spraying. Simultaneously, after three consecutive days of temperatures ≥35°C, 900 Neoseios californicus mites were released per acre for high-temperature biological control. Preparation method of high-temperature special trehalose-nano emulsion dilution: When the mite density in 100g of soil is >5, add 1L of trehalose-nano emulsion to 900L of water before use, and dilute and mix evenly; when the air humidity is >85%, add 1L of trehalose-nano emulsion to 900L of water before use, and dilute and mix evenly.

[0034] (4) When the high temperature of ≥35℃ ends, apply a concentrated spray of high temperature special trehalose-nano emulsion diluted solution once, with a dosage of 25L per mu.

[0035] Comparative Example 1: Conventional methods for controlling root mites under extreme high temperatures (≥35℃) (1) Preparation of conventional anti-mite agents Drug type: Commercially available 20% avermectin emulsifiable concentrate (general formulation, without nano-encapsulation technology).

[0036] Preparation steps: ① Oil phase: Weigh 20 kg of abamectin technical, 800 kg of xylene, and 50 kg of sodium dodecylbenzenesulfonate (emulsifier), and stir at room temperature (200 rpm) until dissolved; ② Aqueous phase: Take 130 kg of deionized water, without adding heat-stabilizing agents such as trehalose; ③ Emulsification: Pour the aqueous phase into the oil phase at a rate of 30 mL / min, and stir for 10 min (300 rpm) to obtain a conventional emulsifiable concentrate (particle size distribution CV value 18%, no high-temperature stable structure).

[0037] Defects: Demulsification rate is 42% (effective ingredient loss rate is 38%) in environments above 35℃, and the effective period is less than 2 days (data from the Journal of Plant Protection, 2025).

[0038] (2) Overall prevention and control measures Physical control: Only 30% shade netting is installed (no axial flow fan), the temperature inside the greenhouse is 1-2℃ higher than the outside temperature (up to 40℃ in extreme cases), and the relative humidity is 55%-90% (with large fluctuations).

[0039] Biological control: No California neoseii mite was released, and there were no natural enemies to help control the mites.

[0040] Spraying of pesticides: ① Dosage: 1000 times dilution (50L per acre), once every 7 days, no additional application was made after more than 7 consecutive days of high temperature; ② Adjustment basis: Chlorpyrifos was only used when the leaf damage rate was ≥20%, and the concentration was not adjusted according to the mite density (>5 mites / 100g soil); ③ Operation: Ventilation was carried out within 1 hour after spraying, resulting in 30% loss of pesticide.

[0041] (3) Drug resistance monitoring 1) The "field visual inspection method" was used, which only counted the number of visible mites and did not detect the G119S mutation frequency; 2) There is no early warning mechanism. When drug resistance is discovered, the mutation frequency has already reached more than 12%.

[0042] (4) Prevention and control effect 1) Density of root mite: After 45 days of continuous high temperature, the density reached 12 mites / 100g soil (In Example 1 of this invention, the density of root mite after high temperature compound control of Anoectochilus roxburghii was ≤2 mites / 100g soil). 2) Drug resistance: The peak mutation frequency of G119S was 12.3%, with a monthly growth rate of 1.8% (the peak mutation frequency of G119S after high-temperature compound control of Anoectochilus roxburghii in Example 1 of this invention was only 4.1%). 3) Growth of Anoectochilus roxburghii: The yield is 82 kg per mu, and the content of Anoectochilus roxburghii glycoside is 1.21 mg / g (In Example 1 of this invention, the yield of Anoectochilus roxburghii after high temperature compound control reached 118 kg per mu, and the content of Anoectochilus roxburghii glycoside reached 1.68 mg / g).

[0043] To verify the effectiveness of the invention, the inventors conducted the following experiments in order to better understand how the invention achieves its purpose and solves related technical problems: Preparation of emulsion for mite control in extreme high-temperature climates using *Anoectochilus roxburghii* 1.1 Preparation of high-temperature specific trehalose-nanoemulsion (≥35℃) 1.1.1 Raw material preparation (based on 1000kg of finished product) Oil phase (265 kg): 220 kg of terpinene-4-ol (purity ≥95%), 5 kg of tea polyphenols (purity ≥98%), and 40 kg of diatomaceous earth (particle size ≤5 μm); Aqueous phase (702kg): Tween-80 40kg (HLB value 15.0±0.5), deionized water 662L (conductivity ≤10μS / cm); Functional additives (33kg): Trehalose 30kg (food grade), Azadirachtin 3kg (content ≥90%); Terpinene-4-ol (220kg): Targeting the sensitive nature of the nervous system of mites under high temperature, it disrupts their nerve conduction through contact killing. A 22% concentration can cause a 90% mortality rate of mites in 24 hours at 35℃ (Journal of Pesticide Science 2025). Tea polyphenols (5kg): inhibit the activity of P450 detoxification enzymes in mites, and synergistically enhance the effect with terpinen-4-ol, reducing the metabolic rate of the agent in mites under high temperature (synergistic ratio 1:1.8). Trehalose (30kg): forms a nano-encapsulation membrane (particle size 30-80nm), preventing the volatilization of active ingredients at high temperatures (≥35℃), thus extending the duration of effectiveness from 3 days to 7 days.

[0044] 1.1.2 Preparation steps Oil phase mixing: Add the oil phase components to the reactor in sequence, and stir at 300 rpm ± 20 rpm under a constant temperature of 65℃ ± 2℃ until completely dissolved (about 20 min). During this period, temperature fluctuations are monitored in real time using a thermometer inside the reactor.

[0045] Aqueous phase treatment: Add the aqueous components to the heating tank, heat to 65℃±2℃, and stir to completely dissolve Tween-80 (200 rpm).

[0046] Mixed addition: The aqueous phase is added dropwise to the oil phase reactor at a rate of 10 mL / min ± 2 mL / min using a peristaltic pump, while stirring at 500 rpm ± 50 rpm. After the addition is complete, continue stirring for 10 min (to ensure that the phase interface is fully integrated).

[0047] Functional ingredient addition: Trehalose and azadirachtin were added, and an ultrasonic emulsifier was used at a power of 200W±10W and a temperature of 60℃±1℃ for 10 minutes. The emulsification state was observed every 2 minutes during the process to form a preliminary emulsion.

[0048] 1.1.3 Quality Inspection and Storage: Use a Malvern particle size analyzer to detect particle size distribution, ensuring a CV value ≤ 7% and a particle size range of 30-80 nm.

[0049] The finished product should be stored at 4℃±1℃ away from light. The shelf life is 6 months. During the storage period, the demulsification rate should be checked monthly (it must be ≤8%).

[0050]

Notice

[0051] 1.1.4 Adaptability Adjustment Specifications Altitude adaptation: For every 1000m increase in altitude, the emulsification temperature is reduced by 3-5℃ (e.g., at an altitude of 2000m, the oil phase / water phase temperature is adjusted to 58-62℃), while the ultrasonic emulsification time is extended by 2min.

[0052] Raw material purity adjustment: When the raw material purity is <95%, it should be purified by vacuum distillation in advance (vacuum degree 0.08MPa±0.01MPa, temperature 70℃±2℃), and the purity of the collected fraction should be ≥95%.

[0053] 2. Experiments on the control of mites, insects, and pesticide resistance in Anoectochilus roxburghii under extreme high-temperature climates 2.1 Experimental Design Experiment on the combined control of pesticides, agronomy and biology and management of herbicide resistance in Anoectochilus roxburghii under extreme high temperature climate (high temperature ≥35℃ for ≥3 consecutive days).

[0054] 2.2 Experimental Period April 2024 - October 30, 2024 (harvested in October 2024).

[0055] 2.3 Experimental Objective Verify the stability and mite control specificity of high-temperature trehalose-nanoemulsion under extreme climate conditions; Quantify the mechanism by which the synergistic control of pesticides, agronomy, and biology improves yield and quality; Establish a complete data chain for dynamic monitoring and regulation of drug resistance (G119S gene mutation).

[0056] 2.4 Experimental Materials and Equipment Test crop: Anoectochilus roxburghii seedlings (local species in Boluo, seedling age 30 days, survival rate ≥90%, 100 plants were randomly sampled for statistical analysis). Specialized medicine: High-temperature specific trehalose-nanoemulsion (prepared by the method in Example 1); Biological control material: 5% Trichoderma harzianum preparation (1×10 8 CFU / g), Neoseui californicum (provided by Institute of Plant Protection, Chinese Academy of Agricultural Sciences, viability ≥95%); Equipment and testing instruments: 50% shade net (ISO 9001 certified), axial flow fan (wind speed 1.2m / s), Malvern particle size analyzer, PCR instrument (ABI 7500), HPLC instrument (Agilent 1260).

[0057] 2.5 Field Design A randomized block design was adopted, with 4 treatments (3 replicates, cell size 20㎡): ① High Temperature Control Group (Extreme High Temperature + High Temperature Special Trehalose-Nanoemulsion + Agronomy + Biological Control); ② Predatory Mite Group (Extreme High Temperature + Agronomy + Biological Control).

[0058] Planting density: 10cm × 15cm spacing between plants, 132 plants per plot (66 plants / ㎡).

[0059] 2.6 Experimental Implementation Record of the High Temperature Control Group 2.6.1 Planting (April 2024 - October 2024, with an emphasis on high temperature control) 2.6.1.1 Pre-planting preparations (April 15 - April 25, 2024) April 15: Before transplanting the seedlings of *Anoectochilus roxburghii*, disinfect them by drenching with a 50% carbendazim solution diluted 500 times, using 25L of solution per square meter. Adjust the EC value to 1.2mS / cm, and then use a 0.1% citric acid solution or wood ash to adjust the pH value to 5.5-6.5, thus obtaining disinfected *Anoectochilus roxburghii* seedlings.

[0060] April 18th: A mixture of humus, perlite, and coconut coir in a 3:1:1 ratio was used as the substrate for transplanting *Anoectochilus roxburghii* seedlings. The substrate was disinfected with a 50% carbendazim solution diluted 500 times, at a rate of 25 L / m³. 2 On the third day after disinfection, inoculate with 5% Trichoderma harzianum at a dosage of 10 g / m³. 2 (The colony coverage reached 60% after 7 days); April 25: Transplanting on a cloudy day (average daily temperature 23℃). Planting spacing is 10cm×15cm, depth is 2-3cm. Water until the substrate moisture is 70% (seedling survival rate is 92%, with 3 replicates and 30 plants randomly sampled from each replicate). The amount of water used is 2L per square meter. The rooting water should be deionized water (conductivity ≤10μS / cm) to avoid residual chlorine in tap water. Check the humidity 2 hours after watering and water again if necessary.

[0061] 2.6.1.2 Implementation of High Temperature Prevention and Control Measures (July 13, 2024 - September 1, 2024) Extreme high temperature definition: When Anoectochilus roxburghii enters the maturity stage and the average daily temperature reaches ≥35℃ for 3 consecutive days (natural high temperature), the first level of prevention and control will be activated; 2.6.1.3 Experimental Record of High Temperature Control Agent Group July 13 (Day 1): Set up 50% shade netting in advance and start the axial flow fan (the greenhouse temperature is 5℃ lower than the outside temperature, and the humidity is 70±3%). July 15 (Day 3): Spray with a 1000-fold diluted solution of high-temperature special trehalose-nano emulsion, 50L per acre (completed at 8:30 am). After spraying, close the ventilation for 2 hours, and start the first ventilation at 10:30 am (half the duration to 30 minutes). July 16 (Day 4): Release California neoseii mites (1000 per acre) and monitor the colonization rate (reaching 60%). July 19 (Day 7): Conduct colonization rate testing for Neoseii calcitoninus in California (reached 85% → meets the standard); July 20 (Day 8, continuous high temperature for more than 7 days): Since it was the same day as the regular spraying (1000 times diluted solution of high temperature special trehalose-nano emulsion, 50L / mu), the regular spraying was carried out according to the 5-day cycle (same dosage as before); July 25 (Day 13): Routine spraying (1000-fold dilution of high-temperature special trehalose-nano emulsion, 50L / mu), PCR detection showed G119S mutation frequency of 3.8% (not reaching the warning value). July 30 (Day 18): Routine spraying (1000-fold dilution of high-temperature special trehalose-nano emulsion, 50L / mu), monitoring the highest temperature inside the greenhouse was 32℃ (ventilation met the standard), and the particle size CV value of the high-temperature special trehalose-nano emulsion was 6.7% (good stability). August 1 (Day 20, more than 13 consecutive days of high temperature): Spray with half the dose (1000 times diluted solution of high temperature special trehalose-nano emulsion, 25L / mu) (to ensure the survival rate of natural enemies), and simultaneously release 500 California neoseii mites / mu (to supplement natural enemies); August 5 (Day 24): Routine spraying (1000x dilution of high-temperature special trehalose-nano emulsion, 50L / mu), HPLC test showed that the pesticide residue on the leaves was 0.003mg / kg (compliant). August 10 (Day 29): Routine spraying (1000-fold dilution of high-temperature special trehalose-nano emulsion, 50L / mu). On the same day, PCR testing showed a G119S mutation frequency of 4.0%, and the azadirachtin concentration was maintained at 0.3% (not reaching the upregulation threshold). August 12 (Day 31): Mite density was 1.5 individuals / 100g soil (0.3 individuals higher than the previous count, which is within the normal range). August 15 (Day 34): Routine spraying (1000 times diluted high-temperature special trehalose-nano emulsion, 50L / mu), axial flow fan switched to high frequency mode (wind speed 1.5m / s) because the outside temperature reached 38℃; August 20 (Day 39): Routine spraying (1000 times diluted high-temperature special trehalose-nano emulsion, 50L / mu), with simultaneous testing of the high-temperature special trehalose-nano emulsion particle size of 30-80nm and demulsification rate of 7.1% (<8%, stable). August 22 (Day 41): PCR detection showed a G119S mutation frequency of 3.6% (effective regulation); August 25 (Day 44): Routine spraying (1000x dilution of high-temperature special trehalose-nano emulsion, 50L / acre), ventilation time half an hour earlier; simultaneous mite density test 0.95 mites / 100g soil; August 27 (Day 1 after the heatwave ends): Daily high temperature below 35℃, marking the beginning of the end of the continuous high-temperature period; August 30 (4th day after the end of high temperature): Although the high temperature has ended, it is still within the control period. Carry out the last spraying within the period (1000 times diluted solution of high temperature special trehalose-nano emulsion, 50L / mu). August 31 (5th day after the end of the high temperature period): The highest temperature has been below 35℃ for 5 consecutive days, ending the continuous high temperature prevention and control period. All tests have met the standards. September 1 (end of high temperature control period): Perform one cycle of consolidation spraying (1000 times diluted high temperature special trehalose-nano emulsion, 25L / mu). On that day, the temperature inside the greenhouse was 32℃ and the relative humidity was 72%. The continuous high temperature control ended and routine management began.

[0062] 2.7 Biocontrol Validation A predatory mite group (which did not use the high-temperature-specific trehalose-nanoemulsion, but used the same methods as the high-temperature control group) was established, achieving a mite control rate of 50% and a predatory mite survival rate of 65% after 3 days. The group using the high-temperature-specific trehalose-nanoemulsion in conjunction with the predatory mite group achieved a mite control rate of 80% (a 40% improvement over the predatory mite group alone). Monitoring showed that the predatory mite survival rate was ≥85% 3 days after spraying, as the agent inhibited the expression of the CYP6 family of mite esterase genes, enhancing predation efficiency against resistant individuals. The mite incidence rate in high-temperature scenarios decreased from 32% to 3.8%, a 62% improvement compared to the original technology.

[0063] 2.8 Harvesting: The harvesting date is October 21, 2024. During the high-temperature season, the yield of Anoectochilus roxburghii is 118 kg per mu. The use of pesticides is reduced by 2.3 tons per 100 mu, and carbon emissions are reduced by 28 tons (calculated according to ISO 14064-1).

[0064] Ten plants were randomly sampled from each plot, and HPLC was used to detect anoectochilin (chromatographic conditions: C18 column, methanol-water 20:80, flow rate 1.0 mL / min, 254 nm).

[0065] The content of rosinase was determined by HPLC (chromatographic conditions: C18 column, methanol-water 20:80, flow rate 1.0 mL / min, 254 nm). The rosinase content of the group using high-temperature trehalose-nanoemulsion was 1.68 mg / g, compared with 1.21 mg / g in the group that only preyed on mites.

[0066] 3. Preparation of PCR system for drug resistance monitoring Configuration table of PCR system for drug resistance monitoring (specifically for G119S gene mutation detection) 3.1 Sample pretreatment system (for DNA extraction) Table 1: Sample Pretreatment System 3.2 PCR amplification reaction system Table 2: PCR amplification reaction system (20 μL total system) 3.3 PCR reaction procedure Table 3: PCR reaction procedure 3.4 Primer Information (Specifically for G119S Gene Mutation Detection) Table 4: Primer Information 3.5 Reagent Specifications and Operating Precautions 3.5.1 Key reagent specifications: Lysis buffer: contains 20 mg / mL proteinase K; Glycerol: analytical grade, 10% concentration (for low-temperature samples only, to prevent DNA degradation); Fluorescent primers: Store at -20℃ away from light to avoid repeated freeze-thaw cycles that could cause labeling failure.

[0067] 3.5.2 Operational Prohibitions: The system configuration must be performed in a sterile operating room to prevent cross-contamination; Each experiment requires the inclusion of a negative control (without template water) and a positive control (a known mutant sample) to verify the effectiveness of the system.

[0068] 3.6 Results: During the high-temperature period, the G119S mutation frequency was controlled at 4.1% (12 months of monitoring, standard deviation ±0.3%), the resistance period was extended to 28 months, the drug resistance index of mites to the agent was reduced by 35% compared with the traditional method without the use of agents, and the inhibition rate of other metabolic resistance genes such as the CYP6 family of esterase genes reached 29%.

[0069] 4. Compatibility testing of biological agents and pharmaceuticals Biological Agent and Pharmaceutical Compatibility Test Report (Specifically for Experiments on Extreme Climate Prevention and Control of Anoectochilus roxburghii) 4.1 Purpose of the test This experiment aimed to verify the compatibility of 5% Trichoderma harzianum preparation, Neoseis caesarea, and high-temperature-specific trehalose-nanoemulsion, ensuring that there is no mutual inhibition when the "biological agent-drug" works synergistically, thus guaranteeing the control effect.

[0070] 4.2 Testing Materials and Equipment 4.2.1 Biological agents: 5% Trichoderma harzianum preparation (1×10 8 CFU / g, same as the main experimental material); Neoseui caesarea (vitality ≥95%, provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, same as the main experimental material).

[0071] 4.2.2 Reagents: High-temperature specific trehalose-nanoemulsion (1000 times dilution, same as the concentration used in the main experiment).

[0072] 4.2.3 Equipment: Incubator (25℃±1℃), stereomicroscope (magnification 40×), petri dish (9cm), counter, temperature and humidity recorder.

[0073] 4.3 Detection methods and operating procedures 4.3.1 Compatibility testing of Trichoderma harzianum with pesticides Experimental design: Two treatment groups (three replicates) were set up, with each group using 500g of sterile substrate (same as the main experimental formula). Control group: Inoculated only with Trichoderma harzianum (10 g / m²) 2 (same as the amount used in the main experiment). High-temperature agent group: 24 hours after inoculation with Trichoderma harzianum, spray with a 1000-fold diluted solution of high-temperature special trehalose-nanoemulsion (50L / acre, same as the main experimental dose).

[0074] Testing indicators and cycle: 1 day, 3 days and 7 days after spraying, 10g of substrate was taken from each group and the number of colonies was counted using the dilution plate method (PDA medium); Observe the mycelial morphology (deformed, broken) and spore germination rate (randomly count 100 spores and calculate the germination rate).

[0075] 4.3.2 Compatibility testing of Neoseui californicus with pesticides Experimental design: Two treatment groups were set up (three replicates). Ten seedlings of Anoectochilus roxburghii with uniform growth were selected in each group (with leaves infested with mites, mite density of 20 per seedling). Control group: Release only Neoseii Californians (1000 per acre, same as the main experiment). High-temperature pesticide group: 24 hours after the release of predatory mites, spray with a 1000-fold diluted solution of high-temperature special trehalose-nanoemulsion (50L / acre, same as the main experimental dose).

[0076] Testing indicators and cycle: 1 day, 3 days and 5 days after spraying, the number of surviving predatory mites in each group was counted under a stereomicroscope, and the survival rate was calculated; Record the activity frequency of predatory mites (moving a distance of ≥5cm per hour is considered active) and the average daily predation amount (the number of harmful mites preyed on by each mite per day).

[0077] 4.4 Test Results 4.4.1 Results of compatibility between Trichoderma harzianum and pesticides Table 5: Compatibility results of Trichoderma harzianum with pesticides Conclusion: The survival rate of Trichoderma harzianum colonies in the high-temperature agent group was ≥91.5%, and there was no significant difference in mycelial morphology and spore germination rate compared with the control group (P>0.05), indicating good compatibility.

[0078] 4.4.2 Results of Neoseii calica compatibility with pesticides Table 6: Results of compatibility between Neoseii calica and pesticides Conclusion: The survival rate of predatory mites in the drug-treated group was ≥81%, and the difference in activity frequency and predation amount between the drug-treated group and the control group was ≤5% (P>0.05). The drug showed no significant toxic effects and good compatibility.

[0079] 5. Test Report on the Toxicity Determination of Golden Thread Lotus Special Agent for Extreme Climate 5.1 Purpose of the Measurement The study aimed to clarify the control efficacy of high-temperature trehalose-nanoemulsion against mites under different G119S mutation frequencies, and to determine the "critical point for decreased control efficacy" and the regulatory threshold for drug resistance monitoring (e.g., G119S mutation frequency ≥ 6%).

[0080] 5.2 Measurement Materials and Design Test reagents: High-temperature trehalose-nanoemulsion containing 0.3% azadirachtin (developed by the team, preparation date: August 2, 2023, number: HT-20230802-01); Low-temperature proline-modified emulsifier containing 0.2% terpinene (developed by the team, preparation date: August 2, 2023, number: LT-20230518-03); Test mites: *Rhizoctonia solani* (collected from Boluo base, sampling date: August 5, 2023, divided into 5 groups by molecular testing, with G119S mutation frequencies of 0%, 3%, 6%, 9%, and 12%, respectively, with 3 replicates per group, numbered A / B / C, 100 individuals per replicate). Handling method: ① High temperature group (35℃ constant temperature): spray with a 1000-fold diluted solution of high temperature special trehalose-nano emulsion, and count the mortality rate after 24 hours; ② Control group: sprayed with an equal amount of water.

[0081] Data statistical methods: Mortality rate = (number of dead mites / total number of mites) × 100%. Results are expressed as mean ± standard deviation. SPSS 26.0 software was used for significance analysis (P<0.05).

[0082] 5.3 Measurement results (mean ± standard deviation) Table 7: Results of Toxicity Testing of *Anoectochilus roxburghii* Special Agent for Extreme Climates Analysis: When the G119S mutation frequency is ≤3%, the mortality rate of the drug decreases by <5% (still ≥85%), and the control effect is stable; when the mutation frequency is ≥6%, the mortality rate drops sharply to below 68.7% (high temperature) and below 65.3% (low temperature), which is more than 23% lower than the sensitive group. This is determined to be the "critical point of significant decrease in control effect", which is completely matched with the threshold of "initiating regulation when the G119S mutation frequency is ≥6%".

[0083] 5.4 Conclusion The high-temperature-specific trehalose-nanoemulsion achieved a control effect of over 91% against sensitive mites (mutation frequency 0%) and maintained an effect of over 85% against low-resistant mites (3%). When the G119S mutation frequency was ≥6%, the control effect of the agent dropped sharply by more than 23%, which is confirmed as the scientific basis for the control threshold.

[0084] 6. Overall Conclusion The 5% Trichoderma harzianum preparation, Neoseii calica, and high-temperature-specific trehalose-nanoemulsion used in this experiment showed good compatibility. The agents did not significantly inhibit the growth, activity, or control effect of the biological agents and can be synergistically applied to the Anoectochilus roxburghii extreme climate control system.

Claims

1. A combined control method for mite and insect resistance and pesticide resistance in *Anoectochilus roxburghii* under extreme high-temperature climates, characterized in that: The aforementioned combined control method involves disinfecting, transplanting, and planting Anoectochilus roxburghii seedlings in April, which then mature in July. Mature seedlings are then sprayed with a high-temperature-specific trehalose-nanoemulsion diluted solution under continuous high-temperature conditions (≥35℃) to control high-temperature resistance. The combined prevention and control method shall be carried out according to the following specific steps: (1) Disinfection treatment before planting: In April, before transplanting the seedlings of Anoectochilus roxburghii, disinfect them by drenching with 50% carbendazim at a dilution of 400-600 times, with a drenching volume of 20-30L per square meter, and adjust the soil EC value to 1.0-1.5mS / cm. Then, use 0.1% citric acid solution or wood ash to adjust the soil pH value to 5.0-7.0, and obtain disinfected Anoectochilus roxburghii seedlings. (2) Transplanting and planting: Use humus:perlite:coconut coir = 2.5-3.5:0.9-1.1:0.9-1.1 as the mixed substrate for transplanting Anoectochilus roxburghii seedlings. Disinfect the mixed substrate with 50% carbendazim at a dilution of 400-600 times. The dosage of 50% carbendazim at a dilution of 400-600 times is 20-30 L / m². 2 Inoculate with 5% Trichoderma harzianum 2-4 days after disinfection. The dosage of 5% Trichoderma harzianum 8-12 g / m³ is as follows. 2 Transplant the seedlings of *Anoectochilus roxburghii* 6-8 days later, with a spacing of 10cm x 15cm and a planting depth of 2-3cm. Plant the seedlings so that the rootstock is level with the substrate surface, with 60-70 seedlings per square meter. Immediately after transplanting, water thoroughly with deionized water (≤10μS / cm) at a rate of 20-30L per square meter, drip-irrigating until the substrate moisture reaches 65-75%. (3) High-temperature composite control: After the golden thread lotus enters the maturity period in July, when the temperature reaches ≥35℃, a 40-60% shade net is erected on the top and axial flow fan is used for forced ventilation to control the high temperature. The wind speed is 0.6-1.5m / s, and ventilation is carried out 2-4 times a day, each time for 1.8-2.2 hours. After ventilation, the highest temperature in the greenhouse should be ≤32℃, the day-night temperature difference should be ≤8℃, the relative humidity should be controlled at 65-75%, and the wind speed difference between the four corners and the central area of ​​the greenhouse should be ≤0.3m / s. After ≥3 consecutive days of high temperature of 35℃, a high-temperature special trehalose-nano emulsion is added. The trehalose-nanoemulsion is diluted with water to obtain a high-temperature-specific trehalose-nanoemulsion solution. This solution is sprayed every 4-6 days to control high-temperature resistance. Each application uses 45-55 L of the high-temperature-specific trehalose-nanoemulsion solution per acre. Spraying should be done before 9:00 AM or between 2:00 PM and 4:00 PM. Before spraying, the solution should be preheated to 22-28°C. After spraying, ventilation should be closed for 1.8-2.2 hours. Simultaneously, after three consecutive days of temperatures ≥35°C, 900-1100 Neoseios californicus mites per acre are released for high-temperature biological control. The method for preparing the high-temperature trehalose-nanoemulsion diluent is to take 1L of high-temperature trehalose-nanoemulsion and add it to 900-1100L of water before use, dilute and mix evenly to obtain the high-temperature trehalose-nanoemulsion diluent.

2. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1, characterized in that: In step (1), disinfection treatment before planting: In April, before transplanting the seedlings of Anoectochilus roxburghii, disinfect them by irrigating with 50% carbendazim at a dilution of 500 times, with an irrigation volume of 25L per square meter, and adjust the soil EC value to 1.2mS / cm. Then, use 0.1% citric acid solution or wood ash to regulate the soil pH value, and control the pH value at 5.5-6.5 to obtain disinfected Anoectochilus roxburghii seedlings.

3. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1 or 2, characterized in that: In step (1), the EC value adjustment method is as follows: when the EC value is <1.0mS / cm, spray a nitrogen, phosphorus and potassium water fertilizer with a concentration of 100mg / L, the mass ratio of nitrogen, phosphorus and potassium is 3:1:2, the amount of each spray is 20-30L per square meter, and the EC value is retested 24 hours after each adjustment until it is adjusted to 1.0-1.5mS / cm.

4. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1, characterized in that: In step (2), transplanting and planting: humus:perlite:coconut coir = 3:1:1 is used as the mixed substrate for transplanting Anoectochilus roxburghii seedlings. The mixed substrate is disinfected with a 50% carbendazim solution diluted 500 times, and the dosage of the 50% carbendazim solution is 25 L / m³. 2 Three days after disinfection, inoculate with 5% Trichoderma harzianum at a dosage of 10 g / m³. 2 Seven days later, transplant the seedlings of Anoectochilus roxburghii. The planting spacing is 10cm×15cm, and the planting depth is 2-3cm. The rhizome should be level with the substrate surface. Plant 75 seedlings per square meter. Immediately after transplanting, water the seedlings with deionized water with an electrical conductivity ≤10μS / cm. Use 25L per square meter and drip irrigate until the substrate moisture reaches 65-75%.

5. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1, characterized in that: In step (3), high-temperature composite control: After *Anoectochilus roxburghii* enters its maturity period in July, and after the temperature reaches ≥35℃, a 50% shade net is erected on the top in conjunction with axial flow fans for forced ventilation to control the high temperature. The wind speed is 0.6-1.5 m / s, and ventilation is carried out 3 times a day for 2 hours each time. After ventilation, the highest temperature inside the greenhouse should be ≤32℃, the day-night temperature difference should be ≤8℃, the relative humidity should be controlled at 65-75%, and the wind speed difference between the four corners and the central area of ​​the greenhouse should be ≤0.3 m / s. After ≥3 consecutive days of ≥35℃ high temperature weather, high-temperature special trehalose is applied. - Dilute the nanoemulsion with water to obtain a trehalose-nanoemulsion solution. Spray the high-temperature-specific trehalose-nanoemulsion solution to control high-temperature resistance. Spray once every 5 days, with each application using 50L of the high-temperature-specific trehalose-nanoemulsion solution per acre. Spray before 9:00 AM or between 2:00 PM and 4:00 PM. Before spraying, preheat the high-temperature-specific trehalose-nanoemulsion solution to 25°C. After spraying, close the ventilation for 2 hours. Simultaneously, after 3 consecutive days of high temperatures ≥35°C, release 1000 Neoseios californica mites per acre for high-temperature biological control.

6. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1 or 5, characterized in that: In step (3), the preparation method of high-temperature special trehalose-nanoemulsion is carried out according to the following steps: Preparation of S1 oil phase: Weigh 210-230 kg of terpinene-4-ol, 4-6 kg of tea polyphenols, and 38-42 kg of diatomaceous earth, mix them, and stir at a constant temperature of 63-67℃ for 8-12 min until completely dissolved. The stirring speed is 400-600 rpm, and the particle size is controlled at CV≤7% to obtain the oil phase for later use. The purity of the terpinene-4-ol is ≥95%, the purity of the tea polyphenols is ≥98%, and the particle size of the diatomaceous earth is ≤5 μm. Preparation of S2 aqueous phase: Weigh 38-52 kg of Tween-80 and add it to 650-670 L of deionized water. Mix and heat to 63-67 °C to obtain the aqueous phase for later use. Preparation of S3 High-Temperature Special Trehalose-Nanoemulsion: The aqueous phase, controlled at 63-67℃, is slowly added dropwise to the oil phase, which is at 63-67℃, at a rate of 8-12 mL / min, while stirring at a stirring rate of 450-550 rpm. After the addition is complete, stirring is continued for 8-12 min. Then, 28-32 kg of trehalose and 2.5-3.5 kg of azadirachtin are added and sonicated for 8-12 min for secondary emulsification. The secondary emulsification is maintained at a temperature of 59-61℃ and an ultrasonic power of 190-210 W to obtain the high-temperature special trehalose-nanoemulsion, which is stored at 3-5℃ away from light for later use.

7. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 6, characterized in that: In step (3), the preparation method of high-temperature special trehalose-nanoemulsion is carried out according to the following steps: Preparation of S1 oil phase: Weigh 220 kg of terpinene-4-ol, 5 kg of tea polyphenols and 40 kg of diatomaceous earth and mix them. Stir at 65℃ for 10 min until completely dissolved. The stirring speed is 500 rpm and the particle size is controlled CV≤7% to obtain the oil phase for later use. The purity of terpinene-4-ol is ≥95%, the purity of tea polyphenols is ≥98%, and the particle size of diatomaceous earth is ≤5 μm. Preparation of S2 aqueous phase: Weigh 40 kg of Tween-80 and add it to 662 L of deionized water. Mix and heat to 63-67 °C to obtain the aqueous phase for later use. Preparation of S3 high-temperature trehalose-nanoemulsion: The aqueous phase, controlled at 65℃, is slowly added dropwise to the oil phase at 65℃ at a rate of 10mL / min, while stirring at a stirring rate of 500rpm. After the addition is complete, stirring is continued for 10min. Then, 30kg of trehalose and 3kg of azadirachtin are added and sonicated for 10min for secondary emulsification. The secondary emulsification is carried out at a temperature of 60℃ and a sonication power of 200w to obtain the high-temperature trehalose-nanoemulsion, which is stored at 4℃ away from light for later use.

8. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1, characterized in that: In step (3), if the continuous high temperature environment of ≥35℃ exceeds 13 days, an additional spraying is applied, and the dosage of the high temperature special trehalose-nano emulsion dilution is halved, and 500 California New Seymite per acre is released again simultaneously.

9. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1 or 5, characterized in that: In step S4, the preparation of the trehalose-nanoemulsion dilution is as follows: when the mite density in 100g of soil is >5, take 1L of trehalose-nanoemulsion and add it to 999L of water before use, and dilute and mix evenly; when the air humidity is >85%, take 1L of trehalose-nanoemulsion and add it to 999L of water before use, and dilute and mix evenly.

10. The combined control method for mite and insect resistance and drug resistance of *Anoectochilus roxburghii* under extreme high-temperature climates as described in claim 1 or 5, characterized in that: In step (3), when the high temperature of ≥35℃ ends, a periodic consolidation spray of high temperature special trehalose-nano emulsion diluted solution is carried out once, with a dosage of 25L per mu.

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