Complex microbial inoculant for preventing and treating carya illinoensis perwing moth and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for preventing and treating carya illinoensis perwing moths and application of the complex microbial inoculant, and belongs to the technical field of pest prevention and treatment. The complex microbial inoculant for preventing and treating the carya illinoensis pernicifuge is prepared from the following raw materials in parts by mass: 2 to 3 parts of beauveria bassiana bacterial liquid, 3 to 5 parts of bacillus thuringiensis bacterial liquid, 4 to 8 parts of brevibacillus laterosporus bacterial liquid, 5 to 6 parts of bacillus cereus bacterial liquid, 8 to 10 parts of sodium alginate, 3 to 5 parts of seaweed extract, 1 to 2 parts of liquorice root extract and 3 to 4 parts of soybean extract. The complex microbial inoculant disclosed by the invention has an excellent prevention and treatment effect on the perfond moth, and the problems that the existing chemical agent is easy to generate drug resistance and pesticide residue, destroys ecological balance, threatens food safety and human health and the like are solved.

Description

Technical Field

[0001] This invention relates to the field of pest control technology, and in particular to a compound microbial agent for controlling the thin-shelled pecan clearwing moth and its application. Background Technology

[0002] Pecan (Carya illinoinensis), also known as American pecan or long pecan, belongs to the genus Carya in the family Juglandaceae. It is a multi-purpose economic and ecological tree species used for fruit production, oil extraction, timber, and landscaping. Pecan fruits are characterized by their large size, thin shells, high kernel yield, high production volume, sweet taste, lack of astringency, and rich nutritional value. It is an important woody oilseed tree species with an oil content as high as 70%, especially unsaturated fatty acids, which can reach 97%. Its wood has a fine and varied grain, beautiful and elegant color, and is tough and hard, making it an ideal material for interior decoration, high-end furniture, handicrafts, construction, and military applications. With its well-developed root system, upright posture, and tall tree shape, it is a popular tree species for shade, ornamental purposes, and street planting. In short, pecan is an excellent economic tree species with high economic benefits, wide applications, a long benefit period, and significant social and ecological benefits. It is world-renowned for its high commercial value and has an economic lifespan of up to 80 years.

[0003] With the continuous expansion of the cultivation area and the increasing number of pecans, various pests that harm pecans have also emerged, especially the clearwing moth, which is more susceptible to damage as the trees age. The damage not only affects tree growth but can also lead to reduced yields, affect fruit quality, and ultimately cause tree death, resulting in huge economic losses for actual production and planting.

[0004] Currently, the control of the thin-shelled pecan clearwing moth mainly employs a combination of manual and chemical control methods. Manual control includes capturing larvae through frass holes and using traps to kill adult moths. Chemical control involves spraying the trees with an 800-fold dilution of 40% dimethoate emulsifiable concentrate two to three times. For larvae that have already bored into branches, small cotton balls soaked in abamectin or a 200-fold dilution of 20% imidacloprid can be inserted into the borer holes, and then sealed with mud or plastic film. However, manual control suffers from drawbacks such as high labor intensity, low efficiency, incomplete control, short-lived effects, limited applicability, and potential mechanical damage to plants. Chemical control poses risks such as the development of pesticide resistance, pesticide residues, disruption of ecological balance, and threats to food safety and human health. Therefore, finding a pest control method that is highly effective, simple to use, and does not cause pesticide residues has become a pressing technical challenge for those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial agent for controlling the thin-shelled pecan clearwing moth and its application, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of the present invention is a compound microbial agent for controlling the thin-shelled pecan clearwing moth, comprising the following raw materials in parts by weight: 2-3 parts of Beauveria bassiana bacterial solution, 3-5 parts of Bacillus thuringiensis bacterial solution, 4-8 parts of Bacillus laterosporus brevis bacterial solution, 5-6 parts of Bacillus cereus bacterial solution, 8-10 parts of sodium alginate, 3-5 parts of seaweed extract, 1-2 parts of licorice extract, and 3-4 parts of soybean extract.

[0008] Beauveria bassiana can invade insects by germinating hyphae upon contact with the insect's body wall, producing toxins that lead to insect death; Bacillus thuringiensis produces toxins that damage the insect's digestive system, causing insect death; the metabolites of Bacillus laterosporus have inhibitory and killing effects on insect eggs; Bacillus cereus can produce lecithinase to destroy the eggshell; combining these beneficial bacteria can achieve excellent pest control results.

[0009] Furthermore, the effective viable count of each of the Beauveria bassiana, Bacillus thuringiensis, Bacillus laterosporus, and Bacillus cereus bacterial suspensions is independently 1.0 × 10⁻⁶. 7 ~1.0×10 9 cfu / mL.

[0010] Furthermore, the method for preparing the seaweed extract includes the following steps:

[0011] Kelp is dried and then pulverized to obtain kelp powder;

[0012] The kelp powder and water were mixed and extracted under high pressure to obtain a crude extract;

[0013] Activated carbon was added to the crude extract for decolorization. After decolorization, the extract was concentrated and precipitated with alcohol to obtain the seaweed extract.

[0014] Furthermore, the mass / volume ratio of the kelp powder to water is 1g:20-30mL;

[0015] The high-pressure extraction is performed at a pressure of 300–350 MPa, a temperature of 50–60 °C, and a time of 10–15 min.

[0016] The concentration is to reduce the volume to 1 / 5 of the original volume.

[0017] Seaweed extract contains abundant polysaccharide components, which can stimulate the primary metabolism of microorganisms, activate their proliferation, and enhance the pest control effect of compound microbial agents.

[0018] Furthermore, the preparation method of the licorice extract includes the following steps:

[0019] The licorice was pulverized and added to a mixed aqueous solution containing ethanol and sodium hydroxide, and then subjected to ultrasonic extraction to obtain the licorice extract.

[0020] Furthermore, the concentration of ethanol in the mixed aqueous solution containing ethanol and sodium hydroxide is 65-70 vol.% and the concentration of sodium hydroxide is 0.8-1.2 wt.%.

[0021] The mass / volume ratio of the licorice and the mixed aqueous solution is 1g:10-15mL;

[0022] The ultrasonic extraction was performed at a power of 800–900 W, a temperature of 45–55 °C, and a time of 40–60 min.

[0023] Licorice extract is rich in glycyrrhizic acid and glycyrrhizin, which can promote the growth of microorganisms, increase their number, and thus enhance the control effect of compound microbial agents on pests.

[0024] Furthermore, the method for preparing the soybean extract includes the following steps:

[0025] Soybeans were pulverized, mixed with an ethanol solution, soaked, and then extracted using microwave to obtain the soybean extract.

[0026] Furthermore, the mass / volume ratio of the soybean and the ethanol solution is 1g:25-30mL;

[0027] The concentration of the ethanol solution is 70–80 vol.%.

[0028] The soaking time is 10-15 hours;

[0029] The microwave extraction power is 350-400W, and the time is 30-45s.

[0030] Sodium alginate, as an adhesive and film-forming agent, can reduce the loss of compound microbial agents, improve the absorption efficiency of compound microbial agents, and thus ensure the control effect of compound microbial agents on pests.

[0031] Soybean extract contains a large amount of saponins, which have excellent emulsifying, foaming and wetting properties. They can reduce the surface tension of compound microbial agents, dissolve epidermal wax, and greatly improve the spreading and adhesion of compound microbial agents on the surface of plants and insects, thereby promoting absorption and improving the control effect.

[0032] The second technical solution of the present invention: a method for preparing the above-mentioned compound microbial agent, comprising the following steps:

[0033] The compound microbial agent is obtained by mixing Beauveria bassiana bacterial suspension, Bacillus thuringiensis bacterial suspension, Bacillus laterosporus brevis bacterial suspension, Bacillus cereus bacterial suspension, sodium alginate, seaweed extract, licorice extract and soybean extract evenly.

[0034] The third technical solution of the present invention: the application of the above-mentioned compound microbial agent in the preparation of a drug for controlling the thin-shelled pecan clearwing moth.

[0035] The present invention discloses the following technical effects:

[0036] The compound microbial agent of the present invention has excellent control effect on clearwing moth, overcoming the problems of existing chemical agents such as easy development of drug resistance, pesticide residues, disruption of ecological balance, and threats to food safety and human health. Detailed Implementation

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0042] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0043] Pecan, also known as American pecan, long pecan, or simply pecan, belongs to the genus Carya in the family Juglandaceae.

[0044] Thin-shelled pecans possess high economic and ornamental value, making them a versatile economic tree species used for fruit, timber, and landscaping. As one of the world's important high-grade nut trees, their kernels are delicious, fragrant, and free of astringency. They are rich in nutrients, containing approximately 72% oil, 13% carbohydrates, and 11% protein. They also contain various amino acids beneficial to the human body and are rich in vitamins B1 and B2. Each kilogram of kernels provides approximately 32 kJ of energy, making them ideal for health foods and food additives. Thin-shelled pecans are also an important oilseed tree species, with an oil content exceeding 70%, superior to camellia (44%), walnut (60%), and *Sapindus mukorossi* (57%). Their unsaturated fatty acid content reaches 97%, superior to camellia oil (91%), walnut oil (89%), peanut oil (82%), cottonseed oil (70%), soybean oil (86%), and corn oil (86%). They also have excellent storage properties, making them a superior cooking oil and salad oil (cold dish oil). Meanwhile, the thin-shelled pecan is also a timber and landscaping tree species. Its wood has a fine and varied texture, beautiful and elegant color, and is tough and very hard, making it an ideal material for interior decoration, high-end furniture, handicrafts, construction, and military applications. With its well-developed root system, upright posture, and tall tree shape, it is a popular tree species for shade, ornamental purposes, and street planting. In short, the thin-shelled pecan is an excellent economic tree species with high economic benefits, wide applications, a long benefit period, and significant social and ecological benefits. It is world-renowned for its high commercial value and has an economic lifespan of up to 80 years.

[0045] With the continuous expansion of the cultivation area and the increasing number of pecans, various pests that harm pecans have also emerged, especially the clearwing moth, which is more susceptible to damage as the tree ages. This damage not only affects tree growth but can also lead to reduced yields, compromised fruit quality, and ultimately, tree death, causing significant economic losses to actual production. In research on pecan pests, domestic and international scholars have reported on Lepidoptera such as wood-boring moths, tussock moths, bagworms, pecan boat moths, inchworms, and peach borers; Coleoptera such as scarab beetles, jewel beetles, longhorn beetles, weevils, and leaf beetles; Hemiptera such as root aphids, yellow aphids, black aphids, stink bugs, and green rice bugs; as well as underground pests and leaf-eating pests that damage pecans.

[0046] Characteristics and control methods of major pests affecting thin-shelled pecans

[0047] (1)Lepidoptera

[0048] The Lepidoptera pests that harm thin-shelled pecans are the most numerous, comprising 26 species from 15 families.

[0049] A. Tussock moths

[0050] The tussock moth is an insect belonging to the family Limacodidae in the order Lepidoptera, commonly known as the "spicy caterpillar." There are three species of tussock moths that damage pecans: the yellow tussock moth, the brown tussock moth, and the double-toothed green tussock moth. The degree of damage varies depending on the region and the population. The larvae feed on the leaves of pecans, creating a net-like pattern that leaves only the veins, thus affecting the tree's vigor. The adults have a relatively short lifespan (male adults generally live for about 7 days, and female adults for about 4 days), causing relatively little damage to pecans. In the Nanjing area, the tussock moth has two generations per year, overwintering as mature moths that spin cocoons on the trunk and branches. The cocoons have a protective layer resembling bark.

[0051] Control methods: Adult moths are attracted to light, so black light lamps can be used to trap and kill them; young larvae of the tussock moth tend to swarm and cause damage, so prune infested branches during the larval stage, or spray with 800-1000 times dilution of trichlorfon or 600 times dilution of 25% phosmet emulsifiable concentrate; in winter, overwintering cocoons are found on the bark and branches, which can be collected and killed manually.

[0052] B. Wood-boring moth

[0053] The wood-boring moth, belonging to the order Lepidoptera and the family Corydalis, is a trunk-boring pest. Its larvae feed on the xylem of the current year's shoots of thin-shelled pecan trees, damaging the vascular tissue and severely affecting the plant's water transport. They excrete brown frass and sawdust, along with a brownish liquid. The bored branches are easily broken by the wind. Affected plants become weak, and in severe cases, the entire tree can die.

[0054] Control methods: Prune infested branches. From early summer to autumn, promptly prune infested and wind-broken branches to eliminate larvae inside. In winter, during orchard sanitation, scraping off the old bark from the lower and middle parts of the trunk and applying whitewash can effectively control overwintering larvae. During the peak adult activity period (usually April to June in Nanjing), use black light lamps or insecticidal lamps to attract and kill them. During the larval hatching period in early to mid-June, spray the branches with pesticides such as acetamiprid, cypermethrin, or 50% fenitrothion at a dilution of 1000 times, once every 7 days, for 2-3 consecutive applications.

[0055] C. Peach borer

[0056] The peach twig borer, also known as the peach spotted twig borer, is an insect belonging to the family Pyralidae in the order Lepidoptera. Its larvae damage fruit. The eggs are laid on the fruit and fruit stalk. After hatching, the larvae bore into the fruit, leaving holes in the outer skin. A yellowish-brown, transparent sap oozes from these holes, often mixed with the blackish-brown excrement, adhering to the fruit surface and making it easily identifiable. When damaging young fruit, this pest can bore into the entire fruit, causing it to fall off. A single larva can often destroy an entire cluster of nuts, significantly impacting yield. For fruits in the hard-shell stage, the larvae bore into the outer pericarp, creating tunnels that severely impair nutrient and water transport, reducing the kernel yield. After maturing, the larvae spin white cocoons inside the fruit or at the junction of the fruit stalk to pupate. The adults then emerge and migrate to other fruit trees or crops to continue their damage. When this pest occurs, borer debris can be found at the base of the fruit.

[0057] Control methods: Spray pesticides during the peak egg-laying period of adults and the period of larval borer damage to fruit. From late August to early September, spray every 10 days, alternating between 50% fenitrothion emulsion at 1000 times dilution, Bacillus thuringiensis emulsion (BT) at 600 times dilution, 35% cypermethrin emulsion at 2500-3000 times dilution, or 2.5% cypermethrin emulsion at 3000 times dilution, etc., for 3 consecutive sprays; at the same time, manually remove and destroy the damaged fruit to eliminate the larvae inside the fruit.

[0058] D. Clearwing Moth

[0059] The pecan clearwing moth (Hemiberlesia lataniae) is a significant pest of pecans. Its larvae damage the lower and middle parts of the trunk of large trees with a diameter of 15 cm or more, boring holes between the phloem and xylem. Affected trees become weak, and in severe cases, the entire tree can die.

[0060] Main control measures: Promptly prune infested branches and trunks, and remove them from the orchard for centralized burning to reduce the insect population; use traps to kill adult insects; locate larvae by examining frass holes and dissecting the stems to kill them; combine with chemical control, during the larval stage, try inserting small cotton balls soaked in a 200-fold dilution of chlorpyrifos or imidacloprid into the insect holes, and then seal them with mud or plastic film. In winter, during orchard sanitation, scrape off the old bark from the lower and middle parts of the trunk with a knife and whitewash it, which is effective in controlling overwintering larvae.

[0061] E. Other Lepidoptera pests

[0062] Some pecan plants have been found to be infested with stem borers, damaging the main trunk and lateral branches. Control methods include promptly pruning and burning affected branches, cleaning the frass from the borer holes in the main trunk, and sealing the holes with cotton soaked in trichlorfon or dichlorvos. In almost all planting areas, larvae of leafminer moths have been found to swarm and damage young fruit. The most effective control method is timely spraying of insecticides to kill the larvae and prevent them from entering the young fruit. Pyralid moths and leafrollers bore into the top shoots and fruit, while noctuid moths damage the roots of the pecan.

[0063] (2) Coleoptera

[0064] Among the Coleoptera pests that damage pecans, the family Cerambycidae is the most abundant, with the spotted longhorn beetle, star longhorn beetle, and thin-winged saw beetle causing the most serious damage, followed by the bark beetles and weevils.

[0065] Longhorn beetles damage the leaves and branches of pecans, and severely affected plants can even die. Larvae of the star longhorn beetle and the thin-winged saw beetle feed on the bark of the root collar, causing the host to die. During the larval stage (July-August), the larvae will excrete feces at the infested areas, making them easily identifiable. At this time, dichlorvos (undiluted or diluted 5-10 times) can be injected into the insect holes, and then sealed with cotton balls or mud for control.

[0066] The main beetle that infests thin-shelled pecans is the green-spotted beetle. Adults feed on leaves, while larvae damage the roots of seedlings. During the peak infestation period (June-July), spray the leaves with 0.4% trichlorfon in the evening; or mix 100g of 50% phoxim emulsifiable concentrate with 50kg of seeds, or mix with 1kg of slag, and then scatter the resulting 5% toxic sand into the sowing furrow to kill the larvae.

[0067] The jewel beetle larvae feed spirally or bore into the bark of branches and trunks for up to a week, excreting brown frass and fine sawdust, and oozing a brown liquid. Infested trees quickly weaken, resulting in significantly reduced fruit yield and extremely poor quality. Damaged branches turn leaves yellow and fail to bear fruit. The peak infestation period is from June to August each year; regular inspection of the canopy and under the trunk for insect frass is necessary. If frass is found, the insect holes should be located and controlled promptly.

[0068] (3) Hemiptera

[0069] The phylloxera is one of the most serious pests of pecans, affecting up to 100% of trees, with most leaves developing galls in severe cases. Seedlings and non-fruiting trees are more severely affected, and should be sprayed in early April, early June, and early August with a 3000-fold dilution of 2.5% deltamethrin EC or a 1000-fold dilution of 80% dichlorvos EC to kill newly hatched aphids climbing the tree, as well as the second and third generations of feeding first-instar female aphids. Spraying the trunk with lime sulfur solution at 3-5 Baume degrees or whitewashing it in winter also provides some control.

[0070] Both the jujube scale and the locust scale, belonging to the scale family, cause damage by sucking sap from nymphs and adult females. The peak period of damage is from mid-April to mid-May, with infestations reaching 100% in severely affected areas. Control measures include pruning dead branches in winter and burning them in the forest, manually scraping off adult females in early spring, and using 5% diesel emulsion for combined control. During the nymph hatching period, chemical control using pesticides such as pyrethroids and chlorpyrifos can effectively control the infestation.

[0071] (4)Orthoptera

[0072] The orthoptera pest that damages pecans is the green-legged grasshopper (Cephalopoda spp.) of the family Acrididae, but its damage is relatively minor. Adults feed directly on pecan leaves; severe infestations can defoliate the tree, leading to a decrease in yield. The green-legged grasshopper has one generation per year, overwintering as eggs in the topsoil. Eggs are laid in clusters along roadsides at the foot of mountains, in areas with little weeds and loose soil. The larvae have four instars; from the second instar onwards, they climb the tree and damage the pecan leaves.

[0073] (5)Hymenoptera

[0074] Hymenoptera pests that damage pecans include black ants (of the Formicidae family) and sawflies (of the Spodidae family). Adult sawflies damage the tender branches and trunks of pecans, while the larvae feed on the leaves. Black ants damage the roots, branches, leaves, and fruit of pecans. Controlling sawflies is similar to controlling leaf-eating pests; black ants are controlled by mixing fipronil with farmyard manure and applying it around the trunk 2-3 times a year.

[0075] (6) Acari order

[0076] Spider mites, belonging to the family Tetranychidae, are among the mites that damage thin-shelled pecans. After these mites pierce and feed on pecan leaves, small chlorotic spots initially appear, which then enlarge and merge, eventually turning the entire leaf yellowish-brown and causing it to fall off. Spider mites can be controlled by spraying with a 3000-fold dilution of 15% pyridaben EC or a 2000-fold dilution of 5% tetradifon EC.

[0077] In the following examples, "parts" refers to "parts by weight".

[0078] In the specific embodiments of this invention, *Beauveria bassiana*, *Bacillus thuringiensis*, *Bacillus laterosporus*, *Bacillus cereus*, and *Bacillus megaterium* were all purchased from the China Industrial Microbial Culture Collection Center. Specifically, the product number for *Beauveria bassiana* is CICC 14071; for *Bacillus thuringiensis*, CICC 20554; for *Bacillus laterosporus*, CICC 22322; for *Bacillus cereus*, C0395; and for *Bacillus megaterium*, CICC20167. The preparation method of the bacterial suspension is as follows:

[0079] (1) Preparation method of Beauveria bassiana bacterial suspension:

[0080] After inoculating Beauveria bassiana into liquid culture medium for activation and followed by fermentation, a bacterial count of 1.0 × 10⁻⁶ was obtained. 7 ~1.0×10 9 Beauveria bassiana culture solution at cfu / mL.

[0081] Preparation of liquid culture medium: Take 200g of peeled potatoes, cut them into small pieces, add 1000mL of water and boil for 30min. Filter out the potato pieces, add 20g of glucose, 0.1g of yeast extract, 3.0g of KH2PO4 and 1.5g of MgSO4·7H2O, then add water to make up to 1000mL, adjust the pH to 6.0, and sterilize at 121℃ for 15min.

[0082] (2) Preparation method of Bacillus thuringiensis bacterial suspension:

[0083] Bacillus thuringiensis was inoculated into liquid culture medium for activation and then fermented to obtain a bacterial count of 1.0 × 10⁻⁶. 7 ~1.0×10 9 Bacillus thuringiensis bacterial suspension at cfu / mL.

[0084] Preparation of liquid culture medium: Take 5g of peptone, 3g of beef extract and 5g of sodium chloride, add water to make up to 1000mL, adjust the pH to 7.0, sterilize at 121℃ for 15min.

[0085] (3) Preparation method of Bacillus retroflexus bacterial suspension:

[0086] After inoculating Bacillus laterosporus with liquid culture medium for activation and followed by fermentation, a bacterial count of 1.0 × 10⁻⁶ was obtained. 7 ~1.0×10 9 CFU / mL of Bacillus lateralis culture.

[0087] Preparation of liquid culture medium: Take 0.5g yeast extract, 20g mannitol, 0.2g KH2PO4, 0.8g K2HPO4, 0.2g MgSO4·7H2O, 0.1g CaSO4·2H2O, 1mg FeCl3, and 1mg Na2MoO4·2H2O, add water to make up to 1000mL, adjust the pH to 7.2, and sterilize at 121℃ for 15min.

[0088] (4) Preparation method of Bacillus cereus bacterial suspension:

[0089] After inoculating Bacillus cereus into liquid culture medium for activation and followed by fermentation, a bacterial count of 1.0 × 10⁻⁶ was obtained. 7 ~1.0×10 9 Bacillus cereus bacterial suspension at cfu / mL.

[0090] Preparation of liquid culture medium: Take 5g of peptone, 3g of beef extract and 5g of sodium chloride, add water to make up to 1000mL, adjust the pH to 7.0, sterilize at 121℃ for 15min.

[0091] (5) Preparation method of Bacillus megaterium culture:

[0092] Bacillus megaterium was inoculated into liquid culture medium for activation and then fermented to obtain a bacterial count of 1.0 × 10⁻⁶. 7 ~1.0×10 9 Bacillus megateria culture at cfu / mL.

[0093] Preparation of liquid culture medium: Take 5g of peptone, 3g of beef extract and 5g of sodium chloride, add water to make up to 1000mL, adjust the pH to 7.0, sterilize at 121℃ for 15min.

[0094] Example 1

[0095] A compound microbial agent for controlling the thin-shelled pecan clearwing moth:

[0096] (1) A compound microbial agent for controlling the thin-shelled pecan clearwing moth, composed of the following raw materials in parts by weight: Beauveria bassiana inoculum solution (containing 1.0 × 10⁻⁶ bacteria). 8 2.5 portions of Bacillus thuringiensis bacterial suspension (cfu / mL) and 1.0 × 10⁻⁶ bacterial count. 8 Five samples of *Bacillus laterosporus* suspension (cfu / mL) and five samples of *Bacillus laterosporus* suspension (containing 1.0 × 10⁻⁶ bacteria). 8 Six samples of *CFU / mL* and *Bacillus cereus* bacterial suspension (containing 1.0 × 10⁻⁶ bacteria) were tested. 8 5 parts (cfu / mL), 9 parts sodium alginate, 5 parts seaweed extract, 1 part licorice extract and 4 parts soybean extract.

[0097] (2) Preparation of seaweed extract:

[0098] Kelp is dried and then pulverized to 40 mesh to obtain kelp powder;

[0099] Kelp powder and water were mixed at a ratio of 1g:25mL and subjected to high-pressure extraction (pressure of 350MPa, temperature of 50℃, and time of 12min) to obtain crude extract.

[0100] Add activated carbon (mass ratio of crude extract to activated carbon is 1:2) to the crude extract for decolorization for 60 min. After decolorization, concentrate to 1 / 5 of the original volume to obtain a concentrated solution. Mix the concentrated solution with anhydrous ethanol at a volume ratio of 1:3 for alcohol precipitation, and dry to obtain seaweed extract.

[0101] (3) Preparation of licorice extract:

[0102] The dried licorice was pulverized to 40 mesh to obtain licorice powder;

[0103] Add a mixed aqueous solution containing ethanol and sodium hydroxide to licorice powder (mass / volume ratio of licorice powder to mixed aqueous solution is 1g:15mL), and then perform ultrasonic extraction (ultrasonic extraction power is 900W, temperature is 55℃, time is 60min). After filtration, concentrate the extract and dry it to obtain licorice extract.

[0104] The concentration of ethanol in the mixed aqueous solution containing ethanol and sodium hydroxide is 70 vol.% and the concentration of sodium hydroxide is 1.0 wt.%.

[0105] (4) Preparation of soybean extract:

[0106] Dry soybeans are ground to 100 mesh to obtain soybean powder;

[0107] Soybean flour was mixed with an ethanol solution (75 vol.% concentration, mass / volume ratio of soybean flour to ethanol solution was 1 g: 30 mL), soaked at room temperature (25℃) for 12 h, and then subjected to microwave extraction (microwave extraction power was 400 W, time was 30 s). After filtration, the extract was concentrated and dried to obtain soybean extract.

[0108] (5) Preparation of compound microbial agents:

[0109] A compound microbial agent was obtained by mixing Beauveria bassiana bacterial suspension, Bacillus thuringiensis bacterial suspension, Bacillus laterosporus brevis bacterial suspension, Bacillus cereus bacterial suspension, sodium alginate, seaweed extract, licorice extract and soybean extract evenly.

[0110] Example 2

[0111] A compound microbial agent for controlling the thin-shelled pecan clearwing moth:

[0112] (1) A compound microbial agent for controlling the thin-shelled pecan clearwing moth, composed of the following raw materials in parts by weight: Beauveria bassiana inoculum solution (containing 1.0 × 10⁻⁶ bacteria). 8 Two samples of Bacillus thuringiensis (CFU / mL) and two samples of Bacillus thuringiensis bacterial suspension (containing 1.0 × 10⁻⁶ bacteria). 8 Three samples of *Bacillus laterosporus* suspension (cfu / mL) and three samples of *Bacillus laterosporus* suspension (containing 1.0 × 10⁻⁶ bacteria). 8 Eight samples of CFU / mL and Bacillus cereus bacterial suspension (containing 1.0 × 10⁻⁶ bacteria) were tested. 8 6 parts (cfu / mL), 8 parts sodium alginate, 3 parts seaweed extract, 2 parts licorice extract and 3 parts soybean extract.

[0113] (2) Preparation of seaweed extract:

[0114] Kelp is dried and then pulverized to 40 mesh to obtain kelp powder;

[0115] Kelp powder and water were mixed at a ratio of 1g:20mL and subjected to high-pressure extraction (pressure of 300MPa, temperature of 60℃, and time of 15min) to obtain crude extract.

[0116] Add activated carbon (mass ratio of crude extract to activated carbon is 1:2) to the crude extract for decolorization for 60 min. After decolorization, concentrate to 1 / 5 of the original volume to obtain a concentrated solution. Mix the concentrated solution with anhydrous ethanol at a volume ratio of 1:3 for alcohol precipitation to obtain seaweed extract.

[0117] (3) Preparation of licorice extract:

[0118] The dried licorice was pulverized to 40 mesh to obtain licorice powder;

[0119] Add a mixed aqueous solution containing ethanol and sodium hydroxide to licorice powder (mass / volume ratio of licorice powder to mixed aqueous solution is 1g:12mL), and then perform ultrasonic extraction (ultrasonic extraction power is 800W, temperature is 50℃, time is 40min). After filtration, concentrate the extract and dry it to obtain licorice extract.

[0120] The concentration of ethanol in the mixed aqueous solution containing ethanol and sodium hydroxide is 65 vol.% and the concentration of sodium hydroxide is 1.2 wt.%.

[0121] (4) Preparation of soybean extract:

[0122] Dry soybeans are ground to 100 mesh to obtain soybean powder;

[0123] Soybean flour was mixed with an ethanol solution (the concentration of the ethanol solution was 80 vol.%, and the mass / volume ratio of soybean flour to ethanol solution was 1 g: 25 mL), and soaked at room temperature (25℃) for 15 h. Then, microwave extraction was performed (microwave extraction power was 350 W, time was 45 s). After filtration, the extract was concentrated and dried to obtain soybean extract.

[0124] (5) Preparation of compound microbial agents:

[0125] A compound microbial agent was obtained by mixing Beauveria bassiana bacterial suspension, Bacillus thuringiensis bacterial suspension, Bacillus laterosporus brevis bacterial suspension, Bacillus cereus bacterial suspension, sodium alginate, seaweed extract, licorice extract and soybean extract evenly.

[0126] Comparative Example 1

[0127] Same as Example 1, except that the Bacillus lateralis bacterial solution is replaced with an equal mass fraction of Bacillus giantis bacterial solution.

[0128] Comparative Example 2

[0129] Same as Example 1, except that the Beauveria bassiana bacterial solution was replaced with an equal mass of Bacillus thuringiensis bacterial solution;

[0130] Replace the Bacillus lateralis culture with an equal number of Bacillus cereus culture.

[0131] Comparative Example 3

[0132] Same as Example 1, except that the compound bacterial agent does not contain sodium alginate and seaweed extract.

[0133] Comparative Example 4

[0134] Same as Example 1, except that the soybean extract is replaced with an equal mass of bellflower extract.

[0135] Preparation of Platycodon grandiflorus extract:

[0136] The dried bellflower root is pulverized to 100 mesh to obtain bellflower root powder;

[0137] Mix the platycodon powder with an ethanol solution (75 vol.% concentration of ethanol solution, mass / volume ratio of platycodon powder to ethanol solution of 1 g: 30 mL), soak at room temperature (25℃) for 12 h, and then perform microwave extraction (microwave extraction power of 400 W, time of 30 s). After filtration, concentrate and dry the extract to obtain platycodon extract.

[0138] Comparative Example 5

[0139] Same as Example 1, except that the method for preparing the licorice extract is as follows:

[0140] The dried licorice was pulverized to 40 mesh to obtain licorice powder;

[0141] Add 65 vol.% ethanol aqueous solution to licorice powder (mass / volume ratio of licorice powder to ethanol aqueous solution is 1 g: 12 mL), and then perform ultrasonic extraction (ultrasonic extraction power is 800 W, temperature is 50℃, time is 40 min). After filtration, concentrate the extract and dry it to obtain licorice extract.

[0142] Example 1

[0143] (1) Select a thin-shelled pecan plantation that had been infested with clearwing moths in the previous year for the experiment. The pecan trees were 15 years old (the rate of infestation in the previous year was about 35%), and the spacing between trees was 6m×8m. The plantation was divided into several plots, with 100 trees in each plot.

[0144] (2) Before the thin-shelled pecan tree sprouts, spray the compound microbial agent prepared in the example or comparison on the trunk, branches and ground surface of the thin-shelled pecan tree (with the trunk as the center and a radius of 2m). The dosage is 300g per mu, diluted with water and sprayed. The water volume is 60L. Use clean water as a control. Do not apply other insecticides or carry out artificial control during the period.

[0145] At the end of May, the compound microbial agent prepared in the example or comparison was sprayed again on the trunk, branches and ground surface of the thin-shelled pecan trees (within a radius of 2m centered on the trunk). The dosage was 250g per acre, diluted with water and sprayed with 80L of water. Clean water was used as a control. No other insecticides were applied during the period, and no artificial control was carried out.

[0146] (3) The number of affected plants was investigated 20 days after the second application of compound microbial agent, and the rate of affected plants was calculated. The results are shown in Table 1.

[0147] The percentage of affected plants is calculated as follows: (Number of affected plants / Total number of plants surveyed) × 100%.

[0148] Table 1. Rate of Affected Plants

[0149] Grouping Percentage of affected plants (%) Example 1 3 Example 2 4 Comparative Example 1 9 Comparative Example 2 15 Comparative Example 3 17 Comparative Example 4 7 Comparative Example 5 8 Comparison 46

[0150] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound microbial agent for controlling the clearwing moth of the pecan, characterized in that, The raw materials include the following parts by weight: 2-3 parts of Beauveria bassiana bacterial solution, 3-5 parts of Bacillus thuringiensis bacterial solution, 4-8 parts of Bacillus laterosporus brevis bacterial solution, 5-6 parts of Bacillus cereus bacterial solution, 8-10 parts of sodium alginate, 3-5 parts of seaweed extract, 1-2 parts of licorice extract, and 3-4 parts of soybean extract.

2. The compound microbial agent according to claim 1, characterized in that, The effective viable count of each of the following bacterial suspensions—Beauveria bassiana, Bacillus thuringiensis, Bacillus laterosporus, and Bacillus cereus—was independently 1.0 × 10⁻⁶. 7 ~1.0×10 9 cfu / mL.

3. The compound microbial agent according to claim 1, characterized in that, The method for preparing the seaweed extract includes the following steps: Kelp is dried and then pulverized to obtain kelp powder; The kelp powder and water were mixed and extracted under high pressure to obtain a crude extract; Activated carbon was added to the crude extract for decolorization. After decolorization, the extract was concentrated and precipitated with alcohol to obtain the seaweed extract.

4. The compound microbial agent according to claim 3, characterized in that, The mass / volume ratio of the kelp powder to water is 1g: 20-30mL; And / or, the high-pressure extraction is performed at a pressure of 300–350 MPa, a temperature of 50–60 °C, and a time of 10–15 min; And / or, the concentration is to be concentrated to 1 / 5 of the original volume.

5. The compound microbial agent according to claim 1, characterized in that, The method for preparing the licorice extract includes the following steps: The licorice was pulverized and added to a mixed aqueous solution containing ethanol and sodium hydroxide, and then subjected to ultrasonic extraction to obtain the licorice extract.

6. The compound microbial agent according to claim 5, characterized in that, The concentration of ethanol in the mixed aqueous solution containing ethanol and sodium hydroxide is 65-70 vol.% and the concentration of sodium hydroxide is 0.8-1.2 wt.%. And / or, the mass / volume ratio of the licorice and the mixed aqueous solution is 1 g: 10-15 mL; And / or, the ultrasonic extraction power is 800-900W, the temperature is 45-55℃, and the time is 40-60min.

7. The compound microbial agent according to claim 1, characterized in that, The method for preparing the soybean extract includes the following steps: Soybeans were pulverized, mixed with an ethanol solution, soaked, and then extracted using microwave to obtain the soybean extract.

8. The compound microbial agent according to claim 7, characterized in that, The mass / volume ratio of the soybean and ethanol solution is 1g: 25-30mL; And / or, the concentration of the ethanol solution is 70-80 vol.%. And / or, the soaking time is 10 to 15 hours; And / or, the power of the microwave extraction is 350-400W, and the time is 30-45s.

9. A method for preparing the compound microbial agent according to any one of claims 1 to 8, characterized in that, Includes the following steps: The compound microbial agent is obtained by mixing Beauveria bassiana bacterial suspension, Bacillus thuringiensis bacterial suspension, Bacillus laterosporus brevis bacterial suspension, Bacillus cereus bacterial suspension, sodium alginate, seaweed extract, licorice extract and soybean extract evenly.

10. The use of the compound microbial agent according to any one of claims 1 to 8 in the preparation of a drug for controlling the thin-shelled pecan clearwing moth.