A biocontrol preparation containing Beauveria bassiana and its application in preventing and controlling leafminers

Abstract

The invention discloses a biological control preparation, system and application thereof for preventing and controlling leafminers, wherein the biological control preparation comprises Beauveria bassiana and a food attractant, wherein the food attractant comprises plant volatile secondary substances and a solvent, wherein the plant volatile secondary substances comprise trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone, linalool and / or caryophyllene. By using the food attractant in combination with Beauveria bassiana, the amount of attraction to leafminers is increased, and at the same time, the germination of conidia of Beauveria bassiana is not affected, thereby improving the transmission efficiency of leafminers to Beauveria bassiana, and achieving a sustained biological control effect.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and particularly relates to a biological control preparation containing Beauveria bassiana and application thereof in preventing and controlling leafminers. Background Art

[0002] The leafminer is an insect of the genus Liriomyza of the subfamily Mylidae of the family Mylidae in the order Diptera. The temperature between 20℃ and 30℃ is suitable for its growth and development, and its mortality rate increases above 30℃. The damage in the greenhouse is more serious than in the open field. The female insect lays eggs in the host leaf tissue. After the larvae hatch, they feed on the mesophyll under the leaf epidermis. The adults mostly suck the juice of the plant leaves at the wound, which slows the growth of the plant, causes the leaves to fall off, and causes a reduction in yield. The main leafminers are the American leafminer, the clover leafminer, and the South American leafminer, and the American leafminer is the most harmful. The main control method is agricultural control, plowing the land, killing the overwintering larvae in the topsoil or dead leaves, and chemical pesticides. The American leafminer Liriomyza sativae Blanchard belongs to the family Agromyzidae of the order Diptera. It is an omnivorous invasive pest that harms a variety of vegetable crops and flower crops, as well as some ornamental plants, causing serious economic losses. It particularly likes to harm vegetable crops such as cucurbits, leguminous plants, and solanaceous plants, causing crop yield reduction or even crop failure. The adults and larvae of the American leafminer will damage plant leaves. The adults pierce the leaves to feed and lay eggs, forming spots. The females lay eggs in the mesophyll tissue, where the eggs hatch into larvae. The larvae feed, grow, and develop in the mesophyll tissue, forming white tunnels, which reduce the photosynthesis capacity of the plant and affect the flowering and fruiting of crops. In 2000, it was listed as a quarantine pest by the European Commission (EU 2000).

[0003] At present, chemical control is more commonly used for the prevention and control of American leafminers. However, the frequent use of pesticides has increased the resistance of American leafminers to insecticides (Mason et al., 1987; Askari Saryazdi et al., 2014), and has an adverse effect on natural enemies, resulting in an increase in the abundance of American leafminers (Oatman & Kennedy, 1976; Matsuda & Saito, 2014). In other non-chemical control methods, such as biological control, parasitic wasps are introduced (Johnson, 1993; Ridland et al., 2020), yellow sticky boards are used to trap American leafminers based on the tendency of leafminers to yellow (Zoebisch & Schuster, 1990; Namvar, 2012), resistant plants resist the damage of American leafminers (Celin et al., 2017), and insect pathogenic nematodes develop and reproduce in the body of American leafminers, reducing the population of American leafminers (Ebrahimi et al., 2017). al., 2016), some bacteria such as Bacillus thuringiensis (Bt) and fungi have also achieved good control effects on the control of American leafminers.

[0004] Insect pathogenic fungi invade the cuticle of insects, and the hyphae penetrate the host cells and proliferate, causing the death of the host (Elkhateeb et al., 2021). The host's mycelium will produce many conidia, which will infect other hosts by spreading, and the chain of infection continues. At present, the most widely used insect pathogenic fungi are Metarhizium and Beauveria bassiana, which are toxic to other dipteran pests such as Mediterranean fruit fly (Quesada-Moraga et al., 2006), Melon fruit fly (Hintènou et al., 2023), and Citrus fruit fly. This technology provides technical guidance for the application of insect pathogenic fungi and devices to control American leafminers.

[0005] However, there is no report on how to use food attractants and insect pathogenic fungi in combination for the control of leafminers. It is of great significance to develop a new method of combining insect pathogenic fungi and food attractants for effective control of insects. Summary of the invention

[0006] In order to overcome the above defects, the present invention combines a fungal product with infectivity and transmission potential with a food attractant that has an attractant effect on leafminers, thereby providing a simple, easy and efficient method for controlling leafminers. Specifically:

[0007] The first aspect of the present invention provides a biocontrol preparation for controlling leafminers, the biocontrol preparation comprising Beauveria bassiana and a food attractant, the food attractant comprising plant volatile secondary metabolites and a solvent, the plant volatile secondary metabolites comprising trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone, linalool and / or caryophyllene.

[0008] Preferably, the biocontrol agent is a mixture, or the Beauveria bassiana and the food attractant are packaged separately.

[0009] Preferably, the biocontrol agent is a mixture, and the Beauveria bassiana and the food attractant are mixed evenly.

[0010] Preferably, the biocontrol agent is packaged separately, that is, Beauveria bassiana and the food attractant are not mixed, but packaged separately, and can be placed separately or mixed when used.

[0011] Preferably, the Beauveria bassiana includes a Beauveria bassiana strain or spore powder prepared therefrom.

[0012] Preferably, the content of Beauveria bassiana is 1-5 (eg, 1, 2, 3, 4, 5) g.

[0013] More preferably, the Beauveria bassiana includes strain XJWLMQ-1 and / or strain LNTL-1 or spore powder prepared therefrom, wherein the strain XJWLMQ-1 is Beauveria bassiana with a preservation number of CGMCC No.40509, and the strain LNTL-1 is Beauveria bassiana with a preservation number of CGMCC No.40508.

[0014] Preferably, the method for preparing the spore powder comprises:

[0015] 1) Preparation of activated spore powder: Inoculate Beauveria bassiana XJWLMQ-1 and / or LNTL-1 on Sabouraud medium SDAY, wherein the Sabouraud medium SDAY contains 1% peptone, 1% yeast extract, 4% glucose and 2% agar. More preferably, the culture conditions are to maintain the temperature at 25-27° C. and culture for 7-10 days to produce activated spore powder of XJWLMQ-1 and / or LNTL-1.

[0016] More preferably, the spore powder culture method further comprises:

[0017] The activated spore powder is prepared by the steps of 2) primary seed liquid culture, 3) secondary seed liquid culture, 4) solid phase fermentation and 5) drying and harvesting the spore powder.

[0018] More preferably,

[0019] 2) Cultivation of primary seed solution

[0020] The activated spore powder of XJWLMQ-1 and / or LNTL-1 was taken and inoculated into Sabouraud culture medium SDY to obtain seed solution. The Sabouraud culture medium SDY contained 1% peptone, 1% yeast extract, and 4% glucose. The initial spore concentration of the seed solution was 1.0×10 6 pc.ml -1 , at 25℃, 120r·min -1 The mixture was shaken and cultured for 48 hours to obtain a primary seed solution.

[0021] 3) Cultivation of secondary seed solution

[0022] The first-level seed solution was inoculated with Sabouraud culture medium SDY culture medium at a 5% inoculation rate and cultured in a 7-liter liquid fermentation tank. 0.5% soybean oil was added to the volume of the fermentation liquid. The culture temperature was 25-27°C, the rotation speed was 100-200rpm, and the initial dissolved oxygen content was above 50%. After culturing for 40 hours, the second-level seed solution was obtained.

[0023] 4) Solid phase fermentation

[0024] Soak the rice in boiling water for 30 minutes, remove and drain excess water, sterilize at 121℃ for 30 minutes, inoculate the rice with 15ml of secondary seed solution per 100g of rice, and use a 20×30cm plastic bag as the fermentation container. After inoculation, lay the rice at 25-26℃ and culture for 7-10 days.

[0025] 5) Drying and harvesting of spore powder

[0026] The solid phase fermented material is dried at 30°C for 3 days, and then spore powder is harvested using a spore harvester, such as a harvester from Mycoharvester Company, UK.

[0027] In a specific embodiment, the plant volatile secondary substances include trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate and α-ionone. Preferably, the mass ratio of trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate and α-ionone is (1-5): (1-5): (1-5): (1-5): (1-5), for example (1, 2, 3, 4, 5): (1, 2, 3, 4, 5): (1, 2, 3, 4, 5): (1, 2, 3, 4, 5): (1, 2, 3, 4, 5): (1, 2, 3, 4, 5).

[0028] In a specific embodiment, the mass ratio of trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate and α-ionone is 4:1:1:3:1.

[0029] Preferably, the solvent comprises one or more of n-hexane, liquid paraffin, oleic acid, isopropyl myristate and / or dioctyl terephthalate. Further preferably, the solvent is paraffin oil.

[0030] More preferably, the volume ratio of the above-mentioned plant volatile secondary substances to the solvent is 1:(1-10), for example 1:(1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0031] Preferably, the food attractant may also include nutrients.

[0032] Preferably, the nutrients include cowpea powder, honey, glucose and / or corn syrup.

[0033] Preferably, the food attractant may further include a slow-release carrier. Further preferably, the slow-release carrier includes a filter paper block, a PVC capillary tube, a butyl rubber head, a cotton swab and / or a hollow rubber rod.

[0034] The food attractant inhibits the germination of Beauveria bassiana, the biocontrol agent is used separately, and the Beauveria bassiana and the food attractant are placed at a certain distance; or,

[0035] The food attractant does not affect the germination of Beauveria bassiana, and the biocontrol agents can be mixed or used separately.

[0036] Preferably, the leafminers include Liriomyza sativae, Liriomyza huidobrensis or Liriomyza trifoliata.

[0037] The second aspect of the present invention provides a device for controlling leafminers, wherein the device can contain any of the above-mentioned biological control agents.

[0038] Preferably, the device comprises a container having a certain internal space, and the biocontrol agent is contained in the internal space of the container.

[0039] More preferably, the container is cylindrical.

[0040] Preferably, holes are provided on the sides of the container to facilitate entry and exit of leafminers.

[0041] Preferably, the bottom of the container is also provided with holes, which are a plurality of small holes to facilitate air circulation.

[0042] Preferably, the top of the container is not connected to the outside.

[0043] Preferably, the device also includes components for fixing the container, such as a hook, a bracket, etc.

[0044] Preferably, the container is made of any material, and further preferably, the material is a waterproof material.

[0045] Preferably, the size of the device is suitable for placing the Beauveria bassiana and the food attractant at a certain distance. Further preferably, the distance between the food attractant and the Beauveria bassiana is 0-20 (e.g., 0, 5, 10, 15, 20) cm.

[0046] According to a third aspect of the present invention, a system for controlling leafminers is provided.

[0047] Preferably, the system comprises the above-mentioned biocontrol agent and the above-mentioned device.

[0048] A fourth aspect of the present invention provides a use of the above-mentioned biological control agent, the above-mentioned device or the above-mentioned system in controlling leafminers.

[0049] Preferably, the replacement time of Beauveria bassiana is more than 3 days, more preferably, more than 6 days, for example, more than 7, 8, 9, 10, 11, 12, 13, 14, 15 days, etc.

[0050] Preferably, the device is placed for no less than 1 day, and the device is placed for no less than 1, 2, 3, 4, 5, 7, 9, 10, 13, or 14 days.

[0051] A fifth aspect of the present invention provides a method for controlling leafminers, the method comprising using the above-mentioned biological control agent, the above-mentioned device or the above-mentioned system for control.

[0052] Preferably, the leafminers include Liriomyza sativae, Liriomyza huidobrensis or Liriomyza trifoliata. Preferably, the biocontrol agent is a mixture, or Beauveria bassiana and the food attractant are packaged separately.

[0053] Preferably, the biocontrol agent is a mixture, and the Beauveria bassiana and the food attractant are mixed evenly.

[0054] Preferably, the biocontrol agent is packaged separately, that is, Beauveria bassiana and the food attractant are not mixed, but packaged separately, and can be placed separately or mixed when used.

[0055] The food attractant inhibits the germination of Beauveria bassiana, the biocontrol agent is used separately, and the Beauveria bassiana and the food attractant are placed at a certain distance; or, the food attractant does not affect the germination of Beauveria bassiana, and the biocontrol agents are mixed or used separately.

[0056] Preferably, the replacement time of Beauveria bassiana is more than 3 days, more preferably, more than 6 days, for example, more than 7, 8, 9, 10, 11, 12, 13, 14, 15 days, etc.

[0057] Preferably, the device or system is placed for no less than 1 day, and the device or system is placed for no less than 1, 2, 3, 4, 5, 7, 9, 10, 13, or 14 days.

[0058] Preferably, the distance between the food attractant and Beauveria bassiana is 0-20 (eg, 0, 5, 10, 15, 20) cm.

[0059] Preferably, the distance between the device or system and the target plant is -20-20 (e.g., -20, -10, 0, 5, 10, 15, 20) cm.

[0060] The term “include” or “comprising” described in the present invention is an open description, which includes the specified components or steps described and other specified components or steps that will not be substantially affected.

[0061] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0062] The advantages of the present invention include but are not limited to:

[0063] 1) The biological control agent of the present invention includes both fungal biological control and chemical attractant, and combines the advantages of the two. By establishing an "attract-infect-disseminate" system, the attractant is used to attract the leafminers to a device where fungal propagules (e.g., spores) are placed, and the leafminers infected by the fungi spread the fungi. Less Beauveria bassiana can be used to control more leafminers, thereby improving the efficiency of fungal use and reducing waste, so as to achieve environmentally friendly green control of leafminers.

[0064] 2) The food attractant increases the amount of the fungal inoculation device to attract Diptera, especially leafminers, so that more leafminers are attracted to the inoculation device containing the food attractant. The biological control preparation of the present invention increases the amount of attraction to leafminers, and at the same time, does not affect the germination of conidia of Beauveria bassiana, and because the inoculation process is automated, the transmission efficiency of leafminers to Beauveria bassiana is improved, thereby achieving a sustained biological control effect.

[0065] 3) The device of the present invention is configured to accommodate the biocontrol agent and facilitate the entry and exit of leafminers, so as to protect the biocontrol agent, especially the fungus Beauveria bassiana, and avoid the adverse effects of the external environment (such as ultraviolet rays, rain, etc.) on the fungal agent. At the same time, the size of the device is suitable for placing the Beauveria bassiana and the food attractant at a certain distance, thereby avoiding the inhibitory effect of the food attractant on the germination rate of Beauveria bassiana, and also prolonging the effective use period of the Beauveria bassiana.

[0066] 4) This combined control system can not only achieve effective control of leafminers, but also reduce the impact of ultraviolet rays and temperature and humidity in the environment on Beauveria bassiana spores. This ensures that the fungal spores have a longer persistence period in the field, thereby improving the durability and stability of the entire control system.

[0067] 5) The present invention uses a combination of spore powder and food attractant to attract and infect leafminers, avoiding the waste caused by flooding spraying of fungal liquid in the prior art. At the same time, the spore powder used in the present invention has a longer survival period and a smaller usage amount than the fungal liquid in the prior art.

[0068] 6) The method for controlling leafminers of the present invention is low in cost, simple to operate, easy to implement and highly effective.

[0069] Strain deposit information:

[0070] 1) Strain XJWLMQ-1

[0071] Depository: China General Microbiology Center (CGMCC);

[0072] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0073] Accession number: CGMCC No.40509;

[0074] Deposit date: February 27, 2023;

[0075] Classification name: Beauveria bassiana.

[0076] 2) Strain LNTL-1

[0077] Depository: China General Microbiology Center (CGMCC);

[0078] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0079] Accession number: CGMCC No.40508;

[0080] Deposit date: February 27, 2023;

[0081] Classification name: Beauveria bassiana. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 : Schematic diagram of the connection of the Y-type olfactometer.

[0083] Figure 2 : Sustained release carrier.

[0084] Figure 3 : The preference of Liriomyza sativae leafminers for different nutrients. CK was the control group.

[0085] Figure 4 : The results of attracting Liriomyza sativae leafminers with different plant volatile secondary substances as food attractants, CK was the control group.

[0086] Figure 5 : The results of attracting Liriomyza sativae leafminers with different secondary biomass combinations, CK was the control group.

[0087] Figure 6 : The results of different combinations of plant secondary volatile substances on the attraction of Liriomyza sativae leafminer, CK was the control group.

[0088] Figure 7 : The attraction results of different addition concentrations of plant secondary volatiles combinations on Liriomyza sativae leafminers, CK was the control group.

[0089] Figure 8 : The attraction results of plant secondary volatile substances combined with different dilution solvents, CK was the control group.

[0090] Fig. 9 : Results of attractant test of different sustained-release carriers.

[0091] Fig.10 : Toxicity of highly virulent strains to adult Liriomyza sativae leafminers, where CK is the control group, a, b, c, d, and e represent significant differences compared with the control when P<0.001, and there are also significant differences between different strains.

[0092] Fig.11 : The spore production amount of 20 strains of Beauveria bassiana.

[0093] Fig.12 : Food attractant and Beauveria bassiana inoculation device.

[0094] Fig.13 :Effect of the distance between food attractant and Beauveria bassiana on conidia germination rate, where different letters on the error bar represent significant differences, one-way ANOVA (P<0.001), R0, R5, R 10 , R 15 , R20 Respectively represent the treatments with the distance between the food attractant and the conidia of Beauveria bassiana being 0, 5, 10, 15, and 20 cm, CK is the control, and a, b, c, d, and e indicate significant differences when P<0.001.

[0095] Fig.14 :Effects of field inoculation devices on the population dynamics of Liriomyza sativae. DETAILED DESCRIPTION

[0096] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0097] Data processing in the embodiment was performed using Excel 2016 to process the raw data of each experiment, infection rate, lethal time LT 50 SAS 9.2 was used to analyze the significant differences of the spore production of the strains, the attraction rate of Liriomyza sativae, and the amount of fungi obtained. 50 Calculated by POLO software.

[0098] Based on bioassay data

[0099] Infection rate = number of infected insects / total number of treated insects × 100%

[0100] Mortality rate = number of dead insects / total number of insects treated × 100%

[0101] Example 1: Test on the tendency of Liriomyza sativae to different food attractant ingredients

[0102] 1. Test of the tropism of Liriomyza sativae to different food attractant ingredients

[0103] Liriomyza sativae Blanchard attractants are compounds that use sugar, plant acid and secondary volatile substances to attract and promote feeding of adult pests. They continue to work in the field with the help of slow-release carriers, thereby attracting and killing target pests. Based on this, the ingredients of the American leafminer attractant composition were screened in the laboratory. The individual substances were mainly screened from the secondary volatile substances and nutrients of the attractants, and then the screened substances were combined to screen the attractant effect, composition ratio, added solubility, dilution solvent components and slow-release carrier materials of the composition. This stage is the initial test and screening stage of this study, which is mainly carried out in the laboratory. The laboratory data provides a basis for the next field test.

[0104] 1.1 Materials and methods

[0105] 1.1.1 Adults of Liriomyza sativae and plants

[0106] The American leafminer population was collected from Fangshan, Beijing in 2023. The adults were raised in a 40cm*40cm*40cm egg-receiving box surrounded by 200-mesh gauze, and one side of the door was covered with black cloth to prevent the adults from escaping. Diluted honey water was spread on the net on the top of the box with a dropper to supplement the nutrition of the adults. The indoor host was dwarf beans (Phaseolus vulgaris), and the bean seedlings with eggs were moved into the appropriate breeding conditions, which were temperature (25±2)℃, relative humidity 60%-70%, and photoperiod L:D=14h:10h. The breeding was purified for more than 3 generations.

[0107] 1.1.2 Test reagents and nutrients

[0108] Secondary attractants: trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone, linalool, caryophyllene (purchased from Beijing Ecoman Biotechnology Co., Ltd.).

[0109] Nutrients: corn syrup, glucose, honey, cowpea powder (purchased from supermarket).

[0110] Solvent: n-hexane, liquid paraffin, oleic acid, isopropyl myristate, dioctyl terephthalate (purchased from Beijing Ecoman Biotechnology Co., Ltd.).

[0111] Sustained-release carrier: filter paper block, PVC capillary tube, butyl rubber head, cotton swab, hollow rubber rod (purchased from Beijing Ecoman Biotechnology Co., Ltd.).

[0112] 1.1.3 Y-type tube test method

[0113] According to previous studies, the highest activity of Liriomyza sativae is between 9:00-11:00 in the morning and 14:00-16:00 in the afternoon (Distribution and daily activity patterns of Liriomyza sativae adults in a solar greenhouse). The main test period of this experiment is between 9:00-11:00 in the morning and 14:00-16:00 in the afternoon, and the test is conducted using a "Y"-shaped tube. Figure 1 As shown, the overall connection method of the "Y"-type tube olfactometer is: air pump → activated carbon → molecular sieve → color-changing silica gel → gas washing bottle → flow meter → "Y"-type tube. The gas connection part is composed of soft sterile silicone tube, and a 15W-6500K energy-saving lamp is installed above the whole. When using, the "Y"-type tube olfactometer is wiped with anhydrous alcohol to remove dust and other messy organic compounds and grease, and ventilated for 30 minutes after drying to allow the system to reach equilibrium.

[0114] The gas flow rate was set to 200 mL / min, and the olfactory selection behavior of each American leafminer in the "Y" tube was tested. A single American leafminer was placed about 5 cm inside the entrance of the "Y" tube, and then the behavioral response of the American leafminer was observed within 5 minutes. If it did not make a choice within 5 minutes, it was considered invalid and a new test insect was placed. A total of 30 American leafminers were tested in each treatment, and repeated 3 times. After each test, the washing bottle needs to be replaced, and the washing bottle should be wiped with anhydrous ethanol and dried before use.

[0115] 1.1.4 Test on the attraction of nutrients to Liriomyza sativae

[0116] The test was conducted using a Y-type olfactometer. A water-diluted solution of different nutrients with a concentration of 50 g / L was prepared. 100 μL of the attractant was dripped into a filter paper block (2*2 cm) and placed in a temperament bottle. The control was distilled water.

[0117] 1.1.5 Test on the attraction of plant secondary volatile substances to Liriomyza sativae

[0118] Prepare a 200 mg / L dilution of n-hexane solution of different attractants, draw 100 μL of the attractant and drop it on a filter paper block (2*2 cm), and put it into a gas chromatography-mass spectrometer. The control is a n-hexane solution.

[0119] 1.1.6 Test on the attraction of plant secondary volatiles to Liriomyza sativae

[0120] The Y-type olfactometer was used for testing. The volatile secondary single substances (trans-2-hexenal Y-1, cis-3-hexenol Y-2, 1-octen-3-ol Y-3, cis-3-hexenol acetate Y-4, α-ionone Y-5) with good results in the initial test were mixed in various combinations according to a 1:1 ratio and prepared into solutions for testing. A dilute n-hexane solution of different attractants with a concentration of 200 mg / L was prepared, 100 μL of the attractant was drawn and dropped into a filter paper block (2*2 cm), and placed in a gas flask. The control was a n-hexane solution.

[0121] 1.1.7 Test on the attraction of different ratios of plant secondary volatile substances to Liriomyza sativae

[0122] The best formula combination selected from the preliminary test results is trans-2-hexenal Y-1, cis-3-hexenol Y-2, 1-octen-3-ol Y-3, cis-3-hexenol acetate Y-4, and α-ionone Y-5. The above test was conducted according to the weight ratio of 1:1:1:1:1. In order to find a better configuration ratio, the five substances were combined in more different ratios in this experiment (Table 1) and configured as solutions for testing. Prepare a dilute n-hexane solution of different attractants with a total concentration of 200 mg / L, draw 100 μL of the attractant and drop it on a filter paper block (2*2 cm), put it into a gas flask, and use n-hexane solution as the control.

[0123] Table 1 Different ratio settings of volatile secondary substances of food attractants

[0124]

[0125] 1.1.8 Attraction test of different concentrations of plant secondary volatile substances

[0126] The best formula combination selected from the preliminary test results is trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, and α-ionone, which are tested according to the weight ratio of 4:1:1:3:1. The concentration in the test is 200mg / L, and the flow rate is 200mL / min. While keeping the flow rate unchanged, different concentrations are set for further exploration and testing. Seven concentrations were set in this experiment, ranging from 0.1g / L to 6.4g / L. After the above concentrations are configured, 100μL of the attractant is dripped into a filter paper block (2*2cm) and placed in a gas chromatography hood. The control is a n-hexane solution.

[0127] 1.1.9 Attraction test of plant secondary volatile substances in different dilution solvents

[0128] In order to test the effect of different solvents on the combination of volatiles, a most suitable solvent type was selected. In this experiment, a total of five reagents were selected for testing: n-hexane, liquid paraffin, oleic acid, isopropyl myristate, and dioctyl terephthalate.

[0129] 1.1.10 Attraction test of plant secondary volatile substances combined with different slow-release carriers

[0130] The sustained-release carrier can affect the effect and duration of the attractant, so the Figure 2As shown, six slow-release materials were selected for testing, including filter paper block, PVC capillary, butyl rubber head, cotton swab, slow-release bottle, and hollow rubber rod. The selected plant secondary volatile substances were added to the slow-release carrier in an amount of 1 ml each, placed in an outdoor ventilated place, and retrieved at 0d, 7d, 14d, 28d, 56d, and 84d to test its attractant effect.

[0131] 1.1.11 Data Statistics and Analysis

[0132] SPSS software was used to conduct one-way analysis of variance on the attraction rates of different treatments of substances and substance combinations.

[0133] 1.2 Results and Analysis

[0134] 1.2.1 Test on the attraction of nutrients to Liriomyza sativae

[0135] The results showed that cowpea powder had a certain attracting effect on the American leafminer, which was significantly different from the control. Corn syrup, glucose and honey had no obvious effect on the American leafminer, and there was no difference from the control ( Figure 3 ).

[0136] 1.2.2 Test on the attraction of plant secondary volatile substances to Liriomyza sativae

[0137] The results of attracting single substances of plant secondary volatiles showed that the five components of trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, and α-ionone showed strong tropism, which was significantly different from the control. Linalool and caryophyllene had basically no attracting effect on the American leafminer ( Figure 4 ).

[0138] 1.2.3 Test on the attraction of plant secondary volatile compounds to Liriomyza sativae leafminers

[0139] The combination test of the five single volatile components (trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone) showed that the American leafminer had different tropism to different combinations. Among them, the tropism of the combination of Y1+2+3+4+5 was the strongest, with a tropism rate of 86.7%, which was significantly higher than the control ( Figure 5 ).

[0140] 1.2.4 Test on the attraction of different ratios of plant secondary volatile substances to Liriomyza sativae

[0141] Through screening, the five components of trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, and α-ionone were prepared in different proportions to test their differences. The results showed that each treatment had a strong tendency, and the difference between different proportions was not very large. Among them, the composition and its ratio of trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone, according to the weight ratio of 4:1:1:3:1, the food attractant had the strongest effect on the attraction of American leafminers ( Figure 6 ).

[0142] 1.2.5 Attraction test of different concentrations of plant secondary volatile substances

[0143] Using n-hexane as solvent, attractant combination ratio (trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone, according to the weight ratio of 4:1:1:3:1) were prepared 0.1-6.4g / L of 7 concentrations of mixed solution to test their differences. The results showed that the differences between the treatments were obvious. When the concentration reached below 0.4g / L and above 3.2g / L, the tendency began to decrease. The optimal concentration range was 0.8g / L to 1.6g / L ( Figure 7 ).

[0144] 1.2.6 Attraction test of plant secondary volatile substances in different dilution solvents

[0145] The attractant combination ratio (trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone, according to the weight ratio of 4:1:1:3:1, the concentration of active ingredients is 1g / L) and 5 chemical solvents were selected for the test of the attraction of the American leafminer, and n-hexane showed the best effect ( Figure 8 ).

[0146] 1.2.7 Attraction test of plant secondary volatile substances combined with different slow-release carriers

[0147] Different sustained-release carriers have different sustained-release effects on the attractant. Using n-hexane solvent, trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone, according to the weight ratio of 4:1:1:3:1, the active ingredient concentration is 1g / L, different sustained-release carriers were screened. The results showed that there was no significant difference in performance on the 0th day, and on the 7th day, there was a decrease in varying degrees, and the decrease in filter paper blocks was the most obvious. After 84 days of testing, the hollow rubber rod had the best sustained-release effect and the longest lasting effect ( Fig. 9 ).

[0148] Example 2: Preparation of conidia of Beauveria bassiana to screen for highly toxic strains

[0149] 1 Test insects

[0150] The American leafminer population was collected from Haikou City, Hainan Province in 2022. The indoor host was dwarf bean (Phaseolus vulgaris), and the breeding conditions were temperature (26±2)℃, relative humidity 50%-60%, and photoperiod L:D=14h:10h. The breeding was purified for more than 3 generations.

[0151] 2 Test strains

[0152] The test strain of Beauveria bassiana was inoculated on Goettel spore-forming medium for activation culture. The formula was 2% corn flour, 1% wheat bran, 0.5% peptone, 0.3% KH2PO4, 0.1% MgSO4.7H2O, 0.1% NH4NO3, 2% agar, and 0.5% cicada shells. Strains with good biological properties were selected for further bioassays. The spore powder of the test strain obtained by culture on Goettel medium was dissolved in 0.05% Tween-80 solution and prepared to a final concentration of 1×10 8 spores / ml of spore suspension; Tween-80 solution was used as blank control.

[0153] 2.1 Strain production

[0154] Conidia were produced in large quantities on rice culture medium. The rice was sterilized by high pressure at 121°C for 1 hour and inoculated with conidial germination solution (Jenkins et al., 1998) that had been germinated for 3 days. The culture was cultured at 20°C to 26°C and 40°C to 70% RH for 21 days and then dried at room temperature for 5 days. The culture medium was sieved (295-μm mesh) to harvest conidia and stored at 4 to 6°C for later use.

[0155] 3. Screening of highly virulent strains

[0156] The leaf dipping method was used for the bioassay of the larvae of the American leafminer. When two true leaves of kidney beans grew, the leaves with the second-instar larvae of the American leafminer were immersed in the prepared spore suspension for 20 seconds. After the leaves were naturally dried, they were placed in insect cages. The control group was treated with 0.05% Tween-80. The culture conditions were 26±1℃, RH 85%, and a light cycle of L:D=14:10. The experiment was observed for 8 consecutive days. The test was repeated 4 times, and the number of dead insects was recorded regularly every day. The lethal median time of each strain to the larvae of the American leafminer was calculated.

[0157] Five strains with high toxicity to the larvae of Liriomyza sativae were used for bioassay of adults. 0.1 g of dry conidia of each strain was evenly distributed on the cotton velvet cloth inside a cylindrical plastic tube (70 mm long × 48 mm diameter). 25 adults of Liriomyza sativae were moved into the cylindrical plastic tube and walked on the velvet cloth for 1 minute. Then, 25 adults were moved from the velvet cloth into a clean and ventilated glass cage (150 × 150 × 200 mm). A cotton ball soaked in 10% sugar solution was placed in the bottom corner of the cage as a food source. Adults in the control group were exposed to the velvet cloth without dry conidia and then transferred to the same cage. The treatment was completely randomized, and the rearing conditions of Liriomyza sativae were maintained at 25℃~27℃, 50℃~70%RH, and 12L:12D photoperiod.

[0158] The number of dead insects was recorded daily, and each test was repeated 4 times for 8 consecutive days. The mortality of each strain against adult leafminers was calculated. The dead insects were surface disinfected with 70% alcohol, rinsed three times in distilled water, and transferred to a petri dish with moist sterile filter paper. Mycelium grew on the surface of the dead insects. The spores grown on the surface of the insects were used for analysis. Five of the 25 leafminers initially infested were taken out for determination of the number of conidia obtained by a single leafminer in each treatment. The insects were transferred to a 2 ml centrifuge tube and 1 ml of sterile 0.05% Tween-80 solution was added. Vortex for 2 to 3 minutes to shake the conidia from the insects, and the conidia concentration was determined using a hemocytometer.

[0159] To determine the spore production of each highly virulent strain, the spores of the highly virulent strains screened above were prepared with 0.05% Tween-80 solution to a concentration of 1×10 6 Spores / ml suspension. Take 0.1ml of the suspension on 90mm Goettle spore medium (corn flour 2%, bran 1%, peptone 0.5%, KH2PO4 0.3%, MgSO4·7H2O 0.1%, NH4NO3 0.1%, agar 2%), and spread it evenly with a sterile triangular glass rod. Repeat 4 times for each strain. The culture medium was cultured in the dark at 26±1℃ for 15 days. Five points were randomly selected from the culture medium with a sterile hole puncher with a diameter of 4mm and placed in 5ml of 0.05% Tween-80 solution. The suspension was ultrasonically treated in an ultrasonic cleaner for 15min to destroy the spore block structure, and then vortexed with a vortex oscillator for 10min to obtain a uniformly dispersed spore suspension. After the suspension was diluted 10 times, the number of spores was determined using a hemocytometer, and each was repeated 3 times, and then the number of spores per square centimeter was calculated.

[0160] 4. Screening results of highly toxic strains

[0161] 4.1 Toxicity of Beauveria bassiana to Liriomyza sativae larvae

[0162] The results are shown in Table 2. The tested strains of Beauveria bassiana have certain toxicity to the larvae of the American leafminer. Most of the insects in the treatments reached the peak of death in 3-5 days, and the burrowing channel no longer extended. Among them, the infection rate of 5 strains was above 75%, namely LNTL-1, HNLY-47, SDDZ-20, XJWLMQ-1, and XJWLMQ-36. The dead larvae were picked out and moisturized for culture, and white hyphae grew. It was identified that they were killed by infection with Beauveria bassiana.

[0163] Table 2 Numbers, collection locations, and median lethal time (LT) of 20 strains of Beauveria bassiana 50 ±SE

[0164]

[0165]

[0166] 4.2 Toxicity of Beauveria bassiana to adults of Liriomyza sativae

[0167] like Fig.10 As shown, the five strains have strong pathogenicity to adult Liriomyza sativae. Five days after inoculation, the average mortality rate of the control group was 5.95±1.1%, and the mortality rate of the experimental group was 85.0%-98.1%. The mortality rates of adults inoculated with strains XJWLMQ-1 and LNTL-1 were the highest, at 98.1% and 96.9%, respectively.

[0168] 4.3 Determination of spore production

[0169] The spore production results of 20 highly toxic strains of Beauveria bassiana are as follows Fig.11 The average spore production is 3.01×10 8 Pieces / cm 2 Among them, the strain with the highest spore production was LNTL-1, with a spore production of 3.85×10 8 Pieces / cm 2 The second was XJWLMQ-1, with a spore production of 3.74×10 8 Pieces / cm 2 In the screening of toxic strains against several pests, LNTL-1 and XJWLMQ-1 were more toxic than other strains, and the lethal time of infection against several pests was shorter. Moreover, these two strains had a higher spore production rate, and the high spore production rate strains played an important role in the vigorous production in the application. In summary, it was shown that Beauveria bassiana XJWLMQ-1, LNTL-1, etc. were entomogenous fungi with great application prospects in biological control of American leafminers.

[0170] Example 3: Indoor experiment to test the control effect of automatic inoculation device on leafminers

[0171] 1 Indoor test of the combined use of food attractants and Beauveria bassiana against Liriomyza sativae leafminers

[0172] The food attractants and Beauveria bassiana inoculation device used in this study were Fig.12 As shown. There are 3 large inlet and outlet holes (5×6 cm) evenly distributed in the middle of the side of the cylinder (15 cm in diameter, 40 cm in height), and 22 small inlet and outlet holes (2 cm in diameter) at the bottom. A plastic cylinder (10 cm in diameter, 15 cm in height) wrapped in velvet cloth is hung in the center of the cylinder, and about 3 g of conidia of Beauveria bassiana XJWLMQ-1 are evenly distributed on the velvet cloth. The food attractants and Beauveria bassiana inoculation device used in this study are shown in Fig.12 The food attractant was located under the lid, at a vertical distance of 15 cm from the spore powder (inner cylinder).

[0173] 1.1 Effects of food attractants on conidia germination of Beauveria bassiana

[0174] The ratio of the food attractant: trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate, α-ionone (each component purchased from Beijing Yikeman Biotechnology Co., Ltd.) was mixed according to the weight ratio of 4:1:1:3:1. The food attractant was mixed evenly with liquid paraffin oil (Beijing Yikeman Biotechnology Co., Ltd.) (v:v = 1:9).

[0175] Weigh 0.01 g of Beauveria bassiana conidia powder, spread it evenly on a cover glass (2.5×2.5 cm), and transfer it to a culture dish (12 cm) containing water agar (2% w / v). 10 , R 15 , R 20 There were five groups, representing the vertical distance between the food attractant and the culture dish (with conidia) of 0, 5, 10, 15, and 20 cm, respectively. The non-woven bag containing the food attractant was hung just above the culture dish, and the control group was a non-hanging food attractant. Each treatment was repeated 3 times.

[0176] Germination rate determination: After 24 hours, transfer the coverslip with conidia to a 15 ml centrifuge tube containing 2 mL of sterile 0.05% Tween-80 solution, vortex for 30 seconds to elute the conidia. Take 500 μL of spore eluate and transfer it to 4.5 mL of sterile germination solution (4% glucose, 1% yeast powder), and seal it with a sealing film. 26°C, RH: 30%, 180rpm, gyroscopic culture for 18 hours. The spore germination rate was counted by the hemocytometer method. The spore germ tube was considered to have germinated if it exceeded 1 / 2 of the spore diameter, and at least 200 spores were counted for germination.

[0177] 1.2 Effect of indoor test device on attraction rate and yield of Liriomyza sativae leafminers

[0178] (1) All bean plants used in this study were planted in 10 cm pots (6 plants per pot) in an insectary room using a mixture of vermiculite and nutrient soil in a ratio of 1:1. Twenty 2-week-old bean plants in pots (30 cm in height) were placed in cages (2 m high × 2.9 m in diameter) in the field. The flannel cloth containing conidia of Beauveria bassiana was replaced with sticky boards, which were hung with ropes at -20, -10, 0, 10, and 20 cm above the plant canopy. 500 adult Liriomyza sativae were placed in the cages, with 1 m between each cage. The sticky boards were replaced every day at 1, 2, 3, 4, and 5 days after release, and the number of captured Liriomyza sativae was accumulated. The experiment was repeated 3 times. The attraction of the inoculation device to Liriomyza sativae was evaluated at different distances from the plant. The experiment lasted for a total of 5 days, with investigations conducted once a day.

[0179] (2) Take 20 2-week-old bean plants (30 cm in height) and place them in a cage. Put 500 adult leafminers in the field cage (2 m in height × 2.9 m in diameter). Hang the automatic inoculation device with a rope at the distance from the plant canopy that attracts the most insects. Collect 30 leafminers on the 1st, 2nd, 3rd, 4th and 5th day after release and transfer them to clean sterile centrifuge tubes (7 mm in diameter, 49 mm in height). Keep the leafminers in 20 plastic tubes in an incubator at 25±2°C and 80±90% RH. Plug the tubes with cotton balls soaked in 10% sugar solution as food source. Replace with fresh cotton balls every 2 days. Record the mortality rate every day until all leafminers die. Dead insects with hyphae growing on the surface of the insects are identified as dead due to fungal infection. For the remaining 10 plastic tubes, transfer the insects to 2 ml centrifuge tubes and add 1 ml of sterile 0.05% Tween-80 solution. Vortex for 2-3 minutes to shake out the conidia from the insect body, and use a hemocytometer to determine the number of conidia obtained by a single fly. No conidia powder was added to the control group. The experiment was repeated 3 times.

[0180] 2 Results of the combined application of Beauveria bassiana and the device to Liriomyza sativae leafminers in the laboratory

[0181] 2.1 Effects of food attractants on conidia germination of Beauveria bassiana

[0182] Effect of the distance between food attractant and Beauveria bassiana on conidia germination Fig.13 The results showed that in the open space indoors, as the distance between the conidia of Beauveria bassiana and the food attractant increased, R0, R5, R 10 , R 15 , R 20(When the distance between the food attractant and conidia was 0, 5, 10, 15, and 20 cm, the germination rate increased to 48.05%, 55.25%, 64.9%, 67.9%, and 80.9%, respectively. When the distance between the food attractant and conidia was 20 cm, the germination rate was 80.9%, which was not significantly different from the control group (the germination rate was 81.6%).

[0183] 2.2 Effect of the measuring device on the attraction and yield of Liriomyza sativae leafminers

[0184] (1) The attraction rates of the device to the American leafminers at five different distances from the top of the bean plant in the experiment are shown in Table 3. In general, the attraction rate of the inoculation device to the adult American leafminers showed an increasing trend with the increase of sampling time. When the distance between the attractant device and the top of the bean plant was 20 cm, the attraction rate of American leafminers on the 5th day was the highest, which was 98.71% (Table 3).

[0185] Table 3 The attraction effect of the device at different distances from the top of the bean plant on the leafminer

[0186]

[0187] (2) After the inoculation device was hung 20 cm from the top of the bean plant, the mortality rate of the treated group ranged from 66.15% to 99.75%, and the mortality rate of the leafminers in the control group ranged from 0 to 29.3%. 50 The value varies with the sampling date. The LT of the contact device on the 3rd day 50 The shortest was 2.03 days. The average number of spores carried by a single leafminer in the treatment group continued to increase with time. On the first day after inoculation, a single leafminer adult carried an average of 5.9×10 5 conidia, and on the 5th day after inoculation, it increased to 38.1×10 5 conidia (Table 4).

[0188] Table 4 Effect of inoculation device on mortality, lethal time and bacterial yield of leafminers

[0189]

[0190] The control effect of strain LNTL-1 is basically the same as that of XJWLMQ-1.

[0191] Example 4: Field experiment to test the control effect of automatic inoculation device on leafminers

[0192] Effect of combined device on field application of leafminers

[0193] The experiment was conducted in two greenhouses with the same severity of leafminer damage in Xinqiao Town, Youxian District, Mianyang City, Sichuan Province. No chemical pesticides were used during the experiment, and other management was normal. The area was 332m 2 The greenhouse is divided into 5 small areas, each with an area of ​​2m 2 , one device was hung in each plot, and the plots were separated by more than 5 m to reduce the interference between different treatments. A 15×15 cm 2 Double-sided yellow sticky boards were hung in the other greenhouse as a control. The other conditions were the same. The survey began in September 2023, and the sticky boards were replaced every 7 days, and the number of adult leafminers on each yellow sticky board was counted.

[0194] Effects of combined device application on the population of leafminers Fig.14 As shown. The population of leafminers in the greenhouse with the Beauveria inoculation device was significantly lower than that in the control greenhouse, and the hanging Beauveria inoculation device significantly delayed the peak of the leafminer outbreak in the greenhouse. The leafminers in the control greenhouse began to show an outbreak trend on September 26, and the average number of leafminers adults on a single yellow board increased from 71 / block to 249 / block. The leafminers in the greenhouse with the hanging Beauveria inoculation device showed an outbreak trend on October 3, and the average number of leafminers adults on a single yellow board increased from 75 / block to 144 / block.

[0195] The field control effect of strain LNTL-1 is basically the same as that of XJWLMQ-1.

[0196] The above experimental results show that in the open space indoors, the spore germination rate increases with the distance between the food attractant and Beauveria bassiana. When the distance is 20 cm, the spore germination rate is 80.9%, which is no different from the control. The attraction rate of the inoculation device to the adult leafminers of the American leafminers shows an increasing trend with the increase of sampling time. On the fifth day after inoculation, the attraction rate of the inoculation device to the adult leafminers of the American leafminers is the highest, reaching 98.71%. After hanging the inoculation device, the average number of spores carried by a single leafminer continued to increase with the increase of time. One day after inoculation, a single leafminer adult carried an average of 5.9×10 5 conidia, while 5 days after inoculation, the number was 38.1×10 5 The number of conidia was 100,000, and the mortality rate ranged from 66.15% to 99.75%. The field application effect of the combined device on leafminers showed that the hanging Beauveria bassiana inoculation device significantly delayed the peak outbreak of leafminers in greenhouses and suppressed the population of leafminers. The combined application of Beauveria bassiana and food attractants can establish an "attraction-infection-transmission" system, laying the foundation for the research and development of new control technologies for leafminers.

[0197] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0198] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0199] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A biological control agent for controlling leafminers, characterized in that: The biocontrol agent comprises Beauveria bassiana and a food attractant, wherein the food attractant comprises plant volatile secondary metabolites and a solvent, the Beauveria bassiana is strain XJWLMQ-1 and / or strain LNTL-1 or spore powder prepared therefrom, and the strain XJWLMQ-1 is Beauveria bassiana with a preservation number of CGMCC No.40509 ( Beauveria bassiana ), the strain LNTL-1 is Beauveria bassiana with a preservation number of CGMCC No.40508 ( Beauveria bassiana ), the plant volatile secondary substances are trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate and α-ionone, and the mass ratio of trans-2-hexenal, cis-3-hexenol, 1-octen-3-ol, cis-3-hexenol acetate and α-ionone is (1-5): (1-5): (1-5): (1-5): (1-5).

2. The biocontrol agent according to claim 1, characterized in that The solvent includes one or more of n-hexane, liquid paraffin, oleic acid, isopropyl myristate and / or dioctyl terephthalate.

3. The biocontrol agent according to any one of claims 1 to 2, characterized in that The volume ratio of the plant volatile secondary substances to the solvent is 1:(1-10).

4. A system for controlling leafminers, characterized in that: The system comprises the biological control agent according to any one of claims 1 to 3 and a device for controlling leafminers, wherein the device can accommodate the biological control agent according to any one of claims 1 to 3, and the device comprises a container having a certain internal space, and the biological control agent is accommodated in the internal space of the container.

5. The system according to claim 4, characterized in that The side of the container is provided with holes to facilitate the entry and exit of leafminers; the bottom of the container is also provided with holes, which are multiple small holes to facilitate air circulation.

6. Use of the biological control agent according to any one of claims 1 to 3 or the system according to any one of claims 4 to 5 in controlling leafminers.

7. A method for controlling leafminers, characterized in that: The method comprises using the biocontrol agent according to any one of claims 1 to 3 or the system according to any one of claims 4 to 5 for control.

8. The method according to claim 7, characterized in that The biocontrol agents are used separately, the distance between the food attractant and Beauveria bassiana is 0-20 cm; the distance between the system and the target plant is -20-20 cm.

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