A biocontrol agent containing Beauveria bassiana and its application in controlling tomato leafminer

Abstract

The invention discloses a biological control preparation, system and application thereof for preventing and controlling tomato leafminer, wherein the biological control preparation comprises Beauveria bassiana and a food attractant, wherein the food attractant comprises an attractant substance and a solvent, wherein the attractant substance comprises tetradecatrienol acetate, ethyl octanoate, 1-nonanol, phenylacetaldehyde and 3-methyl-1-butanol. By using the food attractant in combination with Beauveria bassiana, the amount of attraction to tomato leafminer is increased, and at the same time, the germination of conidia of Beauveria bassiana is not affected, thereby improving the transmission efficiency of tomato leafminer 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 controlling tomato leafminer. Background Art

[0002] The tomato leafminer, scientifically known as Tuta absoluta (Meyrick), is also widely known as the tomato wheat moth or tomato wheat miner. It belongs to the Lepidoptera family Gelechiidae and is a world quarantine invasive pest. After hatching, the larvae of the tomato leafminer will immediately burrow into the tissue of the tomato leaves to feed on the mesophyll, and will also bore into the terminal buds, tender stems, flower buds and fruits, posing a threat to the entire growth period of tomatoes and often causing significant economic losses. Due to the short life cycle of the tomato leafminer, overlapping generations and the hidden "lurking feeding" characteristics of the larvae, the use of chemical agents is not only difficult to kill it, but also easily has a negative impact on natural enemies and pollutes the ecological environment. In Chile, Brazil, Argentina and other countries, it has been reported that it has developed resistance to a variety of insecticides. At the same time, due to its hidden feeding habits, it is also difficult for parasitic and predatory natural enemies to effectively control it. Therefore, it is urgent to find effective green, ecological and sustainable prevention and control technologies for development and utilization.

[0003] Beauveria bassiana is one of the most widely used insect pathogenic fungi in biological pest control. It has the characteristics of easy large-scale production, active infection, broad insecticide spectrum, and strong pathogenicity. It has great potential in replacing chemical pesticides to control a variety of pests. Studies have shown that the strain of Beauveria bassiana Qu-B912 has the best effect, with a maximum mortality rate of 80% for eggs and 68% for third-instar larvae (Entomopathogenic fungi isolates selection for egg control of tomato moth, Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) eggs [J]. Agricultura Tecnica, 2006, 66 (2): 151-158 and Effectiveness of entomopathogenic fungus strains on tomato moth Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) larvae [J]. Agricultura Tecnica, 2006, 6 (2): 159-165). Other studies have also shown that Beauveria bassiana has a strong lethal effect on larvae (corrected mortality rate of 90% to 100%). epiphytic and endophytic activity against the tomato leaf miner Tuta absoluta [J]. Journal of Applied Entomology, 2016, 140 (8): 580-589.). However, insect pathogenic fungi are easily affected by the environment in practical applications, and the pest control effect is unstable. Even if the highly toxic strains are used in indoor bioassays and applied in the field in an inundated manner, the expected control effect is often not achieved. Therefore, how to reduce the inoculation requirements and costs of insect pathogenic fungi and increase the infection rate of tomato leaf miner is the key to promoting the production and application of biocontrol agents.

[0004] In the process of herbivorous insects identifying and locating host plants, various volatile secondary metabolites produced by plants play an important role. Various food attractants developed by simulating chemical signals produced by host plants are green and safe pest control products. Plant volatiles and volatile components of traditional food attractants affect the host selection and oviposition behavior of tomato leaf miners. Studies have found that the main volatiles of tomatoes, such as limonene, β-ocimene, α-terpinene, δ-elemene and (E)-β-caryophyllene, when mixed in a specific ratio, show a strong attraction to tomato leaf miners (Attraction of female tomato leaf miner, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) to shared compounds from hosts. Phytoparasitica, 2021, 49: 153-162.). This mixture can simulate the natural smell of tomatoes, causing tomato leaf miners to mistakenly think it is a suitable food source and approach.

[0005] However, there is no report on how to use food attractants and insect pathogenic fungi together for the control of tomato leafminer. It is of great significance to develop a new method of using insect pathogenic fungi and food attractants together 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 infectious and spreading potential with a food attractant that has an attractant effect on tomato leafminers, thereby providing a simple, easy and efficient method for controlling tomato leafminers. Specifically:

[0007] The first aspect of the present invention provides a biocontrol formulation for controlling tomato leafminer, the biocontrol formulation comprising Beauveria bassiana and a food attractant, the food attractant comprising an attractant and a solvent, the attractant comprising tetradecatriene acetate, ethyl octanoate, 1-nonanol, phenylacetaldehyde and / or 3-methyl-1-butanol.

[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 mass ratio of tetradecatriene acetate, ethyl octanoate, 1-nonanol, phenylacetaldehyde and 3-methyl-1-butanol is (0.1-0.5): (0.1-0.5): (0.1-0.5): (0.05-0.1): (0.10-0.15), for example (0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5): (0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5): (0.1, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5): (0.05, 0.06, 0.07, 0.08, 0.09, 0.1): (0.10, 0.11, 0.12, 0.13, 0.14, 0.15).

[0028] 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 n-hexane.

[0029] More preferably, the volume ratio of the attractant to the solvent is 1:(1-100), for example 1:(10, 20, 30, 40, 50, 60, 70, 80, 90, 100).

[0030] Preferably, the food attractant may also include nutrients.

[0031] Preferably, the nutrients include pine pollen, honey, glucose and / or corn syrup.

[0032] 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 slow-release bottle.

[0033] Preferably, the food attractant inhibits the germination of Beauveria bassiana or the food attractant does not affect the germination of Beauveria bassiana.

[0034] More preferably, the food attractant inhibits the germination of Beauveria bassiana, and the food attractant and Beauveria bassiana are separately packaged.

[0035] More preferably, the food attractant does not affect the germination of Beauveria bassiana, and the food attractant and Beauveria bassiana are mixed or packaged separately.

[0036] A second aspect of the present invention provides a device for controlling tomato leafminer, wherein the device can contain any of the above-mentioned biological control agents.

[0037] Preferably, the device comprises a container having a certain internal space, and the biocontrol agent is contained in the internal space of the container.

[0038] More preferably, the container is in the shape of a triangular prism.

[0039] Preferably, holes (inlet and outlet) are provided on the side of the container to facilitate the entry and exit of tomato leaf miners.

[0040] Preferably, the device also includes components for fixing the container, such as a hook, a bracket, etc.

[0041] Preferably, the container is made of any material, and further preferably, the material is a waterproof material.

[0042] 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.

[0043] A third aspect of the present invention provides a system for controlling tomato leafminer.

[0044] Preferably, the system comprises the above-mentioned biocontrol agent and the above-mentioned device.

[0045] 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 tomato leafminer.

[0046] 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.

[0047] 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.

[0048] A fifth aspect of the present invention provides a method for controlling tomato leafminer, the method comprising using the above-mentioned biological control agent, the above-mentioned device or the above-mentioned system for control.

[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 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.

[0051] Preferably, the biocontrol agent is a mixture, or the Beauveria bassiana and the food attractant are packaged separately.

[0052] Preferably, the biocontrol agent is a mixture, and the Beauveria bassiana and the food attractant are mixed evenly.

[0053] 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.

[0054] Preferably, the distance between the device or system and the target plant is 0-20 (e.g., 0, 5, 10, 15, 20) cm.

[0055] 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.

[0056] 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".

[0057] The advantages of the present invention include but are not limited to:

[0058] 1) The biocontrol 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 tomato leafminer to a device where fungal propagules (e.g., spores) are placed. The tomato leafminer infected by the fungus spreads the fungus, and less Beauveria bassiana can be used to control more tomato leafminers, thereby improving the efficiency of fungal use and reducing waste, so as to achieve environmentally friendly green control of tomato leafminers.

[0059] 2) The biocontrol agent of the present invention increases the amount of attraction to the tomato leafminer while not affecting the germination of conidia of Beauveria bassiana, thereby improving the transmission efficiency of the tomato leafminer to Beauveria bassiana and achieving a sustained biological control effect.

[0060] 3) The device of the present invention is configured to accommodate the biocontrol agent and facilitate the entry and exit of the tomato leafminer, 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.

[0061] 4) 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.

[0062] 5) The method for controlling tomato leafminers of the present invention is low in cost, simple to operate, easy to implement and highly effective.

[0063] Strain deposit information:

[0064] 1) Strain XJWLMQ-1

[0065] Depository: China General Microbiology Center (CGMCC);

[0066] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0067] Accession number: CGMCC No.40509;

[0068] Deposit date: February 27, 2023;

[0069] Classification name: Beauveria bassiana.

[0070] 2) Strain LNTL-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.40508;

[0074] Deposit date: February 27, 2023;

[0075] Classification name: Beauveria bassiana. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 : Toxicity of highly virulent strains to adult tomato leafminers, where CK is the control group, a, b, c, d, and e represent treatment groups with significant differences from the control group, and there are significant differences between the numbers of each treatment group.

[0077] Figure 2 : Determination of spore production of highly virulent strains, where a, b, c, d, and e represent numbers with significant differences.

[0078] Figure 3 : Schematic diagram of the automatic inoculation device for food attractants and Beauveria bassiana. DETAILED DESCRIPTION

[0079] 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.

[0080] Data processing in the examples was performed using Excel 2016 to process the raw data of each experiment, and the infection rate, lethal time LT50, spore production of the strain, and fungus yield of the tomato leafminer were analyzed for significant differences using SAS 9.2, wherein LT50 was calculated using POLO software.

[0081] Based on bioassay data

[0082] Infection rate = number of infected insects / total number of treated insects × 100%

[0083] Mortality rate = number of dead insects / total number of insects treated × 100%

[0084] Example 1: Preparation of Beauveria bassiana conidia and food attractants

[0085] 1 Test insects

[0086] Tomato leafminers were collected from Hohhot, Inner Mongolia Autonomous Region, and placed in insect cages (30 cm × 30 cm × 30 cm) indoors and subcultured with tomato Solanum lycopersicum L. plants in an artificial climate chamber (LTC-1000, Shanghai Santeng Instruments) at a temperature of 27 ± 1 °C, a relative humidity of 60 ± 5%, and a photoperiod of 16L:8D.

[0087] 2 Test strains

[0088] The 20 strains of Beauveria bassiana for testing were collected from insect bodies in different regions of the country. After being isolated and purified indoors, they were inoculated on a slant and stored at 4°C. During the test, the strains of Beauveria bassiana were inoculated on Goettel spore-forming medium for activation culture. The formula is 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 strains 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.

[0089] 3 Food attractant recipes

[0090] According to the food attractant screened in the early stage and the improved ratio: the mass ratio of tetradecatriene acetate, ethyl octanoate, 1-nonanol, phenylacetaldehyde, and 3-methyl-1-butanol is 0.45:0.35:0.2:0.08:0.13. The food attractant is mixed evenly with n-hexane (Shanghai Aladdin Biochemical Technology Co., Ltd.) (v:v=1:90).

[0091] 4 Determination of infection rate of test strains to tomato leafminer

[0092] 4.1 Tomato leafminer larvae

[0093] The second-instar larvae of tomato leafminer on tomato leaves were treated with the leaf dipping method. 2 mL of the prepared spore suspension was taken and the leaves with the second-instar larvae were immersed in the suspension for 30 seconds. The leaves were allowed to dry naturally or the excess water was absorbed with sterilized filter paper. The leaves were transferred to a culture dish (diameter × height = 20 cm × 3.5 cm), the petioles were wrapped in moist cotton balls, and then the culture dish was sealed. The control group was treated with 0.05% Tween-80 in the same way. The number of dead larvae was recorded every day. Each test was repeated 4 times and recorded for 8 consecutive days. The lethal median time of each strain to tomato leafminer larvae was calculated.

[0094] 4.2 Adult tomato leafminer

[0095] Five strains with high toxicity to the larvae of tomato leafminer 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). 20 adults of tomato leafminer were moved into the cylindrical plastic tube and walked on the velvet cloth for 1 minute. Then, 20 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 carried out completely randomly. The rearing conditions of tomato leafminers were maintained at 25℃~27℃, 50℃~70%RH, and 12L:12D photoperiod. The number of dead insects was recorded every day. Each test was repeated 4 times and recorded for 8 consecutive days. The mortality rate of each strain against adult tomato leafminers was calculated. The dead insects were surface disinfected with 70% alcohol, rinsed three times in distilled water, and transferred to a culture dish with moist sterile filter paper. Mycelium grew on the surface of the dead insects.

[0096] 5. Determination of spore production of test strains

[0097] 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 6Spores / 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 15d, and 5 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 sonicated 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.

[0098] Results of the pathogenicity test of 6 test strains to tomato leafminer larvae

[0099] As shown in Table 1, the tested Beauveria bassiana strains have certain toxicity to tomato leafminer, but the median lethality of tomato leafminer larvae is significantly different (F=271.66, P<0.001), and the average median lethality is 5.65d. The shorter ones are LNTL-1 and XJWLMQ-1, which are 4.38d and 4.42d respectively, and the longest one is JLGZL-3, which is 6.89d. The 20 strains in the table have high toxicity to tomato leafminer larvae. Most of the insects in the treatments reach the peak of death at 4-6d, and the burrowing channel no longer extends. The dead larvae are picked out and moisturized for cultivation, and white hyphae can grow. It is identified that they are killed by infection with Beauveria bassiana.

[0100] Table 1 Numbers, collection locations, and median lethal time (LT) of 20 strains of Beauveria bassiana 50 ±SE

[0101]

[0102]

[0103] In summary: The above five strains, LNTL-1, LNTL-3, LNTL-5, XJWLMQ-1, and XJWLMQ-36, have a high infection rate and stable virulence against the larvae of tomato leafminer.

[0104] Results of the pathogenicity test of 7 tested strains to adults of tomato leafminer

[0105] like Figure 1As shown, five strains with higher infection rates to larvae were selected to determine their pathogenicity to adults of the tomato leafminer. Five days after inoculation, the average mortality rate of the control group was 5.1±1.1%, and the mortality rate of the experimental group ranged from 72% to 96%. The mortality rates of adults inoculated with the XJWLMQ-1 strain and the LNTL-1 strain were the highest, at 95.95% and 94.3%, respectively. There was no difference in the mortality rate between the two strains, but there was a significant difference in the mortality rate between the two strains (F=198.6, P<000.1) and the other strains (F=108.7, P<000.1).

[0106] 8 Determination of spore production of highly virulent strains

[0107] like Figure 2 As shown in the figure, the conidia production of the above five strains of Beauveria bassiana ranged from 2.5×10 8 ~3.9×10 8 Pieces / cm 2 There was a significant difference between the two strains (F=32.78, P<0.01). The strain with the highest average spore production was No. 1 XJWLMQ-1, which reached 3.85×10 8 Pieces / cm 2 The lowest spore production was in XJWLMQ-36, No. 20, which was only 2.5×10 8 Pieces / cm 2 Among them, the conidia production of LNTL-1 was 3.71×10 8 Pieces / cm 2 The average spore production was 3.01×10 8 Pieces / cm 2 Artificial classification: conidia production is lower than the average value of 3.02×10 8 Pieces / cm 2 The strains with low spore production were more than 3.02×10 8 Pieces / cm 2 The strain with the highest spore production.

[0108] In summary, in the screening of virulent strains against tomato leafminer, the virulence and lethality of XJWLMQ-1 were superior to those of other strains.

[0109] Example 3: Indoor experiment to test the control effect of automatic inoculation device on tomato leafminer

[0110] 1. Combined effects of indoor food attractants and Beauveria bassiana on tomato leafminer

[0111] The food attractants and Beauveria bassiana inoculation device used in this study were Figure 3A triangular prism-shaped paper box (triangle side length 24 cm, prism length 30 cm), a food attractant is hung in the center of the paper box (20 cm from the bottom), a sticky flannel (24 cm×24 cm) is laid flat on the bottom, and about 5 g of conidia of Beauveria bassiana XJWLMQ-1 are evenly distributed on the flannel.

[0112] 1.1 The effect of the test device on the attraction of tomato leafminers

[0113] 30 adult tomato leafminers were placed in an insect cage (80×80×80cm), and 14-day-old tomato seedlings (about 20cm high) were planted as food sources. The flannel cloth with conidia powder of Beauveria bassiana at the bottom of the automatic inoculation device was replaced with a sticky board, which was hung with a rope at a height of 30cm above the ground. The number of tomato leafminers captured by the sticky board was recorded 1d, 3d, 5d, 7d, and 14d after release. The experiment was repeated four times.

[0114] 1.2 Determination of the amount of fungi captured and the transmission rate of tomato leafminer

[0115] 30 adult tomato leafminers were placed in an insect cage (80×80×80cm), and 14-day-old tomato seedlings (about 20cm high) were planted as food sources. The automatic inoculation device was suspended at a height of 30cm above the ground with a rope, and 10 tomato leafminers were randomly captured at 1d, 3d, 5d, 7d, and 14d after release, and repeated four times. The tomato leafminers were transferred to a 2ml centrifuge tube and 1ml of sterile 0.05% Triton X-100 solution was added. Vortex for 2 to 3 minutes to shake out conidia from the insect body, and the concentration of conidia was determined using a hemocytometer.

[0116] 2. The control effect of indoor automatic inoculation device on tomato leafminer

[0117] As shown in Table 2, the attraction rate of the automatic inoculation device to the tomato leafminer adults showed an increasing trend with the sampling time. The attraction rate of the automatic inoculation device to the tomato leafminer adults was the highest 14 days after treatment. The number of conidia obtained from the inoculation device for a single tomato leafminer was 5.08×10 5 ~6.07×10 5 The average number of spores carried by a single tomato leafminer exposed to the automatic inoculation device continued to increase over time, which may mean that the control effect of the automatic inoculation device will not weaken over time.

[0118] Table 2 The attraction rate of the automatic inoculation device and the amount of fungi captured by the tomato leafminer

[0119]

[0120] Note: The data were processed by SPSS. Different letters in the columns represent significant differences. One-way ANOVA test (attraction rate of tomato leafminer adults P = 6.49e-06 < 0.05, the amount of fungi captured by a single tomato leafminer adult P = 2.01e-05 < 0.05).

[0121] The control effect of strain LNTL-1 is basically the same as that of XJWLMQ-1.

[0122] Example 4: Field experiment to test the control effect of automatic inoculation device on tomato leafminer

[0123] 1. Effect of field combined inoculation device on infection rate and bacterial yield of tomato leafminer

[0124] The experiment was conducted in July 2023 in a tomato greenhouse in Shoubonong Ecological Park, Fangshan District, Beijing. Two greenhouses with the same growth status and damage by tomato leafminers were selected, with an area of ​​80m 2 No chemical pesticides were applied during the experiment, and other management was normal. The greenhouse was divided into 6 small areas, and the small areas were separated by more than 5m to reduce interference between different treatments.

[0125] In the experimental group, one automatic inoculation device was placed in each plot in the greenhouse, and XJWLMQ-1 spores were evenly spread on the flannel of each device, and the device was hung 10 cm above the plant. In the control group, one automatic inoculation device was placed in each plot in the greenhouse, and no spores were spread on the flannel of each device.

[0126] On the 1st, 3rd, 5th, 7th and 14th day after the device was placed, 10 plants were randomly selected from each plot and 20 tomato leafminers were captured in 2ml centrifuge tubes. They were brought back to the laboratory and recorded for 11 consecutive days. The number of dead insects was recorded at regular intervals every day. The dead insects were surface disinfected with 70% alcohol, rinsed three times in distilled water, and transferred to a culture dish with moist sterile filter paper. Mycelium grew on the surface of the dead insects.

[0127] Of the 20 initially captured tomato leafminers, 5 were used to determine the number of conidia obtained per tomato leafminer in each treatment. The insects were transferred to a 2 ml centrifuge tube and 1 ml of sterile 0.05% Triton X-100 solution was added. Vortex for 2 to 3 minutes to shake the conidia out of the insect body, and the conidia concentration was determined using a hemocytometer.

[0128] 2 Results of the control of tomato leafminer by automatic field inoculation device

[0129] As shown in Table 3, the lethal time of tomato leafminers around the experimental group was significantly lower than that of the control group; the average lethal time of the experimental group was 4.56 days, and the average lethal time of the control group was 21.11 days. In addition, the lethal time of tomato leafminers shortened with the increase of time. The dead insects were kept moist and cultivated to grow white hyphae, which was identified as infection by Beauveria bassiana. In the experimental group, the average fungus amount of tomato leafminers on the first day was 5.89×10 5 spores / 5, the average amount of fungi increased with time, in the order of 1st day < 3rd day < 5th day < 7th day < 14th day, and no spores of Beauveria bassiana were detected in the tomato leafminer in the control group.

[0130] Table 3 The median lethal time and spore yield of tomato leafminer in field test

[0131]

[0132] Note: Data were processed by SPSS, and different letters in the columns represent significant differences, one-way ANOVA (P=1.04e-06<0.05). The experimental group was an automatic inoculation device carrying Beauveria bassiana spore powder; the control group was a hanging device flannel only, without Beauveria bassiana spore powder.

[0133] The field control effect of strain LNTL-1 is basically the same as that of XJWLMQ-1.

[0134] The above experimental results show that this study evaluated the pathogenicity of 20 strains of Beauveria bassiana to tomato leafminers in the laboratory. The tomato leafminers were "automatically" infected by the dried conidia of Beauveria bassiana by placing the flannel material in the food attractant device. The results showed that all 20 strains were pathogenic to tomato leafminers, among which LNTL-1 and XJWLMQ-1 were more toxic and lethal than other strains. The lethal time of LNTL-1 strain to larvae was 4.38d, the mortality rate to adults was 94.3%, and the spore production was 3.71×10 8 Pieces / cm 2 The lethal time of XJWLMQ-1 strain to larvae was 4.42 days, the mortality rate to adults was 95.95%, and the spore production was 3.85×10 8 Pieces / cm 2 . The automatic inoculation device was evaluated indoors and in the field, using the highly virulent Beauveria bassiana strains XJWLMQ-1 or LNTL-1. The device was loaded with 5 g of dried conidia of Beauveria bassiana. In the field experiment, the mortality time of tomato leafminer adults captured from the greenhouse with Beauveria bassiana conidia was significantly lower than that from the greenhouse without Beauveria bassiana conidia. The automatic inoculation device with Beauveria bassiana and food attractants can establish an "attraction-infection-transmission" system, which provides the possibility of using fungal self-inoculation devices to control tomato leafminer.

[0135] 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.

[0136] 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.

[0137] 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 tomato leafminer, characterized in that: The biocontrol preparation comprises Beauveria bassiana and a food attractant, wherein the Beauveria bassiana is 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 ( Beauveria bassiana ), the strain LNTL-1 is Beauveria bassiana with a preservation number of CGMCC No.40508 ( Beauveria bassiana ), the food attractant includes an attractant and a solvent, the attractant is tetradecatriene acetate, ethyl octanoate, 1-nonanol, phenylacetaldehyde and 3-methyl-1-butanol, and the mass ratio of tetradecatriene acetate, ethyl octanoate, 1-nonanol, phenylacetaldehyde and 3-methyl-1-butanol is (0.1-0.5): (0.1-0.5): (0.1-0.5): (0.05-0.1): (0.10-0.15).

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 claim 2, characterized in that The volume ratio of the attractant to the solvent is 1:(1-100).

4. The biocontrol agent according to any one of claims 1 to 3, characterized in that The food attractant also includes a slow-release carrier and / or nutrients.

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

6. The system according to claim 5, characterized in that The side of the container is provided with holes to facilitate the entry and exit of tomato leaf miners.

7. Use of the biocontrol agent according to any one of claims 1 to 4 or the system according to any one of claims 5 to 6 in controlling tomato leafminer.

8. A method for controlling tomato leafminer, characterized in that: The method comprises using the biocontrol agent according to any one of claims 1 to 4 or the system according to any one of claims 5 to 6 for control.

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