Beauveria bassiana LNTL-1 and its application in biological control

Abstract

The invention relates to a Beauveria bassiana (Balsamo) Vuillemin strain LNTL-1, spore powder prepared from the strain, and use of the strain or the spore powder in preparing a biocontrol preparation. The strain has a significantly short lethal time and significantly improved infection rate for western flower thrips, American leafminers, tomato leafminers and / or whiteflies. The biocontrol preparation containing the strain or the spore powder can be used for the prevention and treatment of the above-mentioned pests.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a Beauveria bassiana strain LNTL-1 and application thereof in biological control of western flower thrips, leafminers, tomato leafminers and / or whiteflies. Background Art

[0002] There are many pests in the agricultural production process, especially in vegetable planting. The ones that cause serious damage to crops include Frankliniella occidentalis (Pergande), Liriomyza sativae (Blanchard), Tuta absoluta (Meyrick), Bemisia tabaci (Gennadius), and other global invasive pests. These pests have a wide range of hosts, a variety of diets, and are difficult to control. They can not only directly harm host plants, causing poor plant growth, crop yield reduction, or even crop failure; they can also spread a variety of plant viruses, causing large-scale viral diseases, leading to huge agricultural economic losses.

[0003] Currently, chemical agents are widely used for prevention and control, and pests show high levels of resistance to a variety of agents, resulting in decreased prevention and control efficiency, increased dosage of chemical pesticides, and accumulated residues of agents causing environmental pollution, which also poses a hidden danger to people's health.

[0004] As people pay more attention to issues such as insecticide resistance, human health and safety, and environmental pollution, biological control has received more and more attention, among which biological pesticides play an important role. The infection of insect pathogenic fungi on the host generally goes through 10 stages, namely, the attachment of conidia, germination, penetration of the epidermis, proliferation, production of toxins, host death, hyphae invasion of all organs, hyphae penetration of the epidermis, production of conidia, and conidia diffusion. Beauveria bassiana is an excellent entomogenous fungus with high pathogenicity to a variety of pests. It is harmless, safe, and non-polluting to humans and animals. Therefore, Beauveria bassiana is widely used in the integrated pest control system. Summary of the invention

[0005] This application is based on the experience of actual biological control. The virulence of different Beauveria bassiana strains varies greatly, and pests have a long survival time after contact with the fungus before death, and can still cause serious damage to crops. Therefore, it is particularly important to select strains with strong virulence and short lethality; the second is the spore production of the strain. A highly virulent strain must have a higher spore production, thereby reducing production costs, and ultimately achieving large-scale industrial production and commercial applications. Therefore, this application conducts indoor evaluations on the above two aspects, in order to screen out highly effective Beauveria bassiana strains, and ultimately apply them to the comprehensive pest control system. Specifically,

[0006] The present application provides a Beauveria bassiana (Balsamo) Vuillemin strain LNTL-1, which is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 40508 and the deposit date February 27, 2023, belonging to the order Combinosporales, the family Combinosporaceae, and the genus Beauveria bassiana.

[0007] The present invention also provides a method for preparing spore powder of the above strain, which comprises culturing the above strain.

[0008] Preferably, the culturing comprises the following steps:

[0009] 1) Preparation of activated spore powder: Inoculate Beauveria bassiana LNTL-1 on Sabouraud medium SDAY, wherein the Sabouraud medium SDAY contains 1% peptone, 1% yeast extract, 4% glucose and 2% agar. Preferably, the culture conditions are to maintain a temperature of 25-27° C. and culture for 7-10 days to produce activated LNTL-1 spore powder.

[0010] More preferably, the method for preparing the spore powder further comprises:

[0011] The activated LNTL-1 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.

[0012] More preferably,

[0013] 2) Cultivation of primary seed solution

[0014] The activated LNTL-1 spore powder was taken and inoculated into Sabouraud medium SDY to obtain seed solution. The Sabouraud medium SDY contained 1% peptone, 1% yeast extract, and 4% glucose. The initial spore concentration of the seed solution was 1.0×10 6 ml-1, and culture with shaking at 25℃ and 120r·min-1 for 48hr to obtain the first-level seed solution.

[0015] 3) Cultivation of secondary seed solution

[0016] 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.

[0017] 4) Solid phase fermentation

[0018] 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.

[0019] 5) Drying and harvesting of spore powder

[0020] 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.

[0021] The present invention also provides spore powder of strain LNTL-1, and the spore powder is prepared by the above preparation method.

[0022] The present invention also provides an application of the above-mentioned strain or spore powder in preparing a biocontrol agent.

[0023] Preferably, the strain or spore powder is used in combination with other pesticides.

[0024] The present invention also provides a biocontrol preparation, which comprises the above-mentioned strain or spore powder.

[0025] Preferably, the biocontrol agent also includes other insecticides.

[0026] The present invention also provides an application of the above-mentioned biocontrol agent in controlling target insects, wherein the target insects are western flower thrips, American leafminer, tomato leafminer and / or Bemisia tabaci.

[0027] The present invention also provides a method for controlling target insects, comprising spraying the above-mentioned biological control preparation on susceptible crops, wherein the target insects are western flower thrips, American leafminer, tomato leafminer and / or whitefly.

[0028] The term "include" or "comprising" in the present invention is an open description containing the specified components or steps described, and other specified components or steps that will not be substantially affected.

[0029] Beneficial effects of the present invention:

[0030] The strain LNTL-1 screened by the present invention has a significantly higher infection rate to four common invasive pests worldwide, including western flower thrips, American leafminer, tomato leafminer and Bemisia tabaci (Gennadius), than other strains, and can increase the mortality rate to pests, thereby reducing the damage of pests to crops.

[0031] The strain LNTL-1 screened by the present invention has a shorter lethal time to the above four pests compared with other strains. While the infection rate is high, the lethal time is shortened, which can further reduce the amount of damage caused by pests to crops.

[0032] The strain LNTL-1 screened by the present invention has a higher spore production than other strains, thereby reducing production costs and ultimately achieving large-scale industrial production and commercial application.

[0033] Strain deposit information:

[0034] Depository: China General Microbiology Center (CGMCC);

[0035] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0036] Accession number: CGMCC No.40508;

[0037] Deposit date: February 27, 2023;

[0038] Classification name: Beauveria bassiana BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :Toxicity of highly virulent strains to adults of Liriomyza sativae.

[0040] Figure 2 : Toxicity of highly virulent strains to adults of tomato leafminer.

[0041] Figure 3 :Toxicity of highly virulent strains to adults of Bemisia tabaci.

[0042] Figure 4 : The spore production of 19 strains of Beauveria bassiana. DETAILED DESCRIPTION

[0043] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0044] Unless otherwise specified, the materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources.

[0045] Example 1 Screening of highly toxic pathogenic strains

[0046] 1. Materials and Methods

[0047] 1.1 Test insects

[0048] The western flower thrips used in the experiment were collected from the Bikun Vegetable Planting Center in Mentougou, Beijing, and were reared for multiple generations in the laboratory with Phaseolus vulgaris L. after washing and drying. They were purchased from Xingfu Rongyao Supermarket, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. The rearing temperature was 26±1℃, and the photoperiod was L:D=14:10.

[0049] The American leafminer population was collected from Haikou City, Hainan Province. The host for indoor rearing was dwarf bean (Phaseolus vulgaris), and the rearing conditions were temperature (26±2)℃, relative humidity 50%-60%, and photoperiod L:D=14:10. The rearing was purified for more than 3 generations.

[0050] Tomato leafminers were collected from Xinqiao Town, Youxian District, Mianyang City, Sichuan Province, 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 Instrument).

[0051] Bemisia tabaci were collected from tobacco in a greenhouse in Langfang, Hebei Province. Bemisia tabaci were reared for multiple generations in the laboratory using tomato seedlings Solanum lycopersicum L. The tomato seedlings were placed in insect cages (40×40×30 cm) at 26±2℃ and a photoperiod of 12:12 (L:D).

[0052] 1.2 Bioassay

[0053] Determination of the lethal median time of the strain to the nymphs of western flower thrips: First, after the western flower thrips were anesthetized with carbon dioxide, they were poured into the prepared spore suspension, soaked for 3s, poured onto filter paper, and the excess spore suspension was sucked off. Then, the test insects were picked up with a soft brush and put into an insect culture tube (20 cm high, 3 cm in diameter) with a bean placed in it. Each tube was filled with 20 insects and sealed with a 200-mesh gauze and a rubber band. The test was repeated 4 times, and a 0.05% Tween-80 solution was used as a control. The number of dead insects was recorded regularly every day for 8 consecutive days, and the lethal median time of each strain to the nymphs of western flower thrips was calculated.

[0054] Determination of the lethal median time of the strain to the larvae of the American leafminer: The biological test of the larvae of the American leafminer adopts the leaf immersion method. When the kidney bean grows two true leaves, the leaves with the second-instar larvae of the American leafminer are immersed in the prepared spore suspension for 20 seconds; after the leaves are naturally dried, they are placed in the insect cage, and the control group is treated with 0.05% Tween-80 in the same way. The culture conditions are temperature 26±1℃, relative humidity RH 85%, light cycle L:D=14:10, and continuous observation for 8 days. The test is repeated 4 times, and the number of dead insects is recorded regularly every day, and the lethal median time of each strain to the larvae of the American leafminer is calculated.

[0055] Determination of the mortality of adult Liriomyza sativae by strains: Five strains with high toxicity to Liriomyza sativae larvae 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 adult Liriomyza sativae were transferred into the cylindrical plastic tube and walked on the velvet cloth for 1 minute. Then, 20 adults were transferred 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 treatments were carried out completely randomly. The rearing conditions of the American leafminers were maintained at 25℃~27℃, 50℃~70%RH, and 12L:12D photoperiod. The number of dead insects was recorded every day. Each experiment was repeated 4 times and the records were kept for 8 consecutive days. The mortality rate of each strain against adult American leafminers was calculated.

[0056] Determination of the lethal median time of the strain to the larvae of the tomato leafminer: Use the leaf immersion method to treat the second-instar larvae of the tomato leafminer on the tomato leaves. Take 2 mL of the prepared spore suspension, soak the leaves with the second-instar larvae in the suspension for 30 seconds, and let them dry naturally or use sterilized filter paper to absorb excess water. Transfer to a culture dish (diameter × height = 20 cm × 3.5 cm), wrap the petiole in a moist cotton ball, and then seal the culture dish. The control group is treated in the same way with 0.05% Tween-80. Record the number of dead larvae every day, repeat each test 4 times, and record for 8 consecutive days to calculate the lethal median time of each strain to the larvae of the tomato leafminer.

[0057] Determination of the mortality of tomato leafminer adults by strains: Five strains with high toxicity to tomato leafminer larvae were used for bioassay on adults. 0.1 g of dry conidia of each strain was evenly distributed on a cotton velvet cloth on the inside of a cylindrical plastic tube (70 mm long × 48 mm diameter). 20 tomato leafminer adults 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 velvet cloth without dry conidia and then transferred to the same cage. The treatments were completely randomized. 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 experiment was repeated 4 times and recorded for 8 consecutive days. The mortality rate of each strain against tomato leafminer adults was calculated.

[0058] Determination of the lethality of the strain to whitefly nymphs: a single tomato leaf containing 30 4th instar whiteflies was selected and immediately immersed in the prepared 1×10 8 spore / ml suspension for 10s, then take out the leaves and place them on sterile filter paper for 10-20min to dry naturally. The control group was treated in the same way with 0.05% Tween-80. The dried leaves were immediately placed in a culture dish (diameter: 90mm) containing 2% agar, and the culture dish was sealed with a sealing film and had small holes. Cultured in a light incubator at 26±1℃ and 12:12 (L:D). The number of infected whiteflies was recorded every day, and each test was repeated 4 times, and the records were continuously recorded for 8 days, and the lethal median time of each strain to the 4th instar nymphs of whiteflies was calculated.

[0059] Determination of the mortality of tobacco whitefly adults by strains: 5 strains with high toxicity to tobacco whitefly nymphs were used for bioassay on adults. 0.1 g of dry conidia of each strain was evenly distributed on the cotton velvet cloth at the bottom of a culture dish (90 mm diameter). 20 tobacco whitefly adults were transferred to the culture dish and walked on the velvet cloth for 1 minute. Then, 20 adults were transferred from the velvet cloth to clean tomato leaves in a culture dish containing 2% agar. The adults in the control group were contacted with the velvet cloth without dry conidia and then transferred to clean tomato leaves in the same culture dish containing 2% agar. The culture dish was sealed with a sealing film and had small holes. The feeding conditions of tobacco whiteflies were maintained at 26±1℃ and 12:12 (L:D) light cycle. The number of dead insects was recorded every day. Each test was repeated 4 times and recorded for 8 consecutive days. The mortality of each strain against tobacco whitefly adults was calculated.

[0060] 1.3 Test strains

[0061] The 19 strains of Beauveria bassiana for testing were collected from insect bodies in different regions of the country. After indoor separation and purification, 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 traits 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 to a final concentration of 1×10 8 spores / ml of spore suspension; 0.05% Tween-80 solution was used as blank control.

[0062] Conidia were produced in large quantities on rice culture medium. Rice was autoclaved for 1 hour at 121°C and inoculated with 3-day-old blastospores (Jenkins et al., 1998). Culture was carried out 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.

[0063] 1.4 Determination of fungal spore production

[0064] 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. Each repeat was counted 3 times, and then the number of spores per square centimeter was calculated.

[0065] 1.5 Data processing

[0066] Excel 2016 was used to process the raw data of each experiment, and SAS 9.2 was used to analyze the significance of infection rate, lethal time, mortality rate, and conidia production of the strains. LT50 was calculated by POLO software.

[0067] Based on bioassay data

[0068] Infection rate = number of infected insects / total number of treated insects × 100%

[0069] Mortality rate = number of dead insects / total number of insects treated × 100%

[0070] 2 Results and analysis

[0071] 2.1 Toxicity of Beauveria bassiana to nymphs of western flower thrips

[0072] Table 1: Toxicity of 19 strains of Beauveria bassiana to nymphs of western flower thrips

[0073]

[0074]

[0075] The tested strains of Beauveria bassiana had certain toxicity to western flower thrips, and the thrips nymphs in all treatments reached the peak of death at 3-5 days. Within 8 days, only the infection rates of LNTL-1, JXNC-5 and JXNC-11 were above 75%, and the median lethal time was 3.40 days, 3.51 days and 3.71 days respectively. The dead insects were cultured in a moisturizing environment, and white hyphae grew, which was identified as infection and death caused by Beauveria bassiana.

[0076] 2.2 Toxicity of Beauveria bassiana to Liriomyza sativae

[0077] 2.2.1 Toxicity of Beauveria bassiana to Liriomyza sativae larvae

[0078] Observation of the toxicity of Beauveria bassiana to the larvae of Liriomyza sativae using the fungus in Table 1

[0079] Table 2 Toxicity of 19 strains of Beauveria bassiana to larvae of Liriomyza sativae

[0080]

[0081]

[0082] The 19 tested strains of Beauveria bassiana had certain toxicity to the larvae of the American leafminer. Most of the insects in the treatment reached the peak of death in 3-5 days, and the burrowing path was no longer extended. The strains with an infection rate of more than 75% within 8 days were LNTL-1, HNLY-47, SDDZ-20 and XJWLMQ-36, and the median lethal time was 3.72 days, 4.44 days, 4.08 days and 3.75 days respectively. The dead larvae were picked out and moisturized for culture, and white hyphae grew out. It was identified that they were killed by infection with Beauveria bassiana.

[0083] 2.2.2 Toxicity of Beauveria bassiana to adults of Liriomyza sativae

[0084] Four strains, LNTL-1, HNLY-47, SDDZ-20 and XJWLMQ-36, whose infection rates to Liriomyza sativae larvae were above 75%, were selected to evaluate their pathogenicity to Liriomyza sativae adults.

[0085] Results Figure 1 The above four strains all have strong pathogenicity. Five days after inoculation, the average mortality rate of the control group CK was 5.95±0.3%, and the mortality rate of the experimental group was 85.0%-98.1%. Among them, the mortality rate of adults inoculated with strain LNTL-1 was the highest, at 98.1%, which was significantly different from other strains.

[0086] 2.3 Toxicity of Beauveria bassiana to Tomato leafminer

[0087] 2.3.1 Toxicity of Beauveria bassiana to larvae of the tomato leafminer

[0088] Table 3: Toxicity of 19 strains of Beauveria bassiana to larvae of tomato leafminer

[0089]

[0090]

[0091] The tested Beauveria bassiana strains have certain toxicity to the larvae of tomato leafminer. Most of the insects in the treatment reached the peak of death in 4-6 days, and the burrowing path no longer extended. The strains with an infection rate of more than 75% within 8 days were LNTL-1, LNTL-3, LNTL-5, and XJWLMQ-36, and the median lethal time was 4.38 days, 4.78 days, 4.77 days, and 4.97 days, respectively. The dead larvae were picked out and moisturized for cultivation, and white hyphae grew. It was identified that they were killed by infection with Beauveria bassiana.

[0092] 2.3.2 Toxicity of Beauveria bassiana to adults of the tomato leafminer

[0093] Four strains, LNTL-1, LNTL-3, LNTL-5, and XJWLMQ-36, whose infection rates to tomato leafminer larvae were above 75%, were selected to evaluate their toxicity to tomato leafminer adults.

[0094] Results Figure 2 :All four strains have strong pathogenicity to the adults of tomato leafminer. Five days after inoculation, the average mortality of the control group CK was 5.15±1.1%, and the mortality of the experimental group ranged from 71.5% to 95.9%. The mortality of the adults of tomato leafminer inoculated with LNTL-1 strain was the highest, at 95.9%, which was significantly different from the other strains.

[0095] 2.4 Toxicity of Beauveria bassiana to Bemisia tabaci

[0096] 2.4.1 Toxicity of Beauveria bassiana to whitefly nymphs

[0097] Table 4: Toxicity of 19 strains of Beauveria bassiana to whitefly nymphs

[0098]

[0099]

[0100] The tested Beauveria bassiana strains were all toxic to whitefly nymphs, and most of the insects in the treatments reached the peak of death in 4-6 days. The strains with an infection rate of more than 75% within 8 days were LNTL-1, LNTL-5, JXNC-5, and HNLY-46, and the median lethal time was 3.95 days, 4.27 days, 4.46 days, and 4.15 days, respectively. The dead insects were cultured in a moisturizing environment, and white hyphae grew out. It was identified that they were killed by infection with Beauveria bassiana.

[0101] 2.4.2 Toxicity of Beauveria bassiana to adults of Bemisia tabaci

[0102] Four strains, LNTL-1, LNTL-5, JXNC-5 and HNLY-46, whose infection rates to whitefly nymphs were above 75% were selected, and their toxicity to whitefly adults was compared.

[0103] Results Figure 3 All four strains had strong pathogenicity to adults of Bemisia tabaci. Five days after inoculation, the average mortality rate of the control group was 5.65±0.9%, while that of the experimental group ranged from 75.0% to 92.8%. The mortality rate of adults of Bemisia tabaci inoculated with LNTL-1 strain was the highest, at 92.8%, which was significantly different from that of other strains.

[0104] In summary, LNTL-1 has the highest toxicity to the four pests and the shortest lethal time.

[0105] 2.5 Determination of spore production of highly virulent strains

[0106] The results of spore production of 19 highly virulent Beauveria bassiana strains are as follows Figure 4 shown.

[0107] The conidia production of the 19 strains of Beauveria bassiana ranged from 2.4×10 8 ~3.9×10 8 The average spore production was 3.01×10 8 Pieces / cm 2 We artificially defined it as the conidia production lower than the average value of 3.02×10 8 Pieces / cm 2 The strains with low spore production were more than 3.02×108 Pieces / cm 2 The strains with high spore production were classified according to this standard, and the classification results are shown in Table 5. Among them, there were 10 strains with low spore production and 9 strains with high spore production, accounting for 47.3% and 52.6% of the total number of strains, respectively. More than half of the strains had low spore production, which shows that it is necessary to determine the spore production to screen out strains with high spore production.

[0108] Table 5 Classification of Beauveria bassiana based on spore production

[0109]

[0110] According to the spore production, Beauveria bassiana is divided into high spore production strains and low spore production strains. The high spore production strains include LNTL-1, LNTL-3, JXNC-11, HNLY-47, JLGZL-1, JLGZL-2, JLGZL-3, JLGZL-9, and XJWLMQ-33; the low spore production strains include LNTL-5, JXNC-4, JXNC-5, JXNC-12, HNLY-46, SDDZ-13, SDDZ-14, SDDZ-20, SDDZ-21, and XJWLMQ-36. Among them, the strain with the highest spore production is LNTL-1, with a spore production of 3.85×10 8 Pieces / cm 2 .

[0111] A strain with application potential must have strong virulence and high spore production, among which virulence should be the primary condition. In the screening of virulence strains for several pests, LNTL-1 has stronger virulence than other strains, and the lethal time of infection of several pests is shorter. This strain has the highest spore production. The high spore production strain plays an important role in the vigorous production of applications. In summary, Beauveria bassiana LNTL-1 is an entomogenous fungus with great biocontrol application prospects against western flower thrips, American leafminers, tomato leafminer, and whitefly.

[0112] Example 2: Preparation of spore powder of LNTL-1

[0113] (1) Preparation of activated spore powder

[0114] Beauveria bassiana LNTL-1 is inoculated on Sabouraud medium SDAY, wherein the Sabouraud medium SDAY contains 1% peptone, 1% yeast extract, 4% glucose and 2% agar. The culture conditions are to maintain the temperature at 25-27° C. and culture for 7-10 days to produce activated spore powder of LNTL-1.

[0115] (2) Cultivation of primary seed solution

[0116] The activated spore powder of strain LNTL-1 was taken and added to Sabouraud culture medium SDY to obtain seed solution. The Sabouraud culture medium SDY was 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.

[0117] (3) Cultivation of secondary seed solution

[0118] The first-level seed solution was inoculated with 5% of the Sabouraud culture medium SDY culture medium 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, the rotation speed was 100-200 rpm, and the initial dissolved oxygen content was above 50%. After culturing for 40 hours, the second-level seed solution was obtained.

[0119] (4) Solid phase fermentation

[0120] 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.

[0121] (5) Drying and harvesting of spore powder

[0122] The material after solid phase fermentation was dried at 30°C for 3 days, and then spore powder was harvested using a spore harvester.

[0123] Example 3: Field control effect

[0124] At the vegetable production base in Changping District, Beijing, 1×10 7 The suspension of spores / ml was added with 0.05% Tween-80. The vegetable leaves were sprayed and field tests were carried out. The field control effect on whiteflies was 67%, the field control effect on western flower thrips was 72%, the field control effect on tomato leafminers was 77%, and the field control effect on American leafminers was 81%.

[0125] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily conceive of changes or substitutions within the technical scope disclosed by the present invention, and such changes or substitutions shall be within the scope of protection of the present invention.

Claims

1. A Beauveria bassiana ( Beauveria bassiana (Balsamo) Vuillemin) strain LNTL-1, characterized in that The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 40508 and the deposit date February 27, 2023. It belongs to the order Combreales, the family Combreaceae, and the genus Beauveria.

2. A method for preparing spore powder of strain LNTL-1, characterized in that: The preparation method comprises the following steps: Beauveria bassiana (Balsamo) Vuillemin) strain LNTL-1 was cultured.

3. The preparation method according to claim 2, characterized in that: The culture comprises 1) preparing activated spore powder: inoculating the strain LNTL-1 on Sabouraud medium SDAY for culture, wherein the Sabouraud medium SDAY contains 1% peptone, 1% yeast extract, 4% glucose and 2% agar spores.

4. The preparation method according to claim 3, characterized in that: The step 1) comprises inoculating Beauveria bassiana LNTL-1 on Sabouraud medium SDAY, maintaining the temperature at 25-27° C., culturing for 7-10 days, and producing activated spore powder of LNTL-1.

5. The preparation method according to claim 3 or 4, characterized in that: The preparation method further comprises the steps of subjecting the activated LNTL-1 spore powder to 2) primary seed liquid culture, 3) secondary seed liquid culture, 4) solid phase fermentation and 5) drying and harvesting the spore powder.

6. A spore powder of strain LNTL-1, characterized in that: The spore powder is prepared by any preparation method according to claims 2-5.

7. Use of the strain LNTL-1 according to claim 1 or the spore powder according to claim 6 in preparing a biocontrol agent, characterized in that: The biocontrol agent is used for preventing and controlling western flower thrips, American leafminers, tomato leafminers and / or whiteflies.

8. A biocontrol agent, characterized in that: The biocontrol agent comprises the strain LNTL-1 according to claim 1 or the spore powder according to claim 6.

9. Use of the biocontrol agent according to claim 8 in controlling target pests, wherein the target pests are western flower thrips, American leafminer, tomato leafminer and / or Bemisia tabaci.

10. A method for controlling target pests, the method comprising spraying the biocontrol agent of claim 8 onto susceptible crops, wherein the target pests are western flower thrips, American leafminer, tomato leafminer and / or Bemisia tabaci.

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