Beauveria bassiana TYW001 and application thereof

Abstract

The application belongs to the technical field of biological control, and particularly relates to a Beauveria bassiana TYW001 and application thereof. The application provides the Beauveria bassiana TYW001, the Latin name of the Beauveria bassiana TYW001 is Beauveria bassiana, is preserved in the China General Microbiological Culture Collection Center, the address is No. 1, Xibin West Road, Chaoyang District, Beijing, the preservation date is July 30, 2025, and the preservation number is CGMCC NO. 42149. The strain can not only resist high temperature and drought, but also effectively control the bean leafhopper. Moreover, the Beauveria bassiana TYW001 can enhance the predation of the small flower bug on the bean leafhopper, and provides a reliable green control technology for the control of the bean leafhopper.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, and in particular relates to a Beauveria bassiana TYW001 and its application. Background Technology

[0002] The bean thrips, also known as the common thrips or bean flower thrips, is a major pest of leguminous crops. It has a wide host range, damaging leguminous crops such as cowpeas, broad beans, red beans, mung beans, and peanuts, as well as potatoes, eggplants, radishes, sunflowers, loofahs, corn, mangoes, navel oranges, and weeds. The bean thrips is particularly damaging to cowpeas, hiding in the flower stamens, growing points, pods, or the undersides of leaves, sucking sap and pollen. During feeding, it uses its rasping-sucking mouthparts to pierce and suck the sap from the leaves, flowers, or fruits of the host plant. Damaged leaves become wrinkled or deformed, and the affected growing points wither and development stops. In severe cases, it can cause necrosis of the plant's growing points, leading to the plant's death. When it accumulates in large quantities and damages the flower parts, it causes the flower parts to appear scalded, then rot and fall off, affecting yield. After the pods grow, it moves to the pods in large quantities to cause damage, resulting in corking of the pod tip and end surfaces due to rasping and sucking, causing "black head" and "black tail" phenomena, which seriously affect the quality and commercial value of cowpeas.

[0003] Cowpeas are a major vegetable variety, belonging to the indeterminate inflorescence family, meaning they flower and fruit simultaneously. Bean thrips are the most significant pest of cowpeas, reproducing rapidly and in large numbers. This leads to frequent use of chemical pesticides by farmers, resulting in pesticide residues easily exceeding safe levels in cowpeas. This frequently causes poisoning and even death among people who consume cowpeas. In recent years, special campaigns to reduce pesticide use and control pesticide residues in cowpeas and other agricultural products have strongly advocated the use of biological pesticides to control bean thrips. Because bean thrips prefer to hide in flowers to cause damage, natural enemies and pesticides have difficulty reaching them. However, pathogenic fungal spores are more easily accessible to thrips, leading to infection. Therefore, using pathogenic fungi is an important microorganism for effectively replacing chemical pesticides in controlling bean thrips.

[0004] Since bean thrips prefer hot and dry environments, and most current entomopathogenic fungi are intolerant of high temperatures and drought, it is necessary to screen out strains that are resistant to high temperatures and drought from hot and dry regions in order to effectively control the damage caused by bean thrips.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a strain of Beauveria bassiana TYW001 that can effectively control bean thrips.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a Beauveria bassiana TYW001 strain, whose Latin name is Beauveria bassiana. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of July 30, 2025 and accession number CGMCC NO.42149.

[0009] The present invention also provides the application of the aforementioned Beauveria bassiana TYW001 in the control of soybean thrips.

[0010] The present invention also provides a formulation for controlling bean thrips, the formulation comprising the aforementioned Beauveria bassiana TYW001.

[0011] Preferably, the concentration of Beauveria bassiana TYW001 in the formulation is 1.0 × 10⁻⁶. 5 ~1.0×10 7 per mL.

[0012] Preferably, the dosage form of the preparation includes a suspension or a wettable powder.

[0013] The present invention also provides a method for controlling soybean thrips, comprising the following steps: spraying the preparation for controlling soybean thrips onto the field.

[0014] Preferably, the spraying rate of the formulation is 50-70 kg / mu.

[0015] Preferably, the concentration of Beauveria bassiana TYW001 in the formulation is 1.0 × 10⁻⁶. 5 ~1.0×10 7 per mL.

[0016] Preferably, the method further includes the step of releasing small flower bugs into the field.

[0017] Preferably, the release rate of the small flower bug is 800-1200 heads per mu.

[0018] Beneficial effects:

[0019] This invention provides a strain of Beauveria bassiana TYW001, which was screened from the soil and insect bodies of a cowpea base in Yazhou Bay, Sanya. It can not only withstand high temperature and drought, but also effectively control soybean thrips.

[0020] This invention prepares Beauveria bassiana TYW001 into a formulation that can effectively control bean thrips. Furthermore, Beauveria bassiana TYW001 can enhance the predation of small flower bugs on bean thrips, and the predation effect increases with increasing density. Therefore, the combined use of Beauveria bassiana TYW001 and small flower bugs provides a reliable green control technology for the prevention and control of bean thrips. Attached Figure Description

[0021] Figure 1 The indoor control efficacy of Beauveria bassiana TYW001 against adult bean thrips is shown in the figure. C1 in the figure represents a concentration of 1.0 × 10⁻⁶. 5 A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, C2 being a concentration of 1.0 × 10⁶ cells / mL. 6 A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, with C3 being a concentration of 1.0 × 10⁶ cells / mL. 7 A formulation for controlling soybean thrips (number per mL);

[0022] Figure 2 The figure shows the indoor control effect of strain X against adult soybean thrips. C1 in the figure represents a concentration of 1.0 × 10⁻⁶. 5 A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, C2 being a concentration of 1.0 × 10⁶ cells / mL. 6 A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, with C3 being a concentration of 1.0 × 10⁶ cells / mL. 7 A formulation for controlling soybean thrips (number per mL);

[0023] Figure 3 The indoor control effect of strain Y on adult soybean thrips is shown in the figure. C1 in the figure represents a concentration of 1.0 × 10⁻⁶. 5 A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, C2 being a concentration of 1.0 × 10⁶ cells / mL. 6 A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, with C3 being a concentration of 1.0 × 10⁶ cells / mL. 7 A formulation for controlling soybean thrips (number per mL);

[0024] Figure 4 The toxicity of Beauveria bassiana TYW001 to adult soybean thrips;

[0025] Figure 5 The control effect of Beauveria bassiana TYW001 on adult soybean thrips under different wavelengths of ultraviolet light was investigated.

[0026] Figure 6 The control effect of Beauveria bassiana TYW001 on adult soybean thrips at different temperatures;

[0027] Figure 7 The field control effect of Beauveria bassiana TYW001 on soybean thrips is shown in the figure. C1 in the figure represents a concentration of 1.0 × 10⁻⁶. 5A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, C2 being a concentration of 1.0 × 10⁶ cells / mL. 6 A formulation for controlling soybean thrips at concentrations of 1.0 × 10⁶ cells / mL, with C3 being a concentration of 1.0 × 10⁶ cells / mL. 7 The preparation for controlling soybean thrips was 1000 times diluted with Ailvshi, and CK was a negative control group with water.

[0028] Figure 8 The efficacy of combining Beauveria bassiana and Beauveria bassiana TYW001 in controlling soybean thrips.

[0029] Preservation Instructions

[0030] A Beauveria bassiana species, TYW001, with the Latin name Beauveria bassiana, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on July 30, 2025, with accession number CGMCC NO.42149. Detailed Implementation

[0031] This invention provides a Beauveria bassiana TYW001 strain, whose Latin name is Beauveria bassiana. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of July 30, 2025 and accession number CGMCC NO.42149.

[0032] The present invention also provides the application of the aforementioned Beauveria bassiana TYW001 in the control of soybean thrips.

[0033] The present invention also provides a formulation for controlling bean thrips, the formulation comprising the aforementioned Beauveria bassiana TYW001.

[0034] In this invention, the concentration of Beauveria bassiana TYW001 in the formulation is 1.0 × 10⁻⁶. 5 ~1.0×10 7 Cells / mL, preferably 1.0 × 10⁻⁶ 6 per mL.

[0035] In this invention, the dosage form of the preparation includes suspension and wettable powder.

[0036] The present invention also provides a method for controlling soybean thrips, comprising the following steps: spraying the preparation for controlling soybean thrips onto the field.

[0037] In this invention, the spraying amount of the preparation is 50-70 kg / mu, preferably 55-65 kg / mu, and more preferably 60 kg / mu.

[0038] In this invention, the concentration of Beauveria bassiana TYW001 in the formulation is 1.0 × 10⁻⁶. 5 ~1.0×10 7 Cells / mL, preferably 1.0 × 10⁻⁶ 6 per mL.

[0039] In this invention, the method further includes the step of releasing small flower bugs into the field.

[0040] In this invention, the release rate of the small flower bug is 800-1200 heads / mu, preferably 900-1100 heads / mu, and more preferably 1000 heads / mu.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] The thrips test insects used in the embodiments and experimental examples of this invention were collected from the cowpea experimental field of the Plant Protection Station in Haikou City, Hainan Province, and identified as Megalurus usitatus. Adult insects of uniform growth were collected through multiple generations of indoor rearing for indoor bioassay experiments. The test insects were reared in candy jars (2L) using kidney bean pods. Five layers of paper towels and three layers of filter paper were placed at the bottom of the candy jars, and two kidney bean pods were placed in each jar. The jars were sealed with paper towels and gauze and tied with rubber bands. Before use, the kidney bean pods were soaked and washed with dish soap, then rinsed with distilled water to remove the residual dish soap, and dried before use. Every other day, the kidney bean pods were removed from the adult insect jars, and two new kidney bean pods were added. The removed kidney bean pods were placed in another new candy jar to allow the eggs in the kidney bean pods to hatch. The rearing conditions for the test insects were: temperature 26℃, humidity 75%, and light intensity L:D = 16:8.

[0043] Example 1

[0044] The bacterial strain was isolated and purified from the soil of cowpea fields in Yazhou Bay, Sanya, using the large wax moth trapping method, and then subjected to activity screening technology. The specific steps are as follows:

[0045] (1) Taking advantage of the sensitivity of the larvae of the large wax moth to entomopathogenic fungi, the fungi cause the large wax moth to die by infecting it: First, in a warm and humid environment, the larvae of the large wax moth are placed in the soil. The larvae of the large wax moth will be infected by entomopathogenic fungi, which will enter the body through the body surface. The entomopathogenic fungi multiply in the larvae and release toxins, eventually causing the larvae to die. The dead larvae of the large wax moth are placed in PDA medium for culture. After the hyphae grow, single cells are isolated to obtain pure spores, which are then propagated to obtain Beauveria bassiana conidia powder.

[0046] (2) Inoculate thrips with Beauveria bassiana conidia powder. Place the thrips infected with Beauveria bassiana and killed on PDA medium (200g potato, 20g glucose, 15g agar, 1000mL distilled water) and incubate in a 27℃ mold incubator. After 3 days of incubation, prepare a Beauveria bassiana spore suspension with sterile water, shake well, and examine a drop of the spore suspension under a microscope. Adjust the spore concentration to 10 spores per field of view under a 10x objective lens. Spread 200μL evenly on a 2.0% water agar plate and incubate in a 27℃ mold incubator for 8 hours. After spore germination, pick a single spore with a needle to obtain purified Beauveria bassiana. Molecular biological identification of the purified Beauveria bassiana was performed, and the purified Beauveria bassiana was finally identified as Beauveria bassiana TYW001, with the gene sequence as shown in SEQ ID. As shown in No. 1, the purified Beauveria bassiana was placed in a 27°C mold incubator and cultured for 15 days for thrips inoculation.

[0047] The SEQ ID No. 1 is:

[0048] Example 2: A formulation for controlling bean thrips

[0049] Take the Beauveria bassiana PDA plate cultured for 15 days as in Example 1, bend the tip of a sterile 200mL pipette, and gently scrape 1 / 4 of the spore powder from the plate with the bent pipette tip. Place the spore powder into a 50mL centrifuge tube containing 5mL of 0.1% Tween-80. Vortex the liquid in the centrifuge tube until homogeneous, and then use a 1mL pipette tip to draw the liquid into a 1mL pipette tip plugged with glass fibers for filtration. This removes hyphae and culture medium debris, ensuring that the liquid flowing down contains only fungal spores, thus obtaining a spore suspension. If spore blockage is found during the process, the glass fibers should be replaced promptly.

[0050] Cover the counting chamber of a clean, dry hemocytometer with a coverslip. Shake the spore suspension thoroughly. Using a pipette, take an appropriate amount of spore suspension and drop it onto the edge of the coverslip, allowing it to slowly seep in, preventing air bubbles. Absorb any excess spore suspension outside the coverslip with absorbent paper. Let it stand for 5 minutes until the Beauveria bassiana conidia settle to the bottom of the counting chamber. Observe the hemocytometer under an optical microscope and count the spores under 40x magnification. Calculate the concentration of the spore suspension using the hemocytometer's formula and quantitatively dilute it with 0.1% Tween-80 to a concentration of 1.0 × 10⁻⁶. 7 The concentration was calculated as 1.0 × 10⁻⁶ cells / mL. 7 A formulation for controlling soybean thrips (number per mL);

[0051] Then use 1.0×10 7 A spore suspension of 1.0 × 10⁶ spores per mL was prepared by serially diluting the spores in sequence. 6 cells / mL, 1.0×10 5 A suspension of Beauveria bassiana spores at a concentration of 1.0 × 10⁶ spores / mL yields a concentration of 1.0 × 10⁶ spores / mL. 6 cells / mL, 1.0×10 5 A formulation for controlling soybean thrips (number per mL).

[0052] The formula for calculating blood cell counts is as follows:

[0053]

[0054] In the formula, N represents the total number of spores within 80 small cells.

[0055] Experimental Example 1: Indoor Bioassay

[0056] Adult thrips were quickly immersed in suspensions of Beauveria bassiana spores of different concentrations for 15 seconds, then poured onto filter paper to absorb excess water. Live thrips were then carefully transferred using a small brush into 9cm petri dishes containing agar and bean leaves, taking care to avoid mechanical damage. The dishes were sealed with sealing film to prevent the thrips from escaping (the lids of the petri dishes were pre-stamped with a 5cm diameter hole using a soldering iron and then sealed with paper towels for ventilation). Each petri dish contained 30 thrips as one treatment, with four replicates per treatment. The thrips inoculated with Beauveria bassiana were kept in an incubator at 26℃, 75% humidity, and a light intensity of L:D = 16:8. Adult thrips were kept in a separate, clean incubator under the same conditions as a control group. Observations were conducted for 10 consecutive days, with daily records of dead thrips. Dead thrips were promptly removed and examined under a microscope in petri dishes with moisturizing filter paper to determine if they were infected with Beauveria bassiana.

[0057] Statistical results of the control efficacy against soybean thrips are as follows: Figures 1-3 As shown;

[0058] The results showed that strain TYW001 had a good lethal effect on adult soybean thrips and could be used as a strain for field trials.

[0059] Analysis of the indoor virulence and pathogenicity of effective strains in Experiment Example 2

[0060] Cumulative corrected mortality was calculated using the Abbott W.S. formula, expressed as inoculation time (days) or spore suspension concentration (1.0 × 10⁻⁶). 7 Let x be the logarithm of the number of cells / mL, and y be the probability of the cumulative corrected mortality rate. Using probability analysis, the toxicity regression equation (y = 0.4091x + 2.8525, R0) was obtained using Excel. 2 =09405), calculate its lethal concentration LC50 and lethal time LT50, and analyze the virulence of the strain accordingly; perform significant difference analysis using Tukey's multiple comparison method in SPSS 17.0;

[0061] The Abbott formula is:

[0062]

[0063] When the probability value y = 5, the time x = 5.25, which is 10. 7 The lethal time of Beauveria bassiana TYW001 at 1 / mL to adult soybean thrips is 5.25 days. Figure 4 .

[0064] Experimental Example 3: Determination of UV Resistance and High Temperature Resistance

[0065] 1000 μL of TYW001 bacterial culture after liquid culture was transferred to a sterile 1.5 mL centrifuge tube. Mycelia were filtered, and spore suspensions were prepared using 0.05% Tween 80. After mixing, the suspensions were poured onto PDA medium and irradiated with two wavelengths of ultraviolet light (360 nm and 253.7 nm) at a height of 0.35 m for 30 min and 60 min, respectively. After treatment, the spores were washed off with 0.05% Tween 80. The concentration of all spore suspensions was adjusted to 10. 7 1 spore / mL, after treatment, stored at -80℃ for bioassay (refer to Example 1); Beauveria bassiana purchased from Taobao was used as the control CK, and the results are as follows. Figure 5 As shown.

[0066] 1000 μL of TYW001 bacterial culture was transferred to a sterile 1.5 mL centrifuge tube, mycelia were filtered, and spore suspensions were prepared using 0.05% Tween 80. The centrifuge tubes were placed in a water bath and incubated at different temperatures (30℃, 40℃, 45℃) for 4 h, 8 h, and 12 h, respectively. Untreated spore suspensions were kept as controls. The concentration of all spore suspensions was adjusted to 10. 7 1 spore / mL, after treatment, stored at -80℃ for later use in bioassays of its lethal effect on thrips (refer to Example 1); results are as follows Figure 6 As shown.

[0067] The results showed that the TYW001 strain had significantly higher heat resistance and UV resistance than the control strain. Even after treatment at 360 nm for 1 hour, the insecticidal effect of the TYW001 strain could still reach about 80%, which was about twice that of the control strain. After treatment at 40°C for 12 hours, the insecticidal effect of the TYW001 strain could still reach about 80%, which was twice that of the control strain. Therefore, the TYW001 strain has significant stress resistance.

[0068] Experiment Example 4: Field Experiment

[0069] The experiment included five treatments: a negative control of water, and a concentration of 1.0 × 10⁻⁶. 5 A formulation for controlling soybean thrips at a concentration of 1.0 × 10⁶ cells / mL. 6 A formulation for controlling soybean thrips at a concentration of 1.0 × 10⁶ cells / mL. 7 The control agents for soybean thrips, such as a 1000-fold dilution of Aylushi, were applied in 3 replicates per treatment, for a total of 15 plots. Each 7-meter-long row of cowpeas was designated as one plot, and the plots were randomly assigned to a block designation. Backpack-mounted electric sprayers were used to spray the five treatments in sequence, with a spray volume of 60 kg / mu (approximately 40 kg / acre). Application was completed before 10:00 AM.

[0070] The number of adult thrips was randomly surveyed from 10 flowers on cowpea plants at a height of 5m in the middle section. Surveys were conducted once before pesticide application and again at scheduled times in the morning at 3, 5, 7, 9, and 11 days after application. The pest population reduction rate was calculated using Excel, and the control efficacy was corrected. The Tukey multiple comparison method was used in SPSS 17.0 to determine the significance of differences. The results are as follows: Figure 7 As shown;

[0071] The formula for calculating the insect population decline rate is as follows:

[0072]

[0073] The results showed that Beauveria bassiana was significantly effective in controlling cowpea thrips. Overall, high-concentration Beauveria bassiana spore solution provided relatively stable control over cowpea thrips, with rapid lethality and a long duration of efficacy. 7 Both Beauveria bassiana spore suspension / mL and Ivlan reached their peak efficacy on the fifth day after application, but the former had a slightly lower efficacy of (36.18±2.92)%, while the latter reached (41.67±8.56)%, with no significant difference. However, the former had a longer duration of efficacy; at 11 days after application, the corrected efficacy of Ivlan decreased to (0.01±11.65)%, while at 10 days... 7 The corrected efficacy of the treatment with a dose / ml of the formulation could still be maintained at (24.23±11.01)%, such as Figure 7 .

[0074] Experimental Example 5

[0075] Thrips nymphs inoculated with *Beauveria bassiana* were placed in petri dishes, and then adult *Symplocos rubescens* were introduced. The predation behavior of the *Symplocos rubescens* was observed. The results showed that the predation of infected thrips nymphs by the *Symplocos rubescens* was significantly enhanced. Therefore, *Beauveria bassiana* TYW001 can enhance the predation of *Symplocos rubescens* on *Thrips pea*, and the predation effect increases with increasing density. Figure 8 .

[0076] As can be seen from the above embodiments, the present invention provides a Beauveria bassiana TYW001 strain and its application. This strain can not only withstand high temperature and drought, but also effectively control soybean thrips. Moreover, Beauveria bassiana TYW001 can enhance the predation of small flower bugs on soybean thrips, providing a reliable green control technology for the prevention and control of soybean thrips.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for controlling bean thrips, characterized in that, The process includes the following steps: spraying the agent for controlling soybean thrips onto the field and releasing small flower bugs into the field; The formulation includes Beauveria bassiana TYW001; the Latin name of Beauveria bassiana TYW001 is... Beauveria bassiana It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on July 30, 2025, with accession number CGMCC NO.42149.

2. The method according to claim 1, characterized in that, The application rate of the formulation is 50-70 kg / mu.

3. The method according to claim 2, characterized in that, In the formulation, the concentration of Beauveria bassiana TYW001 is 1.0 × 10⁻⁶. 5 ~1.0×10 7 per mL.

4. The method according to claim 3, characterized in that, The release rate of the small flower bugs is 800-1200 per mu.

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