A spore-producing strain of entomogenous fungus and its application in the prevention and treatment of western flower thrips

Abstract

The present application relates to the field of microbial technology, and discloses a spore-producing Metarhizium anisopliae strain and its application in the prevention and treatment of western flower thrips. The Metarhizium anisopliae provided by the present application has a preservation number of CCTCC No. M 20252118, a fast growth rate, a large spore yield, a high spore germination rate, and is easy to be cultured and multiplied in large quantities indoors. The present application can be used as a biocontrol fungus for preventing and treating the invasive agricultural pest western flower thrips, has strong pathogenicity to the western flower thrips nymphs, is safe to the environment, is not easy to make the pests resistant, and has application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a sporulating strain of Metarhizium anisopliae and its application in the control of western flower thrips. Background Technology

[0002] The western flower thrips belongs to the family Thripidae in the order Thysanoptera and accounts for about one-third of the known thrips population. This insect is primarily herbivorous, with a host range covering more than 500 plant species. It sucks plant sap using its rasping-sucking mouthparts, causing leaf chlorosis, curling, flower drop, and plant wilting; damage to fruit leads to scarring, deformities, and fruit drop, severely impacting the quality and economic value of agricultural products. Furthermore, the western flower thrips can transmit various plant viruses, and the viral diseases it causes as a vector result in losses far exceeding those caused by direct consumption.

[0003] Due to their strong adaptability, wide distribution, small size, rapid development, and overlapping generations, both adults and nymphs of the western flower thrips can damage crops, making control extremely difficult. Currently, control mainly relies on chemical pesticides, which are low-cost and fast-acting, but long-term use has led to resistance to many pesticides, reducing their effectiveness. Furthermore, chemical control also poses risks to pesticide residues, human and animal safety, and the ecosystem. Therefore, developing green, safe, efficient, and sustainable control measures has become an urgent priority.

[0004] Chinese patent application CN202510320439.0 discloses a strain of *Metarhizium anisopliae* GZFW22_11 and its applications. This strain was isolated from the larvae of the fall armyworm *Spodoptera frugiperda*. The strain exhibits rapid growth, a short conidia production time, and high conidia yield; after 15 days of culture on SDAY medium, its conidia yield was 3.47 ± 0.31 × 10⁻⁶. 6 / mm 2 Compared to conventional strains, this strain exhibits more efficient spore production, laying the foundation for its large-scale application in biocontrol. However, further research and screening are still needed to find more active biocontrol strains. Summary of the Invention

[0005] The inventors of this application discovered through research that a strain of Metarhizium pemphigi, belonging to the genus Metarhizium, exhibits highly efficient insecticidal properties, 1×10 8 At the specified concentration, the corrected mortality rate of western flower thrips can reach over 70% within 3 days. Therefore, this strain has the potential to be developed into a new type of antifungal and can be applied to biological control strategies for western flower thrips.

[0006] The main objective of this invention is to provide a spore-producing strain of Metarhizium anisopliae for the gall aphid, in order to offer a new approach to the green control of western flower thrips.

[0007] To achieve the above objectives, this invention provides a spore-producing Metarhizium pemphigi strain, specifically strain Mp001, which was deposited on September 25, 2025, at the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province), with accession number CCTCC No. M 20252118 and classification name: Metarhizium pemphigi Mp001.

[0008] Furthermore, the present invention provides a method for culturing the gall-producing Metarhizium anisopliae strain Mp001. The method involves inoculating the gall-producing Metarhizium anisopliae strain Mp001 into SDA solid medium (40g maltose, 10g peptone, 10g yeast extract, 18g agar powder, diluted with distilled water to 1000 mL, and pH adjusted to 7.0) for culture. After the strain produces a large number of conidia, a spore suspension is prepared.

[0009] Furthermore, the cultivation conditions were: temperature 26 ± 1℃, relative humidity 75% ± 5%, and photoperiod L:D = 14 h:10 h.

[0010] Furthermore, the method for preparing spore suspensions is as follows: conidia are added to a sterile aqueous solution of Tween-80 with a concentration of 0.05% to prepare spore suspensions of different concentrations.

[0011] A second aspect of the present invention provides an insecticide containing conidia of the Metarhizium anisopliae strain Mp001.

[0012] Furthermore, the insecticide contains 1.0 × 10⁻⁶ spores of the Metarhizium anisopliae strain Mp001. 5 -1.0×10 8 Spores / mL.

[0013] The application of the *Metarhizium anisopliae* strain Mp001, or any spore suspension obtained by any culture method, or any insecticide described herein, in the control of western flower thrips, provided in the third aspect of this invention.

[0014] Furthermore, the present invention provides a method for controlling western flower thrips, wherein the insecticide is applied to crops damaged by western flower thrips when the western flower thrips are in the nymph stage.

[0015] Furthermore, the crops mentioned include important economic crops such as chili peppers, tomatoes, and tobacco.

[0016] Therefore, this invention provides a strain of Metarhizium anisopliae Mp001, which has a simple cultivation method, rapid growth, high sporulation yield, and high spore germination rate. It is easy to cultivate and reproduce in large quantities indoors. It is a biocontrol fungus that can be used to control western flower thrips, an important economic crop pest that harms peppers, tomatoes, tobacco, and other crops. It can be used as an environmentally friendly green biological control agent to replace chemical pesticides and other control agents that have potential environmental hazards. It can effectively control western flower thrips, an important economic crop pest that harms peppers, tomatoes, tobacco, and other crops. It provides a new type of microbial strain and control strategy for the field of biological control of agricultural pests. This strain has significant environmentally friendly characteristics, has no adverse effects on human health, and exhibits excellent biological control effects against the target pest.

[0017] Prior art, Chinese patent application CN202510320439.0, discloses a strain of *Metarhizium anisopliae* GZFW22_11 and its applications. This strain was isolated from the larvae of the fall armyworm *Spodoptera frugiperda*. The strain exhibits rapid growth, a short conidia production time, and high conidia yield; after 15 days of cultivation on SDAY medium, its conidia yield is 3.47 ± 0.31 × 10⁻⁶. 6 / mm 2 The strain Mp001 of *Metarhizium anisopliae* provided by this invention, when cultured on SDAY medium for 7 days, produced 4.02 ± 0.03 × 10⁻⁶ sporozoites. 6 / mm 2 This study demonstrates the advantages of the Metarhizium anisopliae strain Mp001 in terms of sporulation speed and sporulation quantity. It can achieve a higher number of spores more quickly, which may more effectively control the pest population. It provides a new biocontrol resource for the green control of western flower thrips and delays the development of pesticide resistance, and has better application potential and prospects. Attached Figure Description

[0018] Figure 1 These are photographs of fungal colonies of Metarhizium anisopliae Mp001, where a is the front view and b is the back view.

[0019] Figure 2 The images show the morphology of conidia of Metarhizium anisopliae Mp001, where a and b are two photographs of the morphology.

[0020] Figure 3 Phylogenetic analysis of ITs rDNA for Metarhizium anisopliae Mp001 (Note: Each branch is labeled with: GenBank sequence number + strain name).

[0021] Figure 4 Corrected mortality rate of western flower thrips for Metarhizium anisopliae Mp001.

[0022] Figure 5 This image shows the symptoms of natural infection of western flower thrips nymphs with Metarhizium anisopliae Mp001.

[0023] Information on the preservation of biological materials:

[0024] The preservation date of Metarhizium pemphigium strain Mp001 for the aphid gall midge is September 25, 2025. The depositary institution is the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province), the accession number is CCTCC No. M20252118, and the classification name is Metarhizium pemphigium Mp001. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments. These embodiments are intended to help those skilled in the art to further understand the invention, but do not limit the invention in any way. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0026] Example 1: Isolation and identification of Metarhizium anisopliae strain Mp001

[0027] 1.1 Materials

[0028] 1.1.1 Soil Sample Collection

[0029] A total of 20 samples were collected from different provinces across the country. The specific sampling locations and habitats are shown in Table 1.

[0030] Table 1 Information on tested strains

[0031]

[0032] 1.1.2 Isolation of the test strains

[0033] Soil samples were collected from the above locations and placed in 5 ml sterile EP tubes. They were then transported back to the Hunan Provincial Key Laboratory of Plant Disease and Pest Biology and Control. Using the streak plating method, the samples were cultured on SDA solid medium (40 g maltose, 10 g peptone, 10 g yeast extract, 18 g agar powder, diluted to 1000 mL with distilled water, pH adjusted to 7.0) in an incubator at 26 ± 1℃, 75% ± 5% relative humidity, and a photoperiod L:D = 14 h:10 h. After 7 days of culture, the conidia were transferred to a new medium for purification, resulting in a total of 16 bacterial strains (see Table 2).

[0034] Table 2 Information on tested strains

[0035]

[0036] Subsequently, a spray method was used to spray conidial suspensions (1.0±10 μL) of 15 different strains. 8 Table 3 shows the lethality of different strains of *Thrips przewalskii* after infecting second-instar nymphs. As can be seen from the table, all strains were lethal to second-instar nymphs. However, the lethality rates varied among different strains over the same time period. Five days after spraying, the corrected mortality rate of 10 strains exceeded 50%, with strain Mp001 showing the best performance and the highest lethality rate of 86.75%, indicating a strong ability to infect *Thrips przewalskii* nymphs. Figure 5 Therefore, this strain was selected for further research and the biological material was preserved. This strain was found on April 13, 2024, in a pine forest in Anjia Village, Wuli Town, Lingshan County, Qinzhou City, Guangxi Zhuang Autonomous Region (22.186894°N, 109.225578°E, altitude 93 meters).

[0037] Table 3. Cumulative corrected mortality rate of the tested strains against second-instar nymphs of western flower thrips.

[0038]

[0039] Note: Data in the table are mean ± standard deviation. Different letters after the data in the same column indicate that the difference is significant at the P<0.05 level.

[0040] 1.1.3 Colony morphology observation

[0041] The isolated and purified Mp001 strain was inoculated onto SDA solid medium (40 g maltose, 10 g peptone, 10 g yeast extract, 18 g agar powder, diluted with distilled water to 1000 mL, pH at rest) and incubated for 14 days in an incubator at 26±1℃, 75%±5% relative humidity, and a photoperiod L:D=14 h:10 h. The colony morphology was then observed.

[0042] 1.2 Results

[0043] After obtaining pure culture of the *Metarhizium anisopliae* strain Mp001, its morphological characteristics were identified. This strain was cultured on SDA medium for 14 days. The colonies had white downy hairs on the upper surface, slightly convex in the center, and conidia diffused from the center outwards. Figure 1 (a) The colony is pale yellow on the reverse side; the hyphae are smooth and septate. Figure 1 (b) Conidia are short club-shaped, smooth, and 2.62 μm x 1.70 μm in size. Figure 2(Images a and b show the morphological diagrams of two conidia). Molecular phylogenetic identification was performed using ITS sequence analysis. The ITS gene sequence of the *Metarhizium anisopliae* strain Mp001 is shown in SEQ ID NO. 1. Multiple sequence alignment results are as follows: Figure 3 As shown, the strain has the highest similarity to Metarhizium pemphigi BUM39.4, and is therefore classified as Metarhizium galliformis and named strain Mp001.

[0044] SEQ ID NO.1:

[0045] .

[0046] Example 2: Study on sporulation characteristics of strain Mp001

[0047] 1. Materials

[0048] 1.1 Sporulation rate determination

[0049] The conidia produced by the purified Metarhizium anisopliae strain Mp001 were prepared into 1.0 x 10⁻⁶ spores using 0.1% Tween-80. 7Spores / mL suspension. A 5 mm diameter sterile filter paper was placed in the center of an SDA plate. 5 μL of spore suspension was inoculated onto the filter paper dropwise using a pipette. The plates were incubated at 26 ± 1 °C, 75% ± 5% relative humidity, and a photoperiod L:D = 14 h:10 h, with each incubation repeated three times. On day 7 post-inoculation, the colony diameter was measured using calipers, and the average value was taken using the cross-sectional method. The mycelial growth rate was calculated using the growth diameter of the *Metarhizium anisopliae* strain Mp001. Simultaneously, four mycelial discs were punched from the colony at half the distance from the center using a 5 mm diameter punch, arranged in a cross-shaped pattern from each colony. These discs were placed in 5 ml of 0.1% Tween-80 sterile water and shaken to ensure complete spore dispersion. The spore concentration was determined using a hemocytometer and converted to a unit area (mm²). 2 The sporulation yield of each strain was measured three times.

[0050] 1.2 Spore germination rate determination

[0051] The conidial powder cultured for 7 days was scraped into a centrifuge tube containing 0.05% Tween-80 solution, vortexed to mix, and then counted using a hemocytometer. The conidial concentration was adjusted to 1×10⁻⁶. 8 Spores / mL. Spread 100 μL of the above conidial solution evenly onto SDAY medium and incubate at 28°C. After 16 h of incubation, randomly cut off square sections of medium with sides of 1 cm and observe them under a microscope. Germination is considered complete when the germ tube length is greater than half the length of the spore itself. Spore germination rate = (number of germinating spores / total number of conidia) × 100%. Each set of 100 spores is considered a replicate, and three biological replicates are set up for each group.

[0052] 2 Results

[0053] 2.1 Spore production and spore germination rate of the strain

[0054] Table 4 shows that the growth rate of the Metarhizium anisopliae strain Mp001 was 2.59 cm / d, and the sporulation yield was 1.13 x 10⁻⁶. 5 pcs / cm 2 It has a high germination rate (95.23%) and is easy to cultivate in large quantities in a short period of time.

[0055] Table 4. Spore growth rate, sporulation yield, and spore germination rate of the strains.

[0056]

[0057] Example 3: Study on the lethal time effect of strain Mp001 on western flower thrips

[0058] 1. Materials

[0059] 1.1 Test Insects

[0060] Western flower thrips were continuously reared for several generations indoors by the Hunan Provincial Key Laboratory of Plant Diseases and Insect Pests Biology and Control in an intelligent artificial incubator (RG-300) with a temperature of 26±1℃, relative humidity (RH) of 75±5%, and a photoperiod L:D = 14h:10h. Fresh green beans were used for bioassays.

[0061] 1.2 Preparation of spore suspension

[0062] The isolated strain was inoculated onto SDA solid medium and cultured in an incubator at 26±1℃, 75%±5% relative humidity, and a photoperiod L:D = 14 h:10 h. After a large number of conidia were produced (approximately 7 days), conidia were scraped from the surface of the medium using a sterile inoculation loop and placed in a sterile aqueous solution of 0.05% Tween-80 to prepare a 1.0×10⁻⁶ saturated solution. 4 -1.0×10 8 A spore suspension of 1 spore / mL.

[0063] 1.3 Spraying method

[0064] Healthy western flower thrips nymphs that hatched on the same day, were of uniform size, and inoculated using a spray method. After inoculation, the nymphs were placed in insect rearing boxes, and cowpeas were added daily. Each experiment had 5 replicates, with 30 nymphs per treatment replicate. Larval mortality and infection were observed daily after inoculation. Dead nymphs were promptly removed and placed on 9 cm diameter SDA solid medium, and incubated in a light incubator at (26±1)℃, (75%±5%) relative humidity, and L:D=14 h:10 h to determine if the bacteria growing on the nymphs were from the inoculated treatment.

[0065] 2 Results

[0066] 2.1 Survival curve of western flower thrips

[0067] Strain Mp001 exhibited good lethality against western flower thrips nymphs at different concentrations, and the survival rate of larvae gradually decreased with increasing spore concentration. Figure 4 In western flower thrips nymphs inoculated with different concentrations of spore suspension, the fungus showed varying degrees of death on the second day, and on the third day, 1×10 8 At a spore / mL concentration, the incidence of western flower thrips nymphs decreased to 27.78%, demonstrating a highly effective insecticidal effect.

[0068] 2.2 Lethal time effect of Metarhizium anisopliae Mp001 on western flower thrips

[0069] The infection and pathogenicity time of strain MP001 in cotton bollworm larvae decreased with increasing inoculation concentration (Table 5). At inoculation concentrations of 1.0 x 10⁻⁶, the inoculation time was significantly shorter. 5 1.0 x 10 6 1.0 x 10 7 and 1.0 x 10 8 At spore concentrations of [missing value] / mL, the median LT50 for larval mortality was 11.956 d, 9.384 d, 7.314 d, and 2.682 d, respectively. This indicates that strain MP001 possesses strong infectivity and pathogenicity against western flower thrips worms.

[0070] Table 5. Inoculation with different concentrations of Metarhizium anisopliae strain Mp001.

[0071]

[0072] In summary, the *Metarhizium anisopliae* strain Mp001 provided by this invention exhibits rapid growth, high spore production in a short time, and a high spore germination rate, demonstrating a strong lethal effect on western flower thrips nymphs. *Metarhizium anisopliae* Mp001 is a biocontrol fungus suitable for the green control of western flower thrips nymphs, characterized by strong pathogenicity against western flower thrips, no environmental pollution, and low likelihood of pest resistance, making it widely applicable for the biological control of western flower thrips.

[0073] The above embodiments are only described as preferred embodiments of the present invention and are not intended to limit the scope of the present invention.

Claims

1. A sporulating strain of *Metarhizium pemphigi*, characterized in that, It is strain Mp001 of Metarhizium anisopliae, with the preservation number CCTCC No. M 20252118.

2. A method for culturing the sporulating Metarhizium anisopliae strain according to claim 1, characterized in that, The sporulating Metarhizium anisopliae strain Mp001 was inoculated onto SDA solid medium and cultured until it produced a large number of conidia, at which point a spore suspension was prepared.

3. The cultivation method according to claim 2, characterized in that, The cultivation conditions were: temperature 26±1℃, relative humidity 75%±5%, and photoperiod L:D=14 h:10 h.

4. The cultivation method according to claim 2, characterized in that, The method for preparing spore suspensions is as follows: conidia are added to a sterile aqueous solution of Tween-80 with a concentration of 0.05% to prepare spore suspensions of different concentrations.

5. An insecticide, characterized in that, The insecticide contains conidia of the spore-producing Metarhizium anisopliae strain as described in claim 1.

6. The insecticide according to claim 5, characterized in that, The insecticide contains 1.0 x 10^6 conidia of the sporulating Metarhizium anisopliae strain. 4 -1.0x10 8 Spores / mL.

7. The application of the spore-producing Metarhizium anisopliae strain of claim 1, or the spore suspension prepared by any of the culture methods of claims 2-4, or the insecticide of any one of claims 5-6 in the control of western flower thrips.

8. A method for controlling western flower thrips, characterized in that, The insecticide according to any one of claims 5-6 is applied to crops infested with western flower thrips when the western flower thrips are in the nymph stage.

9. The prevention and control method according to claim 8, characterized in that, The crop in question is a chili pepper, tomato, or tobacco.

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