High-toxicity metarhizium anisopliae strain CIPPMA362 resistant to high temperature and ultraviolet stress and application thereof

Abstract

The invention relates to the field of insect prevention and control, in particular to a high-toxicity metarhizium anisopliae strain IPPMA362 resistant to high temperature and ultraviolet stress and application thereof. The invention aims to screen excellent metarhizium anisopliae strains with high toxicity to bemisia tabaci. Through indoor biological activity determination, the lethal ability of the target strain on the bemisia tabaci nymphs is determined; further, key biological characteristics such as sporulation capability, spore germination rate and ultraviolet resistance of the strain are systematically evaluated, so that the application potential of the strain is judged; and the optimized strain is accurately identified through a molecular biology means, so that a foundation is laid for subsequent strain registration, patent protection and product development.

Description

Technical Field

[0001] This invention relates to the field of insect control, specifically to the highly virulent Metarhizium anisopliae strain CIPPMA362, which is resistant to high temperatures and ultraviolet stress, and its applications. Background Technology

[0002] Whiteflies ( Bemisia tabaci Metarhizium anisopliae is a major agricultural pest with a global distribution. Biological control, due to its high specificity, environmental safety, and low likelihood of resistance development, is considered a key approach to reducing the use of chemical pesticides. Among these, the use of entomopathogenic fungi for biological control is a research hotspot in this field. Entomopathogenic fungi, such as those in the genus *Metarhizium* (… Metarhizium ) and Beauveria bassiana ( Beauveria Fungi (or entomopathogenic fungi) are an important class of entomopathogenic fungi. They transmit conidia to insects, which germinate and penetrate the insect's body wall under suitable conditions. They then multiply rapidly within the hemocoel, ultimately causing the host's death through competition for nutrients, mechanical damage, and toxin production. This unique infection mechanism makes them effective against piercing-sucking pests such as whiteflies and aphids, which are often insensitive to many chemical pesticides and stomach poisons due to their habitat on the undersides of leaves or their protective waxy layer.

[0003] Among numerous entomopathogenic fungi, *Metarhizium anisopliae* is highly favored for its wide host range, strong environmental adaptability, and stable pathogenicity. Different strains of *Metarhizium anisopliae* exhibit significant differences in pathogenicity, environmental adaptability, and reproductive characteristics against specific pests due to variations in their genetic background. Therefore, screening for specific strains with high pathogenicity against whiteflies and systematically evaluating their key biological characteristics are fundamental and prerequisites for developing highly effective *Metarhizium anisopliae* microbial pesticides. Summary of the Invention

[0004] The purpose of this invention is to provide a highly virulent strain of Metarhizium anisopliae that is resistant to high temperatures and ultraviolet stress.

[0005] Another object of the present invention is to provide the application of the above-mentioned Metarhizium anisopliae strain.

[0006] According to the present invention, Metarhizium anisopliae ( Metarhizium anisopliae The strain CIPPMA362 has the accession number CGMCC No.41841.

[0007] This invention provides the above-mentioned Metarhizium anisopliae ( Metarhizium anisopliae Application in the control of tobacco whiteflies.

[0008] This invention screens out Metarhizium anisopliae (a type of beetle). Metarhizium anisopliae CIPPMA362 exhibits high pathogenicity against whitefly nymphs, with a corrected mortality rate of 84.29% in indoor virulence assays and a median lethality of only 4.472 days. This strain also shows advantages in growth rate (2.45 cm / d) and sporulation yield (9.4 × 10⁻⁶). 5spores / cm 2 CIPPMA362 exhibits the best performance in biological characteristics, including spore germination rate (95.3%). Furthermore, it demonstrates significant resistance to environmental stress; after 60 minutes of UV irradiation, its relative germination rate remains at 73.6%, and after 30 minutes of high-temperature stress at 40℃, the relative germination rate reaches 51.4%, both of which are significantly superior to other tested strains (P<0.0001). In summary, CIPPMA362 is a biocontrol potential strain possessing high virulence, good reproductive capacity, and environmental adaptability, providing a reliable microbial resource for the green control of whiteflies. Attached Figure Description

[0009] Figure 1 The following data show the mortality rates of whiteflies treated with different biocontrol strains: AC Metarhizium anisopliae strain and DF Beauveria bassiana strain. Figure 2 Displays CIPPMA362 multiple sequence analysis.

[0010] The *Metarhizium anisopliae* strain CIPPMA362 of this invention was deposited on March 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), and is classified as *Metarhizium anisopliae*. Metarhizium anisopliae The accession number is CGMCC No.41841. Detailed Implementation Example 1: Isolation and Purification of Strains

[0011] Locust larvae and other diseased Lepidoptera and Hemiptera larvae collected from Hebei, Shandong, Inner Mongolia, Beijing, and other regions were inoculated onto SAY medium (40 g glucose, 10 g peptone, 10 g yeast extract powder, 20 g agar, and distilled water to a final volume of 1 L, sterilized at 121°C under high pressure for 15 min) using an inoculation needle in a sterilized laminar flow hood. The culture dishes were then incubated in a constant temperature incubator at 28°C, 75% relative humidity, and a photoperiod of 14 L:10 D. Once single colonies have grown on the plates, select uncontaminated hyphae or spores and transfer them to a new Potato Glucose Yeast Agar (PDAY) medium (200g peeled potatoes boiled in distilled water and filtered to obtain juice, 5g yeast extract powder, 20g sucrose, 15g agar powder, and distilled water to a final volume of 1L, sterilized at 121℃ under high pressure for 15 min) for purification culture. Repeat this process until colonies with consistent morphological characteristics grow on the plates, and number the strains as shown in Table 1 below. After the purified strains have fully sporulated, inoculate them onto slant agar plates and store them at 4℃.

[0012] Table 1. Strain Information strain number strains Storage location CIPPMA362 IPPCAAS IPPMA981 IPPCAAS IPPMA2090 IPPCAAS IPPBb264 IPPCAAS IPPBbJ9 IPPCAAS IPPBbXM12 IPPCAAS . Example 2 Indoor Insecticide Activity Determination

[0013] Bioassays were performed using the Potter spray tower precision spray method. The concentration of pre-prepared spore suspensions for each tested strain was uniformly adjusted to 1×10⁻⁶. 8 Spores / mL (diluted with sterile water containing 0.05% Tween 80). Select tomato leaves with uniform growth and insect infestation, each infested with 15 healthy, uniformly aged second-instar nymphs of the whitefly. Place the petri dish containing the leaves flat in the center of the sample tray of the spray tower. Add 5 mL of the spore suspension to the spray tower's storage tank and spray at a constant pressure of 1 kg / cm², ensuring the droplets settle evenly on the leaf and insect surfaces. After spraying, remove the leaves and allow them to air dry at room temperature for approximately 10 minutes, then transfer them to prepared transparent insect rearing boxes (lined with moist filter paper at the bottom for moisture retention). Each treatment was replicated in triplicate, with 45 insects per treatment, and a control group with the same number of insects was included. All rearing boxes were placed in an artificial climate chamber and cultured at 28°C, 75% relative humidity, and a photoperiod of 14 L:10 D.

[0014] The number of dead whitefly nymphs was investigated and recorded daily for 7 consecutive days after treatment. Mortality was determined by lightly touching the nymph's body with a fine brush needle; if there was no reaction on the nymph's legs and stylet, it was considered dead. The cumulative mortality rate was calculated based on the collected data, and the corrected mortality rate for each treatment was also calculated to evaluate the indoor insecticidal activity of different strains.

[0015] The results of indoor toxicity assays of different strains against whiteflies are shown in Table 2. The study found that the six tested strains exhibited varying degrees of pathogenicity against whitefly nymphs, but significant differences existed among the strains (P<0.05). After 7 days of treatment, the *Metarhizium anisopliae* strain CIPPMA362 showed the strongest insecticidal activity, with the highest corrected mortality rate of 84.29%, significantly higher than the other tested strains (Table 2). Figure 1 Among the Beauveria bassiana strains, IPPBb264 exhibited relatively strong virulence, with a corrected mortality rate of 11.27%. Furthermore, strain CIPPMA362 showed the highest median lethal time (LT). 50 The shortest value is 4.472, indicating that it has the fastest lethal speed.

[0016] Table 2. Lethality of different biocontrol strains against whiteflies (7 days) .

[0017] Molecular identification was performed on the highly virulent strain CIPPMA362 selected through screening. An ITS sequence of approximately 543 bp was obtained by amplification using ITS primers. The sequencing results were then BLAST-aligned in the GenBank database, and a phylogenetic tree was constructed. Figure 2 The results showed that strain CIPPMA362 was similar to Metarhizium anisopliae (…). Metarhizium anisopliae The MeanHN25B01 strain clustered on the same evolutionary clade, with 91% sequence identity, and was identified as *Metarhizium anisopliae*. Metarhizium anisopliae ). Example 3: Biological characteristics and sporulation yield determination of the strain

[0018] To evaluate the reproductive capacity and production potential of the tested strains, the growth rate and sporulation yield of three *Metarhizium anisopliae* strains (CIPPMA362, IPPMA981, IPPMA2090) and three *Beauveria bassiana* strains (IPPBb264, IPPBbJ9, IPPBbXM12) were determined. Conidia of each strain were inoculated into the center of PDAY agar plates using the spot inoculation method, with a cross drawn at the center point. After inoculation, the plates were incubated at 28°C, and the colony diameter was measured daily, along with the colony growth rate. Simultaneously, the tested strains were transferred to the center of PDAY plates for activation and incubated at 28°C for 7 days until full sporulation. Sampling was performed using the perforation method, with five mycelial discs taken at equal intervals at half the radius of a concentric circle from the edge to the center of each colony. Each individual mycelial disc was carefully transferred to a centrifuge tube containing 10 mL of 0.05% Tween 80 solution. Five sterile glass beads were added to each disc, and the tube was vortexed until all spores on the surface of the disc were eluted. The resulting spore suspension was filtered to remove mycelium and culture medium debris. A series of serial dilutions with 0.05% Tween 80 solution were then performed until the estimated spore count was within a suitable range under an optical microscope. Spore counts were then performed using a hemocytometer. Three replicates were performed, and the average value was taken. The formulas for calculating colony growth rate and sporulation yield are as follows: Colony growth rate (cm / d) = new colony diameter / number of growth days; Sporulation yield (spores / cm²) = (average number of spores × dilution factor × total volume of suspension) / (number of mycelial cakes × area of ​​a single mycelial cake). The area of ​​a single mycelial cake is calculated as 0.5024 cm² based on the inner diameter of the punch (0.8 cm).

[0019] The growth rate, sporulation yield, and germination rate of three *Metarhizium anisopliae* strains (CIPPMA362, IPPMA189, IPPMA2090) and three *Beauveria bassiana* strains (IPPBb264, IPPBbJ9, IPPBbXM12) were systematically determined, and the results are shown in Table 3. The results showed that *Metarhizium anisopliae* CIPPMA362 had the highest growth rate (2.45 cm / d), significantly higher than all other strains (P<0.0001); the overall growth rate of *Metarhizium anisopliae* was significantly higher than that of *Beauveria bassiana* (P<0.0001). Sporulation yield determination showed that *Metarhizium anisopliae* CIPPMA362 produced the highest sporulation yield (9.4 × 10⁻⁶). 5 spores / cm 2 The sporulation capacity of *Metarhizium anisopliae* was significantly superior to that of *Beauveria bassiana* (P<0.0001). In germination rate analysis, *Metarhizium anisopliae* CIPPMA362 had the highest germination rate (95.3%), significantly higher than other strains (P<0.0001); the overall germination rate of *Metarhizium anisopliae* was also significantly higher than that of *Beauveria bassiana* (P<0.0001). *Beauveria bassiana* IPPBb264 significantly outperformed other strains in all three indicators (P<0.05). In summary, *Metarhizium anisopliae* generally performed significantly better than *Beauveria bassiana*, with strain CIPPMA362 exhibiting the best potential in all indicators. Example 4 Determination of bacterial germination rate

[0020] To assess the spore viability and infectivity potential of the tested strains, the germination rate of each strain was determined. Three strains of *Metarhizium anisopliae* (CIPPMA362, IPPMA981, IPPMA2090) and three strains of *Beauveria bassiana* (IPPBb264, IPPBbJ9, IPPBbXM12) were transferred to PDAY plates and incubated at 28°C for 7 days until abundant conidia were produced. Fresh conidia were gently scraped from the plate surface and dissolved in 0.05% Tween 80 solution, filtered to remove hyphal fragments, and prepared into a 1×10⁻⁶ concentration. 8 A spore suspension was prepared at a concentration of spores / mL, and the concentration was calibrated using a hemocytometer. Germination was tested using the plate dilution method. 100 μL of spore suspension was evenly spread onto a solid culture medium plate, with three replicates per strain. After incubating the plates at 28℃ for 20 h, the plates were observed under an optical microscope at randomly selected fields of view. Spores clearly showing germination tubes were considered germinated or viable. The spore germination rate was calculated using the following formula: Germination rate (%) = (Number of germinating spores / Total number of observed spores) × 100%. The final result is expressed as the mean of three replicates ± standard error.

[0021] Table 3. Growth rate, sporulation yield, and spore germination rate of different biocontrol strains . Example 5: Determination of UV resistance of strains

[0022] To assess the persistence of the tested strains in the field, the resistance of their conidia to ultraviolet radiation was determined. Spore powder was scraped from six tested strains after 7 days of culture and prepared into a 1×10⁻⁶ solution using 0.05% Tween 80. 8 A spore suspension of 1 spore / mL was prepared. 10 mL of the bacterial suspension was placed in a 9 cm Petri dish and irradiated with a 30W UV lamp at a distance of 30 cm for 15, 30, 45, and 60 min. 10 μL of the suspension was then spread onto PDAY agar plates and incubated in the dark for 3 days. Colony counts were then performed. Each treatment was repeated in triplicate, with the non-UV irradiated group serving as a control. Germination was assessed using the plate dilution method, and the spore germination rate was calculated for different irradiation times.

[0023] Evaluation of UV resistance: Using the germination rate of spores without UV irradiation as a baseline (100%), the relative germination rates at each time point were calculated using the following formula: Relative germination rate (%) = (germination rate after irradiation / control germination rate) × 100%.

[0024] The environmental durability of the strains was evaluated by measuring the relative germination rate of spores after different durations of UV irradiation. As shown in Table 4, the relative germination rate of all strains decreased with increasing UV irradiation time. Strain CIPPMA362 maintained a relative germination rate of 73.6% after 60 min of irradiation, which was significantly higher than that of all other tested strains (P<0.0001).

[0025] Table 4. Relative germination rates of spores from different strains after different UV irradiation times. . Example 6: Screening and determination of the heat resistance phenotype of strains

[0026] To evaluate the survival and adaptability of the tested strains under high temperature stress, their growth phenotypes under high temperature were screened, and the heat resistance characteristics of their conidia were determined.

[0027] Six mycelial blocks of the tested strains were punched using a 5 mm punch and aseptically inoculated into the center of fresh PDAY solid medium. The transferred plates were then incubated for 3 days in constant temperature incubators at 28℃ (control), 30℃, 35℃, and 40℃, respectively. After the high-temperature stress treatment, the plates were immediately transferred to a 28℃ constant temperature environment. Growth status was observed daily thereafter, and the recovery of mycelial growth after stress was systematically recorded. Colony diameter was measured using the cross-crossing method, and the relative diameter increase of *Cordyceps sinensis* fungus was calculated based on the colony diameter. The formula for calculating the relative increase is as follows: relative growth rate SR (%) = ( S A -5) / ( S 0 -5) × 100%. Wherein S A The average diameter (mm) of the sample group; S 0 The average diameter (mm) of the control group.

[0028] Subsequently, after 7 days of culture, spores from the six tested strains were scraped off and diluted with 0.05% Tween 80 solution to prepare a solution with a concentration of 1×10⁻⁶. 8 A spore suspension of 1 spore / mL was prepared, and the concentration was calibrated using a hemocytometer. Two mL of the spore suspension was aliquoted into sterile centrifuge tubes and placed in pre-set water baths at four temperature gradients (28℃ (control), 30℃, 35℃, and 40℃) for 30 min each. Immediately after treatment, the tubes were cooled on ice for 5 min to terminate the heat stress effect. Each treatment was repeated three times. The treated spore suspensions were then subjected to a germination test using the plate dilution method, and the spore germination rate at different irradiation times was calculated. The heat tolerance was evaluated using the spore germination rate at 25℃ as a baseline (100%). The relative germination rate after each temperature treatment was calculated using the following formula: Relative germination rate (%) = (germination rate after temperature treatment / germination rate of 25℃ control) × 100%.

[0029] The strain's tolerance to high temperatures is key to its adaptation to the summer field environment. The results, shown in Table 5, indicate that the relative germination rate of all strains decreased significantly with increasing treatment temperature. After 30 min of high-temperature stress at 40℃, strain CIPPMA362 still maintained a relative germination rate of 51.4%, demonstrating significantly better heat tolerance than all tested strains (P<0.0001). Some strains showed a significant decrease in germination rate at 35℃, indicating that CIPPMA362 has a significant advantage in coping with high-temperature stress.

[0030] Table 5. Relative germination rates of spores of various strains after different temperature stresses. .

[0031] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. Metarhizium anisopliae Metarhizium anisopliae strain CIPPMA362 is characterized by, Its accession number is CGMCC No.41841.

2. The Metarhizium anisopliae according to claim 1 ( Metarhizium anisopliae Application of strain CIPPMA362 in pest control.

3. The application according to claim 2, characterized in that, The pest in question is the whitefly.

4. A method for controlling whiteflies, characterized in that, The method includes spraying the whitefly with the Metarhizium anisopliae as described in claim 1. Metarhizium anisopliae Steps for obtaining spore powder of strain CIPPMA362.

5. A biological agent for controlling whiteflies, characterized in that, The biological agent includes the *Metarhizium anisopliae* as described in claim 1. Metarhizium anisopliae ) Spore powder of strain CIPPMA362.

Images