Beauveria bassiana strain bb-yy for preventing and controlling multiple pests, and derivative products and applications thereof

Abstract

The application provides a Beauveria bassiana strain Bb-YY for preventing and controlling various diseases and pests, a derivative product and application thereof, and belongs to the technical field of functional microorganisms. Beauveria bassiana The strain Bb-YY is screened from the rhizosphere soil of a peanut healthy plant planted in a serious continuous cropping obstacle plot, and is identified as Beauveria bassiana (Bb) by molecular identification, has high efficient prevention and control effects on white grubs, peanut fruit rot pathogens and peanut root rot pathogens, and has certain inhibition effects on Bacterospora coriacea causing tobacco black foot disease and Pyricularia oryzae causing rice blast. It can be seen that the Bb-YY strain can also play a prevention and control effect on tobacco, rice and other crops in addition to peanut planting, and can effectively reduce the use of chemical pesticides, and provides a strong support for green prevention and control of peanut diseases and pests.

Description

Technical Field

[0001] This application belongs to the field of functional microbial technology, specifically relating to a Beauveria bassiana strain that controls various diseases and pests. Beauveria bassiana ) strain Bb-YY and its derivatives and applications. Background Technology

[0002] Peanuts are an important oilseed and cash crop, playing a vital role in ensuring food and oil security. However, peanut cultivation is susceptible to various pests and diseases. The green grub (Gnaphalium affine) is a significant pest in peanut production, feeding on roots and pods, causing seedling loss, pod rot, and reduced yield. It also creates numerous wounds, exacerbating pathogen infection. Peanut root rot leads to root decay, plant wilting, and death, while peanut pod rot causes moldy pods and reduced quality. When these three diseases occur together in continuously cropped fields, a vicious cycle of "pests promoting diseases, and diseases exacerbating yield reduction" is created, seriously threatening peanut production. Therefore, peanut production faces multiple pest and disease threats, necessitating the screening of functional biocontrol bacteria with both pest and disease control capabilities for application in peanut pest and disease management.

[0003] Beauveria bassiana is a broad-spectrum entomopathogenic fungus that plays an important role in the biological control of pests. However, there is currently no Beauveria bassiana that can simultaneously control both insect and fungal diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a Beauveria bassiana strain Bb-YY, which not only has a control effect on a variety of peanut pests, but also has a highly effective control effect on a variety of peanut pathogens.

[0005] This invention provides a Beauveria bassiana strain Bb-YY, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20252340.

[0006] This invention provides a method for culturing the Beauveria bassiana strain Bb-YY, comprising the following steps: Seed culture of Beauveria bassiana strain Bb-YY was inoculated into fermentation medium and fermented to obtain fermentation product; The fermentation medium includes a basal medium containing the following components in the indicated mass percentages: 23%–27% corn flour, 18%–22% soybean meal, 4%–6% rice flour, and 7%–9% fish meal.

[0007] Preferably, the basal culture medium comprises wheat bran and straw; the water content of the basal culture medium is 40% to 60%. The mass ratio of wheat bran to straw is 3.5~4.5:1.

[0008] This invention provides a biocontrol agent comprising the Beauveria bassiana strain Bb-YY or the fermentation product and excipients prepared by the culture method.

[0009] Preferably, the Beauveria bassiana strain Bb-YY comprises hyphae and / or spores.

[0010] Preferably, the mass percentage of the Beauveria bassiana strain Bb-YY is 5% to 90%.

[0011] This invention provides a biological compound formulation, comprising the aforementioned biocontrol agent and at least one of the following: a peanut pest and disease insecticide, a peanut fungicide, a pesticide, and a bio-organic fertilizer.

[0012] This invention provides the application of the Beauveria bassiana strain Bb-YY, the biocontrol agent, or the biological compound preparation in the control of insect pests and / or fungal diseases.

[0013] Preferably, the pests include diseases and pests caused by the green scarab beetle; The fungal diseases include those caused by at least one of the following fungi: Fusarium oxysporum, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, and Pyrethrum indica.

[0014] Preferably, the fungal diseases include at least one of the following: peanut root rot, peanut leaf spot, peanut fruit rot, tobacco black shank, and rice blast fungus.

[0015] This invention provides a Beauveria bassiana strain Bb-YY, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20252340. The strain Bb-YY was obtained from the rhizosphere soil of healthy peanut plants grown in fields with severe continuous cropping obstacles, and molecularly identified as belonging to Beauveria bassiana (…). Beauveria bassiana This invention demonstrates highly effective control of grubs, peanut fruit rot pathogens, and peanut root rot pathogens. Simultaneously, the Bb-YY strain also exhibits good inhibitory effects against *Fusarium solani*, which causes tobacco black shank disease, and a certain degree of inhibitory effect against *Pyrrosia lingua*, which causes rice blast. Therefore, the Bb-YY strain provided by this invention can achieve synergistic control of multiple pests and diseases not only in peanut cultivation but also in crops such as tobacco and rice. Biocontrol agents formulated from the Bb-YY strain can effectively reduce the use of chemical pesticides, providing strong support for the green control of peanut pests and diseases.

[0016] This invention also provides a method for culturing the *Beauveria bassiana* strain Bb-YY. The composition of the fermentation medium for *Beauveria bassiana* strain Bb-YY was optimized. The optimal composition of the basal medium was determined to be a mixture of wheat bran and wheat straw. Adding specific amounts of corn flour, soybean meal, rice flour, and fish meal to the basal medium resulted in the highest sporulation yield, reaching 2.14 × 10⁻⁶ sporulations after 10 days of cultivation. 10 The spores / g provide a material basis for the next step in the preparation of biocontrol agents. Attached Figure Description

[0017] Figure 1 The colony morphology of the isolated Beauveria bassiana strain Bb-YY is shown in the image. Figure 2 The results of bioassay analysis of Beauveria bassiana strain Bb-YY on larvae of the green scarab beetle are shown, where A represents 10. 6 / mL of spore solution was used to soak dead grubs; B is the control group of grubs; Figure 3 Results of the lethality of different concentrations of Beauveria bassiana strain Bb-YY on larvae of the green scarab beetle; P <0.05, P <0.01; Figure 4 Comparative results of the lethal effects of different strains of Beauveria bassiana on larvae of the African scarab beetle; P <0.05; Figure 5 The results of the evaluation of the antifungal effect of Beauveria bassiana strain Bb-YY against five pathogenic fungi are shown below. A is Fusarium oxysporum control; B is the antifungal effect of Beauveria bassiana strain Bb-YY against Fusarium oxysporum; C is Fusarium solani control; D is the antifungal effect of Beauveria bassiana strain Bb-YY against Fusarium solani; E is Colletotrichum gloeosporioides control; F is the antifungal effect of Beauveria bassiana strain Bb-YY against Colletotrichum gloeosporioides; G is Alternaria alternata control; H is the antifungal effect of Beauveria bassiana strain Bb-YY against Alternaria alternata; I is Pyrrosia lingua control; J is the antifungal effect of Beauveria bassiana strain Bb-YY against Pyrrosia lingua. Figure 6 The results of Beauveria bassiana strain Bb-YY controlling peanut root rot.

[0018] Information on the preservation of biological materials A type of Beauveria bassiana ( Beauveria bassianaThe strain Bb-YY is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China. The deposit date is October 24, 2025, and the accession number is CCTCC NO: M 20252340. Detailed Implementation

[0019] This invention provides a Beauveria bassiana strain Bb-YY, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252340.

[0020] In this invention, the Beauveria bassiana strain Bb-YY was isolated from the rhizosphere soil of healthy peanut plants grown in fields with severe continuous cropping obstacles. Molecular identification based on ITS confirmed that it belongs to the Beauveria bassiana family (B. bassiana). Beauveria bassiana The efficacy of strain Bb-YY in controlling *S. aeruginosa* larvae was evaluated. Results showed that strain Bb-YY exhibited good lethality against *S. aeruginosa* larvae, with a mortality rate dependent on spore concentration and time. After 10 days of treatment, the mortality rate was 10%. 8 The spore concentration of / mL achieved a mortality rate of over 80% against the larvae of the green scarab beetle, and its control effect was significantly superior to that of strain Bb-CGMCC-08F04 and the commercial strain Bb-ZK-7680. Meanwhile, strain Bb-YY exhibits inhibitory effects against pathogens of various fungal diseases. For example, using five plant pathogenic fungi as experimental subjects—Fusarium oxysporum causing peanut root rot, Alternaria alternata causing peanut leaf spot, Colletotrichum gloeosporioides causing peanut fruit rot, Fusarium solani causing tobacco black shank, and Pyrrosia oryzae causing rice blast—Bb-YY's inhibitory effect was evaluated. The results showed that strain Bb-YY, compared to strains Bb-CGMCC-08F04 and Bb-ZK-7680, showed stronger inhibitory effects against Fusarium oxysporum, Fusarium solani, Colletotrichum gloeosporioides, Alternaria alternata, and Pyrrosia oryzae. Overall, it exhibited stronger inhibitory effects against Anthracnose and Fusarium species, and relatively weaker inhibitory effects against Alternaria and Pyrrosia oryzae.

[0021] This invention provides a method for culturing the Beauveria bassiana strain Bb-YY, comprising the following steps: Seed culture of Beauveria bassiana strain Bb-YY was inoculated into fermentation medium and fermented to obtain fermentation product; The fermentation medium includes a basal medium containing the following components in the indicated mass percentages: 23%–27% corn flour, 18%–22% soybean meal, 4%–6% rice flour, and 7%–9% fish meal.

[0022] In this invention, the preferred method for preparing the seed culture of the Beauveria bassiana strain Bb-YY is to inoculate Bb-YY mycelial cakes into PD liquid medium and culture by shaking. Three mycelial cakes are inoculated per 100 mL of PD liquid medium. The diameter of the mycelial cakes is preferably 4-6 mm, and can be 5 mm. The shaking speed is preferably 160-200 rpm, and can be 180 rpm. The shaking temperature is preferably 23-27℃, and can be 25℃. The shaking time is preferably 45-50 h. The spore concentration in the seed culture of the Beauveria bassiana strain Bb-YY is preferably (0.5-10) × 10⁻¹⁰. 8 spores / mL, which can be 10 8 Spores / mL.

[0023] In this invention, the basal culture medium preferably comprises wheat bran and straw. The mass ratio of wheat bran to straw is preferably 3.5–4.5:1, and can be 4:1. The straw can be crop straw, such as wheat straw, rice straw, corn straw, etc. The water content of the basal culture medium is preferably 40%–60%, and can be 50%. This invention adjusts the composition of the basal culture medium. Compared with straw and wheat bran alone, the basal culture medium formed by mixing wheat bran and wheat straw is more conducive to increasing spore formation. Furthermore, compared with a wheat bran and straw mass ratio of 2:1, a mixture of wheat bran and straw with a mass ratio of 4:1 is more conducive to increasing spore formation.

[0024] In this invention, the fermentation culture medium preferably contains 24% to 26% corn flour, which can be 25% by weight. Soybean meal is preferably 18% to 22%, which can be 19% to 21%, or 20%. Rice flour is preferably 4% to 6%, which can be 5%. Fish meal is preferably 7% to 9%, which can be 8%.

[0025] In one embodiment of the present invention, the carbon and nitrogen sources in the fermentation medium were optimized and screened. Corn flour, wheat flour, rice flour, or glucose were used as supplementary carbon sources, and soybean meal, broad bean flour, fish meal, or yeast powder were used as supplementary nitrogen sources. Single-factor experiments were conducted, and the results showed that corn flour and soybean flour had sporulation advantages compared to wheat flour and glucose, and could be used as suitable supplementary nitrogen sources. Similarly, soybean meal and fish meal had sporulation advantages compared to fish meal and yeast powder, and could be used as suitable supplementary carbon sources. Furthermore, the present invention conducted orthogonal experiments on the optimized supplementary nitrogen sources and their ratios. The results showed that the factors affecting the sporulation yield of the Bb-YY strain during solid-state fermentation, from largest to smallest, were soybean meal, rice flour, fish meal, and corn flour. The highest sporulation yield was achieved with 25% corn flour, 20% soybean meal, 5% rice flour, and 8% fish meal (formula 1), reaching a sporulation yield of 2.14 × 10⁻⁶. 10 The spores / g concentration is significantly higher than in other formulations.

[0026] This invention provides a biocontrol agent comprising the Beauveria bassiana strain Bb-YY or the fermentation product and excipients prepared by the culture method.

[0027] In this invention, the *Beauveria bassiana* strain Bb-YY preferably comprises hyphae and / or spores. The mass percentage of the *Beauveria bassiana* strain Bb-YY is preferably 5%~90%, more preferably 10%~80%, and can also be 20%~70%, 30%~60%, and further preferably 40%~50%. This invention does not impose any special limitations on the type and amount of excipients; excipients and amounts well-known in the art can be used.

[0028] This invention provides a biological compound formulation, comprising the aforementioned biocontrol agent and at least one of the following: a peanut pest and disease insecticide, a peanut fungicide, a pesticide, and a bio-organic fertilizer.

[0029] In this invention, the peanut pest and disease insecticide preferably includes an insecticide for controlling underground grub pests. The peanut fungal disease fungicide preferably includes a fungicide for controlling at least one of the following diseases: peanut root rot, peanut leaf spot, peanut fruit rot, tobacco black shank, and rice blast. The pesticide includes a pesticide for controlling underground grub pests or a pesticide for controlling at least one of the following diseases: peanut root rot, peanut leaf spot, peanut fruit rot, tobacco black shank, and rice blast, such as azoxystrobin. The bio-organic fertilizer preferably includes organic fertilizer.

[0030] This invention provides the application of the Beauveria bassiana strain Bb-YY, the biocontrol agent, or the biological compound preparation in the control of insect pests and / or fungal diseases.

[0031] In this invention, the insect pests preferably include those caused by the green scarab beetle, i.e., underground grub pests. The fungal diseases preferably include those caused by at least one of the following fungi: *Fusarium oxysporum*, *Alternaria alternata*, *Colletotrichum gloeosporioides*, *Fusarium solani*, and *Pyrophyllus oryzae*. The fungal diseases preferably include at least one of the following: peanut root rot, peanut leaf spot, peanut fruit rot, tobacco black shank, and rice blast fungus.

[0032] In this invention, the Beauveria bassiana strain Bb-YY exhibits significantly improved control over underground pests such as grubs, peanut root rot, peanut leaf spot, and peanut fruit rot compared to other common Beauveria bassiana strains. Moreover, it achieves the same control effect against peanut root rot as azoxystrobin.

[0033] The following detailed description, in conjunction with embodiments, illustrates a Beauveria bassiana strain Bb-YY for controlling various pests and diseases, its derivative products, and applications, but these should not be construed as limiting the scope of protection of this invention.

[0034] Example 1 A method for isolating Beauveria bassiana strain Bb-YY (1) Soil sample treatment In a town in Yuanyang, Henan, where peanuts have been grown for many years and continuous cropping obstacles are severe, healthy peanut plants were selected, the topsoil was removed, and the soil around the roots was taken, placed in sterile self-sealing bags, brought back to the laboratory at 4°C, dried in a fume hood, and then gently crushed in a sterile mortar to remove stones, residual roots, peanut shells and other impurities, and passed through a 2 mm sterile sieve.

[0035] (2) Fungal isolation Weigh 10 g of soil sample and add 90 mL of sterile water (containing 0.05% Tween 80); shake at 200 rpm for 30 min to fully disperse the spores; let stand for 10 min, then take the supernatant and dilute it with sterile water to 10 g / L. -1 10 -2 10 -3 Measure 0.1 mL of each dilution of bacterial suspension and add it dropwise onto a PDA medium plate containing 100 mg / mL streptomycin sulfate. Spread the bacterial suspension evenly across the plate surface using a sterile spreader. Seal the plate and incubate at 28°C. Observe the plate daily for colony growth. Use a sterile inoculation loop to pick fresh hyphae from the edge of a typical, contaminant-free single colony and transfer them to a new PDA medium containing 100 mg / mL streptomycin sulfate. Incubate at 28°C for 5-7 days to obtain the purified primary strain. Figure 1 ).

[0036] (3) Strain identification Molecular identification of the strains was performed using universal fungal primers. Fungal DNA was extracted using the DNAiso Reagent DNA Extraction Kit (TaKaRa). The PCR reaction system consisted of: 10 μl 2×Premix Taq™ (Ex Taq™ Version 2.0 plus dye) (TaKaRa), 0.6 μl each of forward and reverse primers (10 mM), 1 μl DNA template, and double-distilled water to a final volume of 20 μl. The PCR reaction program was: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 55℃ annealing for 10 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 10 min, and storage at 4℃. The PCR primer sequences (ITS1: 3'-TCCGTAGGTGAACCTGCGG-5', SEQ ID NO:1; ITS4: 3'-TCCTCCGCTTATTGATATGC-5', SEQ ID NO:2), primer synthesis, and PCR product sequencing were all performed by Sangon Biotech (Shanghai) Co., Ltd.The original sequence was assembled using MEGA 7.0 software. The assembled sequence (SEQ ID NO:3) was then compared with the submitted sequence using BLASTN on NCBI (http: / / www.ncbi.nlm.nih.gov / ) to find the species corresponding to the most similar sequence.

[0037] The results showed that the top five most similar species were all derived from Beauveria bassiana, with a similarity of 99% (see Table 1), proving that the isolated fungus was Beauveria bassiana. Beauveria bassiana The strain, abbreviated as Bb-YY, has been deposited at the China Center for Type Culture Collection (Wuhan) (No.: CCTCC NO: M 20252340).

[0038] Table 1. ITS sequence alignment results of Beauveria bassiana strain Bb-YY

[0039] Example 2 The control effect of Beauveria bassiana strain Bb-YY on larvae of the green scarab beetle. The Beauveria bassiana strains to be verified include Bb-YY screened in Example 1, the commercially available Bb-ZK-7680 (registration certificate number of Beauveria bassiana from Zhoukou Jinbiao Biotechnology Co., Ltd.: Microbial Fertilizer (2020) Approval No. (7680)) and Bb-CGMCC-08F04 (deposited at China General Microbiological Culture Collection Center, No. 8656).

[0040] Take healthy, uniformly sized third-instar larvae (grubs) of the green scarab beetle. Collect colonies of *Beauveria bassiana* strains (Bb-YY, Bb-ZK-7680, and Bb-CGMCC-08F04) cultured on PDA medium for 10-14 days. Wash off the spores with sterile water containing 0.05% Tween-80, filter through sterile gauze to remove hyphae, count using a hemocytometer, and adjust the concentration to 10. 8 spores / mL, 10 6 Spores / mL, with sterile water + 0.05% Tween-80 as a control. Grub larvae were immersed in spore suspensions of different concentrations for 10 seconds, excess bacterial suspension was blotted off with sterile filter paper, and they were placed in sterile petri dishes (with a layer of moist filter paper at the bottom to maintain humidity). Each replicate contained 15 larvae, reared individually, with three replicates per treatment. They were cultured at 25℃, relative humidity above 85%, in the dark, and fed small pieces of potato daily. Mortality was observed daily for 10 consecutive days; larvae were considered dead when unresponsive to gentle touch with tweezers.

[0041] See results Figure 2 10 6 The grubs treated with spore solution of / mL died, turned black and shrunken, while the grubs in the control group did not show discoloration or shrunkenness.

[0042] The detection results of Bb-YY spore suspension soaking for different times are shown in the figure. Figure 3 10 6 The average mortality rate after 3 days of spore / mL treatment was 40%, after 5 days it was 46.67%, and after 10 days it was 60.00%. 8 The average mortality rate after 3 days was 53.33%, after 5 days it was 66.67%, and after 10 days it was 80.00%. 6 The mortality rate of grubs treated with spores / mL was significantly higher than that of the control. p <0.05), while 10 8 The mortality rate of grubs treated with spores / mL was significantly higher than that of the control. p <0.01). This indicates that the Beauveria bassiana strain Bb-YY has a highly effective control effect on grubs, especially at level 10. 8 Treatment with spore solution at a concentration of / mL yields more significant results.

[0043] The lethality of different strains to *Strombus aeruginosa* larvae is shown in [reference needed]. Figure 4 In 10 8 After 10 days of treatment with spore / mL concentration, the Beauveria bassiana strain Bb-YY showed significantly higher control efficacy against grubs than strains Bb-CGMCC-08F04 and the commercially available strain Bb-ZK-7680.

[0044] Example 3 The antifungal effect of Beauveria bassiana strain Bb-YY against various pathogenic fungi. The antifungal effect of Beauveria bassiana was evaluated on five plant pathogenic fungi: Fusarium oxysporum (causing peanut root rot), Alternaria alternata (causing peanut leaf spot), Colletotrichum gloeosporioides (causing peanut fruit rot), Fusarium solani (causing tobacco black shank), and Pyrethrum orientalis (causing rice blast).

[0045] Using *Fusarium oxysporum*, *Fusarium solani*, *Colletotrichum gloeosporioides*, *Alternaria alternata*, and *Pyrrosia lingua* as target bacteria, the prepared fungal discs were placed upside down in the center of PDA plates. Different strains of *Beauveria bassiana* (Bb-YY, commercially available Bb-ZK-7680, and Bb-CGMCC-08F04) were placed upside down at the four corners of the plate, 30 mm away from the fungal discs. Plates without *Beauveria bassiana* fungal discs served as controls. All plates were incubated at 28°C. When the pathogens on the control plates had fully colonized the plates, the colony diameter was measured using the cross-sectional method, and the mycelial growth inhibition rate was calculated according to Formula I.

[0046] Mycelial growth inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - mycelial cake diameter) × 100% Formula I.

[0047] The Beauveria bassiana strain Bb-YY showed certain inhibitory effects against Fusarium oxysporum, Fusarium solani, Colletotrichum gloeosporioides, Alternaria alternata, and Pyrethrum orientalis. Overall, it exhibited stronger inhibitory effects against Anthracnose and Fusarium species, and weaker inhibitory effects against Alternaria and Blastomyces orientalis. Figure 5 ).

[0048] Table 2 shows that Beauveria bassiana Bb-YY exhibits targeted antagonistic advantages, demonstrating strong inhibitory activity against peanut fruit rot pathogens (Colletotrichum gloeosporioides), tobacco black shank pathogens (Fusarium solani), and peanut root rot pathogens (Fusarium oxysporum), which are serious pests in agricultural production. The mycelial growth inhibition rates reached 43.33%, 38.96%, and 31.25%, respectively, indicating its high application value in the control of plant diseases caused by anthracnose and Fusarium.

[0049] Table 2 Antagonistic effects of Beauveria bassiana strains against different plant pathogenic fungi

[0050] Example 4 Evaluation of the control effect of Beauveria bassiana strain Bb-YY on peanut root rot 1. Test reagent: Beauveria bassiana strain Bb-YY (10 7 spores / ml), azoxystrobin (250 ppm); 2. Target disease: Peanut root rot; 3. Control methods: Inoculate the peanut root rot pathogen, *Fusarium oxysporum*, onto sterilized wheat grains for large-scale culture, and incubate at 25℃ in the dark for 7–10 days for later use. Disinfect peanut seeds with 75% alcohol for 1 min, rinse thoroughly with sterile water, and sow in square flowerpots (19 cm × 14 cm × 10 cm), each pot containing 0.2 kg of sterilized substrate (vermiculite 1–3 mm in diameter). Place all flowerpots in a greenhouse at 25℃, 12 h light, and 70% relative humidity, watering regularly. Apply the pesticide when the peanut seedlings reach the 2–3 leaf stage. Apply 10 mg of the pesticide to each treatment. 7 Spores / mL Bb-YY bacterial suspension, 250 μg / mL azoxystrobin, 5 mL per plant for irrigation. 48 h later, 5 wheat grains pre-cultured with Fusarium oxysporum mycelium were placed at the base of the stems of each peanut plant in each treatment. Each treatment was applied once every 7 days, for a total of 2 applications. The blank control was irrigated with an equal volume of water. Each treatment consisted of 10 peanut plants, with 3 replicates. After the blank control showed full disease development, the disease incidence rate in each treatment was investigated and the control efficacy was calculated.

[0051] Disease incidence rate (%) = Number of diseased plants / Total number of plants × 100% (Formula I) Control effect (%) = (Disease rate in blank control group - Disease rate in treatment group) / Disease rate in blank control group × 100% Formula II.

[0052] Fifteen days after application, the average control effect against peanut root rot was calculated.

[0053] See results Figure 6 Beauveria bassiana strain Bb-YY (10 7 The average control efficacy of spores / ml against peanut root rot was 64.71%, while that of azoxystrobin (250 ppm) was 68.63%. Strawberry strain Bb-YY (10... 7 The average control efficacy of spores / ml against peanut root rot was comparable to that of azoxystrobin (250 ppm), and statistical analysis showed no significant difference in control efficacy between the two.

[0054] Example 5 Optimization of fermentation conditions for Beauveria bassiana strain Bb-YY 1. Preparation of seed culture: 5 mm diameter mycelial discs were collected from the edge of fresh Bb-YY colonies and inoculated into 100 mL of PD liquid medium, with 3 mycelial discs per bottle. The culture was incubated at 25 ℃ and 180 r / min for 48 h with shaking. The culture was then diluted with sterile water to obtain a spore content of 1×10⁻⁶. 8 Seed liquid with spores per mL.

[0055] 2. Screening of basic culture medium: Wheat bran, soybean meal, and wheat straw (crushed into fragments of 2-3 mm × 1-2 mm) were selected as candidate materials for fermentation experiments. The formulas are as follows: (1) wheat straw; (2) wheat bran; (3) wheat bran: wheat straw = 2:1 (wt / wt); (4) wheat bran: wheat straw = 4:1 (wt / wt). The mixture was dispensed into 250 mL conical flasks, 25 g per flask, with a water content of 50%. Seed liquid was inoculated at a 5% inoculation rate and cultured in a constant temperature incubator at 25 ℃ for 10 days. The spore production was then measured, and each treatment was repeated 3 times. The specific method for measuring spore production is as follows: After the solid fermentation product was dried at 40 ℃, 1 g of culture was weighed and 20 mL of sterile 0.1% Tween 80 was added. The mixture was broken up with glass beads, the spore liquid was collected and the volume was adjusted. The spore concentration was investigated using a hemocytometer to screen the optimal basic culture medium.

[0056] The results showed that the spore concentration in the basal medium composed of a mixture of wheat bran and wheat straw (4:10) was the highest and significantly different compared to basal media composed of wheat straw or wheat bran alone, or wheat bran and wheat straw (2:1). Therefore, a mixture of wheat bran and wheat straw (4:1, wt / wt) was selected as the optimal basal medium for promoting sporulation.

[0057] 3. Screening of carbon and nitrogen sources for solid-state fermentation medium: Using the optimal wheat bran and wheat straw mixture (4:1, wt / wt) for promoting sporulation of Bb-YY strain as the basal medium, single-factor experiments were conducted to screen for supplementary carbon and nitrogen sources. Specifically, corn flour, wheat flour, rice flour, or glucose were added at 10% of the optimal basal medium mass as supplementary carbon sources; soybean meal, broad bean flour, fish meal, or yeast powder were added at 10% of the optimal basal medium mass as supplementary nitrogen sources. Using the basal medium formula as a control, single-factor experiments were conducted to screen for the optimal supplementary carbon, nitrogen, and inorganic salts for sporulation of Bb-YY strain. Sporulation yield was investigated after 10 days.

[0058] The results showed that, in terms of carbon source screening, corn flour and rice flour had significant advantages over wheat flour, glucose, and the control in increasing sporulation. In terms of nitrogen source screening, soybean meal and broad bean flour had significant advantages over fish meal, yeast powder, and the control in increasing sporulation.

[0059] 4. Orthogonal Experiment: Based on the results of the single-factor experiments, corn flour and rice flour were selected as the supplementary carbon source, and soybean meal and broad bean flour were selected as the supplementary nitrogen source in the optimal basal culture medium. The moisture content was 50%, the pH value was 7.0, and L25 (5 4 An orthogonal array was used to design a four-factor, five-level orthogonal experiment (Table 3) to determine the optimal ratio of each component.

[0060] Table 3. Orthogonal experimental scheme for the distribution ratio of each supplementary component in solid fermentation medium.

[0061] Optimization of Solid-State Fermentation Conditions for Beauveria bassiana strain Bb-YY: The solid-state fermentation conditions of Beauveria bassiana strain Bb-YY were optimized based on sporulation yield. Screening results for the basal culture medium showed that a mixture of wheat bran and wheat straw (4:1, wt / wt) produced the highest sporulation yield; therefore, this formulation was selected as the basal culture medium. Screening results for supplementing the solid-state fermentation medium with carbon and nitrogen sources showed that corn flour and rice flour were the optimal carbon sources, and soybean meal and fish meal were the optimal nitrogen sources.

[0062] L25 (5 4 The orthogonal experimental results (Table 4) show that the factors affecting the sporulation yield of Beauveria bassiana strain Bb-YY during solid-state fermentation, from largest to smallest, are soybean meal, rice flour, fish meal, and corn flour. Among these, the highest sporulation yield was achieved with 25% corn flour, 20% soybean meal, 5% rice flour, and 8% fish meal (formula 1), reaching 2.14 × 10⁻⁶ sporulations. 10 Spores / g.

[0063] Table 4. Statistical results of the orthogonal experiment for optimizing the distribution ratio of each supplement group in solid fermentation medium.

[0064] The basal culture medium is a crucial factor affecting the solid-state fermentation effect. In this embodiment, inexpensive and readily available wheat bran and wheat straw are used as the basal culture medium raw materials. This not only simplifies the process and reduces fermentation costs but also provides the necessary nutrients for microbial growth and metabolism. This embodiment optimizes the solid-state fermentation conditions of the *Beauveria bassiana* strain Bb-YY using sporulation yield as an indicator. The optimal basal culture medium was determined to be a mixture of wheat bran and wheat straw in a 4:1 ratio. Adding 25% corn flour, 20% soybean meal, 5% rice flour, and 8% fish meal to the basal culture medium yielded the maximum sporulation yield. After 10 days of cultivation, the sporulation yield reached 2.14 × 10⁻⁶. 10 Spores / g.

[0065] 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 type of Beauveria bassiana ( Beauveria bassiana strain Bb-YY, characterized in that, It has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 20252340.

2. A method for culturing the Beauveria bassiana strain Bb-YY as described in claim 1, characterized in that, Includes the following steps: Seed culture of Beauveria bassiana strain Bb-YY was inoculated into fermentation medium and fermented to obtain fermentation product; The fermentation medium includes a basal medium containing the following components in the indicated mass percentages: 23%–27% corn flour, 18%–22% soybean meal, 4%–6% rice flour, and 7%–9% fish meal.

3. The cultivation method according to claim 2, characterized in that, The basal culture medium comprises wheat bran and straw; the water content of the basal culture medium is 40%~60%. The mass ratio of wheat bran to straw is 3.5~4.5:

1.

4. A biocontrol agent, characterized in that, It includes the Beauveria bassiana strain Bb-YY as described in claim 1 or the fermentation product and excipients prepared by the culture method described in claim 2 or 3.

5. The biocontrol agent according to claim 4, characterized in that, The Beauveria bassiana strain Bb-YY comprises hyphae and / or spores.

6. The biocontrol agent according to claim 4, characterized in that, The mass percentage of the Beauveria bassiana strain Bb-YY is 5%~90%.

7. A biological compound preparation, characterized in that, It includes the biocontrol agent described in claim 2 or 3 and at least one of the following: peanut pest and disease insecticide, peanut fungal disease fungicide, pesticide and bio-organic fertilizer.

8. The application of the Beauveria bassiana strain Bb-YY as described in claim 1, the biocontrol agent as described in any one of claims 4 to 6, or the biological compound preparation as described in claim 7 in the control of insect pests and / or fungal diseases.

9. The application according to claim 8, characterized in that, The pests include diseases and pests caused by the green scarab beetle; The fungal diseases include those caused by at least one of the following fungi: Fusarium oxysporum, Alternaria alternata, Colletotrichum gloeosporioides, Fusarium solani, and Pyrethrum indica.

10. The application according to claim 8 or 9, characterized in that, The fungal diseases include at least one of the following: peanut root rot, peanut leaf spot, peanut fruit rot, tobacco black shank, and rice blast fungus.

Images