Tarlaromyces flavus and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to tarlaromyces flavus and application thereof. The invention discloses tarlaromyces flavus H12, and the preservation number of the tarlaromyces flavus H12 is CGMCC No.42336. The tarlaromyces flavus H12 is separated and screened from the nature for the first time, and can generate a remarkable antagonistic effect on a pathogenic bacterium-fusarium oxysporum strain causing the root rot of the fritillaria taipaiensis. Through identification, the fermentation liquor and volatile substances of the tarlaromyces flavus H12 can destroy the structure of pathogenic bacteria filaments to cause malformation and distortion of hyphae, and the inhibition rates of the tarlaromyces flavus H12 on the growth of the hyphae respectively reach 58.54% and 28.02%. Potting and field experiment results show that the morbidity and the disease index of the Fritillaria taipaiensis root rot can be remarkably reduced by applying the H12 fermentation liquor, and the final control effect reaches 56.16% and can be equivalent to that of chemical pesticides.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Bacillus luteus* and its applications. Background Technology

[0002] Fritillaria taibaiense is a perennial herb belonging to the genus Fritillaria in the family Liliaceae. Its dried bulb is one of the sources of Fritillaria cirrhosa listed in all editions of the Chinese Pharmacopoeia. It has the effects of moistening the lungs and relieving cough, resolving phlegm and relieving asthma, and dispersing nodules and reducing swelling. Clinically, it is often used for symptoms such as dry cough due to lung heat and lung yin deficiency. Due to the increasing scarcity of wild resources caused by over-harvesting and habitat destruction, Fritillaria taibaiense has been listed in the "National Key Protected Wild Plants List". Its artificial cultivation has become an effective way to protect and utilize this rare species. However, with the expansion of the cultivation area, soil-borne diseases, especially root rot, have become the main factors restricting the safe production of Fritillaria taibaiense.

[0003] Root rot primarily affects the roots and bulbs of Fritillaria thunbergii, with an extremely high incidence rate in low-lying, waterlogged areas. It can be spread through soil nematodes and water, and in severe cases, leads to bulb rot and even complete crop failure, posing a significant threat to yield and quality. Currently, control of this disease still relies mainly on chemical pesticides, but these methods suffer from short-lasting effectiveness and inconsistent efficacy in the field. Summary of the Invention

[0004] The purpose of this invention is to solve the aforementioned problems in the existing technology and to find a green, safe, efficient, and stable solution for the prevention and control of root rot in Fritillaria thunbergii. To this end, this invention provides a strain of *Bacillus thunbergii* and its application.

[0005] This invention provides a strain of *Bryophytum comosum* (Yellow Blue Bacteria). Talaromyces flavus H12, accession number CGMCCNo.42336.

[0006] The present invention also provides a culture of the bacterium H12 described in the above technical solution.

[0007] Preferably, the culture of *Cymbidium falcatum* H12 includes *Cymbidium falcatum* H12 and its secondary metabolites; The secondary metabolites include volatile substances; the volatile substances include one or more of 4-lactone, hexaketone, 5-hydroxymethylfurfural and methyl 4-carboxy-5-hydroxyphthalimide.

[0008] The present invention also provides a microbial inoculant, wherein the effective components of the microbial inoculant include the *Bacillus chrysophagus* H12 or the culture described in the above-mentioned technical solution.

[0009] Preferably, the effective viable count of *Bacillus chrysogenum* H12 in the microbial agent is ≥2 × 10⁻⁶. 9 CFU / ml.

[0010] The present invention also provides the application of the above-described *Bacillus thunbergii* H12, the above-described culture, or the above-described microbial agent in the prevention and control of plant root rot.

[0011] Preferably, the plant includes Fritillaria thunbergii; The root rot includes root rot caused by Fusarium oxysporum.

[0012] This invention also provides a method for preventing and controlling plant root rot, comprising the following steps: The above-described blue-green bacterium H12, the culture described in the above-described technical solution, or the microbial agent described in the above-described technical solution are applied to the soil around the roots of Fritillaria thunbergii.

[0013] Preferably, the application method includes irrigation with water; The irrigation dosage of the culture or microbial agent is 5 ml / m³. 2 ~15ml / m 2 ; The culture or microbial agent is applied once every 7 days, for a total of 3 applications; The culture or microbial agent contains ≥2×10⁶ viable bacteria. 9 CFU / ml.

[0014] Preferably, the plant includes Fritillaria thunbergii; The root rot includes root rot caused by Fusarium oxysporum.

[0015] Beneficial effects: This invention provides a strain of *Cymbidium falcatum* H12, with the preservation number CGMCC No. 42336. This *Cymbidium falcatum* H12, obtained for the first time from nature, exhibits significant antagonistic activity against *Fusarium oxysporum* strain, the pathogen causing root rot in *Fritillaria taiwanensis*. Identification showed that the fermentation broth and volatile substances (such as 4-lactone, hexazone, 5-hydroxymethylfurfural, and methyl 4-carboxy-5-hydroxyphthalimide) of *Cymbidium falcatum* H12 can disrupt the mycelial structure of the pathogen, leading to mycelial deformities and distortions, with inhibition rates of 58.54% and 28.02%, respectively. Pot and field trials showed that application of the H12 fermentation broth significantly reduced the incidence and disease index of root rot in *Fritillaria taiwanensis*, achieving a final control effect of 56.16%, comparable to chemical pesticides.

[0016] Furthermore, in field trials, the H12 fermentation broth significantly increased chlorophyll content and reduced malondialdehyde accumulation in plants, enhancing their stress resistance and disease resistance. This invention, by isolating and identifying rhizosphere microorganisms of *Fritillaria taibaiense*, clearly identifying the pathogenic bacteria and screening for the highly efficient biocontrol strain H12, and subsequently establishing its fermentation production process and field disease control technology, provides a reliable means for the green control of root rot in *Fritillaria taibaiense*, and has significant practical implications for improving disease control levels and promoting sustainable industrial development.

[0017] Biological Preservation Information Blue-spotted bacterium H12, classified and named Talaromyces flavus It was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 42336. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 The images show colonies of strain B005 on PDA medium and their (40×10) microscopic morphology. Figure 1 In the diagram, A represents the colony morphology of strain B005. Figure 1 In the image, B represents the mycelial micrograph of strain B005. Figure 1 C in the image represents the microscopic morphology of spores from strain B005. Figure 2 Photos of the roots, bulbs, and plants of healthy and diseased Fritillaria thunbergii; among them, Figure 2 A in the image represents real photos of the roots of *Fritillaria thunbergii* in healthy condition (left) and diseased condition (right). Figure 2 B in the image shows real photos of healthy (left) and diseased (right) bulbs of *Fritillaria thunbergii*. Figure 2 C in the image represents real photos of healthy (right side + middle) and diseased (left side) Fritillaria thunbergii plants. Figure 3 Phylogenetic analysis diagram of strain B005; Figure 4 The image shows the antagonistic effect of strain H12 on strain B005; among them, Figure 4 The image on the left shows a plate with only strain B005 coated on it. Figure 4 The image on the right shows a photograph of the plates in which strains H12 and B005 confront each other. Figure 5 The images show colonies of strain H12 on PDA medium and their (40×10) microscopic morphology. Figure 5 The left side shows the colony morphology of strain H12. Figure 5 The right side shows a microscopic morphological image of strain H12; Figure 6 Phylogenetic analysis diagram of strain H12; Figure 7 The graph shows the inhibitory effect of the fermentation broth of strain H12 on strain B005; among them, Figure 7 From left to right: actual photos of plates with only strain B005, plates treated with hymexazol, plates treated with 10% strain H12 fermentation broth, plates treated with 20% strain H12 fermentation broth, and plates treated with 40% strain H12 fermentation broth. Figure 8 The image shows the effect of fermentation broth from strain H12 on the mycelial morphology of strain B005 under a (40×10) microscope; among them, Figure 8 The left side shows a microscopic image of the hyphae of the control group strain B005. Figure 8 The image in the middle is a microscopic morphology of the mycelium of strain B005 treated with hymexazol. Figure 8 The right side shows a microscopic morphology of the mycelium of strain B005 treated with fermentation broth of strain H12. Figure 9 The graph shows the inhibitory effect of volatile substances from strain H12 on strain B005; among them, Figure 9 The image on the left shows a plate with only strain B005 coated on it. Figure 9 The image on the right is a photograph of a plate treated with volatile substances from strain H12. Figure 10 The image shows the effect of volatile substances from strain H12 on the mycelial morphology of strain B005 under a (40×10) microscope; among them, Figure 10 The left side shows a microscopic image of the hyphae of the control group strain B005. Figure 10 The image on the right shows the mycelial morphology of strain B005 treated with volatile substances from strain H12. Detailed Implementation

[0020] This invention provides a strain of *Bryophytum comosum* (Yellow Blue Bacteria). Talaromyces flavusH12, with accession number CGMCC No. 42336, is the first *Cymbidium falcatum* strain isolated and screened from nature. It exhibits significant antagonistic activity against *Fusarium oxysporum* strain, the pathogen causing root rot in *Fritillaria taibaiense*. Identification showed that the fermentation broth and volatile substances (such as 4-lactone, hexane, 5-hydroxymethylfurfural, and methyl 4-carboxy-5-hydroxyphthalimide) of *Cymbidium falcatum* H12 can disrupt the mycelial structure of the pathogen, leading to malformed and distorted hyphae, with inhibition rates of 58.54% and 28.02%, respectively. On PDA medium, the colonies of *Cymbidium falcatum* H12 are initially pale yellow, later turning yellowish-green, with an orange-red reverse side. The colony diameter can reach 8 cm. The hyphae are short and fluffy; under a microscope, the hyphae are smooth and slender, without septa. The conidia are elliptical, clustered at the head of the conidiophore, and odorless. The rDNA-ITS gene sequence of *Chlorella vulgaris* H12 described in this invention is shown in SEQ ID NO.3: 5'--3'. After sequence analysis, the gene sequence was identified as *Chlorophytum comosum* by BLAST homology comparison in the NCBI database, combined with morphological characteristics and molecular sequencing analysis. It was biologically classified as... Talaromyces flavus .

[0021] This invention also provides a culture of *Cymbidium falcatum* H12 as described in the above-mentioned technical solution. As one embodiment, the culture medium for culturing *Cymbidium falcatum* H12 can be potato dextrose agar and / or potato dextrose broth. As one embodiment, the culture of *Cymbidium falcatum* H12 includes *Cymbidium falcatum* H12 and its secondary metabolites; the secondary metabolites include volatile substances; the volatile substances include one or more of 4-lactone, hexane, 5-hydroxymethylfurfural, and 4-carboxy-5-hydroxyphthalimide methyl ester. As one embodiment, *Fusarium oxysporum* hyphae treated with the volatile substances produced by *Cymbidium falcatum* H12 exhibit shriveling and twisting, indicating that the volatile substances produced by *Cymbidium falcatum* H12 have a significant inhibitory effect on the growth of *Fusarium oxysporum*. As one embodiment, the culture of *Cymbidium falcatum* H12 can be a fermentation broth of *Cymbidium falcatum* H12. As one implementation method, the mycelia of Fusarium oxysporum treated with the fermentation broth of the *Fusarium oxysporum* H12 described in this invention exhibit abnormal phenomena such as missing and twisted hyphae, indicating that the H12 fermentation broth has a significant inhibitory effect on the growth of *Fusarium oxysporum* B005.

[0022] This invention also provides a microbial inoculant, the effective component of which includes *Chlorella vulgaris* H12 or the culture described in the above-mentioned technical solutions. As one embodiment, the microbial inoculant of this invention can be a fermentation broth of *Chlorella vulgaris* H12. As one embodiment, the effective viable count of *Chlorella vulgaris* H12 in the microbial inoculant of this invention is ≥2 × 10⁻⁶. 9 CFU / ml.

[0023] This invention also provides the application of the *Cymbidium falcatum* H12, the culture, or the microbial agent described in the above-mentioned technical solutions in the prevention and control of plant root rot. In one embodiment, the plant mentioned in this invention includes *Fritillaria taibai*; the root rot includes root rot caused by *Fusarium oxysporum*. In one embodiment, the *Cymbidium falcatum* H12 described in this invention exhibits a significant antagonistic effect against the pathogen causing root rot in *Fritillaria taibai*—*Fusarium oxysporum* strains. Identification has shown that the fermentation broth and volatile substances (such as 4-lactone, hexaketone, 5-hydroxymethylfurfural, and 4-carboxy-5-hydroxyphthalimide methyl ester, etc.) of the *Cymbidium falcatum* H12 can destroy the pathogenic mycelial structure, leading to mycelial deformities and distortions, with inhibition rates of 58.54% and 28.02%, respectively. As one implementation method, applying the fermentation broth of *Bacillus thuringiensis* H12 described in this invention can significantly reduce the incidence and disease index of root rot in *Fritillaria thunbergii*, with a final control effect of 56.16%, comparable to chemical pesticides. As another implementation method, field trials using the H12 fermentation broth also significantly increased chlorophyll content, reduced malondialdehyde accumulation, and enhanced plant stress resistance and disease resistance.

[0024] This invention also provides a method for preventing and controlling plant root rot, comprising the following steps: The above-described blue-green bacterium H12, the culture described in the above-described technical solution, or the microbial agent described in the above-described technical solution are applied to the soil around the roots of Fritillaria thunbergii.

[0025] This invention utilizes the *Bacillus thunbergii* H12, the culture, or the microbial agent described in the above-mentioned technical solutions, applied to the root soil of *Fritillaria thunbergii*. As one embodiment, the application method includes irrigation; the irrigation dosage of the culture or microbial agent is 5 ml / m³. 2 ~15ml / m 2 The culture or microbial agent is applied once every 7 days, for a total of 3 applications; the number of effective viable bacteria in the culture or microbial agent is ≥2×10⁻⁶. 9 CFU / ml. As one embodiment, the culture or microbial agent described in this invention is applied in the field at a rate of 6 m³ / ml. 2Apply 50 ml. As one embodiment, the plant described in this invention includes *Fritillaria taibai*; the root rot includes root rot caused by *Fusarium oxysporum*. As one embodiment, the culture or microbial agent described in this invention can improve the plant's antioxidant and stress resistance. As one embodiment, improving the plant's antioxidant and stress resistance includes reducing the plant's malondialdehyde content and / or increasing the plant's chlorophyll content. As one embodiment, the culture or microbial agent described in this invention can significantly reduce the incidence and disease index (p<0.05) of *Fritillaria taibai* root rot, reducing them by 25.12% and 28.03%, respectively. As one embodiment, the control effect of the culture or microbial agent described in this invention is higher than that of chemical pesticide treatment. As one embodiment, the chemical pesticide described in this invention includes hymexazol.

[0026] To further illustrate the present invention, the following detailed description of a strain of *Bacillus thunbergii* and its applications, in conjunction with the accompanying drawings and embodiments, is provided but should not be construed as limiting the scope of protection of the present invention.

[0027] Sample Collection: In May 2021, at the *Fritillaria thunbergii* planting base of Chongqing Academy of Traditional Chinese Medicine, Xi'an Village, Lanying Township, Wuxi County, Chongqing (N:31°24′13.25″ E:109°52′42.28″), rhizosphere soil and bulbs of three-year-old healthy and diseased *Fritillaria thunbergii* plants were collected. Diseased *Fritillaria thunbergii* plants showed wilting and yellowing aboveground parts, and rotting underground bulbs covered with white mycelium. Specifically, five sampling points were used. At each sampling point, five healthy and five diseased *Fritillaria thunbergii* plants were selected. After removing the topsoil and excavating the root system, rhizosphere soil was collected using the root-shaking method. Larger stones were removed, and the samples were mixed and placed in sterile self-sealing bags. Bulbs were placed in separate sterile self-sealing bags. The collected samples were stored in ice boxes and quickly transported back to the laboratory for preservation at 4°C for soil microbial isolation.

[0028] Main culture medium: Potato Dextrose Agar (PDA): 200.0 g potato, 20.0 g glucose, 18.0 g agar, natural pH, 1000 mL distilled water, autoclaved at 121℃ for 30 min; Potato Dextrose Broth (PDB): Prepared in the same way as PDA, but without adding agar; Test reagent: 30% aqueous solution of hymexazol, produced by Beijing Weimin Biotechnology Co., Ltd., purchased from Chongqing Beibei District Fuxing Seed Industry Agricultural Materials Co., Ltd.

[0029] Example 1 Isolation and identification of pathogenic strains 1. Isolation and morphological identification of pathogenic strains White mycelia were collected from diseased fritillaria bulbs and inoculated onto PDA medium. After incubation at 28°C for 48 h, the mycelial tips were collected and purified further on PDA medium until a pure culture was obtained, designated B005. The colony characteristics of strain B005 on PDA medium were observed, and the morphology of its mycelia and spores was examined under an optical microscope. Preliminary identification of its colonies, mycelia, and spores was performed according to Wei Jingchao's "Handbook of Fungal Identification".

[0030] Colonies of strain B005 on PDA medium and their (40×10) microscopic morphology images are shown below. Figure 1 As shown. According to Figure 1 It was found that strain B005, isolated from the rotten bulb of Fritillaria cirrhosa, could cover the entire plate on PDA medium at 28℃ in 7 days. The colonies were initially white and later turned pale purple. After 7 days of culture, the colonies were raised and had a diameter of 7-7.8 cm. The hyphae were curly and grew vigorously. Microscopic observation revealed that the large conidia were slender, crescent-shaped, and had 1-3 septa, while the small conidia were oval.

[0031] 2. Pathogenicity detection of pathogenic strains Prepare PDB, sterilize and cool it, then inoculate with B005 mycelial cakes (number of mycelial cakes: medium = 1:100mL) with a diameter of 8 mm, and incubate at 28℃ and 150 r·min. -1 The pathogenic bacteria were cultured in a constant temperature shaker for 5 days to obtain the fermentation broth.

[0032] The pot experiment began in December 2021 in Xi'an Village, Lanying Township, Wuxi County, Chongqing. Healthy 3-year-old fritillaria bulbs were selected and transplanted into pots of 9-12 bulbs each, for a total of 8 pots, and cultivated according to field management practices. The potted plants were divided into two groups. On April 17, 2022, the roots of the fritillaria bulbs were punctured with a sterilized inoculation needle, and each pot was watered with 100 ml of B005 fermentation liquid (Group B). Watering with plain water served as the control (Group A). ​​After 60 days, the bulbs and roots were dug up, the number of rotten bulbs was counted, the disease incidence was calculated, and rhizosphere soil and bulbs were collected. The mycelium from the rotten bulb areas was further isolated, and preliminary identification was performed based on colony and mycelial morphology to compare whether the original strain and the isolated strain were the same.

[0033] The results of the experiment of inoculating potted Fritillaria thunbergii with B005 are shown in Table 1 and Figure 2 As shown in Table 1 and... Figure 2 The results show that after watering with B005 spore suspension (Group B), Fritillaria cirrhosa showed symptoms of root rot, with an incidence rate as high as 85.37%, significantly higher than that after watering with plain water (Group A). P <0.05), an increase of 5.8 times. Observation of the plants revealed ( Figure 2In the C section, healthy fritillaria plants grow straight, with bright green and intact leaves, dark green stems, and a whitish lower part of the stem; diseased fritillaria plants have yellowing and wilting leaves, some leaves are missing, the stems are soft and brown, and the lower part of the stem gradually turns brown. Further observation of the fritillaria root system ( Figure 2 In study A), it was found that healthy fritillaria roots were white, plump, smooth, and had abundant and robust root hairs, while diseased fritillaria roots were brown, shriveled, wrinkled, and had degenerated and withered root hairs. Furthermore, observation of the fritillaria bulbs revealed (…). Figure 2 In the experiment (B), healthy fritillaria bulbs were firm, intact, smooth to the touch, and white, while diseased fritillaria bulbs became soft and rotten, with the rotten areas turning brown and covered with white mycelium. The rotten parts later dissipated into the soil as powder. At the end of the experiment, rotten bulbs of diseased fritillaria were collected, and mycelium from the affected areas was further isolated and purified. The isolated strain showed completely consistent growth characteristics, colony morphology, and mycelial spore morphology on PDA plates with B005.

[0034] Table 1. Disease incidence in Fritillaria taibaiense inoculated with strain B005

[0035] Note: A: Irrigate with clean water; B: Irrigate with a suspension of strain B005 spores; express P<0.05 The differences were significant at the level.

[0036] 3. Molecular biological identification of pathogenic strains Purified strain B005 was inoculated into PDA medium and cultured in the dark at 28°C for 7 days for molecular biological identification. Genomic DNA was extracted from the pure culture strain B005 using the TSINGKE Plant DNA Extraction Kit. PCR amplification was performed using universal primers ITS1 (SEQ ID NO. 1: 5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (SEQ ID NO. 2: 5′-TCCTCCGCTTATTGATATGC-3′). After amplification, the DNA was sent to the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. After DNA extraction from the mycelium of strain B005 and PCR amplification for library construction, the sequencing results were compared for BLAST homology in the NCBI database (blast.ncbi.nlm.nih.gov). A phylogenetic tree was constructed using MEGA 7.0 as follows: Figure 3 As shown. The results indicate that strain B005 is related to Fusarium oxysporum. Fusarium oxysporum (MT032671.1) clusters together, with curly hyphae; large conidia are slender, crescent-shaped, with 1-3 septa; small conidia are oval, consistent with *Fusarium oxysporum*, hence the name: *Fusarium oxysporum*. Fusarium oxysporum B005.

[0037] Example 2 Isolation and identification of biocontrol strains 1. Isolation and purification of biocontrol strains Weigh 10 g of well-mixed rhizosphere soil from healthy (H) and diseased (D) Fritillaria thunbergii plants and place it in a 90 mL Erlenmeyer flask containing sterile water. Shake for 20 min and then let stand. Dilute the soil suspensions from both groups separately with sterile water at a ratio of 10:1. -1 10 -2 10 -3 Serial dilutions were performed in test tubes, and after thorough mixing, 200 μL was transferred to each PDA plate, with three replicates for each dilution. The culture was incubated at 28°C for 4–5 days. Mycelia from the culture medium were then transferred to PDA plates. The above steps were repeated until the strain was purified. The strain was then stored at 4°C.

[0038] 2. Initial screening of biocontrol strains The experiment used the plate confrontation culture method. Pathogenic fungal blocks with a diameter of 8 mm were inoculated into the center of a PDA plate. The fungi isolated above were inoculated into symmetrical parts on the left and right sides of the block. A control group was inoculated with only pathogenic fungi. Each treatment was repeated 3 times. The plates were incubated in the dark at 28 ± 1℃ for 7 to 10 days. The growth of pathogenic fungi in the control and treatment groups was observed.

[0039] The pathogen *Fusarium oxysporum* was screened from the rhizosphere soil of *Fritillaria taibaiense* using dilution plating and confrontation plate methods. F. oxysporum B005 has a better antagonistic effect than strain H12 (such as...). Figure 4 (As shown). Colonies of strain H12 on PDA medium and their (40×10) microscopic morphology were obtained as shown. Figure 5 As shown. It was found that the mycelium of strain H12 could cover the plate after 14 days of culture at 28℃. The colonies were initially pale yellow, then yellowish-green, and orange-red on the reverse side. The colony diameter could reach 8 cm. The mycelium was short and fluffy. Under the microscope, the mycelium was smooth and slender, without septa. The conidia were elliptical and clustered at the head of the conidiophore. They were odorless.

[0040] 3. Biological identification of biocontrol bacteria The colony characteristics of the fungus on PDA medium were observed, and the morphological characteristics of its hyphae and spores were observed under an optical microscope. Preliminary identification was performed according to the descriptions of colony, hyphae, and spore morphology in *Handbook of Fungal Identification* (Wei Jingchao). The isolated biocontrol strain was identified using the same methods as in the molecular biological identification section of the pathogenic strain in Example 1. The rDNA-ITS gene sequence of strain H12 was determined as shown in SEQ ID NO.3. The rDNA-ITS gene sequence of strain H12 was subjected to BLAST homology comparison in the NCBI database, and a phylogenetic tree was constructed using MEGA 7.0 for analysis. Figure 6 As shown. It can be seen that strain H12 and... Talaromyces flavus (ON239605.1) clustered together, and based on the morphological characteristics of the strains, strain H12 was identified as *Bacillus chrysophagus*. Talaromyces flavus H12.

[0041] Example 3 1. Blue-yellow fungus T. flavus H12 fermentation broth on F. oxysporum Effects of B005 growth The experiment used the plate method with live bacteria. The fungal fermentation broth cultured in PDB shake flasks (with a viable cell count ≥ 2 × 10⁻⁶) was... 9 The fermentation broth (CFU / ml) was filtered through a 0.22 μm filter. Different volumes of fermentation broth were added to sterilized and cooled PDA to achieve concentrations of 10%, 20%, and 40% by volume, respectively. 1 mL of hymexazol was added to 99 mL of sterile water and diluted with PDA to a concentration of 0.25 mg / mL. -1 , and prepare a drug-containing culture medium.

[0042] A fungal disc with a diameter of 8 mm was inoculated in the center of a virus-containing plate, with an 8 mm fungal disc inoculated on a PDA plate as a control. The plates were incubated at 28 ± 1 ℃ for 7–10 days. The incubation results are as follows: Figure 7 As shown. The diameter of the pathogenic bacterial cake was measured using the cross-multiplication method, and the inhibition rate was calculated using Equation I. Each concentration was repeated 6 times.

[0043] Inhibition rate = [(control colony area - treated colony area) / control colony area] × 100%, Formula I.

[0044] Pathogen mycelial cakes with a diameter of 8 mm were inoculated onto PDA plates containing fermentation broth and hymexazol. A coverslip was placed 3 mm away from the mycelial cake, and the plates were incubated at 28 ± 1 ℃. When the hyphae reached the coverslip, their morphology was observed under an optical microscope (manufacturer number: L311606210291, made in China, mode 1 MD50). The effect of the fermentation broth of strain H12 on the hyphal morphology of strain B005 under a (40×10) microscope is shown below. Figure 8 As shown.

[0045] according to Figure 7 and Figure 8 The results show that the fermentation broth of *Bacillus thuringiensis* H12 has the effect on... F. oxysporum The growth of B005 was significantly inhibited, and the inhibitory effect increased with increasing fermentation broth concentration, with inhibition rates reaching 38.54% to 58.54%. This is comparable to the effect of hymexazol on... F. oxysporum The inhibition rate of B005 (59.22%) was comparable; observation under an optical microscopeF. oxysporum The mycelia of B005 were found to be smooth and uniform in thickness when growing normally; however, after treatment with the H12 fermentation broth of *Fusarium oxysporum*, abnormal phenomena such as mycelial loss and twisting were observed, indicating that the H12 fermentation broth had a significant inhibitory effect on the growth of *Fusarium oxysporum* B005.

[0046] 2. Blue-yellow bacteria T. flavus H12 volatile substances F. oxysporum Effects of B005 growth The experiment used the flat plate mating method. Inoculations with a diameter of 8 mm were placed in the center of the PDA flat plate. F. oxysporum B005 and T. flavus For H12 mycelial cakes, two plates were inverted and sealed with a sealing film, with only the pathogenic fungus as a control. They were incubated in the dark at 28 ± 1 ℃ for 7–10 days, with each treatment repeated 6 times. The inhibition rate was calculated using the same method as in the experiment on the effect of H12 fermentation broth on the growth of strain B005. The inhibitory effect of volatile substances from strain H12 on strain B005 is shown in the figure. Simultaneously, the mycelial morphology after exposure to volatile gases was observed using the same method as in the experiment on the effect of H12 fermentation broth on the growth of strain B005. The effect of volatile substances from strain H12 on the mycelial morphology of strain B005 under a (40×10) microscope is shown in the figure. Figure 10 As shown.

[0047] according to Figure 9 and Figure 10 The results show that the volatile substances of *Cymbidium falcatum* H12 have an effect on... F. oxysporum The growth of B005 was significantly inhibited, with an inhibition rate of up to 28.02%. This was observed under an optical microscope. F. oxysporum The mycelia of B005 were found to be smooth and uniform in thickness when growing normally; however, the mycelia treated with the volatile substance of *Fusarium oxysporum* H12 showed signs of shriveling and twisting, indicating that the volatile substance of *Fusarium oxysporum* H12 had a significant inhibitory effect on the growth of *Fusarium oxysporum* B005.

[0048] Example 4 1. Test of potted plant efficacy The experiment was conducted in 2023 at the Taibai Fritillaria planting base of Chongqing Academy of Traditional Chinese Medicine, Xi'an Village, Lanying Township, Wuxi County, Chongqing. Three-year-old Fritillaria plants with good growth were selected and transplanted into pots in January, with 8 to 12 plants per pot. The following solutions were prepared: (1) clean water, (2) clean water + pathogens, (3) hymexazol + pathogens, and (4) [unclear]. T. flavus H12 fermentation broth (viable count ≥ 2 × 10⁻⁶) 9 Four treatments were conducted, each consisting of 5 pots, with the treatment consisting of CFU / ml and pathogens.

[0049] According to field management practices, in May, the fritillaria bulbs were irrigated with hymexazol and... T. flavus 50 mL of H12 fermentation broth was applied, followed by irrigation with B005 spore suspension after 3 days. Subsequently, hydrazine and [other fungicides] were applied every 3 days. T. flavus 50 mL of H12 fermentation broth was applied three times. A blank treatment was applied only with water, and a control treatment was applied only with B005 spore suspension. Changes in the fritillaria were observed after inoculation with the pathogen. Three days after treatment, after the control showed symptoms, the total number of leaves and the number of withered leaves were counted. Data were recorded before each subsequent treatment. All samples were dug up in mid-June before the fritillaria collapsed, and the number of rotten bulbs was counted. The disease severity index was determined according to Zheng Doudou's (Zheng Doudou et al., 2019) disease grading standards. The incidence rate, disease severity index, and control effect were calculated.

[0050] Incidence rate = Number of infected plants / Total number of plants × 100%, Formula II; Disease index = ∑ (number of plants at each disease level × representative value at each level) / (total number of plants surveyed × representative value of the highest disease level) × 100%, Equation III; Control effect = [(Disease index in control area - Disease index in treatment area) / Disease index in control area] × 100%, Formula IV.

[0051] Finally obtained T. flavus The potted plant control effects of H12 and hymexazol on root rot are shown in Table 2. The potted plant experiment results showed that both H12 fermentation broth and hymexazol significantly reduced the incidence and disease index of root rot in *Fritillaria thunbergii* (p<0.05). Specifically, in terms of the incidence of root rot in *Fritillaria thunbergii*, the hymexazol treatment reduced the incidence by 40.41%, compared to a 52.43% reduction in the water + pathogen treatment group. T. flavus The H12 fermentation broth treatment group reduced the incidence rate by 45.83%, a reduction of 59.46% compared to the water + pathogen treatment group. Regarding the root rot disease index of *Fritillaria thunbergii*, the hymexazol treatment reduced the disease index by 35.81%, a reduction of 52.34% compared to the water + pathogen treatment group. T. flavus The H12 fermentation broth treatment group reduced the disease index by 38.42%, compared to a 56.15% reduction in the water + pathogen treatment group. Ultimately, the control efficacy of hymexazol against root rot was measured at 52.35%. T. flavus The H12 treatment showed a 56.16% efficacy rate in controlling root rot.

[0052] Table 2 T. flavus The effects of H12 and hymexazol on the prevention and control of root rot in potted plants

[0053] Note: The data in the table are mean ± standard deviation, analyzed by one-way statistical analysis using the least significant difference method. LSD To compare the significance of differences between different treatments, different lowercase letters indicate significant differences at the 0.05 level, and the same applies below.

[0054] 2. Determination of Fritillaria-related biomass and bioindicators When plants are damaged under stress, membrane lipid peroxidation occurs. Malondialdehyde (MDA) is the final product of membrane lipid peroxidation, and its content can reflect the degree of damage caused by the plant under stress. The lower the content, the stronger the plant's antioxidant capacity and stress resistance.

[0055] After the pot experiment, potted Fritillaria thunbergii plants and bulbs were collected, placed in ice boxes, and brought back to the laboratory for storage at 4°C. Changes in plant height before and after the experiment were measured. Changes in chlorophyll and malondialdehyde content before and after the experiment were determined using spectrophotometry and the thiobarbituric acid (TBA) colorimetric method, respectively. The effects of various chemical agents and biocontrol bacteria fermentation broth on plant growth and stress resistance were compared. The results are shown in Table 3.

[0056] As shown in Table 3, the malondialdehyde (MDA) content was significantly lower in the treatment with hymexazol and H12 fermentation broth than in the pathogen treatment (p<0.05), decreasing by 37.50% and 62.50%, respectively. The plant height was significantly higher in the treatment with hymexazol and H12 fermentation broth than in the treatment with pathogen spore suspension (p<0.05), increasing by 43.64% and 30.91%, respectively, but lower than in the water treatment. The chlorophyll content was significantly higher in the treatment with hymexazol and H12 fermentation broth than in the pathogen treatment (p<0.05), increasing by 50% and 33.33%, respectively. Therefore, both H12 fermentation broth and hymexazol can significantly alleviate the damage caused by pathogens to *Fritillaria thunbergii*.

[0057] Table 3 T. flavus The effects of H12 and hymexazol on plant growth

[0058] Example 5 Field efficacy test A field experiment was conducted on a severely diseased plot of Fritillaria cirrhosa cultivation base of Chongqing Academy of Traditional Chinese Medicine in Xi'an Village, Wuxi County. Three treatments were set up, each with a depth of 6 m². 2 Apply 50 mL of water, hymexazol, and a live bacteria count ≥2×10⁻⁶ every 7 days. 9 CFU / ml of *Cyclocarya pallida* ( T. flavus The H12 fermentation broth was applied three times. Before the seedling collapse stage of *Fritillaria thunbergii*, the incidence rate and disease index of *Fritillaria thunbergii* in each treatment were statistically analyzed. The calculation methods for incidence rate, disease index, and control effect were the same as those for the pot control efficacy determination, specifically as described in Example 4, Pot Control Efficacy Determination. T. flavus The field control effects of H12 and hymexazol on root rot are shown in Table 4.

[0059] The field trial results shown in Table 4 indicate that the fermentation broth of strain H12 significantly reduced the incidence and disease index of root rot in *Fritillaria thunbergii* (p<0.05), decreasing by 25.12% and 28.03% respectively, comparable to that of chemical pesticides. Its control efficacy was 28.03%, higher than that of hymexazol treatment.

[0060] Table 4 T. flavus Field control efficacy of H12 and hymexazol against root rot

[0061] In summary, this invention not only identified *Fusarium oxysporum* as the pathogen causing root rot in the *Fritillaria cirrhosa* base in Xi'an Village, Wuxi County, Chongqing, but also... F. oxysporum B005); It also isolated and screened a biocontrol bacterium that effectively antagonizes pathogens—*Bryophyte auricula-judae* (Blancium chrysogenum). T. flavus H12) and proved that its fermentation broth and volatile substances can effectively inhibit pathogens, significantly reduce the incidence and disease index in pot experiments and field experiments, and the control effect is comparable to that of chemical pesticides, showing good application prospects.

[0062] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Cymbidium faberi* ( Talaromyces flavus H12, characterized in that, The accession number is CGMCCNo.42336.

2. The culture of *Cymbidium falcatum* H12 as described in claim 1.

3. The culture according to claim 2, characterized in that, The culture of *Cymbidium falcatum* H12 includes *Cymbidium falcatum* H12 and its secondary metabolites. The secondary metabolites include volatile substances; the volatile substances include one or more of 4-lactone, hexaketone, 5-hydroxymethylfurfural and methyl 4-carboxy-5-hydroxyphthalimide.

4. A microbial inoculant, characterized in that, The effective components of the microbial agent include the *Bacillus thyrsiflora* H12 as described in claim 1 or the culture as described in claim 2 or 3.

5. The microbial agent according to claim 4, characterized in that, The effective viable count of *Bryophyllum luteum* H12 in the microbial agent is ≥2 × 10⁻⁶. 9 CFU / ml.

6. The application of the *Bacillus thyrsiflora* H12 as described in claim 1, the culture as described in claim 2 or 3, or the microbial agent as described in claim 4 or 5 in the prevention and control of plant root rot.

7. The application according to claim 6, characterized in that, The plant includes Fritillaria taibai; The root rot includes root rot caused by Fusarium oxysporum.

8. A method for preventing and controlling plant root rot, characterized in that, Includes the following steps: The *Bacillus thunbergii* H12 as described in claim 1, the culture as described in claim 2 or 3, or the microbial agent as described in claim 4 or 5 is applied to the soil around the roots of *Fritillaria thunbergii*.

9. The method according to claim 8, characterized in that, The application method includes irrigation with water; The irrigation dosage of the culture or microbial agent is 5 ml / m³. 2 ~15ml / m 2 ; The culture or microbial agent is applied once every 7 days, for a total of 3 applications; The culture or microbial agent contains ≥2×10⁶ viable bacteria. 9 CFU / ml.

10. The method according to claim 8, characterized in that, The plant includes Fritillaria taibai; The root rot includes root rot caused by Fusarium oxysporum.

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