Biological control method for cotton fusarium wilt

Abstract

The invention discloses a biological control method for cotton fusarium wilt. The invention provides a combined bacterium or a complex microbial inoculant containing the combined bacterium. The combined bacterium is prepared from AT actinoplanes (Actinoplanes) Q9316 (CGMCC (China General Microbiological Culture Collection Center) No.35443) and Trichoderma asperellum (Trichoderma asperellum) F2609 (CGMCC No.42159). The invention further provides a preparation method of the composite microbial inoculant. The fermentation liquid independently fermented by the trichoderma asperellum F2609 and the AT actinoplanes Q9316 provided by the invention has an inhibition effect on pathogenic bacteria of cotton fusarium wilt, and the bacteriostatic activity strength of the fermentation liquid jointly fermented by the trichoderma asperellum F2609 and the AT actinoplanes Q9316 exceeds that of the fermentation liquid fermented by single bacteria. The invention has important significance for biological prevention and control of cotton fusarium wilt.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a biological control method for cotton wilt disease. Background Technology

[0002] Cotton wilt is one of the most threatening diseases in cotton production, mainly caused by the pathogenic fungus *Fusarium oxysporum*. After infection, cotton plants develop wilting and drying at the leaf margins, and the roots turn brown and necrotic, severely impacting cotton yield and quality. The pathogen, *Fusarium oxysporum*, overwinters in the soil as thick-walled spores and mycelia, and spreads through irrigation water, soil, and infected seeds or seedlings. Furthermore, the disease's development is closely related to temperature, humidity, soil type, and the resistance of the cotton variety, making its control quite challenging.

[0003] Currently, the control of cotton wilt includes chemical pesticide methods and biological control. Chemical pesticides are one of the important means of controlling cotton wilt, but the environmental pollution and increased pesticide resistance of pathogens caused by chemical pesticides are also worrying. Biological control uses microbial agents to inhibit the harm of pathogens. For example, Bacillus subtilis, Bacillus amyloliquefaciens, and other Bacillus biocontrol bacteria can inhibit the growth of pathogens by producing antimicrobial substances and competing for nutrients; Pseudomonas biocontrol bacteria can control cotton wilt by secreting a variety of antimicrobial substances. Therefore, the development of new functional microbial agents will be the main strategy for dealing with cotton wilt.

[0004] Studies show that Trichoderma ( TrichodermaTrichoderma spp. is an important biocontrol fungus for plant diseases (Meng Suling, Tian Yanmei, Gu Xin, et al. Research progress on the synergistic disease control effect of Trichoderma. Chinese Journal of Biological Control, 2022, 38(3): 739-747.; Yao Chenxiao, Li Xiaojie, Liu Chang, et al. Screening and identification of three Trichoderma strains antagonistic to Fusarium oxysporum and evaluation of their growth-promoting and disease-controlling effects. Chinese Journal of Tobacco Science, 2022, 28(4): 96-105.), and can also exert biocontrol potential through various functions, such as parasitism, antibiosis, competition, resistance to adverse stress, enhancement of plant antioxidant defense, alteration of ecological conditions and promotion of plant growth (Tao Lingyun, Zhang Yiwen, Li Yaqian, et al. Detection of volatile secondary metabolites and analysis of antibacterial activity of Trichoderma spp. Chinese Journal of Biotechnology, 2020, 36(6): 1181-1189; Zheng Chuanqi, Linghu Meilin, Shu Zhongze, et al. Isolation and identification of Trichoderma asperellum and its antifungal activity against anthracnose fungus and smut fungus of coix seed. Journal of Southern Agriculture, 2023, 54(7): 2050-2059; Qi W, Zhao L. Study of the siderophore-producing Trichoderma asperellum Q1 oncucumber growth promotion under salt stress. Journal of Basic Microbiology, 2013, 53(4): 355-364; Li YT, Hwang SG, Huang YM, et al. Effects of Trichoderma asperellum on nutrient uptake and Fusarium wilt of tomato. Crop Protection, 2018, 110: 275-282.). Among them, Trichoderma asperellum is the most widely used in biocontrol (Zhang Xiaomeng, Tian Yongqiang, Pan Xiaomei, et al. Antibacterial effect of two Trichoderma strains and their plant growth promotion mechanism. Journal of Southern Agriculture, 2020, 51(11): 2713-2721; Dong Chunxin, Cui Yi, Niu Qichen, et al. Characteristics of Trichoderma asperellum and its biocontrol effect on two turf pathogens. Acta Grasslandica Sinica, 2022, 30(5): 1102-1109; Liu P, Yang R, Wang Z, et al. Biocontrol potential of Trichoderma asperellum CMT10 against strawberry root rotdisease. Horticulturae, 2024, 10(3): 2-16.).

[0005] motile actinomycetes (ActinoplanesRare actinomycetes are a class of actinomycetes with the potential to produce a variety of antibiotics and abundant enzymes and other bioactive substances. They have broad application prospects in medicine, industry, agriculture, forestry, and fisheries. (Ding T, Yang LJ, Zhang WD, Shen YH. The secondary metabolites of rare actinomycetes: chemistry and bioactivity. RSC Adv 2019;9:21964-21988; Vobis G, Schäfer, Kampfer P. Actinoplanes Couch 1950, 89AL emend. Stackebrandt and Kroppenstedt In: Whitman WB, Goodfellow M, Kämpfer P, Busse HJ, Trujillo ME, Ludwig W, Suzuki KI, Parte A (editor). Bergey's Manual of Systematic Bacteriology. New York, NY: Springer; 2012. pp.1058-1088. Phongsopitanun W, Matsumoto A, Inahashi Y, Kudo T, Mori M, Shiomi K, Takahashi Y, TanasupawatS. Actinoplanes lichenis sp. nov., isolated from lichen. Int J Syst EvolMicrobiol 2016; 66:468-473; Zeng J, Iizaka Y, Hamada M, Iwai A, Takeuchi R,Fukumoto A, Tamura T, Anzai Y. Actinoplanes kirromycinicus sp. nov., isolated from soil. J Antibiot (Tokyo) 2024; 77:657-664.).As early as the early 1950s, *Actinomycetes* strains were widely isolated and cultured by scientists both domestically and internationally (Couch JN. *Actinomycetes*. A new genus of the Actinomycetales. Journal of the Elisha Mitchell Scientific Society 1950; 66:87-92.). In 1964, the Institute of Pharmaceutical Biotechnology, Chinese Academy of Medical Sciences, isolated a *Actinomycetes* strain 3945-64 from soil in Jinan, Shandong, my country, and screened it to discover Chuangxinmycin, finding that Chuangxinmycin possesses good antibacterial activity and a novel target of action (Report from the Chuangxinmycin Identification Conference [J]. Pharmaceutical Industry, 1972, (01):24.). *Actinomycetes* strains are Gram-positive bacteria and strictly aerobic (https: / / lpsn.dsmz.de / genus / actinoplanes). On most solid culture media, the colonies exhibit vibrant colors, including orange, yellow, red, violet, and purple. Substrate hyphae are abundant and highly branched, while aerial hyphae are sparse. Aerial hyphae differentiate to form sporangia, which rupture upon maturation to release spores. The spores are spherical to nearly spherical, 1.0-1.5 μm in diameter, and motile via telogen flagella. The main peptidoglycan component of the cell wall of this genus is meso-diaminopimelic acid; the main fatty acid component includes iso-C. 15:0 C 18:1 ω9c and C 16:0 Some strains contain only iso-C as their main fatty acid component. 15:0 and iso-C 16:0 The main methylnaphthoquinone component is MK-9(H4) or MK-9(H6); the G+C content of genomic DNA is approximately 69-74% (Bergey's Manual of Systematic Bacteriology. New York, NY: Springer; 2012.pp.1058-1088.).

[0006] In agriculture, they are mainly used for the development of biopesticides. The various antibiotics and bioactive substances they produce can serve as potential biocontrol agents to inhibit the growth and reproduction of plant pathogens, thereby reducing the use of chemical pesticides and lowering the risks of environmental pollution and pesticide residues to human health. For example, neomycin can be used to control plant diseases. Zooplankton also have potential in environmental remediation. Certain enzymes and metabolites they produce can participate in environmental material cycling and pollutant degradation, helping to improve environmental quality such as soil and water. Summary of the Invention

[0007] The purpose of this invention is to provide a biological control method for cotton wilt disease.

[0008] In a first aspect, the present invention claims protection for a combination of bacteria or a compound bacterial agent containing said combination of bacteria.

[0009] The combined bacteria claimed in this invention consist of AT zooxygenes and Trichoderma hygroscopicum.

[0010] The AT zooplankton is AT zooplankton ( Actinoplanes atrianglus Q9316, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 35443; The *Trichoderma* species is *Trichoderma* ( Trichoderma asperellum F2609, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.42159.

[0011] Secondly, the present invention claims protection for *AT* zooxytrophic bacillus or bacterial agents containing said *AT* zooxytrophic bacillus.

[0012] The AT zooplankton is AT zooplankton ( Actinoplanes atrianglus Q9316, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 35443.

[0013] Thirdly, the present invention claims protection for Trichoderma acicularis or fungal agents containing Trichoderma acicularis.

[0014] The *Trichoderma* species is *Trichoderma* ( Trichoderma asperellum F2609, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.42159.

[0015] In the aforementioned microbial agents, the active ingredient may be the corresponding bacterial strain, its metabolites, and / or its culture. The active ingredient may also contain other biological and / or non-biological components. Other active ingredients in the microbial agents can be determined by those skilled in the art based on the desired effect.

[0016] In addition to the active ingredients, the aforementioned microbial agents, microecological preparations, or biofertilizers may also contain a carrier. The carrier may be a biologically inert carrier commonly used in the pesticide field. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, plant material, or polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of wheat flour, soybean flour, and starch; the polymer compound may be polyvinyl alcohol and / or polyethylene glycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and / or dodecane.

[0017] The above-mentioned microbial agents can be in various formulations, such as liquid, emulsion, suspension, powder, granules, wettable powder or water-dispersible granules.

[0018] Depending on the requirements, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH adjusters, etc. may also be added to the bacterial agent.

[0019] Fourthly, this invention claims protection for a product used to prevent and control cotton wilt disease.

[0020] The product for controlling cotton wilt claimed in this invention may contain the combined bacteria or a compound bacterial agent containing the combined bacteria as described in the first aspect above, or AT zooxygenes as described in the second aspect above, or a bacterial agent containing the AT zooxygenes, or Trichoderma echinosporum as described in the third aspect above, or a bacterial agent containing the Trichoderma echinosporum.

[0021] Fifthly, the present invention claims protection for a product used to inhibit the pathogen of cotton wilt disease.

[0022] The product for inhibiting the pathogen of cotton wilt, as claimed in this invention, may contain the combined bacteria or a compound bacterial agent containing the combined bacteria described in the first aspect above, or AT zooxygenes described in the second aspect above, or a bacterial agent containing the AT zooxygenes, or Trichoderma echinosporum described in the third aspect above, or a bacterial agent containing the Trichoderma echinosporum.

[0023] Furthermore, in the fourth and fifth aspects mentioned above, the product may be a biocontrol agent.

[0024] Sixthly, the present invention claims protection for any of the following applications: (A1) The combined bacteria or compound bacterial agent containing the combined bacteria described in the first aspect above, or the AT zooxygenes described in the second aspect above, or the bacterial agent containing the AT zooxygenes, or the Trichoderma hydatopterii described in the third aspect above, or the bacterial agent containing the Trichoderma hydatopterii, in the preparation of products for controlling cotton wilt disease; (A2) The combined bacteria or compound bacterial agents containing the combined bacteria described in the first aspect above, or the AT zooxygenes described in the second aspect above, or the bacterial agents containing the AT zooxygenes described in the third aspect above, or the bacterial agents containing the Echinococcus spp. described in the third aspect above, in the preparation of products for inhibiting the pathogen of cotton wilt; (A3) The combined bacteria or compound bacterial agents containing the combined bacteria described in the first aspect above, or the AT zooplankton or bacterial agents containing the AT zooplankton described in the second aspect above, or the Trichoderma hygroscopicum or bacterial agents containing the Trichoderma hygroscopicum described in the third aspect above, or the products described in the fourth and fifth aspects above, in the control of cotton wilt disease; (A4) The use of the combined bacteria or compound bacterial agents containing the combined bacteria described in the first aspect above, or the AT zooxycetes or bacterial agents containing the AT zooxycetes described in the second aspect above, or the Trichoderma hygroscopicum or bacterial agents containing the Trichoderma hygroscopicum described in the third aspect above, or the products described in the fourth and fifth aspects above, in inhibiting the pathogen of cotton wilt.

[0025] Seventhly, the present invention claims protection for any of the following methods: Method I: A method for controlling cotton wilt, which utilizes the combined bacteria described in the first aspect above, may include the following steps: (B1) The AT motile actinomycetes and the Trichoderma hydatopsus are mixed and inoculated into a fermentation medium and fermented to obtain a fermentation broth; (B2) Apply the fermentation liquid or the supernatant of the fermentation liquid to the cotton plants to be controlled or the cotton growing environment to control cotton wilt disease.

[0026] Method II: A method for inhibiting the pathogen of cotton wilt, comprising the use of the combined bacteria described in the first aspect above, and including the following steps: (C1) The AT motile actinomycetes and the Trichoderma hydatopsus are mixed and inoculated into a fermentation medium, and fermented to obtain a fermentation broth; (C2) Apply the fermentation liquid or the supernatant of the fermentation liquid to the cotton wilt pathogen, cotton plants suspected of carrying the cotton wilt pathogen, or cotton planting environment suspected of having the cotton wilt pathogen, thereby inhibiting the cotton wilt pathogen.

[0027] Method III: A method for controlling cotton wilt, comprising using *AT* zooplankton as described in the second aspect above or *Trichoderma hydatopsus* as described in the third aspect above, may include the following steps: (D1) The AT motile actinomycetes or the Trichoderma hydatopsus are inoculated into a fermentation medium and fermented to obtain a fermentation broth; (D2) Apply the fermentation liquid or the supernatant of the fermentation liquid to the cotton plants to be controlled or the cotton growing environment to control cotton wilt disease.

[0028] Method IV: A method for inhibiting the pathogen of cotton wilt, comprising the use of *AT* motile actinomycetes described in the second aspect above or *Trichoderma hygroscopicum* described in the third aspect above to inhibit the pathogen of cotton wilt, and may include the following steps: (E1) The AT motile actinomycetes or the Trichoderma hydatopsus are inoculated into a fermentation medium and fermented to obtain a fermentation broth; (E2) Apply the fermentation broth or the supernatant of the fermentation broth to the cotton wilt pathogen, cotton plants suspected of carrying the cotton wilt pathogen, or cotton planting environment suspected of having the cotton wilt pathogen, thereby inhibiting the cotton wilt pathogen.

[0029] Furthermore, the fermentation medium has a pH of 7.2 and a composition ratio of: 5g glucose, 10g malt extract, 5g yeast extract, 10g cottonseed cake, 20g soluble starch, 0.5g potassium dihydrogen phosphate, 5g ammonium sulfate, 3g calcium carbonate, 1g sodium chloride, and 1L deionized water.

[0030] Furthermore, during the fermentation culture, the culture temperature is 30°C and the culture time is 6 days.

[0031] In the aforementioned relevant aspects, the pathogen causing cotton wilt is Fusarium oxysporum.

[0032] In some embodiments of the present invention, the Fusarium oxysporum is Fusarium oxysporum CGMCC 3.11126.

[0033] Experiments have shown that the *Trichoderma hygroscopicum* provided in this invention (…) Trichoderma asperellum F2609 exhibits resistance to the pathogen of cotton wilt—Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum CGMCC 3.11126 has a strong inhibitory effect. The AT zooplankton provided by this invention ( Actinoplanes atrianglusQ9316 exhibits inhibitory activity against Fusarium oxysporum CGMCC3.11126; furthermore, the antibacterial activity of the fermentation broth co-fermented with strain F2609 and strain Q9316 against Fusarium oxysporum CGMCC3.11126 is greater than that of the fermentation broth fermented with a single strain. This invention is of great significance for the biological control of cotton wilt disease.

[0034] Depository Instructions Classification and nomenclature: AT motile actinomycetes ( Actinoplanes atrianglus ); Biomaterials derived from ginseng: Q9316; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: July 30, 2025; Registered with the China National Collection Center (CGMCC) No. 35443.

[0035] Classification and nomenclature: Trichoderma acicularis ( Trichoderma asperellum ); Biomaterial from ginseng: F2609; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: August 7, 2025; Registration number at the Preservation Center: CGMCC No. 42159. Attached Figure Description

[0036] Figure 1 These are colony photos of strain F2609 after being cultured on PDA medium at 30°C for 3 days. Photo A shows the top of the plate; photo B shows the back of the plate.

[0037] Figure 2 The phylogenetic tree constructed based on the ITS gene sequence shows the phylogenetic position of strain F2609.

[0038] Figure 3 The colony formed by strain Q9316 after culturing at 30°C for 72 hours on PYG medium.

[0039] Figure 4 A phylogenetic tree based on the 16S rRNA gene sequence is constructed to show the phylogenetic position of strain Q9316.

[0040] Figure 5The diagram shows the inhibitory effects of various microbial agents in this invention on Fusarium solani CGMCC 3.11126. In the diagram, A represents the fermentation broth of F2609; B represents the fermentation broth of Q9316; C represents the fermentation broth of (F2609+Q9316); and D represents the blank control. Detailed Implementation

[0041] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0043] Example 1: Isolation, identification, and antibacterial activity screening of strains F2609 and Q9316 I. Strain Isolation Both strains F2609 and Q9316 of this invention were isolated from cotton field soil in Liuyuan Town, Aksu Prefecture, Xinjiang Uygur Autonomous Region. The specific procedures for strain isolation are as follows: 2g of soil sample was weighed and dissolved in 18mL of sterile physiological saline to prepare a 10... -1 Soil suspension; then take 1 mL of 10 -1 The soil suspension was placed in 9 mL of sterile physiological saline and mixed thoroughly to obtain 10 -2 Soil suspension; prepared by sequentially diluting with sterile physiological saline to obtain 10 -4 Soil suspension diluted to a certain degree. Take 0.2 mL of 10... -4 A diluted soil suspension was evenly spread onto a PDA culture medium plate (formulation: 3 g / L potato starch). -1 20 g / L glucose -1 15g / L agar powder -1 Mix thoroughly with tap water to a final volume of 1000 mL (pH 6.0), and incubate at 30°C for 3 weeks to isolate filamentous fungi. Take 0.2 mL of 10... -4 The diluted soil suspension was evenly spread on PYG medium plates (formulation: peptone 3 g·L). -1 5 g·L yeast extract -1 10 g·L glycerol -1 betaine 1.25 g·L -1 Sodium pyruvate 1.25 g·L -1 15 g·L agar -1After mixing thoroughly with deionized water, bring the volume to 1000 mL (pH 7) and incubate at 30 °C for 3 weeks to isolate bacterial strains.

[0044] Single fungal colonies were picked from PDA medium and transferred to freshly prepared PDA plates for observation of colony morphology. Strain F2609 was isolated and purified from the PDA medium.

[0045] Single colonies of well-grown actinomycetes were picked from PYG medium and transferred to freshly prepared PYG plates. The strain was purified by the streak method, and a bacterial strain with the number Q9316 was obtained from it. Hereinafter referred to as strain Q9316.

[0046] For subsequent research, the pure strain was preserved in liquid nitrogen and frozen at -80°C using 20% ​​(v / v) glycerol as a protectant.

[0047] II. Strain Identification 1. Identification of strain F2609 (1) Observation of strain morphology Morphological observation of the strain: The morphology of spores and hyphae was observed under a microscope, referring to the Chinese Fungi (Volume 35) - Trichoderma and Bergey's Manual of Identification and the Handbook of Fungal Identification for morphological observation.

[0048] When strain F2609 was cultured on PDA medium at 30°C, the colonies were initially white, but subsequently turned green and whitish-green, producing a large number of green conidia. Figure 1 Under a microscope, 2-5 flask-shaped phialides were observed at the tips of the hyphae. These phialides were ampoules-shaped, swollen in the middle, and terminal to contain conidia. The conidia were spherical or oval, 2-4 μm in diameter. In summary, the morphological characteristics of strain F2609 are consistent with the typical characteristics of Trichoderma fungi. It is speculated that strain F2609 may be a Trichoderma fungus (Handbook of Fungal Identification).

[0049] (2) Identification of ITS gene sequence of strain and construction of phylogenetic tree Strain strain F2609 was transferred to PDA medium without agar and cultured at 30℃ for 160 r / min for 2 days. Mycelia were then collected and subjected to three freeze-thaw cycles at -20℃ and 60℃ to rupture the mycelia. Genomic DNA was extracted. Using this DNA as a template, the ITS gene was amplified by PCR. The PCR reaction mixture consisted of: ITS1 - 0.6 μL, ITS2 - 0.6 μL, template - 1 μL, dNTP Mix - 4 μL, 2×KOD buffer - 10 μL, KOD FX - 0.4 μL, and ddH2O - 4.4 μL. Amplification conditions: Pre-denaturation: 94℃, 5 min; 31 cycles: 98℃, 10 s; 56℃, 45 s; 68℃, 2 min; Extension: 68℃, 5 min. The ITS1 sequence is 5'-TCCGTAGGTGAACCTGCGG-3', and the ITS4 sequence is 5'-TCCTCCGCTTATATGC-3'. The successfully amplified PCR products were sent to Shanghai Biotechnology Co., Ltd. for sequencing.

[0050] The obtained ITS gene sequence was submitted to the NCBI database for comparison and analysis. The results showed that the ITS gene sequence of strain F2609 (SEQ ID NO:1) and that of *Trichoderma echinosporum* in the NCBI database were identical. Trichoderma asperellum The similarity to strain G7 was 100%. Strain F2609 was confirmed to be a member of the *Trichoderma* genus. *Trichoderma echinosporum* (…) was retrieved. Trichoderma asperellum Phylogenetic tree of ITS gene sequences of G7 and related strains (e.g.) Figure 2 As shown in the figure, strain F2609 and *Trichoderma echinococcus* G7 cluster together within the evolutionary branch of the *Trichoderma* genus, and the evolutionary distance between them is zero. Based on the morphological and genetic information characteristics of strain F2609, it can be determined that strain F2609 is *Trichoderma echinococcus*.

[0051] Trichoderma acicularis ( Trichoderma asperellum F2609 was deposited at the China General Microbiological Culture Collection Center on August 7, 2025, with the accession number CGMCC No. 42159. It will be referred to as strain F2609 from now on.

[0052] 2. Identification of strain Q9316 Strain Q9316 was grown on PYG medium at 30°C. Morphological, physiological and biochemical, cytochemical and gene-level studies were conducted on Q9316. Other special cases will be described.

[0053] (1) Observation of cell morphology and detection of physiological and biochemical characteristics of strain Q9316 The growth temperature range for strain Q9316 was 4, 10, 15, 20, 22, 25, 28, 30, 32, 35, 37, 40, and 45 °C; the growth salt concentration (NaCl) range was 0-11% (0-11 g / 100 ml) in 12 gradients (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 g / 100 ml); and the growth pH range was 4-11 in 8 gradients (pH 4, 5, 6, 7, 8, 9, 10, 11). The physiological and biochemical functions of the strain were assessed using API 50CH and API ZYM assay kits, as well as BIOLOG GEN III plates and corresponding procedures. Other physiological and biochemical characteristics of the strains, including Gram staining properties, oxygen requirement, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity, and cellulose hydrolysis activity, were mainly determined by referring to the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. 2001. Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press) and "Systematics of Actinomycetes" (Xu Lihua, Li Wenjun, Liu Zhiheng, et al. Systematics of Actinomycetes. Science Press, 2007.) for strain identification experiments.

[0054] Identification results showed that strain Q9316 is a Gram-negative, aerobic bacterium. After culturing on PYG medium at 30°C for 72 hours, the strain formed dried orange-yellow colonies. Figure 3 The strain's tolerance range for temperature is 22-37℃, its tolerance range for NaCl is 0-7%, and its tolerance range for acid and alkali is pH 6.0-8.0. The optimal growth conditions are 30℃, 0-5% NaCl, and pH 7.0.

[0055] The strain Q9316 tested positive for oxidase and catalase, and also tested positive for starch hydrolysis and gelatin liquefaction. It can utilize dextran, D-maltose, D-mannose, and D-fructose, but cannot utilize D-trehalose, sucrose, raffinose, etc. as its sole carbon and energy source.

[0056] Strain Q9316 and closely related bacteria Actinoplanes palleronii JCM 7626 T The differences in their physiological and biochemical characteristics are shown in Table 1.

[0057] Table 1. Strains Q9316 and closely related strains Actinoplanes palleronii JCM 7626 T Table of differences in physiological and biochemical characteristics

[0058] Note: + indicates positive; - indicates negative.

[0059] (2) Detection of cellular chemical classification characteristics of strain Q9316 The cytochemical components of strain Q9316, including fatty acids, quinone types, and polar lipids, were detected by gas chromatography (GC), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC) (Sasser M. Identification of bacteria by gasghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE:MIDI inc;1990. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye Met al. An integrated procedure for the extraction of bacterial isoprenoidquinones and polar lipids. J Microbiol Methods 1984;2:233–241.). The dominant fatty acid of strain Q9316 was C. 15:0、 C 18:0、 iso-C 15:0、 antesio-C 15:0、 C 17:1 ω9c, these major components are consistent with the major fatty acid components of rare actinomycetes strains, as shown in Table 2. The major fatty acid components of strain Q9316 conform to the taxonomic characteristics of the rare actinomycetes genus (Ding T, Yang LJ, Zhang WD, Shen YH. The secondary metabolites of rare actinomycetes: chemistry and bioactivity). RSC Adv 2019;9:21964-21988;VobisG, Schäfer, Kampfer P. Actinoplanes Couch 1950,89AL emend. Stackebrandt andKroppenstedt In: Whitman WB, Goodfellow M, Kämpfer P, Busse HJ, Trujillo ME, Ludwig W, Suzuki KI, Parte A (editor). Bergey’s Manual of Systematic Bacteriology(New York, NY: Springer; 2012. pp.1058-1088.). The specific contents of each fatty acid component and its closest relatives differ as shown in Table 2. The main respiratory quinone component of strain Q9316 is MK-9(H4), and it also contains trace amounts of MK-9(H6). In the cell membrane of strain Q9316, the polar lipid component is mainly phosphatidylglycerol (PE). The dominant fatty acids, quinones, and main polar lipid components confirm that strain Q9316 is a member of the genus *Actinomyces*. At the same time, the trace components and contents of the fatty acid composition of strain Q9316 can distinguish strain Q9316 from its closest relatives. Actinoplanes palleronii JCM 7626 T Distinguish them from each other.

[0060] Table 2, strain Q9316 and Actinoplanes palleronii JCM 7626 T Cellular fatty acid components

[0061] (3) Determination of the phylogenetic position of strain Q9316 Genomic DNA was extracted from strain Q9316 and sequenced. The 16S rRNA gene sequence (SEQ ID NO:2) was then compared online in an internationally authoritative bacterial taxonomy database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62: 716-721.). The results showed that strain Q9316 of this invention had a similarity of 96.3-97.57% with strains of the genus *Actinomyces*, indicating that strain Q9316 is a strain of the genus *Actinomyces*. Actinoplanes palleronii JCM 7626 TThe highest similarity was 97.57%, far below the 98.65% threshold for distinguishing prokaryotic species, suggesting that strain Q9316 may represent a new species in the genus *Actinomyces* (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int JSyst Evol Microbiol 2014;64:346–351.). A phylogenetic tree was constructed by retrieving the 16S rRNA gene sequences of representative strains of the genus *Actinomyces* with high similarity to strain Q9316. Figure 4 In the phylogenetic tree, strain Q9316 falls within the evolutionary branch of the genus *Actinomyces*, and is related to... Actinoplanes palleronii JCM 7626 T The clusters formed a stable subbranch. This result further supports the view that strain Q9316 is a member of the genus Actinobacteria.

[0062] To further clarify the phylogenetic position of the strain, the whole genome of strain Q9316 was sequenced. The obtained genome sequence was 11.23 Mbp in length, with a G+C content of 71.1%. The mean nucleotide similarity (ANI) between the whole genome sequence of strain Q9316 and that of its closest control strain was compared and calculated using EZbiocloud. Whole genome sequence analysis showed that strain Q9316 and its closest relative, *Actinoplanes palleronii* JCM7626, are closely related. T The average nucleotide similarity (ANI) was 70.2%, far below the 95% threshold for distinguishing prokaryotic gene species (Yoon SH, Ha SM, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity). Antonie van Leeuwenhoek (2017; 110: 1281–1286.). This result indicates that strain Q9316 of the present invention is a novel genera of the genus Actinobacteria.

[0063] In summary, strain Q9316 of this invention exhibits many significant differences from existing *Actinomyces* strains, including in physiological and biochemical aspects, cytochemistry, and genotypic characteristics. The above data fully demonstrate that strain Q9316 of this invention represents a novel species within the *Actinomyces* genus, and is named *AT Actinomyces* (…). Actinoplanes atrianglus ) 。 Strain Q9316 was deposited at the China General Microbiological Culture Collection Center on July 30, 2025, with the accession number CGMCC No. 35443. Hereinafter referred to as strain Q9316.

[0064] Example 2: Detection of antibacterial activity of strains F2609 and Q9316 against the pathogen of cotton wilt. In this embodiment, the specific pathogen of cotton wilt tested was *Fusarium oxysporum* (…). Fusarium oxysporum CGMCC 3.11126.

[0065] The antibacterial activity of strains F2609 and Q9316 was tested using *Fusarium oxysporum* CGMCC 3.11126 as the test bacterium. The inhibition zone method was employed. The specific test steps are as follows: (1) Strains F2609 and Q9316 were fermented separately. Preparation of fermentation broth: 1×10 8 At an inoculum size of CFU / mL, strains F2609 and Q9316 were inoculated into fermentation medium (formula: 5g glucose, 10g malt extract, 5g yeast extract, 10g cottonseed cake, 20g soluble starch, 0.5g potassium dihydrogen phosphate, 5g ammonium sulfate, 3g calcium carbonate, 1g sodium chloride, 1L deionized water, pH 7.2), respectively. The medium was placed on a shaker and cultured at 30℃ for 6 days at 180 rpm. The resulting culture was centrifuged at 4000 rpm to obtain the supernatant of the fermentation broth of strain F2609 and strain Q9316, which were labeled as F2609 fermentation broth and Q9316 fermentation broth, respectively.

[0066] (2) Co-fermentation of strain F2609 and Q9316, preparation of fermentation broth: strain F2609 and Q9316 were prepared at a spore ratio of F2609:Q9316 = 1:1, with a total inoculum of 1×10⁻⁶ spores. 8 CFU / mL was inoculated into the fermentation medium in (1), placed on a shaker, and cultured at 30°C for 6 days at a speed of 180 rpm. The resulting culture was centrifuged at a speed of 4000 rpm to obtain the supernatant of the fermentation broth co-fermented by strains F2609 and Q9316, which was labeled as (F2609+Q9316) fermentation.

[0067] (3) Preparation of blank fermentation broth control: After sterilizing the fermentation medium (formula as above), do not inoculate the culture medium. Place it on a shaker and incubate at 30°C for 6 days at a speed of 180 rpm. Centrifuge at a speed of 4000 rpm to obtain the blank fermentation broth, which is marked as blank control.

[0068] (4) Preparation of assay plates: Fusarium oxysporum CGMCC 3.11126 was used as the assay organism. Using Fusarium oxysporum CGMCC 3.11126 grown to the logarithmic phase, assay plates containing 10... 8 PDA medium plates with a spore concentration of CFU / mL.

[0069] (5) Antibacterial effect test: Take 2 mL each of the prepared F2609 fermentation broth, Q9316 fermentation broth, (F2609+Q9316) fermentation broth, and blank control, and soak a 6 mm diameter filter paper disc in each. Place the filter paper discs soaked in different liquids on the plates prepared in the previous step containing the test bacteria Fusarium oxysporum CGMCC 3.11126. Incubate the culture plates with the filter paper discs at 30℃ for 24 hours and observe the inhibition zone.

[0070] Repeat the above steps a total of 3 times. Record the diameter of the inhibition zone each time.

[0071] (6) Calculate the antibacterial index Calculate the antibacterial index using the formula below.

[0072] Antibacterial index = diameter of transparent ring / diameter of filter paper.

[0073] The average values ​​from three repeated experiments were recorded as the inhibition indices of bacterial agents F2609, Q9316, and (F2609+Q9316), which were 3.53, 1.41, and 4.4, respectively. It is evident that the inhibition index of the (F2609+Q9316) compound bacterial agent was significantly higher than that of the single bacterial agent. See Table 3 for details.

[0074] Table 3. Statistical table of antibacterial index of fungal agents F2609, Q9316, and (F2609+Q9316) against Fusarium oxysporum CGMCC 3.11126.

[0075] Results: Strains F2609 and Q9316 exhibited strong inhibitory activity against Fusarium oxysporum CGMCC 3.11126; the fermentation broth co-fermented with F2609 and Q9316 showed greater inhibitory activity against Fusarium oxysporum CGMCC 3.11126 than the fermentation broth co-fermented with a single strain. Figure 5 ).

[0076] Conclusion: The compound inoculum of strains F2609 and Q9316 of this invention has a significant antagonistic effect on Fusarium oxysporum, the pathogen of cotton wilt.

[0077] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A combination of bacteria or a compound bacterial agent containing said combination of bacteria, characterized in that: The combined bacteria consist of AT zooacti and Trichoderma hydatopsus; The AT zooplankton is AT zooplankton ( Actinoplanes atrianglus Q9316, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 35443; The *Trichoderma* species is *Trichoderma* ( Trichoderma asperellum F2609, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.42159.

2. *AT* motile actinomycetes or an inoculum containing said *AT* motile actinomycetes, characterized in that: The AT zooplankton is AT zooplankton ( Actinoplanes atrianglus Q9316, its registration number at the China General Microbiological Culture Collection Center is CGMCC No. 35443.

3. A fungal agent containing *Trichoderma echinosporum*, characterized in that: The *Trichoderma* species is *Trichoderma* ( Trichoderma asperellum F2609, its registration number at the China General Microbiological Culture Collection Center is CGMCC No.42159.

4. A product for controlling cotton wilt disease, comprising the combined bacteria of claim 1 or a compound bacterial agent containing the combined bacteria, or the AT zooxygenes of claim 2 or a bacterial agent containing the AT zooxygenes, or the Trichoderma hygroscopicum of claim 3 or a bacterial agent containing the Trichoderma hygroscopicum.

5. A product for inhibiting the pathogen of cotton wilt, comprising any one of the following: (a1) The combined bacteria as described in claim 1 or a compound bacterial agent containing said combined bacteria; (a2) The AT motile actinomycetes as described in claim 2 or an agent containing the AT motile actinomycetes; (a3) The Trichoderma hydathodes of claim 3 or a fungal agent containing the Trichoderma hydathodes.

6. Any of the following applications: (A1) The use of the combined bacteria of claim 1 or the compound bacterial agent containing the combined bacteria, or the AT zooxygenes of claim 2 or the bacterial agent containing the AT zooxygenes, or the Trichoderma hydatopsus of claim 3 or the bacterial agent containing the Trichoderma hydatopsus in the preparation of products for controlling cotton wilt; (A2) The use of the combined bacteria of claim 1 or the compound bacterial agent containing the combined bacteria, or the AT actinomycetes of claim 2 or the bacterial agent containing the AT actinomycetes, or the Trichoderma hydatopsus of claim 3 or the bacterial agent containing the Trichoderma hydatopsus in the preparation of a product for inhibiting the pathogen of cotton wilt; (A3) The application of the combined bacteria of claim 1 or the compound bacterial agent containing the combined bacteria, or the AT zooxycetes of claim 2 or the bacterial agent containing the AT zooxycetes, or the Trichoderma hygroscopicum of claim 3 or the bacterial agent containing the Trichoderma hygroscopicum, or the product of claim 4 or 5 in the prevention and control of cotton wilt; (A4) The application of the combined bacteria of claim 1 or the compound bacterial agent containing the combined bacteria, or the AT motile actinomycetes of claim 2 or the bacterial agent containing the AT motile actinomycetes, or the Trichoderma hydatopsus of claim 3 or the bacterial agent containing the Trichoderma hydatopsus, or the product of claim 4 or 5 in inhibiting the pathogen of cotton wilt.

7. Any of the following methods: Method I: A method for controlling cotton wilt disease, comprising the use of the combined bacteria described in claim 1 to control cotton wilt disease, including the following steps: (B1) The AT motile actinomycetes and the Trichoderma hydatopsus are mixed and inoculated into a fermentation medium and fermented to obtain a fermentation broth; (B2) Apply the fermentation liquid or the supernatant of the fermentation liquid to the cotton plants to be controlled or the cotton growing environment to control cotton wilt disease; Method II: A method for inhibiting the pathogen of cotton wilt, comprising the following steps: (The method utilizes the combined bacteria described in claim 1 to inhibit the pathogen of cotton wilt) (C1) The AT motile actinomycetes and the Trichoderma hydatopsus are mixed and inoculated into a fermentation medium, and fermented to obtain a fermentation broth; (C2) Apply the fermentation liquid or the supernatant of the fermentation liquid to the cotton wilt pathogen, cotton plants suspected of carrying the cotton wilt pathogen, or cotton planting environment suspected of having the cotton wilt pathogen, thereby inhibiting the cotton wilt pathogen. Method III: A method for controlling cotton wilt, comprising the following steps: using *AT* zooplankton as described in claim 2 or *Trichoderma hydathodes* as described in claim 3 to control cotton wilt. (D1) The AT motile actinomycetes or the Trichoderma hydatopsus are inoculated into a fermentation medium and fermented to obtain a fermentation broth; (D2) Apply the fermentation liquid or the supernatant of the fermentation liquid to the cotton plants to be controlled or the cotton growing environment to control cotton wilt disease; Method IV: A method for inhibiting the pathogen of cotton wilt, comprising the following steps: *AT* motile actinomycetes as described in claim 2 or *Trichoderma hydatopsus* as described in claim 3, inhibiting the pathogen of cotton wilt. (E1) The AT motile actinomycetes or the Trichoderma hydatopsus are inoculated into a fermentation medium and fermented to obtain a fermentation broth; (E2) Apply the fermentation broth or the supernatant of the fermentation broth to the cotton wilt pathogen, cotton plants suspected of carrying the cotton wilt pathogen, or cotton planting environment suspected of having the cotton wilt pathogen, thereby inhibiting the cotton wilt pathogen.

8. The method according to claim 7, characterized in that: The fermentation medium has a pH of 7.2 and a composition ratio of: 5g glucose, 10g malt extract, 5g yeast extract, 10g cottonseed cake, 20g soluble starch, 0.5g potassium dihydrogen phosphate, 5g ammonium sulfate, 3g calcium carbonate, 1g sodium chloride, and 1L deionized water.

9. The method according to claim 7 or 8, characterized in that: The fermentation culture was carried out at a temperature of 30°C for 6 days.

10. The product, application, or method according to any one of claims 5-9, characterized in that: The pathogen causing cotton wilt is Fusarium oxysporum.

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