Chrysosporium merdarium, biocontrol agent and application thereof

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Chaetomium globosum*, a biocontrol agent, and its applications. This invention provides a strain of *Chaetomium globosum* G3, with accession number CGMCCNO.40837. The *Chaetomium globosum* G3 provided by this invention possesses the ability to produce IAA, cellulase, and protease, and to dissolve inorganic phosphorus. Its mycelium exhibits a broad inhibitory effect on plant pathogens. Example results show that when *Chaetomium globosum* G3 is co-cultured with *Fusarium verticillatum* on plates, the inhibition rate against *Fusarium verticillatum* 'maize' reaches up to 79%, and the inhibition rate of *Fusarium verticillatum* spores reaches 57%. The cell-free fermentation broth of *Chaetomium globosum* G3 shows an inhibition rate of 51.55% against *Fusarium verticillatum* 'maize'. *Chaetomium globosum* G3 can be used as a biocontrol agent for fungal diseases, showing broad application prospects in the control of wilt disease and fungal toxin contamination.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Chaetomium globosum, a biological control agent, and its application. Background Technology

[0002] Fusarium wilt is a significant global disease. Especially in recent years, its incidence has increased rapidly in China's maize-growing areas, and the affected regions are widespread, leading to severe yield losses. *Fusarium verticillioides* is one of the main pathogens causing maize wilt. Besides reducing maize yield, *Fusarium verticillioides* also causes mycotoxin contamination; even in the absence of disease symptoms, it can produce large amounts of fumonisin in infected maize, endangering human and animal health.

[0003] Long-term and excessive use of chemical fungicides can damage soil ecology, aggravate environmental pollution, and is ineffective in controlling fungal diseases. Therefore, disease control strategies have shifted to safe and pollution-free biological control. Screening microorganisms in the rhizosphere soil of healthy plants as biological antagonists is an important way to develop and utilize microbial resources.

[0004] There are no existing reports on the ability of Chaetomium globosum to inhibit the growth of Fusarium verticillatum and simultaneously reduce the production of fungal toxins. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Chaetomium globosum, a biological control agent, and its application. The Chaetomium globosum G3 provided by this invention has a significant disease control effect and can reduce the toxin production of pathogens.

[0006] This invention provides a strain of Chaetomium globosum G3, with accession number CGMCCNO.40837.

[0007] This invention provides a biological control agent, comprising Chaetomium globosa G3 or its metabolites as described in the above technical solution.

[0008] Preferably, the spore concentration of Chaetomium globosa G3 in the biocontrol agent is ≥1×10⁻⁶. 6 CFU / g.

[0009] Preferably, the application form of the *Chaetomium globosa* G3 includes mycelium; the metabolites include cell-free fermentation broth and / or fermentation liquid.

[0010] Preferably, the method for preparing the mycelium includes: inoculating Chaetomium globosa G3 into a culture medium and culturing it to obtain a fermentation broth, then filtering it to obtain the mycelium;

[0011] The method for preparing the cell-free fermentation broth includes: inoculating Chaetomium globosa G3 into a culture medium and culturing it to obtain a fermentation broth; filtering the fermentation broth to obtain a cell-free fermentation broth.

[0012] Preferably, the culture temperature is 18℃~30℃ and the culture time is 2~7 days.

[0013] This invention provides the application of Chaetomium globosa G3 or the biological control agent described in the above-mentioned technical solutions in one or more of the following 1) to 5);

[0014] 1) Prevention and control of plant diseases;

[0015] 2) Inhibit the growth of pathogens and / or reduce spore production;

[0016] 3) Increase the content of defense enzymes in plants;

[0017] 4) Reduce fungal toxin content;

[0018] 5) Promote plant growth.

[0019] Preferably, the plant includes one or more of corn, wheat, watermelon, and tomato; the disease includes soil-borne fungal diseases.

[0020] Preferably, the pathogenic fungus includes one or more of the following: Fusarium verticillatum, Fusarium graminearum, wheat sheath blight fungus, Fusarium pseudograminearum, watermelon wilt fungus, solanum rot fungus, peanut white mold fungus, wheat root rot fungus, watermelon anthracnose fungus, and bitter melon wilt fungus.

[0021] Preferably, the fungal toxin includes fumonisin.

[0022] Beneficial effects of the present invention: The present invention provides a strain of Chaetomium globosum G3, with the preservation number CGMCC NO.40837. The Chaetomium globosum G3 provided by the present invention has the ability to produce IAA, cellulase, and protease, and to dissolve inorganic phosphorus. Its mycelium has a broad inhibitory effect on plant pathogens. Example results show that when Chaetomium globosum G3 is co-cultured with Fusarium verticillatum on plates, the inhibition rate against Fusarium verticillatum reaches up to 79%, and the inhibition rate of Fusarium verticillatum spores reaches 57%; the inhibition rate of Chaetomium globosum G3 fermentation broth against Fusarium verticillatum is 51.55%. This strain significantly reduces the spore production and fumonisin production of Fusarium verticillatum; when applied in pots, this strain can reduce the inhibition of Fusarium verticillatum on corn and increase the content of related defense enzymes in the plant. Chaetomium globosa G3 can be used as a biological control agent for fungal diseases in maize, and has broad application prospects in the control of maize wilt and fungal toxin contamination.

[0023] Biological Preservation Instructions

[0024] Chaetomium globosum G3 was deposited on September 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40837. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0025] 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.

[0026] Figure 1 The image shows the morphology of strain G3 in Example 1. In the image, A represents a plate image of Chaetomium globulum cultured for 4 days; B represents the hyphae of Chaetomium globulum G3 under a light microscope; C represents the spores of Chaetomium globulum G3 under a light microscope; and D represents a micrograph of the ascocarps of Chaetomium globulum G3 under a light microscope.

[0027] Figure 2 This is a phylogenetic tree of ITS sequence analysis for strain G3 in Example 2;

[0028] Figure 3 The image shows the plate inhibition effect of strain G3 with different culture days on Fusarium oxysporum in Example 4.

[0029] Figure 4 The bar graph shows the inhibition rate of cell-free fermentation broth of strain G3 in Example 4 against Fusarium oxysporum.

[0030] Figure 5 The image shows the antagonistic effect of cell-free fermentation broth of strain G3 in Example 4 on Fusarium verticillatum.

[0031] Figure 6 The image shows a comparison of the effects of potted corn in Example 5. In this image, ck represents no application of microorganisms, Cg represents the application of Chaetomium globosa G3, Fv represents the addition of Fusarium verticillatum, and F.v+Cg represents the addition of both Chaetomium globosa G3 and Fusarium verticillatum in the potted corn. Detailed Implementation

[0032] This invention provides a strain of Chaetomium globosum G3, with accession number CG MCCNO.40837. The ITS gene sequence of Chaetomium globosum G3 described in this invention is shown in SEQ ID NO.1. SEQ ID NO.1: ATCCTTTAATTGTGTGGCTGCGGAGGGATCATTACAGA.

[0033] The *Chaetomium globosum* G3 strain described in this invention was screened from healthy farmland with a wheat-maize rotation system that had been continuously fertilized with organic fertilizer for ten years, using wheat rhizosphere soil as the sample. *Chaetomium globosum* G3 can produce IAA, cellulase, and protease, and has the ability to dissolve inorganic phosphorus. *Chaetomium globosum* G3 has simple cultivation requirements, exhibits broad-spectrum antibacterial activity against plant pathogens, and can reduce the spore number and fumonisin production of *Fusarium verticillatum*. On PDA plates, *Chaetomium globosum* G3 presents as pale yellow colonies with petal-shaped edges; aerial hyphae are sparse and fluffy; after 4 days of growth, black spores are scattered on the colonies.

[0034] This invention extracts the ITS sequence of Chaetomium globosum G3 strain, performs a BLAST homology sequence search in GenBank, and constructs a phylogenetic tree of the ITS sequence using the Neighbor-Joining Tree method in MEGA 5.11 software. (See attached image) Figure 2 This indicates that strain G3 is a Chaetomium globosum strain.

[0035] This invention provides a biological control agent, comprising Chaetomium globosa G3 or its metabolites as described in the above technical solution.

[0036] In this invention, the spore concentration of Chaetomium globosa G3 in the biocontrol agent is preferably ≥1×10⁻⁶. 6 CFU / g.

[0037] In this invention, the application form of Chaetomium globosa G3 in the biocontrol agent preferably includes mycelium; the metabolites preferably include cell-free fermentation broth and / or fermentation liquid.

[0038] The preferred method for preparing the cell-free fermentation broth of this invention includes: inoculating *Chaetoceros globosum* into a culture medium, culturing to obtain a fermentation broth; filtering and sterilizing the fermentation broth to obtain a cell-free fermentation broth. The preferred culture temperature is 18℃~30℃, more preferably 25℃~28℃; the preferred culture time is 2~7 days, further preferably 3~6 days, more preferably 4~5 days; the preferred culture rotation speed is 160~200 rpm, further preferably 170~190 rpm, more preferably 180 rpm. The preferred method for filtering the fermentation broth is gauze filtration; the liquid obtained after filtration is the cell-free fermentation broth. Sterilization is preferably performed using a filter membrane, with a preferred pore size of 0.22 μm. This invention utilizes a cell-free fermentation broth containing only the metabolites of *Chaetoceros globosum* G3, which still exhibits antibacterial activity. The cell-free fermentation broth of *Chaetoceros globosum* G3 of this invention has an inhibition rate of 51.55% against *Fusarium verticillatum*.

[0039] The preferred method for preparing mycelia according to the present invention includes: obtaining mycelia after filtering the fermentation broth. The filtration of the fermentation broth is preferably performed using gauze. The liquid obtained after filtering the fermentation broth is cell-free fermentation broth; the solid obtained after filtering the fermentation broth is mycelia. The filtration is preferably performed using a filter membrane. The culture medium is preferably PDB culture medium; the preferred composition of the PDB culture medium is: 200g potato, 20g glucose, and 1000mL deionized water, at natural pH.

[0040] This invention provides the application of *Chaetoceros globosa* G3 or the biocontrol agent described in the above-mentioned technical solutions in one or more of the following 1) to 5):

[0041] 1) Prevention and control of plant diseases;

[0042] 2) Inhibit the growth of pathogens and / or reduce spore production;

[0043] 3) Increase the content of defense enzymes in plants;

[0044] 4) Prevention and control of fungal toxin contamination;

[0045] 5) Promote plant growth.

[0046] This invention provides the application of Chaetomium globosa G3 or the biological control agent described in the above-mentioned technical solutions in the control of plant diseases.

[0047] The plants described in this invention preferably include one or more of corn, wheat, watermelon, and tomato, more preferably corn. The diseases described in this invention preferably include soil-borne fungal diseases, and the soil-borne fungal diseases preferably include wilt. The pathogens of wilt described in this invention preferably include one or more of *Fusarium verticillatum*, *Fusarium graminearum*, *Fusarium graminearum*, *Fusarium pseudograminearum*, *Fusarium fusarium*, *Fusarium solani*, *Fusarium oleraceus ... In this invention, when *Chaetomium globosum* G3 mycelium is applied to the soil in the laboratory stage, the preferred application mass of *Chaetomium globosum* G3 mycelium is 0.7% to 1.5% of the soil mass, more preferably 1%. In the field experiment stage, the preferred application mass of *Chaetomium globosum* G3 mycelium is 2 to 2.5 kg / mu, more preferably 2.3 kg / mu. The preferred application time is 2 to 7 days before corn sowing. When the cell-free fermentation broth or fermentation liquid of *Chaetomium globosum* G3 is used for root irrigation, the preferred application rate is 3780 mL / mu, and the preferred application time is when the corn has grown to the stage of two leaves and one bud.

[0048] In this embodiment of the invention, after the *Chaetoceros globosa* G3 culture was used to obtain mycelia, the mycelia were mixed with soil at a mass ratio of 2%. *Chaetoceros globosa* alleviated the growth-inhibiting effect of *Fusarium oxysporum* on corn and increased the leaf area, stem diameter, and fresh weight of roots, stems and leaves of corn.

[0049] This invention provides the application of *Chaetoceros globosa* G3 or the biocontrol agent described in the above-mentioned technical solutions in inhibiting the growth of pathogens and / or reducing spore production. The preferred forms of application include soil application and / or root irrigation. In this invention, during the laboratory stage, when *Chaetoceros globosa* G3 mycelium is applied to the soil, the preferred application weight is 0.7%–1.5% of the soil mass, more preferably 1%; during the field experiment stage, the preferred application weight is 2–2.5 kg / mu, more preferably 2.3 kg / mu; the preferred application time is 2–7 days before sowing, further preferably 3–6 days, more preferably 4–5 days. When the cell-free fermentation broth or fermentation liquid of *Chaetoceros globosa* G3 is used for root irrigation, the preferred dosage is 3780 mL / mu, and the preferred time is when the corn has grown to the two-leaf-one-heart stage. The application amount of the cell-free fermentation broth or fermentation liquid described in this invention is the amount of the prepared stock solution, without concentration or addition of other nutrients.

[0050] In this embodiment of the invention, a co-culture method was used to verify the effect of *Chaetoceros globosum* G3 in inhibiting the growth of pathogens and / or reducing spore production. The co-culture method preferably includes co-culturing the *Chaetoceros globosum* G3 or the biocontrol agent with the pathogen. The spore count of *Chaetoceros globosum* G3 is preferably ≥10. 6 CFU / mL; the preferred spore count of Chaetomium globosa G3 in the biocontrol agent of this invention is ≥10. 6CFU / mL. The co-culture temperature of the present invention is preferably 18℃~28℃, more preferably 25℃~28℃; the co-culture time is preferably 2~7 days, more preferably 3~6 days, and more preferably 4~5 days. The pathogens described in this invention preferably include one or more of the following: *Furasium graminearum*, *Fusarium verticillioides*, *Rhizoctonia cerealis*, *Fusarium spp.*, *Fusarium oxysporum* f.sp. *niveum*, *Fusarium solani*, *Sclerotium rolfsii*, *Bipolaris sorokiniana*, *Colletorichum lagenerium*, and *Fusarium oxysporum* f.sp. *Momordicae*, more preferably *Furasium graminearum*, *Fusarium verticillioides*, and *Rhizoctonia cerealis*. The fungi causing the following diseases are listed: cerealis, Fusarium oxysporum f.sp. niveum, Fusarium solani, Sclerotium rolfsii, Bipolarissorokiniana, Colletorichum lagenerium, and Fusarium oxysporum f.sp. Momordicae.

[0051] This invention provides the application of *Chaetoceros globosa* G3 or the biocontrol agent described in the above-mentioned technical solutions in increasing the content of defensive enzymes in plants. The defensive enzymes of this invention preferably include one or more of polyphenol oxidase, phenylalanine ammonia-lyase, and peroxidase, more preferably polyphenol oxidase, phenylalanine ammonia-lyase, and peroxidase.

[0052] This invention provides the application of *Chaetoceros globosa* G3 or the biocontrol agent described in the above-described technical solutions in the prevention and control of fungal toxin contamination. The fungal toxins described in this invention preferably include fumonisin.

[0053] The preferred application of this invention includes co-culturing the *Chaetoceros globosum* G3 or the biocontrol agent with the pathogen to reduce the yield of fungal toxins. The spore count of *Chaetoceros globosum* G3 in this invention is preferably ≥10. 6 CFU / mL; the preferred spore count of Chaetomium globosa G3 in the biocontrol agent of this invention is ≥10. 6 CFU / mL. The co-culture temperature described in this invention is preferably 18℃~28℃, more preferably 25℃~28℃; the co-culture time is preferably 2~7 days, further preferably 4~7 days, and more preferably 7 days. The *Chaetoceros globosa* G3 of this invention significantly downregulated the expression levels of FUM1, FUM2, FUM21, FUM7, and FUM8 genes in *Fusarium verticillatum*, thereby reducing fumonisin production. In an embodiment of this invention, co-culturing *Chaetoceros globosa* G3 mycelial cakes with *Fusarium verticillatum* mycelial cakes resulted in a 11.48%~37.98% decrease in fumonisin production from *Fusarium verticillatum*.

[0054] The *Chaetoceros globosa* G3 of this invention exhibits good control effects against diseases caused by *Fusarium verticillatum*. In pot experiments of this invention, *Chaetoceros globosa* G3 reduced the inhibitory effect of *Fusarium verticillatum* on maize. *Chaetoceros globosa* G3 of this invention can be used alone or in combination as a biocontrol agent for maize wilt, and has broad application prospects in the control of soil-borne fungal diseases of maize.

[0055] In existing technologies, *Chaetoceros* can produce various bioactive compounds and is considered a source of novel bioactive metabolites. The effects of metabolites produced by *Chaetoceros* on plant protection have been reported; they can inhibit pathogens such as *Rhizoctonia solani*, *Chaetoceros*, *Alternaria*, *Glucosioides*, and *Alternaria*, thereby reducing the occurrence of soil-borne diseases such as wilt and damping-off. They are considered promising biocontrol agents for plant diseases. However, the control effect of *Chaetoceros* on *Fusarium verticillatum* of maize has not been reported, nor has its effect on fungal toxin production been reported. The *Chaetoceros* G3 of this invention has broad application prospects in the control of soil-borne fungal diseases in maize. Its mycelium, fermentation broth, and cell-free fermentation broth all have strong antibacterial effects, inhibiting spore production of pathogens and reducing the yield of fungal toxins, thus demonstrating broad application potential.

[0056] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1: Isolation of Chaetomium globosum

[0058] During the wheat heading stage, healthy wheat was collected from soils in Nanyang City, Henan Province that had been fertilized with organic fertilizer for 10 consecutive years. The collected wheat was placed in bags, placed on ice, and transferred to the laboratory where the rhizosphere soil was separated under sterile conditions.

[0059] The resulting rhizosphere soil suspension was serially diluted to 10. -2 10 -3 10 -4 10 μL of soil dilution was evenly spread onto Bengal red agar using a sterile spreader. After incubation at 28°C in the dark for 4 days, single colonies were picked, and fungi antagonistic to *Fusarium verticillatum* were screened using the plate confrontation method, yielding 5 strains, one of which was named G3. The culture characteristics of strain G3 on potato dextrose agar (PDA) medium are shown in the figure below. Figure 1 .

[0060] Example 2: DNA extraction and identification of bacterial strains

[0061] The strain obtained in Example 1 was inoculated into PDA medium and cultured at 28°C for 7 days to obtain hyphae. The hyphae were scraped off, flash-frozen in liquid nitrogen, and then pulverized in a high-speed tissue homogenizer. Fungal DNA (Solepro product number D2300) was extracted according to the kit instructions and stored at -80°C for later use. The ITS sequence of the strain was amplified by PCR using primers ITS1 (SEQ ID NO.2: TCCGTAGGTGAACCTGCGG) and ITS4 (SEQ ID NO.3: TCCTCGCTTATTGATATGC). The PCR reaction system (25 μL) consisted of: 12.5 μL of 2×F5 Taq PCR MasterMix (Beijing Zhuangmeng International Biotechnology Co., Ltd.), 1 μL of DNA template, 1 μL of each primer pair, and 9.5 μL of ddH2O. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, and 72℃ extension for 1 min, with 35 cycles of stages 2–4; and a final extension at 72℃ for 10 min.

[0062] The amplified products were analyzed by 1% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The specific ITS sequence is shown in SEQ ID NO.1. The obtained ITS sequences were searched for homology using BLAST in GenBank. A phylogenetic tree of the ITS sequences was constructed using the Neighbor-Joining Tree method in MEGA 5.11 software with 1000 repeated samplings. The results are shown in […]. Figure 2 .according to Figure 2It can be seen that strain G3 and NR 144851.1 *Chaetomium globosum* belong to the same evolutionary branch, indicating that the two strains are most closely related. Furthermore, based on morphological observation and physiological and biochemical characteristic analysis, the isolate was identified as *Chaetomium globosum*, specifically *Chaetomium globosum* G3.

[0063] Example 3: Detection of the growth-promoting ability of Chaetomium coccidioides

[0064] G3 culture of Chaetomium globosum: The strain obtained in Example 1 was inoculated on PDA medium and cultured at 28°C for 7 days. The mycelium spread throughout the culture dish and was punched into mycelial blocks of the same size using a 6 mm puncher for the following experiments.

[0065] 1. IAA production capacity

[0066] Treatment group: Three G3 bacterial blocks were inoculated into 100 mL Erlenmeyer flasks containing PDB liquid medium and tryptophan. The concentration of tryptophan in the PDB liquid medium was 3 mM. The flasks were shaken at 180 rpm and incubated at 28℃ for 7 days. Three parallel experiments were set up.

[0067] Control group: Erlenmeyer flasks contained PDB liquid culture medium and tryptophan. The concentration of tryptophan in the PDB liquid culture medium was 3 mM. No G3 bacterial blocks were inoculated. The flasks were shaken at 180 rpm and incubated at 28°C for 7 days. Three parallel experiments were set up.

[0068] On days 1 to 7 of culture, 2 mL of culture from both the treatment and control groups was centrifuged at 12,000 rpm and 4°C for 10 min to obtain the supernatant. The supernatant was mixed with an equal volume of Salkowski's reagent and reacted at 25°C in the dark for 30 min. The absorbance was measured at 530 nm. All values ​​are the average of three parallel experiments. Serially diluted pure IAA (5–100 mg / L) was used as the calibration curve for quantification: y = 0.0226x - 0.1596, R0. 2 =0.9954. The net IAA yield in the supernatant from day 2 to day 7 of culture was 1.79 mg / L, 3.39 mg / L, 5.86 mg / L, 7.71 mg / L, 9.32 mg / L, and 10.26 mg / L, respectively.

[0069] 2. Enzyme production capacity

[0070] The ability of strain G3 to produce amylase, cellulase, xylanase, pectinase and protease was detected by referring to existing technology (Wang Yan, Wang Liwei, Suo Meng, Qiu Zhijie, Wu Hao, Zhao Min, Yang Hongyan. Regulating Root Fungal Community Using Mortierella alpina for Fusarium oxysporum Resistance in Panax ginseng[J]. Frontiers in Microbiology, 2022, 13).

[0071] The culture medium for detecting amylase was prepared as follows: soluble starch was added to PDA medium, with a mass concentration of 0.2%. G3 bacterial pellets were inoculated into the above medium and cultured at 28°C for 7 days. The medium containing 0.2% soluble starch was stained with 1% iodine solution; a clear zone around the colony indicated the presence of amylase activity.

[0072] The formulation of the cellulase detection medium was as follows: Carboxymethyl cellulose was added to PDA medium, with a carboxymethyl cellulose concentration of 0.5%. G3 bacterial pellets were inoculated into the above medium and cultured at 28°C for 7 days. The medium containing 0.5% carboxymethyl cellulose was stained with 0.2% Congo red solution, followed by washing with 1M NaCl. A yellow halo around the colonies indicated the presence of cellulase activity.

[0073] The formulation of the medium for detecting xylanase was as follows: Xylan was added to PDA medium, with a xylan concentration of 0.5%. G3 bacterial pellets were inoculated into the above medium and cultured at 28°C for 7 days. The medium containing 0.5% xylan was stained with 0.2% Congo red solution, followed by washing with 1M NaCl. A yellow halo around the colony indicates the presence of xylanase activity.

[0074] Formula for pectinase detection medium: Add pectin to PDA medium, with a pectin concentration of 1%. Inoculate G3 bacterial pellets into the above medium and incubate at 28°C for 7 days. Add 1% CTAB (hexadecyltrimethylammonium bromide) to the 1% pectin medium. The presence of a clear zone around the colony indicates the presence of pectinase activity.

[0075] The formulation of the protease detection medium was as follows: Skim milk powder was added to PDA medium, with a mass concentration of 1% skim milk powder. G3 bacterial pellets were inoculated into the above medium and cultured at 28°C for 7 days. A clear zone was observed around the colonies on the 1% skim milk powder medium, indicating the presence of protease activity.

[0076] The results showed that *Chaetoceros globosa* G3 had the ability to produce IAA, cellulase, amylase, xylanase and protease, as shown in Table 1.

[0077] 3. Phosphorus solubility: The ability of G3 to dissolve inorganic phosphorus was evaluated according to existing technology (Wang Yan, Wang Liwei, Suo Meng, Qiu Zhijie, Wu Hao, Zhao Min, Yang Hongyan. Regulating Root Fungal Community Using Mortierella alpina for Fusarium oxysporum Resistance in Panax ginseng[J]. Frontiers in Microbiology, 2022, 13).

[0078] The inorganic phosphorus medium consisted of: 10g glucose, 0.3g NaCl, 0.3g KCl, 0.5g (NH4)2SO4, 0.3g MgSO4·7H2O, 0.03g MnSO4·4H2O, 0.03g ferrous sulfate, 5g Ca3(PO4)2, 18g agar, and 1L distilled water, with a pH of 7.0–7.2. G3 bacterial pellets were inoculated onto inorganic phosphorus medium plates and incubated at 28°C for 7 days. The presence of a clear zone around the colonies indicated their ability to dissolve inorganic phosphorus.

[0079] The results showed that *Chaetoceros globosa* G3 had the ability to dissolve inorganic phosphorus, as shown in Table 1.

[0080] Table 1. Ability of *Chaetoceros* to produce IAA and enzymes

[0081]

[0082] Note: In the table, "-" indicates no ability to produce the substance, "+" indicates a transparent ring diameter of 0-1cm, "++" indicates a transparent ring diameter of 1-2cm, and "+++" indicates a transparent ring diameter of 2-3cm.

[0083] Example 4: Inhibitory effect of Chaetomium globosum and fermentation broth on pathogens

[0084] 1. Determination of the inhibitory effect of Chaetomium globosum on pathogens using the plate confrontation method.

[0085] (1) Microbial *Chaetoceros globosa* G3 was cultured on PDA medium at 25℃ for 5 days. Agar cakes with mycelial growth were directly drilled in the middle of the medium using a 6mm punch. (2) Co-culture: Pathogens listed in Table 2 were placed on one side of a PDA medium plate. One pathogen was placed in each PDA medium plate. G3 agar blocks obtained in step (1) were placed 2cm away from the pathogens for co-culture. After co-culture at 28℃ for 4 days, the colony radius of the pathogens was measured. PDA medium dishes with blank agar blocks but inoculated with pathogens were used as the control group. The control group was the pathogen cultured alone. Two parallel experiments were set up for each pathogen co-culture and control group. The inhibition rate was calculated as follows: Inhibition rate (%) = (Control colony radius - Treatment colony radius) / Control colony radius × 100. The results are shown in Tables 2-1 and 2-2. Figure 3 The growth radius and inhibition rate of *Fusarium verticillatum* Fv were measured in the control group and during co-culture. During 7 days of co-culture, the inhibition rate of *Chaetoceros globosa* G3 against *Fusarium verticillatum* increased from 14% to 79%, causing *Fusarium verticillatum* mycelia to shrink and deform. Table 2-1 shows that *Chaetoceros globosa* G3 has an inhibitory effect on pathogens of different crops such as corn, wheat, watermelon, and tomato. G3 exhibits rapid growth on plates and has a significant advantage in nutrient and space competition. During the 3-day culture period, the inhibition rate of G3 against pathogens ranged from 38% to 82%. There were no restrictions on the source of pathogens; all pathogens were existing species obtained directly from the Pathology Laboratory of the College of Plant Protection, Henan Agricultural University, for ease of experimental operation. The inhibition rate was calculated as the mean ± standard deviation after calculating the inhibition rate in parallel experiments.

[0086] Table 2-1 Culture radius and inhibition rate of *Chaetoceros globosum* against 11 plant pathogens

[0087]

[0088]

[0089] Table 2-2 Culture radius and inhibition rate of *Chaetoceros globosum* against *Fusarium verticillatum*

[0090]

[0091] 2. Spore counting

[0092] Control group: Fusarium oxysporum was cultured separately in PDA medium for 4 and 7 days, and mycelial cakes were prepared using a 6 mm diameter punch. Three parallel experiments were set up for each.

[0093] Treatment group: The pathogen *Fusarium verticillatum* was placed on one side of a PDA medium culture dish, and a G3 agar block was placed 2 cm away from the pathogen for co-cultivation. After co-cultivation at 28℃ for 4 days and 7 days, mycelial discs were prepared using a punch. Three parallel experiments were set up for each treatment group.

[0094] The PDA medium consists of 200g potato, 20g glucose, 20g agar, and 1000mL deionized water at natural pH.

[0095] (1) Take fungal cakes of the same size as the control group and the treatment group, wash off the spores with 1 mL of sterile water, and then serially dilute to 10. -1 10 -2 The samples were counted using a microscope, and the counts were repeated twice. The results are shown in Table 3.

[0096] (2) Determination of toxin content: Take the same area of ​​fungal cakes from the control group and the treatment group, dry them in an oven at 60℃ until constant weight, then mix them with 1 mL of 60% methanol for 5 min, and then determine the total amount of fumonisins using the Pribolab Fumonisins ELISA kit (EKT-051-48 / 96T). The results are shown in Table 3.

[0097] Table 3 shows that the number of *Fusarium verticillatum* spores on the fourth day of *Chaetoceros globosa* G3 culture increased from 1.07 × 10⁻⁶ to 1.07 × 10⁻⁶. 6 Reduced to 0.64×10 6 The inhibition rate was 40.23%; the number of Fusarium verticillatum spores on the seventh day of culture increased from 3.83 × 10⁻⁶ to 40%. 6 Reduced to 1.61 × 10 6 The inhibition rate was 57.84%; the fumonisin production of Fusarium oxysporum decreased by 28.53% to 36.19%. The inhibition rates in Table 3 were obtained by first calculating the average value of parallel experiments and then calculating the inhibition rate, so there is no positive or negative error.

[0098] Table 3. Spore count and toxin content of Fusarium verticillatum after different culture days before and after treatment with Chaetomium globosum.

[0099]

[0100] Note: The independent samples t-test was used to analyze the significance of the treatments at a significance level of 0.05.

[0101] 3. Determination of the antibacterial rate of cell-free fermentation broth of strain G3 using the Oxford cup method.

[0102] (1) Preparation of fermentation broth: *Chaetoceros* was inoculated into potato dextrose liquid medium (composed of 200g potato, 20g glucose, and 1000mL deionized water, natural pH), and cultured on a shaker at 28℃ and 180rpm for 7 days. Fermentation broth was collected daily, and the concentration of the broth after 7 days was 0.032g / mL (the concentration was determined by pipetting 1ml of broth into a clean, dried aluminum box and drying it to constant weight in a 60℃ oven). The fermentation broth was filtered through gauze to remove mycelium, and the supernatant was then passed through a 0.22μm filter membrane to remove microorganisms. The supernatant was then placed in a sterile container as sterile fermentation broth for subsequent experiments and stored at 4℃. Two parallel experiments were set up.

[0103] (2) Antibacterial tests were conducted using G3 cell-free fermentation broth with different incubation times via the Oxford cup method: Pathogens were placed on one side of a PDA medium, and sterile Oxford cups were placed 2 cm away from the pathogens. 100 μL of sterile fermentation broth was added to each Oxford cup. Only one type of sterile fermentation broth was added to each PDA medium plate. After incubation at 28°C for 4 days, the colony radius of the pathogens was measured. A culture dish with added sterile water was used as a control. The antibacterial rate was calculated according to the formula in Example 4. The results are shown below. Figure 4 , Figure 5 As shown in Table 4, the colony radius decreases with increasing fermentation time. When using fermentation broth cultured for 7 days, the growth radius of the pathogen is 10.95 mm, and the inhibition rate reaches a maximum of 51.55% at this time. This indicates that G3 secretes antimicrobial substances that inhibit pathogens during the culture process. Two parallel experiments were set up. Figure 5 From left to right, the numbers represent cultivation day 0, day 1, day 2, day 3, day 4, day 5, day 6, and day 7.

[0104] Table 4. Antimicrobial activity of cell-free fermentation broth of strain G3 determined by the Oxford cup method.

[0105]

[0106]

[0107] Example 5: Effect of G3 on the expression of genes related to fumonisin synthesis in Fusarium oxysporum

[0108] Fumonisins are synthesized through proteins encoded by the fumonisin biosynthesis (FUM) gene cluster, which is composed of 15 co-regulated genes. Furthermore, the FUM21 gene, encoding a zinc cluster transcription factor, regulates the expression of almost all FUM genes. To investigate the mechanism by which G3 reduces fumonisin levels, key genes regulating FUM synthesis, including FUM1, FUM2, FUM21, FUM7, and FUM8, were selected for reverse transcription quantitative PCR.

[0109] The specific process is as follows:

[0110] 1. RNA Extraction and cDNA Preparation: Fv hyphae cultured alone and Fv+Cg co-cultured on PDA medium plates at 28℃ for 4 days were collected and ground into powder in a mortar pre-cooled by liquid nitrogen. Total RNA was extracted from the hyphae using RNApure trizol (Zhuangmeng ZP401-1). The RNA integrity concentration was detected by nucleic acid gel electrophoresis and quantified using a Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, Wilmington, Delaware, USA). The RNA was diluted to the same concentration of 1000 ng. First-strand cDNA was synthesized from 1000 ng of total RNA using the gDNAremover EX RT kit (gDNAremover, ZR108). Fv represents *Fusarium verticillatum*, and Cg represents *Chaetoceros globosa* G3.

[0111] 2. Quantitative Real-Time PCR: The total reaction volume for analysis was 20 μL, including 3 μL cDNA, 0.2 μL each of specific primers, and 10 μL 2×HQ SYBR qPCR Mix (Low ROX) (Beijing Zoman Biotechnology Co., Ltd., ZF502), which was then brought to 20 μL with dd H2O. Relative quantification was performed using Applied Biosystems QuantStudio 3 (ThermoFisher Scientific Inc.). The initial step was performed at 95°C for 30 s, followed by 40 cycles at 95°C for 10 s and 60°C for 10 s. The melting curve analysis range was set according to the recommended program of the quantitative PCR instrument used. The FvActin gene was used as a reference gene for relative quantification normalization. The relative expression level of each gene was calculated using 2... -ΔΔCt Methods were performed, with at least three biological replicates for each expression analysis and three technical replicates for each biological replicate. Primers used in quantitative real-time PCR are listed in Table 6, with FvActin listed as the reference gene.

[0112] The results are shown in Table 5. When co-cultured with G3, the expression levels of FUM1, FUM2, FUM21, FUM7, and FUM8 genes of Fusarium tumefaciens were significantly downregulated, reducing fumonisin production.

[0113] Table 5. Expression levels of fumonisin synthesis-related genes in Fusarium oxysporum cultured alone and in co-culture.

[0114]

[0115] Note: The independent samples t-test was used to analyze the significance of the treatments at a significance level of 0.05.

[0116] Table 6 Primers for quantitative real-time PCR of fumonisin synthesis-related genes (from 5' to 3')

[0117]

[0118] Example 6: Control effect of Chaetomium globosum on maize wilt disease

[0119] 1. Preparation of pathogenic soil: Fusarium oxysporum culture was added to the soil at a mass ratio of 5%. The Fusarium oxysporum culture was cultured on millet medium for 7 days. Pathogenic soil was set up as a control group (Fv). Twenty pots were set up for parallel experiments.

[0120] 2. Inoculate *Chaetoceros globosa* G3 into potato dextrose liquid medium and culture on a shaker at 28°C and 180 rpm for 7 days. Filter the fermentation broth using qualitative filter paper in a vacuum filter until the mycelia are filtered to a constant weight. Then, mix 1% by weight of pathogenic soil as the experimental group C.g+Fv. Set up 20 parallel experiments.

[0121] 3. CK group: Normal soil without the application of Fusarium oxysporum and Chaetomium globosa G3. Twenty pots were used in parallel experiments.

[0122] 4. Cg group: *Chaetomium globosa* G3 was inoculated into potato dextrose liquid medium and cultured on a shaker at 28°C and 180 rpm for 7 days. The fermentation broth was filtered off, leaving the mycelium. Then, it was mixed into normal soil at a ratio of 2% by mass, which was used as the experimental group Cg. Twenty pots were set up for parallel experiments.

[0123] The control group, experimental group, CK group, and Cg group were all cultured in a 28℃ light incubator for 28 days (light cycle of 16 hours per day and 8 hours per day in darkness). During the experiment, samples were taken from the experimental group and the control group every 7 days to measure growth indicators. The results are shown in Table 5.

[0124] The activities of defense enzymes in maize roots were measured on days 14 and 28 of culture, as shown in Table 6. The method was as follows: samples were taken on days 14 and 28 of culture. Maize roots were washed with distilled water, and 1 g of the sample was ground in a liquid nitrogen-cooled tissue homogenizer. The powder was transferred to sterile, enzyme-free centrifuge tubes and stored at -80°C for later use. The activities of polyphenol oxidase (PPO), lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL), and peroxidase (POD) were measured using kits from Beijing Solarbio Science & Technology Co., Ltd.

[0125] Table 7 shows that *Chaetoceros globosum* alleviated the growth-inhibiting effect of *Fusarium verticillatum* Fv on maize, and increased leaf area, stem diameter, and fresh weight of roots, stems, and leaves at different culture times. Figure 6 . Figure 6 The scale in the image is 10cm. Figure 6 From left to right, they are Ck, Cg, Fv, and C.g+Fv.

[0126] Table 7. Control efficacy of *Chaetoceros globosum* against maize wilt.

[0127]

[0128]

[0129] Note: The treatment groups in Table 7 were analyzed using Duncan's method with a significance level of 0.05 through one-way ANOVA.

[0130] Table 8. Results of defense enzyme activity assays in maize roots on days 14 and 28 of culture.

[0131]

[0132] Note: The treatment groups in Table 8 were analyzed using Duncan's method with a significance level of 0.05 through one-way ANOVA.

[0133] Table 8 shows that on day 14 of culture, Fv reduced the activities of lipoxygenase, phenylalanine ammonia-lyase, and peroxidase in maize roots, while C.g+Fv treatment significantly increased the activities of these enzymes. On day 28 of culture, Fv reduced the activities of polyphenol oxidase, phenylalanine ammonia-lyase, and peroxidase in maize roots, while C.g+Fv treatment significantly increased the activities of these three enzymes.

[0134] In summary, the *Chaetoceros globosa* G3 of this invention has broad application prospects in the prevention and control of soil-borne fungal diseases. Its mycelium, fermentation broth, and cell-free fermentation broth all have strong antibacterial effects and can be used alone or in combination as a biocontrol agent for maize wilt.

[0135] 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 Chaetomium globosum G3, characterized in that, The accession number is CGMCCNO.40837.

2. A biological control agent, characterized in that, Includes the *Chaetoceros globosa* G3 or its cell-free fermentation broth as described in claim 1.

3. A biological control agent, characterized in that, Includes the *Chaetomium globosa* G3 or its fermentation broth as described in claim 1.

4. The biological control agent according to claim 2 or 3, characterized in that, The spore concentration of Chaetoceros globosa G3 in the biocontrol agent is ≥1×10⁻⁶. 6 CFU / g.

5. The biological control agent according to claim 2 or 3, characterized in that, The application of Chaetomium globulus G3 includes hyphae.

6. The biological control agent according to claim 5, characterized in that, The method for preparing the mycelium includes: inoculating Chaetomium globosa G3 into a culture medium and culturing it to obtain a fermentation broth, which is then filtered to obtain the mycelium.

7. The biological control agent according to claim 2, characterized in that, The method for preparing the cell-free fermentation broth includes: inoculating Chaetomium globosa G3 into a culture medium and culturing it to obtain a fermentation broth; filtering the fermentation broth to obtain a cell-free fermentation broth.

8. The biological control agent according to claim 6 or 7, characterized in that, The culture temperature is 18℃~30℃, and the culture time is 2~7 days.

9. The use of Chaetomium globosa G3 as described in claim 1 or the biocontrol agent as described in any one of claims 2 to 8 in at least one of the following 1) to 5); 1) Prevention and control of plant diseases; 2) Inhibit the growth of pathogens and / or reduce spore production; 3) Increase the content of defense enzymes in plants; 4) Reduce fungal toxin content; 5) Promote plant growth.

10. The application according to claim 9, characterized in that, The plants include one or more of corn, wheat, watermelon, and tomato; the diseases include soil-borne fungal diseases.

11. The application according to claim 9, characterized in that, The pathogens include at least one of the following: Fusarium verticillatum, Fusarium graminearum, wheat sheath blight fungus, Fusarium pseudograminearum, watermelon wilt fungus, solanum rot fungus, peanut white mold fungus, wheat root rot fungus, watermelon anthracnose fungus, and bitter melon wilt fungus.

12. The application according to claim 9, characterized in that, The fungal toxins include fumonisins.

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