Fecalibacterium sp. yn2601 and application thereof

Abstract

This invention relates to a strain of *Faecalibacterium* YN2601 and its applications, wherein the *Faecalibacterium* ( Bacillus stercoris The preservation number of YN2601 is: CCTCC NO: M 2026289. The *Femtobacterium clavatum* YN2601 provided by this invention is specific for *Fusarium oxysporum* cuboid strain […]. Fusarium oxysporum f. sp. cubense ( Foc The 4th physiological race of banana wilt disease has a good control effect and can significantly promote the growth of banana plants.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a strain of *Femtobacterium faecium* YN2601 and its applications. Background Technology

[0002] Banana wilt, also known as Panama disease, is caused by Fusarium oxysporum Cuban variant (Fusarium oxysporum). Fusarium oxysporum f.sp. from Cuba , Fire This is a devastating soil-borne vascular disease caused by *Fusarium oxysporum*. Since its first report in Australia in 1874, it has rapidly spread to almost all banana-growing regions worldwide, posing a serious threat to the banana industry in major producing areas of my country, including Guangdong, Guangxi, Hainan, and Yunnan. Among the most prevalent diseases is infection with *Fusarium oxysporum* race 1 Cuban specific type (…). Fire 1) The wilt disease caused by this disease led to the global extinction of the 'Gros Michel' variety; while the currently prevalent tropical physiological race 4 ( Fire TR4 can infect most major banana varieties, including Cavendish, and is more damaging and difficult to control.

[0003] Currently, the prevention and control of banana wilt disease mainly faces the following difficulties and challenges: 1. Lack of highly resistant varieties and poor efficacy of chemical control: Despite ongoing breeding efforts, existing commercial banana varieties (especially the main 'Cavendish' series) generally lack resistance to Foc TR4. Furthermore, because the pathogen can survive in the soil for extended periods (thick-walled spores can survive for decades) and infect through the host's vascular system, chemical pesticides struggle to effectively reach their sites of action. Long-term use of chemical pesticides not only offers limited control but also leads to increased pathogen resistance, damage to the soil microecology, and environmental residues.

[0004] 2. Agricultural pest control measures are costly and incomplete: Agricultural measures such as crop rotation, soil disinfection, and removal of diseased plants can mitigate disease occurrence to some extent. However, due to bananas being a perennial crop and limited land resources, crop rotation is difficult to implement. Soil fumigation (using agents such as chloropicrin and dazomet) is costly and has a non-specific killing effect on the soil structure and beneficial microbial communities in the field, making it difficult to promote on a large scale as a routine method.

[0005] Currently, biological control is the mainstream approach, and the use of antagonistic microorganisms for biological control is considered one of the most promising ways to control banana wilt disease due to its environmental friendliness and strong sustainability. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a strain of *Femtobacterium faecium* YN2601 and its applications.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of this invention provides a strain of *Femtobacterium* YN2601, wherein *Femtobacterium* ( Dung bacillus The accession number of YN2601 is: CCTCC NO: M 2026289.

[0008] A second aspect of the present invention provides a microbial agent, wherein the active ingredient of the microbial agent is the *Femtobacterium clavatum* YN2601 as described in claim 1.

[0009] Furthermore, the bacterial agent is a liquid bacterial agent, and the effective viable count of *Femtobacterium clavatum* YN2601 in the liquid bacterial agent is not less than 1 × 10⁻⁶. 8 CFU / mL.

[0010] The third aspect of this invention provides the application of the *Faecalibacterium cladobacterium* YN2601 of the first aspect or the bacterial agent of the second aspect in the prevention and control of banana wilt disease and the promotion of banana plant growth; wherein the pathogen of banana wilt disease is *Fusarium oxysporum* var. *cubicans*. Fusarium oxysporum f. sp. from Cuba ( Fire )]4th physiological race.

[0011] Beneficial effects: The *Femtobacterium clavatum* YN2601 provided by this invention is a specific strain of *Fusarium oxysporum* var. *cubicans*. Fusarium oxysporum f. sp. from Cuba ( Fire The 4th physiological race of banana wilt disease has a good control effect and can significantly promote the growth of banana plants. Attached Figure Description

[0012] Figure 1 This is the phylogenetic tree of *Faecalibacterium* in this invention; Figure 2 This is a diagram showing the antibacterial effect of *Faecalibacterium tumefaciens* against *Fusarium wilt* of banana in this invention. The left image is a plate contrast diagram of *Faecalibacterium tumefaciens* against *Fusarium wilt* of banana, and the right image is the control (CK). Figure 3 This invention demonstrates the control effect of Bacillus faecalis fermentation broth on TR4, the pathogen of banana wilt. In the figure, a is a physical image of the product after YN2601+TR4 treatment, and b is a physical image of the product after CK+TR4 treatment. Figure 4 This is a diagram of the plant growth promotion experiment using Bacillus faecalis fermentation liquid in this invention. In the diagram, a is the treatment of watering with YN2601, and b is the control.

[0013] Biological Preservation The present invention provides fecal bacteria ( Dung bacillus YN2601, with accession number CCTCC NO: M2026289, is classified as follows: Dung bacillus YN2601 was deposited at the China Center for Type Culture Collection on January 29, 2026, at Wuhan University, Wuhan, Hubei Province, 430072, China. Detailed Implementation

[0014] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention. Example

[0015] , fecal bacteria ( Dung bacillus Acquisition, identification and cultivation of ) 1.1 Source of Materials fecal bacteria ( Dung bacillus YN2601, accession number: CCTCC NO: M 2026289, was isolated from mature apple fruit from Zhaotong, Yunnan Province.

[0016] 1.2 Culture medium NA medium: Medium formula: glucose 10g, agar 20g, beef extract 3.0g, yeast extract 1.0g, peptone 10g, distilled water 1000mL, pH 7.0. Liquid NA medium is prepared without agar.

[0017] 1.3 Separation Method Sample disinfection: After rinsing the apples with sterile water, surface disinfect them with 75% ethanol for 60 seconds, and finally rinse with sterile water and air dry. The percentages mentioned are mass fractions.

[0018] Isolation and culture: After the apples were disinfected, the apples were cut open with a sterile knife under aseptic conditions, and the internal pulp was used as the isolation material. The pathogens were isolated by streak plating on NA medium and cultured at 30°C for 1 day. After purifying the single colonies three times, the bacteria were stored at 4°C for later use.

[0019] 1.4 Classification and Identification The culture characteristics and morphological features of the strain were observed, and 16S rDNA amplification and sequence analysis were performed.

[0020] The observation and identification of culture traits and morphological characteristics were carried out in accordance with the "Methods for Plant Disease Research".

[0021] Molecular identification: Genomic DNA was extracted using a bacterial genomic DNA extraction kit. A partial 16S rDNA sequence of the strain was amplified by PCR using universal primers 27F / 1492R (5'-AGAGTTTGATCCTGGCTCAG3' / 5'-GGTTACCTTGTTACGACTT-3'). The primer sequences were synthesized by Shanghai Sangon Biotech Co., Ltd., and the PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. This product was then used as a template for further PCR amplification.

[0022] The PCR reaction system consisted of 50 μL: 0.5 μL TaKaRa LA-Taq, 2 μL DNA template, 5 μL 10 × PCR Buffer, 8 μL dNTP™ imprint, 2 μL each of forward and reverse primers, and 30.5 μL double-distilled water. The PCR program was: 94 °C pre-denaturation for 5 min, 94 °C for 30 s, 54 °C for 30 s, 72 °C for 1 min, for 30 cycles; extension at 72 °C for 7 min. After sequencing the PCR products, the sequencing results were BLASTed on the NCBI website. The sequence results are shown in SEQ ID NO.1.

[0023] Based on culture characteristics, morphological observation, physiological and biochemical tests, and 16S rDNA sequence analysis, this strain was identified as *Faecalibacterium faecium*. Dung bacillus Its code name is YN2601.

[0024] SEQ ID NO.1: 1.5 Cultivation and Preservation Methods Short-term culture and preservation: Inoculate on NA solid slant medium, and after the bacteria have grown sufficiently, seal the cotton plug with sealing film and store in a refrigerator at 2-8℃.

[0025] Long-term culture and preservation: The cells were preserved in an ultra-low temperature freezer using the glycerol preservation method.

[0026] , fecal bacteria ( Dung bacillus Test on the antibacterial activity of YN2601 against Fusarium wilt of banana Fusarium wilt of bananas, race 4 (abbreviated) Fire 4) As indicator bacteria, conventional confrontation culture methods were used to culture *Faecalibacterium* (…). Dung bacillus The antibacterial activity of strain YN2601 was determined. The pathogen causing banana wilt is strain YN2601. Fire 15-1 was isolated and preserved by the banana research team of the Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences. The culture medium for the pathogen of banana wilt disease was PDA medium.

[0027] PDA culture medium formula: 200 g potato, 20 g glucose, 15 g agar, 1 L water, pH 7.0.

[0028] The inhibition rate of the strains was determined using the plate confrontation method. The pathogen of banana wilt was transferred to PDA plates and incubated at 28℃ for 7 days. A 5 mm diameter mycelial cake was then punched along the edge of the colony using a sterilized punch and inoculated into the center of the PDA plate. Finally, fecaliths were picked up using an inoculation needle. Dung bacillus Mycelia were spotted onto the edge of PDA plates 25 mm from the center, with 4 spots symmetrically placed per plate. Each treatment was repeated 3 times. PDA plates inoculated only with the tested pathogen served as a control. The antibacterial effect was observed after incubation at 28 °C for 7 days, as shown in the attached figure. Figure 2 As shown; the diameter of the banana wilt pathogen (Foc 15-1) was determined using the cross-cross method.

[0029] Inhibition rate (%) = (Coronavirus colony diameter of control pathogen - Coronavirus colony diameter of treatment pathogen) / Control diameter × 100.

[0030] Experimental data: The colony diameters of the pathogen in the three treatments were 2.50 cm, 2.40 cm, and 2.40 cm, respectively; the colony diameters of the pathogen in the three control treatments were 8.85 cm, 8.90 cm, and 8.85 cm, respectively. The inhibition rates of the three replicates were 71.75%, 73.03%, and 72.88%, respectively. The average inhibition rate of *Faecalibacterium tumefaciens* against the pathogen of banana wilt was 72.55%. It has a strong inhibitory effect on the growth of the pathogen of banana wilt.

[0031] Determination of the control efficacy and growth-promoting effect of *Faecalibacterium tumefaciens* on potted banana wilt disease. Preparation of experimental banana seedlings: In a plastic greenhouse, tissue-cultured Brazilian banana seedlings (tissue-cultured seedlings from the Banana Research Laboratory of the Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences) were washed of the culture medium from their roots and transplanted into seedling bags. After the seedlings had grown 3-4 leaves (approximately one month), they were transplanted into plastic pots with a diameter of 11 cm and a height of 12 cm using vermiculite as the substrate. After transplanting, the seedlings were frequently watered to maintain moisture. Fertilizer was applied weekly (2g of compound fertilizer per plant dissolved in water, with a mass fraction of N-P2O5-K2O of 15-15-15) 1-2 times. Once the seedlings had grown 4-5 leaves (approximately one month), they were ready for use.

[0032] Preparation of the fermentation broth of the tested strain *Bacillus faecalis* in this invention: The cryopreserved strain YN2601 was streaked onto a solid NA medium plate and activated in a 28 ℃ incubator for 2 days. The activated antagonistic bacteria were then inoculated into liquid NA medium and cultured at 28 ℃ and 250 r / min for 5 days. The culture broth was filtered through four layers of sterile gauze to obtain the fermentation broth. A 1×10⁻⁶ concentration was prepared using sterile water. 8 Prepare fermentation broth containing antagonistic fungi at CFU / mL.

[0033] Preparation of spore suspension of *Fusarium wilt* spores: *Fusarium wilt* spores were streaked onto solid PDA agar plates and activated in a 28 ℃ incubator for 5 days. The activated *Fusarium wilt* mycelial cakes were then inoculated into liquid PDA agar and cultured at 28 ℃ and 250 r / min for 7 days. The culture medium was filtered through four layers of sterile gauze to obtain a spore suspension. The spores were then diluted with sterile water to a concentration of 1×10⁻⁶. 6 Prepare a solution of banana wilt spores at CFU / mL.

[0034] Banana Fusarium wilt control experiment in pots Control test method: The fermentation broth of the strain of the present invention resistant to banana wilt (Factobacillus fermentation broth: concentration 1×10⁻⁶) was used. 8 The solution (CFU / mL) was applied to the roots of the potted banana plants with uniform growth, 50 mL per plant, while the control plants were each treated with 50 mL of NA liquid culture solution. After 7 days, the roots of the potted banana plants were treated to prevent root damage, and a solution of 1×10⁻⁶ CFU / mL was applied to the roots. 6 Apply 50 mL of CFU / mL banana wilt spore solution (TR4) to the roots of potted banana plants, with a control of applying PDA liquid culture solution.

[0035] Control and investigation methods: Disease index was investigated 45 days after TR4 inoculation, examining both external symptoms (leaves) and internal symptoms (coronavirus tubers) for each treatment. Disease severity was determined based on the degree of browning in the longitudinal sections of banana leaves and corms, and the disease index and control effect were calculated. The disease index grading criteria are shown in Figure 1.

[0036] Table 1 Grading Standards for Banana Fusarium Disease

[0037] The potted plant control experiment included two treatments: A1: irrigation with NA liquid culture medium + Fusarium wilt spore liquid of banana (CK+TR4); A2: irrigation with Bacillus faecalis fermentation broth + Fusarium wilt spore liquid of banana (YN2601+TR4). The disease incidence of each treatment was investigated, the disease index of each treatment was calculated, and the control effect was calculated. The treatment results are shown in Table 2.

[0038] Table 2 Treatments in pot experiments

[0039] Potted plant control effect: Through pot experiment, 45 days after inoculation with TR4 spore solution, as... Figure 3 As shown, the leaves of banana plants inoculated only withered and turned yellow, with fewer leaves and shorter plants; while the leaves of banana plants inoculated with coccidioides fermentation broth remained mostly healthy, with more leaves and better plant growth. Furthermore, after dissecting the corms, it was observed that the corms inoculated with the antagonistic bacteria were almost completely unaffected by TR4 mycelia, with only a few corms showing slight browning, while the corms inoculated only with TR4 turned brown and showed more brown mycelia.

[0040] Forty-five days after inoculation with TR4, the disease index of the treatment treated with Bacillus faecalis fermentation broth was investigated, and the control effect was calculated. The results, shown in Table 3, indicate a significant difference in the bulb disease index between the treatment treated with Bacillus faecalis fermentation broth and the control. The bulb disease index of the treatment treated with Bacillus faecalis fermentation broth (YN2601+TR4) was 10.42, and the leaf disease index was 21.88; while the control bulb disease index was 45.84, and the leaf disease index was 53.13. The control efficacy of Bacillus faecalis fermentation broth (YN2601+TR4) for bulbs was 77.45%, and for leaves it was 57.59%. The strain Bacillus faecalis (YN2601) showed good control efficacy against banana wilt.

[0041] Table 3. Control efficacy of YN2601 against banana wilt disease.

[0042] Plant growth promotion experiment Plant growth promotion test method: The fermentation broth of the strain of the present invention that is resistant to banana wilt (Factobacillus fermentation broth: concentration 1×10⁻⁶) was used. 8 The solution (CFU / mL) was applied to the roots of potted banana plants with uniform growth, with 50 mL applied to each plant. The control group was treated with 50 mL of NA liquid culture solution per plant.

[0043] Plant growth promotion survey method: On the day of irrigation with Bacillus foetida ferment broth (0 dpi) and 45 days after irrigation with Bacillus foetida ferment broth (45 dpi), bioinformatics indicators such as plant height, pseudostem diameter, number of leaves, leaf length and leaf width of all treated banana plants were measured and recorded. In addition, bioinformatics indicators such as fresh weight of aboveground and underground parts of the plants were measured 45 days after irrigation with Bacillus foetida ferment broth.

[0044] Measurement and recording methods: Plant height: Measured from the ground to the point where the petioles of the two top leaves intersect; Leaf length: Measured from the tip of the first leaf at the top of the plant to the tip of the leaf; Leaf width: Measure the width of the widest part of the first leaf at the top of the plant; Pseudostem diameter: Measure the diameter of the base of the pseudostem about 1 cm above the ground using vernier calipers; Number of leaves: Record the number of green leaves per plant; Fresh weight of the above-ground parts: The weight of the banana plant above the base of the pseudostem, about 1 cm above the ground; Fresh weight of the underground part: The weight of the banana plant below the base of the pseudostem, about 1 cm above the ground.

[0045] Table 3 shows the bioinformatics indicators of banana plants treated with Bacillus faecalis fermentation broth on the same day (0 dpi) (0 dpi CK and 0 dpi YN2601). There were no significant differences in the five bioinformatics indicators between the two treatments.

[0046] The plant growth promotion experiment included two treatments: B1: irrigation with NA liquid culture medium, serving as the plant growth promotion experiment control (CK); B2: irrigation with fecal microbiota fermentation broth (YN2601). The treatment details are shown in Table 2.

[0047] Forty-five days after irrigating with the Bacillus foetida ferment broth, bioinformatics indicators of the treated banana plants were measured. Specific results are shown in Table 4 and... Figure 4As shown, the plant height, stem diameter, and number of leaves treated with ferrous sulfate fermentation broth (45 dpi YN2601) were significantly different from the control (45 dpi CK). Leaf length, leaf width, aboveground fresh weight, and underground fresh weight were all higher in the ferrous sulfate treatment than in the control. These results indicate that ferrous sulfate fermentation broth (45 dpi YN2601) treatment promoted plant height, leaf length, leaf width, number of leaves, stem diameter, and aboveground and underground fresh weight compared to the control, with some indicators showing significant increases. This suggests that ferrous sulfate fermentation broth (YN2601) has a growth-promoting effect on banana plants.

[0048] Table 4. Growth-promoting effects of *Bacillus foetida* (YN2601) on banana plants.

[0049] This strain is a functional bacterium with disease prevention and growth promotion effects. It can provide excellent strain resources in the fields of biological growth promotion and disease resistance in agriculture, and can lay the foundation for the development of microbial fertilizers for banana wilt disease.

[0050] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A strain of *Faecalibacterium* YN2601, characterized by: The fecal bacteria ( Bacillus stercoris The accession number of YN2601 is CCTCC NO: M 2026289, and its classification name is: Bacillus stercoris YN2601.

2. A microbial agent, wherein the active ingredient of the microbial agent is the *Femtobacterium clavatum* YN2601 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that: The bacterial agent is a liquid bacterial agent, and the effective viable count of *Femtobacter faecium* YN2601 in the liquid bacterial agent is not less than 1 × 10⁻⁶. 8 CFU / mL.

4. The application of the *Faecalibacterium tumefaciens* YN2601 as described in claim 1, the microbial agent as described in claim 2, or the microbial agent as described in claim 3 in the prevention and control of banana wilt and the promotion of banana plant growth; wherein the pathogen of banana wilt is *Fusarium oxysporum* var. *cubicans*. Fusarium oxysporum f. sp. cubense ( Foc )]4th physiological race.

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