Antagonistic bacteria yf lys026 and application thereof

Abstract

The application discloses antagonistic bacteria YFLYS026 and application thereof, relates to the field of biotechnology, and has a preservation number of CGMCC NO.35236 and a classification name of Burkholderia pyrrocinia Burkholderia pyrrocinia , and is preserved in the China General Microbiological Culture Collection Center on July 15, 2025. The antagonistic bacteria YFLYS026 are isolated and screened from rhizosphere soil of Lilium auratum plants, can effectively prevent and treat leaf anthracnose of Machilus japonica and root rot of Lilium auratum, play a great role in the prevention and treatment of anthracnose and root rot, and improve planting benefits of Machilus japonica plants and Lilium auratum.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antagonistic bacterium YFLYS026 and its applications. Background Technology

[0002] Mountain fragrant round leaves belong to the oil family ( Staphyleaceae ) Plant Mountain Fragrance Round ( Turpinia arguta The dried leaves of *Hedyotis diffusa*, also known as Qian Da Chui, Qi Cun Ding, E Zi Yao, She She Cao, and Bai Hua She She Cao, are mainly produced in Jiangxi, Fujian, Guangdong, and Guangxi provinces. It is cold in nature and bitter in taste, and has the effects of clearing heat and detoxifying, relieving sore throat and swelling, and promoting blood circulation and relieving pain. In Xinmin Township, Anyi County, Jiangxi Province, its cultivation scale has been continuously expanding, bringing significant economic and ecological benefits to the local area. However, recently, anthracnose has been found on the leaves of *Hedyotis diffusa* in this area. Small, round or oval reddish-brown spots appear on the surface of the leaves. As the spots expand and merge, large areas of the leaves wither and fall off, severely damaging the physiological functions of the *Hedyotis diffusa* plant and affecting the quality of the medicinal material. Similarly, *Lilium tigrinum* is slightly cold in nature and can clear the heart and relieve irritability, calm the mind and soothe the nerves. It is used for symptoms such as lingering fever after febrile diseases, mental confusion, insomnia, and depression. At the same time, it can also moisten the lungs and relieve coughs, and has a therapeutic effect on dry or hot coughs. In recent years, with the expansion of cultivation area and the increase in continuous cropping years, the occurrence of diseases in *Lilium davidii* has gradually become more severe, especially rot in medicinal parts, which seriously affects the yield and quality of *Lilium davidii* and is difficult to control, becoming a major factor limiting the large-scale planting of *Lilium davidii*. Currently, the control of anthracnose and root rot mainly relies on chemical pesticides. Long-term improper use not only easily pollutes the environment but also easily leads to the development of pesticide resistance in pathogens under strong selective pressure. Therefore, environmentally friendly biological control technologies will become a hot topic in plant disease control research. Summary of the Invention

[0003] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide an antagonistic bacterium YFLYS026 and its application.

[0004] The technical solution of the present invention is as follows:

[0005] An antagonistic bacterium, YFLYS026, with accession number CGMCC NO.35236, is classified as Burkholderia pyrrole. Burkholderia pyrrocinia It was deposited at the China General Microbiological Culture Collection Center on July 15, 2025.

[0006] Preferably, the antagonistic bacterium YFLYS026 has an inhibitory effect on the pathogens of anthracnose of Rhododendron simsii and root rot of Lilium acutum.

[0007] Preferably, the antagonistic bacterium YFLYS026 is resistant to anthrax bacteria. Colletotrichum fructicola Shared Fusarium Fusarium commune Fusarium circumflexum Fusarium annulatum, Fusarium verticillata Fusarium verticillioides It has an inhibitory effect.

[0008] Preferably, the antagonistic bacterium YFLYS026 is isolated and screened from the rhizosphere soil of the Lilium longiflorum plant.

[0009] Preferably, the separation and screening method includes the following steps:

[0010] S1: Collect rhizosphere soil from healthy dragon tooth lily plants, add sterile water, disperse, let stand, and take the soil supernatant for stepwise dilution to obtain soil suspension.

[0011] S2: Spread the soil suspension evenly on LB solid medium for culture and observe the growth of the colonies;

[0012] S3: Select single colonies with consistent morphological characteristics from LB solid medium, perform streak purification, and obtain purified strains;

[0013] S4: Inoculate the purified strain into LB liquid medium and culture to obtain a bacterial suspension of the purified strain;

[0014] S5: Plant pathogens and bacterial suspensions were co-cultured using the plate confrontation method to screen out bacterial strains that antagonize plant pathogens.

[0015] Preferably, the plant pathogens include at least one of the following: *Anthracnose causal agent*, *Lilium longiflorum* root rot pathogen, *Corn ear rot* pathogen, and *Achyranthes bidentata* leaf spot pathogen.

[0016] This invention also discloses the application of an antagonistic bacterium YFLYS026 or its culture medium or its bacterial culture or its fermentation broth in the preparation of products for the prevention and control of anthracnose in *Lilium lancifolium* or for the prevention and control of root rot in *Lilium longiflorum*.

[0017] Preferably, the product is a microbial agent or a pesticide.

[0018] Preferably, the product is applied by foliar spraying or root irrigation, or applied to the soil before planting.

[0019] The beneficial effects of this invention are: YFLYS026, an antagonistic bacterium isolated and screened from the rhizosphere soil of healthy Lilium longiflorum plants, can effectively prevent and control anthracnose of Lilium semperflorens and root rot of Lilium longiflorum. It plays a significant role in the prevention and control of anthracnose and root rot, thereby improving the planting benefits of Lilium semperflorens and Lilium longiflorum. Attached Figure Description

[0020] Figure 1The inhibitory effect of antagonistic strain YFLYS026 on *Anthracnose stenoptera* is shown. In the figure, A is the control graph for *Anthracnose stenoptera*, B is the inhibition graph for antagonistic *Anthracnose stenoptera*, and C is the inhibition diameter result. ,express P <0.01).

[0021] Figure 2 Morphological characteristics of the antagonistic strain YFLYS026.

[0022] Figure 3 Gram staining results for the antagonistic strain YFLYS026.

[0023] Figure 4 Phylogenetic tree of antagonistic strain YFLYS026.

[0024] Figure 5 To investigate the effect of antagonistic strain YFLYS026 on the mycelial growth of six plant pathogens, namely: 1: Fusarium oxysporum, 2: Fusarium circumsus, 3: Fusarium solanum, 4: Fusarium spp., 5: Fusarium verticillatum, and 6: Alternaria alternata.

[0025] Figure 6 The growth of the antagonistic strain YFLYS026 on PVK medium.

[0026] Figure 7 The growth of the antagonistic strain YFLYS026 on nitrogen-fixing bacteria Assumption solid medium.

[0027] Figure 8 The growth of the antagonistic strain YFLYS026 on CAS medium.

[0028] Figure 9 To determine the IAA production activity of the antagonistic strain YFLYS026; where CK: blank control; YFLYS026: color change of the culture medium of the antagonistic strain YFLYS026 after incubation in the dark for 30 min with Salkowski reagent.

[0029] Figure 10 This is the IAA standard curve.

[0030] Figure 11 Potted Sedum sarmentosum round-leaved plants under different treatments; A: CK group; B: anthracnose treatment group; C: antagonistic strain YFLYS026 treatment group.

[0031] Figure 12 Potted Lilium 'Dragon Tooth' plants under different treatments: A: CK group; B: root rot treatment group; C: antagonistic strain YFLYS026 treatment group. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] Example 1: Screening of bacterial strains

[0035] 1. Soil sampling

[0036] Preparation of LB solid medium: Weigh 10.0 g peptone, 5.0 g yeast extract, 5.0 g sodium chloride, 1.0 g glucose, and 15.0 g agar, adjusting the final pH to 7.0 ± 0.2. Add 1 L of deionized water, stir, heat to boiling until completely dissolved, and then autoclave at 121℃ for 15 min. All liquid culture media used in the experiment were based on the solid medium formulation without agar.

[0037] Healthy *Lilium longiflorum* plants were collected from the *Lilium longiflorum* planting area in Yongfeng County, Ji'an City, Jiangxi Province, using a five-point sampling method. These plants were transported to the laboratory at low temperatures and stored at -20°C. The soil adhering to the surface of the healthy *Lilium longiflorum* roots was shaken off, and the plants were air-dried and passed through a No. 4 pharmacopoeia sieve to remove impurities before use.

[0038] 2. Strains Isolation and Purification

[0039] Accurately weigh 1g of rhizosphere soil from each plant into a 15mL sterile centrifuge tube, add 10mL of sterile water, and incubate on a shaker (30℃, 140 r / min) for 30min. Sonicate for 30s to fully mix the soil sample with the water, thereby dispersing the rhizosphere microorganisms in the soil. Let stand for 15min.

[0040] Take 1 mL of soil supernatant and dilute it stepwise to 10. -6 Take 100 μL of 10 -4 ~10 -6A concentrated soil suspension was evenly spread onto LB solid medium. After the surface liquid dried, the medium was inverted and incubated at 37°C for 2-3 days. Each day, based on colony morphology (color, size, elevation, edge characteristics, etc.), single colonies of different morphologies were selected for further purification using the streak plate method until the colony characteristics on each plate were essentially consistent, thus obtaining a pure culture strain. The pure culture strain was inoculated into 2 mL of LB liquid medium and incubated on a shaker (37°C, 220 rpm / min) for 18-24 h. Finally, the bacterial suspension was mixed with 50% glycerol in a cryovial at a 1:1 volume ratio and stored at -80°C.

[0041] Example 2: Culture of pathogenic bacteria

[0042] PDA solid culture medium preparation: 5g potato extract powder, 20g glucose, 15g agar, pH at rest, autoclave at 121℃ for 15min.

[0043] Tested plant pathogens: Anthracnose of *Sedum morganianum* (anthracnose fungus: *Sedum morganianum*) Colletotrichum fructicola ), root rot of Lilium acutum (Fusarium oxysporum: Fusarium oxysporum Fusarium solani: Fusarium solani Shared Fusarium: Fusarium commune Fusarium circumflexum: Fusarium annulatum Corn ear rot (Fusarium verticillata: Fusarium verticillioides ), Leaf spot disease of Alternaria alternifolia (Alternaria: Alternaria alternata The aforementioned plant pathogens were isolated and purified in our laboratory from infected leaves of *Ligustrum lucidum*, bulbs of *Lilium tigrinum*, and leaves of *Heliotropium indicum*, and have undergone molecular identification and pathogenicity testing. The strains were activated and cultured on PDA solid medium at 28°C before testing.

[0044] Example 3: Screening and Identification of Antagonistic Strains

[0045] 1. Preparation of bacterial suspension

[0046] The purified bacterial colonies were picked and inoculated into LB liquid medium. The culture was carried out overnight at 37°C and 200 rpm / min on a constant temperature air bath shaker for 18 hours to obtain the OD. 600 A bacterial suspension of a purified strain with a concentration of approximately 0.8.

[0047] 2. Screening of antagonistic strains

[0048] The plate confrontation method was used to co-culture plant pathogens with multiple bacterial strains in the suspension of purified bacterial strains to observe and screen bacterial strains antagonistic to plant pathogens. The specific experimental procedure is as follows: Using a sterilized punch, a 5mm diameter mycelial cake was collected from the edge of the activated *Anthracnose* colony as the target pathogen. This cake was inoculated into the center of a PDA solid medium plate, approximately 3 cm away from the mycelial cake. A thin line was drawn symmetrically on each side of the plate using an inoculation loop dipped in bacterial culture solution incubated for 18 hours. Plates inoculated only with the pathogen served as controls. This process was repeated three times. The plates were placed upright in a 28℃ incubator for 7 days. The growth of the pathogen mycelium in the experimental group was observed by monitoring the growth of the pathogen in the control plates. The antagonistic strain with the best inhibitory effect on *Anthracnose rotundifolia* was screened. The diameter of the pathogen mycelium after 7 days of growth was measured using the cross-sectional method, and the growth inhibition rate was calculated using the following formula:

[0049] ;

[0050] See results Figure 1 The results showed that a total of 7 bacterial strains had an inhibitory effect on Bacillus anthracis, among which strain YFLYS026 had the best inhibitory effect. The average hyphal diameter of the control plate was approximately 74.79 mm, while the average inhibition diameter of the treatment group was approximately 44.14 mm, with an inhibition rate of 59.02%. This indicates that strain YFLYS026 can significantly inhibit the hyphal growth of Bacillus anthracis (see...). Figure 1 ).

[0051] 3. Identification of antagonistic strain YFLYS026

[0052] (1) Morphological identification

[0053] Morphological identification of strain YFLYS026 after incubation in TSA medium at 30°C for 24 h is shown in the figure. Figure 2 ;

[0054] Gram staining results of strain YFLYS026 are shown below Figure 3 .

[0055] (2) Physiological and chemical classification and identification

[0056] The fatty acid composition of strain YFLYS026 was detected using the Microbial Fatty Acid Rapid Identification System (MIDI), as shown in Table 1.

[0057] Table 1. Fatty acid composition of strain YFLYS026

[0058] In the table, / indicates: none.

[0059] Table 1 shows that the main fatty acid of the strain is C. 16:0 C17:0 cyclo, C 16:0 3OH and C 19:0 Cyclo ω8c, with contents of 24.86%, 16.48%, 5.56%, and 4.22%, respectively. This conforms to the genus Burkholderia (…). Burkholderia The main cellular fatty acid characteristics.

[0060] The API 50CH data for strain YFLYS026 are shown in Table 2.

[0061] Table 2. API 50CH data for strain YFLYS026

[0062]

[0063]

[0064] Note: + indicates positive; - indicates negative; w indicates weak positive.

[0065] Table 2 shows the API 50 CH test results: Positive reactions included aesculin, maltose, lactose, D-rockose, and D-arabinitol; weakly positive reactions included D-arabinose, L-arabinose, galactose, cellobiose, trehalose, and L-rockose; negative reactions included control, glycerol, erythrose, ribose, D-xylose, L-xylose, azircol, β-methyl-D-xyloside, glucose, fructose, mannose, sorbitol, rhamnose, and euonymus alcohol. Inositol, mannitol, sorbitol, α-methyl-D-mannoside, α-methyl-D-glucoside, N-acetyl-glucosamine, amygdalin, arbutin, salicylic acid, melibiose, sucrose, inulin, pinosyl, raffinose, starch, glycogen, xylitol, geraniol, D-pinobiose, D-lysose, D-tagatose, L-arabinitol, gluconate, 2-keto-gluconate, and 5-keto-gluconate. Conforms to Burkholderia spp. ( Burkholderia The biochemical metabolic characteristics of ).

[0066] (3) Molecular biological identification

[0067] 16S rRNA gene analysis of strain YFLYS026: Using universal 27F (see SEQ ID NO. 1) and 1492R (see SEQ ID NO. 2) primers, the 16S rRNA gene sequence was determined (see SEQ ID NO. 3), yielding a 1429 bp gene fragment. The sequence was compared with known type bacteria via the website (http: / / www.ezbiocloud.net / eztaxon / ), and the results are shown in Table 3 below. (Strain and known species...) Burkholderia ambifaria AMMD TThe highest similarity was 99.78%. For the phylogenetic analysis of the 16S rRNA of strain YFLYS026, please refer to [link to relevant documentation]. Figure 3 .

[0068] Table 3 shows the results of the comparison analysis of the 16S rRNA gene sequence with strain LEB26.

[0069]

[0070] strain YFLYS026 gyrB Gene sequence analysis: using bacteria gyrB Using universal UP-1 (see SEQ ID NO.4) and UP-2r (see SEQ ID NO.5) as primers, the determination was performed. gyrB A conserved gene fragment of 1207 bp was obtained. Comparison with NCBI revealed that strain YFLYS026 is related to a known type bacterium. Burkholderia pyrrocinia DSM 10685 T The highest similarity was 98.50%. A phylogenetic tree was constructed between strain LEB26 (see SEQ ID NO.6) and its closely related species. It was observed that strain YFLYS026 showed the highest similarity. Burkholderia pyrrocinia ATCC 15958 T They cluster into the same branch.

[0071] Identification results: Based on the above 16S rRNA gene sequence and... gyrB Conserved gene sequence analysis, Gram staining, colony morphology, and physiological and biochemical characteristics (whole-cell fatty acids and API 50 CH) analysis led to the identification of strain YFLYS026 as... Burkholderia pyrrocinia (Burkholderia pyrrole), this identification result is limited to the current state of this strain.

[0072] (4) Preservation of strains

[0073] strain YFLYS026 (Burkholderia pyrrole) Burkholderia pyrrocinia It was deposited on July 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.35236. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0074] (5) Determination of the antibacterial spectrum and analysis of the growth-promoting properties of the antagonistic strain YFLYS026

[0075] Antimicrobial spectrum of antagonistic strain YFLYS026: To understand whether strain YFLYS026 has a broad-spectrum antimicrobial effect against plant pathogens, the plate confrontation method was used to observe whether this strain inhibited the growth of mycelia of six tested plant pathogens (Fusarium oxysporum, Fusarium circumcisionum, Fusarium solanum, Fusarium spp., Fusarium verticillatum, and Alternaria alternata). 5 mm diameter fungal discs were punched at the edge of pathogen colonies using a puncher as target fungal discs. Single colonies of strain YFLYS026 were collected with an inoculation loop and incubated overnight in LB broth to allow the OD of the culture solution to reach the target concentration. 600 The bacterial suspension of strain YFLYS026 was obtained with a concentration of approximately 0.8. A small amount of pathogenic fungal mycelium was placed in the center of a 90 mm diameter PDA solid medium plate, and the bacterial suspension of strain YFLYS026 was streaked 3 cm away from the mycelium. Plates inoculated only with pathogenic fungal mycelium served as controls. Each treatment was repeated in triplicate. The plates were incubated at 28°C for 7 days, and the mycelial diameter of the pathogenic fungi was measured using the cross-hatching method after 7 days. The effect of strain YFLYS026 on the mycelial growth of the six pathogenic fungi was calculated according to the aforementioned formula for fungal growth inhibition rate.

[0076] Experimental results showed that this strain inhibited the growth of all six tested plant pathogens to varying degrees, with an average growth inhibition rate of 46.62%–59.02% (see...). Figure 5 ).

[0077] Analysis of the growth-promoting characteristics of antagonistic strain YFLYS026:

[0078] Assay for the inorganic phosphorus solubility activity of the strain: The test strain YFLYS026 was inoculated onto PVK (inorganic phosphorus solid medium, Beijing Kulaibo Technology Co., Ltd.) medium, repeated three times, and incubated at 37℃ for 4 days. Colonies with clear zones (see...) Figure 6 This indicates that the strain has the ability to dissolve inorganic phosphorus. The results show that a white transparent ring forms around strain YFLYS026, indicating that the strain has phosphorus-dissolving activity.

[0079] Nitrogen fixation activity assay: The nitrogen fixation ability of the test strains was determined using Assumption solid medium (Beijing Kulaibo Technology Co., Ltd.). The test strain YFLYS026 was inoculated onto this medium and cultured at 28℃ for 5 days. Strains that could grow normally on the medium possessed nitrogen fixation ability. The results showed that strain YFLYS026 could grow on this medium, indicating that this strain has nitrogen fixation ability (see...). Figure 7 ).

[0080] Determination of the ability of the strain to produce ferrophosphate: CAS (chromium azure) medium: (1) CAS detection plate formulation: CAS staining solution contains 1 mmol / L CAS, 0.1 mmol / L FeCl3, 4 mmol / L hexadecyltrimethylammonium bromide (HDTMA), 0.1 mol / L phosphate buffer (pH = 6.0); each 100 mL contains 2.427 g of disodium hydrogen phosphate (Na2HPO4·12H2O), 0.5905 g of sodium dihydrogen phosphate (NaH2PO4·2H2O), 0.075 g of KH2PO4, 0.250 g of NH4Cl, and 0.125 g of sodium chloride. Dilute 10 times with sterile water before use. (2) Preparation of CAS detection plates: For every 100 mL of plate, add 1 mL of 20% sucrose solution, 3 mL of 10% acid-hydrolyzed casein, 100 μL of 1 mmol / L CaCl2, 2 mL of 1 mmol / L MgSO4, and 1.8 g of agar. Slowly add 5 mL each of salt solution and CAS staining solution at approximately 60°C to obtain the blue detection medium. When the iron ions in the blue detection plate are removed by the ferrophiles secreted by the microorganisms, an orange-yellow halo will form around the colonies where the microorganisms grow.

[0081] The heparin-producing ability of strain YFLYS026 was analyzed using CAS medium. 2 μL of bacterial suspension (OD) was collected via spot inoculation. 600 = 0.8) was inoculated onto CAS solid medium and incubated in the dark at 37°C for 4 days. The presence of an orange-yellow halo around the colonies was then observed to determine whether the strain possessed the ability to produce ferroptosis. The results showed that strain YFLYS026 had an orange-yellow halo around its colonies, indicating its ability to produce ferroptosis (see...). Figure 8 ).

[0082] Determination of the strain's ability to produce indole-3-acetic acid (IAA): The Salkowski colorimetric solution was prepared by adding 1 mL of 0.5 mol / L FeCl3 to 50 mL of 35% perchloric acid. The Salkowski colorimetric method was used for qualitative analysis of IAA production by the growth-promoting strain. Strain YFLYS026 was inoculated into LB medium containing 0.5 g / L tryptophan, with a blank control. The culture was incubated at 37℃ with shaking at 220 r / min for 48 h. 1 mL of the culture was centrifuged at 12000 r / min for 5 min, and 500 μL of the supernatant was mixed with an equal volume of Salkowski reagent. After color development in the dark at room temperature for 30 min, the color change was observed. A red color indicated that the strain had the ability to produce IAA. The absorbance was measured at 530 nm, using the blank medium as a control. A standard absorbance curve of IAA was plotted by setting different concentration gradients of IAA standard solutions (10, 20, 60, 70, 80 μg / mL), and the IAA yield was then calculated.

[0083] The experimental results showed that the color of the experimental group did not change significantly compared with the control group (see...). Figure 9 By setting up IAA standard solutions with different concentration gradients, the standard curve for IAA was obtained as follows: Y = 0.0045X + 0.0661, RA 2 =0.9987, see standard curve. Figure 10 Calculations showed that the IAA yield of strain YFLYS026 was -4.61 μg / mL, indicating that strain YFLYS026 cannot synthesize auxin IAA using L-tryptophan.

[0084] Example 4: Efficacy test of antagonistic strain YFLYS026 against anthracnose of Sedum morganianum.

[0085] Inoculation with wounds: To prevent the waxy leaves of the Rhododendron simsii from potentially hindering the attachment and infection of bacterial suspension or spores, this experiment used cotton balls soaked in a small amount of ethanol to gently wipe the leaf surface, removing part of the waxy layer. First, a sterile needle or blade was used to gently make tiny wounds on the leaf surface. Then, 10 mL of bacterial suspension (OD) of strain YFLYS026 was injected into a sterile spray bottle. 600 = 0.8) Spray evenly on the leaf surface, with sterile water treatment as a blank control (CK group). After inoculation, the *Syzygium stenoptera* round-leaved plants were placed in a dark environment at 28℃. After 24 hours, 10 mL of anthracnose conidial suspension (1×10⁻⁶) was evenly sprayed on the wound. 6~7 After the leaves were dry (CFU / mL), the plants were placed in a light incubator (28℃, 12h light / 12h dark, 3000lx, 80% RH) and cultured until there was no obvious liquid on the leaf surface. Simultaneously, 10ml of anthracnose conidial suspension was sprayed onto the leaves of plants not inoculated with strain YFLYS026 as an anthracnose treatment group. Observations began on the second day and were conducted daily to investigate the leaf infection status of *Ligustrum lucidum* roundleaf anthracnose and the control effect of antagonistic strains. Five plants were used for each treatment, and three leaves were treated on each plant.

[0086] After the treated Rhododendron simsii leaves were placed in an incubator for 14 days, the experimental results showed that ( Figure 11 The leaves of the *Sedum morganianum* round-leaf plants treated with sterile water showed a healthy green color overall, with good growth and no obvious discoloration or spots, while those inoculated with anthracnose... C. fructicolaIn the early stages, the leaves of the affected plants develop small, round, oval, or irregularly shaped reddish-brown spots with yellowish halos around the edges. As the culture time increases, these spots gradually enlarge, turning dark brown or grayish-brown. Later, multiple spots may merge, forming larger necrotic areas, leading to localized leaf wrinkling, curling, and drying. These spots often appear on the leaf margins, tips, or near the veins. Plants treated with a suspension of strain YFLYS026 showed smaller, fewer, and lighter-colored spots on their leaves, with leaf morphology similar to the control group, and no obvious wrinkling, perforation, or drying. Therefore, inoculation with strain YFLYS026 alleviated the disease symptoms on the leaves, indicating that the antagonistic strain YFLYS026 is effective against anthracnose. C. fructicola It has a significant preventive and therapeutic effect on anthracnose caused by *Sedum morganianum*.

[0087] Example 5: Efficacy test of antagonistic strain YFLYS026 against root rot in Lilium longiflorum

[0088] Microorganisms were inoculated using soaking and root drenching methods. The retained strains of *Fusarium oxysporum*, *Fusarium solanum*, *Fusarium spp.*, and *Fusarium circumsporum* were activated on PDA plates and then transferred to PDB medium. The medium was incubated on a shaker at 28°C for 7 days. The mycelia were then removed by filtering the cultured strains from the liquid medium through four layers of gauze. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was mixed with an appropriate amount of sterile water to prepare a solution with a concentration of approximately 1×10⁻⁶. 6~7 Prepare a CFU / mL spore suspension. Transplant 2-week-old *Lilium tigrinum* tissue culture seedlings into pots and cultivate outdoors for 2 weeks. Then, dig them up, spray the bulb area with 75% ethanol for surface disinfection, rinse five times with sterile water, and wipe away surface moisture with filter paper. Make incisions on the disinfected bulb surface with a sterile blade and place them in a sterilized beaker. Inject a bacterial suspension of YFLYS026 (OD600 = 0.8) and soak for 1 hour. Immediately after treatment, plant the seedlings in plastic pots filled with sterilized soil, inject 10 ml of YFLYS026 bacterial suspension at the base, and incubate at room temperature for 24 hours. Then, inject another 10 ml of a 1×10⁻⁶ concentration of spore suspension into the base. 6~7 CFU / mL spore suspension was used as the control group (CK group) with 10 ml of sterile water added, and the root rot treatment group had 15 plants added to each treatment. The treated plants were placed in a light incubator (28℃, 12h light / 12h dark, 3000 lx, 80% RH) and observed from the second day onwards, once a day, to investigate the root rot of the Lilium draco plants and the control effect of the antagonistic strain.

[0089] After 3 days of cultivation, the experimental results showed that the leaves of the plants inoculated with the pathogen began to turn yellow in some areas, while the plants in the other two treatments grew well and had bright green leaves. After 7 days of cultivation, it was found that the growth of the plants inoculated with the pathogen was inhibited, more than 2 / 3 of the leaves showed wilting and discoloration, brown lesions appeared on the root bulbs, and even rotten symptoms appeared at the junction of the root hairs and the bulbs. Figure 12 (B in the text), while the plants inoculated with sterile water and the antagonistic strain YFLYS026 showed significantly better growth than those inoculated only with the pathogen, with brighter green leaves and similar growth characteristics. Figure 12 (A and C in the text). Meanwhile, plants inoculated with the antagonistic strain YFLYS026 showed some brown spots on their roots, but the roots did not rot. Figure 12 (C in the text). This indicates that inoculation with the antagonistic strain YFLYS026 can effectively reduce the occurrence of root rot in Lilium acutum caused by Fusarium oxysporum, Fusarium solanum, Fusarium spp., and Fusarium circumsus, demonstrating its potential biocontrol effect.

[0090] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. An antagonistic bacterium YFLYS026, characterized in that, Its accession number is CGMCC NO.35236, and its classification name is Burkholderia pyrrole. Burkholderia pyrrocinia It was deposited at the China General Microbiological Culture Collection Center on July 15, 2025; it has an inhibitory effect on the pathogens of anthracnose of Rhizoma Sedum sarmentosum and root rot of Lilium acutum; It is effective against anthrax bacteria. Colletotrichum fructicola Shared Fusarium Fusarium commune Fusarium circumflexum Fusarium annulatum and Fusarium verticillata Fusarium verticillioides It has an inhibitory effect; It is effective against Fusarium oxysporum Fusarium oxysporum Fusarium solani Fusarium oxysporum It has an inhibitory effect on root rot of Lilium acutum caused by Fusarium spp. and Fusarium circumsii; The antagonistic bacterium YFLYS026 was isolated and screened from the rhizosphere soil of Lilium longiflorum plants.

2. The application of the antagonistic bacterium YFLYS026 or its bacterial solution as described in claim 1 in the preparation of products for the prevention and control of anthracnose in *Lilium lancifolium* or for the prevention and control of root rot in *Lilium longiflorum*, characterized in that, The anthracnose of the mountain jasmine round-leaf variety is caused by anthracnose bacteria. Colletotrichum fructicola The root rot of *Lilium lancifolium* is caused by *Fusarium oxysporum*, *Fusarium solanum*, *Fusarium spp.*, and *Fusarium circumcisionum*.

3. The application according to claim 2, characterized in that, The product is a microbial agent or pesticide.

4. The application according to claim 3, characterized in that, The product is applied by foliar spraying or root irrigation, or by applying it to the soil before planting.

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