Stenotrophomonas CLX-022 strains and uses thereof

Abstract

The invention provides a stenotrophomonas sp. CLX-022 strain which has a good antagonistic effect on colletotrichum gloeosporioides and fusarium fusiforme, and the strain is preserved in China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) and has a good antagonistic effect on colletotrichum gloeosporioides and fusarium fusiforme. The strain is preserved in China Center for Type Culture Collection (CCTCC) NO: M20252110 on September 24, 2025, the postal code is 430072, and the preservation number is CCTCC NO: M20252110. The 16S rDNA (ribosomal deoxyribonucleic acid) sequence of the strain is as shown in SEQ ID NO. 1. The stenotrophomonas CLX-022 strain is high in antibacterial property and can be used for preparing a microbial preparation for treating / preventing strawberry anthracnose and rice bakanae disease.

Description

[0001] This invention relates to an oligotrophic monotypic bacterium strain that exhibits strong antibacterial and antagonistic effects against *Colletotrichum gloeosporioides* and *Fusarium oxysporum*, and also has a good growth-promoting effect on plants, belonging to the field of microbial technology. Background Technology

[0002] Strawberry anthracnose is caused by Colletotrichum gloeosporioides (C. gloeosporioides) Colletotrichum gloeosporioides Anthracnose is a disease of strawberries caused by infection with *Anthracnose causalus* or *Anthracnose spp.*. Two types of anthracnose fungi infect strawberry leaves: one is anthracnose leaf spot, also called black spot, caused by *Colletotrichum gloeosporioides* and *Anthracnose spp.*; the other is irregular leaf spot, caused by *Anthracnose causalus*. Strawberry anthracnose mainly occurs on the leaves, petioles, stolons, rhizomes, flowers, and fruits of strawberries. Strawberry anthracnose occurs in strawberry-growing regions worldwide, but the specific pathogens vary from region to region. The high temperature and humidity environment in greenhouses has exacerbated the occurrence of strawberry anthracnose, often causing yield reductions of 25-30%, severely impacting strawberry yield and quality. Strawberry anthracnose has become the third largest disease restricting the development of China's strawberry industry, after strawberry gray mold and strawberry powdery mildew.

[0003] Rice seedling blight, also known as excessive growth disease, is caused by Fusarium oxysporum (Fusarium oxysporum). Fusarium fujikuroi This disease, caused by infection, occurs in all rice-growing areas of China. Diseased grains often fail to germinate or emerge from the soil after sowing. During the seedling stage, infected seedlings are thinner and taller than healthy seedlings, with slender leaf sheaths, pale yellow leaves, and poor root development; some seedlings die before transplanting. A pale red or white powdery mold, the conidia of the pathogen, is present on dead seedlings. Under high humidity, the surface of dead plants is covered with a pale brown or white powdery mold, later developing into small black dots, the fungal cysts. Mildly infected plants produce early heading, but the panicles are small and empty. Grains can also be affected during the heading stage; severely infected grains turn brown, fail to set grain, and develop pale red mold in the crevices of the glumes. Mild cases may not show symptoms, but mycelium may already be latent inside.

[0004] Biological control methods, due to their advantages of low cost, high efficiency, and low pollution, have gradually become a key research area for agricultural disease control. The screening and application of antagonistic bacteria in biological control is currently a major measure for dealing with plant diseases. Endophytic bacteria, which are bacteria that exist long-term within healthy plants without causing obvious diseases in the host plant, possess a certain degree of biosafety. Endophytic bacteria are also easy to cultivate and ferment, requiring less time and having simpler methods for extracting secondary metabolites, thus gradually becoming a major source of biocontrol bacteria. Screening antagonistic bacteria with good control efficacy against strawberry anthracnose, banana wilt, and rice bakanae disease can provide more strain resources and measures for the biological control of these plant diseases, enabling better application in the large-scale control of strawberry anthracnose, banana wilt, and rice bakanae disease.

[0005] At present, there are many related researches on the antagonistic bacteria of Colletotrichum gloeosporioides and rice seedling blight, but there is no related report on the endophytic bacteria of Stenotrophomonas sp. Stenotrophomonas sp. for antagonizing Colletotrichum gloeosporioides and Fusarium fujikuroi in the prior art. SUMMARY

[0006] The present application solves the problems in the background art and provides an endophytic bacteria Stenotrophomonas sp. CLX-022 strain with good antagonistic effect on Colletotrichum gloeosporioides and Fusarium fujikuroi. Stenotrophomonas The endophytic bacteria has strong antibacterial property and can be used in the preparation of microbial agents for treating / preventing these plant diseases.

[0007] The applicant screens a new strain from the endophytic bacteria of Paris polyphylla, which is a Stenotrophomonas CLX-022 strain. The strain is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a postal code of 430072, on September 24, 2025, with a preservation number of CCTCC NO: M 20252110. The 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.

[0008] The Stenotrophomonas CLX-022 strain provided in the present application can be used in the preparation of an antagonistic bacterial agent of Colletotrichum gloeosporioides, and in the preparation of a microbial agent for treating / preventing strawberry anthracnose.

[0009] The Stenotrophomonas CLX-022 strain provided in the present application can be used in the preparation of an antagonistic bacterial agent of Fusarium fujikuroi, and in the preparation of a microbial agent for treating / preventing rice seedling blight.

[0010] The Stenotrophomonas CLX-022 strain provided in the present application can also promote the growth of wheat seedlings.

[0011] Compared with the prior art, the present application has the following advantages: The inhibition rate of the CLX-022 strain provided in the present application on the pathogen of strawberry anthracnose, Colletotrichum gloeosporioides, can reach 17.15%, and the inhibition rate on the pathogen of rice seedling blight, Fusarium fujikuroi, can reach 11.13%. The CLX-022 strain can be further used to develop microbial pesticides for preventing these plant diseases, and the prevention effect is remarkable. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 FIG. 1 is a graph showing the antagonistic effect of the CLX-022 strain on the pathogen of strawberry anthracnose, Colletotrichum gloeosporioides (A), the pathogen of strawberry fusarium wilt, Fusarium oxysporum (B), the pathogen of rice seedling blight, Fusarium fujikuroi (C), and the pathogen of banana fusarium wilt, Fusarium oxysporum f. sp. cubense (D) in the embodiments of the present application; Figure 2Examples of embodiments in this application Figure 2 Figure 2 shows the antagonistic effect of fermentation broth (A), intracellular material (B), bacterial cells (C), and control (D) of Oligotrophomonas CLX-022 on the anthracnose pathogen of strawberry, Colletotrichum gloeosporioides (2 days). Figure 3 The graph shows the inhibition rate (3d) of methanol, ethanol and n-butanol extracts of Oligotrophomonas CLX-022 fermentation broth against the strawberry anthracnose pathogen, Colletotrichum gloeosporioides. Figure 4 The following figures show the disease incidence of strawberry leaves after spraying CLX-022 ethanol extract in the embodiments of this application (5 days): (A): Inoculation with pathogen; (B): Ethanol (extract solvent) treatment (control group); (C): Ethanol extract treatment 24 h after inoculation; (D): Ethanol extract treatment 0 h after inoculation. Detailed Implementation

[0013] The present invention will now be described in detail with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0014] Oligotrophomonas CLX-022 was isolated from the rhizome of Paris polyphylla.

[0015] After rinsing the surface soil of the Paris polyphylla rhizomes with tap water, the outer skin was removed under aseptic conditions. The rhizomes were then soaked in 1.0% NaClO solution for 5 min, rinsed with sterile water for 5 min, soaked in 75% (v / v) ethanol for 1 min, and rinsed with sterile water for 3 min. 0.1 g of rhizome tissue was then gently ground into a suspension in 2 mL of physiological saline and spread onto LA medium. The suspension was incubated at 28±2 ℃ for 7 days. During the incubation period, newly grown endophytic bacteria were continuously picked and transferred to fresh medium, and single clones were purified using the streak plating method. The applicant used a plate confrontation experiment to analyze the antagonistic effects of 100 strains of Paris polyphylla endophytic bacteria on plant pathogens such as Colletotrichum gloeosporioides, Fusarium oxysporum, Fusarium oxysporum f. sp. coli, and Fusarium oxysporum f. sp. coli Cuban-specific strain. The applicant also analyzed the growth-promoting ability of these endophytic bacteria on wheat seedlings through hydroponic and pot experiments. The results showed that an endophytic bacterium with the number CLX-022 had good antagonistic and growth-promoting effects.

[0016] Genomic DNA was extracted from the endophytic bacterium CLX-022, and its 16S rDNA sequence was amplified using CLX-022 as a template. After recovery, the amplified product was ligated into the pGEM-T vector, transformed into competent Escherichia coli cells, identified by colony PCR, and then sequenced and compared. The 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0017] Endophytic bacteria CLX-022 and Stenotrophomonas sp.The 16S rDNA sequence (GenBank no. MH232037.1) of strain 2.3SPb8 showed 99.71% identity, and was consistent with... Stenotrophomonas rhizophila The 16S rDNA sequence of strainRb2D (GenBank no. PQ803940.1) showed 99.61% identity, and was consistent with... Stenotrophomonas rhizophila The 16S rDNA sequence of strain MAIDO-R16b-10 (GenBank no. MW631981.1) showed 99.90% identity, and was consistent with... Stenotrophomonas sp. The 16S rDNA sequence of strain B1P3 (GenBank no. OQ221849.1) showed 99.81% identity. Based on this, and combined with its 16S rDNA phylogenetic tree, strain CLX-022 was identified as belonging to the genus *Oligotrophomonas*. Stenotrophomonas sp. ).

[0018] The strain was deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China 430072, on September 24, 2025, with accession number CCTCC NO: M 2025 2110.

[0019] Experimental materials: Plant pathogens: including the strawberry anthracnose pathogen—Colletotrichum gloeosporioides (… Colletotrichum gloeosporioides ), the pathogen of strawberry wilt—Fusarium oxysporum ( Fusarium oxysporum Rice seedling blight pathogen—Fusarium oxysporum ( Fusarium fujikuroi ); Fusarium oxysporum, the pathogen of banana wilt, is a Cuban-specific strain ( Fusarium oxysporum f. sp. cubense). These plant disease strains were provided by the Institute of Economic Crops, Hubei Academy of Agricultural Sciences.

[0020] Plant materials: Red Beauty strawberry plants were provided by the Institute of Economic Crops, Hubei Academy of Agricultural Sciences.

[0021] Experimental methods: 1. Determination of the antagonistic effect of Oligotrophomonas CLX-022 The antagonistic effect of *Oligotrophomonas CLX-022* was assessed using the plate confrontation method. Plant pathogen agar plates cultured for 7 days were used to remove mycelial growth using a 5mm punch and inoculate the growth into the center of the PDA plate. *Oligotrophomonas CLX-022* was then inoculated 2.5 cm away from the mycelial growth. The plates were incubated in the dark at 28℃. Six biological replicates were used for each treatment group. Recording began when the plant pathogen and endophytic bacteria came into contact. PDA plates inoculated only with the plant pathogen served as the control group. The inhibition rate was calculated as follows: Inhibition rate % = (Control group colony diameter - Treatment group colony diameter) / (Control group colony diameter) × 100%.

[0022] Figure 1 The antagonistic effects (3 days) of strain CLX-022 against *Colletotrichum gloeosporioides* (A), *Fusarium oxysporum* (B), *Fusarium oxysporum* (C), and *Fusarium oxysporum* Cuban-specific type (D), the pathogens of strawberry anthracnose, strawberry wilt, and rice bakanae disease, were demonstrated. Table 1 shows the inhibition rates of strain CLX-022. Strain CLX-022 exhibited good antagonistic activity against *Colletotrichum gloeosporioides*, with an inhibition rate of 17.15%. It also showed significant antagonistic effects against *Fusarium oxysporum* Cuban-specific type and *Fusarium oxysporum*, with inhibition rates of 15.22% and 11.13%, respectively. It also showed antagonistic activity against *Fusarium oxysporum*, with an inhibition rate of 2.51%. These results indicate that Oligotrophomonas CLX-022 has potential application value in the control of collodion anthracnose, banana wilt, and rice bakanae disease.

[0023] Table 1. Inhibition rate of oligotrophomonas CLX-022 against four pathogenic fungi (3 days)

[0024] 2. Antibacterial activity analysis of metabolites from Oligotrophomonas CLX-022: Oligotrophomonas CLX-022 was inoculated into 100 mL LB medium and fermented for 5 days. After centrifugation at 15000 rpm for 20 min, the supernatant was collected as treatment group A. The bacterial pellet was divided into two parts. One part of the bacterial cells was resuspended in 50 mL PBS buffer, sonicated, centrifuged at 15000 rpm for 20 min, and the supernatant was collected and filtered through a 0.22 μm microporous membrane. The filtrate was collected as treatment group B. The remaining bacterial cells were resuspended in 50 mL sterile PBS buffer as treatment group C. PBS buffer was also set up as a control group D. *Colletotrichum gloeosporioides* was inoculated into the center of a PDA plate. Four sterile Oxford cups were placed 2.5 cm away from the bacterial growth, and 200 μL of each treatment solution was added to each. The plates were incubated in the dark at 28 °C until an interaction occurred between the pathogen and the treatment solution. The antagonistic effect was observed and recorded.

[0025] Figure 2 This study demonstrates the antagonistic effects (2 days) of the fermentation broth (treatment group A), intracellular components (treatment group B), bacterial cells (treatment group C), and control group D (PBS buffer) of *Colletotrichum gloeosporioides* CLX-022 on *Colletotrichum gloeosporioides*. The fermentation broth (treatment group A) inhibited *Colletotrichum gloeosporioides* growth by 12.12%, the bacterial cells (treatment group C) by 10.33%, and the intracellular components (treatment group B) showed no inhibitory effect. This indicates that the antagonistic substances produced by *Colletotrichum gloeosporioides* CLX-022 are mainly present in the fermentation broth.

[0026] Subsequently, 50 mL of the Oligotrophomonas CLX-022 fermentation broth was extracted with equal volumes of n-butanol, ethyl acetate, chloroform and petroleum ether, ethanol and methanol, respectively. The extracts were then evaporated to dryness at 40 °C and dissolved in 2 mL of methanol. The antagonistic effects of each extract were analyzed using the Oxford cup plate confrontation experiment. Figure 3 The results showed that chloroform, ethyl acetate, and petroleum ether extracts did not significantly inhibit *Colletotrichum gloeosporioides*, while methanol, ethanol, and n-butanol extracts did significantly inhibit *Colletotrichum gloeosporioides*, with inhibition rates of 20.19%, 24.98%, and 16.02%, respectively. This indicates that the antagonistic substances produced by *Oligotrophomonas CLX-022* are soluble in methanol, ethanol, and n-butanol, and the ethanol extract has a better antibacterial effect.

[0027] 3. Experiments on detached strawberry leaves to verify the disease-resistant effect of CLX-022 ethanol extract: Select healthy, uniformly growing strawberry compound leaves, wash them with sterile water, disinfect the surface with 75% ethanol, clean them with sterile water, and let them air dry. Wrap a damp paper towel around the petiole 2-3 cm deep.

[0028] Activated *Colletotrichum gloeosporioides* was inoculated onto PDL medium and cultured at 28°C and 150 rpm for 2 days. Conidia were collected by filtration, counted using a hemocytometer, and then diluted to 1×10⁻⁶. 6CFU / mL, inoculated by leaf dipping, with a control group consisting of strawberry leaves dipped in sterile water, 8-10 leaves per group. The ethanol extract of CLX-022 was sprayed onto detached strawberry leaves inoculated with conidia of *Colletotrichum gloeosporioides* to analyze the inhibitory effect of the CLX-022 ethanol extract on strawberry anthracnose, with simultaneous inoculation and 24-hour interval inoculation groups. Strawberry leaves were cultured in a light incubator at 28℃, a light / dark cycle of 12h / 12h, and 85% humidity. Disease lesions on strawberry leaves were observed and recorded after 7 days. The percentage of diseased area on strawberry leaves (%) = diseased area / total leaf area × 100%. The control rate of diseased area on strawberry leaves (%) = (ab) / a × 100%, where a is the percentage of diseased area on strawberry leaves in the control group and b is the percentage of diseased area on strawberry leaves in the treatment group.

[0029] The experimental results are shown in Figure 4 And Table 2, Figure 4 In the diagram, (A): Inoculation with pathogenic bacteria; (B): Ethanol treatment (control group); (C): Ethanol extract treatment 24 hours after inoculation; (D): Ethanol extract treatment 0 hours after inoculation. Figure 4 As shown in Table 2, the control (ethanol) treatment group did not inhibit the lesions on detached strawberry leaves. Spraying the ethanol extract of Oligotrophozoites CLX-022 fermentation broth effectively inhibited the growth of Colletotrichum gloeosporioides on detached strawberry leaves, and the inhibitory effect of spraying the extract was more obvious 24 hours after inoculation with Colletotrichum gloeosporioides.

[0030] Table 2. Experimental verification of the control effect of CLX-022 ethanol extract on *Colletotrichum gloeosporioides* on detached strawberry leaves.

[0031] 4. Growth-promoting effect of Oligotrophomonas CLX-022 Hydroponic Experiment: Jimai 22 seeds were soaked in sterile water for 1 min, followed by surface disinfection with 75% ethanol for 1 min. The disinfected seeds were placed in petri dishes lined with moist gauze and incubated in the dark at 28℃ for 2 days. After germination, 25 seeds were transferred to tissue culture flasks containing 200 mL of sterile water per flask. Three biological replicates were set up for each group. Oligotrophozoites CLX-022 were inoculated into LB medium and cultured at 28℃ and 150 rpm for 36 h. The bacterial cells were collected by centrifugation and resuspended in sterile water to OD600=1.0. 1 mL of bacterial suspension was inoculated into each tissue culture flask, sealed, and placed in a light incubator at 28℃ with a 12 h / 12 ​​h light / dark cycle. The hydroponic experiment results showed that, compared with the control group, the wheat plant height of the CLX-022 strain increased by 27.94% (p<0.05) and the root length increased by 35.76% (p<0.05) at the sampling time point on the 14th day.

[0032] Pot experiment: Newly germinated wheat seeds were transplanted into experimental boxes (33 cm × 21 cm × 16 cm) filled with substrate, with 80 seeds per box, and 3 biological replicates were set up. The fermentation broth of Oligotrophozoites CLX-022 was adjusted to OD using sterile water. 600 =1.0, 50 mL per box. An equal volume of sterile water was sprayed as a control group. Growth indicators were measured at 7, 14, 21, and 28 days. The results showed that after applying endophytic bacteria CLX-022, wheat plant height increased by 6.00% ( ) at 14 and 21 days compared to the control group. p <0.01) and 4.06% ( p <0.01), and the root length at sampling points on days 14 and 21 increased by 9.20% compared to the control group. p <0.05) and 19.10% ( p <0.05), the fresh weight at the sampling time point on day 21 increased by 28.35% compared with the control group ( p <0.05).

[0033] The hydroponic and potted experiments showed that the oligotrophomonas CLX-022 can promote the growth of plants (wheat).

Claims

1. A strain of oligotrophomonas ( Stenotrophomonas The strain sp.) CLX-022, with accession number CCTCCNO: M 20252110, has a 16S rDNA sequence as shown in SEQ ID NO.

1.

2. Use of the oligotrophomonas CLX-022 of claim 1 in the preparation of an antagonistic agent against collodion.

3. Use of the oligotrophomonas CLX-022 of claim 1 in the preparation of a drug for the prevention and treatment of collodion anthrax.

4. The use according to claim 3, characterized in that: The anthracnose mentioned includes strawberry anthracnose.

5. The use according to claim 3, characterized in that: The fermentation broth of Oligotrophozoites CLX-022 was used in the preparation of a drug for the prevention and treatment of collodion anthrax.

6. The use according to claim 5, characterized in that: The fermentation broth of Oligotrophomonas CLX-022 was extracted with methanol, ethanol or n-butanol to obtain methanol, ethanol or n-butanol phase extracts, which were then used in the preparation of drugs for the prevention and treatment of collodion anthrax.

7. Use of the oligotrophomonas CLX-022 of claim 1 in the preparation of an antagonistic agent against Fusarium oxysporum.

8. Use of the oligotrophomonas CLX-022 of claim 1 in the preparation of a drug for the prevention and control of rice bakanae disease.

9. The use according to claim 8, characterized in that: Oligotrophomonas CLX-022 was prepared into an aqueous suspension agent and used in the preparation of drugs for the prevention and control of rice bakanae disease.

10. Use of the oligotrophomonas CLX-022 of claim 1 in the preparation of a wheat growth promoter.

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